Modifying the regioselectivity of glycosynthase reactions through changes in the acceptor

Article abstract of DOI:10.1071/CH04025

Successful glycosynthase-mediated reactions have been performed on 6-O-benzyl-, 6-O-(4-nitrobenzyl)-, and 6-O-benzoyl-D-glucopyranose to give 1,2-β- and 1,3-β-D-glycosylated products; 4-O-benzyl-D-xylopyranose gave only a 1,2-β-glycosylated product. A rat

Full text of DOI:10.1071/CH04025

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2004, 57, 779–786
Modifying the Regioselectivity of Glycosynthase Reactions
Through Changes in the Acceptor∗
Robert V. Stick,A,B Keith A. Stubbs,A and Andrew G. WattsA
A Chemistry, School of Biomedical and Chemical Sciences M313, University of Western Australia,
Crawley WA 6009, Australia.
B Author to whom correspondence should be addressed (e-mail: rvs@chem.uwa.edu.au).
Successful glycosynthase-mediated reactions have been performed on 6-O-benzyl-, 6-O-(4-nitrobenzyl)-, and 6-O-
benzoyl-d-glucopyranose to give 1,2-β- and 1,3-β-d-glycosylated products; 4-O-benzyl-d-xylopyranose gave only
a 1,2-β-glycosylated product. A rationale is presented for these rather unusual results.
Manuscript received: 4 February 2004.
Final version: 26 March 2004.
Introduction
It occurred to us that it might be possible to increase this
‘structural infidelity’ of glycosynthases by employing aryl
ethers or esters as acceptors, rather than aryl glycosides.
Our premise was that a molecule such as 6-O-benzyl-d-
glucopyranose would perhaps present an alternative hydroxyl
group (from an aryl β-d-glucopyranoside) for functionaliza-
tion by the glycosyl donor, that is, if it actually did bind to
the active site of a glycosynthase (Scheme 2).
In order to test our hypothesis, we prepared 6-O-benzyl-
1, 6-O-benzoyl- 2, and 6-O-(4-nitrobenzyl)-d-glucopyranose
3 (Scheme 3). It was found that it was best to isolate the
two ethers (1 and 3) as their tetraacetates (4 and 5), a
feat which was not possible with ester 2. To our delight,
the treatment of all three of the free sugars (1, 2, and 3)
with α-d-galactopyranosyl fluoride (to prevent higher sac-
charide formation[3]) in the presence of the glycosynthase
Abg E358S resulted in the formation of mixtures of disac-
charides that had either β-1,3- or β-1,2-linkages (Table 1)
rather than the characteristic β-1,4-linkage observed when
an aryl β-d-glucopyranoside was the acceptor.[3] Once again,
Glycosynthases, which are mutant glycoside hydrolases that
arecapableofmakingbutnotbreakingtheglycosidiclinkage,
are currently available for the construction of β-1,3-, β-1,4-,
β-1,6-, and α-1,4-linked oligosaccharides.[1–11] Generally,
the choice of donor sugar in the glycosylation process is
guided by the structure of the natural substrate for the original
glycoside hydrolase, while the acceptor is often an aryl glyco-
side that exhibits good binding in both the +1 and +2 subsites
of the original glycoside hydrolase (and glycosynthase).[1]
Like glycoside hydrolases, which are used in the trans-
glycosylation mode for the construction of the glycosidic
linkage, glycosynthases are capable of constructing linkages
for which they were not designed. For example, although the
glycosynthase (Abg E358S) derived from a β-glucosidase
isolated from anAgrobacterium sp. predictably presided over
the construction of a β-1,4-linkage when α-d-glucopyranosyl
fluoride was treated with 4-nitrophenyl β-d-glucopyranoside,
when the acceptor was changed to 4-nitrophenyl β-d-
xylopyranoside, a β-1,3-linkage was obtained (Scheme 1).[3]
OH
O
HO
HO
OH
OC6H4NO2-4
OH
O
OH
HO
O
OC6H4NO2-4
HO
HO
(a)
O
OH
HO
OH
O
HO
HO
HO
OH
F
(a)
O
O
HO
HO
HO
OC6H4NO2-4
O
O
HO
HO
OH
OC6H4NO2-4
HO
OH
Scheme 1. (a) Abg E358S, NH4HCO3, H2O.
∗ Dedicated to the memory of Christian Pedersen.
© CSIRO 2004
10.1071/CH04025
0004-9425/04/080779

780
R. V. Stick, K. A. Stubbs and A. G. Watts
OH
OH
OAc
O
O
O
(a)
HO
HO
HO
HO
AcO
AcO
(b)
O
OBn
OH
HO
NO2
NHAc
NH3Cl
OBz
OBz
ϩ1
ϩ2
O
O
HO
HO
HO
HO
(c)
OBn
HO
OH
NHAc
NHAc
OH
HO
HO
O
6
O
Scheme 5. (a) Et2NH, EtOH, then Ac2O, pyridine, then BnOH,
TMSOTf, CH2Cl2; (b) Na, MeOH, then BzCl, pyridine, CH2Cl2,
−20◦C; (c) H2, Pd/C, MeOH.
ϩ1
ϩ2
Scheme 2.
OH
OH
OH
HO
HO
RO
HO
O
O
O
BnO
HO
HO
BnO
HO
HO
OH
O
OH
O
OH
OH
OH
HO
HO
(a)
(b)
BnO
O
O
8
9
10
O
O
Scheme 6.
R ϭ Bn, Bz, or CH2C6H4NO2-4
O
OR
OR
O
O
O
AcO
AcO
HO
(a)
(c)
(b)
OAc
SPh
O
O
(c)
HO
HO
AcO
AcO
OAc
OH
OAc
HO
AcO
1 R ϭ Bn
2 R ϭ Bz
3 R ϭ CH2C6H4NO2-4
4 R ϭ Bn
5 R ϭ CH2C6H4NO2-4
O
O
BnO
HO
BnO
HO
SPh
OH
OH
OH
Scheme 3. (a) (Bu3Sn)2O, PhMe, then BnBr, BzCl or
4-NO2C6H4CH2Br; (b) CF3CO2H/H2O (4 : 1); (c) Ac2O, pyridine for
1 and 3.
11
Scheme 7. (a) PhSH, Et2OBF3, CH2Cl2, then Na, MeOH, then
2-methoxypropene, CSA, DMF; (b) BnBr, NaH, DMF, then H3O+,
MeOH; (c) NBS, H3O+, MeCN.
HO
OBn
OBz
O
O
HO
HO
4-O-benzyl- 8,[14] 3-O-benzyl- 9,[15] and 2-O-benzyl-d-
glucopyranose 10[16] (Scheme 6), but once again, none
of these free sugars acted as acceptors in glycosynthase-
mediated reactions.
