A robust synthetic route to 2'-deoxy-3'-nucleotide derivatives of modified nucleobases

Full text of DOI:10.1021/jo982473i

7628
J . Org. Chem. 1999, 64, 7628-7632
phosphorylating agents and maintenance of strictly
A Robu st Syn th etic Rou te to
2′-Deoxy-3′-n u cleotid e Der iva tives of
Mod ified Nu cleoba ses
anhydrous reaction conditions, and yields may be highly
variable.11,12 Hence, the procedures described here should
find wide applicability in the syntheses of modified 3′-
deoxynucleotides and oligonucleotides containing modi-
fied bases at specific sites.
C. M. Prusiewicz,† R. Sangaiah, K. B. Tomer,‡ and
A. Gold*
Department of Environmental Sciences and Engineering,
University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599-7400,
National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina 27709
Resu lts a n d Discu ssion
The key step in the strategy is phosphitylation of
2-fluoro-6-benzyloxypurine-9-(5′-O-[4,4′-dimethoxytrityl]-
2′-deoxyribose) (1), summarized in Scheme 1.
Received December 18, 1998
Salicylchlorophosphine has been reported to phosphi-
tylate O6- and N2-protected 2′-deoxyguanosine in high
yield in a robust procedure.8,9 On the basis of this report,
the reaction was applied to the O6-blocked fluorodeoxy-
inosine (1) to give the corresponding fluoroinosine 3′-H-
phosphonate (2) in 81% yield as the triethylammonium
salt. In accord with expectation, 2 could be readily
identified by the characteristically large P-H coupling
In tr od u ction
We required pure diastereomeric cis- and trans-3-(2′-
deoxyguanosine-3′-phosphate-N2-yl)-4-hydroxy-3,4-dihy-
drocyclopenta[cd]pyrene (7a ,b) as chromatographic stan-
dards to confirm the structures of the major in vivo DNA
adducts of the ubiquitous environmental carcinogen
cyclopenta[cd]pyrene (CPP)1-6 detected by 32P-postlabel-
ing techniques. Although 32P-postlabeling of DNA ad-
ducts followed by chromatographic separation is an
extremely sensitive analytical technique,7 only limited
structural information about adducts can be derived in
the absence of such standards. This need prompted us
to pursue a convenient and robust synthetic route to 3′-
deoxynucleotide derivatives of base-modified nucleosides.
We report here the synthesis of diastereomeric cis- and
trans-deoxyguanosine-3′-nucleotide adducts by a conve-
nient route, using the stable, commercially available
phosphitylating reagent 2-chloro-5,6-benzo-1,3,2-dioxa-
phosphorin-4-one (salicylchlorophosphine).8,9 An advan-
tage of the synthetic scheme is that 3′-H-phosphonate
products of the phosphitylation reaction can be used
directly as reagents for automated oligonucleotide syn-
theses due to their stability and ease of handling.10
However, despite the importance of 3′-H-phosphonate
and 3′-nucleotide derivatives of DNA adducts, synthetic
routes to such derivatives are not generally available.
Current approaches require the preliminary synthesis of
1
constant (∼600 Hz), which was confirmed in both H and
31P NMR spectra.
Cis- and trans N2-adducted deoxynucleoside 3′-H-
phosphonate diastereomers 6a ,b were obtained by cou-
pling the corresponding racemic 3-amino-4-hydroxy-3,4-
dihydro-CPP (CPP-aminohydrin) with O6-deblocked phos-
phonate 5. Coupling the racemic cis-CPP-aminohydrin
with O6-blocked fluoroinosine 3′-H-phosphonate 3 yielded
the O6-benzylated cis N2-adducted 3′-H-phosphonate di-
astereomeric mixture 4. The coupling reaction, which
required no phosphorus protection, was accomplished by
published procedures.13 However, because the aminohy-
drin derivatives of CPP appear to degrade rapidly in an
aerobic environment, this reaction was performed with
the strict exclusion of air. Coupling via 3 provides O6-
protected phosphonate intermediates suitable for use in
preparation of oligonucleotides containing the modified
deoxyguanosine.
