The photoreactions of simple amides with NO. Gaining insight into radical bio-damages through an EPR case study

Article abstract of DOI:10.1016/j.tet.2012.01.066

Eight simple amides have been subjected to UV irradiation in the presence of either MNP or NO. In all cases radical species were generated: these were detected by means of EPR spectroscopy in the form of different nitroxides resulting from the trapping of the primary radicals. NO acted as a double spin trap, scavenging a radical to afford a diamagnetic nitroso derivative that in turn acted as trap towards another radical unit. As amido-groups are present in components of skin tissue and may be present in many therapeutic or cosmetic products used as skin sunscreen, and NO is a ubiquitous endogenous reactive species, the nitroxides detected in the present studies might participate in radical processes triggered by sun exposure and resulting in damages, even severe, of biological tissues.

Full text of DOI:10.1016/j.tet.2012.01.066

Tetrahedron 68 (2012) 2662e2670
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
The photoreactions of simple amides with NO. Gaining insight into radical
bio-damages through an EPR case study
,
a
Angelo Alberti a, Loris Grossi b , Dante Macciantelli
*
a ISOF, Area della Ricerca del CNR, Via P. Gobetti 101, 40129 Bologna, Italy
Dipartimento di Chimica Organica “A. Mangini”, Universita di Bologna, Viale Risorgimento 4, 40136 Bologna, Italy
b
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight simple amides have been subjected to UV irradiation in the presence of either MNP or NO. In all
cases radical species were generated: these were detected by means of EPR spectroscopy in the form of
different nitroxides resulting from the trapping of the primary radicals. NO acted as a double spin trap,
scavenging a radical to afford a diamagnetic nitroso derivative that in turn acted as trap towards another
radical unit. As amido-groups are present in components of skin tissue and may be present in many
therapeutic or cosmetic products used as skin sunscreen, and NO is a ubiquitous endogenous reactive
species, the nitroxides detected in the present studies might participate in radical processes triggered by
sun exposure and resulting in damages, even severe, of biological tissues.
Received 27 September 2011
Received in revised form 9 January 2012
Accepted 23 January 2012
Available online 30 January 2012
Keywords:
Nitric oxide
Amides
Ó 2012 Elsevier Ltd. All rights reserved.
Photolysis
Nitroxides
EPR spectroscopy
1. Introduction
In addition to the ROS and the above mentioned radicals, other
reactive species participate in chemical processes taking place in
The effects of UV radiation on the skin have been described, but
only the first reaction-step of the ‘chemistry’ underneath the in-
duced skin sensitization due to endogenous or exogenous stimulus
seems to have been ascertained, whilst the molecular mechanisms
by which secondary effects are produced have not been clarified.
Indeed, photo-excited states of endogenous UVA chromophores,
such as porphyrins, melanin precursors, and crosslink-fluorophores
of skin collagen can exert a negative action, giving rise to reactive
oxygen species, ROS, by direct reaction with substrate molecules
(type I photosensitization) or with molecular oxygen (type II). Some
of these species, including alkoxy, peroxy radicals and the super-
oxide anion, are actually involved in the first step of the process
induced by UVA, and have been recognized as being responsible for
skin damage.1,2
The different amido-groups belonging to the peptides and
ceramides present in the epidermis, are among the many functional
groups liable to generate radicals following UV irradiation; in fact,
the photoexcitation of the amido-group can lead to the formation of
both carbon centered and nitrogen centered radicals. Oxygen cen-
tered radicals can also be formed by further reaction of the carbon
centered species with oxygen, while the oxygen end of the pho-
toexcited triplet carbonyl group may act as an alkoxy radical.
