Convenient synthesis of nucleoside borane diphosphate analogues: Deoxy- and ribonucleoside 5′-Pα-boranodiphosphates

Article abstract of DOI:10.1021/jo049094b

The nucleoside boranophosphates, having one of the nonbridging phosphate oxygens substituted with a borane (BH₃) group, have shown potential therapeutical applications as aptamers, antisense agents, and antiviral prodrugs. An oxathiaphospholane

Full text of DOI:10.1021/jo049094b

Con ven ien t Syn th esis of Nu cleosid e Bor a n e Dip h osp h a te
An a logu es: Deoxy- a n d Ribon u cleosid e 5′-P r-Bor a n od ip h osp h a tes
Ping Li and Barbara Ramsay Shaw*
Department of Chemistry, Box 90346, Duke University, Durham, North Carolina 27708-0346
brshaw@chem.duke.edu
Received May 30, 2004
The nucleoside boranophosphates, having one of the nonbridging phosphate oxygens substituted
with a borane (BH3) group, have shown potential therapeutical applications as aptamers, antisense
agents, and antiviral prodrugs. An oxathiaphospholane approach, which does not require exocyclic
amine protection of the nucleobase, has been successfully developed to efficiently synthesize 5′-
PR-boranodiphosphates of 2′-deoxythymidine, adenosine, guanosine, and uridine. The approach
involves a key intermediate, the borane complex of nucleoside 5′-O-1,3,2-oxathiaphospholane 16,
that undergoes a ring-opening reaction catalyzed by 1,4-diazabicyclo[5.4.0]-undec-7-ene to form
the protected nucleoside 5′-PR-boranodiphosphate 18. Treatment of 18 with ammonium hydroxide
yielded diastereoisomeric mixtures of nucleoside 5′-PR-boranodiphosphates 5. This oxathiaphos-
pholane approach ensures the availability of nucleoside 5′-PR-boranodiphosphate analogues needed
for antiviral drug research.
In tr od u ction
Nucleotide analogues with modifications in the base,
sugar, or phosphate residues1,3a,b can be used as probes
for substrate properties, kinetic pathways, and transition
states of enzymes involved in nucleic acid metabolism.2
For example, nucleotide analogues of diphosphates and
triphosphates have been successfully and widely used as
biochemical tools to unravel functions and mechanisms
of nucleoside kinases and polymerases.3 In particular,
phosphorothioate analogues 3 (Figure 1), in which a
sulfur atom is exchanged for a nonbridging oxygen at a
phosphate group, have been well-studied and employed
to determine the stereochemical course of a large number
of enzymatic nucleotidyl and phosphoryl transfer reac-
tions.4
F IGURE 1. Phosphate and its modified analogues.
The substitution of one of the nonbridging oxygens at
a phosphate group by a borane (BH3) group results in a
new class of phosphate-modified nucleotides known as
boranophosphates 2.5 Because the BH3 group is isoelec-
tronic with oxygen, isolobal with sulfur, and isosteric with
the methyl group, the boranophosphate 2 can be consid-
ered as a “hybrid” of three types of modified phosphates,
(4) (a) Eckstein, F. Biochimie 2002, 84, 841. (b) Eckstein, F. Angew.
Chem., Int. Ed. Engl. 1983, 22, 423. (c) Gerlt, J . A.; Coderre, J . A.;
Mehdi, S. Adv. Enzymol. 1983, 55, 291. (d) Eckstein, F.; Romaniuk, P.
J .; Connolly, B. A. Methods Enzymol. 1982, 87, 197. (e) Frey, P. A.;
Richard, J . P.; Ho, H. T.; Brody, R. S.; Sammons, R. D.; Sheu, K. F.
Methods Enzymol. 1982, 87, 213. (f) Webb, M. R. Methods Enzymol.
1982, 87, 301. (g) Buchwald, S. L.; Hansen, D. E.; Hassett, A.; Knowles,
J . R. Methods Enzymol. 1982, 87, 279. (h) Knowles, J . R. Annu. Rev.
Biochem. 1980, 49, 877. (i) Eckstein, F. Acc. Chem. Res. 1979, 12, 204.
(5) (a) Sood, A.; Shaw, B. R.; Spielvogel, B. F. J . Am. Chem. Soc.
1990, 112, 9000. (b) Tomasz, J .; Shaw, B. R.; Porter, K.; Spielvogel, B.
F.; Sood, A. Angew. Chem., Int. Ed. Engl. 1992, 31, 1373. (c) Shaw, B.
R.; Madison, J .; Sood, A.; Spielvogel, B. F. Methods Mol. Biol. 1993,
20, 225. (d) Porter, K. W.; Briley, J . D.; Shaw, B. R. Nucleic Acids Res.
1997, 25, 1611. (e) Krzyzanowska, B. K.; He, K. Z.; Hasan, A.; Shaw,
B. R. Tetrahedron 1998, 54, 5119. (f) He, K. Z.; Hasan, A.; Krzyza-
nowska, B.; Shaw, B. R. J . Org. Chem. 1998, 63, 5769. (g) Shaw, B.
R.; Sergueev, D.; He, K. Z.; Porter, K.; Summers, J . S.; Sergueeva, Z.;
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Wang, X.; Dobrikov, M.; He, K.; Porter, K.; Lin, J . L.; Rait, V.; Sergueev,
D.; Sergueeva, Z. In Chemistry of Nucleic Acid Components; Tocik, Z.,
Hocek, M., Eds; Academy of Sciences of the Czech Republic: Prague,
2002; Vol. 5, p 169. (i) Summers, J . S.; Shaw, B. R. Curr. Med. Chem.
2001, 8, 1147. (j) Shaw, B. R.; Dobrikov, M.; Wang, X.; Wan, J .; He, K.
Z.; Lin, J . L.; Li, P.; Rait, V.; Sergueeva, Z.; Sergueev, D. Ann. N.Y.
