The preparation of new phosphorus-centered functional groups for modified oligonucleotides and other natural phosphates

Article abstract of DOI:10.3390/10091048

Efforts to develop synthetic methodologies allowing the preparation of α,α-difluorophosphonothioates, α,α- difluorophosphonodithioates, α,α-difluorophosphonotrithioates, and α,α-difluorophosphinates are reviewed in the light of applications in the field o

Full text of DOI:10.3390/10091048

Molecules 2005, 10, 1048-1073
molecules
ISSN 1420-3049
http://www.mdpi.org
The Preparation of New Phosphorus-Centered Functional
Groups for Modified Oligonucleotides and Other Natural
Phosphates
#
‡
§
¶
Arnaud Gautier, Chrystel Lopin , Goulnara Garipova , Olivier Dubert , Irina Kalinina ,
Carmen Salcedo, Sébastien Balieu, Stéphane Glatigny, Jean-Yves Valnot, Géraldine Gouhier
and Serge R. Piettre*
Laboratoire des Fonctions Azotées et Oxygénées Complexes, UMR CNRS 6014, IRCOF-Université
de Rouen, Rue Tesnières, F-76821 Mont Saint Aignan, France.
*
Author to whom correspondence should be addressed; e-mail: Serge.Piettre@univ-rouen.fr
Received: 23 April 2005 / Published: 30 September 2005
Abstract: Efforts to develop synthetic methodologies allowing the preparation of α,α–
difluorophosphonothioates, α,α–difluorophosphonodithioates, α,α–difluorophosphono-
trithioates, and α,α–difluorophosphinates are reviewed in the light of applications in the
field of modified oligonucleotides and cyclitol phosphates. Two successful approaches
have been developed, based either on the addition of phosphorus-centered radicals onto
gem–difluoroalkenes or on a process involving the addition of lithiodifluorophosphono-
thioates 91 onto a ketone and the subsequent deoxygenation reaction of the adduct. The
radical route successfully developed a practical route to α,α–difluoro–H–phosphinates
which proved to be useful intermediates to a variety of phosphate isosters. The ionic
route led to the first preparation of phosphonodifluoromethyl analogues of nucleoside–
3’–phosphates.
Keywords: Phosphate isoster; difluorophosphonates; difluoro-H-phosphinates;
nucleotide analogue; cyclitol analogue.
#
Present Address: Institut de Chimie Organique et Analytique, UMR 6005 CNRS, Université d’Orléans – UFR de
Sciences, rue de Chartres, B. P. 6759, F – 45067 Orléans cedex 2, France.
Present Address: Laboratoire de Chimie Macromoléculaire, UMR 7610 CNRS, Université Pierre et Marie Curie, 4 Place
Jussieu, F – 75252 Paris cedex, France.
Present Address: Department of Chemistry, University of California, Riverside, CA 92521-403, USA.
Present Address: Department of Chemistry, Loughborough University, Leicestershire LE 11 3TU, UK.
‡
¶
§

Molecules 2005, 10
1049
Introduction
The ubiquitous presence of the phosphate group in molecules involved in life processes has
focused the attention of many organic and medicinal chemists in their efforts to design and generate
non natural molecules to interfere with biochemical transformations. The phosphate group is mainly
found in biomolecules in the form of its monoesters 1 or diesters 2 (Scheme 1), which are produced
and broken down by the action of enzymes such as kinases, phosphorylases, phosphatases or
nucleases, for instance. Hence the early design of efficient enzyme inhibitors has often relied on the
use of phosphonates 3a, in which the esterified oxygen atom of the phosphate is replaced with a CH
2
unit [1]. The pioneering work of Blackburn and McKenna in the early eighties resulted in the design
and use of the α,α–difluoromethylphosphonates 3b as closer isosters of the phosphate [2]. Structural
and electronic studies by Chambers and O’Hagan on the zwitterionic species 6 have since then
confirmed this fact and firmly established the monofluorophosphonates and difluorophosphonates as
closely related, hydrolytically and enzymatically stable mimics of the phosphate (Table 1) [3].
Scheme 1
O
P
O
RO
OH
R
CX2
P
OH
Y
P
OH
OH
R
CF2
Z
1
3a: X = H
W
3
b: X = F
5
5
5
a: Y = S; W = Z = OR2
b: Y = O; W = Z = SR
c: Y = S; W = Z = SR2
2
O
O
P
OR
R1 CX2
P
CY2 R2
RO
OH
2
OH
4a: X = Y = H
b: X = F; Y = H
4
2
Thus, the pKa as well as the bond lengths and angles obtained from X–ray crystallography for the
various functional groups clearly indicated the closer analogy between both 6c and 6d, on the one
hand, and the phosphate 6a, on the other hand. Indeed, the literature reports numerous examples of
enzyme inhibitors encompassing the above fluorinated phosphonates. Among these, analogues
targeting phospholipase C (PLC), purine nucleoside phosphorylase (PNP) and protein
phosphotyrosine, phosphoserine or phosphothreonine phosphatases have been published [4]. This
functional group has also been successfully used to mimic the phosphate in nucleotide
monophosphates and trisphosphates: analogues of adenosine monophosphate, cyclic adenosine
monophosphate, adenosine triphosphate and adenosyl adenosine triphosphate, as well as structurally
related potent inhibitors of the reverse transcriptase of Human Immunodeficiency Virus (HIV), have
been described in literature [5].