human body, and among these NO, a ubiquitous and endogenously
formed radical has a major relevance. In particular, nitric oxide
plays a key role in the dermal response to external stimuli: it is
present in lesional psoriatic skin, in squamous cell carcinomas,
during wound healing and contributes to the formation of sunburn
erythema.3 On the favorable side, NO is an effective inhibitor of
lipid peroxidation and its coordinated action on gene expression
and preservation of membrane function effectively protects against
UV-A or ROS induced apoptotic and necrotic cell death.4
Significant quantities of NO are continuously released from
human skin5 and it has been repeatedly shown that the NO-
synthase dependent production of NO potentially occurs in all
dermal cell types.4 Nitric oxide is also produced at the skin surface
by bacterial and chemical reduction of sweat nitrate.5 In addition, it
has also been proved that NO release is significantly increased
following exposure to UV radiation, as in the case of sun
exposure.5,6
The most reliable mechanisms of skin damage are considered to
involve radical intermediates: thus, the detection and identification
of these species would greatly help the understanding of the
mechanism of interaction. EPR spectroscopy, especially when used
in combination with the spin trapping technique, has long since
been recognized as the most powerful tool for the detection and
identification of free radicals, normally very short lived in-
termediates. Its application to the study of processes taking place
* Corresponding author. Tel.: þ39 (0)51 2093627; fax: þ39 (0)51 2093654; e-mail
address: loris.grossi@unibo.it (L. Grossi).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.01.066

A. Alberti et al. / Tetrahedron 68 (2012) 2662e2670
2663
even in a very complex ‘reaction pot’ such as the skin has been
largely exploited either directly, especially through the use of
nitrones (e.g., 5,5-dimethyl-1-pyroline N-oxide, DMPO, and N-tert-
solution after adding PILA 124. Under these conditions PILA 124
abstracts one hydrogen from a methyl group of 1 through its ex-
cited carbonyl group leading again to 1a.
butyl-
a
-phenyl nitrone, PBN) able to trap carbon or oxygen
Room temperature photolysis of 1 in the presence of NO, led to
the EPR detection of only one radical species that was identified as
nitroxide 1b, its spectral parameters being consistent with those
reported in the literature.12 Although unexpected, the detection of
nitroxide 1b can be justified on the basis of the two competing
photocleavage paths of dimethylformamide; that is cleavage of the
C(O)eN bond and cleavage of an N-methyl bond.13
centered radicals forming detectable EPR nitroxides, or indirectly
monitoring the decay of nitroxidic spin probes such as 2,2,6,6-
tetramethylpiperidine 1-oxyl (TEMPO), 3-carbamoyl-2,2,5,5-
tetramethylpyrrolidine-1-oxyl (PCM), and 3-carboxy-2,2,5,5-
tetramethylpyrrolidine-1-oxyl (PCA) to show the presence of ROS,
which are known to convert nitroxides to EPR silent hydroxyl-
amines. Unfortunately, the results were not always unequivocal. In
particular, the finding that in several cases the decay of the EPR
signal of the nitroxides could only be observed in the presence of
thiols7e10 or of NADH,11 made the actual formation of ROS un-
certain, let alone their direct interaction with the spin probes.
On the other hand, the presence of ROS and other radicals along
with that of nitric oxide may result in adventitious radical species.
First NO, being a radical itself, might scavenge other paramagnetic
species (carbon centered radicals or ROS) to give diamagnetic
nitroso derivative. In a second stage, these would act as spin traps
leading to the formation of EPR detectable nitroxides.
Thus, it may be tentatively envisaged a CeΝ b-fragmentation of
photoexcited 1, followed by coupling of the resulting formyl radical
with nitric oxide to give HC(O)NO. This in turn might trap a methyl
radical from the other type of fragmentation of DMF.
