Ni-to-Ni + 3-ethylene-bridged partially modified retro-inverso tetrapeptide β-turn mimetic: Design, synthesis, and structural characterizationt

Article abstract of DOI:10.1021/jo011041w

A 10-membered heterocyclic ring system 1,3,8-trisubstituted 2,5,7-trioxo-1,4,8-triazadecane that represents a N_i-to-N_i + ₃-ethylene-bridged partially modified retro-inverso tetrapeptide β-turn mimetic (EBRIT-BTM) has been designed, synthesized, and structurally analyzed. These compounds utilize an ethylene bridge to replace the CO_i · · · HN_i + ₃ 10-membered hydrogen bond of standard β-turns. The N, N'-ethylene-bridged dimer was obtained in 90% yield by reductive alkylation of phenylalanylamide with a tert-butyl N-(9-fluorenylmethyloxycarbonyl), N-(2-formylmethyl)-glycinate. An orthogonal protection strategy and HATU-mediated couplings allowed efficient stepwise additions of monomeric building blocks leading to a N_i-to-N_i+3-ethylene-bridged linear precursor: H-Asp-(OcHex)-NGly-OBu^t HO₂CCH₂CO-NPhe-NH₂. Further elaboration of the linear precursor generated the ethylene-bridged model compounds H2N-rPheN-mGly-Asp-NGly-OH (16) and Ac-gPheN-mGly-Asp-NGly-OH (18) (g, gem-diaminoalkyl; m, malonyl; and r, direction-reversed amino acid residue) in 44 and 39% yields, respectively. The structural features of the two EBRIT-BTM compounds were determined using ¹H NMR and extensive computer simulations. The results indicate that the 10-membered rings are conformationally constrained with well-defined structural features and that the three amide bonds in the ring are in the trans orientation. The topological arrangement of the residues in the ring system closely resembles a type II' β-turn. Transformation of CONH₂ in the N-terminal amino acid residue of 16 into NHCOCH₃ in 18 resulted in the formation of a hydrogen bond between the NH ofgPhe-COCH₃ and the C-terminal carboxyl of Gly, initiating an antiparallel β-sheet. The formulation of the concept applying a minimalistic structural elaboration approach and the synthetic exploration, together with the conformational analysis, offer a new molecular scaffolding system and a true tetrapeptide secondary structure mimetic that can be used to generate peptidomimetics of biological interest.

Full text of DOI:10.1021/jo011041w

Ni-to-Ni + 3-Eth ylen e-Br id ged P a r tia lly Mod ified Retr o-In ver so
Tetr a p ep tid e â-Tu r n Mim etic: Design , Syn th esis, a n d Str u ctu r a l
Ch a r a cter iza tion †
Yinglin Han,‡ Craig Giragossian,§ Dale F. Mierke,§,| and Michael Chorev*,‡
Department of Medicine, Bone and Mineral Metabolism Unit, Charles A. Dana and
Thorndike Laboratories, Beth Israel Deaconess Medical CentersHarvard Medical School,
Boston, Massachusetts 02215, Department of Chemistry and Department of Molecular Pharmacology,
Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912
chorev@hms.harvard.edu
Received October 29, 2001
A 10-membered heterocyclic ring system 1,3,8-trisubstituted 2,5,7-trioxo-1,4,8-triazadecane that
represents a Ni-to-Ni
3-ethylene-bridged partially modified retro-inverso tetrapeptide â-turn
+
mimetic (EBRIT-BTM) has been designed, synthesized, and structurally analyzed. These compounds
utilize an ethylene bridge to replace the COi‚‚‚HNi 10-membered hydrogen bond of standard
+
3
â-turns. The N,N′-ethylene-bridged dimer was obtained in 90% yield by reductive alkylation of
phenylalanylamide with a tert-butyl N-(9-fluorenylmethyloxycarbonyl),N-(2-formylmethyl)-glycinate.
An orthogonal protection strategy and HATU-mediated couplings allowed efficient stepwise additions
of monomeric building blocks leading to a Ni-to-Ni+3-ethylene-bridged linear precursor: H-Asp-
(OcHex)-NGly-OBut HO2CCH2CO-NPhe-NH2. Further elaboration of the linear precursor generated
the ethylene-bridged model compounds H2N-rPheN-mGly-Asp-NGly-OH (16) and Ac-gPheN-mGly-
Asp-NGly-OH (18) (g, gem-diaminoalkyl; m, malonyl; and r, direction-reversed amino acid residue)
in 44 and 39% yields, respectively. The structural features of the two EBRIT-BTM compounds
1
were determined using H NMR and extensive computer simulations. The results indicate that
the 10-membered rings are conformationally constrained with well-defined structural features and
that the three amide bonds in the ring are in the trans orientation. The topological arrangement
of the residues in the ring system closely resembles a type II′ â-turn. Transformation of CONH2 in
the N-terminal amino acid residue of 16 into NHCOCH3 in 18 resulted in the formation of a hydrogen
bond between the NH of gPhe-COCH3 and the C-terminal carboxyl of Gly, initiating an antiparallel
â-sheet. The formulation of the concept applying a minimalistic structural elaboration approach
and the synthetic exploration, together with the conformational analysis, offer a new molecular
scaffolding system and a true tetrapeptide secondary structure mimetic that can be used to generate
peptidomimetics of biological interest.
In tr od u ction
II,1 gonadotrophin releasing hormone (GnRH),2,3 and
somatostatin,4,5 as well as other bioactive peptides such
as Arg-Gly-Asp-based integrin antagonists,6,7 fibrinopep-
tide A,8 prohormones,9,10 and a CD4-derived motif that
Reverse turns, such as â-turns, are documented to be
important structural elements in bioactive conformations
of many peptides. Peptide hormones such as angiotensin
(2) Nikiforovich, G.; Marshall, G. R. Int. J . Pept. Protein Res. 1993,
42, 171-180.
* To whom correspondence should be addressed. Phone: (617) 667-
0901. Fax: (617) 667-4432.
(3) Nikiforovich, G. V.; Marshall, G. R. Int. J . Pept. Protein Res.
1993, 42, 181-193.
† Prefixes g and m preceding the standard three-letter notation for
amino acid residues indicate the gem-diaminoalkyl and 2-alkyl-
substituted malonyl residues corresponding to the side chain charac-
terizing the amino acid specified by the three-letter notation. The prefix
r preceding the standard three-letter notation for amino acid residues
indicates the incorporation of this amino acid in the reversed-sense
orientation, namely, NHrCO
(4) Nutt, R. F.; Veber, D. F.; Saperstein, R. J . Am. Chem. Soc. 1980,
102, 6539-6545.
(5) Brady, S. F.; Paleveda, W. J ., J r.; Arison, B. H.; Saperstein, R.;
Brady, E. J .; Raynor, K.; Reisne, T.; Veber, D. F.; Freidinger, R. M.
Tetrahedron 1993, 3449-3466.
(6) Bach, A. C., II; Espina, J . R.; J ackson, S. A.; Stouten, P. F. W.;
Duke, J . L.; Mousa, S. A.; DeGrado, W. F. J . Am. Chem. Soc. 1996,
118, 293-294.
‡ Beth Israel Deaconess Medical CentersHarvard Medical School.
§ Department of Chemistry, Brown University.
| Department of Molecular Pharmacology, Division of Biology and
Medicine, Brown University.
(7) Haubner, R.; Schmitt, W.; Holzemann, G.; Goodman, S. L.;
J onczyk, A.; Kessler, H. J . Am. Chem. Soc. 1996, 118, 7881-7891.
(8) Nakanishi, H.; Chrusciel, R. A.; Shen, R.; Bertenshaw, S.;
J ohnson, M. E.; Rydel, T. J .; Tulinsky, A.; Kahn, M. Proc. Natl. Acad.
Sci. U.S.A. 1992, 89, 1705-1709.
(1) Plucinska, K.; Kataoka, T.; Yodo, M.; Cody, W. L.; He, J . X.;
Humblet, C.; Lu, G. H.; Lunney, E.; Major, T. C.; Panek, R. L.;
Schelkun, P.; Skeean, R.; Marshall, G. R. J . Med. Chem. 1993, 36,
1902-1913.
(9) Rholam, M.; Nicolas, P.; Cohen, P. FEBS Lett. 1986, 207, 1-6.
10.1021/jo011041w CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/19/2002
J . Org. Chem. 2002, 67, 5085-5097
5085

