Solid-phase synthesis of hydrazinopeptides in Boc and Fmoc strategies monitored by HR-MAS NMR

Article abstract of DOI:10.1016/j.tet.2007.07.038

With the aim of finding good conditions to incorporate hydrazino moieties into peptidic sequences of biological interest, we developed two strategies towards solid-phase synthesis of hydrazinopeptides: a stepwise methodology in which a hydrazinoacid was introduced as a normal aminoacid and a semi-convergent one in which the hydrazino moiety is incorporated as a pseudodipeptide building block. HR-MAS NMR analyses were run in order to monitor the reaction and to improve the reaction conditions.

Full text of DOI:10.1016/j.tet.2007.07.038

Tetrahedron 63 (2007) 9635–9641
Solid-phase synthesis of hydrazinopeptides in Boc and Fmoc
strategies monitored by HR-MAS NMR
*
ꢀ
Isabelle Bouillon, Regis Vanderesse, Nicolas Brosse, Olivier Fabre and Brigitte Jamart-Gregoire
ꢀ
´
´
Laboratoire de Chimie Physique Macromoleculaire, UMR CNRS-INPL 7568, Nancy-Universite,
ENSIC 1, rue grandville BP 451, 54001 Nancy, France
Received 5 June 2007; revised 12 July 2007; accepted 12 July 2007
Available online 19 July 2007
Abstract—With the aim of finding good conditions to incorporate hydrazino moieties into peptidic sequences of biological interest, we
developed two strategies towards solid-phase synthesis of hydrazinopeptides: a stepwise methodology in which a hydrazinoacid was intro-
duced as a normal aminoacid and a semi-convergent one in which the hydrazino moiety is incorporated as a pseudodipeptide building block.
HR-MAS NMR analyses were run in order to monitor the reaction and to improve the reaction conditions.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
such as diketopiperazine analogues or oligomers.4 Unfortu-
nately, very few methods allow the synthesis of orthogonally
protected hydrazinoacid derivatives with good yield.
The replacement of an amide bond with that of a hydrazide
bond in a peptide chain leads to the formation of novel com-
pounds called hydrazinopeptides. It has been demonstrated
that the presence of hydrazide bonds in a peptide chain can
induce a specific folding conformation called a hydrazino-
turn.1 Moreover, Lelais and seebach demonstrated that hy-
drazinomers were very resistant to protease activity.2 Both
remarks allow consideration of the use of these pseudopep-
tides to mimic endogenous peptides in biological processes.
In fact, only a few hydrazinopeptides have been described
in the literature; Niedrich et al. synthesized nine analogues
of eledoisin,3 an octapeptide involved in vasodilatation pro-
cesses and Guy et al. described some human leukocyte elas-
tase (HLE) inhibitors containing hydrazino moiety.4 In both
cases, the overall yields varied from low to moderate. More
recently, Lelais et al. synthesized a hexahydrazinopeptide in
solution with an overall yield of 14%.2
In previous papers,7 we showed that chiral orthogonally pro-
tected hydrazinoacid derivatives could be easily prepared via
a Mitsunobu reaction. More recently, we demonstrated that
Nb-Fmoc- and Nb-Boc-Na-Z-hydrazinoacid derivatives, di-
rectly suitable for SPPS, could be easily prepared in six steps
with good yields starting from the corresponding a-amino-
acids.7c The coupling reaction assays performed between
Na-Z-hydrazinoesters and N-Fmoc-a-aminoacids demon-
strated the low reactivity of a-hydrazinoester derivatives
when the Na is protected by a benzyloxycarbonyl group.
However, among the numerous coupling methods tested,
we found that the acid fluoride method allowed the forma-
tion of hydrazinodipeptides almost quantitatively. Consider-
ing that one of the goals of this study was to incorporate the
hydrazino moiety into sequences of biological interest, we
decided to find conditions, which allowed the synthesis of hy-
drazinopeptides on a solid-phase support. To date, only the H-
Asp-Tyr-Glyc[CO-NH-NH]Ile-Leu-Gln-Ile-Asn-Ser-Arg-OH
(hydrazide) decapeptide, an analogue of an HEL T-epitope,
has been prepared in poor yield by a solid-phase procedure,
using the step-by-step Boc/TFA/HF strategy.8 Bocc[CO-
NH-NH]Ile-OH was incorporated using DCC/HOBt, and
the coupling step was repeated three times with 3 equiv
each time. It is noteworthy that, due to the presence of the
unprotected a-nitrogen in the growing (hydrazide) peptide,
capping with Ac2O is precluded.
