Bip: A C(α)-tetrasubstituted, axially chiral α-amino acid. Synthesis and conformational preference of model peptides

Article abstract of DOI:10.1016/S0040-4020(00)00801-2

By using the recently proposed biphenyl-based, C(α)-tetrasubstituted, cyclic, axially chiral α-amino acid Bip we synthesised by solution methods a large set of model peptides, including the homo-oligomer series, to the pentamer level. All of the peptides were fully characterised and their preferred conformation was assessed in solution by means of a FT-IR absorption and ¹H NMR study. Results of X-ray diffraction analyses of two Bip derivatives and a terminally protected tripeptide with the sequence -Gly-Bip-Gly- are also presented. Our findings indicate that Bip tends to support β-turn and 3₁₀-helical structures, although in short peptides the fully-extended (C₅) conformation would also be populated to some extent. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00801-2

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 8721±8734
Bip: a Ca-Tetrasubstituted, Axially Chiral a-Amino Acid.
Synthesis and Conformational Preference of Model Peptides
b
b
Fernando Formaggio,a Marco Crisma,a Claudio Toniolo,a, Luba Tchertanov, Jean Guilhem,
*
Jean-Paul Mazaleyrat,c Anne Gaucherc and Michel Wakselmanc
aBiopolymer Research Centre, C.N.R., Department of Organic Chemistry, University of Padova, 35131 Padova, Italy
bInstitut de Chimie de Substances Naturelles, UPR 2301, 91198 Gif-sur-Yvette Cedex, France
c
Ã
SIRCOB, Bat. Lavoisier, University of Versailles, 78035 Versailles Cedex, France
Received 23 June 2000; revised 14 August 2000; accepted 30 August 2000
AbstractÐBy using the recently proposed biphenyl-based, Ca-tetrasubstituted, cyclic, axially chiral a-amino acid Bip we synthesised by
solution methods a large set of model peptides, including the homo-oligomer series, to the pentamer level. All of the peptides were fully
characterised and their preferred conformation was assessed in solution by means of a FT-IR absorption and 1H NMR study. Results of X-ray
diffraction analyses of two Bip derivatives and a terminally protected tripeptide with the sequence ±Gly±Bip±Gly± are also presented. Our
®ndings indicate that Bip tends to support b-turn and 310-helical structures, although in short peptides the fully-extended (C5) conformation
would also be populated to some extent. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
acid (Ac7c)5 (Fig. 1) exhibit a very high tendency to fold
into b-turns4a,6 and 310-helices.7
The last twenty years have witnessed a steady stream of new
synthetic approaches to and conformational investigations
of peptidomimetics, compounds that replace physiologi-
cally vulnerable peptide functionalities with chemical
modules of increased stability and cellular penetration.1 A
generally applicable method for development of peptidomi-
metics involves preparation of conformationally restricted
analogues. At the local level this target can be achieved by a
modi®cation of individual residues via incorporation of
rigid structural elements, e.g. Ca-tetrasubstituted a-amino
acids.1g In particular, in our ongoing study of the conforma-
tional preferences of peptides rich in this type of sterically
demanding building block2 we have already shown that
peptides based on Ca,a-dibenzylglycine (Dbzg)3 (Fig. 1)
strongly prefer the fully-extended (C5) conformation,4
whereas those rich in 1-aminocycloheptane-1-carboxylic
In this work we describe the synthesis and preferred confor-
mation of peptides characterised by Bip (20,10: 1,2; 100,200:
3,4-dibenzcyclohepta-1,3-diene-6-amino-6-carboxylic acid)
(Fig. 1), a Ca-tetrasubstituted a-amino acid which combines
structural features of both Dbzg and Ac7c.8 More speci®-
cally, model peptides based on Bip/(S)-Ala and Bip/Gly
sequences, along with Bip homo-oligomers, to the penta-
peptide level have been synthesised by solution methods
and fully characterised, and their conformation investigated
1
in solution by FT-IR absorption and H NMR techniques.
X-Ray diffraction structures of two Bip derivatives and a
terminally protected tripeptide with the sequence -Gly-Bip-
Gly- have also been solved. In Bip, a mimic of the coded
Phe residue, the chirality imposed by the biaryl axis
(atropoisomerism) represents an additional, versatile tool
Figure 1. Chemical structure of Bip compared to those of Ac7c and Dbzg.
Keywords: amino acids and derivatives; NMR; peptides; X-ray crystal structures.
