Chemoselective nitration of phenols with iert-butyl nitrite in solution and on solid support

Article abstract of DOI:10.1021/ol901731w

test-Butyl nitrite was identified as a safe and chemoselective nitrating agent that provides preferentially mononltro derivatives of phenolic substrates In the presence of potentially competitive functional groups. On the basis of our control experiments, we propose that the reaction proceeds through the formation of O-nltrosyl Intermediates prior to C-nitration via homolysis and oxidation. The reported nitration method Is compatible with tyroslne-containlng peptides on solid support In the synthesis of fluorogenic substrates for characterization of proteases.

Full text of DOI:10.1021/ol901731w

ORGANIC
LETTERS
2009
Vol. 11, No. 18
4172-4175
Chemoselective Nitration of Phenols
with tert-Butyl Nitrite in Solution and on
Solid Support
Dipankar Koley, Olvia C. Colo´n, and Sergey N. Savinov*
Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907-2084
ssaVinoV@purdue.edu
Received July 28, 2009
ABSTRACT
tert-Butyl nitrite was identified as a safe and chemoselective nitrating agent that provides preferentially mononitro derivatives of phenolic
substrates in the presence of potentially competitive functional groups. On the basis of our control experiments, we propose that the reaction
proceeds through the formation of O-nitrosyl intermediates prior to C-nitration via homolysis and oxidation. The reported nitration method is
compatible with tyrosine-containing peptides on solid support in the synthesis of fluorogenic substrates for characterization of proteases.
The technique of fluorescence resonance energy transfer
(FRET) is currently a common method for monitoring
structural, functional, or aggregation changes in evaluated
biomolecules.1 Real-time monitoring of hydrolase activity
is a particularly useful application of FRET because the
substrate can be chemically modified at sites remote to
the scissile bond, minimizing probe interferences with
substrate recognition. The 2-aminobenzoic acid (2-Abz)
fluorophore and 3-nitrotyrosine quencher pair has been
utilized extensively in determining proteolytic activities of
endoproteases.2 As amino acids, these agents represent a
convenient fluorophore-quencher combination that can be
readily incorporated into a conventional solid-phase peptide
synthesis protocol. As a part of a program in evolution of
site-specific proteases as therapeutic agents, we are interested
in developing a flexible and practical platform for kinetic
and thermodynamic characterization of evolved enzymes.
Thus, we envisioned a unified synthetic strategy for accessing
both internally quenched substrates, useful for real-time
kinetic analysis of active enzymes, as well as unquenched
variants, suitable for fluorescence quenching and anisotropy
studies with inactive mutants. We were very keen, therefore,
to identify a reagent that could perform a nitration reaction
on solid-phase-immobilized peptides containing tyrosines
with high efficiency and selectivity. Unfortunately, the
existing nitrating agents, such as mixed acids, superacids,
and metal nitrates, are not suitable for polymer-supported
substrates because of their polymer-reactive or heterogeneous
nature.3,4 In addition, many of the existing reagents can lead
to overnitration of phenols4a,g,h and other unwanted reactions
with a variety of functional groups found in peptides.5 Thus,
development of a methodology enabling chemoselective
nitration of polymer-immobilized tyrosine would be valued.
(3) Ridd, J. H. Chem. Soc. ReV. 1991, 20, 149–165
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9071–9073. (h) Selvam, J. J. P.; Rajesh, V. S. K.; Reddy, S. R.;
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therein
.
10.1021/ol901731w CCC: $40.75
Published on Web 08/21/2009
 2009 American Chemical Society

