Synthesis of enzymatically stable analogues of GDP for binding studies with transducin, the G-protein of the visual photoreceptor

Article abstract of DOI:10.1021/jo9806207

The synthesis of five enzymatically stable analogues of guanosine diphosphate (GDP) has been carried out. The pyrophosphate moiety was mimicked in turn by the malonate, the acetophosphonate, the phosphonoacetate, the methylene-bis-phosphonate, and the imidodiphosphate groups. All the compounds were prepared via the synthesis of a transient fully protected nucleoside diphosphate analogue, and the final deprotection step was achieved by catalytic hydrogenolysis. The biological properties of the compounds have been evaluated toward transducin, the G-protein of the visual photoreceptor. Three guanosine imidodiphosphate derivatives bearing a linker at different positions on the sugar and on the base were then prepared and evaluated, giving some insight into the GDP binding site of transducin.

Full text of DOI:10.1021/jo9806207

7244
J . Org. Chem. 1998, 63, 7244-7257
Syn th esis of En zym a tica lly Sta ble An a logu es of GDP for Bin d in g
Stu d ies w ith Tr a n sd u cin , th e G-P r otein of th e Visu a l
P h otor ecep tor
Ste´phane Vincent,† Sonya Grenier,‡ Alain Valleix,§ Christian Salesse,‡ Luc Lebeau,*,† and
Charles Mioskowski*,†
Universite´ Louis Pasteur de Strasbourg, Laboratoire de Synthe`se Bioorganique associe´ au CNRS,
Faculte´ de Pharmacie, 74, route du Rhin - BP 24 - 67 401 Illkirch Cedex, France, Universite´ du
Que´bec a` Trois-Rivie`res, De´partement de chimie-biologie, Trois-Rivie`res (Que´bec) Canada, G9A 5H7, and
CEA - CE Saclay, Service des Mole´cules Marque´es, Baˆt. 547, De´partement de Biologie Mole´culaire et
Cellulaire, 91 191 Gif sur Yvette, France
Received April 3, 1998
The synthesis of five enzymatically stable analogues of guanosine diphosphate (GDP) has been
carried out. The pyrophosphate moiety was mimicked in turn by the malonate, the acetophos-
phonate, the phosphonoacetate, the methylene-bis-phosphonate, and the imidodiphosphate groups.
All the compounds were prepared via the synthesis of a transient fully protected nucleoside
diphosphate analogue, and the final deprotection step was achieved by catalytic hydrogenolysis.
The biological properties of the compounds have been evaluated toward transducin, the G-protein
of the visual photoreceptor. Three guanosine imidodiphosphate derivatives bearing a linker at
different positions on the sugar and on the base were then prepared and evaluated, giving some
insight into the GDP binding site of transducin.
In tr od u ction
phenomenon involved in the visual cascade, and the way
the receptor activates the effector still remains unknown.
Therefore, many efforts are directed toward structural
analysis of intermediate complexes between the different
protagonists of the visual cascade. In particular, our
group is interested in the GRâγ-GDP-rhodopsin com-
plex and aim to study its structure by electron crystal-
lography.18,19
Electron crystallography was initially developed for
structural analysis of membrane proteins, and perfor-
mance of the technique (minute amount of biological
material needed, no molecular weight limitation for the
protein to be studied, resolution reached...) rapidly
extended to the structural analysis of water soluble
proteins.20-22 The key point of the technique consists of
the concentration and organization into periodic two-
dimensional (2D) arrays of the investigated macromol-
ecule, mostly at the air-water interface. This can be
simply achieved by using Coulombic interactions between
the protein and charged lipids spread at the interface.23-26
Cells communicate with each other by a variety of
signal molecules that are detected by specific receptors
on the plasma membrane of the responding cell. Hun-
dreds of cell-surface receptors employ G-proteins to
initiate intracellular signaling chain reactions.1-6 These
G-proteins are heterotrimeric assemblies (GRâγ) and are
under the control of both GDP and GTP.7,8
One of the best characterized heterotrimeric G-protein-
coupled pathways is the visual cascade of the rod cell.9,10
In retinal rod outer segment, conversion of the light
signal into an electrical signal involves in series rhodop-
sin (the photon receptor), transducin (the retinal guanine
nucleotide-binding protein), and a specific phosphodi-
esterase (PDE, the effector) hydrolyzing the cytosolic
cGMP.11,12 Recently, structural data at near atomic
resolution were obtained from crystals of a modified
heterotrimeric transducin and independent subunits.13-17
These remarkable results, however, do not clarify every
* Corresponding author. lebeau@bioorga.u-strasbg.fr/mioskowski@
bioorga.u-strasbg.fr.
(13) Gaudet, R.; Bohm, A.; Sigler, P. B. Cell 1996, 87, 577-588.
(14) Lambright, D. G.; Sondek, J .; Bohm, A.; Skiba, N. P.; Hamm,
H. E.; Sigler, P. B. Nature 1996, 379, 311-319.
(15) Noel, J . P.; Hamm, H. E.; Sigler, P. B. Nature 1993, 366, 654-
662.
† Universite´ Louis Pasteur de Strasbourg.
‡ Universite´ du Que´bec a` Trois-Rivie`res.
§ CEA - CE Saclay, Service des Mole´cules Marque´es.
(1) Hille, B. Neuron 1992, 9, 187-195.
(16) Sondek, J .; Lambright, D. G.; Noel, J . P.; Hamm, H. E.; Sigler,
P. B. Nature 1994, 372, 276-279.
(2) Birnbaumer, L.; Abramowitz, J .; Brown, A. M. Biochim. Biophys.
Acta 1990, 1031, 163-224.
(17) Sondek, J .; Bohm, A.; Lambright, D. G.; Hamm, H. E.; Sigler,
P. B. Nature 1996, 379, 369-374.
(3) Neer, E. J . Cell 1995, 80, 249-257.
(4) Neubig, R. R. FASEB J . 1994, 8, 939-946.
(5) Yarfitz, S.; Hurley, J . B. J . Biol. Chem. 1994, 269, 14329-14332.
(6) Hamm, H. E.; Gilchrist, A. Curr. Op. Cell Biol. 1996, 8, 189-
196.
(18) Unwin, P. N. T.; Glaeser, R. M. Annu. Rev. Phys. Chem. 1985,
36, 243.
(19) Chiu, W.; Schmid, M. F.; Prasad, B. V. V. Biophys. J . 1993, 64,
1610-1625.
(20) Uzgiris, E. E.; Kornberg, R. D. Nature 1983, 301, 134-136.
(21) Lebeau, L.; Schultz, P.; Ce´lia, H.; Me´sini, P.; Nuss, S.; Klinger,
C.; Olland, S.; Oudet, P.; Mioskowski, C. In Handbook of nonmedical
applications of liposomes; Barenholz, Y., Lasic, D. D., Eds.; CRC
Press: Boca Raton, 1996; Vol. II, pp 153-186.
(22) J ap, B. K.; Zulauf, M.; Scheybani, T.; Hefti, A.; Baumeister,
W.; Aebi, U.; Engel, A. Ultramicroscopy 1992, 46, 45-84.
(23) Darst, S. A.; Ribi, H. O.; Pierce, D. W.; Kornberg, R. D. J . Mol.
Biol. 1988, 203, 269-273.
(7) Hepler, J . R.; Gilman, A. G. Trends Biochem. Sci. 1992, 17, 383-
387.
(8) Lamb, T. D.; Pugh, E. N. J . Trends Neurosci. 1992, 15, 291-
298.
(9) Pfister, C.; Bennet, N.; Bruckert, F.; Catty, P.; Clerc, A.; Pages,
F.; Deterre, P. Cell. Signalling 1993, 5, 235-241.
(10) Hurley, J . B. J . Bioenerg. Biomem. 1992, 24, 219-226.
(11) Chabre, M.; Deterre, P. Eur. J . Biochem. 1989, 179, 255-266.
(12) Stryer, L. J . Biol. Chem. 1991, 266, 10711-10714.
S0022-3263(98)00620-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/26/1998

Enzymatically Stable Analogues of GDP
J . Org. Chem., Vol. 63, No. 21, 1998 7245
Nevertheless, that approach is hardly successful as it is
usually difficult to select a discrete number of orienta-
tions of the protein when binding onto the lipid film. This
process mainly results in obtaining noncrystalline 2D
close-packing of proteins. By selecting unique and
unambiguous interactions between proteins and the lipid
layer, it is possible to increase the number of successful
results in 2D crystallization experiments. Such unique
interactions can be achieved by anchoring a specific
ligand of the protein to the lipid molecule.20,27-32
In particular, for the structural analysis of G-proteins,
we were led to investigate the possible ways to im-
mobilize GDP on lipid supports. Due to the relative poor
stability of the pyrophosphate bridge in nucleotides, our
efforts were consecutively directed in two directions: (i)
synthesis of a GDP analogue stable to enzymatic hy-
drolysis and recognized by transducin; (ii) functionaliza-
tion of that GDP analogue (for further immobilization
purpose) in a manner preserving its affinity for the
protein.
Resu lts a n d Discu ssion
Syn th esis of Non h yd r olyza ble An a logu es of GDP .
The lack of stability in nucleoside polyphosphates results
from the presence of pyrophosphate bridges in the
structure. The pyrophosphate moiety is highly sensitive
to both enzymatic and chemical hydrolysis and is prone
to dismutation reactions.33,34 It is thus of outmost
interest to use stable analogues of nucleotides in biologi-
cal experiments.35 There are very few reports in the
literature on the synthesis of GDP analogues. The bis-
thiophosphate analogue of GDP (GDPRSâS) has been
described by Ludwig and Eckstein a few years ago.36
Guanosine phosphonoacetate (compound 3, Figure 1) has
been claimed in an early patent by Heimer and Nuss-
baum in 1978 and was obtained via phosphorylation of
an appropriate protected nucleoside with phosphonoace-
tic acid ethyl ester.37 Last, the synthesis of guanosine
imidodiphosphate (compound 5) has been described via
the reaction of guanosine with trichloro[(dichlorophos-
phoryl)imido]phosphorane, followed by careful hydrolysis
and tricky purification.38 We prepared five different GDP
F igu r e 1.
analogues (including compounds 3 and 5) that could be
recognized and bound by transducin for further function-
alization and immobilization experiments (Figure 1). In
compounds 1-4, the oxygen atom in the pyrophosphate
bridge is replaced with a methylene group and, for
compounds 1-3, one or both phosphate group(s) is (are)
replaced with carboxylate(s). The rationale for these
structural modifications is based on documented biologi-
cal properties of such diphosphate analogues of other
nucleosides.35,39-41 In compound 5, an imidodiphosphate
moiety stands for the pyrophosphate group. The imido-
diphosphate group has been widely used to mimic pyro-
phosphates in nucleosides triphosphates,35,42-44 and the
resulting nucleotide analogues are usually well recog-
nized by their target enzymes, but are generally poor
substrates.
Compounds 1 and 2 were conveniently prepared by
acylation of 2′,3′-O-benzylidene guanosine 645,46 with the
corresponding carboxylic acids, and further debenzylation
by catalytic hydrogenolysis (Figure 2). Compounds 3-5
could not be obtained by following the same reaction
sequence as phosphonic acid monoesters and essentially
remain unreacted in the presence of carbodiimides and
alcohols. Only rare examples of such coupling reactions
are reported in the literature.47-50 Though phosphonic
acid monoesters are poor phosphonylating agents toward
(24) Schnyder, T.; Cyrklaff, M.; Fuchs, K.; Wallimann, T. J . Struct.
Biol. 1994, 112, 136-147.
(25) Schultz, P.; Ce´lia, H.; Riva, M.; Darst, S. A.; Colin, P.; Kornberg,
R. D.; Sentenac, A.; Oudet, P. J . Mol. Biol. 1990, 216, 353-362.
(26) Schultz, P.; Ce´lia, H.; Riva, M.; Sentenac, A.; Oudet, P. EMBO
J . 1993, 12, 2601-2607.
(27) Ahlers, M.; Mu¨ller, W.; Reichert, A.; Ringsdorf, H.; Venzmer,
J . Angew. Chem., Int. Ed. Engl. 1990, 29, 1269-1285.
(28) Ahlers, M.; Blankenburg, R.; Haas, H.; Mo¨bius, D.; Mo¨hwald,
H.; Mu¨ller, W.; Ringsdorf, H.; Siegmund, H. U. Adv. Mater. 1991, 3,
39-46.
(29) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Bio-
chemistry 1989, 28, 8214-8221.
(30) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.;
Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys.
J . 1991, 59, 387-396.
(39) Lambert, R. W.; Martin, J . A.; Thomas, G. J .; Duncan, I. B.;
Hall, M. J .; Heimer, E. P. J . Med. Chem. 1989, 32, 367-374.
(40) Griengl, H.; Hayden, W.; Penn, G.; De Clercq, E.; Rosenwirth,
B. J . Med. Chem. 1988, 31, 1831-1839.
(31) Lebeau, L.; Regnier, E.; Schultz, P.; Wang, J . C.; Mioskowski,
C.; Oudet, P. FEBS Lett. 1990, 267, 38-42.
(41) Blackburn, G. M. Chem. Ind. 1981, 134-138.
(42) Yanachkov, I.; Pan, J . Y.; Wessling-Resnick, M.; Wright, G. E.
Mol. Pharmacol. 1997, 51, 47-51.
(32) Ribi, H. O.; Reichard, P.; Kornberg, R. D. Biochemistry 1987,
26, 7974-7979.
(33) Admiraal, S. J .; Herschlag, D. Chem. Biol. 1995, 2, 729-739.
(34) Verheyden, D. L. M.; Wehrli, W. E.; Moffatt, J . G. J . Am. Chem.
Soc. 1965, 87, 2257-2265.
(43) Yount, R. G.; Babcock, D.; Ballantyne, W.; Ojala, D. Biochem-
istry 1971, 10, 2484-2489.
(44) Ma, Q. F.; Babbitt, P. C.; Kenyon, G. L. J . Am. Chem. Soc. 1988,
110, 4060-4061.
(35) Yount, R. G. Adv. Enzymol. 1975, 43, 1-56.
(36) Ludwig, J .; Eckstein, F. J . Org. Chem. 1991, 56, 5860-5865.
(37) Heimer, E. P.; Nussbaum, A. L. Phosphonoacetic acid deriva-
tives of nucleosides Chem. Abstr. 1977, 88, 51130s.
(38) Tomasz, J .; Vaghefi, M. M.; Ratsep, P. C.; Willis, R. C.; Robins,
R. K. Nucleic Acids Res. 1988, 16, 8645-8664.
(45) Gulland, M. J .; Overend, W. G. J . Chem. Soc. 1948, 1380-1382.
(46) Lipkin, D.; Phillips, B.; Hunter, W. H. Tetrahedron Lett. 1959,
18-21.
(47) Kamikawaji, Y.; Sakoda, R.; Seto, K. Chem. Pharm. Bull. 1995,
43, 315-317.