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10.1021/jo049094b CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/14/2004
J . Org. Chem. 2004, 69, 7051-7057
7051

Li and Shaw
SCHEME 1. Syn th esis of Deoxyth ym id in e
i.e., the normal phosphate 1, the phosphorothioate 3, and
the methylphosphonate 4 shown in Figure 1. Thus,
boranophosphates would be expected to share a number
of chemical and biochemical properties with phospho-
rothioate and methylphosphonate analogues, such as
good aqueous solubility, increased lipophilicity, and nu-
clease resistance. Additionally, boranophosphates may be
unique and useful in transporting boron to tumor tissue
selectively for a type of radiation therapy known as
BNCT (boron neutron capture therapy).6
5′-P r-Bor a n od ip h osp h a te 5a a n d
5′-P r-Bor a n otr ip h osp h a te 13 w ith Sa licyl
Ch lor op h osp h ite
(2-Ch lor o-4H-1,3,2-ben zod ioxa p h osp h or in -4-on e)
Boranophosphates of nucleosides and oligonucleosides
have recently attracted attention due to their possible
therapeutic applications as antiviral prodrugs,7 aptam-
ers,8 and antisense agents.9 For example, the most widely
used antiviral drugs are nucleosides that lack a 3′-OH
group and exert their biological functions through a series
of phosphorylations to their corresponding mono- (dNMP),
di- (dNDP), and triphosphate (dNTP) analogues by in-
tracellular kinases.10a-c The biologically active nucleoside
triphosphate (dNTP) analogues are then incorporated
into the viral DNA and subsequently cause the chain
termination of the growing viral DNA. It has been found
in this laboratory that introduction of the boranophos-
phate group into dNTPs (as dNTPRB) increases their
selectivity to viral reverse transcriptases (RT) relative
to bacterial DNA polymerases.7a,b The presence of the
R-boranophosphate group in AZTDP,7c D4TDP,7d and
ddADP7e improves phosphorylation of these diphosphates
by nucleoside diphosphate kinase as well as the incor-
poration of their corresponding nucleoside triphosphates
by wild type7c and mutant multidrug-resistant HIV-1
RT.7d,e Moreover, after these nucleoside boranophosphate
analogues were incorporated into DNA, repair of the
blocked DNA chains by pyrophosphorolysis was reduced
significantly by mutant HIV-1 RT enzymes from drug-
resistant viruses.7b These results suggest that borano-
phosphates may be potentially useful in a prodrug
delivery10d-e of R-P-borane modified nucleotide analogues.
Therefore, to study the substrate properties of borano-
phosphate analogues, the development of new methods
for synthesizing these new compounds is warranted.
Because of the challenges in conveniently obtaining
phosphate-modified nucleotides,11 including R-P-borano-
diphosphate analogues in large quantity, here we focused
on the synthesis of 5′-PR-boranodiphosphates 5 of 2′-
deoxythymidine (dTDPRB), adenosine (ADPRB), gua-
nosine (GDPRB), and uridine (UDPRB).
Resu lts a n d Discu ssion
Stu d ies on th e Syn th esis of 2′-Deoxyth ym id in e 5′-
P r-Bor an odiph osph ate (dTDP rB) with Salicyl Ch lo-
r op h osp h ite. Our group recently reported the first
synthesis of
a
nucleoside 5′-PR-boranodiphosphate
(NDPRB) using a phosphoramidite approach.12 Due to the
lengthy experimental procedure and relatively low yield,
we decided here to explore alternative synthetic methods
and increase the overall yield. Our first attempt to
prepare dTDPRB 5a , based on an approach for 2′-de-
oxythymidine thiodiphosphate reported by Ludwig and
Eckstein,13 is outlined in Scheme 1. As shown earlier, 3′-
O-acetylthymidine was phosphitylated by 2-chloro-4H-
1,3,2-benzodioxaphosphorin-4-one (salicyl chlorophos-
phite) to form two diastereomers of the intermediate 2′-
deoxythymidine 5′-(4H-,3,2-benzodioxaphosphorin-4-one)
7. They were identified by the appearance of two signals,
at δ 128.12 and 126.18 ppm, observed in the 31P NMR
spectra of the reaction mixture. Due to the bifunctionality
(6) Hawthorne, M. F. Angew. Chem., Int. Ed. Engl. 1993, 32, 950.
(7) (a) Dobrikov, M. I.; Sergueeva, Z. A.; Shaw, B. R. Nucleosides
Nucleotides Nucleic Acids 2003, 22, 1651. (b) Dobrikov, M. I.; Grady,
K. M.; Shaw, B. R. Nucleosides Nucleotides Nucleic Acids 2003, 22,
275. (c) Meyer, P.; Schneider, B.; Sarfati, S.; Deville-Bonne, D.;
Guerreiro, C.; Boretto, J .; J anin, J .; Veron, M.; Canard, B. EMBO J .
2000, 19, 3520. (d) Selmi, B.; Boretto, J .; Sarfati, S. R.; Guerreiro, C.;
Canard, B. J . Biol. Chem. 2001, 276, 48466. (e) Deval, J .; Selmi, B.;
Boretto, J .; Egloff, M. P.; Guerreiro, C.; Sarfati, S.; Canard, B. J . Biol.
Chem. 2002, 277, 42097. (f) Li, P.; Dobrikov, M.; Liu, H. Y.; Shaw, B.
R. Org. Lett. 2003, 5, 2401.
(8) Lato, S. M.; Ozerova, N. D. S.; He, K. Z.; Sergueeva, Z.; Shaw,
B. R.; Burke, D. H. Nucleic Acids Res. 2002, 30, 1401.
(9) (a) Rait, V. K.; Shaw, B. R. Antisense Nucleic Acid Drug Dev.
