Synthesis of apiose-containing oligosaccharide fragments of the plant cell wall: Fragments of rhamnogalacturonan-II side chains A and B, and apiogalacturonan

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PAPER

Synthesis of apiose-containing oligosaccharide fragments of the plant cell

wall: fragments of rhamnogalacturonan-II side chains A and B, and

apiogalacturonan†

Sergey A. Nepogodiev,*^a,bMargherita Fais,^a,bDavid L. Hughes^band Robert A. Field*^a,b

Received 12th April 2011, Accepted 20th June 2011

DOI: 10.1039/c1ob05587a

Fragments of pectic polysaccharides rhamnogalacturonan-II (RG-II) and apiogalacturonan were

synthesised using p-tolylthio apiofuranoside derivatives as key building blocks. Apiofuranose

thioglycosides can be conveniently prepared by cyclization of the corresponding dithioacetals possessing

a 2,3-O-isopropylidene group, which is required for preservation of the correct (3R) conﬁguration of the

apiofuranose ring. The remarkable stability of this protecting group in apiofuranose derivatives

requires its replacement with a more reactive protecting group, such as a benzylidene acetal which was

used in the synthesis of trisaccharide b-Rhap-(1→3¢)-b-Apif -(1→2)-a-GalAp-OMe. The X-ray crystal

structure of the protected precursor of this trisaccharide has been elucidated.

as a point of cross-linking of RG-II molecules in the cell wall.

Introduction

This chemical event has important biological consequences: plant

Understanding the sequence of biochemical events leading to

development is severely compromised in mutant plant lines with

assembly of polysaccharides present in the plant cell wall¹requires

access to a variety of oligosaccharide substrates which can help

to unravel the identity and speciﬁcity of the enzymes involved.²

Among the plant cell wall polysaccharides, a family of pectins is

characterized by its particular structural complexity. This family

consists of several structurally unrelated macromolecules which

may be covalently and non-covalently linked to each other, form-

ing highly branched polysaccharide networks.³One of the major

pectic polysaccharides is a-(1→4)-linked polygalacturonic acid

(homogalacturonan or HG), which plays the role of a backbone

to which a number of mono-, oligo- and poly-saccharides are

attached in apparently random order. Most of the HG side chains

are heterogeneous but there is one homogeneous and conserved

structure, rhamnogalacturonan-II (RG-II), that is comprised of

four discrete oligosaccharide chains attached to HG, as shown in

Fig. 1A.⁴

reduced ability to form such borate cross-links.⁷Apart from RG-II,

apiofuranose occurs in other types of pectins that are characteristic

for aquatic plants such as the sea grasses Zosteraceae⁸and

duckweed Lemna minor.⁹In these cases b-apiofuranosyl residues

are attached to either the 2 or 3 positions, or both positions, of

selected GalAp residues of HG chains in the form of mono- or

sometimes short oligo-saccharides (Fig. 1B).

Continuing our efforts in the chemical synthesis of RG-II

fragments,^2b,10we describe here the synthesis of trisaccharide

fragment 1 of RG-II, which is common to both side chains A and

B, and oligosaccharide fragments 2 and 4 related to apiogalactur-

onans (Fig. 2). In particular, this study addresses issues associated

with the inherent chemical reactivity of furanosides and protecting

group compatibility with 2,3-cis-conﬁgured furanoses.

Results and discussion

The two most complex side chains of RG-II, A and B, have the

same structure at the “reducing end”, represented by disaccharide

b-Rhap-(1→3¢)-b-Apif , which in turn is attached to the HG

backbone through a (1→2)-linkage. Apiofuranose⁵(Apif ) is a

branched pentose capable of borate diester formation⁶and serves

Synthesis of RG-II trisaccharide 1: ﬁrst approach

Disaccharide thioglycoside donor. A key step common for

synthesis of oligosaccharides 1–4 consists of the construction of

b-D-apiofuranosyl linkages which requires suitable apiofuranosyl

donors. As glycosyl donors, apiofuranose derivatives have been

employed in the form of thioglycosides,¹¹1-O-acetates,¹²glyco-

syl bromide,¹³trichloroacetimidate¹⁴and 1,2-O-cyanoethylidene

derivatives.¹⁵In our study we focused attention on thioglycosides

as stable and versatile reagents which have been widely used

for the synthesis of 1,2-trans-glycofuranosides.¹⁶In the initial

approach to target trisaccharide 1, disaccharide b-L-Rhap-(1-3¢)-

Apif, previously synthesised by us as protected methyl glycoside

^aDepartment of Biological Chemistry, John Innes Centre, Norwich Re-

search Park, Norwich, UK, NR4 7UH. E-mail: sergey.nepogodiev@

bbsrc.ac.uk, rob.ﬁeld@bbsrc.ac.uk

^bSchool of Chemistry, University of East Anglia, Norwich Research Park,

Norwich, UK, NR4 7TJ

† Electronic supplementary information (ESI) available: X-Ray crystallo-

graphic data for 41, NMR spectra. CCDC reference numbers 821796. For

ESI and crystallographic data in CIF or other electronic format see DOI:

10.1039/c1ob05587a

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Fig. 1 (A) Structure of rhamnogalacturonan-II backbone and side chains A–D (the exact position of each side chain attachment is ambiguous^4e). (B)

Tentative structure of apiogalacturonan showing various types of branch point. The target fragments are shown in boxes.

Fig. 2 Apiose-containing oligosaccharide fragments of pectic polysaccharides RG-II (1 and 2) and apiogalacturonan (2, 3 and 4).

5,¹⁷was considered as a precursor for the disaccharide thioglyco-

side donor 6. However, attempts to convert 5 into arylthio glyco-

side 6 (Scheme 1) under conventional (4-MeC₆H₄SH, BF₃·OEt₂,

Sequential glycosylation. b-D-Apiofuranose derivatives are

not readily available, mostly because of the rare natural oc-

currence of this branched-chain sugar and its ability to exist

in four different cyclic forms, which complicates apiofuranose

derivatisation.⁵The desired form of apiofuranose needs to have

the correct (3R)-stereochemistry, as present in natural apiofu-

ranosides. Such apiofuranose derivatives have been synthesised

in the form of compounds with ﬁxed 2,3-cis-conﬁguration, for

instance O-isopropylidene derivative 7. Compound 7 can be

prepared by a number of methods, including by protecting

group manipulations of expensive 1,2:3,3¢-di-O-isopropylidene-

a-D-erythro-apiofuranose^11aor by multistep procedures utilizing

either D-mannose,²⁰L-arabinose²¹or D-xylose^20c,22as starting

materials. We employed compound 7 as a precursor of the isomeric

apiofuranosyl donors 9a and 9b, which were prepared from 7 in

two successive steps, including acetylation and thioglycosidation

with thiocresol in 86% overall yield (Scheme 2).

◦

20 C)¹⁸or more forceful (Me₃SiSPh, Me₃SiOTf)¹⁹conditions

were largely unsuccessful and resulted in the decomposition

of starting material. Therefore, methyl apiofuranoside, which

has been used for the synthesis of disaccharide 5, was not

suitable for the preparation of trisaccharide 1 and building

blocks based on other types of apiose derivatives needed to be

designed.

Galacturonide derivative 12, having an unprotected 2-OH

group, served as the “reducing” end building block for the target

RG-II trisaccharide 1. The former was prepared from readily

accessible methyl (methyl a-D-galacturonopyranosid)uronate 11²³

Scheme 1 Attempted synthesis of arylthio glycoside 6. Reagents and

conditions: (a) TolSH, BF₃·OEt₂for Ar = Tol; (b) Me₃SiSPh, Me₃SiOTf for

Ar = Ph.

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Scheme 2 Thioapiofuranoside donors 9a and 9b. Reagents and conditions: (a) Ac₂O, C₅H₅N; (b) p-MeC₆H₄SH, BF₃·OEt₂, CH₂Cl₂.

Scheme 3 Synthesis of trisaccharide derivative 17. Reagents and conditions: (a) MeOH–HCl, reﬂux 18 h; (b) Me₂C(OMe)₂, TsOH; (c) NIS–TfOH,

CH₂Cl₂, -30 ^◦C; (d) NaOMe, MeOH; (e) Ag₂O, MS 4 A, CH₂Cl₂; (f) Amberlite IR120 (H ).

+

˚

by a known procedure²⁴(Scheme 3). Glycosylation of 12 with

p-tolylthio apiofuranoside 9b in the presence of NIS–TMSOf

as a promoter afforded disaccharide 13 in 80% yield. The lack

of a participating group in the glycosyl donor did not interfere

with expected 1,2-trans-glycosylation, which is generally a pref-

erential stereochemical outcome of reactions with glycofuranosyl

donors.²⁵Disaccharide 13 was deacetylated to form glycosyl ac-

ceptor 14, which was coupled with the 2,3-O-carbonate-protected

rhamnopyranosyl bromide 15²⁶in the presence of molecular

sieves and Ag₂O as an insoluble promoter to give fully protected

trisaccharide 16 in 64% yield. This direct b-rhamnosylation

Scheme 4 Ethyl thioapiofuranoside donor.^12aReagents and conditions: (a)

technique,²⁶applied in this case as well as in the synthesis of 5,¹⁷

80% HCO₂H, 60 ^◦C then Ac₂O, C₅H₅N; (b) EtSH, SnCl₄.

proved to be practical for stereoselective glycosylation of reactive

alcohols, although its efﬁciency was moderate. Deprotection of

for compound 17 were made; rather a slightly different synthetic

trisaccharide 16 was planned by sequential de-O-esteriﬁcation and

strategy was developed (vide infra).

de-O-acetalation in basic and acidic conditions, respectively. The

ﬁrst stage of the deprotection of trisaccharide 16 proceeded as

expected, but acid hydrolysis of isopropylidene groups catalysed

by Amberlite IR120 (H⁺) resin did not go to completion,

Synthesis of apiogalacturonan fragments 2–4

The extreme stability of the O-isopropylidene group in positions

leading instead to 2¢,3¢-mono-O-isopropylidene derivative 17 in

86% yield. Further attempts at the cleavage of the remaining

O-isopropylidene group in 17 using more vigorous conditions

involving 90% CF₃CO₂H in water were unsuccessful: either no

changes of starting material were observed or decomposition

occurred when slightly elevated temperature (30 ^◦C) was applied.

High resistance to hydrolysis of 2,3-O-isopropylidene acetals of C-

3-branched furanoses, including molecules with apiofuranosyl^11b

and streptofuranosyl²⁷residues, has been documented in the liter-

ature. It has also been observed that cleavage of the cyclic acetal in

apiofuranosides 18 and 19 requires forcing hydrolysis conditions

(80% HCO₂H, 60 ^◦C, Scheme 4), which are accompanied by

a complete cleavage of O-glycosidic linkages.^11a,15Therefore, no

further attempts to improve de-O-isopropylidenation conditions

2,3 of the apiofuranose ring limits usability of this particular acetal

as a protecting group in apiofuranose-containing oligosaccharide

synthesis. Since presence of the 2,3-O-isopropylidene protecting

group is essential for construction of initial building blocks such

as acetal 7, ﬁnding a practical alternative for the installation

of isopropylidene protection at an earlier stage in the synthesis

is complicated. Therefore, it is more practical to carry out the

replacement of the 2,3-O-isopropylidene group with more easily

cleavable protecting groups after the apiofuranose building block

is already assembled. To preserve the (3R)-conﬁguration of apiofu-

ranose during the hydrolytic removal of the 2,3-O-isopropylidene

group, the 3¢-OH group has previously been protected with

relatively stable benzyl ether.¹⁵Thus, the 3¢-O-benzyl ether survives

acid hydrolysis in benzyl or methyl apiofuranoside derivatives

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18 and 19, which have been used for preparation of triacetate

20 and its subsequent conversion to anomeric mixture of ethyl

thioapiofuranosides 21 (Scheme 4).^11a

compound 11, followed by selective ring opening of the cyclic

orthoester by careful hydrolysis of 24 or its 2-O-benzoate 25,

leading to diol 27 and alcohol 26, respectively (Scheme 6).

Glycosylation of compounds 26 and 27 with thioglycoside 22

under the same conditions as described for glycosylation of

alcohol 12 led to di- and tri-saccharides 28 and 29, in 74%

and 84% yield, respectively. Ester protecting groups in 23 and

28 were cleaved under basic conditions to afford disaccharides

representing fragments of apiogalacturonan 2 (Scheme 5), and 3

and 4 (Scheme 6).

We exploited the higher hydrolytic stability of S-glycosides

compared to O-glycosides,²⁸which made possible selective removal

of the 2,3-O-isopropylidene group in thioapiofuranosides without

affecting the (3R)-conﬁguration and the cleavage of the S-

glycosidic linkage. Thus, treatment of arylthio glycoside 9b with

90% CF₃CO₂H followed by acetylation afforded triacetates 22 as

a mixture of anomeric thioglycosides in a combined 76% yield

over two steps (Scheme 5). Reversible anomerisation took place

during the preparation of triacetates 22a,b (Scheme 5), which

were formed as a 1 : 4 mixture of a- and b-thioglycosides, even

when anomerically pure 1,2-trans-glycoside 9b was used as a

starting material. This anomeric mixture of triacetates 22a,b was

used for glycosylation of the alcohol 12, to afford exclusively b-

apiofuranoside 23 in 79% yield. This reaction was promoted by

NIS in the presence of HClO₄on silica gel.²⁹Similar results have

been reported for coupling of 12 with ethyl thioglycoside 21 in the

presence of NIS–AgOTf.^11c

Synthesis of RG-II trisaccharide 1: revised approach

Revised thioapiofuranoside donor synthesis. Reﬁnement of the

reaction scheme leading to apiogalacturonan fragment 2 required

additional levels of orthogonal protection for access to RG-

II fragment 1, given the internal apiose residue. This was

achieved employing thioapiofuranoside 38, which was equipped

with cleavable 2,3-O-benzylidene and 3¢-O-chloroacetyl protecting

groups (Scheme 7). Although protecting groups other than the O-

benzylidene group can be envisaged for blocking positions 2 and 3

in the apiofuranose residue (e.g. compound 21^10a), the benzylidene

protection was preferred since it has potential advantages for

the construction of larger RG-II fragments. In the synthesis of

such fragments, benzylidene along with benzyl groups can be

employed as permanent protecting groups which are orthogonal

to temporary ester groups.

