Synthesis and conformational preferences of unnatural tetrapeptides containing l-valine units

Article abstract of DOI:10.1016/j.tetasy.2006.12.006

The stereoselective synthesis of non-proteinogenic tetrapeptides 7, 16 and 17 containing two l-valine units and two modified α-amino acids (proline and aspartic acid) has been accomplished starting from the l-valine derived chiral synthon 1. Investigation

Full text of DOI:10.1016/j.tetasy.2006.12.006

Tetrahedron: Asymmetry 17 (2006) 3273–3281
Synthesis and conformational preferences of unnatural
tetrapeptides containing L-valine units
Daniele Balducci, Andrea Bottoni, Matteo Calvaresi, Gianni Porzi* and Sergio Sandri*
`
Dipartimento di Chimica ‘G.Ciamician’, Universita di Bologna, Via Selmi 2, 40126 Bologna, Italy
Received 31 October 2006; accepted 4 December 2006
Abstract—The stereoselective synthesis of non-proteinogenic tetrapeptides 7, 16 and 17 containing two L-valine units and two modified
a-amino acids (proline and aspartic acid) has been accomplished starting from the L-valine derived chiral synthon 1. Investigations of the
1
conformational preference and structure of these unnatural peptides were carried out using H NMR and IR spectroscopic techniques
and a conformational analysis based on quenched molecular dynamics (QMD).
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
retical investigation based on a ‘quenched molecular
dynamics’ (QMD) approach, has been carried out.
In a previous paper,1 we reported the stereoselective
synthesis of the unusual di- and tripeptides containing
the L-valine unit and a cyclic unnatural a-aminoacid. The
importance of replacing natural amino acids in peptides
with non-proteinogenic counterparts, in order to obtain
drug-like target molecules, has stimulated our interest to
undertake the synthesis of new and more complex unnatu-
ral peptides. In particular, we are interested in the synthesis
of peptidomimetic structures because they exhibit thera-
peutic effects which are similar to those of natural peptides
but with the advantage of metabolic stability. The strategy
followed to accomplish the asymmetric synthesis of the title
pseudotetrapeptide is identical to that previously acquired
on the stereoselective approach to unnatural peptides
containing the L-valine and proline1 or aspartic acid
derivatives.2 In this approach we started from a chiral
monolactim ether, easily obtained from L-valine.
2. Results and discussion
The stereoselective synthesis followed, makes use of chiral
synthon 2 obtained from (5S)-4-benzyl-6-ethoxy-5-isopro-
pyl-3-oxo-2,3,4,5-tetrahydro-pyrazine 1,
a monolactim
ether easily synthesized from L-valine, as described in our
previous papers.3 Chiral sinthon 2, a masked unnatural
cyclic dipeptide (constituted by L-valine and an aspartic
acid derivative), was reacted with another unnatural dipep-
tide 31,2 (containing L-valine and a proline derivative) to
obtain the more complex structure 4. After alkaline hydro-
lysis, acid derivative 5 was converted into amide 6 by
treating with cyclohexylamine in the presence of 4-
(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium
chloride (DMTMM), obtained starting from 2,4-dime-
thoxy-6-chloro-1,3,5-triazine.4
The non-proteinogenic peptides 6, 7, 14, 15, 16 and 17 (see
Schemes 1 and 2) have been examined using spectroscopic
techniques (1H NMR and IR) to understand if the cyclic
unnatural a-aminoacid (a proline derivative) behaves as a
scaffold, promoting a compact conformation and the
reverse turn formation through intra-molecular hydrogen
bonds, that is, a U-shaped structure. Furthermore, to ana-
lyze the conformational features of these peptides, a theo-
The acid hydrolysis under mild conditions of intermediate
6 and subsequent acylation with acetyl chloride allowed us
to obtain tetrapseudopeptide 7, containing L-valine (red),
modified proline (green) and modified aspartic acid (blue),
in a satisfactory overall yield.
In order to synthesize tetrapseudopeptides 16 and 17,
which differ from 7 by the substituent and the configuration
of the proline derivative, we employed pseudopeptides 8 or
9 already synthesized by us.1 In fact, chiral synthon 2 was
*
Corresponding authors. Fax: +39 (0)51 2099512 (S.S.); e-mail: gianni.
porzi@unibo.it
0957-4166/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.12.006

3274
D. Balducci et al. / Tetrahedron: Asymmetry 17 (2006) 3273–3281
O
OEt
OEt
HCl.NH2
N
N
i
N
+
N
CO2Pfp
N
H
COOEt
O
Ph
O
3
2
1
OEt
OEt
O
N
O
N
O
N
iii
COOR
N
O
N
HN
N
H
H2
N
O
N
H3
O
H1
4
5
R = Et
R = H
O
ii
6
O
O
N
O
H4
H2
N
O
H3
iv
N
EtO
N
O
N
H1
O
7
Scheme 1. Reagents and conditions: (i) CH2Cl2 in the presence of Et3N at rt; (ii) 2 M NaOH in EtOH; (iii) cyclohexylamine in THF in the presence of
DMTMM at rt; (iv) 0.5 M HCl, then CH3COCl in CH2Cl2/Et3N.
OEt
OEt
O
O
N
H2N
N
N
N
O
iii
i
+
COOR'
R
HN
N
CO2Pfp
N
H
H
R
COOEt
O
O
8
9
R = H
R = CH3
2
R' = Et, R = CH3
12
10 R'= Et, R= H
11 R' = R = H
ii
ii
13 R' = H, R = CH3
O
O
N
H2
OEt
O
H4
N
O
H3
O
N
N
EtO
R
iv
N
O
N
N
O
R
N
N
H2
O
N
H1
O
H3
H1
O
R = H
R = H
16
14
17 R = CH3
15 R = CH3
Scheme 2. Reagents and conditions: (i) in CH2Cl2 in the presence of Et3N, at rt; (ii) 2 M NaOH in EtOH; (iii) cyclohexylamine in THF in the presence of
DMTMM at rt; (iv) 0.5 M HCl, then CH3COCl in CH2Cl2/Et3N.

