Chemical synthesis of GDP-L-galactose and analogues

Article abstract of DOI:10.1016/S0008-6215(97)10094-5

Succinct syntheses for L-galactose, 3-deoxy-L-xylo-hexose (3-deoxy-L- galactose), 6-deoxy-Lgalactopyranose (L-flucose) and 3,6-dideoxy-L-xylo- hexose (3,6-dideoxy-L-galactose) have been developed starting from commercially available L-galactono-1,4-lactone. L-Galactose and variants were then converted to the guanosine diphosphate derivatives, via the formation of the anomeric phosphates and coupling to guanosine monophosphate morpholidate.

Full text of DOI:10.1016/S0008-6215(97)10094-5

Carbohydrate Research 306 (1998) 409±419
Chemical synthesis of GDP-l-galactose and
analogues
Hayley Binch, Katja Stangier, Joachim Thiem*
Institut fur Organische Chemie, Universitat Hamburg, Martin-Luther-King-Platz 6,
È
È
D-20146 Hamburg, Germany
Received 10 June 1997; accepted in revised form 18 November 1997
Abstract
Succinct syntheses for l-galactose, 3-deoxy-l-xylo-hexose (3-deoxy-l-galactose), 6-deoxy-l-
galactopyranose (l-fucose) and 3,6-dideoxy-l-xylo-hexose (3,6-dideoxy-l-galactose) have been
developed starting from commercially available l-galactono-1,4-lactone. l-Galactose and variants
were then converted to the guanosine diphosphate derivatives, via the formation of the anomeric
phosphates and coupling to guanosine monophosphate morpholidate. # 1998 Elsevier Science
Ltd. All rights reserved
Keywords: l-Galactono-1,4-lactone; GDP-ꢀ-l-galactose and analogues; 3-Deoxy-l-galactose; 3,6-Dideoxy-l-
galactose
1. Introduction
particular importance. In order to elucidate the
role of l-fucose, a series of l-fucose derivatives
have already been prepared for biological tests [2].
The synthesis of guanosine diphosphate (GDP) ꢀ-
l-fucose, the substrate for fucosyl transferases has
also been well documented by enzymatic [3] and
chemical [4] procedures. In this paper, we now
describe the synthesis of GDP-ꢀ-l-galactose, GDP-
3-deoxy-ꢀ-l-galactose and GDP-3,6-dideoxy-ꢀ-l-
galactose, which were prepared as mimics of GDP-
ꢀ-l-fucose for transferase enzymatic studies. l-
Galactose can be favourably compared to l-fucose
with a functional group di€erence at C-6.
Previously, approaches towards such deox-
ygenated derivatives were usually multi-step start-
ing from the parent hexopyranoses [5]. Each route
was expressly designed to give the desired site of
deoxygenation highlighted by a speci®c protecting
group, demanding extensive usage of protection
Oligosaccharides conjugated to lipids or proteins
have an important role in biological systems and are
found in the cytoplasm, on cell membranes and in
extra-cellular ¯uids and matrices. Some of the many
biological functions in which glycoconjugates are
involved include blood clotting, lubrication, struc-
tural support, immunological protection, cell
adhesion, hormone activity and many other recog-
nition events [1]. Oligosaccharides modify the
structure dynamics and physical properties of pro-
teins to which they are covalently bound via nitro-
gen to asparagine (N-linked) or oxygen to serine or
threonine (O-linked).
l-Fucose terminates the oligosaccharide chain of
various glycoconjugates and may therefore be of
* Corresponding author.
0008-6215/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(97)10094-5

410
H. Binch et al./Carbohydrate Research 306 (1998) 409±419
and deprotection chemistry. To obtain the deoxy
sugar, this subsequent step would be a radical or
alternative deoxygenation. The overall yields for
the syntheses of deoxygenated derivatives are in the
range of 10% from expensive starting materials
and therefore an alternative, more direct procedure
is required. A suitable starting point is commer-
cially available l-galactono-1,4-lactone, which can
be manipulated in two steps to the deoxygenated
species, followed by a reduction of the lactone
functionality to a€ord the desired compounds in
80% yield. l-Galactono-1,4-lactone can also be
easily reduced to l-galactose in one step. The acti-
vation of the anomeric centre via the phosphates
®nally gave the GDP derivatives.
and freshly distilled triethylamine should be used
to avoid production of side products and long
reaction times. Lactone (4) was then treated with
the highly selective reducing agent disiamylborane
(DIIAMB) [10], (prepared in situ) to give furanose
(5) in 81% yield. Conversion to the pyranose was
achieved by Zemplen O-deacetylation conditions
and subsequent treatment with benzoyl chloride in
the presence of pyridine, a€ording 1,2,4,6-tetra-O-
benzoyl-3-deoxy-ꢀ-l-galactopyranose (6), a pre-
cursor in the preparation of GDP-3-deoxy-ꢀ-l-
galactose (28).
Preparation of l-fucose and 3,6-dideoxy-l-
galactose.ÐKnown methodology published by
Pedersen et al. allows the transformation of 6-OH
groups in aldonolactone systems to bromides,
which can be further modi®ed by a hydrogenation
reaction to the 6-deoxy derivative [11]. Treatment
of l-galactono-1,4-lactone with HBr in acetic acid,
as described for the d-galactonolactone [12], gave
bromide (7) in good yield. The next step towards
the synthesis of 3,6-dideoxy-l-galactose was a
hydrogenation reaction in the presence of triethy-
lamine, which in one step provided the C-3 and C-6
dideoxy functionality leading to 3,6-dideoxylactone
(8) in 79% yield. To complete the synthesis, lactone
(8) underwent reduction with DIIAMB to give
furanose (9), deprotection of the ester groups, fol-
lowed by benzoylation gave 1,2,4-tri-O-benzoyl-
3,6-dideoxy-ꢀ-l-galactose (10) to be used for the
formation of GDP-3,6-dideoxy-ꢀ-l-galactose (29).
In the fucose case, once the bromide had been
synthesised, it was necessary at this stage to remove
the ester protecting groups (accomplished by addi-
tion of methanol on work up of the bromination
reaction) to prevent any elimination occurring
during the hydrogenation reaction furnishing lac-
tone (11). Hydrogenation of 6-bromolactone (11)
gave lactone (12), which was acetylated and then
reduced with DIIAMB to furnish 2,3,5-tri-O-
acetyl-6-deoxy-ꢀ-l-galactofuranose (13) in 76%
yield. Finally the remaining protecting groups were
removed by the Zemplen O-deacetylation condi-
tions to give l-fucose (Scheme 1).
2. Results and discussion
Preparation of l-galactose.ÐA two step synth-
esis of l-galactose has already been documented,
whereby l-galactono-1,4-lactone (1) is reduced to
L-galactose by the use of sodium amalgam [6].
Alternatively, careful reduction of l-galactono-1,4-
lactone (Amberlite IR-120 H+, pH 3±5, sodium
borohydride) [7] a€orded l-galactose as a mix-
ture with the over-reduced product l-galactitol.
Treatment of this mixture with typical acetylation
conditions gave the desired acetylated l-galactose
(2), in 71% yield after puri®cation.
Preparation of 3-deoxy-l-galactose.ÐUnder
basic conditions, acetylated aldonolactones su€er
elimination reaction to give ꢁ,ꢀ-unsaturated lac-
tones, which can be further transposed to 3-deoxy
sugars, but with the complication of a further
spontaneous elimination [8]. However, a more e-
cient procedure to 3-deoxyaldonolactones has been
described, which involves treatment of aceylated
aldonolactones (generated by acidic acetylation
conditions) with hydrogen using palladium catalyst
(5% Pd/C) in the presence of triethylamine [9].
This route combines ®rstly a base catalysed elim-
ination with a subsequent reduction of the ensuing
ꢁ,ꢀ-unsaturated lactone.