Molecules 2005, 10
1050
Table 1.
O
P
α
-
CH2
O
+
H N
a
b
3
X
OH
6
Entry Compound
X
α (°)
118.7
112.1
113.3
116.5
a
b
a+b
3.02
3.32
3.32
3.35
pKa
6.4
7.6
6.5
5.4
2
1
2
3
4
6a
6b
6c
6d
O
1.43
1.807
1.82
1.59
1.51
1.50
1.496
CH
2
CHF
CF
2
1.85
In the case of phosphate diesters 2, the replacement of both esterified oxygen atoms with two
methylene units generates a phosphinate 4a, a functional group which has been used in the past to
generate analogues of the natural parent phosphates. (Scheme 1) [6].
Our interest in new variants of the phosphonates led us to develop efficient preparations of the
α,α–difluorophosphonothioate 5a, α,α–difluorophosphonodithioate 5b and α,α–difluorophosphono-
trithioate 5c, in which the various oxygen atoms of the parent phosphonate become replaced with
sulfur. More recently, we have focused on the phosphinate functional group, and the hitherto poorly
developed fluorinated analogues 4b.
Among the many possibilities for application are the antisense and antigene strategies, and the
inositol cycle. Briefly, the antisense and antigene strategies rely on the formation of a triplex between
the DNA and a modified oligonucleotide (MON), and of a duplex between the messenger RNA (m–
RNA) and a MON, respectively, to keep the transcription (antigene) or the translation (antisense) from
occurring (Scheme 2) [7]. This should result in the regulation of gene expression and open the door to
a conceptually new therapeutic treatment of diseases. Analysis of the requirements for an optimal
analogue of the monomeric unit in MONs points the finger at the necessity to keep the 3’–endo
conformation 7 of the ribose cycle that favors pairing of the strands (Figure 1), an optimal geometry
and electronic features of the phosphate mimic, and a stability towards the action of nucleases; this
allows the MON to target another molecule of m–RNA molecule, once the first one has been lyzed by
enzymes (antisense strategy; 4’–endo conformation).
Figure 1
O
Base
2'
3
'
O
Base
OH
O
O
O
OH
O
7
8
C -endo
C -exo
3
'
2'

Molecules 2005, 10
1051
Scheme 2
transcription
traduction
Proteins
DNA
m-RNA
(gene)
degradation
of m-RNA
antigene
oligomer
antisense
oligomer
Numerous studies aiming at understanding the inositol cycle have underlied its importance [8]. The
cycle is part of an important cell signalling system which helps regulate the intracellular calcium level.
A very simplified view of the cycle (Scheme 3) shows how the second messenger inositol-1,4,5-
3 2
trisphosphate (1,4,5–IP ) released from phophatidylinositol–4,5–diphosphate (PIP ) by the action of a
phospholipase C, undergoes a sequential series of phosphorylations of the hydroxyl groups and
hydrolysis of the phosphates to produce, among others, three different inositol monophosphates (I–4–
P, I–3–P and I–1–P). These are hydrolysed by a single enzyme, inositol monophosphatase (IMPase) to
2
yield myo–inositol which is then converted into PIP , thereby closing the cycle. Inositol
monophosphatase is a homodimeric enzyme and has been postulated as a possible target to treat manic
depressive illness [9].

Molecules 2005, 10
1052
Scheme 3
Agonist
EXTRACELLULAR
MEMBRANE
C18H37
C19H30
C18H37
C19H30
OH
O
P
OH
G-Protein
DAG
INTRACELLULAR
HO
O
OH
O
H2O3PO
OH
Phospholipase C
OPO3H2
PIP2
OH
OH
HO
OPO3H2
OH
H2O3PO
H2O3PO
OPO3H2
H2O3PO
OH
OPO3H2
OPO3H2
1,4,5-IP3
1,3,4,5-IP4
Calcium concentration
1,4-IP2
3,4-IP2
1,3-IP2
OH
OH
OH
HO
HO
OH
OH
H O PO
OH
OH
OPO H
2
3
3
2
H2O3PO
HO
HO
OH
OH
OH
OH
OH
OH
I-4-P
I-3-P
I-1-P
HO
HO
OH
OH
INOSITOL MONOPHOSPHATASE
Myo-inositol
Results and Discussion
The target molecules we elected to work on are depicted on Figure 2. Thus both inositol-3-
phosphate 9 and nucleoside-3’ phosphate 10 can be related to analogues 11 and 12, respectively,
encompassing phosphono(thio)difluoromethyl units.
Figure 2
HO
Base
O
P
OH
O
HO
O
OH
OH
Inositol
cycle
Nucleic
acids
HO
O
P
OH
OH
HO
O
OH
OH
9
10
Nucleoside-3'-phosphate
Inositol-3-phosphate
HO
Base
OH
O
O
Disubstituted
carbon atom
HO
CF2
OH
P
OH
OH
F2C OH
HO
O
P
OH
OH
OH
1
1a
12a

Molecules 2005, 10
1053
A particular feature of these molecules is the disubstitution of the carbon atoms bearing the CF
2
moiety. A number of methodologies allowing the preparation of compounds characterized by this
particular substitution pattern have been published and are outlined in Scheme 4 [10]. Early on, we
chose to develop our own radical approach, and to use Berkowitz methodology relying on the
sequential addition of phosphonodifluoromethyl anion on ketone and radical mediated deoxygenation
process of the adduct.
Scheme 4
R1
R2
O
P
O
P
EWG
OR3
OR3
OR
OR
CF2
+
LiF C
2
+
R
(Percy, 1995)
(Piettre; Motherwell, 1996)
R1
R2
O
P
OR3
OR3
CF2
R1
R2
O
OR3
OR3
O
+ LiF C
P
2
R1
O
CF P(O)(OR )
2 2
2
(
Berkowitz, 1996)
(
Percy, 2000)
3
CF P(O)(OR )
2
2
3
R1
CF P(O)(OR )
Organometallic-mediated
SN '-like coupling reactions
2
2
R1
or
+
+
2
R2
Percy; Shibuya, Yokomatsu, 1998-2003)
(
Shibuya, Yokomatsu
003)
2
(
A) MONs
a) Radical approach
Retrosynthetic analysis of the target nucleoside–3’–phosphate analogues 12 pointed the finger at
the probable wrong stereodirecting effect of the purine or pyrimidine base in the hydrogen quench of
the radical adducts 15 generated from the addition of phosphonyl or phosphonothioyl radicals 13 onto
difluoroalkene 14 (Scheme 5); this would produce the undesired phosphono(thio)difluoromethyl
derivative 16. Hence it was decided to start from a carefully protected furanose derivative 19. A 1,2–
isopropylidene protecting group was chosen to take advantage of the steric hindrance generated by the
angular methyl group and thus favor the formation of the desired diastereomer 18 [11].

