N-carbamoyl derivatives and their nitrosation by gaseous NOx - A new, promising tool in stepwise peptide synthesis

Article abstract of DOI:10.1002/1099-0690(200203)2002:6<1026::AID-EJOC1026>3.0.CO;2-C

New uses of the N-carbamoyl group in peptide synthesis as an N^α-protecting group in classical peptide coupling methods, and as a preactivating group for stepwise coupling by NCA formation - are presented. In the first application, the N-carbamoyldipeptide esters C-Val-Gly-OEt, C-Leu-Gly-OEt, C-Ala-Gly-OEt, and C-Ala-Phe-OEt were obtained in good yields by treatment of the corresponding N-carbamoylamino acids (CAA) with amino acid esters. Quantitative N-deprotection without racemisation was then achieved in the solid through nitrosation by gaseous NO_x. The extent of racemisation occurring in the coupling step is discussed. In the second application, an easy route to amino acid N-carboxy anhydrides (NCAs) through nitrosation of CAA under the same conditions as above allowed straightforward "one-pot" peptide stepwise coupling, as demonstrated by the formation of Leu-Gly and Val-Gly in good yields and enantiomeric excess. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200203)2002:6<1026::AID-EJOC1026>3.0.CO;2-C

FULL PAPER
N-Carbamoyl Derivatives and Their Nitrosation by Gaseous NOx ؊ A New,
Promising Tool in Stepwise Peptide Synthesis
Olivier Lagrille,[a] Jacques Taillades,*[a] Laurent Boiteau,[a] and Auguste Commeyras[a]
Keywords: Amino acids / N-carbamoyl group / Nitrosation / Peptides / Protecting groups
New uses of the N-carbamoyl group in peptide synthesis − cemisation occurring in the coupling step is discussed. In the
as an Nα-protecting group in classical peptide coupling second application, an easy route to amino acid N-carboxy
methods, and as a preactivating group for stepwise coupling
by NCA formation − are presented. In the first application, same conditions as above allowed straightforward ‘‘one-pot’’
the N-carbamoyldipeptide esters C-Val-Gly-OEt, C-Leu-Gly- peptide stepwise coupling, as demonstrated by the formation
anhydrides (NCAs) through nitrosation of CAA under the
OEt, C-Ala-Gly-OEt, and C-Ala-Phe-OEt were obtained in of Leu-Gly and Val-Gly in good yields and enantiomeric ex-
good yields by treatment of the corresponding N-carbamoyl- cess.
amino acids (CAA) with amino acid esters. Quantitative N-
deprotection without racemisation was then achieved in the
(
Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
solid through nitrosation by gaseous NOx. The extent of ra- 2002)
Introduction
Despite the ease of N-carbamoylation of amino acids and
peptides, the carbamoyl group is hardly or never used as
an Nα-protecting group in stepwise peptide synthesis, the
removal of an N-carbamoyl moiety requiring drastic basic
conditions or the use of enzymes, conditions not convenient
in peptide chemistry. Recent studies in our laboratory, how-
ever, have shown that nitrosation of N-carbamoylpeptides
(CPs) 1, carried out in the solid phase with gaseous NOx
as the nitrosating agent, results in fast, smooth and quantit-
ative decarbamoylation of the N-carbamoyl group, with
neither peptide bond cleavage nor deamination side reac-
tions.[1] Under the same conditions, N-carbamoylamino ac-
ids (CAAs) 2 afford the corresponding N-carboxy anhyd-
rides (NCAs) 3, which can be isolated quantitatively prior
to hydrolysis into the free amino acids (Scheme 1).[2] Both
reactions operate without racemisation, and yield nitrogen
and water as the only waste materials, thus also offering
interesting perspectives as an eco-friendly reaction.
Scheme 1
nitrosation of the N-alkylurea group in the solid phase by
gaseous NOx.
Results and Discussion
N-Carbamoylation Reaction
Both strategies required efficient N-carbamoylation of
amino acids (and of peptides). Although several synthetic
routes exist, such as the hydrolysis of hydantoins by strong
bases[3] or enzymatic systems,[4] or trans-N-carbamoylation
from urea,[5] the simplest and cheapest method for amino
group N-carbamoylation was treatment with aqueous min-
eral cyanate.[6,7] This reaction appeared to be highly de-
pendent on the pH; a kinetic study of cyanate reactivity
allowed optimised conditions to be identified: pH ϭ 8Ϫ8.5
at 40Ϫ50 °C for a few hours.[8] This reaction was reasonably
robust, however, and could be achieved over a wider pH
range (6.5Ϫ10). The CAA or CP was usually isolated in
good yields by precipitation from the chilled, acidified reac-
In this context, we examined two possible uses of the car-
bamoyl group in peptide synthesis, scrutinising the enanti-
oselectivity of the reactions. The first was as an Nα-pro-
tecting group for classical peptide coupling methods, the
second was as a preactivating group for peptide stepwise
coupling by NCA formation. Both strategies rely on the
[a]
´
´
´
Organisation Moleculaire Ϫ Evolution et Materiaux Fluores,
UMR 5073 Ϫ CC017, University of Montpellier 2,
Place Bataillon, 34095 Montpellier Cedex 5, France
Fax: (internat.) ϩ 33-4/67631046
E-mail: jtaillad@univ-montp2.fr
Supporting information for this article is available on the
WWW under http://www.eurjoc.com or from the author.
