The mechanism of selective purine C-nitration revealed: NMR studies demonstrate formation and radical rearrangement of an N7-nitramine intermediate

Article abstract of DOI:10.1021/ja042590x

Modified purine derivatives are of great importance in biomedical sciences, and substitution reactions on the purine skeleton are intensively studied. In our laboratory, an efficient and selective purine C2-nitration reaction was developed using a mixture of tetrabutylammonium nitrate and trifluoroacetic anhydride. The resulting 2-nitro moiety appeared to be a versatile handle to introduce a variety of pharmacophores onto the purine skeleton. Since the mechanism of this selective purine C2-nitration reaction has remained unclear, we now present an extensive NMR study leading to its elucidation, using N9-Boc-protected 6-chloropurine as a model compound. Direct electrophilic aromatic nitration of the highly electron-deficient C2 position was excluded, and we demonstrate that this reaction occurs in a three-step process. Electrophilic attack by trifluoroacetyl nitrate on the purine N7 position results in a nitrammonium species that is trapped by a trifluoroacetate anion furnishing N7-nitramine intermediate 11. This intermediate was characterized at -50°C by ¹H, ¹³C, ¹⁵N, and ¹⁹F NMR. At T > -40°C, the N7-nitramine intermediate undergoes a nitramine rearrangement, which generates a C2-nitro species that immediately eliminates TFA to give 2-nitro-6-chloro-9-Boc purine 10. The involvement of radicals during the nitramine rearrangement was unequivocally established by ¹⁵N-CIDNP. Moreover, the emission signal observed for the rearranged product, 2-nitropurine 10, showed that it is primarily formed in an intermolecular process. A quantitative radical trapping experiment finally disclosed that 65-70% of the nitramine rearrangement takes place intermolecularly.

Full text of DOI:10.1021/ja042590x

Published on Web 03/30/2005
The Mechanism of Selective Purine C-Nitration Revealed:
NMR Studies Demonstrate Formation and Radical
Rearrangement of an N7-Nitramine Intermediate
Boris Rodenko, Melle Koch, Alida M. van der Burg, Martin J. Wanner, and
Gerrit-Jan Koomen*
Contribution from the Van’t Hoff Institute for Molecular Sciences, UniVersiteit Van Amsterdam,
Nieuwe Achtergracht 129, NL-1018 WS Amsterdam, The Netherlands
Received December 9, 2004; E-mail: gjk@science.uva.nl
Abstract: Modified purine derivatives are of great importance in biomedical sciences, and substitution
reactions on the purine skeleton are intensively studied. In our laboratory, an efficient and selective purine
C2-nitration reaction was developed using a mixture of tetrabutylammonium nitrate and trifluoroacetic
anhydride. The resulting 2-nitro moiety appeared to be a versatile handle to introduce a variety of
pharmacophores onto the purine skeleton. Since the mechanism of this selective purine C2-nitration reaction
has remained unclear, we now present an extensive NMR study leading to its elucidation, using N9-Boc-
protected 6-chloropurine as a model compound. Direct electrophilic aromatic nitration of the highly electron-
deficient C2 position was excluded, and we demonstrate that this reaction occurs in a three-step process.
Electrophilic attack by trifluoroacetyl nitrate on the purine N7 position results in a nitrammonium species
that is trapped by a trifluoroacetate anion furnishing N7-nitramine intermediate 11. This intermediate was
characterized at -50 °C by 1H, 13C, 15N, and 19F NMR. At T > -40 °C, the N7-nitramine intermediate
undergoes a nitramine rearrangement, which generates a C2-nitro species that immediately eliminates
TFA to give 2-nitro-6-chloro-9-Boc purine 10. The involvement of radicals during the nitramine rearrangement
was unequivocally established by 15N-CIDNP. Moreover, the emission signal observed for the rearranged
product, 2-nitropurine 10, showed that it is primarily formed in an intermolecular process. A quantitative
radical trapping experiment finally disclosed that 65-70% of the nitramine rearrangement takes place
intermolecularly.
Scheme 1
Introduction
Given the pivotal role of purines in the regulation of many
biological processes and as constituents of DNA and RNA, the
interest in modified purine derivatives is immense in biomedical
sciences. For instance, these compounds have found application
as adenosine receptor ligands,1 antiparasitic agents,2,3 and cyclin-
dependent kinase inhibitors,4 antitumor,4-6 and antiviral com-
pounds.7,8 Substitutions on the purine skeleton are thus subject
to extensive study, and in pursuing this line of research, we
described the selective introduction of a C2-nitro group by
application of the mild tetrabutylammonium nitrate-trifluoro-
acetic anhydride (TBAN-TFAA)9 system to purines of type 1
(Scheme 1).10,11
(1) Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz, K. N.; Linden,
J. Pharmacol. ReV. 2001, 53, 527-552.
The C2-nitro group proved to be a versatile synthetic
functionality. The electrophilicity of the purine C6 position is
greatly enhanced by the nitro substituent, and introduction of
nucleophiles on the C6 position by displacement of the 6-chloro
moiety of 2 can be achieved at temperatures below 0 °C. By
using various amines, 2-nitroadenosine derivatives were obtained
(2) Bressi, J. C.; Verlinde, C. L. M. J.; Aronov, A. M.; Shaw, M. L.; Shin, S.
