Synthesis and Separation of Diastereomers of Ribonucleoside 5′-(α-P-Borano)triphosphates

Article abstract of DOI:10.1021/jo972002g

Nucleoside boranophosphates, in which one of the phosphate oxygens is replaced by a borane group, are isoionic and isoelectronic analogues of naturally occurring nucleotides. Boranophosphates also are biochemically important congeners of phosphorothioates and methylphosphonates. We have developed a convenient one-pot method to synthesize the set of ribonucleoside (A, U, G, and C) 5′-(α-P-borano)triphosphates. Phosphitylation of the 2′,3′-protected ribonucleoside with 2-chloro4H-1,3,2-benzodioxaphosphorin-4-one gives the 5′-phosphite intermediate 2 which undergoes in situ substitution in the presence of pyrophosphate to give the cyclic intermediate, P²,P³-dioxo-P¹-ribonucleosidylcyclotriphosphate 3. Immediate oxidation of compound 3 with amine·borane complex results in ribonucleoside 5′-(α-P-borano)cyclotriphosphate 4. Subsequent reaction of compound 4 with water followed by ammonium hydroxide yields the crude product as a diastereomeric mixture of ribonucleoside 5′-(α-P-borano)triphosphate 6. Pure compound 6 is isolated in 30-45% overall yield using ion-exchange chromatography. The separation of two diastereomers of ribonucleoside 5′-(α-P-borano)triphosphate 6 is achieved by reverse phase HPLC.

Full text of DOI:10.1021/jo972002g

J . Org. Chem. 1998, 63, 5769-5773
5769
Syn th esis a n d Sep a r a tion of Dia ster eom er s of Ribon u cleosid e
5′-(r-P -Bor a n o)tr ip h osp h a tes
Kaizhang He, Ahmad Hasan,† Bozenna Krzyzanowska, and Barbara Ramsay Shaw*
Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University,
Durham, North Carolina 27708
Received October 31, 1997
Nucleoside boranophosphates, in which one of the phosphate oxygens is replaced by a borane group,
are isoionic and isoelectronic analogues of naturally occurring nucleotides. Boranophosphates also
are biochemically important congeners of phosphorothioates and methylphosphonates. We have
developed a convenient one-pot method to synthesize the set of ribonucleoside (A, U, G, and C)
5′-(R-P-borano)triphosphates. Phosphitylation of the 2′,3′-protected ribonucleoside with 2-chloro-
4H-1,3,2-benzodioxaphosphorin-4-one gives the 5′-phosphite intermediate 2 which undergoes in
situ substitution in the presence of pyrophosphate to give the cyclic intermediate, P2,P3-dioxo-P1-
ribonucleosidylcyclotriphosphate 3. Immediate oxidation of compound 3 with amine·borane complex
results in ribonucleoside 5′-(R-P-borano)cyclotriphosphate 4. Subsequent reaction of compound 4
with water followed by ammonium hydroxide yields the crude product as a diastereomeric mixture
of ribonucleoside 5′-(R-P-borano)triphosphate 6. Pure compound 6 is isolated in 30-45% overall
yield using ion-exchange chromatography. The separation of two diastereomers of ribonucleoside
5′-(R-P-borano)triphosphate 6 is achieved by reverse phase HPLC.
Sch em e 1
In tr od u ction
Deoxyribo- and ribonucleoside triphosphates are the
basic building blocks for enzymatic synthesis of DNA and
RNA in vitro and in vivo. Modified nucleoside triphos-
phates have received much attention in searches for
potential diagnostic and therapeutic agents, as well as
for compounds for studying numerous biochemical and
pharmacological processes.1,2
The nucleoside 5′-(R-P-borano)triphosphates comprise
a new class of modified nucleotides in which one of the
R-phosphate oxygens in nucleoside 5′-triphosphate is
replaced by a borane group (BH3) (Scheme 1).3 The
borane moiety in boranophosphate is isoionic and iso-
electronic with oxygen in normal phosphate and phos-
phorothioate4 and isosteric with the methyl group (CH3)
in methylphosphonate.5,6 Consequently, boranophos-
phate analogues have a number of biochemical properties
that are similar to those of the phosphorothioates and
methylphosphonate analogues. The 2′-deoxy boronated
nucleoside triphosphates, like the 2′-deoxyribonucleoside
5′-thiotriphosphates, are substrates for DNA polymerase
enzymes and can be successfully incorporated into DNA.7-9
Once in DNA, the boranophosphate linkage is more
resistant to exo- and endo-nucleases than that of phos-
phate diesters.9-11 The increased nuclease resistance of
boranophosphate may also be useful in stabilizing RNA.