1026
WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1434Ϫ193X/02/0306Ϫ1026 $ 17.50ϩ.50/0 Eur. J. Org. Chem. 2002, 1026Ϫ1032

N-Carbamoyl Derivatives and Their Nitrosation by Gaseous NOx
FULL PAPER
tion mixture (Scheme 2). No racemisation was found to oc- was directly used for the preparation of C-Ala-Phe-OEt (7),
cur under these conditions.
isolated in good yield. No significant change was observed
for the preparation of C-Leu-Gly-OEt (5) either with the
classical or with the alternative procedure, while the results
for the preparation of 6 suggested a high instability of the
mixed anhydride derived from C-Ala, which would then
readily decompose prior to aminolysis in the classical pro-
cedure.
Scheme 2
Epimerisation
The Carbamoyl Moiety as an Nα-Protecting Group
To evaluate the extent of epimerisation during the coup-
ling step, the enantiomeric excesses of peptide residues were
measured after N-decarbamoylation (see next section) and
then after peptide bond hydrolysis with boiling 6  HCl.[11]
The enantiomeric excesses (ees) of the resulting amino acids
were measured by GC analysis after derivatisation with (Ϫ)-
menthyl chloroformate in 2-propanol.[12] To determine the
extent of racemisation occurring during the hydrolysis step,
a phenomenon described in the literature,[13,14] commercial
dipeptide ester samples were submitted to the same treat-
ment, and showed ca. 5% racemisation (Table 1).
Peptide Coupling Reaction
We first investigated the possibility of using the carba-
moyl group as an Nα-protecting group for peptide coupling,
focusing on the mixed-anhydride method, well known in
peptide chemistry for its efficiency in terms of yield and
absence of racemisation.[9,10] The carboxyl group of the N-
protected amino acid is activated to a mixed anhydride by
treatment with isobutyl chloroformate (IBCF) in the pres-
ence of N-methylmorpholine (NMM, Scheme 3). The
mixed anhydride is usually formed prior to the addition of
the O-protected amino acid (a procedure established by
Anderson et al.[10]).
Though Val-Gly or Leu-Gly coupling occurred with (al-
most) no epimerisation (the measured ees remaining very
close to those of commercial dipeptides after the hydrolysis
step), almost complete epimerisation (together with lower
yields) occurred during both Ala-Gly and Ala-Phe coup-
ling, whether the classical or the alternative coupling pro-
cedure was applied. Attempts to use N,N-dimethylacet-
amide (DMAc, known as a low-racemisation medium) in-
stead of DMF as the reaction solvent did not significantly
improve either the yield or the ee for the synthesis of C-
Ala-Gly-OEt. No change/difference was observed with a
change in solvent for the preparation of C-Val-Gly-OEt (4).
Scheme 3
Epimerisation during the peptide coupling step may be
This method was applied to produce N-carbamoyldipep- explained in terms of the formation and racemisation of a
tide esters 4Ϫ7 (Table 1). Coupling of C-Val or C-Leu to cyclic intermediate, as previously observed in the activation
Gly-OEt afforded the respective dipeptides 4Ϫ5 in good of amino acids Nα-protected with groups of the urethane
yields, but this proved poorly efficient when C-Ala was the type by the mixed-anhydride method.[10,15,16] The formation
starting material. A modification of the reagent addition of the oxazolone 8a from the mixed anhydride (Scheme 4)
sequence (amino ester addition prior to IBCF), in order to was found to depend on various factors such as the nature
form the mixed anhydride of C-Ala in the presence of the of the N-protecting group, the amino acid side chain and
amino ester, significantly improved the yield of isolated C- the activating base. Although the nucleophilic attack on 8a
Ala-Gly-OEt (6). Therefore this alternative procedure by the free amino ester also results in the peptide coupling,
(named in contrast with Anderson’s classical procedure) this last reaction is in competition with the reversible depro-
Table 1. Results of coupling amino acid esters to CAA by the mixed-anhydride method (yields are not optimised)
Compound
Yield (%)
ee (%) at the N residue[a]
classical[b]
alternative[b]
classical[b]
alternative[b]
(blank)[c]
4 (C-Val-Gly-OEt)
5 (C-Leu-Gly-OEt)
6 (C-Ala-Gly-OEt)
7 (C-Ala-Phe-OEt)
87
80
52
Ϫ
Ϫ
89
89.6
10
Ϫ
89.6
10
89.6
90.6
90
82
84
91
Ϫ
10
Ϫ
[a]
[b]
[c]
Measured by GC after peptide N-deprotection followed by hydrolysis.
Peptide coupling procedure used (see text). ee measured
after hydrolysis (same method) of the corresponding commercial dipeptide esters.
