Recognition and inhibition of HIV integrase by novel dinucleotides

Article abstract of DOI:10.1021/ja992528d

HIV integrase is involved in the integration of viral DNA into chromosomal DNA, a biological process that occurs by a sequence involving HIV DNA splicing and subsequent integration steps. In the quest for small nucleotide systems with nuclease stability of the internucleotide phosphate bond and critical structural features for recognition and inhibition of HIV-1 integrase, we have discovered novel, nuclease-resistant dinucleotides with defined base sequences that are inhibitors of this key viral enzyme. Synthetic methodologies utilized for the syntheses of the novel dinucleotides include an excellent new phosphorylating agent.

Full text of DOI:10.1021/ja992528d

VOLUME 122, NUMBER 24
J UNE 21, 2000
© Copyright 2000 by the
American Chemical Society
Recognition and Inhibition of HIV Integrase by Novel Dinucleotides
Michael Taktakishvili,† Nouri Neamati,‡ Yves Pommier,‡ Suresh Pal,† and Vasu Nair*,†
Contribution from the Department of Chemistry, The UniVersity of Iowa, Iowa City, Iowa 52242, and
Laboratory of Pharmacology, National Cancer Institute, NIH, Bethesda, Maryland 20892
ReceiVed July 19, 1999
Abstract: HIV integrase is involved in the integration of viral DNA into chromosomal DNA, a biological
process that occurs by a sequence involving HIV DNA splicing and subsequent integration steps. In the quest
for small nucleotide systems with nuclease stability of the internucleotide phosphate bond and critical structural
features for recognition and inhibition of HIV-1 integrase, we have discovered novel, nuclease-resistant
dinucleotides with defined base sequences that are inhibitors of this key viral enzyme. Synthetic methodologies
utilized for the syntheses of the novel dinucleotides include an excellent new phosphorylating agent.
Introduction
specific sequences (5′-ACTG...CAGT-3′) in the long terminal
repeats (LTRs) of viral DNA. In the first step, which involves
a pre-integration process and is referred to as 3′-processing (or
cleavage step), specific endonuclease activity removes two
nucleotides from each end of the double helical viral DNA
(synthesized previously by reverse transcription) producing new
3′-hydroxyl termini at the conserved CA dinucleotide. This
truncated viral DNA is coupled in the next steps to host cell
DNA in the nucleus (integration) which includes the DNA strand
transfer reaction in which IN catalyzes covalent bond formation
between the processed 3′-ends of retroviral DNA and the 5′-
phosphate end of an integrase-cleaved chromosomal DNA. Both
the 3′-processing and strand transfer steps involve transesteri-
fications and can be assayed using recombinant HIV-1 IN and
a model double-helical DNA (21-mer) corresponding to the U5
region of HIV-1 LTR.9 The crystal structure (2.5 Å resolution)
of HIV-1 IN has been reported.7,10
The pol gene of HIV-1 encodes three viral enzymes, reverse
transcriptase (RT), protease, and integrase (IN), that are essential
for viral replication. Two of these enzymes, HIV RT and HIV
protease, have received considerable attention with respect to
the development of inhibitors.1-4 The third enzyme, HIV IN,
which is encoded in the 3′-end of the pol gene of the virus, has
received much less attention. HIV-1 IN is a relatively small
protein (32 000 Da). It is involved in the integration of viral
DNA into host cell DNA, a biological process that occurs by a
sequence involving DNA cleavage (3′-processing) and coupling
(integration) reactions.5-8 The enzyme apparently recognizes
* To whom correspondence should be addressed. Telephone: (319) 335-
1364. Fax: (319) 353-2621. E-mail: vasu-nair@uiowa.edu.
† The University of Iowa.
‡ National Cancer Institute, NIH.
(1) De Clercq, E. J. Med. Chem. 1995, 38, 2491-2417.
(2) De Clercq, E. Clin. Microbiol. ReV. 1997, 10, 674-693.
(3) Moyle, G.; Gazzard, B. Drugs 1996, 51, 701-712.
(4) Molla, A.; Granneman, G. R.; Sun, E.; Kempf, D. J. AntiViral Res.
1998, 39, 1-23.
(5) Drake, R.; Neamati, N.; Hong, H.; Pilon, A. A.; Sunthankar, P.; Hume,
S. D.; Milne, G. W. A.; Pommier, Y. Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 4170-4175.
(6) Katz, R. A.; Skalka, A. M. Annu. ReV. Biochem. 1994, 63, 133-
173.
Although studies on the search for clinically useful anti-
integrase agents are relatively recent, the availability of the
methodology of screening of inhibitors utilizing purified
(8) Vaishnav, Y. N.; Wong-Staal, F. Annu. ReV. Biochem. 1991, 60, 577-
630.
(9) Vink, C.; van Gent, D. C.; Elgersma, Y.; Plasterk, R. H. A. J. Virol.
1991, 65, 4636-4644.
(10) Goldgur, Y.; Dyda, F.; Hickman, A. B.; Jenkins, T. M.; Craigie,
R.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 9150-9154.
(7) Dyda, F.; Hickman, A. B.; Jenkins, T. M.; Engelman, A.; Craigie,
R.; Davies, D. R. Science 1994, 266, 1981-1986.
10.1021/ja992528d CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/03/2000

5672 J. Am. Chem. Soc., Vol. 122, No. 24, 2000
TaktakishVili et al.
Scheme 1. Summary of the Key Steps in the Synthesis of Dinucleotides 6 and 7
recombinant integrase has contributed to the identification of
some interesting lead compounds which include small and large
nucleotides.11-15 In the quest for a small nucleotide system with
resistance to nuclease of the internucleotide phosphate bond and
critical structural features for recognition and inhibition of HIV
integrase, we designed dinucleotides with features that exploited
the ability of integrase to recognize the terminal sequence of
the truncated viral DNA during DNA cleavage and prior to
strand transfer. The molecular design led to the discovery of
conceptually novel, nuclease-resistant, non-natural dinucleotides
with defined base sequences that are inhibitors of this key viral
enzyme. This paper describes our discoveries in this area
involving both chemistry and enzymology.
molecules. The solution phase approach was preferred because
this allowed the synthesis of these compounds in sufficient
quantities (>50 mg) to allow for complete structural studies as
well as reproducible enzyme assays. Thus, isodeoxynucleoside
2 was phosphorylated with the bifunctional phosphorylating
agent, 2-chlorophenylphosphoro-bis-triazolide22,23 to give 3 in
58% yield after purification (Scheme 1).