S.; Nguyen, L. N.; Suresh, S.; Buckner, F. S.; Van Voorhis, W. C.; Kuntz,
I. D.; Hol, W. G. J.; Gelb, M. H. J. Med. Chem. 2001, 44, 2080-2093.
(3) Harmse, L.; van Zyl, R.; Gray, N.; Schultz, P.; Leclerc, S.; Meijer, L.;
Doerig, C.; Havlik, I. Biochem. Pharmacol. 2001, 62, 341-348.
(4) Meijer, L.; Raymond, E. Acc. Chem. Res. 2003, 36, 417-425.
(5) Galmarini, C. M.; Mackey, J. R.; Dumontet, C. Lancet Oncology 2002, 3,
415-424.
(6) Several recent examples include: (a) Prekupec, S.; Svedrui, D.; Gazivoda,
T.; Mrvo-Sermek, D.; Nagl, A.; Grdia, M.; Paveli, K.; Balzarini, J.; De
Clercq, E.; Folkers, G.; Scapozza, L.; Mintas, M.; Rai-Mali, S. J. Med.
Chem. 2003, 46, 5763-5772. (b) Hocek, M.; Votruba, I.; Dvorakova, H.
Tetrahedron 2003, 59, 607-611.
(9) Abbreviations used in the text: Boc (tert-butoxycarbonyl), DMAP (4-N,N-
(dimethylamino)pyridine), TBAN (tetrabutylammonium nitrate), TFA (tri-
fluoroacetic acid), TFAA (trifluoroacetic anhydride), TFAN (trifluoroacetyl
nitrate), OTFA (trifluoroacetoxy).
(10) Deghati, P. Y. F.; Wanner, M. J.; Koomen, G.-J. Tetrahedron Lett. 2000,
41, 1291-1295.
(11) Deghati, P. Y. F.; Biera¨ugel, H.; Wanner, M. J.; Koomen, G.-J. Tetrahedron
Lett. 2000, 41, 569-573.
(7) De Clercq, E.; Neyts, J. ReV. Med. Virol. 2004, 14, 289-300.
(8) Several recent examples include: (a) Diwan, P.; Lacasse, J. J.; Schang, L.
M. J. Virol. 2004, 78, 4431-4434. (b) Chen, X.; Kern, E. R.; Drach, J. C.;
Gullen, E.; Cheng, Y.-C.; Zemlicka, J. J. Med. Chem. 2003, 46, 1531-
1537.
9
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J. AM. CHEM. SOC. 2005, 127, 5957-5963
5957

A R T I C L E S
Chart 1
Rodenko et al.
Scheme 2
that appeared to be potent and selective agonists for the
adenosine receptors.12 In addition, the remaining C2-nitro group,
with a leaving group ability comparable to that of a fluoro
moiety, can simply be displaced by nucleophilic species, which
was applied in the solid-phase synthesis of C2,N6-disubstituted
adenosine analogues 3 (Chart 1, R1 ) ribose).13 Also, the nitro
group itself can be modified in several ways. Raney-Nickel-
catalyzed hydrogenation led to 2-amino-adenosine deriva-
tives.10 More importantly, hydrogenation of the nitro group with
Pd or Pt on carbon completely stops in the NHOH stage, and
by oxidation with sodium periodate, 2-nitroso derivatives 4 are
obtained, which were useful in hetero-Diels-Alder reactions.14
The effective conversion of C2-nitroso purines into triazenes 5
by condensation with hydrazino-functionalized pharmacophores
offered purine derivatives with high selectivity for the adenosine
A1 receptor.15 Introduction of a methyl group in the triazene
side chain gave O6-benzyl-substituted purines 5 with promising
cytotoxic properties.16
The mild TBAN-TFAA nitration that introduces this ver-
satile nitro group on the purine skeleton proved to be substrate
dependent. Purines and 1-deazapurines with 6-chloro, 6-nitro,
protected 6-hydroxyl, and doubly protected 6-amino function-
alities are readily nitrated. Unprotected purines (mono-N6-
acylated), adenosine triacetate, and nebularine (6-H-purine
riboside) triacetate did not give any of the expected nitration
products.10,11,17
with TBAN-TFAA was reported to introduce the nitro group
selectively on the 3 position.20 Apart from aromatic nitrations,
trifluoroacetyl nitrate has also been used for the nitration of
enolacetates,21,22 silylenolethers,23 and the N-nitration of
amides,24,25 uridines, and inosines.26,27 Although generally the
heterolysis of TFAN (eq 2), thereby releasing nitronium ions,
is considered to be the nitrating mode of action, concerted
pathways involving covalent TFAN25 and radical pathways via
homolysis (eq 3) are also believed to be operative.20,23
The selective introduction of the nitro group in the deactivated
purine system at the highly electron-deficient 2 position and
not at the 8 position is remarkable; it makes both direct
electrophilic nitration28 and the alternative mechanism via
electron transfer29 unlikely, taking the high oxidation potential
of purines into account. Moreover, upon TBAN-TFAA nitra-
tion of solid-supported purine nucleosides, no substantial
nitration of phenyl rings in the polystyrene matrix was detected,
indicating the presence of negligible amounts of strongly
electrophilic nitronium ions.13 Therefore, in an earlier publication
from our group, a radical nitration pathway was proposed
(Scheme 2).10
Homolytic cleavage of trifluoroacetyl nitrate (eq 3) as
proposed by Evans et al. generates the trifluoroacetoxyl and
(15) Beukers, M. W.; Wanner, M. J.; Von Frijtag Drabbe Ku¨nzel, J. K.; Klaasse,
E. C.; IJzerman, A. P.; Koomen G.-J. J. Med. Chem. 2003, 46, 1492-
1503.