One of the main obstacles in the synthesis of hydrazinopep-
tides is regioselectivity. The presence of the two nitrogen
atoms makes regioselective acylation of a-hydrazinoacids
difficult to perform without protection. As an example, dur-
ing the study of the protection of the hydrazinoglycine ethyl
ester H2Nb-HNa-CH2-COOEt, Viret et al.5 and Lecoq and
Marraud6 demonstrated that the formation of mono- and
bis-acylated products strongly depends on the nature of the
reactants and the procedure used. Furthermore, coupling of
unprotected hydrazinoacids can give rise to byproducts
We describe in this paper, the comparative study of two
original protocols. The first one, a step-by-step synthesis,
Keywords: Hydrazinoacids; HR-MAS NMR; SPPS; Magic mixture.
* Corresponding author. E-mail: brigitte.jamart@ensic.inpl-nancy.fr
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.07.038

9636
I. Bouillon et al. / Tetrahedron 63 (2007) 9635–9641
O
Z
N
Z
Z
leads us to use HR-MAS NMR (High-Resolution Magic
Angle Spinning) analysis to monitor each step of the synthe-
sis of 5. This NMR technique allows spectral analysis of
peptides that are still linked to their resin support in such a
way that the completion of each coupling and deprotect-
ing steps can be accurately and easily observed. Using
this NMR technique, we were able to demonstrate that the
replacement of NaHCO3/DCM mixture by DIEA/DMF al-
lowed the coupling of Fmoc-Pro-F. It is interesting to note
that during all these assays, an N-Fmoc protected proline
was used instead of a Boc one. The reasoning behind this
is due to the fact that the Fmoc group is more accurately
identified by NMR spectra than the Boc group. This allows
more precise estimation of the yield in the last coupling re-
action by calculation of the ratio between the signals of the
g(CH3)Leu and those of the Fmoc group. After one coupling
run, the incorporation of the proline aminoacid was esti-
mated to 35% yield (Fig. 2). This result confirmed the low
reactivity of the Nb-position and that specific conditions
must be found to increase the yield of hydrazinopeptide
synthesis on solid phase.
FmocHN
CO2H
CH3
N
CO2H
N
CO2H
CH
N
H
BocHN
FmocHN
R
3
1 R = iBu : Boc-h(Z)Leu-OH
2 R = Me: Boc-h(Z)Ala-OH
4 Fmoc-Val-h(Z)Ala-OH
3 Fmoc-h(Z)Ala-OH
Figure 1. Boc- and Fmoc-hydrazinoacid 1–3 and building block 4 used in
this work.
consisting of the incorporation of Na,Nb-protected hydr-
azinoacids 1–3 (Fig. 1) using a classical coupling reaction
generally used for peptide synthesis. In the second one, pre-
formed hydrazinodipeptides 4 (Fig. 1), previously descri-
bed7c are incorporated as building blocks in a peptide chain.
2. Results and discussion
2.1. Step-by-step synthesis
To define the optimal conditions, we decided to synthesize
the pseudotripeptide H-Pro-hLeu-Ala-OH (5) on a Boc-Ala-
PAM resin using a classical Boc strategy. The formation of
the amide bond Leu-Ala was performed by using the classi-
cal TBTU/HOBt/DIEA mixture. As expected, the formation
of the hydrazide bond was more delicate. When using the
acid fluoride method in a NaHCO3/DCM mixture, we did
not observe any formation of the desired product. The only
product obtained was the pseudodipeptide H-h(Z)Leu-Ala-
OH. This result clearly demonstrates that the acid fluoride
coupling method found to be the best in liquid phase proto-
col7c is ineffective on solid support. Furthermore, we noticed
that the low reactivity of the Nb renders the qualitative col-
orimetric tests (ninhydrin, TNBSA) useless. This remark
After this encouraging result, we considered the incorpora-
tion of a hydrazinoacid in a longer sequence to evaluate the
impact of this insertion on the further steps of the synthesis.