The internucleotide coupling was carried out with triisopro-
pylbenzenesulfonyl tetrazolide (TPS-TAZ)24 to give the pro-
tected dinucleotide 4 in 66% yield. The formation of 4 was
confirmed by its NMR and HRMS data. Deprotection with 2%
dichloroacetic acid gave 5 (56% yield), and this key intermediate
1
was fully characterized by H and 31P NMR and HRMS data.
Further deprotection of 5 with NH4OH gave target molecule 7
(66%), after purification by reversed-phase HPLC (C18, ethanol/
water). The product was initially identified by comparison of
HPLC ion-exchange retention times with that of natural di-
nucleotides (Partisil-10 SAX ion-exchange column, phosphate
buffer system, retention time ) 70 min). The complete structure
was established by multinuclear NMR data (1H, 13C, 31P NMR
spectra and COSY, HMQC, and HMBC data), quantitative UV
spectra, CD spectra, and ESI HRMS data [calculated for 7:
539.1404 (M - H)-, found: 539.1402]. The quantitative UV
spectral data (λmax 263 nm, ꢀ 19 400) gave evidence of
hypochromicity in these molecules. This and the CD data
(Figure 1) suggested the existence of base stacking interactions.
For stacking interactions to occur, the carbohydrate moieties
must assume an orthogonal relationship, which results in an
unusual internucleotide phosphate linkage (Figure 2). The
Results and Discussion
Synthesis. The synthesis of two of the target molecules, 6
and 7, are summarized in Scheme 1. These and other molecules
of this paper were designed with a natural D-deoxynucleoside
(3′-OH terminus) bonded through a 5′f3′ internucleotide
phosphate linkage to an isomeric L-related deoxynucleoside.16-18
Dinucleotide 6 and its precursor 7 were synthesized in several
steps from protected (S,S)-isodeoxyadenosine, a structural
analogue of natural 2′-deoxyadenosine. This precursor, although
previously synthesized by us,19 was prepared for these studies
by a more convenient route using the cyclic sulfite 1. Synthesis
of the dinucleotides utilized the solution-phase phosphotriester
approach.20,21 The solid-phase synthesis using a DNA synthe-
sizer was useful only for sub-milligram quantities of target
(11) Mazumder, A.; Neamati, N.; Sommadossi, J.-P.; Gosselin, G.;
Schinazi, R. F.; Imbach, J.-L.; Pommier, Y. Mol. Pharmacol. 1996, 49,
621-628.
(17) Nair, V.; St. Clair, M.; Reardon, J. E.; Krasny, H. C.; Hazen, R. J.;
Paff, M. T.; Boone, L. R.; Tisdale, M.; Najera, I.; Dornsife, R. E.; Everett,
D. R.; Borroto-Esoda, K.; Yale, J. L.; Zimmerman, T. P.; Rideout, J. L.
Antimicrob. Agents Chemother. 1995, 39, 1993-1999.
(18) Nair, V.; Nuesca, Z. M. J. Am. Chem. Soc. 1992, 114, 7951-7953.
(19) Nair, V.; Wenzel, T. Bioconjugate Chem. 1998, 9, 683-690.
(20) Reese, C. B. Tetrahedron 1978, 34, 3143-3179.
(21) Chattopadhyaya, J. B.; Reese, C. B. Nucleic Acids Res. 1980, 8,
2309.
(22) van Boom, J. H.; van der Marel, G. A.; van Boeckel, C. A. A.;
Wille, G.; Hoyng, C. F. In Chemical and Enzymatic Synthesis of Gene
Fragments; Gassen, H. G., Lang, A., Eds.; Verlag Chemie: Weinheim,
1982; pp 53-70.
(12) Mazumder, A.; Cooney, D.; Agbaria, R.; Gupta, M.; Pommier, Y.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5771-5775.
(13) Ojwang, J. O.; Buckheit, R. W.; Pommier, Y.; Mazumder, A.; De
Vreese, K.; Este, J. A.; Reymen, D.; Pallansch, L. A.; Lackman-Smith, C.;
Wallace, T. L.; De Clercq, E.; McGrath, M. S.; Rando, R. F. Antimicrob.
Agents Chemother. 1995, 39, 2426-2435.
(14) Mazumder, A.; Neamati, N.; Ojwang, J. O.; Sunder, S.; Rando, R.
F.; Pommier, Y. Biochemistry 1996, 35, 13762-13771.
(15) Taktakishvili, M.; Neamati, N.; Pommier, Y.; Nair, V. Bioorg. Med.
Chem. Lett. 2000, 10, 249-251.
(16) Nair, V.; Jahnke, T. S. Antimicrob. Agents Chemother. 1995, 39,
1017-1029; Bolon, P. J.; Sells, T. B.; Nuesca, Z. M.; Purdy, D. F.; Nair,
V. Tetrahedron 1994, 50, 7747-7764; Nair, V.; Nuesca, Z. M. J. Am. Chem.
Soc. 1992, 114, 7951-7953; Nair, V.; Jahnke, T. S. Bioorg. Med. Chem.
Lett. 1995, 5, 2235-2238.
(23) Wreesmann, C. T. J.; Fidder, A.; van der Marel, G. A.; van Boom,
J. H. Nucleic Acids Res. 1983, 11, 8389-8405.
(24) 24.Oligonucleotide Synthesis: A Practical Approach; Gait, M. J.,
Ed.; IRL Press: Oxford, 1984.

Recognition and Inhibition of HIV Integrase
J. Am. Chem. Soc., Vol. 122, No. 24, 2000 5673
ethanol, for this transformation (Scheme 2). Treatment of 8a
with 10 in pyridine in the presence of TPS-TAZ at room
temperature for 0.5 h followed by deprotection of the trityl group
with dichloroacetic acid (3 min) gave 11 in 70% overall yield
(for two steps). The yield of the phosphorylation step was
consistently between 80 and 90%. Reagent 10 is an excellent
new phosphorylating agent! Compound 11 was isolated, puri-
fied, and completely characterized. Deprotection with NH4OH
gave target molecule 12. If the immediate product of the reaction
of 8a and 10 is worked up with 10% NH4OH instead of aqueous
pyridine, the overall phosphorylation approach produces a novel
dinucleotide 13, which bears a methylcytosine base, a result of
the ammonolysis of the phosphorylated intermediate involving
the 4-position of thymine. That this reaction had indeed
occurred, was confirmed unequivocally by the NMR and ESI
HRMS data of 13.
