Synthesis of small cyclic peptides via reverse turn induced ring closing metathesis of tripeptides.

Article abstract of DOI:10.1021/jo0200320

A reverse turn induced (gamma/beta-turn) cyclization of tripeptides 1 can be performed in a ring closing metathesis reaction with Grubbs' catalyst to the corresponding cyclic peptides 2. These cyclic peptides may be useful probes as a conformationally con

Full text of DOI:10.1021/jo0200320

tures, we have demonstrated that the later structural
unit can be incorporated into a cyclic peptide obtainable
by ring closing metathesis. Thus the presence of the
known transition state analogue in the cyclic peptide may
make them interesting as potential HIV protease inhibi-
tors. It was reasoned that the tripeptides derived from
sequence where L-proline is in the γ-turn forming i+1
position6 would be an ideal precursor for such cycliza-
tions. This assumption was vindicated as we had shown
that the ring closing metathesis on a tripeptide such as
O-allyl-Xaa-L-proline-â-phenylisoserine-N-allyl 1a (Fig-
ure 1) led to cyclic peptide 2a . We proposed that this
cyclization was dictated by the presence of a γ-turn that
was supported by the fact that tripeptides 1b, consisting
of L-proline residue in the i+2 position, did not yield any
corresponding cyclic peptide on ring closing metathesis.
Clearly the tripeptide 1b lacks the intramolecular hy-
drogen bond and this led us to believe that the preorga-
nization of such structures may be a necessary condition
for ring closing metathesis. To assess the role of the
intramolecular hydrogen bond in these cyclizations, we
have further carried out studies on acyclic peptides
capable of forming an intramolecular ten-membered
hydrogen bond, i.e., a â-turn, and a detailed account of
these findings is given below.
The design of these cyclic peptides is based upon the
concept of mimicking the bioactive conformation by
introducing constraints in the flexible molecules through
their cyclization. We now show that tripeptides 3 derived
from O-allyl-Xaa-L-proline-â-phenylisoserine-N-allyl or
O-allyl-L-proline-Xaa-â-phenylisoserine-N-allyl deriva-
tives are preorganized due to γ- or â-turn and can be
cyclized by ring closing metathesis (Scheme 1 and 2) with
Grubbs’ catalyst.3a It is also demonstrated that irrespec-
tive of the position of L-proline in the tripeptide, the
cyclization is facile only when a γ- or â-turn is present
in such structures. The precursor peptides 3a -d were
prepared from N-cinnamoyl amino acids by conventional
coupling, using mixed anhydride protocol followed by O-
or N-allylation of the C-terminal end of the resulting
peptides.
Syn th esis of Sm a ll Cyclic P ep tid es via
Rever se Tu r n In d u ced Rin g Closin g
Meta th esis of Tr ip ep tid es
E. N. Prabhakaran,† I. Nageswara Rao,‡
Anima Boruah,‡ and J aved Iqbal*,†,‡,§
Department of Chemistry, Indian Institute of Technology,
Kanpur 208 016, India, and Discovery Research,
Dr. Reddy’s Laboratories Ltd., Miyapur,
Hyderabad 500 050, India
javediqbaldrf@hotmail.com
Received J anuary 15, 2002
Abstr a ct: A reverse turn induced (γ/â-turn) cyclization of
tripeptides 1 can be performed in a ring closing metathesis
reaction with Grubbs’ catalyst to the corresponding cyclic
peptides 2. These cyclic peptides may be useful probes as a
conformationally constrained mimic of the bioactive confor-
mation of structurally related HIV protease inhibitors.