* Corresponding author. Tel.: 139-049-827-5247; fax: 139-049-827-5239; e-mail: biop02@mail.chor.unipd.it
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00801-2

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F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
Figure 2. Interconversion between (R) and (S)-Bip.
for conformational restriction. However, the enantiomers of
Bip and its derivatives have already been shown to inter-
convert in solution at room temperature.8b Preliminary
accounts of a limited part of this work have been repor-
ted.8a,9 The synthesis of a ring-dimethylated analogue of
Bip has been published by Zavada and coworkers.10
bis(bromomethyl)-1,10-biphenyl
under
phase-transfer
conditions led to good yields (75%) of the a-amino ester
H-Bip-OtBu. Acidolysis of the amino ester in a 1:1 TFA/
CH2Cl2 (TFA, tri¯uoroacetic acid) solution gave the free
amino acid. The Na-protected derivatives were prepared
by standard procedures.
For the conformationally labile Bip and its derivatives
broadened H NMR signals were generally observed at
1
Results and Discussion
room temperature, indicating a slow interconversion on
the NMR time scale between the two conformers [(R)-
and (S)-enantiomers] resulting from rotation about the
Amino acid and peptide synthesis
C1±C1 bond of the biphenyl moiety. The calculated
0
The synthesis and characterization of Bip and its derivatives
H-Bip-OtBu (OtBu, tert-butoxy), Boc-Bip-OH (Boc, tert-
butyloxycarbonyl) and Z-Bip-OH (Z, benzyloxycarbonyl)
used in this work were already reported.8 Brie¯y, Ca-bis-
alkylation of a Schiff base from H-Gly-OtBu by 2,20-
rotational energy barrier is 14 kcal mol21 (Fig. 2).8b
In this work the Bip/Ala and Bip/Gly peptides have been
prepared via the step-by-step strategy in solution starting
Figure 3. FT-IR absorption spectra (3500±3200 cm21 region) in CDCl3 solution of (A): Boc-(S)-Ala-Bip-(S)-Ala-OMe (3), Boc-[(S)-Ala]2-Bip-(S)-Ala-OMe
(4), and Z-Bip-[(S)-Ala]2-Bip-(S)-Ala-OMe (5); (B): Z-(Bip)3-OtBu (3), Z-(Bip)4-OtBu (4), and Z-(Bip)5-OtBu (5). Peptide concentration: 1.0 mM.

F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
8723
from the C-terminus. Bip±Ala, Bip±Gly, Ala±Ala and
Gly±Gly peptide bond formation has been achieved by
the DCC/HOBt11a (DCC, N,N0-dicyclohexylcarbodiimide;
HOBt, 1-hydroxy-1,2,3-benzotriazole) or the EDC/HOBt
[EDC, N-ethyl,N0-(3-dimethylaminopropyl)-carbodiimide]
method, while Ala±Bip and Gly±Bip couplings have been
performed by the symmetrical anhydride procedure.
Removal of the Na-protecting Z-group was obtained by
treatment with a 33% HBr/AcOH (acetic acid) solution,
while removal of the Boc group was achieved using either
a HCl/EtOAc (ethyl acetate) or a 1:1 TFA/CH2Cl2 solution.
The fully-protected Bip homo-dipeptide has been prepared
by means of the pivaloyl mixed anhydride method.11b,c The
tert-butyl ester moiety has been removed by acidolysis
(TFA/CH2Cl2 1:1). Then, a coupling strategy from the
N-terminus, involving carboxyl group activation through
the 5(4H)-oxazolone intermediate,11b,c has been used.
Acylation of H-Bip-OtBu by the oxazolones from
Z-(Bip)2-4-OH has required several days, but has given
acceptable yields (60%) of the fully protected homo-
oligomers Z-(Bip)n-OtBu (nˆ3±5), with the unreacted
starting oxazolones generally also recovered.