1999, 9, 53. (b) Wang, X.; Dobrikov, M.; Sergueev, D.; Shaw, B. R.
Nucleosides Nucleotides Nucleic Acids 2003, 22, 1151.
(10) (a) De Clercq, E. Nature Rev. Drug Dis. 2002, 1, 13. (b) Meier,
C. Synlett 1998, 233. (c) Wagner, C. R.; Iyer, V. V.; McIntee, E. J . Med.
Res. Rev. 2000, 20, 417. (d) Li, P.; Shaw, B. R. Org. Lett. 2002, 4, 2009.
(e) Li, P.; Shaw, B. R. Nucleosides Nucleotides Nucleic Acids 2003, 22,
699.
(11) Burgess, K.; Cook, D. Chem. Rev. 2000, 100, 2047.
(12) Lin, J . L.; He, K. Z.; Shaw, B. R. Helv. Chim. Acta 2000, 83,
1392.
7052 J . Org. Chem., Vol. 69, No. 21, 2004

Deoxy- and Ribonucleoside 5′-PR-Boranodiphosphates
SCHEME 2. P r op osed Mech a n ism for th e Step s
In volvin g Su bstitu tion w ith Tr ibu tyla m m on iu m
Or th op h osp h a te w ith Use of th e Sa licyl
Ch lor op h osp h ite Ap p r oa ch
produced during the dehydration step (Scheme 2) was
consumed by the addition of trimethylsilane chloride,
driving the reaction toward the formation of P2,P3-dioxo-
P1-thymidinylcyclotriphosphate 10, which was finally
transformed into dTTPRB 13.
Stu d ies on th e Syn th esis of Nu cleosid e 5′-P r-
Bor a n od ip h osp h a te (NDP rB) via a n Oxa th ia p h os-
p h ola n e Ap p r oa ch . Oxathiaphospholanes were first
introduced by Stec15 to be a method of choice for stereo-
controlled synthesis of oligo(nucleoside phosphorothio-
ate)s and were recently employed by Baraniak16 to obtain
nucleoside monophosphate prodrugs in reasonable to
good yields. The oxathiaphospholane approach involves
a tricoordinate phosphorus intermediate, and therefore,
as demonstrated earlier,17 is suitable for the introduction
of the borane moiety. The detailed synthesis of a nucleo-
side R-P-borane modified diphosphate, NDPRB 5, is
depicted in Scheme 3.
Reaction of 2-chloro-1,3,2-oxathiaphospholane 14 with
deoxy- and/or ribonucleoside 6, carried out in anhydrous
acetonitrile, gave nucleoside 5′-O-1,3,2-oxathiaphos-
pholane 15 in the presence of 2 molar equiv of diisopro-
pylethylamine (DIPEA). This phosphorylating reaction
was completed in 15 min as evidenced by 31P NMR
spectra, where the singlet at δ 207 ppm for oxathiaphos-
pholane 14 was transformed into two singlets around δ
172 ppm corresponding to the diastereomers of the PIII
intermediate 15 (ratio 1:1). When 2′-deoxythymidine or
uridine was used, the phosphorylation was very clean.
However, when adenosine or guanosine without nucleo-
base protection was used, 31P NMR spectra of 15b ,c
revealed two extra singlets at δ 165 ppm with a total
integration of 5%. It was assumed that these peaks at δ
165 ppm corresponded to the two diastereomers of
N-phosphorylated exocyclic products of the nucleobase.
Since the side reaction was almost negligible, we did not
seek to protect the nucleobase for the phosphitylation
step. Attempts to purify compound 15 by silica gel column
chromatography were unsuccessful, as the compound
appeared to decompose quickly on the column. Thus,
without purification, the reaction mixtures containing 15
proceeded to the next boronation step.
of the thymidine phosphite 7, it could undergo two
subsequent nucleophilic substitution reactions as de-
picted in Scheme 2. With the assumption that the
carboxyl group is a better leaving group than the phenolic
group, then substitution by the first incoming nucleo-
phile, tributylammonium orthophosphate,14 would yield
2′-deoxythymidine P-phosphoryl-P-(2-carbonylphenolic)
phosphite 8. Subsequently, the phenolic group on inter-
mediate 8 would be replaced with a second incoming
nucleophilic tributylammonium orthophosphate to obtain
the 2′-deoxythymidine P-diphosphoryl phosphite 9 (Scheme
2), which could undergo a reversible dehydration reaction
(loss of H2O) to form P2,P3-dioxo-P1-thymidinylcyclotri-
phosphate 10. The intermediates 8 and 10, when sub-
jected to boronation in the presence of excess borane-
dimethyl sulfide, gave the corresponding boronated com-
plexes 11 and 12 as shown in Scheme 1. Treatment of
the reaction mixtures, first with H2O and then concen-
trated ammonium hydroxide, yielded the final compounds
dTDPRB 5a and dTTPRB 13. The overall yield was only
12% and 17% for the diastereomers of dTDPRB 5a and
dTTPRB 13, respectively, after ion-exchange isolation.
Although steps outlined in Scheme 1 were monitored
by 31P NMR, the complexity of the reaction mixtures
together with the phosphorus peak quadrupole broaden-
ing by the 10B and 11B nuclei made it impossible to assign
the 31P NMR peaks to the corresponding intermediates
8 through 12. However, the isolation of the final products,
dTDPRB 5a and dTTPRB 13, was consistent with our
proposed structures for those intermediates 8 through
10 and the mechanism, as shown in Scheme 2. To further
support the proposed mechanism involving dehydration,
2 molar equiv of chlorotrimethylsilane (TMSCl) were
introduced into the reaction mixture immediately after
the addition of 2 equiv of tributylammonium orthophos-
phate. In a comparison of the final products after ion-
exchange separation, the yield of dTTPRB 13 increased
from 17% (without TMSCl) to 23% (with TMSCl) for
diastereoisomeric mixtures, whereas the yield for dTDPRB
5a decreased from 12% (without TMSCl) to trace amounts
(with TMSCl). These results suggested that the water
Exchange of a borane group between the nucleoside
5′-O-1,3,2-oxathiaphospholane 15 and a boronation re-
agent produced the oxathiaphospholane-borane complex
16. According to valence shell electron pair repulsion
(VSEPR) theory and molecular orbital (MO) theory, the
boron atom in BH3 carries an empty 2p orbital and,
(13) Ludwig, J .; Eckstein, F. J . Org. Chem. 1989, 54, 631.