Synthesis of thioapiofuranosides 38a,b was carried out starting

from readily available L-arabinose dithioacetal by introduction of

a C-3 branch point through a one-pot aldol–Cannizzaro reaction,

followed by cyclisation of acyclic thioapiofuranoside 34. Since

aromatic thioglycosides possess higher hydrolytic stability than

aliphatic analogues,²⁸the dithioacetal precursor was prepared in

the form of ditolylthio derivative 30. The application of a similar

approach to the preparation of apiose diethyldithio acetal has been

reported.³⁰

Scheme 5 Disaccharide 2. Reagents and conditions: (a) 90% TFA, 20 ^◦C,

b) Ac₂O, DMAP, C₅H₅N; (c) NIS, HClO₄/SiO₂, -30 ^◦C; (d) NaOMe,

MeOH then NaOH, H₂O.

Glycosyl acceptors having unprotected 3-OH or both 2- and

3-OH groups in methyl (methyl a-D-galactopyranosid)uronate

derivatives were also prepared and coupled with apiofuranosyl

donor 22. Synthesis of these glycosyl acceptors was carried out

via the installation of the 3,4-O-[1-(ethoxy)ethylidene] group in

The reaction of L-arabinose with water-insoluble p-MeC₆H₄SH

was carried out in 90% CF₃CO₂H according to a literature

method.³¹This reaction gave crystalline dithioacetal 30 in 63%

yield. O-Isopropylidenation of 30 led to dithioacetal 31, which was

hydrolyzed to 2,3-O-isopropylidene derivative 32 in 58% overall

Scheme 6 Synthesis of apiogalacturonan fragments. Reagents and conditions: (a) MeC(OEt)₃, TsOH, THF; (b) BzCl, C₅H₅N; (c) 80% AcOH; (d) NIS,

HClO₄/SiO₂, MS 4 A, -30 ^◦C; (e) NaOMe, MeOH; (f) Et₃N–MeOH–H₂O; (g) NaOH, H₂O–MeOH.

˚

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Scheme 7 Trisaccharide fragment 1 of RG-II. Reagents and conditions: (a) p-MeC₆H₄SH, 90% TFA; (b) Me₂C(OMe)₂, Me₂CO, TsOH; (c) Py·HOTs,

MeOH; d) NaIO₄on SiO₂, CH₂Cl₂; (e) HCHO, NaOH, EtOH, H₂O; (f) NIS, CH₂Cl₂, -60 ^◦C; (g) CClCH₂COCl, collidine, CH₂Cl₂; (h) 90% TFA;

˚

(i) PhCH(OMe)₂, CSA, toluene; (j) NIS, HClO₄on SiO₂, CH₂Cl₂; (k) NaOMe, MeOH; (l) Ag₂O, MS 4 A, CH₂Cl₂; (m) H₂–Pd/C, EtOAc; (n)

Et₃N–H₂O–MeOH. ClAc = ClCH₂CO.

yield. It should be noted that while the former reaction was almost

quantitative, in our hands the latter was accompanied by the

undesirable cleavage of the 2,3-O-isopropylidene group, despite

literature precedent for similar reactions with the diethyl dithioac-

etal analogue of 31.³²In order to improve the regioselectivity of

the de-O-isopropylidenation of 31, a very mild acid—pyridinium

tosylate in MeOH³³—was employed to catalyse the reaction, which

was achieved in 58% yield.

with a slight excess of formaldehyde in the presence of NaOH gave

3-C-hydroxymethyl derivative 34 in 53% yield. Compound 34 is an

acyclic derivative of apiose, which needs to be cyclized to form the

furanose having the desired D-erythro-conﬁguration via cleavage of

one of the STol groups. Cyclization of dithioacetals leading to the

formation of anomeric mixtures of corresponding thioglycosides

is possible under the action of HgCl₂–HgO³⁵or CdCO₃.³⁶More

recently such cyclization processes have been promoted with NIS–

TfOH³⁷or NIS³⁸at low temperature. The latter method involving

NIS as a thiophilic reagent was applied to synthesis of p-tolyl

thioapiofuranosides 35a,b from dithioacetal 34, which proceeded

in 89% yield.

In the following step, 4,5-diol 32 was oxidatively cleaved to

afford aldehyde 33—the precursor of the acyclic apiose derivative

34. Complete insolubility of the starting diol 32 in water pre-

vented application of conventional oxidation procedures for diols

based on aqueous NaIO₄treatment in homogeneous mixtures

with organic solvents. To overcome the water solubility issue,

periodate oxidation of vicinal diols was conducted in CH₂Cl₂

with the help of silica gel-supported NaIO₄.³⁴Careful application

of this procedure for oxidation of 32 allowed the synthesis of

aldehyde 33 in 90% yield with over-oxidation of the substrate

incorporating potentially vulnerable arylthio functionalities being

avoided. The reaction was monitored by silica gel TLC, judged

by the disappearance of the starting material. The product did

not appear as a distinct spot, presumably because of the partial

hydration of the aldehyde on the TLC plate. The NMR spectra of

34 clearly indicated the formation of an aldehyde, showing signals

The anomeric products ratio for the mixture of compounds

35a,b was determined as a : b = 78 : 22 using the H-1 and H-2 signal

1

intensities in H NMR spectra. Pure thioglycosides were easily

isolated in the form of 3¢-chloroacetates 36a and 36b, which were

obtained by chloroacetylation of the mixture 35a,b. Formation of

a-thioapiofuranoside 35a as the major product of cyclization was

in accordance with the known, though not well understood, fact

that cyclization of dithioacetals to thioglycosides proceeds with

the predominant formation of 1,2-cis-thio glycofuranosides, which

are apparently kinetic products of the reaction.^28,35Cleavage of the

isopropylidene acetal in compounds 36a and 36b, used as a mixture

of thioglycosides, with 90% aq. TFA produced 2,3-diols 37a,b as

judged by near complete disappearance of O-isopropylidene group

signals (d 1.47 and 1.40 ppm) in the ¹H NMR spectrum. The crude

1

of the CHO group at d_H9.80 and d_C201.0 ppm in H and ¹³C

NMR spectra, respectively. The aldol–Cannizzaro reaction of 33

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Table 1 Chemical shifts and coupling constants in ¹H NMR spectra (400 MHz, CDCl₃) of thioapiofuranosides 9a,b, 22a,b, 36a,b and 38a,b

Compound

H-1 (J_1,2

)

H-2

H-3¢a (J_3¢a,3¢b

)

H-3¢b

H-4a (J_4a,4b

)

H-4b

9a

4.98 (3.9)

5.60 (0)

4.64

4.54

5.58

5.21

4.65

4.54

4.80

4.62

4.24 (11.7)

4.41 (11.7)

4.59 (12.3)

4.54 (12.2)

4.25 (11.7)

4.40 (10.8)

4.41 (11.8)

4.51 (11.9)

4.14

4.29

4.34

4.37

4.29

4.48

4.47

4.60

4.21 (10.2)

4.17 (10.4)

4.27 (10.2)

4.27 (10.7)

3.61 (10.3)

4.02 (10.4)

3.64 (10.3)

4.22

4.04

4.03

4.21

4.10

4.05

4.15

4.30

4.22

9b

22a^a

22b^a

36a

36b

38a

38b

5.66 (5.7)

5.32 (4.9)

5.03 (3.9)

5.60 (<0.1)

5.07 (4.0)

5.77 (<0.1)

^aThe assignment of the anomeric conﬁgurations of 22a and 22b is ambiguous and may be interchanged.

Table 2 Chemical shifts in ¹³C NMR spectra (100 MHz, CDCl₃) of

70% yield as the only disaccharide product. Therefore 2,3-O-

benzylidene-protected apiofuranose donors, as expected, can serve

as stereoselective glycosylating agents. De-O-chloroacetylation of

39 afforded glycosyl acceptor 40, which was b-rhamnosylated with

4-O-acetyl-2,3-O-carbonyl-a-L-rhamnopyranosyl bromide (15) in

the presence of Ag₂O to afford protected trisaccharide 41 in 59%

yield.

thioapiofuranosides 9a,b, 22a,b, 36a,b and 38a,b

Compound

C-1

C-2

C-3

C-3¢

C-4

9a

91.7

93.3

89.4

90.7

89.8

93.3

91.4

92.6

84.5

87.7

72.9

75.9

84.4

87.6

84.6

87.7

90.0

90.5

82.6

83.6

91.7

90.3

89.8

90.3

64.7

65.1

62.6

66.0

66.5

65.1

65.4

74.0

73.6

70.2

72.6

73.8

73.5

72.8

72.6

9b

22a

22b

36a

36b

38a

38b

X-Ray crystal structure of trisaccharide 41. The structure

of trisaccharide 41, in particular the conﬁguration of the b-

rhamnopyranosidic and b-apiofuranosidic linkages, and the

conﬁguration of the benzylidene acetal protecting group, re-

quired additional conﬁrmation which was obtained by X-ray

crystallographic analysis (Fig. 3). Crystallographic data clearly

demonstrated the endo-orientation of the phenyl group ((S)-

conﬁguration) of the benzylidene protecting group. They also

revealed the anomeric conﬁguration and conformations of indi-

vidual monosaccharide components of the trisaccharide, each of

which incorporates a ﬁve-membered ring protecting group. While

mixture of 37a,b was converted directly into 2,3-O-benzylidene

compounds 38a and 38b by a camphorsulfonic acid-catalysed

reaction with PhCH(OMe)₂. As expected from experience with

thiofuranosides 22a,b, noted earlier, deacetalation of individual

samples of 36a and 36b with 90% TFA afforded anomeric mixtures

of p-tolyl thioglycosides having approximately the same ~1 : 4 1,2-

cis-/1,2-trans-glycoside ratio, as judged from ¹H NMR spectra.

4

a 3,4-O-isopropylidene protecting group doesn’t affect the C₁

Conﬁgurational assignment of thioapiofuranosides by NMR spec-

troscopy. The anomeric conﬁguration assignment of thioapiofu-

ranosides was made using the NMR spectral data for individual 3¢-

O-acylated compounds (Tables 1 and 2). Anomeric thioglycosides

3

showed diagnostic differences between values of the J_H-1,H-2

1

coupling constant in H NMR spectra of 2,3-O-isopropylidene

derivatives: for 1,2-trans-thioglycosides the signal of H-1 appeared

as a characteristic singlet whereas the corresponding signal of

1,2-cis-glycosides was in all cases a doublet with J ~ 4 Hz. It

3

is well established³⁹that J_H-1,H-2coupling constants are usually

1–4 Hz larger for 1,2-cis- than for 1,2-trans-glycofuranosides,

though absolute values of these coupling constants can be higher

for S-glycofuranosides than for O-glycosides.^10dSome deviation

from typical patterns of ³J_H-1,H-2values was observed in ¹H NMR

spectra of a mixture of triacetates 22a,b, where these coupling

constants were 4.9 and 5.7 Hz and unambiguous assignment of

a- and b-conﬁguration for each component of the mixture was

not possible. The conﬁguration of benzylidene acetals 38a and

38b was tentatively assigned as (S), which proved consistent with

subsequent X-ray crystallographic analysis (next section). The

corresponding (R)-diastereoisomers were not puriﬁed as they were

present only in minor quantities.

Sequential glycosylation to give trisaccharide 41. Coupling

either 38a or 38b with methyl (galactopyranoside)uronate ac-

ceptor 12 promoted by NIS in the presence of cat. HClO₄on

Fig. 3 ORTEP representation of the X-ray crystal structure of trisaccha-

ride derivative 41.

SiO₂at -40 ^◦C led to 1,2-trans-linked disaccharide 39 in 62–

29

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conformation of a-D-galactopyranosiduronic acid, the presence

of a 2,3-O-carbonate group on a b-L-rhamnopyranosyl residue led

to signiﬁcant deviation from the normal ⁴C₁chair conformation.

In trisaccharide 41, the rhamnopyranose ring approximates a

followed by heating to 150 ^◦C. Solutions of reaction products were

dried over MgSO₄and solvents were evaporated under reduced

pressure at 25–40 ^◦C. Column chromatography was performed

on a Biotage SP4 puriﬁcation system using silica gel pre-packed

cartridges. Gel permeation chromatography (GPC) was performed

using Toyopearl HW-40S resin (80 ¥ 1.5 cm column) using water

as an eluent and a differential refractometer as a detector. Optical

rotations were measured using a Perkin–Elmer 141 polarimeter.

¹H and ¹³C NMR spectra were recorded at 22 ^◦C with a JEOL

Lambda spectrometer at 400 and 100 MHz, respectively, using

TMS (for solution in CDCl₃) or Me₂CO (d 49.9, for solutions in

D₂O) as internal standards. Resonance assignments were made

with the aid of gCOSY and gHSQC experiments. High resolution

electrospray ionisation mass spectra (HR ESI-MS) were obtained

using positive ionization mode on a Finnigan MAT 900 XLT mass

spectrometer or Thermoﬁsher LTQ Orbitrap instrument.

5

B_1,4boat conformation, which is different to the H_Ohalf-chair

conformation found in the crystal structure of 3b-cholestanyl 4-

O-acetyl-2,3-O-carbonyl-b-L-rhamnopyranoside.⁴⁰The most ob-

vious difference between conformations of the pyranose rings in

these compounds is the orientation of the C1–O1 bond of the b-

rhamnopyranosyl residue, which may be described as pseudo-axial

5

for B_1,4and pseudo-equatorial for H_Oconformations. However,

the solution conformation of the b-rhamnopyranosyl ring of 41

5

3

appears different to either B_1,4or H_O, since values of the J_1,2

(~1 Hz) and ³J_2,3(<3.7 Hz, see the ESI†) coupling constants in ¹H

NMR spectra are smaller than the values of ³J_1,2(>2 Hz) and ³J_2,3

(6–7 Hz) that can be expected for these conformations.

In the crystal structure of 41, the ﬁve-membered fura-

nose ring, the mobility of which is constrained by the

cyclic acetal protecting group, adopts the E_Oconformation.

