Chromogenic oxazines for cyanide detection

Article abstract of DOI:10.1021/jo052096r

We have designed two heterocyclic compounds for the colorimetric detection of cyanide. The skeleton of both molecules fuses a benzooxazine ring to an indoline fragment and can be assembled efficiently in three synthetic steps starting from commercial prec

Full text of DOI:10.1021/jo052096r

Chromogenic Oxazines for Cyanide Detection
Massimiliano Tomasulo,† Salvatore Sortino,*,‡ Andrew J. P. White,§ and
Franc¸isco M. Raymo*,†
Center for Supramolecular Science, Department of Chemistry, UniVersity of Miami, 1301 Memorial DriVe,
Miami, Florida 33146-0431, Dipartimento di Scienze Chimiche, UniVersita´ di Catania,
Viale Andrea Doria 8, Catania, I-95125, Italy, and Department of Chemistry, Imperial College London,
South Kensington, London, SW7 2AZ, United Kingdom
ssortino@unict.it; fraymo@miami.edu
ReceiVed October 6, 2005
We have designed two heterocyclic compounds for the colorimetric detection of cyanide. The skeleton
of both molecules fuses a benzooxazine ring to an indoline fragment and can be assembled efficiently in
three synthetic steps starting from commercial precursors. The two compounds differ in the nature of the
substituent on the carbon atom at the junction of the fused heterocycles, which can be either a methyl or
a phenyl group. In the presence of cyanide, both molecules are converted quantitatively into cyanoamines
with the concomitant appearance of an intense band in the visible region of the absorption spectrum. The
developing absorption is a result of the opening of the benzooxazine ring with the formation of a
4-nitrophenylazophenolate chromophore. Nuclear magnetic resonance spectroscopy and X-ray crystal-
lographic analyses demonstrate that the covalent attachment of a cyanide anion to the indoline fragment
is responsible for these transformations. The chromogenic process is particularly fast for the methyl-
substituted oxazine and can be exploited to detect micromolar concentrations of cyanide in water.
Furthermore, the colorimetric response of this compound to cyanide does not suffer the interference of
the halide anions, which instead are known to complicate the detection of cyanide in conventional sensing
protocols. Thus, our mechanism and compounds for the colorimetric identification of cyanide can lead
to the development of practical strategies for the convenient determination of this toxic anion in aqueous
environments.
Introduction
applications have been developed around the excellent binding
properties of this particular ligand.4,5 The strong affinity of
The cyanide anion is a particularly strong nucleophile and
forms stable complexes with a variety of transition metals in
aqueous solution.1-3 In fact, a wealth of diverse industrial
(2) Verdaguer, M.; Bleuzen, A.; Marvaud, V.; Vaissermann, J.; Seulei-
man, M.; Desplanches, C.; Scuiller, A.; Train, C.; Garde, R.; Gelly, G.;
Lomenech, C.; Rosenman, I.; Veillet, P.; Cartier, C.; Villain, F. Coord.
Chem. ReV. 1999, 192, 1023-1047.
(3) Baraldo, L. M.; Forlano, P.; Parise, A. R.; Slep, L. D.; Olabe, J. A.
Coord. Chem. ReV. 2001, 219, 881-921.
(4) Young, C.; Tidwell, L.; Anderson, C. Cyanide: Social, Industrial,
and Economic Aspects; Minerals, Metals, and Materials Society: Warren-
dale, 2001.
† University of Miami.
‡ Universita´ di Catania.
§ Imperial College London.
(1) Dunbar, K. R.; Heintz, R. A. Prog. Inorg. Chem. 1997, 45, 283-
391.
10.1021/jo052096r CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/09/2005
744
J. Org. Chem. 2006, 71, 744-753

Chromogenic Oxazines for Cyanide Detection
cyanide for transition metals, however, has deleterious conse-
quences on cell metabolism.6-8 Specifically, this anion binds
the active site of cytochrome oxidase and inhibits the mito-
chondrial electron-transport chain. As a result, cyanide is ex-
tremely toxic and even relatively small amounts of this species
(0.5-3.5 mg per kg of body weight) are lethal to humans.9
Unfortunately, cyanide does not easily decompose in the
environment.10 Therefore, the accidental spillage of this toxic
chemical from industrial plants, or even its intentional release,
can contaminate drinking waters and become a serious threat
to human health. Indeed, the concentration of cyanide in drinking
water cannot be greater than 1.9 µM according to the World
Health Organization.11
Numerous standard methods for the detection of micromolar
amounts cyanide in water have been developed relying on a
diversity of experimental protocols and detection techniques.12
Most of these strategies, however, require either multistep
procedures with tedious sample pretreatments or sophisticated
instrumentation. The development of chemosensors13-16 for the
recognition of anions17-31 can facilitate the qualitative, and
perhaps even the quantitative, determination of cyanide. In
particular, the identification of chromogenic compounds that
respond to the presence of cyanide anions with fast and visible
color changes would offer the opportunity to screen rapidly
water samples relying exclusively on the naked eye. Indeed,
few organic molecules and transition metal complexes able to
signal the presence of cyanide with pronounced changes in their
absorption and emission properties have been already identi-
fied.32-37 Their operating principles are based on hydrogen
bonding interactions, metal coordination, or the formation of
covalent bonds between the nucleophilic cyanide anion and
compatible electrophilic centers. Some of these chemosensors
can even detect micromolar amounts of cyanide;36,37 however,
most of them suffer the deleterious interference of other
anions.32-37 Halide anions in particular, and especially fluoride,
tend to mask the response of cyanide.32,34b,35,37c In search for
specific chemosensors for the colorimetric determination of
cyanide, we have designed a series of chromogenic oxazines.
In this article, we illustrate the logic behind our molecular design
and we report the synthesis and characterization of these
compounds together with their colorimetric response to cyanide.
(5) Ullmann’s Encyclopedia of Industrial Chemistry; Wiley-VCH: New
York, 2003.
(6) Vennesland, B. Cyanide in Biology; Academic Press: New York,
1981.