The cis-O6-benzyl-protected phosphonate diastereo-
mers 4 were resolved by reverse phase HPLC. The
diastereomeric mixtures of O6-deblocked CPP-modified
deoxyguanosine 3′-H-phosphonates 6a ,b were not well-
resolved by reverse phase HPLC. However, separation
of 6a ,b was achieved following oxidation to the corre-
sponding 3′-nucleotides 7a ,b. Although phosphonate mo-
noester salts are more difficult than phosphodiesters to
oxidize to phosphates, quantitative oxidation of the
modified 3′-H-phosphonates was achieved by treatment
of the persilylated compounds by I2 in pyridine.14 Use of
triethylammonium bicarbonate buffer proved advanta-
geous in reverse phase HPLC separations of the diaster-
eomeric phosphates, since the buffer salts were easily
removed from the collected HPLC fractions by repeated
evaporation in the presence of water. In the case of the
cis-N2-deoxyguanosine adducts of CPP-oxide, the 3′-H-
† Current address: Glaxo-Wellcome, Inc., Research Triangle Park,
NC 27709.
‡ National Institute of Environmental Health Sciences.
(1) Gold, A.; E. Eisenstadt Cancer Res. 1980, 40, 3940-3944.
(2) Gold, A.; Nesnow, S.; Moore, M.; Garland, H.; Curtis, G.; Howard,
B.; Graham, D.; Eisenstadt, E. Cancer Res. 1980, 40, 4482-4484.
(3) Wood, A. W.; Levin, W.; Chang, R. L.; Huang, M.-T.; Ryan, D.
E.; Thomas, P. E.; Lehr, R. E.; Kumar, S.; Koreeda, M.; Akagi, H.; Ittah,
Y.; Dansette, P.; Yagi, H.; J erina, D. M.; Conney, A. H. Cancer Res.
1980, 40, 642-649.
(4) Gold, A. Anal. Chem. 1975, 47, 1469-1471.
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Sci. Technol. 1975, 9, 143-145.
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Schmel, D., Tomatis, L., Eds.; IARC: Lyon, France, 1977; pp 193-
199.
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Schurdak, M. E.; Reddy, M. V. Environ. Health Persp. 1985, 62, 57-
65.
(8) Hermans, J . P. G.; de Vroom, E.; Elie, C. J . J .; van der Marel,
G. A.; van Boom, J . H. Recl. Trav. Chim. Pays-Bas 1986, 105, 510-
511.
(9) Marugg, J . E.; Tromp, M.; Kuyl-Yeheskiely, E.; van der Marel,
G. A.; van Boom, J . H. Tetrahedron Lett. 1986, 27, 2661-2664.
(10) Froehler, B. C. In Methods in Molecular Biology: Protocols for
Oligonucleotides and Analogues: Agrawal, S., Ed.; Humana Press
Inc.: Totowa, 1993; Vol. 20, Chapter 4, p 63-80.
(11) Pongracz, K.; Bodell, W. J . Chem. Res. Toxicol. 1995, 9, 593-
598.
(12) Han, S.; Harris, C. M.; Harris, T. H.; Kim, H. H.; Kim, S. J . J .
Org. Chem. 1996, 61, 174-178.
(13) Lakshman, M. K.; Sayer, J . M.; Yagi, H.; J erina, D. M. J . Org.
Chem 1992, 57, 4585-4589.
(14) Garegg, P. J .; Regberg, T.; Stawinski, J .; Stromberg, R. Nucleo-
sides Nucleotides 1987, 6, 429-432.
10.1021/jo982473i CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/16/1999

Notes
J . Org. Chem., Vol. 64, No. 20, 1999 7629
Sch em e 1
phosphonate diastereomers appropriate for use as syn-
thons in the preparation of oligodeoxynucleotides were
more easily resolvable as the O6-benzylated derivatives.
In general, whether 3′-H-phosphonate diastereomers for
oligodeoxynucleotide synthesis are more easily separated
as the O6-blocked derivatives or in deblocked form will
depend on the specific polycyclic aromatic hydrocarbon
and the relative cis/trans orientation of the aminohydrin
intermediate.