(16) Wanner, M. J.; Koch, M.; Koomen G.-J. J. Med. Chem. 2004, 47, 6875-
The use of the TBAN-TFAA mixture for electrophilic
aromatic nitration was reported by Masci18 as an adaptation of
Crivello’s nitration system, consisting of TFAA and heteroge-
neous metal or ammonium nitrates in inert solvents.19 The active
species in both methods is trifluoroacetyl nitrate, TFAN, formed
in situ (eq 1).
6883.
(17) The 2-nitration of N6-acetyl-2′-deoxyadenosine with Cu(II)NO3/Ac2O was
published by Kaiya, T.; Tanaka, H.; Kohda, K. Nucleosides Nucleotides
2002, 21, 427-433.
(18) Masci, B. J. Org. Chem. 1985, 50, 4081-4087.
(19) Crivello, J. V. J. Org. Chem. 1981, 46, 3056-3063.
(20) (a) Njoroge, G. F.; Vibulbhan, B.; Pinto, P.; Chan, T.-M.; Osterman, R.;
Remiszewski, S.; Del Rosario, J.; Doll, R.; Girijavallabhan, V.; Ganguly,
A. K. J. Org. Chem. 1998, 63, 445-451. (b) Adriano, A.; Weinstein, J.;
Kelly, J.; Wolin, R.; Rosenblum, S. B.; Connolly, M.; Guzi, T.; James, L.;
Carr, D.; Patton, R.; Bishop, W. R.; Kirshmeier, P.; Liu, M.; Heimark, L.;
Chen, K. J.; Nomeir, A. A. Bioorg. Med. Chem. 1999, 7, 1845-1855. (c)
Njoroge, G. F.; Vibulbhan, B.; Wong, J. K.; White, S. K.; Wong, S.-C.;
Carruthers, N. I.; Kaminski, J. J.; Doll, R. J.; Girijavallabhan, V.; Ganguly,
A. K. Org. Lett. 1999, 1, 1371-1373.
Bu4NNO3 + (CF3CO)2O h
Bu4N+CF3CO2- + CF3CO2NO2 (1)
+
CF3CO2NO2 h CF3CO2- + NO2
(2)
(3)
(21) Dampawan, P.; Zajac, W. W., Jr. Synthesis 1983, 545-546.
(22) Rank, W. Tetrahedron Lett. 1991, 32, 5353-5356.
(23) Evans, P. A.; Longmire, J. M. Tetrahedron Lett. 1994, 35, 8345-8348.
(24) Carvalho, E.; Iley, J.; Norberto, F.; Rosa, E. J. Chem. Res., Synop. 1989,
260-261.
•
•
CF3CO2NO2 h CF3CO2 + NO2
(25) Romea, P.; Aragone`s, M.; Garcia, J.; Vilarrasa, J. J. Org. Chem. 1991, 56,
7038-7042.
For benzenes, high regioselectivity was obtained with this
nitrating reagent. More recently, reaction of substituted pyridines
(26) Ariza, X.; Bou, V.; Vilarrasa, J. J. Am. Chem. Soc. 1995, 117, 3665-
3673.
(27) Ariza, X.; Farra`s, J.; Serra, C.; Vilarrasa, J. J. Org. Chem. 1997, 62, 1547-
1549.
(12) Wanner, M. J.; Von Freitag Drabbe Ku¨nzel, J. K.; IJzerman, A. P.; Koomen,
G.-J. Bioorg. Med. Chem. Lett. 2000, 10, 2141-2144.
(13) Rodenko, B.; Wanner, M. J.; Koomen, G.-J. J. Chem. Soc., Perkin Trans.
1 2002, 1247-1252.
(14) Wanner, M. J.; Koomen, G.-J. J. Chem. Soc., Perkin Trans. 1 2001, 1908-
1915.
(28) Moodie, R. B. Aromatic Nitration; Schofield, K., Ed.; Cambridge University
Press: Cambridge, 1980; Chapter 15.
(29) Esteves, P. M.; Walkimar de M. Carneiro, J.; Cardoso, S. P.; Barbosa, J.
G. H.; Laali, K. K.; Rasul, G.; Prakash, G. K. S.; Olah, G. A. J. Am. Chem.
Soc. 2003, 125, 4836-4849.
