Synthesis and conformational studies of peptidomimetics containing a new bifunctional diketopiperazine scaffold acting as a β-hairpin inducer

Article abstract of DOI:10.1021/jo702072z

(Chemical Equation Presented) A practical synthesis of a new bifunctional diketopiperazine (DKP) scaffold 1, formally derived from the cyclization of L-aspartic acid and (S)-2,3-diaminopropionic acid, is reported. DKP-1 bears a carboxylic acid and an amin

Full text of DOI:10.1021/jo702072z

Synthesis and Conformational Studies of Peptidomimetics
Containing a New Bifunctional Diketopiperazine Scaffold Acting as
a â-Hairpin Inducer
Ana Sofia M. Ressurreic¸a˜o,† Andrea Bordessa,‡ Monica Civera,# Laura Belvisi,§,#
Cesare Gennari,*,§,#, and Umberto Piarulli*,†
Dipartimento di Scienze Chimiche e Ambientali, UniVersita` degli Studi dell’Insubria, Via Valleggio 11,
I-22100 Como, Italy, Institut fu¨r Organische Chemie, UniVersita¨t Regensburg, UniVersita¨tsstrasse 31,
93053 Regensburg, Germany, and Dipartimento di Chimica Organica e Industriale and Centro di
Eccellenza C.I.S.I., UniVersita` degli Studi di Milano, Via Venezian 21, I-20133 Milano, Italy
umberto.piarulli@uninsubria.it; cesare.gennari@unimi.it
ReceiVed October 3, 2007
A practical synthesis of a new bifunctional diketopiperazine (DKP) scaffold 1, formally derived from
the cyclization of L-aspartic acid and (S)-2,3-diaminopropionic acid, is reported. DKP-1 bears a carboxylic
acid and an amino functionalities in a cis relationship, which have been used to grow peptide sequences.
Tetra-, penta-, and hexapeptidomimetic sequences were prepared by solution-phase peptide synthesis
1
(Boc strategy). Conformational analysis of these derivatives was carried out by a combination of H
NMR spectroscopy, IR spectroscopy, CD spectroscopy, and computer modeling, and reveals the formation
of â-hairpin mimics involving 10-membered and 18-membered H-bonded rings and a reverse turn of the
growing peptide chain.
Introduction
Reverse-turn mimics are generally cyclic or bicyclic dipeptide
analogues which, as a result of their constrained structure, force
a peptide chain to fold back upon itself.3 Some of us have
recently prepared several azabicycloalkane amino acid scaffolds
containing a bicyclic lactam unit, and studied their conforma-
tional properties as reverse-turn inducing dipeptide mimics,
where the nature and stereochemistry of the bicyclic lactam
strongly influence their turn-inducing abilities.4
In the field of peptidomimetics much effort has been focused
on the design and synthesis of conformationally constrained
compounds that mimic, or induce, specific secondary structural
features of peptides and proteins.1 In fact, short linear peptides
are inherently flexible molecules, especially in aqueous solution,
and so are often poor mimics of the secondary structures (turns,
R-helices, â-strands) found on the surfaces of folded proteins.
A common motif in protein structure is the reverse-turn, which
is defined as a site where the peptide backbone reverses the
direction of propagation by adopting a U-shaped conformation.2
(2) (a) Rose, G. D.; Gierasch, L. M.; Smith, J. A. AdV. Protein Chem.
1985, 37, 1-109. (b) Boussard, G.; Marraud, M. J. Am. Chem. Soc. 1985,
107, 1825-1828.
(3) (a) Cluzeau, J.; Lubell, W. D. Biopolymers 2005, 80, 98-150. (b)
Belvisi, L.; Colombo, L.; Manzoni, L.; Potenza, D.; Scolastico, C. Synlett
2004, 1449-1471. (c) Lelais, G.; Seebach, D. Biopolymers 2004, 76, 206-
243. (d) Hanessian, S.; McNaughtonSmith, G.; Lombart, H. G.; Lubell, W.
D. Tetrahedron 1997, 53, 12789-12854.
(4) (a) Belvisi, L.; Bernardi, A.; Manzoni, L.; Potenza, D.; Scolastico,
C. Eur. J. Org. Chem. 2000, 2563-2569. (b) Belvisi, L.; Gennari, C.;
Madder, A.; Mielgo, A.; Potenza, D.; Scolastico, C. Eur. J. Org. Chem.
2000, 695-699. (c) Belvisi, L.; Gennari, C.; Mielgo, A.; Potenza, D.;
Scolastico, C. Eur. J. Org. Chem. 1999, 389-400.
† Universita` degli Studi dell’Insubria.
‡ Universita¨t Regensburg.
§ Dipartimento di Chimica Organica e Industriale, Universita` degli Studi di
Milano.
# Centro di Eccellenza C.I.S.I., Universita` degli Studi di Milano.
(1) (a) Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem. ReV.
2001, 101, 3131-3152. (b) Nowick, J. S.; Smith, E. M.; Pairish, M. Chem.
Soc. ReV. 1996, 25, 401-415.
10.1021/jo702072z CCC: $40.75 © 2008 American Chemical Society
Published on Web 12/13/2007
652
J. Org. Chem. 2008, 73, 652-660

Synthesis and Conformational Studies of Peptidomimetics
carboxylic acid and an amino functionalities. As a consequence
of the absolute configuration of the two R-amino acids forming
the cyclic dipeptide unit, the two reactive functionalities (amino
and carboxylic acid) are locked in a cis-configuration. When
inserted into an oligopeptide sequence, the DKP-scaffold acts
as a reverse-turn inducer. In addition, the DKP scaffold 1, while
being derived from R-amino acids [L-aspartic acid and (S)-2,3-
diaminopropionic acid], can be seen as a conformationally
constrained dipeptide formed by two â-amino acids (see Figure
1),12 and in particular a â2 and a â3-amino acids (following
Seebach’s nomenclature).13 A few sequences incorporating the
DKP-scaffold 1 were synthesized (tetrapeptides AA1-DKP-AA2,
pentapeptides AA1-AA2-DKP-AA3, and hexapeptides AA1-AA2-
DKP-AA3-AA4), and their conformations were studied by NMR,
IR, CD spectroscopy, and molecular modeling showing the
formation of a â-hairpin mimic.
FIGURE 1. Structure of the bifunctional diketopiperazine scaffold 1
(DKP-1) highlighting the conformationally constrained â2-â3 dipeptide
sequence.
Diketopiperazines (DKP), the smallest cyclic peptides, are a
common motif found in several natural products with therapeutic
properties.5 In addition, DKP have been used as organic catalysts
in the hydrocyanation of imines,6 and have been shown to be
useful scaffolds for the rational design of drugs and peptido-
mimetics.7 In these cases, advantage can be taken from the
synthesis of symmetrical and unsymmetrical DKP bearing
reactive functionalities in the lateral chains of the amino acids.