In addition, the P-B bond should have a different
polarity than a P-O bond, and thereby, the introduction
of boranophosphate into RNA could be useful for a variety
of studies on RNA structure and function, such as metal-
RNA interaction and the mechanism of RNA cleavage.12
One approach for generating boranophosphate oligori-
bonucleotides is direct chemical synthesis, reported only
for synthesis of diuridine 3′,5′-boranophosphate.13 The
other approach is to employ an enzymatic method, but
this requires synthesis of a set of 5′-(R-P-borano)tri-
phosphate ribonucleosides (NTPRB) as substrates for
polymerases. The availability of NTPRB would also
permit investigations where other backbone-modified
nucleoside triphosphates have been used,14,15 since the
†Current address: Peninsula Laboratories, Inc., 601 Taylor Way,
San Carlos, CA 94070.
(1) Sanghvi, Y. S.; Cook, P. D. In Nucleosides and Nucleotides as
Antitumor and Antiviral Agents; Chu, C. K., Baker, D. C., Eds.; Plenum
Press: New York, 1993, p 311-323.
(2) Mesmaeker, A. D.; Haner, R.; Martin, P.; Moser, H. E. Acc. Chem.
Res. 1995, 28, 366-374.
(3) (a) Sood, A.; Shaw, B. R.; Spielvogel, B. F. J . Am. Chem. Soc.
1990, 112, 9000-9001. (b) Shaw, B. R.; Madison, J .; Sood, A.;
Spielvogel, B. F. Methods Mol. Biol. 1993, 20, 225-243.
(4) Eckstein, F. Annu. Rev. Biochem. 1985, 54, 367-402.
(5) Miller, P. S. In Antisense RNA and DNA; Murray, J . A. H., Ed.;
Wiley-Liss Inc.: New York, 1992; p 241-253.
(8) Porter, K.; Briley, J . D.; Shaw, B. R. In Genome Sequencing and
Analysis Conference VI; Mary Ann Liebert, Inc.: Hilton Head, SC,
1994, p 45.
(9) Porter, K.; Briley, J . D.; Shaw, B. R. Nucleic Acids Res. 1997,
25, 1611-1617.
(10) Huang, F.; Sood, A.; Spielvogel, B. F.; Shaw, B. R. J . Biomol.
Struct. Dyn. 1993, 10, a078.
(11) Huang, F. Ph.D. Dissertation, Duke University, 1994.
(12) Heidenreich, O.; Pieken, W.; Eckstein, F. FASEB J . 1993, 7,
90-96.
(13) Chen, Y.; Qu, F.; Zhang, Y. Tetrahedron Lett. 1995, 36, 745-
748.
(6) Dineva, M.; Petkov, D. Nucleosides Nucleotides 1996, 15, 1459-
1467.
(7) Li, H.; Porter, K.; Huang, F.; Shaw, B. R. Nucleic Acids Res. 1995,
21, 4495-4501.
S0022-3263(97)02002-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/30/1998

5770 J . Org. Chem., Vol. 63, No. 17, 1998
He et al.
Sch em e 2
boronated backbone (P-BH3) resembles several biochemi-
cally important modified backbones (P-O, P-S, and
P-CH3). These potential applications of NTPRB led us
to develop a chemical synthesis for NTPRB. In this
paper, we report for the first time the synthesis and
separation of diastereomers of the four ribonucleoside 5′-
(R-P-borano)triphosphates (NTPRB) of adenine, uracil,
guanine, and cytosine.