2.2. N-Methylacetamide, 2
In contrast with 1, photolysis of N-methylacetamide 2 in the
presence of MNP led to the observation of three distinct radical
species originating via either hydrogen abstraction by the pho-
toexcited carbonyl (radicals 2a and 2b) or its b-scission (radical
Following this hypothesis, we conducted a case study on the
photoexcitation of amides 1e8 in the presence of NO, not so much
to isolate the resulting products but with the aim to identify the
newly formed radicals that might model those formed in the actual
biological processes. As a preliminary support of this study the
photoexcitation of the same amides in the presence of MNP, a well-
known spin trap, was at first carried out. Several nitroxides, de-
2c). While nitroxide 2a can be assimilated to 1a, that analogous to
2b was not observed with DMF, 1. Even if some doubts could be
reasonably cast on the identity of 2b as di-tert-butyl nitroxide, it
exhibits a very similar spectrum, and we base its identification on
the value of the nitrogen splitting constants, which were found to
vary slightly but definitely with every amide (see below) being
somewhat larger than that normally observed for di-tert-butyl
nitroxide in acetonitrile. In principle one might envisage the au-
thentic 2b via photoreaction of N-methylethanolamine with di-
tert-butyl peroxide in the presence of MNP. On the other hand,
the necessary starting amine is commercially unavailable and we
believe that it synthesis would not be worth the effort as its re-
action with tert-butoxy radicals may result in the formation of
several different radicals and hence in complex and un-
informative spectra. The identification of 2b is therefore to be
considered tentative.
riving from the trapping of radicals originating from both the b-
cleavage and intermolecular hydrogen abstraction of the excited
carbonyl group, were identified and compared with those detected
using the nitric oxide as trap.
2. Results
Photolysis of ACN solutions of alkyl amides 1e8 was expected to
lead to several radicals deriving from b-fragmentation (acyl radi-
cals, alkyl radicals, and aminyl radicals) and from intermolecular
hydrogen abstraction by the photoexcited carbonyl group. None of
these ‘primary’ species were directly detected when the amides
were photolyzed, but in the presence of MNP, the corresponding
adducts, whose spectral parameters are collected in Table 1, were
observed. In nearly all cases, signals due to di-tert-butyl nitroxide,
a species whose formation is practically unavoidable when pho-
tolyzing MNP, were also detected.
In principle, doubts might be also cast on the identity of 2c, as
the
a formyl and a dimethylaminyl radical or a methyl and a dimethy-
laminocarbonyl radical. We favor the former kind of -scission, as if
b-scission of photoexcited 2 may result in the formation of both
b
the latter took place, the methyl radical should be readily trapped
by MNP. On the other hand, MNP is much less efficient in trapping
aminyls than alkyl radicals, and the failure to detect tert-butyl
dimethylamino nitroxide is not surprising. In any case, radical 2c
disappeared when PILA 124 was added to the solution, leaving
nitroxides 2a and 2b as the only detectable species.
The prolonged photolysis of a NO saturated ACN solution of 2 led
to a strong clean signal from a radical identified as 2d (see Fig. 1);
yet, it may be worth noting that while the formation of this species
clearly involves two radicals from a hydrogen-abstraction process,
no tert-alkyl nitroxide involving their counterpart was observed.
In the experiments run in the presence of PILA 124 (2-
ethoxythioxanthone) and irradiating with light at
l
ꢀ400 nm, only
MNP adducts of the radicals originating via hydrogen abstraction
from the amides by the photoexcited PILA 124 were observed.
The ‘primary’ radicals could also not be directly detected upon
irradiation of NO-saturated solutions of amides 1e8, which instead
resulted again in the formation of nitroxides (Table 1).
2.1. N,N-Dimethylformamide, 1
2.3. N,N-Dimethylacetamide, 3
Photolysis of 1 in the presence of MNP only led to the detection
of the spectrum from a nitroxide, identified as 1a, overimposed to
that of di-tert-butyl nitroxide. The spectral parameters indicate that
1a results from the trapping by MNP of the radical originating via
an intermolecular hydrogen abstraction from a methyl group by
a photoexcited carbonyl.
Competition between b-scission and hydrogen abstraction was
also evident when photolyzing 3 in the presence of MNP. In this
case tert-butyl acetyl nitroxide 2c could be readily detected.
