Synthesis of some glycosylated derivatives of 2-deoxy-2-fluoro-β- laminaribiosyl fluoride: Another success for glycosynthases

Article abstract of DOI:10.1071/CH06394

The synthesis of 4,6-di-O-acetyl-2-deoxy-2-fluoro-3-O-(tetra-O-acetyl-?-d- glucopyranosyl)-?-d-glucosyl fluoride is described. Upon deacetylation and treatment with ?-d-glucopyranosyl fluoride in the presence of a glycosynthase, three products, all 2-deox

Full text of DOI:10.1071/CH06394

Communication
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2007, 60, 83–88
Synthesis of Some Glycosylated Derivatives of 2-Deoxy-
2-fluoro-β-laminaribiosyl Fluoride: Another Success
for Glycosynthases
Adrian Scaffidi,A Robert V. Stick,A,B and Keith A. StubbsA
AChemistry M313, School of Biomedical, Biomolecular and Chemical Sciences,
University of Western Australia, Crawley WA 6009, Australia.
BCorresponding author. Email: rvs@chem.uwa.edu.au
The synthesis of 4,6-di-O-acetyl-2-deoxy-2-fluoro-3-O-(tetra-O-acetyl-β-d-glucopyranosyl)-β-d-glucosyl fluoride is
described. Upon deacetylation and treatment with α-d-glucopyranosyl fluoride in the presence of a glycosynthase,
three products, all 2-deoxy-2-fluoro-β-glycosyl fluorides, could be isolated: a trisaccharide, a tetrasaccharide, and a
pentasaccharide. An attempt to convert a trisaccharide glycal into a related difluoride, employing Selectfluor, is also
reported.
Manuscript received: 25 October 2006.
Final version: 4 December 2006.
Quite recently we were involved in a piece of work that man-
aged to label the catalytic nucleophile, a glutamic acid residue,
in a family GH26 (retaining) lichenase, an enzyme that favours
the hydrolysis of mixed-linkage substrates such as β-1,3:β-1,4
glucans.[1,2] The successful label was the ‘difluoride’ 1, chosen
for its sufficient reactivity at C1 to induce the labelling pro-
cess, and for the presence of a fluorine atom at C2 that helps
retain the label on the enzyme (Scheme 1).[3] Here we report
the synthesis of the difluoride 1 and the related tetrasaccharide
2 (Fig. 1), both employing glycosynthase technology;[4] as well,
we describe attempts to prepare the related trisaccharide 3, a
putative inhibitor of enzymes that hydrolyse substrates with just
β-1,3 linkages, for example, laminarin. Any synthesis of 3 is
restricted more to a traditional approach as the development of
glycosynthases to generate the β-1,3 linkages lags significantly
behind that for the β-1,4 counterpart.[5]
The synthesis of the molecules in question here seemingly
has to start with laminaribiose, still better prepared via our
published procedure than the rather tedious partial hydrolysis
of laminarin.[6] Thus, towards our first target, the β-1,3 linked
trisaccharide 3, we prepared the laminaribiosyl trichloroacetim-
idate 4 (Scheme 2).[6] A subsequent glycosylation of the alcohol
OH
OH
OH
OH
OH
O
OH
O
O
O
O
O
HO
HO
O
HO
O
HO
HO
HO
HO
HO
O
O
F
OH
OH
F
F
Ϫ
OH
OH
O
O
O
O
1
Enzyme
Scheme 1.
OH
OH
OH
OH
O
O
O
HO
O
HO
HO
HO
O
O
HO
F
O
OH
OH
F
OH
2
OH
OH
OH
O
O
O
HO
HO
HO
HO
O
O
F
OH
OH
F
3
Fig. 1.
© CSIRO 2007
10.1071/CH06394
0004-9425/07/010083

84
A. Scaffidi, R. V. Stick, and K. A. Stubbs
OAc
OAc
Ph
O
(a)
O
O
O
AcO
AcO
AcO
O
ϩ
O
HO
AcO
AcO
BzO
OCCCl3
NH
OMe
4
5
AcO
AcO
AcO
OAc
OAc
OAc
Ph
O
O
(b)
(c)
O
O
O
O
O
AcO
AcO
AcO
AcO
AcO
O
O
O
O
O
AcO
AcO
AcO
AcO
AcO
BzO
AcO
OAc
OMe
6
7
OAc
OAc
OAc
OAc
OAc
OAc
F
(d)
O
O
O
O
O
O
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
O
AcO
AcO
AcO
AcO
OH
8
9
Scheme 2. (a) 4 Å sieves, TMSOTf, CH2Cl2. (b) (i) NaOMe, MeOH; (ii) AcOH/H2O; (iii) Ac2O, pyridine; (iv) H2SO4, Ac2O. (c) (i) HBr, AcOH; (ii) Zn,
NH4Cl, VO(salen). (d) Selectfluor, MeNO2 or MeCN, H2O.
OH
OH
OH
O
O
Glycosynthase
O
HO
HO
HO
HO
HO
ϩ
1
O
F
OH
OH
F
F
10
Scheme 3.
AcO
AcO
AcO
OAc
Ph
O
Ph
O
(a)
(b)
BnO
BnO
O
O
O
O
AcO
AcO
O
O
O
ϩ
HO
OAc
AcO
OMe
OCCCl3
NH
OMe
12
11
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
BnO
(c)
O
O
O
O
AcO
O
O
F
OAc
OAc
F
OH
13
14
Scheme 4. (a) 4 Å sieves, TMSOTf, CH2Cl2. (b) (i) AcOH/H2O; (ii) Ac2O, pyridine; (iii) H2SO4, Ac2O; (iv) BnNH2, THF. (c) (i) Pd-C, H2, AcOH, MeOH;
(ii) DAST, CH2Cl2.
