Synthesis of 2-fluoro and 4-fluoro galactopyranosyl phosphonate analogues of UDP-Gal

Article abstract of DOI:10.1016/j.carres.2012.07.015

Two novel nonisosteric UDP-Gal analogues, (2-deoxy-2-fluoro- and 4-deoxy-4-fluoro-α-D-galactopyranosyl) phosphonoyl phosphates, were synthesized by optimized multistep procedures starting from 3,4,6-tri-O-benzyl- D-galactal and allyl 2,3,6-tri-O-benzyl-α-D-glucopyranoside, respectively. The key steps were a Michaelis-Arbuzov reaction of respective deoxy-fluoro-D-galactopyranosyl acetate with triethyl phosphite followed by a Moffatt-Khorana coupling reaction with UMP-morpholidate. The structure of all new compounds was confirmed by NMR and mass spectroscopies.

Full text of DOI:10.1016/j.carres.2012.07.015

Carbohydrate Research 360 (2012) 31–39
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of 2-fluoro and 4-fluoro galactopyranosyl phosphonate analogues
of UDP-Gal
⇑
ˇ
Irekhjargal Jambal, Karel Kefurt, Martina Hlavácková, Jitka Moravcová
Department of Chemistry of Natural Compounds, Institute of Chemical Technology Prague, Technická 5, Prague 166 28, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 April 2012
Received in revised form 13 July 2012
Accepted 17 July 2012
Available online 2 August 2012
Two novel nonisosteric UDP-Gal analogues, (2-deoxy-2-fluoro- and 4-deoxy-4-fluoro-a-D-galactopyran-
osyl) phosphonoyl phosphates, were synthesized by optimized multistep procedures starting from 3,4,6-
tri-O-benzyl-D-galactal and allyl 2,3,6-tri-O-benzyl- -D-glucopyranoside, respectively. The key steps
a
were a Michaelis-Arbuzov reaction of respective deoxy-fluoro-D-galactopyranosyl acetate with triethyl
phosphite followed by a Moffatt-Khorana coupling reaction with UMP-morpholidate. The structure of
all new compounds was confirmed by NMR and mass spectroscopies..
Keywords:
UDP-Gal
Ó 2012 Elsevier Ltd. All rights reserved.
Deoxyfluorogalactose
Phosphonoylphosphate
1. Introduction
nucleophilic substitutions and the Michaelis–Arbuzov reaction is
generally a suitable tool for the creation of hexopyranosyl phos-
Glycosyltransferases are essential enzymes for the biosynthesis
of cell–surface complex carbohydrates.1 The galactosyltransferase
phonate.14 According to preliminary experiments, the fluorination
was chosen as the first modification and benzyl protecting groups
were preferred over acetyls (Scheme 1).
(GalT) family transfers, in the presence of a metal ion,
pyranosyl residue from uridine-diphosphate- -galactose (UDP-
Gal) to specific hydroxyl groups of a particular acceptor via b1–4,
b1–3, 1–3 and
1–4 linkages.2 Derivatives and mimics of UDP-
D-galacto-
a-
D
The key intermediates, 1-O-acetyl-2,3,6-tri-O-benzyl-4-deoxy-
4-fluoro-D-galactopyranose (7) and 1-O-acetyl-3,4,6-tri-O-benzyl-
a
a
2-deoxy-2-fluoro-D-galactopyranose (8) (Scheme 1) could yield in
Gal have been therefore synthesized very frequently as potential
GalT inhibitors for drug discovery.3,4 The replacement of b-phos-
phate in UDP-Gal with a phosphonate group seems to be a general
strategy for the generation of hydrolytically stable analogues.5–8
the Michaelis–Arbuzov reaction with triethyl phosphite and Lewis
acid the corresponding anomeric diethyl phosphonates 5 and 6,
respectively. After separation and deesterification, the phosphonic
acids 3 and 4 will be coupled with uridine 50-phosphate (UMP) to
the target compounds 1 and 2.
Furthermore, the substitution of a
D-galactopyranose hydroxyl
group with fluorine especially at position 2 can imply dramatic
changes in binding ability.9–12 UDP-Gal2F was found to be a revers-
2.1. Synthesis of acetates 7 and 8
ible inhibitor of a-1,3-GalT, giving Ki 245 l
M.13
As a further study on the synthesis of new UDP-Gal analogues
combining the structural features of a hydrolytically stable C1–P
bond and an activation effect of fluorine attached at the position
2 or 4 of the galactopyranosyl unit, we report herein the synthesis
Methyl 2,3,6-tri-O-benzyl-a-D
-glucopyranoside15 seemed to be
initially a good starting compound for the synthesis of acetate 7
(Scheme 2). However, the final hydrolysis or acetolysis of methyl
galactopyranoside 9 yielded the respective pyranose 12 or 1-ace-
tate 7 only with very low yields. The loss of valuable intermediate
9 urged us to search for a more suitable starting compound. Allyl
of uridine 50-(4-deoxy-4-fluoro-
a-D-galactopyranosyl)phospho-
noyl phosphate (1) and uridine 50-(2-deoxy-2-fluoro-
pyranosyl)phosphonoyl phosphate (2).
a-D-galacto-
2,3,6-tri-O-benzyl-
allyl 2,3,6-tri-O-benzyl-4-O-triflyl-
lated and characterized), which was transformed into allyl
a-
D
-glucopyranoside16 was esterified providing
-glucopyranoside (not iso-
a
-D
2. Results and discussion
4-deoxy-4-fluoro-a-D-galactopyranoside 10 by treatment with
TASF.17 Allyl glycoside 10 was then treated with a catalytic amount
The synthesizing strategy was dictated by a modification that
was accomplished first. Fluorine can be introduced by numerous
of (Ph3P)3RhCl in the presence of DABO18 to give prop-1-en-1-yl
2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-a-D-galactopyranoside (11)
as a mixture of (E)- and (Z)-isomers that was cleaved without
seperation to produce the corresponding galactopyranose 12 with
⇑
Corresponding author. Tel.: +420 220 444 283; fax: +420 220 444 422.
E-mail address: Jitka.Moravcova@vscht.cz (J. Moravcová).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.07.015

32
I. Jambal et al. / Carbohydrate Research 360 (2012) 31–39
R2
HO
OH
O
O
R2
OH
O
NH
O
P
R1
O
HO
N
O
R1
O
P
O
O
O
P
OH
[BH]+O-
O-[BH]+
OH
OH OH
1 R1 = OH, R2 = F
2 R1 = F, R2 = OH
3 R1 = OH, R2 = F
4 R1 = F, R2 = OH
R2
BnO
OBn
O
R2
BnO
OBn
O
R1
R1
O
P
OC2H5
OAc
OC2H5
7 R1 = OBn, R2 = F
8 R1 = F, R2 = OBn
5 R1 = OBn, R2 = F
6 R1 = F, R2 = OBn
Scheme 1. Retrosynthesis.
F
OBn
F
OBn
O
F
OBn
O
F
OBn
O
O
a
c
b
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
OH
OR
O
OAc
9 R = CH3
10 R = All
7
12
11
d
e
OBn
BnO
OBn
BnO
OBn
O
OBn
O
OBn
BnO
OBn
O
g
f
F
F
OAc
OH
8
Scheme 2. Reagents and conditions: (a) EtOH–Tol–H2O (7:3:1), (Ph3P)3RhCl, DABO, 92%; (b) acetone–1 M HCl (10:1), 86%; (c) Py, Ac2O, 91%; (d) 80% AcOH, 1 M HCl, 36%; (e)
AcOH–Ac2O (3:1), CH2Cl2, H2SO4, 15%; (f) CH3NO2, Selectfluor™, H2O; (g) Py, Ac2O, 54%.
an 86% two-step overall yield. In contrast, direct deprotection of al-
lyl glycoside 10 with palladium chloride in diluted acetic acid19
gave only 44% aldose 12. Sequential acetylation of 12 provided ace-
tate 7 with a 91% yield.
Contrary to the 4-deoxy-4-fluorohexose 7, the synthesis of its 2-
deoxy-2-fluoro isomer 8 has been a very active area for many
accomplished by the Michaelis–Arbuzov reaction; the mechanism
of which has been proposed.21–23
Treatment of the anomeric acetates 7 (a/b = 1.4) with triethyl
phosphite and TMSOTf in dichloromethane primarily at 0 °C, later
at rt gave diethyl phosphonates 5 and 13 with a very good yield
of 77%, that is comparable with the 80% described for diethyl
years. Commercially available 3,4,6-tri-O-benzyl-D-lyxo-hex-1-eni-
(2,3,4,6-tetra-O-benzyl-a-D
-galactopyranosyl)phosphonates14
tol (3,4,6-tri-O-benzyl-
D
-galactal) was subjected to the treatment
(Scheme 3).
of Selectfluor™ in a slightly modified protocol.20 Instead of N,N-
dimethylformamide, nitromethane was used as a solvent due to
its greater evaporation rate. 3,4,6-Tri-O-benzyl-2-deoxy-2-fluoro-
Diethyl 4-deoxy-4-fluoro-
a-D-galactopyranosyl phosphonate 5
and its b- -isomer 13 were easily separated by column chromatog-
D
raphy. Both phosphonates 5 and 13 showed in NMR spectra signals
characteristic for the presence of fluorine on C-4 and phosphorus
on C-1. The comparison of the chemical shifts and coupling con-
stants JC,P of the C-1 signals is highly valuable for the determination
of the anomeric configuration in 5 and 13. The coupling constant
D-galactose was not isolated in a pure state but the crude reaction
mixture was immediately acetylated and the target acetate 8 was
obtained with a 54% overall yield, as a mixture of the
in the ratio of ca 1.2 (Scheme 2).
