3712
G. Keceli et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3710–3713
Figure 3. Standard DTNB analyses of
L-cysteine (Cys) or synthetic peptides
(0.3 mM) initially (h), or following treatment with nitrite (( ), 0.3 mM) and AS
((j), 0.3 mM) in 10 mM phosphate buffer with 50 M DTPA (pH 7.4) at 37 °C for
l
30 min. The amounts of free thiol were normalized with respect to that detected in
the initial samples.
ratios of HNO-donor in 10 mM phosphate buffer with 50 lM metal
chelator at pH 7.4 (Fig. 2). The amount of thiol in the unmodified
peptide samples was found to be similar based on a standard
5,50-dithiobis(2-nitrobenzoic acid) (DTNB) assay (data not
shown).16 Following a 30 min-incubation with AS at physiological
pH and temperature, the presence of TrpNO was analyzed by
Figure 4. Selected region of ESI-MS spectra showing CGSAWA (0.1 mM) treated
with (a) nitrite (1 mM) and (b) AS (1 mM) in 10 mM phosphate buffer with 50
UV–visible spectroscopy (e
335 = 6100 Mꢀ1cmꢀ1).17
l
M
TrpNO is observed in all peptides upon treatment of 10-fold
excess or equimolar amount of HNO-donor. As expected, the per-
cent yield of TrpNO is significantly higher in the presence of excess
HNO-donor (ca. 24% vs ca. 3%) (Fig. 2 and Supplementary data).
Side-by-side control experiments conducted with the AS byprod-
uct, nitrite, did not produce any TrpNO peaks in either case (data
DTPA (pH 7.4) at 37 °C for 30 min. The peaks were assigned to the unmodified
peptide (m/z 593.9 0.1), peptide sulfinamide (m/z 624.9 0.1), peptide sulfinam-
ide carrying
a TrpNO modification (m/z 653.7 0.1), peptide disulfide (m/z
1185.4 0.1), and peptide disulfide carrying
1214.1 0.1).
a TrpNO modification (m/z
not shown). Comparisons with N-acetyl-L-tryptophan and AGSA-
detection of protons attached to the 15N nuclei and provides sim-
plified NMR spectra.18,19 Consistent with the expected reactivity
of HNO, the corresponding sulfinamides are observed for all cys-
teine-containing peptides upon treatment with 10-fold excess
H15NO-donor (Supplementary data),10,11,20 except for AGSAWC,
which has a C-terminal cysteine and possesses an atypical reactiv-
ity, which we have recently examined in detail.21 With the use of
an internal standard, 15N-labeled benzamide, the approximate rel-
ative yields of sulfinamides were estimated; they follow the order
AGSCWA ꢁ GSAWCA > ACSAWA > AGCAWA > CGSAWA.
WA indicate that although similar TrpNO yields are obtained for
the samples in the presence of excess AS, the TrpNO yields are sig-
nificantly lower in cysteine-containing peptides upon treatment
with an equimolar amount of AS (Fig. 2). These results suggest that
the reaction of HNO with tryptophan is approximately 1–2 orders
of magnitude slower than the corresponding reaction with cys-
teine. It should also be noted that no significant TrpNO modifica-
tion was observed under anaerobic conditions, consistent with
previous reports (data not shown).13 Although the formation of
TrpNO requires both HNO and oxygen, the mechanism for this
reaction is not well understood, and ongoing efforts are focused
on addressing this issue.
Since HNO is known to be very thiophilic (Scheme 1), the extent
of HNO-derived cysteine modifications in the above samples was
determined by a DTNB assay. As seen in Figure 3, treatment of
the cysteine and tryptophan-containing peptides with equimolar
amounts of AS results in the complete depletion of thiols, indicat-
ing that all cysteine residues are modified to the corresponding
sulfinamide or disulfide species. Consistent with these results, no
free thiol is detected following incubation of the peptides with
excess HNO-donor (data not shown). Importantly, comparable
quantities of free thiol are observed in the untreated and nitrite-
treated samples, suggesting that the depletion in thiol is due to
HNO treatment (Fig. 3). Overall, these results reveal that cysteine
is more reactive towards HNO than tryptophan.
To investigate the presence of doubly modified-peptides, we
examined HNO-treated AGSCWA and CGSAWA by ESI-MS. These
results indicate that the HNO-induced TrpNO modification is
observed on both sulfinamide and disulfide-containing peptides
(Fig. 4 and Supplementary data). Further studies are required to
determine if TrpNO formation has a preference for either one of
these thiol modifications. Treatment with nitrite did not produce
any modifications under these conditions.
Since TrpNO is known to undergo denitrosation upon incubation
with thiols,22–24 we have also examined the aerobic denitrosation of
TrpNO-containing peptides in the presence of excess L-cysteine
(5 mM). The observed rates for TrpNO disappearance under
pseudo-first-order conditions were determined by UV–visible spec-
troscopy. Similar results (kobs ꢁ8 ꢂ 10ꢀ4 sꢀ1) were obtained for all
TrpNO-containing peptides, indicating the presence of a nearby
HNO-derived cysteine modification does not have a significant
effect on denitrosation under these conditions (Table 1 and
Supplementary data). Comparison with kobs for N-acetyl-
N-nitroso-tryptophan (ca. 1.2 ꢂ 10ꢀ3 sꢀ1), which is in agreement
with the reported values for glutathione and other nucleophiles,23,25
demonstrates that the observed rates of denitrosation are slightly
slower for peptide samples examined (Table 1), potentially due to
steric hindrance.23 Under aerobic conditions, the kinetic behavior
Since cysteines were found to be completely modified upon
HNO treatment, all the TrpNO-containing peptides are also
expected to carry an HNO-derived cysteine modification. To gain
more information about the products formed, we first analyzed
the peptide samples with our 15N-edited nuclear magnetic
resonance (NMR) method for sulfinamide detection.18 In this
method, application of an isotope filter for 15N allows the selective
Keceli, Gizem
