Regioselective syntheses of 7-nitro-7-deazapurine nucleosides and nucleotides for efficient PCR incorporation

Article abstract of DOI:10.1021/ol051144r

(Chemical Equation Presented) Substitution at the C⁷ position of purine nucleotides by a potent electron-withdrawing nitro group facilitates the cleavage of glycosidic bonds under alkaline conditions. This property is useful for sequence-specif

Full text of DOI:10.1021/ol051144r

ORGANIC
LETTERS
2005
Vol. 7, No. 18
3865-3868
Regioselective Syntheses of
7-Nitro-7-deazapurine Nucleosides and
Nucleotides for Efficient PCR
Incorporation
Tomohiko Kawate,†,‡ Charles R. Allerson,†,§ and Jia Liu Wolfe*,†,‡
Variagenics, Inc., Cambridge, Massachusetts 02139
wolfe@molbio.mgh.harVard.edu
Received May 16, 2005
ABSTRACT
Substitution at the C7 position of purine nucleotides by a potent electron-withdrawing nitro group facilitates the cleavage of glycosidic bonds
under alkaline conditions. This property is useful for sequence-specific cleavage of DNA containing these analogues. Here we describe the
preparation of 7-deaza-7-NO2-dA and 7-deaza-7-NO2-dG using two different approaches, starting from 2′-deoxy-adenosine and 6-chloro-7-
deaza-guanine, respectively. These modified nucleosides were converted to nucleotide triphosphates, each of which can replace the
corresponding, naturally occurring triphosphate to support PCR amplification.
Modified purines and purine nucleosides have demonstrated
broad utility as pharmaceuticals as well as biochemical tools.
Compounds containing the pyrrolo[2,3-d]pyrimidine system,
which often is referred to as 7-deazapurine, have shown
useful pharmaceutical properties, either as naturally occurring
antibiotics or synthetic derivatives of 7-deazapurine.1 In
addition, oligonucleotides containing 7-deazapurine deriva-
tives often exhibit interesting properties, such as increased
duplex stability,2 diminished nuclease susceptibility,3 and
altered chemical reactitivity,4,5 that are useful for biological
applications.
Recently, 7-nitro-7-deazapurines have been utilized in a
DNA genotyping method (Incorporation and Complete
(2) (a) Seela, F.; Shaikh, K. I. Tetrahedron 2005, 61, 2675-2681. (b)
Balow, G.; Mohan, V.; Lesnik, E.; Johnson, J. F.; Monia, B. P.; Acevedo,
O. L. Nucleic Acids Res. 1998, 26, 3350-3357.
(3) Seela, F.; Roeling, A. Nucleic Acids Res. 1992, 20, 55-61.
(4) Seela, F.; Feiling, E.; Gross, J.; Hillenkamp, F.; Ramzaeva, N.;
Rosemeyer, H.; Zulauf, M. J. Biotechnol. 2001, 86, 269-279.
(5) (a) Min, C.; Cushing, T. D.; Verdine, G. L. J. Am. Chem. Soc. 1996,
118, 6116-6120. (b) Storek, M. J.; Ernst, A.; Verdine, G. L. Nat. Biotechnol.
2002, 20, 183-186.
† Variagenics, Inc.
‡ Current affiliation: Department of Molecular Biology, Massachusetts
General Hospital, Boston, MA 02114.
§ Current affiliation: Isis Pharmaceuticals, Inc., Carlsbad, CA 92008.
(1) Amarnath, V.; Madhav, R. Synthesis 1974, 12, 837-859.
10.1021/ol051144r CCC: $30.25
© 2005 American Chemical Society
Published on Web 08/12/2005