Fig. 3 shows the FT-IR absorption spectra in the conforma-
tionally informative 3500±3250 cm21 (N±H stretching)
region of the Bip/Ala and (Bip)n peptide series, while in
Fig. 4 the spectra of four Bip/Ala and Bip/Gly tripeptides
are illustrated. We assign: (i) The high-frequency band(s)
found at $3420 cm21 to free, solvated N±H groups. (ii) The
medium-frequency band (shoulder) near 3405 cm21 to
weakly intramolecularly H-bonded N±H groups of fully-
extended (C5) conformers. (iii) The low-frequency band
at 3360±3335 cm21 to more strongly, intramolecularly
H-bonded N±H groups of folded conformers.12
No marked differences are observed in the spectra of the
conformationally relevant pentapeptides by heating the
CDCl3 solution to 508C (data not shown). This result may
suggest that Bip isomer inter-conversion and the related
formation of diastereomeric compounds, that are expected
to be facilitated at higher temperature, do not signi®cantly
alter the overall peptide conformational properties. In both
peptide series the intensity of the low-frequency band, rela-
tive to the high-frequency band, tends to increase with
peptide main-chain lengthening (Fig. 3). While this effect
is gradual in the homo-peptide series, an abrupt enhance-
ment is seen in the Bip/Ala series between the tetra- and
pentapeptides. As in this series the intensity enhancement is
rather modest between the tri- and tetrapeptides, these
results, taken together, strongly support the view that the
extent of folding is more enhanced as a consequence of the
incorporation of an N-terminal Bip than an Ala residue.
Concomitantly, the absorption maximum shifts to lower
wavenumbers. In addition, a visual inspection of the spectra
Solution conformational analysis
A solution conformational analysis of the terminally
protected Bip/Ala, Bip/Gly and (Bip)n peptides was carried
out by using FT-IR absorption and 1H NMR techniques in a
structure-supporting solvent (CDCl3). The longest peptides
(pentamers) were also examined as a function of concentra-
tion and heating.
Figure 4. FT-IR absorption spectra (3500±3200 cm21 region) in CDCl3 solution of Z-Bip-[(S)-Ala]2-OMe (A), Z-(S)-Ala-Bip-(S)-Ala-OMe (B), Z-Bip-
(Gly)2-OEt (C), and Z-Gly-Bip-Gly-OEt (D), Peptide concentration: 1.0 mM.

8724
F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
All other NH protons display a behaviour characteristic of
shielded protons, as their chemical shifts appear relatively
insensitive to solvent composition. From our 1H NMR
analysis it is reasonable to conclude that the most populated
conformation adopted by the terminally protected Bip/Ala
pentapeptide is the 310-helix (originated by three consecu-
tive b-turn conformations), as in this ordered secondary
structure only the two N-terminal NH protons do not par-
ticipate in the intramolecular H-bonding scheme. Two
views of a molecular model of the pentapeptide in the
310-helical conformation are shown in Fig. 6. They clearly
illustrate the overlapping of the two Bip side chains one on
top of the other after one complete turn of the ternary helix.
Crystal-state conformational analysis
Figure 5. Plots of NH chemical shifts in the 1H NMR spectra of Z-Bip-[(S)-
Ala]2-Bip-(S)-Ala-OMe at 323 K as a function of: (A) peptide concentra-
tion (between 10 mM and 1 mM), and (B) increasing percentages of DMSO
added to the CDCl3 solution (v/v) (peptide concentration: 1.0 mM).
The molecular and crystal-structure of two Bip derivatives,
H-Bip-OtBu and Boc-Bip-O@, and one terminally
protected Bip tripeptide, Z-Gly-Bip-Gly-OEt (OEt, ethoxy),
were determined by X-ray diffraction. The molecular
structures with the atomic numbering schemes are shown
in Figs. 7 and 8. Selected Na-protecting group, backbone
and side-chain torsion angles14 are given in Table 1. In
Table 2 the intra- and intermolecular H-bond parameters
are listed. Because of the relatively low rotation energy
of the four tripeptides (Fig. 4) clearly indicates that the
amount of folding largely depends on a combination of
type of protein amino acid and position of the Bip residue
in the sequence. Interestingly, a remarkably high population
of fully-extended conformers is seen in the ±Bip±Gly±
Gly± tripeptide.
barrier about the C1±C1 bond the enantiomers of the
0
three compounds could not be separated, but rather they
coexist in the crystals. For consistency, the torsion angles
reported for all three compounds refer to the (S) isomer.
To better understand the conformational tendency of the Bip
residue in CDCl3 solution we performed a 1H NMR
investigation of the pentapeptide Z-Bip-[(S)-Ala]2-Bip-(S)-
Ala-OMe (OMe, methoxy) (Fig. 5). To markedly reduce
the signal broadening effect associated with Bip isomer
interconversion this study was performed at 508C. All NH
proton resonances were unambiguously assigned by means
of 2D ROESY experiments, starting from the urethane
N(1)H proton known to resonate at higher ®elds. The penta-
peptide weakly self-associates above 1.0 mM concentration
and in this process only the N(1)H and N(2)H protons are
involved. A detailed conformational analysis was carried
out at 1.0 mM peptide concentration where self-association
is absent. The participation of speci®c NH groups in
intramolecular H-bonding was assessed by examining the
behaviour of the NH resonances upon addition of the
perturbing agent DMSO (dimethyl sulphoxide), a strong
H-bonding acceptor solvent, to the CDCl3 solution.13 It is
noteworthy that only the N(1)H and N(2)H proton chemical
shifts are signi®cantly sensitive to the addition of DMSO.