(14) Tributylammonium orthophosphate was generated as follows.
Tributylamine (1 equiv) was added dropwise into the solution of
anhydrous orthophosphoric acid (1 equiv) in methylene chloride over
30 min. The mixture was left stirring for another 60 min and the
solvent was removed under reduced pressure. The residue was
re-evaporated with anhydrous pyridine and DMF. The final compound
was dissolved in anhydrous DMF to a concentration of 1 M, and stored
over 4 Å molecular sieves.
(15) (a) Stec, W. J .; Grajkowski, A.; Kobylanska, A.; Karwowski,
B.; Koziolkiewicz, M.; Misiura, K.; Okruszek, A.; Wilk, A.; Guga, P.;
Boczkowska, M. J . Am. Chem. Soc. 1995, 117, 12019. (b) Guga, P.;
Okruszek, A.; Stec, W. J . New Aspects Phosphorus Chem. I 2002, 220,
169.
(16) (a) Baraniak, J .; Kaczmarek, R.; Stec, W. J . Tetrahedron Lett.
2000, 41, 9139. (b) Baraniak, J .; Kaczmarek, R.; Wasilewska, E.; Stec,
W. J . Phosphorus, Sulfur Silicon Relat. Elem. 2002, 177, 1667. (c)
Baraniak, J .; Kaczmarek, R.; Korczynski, D.; Wasilewska, E. J . Org.
Chem. 2002, 67, 7267. (d) Baraniak, J .; Kaczmarek, R.; Wasilewska,
E. Tetrahedron Lett. 2004, 45, 671.
J . Org. Chem, Vol. 69, No. 21, 2004 7053

Li and Shaw
SCHEME 3. Syn th esis of Nu cleosid e
5′-P r-bor a n od ip h osp h a te w ith
2-Ch lor o-1,3,2-oxa th ia p h osp h ola n e
reaction mixture containing complex 16 was immediately
treated with simultaneous addition of 1 molar equiv of
tributylammonium orthophosphate19 in anhydrous DMF
and 5 molar equiv of 1,4-diazabicyclo[5.4.0]undec-7-ene
(DBU). The reaction underwent the ring-opening con-
densation to form the boranophosphate intermediate 17
followed by spontaneous elimination of ethylene episul-
fide to generate protected nucleoside 5′-PR-boranodiphos-
phate 18. A white precipitate was formed during this
step, indicating the formation of boranodiphosphate. A
series of experiments on 3′-O-acetyl-2′-deoxythymidine
6a with increasing equivalents of DBU were carried out
and monitored by TLC.20 It was found that the rate of
DBU-assisted ring-opening condensation varied when
different quantities were used. The completion time for
this ring-opening condensation took more than 12 h when
2 equiv of DBU were added, but decreased to 40 min with
5 equiv of DBU and to 30 min with 10 equiv of DBU.
Hence, 5 equiv of DBU was chosen to be the optimal
amount. Furthermore, the yield of this step was found
to decrease dramatically with the presence of DIPEA
remaining from the previous steps in the reaction mix-
ture, as indicated by TLC and 31P NMR. Although the
exact reason is unknown, it is likely that the DIPEA was
interfering with the DBU-catalyzed ring-opening conden-
sation.15a
The reaction mixture was subsequently purified by ion-
exchange column chromatography. The existence of
protected nucleoside 5′-PR-boranodiphosphate 18 was
identified by the appearance of peaks at δ -6 ppm for
P(â) (doublet, J ) 25-35 Hz) and δ 81 ppm for P(R)
(broad multiplet) in the 31P NMR spectrum. The chemical
shift for P(â) in boranodiphosphates is similar to that in
unmodified diphosphates as well as P(â) in analogues
modified only at the R-position (e.g., R-thiodiphosphates).
Modification of a nucleoside diphosphate at the R-posi-
tion, however, results in a noticeable change of the P(R)
chemical shift: from δ ca. -10 ppm for a normal di-
phosphate to δ ca. 20 ppm for R-methylphosphonyl-â-
monophosphate,21 to δ ca. 40 ppm for R-thiodiphosphate13
and, as we have found in our studies, to δ ca. 81 ppm
(broad multiplet) for R-boranodiphosphate shown in
Figure 2.
The removal of the ribose protecting groups on 5′-PR-
boranodiphosphate 18 was carried out by treating it
with ammonium hydroxide in water (1/1, v/v) for 6 h. The
final product, nucleoside 5′-PR-boranodiphosphate 5, was
isolated by ion-exchange column chromatography on
QA-52 [HCO3-] cellulose. The overall yields (from 6 to
5) were 30-43% and the structures of boranodiphos-
phates 5a -d were confirmed by 1H NMR, 31P NMR,
FAB-MS, and HRMS.
therefore, is susceptible to attack by nucleophiles.18 The
tervalent phosphorus atom could act as a Lewis base
donor of electrons to electron-deficient BH3. The reac-
tivities of a series of boronating reagents to transfer a
borane group to compound 15, including borane-dimeth-
yl sulfide and borane-tetrahydrofurane, were examined,
as well as three types of borane-amine complexes:
aliphatic-borane (borane-N,N-diisopropylethylamine),
aromatic-borane (borane-aniline), and heterocyclic-
borane (borane-pyridine and borane-2-chloropyridine).
