A facile conversion of a 310 helical structure to a cyclic β-turn mimic in dehydrophenylalanine-derived small peptides through ring-closing metathesis

Article abstract of DOI:10.1016/S0040-4039(02)01601-5

Dehydrophenylalanine-derived small peptides can be preorganized in a 3₁₀ helical structure which is transformed into a β-turn mimic during a ring-closing metathesis cyclization.

Full text of DOI:10.1016/S0040-4039(02)01601-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 7621–7625
A facile conversion of a 310 helical structure to a cyclic b-turn
mimic in dehydrophenylalanine-derived small peptides through
ring-closing metathesis
T. V. R. S. Sastry,b Biswadip Banerji,a S. Kiran Kumar,c A. C. Kunwar,c Jagattaran Das,b
Jyoti Prokash Nandya and Javed Iqbala,b,
*
aDepartment of Chemistry, Indian Institute of Technology, Kanpur 208016, India
bDiscovery Research, Dr. Reddy’s Laboratories Ltd., Bollaram Road, Miyapur, Hyderabad 500 050, India†
cIndian Institute of Chemical Technology, Hyderabad 500007, India
Received 19 July 2002; accepted 31 July 2002
Abstract—Dehydrophenylalanine-derived small peptides can be preorganized in a 310 helical structure which is transformed into
a b-turn mimic during a ring-closing metathesis cyclization. © 2002 Elsevier Science Ltd. All rights reserved.
Recent developments in the domain of drug-discovery
have focused attention on the synthesis of small-con-
strained mimics of bioactive conformations of potent
therapeutic molecules.1 It is very important for a pep-
tide molecule to retain its conformational features in
vivo to bind strongly to the target. Thus elements of
constraint (local or global) in a molecule can lock it
into a particular conformation, which may mimic the
exact bioactive conformation. Local constraints in
terms of side-chain modifications2 in the amino acids,
incorporation of protein secondary structures like
turns, helices, etc., into the molecule and cyclization3 as
part of global constraints are commonly practiced.
drophenylalanine (DPhe) residue as a constrained
phenylalanine mimic has gained much importance in
particular because of its turn inducing as well as helix-
forming propensity.4 It was also observed that the DPhe
residue when present as part of a peptide renders
stability towards proteolytic degradation. The sp2-Ca in
the dehydro residue results in specific ƒ and „ angles
which facilitate b-turn formation capability of this
residue. In an ongoing project in our laboratory5 on the
development of potent HIV-protease inhibitors,6 we
developed a strategy to synthesize DPhe-derived small
cyclic b-turn mimics. Recently,7 we have shown that in
a small peptide the presence of a DPhe residue at the
C-terminal (I) or N-terminal (II) of L-proline can con-
These molecules can also be very useful as pharmaceu-
tical probes towards various proteases. The dehy-
strain the molecule to adopt a b-turn (Fig. 1). We have
shown that such preorganization into a b-turn in the
Ph
O
O
O
N
H
N
H
N
H
CO2Me
N
Ph
CO2Me
N
O
O
H
O
N
Ph
( I )
( II )
Ph
Figure 1. Dehydrophenylalanine-derived b-turn mimics.
* Corresponding author. Present address: Director, Regional Research Laboratory, Trivandrum 695019, India. E-mail: javediqbaldrf@
hotmail.com
† DRL Publication No. 238.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01601-5

7622
T. V. R. S. Sastry et al. / Tetrahedron Letters 43 (2002) 7621–7625
molecule can bring the terminal olefin bonds in suffi-
ciently close proximity to undergo a facile ring-closing
metathesis (RCM)5 cyclization. In this communication
we report the synthesis of a DPhe-containing small
cyclic b-turn mimic8 through a RCM reaction.
