Synthesis and conformational analysis of small peptides containing 6-Endo-BT(t)L scaffolds as reverse turn mimetics

Article abstract of DOI:10.1021/jo0202132

Two new dipeptide isosteres derived from L-leucine and meso-tartaric acid derivatives, named 6-endo-BTL and 6-endo-BtL, were inserted in a small peptide by means of SPPS, and the conformational features of the resulting peptides 3 and 4 were studied by NMR, IR, and molecular modeling techniques. The presence of a reverse turn conformation was observed in all the structures, suggesting the key role of the scaffolds as reverse turn promoters. Peptides 3 and 4 did not adopt a preferred conformation as indicated by the presence of equilibria between open turn and intramolecular hydrogen-bonded structures. 6-endo-BTL-peptide 3 showed a 3:1 mixture of conformers. The major conformer adopted mainly an open turn structure in equilibrium with hydrogen-bonded structures. The minor conformer displayed a better organized structure with a 14-membered ring hydrogen-bond typical of a Β-hairpin-like structure, in equilibrium with γ-turn, too. 6-endo-BtL-peptide 4 showed a unique conformer, and did not adopt as good a conformation as 3, due to the bulky equatorial substituent at C-2. Thus, marked structural differences between peptides containing 6-endo-BTL and 6-endo-BtL scaffolds as reverse turn inducers exist.

Full text of DOI:10.1021/jo0202132

Syn th esis a n d Con for m a tion a l An a lysis of Sm a ll P ep tid es
Con ta in in g 6-En d o-BT(t)L Sca ffold s a s Rever se Tu r n Mim etics
Andrea Trabocchi,† Ernesto G. Occhiato,† Donatella Potenza,‡ and Antonio Guarna*,†,§
Dipartimento di Chimica Organica “Ugo Schiff”, Universita` di Firenze, Polo Scientifico di Sesto
Fiorentino, Via della Lastruccia 13, I-50019 Sesto Fiorentino, Firenze, Italy, Istituto di Chimica dei
Composti Organo Metallici, Via J acopo Nardi 39, I-50132 Firenze, Italy, and Dipartimento di Chimica
Organica e Industriale, Universita` di Milano, Via Venezian 21, I-20133 Milano, Italy
antonio.guarna@unifi.it.
Received March 27, 2002
Two new dipeptide isosteres derived from L-leucine and meso-tartaric acid derivatives, named
6-endo-BTL and 6-endo-BtL, were inserted in a small peptide by means of SPPS, and the
conformational features of the resulting peptides 3 and 4 were studied by NMR, IR, and molecular
modeling techniques. The presence of a reverse turn conformation was observed in all the structures,
suggesting the key role of the scaffolds as reverse turn promoters. Peptides 3 and 4 did not adopt
a preferred conformation as indicated by the presence of equilibria between open turn and
intramolecular hydrogen-bonded structures. 6-endo-BTL-peptide 3 showed a 3:1 mixture of
conformers. The major conformer adopted mainly an open turn structure in equilibrium with
hydrogen-bonded structures. The minor conformer displayed a better organized structure with a
14-membered ring hydrogen-bond typical of a â-hairpin-like structure, in equilibrium with a γ-turn,
too. 6-endo-BtL-peptide 4 showed a unique conformer, and did not adopt as good a conformation as
3, due to the bulky equatorial substituent at C-2. Thus, marked structural differences between
peptides containing 6-endo-BTL and 6-endo-BtL scaffolds as reverse turn inducers exist.
In tr od u ction
Reverse turns are structural motifs commonly found
in proteins and bioactive peptides that, besides being
fundamental in protein folding,1 play a central role as
molecular recognition elements for many biological proc-
esses. â-Turns are a subset of reverse turns and consist
of a tetrapeptide sequence in a nonhelical region in which
the chain direction is reversed. These turns are often
stabilized by an intramolecular hydrogen bond between
the carbonyl oxygen of the first residue (i) and the amide
proton of the fourth one (i + 3), as shown in Figure 1.1a,2
Βy virtue of presenting up to four side chains in a well-
defined spatial arrangement, â-turns are often carriers
of biological activity,1a,3 thus being very important as
starting points in the design of the so-called “turn-
mimetics” for the development of new drugs.4 During the
past decade many efforts have been concentrated to
F IGURE 1. â-turn structure (left) and the isosteric replace-
ment of i + 1-i + 2 dipeptidic sequence by 6-endo-BTAa
(right).
synthesize new reverse turn inducers and study the
conformational preferences of turn-analogues within
protein secondary structure models.5 Surprisingly, to our
knowledge the only examples of bicycles belonging to
[x.y.1] family reported so far are based on norbornene
skeleton, which acts as reverse turn inducer and stabi-
lizes â-sheet secondary structures without the aid of any
hydrogen bond by virtue of its “U-shape”.6
In recent years we have developed a new class of aza-
dioxa[3.2.1]bicyclic compounds named BTAa, which de-
rive from the condensation of tartaric acid and amino acid
derivatives.7 We have previously described the potential
* To whom correspondence should be addressed. Tel.: 0039-055-
4573481. Fax: 0039-055-4573569.
† Universita` di Firenze.
‡ Universita` di Milano.
§ Istituto di Chimica dei Composti Organo Metallici.
(1) Rose G. D.; Gierasch, L. M.; Smith J . A. Adv. Prot. Chem. 1985,
37, 1-109. (b) Stanfield, R. L.; Fieser, T. M.; Lerner, R. A.; Wilson, I.
A. Science, 1990, 248, 712-719. (c) Chou, P. Y.; Fasman, G. D. J . Mol.
Biol. 1977, 115, 135-175. (d) Wilmot, C. M.; Thorton, J . M. J . Mol.
Biol. 1988, 203, 221-232. (e) Lewis, P. N.; Momany, F. A.; Scheraga,
H. A. Biochim. Biophys. Acta 1973, 303, 211-229.
(2) Belvisi L.; Gennari C.; Mielgo A.; Potenza D.; Scolastico C. Eur.
J . Org. Chem. 1999, 389-400.
(4) Cho, N.; Harada, M.; Imaeda, T.; Imada, T.; Matsumoto, H.;
Hayase, Y.; Sasaki, S.; Furuya, S.; Suzuki, N.; Okubo, S.; Ogi, K.; Endo,
S.; Onda, H.; Fujino, M. J . Med. Chem. 1998, 41, 4190-4195. (b)
Souers, A. J .; Virgilio, A. A.; Schu¨rer, S. S.; Ellman, J . A.; Kogan, T.
P.; West, H. E.; Ankener, W.; Vanderslice, P. Bioorg. Med. Chem. Lett.
1998, 8, 2297-2302. (c) Souers, A. J .; Virgilio, A. A.; Rosenquist, Å.;
Fenuik, W.; Ellman, J . A. J . Am. Chem. Soc. 1999, 121, 1817-1825.
(3) Richardson, J . S. Adv. Protein Chem. 1981, 34, 167-339. (b)
Mu¨ller, G. Angew. Chem. 1996, 108, 2941-2943; Angew. Chem., Int.
Ed. Engl. 1996, 35, 2767-2769.
10.1021/jo0202132 CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/18/2002
J . Org. Chem. 2002, 67, 7483-7492
7483

Trabocchi et al.
