Synthesis of deoxyadenosine 3′-phosphates bearing cis and trans adducts of 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene: Standards for 32P-postlabeling assays

Article abstract of DOI:10.1021/jo9510898

Deoxyadenosine 3′-phosphates bearing cis and trans N⁶ adducts of (7R) and (7S)-anti 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrenes have been prepared in good yield by reaction of 6-fluoropurinyl 2′-deoxyriboside 3′-(bis(2-[4-nitrophenyl]ethyl )phosphate with the (±)-(7β,8α,9α,10β)- and (±)-7β,8α,9α,10α)-10-amino-7,8,9,10- tetrahydrobenzo[a]pyrene-7,8,9-triols. The protected phosphates are easily prepared as diasteromeric mixtures, readily resolved by reversed phase HPLC, and efficiently deprotected with DBU to give the adducted 3′-phosphates. These nucleotides are of value as standards for the ³²P-postlabeling procedure of Randerath for determination of benzo[a]pyrene adducts in DNA (Reddy et al. Carcinogenesis 1984, 5, 231).

Full text of DOI:10.1021/jo9510898

174
J . Org. Chem. 1996, 61, 174-178
Syn th esis of Deoxya d en osin e 3′-P h osp h a tes Bea r in g Cis a n d Tr a n s
Ad d u cts of
7â,8r-Dih yd r oxy-9r,10r-ep oxy-7,8,9,10-tetr a h yd r oben zo[a ]p yr en e:
Sta n d a r d s for 32P -P ostla belin g Assa ys
Shin Han, Constance M. Harris, Thomas M. Harris,* Hye-Young Hong Kim, and
Seong J . Kim
Chemistry Department and Center in Molecular Toxicology, Vanderbilt University,
Nashville, Tennessee 37235
Received J une 15, 1995X
Deoxyadenosine 3′-phosphates bearing cis and trans N6 adducts of (7R) and (7S)-anti 7â,8R-
dihydroxy-9R,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrenes have been prepared in good yield by
reaction of 6-fluoropurinyl 2′-deoxyriboside 3′-(bis(2-[4-nitrophenyl]ethyl )phosphate with the (()-
(7â,8R,9R,10â)- and (()-(7â,8R,9R,10R)-10-amino-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8,9-triols. The
protected phosphates are easily prepared as diasteromeric mixtures, readily resolved by reversed
phase HPLC, and efficiently deprotected with DBU to give the adducted 3′-phosphates. These
nucleotides are of value as standards for the 32P-postlabeling procedure of Randerath for
determination of benzo[a]pyrene adducts in DNA (Reddy et al. Carcinogenesis 1984, 5, 231).
In tr od u ction
chromatographic separations can be difficult, and the
adducts of interest may be only minor products. Alter-
natively, the adducted 3′-nucleotides can be formed by
reaction of the unadducted 3′-nucleotides with the diol
epoxides. For example, 2-5 can be prepared by the
reaction of deoxyadenosine 3′-phosphate with 7R and 7S
forms of 7â,8R-dihydroxy-9R,10R-epoxy-7,8,9,10-tetrahy-
drobenzo[a]pyrene (1a and 1b, commonly called (+)- and
(-)-anti-BPDE, respectively) (Chemical Abstracts Service
designates BPDE as a derivative of benzo[10,11]chryseno-
[3,4-b]oxirene) as shown in Scheme 1. The procedure
leads to both cis and trans opening of the epoxide, but
the separations are easier since they only involve the
adducts of a single nucleotide.3b With both methods,
hydrolysis of the diol epoxides is a major side reaction.
We report herein an efficient and straightforward method
for preparing regio- and stereospecific deoxyadenosine 3′-
phosphates bearing N6 adducts of the PAH diol epoxides
by a strategy in which the electrophile-nucleophile
relationship of PAH and nucleotide are reversed. The
examples described in this paper involve the cis and trans
opening products 2-5 of (+)- and (-)-anti-BPDE. The
corresponding adducted nucleosides have been isolated
from BPDE-treated calf thymus and plasmid DNA.4 This
indirect method is of particular value for the adenine
adducts which are formed in very low yields by reaction
of the epoxides with DNA; the N2 guanine adduct arising
by trans opening of the anti-BPDE is the predominant
product. Interestingly, there is evidence that despite the
low yields, the adenine adducts may have disproportion-
ate importance in mutagenic and carcinogenic processes.5
The 32P-postlabeling assay developed by Randerath
and co-workers1 is a versatile method for detection of
DNA adducts and is widely used for analysis of adducts
in DNA isolated from living systems.2 In this method
adducted DNA is enzymatically hydrolyzed to nucleoside
3′-phosphates which are converted to radioactive 3′,5′-
diphosphates by [32P] transfer from [γ-32P]ATP catalyzed
by T4 polynucleotide kinase. The diphosphates are
analyzed by high-resolution thin layer chromatography
and visualized by radioautography. [γ-32P]ATP is com-
mercially available in very high specific activity and as
a consequence the method has extraordinarily high
sensitivity such that it is possible to detect adducts at
the level of approximately 1 base in 1010 using no more
than 10 µg of DNA.1 The high sensitivity presents a
problem: adducts can readily be detected at lower levels
than can be assayed by other analytical methods. Con-
sequently, authentic standards of the adducted nucleo-
side 3′-phosphates are required for identification of the
individual adducts. Use of the methodology for the
analysis of polycyclic aromatic hydrocarbon adducts has
been hindered by the poor availability of such standards.