Molecules 2005, 10
1054
Scheme 5
HO
Base
PO
O
O
PO
Base
X
O
OR
(RO)2P
+
OH
OP
(
RO)2P
X
CF2
(RO)2P
X
CF2
F2C
OP
1
1
3a: X = O
3b: X = S
14
12a: X = O
2b: X = S
17a: X = O
17b: X = S
1
PO
O
O
PO
Base
PO
O
O
O
(
RO)2P
X
CF2
O
(
EtO)2P(X)-CF2
OP
(EtO)2P(X)-CF2
O
1
1
8a: X = O
8b: X = S
2
2
0a: X = O
0b: X = S
1
5a: X = O
5b: X = S
1
PO
RO)2P CF2
Base
PO
O
X
O
(
(
RO)2P
+
O
X
H
OP
2
F C
O
1
6a: X = O
6b: X = S
13a: X = O
13b: X = S
19
1
For synthetic purposes, phosphonyl 13a and phosphonothioyl 13b radicals are usually generated
either from the corresponding phosphite 21a and thiophosphite 21b, respectively. These radicals, along
with phosphonodithioyl 13c and phosphonotrithioyl 13d radicals, can also be produced from the
selenophosphate derivatives 22 (Scheme 6) [11l,12].
Scheme 6
X
X
X
Radical initiator
Radical initiator
RO P
H
RY P
RY P SeC H5
6
OR
YR
YR
2
1a: X = O
1b: X = S
13a: X = Y = O
22a: X = Y = O
22b: X = S, Y = O
22c: X = O, Y = S
22d: X = Y = S
2
13b: X = S, Y = O
1
1
3c: X = O, Y = S
3d: X = Y = S
Radicals 13a and 13b have been shown to add onto β,β–disubstitued–α,α–difluoroalkenes under
radical-chain conditions to yield the expected adducts in fair to good yields [10b-c, 10h, 10j]. The use
of thiophosphite usually results in the formation of adducts in substantially higher yields (when
compared to phosphites). Radicals 13c and 13d, however, were found to be unreactive towards these
substrates.
In our case, the readily available glucofuranose derivative 23 was selected as starting material and
oxidized into 24 [13], which was then conveniently transformed into the corresponding difluoroalkene
2
5 using literature methods (Scheme 7) [14]. However, attempts to add phosphorus-centered radicals
3 onto this substrate by either method completely failed to furnish the desired adducts 26, the
1
substrate being recovered [15]. Other cyclic difluoroalkenes (e.g. 19, 27 and 28) displayed a similar
behavior (Figure 3). The successful addition of phosphonyl radicals onto gem-difluoro enol ether

Molecules 2005, 10
1055
derivatives 29-31 by the groups of Motherwell and Sinaÿ may be indicative of the electrophilic nature
of the radicals [10c, 10h, 10j].
Scheme 7
O
O
O
O
O
O
O
O
X
O
OH
O
O
P(OEt)2
O
Dess-Martin
81%
O
O
O
(X = O, S)
O
O
O
F2C
O
O
(EtO) P(X)-CF2
O
2
2
3
24
25
26a: X = O
26b: X = S
(
from α-D-glucose)
Figure 3
DPTBS O
O
PO
O
O
O
OMe
O
O
O
O
F2C
F2C
F2C
O
O
OMe
28
19
27
O
H
O
CF2
O
O
O
BnO
BnO
CF2
CF2
OTBDMS
O
OBn
O
TBDMSO
O
O
OBn
29 (14%)
30 (23%)
31 (56%)
(Sinaÿ, 1999)
(
Motherwell, 1997)
A literature search for alternate precursors of phosphorus-centered radicals quickly led to the
identification of hypophosphorous acid 32a, featuring two P-H bond and only one phosphorus–oxygen
single bond. Indeed, the work of Nifant’ev has demonstrated the possibility of adding
hypophosphorous acid onto alkenes 33 under radical-chain reaction conditions, thereby producing H–
phosphinates 34 (single addition) or phosphinates 35 (double addition) (Scheme 8) [16].
Scheme 8
O
R2
R3
R2
R3
O
P
O
H
P
OR1
+
R2
H
H
R2
P
Radical initiator
OR1
OR1
R3
R3
1
3
3
2a: R = H
33
34
35
1
H-Phosphinates
Phosphinates
2b: R = Na
Nifant’ev also showed that the sodium salt 32b of hypophosphorous acid behaves similarly, a
feature of importance in the context of acid-sensitive protected substrates. In addition, we were
especially interested in testing hypophosphorous acid (or its sodium salt) addition onto gem-
difluoroalkenes, as this would open the door to both hitherto unreported α,α-difluoro–H–phosphinates,
and α,α–difluorophosphinates, a functional group for which no general method of synthesis has been
reported to date.

Molecules 2005, 10
1056
Thus, refluxing a solution of either difluoroalkene 36-39 and 32b in methanol in the presence of a
catalytic amount of tert–butyl peroxypivalate (40) resulted in a complete consumption of the sustrates.
Hydrolysis and work-up led to the isolation of the first α,α–difluoro–H–phosphinates 42-45 in good
yields (Scheme 9). The compounds were found to be both air and room temperature stable for
3
1
extended periods of time and displayed a one-bond P–H coupling constant of about 560 Hz ( P-NMR
spectrometry). Infrared spectra featured both P=O and P–H bonds signals, indicative of a P(V) species.
Other successful radical initiators included commercially available tert-butyl 2-ethylhexyl
peroxycarbonate and the triethylborane/air system [17].
Scheme 9
H C
O
P
H C
3
O
H C
3
H PO Na (32b)
2
2
3
TFA
C
CF2
CH CF2
H
CH CF2
P
H
tBuOOC(O)tBu
40)
ONa
OH
(
3
6
41
42
75%)
(
O
P
O
O
CF2
CF2
H
CF2
CF2
CF2
P
H
CF2
P
H
OH
OH
OH
37
38
39
43
85%)
44
(83%)
45
(79%)
(
Homolytic cleavage of the phosphorus hydrogen single bond present in difluoro–H–phosphinates
was achieved by heating the compounds in the presence of both an alkene (1 equiv) and a radical
initiator. Thus, for instance, H–phosphinate 43 sequentially reacted with either alkene 46 or 47, and
diazomethane (CAUTION) to deliver methyl α,α–difluorophosphinates 50 and 51 in good isolated
yield (Scheme 10).
Scheme 10
O
2 2
CH N
O
tBuOOC(O)tBu
CF2
P
CF2
P
OH
OMe
46
48
50
80%)
O
(
CF2
P
H
OH
4
3
tBuOOC(O)tBu
O
2 2
CH N
O
CF2
P
CF2
P
OH
OMe
49
51
53%)
4
7
(
The documented tautomeric equilibrium between H–phosphonates 52 and phosphites 53 led us to
develop alternate ionic routes to α,α–difluorophosphinates [18]. Treatment of α,α–difluoro–H–
phosphinates 43 and 44 with trimethylsilyl chloride in the presence of pyridine afforded the air-
sensitive O,O–bissilylated phosphites 54 and 57, which were then subjected into interaction with
methyl vinyl ketone 55 and pivalaldehyde 58, respectively (Scheme 11). Hydrolysis and esterification
with diazomethane (CAUTION) cleanly delivered difluorophosphinates 56 and 59 [19,20]. Of
particular note is the fact that the two fluorine atoms present in the O,O–bissilylated phosphites did not
prevent the phosphorus atom from reacting with electrophilic centers.

