A novel azetidinyl γ-lactam based peptide with a preference for β-turn conformation

Article abstract of DOI:10.1039/b511029g

Synthesis of a new azetidinyl γ-lactam based peptides 1-3 with the preference for β-turn conformation is discussed. The tripeptide model compounds, namely the trans and the corresponding cis isomers, were studied by H NMR spectroscopy to measure the temperature coefficients of the two NH photons. It was observed that temperature coefficient NH of glycine was lowest, phenyl alanine-NH has higher temperature coefficient, and for both the cis tripeptides the temperature coefficients of shifts for all the NHs are above 5.0. The results show that the stereochemistry of β-lactam ring plays a key role in controlling the conformation of the peptides.

Full text of DOI:10.1039/b511029g

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C O M M U N I C A T I O N
A novel azetidinyl c-lactam based peptide with a preference for
b-turn conformation†
Amit Basak,*a Subhash C. Ghosh,a Amit K. Dasb and Valerio Bertolasic
a Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology,
Kharagpur, 721302, India
b Department of Biotechnology, Indian Institute of Technology, Kharagpur, 721302, India
c Dipartimento di Chimica, Universita di Ferrara, Ferrara, Italy
Received 4th August 2005, Accepted 28th September 2005
First published as an Advance Article on the web 12th October 2005
Novel azetidinyl c-lactam based peptides 1–3 have been
synthesized with only compound 1 showing a preference
for the b-turn conformation.
H-bond as shown can then be visualized which will ensure the
retention of the b-turn. An alternative structural design C can
also be thought of in which the N of (i + 1) and C(a) amino
acid interchange their positions. This should not disturb the H-
bond and should retain the turn structure as conceived. Since
structure C is more amenable towards synthesis, we had to work
on its synthesis and to study its conformational characteristics.8
The physiological role displayed by a large number of peptides
has stimulated researchers all over the world to design and
synthesize similar molecules, which are now collectively called
peptidomimetics.1 Natural peptides seldom can be used ther-
apeutically as drugs because of the problems associated with
low absorption, rapid metabolism and poor oral bioavailability.2
Peptidomimetics, designed on the basis of modification of the
natural sequence of amino acids in bioactive peptides, have the
advantage of providing new functionalities that can circumvent
these problems.3 However, since small molecules are usually
conformationally flexible and hence have to cross a considerable
entropy barrier to adopt the bioactive conformation, it would
be favourable to design molecules with rigid conformation.
Rigidity in conformation can be achieved by introducing
features in the structure that will induce the molecule to adopt a
particular conformation.4 An intriguing challenge in the design
of peptidomimetics is the development of templates that stabilize
structures resembling secondary structure motifs of peptides.
Reverse turn mimetics5 have so far been the prime target in this
area. The localization of turns on the surface of proteins has led
to the belief that these must play an important role in receptor–
peptide recognition events.6 The b-turn which is the most
common in peptides, is a tetrapeptide sequence in a non-helical
region, in which the distance between Ca(i) to the Ca (i + 3) is
Before embarking on the actual synthesis, a conformational
analysis was performed. Low-energy conformers were generated
by molecular mechanics force field calculations using Spartan’04
VI 0.0 (Fig. 1).9 Only the trans peptide was shown to adopt
a conformation in which the glycine NH and the b-lactam
˚
carbonyl are within a distance of 1.813 A, indicating strong
intramolecular H-bonding possibility. No such preferences
could be seen for the cis peptides.
˚
less than or equal to 7 A and the donor/acceptor distance (i +
3)NH · · · OC(i) of the turn-stabilizing hydrogen bond (typically,
˚
˚
1.8–2.5 A for H · · · O distance and 2.6–3.2 A for N · · · O
distance).7 The induction of b-turns in non-peptide molecules
is driven by the formation of such an intramolecular H-bond.
Herein we report our investigation of the design, synthesis
and conformational characterization of a novel azetidinyl c-
lactam based peptide that illustrates this concept. In addition,
the identification of the structural parameters along with the
stereochemistry involved in the stabilization of lactam-based
peptidomimetics is also reported.
Fig. 1 Energy minimized conformation of 1.
The synthesis of the peptides relied upon the availability
of the 3-pyroglutamylmethyl b-lactam in cis and trans forms,
preferably enanatiomerically pure. To prepare these compounds,
the Kinugasa reaction10 was our method of choice because of
its mild conditions coupled with the easy access to various
propargyl acetylenes used as the 2-electron component for these
reactions. An asymmetric version of the Kinugasa reaction is
also quite well known.11 Our synthetic strategy is shown in
Scheme 1. The propargyl ethyl pyroglutamate 6 was prepared
and the Kinugasa reaction between 6 and the diphenyl nitrone 7