In the absence of such constraints in the crystal structure

of benzyl (2,3,3¢-tri-O-acetyl-b-D-apiofuranosyl)-(1→3)-(2,4-di-

O-benzoyl-a-D-xylopyranoside)^11bthe apiofuranose ring has the

E₂conformation but both in this case and in the structure of 41

the C1–O1 bond is quasi-axial.

p-Tolyl 3-C-acetoxymethyl-2,3-O-isopropylidene-1-thio-a-D-

erythrofuranoside (9a) and p-tolyl 3-C-acetoxymethyl-2,3-O-

isopropylidene-1-thio-b-D-erythrofuranoside (9b)

A

mixture of 3-C-hydroxymethyl-2,3-O-isopropylidene-b-D-

erythrofuranoside (7)^20a(2.00 g, 10.52 mmol) and Ac₂O (5.0 mL)

in pyridine (10 mL) was stirred for 17 h at 22 ^◦C, the reaction

was quenched with MeOH (5.0 mL) at +5 ^◦C, toluene (15 mL)

was added and the mixture was concentrated. The operation

was repeated several times and the residue was puriﬁed by

chromatography. Fractions containing the major faster moving

component were combined and concentrated to give diacetate

8,^12aas an oil, 2.34 g, 81%, R_f0.42 (hexane–EtOAc 7 : 3), ¹H NMR

(400 MHz, CDCl₃): d 6.20 (1 H, s, H-1), 4.47 (1 H, s, H-2), 4.39

(1 H, d, J 11.7 Hz, H-3a), 4.25 (1 H, d, J 11.7 Hz, H-3b), 4.11

(1 H, d, J 10.3 Hz, H-4a), 3.97 (1 H, d, J 10.3 Hz, H-3b), 2.13

(3 H, s, Ac), 2.08 (3 H, s, Ac), 1.50 (3 H, s, CMe₂), 1.42 (3 H, s,

CMe₂). Compound 8 (2.20 g, 8.02 mmol) and thiocresol (885 mg,

8.03 mmol) were dissolved in dry CH₂Cl₂(20 mL), the solution

was cooled to 0 ^◦C, BF₃·OEt₂(0.4 mL, 3.65 mmol) was added and

the mixture was stirred for 2 h at 22 ^◦C. The solution was diluted

with CH₂Cl₂(100 mL), washed with satd NaHCO₃, concentrated

and products were puriﬁed by chromatography. Compound 9b

was eluted ﬁrst (1.80 g, 66%), R_f0.42, then was eluted compound

9a (160 mg, 6%), R_f0.29 (hexane–EtOAc 80 : 20). Compound 9a,

[a]²_D²+43 (c 1.5, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d 7.38 (2

H, d, J 8.1 Hz, SC₆H₄Me), 7.05 (2 H, d, J 8.1 Hz, SC₆H₄Me),

4.98 (1 H, d, 3.9 Hz, H-1), 4.64 (1 H, d, 3.9 Hz, H-2), 4.24 (1 H,

d, J 11.7 Hz, H-3¢a), 4.21 (1 H, d, J 10.2 Hz, H-4a), 4.14 (1 H,

d, J 11.7 Hz, H-3¢b), 4.04 (1 H, d, J 10.2 Hz, H-4b), 2.32 (3 H,

s), 2.03 (3 H, s, Ac), 1.55 (3 H, s, CMe₂), 1.38 (3 H, s, CMe₂);

¹³C NMR (100.5 MHz, CDCl₃): d 170.6 (C O), 137.6, 131.7,

129.9 (SC₆H₄Me), 115.3 (CMe₂), 91.7 (C-1), 90.0 (C-3), 84.5 (C-

2), 74.0 (C-4), 64.7 (C-3¢), 27.5 (¥ 2, CMe₂), 21.0 (SC₆H₄Me), 20.6

(Ac);. ESI MS (+): m/z found 356.20 ([M + NH₄]⁺); calcd for

C₁₇H₂₆NO₅S 356.15. Compound 9b, [a]²_D²-282 (c 1.4, CHCl₃); ¹H

NMR (400 MHz, CDCl₃): d 7.36 (2 H, d, J 8.1 Hz, SC₆H₄Me),

7.12 (2 H, d, J 8.1 Hz, SC₆H₄Me), 5.60 (1 H, s, H-1), 4.54 (1 H, s,

H-2), 4.41 (1 H, d, J 11.7 Hz, H-3¢a), 4.29 (1 H, d, J 11.7 Hz, H-

3¢b), 4.17 (1 H, d, J 10.4 Hz, H-4a), 4.03 (1 H, d, J 10.4 Hz, H-4b),

2.32 (3 H, s, SC₆H₄Me), 2.14 (3 H, s, Ac), 1.48 (3 H, s, CMe₂), 1.41

(3 H, s, CMe₂); ¹³C NMR (100.5 MHz, CDCl₃): d 170.6 (C O),

Deprotection to complete synthesis of target RG-II trisaccharide

42. The trisaccharide 41 was deprotected in a series of reactions

starting with catalytic hydrogenolysis, which allowed removal

of the benzylidene group, as conﬁrmed by NMR spectroscopy

and MS data. Mild acid hydrolysis of the isopropylidene group

followed by standard Zemplen de-acetylation furnished methyl

ester 42 in 61% overall yield. Finally, treatment of 42 with Et₃N

in MeOH–H₂O⁴¹led to de-esteriﬁcation of the GalA residue and

formation of trisaccharide 1.

Conclusions

The biosynthesis of complex pectic glycans remains a largely blank

canvas. To date, few of the enzymes/genes required have been iden-

tiﬁed. In this study, we have elaborated methods for the chemical

synthesis of apiofuranose building blocks and their use for the

preparation of apiose-containing glycans. We have successfully

completed the synthesis of di- and tri-saccharide fragments of RG-

II side-chains A and B, and related apiogalacturonan structures.

The biochemical evaluation of these materials will be reported in

due course.

Experimental

All reagents were used as purchased without further puriﬁcation.

Reactions were carried out in dry solvents using septa and syringes

for addition of reagents. Dry CH₂Cl₂and MeCN were used

freshly distilled from CaH₂. The SiO₂-supported HClO₄-catalyst

(~0.5 mmol of the acid per 1 g of powder) was prepared using

Merck silica gel (15–40 mkm) according to procedure reported

in ref. 42 The SiO₂-supported NaIO₄was prepared according

to the method in ref. 34. TLC was performed on precoated

aluminium plates (Silica Gel 60 F₂₅₄, Merck). Spots were visualized

by exposure to UV light or by immersion into 5% ethanolic H₂SO₄

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138.0, 132.7, 130.0 (SC₆H₄Me), 115.3 (CMe₂), 91.7 (C-1), 90.0 (C-

3), 84.5 (C-2), 74.0 (C-4), 64.7 (C-3¢), 27.5 (CMe₂), 27.4 (CMe₂),

21.0 (SC₆H₄Me), 20.6 (Ac); HRMS (ESI+): m/z found 339.1263

([M + H]⁺); calcd for C₁₇H₂₂O₅S·H 339.1261.

GalA), 89.9 (C-3 Api), 86.4 (C-2 Api), 75.8 (C-2 GalA), 75.1 (C-3

GalA), 74.5 (C-4 Api), 73.9 (C-4 GalA), 67.2 (C-5 GalA), 65.6 (C-

3¢ Api), 56.3 (OMe), 52.7 (CO₂Me), 28.3 (Me₂C), 27.7 (Me₂C), 27.2

(Me₂C).26.5 (Me₂C), 21.0 (MeCO); HRMS (ESI+): m/z found

494.2236 ([M + NH₄]⁺); calcd for C₂₁H₃₂O₁₂·NH₄494.2232.

Methyl (methyl 3,4-O-isopropylidene-a-D-

galactopyranosid)uronate (12)

Methyl (2,3-O-isopropylidene-b-D-erythrofuranosyl)-

(1→2)-(methyl 3,4-O-isopropylidene-a-D-

galactopyranosid)uronate (14)

AcCl (11.2 mL) was carefully added to a stirred solution of

galacturonic acid (10) (12.5 g, 64 mmol) in MeOH (750 mL) in

order to generate 1 w/v% HCl solution. The mixture was reﬂuxed

for 18 h, neutralized with PbCO₃(~21 g), the precipitate was

ﬁltered off and the solution was concentrated. Crystallization of

the resulting residue from EtOH afforded compound 11²³(3.0 g,

21%) as a white powder. The mother liquor contained a mixture

methyl (methyl galactopyranosid)uronates which was recycled as

a starting material for preparation of compound 11. Crystalline 11

(1.0 g, 4.5 mmol) was suspended in a mixture of DMP (10 mL) and

acetone (10 mL), cat. CSA was added and the mixture was stirred

for 4 h at 22 ^◦C until starting material almost disappeared (TLC

EtOAc–MeOH 9 : 1). The mixture was neutralized with Et₃N,

concentrated and the residue was puriﬁed by chromatography

followed by crystallization from _◦hexane–EtOAc to give derivative

12 (910 mg, 77%); m.p. 112–114 C; [a]²_D²+122 (c 1.0, H₂O); Lit.⁴³

A solution of monoacetate 13 (186 mg, 0.39 mmol) in 0.1 M

NaOMe in MeOH (2 mL) was kept for 1 h at 24 ^◦C, carefully

neutralized with Amberlite IR 120 (H⁺) resin and concentrated.

Puriﬁcation of the residue by chromatography gave alcohol 14 as

a white solid (164 mg, 97%); [a]²_D²+10 (c 1.0, CHCl₃); H NMR

1

(400 MHz, CDCl₃): d 5.31 (1 H, s, H-1 Api), 4.92 (1 H, d, J 3.6 Hz,

H-1 GalA), 4.58 (1 H, d, J 2.9 Hz, H-5 GalA), 4.48 (1 H, dd, J

5.5 Hz, 2.9 Hz, H-4 GalA), 4.43 (1 H, s, H-2 Api), 4.27 (1 H,

dd, J 7.9 Hz, 5.5 Hz, H-3 GalA), 3.96 (1 H, d, J 10.0 Hz, H-4a

Api), 3.88 (1 H, d, J 10.0 Hz, H-4b Api), 3.80–3.71 (6 H, m, H-2

GalA, H-3¢a and H-3¢b Api, CO₂Me), 3.41 (3 H, s, OMe), 1.46 (s,

3 H, CMe₂), 1.43 (s, 3 H, CMe₂), 1.34 (s, 3 H, CMe₂), 1.28 (s, 3

H, CMe₂); ¹³C NMR (100 MHz, CDCl₃): d 168.5 (CO₂Me), 113.2

(Me₂C), 109.8 (Me₂C), 107.5 (C-1 Api), 99.6 (C-1 GalA), 91.9

(C-3 Api), 86.1 (C-2 Api), 75.0 (C-2 GalA), 74.8 (C-3 GalA), 74.3

(C-4 Api), 73.7 (C-4 GalA), 67.0 (C-5 GalA), 64.5 (C-3¢ Api), 56.1

(OMe), 52.4 (CO₂Me), 27.9 (Me₂C), 27.4 (Me₂C), 27.2 (Me₂C),

26.2 (Me₂C). HRMS (ESI+): m/z found 435.1861 ([M + NH₄]⁺);

calcd for C₁₉H₃₀O₁₁·NH₄435.1861.

m.p. 113–4 (pet. ether); [a]²_D⁰+117 (H₂O) H NMR. (400 MHz,

1

CDCl₃): d 4.88 (1 H, d, J 3.9 Hz, H-1), 4.63 (1 H, d, J 2.5 Hz,

H-5), 4.52 (1 H, dd, J 6.3 Hz, J 2.5 Hz, H-4), 4.33 (1 H, dd, J

10.4 Hz, 4.3 Hz, H-3), 3.93 (1 H, dd, J 5.8 Hz, 3.9 Hz, H-2), 3.81

(3 H, s, CO₂Me), 3.48 (3 H, s, OMe), 2.62 (1 H, br s, OH), 1.46 (3

H, s, CMe₂), 1.32 (3 H, s CMe₂); ¹³C NMR (100 MHz, CDCl₃):

d 168.9 (C O), 110.4 (Me₂C), 98.5 (C-1), 75.4 (C-5), 73.7 (C-4),

68.8 (C-3), 68.3 (C-2), 56.3 (CO₂Me), 52.6, OMe), 27.4 (Me₂C),

25.9 (Me₂C).

Methyl (4-O-acetyl-2,3-O-carbonyl-b-L-rhamnopyranosyloxy-

methyl)-(1→3)-C-(2,3-O-isopropylidene-b-D-erythrofuranosyl)-

(1→2)-(methyl 3,4-O-isopropylidene-a-D-galactopyranosid)-

uronate (16)

Methyl (3-C-acetoxymethyl-2,3-O-isopropylidene-b-D-

erythrofuranosyl)-(1→2)-(methyl 3,4-O-isopropylidene-a-D-

galactopyranosid)uronate (13)

A mixture of disaccharide 14 (130 mg, 0.30 mmol), Ag₂O (280 mg,

1.21 mmol) and molecular sieves 4 A (1.0 g) in CH₂Cl₂(6 mL) was

˚

stirred for 1 h in the dark at 20 ^◦C and a solution of freshly prepared

rhamnopyranosyl bromide 15²⁶(195 mg, 3.05 mmol) in CH₂Cl₂

(4 mL) was added over 1 h. After stirring the mixture in the dark

for 5 h at 20 ^◦C, it was treated with pyridine (0.3 mL), diluted with

CH₂Cl₂and ﬁltered through Celite. The ﬁltrate was washed with

10% satd aq. Na₂S₂O₃, water, the organic layer concentrated and

the residue was puriﬁed by chromatography to give trisaccharide

16 (85 mg, 44%), R_f0.27 (toluene–EtOAc 50 : 50); [a]²_D²+31 (c 0.9,

CHCl₃); ¹H NMR (400 MHz, CDCl₃): d 5.36 (1 H, dd, J 8.3 Hz,

4.6 Hz, H 4 Rha), 5.27 (1 H, s, H-1 Api), 5.16 (1 H, s H 1 Rha),

4.98 (1 H, d, J 3.4 Hz, H-1 GalA), 4.81–4.78 (2 H, m, H-2, H-3

Rha), 4.58 (1 H, d, J 2.6 Hz, H 5 GalA), 4.48 (1 H, dd, J 5.3 Hz,

2.6 Hz, H-4 GalA), 4.37 (1 H, s, H-2 Api), 4.26 (1 H, dd, J 7.6 Hz,

5.3 Hz, H-3 GalA), 4.10 (1 H, d, J 11.2 Hz, H-3¢a Api), 4.01

(1 H, d, J 10.2 Hz, H-4a Api), 3.94 (1 H, d, J 10.2 Hz, H-3¢b

Api), 3.87 (1 H, d, J 11.2 Hz, H-4b Api) 3.84 (3 H, s, CO₂Me),

3.78–3.73 (2 H, m, H-2 GalA, H-5 Rha), 3.42 (3 H, s), 2.12 (3

H, s, OMe), 1.51 (3 H, s, Ac), 1.47 (s, 3 H, CMe₂), 1.40 (s, 3 H,

CMe₂), 1.34 (s, 3 H, CMe₂), 1.30 (3 H, d, J 6.3 Hz, H-6 Rha);

¹³C NMR (100.5 MHz): d 169.3 (CH₃CO), 168.7 (CO₂Me), 153.6

(C O), 114.1 (Me₂C), 110.0 (Me₂C), 108.2 (C-1 Api), 99.9 (C-1

GalA), 95.1 (C-1 Rha), 91.5 (C-3 Api), 86.3 (C-2 Api), 76.3 (C-2

A mixture of 12 (170 mg, 0.65 mmol), NIS (150 mg, _◦0,67 mmol)