The 3′-nucleotides can be isolated from the reaction of
the PAH diol epoxides with DNA followed by enzymatic
hydrolysis and chromatographic separation of the various
products,3a but the procedure is frequently inefficient, the
X Abstract published in Advance ACS Abstracts, December 15, 1995.
(1) (a) Reddy, M. V.; Gupta, R.; Randerath, E.; Randerath, K.
Carcinogenesis 1984, 5, 231. (b) Randerath, K.; Randerath, E. Drug
Metab. Rev. 1994, 26, 67. (c) Randerath, K.; Randerath, E.; Danna, T.
F.; van Golen, L.; Putman, K. L. Carcinogenesis 1989, 10, 1231.
(2) (a) Schoket, B.; Le´vay, K.; Phillips, D. H.; Vincze, I. Cancer Lett.
1989, 48, 67. (b) Bodell, W. J .; Devanesan, P. D.; Rogan, E. G.;
Cavalieri, E. L. Chem. Res. Toxicol. 1989, 2, 312. (c) Manchester, D.
K.; Wilson, V. L.; Hsu, I. C.; Choi, J . S.; Parker, N. B.; Mann, D. L.;
Weston, A.; Harris, C. C. Carcinogenesis 1990, 11, 553. (d) Hemminki,
K.; Randerath, K.; Reddy, M. V.; Putman, K. L.; Santella, R. M.; Perera,
F. P.; Young, T. L.; Phillips, D. H.; Hewer, A.; Savela, K. Scand. J .
Work Environ. Health 1990, 16, 158. (e) Reddy, A. P.; Pruess-Schwartz,
D.; J i, C.; Gorycki, P.; Marnett, L. J . Chem. Res. Toxicol. 1992, 5, 26.
(f) Roggeband, R.; Wolterbeek, A. P. M.; Melis, O. W. M.; Wittekoek,
M. E.; Rutten, A. A. J . J . L.; Feron, V. J .; Baan, R. A. Carcinogenesis
1994, 15, 661. (g) J ahnke, G. D.; Thompson, C. L.; Walker, M. P.;
Gallagher, J . E.; Lucier, G. W.; DiAugustine, R. P. Carcinogenesis 1990,
11, 205.
(3) (a) Amin, S.; Misra, B.; Desai, D.; Huie, K ; Hecht, S. S.
Carcinogenesis 1989, 10, 1971. (b) Canella, K.; Peltonen, K.; Dipple,
A. Carcinogenesis 1991, 12, 1109.
(4) (a) Cheng, S. C.; Hilton, B. D.; Roman, J . M.; Dipple, A. Chem.
Res. Toxicol. 1989, 2, 334. (b) J effrey, A. M.; Grzeskowiak, K.;
Weinstein, I. B.; Nakanishi, K.; Roller, P.; Harvey, R. G. Science 1979,
206, 1309.
(5) (a) Vousden, K. H.; Bos, J . L.; Marshall, C. J .; Phillips, D. H.
Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 1222. (b) Dipple, A.; Pigott, M.;
Moschel, R. C.; Costantino, N. Cancer Res. 1983, 43, 4132. (c) Wei, S.
J . C.; Chang, R. L.; Wong, C.-Q.; Bhachech, N.; Cui, X. X.; Hennig, E.,
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Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11227-11230.
0022-3263/96/1961-0174$12.00/0 © 1996 American Chemical Society

Deoxyadenosine 3′-Phosphates as 32P-Postlabeling Standards
J . Org. Chem., Vol. 61, No. 1, 1996 175
Sch em e 1
Sch em e 2
relationship of the PAH and nucleoside, to be highly
efficient.6b-h The yields are high, and the stereochemical
arrangement of the PAH functional groups is determined
exclusively by the structure of the amino triol. Excess
amino triol, used to drive the reaction, can be recovered.