a/b anomers
JC1,P for
a-phosphonate 5 was lower than that for b-phosphonate
2.2. Preparation of C1-phosphonates by the Michaelis–Arbuzov
reaction
13 in accord with the data published for other phosphonates.14,24
This assignment corresponds also with the generally accepted rule
that the signal of carbon C-1 of pyranose with
appears at a higher field than the C-1 signal of the corresponding
b-
-anomer.25
a-D-configuration
A direct nucleophilic substitution of the C-1 acetoxy group of
acetates 7 and 8 with a dialkyl phosphonate group could be
D

I. Jambal et al. / Carbohydrate Research 360 (2012) 31–39
33
R2
BnO
OBn
O
R2
BnO
OBn
O
R2
BnO
OBn
O
O
P
a
OC2H5
R1
R1
R1
OC2H5
O
P
OC2H5
OAc
OC2H5
5 R1 = OBn, R2 = F (53%)
6 R1 = F, R2 = OBn (20%)
7 R1 = OBn, R2 = F
8 R1 = F, R2 = OBn
13 R1 = OBn, R2 = F (24%)
14 R1 = F, R2 = OBn (34%)
b
e
R2
HO
OH
O
R2
OH
F
OBn
F
OBn
O
O
O
c
b
HO
BnO
BnO
R1
O
BnO
2 Py
R1
O
BnO
P
OH
P
OC2H5
O
P
OH
O
P
OH
OC2H5
OH
OH
OH
3 R1 = OH, R2 = F (86%)
4 R1 = F, R2 = OH (98%)
15 (59%)
18 R1 = OH, R2 = F (99%)
20 R1 = F, R2 = OH (79%)
16 (38%)
d
c
R2
HO
OH
O
F
BnO
OBn
O
O
N
O
N
R2
HO
OH
O
NH
O
NH
d
O
P
O
R1
O
BnO
O
O
P
O
O
P
O
P
O
2 Py
OH
R1
O
O
O
[BH]+O-
O-[BH]+
[BH]+O-
O-[BH]+
P
OH
OH OH
OH OH
17 B = B1 (41%)
19 R1 = OH, R2 = F (73%)
21 R1 = F, R2 = OH (96%)
1a R1 = OH, R2 = F, B = B1 (70%)
2a R1 = F, R2 = OH, B = B1 (75%)
f
f
1b R1 = OH, R2 = F, B = NH3 (31%)
2b R1 = F, R2 = OH, B = NH3 (63%)
O
N
B1
=
HN
N
Scheme 3. Reagents and conditions: (a) CH2Cl2, (C2H5O)3P, TMSOTf; (b) CH2Cl2, BrSiMe3, acetone–H2O (9:1) (2 steps); (c) Py; (d) Py, UMP-morpholidate, 1H-tetrazole, Hünigs
base; (e) H2, MeOH, 20% Pd(OH)2/C; (f) Dowex 1-X8, HCOOꢀ, elution with 0–1 M NH4HCO3.
Preparation of the isomeric 2-deoxy-2-fluoro-
osyl phosphonate 6 and 2-deoxy-2-fluoro-b-
phosphonate 14 proceeded starting from acetate 8, analogous to
the procedure for phosphonates and 13 described above
(Scheme 3). Unfortunately, we observed a complete loss of stere-
oselectivity resulting in a ratio of 6/14 = 3:5. In addition, the pre-
parative yield was low, reaching only 54% due to the presence of
several by-products. Our results confirm the previous finding that
a ratio of products arising from the Michaelis–Arbuzov reaction
does not depend substantially on the anomeric configuration of
incoming glycosyl acetate.26 For comparison, an anomeric mixture
a
-
D
-galactopyran-
phosphonic acid with an activated form of UMP following the
Moffatt–Wong protocol.27 Deesterification of the diethyl phospho-
nate 5 by treatment with bromotrimethylsilane produced phos-
phonic acid 15 (Scheme 3). Phosphonic acid 15 was then
transformed into pyridinium salt 16 and its coupling with UMP-
morpholidate was finished after 8 days. The phosphonophosphate
17 was isolated as a bis(4-morpholine-N,N0-dicyclohexyl carboxi-
midamide) salt. Subsequently, the salt 17 was debenzylated by cat-
alytic hydrogenation but the deprotection was accompanied with
the partial reduction of the uracil double bond. Because of that,
the deprotected phosphonic acids 3 and 4 had to be used in the
coupling reaction.
D
-galactopyranosyl
5
of 1-O-acetyl-2,3,4,6-tetra-O-benzyl-
gave the corresponding diethyl phosphonates in the ratio
b = 1.5.14 A ratio varying from 20: 1 to 10: 1 in favour of the 1,2-
cis-configurated dimethyl glycosyl phosphonates (i.e., -gluco,
-galacto, or b- -manno), was established for the respective pro-
tected 1-O-acetyl hexopyranoses.24
D-galactopyranose (a/b = 2)
a/
Thus, diethyl phosphonate 5 was hydrogenated on Pd(OH)2/C in
MeOH and produced 4-deoxy-4-fluoro-a-D-galactopyranosyl phos-
phonate 18 (Scheme 3). In the next step ethyl ester groups in 18
were cleaved with trimethylsilyl bromide giving phosphonic acid
a
-D
a-
D
D
3. Finally, bis(pyridinium) (4-deoxy-4-fluoro-a-D-galactopyrano-
syl)phosphonate (19) was obtained after repeated co-evaporation
of 3 with pyridine. Coupling reaction of 19 with UMP-morpholi-
date under activation by 1H-tetrazole was finished after 8 days.
After repeated LC purification on RP-18 phase, only impure
4-morpholine-N,N0-dicyclohexyl carboximidamidium salt 1a
2.3. Preparation of target phosphonoyl phosphates 1 and 2
The phosphono-phosphate bond in 1 and 2 could be created by
the reaction of ammonium or pyridinium salt of the respective

34
I. Jambal et al. / Carbohydrate Research 360 (2012) 31–39
contaminated with UMP-morpholidate was obtained. In 1H NMR
spectra there were characteristic signals of protons on the double
Unless otherwise stated, solvents were evaporated at 40 °C and
2 kPa and compounds were dried at 60 °C and 2 kPa. Reactions
were monitored by thin-layer chromatography (TLC) on the silica
bond of uracil, doublet of the
D-ribofuranose H-1 proton, signal of
H-4 of 4-deoxy-4-fluoro- -galactose and dominant signals belong-
D
gel plates with Merck Stahl 10–40 lm. Compound traces on the
ing to the protons of the base moiety. The further purification was
ineffective therefore the dicyclohexyl carboximidamidium salt 1a
was transformed to ammonium salt 1b using ion-exchange resin
Dowex 1-X8 in the formiate form and ammonium bicarbonate
solution as eluent in a concentration gradient.28 In the first
fractions eluted with water only, 4-morpholine-N,N0-dicyclohexyl
carboximidamidium formiate was identified. Diammonium uri-
TLC plates were visualized by exposure or with an aqueous solu-
tion of Ce(SO4)2 in 10% H2SO4 followed by charring, or with UV
radiation at a wavelength of 254 nm.
3.2. Synthesis of acetates 7 and 8
3.2.1. Methyl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-a-D-
din-50-[(4-deoxy-4-fluoro-
a
-D-galactopyranosyl)phosphonyl]phos-
galactopyranoside (9)
phate (1b) was then obtained with a 31% yield, followed by the
Methyl 2,3,6-tri-O-benzyl-a-D
-glucopyranoside15 (2.22 g, 4.78
spare UMP-morpholidate.
mmol) in a solution of pyridine (2.4 mL) and CH2Cl2 (30 mL) was
converted into the 4-O-triflyl derivative by treatment with triflic
anhydride (1 mL, 5.9 mmol + 2 mL CH2Cl2) at ꢀ10 °C. After 1 h
the reaction was judged by TLC (3:1, petroleum ether–EtOAc) to
be complete. The reaction mixture was then diluted with CH2Cl2
(60 mL), washed with aqueous HCl (2 M, 30 mL), satd NaHCO3
(30 mL), H2O (30 mL), dried and concentrated. To a solution of
raw 4-O-triflyl derivative (2.72 g, 96%) in cold CH2Cl2 (60 mL), TASF
(4 g, 14.52 mmol) was added. The solution was heated under reflux
(60 °C) and the reaction was judged to be complete by TLC after
20 min. Product was obtained in the usual manner and purified
by flash chromatography (3:1, petroleum ether–EtOAc,) to give
The synthesisizing route for the preparation of the analogue 2
followed the same strategy already developed for 1 (Scheme 3).
Benzyl groups in diethyl phosphonate 6 were removed by catalytic
hydrogenation followed by deesterification of 20 and transforma-
tion of phosphonic acid 4 into bis(pyridinium) salt 21. The coupling
reaction of the bis(pyridinium) phosphonate 21 with UMP-mor-
pholidate produced 4-morpholine-N,N0-dicyclohexyl carboximida-
mide salt 2a. The diammonium salt 2b was then prepared with a
63% yield by passing salt 2a through an ion-exchange resin.
In summary, we have synthesized two novel hydrolytically sta-
ble UDP-Gal mimics having fluorine atom at the sensitive positions
of the galactopyranosyl unit. Biological evaluation is currently un-
der investigation and the results will be reported in due course.
syrup 9 (1.68 g, 75%), ½a D20
ꢂ
+63 (c 1.1, MeOH). 1H NMR (500 MHz,
CDCl3): d 7.43–7.29 (m, 15H, aromatic protons), 4.86 (br d, 1H,
2J4,F = 49.8, H-4), 4.84 (d, 1H, J = 12.1, PhCH), 4.83 (d, 1H, J = 12,
PhCH), 4.77 (d, 1H, J = 12, PhCH), 4.70 (d, 1H, J = 12.1, PhCH), 4.67
(d, 1H, J1,2 = 2.9, H-1), 4.60 (d, 1H, J = 11.8, PhCH), 4.56 (d, 1H,
J = 11.8, PhCH), 3.95–3.86 (m, 3H, H-2, H-3, H-5), 3.69 (dd, 1H,
J6a,5 = 7.2, J6a,6b = 9.5, H-6a), 3.62 (dd, 1H, J6b,5 = 6.5, J6b,6a = 9.5, H-
60), 3.40 (s, 3H, OCH3); NMR (125 MHz, CDCl3) d: 138.26, 138.14,
137.79 (C-quart.), 128.4–127.57 (C-arom.), 98.71 (C-1), 87.55 (d,
3. Experimental
3.1. General methods
Melting points were determined with a Kofler apparatus, and
are not corrected. Optical rotations were measured with a Autopol
VI (Rudolph) digital polarimeter in appropriate solvents, at a tem-
perature of 20 °C and the 589 nm sodium line, in 1 dm cuvettes and
2
1JC,F = 182.5, C-4), 75.94 (d, JC-3,F = 17.6, C-3), 75.7 (C-2), 73.84,
2
73.58, 72.74 (3 ꢁ OCH2Ph), 68.01 (d, JC-6,F = 6.8, C-6), 67.92 (d, JC-
are given at 10ꢀ1 deg cm2 gꢀ1. Silica gel (100–160
lm, Merck) was
5,F = 19.5, C-5), 55.52 (–OCH3); 19F NMR (470 MHz) d: ꢀ219.1
(ddd, JF,H-4 = 50.3, JF,H-3 = JF,H-5 = 30.4, F-4); ESI-MS, m/z: calcd for
used for column chromatography, C-18 silica gel (200–400 mesh,
Sigma Aldrich) for chromatography on reversed phase.
C
C
28H31FNaO5 [9+Na]+: 489.20, found: 489.24. Anal. Calcd for
28H31FO5: C, 72.08; H, 6.70. Found: C, 72.11; H, 6.56.