˚

and mol. sieves 4 A (0.5 g) was stirred for 30 min at 22 C, then the

mixture was cooled to -40 ^◦C and thioglycoside 9b (170 mg, 0.50

mmol) in CH₂Cl₂(4.0 mL) and TMSOTf (36 mL, 0.20 mmol) were

sequentially added fro_◦m separate syringes. The stirring continued

for 1 h at -40 to -20 C, before the reaction was quenched with

Et₃N (0.4 mL). The mixture was ﬁltered through a Celite pad, the

ﬁltrate was diluted with CH₂Cl₂(50 mL) and washed with 10%

Na₂S₂O₃and satd NaHCO₃solutions. Solvent was removed under

reduced pressure and the residue was puriﬁed by chromatography

to afford disaccharide 13 (190 mg, 79%), R_f0.51 (hexane–EtOAc

1

70 : 30); [a]²_D⁴-1 (c 1.0, CHCl₃); H NMR (400 MHz, CDCl₃): d

5.29 (1 H, s, H-1 Api), 4.92 (1 H, d, J 3.5 Hz, H-1 GalA), 4.57 (1 H,

d, J 2.8 Hz, H-5 GalA), 4.50– 4.45 (2 H, m, H-4 GalA, H-2 Api),

4.40 (1 H, d, J 11.6 Hz, H-H-3¢a Api), 4.25 (1 H, dd, J 7.7 Hz, J

5.5 Hz, H-3 GalA), 4.19 (1 H, d, J 11.6 Hz, H-3¢b Api), 3.97 (1 H, d,

J 10.0 Hz, H-4a Api), 3.85–3.80 (4 H, m, H-4b Api, CO₂Me), 3.76

(1 H, dd, J 8.0 Hz, 3.5 Hz, H-2 GalA), 3.39 (3 H, s, OMe), 2.10 (3 H,

s, Ac), 1.50 (3 H, s, CMe₂), 1.47 (3 H, s, CMe₂), 1.39 (3 H, s, CMe₂),

1.33 (3 H, s, CMe₂); ¹³C NMR (100 MHz): d 170.7 (CH₃CO), 168.5

(CO₂Me), 113.9 (Me₂C), 109.9 (Me₂C), 108.0 (C-1 Api), 99.9 (C-1

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GalA), 76.2 (C-2* Rha), 75.1 (C-3 GalA), 75.0 (C-4 Api), 74.0 (C-4

GalA), 72.6 (C-3* Rha), 71.4, 71.3 (C-3¢ Api, C-4 Rha), 70.8 (C-5

Rha), 67.2 (C-5 GalA), 56.4 (OMe), 52.8 (CO₂Me), 28.4 (Me₂C),

27.7 (Me₂C), 27.4 (Me₂C), 26.6 (Me₂C), 21.0 (Me CO), 19.8 (C-6

Rha); HRMS (ESI+): m/z found 666.2605 ([M + NH₄]⁺); calcd

for C₂₈H₄₀O₁₇·NH₄666.2604.

s, Ac, a), 2.10 (3 H, s, Ac b), 2.07 (3 H, s, Ac, a), 2.06 (3 H, s, Ac

a), 2.04 (3 H, s, Ac b), 2.02 (3 H, s, Ac b).; ¹³C NMR (100 MHz,

CDCl₃): 170.5, 169.9, 169.4 (MeCO), 138.8, 133.8, 132.9, 130.1,

128.5 (SC₆H₄Me), 90.7 (1a), 89.4 (1b), 83.6 (3b), 82.6 (3a), 75.9

(2b), 72.9 (2a), 72.6 (4b), 70.2 (4a), 62.6 (2 C, 3¢a, 3¢b), 21.5, 21.4

(SC₆H₄Me), 20.9 (2 C), 20.8 (MeCO); HRMS (ESI+): m/z found

400.1424 ([M + NH₄]⁺); calcd for C₁₈H₂₂O₇S·NH₄400.1424.

Methyl (b-L-rhamnopyranosyloxymethyl)-(1→3)-C-(2,3-O-

isopropylidene-b-D-erythrofuranosyl)-(1→2)-(methyl

3,4-O-isopropylidene-a-D-galactopyranosid)uronate (17)

Methyl (3-C-acetoxymethyl-2,3-di-O-acetyl-b-D-erythrofura-

nosyl)-(1→2)-(methyl

3,4-O-isopropylidene-a-D-galactopyra-

nosid)uronate (23). A solution of thioglycoside 9b (390 mg,

1.02 mmol) and alcohol 12 in CH₂Cl₂(20 mL) was stirred with

A solution of trisaccharide 16 (65 mg, 100 mmol) in 0.1 M

˚

mol. sieves 4 A (1.0 g) for 20 min at room temperature and the

methanolic NaOMe solution (1 mL) was allowed to stand for

mixture was cooled to -40 ^◦C. A solution of TMSOTf (18 mL,

0.10 mmol) in CH₂Cl₂(0.18 mL) was added, the mixture was

stirred for 30 min allowing the temperature to rise to -20 ^◦C

and then treated with Et₃N (0.2 mL). After dilution with CH₂Cl₂

and ﬁltration through the Celite, Na₂S₂O₃solution (10%) was

added to the ﬁltrate and the mixture was vigorously stirred for

30 min. The organic layer was separated and washed with aq.

NaHCO₃solution, dried and concentrated. Puriﬁcation of the

product by chromatography afforded disaccharide 23 (410 mg,

77%) as crystalline solid; R_f0.36 (pet. ether–EtOAc 50 : 50); m. p.

146–148 ^◦C; [a]_D²²-2 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃):

d 5.51 (1 H, s, H-2 Api), 5.23 (1 H, s, H-1 Api), 4.94 (1 H, d, J

3.6 Hz, H-1 GalA), 4.92 (1 H, d, J 12.3 Hz, H-3¢a Api), 4.59 (1 H,

d, J 2.8 Hz, H-5 GalA), 4.51 (1 H, d, J 12.3 Hz, H-3¢b Api), 4.48

(1 H, d, J 2.8 Hz, H-4 GalA), 4.30 (1 H, dd, J 5.5 Hz, J 7.7 Hz,

H-3 GalA), 4.17 (2 H, s, H 4a,b Api), 3.84 (3 H, s, OMe), 3.79

(dd, J 3.6 Hz, J 7.80 Hz, H-2 GalA), 3.43 (3 H, s, CO₂Me), 2.10

(3 H, s, Ac), 2.01 (3 H, s, Ac), 1.51 (3 H, s, Me₂C), 1.34 (3 H, s,

Me₂C); ¹³C NMR (100 MHz, CDCl₃): d 170.6, 169.8, 168.9, 168.4

(C O), 109.9 (Me₂C), 105.8 (C-1 Api), 99.6 (C-1 GalA), 84.1

(C-3 Api), 76.2 (C-2 Api), 75.4 (C-2 GalA), 74.8 (C-3 GalA), 73.7

(C-4 GalA), 72.5 (C-4 Api), 67.1 (C-5 GalA), 63.0 (C-3¢ Api),

56.2 (OMe), 52.5 (CO₂Me), 28.1 (CMe₂), 26.3 (CMe₂), 21.1, 20.7,

20.6 (C-Ac); HRMS (ESI+): m/z found 538.2133 ([M + NH₄]⁺);

calcd for C₂₂H₃₂O₁₄·NH₄538.2130.

◦

30 min at 22 C, then neutralized with Amberlite IR-120 (H⁺)

resin, diluted with MeOH (6 mL) and an excess of the same resin

was added. The mixture was stirred for 48 h at 22 C, the resin

◦

was ﬁltered off and the solvent was evaporated to give target

1

trisaccharide 17 (50 mg, 91%); [a]²_D⁵+22.5 (c 1.6, MeOH); H

NMR (400 MHz, CDCl₃): d 5.26 (1 H, s, H-1 Api), 4.94 (1 H, d,

J 3.8 Hz, H-1 GalA), 4.61 (1 H, br s, H-1 Rha), 4.54 (1 H, d, J

1.3 Hz, H-5 GalA), 4.50 (1 H, s, H-2 Api), 4.26 (1 H, dd, J 1.5 Hz,

J 3.4 Hz, H-4 GalA), 4.05 (1 H, d, J 11.0 Hz, H-3¢a Api), 4.02

(1 H, d, J 10.7 Hz, H-4a Api), 3.96 (1 H, br d, J 3.3 Hz, H-2

Rha), 3.93 (1 H, d, J 10.7 Hz, H-4b Api), 3.89 (1 H, d, J 11.0 Hz,

H-3¢b Api), 3.86 (1 H, dd, J 3.4 Hz, J 10.3 Hz, H-3 GalA), 3.77

(1 H, dd, J 3.8 Hz, J 10.3 Hz, H-2 GalA), 3.72 (3 H, s, CO₂Me),

3.50 (1 H, m, H-3 Rha), 3.33 (3 H, s, OMe), 3.27–3.33 (2 H, m,

H-4, H-5 Rha), 1.40 (3 H, s, Me₂C), 1.36 (3 H, s, Me₂C), 1.22 (1

H, d, J 6.5 Hz, H-6 Rha); ¹³C NMR (100 MHz, D₂O): d 171.6

(CO₂Me), 114.7 (Me₂C), 109.1 (C-1 Api), 100.6 (C-1 Rha), 99.5

(C-1 GalA), 91.5 (C-3 Api), 86.2 (C-2 Api), 75.6 (C-2 GalA), 75.0

(C-4 Api), 73.0 (C-2 or C-3 Rha), 72.6, 72.5 (C-4, C-5 Rha), 71.7

(C-3¢ Api), 70.9 (C-3 or C-2 Rha), 70.8 (C-5 GalA), 70.4 (C-4

GalA), 68.2 (C-3 GalA), 55.9 (OMe), 53.3 (CO₂Me), 26.9 (Me₂C),

26.5 (Me₂C), 17.0 (C-6 Rha); HRMS (ESI+): m/z found 558.2393

([M + NH₄]⁺); calcd for C₂₂H₃₆O₁₅·NH₄558.2392.

p-Tolyl 3-C-acetoxymethyl-2,3-di-O-acetyl-1-thio-a,b-D-

erythrofuranoside (22a and 22b)

Methyl-(methyl 3,4-O-(1-ethoxyethylidene)-

a-D-galactopyranosid)uronate (24)

A solution of compound 9b (570 mg, 1.68 mmol) in 90% CF₃CO₂H

was allowed to stand at 20 ^◦C, until TLC (EtOAc) indicated

almost complete disappearance of the starting material (<1 h).

The solution was concentrated in vacuo with toluene, the residues

were dissolved in C₅H₅N (10 mL), Ac₂O (5.0 mL) and cat. DMAP

were added and the mixture was kept for 18 h at 22 ^◦C. After

careful quenching of the reaction by addition of MeOH (5 mL) at

0 ^◦C, the mixture was concentrated and the residue was puriﬁed by

chromatography to give triacetate 22 (490 mg, 76%) as a mixture of

a- and b-glycosides (22a : 22b = 1 : 5, from ¹H NMR integration);

A solution of triol 11 (2.22 g, 10.0 mmol), (EtO)₃CMe (7.3 mL,

40 mmol) and cat. CSA in THF (20 mL) was stirred for 1.5

h at 22 ^◦C. The reaction was quenched by addition of Et₃N

(2 mL), the mixture was concentrated, redissolved in CH₂Cl₂,

washed with satd NaHCO₃and concentrated. The residue was

puriﬁed by chromatography to give the orthoester 24 as a single

diastereomer (2.12 g, 73%); R_f0.33 (EtOAc–hexane 80 : 20); [a]_D²⁰

1

+70 (c 1.0, CHCl₃); H NMR (400 MHz, CDCl₃): d 4.89 (1 H,

1

R_f0.27 (hexane–EtOAc 80 : 20); H NMR (400 MHz, CDCl₃): d

d, J 4.0 Hz, H-1), 4.74 (1 H, dd, J 6.9 Hz, 2.4 Hz, H-4), 4.67 (1

H, d, J 2.4 Hz, H-5), 4.56 (1 H, dd, J 6.9 Hz, 5.1 Hz, H-3), 4.02

(1 H, m, H-2), 3.83 (3 H, s, CO₂Me), 3.58–3.49 (5 H, m, OMe,

OCH₂Me), 1.62 (3 H, s, C(OEt)Me), 1.22–1.16 (3 H, m, J 7.1 Hz,

OCH₂Me); ¹³C NMR (100 MHz, CDCl₃): d 168.4 (CO₂Me), 122.1

(MeCOEt), 97.5 (C-1), 75.1 (C-3), 74.0 (C-4), 68.6 (C-5), 67.0 (C-

2), 58.5 (CH₂Me), 56.0 (OMe), 52.4 (CO₂Me), 21.9 (MeCOEt),

15.4 (CH₂Me); HRMS (ESI+): m/z found 293.1231 ([M + NH₄]⁺);

calcd for C₁₂H₂₀O₈·NH₄293.1231.

7.39 (2.4 H, m, SC₆H₄Me a and b), 7.11 (2.4 H, m, SC₆H₄Me a

and b), 5.66 (0.2 H, d, J 5.7 Hz, H-1 a), 5.58 (0.2 H, d, J 5.7 Hz,

H-2 a), 5.32 (1 H, d, J 4.9 Hz, H-1 b), 5.21 (1 H, d, J 4.9 Hz, H-2

b), 4.59 (0.2 H, d, J 12.3 Hz, H-3¢a a), 4.54 (1 H, d, J 12.2 Hz,

H-3¢a b), 4.37 (1 H, d, J 12.2 Hz, H-3¢b b), 4.34 (0.2 H, d, J

12.3 Hz, H-3¢b a), 4.27 (1.2 H, d, J 10.7 Hz, H-4a b), 4.21 (0.2 H,

br d, J 10.6 Hz, H4a a, H-4a b), 4.10 (1 H, d, J 10.6 Hz, H-4b b),

2.32 (3 H, s, SC₆H₄Me b), 2.31 (1 H, s, SC₆H₄Me, a), 2.17 (3 H,

6678 | Org. Biomol. Chem., 2011, 9, 6670–6684

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Methyl (methyl 2-O-benzoyl-3,4-O-(1-ethoxyethylidene)-a-D-

galactopyranosid)uronate (25)

CH₂Cl₂(15 mL) for 30 min, NIS (225 mg, 1.00 mmol) was added

and the mixture was cooled to -40 ^◦C. Fine powder of HClO₄/SiO₂

(50 mg) was added to the mixture by syringe through a wide

bore needle and stirring continued for 1 h to allow the mixture

to warm up to 20 ^◦C. The reaction was quenched with Et₃N

(0.4 mL), ﬁltered through Celite, diluted with CH₂Cl₂(20 mL)

and vigorously stirred with aq. Na₂S₂O₃(10%, 20 mL) for 30 min.