Inhibition Studies with Recombinant HIV Integrase. HIV
integrase inhibition assays were conducted with purified re-
combinant integrase using a 21-mer oligonucleotide substrate
as described previously by Pommier and co-workers.27 The data
are summarized in Table 1. Compound 6 was found to have
strong inhibitory activity against recombinant wild-type HIV
integrase in reproducible assays (IC50 19 µM for 3′-processing
and 25 µM for strand transfer). The activity is much greater
than for dideoxynucleoside monophosphates5,6 (e.g., (S,S)-
IsoddAMP, AZT, L-ddCMP, Table 1) and is close to the activity
of the corresponding “natural dinucleotide” pdApdC.28 The
significant anti-integrase activity of the unusual dinucleotide 6
and its natural analog suggests base sequence selectivity. This
sequence selectivity is consistent with the catalytic mechanism
of 3′-processing in which endonuclease activity produces a
truncated viral DNA with a terminal CA dinucleotide. Molecular
recognition by the integrase of the ultimate and penultimate
bases at the 5′-end of the minus strand of noncleaved viral DNA
may result in stable complex formation before the strand transfer
reaction.29 Thus, the potent inhibitor activity of 6 may reflect
the affinity that HIV integrase has for this dinucleotide sequence.
In addition, other data28 also suggest that two neighboring bases
may fulfill a substantial part of the essential interaction
requirements when integrase recognizes its viral DNA substrate.
Further support for this comes from the inactivity of pCpIsodA,
that is, the compound in which the sequence is reversed from
compound 6. Additional support comes from the observation
that the AT analogue corresponding to compound 6 (i.e.,
pIsodApT 12) exhibits low activity for the cleavage and strand
transfer steps. The terminal 5′-phosphate appears to be essential
for activity as the precursor of 6, that is, the dinucleotide that
is devoid of the 5′-phosphate group, (i.e., compound 7) is
remarkably less potent. Another interesting observation that
arises from these studies is that HIV integrase tolerates
modifications in the base moiety, which is in contrast to the
nucleotide binding site of HIV reverse transcriptase where there
is little tolerance for modified bases. For example, compound
13 which bears a 5-methylcytosine base shows good activity
against HIV integrase. The active dinucleotides of Table 1
inhibited all enzymatic activities of integrase and DNA-integrase
cross-linking in the same range as indicated in Table 1 when
either Mn2+ or Mg2+ was used as a cofactor (data not shown).
Figure 1. Temperature-dependent CD spectra of dApdC (a) and
IsodApdC (b) in 10 mM sodium-cacodylate (pH 7.0) at 0.2 mM final
concentration.
Figure 2. A Minimum-energy conformational representation of the
anti-integrase inhibitor, pIsodApdC.
consequences of this with respect to nuclease stability are
discussed below.
Target molecule 6 was synthesized by phosphorylation of
intermediate 5 with 2-cyanoethylphosphate25,26 in the presence
of DCC followed by deprotection with NH4OH. This compound
was characterized as described above for 7. The COSY spectrum
with assignments is shown in Figure 3.
Three other dinucleotides were synthesized by multistep
procedures for these studies. Compound 9 was prepared using
methodologies similar to the synthesis of 7. However, 5′-
phosphorylation of 8a with 2-cyanoethylphosphate as well as
other reagents (e.g., phosphoroimidazolides, phosphorotriaz-
olides, phosphoramidites, etc.) was inefficient. We therefore
developed a new reagent (10), a derivative of 2,2′-sulfonyldi-
(27) Mazumder, A.; Neamati, N.; Sundar, S.; Owen, J.; Pommier, Y. In
Methods in Cellular and Molecular Biology: AntiViral EValuation; Kinch-
ington, D., Schinazi, R., Eds.; Humana: Totowa, 1998.
(28) Mazumder, A.; Uchida, H.; Neamati, N.; Sunder, S.; Jaworska-
Maslanka, M.; Wickstrom, E.; Zeng, F.; Jones, R. A.; Mandes, R. F.;
Chenault, H. K.; Pommier, Y. Mol. Pharmacol. 1997, 51, 567-575.
(29) Ellison, V.; Brown, P. O. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
7316-7320.
(25) Torrence, P. A.; Witkop, B. In Nucleic Acid Chemistry; Townsend,
L. B., Tipson, R. S., Eds.; Wiley: New York, 1978; Part 2, pp 977-988.
(26) Smrt, J. In Nucleic Acid Chemistry; Townsend, L. B., Tipson, R.
S., Eds.; Wiley: New York, 1978; pp 885-888.

5674 J. Am. Chem. Soc., Vol. 122, No. 24, 2000
TaktakishVili et al.
1
Figure 3. The 600 MHz H COSY NMR Spectrum of compound 6 with complete assignments.
Scheme 2. New Phosphorylation Methodology in the Synthesis of Dinucleotides 11 and 13
(10)
This suggests that these compounds bind to the catalytic core
of integrase and the inhibition of integrase is metal-independent.
Studies with Exonucleases. Another remarkable aspect of
these dinucleotides with isomeric deoxynucleoside components
is that the internucleotide bond exhibits resistance to cleavage
by mammalian 5′- and 3′-exonucleases [phosphodiesterases
(PDE I and II)]. Cleavage reactions were monitored by HPLC
as described in the Experimental Section. Analysis of these
results revealed that internucleotide phosphate bonds in which
one of the components is an isodeoxynucleoside are resistant
to degradation by exonucleases. For example, for compound 7,
cleavage of the internucleotide phosphate bond is approximately
33% of that for the natural dinucleotide, dApdC, with PDE I
and approximately 20% with PDE II. The results are graphically
represented in Figures 4 and 5.
The resistance to internucleotide phosphate bond cleavage is
not associated with chemical alteration of the phosphate bond
(e.g., thio modification30) or with other structural changes31,32
(e.g., arabino modification) as is found with nuclease-resistant
(30) Marshall, W. S.; Caruthers, M. H. Science 1993, 259, 1564-1570.