We decided to synthesize the PFVhAL sequence (6) for two
main reasons. (1) This pseudopeptide, easily discernible
by HR-MAS NMR analyses, does not have any functional-
ized lateral chain, which necessitates a protective group.
(2) This sequence involved the coupling reaction between
a hindered aminoacid, valine, on the unreactive Nb of the
hydrazinoalanine moiety. First, an assay was run via the
Boc strategy on a Boc-Leu-PAM resin as described in
Scheme 1.
CH2Ph (Z)
Resin
CH2-CH (hLeu)
CH3 (Ala)
CH (Fmoc)
+ CH2 (Fmoc)
(CH3)2 (hLeu)
CHα (hLeu)
Arom. Fmoc
CH2Ph (Z)
Pro
CHα Ala
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
(ppm)
Figure 2. HR-MAS spectrum of Fmoc-Pro-hLeu-Ala-PAM resin.

I. Bouillon et al. / Tetrahedron 63 (2007) 9635–9641
9637
Phe
Val
Pro
h(Z)Ala
Leu
a
b
Boc
H
1
2
Z
Z
BocNH
BocNH
N
OH
a
c
N
Z
Boc
Boc
F
H2N N
3
4
a
b
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHNH]
H
OH
5
6
Boc
Boc
a
b
7
Boc
Boc
OH
H
a
8
9
d
H
H
OH
a : 25%TFA/DCM b : TBTU / HOBt / DIEA 3eq c : DCM / DMF / DIEA 3eq d : TFA/ TFMSA
Scheme 1. Synthetic procedure for the preparation of PFVhAL in Boc strategy.
The coupling and deprotection steps were monitored by HR-
MAS by visualizing the presence of the Boc signal (Fig. 3).
In this assay, we replaced DMF by a 50/50 DCM/DMF mix-
ture for the coupling of fluoride to ensure a better swelling of
the resin.
(iii) After cleavage of the pseudopeptide from the resin and
HPLC purification, we observed as side products, some de-
leted peptides particularly the H-Pro-Phe-Val-OH tripeptide.
The absence of the hAla-Leu sequence could be explained
by a cyclization step occurring after the removal of the Boc
group from the hAla residue resulting in the formation of
the perhydrotriazepine 8. Such cyclization can be compared
to those leading to the well known diketopiperazine9
(Scheme 2). Unfortunately, we were not able to isolate this
compound.
The second spectrum in Figure 3 shows the simultaneous
presence of the Z (at 5.2 ppm) and the Boc (at 1.5 ppm)
groups, which confirms the linkage of the modified amino-
acid Boc-h(Z)Ala-OH (2). Every coupling (spectra 2, 4, 6, 8)
and deprotection (spectra 3, 5, 7, 9) steps can be qualitatively
verified, respectively, by checking the presence or the ab-
sence of the Boc signal.
Z
N
Z
N
O
HN
O
O
HO
+
During these assays, some important information arose. (i)
To reach a reasonable incorporation level of Boc-Val-F into
the sequence, four successive couplings were required. (ii)
Surprisingly, after the formation of the hydrazide bond, the
further deprotection and coupling steps were problematic.
H2N
N
H
NH
O
O
7
8
Scheme 2. Formation of perhydrotriazepane 8.
Boc
CH2 (Z)
Spectrum N°
1
Boc
2
3
4
Boc
5
Boc
6
7
Boc
8
9
ppm
9
8
7
6
5
4
3
2
1
Figure 3. HR-MAS monitoring during synthesis of PFVhAL on a PAM resin.

9638
I. Bouillon et al. / Tetrahedron 63 (2007) 9635–9641
Pro
Phe
Val
h(Z)Ala
OH
H
Leu
Z
Z
a
FmocNH
FmocNH
N
N
1
2
b
Z
c
Fmoc
Fmoc
H2N N
F
3
4
5
6
7
8
9
b
a
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
Fmoc
Fmoc
H
OH
b
a
Fmoc
Fmoc
H
OH H
b
d
H
OH
a : TBTU / HOBt / DIEA 3eq b : 25% piperidine / DMF c : DCM / DMF / DIEA 3eq d : 5% TFA/ DCM
Scheme 3. Synthetic procedure for the preparation of PFVhAL in Fmoc strategy.