This radical, however, was accompanied by two additional MNP
adducts, both exhibiting interaction of the unpaired electron with
a nitrogen and two equivalent hydrogen atoms. These were iden-
tified as the isomeric nitroxides 3a and 3b on the basis of the
similarity of their spectral parameters with those exhibited by 1a
and 2a. Geometrical isomerism in amides is well established and
a large number of NMR studies have addressed this issue.14 It would
then appear that a photoexcited amide abstracts a hydrogen from
The failure to show any trace of tert-butyl-acetyl nitroxide
suggests that at the temperature of operation, hydrogen abstraction
by the triplet carbonyl prevails over
identification of 1a was further substantiated by the finding that an
identical spectrum was observed when irradiating (
ꢀ400 nm) the
b-fragmentation processes. The
l

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A. Alberti et al. / Tetrahedron 68 (2012) 2662e2670
Table 1
Radicals upon photolysis of amides 1e8a

A. Alberti et al. / Tetrahedron 68 (2012) 2662e2670
2665
Table 1 (continued)
a
Hyperfine splitting constants in mT.

2666
A. Alberti et al. / Tetrahedron 68 (2012) 2662e2670
Fig. 1. EPR spectrum observed upon photolysis of a NO saturated ACN solution of 2 at
243 K. Blue: experimental; red: computer simulation.
Fig. 2. EPR spectrum observed upon photolysis of a NO saturated ACN solution of
compound 4 at 233 K. Blue: experimental; red: computer simulation.
As both species showed, beside that with the nitroxidic nitro-
gen, the interaction of the unpaired electron with two equivalent
nitrogen and two equivalent hydrogen atoms, to both were
assigned MeC(O)NHCH(Me)N(O$)CH(Me)NHC(O)Me as the same
general structure, what may be consistent with the presence of two
diasteroisomers (4c and 4d). Indeed, over the investigated tem-
perature range, only the splittings of the two equivalent hydrogens
experienced an appreciable variation increasing from 0.194 mT at
233 K to 0.268 mT at 293 K for one species and from 0.539 mT at
233 K to 0.570 mT at 293 K for the other, while all other parameters
only experienced negligible variations. As in the corresponding
nitroxide from 2 only one isomer, i.e., 2d, was detected, it seems
reasonable attributing this conformational behavior to the pres-
ence of the more sterically demanding ethyl group in 4 than that of
a methyl group in 2.
either of the magnetically non-equivalent methyl groups of other
amide molecules leading to two ‘different’ primary-alkylamino
radicals that, once trapped by MNP, afford 3a and 3b. Also in the
case of 3, no tert-butyl tert-alkyl nitroxide was observed, originat-
ing from the trapping of the counterpart of the primary-alkylamino
radicals leading to 3a and 3b. The main difference to be found in the
spectral parameters of these two nitroxides concerns the couplings
of the b-hydrogen and b-nitrogen atoms that, due to their position,
are more conformation sensitive.
As had been the case with amides 1 and 2, the addition of PILA
124 totally suppresses
disappearance of the acyl nitroxide 2c.
Hydrogen abstraction and -scission were also operative upon
b-fragmentation, resulting in the complete
b
photolysis of solutions of 3 and NO: spectra showed the presence
of four different nitroxides. Nitroxide 3d, results from the cou-
pling of NO with the primary alkylamino radical (hydrogen ab-
straction from an N-methyl group) followed by trapping of an
identical radical by the resulting alkylnitroso derivative. The same
alkylnitroso derivative also traps the other tert-alkylamino radical
formed in the hydrogen abstraction to give nitroxide 3e, whereas
acyl nitroxide 3f is formed when yet the same alkylnitroso de-
2.5. N,N-Diethylacetamide, 5
Photolysis of 5 in the presence of MNP led to the detection of
three different nitroxides. The acetyl tert-butyl nitroxide 2c, which
derives from
b-fragmentation of photoexcited 5, the nitroxides
rivative traps the acetyl radical resulting from
worth noting that the four -hydrogen atoms in nitroxides 3d are
magnetically equivalent only in pairs and that the two -hydro-
gens in 3e are not equivalent, while in nitroxide 2d all the four
-hydrogen atoms are equivalent. It would then appear that the
replacement of the amidic hydrogen with a second methyl group
hampers the rotation about the N(O$)eC bonds. Yet, in radical 3f,
where a less sterically demanding acetyl unit replaces one alky-
lamido moiety, the two methylenic hydrogen atoms are again
equivalent.
b-scission. It is
resulting from the trapping by MNP of the secondary-alkyl (5a),
and tertiary-alkyl radical (5b) generated via intermolecular hy-
drogen abstraction by photoexcited 5. Consistently, addition of PILA
124 to the solution being photolyzed resulted in the disappearance
of nitroxide 2c and in a significant increase of the intensity of the
signal from 5a.
