Synthesis and application of nitro-substituted nucleosides and nucleotides

Article abstract of DOI:10.1055/s-2006-950218

Protocols for the synthesis of C⁷-nitropurine nucleosides and nucleotides are described. Each of these nucleotides can completely replace its naturally occurring counterpart in polymerase chain reaction (PCR), generating amplified duplex DNA containing nitropurines. As predicted, the electron-withdrawing power of the nitro group rendered these modified oligonucleotides susceptible to chemoselective cleavage at the modified bases. Treatment with secondary amines at elevated temperatures produced selective DNA scission at nitropurine sites. Interestingly, this Maxam-Gilbert type of DNA cleavage reaction was facilitated by the addition of tris(2-carboxyethyl) phosphine (TCEP), a strong reducing agent and nucleophile. Mass spectrometry analysis not only confirmed that DNA cleavage occurred at the nitropurines, but also revealed that the fragmented DNA products are appended by a TCEP moiety at its 3′-end. This surprising result provided new insights into the mechanisms of such DNA cleavage reactions. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2006-950218

3280
SPECIAL TOPIC
Synthesis and Application of Nitro-Substituted Nucleosides and Nucleotides
7-Nitropurine
N
o
ucleosides
a
m
nd
N
ucleotides ohiko Kawate, Bing H. Wang,1 Charles R. Allerson,2 Jia Liu Wolfe*
Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge St., Boston, MA 02114, USA
Fax +1(617)6433328; E-mail: jwolfe@ccib.mgh.harvard.edu
Received 28 April 2006
The strong electron-withdrawing power of the nitro group
Abstract: Protocols for the synthesis of C7-nitropurine nucleosides
at C7 position had indeed rendered 7-NO2-dA and 7-NO2-
dG similar lability to basic conditions, which conferred
and nucleotides are described. Each of these nucleotides can com-
pletely replace its naturally occurring counterpart in polymerase
chain reaction (PCR), generating amplified duplex DNA containing
sequence-specific cleavage of DNA at the modified
nitropurines. As predicted, the electron-withdrawing power of the bases.14 Thus, the nitropurine nucleotides are stable
nitro group rendered these modified oligonucleotides susceptible to
enough to withstand high temperatures during PCR am-
chemoselective cleavage at the modified bases. Treatment with sec-
plification, and yet are labile enough to be cleaved by al-
ondary amines at elevated temperatures produced selective DNA
kaline treatments, under conditions similar to that used by
scission at nitropurine sites. Interestingly, this Maxam–Gilbert type
classical Maxam–Gilbert DNA sequencing methods.15
of DNA cleavage reaction was facilitated by the addition of tris(2-
The site of nitro modification is positioned at C7 in 7-NO2-
carboxyethyl)phosphine (TCEP), a strong reducing agent and nu-
dATP and 7-NO2-dGTP. Substituents on C7 of 7-deazapu-
cleophile. Mass spectrometry analysis not only confirmed that DNA
cleavage occurred at the nitropurines, but also revealed that the
rines point toward the major groove of DNA, such that
fragmented DNA products are appended by a TCEP moiety at its 3¢-
they do not interfere with the formation of Watson–Crick
end. This surprising result provided new insights into the mecha-
base pairs with thymidine (T) or 2¢-deoxycytidine (dC)
(Figure 2). As a result, these nitro-modified nucleotides
can be readily incorporated into DNA by PCR, complete-
ly displacing dA and dG, respectively.14 Because 7-deaza-
2¢-deoxy-7-nitroinosine is also capable of forming Wat-
son–Crick base pair with dC, its 5¢-triphosphate (7-NO2-
dITP) was also synthesized and tested.
nisms of such DNA cleavage reactions.
Key words: cleavage, nucleotides, oligonucleotides, nucleophilic
additions, regioselectivity
Chemically modified nucleosides and nucleotides that are
substrates of DNA and RNA polymerases are useful in
many different applications, including DNA sequencing3
and genotyping,4 sequence-specific protein-DNA interac-
tion analysis,5 and functional aptamer generation and se-
lection.6 These DNA and RNA building blocks are used in
a variety of different capacities, including that of intro-
ducing fluorescent labels,7,8 mass tags,9 and diverse chem-
ical functional groups into polynucleotides of interest.10,11
NO2
H
H N
H
NO2
O
N
N
N
H
O
N
O
N
N
O
H
H
N
H
N
dR
N
dR
N
N
N
dR
dR
H
7-NO2-dA:T
7-NO2-dG:dC
dR = 2'-deoxyribo-
Nitro modification has recently been introduced into pu-
rine and purine nucleotides in the forms of 7-deaza-2¢-
deoxy-7-nitroadenosine (7-NO2-dA) and 7-deaza-2¢-
deoxy-7-nitroguanosine (7-NO2-dG).12 They were de-
signed to mimic the effect of N7-methylation (Figure 1) on
2¢-deoxyguanine (dG) that weakens the glycosidic bond
due to the electron-withdrawing N7+-CH3 moiety. At ele-
vated temperatures, such methylated DNA can be cleaved
at the modified dG by piperidine treatment.13
NO2
H
H N
O
N
N
N
H
dR
N
N
O
dR
7-NO2-dI:dC
Figure 2 Watson–Crick base pairs 7-NO2-dA:T, 7-NO2-dG:dC and
7-NO2-dI:dC
Competing electronic effects from neighboring and/or
conjugating functional groups often hamper the efforts of
regioselective electrophilic substitutions of 7-deazapu-
rines and their nucleosides. The process of identifying a
suitable synthetic protocol often involves trial-and-error
rather than rational design. Seela’s group has reported on
7-deaza-dA modifications, and has shown that judicious
selection of protection groups could exert remarkable in-
fluence on the outcome of the reactions.16,17 The relative
stability of postulated intermediate s complexes resulted
from an electrophilic attack at C7 or C8 was used to ex-
plain the experimental results.
O
O
Me
N+
O2N
NH
NH2
NH
NH2
N
dR
N
dR
N
N
N7+-Me-dG
7-NO2-dG
dR = 2'-deoxyribose
Figure 1 7-NO2-dG and N7+-Me-dG
SYNTHESIS 2006, No. 19, pp 3280–3290
Advanced online publication: 04.09.2006
DOI: 10.1055/s-2006-950218; Art ID: C04106SS
© Georg Thieme Verlag Stuttgart · New York
x
x.
x
x
.
2
0
0
6

SPECIAL TOPIC
7-Nitropurine Nucleosides and Nucleotides
3281
NH2
6
NRAc
A systematic examination of differently protected 7-dea-
zapurine derivatives revealed that, to achieve regioselec-
tive nitration, chemically and electronically distinctive
analogues of dA and dG are required. Figure 3 compares
the mesomeric stabilization of electrophilic addition at C7
of 6-NH2-protected 7-deaza-dA and the attack at C8 of un-
protected 7-deaza-dA, through the formation of s-com-
plexes 3A and 3B, respectively.
7
N
N
N
8
2
N
N
N
HO
AcO
5'
AC2O
100%
O
O
1'
4'
2'
3'
OH
OAc
2: R = H (97%)
3: R =Ac (3%)
1
HNO3-H2SO4
91%
O
+NH2
HN
6
R
H
7
O2N
NH2
NHAc
O2N
H
N
O2N
O2N
N1
8
N
N
N
N
2
N+
N
3
9
dR
N
N
dR
N
N
3A
3B
HO
AcO
NaOMe
79%
O
O
Figure 3 s-Complexes 3A and 3B
4
5
OH
OAc
Similar analyses on various 7-deaza-dG analogues, on the
other hand, suggested that 6-chloro-substitution (in 4A)
would promote C7 selectivity, whereas the presence of un-
protected 2-NH2 (in 4B) or 6-OMe (in 4C) favors electro-
philic attack at C8 (Figure 4). In addition, because 2¢-
deoxyguanosine is known to be more susceptible to deg-
lycosylation than 2¢-deoxyadenosine, and a positive
charge is expected to generate on N9 by electrophilic at-
tack at C7 (4A), 7-deazaguanine should be a better starting
compound than 2¢-deoxyguanosine. Analogues of purines
or purine nucleosides that promote selective C7 electro-
philic substitution were thus selected accordingly for ni-
tration reactions.
O
N
O2N
Me2CHCH2CH2ONO
NH
N
RO
O
6: R = H (38%)
7: R = Ac (37%)
OH
Scheme 1
the relative stability of the intermediate s-complexes
shown in Figure 3.