Results and Discussion
Design and Synthesis. Recently, we have discovered that
the [1,3]oxazine 1a (Figure 1) is converted quantitatively into
the hemiaminal 1c upon treatment with Bu4NOH in acetoni-
trile.38 The nucleophilic attack of the hydroxide anion of
Bu4NOH to the indolium cation of the short-lived intermediate
1b is responsible for this transformation. Interestingly, the
bimolecular conversion of 1a into 1c is accompanied by the
appearance of a yellowish color. Indeed, the absorption spectrum
of 1a shows bands in the ultraviolet region only, while the
4-nitrophenolate chromophore of 1c has an intense absorption
centered at 440 nm.
(7) Kulig, K. W. Cyanide Toxicity; U.S. Department of Health and
Human Services: Atlanta, GA, 1991.
(8) Baskin, S. I.; Brewer, T. G. In Medical Aspects of Chemical and
Biological Warfare; Sidell, F. R., Takafuji, E. T., Franz, D. R., Eds.; TMM
Publications: Washington, DC, 1997; pp 271-286.
(9) Muir, G. D. Hazards in the Chemical Laboratory; The Royal
Chemical Society: London, UK, 1977.
(10) Baird, C.; Cann, M. EnVironmental Chemistry; Freeman: New York,
2005.
(11) Guidelines for Drinking-Water Quality; World Health Organiza-
tion: Geneva, Switzerland, 1996.
(12) Ikebukuro, K.; Nakamusa, H.; Karude, I. In Handbook of Water
Analysis; Nollet, L. M., Ed.; Marcel Dekker: New York, 2000; Vol. 102,
pp 367-385.
(13) Desvergne, J.-P.; Czarnik, A. W., Eds. Chemosensors of Ion and
Molecular Recognition; Kluwer Academic Publishers: Dordrecht, The
Netherlands, 1997.
On the basis of these observations, we have tested the
response of 1a to Bu4NCN under otherwise identical conditions.
Once again, the characteristic band of a 4-nitrophenolate
chromophore appears in the visible region of the absorption
(14) Ricco, A. J.; Crooks, R. M., Eds. Chemical Sensors. Acc. Chem.
Res. 1998, 31, 199-324.
(15) Ellis, A. B.; Walt, D. R., Eds. Chemical Sensors. Chem. ReV. 2000,
100, 2477-2738.
(16) Fabbrizzi, L., Ed. Luminescent Sensors. Coord. Chem. ReV. 2000,
205, 1-232.
(27) (a) Gale, P. A. Coord. Chem. ReV. 2000, 199, 181-233. (b) Gale,
P. A. Coord. Chem. ReV. 2001, 213, 79-128.
(28) (a) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V.
Acc. Chem. Res. 2001, 34, 963-972. (b) Lavigne, J. J.; Anslyn, E. V. Angew.
Chem., Int. Ed. 2001, 40, 3119-3130.
(29) Amendola, V.; Fabbrizzi, L.; Mangano, C.; Pallavicini, P.; Poggi,
A.; Taglietti, A. Coord. Chem. ReV. 2001, 219, 821-837.
(30) Mart´ınez-Ma´n˜ez, R.; Sancero´n, F. Chem. ReV. 2003, 103, 4419-
4476.
(31) Suksai, C.; Tuntulani, T. Chem. Soc. ReV. 2003, 32, 192-202.
(32) Miyaji, H.; Sessler, J. L. Angew. Chem., Int. Ed. 2001, 40, 154-
157
(17) Bianchi, A.; Bowman-James, K.; Garcia-Espan˜a, E., Eds. Supramo-
lecular Chemistry of Anions; Wiley-VCH: New York, 1997.
(18) Gale, P. A., Ed. 35 Years of Synthetic Anion Receptor Chemistry
1968-2003. Coord. Chem. ReV. 2003, 240, 1-226.
(19) (a) Schmidtchen, F. P.; Gleich, A.; Schummer, A. Pure Appl. Chem.
1989, 61, 1535-1546. (b) Schmidtchen, F. P.; Berger, M. Chem. ReV. 1997,
97, 1609-1646.
(20) Dietrich, B. Pure Appl. Chem. 1993, 65, 1457-1464.
(21) Czarnik, A. W. Acc. Chem. Res. 1994, 27, 302-308.
(22) Atwood, J. L.; Holman, K. T.; Steed, J. W. Chem. Commun. 1996,
1401-1407.
(23) (a) Scheerder, J.; Engbersen, J. F. J.; Reinhoudt, D. N. Recl. TraV.
Chim. Pays-Bas 1996, 115, 307-320. (b) Antonisse, M. M. G.; Reinhoudt,
D. N. Chem. Commun. 1998, 443-448.
(33) Kim, Y.-H.; Hong, J.-I. Chem. Commun. 2002, 512-513.
(34) (a) Ros-Lis, J. V.; Mart´ınez-Ma´n˜ez, R.; Soto, J. Chem. Commun.
2002, 2248-2249. (b) Jime´nez, D.; Mart´ınez-Ma´n˜ez, R.; Sanceno´n, F.; Soto,
J. Tetrahedron Lett. 2002, 43, 2823-2825.
(24) (a) Beer, P. D.; Smith, D. K. Prog. Inorg. Chem. 1997, 46, 1-96.
(b) Beer, A. D. Acc. Chem. Res. 1998, 31, 71-80. (c) Beer, P. D.; Gale, P.
A.; Chen, G. Z. Coord. Chem. ReV. 1999, 186, 3-36. (d) Beer, P. D.; Gale,
P. A. Angew. Chem., Int. Ed. 2001, 40, 487-516.
(25) (a) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997,
97, 1515-1566. (b) de Silva, A. P.; Fox, D. B.; Huxley, A. J. M.;
McClenaghan, N. D.; Roiron, J. Coord. Chem. ReV. 1999, 186, 297-306.
(26) (a) Gale, P. A.; Sessler, J. L.; Kral, V. Chem. Commun. 1998, 1-8.
(b) Sessler, J. L.; Anzenbacher, P., Jr.; Jursikova, K.; Miyaji, H.; Genge, J.
W.; Tvermoes, N. A.; Allen, W. E.; Shriver, J. A.; Gale, P. A.; Kral, V.
Pure Appl. Chem. 1998, 70, 2401-2408. (c) Gale, P. A.; Anzenbacher, P.,
Jr.; Sessler, J. L. Coord. Chem. ReV. 2001, 222, 57-102.
(35) (a) Anzenbacher, P., Jr.; Tyson, D. S.; Jurs´ıkova´, K.; Castellano, F.