When photolyzed in the presence of NO, 5 led to the detection of
very complex spectra with a fairly significant dependence on
temperature. We interpreted the spectrum recorded at room
temperature as the sum of the signals from the three nitroxides 5c,
5d, and 5e. The formation of the last two nitroxides is easy to ex-
plain, as intermolecular hydrogen abstraction by photoexcited 5
from a methylene moiety of an ethyl group leads to a secondary-
alkyl and a tertiary-alkyl radical; both can couple with NO to give
two adventitious nitroso derivatives that can trap another sec-
ondary- or tertiary-alkyl radical to give 5d and 5e.
b
b
b
The spectral parameters determined for the fourth nitroxide
detected when photolyzing 3 and NO led to its identification as the
acetyl nitroxide 3c. Its formation is an unexpected finding, fairly
intriguing to be accounted for (see Discussion).
2.4. N-Ethylacetamide, 4
As for 5c, while its spectral parameters leave little doubt about
its identity, it is more difficult to rationalize its formation: although
this may be thought to involve the secondary-alkyl nitroso de-
rivative, the presence of the hydrogen atom directly bound to the
nitroxidic nitrogen is difficult to account for.
Only species deriving from a hydrogen abstraction process were
observed when photolyzing a solution of 4 containing some MNP
and PILA 124. These radicals could be identified as the tert-butyl
secondary-alkylamino nitroxide 4a and the tert-butyl tert-alkyla-
mino nitroxide 4b. As expected, in the absence of PILA 124 the acyl
nitroxide 2c could also be observed.
2.6. N-Methylpropionamide, 6
The photoreaction of 4 with NO led to the detection of an EPR
spectrum due to the superimposition of the signals from two
nitroxides exhibiting different couplings with the same groups of
atoms, that is, from two isomeric species (see Fig. 2).
As could have been foreseen, the photoreaction of 6 with MNP
paralleled that of 2 leading to the detection of the two nitroxides 6a
and 6b resulting from intermolecular hydrogen abstraction along

A. Alberti et al. / Tetrahedron 68 (2012) 2662e2670
2667
with that of the acyl-nitroxide 6c originating from the trapping of
a propionyl radical ( -scission) by MNP.
Likewise 2, which upon photolysis in the presence of NO only
led to the detection of nitroxide 2e, 6 only afforded the symmetric
nitroxide 6d where two identical primary-alkyl fragments are
bound to the nitroxidic function.
2.8. N,N-Dimethylisobutyramide, 8
Both intermolecular hydrogen abstraction and
were evident in the photoreaction of 8 with MNP that led to the
detection of five different nitroxides. Also in this case, due to the
intrinsic non equivalence of the amidic methyl groups, hydrogen
abstraction resulted in two isomeric primary-alkyl radicals that
eventually led to nitroxides 8a and 8b along with the tert-butyl
b
b
-scission
2.7. N,N-Dimethylpropionamide, 7
tertiary-alkyl nitroxide 8c.
b-Scission led instead to acyl nitro-
xide 8d, while tert-butyl isopropyl nitroxide (8e) reflects
decarbonylation of some isobutyroyl radicals prior to trapping
by MNP.
The detection of 8g when 8 was photolyzed in the presence of
NO reflects intermolecular hydrogen abstraction, while that of
nitroxide 8f requires the occurrence of both intermolecular hy-
Four different nitroxides could be characterized when photo-
lyzing 7 in the presence of MNP. Indeed, due to the intrinsic mag-
netic non equivalence of the two amidic methyl groups, hydrogen
abstraction led to the two isomeric tert-butyl primary-alkyl nitro-
xides 7a and 7b along with the tert-butyl tert-alkyl nitroxide 7c. As
it had been the case for 6, the tert-butyl propionyl nitroxide 6c was
drogen abstraction and b-scission.
also observed, the b-scission process from which it was originated
being inhibited by the addition of some PILA 124.