Cl
6
The high efficiency of 7-NO2-dA (5) synthesis prompted
our interest in the feasibility of preparing 7-NO2-dI (6) di-
rectly from 5. Derivatives of adenosine can be converted
to corresponding inosines in a single step by hydrolytic
deamination, which has been achieved by using adenosine
deaminase20,21 or alkyl nitrites.21,22 Alkyl nitrites have
been very useful in replacing the 6-amino group of ade-
nosine with various different functional groups, presum-
ably through a 6-diazonium intermediate. Treatment of
triacetylated adenosine with tert-butyl nitrite has been re-
ported to produce similarly protected inosine in good
yields.23
H
O2N
7
1
N
O
8
N+
2
N
3
N
H
R
9
H
4A
O
O+Me
N
O2N
H
O2N
H
NH
O
N
N
N
N+H
N
N
R
H
dR
dR
4B
4C
Figure 4 Various 7-deaza-dG analogues for transition-state analy-
To convert 7-NO2-dA (5) to 7-NO2-dI (6), compound 5
was dissolved in a 1:1 mixture of acetic acid and water and
treated with isoamyl nitrite at room temperature. After re-
action with a total of 2 molar equivalents of isoamyl nitrite
over a period of 40 hours, TLC analysis showed that the
conversion was about 50% complete. Longer reaction
time with additional isoamyl nitrite caused the production
of side products, a significant portion of which was iden-
tified as 5¢-acetyl-7-NO2-dI (7, 37%) after column chro-
matography. The desired 7-NO2-dI (6) was isolated in a
yield of 38%. This result suggests that the nitro substitu-
tion is compatible with the alkyl nitrite dA to dI conver-
sion reaction, and that avoiding acetic acid in the reaction
mixture may improve the conversion yield. However, this
ses
Regioselective C7 nitration (Scheme 1) was accomplished
by using N6-acetylated dA 2/3 as starting material, which
was obtained quantitatively from 7-deaza-dA (1) through
treatment with a large excess of acetic anhydride. Nitra-
tion with 1:1 fuming nitric acid and concentrated sulfuric
acid afforded compound 4 in 91% yield as a single regio-
isomer. Deprotection at N6 afforded desired 7-NO2-dA (5)
in 71% yield after column chromatography. Compared to
a previously reported nitration reaction starting from un-
protected 7-deaza-dA,18 the protection of exocyclic amino
group greatly improved both yield and regioselectivity.19
These results are in agreement with predictions based on
Synthesis 2006, No. 19, 3280–3290 © Thieme Stuttgart · New York

3282
T. Kawate et al.
SPECIAL TOPIC
Cl
direction was not pursued further as the focus was turned
to 7-NO2-dG preparation.
Cl
N
N
O
Me2CHCOCl
70%
N
7-NO2-dG (15) was prepared according to Scheme 2, a
seven-step procedure that afforded 15 in good yields. The
key step was the nitration reaction, in which the regiose-
lectivity can be dramatically influenced by the choice of
starting material. When 2-NH2 protected 7-deaza-2¢-
deoxy-6-methoxyguanosine was used, a complex mixture
of 7- and 8-NO2 derivatives of 7-deaza-dG was generated
that was very difficult to purify.18 By simply rearranging
the synthetic sequence and using an electron-withdrawing
protecting group at C6 (Cl), the nitration regioselectivity
was markedly improved to the extent that only the desired
C7 nitration was observed. These results are consistent
with the transition state analyses depicted in Figure 4, i.e.
a C8 electrophilic attack on the C6-methoxy analogue is
stabilized by the formation of s-complex 4C, whereas a
C7 attack on the C6-Cl analogue resulted in an electroni-
cally favorable intermediate 4A.
N
H
N
N
N
NH2
H
H
8
9
OMe
Cl
O2N
O2N
N
HNO3-H2SO4
78%
NaOCH3
65%
N
O
N
N
H
N
NH2
N
N
H
H
11
10
OMe
N
O2N
(1) NaH
N
p-ClBz
O
N
NH2
p-ClBz
O
O
(2)
12
p-ClBz
O
Cl
O
13
p-ClBz
O
61%
Specifically as shown in Scheme 2, 6-chloro-7-deazagua-
nine (8) was first protected at 2-NH2 by reaction with
isobutyryl chloride to genereate 9 in an isolated yield of
70%. Treatment of 9 with fuming nitric acid provided C7-
selective nitration product 10 in 78% after silica chroma-
tography. Deprotection at N2 and Cl substitution by OMe
(65%), glycosylation with 12 (61%), followed by demeth-
ylation at O6 (73%) and ribose deprotection (79%), pro-
vided the desired product 7-NO2-dG (15). Because of the
relative instability of glycosidic bond in dG derivatives, as
well as the need of transiently using an electron-with-
drawing substituent in place of O6 to alter its electronic
character to favor C7 nitration, the preparation of 7-NO2-
dG (15) was much more lengthier than that of 7-NO2-dA
(5).
O
N
O2N
NH
(1) Me3SiCl/NaI, 73% 14
(2) K2CO3, 79% 15
N
NH2
RO
O
OR
R = p-ClBz, 14
R = H, 15
Scheme 2
Considering that the nitropurine triphosphates can survive
PCR conditions and are much less labile than N7+-CH3-
dGTP, it is not entirely surprising that the standard Max-
am–Gilbert piperidine treatment did not completely
cleave these bases. To address the possibility that piperi-
dine might lose its activity during the course of the reac-
tion through N-oxide formation, we added DTT
(dithiothreitol) or TCEP, two commonly used reducing
agents in molecular biology, to the cleavage reaction mix-
tures. To our surprise, preliminary data indicated that, al-
though DTT did not produce any obvious effect, addition
of TCEP altered the cleavage pattern and generated clean-
er cleavage products.
With these nitro-modified nucleosides in hand, the 5¢-
triphosphates of 7-NO2dA, 7-NO2-dI, and 7-NO2-dG were
prepared in 20–30% yields using a standard synthetic pro-
cedure with several modifications. As reported, 7-NO2-
dATP (16) and 7-NO2-dGTP (17) were each used in place
of dATP and dGTP, respectively, in PCR and generated
DNA products that contain each of these nitropurines. 7-
NO2-dITP (18) was similarly incorporated into DNA by
PCR, where it replaced guanine as base-pairing partner
for cytosine (see Figure 2).
To examine the feasibility to sequence-specifically cleave
these modified DNA at the incorporated nitropurine
bases, the PCR products were fragmented under alkaline
conditions and the products analyzed by gel electrophore-
sis and/or mass spectrometry. In initial cleavage experi-
ments, DNA products were treated with the standard
conditions used for Maxam–Gilbert DNA sequencing,
i.e., heating in 1 M aqueous solution of piperidine at
90 °C.15 In these experiments, although DNA cleavage
was evident and seemed to occur at selected bases, the
cleavage was incomplete and many different discrete
bands were observed by polyacrylamide gel electrophore-
sis (PAGE) analysis (data not shown).
To investigate this unexpected phenomenon more closely,
the human transferrin receptor (TR) gene, which contains
a polymorphic nucleotide A or G at position 424, was cho-
sen as a model system.14 As depicted in Figure 5, Primer I
and Primer II were designed to generate three 7-NO2-dA-
containing PCR products (82 base pairs) using TR DNA
templates of three genotypes: A/A homozygote, G/G ho-
mozygote, or A/G heterozygote. Primer I was designed to
hybridize near the polymorphic base, such that the 7-NO2-
dA specific cleavage would generate an oligonucleotide
product of 25nt and/or 28nt in length, depending on which
alleles are present in a particular PCR product. Complete
site-specific cleavage at 7-NO2-dA residues is expected to
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SPECIAL TOPIC
7-Nitropurine Nucleosides and Nucleotides
3283
generate following primer-containing fragments: a 22nt
fragment comprising Primer II from the cleavage of the
bottom strand DNA, which should be present in all three
samples, a Primer I-containing 25nt fragment from A/A
homozygote and A/G heterozygote samples, and a Primer
I-containing 28nt fragment from G/G and A/G samples
(Figure 5). In addition, an 11nt fragment, which contains
the complementary polymorphic base (T or C) on the bot-
tom strand of DNA, will be generated by cleavage at 7-
NO2-dA as well, as indicated by green arrows in Figure 5.
For PAGE experiments, a small portion of the A/A geno-
type PCR product was labeled at 5¢-end using [g-32P]-ATP
and T4 kinase. The labeled sample was treated with vari-
ous cleavage reagents, and the products were purified/de-
salted and then analyzed by denaturing PAGE along with
32P-labeled control samples and oligonucleotide markers.