N. J. Am Chem. Soc. 2002, 124, 6232-6233. (b) Anzenbacher, P., Jr.;
Jurs´ıkova´, K.; Aldakov, D.; Marquez, M.; Pohl, R. Tetrahedron 2004, 60,
11163-11168.
(36) Chow, C.-F.; Lam, M. H. W.; Wong, W.-Y. Inorg. Chem. 2004,
43, 8387-8393.
(37) (a) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. Anal. Chim. Acta
2004, 522, 9-17. (b) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. Dyes
Pigmen. 2005, 64, 49-55. (c) Badugu, R.; Lakowicz, J. R.; Geddes, C. D.
J. Am. Chem. Soc. 2005, 127, 3635-3641.
(38) (a) Tomasulo, M.; Sortino, S.; Raymo, F. M. Org. Lett. 2005, 7,
1109-1112. (b) Tomasulo, M.; Sortino, S.; White, A. J. P.; Raymo, F. M.
J. Org. Chem. 2005, 70, 8180-8189.
J. Org. Chem, Vol. 71, No. 2, 2006 745

Tomasulo et al.
FIGURE 1. Transformation of the [1,3]oxazine 1a into either the hemiaminal 1c or the cyanoamine 1d.
FIGURE 2. Steady-state absorption spectra (0.1 mM, MeCN, 298 K)
of 1a before (a) and after (b) the addition of Bu4NCN (100 equiv).
spectrum (a and b in Figure 2) after the addition of the
nucleophile. Thus, the cyanide anion can also react with the
intermediate 1b and trap its 4-nitrophenolate chromophore in
the form of the cyanoamine 1d (Figure 1) with the concomitant
appearance of color. It follows that similar transformations can
be a possible operating mechanism for the colorimetric detection
of cyanide. In this context, we have designed the two compounds
7a and 8a (Figure 3). They share a central [1,3]oxazine ring
with 1a, but incorporate a 4-nitrophenylazo appendage in place
of the nitro group. After ring opening and nucleophilic trapping,
the extended conjugation of the resulting 4-nitrophenylazophe-
nolate chromophore should translate into an enhancement of
ca. 18 mM-1 cm-1 in molar extinction coefficient, relative to
the 4-nitrophenolate chromophore of 1c. Thus, 7a and 8a should
be more appropriate than 1a as potential chromogenic probes
for cyanide.
We have synthesized the two [1,3]oxazines 7a and 8a in two
steps (Figure 3) starting from 2-hydroxymethylphenol (2) and
4-nitrobenzenediazonium tetrafluoroborate (3).39 The reaction
of 2 and 3 in aqueous NaOH affords the 4-nitrophenylazophenol
4 in a yield of 96%. The treatment of 4 with PBr3 and the
reaction of the resulting bromide with an excess of the indole
FIGURE 3. Synthesis of the [1,3]oxazines 7a and 8a.
5 or 6 in situ gives the corresponding [1,3]oxazine 7a or 8a in
a yield of 41% or 51%, respectively.
of the C(9)-C(20) bond [the C(20)-C(9)-N(1)-lone pair
dihedral angle is ca. 13°].
X-ray Crystallography. The X-ray analysis of crystals of
the cyanoamine 1d confirmed the opening of the benzooxazine
ring with the addition of a cyano group (Figure 4). The structure
is somewhat similar to that of the hemiaminal 1c,38b including
the incorporation of water molecules hydrogen bonded to the
phenolate anion. The indoline ring of 1d has an envelope-type
geometry, C(9) lying ca. 0.46 Å out of the {N(1) to C(8)} plane,
which is coplanar to within ca. 0.02 Å. The geometry at N(1)
is similar to those seen in related species,38b the nitrogen center
being pyramidal (ca. 0.33 Å out of the plane of its substituents),
and with its 2pz orbital approximately collinear with the σ orbital
As can readily be seen from Figure 4, the packing in the
crystals of 1d is dominated by O-H‚‚‚O hydrogen bonds
involving the phenolate anion and the three included water
molecules. There are also, however, some notable cation‚‚‚anion
contacts with both rings A and B approached by methylene
protons from the tetra-n-butylammonium cation [H‚‚‚A 2.95 Å,
C-H‚‚‚A 178°; H‚‚‚B 2.78 Å, C-H‚‚‚B 175°]. The opposite
faces of both of these rings are involved in a bifurcated C-H‚‚‚π
hydrogen bond from a methyl proton of the cation [H‚‚‚A 3.28
Å, C-H‚‚‚A 119°; H‚‚‚B 3.15 Å, C-H‚‚‚B 129°], the angles
subtended at each ring centroid being ca. 155 and 172° for A
(39) Tomasulo, M.; Raymo, F. M. Org. Lett. 2005, 7, 4633-4636.
746 J. Org. Chem., Vol. 71, No. 2, 2006

Chromogenic Oxazines for Cyanide Detection
ring B [the values for molecule II are given in square brackets].
The terminal nitro group is rotated by ca. 16° [10°] to ring C.
Adjacent molecules are linked by C-H‚‚‚π interactions; ring
A (in molecule I) is approached by a C(4′)-H proton from
molecule II [H‚‚‚A 2.45 Å, C(4′)-H‚‚‚A 176°], ring B in
molecule I is approached by a C(20)-H proton in a neighboring
molecule I [H‚‚‚B 2.91 Å, C(20)-H‚‚‚B 145°], and similarly
ring B′ in molecule II is approached by a C(20′)-H proton
from another molecule II [H‚‚‚B 2.82 Å, C(20′)-H‚‚‚B 153°].
There is also some evidence for a possible weak O‚‚‚π
interaction between the nitro group of molecule II and ring C
in molecule I with an O(30′)‚‚‚C separation of ca. 3.22 Å.
The single-crystal structure of 8a (the phenyl analogue of
7a) is similar, though here there is only one independent
molecule, and the azo-4-nitrophenyl moiety adopts a noticeably
different conformation (Figure 7) where the azo unit is anti with
respect to the C(17) carbon center (in both independent
molecules of 7a the relationship was gauche). The fused
indoline/benzooxazine core of the structure is very similar to
that seen in 7a. The indoline ring adopts an envelope-type
geometry with C(9) lying ca. 0.47 Å out of the C7N plane (which
is coplanar to within ca. 0.05 Å), while the benzooxazine ring
again has a twisted conformation with N(1) ca. 0.35 Å “below”
and C(9) ca. 0.28 Å “above” the C7O plane (which is coplanar
to within ca. 0.03 Å); the C7N and C7O planes are inclined by
ca. 80°. The N(1) is again pyramidal, lying ca. 0.36 Å out of
the plane of its substituents, and the nitrogen 2pz orbital is
approximately collinear with the σ orbital of the C(9)-O(10)
bond, the O(10)-C(9)-N(1)-lone pair dihedral angle being
ca. 8°. The C-NdN-C unit is again flat (coplanar to better
than 0.01 Å), but unlike in 7a, here it is almost coplanar with
both ring B (rotated by ca. 6°) and ring C (rotated by ca. 1°).