7246 J . Org. Chem., Vol. 63, No. 21, 1998
Vincent et al.
F igu r e 2.
F igu r e 3.
alcohols, high yield phosphonylations can be performed
using the Mitsunobu reaction.51-54 However, the activa-
tion of 2′,3′-O-benzylidene guanosine 6 as an alkoxyphos-
phonium species straightforwardly provides a cyclogua-
nosine product resulting from intramolecular displacement
of the nucleofugal group on C5 by N3 (Figure 3).55-57 That
particular reactivity of purine nucleosides in the Mit-
sunobu reaction has been documented in an early work
by Mitsunobu and co-workers on phosphorylation of
nucleosides.58 The intramolecular cyclization reaction
can be efficiently circumvented in the case of adenosine
derivatives by adequate masking of the exocyclic amino
group on adenine resulting in a decreased nucleophilicity
of N3.51 To address the same problem in the case of
guanosine derivatives, we realized a series of phospho-
nylation reactions involving different guanine-protected
compounds (Figure 4). Compounds 9-12 do not suf-
ficiently deactivate N3 to prevent the intramolecular
cyclization reaction upon activation of the 5′-hydroxy
F igu r e 4.
group as an alkoxyphosphonium species. We concluded
that as long as there is a labile hydrogen atom on the
base, the cyclization side-reaction can proceed. This is
argued considering the tautomeric forms of the substi-
tuted bases, as one of them always displays the acidic
proton on the N3 position, which indicates the high
nucleophilicity of that nitrogen atom, at least in the
intramolecular reaction. So, introducing a divalent
protective group on the exocyclic amine of guanine
together with an alkyl substituent at O6 or N1 is expected
to definitely prevent the formation of cycloguanosine.
This proved to be the case as compounds 13, 14, and 15
could be phosphonylated with good to excellent yields
(vide infra).
(48) Richard, J . P.; Frey, P. A. J . Am. Chem. Soc. 1982, 104, 3476-
3481.
(49) Natchev, I. A. Tetrahedron 1988, 44, 6455-6464.
(50) Cevasco, G.; Penco, S.; Thea, S. Phosphorus, Sulfur, Silicon
Relat. Elem. 1993, 84, 257-260.
(51) Saady, M.; Lebeau, L.; Mioskowski, C. Tetrahedron Lett. 1995,
36, 2239-2242.
(52) Saady, M.; Lebeau, L.; Mioskowski, C. Synlett 1995, 643-644.
(53) Campbell, D. A.; Bermack, J . C. J . Org. Chem. 1994, 59, 658-
660.
(54) Campbell, D. A. J . Org. Chem. 1992, 57, 6331-6335.
(55) J enkins, J . D.; Verheyden, J . P. H.; Moffatt, J . G. J . Am. Chem.
Soc. 1976, 98, 3346-3357.
Guanosine alkylation at O6 is well documented and is
used in many transformation sequences of that nucleo-
side. It can be regioselectively achieved via a Mitsunobu
reaction on sugar-protected guanosine derivatives, with
the exocyclic amine either masked59-66 or unmasked.67-70
(56) Verheyden, J . P. H.; Moffatt, J . G. J . Org. Chem. 1970, 35,
2319-2326.
(57) Schattka, K.; J astorff, B. Chem. Ber. 1972, 105, 3824-3832.
(58) Kimura, J .; Fujisawa, Y.; Yoshizawa, T.; Fukuda, K.; Mit-
sunobu, O. Bull. Chem. Soc. J pn. 1979, 52, 1191-1196.

Enzymatically Stable Analogues of GDP
J . Org. Chem., Vol. 63, No. 21, 1998 7247
F igu r e 6.
F igu r e 5.
5′-protective group was achieved in methanolic ammonia
to yield compound 15.
Thus O6-protected benzylidene guanosine 9 was obtained
in three steps from 6 (Figure 5). The sequence involved
acetylation of the 5′-hydroxy group prior to alkylation of
16 at O6 to yield compound 17. Removal of the 5′-acetyl
protective group from 17 by methanolic ammonia pro-
duced the O6-benzyl nucleoside 9. When compound 16
was treated with phenyl chlorothioformate, it provided
the fully protected nucleoside 1871,72 that was directly
transformed into 2′,3′-O-benzylidene N2-benzyloxycar-
bonyl guanosine 11 upon treatment with sodium benzy-
late. That triple transformation was achieved with 90%
yield. Introduction of the N,N-dibenzyl formamidine
protection73 at the N2 position in 17 produced the fully
protected guanosine derivative 19, and removal of the
N,N-Diethyl benzamidine compounds 12 and 13 were
prepared by reacting the sugar-protected guanosine
derivative 16 with N,N-diethyl benzamide diethyl acetal73
to give the intermediate compound 20 (Figure 6). Com-
pound 12 was obtained from 20 via treatment with
methanolic ammonia as described above. Attempted
introduction of an O6-benzyl group on 20 using the
Mitsunobu reaction invariably failed and almost quan-
titatively led to compound 21 resulting from N1-alkyl-
ation as assessed by X-ray crystallography.74 This ob-
servation appears especially interesting as all reported
alkylation of guanine nucleosides through the Mitsunobu
reaction exclusively produced the O6-alkylated com-
pounds.59-70 All of the examples described in the litera-
ture refer either to guanine or N2-acyl guanine deriva-
tives. Thus, this unprecedented regioselectivity of gua-
nine alkylation is clearly due to the amidine protection
at N2 which provides a straightforward and very efficient
access to N1-alkylated guanosine derivatives when com-
pared to those previously described in the literature.75-81
Compound 13 resulted from treatment of 21 with metha-
nolic ammonia.
(59) Loeppky, R. N.; Yu, L.; Gu, F.; Ye, Q. J . Am. Chem. Soc. 1996,
118, 10995-11005.
(60) Gao, X.; Gaffney, B. L.; Hadden, S.; J ones, R. A. J . Org. Chem.
1986, 51, 755-758.
(61) Chollet, A.; Ayala, E.; Kawashima, E. H. Helv. Chim. Acta 1984,
67, 1356-1364.
(62) Lee, H.; luna, E.; Hinz, M.; Stezowski, J . J .; Kiselyov, A. S.;
Harvey, R. G. J . Org. Chem. 1995, 60, 5604-5613.
(63) Dahma, M.; Ogilvie, K. K. J . Org. Chem. 1988, 53, 3710-3722.
(64) Himmelsbach, F.; Schulz, B. S.; Trichtinger, T.; Charubala, R.;
Pfleiderer, W. Tetrahedron 1984, 40, 59-72.
(65) Matsuda, A.; Watanabe, K.; Miyasaka, T.; Ueda, T. Chem.
Pharm. Bull. 1989, 37, 298-303.
(66) Usui, H.; Matsuda, A.; Ueda, T. Chem. Pharm. Bull. 1986, 34,
1961-1967.
(74) Vincent, S.; Mioskowski, C.; Lebeau, L. Manuscript in prepara-
tion.
(67) Resmini, M.; Pfleiderer, W. Helv. Chim. Acta 1993, 76, 158-
167.
(75) Moschel, R. C.; Hudgins, W. R.; Dipple, A. J . Org. Chem. 1980,
45, 533-535.
(68) Steinbrecher, T.; Wameling, C.; Oesch, F.; Siedel, A. Angew.
Chem. 1993, 105, 408-410.
(76) Yamauchi, K.; Nakagima, T.; Kinoshita, M. J . Org. Chem. 1980,
45, 3865-3868.
(69) Woo, J .; Sigurdsson, S. T.; Hopkins, P. B. J . Am. Chem. Soc.
1993, 115, 3407-3415.
(77) Yamauchi, K.; Nakagima, T.; Kinoshita, M. J . Chem. Soc.,
Perkin Trans. 1 1980, 2787-2792.
(70) Schirmeister, H.; Himmelsbach, F.; Pfleiderer, W. Helv. Chim.
Acta 1993, 76, 385-401.
(78) Sekine, M.; Satoh, T. J . Org. Chem. 1991, 56, 1224-1227.
(79) Moschel, R. C.; Hudgins, W. R.; Dipple, A. J . Org. Chem. 1984,
49, 363-372.
(71) Watkins, B. E.; Rapoport, H. J . Org. Chem. 1982, 47, 4471-
4477.
(72) Watkins, B. E.; Kiely, J . S.; Rapoport, H. J . Am. Chem. Soc.
1982, 104, 5702-5708.
(73) Vincent, S.; Mons, S.; Lebeau, L.; Mioskowski, C. Tetrahedron
Lett. 1997, 38, 7527-7530.
(80) McBride, L. J .; Kierzek, R.; Beaucage, S. L.; Caruthers, M. H.
J . Am. Chem. Soc. 1986, 108, 2040-2048.
(81) Leonard, N. J .; Hosmane, R. S.; Agasimundin, Y.; Kostuba, L.
J .; Oakes, F. T. J . Am. Chem. Soc. 1984, 106, 6847-6848.

7248 J . Org. Chem., Vol. 63, No. 21, 1998
Vincent et al.
F igu r e 7.
The N1-benzyl derivatives of guanosine 10 and 14 were
prepared by reacting 16 with N,N-dibenzyl formamide
dimethyl acetal73 to yield 22 that was further regiose-
lectively alkylated at N1 via a Mitsunobu reaction to
produce compound 23 (Figure 7). Treatment of this
compound with potassium hydroxide yielded nucleoside
10, whereas 14 was obtained through standard treatment
with methanolic ammonia.
When compounds 13-15 were treated with triphen-
ylphosphine, diethyl azodicarboxylate, and a phosphonic
acid monoester, the corresponding phosphonic acid di-
esters were obtained with satisfactory yields (i.e. 60-
80%). However, all attempts for the removal of the
benzamidine protective group on 13 either by hydro-
genolysis or hydrolysis failed.73 This is presumably due
to the high conjugation energy of the amidine double bond
with the phenyl ring and the heterocycle. Considering
the overall yields for the syntheses of compounds 14
(Figure 7) and 15 (Figure 5), the subsequent phospho-
rylation reactions with diphosphate analogue building
blocks were realized with the N1-benzyl nucleoside 14
(Figure 8). Interestingly, when the phosphorylation
reactions were run at room temperature, product 30
resulting from N-alkylation of the reduced form of DEAD
was obtained as the major product, once more due to the
above-mentioned poor nucleophilicity of phosphonic acid
monoesters (Figure 9). Neither the use of other phos-
phine compounds (PBu3, (4-Cl-Ph)3P53) nor other azo
derivatives (DIAD, ADDP82-84) prevented such a side
reaction. Finally, the best results were obtained when
conducting the reaction with PPh3 and DEAD at 60 °C.
Hydrogenolysis of compounds 25, 27, and 29 was achieved
using a mixture of Pd/C and Pearlman catalyst85 at pH
8.5, and compounds 3-5 were obtained as their am-
monium or triethylammonium salt.
F igu r e 8.
nized by the protein (vide infra). Consequently, we
modified our previous synthetic schemes so as to intro-
duce a substituent at different positions on this GDP
analogue. This was achieved in order to determine which
ligand modification if any is allowed for further im-
mobilization of transducin.
Functionalization at N1 was realized starting from
compound 22 (Figure 10). Instead of alkylating N1 with
activated benzyl alcohol in the Mitsunobu reaction condi-
tions as described in Figure 7, we used alcohol 31 with
an aim to selectively reduce the azide group into amine
for the eventual coupling to a lipid matrix. After hy-
drolysis of the 5′-acetate of 32, compound 33 was phos-
phorylated into 34 and fully deprotected by catalytic
hydrogenolysis to produce the first functionalized GDP
analogue 35. Functionalization at O6 was achieved using
a different reaction sequence (Figure 11). Compound 22
was first sulfonated at O6 to yield 36 according to a
previously described procedure,86 and the sulfonate was
successively displaced by quinuclidine and azido alcohol
37 to produce the protected nucleoside 38. Deacetylation,
subsequent phosphorylation of 39 into 40, and hydro-
genolysis led to the second functionalized GDP analogue
41.
Syn th esis of F u n ction a lized Der iva tives of Gu a -
n osin e Im id od ip h osp h a te. Binding experiments of
compounds 1-5 with transducin showed that only the
imidodiphosphate compound 5 was satisfactorily recog-
(82) Tsunoda, T.; Otsuka, J .; Yamamiya, Y.; Itoˆ, S. Chem. Lett. 1994,
539-542.
(83) Tsunoda, T.; Yamamiya, Y.; Itoˆ, S. Tetrahedron Lett. 1993, 34,
1639-1642.
(84) Tsunoda, T.; Ito, S. J . Synth. Org. Chem. J pn. 1997, 55, 631-
641.
(85) Pearlman, M. W. Tetrahedron Lett. 1967, 17, 1663-1664.
(86) Hagen, M. D.; Chla´dek, S. J . Org. Chem. 1989, 54, 3189-3195.

Enzymatically Stable Analogues of GDP
J . Org. Chem., Vol. 63, No. 21, 1998 7249
F igu r e 9.
F igu r e 10.
A third functionalized GDP analogue bearing a sub-
stituent on the sugar moiety has been prepared (Figure
12). The synthetic route to 42 originated from the
previously reported reactivity of the 2′-hydroxy group of
nucleosides toward electrophiles.87-90 Ikehara and co-
workers realized the selective alkylation of N2-isobutyryl
guanosine at the 2′-position on the sugar moiety with 29%
yield by reacting this compound with o-nitrobenzyl
bromide and sodium hydride (Figure 13).90 Surprisingly
the treatment of N2-(N,N-dibenzyl formamidine) gua-
nosine 43 with a stoichiometric amount of benzyl bromide
and NaH produced nearly quantitatively compound 44
resulting from the N1-benzylation of the base, leaving the
2′-hydroxy group unreacted (Figure 14). This result is
in contrast with the data of Ikehara and once again
accounts for the unusual reactivity of guanosine protected
at N2 as an amidine. When compound 43 was treated
F igu r e 11.
(87) Takaku, H.; Kamike, K.; Tsuchiya, H. J . Org. Chem. 1984, 49,
51-56.
F igu r e 12.
(88) Quaedflieg, P. J . L. M.; van der Heiden, A. P.; Koole, L. H.;
Coenen, A. J . J . M.; van der Wal, S.; Meijer, E. M. J . Org. Chem. 1991,
56, 5846-5859.
with 2 equiv of sodium hydride and benzyl bromide, the
1-benzyl-2′-O-benzyl compound 45 was obtained with
54% yield. Direct regioselective phosphorylation of 45
with 28 yielded the 3′-hydroxy compound 46 that was
(89) Ohtsuka, E.; Tanaka, T.; Tanaka, S.; Ikehara, M. J . Am. Chem.
Soc. 1978, 100, 4580-4584.
(90) Ohtsuka, E.; Tanaka, S.; Ikehara, M. Synthesis 1977, 453-454.

7250 J . Org. Chem., Vol. 63, No. 21, 1998
Vincent et al.
F igu r e 13.
F igu r e 15.
F igu r e 16.
F igu r e 14.
further acylated with propanoyl chloride to produce the
masked nucleotide 47 (Figure 15). Hydrogenolysis of
compound 47 led to a mixture of the two regioisomers
42 and 48, resulting from partial migration of the acyl
moiety from the 3′- to the 2′-position.
Finally, we investigated the synthesis of a GDP
analogue functionalized at the nitrogen atom bridging
the two phosphorus atoms (Figure 16). The synthetic
route to compound 49 is similar to those described above
except for the use of the diphosphate building block 52
(Figure 17). Attempts to completely debenzylate com-
pound 53 to obtain 49 invariably failed, resulting in an
important hydrolysis of the imidodiphosphate moiety and
formation of a mixture of guanosine monophosphate and
guanosine phosphoramide. This result highlights the fact
that if imidodiphosphate nucleosides are more resistant
to enzymatic hydrolysis than the natural corresponding
nucleotides, they remain much sensitive to chemical
hydrolysis and have to be handled and stored with
care.38,91-93
F igu r e 17.
Bin d in g to Tr a n sd u cin . To our knowledge, up to
now only two GDP analogues have been tested toward
transducin (GDPRS and GDPâS).94 Nevertheless, as they
appear to be a substrate for many enzymes, their use
meets some limitations.95
Binding experiments were realized with compounds
1-5, using membranes of retinal rod outer segments.
(91) Quimby, O. T.; Narath, A.; Lohman, F. H. J . Am. Chem. Soc.
1960, 82, 1099-1108.
(92) Reynolds, M. A.; Gerlt, J . A.; Demou, P. C.; Oppenheimer, N.
J .; Kenyon, G. L. J . Am. Chem. Soc. 1983, 105, 6475-6481.
(93) Pollard, F. H.; Nickless, G.; Bigwood, A. M. J . Chromatogr.
1964, 16, 207-213.