Lanes 5–8 of Figure 6 show the cleavage products using
four different mixtures of reagents as indicated, either at
95 °C for 1 hour (lanes 5, 7, and 8), or at 70 °C for 2 hours
for the use of low boiling ammonia (lane 6). Tris base (5
equiv) was added to each cleavage reaction to neutralize
the acidic TCEP. In this experiment piperidine/Tris
seemed to have cleaved 7-NO2-dA with higher efficiency
than previously observed with 1 M piperidine at 90 °C,
probably due to the presence of Tris base and the higher
reaction temperature (lane 8). However, slower migrating
DNA bands are still visible just above the two major prod-
uct bands. In contrast, lane 5 did not show any incomplete
cleavage products, suggesting that the treatment with a
mixture of 1 M piperidine, 0.2 M TCEP, and 0.5 M Tris
generated complete cleavage products. Interestingly,
these products migrated slightly slower than the major
cleavage products in lane 8. Surprisingly, with the forma-
tion of minor incomplete cleavage products notwithstand-
ing, treatment with NH4OH/TCEP/Tris produced similar
results (lane 6 vs. lane 5). Furthermore, cleavage by pyr-
rolidine/TCEP/Tris produced two sets of cleavage prod-
ucts: one set corresponds to the piperidine cleavage
products, and the other to that from piperidine/TCEP/Tris.
It is noteworthy that the slower migrating products were
only present when TCEP is in the cleavage mixture.
Figure 6 Lane 1: 2nt oligonucleotide ladder containing oligonucleo-
tides of 32nt–14nt in length. Lane 2: Primer I. Lane 3: Primer II. Lane
4: 82bp 7-NO2-dA-modified PCR product with AA genotype. Lanes
5–8: cleavage products from the sample in Lane 4 under the conditi-
ons indicated. Lanes 9–11: cleavage products from AA, GG, or AG
genotypes, using the reagents indicated. Lane 12: 5nt oligonucleotide
ladder containing oligonucleotides of 15nt–80nt in length.
After these exploratory cleavage experiments, piperidine/
TCEP/Tris mixture was selected as the cleavage reagents
for treating three PCR products corresponding to A/A, G/
G, or A/G genotypes. Each cleavage product was desalted
and divided into two portions. A small sample was labeled
at 5¢-end using [g-32P]-ATP and T4 kinase, and then ana-
lyzed by denaturing PAGE. The remainder of each sample
was saved for mass spectrometry analysis. As shown in
lanes 9–11 of Figure 6, two or three distinctive product
bands were observed, in agreement with expected geno-
types of each sample: 25nt represents the presence of A al-
lele in samples A/A and A/G, 28nt represents the presence
of G allele in the samples G/G and A/G, with a common
22nt in all three samples. This experiment demonstrated
the feasibility to genotype a DNA sample using the ana-
logue incorporation and chemical cleavage approach, spe-
cifically (1) using 7-NO2-dATP in place of dATP during
Figure 5 DNA sequence of 82bp PCR products containing 7-NO2-dA, which is denoted as blue-colored A in the sequence. Polymorphic
bases are shown in parentheses. Red arrows indicate 7-NO2-dA residues that are positioned closest to the primers. Green arrows indicate 7-
NO2-dA residues that surround the internal fragment containing a polymorphic base. bp: base pairs; nt: nucleotides.
Synthesis 2006, No. 19, 3280–3290 © Thieme Stuttgart · New York

3284
T. Kawate et al.
SPECIAL TOPIC
PCR amplification, (2) fragmenting the PCR product us- ide to open the five-membered ring of the purine. The re-
ing piperidine/TCEP/Tris mixture, and (3) analyzing the sulting riboside carbinolamine can open to form a Schiff
cleavage products by denaturing PAGE.
base, which is susceptible to attack by piperidine to form
a piperidinium ion. Depending on reaction conditions, this
key intermediate of the cleavage reaction can lead to sev-
eral different products. First, as shown in Scheme 3, a
general base attacks the activated a-hydrogen, resulting in
an E2 reaction with loss of the b-phosphate group to gen-
erate DNA strand scission at what used to be 3¢-phos-
phate. A second E2 reaction causes the loss of g-hydrogen
and d-phosphate, releasing what was the 5¢-end of the
DNA chain. The final oligonucleotide product is the 3¢-
phosphorylated DNA.
To understand the cleavage mechanism and definitively
assign the chemical structures of the cleaved products,
and to investigate the feasibility to utilize this incorpora-
tion-cleavage genotyping strategy in mass spectrometry
platform, the remaining samples of each PCR product was
similarly cleaved and then analyzed using MALDI-TOF
mass spectrometry. The resulting mass spectrum from the
heterozygote G/A sample is shown in Figure 7A, with the
expected and observed major fragments listed in
Figure 7B. As shown in Figure 7A, three fragments were
observed in the mass region between 7000 and 9000 Dal- The second plausible fate of the activated piperidinium in-
tons. By using internal mass calibration, the three species termediate is shown at the top panel of Scheme 4. It could
were determined to be 7189 Da, 8057 Da, and 9005 Da, be hydrolyzed before any E2 reactions could occur, result-
respectively. Based on
a
previously proposed ing in the formation of an apurinic DNA. It has been
mechanism15 of piperidine-induced, Maxim-Gilbert DNA shown that apurinic DNA is susceptible to b-elimination
scission, the cleavage products expected from such cleav- catalyzed by N-terminal b-amino group of peptides,
age are 3¢-phosphorylated DNA (3¢-P-DNA) fragments, through the formation of a Schiff base intermediate simi-
including Primer II-G-PO3 , Primer I-CC-PO3 , and lar to piperidinium intermediate.25 Conversely, it seems
–
–
–
Primer I-CCGGC-PO3 (see Figure 5 and Scheme 3). reasonable to hypothesize that an inadequate availability
The expected m/z of these 3¢-phosphorylated species in of free amines such as piperidine could stall the DNA
positive ion mode are 6841 Da, 7709 Da, and 8657 Da, re- cleavage reaction at the apurinic DNA stage.
spectively. Thus, there is an extra 348 Da in our piperi-
dine/TCEP/Tris cleaved fragments.
When TCEP is added to the cleavage reaction, the other-
wise stagnant apurinic DNA is driven by b-elimination of
Because our experimental data showed an excellent corre- what used to be 3¢-phosphate (Scheme 4), generating a
spondence among the observed higher masses, the slower Michael acceptor that can be readily attacked by the phos-
electrophoresis mobility, and the presence of TCEP in the phine on TCEP, a strong nucleophile. The resulting
cleavage reaction mixtures, it seemed reasonable to postu- Michael addition product is a 3¢-phosphodideoxyribosy-
late that the cleavage products contain a TCEP-generated late-DNA modified by a phosphonium moiety at the 3¢-
moiety. Because the molecular weight of TCEP position of the basic ribose. This proposed product is in
(C9H15O6P) is 250 Da and that of dideoxyribose moiety excellent agreement with the observed mass spectrometry
(C5H10O2) is 102 Da, the fusion of the two with 3¢-phos- data, adding 348 Da mass to 3¢-phosphorylate-DNA. Be-
phorylated DNA should provide the observed extra mass cause piperidine moiety is not observed in the final prod-
of 348 Da.
uct, the piperidinium intermediate must have been
hydrolyzed before Michael addition. This also lends addi-
tional support to the idea that the incomplete cleavage re-
action was stalled at the stage of apurinic DNA species in
the absence of TCEP.