In H-Bip-OtBu the neutral amino group has a pyramidal
structure, the sum of bond angles at nitrogen being h3278.
The Bip c1 torsion angle is typical of a folded conformation.
The ester moiety is trans planar (v1 torsion angle).15a
In the Na-Boc derivative the Bip f1 torsion angle indicates a
folded conformation. The trans, trans arrangement of the
Boc±NH± moiety (v0and u1 torsion angles) is that
commonly reported for Boc-protected peptides (type b
conformation).15b
The 1±2 sequence of the tripeptide is S-shaped. Indeed,
Gly1 is distorted helical, while Bip2 is regular helical, but
the handedness of the turn conformation is opposite in the
two residues. The conformation of the C-terminal Gly
residue is semi-extended. As described above for the Boc-
derivative, the conformation of the related Z-NH- moiety in
the tripeptide is the usual trans, trans (type b).15c The
Figure 6. Molecular model of the 310-helical structure formed by the pentapeptide sequence -(S)-Bip-[(S)-Ala]2-(S)-Bip-(S)-Ala-. (A) Top view; (B) side view.

Figure 7. ortep views of the H-Bip-OtBu molecule (left) and the Boc-Bip-O@ molecule (right) with numbering of the atoms. In both cases only the structure of the (S)-Bip isomer is shown. Anisotropic displacement
ellipsoids are drawn at the 50% probability level.

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F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
Figure 8. ortep view of the Z-Gly-Bip-Gly-OEt molecule with numbering of the atoms. Only the structure of the (S)-Bip isomer is shown. Anisotropic
displacement ellipsoids are drawn at the 30% probability level.
peptide,15d,e and ester15a groups (v1, v2, and v3 torsion
angles) are trans planar.
average ring plane. The puckering parameters17 are
Ê
QTˆ1.028(4) A, f2ˆ287.5(2)8, f3ˆ276.7(16)8, and u2ˆ
Ê
82.6(2)8 for H-Bip-OtBu; QTˆ1.049(3) A, f2ˆ286.8(1)8,
It is worth mentioning that in the Na-Boc derivative and the
tripeptide the sign of the Bip f torsion angle is positive, thus
implying a left-handed screw sense preference for the (S)
isomer.
f3ˆ281.8(11)8, and u2ˆ82.8(1) for Boc-Bip-O@; and
Ê
QTˆ1.058(4) A, f2ˆ289.1(2)8, f3ˆ288.5(20)8, and
u2ˆ82.7(2)8 for the tripeptide. According to literature
data, in the family of conjugated cycloheptadienes the
seven-membered ring adopts a boat or a ¯at boat conforma-
tion.18 The vicinal CvC double bond moiety is intermediate
between the cis and gauche conformations. If two aromatic
rings are vicinally fused to cycloheptane, they are rotated
about the joining C1±C10 bond forming a gauche conforma-
tion with a torsion angle in the range ^45±508. All seven-
membered rings of this type are classi®ed as distorted boats,
An analysis of the geometry of the Bip residues in the three
structures shows that tricyclic skeleton deformations,
compared to the saturated ring of the parent Ac7c, may be
produced by unfavourable steric interactions. More speci®-
cally, in each Bip residue the seven-membered cycle is
pseudo-symmetrical with a non crystallographic C2 axis
passing through Ca and the middle of the opposite bond,
and the two phenyl rings being not coplanar [the dihedral
angle between the planes of the two rings is about 458 in the
two derivatives and 48.0(1)8 in the tripeptide]. The torsion
most of them adopting a conformation with the two torsion
0
angles about the fusion bonds (Cb±C2±C1±C1 and C ±
b
0
2
1
1
0
0
C ±C ±C ) close to 08. Indeed, the absolute values
observed for these two torsion angles in our three
compounds are in the range 0.4±4.98.
angle about the C1±C1 bond is 46.6(5)8 in H-Bip-OtBu,
0
46.7(4)8 in Boc-Bip-O@, and 46.6(3)8 in the tripeptide.