Eur. J. Org. Chem. 2002, 1026Ϫ1032
1027

O. Lagrille, J. Taillades, L. Boiteau, A. Commeyras
FULL PAPER
tonation of 8a at the α position to give the planar cyclic NOϩ. These solid/gas conditions produced a significant
anion 9a. Racemisation of 8a is then possible through 9a.
acidification of the medium due to release of HNO2/HNO3,
thus ensuring a fast equilibrium between the two nitroso
urea adducts 10 and 11 (detailed investigations to be pub-
lished elsewhere). While base-catalysed decomposition of 10
(into diazo compound and deamination products) did not
occur, due to the absence of HOϪ, the unstable 11 spontan-
eously decomposed into an isocyanate that finally under-
went hydrolysis (Scheme 5).
Only traces of nitroso adducts at the peptide bond nitro-
gen atom (compounds 12) were observed in the crude reac-
tion mixtures. Although peptidic nitrogen atoms are prob-
ably less reactive than ureidic nitrogen atoms towards nitro-
sation, the acid-catalysed denitrosation was probably also
involved in this case. That might explain why neither pep-
tide bond cleavage nor deamination were observed, in con-
trast with, for instance, the results of Garcia et al.[17] in a
model study of lung tissue damage caused by polluted
urban air, in which the nitrosating agent was obtained from
an NO2 stream through a sodium acetate suspension in
dichloromethane.
In view of future applications, however, the presence of
nitroso adducts in the final products had to be strictly
avoided, due to their toxicity: numerous studies have exam-
ined the carcinogenic, mutagenic or teratogenic character
of nitroso ureas.[18Ϫ21] Our nitrosation method offers the
advantage that only trace amounts of these undesirable ad-
ducts will have to be removed, but a further denitrosation
procedure will have to be applied anyway. In a preliminary
study, a solution of Nε-nitroso-Nε-carbamoyllysine NCA in
acetonitrile was efficiently and quantitatively denitrosated
by bubbling of dry HCl through the solution. The use of
a suitable scavenger (such as hydrazine, urea, etc.) may be
necessary to remove final traces of nitroso ureas and nitroso
amides, the presence of which being a serious limitation to
this protecting group strategy otherwise. Details regarding
this concern will be discussed in a future paper.
Scheme 4
By structural analogy, such a mechanism was also likely
to occur in our case, and would involve the formation and
racemisation of 8b. However, such 2-amino-5-(4H)-oxazo-
lones (e.g. 8b) have never been reported in the literature. If
such a mechanism were involved, the racemisation rate
should be very powerfully affected by the steric hindrance
of the side chain, explaining why almost complete racemis-
ation was observed upon treatment of C-Ala, while bulky
side chain compounds such as C-Val or C-Leu were not
affected. This explanation, however, is insufficient to ac-
count for epimerisation with C-Ala, in comparison with
such notoriously nonracemising urethane derivatives as Z-
Ala or Boc-Ala, in which the bulky ORЈЈ substituent prob-
ably efficiently prevents α-proton abstraction from 8a des-
pite its expected (from the comparative electronegativities
of ORЈЈ and NH2) higher acidity in comparison with 8b. It
is also possible, however, that the (unhindered) mixed an-
hydride would itself be subject to α-proton abstraction, and
then to racemisation.
Deprotection Reaction
The N-deprotection of N-carbamoylpeptides was carried
out by nitrosation in solid/gas phase:[1] the solid N-carba-
moyldipeptide was exposed (under nitrogen) to a mixture
of nitric oxide and oxygen (in a 2.5:1 ratio). A small amount
of water was necessary to initiate the reaction (the progress
of which was visible by the disappearance of the red-brown
colour of the NO2) and to obtain quantitative conversion
in a reasonable time. The N-deprotected dipeptides were
obtained as their corresponding salts with nitric acid. The
nitrosation probably proceeds through species such as N2O3
or N2O4, although the ultimate intermediate is probably
The N-Carbamoyl Group as a Preactivating Group for
Peptide Coupling via NCAs
An alternative use of ureido derivatives in peptide syn-
thesis is as starting materials for NCA synthesis through
nitrosation:[2] this novel reaction offers much easier access
to NCAs than the classical phosgene route does.[22,23]
NCAs are well-known monomers for polypeptide synthesis;
control of their polymerisation is the object of important
investigations into the obtainment of well-defined homo-
polymers and statistical[24] or Ϫ recently Ϫ block copoly-
mers.[25] NCA reactivity has also been investigated in the
context of controlled oligomerisation and stepwise peptide
coupling: Bailey[26] reported oligopeptide synthesis by treat-
ment of NCAs with amino acid esters at low temperature
in anhydrous organic media, without being able to avoid
the formation of dioxopiperazine. On the basis of a kinetic
study of NCA hydrolysis and reaction with amino groups
in aqueous solution,[27] Denkewalter et al.[28,29] succeeded
in controlling stepwise NCA oligomerisation in aqueous
media.