Enzymatically Stable Analogues of GDP
J . Org. Chem., Vol. 63, No. 21, 1998 7251
8/4, 15 mL) until pH 8.5. Pd/C (10%, 280 mg) and Pd(OH)2/C
(20%, 257 mg) are added, and the mixture is stirred under
hydrogen pressure (6 atm) for 24 h. Catalysts are removed
by centrifugation on a Millipore membrane (Millex GV, 0.22
µM, solvent resistant), and solvents are evaporated in vacuo.
The aqueous residue is lyophilized to dryness to yield the
triethylammonium salt of 1 as a white powder (38 mg, 73%).
1H NMR (D2O, 200 MHz) δ 7.76 (s, 1H); 5.68 (d, J ) 4.8 Hz,
1H); 4.62-4.54 (m, 1H); 4.32-4.14 (m, 4H); 2.96 (q, J ) 9.2
Hz, 6H); 1.02 (t, J ) 9.2 Hz, 9H). 13C NMR (D2O, 75 MHz) δ
170.75; 158.71; 154.03; 151.88; 137.42; 116.50; 87.49; 81.98;
73.54; 70.18; 64.14; 46.76; 39.39; 8.37. IR (KBr) ν 3104; 2626;
1691; 1598; 1364. HRMS: calcd for C13H14N5O8 368.0842,
found 368.0822 [M - H]-. HPLC (H2O/TFA 1000/1) tR 40.0
min.
9-(5′-O -(P h o sp h on oa c e t yl)-â-D -r ib o fu r a n o sy l)g u a -
n in e (2). Compound 2 is obtained as its bis-triethylammo-
nium salt from 8 following the same procedure as for 1 (white
powder, 71 mg, 63%). 1H NMR (D2O, 200 MHz) δ 7.71 (s, 1H);
5.62 (d, J ) 4.8 Hz, 1H); 4.53-4.46 (m, 1H); 4.28-4.23 (m,
1H); 4.18-4.04 (m, 3H); 2.96 (q, J ) 9.2 Hz, 12H); 2.43 (d, J
) 19.4 Hz, 2H); 1.02 (t, J ) 9.2 Hz, 19H). 13C NMR (D2O, 50
MHz) δ 172.57; 163.20; 158.31; 151.79; 136.29; 117.21; 87.08;
81.97; 73.39; 70.39; 63.98; 46.61; 38.08 (d, J ) 106.5 Hz); 8.43.
31P NMR (D2O, 121 MHz, [Et3N] ) 0.5 M) δ 11.49. IR (KBr)
ν 3134; 1691; 1270; 1122; 1033. HRMS: calcd for C12H15N5O9P
404.0607, found 404.0630 [M - H]-. HPLC (H2O/TFA 1000/
1) tR 62.5 min.
9-{5′-O-[(Ca r b oxym et h yl)h yd r oxyp h osp h in yl]-â-D-r i-
bofu r a n osyl}gu a n in e (3). Compound 3 is obtained as its
bis-triethylammonium salt from 25 following the same proce-
dure as for 1 (white powder, 43 mg, 91%). 1H NMR (D2O, 300
MHz) δ 8.00 (s, 1H); 5.74 (d, J ) 6.0 Hz, 1H); 4.70-4.52 (m,
1H); 4.32 (dd, J ) 3.0, 4.6 Hz, 1H); 4.15 (m, 1H); 3.99 (m, 2H);
2.96 (q, J ) 9.2 Hz, 12H); 2.73 (d, J ) 21.7 Hz, 2H); 1.02 (t, J
) 9.2 Hz, 18H). 13C NMR (D2O, 75 MHz) δ 174.27; 158.99;
154.03; 151.88; 137.70; 116.22; 86.79; 84.13 (d, J ) 7.8 Hz);
73.81; 70.58; 64.00 (d, J ) 5.3 Hz); 46.78; 37.01 (d, J ) 121.4
Hz); 8.34. 31P NMR (D2O, 121 MHz, [Et3N] ) 0.5 M) δ 18.20.
IR (KBr) ν 3422; 3135; 1696; 1398; 1172; 1036. HRMS: calcd
for C12H15N5O9P 404.0607, found 404.0633 [M - H]-. HPLC
(H2O/TFA 1000/1) tR 40.7 min.
Assays involve the binding of a radioactive nucleotide
(3H-GDP), and detailed experimental procedures will be
described elsewhere.96 Compounds 1-4 could not dis-
place the radioactive nucleotide even with a 104-fold
excess. Only guanosine imidodiphosphate (compound 5)
could efficiently compete with 3H-GDP for binding to
transducin, and thus was selected for further chemical
transformations. Whatever the structural modification
tested (on the base at N1: compound 35, or O6: compound
41; or on the sugar moiety: compounds 42 and 48), it
definitely prevents any recognition of the compound by
the enzyme. This is markedly in opposition with the
results obtained with other G-proteins of similar struc-
ture that exhibit a higher tolerance for modified nucleo-
tides.97-101 Further analysis is underway.
Con clu sion
The synthesis of 5 analogues of GDP stable to enzy-
matic hydrolysis has been carried out. The carboxylic
esters 1 and 2 were obtained using the DCC/DMAP
methodology for the coupling to the nucleoside of a
precursor of the diphosphate moiety mimicry. The phos-
phonic and phosphoramidic esters 3-5 were prepared
using an alternate procedure for the phosphorylation of
the guanosine moiety, involving a Mitsunobu reaction
and requiring the complete protection of the purine base.
Only compound 5 could bind to transducin, the G-protein
of the visual photoreceptor. Four derivatives of 5 bearing
a linker at different positions were synthesized. None
of these compounds was recognized by the protein,
indicating that transducin is especially highly restrictive
and specific at the GDP binding site, in contrast to
numerous other heterotrimeric G-proteins of closely
related structure.
Exp er im en ta l Section
9-[5′-O-(Meth ylen ebisp h osp h on a te)-â-D-r ibofu r a n osyl]-
gu a n in e (4). Compound 4 is obtained as its tris-ammonium
salt from 27 following a similar procedure to that described
for 1, except that triethylammonium carbonate is replaced with
ammonium carbonate (white powder, 53 mg, 93%). 1H NMR
(D2O, 300 MHz) δ 8.05 (s, 1H); 5.84 (d, J ) 5.5 Hz, 1H); 4.43
(dd, J ) 4.0, 4.4 Hz, 1H); 4.31-3.95 (m, 4H); 2.08 (t, J ) 18.9
Hz, 2H). 13C NMR (D2O, 75 MHz) δ 159.31; 154.95; 152.51;
136.78; 115.30; 87.82; 84.75; 74.57; 71.29; 64.34; 47.64; 25.90
(t, J ) 47.1 Hz); 9.22. 31P NMR (D2O, 121 MHz, [Et3N] ) 0.5
M) δ 19.72 (b, 1P); 15.73 (b, 1P). IR (KBr) ν 3134; 1686; 1406;
1254; 1079. HRMS: calcd for C11H16N5O10P2 440.0372, found
440.0387 [M - H]-. HPLC (H2O/CH3CN/TFA 980/20/1) tR 11.4
min.
Gen er a l. 1H, 13C, and 31P NMR chemical shifts δ are
reported in ppm relative to an internal reference resulting from
incomplete deuteration of the NMR solvent (1H: CHCl3 at 7.27
ppm, HDO at 4.63 ppm, CD2HOD at 3.31 ppm, and DMSO-d5
at 2.50 ppm; 13C: CDCl3 at 77.0 ppm, CD3OD at 49.0 ppm,
DMSO-d6 at 39.5 ppm; 31P: H3PO4 at 0.00 ppm). IR spectra
were recorded in wavenumbers (cm-1). Mass spectra (MS)
were recorded at chemical ionization (CI, NH3). High-resolu-
tion mass spectra (HRMS) were recorded in the negative
electrospray mode. Mass data are reported in mass units (m/
z). Analytical HPLC studies were carried out in the isocratic
mode using a reversed-phase analytical column (Zorbax SBC18,
250 × 4.6 mm, 5 µm) equipped with a photodiode array
detector (detection at 267 nm). Elutions were conducted at
25 °C with a 1 mL/min flow rate.
9-[5′-O-(Im id od ip h osp h a t e )-â-D-r ib ofu r a n osyl]gu a -
n in e (5). Compound 5 is obtained as its tris-triethylammo-
nium salt from 29 following the same procedure as for 1 (white
powder, 75 mg, 91%). 1H NMR (D2O, 300 MHz, [Et3N] ) 0.5
M) δ 8.00 (s, 1H); 5.83 (d, J ) 5.4 Hz, 1H); 4.60 (dd, J ) 5.1,
5.4 Hz, 1H); 4.53 (dd, J ) 4.6, 5.1 Hz, 1H); 4.23 (m, 1H); 4.15-
3.98 (m, 2H). 13C NMR (D2O, 75 MHz, [Et3N] ) 0.5 M) δ
158.15; 154.38; 152.97; 137.35; 117.58; 87.66; 84.66; 74.15;
70.20; 64.06; 46.78; 10.64. 31P NMR (D2O, 121 MHz, [Et3N]
) 0.5 M) δ 3.73 (d, J ) 3.6 Hz, 1P); -0.09 (d, J ) 3.6 Hz, 1P).
IR (KBr) ν 3130; 2351; 1682; 1455; 1064. HRMS: calcd for
9-(5′-O-(Ma lon yl)-â-D-r ibofu r a n osyl)gu a n in e (1). A so-
lution of triethylammonium carbonate in water (0.3 mL, 0.2
M) is added to compound 7 (60 mg) in THF/t-BuOH/H2O (1/
(94) Yamanaka, G.; Eckstein, F.; Stryer, L. Biochemistry 1985, 24,
8094-8101.
(95) Eckstein, F. Angew. Chem., Int. Ed. Engl. 1983, 22, 423-439.
(96) Grenier, S.; Vincent, S.; Lebeau, L.; Salesse, C. Manuscript in
preparation.
(97) Neal, S. E.; Eccleston, J . F.; Webb, M. R. Proc. Natl. Acad. Sci.
U.S.A. 1990, 87, 3562-3565.
C
10H15N6O10P2 441.0325, found 441.0329 [M - H]-. HPLC
(ammonium carbonate 0.5 M, pH 7.0/CH3CN 99/1) tR 4.8 min.
9-[5′-O-(O-Ben zylm a lon yl)-2′,3′-b en zylid en e-â-D-r ib o-
fu r a n osyl]gu a n in e (7). 2′,3′-O-Benzylidene guanosine 645,46
(mixture of 2 diastereomers, 90 mg, 0.24 mmol), malonic acid
monobenzyl ester102 (81 mg, 0.42 mmol), and DCC (104 mg,
0.50 mmol) are sonicated in THF (5 mL) for 2 h at room
temperature. The crude reaction mixture is filtered, reduced
(98) Hingorani, V. N.; Ho Chang, L. F.; Ho, Y. K. Biochemistry 1989,
28, 7424-7432.
(99) Remmers, A. E.; Posner, R.; Neubig, R. R. J . Biol. Chem. 1994,
269, 13771-13778.
(100) Remmers, A. E.; Neubig, R. R. J . Biol. Chem. 1996, 271, 4791-
4797.
(101) Zera, E. M.; Molloy, D. P.; Angleson, J . K.; Lamture, J . B.;
Wensel, T. G. M., J . A. J . Biol. Chem. 1996, 271, 12925-12931.