Finally, we prepared 4-O-benzyl-d-xylopyranose 11
(Scheme 7), which subsequently acted as an acceptor in
the glycosynthase reaction to provide a single β-1,2-linked
disaccharide derivative (Table 1).
HO
OH
HO
5
7
Scheme 4.
the disaccharides described here were best isolated as their
per-acetates. Two of the reactions were repeated with α-d-
glucopyranosyl fluoride as the donor and, again, the β-1,3-
and β-1,2-linked disaccharides were the major products
(Table 1).
Encouraged by these early results, we prepared 6-O-
benzyl-d-galactopyranose 5[12] (Scheme 4), 2-acetamido-
6-O-benzoyl-2-deoxy-d-glucopyranose 6 (Scheme 5), and
6-O-benzoyl-d-glucal 7[13] (Scheme 4). Unfortunately, none
of these molecules acted as acceptors in our glycosynthase
reactions with α-d-galactopyranosyl fluoride. However, it
still seemed worthwhile to investigate the ability of sug-
ars functionalized at positions other than O1 and O6 to act
as acceptors in glycosynthase reactions. Thus, we prepared
Discussion
How can one rationalize the successful glycosynthase-
mediated reactions reported here? For an acceptor such as
4-nitrophenyl β-d-glucopyranoside, binding to the active
site of the glycosynthase is predictable and it is the 4-OH
group that is correctly positioned for glycosylation to occur
(Fig. 1a). For 4-nitrophenyl β-d-xylopyranoside, the mode of
binding is likely to be similar (glycosyl moiety in the +1 site
and aryl group in the +2 site), but the absence of a primary
hydroxyl group allows a certain degree of positional freedom
(through a ‘flip’ or translocation of the pyranose ring), and

Modifying the Regioselectivity of Glycosynthase Reactions
781
Table 1. The glycosylation of various acceptors with glycosynthase Abg E358S
Acceptor Products
Donor
Yield [%]
53
1
33
41
30
47
29
56
2
3
11
1
39
32
43
33
2

782
R. V. Stick, K. A. Stubbs and A. G. Watts
OH
HO
OH
O
O
HO
HO
O
HO
HO
HO
NO2
F
(a)
Ϫ1
ϩ1
ϩ2
OH
HO
O
NO2
HO
O
HO
HO
HO
O
HO
F
F
F
F
(b)
OH
HO
HO
O
OH
HO
HO
O
R
R ϭ Bn, Bz, or CH2C6H4NO2-4
(c)
O
HO
HO
OH
HO
HO
O
O
O
R
HO
HO
OH
R ϭ Bn, Bz, or CH2C6H4NO2-4
(d )
HO
HO
OH
HO
OH
O
HO
HO
O
HO
O
HO
(e)
Fig. 1.
thus allows the 3-OH group to be presented for glycosylation
(Fig. 1b).
glycosylation occurs to give higher oligosaccharides. The
‘unnatural’ β-1,2- and β-1,3-linked products described here
are obviously poor acceptors and so further glycosylation
is discouraged, even in the presence of the ‘natural’ donor,
α-d-glucopyranosyl fluoride.
Our next intention is to apply some of the principles estab-
lished here to extend the scope of the recently developed
thioglycoligases.[17]
The d-glucopyranoses 1–3 (Scheme 3) presumably bind
in an alternative mode, whereby the functional group at O6
occupies the aglycon (+2) site, while the glycosyl moi-
ety is rotated into the +1 site. Although these molecules
have lost any contribution to binding that involves hydrogen
bonding of the 6-OH group, compensation may be provided
through hydrophobic interactions involving the aromatic ring
in the +2 site. Therefore, the glycosyl moiety (which lacks
a primary hydroxyl group) presents either the 2-OH group
(Fig. 1c) or the 3-OH group (Fig. 1d) for glycosylation. The
other positive result, namely the glycosylation of 4-O-benzyl-
d-xylopyranose 11, may be rationalized in similar terms, that
is, the benzyl ether binds in the +2 site and so presents the
2-OH group of the sugar for glycosylation (Fig. 1e).
One final observation in the glycosynthase-mediated
transformations described here is the lack of formation of
higher oligosaccharides (tri- and tetra-saccharides). Nor-
mally, the initial product of glycosylation (usually a disac-
charide) is a good (or even better) acceptor, and so further
Experimental
General experimental procedures have been given previously.[18]
Tetra-O-acetyl-6-O-benzyl-D-glucopyranose 4
(i) Bis(tributyltin) oxide (3.2 g, 5.4 mmol) was added to 1,2-O-
isopropylidene-α-d-glucofuranose (1.0 g, 4.5 mmol) in toluene (50 mL)
and the mixture was heated at reflux for 24 h. Benzyl bromide (1.1 g,
6.3 mmol) was then added and the solution was refluxed for a further
2 h. Concentration of the mixture left a brown residue that, after flash
chromatography (EtOAc/light petroleum 2 : 3), furnished 6-O-benzyl-
1,2-O-isopropylidene-α-d-glucofuranose (1.3 g, 92%) as an oil. The
1H NMR spectrum of this oil was consistent with that reported.[19]

Modifying the Regioselectivity of Glycosynthase Reactions
783
(ii) 6-O-Benzyl-1,2-O-isopropylidene-α-d-glucofuranose (1.3 g,
4.1 mmol) was added to a trifluoroacetic acid/H2O (4 : 1, 15 mL) mix-
tureandthesolutionwasstirredatroomtemperaturefor30 min. Concen-
tration of the solution gave a brown residue that was dissolved in
pyridine (20 mL). Acetic anhydride (20 mL) was then added and after
1 h the solution was quenched with MeOH (10 mL) and concentrated.
Usual workup (EtOAc) followed by flash chromatography (EtOAc/light
petroleum, 3 : 7) gave tetra-O-acetyl-6-O-benzyl-d-glucopyranose 4
(1.7 g, 92%) as an oil. The 1H NMR spectrum of this oil was consistent
with that reported.[20]
and 128.7 (Ph), 101.6 (C1), 75.8, 75.5, and 72.3 (C3, C4, and C5), 71.5
(CH2Ph), 65.1 (C6), 57.4 (C2), 22.9 (Me).