5 with 4 then gave the trisaccharide derivative 6.[7] Removal of
all of the protecting groups from 6, followed by acetylation and
acetolysis of the resulting methyl glycoside, easily formed the
per-acetate 7. Conversion of 7 into the glycosyl bromide, and
application of our recent procedure for the synthesis of glycals,
then gave 8. Much to our surprise, the addition of Selectfluor to
the glycal 8 in either wet nitromethane or acetonitrile furnished
only the d-manno configured fluoride 9; the configuration of
9 was assigned from the small value for J2,3, just 2.6 Hz. This
result was both perplexing and disappointing as earlier studies
had shown that a bulky equatorial substituent at O3 of a gly-
cal virtually guaranteed a preponderance of the product with an
equatorial fluorine at C2.[8]
as the difluoride 10 (Scheme 3). Thus, towards a synthesis of
10, we converted the alcohol 11 into the disaccharide derivative
12; subsequent removal of the benzylidene protecting group,
acetylation, acetolysis of the methyl glycoside, and selective
deacetylation of the anomeric acetate gave the hemiacetal 13
(Scheme 4). Hydrogenolysis of 13 presumably gave the ‘diol’
that, upon treatment with diethylaminosulfur trifluoride, pro-
vided the difluoride 14 in good yield.This sort of transformation
was first performed by Street and Withers[9] and is notable for
the two separate inversion steps that it employs.
The stage was now set for the critical glycosynthase
reaction. Thus, treatment of a mixture of tetra-O-acetyl-α-d-
glucopyranosyl fluoride[10] and 14 with sodium methoxide in
methanol presumably gave α-d-glucopyranosyl fluoride and the
difluoride 10, incubation of which with the glycosynthase, Abg
E358G, rapidly gave a mixture of the glycosylated products 15
(54%) and 16 (29%), both having been acetylated during the
At this stage we decided to abandon a synthesis of 3 and
concentrate on the mixed-linkage target 1; this decision was
prompted by the possession of a glycosynthase that could, in
principle, add d-glucopyranose units to O4 of an acceptor such

Glycosylated Derivatives of 2-Deoxy-2-fluoro-β-laminaribiosyl Fluoride
85
OAc
(a)
O
AcO
AcO
ϩ
14
AcO
F
OAc
OAc
OAc
O
O
O
AcO
O
AcO
AcO
AcO
O
F
OAc
F
OAc
15
OAc
OAc
OAc
OAc
O
O
O
AcO
AcO
AcO
O
AcO
AcO
O
O
F
O
OAc
OAc
OAc
F
16
OAc
OAc
OAc
OAc
O
OAc
O
O
O
O
AcO
O
AcO
O
AcO
AcO
AcO
AcO
O
O
F
OAc
OAc
F
OAc
OAc
17
Scheme 5. (a) (i) NaOMe, MeOH. (ii) Abg E358G, NH4HCO3, H2O; (iii) Ac2O, pyridine, DMAP.
workup procedure (Scheme 5). By employing a longer reaction
time, a new product, the pentasaccharide 17, was obtained but
to the natural detriment of the trisaccharide 15 (16 and 17, 70%
and 17%, respectively).
The deacetylation of 15 was conducted smoothly with tri-
ethylamine in methanol to provide the polyol 1. We were most
pleased to learn that 1 was an effective label of the family
GH26 lichenase from Clostridium thermocellum, enabling an
insight into the mechanism of action of this versatile glycoside
hydrolase.[1]
H4ꢀꢀ), 5.11 (dd, H3ꢀꢀ). δC (125.8 MHz) 20.33–20.74 (CH3), 55.34
(OCH3), 61.62, 61.91, 62.05 (C6,C6ꢀ,6ꢀꢀ), 67.31 (C5), 67.89,
68.00, 70.86, 72.78 (C2-C2ꢀꢀ,C3ꢀꢀ,C4-C4ꢀꢀ), 71.53 (C5ꢀ,C5ꢀꢀ),
75.63 (C3), 79.09 (C3ꢀ), 96.65 (C1), 100.55, 100.99 (C1ꢀ,C1ꢀꢀ),
168.41–170.73 (10C, C=O). m/z (HR-MS FAB) 939.3004;
[M + H]+ requires 939.2981.
(b) The methyl glycoside from (a) was treated with acetic
anhydride and concentrated sulfuric acid to provide the per-
acetate 7.[7]
Di-O-acetyl-1,5-anhydro-2-deoxy-3-O-[tetra-O-acetyl-
β-D-glucopyranosyl-(1→3)-tri-O-acetyl-
Experimental
General experimental procedures have been given previously.[11]
β-D-glucopyranosyl]-D-arabino-hex-1-enitol 8
Hydrogen bromide in AcOH (3.0 mL of 30% w/v, 15 mmol)
was added to the per-acetate 7 (200 mg, 0.21 mmol) in CH2Cl2
(5 mL) and the solution stirred (rt, 12 h). Usual workup (CH2Cl2)
gave a colourless oil (220 mg), presumably the glycosyl bromide.