D. Balducci et al. / Tetrahedron: Asymmetry 17 (2006) 3273–3281
3275
1
matched with nucleophiles 8 or 9 giving compounds 10 or
12, respectively. These intermediates, submitted to the reac-
tions sequence above described to obtain compound 7,
gave tetrapseudopeptides 16 and 17 in satisfactory overall
yields (Scheme 2).
1 and 2). The H NMR spectra assignments of the H1,
H2, H3 and H4 protons (which are not magnetically equiv-
alent) were achieved on the basis of the signal multiplicity
as well as by irradiation and/or NOE measurements.
From the spectroscopic data reported in Table 1, it can be
deduced that in pseudopeptides 6 and 17, the H1 protons
most probably do not form hydrogen bonds because their
chemical shifts are <7 ppm and, more importantly, the sig-
nals are characterized by a significant downfield shift (0.89–
1.13 ppm) upon addition of 20% DMSO. Furthermore, the
IR spectra show sharp bands at m > 3400 cmꢀ1, that is, in
the characteristic region of free NH amide absorbance. In
our opinion also in 7, the H1 proton, in spite of the large
temperature coefficient (5.5 ppb), does not form hydrogen
bonds. This because a remarkable downfield shift (1 ppm)
upon addition of 20% DMSO and an IR band at
m = 3419 cmꢀ1 were observed. The dNH values of H1 in
14 and 15 in connection with the downfield shift upon addi-
tion of 20% DMSO (DdNH = 1.0, 0.7, 0.73 ppm) and the
absolute values of temperature coefficients (5.5, 6.4,
6.0 ppb/°C) are indicative of the existence of a dynamic
equilibrium between hydrogen-bonded and a non-hydro-
gen-bonded structure. On the basis of the chemical shift va-
lue, the downfield shift upon addition of 20% DMSO and
the absolute value of the temperature coefficient (5 ppb/
°C), we believed that also in 16, the H1 proton is involved
in a dynamic equilibrium between a hydrogen-bonded and
a non-hydrogen-bonded structure. However, the moderate
downfield shift upon addition of 20% DMSO (0.3 ppm),
suggests that H1 in 16 is probably involved in a hydrogen
bond, which is stronger than in 14 and 15.
1
3. H NMR and IR studies
In order to determine the structural features of tetra-
pseudopeptides 7, 16, 17 and their precursors 6, 14, 15,
spectroscopic studies using H NMR and IR techniques
1
were carried out.1,5,6 Information on the existence of the
hydrogen bonds was obtained from the chemical shift of
the amide NH bond, the value of the temperature coeffi-
cient (DdNH/DT) and the dNH change after addition of
DMSO, which is a strongly competitive solvent in hydro-
gen bond formation. Further information can be attained
from the infrared stretching absorption of the amide NH
bond. When the spectra are obtained in dilute solutions,
a broad band in the range 3300–3350 cmꢀ1 indicates the
presence of an intra-molecular hydrogen bond, while a
sharp band higher than 3400 cmꢀ1 can be assigned to a free
amide NH bond. In Table 1, we have collected the mean-
ingful 1H NMR and IR data of the various substrates
investigated in diluted solutions. For clarity their amide
protons are labelled as H1, H2, H3 and H4 (see Schemes
1
Table 1. Meaningful H NMR and IR data for substrates 6, 7, 14, 15, 16
and 17
dNH (ppm)
dNH (ppm)
jDdNH/DTj IR (cmꢀ1
)
(2 mM CDCl3) (2 mM CDCl3/ (ppb/°C)
(2 mM CHCl3)
DMSO 4:1)
(in CDCl3)
It is important to stress that the dNH value below 7 ppm,
registered in all substrates for the H2 proton, is almost cer-
tainly due to the shielding effect induced by the proximal
cyclohexane ring. This aspect and the moderate downfield
shift upon addition of 20% DMSO, suggest that in all these
substrates, the H2 proton is involved in an intramolecular
hydrogen bond, in spite of the chemical shift value being
below 7 ppm, as generally reported in the literature.6
6a H1 6.58
H2 6.41
H1 7.47
H2 6.65
H3 7.01
H1 0.6
H2 1.4
H3 1.6
3376, 3394, 3420
3290, 3378, 3419
H3 5.70
7a H1 6.91
H2 6.48
H1 7.91
H2 6.64
H3 7.85
H4 7.72
H1 5.5
H2 0.5
H3 2.6
H4 2.6
H3 8.04
H4 7.35
The spectroscopic data indicate that in 6, 14 and 15, the H3
proton does not participate with intra-molecular hydrogen
bonds. Conversely, in pseudotetrapeptides 7, 16 and 17 we
can assert that both H3 and H4 are involved in hydrogen
bond formation. However, the hydrogen bond involving
H4 is presumably weaker in 16 with respect to 7 and 17,
owing to the larger downfield variation of the chemical
14 H1 7.20
H2 6.64
H1 7.90
H2 6.95
H3 7.14
H1 6.4
H2 2.6
H3 2.6
3345, 3400
H3 5.91
15 H1 7.27
H2 6.43
H1 8.00
H2 6.52
H3 7.20
H1 6.0
H2 2.0
H3 1.1
3290, 3366, 3400
3341, 3426
H3 5.80
shift dNH observed for these two compounds after the
4
16 H1 7.13
H2 6.75
H1 7.43
H2 6.96
H3 7.94
H4 7.77
H1 5.0
H2 2.0
H3 0.4
H4 0.4
addition of 20% DMSO (see Table 1).
H3 7.87
H4 7.06
4. Molecular modelling and conformational analysis
17 H1 6.97
H2 6.58
H1 8.10
H2 6.58
H3 7.80
H4 7.60
H1 2.0
H2 1.4
H3 0.9
H4b
3294, 3363, 3426
To explore the conformational space of pseudopeptides 6,
7, 14, 15, 16 and 17 and obtain useful information for
understanding spectroscopic data, we carried out a high-
temperature QMD. The QMD protocol, which has been
used over the last decade to produce reasonable molecular
structure of a variety of peptides,7,8 generates representa-
tions of families of low-energy conformational structures
H3 7.96
H4 7.30
a dNH Values of the more abundant conformer (see Section 6).
b It was not possible to determine the temperature coefficient, DdNH/DT,
because in the range of temperatures from 30 to 50 °C, the signal of H4 is
hidden by the CHCl3 present in CDCl3.

