Chemical synthesis using enzymatically generated building units for construction of the human milk pentasaccharides sialyllacto-N-tetraose and sialyllacto-N-neotetraose epimer

Article abstract of DOI:10.3762/bjoc.6.18

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 acce

Full text of DOI:10.3762/bjoc.6.18

Chemical synthesis using enzymatically generated
building units for construction of the human milk
pentasaccharides sialyllacto-N-tetraose
and sialyllacto-N-neotetraose epimer
Dirk Schmidt and Joachim Thiem*
Full Research Paper
Open Access
Address:
Beilstein Journal of Organic Chemistry 2010, 6, No. 18.
Faculty of Science, Department of Chemistry, University of Hamburg,
Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
doi:10.3762/bjoc.6.18
Received: 26 November 2009
Accepted: 11 February 2010
Published: 22 February 2010
Email:
Joachim Thiem* - thiem@chemie.uni-hamburg.de
* Corresponding author
Guest Editor: T. K. Lindhorst
Keywords:
© 2010 Schmidt and Thiem; licensee Beilstein-Institut.
License and terms: see end of document.
block synthesis; human milk oligosaccharides;
sialyllacto-N-neotetraose epimer; sialyllacto-N-tetraose; trisaccharide
thioglycoside donors
Abstract
α,2-3- and α,2-6-sialylated lactosaminide precursor structures obtained by various enzymatic procedures could be used for glycosyl-
ations 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.
Introduction
From an inspection of contemporary syntheses of biologically well as the yields. Whilst both approaches can be employed
and medicinally relevant oligosaccharides, it is evident that the advantageously in certain cases, in others this is certainly not
majority is performed either by classical chemical methods or so.
exclusively by enzymatic procedures. Even although consider-
able progress has been reported during the recent decades, every For instance, for structures that contain glycosamino units, it
synthesis of a complex heterooligosaccharide still represents a has been demonstrated that these units can be introduced by
challenge. To arrive at an oligosaccharide structure with classical methods in high yields and with good to excellent
specific patterns of substitution and defined regio- and stereo- stereochemical control. In case of glucosylations and galacto-
chemical layout, all the presently available procedures need to sylations, and in particular for β-galactosylations, classical and
be checked for efficiency with respect to not only all the above enzymatic methods are almost equal in terms of stereose-
points, but also the efforts required at the purification steps as lectivity and transfer efficiency. By contrast, for syntheses of
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Beilstein Journal of Organic Chemistry 2010, 6, No. 18.
sialylated structures, enzymatic procedures are still consider- β-galactosylation of the 2-azidothioglucoside with
ably superior to classical chemical sialylations with respect to p-nitrophenyl β-galactopyranoside and β-galactosidase (Bovin
both stereochemical outcome and preparative input.
testes). The subsequent transsialylation was carried out with
p-nitrophenyl sialoside (pNp-αNeu5Ac) and either sialidase
The use of both procedures in a synergistic mode should also be from Salmonella typhimurium or from Newcastle disease virus
considered. One of the general approaches ideally suited in such [7]. Recently, a more effective higher yielding transfer has been
cases is the block synthesis method. Moreover, in recent years a reported in which sialylation with recombinant transsialidase
number of combined chemical and chemoenzymatic syntheses (Trypanosoma cruzi) gave the trisaccharide 3 in 32% yield [8].
have been reported [1-4].
Treatment of 3 with methanol and acidic ion exchange resin led
to the methyl ester (for the method cf. lit. [9]) which was then
As a proof of principle, we were interested to employ some peracetylated to give trisaccharide 4 as the donor building
trisaccharide building units previously obtained by enzymatic block.
routes in block syntheses en route to interesting structures. To
this end, two human milk pentasaccharides of prominent For formation of the disaccharide acceptor 6, a straight-forward
importance, sialyllacto-N-tetraose (1) and an epimer of sialyl- three-step standard reaction sequence was used [10]. Methyl
lacto-N-neotetraose (2) (Figure 1) were selected as target β-lactoside was isopropylidenated at 3′,4′-position with dimeth-
molecules. Both these pentasaccharides, Neu5Acα2-3Galβ1- oxypropane and p-toluene sulfonic acid in DMF/acetone. Per-
3GlcNAcβ1-3Galβ1-4Glc (1) and Neu5Acα2-6Galβ1- acetylation (Ac2O/Py) and subsequent cleavage of the isoprop-
4GlcNAcβ1-4Galβ1-4Glc (2), are dominant constituents of ylidene group with 80% acetic acid at 80 °C gave the diol
complex human milk oligosaccharides (Figure 1). They are acceptor 6. Since it is known that in galactopyranosyl struc-
considered to play a major role in immuno defense against tures the nucleophilicity of 3-OH considerably exceeds that of
bacterial and viral infections in the gastrointestinal tract of the 4-OH-group, further protecting group manipulations were
infants [5]. It is thought that they effectively inhibit bacterial not required.