The initial approach we considered for preparation of the
nucleotides involved direct introduction of phosphate on
the 3′-hydroxyl group of the N6-adducted deoxyadenos-
ines. However, use of these nucleosides for synthesis of
3′-nucleotides would be somewhat cumbersome since
selective protection of the hydroxyl groups on the PAH
and the 5′-hydroxyl of deoxyribose would be required
before reaction with a phosphorylating agent.
Phosphorylation prior to introduction of the PAH
appeared more attractive. In addition, there would be
considerable advantage to using a protected form of the
phosphate to promote solubility in organic solvents since
the solubility of PAH amino triols in water is very low
and the fluoropurine is subject to hydrolysis in aqueous
solvents. A protective group for phosphate was needed
which was relatively stable to the conditions required for
the preparation and reaction of the 6-fluoro-3′-nucleotide
but could be removed under conditions which did not
perturb the PAH or deoxyribose moieties.
With these requirements in mind, we examined several
possibilities, finally settling on 2-(4-nitrophenyl)ethyl
(NPE) as the protective group for phosphate. NPE esters
are relatively stable but can be removed by â-elimination
using DBU. An uncertainty concerning its use was
whether the group would be stable to the relatively harsh
conditions employed for the condensation of PAH amino
triols with 6-fluoropurine derivatives. However, even
before this potential problem could be addressed, dif-
ficulties arose in the use of bis-NPE phosphorochloridate
to phosphorylate the 3′-hydroxyl group. The phospho-
rochloridate is very sensitive to moisture, and reproduc-
ible yields were difficult to obtain in the phosphorylation
reaction.7
To circumvent the moisture sensitivity problem, the
trivalent bis(NPE-O)(diisopropylamino)phosphine (10)
was used instead. Phosphoramidite 10 is readily pre-
pared from PCl3 via dichloro(diisopropylamino)phosphine
(9).8-10 Phosphitylation of the 5′-(dimethoxytrityl)-
protected 6-chloropurine nucleoside11 11 in the presence
of tetrazole followed by in situ oxidation of the resulting
Resu lts a n d Discu ssion
We have recently developed an attractive procedure for
preparation of the adducted nucleosides 8 involving
reaction of 6-fluoropurine 2′-deoxyribonucleoside (6) with
amino triols 7 derived from ammonolysis of PAH diol
epoxides (Scheme 2).6a Others have also found this
strategy, which reverses the electrophile-nucleophile
(6) (a) Kim, S. J .; Harris, C. M.; J ung, K. Y.; Koreeda, M.; Harris,
T. M. Tetrahedron Lett. 1992, 32, 6073. (b) Lee, H.; Hinz, M.; Stezowski,
J . J .; Harvey, R. G. Tetrahedron Lett. 1990, 31, 6773. (c) Lakshman,
M. K.; Sayer, J . M.; J erina, D. M. J . Am. Chem. Soc. 1991, 113, 6589.
(d) Lakshman, M. K.; Sayer, J . M.; Yagi, H.; J erina, D. M. J . Org.
Chem. 1992, 57, 4585. (e) Lakshman, M. K.; Sayer, J . M.; J erina, D.
M. J . Org. Chem. 1992, 57, 3438. (f) Lakshman, M.; Lehr, R. E.
Tetrahedron Lett. 1990, 31, 1547. (g) Steinbrecher, T.; Becker, A.;
Stezowski, J . J .; Oesch, F.; Seidel, A. Tetrahedron Lett. 1993, 34, 1773.
(h) Sangaiah, R.; Gold, A.; Ball, L. M.; Matthews, D. L.; Toney, G. E.
Tetrahedron Lett. 1992, 33, 5487.
(7) Himmelsbach, F.; Charubala, R.; Pfleiderer, W. Helv. Chim.
Acta 1987, 70, 1286.
(8) Tanaka, T.; Tanatsukuri, T.; Ikehara, M. Tetrahedron Lett. 1986,
27, 199.
(9) Uhlmann, J . E. Tetrahedron Lett. 1986, 27, 1023.
(10) Bannwarth, W.; Trazeciak. A. Helv. Chim. Acta 1987, 70, 175.
(11) Harris, C. M.; Zhou, L.; Strand, E. A.; Harris, T. M. J . Am.
Chem. Soc. 1991, 113, 4328.

176 J . Org. Chem., Vol. 61, No. 1, 1996
Han et al.
Sch em e 3
characterization. The PAH adducted 3′-nucleotides have
only limited solubility in aqueous buffers and cling to
surfaces on account of the hydrophobic PAH moieties, so
care has to be taken in handling them to avoid losses.
