Towards a general synthesis of di-aza-amino acids containing peptides

Article abstract of DOI:10.1039/c8nj03635g

While the incorporation of one aza-amino acid in peptides has been proved to be beneficial for inducing a structure constraint, increasing the resistance towards proteolysis and improving the biological activity, only very rare examples of the incorporation of two or more consecutive aza-amino acids have been reported. In this work, we demonstrate that this fact is probably due to the unsuspected difficulty in synthesizing such peptide analogues, as illustrated by the synthesis of tripeptide derivatives containing two consecutive aza-amino acids. Herein, we report some general guidelines regarding the activation and the coupling of alkyl-hydrazides either mutually or with a natural amino acid, taking into account their nucleophilicity and the nature of their side chains.

Full text of DOI:10.1039/c8nj03635g

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The role of atropisomers on the photo-reactivity and fatigue of
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Towards a general synthesis of di-aza-amino acids containing peptides
Faustine Bizet, Nicolo Tonali, JeanꢀLouis Soulier, Agostino Oliva, Julia Kaffy, Benoit Crousse,
Sandrine Ongeri*
BioCIS, Univ. ParisꢀSud, CNRS, Université Paris Saclay, 5 rue JeanꢀBaptiste Clément, 92296 ChâtenayꢀMalabry
Cedex, France.
Corresponding author: Eꢀmail: sandrine.ongeri@uꢀpsud.fr; Tel : +33 146835737.
ABSTRACT:
While the incorporation of one azaꢀamino acid in peptides has been proved to be beneficial for
inducing a structure constraint, increasing resistance towards proteolysis and improving biological
activity, only very rare examples of the incorporation of two or more consecutive azaꢀamino acids
have been reported. In this work, we demonstrated that this fact is probably due to the unsuspected
difficulty to synthesize such peptide analogues, as illustrated by the synthesis of tripeptide derivatives
containing two consecutive azaꢀamino acids. Herein, we report some general guidelines regarding the
activation and the coupling of alkylꢀhydrazides either mutually or with a natural amino acid, taking
into account their nucleophilicity and the nature of their side chains.
KEYWORDS aza-peptides, aza-amino acids, peptides, peptidomimetics, hydrazine
1. Introduction
The number of peptideꢀbased drugs is dramatically increasing as they generally offer greater efficacy,
high potency and selectivity and a reduced risk of toxicity associated with side effects, compared to
non peptide molecules.1 However, they suffer from rapid proteolysis and suitable small modifications
that increase peptide stability are highly desirable. Currently, more than 60 peptide derivatives have
been approved as commercial drugs and about 140 are in clinical trials.1e Among the numerous
modifications of peptides which include peptide side chain modifications, amino acid substitutions
extensions, deletions, and the inversion of stereochemistry of the αꢀcarbon(s), azaꢀpeptides have
attracted attention. In azaꢀpeptides the α–CH(R) of at least one aminoꢀacid residue is substituted by a
nitrogen atom (Figure 1). This residue, called azaꢀamino acid (azaAA), impart special conformational
properties to the parent peptide structure due to the loss of stereogenicity and reduction of flexibility.2
Thus, the incorporated azaꢀamino acid can introduce some conformational constraint to the parent
peptide and allow the chain to adopt a secondary structure and the proper pharmacophore orientation
for activity and selectivity. Furthermore, the formed urea bond confers better resistance towards
proteolytic and chemical degradation than the amide bond of natural peptides. The replacement of an
αꢀcarbon by a nitrogen in a peptide results in an additional possibility for the formation of hydrogen
bonds and was also found to affect the acidity of the neighbouring amide NꢀH bonds, while decreasing
the electrophilic character of the carbonyl group.3,4 Application of azaꢀpeptides in providing
biologically active peptides has shown significant success.3,4,5ꢀ7 However, we were particularly
intrigued by the fact that only one azaꢀamino acid has been incorporated in an isolated manner and

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only very rare examples have been reported for the incorporation of two or more azaꢀamino acids
consecutively. To our knowledge, very few groups8 including ours 9 have reported the incorporation of
two to five azaꢀamino acids consecutively. Remarkably, in these rare cases, azaꢀGly is the most
representative azaꢀamino acid in such azaꢀpeptides. We suspected in a first attempt the importance of
the additional NH in azaꢀGly for providing supplementary hydrogen bonds and thus for increasing the
binding capacity to the biological target. However, considering the scarcity of azaꢀpeptides including
consecutive azaꢀamino acids bearing a side chain, we also suspected a difficulty of synthesis of this
kind of peptides. Thus, we decided to start by prospecting the feasibility of the synthesis of peptides
containing two consecutive azaꢀamino acids, by varying the nature of these azaꢀamino acids, and we
report here our attempts to find a generalizable strategy.
Figure 1. Comparison of azaꢀpeptide, native peptide and azatide
In this work, the synthesis of six tripeptide analogues containing two azaꢀamino acids IꢀVI have been
studied in order to evaluate the influence of the side chains of both the azaꢀamino acids and the natural
amino acids on the efficiency of the synthesis. For that purpose, we considered the synthesis of Bocꢀ
azaValꢀazaGlyꢀPheꢀNH2 (I), BocꢀazaLysꢀazaGlyꢀPheꢀNH2 (II), BocꢀazaValꢀazaAlaꢀValꢀNH2 (III),
BocꢀazaLeuꢀazaValꢀHisꢀOCH3 (IV) and BocꢀazaPheꢀazaLeuꢀValꢀOCH3 (V) (Figure 2) to compare
them together and also with our previously reported synthesis of BocꢀazaGlyꢀazaGlyꢀValꢀNH2 (VI).9

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NHCbz
O
O
H
N
H
N
NH2
N
N
NH2
BocHN
BocHN
N
H
N
H
BocHN
BocHN
N
N
H
H
O
O
O
O
N
I
II
NH
O
O
H
N
H
N
NH2
N
N
O
N
N
H
N
N
H
O
O
O
O
IV
III
O
O
H
H
H
N
N
NH2
N
N
O
BocHN
N
N
BocHN
N
N
H
H
H
O
O
O
O
V
VI
Figure 2. Structure of the tripeptide analogues containing two consecutive azaꢀamino acids IꢀVI.
2. Results and discussion
The synthesis of azaꢀpeptides containing one isolated azaꢀamino acid has been largely reported and
3ꢀ9
requires hydrazine chemistry and classical peptide synthesis, either in solution or in solidꢀphase.
These azaꢀpeptides have been typically synthesized by two general approaches: either the activation of
the peptide Nꢀterminus (or the natural amino acid) as an isocyanate or active carbamate, followed by
coupling to a hydrazide, or the activation of the hydrazide moiety, usually in the form of Nꢀalkyl
carbazate and the subsequent coupling with the natural amino acid. In both cases, the activation
proceeds using phosgene, triphosgene, carbonyldiimidazole, bis(2,4ꢀdinitrophenyl)carbonate,
phenylchloroformate, 4ꢀnitrophenyl chloroformate or bis(pentafluorophenyl)carbonate as carbonyl
donors.5,6,10ꢀ12 The activation and the coupling of the alkylꢀhydrazides is suspected to be more
challenging since they are poorer nucleophiles than simple amines.13 The strategy for the synthesis of
azaꢀpeptides including consecutive azaꢀamino acids is necessarily based on the activation of protected
alkylꢀhydrazines. Two routes can be considered: A) the activation of the protected 2ꢀalkylꢀhydrazine 1
to obtain 2, followed by the reaction with the protected 1ꢀalkylꢀhydrazine 3 or B) the activation of the
protected 1ꢀalkylꢀhydrazine 3 to obtain 4, followed by the reaction with the protected 2ꢀalkylꢀ
hydrazine 1 (Scheme 1). In our case, for the synthesis of the diꢀazaꢀamino acids containing tripeptide
analogues we had also to study the formation of the urea bond between the second azaꢀamino acid of

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the diazaꢀpeptide 5 and the natural amino acid 6. For both steps, we were aware that the feasibility of
the urea bond formation could be influenced by the side chain of the alkylꢀhydrazine and of the amino
acid residue.
R1
N
O
R3
H
N
R4
BocHN
N
N
H
O
R2
O
I-VI
R3
R4
+
H2N
O
6
R1
N
H
N
BocHN
NH
O
R2
5
Route A
Route B
R
R1
N
H
+
+
NH
X
N
H2N
X
BocHN
N-PG1
R2
N-PG1
R2
BocHN
O
O
2
3
4
1
O
O
R1
H2N
+
N-PG1
NH
Cl
X
+
Cl
X
BocHN
R2
1
3
NH2
BocHN
Scheme 1. General strategy for the synthesis of the tripeptide analogues containing two consecutive
azaꢀamino acids IꢀVI.
9
We have readily prepared BocꢀazaGlyꢀazaGlyꢀValꢀNH2 VI following the route A. Firstly Bocꢀ
hydrazine (tertꢀbutyl carbazate) was activated using phenyl chloroformate in dry DCM in the presence
of pyridine, and secondly the reaction with hydrazine hydrate 3a (R2=PG1=H) at reflux of MeOH gave
the intermediate 5 (where R1= R2= H) in good yields (97% and 72% respectively for the activation and
the coupling). The activation of 5 (where R1= R2= H) by phenyl chloroformate in dry THF, in the
presence of pyridine, and followed by reaction with LꢀValꢀNH2 afforded BocꢀazaGlyꢀazaGlyꢀValꢀNH2