3276
D. Balducci et al. / Tetrahedron: Asymmetry 17 (2006) 3273–3281
and provides statistical indications about selected hydrogen
bonds.
pound 7 (see the previous section for the discussion of the
data reported in Table 1).
Representative conformations of the six peptides, obtained
from the cluster analysis, are depicted in Figures 1–3. These
conformations correspond to the most populated struc-
tures within different sets of clusters. These sets have been
obtained by grouping together the original clusters on the
basis of the similarity of the hydrogen bond pattern (see
Computational details in Section 6). A list of hydrogen
bond lifetimes for each compound is given in Table 2.
The cluster analysis shows that compound 14 can exist in
many different conformations that are characterized by
similar populations (some representative conformations
are reported in Fig. 2). This species is characterized by a
rather undefined helicoidal structure. The lifetimes of the
hydrogen bonds (never larger than 40%) are in agreement
with this unfolded structure. Only H1 and H2 and not
H3 seem to be involved in hydrogen bonds in agreement
1
with the indications of H NMR data.
Inspection of H-bond lifetimes and cluster analysis indicate
6 and 7 as the most stable structures (see Fig. 1) since they
hold their b-turn motif during the whole molecular dynam-
ics. In structure 6, the O4ꢁ ꢁ ꢁH2(N) hydrogen bond has a
lifetime of 98.55% and is responsible for the stability of
the 10-membered cycle of the b-turn. The same hydrogen
bond plays a key-role in structure 7 to maintain the cyclic
skeleton. In this case the O4ꢁ ꢁ ꢁH2(N) hydrogen bond has a
lifetime of 96.20%.
The addition of a methyl group in the five-membered ring
(compound 15) causes the breaking of the helicoidal struc-
ture and the pseudopeptide adopts a b-turn structure. In
this case, two dominant conformations (see Fig. 2) can be
identified from the cluster analysis. In both these confor-
mations, the O4ꢁ ꢁ ꢁH2(N) hydrogen bond can be recognized
(lifetime of 77.65%). This interaction is responsible for the
stability of the 10-membered cyclic structure of the b-turn.
In only one of these two representative conformations, the
H1 proton is engaged in a hydrogen bond (N5ꢁ ꢁ ꢁH1(N))
with a lifetime of 29.30%. This computational evidence is
Furthermore, in structure 6 and 7 H1 is involved in the for-
mation of hydrogen bonds with a rather short lifetime. In
6, H1 interacts with O7 and N5 (lifetimes of 21.35% and
18.10%, respectively), while in 7 this proton forms a hydro-
gen bond with O6 (lifetime of 18.10%). Additional hydro-
gen bonds involving H3 (O5ꢁ ꢁ ꢁH3(N), lifetime of 94.45%)
and H4 (O4ꢁ ꢁ ꢁH4(N), lifetime of 67.05%) are observed in
compound 7. These computational results are in rather
1
consistent with the H NMR results that clearly indicate
the existence of a hydrogen bond involving H2, the exis-
tence of less populated conformations characterized by a
H1 hydrogen bond and the absence of hydrogen bonds
involving H3.
1
good agreement with the H NMR data which indicate
Pseudopeptide 16 is an interesting example of a c-turn
structure. In the most populated conformation (see
Fig. 3), hydrogen bonds involving H4 (O4ꢁ ꢁ ꢁH4(N), life-
the presence of hydrogen bonds involving H2 in compound
6 and hydrogen bonds involving H2, H3 and H4 for com-
˚
Figure 1. Representative conformations of pseudopeptides 6 and 7 (atom distances are in A).

D. Balducci et al. / Tetrahedron: Asymmetry 17 (2006) 3273–3281
3277
˚
Figure 2. Representative conformations of pseudopeptides 14 and 15 (atom distances are in A).
bonds are also present in the second representative confor-
mation of Figure 3. Also in this case the conformational
1
modelling is in agreement with the H NMR data indicat-
ing that all four protons form hydrogen bonds.
Pseudopeptide 17, obtained by the addition of a methyl
group to the five-membered ring in 16, is characterized
by the equilibrium between two main conformations (see
Fig. 3). While the O7ꢁ ꢁ ꢁH2(N) interaction can be recog-
nized only in the former one, the O4ꢁ ꢁ ꢁH4(N) hydrogen
bond is present in both conformations. However, cluster
analysis shows that H2 is engaged in other hydrogen bonds
with lifetimes approximately in the range 33–7%. This is in
1
agreement with the H NMR results that indicate the exis-
tence of hydrogen bonds involving H2 and H4.
5. Conclusions
Herein we have achieved a new synthetic approach for
obtaining optically active tetrapseudopeptides. The synthe-
sis of new peptidomimetic structures was performed in a
satisfactory overall yield starting from chiral synthon 1, a
monolactim ether easily synthesized from L-valine. This
approach represents a new and simple synthetic path for
obtaining tetrapseudopeptides containing two L-valine
residues, one modified proline unit and one modified
aspartic acid unit.
Figure 3. Representative conformations of pseudopeptides 16 and 17
˚
(atom distances are in A).
time of 80.25%), H3 (O5ꢁ ꢁ ꢁH3(N), lifetime of 85.15%), H1
(O6ꢁ ꢁ ꢁH1(N), lifetime of 57.10%) and H2 (O6ꢁ ꢁ ꢁH2(N),
lifetime of 26.75%) are evident. The H4 and H3 hydrogen
1
Spectroscopic investigation using H NMR and IR tech-
niques combined with conformational analysis based on

3278
D. Balducci et al. / Tetrahedron: Asymmetry 17 (2006) 3273–3281
Table 2. Lifetimes for the various hydrogen bonds in substrates 6, 7, 14,
15, 16 and 17
6. Experimental
6.1. General information
Structure
Hydrogen bond
Lifetime (%)
6
O4–H2
N2–H2
O7–H1
O3–H2
N5–H1
98.55
33.85
21.35
15.40
8.30
1H and 13C NMR spectra were recorded on a Gemini spec-
trometer at 300 MHz (in about 15 mM solutions) using
CDCl3 as solvent, unless otherwise stated. Chemical shifts
are reported in ppm relative to CDCl3 while the coupling
constants (J) are in Hz. IR spectra were recorded on a
Nicolet FT 380 spectrophotometer. Optical rotation values
were measured at 25 °C on a Perkin–Elmer 343 polari-
meter. Dry THF was distilled from sodium benzophenone
ketyl. Chromatographic separations were performed with
silica gel 60 (230–400 mesh).