Applying this strategy, l-galactono-1,4-lactone
(1) was converted to tetra-O-acetyl-l-galactono-
1,4-lactone (3) using acetic anhydride and a cata-
lytic amount of acid, which was then subsequently
treated with hydrogen in the presence of 5% Pd/C
and triethylamine for 18 h to furnish 2,4,5-tri-O-
acetyl-3-deoxy-l-galactono-1,4-lactone (4) in 88%
yield. It should be noted that high quality Pd/C
Preparation of GDP-b-l-galactose, GDP-3-
deoxy-b-l-galactose and GDP - 3,6 - dideoxy - b- l-
galactose.ÐThere are many reported routes for
the synthesis of GDP-fucose and GDP-arabinose
[3,4] and we intended to base our strategy for the
targets, GDP-ꢀ-l-galactose, GDP-3-deoxy-ꢀ-l-
galactose and GDP-3,6-dideoxy-ꢀ-l-galactose on
such published work. For such an approach, we

H. Binch et al./Carbohydrate Research 306 (1998) 409±419
411
would couple the sugar containing one of the
phosphorus moieties in an activated form with the
correct anomeric con®guration, to guanosine
monophosphate morpholidate N,N0-dicyclohex-
ylcarboxaminide (GMP). Although this type of
reaction has been well described for GDP-fucose,
the coupling reaction had until recently su€ered
from poor yields in the range of 25 to 48%. Wong
et al. have recently described an improvement in the
synthesis of these GDP-sugars, whereby 1H-tetra-
zole was employed as a catalyst in the coupling
reaction, boosting the yields to 76±91% and mini-
mising reaction times [13]. However, at the time this
improvement was not known and our chemistry
was carried out in accordance with typical coupling
conditions for the formation of GDP-fucose.
The formation of the phosphate must be ste-
reospeci®c, allowing access only to the desired
Scheme 1.

412
H. Binch et al./Carbohydrate Research 306 (1998) 409±419
ꢀ-phosphate. This can be achieved by treating the
sugar, activated at the anomeric position, with the
nucleophilic reagent dibenzyl phosphate in the
presence of silver carbonate [2]. Activation of the
sugar at the anomeric position is usually attained
by the formation of a glycosyl halide. Synthesis of
ꢁ-bromides (14) and (15) was achieved in 85 and
63% yield, respectively, from the preceding acetate
(2) and benzoate (6) using hydrogen bromide in
acetic acid. In the case of the 3-deoxygalactose
compound, the synthesis of 3,6-dideoxy-l-galac-
tose phosphate via bromide has been documented,
but the phosphate was generated as a 5.4:1 ꢀ=ꢁ
anomeric mixture in low yield [4]. Due to both the
instability of the glycosyl bromide and the forma-
tion of both anomers of the subsequent phospho-
nate, the more stable 3,6-dideoxy-l-galactosyl
chloride (16) was prepared following a procedure
using zinc chloride in dichloromethyl-methylether
[5] and used immediately without any puri®cation.
With halides (14), (15) and (16) in hand, conversion
to the desired ꢀ-phosphates (17), (18), and (19) was
achieved by reaction with dibenzyl phosphate in
the presence of silver carbonate, followed by pur-
i®cation by silica gel column chromatography in
89, 90 and 55% yields (over two steps), respec-
tively. Full deprotection was performed by hydro-
genolysis of the benzyl ester groups using Pd/C as
catalyst, followed by basic hydrolysis of the
benzoate or acetate groups also allowing for the
formation of the disodium salt of l-galactosyl
phosphate (20), the dilithium salt for both 3-deoxy-
l-galactosyl phosphate (21) and 3,6-dideoxy-l-
galactosyl phosphate (22) in 69, 79 and 65% yields,
respectively (Scheme 2).
Before the ®nal step, phosphates (20), (21) and
(22) were converted to the tributylammonium salts
to increase solubility in dimethylformamide. GMP
(26) was then allowed to react with phosphates
(23), (24) and (25) under strictly anhydrous condi-
tions for 7±10 days. Puri®cation of the reaction
mixture in each case was performed by ion-
exchange and then desalting of the residue using
Sephadex G-10 chromatography to give GDP-l-
galactose (27) (29%), GDP-3-deoxy-l-galactose
(28) (27%) and GDP-3,6-dideoxy-l-galactose (29)
(19%) (Scheme 3).
3. Experimental
General methods.ÐOptical rotations were mea-
sured using a Perkin±Elmer 241 polarimeter at
22‹2 ꢀC. Analytical TLC was performed on Silica
Gel 60-F254 (E. Merck, Darmstadt) with detection
by UV absorption and/or by charring with H2SO4.
All commercial reagents were used as supplied and
chromatography solvents were distilled prior to
use. Column chromatography was performed on
Silica Gel 60 (40±60 ꢂm, E. Merck, Darmstadt). 1H
NMR spectra were recorded at 500 MHz (Bruker
AMX-500), 360 MHz (Bruker WM-360) or
300 MHz (Bruker AM-300) with either (Me)4Si (ꢃ
0, for solutions in CDCl3 and CD3OD) or DOH (ꢃ
Scheme 2.

H. Binch et al./Carbohydrate Research 306 (1998) 409±419
413
Scheme 3.
4.80, for solutions in D2O) as internal references.
13C NMR spectra were recorded at 75.5 MHz
(Bruker AM-300) and 125.7 MHz (Bruker AMX-
500) with internal (Me)4Si (ꢃ 0, for solutions in
extract was washed with water, sat NaHCO3, dried
(MgSO4) and concentrated under reduced pressure.
The residue was puri®ed by column chromato-
graphy using 1:3 EtOAc±toluene as eluent to give
the title compound (2) (1.54 g, 71%) as an oil.
1
CDCl3 and CD3OD). H NMR data are reported
as though they were ®rst order. Assignments of 13C
chemical shifts are tentative and are based on
comparison with published spectral data. Unless
otherwise stated, all reactions are carried out at
room temperature.
[ꢁ]20d
173.1 (c 2.1 in CHCl3); 1H NMR
(500 MHz, CDCl3): ꢃ 5.74 (d, 1 H, J1,2 7.1 Hz, H-
1), 5.58 (ddꢁt, 1 H, J2,1 7.1, J2,3 6.6 Hz, H-2), 5.48
(d, 1 H, J4,3 3.0 Hz, H-4), 4.73 (dd, 1 H, J3,2 6.6,
J3,4 3.0 Hz, H-3), 4.49 (dd, 1 H, J6a,6b 11.7, J6a,5
5.0 Hz, H-6a), 4.39 (dd, 1 H, J6a,6b 11.7, J6b,5
6.6 Hz, H-6b), 4.25 (m, 1 H, J5,6b 6.6, J5,6a 5.0 Hz,
H-5), 2.30±2.10 (5Âs, 15 H, 5ÂOCOCH3) ppm.;
Anal. Calcd. for C16H22O11 (390.4): C, 49.23; H,
5.68. Found: C, 50.14; H, 5.64.
2,5,6-Tri-O-acetyl-3-deoxy-l-xylo-hexono-1,4-
lactone (4).ÐTo 2,3,5,6-tetra-O-acetyl-l-galac-
tono-1,4-lactone (3) (1.0 g, 2.91 mmol) in EtOAc
(10 mL) containing triethylamine (1 mL) was added
5% Pd/C (0.1 g) and the soln was stirred for 18 h
under an atmosphere of hydrogen (3 atm). Once
the reaction was complete, the reaction mixture
was ®ltered and washed with 2 N HCl (2Â10 mL),
then the organic phase was dried (MgSO4) and
concentrated under reduced pressure to give the
title compound (4) (731 mg, 88%) as an oil. [ꢁ]20d
93 (c 1.7 in CHCl3); 1H NMR (500 MHz,
1,2,3,4,6-Penta-O-acetyl-b-l -galactopyranose
(2).ÐTo l-galactono-1,4-lactone (1.0 g, 5.55 mmol)
in MeOH (6 mL) and water (25 mL) at 0 ꢀC was
added Amberlite IR 120 (H+) resin (5 mL).