(ii) The above benzoate (250 mg, 0.60 mmol) and palladium-on-
carbon (20 mg of 10%) in MeOH (15 mL) were stirred vigorously under
H2 (1 atm) for 24 h. The mixture was then filtered through Celite, the
filtrate was concentrated, and the residue was purified by flash chro-
matography (MeOH/CHCl3, 1 : 4) to give hemiacetal 6 (195 mg) as
an oil, [α]D +37.7◦ (equilibrium) (Found: m/z 326.1224. C15H19NO7
requires[M + H]+• 326.1239). δH (600 MHz)8.03–7.99, 7.60–7.57, and
7.48–7.43 (3 m, Ph), 5.13 (d, J1,2 3.4, H1α), 4.66 (m, H1β and H6α),
4.61 (dd, J5,6 1.9, J6,6 12.7, H6β), 4.47 (m, H6α and H6β), 4.14 (ddd,
J4,5 10.0, J5,6 5.1, H5β), 3.91 (dd, J2,3 8.9, H2α), 3.78 (dd, J3,4 9.0,
H3α), 3.67 (dd, J1,2 ≈ J2,3 9.0, H2β), 3.64 (ddd, J4,5 9.9, J5,6 5.6 and
1.8, H5α), 3.51 (m, H3β, H4α, and H4β), 1.99 (s, Me). δC (150.8 MHz)
174.2, 173.7, 168.0, and 167.9 (2C, C=O), 134.4, 134.3, 132.1, 131.2,
130.5, 130.4, 129.5, and 128.9 (Ph), 97.0 (C1β), 92.6 (C1α), 75.8 (C3β),
75.4 (C5α), 72.5 (C3α and C4β), 72.1 (C4α), 70.8 (C5β), 65.3 (C6α),
65.2 (C6β), 58.7 (C2β), 55.8 (C2α), 22.9 and 22.7 (Me).
6-O-Benzoyl-D-glucopyranose 2
(i) Following the procedure described in (i) above, but using benzoyl
chloride (890 mg) instead of benzyl bromide, gave 6-O-benzoyl-1,2-O-
isopropylidene-α-d-glucofuranose[21] (87%) as an oil.
(ii) 6-O-Benzoyl-1,2-O-isopropylidene-α-d-glucofuranose (1.3 g,
4.0 mmol)wasaddedtoatrifluoroaceticacid/H2O(4 : 1, 15 mL)mixture
and the solution was stirred at room temperature for 30 min. Concentra-
tionofthemixturegaveabrownresiduethat, uponflashchromatography
(MeOH/CHCl3, 3 : 7), gave tetrol 2 (1.1 g, 94%) as an oil. The 1H NMR
spectrum of this oil was consistent with that reported.[22]
4-O-Benzyl-D-xylose 11
(i) Sodium hydride (60% dispersion in mineral oil, 120 mg, 2.8 mmol)
and benzyl bromide (0.34 mL, 2.8 mmol) were added to phenyl 2,3-
O-isopropylidene-1-thio-β-d-xyloside[24] (400 mg, 1.40 mmol) in DMF
(10 mL). The reaction mixture was stirred at room temperature for 1 h
before being quenched by the addition of MeOH (10 mL). Concentration
of the mixture followed by a usual workup (EtOAc) and flash chromato-
graphy (EtOAc/light petroleum 17 : 83) of the crude product gave phenyl
4-O-benzyl-2,3-O-isopropylidene-1-thio-β-d-xyloside (530 mg, 89%)
as a solid, [α]D −32.9◦ (Found: m/z 373.1465. C21H24O4S requires
[M + H]+• 373.1473). δH (600 MHz) 7.56–7.54 and 7.34–7.30 (10H,
2 m, Ph), 4.81 (d, J1,2 9.4, H1), 4.80 and 4.59 (AB, J 11.9, CH2Ph),
4.10 (dd, J4,5 5.1, J5,5 11.9, H5), 3.76 (ddd, J3,4 9.0, J4,5 8.7, H4), 3.66
(dd, J2,3 9.1, H3), 3.28 (dd, H5), 3.24 (dd, H2), 1.49 and 1.46 (6H, 2 s,
Me). δC (150.8 MHz) 137.9, 132.8, 131.9, 128.8, 128.4, 128.0, 127.8,
and 127.7 (Ph), 111.2 (CMe2), 85.3 (C1), 82.5, 75.5, and 75.3 (C2, C3,
and C4), 72.1 (CH2Ph), 68.3 (C5), 26.7 and 26.6 (2C, Me).
Tetra-O-acetyl-6-O-(4-nitrobenzyl)-D-glucopyranose 5
(i) Following the procedure described in (i) above for 4, but using
4-nitrobenzyl bromide (1.4 g) instead of benzyl bromide, gave 1,2-O-
isopropylidene-6-O-(4-nitrobenzyl)-α-d-glucofuranose (91%) as an oil,
[α]D −1.9(Found(FAB):m/z356.1363. C16H21NO8 requires[M + H]+
356.1345). δH (300 MHz) 8.21 and 7.50 (4H, AAꢀBBꢀ, Ar), 5.95 (d, J1,2
3.7, H1), 4.70 (s, CH2Ar), 4.54 (d, H2), 4.36 (d, J3,4 2.8, H3), 4.24 (ddd,
J4,5 6.5, J5,6 5.7 and 3.3, H5), 4.13 (dd, H4), 3.82 (dd, J6,6 10.3, H6),
3.71 (dd, H6), 1.49 and 1.33 (6H, 2 s, Me). δC (75.5 MHz) 148.3, 145.1,
127.8, and 123.8 (6C, Ar), 111.8 (CMe2), 104.9 (C1), 85.1, 79.5, 75.7,
and 69.4 (C2, C3, C4, and C5), 72.3 and 71.8 (C6 and CH2Ar), 26.8
and 26.1 (2C, Me).
(ii) Following the procedure described in (ii) above for 4,
1,2-O-isopropylidene-6-O-(4-nitrobenzyl)-α-d-glucofuranose (1.5 g,
4.2 mmol) gave tetra-O-acetyl-6-O-(4-nitrobenzyl)-D-glucopyranose 5
(1.9 g, 93%)asanoil(Found:C52.3, H5.2, m/z484.1435. C21H25NO12
requires C 52.2, H 5.2%, [M + H]+• 484.1455). δH (600 MHz) 8.19 and
7.49 (4H, AAꢀBBꢀ, Ar), 6.33 (d, J1,2 3.7, H1α), 5.70 (d, J1,2 7.5, H1β),
5.47 (dd, J3,4 10.0, H3α), 5.27–5.20 (m, H3β, H4α, and H4β), 5.13 (dd,
J2,3 9.9, H2β), 5.07 (dd, J2,3 10.0, H2α), 4.65 and 4.57 (AB, J 13.1,
CH2Ar), 4.07 (ddd, J4,5 9.2, J5,6 4.5 and 2.6, H5α), 3.82 (ddd, J4,5 9.3,
J5,6 2.7 and 4.5, H5β), 3.68 (dd, J6,6 11.1, H6α), 3.64 (dd, H6α), 3.60–
3.56 (m, H6β), 2.17, 2.10, 2.03, 2.02, 2.01, 1.98, and 1.97 (12H, 7 s,
Me). δC (150.8 MHz) 170.1, 169.4, 169.2, and 169.0 (4C, C=O), 145.2,
145.2, 127.8, 123.6, and 123.6 (6C, Ar), 91.8 (C1β), 89.1 (C1α), 73.9
(C5β), 72.9 (C4α), 72.4 and 72.3 (CH2Ar), 71.1 (C5α), 70.2 (C2β), 69.9
(C3α), 68.2 (C2α), 68.7 and 68.7 (C6α and C6β), 68.4 and 68.4 (C3β
and C4β), 20.8, 20.8, 20.6, 20.6, 20.5, 20.5, and 20.4 (4C, Me).