Powdered Zn (140 mg, 2.1 mmol), NH4Cl (110 mg, 2.1 mmol)
andVO(salen)[12] (5 mg) were added to the oil in MeOH (10 mL)
and the mixture stirred (rt, 30 min). Filtration followed by
concentration of the filtrate, usual workup (EtOAc) and flash
chromatography (EtOAc/petrol, 3:2) gave the glycal 8 as a
colourless oil (120 mg, 69%), [α]D −50.7◦. δH (600 MHz) 1.97–
Tetra-O-acetyl-β-D-glucopyranosyl-(1→3)-tri-O-acetyl-
β-D-glucopyranosyl-(1→3)-tetra-O-acetyl-
α-D-glucopyranose 7
(a) Sodium methoxide in MeOH (5 mL of 10%) was added
to the benzoate 6[7] (840 mg) in MeOH (5 mL) and the solu-
tion stirred (1 h). Concentration of the mixture left a yellow
residue that was dissolved in AcOH (5 mL), then H2O (1 mL)
was added and the solution was heated (70◦C, 2 h). Concen-
tration of the mixture left a yellow residue that was dissolved
in pyridine (5 mL), then Ac2O (5 mL) was added and the
solution stirred (rt, 12 h). The mixture was quenched by the
addition of MeOH (5 mL) and concentrated. Usual workup
(CH2Cl2) and flash chromatography (EtOAc/petrol, 1:1) gave
methyl tetra-O-acetyl-β-d-glucopyranosyl-(1→3)-tri-O-acetyl-
β-d-glucopyranosyl-(1 → 3)-tri-O-acetyl-α-d-glucopyranoside
as a colourless oil (660 mg, 84%), [α]D +28.2◦. δH (500 MHz)
ꢀꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
2.12 (27H, 9s, CH3), 3.65 (ddd, J4 ,5 10.0, J5 ,6 2.4, 4.5, H5 ),
3.68 (ddd, J4 ,5 8.3, J5 ,6 2.9, 4.0, H5ꢀ), 3.77 (dd, J2 ,3 ≈ J3 ,4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
9.4, H3ꢀ), 4.02 (dd, J5,6 2.8, J6,6 12.2, H6), 4.03 (dd, J6 ,6 12.4,
ꢀꢀ ꢀꢀ
H6ꢀꢀ), 4.09–4.11 (m, H3), 4.13–4.17 (2H, m, H6ꢀ), 4.32–4.35
(m, H5,H6ꢀꢀ), 4.40 (dd, J5,6 8.3, H6), 4.55 (d, J1 ,2 8.1, H1ꢀ),
ꢀ
ꢀ
4.56 (d, J1 ,2 8.1, H1ꢀꢀ), 4.81–4.83 (m, H2), 4.88 (dd, H4ꢀ), 4.91
ꢀꢀ ꢀꢀ
(dd, H2ꢀ), 4.96 (dd, J2 ,3 9.7, H2ꢀꢀ), 5.04 (dd, J3 ,4 9.9, H4ꢀꢀ),
5.11 (dd, H3ꢀꢀ), 5.17–5.19 (m, H4), 6.44 (dd, J1,2 6.4, J1,3 0.6,
H1). δC (150.8 MHz) 20.30–20.82 (CH3), 61.42, 61.69, 62.03
(C6,C6ꢀ,C6ꢀꢀ), 67.86 (C4), 68.03, 68.26 (C2ꢀ,C4ꢀꢀ), 69.04 (C3),
70.96 (C4ꢀ), 71.69, 71.95, 72.60 (C2ꢀꢀ,C5ꢀ,C5ꢀꢀ), 72.92 (C3ꢀ),
73.85 (C5), 78.79 (C3ꢀꢀ), 96.97 (C2), 98.42, 100.89 (C1ꢀ,C1ꢀꢀ),
145.25 (C1), 168.72–171.11 (9C, C=O). m/z (HR-MS FAB)
849.2663; [M + H]+ requires 849.2664.
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
ꢀ
ꢀ
1.96–2.20 (30H, 10s, CH3), 3.40 (s, OCH3), 3.61 (ddd, J4 ,5
9.6, J5 ,6 2.4, 4.5, H5ꢀ), 3.68 (ddd, J4 ,5 9.6, J5 ,6 2.3, 4.0,
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
H5ꢀꢀ), 3.77 (dd, J2 ,3 ≈ J3 ,4 9.5, H3ꢀ), 3.90 (ddd, J4,5 10.2,
ꢀ
ꢀ
ꢀ
ꢀ
J5,6 2.5, 7.1, H5), 4.02 (dd, J6 ,6 12.4, H6ꢀꢀ), 4.05 (dd, J6 ,6
ꢀ
ꢀꢀ ꢀꢀ
ꢀ
12.5, H6ꢀ), 4.08–4.16 (3H, m, H3,H6), 4.28 (dd, H6ꢀ), 4.38
(dd, H6ꢀꢀ), 4.49 (d, J1 ,2 8.0, H1ꢀꢀ), 4.50 (d, J1 ,2 8.1, H1ꢀ),
ꢀꢀ ꢀꢀ
ꢀ
ꢀ
4.85–4.97 (6H, m, H1,H2-H2ꢀꢀ,H4,H4ꢀ), 5.05 (dd, J3 ,4 9.6,
ꢀꢀ ꢀꢀ

86
A. Scaffidi, R. V. Stick, and K. A. Stubbs
4,6-Di-O-acetyl-2-deoxy-2-fluoro-3-O-[tetra-O-acetyl-
β-D-glucopyranosyl-(1→3)-tri-O-acetyl-
3.73 (dd, J1,2 1.7, J2,3 2.9, H2), 3.74–3.78, 4.19–4.25 (2 × m,
H3,H5ꢀ,H6), 3.84 (dd, J6,6 12.3ꢀ, H6), 3.87 (dd, J3,4 10.3, H4),
ꢀ
ꢀ ꢀ ꢀ ꢀ ꢀ ꢀ
4.07 (dd, J6 ,6 10.3, J5 ,6 4.0, H6 ), 4.17 (dd, J5 ,6 3.0, H6 ), 4.61,
β-D-glucopyranosyl]-α-D-mannopyranose 9
ꢀ
ꢀ
ꢀ
4.83 (ABq, J 12.0, CH2Ph), 4.65 (d, H1), 4.74 (d, J1 ,2 7.7, H1 ),
5.06–5.16 (m, H2ꢀ,H3ꢀ,H4ꢀ), 5.60 (s, CHPh), 7.26–7.48 (10H,
m, Ph). δC (125.8 MHz) 20.51, 20.55, 20.58, 20.65 (4C, CH3),
54.80 (OCH3), 61.01, 68.74 (C6,C6ꢀ), 64.01–77.96 (C2-C5,C2ꢀ-
C5ꢀ), 74.00 (CH2Ph), 100.50, 100.88, 101.78 (C1,C1ꢀ,CHPh),
125.87–137.98(Ph), 169.23, 169.30, 170.33, 170.64(4C, C=O).
m/z (HR-MS FAB) 703.2605; [M + H]+ requires 703.2601.