Molecules 2005, 10
1057
Scheme 11
O
P
O-H
O-H
R
H
R
P
O-H
5
2
53
1
)
(55)
O
P
O
OSiMe3
OSiMe3
TMSCl
O
CF2
P
H
CF2
P
CF2
pyridine
2) CH2N2
OMe
O
O-H
4
3
54
56
57%)
(
α,α-Difluorophosphinate
H
O
1)
(58)
O
OSiMe3
TMSCl
P
O
CF2
P
H
CF2
P
CF2
pyridine
OSiMe3
2) CH N
2 2
MeO OH
O-H
44
57
59
64%)
(
α,α-Difluoro-α'-hydroxyphosphinate
Alternatively, treating these bissilylated phosphites with either oxygen or sulfur led to the isolation
of difluorinated phosphonic and phosphonothioic acids, respectively, as exemplified in Scheme 12.
Scheme 12
H3C
O
CH CF2
P
OH
1) O2
2) H2O
OH
TMSCl (3 equiv)
pyridine
H C
O
P
H C
61
3
3
OSiMe3
OSiMe3
CH CF2
H
CH CF2 P
ONa
4
1
60
H3C
CH CF2
S
P
1
) S8
2) H2O
OH
OH
6
2
O
1) O2
2) H2O
CF2
P
OH
TMSCl (3 equiv)
pyridine
OH
O
OSiMe3
OSiMe3
64
CF2
P
H
CF2
P
OH
S
P
43
63
1) S8
CF2
OH
2) H2O
OH
65
More importantly, when hypophosphorous sodium salt 32b was reacted with difluoroalkene 19a (a
substrate inert to the action of both phosphite and thiophosphite) under the above conditions, a clean
1
reaction occurred and adduct 67 was isolated in 78% yield as a single diastereomer (Scheme 13). H-
NMR spectrometry indicated the structure to be as depicted, indicative of the efficiency of the angular
methyl group of the 1,2–isopropylidene unit in keeping hydrogen quenching of radical-adduct 66 from
occurring on the concave face [12]. Transformation into O,O–bissilylated phosphite 68 and sequential
treatment with oxygen and water delivered the expected phosphonic acid, isolated in the form of its
disodium salt 69a in excellent yield. An analogous sequence involving sulfur in place of oxygen
afforded the corresponding phosphonothioate 69b.

Molecules 2005, 10
1058
Scheme 13
O
O
O
O
O
O
O
O
O
O
O
TMSCl,
Pyridine
O
H2PO2Na
O
O
O
O
F2C
O
tBuC(O)OOtBu
0°C
H
CF2
O
H
CF2
O
Me SiO
3
CF2
O
P
6
P
P
NaO
NaO
O
O
Me3SiO
1
9a
66
67
78%; de = 95%)
68
(
1
2
) O2
) H2O
1) S8
2) H2O
O
O
O
O
O
O
O
O
NaO
NaO
O
NaO
NaO
CF2
O
CF2
P
P
O
S
6
9a
69b
(59%)
(
86%)
Similarly, difluoroalkene 19b and hypophosphorous acid sodium salt reacted together at room
temperature in presence of Et B and oxygen (air) as the radical initiator to furnish the α,α–difluoro–
3
H–phosphinate, sodium salt 70a, conveniently purified as its triethylammonium salt 70b by ion
exchange and simple extraction with ethyl acetate (Scheme 14) [11l]. An additional step forward was
accomplished when this compound could be esterified with alcohol 71 in the presence of
trifluoroacetic acid and dicyclohexyl carbodiimide (DCC). Treatment of the resultant H–phosphinate
72 with a mixture of sulfur, TMSCl and pyridine gave difluorophosphonothioate 73, isolated in 50%
yield as a 1:1 diastereomeric mixture at the phosphorus center [17].
Scheme 14
HO
OMe
O
(
71)
ClBz
O
ClBz O
O
O
O
O
O
O
O
ClBz
O
O
ClBz
O
O
F2C
F C
O
O
H PO Na
S8
2
2
2
O
O
P
H
S P OR
O
O
Et3B, O2
5°C
H
O
DCC, TFA
O
OMe
O
TMSCl,
pyridine
CF2
OMe
O
F2C
O
P
O
2
RO
O
O
O
70a: R = Na+
0b: R = Et3NH+
3%
de = 95%)
19b
72
73
(50%)
2 diastereomers
at phosphorus)
7
(
6
(
The possibility of using the Nifant’ev protocol to link two furanoses on position 3 and 5 with a
dimethylphosphinoyl (i.e. generate compound 75, Scheme 15) was also verified. A double
disconnection of the two phosphorus-carbon bonds points the finger at alkenes 75 and 77 as protected
starting substrates and at a synthetic equivalent of diradical 76. As demonstrated earlier, a process
using hypophosphorous acid sodium salt in two sequential radical addition reactions would achieve
this. Two possible syntheses exist, depending on which alkene 75 or 77 is used first. Here again, a
stereocontrol is needed in the hydrogen quench of any radical-adduct generated from alkene 75. The