was carried out in CH3CN solution in presence of triethylamine
and cuprous iodide. Interestingly, the reaction produced three
diastereomers: one trans isomer 8 and a pair of cis isomers 9 and
10. It appears that only one of the cis isomer 10 has epimerized to
the trans compound. The other cis isomer 9 is configurationally
more stable and is resistant to epimerization. The two cis isomers
had similar polarity on Si-gel and we decided to carry out the
synthesis of the peptides with the mixture. However, elaboration
The natural b-turn is represented by structure A in which a
10-membered H-bond network is formed. Our idea to design the
lactam-based peptidomimetic represented by B is based upon the
consideration of the following: a cyclic moiety replaces a C(a)–N
bond of the i + 2 amino acid, (ii) carbonyl of (i + 1) amino acid is
excluded from the backbone and placed in the side chain and (iii)
most importantly, connecting the N of (i + 1) amino acid and
C(a) of amino acid (i) by a methylene bridge. An intramolecular
† Electronic supplementary information (ESI) available: Experimental
procedures, 1H NMR, 13C NMR, VT NMR and NOESY spectra of
representative compounds, energy minimized conformation of 1 and
ORTEP diagram and X-ray crystallographic data for compound 11. See
DOI: 10.1039/b511029g
4 0 5 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 5 0 – 4 0 5 2
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Scheme 1 Synthesis of the tripeptides 1–3.
to the peptide was first carried out with the trans isomer as
this was one pure diastereomer and for that, the ester in the
pyroglutamic acid moiety needed to be hydrolysed. Initially,
considering the sensitivity of b-lactams towards nucleophiles,
enzymatic hydrolysis was considered; both porcine pancreatic
lipase (PPL) and porcine liver esterase (PLE) gave poor yield of
free acid 11. Finally, LiOH in THF–H2O12 was found to provide
the free acid 11 in acceptable yield (75%). Since the configuration
at C-2 was prefixed as we have started from S-pyroglutamic
acid and since the configuration at this centre remains unaltered
as can be seen from the production of only one diastereomer
upon coupling, the absolute configuration of the acid 11 could
be ascertained from the single crystal X-ray structure (ORTEP
diagram shown in Fig. 2).13 The acid was then coupled with
the C-protected dipeptide 14 in presence of EDCI–HOBT to
afford the protected tripeptide 1 as a white solid. Similarly, the
cis isomers were deprotected to the free acids 12/13 and were
coupled to the free amine to prepare the protected tripeptides
2/3 which could only be separated by HPLC.
at d 4.88 with a coupling constant of 2.1 Hz characteristic of
trans configuration. The cis-isomers behaved similarly with the
NH’s appearing as triplet and doublet in the most downfield
region. C-4 hydrogen expectedly appeared as a doublet with a
characteristic coupling constant of 6 Hz.
The tripeptide model compounds, namely the trans and
the corresponding cis isomers, were studied by 1H NMR
spectroscopy to measure the temperature coefficients of the two
NH protons. This method has become a useful tool to determine
the presence of intramolecular H-bonding. The temperature
coefficients of the various NH’s are listed in Table 1. In the
trans peptide 1 the temperature coefficient NH of glycine is the
lowest and is close to the Kessler limit14 of 3 ppb suggesting
strong intramolecular H-bonding. That the phenyl alanine-
NH has a higher temperature coefficient than the glycine-NH
can be clearly seen. With a rise in temperature the phenyl
alanine-NH signal shifted upfield at a higher rate and ultimately
crossed the signal for the glycine NH (Fig. 3). For both the
cis tripeptides 2 and 3, the temperature coefficients of chemical
shifts for all the NHs are above 5.0, which indicate the absence
of a definite b-turn motif in these systems. Thus, it seems that
while a b-turn like-conformation is preferred in trans peptide
1, no such preferences could be seen in the cis peptides 2
or 3. Hence the stereochemistry of the b-lactam ring plays
a key role in controlling the conformation of the peptides.
For the trans peptide 1, in order to know which carbonyl is
involved in intramolecular H-bonding, the truncated amide
4 was synthesized. In this case, intramolecular H-bonding is
present (temperature coefficient is 4.8 ppb) although to a lesser
extent as compared to the tripeptide 1. Presence of some
degree of intramolecular H-bonding indicates that the b-lactam
carbonyl participates in intramolecular H-bond with the glycine
amide NH.
Table 1 NMR Temperature coefficients of NH chemical shifts in
a
DMSO-d6
Fig. 2 ORTEP diagram of 11.
Compound no. NH Glycine NH Phenylalanine NH Benzylamine
The peptides were fully characterized by NMR and mass
spectroscopy. In the 1H NMR of the trans peptide 1 in CDCl3,
the two NH’s were overshadowed by the aromatic signals, which
were, however, clearly visible when the spectrum was recorded in
d6-DMSO. The glycine NH appeared as a triplet at d 8.21 while
the phenyl alanine NH appeared as a doublet at d 8.27. The
C-4 hydrogen in the azetidinone moiety appeared as a doublet
1
2
3
4
3.4
6.0
6.2
5.6
5.7
6.0
4.8
a Dd/DT (ppb K−1).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 0 5 0 – 4 0 5 2
4 0 5 1