To circumvent the postulated formation of perhydrotriaze-
pine, we decided to perform the synthesis on a more hindered
2-chlorotrityl resin as recommended for preventing the for-
mation of diketopiperazine. This kind of resin necessitated
the use of the Fmoc strategy described in Scheme 3. Due to
the mild conditions used for the cleavage of the peptide from
the resin, the protected PFVh(Z)AL pseudopeptide (9) is
expected as the final product.
NMR signal corresponding to the Fmoc protecting group.
The PFVh(Z)AL was obtained after purification with
a moderate yield of 13%. Keeping in mind that hydrazide
bond can lead to a specific structuration called hydrazino-
turn,1 we suspected the structuration of the growing pseudo-
peptide to be responsible for the poor yield by decreasing the
accessibility of the reactants. So, we decided to use magic
mixtures10 as solvent, which were known to have a destruc-
turant effect on peptides. The above synthesis procedure was
repeated by using a mixture of DMF/DCM/NMP (33/33/33,
v/v/v) for the coupling steps and piperidine/DMF/NMP/tol-
uene (25/25/25/25, v/v/v/v) for the deprotection of the Fmoc
group. With the help of these solvent mixtures, the overall
yield of pure pseudopentapeptide rose to 27%. Surprisingly,
the addition of 1% Triton X-10010 results in a dramatic de-
crease in the yield. Aiming to study the influence of the steric
hindrance of the different amino or hydrazinoacids, we
underwent the synthesis of PFh(Z)AVL (10) in place of
PFVh(Z)AL (9) and we obtained the pure pseudopeptide in
36% yield. This result confirms that the coupling reaction
between the hydrazine moiety and an aminoacid is strongly
dependent on the steric hindrance of the side chain of the
This synthesis was monitored by HR-MAS and the spectra
are gathered in Figure 4. The simultaneous presence of the
Z (at 5.2 ppm) and of the Fmoc (at 7.6 and 7.8 ppm) signals
on the spectrum 2 (Fig. 4) confirms the linkage of the hydr-
azinoacid Fmoc-h(Z)Ala-OH (3) on the H-Leu-2-chlorotrityl
resin. The efficiency of every coupling and deprotection
reactions can be monitored by HR-MAS by visualizing the
presence or the absence of the signal of the Fmoc group
(Fig. 4).
HR-MAS analysis demonstrated that the incorporation of
the Fmoc-Val-F was delicate to perform and necessitated
three successive coupling reactions in order to observe an
CH2(Z)
Spectrum N°
Fmoc
1
2
3
4
5
6
7
8
9
ppm
9
8
7
6
5
4
3
2
1
Figure 4. HR-MAS monitoring during synthesis of PFVhAL on a 2-chlorotrityl resin.

I. Bouillon et al. / Tetrahedron 63 (2007) 9635–9641
9639
Val
Pro
Phe
h(Z)Ala
Leu
c
Z
a
N
Fmoc
Fmoc
Fmoc
Fmoc
H
F
H2N
OMe
OMe
OH
b
ψ[CONHN(Z)]
ψ[CONHN(Z)]
H
d
c
ψ[CONHN(Z)]
ψ[CONHN(Z)]
Fmoc
Fmoc
OH
d
c
ψ[CONHN(Z)]
ψ[CONHN(Z)]
H
Fmoc
Fmoc
H
OH
d
ψ[CONHN(Z)]
ψ[CONHN(Z)]
ψ[CONHN(Z)]
e
H
OH
a : NaHCO3-DCM b: NaOH 12 eq in 0.8 M CaCl2 iPrOH/H2O (7/3)
c : TBTU / HOBt / DIEA 3eq DMF or MM d : 25% piperidine DMF or MM
e : 5% TFA/DCM
Scheme 4. Synthetic procedure for the preparation of PFVhAL (9) by semi-convergent methodology.
aminoacid. Considering that the deprotection of the Fmoc
group in our case is a limiting step, this step-by-step strategy
comprises 10 steps, with an average yield of 88% per step for
PFVh(Z)AL (9) and 90% per step for PFh(Z)AVL (10).