(a) Selectfluor (55 mg, 0.15 mmol) was added to the gly-
cal 8 (60 mg, 0.08 mmol) in CH3NO2/H2O (9:1; 5 mL) and
the solution stirred (12 h). Concentration of the mixture fol-
lowed by a usual workup (CH2Cl2) and flash chromatography
(EtOAc/petrol, 3:2) gave the hemiacetal 9 as a colourless oil
(40 mg, 68%), [α]D −15.1◦. δH (500 MHz) 1.97–2.11 (27H,
8s, CH3), 3.18 (s, OH), 3.64–3.70 (m, H5ꢀ,H5ꢀꢀ), 3.90 (dd,
J2 ,3 ≈ J3 ,4 9.5, H3ꢀ), 4.04 (dd, J5 ,6 2.0, J6 ,6 12.3, H6ꢀꢀ),
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
4.08–4.24 (6H, m, H3,H5,H6,H6ꢀ), 4.35 (dd, J5 ,6 4.5, H6ꢀꢀ),
4,6-Di-O-acetyl-2-O-benzyl-3-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-α-D-mannose 13
4.52 (d, J1 ,2 8.0, H1ꢀ), 4.57 (d, J1 ,2 8.1, H1ꢀꢀ), 4.69 (ddd, J2,F
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
49.5, J1,2 ≈ J2,3 2.6, H2), 4.87 (dd, J2 ,3 8.5, H2ꢀꢀ), 4.92–4.97
ꢀꢀ ꢀꢀ
(m, H2ꢀ,H4ꢀ), 5.05 (dd, J3 ,4 ≈ J4 ,5 9.8, H4ꢀꢀ), 5.13 (dd, H3ꢀꢀ),
5.20 (dd, J3,4 ≈ J4,5 9.5, H4), 5.39–5.42 (m, H1). δC (125.8 MHz)
20.37–21.03 (CH3), 61.72, 62.01, 62.33 (C6,C6ꢀ,C6ꢀꢀ), 66.18–
78.56 (C2ꢀ,C2ꢀꢀ,C3ꢀ,C3ꢀꢀ,C4-C4ꢀꢀ,C5-C5ꢀꢀ), 74.33 (d, J3,F 17.1,
C3), 86.23 (d, J2,F 180.0, C2), 91.79 (d, J1,F 29.0, C1), 98.47,
100.91 (C1ꢀ,C1ꢀꢀ), 168.68–170.87 (9C, C=O). m/z (HR-MS
FAB) 907.2480; [M + Na]+ requires 907.2495.
(a) The glycoside 12 (300 mg, 0.52 mmol) in AcOH/H2O (4:1,
10 mL) was heated at 50◦C (2 h). Concentration of the mix-
ture gave a colourless residue, presumably the diol, which
was dissolved in pyridine (5 mL). Acetic anhydride (0.50 mL,
4.9 mmol) was added and the solution heated at 50◦C (1 h)
before being quenched with MeOH (5 mL). A usual workup
(EtOAc) followed by flash chromatography (EtOAc/petrol,
1:4) yielded methyl 4,6-di-O-acetyl-2-O-benzyl-3-O-(tetra-O-
acetyl-β-d-glucopyranosyl)-α-d-mannoside as a colourless gum
(330 mg, 91%), [α]D +6.7◦. δH (300 MHz) 1.75, 1.90, 1.94,
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
(b) Selectfluor (55 mg) was added to the glycal 8 (50 mg) in
MeCN/H2O (2:1; 5 mL) and the solution stirred (12 h). Concen-
tration of the mixture followed by a usual workup (CH2Cl2) and
flash chromatography (EtOAc/petrol, 3:2) gave the hemiacetal 9
as a colourless oil (40 mg, 62%). The 1H and 13C NMR spectra
were consistent with those reported in (a).
ꢀ
ꢀ
1.98, 2.00 (18H, 5s, CH3), 3.24 (s, OCH3), 3.54 (ddd, J4 ,5 9.5,
J5 ,6 2.3, 4.5, H5ꢀ), 3.67 (dd, J1,2 ≈ J2,3 2.6, H2), 3.76 (ddd, J4,5
ꢀ
ꢀ
9.6, J5,6 2.5, 5.6, H5), 3.98 (dd, J6 ,6 12.3, H6ꢀ), 4.00–4.08 (m,
ꢀ
ꢀ
H3,H6), 4.15 (dd, J6,6 12.2, H6), 4.25 (dd, H6ꢀ), 4.52 (d, J1 ,2 7.7,
ꢀ
ꢀ
H1ꢀ), 4.54, 4.62 (ABq, J 11.8, CH2Ph), 4.70 (d, H1), 4.85 (dd,
J2 ,3 9.0, H2ꢀ), 5.00 (dd, J3 ,4 9.1, H4ꢀ), 5.07 (dd, H3ꢀ), 5.25 (dd,
J3,4 9.6, H4), 7.20–7.28 (Ph). δC (75.5 MHz) 20.22–20.48 (CH3),
54.77 (OCH3), 61.50, 62.60 (C6,C6ꢀ), 66.68–75.86 (C2-C5,C2ꢀ-
C5ꢀ), 72.73 (CH2Ph), 98.10, 98.66 (C1,C1ꢀ), 127.40–137.50
(Ph), 168.78–170.47 (6C, C=O). m/z (HR-MS FAB) 699.2478;
[M + H]+ requires 699.2500.