7
O4–H2
O5–H3
O4–H4
N2–H2
O6–H1
O3–H2
96.20
94.45
67.05
33.35
18.10
16.90
The synthesis and spectroscopic data of compounds 1, 3, 8
and 9 are reported in Ref. 3 while in Ref. 2 the data for
compound 2 are reported.
14
N2–H2
N5–H1
O7–H1
O3–H2
O1–H1
O6–H1
39.30
30.25
27.00
19.65
10.50
6.58
6.1.1. 1-{2(S)-[2-(6-Ethoxy-5(S)-isopropyl-2(R)-methyl-3-
oxo-2,3,4,5-tetrahydro-pyrazin-2-yl)-acetylamino]-3-methyl-
butyryl}-2(S)-methyl-4-methylene-pyrrolidine-2-carboxylic
acid ethyl ester, 4. The activated ester 2 (1.8 g, 4.3 mmol)
was added to a solution of 3 (1.3 g, 4.3 mmol) and triethyl-
amine (0.9 mL, 6.43 mmol) in dry CH2Cl2 (10 mL) under
an inert atmosphere. The reaction mixture was stirred at
rt for about 24 h, then the organic phase, after a rapid
washing with 0.1 M HCl, was dried over CaCl2 and evap-
orated under vacuum. The residue was submitted to purifi-
cation by silica gel chromatography eluting with hexane/
ethyl acetate and the pure product was recovered as an
15
16
O4–H2
N2–H2
N5–H1
O3–H2
O6–H1
77.65
38.70
29.30
25.60
8.30
O5–H3
O4–H4
O6–H1
N2–H2
O6–H2
O3–H2
85.15
80.25
57.10
34.90
26.75
19.50
1
oil in an 82% yield. H NMR d: 0.88 (d, 3H, J = 6.9);
0.92 (d, 3H, J = 6.9); 0.99 (m, 6H); 1.26 (t, 3H, J = 7);
1.45 (s, 3H); 1.49 (s, 3H); 2.0 (m, 1H); 2.3 (m, 1H); 2.46
(d, 1H, J = 14.4); 2.52 (d, 1H, J = 14.6); 2.89 (d, 1H,
J = 14.4); 3.07 (d, 1H, J = 14.6); 4.0–4.25 (m, 6H); 4.37
(m, 2H); 4.47 (dd, 1H, J = 7.4, 9.2); 5.07 (m, 2H); 5.73
(s, 1H); 6.61 (d, 1H, J = 9). 13C NMR d: 13.9, 14.0, 16.2,
17.7, 18.4, 19.0, 20.5, 28.3, 30.7, 31.1, 44.8, 47.2, 51.7,
55.2, 58.6, 58.7, 61.1, 61.3, 65.9, 109.0, 141.5, 157.3,
169.9, 170.0, 172.7, 173.6. [a]D = ꢀ31.3 (c 1.4, CHCl3).
Anal. Calcd for C26H42N4O6: C, 61.64; H, 8.36; N, 11.06.
Found: C, 61.80; H, 8.34; N, 11.02.
17
O4–H4
O5–H3
N2–H2
O7–H2
O4–H3
O3–H2
O8–H2
O4–H2
O6–H1
48.20
36.90
33.25
33.05
31.75
26.00
14.60
7.10
5.95
quenched molecular dynamics (QMD) have been demon-
strated to be a valid tool to elucidate the structures of
the various tetrapeptides and to demonstrate the existence
in all cases of intra-molecular hydrogen bonds involving
carbonyl oxygens and amide protons.
6.1.2. 1-{2(S)-[2-(6-Ethoxy-5(S)-isopropyl-2(R)-methyl-3-
oxo-2,3,4,5-tetrahydro-pyrazin-2-yl)-acetylamino]-3-methyl-
butyryl}-2(S)-methyl-4-methylene-pyrrolidine-2-carboxylic
acid, 5. 2 M NaOH (0.6 mL) was added to a solution of 4
(0.2 g, 0.4 mmol) in ethanol (3 mL) and the reaction was
stirred at room temperature and monitored by TLC. After
about 3 h, ethanol was evaporated in vacuo and CH2Cl2
was added. The organic solution was rapidly washed with
0.5 M HCl dried over CaCl2 and then evaporated to dry-
ness under vacuum. The oily residue was pure to the HPLC
It is worth noting that a value of dNH, larger or smaller
than 7, is not enough to indicate the presence or absence
of hydrogen bond interactions, respectively. This is due to
the fact that in complex structures, such as those exam-
ined here, shielding or deshielding effects of neighboring
groups can play an important role, as evidenced by the
signals of the H2 protons in all substrates. To decide
about the existence of hydrogen bonds, other factors
(dNH change upon addition of strongly competitive
solvents such as DMSO, temperature coefficient and
IR stretching of amidic N–H) must be taken into
account.
1
analysis. H NMR d: 0.88 (d, 3H, J = 6.8); 0.9 (d, 3H,
J = 6.8); 0.97 (d, 3H, J = 6.8); 1.03 (s, 3H, J = 6.8); 1.27
(t, 3H, J = 7); 1.47 (s, 3H); 1.51 (s, 3H); 2.0 (m, 1H);
2.51 (d, 1H, J = 14.4); 2.61 (d, 1H, J = 14.4); 3.04 (d,
2H, J = 14.4); 4.0–4.6 (m, 6H); 5.1 (m, 2H); 7.02 (d, 1H,
J = 8.8); 7.19 (s, 1H). 13C NMR d: 14.0, 16.1, 17.9, 18.3,
18.9, 20.5, 28.5, 30.6, 30.9, 44.9, 47.0, 51.9, 55.4, 58.4,
58.6, 61.3, 66.4, 108.8, 141.7, 157.3, 170.0, 170.2, 174.8,

D. Balducci et al. / Tetrahedron: Asymmetry 17 (2006) 3273–3281
3279
176.4. [a]D = ꢀ41.5 (c 1.0, CHCl3). Anal. Calcd for
C24H38N4O6: C, 60.23; H, 8.0; N, 11.71. Found: C, 59.98;
H, 8.03; N, 11.74.