The terminal nitro group is rotated by ca. 10° to ring C.
FIGURE 4. The single-crystal X-ray structure of 1d (cation not
depicted) showing the O-H‚‚‚O hydrogen bonding interactions between
the phenolate anion and the included solvent water molecules. The
O-H‚‚‚O hydrogen bonding geometries, [O‚‚‚O] (Å), [H‚‚‚O] (Å), and
[O-H‚‚‚O] (deg), are the following: (a) 2.718(4), 1.83, 167; (b) 2.719-
(4), 1.83, 172; (c) 2.771(5), 1.92, 158; (d) 2.742(5), 1.87, 163.
and B, respectively. Ring C is not involved in any noteworthy
intermolecular contacts.
The X-ray analysis of crystals of 7a revealed the presence of
two independent molecules, I and II (see Figures 5 and S5 for
pictures of molecules I and II, respectively). With the exception
of the terminal p-nitrophenyl substituents, the two molecules
have very similar conformations (Figure S7), the N(1) to N(22)
portions of the two independent molecules having a root-mean-
square fit of ca. 0.048 Å. In common with 1a,38b both
independent molecules have the expected near orthogonal
relationship between their indoline and benzooxazine ring
systems (ca. 86° in both molecules). The indoline ring in each
independent molecule has a similar envelope-type geometry to
that seen in 1a [in I C(9) is ca. 0.46 Å out of the {N(1) to
C(8)} plane, which is coplanar to within ca. 0.04 Å; in II C(9′)
is ca. 0.51 Å out of the {N(1′) to C(8′)} plane, which is coplanar
to within ca. 0.02 Å]. By contrast, the benzooxazine rings are
more distorted. In 1a, the ring had an envelope-type geometry
with N(1) lying ca. 0.53 Å out of the plane of the remaining
C8O atoms, which were coplanar to within ca. 0.08 Å. In 7a,
however, both independent molecules have twisted benzooxa-
zine rings with, for I, N(1) lying ca. 0.50 Å “below” and C(9)
ca. 0.22 Å “above” the plane of the remaining C7O atoms, which
are coplanar to within ca. 0.08 Å (for II the values are 0.42,
0.30, and 0.06 Å, respectively).40 In each independent molecule,
the N(1) nitrogen centers have pyramidal geometries, the
nitrogen lying ca. 0.36 Å out of the plane of its substituents in
both I and II. As was seen in 1a, here in both independent
molecules of 7a the nitrogen 2pz orbital is approximately
collinear with the σ orbital of the C(9)-O(10) bond, the O(10)-
C(9)-N(1)-lone pair dihedral angles being ca. 8° and 7° in I
and II, respectively. Though flat (with {C(14),N(21),N(22),-
C(23)} being coplanar to within 0.01 Å [0.01 Å]), the plane of
the N2 moiety is twisted with respect to ring B by ca. 16° [18°],
and to ring C by ca. 29° [21°]; these twists are in the same
sense so that ring C is twisted by ca. 46° [39°] with respect to
The extended structure of 8a is dominated by the stacking
of the 4-nitrophenylazophenolate units of adjacent, centrosym-
metrically related molecules linked by a combination of
interactions/contacts (Figure S9). Ring C of one molecule lays
above the azo unit of a Ci related counterpart, and vice versa,
with a centroid‚‚‚centroid separation of ca. 3.40 Å (a in Figure
S9). In the same centrosymmetric pair, ring B of one molecule
overlays the nitro unit of the other, the closest approach being
from O(35) at ca. 3.13 Å to the ring B centroid (b in Figure
S9). This same oxygen atom also approaches the centroid of
ring D in the second molecule, but only at ca. 3.63 Å (c in
Figure S9). These centrosymmetric pairs of molecules are linked
to adjacent pairs across an independent center of symmetry by
a couple of C-H‚‚‚π hydrogen bonds from a nitrophenyl
hydrogen atom [on C(30)] to the centroid of ring A [H‚‚‚A 2.88
Å, C(30)-H‚‚‚A 151°, d in Figure S9].
1H NMR Spectroscopy. The chiral center at the junction of
the two heterocycles in 7a and 8a imposes two distinct
environments on the adjacent pairs of methyl groups (MeO and
Me0 in Figures 7 and S10) and methylene protons (HO and H0).
As a result, the 1H NMR spectra of both compounds show two
distinct singlets for the protons of MeO and Me0 and an AB
system for HO and H0, when recorded in acetonitrile-d3 at 275
K (a in Figures 7 and S10). Upon warming the solution, these
resonances broaden considerably for both compounds (b and c
in Figures 7 and S10) and, eventually, coalesce into single peaks
for 7a (d in Figures 7 and S10). These changes are a conse-
quence of the interconversion between the two enantiomers of
each compound on the 1H NMR time scale. This process
(40) If the benzooxazine ring in 1a is viewed as twisted in a similar
fashion, then N(1) lies ca. 0.48 Å “below” and C(9) ca. 0.14 Å “above”
the plane of the remaining C7O atoms which are coplanar to within ca.
0.04 Å.
J. Org. Chem, Vol. 71, No. 2, 2006 747

Tomasulo et al.
FIGURE 5. The structure of one (I) of the two crystallographically independent molecules present in the crystals of 7a.