The conformation of the seven-membered ring in the three
compounds is close to a twist-boat (TB),16 with a bisectional
arrangement of the Ca±N and Ca±C0 bonds relative to the
The H-bonding scheme in H-Bip-OtBu is determined by a
single weak interaction of the pyramidal a-NH2 group as a
donor with the ester carbonyl O1vC01 as an acceptor.19 The

F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
8727
Table 1. Selected Na-protecting group backbone and side-chain torsion
angles (deg) for the Bip derivatives and peptide (the torsion angles for
rotation about bonds of the peptide backbone (f, c, v) are described in
Ref. 14. For the torsion angles for rotation about bonds of the Boc- and Z-
protecting groups (u1 and u2) see Ref. 15b and 15c, respectively)
molecule forming a two-dimensional in®nite chain along
the a axis. The methanol molecule, coordinated with
Ca11, is H-bonded to the carboxylic O1 atom, which acts
as a double acceptor (three-centre H-bonding).20 Between
the layer-like H-bond network a spacious hydrophobic zone,
formed by the bulky biphenyl moieties, is observed.
Torsion angle
H-Bip-OtBu a
Boc-Bip-
O@a
water/
Z-Gly-Bip-
Gly-Oet a
In the crystal structure of the tripeptide Z-Gly-Bip-Gly-OEt
the molecules are connected through (urethane) N1±
H´´´O1vC01 (peptide) and (peptide) N3±H´´´O2vC02
(peptide) intermolecular H-bonds which give rise to in®nite
rows along the a axis. This structure is further stabilised by
van der Waals interactions between the apolar moieties.
methanol
solvate
Na -protecting group
u2
96.3(5)
2177.5(4)
2173.3(4)
u1
v0
169.7(2)
2168.9(2)
Backbone
f1
c1
v1
f2
c2
v2
f3
58.9(3)
287.3(6)
26.2(7)
2179.8(4)
59.9(6)
In summary, as far as the backbone conformation of Bip is
concerned, this crystal-state analysis clearly indicates a
tendency of this Ca-tetrasubstituted a-amino acid to fold,
as already observed for Aib (a-aminoisobutyric acid), the
prototype of this family, and 1-aminocycloalkane-1-
carboxylic acids, including Ac7c.2,5 However, in the tri-
peptide this propensity is apparently not strong enough to
overcome that of the Gly residue, known to dislike
incorporation into position 1 of a b-turn conformation.
272.5(3)b
176.1(3)c
43.9(6)
171.5(4)
271.6(6)
159.3(5)
178.5(5)
c3
v3
Bip side chain
Ca±Cb±C2±C1
271.1(4)
274.1(4)
3.3(5)
273.3(3)
275.0(3)
4.9(4)
276.4(4)
275.6(4)
4.0(4)
Ca±Cb0 ±C20 ±C1
0
Cb±C2±C1±C1
0
Cb0 ±C20 ±C10 ±C1
1.1(5)
0.4(4)
1.9(4)
C2±C1±C10 ±C2
46.6(5)
37.7(4)
47.9(4)
46.7(4)
37.8(3)
48.3(3)
46.6(3)
42.3(5)
44.6(5)
0
Conclusions
C2±Cb±Ca±Cb
0
C20 ±Cb0 ±Ca±Cb
In this paper we have reported on the synthesis and preferred
conformation of peptides built from Bip, a Ca-tetrasubsti-
tuted a-amino acid which combines the characteristics of
a Torsion angles for the (S)-isomer are reported.
b N±Ca±C0 ±OT torsion angle.
both Ac7c, a Cia$Cai cyclized residue,1g and Dbzg, a Ca,a
-
c Ca±C0 ±OT±CT torsion angle.
symmetrically di-substituted glycine without cyclization
between the two side chains. The former class of amino
acids is known to induce b-turns and 310-helices,5 while
the latter class tend to force the peptide main chain into a
fully-extended disposition.3 Therefore, it is not surprising
that the results described here would indicate that: (i) Bip
is a turn/helix former, although less effective than Ac7c. (ii)
A detectable amount of the fully-extended conformation
populates the equilibrium mixtures of some of the short
Bip peptides.
H-bond forms in®nite chains along the b axis. Between the
chains a hydrophobic zone, packed with the biphenyl and
tert-butyl moieties, is observed.