adhesion to epithelial surfaces and so block the first stages of
infection processes. Thus, these human milk oligosaccharides Glycosylation of 4 by 6 catalyzed by N-iodosuccinimide and
are considered as soluble receptor analogues of epithelial cell trifluoromethane sulfonic acid (as introduced by van Boom et
surfaces [6].
al. [11]) 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
Results and Discussion
Previously, we reported the chemoenzymatic synthesis of the observed as side products and separated by chromatography but
3-sialylated lactosamine derivative 3 obtained by the enzymatic these were not further characterized. Reduction of the 2′′′-azido
Figure 1: Structures of pentasaccharides 1 and 2.
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Beilstein Journal of Organic Chemistry 2010, 6, No. 18.
Scheme 1: Preparation of pentasaccharide 8. 1) MeOH, acidic ion exchange resin; 2) Ac2O, pyridine; 3) 80% HOAc, 90 °C; 4) NIS, CF3SO3H, 61%;
5) NiCl2•6H2O, H3BO3, EtOH, then NaBH4, EtOH and acidic workup.
to the 2′′′-amino group with nickel boride [12,13] followed by by transsialylation with recombinant transsialidase
peracetylation gave 8 in 81% yield (Scheme 1). 1H NMR spec- (Trypanosama cruzi) [8]. Formation of the methyl ester and per-
trum contained a doublet for H-1″ at δ 4.96 (J1″2″ = 8.0 Hz) and acetylation led to the trisaccharide donor building block 10.
a down field shifted doublet for H-4′ at δ 5.37 (J3′4′ = 2.9 Hz).
Synthesis of the disaccharide acceptor in this case started from
A similar approach was employed for the synthesis of the methyl β-lactoside, which was transformed into its 4′,6′-
protected epimer of sialyllacto-N-neotetraose 14. β-Galacto- benzylidene-protected derivative 11 in almost quantitative yield
sylation of 2-azidothioglucoside with p-nitrophenyl by transacetalization with benzaldehyde dimethylacetal in
β-galactopyranoside and β-galactosidase (Bacillus circulans) acetonitrile under p-toluenesulfonic acid catalysis. Subsequent
gave the β,1-3-linked isolactosamine derivative. Further sialyl- peracetylation with acetic anhydride/pyridine, selective
ation at position 3′-OH with pNp-αNeu5Ac and either sialidase cleavage of the benzylidene group with 80% acetic acid at 90
from Vibrio cholerae or Clostridium perfringens afforded the °C and finally treatment with tert-butyldiphenylsilyl chloride
α,2-6-sialylated trisaccharide 9 exclusively [7]. Later studies and imidazole in DMF afforded the disaccharide 12 (cf. refer-
showed that 9 could be obtained in an enhanced yield of 32% ences [14,15]).
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Beilstein Journal of Organic Chemistry 2010, 6, No. 18.
After activation of the trisaccharide donor 10 with N-iodo- of 14 with the precursor tri- and disaccharide units 10 and 12,
succinimide and trifluoromethanesulfonic acid, the disac- the novel characteristic doublet for the anomeric H-1″ of the
charide acceptor unit 12 could be glycosylated to give the β,1-4- β-GlcNAc unit at δ 5.12 (J1″2″ = 8.2 Hz) as well as the down-
linked pentasaccharide derivative 13 in 53% yield. In addition, field shift Δδ 0.15 of H-4′ to δ 4.14 compared to 12 were in
the corresponding α,1-4-linked pentasaccharide was obtained in accord with structure of the target pentasaccharide.
8% yield.