Molecules 2005, 10
1059
demonstrated efficiency of a 1,2–isopropylidene unit (see above) led us to consider furanose 80, along
with 32b and 81.
Scheme 15
R1
PO
O
O
P
H
H
OH
77
H C OP
2
O P OH
H
O
O
78
O
+
R1
PO
O
R1
PO
O
O
7
5
80
O
OP
OH
H C OP
2
O
P
H
H
O P OH
O
P
76
ONa
+
32b
R2
H C
2
O
R2
O
OMe
O
7
7
8
1
PO OP
PO OP
7
4
O
O
H
P
O
OH
R2
H C
7
5
2
O
O
H
P
OH
H
PO OP
7
9
Reaction of a solution of alkene 80 in EtOH with hypophosphorous acid sodium salt 32b (5 equiv)
in the presence of di-tert–butyl peroxide at 120°C resulted in the quantitative formation of H–
phosphinate 82a isolated in the form of its methyl ester 82c (diazomethane) as a single disatereomer
(
[
Scheme 16). Similarly, alkene 81 generated the expected adduct 83c in virtually quantitative yield
21].
Scheme 16
O
O
O
O
O
P
O
H
H
O
ONa (5 equiv)
O
O
O
O
t-BuOOt-Bu
O
H
P
EtOH, 120°C
OR
80
82a: R=Na
8
2b: R=H
82c: R=Me ( 97%; de>95%)
OMe
O
H
P
O
P
OMe
O
RO
O
H
H
O
ONa (5 equiv)
OMe
O
O
O
RO
P O
O
+
O
t-BuOOt-Bu
OMe
EtOH, 120°C
O
O
O
O
8
1
83a: R=Na
84a: R=Na
8
8
3b: R=H
3c: R=Me (98%)
84b: R=H
84c: R=Me (traces)

Molecules 2005, 10
1060
Both H–phosphinate 82c and 83c were then subjected to the action of tert–butyl peroxopivalate in
the presence of alkene 81 and 80, respectively. A 1:3 mixture of the expected methyl phosphinate 85
and phosphinic acid 86 were obtained in each case, which was treated with diazomethane (CAUTION)
to deliver methyl ester 85 in 82 and 70% isolated yield (Scheme 17). Phosphinic acid 86 was probably
formed through internal hydrogen quenching of the radicals–adducts by the methyl group of
phosphinic ester, loss of formaldehyde, hydrogen quenching of the thereby-formed phosphorus radical
and oxidation of the resultant H–phosphine oxide (through its P(III) tautomer) during work-up. These
results thus demonstrated that the Nifant’ev protocol can be efficiently exploited to prepare 3–
furanosyl–5’–furanosylphosphinate through a tandem sequential radical process.
Scheme 17
H
MeO
P
O
O
O
OMe
O
O
tBuC(O)OOtBu
O
+
16h, 55°C
O
O
O
O
O
O
(82%)
O
O
O
8
3c
80
O
O
MeO
O
HO
O
P
+
P
O
O
OMe
OMe
O
O
O
O
O
O
O
O
OMe
O
O
O
tBuC(O)OOtBu
+
8
5
86
O
O
16h, 55°C
(70%)
O
O
H
P
CH N
2
2
MeO
8
2c
81
b) Ionic approach
An alternate approach, relying on the ionic introduction of the phosphonodifluoromethyl unit was
also considered. The retrosynthetic analysis of our target molecules 12a is outlined in Scheme 18.
Based on Berkowitz’ work, a two-step process calling for (i) the addition of reagent 87a onto a keto
group and (ii) a radical-based deoxygenation of the resultant adduct was developed [10d]. Here again,
the stereodirecting effect of the base was expected to result in a predominant attack of nucleophilic
species 87a on the convex face of the molecule to generate 88. However, the same stereodirecting
effect acting on the hydrogen quenching of radical intermediate 15a during the deoxygenation step
would probably provide the undesired diastereomer 16a. As this last step would probably play a
crucial role in installing the requisite configuration on carbon C3’, it was decided to start from a
furanose derivative featuring a hindered concave face to force the hydrogen quenching to occur on the
convex f ace (thereby positioning the phosphonodifluoromethyl unit on the desired face of the ring),
and to introduce the base in a subsequent step, thus paralleling the radical approach (see above).
However, interacting either 87a or the analogous organomagnesium reagent 87b [22], with ketone 24
never resulted in a clean transformation, and product 89 was isolated in yields lower than 20%, along
with substantial amounts of bis(O,O–diethylphosphono)difluoromethane and the hydrate of the starting
ketone (Scheme 19) [15].

Molecules 2005, 10
1061
Scheme 18
PO
Base
O
PO
Base
O
+
-
+
Li CF -P(O)(OR)2
2
OP
(RO)2P CF2
O
OP
87a
O
12a
P = Protecting group
PO
Base
PO
HO
Base
O
O
(
RO)2P CF2
O
OP
5a
OP
(
RO)2P CF2
O
1
88
PO
Base
O
(
RO)2P CF2
O
H
OP
6a
1
Scheme 19
O
O
O
CF -P(O)(OEt)
2
O
2
O
O
1
) LiCF -P(O)(OEt) (87a)
2 2
O
or
ClMgCF -P(O)(OEt) (87b)
O
O
O
HO
O
2
2
24
89
< 20%; difficult to purify)
(
We next considered the thioanalogues 91a-91c of reagent 87a (Scheme 20, Figure 4). The
thermally more stable lithiated reagent 91a is conveniently prepared either by treatment of
difluoromethylphosphonothioate 90 with lithium diisopropylamide, or by interacting bromodifluoro-
phosphonothioate 92 and tert–butyllithium [23]. Reagent 91a has been shown to be stable at
temperatures up to –20°C, thus favorably comparing to phosphonate 87a, stable only at –78° C or
below [24].
Scheme 20
S
P
S
P
S
P
LDA
t-BuLi
OEt
OEt
OEt
OEt
OEt
OEt
HF C
LiF C
BrF C
2
2
2
90
91a
92
1
R -C(O)R2
S
P
HO
1
OEt
OEt
R
CF2
R2
9
3
1
2
(
R = alkyl, aryl; R = H, alkyl;
yields: 63-85%)