9
5958 J. AM. CHEM. SOC. VOL. 127, NO. 16, 2005

Mechanism of Selective Purine C-Nitration
A R T I C L E S
Scheme 3
nitrogen dioxide radicals.23 Addition of the reactive trifluoro-
acetoxyl radical to the purine C-8 of 1 gives a highly delocalized
radical that is stabilized by the substituent at C-6. Subsequent
combination of 6 with NO2 at C-2 forms intermediate 7 and is
followed by elimination of trifluoroacetic acid, which affords
2-nitro product 2.
A drawback in the mechanism proposed in Scheme 2 is the
generation of the very unstable trifluoroacetoxyl radical, which
rapidly decomposes to CO2 and the trifluoromethyl radical with
a reported dissociation constant of k > 5 × 104 s-1 30
.
During
Figure 1. Aromatic region of 1H NMR spectra of the nitration of 6-chloro-
9-Boc-purine 9 at -10 °C in CD2Cl2.
TBAN-TFAA purine nitrations, we did not observe formation
of species such as CO2 or CF3H that might indicate homolytic
cleavage of trifluoroacetyl nitrate as the initiating step. Puzzled
by these facts, we further investigated the mechanism of purine
nitration and looked for reaction intermediates with NMR, and
we were able to identify and characterize an N7-nitropurine
intermediate; its rearrangement into the C2-nitro product ac-
companied by 15N-CIDNP effects will be discussed in this paper
as evidence for a new three-step purine nitration mechanism.
Results and Discussion
Detection of a 7-Nitro-8-trifluoroacetoxy Purine Inter-
mediate. To facilitate the interpretation of NMR data, a simple
purine substrate was required which would offer a clean and
complete nitration with the TBAN-TFAA mixture. In our
hands, 6-chloro-9-Boc-purine 9, a compound not reported before
in literature, appeared to be the most suitable candidate. This
crystalline compound was easily synthesized in 89% yield by
reaction of 6-chloropurine 8 with Boc2O (1.4 equiv) and catalytic
DMAP (Scheme 3, step a).31 On preparative scale, the nitration
of 9 with TBAN-TFAA (1.6 equiv) was carried out in CH2Cl2
at 0 °C and furnished 2-nitro-6-chloro-9-Boc purine 10 in 86%
isolated yield. Here, further indication that the rate-determining
step is not electrophilic is obtained from the observation that
an electron-withdrawing N9-Boc group increases both the rate
and yield of the nitration reaction compared to an N9-ribosyl
or N9-methyl substituent.
Monitoring the nitration of 6-chloro-9-Boc-purine 9 with 1H
NMR at -10 °C revealed, besides product formation, the
presence of a purine intermediate. Figure 1 (t0) shows part of
the 1H spectrum of a mixture of 6-chloro-9-Boc purine 9 (0.13
M) and TBAN (0.26 M) in CD2Cl2 at -10 °C. When TFAA (2
equiv) was added to this mixture, the appearance of the H8
signal (8.92 ppm) of the product 10 and the disappearance of
the H2 (8.86 ppm) and H8 (8.63 ppm) signals of starting material
9 were observed as expected (Figure 1, t10 min). In addition, the
fast formation and gradual decrease of a set of signals was seen
that contained a singlet at 8.82 ppm (integrated as 1H), a singlet
at 8.78 ppm (integrated as 1H), and a singlet at 1.48 ppm
Figure 2. Progress of the nitration by plotting the 1H integral values against
time (T ) -10 °C). Triangles, H2 of starting material 9; closed circles,
(H2 + H8)/2 of intermediate 11; open circles, H8 of product 10. During
the reaction, the signals of H2 and H8 of intermediate 11 partly overlap;
hence, they were jointly integrated as 2H for this graphical representation.
(integrated as 9H). These signals were assigned to intermediate
11. Additional minor peaks were detected, which were ascribed
to side products (see below and Supporting Information). After
5 h, all of the starting material and intermediary peaks had
disappeared, while in the aromatic region, only the H8 signal
of the product remained (Figure 1, t5 h). Three corresponding
tert-butyl signals (see Supporting Information) were observed:
starting material 9 (1.65 ppm), intermediate 11 (1.48 ppm), and
product 10 (1.72). The progress of the nitration is represented
graphically in Figure 2 as a plot of the normalized integral values
against time.
A successful attempt to “freeze” the nitration reaction in the
intermediate stage allowed extensive spectroscopic characteriza-
tion of 11. The progress of intermediate formation was
monitored at -50 °C, and complete conversion of 6-chloro-
purine 9 into intermediate 11 over an 8 h period was observed,
while formation of 2-nitro product 10 was suppressed to less
than 3%.32
The 15N NMR spectrum of intermediate 11 formed from 98%
15N-labeled TBAN revealed a doublet at 339.6 ppm with JNH
) 2.7 Hz (Table 1). In the corresponding 1H spectrum, a doublet
(30) (a) Francisco, J. S. Chem. Phys. Lett. 1992, 191, 7-12. (b) Maricq, M.
M.; Szente, J. J.; Khitrov, G. A.; Francisco, J. S. J. Phys. Chem. 1996,
100, 4514-4520.