SCHEME 1
SCHEME 2
applications of these scaffolds as dipeptide isosteres when
inserted in an open chain peptide.7a If meso-tartaric acid
is used for the preparation of BTAa, the substituent at
C-6 of BTAa scaffolds adopts an endo configuration and
a subset of potential reverse turn inducers is obtained:
in fact, the chair conformation of the six-membered ring
locked in the bicyclic structure, in conjunction with the
endo configuration at C-6 gives the right shape for
imposing a reverse turn, thus replacing the i + 1-i + 2
dipeptidic sequence of a â-turn (Figure 1). Having
demonstrated in a previous work that 6-endo-scaffolds
are able to mimic the turn of a cyclic constrained peptide
by replacing the X-Pro dipeptidic sequence,8 we focused
our attention on the possibility of BTAa to induce a
reverse turn when inserted in a linear peptide chain
without any further constraints.
The diastereomeric 6-endo-BTL and 6-endo-BtL scaf-
folds are obtained from L-leucinol and (RS,SR)-tartaric
acid monomethyl ester derivative (Scheme 1) and show
differences both in the absolute configuration at C-5 and
C-6 carbon atoms of the meso-tartaric acid moiety, and
in the relative disposition of leucine side chain at C-2
that is axial in the case of 6-endo-BTL and equatorial in
the 6-endo-BtL scaffold. The two scaffolds were prepared
as Fmoc-amino acids 1 and 2 (Scheme 2) and inserted in
a linear hexapeptidic sequence by means of SPPS to give
peptides Ac-Val-Ala-6-endo-BTL-Val-Gly-OMe (3) and
Ac-Val-Ala-6-endo-BtL-Val-Gly-OMe (4). Successively,
the conformational analysis of peptides 3 and 4 was
carried out by NMR and IR spectroscopy and by molec-
ular modeling. Many examples are present in the litera-
ture about NMR experiments in which the hydrogen-
bonding ability of amide NH protons is elucidated by
dilution and temperature effects on chemical shift and
by NOE effects which are diagnostic of structural prefer-
ences of reverse-turn peptides in solution.9 Valine, ala-
nine, and glycine were chosen as the amino acids to form
the two â-strands on both sides of 6-endo-BT(t)L scaffolds
since they satisfy the requirements of solubility in organic
solvents (i.e. CDCl3), lack of polar groups as possible
competitors on intramolecular hydrogen bond formation,
and absence of aromatic groups that might interfere
during the study of amide protons by NMR.10
(5) Nagai, U.; Sato, K. Tetrahedron Lett. 1985, 26, 647-650. (b)
Kemp, D. S.; Sites, W. E. Tetrahedron Lett. 1988, 29, 5057-5060. (c)
Nagai, U.; Sato, K.; Nakamura, R.; Kato, R. Tetrahedron 1993, 49,
3577-3592. (d) Gardner, B.; Nakanishi, H.; Kahn, M. Tetrahedron
1993, 49, 3433-3448. (e) Giannis, A.; Kolter, T. Angew. Chem., Int.
Ed. Engl. 1993, 32, 1244-1267. (f) Virgilio, A. A.; Ellman, J . A. J . Am.
Chem. Soc. 1994, 116, 11580-11581. (g) Liskamp, R. M. J . Recl. Trav.
Chim. Pays-Bas 1994, 113, 1-19. (h) Lombart, H.-G.; Lubell, W. D. J .
Org. Chem. 1994, 59, 6147. (i) Gante, J . Angew. Chem., Int. Ed. Engl.
1994, 33, 1699-1720. (j) Schneider, J . P.; Kelly, J . W. Chem. Rev. 1995,
95, 2169-2187. (k) Gardner, R. R.; Liang, G.-B.; Gellman, S. H. J . Am.
Chem. Soc. 1995, 117, 3280-3281. (l) Hanessian, S.; McNaughton-
Smith, G.; Lombart, H.-G.; Lubell, W. D. Tetrahedron 1997, 53, 12789-
12854. (m) Krautha¨user, S.; Christianson, L. A.; Powell, D. R.; Gellman,
S. H. J . Am. Chem. Soc. 1997, 119, 11719-11720. (n) Feng, Y.;
Pattarawarapan, M.; Wang, Z.; Burgess, K. Org. Lett. 1999, 1, 121-
124. (o) Soth, M. J .; Nowick, J . S. J . Org. Chem. 1999, 64, 276-281.
(p) Alonso, E.; Lo´pez-Ortiz, F.; Del Pozo, C.; Peralta, E.; Mac´ıas, A.;
Gonza´lez, J . J . Org. Chem. 2001, 66, 6333-6338.
(6) Ranganathan, D.; Haridas, V.; Kurur, S.; Thomas, A.; Madhusu-
danan, K. P.; Nagaraj, R.; Kunwar, A. C.; Sarma, A. V. S.; Karle, I. L.
J . Am. Chem. Soc. 1998, 120, 8448-8460. (b) Hibbs, D. E.; Hursthouse,
M. B.; J ones, I. G.; J ones, W.; Abdul Malik, K. M.; North, M. J . Org.
Chem. 1998, 63, 1496-1504. (c) J ones, I. G.; J ones, W.; North, M. J .
Org. Chem. 1998, 63, 1505-1513.
(7) Guarna, A.; Guidi, A.; Machetti, F.; Menchi G.; Occhiato E. G.;
Scarpi D.; Sisi S.; Trabocchi A. J . Org. Chem. 1999, 64, 7347-7364.
(b) Guarna, A.; Cini, N.; Machetti, F.; Menchi, G.; Occhiato, E. G. Eur.
J . Org. Chem. 2002, 873-880.
(8) Scarpi, D.; Occhiato E. G.; Trabocchi, A.; Leatherbarrow R. J .;
Brauer A. B. E.; Nievo M.; Guarna A. Bioorg. Med. Chem. 2001, 9,
1625-1632.
(9) See, for example: (a) Stevens, E. S.; Sugawara, N.; Bonora, G.
M.; Toniolo, C. J . Am. Chem. Soc. 1980, 102, 7048-7050. (b) Boussard,
G.; Marraud, M. J . Am. Chem. Soc. 1985, 107, 1825-1828. (c) Gellman,
S. H.; Dado, G. P.; Liang, G.-B.; Adams, B. R. J . Am. Chem. Soc. 1991,
113, 1164-1173. (d) Liang, G.-B.; Rito, C. J .; Gellman, S. H. J . Am.
Chem. Soc. 1992, 114, 4440-4442. (e) Haque, T. S.; Little, J . C.;
Gellman, S. H. J . Am. Chem. Soc. 1996, 118, 6975-6985. (f) Yang, J .;
Gellman, S. H. J . Am. Chem. Soc. 1998, 120, 9090-9091.
(10) Huck B. R.; Fisk, J . D.; Gellman S. H. Org. Lett. 2000, 2, 2607-
2610.
7484 J . Org. Chem., Vol. 67, No. 21, 2002

Peptide Endo-BT(t)L Scaffolds as Reverse Turn Mimetics
SCHEME 3 a
a
Key: (a) LiAlH4, THF, reflux, 2 h; (b) H2, Pd(OH)2/C, MeOH, 12 h; (c) Fmoc-O-Su, H2O, acetone, Na2CO3, 0 °C, then 25 °C, 18 h; (d)
J ones reagent, acetone, 0 °C, the 25 °C, 18 h.
Resu lts a n d Discu ssion
the drastic conditions employed during the synthesis, the
preparation of peptide 3 afforded, after TFA cleavage
from the resin, a mixture of the desired compound as
peptide acid and the Ac-6-endo-BTL-Val-Gly-OH byprod-
uct due to incomplete coupling, as observed by ESI-MS
and HPLC. The two compounds were separated by
semipreparative HPLC. Final esterification with diazo-
methane and further HPLC purification afforded pure
3. In the case of 4 the crude peptide was cleaved off the
resin, and then it was directly esterified with diazo-
methane and purified by flash chromatography, which
furnished a peptide with a better purity grade, although
in lower yield.