sidylcyclotriphosphate 3. In the 31P NMR spectrum of
the reaction mixture containing compound 3, the upfield
shift of peaks from δ 128.12 and 126.18 to a triplet at δ
106.72 (J ) 40.48 Hz) along with a doublet at δ -18.55
and -18.81 (J ) 42.09 Hz) supported the formation of
intermediate 3.17
An in situ boronation of intermediate 3 by the exchange
reaction with borane-N,N-diisopropylethylamine com-
plex (DIPEA·BH3) at room temperature for 6 h resulted
in the boranophosphate intermediate, ribonucleoside 5′-
(R-P-borano)cyclotriphosphate 4. According to valence
shell electron pair repulsion (VSEPR) theory and molec-
ular orbital (MO) theory, the boron atom in BH3 carries
an empty 2p orbital and, therefore, is extremely suscep-
tible to attack by nucleophiles.18 Thus, monoborane is
willing to accept a pair of electrons. These electrons are
provided by a Lewis base, such as an amine or a
phosphine. As the Lewis base strength of the tertiary
phosphite ((RO)3P) is much greater than that of trialkyl-
amine toward BH3,19 the tertiary phosphite intermediate
3 displaced N,N-diisopropylethylamine in DIPEA·BH3 to
form the boron-phosphorus coordination compound, the
phosphite borane 4. The formation of a P f B bond in
intermediate 4 was confirmed on the basis of 31P NMR
(Figure 1A), where the triplet at δ 106.72 in compound
3 was completely shifted to a broad peak centered at δ
86.72 in compound 4. A slight upfield shift of the doublet
at δ -21.64 and -21.92 (J ) 45.33 Hz) of P2 and P3 peaks
in compound 4 was also observed. We tried several
amine·borane complexes for the boronation step and
found that the borane-pyridine complex caused decom-
position of intermediate 3 and resulted in nonboronated
triphosphate, observed with 31P NMR. In the 31P NMR
spectrum, the peak at δ 106.72 in intermediate 3 disap-
peared, and new peaks at δ -8.40 (pyrophosphate) and
at δ -5.90, -12.56, and -20.96 (normal triphosphate)
were observed. Furthermore, treatment with the more
Resu lts a n d Discu ssion
We initially attempted to synthesize NTPRB by modi-
fication of the phosphoramidite approach reported for the
synthesis of the deoxy analogues of 5′-(R-P-borano)-
triphosphates (dNTPRB).16 Although one can synthesize
NTPRB by using this method, we encountered certain
limitations and disadvantages. For example, this method
required exocyclic amine protection, isolation of one
intermediate compound, and two ion-exchange column
chromatography steps, resulting in low overall yield.
These limitations led us to explore an alternative syn-
thetic method to increase the yield as well as to decrease
the time and cost. Here we have synthesized NTPRB as
outlined in Scheme 2 by modification of the method
reported by Ludwig and Eckstein for nucleoside 5′-
thiotriphosphates.17
Phosphitylation of ribonucleoside 1 with 2-chloro-4H-
1,3,2-benzodioxaphosphorin-4-one yielded the two dia-
stereomers of ribonucleoside 5′-(4H-1,3,2-benzodioxaphos-
phorin-4-one) 2. They were identified by the appearance
of two signals at δ 128.12 and 126.18, observed in the
31P NMR spectra of the reaction mixture. Instead of
introduction of the borane group (BH3) after the phos-
phitylation stage, as in the phosphoramidite approach,16
intermediate 2 was treated directly with pyrophosphate
to form a cyclic intermediate P2,P3-dioxo-P1-ribonucleo-
(14) Eckstein, F. Angew. Chem., Int. Ed. Engl. 1983, 22, 423-439.
(15) Gish, G.; Eckstein, F. Trends Biochem. Sci. 1989, 14, 97-100.
(16) Tomasz, J .; Shaw, B. R.; Porter, K.; Spielvogel, F.; Sood, A.
Angew. Chem., Int. Ed. Engl. 1992, 31, 1373-1375.
(18) Housecroft, C. E. Boranes and Metalloboranes Structure, Bond-
ing and Reactivity; J ohn Wiley & Sons: New York, 1990; p 18-30.
(19) Reetz, T. J . Am. Chem. Soc. 1960, 82, 5039-5042.
(17) Ludwig, J .; Eckstein, F. J . Org. Chem. 1989, 54, 631-635.

Ribonucleoside 5′-(R-P-Borano)triphosphates
J . Org. Chem., Vol. 63, No. 17, 1998 5771
F igu r e 1. (A) Selected regions of the 31P NMR spectrum (ppm) of 4a . (B) Selected regions of the 31P NMR spectrum (ppm) of 5a .
(C) 31P NMR spectrum (ppm) of 6a .
Sch em e 3
reactive borane-tetrahydrofuran complex under the
same conditions resulted in the reduction of nucleoside
(unpublished results).
