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Exposure of tetrapeptide AcNHÀGlyÀGlyÀGlyÀPheÀCO2Me
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(16) to NO2 /N2O4 produced three cleavage products: tripep-
tide AcNHÀGlyÀGlyÀPheÀCO2Me (11), dipeptide AcNHÀGlyÀ
PheÀCO2Me (8), and protected phenylalanine 7, in a 2:3:5
ratio, respectively (Scheme 5). 1H NMR analysis showed that
these contributed to about 80% of the product mixture,
whereas the remaining 20% constituted various degradation
products, which could not be identified.
Tripeptide 11 resulted from one single N-nitrosation process
(at either the N-1, N-2, or N-3 atom), whereas the shorter prod-
ucts required several successive N-nitrosation and fragmenta-
tion–rearrangement sequences. This caused depletion of the
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Scheme 6. Mechanistic studies of the reaction of dipeptide 1 with NO2 /
N2O4.
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NO2 /N2O4 concentration before the fragmentation of tetrapep-
tide 16 to give protected phenylalanine 7 was quantitative,
and it explains the mixture of shorter peptides that was ob-
tained in this reaction.
copy), in addition to some unidentifiable decomposition prod-
As expected, protection of the N-terminus as phthalimide re-
duced the number of fragmentation–rearrangement sequen-
ces. Exposure of tetrapeptide PhthNÀGlyÀGlyÀGlyÀPheÀCO2Me
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CO2Me (12), which resulted from nitrosation of the N-2 or N-3
atom, and dipeptide PhthNÀGlyÀPheÀCO2Me (19), which was
formed through nitrosation of product 12 at the N-3 atom.
Steric hindrance at the N-1 and N-2 positions increases the
resistance of the tetrapeptide AcNHÀValÀValÀGlyÀPheÀCO2Me
(18) towards backbone rearrangement; consequently, no reac-
ucts. The complete absence of protected phenylalanine 7 in
the H NMR spectrum of the product mixture, even after pro-
1
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longed reaction with NO2 /N2O4, is a clear indication of the
(17) to NO2 /N2O4 led to the tripeptide PhthNÀGlyÀGlyÀPheÀ high stability of N-nitrosated peptides in an acidic, non-aque-
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ous environment, which is provided by NO2 /N2O4 in acetoni-
trile. Therefore, we can conclude that fragmentation of the N-
nitrosated peptide backbone requires an aqueous environment
at a physiologically-relevant22 near-neutral pH, which can be
achieved by neutralizing the reaction system with aqueous
sodium bicarbonate.
tion occurred within 20 min of exposure to NO2 /N2O4. Howev-
Because the 1H NMR data of the fragmentation studies in
part one of our research did not reveal any product signals
that could be traced back to the excised amino acid, it is likely
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er, the high-resolution mass spectrum of the crude reaction
mixture, which was obtained after a reaction time of 4.5 h, in-
dicated the formation of the tripepeptide AcNHÀValÀValÀPheÀ that this byproduct was extracted into the aqueous phase
CO2Me (21) as a minor component of a complex mixture of de-
composition products (HRMS, ESI) for tripeptide 21: m/z calcd.
for C22H34N3O5 [M+H]+: 420.2493, found: 420.2498; m/z calcd.
for C22H33NaN3O5 [M+Na]+: 442.2312, found: 442.2312). Tri-
peptide 21 was formed from tetrapeptide 18 by N-nitrosation
of the sterically least hindered peptide bond (N-3) and expul-
sion of the glycine residue.
upon neutralization. Therefore, we performed a reaction in
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which dipeptide 1 was treated with excess NO2 /N2O4 in aceto-
nitrile for 20 mins. The reaction mixture was neutralized with
aqueous sodium bicarbonate and subsequently acidified with
hydrochloric acid (1m), followed by extraction with ethyl ace-
tate (Scheme 6b). 1H NMR analysis of the resultant product
mixture showed, as well as unconsumed dipeptide 1, the ex-
pected phenylalanine derivative 7 and a second product
formed in equal amount, which could be unequivocally identi-
fied as 2-hydroxy-3-phenyl propionic acid (23) by comparison
with literature data.[23] The latter byproduct likely relates to the
expelled amino acid moiety.
2. Mechanistic studies
To identify the chemical nature of the excised amino acid
moiety, we performed independent mechanistic investigations
on the previously studied reaction of dipeptide 1[9b] with an
Based on these findings, we have proposed a revised mech-
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excess of NO2 /N2O4 in [D3]acetonitrile, but the reaction mixture
anism for the NO2 mediated fragmentation–rearrangement,
1
was not neutralized. Quantitative H NMR analysis of the reac-
which is shown in Scheme 7, by using the reaction of dipep-
tide 1 as an example.
tion progress at different times (at 208C, using dimethyl ter-
ephthalate as internal standard) revealed that N-nitrosation of
1 occurred rapidly at the less hindered N-terminal amide and
led to the mono-nitrosated dipeptide 2 in 57% yield (deter-
Thus, under non-aqueous conditions, the N-nitroso peptide
2, the cyclized nitroso amide 3, and the rearranged diazotic
acid 4 exist in equilibrium, which lies on the side of peptide 2.
Upon neutralization in an aqueous environment, irreversible
decomposition of compound 4 to give the protected phenyl-
alanine 7 and the a-hydroxylated acid 23 occurs, which could
proceed through various pathways. Thus, as outlined in
Scheme 1, intermediate 4 could rearrange to give a-diazo
imide 5; subsequent regioselective imide hydrolysis and re-
lease of the protected phenylalanine 7 produced the a-diazo
acid anion 6À. The latter could also be formed through intra-
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mined by H NMR spectroscopy) after 20 min (Scheme 6a). No
peptide fragmentation to produce the protected phenylalanine
7 occurred; elongation of the reaction time only resulted in ex-
haustive N-nitrosation, which included reaction of the sterically
more hindered “internal” amide bond. Thus, the reaction mix-
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ture, after 17 h of exposure to NO2 /N2O4, contained only the
bis-nitrosated dipeptide 22 and the mono-nitrosated 2 in 57
and 22% yield, respectively (determined by H NMR spectros-
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Chem. Eur. J. 2015, 21, 14924 – 14930
14928
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