7252 J . Org. Chem., Vol. 63, No. 21, 1998
Vincent et al.
in vacuo, and purified by chromatography on silica gel (AcOEt/
EtOH: 100/0 to 60/40) to yield 7 as a white powder (mixture
of two diastereomers, 85 mg, 65%). 1H NMR (CDCl3/CD3OD:
9/1, 200 MHz) δ 7.57 and 7.55 (2s, 1H); 7.42-7.15 (m, 10H);
6.01 and 5.85 (2s, 1H); 5.98 and 5.93 (2d, J ) 1.8 Hz, 1H);
5.24 (dt, J ) 1.8, 7.7 Hz, 1H); 5.11-4.99 (m, 3H); 4.64-4.35
(m, 2H); 4.23-4.11 (m, 1H). 13C NMR (CD3OD/CDCl3: 3/1,
50 MHz) δ 167.27; 167.24; 158.69; 154.31; 151.37; 137.61;
136.19; 135.66; 130.71; 130.43; 129.01; 128.74; 127.33; 127.25;
117.91; 108.25 and 104.76; 90.51; 85.81 and 85.23; 84.35 and
84.18; 82.93 and 82.05; 67.93; 65.31; 61.13. IR (CHCl3) ν 2926;
2372; 1670 1090. MS (CI/NH3): 548 [M + H]+. Anal. Calcd
for C27H25N5O8: C, 59.22; H, 4.60; N, 12.79. Found: C, 59.01;
H, 4.50; N, 12.74.
°C. The mixture is stirred at room temperature for 1 h before
it is dropwise added to compound 18 (7.6 g, 10.2 mmol) in THF
(80 mL) at 0 °C. The solution is stirred for 2 h at that
temperature and then is treated with a saturated NH4Cl
aqueous solution. THF is removed in vacuo, and the residue
is extracted with chloroform. The organic layer is dried over
MgSO4, concentrated, and purified by silica gel chromatogra-
phy (Et2O/n-C6H14: 4/1) to yield 11 as a white powder (mixture
of two diastereomers, 5.48 g, 90%). 1H NMR (CDCl3, 200 MHz)
δ 789 and 7.83 (2s, 1H); 7.71-7.15 (m, 15H); 6.23 and 6.05
(2s, 1H); 6.09 and 6.06 (2d, J ) 2.6 Hz, 1H); 5.76-5.20 (m,
6H); 4.60 (m, 1H); 3.90 (m, 2H). 13C NMR (CDCl3, 50 MHz) δ
181.65; 160.22; 151.61; 151.23; 140.78; 135.82; 135.59; 135.41;
129.25 and 129.07; 127.97; 127.92; 127.85; 127.71; 127.62;
127.59; 127.57; 126.21 and 126.07; 117.80 and 117.71; 106.97
and 103.82; 90.38 and 89.78; 86.87 and 85.57; 84.27 and 83.55;
82.01 and 80.73; 68.25; 66.64; 61.78. IR (CHCl3) ν 3289; 3016;
1757; 1607; 1219; 1095. MS (CI/NH3): 596 [M + H]+. Anal.
Calcd for C32H29N5O7: C, 64.53; H, 4.91; N, 11.76. Found: C,
64.70; H, 4.99; N, 11.91.
2-N-(N′,N′-Dieth ylben za m id in e)-9-(2′,3′-O-ben zylid en e-
â-D-r ibofu r a n osyl)gu a n in e (12). Compound 12 is obtained
as a white solid (mixture of two diastereomers, 1.03 g, 97%),
starting from 20 and following the same procedure as for 9.
1H NMR (CDCl3, 300 MHz) δ 7.54-7.11 (m, 11H); 6.19 and
6.01 (2s, 1H); 5.68 and 5.65 (2d, J ) 3.0 Hz, 1H); 5.08 and
5.03 (2dd, J ) 1.8, 4.3 Hz, 1H); 4.59 and 4.52 (m, 1H); 4.45-
4.40 (m, 1H); 3.91-3.86 (m, 2H); 3.77-3.72 (m, 1H); 3.53-
3.38 (m, 1H); 3.29-3.09 (m, 2H); 1.38-1.30 (m, 3H); 1.15-
1.06 (m, 3H). 13C NMR (CDCl3, 75 MHz) δ 164.27 and 164.15;
158.48; 157.18; 149.08 and 149.02; 137.94 and 137.74; 136.85;
133.93; 133.26 and 133.21; 129.82; 129.64; 129.58; 128.55;
128.47; 126.41 and 126.23; 107.49 and 104.38; 92.49 and 91.03;
85.45 and 85.13; 83.37 and 83.01; 82.33 and 80.29; 63.30 and
63.03; 44.43 and 42.45; 14.21 and 12.23. IR (CHCl3) ν 3178;
2934; 1681; 1525; 1459; 1104. MS (CI/NH3): 531 [M + H]+.
Anal. Calcd for C28H30N6O5: C, 63.38; H, 5.70; N, 15.84.
Found: C, 63.36; H, 5.51; N, 15.66.
1-Ben zyl-2-N-(N′,N′-dieth ylben zam idin e)-9-(2′,3′-O-ben -
zylid en e-â-D-r ibofu r a n osyl)gu a n in e (13). Compound 13 is
obtained as a white solid (mixture of two diastereomers, 1.20
g, 96%), starting from 21 and following the same procedure
as for 9. 1H NMR (CDCl3, 200 MHz) δ 7.70-7.61 (m, 1H);
7.54-7.17 (m, 13H); 7.09 (t, J ) 7.7 Hz, 1H); 6.86 (t, J ) 7.7
Hz, 1H); 6.18 and 5.98 (2s, 1H); 5.91 and 5.85 (2d, J ) 3.2 Hz,
1H); 5.63 (dd, J ) 4.4, 7.3 Hz, 1H); 6.15-4.98 (m, 2H); 4.49-
4.30 (m, 2H); 4.10 and 4.03 (2q, J ) 6.8 Hz, 1H); 3.79 (AB
part of ABX syst, ∆δ ) 58.7 Hz, J AB ) 12.4 Hz, J AX ) 4.0 Hz,
J BX ) 3.0 Hz, 2H); 3.46 and 3.43 (2q, J ) 6.8 Hz, 1H); 1.42-
1.32 (m, 3H); 1.03 and 1.00 (2t, J ) 6.9 Hz, 3H). 13C NMR
(CDCl3, 50 MHz) δ 163.44; 158.06; 156.77; 146.82; 138.10;
137.76 and 137.61; 136.70; 135.82; 132.70; 132.56; 132.00;
131.79; 129.07; 129.44; 129.25; 129.16; 128.73; 128.49; 128.42;
128.34; 128.20; 127.22; 126.32; 126.05; 120.30; 107.36 and
104.20; 92.12 and 90.76; 85.27 and 84.94; 83.31 and 82.59;
81.97 and 80.28; 63.20 and 62.97; 45.75; 44.09; 42.26; 14.22;
12.72. IR (CHCl3) ν 3306; 2934; 1691; 1519; 1295; 1115; 732.
MS (CI/NH3): 621 [M + H]+. Anal. Calcd for C35H36N6O5:
C, 67.72; H, 5.85; N, 13.54. Found: C, 67.80; H, 6.03; N, 13.80.
1-Ben zyl-2-N-(N′,N′-d ib en zylfor m a m id in e)-9-(2′,3′-O-
ben zylid en e-â-D-r ibofu r a n osyl)gu a n in e (14). Compound
14 is obtained as a white solid (mixture of two diastereomers,
0.41 g, 99%), starting from 23 and following the same
procedure as for 9. 1H NMR (CDCl3, 300 MHz) δ 8.72 and
8.69 (2s, 1H); 7.73 and 7.67 (2s, 1H); 7.60-7.15 (m, 20H); 6.19
and 6.03 (2s, 1H); 5.98 and 5.95 (2d, J ) 4.4 Hz, 1H); 5.52
(AB syst, ∆δ ) 135.0 Hz, J AB ) 14.6 Hz, 2H); 5.48 and 5.42
(2dd, J ) 2.2, 4.4 Hz, 1H); 5.15 (m, 1H); 4.75-4.55 (m, 2H);
4.52-4.35 (m, 3H); 4.00 (m, 1H); 3.77 (m, 1H). 13C NMR
(CDCl3, 50 MHz) δ 158.21; 158.16 and 157.97; 157.55; 147.20;
137.94; 136.24; 135.92; 134.85; 134.71; 129.80; 129.60; 128.90;
128.73; 128.50; 128.41; 128.15; 127.81; 127.51; 126.76; 128.48;
126.33; 120.95; 107.50 and 104.26; 91.88 and 90.52; 85.28 and
83.93; 85.08 and 83.64; 83.34 and 80.37; 62.65 and 62.32; 54.83;
48.02 and 47.92; 45.56. IR (CHCl3) ν 3306; 2924; 1687; 1612;
9-(5′-O-Diben zylp h osp h on oa cetyl-2′,3′-O-ben zylid en e-
â-D-r ibofu r a n osyl)gu a n in e (8). Compound 8 is obtained as
a white powder (mixture of two diastereomers, 163 mg, 77%),
starting from 6 and R-dibenzylphosphonoacetic acid,103 and
following the same procedure as for 7. 1H NMR (CDCl3, 200
MHz) δ 7.70 and 7.64 (2s, 1H); 7.50-7.15 (m, 15H); 6.03 and
5.98 (2s, 1H); 5.35-5.27 (m, 1H); 5.11-4.94 (m, 6H); 4.56-
4.25 (m, 2H); 3.06 (d, J ) 21.4 Hz, 2H). 13C NMR (CDCl3, 50
MHz) δ 157.81; 153.28; 150.36; 136.76; 135.21 and 135.14;
135.10 and 135.01; 129.70; 129.59; 128.36; 128.29; 128.17;
127.64; 126.42; 126.34; 117.18; 107.31 and 103.90; 89.65; 84.93
and 84.35; 83.48 and 82.33; 82.13 and 81.40; 68.15 (d, J ) 6.5
Hz); 64.38; 33.42 (d, J ) 132.1 Hz). 31P NMR (CDCl3, 121
MHz) δ 21.50. IR (CHCl3) ν 3420; 2927; 1682; 1260; 995. MS
(CI/NH3): 674 [M + H]+. Anal. Calcd for C33H32N5O9P: C,
58.84; H, 4.79; N, 10.40. Found: C, 58.80; H, 4.43; N, 10.44.
6-O-Ben zyl-9-(2′,3′-O-b en zylid en e-â-D-r ib ofu r a n osyl)-
gu a n in e (9). Compound 17 (100 mg, 0.20 mmol) is stirred in
methanolic ammonia (3 mL) for 1 h at 0 °C. The crude mixture
is evaporated to dryness to yield 9 as a white powder (mixture
of two diastereomers, 91 mg, 99%). 1H NMR (CDCl3, 200 MHz)
δ 7.70 and 7.61 (2s, 1H); 7.61-7.15 (m, 10H); 6.29 and 6.05
(2s, 1H); 5.95 and 5.90 (2d, J ) 4.5 Hz, 1H); 5.56 (s, 2H); 5.34
(m, 2H); 5.18 (m, 2H); 4.64 and 4.52 (2m, 1H); 3.98 (t, J )
12.1 Hz, 1H); 3.81 (t, J ) 9.2 Hz, 1H). 13C NMR (CDCl3/CD3-
OD: 2/1, 50 MHz) δ 161.21; 159.03; 152.84; 138.79 and 138.52;
136.17; 135.84 and 135.66; 132.82 and 132.64; 129.73 and
129.50; 128.23; 127.97; 127.08; 126.35 and 126.15; 125.51 and
125.21; 116.14 and 115.05; 107.47 and 104.42; 92.85 and 90.98;
85.31 and 85.12; 83.58 and 83.27; 82.75 and 80.26; 68.10; 62.92
and 62.59. IR (CHCl3) ν 3364; 2929; 1612; 1257; 1093. MS
(CI/NH3): 462 [M + H]+. Anal. Calcd for C24H23N5O5: C,
62.46; H, 5.03; N, 15.18. Found: C, 62.17; H, 4.98; N, 15.00.
1-Ben zyl-9-(2′,3′-O-ben zylid en e-â-D-r ibofu r a n osyl)gu a -
n in e (10). Compound 23 (46 mg, 0.06 mmol) and K2CO3 (48
mg, 0.35 mmol) in methanol (2 mL) are stirred for 2 h at room
temperature. The solvent is removed under reduced pressure,
and the residue is purified by preparative TLC (AcOEt/EtOH:
9/1) to yield 10 as a white powder (mixture of two diastereo-
mers, 20 mg, 66%). 1H NMR (CD3OD, 200 MHz) δ 7.99 and
7.97 (2s, 1H); 7.55-7.15 (m, 10H); 6.20 and 6.17 (2d, J ) 2.2
Hz, 1H); 6.18 and 6.01 (2s, 1H); 5.49 and 5.42 (2dd, J ) 2.2,
6.6 Hz, 1H); 5.31 (s, 2H); 5.22 and 5.16 (2dd, J ) 4.0, 6.6 Hz,
1H); 4.47 and 4.36 (2m, 1H); 3.79 (m, 2H). 13C NMR (CD3OD,
50 MHz) δ 158.91; 155.61; 150.57 and 150.50; 138.92; 137.74
and 137.59; 136.79; 130.85 and 130.71; 129.90 and 129.76;
129.43 and 129.36; 129.20 and 128.58; 128.07 and 127.94;
127.58; 116.87; 108.77 and 105.25; 91.54 and 90.87; 88.53 and
86.77; 86.31 and 85.21; 84.09 and 82.47; 63.44; 45.33. IR (KBr)
ν 3332; 2923; 1690; 1630; 1584; 1535; 1092. MS (CI/NH3): 462
[M + H]+. Anal. Calcd for C24H23N5O5: C, 62.46; H, 5.03; N,
15.18. Found: C, 62.31; H, 4.98; N, 14.88.
6-O-Ben zyl-2-N-(O-ben zylca r ba m oyl)-9-(2′,3′-O-ben zyl-
id en e-â-D-r ibofu r a n osyl)gu a n in e (11). Benzyl alcohol (9.4
mL, 90.4 mmol) is slowly added to a suspension of sodium
hydride (60% in oil, 3.60 g, 86.4 mmol) in THF (90 mL) at 0
(102) Fritsch, W.; Stache, U.; Ruschig, H. Liebigs Ann. Chem. 1966,
195-205.
(103) Koppel, G. A.; Kinnick, M. D. Tetrahedron Lett. 1974, 711-
713.