The cyclic intermediate 4 underwent hydrolytic open-
ing by treatment with water to give ribonucleoside 5′-
(R-P-borano)triphosphate 5. Although the nucleophilic
attack on intermediate 4 by water might occur at the
boranophosphate (paths A and C in Scheme 3) or at the
phosphate (paths B and D in Scheme 3), we have not
detected any branched triphosphate20 derived from open-
ing of the pyrophosphate bond (path D). This was to be
(20) Kozarich, J . W.; Chinault, A. C.; Hecht, S. M. Biochemistry
1973, 12, 4458-4463.

5772 J . Org. Chem., Vol. 63, No. 17, 1998
He et al.
F igu r e 2. Isocratic separation of diastereomers of cytidine 5′-(R-P-borano)triphosphate 6d by HPLC (A) and analysis of purity
of HPLC-isolated isomer I (B) and isomer II (C). The elution was carried out on the Delta Pak C18 column with 100 mM TEAA
(pH 6.80) and methanol at a flow rate of 3.0 mL/min. The retention times for two diastereomers were 8.55 and 12.24 min,
respectively. Peak 1: isomer I of CTPRB. Peak 2: isomer II of CTPRB.
Ta ble 1. HP LC P r ofiles
expected, since the boranophosphate as a monoanion has
a pKa of 1.511 and is thus a better leaving group than the
retention time (min)
phosphate dianion with a pKa of 6.8.17 From the 31P NMR
spectrum (Figure 1B) of the reaction mixture, the ex-
pected intermediate 5 formed by paths A and B showed
a broad peak centered at δ 85.27 (R-P), two quartets (â-
P) at δ -21.25 (J Râ ) 32.87 Hz, J âγ ) 20.56 Hz) and δ
-21.30 (J Râ ) 33.51 Hz, J âγ ) 19.75 Hz), and a doublet
at δ -8.50 and -8.63 (J ) 21.05 Hz, γ-P). Another triplet
at δ -20.65 and two doublets at δ -8.04 and -3.06
resulted from the normal triphosphate (path C). The
singlet at δ -7.02 was due to pyrophosphate. Another
singlet at δ 8.12 was attributed to H-phosphonate derived
from the hydrolysis of excess phosphitylating agent.21
The protecting groups of intermediate 5 were removed
by ammonia hydrolysis to afford ribonucleoside 5′-(R-P-
borano)triphosphate 6 (Figure 1C) which was isolated in
30-45% yield by ion-exchange column chromatography
on QA-52 (HCO3-) cellulose. The structure, purity, and
homogeneity of compound 6 was confirmed by 31P NMR,
1H NMR, and FAB-MS. In this reaction, in addition to
the desired product, three byproducts were also isolated.
One was identified as H-phosphonate (about 20% yield)
derived from the hydrolysis of excess phosphitylating
agent. The other two byproducts were identified as
ribonucleoside 5′-monophosphate (about 30% yield) and
free pyrophosphate, which likely resulted from decom-
position of normal ribonucleoside 5′-triphosphate formed
via path C (Scheme 3).
compound
buffera
isomer I (%) isomer II (%)
8.32 (46.4) 11.80 (53.6)
UTPRB
ATPRB
GTPRB
CTPRB
92% TEAA/8% MeOH
90% TEAA/10% MeOH 11.04 (42.7) 17.82 (57.3)
92% TEAA/8% MeOH
94% TEAA/6% MeOH
9.31 (47.5) 14.56 (52.5)
8.55 (51.0) 12.24 (49.0)
a
100 mM TEAA, pH 6.80, and methanol.
and after preparative HPLC separation are presented in
Figure 2. The isolated yields of isomers I and II are
presented in Table 1. The homogeneity and structure
1
were confirmed by H NMR, 31P NMR, and FAB-MS.
Con clu sion s
We have successfully synthesized the set of four 5′-(R-
P-borano)triphosphate ribonucleosides (A, U, G, and C)
using a one-pot procedure. This method does not require
exocyclic amine protection of the nucleobase. Introduc-
tion of the borane group (BH3) was achieved under mild
conditions, eliminating the risk of possible reduction of
nucleoside. Compared to the previously reported method
for synthesis of the deoxy NTPRB analogues, this is a
time- and cost-effective approach which resulted in high
yields. A two-step purification, ion-exchange chroma-
tography followed by reverse phase HPLC, gave two
diastereomers of NTPRB in high purity.