The organic layer was separated, washed with satd aq. NaHCO₃,

concentrated and dried. In order to facilitate the chromatographic

puriﬁcation, the residual alcohol was acetylated by treatment of

the product mixture with Ac₂O–pyridine mixture (1 : 3, 10 mL)

for 6 h at 22 ^◦C, then the reaction was quenched with MeOH

(3 mL) and concentrated with toluene. The residue was puriﬁed

by chromatography to give disaccharide 28 (325 mg, 74%); R_f0.38

(hexane–EtOAc 50 : 50), m.p. 151–152 ^◦C; [a]_D²²99 (c 1.1 CDCl₃);

¹H NMR (400 MHz, CDCl₃): d 8.04 (2 H, m, Ph), 7.58, (1 H,

m, Ph), 7.46 (2 H, m, Ph), 5.79 (1 H, br d, J 2.2 Hz, H-4 GalA),

5.33 (2 H, dd, J 10.5 Hz, J 3.5 Hz, H-2 GalA), 5.28 (1 H, d, J

3.5 Hz, H-1 GalA), 5.26 (1 H, s, H-1 Api), 4.58 (1 H, br s, H-5

GalA), 4.50 (1 H, d, J 12.5 Hz, H-3¢a Api), 4.45 (1 H, d, J 12.5 Hz,

H-3¢b Api), 4.33 (1 H, d, J 10.7 Hz, H-4a Api), 4.18 (1 H, d, J

10.7 Hz, H-4b Api), 3.77 (3 H, s, CO₂Me), 3.43 (3 H, s, OMe), 293

(3 H, s, Ac), 2.99 (3 H, s, Ac), 2.00 (3 H, s, Ac), 1.85 (3 H, s, Ac);

¹³C NMR (100 MHz, CDCl₃): d 170.5, 170.1, 169.7, 168.6, 167.8,

165.9 (C O), 133.4, 129.9, 129.5, 128.5 (Ph), 107.5 (C-1 Api), 97.8

(C-1 GalA), 83.9 (C-3 Api), 76.3 (C-2 Api), 73.0 (C-4 Api), 72.0

(C-3 GalA), 70.8 (C-4 GalA), 70.5 (C-2 GalA), 68.7 (C-5 GalA),

63.0 (C-3¢ Api), 56.2 (OMe), 52.5 (CO₂Me), 20.9, 20.5, 20.5, 20.0

(Ac); HRMS (ESI+): m/z found 644.2184 ([M + NH₄]⁺); calcd for

C₂₈H₃₄O₁₆·NH₄644.2185.

Benzoyl chloride (0.81 mL, 7.00 mmol) was added to a solution

of orthoester 24 (2.20 g, 5.56 mmol) in pyridine (10 mL) and the

mixture was stirred for 17 h at 21 ^◦C. After quenching the excess

of BzCl with MeOH, the mixture was diluted with CH₂Cl₂and

washed with satd NaHCO₃, the organic layer was concentrated

and the product 25 (1.73 g, 78%) was isolated by chromatography.

R_f0.30 (hexane–EtOAc 4 : 1); [a]²_D⁰+112.5 (c 1.4, CHCl₃); ¹H NMR

(400 MHz, CDCl₃): d 8.03 (2 H, m, Ph), 7.56 (1 H, m, Ph), 7.40 (2

H, m, Ph), 5.17 (1 H, dd, J 7.4 Hz, 3.7 Hz, H-2), 5.10 (1 H, d, J

3.7 Hz, H-1), 4.79 (1 H, dd, J 6.0 Hz, 2.9 Hz, H-4), 4.67 (1 H, dd,

J 7.4 Hz, J 6.0 Hz, H-3), 4.66 (1 H, d, J 2.9 Hz, H-5), 3.83 (3 H,

s, CO₂Me), 3.56–3.47 (2 H, m, CH₂Me), 3.39 (3 H, s, OMe), 1.62

(3 H, s, EtOCMe), 1.15 (3 H, t, J 9.3 Hz, OCH₂Me); ¹³C NMR

(100 MHz, CDCl₃): d 168.1 (C O), 165.9 (CO₂Me), 133.6, 130.1,

129.5, 128.6 (2C) (Ph), 122.0 (MeCOEt), 97.5 (C-1), 74.2, 74.0 (C-

3, C-4), 71.2 (C-2), 67.4 (C-5), 58.8 (CH₂Me), 56.6 (OMe), 52.8

(CO₂Me), 23.0 (MeCOEt), 15.5 (CH₂Me); HRMS (ESI+): m/z

found 414.1763 ([M + NH₄]⁺); calcd for C₁₉H₂₄O₉·NH₄414.1759.

Methyl (methyl 4-O-acetyl-2-O-benzoyl-a-D-

galactopyranosid)uronate (26)

A solution of orthoester 25 in 80% AcOH (20 mL) was kept for

30 min at 22 ^◦C, then concentrated repeatedly with toluene

to remove all solvents and the residue was crystallized from a

toluene–hexane mixture to give acetate 26, (1.25 g (79%), R_f0.25

◦

(hexane–EtOAc 60 : 40); m.p. 144.5–145 C; [a]²_D⁰+141 (c 1.1,

CHCl₃); ¹H NMR (400 MHz, CDCl₃): d 8.05–8.01 (2 H, m, Ph),

7.54 (1 H, m, Ph), 7.43–7.38 (2 H, m, Ph), 5.67 (1 H, dd, J 3.6 Hz,

J 1.4 Hz, H-4), 5.26 (1 H, dd, J 10.4 Hz, 3.7 Hz, H-2), 5.18 (1 H,

d, J 3.7 Hz, H-1), 4.56 (1 H, d, J 1.4 Hz, H-5), 4.42–4.35 (1 H,

m, H-3), 3.73 (3 H, s, CO₂Me), 3.40 (3 H, s, OMe), 2.93 (1 H, d,

J 5.9 Hz, OH), 2.10 (3 H, s, Ac); ¹³C NMR (100 MHz, CDCl₃):

d 170.9, 168.2, 166.8 (C O), 133.7, 130.1, 129.5, 128.6 (Ph), 98.1

(C-1), 71.8 (C-4), 71.4 (C-2), 68.8 (C-5), 66.8 (C-3), 56.5 (OMe),

52.9 (CO₂Me), 20.9 (Ac); HRMS (ESI+): m/z found 369.1180 ([M

+ H]⁺); calcd for C₁₇H₂₁O₉369.1180.

Methyl (3-C-acetoxymethyl-2,3,di-O-acetyl-b-D-erythrofurano-

syl)-(1→2)-[(3-C-acetoxymethyl-2,3-di-O-acetyl-b-D-erythrofu-

ranosyl)-(1→3)]-(methyl 4-O-acetyl-a-D-galactopyranosid)uronate

(29)

The title compound was synthesized by essentially the same

method as disaccharide 28 using the following proportions of

reagents: diol 27 (79 mg, 0.30 mmol), thioglycoside 22a,b (250 mg,

˚

0.65 mmol), mol. sieves 4 A (500 mg), NIS (225 mg, 1.0 mmol),

Methyl (methyl 4-O-acetyl-a-D-galactopyranosid)uronate (27)

CH₂Cl₂(15 mL), HClO₄/SiO₂(50 mg). The product was puriﬁed

by chromatography to give trisaccharide 29 (196 mg, 84%), R_f

0.41 (hexane–EtOAc 40 : 60); [a]²_D⁰+12.5 (c 1.0, CHCl₃); ¹H NMR

(400 MHz, CDCl₃): d 5.64 (1 H, dd, J 3.6 Hz, 1.5 Hz, H-4 GalA),

5.34 (1 H, s, H-2 Api), 5.27 (1 H, s, H-1 Api), 5.21 (1 H, s, H-2

Api), 5.20 (1 H, s, H-1 Api), 4.98 (1 H, d, J 3.7, H-1 GalA), 4.86

(1 H, d, J 12.3 Hz, H-3¢ Api), 4.575 (1 H, d, J 12.3 Hz, H-3¢

Api), 4.570 (1 H, d, J 12.3 Hz, H-3¢ Api), 4.48 (1 H, d, J 1.5 Hz,

H-5 GalA), 4.41 (1 H, d, J 12.3 Hz, H-3¢ Api), 4.31 (1 H, d, J

10.8 Hz, H-4 Api), 4.20–4.08 (4 H, m, H-3 GalA, H-4 Api ¥ 3),

3.97 (1 H, dd, J 10.2 Hz, 3.7 Hz, H-2 GalA), 3.73 (3 H, s, CO₂Me),

3.42 (3 H, s, OMe), 2.11 (3 H, s, Ac), 2.09 (3 H, s, Ac), 2.08 (6

H, s, Ac), 2.07 (3 H, s, Ac), 2.03 (3 H, s, Ac), 2.02 (3 H, s, Ac);

¹³C NMR (100 MHz, CDCl₃): d 174.9, 170.6, 170.1, 169.9, 169.9,

169.3, 169.1, 167.9 (C O ¥ 8), 107.4 (C-1 Api), 106.6 (C-1 Api),

99.8 (C-1 GalA), 84.1 (C-3 Api), 83.9 (C-3 Api), 76.2 (C-2 Api),

76.0 (C-2 Api), 74.5 (C-2 GalA), 73.2 (C-4 Api), 72.6 (C-3 GalA),

71.7 (C-4 Api), 70.8 (C-2 GalA), 68.5 (C-5 GalA), 63.5 (C-3¢ Api),

62.7 (C-3¢ Api), 56.0 (OMe), 52.4 (CO₂Me), 20.9, 20.9, 20.5, 20.4,

A solution of orthoester 24 (430 mg, 1.47 mmol) in 80% AcOH

(10 mL) was kept for 40 min at 22 ^◦C, then concentrated repeatedly

with toluene to remove solvents and the residue was puriﬁed by

chromatography to give diol 27 as a white solid (370 mg, 95%),

R_f0.16 (EtOAc); [a]²_D⁰+200 (c 0.75, CHCl₃); ¹H NMR (400 MHz,

CDCl₂, D₂O-exchange): d 5.61 (1 H, d, J 3.4 Hz, H-4), 4.98 (1 H,

d, J 3.8 Hz, H-1), 4.51 (1 H, s, H-5), 4.02 (1 H, dd, J 10.0 Hz,

3.4 Hz, H-3), 3.86 (1 H, dd, J 10.0 Hz, 3.8 Hz, H-2), 3.76 (3

H, s, CO₂Me), 3.48 (3 H, s, OMe), 2.11 (3 H, s, Ac); ¹³C NMR

(100 MHz, CDCl₃): d 170.4, 168.0 (C O), 99.8 (C-1), 71.2 (C-4),

68.6, 68.2, 67.9 (C-2, C-3, C-5), 55.8 (OMe), 52.2 (CO₂Me), 20.2

(Ac).

Methyl (3-C-acetoxymethyl-2,3,di-O-acetyl-b-D-erythrofurano-

syl)-(1→3)-(methyl 4-O-acetyl-2-O-benzoyl-a-D-

galactopyranosid)uronate (28)

Glycosyl acceptor 26 (258 mg, 0.70 mmol), glycosyl donors 22a,b

˚

(250 mg, 0.96 mmol) and mol. sieves 4 A (500 mg) were stirred with

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20.3, 20.2 (Ac ¥ 7); HRMS (ESI+): m/z found 798.2660 ([M +

NH₄]⁺); calcd for C₃₂H₄₄O₂₂·NH₄798.2662.

residue was puriﬁed by chromatography to give compound 32 (4.35

g, 58%); R_f0.40 (hexane–EtOAc 3 : 2); [a]²_D³+76 (c 1.05, CHCl₃);

¹H NMR (400 MHz, CDCl₃): d 7.36–7.31 (4 H, m, SC₆H₄Me),

7.07 (4 H, dd, J 7.7 Hz, J 0.7 Hz, SC₆H₄Me), 4.68 (1 H, d, J

2.6 Hz, H-1), 4.38 (1 H, dd, J 7.2 Hz, 2.6 Hz, H-2), 4.24 (1 H,

dd, J 7.3 Hz, J 7.2 Hz, H3), 3.79–3.59 (3 H, m, H-4, H-5a, H-5b),

3.05 (1 H, br d, J 4.7 Hz, OH-4), 2.67 (1 H, br s, OH-5), 2.31

L-Arabinose di(p-tolyl) dithioacetal (30)

Powdered L-arabinose (13.95 g, 93.0 mmol) was added portion-

wise to vigorously stirred solution of thiocresol (25.4 g, 204.6

mmol) in 90% CF₃CO₂H (55 ml), the mixture heated to 50–60 ^◦C

and stirring continually until the sugar was completely dissolved.

The solution was allowed to stand for 30 min and concentrated

under reduced pressure at 40 ^◦C to give a viscous syrup which was

crystallized after addition of EtOH (150 mL). The crystals were

ﬁltered off, washed extensively with EtOH and _◦dried to afford

dithioacetal 30 (24.0 g, 68%); m.p. 168.5–169.5 C; [a]²_D⁴-34 (c

1.05, DMF); ¹H NMR (400 MHz, CD₃OD): d 7.36–7.30 (4 H, m,

SC₆H₄Me), 7.12–7.05 (4 H, m, SC₆H₄Me), 4.60 (1 H, d, J 7.7 Hz,

H-1), 4.12 (1 H, dd, J 8.1 Hz, 1.9 Hz, H-3), 3.96 (1 H, dd, J 7.7 Hz,

1.9 Hz, H-2), 3.77 (1 H, dd, J 10.9 Hz, 3.1 Hz, H-5a), 3.72–3.64

(1 H, m, H-4), 3.62 (1 H, dd, J 10.9 Hz, 6.0 Hz, H-5b), 2.31 (3

H, s, SC₆H₄Me), 2.30 (3 H, s, SC₆H₄Me); ¹³C NMR (100 MHz,

CD₃OD): d 138.7, 134.2 (2 C), 131.4, 130.2 (2 C) (SC₆H₄Me), 72.8

(C-4), 72.2, 71.6 (C-2 and C-3), 65.1 (C-1), 64.4 (C-5), 20.8 (2 C,

SC₆H₄Me); HRMS (ESI+): m/z found 398.1461 ([M + NH₄]⁺);

calcd for C₁₉H₂₄O₄S₂·NH₄398.1454.

(6 H, s, SC₆H₄Me), 1.52 (3 H, s, CMe₂), 1.39 (3 H, s, CMe₂); ¹³

C

NMR (100 MHz, CDCl₃): d 137.9, 137.8, 133.0, 132.6, 1, 129.8,

129.7 (SC₆H₄Me), 110.2 (CMe₂), 81.4 (C-3), 78.4 (C-2), 72.9 (C-

4), 63.9 (C-5), 61.6 (C-1), 27.3 (CMe₂), 27.0, (CMe₂), 21.1 (¥ 2,

SC₆H₄Me); HRMS (ESI+): m/z found 421.1503 ([M + NH₄]⁺);

calcd for C₂₂H₂₈O₄S₂·NH₄421.1502.