purification. Reactions were monitored by thin-layer chro-
matography (TLC) using aluminium-backed silica gel plates
(Macherey–Nagel ALUGRAMÒ SIL G/UV254). TLC spots
were viewed under ultraviolet light and by heating the plate
after treatment with a staining solution of phosphomolybdic
acid. Product purifications were performed using Geduran
60 H Silica Gel (63–200 mesh). Reagent grade solvents
were used as received. The Boc-Ala-Merrifield resin, Boc-
Leu-PAM resin, H-Leu-chlorotrityl resin, Boc-aminoacids,
Fmoc-aminoacids, 1-hydroxybenzotriazole (HOBt), O-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium tetrafluoro-
borate (TBTU) and diisopropylethylamine (DIEA) were
purchased from Senn Chemicals or Novabiochem. Boc-h(Z)-
Ala-OH and Fmoc-h(Z)Ala-OH were prepared as previously
described in our laboratory.7c Solid-phase hydrazinopeptide
syntheses were performed on a multichannel peptide synthe-
sizer PSP 400011 according to a classical Fmoc/tBu and Boc
methodology. The peptides were purified by reverse phase
high-performance liquid chromatography (HPLC) using a
preparative HPLC system (Waters Corp., Milford, MA,
USA) on an Interchrom UP5 ODB.25 M Uptisphere 5 mm
column (250ꢀ10 mm). Peptides were eluted with a gradient
of solution A (water containing 0.1% of TFA) and solution B
(20% of water in acetonitrile with 0.1% of TFA). The purity
of the peptide was checked by analytical HPLC, which was
run on a Merck apparatus (Darmstadt, Germany) using an
Interchrom UP10 ODB.25 K Uptisphere 5 mm column
2.2. Semi-convergent synthesis
Hydrazinopeptides could be obtained using another strategy
based on the incorporation of a preformed synthon of general
formula Fmoc-Xaa-h(Z)Xbb-OH into the growing pseudo-
peptide. As we previously described, these building blocks
can be easily synthesized in liquid phase.7c To test the
efficiency of this strategy, the synthesis of 9 was run on a
Leu-Wang resin as depicted in Scheme 4.
The building block Fmoc-Val-(Z)Ala-OH (4) can be an-
chored by using a classical coupling method. When DMF
was used as solvent, an overall 32% yield was obtained while
the use of magic mixtures led to 46% (regardless of the resin
substitution) of pure pseudopentapeptide (9). This overall
yield corresponds to an average yield of 87% per step.
3. Conclusion
We demonstrated that the incorporation of hydrazino bond in
a growing peptide chain can be performed on solid phase by
a step-by-step strategy or by direct incorporation of a hydr-
azinodipeptides. With the help of HR-MAS NMR analyses,
we were able to follow the progression of the synthesis and
demonstrated that the use of magic solvents improves the
overall yield of the synthesis.
1
(250ꢀ4.6 mm). H and 13C NMR (300 MHz) spectra were
recorded on a Bruker Advancer 300. Multiplicities are re-
ported as follow: s¼singlet, t¼triplet, q¼quadruplet, m¼
multiplet. Electron Impact Mass Spectra were performed
on a ProMALDI/FTMS apparatus in the ‘Laboratoire de
ꢀ
ꢀ
Spectrometrie de Masse et de Chimie Laser—Universite
de Metz—France’. Electron spray ionization mass spectra
(ESI-MS) were recorded on an SCIEX API 150EX single
quadrupole spectrometer (Sciex, Toronto, Canada).
4. Experimental
4.1. General
4.2. HR-MAS NMR experiments
Tetrahydrofuran was dried by distillation over sodium ben-
zophenone ketyl. Unless otherwise stated, reagents were
purchased from chemical companies and used without prior
Resin was washed twicewith CH2Cl2 and filtered. Then 5 mg
of the resin was loaded in 4 mm rotor and swollen with

9640
I. Bouillon et al. / Tetrahedron 63 (2007) 9635–9641
Table 2. 1H NMR (DMSO-d6): d(ppm) PFVhAL (6)
CDCl3 in which tetramethylsilane (TMS) was used as
internal reference. All NMR experiments were performed
at 298 K on a 300 MHz spectrometer equipped with
NbH
NaH
a
b
g
d
1
Pro
Phe
Val
hAla
Leu
3.97
4.69
4.02
3.35
4.21
2.19
1.72
3.61
a 4 mm HR-MAS probe using a 2500 Hz spinning rate. H
NMR spectra were recorded with Carr–Purcell–Meiboom–
8.61
8.18
5.16
8.15
3.05, 2.80
1.82
1.11
0.81
1.56
Gill experiments (CPMG)12 with 64 scans.