The structure of Boc-Bip-O@ is a centrosymmetrical
Ca-complex with a six-coordinated cation. Four coordina-
tion sites are provided by co-crystallised solvent molecules:
Ê
one water molecule [Ca´´´OW 2.340(2) A] and one methanol
Ê
molecule [Ca´´´OM 2.364(2) A], while two coordination
sites are given by the carboxylate ligand uniquely via the
In protein amino acids the chirality depends exclusively on
the presence of at least one stereogenic centre (the asym-
metric a-carbon atom). By contrast, the chirality of Bip, the
biphenyl-based a-amino acid investigated in this work,
results from a different kind of molecular dissymmetry,
the biaryl axis. In this work we have been able to show
that in the crystal state, in contrast to protein amino acids,
a (S)-Bip residue tends to adopt a left-handed turn/helical
structure. However, a slow interconversion between the two
enantiomers of Bip does take place in solution at room
Ê
oxygen O2 atom [Ca´´´O2 2.356(2) A]. The orientation of
the Boc-NH± fragment (see above) is not favourable for
other cation-ligand interactions. In the crystal structure
both water H-atoms participate in strong H-bonds with the
carbonyl groups O1 and O0. The H-bond with O1 generates
a planar six-membered pseudo-ring [the deviations of atoms
from the plane O1´´´H2±OW´´´Ca´´´O2±C01 are less than
Ê
0.08 A], while the second H-bond involves the O0 atom
of the urethane carbonyl function of a symmetry-related
Table 2. Intra- and intermolecular H-bond parameters for the Bip derivatives and peptide
Ê
Distance (A)
D´´´A
Ê
Distance (A)
H´´´A
Compound
Donor D-H
Acceptor A
Symmetry operation
Angle (deg)
D-H´´´A
H-Bip-OtBu
Boc-Bip-O@ water/methanol solvate
N1±H
O1
O0
O1
O1
O1
O2
x21/2, 2y13/2, z
12x,2y,2z
x, y, z
11x, y, z
x21/2, 2y13/2, z
x21/2, 2y13/2, z
3.144(4)
2.788(3)
2.697(3)
2.621(3)
2.756(5)
2.920(5)
2.34(3)
2.04(2)
1.85(2)
1.73(2)
2.14
139(2)
169(2)
166(1)
175(1)
128
O
O
W±H1
W±H2
OM±H
N1±H
N3±H
Z-Gly-Bip-Gly-OEt
2.08
165

8728
F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
temperature, a clear indication of a residual mobility in the
biphenyl system. This property, in turn, tends to dramati-
cally complicate the structural assignment of Bip-rich
peptides, as numerous diastereomeric species may con-
comitantly be present in these compounds. For this reason
we are currently synthesizing and investigating the
preferred conformation of peptides based on rigidi®ed Bip
congeners.
was stirred at room temperature for 2 h and evaporated in
vacuo. The residue was dissolved in EtOAc and the solution
was extracted with 5% NaHCO3 (50 ml), H2O (100 ml),
dried over MgSO4, ®ltered and evaporated in vacuo. The
crude product (0.068 g, 96%) was chromatographed on a
1.5£37 cm column of silica gel with eluent (B) to give
0.055 g (78%) of pure title dipeptide as a glass. Found: C,
69.73; H, 6.57; N, 7.99%; C20H22N2O´30.3 H2O (347.796)
25
requires C, 69.87; H, 6.62; N, 8.15%. [a] ˆ253;
589
25
578
25
546
25
436
25
365
[a] ˆ255; [a] ˆ264; [a] ˆ2123; [a] ˆ2227 (c
1
Experimental
0.5; MeOH). Rfˆ0.40 (B). H NMR: 7.80 (d, 1H, NH Ala,
Jˆ7.9 Hz); 7.45±7.25 (m, 8H, ArH); 4.58 (dq, 1H, Ha Ala,
Jˆ7.9; 7.2 Hz); 3.77 (s, 3H, OMe); 3.14 (broad d, 2H, Hb
Bip); 2.33 (d, 2H, Hb Bip); 1.84 (broad s, 2H, NH Bip); 1.46
(d, 3H, Hb Ala, Jˆ7.2 Hz). 13C NMR: 175.2, 173.5 (CvO
Ala and Bip), 140.5±127.3 (CAr), 67.8 (Ca Bip), 52.3
(OMe), 47.7 (Ca Ala), 44.1 (Cbb0 Bip), 18.1 (Cb Ala).