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VI in good yield (91% and 93% respectively for the activation and the coupling).9 Janda et al. were
able to prepare BocꢀazaAAꢀazaAABoc (with azaGly, azaAla and azaLeu coupled to azaGly, azaAla,
azaPhe, azaLeu or azaVal) in good yields (82ꢀ92%), following the route B mentioned above, i.e.
activation of the protected 1ꢀalkylꢀhydrazine 3 followed the reaction with the protected 2ꢀalkylꢀ
hydrazine 1.8 They also mentioned the preparation of the tetraꢀazaꢀamino acids containing
pentapeptide TyrꢀazaAlaꢀAzaGlyꢀazaPheꢀazaLeu in 50% yield.8b The synthesis of our new target
compounds IꢀV started by the preparation of the protected 2ꢀalkylꢀhydrazine 1 and of the protected 1ꢀ
alkylꢀhydrazine 3. In the literature, the condensation of Nꢀprotected carbazate with the appropriate
aldehyde or ketone is reported to yield the hydrazone which is immediately reduced by catalytic
hydrogenation or hydride addition using NaBH3CN to give protected 2ꢀalkylꢀhydrazine 1. 10,14,15 In our
hands, the Bocꢀprotected 2ꢀisopropylꢀ, Cbzꢀ4ꢀaminoꢀbutylꢀ, isobutylꢀ and benzylꢀhydrazines 1a-d
were obtained by reaction of the commercially available tertꢀbutyl carbazate with acetone, benzyl 2ꢀ
hydroxypyrrolidineꢀ1ꢀcarboxylate (prepared according to references 10, 14 and 15), butyraldehyde and
benzaldehyde respectively, in dry THF, to give the corresponding hydrazone intermediates which were
reduced in a one pot manner without isolation, in the presence of NaBH3CN in satisfactory yields
(52%, 58%, 66% and 70% respectively). The direct protection of the commercial methylhydrazine
with benzyl chloroformate yielded the Cbzꢀ1ꢀmethylꢀhydrazine 3b in 47 % yield.12 The Bocꢀ2ꢀ
isopropyl-hydrazine 1a was protected with benzyl chloroformate to afford a diprotected intermediate
that provided the hydrochloride salt of the Cbzꢀ1ꢀisopropyl hydrazine 3c after acidic cleavage of the
Boc moiety in 90% yield from 1a. The protection of the Bocꢀ2ꢀisobutylꢀhydrazine 1c by the Alloc
group followed by the acidic cleavage of the Boc moiety provided the hydrochloride salt of the Allocꢀ
1ꢀisobutyl hydrazine 3d with a quantitative yield (Scheme 2).

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R1
R
i
ii
H
Boc-NHNH2
NH
N
BocHN
Boc
N
R'
1a R1 = iPr (52%)
1b R1 = (CH2)4NHCbz (58%)
1c R1 = iBu (66%)
1d R1 = Bn (70%)
H
N
iii
N
O
H2N
H2N
O
3b (47%)
O
iv
H
N
H3N
N
O
Cl
Boc
N
H
1a
3c (90%)
v
H
N
N
O
BocHN
Cl
H3N
O
1c
3d (quantitative)
Scheme 2: Preparation of Bocꢀ2ꢀalkylꢀhydrazine 1a-d, Cbzꢀ1ꢀalkylꢀhydrazine 3b and 3c and Allocꢀ1ꢀ
isobutylꢀhydrazine 3d.
Reagents and conditions: i) acetone, benzyl 2ꢀhydroxypyrrolidineꢀ1ꢀcarboxylate 10,14 butyraldehyde, or
benzyaldehyde, acetic acid, THF, r.t., 5 h to overnight ii) NaBH3CN, acetic acid or PTSA THF, r.t, 1h
to 24h; iii) benzyl chloroformate, NaOH, DCM, r.t, 4h, 47%; iv) a) benzyl chloroformate, TEA, DCM,
r.t, overnight, b) HCl 4M dioxane, r.t, overnight; v) a) allyl chloroformate, Na2CO3, water, dioxane, r.t,
overnight, b) HCl 4M dioxane, r.t, overnight
Having in hand the appropriate protected 2ꢀalkylꢀhydrazine 1 and 1ꢀalkylꢀhydrazine 3, we first chose
to evaluate the route A described above, i.e. the activation of the protected 2ꢀalkylꢀhydrazine 1
followed by the reaction with the protected 1ꢀalkylꢀhydrazine 3 (Scheme 3). The activation of Bocꢀ2ꢀ
alkylꢀhydrazines 1a-d in dry DCM in the presence of pyridine, using the 4ꢀnitrophenyl chloroformate
as carbonyl donor, afforded the intermediates 2a-d in good yields (80ꢀ89%). Noteworthy, the steric
hindrance of the alkyl groups of Bocꢀprotected hydrazines 1 does not seem to prevent the nucleophilic
attack of Bocꢀ2ꢀalkylꢀhydrazines 1a-d. The following reaction of 2a and 2b with hydrazine
monohydrate 3a in MeOH overnight at room temperature gave 5a and 5b respectively, in excellent
yields (82%). To activate 5a and 5b we chose phenyl chloroformate, hypothesizing that the amine of
the natural amino acid would be sufficiently nucleophilic to react with the carbonyl of the phenyl

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formate, as observed for the synthesis of BocꢀazaGlyꢀazaGlyꢀValꢀNH2 VI (see above). However,
while we obtained 7a and 7b in good yields (respectively 80 and 96%), the final coupling of 7a and 7b
with LꢀPheꢀNH2 afforded I in good yield (85%) but II in modest yield (35 %). We hypothesized that
the presence of the long and bulky Cbzꢀ4ꢀaminoꢀbutyl in 7b may prevent the efficiency of the
coupling. Unfortunately, the reaction of 2a, 2c and 2d with 3b, 3c and 3d respectively in order to
obtain 8a, 8b and 8c did not occur under the conditions previously used for adding hydrazine hydrate
3a to 2a and 2b (MeOH overnight at room temperature). Other conditions have been tried by changing
either the solvent (DMF or DCM), or the temperature (room temperature or at 40°C), and in the
presence or in the absence of DMAP. None of these conditions allowed to obtain the expected diazaꢀ
peptides 8aꢀ8c. We thus suspected that this failure could be due to the lower nucleophilicity of the
protectedꢀ1ꢀalkylꢀhydrazines 3bꢀd compared to that of hydrazine hydrate 3a and also to the steric
hindrance of their protecting and alkyl groups.
R1
R1
N
(ii)
H
R1
(i)
NH2
N
N
O
+
PG1-N
R2
NH
BocHN
NPG1
R2
BocHN
BocHN
O
3b R2 = CH3, PG1= Cbz
3c R2 = iPr, PG1= Cbz
3d R2 = iBu, PG1= Alloc
O
NO2
3
1a R1 = iPr
1b R1 = (CH2)4NHCbz
1c R1 = iBu
2a R1 = iPr (80% from 1a)
2b R1 = (CH2)4NHCbz (85% from 1b)
2c R1 = iBu (89% from 1c)
8a R1 = iPr , R2 = CH3, PG1= Cbz (from 2a and 3b)
8b R1 = iBu, R2 = iPr, PG1= Cbz (from 2c and 3c)
8c R1 = Bn, R2 = iBu, PG1= Alloc (from 2d and 3d)
(ii)
1d R1 = Bn
2d R1 = Bn (82% from 1d)
3a R2 = PG1 = H
R1
H
N
N
BocHN
NH2
O
5a R1 = iPr (82% from 2a and 3a)
5b R1 =(CH2)4NHCbz (82% from 2b and 3a)
(iii)
R1
N
O
R1
N
O
H
(iv)
H
N
N
NH2
BocHN
N
O
BocHN
N
N
H
H
H
O
O
O
I R1 = iPr (85% from 7a)
II R1 = (CH2)4NHCbz (35% from 7b)
7a R1 = iPr (80% from 5a)
7b R1 = (CH2)4NHCbz (96% from 5b)
Scheme 3. Strategy A): activation of the protected 2ꢀalkylꢀhydrazine 1 followed the reaction with the
protected 1ꢀalkylꢀhydrazine 3. Access to BocꢀazaValꢀazaGlyꢀPheꢀNH2 (I) and BocꢀazaLysꢀazaGlyꢀ
PheꢀNH2 (II).
Reagents and conditions: i) 4ꢀnitrophenyl chloroformate, pyridine, DCM, r.t., overnight; ii) MeOH, r.t.
overnight; iii) phenyl chloroformate, pyridine, DCM, r.t., 20 min; iv) LꢀPheꢀNH2, triethylamine, ACN,
r.t., 3 days.
Thus, in order to access to BocꢀazaValꢀazaAlaꢀValꢀNH2 (III), BocꢀazaLeuꢀazaValꢀHisꢀOCH3 (IV) and
BocꢀazaPheꢀazaLeuꢀValꢀOCH3 (V), we evaluate the second route B, i.e. the activation of the protected
1ꢀalkylꢀhydrazine 3 followed by the reaction with the protected 2ꢀalkylꢀhydrazine 1 (Scheme 4). The

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activation of the protectedꢀ1ꢀalkylꢀhydrazines 3b, 3c and 3d using 4ꢀnitrophenyl chloroformate
afforded the intermediates 4a, 4b and 4c in good yields (93% and 82% and 73% respectively). The
subsequent reaction of 4a and 4c with Bocꢀ2ꢀalkylꢀhydrazines 1a and 1d afforded 8a and 8c
respectively in good yields (80% and 62%, respectively). However, the yield of coupling between 4b
and 1c to afford 8b was very modest (28%). We decided to test the activation of 3c with
bis(pentafluorophenyl)carbonate, in order to increase the electrophilic character of the carbonyl group,
but the yield of the subsequent reaction with 1c remained similarly low. However, overall it is
noteworthy that activating first the protected 1ꢀalkylꢀhydrazine 3 followed by the reaction with the
protected 2ꢀalkylꢀhydrazine 1 (route B) is more convenient than the previous route A in the case of
substituted and hindered alkyl hydrazines, maybe because the protected 2ꢀalkylꢀhydrazines 1 are more
nucleophilic than the protected 1ꢀalkylꢀhydrazines 3. We continued the synthesis from 8a and 8c to
obtain the final compounds III and V. Thus, the cleavage of the Cbz group of 8a and of the Alloc
group of 8c afforded 5c and 5d in satisfactory yields (95% and 51% respectively, scheme 4). Then, in
a first attempt, we followed our previous strategy by activating first 5c and reacting then with the
natural amino acid LꢀValꢀNH2 6a. However, while the activation of 5c using phenyl chloroformate or
4ꢀnitrophenyl chloroformate was satisfactory, the reaction with the amino acid ValꢀNH2 6a in the
presence of TEA and in dry acetonitrile as described previously (in scheme 3 for I and II), or by
adding also DMAP, did not afford the final compound III. Thus, we evaluated the activation firstly of
LꢀValꢀNH2 6a with 4ꢀnitrophenyl chloroformate in dry DMF to afford 10a that was not isolated and
directly reacted in a one pot manner with 5c in the presence of DMAP. The final azaꢀtripeptide III was
obtained with a satisfactory yield of 60%. Similarly, LꢀValꢀOCH3 6b was activated to afford 10c that
reacted with 5d to give the final azaꢀtripeptide V in modest yield (35%, Scheme 4).