aziridine to be 2S,3R.7 The compound 3 was then
subjected to a stereoselective conversion to the corre-
sponding DPhe-containing peptide 4 using our novel
Me3SiI/Et3N mediated methodology.7 Subsequently,
the peptide 4 was hydrolyzed (LiOH–MeOH/H2O) and
extended at its C-terminus with
L
-LeuOMe (ClCO2Bui/
We synthesized tripeptide 6 having two terminal olefinic
bonds at the C- and N-termini with the aim of cyclizing
them by a RCM reaction. Accordingly, N-pent-4-enoyl-
Et3N)9 to afford the corresponding peptide 5. An alka-
line hydrolysis (LiOH–MeOH/H2O) of
5
and
subsequent coupling with allylamine (ClCO2Bui/Et3N)
afforded the peptide 6 in an overall yield of 68%
L
-proline 1 was used to resolve the racemic aziridine 2
as described earlier7 to get the optically pure dipeptide
3 in 41% yield (Scheme 1). We have recently proved the
stereochemistry of the stereogenic centers in the
(Scheme 1). The solution H NMR data of compound
1
6 is presented in Table 1. The presence of two
intramolecular hydrogen bonds10 was revealed by sol-
Scheme 1. Synthesis of cyclic peptide 7 as a b-turn mimic.
Table 1. 1H chemical shifts (l in ppm), coupling constants (J in Hz) of 6 in CDCl3 at 500 MHz
Protons
Pro
DPhe
Leu
Allyl
NH
–
7.62 (s)
–
7.40 (s)
7.17 (d, JNH–aH=8.2)
4.59 (ddd, JaH–bH=4.3, JaH–b%H=10.4) 3.90 (m)
1.93 (m)
1.70 (m)
1.68 (m)
–
7.08 (t, J=5.5)
CaH
CbH
Cb%H
CgH
Cg¦H
CdH
4.44 (t, J=6.8)
2.22 (m)
2.22 (m)
2.07 (m)
2.07 (m)
3.57 (dt, J=7.2, 9.8)
3.62 (ddd, J=5.6, 7.2, 9.8)
–
5.87 (tdd, J=5.3, 10.4, 17.1)
–
5.22 (qd, J=1.6, 17.2)
5.09 (qd, J=1.6, 10.4)
–
–
–
0.96 (d, J=6.3)
–
Cd%H
–
0.95 (d, J=6.3)
–
Others: 2.43–2.37 (m, 4H, 2CH2), 5.79 (tdd, J=6.2, 10.3, 17.2 Hz), 4.99 (qd, J=1.5, 17.2), 4.92 (qd, J=1.5, 10.3), 7.32–7.42 (m, 5H)

T. V. R. S. Sastry et al. / Tetrahedron Letters 43 (2002) 7621–7625
7623
vent titration studies. Titrating with DMSO-d6 in
CDCl3 showed that the variation of the chemical shift
of the allyl-NHb (<0.36 ppm) and Leu-NHa (<0.70
ppm) is very small when 33% v/v DMSO-d6 was added
to the CDCl3 solution. The appearance of the Leu NHa
(7.17 ppm) as well as allyl-NHb (7.08 ppm) at low field
in the proton spectrum confirms their participation in
H-bonding. The 1H NMR of 6 in CDCl3 solution
showed only one isomer with a trans imide bond pre-
ceding the proline residue, as shown by the cross peak
between the Prod-Hd/pentenoyl-Hc in the NOESY spec-
trum. The appearances of NOE cross peaks between
He/Ha, Ha/Hj and He/Hj strongly indicates a ten-mem-
bered hydrogen bonding involving the leucine NHa and
the pentenoyl carbonyl in 6 (Fig. 2). The presence of
strong NOE peaks between Ha/Hb, Hb/Hj and Hi/Hb
along with the observation of the allyl-NHb being
hydrogen bonded (DMSO-d6 study), confirms the pres-
ence of a second b-turn in 6 (Fig. 2). Also the strong
ROE peak between the DPhe-b-Hf/leu NHa confirms
the Z-geometry of the double bond.
cyclization to afford the corresponding cyclic peptide 7
as an E-isomer in good yields (Scheme 1). In the
process of cyclization of 6, one new unnatural v-amino
acid, 6-aminohex-4-enoic acid (Aha) has been created
and 7 can be considered as a cyclic tetrapeptide with
two natural (Pro and Leu) and two unnatural (DPhe
and Aha) residues arranged in an alternating manner.