J = 14.4); 3.23 (A) (d, 1H, J = 15.3); 3.73 (m, 1H); 4.1–
4.6 (m, 6H); 5.08 (m, 2H); 6.5 (A) (d, 1H, J = 8.1); 6.53
(B) (d, 1H, J = 8.1); 6.87 (B) (d, 1H, J = 9); 6.96 (A) (d,
1H, J = 8.1); 7.36 (B) (s, 1H); 7.39 (A) (s, 1H); 7.98 (A)
(d, 1H, J = 8.7); 8.26 (B) (d, 1H, J = 7.8). 13C NMR d:
14.1, 17.7, 18.1, 19.0, 19.3, 21.4, 22.9, 24.0, 24.7, 25.5,
30.5, 30.8, 32.7, 32.8, 42.3, 44.9, 48.4, 52.9, 56.8, 57.8,
58.7, 61.0, 68.4, 108.4, 141.4, 170.6, 170.8, 171.1, 171.6,
171.8, 173.5. The product was not isolated in sufficiently
pure form for elemental analysis and to measure the spe-
cific rotation.
6.1.3. 1-{2(S)-[2-(6-Ethoxy-5(S)-isopropyl-2(R)-methyl-3-
oxo-2,3,4,5-tetrahydro-pyrazin-2-yl)-acetylamino}-3-methyl-
butyryl}-2(S)-methyl-4-methylene-pyrrolidine-2-carboxylic
acid cyclohexyl amide, 6. Cyclohexylamine (0.1 mL,
1 mmol) was added to a solution of 5 (0.48 g, 1 mmol) dis-
solved in 6 mL of dry THF. After 10 min, DMTMM4
(0.28 g, 1.2 mmol) was added and the reaction mixture stir-
red at room temperature for 12 h. The reaction was con-
centrated under vacuum and the residue was dissolved
with ethyl acetate. The organic solution was firstly washed
with 2 M NaOH and secondly with 0.5 M HCl and then
concentrated in vacuo. The residue was submitted to puri-
fication by silica gel chromatography eluting with hexane/
ethyl acetate and the pure product was recovered as an oil
6.1.5. 1-{2(S)-[2-(6-Ethoxy-5(S)-isopropyl-2(R)-methyl-3-
oxo-2,3,4,5-tetrahydro-pyrazin-2-yl)-acetylamino]-3-methyl-
butyryl}-4-methylene-pyrrolidine-2(R)-carboxylic acid ethyl-
ester, 10. Compound 10 was prepared by reacting the
activated ester 2 (1.27 g, 3 mmol) with 8 (0.87 g, 3 mmol)
and following the procedure used for 4. The product was
recovered as an oil in an 85% yield after elution by silica
1
in an 85% yield. H NMR (as a 4:1 mixture of conformers
1
A/B) d: 0.9 (d, 3H, J = 6.9); 0.93 (d, 3H, J = 6.9); 0.99 (d,
3H, J = 6.6); 1.02 (d, 3H, J = 6.6); 1.16–1.95 (m, 10H); 1.3
(A) (t, 3H, J = 6.9); 1.31 (B) (t, 3H, J = 6.9); 1.46 (s, 3H);
1.6 (s, 3H); 2.01 (m, 1H); 2.3 (m, 1H); 2.4 (A) (dd, 1H,
J = 1.2, 15.3); 2.55 (A) (d, 1H, J = 15); 2.7 (B) (d, 1H,
J = 15); 2.87 (B) (d, 1H, J = 15); 3.07 (A) (d, 1H, J =
14.7); 3.19 (A) (d, 1H, J = 15.3); 3.74 (m, 1H); 4.02–4.30
(m, 3H); 4.24 (d, 1H, J = 13.5); 4.45 (d, 1H, J = 13.5);
4.53 (dd, 1H, J = 6.9, 8.7); 5.05 (m, 2H); 5.92 (A) (s,
1H); 6.03 (B) (s, 1H); 6.44 (A) (d, 1H, J = 7.8); 6.59 (B)
(d, 1H, J = 7.8); 6.7 (A) (d, 1H, J = 9.3); 7.9 (B) (d, 1H,
J = 9.3). 13C NMR d: 14.1, 16.2, 17.6, 18.4, 19.3, 21.6,
24.7, 25.5, 28.5, 30.7, 31.2, 32.7, 32.8, 45.0, 47.3, 48.3,
52.9, 55.5, 58.6, 58.8, 61.4, 68.3, 108.5, 141.2, 157.4,
170.0, 171.2, 171.7, 173.6. [a]D = ꢀ34.3 (c 0.7, CHCl3).
Anal. Calcd for C30H46N5O5: C, 64.72; H, 8.33; N, 12.58.
Found: C, 64.48; H, 8.34; N, 12.60.
gel chromatography with hexane/ethyl acetate. H NMR
d: 0.89 (d, 3H, J = 6.9); 0.91 (d, 3H, J = 6.9); 0.94 (d,
3H, J = 6.9); 1.01 (d, 3H, J = 6.9); 1.29 (m, 6H); 1.48 (s,
3H); 2.0 (m, 1H); 2.29 (m, 1H); 2.54 (d, 1H, J = 14.1);
2.68 (m, 1H); 2.96 (m, 1H); 3.06 (m, 1H, J = 14.1); 4.05
(m, 1H); 4.18 (m, 2H); 4.26 (d, 1H, J = 14.1); 4.36 (d,
1H, J = 14.1); 4.62 (m, 1H); 5.09 (m, 2H); 6.01 (s, 1H);
6.72 (d, 1H, J = 8.1). 13C NMR d: 13.9, 14.0, 16.1, 17.6,
18.2, 19.2, 28.1, 30.7, 31.4, 35.2, 47.2, 51.1, 54.9, 58.4,
58.6, 61.0, 61.2, 108.8, 141.8, 157.2, 169.7, 170.6, 171.0,
173.3. The product was not isolated in a sufficiently pure
form for elemental analysis and to measure the specific
rotation.