The synthesis and screening of huge peptide libraries
has led to the emergence of small peptides as important
lead structures for the development1 of potential thera-
peutic agents. It is known that the linear peptide frag-
ments are flexible and exhibit numerous conformations
in solution; however, if one can restrict the conforma-
tional freedom of these linear peptides by introducing2
some constraints in the structure, it may render a bio-
logically active peptide more potent, more specific, and
orally active and this may give rise to species which are
therapeutically useful. Thus, small cyclic peptides are of
great interest for the elucidation of bioactive conforma-
tions due to their restricted conformational flexibility. In
view of the importance of constrained conformations,
there have been several attempts3 to lock peptides into
turn configurations and to synthesize molecules that
might mimic a reverse turn present in proteins. Several
types of turns are found and among them the type I and
type II â-turns are most commonly observed in protein
secondary structures. Therefore the understanding of the
conformation of type I and II â-turn is very crucial to the
development of inhibitors of HIV protease.4
These tripeptides were found to exist as preorganized
structures due to the presence of a γ- or â-turn formed
by intramolecular hydrogen bonds. The presence of the
In a previous communication5 relating to the work on
the search for HIV protease inhibitors based on pyrroli-
dine-containing R-hydroxy â-amino amide core struc-
1
intramolecular hydrogen bond was studied by H NMR
of the tripeptides 3a -d in dilute CDCl3 solution in the
presence of different concentrations of DMSO-d6. The
δNHa and δNHb in 3a and 3b indicate (Table 1) that these
amide protons appear at lower field as compared to δNHa
for 3d , suggesting a possible intramolecular hydrogen
† Indian Institute of Technology.
‡ Discovery Research, Dr. Reddy’s Laboratories Ltd.
§ Present Address: Director, Regional Research Laboratory, Trivan-
drum, India 695 019.
(1) (a) Thompson, L. A.; Ellman, J . A. Chem Rev., 1996, 96, 555. (b)
Fruchtel, J . S.; J ung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17. (c)
Terrett, N. K.; Gardner, M.; Gordon, D. W.; Kobylecki, R. J .; Steele, J .
Tetrahedron, 1995, 51, 8135.
(2) For â-turn see: (a) Ball, J . B.; Hughes, R. A.; Alewood, P. L.;
Andrews, P. R. Tetrahedron 1993, 49, 3467 and references therein.
(b) Haubner, R.; Finsinger, D.; Kessler, H. Angew. Chem., Int. Ed. Engl.
1997, 36, 1375. (c) Kim. K.; Germanas, J . P. J . Org. Chem. 1997, 62,
2853. (d) J ones, I. G.; J ones, W.; North, M. J . Org. Chem. 1998, 63,
1505. (e) Krauthauser, S.; Christianson, L. A.; Powell, D. R.; Gellman,
S. H. J . Am. Chem. Soc. 1997, 119, 11719.
(4) (a) Slee, D. H.; Laslo, K. L.; Elder, J . H.; Ollmann, I. R.;
Gustchina, A.; Kervinen, J .; Zdanov, A.; Wlodawer, A.; Wong, C. J .
Am. Chem. Soc. 1995, 117, 11867. (b) Lam, P. Y. S.; J adhav, P. K.;
Eyermann, C. J .; Hodge, C. N.; Ru, Y.; Bacheler, L. T.; Meek, J . L.;
Otto, M. J .; Rayner, M. M.; Wong, Y. N.; Chang, C.-H.; Weber, P. C.;
J ackson, D. A.; Sharpe, T. R.; Erickson-Viitanen, S. Science 1994, 263,
380.
(5) (a) Prabhakaran, E. N.; Nandy, J . P.; Shukla, S.; Iqbal, J .
Tetrahedron Lett. 2001, 42, 333. (b) Prabhakaran, E. N.; Rajesh, V.;
Dubey, S.; Iqbal, J . Tetrahedron Lett. 2001, 42, 339.
(3) (a) Miller, S. J .; Blackwell, H. E.; Grubbs, R. H. J . Am. Chem.
Soc. 1996, 118, 9606. (b) Fink, B. E.; Kym, P. R.; Katzenellenbogen, J .
A. J . Am. Chem. Soc. 1998, 120, 4334. (c) Li, W.; Burgess, K.
Tetrahedron Lett. 1999, 40, 6527.
(6) For the turn-inducing role of proline in the cyclization of small
peptides see: Sager, C.; Mutter, M.; Dumy, P. Tetrahedron Lett. 1999,
40, 7987.