Molecules 2005, 10
1062
The reactivity of 91a parallels that of 87a, and was found to smoothly react with ketones and
aldehydes to deliver the expected adducts 93 in good yields. Lithiated reagents 91b and 91c were
however found to be thermally unstable, even at –78°C; compounds encompassing these two last
functional groups were therefore prepared through phosphorus group manipulation [25].
Figure 4
O
P
S
P
SEt
SEt
SEt
SEt
LiF C
LiF C
2
2
9
1b
91c
Reacting ketone 24 with 91a resulted in a clean consumption of the starting material and the
formation of a single product 94, isolated in 78% yield (Scheme 21). NMR Spectrometry analysis
proved the diastereoselection of the process to be over 99%. Again, we assumed that the addition of
the nucleophile had occurred on the convex face, i.e. opposite to the angular methyl group.
Deoxygenation of carbon 3 was achieved by sequential treatment of alcohol 94 with first n–butyl-
lithium and O–methyl oxalyl chloride, then tri–n–butyltin hydride in the presence of azobis-
isobutyronitrile (catalytic amount). Product 97 was isolated in 79% yield as a single diastereomer.
Analysis of the NMR data confirmed the stereochemical inversion of the phosphonothiodifluoromethyl
unit from the convex face to the concave one, the result of hydrogen quenching of the radical adduct
96 exclusively from the convex face. Carbon 6 was then removed from the glucofuranose skeleton by
selective deprotection, oxidation and reduction to furnish alcohol 100 which was transformed into
triacetate 101 in 13% overall yield from ketone 24.
Scheme 21
O
O
O
O
O
O
LiCF P(S)(OEt)
CF2-P(S)(OEt)2
O
CF -P(S)(OEt)2 n-Bu SnH
2
2
n-BuLi
ClCOCOOMe
2
O
3
O
AIBN
9
1a
O
76-80%
O
O
O
O
HO
O
O
MeO C-COO
2
2
4
94
95
(de > 99%)
(de > 99%)
O
O
O
O
HO
HO
O
O
O
NaIO , 25°C
AcOH, 75°C
4
O
79%
2 steps)
O
O
(
(
EtO) P(S)-CF2
O
(EtO) P(S)-CF2
O
(EtO) P(S)-CF2
O
2
2
2
96
97
de > 99%)
98
(
CHO
O
AcO
OAc
HO
Ac2O, AcOH,
O
O
NaBH , 25°C
H SO , 25°C
2
4
4
O
O
4
2%
58%
(
3 steps)
(EtO) P(S)-CF2 OAc
2
(
EtO) P(S)-CF2
O
(EtO) P(S)-CF2
O
2
2
99
100
101

Molecules 2005, 10
1063
Attempts to improve both the synthesis and the overall yield started with a critical analysis of this
route. This led to the identification of several drawbacks impeding its use in larger scale preparation of
101. Thus, LDA or tert–butyllithium were used as base, and chromatography had to be used to obtain
several intermediates in the pure form. The involvement of the somewhat hazardous Dess–Martin
periodane was also considered as a limitation to the development of a larger scale preparation.
Moreover, the 6–carbon starting substrate implied additional steps to obtain the five–carbon skeleton
of ribofuranose 101. An improved synthesis was thus worked out, starting from α–D–xylose derivative
102 (Scheme 22). The required 1,2–isopropylidene protection was completed by the installation of a
para–chlorobenzoyl unit on the 5’–hydroxyl group. An additional feature of para–chlorobenzoate
derivatives lies in their propensity to crystallize (see below) [26].
Scheme 22
BrCF -P(S)(OEt) (92)
2
2
i-PrMgCl
HO
4-ClBzO
Trichlorocyanuric acid 4-ClBzO
O
OH
O
OH
O
ClMgCF -P(S)(OEt) (91b)
4
-ClBzCl, NEt3
79%
TEMPO (cat)
2
2
O
O
77%
72%
O
O
O
O
O
1
02
103
104
4
-ClBzO
1) i-PrMgCl, THF, -45°C,
CF -P(S)(OEt)2 ClC(O)C(O)OMe
Ac O, AcOH,
H2SO4, 25°C
2
O
2
4
-ClBzO
4-ClBzO
OAc
2) n-Bu SnH, AIBN
O
O
3
O
64%
O
77%
HO
O
(EtO) P(S)-CF2
O
(EtO) P(S)-CF2 OAc
2
2
1
05
106
(de > 99%)
107
(
de > 99%)
Alcohol 103 was oxidized by a mixture of trichlorocyanuric acid and 2,2,6,6–tetramethylpiperidine
N–oxide (TEMPO) in catalytic amount to deliver ketone 104 in 77% isolated yield [27]. Lithium
reagent 91a was replaced with the organomagnesium reagent 92b, readily prepared by treating
bromide 92 with 1.1 equivalents of freshly prepared iso–propyl magnesium chloride in diethyl ether
[
22]. For the reason cited above, adduct 105 was obtained as a single diastereomer. Again, radical
deoxygenation of this adduct resulted in a clean stereochemical inversion of the phosphonothio-
difluoromethyl moiety, and phosphonothioate 106 was obtained in 64% isolated yield (two steps) as a
single diastereomer. Treatment of this product with acetic anhydride in a mixture of acetic acid and
sulfuric acid removed the 1,2–acetonide and furnished triester 107 as a single stereoisomer. This
scheme resulted in the formation of a slightly different precursor with an improved overall yield of
21% from xylose derivative 102. Noteworthy is the fact that no chromatography separation is needed,
and that the preparation of 107 has been carried out on a 10-gram scale [28].
Stereoselective installment of the pyrimidine bases was achieved by using Vorbruggen’s
modification of the Hilbert–Johnson protocol while the purine bases were introduced by using the
procedures of Saneyoshi and Robins (Table 2) [29,30]. Isolated yields were good to excellent and a
complete diastereoselectivity was observed, as a result of the steric hindrance generated by both the 2–
acetoxy group and the 3–phosphonothiodifluoromethyl unit. It might be suggested that the longer P=S
bond (1.90 Å versus 1.53 Å for the P=O bond) allows the latter group to participate in the stabilisation