6.1.6. 1-{2(S)-[2-(6-Ethoxy-5(S)-isopropyl-2(R)-methyl-3-
oxo-2,3,4,5-tetrahydro-pyrazin-2-yl)-acetylamino]-3-methyl-
butyryl}-4-methylene-pyrrolidine-2(R)-carboxylic acid, 11.
Compound 11 was obtained from intermediate 10 and
following the procedure described for 5. The product was
recovered as an oil in an 80% yield after elution by silica
6.1.4.
1-[(1-Carbonyl-2(S)-isopropyl-3,8-diaza-4,7-dioxo-
6(R)-N-acetamido-6-methyl-9(S)-isopropyl)ethyl decanoate]-
2(S)-methyl-4-methylene-2-cyclohexylcarbamoylpyrrolidine
hydrochloride, 7. HCl 0.5 M (1 mL) was added to a solu-
tion of 6 (0.134 g, 0.24 mmol) dissolved in ethanol (2 mL)
and the reaction mixture was stirred at room temperature.
After 12 h ethanol was evaporated, the residue extracted
with CH2Cl2 and the organic solution dried over CaCl2.
After filtration, to the organic solution was added triethyl-
amine (0.14 mL, 0.96 mmol) and then cooled to ꢀ10 °C.
Acetyl chloride (0.03 mL, 0.42 mmol) was added and after
10–15 min the cooling bath removed. The reaction mixture
was stirred for 3 h and then the organic solvent was evap-
orated under vacuum. The residue was dissolved in ethyl
acetate, the organic solution washed with 2 M HCl and
then dried over Na2SO4. After evaporation in vacuo to dry-
ness, the residue was submitted to silica gel chromatogra-
phy eluting with hexane/ethyl acetate and the product
1
gel chromatography with hexane/ethyl acetate. H NMR
(CD3OD) d: 0.93 (d, 3H, J = 6.9); 0.94 (d, 3H, J = 6.9);
0.96 (d, 3H, J = 6.9); 1.04 (d, 3H, J = 6.9); 1.29 (t, 3H,
J = 6.9); 1.43 (s, 3H); 1.54 (s, 3H); 2.03 (m, 1H); 2.5 (m,
1H); 2.63 (d, 2H, J = 15); 2.92 (d, 1H, J = 15); 2.98 (d,
1H, J = 15); 4.02 (d, 1H, J = 3.3); 4.05–4.25 (m, 2H);
4.35 (dd, 1H, J = 1.5, 13.8); 4.44 (d, 1H, J = 8.4); 4.52
(d, 1H, J = 13.8); 5.25 (m, 2H). 13C NMR (CD3OD) d:
14.9, 17.5, 18.9, 19.0, 19.7, 21.8, 29.4, 32.3, 32.5, 46.0,
53.7, 57.2, 59.8, 59.9, 62.5, 67.5, 109.4, 143.6, 159.4,
171.5, 172.4, 176.1, 176.3. [a]D = ꢀ16.9 (c 0.9, CHCl3).
Anal. Calcd for C23H36N4O6: C, 59.46; H, 7.81; N, 12.06.
Found: C, 59.23; H, 7.84; N, 12.10.
6.1.7. 1-{2(S)-[2-(6-Ethoxy-5(S)-isopropyl-2(R)-methyl-3-
oxo-2,3,4,5-tetrahydro-pyrazin-2-yl)-acetylamino]-3-methyl-
butyryl}-2(R)-methyl-4-methylene-pyrrolidine-2-carboxylic
acid ethylester, 12. Compound 12 was prepared by react-
ing the activated ester 2 with 9 and following the procedure
used for 10. The product was recovered as an oil in an 80%
yield after elution by silica gel chromatography with hex-
1
was recovered as a wax in a 70% overall yield. H NMR
(as a 4:1 mixture of conformers A/B) d: 0.96 (d, 3H,
J = 6.6); 0.98 (d, 3H, J = 6.6); 1.0 (d, 3H, J = 6.6); 1.04
(d, 3H, J = 6.6); 1.1–2.0 (m, 10H); 1.29 (t, 3H, J = 6.9);
1.62 (s, 3H); 1.67 (s, 3H); 2.04 (s, 3H), 2.05 (m, 1H); 2.22
(m, 1H); 2.41 (A) (d, 1H, J = 14.7); 2.66 (A) (d, 1H,
J = 14.4); 3.04 (A) (d, 1H, J = 14.4); 3.16 (B) (d, 1H,
1
ane/ethyl acetate. H NMR d: 0.88 (d, 3H, J = 7); 0.89
(d, 3H, J = 6.6); 0.93 (d, 3H, J = 6.6); 1.0 (d, 3H, J = 7);

3280
D. Balducci et al. / Tetrahedron: Asymmetry 17 (2006) 3273–3281
1.24 (t, 3H, J = 7); 1.28 (t, 3H, J = 7); 1.48 (s, 3H); 1.53 (s,
3H); 1.95 (m, 1H); 2.3 (m, 1H); 2.5 (d, 1H, J = 15.2); 2.53
(d, 1H, J = 14.4); 2.88 (d, 1H, J = 15.2); 3.05 (d, 1H,
J = 14.4); 3.98–4.30 (m, 6H); 4.5 (m, 2H); 5.06 (m, 2H);
5.94 (s, 1H); 6.63 (d, 1H, J = 9.2). 13C NMR d: 14.1,
16.1, 17.7, 18.3, 19.1, 21.1, 28.4, 30.7, 31.7, 44.6, 47.5,
52.2, 55.1, 58.6, 58.8, 61.1, 61.4, 65.9, 108.8, 141.6, 169.7,
169.9, 172.7, 173.4. The product was not isolated in a suf-
ficiently pure form for elemental analysis and to measure
the specific rotation.