Recognition and Inhibition of HIV Integrase
J. Am. Chem. Soc., Vol. 122, No. 24, 2000 5675
Experimental Section
Table 1. Anti-Integrase Inhibition Data for Novel Dinucleotides
and Some Selected Compounds
General. NMR spectra were recorded on a Bru¨ker model AC-300
and AMX-600 spectrometers. Chemical shifts (δ, ppm) are relative to
TMS (1H and 13C) or H3PO4 (31P). High-resolution FAB and ESI mass
spectra were determined on VG ZAB-HF and Micromass, Inc. Autospec
high-resolution mass spectrometers. UV spectra were recorded on a
Cary 3 UV-visible spectrophotometer. Preparative layer chromatog-
raphy used plates prepared with E. Merck PF254 silica gel. Column
chromatography was carried out on columns packed with 240-400
mesh silica gel. HPLC separations were performed on a Waters
automated 600E system or a Beckman System Gold HPLC using C18
columns or Partisil-10 SAX ion-exchange columns.
compound
3′-processing (µM)
strand transfer (µM)
pIsodApdC 6
IsodApdC 7
pIsodApT 12
pIsodAp5MeC 13
pCpIsodA
19
200
150
60
>300
>200
>110
50
25
200
150
50
>300
>200
140
45
(S,S)-IsoddAMP
AZTMP12
L-ddCMP11
pdApdC28
6
3
Materials. Thymidine, 2′-deoxycytidine hydrochloride, 2,2′-sulfo-
nyldiethanol, TPS-TAZ, 4,4′-dimethoxytrityl chloride, 2-chlorophenyl-
dichlorophosphate, imidazole, 2-cyanoethylphosphate (Ba salt) were
purchased from Aldrich or Sigma, and CPG-immobilized T and C from
Applied Biosystems Inc. PDE I (bovine intestinal mucosa) and PDE II
(bovine spleen) and pAdpC were purchased from Sigma Chemical Co.
N-Benzoyl-3′-O-acetylcytidine24,33 and 3′-O-acetyl- (or benzoyl) thy-
midine24,34 were prepared by reported methods.
Synthesis. N-Benzoyl-5′-O-dimethoxytritylisodeoxyadenosine-3′-
(2-chlorophenyl) Sodium Phosphate (3). Compound 2 (1.784 mmol
1.176 mg) was kept overnight under vacuum over P2O5 to remove traces
of water. 2-Chlorophenyldichlorophosphate (2-ClPhOPOCl2) (2 mmol,
0.32 mL) and 1,2,4-triazole (4.0 mmol, 276 mg)22,23 were dissolved
under nitrogen in anhydrous THF (15-20 mL) and cooled on ice. TEA
(4 mmol, 545 mL) was added dropwise, resulting in a copious
precipitate. The reaction mixture was stirred for 1-2 h and then filtered
into the flask containing 2. It was kept for 2 h on ice and then quenched
with water (2 mL). After 0.5 h, ethyl acetate (200 mL) was added, and
the organic layer was washed with saturated aqueous NaHCO3 (10 mL)
and brine (10 mL). The solvent was evaporated, and pyridine was
removed with toluene. The residue was purified by column chroma-
tography (CH2Cl2/10% MeOH) to give 3 (227 mg, 58%) as a white
Figure 4. Phosphodiesterase I activity. These assays were carried out
using various concentrations of dApdC and isodApdC as substrates.
The reactions were analyzed by HPLC using a methanol-water gradient
as described in the Experimental Section.
1
foam (Rf ) 0.1, CH2Cl2/10% MeOH). H NMR (CDCl3): 8.64 (br s,
1H, NHBz), 8.33 (s, 1H, H-8), 7.96 (s, 1H, H-2), 7.95 (s), 7.58-7.12
(m) and 6.75-6.70 (d) (22H, Arom-H), 5.44 (br s, 1H, 2′), 5.25 (br s,
1H, 3′), 4.27 (br s, 2H, 1′), 3.70 (s, 7H, CH3O, 4′), 3.46 (br s, 2H, 5′).
13C NMR (CDCl3): 167.2 (CO), 153.1 (C-6), 152.2 (C-2), 151.1 (C-
4), 142.0 (C-8), 145.5, 138.4, 136.4, 135.8, 135.4, 134.1, 133.1, 130.1,
128.9, 127.9, 127.6 (Arom), 128.2 (C-5), 115.0, 114.8 (DMTr), 86.0
(Ph3C), 84.9 (4′), 75.3 (2′), 70.1 (1′), 62.8 (5′), 62.4 (3′), 55.0 (OCH3).
ESI HRMS: (M + H)+ calcd for C44H40ClN5NaO9P 848.2243, found
848.2250.
N-Benzoyl-1′-deoxy-2′-isoadenosine-3′-[(2-chlorophenyl)(N-ben-
zoyl-3′-O-acetyl-cytidine-5′-yl)] Phosphate (5). A mixture of 3 (0.770
mmol, 670 mg) and N-benzoyl-3′-O-acetyl-cytidine (0.770 mmol, 270
mg) was repeatedly evaporated with anhydrous pyridine and then
dissolved in pyridine (3 mL) under nitrogen. 2,4,6-Triisopropylben-
zensulfonyltetrazolide (TPS-TAZ)24 (1.54 mmol, 555 mg) was added.
After 1 h at room temperature, the reaction mixture was quenched with
water (0.5 mL) and then left to stand for 1.5 h and concentrated. The
residue was dissolved in ethyl acetate (100 mL) and washed with
saturated aqueous NaHCO3 (2 × 20 mL). Solvents were removed by
evaporation and coevaporation with toluene, and the residue was
purified by column chromatography (CH2Cl2/5% MeOH) to give 600
mg (66%) of N-benzoyl-5′-O-dimethoxytrityl-1-deoxy-2-isoadenosine-
3′-[(2-chlorophenyl)(N-benzoyl-3′-O-acetylcytidine-5′-yl)] phosphate (4)
Figure 5. Phosphodiesterase II activity. These assays were carried out
using various concentrations of dApdC and isodApdC as substrates.
The reactions were analyzed by HPLC using a methanol-water gradient
as described in the Experimental Section.
compounds, but with the structural distortion of this phosphate
bond as illustrated in Figure 2. It appears that distortion of the
internucleotide phosphate bond in compound 7 results in binding
to the active site in a less productive mode than the natural
counterpart.
In summary, we have synthesized novel, sequence-specific
dinucleotides that are inhibitors of HIV integrase. The inhibition
data give evidence for sequence selectivity when HIV integrase
recognizes its viral DNA substrate, which is consistent with the
mechanism of action of integrase. Interestingly, these dinucle-
otides that possess distorted internucleotide phosphate linkages
are more stable with respect to cleavage by both 5′- and 3′-
exonucleases. Further studies of conceptually new, nuclease-
resistant, sequence-specific dinucleotides as inhibitors of HIV
integrase are in progress.