Also, being a cyclic peptide with a Pro-DPhe linkage, 7
can potentially belong to a new class of structural
1
analogues of HIV protease inhibitors. The solution H
NMR study on the cyclic peptide 7 revealed interesting
conformational properties. It was observed that after
cyclization the 310 helical structure in peptide 6 has
been transformed into a single cyclic b-turn mimic 7.
An almost equal abundance of both cis and trans
conformational isomers are observed in the DMSO-d6
medium. The low temperature coefficient (Dl/DT) for
the Aha-NHb chemical shifts (−2.4 ppb/K for the trans-
7 and −1.8 ppb/K for cis-7) indicates its participation in
intramolecular hydrogen bonding. The ROE cross
peaks between Ha/Hb, Hb/Hi, Ha/He confirms the pres-
ence of b-turn involving the DPhe/leu residue. The
energy minimized structures for both cis-7 and trans-7
show the presence of intramolecular hydrogen bonding
involving the proline carbonyl and the Aha-NHb (Fig.
2).
1
The H NMR studies thus clearly support the presence
of two consecutive b-turns, which suggests that the
acyclic peptide 6 is organized as a 310 helical structure.
Molecular dynamics simulation studies on 6 also show
the presence of a 310 helical structure in this peptide11
(Fig. 2). In a recent study from our laboratory12 we
have shown that peptides folded in a 310 helical struc-
ture undergo facile RCM cyclization leading to the
corresponding cyclic 310 helical structure. In order to
probe this we have subjected peptide 6 to RCM condi-
tions. To our gratification, when the tripeptide 6 was
subjected to Ru-carbene (Grubbs’ catalyst) catalyzed
RCM reactions,3,13 it indeed underwent a smooth
To ensure the role of the DPhe in the cyclization of 6,
we synthesized two analogous peptides 8 and 9 by
standard amide coupling procedures9 replacing the
DPhe with
L-Phe and D-Phe (Fig. 3), respectively,
whose solution NMR did not show any well defined
structure. When subjected to RCM condition, peptide 8
did not undergo any cyclization thereby rendering a
strong support to our hypothesis.
Figure 2. Energy minimized structures and different NOEs observed in trans-6, cis-7 and trans-7.

7624
T. V. R. S. Sastry et al. / Tetrahedron Letters 43 (2002) 7621–7625
N
H
N
H
HN
HN
Ph
Ph
O
O
O
O
H
H
N
N
N
N
O
O
O
O
9
8
Figure 3. The dehydrophenylalanine residue has been replaced by L-Phe and D-Phe in 8 and 9 respectively.
Chem., Int. Ed. Engl. 1997, 36, 2036–2056 and references
cited therein; (g) Furstner, A. Angew. Chem., Int. Ed.
2000, 39, 3012–3043 and references cited therein; (h)
Miller, S. C.; Blackwell, H. E.; Grubbs, R. H. J. Am.
Chem. Soc. 1996, 118, 9606–9614.
On the other hand peptide 9 was cyclized in a very poor
yield (5–10%). These results clearly indicate that the
close proximity of the terminal olefins, a prerequisite
for RCM cyclization, is missing in peptides 8 and 9.
Furthermore absence of the b-turn in 8 and 9, also
supports the role of the DPhe induced b-turn preorgani-
zation of 6 leading to the proximity of terminal alkenes
for a successful RCM reaction.
4. (a) Patel, H. C.; Singh, T. P.; Chauhan, V. S.; Kaur, P.