TABLE 1. Kinetic Parameters Associated with the Ring Opening of 7a and 8a at 298 Ka
solvent
compd
k (s-1
)
∆Gq (kcal mol-1
)
∆Hq (kcal mol-1
)
-∆Sq (kcal mol-1 K-1
)
acetonitrile-d3
7a
8a
7a
25 ( 2
15.54 ( 0.04
18.82 ( 0.11
20.17 ( 0.23
15.0 ( 0.2
18.5 ( 0.9
21.4 ( 1.3
0.002 ( 0.001
0.001 ( 0.003
-0.004 ( 0.004
0.10 ( 0.02
b
toluene-d8
0.011 ( 0.004
a The rate constant (k), free energy (∆Gq), enthalpy (∆Hq), and entropy (∆Sq) of activation were determined by variable-temperature 1H NMR spectroscopy
(ref 41). b In toluene-d8, the line widths of the singlets associated with the pair of methyl protons of 8a remain approximately constant in the examined
temperature range (275-363 K). As a result, the kinetic parameters for the ring opening of this compound could not be determined.
with Bu4NOH results in the formation of the corresponding
hemiaminals 7c and 8c (Figures S11 and S12). In both instances,
the chemical shift of the diastereotopic methylene protons
decreases, but their AB system is maintained, confirming the
presence of a chiral center in 7c and 8c. A second product is
also formed in the case of 7. Indeed, the methyl group on its
chiral center is relatively acidic and is partially deprotonated
upon treatment with Bu4NOH to form 7d (Figure S11). The
ratio between 7c and 7d can be estimated to be 70:30 from the
integrals of the resonances associated with the methylene
protons. The formation of the two hemiaminals 7c and 8c is
further confirmed by the appearance of peaks at m/z 431 and
493 in the corresponding fast atom bombardment mass spectra.
1
The changes imposed on the H NMR spectra of 7a and 8a
by the addition of Bu4NOH can be replicated with Bu4NCN. In
both instances, the cyanide anion attacks the indolium cations
of 7b and 8b to form quantitatively 7e and 8d, respectively
FIGURE 6. The single-crystal X-ray structure of 8a.
(Figures 8 and S13). As a result of these transformations, the
chemical shifts of most aromatic protons decrease (a and b in
Figures 8 and S13). For both compounds, the largest change
involves the thermal opening of the [1,3]oxazine ring with the
formation of 7b and 8b and their re-isomerization to 7a and
8a, respectively. The kinetic parameters (Table 1) associated
(-0.45 ppm for 7a and -0.61 ppm for 8a) is observed for the
with the ring-opening step can be extracted from the analysis
proton in the ortho position relative to the phenolate oxygen
of the temperature dependence of the line widths associated with
the singlets for the protons of MeO and Me0 in the slow-
(41) Below the coalescence temperature, two well-separated singlets can
exchange regime.41 In acetonitrile-d3, the rate constants (k) are
be observed for the protons of MeO and Me0 in the 1H NMR spectra (a-c
ca. 25 and 0.1 s-1 for 7a and 8a, respectively. These values
in Figures 7 and S10). Under these conditions, the line width (∆ν) of either
correspond to free energy barriers (∆Gq) of ca. 16 and 19 kcal
one of the two singlets is related to the rate constant (k) of the degenerate
site-exchange process according to eq 1, where ∆ν0 is the line width at the
mol-1, respectively. Interestingly, the ∆Gq values are dominated
stopped-exchange limit (Nelson, J. H. Nuclear Magnetic Resonance
by their enthalpic terms (∆Hq), while the entropic contributions
Spectroscopy; Prentice Hall: Upper Saddle River, NJ, 2003). Following
are negligible (∆Sq). In toluene-d8, the ring-opening process is
this protocol, k can be determined at any temperature (T) within the slow-
exchange regime. A plot of ln(k T-1) against T-1 can then be fitted to eq
significantly slower. The k value for 7a decreases by 4 orders
2, where R is the gas constant, to extract the enthalpy (∆Hq) and entropy
of magnitude with a concomitant increase of ca. 4.5 kcal mol-1
(∆Sq) of activation. Finally, the free energy (∆Gq) of activation can be
calculated at any T by using eq 3.
1
in ∆Gq. In the case of 8a, the process is so slow that the H
NMR spectrum remains virtually unchanged over a broad range
of temperatures.
k ) π (∆ν - ∆ν0)
(1)
k
T
∆Hq
RT
∆Sq
R
The quantitative transformation of 1a into 1c (Figure 1) causes
ln
)
-
+
+ 23.76
(2)
(3)
1
drastic changes in the H NMR spectrum.38 The two oxazines
7a and 8a show essentially the same behavior. Their treatment
∆Gq ) ∆Hq - T∆Sq
748 J. Org. Chem., Vol. 71, No. 2, 2006

Chromogenic Oxazines for Cyanide Detection
1
FIGURE 7. Partial H NMR spectra (500 MHz, acetonitrile-d3, 5 mM) of 7a at 275 (a), 300 (b), 310 (c) and 346 K (d).
1
FIGURE 8. Partial H NMR spectra (400 MHz, acetonitrile-d3, 10 mM) of 7a before (a) and after (b) the addition of Bu4NCN (10 equiv).
atom (Hi for 7a and Hl for 8a). Once again, the AB system for
the methylene protons is maintained with the transformation of
7a into 7e and of 8a into 8d, but moves by -0.57 ppm for 7
and -0.69 ppm for 8. Furthermore, the fast atom bombardment
mass spectra show the appearance of peaks at m/z 441 and 503
in support of the formation of 7e and 8d, respectively.
Steady-State Absorption Spectroscopy. The steady-state
absorption spectra of the two [1,3]oxazines 7a and 8a (a in
Figures 9 and S14) resemble the sum of those of the model
compounds 9 or 10 (b in Figures 9 and S14) and 11 (c in Figures
9 and S14). In particular, an intense band for the π f π*
transition of the 4-nitrophenylazophenyl chromophore is evident
J. Org. Chem, Vol. 71, No. 2, 2006 749

Tomasulo et al.
FIGURE 9. Steady-state absorption spectra (0.1 mM, MeCN, 298 K)
of 7a (a), 9 (b), 11 (c), 7a before (d) and after (e) the addition of
Bu4NOH (1 equiv), and 12 (f).
FIGURE 10. Steady-state absorption spectra (0.1 mM, MeCN, 298
K) of 7a before (a) and after (b) the addition of Bu4NCN (15 equiv).
Evolution of the absorbance at 581 nm for solutions (0.1 mM, MeCN,
298 K) of 7a (c) and 8a (d) after the addition of Bu4NCN (1.5 equiv
for 7a and 15 equiv for 8a).