Exp er im en ta l Section
Introduction of a borane group (BH3) to replace one of
the oxygen atoms on the R-phosphate produces a pair of
NTPRB diastereomers. These two diastereomers, whose
absolute configuration is not yet known, have been
separated by preparative reverse phase HPLC and have
arbitrarily been named isomer I and isomer II (Table 1).
The isomeric purity of each of the individual diastereo-
mers was checked by reverse phase HPLC under the
conditions used for separation. The HPLC profiles before
All solvents, chemicals, and reagents were used without
further purification unless otherwise indicated. 2′,3′-Diacetyl-
adenosine, 2′,3′-diacetylguanosine, and 2′,3′-dibenzoyluridine
were purchased from Sigma and dried before use in a desic-
cator over P2O5. 2′,3′-Diacetyl-N4-benzoylcytidine was pre-
pared from acetylation of 5′-dimethoxytrityl-N4-benzoylcytidine
(ChemGenes) followed by detritylation of the 5′-dimethoxytri-
tyl group with 80% acetic acid; the desired compound was
isolated in 58% overall yield after chromatography on silica
gel. Borane-N,N-diisopropylethylamine complex, borane-
pyridine complex, and borane-tetrahydrofuran complex were
purchased from Aldrich and stored over 4 Å molecular sieves.
(21) Marrug, J . E.; Tromp, M.; Kuyl-Yeheskiely, E.; Van Del Marel,
G. A.; Van Boom, J . H. Tetrahedron Lett. 1986, 27, 2661-2664.

Ribonucleoside 5′-(R-P-Borano)triphosphates
J . Org. Chem., Vol. 63, No. 17, 1998 5773
QA-52 quaternary ammonium cellulose was purchased from
Whatman International. Triethylammonium acetate was
prepared from triethylamine and acetic acid. 1H and 31P NMR
spectra were acquired at 400.0 and 161.9 MHz, respectively.
Ultraviolet (UV) and mass spectroscopy, ion-exchange chro-
matography, and reverse phase HPLC were performed as
described.22
4.57 (m, 1H), 4.25 (m, 1H), 4.16, 4.10 (2m, 3H), -0.20 to +0.80
(2br, 3H); 31P NMR (D2O) δ 85.20 (br, 1P, R-P), -21.26 (m,
1P, â-P), -8.92 (d, J ) 18.29 Hz, 1P, γ-P); UV (H2O) λmax 272.5
nm; FAB-MS m/z 480.0 (M-, calcd 479.96).
Rever se P h a se HP LC Sep a r a tion of Dia ster eom er s of
Ribon u cleosid e 5′-(r-P -bor a n o)tr ip h osp h a tes. The sepa-
ration of diastereomers of each NTPRB was carried out by ion-
pairing chromatography on a reverse phase column (Delta Pak
C18, 7.8 × 300 mm, 15 µm, 300 Å) using isocratic elution (100
mM triethylammonium acetate (TEAA), pH 6.80, and metha-
nol as buffer components) with a flow rate 3.0 mL/min.
Fractions containing the same isomer (similar retention time)
were combined, and the solvent was removed under reduced
pressure. The buffer components, triethylammonium acetate
(TEAA) and methanol, were removed by repeated lyophiliza-
tion.
Ribon u cleosid e 5′-(r-P -bor a n o)tr ip h osp h a tes. Gen -
er a l P r oced u r e. One of the protected nucleosides [2′,3′-
dibenzoyluridine, 2′,3′-diacetyladenosine, 2′,3′-diacetylgua-
nosine, 2′,3′-diacetyl-N4-benzoylcytidine] (0.50 mmol) was
dissolved in anhydrous DMF (1.0 mL) and anhydrous pyridine
(0.25 mL) under an argon atmosphere. A freshly prepared
solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in
anhydrous DMF (0.50 mL, 1.0 M) was added with a syringe.
After 10 min, tributylamine (0.30 mL) was added, followed by
a solution of tributylammonium pyrophosphate in anhydrous
DMF (1.0 mL, 0.5 M). The mixture was stirred for 10 min,
borano-N,N-diisopropylethylamine complex (2.0 mL) was
added, and the stirring was continued for 6 h. Deionized water
(5.0 mL) was added, and the mixture was stirred for 1 h. The
solvent was removed under vacuum, and the resulting residue
was treated with a mixture of ammonium hydroxide and
methanol (1:1, v/v, 10.0 mL) for 24 h. The solvent was
removed, and the residue was taken in water (50 mL) and
extracted with diethyl ether (50 mL). The water layer was
evaporated, and the residue was applied to a column packed
with QA-52 cellulose (HCO3-). The column was eluted with a
linear gradient of 0.005 and 0.2 M ammonium bicarbonate
buffer, pH 9.56 (800 mL each). The desired fraction was dried
by lyophilization, and excess salt was removed by repeated
lyophilization with deionized water to yield the ammonium
salt of ribonucleoside 5′-(R-P-borano)triphosphate.