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R1
N
H
R1
(i)
H
(ii)
NH2
N
O
N
NH
PG1-N
R2
PG1-N
R2
BocHN
N-PG1
R2
+
BocHN
O
O
NO2
8a R1 = iPr , R2 = CH3, PG1= Cbz (80% from 4a and 1a)
8b R1 = iBu, R2 = iPr, PG1= Cbz (28% from 4b and 1c)
8c R1 = Bn, R2 = iBu, PG1= Alloc (62% from 4c and 1d)
3b R2 = CH3, PG1= Cbz
3c R2 = iPr, PG1= Cbz
3d R2 = iBu, PG1= Alloc
4a R2 = CH3, PG1= Cbz (93% from 3b)
4b R2 = iPr, PG1= Cbz (82% from 3c)
4c R2 = iBu, PG1= Alloc (73% from 3d)
1a R1 = iPr
1c R1 = iBu
1d R1 = Bn
(iii)
NO2
O
R1
N
O
R1
H
(iv)
H
H2N
R4
N
N
N
+
BocHN
N
O
BocHN
NH
R2
R3
O
R2
O
5c R1 = iPr , R2 = CH3 (95% from 8a)
5d R1 = Bn, R2 = iBu (51% from 8c)
9a. R1 = iPr , R2 = CH3 (95% from 5c)
6a R3 = iPr, R4 = NH2
(vii)
(v)
O
H
O
O
N
(vi)
R4
H2N
R3
O
+
R1
R4
H
O
R3
N
N
R4
O2N
R3
BocHN
N
N
H
O
R2
O
III, V
6a R3 = iPr, R4 = NH2
6b R3 = iPr, R4 = OCH3
10a R3 = iPr, R4 = NH2
10b R3 = iPr, R4 = OCH3
III R1 = iPr , R2 = CH3, R3 = iPr, R4 = NH2 (60% for two steps from 6a and 5c)
V R1 = Bn, R2 = iBu , R3 = iPr, R4 = OCH3 (35% for two steps from 6b and 5d)
Scheme 4: Strategy B): activation of the protected 1ꢀalkylꢀhydrazine followed the reaction with the
protected 2ꢀalkylꢀhydrazine. Access to BocꢀazaValꢀazaAlaꢀValꢀNH2 (III) and BocꢀazaPheꢀazaLeuꢀ
ValꢀOCH3 (V).
Reagents and conditions: i) 4ꢀnitrophenyl chloroformate, pyridine, DCM, r.t., overnight; ii) DMAP,
DMF, r.t., overnight; iii) Pd/C 10%, H2, MeOH, r.t., overnight from 8a or [Pd(PPh3)4]/NDMBA,
DCM, 35°C,4h from 8c; iv) 4ꢀnitrophenyl chloroformate, pyridine, DCM, r.t., overnight; v)
triethylamine, ACN, r.t., 3 days; vi) 4ꢀnitrophenyl chloroformate, pyridine, DMF, r.t., overnight; vii)
DMAP, DMF, r.t., overnight
Due to the poor yield for the formation of the BocꢀazaLeuꢀazaVal intermediate 8b, the strategy B was
given up for the synthesis of the final compound BocꢀazaLeuꢀazaValꢀHisꢀOCH3 IV. Thus, we
evaluated a third and alternative route to synthesize IV, starting from the preparation of the Cꢀterminal
azaValꢀHisꢀOCH3 dipeptide 12 and its final coupling with the Nꢀterminal BocꢀazaLeu 1a (Scheme 5,
strategy C).

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O
H
O
O
O
H
O
N
(i)
(ii)
N
N
NH2
N
O
NH
BocHN
O
+
BocHN
O
NO2
O
NH
NH
N
NH
N
1a
12 (49%)
6c
11
(iii)
N
O
F
F
NH
F
F
F
O
H
O
O
H
(iv)
N
N
O
+
NH
BocHN
N
N
N
N
O
BocHN
O
N
H
O
H
O
NH
N
13
1c
IV (53%)
Scheme 5: Strategy C): activation of the natural amino acid followed by the reaction with the
protected 2ꢀalkylꢀhydrazine. Access to BocꢀazaLeuꢀazaValꢀHisꢀOCH3 (IV).
Reagents and conditions: i) 4ꢀnitrophenyl chloroformate, pyridine, DMF, 40° C, overnight; ii) DMAP,
DMF, r.t., overnight; iii) a) HCl 4M dioxane, r.t., overnight; b) bis(pentafluorophenyl)carbonate,
DIPEA, DMAP, DCM, r.t., 40 min.; iv) DMAP, DCM, r.t., 4 days.
The activation of the natural amino acid LꢀHisꢀOCH3 6c was more efficient when performed at 40°C
than at room temperature, and the subsequent coupling with 1a in a one pot manner gave BocꢀazaValꢀ
HisꢀOCH3 12 in 49% yield. We also tried the activation of 6c using bis(pentafluorophenyl)carbonate,
but the yield of the coupling reaction with 1a was even lower (15%). The inverse pathway i.e. the
reaction of the activated 2a (obtained from 1a, see scheme 3) with LꢀHisꢀOCH3 6c did not afford Bocꢀ
azaValꢀHisꢀOCH3 12. After the cleavage of the Boc moiety of 12 in acidic conditions, the activation of
the hydrazine moiety using bis(pentafluorophenyl)carbonate in dry DCM in the presence of DMAP
and DIPEA, and the subsequent reaction with 1c gave the final compound IV with a satisfactory yield
of 53% (for 3 steps). The activation with 4ꢀnitrophenyl chloroformate did not allow us to obtain the
desired compound IV. Overall, this third strategy of synthesis, starting from the preparation of first the
Cꢀterminal azaValꢀHisꢀOCH3 dipeptide and its final coupling with the Nꢀterminal BocꢀazaLeu was
more convenient than the previous route starting from BocꢀazaLeuꢀazaVal and coupling with the Cꢀ
terminal LꢀHisꢀOCH3. However, it is important to note that this strategy is substrateꢀdependent. In
fact, we tried to apply this third strategy to obtain the final compound V. We prepared BocꢀazaLeuꢀ
ValꢀOCH3 in satisfactory yield (48%) but unfortunately the last coupling with BocꢀazaPhe to obtain
BocꢀazaPheꢀazaLeuꢀValꢀOCH3 V was unsuccessful (the activation was done using either
bis(pentafluorophenyl)carbonate or 4ꢀnitrophenyl chloroformate).
3. Conclusion

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As mentioned in the introduction, before starting this study, we wondered about the scarcity of
examples of the incorporation of two or more azaꢀamino acids consecutively in azaꢀpeptides while the
introduction of one azaꢀamino acid has demonstrated increased interest in biologically active peptides.
As demonstrated in this work, the reactivity of alkylꢀhydrazides is highly susceptible to the nature and
the bulkiness of the alkyl chain. This capricious reactivity has been observed both in the case of the
synthesis of azaAAꢀazaAA and also for the final coupling with the natural amino acid at the Cꢀ
terminal position of the diꢀazaꢀamino acids containing tripeptides. However, from this work, we can
draw some general guidelines. i) Concerning the synthesis of the diazaꢀpeptides (5), the activation of
the protected 1ꢀalkylꢀhydrazine 3 followed by the reaction with the protected 2ꢀalkylꢀhydrazine 1
(route B) is more efficient than the activation of the protected 2ꢀalkylꢀhydrazine 1 followed the
reaction with the protected 1ꢀalkylꢀhydrazine 3 (route A), because the protected 2ꢀalkylꢀhydrazines 1
are more nucleophilic than the protected 1ꢀalkylꢀhydrazines 3. ii) Concerning the synthesis of the final
tripeptide analogues, the activation of the natural amino acid followed by the reaction with the diazaꢀ
peptide is more efficient than the activation of the diazaꢀpeptide followed by the reaction with the
amino acid. This observation indicates that unless expected, the nucleophilicity of the alkylꢀhydrazides
is not a real restraint. iii) Also concerning the synthesis of final tripeptide analogues, the strategy of
coupling the Nꢀterminal azaAAꢀazaAA dipeptide analogue to a Cꢀterminal natural amino acid is more
efficient than coupling the Nꢀterminal azaAA to the Cꢀterminal azaAAꢀAA dipeptide analogue, but
this last strategy was more efficient for IV. The bulkiness of the alkylꢀhydrazides is a real constraint,
as illustrated by the easy synthesis of the two azaꢀGly containing tripeptide VI and the easier
preparation of one azaꢀGly containing tripeptides I and II compared to that of III, IV and V.
Concerning the activation, the use of 4ꢀnitrophenyl chloroformate, in dry DMF with DMAP is
efficient for the more challenging coupling reactions. However, when this strategy is not successful,
the replacement of 4ꢀnitrophenyl chloroformate by bis(pentafluorophenyl)carbonate, in dry DCM and
in the presence of DMAP, can be more efficient.
4. Experimental
4.1.
General Experimental Methods.
Usual dry solvents were purchased from commercial sources. 4ꢀnitrophenyl chloroformate, hydrazine
monohydrate, phenyl chloroformate, benzyl chloroformate, bis(pentafluorophenyl) carbonate, 4ꢀ
aminobutyraldehyde diethylacetal, LꢀPheꢀNH2 LꢀHisꢀOMe, LꢀValꢀNH2, and L-ValꢀOCH3 were
purchased from commercial sources. Benzyl 2ꢀhydroxypyrrolidineꢀ1ꢀcarboxylate was prepared
according to published methods.10,14 Pure products were obtained after liquid chromatography using
Merck silica gel 60 (40−63 ꢁm). TLC analyses were performed on silica gel 60Fꢀ250 (0.26 mm
thickness) plates. The plates were visualized with UV light (λ = 254 nm) or revealed with a 5%
solution of phosphomolybdic acid in EtOH or with a solution of ninhydrin in EtOH. Melting points