We initially investigated the 2,2,2-trichloroethyl group
for protection of the phosphate; the bis-ester can be
prepared from the commercially available bis(2,2,2-
trichloroethyl) phosphorochloridate.15 The 5′-(dimethoxy-
trityl) derivative 11 of 6-chloropurine 2′-deoxyriboside11
was acylated with the phosphorochloridate to give the
protected nucleotide; the dimethoxytrityl group was
removed by treatment with acetic acid, the chloro sub-
stituent replaced by fluoro by treatment with trimethy-
lamine followed by anhydrous KF.12 The bis(2,2,2-
trichloroethyl)phosphate group was fully resistant to the
conditions used to prepare the 6-fluoro synthon. Reaction
with racemic cis-amino triol 15 to give diastereomeric
adducts analogous to 17/18 went efficiently but difficul-
ties arose with the deprotection step. In literature
examples, bis(2,2,2-trichloroethyl) groups have been re-
moved from phosphates by reductive cleavage using Zn-
Cu couple or tributylphosphine.16-18 The procedure of
Imai18 using a highly activated Zn-Cu couple was
successful in model deprotection reactions with bis(2,2,2-
trichloroethyl)-protected 3′-phosphate of thymidine and
bis(2,2,2-trichloroethyl)-protected 3′-phosphate of N6-
(styrene oxide)-adducted deoxyadenosine. Unfortunately,
the reported deprotection methods were unsuccessful
with the bis(2,2,2-trichloroethyl)-protected 3′-phosphate
of benzo[a]pyrene-adducted deoxyadenosine. One trichlo-
roethyl group could be removed, but the second was
resistant to reductive elimination.
6-Fluoropurine 3′-nucleotide 14 represents a valuable
synthon for preparation of the N6-PAH-adducted deoxy-
adenosine 3′-nucleotides since the adduction reaction
should proceed equally well with amino alcohols derived
from the epoxides and diol epoxides of many other PAHs.
Indeed, with sterically unencumbered amines, the chlo-
ropurine nucleotide would be equally satisfactory. The
6-chloronucleoside is significantly easier to prepare since
moisture must be scrupulously excluded during the KF
reaction used to prepare 14.
The reactions described herein have been carried out
with racemic amino triols. The use of racemic diol
epoxides is much more cost-effective than the individual
enantiomers, only a few of which are commercially
available, and even those are often in short supply. In
the cases presented here, separation of the resulting
diastereomers has been relatively straightforward during
the HPLC purification of the adducts.
phosphite ester with aqueous iodine afforded phospho-
triester 12 in 86% yield (Scheme 3). After detritylation
with aqueous AcOH (86% yield), 6-chloronucleotide 13
was converted to the 6-fluoro analog 14 in 85% yield by
treatment with trimethylamine in glyme followed by KF
in DMF.12
Condensation of 14 with cis and trans amino triols (15
and 16) derived from racemic anti-BPDE6a,c,13 afforded the
corresponding diastereomeric mixtures of N6-adducted
deoxyadenosine 3′-phosphotriesters 17/18 and 19/20,
respectively, in yields of ∼50% (Scheme 4). The diaster-
eomeric mixtures were separated by preparative reversed-
phase HPLC. The NPE protecting groups were removed
with DBU in pyridine to give adducts 2-5 in yields of
∼78%. Final purification of the deprotected 3′-phos-
phates was accomplished by reversed-phase HPLC. A
cursory examination was made of whether it would be
preferable to separate the diastereomeric mixtures after
deprotection, but separations appeared to be easier with
the NPE-protected nucleotides.
Exp er im en ta l Section
Gen er a l. (()-7â,8R-Dihydroxy-9R,10R-epoxy-7,8,9,10-tet-
rahydrobenzo[a]pyrene used for synthesis of racemic benzo-
[a]pyrenetriol amines (15 and 16) was purchased from Mid-
west Research Institute or Chemsyn. 1H NMR spectra were
obtained at 300 and 400 MHz. Phosphorus spectra were
standardized to external 85% H3PO4 (δ ) 0). Fluorine spectra
were standardized to CFCl3 (δ ) 0). Low and high resolution
mass spectra were obtained in either negative or positive FAB
mode; matrices are indicated with individual compounds.
The adducted nucleotides were characterized by FAB
mass spectrometry and 1H and 31P NMR. Stereochemical
assignments were made by comparison of circular dichro-
ism spectra with published spectra.4,14 For both the trans
and cis adducts the isomers with the 10S configuration
(2 and 5) eluted first on HPLC. To our knowledge, PAH-
adducted 3′-nucleotides have never been prepared previ-
ously on sufficient scale to permit extensive spectroscopic
(15) (a) Bannwarth, W.; Kung, E. Tetrahedron Lett. 1989, 30, 4219.
(b) Adamiak, R. W.; Biata, E. K.; Grzeskowiak, K.; Kierzek, R.;
Kraszewski, A.; Markiewicz, W. T.; Stawinski, J .; Wiewiorowski, M.