Ur id in e 5′-(r-P -bor a n o)tr ip h osp h a te (UTP rB) (6a ).
Compound 6a was prepared in 46% yield (125.4 mg) following
the general procedure by using 2′,3′-dibenzoyluridine 1a (225.5
mg, 0.50 mmol): 1H NMR (D2O) δ 7.89 (m, 1H), 5.83 (m, 2H),
4.29 (m, 1H), 4.22 (m, 1H), 4.12, 4.08 (2m, 3H), 0.39, 0.13 (2br,
3H); 31P NMR (D2O) δ 85.05 (br, 1P, R-P), -21.03 (m, 1P, â-P),
-7.87 (m, 1P, γ-P); UV (H2O) λmax 264.0 nm; FAB-MS m/z
480.97 (M-, calcd 480.97).
Ad en osin e 5′-(r-P -bor a n o)tr ip h osp h a te (ATP rB) (6b).
Compound 6b was prepared in 31% yield (88.9 mg) following
the general procedure by using 2′,3′-diacetyladenosine 1b
(175.8 mg, 0.50 mmol): 1H NMR (D2O) δ 8.42, 8.40 (2s, 1H),
8.09 (s, 1H), 5.98 (d, J ) 5.2 Hz, 1H), 4.57 (s, 1H), 4.45, 4.37
(2m, 1H), 4.23 (m, 1H), 4.12, 4.00 (2m, 2H), 0.37, 0.13 (2br,
3H); 31P NMR (D2O) δ 85.36 (br, 1P, R-P), -21.15 (dd, J )
21.37 Hz, 30.52 Hz, 1P, â-P), -8.85 (d, J ) 18.29 Hz, 1P, γ-P);
UV (H2O) λmax 260.2 nm; FAB-MS m/z 504.0 (M-, calcd 504.0).
UTP rB, isom er I: 1H NMR (D2O) δ 7.91 (d, J ) 8.4 Hz,
1H), 5.84 (m, 2H), 4.29 (m, 1H), 4.22 (m, 1H), 4.12, 4.05 (2m,
3H), -0.20 to +0.80 (br, 3H); 31P NMR (D2O) δ 85.40 (br, 1P,
R-P), -21.47 (m, 1P, â-P), -9.16 (m, 1P, γ-P); FAB-MS m/z
480.91 (M-, calcd 480.95). Isom er II: 1H NMR (D2O) δ 7.89
(d, J ) 8.8 Hz, 1H), 5.83 (m, 2H), 4.21 (m, 2H), 4.12 (m, 1H),
4.07 (m, 2H), -0.20 to +0.80 (br, 3H); 31P NMR (D2O) δ 85.12
(br, 1P, R-P), -21.28 (m, 1P, â-P), -9.14 (m, 1P, γ-P); FAB-
MS m/z 480.91 (M-, calcd 480.95).
ATP rB, isom er I: 1H NMR (D2O) δ 8.43 (s, 1H), 8.09 (s,
1H), 5.98 (d, J ) 5.6 Hz, 1H), 4.56 (m, 1H), 4.44 (m, 1H), 4.23
(m, 1H), 4.13, 4.00 (2m, 2H), 0.42, 0.18 (2br, 3H); 31P NMR
(D2O) δ 84.79 (br, 1P, R-P), -21.14 (m, 1P, â-P), -8.86 (m, 1P,
γ-P); FAB-MS m/z 503.9 (M-, calcd 503.99). Isom er II: 1H
NMR (D2O) δ 8.40 (s, 1H), 8.06 (s, 1H), 5.96 (d, J ) 6.0 Hz,
1H), 4.56 (m, 1H), 4.37 (m, 1H), 4.21 (m, 1H), 4.11, 4.00 (2m,
2H), 0.39, 0.12 (2br, 3H); 31P NMR (D2O) δ 84.40 (br, 1P, R-P),
-20.98 (m, 1P, â-P), -7.71 (m, 1P, γ-P); FAB-MS m/z 503.95
(M-, calcd 503.99).