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were determined on a Kofler melting point apparatus. Element analyses (C, H, and N) were performed
on a PerkinElmer C, H, N Analyzer 2400 at the Microanalyses Service of the Faculty of Pharmacy at
ChâtenayꢀMalabry (BioCIS, France). NMR spectra were recorded on an Ultrafield Bruker AVANCE
300 (1H, 300 MHz, 13C, 75 MHz) or on a Bruker Avance 400 (1H, 400 MHz, 13C, 100 MHz). Chemical
shifts δ are in ppm, and the following abbreviations are used: singlet (s), broad singlet (bs), doublet
(d), doublet of triplet (dt), triplet (t), multiplet (m). IR spectra were recorded on a Bruker Vector 22
FTꢀIR spectrometer. Mass spectra were obtained using a Bruker Esquire electrospray ionization
apparatus. HRMS were obtained using a TOF LCT Premier apparatus (Waters), with an electrospray
ionization source. The purity of compounds was determined by HPLC using the 2695 Alliance system
(Waters) and a Sunfire column (C18, 3.5 ꢁm, 150 mm × 2.1 mm); mobile phase, MeCN/H2O + 0.1%
formic acid from 5 to 100% in 20 min; detection at 257 nm; flow rate 0.25 mL/min.
4.2.
Synthesis
Strategy A:
A1/ General procedure for the preparation of 2a-d: To a solution of the carbazate 1a, 1b, 1c or 1d
in dry DCM (1eq.), was added pyridine (5 eq.) at 0 °C under argon atmosphere. After 10 minutes, 4ꢀ
nitrophenyl chloroformate (1.1 eq.) was introduced slowly and the reaction was stirred at room
temperature overnight. After evaporation of the volatiles under vacuum, the residue obtained was
taken up with EtOAc. The organic phase was successively washed with 10% aqueous citric acid,
distilled water, 10% aqueous K2CO3, brine, dried over Na2SO4, filtered and concentrated. The
intermediate 2a, 2b, 2c or 2d could be used in the next step without any further purification.
A2/ General procedure for preparation of 5a-b: To a solution of 2a or 2b (1 eq.) in dry MeOH was
added hydrazine monohydrate (6 eq.). The reaction mixture was stirred at room temperature for one
night. After evaporation of the volatiles under vacuum, the crude material obtained was purified by
column chromatography on silica gel using the appropriate solvent to yield the pure compound.
A3/ General procedure to preparation of 7a-b: To a solution of 5a or 5b (1eq.) in dry THF under
argon atmosphere was added at 0 °C pyridine (2.5 eq.). After 10 minutes, phenyl chloroformate (1.1
eq.) was introduced slowly and the reaction was stirred at room temperature 20 minutes. After
evaporation of the volatiles, the crude residue obtained was taken up with EtOAc. The organic phase
was successively washed with aqueous 10% citric acid, distilled water, 10% aqueous K2CO3, brine,
dried over Na2SO4, filtered and concentrated. After evaporation under vacuum, the crude residue
afforded was purified by column chromatography on silica gel with the appropriate solvent to afford
the pure compound.

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A4/ General procedure for preparation of I-II: To a solution of 7a or 7b (1eq.) in dry ACN under
argon atmosphere was added the LꢀPheꢀNH2 hydrochloride (1.1 eq.). Triethylamine (6 eq.) was added
and the mixture was stirred at room temperature for three days. The volatiles were removed under
vacuum and the crude material was purified by column chromatography on silica gel using the
appropriate eluent to yield the pure compound.
Strategy B:
General procedure for preparation of 9a, 4a-c: prepared according to the general procedure A1
B1/ General procedure for preparation of 8a-c: To a solution of 4a, 4b or 4c in dry DMF, were
successively added at 0 °C under argon atmosphere, 1a, 1c or 1d (1.0 eq.) and DMAP (1.0 eq.) The
reaction was stirred overnight at room temperature. After evaporation of the volatiles under vacuum,
the crude residue was purified by chromatography on silica gel using the appropriate eluent to yield
the pure compound.
B2/ General procedure preparation of III, V: To a solution of corresponding aminoꢀacid 6a or 6b
(1.1 eq.) in dry DMF at 0 °C were successively added under argon atmosphere DIPEA (2.2 eq.) and 4ꢀ
nitrophenyl chloroformate (1.1 eq.). The solution was stirred at room temperature for one night and
after formation of carbamate 10a, or 10b monitored by TLC, a solution of the previously prepared 5c
or 5d (1.0 eq.) and DMAP (1.0 eq.) were added to the reaction mixture. After stirring overnight at
room temperature, the volatiles were removed under vacuum, and the crude material obtained was
purified by chromatography on silica gel using the appropriate eluent to yield the pure compound.
Tert-butyl 2-isopropylhydrazine-1-carboxylate (1a): To a solution of tertꢀbutylꢀcarbazate (2 g, 15.1
mmol, 1.0 eq.) in 20 mL of dry THF under argon atmosphere was successively added under argon
atmosphere acetone (3.3 mL, 45.3 mmol, 3 eq.) and acetic acid (1.05 mL, 13.6 mmol, 0.9 eq.). After 3
h the volatiles were removed under vacuum and the remaining oil was dissolved in 100 mL of dry
THF. NaBH3CN was then added with some seeds of bromocresol green. A solution of paraꢀtolueneꢀ
sulfonic acid (2.86 g, 16.6 mmol, 1.1 eq.) in 4 mL of dry THF was added dropwise and the mixture
became yellow. After 1 h the resulting suspension was removed under vacuum. The remaining
colourless powder was dissolved in a mixture of EtOAc/NaClaq, extracted with EtOAc and the organic
layer washed with a mixture of aqueous NaCl/NaHCO3. 1/1. The organic phase was dried over
Na2SO4, filtered and concentrated under reduced pressure. dried over Na2SO4, filtered and
concentrated. The crude material obtained was then dissolved in 10 mL of MeOH and NaOH 1M (18
mL, 18.1 mmol, 1.2 eq.). At that time, the solution became successively blue and colourless. After
stirring 1 hour at room temperature and evaporation of the volatiles under vacuum, the oil afforded

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was taken up in EtOAc, washed with brine, dried over Na2SO4 filtrated and concentrated. Purification
by column chromatography on silica gel using DCM/MeOH 98:2 as eluent gave 1a (1.37g, 7.9 mmol,
1
52%) as an oil which crystallized by cooling. Rf = 0.50 (DCM/MeOH 98:2); H NMR (300 MHz,
CDCl3): δ = 5.31 (2H, bs), 3.28 (1H, dt, J = 12.26 and 6.3 Hz), 1.47 (9H, s), 1.12 (6H, d, J = 6.4 Hz),
13C NMR (75 MHz, CDCl3): δ = 156.9, 80.3, 50.7, 28.3, 20.6; HRMS (TOF ESI, ion polarity
positive): m/z 197.1266 calcd for [C8H18N2O2 +Na]+, found: 197.1266.
Tert-butyl 2-(4-(((benzyloxy)carbonyl) amino) butyl) hydrazinecarboxylate (1b): 1b was
synthesized following the same procedure described for 1a from tertꢀbutylꢀcarbazate (450 mg, 3.4
mmol, 1.0 eq.) and benzyl 2ꢀhydroxypyrrolidineꢀ1ꢀcarboxylate (750 mg, 3.4 mmol) to give after
purification by flash chromatography on silica gel using EtOAc/hexane 6:4 as eluent a white solid
(562 mg, 1.7 mmol, 49%). Rf = 0.85 (EtOAc/hexane 6:4); 1H NMR (300 MHz, CDCl3): δ = 7.31 (5H,
m), 5.74 (1H, bs), 5.21 (1H, bs), 5.07 (2H, s), 3.44 (1H, bs), 3.18 (2H, d, J = 5.8 Hz), 2.87 (2H, t, J =
6.5 Hz), 1.51 (4H, m), 1.44 (9H, s); 13C NMR (75 MHz, CDCl3): δ = 203.5, 156.6, 136.6, 128.5,
128.1, 84.0, 66.7, 51.1, 40.3, 28.1, 26.7, 21.5; HRMS (TOF ESI, ion polarity positive): m/z 244.0950
calcd for [C17H27N3O4 +Na]+: found: 244.0942.
Tert-butyl 2-isobutylhydrazine-1-carboxylate (1c): 1c was synthesized following the same
procedure described for 1a from tertꢀbutylꢀcarbazate (1 g, 7.6 mmol, 1.0 eq.) in 15 mL of dry THF and
isobutyraldehyde (1.0 mL, 11.4 mmol, 1.5 eq.) to afford after purification by flash chromatography on
silica gel using DCM 100% as eluent as a colourless oil (943 mg, 5.0 mmol, 66%). Rf = 0.10 (DCM
1
100%); H NMR (300 MHz, CDCl3): δ = 7.3 (1H, bs), 5.25 (1H, bs), 2.65 (2H, d, J = 6.8 Hz), 1.73
(1H, dd, J = 13.6 and 6.7 Hz), 1.44 (9H, s), 0.91 (6H, d, J = 6.7 Hz); 13C NMR (75MHz, CDCl3): δ =
157.3, 81.0, 60.4, 28.8, 27.1, 21.0; HRMS (TOF ESI, ion polarity positive): m/z 189.1603 calcd for
[C9H20N2O2+H]+, found: 189.1600
Tert-butyl 2-benzylhydrazine-1-carboxylate (1d): 1d was synthesized following the same procedure
described for 1a from tertꢀbutylꢀcarbazate (1 g, 7.6 mmol, 1 eq.) in 10 mL of dry THF and
benzaldehyde (0.77 mL, 7.60 mmol, 1 eq.) to afford after purification by flash chromatography on
silica gel using cꢀHex/AcOEt 8:2 as eluent a colourless oil (1.19 g, 5.32 mmol, 71%). Rf = 0.35 (cꢀ
1
Hex /EtOAc 9:1); H NMR (300 MHz, CD3OD): δ = 7.36ꢀ7.26 (5H, m); 3.90 (2H, s); 1.44 (9H, s)
ppm; 13C NMR (75 MHz, CD3OD): δ = 156.6, 137.5, 129.0, 128.5, 127.5, 80.6, 55.9, 28.3 ppm;
HRMS (TOF ESI, ion polarity positive): m/z 245.1266 calcd. for [C12H18N2O2 +Na]+, found:
245.1255.
2-(Tert-butyl) 1-(4-nitrophenyl)1-isopropylhydrazine-1,2-dicarboxylate (2a): 2a was obtained
from 1a (500 mg, 2.87 mmol) as a yellow powder (779 mg, 2.30 mmol, 80%) following the general