For instance, Wennemers and co-workers have prepared a few
symmetrical diketopiperazine two-armed receptors derived from
4-aminoproline where the two amino groups (with a cis
disposition) were functionalized with two tripeptide side chains.8
The resulting two-armed receptors were screened toward a
tripeptide library and showed highly selective binding properties
which were attributed to the specific turn geometry of the
receptor. Alternatively, two different functionalities can be
created in the lateral chains of the two amino acids forming the
DKP core, such as an amine (e.g., derived from Lys, Orn, or
diaminobutyric acid) and a carboxylic acid (e.g., derived from
Asp or Glu). In this case, a new peptidomimetic structure is
formed, possessing a fixed conformation (due to the cyclic DKP
core and the configuration of the two amino acids), and which
can now be inserted into oligopeptide sequences.9 For instance,
Royo, Albericio, et al. reported the synthesis of cyclic pepti-
domimetics containing a bifunctional DKP (cyclo-Lys-Glu) and
a RGD sequence and measured their binding affinity to the RVâ3
integrin receptor.10 Robinson and co-workers have synthesized
a novel bicyclic template, comprising a diketopiperazine derived
from L-aspartic acid and (2S,3R,4R)-diaminoproline, which in
the context of a cyclic peptide mimic can stabilize â-hairpin
conformations.11
Results and Discussion
The synthesis of DKP-1 was conveniently obtained according
to Scheme 1, starting from suitably protected N-(tert-butoxy-
carbonyl)-(2S)-aspartic acid â-allyl ester14 and (S)-N-ben-
zylserine methyl ester,15 which were coupled to form dipeptide
2. Dipeptide 2 was then deprotected and its trifluoroacetate salt
cyclized, in good yields, to the diketopiperazine 3, in a basic
biphasic system (EtOAc/NaHCO3 aq).16 These conditions were
selected to minimize the epimerization of the serine methyl ester
and the formation of the diastereomeric trans-DKP (<10%),
which could, however, be separated by a chromatographic
purification. Other conditions, such as the use of tertiary amines
(Et3N or iPr2EtN) as base or of other solvents (e.g., dichlo-
romethane), gave increased proportions of the epimeric trans-
DKP. The stereochemistry of the cis-DKP 3 was unequivocally
established by X-ray diffraction.
The introduction of the nitrogen functionality was then
realized through a Mitsunobu-type reaction, using HN3‚Tol in
a toluene/dichloromethane solution, thus obtaining azide 4 in a
moderate yield (48%). This procedure had been reported for
the successful synthesis of 2,3-diaminopropionic acid starting
from serine derivatives.17 Other methodologies, involving the
activation of the hydroxyl group of serine, were hampered by
the concurrent elimination reaction leading to the dehydroalanine
derivative as the major reaction product. The same Mitsunobu-
HN3 reaction run on dipeptide 2 gave a higher yield of the azide
derivative, but, unfortunately, all attempts to cyclize this
derivative met with no success. Finally, a one-pot Staudinger-
Boc protection18 yielded the DKP scaffold allyl ester 5, which
was de-allylated19 to give the amino acid derivative 1 in
quantitative yield.
In this paper we report the synthesis of a new bifunctional
DKP scaffold 1 (DKP-1, Figure 1), formally derived from
L-aspartic acid and (S)-2,3-diaminopropionic acid, bearing a
(5) (a) Martins, M. B.; Carvalho, I. Tetrahedron, 2007, 63, 9923-9932.
(b) Fischer, P. M. J. Peptide Sci. 2003, 9, 9-35.
(6) Becker, C.; Hoben, C.; Schollmeyer, D.; Scherr, G.; Kunz, H. Eur.
J. Org. Chem. 2005, 1497-1499 and references cited therein.
(7) (a) Horton, D. A.; Bourne, G. T.; Smythe, M. L. Mol. DiVersity 2000,
5, 289-304. (b) Falorni, M.; Giacomelli, G.; Porcheddu, A.; Taddei, M.
Eur. J. Org. Chem. 2000, 1669-1675. (c) Falorni, M.; Giacomelli, G.;
Nieddu, F.; Taddei, M. Tetrahedron Lett. 1997, 38, 4663-4666. (d) del
Fresno, M.; Alsina, J.; Royo, M.; Barany, G.; Albericio, F. Tetrahedron
Lett. 1998, 39, 2639-2642.
(12) For recent reviews on the structure and function of â-peptides see:
(a) Seebach, D.; Hook, D. F.; Gla¨ttli, A. Biopolymers 2006, 84, 23-37. (b)
Fulop, F.; Martinek, T. A.; To´th, G. K. Chem. Soc. ReV. 2006, 35, 323-
334. (c) Steer, D. L.; Lew, R. A.; Perlmutter, P.; Smith, A. I.; Aguilar, M.
I. Curr. Med. Chem. 2002, 9, 811-822. (d) Cheng, R. P.; Gellman, S. H.;
DeGrado, W. F. Chem. ReV. 2001, 101, 3219-3232.
(13) The superscripted number after â specifies the position of the side
chain on the corresponding â-amino acid, see: Hintermann, T.; Seebach,
D. Synlett 1997, 437-438.
(14) Webster, K. L.; Maude, A. B.; O’Donnell, M. E.; Mehrotra, A. P.;
Gani, D. J. Chem. Soc., Perkin Trans. 1 2001, 1673-1695.
(15) Thompson, C. M.; Frick, J. A.; Green, D. L. C. J. Org. Chem. 1990,
55, 111-116.
(8) (a) Krattiger, P.; Wennemers, H. Synlett 2005, 706-708. (b)
Wennemers, H.; Nold, M. C.; Conza, M. M.; Kulicke, K. J.; Neuburger,
M. Chem. Eur. J. 2003, 9, 442-448. (c) Conza, M.; Wennemers, H. J.
Org. Chem. 2002, 67, 2696-2698. (d) Wennemers, H.; Conza, M.; Nold,
M.; Krattiger, P. Chem. Eur. J. 2001, 7, 3342-3347.
(9) Rodionov, I. L.; Rodionova, L. N.; Baidakova, L. K.; Romashko, A.
M.; Balashova, T. A.; Ivanov, V. T. Tetrahedron 2002, 58, 8515-8523.
(10) Royo, M.; Van den Nest, W.; del Fresno, M.; Frieden, A.; Yahalom,
D.; Rosenblatt, M.; Chorev, M.; Albericio, F. Tetrahedron Lett. 2001, 42,
7387-7391.
(11) Pfeifer, M. E.; Moehle, K.; Linden, A.; Robinson, J. A. HelV. Chim.
Acta 2000, 83, 444-464 and references cited therein.
(16) Veerman, J. J. N.; Bon, R. S.; Hue, B. T. B.; Girones, D.; Rutjes,
F. P. J. T.; van Maarseveen, J. H.; Hiemstra, H. J. Org. Chem. 2003, 68,
4486-4494.
(17) (a) Boger, D. L.; Lee, J. K. J. Org. Chem. 2000, 65, 5996-6000.
(b) Pickersgill, I. F.; Rapoport, H. J. Org. Chem. 2000, 65, 4048-4057.
J. Org. Chem, Vol. 73, No. 2, 2008 653

Ressurreic¸a˜o et al.
SCHEME 1. Synthesis of the Diketopiperazine Scaffold DKP-1
SCHEME 2. Peptidomimetics Containing DKP-1a
As we anticipated in the introduction, the diketopiperazine
scaffold 1 can be seen as a conformationally constrained
dipeptide formed by â2 and â3-amino acids (see Figure 1).