Han et al.
F IGURE 1. Schematics of a â-turn and its dipeptido- and tetrapeptidomimetics are accompanied by selected examples of both
modes of mimetics.
binds to gp120,11 are only a representative sample of
â-turn-containing peptides. In these peptides, the â-turns
act as a molecular scaffold, presenting recognition motifs
and acceptor interaction sites.12,13
A â-turn consists of four amino acid residues i-to-i + 3
leading to a reversal in the backbone direction, with
amino acid residues i +1 and i + 2 found in the two
corners of the turn. A CdO‚‚‚HN hydrogen bond between
the i and the i + 3 residues, respectively, stabilizes almost
all of these turns forming a 10-membered intramolecular
hydrogen-bonded ring. Different â-turns are distin-
guished on the basis of the φ and ψ torsion angles of the
i + 1 and i + 2 amino acid residues12,14 or the topology of
the side chains involved.15
To elucidate the contribution of â-turns to the bioactive
conformation of a minimal bioactive sequence, which
otherwise is highly flexible, and to enhance and select
for a specific profile of pharmacological activity, rigidified
â-turn mimetics are developed.14,16-18 These â-turn-
mimicking scaffolds are designed to have a higher avidity
for the acceptor by overcoming what otherwise is the
inherent entropic cost paid for â-turn formation upon
binding to the acceptor. At the same time, in some cases,
scaffolding via a fused bicyclic system may cause over-
rigidification leading to compromised ligand-acceptor
interactions. Nevertheless, these peptidomimetics are
potential conformational probes, thus enabling biological
activity to be more effectively correlated with structure,
and prospective therapeutics overcoming drawbacks such
as proteolytic susceptibility and poor bioavailability,
typical to many important bioactive peptides.
Many of the â-turn mimetics are mono- and bicyclic
systems designed to be dipeptide mimetic replacements
of the i + 1 and i + 2 residues (Figure 1).14,19-23 In these,
attempts are made to correctly position the amino- and
carboxyl-termini of the dipeptidomimetic unit to force a
reversal of the backbone. However, in general, side chains
Ri + 1 and Ri + 2 are not maintained. Several efforts have
been directed toward the design of large heterocyclic
systems as tetrapeptide mimetics that carry some of the
four side chains presented by the original â-turn.24-26 The
restricted conformational space of cyclic peptides and
peptidomimetics can be characterized through the use
of NMR and computational techniques such as distance
geometry, energy minimization, and molecular dy-
namics.24,27-30
(19) Nagai, U.; Sato, K. Tetrahedron Lett. 1985, 26, 647-650.
(20) Estiarte, M. A.; Rubiralta, M.; Diez, A. J . Org. Chem. 2001,
65, 6992-6999.
(21) Hinds, M. G.; Richards, N. G. J .; Robinson, J . A. J . Chem. Soc.
1988, 1447, 131-134.
(22) Freidinger, R. M.; Perlow, D. S.; Veber, D. F. J . Org. Chem.
1982, 47, 104-109.
(10) Bek, E.; Berry, R. Biochemistry 1990, 29, 178-183.
(11) Chen, S.; Chrusciel, R. A.; Nakanishi, H.; Raktabutr, A.;
J ohnson, M. E.; Sato, A.; Weiner, D.; Hoxie, J .; Saragovi, H. U.; Greene,
M. I.; Kahn, M. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5872-5876.
(12) Rose, G. D.; Gierasch, L. M.; Smith, J . A. Adv. Protein Chem.
1985, 37, 1-109.
(23) Kemp, D. S.; McNamara, P. E. J . Org. Chem. 1985, 50, 5834-
5838.
(24) Souers, A. J .; Virgilio, A. A.; Rosenquist, A.; Fenuik, W.; Ellman,
J . A. J . Am. Chem. Soc. 1999, 121, 1817-1825.
(25) Ripka, W. C.; DeLucca, G. V.; Bach, A. C., II; Pottorf, R. S.;
Blaney, J . M. Tetrahedron 1993, 3593-3608.
(26) Gardner, B.; Nakanishi, H.; Kahn, M. Tetrahedron 1993, 49,
3433-3448.
(27) Graf von Roedern, E.; Lohof, E.; Hessler, G.; Hoffmann, M.;
Kessler, H. J . Am. Chem. Soc. 1996, 118, 10156-10167.
(28) Mierke, D. F.; Geyer, A.; Kessler, H. Int. J . Pept. Protein Res.
1994, 44, 325-31.
(13) Muller, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 2767-2769.
(14) Hanessian, S.; McNaughton-Smith, G.; Lombart, H.-G.; Lubell,
W. D. Tetrahedron 1999, 426, 12789-12852.
(15) Ball, J . B. Tetrahedron 1993, 3467-3478.
(16) Holzemann, G. Kontakte (Darmstadt) 1991, 1, 3-12.
(17) Ball, J . B.; Alewood, P. J . Mol. Recog. 1990, 3, 55-64.
(18) Holzemann, G. Kontakte (Darmstadt) 1991, 2, 55.
5086 J . Org. Chem., Vol. 67, No. 15, 2002

Retro-Inverso â-Turn Peptidomimetic
F IGURE 2. Schematics of the structural transformations relating a tetrapeptide to an ethylene-bridged partially modified retro-
inverso â-turn mimetic (EBRIT-BTM). Sequential singular retro-inverso transformation of COiNHi
followed by Coi + 1NHi
+ 1
+ 2
generates partially modified retro-inverso tetrapeptides I and II (PMRI tetrapeptides I and II), respectively. Introduction of an
ethylene bridge between N′i and Ni transforms PMRI tetrapeptides I and II into the corresponding EBRIT-BTMs I and II.
+
3
An ideal â-turn mimetic should be a modular unit, in
which the characteristics of the critical side chains are
maintained. In addition, it should carry amino- and
carboxyl-functions, which will allow its incorporation as
a synthon into a peptide chain of choice. Moreover, it
should allow the presentation of a wide array of side
chains in positions i-to-i + 3. Ideally, the â-turn mimetic
should resemble closely the backbone of the turn, repro-
duce closely the side chain topology, and be devoid of
excessive bulk and/or hydrophobicity. Most importantly,
the tetrapeptide-like â-turn mimetic should be con-
structed from readily available building blocks. We also
believe that divergence from the perfect topologic mimicry
should be compensated by building in sufficient flexibility
to allow subtle adjustments to adapt to specific macro-
molecular targets.
extensive molecular dynamics (MD) simulations. The
salient structural features of the peptidomimetics with
respect to their use as molecular scaffolds are presented.
Resu lts a n d Discu ssion
Design of th e Mod el Ni-to-Ni + 3-Eth ylen e-Br id ged
P a r tia lly Mod ified Retr o-In ver so â-Tu r n Tetr a p ep -
tid e Mim etic. Our design sought to transform the
putative transient hydrogen-bonded 10-membered ring,
characteristic of many â-turn structures, into a 10-
membered monoheterocyclic ring. We were committed to
maintain the polar nature of the backbone, the charac-
teristic side chains, and their chiral disposition and to
accomplish all of the above by employing readily available
amino acid derivatives as starting materials. To this end,
we utilized the well-established retro-inverso pseudopep-
tide approach, in which reversal of the amide bond
direction in the nonpalindromic backbone is accompanied
by the inversion of chiralities at the CR carbons.31-33
Specifically, we employed the partial retro-inverso modi-
fication in which reversal of a single peptide bond within
To this end, we report the design, synthesis, and
conformational analysis of novel Ni-to-Ni 3-ethylene-
+
bridged partially modified retro-inverso tetrapeptide
â-turn mimetics (EBRIT-BTM).31-33 This design applies
a minimalistic structural modification approach and
fulfills very closely the above criteria for a tetrapeptide-
based â-turn mimetic. This tetrapeptide mimetic incor-
porates a reversal of only a single peptide bond, namely,
the CiO‚‚‚Ni + 1H amide bond, and therefore represents a
partial retro-inverso modification.31,32 The EBRIT-BTM
a sequence, for example, the CiO-Ni 1H amide bond,
+
generates a partially modified retro-inverso peptide
(Figure 2, PMRI Tetrapeptide I).31,32 This partial retro-
inverso transformation requires the conversion of the ith
amino acid residue into the corresponding gem-diami-
noalkyl residue and the replacement of amino acid
residue in position i + 1 with a 2-alkyl malonyl residue.
In principle, this partial retro-inverso transformation can
be extended to the adjacent amide bond, Ci + 1O-Ni + 2H,
reversal of which will result in the incorporation, in the
reversed sense of direction, of the enantiomeric form of
amino acid residue i + 1 and the replacement of amino
acid residue i + 2 with the corresponding 2-alkyl malonyl
moiety (Figure 2, PMRI Tetrapeptide II). Introduction
of the gem-diamino alkyl in the ith position presents N′i
as a potential site for N-alkylation. Therefore, it allows
the replacement of the original CidO‚‚‚HNi + 3 hydrogen
bond with an N′i-CH2-CH2-Ni + 3 ethylene bridge thus
locking a 10-membered heterocyclic ring as a potential
structures, H2N-rPheN-mGly-Asp-NGly-OH (16) and the
H2N-rPhe-to-Ac-gPhe-rearranged Ac-gPheN-mGly-Asp-
NGly-OH (18) (g, gem-diaminoalkyl; m, malonyl; and r,
direction-reversed amino acid residue) (see Scheme 5),
were characterized by proton and carbon NMR and
refined with metric-matrix distance geometry (DG) and
(29) Pellegrini, M.; Royo, M.; Chorev, M.; Mierke, D. F. J . Pept. Res.
1997, 49, 404-414.
(30) Porcelli, M.; Casu, M.; Lai, A.; Saba, G.; Pinori, M.; Cappelletti,
S.; Mascagni, P. Biopolymers 1999, 50, 211-9.
(31) Goodman, M.; Chorev, M. Acc. Chem. Res. 1979, 12, 1-7.
(32) Chorev, M.; Goodman, M. Acc. Chem. Res. 1993, 26, 266-273.
(33) Fletcher, M. D.; Campbell, M. M. Chem. Rev. 1998, 98, 763-
795.
J . Org. Chem, Vol. 67, No. 15, 2002 5087

Han et al.
F IGURE 3. Retrosynthesis of ethylene-bridged partially modified retro-inverso â-turn tetrapeptide mimetic I (EBRIT-BTM I).
Pathways a and b represent synthetic strategies in which ring closure of linear precursors is carried out either between residues
i + 1 and i + 2 of the (2 + 2) partially modified retro-inverso ethylene-bridged tetrapeptide I or between residues i + 2 and i +
3 of the (1 + 3) partially modified retro-inverso ethylene-bridged tetrapeptide II, respectively. Both pathways share a common (1
+ 1) ethylene-bridged dipeptide III [(1 + 1)EB-dipeptide].
SCHEME 1. P r ep a r a tion of
ethylene-bridged PMRI tetrapeptide â-turn mimetic (Fig-
ure 2, EBRIT-BTM I). In this paper, we report the
synthesis of a model EBRIT-BTM I system, Ac-NHCH-
N-(ter t-Bu toxyca r bon ylm eth yl)-N-F m oc S-Eth yl
Th ioglycin a te (4)a
(Bzl)N-mGly-Asp-N-CH2-CO2H (18), which is structurally
related to the linear PMRI tetrapeptide Ac-gPhe-mGly-
Asp-Gly-OH1.
Syn t h esis of Mod el E BR IT-BTM I. Our retro-
synthetic analysis, outlined in Figure 3, requires the
construction of the linear precursors I or II, which differ
in the designated site for ring closure. End-to-end cy-
clization of these linear PMRI-ethylene-bridged (EB)
tetrapeptides I and II by forming either peptide bond a
or b, respectively, will accomplish the synthesis of the
DMF, 95 °C, 4 h, 86%; (b) 10% Pd-C, H2 in 95% EtOH, 40 psi, 2
a
Reaction conditions: (a) tert-butyl bromoacetate, K2CO3, in
h, 97%; (c) LiOH in dioxane-H2O, 4oC, overnight; (d) Fmoc-Cl,
NaHCO3, room temperature, 4 h, 86%; (e) EtSH, DCC, DMAP in
DCM, room temperature, 2 h, 87%.
trisubstituted 2,5,7-trioxo-1,4,8-triazadecane as the
EBRIT-I. Intuitively, macrocyclization via pathway a ,
generating a new secondary amide bond between the
malonyl residue and the amino acyl moiety, should be
easier to carry out than the ring closure via pathway b,
which generates a tertiary amide bond between an
N-alkylated ethylene amino acyl moiety and the carboxyl
of an N-acylated amino acid residue. In addition, while
pathway a represents stepwise addition of monomeric
units leading to the assembly of the (2 + 2)PMRI-EB-
tetrapeptide I, pathway b represents a convergent syn-
thesis in which a pseudodipeptide IV is condensed with
a (1 + 1)EB-dipeptide III to generate the (1 + 3)PMRI-
EB-tetrapeptide II. In both pathways, the reductive
alkylations generating the N′i-CH2-CH2-Ni + 3 ethylene
bridge comprise the key steps in the synthesis. Both
syntheses utilize readily available protected amino acid
derivatives as building blocks for the construction of the
linear PMRI tetrapeptides I or II.
The first building block for pathways a and b is N-(tert-
butyloxycarbonylmethyl)-N-Fmoc S-ethyl thioglycinate
(4), which was obtained in an overall yield of 62%
(Scheme 1). Alkylation of ethyl N-benzyl glycinate by tert-
butyl bromoacetate yielded the orthogonally protected
ethyl tert-butyl N-benzyliminoacetate 1 that was further
manipulated to yield the free acid 3. DCC-mediated
thioesterification of 3 with ethanethiol35 generated the
thioester 4 in 87% yield.
(34) Bergmeier, S. C.; Cobas, A. A.; Rapoport, H. J . Org. Chem. 1993,
58, 2369-2376.
5088 J . Org. Chem., Vol. 67, No. 15, 2002