Molecules 2005, 10
1064
of the intermediate cation. Such an analogous participation has recently been described with 2,3–
dideoxy–3–phosphonothiofuranose derivatives [31]. Of particular note is the excellent regiochemical
control observed in the induction of the purine bases: a single regioisomer was found to have formed.
Table 2
AcO
OAc
Reagents,
Conditions
AcO
Base
AcO
Base
O
O
m-CPBA
O
(
EtO) P(S)-CF2 OAc
(EtO) P(S)-CF2 OAc
(EtO) P(O)-CF2 OAc
2
2
2
1
01
108
109
Yields
Yields
(%)
Entry
Base
Reagent
Conditions Products
Products
109a
(
%)
TMSOTf,
O
OSiMe3
N
NH
ClCH CH C
2
2
(
T)
N
O
1
2
3
l,
108a
108b
108c
92
77
54
91
N
OSiMe3
2
5°C
TMSOTf,
O
OSiMe3
N
NH
ClCH CH C
2
2
(
U)
N
O
l,
81
95
109b
109c
N
OSiMe3
2
5°C
NH2
N
TMSOTf,
2
ClCH CH C
NHSiMe3
N
(
C)
2
N
O
l,
N
OSiMe3
8
0°C
NH2
NH2
N
SnCl4,
CH CN,
25°C
TMSOTf,
N
N
N
N
(
A)
3
N
N
H
N
4
5
108d
108e
87
66
109d
109e
73
66
OCON(C H )
6
5
2
O-C(O)-N(C H )
6 5 2
N
N
ClCH CH C
N
(G)
NHAc
N
2
2
N
N
N
H
N
NHAc
l,
80°C
Interconversion of difluorophosphonates 5d and difluorophosphonothioates 5a can easily be
achieved in high yields and require either the action of Lawesson’s reagent 110 (P=O→P=S) or a mild
oxidating agent (P=S→P=O) such as dimethyl dioxirane 111, perfluorooxaziridine 112 or m–CPBA
(
Scheme 23) [32].

Molecules 2005, 10
1065
Scheme 23
S
S
MeO
P
P
OMe (110)
S
S
O
71-93%
S
OR2
OR2
OR2
OR2
1
1
R CF P
R CF P
2
2
n-C3F7
F
5d
(111) or
N
O
(112) or
C(O)OOH
5a
O
n-C4F9
O
Cl
7
7-97%
In this program, nucleotide analogues 108, encompassing the phosphonothiodifluoromethyl unit,
were easily transformed into the corresponding phosphonates 109 in good to excellent yields by simple
treatment with m–CPBA (Table 2). Pyrimidine derivatives 109a-109c were then fully deprotected by
reaction with trimethylsilyl bromide, hydrolysis and treatment with methanolic ammonia; the final
products 114a-114c were isolated as their disodium salts in yields ranging from 70-77% (Scheme 24).
Scheme 24
AcO
Base
1) TMSBr (6 equiv)
0°C, 1 h
AcO
Base
HO
Base
O
O
O
8
NH3, MeOH
7
1-77%
(
two steps)
2
) H O
2
F2C OAc
F2C OAc
F2C OH
O
P
O
P
O
P
OEt
OH
OH
EtO
HO
HO
1
09
113
114
O
NH2
N
O
NH
NH
a: Base =
; b: Base =
; c: Base =
N
O
N
O
N
O
Comparison of both radical and ionic approaches shows them to be equally effective in
stereoselectively producing a protected furanose derivative bearing a phosphono(thio)difluoromethyl
unit on carbon 3 (Scheme 25).
Scheme 25
CF -P(S)(OEt)
2
O
2
PO
Overall yield = 44%
LiCF P(S)(OEt)2
2
O
PO
PO
O
O
HO
O
O
O
O
O
O
2
(EtO) P(X)-CF2
O
PO
O
H PO Na
2
2
X = O, S
O
CF Br , HMPT
Radical
iniator
2
2
Overall yield = 43%
F2C

Molecules 2005, 10
1066
Cyclitols
Radical approach
L–Quebrachitol (115) is a waste-product of the natural rubber industry and can be obtained from
the serum (aqueous phase) of the latex [33]. Protection of the natural product as biscyclohexylidene
acetal derivative 116 and oxidation of the remaining hydroxyl group under the conditions of Swern
delivered ketone 117 with five of the six stereocenters present in inositol-3-phosphate. Ketone 117 was
next transformed into gem-difluoroalkene 118 by using the three-step procedure published by
McCarthy [34]. When a mixture of hypophosphorous acid sodium salt 32b and substrate 118 were
heated at 55°C in the presence of tert–butyl peroxopivalate as radical initiator, a complete
consumption of the substrate was observed and products 119a and 119b were formed (9:1
diastereomeric ratio) (Scheme 26). The neighboring axial ether group was thus efficient in directing
the hydrogen quenching to occur on the face opposite to that carbon-oxygen bond. The major product
119a constitutes a key intermediate en route to the phosphono(thio)difluoromethyl analogues of
inositol–3–phosphate.
Scheme 26
OMe
OH
O
O
Swern
OH
OH
O
O
O
O
HO
HO
O
oxidation
H+
96%
MeO
MeO
MeO
OH
95%
O
O
1
15
116
117
L-Quebrachitol)
O
O
1
2
3
) LiCF2SO2C6H5 (60%)
) MsCl (70%)
) SmI2 (91%)
H2PO2Na
tBuC(O)OOtBu
55°C
O
O
P
O
H
O
O
H
P
CF2
O
O
CF2
O
O
F2C
NaO
NaO
+
MeO
MeO
MeO
O
O
O
118
119a
119b
9
:1
(
76%)
Quinic acid 120 also represents a useful chiral source for the preparation of modified cyclitols
(Scheme 27) [35]. Protection of the three secondary hydroxyl groups was accomplished using
literature procedure and the resultant methyl ester 122 was subjected to a decarboxylating oxidation to
deliver ketone 123. Transformation into gem–difluoroalkene 124 yielded the substrate on which the
radical methodology was applied. The phosphorous-centered radical generated from 32b by the
standard procedure once again delivered the expected difluoro–H–phosphinates 125, this time as a 7:3
diastereomeric mixture due to the relative remoteness of the stereodirecting, axial silyl ether group.