The conversion of nucleoside 5′-O-1,3,2-oxathiaphos-
pholane 15 to its oxathiaphospholane-borane complex
16 was monitored by 31P NMR spectra, which showed a
disappearance of two singlets at δ 172 ppm for compound
15 and formation of a broad signal centered at δ 160 ppm
for oxathiaphospholane-borane complex 16. The results
are summarized in Table 1, where the most active
boronation reagent is seen to be borane-dimethyl sulfide.
Thus, after removing solvent and excess DIPEA under
reduced pressure by oil pump, the crude product 15 was
redissolved in anhydrous DMF and reacted with 5 to 8
molar equiv of borane-dimethyl sulfide at room temper-
ature for 30 min. This exchange reaction was very clean,
producing only the oxathiaphospholane-borane complex
16 according to 31P NMR.
(17) Okruszek, A.; Sierzchala, A.; Zmudzka, K.; Stec, W. J . Nucleo-
sides Nucleotides Nucleic Acids 2001, 20, 1843.
(18) Housecroft, C. E. Boranes and Metalloboranes Structure,
Bonding and Reactivity; J ohn Wiley & Sons: New York, 1990; pp
18-30.
(19) We initially used commercially available tetrabutylammonium
dihydrogenphosphate as the phosphate nucleophile. However, due to
its low solubility in DMF, we switched to freshly prepared tributyl-
ammonium orthophosphate.
(20) TLC developing system: CH2Cl2/EtOAc ) 1/4 (v/v). Reaction
was monitored by the disappearance of oxathiaphospholane-borane
complex 16a .
(21) Higuchi, H.; Endo, T.; Kaji, A. Biochemistry 1990, 29, 8747-
8753.
Oxathiaphospholane-borane complex 16 was unable
to be isolated by silica gel column chromatography due
to its instability in the presence of moisture. Hence, the
7054 J . Org. Chem., Vol. 69, No. 21, 2004

Deoxy- and Ribonucleoside 5′-PR-Boranodiphosphates
TABLE 1. Rea ctivity of Bor on a tin g Rea gen tsa
borane-amine complex
borane complexb
BH3:Me2S
100%
BH3:THF
none
BH3:DIPEA
30%
BH3:An
28%
BH3:Py
none
BH3:Py-2-Cl
c,d
conversion of 15f16
85%
a
b
c
3′-O-Acetyl-2′-deoxythymidine was used as model compound. Five molar equivalents of borane complex were used. 31P NMR
d
samples were prepared at 30 min after addition of the borane complex. Conversion was calculated as the integration percentage of
compound 16 relative to the total integration of compounds 15 and 16 in the 31P NMR spectra.
F IGURE 2. 31P NMR spectrum of diastereomers of adenosine 5′-PR-boranodiphosphate (ADPRB, 5b).
TABLE 2. RP -HP LC P r ofiles for NDP rB 5a
obtain a set of four compounds, including dTDPRB 5a ,
ADPRB 5b, GDPRB 5c, and UDPRB 5d , in good yields.
Since this method does not require the exocyclic amine
protection of the nucleobase, it eliminates the risk of
possible nucleobase reduction during the boronation
step.22 Comparative studies on different boronating
reagents and the optimal conditions for DBU-catalyzed
ring-opening condensation were carried out. The two
diastereomers of NDPRB 5 were successfully separated
by RP-HPLC by using isocratic elution and their struc-
tures were confirmed by spectroscopic methods. This
oxathiaphospholane approach ensures the availability of
nucleoside 5′-PR-boranodiphosphate analogues, which are
needed for antiviral drug research.
retention time (min) [area %]
compd
bufferb
RP isomer
SP isomer
dTDPRB 5a 92% TEAB/8% MeOH 18.50 [45.5]
ADPRB 5b 92% TEAB/8% MeOH 15.22 [56.1]
25.48 [54.5]
27.85 [43.9]
12.47 [48.9]
10.20 [57.0]
GDPRB 5c 95% TEAB/5% MeOH
UDPRB 5d 94% TEAB/6% MeOH
7.62 [51.1]
6.64 [43.0]
a
Waters Delta-Pak C18 column: 15 µm, 100 Å, 25 mm × 100
b
mm, with Z-module. 100 mM TEAB (triethylamonium bicarbon-
ate, pH 8.0) and methanol
Stu d ies on th e HP LC Sep a r a tion of Dia ster eo-
m er s of Nu cleosid e 5′-P r-Bor a n od ip h osp h a tes. The
presence of a borane group replacing a nonbridging
oxygen at the P(R) position in NDPRB 5 produces a pair
of diastereomers, whose absolute configurations have
been recently determined by cocrystalization with nu-
cleoside diphosphate kinase.7c The two diastereomers
were resolved by semipreparative RP-HPLC, using iso-
cratic elution as described in Table 2. The first and second
eluting diastereomers are the RP and SP isomers, respec-
tively. The isomeric purity of each of the individual
diastereomers was determined by RP-HPLC under the
same conditions used for separation. The HPLC profiles
for adenosine 5′-PR-boranodiphosphate 5b before and
after semipreparative HPLC separation are shown in
Figure 3. The structure for each diastereomer was
Like nucleoside phosphorothioates,4 the two diastere-
omers of NDPRB are expected to have different substrate
properties toward nucleosidyl transferases and hydro-
lases, and should be useful for investigating the roles of
phosphate and metal ions in biological processes to
elucidate the stereochemical and metal requirements of
the enzymatic reactions involving nucleoside diphos-
phates. Moreover, when compared to the natural nucle-
otide, the improved substrate properties,7 increased
lipophilicity,5 and increased nuclease resistance5 im-
parted by the borane group, in conjunction with the
potential utility as a carrier of 10B in BNCT,6 make the
nucleoside boranodiphosphate a useful compound and
tool in antiviral drug research.
confirmed by H NMR, 31P NMR, FAB-MS, and HRMS.