10.1021/jo0200320 CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/25/2002
J . Org. Chem. 2002, 67, 8247-8250
8247

F IGURE 1.
SCHEME 1
SCHEME 2
bond. On the other hand, the δNHa values in 3c and 3d
suggest that these amide protons are not involved in
hydrogen bonding whereas the δNHb chemical shifts are
supportive of an intramolecular hydrogen bond. Also the
value in variable-temperature 1H NMR suggests that
peptide 3c lacks the intramolecular hydrogen bonding
while for 3a , 3b, and 3d it falls in a range indicative of
the presence of a â- or γ-turn. The FT-IR spectra (Table
1) of peptides 3a -d (3250-3350 cm-1) also reveal that
3a , 3b, and 3d possess an intramolecular hydrogen bond
(80%) as the trans rotamer. The leu NHa in 3a appeared
at lower field (compared to NHa in 3b) with a δ value of
7.67 ppm, indicating its possible participation in H-
bonding. Additional support for such a H-bond came from
solvent titration studies where addition of up to 33%
DMSO (v/v) in CDCl3 shifted the resonance signal only
by 0.4 ppm. The most likely donor/acceptor in 3a would
be NHa and the carbonyl of the cinnamoyl group. A strong
proRHc-leu NHa NOESY peak in addition to the above-
mentioned H-bonding implies the propensity of a γ-turn
in the molecule (Table 1). The presence of a â-turn in 3b
is quite evident from variable-temperature experiment
1
whereas 3c (∼3550 cm-1) is devoid of it. The H NMR in
CDCl3 solution showed that 3a exists predominantly
8248 J . Org. Chem., Vol. 67, No. 23, 2002

F IGURE 2. CD spectra of 1c in acetonitrile.
TABLE 1. Am id e Ch em ica l Sh ifts (p p m ) in CDCl3, F T-IR
(cm -1, CH2Cl2), a n d NOE’s in 3a , 3b, a n d 3d
catalytic amount of polyaniline supported cobalt(II) salen
to yield the corresponding epoxides 4a and 4b, respec-
tively, obtained after purification by chromatography as
a single diastereomer in good yields. The absolute ster-
eochemistry (2R,3S) for these epoxides was assigned
based on the correlation studies as described in an
earlier5a communication. The opening of epoxides 4a and
4b was achieved with N-allyl anisidine in the presence
of a catalytic amount of cobalt(II) chloride to afford the
corresponding tripeptide derivatives 1a and 1c, respec-
tively, mainly as the anti diastereomer in 55-60% yields.
We have shown7b,c earlier that the cobalt-catalyzed open-
compd
δNH
δNH
Ir-Ha
Ir-Hb
a
b
3a
3b
3d
7.67
7.78
6.34
3324
3265
3506
7.98
7.56
3305
3354
2
ing of cinnamoyl epoxide takes place by an SN pathway
leading to the anti diastereomer as the predominant
product.