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procedure A1. Rf = 0.6 (cꢀHex /EtOAc 7:3); 1H NMR (300 MHz, CDCl3): δ = 8.25 (2H, d, J = 9.0 Hz),
7.30 (2H, d), 6.36 (1H, bs,), 4.5 (1H, dt, J = 12.0 and 6.0 Hz), 1.47 (9H, s), 1.23 (6H, d, J = 6.3 Hz) ;
13C NMR (75 MHz, CDCl3): δ = 125.7, 125.3, 122.5, 82.1, 50.4, 28.2, 19.6; MS (ESI, ion polarity
positive): m/z 340.14 calcd for [C15H21N3O6 +H]+, found: 340.14.
2-(Tert-butyl1-(4-nitrophenyl)1-(4-(((benzyloxy)carbonyl)amino)butyl)hydrazine1,2-dicar-
boxylate (2b): 2b was obtained from 1b (443.1 mg, 1.31 mmol) as a white solid (560 mg, 1.12 mmol,
1
85%), following the general procedure A1. Rf = 0.85 (EtOAc/hexane 6:4); H NMR (300 MHz,
CDCl3): δ = 8.24 (2H, d, J = 9.0 Hz), 7.32 (7H, m),6.83 (1H, bs), 5.13 (2H, s), 4.92 (1H, bs), 3.71 (2H,
m), 3.24 (2H, s), 1.68 (4H, m), 1.46 (9H, s); 13C NMR (75MHz, CDCl3): δ = 156.4, 155.3, 154.9,
153,8, 145.2,136.5, 128.5, 128.1, 125.1, 122.3, 122.1, 82.0, 66.8, 50.1, 40.5, 28.1, 27.0, 24.0; HRMS
(TOF ESI, ion polarity positive): m/z 525.1961 calcd for [C24H30N4O8 +Na]+, found: 525.1964.
2-(Tert -butyl) 1-(4-nitrophenyl) 1-isobutylhydrazine-1,2-dicarboxylate (2c). 2c was obtained from
1c (200 mg, 1.06 mmol) as a colourless oil (335 mg, 0.95 mmol, 89%) following the general
procedure A1. Rf = 0.3 (EtOAc/Cyclo 2:8); 1H NMR (300 MHz, CDCl3): δ = 8.34 (2H, d, J =9.7 Hz),
7.49 (2H, d, J = 9.7 Hz), 7.30 (1H, bs), 3.46 (2H, m), 2.01 (1H, m), 1.48 (9H, s), 0.98 (6H, d, J = 6.4
Hz); 13C NMR (75 MHz , CDCl3): δ = 155.0, 150.2, 146.0, 125.7, 122.4, 77.4, 58.2, 28.31, 26.8, 20.1 ;
HRMS (TOF ESI, ion polarity positive): m/z 376.1485 calcd for [C16H23N3O6 +Na]+, 376.1485, found:
376.1479.
2-(Tert-butyl ) 1-(4-nitrophenyl) 1-benzylhydrazine-1,2-dicarboxylate (2d): 2d was obtained from
1d (67 mg, 0.30 mmol) as a yellow oil (95 mg, 0.25 mmol, 82%) following the general procedure A1.
Rf = 0.55 (7:3 cꢀHex /AcOEt) ; 1H NMR (300 MHz, CDCl3): δ = 8.28 (2H, d, J = 9.0 Hz), 7.37 (7H,
m), 6.52 (1H, bp), 4.83 (2H, m), 1.26 (9H, s); 13C NMR (300 MHz, CDCl3): δ = 151.5, 129.3, 128.96,
128.7, 128.3, 127.9, 125.3, 122.4, 82.5, 52.5, 28.3.
Benzyl 1-methyl hydrazinecarboxylate (3b): To a solution of methyl hydrazine (2.0 mL, 38.0 mmol,
1.0 eq.) in DCM (20 mL) cooled at 0 °C was added slowly an aqueous solution of NaOH (30.4 mL,
30.4 mmol, 0.8 eq.). The biphasic mixture was stirred rapidly for 5 min and at that time, benzyl
chloroformate (4.3 mL, 30.4 mmol, 0.8 eq.) was added dropwise. The reaction was gradually warmed
to room temperature and stirred for 4h. After separation of the biphasic mixture, the isolated organic
layer was successively washed with water, brine, dried over Na2SO4, filtered and concentrated under
vacuum. Purification of the crude oil by distillation (bp 104ꢀ134 °C, 0.2 mmHg) provided pure product
1
3b as colourless oil (2.56 g, 14.2 mmol, 74%); H NMR (300 MHz, CDCl3): δ = 7.33 (5H, m), 5.12
(2H, s), 4.38 (2H, s), 3.13 (3H, s); 13C NMR (300 MHz, CDCl3): δ = 136.4, 128.5, 128.2, 128.0, 67.7,

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38.4; HRMS (TOF ESI, ion polarity positive): m/z 181.0977 calcd for [C9H12N2O2 +H]+, found:
181.0974; m/z 203.0796 calcd for [C9H12N2O2 +Na]+, found: 203.0798.
Benzyl 1-isopropylhydrazinecarboxylate, HCl (3c): To a solution of 1a (500 mg, 2.87 mmol, 1 eq.)
in 10 mL of dry DCM was successively added at 0 °C TEA (0.774 mL, 5.74 mmol, 2eq.) then benzyl
chloroformate (0.819 mL, 5.74 mmol, 2 eq.). A precipitate was formed. The reaction was stirred
overnight at room temperature. The volatiles were evaporated under vacuum and the crude material
obtained was taken up with EtOAc. The organic layer was washed successively with an aqueous
saturated solution of NH4Cl, brine, dried over Na2SO4, filtered and concentrated under vacuum. The
resulting crude material was purified by column chromatography on silica gel using cꢀHex /EtOAc 9:1
as eluent to afford a colourless oil (0.800 g, 2.60 mmol, 1 eq., 90% yield). To a solution of this oil in 5
mL of dry dioxane under argon atmosphere, was added dropwise at 0 °C, HCl 4M in dioxane (13.0
mL, 52.0 mmol, 20 eq.). The reaction was let under stirring overnight at room temperature. After
removing the volatiles under vacuum and drying, the hydrochloride salt 3c was afforded as a
1
colourless oil (636 mg, 2.60 mmol, quantitative). Rf = 0.1 (cꢀHex /EtOAc 8:2; H NMR (400 MHz,
CD3OD): δ = 7.38 (5H, m); 5.28 (2H, s), 3.62 (1H, m), 1.32 (3H, d, J = 6.6 Hz), 1.29 (3H, d, J = 6.9
Hz); 13C NMR (100 MHz, CD3OD): δ = 155.6, 136.9, 129.5, 129.4, 129.1, 70.0, 55.4, 19.2, 17.2;
HRMS (TOF ESI, ion polarity positive): m/z 209.1290 calcd for [C11H16N2O2 +H]+, found: 209.1284.
Allyl 1-isobutylhydrazine-1-carboxylate, HCl (3d): To a solution of 1c (0.300 g, 1.60 mmol, 1.0
eq.) in dioxane, was added Na2CO3 (450 mg, 4.25 mmol, 2.6 eq.) dissolved in 3 mL of water. At that
°
time, allyl chloroformate (0.153 mL, 1.44 mmol, 0.9 eq.) was added dropwise at 0 C. The mixture
was stirred 30 min at this temperature and then overnight at room temperature. Water was added, and
the organic phase was extracted with diethyl ether. The organic layer was dried over Na2SO4, filtrated
and the volatiles were removed under reduced pressure. The resulting crude material was purified by
column chromatography on silica gel using cꢀHex /EtOAc 8:2 as eluent to afford a white powder of
the Boc allyl intermediate (425 mg, 1.5 mmol, 98%). To a solution of this powder (39 mg,1.44 mmol,
1.0 eq.) in dioxane under argon atmosphere was added dropwise at 0 °C HCl 4M in dioxane (7.2 mL,
28.8 mmol, 20 eq.). The reaction was let under stirring overnight at room temperature. The volatiles
were removed under reduced pressure and the powder was dried under vacuum to yield the
hydrochloride salt 3d as a colourless oil (300 mg, 1.44 mmol, quantitative). Rf = 0.1 (cꢀHex /EtOAc
1
8:2); H NMR (300 MHz, CD3OD): δ = 5.97 (1H, dq, J = 10.7, 5.8 Hz), 5.32 (2H, dd, J = 27.7, 13.9
Hz), 4.71 (2H, d, J = 5.7 Hz), 3.42 (2H, d, J = 7.6 Hz), 1.97 (1H, m), 0.94 (6H, d, J = 6.6 Hz); 13C
NMR (75 MHz, CD3OD): δ = 132.9, 119.5, 69.1, 57.2, 28.1, 19.8; HRMS (TOF ESI, ion polarity
positive): m/z 173.1290 calcd. for [C8H16N2O2 +H]+, found: 173.1290