TABLE 2. Absorption Wavelengths (λ) and Molar Extinction
Coefficients (E) of the Oxazines 7a and 8a and of the Model
Compounds 9-12 in MeCN at 298 Ka
compd
λ (nm)
ꢀ (mM-1 cm-1
)
7a
8a
9
10
11
12
380
371
283
281
371
576
23.0 ( 1.2
22.0 ( 1.1
2.2 ( 0.1
3.9 ( 0.2
26.1 ( 1.3
50.4 ( 1.1
a The model compounds are shown in the following diagram. The λ and
ꢀ of the phenolate 12 were determined by recording the absorption spectrum
of the corresponding phenol in the presence of Bu4NOH (4 equiv).
FIGURE 11. Steady-state absorption spectra (0.1 mM, 550 µL, MeCN,
298 K) of 7a after the addition of sodium phosphate buffer (550 µL,
pH 7.6) without (b) or with 10 mM of NaCN (a), NaF (c), NaCl (d),
NaBr (e), or NaI (f).
addition of only 1.5 equiv of Bu4NCN to 7a (c in Figure 10).
Instead, more than 30 min are required to complete the
transformation of 8a even in the presence of up to 15 equiv of
Bu4NCN (d in Figure 10). These observations are in full
agreement with the kinetic parameters (Table 1) determined for
the ring opening of 7a and 8a by 1H NMR spectroscopy. Indeed,
these data show that the ring opening of 8a is 2 orders of
magnitude slower than that of 7a.
The relatively fast colorimetric response of 7a to cyanide can
be exploited to sense this particular anion in aqueous environ-
ments. In fact, the addition of NaCN in sodium phosphate buffer
(pH 7.6) to an acetonitrile solution of 7a results in the
appearance of the 4-nitrophenylazophenolate absorption (a in
Figure 11). This band cannot be observed if the acetonitrile
solution of 7a is treated with sodium phosphate buffer lacking
NaCN (b in Figure 11). Similarly, halide salts have essentially
no influence on 7a under otherwise identical conditions. Indeed,
at 380 nm for 7a and at 371 nm for 8a (Table 2). Upon addition
of Bu4NOH, this absorption disappears with the concomitant
appearance of a band at ca. 575 nm (d and e in Figures 9 and
S14) for the 4-nitrophenylazophenolate chromophores of 7c/
7d and 8c. Indeed, the spectrum of the model 4-nitrophenyl-
azophenolate 12 (f in Figures 9 and S14) shows an absorption
at 576 nm with a molar extinction coefficient close to 50 mM-1
cm-1 (Table 2).
The addition of Bu4NCN to acetonitrile solutions of 7a and
8a causes similar absorption changes (a and b in Figures 10
and S15). Once again, the characteristic absorption of a
4-nitrophenylazophenolate chromophore can be observed at ca.
580 nm only in the presence of the nucleophile. The chromoge-
nic transformation of 7a, however, is significantly faster than
that of 8a. The 4-nitrophenylazophenolate absorbance of the
product reaches a stationary value in less than 1 min after the
750 J. Org. Chem., Vol. 71, No. 2, 2006

Chromogenic Oxazines for Cyanide Detection
FIGURE 12. Solutions of 7a (1 mM, 200 µL, dichloroethane, 298 K)
and Bu4NCl (1 M) with overlaid sodium phosphate buffer (500 µL,
pH 9.0) without (a) or with 10 (b) or 100 µM (c) of NaCN.
FIGURE 14. Transient absorption spectrum (a) of 8a recorded 4 µs
after the laser pulse (355 nm, 6 ns, 8 mJ, 0.1 mM, MeCN, 295 K) and
evolution of the absorbance at 290 (b) and 380 (c) nm upon laser
excitation.
transfer of cyanide anions from the aqueous to the organic phase
and encourages the chromogenic transformation. Furthermore,
the chromogenic process is particularly sensitive to the pH of
the aqueous phase and has an optimal response to cyanide at a
pH of 9.0. Indeed, the absorbance of the organic solution at
581 nm remains negligible up to this particular pH value (a in
Figure 13) in the absence of cyanide salts in the aqueous phase.
Instead, the absorbance increases significantly with the pH of
the aqueous solution when this particular phase contains cyanide
anions (b in Figure 13). At a pH of 9.0, even micromolar
concentrations of cyanide in the aqueous phase are sufficient
to impose a detectable change on the absorbance of the organic
phase (c and d in Figure 13).
Transient Absorption Spectroscopy. The laser excitation
of the [1,3]oxazine 1a induces the formation of the ring-opened
isomer 1b in less than 6 ns with a quantum yield of 0.1 in
aerated acetonitrile.38 The photogenerated species reverts to the
original form with a lifetime of 22 ns. In principle, a similar
process can also occur upon excitation of the [1,3]oxazines 7a
and 8a. To explore this possibility, we have analyzed 8a by
laser flash photolysis. In contrast to the behavior of 1a, the
characteristic absorption of 8b cannot be detected in the resulting
transient absorption spectra. Instead, an increase in absorbance
at 290 nm and the bleaching of the π f π* transition of the
4-nitrophenylazophenyl chromophore at 380 nm are evident in
the spectrum recorded after 4 µs from the laser pulse (a in Figure
14). Both transient bands remain constant in the microsecond
domain (b and c in Figure 14), but eventually disappear with
millisecond-second time scales.42 In fact, the steady-state
absorption spectra recorded before and after the laser flash
photolysis experiment are virtually indistinguishable. These
observations are consistent with the photoinduced trans f cis
FIGURE 13. Absorbance at 581 nm of a solution of 7a (1 mM, 200
µL, dichloroethane, 298 K) and Bu4NCl (1 M) after treatment with
sodium phosphate buffer (300 µL) without (a) or with (b) NaCN (0.1
mM) and dilution with dichloroethane (470 µL). Steady-state absorption
spectra of a solution of 7a (1 mM, 200 µL, dichloroethane, 298 K)
and Bu4NCl (1 M) after treatment with sodium phosphate buffer
(100 µL, pH 9.0) without (c) or with (d) increasing amounts of NaCN
(1-100 µM) and dilution with dichloroethane (120 µL).
the absorption spectra of acetonitrile solutions of 7a do not show
any increase in absorbance in the visible region even after the
addition of sodium phosphate buffer containing large amounts
(10 mM) of NaF (c in Figure 11), NaCl (d), NaBr (e), or NaI
(f).