Sodium borohydride (0.22 g, 5.61 mmol) was added
portionwise, keeping the pH between 3 and 5. Once
the addition of NaBH4 was complete, the reaction
mixture was stirred for 1 h, warming to room tem-
perature, then the resin was removed by ®ltration.
Methanol was removed under reduced pressure
and the resulting solid was dissolved in MeOH
(50 mL) and then concentrated under reduced
pressure. This procedure was repeated twice. The
residue was then dissolved in Ac2O (30 mL), 60%
HclO4 (0.5 mL) was added and the soln stirred for
1 h. Ice water was added and, after 0.5 h, the mix-
ture was extracted with CH2Cl2. The organic

414
H. Binch et al./Carbohydrate Research 306 (1998) 409±419
CDCl3): ꢃ 5.48 (dd, 1 H, J2,3b 10.2, J2,3a 9.0 Hz, H-
2), 5.16 (dt, 1 H, J5,6b 6.4, J5,6a 4.5, J5,4 4.5 Hz, H-
5), 4.64 (ddd, 1 H, J4,3b 11.4, J4,3a 6.0, J4,5 4.5 Hz,
H-4), 4.34 (dd, 1 H, J6a,6b 12.2, J6a,5 4.5 Hz, H-6a),
4.15 (dd, 1 H, J6b,6a 12.2, J6b,5 6.4 Hz, H-6b), 2.72
(ddd, 1 H, J3a,3b 13.0, J3a,2 9.0, J3a,4 6.0 Hz, H-3a),
2.09, 2.00 (2 Â s, 6 H, 2 Â OCOCH3), 1.95 (m, 4 H,
H-3b, 3 Â OCOCH3); 13C NMR (125.76 MHz,
CDCl3): ꢃ 171.4 (C-1), 74.34 (C-4), 70.53 (C-2),
67.66 (C-5), 61.90 (C-6), 30.43 (C-3) ppm. Anal.
Calcd. for C12H16O8 (288.3):C, 50.00; H, 5.59.
Found: C, 49.81; H, 5.69.
form) the mixture was ®ltered, concentrated under
reduced pressure to yield 3-deoxy-l-xylo-hexopyr-
anose. This residue (0.5 g, 3.05 mmol) was dis-
solved in dry pyridine (10 mL) and dry CH2Cl2
(2 mL), cooled to 40 ꢀC and benzoyl chloride
(390 ꢂL, 3.35 mmol) was added dropwise over 5 h
under an atmosphere of argon. The reaction was
quenched with water (10 mL), the mixture extrac-
ted with CH2Cl2 and the organic layer con-
centrated under reduced pressure. The residue was
then puri®ed by column chromatography using as
eluent, 1:10 EtOAc±toluene, to give the title com-
pound (6) (1.39 g, 58%) as colourless syrup. [ꢁ]20d
168.6 (c 1.9 in CHCl3); 1H NMR (500 MHz,
CDCl3): ꢃ 8.20 7.14 (m, 20 H, Aryl-H), 6.24 (d, 1
H, J1,2 8.0 Hz, H-1), 5.70 5.52 (m, 2 H, H-2, H-4),
4.79 (m, 1 H, H-5), 4.73 (dd, 1 H, J6a,6b 13.8, J6a,5
3.8 Hz, H-6a), 4.60 (dd, 1 H, J6b,6a 13.8, J6b,5
7.0 Hz, H-6b), 2.81 (m, 1 H, H-3e), 2.21 (m, 1 H,
H-3a) ppm.; Anal. Calcd. for C34H28O9 (580.6): C,
70.34; H, 4.86. Found: C, 70.31; H, 4.97.
1
The H and 13C data for (4) are consistent with
the reported data for 2,3,6-tri-O-acetyl-3-deoxy-d-
galactono-1,4-lactone [9].
2,5,6-Tri-O-acetyl-3-deoxy-l-xylo-hexofuranose
(5).ÐTo a solution of borane±dimethyl sul®de
complex (3.2 mL, 0.03 mol) in CH2Cl2 (10 mL) was
added dropwise under an atmosphere of N2, a soln
of 2-methyl-2-butene (3.8 mL, 0.07 mol) in CH2Cl2
(15 mL) and then stirred for 3 h. After this time, the
reaction mixture was cooled to 0 ꢀC, a soln of 2,5,6-
tri-O-acetyl-3-deoxy-l-xylo-hexono-1,4-lactone (4)
(731 mg, 2.53 mmol) in CH2Cl2 (10 mL) was added
and the reaction mixture stirred for 18 h. Water
(10 mL) was added, the reaction mixture was stir-
red for a further 1 h and then concentrated under
reduced pressure. The residue was co-evaporated
with water (3Â15 mL) and then MeOH (3Â15 mL)
and then puri®ed by column chromatography
using 1:2 EtOAc±toluene as eluent to give the title
compound (5) (596 mg, 81%) as an oil (ꢁ=ꢀ 6:1).
1H NMR (500 MHz, CDCl3) ꢁ-anomer: ꢃ 5.33
(ddꢁt, 1 H, J1,OH 3.6, J1,2 3.1 Hz, H-1), 5.11 (m, 1
H, H-5), 4.96 (ddd, 1 H, J2,3a 7.0, J2,1 3.1, J2,3b
2.5 Hz, H-2), 4.40 (dddꢁdt, 1 H, J4,3a 8.2, J4,5 5.7,
J4,3b 5.4 Hz, H-4), 4.29 (dd, 1 H, J6a,6b 12.1, J6a,5
3.4 Hz, H-6a), 4.11 (dd, 1 H, J6b,6a 12.1, J6b,5
8.5 Hz, H-6b), 3.96 (d, 1 H, JOH,1 3.6 Hz, OH), 2.49
(ddd, 1 H, J3a,3b 14.8, J3a,4 8.2, J3a,4 7.0 Hz, H-3a),
2.10, 2.01, 1.96 (3 Â s, 9 H, 3 Â OCOCH3), 1.69
(ddd, 1 H, J3b,3a 14.8, J3b,4 5.4, J3b,2 2.5 Hz, H-3b);
13C NMR (125.76 MHz, CDCl3) ꢀ-anomer: ꢃ
100.59 (C-1), 77.91 (C-4), 76.03 (C-2), 71.58 (C-5),
62.96 (C-6), 31.38 (C-3) ppm. Anal. Calcd. for
C12H18O8 (290.3):C, 49.65; H, 6.25. Found: C,
49.12; H, 6.43.
2,3,5-Tri-O-acetyl-6-bromo-6-deoxy-l-galactono-
1,4-lactone (7).Ðl-Galactono-1,4-lactone (1) (1.0 g,
5.61 mmol) was dissolved in HBr (30 mL, 30% in
glacial acetic acid) and stirred for 4 h. Acetic
anhydride (10 mL) was added, and the mixture was
stirred for a further 1 h. The reaction mixture was
poured on ice water and after 30 min, the mixture
was extracted with CH2Cl2. The organic extract
was washed with water, sat NaHCO3 soln, dried
(MgSO4) and concentrated under reduced pressure
to give the title compound (7) (1.87 g, 91%) as
colourless crystals. mp 97 ꢀC; [ꢁ]20d 67 (c 1.4 in
1
CHCl3); H NMR (500 MHz, CDCl3): ꢃ 5.59 (d, 1
H, J2,3 7.0 Hz, H-2), 5.36 (ddꢁt, 1 H, J3,2 7.0, J3,4
6.7 Hz, H-3), 5.18 (dt, 1 H, J5,6 6.5, J5,4 2.3 Hz, H-
5), 4.80 (dd, 1 H, J4,3 6.7, J4,5 2.3 Hz, H-4), 3.50 (d,
2 H, J5,6 6.5 Hz, H-6), 2.09, 2.02, 1.96 (3Âs, 9 H,
3ÂOCOCH3); 13C NMR (125.76 MHz, CDCl3): ꢃ
171.4 (C-1), 77.27 (C-4), 72.33 (C-3), 72.00 (C-2),
70.14 (C-5), 27.25 (C-6) ppm.; Anal. Calcd. for
C12H15 BrO8 (367.2): C, 39.26; H, 4.12; Br, 21.76.