(31) For the reaction of adenine and guanine with Boc2O, see: Dey, S.; Garner,
P. J. Org. Chem. 2000, 65, 7697-7699.
(32) The composition of the sample did not change when placed in a freezer at
-80 °C; after 5 months, still no conversion of the intermediate to the end
product had occurred.
9
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A R T I C L E S
Rodenko et al.
Table 1. Selected NMR Data from the Nitration of
6-Cl-9-Boc-purine 9 with 15N-Labeled TBANa
Scheme 4
6-Cl-9-Boc-purine 9
(ppm) J (Hz)
Intermediate 11
(ppm) J (Hz)
2-NO2
−
6-Cl-9-Boc-purine 10
δ
δ
δ
(ppm)
J (Hz)
1H-2
1H-8
8.86 (s)
8.63 (s)
8.78 (s)
3
8.82 (d) JHN ) 2.7
13C-2 153.5 (d) JCH ) 211 157.7 (d) JCH ) 212 154.2 (d) JCN ) 29.4
1
1
1
13C-8 144.3 (d) JCH ) 222 93.0 (dd) JCH ) 194 148.3 (d) JCH ) 223
1
1
1
2JCN ) 1.8
15NO2
19F
339.6 (d) JNH ) 2.7 365.0 (s)
-75.86 (s)
3
a Recorded in CD2Cl2 at -50 °C.
at 8.82 ppm with JNH ) 2.7 Hz was observed instead of a singlet.
A 1H decoupling experiment confirmed 1H-15N coupling. The
3
value of the coupling constant points toward a JNH coupling.
Also, the 15N NMR spectrum revealed that intermediate 11
contains an N-nitro group and no C-nitro or -nitrato moiety.
The 15N chemical shift of the doublet of intermediate 11
typically lies within the range of N-nitro compounds, which is
generally shifted about 20 ppm upfield relative to that of C-nitro
compounds.33 Peaks derived from covalently bound nitrates,
which appear at even higher field values relative to N-nitro
derivatives, were not detected.34 The 15N signal of the nitro
group in product 10, a singlet at 365.0 ppm, displays a chemical
shift value characteristic for aromatic C-NO2 compounds.
In the 13C spectrum of starting material 9, C2 was found at
153.5 ppm with 1JCH ) 211 Hz and C8 at 144.3 ppm with 1JCH
) 222 Hz (see Table 1). In the spectrum of intermediate 11,
the values for C2 (δ ) 157.7 ppm, 1JCH ) 212 Hz) were similar
to those of the starting material, but C8 showed a remarkable
The N-Nitration-Nitramine Rearrangement Mechanism.
The radical addition mechanism proposed in Scheme 2 appeared
to be inconsistent with the obtained NMR results, and a new
N-nitration-addition-nitramine rearrangement pathway was
suggested, as is depicted in Scheme 4. The purine ring system
is N-nitrated in the imidazole ring on nitrogen atom 7 by
electrophilic attack of TFAN. The highly electrophilic imida-
zolium cation 12 is rapidly trapped by a suitable nucleophile,
in this case, trifluoroacetate, furnishing the observed nitramine
intermediate 11. This step emphasizes the essential requirement
of a nucleophilic species, such as the trifluoroacetate anion, for
C-nitration to occur; the mere reaction of 6-chloropurine 9 with
nitronium ions results only in the recovery of starting material
as was shown in a control experiment with nitronium tetrafluo-
roborate.10 Subsequently, from 11, a nitramine rearrangement
takes place in which the nitro group moves to the “para” carbon
atom C2.35 The classical nitramine rearrangement refers to the
migration of the nitro group of N-nitroaniline to the ortho and
para positions to yield a mixture of o- and p-nitroaniline.36 In
the case of purine nitramine intermediate 11, both ortho
positions C4 and C6 are blocked, leaving para carbon atom 2
as the only available position for migration of the nitro group.
The product of this rearrangement, intermediate 13, was not
detected because the subsequent TFA elimination leading to
2-nitropurine 10 is too fast on the NMR time scale.
1
upfield shift to 93.0 ppm and a decreased JCH value of 194
Hz. Moreover, C-N coupling with 2JCN ) 1.8 Hz was observed
for C8 in experiments with 15N-labeled TBAN. These results
indicated that aromaticity was retained in the pyrimidine part,
but not in the imidazole ring. In addition, the presence of a
trifluoroacetoxy group in intermediate 11 was observed with
13C NMR, as indicated by a quartet at 114.2 ppm (1JCF ) 290
3
Hz) and a double quartet at 153.8 ppm (2JCF ) 44 Hz, JCH
)
2.9 Hz). The 19F NMR spectrum verified the presence of the
trifluoroacetoxy group, which appeared as a singlet at -75.86
ppm. With the help of CH correlation spectra and the observed
1
long-range couplings, the intermediary singlet in H NMR at
8.78 ppm could be assigned to H2 and the doublet at 8.82 ppm
to H8. The extremely high chemical shift of 8.82 ppm for H8,
a proton attached to an sp3 carbon atom, can be explained by
the three heteroatoms attached to C8, the electron-withdrawing
effect of the nitro, Boc, and trifluoroacetoxy groups and the
anisotropic effect of the carbonyl and nitrosyl moieties. During
the reaction, the concentration of TFA increases and H8 of 11
showed a gradual upfield shift of about 0.2 ppm as a conse-
quence of partial protonation, which disturbs the anisotropic
effect.