Retro-Inverso â-Turn Peptidomimetic
SCHEME 2. Bu ild in g th e Eth ylen e Br id ge th r ou gh
Red u ctive Alk yla tion
SCHEME 3. P r ep a r a tion of N-Ma lon yl
r-(9-F lu or en ylm eth yl)-â-cycloh exyl Asp a r ta tea
a
Reaction conditions: (a) HOOCCH2COOBut, DCC in DCM,
room temperature, 2 h, 92%; (b) saturated HCl in Et2O, room
temperature, overnight, 100%.
SCHEME 4. Syn th esis of Mod el ERBIT-BTM via
P a th w a y a Ap p lyin g 2 + 2 F r a gm en t Con d en sa tion
The (1 + 1)EB-dipeptide 6 (Scheme 2) is the common
intermediate for pathways a and b represented by
compound III in the retrosynthetic scheme (Figure 3).
Alternative approaches to prepare N-(tert-butoxycarbo-
nylmethyl)-N-Fmoc S-ethyl thioacetate (4) failed. These
included attempts to ring-open N-Fmoc-iminodiacetic
acid anhydride34 with either tert-butyl alcohol or ethaneth-
iol and to N-alkylate tert-butyl glycinate with benzyl
bromoacetate in the presence of K2CO3 and tetrabutyl-
ammonium hydrogensulfate (TBA).
The formation of the N′i-CH2-CH2-Ni
ethylene
+
3
bridge in 6 was accomplished by a two-step procedure,35-37
which was found to be superior to our previously pub-
lished one-pot reductive alkylation.38 In the first step,
thioglycinate 4 was reduced to the corresponding alde-
hyde 5 in 93% yield. Due to the limited stability of
aldehyde 5 at room temperature, it was used shortly after
synthesis. In the following step, sodium triacetoxyboro-
hydride [NaB(OAc)3H]-mediated reductive alkylation of
phenylalaninamide with the aldehyde 5 generated the
ethylene-bridged intermediate in 90% yield, bringing the
total yield of this procedure to about 84%.38,39 In com-
parison, the one-pot reductive alkylation procedure yielded
less than 50% of the ethylene-bridged compound 6.38
Pathway b represents a convergent synthesis in which
a pseudodipeptide IV is coupled to a (1 + 1)EB-dipeptide
III to yield the linear (1 + 3)EB-tetrapeptide II, the
precursor for the final macrocyclization (Figure 3). The
synthesis of fully protected model â-turn mimetic pseudo-
dipeptide N-(tert-butyloxycarbonylmethylcarbonyl) R-(9-
fluorenylmethyl) and â-cyclohexyl L-aspartate (tert-butyl
mGly-Asp(OcHex)OFm) (7) and its partially protected
form 8 are outlined in Scheme 3.
removed the N-Fmoc/OFm protecting groups (1 equiv of
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) in dimethyl-
formamide (DMF)) to a solution of HATU and DIPEA in
DMF generated a very complicated reaction mixture.
LC-ESIMS analysis of this mixture revealed that in-
tramolecular coupling occurred to a very limited extent
and the anticipated fully protected model EBRIT-BTM
10 was only a minor product (5-7% yield) (Scheme 4).
Attempts to carry out this macrocyclization in a highly
diluted solution42 yielded similarly disappointing results.
This apparently difficult intramolecular coupling is caused
by a combination of the low reactivity of the secondary
amine, the steric hindrance imposed by the N-tert-
butyloxycarbonylmethyl substituent on the secondary
amine, and the structural strain involved in the ring
closure to obtain the 2,5,7-trioxo-1,4,8-triazadecane sys-
tem.
The alternative synthetic pathway a employed the
same (1 + 1)EB-dipeptide 6 used in pathway b, which is
extended by the monomeric units in a stepwise manner
(Scheme 5). The overall yield of the fully protected (2 +
2)EB-tetrapeptide 13 after two HATU-mediated cou-
plings and an intervening DBU-mediated deprotection
of the N-Fmoc from the secondary amine in the (1 + 2)-
EB-tripeptide 11 was 71%. Catalytic hydrogenation of 13
in which N-Cbz and O-Bzl protecting groups were quan-
titatively removed generated the partially protected
linear (2 + 2)EB-tetrapeptide 14. Importantly, the linear
precursor was labile upon storage. After several days at
room temperature, it spontaneously cyclized to the cor-
responding diketopiperazine 15 as confirmed by LC-
The O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethylu-
ronium hexafluorophosphate (HATU)40,41-mediated cou-
pling between the secondary amine in the (1 + 1)EB-
dipeptide 6 and the PMRI dipeptide HO-mGly-Asp-
(OcHex)-OFm (8) generated the fully protected linear (1
+ 3)EB-tetrapeptide partially deprotected 9, in 92% yield
(Scheme 4). However, macrocyclization carried out by
dropwise addition of 9 in the deprotection mixture that
(35) Kim, H. O.; J i, X. D.; Melman, N.; Olah, M. E.; Stiles, G. L.;
J acobson, K. A. J . Med. Chem. 1994, 37, 3373-3382.
(36) Fukuyama, T.; Lin, S. C.; Li, L. J . Am. Chem. Soc. 1990, 112,
7050-7051.
(37) Ho, P. T.; Ngu, K. Y. J . Org. Chem. 1993, 58, 2313-2316.
(38) Han, Y.; Chorev, M. J . Org. Chem. 1999, 64, 4, 1972-1978.
(39) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C.
A.; Shah, R. D. J . Org. Chem. 1996, 61, 3849-3862.
(40) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397-4398.
(41) Carpino, L. A.; El-Faham, A. J . Org. Chem. 1994, 59, 695-
698.
1
ESIMS and H NMR (data not shown). HATU-mediated
(42) Weitz, I. S.; Pellegrini, M.; Mierke, D. F.; Chorev, M. J . Org.
Chem. 1997, 62, 2527-2534.
J . Org. Chem, Vol. 67, No. 15, 2002 5089

Han et al.
SCHEME 5. Syn th esis of Mod el ERBIT-BTM Com p ou n d s
cyclization of 14 generated a new secondary amide bond
between a carboxyl of the malonyl residue and a free
amino of the aspartic acid at a yield of 56%. The yield of
the fully protected ERBIT-BTM 10, obtained through
pathway a , was 8 to 11-fold higher than the one obtained
via pathway b. Intermolecular coupling was effectively
overcome by the slow dropwise addition (>2 h) of the
DMF solution of 14 to the solution containing the
coupling reagent. Removal of the side chain protecting
groups, O-cHex and O-But, from 10 was carried out in
liquid HF at 0 °C and generated the model ERBIT-BTM
16 in 82% yield.
sense of direction. The rearrangement reaction was very
fast and efficient, and the generated product was stable
in the reaction mixture for several hours (as monitored
by LC-ESIMS). Long-term stabilization was achieved by
acetylation of the free amino group in the gem-diami-
noalkyl moiety, generating the N′i-to-Ni + 3 ERBIT-BTM
Ac-gPheN-mGly-Asp(OcHex)-NGly-OBut (17). Final re-
moval of the O-But and O-cHex protecting group is
accomplished in liquid HF generating the model N′i-to-
N
i + 3 ERBIT-BTM Ac-gPheN-mGly-Asp-NGly-OH (18) in
86% yield.
The presence of the carboxamide function at the amino
acid residue in the ith position of the ERBIT-BTM 10
was aimed at allowing the conversion of this residue into
the corresponding gem-diaminoalkyl by a mild oxidative
Hofmann-type rearrangement with iodobenzene bis-
(trifluoroacetate) (IBTFA) in aqueous solution.43 The
introduction of the gem-diaminoalkyl residue offers the
opportunity to resume chain elongation toward the
amino-terminus with the backbone recovering the correct
A closely related approach to synthesize a γ-turn
mimetic, as a fibrinogen receptor antagonist, was re-
ported by Callahan et al.44 (Figure 4). Reversal of the
i-to-i + 1 amide bond followed by ethylene bridging of
Ni-to-Ni
yielded a seven-membered PMRI γ-turn
+
2
mimetic. The major shortcoming of this design is the
absence of a carboxyl function that will allow the exten-
sion of the main chain with a C-terminus sequence, and
the method therefore does not generate a synthon that
can be incorporated as a γ-turn mimetic building block
within a sequence. The use of an ethylene-bridged
constraint was reported as a means to link Ni and Ni + 1
generating 2-oxopiperazines as dipeptidomimetics (Fig-
ure 4).45 However, this conformationally constrained
system was not designed to mimic a known element of
secondary structure.
(43) Radhakrishna, S. A.; Parham, M. E.; Riggs, R. M.; Loudon, G.
M. J . Org. Chem. 1979, 44, 1746-1747.
(44) Callahan, J . F.; Newlander, K. A.; Brugess, J . L.; Eggleston,
D. S.; Nichols, A.; Wong, A.; Huffman, W. F. Tetrahedron 1993, 3479-
3488.
F IGURE 4. Examples of ethylene-bridged peptidomimetics
reported in the literature. In these structures, the bridging is
either Ni-to-Ni + 2 or Ni-to-Ni + 1
.
5090 J . Org. Chem., Vol. 67, No. 15, 2002