Found: C, 38.99; H, 4.07; Br, 21.59.
The 1H and 13C data found for (7) are consistent
with the reported data for 2,3,5-tri-O-acetyl-6-
bromo-6-deoxy-d-galactono-1,4-lactone [9].
2,5-Di-O-acetyl-3,6-dideoxy-l-xylo-hexono-1,4-
lactone (8).Ð2,3,5-Tri-O-acetyl-6-bromo-6-deoxy-
l-galactono-1,4-lactone (7) (0.5 g, 1.36 mmol) in
EtOAc (15 mL) and triethylamine (0.75 mL) were
stirred for 20 h under an atmosphere of hydrogen
(5 atm) in the presence of 5% Pd/C (0.1 g). Once
1,2,4,6-Tetra-O-benzoyl-3-deoxy-b-l-xylo-hexo-
pyranose (6).Ð2,5,6-Tri-O-acetyl-3-deoxy-l-xylo-
hexofuranose (5) (1.2 g, 4.10 mmol) was treated
with 0.1 M NaOMe in dry MeOH (10 mL) for 24 h.
After neutralisation with Amberlite IR 120 (Cl

H. Binch et al./Carbohydrate Research 306 (1998) 409±419
415
the reaction was complete, the reaction mixture
was ®ltered and washed with 2 N HCl (2Â10 mL),
the organic phase was dried (MgSO4) and con-
centrated under reduced pressure to give the title
compound (8) (246 mg, 79%) as colourless crystals.
argon. The reaction was quenched with water
(10 mL), the mixture extracted with CH2Cl2 and the
organic layer concentrated under reduced pressure.
The residue was then puri®ed by column chromato-
graphy using as eluent, 1:10 EtOAc±toluene to give
the title compound 10 (2.44g, 64%) as a colourless
mp 82±84 ꢀC; [ꢁ]20d 33.4 (c 1.0 in CHCl3); H
1
1
NMR (500 MHz, CDCl3): ꢃ 5.48 (dd, 1 H, J2,3b
10.3, J2,3a 8.9 Hz, H-2), 5.00 (dqꢁm, 1 H, J5,6 6.5,
J5,4 5.4 Hz, H-5), 4.42 (ddd, 1 H, J4,3b 10.3, J4,3a
6.1. J4,5 5.4 Hz, H-4), 2.69 (ddd, 1 H, J3a,3b 12.6,
J3a,2 8.9, J3a,4 6.1 Hz, H-3a), 2.12, 2.08, 2.01 (3Âs, 9
H, 3ÂOCOCH3), 1.94 (td, 1 H, J3b,3a 12.6, J3b,2
10.3, J3b,4 10.3 Hz, H-3b), 1.31 (dd, 3 H, J6,5
6.5 Hz, H-6); 13C NMR (125.76 MHz, CDCl3): ꢃ
171.52 (C-1), 77.41 (C-4), 77.00 (C-2), 68.02 (C-5),
30.76 (C-3), 15.64 (C-6) ppm.; Anal. Calcd. for
C10H14O6 (230.2): C, 52.17; H, 6.13. Found: C,
52.11; H, 6.10.
syrup;[ꢁ]20d 184.3 (c 1.9 in CHCl3); H NMR
(500MHz, CDCl3): ꢃ 8.25±7.22 (m, 15 H, Aryl-H),
6.17 (d, 1 H, J1,2 8.2 Hz, H-1), 5.61 (m, 1 H, H-2),
5.40 (m, 1 H, H-4), 4.62 (dq, 1 H, J5,6 6.4, J5,4 4.7 Hz,
H-5), 2.79 (ddd, 1 H, J3a,3e 15.1, J3a,2 7.5, J3a,4
5.8 Hz, H-3a), 2.13 (ddd, 1 H, J3a,3e 15.1, J3e,2 5.8,
J3e,4 3.5 Hz, H-3e), 1.43 (d, 3 H, H-6) ppm.; Anal.
Calcd. for C27H24O7 (460.5): C, 70.43; H, 5.25.
Found: C, 70.08; H, 5.33.
6-Bromo-6-deoxy-l-galactono-1,4-lactone (11).Ð
To l-galactono-1,4-lactone (1) (1.0 g, 5.61 mmol)
was added HBr (15 mL, 30% in glacial acetic acid)
and the reaction mixture was stirred for 4 h.
Methanol (40 mL) was then added and the reaction
mixture stirred for additional 12 h. After con-
centration and co-evaporation twice with MeOH
(30 mL) and water (30 mL), the title compound
(11) (1.13 g, 84%)ꢀwas obtained as colourless crys-
tals. mp 105±106 C; [ꢁ]20d 105 (c 2.3 in H2O);
13C NMR (125.76 MHz, D2O): ꢃ 176.22 (C-1),
81.33 (C-4), 74.54 (C-2), 74.04 (C-3), 70.40 (C-5),
33.74 (C-6) ppm.; Anal. Calcd. for C6H9O5Br
(241.1): C, 29.90; H, 3.76; Br, 33.15. Found: C,
30.17; H, 3.83; Br, 33.03.
The 1H and 13C data found for (8) are consistent
with the reported data for 2,5-di-O-acetyl-3,6-
dideoxy-d-galactono-1,4-lactone [9].
2,5-Di-O-acetyl-3,6-dideoxy-l-xylo-furanose (9).Ð
Lactone (8) (246 mg, 1.07 mmol) was reduced in a
similar manner to that described for the prepara-
tion of (5). The residue was puri®ed by column
chromatography using 1:3 EtOAc±toluene as
eluent to give the title compound (9) (191 mg, 77%)
as a colourless syrup (ꢁ:ꢀ 5:1). 1H NMR
(500 MHz, CDCl3) ꢁ-anomer: 5.36 (t, 1 H, J1,2 3.0,
J1,OH 3.0 Hz, H-1), 5.00 (ddd, 1 H , J2,3a 6.7, J2,1
3.0, J2,3b 2.1 Hz, H-2), 4.90 (ddꢁt, 1 H, J5,6 6.3, J5,4
5.6 Hz, H-5), 4.26 (dddꢁdt, 1 H, J4,3a 7.9, J4,3b 6.0,
J4,5 5.6 Hz, H-4), 3.69 (d, 1 H, JOH,1 3.0 Hz, OH),
2.49 (ddd, 1 H, J3a,3b 14.2, J3a,4 7.9, J3a,2 6.7 Hz, H-
3a), 2.01, 2.00 (2Âs, 6 H, 2ÂOCOCH3), 1.62 (ddd,
1 H, J3b,3a 14.2, J3b,4 6.0, J3b,2 2.1 Hz, H-3b), 1.19
(d, 3 H, J6,5 6.3 Hz, H-6); 13C NMR (125.76 MHz,
CDCl3): ꢃ 100.84 (C-1), 79.64 (C-4), 78.17 (C-2),
71.64 (C-5), 32.00 (C-3), 16.34 (C-6) ppm.; Anal.
Calcd. for C10H16O6 (232.2): C, 51.72; H, 6.94.
Found: C, 52.15; H, 6.81.
1,2,4-Tri-O-benzoyl-3,6-dideoxy-b-l-xylo-hexopy-
ranose (10).Ð2,5-Di-O-acetyl-3,6-dideoxy-l-xylo-
furanose (9) (1.9 g, 8.32 mmol) was treated with
0.1 M NaOMe in dry MeOH (10 mL) for 24 h.
After neutralisation with Amberlite 120 (Cl- form)
the mixture was ®ltered, concentrated under reduced
pressure to yield 3,6-dideoxy-l-xylo-hexopyranose.
This residue (1.0 g, 6.72 mmol) was dissolved in dry
pyridine (10 mL) and dry CH2Cl2 (2 mL), cooled to
40ꢀC and benzoyl chloride (860ꢂL, 7.30mmol)
was added dropwise over 5 h under an atmosphere of
The 13C data found for (11) was consistent with
the reported data for 6-bromo-d-galactono-1,4-
lactone [12].