1
Exp er im en ta l Section
Con clu sion s
2-Chloro-1,3,2-oxathiaphospholane was synthesized accord-
ing to the reported procedure.15a All solvents were freshly
distilled under argon from calcium hydride. All boronating
We developed a new method to efficiently synthesize
nucleoside 5′-PR-boranodiphosphate 5 via the boronation
of the P(III) intermediate, nucleoside 5′-O-1,3,2-oxathia-
phospholane 15. This convenient method was applied to
(22) Sergueeva, Z. A.; Sergueev, D. S.; Shaw, B. R. Nucleosides
Nucleotides Nucleic Acids 2000, 19, 275-282.
J . Org. Chem, Vol. 69, No. 21, 2004 7055

Li and Shaw
added. After 30 min, the reaction was evaporated to dryness
and the residue was treated with concentrated ammonium
hydroxide at room temperature for 5 h. The reaction mixture
was evaporated to dryness again and the residue was parti-
tioned between water (20 mL) and diethyl ether (20 mL). The
water layer was subjected to ion-exchange chromatography
with QA-52 cellulose ([HCO3-]), using a linear gradient of
5 mM and 200 mM ammonium bicarbonate buffer (pH 8.0,
800 mL each). The desired fractions of dTDPRB and dTTPRB
were collected and dried by lyophilization, and excess salt was
removed by repeated lyophilization with deionized water. The
yields were 12% (27.1 mg) and 17% (46.59 mg) for dTDPRB
and dTTPRB, respectively.
Syn th esis of Deoxy- a n d Ribon u cleosid e 5′-P r-Bor a -
n od ip h osp h a t es w it h 2-Ch lor o-1,3,2-oxa t h ia p h osp h o-
la n e: Gen er a l P r oced u r e. Protected nucleoside (3′-O-acetyl-
2′-deoxythymidine, 2′,3′-O-diacetyladenosine, 2′,3′-O-diacetylgua-
nosine, or 2′,3′-O-dibenzoyluridine) (0.5 mmol, 1 equiv) and
DIPEA (1.0 mmol, 2 equiv) were dissolved in acetonitrile
followed by the addition of 2-chloro-1,3,2-oxathiaphospholane
14 (0.55 mmol, 1.1 equiv). After 15 min, solvent was removed
under vacuum and the residue was redissolved in DMF.
Borane-dimethyl sulfide complex (2.5 mmol, 5-8 equiv) was
then added and stirred for 30 min. A freshly prepared 1 M
solution of tributylammonium orthophosphate in DMF (0.5
mmol, 1 equiv) and DBU (2.5 mmol, 5 equiv) and were
simultaneously injected into the reaction mixture by syringe,
which was continually stirred for 2 h. Solvent was removed
and the residue was partitioned between 20 mL of water and
10 mL of ethyl acetate. The aqueous layer was concentrated
and purified by ion-exchange chromatography eluted with a
linear gradient of 5 mM and 200 mM ammonium bicarbonate
buffer (pH 8.0, 800 mL each) to afford the ammonium salt of
protected nucleoside 5′-PR-boranodiphosphate 18. The desired
fractions were collected, concentrated, and treated with con-
centrated ammonium hydroxide in water (10 mL, 1/1, v/v) for
6 h. After the removal of solvent, the residue was applied to
the ion-exchange chromatography column and eluted with a
linear gradient of 5 mM and 200 mM ammonium bicarbonate
buffer (pH 8.0, 800 mL each) to afford the ammonium salt of
deoxy- and ribonucleoside 5′-PR-boranodiphosphate 5. The
excess salt was removed by repeated lyophilization with
deionized water.
F IGURE 3. Isocratic separation of diastereomers of adenosine
5′-PR-boranodiphosphate 5b by HPLC (A) and analysis of
purity of HPLC-isolated RP isomer (B) and SP isomer (C). The
separation was carried out on the Waters Delta-Pak C18
column with 92% 100 mM TEAB (pH 8.0) and 8% methanol
at a flow rate of 5.0 mL/min. The absorbance was monitored
at λ 260 nm. The retention times for the two diastereomers
were 15.22 and 27.85 min, respectively. Peak 1: RP isomer of
ADPRB. Peak 2: SP isomer of ADPRB.
2′-Deoxyth ym idin e 5′-P r-Bor an odiph osph ate (dTDP rB)
(5a ). The diastereomeric mixture of compound 5a was pre-
pared in 43% yield (97 mg) following the general procedure
by using 3′-O-acetyl-2′-deoxythymidine and 5 molar equiv of
borane-dimethyl sulfide. 1H NMR (D2O) δ 7.61, 7.59 (2s, 2
isomers, 1H), 6.21, 6.19 (2t, 2 isomers, J ) 6.0 Hz, 1H), 4.48
(m, 1H), 4.10-3.90 (m, 3H), 2.21 (m, 2H), 1.80 (s, 3H), (+)0.84
to (-)0.17 (br, 3H); 31P NMR (D2O) δ 83.45 (br, 1P, R-P),
-10.03 (d, 1P, â-P, J Râ ) 27.52 Hz); UV (H2O) λmax 267.6 nm;
FAB-MS m/z 399.05 (M-); HRMS found m/z 399.0541 (for 11B),
reagents except borane-dimethyl sulfide complex, diisopro-
pylethylamine (DIPEA), and 1,4-diazabicyclo[5.4.0]undec-7-ene
(DBU) were dried by 4 Å molecular sieves overnight. Nucleo-
sides and tributylammonium orthophosphate were dried by
P2O5 overnight under vacuum prior to use. Triethylammonium
bicarbonate was prepared from triethylamine, H2O, and CO2.
All the reactions were carried out under an argon atomosphere
unless otherwise stated.