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1-Benzyl 2-(4-nitrophenyl) 1-methylhydrazine-1,2-dicarboxylate (4a): 4a was obtained as a yellow
oil (886.3 mg, 2.57 mmol, 93%) following the general procedure A1 from 3b (500 mg, 2.77 mmol). Rf
1
= 0.40 (cꢀHex /EtOAc 6:4); H NMR (300 MHz,CDCl3): δ = 8.23 (2H, d, J = 7.9 Hz), 7.32 (8H, m
and bs), 5.21 (2H, s), 3.29 (3H, s); 13C NMR (75MHz, CDCl3): δ = 155.1, 145.2, 135.6, 128.6, 125.2,
121.9, 128.5, 128.1, 68.6, 38.1; HRMS (TOF ESI, ion polarity positive): m/z 368.0859 calcd for
[C16H15N3O6 +Na]+, found: 368.0852.
1-Benzyl 2-(4-nitrophenyl) 1-isopropylhydrazine-1,2-dicarboxylate (4b): 4b was obtained as a
colourless oil (133 mg, 0.35 mmol, 89%) following the general procedure A1 from 3c (0.1 g, 0.4
1
mmol, 1 eq). Rf = 0.2 (cꢀHex /EtOAc 8:2); H NMR (300MHz, CDCl3): δ = 8.23 (2H, m), 7.36 (7H,
m), 7.17 (1H, bp), 5.21 (2H, s), 4.53 (1H, m), 1.23 (6H, m); 13C NMR (75 MHz, CDCl3): δ = 128.8,
128.3, 125.2, 122.0, 121.8, 68.5, 19.8; HRMS (TOF ESI, ion polarity positive): m/z 396.1172 calcd
for [C18H19N3O6 +Na]+, found: 369.1165.
1-Allyl 2-(4-nitrophenyl) 1-isobutylhydrazine-1,2-dicarboxylate (4c): 4c was obtained as a
colourless oil (354 mg, 1.05 mmol, 73%) following the general procedure A1 from 3d (0.3 g, 1.44
mmol, 1 eq.). Rf = 0.1 (cꢀHex /EtOAc 8:2), 1H NMR (300 MHz, CDCl3): δ = 8.26 (2H, d, J = 9.0 Hz),
7.36 (2H, d, J = 8.7 Hz), 7.07 (1H, bp), 5.93 (1H, ddd, J = 22.3, 10.5, and 5.4 Hz), 5.29 (2H, dd, J =
25.1, 13.9 Hz), 4.67 (2H, d, J = 5.5 Hz), 3.42 (2H, d, J = 7.1 Hz), 1.68 (1H, m), 0.97 (6H, d, J = 6.7
Hz); 13C NMR (75 MHz, CDCl3): δ = 155.3, 140.9, 132.1, 125.4, 122.0, 118.4, 67.4, 58.0, 27.0, 20.1.
MS (ESI, ion polarity positive): m/z 697.26 calcd for [2xC15H19N3O6 +Na]+, found: 697.26.
Tert-butyl-2-(hydrazinecarbonyl)-2-isopropylhydrazine-1-carboxylate (5a): 5a was obtained as a
white powder (765 mg, 3.29 mmol, 86%) following the general procedure A2 from 2a (1.3 g, 3.83
mmol) and 3a (1.38 ml, 22.9 mmol) after purification by column chromatography on silica gel using
1
EtOAc/MeOH 95:5 as eluent. Rf = 0.3 (EtOAc/MeOH 96:4); H NMR (300 MHz, CDCl3): δ = 6.52
(1H, bs), 6.21 (1H, bs), 4.6 (1H, dt, J = 13.4 and 6.7 Hz), 3.01 (2H, bs), 1.47 (9H, s), 1.11 (6H, d, J =
6.3 Hz; 13C NMR (75 MHz, CDCl3): δ = 158.2, 158.1, 80.0, 49.3, 28.3, 19.4, 19.2,; HRMS (TOF ESI,
ion polarity positive): m/z 233.1614 calcd for [C9H20N4O3 +H]+, found: 233.1604.
Tert-butyl 2-(4-(((benzyloxy)carbonyl) amino)butyl)-2-(hydrazinecarbonyl)hydrazinecarboxylate
(5b): 5b was obtained as a white solid (130 mg, 0.33 mmol, 82%) following the general procedure A2
from 2b (200 mg, 0.4 mmol) and 3a (0.12 mL, 2.4 mmol) after purification by column
1
chromatography on silica gel using EtOAc/MeOH 9:1 as eluent. Rf = 0.60 (EtOAc/MeOH 9:1; H
NMR (300 MHz, Acetone–d6): δ = 8.52 (2H, bs); 7.31 (5H, m), 6.37 (1H, bs), 5.01 (2H, s), 3.43 (2H,
m), 3.12 (2H, s), 2.83 (2H, bs), 1.58 (4H, m), 1.44 (9H, s); 13C NMR (75 Hz, Acetone–d6): δ = 204.9,

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155.9, 155.4, 137.6, 128.2, 127.6, 80.5, 65.4, 47.3, 40.2, 27.5, 26.9, 24.4; HRMS (TOF ESI, ion
polarity positive): m/z 396.2247 calcd for [C18H29N5O5 +H]+, found: 396.2248.
Tert-butyl 2-isopropyl-2-(2-methylhydrazinecarbonyl) hydrazine carboxylate (5c): 8a (720 mg,
1.89 mmol, 1.0 eq.) was dissolved in MeOH (15 mL). At that time, Pd/C 10% (144 mg, 20% mass)
was added and the reaction mixture was kept under stirring overnight at room temperature under
hydrogen atmosphere. The reaction mixture was filtered through a pad of Celite and after washing the
pad of Celite several times with MeOH, the filtrate was evaporated under vacuum to afford compound
1
5c (441 mg, 1.79 mmol, 95%) as white solid. H NMR (300 MHz, CDCl3): δ = 6.79 (2H, bp), 4.57
(1H, dt, J = 13.4, 6.7 Hz), 4.35 (1H, bp), 2.58 (3H, s), 1.45 (9H, s), 1.09 (6H, s); 13C NMR (75 MHz,
CDCl3): δ = 158.2, 158.2, 81.9, 48.5, 39.7, 28.1, 19. 4, 19.2; HRMS (TOF ESI, ion polarity positive):
m/z 257.1770 calcd. for [C10H22N4O3 +H]+, found: 257.1775; m/z 269.1590 calcd. for [C10H22N4O3
+Na]+, found: 269.1602
Tert-butyl 2-benzyl-2-(2-isobutylhydrazine-1-carbonyl)hydrazine-1-carboxylate (5d): After
mixing Tetrakis (triphenylphosphine) palladium (0) (40 mg, 0.034 mmol, 0.05 eq.) and N,Nꢀ
dimethylbarbituric acid (323 mg, 2.07 mmol, 3 eq.) under argon atmosphere, 8c (290 mg, 0.69 mmol,
1.0 eq.) in DCM was added dropwise. After stirring 4h at 35°C, the volatiles were removed under
reduced pressure and the remaining oil was taken up in EtOAc. The organic layer was then
successively washed with aqueous solution of Na2CO3, dried over Na2SO4 and filtrated. The volatiles
were removed under reduced pressure and the crude material was purified by column chromatography
on silica gel using cꢀHex / EtOAc 6:4 as eluent to afford 5d (119 mg, 0.35 mmol, 51%) as a colourless
1
oil. Rf = 0.3 (cꢀHex /EtOAc 6:4); H NMR (300 MHz,CDCl3): δ = 7.30 (5H, m), 4.66 (2H, bs), 3.74
(3H, m), 3.01 (2H, d, J = 6.5 Hz), 2.05 (1H, m), 1.38 (9H, s), 1.02 (6H, d, J = 6.6 Hz); 13C NMR
(75MHz, CDCl3): δ = 129.3, 128.8, 128.2, 82.6, 59.4, 28.2, 24.8, 20.4; HRMS (TOF ESI, ion polarity
positive): m/z 337.2240 calcd for [C17H28N4O3 +H]+, found: 337.2244.
Phenyl N-[[(tert-butoxycarbonylamino)-isopropyl-carbamoyl]amino]carbamate (7a): 7a was
obtained as a white powder (995 mg, 2.83 mmol, 90%) following the general procedure A3 from 5a
(730 mg, 3.14 mmol) after purification by column chromatography on silica gel using cꢀHex /EtOAc
1
4:6 as eluent. Rf = 0.5 (cꢀHex /EtOAc 4:6); H NMR (300 MHz,CDCl3): δ = 7.33 (2H, m), 7.18 (4H,
m), 6.98 (1H, s), 6.54 (1H, bs), 4.61 (1H, dt, J = 13.5 and 6.9 Hz), 1.48 (9H, s), 1.14 (6H, d, J = 6.3
Hz); 13C NMR (75MHz, CDCl3): δ = 157.3, 155.7, 150.8, 129.5, 125.8, 121.6, 82.4, 49.5, 28.2, 19.2,
19.6; HRMS (TOF ESI, ion polarity positive): m/z 353.1825 calcd for [C16H24N4O5 +H]+, found:
353.1816, m/z 375.1644 calcd for [C16H24N4O5 +Na]+, found: 375,1649.