1.02 (m, 9H); 1.3 (t, 3H, J = 7); 1.5 (s, 3H); 1.7 (s, 3H);
1.1–1.9 (m, 10H); 2.0 (m, 1H); 2.15 (m, 1H); 2.52 (d, 1H,
J = 15); 2.69 (d, 1H, J = 14.6); 2.86 (d, 1H, J = 14.6);
3.02 (d, 1H, J = 15); 3.65 (m, 1H); 4.0–4.4 (m, 5H); 4.55
(d, 1H, J = 13.5); 5.02 (m, 2H); 6.02 (s, 1H); 6.44 (d, 1H,
J = 8). 13C NMR d: 14.0, 16.4, 18.4, 18.8, 18.9, 21.9,
24.9, 25.0, 25.5, 27.7, 30.8, 31.2, 32.6, 46.2, 46.7, 48.6,
53.2, 57.3, 58.6, 61.5, 68.4, 108.4, 141.1, 157.7, 169.0,
171.3, 171.8, 173.1. The product was not isolated in a suf-
ficiently pure form for elemental analysis and to measure
the specific rotation.
6.1.8. 1-{2(S)-[2-(6-Ethoxy-5(S)-isopropyl-2(R)-methyl-3-
oxo-2,3,4,5-tetrahydro-pyrazin-2-yl)-acetylamino]-3-methyl-
butyryl}-2(R)-methyl-4-methylene-pyrrolidine-2-carboxylic
acid, 13. Compound 13 was obtained from intermediate
12 and following the procedure described for 11. The pure
product was recovered as an oil in an 80% yield after elu-
tion by silica gel chromatography with hexane/ethyl ace-
tate. 1H NMR d: 0.86 (d, 3H, J = 6.8); 0.92 (d, 6H,
J = 6.8); 1.03 (d, 3H, J = 6.8); 1.26 (t, 3H, J = 7.2); 1.45
(s, 3H); 1.54 (s, 3H); 2.02 (m, 1H); 2.25 (m, 1H); 2.47 (d,
1H, J = 15.8); 2.6 (d, 1H, J = 15.8); 2.96 (d, 1H,
J = 15.8); 3.04 (s, 1H, J = 15.8); 3.96–4.35 (m, 4H); 4.45
(m, 2H); 5.02 (m, 2H); 7.19 (s, 1H); 7.5 (d, 1H, J = 9.2).
13C NMR d: 13.9, 15.9, 17.8, 18.1, 18.9, 20.8, 20.9, 28.6,
30.3, 30.9, 44.4, 46.5, 52.3, 55.6, 58.0, 58.4, 61.1, 66.0,
108.4, 141.7, 157.8, 170.1, 170.5, 174.9. [a]D = ꢀ48.6 (c
0.7, CHCl3). Anal. Calcd for C24H38N4O6: C, 60.23; H,
8.0; N, 11.71. Found: C, 60.44; H, 7.98; N, 11.69.
6.1.11. 1-[(1-Carbonyl-2(S)-isopropyl-3,8-diaza-4,7-dioxo-
6(R)-N-acetamido-6-methyl-9(S)-isopropyl)ethyl decanoate]-
4-methylene-2(R)-cyclohexylcarbamoylpyrrolidine
hydro-
chloride, 16. Compound 16 was obtained from intermedi-
ate 14 and following the procedure described for 7. The
pure product was recovered as a wax in an 85% yield after
elution by silica gel chromatography with hexane/ethyl ace-
1
tate. H NMR d: 0.9–1.05 (m, 12H); 1.29 (t, 3H, J = 7.2);
1.71 (s, 3H); 2.07 (s, 3H); 1.05–2.0 (m, 10H); 2.08 (m,
1H); 2.27 (m, 1H); 2.63 (d, 1H, J = 14.2); 2.83 (m, 1H);
2.96 (d, 1H, J = 14.2); 3.13 (d, 1H, J = 14.2); 3.75 (m,
1H); 4.18–4.38 (m, 4H); 4.43 (m, 2H); 4.73 (m, 1H); 5.09
(m, 2H); 6.75 (d, 1H, J = 8.4); 7.07 (s, 1H); 7.13 (d, 1H,
J = 6.3); 7.88 (d, 1H, J = 8, 7). 13C NMR d: 14.1, 17.7,
18.4, 19.0, 19.3, 23.6, 23.8, 24.9, 25.4, 29.9, 30.9, 32.6,
32.8, 35.8, 42.9, 48.5, 51.3, 57.4, 57.7, 59.2, 60.0, 61.1,
108.8, 142.0,1, 169.6, 171.0, 171.1, 171.7, 171.8, 173.7.
[a]D = +75.7 (c 0.5 CHCl3). Anal. Calcd for C31H47N5O7:
C, 61.88; H, 7.87; N, 11.64. Found: C, 62.15; H, 7.85; N,
11.62.
6.1.9. 1-{2(S)-[2-(6-Ethoxy-5(S)-isopropyl-2(R)-methyl-3-
oxo-2,3,4,5-tetrahydro-pyrazin-2-yl)-acetylamino}-3-methyl-
butyryl}-4-methylene-pyrrolidine-2(R)-carboxylic acid cyclo-
hexyl amide, 14. Compound 14 was obtained by treating
intermediate 11 (0.93 g, 2 mmol) with cyclohexylamine
(0.25 mL, 2.2. mmol) in the presence of DMTMM4
(0.72 g, 2.6 mmol) and following the procedure described
for 6. The pure product was recovered as an oil in an
83% yield after elution by silica gel chromatography with
6.1.12. 1-[(1-Carbonyl-2(S)-isopropyl-3,8-diaza-4,7-dioxo-
6(R)-N-acetamido-6-methyl-9(S)-isopropyl)ethyl decanoate]-
2(R)-methyl-4-methylene 2-cyclohexylcarbamoylpyrrolidine
hydrochloride, 17. Compound 17 was obtained from
intermediate 15 and following the procedure described
for 7. The product was recovered as a wax in an 83% yield
after elution by silica gel chromatography with hexane/
1
hexane/ethyl acetate. H NMR d: 0.90 (d, 3H, J = 6.8);
1
0.97 (d, 3H, J = 6.8); 0.99 (d, 3H, J = 6.8); 1.01 (d, 3H,
J = 6.8); 1.1–1.9 (m, 10H); 1.33 (t, 3H, J = 7.2); 1.5 (s,
3H); 2.02 (m, 1H); 2.25 (m, 1H); 2.69 (d, 1H, J = 14.6);
2.75 (m, 1H); 2.86 (d, 1H, J = 14.8); 2.95 (d, 1H, J = 15);
3.67 (m, 1H); 4.01 (m, 1H); 4.05–4.35 (m, 4H); 4.42 (d,
1H, J = 14.2); 4.7 (d, 1H, J = 8.8); 5.08 (m, 2H); 6.14 (s,
1H); 6.67 (d, 1H, J = 8.6); 7.38 (d, 1H, J = 7.2). 13C
NMR d: 14.1, 16.5, 18.4, 18.6, 19.2, 24.2, 24.9, 25.4, 27.9,
29.7, 30.5, 30.9, 31.2, 32.5, 35.6, 47.0, 48.7, 51.4, 56.9,
58.8, 60.0, 61.6, 109.0, 142.1, 157.7, 169.7, 171.3, 173.2.