consistent with assigned structures. Formation of the 3′-
H-phosphonates and oxidation to the corresponding 3′-
phosphates is easily demonstrated by 31P NMR. 31P NMR
signals of the 3′-H-phosphonates appear as doublets (J
= 600 Hz) centered at 3.79 ppm downfield from the H3-
PO4 standard, and the phosphate signals as singlets at
1
0.01 ppm. In the H NMR spectra of both cis- and trans-
diastereomers, the proton resonances of the 3,4-dihydro-
cyclopenta[cd]pyrenyl moieties of the early and late-
eluting nucleotides are identical. Peak assignments were
made by comparison to the spectra of the corresponding
nucleoside adducts.15,16 Peak assignments for the sugar
The UV-vis spectra of both sets of 3′-phosphate
diastereomers are dominated by the pyrene chromophore
and are indistinguishable. Both 31P and 1H NMR are

7630 J . Org. Chem., Vol. 64, No. 20, 1999
Notes
stituted benzylic carbon atom of bay region diolepoxide
adducts has been reported on the basis of the sign of the
major Cotton effect.18 However, the cyclopenta[cd]pyrene-
deoxyguanosine-3′-phosphate adducts lack the trans-
dihydrodiol feature adjacent to the amine alcohol gener-
ated in bay region diol epoxide adducts, and there is no
evidence to support application of this correlation in the
absence of the dihydrodiol structural feature. Absolute
stereochemical configurations can therefore not be as-
signed on the basis of CD spectroscopy of the adducts
alone. The CD spectra of the cis diastereomers are
virtually mirror images, indicating that the coupled
chromophores are related by reflection in a mirror plane
and that the relative orientation of the chromophores is
not sensitive to the configuration at C3. The CD spectra
of the trans diastereomers are similar to those of the cis
diastereomers.
Exp er im en ta l Section
Gen er a l In for m a tion . 1H NMR spectra were recorded at 500
MHz, and chemical shifts are reported in ppm relative to
trimethylsilane. 31P NMR spectra were recorded at 202.5 MHz
using 5% H3PO4 in D2O (A) or 0.1% H3PO4 in D2O (B) as the
external standard. Adducts were lyophilized prior to analysis.
Electrospray mass spectra (ESMS) were acquired on a Finnegan
4000 quadrupole mass spectrometer equipped with an electro-
spray ionization (ESI) source (nonpneumatically assisted elec-
trospray ionization) operating in the negative ion mode. The ESI
voltage and current were -2.5-3 kV and 0.06-0.1 µA, respec-
tively. Samples were run in the infusion mode and the flow rate
of the solution was controlled using a syringe pump (Kd
Scientific, Model 100, Fisher Scientific) at 1.5-2.0 µL/min.
Coaxial flow of SF6 was applied (2 psi) on the ESI needle to
stabilize the electrospray. Mass spectra were acquired over a
mass range of m/z 0-800 in 2 s and processed by a Technivet
Vector/2 (St. Louis, MO) data system. The exact mass measure-
ment was performed under negative ion electrospray conditions
on a Micromass Q-TOF hybrid quadrupole/time-of-flight mass
spectrometer (Altrincham, UK) using a nanoflow electrospray
interface. The capillary voltage was 2895 V and the desolvation
(cone) voltage was 30 eV. The solvent was 50:50 methanol/0.1%
formic acid in water. The instrument was calibrated with the
formic acid dimer (-H) and the 2′-deoxyguanosine-3′-monophos-
phate (M-H)- and (2M-H)- ions. Fast atom bombardment mass
spectra (FABMS) were obtained on a VG 70-250SEQ mass
spectrometer operating in the positive ion mode, with samples
suspended in a 1:1 dithioerythritol/dithiothreitol matrix. Reverse
phase HPLC was carried out using a Zorbax C18 9.6 × 250 mm
column (Dupont) eluted at a flow rate of 2.5 mL/min with a
gradient program: 40% MeOH/60% 50 mM triethylammonium
bicarbonate to 80% MeOH/20% 50 mM triethylammonium
bicarbonate over 80 min (gradient A) or 30% MeOH/70% 50 mM
triethylammonium bicarbonate to 60% MeOH/40% 50 mM
triethylammonium bicarbonate over 60 min (gradient B). The
UV absorbance of the HPLC eluate was monitored at 254 nm.
Electronic (UV-vis) spectra were scanned between 400 and 200
nm. Circular dichroism spectra were recorded at 20 °C in
methanol and normalized to 1.0 AUFS at 242 nm.