Extensive investigation on â-peptides indicated that these are
able to adopt stable secondary structures such as helices and
sheets. The stabilization of â-peptide-hairpins sequences was
also studied,12 and in particular Seebach and co-workers
described the formation of turn-like secondary structures in
oligo-â-peptides containing the dipeptide sequence formed by
a â2-amino acid (C2-substituted) followed by a â3-amino acid
(C3-substituted).20 Gellman and co-workers reported the forma-
tion of a hairpin conformation when a heterochiral dinipecotic
acid â-peptide unit was introduced in a tetrapeptide.21 The
intramolecular hydrogen-bonding pattern in these two cases is
different: in the first case a 10-membered H-bonded ring is
formed involving the CdO of the â3-amino acid and the NH
of the â2-amino acid, while in the second case a 12-membered
H-bonded ring can be identified, which is a two-term homologue
of the â-turn structure formed by R-amino acids. â-Hairpins
containing both R- and â-amino acids have also been reported
to be very stable.22
In view of these potential properties, we decided to study
the ability of DKP-1 to form well-defined folded structures,
when introduced in peptide sequences. We realized the synthesis
of several peptidomimetics (6-11, Scheme 2) by solution-phase
peptide synthesis (Boc strategy) starting from the C-terminus.23
Good yields were obtained in the coupling of the amino acids
(18) Ariza, X.; Urp´ı, F.; Viladomat, C.; Vilarrasa, J. Tetrahedron Lett.
1998, 39, 9101-9102.
a Synthetic schemes, experimental procedures and characterization of
compounds 6-11 are reported in the Supporting Information.
(19) David, C.; Bischoff, L.; Meudal, H.; Mothe´, A.; De Mota, N.;
DaNascimento, S.; Llorens-Cortes, C.; Fournie´-Zaluski, M.-C.; Roques, B.
P. J. Med. Chem. 1999, 42, 5197-5211.
to the amino terminus of DKP-1, using EDC (N-ethyl,N′-[3′-
(dimethylamino)propyl]carbodiimide)/HOAt (7-aza-1-hydroxy-
1,2,3-benzotriazole) or HATU {[(dimethylamino)([1,2,3]triazolo-
[4,5b]pyridin-3-yloxy)methylene]dimethylammonium-
hexafluorophosphate},24 in a methylene chloride or DMF
(20) Seebach, D.; Abele, S.; Gademann, K.; Jaun, B. Angew. Chem.,
Int. Ed. 1999, 38, 1595-1597.
(21) Chung, Y. J.; Christianson, L. A.; Stanger, H. E.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 10555-10556.
(22) Seebach, D.; Jaun, B.; Sebesta, R.; Mathad, R. I.; Flo¨gel, O.;
Limbach, M. HelV. Chim. Acta 2006, 89, 1801-1825.
(23) Synthetic schemes, experimental procedures, and characterization
of compounds 6-11 are reported in the Supporting Information.
(24) Carpino, L. A.; El-Faham, A.; Albericio, F. J. Org. Chem. 1995,
60, 3561-3564.
654 J. Org. Chem., Vol. 73, No. 2, 2008

Synthesis and Conformational Studies of Peptidomimetics
TABLE 1. 1H NMR Data for the Amide Protons in Compounds 7
and 8
exchange of H4/D upon addition of CD3OD is quite slow (ca.
960 min). In the case of proton NH1, the same parameters (i.e.,
values of chemical shift, temperature dependence, ∆δ upon
addition of CH3OH, and rate of exchange of H1/D upon addition
of CD3OD) are indicative of an equilibrium between an
intramolecularly hydrogen-bonded and a non-hydrogen-bonded
status for both 7 and 8. A similar equilibrium is also partially
displayed by proton NH2, although in this case the NMR
parameters reflect a looser intramolecular hydrogen bond.
NOE contacts can be highly indicative of the formation of a
â-hairpin mimic when interstrand contacts are visible. Unfor-
tunately, in the case of the tetrapeptide mimics 7 and 8 we could
not detect this kind of contacts, and only strong intrastrand
contacts were observed, which are indicative of an extended
conformation for the amino acid residues. The FT-IR spectrum
of the tetrapeptide mimic 7 (2 mM solution in CHCl3) is
characterized by two bands at 3427 and 3395 cm-1 (free NH
groups) and two prominent bands at 3321 and 3291 cm-1 (H-
bonded NH groups), respectively.26 In the case of 8, only two
7a
8b
∆δe
(added
(ppm) (ppb/K) (ppm) (ppb/K) CH3OH)
NH/ND
exchangef
(min)
δc
∆δ/∆Td
δc
∆δ/∆Td
NH1
NH2
NH3
NH4
NH5
7.81
7.03
6.28
8.10
5.41
-7.6
-8.2
-9.1
-4.1
-1.8
8.21
7.21
6.61
8.18
6.40
-4.6
-5.3
-15.5
-2.7
-2.3
-0.07
0.53
1.25
0.03
g0.7
300
160
<10
960
g
a Concentration 0.5 mM in CDCl3. b Concentration 2.0 mM in CDCl3.
c At 298 K. d Determined between 238 and 288 K. e Measured in CDCl3/
CH3OH 4/1. f Measured in CDCl3/CD3OD 4/1. g Not determined due to
overlap with other resonances.
bands can be recognized, one in the free NH region (3410 cm-1
)
and one at 3291 cm-1 indicative of H-bonded NH’s. These data
support the formation of a â-hairpin mimic involving 10-
membered and 18-membered H-bonded rings and a reverse turn
of the growing peptide chain. The weak hydrogen-bonded
character of NH2 might indicate that a different â-hairpin mimic
involving 12-membered and 16-membered H-bonded rings (vide
infra the molecular modeling discussion) is present as a minor
conformer at the equilibrium.
solution, and in the presence of a tertiary amine (N-methyl-
morpholine or N,N-diisopropylethylamine).
The tendency of the DKP-1-containing peptidomimetics 6-11
to adopt a â-hairpin conformation was then evaluated. Char-
acteristic differences in the NMR spectral parameters for
unstructured peptides and peptides in extended and intramo-
lecularly hydrogen bonded conformations have been reported
in organic solvents.4c,25 Chemical shifts and coupling constants
for the CR hydrogens reflect the average conformations of
individual amino acid residues, while the chemical shifts of the
NH hydrogens and their temperature dependence reveal whether
they are solvent exposed or hydrogen bonded intramolecularly.
The dipeptide mimic 6, in CDCl3, showed some degree of
concentration dependence and a strong temperature dependence
of the chemical shifts of all the NH’s (>20 ppb/K) at a 2 mM
concentration, while the chemical shift values were only slightly
deshielded (6.25-7.02 ppm) with respect to the average values
for non-hydrogen-bonded NH protons (ca. 6.0 ppm). These data
are in agreement with an equilibrium between a non-hydrogen-
bonded and an intermolecularly H-bonded status (aggregation).