Retro-Inverso â-Turn Peptidomimetic
TABLE 1. 1H a n d 13C Ch em ica l Sh ifts (p p m , r ela tive to TMS) of Mod el EBRIT-BTM Str u ctu r es
16
18
H
13C
13CdO
H
13C
13CdO
170.0
Ac
HN
CH3
R
8.37
1.79
5.74
2.76a
3.05
9.02
5.08
2.36
3.82
8.37
1.79
3.11
3.49a
22.7
62.1
39.2
45.8
Phe
4.64
3.05a
3.11
9.04
5.01
2.33
3.74a
60.5
34.3
45.4
171.3
â
3.23
2.82a
3.71
mGly
Asp
R
3.73a
166.8, 167.8
165.1, 167.5
HN
R
47.4
36.1
48.9
170.2
171.5
169.8
47.7
36.2
49.2
171.3
172.5
173.2
170.0
â
R
2.66
3.94
2.71a
4.03
Gly
Ac
HN
CH3
C1
C2
22.7
46.9
42.3
Eth
3.06a
3.26a
3.26a
3.55
46.6
46.8
3.56a
3.64
a
Overlapped resonances.
TABLE 2. Exp er im en ta l Dista n ces in Mod el
EBRIT-BTM Str u ctu r es Der ived fr om ROESY Sp ectr a a ,b
TABLE 3. 3J P r oton -P r oton Cou p lin g Con sta n ts (Hz)
in th e Mod el EBRIT-BTM Str u ctu r es Obta in ed fr om
P E-COSY Sp ectr a a ,b
distance
(Å)
distance
(Å)
cross peaks
cross peaks
16
18
16 18
16 18
Phe HR-Phe HâR
Phe HR-Phe HâS
Asp HN-Asp HR
Asp HR-Asp HâR
Asp HR-Asp HâS
Eth H1S-Eth H2R
Eth H1R-Eth H2R
Eth H1S-Eth H2S
Eth H1R-Eth H2S
8.4
6.4
9.6
9.2
3.6
7.6
1.2
8.2
Phe HR
Phe HâR
Phe HâS
Phe Hγ
Ac HN
Eth H2R
Eth H2S
Phe Hγ
Ac HN
3.3 2.6* Asp HN Eth H2R
2.7 3.0* Asp HN Eth H2S
2.6 2.6
3.6 3.9
2.7 2.5*
2.3 2.4*
10.0
8.4*
4.4*
Phe HR
Phe HR
Phe HR
Phe HR
Phe HR
Phe HâR
Phe HâR
Phe HâR
Phe HâS
Phe HâS
3.7
Asp HR
3.2 Asp HR
3.4 Asp HR
2.6 Asp HR
Asp HâR
Asp HâS
mGly HRR 3.3
Eth H1R
Eth H1S
2.0
3.4 4.1* Asp HR
3.0
3.3
2.2 3.3
2.8 3.2
2.7
2.8
2.9 2.7
2.6
3.2* Gly HRR Eth H1R
4.0* Gly HRR Eth H2R
3.6* Gly HRR Eth H1S
3.4 4.1* Gly HRS Asp HR
2.2 Gly HRS Eth H1R
3.8 Gly HRS Eth H1S
2.9 3.0 Ac HN
3.3 3.5* Ac HN
3.0 3.1* Eth H1R Eth H2R
Eth H1S
Eth H1S
Phe Hγ
a
R and S indicate pro-R and pro-S stereospecific assignments,
b
respectively. Asterisk indicates nonstereospecific assignment.
mGly HRS Eth H2R
mGly HRS Eth H2S
Asp HN
Asp HN
Asp HN
Asp HN
rPhe of 16. Overlapped 13C resonances between the amide
carbon and the carboxyl carbon of Asp precluded the
stereospecific assignment of the Asp â protons of 18.
Asp HR
Asp HâR
Asp HâS
Eth H2S
Ac CH3
3.5
2.2
mGly HRS 2.2 2.3
Random metrization and distance- and angle-derived
dynamics (DADD) simulations utilizing two-dimensional
rotating frame NOE (ROE) restraints produced 95-100
conformations with small violations (less than 0.3 Å for
ROEs). With the exception of the backbone dihedral
angles of r,gPhe (φ1 and ψ1), the dihedral angles of 16
and 18 populated the same conformational space. The
three amide bonds in the 10-membered ring were pref-
erentially oriented in the trans configuration, and the
projecting r,gPhe, Asp, and Gly residues displayed vary-
ing degrees of conformational freedom. A comparison of
the φ1 and ψ1 dihedral angles of rPhe and gPhe revealed
marked differences in their conformational preference
(Figure 5). The φ1 and ψ1 dihedral angles of rPhe were
dispersed, as expected for this exo-cyclic residue, while
the corresponding dihedral angles of gPhe were tightly
clustered. Within the cyclic ring system, the φ2 and ψ2
dihedral angles of mGly of both compounds were more
dispersed than the other φ,ψ pairs.
The conformations of the DG structures were analyzed,
and a structure that was representative of the distribu-
tion of dihedral angles, energies, and distance restraint
violations was selected for each mimetic and further
refined using EM and MD simulations (Figures 6 and
7). The DG algorithm generated structures that satisfied
the experimentally derived and holonomic distance re-
straints without regard for intra- and intermolecular
interactions (e.g., partial charges, Coulombic interactions,
a
R and S indicate pro-R and pro-S stereospecific assignments,
b
respectively. Asterisk indicates nonstereospecific assignment.
Con for m a tion a l An a lysis. The 1H and 13C NMR
spectra display one complete set of signals for each
peptide mimetic, indicating the lack of cis/trans isomer-
ization about the amide bonds. The H and 13C chemical
1
shifts obtained from total-correlation spectroscopy
(TOCSY), 1H-13C heteronuclear multiple-quantum shift
correlation (HMQC), and heteronuclear multiple bond
connectivity (HMBC) spectra are provided in Table 1.
Nuclear Overhauser enhancement spectroscopy (NOESY)
spectra yielded very few structurally informative cross-
peaks, as would be expected for a molecule of this size in
dimethyl sulfoxide (DMSO) at 298 K. All distance re-
straints were generated from two-dimensional rotating
frame NOE spectroscopy (ROESY) spectra (Table 2). The
3J HH coupling constants obtained from primitive exclusive
correlation spectroscopy (PE-COSY) spectra are provided
in Table 3. These coupling constants were used along
with the HMBC long-range heteronuclear couplings for
the diastereotopic assignment of the â-protons of Asp and
(45) (a) DiMaio J .; Bernard, B. J . Chem. Soc., Perkin Trans. 1 1989,
1687-1689. (b) Kojima, Y.; Ikeda, Y.; Kumata, E.; Maruo, J .; Okamoto,
A.; Hirostu, K.; Shibata, K.; Ohsuka, A. Int. J . Pept. Protein Res. 1991,
37, 468-475. (c) Pohmann, A.; Schanen, V.; Guillaume, D.; Quirion,
J .-C.; Husson, H.-P. J . Org. Chem. 1997, 62, 1016-1022.
J . Org. Chem, Vol. 67, No. 15, 2002 5091

Han et al.
F IGURE 6. Energy-minimized structures of model EBRIT-
BTM H2N-rPheN-mGly-Asp-NGly-OH 16 (top) and Ac-gPhe-
N-mGly-Asp-NGly-OH 18 (bottom) in DMSO. ROEs are indi-
cated by dashed lines.
tional changes, as illustrated by the superposition of
structures taken from the MD trajectory (Figure 7).
The ring structures of EBRIT-BTMs 16 and 18 were
derived from ethylene-mediated cyclization of the amide
nitrogens of rPhei and Glyi + 3. Cyclization of the peptide
mimetics resulted in a constrained ring system with a
limited number of conformations. As anticipated, the
structures of the 10-membered ring system for EBRIT-
BTM 16 and 18 were nearly identical (Table 4). The
results from the MD simulations also indicate a low
degree of flexibility in the core ring system (Figure 7), a
desirable feature for maintaining the optimal topological
display of the pharmacophores. As expected, the side
chains, with unhindered rotation about the CR-Câ
bonds, displayed a much higher degree of flexibility.
Superposition of the heavy backbone atoms of the 10-
membered ring, excluding the ethylene bridge, with the
standard â-turn structures (Figure 8) indicated that the
EBRIT-BTM system most closely resembled a type II′
â-turn (RMSD 0.3 Å). Superposition of compound 18 with
a model âII′-turn (φ,ψ values of +60°, -120°, -80°, and
0°) is illustrated in Figure 8. The distances between the
F IGURE 5. Ramanchandran plots indicating the conforma-
tional space of the φ1 and ψ1 of r,gPhe, the φ2 and ψ2 of mGly,
and the φ3 and ψ3 of Asp, during the DG simulation. Open (O)
and closed (b) circles depict dihedral angle pairs for structures
16 and 18, respectively.
CRi and CRi
atoms of the model EBRIT-BTM II,
+
3
namely, compound 18 (5.5 Å), and the âII′-turn (5.1 Å)
were comparable, indicating a propensity for the â-turn
mimetic to nucleate the formation of a â-sheet.
Rearrangement of the carboxamide group -CONH2 in
16 and concomitant acetylation transformed it into the
acetamido group -NHCOCH3 (compound 18). This modi-
fication resulted in the formation of an intramolecular
hydrogen bond between the NRH of gPhe-COCH3 and the
carboxyl of Gly, as observed by the DG, EM, and MD
calculations. The distance between the hydrogen bond
donor and the acceptor was 2.2 Å, and the distance
between the nitrogen and oxygen atoms was 2.9 Å. This
hydrogen bond is consistent with the onset of an anti-
parallel â-sheet. The model EBRIT-BTM 18 mimics the
â-turn often found at the center of an antiparallel â-sheet,
and Lennard-J ones interactions). Through the use of
restrained EM and MD simulations, the conformational
space of the EBRIT-BTM scaffolds with respect to the
potential energy surface was assessed in DMSO, the
same solvent system that was used in the NMR experi-
ments. The dihedral angles of the representative energy-
minimized structures of EBRIT-BTM 16 and 18 were
consistent between the two structures (Table 4). During
the MD simulation, the backbone atoms of the core 10-
membered ring system did not undergo major conforma-
5092 J . Org. Chem., Vol. 67, No. 15, 2002