F igu r e 1. Circular dichroism spectra (methanol, 20 °C) of
early (s) and late (- - -) eluting diastereomers of cis-3-(2′-
deoxyguanosine-3′-phosphate-N2-yl)-4-hydroxy-3,4-dihydrocy-
clopenta[cd]pyrene.
and nucleobase resonances were made by comparison to
spectra of nucleoside adducts, the most significant dif-
ference being a shift of the H3′ signal by ∼0.4 ppm
downfield in the nucleotides. Mass spectra of the target
nucleotides, obtained by electrospray ionization operated
in the negative mode, are characterized by the expected
peak at m/z 588, corresponding to loss of a hydrogen from
the molecular ion (M•-). Major fragment ions are consis-
tent with the assigned structures: m/z 392, correspond-
ing to loss of deoxyribose-3-phosphate; m/z 374, corre-
sponding to loss of deoxyribose-3-phosphate and water;
and m/e 265, corresponding to loss of both 4-hydroxy-3,4-
dihydrocyclopenta[cd]pyrenyl and PO3.
The CD spectra of the adducts (Figure 1) show Davy-
dov-split Cotton effects17 arising from coupling of the in-
plane purine and pyrene transitions. However, inspection
of the CD traces suggests that exciton coupling may
involve more than two bands for both cis and trans sets
of diastereomers and identification of a first Cotton effect
is not straightforward. Moreover, the relative orientation
of the transition dipoles of the coupled bands cannot be
unambiguously established. As a result, application of
exciton chirality rules to assign configuration at the
N-bonded benzylic carbon does not appear warranted.
Tentative assignment of stereochemistry at the N-sub-
CPP, cis- and trans-3-amino-4-hydroxy-3,4-dihydro-CPP, and
2-fluoro-O6-benzyl-2′-deoxyinosine were synthesized according
to published procedures.16,19-24 Chemicals were obtained from
(18) Agarwal, S. K.; Sayer, J . M.; Herman, J . C. Y.; Pannell, L. K.;
Hilton, B. D.; Pigott, M. A., Dipple, A.; Yagi, H.; J erina, D. M. J . Am.
Chem. Soc. 1987, 109, 2497-2504.
(19) Lakshman, M. K.; Sayer, J . M.; J erina, D. M. J . Org. Chem.
1992, 57, 3438-3443.
(15) Hsu, C. H.; Skipper, P. L.; Harris, T. M.; Tannenbaum, S. R.
Chem. Res. Toxicol. 1997, 10, 248-253.
(20) Konieczny, M.; Harvey, R. G. J . Org. Chem. 1979, 44, 2158-
2160.
(16) Sangaiah, R.; Gold, A.; Ball, L. M.; Matthews, D. L.; Toney, G.
E. Tetrahedron Lett. 1992, 33, 5487-5490.
(21) Sangaiah, R.; Gold, A. J . Org. Chem. 1988, 53, 2620-2622.
(22) Robins, M. J .; Robins, R. K. J . Org. Chem. 1969, 34, 2160-
2163.
(23) McManus, S. P.; Larson, C. A.; Hearn, R. A. Synth. Commun.
1973, 3, 177-180.
(17) Gaffield, W. In Bioactive Natural Products. Detection, Isolation
and Structural Determination; Colegate, S. M., Molyneux, R. J ., Eds.;
CRC Press: Boca Raton, FL, 1993; pp 148-156.

Notes
J . Org. Chem., Vol. 64, No. 20, 1999 7631
commercial sources (Aldrich Chemical Co. or Sigma Chemical
Co.). HPLC solvents and normal phase and reverse phase
preparative TLC plates (20 × 20 cm, 1000 µm, Whatman) were
obtained from Fisher Scientific (Pittsburgh, PA). All solvents
used were anhydrous unless otherwise noted.