Based on the Maxam–Gilbert cleavage mechanism of
N7+-Me-dG-DNA,24 a piperidine cleavage mechanism for
7-NO2-dA-DNA is proposed in Scheme 3. As illustrated,
the electron-withdrawing capacity of nitro group facili-
tates attack by hydroxide on the neighboring carbon and In short, two competitive cleavage pathways exist for the
yields a carbinolamine, which further reacts with hydrox- piperidinium intermediate: b-elimination/hydrolysis,
Figure 7 Mass spectrum of a heterozygote G/A sample
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SPECIAL TOPIC
7-Nitropurine Nucleosides and Nucleotides
3285
O–
O–
O–
N+
NH2
NH2
NH2
NH2
N+
O2N
O2N
OHC
O
HO–
DNA
H
HO–
N
N
N
N
OHC
HN
O
DNA
O
DNA
O
DNA
O
H
N
N
N
O
N
N
N
N+
O
P
O–
O
O
P
O–
O
O
P
O–
O
O
P
O–
O
OH
O
O
O
1'
4'
3'
2'
O
P
O
P
O
P
O
P
N
O
O–
O
O–
O
O–
O
O–
O
O
O
O
DNA
O2N
DNA
DNA
DNA
Schiff base intermediate
NH2
DNA
O
DNA
O
N
DNA
O
OHC
H
DNA cleavage
at 5'-phosphate
DNA
O
O
P
O-
O
OH
N+
DNA cleavage
at 3'-phosphate
N
O
P
O–
N
O
P
O–
δ-elimination
O
P
O
2'
O
OH
O–
OH
O
H
N+
O–
N
NH2
N
β-elimination
H
O2N
O
O–
_
4'
P
:B
3'-Phosphorylated
DNA
O
P
OHC
H2N
O
O
O–
DNA
N
:B
O
Piperidinium intermediate
DNA
Scheme 3
which lead to d-elimination followed by formation of 3¢- the same mobility as the piperidine cleavage products, the
phosphorylated DNA (Scheme 3), or apurinic DNA for- other the same as the cleavage products by piperidine/
mation followed by Michael addition of a strong nucleo- TCEP/Tris. The higher pKa of pyrrolidine likely promoted
phile (TCEP) to yield a 3¢-phosphodideoxyribosylated the formation of pyrrolidinium intermediate, ensuring
DNA adduct (Scheme 4). These proposed pathways pre- subsequent b- and d-elimination to yield 3¢-phosphate-
dict that the final cleavage outcome could be influenced DNA product, which was absent when TCEP/piperidine/
by the strength of basicity or nucleophilicity of the re- Tris is used (lane 5 in Figure 6). NH4OH, on the other
agents used; giving rise to 3¢-phosphorylate-DNA and/or hand, with a lower pKa than piperidine and pyrrolidine,
the TCEP adduct of 3¢-phosphoribosylated-DNA. Indeed, could not cleave 7-NO2-dA completely (lane 6 in
in the mixture of pyrrolidine/TCEP/Tris where piperidine Figure 6). However, the fact that NH4OH/TCEP/Tris ac-
is replaced by pyrrolidine (lane 7 of Figure 6), two sets of tually could cleave 7-NO2-dA-DNA quite efficiently sug-
distinctive cleavage products were observed. One set with gests that the formation of piperidinium or pyrrolidinium
DNA
O
DNA
O
DNA
O
O
P
O–
O
O
P
O–
O
O
P
O–
O
OH
OH
O
N+
OH
O
β-elimination
H2O
2'
2'
O
P
H
O–
O
P
O
P
H
H
O–
O
O–
O
O
O
O
O
DNA
DNA
DNA
Apurinic DNA
Piperidinium intermediate
DNA
O
DNA
O
DNA
O
P
5'-Phosphorylated
DNA
O
P
O–
O
P
O–
O
O–
nucleophilic
addition
O
O
O
OH
O–
OH
O
OH
O
O
O
P
O–
+
TCEP
P+
P+
O
DNA
P
–OOC
–OOC
COO–
COO–
–OOC
COO–
–OOC
–OOC
–OOC
TCEP-modified 3'-phosphodideoxyribosylated DNA
Scheme 4
Synthesis 2006, No. 19, 3280–3290 © Thieme Stuttgart · New York

3286
T. Kawate et al.
SPECIAL TOPIC
intermediates can be bypassed; the hydrolysis of Schiff addition of a potent nucleophile such as TCEP could drive
base intermediate could directly yield apurinic DNA, the cleavage reaction of 7-NO2-dA-DNA to completion.
which is a good Michael acceptor for phosphine.
Quite unexpectedly, a different end product, i.e., a TCEP
modified 3¢-phosphodidepxyribosylate DNA species, was
obtained. The identity of the cleavage products was con-
firmed by MALDI-TOF mass spectrometry analysis, and
a cleavage mechanism was proposed based on the product
structure and the previously articulated mechanism for
N7+-Me-dG-DNA cleavage. Based on the proposed mech-
anism, additional cleavage reagents were tested and found
to be superior in generating complete cleavage products.
Nitropurines turned out to be an excellent choice for PCR
incorporation and chemical cleavage.14 The facility with
which the TCEP adduct was formed during cleavage sug-
gests that this reaction may be useful for introducing other
functional groups, such as fluorescent labels, into cleaved
DNA in situ.
Mass spectrometry analysis (Figure 7) not only provided
exact mass of each major cleavage fragments that led to
the proposal of cleavage mechanisms, but also provided
information on the smaller, internal cleavage fragments
that are genotype-specific. Four m/z peaks were observed
in the m/z region of 3500–5000 (Figure 7A and inset
therein), corresponding to one 11nt fragment from top
strand DNA (located near Primer II in Figure 5), one 13nt
fragment from bottom strand DNA (located near Primer
I), and two 11nt fragments that contained genotype infor-
mation on SNP 424 (highlighted in Figure 5). These 11nt
long fragments clearly showed the mass difference of 15
Da, in agreement with expected mass difference between
T allele and C allele. The observed m/z values, 3912 Da
and 3927 Da, are consistent with molecular structures of a
protonated 5¢-PO3H2-TGG(T/C)GCTGGTG-PO2H-di-
deoxyribosylate-TCEP. This result provides additional
support for the cleavage mechanism shown in Scheme 4,
which suggests the formation of 5¢-phosphorylated C-ter-
minus cleavage fragment.
Reagents and solvents were purchased from Sigma-Aldrich (St.
Louis, MO) or J. T. Baker (Phillipsburg, NJ) unless otherwise spec-
ified. The 1H and 31P NMR spectra were recorded on a Bruker DPX-
400 NMR spectrometer using either TMS as an internal standard
(1H spectra) or 80% H3PO4 as an external standard (31P spectra).26
Chemical shifts (d) are reported in ppm downfield from TMS or
H3PO4. Column chromatography was performed with silica gel 60
(230–400 mesh, Merck 9385) using standard flash methods. Ana-
lytical TLC was carried out on Merck silica gel 60 F254 precoated
plates.
The proposed cleavage mechanisms also suggest that the
cleavage efficiency could be improved using reagents and
conditions with increased basicity and nucleophilicity. To
this end, pyrrolidine was first used to cleave 7-NO2-dA-
DNA at 95–98 °C and resulted in better cleavage than pi-
peridine. Further improvements were accomplished with
the use of 3-pyrrolidinol, a pyrrolidine derivative with
high boiling point, excellent water miscibility, and pre-
sumably comparable pKa to piperidine. Because of its
high boiling point, reagent loss during the cleavage reac-
tion was minimized, which both improves reaction effi-
ciency and eliminates the unpleasant odor typical of
amines. Due to its high miscibility with water, it can be
3¢,5¢,N6-Triacetyl-7-deaza-2¢-deoxyadenosine (2) and
3¢,5¢,N6,N6-Tetraacetyl-7-deaza-2¢-deoxyadenosine (3)
2¢-Deoxy-7-deazaadenosine (1; 2.092 g, 8.36 mmol, from Chem-
Genes) and 4-dimethylaminopyridine (30 mg, 0.36 mmol) were
placed in a 100-mL flask. Anhyd pyridine (20 mL) and Ac2O (20
mL, 0.21 mol) were added and the mixture was stirred at r.t. for 7.5
h. The mixture was evaporated to near dryness and the residue was
chromatographed on silica gel (Merck 9385, 50 g, 2% MeOH–
CH2Cl2) to give a mixture of acetylated 7-deaza-dA (3.328 g, quan-
titative). The ratio of triacetylated 7-deaza-dA (2: R = H) and tet-
1
used at higher concentrations than piperidine and pyrroli- raacetylated 7-deaza-dA (3: R = Ac) was 34:1 based on H NMR
dine. Eventually it was found that a 1.46 M aqueous solu-
tion of 3-pyrrolidinol could consistently induce complete
spectroscopy. The following spectral data were obtained from each
isolated material.
cleavage at 7-NO2-dA by heating at 98 °C for 1 hour.
Consequently, these conditions were used in the proof-of-
Compound 2
1H NMR (400 MHz, CDCl3): d = 9.09 (br s, 1 H, NH), 8.54 (s, 1 H,
concept experiments for a mass spectrometry based geno-
H-2), 7.31 (d, J = 3.9 Hz, 1 H, H-8), 7.01 (d, J = 3.5 Hz, 1 H, H-7),
6.77 (dd, J = 5.9, 8.6 Hz, 1 H, H-1¢), 5.37 (td, J = 2.0, 6.2 Hz, 1 H,
H-3¢), 4.32 (m, 3 H, H-4¢, H-5¢), 2.68 (ddd, J = 6.2, 8.6, 14.1 Hz, 1
H, H-2¢b), 2.52 (ddd, J = 2.0, 5.9, 14.1 Hz, 1 H, H-2¢a), 2.34 (s, 3 H,
COCH3), 2.13 (s, 3 H, COCH3), 2.12 (s, 3 H, COCH3).
typing methodology.14
In summary, we have developed synthetic methods that
support regioselective nitration of purine derivatives
based on electronic considerations of transition states.