9.47
1.65
0.85
4.3. Synthesis of the pseudotripeptide PhLA (5)
purified by HPLC with a gradient from 5 to 40% of solution B
for 25 min at a flow rate of 4 ml/min with UV detection at
230 nm. After removal of the solvents, the purified compound
was lyophilized and analyzed by mass spectrometry and
NMR. A single peptide peak was detected after HPLC and
mass spectrometry revealed a peptide mass of 560.16 (for
C28H45N6O6 [M+H]+), virtually identical to the calculated
The synthesis was performed using Boc-Ala-Merrifield resin
(0,7 mmol/g). Boc-h(Z)Leu-OH (1) (3 equiv) was activated
using HBTU/HOBT/DIEA (3 equiv, 3 equiv, 9 equiv) in
DMF. After the deprotection of the Boc group using 25%
TFA in DCM, Boc-Pro-F (3 equiv) was added with DIEA
(9 equiv) in DMF. HR-MAS spectrum was recorded. After
the N-terminal deprotection, the peptide resin was washed
twice with dichloromethane and dried under vacuum. A
standard cleavage with a mixture of 5% of TFMSA in tri-
fluoroacetic acid (TFA) for 2 h afforded the crude peptide,
which was lyophilized and purified by HPLC with a gradient
from 0 to 50% of solution B for 40 min at a flow rate of 2 ml/
min with UV detection at 254 nm. After removal of the sol-
vents, the purified compound was lyophilized and analyzed
by mass spectrometry and NMR. A single peptide peak was
detected after HPLC and mass spectrometry revealed a
peptide mass of 314.88 (for C14H27N4O4 [M+H]+), virtually
1
mass of 560.33. H NMR (1D, COSY and TOCSY) was in
agreement with the sequence of the peptide, see Table 2.
4.5. Synthesis of pseudopentapeptides PFVh(Z)AL (9)
and PFh(Z)AVL (10) in Fmoc strategy
The synthesis was performed using H-Leu-2-chlorotrityl
resin (0.99 mmol/g). The aminoacids were activated using
HBTU/HOBT/DIEA (3 equiv, 3 equiv, 3 equiv) in DMF
or in magic mixture DMF/DCM/NMP (33/33/33, v/v/v),
except for the acid fluoride, which was added with DIEA
(9 equiv) in DMF/DCM or in magic mixture. Each coupling
was tripled to prevent deletion. For the semi-convergent syn-
thesis, the coupling of the building block was achieved as for
a usual aminoacid. Typically, the coupling reactions were
complete within 6 h (three coupling reaction of 2 h). The de-
protection of the Fmoc group was achieved with 25% piper-
idine in DMF or in magic mixture piperidine/DMF/NMP/
toluene (25/25/25/25, v/v/v/v). Each deprotection was qua-
drupled. After N-terminal deprotection, the pseudopeptide
resin was washed twice with dichloromethane and dried un-
der vacuum. A standard cleavage with a mixture of 5% TFA
in DCM for 2 h afforded the crude peptide, which was lyoph-
ilized and purified by HPLC with a gradient from 5 to 100%
of solution for 35 min at a flow rate of 4 ml/min with UV de-
tection at 230 nm. After removal of the solvents, the purified
compound was lyophilized and analyzed by mass spectro-
metry and NMR.
1
identical to the calculated mass of 315.20. H NMR (1D,
COSY and TOCSY) was in agreement with the sequence of
the peptide, see Table 1.