Syn th esis of d TDP rB a n d d TTP rB w ith Sa licyl Ch lo-
r op h osp h ite. 3′-O-Acetyl-2′-deoxythymidine 6a (0.5 mmol, 1
equiv) was dissolved in anhydrous DMF and anhydrous
pyridine (1.5 mmol, 3 equiv). A freshly prepared 0.5 M solution
of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in anhydrous
DMF (0.55 mmol, 1.1 equiv.) was added. After 10 min, a 1 M
solution of tributylammonium orthophosphate in anhydrous
DMF (1.25 mmol, 2.5 equiv) and tributylamine (2.5 mmol, 5.0
equiv) were simultaneously injected into the reaction mixture.
A white precipitate was formed but quickly disappeared and
30 min later a solution of 2 M borane-dimethyl sulfide in THF
was added (5 mmol, 10 equiv). After the mixture was stirred
for 60 min, the septum was removed and water (2 mL) was
-
calcd for C10H18BN2O10P2 399.0530.
Ad en osin e 5′-P r-Bor a n od ip h osp h a te (ADP rB) (5b).
The diastereomeric mixture of compound 5b was prepared in
35% yield (83.3 mg) following the general procedure by using
2′,3′-O-diacetyladenosine and 7 molar equiv of borane-di-
methyl sulfide. 1H NMR (D2O) δ 8.45, 8.43 (2s, 2 isomers, 1H),
8.11 (s, 1H), 6.00 (d, J ) 5.2 Hz, 1H), 4.59 (m, 1H), 4.47, 4.39
(2m, 2 isomers, 1H), 4.25 (m, 1H), 4.14 (m, 1H), 4.03 (m, 1H),
(+)0.73 to (-)0.15 (br, 3H); 31P NMR (D2O) δ 81.90 (br, 1P,
R-P), -7.20 (d, 1P, â-P, J Râ ) 30.13 Hz); UV (H2O) λmax 259.3
nm; FAB-MS m/z 424.08 (M-); HRMS found m/z 424.0605 (for
-
11B), calcd for C10H17BN5O9P2 424.0595.
Gu a n osin e 5′-P r-Bor a n od ip h osp h a te (GDP rB) (5c).
The diastereomeric mixture of compound 5c was prepared in
30% yield (73.8 mg) following the general procedure by using
2′,3′-O-diacetylguanosine and 8 molar equiv of borane-di-
methyl sulfide. 1H NMR (D2O) δ 8.05, 8.02 (2s, 2 isomers, 1H),
5.79 (d, J ) 5.2 Hz, 1H), 4.60 (m, 1H), 4.46, 4.38 (2m, 2 isomers,
1H), 4.22 (m, 1H), 4.13 (m, 1H), 4.03 (m, 1H), (+)0.81 to (-)0.14
7056 J . Org. Chem., Vol. 69, No. 21, 2004

Deoxy- and Ribonucleoside 5′-PR-Boranodiphosphates
(br, 3H); 31P NMR (D2O) δ 83.10 (br, 1P, R-P), -9.06 (d, 1P,
â-P, J Râ ) 29.14 Hz); UV (H2O) λmax 253.5 nm; FAB-MS m/z
â-P, J Râ ) 30.60 Hz); UV (H2O) λmax 259.3 nm; FAB-MS m/z
424.08 (M-); HRMS found m/z 424.0599 (for 11B), calcd for
440.08 (M-); HRMS found m/z 440.0541 (for 11B), calcd for
C
10H17BN5O9P2 424.0595.
-
-
C
10H17BN5O10P2 440.0544.
ADP rB, SP isom er : 1H NMR (D2O) δ 8.43 (s, 1H), 8.06 (s,
Ur id in e 5′-P r-Bor a n od ip h osp h a te (UDP rB) (5d ). The
1H), 5.96 (d, J ) 5.2 Hz, 1H), 4.57 (m, 1H), 4.41(m, 1H), 4.20
(m, 1H), 4.09 (m, 1H), 3.96 (m, 1H), (+)0.80 to (-)0.17
(br, 3H); 31P NMR (D2O) δ 80.12 (br, 1P, R-P), -5.55 (d, 1P,
â-P, J Râ ) 28.17 Hz); UV (H2O) λmax 259.3 nm; FAB-MS m/z
diastereomeric mixture of compound 5d was prepared in 39%
yield (88.4 mg) following the general procedure by using 2′,3′-
O-dibenzoyluridine and 5 molar equiv of borane-dimethyl
sulfide. 1H NMR (D2O) δ 7.92, 7.91 (2d, 2 isomers, J ) 8.0 Hz,
1H), 5.85 (d, J ) 4.4 Hz, 1H), 5.81 (d, J ) 8.0 Hz, 1H), 4.24
(m, 1H), 4.20 (m, 1H), 4.15 (m, 1H), 4.08-3.95 (m, 2H), (+)0.87
to (-)0.20 (br, 3H); 31P NMR (D2O) δ 83.53 (br, 1P, R-P),
-10.03 (d, 1P, â-P, J Râ ) 24.45 Hz); UV (H2O) λmax 262.9 nm;
FAB-MS m/z 401.07 (M-); HRMS found m/z 401.0310 (for 11B),
424.08 (M-); HRMS found m/z 424.0588 (for 11B), calcd for
-
C
10H17BN5O9P2 424.0595.
GDP rB, RP isom er : 1H NMR (D2O) δ 8.04 (s, 1H), 5.77 (d,
J ) 4.8 Hz, 1H), 4.53 (m, 1H), 4.50 (m, 1H), 4.13 (m, 1H), 4.10
(m, 1H), 3.93 (m, 1H), (+)0.78 to (-)0.18 (br, 3H); 31P NMR
(D2O) δ 79.48 (br, 1P, R-P), -5.63 (d, 1P, â-P, J Râ ) 28.98 Hz);
UV (H2O) λmax 253.5 nm; FAB-MS m/z 440.08 (M-); HRMS
-
calcd for C9H16BN2O11P2 401.0322.
found m/z 440.0532 (for 11B), calcd for C10H17BN5O10P2
-
Rever se-P h a se HP LC Sep a r a tion of Dia ster eom er s of
Deoxy- a n d Ribon u cleosid e 5′-P r-Bor a n od ip h osp h a tes.