Methyl 2-O-Benzyl-4,6-O-benzylidene-
α-D-mannoside 11
ꢀ
ꢀ
ꢀ
ꢀ
tert-Butyldimethylsilyl chloride (3.0 g, 20 mmol) was added to
methyl 4,6-O-benzylidene-α-d-mannoside (5.0 g, 18 mmol) and
2,6-lutidine (5.0 mL, 43 mmol) in DMF (50 mL) and the solu-
tion kept (24 h). Concentration of the mixture followed by a
usual workup (CH2Cl2) gave a gum that was dissolved in DMF
(40 mL) and treated with sodium hydride (60% dispersion in
mineral oil; 800 mg, 20.0 mmol) and BnBr (2.3 mL, 20 mmol).
The resulting mixture was stirred (2 h) before being quenched
with MeOH (10 mL). Concentration of the mixture followed by
a usual workup (CH2Cl2) gave a light yellow gum that was dis-
solved in THF (20 mL) and treated with tetrabutylammonium
fluoride (1 M in THF; 10 mL; 2 h). Concentration of the mix-
ture followed by flash chromatography (EtOAc/toluene, 1:9)
(b) Concentrated H2SO4 (10 drops) was added to the
glycoside from (a) (1.5 g, 2.1 mmol) in Ac2O (10 mL) and
the solution kept (0.5 h). The reaction was quenched with
saturated NaHCO3 solution (100 mL, 1 h). A usual workup
(CH2Cl2) followed by flash chromatography (EtOAc/petrol,
2:3) gave 1,4,6-tri-O-acetyl-2-O-benzyl-3-O-(tetra-O-acetyl-β-
d-glucopyranosyl)-α-d-mannose as a colourless solid (900 mg,
58%), mp 64–66◦C, [α]D +6.6◦. δH (300 MHz) 1.87–2.13 (21H,
7s, CH3), 3.50 (ddd, J4 ,5 9.7, J5 ,6 2.4, 4.4, H5ꢀ), 3.76 (dd,
J1,2 ≈ J2,3 2.8, H2), 3.96 (ddd, J4,5 9.5, J5,6 2.5, 5.0, 2.5, H5),
ꢀ
ꢀ
ꢀ
ꢀ
1
returned the alcohol 11 as an oil (3.0 g, 46%). The H NMR
4.05–4.19 (5H, m, H2ꢀ,H3,H6,H6ꢀ), 4.24 (dd, J6 ,6 12.3, H6ꢀ),
spectrum was consistent with that previously reported.[13]
ꢀ
ꢀ
4.50 (d, J1 ,2 7.6, H1ꢀ), 4.64, 4.68 (ABq, J 11.8, CH2Ph), 4.87–
4.95, 5.04–5.13 (2 × m, H1ꢀ,H3ꢀ,H4ꢀ), 5.36 (dd, J3,4 9.5, H4),
6.20 (d, H1), 7.30–7.38 (Ph). δC (75.5 MHz) 20.46–21.01 (CH3),
61.69, 62.43 (C6,C6ꢀ), 67.21–74.65 (C2-C5,C2ꢀ-C5ꢀ), 72.56
(CH2Ph), 91.19, 97.75 (C1,C1ꢀ), 127.94–137.06 (Ph), 168.71–
170.77 (7C, C=O). m/z (HR-MS FAB) 727.2467; [M + H]+
requires 727.2449.
ꢀ
ꢀ
Methyl 2-O-Benzyl-4,6-O-benzylidene-3-O-(tetra-
O-acetyl-β-D-glucopyranosyl)-α-D-mannoside 12
Tetra-O-acetyl-α-d-glucopyranosyl
trichloroacetimidate[14]
(1.2 g, 2.4 mmol), the alcohol 11 (630 mg, 1.7 mmol) and
crushed molecular sieves (4 Å) in dry CH2Cl2 (25 mL) were
stirred under an atmosphere of nitrogen (1 h) and then cooled
to 0◦C. Trimethylsilyl trifluoromethanesulfonate (0.1 mL) was
added and the mixture warmed to room temperature (1 h). The
reaction was quenched with Et3N (0.50 mL, 3.6 mmol) and
filtered through Celite, washing with EtOAc. The combined fil-
trate/washings were concentrated to leave a pale yellow residue
that was subjected to flash chromatography (EtOAc/petrol, 3:7)
to afford the glycoside 12 as a colourless solid (680 mg, 69%),
mp 80–82◦C, [α]D +11.2◦. δH (500 MHz) 1.86, 1.97, 1.98 (12H,
3s, CH3), 3.33 (s, OCH3), 3.45 (ddd, J4,5 9.4, J5,6 2.2, 3.8, H5),
(c) Benzylamine (0.50 mL, 4.6 mmol) was added to the prod-
uct from (b) (300 mg, 0.41 mmol) inTHF (5 mL) and the solution
heated at 50◦C (0.5 h). Concentration of the mixture followed by
flash chromatography (EtOAc/petrol, 1:1) afforded a solid that
was recrystallized to furnish the hemiacetal 13 as small needles
(200 mg, 71%), mp 149–151◦C (EtOAc/petrol), [α]D −4.5◦. δH
(500 MHz) 1.85–2.08 (18H, 6s, CH3), 3.30 (d, J1,OH 3.1, OH),
3.64 (ddd, J4 ,5 9.8, J5 ,6 2.6, 4.5, H5ꢀ), 3.76 (dd, J1,2 ≈ J2,3 2.6,
ꢀ
ꢀ
ꢀ
ꢀ
H2), 4.06–4.12, 4.17–4.24 (4H, 2 × m, H3,H5,H6), 4.16 (dd,
ꢀ
ꢀ
ꢀ
ꢀ
J6 ,6 12.2, H6 ), 4.33 (dd, H6 ), 4.63, 4.72 (ABq, J 11.7, CH2Ph),

Glycosylated Derivatives of 2-Deoxy-2-fluoro-β-laminaribiosyl Fluoride
87
ꢀ
4.64 (d, J1 ,2 7.8, H1ꢀ), 4.96 (dd, J2 ,3 9.2, H2ꢀ), 5.10 (dd, J3 ,4
H5ꢀꢀ), 3.79 (dd, J3 ,4 ≈ J4 ,5 , H4 ), 3.81 (dd, J4,5 9.5, J5,6 3.1,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
9.8, H4ꢀ), 5.17 (dd, H3ꢀ), 5.26 (dd, H1), 5.34 (dd, J3,4 ≈ J4,5
9.2, H4), 7.28–7.36 (Ph). δC (125.8 MHz) 20.48–20.78 (CH3),
61.78, 62.89 (C6,C6ꢀ), 67.23–75.85 (C2-C5,C2ꢀ-C5ꢀ), 73.11
(CH2Ph), 92.55, 98.65 (C1,C1ꢀ), 127.69–137.74 (Ph), 169.11–
170.92 (6C, C=O). m/z (HR-MS FAB) 685.2320; [M + H]+
requires 685.2344.