Conclusion
Finally, the azido group was reduced by the nickel boride In this contribution chemoenzymatically generated sialyl α,2-3-
method with sodium borohydride, nickel chloride and boric acid and sialyl α,2-6-glycosylated thiophenol 2-azido-lactose deriva-
[12,13]. During this step partial cleavage of the tert- tives were employed as precursors for sialylated lactosaminide
butyldiphenylsilyl groups was also observed. Complete removal donor substituents in triflic acid/N-iodosuccinimide glycosyl-
was achieved with trifluoroacetic acid in dichloromethane. For ations. With easily accessible selectively unprotected lactose
characterization purposes, peracetylation was carried out to give acceptor glycosides the pentasaccharide structures sialyllacto-N-
the completely protected pentasaccharide 14 in 67% yield tetraose and the epimer of sialyllacto-N-neotetraose could be
(Scheme 2). As evident from a comparison of the 1H NMR data obtained in good yields, and subsequently transformed into their
Scheme 2: Preparation of pentasaccharide 14. 1) MeOH, acidic ion exchange resin; 2) Ac2O, pyridine; 3) 80% HOAc, 90 °C; 4) TBDPSCl, imidazole,
DMF; 5) NIS, CF3SO3H, 53%; 6) NiCl2•6H2O, H3BO3, EtOH, then NaBH4, EtOH, then acidic workup; 7) CF3CO2H, CH2Cl2.
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Beilstein Journal of Organic Chemistry 2010, 6, No. 18.
peracetylated derivatives for structure elucidation. Thus, a = 2.8 Hz, 1 H, H-3IV), 4.51 (d, J1,2 = 7.8 Hz, 1 H, H-1I), 4.49
combination of enzymatic and purely chemical procedures was (dd, 1 H, H-6V), 4.39 (d, J1,2 = 8.0 Hz, 1 H, H-1II), 4.37 (d, J1,2
shown to be advantageous in the preparation of complex = 8.1 Hz, 1 H, H-1III), 4.29 (dd, J9a,9b = 12.4 Hz, J8,9b = 2.4 Hz,
oligosaccharides.
H-9bV), 4.24 (dd, J6a,6b = 12.6 Hz, J5,6a = 6.6 Hz, 1 H, H-6aIII),
4.17 (dd, J5,6 = 10.2 Hz, H-5V), 4.13 (d, 1 H, NH), 3.75 (s, 3 H,
COOCH3V), 4.00 (dd, J8,9a = 5.8 Hz, 1 H, H-9aV), 3.72 (bt, 1
Experimental
For general methods cf. reference [16]. The NMR data for the H, H-5IV), 3.59 (m, 1 H, H-5I), 3.47–3.44 (m, 2 H, H-3II,
saccharide rings in the pentasaccharides 7, 8, 13 and 14 are H-6aIV), 3.39 (dd, J6a,6b = 11.0 Hz, J5,6b = 7.8 Hz, H-6bIV),
denoted according to the Roman numberals I-V from the redu- 2.61 (dd, J3ax,3eq = 12.4 Hz, J3eq,4 = 4.8 Hz, 1 H, H-3eqV),
cing end, as depicted for compounds 8 and 14 (Figure 2):
2.17–1.33 (15s, 45 H, 14 OAc, 1 NAc), 1.91 (dd, J3ax,4 = 11.8
Hz, 1 H, H-3axV). C67H92N4O43 (1641.45): Found C, 49.33; H,
Methyl O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- 5.59; N, 3.62. Calculated C; 49.02; H, 5.65; N, 3.41. MALDI-
dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2- TOF: 1664.44 (M+Na)+; 1680.59 (M+K)+.