where the low magnitude of the chemical shift/temper-
ature coefficient (∆δ/∆T) for the amide proton (NHb) is
an indicator of its participation in H-bonding. Expectedly
the (∆δ/∆T) values for the cis isomer of 3b have a large
magnitude ((∆δ/∆T ) -6 ppb/°C) for both the NH’s while
it is moderately small for the trans isomer (∆δ/∆T ) -2.4
ppb/°C for allyl NHb and -4.5 ppb/°C for leu NHa). This
suggests that a sizable fraction of the trans isomer has
allyl NHb participating in H-bonding. The presence of
such a H-bond coupled with the existence of NOESY
peaks between leu NHa-allyl NHb and strong Pro CRHc-
leu NHa suggests the preference of a â-turn around pro-
The tripeptides 1a and 1c also showed the presence of
intramolecular hydrogen bonding as indicated by the
appearance of a low-field NMR signal at 7.43 (J ) 8 Hz)
for 1a and 7.16 (J ) 8.2 Hz) for 1c. Also the support for
the intramolecular hydrogen bond came from solvent
titration studies where addition of up to 33% DMSO
(v/v) in CDCl3 shifted the resonance signal NHa in 1a by
0.35 ppm and that of NHb in 1c by 0.4 ppm. A small shift
in amide (NHb) signal in 1c is indicative of a hydrogen-
bonded/solvent-shielded proton like those involved in C10
â-turn conformations. The CD spectra8 of 1c also indi-
cated the presence of a â-turn as it most closely resembled
a type II â-turn with a maxima at 195.5 nm and a
minima at 216.5 nm (Figure 2). The presence of an
intramolecular hydrogen bond in 1a and 1c suggests that
these molecules are preorganized due to the presence of
a γ-turn (i.e., 1a ) and a â-turn (i.e., 1c) which may
facilitate the cyclization of these acyclic peptides via ring
closing metathesis with Grubbs’ catalyst.3a This indeed
was found to be the case, as subjecting 1a and 1c to
heating in the presence of 10 mol % of ruthenium
alkylidene (Grubbs’ catalyst) in dichloromethane (0.6
mM) for 12-30 h yielded the cyclized peptides 2a and
2c, respectively, in 40-45% yields after column chroma-
1
leu in 3b (Table 1). Interestingly, the H NMR of 3d in
CDCl3 showed the presence of a mixture of conformations
and it was not possible to correlate any NOESY peaks
from the spectrum. However, the solvent titration studies
upon addition of up to 33% DMSO (v/v) in CDCl3 showed
the shift of NHb resonance signal only by 0.3 ppm,
indicating the presence of an intramolecular hydrogen
bond in 3d .
The tripeptide derivatives 1 were prepared by our
polyaniline supported cobalt catalyzed7 aerobic epoxida-
tion and its opening protocol as described earlier (Scheme
1). The peptides 3a and 3b were subjected to aerobic
epoxidation in the presence of 2-methylpropanal and a
(7) For the synthesis and reaction of this catalyst see: (a) Das, B.
C.; Iqbal, J . Tetrahedron Lett. 1997, 38, 2903. (b) Punniyamurthy, T.;
Iqbal, J .; Tetrahedron Lett. 1997, 38, 4463. (c) De, A.; Basak, P.; Iqbal,
J . Tetrahedron Lett. 1997, 38, 8383.
(8) For CD spectra of the â-turn derived from small peptides see:
(a) Macdonald, M.; Aube, J . Curr. Org. Chem. 2001, 5, 417 and
references cited therein. (b) Park, C.; Burgess, K. J . Comb. Chem. 2001,
3, 257.
J . Org. Chem, Vol. 67, No. 23, 2002 8249

tography. It is noteworthy though not particularly sur-
prising that the tripeptide 1c underwent smooth ring
closing metathesis in a relatively shorter period (12 h)
as compared with the reaction time taken (30 h) by 1a
for similar cyclization. This difference in the reaction time
suggests that the cyclization of 1c is facile due to the
presence of a â-turn, which appears to preorganize these
structures much more efficiently than the γ-turn present
in 1a . These cyclic peptides were obtained as a mixture
of E:Z (4:1) isomers as indicated by the 1H NMR. The
reaction mixture also consisted of some minor oligomeric
products and ∼20% of the tripeptides 1 were recovered
1
unchanged. The H NMR titration with DMSO-d6 (δ 7.45
ppm, J ) 8.23 Hz) of the cyclic peptides 2c revealed the
presence2 of the intramolecular hydrogen bonding, which
clearly suggests that a â-turn is also present in the cyclic
form and might have been responsible for the cyclization.
That the presence of a γ-turn/â-turn may be respon-
sible for such cyclization is evident from the ring closing
studies on the acyclic tripeptides 1b and 1d containing
L-proline at the i+2 position of the residue (Scheme 2).
The tripeptides 1b and 1d were synthesized by the
protocol as described earlier and accordingly 3c,d were
transformed to the corresponding N-cinnamoyl epoxides
4c,d which were subjected to cobalt(II) chloride catalyzed
opening by N-allyl anisidine to afford the tripeptides 1b
and 1d , respectively (Scheme 2). The tripeptides 1b and
1d were subjected (0.6 mM in dichloromethane) to ring
closing metathesis with Grubbs’ catalyst and as expected
the corresponding cyclic peptide from 1b was not ob-
served, instead the reaction mixture consisted of intrac-
table oligomeric material apart from the unreacted 1b
(20-30%), whereas 1d afforded the corresponding cyclic
peptide 2d in 35% yield after column chromatography.