2,3,5-Tri-O-acetyl-6-deoxy-l-galactono-1,4-
lactone (12).Ð6-Bromo-6-deoxy-l-galactono-1,4-
lactone (11) (0.5 g, 2.07 mmol) in EtOAc (10 mL)
and triethylamine (1 mL) was stirred in the pre-
sence of 5% Pd/C (0.1 g) under an atmosphere of
H2 (3 atm) for 16 h. Once the reaction was com-
plete, the reaction mixture was ®ltered and washed
with 2 N HCl (2Â10 mL), the organic phase was
dried (MgSO4) and concentrated under reduced
pressure. Acetic anhydride (20 mL) and 60%
HClO4 (0.5 mL) were added and the soln stirred for
1 h. Ice water was added and the reaction mixture
was extracted with CH2Cl2. The organic extract
was washed with water, saturated NaHCO3 soln,
dried (MgSO4) and concentrated under reduced
pressure. The residue was puri®ed by column chro-
matography using 1:2 EtOAc±toluene as eluent to
give the title compound (12) (482 mg, 81%) as an
oil; [ꢁ]20d 29.8 (c 1.3 in CHCl3); 1H NMR

416
H. Binch et al./Carbohydrate Research 306 (1998) 409±419
(500 MHz, CDCl3): ꢃ 5.71 (d, 1 H, J2,3 6.8 Hz, H-
2), 5.38 (t, 1 H, J3,2 6.8, J3,4 6.7 Hz, H-3), 5.11 (dq,
1 H, J5,6 6.6, J5,4 3.1 Hz, H-5), 4.31 (dd, 1 H, J4,3
6.7, J4,5 3.1 Hz, H-4), 2.11, 2.08, 2.06 (3Âs, 9 H,
3ÂOCOCH3), 1.39 (d, 3 H, J6,5 6.6 Hz, H-6); 13C
NMR (125.76 MHz, CDCl3): ꢃ 168.9 (C-1), 80.4
(C-4), 72.1 (C-2), 71.9 (C-3), 67.8 (C-5), 15.4 (C-6)
ppm.; Anal. Calcd. for C12H16O8 (288.3): C, 50.00;
H, 5.59. Found: C, 49.77; H, 5.11.
1 H, J4,3 3.2, J4,5 1.0 Hz, H-4), 5.24 (dd, 1 H, J3,2
10.6, J3,4 3.2 Hz, H-3), 4.86 (dd, 1 H, J2,3 10.6, J2,1
3.9 Hz, H-2), 4.29 (dddꢁt, 1 H, J5,6b 6.8, J5,6a
6.2 Hz, H-5), 4.02 (dd, 1 H, J6a,6b 11.5, J6a,5 6.2 Hz,
H-6a), 3.97 (dd, 1 H, J6b,6a 11.5, J6b,5 6.8 Hz H-6b)
ppm.; Anal. Calcd. for C14H19BrO9 (411.2): C,
40.89; H, 4.66; Br 19.43. Found: C, 40.47; H, 4.53;
Br, 19.24.
Dibenzyl-(2,3,4,6-tetra-O-acetyl-b-l-galactopyr-
anosyl)-phosphate (17).Ð2,3,4,6-Tetra-O-acetyl-ꢁ-
2,3,5-Tri-O-acetyl-6-deoxy-l-galactofuranose
(13).ÐA solution of diisoamylborane was pre-
pared by addition of 2-methyl-2-butene (3.8 mL,
68 mmol) in CH2Cl2 (15 mL) to the borane±
dimethylsul®de complex (3.2 mL, 34 mmol) at
0 ꢀC under nitrogen atmosphere. After 3 h, 2,3,5-
tri-O-acetyl-6-deoxy-l-galactono-1,4-lactone (12)
(482 mg, 1.68 mmol) was added and the mixture
was stirred for 18 h at room temperature. Water
(15 mL) was added and the mixture was stirred for
1 h, the soln concentrated and the residue was co-
evaporated three times with water at 40 ꢀC and
three times with MeOH leaving the reduced
hydroxyaldehyde. This residue was puri®ed by
column chromatography using as eluent 1:2
EtOAc±toluene to give the title compound (13)
l-galactopyranosyl
bromide
(14)
(670 mg,
1.63 mmol) was dissolved in a mixture of dry
CH2Cl2 (10 mL), dry CH3CN (10 mL) and dry
Et2O (10 mL) containing crushed 3 A molecular
sieves (1.0 g) and the soln was stirred over 20 min.
To the slurry was added dibenzyl phosphate
(873 mg, 3.14 mmol) and silver carbonate (865 mg,
3.14 mmol) and this mixture was stirred for 16 h
with exclusion of light. The reaction mixture was
then ®ltered, concentrated under reduced pressure
and the residue puri®ed by column chromato-
graphy using as eluent 1:2 EtOAc±toluene + 2%
triethylamine to give the title compound (17)
(839 mg, 89%) as a colourless syrup. [ꢁ]20d 67 (c
2.4 in CHCl3); 1H NMR (500 MHz, C6D6): ꢃ 7.45±
7.20 (m, 10 H, Ar-H), 5.97 (dd, 1 H, J2,3 10.0, J2,1
1
(368 mg, 76%) as a colourless oil (ꢁ : ꢀ 5:1). H
3
NMR (500 MHz, CDCl3) ꢁ-anomer: 5.38 (t, 1 H,
J1,OH 3.7, J1,2 3.2 Hz, H-1), 5.12 (m, 1 H, H-5), 5.02
(m, 2 H, H-2, H-3), 4.22 (t, 1 H, J4,5 5.1, J4,3
5.0 Hz, H-4), 3.00 (d, 1 H, JOH,1 3.7 Hz, OH), 2.09,
2.07, 2.06 (3Âs, 9 H, 3ÂOCOCH3), 1.29 (d, 3 H,
J6,5 6.5 Hz, H-6); 13C NMR (125.76 MHz, CDCl3):
ꢁ 100.57 (C-1), 83.65 (C-4), 82.14 (C-2), 77.78 (C-
3), 69.07 (C-5), 16.14 (C-6) ppm.; Anal. Calcd. for
C12H18O8 (290.3): C, 49.65; H, 6.25. Found: C,
49.57; H, 6.21.
7.6 Hz, H-2), 5.70 (t, 1 H, J1,2 7.6, J1,P 7.6 Hz, H-
1), 5.60 (dd, 1 H, J4,3 3.0, J4,5 1.0 Hz, H-4), 5.34
(dd, 1 H, J3,2 10.0, J3,4 3.0 Hz, H-3), 5.30±5.15 (m,
4 H, 2ÂCH2Ph), 4.24 (d, 2 H, J6,6 6.6 Hz, 2ÂH-6),
3.52 (dt, 1 H, J5,6 6.6, J5,4 1.0 Hz, H-5), 1.92, 1.90,
1.78, 1.76 (4Âs, 12 H, 4ÂOCOCH3); 13C NMR
(125.76 MHz, CDCl3): ꢃ 96.2 (C-1), 71.1 (C-5), 69.8
(C-3), 69.1, 69.0 (2ÂPhCH2), 68.1 (C-2), 66.1 (C-
4), 60.5 (C-6) ppm.
b-l-Galactopyranosylphosphate disodium salt
(20).ÐA soln of dibenzyl-(2,3,4,6-tetra-O-acetyl-
b-l-galactopyranosyl)phosphate (17) (950 mg,
1.64 mmol) in EtOH (10 mL) and 1 M NaHCO3
(2 mL) was treated with 10% Pd/C (0.2 g) and
stirred under an atmosphere of hydrogen for 24 h.