We disclose in this paper a novel nitration procedure using
tert-butyl nitrite, a reagent that displays exquisite chemose-
lectivity for phenols, yields only tert-butyl alcohol as a
byproduct, and is relatively safe, volatile, and soluble in a
variety of organic solvents.
Table 1. Optimization Studies of the Reaction between 1a and
t-BuONOa
yieldb (%)
In our attempt to perform diazotization of tyrosine with
t-BuONO,6 we observed the formation of unexpected yellow
products. To explore this finding further, we subjected Boc-
Tyr-OH (1a) to the nitrite treatment in dichloromethane at
room temperature (Scheme 1). After 3 h of agitation, we
entry
solvent
MeOH
CHCl3
CHCl3
DMSO
DMSO
DMF
DMF
acetonitrile
acetonitrile
diethyl ether
diethyl ether
THF
THF
THF
THF
time (h)
2a
3a
4a
1
2
3
4
5
6
7
8
16
3
16
3
16
3
16
1.5
16
16
3
no reaction
>95
19
43
>95
>95
58
66
37
68
95
nd
50
nd
nd
nd
16
23
37
nd
nd
nd
nd
nd
nd
nd
31
nd
nd
nd
25
10
24
nd
nd
nd
nd
4
Scheme 1. Nitration of Tyrosine Derivatives with t-BuONO
9
10
11c
12
13
14
15
1
3
6
16
93
>95
95
85
14
a Unless specified otherwise, all reactions were carried out at room
temperature in 0.2 M solutions of 1a in THF with 3 equiv of t-BuONO.
b
All yields were measured by H NMR. c Under reflux conditions, nd )
1
not detected.
detected a complete consumption of 1a and quantitative
(>95%) formation of Boc-Tyr(3-NO2)-OH (2a) and corre-
sponding N-nitroso derivative 3a (2a:3a 57:43). While there
have been reports of C-nitro products emerging from the
exposure of electron-rich aromatic compounds to inorganic7
or organic8 nitrites, no mechanistic or scope studies have
been carried out. In 1H NMR spectrum of the crude reaction
mixure we also identified a trace ammount of bisnitrated
product 4a (<1%), and we confirmed that both 3a and 4a
can arise from 2a by re-exposing the latter to a larger excess
of t-BuONO. The presence of a nitro rather than a nitrosyl
protic solvents appeared to prevent nitration altogether (entry
1), use of chloroform noticeably improved the chemoselec-
tivity of this reaction (entry 2). Upon prolonged exposure,
however, undesired byproducts 3a and 4a became dominant
(entry 3), limiting the utility of this solvent. In DMSO, the
reaction was noticeably slower than in the halogenated
solvents but provided exclusively the desired product 2a
(entries 4 and 5). The reaction proceeded in DMF at a similar
rate as in chloroform (entry 6) but displayed higher chemose-
lectivity for mononitration, since byproducts 3a and 4a
emerged only upon extended incubation with t-BuONO
(entry 7). The use of acetonitrile, on the other hand, severely
compromised chemoselectivity of the reaction with byprod-
ucts emerging early in the reaction course (entry 8) and
accumulating significantly after 16 h (entry 9). While the
use of diethyl ether led to a relatively slow nitration progress
(entry 10), a temperature increase led to nearly quantitative
conversion to 2a in 3 h (entry 11). Finally, by employing
THF we obtained a nitration procedure that is both highly
selective and reasonably fast. Within 1 h, reaction
conversion reached 93% (entry 12) and became nearly
quantitative after 3 h (entry 13). While overnitration
product 4a started to form after a 6-h incubation (entry
14), no detectable N-nitrosylated product emerged even
after 16 h (entry 15). These results demonstrate that the
rates of neither the desired C-nitration nor the undesired
N-nitrosylation and bisnitration reactions appear to cor-
relate with solvent polarities. Overnitration has occurred
in both polar and nonpolar solvents, albeit at significantly
distinct rates: faster in dichloromethane (ε ) 8.910) and
acetonitrile (ε ) 36.6), but slower in DMF (ε ) 38.3)
1
group in 2a was corraborated by mass spectrometry, H
NMR,9 and UV-vis spectroscopy (λmax ≈ 430 nm). Al-
though N-nitrosyl compound 3a rapidly decomposed upon
isolation to form 4a along with several other compounds,
we confirmed its structure by comparison of 1H NMR
resonances with those of the more stable ester variant 2b,
prepared from Boc-Tyr-OMe (1b) along with 3b and 4b (2b:
3b:4b 47:32:21, >95%) under identical conditions (Scheme
1). On the basis of these results, we speculated that a careful
optimization of reaction conditions could provide a selective
mononitration procedure.
To identify a more practical nitration procedure, we
proceeded to screen a variety of solvents (Table 1). While
(5) (a) Bruice, T. C.; Gregory, M. J.; Walters, S. L. J. Am. Chem. Soc.
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Org. Lett., Vol. 11, No. 18, 2009
4173