Chemical Cleavage, ICCC)6 and a footprinting technique
(Template Directed Interferance, TDI) that maps DNA-
protein interactions.5 Both technologies are based on incor-
poration of a chemically labile nucleotide by PCR followed
by specific chemical cleavage of the resulting amplicon at
modified bases. Whereas TDI uses each modified nucleotide
to partially substitute a particular base, ICCC requires the
complete displacement of a selected base by its cleavable
analogue for subsequent, complete cleavage at the sites of
modification. Modeled after N7-methyl-dGTP, which is labile
under alkaline conditions as a result of its electron-withdraw-
ing CH3-N7+ moiety,7 7-deaza-7-nitro-dATP (7-NO2-dATP)
and 7-deaza-7-nitro-dGTP (7-NO2-dGTP) were designed to
provide similar sensitivity to alkaline treatment while gaining
thermal stability to survive PCR cycles.5,6
improved procedure was the use of a large molar excess of
acetic anhydride to effect acetylation at the exocyclic amino
group of 1 in addition to the two hydroxyl groups. This
change resulted in quantitative conversion of 1 to a 97:3
mixture of tri-acetylated and tetra-acetylated 7-deaza-dA (2).
Nitration of 2 with 1:1 fuming nitric acid and concentrated
sulfuric acid yielded 91% of 3 after purification by silica
column chromatography. Removal of acetyl protecting
groups generated 4 in a purified yield of 79%. It should be
noted here that Seela et al. have also described a similarly
improved procedure for the preparation of 4 and definitively
assigned its structure using X-ray crystallography.8
The much improved synthesis due to additional acetylation
of exocyclic amino group may be attributable to reduced
electrophilic attack at C8. As illustrated in Figure 1,
Although nonsubstituted 7-deaza-dATP and 7-deaza-dGTP
have been successfully incorporated by PCR, only the latter
could completely replace the corresponding native nucleotide
dGTP.3 In our initial PCR attempts, it was observed that even
though both 7-NO2-dATP and 7-NO2-dGTP can partially
replace dATP and dGTP, respectively, it was challenging to
incorporate 7-NO2-dGTP without the dGTP supplement.
Since it is well-known that the quality of nucleotide
triphosphates is important for the success of PCR, we set
out to improve the synthesis and purification process for
7-NO2-dATP and 7-NO2-dGTP. Described in this report are
our optimized synthetic procedures that led to their successful
application in ICCC.6
Figure 1. Mesomeric stabilization of electrophilic attack at C8 (A)
and protonation of N3 (B) of 7-deaza-dA (1).
7-NO2-dA (4) was prepared according to a procedure
described by the Verdine group5a with some changes that
are beneficial to isolation and purification of the intermedi-
ates. As summarized in Scheme 1, 4 was conveniently
unprotected NH2 may direct electrophilic attack at C8 and/
or increase the likelihood of N3 protonation and subsequent
depurination, resulting from mesomeric stabilization of
σ-complexes A and B, respectively. Acetylation of NH2
reduces the electron density on the nitrogen and thus favors
the nitration at 7-position.
Scheme 1. Synthesis of 7-NO2-7-deaza-dA (4)
The synthesis of 7-NO2-dG (12) has been reported by
Storek et al., starting from 6-Cl-7-deazaguanine and involv-
ing glycosylation, 6-methoxylation, and exocyclic amine
protection, followed by nitration. Unfortunately a mixture
of 7- and 8-NO2 substituted 7-deaza-dG analogues were
obtained that were difficult to separate.5b To improve the
regioselectivity of the nitration reaction, we attempted to
predict what synthetic intermediates could promote C7-
nitration based on a stabilized transition state. Shown in
Figure 2 are a few postulated σ-complexes that would be
involved in the nitration of a particular synthetic intermediate.
Structure A illustrates that electrophilic attack at the 8-posi-
tion is promoted by 2-NH2 in 7-deaza-dG, which was first
recognized by the Seela group.9 Structure B shows that the
6-OMe group could direct electrophilic attack to the 8-posi-
tion of 6-OMe-7-deaza-dG, even though 2-NH2 is protected.
Structure C shows how N9 could help direct an attack by an
electrophile (E) to the desired 7-position, as previously
proposed by Seela et al.9 Both σ-complexes B and C could
be involved in the aforementioned nitration procedure5b that
prepared in three steps from now commercially available
7-deaza-dA (1, also known as 2′-deoxytubercidin), in sub-
stantially better yields (72% overall) than what was previ-
ously reported (33% overall). One key feature of this
(6) 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,
11073-11078.
(7) Hayashibara, K. C.; Verdine, G. L. J. Am. Chem. Soc. 1991, 113,
5104-5106.
(8) Seela, F.; Rosemeyer, H.; Zulauf, M.; Chen, Y.; Kastner, G.; Reuter,
H. Liebigs Ann./Recl. 1997, 2525-2530.
(9) (a) Ramzaeva, N.; Seela, F. HelV. Chim. Acta 1995, 78, 1083-1090.
(b) Seela, F.; Peng, X. Synthesis 2004, 8, 1203-1210.
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Org. Lett., Vol. 7, No. 18, 2005