To validate that formation of 2-nitropurine 10 takes place
via the monophasic process of rearrangement of the N7-nitro
intermediate and not via other routes, we preformed nitramine
intermediate 11 at -50 °C in CDCl3 and monitored its
conversion into 2-nitro purine 10 with 1H NMR at -10 °C. In
Figure 3, the progress of the normalized integral values of H8
of nitramine intermediate 11 and 2-nitro purine 10 is represented
graphically (for real-time spectra, see Supporting Information).
These combined NMR data led us to the structure assignment
for the N-nitro intermediate 11, as shown in Figure 1.
(33) Berger, S.; Braun, S.; Kalinowski, H.-O. NMR Spectroscopy of the Non-
Metallic Elements; J. Wiley & Sons: Chichester, U.K., 1997; pp 207-
212.
(34) (a) Moodie, R. B.; Stephens, R. J. J. Chem. Soc., Perkin Trans. 2 1987,
1059-1064. (b) Moodie, R. B.; Sanderson, A. J.; Willmer, R. J. Chem.
Soc., Perkin Trans. 2 1991, 645-650. (c) Titov, A. I. Tetrahedron 1963,
19, 557-580.
(35) Shine, H. J. Aromatic Rearrangements; Elsevier: Amsterdam, 1967; pp
235-249.
(36) Bamberger, E.; Landersteiner, K. Ber. 1893, 26, 482-495.
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Mechanism of Selective Purine C-Nitration
A R T I C L E S
spectra as an abnormal intensity of the NMR signals; the signals
display either enhanced absorption or emission. The phase of
the net polarization observed, Γne (positive for enhanced
absorption signals, negative for emission signals), can be
predicted with Kaptein’s rules.47 When applied to the polariza-
tion of 15N nuclei, the equation takes the following form:48
Γne ) - µꢀai(gA - gB)
Here, µ is derived from how the radical pair is formed
(positive when formed from a triplet precursor or by the
encounter of free radicals, negative when formed from a singlet
precursor); ꢀ is derived from how the products are formed
(positive for reaction within the radical pair, negative for reaction
after separation of the radical pair); ai is the sign of the hyperfine
coupling constant for the observed nucleus i in radical A, and
(gA - gB) is the difference between the g values (obtained from
EPR data) of radicals A and B.
Figure 3. Conversion of N-nitro intermediate 11 to C2-nitro product 10
followed with H NMR at -10 °C. Closed circles, nitramine intermediate
11; open circles, product 10. kN, nitramine decrease; kP, product formation.
1
The excellent first-order kinetics confirm the expected uni-
molecular process. First-order rate coefficients, kN ) 1.5 × 10-3
s-1 (nitramine 11 decrease) and kP ) 1.9 × 10-3 s-1 (product
10 increase), were determined over about 4 half-lives with a
high R2 value and good reproducibility. The slight difference
in the values of kN and kP is explained by the occurrence of
side reactions (vide infra); a subsequent reaction would decrease
the magnitude of the signal for the product at the end of the
reaction and thus cause the extent of reaction at earlier times to
be overestimated.37
As both thermal38-40 and acid-catalyzed36,41 nitramine rear-
rangements of nitroanilines have been reported, we also allowed
the rearrangement to proceed in the presence of the base DIPEA,
and identical reaction rates were found (see Supporting Informa-
tion). This proved that the rearrangement was thermal and not
acid catalyzed. Moreover, if the rearrangement would be acid
catalyzed, the reaction rate would increase in the course of the
reaction, as TFA is generated upon product formation. Deviation
from a first-order correlation was not observed. To our
knowledge, this is the first identified example of a nitramine
rearrangement in a purine/pyrimidine system.
15N-CIDNP in Purine Nitration. With the unimolecularity
of the reaction now being established, we chose to study the
mechanism of the rearrangement with 15N NMR, which has been
a valuable tool in elucidating reaction pathways in nitration
reactions.42 Several mechanisms have been put forward for the
nitramine rearrangement, both heterolytic43 and homolytic
pathways.41 The observation of CIDNP effects in 15N NMR
spectra during the rearrangement of nitroaniline derivatives
offered convincing evidence that radicals were involved, thus
supporting a homolytic rearrangement mechanism.37,44 Chemi-
cally Induced Dynamic Nuclear Polarization refers to the
perturbation of the nuclear spins away from the expected
Boltzmann distribution.45,46 The effect is observed in NMR
In the case of the rearrangement of nitramine intermediate
11, the above equation can be reduced to the extent that the
sign of Γne discloses whether the rearrangement takes places in
an inter- or intramolecular fashion. In the NO2 radical, the
negative magnetogyric ratio of the 15N nucleus also causes aN
to be negative,49 while the analysis can be further simplified if
the reasonable assumption is made that the g value of the organic
purinyl radical50 is larger than the g value of NO2 (2.0000).49
The conclusions of Kaptein’s rules can now simply be related
to how the radicals are formed and how they react to form the
product. In a thermal radical rearrangement, the radical pair is
generated from a singlet precursor, so µ is negative. Thus, from
the phase of the polarized NMR signals, one can then conclude
whether the rearrangement occurs intra- or intermolecularly. If
the product is formed in an intramolecular fashion, it is the result
of a recombination reaction within the radical pair (ꢀ is positive)
and enhanced absorption is observed. If the product is formed
in an intermolecular fashion, it is the result of recombination
of the radicals after separation of the original pair (ꢀ is negative)
and emission is observed. Conclusions are less obvious if the
rearrangement has both an intra- and an intermolecular coun-
terpart. Since the observed NMR signal matches the sum of
the unpolarized and polarized (positive and/or negative) material
formed, even no net polarization at all can be the consequence.