Different synthetic strategies were developed for the ni-
Compound 3
tration of 7-deazaadenosine and 7-deaza-guanine, with 1H NMR (400 MHz, CDCl3): d = 8.87 (s, 1 H, H-2), 7.49 (d, J = 3.9
excellent yields and regioselectivity.12 7-NO2-dA can be
Hz, 1 H, H-8), 6.77 (dd, J = 5.9, 8.6 Hz, 1 H, H-1¢), 6.47 (d, J = 3.9
Hz, H-7), 5.39 (td, J = 2.0, 6.6 Hz, 1 H, H-3¢), 4.36 (m, 3 H, H-4¢,
converted to 7-NO2-dI by hydrolytic deamination, al-
though the procedure has not been optimized. Corre-
sponding nucleotide triphosphates were prepared and can
3 H, COCH3), 2.11 (s, 3 H, COCH3).
be incorporated into DNA by PCR, substituting corre-
sponding natural nucleotides. Such modified DNA is sus-
ceptible to chemical cleavage under alkaline conditions
H-5¢), 2.74 (ddd, J = 6.8, 8.6, 14.1 Hz, 1 H, H-2¢b), 2.58 (ddd,
J = 2.0, 5.9, 14.1 Hz, 1 H, H-2¢a), 2.32 (s, 6 H, 2 × COCH3), 2.15 (s,
ES+-MS: m/z [M + Na+] calcd for C19H22N4O7 + Na: 441; found:
441.35.
3¢,5¢,N6-Triacetyl-7-deaza-2¢-deoxy-7-nitroadenosine (4)
A mixture of acetylated 7-deaza-dA (2/3, 3.325 g, ~8.36 mmol, pre-
pared as above) was dissolved in CH2Cl2 (400 mL, HPLC grade) in
that are similar to what has been used by Maxam–Gilbert
DNA sequencing.15 Although the resulting DNA resisted
complete cleavage by piperidine, it was discovered that
Synthesis 2006, No. 19, 3280–3290 © Thieme Stuttgart · New York

SPECIAL TOPIC
7-Nitropurine Nucleosides and Nucleotides
3287
a 1-L flask. The solution was cooled in an ice-bath and stirred vig-
orously. A mixture of fuming HNO3 (6.2 mL) and concd H2SO4 (6.2
mL) was prepared by dropwise addition of H2SO4 to ice-cooled and
stirred HNO3, and immediately added dropwise to the CH2Cl2 solu-
tion of 2/3 over a 10 min period. After stirring for another 20 min,
the reaction was quenched by the addition of chilled 20% aq K2CO3
(150 mL). The resulting organic phase was saved, and the aqueous
phase was extracted with CH2Cl2 (3 × 50 mL). The combined or-
ganic solutions were washed with H2O (100 mL) and brine (100
mL) and dried (Na2SO4). After rotary evaporation, the residue was
chromatographed on silica gel (Merck 9385, 50 g, 2% MeOH–
CH2Cl2] to give 4; yield: 3.209 g (91% from 1).
1H NMR (400 MHz, DMSO-d6): d = 8.58 (s, 1 H, H-8), 8.07 (s, 1
H, H-2), 6.49 (t, J = 6.6 Hz, 1 H, H-1¢), 5.34 (br s, 1 H, OH), 5.17
(br s, 1 H, OH), 4.36 (m, 1 H, H-3¢), 3.87 (m, 1 H, H-4¢), 3.64 (dd,
J = 3.9, 11.7 Hz, 1 H, H-5¢b), 3.57 (dd, J = 3.4, 11.7 Hz, 1 H, H-5¢a),
2.47 (m, 1 H, H-2¢b, overlapped with DMSO), 2.30 (ddd, J = 3.4,
5.9, 13.2 Hz, 1 H, H-2¢a).
Compound 7
1H NMR (400 MHz, CD3OD): d = 8.36 (s, 1 H, H-8), 8.03 (s, 1 H,
H-2), 6.61 (t, J = 6.2 Hz, 1 H, H-1¢), 4.48–4.56 (m, 1 H, H-3¢), 4.34
(m, 2 H, H-5¢), 4.16 (m, 1 H, H-4¢), 2.42–2.63 (m, 2 H, H-2¢), 2.13
(s, 3 H, COCH3).
1H NMR (400 MHz, CDCl3): d = 10.67 (br s, 1 H, NH), 8.74 (s, 1
H, H-8), 8.53 (s, 1 H, H-2), 6.73 (t, J = 6.6 Hz, 1 H, H-1¢), 5.36 (m,
1 H, H-3¢), 4.42 (m, 3 H, H-4¢ and H-5¢), 2.75 (ddd, J = 3.1, 6.2, 14.4
Hz, 1 H, H-2¢b), 2.59 (td, J = 7.0, 14.4 Hz, 1 H, H-2¢a), 2.54 (s, 3 H,
COCH3), 2.19 (s, 3 H, COCH3), 2.15 (s, 3 H, COCH3).
6-Chloro-7-deaza-N2-isobutyrylguanine (9)
6-Chloro-7-deazaguanine (8; 6.7228 g, 39.88 mmol, from Chem-
Genes) was dissolved in anhyd pyridine (280 mL). The solution was
chilled in an ice/water bath. To this solution was added freshly dis-
tilled isobutyryl chloride (43.87 mmol, 4.6 mL, 1.1. equiv, from
ICN) dropwise with stirring. After stirring for 20 min at 0 °C, the re-
action was quenched with MeOH (600 mL), followed by an addi-
tional 10 min of stirring. The mixture was concentrated in vacuo to
give a yellow solid, and then resuspended in Et2O (20 mL). The sol-
id was collected by vacuum filtration, washed with ice-cold 80%
MeOH–20% Et2O (50 mL), ice-cold MeOH (10 mL), and then Et2O
(10 mL). Drying in vacuo yielded 6.6794 g (70%) of 9 as pale yel-
low solid.
1H NMR (400 MHz, DMSO-d6): d = 12.34 (br s, 1 H, NH), 10.54
(br s, 1 H, NH), 7.49 (dd, J = 2.4 Hz, 3.2 Hz, 1 H, H-8), 6.49 (dd,
J = 1.6 Hz, 3.2 Hz, 1 H, H-7), 2.78 [sept, J = 6.8 Hz, 1 H,
CH(CH3)2], 1.08 [d, J = 6.8 Hz, 6 H, CH(CH3)2].
13C NMR (100 MHz, DMSO-d6): d = 174.7, 152.8, 151.2, 150.3,
127.1, 112.9, 98.9, 34.4, 19.3.
7-Deaza-2¢-deoxy-7-nitroadenosine (5)
Compound 4 (3.205 mg, 7.61 mmol) was dissolved in anhyd MeOH
(38 mL) and cooled in an ice bath. A solution of NaOMe in MeOH
(0.5 M, 76 mL) was added under ice cooling. The ice bath was re-
moved and the mixture was stirred for 5 h at r.t. After neutralization
with AcOH (~1.2 mL) to pH ~8, silica gel (10 g) was added. The
solvent was evaporated and the residue was loaded on to a silica gel
column (Merck 9385, 40 g). The column was eluted with 7.5–10%
MeOH–CH2Cl2 and fractions containing 7-NO2-dA (5) were com-
bined and evaporated. The yellow residue was triturated in MeOH
(10 mL) and the solid was collected by filtration. After washing
with MeOH (2 × 3 mL) and drying under vacuum overnight, com-
pound 5 was obtained as light yellow powder (1.778 mg, 79%).
Evaporation of the filtrate produced additional 212 mg of product as
yellow caramel (9%), which was almost as pure as the yellow pow-
der.
6-Chloro-7-deaza-2-isobutyrylamino-7-nitroguanine (10)
Solid 9 (6.2651 g, 26.25 mmol) was added to stirred, ice-chilled
fuming HNO3 (25 mL). This solution was stirred under ice-bath
cooling for 2 h, during which time the reaction took on a bright yel-
low color. This solution was then added dropwise to stirred ice wa-
ter (600 mL). Within 5 min, a pale-yellow precipitate formed. This
solution was allowed to warm to r.t., with continued stirring, for 3 h.
The precipitate was collected by filtration using a fine-fritted filter
funnel, washed with H2O until the filtrate was no longer acidic, and
then dried in vacuo to yield 5.8055 g (78%) of 10 as pale yellow sol-
id.
1H NMR (400 MHz, DMSO-d6): d = 13.69 (br s, 1 H, NH), 10.87
(br s, 1 H, NH), 8.81 (s, 1 H, H-8), 2.79 [sept, J = 6.8, 1 H,
CH(CH3)2], 1.09 [d, J = 6.8Hz, 6 H, CH(CH3)2].