1
as a white foam (Rf ) 0.35, CH2Cl2/5% MeOH). H NMR (CDCl3):
8.67 (s, 1H, A H-8), 8.22 (s, 1H, A H-2), 8.05-8.03 (d, 2H, C H-6,
Bz), 7.85-7.83 (d, 1H, C H-5), 7.83-7.80 (m, 3H, Arom), 7.55-7.10
(m, 14H, Arom), 6.78-6.70 (m, 5H, DMTr), 6.20 (q, 1H, C 1′), 5.62-
5.50 (m, 1H, A 2′), 5.48-5.38 (m, 1H, C 3′), 5.27-5.22 (d, 1H, A 3′),
4.52-4.35 (m, 2H, A 1′) 4.30 (m. 1H, C 4′), 4.25-4.13 (m, 2H, C H
5′), 3.70 (s, 6H, CH3O), 3.53-3.35 (m, 2H, A 5′).
The protected dinucleotide 4 (500 mmol, 600 mg) was dissolved in
3 mL of anhydrous methylene chloride and cooled on an ice-bath. Cold
(31) Torrence, P. F.; Imai, J.; Johnston, M. I. Anal. Biochem. 1993, 129,
103-110.
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Z. A. Nucleosides Nucleotides 1990, 9, 515-531.
(33) Ishido Y. Chem. Abstr. 1990, 112, 700.
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Soc 1963, 85, 3821-3827.

5676 J. Am. Chem. Soc., Vol. 122, No. 24, 2000
TaktakishVili et al.
2% dichloroacetic acid (DCA) in methylene chloride (10-15 mL) was
added. The crimson red reaction mixture was kept for 3 min on the
ice-bath, poured onto saturated aqueous NaHCO3 (20 mL), and extracted
with methylene chloride (2 × 50 mL), and then the solvent was
evaporated. The residue was purified by column chromatography (CH2-
Cl2/7% MeOH) to give 5 (225 mg, 56%) as a white foam (Rf ) 0.35,
CH2Cl2/7% MeOH). 1H NMR (D2O): 8.31 (br s, 1H, A H-8), 8.11 (s,
1H, A H-2), 7.43 (d, J 7.8 Hz, 1 H, C H-6), 5.97 (t, J 6.0 Hz, 1 H, C
1′), 5.75 (br s, 1H, C H-5), 5.24 (q, J 5.4 Hz, 1H, A 2′), 4.76-4.75 (m,
1H, A 3′), 4.40-4.37 (m, 1H, A 1′), 4.36-4.34 (m, 1H, C 3 ′), 4.31-
4.28 (m, 1H, A 1′), 4.22 (s, 1H, A 4′), 4.14 (dt, 1H, A 5′a), 4.10 (dt,
1H, A 5′b), 3.95 (s, 1H, C 4′), 3.35 (s, 1H, C 5′a), 2.95 (s, 1H, C 5′b),
2.24-2.20 (m, 1H, C 2′a), 2.01-1.97 (m, 1H, C 2′b). 31P NMR (CHCl3)
-11.13 (s), -12.22 (s), ratio 1:1. ESI HRMS: (M + H)+ calcd for
C41H39ClN8O12P 901.2105, found 901.2121.
2.25-2.21 (dt, J1 ) 13.8 Hz, J2 ) 6.0 Hz, 1H, C 2′a) and 1.98-1.93
(dt, J1 ) 13.8 Hz, J2 ) 6.0 Hz, 1H, C 2′b). 13C NMR, (D2O): 164.2 (C
C-4), 157.5 (C C-2), 157.4 (A C-6), 154,6 (A C-2), 154.0 (A C-4),
143.5 (A C-6), 143.4 and 143.0 (A C-8), 120.8 (A C-5), 97.5 (C C-5),
87.7 and 87.5 (A 4′), 87.5 (C 1′), 87.1 and 87.0 (C 4′), 82.0 and 82.00
(A 3′), 73.00 (A 1′), 71.7 (C 3′), 66.7 and 66.6 (C 5′), 64.1 and 64.00
(A 2′), 62.7 (A 5′), 42.2 (C 2′). 31P NMR (D2O): -0.85 (s). λmax 263
(ꢀ ) 19400). ESI HRMS: (M - H)- calcd for C19H24N8O9P 539.1404,
found 539.1402.
N-Benzoyl-1′-deoxy-2′-isoadenosine-3′-[(2-chlorophenyl)(3′-O-
acetylthymidine-5′-yl)] Phosphate (8a). The 5′-O-dimethoxytrityl
derivative of 8a (423 mg, 50%) was prepared using 3 (770 mmol, 670
mg), 3′-O-acetyl-thymidine (770 mmol, 210 mg), and TPS-TAZ (1.54
mmol, 555 mg) in a procedure similar to that described for 4.
Detritylation with methylene chloride/2% DCA furnished 8a (261 mg,
85% yield) as a mixture of diastereomers. It was purified by column
chromatography (CH2Cl2/7% MeOH). The slower moving diastereomer
of 8a [125 mg, 40%, Rf ) 0.40 (CH2Cl2/MeOH)]: 1H NMR (CDCl3):
8.37 (s, 1H A H-2), 8.10 (d, 2H, Bz), 7.57-7.10 (m, 8H, Arom), 7.51
(s, 1H, T H-6), 6.19 (dd, J1 ) 5.8 Hz, J2 ) 2.9 Hz, T H-1′), 5.42-5.39
(m, 2H, A 2′ and T 3′), 5.12-5.10 (m, 1H, A 3′), 4.70-4.64 (m, 1H,
A 1′a), 4.59-4.52 (m, 1H A 1′b), 4.41-4.37 (m, 1H A 4′), 4.20-4.11
(m, 2H, A 5′), 4.06-4.01 (m, 1H, T 4′), 3.93-3.83 (m, 2H, T 5′),
2.24 (dd, J1 ) 9 Hz, J2 ) 5.7 Hz, T 2′a), 2.01-1.96 (m, 1H, T 2′b),
2.09 (s, 3H, Ac), 1.64 (s, 1H, T CH3). 31P NMR (CDCl3): -0.50. The
faster migrating diastereomer of 8a [136 mg, 45% yield, Rf ) 0.41
(CH2Cl2/MeOH)]: 1H NMR (CDCl3): 10.08 (br s, 1H, NH), 8.78 (s,
1H, A H-8), 8.46 (s, 1H, A H-2), 8.16 (d, 2H Bz), 7.56 (s, 1H, T H-6),
7.54-7.17 (m, 8H, Arom), 5.89 (dd, J1 ) 5.8 Hz, J2 ) 2.9 Hz, T 1′),
5.46-5.44 (m, 1H A 2′), 5.25-5.21 (m, 1H, T 3′), 5.09-5.07 (m, 1H,
A 3′), 4.53-4.44 (m, 2H, A 1′), 4.22-4.21 (m, 1H, A 4′), 4.30-4.29
(m, 2H, A 5′), 3.95-3.88 and 3.81-3.76 (m, 3H, T 4′ and 5′), 2.23-
2.16 (m, 1H, T H-2′a), 2.02-1.96 (m, 1H, T H-2′b), 2.10 (s, 3H, Ac),
1.73 (s, 3H, T CH3). 31P NMR (CDCl3): -0.72. ESI HRMS: (M +
H)+ calcd for C35H36ClN7O12P 812.1840, found 812.1845.