Scheme 5
1028
Eur. J. Org. Chem. 2002, 1026Ϫ1032

N-Carbamoyl Derivatives and Their Nitrosation by Gaseous NOx
FULL PAPER
In a reactivity study, Iwakura et al.[30] showed that the
propagation reaction in acetonitrile proceeded only through
a nucleophilic attack on the NCA at C-5 by the amino end-
group of the growing chain. He proposed a phase-transfer
method for the preparation of sequential oligopeptides (up
to 4 residues) in good yields without isolation of the inter-
mediates: the coupling occurred at Ϫ15 °C at the interface
between an acetonitrile solution of the NCA and an aque-
ous solution of the free amino acid (or peptide). Saturation
of the aqueous phase with sodium carbonate stabilised the
carbamate intermediate 13, thus preventing further NCA
coupling (Scheme 6). After separation of the two layers and
N-decarboxylation of 13 by simple warming to 40 °C, the
elongated peptide could be treated with a further batch of
NCA, or recovered after acidification as the free peptide.
The phase-transfer system efficiently prevented side reac-
tions such as hydrolysis of the NCA, which could therefore
be used in excess to obtain complete conversion.
Conclusion
The solid-phase nitrosation of N-carbamoylpeptides or
of CAAs by gaseous NOx, affording N-deprotected pep-
tides or NCAs, respectively, offers interesting possibilities
in stepwise peptide synthesis. We have shown that the N-
carbamoyl group may be used as an N-protecting group in
peptide coupling by the mixed-anhydride method. Further-
more, complementary studies to evaluate this protecting
group strategy with other peptide-coupling methods and to
overcome its major limitations, namely the presence of trace
nitroso adducts in coupling products and α-C epimeris-
ation, remain to be done. This last concern might be favour-
ably addressed by use of improved IBCF/NMM operating
procedures.[31]
On the other hand, the easy route to NCAs offered by
CAA nitrosation allows stepwise peptide elongation to be
carried out without protecting groups in a simple, one-pot
procedure. Both strategies share the important advantage
of cheapness of starting N-carbamoylamino acids, in com-
parison with the usual N-protected amino acids.
Experimental Section
Hazardous Compounds: Nitric oxide is skin- and mucous-tissue-ir-
ritant. Nitroso ureas are potent carcinogens. All operations invol-
ving the handling of these compounds or their solutions were car-
ried out in a fume hood, while wearing gloves. Effluents were de-
stroyed by treatment with concentrated sodium hydroxide.
Scheme 6
Materials and Methods: Methanol, acetonitrile, chloroform, and
ethyl acetate were obtained from Baeckeroot (France). Alanine, va-
line, leucine, dimethylformamide (DMF), (Ϫ)-menthyl chloro-
formate, isobutyl chloroformate (IBCF), pyridine, and sodium bor-
ate were obtained from Aldrich. Glycine ethyl ester hydrochloride
and phenylalanine ethyl ester hydrochloride were obtained from
Acros Chemicals. N-Methylmorpholine was obtained from Fluka.
Sodium glycinate was obtained from Sigma. Sodium carbonate and
potassium cyanate were obtained from Prolabo. Hydrochloric acid
(36%) was obtained from Carlo Erba. Nitric oxide and oxygen were
obtained from L’Air Liquide (France). Acetonitrile, DMF, and
N,N-dimethylacetamide (DMAc) were dried with molecular sieves
prior to use. Potassium cyanate was washed with methanol and
dried in vacuo prior to use. All other reagents and solvents were
used as received. Melting points were taken with a Büchi 520 ap-
paratus. 1H NMR spectra were recorded with Bruker AC 200 or
AC 250 spectrometers (200 or 250 MHz) in [D6]DMSO solution.
The pH regulation was performed with a Methrom Titrino 719S
autotitrator. HPLC analyses were carried out with a Varian appar-
atus set up with a Varian 2510 pump, a Varian 2550 UV detector
and a Varian 4290 integrator. Nucleosil RP-C18 (5 µm, 250 ϫ
4.1 mm i.d.) analytical columns (obtained from Shandon, France)
were used at room temperature under isocratic conditions (detailed
conditions are provided below). Gas Chromatography (GC) ana-
lyses were carried out with a Shimadzu GC-14A apparatus
equipped with a chromatopac C-9R 6A integrator, with OV-1701
silica (30 m ϫ 0.25 mm i.d. film thickness 0.25 µm) capillary col-
umns (obtained from The Quadrexp Corp., New Haven, CT). Op-
tical rotation measurements were carried out with a PerkinϪElmer
241 polarimeter at the sodium emission wavelength (589 nm) in a
10-cm glass cell. The preparation of the N-carbamoylamino acids
Our NCA synthetic route offered the opportunity to ad-
apt the method of Iwakura through a straightforward ‘‘one-
pot’’ procedure. The NCA was prepared by nitrosation of
the CAA by a mixture of NO and O2 (4:1) at room temper-
ature and under atmospheric pressure, in the same way as
the decarbamoylation of C-peptides 4Ϫ7;[2] the crude NCA
was then directly used in the peptide coupling reaction, by
Iwakura’s procedure.
The dipeptides Val-Gly and Leu-Gly were thus obtained
in good yields (Table 2). On the basis of preliminary experi-
ments on the nitrosation step, which showed about 90%
conversion, the initially reacting CAA amounted to ca.
1.2Ϫ1.3 equiv. of the glycine involved in the coupling step,
hence ensuring a sufficient NCA/amino acid stoichiometric
excess. Optical rotation measurement, with comparison
with commercial authentic samples, showed that almost no
racemisation occurred (ca. 97% ee in final products).