Enzymatically Stable Analogues of GDP
J . Org. Chem., Vol. 63, No. 21, 1998 7253
1493; 1094. MS (CI/NH3): 669 [M + H]+. Anal. Calcd for
4.22 (m, 2H); 1.98 and 1.96 (2s, 3H). 13C NMR (CDCl3, 50
MHz) δ 173.11; 169.76 and 169.63; 168.81; 163.09; 149.73;
149.12; 144.05; 135.69 and 135.52; 135.15; 131.10; 131.05;
129.81; 129.67; 129.45; 128.98; 128.38; 128.33; 127.31; 126.54;
126.39; 123.46; 107.74 and 104.74; 90.36; 84.66 and 83.51;
84.71 and 83.14; 82.25 and 81.56; 63.66 and 63.54; 20.28. IR
(CHCl3) ν 3425; 1745; 1680; 1563; 1369; 1196. MS (CI/NH3):
778 [M + H]+. Anal. Calcd for C39H31N5O7S3: C, 60.22; H,
4.02; N, 9.00. Found: C, 60.65; H, 4.27; N, 9.16.
6-O-Ben zyl-2-N-(N′,N′-d iben zylfor m a m id in e)-9-(2′,3′-O-
ben zylid en e-5′-O-a cetyl-â-D-r ibofu r a n osyl)gu a n in e (19).
Compound 19 is obtained as a white solid (mixture of two
diastereomers, 0.53 g, 77%), starting from 17 and following
the same procedure as described for 22. 1H NMR (CDCl3, 300
MHz) δ 9.00 and 8.99 (2s, 1H); 7.88 and 7.87 (2s, 1H); 7.56-
7.22 (m, 20H); 6.34 and 6.32 (2d, J ) 1.9 Hz, 1H); 6.19 and
6.04 (2s, 1H); 5.66 (s, 2H); 5.64 and 5.62 (dt, J ) 1.9, 6.4 Hz,
0.5H); 5.53 (dd, J ) 1.9, 6.8 Hz, 0.5H); 5.22-5.15 (m, 1H); 4.91
(AB syst, ∆δ ) 13.0 Hz, J AB ) 6.8 Hz, 1H); 4.73 (AB syst, ∆δ
) 14.4 Hz, J AB ) 3.0 Hz, 1H); 4.62-4.56 and 4.53-4.47 (2m,
1H); 4.43-4.38 (m, 2H); 4.34-4.26 (m, 2H); 2.02 and 1.99 (2s,
3H). 13C NMR (CDCl3, 75 MHz) δ 170.41; 162.54; 160.46;
159.23; 153.33 and 153.16; 139.45 and 139.12; 136.65; 135.95;
135.62; 135.57; 129.95; 129.84; 128.91; 128.63; 128.47; 128.36;
128.18; 127.94; 127.87; 127.78; 127.66; 118.34; 107.90 and
104.39; 89.53 and 88.94; 85.06 and 84.26; 83.73 and 82.22;
82.11 and 81.25; 68.21; 63.95 and 63.84; 54.38; 48.05; 20.64.
IR (KBr) ν 2927; 1743; 1376; 1228; 1090. MS (CI/NH3): 710
[M + 1]+. Anal. Calcd for C41H38N6O6: C, 69.28; H, 5.39; N,
11.82. Found: C, 69.31; H, 5.45; N, 11.83.
2-N-(N′,N′-Dieth ylben za m id in e)-9-(5′-O-a cetyl-2′,3′-O-
ben zylid en e-â-D-r ibofu r a n osyl)gu a n in e (20). N,N-Diethyl
benzamide (5.04 g, 28.4 mmol) is added to triethyloxonium
tetrafluoroborate (5.40 g, 30.5 mmol) in anhydrous ether (10
mL) at 0 °C. The reaction mixture is stirred at room temper-
ature for 1 h before sodium ethylate (2.26 g, 33.3 mmol) in
ethanol (15 mL) is added. The solution is stirred for 3 h and
filtered, and the filtrate is reduced in vacuo. The crude N,N-
diethyl benzamide diethyl acetal is then added to compound
16 (2.35 g, 5.7 mmol) in anhydrous DMF (25 mL). The reaction
mixture is stirred at room temperature for 16 h, reduced in
vacuo, and purified by chromatography on silica gel (CH2Cl2/
EtOH: 100/0 to 95/5) to yield 20 as a white solid (mixture of
two diastereomers, 2.48 g, 76%). 1H NMR (CDCl3, 200 MHz)
δ 7.80-7.15 (m, 11H); 6.21 and 6.14 (2d, J ) 2.2 Hz, 1H); 6.06
and 6.00 (2s, 1H); 4.86 and 4.80 (2dd, J ) 2.2, 6.8 Hz, 1H);
4.68 (m, 1H); 4.35 (m, 2H); 4.08 (m, 1H); 3.78 (m, 1H); 3.50
(m, 1H); 3.18 (m, 2H); 1.97 and 1.96 (2s, 3H); 1.37 (m, 3H);
1.11 (m, 3H). 13C NMR (CDCl3, 50 MHz) δ 169.75 and 169.69;
163.43 and 163.32; 158.53; 156.15; 149.09; 136.97 and 136.78;
135.42; 133.25 and 133.17; 129.38; 128.78; 127.97; 127.27;
127.09; 126.17; 126.04; 119.69; 107.03 and 103.43; 89.38; 84.18
and 82.58; 83.77 and 82.04; 81.96 and 81.28; 63.40 and 63.28;
43.77; 41.77; 20.04; 13.71; 11.76. IR (CHCl3) ν 3067; 2935;
1743; 1681; 1640; 1223; 1104. MS (CI/NH3): 573 [M + H]+.
Anal. Calcd for C30H32N6O6: C, 62.92; H, 5.64; N, 14.67.
Found: C, 62.79; H, 5.60; N, 14.61.
C
39H36N6O5: C, 70.04; H, 5.43; N, 12.57. Found: C, 70.04; H,
5.49; N, 12.66.
6-O-Ben zyl-2-N-(N′,N′-d iben zylfor m a m id in e)-9-(2′,3′-O-
ben zylid en e-â-D-r ibofu r a n osyl)gu a n in e (15). Compound
15 is obtained as a white solid (mixture of two diastereomers,
1.87 g, 98%), starting from 19 and following the same
procedure as for 9. 1H NMR (CDCl3, 200 MHz) δ 8.90 and
8.89 (2s, 1H); 7.87 and 7.77 (2s, 1H); 7.61-7.21 (m, 20H); 6.31
and 6.06 (2s, 1H); 6.07 and 6.00 (2d, J ) 4.4 Hz, 1H); 5.65 (s,
2H); 5.46 (dd, J ) 4.8, 6.2 Hz, 1H); 5.27-5.21 (m, 1H); 4.93
and 4.86 (2d, J ) 5.1 Hz, 1H); 4.71-4.68 (m, 1H); 4.64 and
4.53 (2d, J ) 1.6 Hz, 1H); 4.41 (m, 2H); 4.08-4.01 (m, 1H);
3.87-3.75 (m, 1H). 13C NMR (CDCl3, 50 MHz) δ 162.50 and
162.39; 160.60; 159.16 and 159.10; 152.63 and 152.46; 140.27
and 139.92; 136.63 and 136.52; 136.05 and 136.00; 135.53;
129.84; 129.56; 128.92; 128.86; 128.57; 128.46; 128.37; 128.17;
127.94; 127.59; 126.90; 126.49; 126.23; 119.33 and 119.13;
107.44 and 104.61; 93.46 and 91.38; 85.78 and 85.49; 83.79
and 82.77; 80.41; 68.28; 63.34 and 62.99; 54.39; 48.00. IR
(CHCl3) ν 3273; 2928; 1592; 1377; 1215; 1071. MS (CI/NH3):
669 [M + H]+. Anal. Calcd for C39H36N6O5: C, 70.04; H, 5.43;
N, 12.57. Found: C, 70.26; H, 5.55; N, 12.59.
9-(5′-O-Acet yl-2′,3′-O-b en zylid en e-â-D-r ib ofu r a n osyl)-
gu a n in e (16). 2′,3′-O-Benzylidene guanosine 645,46 (3.53 g,
12.2 mmol), 4-DMAP (0.10 g, 0.90 mmol), and triethylamine
(2.90 mL, 20.80 mmol) in acetonitrile (120 mL) are treated
dropwise at 0 °C with acetic anhydride (1.60 mL, 16.90 mmol).
The mixture is stirred at room temperature for 1 h before it is
reduced in vacuo. The residue is washed twice with water and
recrystallized in EtOH/H2O (8/2) to yield 16 as a white powder
(mixture of two diastereomers, 4.42 g, 88%). 1H NMR (DMSO-
d6, 200 MHz) δ 7.88 (s, 1H); 7.67-7.15 (m, 5H); 6.57 (s, 2H);
6.11 (m, 2H); 5.39 (m, 2H); 4.23 (m, 3H); 1.99 and 1.98 (2s,
3H). 13C NMR (DMSO-d6/CDCl3: 1/2, 50 MHz) δ 170.45;
156.98; 153.86; 149.94; 136.34; 135.16; 129.83; 128.48 and
128.38; 126.91; 117.04; 106.86 and 104.05; 88.41; 84.75 and
82.89; 84.01 and 82.06; 82.04 and 80.97; 64.07 and 64.04; 20.49.
IR (KBr) ν 3260; 2978; 1675; 1316; 1094. MS (CI/NH3): 414
[M + H]+. Anal. Calcd for C19H19N5O6: C, 55.20; H, 4.64; N,
16.94. Found: C, 55.10; H, 4.62; N, 16.80.
6-O-Ben zyl-9-(5′-O-a cetyl-2′,3′-O-ben zylid en e-â-D-r ibo-
fu r a n osyl)gu a n in e (17). Diethyl azodicarboxylate (420 µL,
2.68 mmol) is added to a mixture of compound 16 (550 mg,
1.33 mmol), triphenylphosphine (700 mg, 2.70 mmol), and
benzyl alcohol (280 µL, 2.73 mmol) in THF (10 mL) at room
temperature. The solution is stirred for 2 h and concentrated
under reduced pressure, and the crude residue is purified by
chromatography on silica gel (Et2O/n-C6H14: 1/1 to 1/0).
Compound 17 is obtained as a white powder (mixture of two
diastereomers, 281 mg, 42%). 1H NMR (CDCl3, 200 MHz) δ
7.69 and 7.68 (2s, 1H); 7.71-7.15 (m, 10H); 6.16 and 6.09 (2d,
J ) 1.8 Hz, 1H); 6.16 and 5.99 (2s, 1H); 5.56 (s, 2H); 5.49 (m,
2H); 5.17 (m, 3H); 4.62 (m, 2H); 4.12 (m, 1H); 2.06 and 2.04
(2s, 3H). 13C NMR (CDCl3, 50 MHz) δ 170.31; 160.84; 159.09;
152.82; 138.23 and 138.11; 136.04 and 135.67; 135.50 and
135.38; 129.68; 129.55; 128.22; 128.07; 127.89; 127.68; 127.26;
115.76; 107.31 and 104.05; 89.74 and 89.67; 84.80 and 84.56;
83.37 and 82.61; 82.32 and 81.68; 67.75; 63.61 and 63.43; 20.38.
MS (CI/NH3): 504 [M + H]+. Anal. Calcd for C26H25N5O6:
C, 62.02; H, 5.01; N, 13.91. Found: C, 62.20; H, 5.09; N, 14.16.
6-Th iop h en yl-2-(N,N-d ith iop h en ylca r boxy)-9-(5′-O-a c-
etyl-2′,3′-O-ben zylid en e-â-D-r ibofu r a n osyl)gu a n in e (18).
Compound 16 (6.0 g, 14.5 mmol) in pyridine (60 mL) is treated
with phenyl chlorothioformate (50.0 g, 145.0 mmol) at 0 °C in
the dark. The mixture is stirred for 5 h at room temperature,
and then it is poured on iced water (200 mL), stirred for 30
min, and extracted with chloroform. The organic layer is dried
over MgSO4 and reduced in vacuo, and the residue is purified
by chromatography on silica gel (Et2O/n-C6H14: 1/1 to 1/0) to
yield 18 as a pale yellow powder (mixture of two diastereomers,
7.9 g, 74%). 1H NMR (CDCl3, 200 MHz) δ 8.33 (s, 1H); 7.69-
7.27 (m, 20H); 6.34 and 6.28 (2d, J ) 1.8 Hz, 1H); 6.22 and
6.08 (2s, 1H); 5.66 and 5.60 (2dd, J ) 1.7, 6.4 Hz, 1H); 5.25
and 5.14 (2dd, J ) 3.3, 6.4 Hz, 1H); 4.73-4.58 (m, 1H); 4.41-
1-Ben zyl-2-N-(N′,N′-dieth ylben zam idin e)-9-(5′-O-acetyl-
2′,3′-O-ben zylid en e-â-D-r ibofu r a n osyl)gu a n in e (21). Com-
pound 21 is obtained as a white solid (mixture of two
diastereomers, 1.16 g, 91%), from 20 and following the same
procedure as for 17. 1H NMR (CDCl3, 200 MHz) δ 7.80-7.15
(m, 16H); 6.21 and 6.14 (2m, 1H); 6.05 and 6.01 (2s, 2H); 5.30
(m, 1H); 4.92 (m, 1H); 4.75-3.80 (m, 5H); 3.55 (m, 1H); 3.14
(m, 2H); 1.98 and 1.97 (2s, 3H); 1.31 (m, 3H); 1.06 (m, 3H).
13C NMR (CDCl3, 50 MHz) δ 169.91 and 169.85; 163.16 and
163.11; 157.91; 155.87; 146.88 and 146.82; 135.55; 138.20;
137.08 and 136.77; 135.55; 133.18; 132.90; 132.80; 131.76;
131.57; 131.11; 129.59; 129.52; 128.82; 128.19; 128.02; 127.93;
127.28; 127.03; 126.85; 126.29; 126.14; 119.62 and 119.54;
107.18 and 103.62; 89.84 and 89.74; 84.26 and 83.89; 82.61
and 82.36; 81.66; 63.60 and 63.44; 45.32; 43.78; 41.83; 20.23;
13.96; 12.64. MS (CI/NH3): 693 [M + H]+. Anal. Calcd for
C
37H38N6O6: C, 67.05; H, 5.78; N, 12.68. Found: C, 66.82; H,
5.70; N, 12.51.

7254 J . Org. Chem., Vol. 63, No. 21, 1998
Vincent et al.
2-N-(N′,N′-Diben zylfor m a m id in e)-9-(5′-O-a cetyl-2′,3′-O-
ben zylid en e-â-D-r ibofu r a n osyl)gu a n in e (22). Freshly dis-
tilled dibenzylamine (7.5 g, 38.1 mmol) and N,N-dimethyl-
formamide dimethylacetal (1.7 mL, 12.8 mmol) are refluxed
in acetonitrile (15 mL) for 24 h. Anhydrous toluene (15 mL)
is added, and the solution is reduced in vacuo. That operation
is repeated twice before the crude residue in anhydrous THF
(10 mL) is added to compound 16 (1.73 g, 4.2 mmol) in THF
(20 mL). The resulting solution is stirred for 16 h at room
temperature. THF is removed in vacuo, and the residue is
chromatographed on silica gel (AcOEt/EtOH: 100/0 to 95/5) to
yield compound 22 (mixture of two diastereomers, 2.45 g, 94%)
as a white solid. 1H NMR (CDCl3, 200 MHz) δ 8.94 and 8.93
(2s, 1H); 7.73 (s, 1H); 7.60-7.15 (m, 15H); 6.24 and 6.17 (2d,
J ) 2.5 Hz, 1H); 6.19 and 6.04 (2s, 1H); 5.45 and 5.41 (2dd, J
) 2.5, 6.6 Hz, 1H); 5.07-4.97 (m, 1H); 4.71 and 4.69 (2s, 2H);
4.65-4.39 (m, 2H); 4.47 (s, 2H); 4.28-4.18 (m, 1H); 2.10 and
2.01 (2s, 3H). 13C NMR (CDCl3, 50 MHz) δ 170.45; 158.27;
157.77; 156.86; 149.47; 136.96 and 136.86; 135.44 and 134.08;
134.71 and 134.65; 130.01; 129.87; 129.08; 128.79; 128.56;
128.50; 127.99; 127.84; 126.63; 126.48; 121.26; 107.83 and
104.37; 89.58; 85.25 and 83.88; 83.86 and 82.36; 82.28 and
81.38; 63.88 and 63.77; 54.87; 48.45; 20.63. IR (KBr) ν 2928;
1740; 1684; 1616; 1527; 1358; 1218. MS (CI/NH3): 621 [M +
H]+. Anal. Calcd for C34H32N6O6: C, 65.79; H, 5.20; N, 13.54.
Found: C, 66.00; H, 5.39; N, 13.69.
1738; 1272; 1016. MS (CI/NH3): 338 [M + NH4]+. Anal.
Calcd for C16H17O5P: C, 60.00; H, 5.35. Found: C, 60.43; H,
5.54.
1-Ben zyl-2-N-(N′,N′-d ib en zylfor m a m id in e)-9-[2′,3′-O-
ben zyliden e-5′-O-(O-ben zylacetyl(ben zyloxy)ph osph or yl)-
â-D-r ibofu r a n osyl]gu a n in e (25). Compounds 14 (100 mg,
0.15 mmol) and 24 (96 mg, 0.30 mmol) and DEAD (72 µL, 0.37
mmol) are dissolved in anhydrous THF (2 mL). The solution
is warmed to 60 °C, and triphenylphosphine (79 mg, 0.30
mmol) in solution in anhydrous THF (0.6 mL) is added in one
portion. The reaction mixture is stirred for 2 h at that
temperature, reduced in vacuo, and chromatographed on silica
gel (AcOEt/EtOH: 100/0 to 95/5) to yield 25 (mixture of four
diastereomers, 101 mg, 69%) as a colorless glassy solid. 1H
NMR (CDCl3, 300 MHz) δ 8.80 and 8.53 (2s, 1H); 7.80, 7.78,
7.77 and 7.74 (4s, 1H); 7.60-7.15 (m, 30H); 6.22, 6.19 and 6.12
(3d, J ) 2.6 Hz, 1H); 6.15, 6.14, 5.97 and 5.94 (4s, 1H); 5.55
(AB syst, ∆δ ) 9.0 Hz, J AB ) 14.7 Hz, 2H); 5.38, 5.30 and 5.21
(3dd, J ) 2.6, 6.4 Hz, 1H); 5.15-4.85 (m, 5H); 4.75-4.10 (m,
7H); 2.99, 2.98 and 2.95 (3d, J ) 21.4 Hz, 2H). 13C NMR
(CDCl3, 50 MHz) δ 165.25 and 165.14; 158.05; 157.28; 147.52;
138.22; 136.75; 136.65; 135.01; 129.86; 129.07; 128.66; 128.46;
128.32; 128.17; 127.94; 127.74; 127.62; 126.74; 126.62; 120.47;
107.72 and 104.41; 89.14, 88.98 and 88.79; 85.09 and 84.93;
83.83, 83.71 and 82.64; 81.92 and 80.65; 68.51 and 67.52; 65.22;
54.69; 48.41; 45.50; 34.24 (d, J ) 135.8 Hz). 31P NMR (CDCl3,
121 MHz) δ 21.72 (s, 0.25P), 21.70 (s, 0.25P), 21.67 (s, 0.25P)
and 21.64 (s, 0.25P). IR (CHCl3) ν 2924; 1733; 1689; 1612;
1-Ben zyl-2-N-(N′,N′-d iben zylfor m a m id in e)-9-(5′-O-a c-
etyl-2′,3′-O-ben zylid en e-â-D-r ibofu r a n osyl)gu a n in e (23).
Compound 23 is obtained as a white solid (mixture of two
diastereomers, 456 mg, 76%), from 22 and following the same
procedure as for 17. 1H NMR (CDCl3, 300 MHz) δ 8.84 and
8.81 (2s, 1H); 7.73 and 7.72 (2s, 1H); 7.60-7.15 (m, 20H); 6.23
and 6.17 (2d, J ) 2.3 Hz, 1H); 6.19 and 6.03 (2s, 1H); 5.57
(AB syst, ∆δ ) 9.0 Hz, J AB ) 14.3 Hz, 2H); 5.48 and 5.42 (2dd,
J ) 2.3, 6.4 Hz, 1H); 5.03 and 4.98 (2dd, J ) 4.8, 6.4 Hz, 1H);
4.66 (s, 2H); 4.62 and 4.53 (2q, J ) 4.5 Hz, 1H); 4.43-4.37 (m,
3H); 4.25 (dd, J ) 6.0, 11.7 Hz, 1H); 2.00 (s, 3H). 13C NMR
(CDCl3, 50 MHz) δ 170.41; 157.99; 157.79 and 157.69; 157.13;
147.27; 138.14; 136.82 and 136.62; 135.55; 135.47 and 134.89;
134.78 and 134.71; 132.09 and 131.89; 129.96 and 129.81;
129.06; 128.77; 128.46; 128.24; 128.13; 127.92; 127.61; 127.56;
126.71; 126.60; 126.42; 120.80; 107.83; 104.38; 89.49; 85.26 and
83.81; 83.73 and 82.34; 82.16 and 81.35; 63.91 and 63.80; 54.21;
48.47; 45.44; 20.59. IR (CHCl3) ν 2929; 1743; 1689; 1611; 1220;
1493; 1455; 1267; 1073; 1016; 999. Anal. Calcd for C55H51
-
N6O9P: C, 68.03; H, 5.30; N, 8.65. Found: C, 68.00; H, 5.19;
N, 8.58.
1-Ben zyl-2-N-(N′,N′-d ib en zylfor m a m id in e)-9-{2′,3′-O-
ben zylid en e-5′-O-[P ,P ′,P ′-t r ib en zyl-m et h ylen eb is(p h os-
p h on a te)]-â-D-r ibofu r a n osyl}gu a n in e (27). Compound 27
is obtained as a white solid (mixture of four diastereomers,
116 mg, 71%), starting from 14 and 26104,105 and following the
same procedure as for 25. 1H NMR (CDCl3, 300 MHz) δ 8.87,
8.86, 8.85 and 8.84 (4s, 1H); 7.95, 7.91, 7.86 and 7.83 (4s, 1H);
7.60-7.15 (m, 35 H); 6.24, 6.18, 6.15 and 6.11 (4d, J ) 2.6 Hz,
1H); 6.13, 6.11, 5.95 and 5.92 (4s, 1H); 5.53 (AB syst, ∆δ )
12.0 Hz, J AB ) 14.3 Hz, 2H); 5.17 (m, 2H); 5.40-5.14 (m, 2H);
5.08-4.91 (m, 6H); 4.61-4.06 (m, 7H); 2.46, 2.45, 2.42 and 2.37
(4t, J ) 21.1 Hz, 2H). 13C NMR (CDCl3, 50 MHz) δ 158.09
and 158.00; 157.22; 147.63 and 147.52; 136.83 and 136.66;
135.65; 135.59; 135.50; 135.01; 134.90; 133.45; 132.12; 131.92;
131.86; 1311.40; 129.79; 129.03; 128.78; 128.54; 128.46; 128.29;
128.08; 127.88; 127.71; 127.59; 126.70; 126.61; 126.49; 120.46,
120.37 and 120.26; 107.67 and 104.32; 88.98, 88.75, 88.66 and
88.52; 85.14, 84.91, 84.22 and 84.05; 83.84, 83.73, 82.75 and
83.66; 82.00, 81.76, 80.67 and 80.58; 68.51, 68.39, 68.28 and
68.16; 65.33 and 65.22; 54.65; 48.32; 45.47; 25.83 (t, J ) 137.2
Hz). 131P NMR (CDCl3, 121 MHz) δ 20.99 (AB syst, ∆δ ) 76.8
1093. MS (CI/NH3): 711 [M + H]+. Anal. Calcd for C41H38
-
N6O6: C, 69.28; H, 5.39; N, 11.82. Found: C, 69.38; H, 5.44;
N, 12.01.
(Ben zyloxyh yd r oxyp h osp h or yl)a cetic Acid Ben zyl Es-
ter (24). R-Dibenzylphosphonoacetic acid103 (5.45 g, 17.0
mmol), DCC (5.14 g, 25.0 mmol), and benzyl alcohol (2.0 mL,
19.0 mmol) are stirred at room temperature in dichlo-
romethane (60 mL) for 6 h. The reaction mixture is filtered,
and the filtrate is washed with 5% HCl. The organic layer is
dried over MgSO4, reduced in vacuo, and purified by chroma-
tography on silica gel (Et2O/n-C6H14: 3/7 to 10/0) to yield the
intermediate (dibenzyloxyphosphoryl)acetic acid benzyl ester
(5.30 g, 76%). 1H NMR (CDCl3, 200 MHz) δ 7.40-7.20 (m,
Hz, J AB ) 6.1 Hz, 0.5P); 20.98 (AB syst, ∆δ ) 76.8 Hz, J AB
)
6.1 Hz, 0.5P); 20.95 (AB syst, ∆δ ) 78.0 Hz, J AB ) 5.8 Hz,
0.5P); 20.91 (AB syst, ∆δ ) 77.4 Hz, J AB ) 6.2 Hz, 0.5P). IR
(CHCl3) ν 3063; 2925; 1689; 1612; 1493; 1259; 1066; 998. Anal.
Calcd for C61H58N6O10P2: C, 66.78; H, 5.33; N, 7.66. Found:
C, 67.02; H, 5.47; N, 7.81.
15H); 5.13 (s, 2H); 5.04 (m, 4H); 3.04 (d, J ) 21.5 Hz, 2H). 13
C
NMR (CDCl3, 50 MHz) δ 165.33 (d, J ) 5.7 Hz); 135.72 (d, J
) 5.8 Hz); 135.06; 128.39 (b); 128.29 (b); 127.87; 67.95 (d, J )
5.8 Hz); 57.28; 34.58 (d, J ) 134.4 Hz). 31P NMR (CDCl3, 121
MHz) δ 20.86 (s). IR (CHCl3) ν 3065; 3033; 2948; 1734; 1456;
1381; 1270; 1215; 1115; 997. MS (CI/NH3): 411 [M + H]+.The
latter compound (1.20 g, 2.9 mmol) is refluxed in toluene (15
mL) with DABCO (0.36 g, 3.2 mmol) for 4 h. The solution is
reduced in vacuo, and the residue is gently stirred with Dowex
50 × 8 resin (H+ form) in water/methanol (9/1, 100 mL) for 12
h. The ion-exchange resin is filtered off, and the filtrate is
reduced in vacuo and dried by azeotropic distillation with
toluene to yield 24 (0.90 g, 97%) as a colorless oil. 1H NMR
(CDCl3, 200 MHz) δ 7.40-7.20 (10H); 5.14 (s, 2H); 5.07 (d, J
) 7.9 Hz, 2H); 3.05 (d, J ) 21.6 Hz, 2H). 13C NMR (CDCl3, 50
MHz) δ 165.47; 135.84 (d, J ) 5.8 Hz); 135.22; 128.3 (b); 127.80
(b); 67.67 (d, J ) 5.8 Hz); 57.34; 34.59 (d, J ) 137.9 Hz). 31P
NMR (CDCl3, 121 MHz) δ 22.69 (s). IR (CHCl3) ν 3500; 2941;
1-Ben zyl-2-N-(N′,N′-d ib en zylfor m a m id in e)-9-{2′,3′-O-
ben zyliden e-5′-O-[N-ben zyl-(P ,P ′,P ′-tr iben zylim idodiph os-
p h a te)]-â-D-r ibofu r a n osyl}gu a n in e (29). Compound 29 is
obtained as a glassy solid (mixture of four diastereomers, 83
mg, 75%), starting from 14 and 28,105 and following the same
procedure as for 25. 1H NMR (CDCl3, 300 MHz) δ 8.86 and
8.83 (2s, 1H); 7.80, 7.77, 7.74, and 7.72 (4s, 1H); 7.60-7.15
(m, 40H); 6.16 and 6.15 (2d, J ) 1.9 Hz, 0.5H), 6.08 and 6.07
(2d, J ) 3.0 Hz, 0.5H); 6.09, 5.89 and 5.86 (3s, 1H); 5.55 (AB
syst, ∆δ ) 10.5 Hz, J AB ) 13.9 Hz, 2H); 5.17 (m, 1H); 5.05-
4.31 (m, 14H); 4.25-3.95 (m, 2H). 13C NMR (CDCl3, 50 MHz)
δ 158.07; 158.06; 157.20; 147.54; 138.25 and 137.84; 136.52;
(104) Saady, M.; Lebeau, L.; Mioskowski, C. Helv. Chim. Acta 1995,
78, 670-678.
(105) Saady, M.; Lebeau, L.; Mioskowski, C. J . Org. Chem. 1995,
60, 2946-2947.