Syn th esis of N-F m oc-a m in o Acid s. Compounds 1
and 2 required for the synthesis of the target peptides 3
and 4, respectively, were prepared following the general
procedure for the synthesis of BTAa7a (Scheme 3). The
starting amido esters 5 and 611 were completely reduced
with LiAlH4 to give amino alcohols 7 and 8, respectively,
in good yields. Successively, debenzylation by Pd(OH)2/C
catalyzed hydrogenolysis and subsequent protection of
the amine function of the resulting compounds 9 and 10
with Fmoc-O-Su afforded the corresponding N-Fmoc-
amino alcohols 11 and 12. The final oxidation was carried
out with J ones reagent. The crude N-Fmoc-amino acids
1 and 2 were carefully purified by FCC thus obtaining
the title compounds in fair yields and without any trace
of epimers at C-6, as evidenced by NMR.
P ep tid e Syn th esis. Peptides 3 and 4 (Scheme 2) were
prepared by means of a solid-phase peptide synthesis on
a Wang resin using HBTU/HOBt chemistry except for
the coupling of the amine and carboxyl functions of the
6-endo-BT(t)L scaffolds, which required different condi-
tions due to their relative low reactivity, as already
observed for similar BTAa.8 Specifically, DIPC/HOBt
activators were employed, and DIPEA was replaced by
2,4,6-collidine for alanine coupling to avoid C-R epimer-
ization.12 Peptide couplings on the amine and carboxyl
functions of the scaffolds were repeated twice, and the
reaction times were prolonged up to 3 days. All the
peptide syntheses were monitored with ninhydrin fol-
lowing the Kaiser’s method;13 in the case of the secondary
amine of the bicyclic scaffolds, bromophenol blue14 and
chloranil15 tests were found to be not reliable. Despite
Con for m a tion a l An a lysis of P ep tid e 3. Conforma-
tional studies on peptides 3 and 4 were performed by
NMR, using a relatively nonpolar solvent (i.e. CDCl3) in
order not to interfere in the hydrogen bonding of amide
protons. For ease of discussion only C-R and amide
protons were numerated as well as the hydrogens of the
scaffold. TOCSY and NOESY spectra were recorded to
assign proton resonances and investigate both sequential
and long-range NOE's that provide evidences of preferred
conformations and give insight into stable â-hairpinlike
conformations.16 Temperature dependence experiments
were carried out since the amide proton chemical shifts
are sensitive to temperature and dilution variations, thus
giving further insight into the conformational preferences
of peptides. The combination of chemical shift and ∆δ/
∆T coefficient of amide protons provides information on
the extent of hydrogen bonding.2,9
1
The H NMR of peptide 3 showed two distinct sets of
proton resonances in a 3:1 ratio clearly visible in the
chemical shift range of the amide hydrogens. This is due
to the presence of rotamers about the alanine-6-endo-BTL
amide bond that do not interconvert at room tempera-
ture, as evinced in a previous work for similar com-
pounds.17 By comparison of chemical shift values of
(11) The preparation of scaffolds 5 and 6 for the synthesis of amino
acids 1 and 2 was accomplished according to the general procedure
previously reported (see ref 7a), having introduced some improvements
concerning the oxidation of the alcohol obtained from the condensation
of N-benzylleucinol with meso-tartaric acid monoester derivative. This
was carried out by Dess-Martin periodinane, that was found to be
more efficient than the Swern methodology used so far because of both
easier synthetic procedure and absence of epimerization at C-R of the
leucine mojety.
(12) Gennari, C.; Mielgo, A.; Potenza, D.; Scolastico, C.; Piarulli, U.;
Manzoni, L. Eur. J . Org. Chem. 1999, 379-388.
(13) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595-598.
(14) Krchna´k, V.; Va´gner, J .; Safa´r, P.; Lebl, M. Collect. Czech.
Chem. Commun. 1988, 53, 2542-2548.
(15) Christensen, T. Acta Chem. Scand. 1979, B33, 763-766.
(16) Sieber, V.; Moe, G. R. Biochemistry 1996, 35, 181-188.
(17) Machetti, F.; Ferrali, A.; Menchi, G.; Occhiato, E. G.; Guarna,
A. Org. Lett. 2000, 2, 3987-3990.
J . Org. Chem, Vol. 67, No. 21, 2002 7485

Trabocchi et al.
TABLE 1. NOESY Cr oss-P ea k s Obser ved for P ep tid es 3 a n d 4
Ac-Val1-Ala-6-endo-BT(t)L-Val2-Gly-OMe
3
major conformer
minor conformer
NOE's
4
residue
valine 1
proton
NOE's
proton
proton
NOE's
N-H
N-H
N-H
N-H
N-H
N-H
alanine
Ala R-H
Val1 R-H, â-H
Ala R-H; Val1 R-H, â-H
Ala R-H, â-H
Val1 R-H
Ala R-H, â-H; Val2 N-H
6-endo-BT(t)L
H-1
H-2
H-4
H-5
H-1
H-2
H-4
H-5
H-1
H-2
H-4
H-5
Ala R-H
Ala R-H
H-6
N-H
N-H
H-6
N-H
N-H
H-6
N-H
N-H
valine 2
glycine
H-6
Val2 R-H; Leu â-H, γ-H
Val2 R-H
a better organized structure. The NOESY spectrum
showed sequential NOE cross-peaks between 8-H, 7-H,
and 9-H indicating a â-strand organization of the N-
terminal main chain (Table 1 and Figure 4a, left). Amide
11-H was found to be internal to the peptide turn in
accordance with the cross-strand NOE cross-peak be-
tween 11-H and 8-H, that demonstrates the presence of
a tight reverse turn. The temperature dependence ex-
periments (see Table 2 and Figure 4b) suggested the
presence of equilibrating structures, too. Amide 8-H is
involved in an equilibrium between a non-hydrogen-
bonded state and a strong intramolecular hydrogen-
bonded 14-membered ring of â-hairpinlike structure, with
rigidity like that observed in a classical â-hairpin hydro-
gen-bonded 10-membered ring. Glycine amide proton
13-H displayed weak hydrogen bonding in equilibrium
with a non-hydrogen-bonded state, according to relatively
large chemical shift and ∆δ/∆T values, probably generat-
ing a γ-turn with the CdO group of the scaffold, in
analogy with Z rotamer (Figure 4a, right).
F IGURE 2. E and Z rotamers at alanine-6-endo-BTL amide
bond, and CdO effect on 2-H and 4-H chemical shifts.
protons at C-2 and C-4 in the two rotamers it was
possible to establish that the major conformer possesses
a Z configuration at the alanine-6-endo-BTL bond. In fact,
the amide CdO bond determines a strong downfield shift
of the proton at C-2 in the Z rotamer and the protons at
C-4 in the E rotamer (Figure 2). Despite the isocronousity
of the 7-H proton of the two rotamers in the NOESY
spectrum, the presence of NOE cross-peaks between 4-H
and 7-H in the major conformer and between 2-H and
7-H in the minor one, and the absence of NOE's between
2-H and 7-H in the former rotamer and between 4-H and
7-H in the latter, confirmed that the major conformer
possesses a Z configuration at alanine-6-endo-BTL amide
bond (Figures 3a and 4a and Table 1). The absence of
significant NOE's in the NOESY spectrum indicated that
in the Z rotamer the structure is mainly in an open
reverse turn conformation which is operated by the
particular structural asset of 6-endo-BTL scaffold. NOE
cross-peaks between 11-H and 6-H suggested that the
CdO group of the scaffold has a certain rotational
freedom and points inside the turn (Figure 3a). The
IR spectroscopy was taken into account to distinguish
between amide hydrogen-bonded and non-hydrogen-
bonded states. The Ac-6-endo-BTL-NHMe monomer was
chosen as reference compound, as it cannot experience
intramolecular hydrogen bonding. The N-H stretching
region for this compound displays a single N-H band,
at 3430 cm-1. At room temperature, in the infrared
spectra of a 7 mM sample of compound 3 in CHCl3 the
non-hydrogen-bonded NH stretching vibrations appear
as a narrow band at 3418 cm-1, while the hydrogen-
bonded NH stretching vibrations appear as a broad band
at 3307 cm-1
.