2′-Deoxycytidylyl(5′f3′)-1′-deoxy-2′-isoadenosine 5′-phosphate
(6). Compound 5 (0.16 mmol, 150 mg) and â-cyanoethylphosphate25,26
(0.8 mmol, 0.6 mL) (prepared from its Ba salt), were evaporated with
anhydrous pyridine three times. Dicyclohexylcarbodiimide (DCC) (110
mmol, 230 mg) was added together with 4 mL of pyridine. A copious
white precipitate of dicyclohexylurea formed within 10 min. After 48
h, the reaction mixture was filtered, the precipitate was washed with
pyridine, and the combined filtrate was evaporated and coevaporated
with water to remove pyridine. Cold NH4OH (29%, d 0.88, 40 mL)
was added, and the suspension was heated in a Hastelloy bomb reactor
at 60-65 °C for 6 h. The reactor was cooled and opened, and the
reaction mixture was filtered. The filtrate volume was reduced by one-
fifth to remove excess NH3. The pH was adjusted to 10 by the addition
of 29% NH4OH (a drop). The reaction mixture was extracted with
diethyl ether (2 × 20 mL) and the aqueous layer was evaporated to a
small volume and filtered, and the filtrate was treated with LiOH (to
saturation). Anhydrous ethanol (10 volumes) was added to the resulting
slurry which was centrifuged at 10 000 rpm, 0 °C for 15 min. The
supernatant was discarded, and the precipitate was stirred with Dowex
50 H+ resin (5 mL) and filtered through the same resin (20 mL). The
UV-absorbing fractions (60 mL) were concentrated and filtered. The
filtrate was purified by reversed-phase HPLC (flow rate 5 mL/min,
H2O) to give 6 (49 mg, 49%) (retention time 35 min, single peak) as
a fluffy white material after lyophilization. Ion-exchange HPLC used
the following buffers: A 2mM KH2PO4, 8 mM KCl, 0.05 M MgCl2,
0.05% acetonitrile, pH 3.0; B: 0.2 M KH2PO4, 0.9 M KCl, 0.8 M
MgCl2, 5% acetonitrile, pH 3.0; retention time 70 min (cf., ref 17). 1H
NMR (D2O): 8.31 (br s, 1H, A H-8), 8.11 (s, 1H, A H-2), 7.43 (d, J
) 7.8 Hz, 1 H, C H-6), 5.97 (t, J ) 6.0 Hz, 1 H, C 1′), 5.75 (br s, 1H,
C H-5), 5.24 (q, J ) 5.4 Hz, 1H, A 2′), 4.76-4.75 (m, 1H, A 3′),
4.40-4.37 (m, 1H, A 1′), 4.36-4.34 (m, 1H, C 3′), 4.31-4.28 (m,
1H, A 1′), 4.22 (s, 1H, A 4′), 4.14 (dt, 1H, A 5′a), 4.10 (dt, 1H, A 5′b),
3.95 (s, 1H, C 4′), 3.35 (s, 1H, C 5′a), 2.95 (s, 1H, C 5′b), 2.24-2.20
(m, 1H, C 2′a), 2.01-1.97 (m, 1H, C 2′b). 13C NMR, D2O: 166.6 (C
C-4), 157.6 (C C-2), 157.6 (A C-6), 154.5 (A C-2), 154.0 (A C-4, not
observable), 143.0 (C C-6), 142.8 (A C-8), 120.0 (A C-5) 98.1 (C C-5),
87.5 (A 4′), 87.1 (C 1′), 87.0 (C 4′), 82.3 (A 3′), 73.3 (A 1′), 71.8 (C
3′), 66.8 (C 5′), 66.1 (A 2′), 63.4 (A 5′), 42.0 (C 2′). 31P NMR (D2O):
8.8 (s, phosphomonoester), -0.9 (s, phospodiester), ratio 1:1. λmax263
(ꢀ ) 19500). ESI HRMS: (M - H)- calcd for C19H25N8O12P2 619.1067,
found 619.1056; (M - 2H + Na)- calcd for C19H23N8NaO18P2
641.0887, found 641.0906.
N-Benzoyl-1-deoxy-2-isoadenosine-3′-[(2-chlorophenyl)(3′-O-ben-
zoylthymidine-5′-yl)] Phosphate (8b). The 5′-O-dimethoxytrityl de-
rivative of 8b (118 mg, 100%) was prepared using 3 (0.12 mmol, 109
mg), 3′-O-benzoylthymidine (0.1 mmol, 35 mg), and TPS-TAZ (0.3
mmol, 100 mg) in a procedure similar to that described for 4.
Detritylation (CH2Cl2/2% DCA,15 mL) and purification by column
chromatography (CH2Cl2/5%MeOH) gave 8b (30 mg, 35%) (mixture
of diastereomers, ratio 1:1) as a white foam, Rf ) 0.35 (CH2Cl2/
1
5%MeOH). H NMR (CDCl3): 11.06 (br s, 1H, NHBz), 10.17 (br s,
1H, T NH), 8.76 and 8.74 (s and s, 1H, A H-8), 8.39 and 8.37 (s and
s, 1H, A H-2), 8.04-7.99 (m, 4H, Bz), 7.62 and 7.59 (s and s, 1H,
T-6), 7.69-7.14 (m, 10H, Arom), 6.54-6.37 (m, 1H, T 1′), 5.88 (br s,
1H, 5′-OH), 5.58 (br s, 2H, A 2′ and T 3′), 4.66-4.60 and 3.99-3.91
(9H, unresolved sugar protons signal), 2.66-2.36 (m, 2H, T 2′), 1.84
and 1.82 (d and d, 3H, CH3). 31P NMR (CDCl3): -8.95 (s), -9.93 (s).
ESI HRMS: (M + H)+ calcd for C40H38ClN7O12P 874.1996, found
874.2021.