(ii) Concentrated HCl (0.2 mL) was added to phenyl 4-O-benzyl-2,3-
O-isopropylidene-1-thio-β-d-xyloside (500 mg, 1.3 mmol) in MeOH
(10 mL) and the solution was stirred at room temperature for 1 h.
The solution was concentrated and the residue was purified by flash
chromatography (EtOAc/light petroleum 1 : 1) to give phenyl 4-O-
benzyl-1-thio-β-D-xyloside (430 mg, 97%) as plates, mp 119–120◦C
(EtOAc/light petroleum), [α]D −57.1◦ (Found: C 65.1, H 6.0, m/z
333.1141. C18H20O4S requires C 65.0, H 6.1%, [M + H]+• 333.1160).
δH (300 MHz) 7.51–7.47 and 7.35–7.25 (10H, 2 m, Ph), 4.73 and 4.60
(AB, J 11.7, CH2Ph), 4.54 (d, J1,2 9.4, H1), 3.99 (dd, J4,5 4.9, J5,5 11.3,
H5), 3.50 (dd, J2,3 8.8, H2), 3.38 (ddd, J3,4 8.7, J4,5 8.6, H4), 3.22–
3.18 (m, H3 and H5). δC (75.5 MHz) 139.8, 134.7, 133.1, 129.9, 129.3,
128.9, 128.7, and 128.5 (Ph), 89.9 (C1), 78.6, 78.4, and 74.0 (C2, C3,
and C4), 73.7 (CH2Ph), 68.3 (C5).
(iii) N-Bromosuccinimide (170 mg, 0.94 mmol) was added to a solu-
tionofphenyl4-O-benzyl-1-thio-β-d-xyloside(310 mg, 0.94 mmol)and
concentrated HCl (0.1 mL) in CH3CN/H2O (19 : 1, 16 mL).The mixture
was then stirred for 30 min and subsequently quenched with pyridine
(10 mL). Evaporation of the solvents followed by flash chromatography
(MeOH/CHCl3, 1 : 9) of the residue gave hemiacetal 11 (180 mg, 80%)
as colourless crystals. The 13C NMR spectrum was consistent with that
reported.[25]
2-Acetamido-6-O-benzoyl-2-deoxy-D-glucopyranose 6
(i) Benzoyl chloride (145 mg, 1.10 mmol) in DMF (5 mL) was added
dropwise to benzyl 2-acetamido-2-deoxy-β-d-glucopyranoside[23]
(250 mg, 0.810 mmol) and pyridine (0.10 mL, 1.1 mmol) in DMF
(15 mL) at −30◦C and the solution was stirred for 30 min. The mixture
was quenched by the addition of MeOH (10 mL) and then concentra-
ted. Flash chromatography of the residue (MeOH/CHCl3, 1 : 9) gave
benzyl 2-acetamido-6-O-benzoyl-2-deoxy-β-D-glucopyranoside (290 mg,
87%) as colourless crystals, mp 187–190◦C (EtOH), [α]D +26.1◦
(Found: C 63.3, H 6.2, m/z 416.1723. C22H25NO7 requires C 63.6,
H 6.1%, [M + H]+• 416.1709). δH (500 MHz) 8.09–8.06, 7.63–7.59,
7.51–7.47, and 7.27–7.23 (10H, 4 m, Ph), 4.77 and 4.55 (AB, J 12.2,
CH2Ph), 4.69 (dd, J5,6 3.7, J6,6 12.3, H6), 4.51 (dd, J5,6 5.9, H6), 4.49
(d, J1,2 8.4, H1), 3.75 (ddd, J2,3 8.0, J2,NH 2.7, H2), 3.58 (ddd, J4,5
11.0, H5), 3.49 (m, H3 and H4), 1.95 (s, Me). δC (125.7 MHz) 173.7
and 167.9 (2C, C=O), 138.9, 134.4, 131.4, 130.6, 129.6, 129.3, 128.9,
General Procedure for the Glycosynthase (E358S)-Mediated
Glycosylation of 6-O-Benzyl-D-glucopyranose 1
and 6-O-(4-Nitrobenzyl)-D-glucopyranose 3
A few drops of NaOMe in MeOH (1.5 M) were added to a mixture
of tetra-O-acetyl-α-d-galactopyranosyl or -glucopyranosyl fluoride and
tetra-O-acetyl-6-O-benzyl-d-glucopyranose 4 or tetra-O-acetyl-6-O-(4-
nitrobenzyl)-d-glucopyranose 5 in MeOH and the solution was stirred
for 30 min. The mixture was quenched with resin (Amberlite IR-120,
H+), filtered, and the filtrate was concentrated. The residue was dis-
solved in NH4HCO3 solution (3 mL of 150 mM), Abg E358S (2 mg)

784
R. V. Stick, K. A. Stubbs and A. G. Watts
was added, and the solution was kept at 25◦C for 7 days. The solvent
was then removed under reduced pressure, the residue was dissolved
in Ac2O (5 mL) that contained NaOAc (50 mg), and the solution was
heated at reflux for 10 min. The reaction was quenched by the addition
of ice/water. Usual workup (CH2Cl2) followed by flash chromatography
(EtOAc/lightpetroleum, 1 : 4to1 : 1)gavetheappropriateper-acetylated
disaccharides.