Table 1. 1H NMR (DMSO-d6): d(ppm) PhLA (5)
NbH
NaH
a
b
g
d
Pro
h(Z)Leu
Ala
4.33
4.75
4.51
2.17
1.63
1.42
1.77
1.67
3.16
0.90, 0.87
8.67
8.03
7.91
4.4. Synthesis of the pseudopentapeptide PFVhAL (6)
in Boc strategy
The synthesis was performed using Boc-Leu-PAM resin
(0.7 mmol/g). The aminoacids were activated using HBTU/
HOBT/DIEA (3 equiv, 3 equiv, 9 equiv) in DMF except for
the acid fluoride, which was added with DIEA (9 equiv) in
DMF/DCM. Each coupling was tripled to prevent deletion.
Typically, the coupling reactions were complete within 6 h
(three coupling reaction of 2 h). The deprotection of the
Boc group was achieved with 25% TFA in DCM and each
deprotection was tripled. After N-terminal deprotection,
the pseudopeptide resin was washed twice with dichlorome-
thane and dried under vacuum. A standard cleavage with a
mixture of 5% of TFMSA in trifluoroacetic acid (TFA) for
2 h afforded the crude peptide, which was lyophilized and
For PFVh(Z)AL (9), mass spectrometry revealed a peptide
mass of 695.60 (for C36H51N6O8 [M+H]+), virtually identi-
cal to the calculated mass of 695.37. H NMR (1D, COSY
1
and TOCSY) was in agreement with the sequence of the
peptide, see Table 3.
For PFh(Z)AVL (10), mass spectrometry revealed a peptide
mass of 695.40 (for C36H51N6O8 [M+H]+), virtually identi-
cal to the calculated mass of 695.37. H NMR (1D, COSY
1
Table 3. 1H NMR (DMSO-d6): d(ppm) PFVh(Z)AL (9)
NbH
NaH
a
b
g
d
Pro
Phe
Val
h(Z)Ala
Leu
4.00
4.67
4.27
4.29, 4.46, 4.70
4.17
2.17
3.04
1.97
1.23
1.57
1.77
3.06
8.65
8.30
0.94, 0.80
1.50, 1.49
8.67
CH2(Z) 5.06, Ph(Z) 7.33
8.30
0.84, 0.76

I. Bouillon et al. / Tetrahedron 63 (2007) 9635–9641
Table 4. 1H NMR (DMSO-d6): d(ppm) PFh(Z)AVL (10)
9641
NbH
NaH
a
b
g
d
Pro
Phe
h(Z)Ala
Val
4.00
4.67
4.29, 4.46, 4.70
4.10
2.17
3.04
1.23
1.97
1.77
3.06
CH2(Z) 5.06, Ph(Z) 7.33
8.10
0.94, 0.80
(d) Niedrich, H. Chem. Ber. 1965, 98, 3451–3461; (e)
Niedrich, H. Chem. Ber. 1967, 100, 3273–3282; (f) Niedrich,
H. Chem. Ber. 1969, 102, 1557–1569; (g) Niedrich, H.;
Knobloch, W. J. Prakt. Chem. 1962, 17–27; (h) Niedrich, H.;
€
Koller, G. J. Prakt. Chem. 1974, 316, 729–740; (i) Niedrich,
H.; Pirrwitz, J. J. Prakt. Chem. 1972, 314, 735–750.
and TOCSY) was in agreement with the sequence of the
peptide, see Table 4.
Acknowledgements
We thank Graduiertenkolleg 532 for a grant and Research
National Agency (ANR) for financial support (N NT05_4_
42848).
4. Guy, L.; Vidal, J.; Collet, A. J. Med. Chem. 1998, 41, 4833–
4843.
5. Viret, J.; Collet, A.; Pichon-Pesme, V.; Aubry, A. New J. Chem.
1988, 12, 253–256.
6. Lecoq, A.; Marraud, M. Tetrahedron Lett. 1991, 24, 2765–
2768.
Supplementary data
ꢀ
7. (a) Bouillon, I.; Brosse, N.; Vanderesse, R.; Jamart-Gregoire,
B. Tetrahedron Lett. 2004, 45, 3569–3572; (b) Brosse, N.;
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2007.07.038.
ꢀ
Pinto, M.-F.; Jamart-Gregoire, B. Eur. J. Org. Chem. 2003,
4757–4764; (c) Bouillon, I.; Brosse, N.; Vanderesse, R.;
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