NMR spectra were recorded with a Bruker DRX 500 Avance
spectrometer operating at 500.1 MHz for 1H, 125.8 MHz for 13C,
470.4 MHz for 19F and 202.4 MHz for 31P, or with a Varian Gemini
300HC spectrometer operating at 299.9 MHz for 1H, 282.2 MHz for
19F and 121.4 MHz for 31P, in the solvents CDCl3, CD3OD, or D2O.
Chemical shifts were stated in ppm and referenced to tetramethyl-
silane, trichlorofluoromethane or phosphoric acid, respectively
(d = 0 ppm). Coupling constants (J) are reported in Hertz (Hz).
Assignment of the signals was accomplished by means of COSY,
HETCOR, APT and HMQC experiments. In the description of spectra
3.2.2. Allyl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-a-D-
galactopyranoside (10)
A
solution of allyl 2,3,6-tri-O-benzyl-a-D
-glucopyranoside16
(475 mg, 0.97 mmol) in pyridine (1.2 mL) and CH2Cl2 (10.7 mL)
was converted into the 4-O-triflyl derivative by treatment with
Tf2O (0.53 mL, 3.1 mmol + 1.06 ml CH2Cl2) at ꢀ10 °C. After 1 h,
the reaction was completed as checked by TLC (3:1, n-hexane–
EtOAc). The reaction mixture was then diluted with CH2Cl2
(13 mL), the solution stepwise washed with 2 m aq HCl (7 mL),
satd NaHCO3 (7 mL), H2O (7 mL) and dried (MgSO4). After filtration
and evaporation of the solvent, the residual syrup triflate (584 mg,
yield 97%) was diluted with cold CH2Cl2 (15 mL) and TASF (878 mg,
3.18 mmol) was added. The solution was heated at 60 °C and the
reaction was judged to be complete by TLC (3:1, n-hexane–EtOAc)
after 20 min and then concentrated. Purification by column chro-
matography (3:1, n-hexane–EtOAc) gave 10 as a syrup (436 mg,
of the potential inhibitors, H or C nuclei of the D-ribose residue are
apostrophized, nuclei on the uracil moiety are marked in brackets
as (U); the nuclei of the deoxyfluorogalactose are presented with-
out special marking.
Mass spectra were measured with a Q-Tof Micro (Waters, USA)
or LTQ Velos Orbitrap (Thermo Fisher Scientific, UK) instruments
equipped with LockSpray in ES+ and ESꢀ modes with the mobile
phase of methanol and a flow rate of 100 l .
L minꢀ1
91%), ½a 2D0
ꢂ
+37 (c 1.4, CHCl3). 1H NMR (500 MHz, CDCl3): d 7.40–
HPLC-MS system consisted of a Hewlett-Packard HP/Agilent
Technologies (USA) 1100 HPLC instrument which was coupled on
line to HP mass selective single quadrupole detector (model
G1946 A) and controlled by ChemStation software (revision
7.26 (m, 15H, aromatic protons), 5.91 (m, 1H, –CH@), 5.30 (m,
1H, @CH2), 5.20 (m, 1H, @CH2), 4.86 (dd, 1H, JF,4 = 49.6, H-4), 4.85
(d, 1H, overlap., H-1), 4.83 (d, 1H, J = 11.5, PhCH), 4.82 (d, 1H,
J = 10.8, PhCH), 4.80 (d, 1H, J = 10.2, PhCH), 4.75 (d, 1H, J = 10.8,
PhCH), 4.66 (d, 1H, J = 12, PhCH), 4.55 (d, 1H, J = 11.6, PhCH), 4.15
(m, 1H, OCH2), 4.02 (m, 1H, OCH2), 4.00–3.88 (m, 3H, H-2, H-3,
H-5), 3.67 (dd, 1H, J = 9.4, 7.2, H-6a), 3.59 (dd, 1H, J = 9.5, 6.5, H-
B.02.01). Column RP C18 (30 ꢁ 2.1 mm) Zorbax SB-C18 (3.5
lm
particle size) was used with mobile phases of 50% methanol (A)
and 100% methanol (B), each containing 5 mmol ammonium form-
iate and a flow rate 0.3 mL minꢀ1. Capillary voltage for electrospray
ionization was set at 3.5 kV.
6b); 13C NMR (125 MHz, CDCl3):
d 138.35, 138.23, 137.83

I. Jambal et al. / Carbohydrate Research 360 (2012) 31–39
35
1
(C-quart.), 133.66 (–CH@), 128.41–127.64 (C-arom.), 118.17
(CH2@), 96.28 (C-1), 87.54 (d, JC,F = 182.5, C-4), 76.14 (d,
(C-arom.), 97.36 (C-1b), 91.84 (C-1
a
), 87.17 (d, JC,F = 183.2, C-
), 86.74 (d, JC,F = 183.0, C-4b), 75.68 (C-3 ), 73.83 (PhCH2 ),
75.32 (PhCH2b), 73.72 (PhCH2b), 73.59 (PhCH2 ), 72.37 (PhCH2
PhCH2b), 68.19 (C-5 ), 68.01 (C-6
), 67.9 (C-6 b); 19F NMR
(470 MHz, CDCl3) d:-219.4 (ddd, JF,4 = 50.5, JF,3 = JF,5 = 30.5, F-4 );
1
1
4a
a
a
a
a/
2J = 17.6, C-3), 75.76 (C-2), 73.64, 73.57 and 72.77 (3 ꢁ PhCH2),
68.50 (–OCH2–), 68.11 (d, 2J = 17.8, C-5), 67.96 (d, JC-6,F = 5.5, C-
6); 19F NMR (470 MHz, CDCl3) d: ꢀ218.04 (ddd, JF,3 = JF,5 = 29.9,
JF,4 = 49.6); ESI-MS, m/z: calcd for C30H33FNaO5 [10+Na]+: 515.22,
found: 515.30. Anal. Calcd for C30H33FO5: C, 73.15; H, 6.75. Found:
C, 72.71; H, 6.57.
a
a
a
ꢀ216.7 (ddd, JF,4 = 50.1, JF,3 = JF,5 = 27.9, F-4b); ESI-MS, m/z: calcd
for C27H30FO5 [12+H]+ 453.20, found: 453.21. Anal. Calcd for
C
27H29FO5: C, 71.66; H, 6.46. Found: C, 71.95; H, 6.78.
3.2.3. Prop-1-en-1-yl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-
galactopyranoside (11)
a-
D-
3.2.4.3. From prop-1-en-1-yl 2,3,6-tri-O-benzyl-4-deoxy-4-flu-
oro- -galactopyranoside (11). Galactopyranoside 11
a-D
To a solution of allyl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-
a
-
D
-
(4.035 g, 8.19 mmol) was dissolved in acetone-aq 1 M HCl (10:1,
238 mL) and stirred under reflux at 100 °C for 40 min. The course
of the reaction was monitored by TLC (3:1, n-hexane–EtOAc). After
cooling the solution was neutralized with NaHCO3 and then evap-
orated. The residue was diluted with water and then extracted
with diethylether, the organic phase was dried and concentrated.
Column chromatography (3:1, n-hexane–EtOAc) gave 12 (2.96 g,
86%). 1H NMR data were consistent with those obtained in
Section 3.2.4.2.
galactopyranoside (10) (1.55 g, 3.14 mmol) in EtOH–toluene–H2O
(7:3:1) (185 mL), tris(triphenylphosphine)-rhodium(I)chloride
(239 mg) and 1,4-diazabicyclo[2.2.2]octane (77 mg) were added.
The mixture was stirred under reflux at 100 °C until the starting
compound was consumed (ca 5 h). After concentration the residue
(1.94 g) was extracted between water and diethyl ether. The organ-
ic phase was dried (MgSO4) and the solvent evaporated. The residue
(1.71 g) was purified by chromatography (3:1, n-hexane–EtOAc) to
give prop-1-en-1-yl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-a-D-
galactopyranoside (11) (1.57 g, 91.8%, mixture of (E) and (Z) iso-
mers in a ratio about 1.6:1) which was directly used in the next
reaction for the preparation of free aldose 12. 1H NMR (300 MHz,
CDCl3) d: 7.41–7.25 (m, aromatic protons), 6.14 (dq, J = 12.3, 1.7, –
3.2.5. 1-O-Acetyl-2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-D-
galactopyranose (7)
3.2.5.1. From methyl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-
galactopyranoside (9). To a solution of methyl glycoside 9
a-D-
O–CH@ (E)), 6.03 (dq, J = 6.2, 1.7, –O–CH@ (Z)), 5.16 (m,
R
J = 33.1,
(200 mg, 0.43 mmol) in AcOH–Ac2O, 3:1 (3 mL) and CH2Cl2
(30 mL), concentrated H2SO4 (0.1 mL) was added dropwise. After
vigorous stirring for 1 h at room temperature the reaction mixture
was monitored by TLC (2:1, n-hexane–EtOAc; 9: Rf = 0.54, 7:
Rf = 0.71). The reaction was quenched by the addition of ice
(15 g) and the mixture was extracted with CH2Cl2 (3 ꢁ 10 mL),
the extract was washed with satd NaHCO3 soln (3 ꢁ 10 mL) and
H2O (3 ꢁ 10 mL), concentrated and dried. The raw product
(151 mg) was purified by flash chromatography (3:1, petroleum
ether–EtOAc) to give acetate 7 (30 mg, 15%) as a syrup, the TLC
and NMR data were consistent with the product obtained in
Section 3.2.5.2.
@HCCH3 (E)), 5.0–4.58 (m, H-4, H-1, @HC–CH3 (Z) and 3 ꢁ OCH2Ph),
4.19–3.86 (m, H-2, H-3, H-5), 3.69–3.42 (m, H-6a, H-6b), 1.61 (dd,
J = 7, 1.7, CH3–C@ (Z)), 1.54 (dd, J = 7.9, 1.5, CH3–C@ (E)). ESI-MS,
m/z: calcd for C30H34FO5 [11+H]+: 493.23, found: 493.24. Anal. Calcd
for C30H33FO5: C, 73.15; H, 6.75. Found: C, 72.95; H, 6.78.