Nucleic Acids Res. 1977, 4, 2321.
(16) Letsinger, R. L.; Finnan, J . L. J . Am. Chem. Soc. 1975, 97, 7197.
(17) Letsinger, R. L.; Groody, N. L. Tetrahedron 1983, 40, 137.
(18) Imai, J .; Torrence, P. F. J . Org. Chem. 1981, 46, 4015.
(12) Robins, M. J .; Basom, G. L. Nucleic Acid Chem. 1978, 2, 601.
Robins, M. J .; Basom, G. L. Can. J . Chem. 1973, 51, 3161.
(13) J hingan, A. K.; Meehan, T. J . Chem. Res. (S) 1991, 122.
(14) Cheh, A. M.; Chadha, A.; Sayer, J . M.; Yeh, H. J . C.; Yagi, H.;
Pannell, L. K.; J erina, D. M. J . Org. Chem. 1993, 58, 4013.

Deoxyadenosine 3′-Phosphates as 32P-Postlabeling Standards
Sch em e 4
J . Org. Chem., Vol. 61, No. 1, 1996 177
Reactions were monitored by TLC on silica gel plates (EM
Science, Kieselgel 60, F254). TLC plates were visualized by
UV or anisaldehyde stain.19 Column chromatography was
conducted on silica gel 60 (70-230 mesh) from EM Science.
HPLC analyses and isolations of nucleosides were carried out
on a gradient HPLC system equipped with a diode array
detector. Analytical runs were performed on a C-18 reverse-
phase column (4.6 × 250 mm) at a flow rate of 1.0 mL/min;
preparative scale isolations were carried out on a 10 × 250
mm C-18 column at a flow rate of 2.0 mL/min.
was filtered to remove amine hydrochloride. The filtrate was
diluted with 200 mL of acid-free ethyl acetate, and the organic
layer was extracted three times with 30 mL of phosphate
buffer, pH 7, dried over sodium sulfate, and concentrated in
vacuo. The oily product was purified by short column chro-
matography in the presence of Et3N in 94% yield. 1H NMR
(CDCl3) δ 8.10-8.15 (d, 4H), 7.33-7.36 (d, 4H), 3.72-3.84 (m,
4H), 3.46-3.56 (m, 1H), 2.95-2.98 (t, 4H), 1.07 (s, 6H), 1.05
(s, 6H). 31P NMR (CDCl3) δ 147; lit.9 (no solvent given) δ 149.
6-Ch lor op u r in yl-5′-O-(d im eth oxytr ityl)-2′-d eoxyr ibo-
sid e 3′-(Bis(2-[4-n itr op h en yl]eth yl) p h osp h a te) (12). To
0.5 g (0.87 mmol) of 5′-O-(dimethoxytrityl)-6-chloro-2′-deox-
yriboside 1111 and 0.57 g (1.32 mmol) of 5 was added 3.0 mL
(0.5 M) of 1H-tetrazole in CH3CN at room temperature. After
addition of 20 mL of CH3CN and 10 mL of CH2Cl2 at room
temperature, the mixture was stirred for 1 h at which time
TLC showed complete disappearance of 11 (silica gel, EtOAc/
CH2Cl2 70:30, Rf 0.68). For oxidation, a solution of 16 mL of
iodine (0.5 M in pyridine/water/acetonitrile 3:1:1) was added
to the mixture over 5 min at room temperature. TLC (silica
gel, EtOAc/CH2Cl2 70:30, Rf 0.22) after 2 h showed complete
conversion to compound 12. The pure product was obtained
by short column chromatography in 86% yield. 1H NMR
(CDCl3) δ 8.61 (s, 1H), 8.2 (s, 1H), 6.74-8.14 (m, 13H), 6.37-
6.41 (dd, 1H), 5.12-5.16 (t, 1H), 4.30 (br, 1H), 4.15-4.26 (m,
4H), 3.76 (s, 6H), 2.96-3.05 (m, 4H), 2.92 and 2.68 (m, 2H).
31P NMR δ -1.5. LRMS (FAB-, Et3N/DMSO) 950 (M•-);
(Diisop r op yla m in o)d ich lor op h osp h in e (9).8 To a solu-
tion of phosphorus trichloride (1.83 mL, 21 mmol) in anhydrous
ether (20 mL) was added a solution of diisopropylamine (5.8
mL, 42 mmol) in anhydrous ether (10 mL) with stirring at -10
°C for 2.5 h followed by an additional 1 h at room temperature.
After the precipitated salt was filtered off, the reaction mixture
was distilled at ambient temperature and 0.5 mmHg to give 9
in 76% yield. 31P NMR (CDCl3) δ 177; lit.8 31P NMR (CDCl3)
δ 164.3.