[a]D = +41.0 (c 0.4, CHCl3). Anal. Calcd for
C29H44N5O5: C, 64.18; H, 8.17; N, 12.9. Found: C, 63.95;
H, 8.19; N, 12.95.
ethyl acetate. H NMR d: 0.92 (d, 3H, J = 6.9); 0.96 (d,
3H, J = 6.9); 0.98 (d, 6H, J = 6.6); 1.27 (t, 3H, J = 7.2);
1.64 (s, 3H); 1.68 (s, 3H); 1.1–1.9 (m, 10H); 2.02 (m, 1H);
2.23 (m, 1H); 2.53 (d, 1H, J = 15.6); 2.64 (d, 1H,
J = 14.4); 2.97 (m, 2H); 3.7 (m, 1H); 4.16 (m, 3H); 4.27
(d, 1H, J = 13.8); 4.4 (m, 1H); 4.57 (d, 1H, J = 13.8);
5.04 (m, 2H); 6.54 (d, 1H, J = 8.1); 7.24 (d, 1H, J = 6.6);
7.46 (s, 1H); 7.86 (d, 1H, J = 8.1). 13C NMR d: 14.1,
17.6, 18.5, 18.9, 19.0, 21.7, 23.9, 24.9, 25.5, 29.9, 30.8,
32.6, 32.7, 43.4, 46.2, 48.6, 53.1, 57.6, 57.8, 59.5, 61.1,
65.8, 68.4, 108.6, 140.9, 170.7, 171.2, 171.6, 171.7, 172.0,
173.9. The product was not isolated in a sufficiently pure
form for elemental analysis and to measure the specific
rotation.
6.1.10. 1-{2(S)-[2-(6-Ethoxy-5(S)-isopropyl-2(R)-methyl-3-
oxo-2,3,4,5-tetrahydro-pyrazin-2-yl)-acetylamino}-3-methyl-
butyryl}-2(R)-methyl-4-methylene-pyrrolidine-2-carboxylic
acid cyclohexyl amide, 15. Compound 15 was obtained
from intermediate 13 and following the procedure de-
scribed for 14. The product was recovered as an oil in an
85% yield after elution by silica gel chromatography with
6.2. Computational details
The computational strategy used here is a high-tempera-
ture quenched molecular dynamics (QMD) approach.
The QMD protocol, which has been used over the last dec-
ade to produce a reasonable molecular structure of a vari-
ety of peptides, allows us to search the conformational
1
hexane/ethyl acetate. H NMR d: 0.94 (d, 3H, J = 6.8);

D. Balducci et al. / Tetrahedron: Asymmetry 17 (2006) 3273–3281
3281
space of the pseudopeptides and to locate the lowest energy
Acknowledgement
minimum.
Thanks are due to the University of Bologna for financial
support (Ricerca Fondamentale Orientata, ex 60%).
The 3D structures of the molecules were built with CORI-
NA.9 All the calculations were performed at the molecular
mechanics level using the AMBER 8.0 package.10 Simula-
tions were carried out using the Gaff force field.11 Charges
were assigned to atoms using the AM1-BCC method12 as
implemented in the Antechamber package.13 Solvation
effects were incorporated using the Generalized Born
Model.14 Thus dynamics were performed with a dielectric
constant e = 4.9 to simulate the electrostatic effects of chlo-
roform (the solvent where 1H NMR data have been
recorded).
References
1. Balducci, D.; Emer, E.; Piccinelli, F.; Porzi, G.; Recanatini,
M.; Sandri, S. Tetrahedron: Asymmetry 2005, 16, 3785.
2. Balducci, D.; Porzi, G.; Sandri, S. Tetrahedron: Asymmetry
2006, 17, 1521, and references cited therein.
3. Balducci, D.; Grandi, A.; Porzi, G.; Sandri, S. Tetrahedron:
Asymmetry 2005, 16, 1453.
4. Kunishima, M.; Kawachi, C.; Morita, J.; Terao, K.; Iwasaki,
F.; Tani, S. Tetrahedron 1999, 55, 13159.
To locate the lowest energy structure, without being
trapped in local minima, we first employed a preliminary
QMD simulation where the molecules were heated from
0 to 600 K in 100 ps and then, a trajectory of 5 ns was car-
ried out at constant temperature (600 K) and constant
pressure (1 atm) with an integration step of 2 fs. The
SHAKE algorithm15 was used to constrain the stretching
of bonds involving hydrogen atoms. The coordinates of
the pseudopeptides were saved on a trajectory file every
1 ps, giving a total of 5000 conformations for further anal-
ysis. Each of the structures obtained was energy minimized
till the root mean square of the Cartesian elements of the
gradient was less than 0.001 kcal molꢀ1 using a full conju-
gate gradient minimization and GB/SA model.14,16
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˚
(distance between acceptor and donor shorter than 4 A,
and a bond angle larger than 109°). For structure 6 we have
compared the results obtained with the 1 ns trajectory to
those obtained with a 10 ns trajectory. Since the difference
in the lifetimes was not larger than 3%, we have used tra-
jectories of 1 ns for all the six pseudopeptides examined
here.
To visualize the most important conformations of the pep-
tides, we have carried out a cluster analysis (the MMTSB
toolset was used).17 We have clustered the conformations
obtained from the dynamics by a structural similarity
˚
(using kclust and a fixed radius clustering of 1.5 A on carte-
sian coordinate RMSD of heavy atoms). Clusters have
been grouped together, if possible, on the basis of the sim-
ilarity of the hydrogen bond pattern. For each set of clus-
ters the most populated structures have been selected as the
representative conformations of each pseudopeptide.