The reaction mixture was then ®ltered, con-
centrated under reduced pressure, diluted in
water (40 mL) and washed with CH2Cl2
(2Â40 mL). The aq layer was separated, cooled to
0 ꢀC and NaOH (2 M) was added until pH 12. The
soln was stirred at this pH for 4 h and was then
carefully neutralised with Dowex 50WX H+ resin,
®ltered and lyophilised. The residue was puri®ed by
Sephadex G10 chromatography using water as
eluent ar a rate of 10 mL/h. The appropriate frac-
tions were pooled and lyophilised to yield the title
2,3,4,6 - Tetra - O - acetyl - a - l - galactopyranosyl
bromide (14).Ð1,2,3,4,6-Penta-O-acetyl-b-l-galac-
topyranose (2) (750 mg, 1.92 mmol) was dissolved
in HBr (10 mL, 30% in glacial acetic acid) and
acetic anhydride (10 mL) and the soln stirred for
2 h. The reaction mixture was then poured onto
ice water (20 mL) and extracted with CH2Cl2
(2Â100 mL). The combined organic extract was
washed with sat aq NaHCO3 (3Â50 mL), brine
soln (50 mL), dried (Na2SO4) and concentrated
under reduced pressure. The resulting yellow oil
was puri®ed by column chromatography using as
1:2 EtOAc±toluene + 2% triethylamine to give the
title compound (14) (670 mg, 85%) as an oil. [ꢁ]20d
164 (c 2.4 in CHCl3); 1H NMR (500 MHz,
CDCl3): d 6.51 (d, 1 H, J1,2 3.9 Hz, H-1), 5.31 (dd,

H. Binch et al./Carbohydrate Research 306 (1998) 409±419
417
compound (20) (309 mg, 69%) as an amorphous
1
6b), 4.36 (dd, 1 H, J5,6a 7.1, J5,6b 5.5 Hz, H-5), 2.76
(m, 1 H, H-3e), 2.07 (ddd, 1 H, J3a,3e 14.1, J3a,2
12.0, J3a,4 2.8 Hz, H-3a) ppm.
lather. H NMR (400 MHz, D2O): ꢃ 4.84 (t, 1 H,
J1,2 7.6, 3J1,P 7.6 Hz, H-1), 3.90 (d, 1 H, J4,3 3.0 Hz,
H-4), 3.73±3.65 (m, 4 H, H-3, H-5, 2ÂH-6), 3.52
(dd, 1 H, J2,3 9.9, J2,1 7.6 Hz, H-2) ppm.
2,4,6-Tri-O-benzoyl-3-deoxy-a-l-xylo-hexopyr-
anosyl bromide (15).Ð1,2,4,6-Tetra-O-benzoyl-3-
3 - Deoxy - b - l - xylo - hexopyranosylphosphate
dilithium salt (21).ÐA soln of dibenzyl-(2,3,4,6-
tri-O-benzoyl-3-deoxy-b-l-xylo-hexopyranosyl)-
phosphate (18) (500 mg, 0.71 mmol) in EtOH
(10 mL) and 1 M NaHCO3 (2 mL) was treated with
5% Pd/C (0.2 g) and stirred under an atmosphere
of H2 for 1 h. The reaction mixture was then ®l-
tered, concentrated under reduced pressure, diluted
with CH2Cl2 (40 mL) and washed with water
(2Â40 mL). The organic layer was separated, con-
centrated under reduced pressure, dissolved in 2:1
water±MeOH and aq NaOH was added until pH
12. The reaction mixture was stirred for 3 h, then
neutralised with AcOH (1M) and this soln was
further diluted with water (200 mL) and absorbed
onto a resin ion exchange column Dowex 2Â8
(1.5Â20 cm, Cl ). The column was washed with
water (200 mL) then with a gradient eluent of LiCl
solution (0±0.8 M) at a ¯ow rate of 1.5 mL/min.
Fractions containing the desired product were
pooled and lyophilised and then the residue was
desalted using Sephadex G10 chromatography
(100Â1.6 cm) with a ¯ow rate of 1 mL/min using
water as eluent. The appropriate fractions were
pooled and lyophilised to yield the title compound
(21) (143 mg, 79%) as a colourless syrup. 1H NMR
deoxy-b-l-xylo-hexopyranose
(6)
(1.0 g,
1.72 mmol) was dissolved in HBr (5 mL, 30% in
glacial acetic acid) and Ac2O (5 mL) and the soln
stirred for 2 h. The reaction mixture was then
poured onto ice water (20 mL) and extracted with
CH2Cl2 (2Â100 mL). The combined organic
extract was washed with sat NaHCO3 (3Â50 mL),
brine soln (50 mL), dried (MgSO4) and con-
centrated under reduced pressure. The resulting oil
was puri®ed by column chromatography using as
1:2 EtOAc±toluene + 2% triethylamine to give the
title compound (15) (583 mg, 63%) as an oil. [ꢁ]20d
147.3 (c 1.8 in CHCl3); 1H NMR (500 MHz,
CDCl3): ꢃ 8.15±7.18 (m, 15 H, Ar-H), 6.93 (d, 1 H,
J1,2 2.7 Hz, H-1), 5.64 (d, 1 H, J4,3a 2.8 Hz, H-4),
5.35 (ddd, 1 H, J2,3a 11.8, J2,3e 4.2, J2,1 2.7 Hz, H-
2), 4.66 (dd, 1 H, J5,6a 7.1, J5,6b 5.7 Hz, H-5), 4.57
(dd, 1 H, J6a,6b 11.6, J6a,5 7.1 Hz, H-6a), 4.48 (dd, 1
H, J6b,6a 11.6, J6b,5 5.7 Hz, H-6b), 2.56 (ddd, 1 H,
J3a,3e 14.0, J3a,2 11.8, J3a,4 2.8 Hz, H-3a), 2.42 (m, 1
H, H-3e) ppm.; Anal. Calcd. for C27H23BrO7
(539.4): C, 60.12; H, 4.30; Br, 14.81. Found: C,
57.41; H, 4.11; Br, 14.53 (labile compound).
3
(500 MHz, D2O): ꢃ 4.89 (t, 1 H, J1,2 7.6, J1,P
Dibenzyl-(2,4,6-tri-O-benzoyl-3-deoxy-b-l-xylo-
hexopyranosyl)-phosphate (18).Ð2,4,6-Tri-O-ben-
zoyl-3-deoxy-ꢁ-l-xylo-hexopyranosyl bromide (15)
(583 mg, 1.08 mmol) was dissolved in a mixture of
dry CH2Cl2 (10 mL), dry CH3CN (10 mL) and dry
Et2O (10 mL) containing crushed 3 A molecular
sieves (1.0 g) and the soln was stirred over 20 min.
To this slurry was added dibenzyl phosphate
(585 mg, 2.10 mmol) and silver carbonate (580 mg,
2.10 mmol) and this mixture was stirred for 18 h
with exclusion of light. The reaction mixture was
then ®ltered, concentrated under reduced pressure
and the residue puri®ed by column chromato-
graphy using as eluent 1:2 EtOAc±toluene + 2%
triethylamine to give the title compound (18)
7.6 Hz, H-1), 4.03 (m, 1 H, H-4), 3.85±3.75 (m, 3
H, H-5, H-6a, H-6b), 3.73 (m, 1 H, H-2), 2.21
(ddd, 1 H, J3e,3a 13.8, J3e,4 4.5, J3e,2 3.8, Hz, H-3e),
1.79 (ddd, 1 H, J3a,3e 13.8, J3a,2 12.2, J3a,4 2.9 Hz
H-3a) ppm.