In contrast with what observed with the structurally related
amide 3, both the two nitroxides detected when photolyzing 7 in
3. Discussion
the presence of NO reflected a
b
-scission process. Thus, the for-
As the action of the UVA and UVB components of solar radiation
on carbonyl compounds, and in particular amides, that are common
components of skin proteins as well as therapeutic or cosmetic
salves, balms, and soothing ointments used as skin sunscreens, and
normally results in the onset of radical processes, we were
prompted to investigate the photo behavior of some alkyl amides in
the presence of NO, a reactive species naturally occurring in the
human body.
mation of the propionyl nitroxide 7d may be thought to proceed as
proposed in Scheme 4 (see Discussion) for the formation of 3c and
5c, while in principle that of 7e may involve either trapping of
a propionyl radical by EtC(O)NMeCH2NO (coupling of NO with the
primary-alkyl radical from hydrogen abstraction) or that of the
primary radical EtC(O)NMeCH2ꢁ by EtC(O)NO (coupling of NO with
the propionyl radical from b-scission).
Scheme 1.
Scheme 2.

2668
A. Alberti et al. / Tetrahedron 68 (2012) 2662e2670
Scheme 3.
Scheme 4.
The photochemical behavior of alkyl amides has long since been
abstracts a hydrogen atom from the alkyl group of a nearby amide
molecule. This process is outlined in Scheme 1a for amides 1e3
and 6e8.
The detection of nitroxides E and of acyl nitroxides G provides
evidence that radicals B are formed via hydrogen abstraction by the
triplet of the photoexcited amide rather than by the acyl radical
the subject of investigation, and the initial proposal15 of a type II
photodecomposition of these substances with formation of alkenes
and lower amides has been later reassessed16,17 in favor of a type I
photodecomposition involving acyl, alkyl, and aminyl radicals in
the case of N,N-dialkyl amides, whereas unsubstituted alkyl amides
were found to be stable to photolysis. Subsequent spin trapping
studies carried out either in the presence or in the absence of H2O2
confirmed these results, and also led to the indirect detection of
radicals resulting from hydrogen abstraction from the amides by
photogenerated hydroxyl radicals.18,19
formed in the
b
-scission of A . For N-ethyl- and N,N-diethyl-acet-
*
amide 4 and 5, respectively, hydrogen abstraction takes place from
one of the methylenic groups (see Scheme 1b).
Photolysis of amides 1e8 in the presence of NO also led in all
cases to the EPR detection of nitroxides. Because of the replacement
of MNP with NO, on the other hand, these nitroxides differed from
those detected with MNP despite the fact that the radicals involved
in their formation were the same, reflecting the occurrence of the
The occurrence of a type I process is confirmed also in the
present case based on the detection of tert-butyl acetyl nitroxide 2c
in the photoreaction of 2, 3, 4, and 5, of tert-butyl propionyl
nitroxide 6c in the photoreaction of 6 and 7, and of tert-butyl iso-
butyroyl nitroxide 8d in the photoreaction of 8. The corresponding
acyl nitroxides were instead not detected upon photolysis of 1 in
the presence of either MNP or NO.
The detection of nitroxides 1a, 2a,b, 3a,b, 4a,b, 5a,b, 6a,b, 7a,c,
and 8aec upon photolysis of amides 1e8 in the presence of MNP
as a spin trap indicates that in all cases hydrogen abstraction from
an N-alkyl group takes place. This must of course be an in-
termolecular process whereby the photoexcited (triplet) carbonyl
same processes, i.e., intermolecular hydrogen abstraction and b-
scission. Indeed, being a radical, NO acts as a radical scavenger and
reacts very quickly, coupling with the radicals formed in the pho-
tolysis of 1e8 giving rise to adventitious alkyl- or acyl-nitroso
compounds, which in turn behave as spin traps for other radicals
being generated.