4.9, H5), 3.97 (ddd, J3,F2 15.3, J2,3 ≈ J3,4 9.0, H3), 4.05 (dd,
J6 ,6 12.5, H6ꢀꢀ), 4.12 (dd, J6 ,6 12.1, H6ꢀ), 4.14–4.16, 4.18–
4.21 (2H, 2 × m, H6), 4.30–4.45 (m, H2), 4.35 (dd, H6ꢀꢀ), 4.47
ꢀꢀ ꢀꢀ
ꢀ
ꢀ
(dd, H6ꢀ), 4.52 (d, J1 ,2 7.9, H1ꢀꢀ), 4.65 (d, J1 ,2 7.9, H1ꢀ), 4.87
ꢀꢀ ꢀꢀ
ꢀ
ꢀ
(dd, J2 ,3 9.6, H2ꢀ), 4.91 (d, J2 ,3 9.3, H2ꢀꢀ), 5.02 (dd, H4), 5.05
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
(dd, J3 ,4 9.8, H4ꢀꢀ), 5.13 (dd, H3ꢀꢀ), 5.17 (dd, H3ꢀ), 5.35 (ddd,
ꢀꢀ ꢀꢀ
J1,F1 51.8, J1,2 6.2, J1,F2 4.2, H1). δC (125.8 MHz) 20.40–20.74
(CH3), 61.49, 61.79, 61.88 (C6,C6ꢀ,C6ꢀꢀ), 66.91 (d, J4,F2 7.5,
C4), 67.72–75.98 (C2ꢀ-C5ꢀ,C2ꢀꢀ-C5ꢀꢀ), 72.20 (dd, J5,F1 3.5, C5),
78.92 (dd, J3,F2 18.9, J3,F1 8.9, C3), 91.09 (dd, J2,F2 187.9, J2,F1
26.8, C2), 100.59, 101.23 (C1ꢀ,C1ꢀꢀ), 105.90 (dd, J1,F1 219.3,
J1,F2 27.3, C1), 168.96–170.52 (9C, C=O). m/z (HR-MS FAB)
887.2632; [M + H]+ requires 887.2633.
4,6-Di-O-acetyl-2-deoxy-2-fluoro-3-O-(tetra-O-acetyl-β-
D-glucopyranosyl)-β-D-glucosyl Fluoride 14
Palladium-on-activated-charcoal (20 mg, 10% w/w) was added
to the hemiacetal 13 (120 mg, 0.17 mmol) in THF/AcOH (9:1,
3 mL) and the mixture heated at 50◦C under an atmosphere of
hydrogen (24 h). Filtration through Celite followed by concentra-
tion of the filtrate gave a powder. Diethylaminosulfur trifluoride
(0.1 mL, 0.76 mmol) was then added dropwise to this powder
dissolved in CH2Cl2 (2 mL) at −78◦C and the solution was kept
at room temperature (24 h).The reaction was then quenched with
saturated NaHCO3 solution (10 mL). A usual workup (CH2Cl2)
followed by flash chromatography (EtOAc/petrol, 2:3) gave
the difluoride 14 as a powder (70 mg, 66%), mp 148–150◦C
(EtOAc/petrol), [α]D +1.5◦. δH (500 MHz) 1.97–2.08 (18H, 6s,
Next to elute was the tetrasaccharide 16 as a powder (25 mg,
29%), mp 108–110◦C, [α]D −6.8◦. Partial δH (500 MHz)A 1.97–
2.15 (36H, 11s, CH3), 3.57 (ddd, J5,6 1.9, 5.1, H5), 3.61 (ddd,
J5,6 1.9, 5.6, H5), 3.65 (ddd, J4,5 9.9, J5,6 1.9, 5.1, H5), 3.82
(ddd, J4,5 9.4, J5,6 3.1, 4.8, H5), 3.96 (ddd, J3,F2 15.2, J2,3 ≈ J3,4
8.4, H3), 4.04 (dd, J5,6 2.2, J6,6 12.4, H6), 4.14 (dd, J3,4 ≈ J4,5
7.2, H4), 4.36 (dd, J5,6 4.8, J6,6 12.6, H6), 4.64 (d, J1,2 8.0,
H1), 4.84 (dd, J1,2 7.9, J2,3 9.4, H2), 4.86 (dd, J1,2 8.0, J2,3 9.5,
H2), 4.90 (dd, J1,2 7.9, J2,3 9.2, H2), 5.34 (ddd, J1,F1 51.9, J1,2
6.3, J1,F2 4.2, H1). δC (125.8 MHz) 20.40–20.78 (CH3), 61.49,
61.80, 61.85, 62.05 (C6-C6ꢀꢀꢀ), 66.92 (d, J4,F2 7.7, C4), 67.72–
76.07 (C2ꢀ-C5ꢀ,C2ꢀꢀ-C5ꢀꢀ,C2ꢀꢀꢀ-C5ꢀꢀꢀ), 72.19 (d, J5,F1 4.3, C5),
78.94 (dd, J3,F2 19.0, J3,F1 9.5, C3), 90.96 (dd, J2,F2 188.1, J2,F1
26.7, C2), 100.31, 100.79, 101.21 (C1ꢀ,C1ꢀꢀ,C1ꢀꢀꢀ), 105.89 (dd,
J1,F1 218.9, J1,F2 27.2, C1), 169.00–170.53 (C=O). m/z (HR-MS
FAB) 1175.3488; [M + H]+ requires 1175.3478.