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Phenyl-5-((tert-butoxycarbonyl) amino)-4,11-dioxo-13-phenyl-12-oxa-2,3,5,10-tetraazatridecan-
1-oate (7b): 7b was obtained as white solid (158 mg, 0.31 mmol, 96%) following the general
procedure A3 from 5b (128 mg, 0.32 mmol) after purification by column chromatography on silica gel
1
using EtOAc/ cꢀHex 6:4 as eluent. Rf = 0.35 (EtOAc/ cꢀHex 6:4); H NMR (300 MHz, CDCl3): δ =
7.48 (2H, bs),7.26 (10H, m), 5.31 (1H, bs), 5.08 (2H, s), 3.68 (2H, m), 3.14 (2H, s), 2.53 (1H, bs), 1.33
(13H, s); 13C NMR (75 MHz, CDCl3): δ = 157.9, 156.8, 155.7, 154.8, 150.6, 136.6, 129.3, 128.4,
128.0, 125.7, 121.4, 82.4, 65.6, 48.3, 40.6, 28.1, 26.7, 23.9 ; HRMS (TOF ESI, ion polarity positive):
m/z 538.2278 calcd for [C25H33N5O7 +Na]+: M/z 538.2278, found: 538.2277.
Benzyl 2-(2-(tert-butoxycarbonyl)-1-isopropylhydrazinecarbonyl)-1-methylhydrazinecarbo-
xylate (8a): 8a was obtained as white solid (777,5 mg, 2.04 mmol, 88%) following the general
procedure B1 from 1a (404 mg, 2.32 mmol) and 4a (800 mg, 2.32 mmol) after purification by column
1
chromatography on silica gel using EtOAc/ cꢀHex 1:1 as eluent. Rf = 0.55 (EtOAc/ cꢀHex 1:1); H
NMR (300 MHz, CDCl3): δ = 7.33 (5H, s), 6.58 (1H, bs), 6.12 (1H, bs),5.15 (2H, s), 4.58 (1H, dt, J =
13.3, 6.6 Hz), 3.21 (3H, s), 1.47 (9H, s), 1.13 (6H, s); 13C NMR (75 MHz, CDCl3): δ = 163.3, 157.1,
155.3, 136.0, 128.5, 128.1, 127.9, 82.2, 68.1, 48.9, 38.5, 28.1, 19.5, 19.1; HRMS (TOF ESI, ion
polarity positive): m/z 403.1957 calcd for [C18H28N4O5 +Na]+, found: 403.1958.
Benzyl 2-(2-(tert-butoxy carbonyl)-1-isobutyl hydrazine-1-carbonyl)-1-isopropyl hydrazine
carboxylate (8b): 8b was obtained as a colourless oil (62 mg, 0.02 mmol, 28%) following the general
procedure B1 from 1c (0.126 g, 0.67 mmol) and 4b (0.25 g, 0.67 mmol) after purification by column
1
chromatography on silica gel using cꢀHex /EtOAc7:3 as eluent. Rf = 0.4 (EtOAc 100%); H NMR
(400 MHz, CDCl3): δ = 7.31 (5H, m), 5.12 (2H, s, H12), 5.12 (2H, s); 4.40 (1H, m), 3.31 (2H, m),
1.86 (1H, m), 1.48 (9H, s), 1.14 (6H, d, J = 6.4 Hz), 0.88 (6H, m); 13C NMR (100 MHz, CDCl3): δ =
158.2, 156.1, 154.4, 136.6, 128.5, 128.0, 127.7, 82.1, 67.7, 56.1, 50.4, 29.6, 28.1, 26.4, 20.0; HRMS
(TOF ESI, ion polarity positive): m/z 423.2607 calcd. for [C21H34N4O5 +H]+, found: 423.2613, m/z
440.2873 calcd. for [C21H34N4O5 +NH4]+, found: 440.2871, m/z 445.2427 calcd. for [C21H34N4O5
+Na]+: found: 445.2421.
Allyl 2-(1-benzyl-(2-(tert-butoxy carbonyl) hydrazine-1-carbonyl)-1-isobutyl hydrazine-1-
carboxylate (8c): 8c was obtained as a colourless oil (94 mg, 0.02 mmol, 62%) following the general
procedure B1 from 1d (80 mg, 0.36 mmol,) and 4c (121 mg, 0.36 mmol) after purification by column
chromatography on silica gel using cꢀHex /EtOAc7:3 as eluent. Rf = 0.2 (cꢀHex /EtOAc 7:3); 1H NMR
(400 MHz, CDCl3): δ = 7.34 (5H, m), 5.90 (1H, ddd, J = 22.3, 10.5, 5.4 Hz); 5.31 (1H, d, J = 17.1
Hz), 5.21 (1H, d, J = 10.3 Hz), 4.61 (2H, m), 3.39 (2H, d, J = 7.0 Hz), 1.93 (1H, m), 1.48 (9H, s), 0.92
(6H, d, J = 6.4 Hz); 13C NMR (100 MHz, CDCl3): δ = 156.5, 154.1, 138.4, 135.0, 132.5, 129.0, 118.0,

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82.1, 67.0, 57.9, 51.1, 28.3, 26.9, 20.1; HRMS (TOF ESI, ion polarity positive): m/z 443.2267 calcd.
for [C21H32N4O5 +Na]+, found: 443.2270.
Strategy C:
(S)-tert-butyl-2-((3-(1H-imidazol-5-yl)-1-methoxy-1-oxopropan-2-yl) carbamoyl)-2-isopro-
pylhydrazinecarboxylate (12): To a solution of histidineꢀmethyl ester (0.50 g, 2.07 mmol, 1.0 eq.) in
◦
10 mL of dry DMF under argon atmosphere was added at 0 C, pyridine (0.54 mL, 6.60 mmol, 3.2
eq.). The mixture was stirred 30 min at 0 °C and at this time, 4ꢀnitrophenyl chloroformate (0.46 g,
2.28 mmol, 1.1 eq.) in 3 mL of dry DMF was added dropwise. After 5 hours stirring at 40 °C, the
reaction was stopped having checked completion by TLC (8:2 EtOAc/MeOH + 0.2
NH4OH20%Aq.). At this time, DMAP (0.25 g, 2.07 mmol, 1.0 eq.) was added to the previous mixture
cooling to 0 °C which became immediately yellow. After stirring for 10 min, 1a (0.36 g, 2.07 mmol,
1.0 eq.) in dry DMF was added slowly to the reaction and let stirring for 4 days at room temperature
under argon atmosphere. After evaporation of the volatiles under reduced pressure, the crude residue
was purified by column chromatography on silica gel using first EtOAc 100% then EtOAc/MeOH 8:2
as eluent to give 12 (381 mg, 1.03 mmol, 49%) as a white powder.
Rf = 0.44 (EtOAc/MeOH/NH4OH20%Aq 8:1.8:0.2); 1H NMR (400 MHz, CD3OD): δ = 7.58 (1H, s), 6.62
(1H, s), 4.48 (2H, m), 3.68 (3H, s), 3.06 (2H, m), 1.47 (9H, s), 1.04 (6H, m); 13C NMR (100 MHz,
CD3OD): δ = 181.2, 173.6, 159.1, 136.2, 118.7, 118.2, 82.2, 54. 8, 52.8, 49.7, 30.6, 28.5, 19.9; HRMS
(TOF ESI, ion polarity positive): m/z 370.2090 calcd. for [C16H27N5O5 +H]+, found: 370.2092.
(1-amino-1-oxo-3-phenylpropan-2-yl)-2-(1-isopropylhydrazine-1-carbonyl)hydrazine-1-car-
boxamide (I): I was obtained as a white powder (850 mg, 2.01 mmol, 86%) following the general
procedure A4 from 7a (823 mg, 2.34 mmol) and LꢀPheNH2 hydrochloride (474 mg, 2.36 mmol) after
purification by column chromatography on silica gel using EtOAc/ cꢀHex01 1:1 as eluent. Rf = 0.2
1
(EtOAc/MeOH 95:5); mp = 130–132 °C; H NMR (400 MHz, DMFꢀd7): 2 conformers, δ = 9.16
(0.5H, bs), 9.03 (1H, bs), 8.82 (0.5H, bs), 7.66 (2H, bs), 7.36ꢀ7.22 (5H, m), 7.13 (1H, bs), 7.04 (1H,
bs), 6.50 (1H, d, J = 7.8 Hz), 4.52 (1H, m), 4.44 (0.5H, m), 4.34 (0.5H, m), 3.28 (0.5H, d, 13.1 Hz),
3.15 (0.5H, d, 13.1Hz), 2.92ꢀ2.81 (1H, m), 1.44 (9H, s),1.09 (6H, m); 13C NMR (100 MHz, DMFꢀd7):
δ = 174.1, 158.9, 158.5, 156.2, 138.7, 129.2, 127.9, 125.9, 79.9, 55.1, 48.5, 37.6, 27.5, 18.6, 19.2; IR:
3255 (ν N–H); 1659 (ν C=O); 1532 (ν C=C); 1155 (ν C–O) cm−1. HRMS (TOF ESI, ion polarity
positive): m/z 423.2346 calcd for [C19H30N6O5 +H]+, found: 423.2346, m/z 445.2175 calcd for
[C19H30N6O5 +Na]+, found: 445.2169. HPLC purity (XBridge C18, 3.5µm, H2O + 0.1% form.
ac./ACN, gradient 5–100% in 20 min): TR =12.97 min, 100%.