Next to elute was 1,2,4-tri-O-acetyl-6-O-(4-nitrobenzyl)-3-O-(tetra-
O-acetyl-β-d-galactopyranosyl)-β-d-glucose (115 mg, 29%), which
was isolated as an oil (Found: m/z 772.2276. C33H41NO20 requires
[M + H]+• 772.2300). δH (600 MHz) 8.16 and 7.47 (4H, AAꢀBBꢀ, Ar),
5.59 (d, J1,2 8.4, H1), 5.33 (dd, J3 ,4 3.5, J4 ,5 1.0, H4ꢀ), 5.08–5.05 (m,
ꢀ
ꢀ
ꢀ
ꢀ
H2 and H2ꢀ), 5.00 (dd, J3,4 9.5, J4,5 9.9, H4), 4.94 (dd, J2 ,3 10.4, H3ꢀ),
ꢀ
ꢀ
4.60 and 4.58 (AB, J 13.4, CH2Ar), 4.55 (d, J1 ,2 8.0, H1ꢀ), 4.15 (dd,
ꢀ
ꢀ
J5 ,6 3.9, J6 ,6 11.1, H6ꢀ), 4.04 (dd, J5 ,6 6.4, H6ꢀ), 3.95 (dd, J2,3 9.4,
H3), 3.90 (ddd, H5ꢀ), 3.76 (ddd, J5,6 2.6 and 2.5, J6,6 11.3, H5), 3.62
(dd, H6), 3.56 (dd, H6), 2.16, 2.11, 2.08, 2.02, 2.00, 1.99, and 1.94 (21H,
7 s, Me). δC (150.8 MHz) 170.3, 170.1, 170.1, 170.0, 169.3, 169.1, and
168.9 (7C, C=O), 147.3, 145.3, 127.8, and 123.5 (6C, Ar), 101.1 (C1ꢀ),
91.8 (C1), 78.3 (C3), 74.2 (C5), 72.3 (CH2Ar), 70.9 (C3ꢀ), 70.5 (C5ꢀ),
69.0 (C6), 71.3, 68.7, and 68.3 (C2, C4, and C2ꢀ), 66.7 (C4ꢀ), 60.9 (C6ꢀ),
20.8, 20.7, 20.7, 20.5, 20.4, 20.4, and 20.3 (7C, Me).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1,3,4-Tri-O-acetyl-6-O-benzyl-2-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
and 1,2,4-Tri-O-acetyl-6-O-benzyl-3-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
Utilizing the above general procedure, tetra-O-acetyl-α-d-galactopyra-
nosyl fluoride (275 mg, 0.79 mmol) and tetra-O-acetyl-6-O-benzyl-
d-glucopyranose
4 (230 mg, 0.52 mmol) firstly gave 1,3,4-tri-
O-acetyl-6-O-benzyl-2-O-(tetra-O-acetyl-β-d-galactopyranosyl)-β-d-
glucose (200 mg, 53%) as an oil (Found: m/z 727.2385. C33H42O18
requires [M + H]+• 727.2449). δH (600 MHz) 7.31–7.25 (m, Ph), 5.63
1,3,4-Tri-O-acetyl-6-O-benzyl-2-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-β-D-glucose
and 1,2,4-Tri-O-acetyl-6-O-benzyl-3-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-β-D-glucose
(d, J1,2 8.0, H1), 5.33 (dd, J4 ,5 1.1, J3 ,4 3.5, H4ꢀ), 5.17 (dd, J2,3 9.3,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J3,4 10.0, H3), 5.10 (dd, J4,5 9.8, H4), 5.08 (dd, J1 ,2 8.0, J2 ,3 10.4,
H2ꢀ), 4.91 (dd, H3ꢀ), 4.58 (d, H1ꢀ), 4.55 and 4.40 (AB, J 12.2, CH2Ph),
Utilizing the above general procedure, tetra-O-acetyl-α-d-glucopyra-
nosyl fluoride (180 mg, 0.52 mmol) and tetra-O-acetyl-6-O-benzyl-
d-glucopyranose 4 (230 mg, 0.52 mmol) firstly gave 1,3,4-tri-O-acetyl-
6-O-benzyl-2-O-(tetra-O-acetyl-β-d-glucopyranosyl)-β-d-glucose
(147 mg, 39%) as an oil (Found: m/z 727.2455. C33H42O18 requires
[M + H]+• 727.2449). δH (600 MHz) 7.31–7.25 (m, Ph), 5.61 (d, J1,2
8.1, H1), 5.17–5.16 (m, H3 and H3ꢀ), 5.10–5.07 (m, H4 and H4ꢀ), 4.89
4.11 (dd, J5 ,6 6.1, J6 ,6 11.8, H6ꢀ), 4.06 (dd, J5 ,6 9.9, H6ꢀ), 3.92 (ddd,
H5ꢀ), 3.81 (dd, H2), 3.70 (ddd, J5,6 3.9 and 2.6, H5), 3.56 (dd, J6,6 11.1,
H6), 3.47 (dd, H6), 2.12, 2.09, 2.06, 2.02, 1.99, 1.94, and 1.84 (21H,
7 s, Me). δC (150.8 MHz) 170.3, 170.1, 170.1, 169.9, 169.6, 169.3, and
168.8 (7C, C=O), 137.4, 128.3, 127.9, and 127.7 (Ph), 101.1 (C1ꢀ), 91.7
(C1), 76.9 (C2), 75.0 (C5ꢀ), 74.7 (C3), 73.5 (C5), 73.4 (CH2Ph), 70.9
(C3ꢀ), 68.6 and 68.5 (C4 and C2ꢀ), 67.3 (C6), 66.8 (C4ꢀ), 61.2 (C6ꢀ),
20.9, 20.7, 20.7, 20.5, 20.4, 20.4, and 20.4 (7C, Me).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(dd, J1 ,2 8.0, J2 ,3 9.9, H2ꢀ), 4.55 (d, H1ꢀ), 4.45 and 4.40 (AB, J 12.5,
ꢀ
ꢀ
ꢀ
ꢀ
CH2Ph), 4.12 (dd, J5 ,6 2.7, J6 ,6 11.9, H6ꢀ), 4.06 (dd, J5 ,6 4.4, H6ꢀ),
3.83 (dd, J2,3 9.9, H2), 3.71 (ddd, J4,5 10.0, J5,6 3.6 and 2.8, H5), 3.64
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Next to elute was 1,2,4-tri-O-acetyl-6-O-benzyl-3-O-(tetra-O-
acetyl-β-d-galactopyranosyl)-β-d-glucose (120 mg, 33%), which was
isolated as an oil (Found: m/z 727.2445. C33H42O18 requires [M + H]+•
727.2449). δH (600 MHz) 7.31–7.23 (m, Ph), 5.60 (d, J1,2 8.2, H1), 5.32
(ddd, J4 ,5 8.2, H5ꢀ), 3.58 (dd, J6,6 11.0, H6), 3.46 (dd, H6), 2.14, 2.11,
2.07, 2.02, 1.99, 1.96, and 1.94 (21H, 7 s, Me). δC (150.8 MHz) 170.3,
170.1, 169.9, 169.2, and 168.6 (7C, C=O), 137.2, 128.1, 127.5, and
127.3 (Ph), 100.3 (C1ꢀ), 91.9 (C1), 76.8 (C2), 75.2, 68.9, 68.2, and
68.1 (C3, C4, C3ꢀ, and C4ꢀ), 73.9 (C5), 73.1 (CH2Ph), 71.8 (C5ꢀ), 71.0
(C2ꢀ), 67.1 (C6), 61.6 (C6ꢀ), 20.7, 20.7, 20.6, 20.5, 20.4, 20.4, and 20.2
(7C, Me).