Enzymatically Stable Analogues of GDP
J . Org. Chem., Vol. 63, No. 21, 1998 7255
135.67; 135.55; 135.41; 135.28; 135.03; 134.98; 134.90; 129.86;
129.80; 129.02; 128.76; 128.31; 128.13; 128.01; 127.96; 127.90;
127.75; 127.64; 126.70; 126.61; 126.49; 120.50; 107.66, 104.36
and 104.31; 88.65 and 88.45; 84.87 and 84.72; 83.70, 83.42 and
82.24; 82.10, 81.98 and 80.61; 69.19; 69.07; 68.98 and 68.88;
66.13 and 66.04; 54.62; 50.81; 48.32; 45.46. 31P NMR (CDCl3,
121 MHz) δ 4.71 (AB syst, ∆δ ) 65.3 Hz, J AB ) 20.4 Hz, 0.5P);
4.67 (AB syst, ∆δ ) 67.8 Hz, J AB ) 20.4 Hz, 0.5P); 4.66 (AB
syst, ∆δ ) 54.4 Hz, J AB ) 20.7 Hz, 0.5P); 4.56 (AB syst, ∆δ )
59.9 Hz, J AB ) 20.4 Hz, 0.5P). IR (CHCl3) ν 2926; 1691; 1612;
1493; 1275; 1016. Anal. Calcd for C67H63N7O10P2: C, 67.72;
H, 5.35; N, 8.25. Found: C, 67.58; H, 5.29; N, 8.23.
1-Ben zyl-2-N-(N′,N′-d ib en zylfor m a m id in e)-9-[2′,3′-O-
b en zylid en e-5′-N-(N,N′-d iet h ylca r b oxyd ia za )-â-D-r ib o-
fu r a n osyl]gu a n in e (30). Compound 30 is a side product
obtained as a white solid in the different coupling reactions
(mixture of two diastereomers, 32-58%). 1H NMR (CDCl3, 300
MHz) δ 8.85 (s, 1H); 7.64 and 7.62 (2s, 1H); 7.60-7.15 (m,
20H); 6.15 and 5.98 (2s, 1H); 6.09 and 6.04 (2d, J ) 4.4 Hz,
1H); 5.55 (AB syst, ∆δ ) 16.5 Hz, J AB ) 14.2 Hz, 2H); 4.92
(m, 1H); 4.80-4.40 (m, 6H); 4.35-3.98 (m, 6H); 1.18 (m, 6H).
13C NMR (CDCl3, 50 MHz) δ 158.03; 158.02; 157.22; 156.78
and 156.64; 147.24; 138.14; 137.57; 135.55; 134.96; 132.15;
131.95; 129.84; 129.05; 128.79; 128.59; 128.48; 128.28; 128.16;
128.01; 127.96; 127.91; 127.75; 127.64; 126.75; 126.60; 126.52;
126.45; 120.98; 107.50 and 104.32; 89.18; 83.61; 83.06 and
81.97; 80.63; 62.73 and 62.06; 54.63; 51.33; 48.44; 45.54; 14.31.
IR (KBr) ν 3237; 2981; 1694; 1612; 1493; 1092. MS (CI/NH3):
828 [M + H]+. Anal. Calcd for C45H46N8O8: C, 65.36; H, 5.61;
N, 13.55. Found: C, 65.41; H, 5.67; N, 13.60.
1-{2-[2-(2-Azid oeth oxy)eth oxy]eth yl}-2-N-(N′,N′-d iben -
zylfor m a m id in e)-9-(5′-O-a cetyl-2′,3′-O-ben zylid en e-â-D-r i-
bofu r a n osyl)gu a n in e (32). Compound 32 is obtained as a
white solid (mixture of two diastereomers, 603 mg, 55%),
starting from 22 and 31,106 and following the same procedure
as for 17, except that triphenylphosphine and DEAD are mixed
together before they are added to 22 and 31. 1H NMR (CDCl3,
200 MHz) δ 8.91 and 8.88 (2s, 1H); 7.70 (s, 1H); 7.60-7.15
(m, 15H); 6.23 and 6.14 (2d, J ) 6.4 Hz, 1H); 6.19 and 6.03
(2s, 1H); 5.47 and 5.42 (2dd, J ) 2.3, 6.4 Hz, 1H); 5.03 (2dd, J
) 3.2, 6.4 Hz, 1H); 4.71 (s, 2H); 4.60 (t, J ) 6.3 Hz, 2H); 4.62-
4.36 (m, 2H); 4.48 (s, 2H); 4.28-4.19 (m, 1H); 3.74 (t, J ) 6.3
Hz, 2H); 3.69-3.54 (m, 6H); 3.28 (t, J ) 5.2 Hz, 2H); 2.00 (s,
3H). 13C NMR (CDCl3, 50 MHz) δ 170.34; 157.73; 157.64;
156.97; 147.04; 136.75 and 136.54; 135.48 and 135.40; 135.10;
134.68; 129.90; 129.74; 129.02; 128.77; 128.40; 128.07; 127.89;
127.67; 126.54; 126.36; 120.70; 107.72 and 104.27; 89.50; 85.16;
83.72; 82.34; 82.18 and 81.36; 70.37; 70.01; 69.71; 68.32; 63.89;
54.85; 50.43; 48.56; 41.16; 20.53. IR (CHCl3) ν 2923; 2106;
1743; 1690; 1613; 1493; 1223; 1097. MS (CI/NH3): 779 [M +
H]+. Anal. Calcd for C40H43N9O8: C, 61.77; H, 5.58; N, 16.21.
Found: C, 61.29; H, 5.31; N, 15.84.
1-{2-[2-(2-Azid oeth oxy)eth oxy]eth yl}-2-N-(N′,N′-d iben -
zylfor m a m id in e)-9-(2′,3′-O-b en zylid en e-â-D-r ib ofu r a n o-
syl)gu a n in e (33). Compound 33 is obtained as a white solid
(mixture of two diastereomers, 1.33 g, 99%), starting from 32
and following the same procedure as for 9. 1H NMR (CDCl3,
200 MHz) δ 8.82 and 8.79 (2s, 1H); 7.77 and 7.72 (2s, 1H);
7.60-7.15 (m, 15H); 6.25 and 6.08 (2s, 1H); 6.23 and 6.14 (2d,
J ) 2.4 Hz, 1H); 5.42 (m, 1H); 5.20 (m, 1H); 4.77-4.45 (m,
7H); 4.08-3.97 (m, 1H); 3.85-3.52 (m, 9H); 3.27 (t, J ) 4.9
Hz, 2H). 13C NMR (CDCl3, 50 MHz) δ 158.13; 157.64; 157.24;
146.97; 137.61; 136.08 and 135.80; 134.99; 134.56; 129.61;
129.43; 128.64; 128.24; 128.05; 127.95; 127.84; 127.68; 126.67;
126.39; 125.03; 120.55; 107.28 and 103.98; 91.42 and 90.12;
85.27 and 84.96; 83.84 and 82.71; 83.14 and 80.25; 70.21; 69.96;
69.61; 68.11; 62.41; 62.08; 54.88; 50.30; 48.04; 41.19. IR
(CHCl3) ν 3338; 2923; 2106; 1688; 1613; 1494; 1097. MS (CI/
NH3): 736 [M + H]+. Anal. Calcd for C38H41N9O7: C, 62.03;
H, 5.62; N, 17.13. Found: C, 62.14; H, 5.65; N, 17.20.
1-{2-[2-(2-Azid oeth oxy)eth oxy]eth yl}-2-N-(N′,N′-d iben -
zylfor m a m id in e)-9-{2′,3′-O-b en zylid en e-5′-O-[N-b en zyl-
(P ,P ′,P ′-tr iben zylim id od ip h osp h a te)]-â-D-r ibofu r a n osyl}-
gu a n in e (34). Compound 34 is obtained as a glassy solid
(mixture of four diastereomers, 121 mg, 97%), starting from
33 and 28,105 and following the same procedure as for 25,
except that triphenylphosphine and DEAD are mixed together
before they are added to 28 and 33. 1H NMR (CDCl3, 200
MHz) δ 8.93 and 8.91 (2s, 1H); 7.80, 7.75, 7.72, and 7.68 (4s,
1H); 7.60-7.15 (m, 35H); 6.16 and 6.08 (2m, 1H); 6.10, 6.06,
5.90, and 5.87 (4s, 1H); 5.17 (m, 1H); 5.13-4.43 (m, 15H); 4.68
(s, 2H); 4.32 (m, 1H); 4.19-4.02 (m, 1H); 3.72 (t, J ) 6.4 Hz,
2H); 3.64-3.45 (m, 6H); 3.27 (t, J ) 5.0 Hz, 2H). 13C NMR
(CDCl3, 50 MHz) δ 158.07; 157.89; 157.11; 147.54 and 147.48;
137.84; 136.46; 135.73; 135.56; 135.42; 135.30; 134.93; 129.81;
129.06; 128.86; 128.72; 128.46; 128.31; 128.23; 128.08; 127.97;
127.88; 127.74; 126.61; 126.50; 120.55; 107.76 and 104.30;
88.47; 83.70 and 83.44; 82.31 and 82.14; 81.99 and 80.67; 70.50;
70.13; 69.84; 68.97 (b); 68.42; 66.14; 54.88; 50.83; 50.58; 48.50;
41.25. 31P NMR (CDCl3, 121 MHz) δ 4.78 (AB syst, ∆δ ) 65.3
Hz, J AB ) 20.1 Hz, 0.5P); 4.76 (AB syst, ∆δ ) 65.3 Hz, J AB
)
20.1 Hz, 0.5P); 4.63 (AB syst, ∆δ ) 64.1 Hz, J AB ) 20.7 Hz,
0.5P); 4.60 (AB syst, ∆δ ) 61.7 Hz, J AB ) 20.1 Hz, 0.5P). IR
(KBr) ν 2926; 2105; 1692; 1611; 1493; 1454; 1273; 1216. Anal.
Calcd for
C66H68N10O12P2: C, 67.15; H, 5.46; N, 11.16.
Found: C, 63.29; H, 5.49; N, 11.22.
1-{2-[2-(2-Azid oet h oxy)et h oxy]et h yl}-9-[5′-O-(im id o-
d ip h osp h a te)-â-D-r ibofu r a n osyl]gu a n in e (35). Compound
35 is obtained as its bis-triethylammonium salt (white powder,
25 mg, 97%), starting from 34 and following the same
procedure as for 1. 1H NMR (D2O, 300 MHz) δ 8.04 (s, 1H);
5.83 (d, J ) 6.6 Hz, 1H); 4.40 (m, 1H); 4.25-3.95 (m, 5H); 3.81
(t, J ) 5.1 Hz, 2H); 3.65-3.45 (m, 6H); 3.11 (q, J ) 7.3 Hz,
12H); 3.01 (t, J ) 4.4 Hz, 2H); 1.19 (t, J ) 7.3 Hz, 18H). 13C
NMR (D2O/CD3OD: 9/1, 75 MHz) δ 159.37; 149.84; 138.15;
116.53; 86.93; 84.24; 73.63; 70.64; 70.18; 69.76; 68.06; 66.46;
64.46; 46.80; 42.37; 39.18. 31P NMR (D2O, 121 MHz, [Et3N]
) 0.5 M) δ 3.73 (d, J ) 2.4 Hz, 1P), -0.11 (d, J ) 2.4 Hz, 1P).
IR (KBr) ν 3338; 3216; 2947; 2602; 2493; 1694; 1537; 1198;
1037. HRMS: calcd for
C16H28N7O12P2 572.1271, found
572.1260 [M - H]-. HPLC (ammonium carbonate 0.5 M, pH
7.0/CH3CN 99/1) tR 14.2 min.
6-O-(2,4,6-Tr iisop r op ylben zen esu lfon yl)-2-N-(N′,N′-d i-
ben zylfor m a m id in e)-9-(2′,3′-O-ben zylid en e-5′-O-a cetyl-â-
D-r ibofu r a n osyl)gu a n in e (36). 2,4,6-Triisopropylbenzene-
sulfonyl chloride (TPSCl) (710 mg, 3.00 mmol) is added at 0
°C to a solution containing compound 22 (720 g, 1.16 mmol),
4-DMAP (35 mg, 0.29 mmol), triethylamine (0.6 mL, 4.30
mmol), and CH2Cl2 (5 mL). The reaction mixture is refluxed
for 30 min. Then it is washed with aqueous NaHCO3, and
the organic layer is dried over MgSO4 and reduced in vacuo.
The crude residue is purified by chromatography on silica gel
(CH2Cl2/Et2O: 1/0 to 0/1) to yield compound 36 as a white solid
(mixture of two diastereomers, 1.011 g, 98%). 1H NMR (CDCl3,
200 MHz) δ 8.74 and 8.73 (2s, 1H); 8.02 and 8.00 (2s, 1H);
7.60-7.15 (m, 17H); 6.35 and 6.34 (2d, J ) 2.2 Hz, 1H); 6.18
and 6.04 (2s, 1H); 5.60 and 5.48 (2dd, J ) 2.2, 4.9 Hz, 1H);
5.17 (td, J ) 2.5, 4.5 Hz, 1H); 4.85 and 4.81 (2d, J ) 4.0 Hz,
1H); 4.66-4.28 (m, 6H); 4.53 (h, J ) 6.9 Hz, 2H); 2.83 (h, J )
6.9 Hz, 1H); 2.04 and 2.00 (2s, 3H); 1.28 (d, J ) 6.9 Hz, 12H);
1.19 (d, J ) 6.8 Hz, 6H). 13C NMR (CDCl3, 50 MHz) δ 170.25;
162.12; 159.82; 156.12 and 156.05; 154.31; 153.93; 150.67;
141.64 and 141.28; 135.43 and 135.17; 131.88; 129.80; 128.86;
128.57; 128.41; 128.20; 127.68; 126.60; 126.54; 123.71; 119.91
and 119.83; 107.96 and 104.42; 89.54 and 88.75; 84.86 and
83.64; 84.16 and 81.99; 81.98 and 80.92; 63.81; 54.41; 47.65;
34.08; 29.80; 24.57; 23.35; 20.60. IR (CHCl3) ν 2959; 2869;
1745; 1596; 1381; 1198; 1073. MS (CI/NH3): 887 [M + H]+.
Anal. Calcd for C49H54N6O8S: C, 66.34; H, 6.14; N, 9.47.
Found: C, 66.12; H, 6.02; N, 9.39.
6-O-[2-(2-Azid oeth oxy)eth yl]-2-N-(N′,N′-d iben zylfor m -
a m id in e)-9-(2′,3′-O-ben zylid en e-5′-O-a cetyl-â-D-r ibofu r a -
n osyl)gu a n in e (38). Quinuclidine (74 mg, 0.66 mmol) and
compound 37 (527 mg, 0.59 mmol) in anhydrous dichlo-
romethane (4 mL) are stirred at room temperature for 2 h.
DBU (100 µL, 0.67 mmol) and (2-azidoethoxy)ethanol 37106 in
dichloromethane (2 mL) are added, and the reaction mixture
(106) Lebeau, L.; Olland, S.; Oudet, P.; Mioskowski, C. Chem. Phys.
Lipids 1992, 62, 93-103.