Table 2. Dipeptide coupling by NCA formation
Dipeptide
Yield (%)[a]
ee (%)
[α]D
[α]D ref.[b]
Val-Gly
Leu-Gly
86 (90)[c]
82 (87)[c]
96.8
97.0
ϩ42.9
ϩ39.0
ϩ44.3
ϩ40.2
[a] Overall yield. [b] Of commercial authentic samples. [α]D measured
[c]
at 20 °C (c ϭ 2.5, 2  HCl). Values in parentheses: yield after
first step.
Eur. J. Org. Chem. 2002, 1026Ϫ1032
1029

O. Lagrille, J. Taillades, L. Boiteau, A. Commeyras
FULL PAPER
C-Val, C-Ala, and C-Leu was carried out according to our previ-
ously published procedure.[8]
anol, 9:1) and then recrystallised from methyl acetate/methanol to
afford 0.56 g (0.26 mmol, 52%) of pure material. M.p. 145Ϫ147 °C.
Ala
NMR: δH ϭ 1.17 (d, 3 H, HβAla, Jαβ
ϭ 7.0 Hz), 1.20 (t, 3 H,
Peptide Coupling by the Mixed-Anhydride Method[9]
Gly
OϪCH2ϪCH3, J1 ϭ7.0 Hz), 3.82 (2 H, ABX system, Hα
,
Gly
Gly
Gly
JααЈ
ϭ 17.5 and Jα-NH
ϭ JαЈ-NH
ϭ 6.0 Hz), 4.09 (q, 2 H,
C-Val-Gly-OEt (4). ؊ Classical Procedure: N-Methylmorpholine
(0.6 mL, 5.5 mmol, 1.1 equiv.) was added to a chilled (Ϫ15 °C,
liquid nitrogen/ethanol bath), stirred solution of N-carbamoylva-
line (0.80 g, 5 mmol) in DMF (30 mL) under an inert gas; after
3Ϫ5 min of stirring, IBCF (0.7 mL, 5.5 mmol) was added to the
mixture, whereupon a precipitate appeared. After another 1Ϫ2 min
of stirring, a solution of glycine ethyl ester hydrochloride (0.70 g,
5 mmol) and N-methylmorpholine (0.6 mL, 5 mmol) in DMF
[10 mL, this solution was prepared by dissolving the glycine ester
hydrochloride in warm DMF, and then adding the N-methylmor-
pholine to the cooled (room temp.) solution and swirling] was ad-
ded. After 20 min of stirring at Ϫ15 °C, the mixture was allowed
to warm up to room temperature, and then stirred again for an-
other 60 min at room temp. The solvent was then evaporated in
vacuo, and the residue was recrystallised from methanol to afford
OϪCH2ϪCH3, J1 ϭ7.0 Hz), 4.16 (m, 1 H, Hα Ala, Jα-NH
ϭ
ALa
7.5 Hz), 5.59 (s, 2 H, COϪNH2), 6.18 (d, 1 H, CHAlaϪNH, Jα-
ALa
Gly
ϭ 7.5 Hz), 8.34 (t, 1 H, ϪCH2GlyϪNHϪ, Jα-NH
ϭ
NH
6.0 Hz); δC ϭ 14.9 (OϪCH2ϪCH3), 20.4 (CβAla), 41.44 (CαGly),
49.1 (CαAla), 61.2 (OϪCH2ϪCH3), 158.8 (ϪNHϪCOϪNH2), 170.6
(CαAlaϪCOϪNHϪ), 174.7 (ϪCOϪOϪ). Ϫ Alternative Procedure:
The reaction was carried out with N-carbamoylalanine (0.66 g,
5 mmol), the crude product was purified as above to afford 0.91 g
(4.2 mmol, 84%) of pure material, exhibiting the same physical
characteristics as above.
C-Ala-Phe-OEt (7). ؊ Alternative Procedure: The reaction was car-
ried out with N-carbamoylalanine (0.66 g, 5 mmol) and -phenylal-
anine ethyl ester hydrochloride (1.15 g, 0.5 mmol). The crude prod-
uct was purified by silica gel chromatography (eluent ethyl acetate/
methanol, 9:1) and recrystallisation from methyl acetate/methanol
to afford 1.40 g (4.55 mmol, 91%) of pure material. M.p. 184Ϫ186
°C. NMR: δH ϭ 0.96 (d, 3 H, HβAla, Jαβ ϭ 7.0 Hz), 1.13 (dd, 3 H,
OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 2.93 (m, 2 H, HβPhe), 4.04 (q, 2 H,
1.07 g (4.35 mmol, 87%) of pure material. M.p. 227Ϫ229 °C.