Biopolymer 1990, 29, 509; (b) Ciajolo, M. R.; Tuzi, A.;
Pratesi, C. R.; Fissi, A.; Pieroni, O. Biopolymer 1992, 32,
727; (c) Rajashankar, K. R.; Ramakumar, S.; Chauhan,
V. S. J. Am. Chem. Soc. 1992, 114, 9225; (d) Pieroni, O.;
Fissi, A.; Pratesi, C.; Temussi, P. A.; Ciardelli, F. Bio-
polymer 1994, 33, 1.
In conclusion, we have demonstrated that the DPhe
residue present in peptide 6 can invoke a 310 helical
structure which changes into a simple b-turn structure
after RCM cyclization. We are currently pursuing the
cyclization studies on the related helical structures.
5. Prabhakaran, E. N.; Rajesh, V.; Dubey, S.; Iqbal, J.
Tetrahedron Lett. 2001, 42, 339–342.
6. (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.; Jadhav, 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.;
Jackson, D. A.; Sharpe, T. R.; Erickson-Vitanen, S.
Science 1994, 263, 380.
References
1. (a) Smith, A. B.; Hirschmann, R.; Pasternak, A.; Guz-
man, M. C.; Yokohama, A.; Sprengeler, P. A.; Darke, P.
L.; Emini, E. A.; Schleif, W. A. J. Am. Chem. Soc. 1995,
117, 11113; (b) Martin, S. F.; Austin, R. E.; Oalmann, C.
J.; Baker, W. R.; Condon, S. L.; DeLara, E.; Rosenberg,
S. N.; Spina, K. P.; Stein, H. H.; Cohen, J.; Kleinert, H.
D. J. Med. Chem. 1992, 35, 1710; (c) Kim, E. E.; Baker,
C. T.; Dwyer, M. D.; Murcko, M. A.; Rao, B. G.; Tung,
R. D.; Navia, M. A. J. Am. Chem. Soc. 1995, 117, 1181;
(d) Martin, S. F.; Dorsey, D. O.; Gane, T.; Hillier, M.;
Kessler, H.; Baur, M.; Matha, B.; Erickson, J.; Bhat, T.
N.; Munshi, S.; Gulnik, S. V.; Topol, I. A. J. Med. Chem.
1998, 41, 1581.
2. For peptides derived from dehydro amino acids, see: (a)
Stammer, C. H. Chemistry and Biochemistry of Amino
Acids, Peptides and Protein; Weinstien, B., Ed.; Marcel
Dekker: New York, 1982; Vol. IV, p. 3; (b) Schmidt, U.;
Lieberknecht, A.; Wild, J. Synthesis 1988, 159; (c)
Nunami, K.; Hirmatsu, K.; Hayashi, K.; Matsumoto, K.
Tetrahedron 1988, 44, 5467. Also see: (d) Hruby, V. J.;
Yamamura, H. I.; Porreca, F. Ann. NY Acad. Sci. 1995,
757, 7–22 and references cited therein.
7. Prabhakaran, E. N.; Nandy, J. P.; Shukla, S.; Tewari, A.;
Das, S. K.; Iqbal, J. Tetrahedron Lett. 2002, 43, 6461.
8. (a) Rose, G. D.; Gierasch, L. M.; Smith, J. A. In Turns in
Peptides and Proteins; Advances in Protein Chemistry;
Academic Press: New York, 1985; (b) Farmer, P. S. In
Drug Design; Ariens, E. J., Ed.; Academic: New York,
1980; Vol. 10, pp. 119–143; (c) Feng, Y.; Pattarawarapan,
M.; Wang, Z.; Burgess, K. Org Lett. 1999, 1, 121; (d)
Belvisi, L.; Bernardi, A.; Manzoni, L.; Potenza, D.; Sco-
lastico, C. Eur. J. Org. Chem. 2000, 2563–2569; (e) Kaul,
R.; Angeles, A. R.; Jager, M.; Powers, E. T.; Kelly, J. J.