13C NMR (100 MHz, DMSO-d6): d = 174.9, 152.9, 152.6, 151.0,
132.1, 127.2, 104.7, 34.6, 19.2.
1H NMR (400 MHz, CD3OD): d = 8.72 (s, 1 H, H-8), 8.19 (s, 1 H,
H-2), 6.59 (t, J = 6.4 Hz, 1 H, H-1¢), 4.53 (td, J = 3.1, 6.2 Hz, H-3¢),
4.04 (td, J = 3.1, 3.1 Hz, 1H, H-4¢), 3.85 (dd, J = 3.1, 12.1 Hz, 1 H,
H-5¢b), 3.76 (dd, J = 3.5, 12.1 Hz, 1 H, H-5¢a), 2.79 (td, J = 6.6, 13.2
Hz, 1 H, H-2¢b), 2.45 (ddd, J = 3.5, 6.2, 13.3 Hz, 1 H, H-2¢a).
1D NOE between H-1¢ and H-8 signals: irradiating at d = 6.59 (H-
1¢) caused 4% enhancement on the signal at d = 8.72 (H-8); irradi-
ating at d = 8.72 caused 5% enhancement on the signal at d = 6.59.
7-Deaza-2¢-deoxy-7-nitroinosine (6)
Solid 5 (61.6 mg, 0.21 mmol) was dissolved in a mixture of AcOH
(4 mL) and H2O (4 mL). To the solution, isoamyl nitrite (140 mL,
1.04 mmol) was added. After stirring at r.t. for 15 h, the mixture was
analyzed using TLC by developing in 5:1 CH2Cl2–MeOH. To the
mixture an additional amount of isoamyl nitrate (140 mL) was add-
ed, and the resulting mixture was allowed to react for 25 h. TLC
analysis suggested that the product contained a mixture of ~1:1 7-
NO2-dA (5) and 7-NO2-dI (6). To this mixture an additional amount
of isoamyl nitrite (140 mL) was added and the mixture stirred for
3 days. TLC results indicated the formation of side products in ad-
dition to compound 6. The mixture was concentrated to dryness,
and the residue was purified using preparative TLC. Beside the re-
covered starting material 5, compound 7 was obtained as a side
product (26.2 mg, 37%). The desired product 6 was obtained as yel-
low caramel (23.4 mg, 38%).
Compound 10 was methylated at N9 position and the resulting com-
pound was analyzed by 1D NOE experiments between H8 and N9-
CH3 signals.
Methylated 10
1H NMR (400 MHz, DMSO-d6): d = 10.93 (1 H, s, NH), 8.92 (1 H,
s, H-8), 3.82 (3 H, s, NCH3), 2.83 (1 H, m, COCH), 1.10 (6 H, d,
J = 6.8 Hz, 2 CH3).
Irradiation at d = 8.92 (H-8) caused 5% enhancement on the signal
at d = 3.82 (NCH3); irradiation at d = 3.82 caused 4% enhancement
on the signal at d = 8.92.
Compound 6
1H NMR (400 MHz, CD3OD): d = 12.4 (br s, 1 H, NH), 8.56 (s, 1
H, H-8), 8.02 (s, 1 H, H-2), 6.63 (t, J = 6.6 Hz, 1 H, H-1¢), 4.52 (m,
1 H, H-3¢), 4.02 (m, 1 H, H-4¢), 3.83 (dd, J = 3.5, 12.1 Hz, 1 H, H-
5¢b), 3.76 (dd, J = 3.9, 12.1 Hz, 1 H, H-5¢a), 2.54 (m, 1 H, H-2¢b),
2.46 (ddd, J = 3.5, 6.2, 13.7 Hz, 1 H, H-2¢a).
7-Deaza-O6-methyl-7-nitroguanine (11)
A solution of 10 (4.6497 g, 16.39 mmol) in 0.5 M NaOMe in MeOH
(100 mL) was refluxed for 8 h. Following the removal of the heating
source, the mixture was cooled to 0 °C in an ice-water bath,
Synthesis 2006, No. 19, 3280–3290 © Thieme Stuttgart · New York

3288
T. Kawate et al.
SPECIAL TOPIC
quenched with glacial AcOH (2 mL), and then evaporated onto sil-
ica gel. Silica gel flash chromatography (~10% MeOH in CH2Cl2)
yielded 2.2183 g (65%) of 11 as a bright yellow solid.
1H NMR (400 MHz, DMSO-d6): d = 12.44 (br s, 1 H, NH), 8.18 (s,
1 H, H-8), 6.57 (br s, 2 H, NH2), 3.95 (s, 3 H, OCH3).
13C NMR (100 MHz, DMSO-d6): d = 162.4, 160.5, 154.7, 128.5,
125.8, 89.6, 53.4 (OCH3).
mL). This solution was washed with Et2O (3 × 100 mL), and then
evaporated to afford 15 as a bright yellow solid (79%).
1H NMR (400 MHz, DMSO-d6): d = 10.80 (br s, 1 H, NH), 8.25 (s,
1 H, H-8), 6.65 (br s, 2 H, NH2), 6.34 (dd, J = 5.9, 7.3 Hz, 1 H, H-
1¢), 5.27 (m, 1 H, OH), 5.04 (m, 1 H, OH), 4.32 (m, 1 H, H-3¢), 3.82
(m, 1 H, H-4¢), 3.58 (m, 2 H, H-5¢a and H-5¢b), 2.38 (ddd, J = 5.9,
7.3, 13.2 Hz, 1 H, H-2¢b), 2.20 (ddd, J = 2.9, 5.9, 13.2 Hz, 1 H, H-
2¢a).
3¢,5¢-Bis[O-(4-chlorobenzoyl)]-7-deaza-2¢-deoxy-O6-methyl-7-
nitroguanosine (13)
7-Deaza-2¢-deoxy-7-nitroadenosine 5¢-O-Triphosphate (16)
Compound 5 (32.3 mg, 0.109 mmol) was co-evaporated twice from
anhyd pyridine and dried overnight under high vacuum. Its suspen-
sion in trimethyl phosphate (0.25 mL) was sonicated for 20 sec and
cooled in an ice-bath. To the mixture was added POCl3 (12 mL,
0.129 mmol) and stirred for 3.5 h at 0 °C. To this mixture was added
simultaneously a 0.5 M solution of (Bu3NH)2H2P2O7 in DMF
(1 mL, 0.5 mmol) and Bu3N (0.13 mL, 0.54 mmol). After stirring
vigorously for 1 h at 0 °C, the mixture was poured into ice-cold 1.5
M triethylammonium bicarbonate (TEAB, pH 7.5, 10 mL) and lyo-
philized. The yellow residue was dissolved in H2O (10 mL) and pu-
rified by ion-exchange chromatography [DEAE Sephadex A25,
9 × 2.5 cm, linear gradient of TEAB (0.05 to 1.5 M, 1500 mL), flow
5 mL/min]. The triphosphate fractions, eluted at 0.6–0.75 M TEAB,
were pooled and lyophilized. The residue was purified by reversed
phase HPLC [Hamilton PRP-1, f 10 mm × l 250 mm, flow 4 mL/
min] with a gradient of MeOH mixed with 0.1 M triethylammonium
acetate (TEAA, pH 8.5). The triphosphate product was eluted at 39–
41% MeOH. Lyophilization (3 times from H2O) gave triethylam-
monium salt of 16 as a white solid. UV quantitation indicated that
33 mmoles of product was obtained, corresponding to 30% yield. 1H
NMR showed this material contains ~3 moles of Et3N per mole of
triphosphate.
A solution of 11 (1.4765 g, 7.059 mmol) in anhyd DMF (14 mL)
was added dropwise to a stirred, ice-water-chilled suspension of
NaH (60% w/w mineral oil dispersion, 1.3 equiv, 9.275 mmol, 371
mg) in anhyd DMF (10 mL). During and after the addition, the vig-
orous evolution of gas (H2) was observed. After stirring for 1 h, this
solution was drawn into a dry syringe, and then was added dropwise
to a ice-chilled, stirring suspension of 12 (3.179 g, 7.398 mmol, 1.05
equiv) in anhyd MeCN (150 mL). This suspension was warmed to
r.t. and stirred for an additional 2 h. The mixture was then poured
into CH2Cl2 (I L) and the CH2Cl2 layer was washed with H2O
(2 × 300 mL), 10% (w/v) aq LiBr (2 × 200 mL), and brine (400
mL). The resulting organic layer was dried (Na2SO4), and then
evaporated. During concentration, a yellow precipitate formed. This
precipitate was collected by filtration, washed with Et2O, and
proved to be pure product by TLC (1:2:7 EtOAc–hexane–CH2Cl2)
and 1H NMR spectroscopy. The remaining solution was evaporated
onto silica gel and flash-chromatographed with EtOAc–hexane–
CH2Cl2 (1:2:7) to isolate the remaining product. The combination of
filtration and chromatography yielded 2.5973 g (61%) of 13.