Molecular modeling calculations using AMBER*18 as
a force field were carried out to gain further insight into
the conformational preferences of peptides 3 and 4. Two
different Monte Carlo conformational searches were
performed for the E and Z rotamers of compounds 3 and
4. The distance d and the virtual torsion angle â were
computed to investigate the turn propensity. The distance
d between the C-R of the first and fourth residue of a
â-turn is diagnostic of the presence of a reverse turn
when its value is lower than 7 Å,1a and the virtual torsion
angle â is indicative of a reverse turn when it assumes a
value within the range of 0° ( 30°.19 Considering the
1
temperature dependence H NMR experiments showed
the presence of equilibrating structures (Figure 3b and
Table 2). Amide 11-H should not form any hydrogen-bond
because of low values of both chemical shift and ∆δ/∆T.
The chemical shift and ∆δ/∆T values of glycine amide
proton 13-H indicated an equilibrium between the hy-
drogen-bonded state and non-hydrogen-bonded states,
suggesting either a γ-turn conformation as a consequence
of weak interactions, or a hydrogen bond with an acetyl
group that determines a strand orientation in accordance
with NOE’s. The data concerning the other amide protons
8-H and 10-H indicated the predominance of non-
hydrogen-bonded states.
(18) Mohamadi, F.; Richards, N. G.; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J .
Comput. Chem. 1990, 11, 440-467.
The minor conformer of peptide 3, having a E config-
uration at the alanine-6-endo-BTL amide bond, showed
7486 J . Org. Chem., Vol. 67, No. 21, 2002

Peptide Endo-BT(t)L Scaffolds as Reverse Turn Mimetics
F IGURE 3. (a) Equilibrating structures of 3, major conformer; the arrows indicate the NOE correlations. (b) Amide proton chemical
shifts of peptide 3, major conformer as a function of temperature.
TABLE 2. 1H NMR Da ta for Am id e P r oton s of P ep tid es
global minimum conformer displayed an open turn
structure with no intramolecular hydrogen bonds, and
3 a n d 4 Recor d ed Usin g CDCl3 Solu tion sa ,b
this structure was found in 54% of conformers (Figure
5a). In 12% of the conformers a γ-turn between 13-H and
carbonyl group at C-6 of the scaffold was observed, in
accordance with hydrogen-bonding of 13-H which is
stabilized when the rotational freedom is reduced at low
temperatures (Figure 5b). Moreover, in 34% of conformers
with higher energy relative to the global minimum
conformer, a structure having two hydrogen bonds formed
by 8-H and 13-H was present (Figure 5c), that is
congruent with NOE cross-peak between 6-H and 11-H
(calculated distance of 2.20 Å), and with the presence of
weak interactions stabilized at lower temperatures. As
illustrated in Figure 5d,e, in which is represented the
distribution of all the conformers found in the Monte
Carlo conformational search versus â and d values,20 all
the structures found showed a turn conformation, as
evidenced by the d value, but only 35% displayed â values
in the range of 0° ( 30° and 92% in 0° ( 60° range, in
agreement with the open turn structure. The molecular
mechanic calculations on E rotamer confirmed the pres-
ence of a reverse turn and a better organized structure.
Ac-Val-Ala-6-endo-BT(t)L-Val-Gly-OMe
3, major conformer 3, minor conformer
4
δ
∆δ/∆T
δ
∆δ/∆T
δ
∆δ/∆T
-5.6
8-H
6.42
6.10
6.91
6.59
-6.0
-5.0
-1.0
-3.5
7.88
6.42
6.83
7.16
-5.25
-1.5
6.75
10-H
11-H
13-H
6.25 -14.86
7.21 -1.8
6.87 -23.2
-5.0
-6.25
a
The chemical shifts δ are expressed in ppm, and ∆δ/∆T
b
coefficients in ppb/K. The ∆δ/∆T coefficients were determined
between 260 and 300 K for peptide 3, and between 250 and 300 K
for peptide 4.
6-endo-BT(t)L scaffolds as mimics of the i + 1-i + 2
central elements of a â-turn, the dihedral angle formed
by the alanine carbonyl carbon, C-2 and C-6 of the
scaffolds and nitrogen of valine next to the scaffold was
taken into account. The conformational search on Z
rotamer of peptide 3 (i.e. the major conformer) resulted
in all the conformers having a marked tendency of
adopting a reverse turn conformation, in which the
scaffold occupies the central turn position (Figure 5). The
(19) Ball, J . B.; Hughes, R. A.; Alewood, P. F.; Andrews, P. R.
Tetrahedron 1993, 49, 3467-3478.
(20) Mu¨ller, G.; Hessler, G.; Decornez, H. Y. Angew. Chem., Int. Ed.
2000, 39, 894-896.
J . Org. Chem, Vol. 67, No. 21, 2002 7487

Trabocchi et al.
F IGURE 4. (a) Equilibrating structures of 3, minor conformer; the arrows indicate the NOE correlations. (b) Amide proton
chemical shifts of peptide 3, minor conformer as a function of temperature.
F IGURE 6. 3D structures of peptide 3, minor conformer. (a)
Global minimum conformer. (b) γ-Turn structure. (c) Distribu-
tion profile of â values. (d) Distribution profile of d values.
that the 10-membered ring hydrogen-bond formed by
11-H and the alanine CdO group cannot exist because
of steric restrictions, and that in all the conformers the
F IGURE 5. 3D structures of peptide 3, major conformer. (a)
C-6 carbonyl group is fixed in an anti orientation.
Con for m a tion a l An a lysis of P ep tid e 4. A single set
of amide hydrogens was found, which is consistent with
the presence of a unique conformer. NOE cross-peaks
between 7-H and 4-H endo, and between 4-H exo and
methyl group of alanine, were indicative of Z configura-
tion at alanine-6-endo-BtL amide bond (Table 1 and
Figure 7a). This conformational restriction is probably
due to a steric hindrance between the equatorial sec-butyl
group at C-2 of 6-endo-BtL scaffold and the methyl group
of alanine, thus resulting in a destabilization of the E
rotamer. NOESY spectrum of peptide 4 showed several
sequential NOE cross-peaks (Table 1 and Figure 7a)
indicating that the two halves are organized into â-strands.
The absence of any cross-strand NOE peaks suggested
that the peptide folds in an open turn, probably because
Global minimum conformer. (b) γ-turn structure. (c) â-Hair-
pinlike structure. (d) Distribution profile of â values. (e)
Distribution profile of d values.
In 86% of conformers a 14-membered ring hydrogen bond
between 8-H and the valine carbonyl group existed
(Figure 6a). The calculated distance between 8-H and
11-H in the global minimum conformer was 2.35 Å, in
agreement with the presence of a cross-strand NOE.