Molecules 2005, 10
1067
Scheme 27
O
HO COOH
HO
HO
COOMe
COOMe
O
TBDMS-Cl, NEt3
NaIO4
O
HO
OH
MeOH, CSA
HO
O
TBDMSO
OMe
OMe
OH
O
O
MeO
MeO
1
20
121
122
Quinic acid
O
O
H
H
H2PO2Na
tBuC(O)OOtBu
P
P
O
CF2
CF2
CF2
O
NaO
NaO
CF2Br2, HMPT
55°C
+
TBDMSO
O
TBDMSO
O
TBDMSO
O
TBDMSO
O
OMe
OMe
OMe
OMe
O
O
O
MeO
MeO
MeO
MeO
123
124
125a
125b
7
:3
(
70%)
Ionic approach
The ionic approach was also tested on a pyranose derivative. Indeed, the possible use of the Ferrier
reaction makes of pyranose derivatives a potentially useful entry into the preparation of cyclitol
analogues [36]. Thus, α–D–glucose derivative 126 was protected and oxidized using a three-step
sequence of transformations depicted in Scheme 28. The resultant ketone 129 was subjected to the
action of lithiated reagent 91a at -78°C, and the corresponding alcoholate was quenched with O–
methyl oxalyl chloride. Difluorophosphonothioate 130 was isolated in 70% yield as a single
diastereomer. Treatment of 130 with tri-n–butyltin hydride and AIBN in toluene at 100°C gave
exclusively product 131 in 57% isolated yield. No stereochemical inversion of the phosphonothio-
difluoromethyl group occurred this time, however. Apparently, the steric hindrance generated by the
methoxy group forces the radical-adduct to adopt conformation 133 rather than 132, thereby leading to
131 and not 134 (Scheme 29).
Scheme 28
C H COCl
6
5
C H CH(OEt)
APTScat, DMF
Me2SnCl2(cat), K2CO3
THF, 25°C
6
5
2
Swern
oxidation
O
OMe
OH
O
OMe
OH
O
OMe
OCOC H
6 5
HO
HO
O
O
6
C H
5
O
C H
6
O
5
OH
OH
OH
1
26
127
79%)
128
(99%)
(
1
2
) LiCF P(S)(OEt) ,
THF, -78°C
2
2
n-Bu SnH, AIBN
toluene, 100°C
3
O
O
OMe
OCOC H5
O
OMe
O
OMe
) ClCOCO Me
2
O
O
O
O
C H
6
O
C H
6
OCOC H
6 5
C6H5
O
OCOC6H5
5
6
5
MeO2COCO CF2P(S)(OEt)2
CF P(S)(OEt)
2 2
1
29
130
(70%)
131
(57%)
(
99%)

Molecules 2005, 10
1068
Scheme 29
S
EtO
EtO
O
O
F
P
P
F
F
RO
OMe
RO
OMe
EtO
OEt
F
S
1
32
133
O
OMe
OCOC H5
O
OMe
OCOC H5
O
O
C H
6
O
5
6
C H
6
O
5
6
CF P(S)(OEt)2
2
CF P(S)(OEt)2
2
134
131
X–ray analyses
Three of the above α,α–difluorophosphonothioates (105, 131, and the benzyl analogue 135 of 131)
were crystalline solids. Careful crystallization led to the growth of monocrystals suitable for X–ray
analysis. Figure 5 shows the structures and ORTEP graphics of these three compounds and Table 3
include
selected
data
obtained
from
the
X–ray
analyses,
along
with
data published by Chambers and O’Hagan on compounds [3a,3c].
Figure 5
4-ClBzO
O
OMe
OCOC H
O
OMe
OCH C H
CF -P(S)(OEt)
2
2
O
O
O
C H
6
O
C H
6
O
5
6
5
5
2
6
5
O
CF P(S)(OEt)
CF P(S)(OEt)2
2
2
2
HO
O
131
135
105
C12
C13
C12
C14
C15
C10
C13
C11
C14
C15
C10
O6
C11
C9
O5
C8
C8
O4
O4
C9
O5
C6
C5
C7
C7
C26
O8
C6
C25
C26
O3
C3
C2
F1
O3
C3
F2
C25
O7
P1
C22
C5
C4
C22
F1
P1
C4
S1
S1
F2
C2
O2
O6
O2
O7
O1
O1
C23
C24
C1
C1
C16
C23
C16
C24
C21
C20
C17
C17
C21
C20
C18
C18
C19
C19

Molecules 2005, 10
1069
Although the three phosphonothioates are structurally and electronically different than zwitterions
a and 6d, the data clearly indicate a widening of the angle formed by C–CF and CF –P bonds (Table
). The bond lengths a and b themselves do not vary much from the values reported for
6
2
2
3
difluorophosphonates (e.g. 6d). The length c of the phosphorus-sulfur double bond is, expectedly
much larger than the oxygenated counterpart in 6a and 6d.
Table 3
Y
c
1
2
a
b
6a: R = H NCH ; X = O; R = H
2 2
R1
OR2
X
P
1
2
6
b: R = H NCH ; X = CF ; R = H
2
2
2
OR2
α
Compound
X
Y
S
a (Å)
1.88
1.86
1.86
1.59
1.85
b (Å)
1.51
1.52
1.55
1.43
1.50
c (Å)
1.90
1.91
1.91
1.52
1.53
α (°)
120.3
122.5
117.5
118.5
116.5
105
131
135
CF
CF
CF
O
2
2
2
S
S
6a
O
O
6
d
CF
2
Conclusions
Our journey into the field of new phosphate isosters related to the difluorophosphonates has
allowed the preparation of new functional entities such as the difluorinated phosphonothioates,
phosphonodithioates and phosphonotrithioates, in which the sulfur atom(s) may either play a positive
role in the course of a reaction, or have a deleterious effect on the outcome of a desired transformation.
Thus the presence of the double-bonded sulfur atom in reagents 91 induced a dramatic, positive effect
in the condensation on ketone 19, 24 and 129, which ultimately translated into a stereocontrolled
synthesis of the phosphonodifluoromethyl analogues of nucleoside-3’-phosphates. Despite the positive
effect of sulfur on the addition of phosphonothioyl radical – when compared to the fully oxygenated
analogues –, it was simply not enough to induce an addition reaction on functionalized, cyclic gem–
difluoroalkenes (e.g. 19, 27 or 28). Hypophosphorous acid sodium salt 32b brings a solution to this
problem and displays a reactivity that allows the easy preparation of a variety of phosphate isosters
through the intermediary of α,α–difluoro-H-phosphinates. It also allows to stereoselectively link two
furanose units on carbon 3 and 5’ with a phosphinyl or a difluorophosphonothioyl unit, and to produce
new fully protected analogues of inositol phosphates. Work is in progress in this laboratory to fully
explore the possibilities offered by this chemistry.
Acknowledgements
We are grateful to Prof Alexandre Boutorine for helpful discussions. C. S. thanks the European
Union for a grant (Erasmus Program).

Molecules 2005, 10
1070
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