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4 M. Goodman and S. Ro, Burger’s Medicinal Chemistry and Drug
Discovery, ed. M. E. Wolff, New York, John Wiley & Sons, Inc.,
1995, 803.
5 C. Palomo, J. M. Aizpurua, A. Benito, R. Galarza, U. K. Khamrai,
J. Vazquez, B. Pascual-Teresa, P. M. Nieto and A. Linden, Angew.
Chem., Int. Ed, 1999, 38, 3056.
6 J. Gante, Angew. Chem., Int. Ed. Engl., 1994, 33, 1699; A. Giannis
and T. Kolter, Angew. Chem., Int. Ed. Engl., 1993, 32, 1244.
7 G. Muo¨ller, G. Hessler and H. Y. Decornez, Angew. Chem., Int. Ed.,
2000, 39, 894; M. Kahn, Synlett, 1993, 821.
8 For c-lactam based peptidomimetics, see E. M. Khalil, A. Pradhan,
W. H. Ojala, W. B. Gleason, R. K. Mishra and R. L. Johnson, J. Med.
Chem., 1999, 42, 4927; N. L. Subasinghe, R. J. Bontems, E. McIntee,
R. K. Mishra and R. L. Johnson, J. Med. Chem., 1993, 36, 2356.
9 SPARTAN MM2 software, presented to on a limited day trial basis.
10 J. Marco-Contelles, Angew. Chem., Int. Ed., 2004, 43, 2198; A. Basak
and R. Pal, Bioorg. Med. Chem. Lett., 2005, 15, 2015; A. Basak and
S. C. Ghosh, Synlett, 2004, 1637.
Fig. 3 1H-NMR signals of phenylalanine and glycine NHs of peptide
1 at various temperatures.
We have thus successfully designed and synthesized a new
class of azetidinyl c-lactam-based peptide. From VT-NMR
experiments it has been shown that the peptide with trans
stereochemistry in the azetidinone moiety adopts a b-turn like
conformation unlike the corresponding cis isomers for which the
NHs are either mostly intermolecularly H-bonded or exposed
to the solvent. Current attempts are aimed towards making the
water-soluble analogs of 1 suitable for study under biological
conditions.
11 G. C. Fu, Angew. Chem., Int. Ed., 2003, 42, 4082; A. Basak, S. C.
Ghosh, T. Bhowmich, A. K. Das and V. Bertolasi, Tetrahedron Lett.,
2002, 43, 5499.
12 D. B. Smith, Ann M. Waltos, D. G. Loughhead, R. J. Weikert, D. J.
Morgans, Jr., J. C. Rohloff, J. O. Link and R. Zhu, J. Org. Chem.,
1996, 61, 2236.
13 Crystal data: chemical formula C21H20N2O4, H2O, formula weight
˚
382.41, crystal system monoclinic, unit cell dimensions a, b, c (A), b
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cell (Z) 2, linear absorption coefficient (l, mm−1) 0.093, number of
reflections measured 4322, number of independent reflections 2369,
Rint 0.064, final R 0.0517. CCDC reference number 280282. For
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