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(S)-tert-butyl2-(2-((1-amino-1-oxo-3-phenylpropan-2-yl)carbamoyl)hydrazinecarbonyl)-2-(4-
(((benzyloxy)carbonyl)amino)butyl)hydrazinecarboxylate (II): II was obtained as a white solid
(143 mg, 0.25 mmol, 35%) following the general procedure A4 from 7b (360 mg, 0.7 mmol) and Lꢀ
PheꢀNH2 hydrochloride (160.5 mg, 0.8 mmol) after purification by column chromatography on silica
gel using EtOAc /MeOH 95:5 as eluent. Rf = 0.70 (EtOAc/MeOH 95:5); mp = 136–138 ° C ;.1H
NMR (400 MHz, DMSOꢀd6): δ = 9.14 (1H, bs), 8.65 (1H, bs), 7.75 (3H, bs), 7.35 (5H, m), 7.17ꢀ7.23
(5H, m), 7.10 (1H, s), 6.18 (1H, s), 5.01 (2H, m), 4.26 (1H, s), 3.35 (2H, m), 3.00 (2H, m), 2.76ꢀ3.06
(2H, m), 1.43 (2H, m), 1.42 (11H, m); 13C NMR (100 MHz, DMSOꢀd6): δ = 173.7, 158.6, 157.9,
156.1, 155.0, 138.1, 129.1, 128.0, 126.1, 137.3, 128.3, 127.7, 80.1, 65.1, 54.4, 47.8, 40.1, 37.8, 28.1,
26.6, 24.0; IR: 3292 (NꢀH stretch), 2935 (CꢀH stretch), 1661 (C=O stretch), 1524 (NꢀH bend), 1454ꢀ
1368 (C=C stretch and CꢀN stretch), 1249ꢀ1156 (CꢀC stretch) cm−1; Elemental analysis: Calcd. for
C28H39N7O7·0.75H2O: C 56.12, H 6.83, N 16.37; found: C 56.33, H 6.95, N 16.03. HRMS (TOF ESI,
ion polarity positive): m/z 608.2809 calcd for [C28H39N7O7 +Na]+, found: 608.2808. HPLC purity
(XBridge C18, 3.5 ꢁm, H2O + 0:1% form. ac./ACN, gradient 5–100% in 20 min): TR = 14.72 min,
98%.
(S)-tert-butyl 2-(2-((1-amino-3-methyl-1-oxobutan-2-yl)carbamoyl-2-methylhydrazinecarbonyl)-
2-isopropylhydrazinecarboxylate (III) : III was obtained as a white solid (95 mg, 0.24 mmol, 60%)
following the general procedure B2 from 6a (68.7 mg, 0.45 mmol) and 5c (100 mg, 0.41 mmol) after
purification by column chromatography on silica gel using EtOAc /MeOH 90:10 as eluent.) . Rf =
1
0.30 (EtOAc/MeOH 90:10); mp = 283–285 °C H NMR (400 MHz, DMSO–d6): δ = 9.14 (1H, bs),
9.01 and 8.92 (1H, 2s), 7.04 and 6.96 (2H, 2 bs), 6.05 (1H, bs), 4.36 (1H, m), 3.89 (1H, m), 2.90 (3H,
s), 2.02 (1H, m),1.44 (9H, s), 1.00 (6H, m), 0.84 (6H, m); 13C NMR (DMSO–d6, 100 MHz): δ =
173.8, 158.1, 157.8, 156.1, 80.0, 58.7, 48.4, 35.6, 29.6, 28.0, 19.9, 19.4, 18.8, 17.2 ppm; mp = 283–
285 °C; IR: 3417ꢀ3197 (NꢀH stretch); 2978 (CꢀH stretch); 1710ꢀ1653 (C=O stretch); 1533 (NꢀH bend);
1393ꢀ1333 (CH3 bend); 1275ꢀ1157 (CꢀN stretch) cm−1; Anal.calcd. for C16H32N6O5: C 49.47, H 8.30,
N 21.63, found: C 50.39, H 8.31, N 21.01; HRMS (TOF ESI, ion polarity positive): m/z 389.2512
calcd for [C16H32N6O5 +H]+, found: 389.2505. HPLC purity (XBridge C18, 3.5 ꢁm, H2O + 0:1% form.
ac./ACN, gradient 5–100% in 20 min): TR = 11.78 min, 100%.
Tert-butyl (S)-2-(2-(1H-imidazol-4-yl)-1-methoxy-1-oxopropan-2-yl)-carbamoyl)-2-isopro-
pylhydrazine-1-carbonyl)-2-isobutylhydrazine-1-carboxylate (IV) : To a solution of 12 (0.35 g,
0.95 mmol, 1.0 eq.) in 5 mL of dry dioxane under argon atmosphere, was added dropwise at 0 °C, HCl
4M in dioxane (4.80 mL, 19.0 mmol, 20 eq). After 3h at room temperature, the reaction was stopped
having checked completion by TLC (EtOAc/MeOH 8:2). After removing the volatiles under vacuum
and drying, the hydrochloride salt obtained as a white power was used immediately.

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To a solution of this salt (0.327 g, 0.96 mmol, 1.0 eq.) in dry DCM under argon atmosphere were
added successively DIPEA (0.45 mL, 2.57 mmol, 3.2 eq.) and DMAP (0.117 g, 0.96 mmol,1eq.). At
this time, a solution of bis(pentafluotophenyl)carbonate (0.416 g, 1.06 mmol, 1.1 eq.) in dry DCM was
added dropwise. The mixture was stirred 40 min at room temperature and then a solution of compound
1c (0.181 g, 0.96 mmol, 1.0 eq.) and DMAP (0.117 g, 0.96 mmol, 1.0 eq.) in dry DCM was added
dropwise to the previous solution. The mixture was let stirring for 4 days at room temperature under
argon atmosphere. After concentration of the compound under reduced pressure, the crude residue
was purified by column chromatography on silica gel eluting with EtOAc 100% to afford IV (247 mg,
0.51 mmol, 33%) as a colorless oil. δ = 8.09 (1H, bs); 7.67 (1H, bs), 7.25ꢀ7.15 (2H, m), 4.71 (1H, m),
4.64 (1H, m), 3.71 (3H, s), 3.48 (1H, m), 3.40 (1H, m), 3.06 (2H, d, J=5.6 Hz), 2.02 (1H, m), 1.36
(9H, s), 1.07 (6H, d, J = 6.7 Hz), 0.98 (6H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3): δ = 173.2,
171.2, 158.7, 158.6, 137.6, 135.4, 116.3, 83.1, 58.5, 53.0, 52.4, 45.7, 30.8, 28.2, 26.2, 20.3, 18.6; IR:
3233 (NꢀH stretch); 2964 (CꢀH stretch); 1732ꢀ1647 (C=O stretch); 1521 (NꢀH bend); 1438ꢀ1369 (CH3
bend); 1260ꢀ1148 (CꢀN stretch) cm−1 HRMS (TOF ESI, ion polarity positive): m/z 484.2884 calcd for
[C21H37N7O6 +H]+, found: 484.2892. HPLC purity (XBridge C18, 3.5µm, H2O + 0.1% form. ac./ACN,
gradient 5–100% in 20 min): tR = 13.89 min, 100%.
Tert-butyl
(S)-2-benzyl-2-(2-isobutyl-2-((1-methoxy-3-methyl-1-oxobutan-2-yl)
carbamoyl)
hydrazine-1-carbonyl) hydrazine-1-carboxylate (V): To a solution of 6b (42 mg, 0.25 mmol, 1.0
◦
eq.) in dry DMF under argon atmosphere was added at 0 C pyridine (80 µL, 1.0 mmol, 4.0 eq)
followed by 4ꢀnitrophenyl chloroformate (55 mg, 0.28 mmol, 1.1 eq.). After stirring overnight at room
temperature, 5d (85 mg, 0.25 mmol, 1 eq.) and DMAP (30 mg, 0.25 mmol, 1 eq.) in dry DMF were
immediately added dropwise together to the previous mixture and the reaction was kept on stirring
overnight at room temperature. After evaporation of the volatiles under reduced pressure, the crude
material obtained was purified by column chromatography on silica gel eluting with EtOAc/ cꢀHex
4:6. to yield V as a colorless oil (43 mg, 0.09 mmol, 35%).
1
Rf = 0.4 (EtOAc/CyH 4:6); H NMR (300 MHz, CDCl3): δ = 7.38ꢀ7.25 (6H, m), 6.22 (1H, s), 5.93
(1H, bs), 4.39 (1H, dd, J = 8.2, 5.4 Hz), 3.69 (3H, s), 3.57 (1H, m), 3.25 (1H, m), 2.13 (1H, m), 1.84
(1H, m), 1.45 (9H, s), 0.92 (12H, m); 13C NMR (75 MHz, CDCl3): δ = 173.6, 158.3, 156.1, 154.5,
135.3, 129.2, 129.0, 128.3, 83.2, 58.8, 55.7, 52.0, 51.6, 31.3, 28.1, 26.9, 20.2, 19.1, 18.0; IR: 3264 (Nꢀ
H stretch); 2964 (CꢀH stretch); 1724ꢀ1648 (C=O stretch); 1520 (NꢀH bend); 1468ꢀ1337 (CH3 bend);
1254ꢀ1155 (CꢀN stretch) cm−1; HRMS (TOF ESI, ion polarity positive): m/z 494.2979 calcd for
[C24H39N5O6 +H]+, found: 494.2881, m/z 516.2798 calcd for [C24H39N5O6 +Na]+, found: 516.2800.
HPLC purity (XBridge C18, 3.5µm, H2O + 0.1% form. ac./ACN, gradient 5–100% in 20 min): tR =
17.81 min, 63%.
Acknowledgment

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DOI: 10.1039/C8NJ03635G
The Laboratory of Excellence LERMIT is thanked for financial support for F. Bizet (LERMIT is
supported by a Grant from ANR, ANRꢀ10ꢀLABXꢀ33). The Ministère de l’Enseignement Supérieur et
de la Recherche (MESR) is thanked for financial support for N. Tonali. The European Union is
thanked for funding the research training of Agostino Oliva (Master student from the Università degli
Studi di Milano) in the frame of the European student exchange Erasmus programme. Camille Dejean
and Karine Leblanc (BioCIS, UMR 8076) are thanked for their help with the NMR experiments and
the HPLC analysis respectively.
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Page 25 of 25
New Journal of Chemistry
Three synthetic routes are studied and compared to introduce into peptides two consecutive aza-amino
acids bearing various side chains.
NHCbz
O
O
H
N
H
N
NH2
N
N
NH2
BocHN
BocHN
N
H
N
H
BocHN
BocHN
N
N
H
H
O
O
O
O
N
NH
O
O
H
N
H
N
NH2
N
N
O
N
N
H
N
N
H
O
O
O
O
O
O
H
H
H
N
N
N
NH2
N
O
BocHN
N
N
BocHN
N
N
H
H
H
O
O
O
O