ꢀ
ꢀ
(dd, J3 ,4 3.5, J4 ,5 1.0, H4ꢀ), 5.09 (dd, J2,3 7.9, H2), 5.05 (dd, J1 ,2 8.0,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J2 ,3 10.4, H2ꢀ), 4.99 (dd, J3,4 7.9, J4,5 9.8, H4), 4.91 (dd, H3ꢀ), 4.54
ꢀ
ꢀ
(d, H1ꢀ), 4.51 and 4.49 (AB, J 12.0, CH2Ph), 4.11 (dd, J5 ,6 6.8, J6 ,6
ꢀ
ꢀ
ꢀ
ꢀ
11.0, H6ꢀ), 4.06 (dd, J5 ,6 8.8, H6ꢀ), 4.00 (ddd, H5ꢀ), 3.86 (dd, H3), 3.75
(ddd, J5,6 4.9 and 2.9, H5), 3.56 (dd, J6,6 11.0, H6), 3.49 (dd, H6), 2.11,
2.07, 2.05, 2.03, 1.99, 1.94, and 1.93 (21H, 7 s, Me). δC (150.8 MHz)
170.3, 170.1, 170.1, 170.1, and 168.7 (7C, C=O), 137.5, 128.2, 127.8,
and 127.6 (Ph), 101.1 (C1ꢀ), 91.8 (C1), 78.4 (C5ꢀ), 74.1 (C5), 73.4
(CH2Ph), 72.0 (C2), 70.9 (C3ꢀ), 70.4 (C3), 68.5 and 68.5 (C4 and C2ꢀ),
68.3 (C6), 66.7 (C4ꢀ), 60.9 (C6ꢀ), 20.9, 20.7, 20.7, 20.5, 20.4, 20.4, and
20.3 (7C, Me).
ꢀ
ꢀ
Next to elute was 1,2,4-tri-O-acetyl-6-O-benzyl-3-O-(tetra-O-
acetyl-β-d-glucopyranosyl)-β-d-glucose (120 mg, 32%), which was
isolated as an oil (Found: m/z 727.2445. C33H42O18 requires [M + H]+•
727.2449). δH (600 MHz) 7.38–7.25 (m, Ph), 5.62 (d, J1,2 8.2, H1), 5.14
(dd, J2 ,3 10.0, J3 ,4 10.0, H3ꢀ), 5.10–5.08 (m, H2 and H4ꢀ), 4.99 (dd,
ꢀ
ꢀ
ꢀ
ꢀ
J3,4 7.9, J4,5 9.9, H4), 4.88 (dd, J1 ,2 7.9, H2ꢀ), 4.59 (d, H1ꢀꢀ), 4.48 and
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4.44 (AB, J 12.3, CH2Ph), 4.19 (dd, J5 ,6 2.9, J6 ,6 11.3, H6 ), 4.05 (dd,
J5 ,6 4.3, H6ꢀ), 3.84 (dd, J2,3 8.2, H3), 3.73 (ddd, J5,6 4.9 and 2.7, H5),
ꢀ
ꢀ
3.65 (ddd, J4 ,5 8.8, H5ꢀ), 3.53 (dd, J6,6 11.2, H6), 3.51 (dd, H6), 2.09,
2.06, 2.05, 2.01, 1.99, 1.96, and 1.94 (21H, 7 s, Me). δC (150.8 MHz)
170.8, 170.4, 170.2, 169.9, 169.6, 169.0, and 168.6 (7C, C=O), 137.4,
128.8, 128.0, and 127.1 (Ph), 100.7 (C1ꢀ), 91.9 (C1), 74.3 (C5), 73.3
(CH2Ph), 70.9 (C3), 70.5 (C2ꢀ), 68.6 (C4), 68.4 (C6), 68.2 (C3ꢀ), 72.2
and 67.9 (C2, C4ꢀ, and C5ꢀ), 61.8 (C6ꢀ), 20.8, 20.7, 20.6, 20.5, 20.4,
20.3, and 20.2 (7C, Me).
ꢀ
ꢀ
1,3,4-Tri-O-acetyl-6-O-(4-nitrobenzyl)-2-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
and 1,2,4-Tri-O-acetyl-6-O-(4-nitrobenzyl)-3-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
Utilizing the above general procedure, tetra-O-acetyl-α-d-
galactopyranosyl fluoride (275 mg, 0.79 mmol) and tetra-O-acetyl-
6-O-(4-nitrobenzyl)-d-glucopyranose 5 (250 mg, 0.52 mmol) firstly
gave 1,3,4-tri-O-acetyl-6-O-(4-nitrobenzyl)-2-O-(tetra-O-acetyl-β-d-
galactopyranosyl)-β-d-glucose (190 mg, 47%) as an oil (Found: m/z
772.2224. C33H41NO20 requires [M + H]+• 772.2300). δH (600 MHz)
General Procedure for the Glycosynthase (E358S)-Mediated
Glycosylation of Hemiacetals 2 and 11
8.19 and 7.46 (4H, AAꢀBBꢀ, Ar), 5.65 (d, J1,2 8.0, H1), 5.35 (dd, J3 ,4
A few drops of NaOMe in MeOH (1.5 M) were added to tetra-O-acetyl-
α-d-galactopyranosyl or -glucopyranosyl fluoride in MeOH (5 mL) and
the resultant solution was stirred for 30 min. The mixture was quenched
with resin (Amberlite IR-120, H+), filtered, and the filtrate was con-
centrated. The residue was dissolved in NH4HCO3 solution (3 mL of
150 mM), the hemiacetal and Abg E358S (2 mg) were added, and the
solution was kept at 25◦C for 7 days. The solution was then concen-
trated, the residue was dissolved in Ac2O (5 mL) that contained NaOAc
(50 mg), and the solution was heated at reflux for 10 min. The reaction
was quenched by the addition of ice/water. Usual workup (CH2Cl2) fol-
lowed by flash chromatography (EtOAc/light petroleum, 1 : 4 to 1 : 1)
gave the appropriate per-acetylated disaccharide(s).
ꢀ
ꢀ
ꢀ
ꢀ
3.5, J4 ,5 1.1, H4ꢀ), 5.24 (dd, J2,3 8.9, J3,4 9.3, H3), 5.14 (dd, J1 ,2
ꢀ
ꢀ
8.0, J2 ,3 9.9, H2ꢀ), 5.06 (dd, J4,5 8.4, H4), 4.94 (dd, H3ꢀ), 4.65 and
ꢀ
ꢀ
4.63 (AB, J 13.2, CH2Ar), 4.59 (d, H1ꢀ), 4.13 (dd, J5 ,6 3.4, J6 ,6 10.9,
ꢀ
ꢀ
ꢀ
ꢀ
H6ꢀ), 4.05 (dd, J5 ,6 6.4, H6ꢀ), 3.98 (ddd, H5ꢀ), 3.84 (dd, H2), 3.68 (dd,
J5,6 2.4, J6,6 11.1, H6), 3.65 (ddd, J5,6 4.1, H5), 3.56 (dd, H6), 2.11,
2.09, 2.08, 2.05, 2.01, 1.99, and 1.95 (21H, 7 s, Me). δC (150.8 MHz)
170.4, 170.3, 170.1, 170.1, 169.9, 169.3, and 168.8 (7C, C=O), 147.4,
145.2, 127.8, and 123.6 (6C,Ar), 101.3 (C1ꢀ), 91.8 (C1), 75.3 (C5), 74.4
(C5ꢀ), 73.5 (C3), 72.3 (CH2Ar), 70.9 (C3ꢀ), 69.1 (C4), 68.6 (C2ꢀ), 68.5
(C6), 67.8 (C2), 66.8 (C4ꢀ), 61.1 (C6ꢀ), 20.8, 20.7, 20.6, 20.5, and 20.4
(7C, Me).