The separation of diastereomers of each NDPRB was carried
out by ion-pairing chromatography on a semipreparative
reverse-phase column (Waters Delta-Pak C18, 15 µm, 100 Å,
25 × 100 mm, with Z-module) at a flow rate of 5 mL/min, using
an isocratic elution [100 mM triethylamonium bicarbonate
(TEAB) buffer, pH 8.0, and methanol]. Fractions containing
the same isomer (similar retention time) were combined, and
the solvent was removed under reduced pressure. The excess
of TEAB and methanol were removed by repeated lyophi-
lization with deionized water. The retention time and ratio
for different diastereomers of NDPRB are summarized in
Table 2.
440.0532.
GDP rB, SP isom er : 1H NMR (D2O) δ 8.01 (s, 1H), 5.77 (d,
J ) 5.6 Hz, 1H), 4.54 (m, 1H), 4.41 (m, 1H), 4.17 (m, 1H), 4.07
(m, 1H), 3.98 (m, 1H), (+)0.78 to (-)0.18 (br, 3H); 31P NMR
(D2O) δ 80.50 (br, 1P, R-P), -5.58 (d, 1P, â-P, J Râ ) 31.25 Hz);
UV (H2O) λmax 253.5 nm; FAB-MS m/z 440.08 (M-); HRMS
-
found m/z 440.0541 (for 11B), calcd for C10H17BN5O10P2
440.0542.
UDP rB, RP isom er : 1H NMR (D2O) δ 7.96 (d, J ) 8.0 Hz,
1H), 5.81 (d, J ) 4.0 Hz, 1H), 5.78 (d, J ) 8.0 Hz, 1H), 4.32
(m, 1H), 4.20 (m, 1H), 4.13 (m, 1H), 4.09 (m, 1H), 4.02 (m,
1H), (+)0.80 to (-)0.20 (br, 3H); 31P NMR (D2O) δ 78.91 (br,
1P, R-P), -5.70 (d, 1P, â-P, J Râ ) 28.98 Hz); UV (H2O) λmax
262.9 nm; FAB-MS m/z 401.07 (M-); HRMS found m/z
d TDP rB, RP isom er : 1H NMR (D2O) δ 7.52 (d, J ) 0.8
Hz, 1H), 6.18 (t, J ) 6.4 Hz, 1H), 4.53 (m, 1H), 4.08 (m, 1H),
3.97-3.87 (m, 2H), 2.16 (dd, J ) 5.6, 6.4 Hz, 2H), 1.76 (d, J )
1.2 Hz, 3H), (+)0.60 to (-)0.20 (br, 3H); 31P NMR (D2O) δ 79.06
(br, 1P, R-P), -5.72 (d, 1P, â-P, J Râ ) 31.89 Hz); UV (H2O)
λmax 267.6 nm; FAB-MS m/z 399.05 (M-); HRMS found m/z
401.0313 (for 11B), calcd for C9H16BN2O11P2 401.0322.
-
UDP rB, SP isom er : 1H NMR (D2O) δ 7.80 (d, J ) 8.0 Hz,
1H), 5.86 (d, J ) 4.4 Hz, 1H), 5.73 (d, J ) 8.0 Hz, 1H), 4.24
(m, 1H), 4.19 (m, 1H), 4.10-4.05 (m, 2H), 4.01 (m, 1H), (+)0.78
to (-)0.18 (br, 3H); 31P NMR (D2O) δ 80.03 (br, 1P, R-P),
-5.56 (d, 1P, â-P, J Râ ) 30.60 Hz); UV (H2O) λmax 262.9 nm;
FAB-MS m/z 401.07 (M-); HRMS found m/z 401.0320 (for 11B),
399.0531 (for 11B), calcd for C10H18BN2O10P2 399.0530.
-
d TDP rB, SP isom er : 1H NMR (D2O) δ 7.62 (d, J ) 1.2 Hz,
1H), 6.20 (t, J ) 6.8 Hz, 1H), 4.48 (m, 1H), 4.05-3.94 (m, 3H),
2.20 (m, 2H), 1.79 (d, J ) 0.8 Hz, 3H), (+)0.60 to (-)0.20
(br, 3H); 31P NMR (D2O) δ 81.61 (br, 1P, R-P), -7.96 (d, 1P,
â-P, J Râ ) 25.90 Hz); UV (H2O) λmax 267.6 nm; FAB-MS m/z
-
calcd for C9H16BN2O11P2 401.0322.
Ack n ow led gm en t. This work was supported by
NIH grant R01 AI52061 to B.R.S.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR and 31P
NMR spectra for each diastereomer of compounds 5a -d and
the corresponding HPLC profiles. This material is available
free of charge via the Internet at http://pubs.acs.org.
399.05 (M-); HRMS found m/z 399.0518 (for 11B), calcd for
-
C
10H18BN2O10P2 399.0530.
ADP rB, RP isom er : 1H NMR (D2O) δ 8.46 (s, 1H), 8.06 (s,
1H), 5.96 (d, J ) 4.0 Hz, 1H), 4.54 (m, 1H), 4.51(m, 1H), 4.18
(m, 1H), 4.13 (m, 1H), 3.96 (m, 1H), (+)0.80 to (-)0.17
(br, 3H); 31P NMR (D2O) δ 79.35 (br, 1P, R-P), -5.63 (d, 1P,
J O049094B
J . Org. Chem, Vol. 69, No. 21, 2004 7057