Acetonitrile solutions of 7a respond to aqueous solutions of
cyanide with a detectable absorbance change only if the cyanide
concentration is greater than 0.1 mM. The chromogenic
response, however, improves considerably when 7a is dissolved
in dichloroethane together with Bu4NCl. The resulting organic
solutions change color when treated with aqueous solutions
containing micromolar concentrations of cyanide (a-c in Figure
12). Presumably, the tetrabutylammonium salt facilitates the
J. Org. Chem, Vol. 71, No. 2, 2006 751

Tomasulo et al.
2-(4′-Nitrophenylazo)-5a,6,6-trimethyl-5a,6-dihydro-12H-in-
dolo[2,1-b][1,3]benzooxazine (7a). A solution of PBr3 in CH2Cl2
(1:20 v/v, 360 µL) was added over 20 min to a solution of 4 (117
mg, 0.4 mmol) in CH2Cl2 (25 mL) maintained at 0 °C under N2.
The mixture was stirred for a further 3 h. During this time, the
temperature was allowed to raise to ambient conditions. At this
point, 5 (345 µL, 2 mmol) was added and the mixture was stirred
for a further 1 h. After filtration over a plug of SiO2, the solvent
was distilled off under reduced pressure and the residue was purified
by HPLC [semipreparative, MeCN/H2O (95:5 v/v)] to give 7a (73
mg, 41%) as an orange-red solid. HPLC [analytical, MeCN/H2O
(90:10 v/v)] RT ) 4.1 min, PA ) 1.4, APP ) 310.4 ( 1.3 nm; mp
156 °C; FABMS m/z 415 [M + H]+; 1H NMR (500 MHz,
chloroform-d) δ 1.25 (3H, s), 1.51 (3H, s), 1.61 (3H, s), 4.66 (2H,
s), 6.59 (1H, d, J ) 8 Hz), 6.80 (1H, d, J ) 9 Hz), 6.82 (1H, t,
J ) 7 Hz), 7.08 (1H, td, J ) 1 and 8 Hz), 7.13 (1H, dd, J ) 1 and
7 Hz), 7.74 (1H, dd, J ) 2 and 9 Hz), 7.80 (1H, d, J ) 2 Hz), 7.96
(2H, d, J ) 9 Hz), 8.37 (2H, d, J ) 9 Hz); 13C NMR (100 MHz,
chloroform-d) δ 16.7, 19.1, 26.2, 40.5, 48.2, 103.8, 108.6, 118.8,
119.4, 120.4, 121.5, 122.4, 122.7, 123.3, 124.9, 127.7, 138.4, 146.4,
147.4, 148.6, 156.5, 158.1.
isomerization of the 4-nitrophenylazophenyl chromophore of
8a, followed by the thermal cis f trans re-isomerization, and
exclude the photoinduced ring opening observed for 1a.
Conclusions
The cyanide anion can be detected colorimetrically relying
on the opening of a [1,3]oxazine ring and the concomitant
formation of a 4-nitrophenylazophenolate chromophore. Chro-
mogenic molecules able to satisfy these design requirements
can be prepared by fusing a benzooxazine ring to an indoline
fragment. The [1,3]oxazine ring of the resulting compounds
opens and closes rapidly on the 1H NMR time scale in
acetonitrile-d3 at ambient temperature. The free energy barrier
for the ring-opening process increases when either (1) the
substituent on the carbon atom at the junction of the two
heterocycles changes from a methyl to a phenyl group or (2)
the solvent varies from acetonitrile-d3 to toluene-d8. The ring-
opened isomer is short-lived, but can be trapped with the
addition of a nucleophile. For example, a cyanide anion can
attack the electrophilic indolium cation of this species preventing
the ring-closing process. The result is the quantitative formation
of a cyanoamine and the appearance of an intense band in the
visible region of the absorption spectrum, corresponding to a
4-nitrophenylazophenolate chromophore. The chromogenic re-
sponse of the phenyl-substituted oxazine to cyanide requires
several minutes to reach a steady state. Instead, the coloration
of the methyl-substituted oxazine occurs on a time scale of
seconds. Furthermore, this particular compound is not affected
by fluoride, chloride, bromide, and iodide anions, which are
common interferents in conventional sensing schemes for
cyanide.32-37 Finally, dichloroethane solutions of this oxazine
and a phase-transfer catalyst respond to aqueous solutions
containing micromolar amounts of cyanide with a noticeable
absorbance increase in the visible region, offering detection
limits comparable to those of the best chemosensors available
for this anion.36,37 Thus, our operating principles for the
colorimetric detection of cyanide can eventually evolve into fast
and simple assays for the determination of relatively small
amounts of this toxic analyte in water without suffering from
the deleterious interference of common anions.
2-(4′-Nitrophenylazo)-5a-phenyl-6,6-dimethyl-5a,6-dihydro-
12H-indolo[2,1-b][1,3]benzooxazine (8a). A solution of PBr3 in
MeCN (1:10 v/v, 190 µL) was added over 20 min to a solution of
4 (185 mg, 0.6 mmol) in MeCN (25 mL) maintained at 0 °C under
Ar. After addition of Et3N (80 µL), the mixture was stirred for a
further 1 h at 0 °C and then heated under reflux for 4 h. At this
point, 6 (400 mg, 1.8 mmol) was added and the temperature was
allowed to lower to ambient conditions. After 36 h, the solvent
was distilled off under reduced pressure and the residue was purified
by column chromatography [SiO2, hexane/CH2Cl2 (1:1 v/v)] to give
8a (135 mg, 51%) as an orange-red solid. HPLC [analytical, MeCN/
H2O (90:10 v/v)] RT ) 4.1 min, PA ) 1.6, APP ) 303.2 ( 1.0
1
nm; mp 200 °C; FABMS m/z 476 [M]+; H NMR (400 MHz,
chloroform-d) δ 0.85 (3H, s), 1.60 (3H, s), 4.57 (1H, d, J ) 11
Hz), 4.67 (1H, d, J ) 11 Hz), 6.75 (1H, d, J ) 8 Hz), 6.90 (1H,
t, J ) 7 Hz), 6.97 (1H, d, J ) 9 Hz), 7.16-7.19 (2H, m), 7.35-
7.42 (3H, m), 7.64-7.73 (4H, m), 7.93 (2H, d, J ) 9 Hz), 8.34
(2H, d, J ) 9 Hz); 13C NMR (100 MHz, chloroform-d) δ 22.9,
31.8, 41.2, 49.8, 104.8, 109.2, 118.7, 120.6, 120.7, 122.4, 122.6,
123.3, 124.3, 124.9, 127.8, 128.3, 128.7, 129.0, 137.0, 138.0, 146.6,
147.5, 148.4, 156.2, 157.9.