ꢀ
ꢀ

Modifying the Regioselectivity of Glycosynthase Reactions
785
1,3,4-Tri-O-acetyl-6-O-benzoyl-2-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
71.9 (C4), 70.8 (C2ꢀ), 68.1 (C2 and C3ꢀ), 67.8 (C4ꢀ), 62.7 (C6), 61.5
(C6ꢀ), 20.7, 20.5, 20.5, 20.5, 20.4, 20.3, and 20.2 (7C, Me).
and 1,2,4-Tri-O-acetyl-6-O-benzoyl-3-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucose
1,3-Di-O-acetyl-4-O-benzyl-2-O-(tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-xylose
Utilizing the above general procedure, tetra-O-acetyl-α-d-galactopyra-
nosyl fluoride (270 mg, 0.77 mmol) and 6-O-benzoyl-d-glucopyranose
2 (140 mg, 0.51 mmol) firstly gave 1,3,4-tri-O-acetyl-6-O-benzoyl-2-O-
(tetra-O-acetyl-β-d-galactopyranosyl)-β-d-glucose (87 mg, 41%) as an
oil (Found: m/z 741.2275. C33H40O19 requires [M + H]+• 741.2242).
δH (600 MHz) 8.04–8.00, 7.57–7.54, and 7.45–7.43 (3 m, Ph), 5.72 (d,
Utilizing the above general procedure, tetra-O-acetyl-α-d-galacto-
pyranosyl fluoride (110 mg, 0.31 mmol) and 4-O-benzyl-d-xylose 11
(50 mg, 0.20 mmol) gave 1,3-di-O-acetyl-4-O-benzyl-2-O-(tetra-O-
acetyl-β-d-galactopyranosyl)-β-d-xylose (76 mg, 56%) as an oil
(Found: m/z 655.2229. C30H38O16 requires [M + H]+• 655.2238). δH
(600 MHz) 7.35–7.28 and 7.25–7.33 (2 m, Ph), 5.59 (d, J1,2 7.3, H1),
J1,2 7.9, H1), 5.35 (dd, J3 ,4 3.5, J4 ,5 1.1, H4ꢀ), 5.25 (dd, J2,3 9.3, J3,4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
5.33 (dd, J3 ,4 3.4, J4 ,5 1.1, H4ꢀ), 5.18 (dd, J2,3 9.0, J3,4 8.9, H3), 5.10
ꢀ
ꢀ
ꢀ
ꢀ
9.3, H3), 5.13 (dd, J1 ,2 8.0, J2 ,3 9.0, H2 ), 5.09 (dd, J4,5 9.3, H4), 4.94
ꢀ
ꢀ
ꢀ
ꢀ
(dd, H3ꢀ), 4.60 (d, H1ꢀ), 4.45 (dd, J5,6 2.3, J6,6 12.4, H6), 4.38 (dd, J5,6
(dd, J1 ,2 9.8, J2 ,3 10.5, H2ꢀ), 4.92 (dd, H3ꢀ), 4.55 (d, H1ꢀ), 4.57 and
ꢀ
ꢀ
ꢀ
ꢀ
4.4, H6), 4.16 (dd, J5 ,6 6.6, J6 ,6 11.3, H6ꢀ), 4.06 (dd, J5 ,6 6.8, H6ꢀ),
3.96 (ddd, H5), 3.87 (ddd, H5ꢀ), 3.85 (dd, H2), 2.18, 2.15, 2.06, 2.03,
2.02, 2.01, and 1.96 (21H, 7 s, Me). δC (150.8 MHz) 170.4, 170.2, 170.1,
169.8, 169.6, 169.0, 168.7, and 166.0 (8C, C=O), 133.2, 129.7, 129.5,
and 128.4 (Ph), 101.2 (C1ꢀ), 92.0 (C1), 77.0 (C2), 74.5 (C3), 72.4 (C5),
70.9 (C3ꢀ), 70.6 (C5ꢀ), 68.6 (C4), 68.3 (C2ꢀ), 66.8 (C4ꢀ), 62.2 (C6), 61.1
(C6ꢀ), 20.7, 20.6, 20.6, 20.6, 20.5, 20.4, and 20.4 (7C, Me).
4.49 (AB, J 12.2, CH2Ph), 4.14 (dd, J5 ,6 6.9, J6 ,6 11.2, H6ꢀ), 4.04
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(dd, J5 ,6 6.5, H6ꢀ), 3.95 (dd, J4,5 4.9, J5,5 11.7, H5), 3.85 (ddd, H5ꢀ),
3.66 (dd, H2), 3.52 (ddd, J4,5 9.8, H4), 3.42 (dd, H5), 2.13, 2.73, 2.06,
2.04, 2.03, and 1.96 (18H, 6 s, Me). δC (150.8 MHz) 170.4, 170.2, 169.4,
169.4, and 168.8 (6C, C=O), 137.5, 128.5, 128.0, and 127.7 (Ph), 101.2
(C1ꢀ), 92.8 (C1), 76.8 (C2), 74.6 (C4), 74.4 (C3), 72.4 (CH2Ph), 70.9
(C3ꢀ), 70.5 (C5ꢀ), 68.5 (C2ꢀ), 66.8 (C4ꢀ), 64.0 (C5), 61.1 (C6ꢀ), 21.0,
20.9, 20.9, 20.7, 20.6, and 20.5 (6C, Me).
ꢀ
ꢀ
Next to elute was 1,2,4-tri-O-acetyl-6-O-benzoyl-3-O-(tetra-O-
acetyl-β-d-galactopyranosyl)-β-d-glucose (64 mg, 30%), which was
isolated as an oil (Found: m/z 741.2235. C33H40O19 requires [M + H]+•
741.2242). δH (600 MHz) 8.05–8.03, 7.58–7.54, and 7.46–7.43 (3 m,
Acknowledgments
Ph), 5.65 (d, J1,2 7.9, H1), 5.34 (dd, J3 ,4 3.5, J4 ,5 1.1, H4ꢀ), 5.14 (dd,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J3,4 9.4, J4,5 8.3, H4), 5.09 (dd, J2,3 9.1, H2), 5.05 (dd, J1 ,2 8.0, J2 ,3
We thank Professor Stephen Withers for the generous gift
of the glycosynthase. K.A.S. thanks the University of West-
ern Australia for the assistance of a Hackett Postgraduate
Scholarship.