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- Methyl O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1-4)-2,3,6-tri-O- dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-
acetyl-β-D-glucopyranoside (7): Glycosylation was carried out 3)-O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1-3)-O-(4,6-
as described for the synthesis of compound 13 from compound di-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1-3)-
4 (95 mg, 83 μmol) as donor and compound 6 (50 mg, 86 μmol) O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1-4)-2,3,6-tri-
as acceptor. The pentasccharide derivative 7 was obtained as a O-acetyl-β-D-glucopyranoside (8): Reduction and peracetyl-
colorless amorphous solid; 83 mg (61%); [α]D20 = −3.5 (c 0.1, ation of compound 7 was carried out as described for 14. Thus,
CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.75 (dt, 1 H, H-8V), from 80 mg (49 μmol) of 7, 65 mg (81%) of 8 was obtained as a
5.72 (d, 1 H, NH), 5.39 (d, J3,4 = 2.8 Hz, 1 H, H-4II), 5.36 (dd, colorless amorphous solid; [α]D20 = −12.7 (c 0.5, CHCl3); 1H
J7,8 = 9.3 Hz, 1H, H-7V), 5.15 (dd, J2,3 = J3,4 = 9.4 Hz, 1 H, NMR (500 MHz, CDCl3) δ 5.74 (dt, 1 H, H-8V), 5.72 (d, 1 H,
H-3I), 5.06 (dd, J1,2 = 8.0 Hz, J2,3 = 10.2 Hz, H-2IV), 4.99–4.97 NH), 5.38 (d, J3,4 = 2.8 Hz, 1 H, H-4II), 5.33 (dd, J7,8 = 9.3 Hz,
(m, 2 H, H-2II, H-4III), 4.96 (dd, J1,2 = 7.8 Hz, J2,3 = 9.6 Hz, 1 1H, H-7V), 5.12 (dd, J2,3 = J3,4 = 9.6 Hz, 1 H, H-3I), 5.05 (dd,
H, H-2I), 4.91 (dd, J1,2 = 8.0 Hz, J2,3 = 10.2 Hz, 1 H, H-1IV), J1,2 = 8.0 Hz, J2,3 = 10.2 Hz, H-2IV), 4.99 (dd, J1,2 = 8.0 Hz,
4.87 (d, J3,4 = 2.6 Hz, H-4IV), 4.83 (dt, J3eq,4 = 4.4 Hz, J3ax,4 = J2,3 = 9.7 Hz, 1 H, H-2II), 4.97 (m, 2 H, H-4III), 4.95 (dd, J1,2 =
J4,5 = 12.1 Hz, 1 H, H-4V), 4.75 (dd, J5,6 = 10.1 Hz, J6,7 = 2.2 8.0 Hz, J2,3 = 9.8 Hz, 1 H, H-2I), 4.91 (dd, J1,2 = 7.9 Hz,1 H,
Hz, H-6V), 4.62 (dd, 1 H, H-3III), 4.57 (dd, J2,3 = 10.0 Hz, J3,4 H-1IV), 4.86 (dt, J3eq,4 = 4.6 Hz, J3ax,4 = J4,5 = 12.0 Hz, 1 H,
Figure 2: Roman numbering of saccharide units in all pentasaccharides for NMR assignment.
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Beilstein Journal of Organic Chemistry 2010, 6, No. 18.
H-4V), 4.83 (d, J3,4 = 2.6 Hz, H-4IV), 4.75 (dd, J5,6 = 9.9 Hz, H-5IV), 3.83 (m, 2 H, H-5I, H-4III), 3.78 (s, 3 H, OCH3), 3.77
J6,7 = 1.8 Hz, H-6V), 4.61 (dd, 1 H, H-3III), 4.57 (dd, J2,3 = 9.8 (t, J3,4 = 9.9 Hz, 1 H, H-4I), 3.62 (ddd, J4,5 = 10.0 Hz, J5,6a =
Hz, J3,4 = 2.6 Hz, 1 H, H-3IV), 4.53 (dd, 1 H, H-6I), 4.51 (d, 5.5 Hz, J5,6b = 2.0 Hz, 1 H, H-5I), 3.61 (dd, 1 H, H-6bIV), 3.59
J1,2 = 8.0 Hz, 1 H, H-1I), 4.39–4.37 (m, 2 H, H-1II, H-1III), 4.32 (ddd, 1 H, H-5III), 3.56 (dd, J2,3 = 10.2 Hz, 1 H, H-2III), 3.33 (s,
(dd, J9a,9b = 12.3 Hz, J8,9b = 2.1 Hz, H-9bV), 4.26 (dd, J6a,6b = 3 H, CH3V), 2.69 (dd, J3eq,3ax = 12.7 Hz, J3eq,4 = 4.6 Hz, 1 H,
12.2 Hz, J5,6a = 6.1 Hz, 1 H, H-6aIII), 4.13 (dd, J5,6 = 9.8 Hz, H-3eqV), 2.15–1.36 (14s, 42 H, 13 OAc, 1 NAc), 2.03 (dd,
H-5V), 4.13 (d, 1 H, NH), 4.00 (dd, J8,9a = 5.8 Hz, 1 H, H-9aV), J3ax,4 = 12.0 Hz, 1 H, H-3axV), 1.01 (s, 9 H, SiCCH3).
3.75 (s, 3 H, COOCH3V), 3.71 (bt, 1 H, H-5IV), 3.55 (m, 1 H, C81H108N4O42Si (1837.81): Found C, 53.89; H, 6.34; N, 2.66.