Retro-Inverso â-Turn Peptidomimetic
F IGURE 8. Superposition of the heavy backbone atoms of
the 10-membered ring system of model EBRIT-BTM Ac-
gPheN-mGly-Asp-NGly-OH (18) with the amino acid residues
of an ideal âII′ turn.
protein consisting of two subunits linked through a two-
stranded antiparallel â-sheet.46 Therefore, in addition to
providing a structural role, â-turn mimetics may poten-
tially provide a functional role as well. The EBRIT-BTM
scaffolds and their structural features as determined here
will greatly assist in these efforts.
Con clu sion
The design, synthesis, and conformational analysis of
novel model EBRIT-BTMs 16 and 18 presented in this
report offer new forms of backbone-to-backbone confor-
mational constraints. This approach not only spares the
integrity of the original side chains and maintains the
overall polar nature of the backbone but also avoids the
introduction of extra bulky and hydrophobic aromatic or
aliphatic ring moieties. The lack of these extra structural
features eliminates sites that may contribute to nonspe-
cific interactions at the macromolecular acceptor and
compromise solubility. The design and synthesis employ
readily available building blocks and allow a large degree
of diversification that includes the choice of the peptide
bond reversal site and the selection of side chains and
chirality at the CR-carbons comprising the ring. The
EBRIT-BTM can be used either in isolation as a molec-
ular scaffold to present pharmacophores of choice at a
restricted topology, or as a conformational element
mimetic when incorporated into peptide sequences. Evi-
dently, the model EBRIT-BTM compounds 16 and 18
include achiral residues in positions i + 1 and i + 3, mGly
and Gly, respectively. We believe that the methodology
described in this report will generate the EBRIT-BTM
compounds containing chiral residues at these positions
but probably in a lower overall yield. In addition,
incorporating C-2-substituted-malonyl residues may in-
troduce configurational lability at the malonyl chiral
carbon, which may lead to enhanced conformational
heterogeneity. As previously reported, the configurational
lability of the chiral malonyl residue depends on the
nature of the C-2 substituent and the nature of the
flanking sequences.31-33 Taken together, these EBRIT-
BTM systems offer new tools to investigators interested
in elucidating the biologically relevant conformation of
F IGURE 7. Results from the molecular dynamics simulations
of model EBRIT-BTM H2N-rPheN-mGly-Asp-NGly-OH 16
(top) and Ac-gPheN-mGly-Asp-NGly-OH 18 (bottom) in DMSO.
Molecules have been superimposed using the heavy backbone
atoms of the ring system. The degree of overlap among the
structures is indicative of the conformational flexibility in the
system.
TABLE 4. Dih ed r a l An gles of Mod el EBRIT-BTM
Str u ctu r es 16 a n d 18 F ollow in g En er gy Min im iza tion of
Selected DG Str u ctu r es
16
18
16
63
-172
-169
55
-91
153
-112
18
65
-75
173
159
-97
166
-92
φ1
ψ1
ø1
ω1
φ2
ψ2
ω2
φ3
-111
-89
-71
170
79
-118
141
-166
105
-145
174
60
-116
149
ψ3
ø3
ω3
φ4
µ1
µ2
µ3
-122
-113
and with the correct orientation of the attached amide
and carbonyl groups, the second hydrogen bond is formed.
Therefore, compound 18 may find use in the stabilization
of â-sheets. In contrast, compound 16 without the ability
to form this hydrogen bond, although it maintains the
correct overall topology of a â-turn, does not stabilize the
propagation of a â-sheet. The stabilization provided by
this exo-cyclic hydrogen bond can be seen in the φ,ψ
dihedral angles of Phe (Figure 5).
Recently, a conformationally constrained â-sheet mi-
metic was shown to inhibit dimerization of human
immunodeficiency virus type 1 protease, a homodimeric
(46) Bouras, A.; Boggetto, N.; Benatalah, Z.; de Rosny, E.; Sicsic,
S.; Reboud-Ravaux, M. J . Med. Chem. 1999, 42, 957-962
J . Org. Chem, Vol. 67, No. 15, 2002 5093

Han et al.
N-(Eth oxyca r bon ylm eth yl)glycin e ter t-Bu tyl Ester (2).
A mixture of 1 (21.5 g, 70 mmol) and 10% Pd-C (2 g) in 95%
ethanol (200 mL) was hydrogenated at 40 psi for 2 h and then
filtered through a Celite bed, concentrated in vacuo, and
vacuum-dried to afford 14.9 g of 2 (97% yield) as a colorless
oil. This crude product was used in the next step without
further purification: Rt ) 11.72 min (0-100% B in A, 30 min);
1H NMR (CDCl3) δ 5.76 (broad, 1H), 4.16 (q, J ) 7.5 Hz, 2H),
3.46 (s, 2H), 3.36 (s, 2H), 1.46 (s, 9H), 1.26 (t, J ) 7.5 Hz, 3H);
13C NMR δ 81.5, 57.3, 56.0, 55.1, 28.2, 14.3; ESIMS calcd for
peptides and will help molecular designers of peptide-
based drugs in their search for smaller, more selective,
pharmacokinetically more favorable peptide mimetics.
Our future efforts will be directed to improve and
implement the synthetic methodology and expand the
diversity of these EBRIT-BTMs. We will also use these
systems as molecular scaffolds and as conformational
determinants of secondary structure elements to generate
peptidomimetics of biological interest.
C
10H19NO4 217.26, found m/z ) 218 (M + H)+.
N-(Ca r boxym eth yl)-N-F m oc-glycin e ter t-Bu tyl Ester
Exp er im en ta l Section
(3). Compound 2 (13.0 g, 60 mmol) was dissolved in dioxane
(200 mL) and cooled with an ice-water bath. LiOH‚H2O (2.94
g, 70 mmol) in water (200 mL) was then added, and the
resulting mixture was stirred at 5 °C overnight. Addition of
NaHCO3 (5.9 g, 70 mmol) was followed by introduction of small
aliquots of Fmoc-Cl (18.1 g, 70 mmol). The resulting mixture
was slowly warmed to room temperature and stirred for 6 h
and then neutralized with saturated KHSO4 to pH ) 3,
concentrated in vacuo to ∼300 mL, and extracted with EtOAc
(3 × 200 mL). The combined extract was washed with water
(200 mL), dried, and concentrated in vacuo. Purification by
column chromatography afforded 21.2 g of 3 (86% yield): Rf )
0.43 (35:64:1 EtOAc/hexane/HOAc); 1H NMR (CDCl3) δ (cis/
trans isomers) 7.78-7.30 (m, 8H), 4.47 (d, J ) 5.5 Hz, 2H),
4.28-4.15 (m, 5H), 4.02 (s, 1H), 3.98 (s, 1H), 1.47 (s, 9H), 1.25
(t, J ) 7.5 Hz, 1.5H), 1.24 (t, J ) 7.5 Hz, 1.5H); 13C NMR δ
(cis/trans isomers) 198.0, 168.5, 156.2, 143.8, 141.4, 128.0,
127.3, 125.2, 120.1, 82.6, 68.6, 57.6, 57.4, 50.6, 50.5, 47.3, 47.2,
28.2, 23.4, 14.4; ESIMS calcd for C23H25NO6 411.45, found 412
(M + H)+. Anal. Calcd for C23H25NO6: C, 67.14; H, 6.12; N,
3.40. Found: C, 66.96; H, 6.17; N, 3.52.
N-(S-Eth yl Th ioca r bon ylm eth yl) N-F m oc-Glycin e ter t-
Bu tyl Ester (4). N,N′-Dicyclohexylcarbodiimide (DCC) (11.3
g, 55 mmol) was added to an ice-cold solution of 3 (20.5 g, 50
mmol), EtSH (7.7 mL, 100 mmol), and 4-(dimethylamino)-
pyridine (630 mg, 5 mmol) in dichloromethane (DCM) stirred
at room temperature for 2 h, filtered, concentrated in vacuo,
and purified by flash column chromatography to afford 19.5 g
of 4 (87% yield) as a colorless oil: Rf ) 0.42 (25:75 EtOAc/
hexane); 1H NMR (CDCl3) δ (cis/trans isomers) 7.78-7.29 (m,
8H), 4.44 (d, J ) 8 Hz, 2H), 4.29 (s, 1H), 4.20 (s, 1H), 4.28-
4.24 (m, 1H), 4.20 (s, 1H), 4.07 (s, 1H), 4.04 (s, 1H), 2.91 (q, J
) 7.5 Hz, 1H), 2.90 (q, J ) 7.5 Hz, 1H), 2.90 (m, 2H), 1.47 (s,
3H), 1.26 (t, J ) 7.5 Hz, 1.5H), 1.25 (t, J ) 7.5 Hz, 1.5H); 13C
NMR δ (cis/trans isomers) 198.0, 197.8, 168.4, 168.3, 156.2,
156.0, 143.9, 141.4, 129.0, 127.3, 125.2, 120.1, 82.4, 68.6, 57.6,
57.3, 50.5, 50.3, 47.2, 28.2, 23.2; ESIMS calcd for C25H29NO5S
455.57, found 456 (M + H)+. Anal. Calcd for C25H29NO5S: C,
65.91; H, 6.42; N, 3.07. Found: C, 66.20; H, 6.45; N, 3.13.
Gen er a l Meth od s. Thin-layer chromatography (TLC) was
performed on plastic plates coated with (0.20 mm) silica gel
60 F254. Open column chromatography was carried out on
silica gel 60 (200-400 mesh) using the solvent mixture as
indicated for TLC. All organic phases obtained from extraction
of aqueous solutions were dried over MgSO4 prior to evapora-
tion in vacuo. Analytical reversed-phase high-performance
liquid chromatography (RP-HPLC) was carried out on
a
Waters 100 RP-18 column (5 µm, 19 mm × 150 mm) column
at a flow rate of 1 mL/min. Semipreparative RP-HPLC was
carried out on a Vydac 90 Å C-18 column (15-20 µm, 10 ×
250 mm) at a flow rate of 20 mL/min. Preparative RP-HPLC
was carried out on a Vydac 300 Å C-18 column (15-20 µm, 47
× 300 mm) at a flow rate of 70 mL/min. The solvent system
used in RP-HPLC included the following: solvent A, 0.1% (v/
v) TFA in H2O; solvent B, 0.1% TFA in CH3CN. The effluent
was monitored at 220 nm. 1H and 13C NMR spectra were
collected on a 500 MHz instrument. NMR chemical shifts are
reported in parts per million. 1H chemical shifts were cali-
brated using tetramethylsilane (TMS) as an internal standard
(0 ppm). 13C chemical shifts were calibrated using the CDCl3
resonance (77.2 ppm) or DMSO-d6 resonance (39.5 ppm) as
an internal standard unless otherwise noted. The coupling
constants in hertz were determined from the one-dimensional
spectra unless otherwise noted. Melting points were deter-
mined with a capillary melting point apparatus and are
uncorrected. Elementary microchemical analysis was carried
out at E+R Microanalytical Laboratory, Inc., NY. Analytical
results were within 0.3% of the theoretical values. Mass
spectroscopy was performed in our laboratory using an elec-
trospray ionization (ESI) source. N,N-Dimethylformamide
(DMF) was dried with 4 Å molecular sieves for 48 h before
use. Other commercially available chemicals were used with-
out further treatment. Monobenzylmalonate47 and N-benzyl-
oxycarbonyl â-cyclohexyl aspartate48 were prepared according
to published procedures.
N-Ben zyl-N-(eth oxyca r bon ylm eth yl)glycin e ter t-Bu tyl
Ester (1). A mixture of N-benzylglycine ethyl ester (19.3 g,
100 mmol), tert-butyl bromoacetate (21.4 g, 110 mmol), and
K2CO3 (13.8 g, 100 mmol) in DMF (100 mL) was heated at
95-100 °C for 4 h and then concentrated in vacuo. The residue
was extracted with EtOAc (500 mL), washed with water (2 ×
300 mL) and brine (100 mL), dried, and concentrated in vacuo.
Purification by flash column chromatography on silica gel
afforded 26.4 g of 1 (86% yield) as a colorless oil: Rf ) 0.43
(15:85 EtOAc/hexane); Rt ) 18.05 min (0-100% B in A 30 min);
1H NMR (CDCl3) δ 7.40-7.30 (m, 5H, ArH), 4.16 (q, 2H, J )
7.3 Hz, CH2CH3), 3.91 (s, 2H, CH2Ph), 3.53 (s, 2H, CH2-
COOBut), 3.44 (s, 2H, CH2COOEt), 1.46 (s, 9H, But), 1.26 (t,
3H, J ) 7.3 Hz, CH2CH3); 13C NMR δ 171.3, 170.5, 138.3,
129.2, 128.4, 127.4, 81.1, 60.4, 57.7, 55.1, 54.3, 28.2, 14.3;
ESIMS calcd for C17H25NO4 307.38, found m/z ) 308 (M + H)+.
Anal. Calcd for C17H25NO4: C, 66.43; H, 8.20; N, 4.56; Found:
C, 66.21; H, 8.17; N, 4.74.
N-(F or m ylm eth yl) N-F m oc-Glycin e ter t-Bu tyl Ester
(5). Triethylsilane (31.9 mL, 200 mmol) was rapidly added to
an ice-cold mixture of 4 (18.2 g, 40 mmol) and 10% Pd-C (2.0
g) in acetone (200 mL) and stirred at 10-20 °C for 30 min.
The reaction mixture was filtered through a Celite bed, and
the cake was washed with acetone (50 mL). The combined
filtrates were concentrated in vacuo and purified by flash
column chromatography to afford 14.6 g of 5 (93% yield) as a
colorless oil: Rf ) 0.38 (35:65 EtOAc/hexane); 1H NMR (CDCl3)
δ (cis/trans isomers) 9.67 (s, 06H), 9.37 (s, 0.4H), 9.78-9.73
(m, 8H), 4.52 (d, J ) 5.5 Hz, 1H), 4.44 (d, J ) 7 Hz, 1H), 4.29-
4.23 (m, 1H), 4.11 (s, 1H), 4.01 (s, 1H), 4.00 (s, 1H), 3.83 (s,
1H), 1.47 (s, 9H); 13C NMR δ (cis/trans isomers) 198.7, 168.6,
156.3, 156.0, 143.8, 141.4, 128.0, 127.3, 125.2, 124.8, 120.2,
82.6, 68.6, 68.1, 58.4, 57.7, 50.7, 47.2, 28.2; ESIMS calcd for
C
23H25NO5 395.45, found 396 (M + H)+.
3-P h en yl-1(S)-[[2′-[N′′-(9-flu or en ylm et h oxyca r b on y)-
N′′-(ter t-b u t yloxyca r b on ylm et h yl)]a m in o]et h yla m in o]-
p r op ion a m id e (6). Sodium triacetoxyborohydride [NaB-
(OAc)3H] (9.54 g, 45 mmol) was added to a solution of 5 (11.9
g, 30 mmol) and phenylalanylamide hydrochloride (7.0 g, 35
(47) Chorev, M.; Rubini, E.; Gilon, C.; Wormser, U.; Selinger, Z. J .
Med. Chem. 1983, 26, 129-135.
(48) Sennyey, G.; Barcelo, G.; Senet, J . P. Tetrahedron Lett. 1986,
27, 5375-5376.
5094 J . Org. Chem., Vol. 67, No. 15, 2002