We next turned our attention to the tetrapeptide mimics 7
and 8 (Scheme 2). The NMR studies for these compounds were
performed in CDCl3 (see Table 1). Dilution studies indicated
that in both cases no aggregates are formed in the concentration
range 0.5-10 mM. From the NMR data summarized in Table
1 it appears that the amide protons NH4 are in an intramolecu-
larly hydrogen-bonded status. In fact, (a) their resonance is
shifted substantially downfield (8.10 and 8.18 for 7 and 8,
respectively), (b) the temperature dependence of the NH4
chemical shift falls within the typical values for intramolecularly
hydrogen-bonded protons (-2.7 ppb/K for 8 and slightly higher
(-4.1 ppb/K) for 7), (c) the ∆δ (NH4) upon addition of CH3-
OH (obtained measuring the spectrum in a CDCl3/CH3OH, 4/1
mixture) is small (0.03 in the case of 8), and (d) the rate of
Computational studies designed to investigate the ability of
the DKP-1 scaffold to induce â-hairpin conformations were
performed on the tetrapeptide mimic 8. The molecule was
subjected to an extensive, unconstrained Monte Carlo/Energy
Minimization (MC/EM) conformational search27 by molecular
mechanics methods, using the AMBER* force field28 and the
implicit CHCl3 GB/SA solvent model.29
Only two types of conformations, both featuring a â-hairpin-
like arrangement, are predominant among the structures found
within 3 kcal/mol from the global minimum. The lowest energy
conformer features an intramolecular hydrogen-bonding pattern
involving the formation of 10-membered and 18-membered
H-bonded rings (Figure 2, 8a). The 10-membered ring of this
conformer features a gauche orientation of the NH and CdO
groups around the C2-C3 bond (â-amino acid numbering),13
with the â2 and â3 amino acid torsion angles (θ) of -87° and
81°, respectively.30 This cross-strand hydrogen-bonding pattern
is in agreement with the structure proposed on the basis of the
NMR experiments (see Table 1 and the discussion above).
A second â-hairpin-like conformer was found at 1.09 kcal/
mol from the global minimum, involving the formation of 12-
membered and 16-membered H-bonded rings (Figure 2, 8b).
The 12-membered ring requires anti C2-C3 torsion angles, with
θ values of -171° and -177° for the corresponding â2 and â3
amino acids. As also suggested by the NMR data reported in
(26) (a) Liang, G.-B.; Desper, J. M.; Gellman, S. H. J. Am. Chem. Soc.
1993, 115, 925-938. (b) Gellman, S. H.; Dado, G. P.; Liang, G.-B.; Adams,
B. R. J. Am. Chem. Soc. 1991, 113, 1164-1173.
(27) Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc. 1989,
111, 4379-4386.
(25) (a) Phillips, S. T.; Blasdel, L. K.; Bartlett, P. A. J. Org. Chem. 2005,
70, 1865-1871. (b) Nowick, J. S.; Lam, K. S.; Khasanova, T. V.; Kemnitzer,
W. E.; Maitra, S.; Mee, H. T.; Liu, R. J. Am. Chem. Soc. 2002, 124, 4972-
4973. (c) Nowick, J. S.; Smith, E. M.; Pairish, M. Chem. Soc. ReV. 1996,
25, 401-415. (d) Steffel, L. R.; Cashman, T. J.; Reutershan, M. H.; Linton,
B. R. J. Am. Chem. Soc. 2007, 129, 12956-12957.
(28) Weiner, S. J.; Kollman, P. A.; Nguyem, D. T.; Case, D. A. J.
Comput. Chem. 1986, 7, 230-252.
(29) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J. Am.
Chem. Soc. 1990, 112, 6127-6129.
(30) This is in agreement with the â-peptide sheetlike conformations
proposed by Gellman and co-workers (see ref 12d).
J. Org. Chem, Vol. 73, No. 2, 2008 655

Ressurreic¸a˜o et al.
SCHEME 4. Selected Intrastrand (Dashed Black Arrows)
and Interstrand (Dashed Red Arrows) NOE Contacts for
Hexapeptides 10 and 11
NMR analysis in DMSO-d6 suggests that both compounds 10
and 11 adopt a â-hairpin-type conformation with a 10-membered
H-bonded ring similar to that observed in the previous structures.
In fact, the ROESY spectra show a set of NOE cross-peaks
that support this conclusion (Scheme 4).
In particular, in the case of 10, several interstrand NOE
contacts were observed: a strong contact between the CRH of
the Val1 and CRH of the Ala2 residue (see also Figure 3a), a
contact between NH2 and the CRH of the Ala2 residue (see also
Figure 3b), and a weak contact between NH2 and NH7 (see also
Figure 3c). The same contacts were also observed when the
ROESY spectrum of 10 was collected in 5% CD3OH-CDCl3
(see the Supporting Information). A similar pattern is also shown
by the spectrum of 11 in DMSO-d6, with the exception of the
cross-peak between NH2 and NH7, which is too weak to be
detected in this case.
FIGURE 2. Structures of low-energy conformers (MC/EM, AMBER*,
CHCl3 GB/SA) calculated for compound 8: upper row, Global
minimum (8a); lower row, conformer with relative energy of 1.09 kcal/
mol (8b). Hydrogen bonds are indicated with dotted lines and for clarity
all nonpolar hydrogen atoms and phenyl groups have been omitted.
SCHEME 3. Proposed H-Bonded Structure for
Pentapeptide 9
The analysis of the 1H NMR spectrum of hexapeptide mimic
10 in 5% CD3OH-CDCl3 (Table 2) showed that protons NH2
and NH5 are notably shifted downfield with respect to the other
NH protons. The temperature dependency is indicative of a
hydrogen-bonded status for both NH2 and NH5, and of an
equilibrium between an intramolecularly hydrogen-bonded and
3
a non-hydrogen-bonded status for NH7. The J values for the
NH-CRH in DMSO-d6 for both compounds 10 and 11 (Table
2) are in agreement with the typical values for peptides showing
a â-hairpin secondary structure (7.5-8.5 Hz).25a Similar values
were also found for the spectrum of 10 in 5% CD3OH-CDCl3.
The ability of peptidomimetics 7, 8, 10, and 11 to adopt an
ordered secondary structure in solution was also evaluated by
CD spectroscopy (Figure 4). The spectra were measured in
methanol (0.5 mM) and showed a similar behavior: two
negative minima, one at 200-205 nm (201 nm for compound
11) and a second one at about 220 nm (220 nm for compound
11), and a negative maximum at 209-215 nm (209 nm for 11),
were displayed by all these compounds. Unfortunately, while
several CD studies have been reported for â-peptides adopting
helical conformations (and in particular 12- and 14-helices),12d,31
no conclusive data on â-peptides assuming hairpin-type con-
formations have appeared in the literature. A hexapeptide
consisting of â3-homo-amino, â2-homo-amino, and R-amino
acids with a central â2-â3 segment was recently reported by
Seebach and co-workers to adopt a turn-like conformation with
a 10-membered H-bonded ring induced by the â2-â3 unit.22
Its CD spectrum (0.2 µM in CH3OH) displayed a similar
Table 1, this second type of â-hairpin structure, or at least its
12-membered H-bonded ring portion, might participate as a
minor conformer to the conformational equilibrium.
The solution structure of the pentapeptide mimic 9 was also
studied by determining the temperature dependence of the NH
chemical shifts at 2 mM in CDCl3 (no aggregation was detected
at this concentration). An equilibrium between an intramolecu-
larly hydrogen-bonded and a non-hydrogen-bonded status is
suggested by the various parameters for protons NH2 and NH5
(δ ) 7.71 and 7.77; ∆δ/∆T ) -8.3 and -6.8 ppb/K,
respectively; see the Supporting Information for the complete
set of parameters), which might be indicative of the presence
of a â-hairpin conformation with 10-membered and 18-
membered H-bonded rings, in analogy to the tetrapeptide-mimics
7 and 8 (Scheme 3).