7256 J . Org. Chem., Vol. 63, No. 21, 1998
Vincent et al.
is stirred for 12 h, reduced in vacuo, and chromatographed
(Et2O/n-C6H14: 6/4 to 10/0) to yield 38 as a white solid (330
mg, 77%). 1H NMR (CDCl3, 300 MHz) δ 9.04 (s, 1H); 7.88 and
7.86 (2s, 1H); 7.60-7.15 (m, 15H); 6.32 and 6.28 (2d, J ) 2.5
Hz, 1H); 6.14 and 5.98 (2s, 1H); 5.59 and 5.50 (2dd, J ) 2.5,
5.0 Hz, 1H); 5.17 (m, 1H); 4.92-4.18 (m, 9H); 3.91 (t, J ) 5.0
Hz, 2H); 3.32 (t, J ) 5.0 Hz, 2H); 1.94 and 1.96 (2s, 3H). 13C
NMR (CDCl3, 75 MHz) δ 170.07; 162.24; 160.25; 158.81; 153.00
and 152.86; 139.37 and 139.11; 135.71; 135.41; 135.33; 129.67;
129.56; 128.63; 128.34; 128.23; 128.17; 127.88; 127.50; 127.36;
126.44; 126.35; 117.92; 107.52 and 104.03; 89.27 and 88.72;
84.77 and 83.38; 84.01 and 82.02; 81.88 and 81.01; 69.87 and
68.94; 65.56; 63.69 and 63.57; 54.07; 50.37; 47.74; 20.37. IR
(CHCl3) ν 2934; 2104; 1742; 1591; 1377; 1230; 1072. MS (CI/
NH3): 734 [M + H]+. Anal. Calcd for C38H39N9O7: C, 62.20;
H, 5.36; N, 17.18. Found: C, 62.65; H, 5.63; N, 17.38.
6-O-[2-(2-Azid oeth oxy)eth yl]-2-N-(N′,N′-d iben zylfor m -
a m id in e)-9-(2′,3′-O-b en zylid en e-â-D-r ib ofu r a n osyl)gu a -
n in e (39). Compound 39 is obtained as a white solid (273
mg, 92%), starting from 38 and following the same procedure
as for 9. 1H NMR (CDCl3, 200 MHz) δ 8.97 and 8.96 (2s, 1H);
7.90 and 7.79 (2s, 1H); 7.80-7.15 (m, 15H); 6.09 and 6.01 (2d,
J ) 4.8 Hz, 1H); 6.29 and 6.04 (2s, 1H); 5.47 and 5.40 (2dd, J
) 4.8, 6.2 Hz, 1H); 6.29 and 6.04 (2s, 1H); 5.47 and 5.40 (2dd,
J ) 4.8, 6.2 Hz, 1H); 5.23 (2d, J ) 6.2 Hz, 1H); 5.01-4.35 (m,
5H); 4.29 and 4.04 (2s, 2H); 4.08-3.75 (m, 2H); 3.96 (t, J )
5.1 Hz, 2H); 3.76 (t, J ) 4.9 Hz, 2H); 3.39 (t, J ) 4.9 Hz, 2H).
13C NMR (CDCl3, 50 MHz) δ 162.77 and 162.63; 160.97 and
160.95; 159.22 and 159.16; 152.81 and 152.67; 140.50 and
140.15; 136.86; 136.20; 135.75; 129.76; 129.03; 128.76; 128.69;
128.49; 128.33; 128.15; 127.79; 127.31; 126.43; 119.02 and
118.79; 107.35 and 104.43; 93.14 and 90.97; 85.60 and 85.43;
83.73 and 83.58; 82.80 and 80.35; 70.07; 69.06; 65.73; 63.14
and 62.76; 54.30; 50.55; 47.89. IR (CHCl3) ν 3277; 2922; 2104;
1592; 1377; 1239; 1072. MS (CI/NH3): 692 [M + H]+. Anal.
Calcd for C36H37N9O6: C, 62.51; H, 5.39; N, 18.22. Found: C,
62.50; H, 5.36; N, 18.22.
9-[5′-O-(Im id od ip h osp h a t e)-3′-O-p r op ion yl-â-D-r ib o-
fu r a n osyl]gu a n in e (42). Compound 42 is obtained as its
tris-ammonium salt from 41 following the same procedure as
for 1, but replacing triethylamine with ammonium carbonate
(white powder, 23 mg, 94%). Analytical sample contains 30%
of the 2′-propionyl isomer 48, resulting from partial isomer-
ization of 42. 1H NMR (D2O, 300 MHz) δ 8.20 (s, 1H); 6.03 (d,
J ) 4.1 Hz, 0.3H); 5.90 (d, J ) 7.2 Hz, 0.7H); 5.56 (dd, J )
4.1, 5.1 Hz, 0.3H); 5.41 (d, J ) 5.1 Hz, 0.7H); 4.98 (dd, J )
5.1, 7.2 Hz, 0.7H); 4.70-4.62 (m, 0.7H); 4.43 (m, 0.7H); 4.29
(m, 0.3H); 4.19-4.05 (m, 2H); 2.47 (q, J ) 7.2 Hz, 1.4H); 2.41
(q, J ) 7.2 Hz, 0.6H); 1.09 (t, J ) 7.2 Hz, 2.1H); 1.01 (t, J )
7.2 Hz, 0.9H). 31P NMR (D2O, pH ) 7.0, 121 MHz) δ 1.36 (s,
1P); 0.19 (s, 1P). HRMS: calcd for C13H19N6O11P2 497.0587,
found 497.0577 [M - H]-. HPLC (ammonium carbonate 0.5
M, pH 7.0) tR 3.1 min (42), 6.6 min (48).
2-N-(N′,N′-Diben zylfor m am idin e)-9-(â-D-r ibofu r an osyl)-
gu a n in e (43). Freshly distilled dibenzylamine (7.5 g, 38.1
mmol) and N,N-dimethylformamide dimethylacetal (1.7 mL,
12.8 mmol) are refluxed in acetonitrile (15 mL) for 24 h.
Anhydrous toluene (15 mL) is added, and the solution is
reduced in vacuo. That operation is repeated twice before the
crude residue in anhydrous acetonitrile (10 mL) is added to
guanosine (1.19 g, 4.2 mmol) in acetonitrile (20 mL). The
resulting solution is stirred for 24 h at 45 °C and poured in
Et2O (65 mL). The precipitate is collected by filtration, and
the filtrate is reduced in vacuo and once again poured into
Et2O (65 mL). The solids are combined and purified by
chromatography on silica gel (CH2Cl2/EtOH: 99/1 to 80/20) to
yield 43 as a white powder (1.44 g, 70%). 1H NMR (CD3OD,
200 MHz) δ 9.05 (s, 1H); 8.10 (s, 1H); 7.40-7.20 (m, 10H); 5.99
(d, J ) 5.9 Hz, 1H); 4.73 (dd, J ) 5.5, 5.9 Hz, 1H); 4.68 (s,
2H); 4.53 (s, 2H); 4.37 (dd, J ) 3.3, 5.5 Hz, 1H); 4.14 (m, 1H);
3.82 (AB part of ABX syst, ∆δ ) 13.0 Hz, J AB ) 12.2 Hz, J AX
) 2.7 Hz, J BX ) 2.7 Hz, 2H). 13C NMR (CD3OD, 50 MHz) δ
160.75; 160.29; 159.34; 151.80; 139.84; 136.99; 136.79; 130.26;
130.03; 129.68; 129.59; 129.10; 121.74; 90.50; 87.58; 75.91;
72.51; 63.32; 56.13; 49.15. IR (CHCl3) ν 3282; 2922; 1691;
1618; 1536; 1360; 1193. MS (CI/NH3): 491 [M + H]+. Anal.
Calcd for C25H26N6O5: C, 61.21; H, 5.35; N, 17.13. Found: C,
61.29; H, 5.37; N, 17.17.
1-Ben zyl-2-N-(N′,N′-d iben zylfor m a m id in e)-9-(â-D-r ibo-
fu r a n osyl)gu a n in e (44). Sodium hydride (60% in oil, 183
mg, 4.5 mmol) is added to compound 43 (1.74 g, 3.6 mmol) in
anhydrous DMF (35 mL) at -10 °C. The reaction mixture is
stirred for 2 h at that temperature. Then benzyl bromide (620
mg, 3.6 mmol) in DMF (5 mL) is added dropwise in 20 min,
and the solution is stirred for 12 h at -10 °C. Aqueous NH4-
Cl (1 mL) is added, the mixture is reduced in vacuo, and the
residue is purified by chromatography on silica gel (CH2Cl2/
EtOH: 99/1 to 90/10) to yield 44 as a white powder (1.69 g,
81%). 1H NMR (CDCl3,/CD3OD: 1/3, 200 MHz) δ 8.81 (s, 1H);
7.94 (s, 1H); 7.40-7.10 (m, 15H); 5.91 (d, J ) 6.4 Hz, 1H);
5.47 (s, 2H); 4.74 (dd, J ) 5.3, 6.4 Hz, 1H); 4.57 (s, 2H); 4.45
(s, 2H); 4.34 (dd, J ) 3.0, 5.3 Hz, 1H); 4.16 (m, 1H); 3.79 (AB
part of ABX syst, ∆δ ) 15.0 Hz, J AB ) 12.4 Hz, J AX ) 2.6 Hz,
J BX ) 2.6 Hz, 2H). 13C NMR (CD3OD, 50 MHz) δ 159.11;
159.10; 158.28; 148.66; 139.16; 138.39; 135.61; 135.47; 129.53;
129.33; 128.99; 128.85; 128.69; 128.39; 127.72; 127.49; 120.69;
89.95; 86.88; 74.85; 71.82; 62.69; 55.49; 48.52; 43.35. IR
(CHCl3) ν 3384; 1674; 1611; 1492; 1118. MS (CI/NH3): 581
[M + H]+. Anal. Calcd for C32H33N6O5: C, 66.08; H, 5.72; N,
14.45. Found: C, 66.20; H, 5.74; N, 14.79.
1-Ben zyl-2-N-(N′,N′-d iben zylfor m a m id in e)-9-(2′-O-ben -
zyl-â-D-r ibofu r a n osyl)gu a n in e (45). Sodium hydride (60%
in oil, 39 mg, 0.98 mmol) is added to compound 43 (201 mg,
0.41 mmol) in anhydrous DMF (8 mL) at -15 °C. The reaction
mixture is stirred for 3 h at that temperature. Then benzyl
bromide (144 mg, 0.84 mmol) in DMF (3 mL) is added
dropwise, and the solution is stirred for 16 h at -15 °C.
Aqueous NH4Cl (1 mL) is added, the mixture is reduced in
vacuo, and the residue is purified by chromatography on silica
gel (CH2Cl2/EtOH: 99/1 to 95/5) to yield 45 as a white solid
(148 mg, 54%). 1H NMR (CD3OD, 300 MHz) δ 8.08 (s, 1H);
7.67 (s, 1H); 7.40-6.76, (m, 20H); 5.81 (d, J ) 7.3 Hz, 1H);
6-O-[2-(2-Azid oeth oxy)eth yl]-2-N-(N′,N′-d iben zylfor m -
a m id in e)-9-{2′,3′-O-b en zylid en e-5′-O-[N-b en zyl-(P ,P ′,P ′-
t r ib e n zylim id od ip h osp h a t e )]-â-D-r ib ofu r a n osyl}gu a -
n in e (40). Compound 40 is obtained as a white solid (mixture
of four diastereomers, 53 mg, 32%), starting from 39 and 28,105
and following the same procedure as for 34. 1H NMR (CDCl3,
300 MHz) δ 9.10, 9.09, 9.08, and 9.07 (4s, 1H); 7.99, 7.98, 7.94,
and 7.91 (4s, 1H); 7.74-7.71 (m, 2H); 7.56-7.20 (m, 33H); 6.36,
6.35, 6.32, and 6.31 (4d, J ) 3.6 Hz, 1H); 6.09, 6.08, 5.90, and
5.86 (4s, 1H); 5.23-5.12 (m, 1H); 5.05-4.76 (m, 9H); 4.56 (t, J
) 20.5 Hz, 2H); 4.40 and 4.39 (2s, 2H); 4.32 (t, J ) 6.6 Hz,
2H); 4.28-4.05 (m, 3H); 3.97 (t, J ) 5.4 Hz, 2H); 3.77 (t, J )
4.9 Hz, 2H); 3.38 (t, J ) 4.9 Hz, 2H). 13C NMR (CDCl3, 50
MHz) δ 162.48; 160.45; 159.27; 153.61 and 153.44; 139.00;
138.82; 137.87; 135.88; 135.62; 135.48; 135.33; 133.40; 132.13;
131.92; 131.35; 130.02; 129.81; 129.73; 128.86; 128.78; 128.57;
128.43; 128.00; 127.74; 127.59; 126.67; 126.61; 117.86 and
117.74; 107.90, 107.74, 107.90 and 107.81; 88.55, 88.52, 87.25
and 87.16; 84.59 and 83.64; 84.44 and 81.82; 81.67 and 79.98;
70.36; 69.66; 69.23 and 69.14; 69.02 and 68.94; 66.57, 66.31,
66.20 and 66.13; 65.80; 54.30; 50.81; 50.78; 47.89. 31P NMR
(CDCl3, 121 MHz) δ 5.90 (m, 1P); 5.07 (m, 1P). IR (CHCl3) ν
2926; 2104; 1706; 1595; 1252; 1014. Anal. Calcd for C64H64
10O11P2: C, 63.46; H, 5.33; N, 11.56. Found: C, 63.64; H,
5.40; N, 11.62.
-
N
6-O-[2-(2-Am in oe t h oxy)e t h yl]-9-[5′-O-(im id od ip h os-
p h a te)-â-D-r ibofu r a n osyl]gu a n in e (41). Compound 41 is
obtained as its bis-triethylammonium salt from 40 following
the same procedure as for 1 (white powder, 15 mg, 62%). 1H
NMR (D2O, 200 MHz) δ 8.04 (s, 1H); 5.83 (d, J ) 6.6 Hz, 1H);
4.68 (m, 1H); 4.46 (m, 1H); 4.26 (m, 1H); 4.10-3.77 (m, 8H);
3.11 (q, J ) 7.5 Hz, 12H); 2.97 (m, 2H); 1.18 (t, J ) 7.5 Hz,
18H). 13C NMR (CDCl3, 75 MHz) δ 162.00; 157.26; 152.92;
129.79; 116.71; 87.80; 84.97; 74.72; 71.58; 69.18; 66.95; 66.46;
64.82; 39.17. 31P NMR (D2O, 121 MHz, [Et3N] ) 0.5 M) δ 4.57
(s, 1P); -0.06 (s, 1P). HRMS: calcd for
C14H24N7O11P2
528.1009, found 528.1031 [M - H]-. HPLC (ammonium
carbonate 0.5 M, pH 7.0/CH3CN 99/1) tR 12.6 min.