85° C. The brown residue from lyophilization was chromato-
graphed by reverse phase TLC (70:30:1 MeOH/H2O/Et3N). The
mixture of diastereomeric 3′-H-phosphonate adducts was col-
lected as a band at Rf ) 0.44. Separation by HPLC using
gradient program A gave diastereomers 4 (triethylammonium
salts) eluting at 79.5 (2.4 mg, 5.9%) and 80.5 (2.1 mg, 5.2%) min,
respectively. Late-eluting diastereomer (identical to early eluting
diastereomer): 1H NMR (DMSO-d6, 50 °C) δ 8.33 (d, 1H J )
7.7 Hz, H8,), 8.25 (d, 1H, J ) 7.7 Hz, H6 or H1), 8.24 (d, 1H, J
) 7.7 Hz, H1 or H6), 8.17 (d, 1H, J ) 9.3 Hz, H9 or H10), 8.15
(d, 1H, J ) 9.3 Hz, H10 or H9), 8.13 (s, 1H, H5), 8.12 (d, 1H, J
) 7.7 Hz, H2), 8.06 (t, 1H, J ) 7.7 Hz, H7), 8.08 (bs, 1H, H8dG),
7.46 (d, 2H, J ) 7.3 Hz, m-phenyl), 7.40-7.32 (m, 3H, phenyl),
6.79 (d, 1H, C3NH, J ) 8.5 Hz), 6.56 (d, 1H, J ) 579 Hz, PH),
6.28, (t, 1H, J ) 7.1 Hz, H1′), 5.84 (bt, 1H, J ) 7.1 Hz, H3), 5.75
(bs, 1H, H4), 5.57 (d, 1H, J ) 12.4 Hz, CH2-Bz), 5.52 (d, 1H, J
) 12.4 Hz, CH2-Bz), 4.86 (bs, 1H, H3′), 3.96-3.94 (m, 2H, H5′,
H5′′), H2′, H2′′ obscured by water and residual proton signal
from DMSO-d6; ESMS m/z 662 (M - N(Et3)H)-, 482 (M - H -
[phosphonylated deoxyribose]), 373 (M - H - OBz - [phospho-
nylated deoxyribose]).
5′-O-(4,4′-Dim eth oxytr ityl)-2-flu or o-O6-ben zyl-2′-d eoxy-
in osin e (1). 2-Fluoro-O6-benzyl-2′-deoxyinosine (275 mg, 0.76
mmol) was coevaporated three times with dry pyridine and then
dissolved in 5 mL of dry pyridine under an argon atmosphere
at room temperature. Dimethoxytrityl chloride (414 mg, 1.2
mmol) and triethylamine (500 µL) were added, and the resulting
solution was stirred at room temperature for 12 h, at which time
TLC analysis (10% MeOH/CH2Cl2) indicated that no more
starting material was present. Ethanol (1 mL) was added and
the solution stirred for 10 min. Water (20 mL) was added and
the mixture extracted with CH2Cl2. The combined CH2Cl2
extracts were washed with 10% K2CO3, dried, and concentrated
to produce an orange oil. Purification by column chromatography
(98:2 CH2Cl2/Et3N to 96:2:2 CH2Cl2/Et3N/MeOH) yielded 1 as a
yellow foamy solid (350 mg, 70% yield): Rf ) 0.41 by TLC in
1
96:2:2 CH2Cl2/Et3N/MeOH; H NMR (acetone-d6) δ 8.31(s, 1H,
2-F lu or o-2′-d eoxyin osin e-3′-H-p h osp h on a te (5). A solu-
tion of 2 (167 mg, 0.202 mmol, triethylammonium salt) and
methanol (20 mL) containing 50 mg of 5% Pd/C was hydroge-
nated for 60 min at room temperature. The catalyst was filtered
off and the filtrate, on concentration, gave a clear oil, which was
purified by reverse phase TLC using 70:30:1 MeOH/H2O/Et3N.
The product, eluting with the solvent front, was extracted with
methanol, treated with decolorizing carbon, and concentrated
to give the triethylammonium salt 5 as a clear oil (53 mg, 60%
yield): 1H NMR (DMSO-d6) δ 7.74 (s, 1H, H8), 6.58 (d, J ) 575
Hz, 1H, PH), 6.00 (t, J ) 7 Hz, 1H, H1′), 5.83 (m, 1H, 5′-OH),
4.65 (m, 1H, H3′), 3.88 (m, 1H, H4′), 3.45 (m, 2H, H5′and 5′′),
2.58 (m, 1H, H2′), 2.23 (m, 1H, H2′′); 31P NMR (MeOH/A) δ 1.01
(dd, J PH ) 575, J PH3′ ) 9.32 Hz, PH); ESMS m/z 333 (M -
N(Et3)H)-, 166.