Val
NMR: δH ϭ 0.84 (d, 3 H, HγVal, Jγδ ϭ 7.0 Hz), 0.88 (d, 3 H, Hγ
,
Jγδ ϭ 7.0 Hz), 1.20 (t, 3 H, OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 1.93 (m,
1 H, HβVal), 3.82 (2 H, ABX system, HαGly, Jα-NHGly ϭ JαЈ-NHGly ϭ
6.0 Hz, JααЈGly ϭ 17.5 Hz), 4.08 (dd, 1 H, HαVal, Jα-NHGly ϭ 9.0 Hz,
Jα-βGly ϭ 7 Hz), 4.09 (q, 2 H, OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 5.60 (s,
OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 4.13 (m, 1 H, HαALa), 4.45 (m, 1 H,
HαPhe), 5.57 (s, 2 H, NH2), 6.13 (d, 1 H, CHAlaϪNH, Jα-NH
ϭ
Ala
2 H, ϪNH2), 6.11 (d, 1 H, ϪCHαValϪNHϪ, J1 ϭ 9.0 Hz), 8.36 (t,
8.0 Hz), 7.25 (m, 5 H, HAr), 8.38 (d, 1 H, ϪCHPheϪNHϪ, Jα-
Gly
1
H, ϪCHαGlyϪNHϪ, Jα-NH
ϭ
5.5 Hz). δC
ϭ
14.9
Phe
ϭ 8.0 Hz); δC ϭ 14.8 (OϪCH2ϪCH3), 20.6 (CβAla), 37.5
NH
(OϪCH2ϪCH3), 18.5 (CγVal), 20.0 (CγЈVal), 32.0 (CβVal), 41.47
(CβPhe), 49.0 (CαAla), 54.1 (CαPhe), 61.4 (OϪCH2ϪCH3), 127.4
(CδAr), 129.1 (CγAr), 130.1 (CβAr), 138.0 (CαAr), 158.8
(ϪNHϪCOϪNH2), 172.3 (CαAlaϪCOϪNHϪ), 174.2 (COϪOϪ).
(CαGly),
58.3
(CαVal),
61.2
(OϪCH2ϪCH3),
159.2
(ϪNHϪCOϪNH2), 170.6 (CαValϪCOϪNH-), 173.5 (ϪCOϪOϪ).
C-Leu-Gly-OEt (5). ؊ Classical Procedure: The reaction was car-
ried out with C-Leu (0.87 g, 5 mmol) and glycine ethyl ester hydro-
N-Decarbamoylation of N-Carbamoyldipeptide Esters
chloride (0.70 g, 5 mmol). The crude product was purified by silica H-Val-Gly-OEt. ؊ General Procedure: A 100-mL flask was fitted
gel chromatography (eluent ethyl acetate/methanol, 9:1) and then
recrystallised from methyl acetate to afford 1.04 g (4 mmol, 80%)
of pure material. M.p. 150Ϫ152 °C. NMR: δH ϭ 0.88 (d, 3 H,
with a magnetic stirrer and capped with a silicon rubber septum,
and finely powdered C-Val-Gly-OEt (100 mg, 0.41 mmol) was ad-
ded, spread over 2-mm glass beads and flushed with nitrogen. Ni-
HδLeu, Jγδ ϭ 6.5 Hz), 0.92 (d, 3 H, HδLeu, Jγδ ϭ 6.5 Hz), 1.20 (t, 3 tric oxide (27.5 mL, 1.23 mmol,
3 equiv.), oxygen (11 mL,
H, OϪCH2ϪCH3, J1 ϭ7.0 Hz), 1.40 (m, 2 H, HβLeu), 1.63 (m, 1 0.49 mmol, 1.2 equiv.), and then pure water (22 µL, 1.22 mmol, 3
Gly
Gly
H, Hγ), 3.80 (2 H, ABX system, HαGly, Jα-NH
6.0 Hz, JααЈ
ϭ JαЈ-NH
ϭ
equiv.) were introduced through separate glass syringes. After
30 min of stirring, the flask was rapidly flushed with nitrogen to
Gly
ϭ 17.0 Hz), 4.08 (q, 2 H, OϪCH2ϪCH3, J1
ϭ
7.0 Hz), 4.18 (1 H, ABX system, HαLeu), 5.55 (s, 2 H, ϪNH2), 6.13 remove the unchanged NOx, and the crude product was then dried
(d,
1
H, CHβLeuϪNHϪ, Jα-NH
ϭ 8.5 Hz), 8.38 (t, 1 H, in vacuo in a heating desiccator (30 °C) to afford 108 mg
Gly
ϪCHαGlyϪNHϪ, Jα-NH
ϭ 6.0 Hz); δC ϭ 14.9 (OϪCH2ϪCH3), (0.41 mmol, 100%) of pure H-Val-Gly-OEt (salt with nitric acid).