Thymidylyl(5′f3′)-1′-deoxy-2′-isoadenosine-5′-phosphate (12) was
prepared from 8a in four steps. Sulfonyldiethanol (20 mmol, 3.08 g)
was evaporated with anhydrous pyridine several times and dissolved
in 10 mL of pyridine under nitrogen. 4,4′-Dimethoxytrityl chloride (10
mmol, 3.39 g) was added, and the reaction was left overnight at room
temperature. The reaction mixture was partitioned between cold
methylene chloride (300 mL) and saturated aqueous NaHCO3 (100 mL),
and the organic phase was concentrated. The residue was purified by
column chromatography (CH2Cl2/3% MeOH) to give 2-O-(4,4′-
2′-Deoxycytidylyl(5′f3′)-1′-deoxy-2′-isoadenosine (7) (21 mg,
66%) was prepared using 5 (0.06 mmol, 54 mg) and concentrated NH4-
OH (15 mL) in a procedure similar to that described for 6. Reversed-
phase HPLC purification of 7 was done at a flow rate 5 mL/min, using
water (solvent A) and ethanol (solvent B) in the following linear binary
gradient: 100% A (100 min), 0-60% B (160 min); retention time 150
min, single peak. Ion-exchange HPLC: retention time 22 min (cf. ref
17). 1 H NMR, (D2O): 8.25 (s, 1H, A H-8), 8.10 (s, 1H, A H-2), 7.40-
7.39 (d, J ) 7.2 Hz, 1H, C H-6), 5.94-5.92 (t, J ) 6.3 Hz, 1H, C 1′),
5. 66-5.65 (d, J ) 7.2 Hz, 1H, C H-5), 5.26-5.24 (qn, J ) 3.6 Hz,
1H, A 2′), 4.84-4.82 (m, 1H, A 3′), 4.39-4.36 (m, 1H, A 1′), 4.29-
1
dimethoxytrityl) sulfonyldiethanol (4.0 g, 87%) as a colorless oil. H
NMR (CDCl3): 7.43-7.24 (9H, Arom), 6.35-6.30 (m, 4H, Arom),
4.12 (m, 2H, DMTrOCH2), 3.81 (s, 6H, CH3O), 3.68 (t, J ) 6 Hz, 2H,
CH2OH), 3.40 (t, J ) 6 Hz, 2H, DMTrOCH2CH2), 3.10-3.05 (m, 2H,
CH2CH2OH). ESI HRMS: (M + Na)+ calcd for C25H28O6NaS
479.1497, found 479.1510.
The compound from the previous step (5 mmol, 2.28 g) was
evaporated with anhydrous pyridine several times and dissolved in 5
mL of pyridine. 1,2,4-Triazole (40 mmol, 276 mg) and TEA (40 mmol,
545 mL) were dissolved in 100 mL of anhydrous THF under nitrogen
4.26 (q, J ) 5.6 Hz, C 3′), 4.24-4.22 (dd, J1 ) 6.6 Hz, J2 4.2 Hz,
)
A 1′), 4.12-4.09 (m, 1H, A 4′), 4.04-4.02 (m, 2H, A 5′a and C 5′a)
and 3.97-3.924 (m, 2H, A 5′b and C 5′b), 3.89-3.88 (m, 1H, C 4′),

Recognition and Inhibition of HIV Integrase
J. Am. Chem. Soc., Vol. 122, No. 24, 2000 5677
and cooled. POCl3 (10 mmol, 0.092 mL) was added dropwise at -5
°C. After stirring for 10 min, the reaction mixture was filtered under
nitrogen into the solution prepared above. After 1 h at 0 °C, the reaction
mixture was partitioned between cold ethyl acetate (300 mL) and
NaHCO3 (saturated, 100 mL), washed with brine (1 × 100 mL), and
concentrated. The residue was purified by column chromatography
(CH2Cl2/12%MeOH) to give 2-O-(4,4′-dimethoxytrityl) sulfonyldietha-
nol disodium phosphate (10) as a colorless glassy material (3.0 g,
100%). 1H NMR (CDCl3): 7.45-7.25 (m, 9H, Arom), 6.35-6.30 (m,
4H, Arom), 4.20-4.10 (m, 2H, DMTrOCH2), 3.65-3.45 (m, 2H, CH2-
OP), 3.20-3.15 (m, 2H, DMTrOCH2CH2), 3.10-3.05 (m, 2H, CH2-
CH2OP). 31P (CDCl3): -4.90 (br s).
The compound from the previous step (0.5 mmol, 300 mg) (10) and
8a (0.1 mmol, 80 mg) were evaporated several times with anhydrous
pyridine and then dissolved in 4 mL of pyridine under nitrogen. TPS-
TAZ (1 mmol, 340 mg) was added and the reaction mixture was kept
for 0.5 h at room temperature. Workup of the reaction mixture and the
following steps were done as described for 5 to give 11 as a colorless
glassy material (0.85 mg, 70% from 8a). Compound 11 (0.7 mmol, 85
mg) was fully deprotected as described for 5, and worked up as
described for 6. Reversed-phase HPLC purification (flow rate 5 mL/
min, H2O) gave 12 (retention time 30 min, major peak) as a white
fluffy material after lyophilization (21 mg, 48%). 1H NMR (D2O): 8.34
(s, 1H, A H-8), 8.10 (s, 1H, A H-2), 7.24 (s, 1H, T H-6), 6.01 (t, J )
6.1 Hz, 1H, T 1′), 5.27 (qn, J ) 3.6 Hz, A 2′), 4.98 (qn, J ) 3.6 Hz,
A 3′), 4.41-4.32, 4.25-4.09 and 4.02-3.95 (m,m,m, 9H, unresolved
sugar signals), 2.21-2.14 (m, 1H, T 2′a), 1.97-1.89 (m, 1H, T 2′b),
1.67 (s, 1H, CH3-T). 31P NMR (D2O): -0.19 (br s, phosphomonoester),
-1.94 (s, phosphodiester), ratio 1:1. λmax 263 (ꢀ ) 18000). ESI
HRMS: (M - H)- calcd for C20H25N7O10P 635.1051, found 635.1078.
Minor peak (retention time 35 min) gave 13 (1.5 mg, 3%, see data
below).
(ꢀ ) 17990). ESI HRMS: (M - H)-calcd for C20H26N7O13P2:
554.0995; found 554.0980.