Moreover, the presence of γ-turn conformers with intra-
molecular hydrogen-bonding was consistent with ∆δ/∆T
and chemical shift values of 13-H (Figure 6b). As il-
lustrated in Figures 6c-d, in all the conformers the
distance d is less than 7 Å, and 82% of the structures
showed a turn conformation resulting in a virtual torsion
angle below 30° in absolute value. Finally, it was found
7488 J . Org. Chem., Vol. 67, No. 21, 2002

Peptide Endo-BT(t)L Scaffolds as Reverse Turn Mimetics
F IGURE 7. (a) Representation of peptide 4 equilibrating structures with selected NOE's. (b) Amide proton chemical shifts of
peptide 4 as a function of temperature.
of the steric hindrance of bulky equatorial C-2 substituent
that forces the six-membered ring of the scaffold into half-
chair conformation, thus taking the two peptide halves
away. The amide proton 11-H, despite its relatively high
chemical shift and low ∆δ/∆T value (Table 2 and Figure
7b), seemed not to form a hydrogen-bond but to remain
in a fixed position as a consequence of antiperiplanar
orientation of CdO group of the scaffold relative to C-6-
O-7 bond. Alanine 8-H proton exhibited a weak hydrogen-
bond probably involved in a γ-turn, and in equilibrium
with a non hydrogen-bonded state, with the former being
stabilized at lower temperatures (Figure 7a, left). The
high ∆δ/∆T coefficient calculated for 10-H and 13-H may
indicate an equilibrium with a â-hairpinlike structure,
in which two hydrogen bonds are formed at lower
temperatures (Figure 7a, right). Thus, peptide 4 showed
a unique conformer by virtue of steric effect produced by
6-endo-BtL, and it seemed to adopt an extended â-strands
reverse turn conformation in analogy with the corre-
sponding 6-endo-BTL modified peptide 3.
Comparison of the N-H stretch region IR data for
F IGURE 8. 3D structures of peptide 4. (a) γ-Turn structure.
(b) â-Hairpinlike structure. (c) Distribution profile of â values.
(d) Distribution profile of d values.
peptide mimics 4 and reference compound Ac-6-endo-BtL-
NHMe indicated evidence of intramolecular hydrogen
bonding in compound 4. We assigned the hydrogen-
bonded NH stretching vibrations at 3316 cm-1 while the
of the two amide protons, thus indicating the stabilization
of hydrogen bonding when rotational freedom is reduced
at low temperatures. The plots of â and d values showed
a broad range of populated values (Figure 8c,d) suggest-
ing that peptide 4 does not exhibit a turn conformation
as good as isomer 3.
free N-H appears at 3418 cm-1
.
The molecular mechanic resulted in a variety of
conformers, all displaying a turn propensity. Only the Z
conformer seemed to agree with NMR experiments, with
the two peptide halves adopting an antiparallel confor-
mation. All the conformers found showed an anti orienta-
tion of the scaffold’s CdO group and the absence of
hydrogen-bonding at 11-H amide proton, thus confirming
that low ∆δ/∆T and relatively high chemical shift values
of 11-H amide proton (Table 2) are due to steric restric-
tions imposed by the scaffold that fixes 11-H in a well
defined chemical environment. The global minimum
conformer has the scaffold that occupies the central turn
position, and 13.5% of the conformers found presented a
γ-turn generated by a hydrogen bond between 8-H and
the CdO of acetyl group (Figure 8a). Higher energy
conformers showed a â-hairpinlike structure in which
10-H and 13-H are involved in two hydrogen bonds
(Figure 8b), in accordance with the high ∆δ/∆T values
Con clu sion s
In this paper we have described the synthesis of two
new 6-endo-BTL and 6-endo-BtL scaffolds having poten-
tial turn-inducing properties. We inserted the two mol-
ecules in a linear peptide in order to evaluate the ability
of these scaffolds to constrain the peptide conformation
in a reverse turn. We observed that the 6-endo-BTL
molecule, having the leucine side chain in axial position,
is a better reverse turn inducer since it can promote a
tighter turn than the corresponding isomer which pos-
sesses the sec-butyl group of leucine in equatorial posi-
tion. The two peptides 3 and 4 showed some hydrogen
J . Org. Chem, Vol. 67, No. 21, 2002 7489

Trabocchi et al.
AMBER* as a force field and the implicit chloroform GB/SA
solvation model.22 The Monte Carlo conformational search was
carried out without imposing any constraint and including
amide bonds among all rotatable bonds. A ring closure bond
was defined for the six- and seven-membered rings of the
bicyclic 6-endo-BT(t)L scaffolds. Two thousand structures were
generated and minimized. All the conformers having an energy
of 6 kcal/mol above the global minimum conformer were
discarded. The E and Z rotamers of each compound were
separately analyzed.
(1S,2S,5S,6R)-3-Ben zyl-2-exo-sec-bu tyl-6-en d o-h yd r oxy-
m eth yl-7,8-d ioxa -3-a za bicyclo[3.2.1]octa n e (7). To a sus-
pension of LiAlH4 (232 mg, 6.12 mmol) in anhydrous THF (10
mL) was added dropwise at 0 °C under a nitrogen atmosphere
a solution of 5 (680 mg, 2.04 mmol) in anhydrous THF (10
mL). The mixture was refluxed for 2 h, and then, after cooling
at 0 °C, aqueous 0.4 N KOH (2 mL) and water (1 mL) were
added and the suspension was refluxed for 30 min. The hot
mixture was finally filtered through Celite and, after evapora-
tion of the solvent, the crude product was purified by flash
chromatography (CH2Cl2-MeOH, 30:1, Rf 0.4) giving pure 7
as a colorless oil (420 mg, 71%): 1H NMR (CDCl3) δ 7.32-
7.20 (m, 5 H), 6.50 (br, 1 H), 5.37 (d, J ) 1.5 Hz, 1 H), 4.26 (m,
1 H), 4.11 (d, J ) 9.9 Hz, 1 H), 4.09 (s, 1 H), 3.84 (d, J ) 10.6
Hz, 1 H), 3.58 (d, J ) 2.2 Hz, 1 H), 2.92 (dd, J ) 12.4, 2.2 Hz,
1 H), 2.78-2.72 (m, 2 H), 1.63-1.30 (m, 3 H), 0.87 (d, J ) 6.3
Hz, 3 H), 0.59 (d, J ) 6.2 Hz, 3 H); 13C NMR (CDCl3) δ 135.3
(s), 129.3 (d, 2 C), 128.3 (d, 2 C), 127.5 (d), 100.4 (d), 77.9 (d),
74.5 (d), 59.1 (t), 56.7 (t), 56.6 (t), 49.0 (t), 30.7 (t), 24.6 (d),
24.0 (q), 21.1 (q); MS m/z 291 (M+, 20), 246 (100), 91 (100); IR
(CHCl3) 3142 (br) cm-1. Anal. Calcd for C17H25NO3: C, 70.07;
H, 8.65; N, 4.81. Found: C, 69.47; H, 8.45; N, 4.68.
bonding different from common â-turn structures, though
this requirement was found to be not crucial for turn
promotion. The absence of a 10-membered ring hydrogen-
bond characteristic of â-turn structures indicates that the
principal driving force for reverse turn formation is
operated by the particular structural asset of the 6-endo-
BT(t)L scaffold. Further stabilization is provided by
intramolecular hydrogen bonding and hydrophobic in-
teractions that contribute to the overall organization of
a peptide into â-strand conformations.