When we followed the rearrangement of nitramine 11 at 0
°C with 15N NMR, indeed, CIDNP effects were observed (Figure
4). During several half-lives of the rearrangement, the doublet
of nitramine intermediate 11 at 339.6 ppm showed enhanced
absorption (see Supporting Information for real-time spectra).
In the early stage of the reaction at t ) 2 min, the singlet of
2-NO2 product 10 at 365.0 ppm showed an emission signal,
while during the remainder of the rearrangement, a reduced
(37) A similar discrepancy in the values of kN and kP was found by Ridd and
co-workers during the acid-catalyzed nitramine rearrangement of 2,6-
dichloro-N-nitroaniline and 2,6-dibromo-N-nitroaniline: Abu-Namous, A.
M. A.; Ridd, J. H.; Sandall, J. P. B. Can. J. Chem. 1986, 64, 1124-1129.
(38) Barnes, T. J.; Hickinbottom, W. J. J. Chem. Soc. 1961, 2616-2620.
(39) Naud, D. L.; Brower, K. R. J. Org. Chem. 1992, 57, 3303-3308.
(40) Naud, D. L. J. Chem. Soc., Perkin Trans. 2 1996, 1321-1324.
(41) (a) White, W. N.; Klink, J. R. J. Org. Chem. 1977, 42, 166. (b) White, W.
N.; White, H. S.; Fentiman, A. J. Org. Chem. 1976, 41, 3166-3170 and
earlier papers in this series.
(45) Lepley, A. R.; Closs, G. L. Chemically Induced Magnetic Polarization; J.
Wiley & Sons: New York, 1973.
(46) Muus, L. T.; Atkins, P. W.; McLaughlan, K. A.; Pedersen, J. B. Chemically
Induced Magnetic Polarization; D. Reidel Publishing Company: Dordrecht,
The Netherlands, 1977.
(47) Kaptein, R. J. Chem. Soc., Chem. Commun. 1971, 732-733.
(48) (a) Clemens, A. H.; Ridd, J. H.; Sandall, J. P. B. J. Chem. Soc., Perkin
Trans. 2 1984, 1659-1665. (b) The additional negative sign must be added
because of the negative magnetogyric ratio of the 15N nucleus. See: Porter,
N. A.; Dubay, G. R.; Green, J. G. J. Am. Chem. Soc. 1978, 100, 920-925.
(49) Morton, J. R.; Preston, K. F.; Strach, S. J. J. Phys. Chem. 1979, 83, 533-
536.
(50) Hellwege, K.-H.; Fischer, H. Landolt-Bo¨rnstein: Numerical Data and
Functional Relationships in Science and Technology; Springer-Verlag:
Berlin, 1977.
(42) Ridd, J. H. Chem. Soc. ReV. 1991, 20, 149-165.
(43) (a) Banthorpe, D. V.; Hughes, E. D.; Williams, D. L. H. J. Chem. Soc.
1964, 5349-5361. (b) Banthorpe, D. V.; Thomas, J. A.; Williams, D. L.
H. J. Chem. Soc. 1965, 6135-6140. (c) Brownstein, S.; Bunton, C. A.;
Hughes, E. D. J. Chem. Soc. 1958, 4354-4357.
(44) Ridd, J. H.; Sandall, J. P. B. J. Chem. Soc., Chem. Commun. 1982, 261-
262.
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A R T I C L E S
Rodenko et al.
Figure 5. CIDNP effects when the intermolecular rearrangement is
prevented by diverting the free radicals with hydroquinone. S: nitromethane
standard; nitramine intermediate 11 (doublet); 2-nitro product 10 (singlet).
Figure 4. 15N-CIDNP NMR spectra of the rearrangement of 0.45 M
nitramine. S: 15N nitrobenzene standard; nitramine intermediate 11
(doublet); 2-nitro product 10 (singlet).
the lowest energy and most stable average configuration, leading
to C-NO2 product formation.51 During the intramolecular
recombination, N-NO2 formation (high-energy conformation,
kinetically favored) is in competition with C-NO2 formation.
This is expressed in the enhanced absorption for N-NO2
intermediate 11. Accordingly, intermolecular recombination
primarily leads to the thermodynamically more stable C-NO2
compound, accounting for the observed emission signal of 10.