Dibenzyl-(2,4-di-O-benzoyl-3,6-dideoxy-b-l-xylo-
hexopyranosyl)-phosphate (19).ÐTo a soln of 1,2,4-
tri-O-benzoyl-3,6-dideoxy-b-l-xylo-hexopyranose
(10) (481 mg, 1.05 mmol) in dichloromethyl
methyl ether (2.5 mL) was added a catalytic
amount of zinc chloride (50 mg, 0.36 mmol), and
the reaction mixture was stirred for 2 h under an
atmosphere of argon. The reaction mixture was
quenched by adding toluene (5 mL) and then
concentrated under reduced pressure. The residue
was extracted with CH2Cl2, washed with 2 N
H2SO4, sat NaHCO3, water, dried (MgSO4) and
concentrated under reduced pressure. Chloride
(16) was used directly in the preparation of the
glycosyl phosphate (19) without any further pur-
i®cation and was dissolved in a mixture of dry
CH2Cl2 (10 mL), dry CH3CN (10 mL) and dry
1
(685 mg, 90%) as a colourless syrup. H NMR
(500 MHz, CDCl3): ꢃ 8.14±7.30 (m, 25 H, Ar-H),
3
5.67 (t, 1 H, J1,2 7.5, J1,P 7.5 Hz, H-1), 5.54 (d, 1
H, J4,3a 2.8 Hz, H-4), 5.45 (ddd, 1 H, J2,3a 12.0, J2,1
7.5, J2,3e 5.3 Hz, H-2), 5.07±4.81 (m, 4 H,
2ÂCH2Ph), 4.54 (dd, 1 H, J6a,6b 11.4, J6a,5 7.1 Hz,
H-6a), 4.46 (dd, 1 H, J6b,6a 11.4, J6b,5 5.5 Hz, H-

418
H. Binch et al./Carbohydrate Research 306 (1998) 409±419
Et2O (10 mL) containing crushed 3 A molecular
sieves (1.0 g) and stirred for 20 min. To this slurry
was added dibenzyl phosphate (584 mg,
2.10 mmol) and silver carbonate (580 mg,
2.10 mmol) and this mixture was stirred for 18 h
with exclusion of light. The reaction mixture was
then ®ltered, concentrated under reduced pressure
and the residue puri®ed by column chromato-
graphy using as eluent 1:2 EtOAc±toluene + 1%
triethylamine to give the title compound (19)
4.9 Hz, H-2), 3.41 (m, 1 H, H-4), 2.14 (ddd, 1 H,
J3e,3a 14.0, J3e,2 4.9 Hz, J3e,4 4.0 Hz, H-3e), 1.75
(ddd, 1 H, J3a,3e 14.0, J3a,2 12.0, J3a,4 3.0 Hz, H-3a),
1.23 (d, 3 H, J6,5 6.5 Hz, H-6) ppm.
1H NMR data are consistent with that reported
in the literature [4].
Guanosine-50-(b-l-galactopyranosyl)-diphosphate
dilithium salt (27).ÐA soln of b-l-galactopyranosyl-
phosphate disodium salt (20) (264 mg, 0.87 mmol)
in water (50 mL) was slowly adsorbed onto a resin
bed of Dowex 50W Â 8 (tributylammonium-form,
2.4Â28 cm) and slowly washed with water. Frac-
tions containing the desired compound were
pooled and lyophilised. Galactopyranosyl-phos-
phate bis-(tributylammonium) salt (23) and gua-
nosine-50-monophosphate morpholidate (26) were
combined in dry 1:1 pyridine±dry DMF (10 mL)
and evaporated under reduced pressure in order to
dry the compounds. This procedure was repeated
twice. The heterogeneous reaction was then stirred
for 8 days under an atmosphere of argon. Eva-
poration of the solvent gave a syrup, which was
diluted to 200 mL with water and passed through a
column of Dowex 1 Â 8 (200±400 mesh) resin (Cl ,
2.4Â28 cm). The column was washed with water
(200 mL) and then eluted with a linear gradient of
LiCl (0±0.8 M) using a ¯ow rate of 4 mL/min.
GDP-l-galactose eluted at a concentration of 0.17±
0.19 M LiCl. The relevant fractions were pooled,
concentrated to 6 mL and passed through a
Sephadex G10 column (100Â1.6 cm) using water as
eluent at a rate of 10 mL/h. Fractions containing
the product were pooled and lyophilised, a€ording
the title compound (27) (155 mg, 29%) as an
1
(356 mg, 55%) as a colourless syrup. H NMR
(500 MHz, CDCl3): ꢃ 8.25±7.11 (m, 20 H, Aryl-
3
H), 5.62 (dd, 1 H, J1,2 7.9, J1,P 7.0 Hz, H-1), 5.40
(ddd, 1 H, J2,3a 12.2, J2,1 7.9, J2,3e 5.5 Hz, H-2),
5.31 (dd, 1 H, J4,3e 3.5, J4,3a 3.1, J4,5 1.5 Hz, H-4),
5.13±4.81 (m, 4 H, 2ÂCH2Ph), 4.11 (dq, 1 H, J5,6
6.5 Hz, H-5), 2.69 (ddd, 1 H, J3e,3a 14.0, J3e,2 5.5,
J3e,4 3.5 Hz, H-3e), 2.08 (dddꢁdt, 1 H, J3a,3e 14.0,
J3a,2 12.2, J3a,4 3.1 H-3a), 1.26 (d, 3 H, J6,5 6.5 Hz,
H-6) ppm.
1
The H data found for (19) was consistent with
that reported for dibenzyl-(2,4-di-O-benzoyl-3,6-
dideoxy-b-l-xylo-hexopyranosyl)- phosphate [2,5].
3,6-Dideoxy-b-l-xylo-hexopyranosylphosphate
dilithium salt (22).ÐDibenzyl-(2,4-di-O-benzoyl-
3,6-dideoxy-b-l-xylo-hexopyranosyl)-phosphate
(19) (356 mg, 0.57 mmol) in EtOH (10 mL) and 1
M NaHCO3 (2 mL) was treated with 5% Pd/C
(0.2 g) and stirred under an atmosphere of H2 for
1 h. The reaction mixture was then ®ltered, con-
centrated under reduced pressure, diluted in
CH2Cl2 (40 mL) and washed with water
(2Â40 mL). The organic layer was separated, con-
centrated under reduced pressure, dissolved in 2:1
water±MeOH and aq NaOH was added until pH
12. The reaction mixture was stirred for 3 h, then
neutralised with acetic acid (1M) and this soln was
further diluted with water (200 mL) and absorbed
onto a resin ion exchange column Dowex 2Â8
(1.5Â20 cm, Cl ). The column was washed with
water (200 mL) then with a gradient eluent of LiCl
solution (0±0.8 M) at a ¯ow rate of 1.5 mL/min.
Fractions containing the desired product were
pooled and lyophilised and then the residue was
desalted using Sephadex G10 chromatography
(100Â1.6 cm) with a ¯ow rate of 1 mL/min using
water as eluent. The appropriate fractions were
pooled and lyophilised to yield the title compound
1
amorphous lather. H NMR (400 MHz, D2O): ꢃ
8.10 (s, 1 H, H-8), 5.97 (d, 1 H, J1 ,2 6.2 Hz, H-10),
0
0
4.95 (t, 1 H, J1 2 7.8, 3J1 ,P 7.8 Hz, H-100), 4.80 (m,
00 00
00
1 H, H-20), 4.55 (m, 1 H, H-30), 4.35 (m, 1 H, H-40),
4.22±4.18 (m, 2 H, 2ÂH-50), 4.01 (d, 1 H, J4 ,3
00 00
3.0 Hz, H-400), 3.89 (dd, 1 H, J3 ,2 10.6, J3 ,4
3.0 Hz, H-300), 3.84±3.73 (m, 3 H, H-500, 2ÂH-600),
00 00
00 00
3.70 (dd, 1H, J2 ,3 10.6, J2 ,1 7.8 Hz, H-200); 13C
NMR (125.76 MHz, D2O): ꢃ 159.93 (C-6), 154.85
(C-2), 152.72 (C-4), 138.73 (C-8), 117.13 (C-5), 99.36
00 00
00 00
(d, J1 ,P 6.3 Hz, C-100), 87.60 (C-10), 84.68 (d, J4 ,P
00
0
8.9 Hz, C-40), 76.70 (C-400), 74.49 (C-20), 73.15 (C-
300), 72.12 (d, J2 ,P 7.6 Hz, C-200), 71.33 (C-30), 69.48
00
(C-500), 66.17 (d, J5 ,P 5.6 Hz, C-50), 62.05 (C-600)
0
1
(22) (91 mg, 65%) as an amorphous lather. H
ppm.