NMR, IR, and molecular modeling analysis showed the
presence of equilibrating structures due to amide hydro-
gens involved in equilibria between hydrogen-bonded and
non-hydrogen-bonded states, with the former being sta-
bilized by lowering the temperature. All the analyses led
to the conclusion that 6-endo-BTAa scaffolds are able not
only to retain a turn geometry, as previously demon-
strated,8 but also to provoke dramatic structural changes
to a linear peptide chain due to their particular molecular
construction. This effect can be modulated by either the
position or the kind of functional group at C-2, as marked
differences between the two 6-endo-BtL and 6-endo-BTL
were observed. More precisely, the 6-endo-BTL scaffold
seemed to act better as a reverse turn inducer, while
6-endo-BtL proved to impose severe structural restric-
tions resulting in a single preferred conformer irrespec-
tive of the stabilizing interactions produced by the two
strands of the peptide.
(1R ,2S ,5R ,6S )-3-Be n zyl-2-en d o-sec-b u t yl-6-en d o-h y-
dr oxym eth yl-7,8-dioxa-3-azabicyclo[3.2.1]octan e (8). Amino
alcohol 8 was synthesized from 6 (579 mg, 1.74 mmol) as
reported for 7. Pure 8 (352 mg, 69%) was obtained after
purification by flash chromatography as a yellow oil (CH2Cl2-
MeOH, 30:1, Rf 0.35): 1H NMR (CDCl3) δ 7.31-7.19 (m, 5 H),
5.90 (br, 1 H), 5.34 (s, 1 H), 4.23 (m, 1 H), 4.05 (d, J ) 14.6
Hz, 1 H), 4.02 (s, 1 H), 3.98 (d, J ) 11.0 Hz, 1 H), 3.68 (d, J )
11.0 Hz, 1 H), 3.11 (d, J ) 12.8 Hz, 1 H), 2.76 (d, J ) 12.1 Hz,
1 H), 2.54 (dd, J ) 12.1, 2.6 Hz, 1 H), 2.44 (dd, J ) 9.2, 2.6
Hz, 1 H), 1.79-1.50 (m, 3 H), 0.95 (pseudo t, J ) 6.2 Hz, 6 H);
13C NMR (CDCl3) δ 135.6 (s), 129.6 (d, 2 C), 128.3 (d, 2 C),
127.7 (d), 100.4 (d), 77.8 (d), 74.3 (d), 61.8 (d), 59.3 (t), 58.3 (t),
51.9 (t), 38.2 (t), 25.2 (d), 24.0 (q), 22.0 (q); MS m/z 291 (M+,
Exp er im en ta l Section
Melting points are uncorrected. Chromatographic separa-
tions were performed on silica gel using flash-column tech-
niques; Rf values refer to TLC carried out on 25-mm silica gel
60 F254 plates with the same eluant indicated for column
chromatography. 1H and 13C NMR spectra of all compounds
but peptides 3 and 4 were recorded at 200 and 50.33 MHz,
respectively. EI mass spectra were carried out at 70 eV ionizing
voltage. THF was distilled from Na/benzophenone. CH2Cl2 was
distilled from CaH2. All reactions requiring anhydrous condi-
tions were performed in oven-dried glassware. Acid silica gel
(H2SO4/SiO2) was prepared as reported.21 All the solid-phase
reactions were carried out on a shaker, using solvents of HPLC
quality. All the compounds 5-12 were obtained with variable
amounts (5-15%) of unseparable epimers at C-6. Only the
final N-Fmoc-amino acids 1 and 2 were obtained as pure
products and were fully characterized. Peptides 3 and 4 were
purified by HPLC system equipped with semipreparative C18
10 µm, 250 × 10 mm, reverse-phase column using a H2O-
CH3CN gradient eluant buffered with 0.1% TFA, and were
characterized by ESI-MS and HPLC equipped with an analyti-
cal C18 10 µm, 250 × 4.6 mm, reverse-phase column.
6), 91 (100); IR (CHCl3) 3168 (br) cm-1. Anal. Calcd for C17H25
-
NO3: C, 70.07; H, 8.65; N, 4.81. Found: C, 69.95; H, 9.19; N,
4.75.
(1S,2S,5S,6R)-2-exo-sec-Bu t yl-6-en d o-h yd r oxym et h yl-
7,8-d ioxa -3-a za bicyclo[3.2.1]octa n e (9). Compound 7 (403
mg, 1.38 mmol) was dissolved in MeOH (50 mL), 20%
Pd(OH)2/C (100 mg) was added, and the resulting suspension
was left stirring at room temperature under a hydrogen
atmosphere overnight. The catalyst was then removed by
careful filtration over Celite and washed with MeOH. The
solution was passed through a column filled with Amberlyst
A-21 resin giving, after solvent evaporation, pure 9 (280 mg,
99%) as a viscous oil: 1H NMR (CDCl3) δ 6.05 (br, 2 H), 5.30
(s, 1 H), 4.31 (m, 1 H), 4.22-4.06 (m, 1 H), 4.11 (d, J ) 12.8
Hz, 1 H), 3.92 (d, J ) 12.8 Hz, 1 H), 3.42-3.10 (m, 3 H), 2.12-
1.39 (m, 3 H), 0.91 (pseudo t, J ) 6.4 Hz, 6 H); 13C NMR
(CDCl3) δ 100.9 (d), 77.6 (d), 73.8 (d), 58.0 (t), 54.5 (d), 41.5
(t), 36.9 (t), 24.7 (d), 22.5 (q), 22.4 (q); MS m/z 201 (M+, 3), 156
(100); IR (CHCl3) 3198 (br) cm-1. Anal. Calcd for C10H19NO3:
C, 59.68; H, 9.51; N, 6.96. Found: C, 59.45; H, 9.48; N, 6.91.
(1R,2S,5R,6S)-2-en d o-sec-Bu tyl-6-en d o-h yd r oxym eth yl-
7,8-d ioxa -3-a za bicyclo[3.2.1]octa n e (10). Prepared from 8
(352 mg, 1.21 mmol) as reported for 9, affording pure 10 (240
NMR a n d IR Meth od s. NMR spectra of compounds 3 and
4 were acquired in CDCl3 with an Avance 400 Bruker
spectrometer. Solutions of 7 and 4.5 mM were employed for
all conformational analyses of peptides 3 and 4, respectively.
1H NMR spectra for determining temperature coefficients were
obtained at 260-300 K for 3 and at 250-300 K for 4, with
increments of 10 K. Complete proton resonance assignments
of compounds 3 and 4 were made with TOCSY and NOESY
experiments. IR spectra were recorded at room temperature
with a FT-IR instrument.
Com p u ta tion a l Meth od s. Molecular mechanic calcula-
tions were carried out on a SGI IRIX 6.5 workstation, using
Macromodel (v6.5) molecular modeling software, with
(21) Guidi, A.; Guarna, A.; Macherelli, M.; Menchi, G. Arch. Pharm.
Med. Chem. 1997, 330, 201-202.
(22) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J .
Am. Chem. Soc. 1990, 112, 6127-6129.