The hexapeptides 10 and 11 (Scheme 2) were then prepared;
the first dramatic difference with respect to the shorter homo-
logues was the insolubility of these products, in particular 11.
In fact 11 was only soluble in DMSO and in hot methanol,
while compound 10 was also sparingly soluble in CHCl3 and
soluble in methanol. For this reason the NMR studies of these
compounds were performed in DMSO-d6 and, in the case of
10, also in 5% CD3OH-CDCl3. All the proton resonances could
be assigned by means of COSY and ROESY spectra. The 2D-
(31) (a) Hart, S. A.; Bahadoor, A. B. F.; Matthews, E. E.; Qiu, X. J.;
Schepartz, A. J. Am. Chem. Soc. 2003, 125, 4022-4023. (b) Ahmed, S.;
Beleid, R.; Sprules, T.; Kaur, K. Org. Lett. 2007, 9, 25-28.
656 J. Org. Chem., Vol. 73, No. 2, 2008

Synthesis and Conformational Studies of Peptidomimetics
FIGURE 3. Sections of the ROESY spectrum of 10 (2 mM in DMSO-d6) showing (a) interstrand NOE’s in the CRH region, (b) interstrand NOE’s
for CRH and NH, and (c) interstrand NOE’s for the NH.
TABLE 2. 1H NMR Data for the Amide Protons in Compounds 10
and 11
10
11
δa,b
∆δ/∆Ta,c
δ
a,d
e,f
3
e,f
(ppm) (ppb/K) 3JNHCHR
3JNHCHR
(ppm)e,f JNHCHR
NH1 6.99
NH2 8.24
NH3 7.61
NH4 7.68
NH5 7.97
NH6 7.42
NH7 5.76
-8.0
-2.5
-15.5
-16.5
-1.3
-8.5
-6.0
5.7
7.7
8.0
g
g
8.0
6.8
5.6
7.4
8.1
g
g
7.8
9.1
7.83
8.19
8.29
8.28
8.25
8.18
7.96
5.6
7.9
7.2
g
g
7.8
8.9
FIGURE 4. CD spectra of peptidomimetics 7, 8, 10, and 11 (0.5 mM
in methanol). The data are normalized for peptide concentration and
for the number of residues.
TABLE 3. Relevant Proton Distances of the Low-Energy
Conformers (MC/EM, AMBER*, in vacuo) Calculated for
Compound 11
a Concentration 2.0 mM in 5% CD3OH-CDCl3. b At 288 K. c Deter-
mined between 248 and 288 K. d At 278 K. e Concentration 2.0 mM in
DMSO-d6. f At 298 K. g Broad signal.
proton distance (Å)
∆E
conformer (kcal/mol) CRH(Val1)-CRH(Ala2) CRH(Ala2)-NH2
11a
11b
0.0
0.26
7.56
2.49
7.92
3.95
behavior with respect to our derivatives, with a minimum at
197 nm, a shoulder at 205 nm, a negative maximum at about
215 nm, and a less pronounced minimum at ca. 220 nm. In
addition, both minima and the maximum showed negative molar
ellipticities of comparable intensity to those of our compounds.
Molecular mechanics calculations were performed on the
hexapeptide mimic 11, similarly to the tetrapeptide mimic 8, to
investigate the ability of the DKP-1 scaffold to induce â-hairpin
conformations. The molecule was subjected to an unconstrained
Monte Carlo/Energy Minimization (MC/EM) conformational
search27 in vacuo (the implicit DMSO solvation model is not
available in the software employed) with a distance dependent
dielectric constant of 4r to generate a suitable starting confor-
mation for the following restrained simulation in explicit DMSO
solvent (see below). Two different, almost energetically equiva-
lent, â-hairpin conformations were found within 3 kcal/mol from
the global minimum (Figure 5). The lowest energy conformer
is characterized by the presence of hydrogen bonds involving
the amide protons NH3, NH6, and NH1 and forming respectively
12-membered, 16-membered, and 24-membered rings (Figure5,
11a). However, no experimental evidence is provided by the
NMR data in solution (Scheme 4, Table 2) for such an
intramolecular hydrogen-bonding pattern (resembling conformer
8b of the tetrapeptide mimic 8, see the discussion above).
The second conformer (Figure 5, 11b) shows a â-hairpin
conformation in agreement with the structure proposed on the
basis of the spectroscopic data (Scheme 4, Table 2). This
conformer features 10-membered and 18-membered H-bonded
rings that resemble the hydrogen-bonding pattern observed in
conformer 8a of the tetrapeptide mimic 8, and an additional
22-membered H-bonded ring involving the NH7 amide proton
and the Ala1 carbonyl group.
Furthermore, comparing the calculated interstrand distances
in conformers 11a and 11b between protons CRH of the Val1
and CRH of the Ala2 residues and between the proton CRH of
J. Org. Chem, Vol. 73, No. 2, 2008 657

Ressurreic¸a˜o et al.
FIGURE 6. Simulated annealing superimposed solutions. Hydrogen
bonds are indicated with dotted lines and all nonpolar hydrogen atoms,
except CRH of Val1 and Ala2 residues, have been omitted for clarity.
Experimental Section
(S)-N-Benzyl-3-tert-butoxycarbonylamino-N-[(S)-2-hydroxy-
1-methoxycarbonylethyl]succinamic Acid Allyl Ester (2). To a
solution of â-allyl (2S)-N-(tert-butoxycarbonyl)aspartate ester (329
mg, 1.2 mmol) in CH2Cl2 (8 mL), under a nitrogen atmosphere
and at 0 °C, was added HATU (510 mg, 1.3 mmol, 1.1 equiv) and
DIPEA (417 µL, 2.4 mmol, 2 equiv). After 30 min, a solution of
(S)-N-benzylserine methyl ester (251 mg, 1.2 mmol, 1 equiv) in
CH2Cl2 (1.6 mL) was added and the reaction was stirred at 0 °C
for 1 h and at rt for 24 h. The mixture was then diluted with EtOAc
(100 mL) and the organic phase was washed in order with 1 M
KHSO4 (2 × 20 mL), aqueous NaHCO3 (2 × 20 mL), and brine
(2 × 20 mL) and dried over Na2SO4, then volatiles were removed
under reduced pressure. The residue was purified by flash chro-
matography on silica gel (petroleum ether/EtOAc, 75:25) to afford
FIGURE 5. Structures of the lowest energy conformers (MC/EM,
AMBER*, in vacuo) calculated for compound 11: upper row, Global
minimum (11a); lower row, conformer with relative energy of 0.26
kcal/mol (11b). Hydrogen bonds are indicated with dotted lines and
for clarity all nonpolar hydrogen atoms, except CRH of Val1 and Ala2
residues, and the phenyl residues, have been omitted.
the Ala2 residue and the NH2 amide proton (Table 3), only the
11b conformer shows distance values consistent with the NOE
contacts observed in DMSO solution (Scheme 4).