3-C-Hydroxymethyl-2,3-O-isopropylidene-D-glycero-tetrose

di(p-tolyl) dithioacetal (34)

Silica gel-supported NaIO₄was prepared by addition of a hot

(70 ^◦C) solution of NaIO₄(2.56 g, 12.0 mmol) in H₂O (6.1 mL) to

silica gel (12.0 g, Fluorochem, 40–70 mkm) with vigorous swirling

and shaking as described.³⁴This mixture was added at room

temperature to a vigorously stirred solution of diol 32 (3.69 g,

8.77 mmol) in CH₂Cl₂(45 mL) co_◦ntaining Et₃N (1.2 mL) and

stirring continued for 30 min at 22 C. Silica gel was ﬁltered off,

washed with CH₂Cl₂(50 mL) and at 22 ^◦C combined ﬁltrates

were concentrated and dried to give crude 2,3-O-isopropylidene-

L-threo-tetrodialdose di(p-tolyl)dithio acetal (33, 3.06 g), R_f0.20–

0.40 (hexane–EtOAc 80 : 20); ¹H NMR (400 MHz, CDCl₃): d 9.82

(1 H, d, J 1.2 Hz, CHO), 7.37 (m, 4 H, SC₆H₄Me), 7.12 (4 H,

m, SC₆H₄Me), 4.65 (1 H, d, J 5.5 Hz, H-2), 4.50–4.44 (2 H, m,

CH(STol)₂, H-3), 2.35 (6 H, s, SC₆H₄Me), 1.59 (3 H, s, CMe₂), 1.39

(3 H, s, CMe₂); ¹³C NMR (100.5 MHz, CDCl₃): d 200.9 (CHO),

138.6, 133.6, 133.5, 130.1 (SC₆H₄Me), 112.6 (CMe₂), 83.0 (C-2),

78.8 (C-3), 62.0 (C-1), 26.9, 26.6 (CMe₂), 21.4 (¥ 2, SC₆H₄Me).

The product 33 was dissolved in EtOH (20 ml), 37% aq. CH₂O (2

mL) and a solution of NaOH (930 mg) in H₂O (14 mL) were added

2,3:4,5-Di-O-isopropylidene-L-arabinose di(p-tolyl) dithioacetal

(31)

Tetraol 30 (15.0 g, 39.5 mmol) was dissolved in a mixture of 2,2-

dimethoxypropane (20.0 mL) and acetone (90 mL) in the presence

of CSA (0.6 g) which was stirred for 24 h at 22 ^◦C. The solution

was treated with Et₃N (10 mL), concentrated and dried to afford

compound 31 (18.0 g, 99%) which was used in the next step without

further puriﬁcation. An analytical sample of 31 was obtained

by chromatographic puriﬁcation, R_f0.67 (hexane–EtOAc 80 : 40);

[a]²_D⁴47 (c 1.3, CHCl₃); (400 MHz, CDCl₃): d 7.37 (2 H, d, J

8.2 Hz, SC₆H₄Me), 7.30 (2 H, d, J 8.2 Hz, SC₆H₄Me), 7.08 (2

H, d, J 8.2 Hz, SC₆H₄Me), 7.06 (2 H, d, J 8.2 Hz, SC₆H₄Me),

4.70 (1 H, d, J 1.8 Hz, H-1), 4.32 (1 H, dd, J 7.5 Hz, J 1.8 Hz,

H-2), 4.19 (1 H, dd, J 7.5 Hz, J 7.9 Hz, H-3), 4.12 (1 H, dd,

J 8.4 Hz, J 6.1 Hz, H-5a), 4.00 (1 H, ddd, J 8.2 Hz, J 6.0 Hz,

5.8 Hz, H-4), 3.91 (1 H, dd, J 8.4 Hz, J 5.6 Hz, H-5b), 2.32 (3

H, s, SC₆H₄Me), 1.52 (2 H, s, CMe₂), 1.38 (2 H, s, CMe₂), 1.31 (1

H, s, CMe₂), 1.28 (2 H, s, CMe₂); ¹³C NMR (100 MHz, CDCl₃):

d 137.8, 137.5, 132.5, 132.2, 131.6, 131.3129.9, 129.8 (SC₆H₄Me),

110.7 (CMe₂), 109.9 (CMe₂), 82.3 (C-2), 79.0 (C-3), 77.2 (C-4),

68.2 (C-5), 60.4 (C-1), 27.4, 27.2, 26.7, 25.5 (4 C, CMe₂), 21.4

(SC₆H₄Me); HRMS (ESI+): m/z found 478.2080 ([M + NH₄]⁺);

calcd for C₂₅H₃₂O₄S₂·NH₄478.2080.

◦

and the mixture was stirred for 17 h at 22 C. The mixture was

acidiﬁed by addition of AcOH and then concentrated to remove

EtOH. The residue was suspended in CH₂Cl₂, washed with H₂O

and satd NaCl solution, the organic layer was separated and the

solvent was evaporated in vacuo. Puriﬁcation of the residue by

chromatography afforded compound 34 (1.76 g, 48%), R_f0.30

(hexane–EtOAc 60 : 40); m.p. 86–89 ^◦C (Et₂O–hexane); [a]_D²⁰+136

1

(c 1.0, CHCl₃); H NMR (400 MHz, CDCl₃) 7.44 (2 H, d, J

8.1 Hz, SC₆H₄Me), 7.35 (2 H, d, J 8.1 Hz, SC₆H₄Me), 7.13 (4

H, m, SC₆H₄Me), 4.55 (1 H, d, J 8.6 Hz, H-1), 4.18 (1 H, d, J

8.6 Hz, H-2), 3.85 (1 H, d, J 12.1 Hz, H-3¢a), 3.82 (2 H, s, H-4a,

4b), 3.77 (1 H, d, J 12.1 Hz, H-3¢b), 2.34 (6 H, s, SC₆H₄Me),

1.53 (3 H, s, CMe₂), 1.43 (3 H, s, CMe₂); ¹³C NMR (100.5 MHz,

CDCl₃): d 138.7, 138.5, 134.7, 133.1, 130.0, 129.56 (SC₆H₄Me),

108.0 (CMe₂), 84.5 (C-3), 78.7 (C-2), 64.1 (C-3¢), 62.5 (C-4), 28.3

(CMe₂), 26.4 (CMe₂), 21.2 (2 C, SC₆H₄Me); HRMS (ESI+): m/z

found 421.1507 ([M + H]⁺); calcd for C₂₂H₂₈O₄S₂·H 421.1502.

2,3-O-Isopropylidene-L-arabinose di(p-tolyl) dithioacetal (32)

A solution of compound 31 (8.2 g, 17.8 mmol) and pyridinium

tosylate (4.5 g, 17.9 mmol) in MeOH (82 mL) was stirred at 55 ^◦C

and the progress of the reaction was monitored by TLC (hexane–

EtOAc 3 : 2). After 18 h TLC indicated the presence of a major

product (R_f0.40), a minor product (R_f0.50), unreacted 32 (R_f

0.90) and a polar material (R_f~ 0). After quenching the reaction

with pyridine (5 mL) and addition of toluene (100 mL), a white

precipitate was ﬁltered off. The ﬁltrate was concentrated and the

p-Tolyl 3-C-chloroacetoxymethyl-2,3-O-isopropylidene-1-thio-a-

D-erythrofuranoside (36a) and p-tolyl 3-C-chloroacetoxy-

methyl-2,3-O-isopropylidene-1-thio-b-D-erythrofuranoside (36b)

A solution of dithioacetal 34 (1.50 g, 3.57 mmol) in MeCN

(30 mL) was cooled to -30 ^◦C and NIS (800 mg, 3.57 mmol)

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was added. The mixture was stirred and allowed to warm to 0 ^◦C,

and after 10 min the reaction was quenched with Et₃N (0.5 mL),

the mixture was diluted with CH₂Cl₂(40 mL) and washed with

10% Na₂S₂O₃and satd NaHCO₃solution. The organic layer was

concentrated to give brown residue which was puriﬁed by column

chromatography to give a mixture of p-tolyl 3-C-hydroxymethyl-

2,3-O-isopropylidene-1-thio-a,b-D-erythrofuranoside (35a,b, 940

mg); R_f0.57 (minor spot) and 0.41 (major spot) (hexane–EtOAc

selected ¹H NMR (400 MHz, CDCl₃): d 5.60 (0.25 H, d, J 3.9 Hz,

H-1 a), 5.38 (1 H, d, J 3.9 Hz, H-1 b). This product was dissolved

in toluene (2.0 mL), PhCH(OMe)₂(2.0 ml, 13.3 mmol) and a cat.

amount of CSA were added, and the mixture was stirred for 17 h at

22 ^◦C. The reaction was quenched with Et₃N (0.5 mL), the mixture

was concentrated and the residue was puriﬁed by chromatography.

The ﬁrst eluted product was benzylidene derivative 38b (690 mg,

61%) and the second was 38a (200 mg, 18%). Compound 38a: R_f

1

50 : 50); H NMR (400 MHz, CDCl₃): d 7.41 (2 H, d, J 8.1 Hz,

0.41 (hexane–EtOAc 9 : 1), [a]²_D²+16 (c 1.01, CHCl₃); H NMR

SC₆H₄Me a), 7.35 (0.46 H, d, J 8.0 Hz, SC₆H₄Me b), 7.10 (2.46

H, m, SC₆H₄Me a and b), 5.60 (0.23 H, s, H-1 b), 5.07 (1 H, d, J

3.8 Hz, H-1 a), 4.70 (1 H, d, J 3.8 Hz, H-2 a), 4.50 (0.23 H, s, H-2

b), 4.10 (0.23 H, d, J 10.6 Hz, H-4a b), 4.08 (1 H, d, J 10.3 Hz,

H-4a a), 4.00 (0.23 H, d, J 10.6 Hz, H-4b b), 3.83 (0.48 H, br s,

H-3¢ b), 3.72 (2 H, m, H-3¢ a), 3.62 (1 H, d, J 10.3 Hz, H-4b a),

2.31 (3 H, s, SC₆H₄Me a), 2.03 (0.69 H, s, SC₆H₄Me b), 1.62 (3

H, s, CMe₂a), 1.48 (0.69 H, s, CMe₂b), 1.45 (3 H, s, CMe₂a),

1.41 (0.69 H, s, CMe₂b); HRMS (ESI+): m/z found 314.1423 ([M

+ NH₄]⁺); calcd for C₁₅H₂₀O₄·NH₄314.1421. To a solution of a

mixture of compounds 35a,b (940 mg, 3.17 mmol) in CH₂Cl₂(30

mL) collidine (0.5 mL, 3.49 mmol) and chloroacetyl chloride (0.28

mL, _◦3.50 mmol) were added successively at -40 ^◦C. After stirring

at 0 C for 30 min the solution was washed with satd NaHCO₃,

concentrated and the residue was puriﬁed by chromatography to

give chloroacetate 36a,b as a mixture of a- and b-thioglycosides

(1.08 g, 81% overall, a : b ~ 67 : 33). Further chromatography

puriﬁcation afforded analytical samples of pure 36a and 36b.

Compound 36a: R_f0.46 (hexane–EtOAc 80 : 20); [a]²_D⁸-255 (c 1.2

(400 MHz, CDCl₃): d 7.68–7.58 (2 H, m), 7.50–7.35 (5 H, m),

7.13 (2 H, d, J 7.9 Hz, PhCH), 6.06 (1 H, s, PhCH), 5.15 (1 H,

d, J 3.9 Hz, H-1), 4.80 (1 H, d, J 3.9 Hz, H-2), 4.55 (1 H, d, J

11.9 Hz, H3¢a), 4.43 (1 H, d, J 11.9 Hz, H-3¢b), 4.30 (1 H, d, J

10.5 Hz, H-4a), 4.13 (2 H, s, CH₂Cl), 3.67 (1 H, d, J 10.5 Hz,

H-4b), 2.33 (4 H, s, SC₆H₄Me); ¹³C NMR (100 MHz, CDCl₃): d

167.0 (C O), 137.7, 135.5, 131.8, 131.3, 130.2, 129.9, 128.5, 127.4

(aromatics), 107.8 (PhCH), 91.4 (C-1), 89.8 (C-3), 84.6 (C-2), 72.8

(C-4), 65.1 (C-3¢), 40.4 (CH₂Cl), 21.0 (SC₆H₄Me); HRMS (ESI+):

m/z found 438.1131 ([M + NH₄]⁺); calcd for C₂₁H₂₁ClO₅S·NH₄

438.1136. Compound 38b: R_f0.49 (hexane–EtOAc 9 : 1), [a]²_D⁰-201

(c 1.1, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d 7.53–7.48 (2 H, m,

aromatics), 7.40–7.34 (5 H, m, aromatics), 7.12 (2 H, d, J 8.3 Hz,

aromatics), 5.93 (1 H, s, PhCH), 5.78 (1 H, s, H-1), 4.60 (1 H, s,

H-2), 4.59 (2 H, d, J 11.8 Hz, H-3¢a), 4.49 (1 H, d, J 11.8, H-3¢b),

4.26–4.17 (2 H, m, H-4a,b), 4.15 (2 H, s, CH₂Cl), 2.32 (3 H, s,

SC₆H₄Me); ¹³C NMR (75 MHz, CDCl₃): d 167.1 (C O), 138.3,

135.7, 133.0, 130.2, 130.1, 128.9, 128.6, 127.2 (Ph), 106.7 (PhCH),

92.6 (C-1), 90.3 (C-3), 87.7 (C-1), 72.6 (C-4), 65.4 (C-3¢), 40.6

(CH₂Cl), 21.0 (SC₆H₄Me); HRMS (ESI+): m/z found 438.1136

([M + NH₄]⁺); calcd for C₂₁H₂₁ClO₅S·NH₄438.1136.