Bis(2-[4-n itr op h en yl]eth yl) N,N-Diisop r op ylp h osp h or -
a m id ite (10).9 A solution of 2-(4-nitrophenyl)ethanol (3.34
g, 0.02 mol) and diisopropylethylamine (3.87 g, 0.03 mol) in
anhydrous THF (10 mL) was cooled to 0 °C. To this solution
was added 1.52 g (0.01 mol) of (diisopropylamino)dichloro-
phosphine (9) with vigorous stirring over 15 min. After
stirring for a further 30 min at room temperature, the solution
HRMS (FAB-, Et3N/DMSO) 950.2456; calcd for C47H44
ClN6O12P 950.2443 (M•-).
-
(19) Thin Layer Chromatography; Stahl, E., Ed.; Springer-Verlag:
New York, 1969; p 587.

178 J . Org. Chem., Vol. 61, No. 1, 1996
Han et al.
6-Ch lor op u r in yl-2′-Deoxyr ib osid e 3′-(Bis(2-[4-n it r o-
p h en yl]eth yl) p h osp h a te) (13). Compound 12 was stirred
with aqueous 85% AcOH for 3 h at room temperature. After
neutralization with aqueous Na2CO3, the mixture was ex-
tracted with CHCl3 (50 mL × 3), followed by rotary evapora-
tion. Column chromatography afforded 13 as a yellow oil in
86% yield: Rf 0.21 (8% MeOH/CH2Cl2). 1H NMR (CDCl3) δ
8.73 (s, 1H), 8.17 (s, 1H), 8.15 (d, 4H), 7.38 (d, 4H), 6.23-6.28
(dd, 1H), 5.04 (t, 1H), 4.98 (d, 1H), 4.21-4.29 (m, 4H), 3.83
and 3.65 (dd, 2H), 3.08 (t, 4H), 3.0 and 2.5 (m, 2H). 31P NMR
(CDCl3) δ -5.3. LRMS (FAB-, Et3N/DMSO) 648.1 (M•-);
(7S)- a n d (7R)-(7â,8r,9r,10r)-2′-Deoxy-N6-(7,8,9,10-tet-
r a h yd r o-7,8,9-t r ih yd r oxyb e n zo[a ]p yr e n -10-yl)a d e n o-
sin e 3′-P h osp h a te (2 a n d 3, r esp ectively). Compound 17
(1 mg) was treated with 0.4 mL of 0.5 M DBU in pyridine for
24 h at room temperature; TLC analysis showed complete
disappearance of the protecting groups. The mixture was
neutralized by addition of 0.1 mL of 1 M AcOH in anhydrous
pyridine and evaporated. The residue was purified by HPLC
on a preparative reversed-phase C-18 HPLC column eluted
at 3.0 mL/min with a gradient of 15-35% B over 20 min
(solvent A, 0.05 M triethylammonium bicarbonate buffer, pH
7.45, and solvent B, CH3CN). The elution profile exhibited
one major peak eluting at 18.2 min, and this component was
collected (75% yield). The fraction was evaporated and co-
evaporated 4-5 times with H2O to remove the buffer. Lyo-
philization gave 2, which was assigned the 10S configuration
on the basis of the positive CD signal at 282 nm.4,14 NMR
(DMSO-d6 + D2O) δ 8.80 (d, 1H), 8.53 (s, 1H), 8.30 (s, 1H),
8.00-8.25 (m, 7H), 6.67 (s, 1H), 6.30 (t, 1H), 5.10 (d, 1H), 4.76
(br, 1H), 4.55 (d, 1H), 4.20 (br, 1H), 4.04 (dd, 1H), 3.42-3.57
(m, 2H), 2.49-2.70 (m, 2H). 31P NMR (DMSO-d6 +D2O) δ 0.1.
LRMS (FAB-, glycerol/DMSO) 632.3 (M - H+). HRMS (FAB-,
3-nitrobenzyl alcohol/DMSO/PEG) 632.1546 (M - H+); calcd
for C30H27N5O9P 632.1546 (M - H+). The adduct 3 was
prepared from 18 by the same procedure. The configuration
at C10 was assigned as R on the basis of the negative CD peak
at 282 nm. 1H NMR (DMSO-d6 + D2O) δ 8.80 (d, 1H), 8.53 (s,
1H), 8.30 (s, 1H), 8.00-8.25 (m, 7H), 6.67 (s, 1H), 6.30 (t, 1H),
5.10 (d, 1H), 4.76 (br, 1H), 4.55 (d, 1H), 4.20 (br, 1H), 4.04
(dd, 1H), 3.42-3.57 (m, 2H), 2.49-2.70 (m, 2H). 31P NMR
(DMSO-d6 + D2O) δ -0.2. LRMS (FAB-, glycerol/DMSO)
632.7 (M - H+); HRMS (FAB-, 3-nitrobenzyl alcohol/DMSO/
PEG) found 632.1546 (M - H+); calcd for C30H27N5O9P
632.1546 (M - H+).