Having been reported that photolysis of DMF 1, besides giving
formyl and dimethylaminyl radicals (type I
b-scission), may also
afford methyl radicals,13 the two converging routes outlined in

A. Alberti et al. / Tetrahedron 68 (2012) 2662e2670
2669
Scheme 2 can be envisaged to account for the detection of acyl
nitroxide 1b.
the well documented HN(Oꢁ)C(O)CH3,22 of the alleged, and in our
knowledge still elusive diaminonitroxide (CH3)2NN(Oꢁ)N(CH3)2,
identified on the basis of an EPR spectrum that was actually simply
due to dimethylaminoxyl radical itself,23,24 and of diacyl nitroxides
RC(O)N(Oꢁ)C(O)R for which spectral parameters are given that are
Compound 1b was in fact the only detected radical, and we
could not gather any evidence of the formation of N-nitroso-amines
nor of the trapping of aminyl radicals. Incidentally, this failure to
intercept alkylaminyls applies to all the substrates of the present
study.
instead in line with acyl tert-alkyl nitroxides RC(O)N(Oꢁ)R0.25
A
more detailed discussion on the erroneous identification of these
radicals is available in Supplementary data.
Nitroxide 2d, the only radical detected upon photolysis of 2 in
the presence of NO, is unmistakable evidence of intermolecular
It may be worth re-emphasizing that NO, being a paramagnetic
species, acts as a scavenger towards the radicals produced in the
photoreactions of amides 1e8, affording diamagnetic nitroso
compounds that in turn can act as spin traps towards other radicals.
In other words, each unit of NO reacts directly or indirectly with
two radical units, and the formation of the observed nitroxides may
be seen as a process akin to the one taking place when nitric oxide
is used to double-trap biradicals leading to cyclic nitroxides.26,27
Sunlight induced skin reactions are well known and are com-
monly targeted by biological end points such as erythema, persis-
tent pigment darkening or immunosuppression. These processes
are claimed to involve free radicals, which can be formed at all
wavelengths and in different skin layers, from the horny layer up to
a depth of 30 mm in the case of near-IR irradiation.2
The EPR spin-trapping technique allows the study and detection
of these species, even if the effect of the spin traps on the cells and
their potential toxicity must be taken into account. However, the
use of spin traps for direct measurements in biological systems has
been limited due to the poor stability of the resulting adducts in
viable systems where the aminoxyls are readily converted to EPR
silent products.28
hydrogen abstraction whereby a photoexcited molecule 2 ab-
*
stracts a hydrogen from an N-methyl group of a nearby molecule
giving a primary-alkylamino radical, which couples with NO. The
resulting nitroso compound traps another primary-alkylamino
radical leading to the symmetric nitroxide 2d (see Scheme 3a and
b). The same mechanism can be applied to the photoreaction of
the other mono- or di-N-alkyl amides, in particular to account for
the formation of 3d from 3, 4c and 4d from 4, 5e from 5, and 6d
from 6. As hydrogen abstraction simultaneously generates two-
carbon centered radicals, the formation of mixed nitroxides is also
possible: this is the case in the formation of 3e from 3, 5d from 5,
and 8g from 8. The detection of two symmetric nitroxides in the
photolysis of 4 deserves a special comment. Indeed, the spectral
parameters of 4c and 4d suggest that they are diastereoisomers
with the methine hydrogen atoms lying closer the CeNeO plane in
the case of 4d than in the case of 4e. No such isomerism was
exhibited by the structurally related nitroxide 5e, possibly because
the ethyl group replacing the NH hydrogen destabilizes the con-
formation where the methine hydrogen atoms lie close to the
CeNeO plane.
Two kinds of acyl nitroxides were also identified among the
radicals detected when photolyzing amides 1e8 in the presence of
NO. Compounds 3f, 7e, and 8f are alkyl acyl nitroxides whereas 3c
and 7d are simple acyl nitroxides. The formation of the three for-
mer radicals can be readily accounted for as outlined in Scheme 3a
Since it is reported that NO participates in the regulation of skin
functions such as circulation, UV-mediated melanogenesis,25 sun-
burn erythema, and the maintenance of the protective barrier
against microorganisms we thought it interesting to investigate
whether it could also exert a protective-role acting as spin trap
towards radicals induced by UV radiations.
by admitting the involvement of acyl radicals originated via
b-
scission (F) and carbon centered radicals from hydrogen abstrac-
tion (B).