H-5I), 3.46–3.44 (m, 2 H, H-6aIV, H-3II), 3.43 (dd, J6a,6b = 11.0 Calculated C, 52.94; H, 5.92; 3.04. MALDI-TOF: 1860.80
Hz, J5,6b = 7.8 Hz, H-6bIV), 2.59 (dd, J3ax,3eq = 12.2 Hz, J3eq,4 (M+Na)+; 1876.91 (M+K)+. The α,1III-4II-anomer of 7 was
= 4.6 Hz, 1 H, H-3eqV), 2.21–1.35 (17s, 51 H, 15 OAc, 2 NAc), obtained as colorless syrup (9 mg, 8%) and not further charac-
1.93 (dd, J3ax,4 = 12.0 Hz, 1 H, H-3axV). C69H96N2O44 terized.
(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)+; Methyl O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
1696.59 (M+K)+.
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-
Methyl O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- di-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1-4)-
dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2- O-(2,3,6-tri-O-acetyl-β-D-galactopyranosyl)-(1-4)-O-2,3,6-
6)-O-(2,3,4-tri-O-acetyl-β-D-galactopyranosyl)-(1-4)-O-(3,6- tri-O-acetyl-β-D-glucopyranoside (14): Compound 13 (53
di-O-acetyl-2-azido-2-deoxy-β-D-glucopyranosyl)-(1-4)-O- mg, 29 μmol), NiCl2·6H2O (105 mg, 450 μmol) and boric acid
(2,3-di-O-acetyl-6-O-tert-butyldiphenylsilyl-β-D- (55 mg, 900 μmol) were dissolved in ethanol (3 mL). Under
galactopyranosyl)-(1-4)-O-2,3,6-tri-O-acetyl-β-D-gluco- vigorous stirring a suspension of sodium borohydride (28 mg,
pyranoside (13): A solution of trisaccharide 10 (68 mg, 60 750 μmol) in ethanol (1 mL) was added with the temperature
μmol) and disaccharide 12 (56 mg, 70 μmol) in anhydrous maintained at 20 °C. After 30 min ethanol (6 mL) and acetic
toluene (2 mL) was cooled to −40 °C. N-Iodosuccinimide (20 acid (3 mL) were added. Then the mixture was co-distilled three
mg, 94 μmol), molecular sieves (4 Å, 200 mg) were added, and times with toluene (5 mL each), and then the residue dissolved
after cooling a saturated solution of trifluoromethane sulfonic in dichloromethane (10 mL). After washing with diluted
acid in CCl4 (ca. 2 M, 50 μL) was added with vigorous stirring. aqueous KHSO4 solution (5 mL), saturated aqueous NaHCO3
The mixture was gradually warmed over 2.5 h to −10 °C. Ethyl solution (5 mL), and water (5 mL), the organic phase was dried
acetate (20 mL) was added and the reaction quenched by add- (MgSO4) and evaporated to dryness. The resulting material was
ition of a saturated aqueous NaHCO3 solution (10 mL). After treated with dichloromethane/trifluoroacetic acid (9:1, 2 mL)
filtration through Celite, the phases were separated. The organic for 1 h at room temperature, then co-distilled three times with
phase was washed with aqueous Na2S2O3 solution (10 mL), toluene (5 mL each) and dried under high vacuum. The residue
dried over MgSO4, evaporated and the residue purified by flash was treated with acetic anhydride (1 mL) and pyridine (5 mL)
chromatography with petroleum ether/ethyl acetate 2:1. Com- for 10 h, then co-distilled three times with toluene (5 mL each).