Am. Chem. Soc. 2001, 123, 5206–5212; (f) Feng, Y.;
Wang, Z.; Jin, S.; Burgess, K. J. Am. Chem. Soc. 1998,
120, 10768; (g) Feng, Y.; Pattarawarapan, M.; Wang, Z.;
Burgess, K. J. Org. Chem. 1999, 64, 9175–9177; (h)
Burgess, W. L. Tetrahedron Lett. 1999, 40, 6527–6530; (i)
Zhang, A. J.; Khare, S.; Kuppan, G. D.; Linthicum, S.;
Burgess, K. Bioorg. Med. Chem. Lett. 2001, 11, 207–210.
9. Standard amide coupling procedure: To an ice-cold stirred
solution of N-pentenoyl proline acid (1 equiv.) in dry
dichloromethane (5 mL) was added triethylamine (1
equiv.) followed by isobutyl chloroformate (1 equiv.).
The resulting mixture was stirred vigorously for 5 min
and then XAA-leucine allyl amide (1 equiv.) was added
followed by 1 equiv. of triethylamine. It was stirred for 5
h. After that the reaction mixture was washed thoroughly
3. (a) Flink, B. E.; Kym, P. R.; Katzenellenbogen, J. A. J.
Am. Chem. Soc. 1998, 120, 4334–4344; (b) Ripka, A. S.;
Bohacek, R. S.; Rich, D. H. Bioorg. Med. Chem. Lett.
1998, 8, 357; (c) Nguyen, S. T.; Johnson, L. K.; Grubbs,
R. H.; Ziller, J. W. J. Am. Chem. Soc. 1992, 114, 3974;
(d) Herisson, J. L.; Chauvin, Y. Makromol. Chem. 1970,
141, 161; (e) Phillips, A. J.; Abell, A. J. Aldrichim. Acta
1999, 32, 75–90; (f) Schuster, M.; Blechert, S. Angew.

T. V. R. S. Sastry et al. / Tetrahedron Letters 43 (2002) 7621–7625
7625
,
with saturated sodium bicarbonate solution, followed by
citric acid solution and water (3×10 mL). Drying and
concentration in the vacuum yielded the crude peptide
which on column chromatography (silica gel,
EtOAc:hexane) afforded the desired peptide in good
yield. Spectral data of some other compounds. Compound
6: MS m/z: 495 (M+1)+, 438, 407, 350, 325, 180, 172.
30 and 5 kcal/A were employed for H-bond distance and
dihedral angle constraints, respectively. The energy-mini-
mized structures were subjected to constrained MD simu-
lations for duration of 120 ps (20 cycles each of 6 ps
period, of the Simulated Annealing protocol). The atomic
velocities were applied following Boltzmann distribution
about the center of mass, to obtain a starting temperature
of 700 K. After simulating for 1.5 ps at high temperature,
the system temperature was reduced stepwise over a 4.5
ps period to reach a final temperature of 300 K. Resulting
structures were sampled for each and every cycle, leading
to an ensemble of total 20 structures. The samples were
minimized using the above-mentioned protocol, and one
of the lowest energy conformations of trans-7, cis-7 and
trans-6 is shown in Fig. 2; (b) Kessler, H.; Griesinger, C.;