(FAB+, glycerol/DMSO) 649.2 (M + H+); calcd for C26H26
-
ClN6O10P 648.
6-F lu or op u r in yl-2′-Deoxyr ib osid e 3′-(Bis(2-[4-n it r o-
p h en yl]eth yl) p h osp h a te) (14). To a solution of 127 mg (0.2
mmol) of 6-chloropurinyl 2′-deoxyriboside 3′-(bis(2-[4-nitro-
phenyl]ethyl)phosphate) (13) in glyme (5.0 mL) was added 0.5
mL of liquid Me3N.12 The reaction mixture was stirred for 40
min at room temperature, evaporated, and dried to give a
yellow foam. The foam was dissolved in dry DMF, and 116
mg (2 mmol) of anhydrous KF was added at room temperature.
The mixture was stirred for 30 min at room temperature under
a nitrogen atmosphere, followed by rotary evaporation. The
residue was purified by column chromatography to afford 14
as a yellow solid in 85% yield: Rf 0.28 (MeOH/CH2Cl2, 10:90).
1H NMR (CDCl3) δ 8.63 (s, 1H), 8.18 (s, 1H), 8.17 (d, 4H), 7.38
(d, 4H), 6.25-6.30 (dd, 1H), 5.05 (t, 1H), 5.01 (br, 1H), 4.21-
4.29 (m, 4H) 3.88 and 3.67 (m, 2H), 3.1 (t, 4H), 3.0 and 2.5 (m,
2H). 31P NMR (CDCl3) δ -1.5. 19F (CDCl3) δ -66.8. LRMS
(FAB-, Et3N/DMSO) 632.2 (M•-); HRMS (FAB-, Et3N/DMSO)
632.1441 (M•-); calcd for C26H26FN6O10P 632.1432 (M•-).
(7S)- a n d (7R)-(7â,8r,9r,10r)-2′-Deoxy-N6-(7,8,9,10-tet-
r a h yd r o-7,8,9-t r ih yd r oxyb e n zo[a ]p yr e n -10-yl)a d e n o-
sin e 3′-(Bis(2-[4-n itr op h en yl]eth yl) p h osp h a te) (17 a n d
18, r esp ectively). (()-Amino triol 15 was prepared from anti-
BPDE via an azide intermediate.13 A solution of 15 (6 mg,
0.02 mmol) and excess nucleotide 14 (17.8 mg, 0.03 mmol) in
anhydrous dimethylacetamide (0.3 mL) containing distilled
triisobutylamine (0.01 mL, 0.04 mmol) was stirred at 60 °C
for 5 days under nitrogen. The reaction mixture was evapo-
rated to give a brown residue which was purified by column
chromatography on silica gel (MeOH/CH2Cl2, 3:97) to give 17/
18 in 55% yield: Rf 0.58 (MeOH/CHCl3, 20:80). 1H NMR
(DMSO-d6 + D2O) δ 8.81 (s, 1H), 7.99-8.53 (m, 13H), 7.53 (d,
4H), 6.67 (s, 1H), 6.30 (t, 1H), 5.11 (d, 1H), 5.00 (br, 1H), 4.56
(d, 1H), 4.23 (m, 5H), 4.02 (s, 1H), 3.56 and 3.52 (m, 2H), 3.05
(t, 4H), 2.98 and 2.43 (m, 2H). 31P NMR (DMSO-d6) δ -0.1.
LRMS (FAB+, glycerol/DMSO/TFA) 932.3 (M + H+); HRMS
(FAB-, Et3N/DMSO) calcd for C46H42N7O13P 931.2578 (M•-);
found 931.2580 (M•-). The diastereomeric mixture was re-
solved by preparative reversed phase HPLC (C-18 column)
with isocratic elution (H2O-acetonitrile, 48:52). The early and
late eluting adducts had retention times of 39.0 min (17) and
43.4 min (18).