Enzymatically Stable Analogues of GDP
J . Org. Chem., Vol. 63, No. 21, 1998 7257
5.41 (AB syst, ∆δ ) 10.7 Hz, J AB ) 14.3 Hz, 2H); 4.70 (dd, J
) 4.9, 7.3 Hz, 1H); 4.57 (m, 3H); 4.40-4.19 (m, 5H); 3.69 (AB
part of ABX syst, ∆δ ) 27.0 Hz, J AB ) 12.8 Hz, J AX ) 2.6 Hz,
J BX ) 2.6 Hz, 2H). 13C NMR (CD3OD/CDCl3: 3/1, 50 MHz) δ
158.69; 158.51; 157.98; 147.67; 139.39; 138.42; 137.22; 135.42;
129.52; 129.38; 129.04; 128.79; 128.60; 128.38; 127.89; 127.70;
121.61; 88.72; 87.70; 78.54; 72.77; 63.19; 55.32; 48.60; 46.26.
IR (CHCl3) ν 3306; 1682; 1614; 1495; 1091. MS (CI/NH3): 671
[M + H]+. Anal. Calcd for C39H38N6O5: C, 69.83; H, 5.71; N,
12.53. Found: C, 70.19; H, 5.80; N, 12.73.
1-Ben zyl-2-N-(N′,N′-d iben zylfor m a m id in e)-9-{2′-O-ben -
zyl-5′-O-[N-ben zyl-(P ,P ′,P ′-tr iben zyl-im id od ip h osp h a te)]-
â-D-r ibofu r a n osyl}gu a n in e (46). Compound 46 is obtained
as a white solid (mixture of two diastereomers, 76 mg, 68%),
starting from 45 and 28,105 and following the same procedure
as for 25. 1H NMR (CDCl3, 300 MHz) δ 8.81 and 8.80 (2s,
1H); 7.80 and 7.74 (2s, 1H); 7.60-7.10 (m, 40H); 6.03 (d, J )
2.6, Hz, 0.5H); 6.02 (d, J ) 4.1 Hz, 0.5H); 5.58 (s, 2H); 5.07-
4.77 (m, 6H); 4.68-4.51 (m, 6H); 4.20-4.06 (m, 7H). 13C NMR
(CDCl3, 50 MHz) δ 158.10; 157.86; 157.06; 147.60; 138.42;
137.82; 136.75; 136.68; 136.24 and 136.00; 135.45; 135.06;
134.85; 132.15; 132.02; 131.92; 129.03; 128.80; 128.69; 128.50;
128.39; 128.28; 128.16; 127.96; 127.77; 127.69; 126.74; 126.11;
120.47; 92.91; 82.01 and 81.70 (2d, J ) 7.9 Hz); 81.00 and
80.43; 73.04; 69.95 and 69.75; 69.20; 69.19; 69.18; 69.13; 66.38
and 66.05 (2d, J ) 5.6 Hz); 54.65; 50.87; 48.26; 45.43. 31P NMR
(CDCl3, 121 MHz) δ 5.15 (AB syst, ∆δ ) 103.5 Hz, J AB ) 22.2
Hz, 1P); 5.13 (AB syst, ∆δ ) 61.7 Hz, J AB ) 20.7 Hz, 1P). IR
(CHCl3) ν 2930; 1686; 1610; 1495; 1270; 1021. Anal. Calcd
for C67H65N7O10P2: C, 67.61; H, 5.51; N, 8.24. Found: C, 67.73;
H, 5.58; N, 8.28.
1-Ben zyl-2-N-(N′,N′-d iben zylfor m a m id in e)-9-{2′-O-ben -
zyl-3′-O-p r op ion yl-5′-O-[N-ben zyl-(P ,P ′,P ′-tr iben zylim id -
od ip h osp h a t e)]-â-D-r ib ofu r a n osyl}gu a n in e (47). Pro-
panoyl chloride (15 µL, 172 µmol) is added to alcohol 46 (70
mg, 59 µmol), triethylamine (25 µL, 180 µmol), and 4-DMAP
(5 mg, 82 µmol) in dichloromethane (3 mL) at room temper-
ature. The reaction mixture is refluxed for 3 h and reduced
in vacuo, and the crude residue is chromatographed on silica
gel (CH2Cl2/Me2CO: 95/5 to 80/20) to yield 47 as a white solid
(mixture of two diastereomers, 57 mg, 78%). 1H NMR (CDCl3,
200 MHz) δ 8.85 and 8.84 (2s, 1H); 7.66 and 7.61 (2s, 1H);
7.60-7.10 (m, 40H); 5.80 and 5.79 (2d, J ) 4.4 Hz, 1H); 5.52-
5.35 (m, 3H); 5.01-4.66 (m, 7H); 4.58-4.06 (m, 11H); 2.30-
2.19 (m, 2H); 1.02 and 1.01 (2t, J ) 7.3 Hz, 3H). 13C NMR
(CDCl3, 50 MHz) δ 173.16; 158.19; 158.10; 156.53; 157.07;
147.74; 138.36; 137.89; 137.82; 137.38 and 137.19; 136.83;
136.78; 135.62; 135.49; 135.24; 135.15; 132.16; 132.02; 131.93;
129.02; 128.78; 128.71; 128.57; 128.43; 128.29; 128.15; 128.02;
127.92; 127.92; 127.87; 127.69; 127.62; 126.75; 120.79; 86.85
and 86.84; 79.99 and 79.98; 78.32; 73.33; 71.46 and 71.12;
69.23; 69.15; 68.96; 68.89; 66.48 and 66.39; 54.39; 50.80; 48.24;
45.49; 27.12; 8.85. 31P NMR (CDCl3, 121 MHz) δ 4.65 (AB syst,
∆δ ) 81.7 Hz, J AB ) 21.1 Hz, 1P); 4.57 (AB syst, ∆δ ) 37.5
Hz, J AB ) 20.7 Hz, 1P). IR (CHCl3) ν 2941; 1741; 1693; 1610;
1495; 1271; 1019. Anal. Calcd for C70H69N7O11P2: C, 67.46;
H, 5.58; N, 7.87. Found: C, 67.01; H, 5.36; N, 7.77.
-78 °C, and sodium benzylate [prepared from benzyl alcohol
(2.020 g, 18.7 mmol) and sodium hydride (60% in oil, 750 mg,
18.7 mmol)] in THF (80 mL) are added. The solution is stirred
for 1 h at -78 °C and then for 1 h at room temperature.
Aqueous NH4Cl is added, and the solution is extracted with
AcOEt. The organic layer is dried over MgSO4, reduced in
vacuo, and purified by chromatography on silica gel (Et2O/n-
C6H14: 75.25 to 100/0) to yield 51 as a slightly yellow oil (726
mg, 26%). 1H NMR (CDCl3, 300 MHz) δ 7.35-7.20 (m, 20H);
5.03 (m, 8H); 3.59 (m, 4H); 3.40 (q, J ) 7.1 Hz, 2H); 1.09 (t, J
) 7.1 Hz, 3H). 13C NMR (CDCl3, 75 MHz) δ 135.71 (t, J ) 3.4
Hz); 128.33; 128.20; 127.78; 69.00; 68.81, 66.13; 46.47; 14.97.
31P NMR (CDCl3, 121 MHz) δ 4.69 (s). IR (CHCl3) ν 2977;
1456; 1272; 1014. MS (CI/NH3): 627 [M + NH4]+; 610 [M +
H]+. Anal. Calcd for C32H37NO7P2: C, 63.05; H, 6.12; N, 2.30.
Found: C, 62.81; H, 5.99; N, 2.18.
N-(2-E t h oxyet h yl)-P ,P ′,P ′-t r ib en zylim id od ip h osp h o-
r ic Acid (52). Compound 52 is obtained as a yellow oil (616
mg, 97%), starting from 51 and following the procedure used
to produce 24 from (dibenzyloxyphosphoryl)acetic acid benzyl
ester. 1H NMR (CDCl3, 300 MHz) δ 7.40-7.15 (m, 15H); 5.05
(m, 6H); 3.60 (t, J ) 6.0 Hz, 2H); 3.47 (m, 2H); 3.33 (q, J ) 6.9
Hz, 2H); 1.09 (t, J ) 6.9 Hz, 3H). 13C NMR (CDCl3, 75 MHz)
δ 136.73 (b); 135.63 (b); 128.37; 128.24; 127.86; 127.53; 68.97;
68.91; 68.72; 67.97; 66.25; 45.58; 14.91. 31P NMR (CD3OD, 121
MHz) δ 5.85 (d, J ) 23.0 Hz, 1P); 3.11 (d, J ) 23.0 Hz, 1P). IR
(CHCl3) ν 2971; 1253; 1025. MS (CI/NH3): 537 [M + NH4]+.
Anal. Calcd for C25H31NO7P2: C, 57.80; H, 6.02; N, 2.70.
Found: C, 57.87; H, 6.03; N, 2.86.
1-Ben zyl-2-N-(N′,N′-d ib en zylfor m a m id in e)-9-{2′,3′-O-
ben zylid en e-5′-O-[N-(2-eth oxyeth yl)-(P ,P ′,P ′-tr iben zylim -
id od ip h osp h a te)]-â-D-r ibofu r a n osyl}gu a n in e (53). Com-
pound 53 is obtained as a white solid (mixture of four
diastereomers, 106 mg, 68%), starting from 14 and 52 and
following the same procedure as for 25. 1H NMR (CDCl3, 300
MHz) δ 8.85 and 8.83 (2s, 1H); 7.85, 7.84, 7.80, and 7.72 (4s,
1H); 7.60-7.15 (m, 35H); 6.21-5.88 (m, 2H); 5.63-5.48 (m,
3H); 5.34-5.23 (m, 1H); 5.18-4.98 (m, 7H); 4.65-4.58 (m, 2H);
4.54-3.93 (m, 5H); 3.65-3.25 (m, 6H); 1.15 (m, 3H). 13C NMR
(CDCl3, 50 MHz) δ 162.21; 158.05 (b); 157.12 (b); 147.53 and
147.23; 136.60 and 136.46; 138.25; 137.55; 136.60 and 136.46;
135.68 (b); 135.00 (b); 131.92; 131.85; 129.79; 129.71; 128.96;
128.72; 128.43; 128.37; 128.22; 128.07; 127.86; 127.82; 127.70;
127.57; 126.63; 126.57; 126.44; 120.96, 120.42; 120.38 and
120.30; 107.69, 107.64, 104.34 and 104.27; 88.80 and 88.53;
83.59, 83.44, 83.05 and 82.35; 82.23, 82.08; 80.73 and 77.63;
69.30; 69.23; 69.17; 69.09; 69.02; 68.95; 68.88; 66.21; 62.96 and
62.63; 54.60; 48.34 and 48.29; 46.67; 45.51 and 45.42; 14.98.
31P NMR (CDCl3, 121 MHz) δ 4.85 (AB syst, ∆δ ) 59.9 Hz,
J AB ) 21.1 Hz, 0.5P); 4.84 (AB syst, ∆δ ) 59.3 Hz, J AB ) 21.1
Hz, 0.5P); 4.81 (AB syst, ∆δ ) 58.7 Hz, J AB ) 20.7 Hz, 0.5P);
4.78 (AB syst, ∆δ ) 56.7 Hz, J AB ) 20.7 Hz, 0.5P). IR (CHCl3)
ν 2927; 1730; 1691; 1608; 1489; 1269; 1212; 1063. Anal. Calcd
for C64H65N7O11P2: C, 65.69; H, 5.60; N, 8.38. Found: C, 65.84;
H, 5.66; N, 8.40.
Ack n ow led gm en t. This work was supported by a
grant from MENESR (S.V.), as well as by NSERC, the
Fonds FCAR and FRSQ (C.S.), and the Coope´ration
Franco-Que´becoise (DDCSTE). The authors thank
Ste´phane Mons for complementary analytical work.
N-(2-E t h oxyet h yl)-P ,P ,P ′,P ′-t et r a b en zylim id od ip h os-
p h a te (51). A solution of 2-ethoxyethylamine (792 mg, 4.7
mmol) in THF (15 mL) is added dropwise over 4 h to
phosphorus oxychloride (0.90 mL, 9.6 mmol) and anhydrous
triethylamine (1.40 mL, 10.1 mmol) in THF (15 mL) at -78
°C. The reaction mixture is stirred at room temperature for
30 min before it is filtered on Celite. The filtrate is cooled to
J O9806207