General
Melting points were determined with a temperature raise of
38C/min and are uncorrected. 1H NMR and 13C NMR spec-
tra were recorded in CDCl3 at 300 MHz and 77 MHz,
respectively, on a Bruker model AC-300. Splitting patterns
are abbreviated as follows: (s) singlet, (d) doublet, (t) triplet,
(q) quartet, (m) multiplet. The optical rotations were
measured with a Perkin±Elmer model 241 polarimeter
equipped with a 1 dm thermostated cell. Analytical thin-
layer chromatography (TLC) and preparative column
chromatography were performed on Kieselgel F 254 and
Kieselgel 60 (0.040±0.063 mm) (Merck), respectively,
with the following eluent systems: 2.5% MeOH-97.5%
CH2Cl2 (A); 5% MeOH-95% CH2Cl2 (B); 10% MeOH-
90% CH2Cl2 (C); CH2Cl2 (D). UV light (254 nm) allowed
visualisation of the spots after TLC runs for all compounds,
even at low concentrations.
Z-(S)-Ala-Bip-(S)-Ala-OMe. To an ice-cold solution of
Z-(S)-Ala-OH (0.135 g, 0.6 mmol) in CH3CN (2 ml) DCC
(0.061 g, 0.3 mmol) was added. The mixture was stirred at
08C for 1 h, ®ltered through glass wool for elimination of the
DCU precipitate, and added to an ice-cold solution of
H-Bip-(S)-Ala-OMe (0.050 g, 0.15 mmol) in CH3CN
(2 ml). The resulting solution was stirred from 08C to
room temperature overnight, and then evaporated in
vacuo. The residue was dissolved in EtOAc and the solution
was extracted with 0.5 M HCl (2£100 ml), H2O (100 ml),
5% NaHCO3 (2£100 ml), H2O (2£100 ml), dried over
MgSO4, ®ltered and evaporated in vacuo. The crude product
was chromatographed on a 1.5£39 cm column of silica gel
with eluent (C) to give 0.078 g of pure title tripeptide as a
glass. Crystallization from EtOAc (ca 2 ml) led to crystals
which were ®ltered, washed with hexane and air dried.
Yield 0.076 g (94%). Mp 2108C. Found: C, 68.09; H,
6.33; N, 7.56%; C31H33N3O´60.3 H2O (549.003) requires C,
Synthesis of Bip/(S)-Ala peptides
Boc-Bip-(S)-Ala-OMe. To a suspension of Boc-Bip-OH8b
(0.088 g, 0.25 mmol), HCl´H-(S)-Ala-OMe (0.070 g,
0.5 mmol) and HOBt (0.068 g, 0.5 mmol) in a mixture of
THF (tetrahydrofuran) (2 ml) and CH2Cl2 (2 ml), a solution
of TEA (triethylamine) (0.052 g, 0.5 mmol) in CH2Cl2
(0.5 ml) was added, followed by a solution of DCC
(0.062 g, 0.3 mmol) in CH2Cl2 (0.5 ml). The reaction
mixture was magnetically stirred at room temperature over-
night, and then evaporated in vacuo. The residue was stirred
for a few minutes in the presence of EtOAc (50 ml) and the
insoluble solid (N,N0-dicyclohexylurea, DCU) was ®ltered
off. The solution was extracted with 5% NaHCO3
(2£50 ml), H2O (100 ml), 0.5 N HCl (2£50 ml), H2O
(2£100 ml), dried over MgSO4, ®ltered and evaporated in
vacuo. The crude product was chromatographed on a
1.5£39 cm column of silica gel with eluent (B) to give
0.103 g (94%) of pure title dipeptide. Crystallization of a
sample from abs EtOH gave very ®ne needles. Mp 2078C.