H8), 7.58 (m, 2H, aromatic), 7.14-7.43 (m, 12H, aromatic), 6.78
(m, 4H, aromatic), 6.45 (t, 1H, J ) 6.5 Hz, H′1), 5.65 (s, 2H,
CH2), 4.69 (m, 1H, 3′-OH), 4.58 (m, 1H, H3′), 4.16 (m, 1H, H4′),
3.75 (s, 6H, OCH3), 3.12 (m, 2H, H5′ and 5′′), 2.94 (m, 1H, H2′),
2.50 (m, 1H, H2′′); FABMS m/z 663 (M + H)+.
5′-O-(4,4′-Dim eth oxytr ityl)-2-flu or o-O6-ben zyl-2′-d eoxy-
in osin e-3′-H-p h osp h on a te (2). To a stirred solution of 5′-O-
(4,4′-dimethoxytrityl)-2-fluoro-O6-benzyl-2′-deoxyinosine (1) (187
mg, 0.282 mmol) in dioxane (1 mL) and pyridine (0.5 mL) under
an argon atmosphere at room temperature was added salicyl-
chlorophosphine reagent (69 mg, 0.362 mmol) dissolved in
dioxane (1 mL). After 15 min, 31P NMR analysis of an aliquot of
the reaction mixture showed strong resonances at 123.82 and
125.35 ppm, confirming the presence of an intermediate contain-
ing phosphorus. Pyridine/water (1:1) (1 mL) was then added to
the reaction mixture and a further aliquot analyzed by 31P NMR.
A doublet at 3.72 ppm (J ) 725 Hz) indicated formation of the
3′-H-phosphonate. Triethylamine (500 µL) was added and the
reaction mixture concentrated under a stream of argon. The
resulting crude residue was purified by preparative reverse
phase TLC (70:30:1 MeOH/H2O/Et3N) to give 2 (triethylammo-
nium salt) as a white foamy oil (167 mg, 72% yield): Rf ) 0.35;
1H NMR (acetone-d6) δ 8.29 (s, 1H, H8), 7.57-7.18 (m, 18H,
benzyl and DMT aromatic), 7.05 (d, 1H, J ) 725 Hz, PH), 6.43
(t, 1H, J ) 6.5 Hz, H1′), 5.60 (s, 2H, CH2), 4.97 (m, 1H, H3′),
4.33 (m, 1H, H4′), 3.77 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.34
(m, 2H, H5′and 5′′, 2.98 (m, 1H, H2′), 2.67 (m, 1H, H2′′), 31P
NMR (pyridine/dioxane/A) δ 3.72 (d, J ) 725 Hz, PH); ESMS
m/z 725 (M - N(Et3)H)-, 423 (M - DMT), 243 (M - [3′-H-
phosphityl-5′-O-DMT-deoxyribose]).
2-Flu or o-O6-ben zyl-2′-deoxyin osin e-3′-H-ph osph on ate (3).
Compound 2 (75 mg, 0.91 mmol, triethylammonium salt) was
treated with 80% acetic acid (3 mL) and stirred at room
temperature for 60 min. The resulting orange solution was
concentrated under reduced pressure at 30 °C. Water (10 mL)
was added along with triethylamine until neutral pH. Excess
triethylamine was removed under a stream of argon. Lyophiliza-
tion followed by preparative reverse phase TLC using 70:30:1
MeOH/H2O/Et3N gave 3 (triethylammonium salt) as a clear oil
(42 mg, 88% yield): Rf ) 0.67; 1H NMR (DMSO-d6) δ 8.57 (s,
1H, H8), 7.55-7.35 (m, 5H, aromatic), 6.67 (d, 1H, J ) 622 Hz,
PH), 6.26 (t, 1H, J ) 6.5 Hz, H1′), 5.59 (s, 3H, CH2 and 5′-OH),
4.76 (m, 1H, H3′), 3.95 (m, 1H, H4′), 3.54 (m, 2H, H5′and 5′′),
2.69 (m, 1H, H 2′), 2.47 (m, 1H, H2′′); 31P NMR (MeOH/B) δ
3.31 (d, J ) 622 Hz, PH); ESMS m/z 423 (M - N(Et3)H)-, 243
(M - [3′-H-phosphityldeoxyribose]).