Lautz, J.; Muller, A.; VanGunsteren, W. F.; Berendsen,
H. J. C. J. Am. Chem. Soc. 1988, 110, 3393–3396.
1
Compound 8: H NMR (200 MHz, CDCl3): l 7.30–7.14
(m, 5H), 6.98 (d, J=7.81 Hz, 1H), 6.79 (bs, 1H), 6.28 (d,
J=7.2 Hz, 1H), 5.90–5.74 (m, 1H), 5.23–4.99 (m, 4H),
4.59–4.53 (m, 1H), 4.36–4.26 (m, 1H), 4.23–3.85 (m, 2H),
3.48–3.32 (m, 2H), 3.24–3.15 (m, 1H), 2.97 (dd, J=5.8
Hz, 13.8 Hz, 1H), 2.4–1.86 (m, 8H), 1.8–1.61 (m, 2H),
1.38–1.29 (m, 1H), 0.96 (d, J=6.2 Hz, 3H), 0.90 (d,
J=6.2 Hz, 3H); MS (CI) m/z: 497 (M+1)+, 440, 412, 384,
327, 229, 180. Compound 9: 1H NMR (200 MHz,
CDCl3): l 7.36–7.10 (m, 5H), 6.99 (d, J=8.3 Hz, 1H),
6.77 (bs, 2H), 5.89–5.75 (m, 2H), 5.21–4.97 (m, 2H),
4.57–4.38 (m, 2H), 4.2 (bs, 1H), 3.84 (bs, 2H), 3.70–3.28
(m, 2H), 3.21–3.18 (m, 2H), 2.37 (s, 4H), 2.2–2.15 (m,
11H), 0.88–0.85 (m, 6H); MS (CI) m/z: 497 (M+1)+, 440,
412, 327, 180.
12. Banerji, B.; Mallesh, B.; Kiran Kumar, S.; Kunwar, A.
C.; Iqbal, J. Tetrahedron Lett. 2002, 43, 6479–6483.
13. General procedure for ring-closure metathesis: To a stir-
ring solution of ruthenium methylidene catalyst (Grubbs
catalyst) (10 mol%) in dichloromethane (60 mL) under
10. Ravi, A.; Balram, P. Tetrahedron 1984, 2577–2583.
11. (a) Molecular mechanics/dynamics calculations were car-
ried out using Sybyl 6.8 programme on a Silicon Graph-
ics O2 workstation. The tripos force field, with default
parameters were used throughout the simulations. In
order to mimic DMSO solvent a dielectric constant of 47
Debye was used in all minimizations as well as MD runs.
Minimizations were done first with steepest decent, fol-
lowed by conjugate gradient methods for a maximum of
2000 iterations each or RMS deviation of 0.005 kcal/mol,
whichever was earlier. The energy-minimized structures
were then subjected to MD. A number of inter atomic
distances and torsional angle constraints obtained from
NMR data were used. Distance constraints with a force
nitrogen, the diene
6
(1 mmol) dissolved in
dichloromethane (40 mL) was added and the mixture was
refluxed for 10–12 h. At this stage an additional ruthe-
nium methylidine catalyst (10 mol%) was added and the
mixture was further refluxed for 8 h. The solvent was
evaporated to yield
a
residue which was chro-
matographed over silica gel (EtOAc: hexane) to afford 7
1
(55%) as a gum. H NMR (500 MHz, DMSO-d6) trans-7:
1H NMR (200 MHz, CDCl3): l 10.09 (s, 1H), 8.62 (d,
J=7.8 Hz, 1H), 7.58 (dd, J=2.0, 7.9 Hz, 1H), 7.52–7.28
(m, 5H), 6.30 (s, 1H), 5.46 (m, 1H), 5.27 (m, 1H), 4.65
(dd, J=3.5, 8.2 Hz), 4.05 (m, 1H), 4.02 (m, 1H), 3.56 (m,
2H), 3.23 (m, 1H), 2.55 (m, 1H), 2.33 (m, 1H), 2.19 (m,
2H), 2.14 (m, 2H), 1.89 (m, 2H), 1.74 (m, 1H), 1.67 (m,
1H), 1.57 (m, 1H), 0.91 (d, J=6.4 Hz, 3H), 0.85 (d,
J=6.4 Hz); MS (CI) m/z: 468 (M+1, 41%)+, 467 (M+,
100%), 379, 339, 322, 297, 281, 223, 197, 180.
,
constant of 15 kcal/A were applied in the form of flat
bottom potential well with a common lower bound of 2.0
,
,
A and the upper bound of 2.8, 3.5, 4.0 and 4.5 A, in
accordance with the NOE intensities. Force constants of