7490 J . Org. Chem., Vol. 67, No. 21, 2002

Peptide Endo-BT(t)L Scaffolds as Reverse Turn Mimetics
TABLE 3. 1H NMR Ch em ica l Sh ifts of P ep tid es 3 a n d 4 Recor d ed in CDCl3 Solu tion s
Ac-Val1-Ala-6-endo-BT(t)L-Val2-Gly-OMe
3, major
C-â
3, minor
C-â
4
sequence
NH
C-R
C-γ
C-δ NH
C-R
C-γ
C-δ NH
C-R
C-â
C-γ
C-δ
Val 1
Ala
Val 2
Gly
6.10 4.24 2.03
6.42 4.62 1.27
6.91 3.92 2.10
0.92
-
1.00
-
-
-
-
6.42 4.49 2.26
7.88 4.62 1.30
6.84 4.01 2.26
0.95
-
1.02
-
7.21 4.40
6.75 4.83
6.25 4.32
2.16
1.35
2.05
-
1.75
1.01
-
0.93
-
-
-
-
-
-
-
6.59 4.05
-
-
7.16 4.14
-
-
6.87 4.17-4.00
-
Leu (BTtL)
-
-
1.55-1.26 1.27 0.94
-
-
1.33-1.85 1.68 1.01
-
-
1.57 1.01
H1
4.73
4.70
4.88
H2
4.22
3.85
4.37
H4
H5
3.64-3.83
5.50
4.28-3.18
5.47
4.02-3.25
5.61
H7
4.52
4.47
4.46
Ac
OMe
2.04
3.77
1.95 22.1
3.77
2.00
3.76
mg, 99%) as an oil: 1H NMR (CDCl3) δ 6.32 (br, 2 H), 5.25 (s,
1 H), 4.34 (m, 1 H), 4.17-4.04 (m, 2 H), 3.91 (d, J ) 12.4 Hz,
1 H), 3.30 (AB system, J ) 12.5 Hz, 2 H), 3.10 (t, J ) 7.0 Hz,
1 H), 1.66 (m, 1 H), 1.38 (t, J ) 7.3 Hz, 2 H), 0.88 (d, J ) 2.9
Hz, 3 H), 0.85 (d, J ) 2.6 Hz, 3 H); 13C NMR (CDCl3) δ 100.3
(d), 78.3 (d), 72.9 (d), 58.1 (d), 55.7 (d), 45.2 (t), 39.1 (t), 23.7
(d), 22.6 (q), 22.5 (q); MS m/z 201 (M+, 9), 156 (100); IR (CHCl3)
3207 (br) cm-1. Anal. Calcd for C10H19NO3: C, 59.68; H, 9.51;
N, 6.96. Found: C, 59.63; H, 9.49; N, 6.93.
(1S,2S,5S,6R)-3-(9-F lu or en ylm eth oxyca r bon yl)-2-exo-
sec-bu tyl-6-en d o-h yd r oxym eth yl-7,8-d ioxa -3-a za bicyclo-
[3.2.1]octa n e (11). Compound 9 (260 mg, 1.30 mmol) was
suspended in a mixture of water (10 mL) and acetone (5 mL)
containing Na2CO3 (138 mg, 1.30 mmol), and, after cooling to
0 °C, a solution of Fmoc-O-Su (482 mg, 1.43 mmol) in acetone
(10 mL) was added. The mixture was stirred at room-
temperature overnight, water (10 mL) was added, the aqueous
phase was saturated with NaCl, and the organic products were
extracted with CH2Cl2. After solvent evaporation, a solid
product was obtained which was purified by flash chromatog-
raphy (CH2Cl2-MeOH, 30:1, Rf 0.37), affording pure 11 (451
mg, 82%): 1H NMR (CDCl3) (mixture of rotamers) δ 7.67 (d, J
) 7.0 Hz, 2 H), 7.51 (d, J ) 7.0 Hz, 2 H), 7.35-7.20 (m, 4 H),
5.18 (s, 1 H), 4.66-3.07 (m, 10 H), 2.64 (br, 1 H), 1.60-1.10
(m, 3 H), 0.89-0.67 (m, 6 H); 13C NMR (CDCl3) δ 155.4 (s),
154.7 (s), 143.5 (s), 143.4 (s), 143.3 (s), 141.0 (s), 127.4 (d), 126.8
(d), 124.4 (d), 119.7 (d), 100.6 (d), 99.8 (d), 77.1 (d), 72.8 (d),
72.0 (d), 66.6 (t), 66.4 (t), 60.4 (t), 54.9 (d), 54.6 (d), 47.2 (d),
40.5 (t), 40.2 (t), 37.6 (t), 24.4 (d), 24.3 (d), 23.0 (q), 22.1 (q),
21.7 (q); ESI-MS m/z amu 424 (M+ + 1, 100); IR (CHCl3) 3163
(br), 1740 cm-1. Anal. Calcd for C25H29NO5: C, 70.90; H, 6.90;
N, 3.31. Found: C, 70.96; H, 6.93; N, 3.28.
(1R,2S,5R,6S)-3-(9-Flu or en ylm eth oxyca r bon yl)-2-en d o-
sec-bu tyl-6-en d o-h yd r oxym eth yl-7,8-d ioxa -3-a za bicyclo-
[3.2.1]octa n e (12). Prepared from 10 (240 mg, 1.20 mmol) as
reported for 11, affording after purification by flash chroma-
tography (CH2Cl2-MeOH, 30:1, Rf 0.4), pure 12 (421 mg, 83%)
as a yellow oil: 1H NMR (CDCl3) δ 7.74 (d, J ) 6.6 Hz, 2 H),
7.56 (d, J ) 7.0 Hz, 2 H), 7.42-7.25 (m, 4 H), 5.38 (d, J ) 3.3
Hz, 1 H), 4.61-4.41 (m, 2 H), 4.19 (t, J ) 6.1 Hz, 1 H), 4.07-
3.96 (m, 2 H), 3.87-3.79 (m, 1 H), 3.66-3.41 (m, 4 H), 2.71
(br, 1 H), 1.77-1.36 (m, 3 H), 0.84 (d, J ) 6.2 Hz, 6 H); 13C
NMR (CDCl3) δ 156.9 (s), 143.6 (s), 141.1 (s), 127.5 (d, 2 C),
126.8 (d, 2 C), 124.5 (d, 2 C), 119.8 (d, 2 C), 99.3 (d), 76.5 (d),
71.2 (d), 66.8 (t), 60.9 (t), 56.0 (d), 47.1 (d), 43.6 (t), 38.9 (t),
24.4 (d), 23.8 (q), 21.2 (q); ESI-MS m/z amu 424 (M+ + 1, 100);
IR (CHCl3) 3375 (br), 1740 cm-1. Anal. Calcd for C25H29NO5:
C, 70.90; H, 6.90; N, 3.31. Found: C, 70.92; H, 6.95; N, 3.24.
(1S,2S,5S,6S)-3-(9-F lu or en ylm et h oxyca r b on yl)-2-exo-
sec-bu tyl-7,8-d ioxa -3-a za bicyclo[3.2.1]octa n e-6-en d o-ca r -
boxylic Acid (1). To a solution of 11 (440 mg, 1.04 mmol) in
acetone (40 mL) was added J ones reagent23 (7.7 mL, 6.24
mmol) dropwise at 0 °C, and the mixture was stirred at 0 °C
for 40 min and at room temperature overnight. 2-Propanol was
added until the color of the mixture turned green, and then it
was filtered and evaporated to give a crude product which was
directly purified by flash chromatography (CH2Cl2-MeOH-
TFA, 30:1:0.03, Rf 0.13), affording pure 1 (422 mg, 93%) as a
viscous oil: [R]23D -2.65 (c 0.49, CHCl3); 1H NMR (CDCl3) (2:1
mixture of rotamers) δ 7.96-7.66 (m, 2 H), 7.62-7.48 (m, 2
H), 7.38-7.19 (m, 4 H), 5.41 (s, 0.3 H), 5.34 (s, 0.6 H), 4.66-
3.69 (m, 7 H), 3.40-3.18 (m, 1 H), 1.69-1.19 (m, 3 H), 0.94-
0.74 (m, 6 H); 13C NMR (CDCl3) δ 170.5 (s), 155.8 (s), 144.2
(s), 143.4 (s), 141.2 (s), 127.5 (d), 127.4 (d), 127.0 (d), 126.9
(d), 125.1 (d), 124.8 (d), 119.7 (d), 102.3 (d), 101.4 (d), 75.6 (d),
74.6 (d), 74.0 (d), 67.5 (d), 55.1 (d), 47.2, 41.7 (t), 41.3 (t), 37.8
(t), 24.6 (d), 23.0 (q), 22.1 (q); ESI-MS m/z amu 460 (M+ + Na,
100), 438 (M+ + 1, 70); IR (CHCl3) 3435 (br), 1754, 1692, 1671
cm-1. Anal. Calcd for C25H27NO6: C, 68.63; H, 6.22; N, 3.20.