tr a n s-CP P -N2-d eoxygu a n osin e-3′-H-p h osp h on a te Dia -
ster eom er s (6b). trans-CPP-aminohydrin (25 mg, 0.096 mmol)
and
5 (25 mg, 0.058 mmol, triethylammonium salt) were
dissolved in DMSO (700 µL) and 2,6-lutidine (40 µL) under
nitrogen. The reaction flask, degassed by three cycles of freeze-
thaw under high vacuum, was stirred at 80 °C for 16-20 h under
a nitrogen atmosphere. Lyophilization of the resulting brown
solution and purification by reverse phase TLC (70:30:1 MeOH/
H2O/Et3N) afforded the diastereomeric product mixture 6b
(triethylammonium salts) as a light yellow oil (4 mg, 10% yield
based on 5): Rf ) 0.77. Definitive NMR (1H NMR, DMSO-d6)
peak assignments were hindered by an inability to resolve the
diastereomeric product mixture by reverse phase HPLC; 1H
NMR (DMSO-d6) δ 8.31 (d, 1H, H8), 8.24-8.17 (m, 2H, H1 and
H6), 8.15 (s, 2H, H9 and H10), 8.06-7.96 (m, 3H, H2, H5 and
H7), 7.90-7.80 (m, 2H, C3NH and H8dG), 6.31-5.9 (m, 2H, H1′
and H3), 5.70-5.55 (m, 1H, H4), 4.97 (m, 1H, H3′), 3.73 (m, 1H,
H4′), 3.5 (m, 2H, H5′ and H5′′); 31P NMR (MeOH/B) δ 3.79 (d, J
) 614 Hz, PH); ESMS m/z 572 (M - N(Et3)H)-, 392 (M -
deoxyribose), 374 (M - deoxyribose - H2O); UV-vis (MeOH)
λmax 342, 326, 277, 266, 242 nm.
tr a n s-CP P -N2-d eoxygu a n osin e-3′-p h osp h a te Dia ster e-
om er s (7b). trans-N2-CPP-deoxyinosine-3′-H-phosphonate (1.5
mg, 0.0022 mmol, triethylammonium salt) was lyophilized and
dissolved in pyridine (500 µL). TMSCl (200 µL) was added at
room temperature under nitrogen and the mixture stirred for 5
min. An iodine solution (200 µL) (2 mg dissolved in 500 µL
pyridine) was added and the mixture stirred for 5 min. Pyridine/
water (1:1, 100 µL) was added followed by solid sodium sulfite
until the yellow color dissipated. The resulting solution was
concentrated at room temperature under a stream of argon.
HPLC (gradient program B) showed no residual starting mate-
rial and the two diastereomeric phosphates 7b as the only
products present, indicating complete conversion. The pure
diastereomers were collected, eluting at 45.0 and 47.5 min.
Early-eluting diastereomer (identical to late-eluting diastere-
omer):1H NMR (DMSO-d6, 50 °C) δ 8.34 (d, 1H, J ) 7.5 Hz, H8),
8.28 (d, 1H, J ) 7.5 Hz, H1 or H6), 8.24 (d, 1H, J ) 7.5 Hz, H6
or H1), 8.17 (s, 2H, H9 and H10), 8.08 (s, 1H, H5), 8.06 (t, 1H,
J ) 7.5 Hz, H7), 8.02 (d, 1H, J ) 7.5 Hz, H2), 7.96 (s, 1H, H8dG),
7.47 (d, 1H, J ) 8 Hz, C3NH), 6.18 (t, 1H, J ) 7 Hz, H1′), 5.99
cis-3-(O6-Ben zyl-2′-d eoxygu a n osin e-3′-H -p h osp h on a t e-
N2-yl)-4-h ydr oxy-3,4-dih ydr ocyclopen ta[cd]pyr en e (4). Phos-
phonate 3 (28 mg, 0.053 mmol, triethylammonium salt) and
cyclopenta[cd]pyrene cis aminohydrin (20 mg, 0.077 mmol) in 1
mL of anhydrous DMSO and 40 µL of collidine were degassed
by freeze-thaw (three cycles) and stirred under N2 for 5 d at
(24) Prusiewicz, C. M.; Sangaiah, R.; Gold, A. Nucleosides Nucle-
otides. In press.