In principle the nitroxides may be formed in any of the two ways
indicated in Scheme 3a, although we favor that involving the
alkylnitroso instead of the acyl nitroso derivatives, the former being
the more stable species.
Actually, not much has been reported on the excitation mech-
anisms triggered in the skin by radiations, on the formation of
radical species, and on the molecules that may be involved in the
initial photoexcitation process. Since the outermost layer of the
epidermis, the stratum corneum (SC), consists of dead, flattened
cells embedded in lipid lamellar regions where a series of ceram-
ides is present, which are characterized by the presence of amido
groups in their structure, these species could be involved in the
initial photoexcitation process.29 Peptides, critical component of
the innate immune response in the skin as antimicrobial,30 or
species such as nicotinamide (vitamin B3)31 are also present in the
epidermis. In all these species the amide functions are present, and
it is sensible hypothesizing their involvement in photo-activation
processes. Moreover, in skin epidermis, NO is also produced by
keratinocytes in response to UV radiation.32
In this light, the study of the photolysis of alkyl amides in the
presence of NO, that could act as endogenous-type spin trap, could
provide valuable information mimicking in vivo conditions.
In conclusion, in what we are aware to be a very optimistic
scenario, the use of NO as an ‘indirect’ spin trap might appear po-
tentially useful for studies in living cells, making unnecessary the
preliminary stability and toxicity trials to determine the appropri-
ateness of exogenous spin traps.
The formation of 3c and 7d must also involve
b-scission of
photoexcited 3 or 7 to give the appropriate acyl radicals P, the
*
*
coupling of which with NO would lead to acyl nitroso derivatives,
an overall possible mechanism being exemplified in Scheme 4.
Although we only consider the last steps of the route to 3c and
7d shown in this scheme to be hypothetic al, we wish to note that
the detection of acyl nitroxides upon photolysis of acyl nitroso
derivatives has actually been reported.20
With radical 5c, its formation must involve intermolecular hy-
drogen abstraction from the methylenic group of 5 by a second
photoexcited 5 molecule with subsequent formation of a second-
*
ary-alkyl nitroso derivative by coupling with NO (see H0). Al-
though nitroso compounds are normally inefficient hydrogen
abstractors, it may be tentatively hypothesized that 5c is eventually
formed through a route formally akin to the last two steps of
Scheme 4.
Finally, we wish to note that the present results reassess what
was wrongly reported in a paper on the EPR characterization of the
nitroxides formed in the photoreactions of some amides with NO.21
Although there seems to be a reasonable consistency between the
spectral parameters we determined for 3a, 3b, 6a, 6b, 7a, 7b, 8a, 8b
and those reported by these authors for some structurally related
nitroxides, we cannot avoid noting that many of the species re-
ported in that paper have been clearly mis-identified. In particular
this is the case of nitroxide CH3CH(OH)N(Oꢁ)C(O)CH3 that in fact is
4. Experimental
4.1. Chemicals
2-Methyl-2-nitrosopropane (MNP), amides 1e8, 2-ethox-
ythioxanthone (PILA 124), and all other reagents were purchased

2670
A. Alberti et al. / Tetrahedron 68 (2012) 2662e2670
from Aldrich in the highest purity grade commercially available,
and were used as received. NO gas, 99%, was supplied by Matheson.
Supplementary data
All relevant EPR spectra and simulations along with a critical
discussion of the misassignments reported in Ref. 21 are available
as Supplementary data. Supplementary data related to this article
can be found online at doi:10.1016/j.tet.2012.01.066.
4.2. Apparatus
All EPR spectra were recorded on an upgraded X-band Bruker
ER200/ESP300 spectrometer equipped with a NMR gaussmeter for
field calibration and a frequency counter for the determination of g-
factors that were corrected with respect to that of perylene radical
cation in concentrated sulfuric acid (g¼2.00258).
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Acknowledgements
This work was financially supported by MIUR, Rome, Fund PRIN
2009.
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