The cyclic peptide 2d was obtained as a mixture of E:Z
(4:1) isomers as indicated by NMR. The reaction mixture
also consisted of some minor oligomeric products and
∼20% of the tripeptide 1d was recovered unchanged. This
study clearly suggests that absence of a γ-turn in 1b will
not render these tripeptides preorganized for cyclization
under ring closing metathesis conditions. On the contrary
the tripeptide containing an allyl amide 1d underwent
ring closing metathesis leading to the synthesis of the
cyclic peptide in moderate yields. The cyclic peptide 2d
showed the presence of the intramolecular hydrogen bond
F IGURE 3. Simulated low-energy conformer for 1c and 2c.
1c are in close proximity for cyclization during ring
closing metathesis.
In conclusion, we have synthesized cyclic peptides
consisting of Xaa-L-proline-â-phenylisoserine tripeptides
from the acyclic precursors having an olefinic group at
both ends via ring closing metathesis using Grubbs’
catalyst. These cyclizations are controlled by the presence
of a γ-turn/â-turn in the acyclic precursor and the cyclic
peptides thus obtained may be a useful probe for under-
standing the role of constrained structures in the search
for a bioactive conformation of species related to HIV
protease inhibitors.
Ack n ow led gm en t. We thank DST, New Delhi and
Dr. Reddy’s Laboratories Ltd., Hyderabad for the finan-
cial support of this work. We are grateful to Dr. Ram
Thaimattam for helping us on molecular dynamics
simulation studies.
Su p p or tin g In for m a tion Ava ila ble: Spectroscopic and
analytical data for 1a , 1b, 1c, 1d , 2a , 2c, 2d , 1H NMR titration
spectra for 1c and 2c, along with the NOESY spectra of 3a
and 3b. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
1
in H NMR and FT-IR. A careful analysis of the H NMR
of 2d revealed the presence of two intramolecular hy-
drogen bonded species which were found to be in equi-
librium. The equilibrium ratio was altered upon addition
of DMSO-d6, indicating that these hydrogen bonded
species rapidly equilibrate between different bonded and
nonbonded structures. These observations also reflect the
moderate yield of the cyclic peptide 2d as the presence
of the different bonded and nonbonded species in 1d is
likely to yield products arising due to intra- as well as
intermolecular reactions during ring closing metathesis
with Grubbs’ catalyst.
J O0200320
(9) Molecular dynamics (MD) calculations for compounds 1c and 2c
were carried out with the Cerius2 program on a Silicon graphics Indigo2
workstation. Charges were calculated by using the charge-equilibration
method. The CFF9 force field with default parameters was used
throughout the simulations. To understand the conformational free-
dom, simulated annealing molecular dynamics calculations were
carried out. The temperature was varied between 300 and 1200 K in
steps of 50 K for 100/150 cycles. The molecules were allowed to
equilibrate for 0.5 ps for every change in temperature. Minimizations
were done first with steepest descent, followed by conjugate gradient
methods for a maximum of 1000 iterations each or a root-mean-square
deviation of 0.01 kcal/mol, whichever was earlier. The energy-
minimized structures were then subjected to MD simulations. Various
conformers obtained in each MD run were minimized by using the
above-mentioned minimization protocol. The energy difference between
the lowest energy conformer and the next higher energy conformer
was 5.6 kcal/mol for 1c and 8.35 kcal/mol for 2c.
The molecular dynamics simulation studies9 on 1c and
2c also supported the presence of an intramolecular
hydrogen bond leading to the â-turn. The bond distance
of 2.1 Å clearly indicates that 1c has the propensity to
exist in the form of a â-turn (Figure 3). It is also evident
from these structures that the terminal double bonds in
8250 J . Org. Chem., Vol. 67, No. 23, 2002