7632 J . Org. Chem., Vol. 64, No. 20, 1999
Notes
(br m, 1H, H3), 5.60 (br s, 1H, H4), 4.89 (br s, 1H, H3′), 4.00 (br
s, 1H, H4′), 3.50 (m, 2H, H5′, H5′′) (H2′, and H2′′ obscured by
DMSO and water); 31P NMR (MeOH/B) δ 0.01 (s); ESMS m/z
588 (M - H)-, 392 (M - [deoxyribose-3′-phosphate]), 374 (M -
[deoxyribose-3′-phosphate] - H2O), 265 (deoxyguanosine); UV-
vis (MeOH) λmax 342, 326, 277, 266, 242 nm.
mixture of diastereomers 7a . Separation by reverse phase HPLC
using gradient program B gave the products eluting at 36.0 and
41.7 min. Late-eluting diastereomer (identical to the early-
eluting diastereomer): 1H NMR (DMSO-d6) 8.35 (d, 1H, J ) 8
Hz, H8), 8.28 (d, 1H, J ) 8 Hz, H1), 8.26 (d, 1H, J ) 8.6 Hz,
H6), 8.18 (bs, 2H, H9, H10), 8.17 (d, 1H, J ) 8 Hz, H2), 8.13 (s,
1H, H5), 8.07 (dd, 1H, J H7H6 ) 8.6 Hz, J H7H8 ) 8 Hz, H7), 7.97
(s, 1H, H8dG), 7.08 (d, 1H, J ) 7 Hz, C3NH), 6.23 (t, 1H, J ) 6
Hz, H1′), 6.16 (m, 1H, H3), 5.88 (d, 1H, J ) 6 Hz, H4), 4.72 (bs,
1H, H3′), 3.95 (bs, 1H, H4′), 3.53 (m, 2H, H5′, H5′′), 2.72 (m,
1H, H2′), 2.06 (m, 1H, H2′′); 31P NMR (MeOH/B) δ 0.01 (s);
HRESMS 588.1281 (M-), calcd for C28H24O8N5P 588.1284; ESMS
m/z 588 (M - H)-, 392 (M - [deoxyribose-3′-phosphate]), 374
(M - [deoxyribose-3′-phosphate] - H2O), 265 (deoxyguanosine);
UV-vis (MeOH) λmax 342, 326, 277, 266, 242 nm.
cis-CP P -N2-d eoxygu a n osin e-3′-H-p h osp h on a te Dia ster -
eom er s (6a ). Coupling of cis-CPP-aminohydrin (25 mg, 0.096
mmol) and 5 (25 mg, 0.057 mmol, triethylammonium salt) was
carried out as described above for 6b, with stirring for 18 h, to
give the diastereomeric mixture 6a as a light yellow solid (6.1
mg, 16% yield) Rf ) 0.83. Diastereomeric mixture: 1H NMR
(DMSO-d6) δ 8.36 (d, 1H, H8), 8.29-8.25 (m, 2H, H1 and H6),
8.18 (s, 2H, H9 and H10), 8.16-8.06 (m, 3H, H2, H5, H7), 7.96
(s, 1H, H8dG), 7.06 (m, 1H, C3NH), 6.56 and 6.39 (2 d, 1H total,
J ) 578 Hz, PH), 6.21 (m, 2H, H1′ and H3), 5.88 (m, 1H, H4),
4.84 (br s, 1H, H3′), 3.89 (m, 1H, H4′), 3.53 (m, 2H, H5′and H5′′),
2.79 (m, 1H, H2′), 2.40 (m, 1H, H2′′); 31P NMR (MeOH/B) δ 3.79
(d, J ) 578 Hz, PH) ppm; ESMS m/z 572 (M - N(Et3)H)-, 392
(M - [deoxyribose-3′-phosphonate]), 374 (M - [deoxyribose-3′-
phosphonate] - H2O); UV-vis (MeOH) λmax 342, 326, 277, 266,
242 nm.
Ack n ow led gm en t. This work was supported in part
by USPHS grant ES06692. The authors thank R.Yen
and H. Koc for ESI/MS and G. Young for help with
acquisition of NMR spectra.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra are
available for compounds 4, 5, 6a , 6b, 7a and 7b. This material
is available free of charge via the Internet at http://pubs.acs.org.
cis-CP P -N2-d eoxygu a n osin e-3′-p h osp h a t e Dia st er eo-
m er s (7a ). Oxidation of cis-N2-CPP-deoxyinosine-3′-H-phospho-
nate (6.0 mg, 0.0089 mmol, triethylammonium salt) was carried
out as described for 7b, giving complete conversion to the
J O982473I