NMR (500 MHz, D2O): ꢀ 4.80 (t, 1 H, J1,2 7.5,
3J1,P 7.5 Hz, H-1), 3.81 (dq, 1H, J5,6 6.5, J5,4 1 Hz,
H-5), 3.63 (ddd, 1 H, J2,3a 12.0, J2,1 7.5, J2,3e
Guanosine-50-(300-deoxy-b-l-xylo-hexopyranosyl)-
diphosphate dilithium salt (28).Ð3-Deoxy-ꢀ-l-xylo-
hexopyranosylphosphate dilithium salt (21)

H. Binch et al./Carbohydrate Research 306 (1998) 409±419
(143 mg, 0.49 mmol) was treated as described in the References
419
synthesis of (27). Ion-exchange chromatography
and subsequent desalting on Sephadex G 10 col-
umn a€orded the title compound (28) (80 mg,
27%) as an amorphous lather. 1H NMR
[1] T.W. Rademacher, R.B. Parekh, and R.A. Dwek,
Annu. Rev. Biochem., 1988, 57, 785±838; R.U.
Lemieux, Chem. Soc. Rev., 1989, 18, 347±374.
[2] (a) T.K. Lindhorst and J. Thiem, Carbohydr. Res.,
1991, 209, 119±129; (b) B. Leon, T.K. Lindhorst,
A. Rieks-Everdiking and W. Kla€ke, Synthesis,
1994, 689±691.
(500 MHz, D2O): ꢃ 8.13 (s, 1 H, H-8), 5.97 (d, 1 H,
J1 ,2 6.1 Hz, H-10), 5.01 (t, 1 H, J1 ,2 7.9, J1 ,P
7.9 Hz, H-100), 4.83 (m, 1 H, H-20), 4.57 (m, 1 H, H-
30), 4.40 (m, 1 H, H-40), 4.25 (m, 2 H, 2ÂH-50), 4.20
(m, 1 H, H-400), 3.85±3.76 (m, 3 H, H-500, H-6a00, H-
3
0
0
00 00
00 00
[3] R. Stiller and J. Thiem, Liebigs Ann. Chem., 1992,
467±471; V. Ginsburg, J. Biol. Chem., 1960, 235,
2196±2201; V. Ginsburg, J. Biol. Chem., 1961, 236,
2389±2393; H. Ishihara, D.J. Massaro, and E.C.
Heath, J. Biol. Chem., 1968, 243, 1103±1109; I.
Jabbal and H. Schachter, J. Biol. Chem., 1971, 246,
5154±5161; H. Schachter, H. Ishihara, and E.C.
Heath, Methods Enzymol., 1972, 28, 285±287.
[4] (a) H.A. Nunez, J.V. OyConnor, P.R. Rosevear,
and R. Barker, Can. J. Chem., 1981,59, 2086±2095;
(b) P. Westerduin, G.H. Veeneman, J.E. Marugg,
G.A. van der Mare, and J.H. van Boom, Tetra-
hedron Lett., 1986, 27, 1211±1214; (c) R.R.
Schmidt, B. Wegmann, and K.H. Jung, Liebigs
Ann. Chem., 1991, 121±124; (d) U.B. Gokhale, O.
Hindsgaul, and M.M. Palcic, Can. J. Chem., 1990,
68, 1063±1071; (e) K. Adelhorst and G.M. White-
sides, Carbohydr. Res., 1993, 242, 69±76; (f) B.M.
Heskamp, H.J.G. Broxterman, G.A. van der
Marel, and J.H. van Boom, J. Carbohydr. Chem.,
1996, 15, 611±622.
6b00), 3.73 (ddd, 1 H, J2 ,3a 11.5, J2 ,1 7.9, J2 ,3e
00
00
00 00
00
00
3.8 Hz, H-200), 2.22 (ddd, 1 H, J3e ,3a 13.5, J3e ,4
00
00
00 00
4.7, J3e ,2 3.8 Hz, H-3e00), 1.77 (ddd, 1 H, J3a ,3e
00 00
00
00
13.5, J3a ,2 11.5, J3a ,4 3.4 Hz H-3a00); 13C NMR
(125,76 MHz, D2O): ꢃ 159.84 (C-6), 154.84 (C-2),
00 00
00 00
152.58 (C-4), 138.37 (C-8), 101.09 (d, C-100, J1 ,P
00
6.3 Hz), 87.53 (C-10), 84.55 (d, C-40, J4 ,P 8.8 Hz),
0
79.78 (C-400), 74.37 (C-20), 71.19 (C-30), 66.68 (d, C-
200, J2 ,P 7.6 Hz), 66.36 (C-500), 66.07 (d, C-50, J5 ,P
6.3 Hz), 62.15 (C-600), 37.13 (C-300) ppm.
00
0
Guanosine-50 -(300,600 -dideoxy-b-l-xylo-hexopyr-
anosyl)-diphosphat dilithium salt (29).Ð3,6-
Dideoxy-ꢀ-l-xylo-hexopyranosylphosphate
dili-
thium salt (22) (91 mg, 0.43 mmol) was treated as
described in the synthesis of (27). Ion-exchange
chromatography and desalting on Sephadex G 10
a€orded the title compound (29) (47 mg, 19%) as
1
an amorphous lather. H NMR (500 MHz, D2O):
0
[5] (a) T. K. Lindhorst and J. Thiem, Liebigs Ann.
Chem., 1990, 1237±1241; (b) D.Horton, T.M.
Cheung and W. Weckerle, Methods Carbohydr.
Chem., 1980, 8, 201±205.
[6] H.L. Frush and H.S. Isbell, Methods Carbohydr.
Chem., 1962, 1, 127±130.
0
ꢀ 8.11 (s, 1 H, H-8), 5.96 (d, 1 H, J1 ,2 6.0 Hz, H-
10), 5.03 (t, 1 H, J1 ,2 8.0, J1 ,P 8.0 Hz, H-100),
4.79 (m, 1 H, H-20), 4.53 (m, 1 H, H-30), 4.37 (m, 1
H, H-40), 4.21 (m, 2 H, 2ÂH-50), 3.82±3.65 (m, 3 H,
3
00 00
00 00
H-200, H-400, H-500), 2.19 (ddd, 1 H, J3e ,3a 13.5,
00
00
J3e ,2 5.0, J3e ,4 4.1 Hz, H-3e00), 1.69 (ddd, 1 H,
00 00
00 00
[7] K. Bock, I. Lundt, and C. Pedersen, Carbohydr.
Res., 1981, 90, 7±16.
J3a ,3e 13.5, J3a ,2 12.0, J3a ,4 3.1 Hz H-3a00), 1.19
00
00
00 00
00 00
(d, 3H, J6 ,5 6.4 Hz, H-600) ppm.
The analytical data found for (29) are consistent
with those reported [4].
[8] O.J. Varela, A. Fernandez-Cirelli, and R.M. De
Lederkremer, Carbohydr. Res., 1980, 79, 219±224.
[9] K. Bock, I. Lundt, and C. Pedersen, Acta Chem.
Scand., 1981, 35B, 155±162.
00 00
[10] (a) P. Kohn, L.M. Lerner, A. Chan, S.D. Ginoc-
chio, and C.A. Zitrin, Carbohydr. Res., 1968, 7, 21±
26; (b) C. Pedersen and H.S. Jensen, Acta Chem.
Scand., 1994, 48, 222±227.
Acknowledgements
[11] C. Pedersen, K. Bock, and I. Lundt, Pure Appl.
Chem., 1978, 50, 1385±1400.
[12] K. Bock, I. Lundt, and C. Pedersen, Carbohydr.
Res., 1979, 68, 313±319.
[13] V. Wittmann and C.H. Wong, J. Org. Chem., 1997,
62, 2144±2147.
Support of this work by the Deutsche For-
schungsgemeinschaft, the Fonds der Chemischen
Industrie and the Commission of the European
Communities, GD XII (Programme CARENET) is
gratefully acknowledged.