GTP rB, isom er I: 1H NMR (D2O) δ 8.03 (s, 1H), 5.77 (d,
J ) 6.0 Hz, 1H), 4.57 (m, 1H), 4.47 (m, 1H), 4.17 (m, 1H), 4.13,
3.98 (2m, 2H), -0.10 to +0.75 (br, 3H); 31P NMR (D2O) δ 84.38
(br, 1P, R-P), -20.86 (m, 1P, â-P), -7.33 (m, 1P, γ-P); FAB-
MS m/z 520.0 (M-, calcd 520.0). Isom er II: 1H NMR (D2O) δ
8.00 (s, 1H), 5.76 (d, J ) 6.0 Hz, 1H), 4.57 (m, 1H), 4.38 (m,
1H), 4.18 (m, 1H), 4.10, 4.03 (2m, 2H), -0.10 to +0.75 (br, 3H);
31P NMR (D2O) δ 85.41 (br, 1P, R-P), -20.99 (m, 1P, â-P), -6.97
(m, 1P, γ-P); FAB-MS m/z 520.0 (M-, calcd 520.0).
CTP rB, isom er I: 1H NMR (D2O) δ 7.93 (d, J ) 7.6 Hz,
1H), 5.97 (m, 1H), 5.84 (d, J ) 4.0 Hz, 1H), 4.55 (m, 1H), 4.26
(m, 1H), 4.15, 4.09 (2m, 3H), -0.20 to +0.80 (2br, 3H); 31P
NMR (D2O) δ 84.30 (br, 1P, R-P), -21.21 (m, 1P, â-P), -8.68
(m, 1P, γ-P); FAB-MS m/z 480.0 (M-, calcd 479.96). Isom er
II: 1H NMR (D2O) δ 7.91 (d, J ) 6.4 Hz, 1H), 5.98 (d, J ) 8.0
Hz, 1H), 5.84 (d, J ) 3.6 Hz, 1H), 4.57 (m, 1H), 4.19 (m, 1H),
4.15, 4.10 (2m, 3H), -0.20 to +0.80 (2br, 3H); 31P NMR (D2O)
δ 84.90 (br, 1P, R-P), -21.16 (m, 1P, â-P), -8.63 (m, 1P, γ-P);
FAB-MS m/z 480.0 (M-, calcd 479.96).
Gu a n osin e 5′-(r-P -bor a n o)tr ip h osp h a te (GTP rB) (6c).
Compound 6c was prepared in 29% yield (85.2 mg) following
the general procedure by using 2′,3′-diacetylguanosine 1c
(183.3 mg, 0.50 mmol): 1H NMR (D2O) δ 8.02, 7.99 (2s, 1H),
5.77 (m, 1H), 4.57 (s, 1H), 4.44, 4.36 (2m, 1H), 4.19 (m, 1H),
4.10, 4.00 (2m, 2H), 0.43, 0.19 (2br, 3H); 31P NMR (D2O) δ 85.72
(br, 1P, R-P), -21.12 (dd, J ) 20.56 Hz, 28.33 Hz, 1P, â-P),
-8.70 (d, J ) 19.91 Hz, 1P, γ-P); UV (H2O) λmax 255.8 nm;
FAB-MS m/z 520.0 (M-, calcd 520.0).
Cytid in e 5′-(r-P -bor a n o)tr ip h osp h a te (CTP rB) (6d ).
Compound 6d was prepared in 26% yield (71.0 mg) following
the general procedure by using 2′,3′-diacetyl-N4-benzoylcyti-
dine 1d (216.0 mg, 0.50 mmol): 1H NMR (D2O) δ 7.95 (d, J )
7.6 Hz, 1H), 6.01 (d, J ) 6.0 Hz, 1H), 5.84 (d, J ) 4.4 Hz, 1H),
Ack n ow led gm en t. This work was supported by
DOE grant DE-FG05-94ER61882 and 97ER62376 to
B.R.S.
Su p p or tin g In for m a tion Ava ila ble: 31P NMR and 1H
NMR spectra for the new compounds 6a , 6b, 6c, and 6d and
for the two diastereomers of 6a , 6b, 6c, and 6d (24 pages).
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(22) Krzyzanowska, B.; He, K.; Hasan, A.; Shaw, B. R. Tetrahedron
1998, 54, 5119-5128.
J O972002G