1

CHCl₃); H NMR (400 MHz, CDCl₃): d 7.40 (1 H, d, J 8.0 Hz,

SC₆H₄Me), 7.09 (2 H, d, J 8.0 Hz, SC₆H₄Me), 5.03 (2 H, d, J

3.9 Hz, H-1), 4.65 (1 H, d, J 3.9 Hz, H-2), 4.29 (1 H, d, J 11.7 Hz,

H-4a), 4.25 (1 H, d, J 11.7 Hz, H-4b), 4.05 (1 H, d, J 10.3 Hz,

H-3¢a), 4.04 (2 H, s, ClCH₂), 3.61 (1 H, d, J 10.3 Hz, H-3¢b),

2.30 (MeC₆H₄S), 1.60 (CMe₂), 1.44 (CMe₂); ¹³C NMR (100 MHz,

CDCl₃): d 167.0 (C O), 137.6, 131.7, 129.9 (SC₆H₄Me), 115.5

(CMe₂), 91.7 (C-3), 89.8 (C-1), 84.4 (C-2), 73.8 (C-4), 66.0 (C-3¢),

40.5 (CH₂Cl), 27.5 (2 C, CMe₂), 20.9 (SC₆H₄Me); HRMS (ESI+):

m/z found 390.1138 ([M + NH₄]⁺); calcd for C₁₇H₂₁ClO₅S·NH₄

390.1136. Compound 36b: R_f0.36 (hexane–EtOAc 80 : 20); [a]_D²⁸

+41 (c1.1 CHCl₃); ¹H NMR (400 MHz, CDCl₃): d 7.35 (2 H, d, J

8.0 Hz, SC₆H₄Me), 7.11 (2 H, d, J 8.0 Hz, SC₆H₄Me), 5.60 (1 H, s,

H-1), 4.54 (1 H, s, H-2), 4.48 (1 H, d, J 11.7 Hz, H-3¢a), 4.41 (1 H, d,

J 11.6 Hz, H-3¢b), 4.15 (1 H, d, J 10.4, H-4a), 4.15 (2 H, s, ClCH₂),

4.02 (1 H, d, J 10.4, H-4b), 2.31 (2 H, s, SC₆H₄Me), 1.47 (3 H, s,

CMe₂), 1.40 (3 H, s, CMe₂); ¹³C NMR (100 MHz, CDCl₃): d 167.1

(C O), 138.2, 133.0, 130.0, 129.2 (SC₆H₄Me), 114.5 (CMe₂), 93.3

(C-3), 90.3 (C-1), 87.6 (C-2), 73.5 (C-4), 66.5 (C-3¢), 40.6 (CH₂Cl),

27.5, 27.4 (CMe₂), 21.0 (SC₆H₄Me); HRMS (ESI+): m/z found

390.1138 ([M + NH₄]⁺); calcd for C₁₇H₂₁ClO₅S·NH₄390.1136.

Methyl (2,3-O-(S)-benzylidene-3-C-chloroacetoxymethyl-b-D-

erythrofuranosyl)-(1→2)-(methyl 3,4-O-isopropylidene-a-D-

galactopyranosid)uronate (39)

A solution of thioglycoside 38b (700 mg, 1.67 mmol), glycosyl

˚

acceptor 12 (440 mg, 1.67 mmol) and mol. sieves 4 A (1.4 g) in

CH₂Cl₂(45 mL) was stirred for 20 min at room temperature, NIS

(395 mg, 1.7 mmol) was added and the mixture was cooled to

-40 ^◦C. A solution of TMSOTf (30 mL, 0.17 mmol) in CH₂Cl₂

(0.3 mL) was added to the mixture w_◦hich was stirred for 30 min

to allow temperature to raise to -20 C, then treated with Et₃N

(0.3 mL) and ﬁltered through Celite. The ﬁltrate was vigorously

stirred for 30 min with aq. Na₂S₂O₃solution (50 mL), the organic

layer was separated and washed with aq. NaHCO₃solution, dried

and concentrated. Puriﬁcation of the product by chromatography

afforded disaccharide 39 (710 mg, 76%). R_f0.55 (hexane–EtOAc

1

60 : 40); [a]²_D¹+2 (c 0.8 CHCl₃); H NMR (400 MHz, CDCl₃): d

7.50 (1 H, m, Ph), 7.39 (1 H, m, Ph), 5.93 (1 H, s, CHPh), 5.45

(1 H, s, H-1 Api), 4.98 (1 H, d, J 3.5 Hz, H-1 GalA), 4.61 (1 H,

s, H-2 Api), 4.59 (1 H, d, J 2.7 Hz, H-5 GalA), 4.52 (2 H, broad

s, H-3¢a,b Api), 4.50 (1 H, dd, J 2.7 Hz, J 5.4 Hz, H-4 GalA),

4.27 (1 H, dd, J 5.4 Hz, J 8 Hz, H-3 GalA), 4.14–4.17 (3 H, m,

H-4a Api, COCH₂Cl), 3.91 (1 H, d, J 10.1 Hz, H-4b Api), 3.84

(3 H, s, CO₂Me), 3.81 (1 H, dd, J 3.5 Hz, J 8 Hz, H-2 GalA),

p-Tolyl 3-C-chloroacetoxymethyl-2,3-O-(S)-benzylidene-1-thio-a-

D-erythrofuranoside (38a) and p-tolyl 3-C-chloroacetoxy-

methyl-2,3-O-(S)-benzylidene-1-thio-b-D-erythrofuranoside (38b)

A mixture of glycosides 36a,b (1.00 g, 2.68 mmol, a : b ~ 67 : 33)

was dissolved in CF₃CO₂H (10 mL), H₂O (1.1 mL) was added

and the solution was allowed to stand at 20 ^◦C for 90 min before

being concentrated with toluene (3 ¥ 20 mL) at reduced pressure at

30 ^◦C to give a yellow syrup containing a crude mixture of 37a,b;

3.43 (3 H, s, OMe), 1.51 (3 H, s, CMe₂), 1.34 (3 H, s, CMe₂); ¹³

C

NMR (100 MHz, CDCl₃): d 168.5, 167.1 (C O), 128.5, 127.1,

130.1 (Ph), 110.0 (Me₂C), 107.6 (C-1 Api), 106.4 (PhCH), 99.7

(C-1 GalA), 89.5 (C-3 Api), 86.9 (C-2 Api), 75.9 (C-2 GalA), 74.8

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(C-3 GalA), 73.7 (C-4 GalA), 72.9 (C-4 Api), 67.0 (C-5 GalA),

65.5 (C-3¢ Api), 56.1 (OMe), 52.5 (CO₂Me), 40.5 (CH₂Cl), 28.0

(Me₂C), 26.2 (Me₂C); HRMS (ESI+): m/z found 557.1420 ([M +

H]⁺); calcd for C₂₅H₃₀ClO₁₂557.1421.

(C-2 GalA), 74.7 (C-3 GalA), 73.6 (C-4 GalA), 73.2 (C-4 Api),

72.2 (C-2 or C-3 Rha), 71.2 (C-4 Rha), 70.0 (C-5 Rha), 69.1 (C-3¢

Api), 66.9 (C-5 GalA), 56.0 (OMe), 52.3 (CO₂Me), 27.9 (CMe₂),

26.1 (CMe₂), 20.5 (Ac), 19.2 (C-6 Rha); HRMS (ESI+): m/z found

714.2611 ([M + NH₄]⁺); calcd for C₃₂H₄₀O₁₇·NH₄714.2604.

Methyl (2,3-O-(S)-benzylidene-3-C-hydroxymethyl-b-D-

erythrofuranosyl)-(1→2)-(methyl 3,4-O-isopropylidene-a-D-

galactopyranosid)uronate (40)

Methyl C-(b-L-rhamnopyranosyloxymethyl)-(1→3)-b-D-erythro-

furanosyl-(1→2)-(methyl a-D-galactopyranosid)uronate (42)

To a solution of the trisaccharide 41 (150 mg, 0.22 mmol)

in EtOAc (4 ml) was added 10% Pd/C, the mixture was

degassed and then stirred for 12 h at 35 ^◦C under H₂. The

catalyst was separated by ﬁltration through a Celite pad, the

ﬁltrate was concentrated and the residue was puriﬁed by ﬂash

chromatography to afford methyl C-(4-O-acetyl-2,3-O-carbonyl-

b-L-rhamnopyranosyloxymethyl)-(1→3)-(b-D-erythrofuranosyl)-

A solution of disaccharide 39 (640 mg, 1.15 mmol) in 0.01 M

MeOH (6 mL) was stirred for 10 min at 21 ^◦C and then neutralized

and concentrated. The residue was puriﬁed by chromatography

(hexane–hexane 30 : 70→10 : 90) to give alcohol 40 (470 mg, 85%);

R_f0.20 (hexane–EtOAc 30 : 70); [a]²_D²-4 (c 1.0 CHCl₃); ¹H NMR

(400 MHz, CDCl₃): d 7.51 (2 H, m, Ph), 7.40 (3 H, m, Ph), 5.91

(1 H, s, H-1 Api), 5.45 (1 H, s, CHPh), 4.97 (1 H, d, J 3.5 Hz,

H-1 GalA), 4.59 (1 H, d, J 2.7 Hz, H-5 GalA), 4.54 (1 H, d, J <

1 Hz, H-2 Api), 4.49 (1 H, dd, J 2.7, 5.4 Hz, H-4 GalA), 4.29 (1 H,

dd, J 5.4, 7.6 Hz, H-3 GalA), 4.13 (1 H, d, J 10.1 Hz, H-4a Api),

3.82–3.98 (7 H, m, H-3¢a, H-3¢b, H-4b Api, H-2 GalA, CO₂Me),

(1→2)-(methyl

3,4-O-isopropylidene-a-D-galactopyranosid)-

1

uronate (110 mg, 84%); H NMR (400 MHz, CDCl₃): d 5.32 (1

H, dd, J 8.6 Hz, 5.7 Hz, 4 Rha), 5.18 (1 H, d, J 1.5 Hz, H-1

Api), 5.02 (1 H, d, J 2.5 Hz, H-1 Rha), 4.96 (1 H, d, J 3.5 Hz,

H-1 GalA), 4.85 (2 H, m, 2 Rha, H-3 Rha), 4.59 (1 H, d, J

2.8 Hz, H-5 GalA), 4.49 (1 H, dd, J 5.4 Hz, 2.9 Hz, H-4 GalA),

4.29 (1 H, dd, J 7.9 Hz, 5.4 Hz, H-3 GalA), 4.07–3.97 (2 H, m,

H-2 Api, 4¢a Api), 3.90 (2 H, s, H-3¢a, H-3¢b Api), 3.83 (3 H, s,

CO₂Me), 3.77 (3 H, m, H-4¢b Api, H-2 GalA, H-5 Rha), 3.42 (3

H, s, OMe), 2.12 (3 H, s, Ac), 1.51 (3 H, s, CMe₂), 1.31 (3 H, s,

CMe₂), 1.24 (3 H, d, J 6.3 Hz, H-6 Rha). ¹³C NMR (100 MHz,

CDCl₃): d 169.3 (MeCO), 168.5 (CO₂Me), 110.1 (Me₂C), 109.8

(C-1 Api), 99.9 (C-1 Gal); 95.8(C-1 Rha), 78.7, 77.5, 76.5, 76.2,

74.9, 74.1, 73.8, 72.7, 72.4, 71.3, 70.3, 67.0, 56.2 (OMe), 52.5

(CO₂OMe), 28.01(CMe₂), 26.3 (CMe₂), 20.8 (Ac), 19.2 (C-6 Rha);

MALDI TOF(+) MS, m/z found 631.08 ([M + Na]⁺); calcd

for C₂₅H₃₆O₁₇·Na 631.53. The latter compound was dissolved in

80% AcOH, the solution was heated for 25 min at 60 ^◦C and

concentrated. After drying the residue by repeated concentration

of its solution in toluene in vacuo, the residue was dissolved in

0.05 M NaOMe in MeOH (1 mL) and the solution was stirred

3.43 (3 H, s, OMe), 1.52 (3 H, s, CMe₂), 1.34 (3 H, s, CMe₂), ¹³

C

NMR (100 MHz, CDCl₃): d 168.5 (CO₂Me), 136.1 (Ph), 130.0

(Ph), 128.5 (Ph), 127.1 (Ph), 109.9 (Me₂C), 107.2 (PhCH), 106.2

(C-1 Api), 99.7 (C-1 Gal), 92.1 (C-3 Api), 86.7 (C-2 Api), 75.3

(C-2 GalA), 74.9 (C-3 GalA), 73.7 (C-4 GalA), 73.3 (C-4 Api),

67.1 (C-5 GalA), 63.7 (C-3¢ Api), 56.1 (OMe), 52.4 (CO₂Me), 28.0

(CMe₂), 26.2 (CMe₂); HRMS (ESI+): m/z found 500.2129 ([M +

NH₄]⁺); calcd for C₂₃H₃₀O₁₁·NH₄500.2126.

Methyl C-(4-O-acetyl-2,3-O-carbonyl-b-L-rhamnopyranosyloxy-

methyl)-(1→3)-(2,3-O-(S)-benzylidene-b-D-erythrofuranosyl)-

(1→2)-(methyl 3,4-O-isopropylidene-a-D-galactopyranosid)-

uronate (41)

To a stirred solution of disaccharide 40 (470 mg, 0.98 mmol),

˚

bromide 15 (540 mg, 1.84 mmol) and mol. sieves 4 A (2 g) in

◦

CH₂Cl₂(10 mL) was added Ag₂O (460 mg, 2.0 mmol) and stirring

was continued for 17 h at 21 ^◦C. The mixture was ﬁltered through

Celite and the ﬁltrate was diluted with CH₂Cl₂, washed successively

with aq. Na₂S₂O₃and water and concentrated. The product was

puriﬁed by chromatography (hexane–EtOAc 80 : 20→20:80) to

give compound 41 (403 mg, 59%). Crystals suitable for X-ray

analysis were obtained by crystallization from hexane–CH₂Cl₂.