3.2.4. 2,3,6-Tri-O-benzyl-4-deoxy-4-fluoro-
3.2.4.1. From methyl glycoside 9 by treatment with AcOH–HCl–
H2O. Methyl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro- -galac-
D-galactopyranose (12)
a
-D
topyranoside (10) (107 mg, 0.23 mmol) was heated in a steam bath
at 100 °C with a mixture of 80% acetic acid (2 mL) and hydrochloric
acid (1 m, 0.65 mL). The reaction mixture was monitored by TLC
(2:1, n-hexane–EtOAc; 9: Rf = 0.86; 12: Rf = 0.65). The mixture
was cooled after 20 h, diluted with CHCl3 (12 mL) and washed with
aq NaHCO3 (3 ꢁ 20 mL) and brine (10 mL). After drying and con-
centration, a dark yellow syrup (118 mg) resulted, which after
purification by column chromatography on silica gel (3:1, n-hex-
ane–EtOAc) gave 21.7 mg (36%) of the hexose 12. 1H NMR
3.2.5.2. From 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-D-galactopyr-
anose (12). A solution of aldose 12 (396 mg, 0.876 mmol) in
pyridine (2 mL) with Ac2O (2 mL) was stirred at room temperature
for 1 h, then was concentrated and dried (407 mg). The residue was
purified by flash chromatography on silica gel (3:1, n-hexane–
EtOAc) to give acetate 7 as a colourless syrup, (390 mg, 91%,
a/
(300 MHz, CDCl3) d: 7.42–7.25 (m, aromatic protons), 5.24 (d, 1H,
b = 1.4). ½a 2D0
ꢂ
+63 (c 1.06, MeOH); 1H NMR (500 MHz, CDCl3) d:
), 5.58 (d,
), 4.89 (bdd,
J4,3 = 2.1, JH,F = 50, H-4b), 4.84 (d, J = 11.2, PhCH–), 4.79–4.68 (m,
PhCH–), 4.56–4.50 (m, PhCH–), 4.08–3.95 (m, H-5 ), 4.01 (dd,
J2,1 = 3.5, J2,3 = 10.6, H-2 ), 3.84 (ddd, J3,4 = 2.2, J3,2 = 9.9,
JH,F = 28.3, H-3 ), 3.84 (dd, J2,3 = 9.7, J2,1 = 8, H-2b), 3.78–3.54 (m,
2
J1,2 = 2.7, H-1
a
), 4.95–4.68 (m, 7H, 6 ꢁ PhCH, H-4, J4,F =50,6),
7.39–7.25 (m, aromatic protons), 6.33 (d, J1,2 = 3.5, H-1a
4.63 (d, 1H, J1,2 = 7.4, H-1b), 4.25–4.16 (ddd, 1H, H-5, J5,F = 30 ).
J1,2 = 8.0, H-1b), 4.94 (bdd, J4,3 = 2.2, JH,F = 50, H-4a
3.2.4.2. From allyl glycoside 10 by treatment with PdCl2.
Pal-
a
ladium chloride (496 mg, 3.12 mmol) was added to a solution of
glycoside 10 (492 mg, 1.00 mmol) in 95% aq AcOH (9.95 mL) con-
taining AcONa (312 mg).19 The dark suspension was stirred until
TLC evidenced complete conversion of the reactant into a slower
moving spot (ca 4 h). The reaction mixture was filtered through a
bed of Celite, diluted with CH2Cl2 (15 mL) and washed with water
(8 mL), satd NaHCO3 soln (2 ꢁ 8 mL) and water (5 mL), dried
(MgSO4) and concentrated. The resulting dark yellow syrup was
purified by flash chromatography n-hexane–EtOAc, 3:1 to provide
a
a
H-6ab, H-6bb, H-6aa, H-6ba, H-5b, H-3b), 2.11 (s, COCH3a), 2.04
(s, COCH3b); 13C NMR (125 MHz, CDCl3) d: 169.30 (COMe),
169.17 (COMe), 138.1–137.53 (C-arom. quart.), 128.45–127.69
1
(C-arom.);
a
anomer: 90.37 (C-1), 86.68 (d, JC,F = 183.75, C-4),
2
75.53 (d, JC,F = 17.6, C-3), 74.62 (C-2), 73.69, 73.63, 72.52
2
(3 ꢁ PhCH2–), 70.34 (d, JC,F = 18, C-5), 67.4 (d, JC,F = 5.5, C-6),
1
21.06 (COCH3); b anomer: 93.73 (C-1), 85.32 (d, JC,F = 183.75, C-
/b 2.5). ½a 2D0
ꢂ
+56 (c 0.84, acetone); 1H
4), 79.37 (d, JC,F = 18.1, C-3), 77.53 (C-2), 75.44, 73.63, 72.28
2
aldose 12 (100 mg, 44%,
a
2
NMR (500 MHz, CDCl3): d 7.25–7.40 (m, aromatic protons), 5.22
(3 ꢁ PhCH2–), 72.74 (d, JC,F = 18.4, C-5), 66.95 (d, JC,F = 5.5, C-6),
(d, J = 2.6, H-1
a
), 4.88–4.66 (m, PhCH2), 4.84 (d, 2J = 50.4, H-4
a
),
20.9 (COCH3); 19F NMR data (470 MHz, CDCl3) d:
ꢀ214.76 (ddd, JF,H-3 = JF,H-5 = 28.6, JF,H-4 = 50.2);
a
anomer
4.63 (dd, J = 7.4, H-1b), 4.56 (d, J = 11.9, PhCH2), 4.52 (d, J = 11.9,
b
anomer:
0
PhCH2), 4.16 (ddd, J5,F = 29.73, J5,6 = J5,6 = 6.5, H-5
a), 3.90–3.86
ꢀ213.32 (ddd, JF,H-3 = JF,H-5 = 27.6, JF,H-4 = 49.6); ESI-MS, m/z: calcd
(m, J3,4 = 3.2, J3,2 = 10.5, H-2, H-3), 3.67–3.62 (m, H-6a, H-6b),
for C29H31FNaO6 [7+Na]+: 517.20, found: 517.32. Anal. Calcd for
3.16 (s, OH); 13C NMR (125 MHz, CDCl3) d: 128.59–127.55
C29H31FO6: C, 70.43; H, 6.32. Found: C, 70.11; H, 6.56.

36
I. Jambal et al. / Carbohydrate Research 360 (2012) 31–39
2
3.2.6. 1-O-Acetyl-3,4,6-tri-O-benzyl-2-deoxy-2-fluoro-
galactopyranose (8)
D
-
protons), 4.92 (br d, 1H, H-4, J4,F = 49.8), 4.79 (m, 4H, 2 ꢁ PhCH),
4.61–4.54 (m, 2H, PhCH), 4.47–4.30 (m, 3H, H-1, H-3, H-5), 4.28–
4.09 (m, 4H, H-2, 3 ꢁ CH3CH), 4.03 (m, 1H, CH3CH) 3.70 (dd, 1H,
J6a,6b = 10, J6a,5 = 6.6, H-6a), 3.64 (dd, 1H, J6b,5 = 10, J6b,5 = 5.8, H-
6b), 1.26 (t, 3H, J = 7.1, CH3), 1.24 (t, 3H, J = 7.1, CH3); 13C NMR
(125 MHz, CDCl3) d: 138.08, 137.90, 137.86 (C-arom. quart.),
A suspension of water (5 mL) and 1-chloromethyl-4-fluoro-1,4-
diazoniabicyclo[2,2,2]octane bis(tetrafluoroborate) (Selectfluor™,
1.7 g, 4.76 mmol) were added to a solution of 3,4,6-tri-O-benzylga-
lactal (1.084 g, 2.66 mmol) in CH3NO2 (25 mL) and the reaction
was allowed to proceed for 15 min at 100 °C. The reaction was
monitored by TLC (4:1, n-hexane–EtOAc) and quenched with water
(20 mL). Then the mixture was extracted with EtOAc (3 ꢁ 30 mL).
The combined organic extracts were dried with MgSO4 and con-
centrated (1.256 g, 2.78 mmol). The residue was dissolved in pyri-
dine (4 mL) and Ac2O (4 mL) was added. After 16 h, the reaction
was finished by the addition of MeOH (8 mL) and CH2Cl2 (40 mL).
After washing with 5% H2SO4 (2 ꢁ 10 mL) and satd NaHCO3 soln
(2 ꢁ 10 mL) the organic phase was dried (MgSO4). Purification on
1
128.34–127.61 (C-arom.), 87.21 (d, JC,F = 182.7, C-4), 76.31 (d,
2JC,F = 17.3, C-3), 74.62 (C-2), 73.94, 73.36, 72.63 (3 ꢁ PhCH2–),
2
1
73.57 (d, JC,F = 18.4, C-5), 71.52 (d, JC,P = 153.7, C-1), 68.15 (d,
JC,F = 4.8, C-6), 62.72 (d, JC,P = 6.4, CH3CH2), 62.14 (d, JC,P = 6.9,
CH3CH2), 16.40 (d, JC,P = 6.3, CH3), 16.33 (d, JC,P = 6.0, CH3); 19F
NMR (470 MHz, CDCl3, dec) d: ꢀ212.7 (F-4); 31P NMR
(202.5 MHz, CDCl3, dec) d: 21.6 (P-1); ESI-MS, m/z: calcd for
C31H38FNaO7P [5+Na]+: 595.22, found: 595.35. Anal. Calcd for
C
31H38FO7P: C, 65.02; H, 6.69. Found: C, 64.96; H, 6.52.
silica gel column (7:1, n-hexane–EtOAc) gave 8 (610 mg, 54%,
a/b
b-Anomer 13 (34.4 mg, 24%), syrup, ½a D20
ꢂ
+40 (c 1.0, acetone). 1H
1) as a colourless oil, ½a D20
ꢂ
+48 (c 3.0, CHCl3). 1H NMR (500 MHz,
CDCl3) d: 7.30–7.19 (m, aromatic protons), 6.32 (d, J1,2 = 3.6, H-
NMR (500 MHz, CDCl3) d: 7.39–7.27 (m, 15H, H-aromatic protons),
2
4.93 (d, 1H, J = 10.3, PhCH), 4.91 (br d, 1H, J4,F = 46.5, H-4, over-
1
a
), 5.59 (dd, J1,2 = 7.8, J1,F = 4.8, H-1b), 5.01 (ddd, J2,1 = 3.8,
lap.), 4.86 (d, 1H, J = 10.3, PhCH), 4.78 (d, 1H, J = 11.7, PhCH), 4.69
(d, 1H, J = 11.7, PhCH), 4.57–4.51 (m, 2H, 2 ꢁ PhCH), 4.25–4.2 (m,
5H, H-2, 4 ꢁ CH3CH), 3.75–3.5 (m, 5H, H-1, H-3, H-5, H-6a, H-6b),
1.27 (t, 3H, J = 7, CH3), 1.23 (t, 3H, J = 7, CH3); 13C NMR (125 MHz,
CDCl3): d 138.21, 137.63, 137.57 (C-arom. quart.), 128.49–127.58
(C-arom.), 85.63 (d, JC,F = 183.6, C-4), 81.38 (dd, JC,F = 17.6,
JC,P = 17.7, C-3), 77.46 (d, JC,P = 16.9, C-5), 75.42 (PhCH2), 74.96 (d,
1JC,P = 171.4, C-1), 74.91 (C-2), 73.67, (PhCH2), 71.84 (PhCH2),
67.64 (d, JC,F = 5.4, C-6), 63.27 (d, JC,P = 5.8, CH3CH2), 62.45 (d,
JC,P = 6.5, CH3CH2), 16.38 (d, JC,P = 5.8, CH3), 16.27 (d, JC,P = 6.4,
CH3); 19F NMR (470 MHz, CDCl3) d: ꢀ218.40 (ddd, J = 47, 27.6, F-
4); 31P NMR (202.5 MHz, CDCl3) d: 19.60 (m, P-1); TOF-ESI: m/z
calcd for C31H38FNaO7P [13+Na]+: 595.22, found: 594.93. Anal.