CH3), 3.67 (ddd, J4 ,5 10.0, J5 ,6 2.4, 4.2, H5ꢀ), 3.78–3.83 (m,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
H5), 3.97 (ddd, J3,F2 15.1, J2,3 ≈ J3,4 8.7, H3), 4.06 (dd, J6 ,6
12.4, H6ꢀ), 4.17–4.20 (2H, m, H6), 4.30–4.48 (m, H2), 4.32 (dd,
H6ꢀ), 4.67 (d, J1 ,2 8.0, H1ꢀ), 4.94 (dd, J2 ,3 9.4, H2ꢀ), 5.02 (dd,
ꢀ
ꢀ
ꢀ
ꢀ
J4,5 9.4, H4), 5.07 (dd, J3 ,4 9.7, H4ꢀ), 5.17 (dd, H3ꢀ), 5.33 (ddd,
J1,F1 51.9, J1,2 6.4, J1,F2 4.2, H1). δC (125.8 MHz) 20.40–20.67
(6C, CH3), 61.66, 61.76 (C6,C6ꢀ), 66.96 (d, J5,F1 7.7, C5), 67.92,
71.32, 71.85, 72.55 (C2ꢀ-C5ꢀ), 72.18 (d, J4,F2 4.7, C4), 78.97 (dd,
J3,F2 18.6, J3,F1 9.3, C3), 91.20 (dd, J2,F2 188.9, J2,F1 26.5, C2),
101.33 (C1ꢀ), 105.89 (dd, J1,F1 219.2, J1,F2 26.9, C1), 169.03–
170.54 (6C, C=O). m/z (HR-MS FAB) 599.1772; [M + H]+
requires 599.1787.
ꢀ
ꢀ
4,6-Di-O-acetyl-2-deoxy-2-fluoro-3-O-[tetra-O-acetyl-
β-D-glucopyranosyl-(1→4)-tri-O-acetyl-β-D-
glucopyranosyl-(1→4)-tri-O-acetyl-β-D-glucopyranosyl]-
β-D-glucosyl Fluoride 16 and
4,6-Di-O-acetyl-2-deoxy-2-fluoro-3-O-[tetra-O-acetyl-β-D-
glucopyranosyl-(1→4)-tri-O-acetyl-β-D-glucopyranosyl-
(1→4)-tri-O-acetyl-β-D-glucopyranosyl-(1→4)-tri-O-
acetyl-β-D-glucopyranosyl]-β-D-glucosyl Fluoride 17
4,6-Di-O-acetyl-2-deoxy-2-fluoro-3-O-[tetra-O-acetyl-β-D-
glucopyranosyl-(1→4)-tri-O-acetyl-β-D-glucopyranosyl]-
β-D-glucosyl Fluoride 15 and
4,6-Di-O-acetyl-2-deoxy-2-fluoro-3-O-[tetra-O-acetyl-β-D-
glucopyranosyl-(1→4)-tri-O-acetyl-β-D-glucopyranosyl-
(1→4)-tri-O-acetyl-β-D-glucopyranosyl]-β-D-
glucosyl Fluoride 16
Tetra-O-acetyl-α-d-glucopyranosyl
fluoride
(150 mg,
0.41 mmol) and the difluoride 14 (50 mg, 0.08 mmol) were
treated first with NaOMe in MeOH and then with Abg E358G
as above (4 days) to furnish the tetrasaccharide 16 as a powder
(50 mg, 70%).
A small piece of Na was added to MeOH (5 mL) and the result-
ing solution was added to tetra-O-acetyl-α-d-glucopyranosyl
fluoride[10] (130 mg, 0.37 mmol) and the difluoride 14 (44 mg,
0.07 mmol) in MeOH (5 mL) and the solution stirred (1 h) before
being neutralized with resin (Amberlite IR-120, H+), filtered,
and concentrated to return a colourless residue. This residue
was then dissolved in NH4HCO3 solution (150 mM, 25 mL),
followed by the addition of Abg E358G[15] (0.2 mg), and the
solution kept at 25◦C (2 days). The solvent was removed under
reduced pressure and the residue in Ac2O (2 mL, 21 mmol) and
pyridine (5 mL) containing DMAP (5 mg) was stirred at 50◦C
(3 h). The reaction was quenched with MeOH (10 mL) and a
usual workup (CH2Cl2) gave a brown residue that was subjected
to flash chromatography (EtOAc/petrol, 2:3–3:2) to furnish the
trisaccharide 15 as a powder (35 mg, 54%), mp 93–95◦C, [α]D
Next obtained was the pentasaccharide 17 as a powder
(20 mg, 17%), mp126–128◦C, [α]D −8.2◦. PartialδH (600 MHz)
3.95 (ddd, J3,F2 15.1, J2,3 ≈ J3,4 8.6, H3), 5.32 (ddd, J1,F1 51.8,
J1,2 6.2, J1,F2 4.2, H1). Partial δC (150.9 MHz) 61.47, 61.85,
61.98, 62.04 (C6-C6ꢀꢀꢀꢀ), 66.91 (d, J4,F2 7.6, C4), 67.71–76.09
(C2ꢀ-C5ꢀ,C2ꢀꢀ-C5ꢀꢀ,C2ꢀꢀꢀ-C5ꢀꢀꢀ), 72.19 (J5,F1 4.2, C5), 78.92 (dd,
J3,F2 19.0, J3,F1 9.2, C3), 91.04 (dd, J2,F2 187.9, J2,F1 26.9, C2),
100.22, 100.52, 100.77, 101.19 (C1ꢀ-C1ꢀꢀꢀꢀ), 105.81 (dd, J1,F1
218.9, J1,F2 27.2, C1). m/z (HR-MS FAB) 1463.4319; [M + H]+
requires 1463.4323.