pound 7 was obtained as a colorless amorphous solid; 58 mg Purification by flash chromatography (toluene/acetone 3:1)
(53%). [α]D20 = −21.6 (c 0.3, CHCl3); 1H NMR (500 MHz, gave 14 (35 mg, 67%) as a colorless amorphous solid; [α]D20 =
CDCl3) δ 7.70–7.24 (m, 10, Ph), 5.74 (dd, 1 H, H-4IV), 5.70 −31.2 (c 0.4, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.72–7.25
(ddd, 1 H, H-8V), 5.61 (dd, J2,3 = 10.2 Hz, 1 H, H-2IV), 5.44 (m, 10, Ph), 5.72 (ddd, 1 H, H-8V), 5.70 (dd, 1 H, H-4IV), 5.59
(dd, J7,8 = 9.0 Hz, 1 H, H-7V), 5.26 (dd, 1 H, H-3IV), 5.22 (t, (dd, J2,3 = 9.9 Hz, 1 H, H-2IV), 5.43 (dd, J7,8 = 8.8 Hz, 1 H,
J2,3 = J3,4 = 9.8 Hz, 1 H, H-3I), 5.10 (dd, J1,2 = 8.0 Hz, J2,3 = H-7V), 5.32 (d, J3,4 = 4.0 Hz, H-4II), 5.23 (dd, 1 H, H-3IV), 5.21
9.9 Hz, 1 H, H-2II),4.93 (t, J2,3 = 9.8 Hz, 1 H, H-2I), 4.87 (2d, 2 (t, J2,3 = J3,4 = 10.2 Hz, 1 H, H-3I), 5.11 (dd, J1,2 = 7.8 Hz, J2,3
H, H-1IV, H-3III), 4.85 (ddd, J4,5 = 10.2 Hz, 1 H, H-4V), 4.65 = 9.8 Hz, 1 H, H-2II), 4.90 (t, J2,3 = 10.0 Hz, 1 H, H-2I), 4.88
(dd, 1 H, H-3II), 4.61 (d, J3,4 = 4.0 Hz, H-4II), 4.59 (dd, J9a,9b = (d, 1 H, H-1IV), 4.86 (m, 2 H, H-3III, H-4V), 4.63 (dd, 1 H,
12.2 Hz, 1 H, H-9aV), 4.51 (d, 1 H, H-1III), 4.49 (d, J1,2 = 8.0 H-3II), 4.59 (dd, J9a,9b = 11.9 Hz, 1 H, H-9aV), 4.53 (d, J1,2 =
Hz, 1 H, H-1II), 4.48 (dd, 1 H, H-6bIII), 4.46 (d, J1,2 = 10.0 Hz, 8.0 Hz, 1 H, H-1II), 4.50 (d, 1 H, H-1III), 4.47 (m, 2 H, H-6bIII,
1 H, H-1I), 4.42 (dd, 1 H, H-6bI), 4.34 (dd, 1 H, H-9bV), 4.32 H-1I), 4.39 (dd, 1 H, H-6bI), 4.32 (d, 1 H, NH), 4.31 (dd, 1 H,
(d, 1 H, NHV), 4.29 (ddd, J5,6 = 10.6 Hz, 1 H, H-5V), 4.20 (dd, H-9bV), 4.27 (ddd, J5,6 = 10.3 Hz, 1 H, H-5V), 4.18 (dd, 1 H,
1 H, H-6bIII), 4.13 (m, 2 H, H-6V, H-6bII), 4.10 (dd, J5,6a = 5.6 H-6bIII), 4.11 (dd, 1 H, H-6V), 4.09 (dd, J5,6a = 5.8 Hz, J6a,6b =
Hz, J6a,6b = 12.4 Hz, 1 H, H-6aI), 4.08 (dd, J5,6a = J6a,6b = 6.0 12.2 Hz, 1 H, H-6aI), 4.01 (dd, 1 H, H-6bIV), 3.92 (ddd, 1 H,
Hz, 1 H, H-6aII), 4.06 (dd, 1 H, H-6bIV), 3.88 (ddd, 1 H, H-5IV), 3.87 (dd, J5,6a = J6a,6b = 6.0 Hz, 1 H, H-6aII), 3.85 (dd,
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Beilstein Journal of Organic Chemistry 2010, 6, No. 18.
1 H, H-6bII), 3.82 (m, 2 H, H-5II, H-4III), 3.77 (s, 3 H, OCH3),
3.75 (t, J3,4 = 10.1 Hz, 1 H, H-4I), 3.65 (ddd, J4,5 = 9.8 Hz, J5,6a
= 5.6 Hz, J5,6b = 1.8 Hz, 1 H, H-5I), 3.59 (m, 2 H, H-6bIV,
H-5III), 3.57(dd, J2,3 = 10.0 Hz, 1 H, H-2III), 3.31 (s, 3 H,
CH3V), 2.70 (dd, J3eq,3ax = 12.5 Hz, J3e,4 = 4.4, 1 H, H-3eqV),
2.17–1.33 (16s, 48 H, 14 OAc, 2 NAc), 2.01 (dd, J3ax,4 = 12.0
Hz, 1 H, H-3axV), 1.00 (s, 9 H, SiCCH3). C69H96N2O44
(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)+.
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Acknowledgements
Support of these studies by the Deutsche Forschungsge-
meinschaft (SFB 470, A5) and the Fonds der Chemischen
Industrie is gratefully acknowledged.
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.6.18
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