Retro-Inverso â-Turn Peptidomimetic
mmol) in DMF (100 mL) and stirred at room temperature for
6 h. The reaction mixture was poured into water (300 mL) and
extracted with EtOAc (3 × 200 mL). The combined extracts
were washed with water (100 mL), dried, and concentrated in
vacuo. Purification by flash column chromatography afforded
14.6 g of 6 (90% yield) as a viscous oil. An analytical sample
was further purified by semipreparative RP-HPLC on a Vydac
C-18 column, λ ) 220 nm, employing a linear gradient of
Anal. Calcd for C59H64N4O11: C, 70.50; H, 6.42; N, 5.57.
Found: C, 70.06; H, 6.27; N, 5.38.
2(S)-[N-[[(N′-(9-F lu or en ylm et h oxyca r b on yl)-N′-(ter t-
bu tyloxyca r bon ylm eth yl)]a m in oeth yl]-N-[(1′,3′-d ioxo-3′-
ben zyloxy)p r op yl]]-2-a m in o-3-p h en ylp r op ion a m id e (11).
HATU (380 mg, 10 mmol) was added to a solution of 6 (2.72
g, 5 mmol), monobenzylmalonate (1.94 g, 10 mmol), and
DIPEA (2.6 mL, 15 mmol) in DMF (20 mL) and stirred at room
temperature for 2 h. The reaction mixture was concentrated
in vacuo, and the residue was dissolved in EtOAc (200 mL).
The solution was washed with water (100 mL), dried, and
concentrated in vacuo. The residue obtained was purified by
flash chromatography affording 3.38 g of 11 (94% yield) as a
viscous oil. An aliquot was dissolved in H2O-CH3CN (30:70,
v/v) and lyophilized to afford a white powder: mp 63-65 °C;
Rf ) 0.42 (1:1 EtOAc/hexane); Rt ) 23.84 (20-100% B in A,
30 min); [R]D20) -20.5 (c 0.74, MeOH); 1H NMR (CDCl3) δ (cis/
trans isomers) 7.77-7.05 (m, 18H), 5.16 (s, 2H), 5.09 (s, 1.1H),
5.05 (s, 0.9H), 4.57-2.86 (m, 12H), 1.46 (s, 9H); 13C NMR δ
(cis/trans isomers) 172.4, 168.8, 168.6, 168.3, 156.1, 143.8,
141.4, 135.3, 129.3, 129.0, 128.8, 128.7, 128.5, 128.0, 127.9,
127.4, 127.0, 125.3, 124.7, 120.2, 82.5, 82.1, 68.2, 67.6, 51.3,
48.9, 47.4, 41.2, 38.9, 34.1, 28.2; ESIMS calcd for C42H45N3O8
719.82, found 720 (M + H)+. Anal. Calcd for C42H45N3O8: C,
70.08; H, 6.30; N, 5.84. Found: C, 69.72; H, 6.43; N, 5.76.
0-70% (v/v) B in A over 100 min and then lyophilized: mp
20
60-62 °C; Rf ) 0.36 (80:19:1 EtOAc/hexane/TEA); [R]D
)
-20.3° (c 0.76, MeOH); 1H NMR (CDCl3) δ (cis/trans isomers)
7.74-7.15 (m, 13H), 6.00 (m, 1H), 4.34 (d, J ) 7.0 Hz, 1H),
4.18 (m, 1H), 3.73 (d, J ) 2.5 Hz, 1H), 3.47 (m, 1H), 3.31 (dd,
J ) 9.0, 5.0 Hz, 1H), 3.24-3.15 (m, 3H), 2.77-2.62 (m, 2H),
1.44 (s, 9H); 13C NMR δ (cis/trans isomers) 177.1, 176.8, 168.9,
156.3, 143.9, 141.3, 137.6, 129.1, 128.8, 127.8, 127.2, 126.9,
125.1, 124.8, 120.0, 82.2, 82.0, 68.0, 67.4, 64.3, 64.1, 50.5, 50.1,
49.2, 48.7, 47.3, 47.0, 46.8, 39.4, 39.2, 28.1; ESIMS calcd for
C
C
32H37N3O5 543.65, found 544 (M + H)+. Anal. Calcd for
32H37N3O5‚1/3TFA: C, 67.39; H, 6.47; N, 7.22. Found: C,
67.02; H, 6.68; N, 7.09.
N-(ter t-Bu tyloxyca r bon ylm eth ylca r bon yl) r-(9-F lu o-
r en ylm eth yl), â-Cycloh exyl L-Asp a r ta te (7). DCC (4.5 g,
22 mmol) was added to a stirred, ice-cold solution of l-R-(9-
fluorenylmethyl)-â-cyclohexyl-aspartate hydrochloride [H-Asp-
(OcHex)-OFm]38 (4.3 g, 20 mmol), diisopropylethylamine
(DIPEA) (3.5 mL, 20 mmol), and mono-tert-butylmalonate (3.2
g, 20 mmol) in DCM (50 mL). After stirring for 30 min at room
temperature, the reaction mixture was filtered, and the filtrate
was concentrated in vacuo. The residue obtained was purified
by flash column chromatography to afford 9.8 g of 7 (92% yield)
2(S )-[N -[[N ′-(t er t -Bu t yloxyca r b on ylm e t h yl)]a m in o-
ethyl]-N-[(1′,3′-dioxo-3′-benzyloxy)propyl]]-2-amino-3-phenyl-
p r op ion a m id e (12). Compound 11 (2.88 g, 4 mmol) was
added to 2% DBU in acetonitrile (10 mL) and stirred at room
temperature for 30 min. The reaction mixture was concen-
trated in vacuo and purified by column chromatography
affording 2.10 g of 12 (87% yield) as an oil. An aliquot was
dissolved in H2O-CH3CN (30:70, v/v) and lyophilized to afford
a white powder: mp 75-76 °C; Rf ) 0.37 (80:19:1 EtOAc/
1
as a viscous oil: Rf ) 0.44 (25:75 EtOAc/hexsane); H NMR
(CDCl3) δ 7.77-7.25 (m, 8H), 4.80-4.65 (m, 4H), 4.41 (m, 2H),
4.21 (t, J ) 7 Hz, 1H), 3.04-3.02 (m, 1H), 2.81-2.78 (m, 1H),
1.20-1.79 (m, 28H); 13C NMR δ 171.6, 171.3, 170.6, 167.3,
155.6, 143.8, 141.3, 128.1, 127.4, 125.3, 120.1, 81.4, 73.8, 68.2,
20
hexane/TEA); Rt ) 18.91 min (0-100% B in A, 30 min); [R]D
1
) -19.7° (c 0.82, MeOH); H NMR (CDCl3) δ 7.35-7.15 (m,
66.4, 50.1, 46.9, 31.6, 28.2, 25.4, 23.9; ESIMS calcd for C31H37
-
10H), 5.66 (m, 1H), 5.15 (m, 2H), 3.56 (s, 2H), 3.42-3.35 (m,
2H), 3.25-3.20 (m, 2H), 3.11-2.95 (m, 2H), 2.83-2.72 (m, 1H),
2.53-2.49 (m, 2H), 1.42 (s, 9H); 13C NMR δ 172.6, 171.0, 167.8,
166.9, 138.5, 135.2, 129.2, 128.9, 128.7, 128.5, 128.4, 126.6,
81.6, 67.5, 51.1, 47.1, 41.7, 33.6, 28.1; ESIMS calcd for
NO7 535.63, found 536 (M + H)+.
N-(Ca r boxylm eth ylca r bon yl) r-(9-F lu or en ylm eth yl),
â-Cycloh exyl l-Asp a r ta te (8). Compound 7 (8.0 g, 15 mmol)
was dissolved in Et2O (200 mL) and then saturated with HCl
(g). The mixture was stored at room temperature overnight,
concentrated in vacuo, and vacuum-dried to afford 7.1 g of 8
(100% yield) as a light yellow semisolid. The product became
a glasslike solid after storage in the refrigerator for several
days. This crude product was used in the subsequent reaction
without further purification: Rf ) 0.39 (35:65 EtOAc/hexane);
1H NMR (CDCl3) δ 7.77-7.25 (m, 8H), 4.79-4.65 (m, 2H), 4.37
(m, 2H), 4.25-4.22 (m, 1H), 3.04-3.02 (m, 1H), 2.81-2.78 (m,
1H), 1.20-1.79 (m, 19H); 13C NMR δ 171.6, 170.2, 170.6, 167.3,
155.6, 143.8, 141.5, 128.1, 127.4, 125.3, 120.1, 73.8, 67.2, 63.4,
51.1, 45.6, 31.6, 25.4, 23.9; ESIMS calcd for C27H29NO7 479.52,
found 480 (M + H)+.
1-(9-F lu or en ylm eth yl), 4-Cycloh exyl 2(S)-{N-[3′-[N′-[2′′-
[[N′′-(9-F lu or en ylm et h oxyca r b on yl)-N′′-(ter t-b u t yloxy-
ca r bon ylm eth yl)]a m in oeth yl]-N′-[2′-[2′(S)-3′-p h en ylp r o-
p ion a m id e]]-1′,3′-d ioxop r op yl]-a m in o} Su ccin n a t e (9).
HATU (1.14 g, 3 mmol) was added to a solution of 6 (1.08 g, 2
mmol), 8 (1.44 g, 3 mmol), and DIPEA (ml, 5 mmol) in DMF
(20 mL) and stirred at room temperature for 2 h. This was
followed by another portion of HATU (1 mmol), and the
mixture was stirred for 2 h. The reaction mixture was
concentrated in vacuo, and the residue was dissolved with
EtOAc (2 × 50 mL). The solution was washed with NaHCO3
(30 mL) and brine (30 mL), dried, and concentrated in vacuo.
Purification of the residue by flash column chromatography
afforded 1.85 g of 9 (92% yield) as a viscous oil. An aliquot
was dissolved in H2O-CH3CN (30:70, v/v) and lyophilized to
afford a white powder: mp 69-71 °C; Rf ) 0.38 (1:1 EtOAc/
hexane); [R]D20 ) -18.7° (c 0.63, MeOH); 1H and 13C NMR were
very complicated due to cis/trans isomers (data not shown);
ESIMS calcd for C59H64N4O11 1105.16, found 1105 (M + H)+.
C
30H44N4O9 604.69, found m/z 605 (M + H)+.
2(S)-[N-[[(N′-(Cycloh exyl 3(S)-3-N′′-Ben zyloxycar bon yl-
am in o-4-oxo-bu tyr ate)]-N′-(ter t-bu tyloxycar bon ylm eth yl)]-
aminoethyl]-N-[(1′,3′-dioxo-3′-benzyloxy)propyl]]-2-amino-3-
p h en ylp r op ion a m id e (13). HATU (228 mg, 6 mmol) was
added to a solution of 12 (1.81 g, 3 mmol), â-cyclohexyl
N-benzyloxycarbonylaspartate [N-Cbz-Asp(OcHex)-OH] (1.89
g, 5 mmol), and DIPEA (1.3 mL, 10 mmol) in DMF (10 mL).
The resulting mixture was stirred at room temperature for 4
h and concentrated in vacuo. The residue was extracted with
EtOAc (2 × 200 mL), washed with water (100 mL), dried, and
concentrated in vacuo. Purification by flash column chroma-
tography afforded 2.15 g of 13 (87% yield) as a viscous oil. An
aliquot was dissolved in H2O-CH3CN (30:70, v/v) and lyoph-
ilized to afford a white powder: Rt ) 21.36 (20-100% B in A,
30 min) [R]D20) -21.5° (c 0.53, MeOH); 1H and 13C NMR were
very complicated due to cis/trans isomers (data not shown);
ESIMS calcd for C45H56N4O11 828.95, found 829 (M + H)+.
Anal. Calcd for C45H56N4O11
: C, 65.20; H, 6.81; N, 6.76.
Found: C, 65.01; H, 6.67; N, 6.42.
2(S)-[N-[[(N′-(Cycloh exyl 3(S)-3-Am in o-4-oxo-bu tyr ate)]-
N′-(ter t-bu tyloxyca r bon ylm eth yl)]a m in oeth yl]-N-(3′-oxo-
p r op ion ic a cid )-2-a m in o-3-p h en ylp r op ion a m id e (14). A
mixture of 13 (1.66 g, 2 mmol) and 10% Pd-C (160 mg) in
95% methanol (50 mL) was hydrogenated at 40 psi for 2 h,
filtered through a Celite bed, concentrated in vacuo, and
vacuum-dried to afford 1.17 g of 14 (97% yield) as a white solid.
This crude product was used immediately in the subsequent
reaction without further purification: Rt ) 18.37 min (0-100%
B in A, 30 min); ESIMS calcd for C30H44N4O9 604.69, found
605 (M + H)+.
J . Org. Chem, Vol. 67, No. 15, 2002 5095