Hydrolytic Cleavage Catalyzed by Phosphodiesterases (PDE I
and II). Phosphodiesterase I Assays. The reaction mixtures were
prepared using 20 µL (0.32 mM), 40 µL (0.64 mM), 60 µL (0.96 mM),
and 80 µL (1.28 mM) of dApdC and isodApdC (and other modified
dinucleotides) substrate concentrations in 250 µL of 50 mM Tris-Cl
buffer (pH 7.6) containing 10 mM MgCl2. Reactions were initiated by
the addition of 40 µL of enzyme (5 units/mL) to each reaction mixture
at 37 °C for 1 h. The reactions were stopped by addition of 250 µL of
1 M phosphate buffer pH 4.0 containing 5 mM EDTA. The reactions
were analyzed by HPLC using a C18 column (3.9 × 300 mm) with a
gradient of the following: 0-20 min, 100% water; 20-40 min, 90:10
water:methanol; 40-60 min, 80:20 water:methanol, and 60-70 min,
70:30 water:methanol. The flow rate was 0.6 mL/min. The retention
times for both PDE assays were as follows: 2′-deoxyadenosine and
2′-isodeoxyadenosine, 52 min; 2′-deoxycytidine, 28 min; 2′-deoxycy-
tidine 5′-monophosphate, 9 min; 2′-deoxyadenosine 3′-monophosphate,
2′-isodeoxy-adenosine 3′-monophosphate, 26 min; dApdC and IsodAp-
dC, 46 min.
Phophodiesterase II Assays. The assay mixtures were prepared
using 20 µL (0.32 mM), 40 µL (0.64 mM), 60 µL (0.96 mM), and 80
µL (1.28 mM) of dApdC and isodApdC and other substrate concentra-
tions in the 250 µL of 0.1 M acetate buffer (pH 6.0) containing 10
mM MgCl2. The reactions were initiated by the addition of 5 µL of
enzyme (5 units/mL) to the reaction mixture at 37 °C for 1 h. The
reactions were stopped by addition of 250 µL of 0.5 M Tris-Cl buffer
(pH 10.0) containing 5 mM EDTA and analyzed by HPLC as described
above.
Anti-Integrase Studies. Anti-integrase studies were carried out as
described previously by Pommier and co-workers.27
In brief, IN was preincubated at a final concentration of 200 nM
with the inhibitor in reaction buffer (50 mM NaCl, 1 mM HEPES,
[pH 7.5], 50 µM EDTA, 50 µM dithiothreitol, 10% glycerol [wt/vol],
7.5 mM MnCl2, 0.1 mg of bovine serum albumin per ml, 10 mM
2-mercaptoethanol, 10% DMSO, and 25 mM MOPS [morpholinepro-
panesulfonic acid] [pH 7.2]) at 30 °C for 30 min. Then, the 5′-end
32P-labeled linear oligonucleotide substrate (20 nM) was added, and
incubation was continued for 1 h. Reactions were quenched by the
addition of an equal volume (16 µL) of loading dye (98% deionized
formamide, 10 mM EDTA, 0.025% xylene cyanol, 0.025% bromophen-
ol blue). An aliquot (5 µL) was electrophoresed on a denaturing 20%
polyacrylamide gel (0.09 M Tris-borate [pH 8.3], 2 mM EDTA, 20%
acrylamide, 8 M urea). Gels were dried, exposed in a PhosphorImager
cassette, and analyzed with a Molecular Dynamics PhosphorImager
(Molecular Dynamics, Sunnyvale, CA). Percent inhibition was calcu-
lated using the following equation: inhibition ) 100[1 - (D - C)/(N
- C)], where C, N, and D are the fractions of 21-mer substrate
converted to 19-mer (3′-processing product) or strand transfer products
for DNA alone, DNA plus IN, and IN plus drug, respectively. The
50% inhibitory concentrations (IC50) were determined by plotting the
log of drug concentration versus percent inhibition and identifying the
concentration which produced an inhibition of 50%.
5-Methyl-2′-deoxycytidinylyl (5′f3′)-1′-deoxy-2′-isoadenosine-5′-
phosphate (13). This compound was prepared starting with the same
procedure as for 12, that is, with 8a (0.1 mmol), the phosphorylating
reagent (0.5 mmol), and TPS-TAZ (1 mmol). However, the workup
after the first step was different and involved treatment with cold 15%
NH4OH (5 mL) followed by 2 h at room temperature. The reaction
mixture was concentrated and partitioned between ethyl acetate (100
mL) and brine (30 mL). The organic phase was separated and
concentrated, and the residue was purified by column chromatography
(CH2Cl2/12%MeOH/1%TEA) to give a colorless glassy material (110
mg, 85%) that was deprotected as described for 6. Reversed-phase
HPLC purification (5 mL/min, H2O) gave 13 (retention time 35 min)
as a white fluffy material after lyophilization (25 mg, 46%). 1H NMR
(D2O): 8.29 (s, 1H, A H-8), 8.03 (s, 1H, A H-2), 7.18 (d, J ) 1.0 Hz,
1H, T H-6), 5.94 (t, J ) 6.3 Hz, 1H, T H-1′), 5.19 (qn, J ) 2.4 Hz, A
2′), 4.91-4.87 (m, 1H, A 3′), 4.33-4.24, 4.19-4.03 and 3.97-3.88
(m,m,m, 9H, unresolved sugar signals), 2.15-2.07 (m, 1H, T 2′a), 1.93-
1.84 (m, H, T 2′b), 1.59 (s, 3H, T CH3). 31P NMR (D2O): 9.17 (br s,
phosphomonoester, -1.49 (s,) phospodiester). λmax 263 (ꢀ ) 18500).
ESI HRMS: (M - H)- calcd for C20H27N8O12P2: 633.1161, found
633,1221; (M - 2H)2-, calcd for C20H26N8O12P2: 316.5030; found
316.5015.
Thymidylyl(5′f3′)-1′-deoxy-2′-isoadenosine (9). Compound 9 (15
mg, 75%) was prepared using 8b (0.035 mmol, 30 mg) and 29% NH4-
OH (10 mL) and purified by reversed-phase HPLC as described for 6
(retention time 120 min, single peak). 1H NMR (D2O): 8.30 (s, 1H, A
H-8), 8.13 (s, 1H, A H-2), 7.21 (s, 1H, T H-6), 5.98 (t, J ) 7.5 Hz,
1H, T 1′), 5.30-5.26 (m, 1H, A 2′), 4.25-4.19 and 4.10-3.92 (m,m,m,
10H, unresolved sugar proton signals), 2.21-2.15 (m, 1H, T 2′a), 2.08-
1.99 (m, 1H, T 2′b), 1.67 (s, 3H, T CH3). 31P (D2O): 1.93 (s). λmax 263
Acknowledgment. We thank Dr. Lynn Teesch for the ESI
and FAB HRMS data and Mr. John Snyder for help with the
COSY, HMBC, and HMQC NMR experiments. We gratefully
acknowledge support of this research by the National Institutes
of Health.
JA992528D