and THF (ε ) 7.5). Widely varying ratios of the
byproducts (cf. entries 3, 7, 8, and 15) suggest distinct
mechanistic pathways responsible for their formation. The
preference for N-nitrosylation in acetonitrile could be
rationalized by the formation of a solvent-nitrosonium
adduct,11 capable of the subsequent N-nitrosylation of the
carbamate. No byproducts were observed in the aprotic
solvents at the extremes of polarity: DMSO (ε ) 47.2)
and diethyl ether (ε ) 4.3). Unlike these solvents,
however, THF maintained a robust nitration rate, providing
a procedure that was fast, selective, and more suitable for
solid-phase synthesis, considering favorable swelling
properties of cross-linked polystyrene resins in THF.12
To establish the scope of this reaction, we exposed a series
of phenols to t-BuONO in THF. We found that this procedure
was effective at converting electron-rich compounds in a
variety of steric contexts into C-nitro derivatives (Table 2).
oxyphenol and 2,6-dimethoxyphenol as substrates (entries
4 and 5) highlighted a dominant role of hydroxyl groups
over alkoxy substituents in directing the reaction regiochem-
istry.
Next, we determined the extent of chemoselectivity
displayed by t-BuONO with respect to functional groups
present in peptides. Gratifyingly, aromatic ethers (anisole,
Ac-Tyr(Bn)-OMe) and N-formylindole (Ac-Trp(CHO)-OMe)
were unreactive toward the organic nitrite (3 equiv of
t-BuONO, THF, 25 °C, 24 h) confirming the strict require-
ment for an aromatic hydroxyl group in this reaction. In
addition, no nitrosylation of primary or secondary amides
was detected upon prolonged incubation (>6 h) of Ac-Ala-
OMe and Ac-Gln-OMe. Of all functionalities tested, only
the sulfide-containing compounds Boc-Cys(Tr)-OH and Boc-
Met-OH yielded complex mixtures, as seen in earlier
diazotization attempts of related compounds.13
To unravel the role that the phenolic hydroxyl group plays
in this nitration, we subjected 2,4,6-tri-tert-butylphenol 5 to
a large excess of t-BuONO (Scheme 2, part A). Within 10
Table 2. Nitration on Phenols with t-BuONOa
Scheme 2
.
Mechanistic Analysis and Proposed Pathway for the
Nitration Reaction with t-BuONO
min, we observed complete consumption of 5 and concomi-
tant formation of a new compound that we have assigned as
O-nitroso derivative 6 on the basis of H NMR and FTIR
a Unless specified otherwise, all reactions were carried out at room
1
temperature using 3 molar equiv of t-BuONO and 5 mL of solvent per
data. Furthermore, in t-BuOH as a solvent, 6 reverted to 5
with concomintant formation of t-BuONO, confirming both
the reversibility of the O-nitrosylation process and the
chemical nature of 6 as aromatic nitrite. Upon storing at room
temperature in CDCl3, 6 slowly transformed into a complex
mixture of C-nitro derivatives14 through apparent interme-
diacy of a phenoxyl radical of 5, detected by its characteristic
blue color.15 The isolation of the O-nitroso species, the lack
of reactivity in protic solvents, and the reaction kineticss
first order with respect to both phenol and t-BuONO (see
the Supporting Information)sallowed us to propose that the
reaction is initiated by a rate-limiting nitrosyl exchange
between the alkyl nitrite and aromatic hydroxyl groups
c
mmol of phenol. b Isolated yields, unless specified otherwise. 1H NMR
yields (18% starting material recovered). d 6 equiv of reagent.
When both ortho and para positions are available, a mixture
of mononitrated regioisomers was obtained (entry 1). The
resistance to overnitration in THF was also confirmed by
the lack of reactivity of both o- and p-nitrophenols toward
t-BuONO (1.5 equiv, 2 h). As expected, ortho- and para-
substituted phenols (entries 2 and 3) provided exclusively
the corresponding para- and ortho-nitro derivatives, respec-
tively. Notably, single regiochemical outcomes with 4-meth-
(11) Darbeau, R. W.; Pease, R. S.; Perez, E. V. J. Org. Chem. 2002,
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8951–8954.
4174
Org. Lett., Vol. 11, No. 18, 2009