(7R)- a n d (7S)-(7â,8r,9r,10â)-2′-Deoxy-N6-(7,8,9,10-tet-
r a h yd r o-7,8,9-t r ih yd r oxyb e n zo[a ]p yr e n -10-yl)a d e n o-
sin e 3′-P h osp h a te (4 a n d 5). Similarly, treatment of 19 with
DBU gave 5. The compound was purified by HPLC on a
preparative reversed-phase C-18 HPLC column eluted at 3.0
mL/min with a gradient of 15-35% over 20 min (solvent A,
0.05 M triethylammonium bicarbonate buffer, pH 7.45, and
solvent B, CH3CN). The elution profile showed one major peak
eluting at 17.8 min (75% yield). The collected fraction was
evaporated and coevaporated 4-5 times with H2O to remove
the buffer. Compound 5 exhibited a positive CD signal at 286
nm and was assigned the S configuration at C10 by comparison
with literature spectra.4,14 1H NMR (DMSO-d6 + D2O) δ 8.50
(s, 1H), 7.99-8.25 (m, 9H), 6.41 (s, 1H), 6.31 (t, 1H), 4.97 (d,
1H), 4.77 (br, 1H), 4.21-4.25 (m, 2H), 4.02 (dd, 1H), 3.43-
3.55 (m, 2H), 2.38-2.64 (m, 2H). 31P NMR (DMSO-d6 + D2O)
δ 0.1. LRMS (FAB-, Et3N/DMSO) 632.3 (M - H+). HRMS
(FAB-, Et3N/DMSO) found 632.1556 (M - H+); calcd for
C30H27N5O9P 632.1546 (M - H+). Compound 4 was prepared
from 20 by the same procedure. It showed a negative CD peak
at 286 nm and was assigned the 10R configuration. 1H NMR
(DMSO-d6 + D2O) δ 8.50 (s, 1H), 7.99-8.25 (m, 9H), 6.41 (s,
1H), 6.31 (t, 1H), 4.97 (d, 1H), 4.77 (br, 1H), 4.21-4.25 (m,
2H), 4.02 (dd, 1H), 3.43-3.55 (m, 2H), 2.38-2.64 (m, 2H). 31P
NMR (DMSO-d6 + D2O) δ 0.1. LRMS (FAB-, glycerol/DMSO)
632.0 (M - H+); HRMS (FAB-, 3-nitrobenzyl alcohol/PEG)
632.1550 (M - H+); calcd for C30H27N5O9P 632.1546 (M - H+).
(7R)- a n d (7S)-(7â,8r,9r,10â-2′-Deoxy-N6-(7,8,9,10-tet-
r a h yd r o-7,8,9-t r ih yd r oxyb e n zo[a ]p yr e n -10-yl)a d e n o-
sin e 3′-(Bis(2-[4-n itr op h en yl]eth yl)p h osp h a te) (19 a n d
20, r esp ectively). (()-Amino triol 16 was prepared from (()-
anti-BPDE by ammonolysis.6a,c Amino triol 16 (12 mg, 0.04
mmol) and excess nucleotide 14 (35.6 mg, 0.06 mmol) in
anhydrous dimethylacetamide (0.6 mL) containing distilled
triisobutylamine (0.02 mL, 0.08 mmol) was stirred at 55 °C
for 4-5 days under nitrogen. The reaction mixture was
evaporated to give a brown residue. Column chromatography
(silica gel, CH3OH/CH2Cl2, 3:97) afforded 19 and 20 in 46%
Ack n ow led gm en t. We wish to acknowledge the
generous financial support from the National Institute
for Environmental Health Sciences (ES-05355 and ES-
00267). We gratefully acknowledge the help of Brian
Nobes and Dr. Ian Blair in obtaining mass spectra.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 2-5, 9, 10, 12-14, 17/18, and 19/20 and 31P NMR
spectra of 2-5, 10, 12-14, 17/18, and 19/20 (18 pages). This
material is contained in libraries on microfiche, immediately
follows this article in the microfilm version of the journal, and
can be ordered from the ACS; see any current masthead page
for ordering information.
yield: Rf 0.58 (MeOH/CHCl3, 20:80). 1H NMR (DMSO-d6
+
D2O) δ 8.52 (s, 1H), 7.99-8.25 (m, 13H), 7.54 (d, 4H), 6.42 (s,
1H), 6.30 (t, 1H), 4.99 (d, 1H), 4.88 (br, 1H), 4.89 (br, 1H), 4.24
(m, 6H), 4.02 (s, 1H), 3.54 and 3.40 (m, 2H), 3.06 (t, 4H), 2.87
and 2.42 (m, 2H). 31P NMR (DMSO-d6) δ -1.4. LRMS (FAB+,
glycerol/DMSO) 932 (M + H+); HRMS (FAB-, Et3N/DMSO)
931.2593 (M•-); calcd for C46H42N7O13P 931.2578 (M•-). The
diastereomeric mixture was resolved by preparative reversed
phase HPLC (C-18 column) with isocratic elution (H2O/
acetonitrile, 48:52). The early and late eluting adducts had
retention times of 39.6 min (19) and 42.5 min (20).
J O9510898