Calcd for C31H38FO7P: C, 65.02; H, 6.69. Found: C, 65.36; H, 6.74.
J2,3 = 9.8, J2,F = 49.5, H-2 ), 4.86 (d, J = 8.7, PhCH), 4.84 (d, J = 8.7,
a
PhCH), 4.75 (ddd, J2,1 = 8.7, J2,3 = 9.1, J2,F = 51.9, H-2b), 4.73 (d,
J = 11.9, PhCH–), 4.68 (d, J = 11.5, PhCH), 4.65–4.59 (m, 2 ꢁ PhCH),
4.53 (d, J = 11.5, PhCH), 4.49 (d, J = 11.3, PhCH), 4.41–4.30 (m,
1
2
4 ꢁ PhCH), 4.0 (br s, H-4
a
), 3.96–3.87 (m, H-5
a
, H-4b, H-3
a
),
3.64 (dd, J5,6a = J5,6b = 6.7, H-5b), 3.60 (ddd, J3,4 = 2.6,
J3,2 = J3,F = 9.6, H-3b), 3.55–3.43 (m, H-6ab, H-6bb, H-6aa, H-6ba
,),
2.06, 2.04 (2 ꢁ s, 2 ꢁ COCH3); 13C NMR (125 MHz, CDCl3) d:
169.27, 169.12 (2 ꢁ COMe), 138.2–137.58 (6 ꢁ C-quart. arom.),
2
128.44–127.45 (C-arom.); 91.99 (d, JC,F = 25.4, C-1b), 90.55 (d,
1JC,F = 183.6, C-2b), 89.73 (d, JC,F = 22.5, C-1
a
), 88.07 (d,
2
1JC,F = 188.0, C-2
a
), 79.97 (d, JC,F = 15.6, C-3b), 76.87 (d,
2
2JC,F = 15.8, C-3
), 74.27 (C-5b), 73.87 (d, JC,F = 8.4, C-4b), 73.58, 73.50, 72.86,
a
), 75.06, 74.9 (2 ꢁ PhCH2), 74.93 (d, JC,F = 8.3, C-
4a
72.80 (4 ꢁ PhCH2), 71.78 (C-5
a
), 67.93 (C-6
a
), 67.55 (C-6b),
3.3.1.2. Diethyl (3,4,6-tri-O-benzyl-2-deoxy-2-fluoro-
topyranosyl)phosphonate (6) and diethyl (3,4,6-tri-O-benzyl-2-
deoxy-2-fluoro-b- -galactopyranosyl)phosphonate
(14). From acetate 8 (550 mg, 1.11 mmol) a raw mixture of
a-D-galac-
20.93, 20.86 (2 ꢁ CH3CO–); 19F NMR (282 MHz, CDCl3) d:
a ano-
mer: ꢀ208.9 (ddd, J = 3.8, 10.0, 48.8); b anomer: ꢀ207.3 (ddd,
J = 3, 10.5, 52.5); ESI-MS, m/z: calcd for C29H35FNO6 [8+NH4]+:
512.24, found: 512.24. Anal. Calcd for C29H31FO6: C, 70.43; H,
6.32. Found: C, 69.99; H, 6.35.
D
the phosphonates 5 and 14 (743 mg) were obtained, (Rf = 0.09,
0.19; n-hexane:EtOAc 4:1). Separation on silica gel column (elution
with n-hexane:EtOAc 2:1) yielded:
3.3. Diethyl galactosyl phosphonates 5, 13, 6 and 14.
Debenzylated diethyl galactosyl phosphonates 18 and 20
a
-Anomer 6 (128 mg, 20%), syrup, ½a D20
ꢂ
+26 (c 1.1, CHCl3). 1H
NMR (500 MHz, CDCl3) d: 7.26–7.17 (m, 15H, aromatic protons),
4.99 (dddd, 1H, J2,F = 48.0, J2,P = 12.8, J2,3 = 6.0, J2,1 = 3.8, H-2),
2
3.3.1. General procedure for phosphonylation of galactosyl
acetates 7 and 8
4.64–4.62 (m, 3H, 3 ꢁ PhCH), 4.49–4.39 (m, 3H, 3 ꢁ PhCH), 4.34–
2
4.31 (m, 1H, H-5), 4.29 (ddd, J1,2 = 3.7, J1,P = 14.3, J1,F = 23.3, H-1),
To a solution of anomeric acetates 7 or 8 (0.2–1.2 mmol) in
CH2Cl2 (4 mL/mmol of substrate), (EtO)3P (2.6 mol equiv) was
added under Ar. After cooling to ꢀ1 °C, TMSOTf (2 mol equiv)
was added dropwise for 1 h and the mixture stirred at room tem-
perature for 1 day and monitored by TLC until all the starting
material was consumed and two products had been formed. The
reaction was finished by the addition of a mixture of water and
EtOAc (1:10, 25 mL/mmol of substrate). After washing two times
with a satd soln of NaHCO3 and two times with a satd soln of NaCl,
the organic phase was dried (MgSO4), evaporated and the residue
was separated on a silica gel column.
4.15–4.03 (m, 5H, H-3, 4 ꢁ CH3CH), 3.93 (ddd, 1H, J = 3.5, H-4),
3.79 (dd, 1H, J6a,6b = 11.1, J6a,5 = 8.1, H-6a), 3.55 (dd, 1H,
J6b,6a = 11.1, J6b,5 = 4.0, H-6b), 1.2 (m, 6H, 2 ꢁ CH3); 13C NMR
(125 MHz, CDCl3) d: 138.19, 137.96, 137.87 (C-arom. quart.),
1
2
128.41–127.59 (C-arom.), 88.67 (dd, JC,F = 182.4, JC,P = 2.5, C-2),
2
75.56 (d, JC,P = 8.6, C-5), 75.35 (br d, JC,F = 24.4, C-3), 73.92 (br d,
1
JC,F = 3.4, C-4), 73.42, 73.21 (3 ꢁ PhCH2), 67.52 (dd, JC,P = 166.6,
2JC,F = 22.9, C-1), 66.51 (C-6), 63.24 (d, JC,P = 6.2, CH3CH2), 62.63
(d, JC,P = 6.7, CH3CH2), 16.41 (d, JC,P = 8.19, CH3), 16.34 (d, JC,P = 8.6,
CH3); 19F NMR (282 MHz, CDCl3) d: ꢀ200.2 (m, F-2); 31P NMR
(202.5 MHz) d: 19.01 (m, P-1); ESI-MS, m/z: calcd for C31H42FNO7P
[6+NH4]+: 590.27, found: 590.23. Anal. Calcd for C31H38FO7P: C,
65.02; H, 6.69. Found: C, 65.01; H, 6.61.
3.3.1.1. Diethyl (2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-
topyranosyl)phosphonate (5) and diethyl (2,3,6-tri-O-benzyl-4-
deoxy-4-fluoro-b- -galactopyranosyl)phosphonate
(13). From acetate 7 (128 mg, 0.25 mmol) a crude mixture of
a-D-galac-
b-Anomer 14 (218 mg, 34%), syrup, ½a D20
ꢂ
+9 (c 1.2, CHCl3). 1H
NMR (500 MHz, CDCl3) d: 7.28–7.17 (m, 15H, aromatic protons),
D
-
D
2
5.05 (dddd, 1H, J2,F = 50.7, J2,P = J2,1 = J2,3 = 9.8, H-2), 4.85, 4.74,
the phosphonates 5 and 13 (198 mg) was obtained, (Rf = 0.15, 0.45;
3:1, n-hexane–EtOAc). Separation on silica gel column (elution
with n-hexane: EtOAc 3:1) yielded:
4.61, 4.48 (4 ꢁ d, 4H, 4 ꢁ PhCH), 4.37–4.31 (m, 2H, 2 ꢁ PhCH),
4.14–4.06 (m, 4H, 4 ꢁ CH3CH), 3.90 (dd, 1H, J4,3 = J4,5 = 2.7, H-4),
2
3.60 (ddd, 1H, J1,2 = J1,F = 9.8, J1,P = 4.5, H-1), 3.57–3.45 (m, 4H, H-
a
-Anomer 5 (75.6 mg, 53%), mp 103–105 °C; ½a D20
ꢂ
+53 (c 1.0,
3, H-5, H-6a, H-6b), 1.22 (t, 3H, J = 7.07, CH3), 1.18 (t, 3H, J = 7.08,
CHCl3). 1H NMR (500 MHz, CDCl3) d: 7.4–7.28 (m, 15H, aromatic
CH3); 13C NMR (125 MHz, CDCl3) d: 138.5, 138.06, 137.76 (C-arom.

I. Jambal et al. / Carbohydrate Research 360 (2012) 31–39
37
quart.), 128.44–127.51 (C-arom.), 88.91 (dd, 1JC,F = 182.5, 2JC,P = 3.0,
3.4. Glycosylphosphonic acids and bis(pyridinium) salts
2
C-2), 81.36 (dd, JC,F = JC,P = 15.9, C-3), 79.05 (d, JC,P = 16.0, C-5),
1
74.76 (PhCH2), 74.67 (d, JC,F = 9.6, C-4), 73.84 (dd, JC,P = 171.54,
3.4.1. General procedure for deesterification of diethyl esters 5,
18 and 20 and for preparation of bis(pyridinium) salts 16, 19
and 21
2JC,F = 25.3, C-1), 73.53 (PhCH2), 72.91 (PhCH2), 68.22 (C-6), 63.24,
63.19 (2 ꢁ ꢁ d, JC,P = 6.5, 2 ꢁ CH3CH2), 16.37 (d, JC,P = 9.32, CH3),
16.29 (d, JC,P = 9.07, CH3); 19F NMR (282 MHz, CDCl3) d: ꢀ200.70
(m, F-2); 31P NMR (202.5 MHz, CDCl3) d: 17.71 (m, P-1); ESI-MS,
m/z: calcd for C31H42FNO7P [14+NH4]+: 590.27, found: 590.16.
Anal. Calcd for C31H38FO7P: C, 65.02; H, 6.69. Found: C, 64.86; H,
6.64.