22.8 (CδLeu), 23.8 (CδЈLeu), 24.9 (CγLeu), 41.4 (CβLeu), 43.1 (CαGly), NMR: δH ϭ 0.98 (d, 3 H, HγVal, Jγδ ϭ 7.0 Hz), 1.00 (d, 3 H, Hγ
,
Val
51.9 (CαLeu), 61.2 (OϪCH2ϪCH3), 158.9 (ϪNHϪCOϪNH2), Jγδ ϭ 7.0 Hz), 1.21 (t, 3 H, OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 2.10 (m,
170.6 (CαLeuϪCOϪNHϪ), 174.5 (ϪCOϪOϪ). Ϫ Alternative Pro- 1 H, HβVal), 3.82 (d, 1 H, CαVal, Jαβ ϭ 5.5 Hz), 3.97 (2 H, ABX
Val
cedure: N-Methylmorpholine (0.6 mL, 5.5 mmol, 1.1 equiv.) was system, HαGly, Jα-NH
ϭ JαЈ-NH
ϭ 6.0 Hz, JααЈ
ϭ 16.0 Hz),
Gly
Gly
Gly
added under an inert gas to a chilled (Ϫ15 °C), stirred solution of
C-Leu (0.87 g, 5 mmol) in DMF (30 mL). After 3Ϫ5 min of stir-
4.13 (q, 2 H, OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 8.13 (s, 3 H, ϪNH3),
Gly
8.86 (t, 1 H, ϪCHαGlyϪNHϪ, Jα-NH
ϭ 6.0 Hz). δC ϭ 14.8
ring, a solution of glycine ethyl ester hydrochloride (0.70 g, 5 mmol) (OϪCH2ϪCH3), 18.4 (CγVal), 18.94 (CγЈVal), 30.7 (CβVal), 41.6
and N-methylmorpholine (0.6 mL, 5 mmol) in 10 mL of DMF
(prepared as above) was added, followed (after another 3Ϫ5 min)
by IBCF (0.7 mL, 5.5 mmol). After 20 min of stirring at Ϫ15 °C,
the mixture was allowed to warm up to room temperature, and
then stirred for another 60 min at room temp. After in vacuo evap-
oration of the solvent, the residue was purified as above to afford
1.06 g (0.41 mmol, 82%) of pure material, exhibiting the same phys-
ical characteristics as above.
(CαGly),
58.2
(CαVal),
61.5
(OϪCH2ϪCH3),
169.2
(CαValϪCOϪNHϪ), 170.1 (ϪCOϪOϪ).
H-Leu-Gly-OEt: The reaction was carried out with (finely pow-
dered) C-Leu-Gly-OEt (100 mg, 0.39 mmol), nitric oxide (26 mL,
1.16 mmol, 3 equiv.), oxygen (10.5 mL, 0.47 mmol, 1.22 equiv.),
and pure water (21 µL, 1.17 mmol, 3 equiv.) to afford 107 mg (0.39
mmol, 100%) of pure H-Leu-Gly-OEt. NMR: δH ϭ 0.97 (d, 3 H,
HδLeu, Jγδ ϭ 6.0 Hz), 0.99 (d, 3 H, HδLeu, Jγδ ϭ 6.0 Hz), 1.20 (t, 3
H, OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 1.58 (m, 2 H, HβLeu), 1.71 (m, 1
H, HγVal), 3.81 (1 H, ABX system, HαLeu), 3.95 (2 H, ABX system,
C-Ala-Gly-OEt (6). ؊ Classical Procedure: The reaction was car-
ried out with N-carbamoylalanine (0.66 g, 5 mmol) and glycine
ethyl ester hydrochloride (0.70 g, 5 mmol). The crude product was HαGly, Jα-NH
Gly
Gly
Gly
ϭ JαЈ-NH
ϭ 6.0 Hz, JααЈ
ϭ 10.5 Hz), 4.13 (q,
purified by silica gel chromatography (eluent ethyl acetate/meth-
2 H, OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 8.26 (s, 3 H, ϪNH3), 8.95 (t, 1
1030
Eur. J. Org. Chem. 2002, 1026Ϫ1032

N-Carbamoyl Derivatives and Their Nitrosation by Gaseous NOx
FULL PAPER
Gly
H, ϪCHαGlyϪNHϪ, Jα-NH
ϭ
6.0 Hz); δC
ϭ
14.9 of the precipitated salts, the dipeptide was precipitated by ether
(OϪCH2ϪCH3), 22.6 (CδLeu), 23.4 (CδЈLeu), 24.9 (CγLeu), 40.8 addition and collected by filtration. The crude product was recrys-
(CβLeu), 41.0 (CαGly), 51.6 (CαLeu), 61.5 (OϪCH2ϪCH3), 170.1 tallised from ethanol/ether to afford 251 mg (1.34 mmol, 82%) of
(CαLeuϪCOϪNHϪ), 170.4 (ϪCOϪOϪ).
pure material. NMR: δH ϭ 0.92 (dd, 6 H, HδLeu, Jγδ ϭ 6.0 Hz),
1.60 (m, 2 H, HβLeu), 1.70 (m, 1 H, HγVal), 3.88 (2 H, ABX system,
H-Ala-Gly-OEt: The reaction was carried out with finely powdered
C-Ala-Gly-OEt (100 mg, 0.46 mmol), nitric oxide (31 mL,
1.38 mmol, 3 equiv.), oxygen (12.5 mL, 0.55 mmol, 1.19 equiv.) and
pure water (25 µL, 1.38 mmol, 3 equiv.) to afford 109 mg (0.46
mmol, 100%) of pure H-Ala-Gly-OEt. NMR: δH ϭ 1.21 (t, 3 H,
OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 1.38 (d, 3 H, HβAla, JαβAla ϭ 7.0 Hz),
3.85 (m, 1 H, HαAla), 3.95 (2 H, ABX system, HαGly), 4.10 (q, 2 H,
Gly
Hα and 1 H, ABX system, HαLeu), 8.15 (s, 3 H, ϪNH3), 8.85 (t,
1 H, ϪCHαGlyϪNHϪ, Jα-NHGly ϭ 5.5 Hz); δC ϭ 22.7 (CδLeu), 23.4
(CδЈLeu), 24.3 (CγLeu), 40.9 (CβLeu), 41.15 (CαLeu), 51.6 (CαLeu),
170.3 (CαLeuϪCOϪNHϪ), 171.5 (ϪCOOH).