and concentrated sulfuric acid generated the single regio-
isomer 6-Cl-7-nitro-7-deaza-guanine (7), which readily pre-
cipitated from the reaction mixture and was isolated in 78%
yield. This excellent regioselectivity made chromatographic
purification unnecessary and was likely the result of a
favorable conjugation shown in Figure 2D. Treating 7 with
sodium methoxide displaced the 6-chloro substituent with a
methoxy group while concurrently removed the isobutyryl
protection group of the exocylclic amine to afford compound
8.
The nucleobase-anion of 8 was generated using NaH and
was glycosylated in situ with 2-deoxy-3,5-di-O-(p-chloro-
benzoyl)-R-D-ribofuranosyl chloride (9) to produce the
desired â-isomer 10 in 61% isolated yield. Compound 9 was
prepared from 2-deoxy-D-ribose in three steps, following a
literature procedure.10 Demethylation of 10 using iodotri-
methylsilane liberated the 6-oxo group in 73% yield after
flash column chromatography. Deprotection of hydroxyl
groups by methanolysis generated 7-NO2-dG (12) in 79%
after column chromatography purification. This synthetic
procedure is a significant improvement over the existing
method,5b which required the separation of 7- and 8-substi-
tuted products using preparatory TLC. In comparison,
conversion of 6 to 7 was performed at a 3.3-g scale and did
not require chromatographical purification. The regiochem-
istry of the nitro group was confirmed by differential NOE
experiments on the N9-methyl derivative of 7 between the
proton at C8 and the protons of N9-methyl.
Figure 2. Mesomeric stabilization of electrophilic attack at C8 of
7-deaza-dG (A), C8 of 2-NH2-protected 6-OMe-7-deaza-dG (B),
C7 of 2-NH2-protected 6-OMe-7-deaza-dG (C), and C7 of 2-NH2-
protected 6-OMe-7-deazaguanine (D).
resulted in both 7- and 8-substitution. On the basis of these
considerations and the commercial availability of 6-Cl-7-
deaza-guanine, it was used as the starting material for
nitration. In addition, we reasoned that although the free
electron pair on N9 in structure C seems to be helpful for
7-substitution, an unglycosylated guanine might be an even
better choice (structure D), because the unsubstituted N9
could direct electrophilic attack to the 7-position without
increasing the risk of deglycosylation.
Scheme 2 depicts the six-step synthetic procedure for the
preparation of 7-NO2-dG (12). First the exocyclic amine of
6-Cl-7-deaza-guanine (5) was protected in 70% yield to
afford 6. Treatment with a 1:1 mixture of fuming nitric acid
7-NO2-dATP (13) and 7-NO2-dGTP (14) were prepared
from 7-NO2-dA (4) and 7-NO2-dG (12), respectively, ac-
cording to Scheme 3. Initially, we attempted to use a
Scheme 2. Synthesis of 7-NO2-7-deaza-dG (12)
Scheme 3. Preparation of Nucleotide Triphosphates
7-NO2-dATP (13) and 7-NO2-7-deaza-dGTP (14)
commonly used procedure that involves phosphorylation
using POCl3 in (MeO)3PO followed by in situ reaction with
pyrophosphate and subsequent anion exchange column
chromatography.5b,3 This process, however, generated nu-
(10) Eger, K.; Jalalian, M.; Schmidt, M. Tetrahedron 1994, 50, 8371-
8380.
Org. Lett., Vol. 7, No. 18, 2005
3867

cleotide triphosphates of low purity and in poor yields.
Consequently, several modifications to the procedure were
implemented. To maintain the neutrality of the phosphoryl-
ation reaction, during which HCl is released as a byproduct,
1,8-bis(dimethylamino)naphthalene (proton sponge), a strong
base with weak nucleophilic character due to steric effects,
was used to balance the pH in place of the more commonly
used alkylamines. In addition, we found that anion exchange
column chromatography purification alone did not provide
sufficiently pure nucleotide triphosphates. Further fraction-
ation by reversed-phase HPLC was necessary to remove
impurities that had comigrated with the triphosphates during
anion exchange column chromatography. After simple
conversion to the Na+ salt using Dowex-50W×8 resin (Na+
form), The final yields were 30% and 28% for 7-NO2-dATP
(13) and 7-NO2-dGTP (14), respectively. These highly pure
purine nucleotide analogues can completely replace dATP
and dGTP, respectively, in supporting PCR amplification of
DNA samples.6 These results suggest that (i) 7-NO2-dATP
and 7-NO2-dGTP can be efficiently incorporated by DNA
polymerase, and (ii) DNA products containing 7-NO2-dA
or 7-NO2-dG can also act as templates for polymerase
extension.
interactions by TDI footprinting.5 The synthesis and purifica-
tion principles discussed herein may also be generally
applicable for the preparation of other modified purine
analogues. For example, to our knowledge, the improved
three-step synthesis of 7-NO2-7-deaza-dA from 7-deaza-dA
is the most efficient route that can introduce an electrophilic
substitution at the 7-position of 7-deazapurine-2′-deoxy-
nucleoside.11 In addition, the discovery that 2-NH2-protected
6-chloro-7-deazaguanine is the most suitable substrate for
electrophilic attack at the 7-position may be instructive for
the preparation of other 7-substituted 7-deazapurine-2′-
deoxynucleosides, such as 7-halogenated and 7-alkylated
7-deazapurine-2′-deoxynucleosides.
Acknowledgment. This work was supported by Variagen-
ics, Inc. We thank Dr. Gregory Verdine and Dr. Alexander
Ernst of Harvard University for sharing experimental results
prior to publication.
Supporting Information Available: Experimental pro-
cedure and spectral data for synthesized compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
In conclusion, we have developed synthetic procedures
that can generate highly pure 7-NO2-dA and 7-NO2-dG, as
well as their nucleotide triphosphates. These reagents are
critical components for polymorphism analysis using ICCC,6
as well as studies of sequence-specific protein-DNA
OL051144R
(11) (a) Seela, F.; Zulauf, M. Synthesis 1996, 6, 726-730. (b) Buhr, C.
A.; Wagner, R. W.; Grant, D.; Froehler, B. C. Nucleic Acids Res. 1996,
24, 2974-2980.
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