Finally, a simulated annealing protocol in explicit DMSO
solvent32 with the NMR restraints derived from the NOE
contacts (see the Supporting Information for computational
details) was performed starting from conformer 11b. The
simulation converged to a unique â-hairpin structure (Figure
6). Consistent with the NMR analysis, this â-hairpin arrange-
ment features 10-membered and 18-membered H-bonded rings
involving the NH5 and NH2 amide protons, respectively, while
the NH7 amide proton does not form any intramolecular
hydrogen bonds.
the desired product as a yellow oil (401 mg, 72%). [R]21 -2.65
D
1
(c 1.0, CHCl3); H NMR (CDCl3) δ 7.24-7.34 (m, 5H), 5.83-
5.93 (m, 1H), 5.48 (d, 1H, J ) 8.2 Hz), 5.30 (d, 1H, J ) 17.2 Hz),
5.23 (d, 1H, J ) 10.4 Hz), 4.51-4.61 (m, 3H), 4.32-4.48 (m,
2H), 3.88 (d, 1H, J ) 13.1 Hz), 3.75 (s, 3H), 3.73 (d, 1H, J ) 13.1
Hz), 3.55 (t, 1H, J ) 4.7 Hz), 2.99 (dd, 1H, J1 ) 17.0 Hz, J2 ) 4.3
Hz), 2.85 (dd, 1H, J1 ) 17.0 Hz, J2 ) 4.7 Hz), 2.21 (br s, 1H),
1.45 (s, 9H). Two sets of signals were observed in the 13C spectrum
due to the presence of two rotational isomers A:B (20:1 ratio): 13C
NMR (CDCl3) δ 172.8 (A), 172.1 (B), 171.0 (A), 170.3 (B), 155.7
(A), 155.0 (B), 139.6 (A), 138.9 (B), 132.1 (A), 131.4 (B), 128.9
(A), 128.7 (A), 128.1 (B), 127.9 (B), 127.6 (A), 126.9 (B), 119.1
(A), 118.4 (B), 80.6 (A), 79.9 (B), 66.2 (A), 66.1 (A), 65.5 (B),
65.4 (B), 59.5 (A), 58.8 (B), 52.7 (A), 52.2 (A), 51.9 (B), 51.4
(B), 50.3 (A), 49.6 (B), 37.1 (A), 36.4 (B), 28.7 (A), 27.9 (B); IR
(CHCl3) νmax 3438, 3338, 3026, 2983, 2953, 2857, 1739, 1500,
1453, 1378, 1341, 1279, 1247, 1176; HRMS (ESI) m/z calcd for
[C23H33N2O8]+ 465.22314 [M + H]+, found 465.22326. Anal. Calcd
for C23H32N2O8: C 59.47, H 6.94, N 6.03. Found: C 59.07, H
7.01, N 5.91.
Conclusions
In this paper, we reported the synthesis of a new bifunctional
diketopiperazine (DKP) scaffold 1, derived from L-aspartic acid
and (S)-2,3-diaminopropionic acid. DKP-1 bears amino and
carboxylic acid functionalities in a cis relationship. As a
consequence, DKP scaffold 1 can be seen as a conformationally
constrained mimic of a dipeptide formed by two â-amino acids
(namely â2 and â3 amino acids). When inserted into a peptidic
sequence, involving R-amino acids, DKP-1 is accommodated
into the turn position of a â-hairpin. IR, NMR, and CD
experiments provide strong support to this conclusion, strength-
ened by molecular modeling and molecular dynamics calcula-
tions.
[(2S,5S)-4-Benzyl-5-hydroxymethyl-3,6-dioxopiperazin-2-yl]-
acetic Acid Allyl Ester (3). Dipeptide 2 (1.95 g, 4.2 mmol) was
dissolved in TFA (32 mL) and stirred for 3 h at rt. The solvent
was evaporated, methanol (3 × 50 mL) was added followed by
evaporation, and then Et2O (35 mL) was added and evaporated to
give the TFA salt of the dipeptide 2 as a white solid. This salt was
dissolved in a mixture of saturated aqueous NaHCO3/EtOAc (0.1
M, 1:1 v/v) and stirred at room temperature for 24-48 h.
Subsequently, the layers were separated and the aqueous layer was
(32) Fox, T.; Kollman, P. A. J. Phys. Chem. B 1998, 102, 8070-8079.
658 J. Org. Chem., Vol. 73, No. 2, 2008

Synthesis and Conformational Studies of Peptidomimetics
extracted with EtOAc (4×). The combined organic layers were
washed with brine and dried over Na2SO4, then volatiles were
removed under reduced pressure. The residue was purified by flash
chromatography on silica gel (CH2Cl2/CH3OH, 97:3) to afford the
Hz, J2 ) 11.1 Hz), 1.46 (s, 9H); 13C NMR (CDCl3) δ 171.5, 166.7,
164.9, 156.2, 135.6, 131.8, 129.4, 128.9, 128.5, 119.3, 80.8, 66.4,
59.2, 52.4, 47.2, 40.8, 40.6, 28.7; IR (Nujol) νmax 3323, 3308, 1716,
1684, 1658, 1339, 1272, 1167, 1127; MS (FAB+) m/z 432 ([M +
1]+, 12%), 376 (49%), 332 (41%), 302 (16%), 91 (100%). Anal.
Calcd for C22H29N3O6: C 61.24, H 6.77, N 9.74. Found: C 61.47,
H 6.93, N 9.56.
desired product as a white solid (1.13 g, 81%). Mp 119-120 °C;
1
[R]25 -72.1 (c 1.0, CHCl3); H NMR (CDCl3) δ 7.25-7.37 (m,
D
5H), 7.05 (br s, 1H), 5.84-5.94 (m, 1H), 5.25-5.34 (m, 3H), 4.55-
4.66 (m, 2H), 4.49-4.51 (m, 1H), 4.07 (d, 1H, J ) 15.0 Hz), 3.98
(d, 1H, J ) 11.1 Hz), 3.85-3.89 (m, 2H), 3.21 (dd, 1H, J1 ) 17.5
Hz, J2 ) 2.8 Hz), 3.16 (br s, 1H), 3.13 (dd, 1H, J1 ) 17.5 Hz, J2
) 10.4 Hz); 13C NMR (CDCl3) δ 171.9, 167.0, 166.3, 135.6, 131.9,
129.5, 128.6, 119.5, 66.4, 61.3, 60.5, 52.8, 47.6, 40.7; IR (CHCl3)
[(2S,5S)-4-Benzyl-5-(tert-butoxycarbonylaminomethyl)-3,6-di-
oxopiperazin-2-yl]acetic Acid, DKP-1 (1). To a solution of 5 (242
mg, 0.56 mmol) in CH2Cl2 (3.0 mL), under nitrogen atmosphere
and at 0 °C, was added pyrrolidine (56 µL, 0.67 mmol, 1.2 equiv),
PPh3 (26 mg, 0.10 mmol, 0.18 equiv), and then [Pd(PPh3)4] (24
mg, 0.02 mmol, 0.04 equiv). After the mixture was stirred for 1 h
at 0 °C, EtOAc (25 mL) was added and the solution was extracted
with aqueous NaHCO3 (4 × 10 mL). The combined aqueous phases
were acidified to pH 2 with a 1 M KHSO4 solution and then
extracted with CH2Cl2. The resulting organic phase was dried over
Na2SO4 and the solvent was evaporated to afford the desired product
as a fluffy white solid (209 mg, 95%). [R]26D -69.9 (c 1.0, CHCl3);
1H NMR (CDCl3, 50 °C) δ 10.02 (br s, 1H), 8.05 (br s, 1H), 7.25-
7.37 (m, 5H), 5.59 (d, 1H, J ) 14.2 Hz), 5.36 (br s, 1H), 4.52 (d,
1H, J ) 11.4 Hz), 4.03 (br s, 1H), 3.88 (s, 1H), 3.79-3.85 (m,
1H), 3.49-3.54 (m, 1H), 3.28 (dd, 1H, J1 ) 17.7 Hz, J2 ) 2.3
ν
max 3388, 3275, 3031, 3017, 2945, 1728, 1680, 1452, 1379, 1336,
1276, 1183, 1124; MS (FAB+) m/z 333 ([M + 1]+, 80%), 275
(11%), 154 (57%), 136 (48%), 91 (100%). Anal. Calcd for
C17H20N2O5: C 61.44, H 6.07, N 8.43. Found: C 61.23, H 5.97, N
8.24.