1H NMR (400 MHz, DMSO-d6): d = 8.46 (s, 1 H, H-8), 8.04 (d,
J = 8.6 Hz, 2 Harom), 7.96 (d, J = 8.6 Hz, 2 Harom), 7.65 (d, J = 8.6
Hz, 2 Harom), 7.57 (d, J = 8.6 Hz, 2 Harom), 6.83 (br s, 2 H, NH2), 6.56
(dd, J = 6.4, 8.0 Hz, 1 H, H-1¢), 5.72 (m, 1 H, H-3¢), 4.68 (dd,
J = 7.4, 14.1 Hz, 1 H, H-5¢b), 4.57 (m, 2 H, H-5¢a and H-4¢), 3.96 (s,
3 H, OCH3), 3.09 (td, J = 7.8, 14.4 Hz, 1 H, H-2¢b), 2.73 (ddd,
J = 2.3, 6.2, 14.4 Hz, 1 H, H-2¢a).
Compound 16·3N(CH2CH3)3
1H NMR (400 MHz, D2O): d = 8.63 (s, 1 H, H-2), 8.16 (s, 1 H, H-
8), 6.57 (t, J = 5.6 Hz, 1 H, H-1¢), 4.76 (m, 1 H, H-3¢, overlapped
with HOD), 4.28 (br, 1 H, H-4¢), 4.18 (m, 2 H, H-5¢), 3.18 (q, J = 7.1
Hz, 18 H, NCH2), 2.70 (m, 1 H, H-2¢), 2.59 (m, 1 H, H-2¢), 1.26 (s,
27 H, NCH2CH3).
3',5'-Bis[O-(4-chlorobenzoyl)]-7-daza-2'-deoxy-7-nitrogua-
nosine (14)
31P NMR (162 MHz, D2O): d = –10.9 (m, g), –11.4 (d, J = 17.2 Hz,
a), –23.3 (m, b).
To a suspension of 13 (2.2519 g, 3.738 mmol) and NaI (1.12 g,
7.476 mmol, 2 equiv) in anhyd MeCN (150 mL) was added freshly
distilled (from K2CO3) chlorotrimethylsilane (0.72 mL, 1.5 equiv).
After refluxing the resulting suspension for 4 h, the mixture was
cooled to 0 °C with ice-water, quenched with MeOH (2 mL), and
then evaporated to dryness. The residue was redissolved in CH2Cl2
(800 mL) washed with half-saturated NaCl (200 mL), 10% (w/v) aq
Na2SO3 (200 mL), and brine (200 mL), dried (Na2SO4), and then
evaporated onto silica gel. Silica gel flash chromatography with
CH2Cl2–MeOH (19:1) gave 1.6121 g (73%) of 14 as a bright yellow
solid.
1H NMR (400 MHz, DMSO-d6): d = 10.87 (br s, 1 H, NH), 8.25 (s,
1 H, H-8), 8.03 (d, J = 8.2 Hz, 2 Harom), 7.98 (d, J = 8.2 Hz, 2 Harom),
7.65 (d, J = 8.2 Hz, 2 Harom), 7.58 (d, J = 8.2 Hz, 2 Harom), 6.68 (br
s, 2 H, NH2), 6.47 (dd, J = 6.2, 8.2 Hz, 1 H, H-1¢), 5.70 (m, 1 H, H-
3¢), 4.66 (dd, J = 6.6, 13.3 Hz, 1 H, H-5¢b), 4.56 (m, 2 H, H-4¢ and
H-5¢a), 3.02 (m, 1 H, H-2¢b), 2.72 (ddd, J = 2.3, 6.2, 14.4 Hz, 1 H,
H-2¢a).
The sample was reconstituted in H2O (~1 mL) and treated with
Dowex-50W × 8 resin (Na+ form, 600 mg) to exchange the counter
ion to Na+. The resin was removed by filtration and washed with
H2O (600 mL total). The combined aqueous solution was lyo-
philized to afford 16.
Compound 16
1H NMR (D2O, 400 MHz): d = 8.68 (s, 1 H, H-2), 8.21 (s, 1 H, H-
8), 6.64 (t, J = 6.7 Hz, 1 H, H-1¢), 4.77 (m, 1 H, H-3¢, overlapped
with HOD), 4.28 (m, 1 H, H-4¢), 4.18 (m, 2 H, H-5¢), 2.70 (m, 1 H,
H-2¢), 2.58 (m, 1 H, H-2¢).
31P NMR (162 MHz, D2O): d = –9.7 (m, g), –11.4 (d, J = 20.8 Hz,
a), –22.9 (t, J = 18.9 Hz, b).
7-Deaza-2¢-deoxy-7-nitroguanosine 5¢-O-Triphosphate (17)
A mixture of Proton-Sponge® [1,8-bis(dimethylaminonaphthalene,
34.7 mg, 151 mmol] and 15 (25.1 mg, 81 mmol) in PO(OMe)3 (250
mL) was sonicated for 1 min and cooled in an ice-bath. POCl3 (12
mL, 129 mmol) was added and the mixture was stirred under ice
cooling for 5 h. A solution of (Bu3NH)2H2P2O7 in DMF (0.5 M, 0.81
mL, 405 mmol) and Bu3N (96 mL, 403 mmol) were added and the
whole was stirred for another 1 h. The mixture was poured into 1 M
TEAB (pH 7.5, 20 mL) and the mixture was vortexed for 2 min and
kept for 1 h at r.t.. The resulting solution was washed with Et2O
(3 × 5 mL) and lyophilized. The residue was dissolved with H2O
7-Deaza-2¢-deoxy-7-nitroguanosine (15)
To a solution of 14 (161.6 mg, 0.2747 mmol) in anhyd CH2Cl2 (10
mL) was added a 50 mM solution of K2CO3 (20 mL) in anhyd
MeOH. This solution was stirred at r.t. for 1 h, during which time it
turned bright orange. The reaction was neutralized by the addition
of Dowex 50WX-100 (H+ form), which was later removed by vac-
uum filtration. The solution was evaporated to dryness, and the res-
idue was resuspended in MeOH (30 mL) and diluted with H2O (150
Synthesis 2006, No. 19, 3280–3290 © Thieme Stuttgart · New York

SPECIAL TOPIC
7-Nitropurine Nucleosides and Nucleotides
PCR Incorporation of 16, 17 or 18
3289
(30 mL) and fractionated on a Sephadex-A-25 DEAE column (f 2.5
cm × l 10 cm, 10 g resin, ca 4 mL/min) eluted with H2O (200 mL)
followed by 0 → 1 M linear gradient of TEAB (pH 7.5, 1000 mL).
Fractions corresponding to 0.69–0.82 M TEAB were pooled and
lyophilized. The residue was purified with reversed phase HPLC
(Hamilton PRP-1, f 10 mm × l 250 mm, flow 4 mL/min) with a gra-
dient of MeOH mixed with 0.1 M TEAA, pH 8.5. The triphosphate
product was eluted at 38–50% MeOH. Fractions containing triphos-
phate were pooled and lyophilized. The residue was lyophilized re-
peatedly from H2O to give a triethylammonium salt of 16 as yellow
solid. 1H NMR showed this material contains ~2 moles of Et3N per
mole of triphosphate.
Primers were purchased from Sigma-Genosys (Woodlands, TX).
Nucleotide dNTPs were from Amersham Biosciences (Piscataway,
NJ), AmpliTaq Gold polymerase, 10 × Taq buffer with 15 mM
MgCl2 were from Applied Biosystems (Foster City, CA). Typical
PCR reactions (20 mL or 500 mL) contained 1 × PCR buffer; 1.5
mM MgCl2; 200 mM 7-NO2-dATP (16), 7-NO2-dGTP (17), or 7-
NO2-dITP (18); 200 mM each remainder three unmodified nucle-
otides, i.e. d(CGT)TPs or d(ACT)TPs; 1 mM Primer I, 1 mM Primer
II, and 2 ng/mL plasmid DNA template.14,27 A typical PCR cycling
protocol was one cycle of (94 °C, 120 s) followed by 40 cycles of
(94 °C, 60 s; 55 °C, 60 s; 72 °C, 120 s) followed by one cycle of (72
°C, 600 s). Each amplified DNA sample was purified using Sepha-
dex G50 spin columns (Amersham). The collected eluates were
dried on a Savant speedvac (Farmingdale, NY), followed by recon-
stitution in 50 mL H2O.