X-ray Crystallography. Single crystals of 1d were grown from
an equimolar solution of 1a and Bu4NCN in chloroform/hexane
(2:1 v/v) maintained in the refrigerator. Single crystals of 7a and
8a were grown by vapor diffusion of MeOH into a solution of the
corresponding compound in chloroform/hexane (2:3 v/v for 7a and
2:1 v/v for 8a).
Experimental Section
2-Hydroxymethyl-4-(4′-nitrophenylazo)phenol (4). A solution
of 3 (1.12 g, 5 mmol) in H2O (15 mL) was added over 90 min to
a solution of 2 (535 mg, 4.3 mmol) in aqueous NaOH (1 M, 5 mL)
and H2O (10 mL) maintained at 0 °C. The mixture was stirred for
a further 45 min. During this time, the temperature was allowed to
raise to ambient conditions and the pH was maintained at ca. 8 by
adding aliquots of aqueous NaOH (1 M). After the addition of
aqueous HCl (1 M, 5 mL), the mixture was cooled to -5 °C and
maintained at this temperature for 1 h. The resulting precipitate
was filtered, dissolved in MeCO2Et (50 mL), and dried (MgSO4).
After filtration, the solvent was distilled off under reduced pressure
to afford 4 (1.13 g, 96%) as a bright-orange solid. Mp 178-179
°C; FABMS m/z 274 [M + H]+; 1H NMR (500 MHz, chloroform-
d) δ 2.43 (1H, br s), 5.03 (2H, s), 7.05 (1H, d, J ) 9 Hz), 7.73
(1H, d, J ) 2 Hz), 7.85 (1H, dd, J ) 2 and 9 Hz), 7.97 (2H, d,
J ) 9 Hz), 8.15 (1H, br s), 8.36 (2H, d, J ) 9 Hz); 13C NMR (100
MHz, chloroform-d) δ 60.4, 116.3, 123.3, 123.8, 125.6, 125.7,
130.4, 147.1, 149.1, 157.1, 160.4.
Crystal data for 1d: [C24H20N3O3](C16H36N)‚3H2O, M )
694.94, monoclinic, P21/n (no. 14), a ) 9.5783(9) Å, b ) 25.749-
(3) Å, c ) 16.5586(17) Å, â ) 96.163(8)°, V ) 4060.3(7) Å3, Z )
4, Dc ) 1.137 g cm-3, µ(Cu KR) ) 0.606 mm-1, T ) 173 K,
yellow/brown needles; 7678 independent measured reflections, F2
refinement, R1 ) 0.103, wR2 ) 0.159, 5514 independent observed
absorption-corrected reflections [|Fo| > 4σ(|Fo|), 2θmax ) 142°],
468 parameters. CCDC 283555.
Crystal data for 7a: C24H22N4O3, M ) 414.46, monoclinic,
P21/c (no. 14), a ) 22.661(4) Å, b ) 14.4660(18) Å, c ) 13.077-
(6) Å, â ) 102.33(3)°, V ) 4188(2) Å3, Z ) 8 (two independent
molecules), Dc ) 1.315 g cm-3, µ(Mo KR) ) 0.089 mm-1, T )
203 K, orange platy needles; 7353 independent measured reflec-
tions, F2 refinement, R1 ) 0.064, wR2 ) 0.127, 3957 independent
observed reflections [|Fo| > 4σ(|Fo|), 2θmax ) 50°], 560 parameters.
CCDC 261143.
Crystal data for 8a: C29H24N4O3, M ) 476.52, monoclinic,
P21/c (no. 14), a ) 8.7976(12) Å, b ) 18.011(6) Å, c ) 15.680(4)
Å, â ) 104.74(2)°, V ) 2402.7(10) Å3, Z ) 4, Dc ) 1.317 g cm-3
,
(42) The transient absorption spectra of 8a trapped in poly(methyl
methacrylate) matrixes show essentially the same temporal evolution (Figure
S16).
µ(Cu KR) ) 0.704 mm-1, T ) 293 K, orange/yellow blocks; 3566
independent measured reflections, F2 refinement, R1 ) 0.060,
752 J. Org. Chem., Vol. 71, No. 2, 2006

Chromogenic Oxazines for Cyanide Detection
wR2 ) 0.168, 2719 independent observed reflections [|Fo| >
4σ(|Fo|), 2θmax ) 120°], 314 parameters. CCDC 261144.
Supporting Information Available: General methods and
experimental procedures for the synthesis of 11; high-performance
liquid chromatograms of 7a and 8a; single-crystal X-ray structures
of 1d, 7a, and 8a; partial 1H NMR spectra of 8a at various
temperatures; partial 1H NMR spectra of 7a and 8a before and after
the addition of Bu4NOH; partial 1H NMR spectra of 8a before and
after the addition of Bu4NCN; steady-state absorption spectra of
8a before and after the addition of Bu4NOH; steady-state absorption
spectra of 8a before and after the addition of Bu4NCN; kinetic trace
for 8a in poly(methyl methacrylate) upon laser excitation. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Absorption Spectroscopy. The steady-state spectra were re-
corded in aerated MeCN, using quartz cells with a path length of
0.5 cm. The transient spectra were recorded either in aerated MeCN,
using quartz cells with a path length of 1.0 cm, or in poly(methyl
methacrylate) (PMMA). The excitation source was a Nd:YAG laser
(355 nm, 6 ns, 8 mJ). The PMMA films were prepared by spin-
coating aliquots of CH2Cl2 solutions of the polymer (160 mg mL-1
)
and 8a (8 mg mL-1) on glass plates at 420 rpm for 9 s. The
thickness of the resulting films (ca. 6 µm) was measured with a
digital micrometer.
Acknowledgment. We thank the National Science Founda-
tion (CAREER Award CHE-0237578), University of Miami,
and MIUR (Rome, Italy) for financial support.
JO052096R
J. Org. Chem, Vol. 71, No. 2, 2006 753