X-ray crystallographic data of 3: C17H20N2O5; MW ) 332.35
g mol-1; T ) 293 K; λ(Mo KR) ) 0.71073 Å, monoclinic, space
group P21, a ) 7.394(4) Å, b ) 10.764(19) Å, c ) 10.800(5) Å,
â ) 99.71(4)°, V ) 847(2) Å,3 Fcalc ) 1.303 g cm-3, Z ) 2; µ(Mo
KR) ) 1.0 cm-1. R and wR2 0.086 and 0.155, respectively, for
1230 unique data collected in the 3-25.3° 2θ range.
Hz), 2.74 (dd, 1H, J1 ) 17.7 Hz, J2 ) 11.4 Hz), 1.50 (s, 9H); 13
C
[(2S,5S)-5-Azidomethyl-4-benzyl-3,6-dioxopiperazin-2-yl]ace-
tic Acid Allyl Ester (4). To a solution of 3 (565 mg, 1.7 mmol) in
CH2Cl2/toluene (6.6 mL/12.2 mL), under nitrogen atmosphere and
at -20 °C, was added PPh3 (530 mg, 2.0 mmol, 1.2 equiv) and the
mixture was stirred until a solution was obtained. Hydrazoic acid
(0.45 M in toluene,33 7.6 mL, 3.4 mmol, 2 equiv) was added
followed by a dropwise addition of DIAD (0.41 mL, 2.0 mmol,
1.2 equiv) and the reaction was stirred at -20 °C for 3.5 h. After
evaporation of the solvent under reduced pressure, a quick
chromatographic purification (petroleum ether/EtOAc, 6:4) was
performed to remove the hydrazo-derivative and the resulting crude
residue was then purified by flash chromatography on silica gel
(CH2Cl2/CH3OH, 99:1) to afford the desired product as a colorless
oil (291 mg, 48%). [R]23D -72.7 (c 1.9, CHCl3); 1H NMR (CDCl3)
δ 7.26-7.39 (m, 5H), 6.91 (br s, 1H), 5.89-5.99 (m, 1H), 5.36 (d,
1H, J ) 17.2 Hz), 5.30 (d, 1H, J ) 10.4 Hz), 5.18 (d, 1H, J )
15.0 Hz), 4.62-4.71 (m, 2H), 4.51-4.54 (m, 1H), 4.20 (d, 1H, J
) 15.0 Hz), 3.95 (br s, 1H), 3.89 (dd, 1H, J1 ) 12.7 Hz, J2 ) 1.7
Hz), 3.68 (dd, 1H, J1 ) 12.7 Hz, J2 ) 3.4 Hz), 3.31 (dd, 1H, J1 )
17.7 Hz, J2 ) 2.2 Hz), 3.08 (dd, 1H, J1 ) 17.7 Hz, J2 ) 11.2 Hz);
13C NMR (CDCl3) δ 171.6, 165.7, 165.1, 135.3, 131.8, 129.6, 128.8,
128.6, 119.6, 66.5, 58.8, 52.6, 51.1, 48.0, 40.7; IR (thin film) νmax
2984, 2929, 2853, 2119, 1734, 1686, 1667, 1451, 1336, 1274, 1181;
MS (FAB+) m/z 358 ([M + 1]+, 12%), 330 (2%), 149 (16%), 109
(27%), 91 (100%). Anal. Calcd for C17H19N5O4: C 57.14, H 5.36,
N 19.60. Found: C 57.39, H 5.28, N 19.25.
NMR (CDCl3, 50 °C) δ 175.1, 168.1, 164.9, 157.0, 135.4, 129.4,
128.8, 128.6, 81.4, 59.5, 52.4, 47.3, 40.9, 40.6, 28.7; IR (Nujol)
ν
max 3382, 3325, 3227, 1715, 1659, 1647, 1272, 1162, 1125; HRMS
(ESI) m/z calcd for [C19H25N3NaO6]+ 414.16356 [M + Na]+, found
414.16367.
Solution-Phase Synthesis of Peptidomimetics. Representative
Procedure for the Coupling with HOBT/EDC: Boc-(S,S)-DKP-
1-(S)-Ala-NH-CH2-Ph. To a solution of Boc-(S)-Ala-NH-CH2-Ph
(67 mg, 0.24 mmol) in CH2Cl2 (1.85 mL; 0.13 M) was added an
equal volume of TFA and the reaction was stirred at rt for 3 h.
The solvent was evaporated, methanol (3 × 2 mL) was added
followed by evaporation, and then ether was added and evaporated
to afford the corresponding TFA salt. This was dissolved in DMF
(2.4 mL, 0.1 M), and 1 (98 mg, 0.25 mmol, 1.05 equiv) was added
followed by HOBt (36 mg, 0.26 mmol, 1.1 equiv) and DIPEA (84
µL, 0.48 mmol, 2 equiv). The solution was cooled in an ice bath
and treated with EDC (40 mg, 0.26 mmol, 1.1 equiv). The reaction
was stirred at 0 °C for 1 h and at rt overnight. The mixture was
diluted with EtOAc (15 mL) and consecutively extracted with 1 M
KHSO4 (2 × 3 mL), aqueous NaHCO3 (2 × 3 mL), and brine (2
× 3 mL) and dried over Na2SO4, then the solvent was evaporated
under reduced pressure. The residue was purified by flash chro-
matography (CH2Cl2/CH3OH, 95/5) to afford the product (122 mg,
92%) as a white solid. Mp 112-113 °C; [R]28 -98.4 (c 0.50,
D
1
CDCl3); H NMR (CDCl3, 40 °C) δ 7.48 (br s, 1H), 7.18-7.34
[(2S,5S)-4-Benzyl-5-(tert-butoxycarbonylaminomethyl)-3,6-di-
oxopiperazin-2-yl]acetic Acid Allyl Ester (5). To a solution of
azide 4 (268 mg, 0.75 mmol) in THF (2.5 mL), under nitrogen
atmosphere and at -20 °C, was added Me3P (830 µL of a 1 M
solution in THF, 0.83 mmol, 1.1 equiv) and 2-(t-butoxycarbony-
loxyimino)-2-phenylacetonitrile (Boc-ON, 206 mg, 0.83 mmol, 1.1
equiv). After the mixture was stirred for 5 h at rt, CH2Cl2 (60 mL)
was added and the solution was washed with H2O (3 × 30 mL)
and brine. The organic phase was dried over Na2SO4 and volatiles
were removed under reduced pressure. The residue was purified
by flash chromatography on silica gel (CH2Cl2/CH3OH, 99:1) to
(m, 12H), 5.83 (br s, 1H), 5.41 (d, 1H, J ) 15.0 Hz), 4.51-4.59
(m, 1H), 4.35-4.44 (m, 3H), 3.99 (d, 1H, J ) 15.0 Hz), 3.78 (br
s, 1H), 3.61-3.72 (m, 1H), 3.49-3.59 (m, 1H), 3.05 (dd, 1H, J1
) 15.1 Hz, J2 ) 3.9 Hz), 2.74 (dd, 1H, J1 ) 15.1 Hz, J2 ) 8.8
Hz), 1.42 (s, 9H), 1.36 (d, 3H, J ) 6.9 Hz); 13C NMR (CDCl3, 40
°C) δ 172.5, 170.4, 166.4, 166.1, 156.4, 138.6, 135.6, 129.3, 128.9,
128.8, 128.5, 128.0, 127.7, 80.6, 59.2, 53.2, 49.6, 47.5, 43.9, 41.7,
41.4, 28.8, 18.5; IR (Nujol) νmax 3354, 3320, 3240, 1717, 1658,
1639, 1552, 1532, 1249, 1173, 1076; MS (FAB+) m/z 552 ([M +
1]+, 4%), 452 (18%), 369 (6%), 147 (31%), 109 (54%), 91 (100%).