Compound 41, R_f0.22 (hexane–EtOAc 30 : 70); m.p. 142–144 ^◦C;

[a]_D+16 (c 1.08, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d 7.54 (2

H, m, Ph), 7.38 (3 H, m, Ph), 5.92 (1 H, s, H-1 Api), 5.42 (1 H, s,

CHPh), 5.39 (1 H, dd, J 3 Hz, J 5.5 Hz, H-4 Rha), 5.15 (1 H, broad

s, H-1 Rha), 5.02 (1 H, d, J 3.5 Hz, H-1 GalA), 4.79–4.75 (2 H, m,

H-2, H-3 Rha), 4.60 (1 H, d, J 2.8 Hz, H-5 GalA), 4.52 (1 H, s, H-2

Api), 4.48 (1 H, dd, J 2.8 Hz, J 5.5 Hz, H-4 GalA), 4.28 (1 H, dd,

J 5.5 Hz, J 7.8 Hz, H-3 GalA), 4.19 (1 H, d, J 3 Hz, H-3¢a Api),

4.15 (1 H, d, J 2 Hz, H-4a Api), 3.95 (1 H, broad s, H-4b Api), 3.91

(1 H, d, J 3 Hz, H-3¢b Api), 3.84 (1 H, s, CO₂Me), 3.72–3.82 (2 H,

m, H-2 GalA, H-5 Rha), 3.43 (1 H, s, OMe), 2.08 (1 H, s, OAc),

1.51 (1 H, s, Me₂C), 1.33 (1 H, s, Me₂C), 1.31 (1 H, m, H-6 Rha);

¹³C NMR (100.5 MHz, CDCl₃): d 169.1 (MeCO), 168.4 (CO₂Me),

153.5 (Ph), 136.2 (Ph), 129.7 (Ph), 128.3 (Ph), 127.2 (Ph), 109.7

(Me₂C), 107.7 (PhCH), 106.2 (C-1 Api), 99.6 (C-1 Gal), 95.1 (C-1

Rha), 91.0 (C-3 Api), 86.5 (C-2 Api), 76.1 (C-2 or C-3 Rha), 75.9

for 30 min at 22 C. The mixture was carefully neutralised with

Amberlite IR120 (H⁺), the resin was removed and the solution

was concentrated. The residue was redissolved in water, ﬁltered

through C18 cartridge and freeze-dried. Compound 42 (60 mg,

1

56%); [a]²_D⁰+61 (c 1.2, H₂O); H NMR (400 MHz, D₂O): d 5.10

(1 H, d, J 3.0 Hz, H-1 Api), 4.94 (1 H, d, J 3.8 Hz, H-1 GalA),

4.56 (1 H, s, H-1 Rha), 4.55 (1 H, d, J 1.0 Hz, H-5 GalA), 4.26

(1 H, dd, J 3.3, 1.0 Hz, H-4 GalA), 4.02 (1 H, d, J 3.0 Hz, H-2

Api), 3.98–3.94 (2 H, m, H-2 Rha, H-3¢a Api), 3.90–3.82 (3 H,

m, H-3¢b and H-4a Api, H-3 GalA), 3.75–3.70 (4 H, m, H-2

GalA, OMe), 3.67 (1 H, d, J 10.7, H-4b Api), 3.53–3.47 (1 H, m,

H-3 Rha), 3.36–3.26 (5 H, m, H-4 and H-5 Rha, CO₂Me), 1.22

(3 H, J 5.4 Hz, H-6 Rha). ¹³C NMR (100 MHz, D₂O): d 171.1

(C-6 GalA), 110.2 (C-1 Api), 100.4 (C-1 Rha), 99.3 (C-1 GalA),

78.6 (C-3 Api), 77.4 (C-2 Api), 75.9, 73.8, 72.5, 72.2, 72.0, 71.3,

70.5, 70.3, 69.9, 67.8, 55.5 (OMe), 52.9 (CO₂Me), 16.7 (C-6 Rha);

MALDI TOF(+) MS, m/z found 522.99 ([M + Na]⁺); calcd for

C₁₉H₃₂O₁₅·Na 523.44.

Methyl C-(b-L-rhamnopyranosyloxymethyl)-(1→3)-b-D-

erythrofuranosyl-(1→2)-(a-D-galactopyranosid)uronic acid (1)

A solution of methyl ester 42 (23 mg, 46 mmol) in H₂O–MeOH–

Et₃N (3 : 2 : 1, 10 mL) was kept for 17 h at 22 ^◦C, diluted with water

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(10 mL), concentrated in vacuo, and the residue was puriﬁed by

GPC to give the title compound 1 (18 mg, 78%); [a]²_D²+ 34 (c 1.0,

H₂O); ¹H NMR (400 MHz, D₂O): d 5.12 (1 H, J 3.0 Hz, H-1 Api),

4.90 (1 H, J 3.8 Hz, H-1 GalA), 4.58 (1 H, s, H-1 Rha), 4.21 (dd,

J 1.2 Hz, J 3.4 Hz, H-4 GalA), 4.13 (1 H, J 1.2 Hz, H-5 GalA),

4.00–3.96 (2 H, m, H-2 Rha, H-3¢a Api), 3.90–3.84 (3 H, H-3¢b

and H-4a Api, H-3 GalA), 3.74 (1 H, J 3.8 Hz, J 10.2 Hz, H-2

GalA), 3.69 (1 H, J 10.8 Hz, H-4b Api), 3.50 (1 H, m, H-3 Rha),

3.34–3.27 (5 H, H-4 and H-5 Rha, OMe), 1.23, (3 H, d, J 5.9 Hz,

H-6 Rha); ¹³C NMR (100 MHz, D₂O): d 175.6 (C O), 110.2 (C-1

Api), 100.4 (C-1 Rha), 98.9 (C-1 GalA), 78.6 (C-3 Api), 77.4 (C-2

Api), 76.3 (C-2 GalA), 73.8 (C-3¢ Api), 72.5 (C-3 Rha), 72.1, 72.0

(C-4 and C-5 Rha), 71.4 (C-4 Rha), 71.0 (C-5 GalA), 70.6 (C-4

GalA), 70.4 (C-2 Rha), 68.7 (C-3 GalA), 55.0 (OMe), 16.6 (C-6

Rha). HRMS (ESI+): m/z found 504.1922 ([M + NH₄]⁺); calcd

for C₁₈H₃₀O₁₅·NH₄504.1923.

(CO₂H), 110.1 (C-1 Api), 99.3 (C-1 GalA), 79.4 (C-3 Api), 77.5

(C-3 GalA), 77.1 (C-2 Api), 73.7 (C-4 Api), 70.5 (C-5 GalA), 69.9

(C-4 GalA), 66.7 (C-2 GalA), 63.5 (C-3¢ Api), 55.4 (OMe). HRMS

(ESI+): m/z found 363.0897 ([M + NH₄]⁺); calcd for C₁₂H₂₀O₁₁·Na

363.0897

Methyl (3-C-hydroxymethyl-b-D-erythrofuranosyl)-(1→2)-[(3-C-

hydroxymethyl-b-D-erythrofuranosyl)-(1→3)]-(a-D-galactopyra-

nosid)uronic acid (4)

A solution of trisaccharide 29 (45 mg, 0.058 mmol)_◦in 0.02 M

methanolic NaOMe (0.5 mL) was kept for 1 h at 22 C, diluted

with MeOH and neutralised with Amberlite IR120 (H⁺) resin.

The resin was ﬁltered off and the ﬁltrate was concentrated to give

methyl ester of 4 (26 mg, 93%); [a]²_D⁰- 20 (c 0.8, H₂O); ¹H NMR

(400 MHz, D₂O): d 5.09 (1 H, d, J 2.9 Hz, H-1 Api), 5.06 (1 H, d, J

2.9 Hz, H-1 Api), 4.86 (1 H, d, J 3.7 Hz, H-1 GalA), 4.49 (1 H, br

s, H-5 GalA), 4.30 (1 H, br d, H-4 GalA), 3.95–3.74 (6 H, H-4 Api,

H-2 and H-3 GalA), 3.65 (3 H, s, OMe), 3.55–3.46 (4 H, m, H-3¢

Api), 3.27 (3 H, s. CO₂Me). ¹³C NMR (100 MHz, D₂O): d 170.8

(CO₂Me), 110.2, 110.0 (C-1 Api), 99.1 (C-1 GalA), 79.55, 79.5 (C-

3 Api), 77.1 (2 C, C-2 Api), 73.4, 74.4 (C-2 and C-3 GalA), 73.7 (2

C, C-4 Api), 70.1 (C-5 GalA), 69.8 (C-4 GalA), 63.65, 63.6 (C-3¢

Api), 55.4 (OMe), 52.8 (CO₂Me). A solution of the methyl ester

(26 mg, 0.054 mmol) in a mixture of H₂O–MeOH–Et₃N (3 : 2 : 1, 3

mL) was stirred for 24 h at 22 ^◦C, then concentrated in vacuo and

the residue was puriﬁed by GPC to give the title compound 4 (20

mg, 73%). ¹H NMR (400 MHz, D₂O): d (400 MHz, D₂O) 5.11 (1

H, d, J 3.0 Hz, H-1 Api), 5.06 (1 H, d, J 2.8 Hz, H-1 Api), 4.79

(1 H, d, J 3.2 Hz, H-1 GalA), 4.23 (1 H, dd, J 2.7 Hz, J 1.2 Hz,

H-4 GalA), 4.05 (1 H, d, J 1.2 Hz, H-5 GalA), 3.95 (1 H, d, J

10.2 Hz, H-4a Api), 3.90 (1 H, d, J 3.0 Hz, H-2 Api), 3.89 (1 H, d,

J 10.2 Hz, H-4a Api), 3.88 (1 H, d, J 2.8 Hz, H-2 Api), 3.81–3.77

(2 H, m, H-2 and H-3 GalA), 3.76 (1 H, d, J 10.2 Hz, H-4b Api),

3.73 (1 H, d, J 10.2 Hz, H-4b Api), 3.53 (s, 2 H, H-3¢ Api) 3.52 (4

H, s, H-3¢ Api), 3.25 (3 H, s, OMe). ¹³C NMR (100 MHz, D₂O): d

173.8 (CO₂H), 110.2, 109.9 (C-1 Api), 98.8 (C-1 GalA), 79.6 and

79.5 (C-3 Api), 77.1 and 77.05 (C-2 Api), 76.3, 74.9 (C-2 and C-3

GalA), 73.7 and 73.6 (C-4 Api), 70.9 (C-5 GalA), 70.5 (C-4 GalA),

63.7 and 63.6 (C-3¢ Api), 55.1 (OMe). HRMS (ESI+): m/z found

495.1319 ([M + Na]⁺); calcd for C₁₇H₂₈O₁₅·Na 495.1320.

Methyl (3-C-hydroxymethyl-b-D-erythrofuranosyl)-(1→2)-(a-D-

galactopyranosid)uronic acid (2)

A solution of disaccharide 29 (120 mg, 0.23 mmol) in 80%

aq. AcOH was stirred for 30 min at 60 ^◦C, the mixture was

concentrated to dryness, the residue was dissolved in MeOH–H₂O

(2 mL, 1 : 1) and conc. NaOH solution was added to pH 10. The

mixture was stirred at 22 ^◦C for 1 h, and then carefully neutralized

with Amberlite IR120 (H⁺) resin, the resin was removed and

the solution was concentrated. The residue was puriﬁed by ﬂash

chromatography (H₂O–MeCN, 100 : 0→80:20) to give the title

1

compound (63 mg, 80%); [a]²_D⁰+ 24.5 (c 1.1, H₂O); H NMR

(400 MHz, D₂O): d 5.10 (1 H, d, J 3.0, H-1 Api), 4.90 (1 H, d,

J 3.6, H-1 GalA), 4.22–4.18 (2 H, m, H-4, H-5 GalA), 3.95 (1 H,

s, H-2 Api) 3.94 (1 H, d, J 8.3 Hz, H-4a Api), 3.85 (1 H, dd, J

11.4 Hz, 3.5 Hz, H-3 GalA), 3.82 (1 H, d, J 10.2 Hz, H-4b Api),

3.72 (1 H, dd, J 10.3 Hz, 3.6 Hz), 3.61–3.56 (2 H, m, H-3¢a, H-3¢b

Api), 3.31 (3 H, s, OMe). ¹³C NMR (100 MHz, D₂O): d 176.7

(CO₂H), 111.2 (C-1 Api), 99.9 (C-1 GalA), 80.4 (C-3 Api), 78.0

(C-2 Api), 77.0 (C-2 GalA), 74.5 (C-4 Api), 71.9 (C-5 Gal), 71.3

(C-4 GalA), 69.3 (C-3 GalA), 64.5 (C-3¢ Api), 56.0 (OMe). HRMS

(ESI+): m/z found 363.0897 ([M + NH₄]⁺); calcd for C₁₂H₂₀O₁₁·Na

363.0898.

Methyl (3-C-hydroxymethyl-b-D-erythrofuranosyl)-(1→3)-(a-D-

galactopyranosid)uronic acid (3)

Crystal structure analysis of protected trisaccharide 41

A solution of disaccharide 28 (40 mg) in 0.05 M methanolic

NaOMe (0.5 mL) was stirred for 3 h at 22 ^◦C. The solvent was

evaporated, the residue was dissolved in water (0.5 mL) and pH

was adjusted to 10 by addition of 1 M NaOH solution. After 1 h at

22 ^◦C the mixture was neutralised by careful addition of Amberlite

IR120 (H⁺) resin, the resin was ﬁltered off of and the ﬁltrate was

concentrated. The residue was puriﬁed by GPC and freeze-dried

Crystal data: C₃₂H₄₀O₁₇, CH₂Cl₂, M = 781.6. Orthorhombic, space

group P2₁2₁2₁(no. 19), a = 11.2799(6), b = 15.1788(9), c =

3

-3

˚

21.4815(13) A, V = 3678.0(4) A . Z = 4, Dc = 1.411 g cm ,

F(000) = 1640, T = 140(1) K, m(Mo-Ka) = 2.5 cm^-1, l(Mo-Ka) =

0.71069 A. Crystals are beautiful, colourless prisms. Intensity

˚

data were measured on an Oxford Diffraction Xcalibur-3 CCD

diffractometer (Mo-Ka radiation, graphite monochromator).

Total number of reﬂections recorded, to q_max= 27.5^◦, was 49119

of which 8410 were unique (R_int= 0.045); 7002 were ‘observed’

with I > 2s_I. Structure was determined by direct methods in

SHELXS, and reﬁned in SHELXL.⁴⁴Non-hydrogen atoms were

reﬁned anisotropically. Hydrogen atoms were included in riding

mode. Final R-values: wR₂= 0.093 and R₁= 0.049⁴⁴for all data;

R₁= 0.037 for the ‘observed’ data.

1

to give disaccharide 3 (18 mg, 75%); [a]²_D⁰+72 (c 1.1, H₂O); H

NMR (400 MHz, D₂O): d 5.18 (1 H, d, J 3.2 Hz, H-1 Api), 4.79

(1 H, d, J 3.7 Hz, H-1 GalA), 4.33 (1 H, dd, J 3.2 Hz, J 1.3 Hz,

H-4 GalA), 4.14 (1 H, d, J 1.3 Hz, H-5 GalA), 4.04 (1 H, d, J

10.3 Hz, H-4a Api), 3.99 (1 H, d, J 3.2 Hz, H-2 Api), 3.87 (1 H,

dd, J 3.7 Hz, J 10.2 Hz, H-2 GalA), 3.82 (1 H, d, J 10.3 Hz, H-4b

Api), 3.80 (1 H, dd, J 3.3 Hz, J 10.2 Hz, H-3 GalA), 3.61 (2 H, s,

H-3¢ Api), 3.33 (2 H, s, OMe). ¹³C NMR (100 MHz, D₂O): d 173.5

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Nakamura and S. Takeuchi, Tetrahedron Lett., 2009, 50, 939–

942.

Acknowledgements

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Article Doi

Synthesis of apiose-containing oligosaccharide fragments of the plant cell wall: Fragments of rhamnogalacturonan-II side chains A and B, and apiogalacturonan

DOI: 10.1039/c1ob05587a

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