8.8, H2ꢀ), 4.95 (dd, H3ꢀ), 4.57 (d, H1ꢀ), 4.50 (dd, J5,6 2.6, J6,6 12.1,
H6), 4.29 (dd, J5,6 4.4, H6), 4.24 (dd, J5 ,6 5.9, J6 ,6 11.0, H6ꢀ), 4.19
ꢀ
ꢀ
ꢀ
ꢀ
(ddd, J5 ,6 3.0, H5ꢀ), 4.05 (dd, H3), 4.01 (dd, H6ꢀ), 3.93 (ddd, H5), 2.15,
2.13, 2.06, 2.03, 2.02, 1.99, and 1.95 (21H, 7 s, Me). δC (150.8 MHz)
170.3, 170.2, 170.1, 169.3, 169.1, 169.0, 168.8, and 166.1 (8C, C=O),
133.1, 129.8, 129.5, and 128.4 (Ph), 101.2 (C1ꢀ), 91.8 (C1), 78.4 (C3),
72.8 (C5), 71.9 (C4), 71.0 (C3ꢀ), 70.2 (C5ꢀ), 68.7 (C2ꢀ), 68.1 (C2), 66.8
(C4ꢀ), 62.4 (C6), 60.9 (C6ꢀ), 20.7, 20.6, 20.5, 20.5, 20.5, 20.5, and 20.4
(7C, Me).
ꢀ
ꢀ
References
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[8] M. Okuyama, H. Mori, K. Watanabe, A. Kimura, S. Chiba,
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ANIE417>3.3.CO;2-M
1,3,4-Tri-O-acetyl-6-O-benzoyl-2-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-β-D-glucose
and 1,2,4-Tri-O-acetyl-6-O-benzoyl-3-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-β-D-glucose
Utilizing the above general procedure, tetra-O-acetyl-α-d-glucopyra-
nosyl fluoride (180 mg, 0.51 mmol) and 6-O-benzoyl-d-glucopyranose
2 (140 mg, 0.51 mmol) firstly gave 1,3,4-tri-O-acetyl-6-O-benzoyl-2-
O-(tetra-O-acetyl-β-d-glucopyranosyl)-β-d-glucose (91 mg, 43%) as an
oil (Found: m/z 741.2239. C33H40O19 requires [M + H]+• 741.2242).
δH (600 MHz) 8.03–7.99, 7.58–7.54, and 7.44–7.42 (3 m, Ph), 5.71 (d,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J1,2 7.8, H1), 5.47 (dd, J2,3 9.4, J3,4 9.5, H3), 5.16 (dd, J2 ,3 9.6, J3 ,4
9.5, H3ꢀ), 5.12 (dd, J4,5 8.2, H4), 5.08 (dd, J4 ,5 8.6, H4 ), 4.89 (dd, J1 ,2
ꢀ
ꢀ
ꢀ
7.9, H2ꢀ), 4.59 (d, H1ꢀ), 4.46 (dd, J5,6 2.2, J6,6 12.1, H6), 4.39 (dd, J5,6
4.5, H6), 4.09–4.05 (3H, m, H5 and H6ꢀ), 3.86 (dd, H2), 3.64 (ddd, J5 ,6
ꢀ
ꢀ
4.4 and 2.8, H5ꢀ), 2.15, 2.11, 2.06, 2.03, 1.99, 1.97, and 1.96 (21H, 7 s,
Me). δC (150.8 MHz) 170.7, 170.3, 170.2, 169.8, 169.6, 169.2, 168.6,
and 166.0 (8C, C=O), 133.1, 129.6, 129.5, and 128.3 (Ph), 100.9 (C1ꢀ),
92.0 (C1), 78.3 (C3), 72.7 (C5), 72.0 (C4), 71.8 (C5ꢀ), 70.9 (C2ꢀ), 68.2
(C2), 68.2 (C3ꢀ), 68.0 (C4ꢀ), 62.3 (C6), 61.5 (C6ꢀ), 20.7, 20.65, 20.5,
20.5, 20.5, 20.4, and 20.3 (7C, Me).
[10] M. Hrmova, T. Imai, S. J. Rutten, J. K. Fairweather, L. Pelosi,
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[11] M. Jahn, D. Stoll, R. A. J. Warren, L. Szabó, P. Singh,
H. J. Gilbert, V. M.-A. Ducros, G. J. Davies, S. G. Withers,
Chem. Commun. 2003, 1327.
Next to elute was 1,2,4-tri-O-acetyl-6-O-benzoyl-3-O-(tetra-O-
acetyl-β-d-glucopyranosyl)-β-d-glucose (70 mg, 33%), which was iso-
lated as an oil (Found: m/z 741.2237. C33H40O19 requires [M + H]+•
741.2242). δH (600 MHz) 8.05–8.03, 7.55–7.53, and 7.47–7.43 (3 m,
Ph), 5.64 (d, J1,2 8.1, H1), 5.16–5.14 (m, H3ꢀ and H4), 5.10–5.08 (m,
H2 and H4ꢀ), 4.88 (dd, J1 ,2 8.0, J2 ,3 9.4, H2ꢀ), 4.58 (d, H1ꢀ), 4.50 (dd,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
[12] E. F. L. J. Anet, Carbohydr. Res. 1968, 7, 84. doi:10.1016/S0008-
6215(00)81438-X
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J5,6 2.6, J6,6 12.2, H6), 4.29 (dd, J5,6 4.4, H6), 4.21 (dd, J5 ,6 2.9, J6 ,6
11.3, H6ꢀ), 4.10–4.06 (2H, m, H3 and H6ꢀ), 3.93 (ddd, J4,5 8.3, H5),
3.63 (ddd, J4 ,5 8.5, J5 ,6 4.4, H5ꢀ), 2.12, 2.09, 2.04, 2.03, 2.02, 1.99,
and 1.95 (21H, 7 s, Me). δC (150.8 MHz) 170.4, 170.3, 170.1, 169.2,
169.0, 169.0, 168.7, and 166.2 (8C, C=O), 133.2, 129.8, 129.7, and
128.4 (Ph), 100.8 (C1ꢀ), 92.0 (C1), 78.4 (C3), 72.7 (C5), 71.9 (C5ꢀ),
ꢀ
ꢀ
ꢀ
ꢀ

786
R. V. Stick, K. A. Stubbs and A. G. Watts
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200390114
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