Han et al.
1-(ter t-Bu tyloxyca r bon ylm eth yl)-3(S)-cycloh exyloxy-
ca r b on ylm et h yl-9-[2′(S)-3′-p h en ylp r op ion a m id e]-2,5,7-
tr ioxo-1,4,8-tr ia za d eca n e (10). Compound 14 (907 mg, 1.5
mmol) and DIPEA (0.65 mL, 5 mmol) were dissolved in DMF
(50 mL) and added dropwise over 2 h to a solution of HATU
(114 mg, 3 mmol) in DMF (100 mL) at room temperature. After
2 h of additional stirring, the reaction mixture was concen-
trated in vacuo. The residue was extracted with EtOAc (2 ×
100 mL) and washed with water (50 mL), and the organic
phase was dried and concentrated in vacuo. The residue that
was purified by preparative RP-HPLC on a Vydac C-18
column, λ ) 220 nm, employing a linear gradient of 0-70%
(v/v) B over 100 min, afforded 490 mg of 10 (56% yield) as a
RPipe.49 ROE cross-peaks were integrated using Felix (Biosym
Technologies, Inc.; San Diego, CA). All spectra were referenced
to tetramethylsilane.
Proton and carbon assignments were obtained using PE-
55
COSY,50 TOCSY,51,52 NOESY,53,54 and ROESY
spectra,
recorded in the phase-sensitive mode using the method of
States et al.56 NOESY and ROESY spectra were collected at
298 and 308 K, respectively, with mixing times varying from
150 to 500 ms; the temperature for all other experiments was
298 K. In the ROESY experiments, a spin lock field of 2500
Hz was realized by a series of short pulses as described by
Kessler et al.57 sandwiched between two 90° pulses to com-
pensate for offset effects (compensated ROESY). The typical
spectral width was 5200 Hz in both dimensions with 2048
points in f2 and 600 in f1, and 32-64 scans were collected at
each increment. HMQC58 spectra were used for the assignment
of carbons with directly bonded protons, with 1024 data points
collected in the proton dimension and 10 000 Hz and 200 data
points for the carbon dimension. Forward linear prediction to
1024 points and zero-filling to 2048 points were applied to the
incremented dimension. Gaussian apodization was applied in
both the f2 and f1 dimensions. The PE-COSY pulse sequence
was used for the determination of homonuclear coupling
constants. Spectra were acquired at 298 K with 4096 data
points in f2 and 640 incremental data points. HMBC59 spectra
were recorded to determine the magnitude of long-range
20
white powder: Rt ) 21.21 min (0-100 B in A 30 min); [R]D
) +6.5° (c 0.12, MeOH); 1D 1H and 13C NMR were very
complicated due to cis/trans isomers (data not shown; ESIMS
calcd for C30H42N4O8 586.68, found 587 (M + H)+. Anal. Calcd
for C30H42N4O8: C, 61.42; H, 7.22; N, 9.55. Found: C, 61.07;
H, 7.47; N, 9.28.
1-(ter t-Bu tyloxyca r bon ylm eth yl)-3(S)-cycloh exyloxy-
ca r bon ylm eth yl-9-[2′(S)-3′-p h en yl-p r op ion a m id e]-2,5,7-
tr ioxo-1,4,8-tr ia za d eca n e (16). Compound 10 (59 mg, 0.1
mmol) and Me2S (1 mL) were placed in a Teflon reactor, which
was connected to a HF-reaction apparatus. Liquid HF (∼10
mL) was introduced, and the resulting mixture was stirred at
0 °C for 4 h. A residue was obtained after removing the volatile
components under vacuum. The residue obtained was dis-
solved in water (20 mL) and purified by semipreparative RP-
HPLC on a Vydac C-18 column, λ ) 220 nm, employing a linear
gradient of 0-10% (v/v) B over 100 min. Purification afforded
36 mg of 16 (82% yield) as a white powder: mp 72-74 °C; Rt
3
heteronuclear coupling constants, J CH, with a spectral width
of 20 000 Hz and 2048 data points.
Com p u ta tion a l Meth od s. Metric matrix DG calculations
were carried out with
a home-written program utilizing
random metrization.60 Experimental distance constraints that
were more restrictive than the geometric distance bounds
(holonomic restraints) were used to create the final distance
matrix.61 The structures were first embedded in four dimen-
sions and then partially minimized using conjugate gradients
followed by distance- and angle-driven dynamics (DADD);28,62,63
the DADD simulation was carried out at 1000 K for 50 ps with
a gradual reduction in the temperature over the next 30 ps.
The DADD procedure utilized holonomic and experimental
distance constraints plus a chiral penalty function for the
generation of the violation “energy” and forces. A distance
matrix was calculated from each structure, and the EMBED
algorithm61 was used to calculate coordinates in three dimen-
sions.
1
) 22.92 min (0-15% B in A 30 min); detailed H and 13C NMR
analysis is reported in Tables 1-4; ESIMS calcd for C20H24N4O8
448.43, found 449 (M + H)+.
1-(ter t-Bu tyloxyca r bon ylm eth yl)-3(S)-cycloh exyloxy-
ca r bon ylm eth yl-9-[1′(R)-a ceta m id e-2′-p h en yleth yl]-2,5,7-
t r ioxo-1,4,9-t r ia za d eca n e (17). Iodobenzene bis(trifluoro-
acetate) (IBTFA, 107 mg, 0.25 mmol) was added to a solution
of 10 (59 mg, 0.1 mmol) in THF-water (2:1, 1.5 mL) at room
temperature. Monitoring the reaction by LC-MS revealed that
the rearrangement proceeded quantitatively and that the
product 1-(tert-butyloxycarbonylmethyl)-3(S)-cyclohexyloxy-
carbonylmethyl-9-[1′(R)-amino-2′-phenylethyl]-2,5,7-trioxo-
1,4,8-triazadecane was stable in solution for hours. Rt ) 18.75
(0-100% B in A 30 min); ESIMS calcd for C29H42N4O7 558.31,
found 558.67 (M + H)+.
After the reaction mixture was stirred for 2 h, acetic
anhydride (0.94 mL, 1 mmol) was added followed by TEA (1.39
mL, 1 mmol). The reaction mixture was stirred for an ad-
ditional 2 h and then concentrated in vacuo. Purification of
the residue by semipreparative RP-HPLC on a Vydac C-18
column, λ ) 220 nm, employing a linear gradient of 0-70%
(v/v) B over 100 min, afforded 50 mg of 17 (84% yield) as a
white powder: Rt ) 21.72 min (0-100% B in A in 30 min); 1H
NMR was very complicated due to cis/trans isomers (data not
shown); ESIMS calcd for C31H44N4O8 600.70, found 601 (M +
H)+. Anal. Calcd for C31H44N4O8: C, 61.98; H, 7.38; N, 9.33.
Found: C, 61.43; H, 7.22; N, 9.24.
1,3(S)-Bis(car boxym eth yl)-9-[1′(R)-acetylam ide-2′-ph en -
yleth yl]-2,5,7-tr ioxo-1,4,8-tr ia za d eca n e (18). Compound 17
(30.0 mg, 0.05 mmol) and Me2S (1 mL) were placed in a Teflon
reactor and treated with liquid HF as described for 16.
Purification by semipreparative RP-HPLC on a Vydac C-18
column, λ ) 220 nm, employing a linear gradient of 0-10%
(v/v) B over 100 min, afforded 18.5 mg of 18 (86% yield) as a
white powder: Rt ) 26.73 min (0-15% B in A in 30 min);
detailed 1H and 13C NMR analysis is shown in Tables 1-4;
ESIMS calcd for C21H26N4O8 462.45, found 463 (M + H)+.
Energy minimization and MD simulations were performed
using Discover (Consistent Valence Force Field) and Insight
II (Molecular Simulations, Inc.). Structures were minimized
with the steepest descents and conjugate gradients for 2000
iterations. During the MD simulations, the temperature was
gradually raised in 50 K increments from 50 to 300 K with
(49) Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J .;
Bax, A. J . Biomol. NMR 1995, 6, 277-293.
(50) Muller, L. J . Magn. Reson. 1987, 72, 191-196.
(51) Braunschweiler, L.; Ernst, R. R. J . Magn. Reson. 1983, 53, 521-
528.
(52) Bax, A.; Davis, D. G. J . Magn. Reson. 1985, 65, 355-360.
(53) J eener, J .; Meier, B. H.; Bachmann, P.; Ernst, R. R. J . Chem.
Phys. 1979, 71, 4546-4553.
(54) Macura, S.; Huang, Y.; Suter, D.; Ernst, R. R. J . Magn. Reson.
1981, 43, 259-281.
(55) Bothner-By, A. A.; Stephens, R. L.; Lee, J .; Warren, C. D.;
J eanloz, R. W. J . Am. Chem. Soc. 1984, 106, 811-813.
(56) States, D. J .; Haberkorn, R. A.; Ruben, D. J . J . Magn. Reson.
1982, 48, 286-292.
(57) Kessler, H.; Griesinger, C.; Kerssebaum, R.; Wagner, K.; Ernst,
R. R. J . Am. Chem. Soc. 1987, 109, 607-609.
(58) Muller, L. J . Am. Chem. Soc. 1979, 101, 4481-4484.
(59) Bax, A.; Summers, M. F. J . Am. Chem. Soc. 1986, 108, 2093-
2094.
(60) Havel, T. F. Prog. Biophys. Mol. Biol. 1991, 56, 43-78.
(61) Crippen, G. M.; Havel, T. F. Distance Geometry and Molecular
Conformation; J ohn Wiley: New York, 1988.
(62) Kaptein, R.; Boelens, R.; Scheek, R. M.; van Gunsteren, W. F.
Biochemistry 1988, 27, 5389-5395.
(63) Mierke, D. F.; Kessler, H. Biopolymers 1993, 33, 1003-1017.
Nu clea r Ma gn etic Reson a n ce. Peptide solutions were
prepared in DMSO (99.9% DMSO-d6) at a concentration of 5
mM. Proton and carbon spectra were recorded on 400 and 600
MHz spectrometers, respectively, and processed using NM-
5096 J . Org. Chem., Vol. 67, No. 15, 2002