Compound 17·2N(CH2CH3)3
1H NMR (400 MHz, D2O): d = 8.28 (s, 1 H, H-8), 6.45 (t, J = 6.9
Hz, 1 H, H-1¢), 4.76 (m, 1 H, H-3¢), 4.24 (br, 1 H, H-4¢), 4.17 (m, 2
H, H-5¢), 3.18 and 3.06 (q, J = 7.2 Hz, 12 H, NCH2), 2.70 (m, 1 H,
H-2¢), 2.47 (m, 1 H, H-2¢), 1.26 and 0.94 (s, 18 H, NCH2CH3).
Chemical Cleavage
Each DNA sample (25 mL out of the 50 mL of purified PCR product,
lyophilized) was subject to chemical cleavage at 95 °C for 60 min
in a thermocycler with heated lid. A typical alkaline cleavage reac-
tion mixture contained DNA sample in 100 mL of 1 M piperidine,
1 M pyrrolidine, or 1 M 3-pyrrolidinol. A piperidine/TCEP/Tris
cleavage reaction contained a DNA sample in 100 mL of 1 M pipe-
ridine, 0.2 M TCEP [tris(2-carboxyethyl)phosphate], and 0.5 M
Tris base [tris(hydroxymethyl)aminomethane]. A pyrrolidine/
TCEP/Tris cleavage reaction contained a DNA sample in 100 mL of
1 M pyrrolidine, 0.2 M TCEP, and 0.5 M Tris base. An NH4OH/
TCEP cleavage reaction contained a DNA sample in 100 mL of 0.2
M TCEP and ~30% NH4OH. The cleavage samples were concen-
trated to dryness using speedvac, and each was dissolved in 500 mL
of 0.2 M TEAA (triethylammonium acetate). The resulting aqueous
solutions were desalted using OASIS® columns from Waters (Mil-
ford, MA), using procedures recommended by the manufacturer.
The collected MeCN eluates containing DNA were concentrated to
dryness, and then dissolved in 100 mL of H2O.
The sample was reconstituted in H2O (~1 mL) and treated with
Dowex-50W × 8 resin (Na+ form, 600 mg) to exchange the counter
ion to Na+. The resin was removed by filtration and washed with
H2O (600 mL total). The combined aqueous solution was lyo-
philized to afford 17 (14.6 mg, 28%) as orange-yellow foam.
Compound 17
1H NMR (400 MHz, D2O): d = 8.32, (s, 1 H, H-8), 6.48 (t, J = 6.8
Hz, 1 H, H-1¢), 4.80 (m, 1 H, H-3¢, overlapped with HOD), 4.17–
4.32 (m, 3 H, H-4¢ and H-5¢), 2.72 (m, 1 H, H-2¢b), 2.50 (ddd,
J = 3.9, 6.2, 14.1 Hz, 1 H, H-2¢a).
31P NMR (162 MHz, D2O): d = –6.22 (d, J = 18.3Hz, g), –10.9 (d,
J = 19.5 Hz, a), –21.91 (t, J = 19.5 Hz, b).
7-Deaza-2¢-deoxy-7-nitroinosine 5¢-O-Triphosphate (18)
A mixture of Proton-Sponge® (9.3 mg, 43.4 mmol) and 7-NO2-dI (6,
6.5 mg, 21.9 mmol) was suspended in H2O (0.5 mL) and lyophilized.
The residue was dissolved in PO(OMe)3 (100 mL) and cooled in an
ice-bath. POCl3 (2.5 mL, 26.8 mmol) was added to the yellow col-
ored solution, and the resulting purple colored mixture was stirred
under ice cooling for 3 h. A solution of (Bu3NH)2H2P2O7 in DMF
(0.5 M, 0.22 mL, 110 mmol) and Bu3N (26 mL, 109 mmol) were add-
ed and the mixture was stirred under ice cooling for another 1 h. The
mixture was poured into 1 M TEAB (pH 7.5, 10 mL) and the mix-
ture was stirred for 1 h at r.t.. The resulting solution was washed
with Et2O (3 × 10 mL) and lyophilized. The residual white solid
was dissolved with H2O (15 mL) and fractionated on a Sephadex-
A-25 DEAE column (f 2.5 cm × l 10 cm, 10 g resin, ca. 3.5 mL/
min), eluted with H2O (150 mL) followed by 0 → 1 M linear gradi-
ent of TEAB (pH 7.5, 1000 mL). Fractions corresponding to 0.65–
0.82 M TEAB were pooled and lyophilized. The residue was puri-
fied with reversed phase HPLC (Hamilton PRP-1, f 10 mm × l 250
mm, flow 4 mL/min) with a gradient of MeOH mixed with 0.1 M
TEAA, pH 8.5. The triphosphate product was eluted at 38–39%
MeOH; the corresponding fractions were pooled and lyophilized.
The residue was lyophilized again from H2O (3 mL) to remove tri-
ethylamine salts. The sample was reconstituted in H2O (0. 4 mL)
and treated with 400 mg of Dowex-50W × 8 resin (Na+ form) to ex-
change the counter ion to Na+. The resin was removed by filtration
and washed with H2O (3 × 400 mL). The combined aqueous solution
was lyophilized to afford 18 as tetrasodium salt (4.66 mg, 34%).
PAGE Analysis of 32P-Labeling of Primers, PCR Products, and
Cleavage Products
T4 DNA polynucleotide kinase and 10 × T4 kinase buffer were
from New England Biolabs (Beverly, MA). [g-32P]-ATP was from
PerkinElmer (Boston, MA). A typical labeling reaction contained 1
mL 10 × T4 kinase buffer, 1 mL 20 mM primer (or 1 mL purified PCR
product, or 1 mL cleavage product), 0.5 mL 1.67 mM [g-32P]-ATP
(6000 Ci/mmol), 6.5 mL distilled H2O, and 1 mL 10 U/mL T4 kinase.
After incubation at 37 °C for 60 min, to each labeling reaction mix-
ture was added 20 ml of 1 × TE (10 mM Tris/1 mM EDTA) pH 8
buffer. The resulting aqueous solutions were purified using Sepha-
dex G50 columns and the resulting eluates were adjusted to 50 mL
using distilled water. Each 32P-labeled sample (~1 mL) was mixed
with formamide loading dye, separated on a 12% denaturing poly-
acrylamide gel, and subjected to autoradiography.
MALDI-TOF Mass Spectrometry Analysis
The remainder of each purified cleavage sample (~99 mL) was con-
centrated to dryness. After resuspending in 3 mL of 3-hydroxypi-
colinic acid/diammonium citrate matrix solution a 0.6 mL aliquot of
the sample was deposited on a stainless steel target plate and al-
lowed to air dry. The sample was analyzed with a Voyager DE Lin-
ear mass spectrometer equipped with a 337 nm nitrogen laser
(Applied Biosystems). Analysis was carried out in the positive ion
mode with delayed extraction. The voltage for acceleration was 20
kV, while the grid voltage was at 92.5%. The guide wire voltage
was set at 0.1% and the delay time was 250 ns. Typically 20 indi-
vidual spectra were added to generate a summed spectrum.
1H NMR (400 MHz, D2O): d = 8.61 (s, 1 H, H-8 or H-2), 8.19 (s, 1
H, H-2 or H-8), 6.67 (t, J = 6.6 Hz, 1 H, H-1¢), 4.80 (m, 1 H, H-3¢,
overlapped with H2O), 4.30 (m, 1 H, H-4¢), 4.17–4.26 (m, 2 H, H-
5¢), 2.74 (m, 1 H, H-2¢b), 2.59 (ddd, J = 4.3, 6.6, 14.1 Hz, 1 H, H-
2a¢).
31P NMR (162 MHz, D2O): d = –6.27 (d, J = 19.5 Hz, g), –10.95 (d,
J = 19.5 Hz, a), –21.85 (t, J = 19.5 Hz, b).
Synthesis 2006, No. 19, 3280–3290 © Thieme Stuttgart · New York

3290
T. Kawate et al.
SPECIAL TOPIC
(12) Kawate, T.; Allerson, C. R.; Wolfe, J. L. Org Lett. 2005, 7,
3865.
(13) Hayashibara, K. C.; Verdine, G. L. J. Am. Chem. Soc. 1991,
113, 5104.
Acknowledgment
This research was supported by Variagenics, Inc. We thank our col-
leagues at Variagenics for their assistance and helpful discussions.
(14) Wolfe, J. L.; Kawate, T.; Sarracino, D. A.; Zillmann, M.;
Olson, J.; Stanton, V. P. Jr.; Verdine, G. L. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 11073.
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Synthesis 2006, No. 19, 3280–3290 © Thieme Stuttgart · New York