The large emittive CIDNP signal of 10 suggested a major
contribution of the intermolecular route toward its formation.
In a radical trapping experiment with hydroquinone, which only
reacts with free radicals and not with paired radicals, we
examined the exact contributions of the inter- and intramolecular
counterparts of the rearrangement. By diverting the free NO2
radicals that have escaped from the original radical pair through
reduction with hydroquinone, the intermolecular route toward
intermediate 13 is efficiently blocked. Using this method, White
has shown that the nitramine rearrangement of, for example,
N-methyl-N-nitroaniline, has both an intra- and intermolecular
counterpart.51,52
Scheme 5
Monitoring the rearrangement of nitramine intermediate 11
in the presence of 3 equiv of hydroquinone with 15N NMR
revealed that only 30-35% of 2-nitro product 10 was generated.
This indicated that 65-70% of the purine nitramine rearrange-
ment occurred intermolecularly. As expected, a greatly enhanced
absorption was observed for product 10 due to the absence of
the emittive contribution to the NMR signal (Figure 5). In
agreement, the signal of nitramine intermediate 11 still displayed
enhanced absorption.
8-Oxo Purine Formation. While monitoring the nitration
reaction with NMR, a slight excess of TFAN was used, and in
the spectra, a minor set of peaks was observed that deserves
more comment. These signals were observed both during direct
nitration of 6-chloro-9-Boc-purine 9 and during the nitramine
rearrangement after preformation of nitramine intermediate 11.
The peaks disappeared after warming the sample to room
temperature. In the 15N NMR spectra, a set of signals composed
absorption signal was observed until after about 4 half-lives
(reaction nearly complete) no CIDNP effects were observed any
longer. These results can be explained with the radical mech-
anism shown in Scheme 5. The enhanced absorption of
nitramine intermediate 11 indicates the reverse reaction of the
radical pair to re-form nitramine 11 by immediate collapse of
the primary radical pair. The emission signal for product 10
points to an intermolecular process in which the paired radicals
separate and become free radicals. The latter re-encounter to
re-form the radical pair and, consequently, the secondary
recombination intermediate 13, which immediately eliminates
TFA to render product 10. In the beginning of the reaction,
this contribution to the NMR signal is larger since the
concentration of the escaped (i.e., free) radicals is higher as is
the chance of random free-radical encounters.
of a singlet at 363.4 ppm and a doublet at 335.9 with JNH
)
1
2.7 Hz corresponded in the H NMR spectra to a doublet at
9.10 ppm with JNH ) 2.7 Hz (integrated as 1H) and a singlet at
1.54 ppm (integrated as 9H) and in the 19F NMR spectra to a
Since the reassociation of free radicals to a radical pair is an
equilibrium process, the resulting radical pair probably possesses
(51) White, W. N.; White, H. S. J. Org. Chem. 1970, 35, 1803-1805.
(52) White, W. N.; Golden, J. T. J. Org. Chem. 1970, 35, 2759-2762.
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5962 J. AM. CHEM. SOC. VOL. 127, NO. 16, 2005

Mechanism of Selective Purine C-Nitration
A R T I C L E S
Scheme 6
reactions, especially when a large excess of nitrating agent is
used.
Conclusion
In summary, extensive monitoring of the TBAN-TFAA
purine nitration reaction with NMR spectroscopy excluded direct
nitration of the highly electrophilic C2 position and demonstrated
that this reaction occurs via an N-nitration-addition-nitramine
rearrangement pathway. Electrophilic attack by TFAN on the
purine N7 position results in a nitrammonium species that is
trapped by a nucleophile, in this case, the trifluoroacetate anion,
furnishing nitramine intermediate 11. A subsequent purine
nitramine rearrangement generates C2-nitro species 13 that
eliminates TFA to give the product 2-nitro-6-chloro-9-Boc-
purine 10. Moreover, the involvement of radicals during the
nitramine rearrangement was unequivocally established by 15N-
CIDNP. A radical trapping experiment disclosed that 65-70%
of the nitramine rearrangement takes place intermolecularly.
singlet at -75.95 ppm. These NMR signals were interpreted as
the consequence of a consecutive addition of TFAN to 2-ni-
tropurine 10 leading to the observed dinitro intermediate 14, as
depicted in Scheme 6. Since the para position is already
substituted, nitramine rearrangement is not possible and either
retroreaction to 2-nitro-6-chloro purine 10 occurs or, alterna-
tively, loss of nitrous acid gives rise to 2-nitro-6-chloro-8-
trifluoroacetoxy purine 16. Hydrolysis of this latter compound
upon aqueous workup explains the formation of 8-oxo purines,
such as 17, which are incidentally isolated after purine nitration
Acknowledgment. The authors thank Jan Geenevasen and
Jan Meine Ernsting for their help and commitment during the
NMR measurements.
Supporting Information Available: Experimental details and
NMR spectra (1H, 13C, [13C,1H]-COSY, 15N-CIDNP, 19F) of
nitration reactions. This material is available free of charge via
the Internet at http://pubs.acs.org.
JA042590X
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