2-Deoxy-2-fluoro-3-O-[β-D-glucopyranosyl-(1→4)-
β-D-glucopyranosyl]-β-D-glucosyl Fluoride 1
−4.5◦. δH (500 MHz) 1.99–2.14 (27H, 8s, CH3), 3.62 (ddd, J4 ,5
Triethylamine (0.50 mL, 3.6 mmol) was added to 15 (10 mg,
ꢀ
ꢀ
9.9, J5 ,6 1.9, 4.7, H5ꢀ), 3.65 (dd, J4 ,5 9.9, J5 ,6 2.2, 4.4,
0.01 mmol) in MeOH (2 mL) and the solution stirred at 50◦C
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
A
Some of the ring protons could not be unambiguously assigned.

88
A. Scaffidi, R. V. Stick, and K. A. Stubbs
(24 h). Concentration of the mixture gave a colourless residue
that was passed through Sephadex LH-20 (H2O) to return 1 as a
paste (5.5 mg, 95%). Partial δH (500 MHz, D2O)A 3.47 (ddd, J4,5
9.36, J5,6 2.5, 4.6, H5), 3.53 (ddd, J4,5 9.8, J5,6 2.1, 5.1, H5), 3.68
(dd, J6,6 11.9, J5,6 5.6, H6), 3.75 (dd, J6,6 12.2, J5,6 5.2, H6), 3.98
(ddd, J3,F2 15.9, J2,3 ≈ J3,4 8.5, H3), 4.38–4.55 (m, H2), 4.43
(d, J1,2 7.8, H1), 4.58 (d, J1,2 7.8, H1), 5.43 (ddd, J1,F1 53.3, J1,2
6.9, J1,F2 3.6, H1). δC (125.8 MHz, D2O) 61.25, 61.75, 62.15
(C6,C6ꢀ,C6ꢀꢀ), 68.60 (d, J4,F2 7.6, C4), 71.08–80.04 (C5,C2ꢀ-
C5ꢀ,C2ꢀꢀ-C5ꢀꢀ), 82.45 (dd, J3,F2 26.8, J3,F1 10.0, C3), 93.07 (dd,
J2,F2 186.6, J2,F1 24.9, C2), 103.77, 104.33 (C1ꢀ,C1ꢀꢀ), 107.75
(dd, J1,F1 213.2, J1,F2 26.2, C1). m/z (HR-MS FAB) 509.1687;
[M + H]+ requires 509.1682.
[3] S. J. Williams, S. G. Withers, Carbohydr. Res. 2000, 327, 27.
doi:10.1016/S0008-6215(00)00041-0
[4] S. J. Williams, S. G. Withers, Aust. J. Chem. 2002, 55, 3.
doi:10.1071/CH02005
[5] M. Hrmova, T. Imai, S. J. Rutten, J. K. Fairweather, L. Pelosi,
V. Bulone, H. Driguez, G. B. Fincher, J. Biol. Chem. 2002, 277, 30102.
doi:10.1074/JBC.M203971200
[6] J. M. Macdonald, M. Hrmova, G. B. Fincher, R.V. Stick,Aust. J. Chem.
2004, 57, 187. doi:10.1071/CH03227
[7] E. B. Rodriguez, R. V. Stick, Aust. J. Chem. 1990, 43, 665. doi:
10.1071/CH99000665
[8] J. Ortner, M. Albert, H. Weber, K. Dax, J. Carbohydr. Chem. 1999,
18, 297.
[9] I. P. Street, S. G. Withers, Can. J. Chem. 1986, 64, 1400. doi:10.1139/
V86-231
[10] L. F. Mackenzie, Q. Wang, R.A. J. Warren, S. G. Withers, J.Am. Chem.
Soc. 1998, 120, 5583. doi:10.1021/JA980833D
Acknowledgments
We again thank Steve Withers for the generous supply of a range of glyco-
synthases, without which this work would not have been possible. K.A.S.
thanks the University of Western Australia for the assistance of a Hackett
Postgraduate Scholarship.
[11] A. Scaffidi, B. W. Skelton, R. V. Stick, A. H. White, Aust. J. Chem.
2006, 59, 426. doi:10.1071/CH06137
[12] R. V. Stick, K. A. Stubbs, D. M. G. Tilbrook, A. G. Watts, Aust. J.
Chem. 2002, 55, 83. doi:10.1071/CH02011
[13] S. C. Ennis, I. Cumpstey, A. J. Fairbanks, T. D. Butters, M. Mackeen,
M. R. Wormald, Tetrahedron 2002, 58, 9403. doi:10.1016/S0040-
4020(02)01221-8
[14] M. Koketsu, M. Kuwahara, H. Sakurai, H. Ishihara, Synth. Commun.
2004, 34, 239. doi:10.1081/SCC-120027259
[15] C. Mayer, D. L. Jakeman, M. Mah, G. Karjala, L. Gal,
R. A. J. Warren, S. G. Withers, Chem. Biol. 2001, 8, 437. doi:10.1016/
S1074-5521(01)00022-9
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[2] E. J.Taylor,A. Goyal, C. I. P. D. Guerreiro, J.A. M. Prates,V.A. Money,
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