Found: C, 68.59; H, 6.19; N, 3.25.
(1R,2R,5R,6R)-3-(9-Flu or en ylm eth oxycar bon yl)-2-en do-
sec-bu tyl-7,8-d ioxa -3-a za bicyclo[3.2.1]octa n e-6-en d o-ca r -
boxylic Acid (2). Prepared from 12 (415 mg, 0.98 mmol) as
reported for 1, giving after purification by flash chromatog-
raphy (CH2Cl2-MeOH-AcOH, 40:1:0.03, Rf 0.2) pure 2 as a
white solid (406 mg, 95%): mp 61-65 °C; [R]23D +65.7 (c 0.49,
1
CHCl3); H NMR (CDCl3) δ 7.70 (d, J ) 7.0 Hz, 2 H), 7.50 (d,
J ) 7.0 Hz, 2 H), 7.45-7.22 (m, 4 H), 5.46 (d, J ) 3.7 Hz, 1
H), 4.74 (br s, 1 H), 4.48-4.33 (m, 2 H), 4.16 (m, 1 H), 3.69
(m, 2 H), 3.33 (d, J ) 13.5 Hz, 1 H), 1.76-1.67 (m, 3 H), 0.81
(m, 6 H); 13C NMR (CDCl3) δ 170.5 (s), 156.9 (s), 143.7 (s),
141.2 (s), 127.6 (d, 2 C), 127.0 (d, 2 C), 124.8 (d, 2 C), 119.9 (d,
2 C), 101.1 (d), 76.7 (d), 71.4 (d), 67.3 (t), 56.3 (d), 47.2 (d),
43.6 (t), 38.0 (t), 24.6 (d), 23.8 (q), 21.5 (q); ESI-MS m/z amu
460 (M++Na, 100), 438 (M++1, 61); IR (CHCl3) 3340 (br), 1755,
1703, 1690 cm-1. Anal. Calcd for C25H27NO6: C, 68.63; H, 6.22;
N, 3.20. Found: C, 68.59; H, 6.18; N, 3.27.
Ac-Val-Ala-6-en do-BTL-Val-Gly-OMe (3). Fmoc-Gly-Wang
resin (86 mg, 64.5 µmol) was used as starting reagent. Fmoc
deprotections were performed twice with 30% piperidine in
DMF for 5 min, followed by resin washings with DMF. In case
of primary amino groups coupling reactions were monitored
with ninhydrin test.13 Coupling of valine was performed using
a 5-fold amino acid excess (110 mg, 0.32 mmol), a mixture of
0.5 M HBTU and 0.5 M HOBt as coupling reagents (0.65 mL,
0.32 mmol), and 1 M DIEA (0.65 mL, 0.65 mmol) as base,
allowing the mixture to shake for 30 min. After Fmoc depro-
tection, peptide bound to resin was treated with an activating
mixture of 0.14 M HOBt and 0.14 M DIPC in DMF (1.4 mL,
0.20 mmol), compound 1 (42 mg, 96 µmol), and 1 M DIEA (0.2
mL, 0.20 mmol), the mixture was allowed to shake for 3 days,
and then the resin was washed with DMF and the coupling
was repeated. After Fmoc deprotection, alanine was mounted
(23) Prepared by slow addition of concentrated H2SO4 (1.2 mL) to a
cold solution of CrO3 (624 mg) in H2O (7.7 mL).
J . Org. Chem, Vol. 67, No. 21, 2002 7491

Trabocchi et al.
on peptide resin using 5-fold amino acid excess (100 mg, 0.32
mmol), 0.14 M HOBt, and 0.14 M DIPC in DMF (1.4 mL, 0.20
mmol) as activating mixture and 2,4,6-collidine as base (43
µL, 0.32 mmol), and the mixture was allowed to shake for 3
days. The free amino groups where then capped by treating
the peptide bound to resin with Ac2O (53 µL, 0.65 mmol) and
1 M DMAP in DMF (65 µL, 6.5 µmol) at room-temperature
overnight. After resin washings with DMF and Fmoc depro-
tection, valine coupling was performed using a 5-fold amino
acid excess (110 mg, 0.32 mmol), a mixture of 0.5 M HBTU
and 0.5 M HOBt (0.65 mL, 0.32 mmol), and 1 M DIEA (0.65
mL, 0.65 mmol), leaving the mixture shaking for 30 min. After
Fmoc deprotection and resin washings with DMF, resin was
suspended in DMF (1 mL) and it was treated with Ac2O (53
µL, 0.65 mmol) and 1 M DMAP in DMF (65 µL, 6.5 µmol) at
room-temperature overnight. After resin washings with DMF,
MeOH, and CH2Cl2, peptide acid was cleaved off the resin with
95% TFA (1.5 mL, 1 × 5 min, 1 × 2 h, 1 × 15 min), and then
the mixtures were filtered, combined, and concentrated in
vacuo to give crude peptide acid which was separated from
the truncated Ac-6-endo-BTL-Val-Gly-OH peptide by semi-
preparative HPLC using 10-90% acetonitrile/55 min as gradi-
ent (tR ) 22.4 min). Peptide acid was dissolved in EtOAc and
treated at 0 °C with a freshly prepared solution of diazo-
methane in Et2O, the mixture was left stirring 1 h at room
temperature, and excess of diazomethane was destroyed by
AcOH addition. Crude peptide was purified by semipreparative
HPLC using 10-90% acetonitrile/55 min as gradient (tR ) 26.6
min), giving pure 3 (8 mg, 21%), which was characterized by
ESI-MS, and analytical HPLC using 10-90% acetonitrile/20
min as gradient (tR ) 11.6 min). ESI-MS m/z 620.4 (M+ + Na,
1
100), 598.6 (M+ + 1, 84). H NMR data are shown in Table 3.
Ac-Va l-Ala -6-en d o-BtL-Va l-Gly-OMe (4). Peptide 4 was
prepared as reported for 3, starting from Fmoc-Gly-Wang resin
(203 mg, 0.15 mmol) and compound 2 (100 mg, 0.17 mmol)
and using a different purification protocol. After cleavage from
the resin, crude peptide acid was directly esterified with
diazomethane and successively purified by flash chromatog-
raphy (CH2Cl2-MeOH, 30:1, Rf 0.11), yielding pure 4 (7.5 mg,
8%), which was characterized by MS and analytical HPLC
using 10-90% acetonitrile/20 min as gradient (tR ) 12.7 min).
1
MS m/z 597 (M+ + 1, 0.3). H NMR data are shown in Table
3.
Ackn owledgm en t. The authors thank COFIN 2000-
2002 for financial support.
J O0202132
7492 J . Org. Chem., Vol. 67, No. 21, 2002