Retro-Inverso â-Turn Peptidomimetic
500 iterations of dynamics in each increment. The simulations
were continued at 300 K for 5000 iterations. Structures were
collected every 1000 iterations and energy minimized with the
steepest descents and conjugate gradients for 2000 iterations.
EB, ethylene-bridged; ESI, electron spray ionization; EBRIT-
BTM, Ni-to-Ni ethylene-bridged partially modified retro-
+
3
inverso tetrapeptide â-turn mimetic; EM, energy minimization;
HATU, O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroni-
um hexafluorophosphate; HMBC, heteronuclear multiple-bond
1
connectivity; HMQC H-13C heteronuclear multiple-quantum
Ack n ow led gm en t. This work was supported in part
by the National Institute of Arthritis and Musculosk-
eletal Disease, National Institutes of Health (AR42833),
Research Foundation through the Cottrell Scholars
Program (D.F.M.) and by the Dreyfus Foundation
through a Camile and Henry Dreyfus Scholar-Teacher
Award (D.F.M.).
shift correlation; IBTFA, iodobenzene bis(trifluoroacetate);
LC-ESIMS, liquid chromatography-electron spray ionization
mass spectrometry; MD, molecular dynamics; NMR, nuclear
magnetic resonance; NOE, nuclear Overhauser enhancements;
NOESY, nuclear Overhauser enhancement spectroscopy; PE-
COSY, primitive exclusive COSY; PMRI, partially modified
retro-inverso; RMSD, root-mean-square deviation; ROE, two-
dimensional rotating frame NOE; ROESY, two-dimensional
rotating frame NOE-spectroscopy; RP-HPLC, reversed-phase
liquid chromatography; TBA, tetrabutylammonium hydrogen-
sulfate; TEA, triethylamine; TLC, thin-layer chromatography;
TMS, tetramethylsilane; TOCSY, total-correlation spectroscopy.
Abbr evia tion s Used : COSY, correlation spectroscopy;
DADD, distance- and angle-derived dynamics; DBU, 1,8-
diazabicyclo[5,4,0]undec-7-ene; DCC, N,N′-dicyclohexyl car-
bodiimide; DCM, dichloromethane; DG, distance geometry;
DIPEA, N,N-diisopropylethylamine; DMAP, N,N-dimethy-
laminopyridine; DMF, N,N-dimethylformamide; DMSO, dim-
ethyl sulfoxide; DQF-COSY, double-quantum-filtered COSY;
J O011041W
J . Org. Chem, Vol. 67, No. 15, 2002 5097