H-Val-Gly-OH: The procedure was the same as above, with C-Val
(370 mg, 2.3 mmol) and the same quantities of other reagents. The
crude product was recrystallised from ethanol/ether to afford
244 mg (1.40 mmol, 86%) of pure material. NMR: δH ϭ 0.98 (dd,
6 H, HγVal, Jγδ ϭ 7.0 Hz), 2.10 (m, 1 H, HβVal), 3.65 (m, 1 H, CαVal),
OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 8.12 (s, 3 H, ϪNH3), 8.83 (t, 1 H,
ϪCH2GlyϪNHϪ, Jα-NH
ϭ 6.0 Hz); δC ϭ 14.6 (OϪCH2ϪCH3),
Gly
17.7 (CβAla), 41.3 (CαGly), 48.6 (CαAla), 61.2 (OϪCH2ϪCH3), 169.9
(CαAlaϪCOϪNHϪ), 170.6 (ϪCOϪOϪ).
Gly
Gly
3.89 (2 H, ABX system, HαGly, Jα-NH
ϭ JαЈ-NH
ϭ 5.5 Hz,
Gly
JααЈ
ϭ 17.5 Hz), 8.11 (s, 3 H, ϪNH3), 8.75 (t, 1 H,
H-Ala-Phe-OEt: The reaction was carried out with finely powdered
C-Ala-Phe-OEt (100 mg, 0.33 mmol), nitrogen dioxide (22 mL,
0.98 mmol, 3 equiv.), oxygen (9 mL, 0.39 mmol, 1.2 equiv.), and
pure water (18 µL, 1 mmol, 3 equiv.) to afford 106 mg (0.33 mmol,
100%) of pure H-Ala-Phe-OEt. NMR: δH ϭ 1.12 (t, 3 H,
OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 1.35 (d, 3 H, HβAla, Jαβ ϭ 7.0 Hz),
3.82 (m, 1 H, HαALa), 2.98 (m, 2 H, HβPhe), 4.11 (q, 2 H,
OϪCH2ϪCH3, J1 ϭ 7.0 Hz), 4.59 (m, 1 H, HαPhe), 7.31 (m, 5 H,
HAr), 8.05 (s, 3 H, ϪNH3), 8.85 (t, 1 H, ϪCHPheϪNHϪ, Jα-NHPheϭ
9.0 Hz); δC ϭ 14.8 (OϪCH2ϪCH3), 18.0 (CβAla), 37.2 (CβPhe), 48.9
(CαAla), 54.2 (CαPhe), 61.6 (OϪCH2ϪCH3), 127.6 (C4Ar), 129.1
(C3Ar), 130.0 (C2Ar), 137.6 (C1Ar), 170.2 (CαAlaϪCOϪNHϪ),
171.9 (COϪOϪ).
ϪCHαGlyϪNHϪ, Jα-NH
ϭ 5.5 Hz); δC ϭ 17.2 (CγVal), 17.8
Gly
(CγЈVal), 29.4 (CβVal), 40.3 (CαGly), 56.9 (CαVal), 167.9
(CαValϪCOϪNHϪ), 171.3 (ϪCOOH).
Acknowledgments
This work was financially supported by the CNRS, the French
Ministries of Research and Education, and the Degussa-Hüls AG
(Germany). We also thank the referees for helpful comments.
Peptide Hydrolysis[11] and Derivatisation[12] for GC Analysis: A solu-
tion of (N-deprotected) dipeptide ethyl ester (100 mg) in 6  HCl
(10 mL) was heated to 110Ϫ120 °C (oven) in a sealed Pyrex tube.
After cooling and concentration to dryness in a rotary evaporator,
the residue was dissolved in aqueous sodium borate (50 m, 2 mL,
pH ϭ 9.0), and then diluted with sodium borate (0.2 , 20 mL,
pH ϭ 7.7). The pH of the mixture was checked and adjusted if
necessary to 7.5Ϫ8 with HCl. To this solution (50 µL) in a 1-mL
vial tube was added a pyridine/methanol mixture (40 µL, 20:80, v/
v), and then (Ϫ)-menthyl chloroformate (10 µL). After occasional
shaking for 10 min at room temp., the mixture was extracted once
with chloroform (100 µL). GC analysis of the chloroform layer was
performed with nitrogen as the carrier gas (10 mL·minϪ1), 300 °C
at inlet and outlet, and a temperature program (190 °C to 230 °C
at 4 °C·minϪ1, and then 30 min at 230 °C). tR [min] ϭ 12.8 (-
Leu), 12.9 (-Leu), 6.3 (-Ala), 6.4 (-Ala), 9.6 (-Val), and 9.9
(-Val).
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