To a solution of diethyl phosphonate 5, 18 or 20 in CH2Cl2
(10 mL/mmol substrate), stirred under Ar at 0 °C, BrSiMe3
(15 mmol/mmol substrate) was added within 30 min. The reaction
mixture was then stirred at rt under Ar for ca 4 h. The course of the
reaction was monitored by TLC (1:1, n-hexane–EtOAc). After the
disappearance of the starting ester, the mixture was concentrated
to give bis(trimethylsilyl) phosphonate which was directly hydro-
lysed in an acetone–water mixture (9:1, 20 mL/mmol of substrate)
under stirring at rt for 2 h (monitoring by TLC, 1:1, n-hexane–
EtOAc), then the mixture was concentrated. By preparative LC on
RP-18 (1:1 ? 3:1, MeOH–H2O), the target phosphonic acid 15, 3
or 4 was obtained.
3.3.2. General procedure for debenzylation of galactosyl
phosphonates 5 and 6
A vigorously stirred mixture of diethyl glycosyl phosphonate 5
or 6 (0.5–6 mmol), 20% palladium hydroxide on carbon (100 mg/
1 mmol of substrate) and MeOH (10 mL/1 mmol of substrate)
was degassed under vacuum and saturated with hydrogen. The
suspension was stirred at rt for 1 day under a slight overpressure
of hydrogen. On TLC (10:1, CHCl3–MeOH) one spot was observed
with Rf ꢃ0.14 under the spot of the starting compound (Rf ꢃ0.9).
Reaction mixture was filtered through Super-Cel. The filter cake
was washed with toluene (3 ꢁ 20 mL) and EtOH (3 ꢁ 25 mL). The
combined filtrate was evaporated to dryness to give corresponding
debenzylated diethyl glycosyl phosphonate 18 or 20 of sufficient
purity.
For preparation of the corresponding bis(pyridinium) salts, the
phosphonic acid 15, 3 or 4 was repeatedly evaporated with dry
pyridine.
3.4.2. (2,3,6-Tri-O-benzyl-4-deoxy-4-fluoro-a-D-
galactopyranosyl)phosphonic acid (15) and its bis(pyridinium)
salt 16
From the diethyl phosphonate 5 (296 mg, 0.52 mmol) the phos-
phonic acid 15 was obtained (156 mg, 59%), ½a D20
ꢂ
+35 (c 0.3, MeOH).
3.3.2.1. Diethyl (4-deoxy-4-fluoro-
phonate (18). From diethyl (2,3,6-tri-O-benzyl-4-deoxy-4-
fluoro- -galactopyranosyl)phosphonate (5) (1.83 g, 6.04 mmol),
a
-D
-galactopyranosyl)phos-
1H NMR (500 MHz, CD3OD) d: 7.42–7.16 (m, 15H, aromatic pro-
2
tons), 4.92 (br d, 1H, J4,F = 50, H-4), 4.86–4.55 (m, 6H, 6 ꢁ PhCH),
a-
D
4.38–4.29 (m, 3H, H-1, H-3, H-5), 4.13 (ddd, 1H, J2,P = 27, J2,3 = 9,
J2,1 = 6, H-2), 3.71 (dd, 1H, J6a,6b = 9.7, J6a,5 = 6.7, H-6a), 3.62 (dd,
1H, J6b,6a = 10, J6b,5 = 6, H-6b); 13C NMR (125 MHz, CD3OD) d:
139.73, 139.66, 139.25 (arom. quart. C), 129.54–128.71 (C-arom.),
diethyl (4-deoxy-4-fluoro-a-D-galactopyranosyl)phosphonate (18)
was obtained (950 mg, 99%) as white crystals, mp 77–79 °C; ½a D20
ꢂ
+82 (c 1.0, MeOH). 1H NMR (500 MHz, CD3OD) d: 4.80 (br d, 1H,
2
2
1
2
H-4, J4,F = 49.8), 4.38 (dd, 1H, J1,2 = 6.7, J1,P = 11.4, H-1), 4.23
88.65 (d, JC,F = 181.4, C-4), 77.57 (d, JC,F = 16.9, C-5), 76.23 (C-2),
4
2
(ddd, 1H, J3,2 = 9.7, J3,P =2.7, J3,F = 25, H-3), 4.20–4.08 (m, 5H,
74.42 (PhCH2), 74.39 (PhCH2), 74.25 (d, JC,F = 16.6, C-3), 73.73
1
4 ꢁ CH3CH, H-2), 4.04 (ddd, 1H, J5,6a = J5,6b = 6.7, JF,5 = 27, H-5),
3.68 (dd, 1H, J6a,6b = 11.4, J6a,5 = 6.7, H-6a), 3.63 (dd, 1H,
J6b,6a = 11.4, J6b,5 = 6.5, H-6b), 1.34 (t, 3H, J = 7, CH3), 1.31 (t, 3H,
(PhCH2), 72.75 (d, JC,P = 155.9, C-1), 69.12 (d, J = 5.8, C-6); 19F
NMR (470 MHz) d:-216.93 (dec, F-2); 31P NMR (202.5 MHz) d:
18.32 (m, P-1); ESI-MS, m/z: calcd for C27H29FO7P [15–H]ꢀ:
515.16, found: 515.14. Anal. Calcd for C27H30FO7P: C, 62.79; H,
5.85. Found: C, 62.35; H, 5.31.
1
J = 7, CH3); 13C NMR (125 MHz, CD3OD) d: 90.41 (d, JC,F = 180.2,
2
1
C-4), 76.90 (d, JC,F = 18.2, C-5), 74.63 (d, JC,P = 151.5, C-1), 70.59
2
(d, JC,F = 17.7, C-3), 68.77 (C-2), 63.91 (d, JC,P = 7, CH3CH2), 63.77
(d, JC,P = 7, CH3CH2), 61.16 (d, J = 5.9, C-6), 16.77 (d, JC,P = 5.4,
CH3), 16.73 (d, JC,P = 5.3, CH3); 19F NMR (282.2 MHz, CD3OD): d
ꢀ221.3 (ddd, J = 27.9, 49.7, F-4); 31P NMR (202.5 MHz, CD3OD): d
24.0 (m, P-1); ESI-MS, m/z: calcd for C10H20FNaO7P [18+Na]+:
325.08, found: 325.06. Anal. Calcd for C10H20FO7P: C, 39.74; H,
6.67. Found: C, 39.63; H, 6.56.
3.4.2.1. Bis(pyridinium) salt 16.
Syrup, ½a 2D0
ꢂ
+51 (c 1.0,
MeOH). 1H NMR (500 MHz, CD3OD) d: 8.61 (br s, 2H, Py), 8.12 (br
t, 2H, J = 7.5, Py), 7.63 (m, 2H, Py) 7.49–7.23 (m, 15H, H-aromatic
2
protons), 4.93 (br d, 1H, J4,F = 50.0, H-4), 4.83 (d, 1H, J = 11.6,
PhCH), 4.70 (m, 2H, 2 ꢁ PhCH), 4.63 (d, 1H, J = 11.6, PhCH), 4.54
(m, 2H, 2 ꢁ PhCH), 4.43–4.31 (m, 3H, H-1, H-3, H-5), 4.14 (ddd,
1H, J2,3 = 9.1 J2.1 = 6.7, J2,P = 26.9, H-2), 3.71 (dd, 1H, J6a,6b = 9.7,
J6a,5 = 6.8, H-6a), 3.62 (dd, 1H, J6b,6a = 9.8, J6b,5 = 5.9, H-6b); 13C
NMR (125 MHz, CD3OD) d: 147.25 (Py), 142.03 (Py), 139.75,
139.69, 139.31 (C-arom. quart.), 126.69 (Py) 129.54–128.71 (C-
3.3.2.2. Diethyl (2-deoxy-2-fluoro-
phonate (20). From diethyl (3,4,6-tri-O-benzyl-2-deoxy-2-
fluoro- -galactopyranosyl)phosphonate (6) (310 mg, 0.54 mmol),
a-D-galactopyranosyl)phos-
a-D
1
2
diethyl (2-deoxy-2-fluoro-
a
-
D
-galactopyranosyl)phosphonate (20)
arom.), 88.66 (d, JC,F = 181.6, C-4), 77.54 (d, JC,F = 16.7, C-5),
was obtained (129 mg, 79%) as a colourless syrup, ½a D20
ꢂ
+46 (c
76.44 (C-3), 74.46, 74.38 (PhCH2), 74.25 (d, JC,P = 18.3, C-2),
2
1.0, CHCl3). 1H NMR (500 MHz, CD3OD) d: 4.90 (dddd, 1H,
73.65 (PhCH2), 72.78 (d, JC,F = 152.4, C-1), 69.11 (d, J = 5, C-6);
1
2
J2,1 = 5.4, J2,3 = 7.3, J2,P = 20.5, J2,F = 49.0, H-2), 4.53 (ddd, 1H,
19F NMR (470 MHz, CD3OD, dec) d: ꢀ216.74 (F-4); 31P NMR
(202.5 MHz, CD3OD) d: 17.96 (m, P-1). ESI-MS, m/z: calcd for
2
J1,2 = 5.3, J1,P = J1,F = 13.7, H-1), 4.25–4.14 (m, 5H, 2 ꢁ CH3CH2, H-
3), 4.05–3.98 (m, 2H, H-5, H-4), 3.83 (dd, 1H, J6a,5 = 7.2,
J6a,6b = 12.1, H-6a), 3.69 (dd, 1H, J6b,5 = 4.4, J6b,6a = 12.1, H-6b),
1.33 (m, 6H, 2 ꢁ CH3); 13C NMR (125 MHz, CD3OD) d: 90.68 (dd,
C
C
27H29FO7P [15–H]ꢀ: 515.16, found 515.17. Anal. Calcd for
37H40FN2PO7: C, 65.87; H, 5.98; N, 4.15. Found: C, 65.82; H,
6.19; N, 4.03.
2
1JC,F = 180.6, JC,P = 1.6, C-2), 78.87 (d, JC,P = 5.7, C-5), 69.95 (dd,
2JC,F = 20.8, JC,P = 3.8, C-3), 69.86 (dd, JC,P = 161.1, JC,F = 23.5, C-1),
69.32 (d, JC,F = 5.7, C-4), 64.63 (d, JC,P = 6.8, CH3CH2), 64.15 (d,
JC,P = 7.2, CH3CH2), 61.02 (C-6), 16.72 (dd, JC,P = 6.3, CH3); 19F NMR
(282.2 MHz, CD3OD) d: ꢀ204.75 (ddd, JF,H-2 = 49.1, F-2); 31P NMR
(CD3OD, 202.5 MHz, dec) d: 21.25 (d, JP,F = 4.8, P-1); ESI-MS, m/z:
calcd for C10H21FO7P [20+H]+: 303.10, found: 303.14. Anal. Calcd
for C10H20FO7P: C, 39.74; H, 6.67. Found: C, 39.74; H, 7.14.