(Scheme 2, part B), as shown previously for aliphatic
alcohols.16 The aromatic O-nitroso derivatives undergo
subsequent thermal homolysis to furnish resonance-stabilized
phenoxyl radicals and nitric oxide.17 Our attempts to detect
any C-nitroso intermediates failed, and we, therefore, propose
that the final steps of this reaction involve rapid oxidation
of nitric oxide with molecular oxygen,18 prior to coupling
of the resultant nitrogen dioxide with phenoxyl radicals.19
A crossover experiment with 6 as a nitrating reagent
furnished the mononitro product (4-methoxy-2-nitrophenol)
with 4-methoxyphenol as a substrate (see the Supporting
Information), confirming the intermediacy of aromatic nitrites
in this process. Use of anisole in this experiment, however,
left the aromatic ether unmodified, establishing that the
formation of phenoxyl radicals is the prerequisite for the
observed C-nitration. Participation of phenoxyl radicals has
been postulated for nitration reactions mediated by
tetranitromethane5a and peroxynitrite20 in polar protic media,
on the basis of isolation of biaryl ethers and biphenols as
dominant byproducts. We do not observe these products,
presumably due to the improved solvation of phenoxyl
radicals in the aprotic solvents, making the bimolecular cross-
coupling less likely.
amide, and aromatic ether functionalities. The sole product
isolated after the treatment of 7 with t-BuONO in THF and
subsequent hydrolysis was Tyr(3-NO2)-containing derivative 8.
Finally, we accomplished efficient debenzylation of 8 in the
presence of a nitro group under transfer hydrogenation condi-
tions to provide Boc-Tyr(3-NO2)-Tyr-OH (9).
Our interest in tobacco etch virus protease (TEV-Pr) as a
scaffold for the evolution of enzymes with novel properties
prompted us to explore synthetic approches toward fluoro-
genic substrates of TEV-Pr. In a search for the shortest TEV-
Pr substrate, a tripeptide truncate (FQG) of the optimal linear
epitope ENLYFQG,21 flanked by the C-terminal Asp-Tyr
dipeptide and N-terminal 6-aminohexanoic acid spacer-
fluorophore (Ahx-Abz) unit (10), was produced as a test
compound for the solid-phase nitration procedure (Scheme
3, part B). Reaction of 10 with t-BuONO, followed by
hydrolysis, yielded a selectively mononitrated peptide fur-
nished with a 3-nitrotyrosine chromophore in >90% purity,
as detected by reversed-phase HPLC, spectrophotometric
analysis, and mass spectrometry (see the Supporting Infor-
mation). While the truncated substrate was not recognized
by TEV-Pr, this stringently quenched peptide (F0/F∞ ≈ 0.9%)
was processed effectively by chymotrypsin, as detected in
real time through the release of a fluorescent anthranilamide-
containing fragment (see the Supporting Information).
In conclusion, we have presented here a novel procedure
for selective and efficient nitration of phenols in aprotic
media, which is compatible with polymer-supported sub-
strates. We expect that the potential of this methodology to
provide access to both internally quenched and unquenched
peptides will greatly facilitate characterization of novel
proteolytic activities. Finally, the facile and selective intro-
duction of a nitro group described herein may also initiate
other synthetic opportunities, such as late-stage chromophore
installation and formation of photolabile groups.
Having developed an efficient nitration procedure for solu-
tion-phase transformations, we sought to implement this meth-
odology on polymer-supported phenolic substrates. We gener-
ated test dipeptide 7, containing both protected and unprotected
tyrosine residues on Merrifield resin (Scheme 3, part A) to
Scheme 3
.
On-Bead Nitration and Synthesis of a Fluorogenic
Peptide
Acknowledgment. This work was funded by NIH Grant
No. 1R21AG031437-01.
Supporting Information Available: Experimental pro-
cedures and compound characterization data. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL901731W
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