Anal. Calcd for C29H37N5O6: C 63.14, H 6.76, N 12.70. Found: C
62.84, H 6.75, N 12.53.
Solution-Phase Synthesis of Peptidomimetics. Representative
Procedure for the Coupling with HATU: Boc-(S)-Ala-(S,S)-
DKP-1-(S)-Ala-NH-CH2-Ph (7). To a solution of Boc-(S,S)-DKP-
1-(S)-Ala-NH-CH2-Ph (61 mg, 0.11 mmol) in CH2Cl2 (0.85 mL)
was added an equal volume of TFA and the reaction was stirred at
rt for 3 h. The solvent was evaporated, methanol (3 × 2 mL) was
added followed by evaporation, and then ether (3 mL) was added
and evaporated to afford the corresponding TFA salt. To a solution
afford the desired product as a white solid (253 mg, 78%). [R]28
D
1
-123.7 (c 1.0, CHCl3); H NMR (CDCl3) δ 7.28-7.36 (m, 5H),
7.06 (br s, 1H), 5.86-5.96 (m, 1H), 5.56 (d, 1H, J ) 15.1 Hz),
5.25-5.36 (m, 3H), 4.60-4.69 (m, 2H), 4.48-4.51 (m, 1H), 4.09
(d, 1H, J ) 15.1 Hz), 3.80-3.86 (m, 2H), 3.45-3.49 (m, 1H),
3.27 (dd, 1H, J1 ) 17.6 Hz, J2 ) 1.7 Hz), 2.85 (dd, 1H, J1 ) 17.6
(33) Equi, A. M.; Brown, A. M.; Cooper, A.; Ner, S. K.; Watson, A. B.;
Robins, D. J. Tetrahedron 1991, 47, 507-518.
J. Org. Chem, Vol. 73, No. 2, 2008 659

Ressurreic¸a˜o et al.
of Boc-(S)-Ala-OH (21 mg, 0.11 mmol, 1 equiv), in CH2Cl2 (0.55
mL), under nitrogen atmosphere and at 0 °C, was added HATU
(46 mg, 0.12 mmol, 1.1 equiv) and DIPEA (38 µL, 0.22 mmol, 2
equiv). After 30 min, a solution of the TFA salt of the peptide in
CH2Cl2 (0.55 mL) and DIPEA (19 µL, 0.11 mmol, 1 equiv) was
added and the reaction mixture was stirred at 0 °C for 1 h and at
rt overnight. The mixture was diluted with EtOAc (10 mL) and
consecutively extracted with 1 M KHSO4 (2 × 3 mL), aqueous
NaHCO3 (2 × 3 mL), and brine (2 × 3 mL) and dried over Na2-
SO4, then the solvent evaporated under reduced pressure. The
residue was purified by flash chromatography (CH2Cl2/CH3OH, 95/
C32H42N6O7: C 61.72, H 6.80, N 13.50. Found: C 61.42, H 6.78,
N 13.35.
Acknowledgment. We thank the European Commission
(Marie Curie Early Stage Research Training Fellowship “Fol-
damers” MEST-CT-2004-515968) for financial support and for
Ph.D. Fellowships (to A.S.M.R. and A.B.). We also gratefully
acknowledge Merck Research Laboratories (Merck’s Academic
DevelopmentProgramAwardtoC.G.)andMinisterodell’Universita`
e della Ricerca (PRIN prot. 2006030449) for financial support
and CILEA for computing facilities. We are grateful to Prof.
N. Masciocchi and Dr. S. Galli (University of Insubria) for the
X-ray structural determination of compound 3. We also would
like to thank Dr. D. Potenza (University of Milano) and Dr. C.
Cabrele (University of Regensburg) for helpful discussions.
5) to afford 7 (54 mg, 82%) as a white solid. Mp 127-128 °C;
1
[R]25 -42.5 (c 0.41, CH3OH); H NMR (CDCl3) δ 8.20 (br s,
D
1H), 8.06 (br s, 1H), 7.75 (d, 1H, J ) 6.9 Hz), 7.55 (br s, 1H),
7.26-7.35 (m, 7H), 7.15-7.21 (m, 3H), 5.52 (d, 1H, J ) 7.5 Hz),
5.39 (d, 1H, J ) 15.0 Hz), 4.67 (t, 1H, J ) 6.0 Hz), 4.47 (t, 1H,
J ) 6.7 Hz), 4.41 (br s, 2H), 4.08 (br s, 1H), 3.99 (br s, 1H), 3.96
(d, 1H, J ) 15.0 Hz), 3.75-3.82 (m, 2H), 3.16 (d, 1H, J ) 15.2
Hz), 2.80 (d, 1H, J ) 15.2 Hz), 1.42 (d, 3H, J ) 6.0 Hz), 1.29 (br
s, 12H); 13C NMR (CDCl3) δ 173.9, 173.7, 170.3, 166.1, 165.7,
155.9, 138.4, 135.5, 129.4, 129.0, 128.7, 128.5, 127.6, 127.2, 80.1,
57.1, 52.4, 49.8, 46.9, 43.6, 39.5, 38.3, 28.7, 20.8, 19.4; IR (CHCl3)
Supporting Information Available: Synthetic schemes, ex-
perimental procedures, computational methods, and characterization
of compounds 6-11, 1H and 13C NMR spectra of all reported
compounds, and conformational studies of compounds 7-11, X-ray
crystallographic data for compound 3. This material is available
free of charge via the Internet at http://pubs.acs.org.
ν
max 3429, 3395, 3330, 3295, 2930, 1689, 1657, 1556, 1506, 1449,
1368, 1332, 1255, 1166; HRMS (ESI) m/z calcd for [C32H42N6-
NaO7]+ 645.30072 [M + Na]+, found 645.29916. Anal. Calcd for
JO702072Z
660 J. Org. Chem., Vol. 73, No. 2, 2008