3.4.3. (4-Deoxy-4-fluoro-a-D-D-galactopyranosyl)phosphonic
acid (3) and its bis(pyridinium) salt 19
1
2
From the diethyl phosphonate 18 (813 mg, 2.69 mmol), phos-
phonic acid 3 was obtained (572 mg, 86%) as a foamy syrup, ½a D20
ꢂ
+50 (c 1, MeOH). 1H NMR (500 MHz, D2O) d: 4.88 (br d, 1H,
2
2J4,F = 49.6, H-4), 4.30 (dd, 1H, J1,2 = 6.5, J1,P = 11.7, H-1), 4.22
4
(ddd, 1H, J3,P = 2.6, J3,2 = 9.6, J3,F = 28.8, H-3), 4.21–4.10 (m, 2H,

38
I. Jambal et al. / Carbohydrate Research 360 (2012) 31–39
H-2, H-5), 3.75 (dd, 1H, J6a,5 = 7.3, J6a,6b = 11.4, H-6a), 3.69 (dd, 1H,
J6b,5 = 5.3, J6b,6a = 11.5, H-6b); 13C NMR (125 MHz, D2O) d: 91.38 (d,
1JC,F = 177.4, C-4), 75.99 (d, 2J = 18.0, C-5), 74.56 (d, 1JC,P = 148.1, C-
base and water (0.8 and 25 mL resp.,/mmol of substrate) was
added, and then the solution was concentrated in vacuo. The resi-
due was coevaporated again with the same amount of Hünigs
base–water, dried under vacuum and then purified by preparative
LC on RP-18 (1:1 ? 3:1, MeOH–H2O). The products were obtained
as salts with the base 4-morpholine-N,N0-dicyclohexyl carboximi-
damide 17, 1a or 2a.
1), 70.34 (d, 2JC,F = 17.5, C-3), 68.79 (C-2), 61.41 (d, J = 5.3, C-6); 19
F
NMR (470 MHz, D2O) d: ꢀ219.9 (dec, F-4), 31P NMR (202.5 MHz,
D2O) d: 17.97 (dd, J = 26.8, 10.7, P-1). ESI-MS, m/z: calcd for
C6H11FO7P [3–H]ꢀ: 245.02, found: 245.04. Anal. Calcd for
C6H12FO7P: C, 29.28; H, 4.91. Found: C, 29.12; H, 5.16.
3.5.2. Bis(4-morpholine-N,N0-dicyclohexyl carboximidamide-
3.4.3.1. Bis(pyridinium) salt 19.
Syrup, ½a 2D0
ꢂ
+68 (c 1.0,
ium) uridine-50-[(2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-
a-D-
MeOH); 1H NMR (500 MHz, D2O) d: 8.59 (br s, 4H, Py), 8.19 (2H,
galactopyranosyl)phosphonoyl]phosphate (17)
2
Py), 7.71 (4H, Py), 4.87 (d, 1H, J4,F = 49.9, H-4), 4.33–4.04 (m, 4H,
Starting from pyridinium phosphonate 16 (412 mg. 0.61 mmol),
H-1, H-2, H-3, H-5), 3.76 (dd, 1H, J6a,6b = 11.9, J6a,5 = 7.3, H-6a),
the salt 17 (351 mg, 41%) was obtained as white yellow solid, ½a D20
ꢂ
3.65 (dd, 1H, J6b,6a = 11.9, J6b,5 = 5, H-6b); 13C NMR (125 MHz,
+19 (c 1.1, MeOH); 1H NMR (500 MHz, CD3OD) d: signals of the
nucleotide moiety: 8.11 (d, 1H, J = 8.1, H-6(U)), 7.48–7.21 (m,
15H, aromatic protons), 5.94 (d, 1H, J = 4.9, H-10), 5.84 (d, 1H,
J = 8.1, H-5(U)), 5.04 (d, 1H, J = 12.2, PhCH), 4.93 (br d, 1H, H-4),
4.77 (d, 1H, J = 11.6, PhCH), 4.72 (d, 1H, J = 10.2, PhCH), 4.67 (d,
1H, J = 10.5, PhCH), 4.65 (d, 1H, J = 11.6, PhCH), 4.60 (d, 1H, J = 12,
1
D2O) d: 146.21, 143.88, 127.37 (Py), 91.48 (d, JC,F = 177.4, C-4),
2
1
75.51 (d, JC,F = 17.9, C-5), 74.30 (d, JC,P = 145.1, C-1), 70.49 (d,
2JC,F = 17.2, C-3), 69.41 (C-2), 61.2 (d, JC,F = 4.6, C-6); 19F NMR
(470 MHz, D2O) d: ꢀ219.75 (m, F-4); 31P NMR (202 MHz, D2O) d:
14.81 (dec, P-1); ESI-MS, m/z: calcd for C6H11FO7P [3–H]ꢀ:
245.02, found: 245.06. Anal. Calcd for C16H22FN2O7P: C, 47.53; H,
5.48; N, 6.93. Found C, 47.20; H, 5.61; N, 7.07.
0 0
0 0
PhCH), 4.42 (dd, 1H, J = J = 4.6, H-30), 4.36 (dd, J = 10.5, 6.0,
2;3
3;4
H-1), 4.25 (dd, 1H, J = J = 4.9, H-20), 4.22 (dd, 1H, J = 11.7, H-
0 0
0 0
2;1
2;3
50), 4.08 (br s, 1H, H-40), 3.78 (bdd, 1H, J = 9.0, 5.0, H-6a), 3.69
(bdd, overlapped, H-5), 3.57 (bdd, 1H, J = 9.0, 8.0, H-6b); other sig-
nals (nuclei of the base moieties): 3.73 (dd, 8H, J = 9.1, 4 ꢁ CH2O),
3.41 (dd, 8H, J = 9.1, 4 ꢁ CH2N), 3.30 (overlap., CHN), 1.93 (m, 8H,
4 ꢁ CH2), 1.82 (m, 8H, 4 ꢁ CH2), 1.67 (m, 4H, 2 ꢁ CH2), 1.44–1.30
(m, 20H, 10 ꢁ CH2); 13C NMR (125 MHz, CD3OD) d: signals of the
nucleotide moiety: 159.37 (C@O(U)), 143.01 (C-6(U)), 129.38–
128.21 (C-arom.), 103.24 (C-5(U)), 89.74 (C-10)), 88.3 (d,
1J4,F = 181.1 Hz, C-4), 78.96 (C-5), 77.15 (C-3), 75.79 (C-20), 74.14,
74.0 (3 ꢁ CH2Ph), 71.12 (C-30); other signals: 67.29 (CH2O
(morph.)), 56.03 (N@CH), 49.52 (CH2N (morph.)), 34.46, 26.3,
26.14 (CH2 (cyclohexyl)); 19F NMR (470 MHz, CD3OD) d: ꢀ217.28
3.4.4. (2-Deoxy-2-fluoro-a-D-galactopyranosyl)phosphonic acid
(4) and its bis(pyridinium) salt 21
From the diethyl phosphonate 20 (102 mg, 0.34 mmol), phos-
phonic acid 4 (81 mg, 98%) was obtained as a syrup, ½a D20
ꢂ
+45 (c
1.3, MeOH). 1H NMR (500 MHz, D2O) d: 4.89 (dddd, 1H, J2,1 = 5.1,
J2,3 = 8.4, J2,P = 18.3, 2J2,F = 48.9, H-2), 4.34 (ddd, 1H,
2J1,P = J1,F = 13.0, J1,2 = 5.1, H-1), 4.22 (bdd, J3,2 = 8.4, J3,F = 10.1, H-
3), 4.02 (m, 2H, H-4, H-5), 3.78 (dd, 1H, J6a,5 = 7.7, J6a,6b = 12, H-
6a), 3.61 (dd, 1H, J6b,5 = 2.8, H-6b); 13C NMR (125 MHz, D2O) d:
1
1
89.54 (d, JC,F = 177.8, C-2), 76.50 (C-5), 69.02 (dd, JC,P = 163.5,
2JC,F = 24.1, C-1), 68.36 (d, JC,F = 20.5, C-3), 67.94 (d, JC,F = 5.9, C-
2
4), 59.66 (C-6); 19F NMR (282 MHz, D2O) d: ꢀ203.65 (br d, JF,H-
2 = 49.1, F-2); 31P NMR (202.5 MHz, D2O, dec) d: 16.33 (P-1). ESI-
MS, m/z: calcd for C6H16FNO7P [4+NH4]+: 264.06, found: 264.08;
calcd for C6H11FO7P [4ꢀH]ꢀ: 245.02, found: 244.8. Anal. Calcd for
C6H12FO7P: C, 29.28; H, 4.91. Found: C, 28.99; H, 5.11.
(dec, F-4); P NMR (202.5 MHz, CD3OD) d: ꢀ10.73 (d, J0P;P = 29.5,
31
phosphate), 5.95 (d, J0P;P = 29.5, phosphonate); ESI-MS, m/z: calcd
for C36H40FN2O15P2 [17ꢀ2BꢀH]ꢀ: 821.2, found: 821.2; calcd for
C
C
17H32N3O [B+H]+: 294.3, found: 294.3. Anal. Calcd for
70H103FN8O17P2: C, 59.65; H, 7.37; N, 7.95. Found: C, 59.27; H,
7.30; N, 7.65.
3.4.4.1. Bis(pyridinium) salt 21.
Syrup, ½a 2D0
ꢂ
+38 (c 1.4,
MeOH); 1H NMR (500 MHz, D2O) d: 8.66 (br s, 4H, Py), 8.38 (2H,
Py), 7.86 (4H, Py), 4.88 (dddd, 1H, J2,1 = 5.8, J2,3 = 6.3, J2,P = 15.5,
J2,F = 49.1, H-2), 4.28–4.18 (m, 2H, H-1, H-3), 4.08–4.03 (m, 2H,
H-4, H-5), 3.84 (dd, 1H, J6a,6b = 12.2, J6a,5 = 8.3, H-6a), 3.62 (dd,
1H, J6b,6a = 12.2, J6b,5 = 2.8, H-6b); 13C NMR (125 MHz, D2O) d:
3.5.3. Bis(4-morpholine-N,N0-dicyclohexyl carboxamidimidium)
uridine-50-[(4-deoxy-4-fluoro-
a-D-galactopyranosyl)phosphonoyl]
phosphate (1a)
Starting from the pyridinium salt 19 (512 mg, 1.27 mmol) the
salt 1a was obtained as colourless syrup (1.01 g, 70%), ½a D20
ꢂ
+11 (c
1
144.73, 143.06, 126.68 (Py), 89.92 (d, JC,F = 177.8, C-2), 76.25 (C-
1.1, water). 1H NMR (500 MHz, D2O) d: signals of the nucleotide
moiety: 7.92 (d, 1H, J = 8.1, H-6(U)), 5.94 (d, 1H, J = 4.9, H-10),
2
1
5), 68.95 (dd, JC,F = 21.7, JC,P = 161.1, C-1), 68.32 (d, JC,F = 21.7, C-
3), 67.82 (d, JC,F = 5.5, C-4), 59.34 (C-6); 19F NMR (282 MHz, D2O)
d: ꢀ202.9 (br d, JF,H-2 = 49.2, F-2); 31P NMR (202 MHz, D2O) d:
13.8 (dec, P-1); ESI-MS, m/z: calcd for C6H11FO7P [4ꢀH]ꢀ: 245.02,
found: 245.04. Anal. Calcd for C16H22FN2O7P: C, 47.53; H, 5.48; N,
6.93. Found C, 47.61; H, 5.55; N, 6.97.
2
5.90 (d, 1H, J = 8.1, H-5(U)), 4.89 (br d, 1H, J4,F = 49.9, H-4), 4.34–
4.19 (m, 8H, H-20, H-30, H-40, H-1, H-2, H-3, H-4, H-5), 4.10 (ddd,
1H, J = 2.4, 4.2, 11.5, H-5a0), 4.02 (ddd, 1H, J = 2.9, 5.3, 11.6, H-
5b0), 3.78 (dd, 1H, J = 7.5, 12.1, H-6a), 3.68 (dd, 1H, J = 4.3,
12.1 Hz, H-6b); other signals: 7.87 (d), 5.89 (d), 3.90 (dd), 3.74
(dd), 3.66 (dddd), 3.40 (dd), 3.25 (dd), 3.15 (q), 1.88 (bd), 1.56
(bd), 1.29(m); 13C NMR (125 MHz, D2O) d: signals of the nucleotide
moiety: 167.65 (C-4 (U)), 159.24 (C-2 (U)), 143.06 (C-6 (U)), 103.98
(C-5 (U)), 91.49 (d, 1JC,F = 177.3, C-4), 89.9 (C-10), 84.83 (d, J = 8.9, C-
40), 75.54 (d, 2JC,F = 17.6, C-5), 75.26 (C-20), 74.39 (d, 1JC,P = 145.5, C-
3.5. Coupling reaction of the bis(pyridinium) salts 16, 19 and 21
with UMP-morpholidate
3.5.1. General procedure
2
2
The crude salt 16, 19 or 21 was coevaporated 3 times with dry
pyridine (10 mL/mmol of substrate). After addition of UMP-mor-
pholidate (1 mol equiv), the mixture was again coevaporated 3
times with dry pyridine (10 mL/mmol of substrate) and dried un-
der high vacuum for 5 h. Subsequently, the solid was taken up in
dry pyridine (25 mL/mol of substrate) under Ar, and to this solu-
tion dried 1H-tetrazole (6 mL/mmol of substrate) was added.
Thereafter, the solution was stirred at room temperature for 7 days
under Ar. For the termination of the reaction a mixture of Hünigs
1), 71.11 (d, JC,P = 14, C-2), 70.47 (d, JC,F = 16.9, C-3), 69.36 (C-30),
65.52 (C-50), 61.23 (C-6); other signals: 153.24, 67.37, 65.02, 55.87,
55.80, 49.42, 44.58, 43.97, 34.30, 26.05, 19.15, 17.68, 13.54; 19F
NMR (470 MHz, D2O, 1H dec) d: ꢀ218.2 (F-4); 31P NMR
(202.5 MHz, D2O, 1H dec) d: 14.8, 0.84, ꢀ10.8; ESI-MS, m/z: calcd
for C15H22FN2O15P [1aꢀ2BꢀH]ꢀ: 551.05, found: 551.11; calcd for
C9H12N2O9P [UMPꢀH]ꢀ: 323.03, found: 323.05; calcd for
C6H11FO7P [3ꢀH]ꢀ: 245.02, found: 245.04; calcd for C17H32N3O
[B+H]+: 294.25, found: 294.70.

I. Jambal et al. / Carbohydrate Research 360 (2012) 31–39
39
3.5.4. Bis(4-morpholine-N,N0-dicyclohexylcarboxamidimidium)
3.5.7. Diammonium uridine-50-[(2-deoxy-2-fluoro-
galactopyranosyl)phosphonoyl] phosphate (2b)
From the salt 2a (203 mg, 0.178 mmol) the diammonium salt 2b
(129 mg, 63%) was obtained as a colourless syrup, [ +6 (c 0.9,
a-D-
uridine-50-[(2-deoxy-2-fluoro-
phosphate (2a)
a-D-galactopyranosyl)phosphonoyl]
Starting from the pyridinium salt 21 (102 mg, 0.25 mmol), salt
a]
D
2a (217 mg, 75%) was obtained as a colourless syrup, ½a D20
ꢂ
+5 (c
water). 1H NMR (500 MHz, D2O) d: 7.92 (d, 1H, J = 8.1, H-6(U)),
5.96–5.94 (m, 2H, H-10, H-5(U)), 4.94 (dddd, 1H, J2,1 = 5.3,
J2,3 = 7.2, J2,P = 18.0, 2J2,F = 48.8, H-2), 4.48 (ddd, J1,2 = 5.3,
2J1,P = J1,F = 14.6, H-1), 4.37–4.33 (m, 3H, H-20, H-30, H-3), 4.28 (br
s, 1H, H-40), 4.23 (ddd, H, J = 2.5, 4.3, 11.8, H-5a0), 4.19 (ddd, 1H,
J = 3.0, 5.4, 11.8, H-5b0), 4.16 (dd, J5,6a = 8.3, J5,6b = 3.5, H-5), 4.12
(ddd, 1H, J4,5 = J4,3 = J4,F = 3.2, H-4), 3.92 (dd, 1H, J6a,5 = 8.3,
J6a,6b = 12.4, H-6a), 3.66 (dd, 1H, J6b,5 = 3.6, J6b,6a = 12.4, H-6b); 13C
NMR (125 MHz, D2O) d: 166.28 (C-4(U)), 151.91 (C-2(U)), 141.76
1.4, MeOH). 1H NMR (500 MHz, D2O) d: signals of the nucleotide
moiety: 7.85 (d, 1H, J = 8.1, H-6(U)), 5.91 (m, 2H, H-10, H-5(U)),
2
4.92 (dddd, 1H, J2,1 = 5, J2,3 = 7 Hz, J2,P = 17.5, J2,F = 47.5, H-2),
4.45 (ddd, J1,2 = 5, J1,P = J1,F = 15, H-1), 4.34– 4.05 (m, 8H, H-20,
H-30, H-40, H-5a0, H-5b0, H-3, H-4, H-5), 3.85 (H-6a, overlap.),
3.59 (H-6b, overlap.); other signals: 7.90 (d), 5.88 (m), 3.88
(dd), 3.64 (dddd), 3.24 (dd), 3.12 (q), 1.26 (m); 13C NMR
(125 MHz, D2O) d: signals of the nucleotide moiety: 166.05 (C-4
(U)), 151.78 (C-2(U)), 141.66 (C-6 (U)), 102.68 (C-5 (U)), 89.56
1
(C-6(U)), 102.75 (C-5(U), 89.77 (d, JC,F = 178.5, C-2), 88.58 (C-10),
1
(dd, JC,P = 13, JC,F = 179.1, C-2), 88.4 (C-10), 83.67 (d, J = 8.7, C-
83.36 (d, JC,P = 8.8, C-40), 76.42 (d, JC,P = 5.3, C-5), 73.83 (C-20),
1
2
1
2
40), 76.31 (C-5), 73.80 (C-20), 69.01 (dd, JC,P = 162.9, JC,F = 19.2,
C-1), 68.37 (d, J = 21.1, C-3), 67.9 (d, J = 12.8, C-4), 64.89 (br s,
C-50), 59.44 (d, J = 8.2, C-6); other signals: 166.18, 151.73,
141.62, 102.62, 88.3, 83.18, 73.75, 54.40, 54.32, 43.19, 43.12,
42.57, 42.51, 17.77, 17.69, 16.31, 16.23; 19F NMR (470 MHz,
D2O) d: ꢀ217.28 (dec, 1F); 31P NMR (202.5 MHz, D2O, 1H dec) d:
ꢀ10.75, 5.57; ESI-MS, m/z: calcd for C15H22FN2O15P [1aꢀ2BꢀH]ꢀ:
551.05, found: 551.11; calcd for C17H32N3O [B+H]+: 294.25,
found: 294.29.
69.77 (C-30), 68.90 (dd, JC,P = 164.1, JC,F = 23.0, C-1), 68.19 (d,
J = 19.8, C-3), 67.97 (d, JC,F = 5.7, C-4), 64.96 (d, JC,P = 4.9, C-50),
59.52 (C-6); 19F NMR (470 MHz, D2O, dec) d: ꢀ202.8 (F-2); 31P
NMR (202.5 MHz, D2O, 1H dec) d: ꢀ10.61 (d, J = 28.8,), 5.75 (d,
J = 28.8 ); ESI-MS, m/z: calcd for C15H22FN2O15P2 [2bꢀ2BꢀH]ꢀ:
551.05, found: 551.29; calcd for C9H12N2O9P [UMPꢀH]ꢀ: 323.03,
found: 323.56;.
Anal. Calcd for C15H29FN4O15P2.H2O: C, 29.81; H, 5.17; N, 9.27.
Found: C, 29.53; H, 5.42; N, 8.96.
Acknowledgment
3.5.5. General procedure for transformation of the bis(4-
morpholine-N,N0-dicyclohexyl carboximidamide-ium) salts 1a
and 2a to the diammonium salts 1b and 2b
This research was supported by the Ministry of Education, Youth
and Sports of the Czech Republic (project MSM 6046137305).
The salt 1a or 2a (0.145–0.180 mmol) were applied in water
solution (10 mL) on a colꢀumn of ion-exchange resin Dowex 1-
X8 (200–400 mesh, HCO2 form, 1.2 ꢁ 10 cm). The column was
eluted with aq ammonium bicarbonate in the gradient of 0–
1 M.28 In the first fraction eluted with water only 4-morpho-
line-N,N0-dicyclohexyl carboxamidimidium formiate was sepa-
rated. The fractions eluted with ca 0.4 M NH4HCO3 were
combined and evaporated to yield the desired diammonium salts
1b or 2b. Elution with 0.8–1 M NH4HCO3 produced unreacted
UMP salt.
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C
15H22FN2O15P2 [1bꢀ2BꢀH]ꢀ: 551.05, found: 551.17; calcd
for C9H12N2O9P [UMPꢀH]ꢀ: 323.03, found: 323.59; calcd for
C6H11FO7P [3ꢀH]ꢀ: 245.02, found: 245.33; calcd for
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C
15H24FN2O15P2 [1b-2B+H]+: 553.06, found: 553.06. Anal. Calcd
for C15H29FN4O15P2.4H2O: C, 27.36; H, 5.66; N, 8.51. Found: C,
27.35; H, 5.76; N, 8.25.