25
589
25
578
67.81; H, 6.17; N, 7.65%. [a] ˆ2153; [a] ˆ2159;
25
546
25
436
25
365
[a] ˆ2186; [a] ˆ2356; [a] ˆ2670 (c 0.5;
MeOH). Rfˆ0.75 (C). H NMR (Z-Ala1-Bip-Ala2-OMe):
7.45±7.22 (m, 13H, ArH); 7.25 (masked d, 1H, NH Ala2);
6.53 (s, 1H, NH Bip); 5.43 (d, 1H, NH Ala1, Jˆ6.4 Hz); 5.06
(m, 2H, CH2 Z); 4.54 (dq, 1H, Ha Ala2, Jˆ7.3; 7.2 Hz); 4.09
(dq, 1H, Ha Ala1, Jˆ6.4; 7.2 Hz); 3.72 (s, 3H, OMe); 3.25
(broad d, 1H, Hb Bip); 3.17 (d, 1H, Hb Bip); 2.98±2.64
(broad m, 2H, Hb Bip); 1.39 (d, 6H, Hb Ala1 and Ala2,
Jˆ7.2 Hz). 1H NMR (213 K; two diastereoisomers D1
,75% and D2 ,25%, are observed): 7.13 (d, ,0.3H, NH
Ala2 D2, Jˆ6.7 Hz); 7.04 (d, ,0.7H, NH Ala2 D1,
Jˆ7.1 Hz); 6.85 (s, ,0.25H, NH Bip D2); 6.32 (s,
,0.75H, NH Bip D1); 5.68 (broad s, ,0.7H, NH Ala1
D1); 5.63 (broad s, ,0.3H, NH Ala1 D2); 5.10 and 4.88
(two d, ,1.4H, CH2 Z D1, Jˆ12.0 Hz); 5.03 (m, ,0.6H,
CH2 Z D2); 4.50 (m, 1H, Ha Ala2 D1 D2); 4.07 (m, ,0.3H,
Ha Ala1 D2); 3.91 (m, ,0.7H, Ha Ala1 D1); 3.78 (s,
1
Found: C, 68.09; H, 6.91; N, 6.38%; C25H30N2O5 (438.506)
25
requires C, 68.47; H, 6.90; N, 6.39%. [a] ˆ2112;
589
25
25
25
25
[a]578ˆ2117; [a]546ˆ2137; [a]436ˆ2263; [a] ˆ2498
365
,2.25H, OMe D1); 3.68 (s, ,0.75H, OMe D2). H NMR
1
1
(c 1; MeOH). Rfˆ0.75 (B). H NMR: 7.45±7.30 (m, 8H,
(333 K): 7.22 (d, 1H, NH Ala2, Jˆ7.1 Hz); 6.34 (s, 1H, NH
Bip); 5.17 (d, 1H, NH Ala1, Jˆ6.6 Hz); 5.10 (m, 2H, CH2
Z); 4.58 (dq, 1H, Ha Ala2, Jˆ7.1; 7.2 Hz); 4.12 (dq, 1H, Ha
Ala1, Jˆ6.6; 7.2 Hz); 3.74 (s, 3H, OMe);); 3.29 (d, 1H, Hb
Bip); 3.23 (d, 1H, Hb Bip); 2.84 (broad d, 1H, Hb Bip); 2.68
(d, 1H, Hb Bip); 1.42 (d, 3H, Hb Ala, Jˆ7.2 Hz); 1.41 (d,
3H, Hb Ala, Jˆ7.2 Hz). 13C NMR: 173.5, 172.3, 171.4
(CvO Ala1, Ala2 and Bip), 156.3 (CvO Z), 140.6±127.5
ArH); 7.12 (broad m, 1H, NH Ala); 4.88 (s, 1H, NH Bip);
4.64 (dq, 1H, Ha Ala, Jˆ7.3; 7.2 Hz); 3.76 (s, 3H, OMe);
3.22 (broad m, 2H, Hb Bip); 2.58 (broad m, 2H, Hb Bip);
1.48 (s, 9H, Boc); 1.44 (d, 3H, Hb Ala, Jˆ7.2 Hz).
H-Bip-(S)-Ala-OMe. To Boc-Bip-(S)-Ala-OMe (0.092 g,
0.21 mmol), dissolved in EtOAc (4 ml), a 4.8 N solution
of HCl in EtOAc was added (4 ml). The resulting solution

F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
8729
(CAr), 70.0 (Ca Bip), 67.1 (CH2 Z), 52.2 (OMe), 51.2, 48.2
(Ca Ala1, Ala2), ,40 (broad, Cbb0 Bip), 17.7,17.5 (Cb
Ala1, Ala2).
3.23 (d, 1H, Hb Bip); 2.75 (broad d, 1H, Hb Bip); 2.68
(broad d, 1H, Hb Bip); 1.39 (d, 3H, Hb Ala, Jˆ7.2 Hz).
13C NMR: 173.5, 171.8 (CvO Ala, Bip), 155.2 (CvO Z),
140.5±127.6 (CAr), 69.8 (Ca Bip), 67.0 (CH2 Z), 52.3
(OMe), 48.2 (Ca Ala), ,40 (broad, Cbb0 Bip), 18.1 (Cb
Ala).
Z-Bip-(S)-Ala-(S)-Ala-OMe. To a suspension of Z-Bip-
OH8b (0.097 g, 0.25 mmol), HCl´H-(S)-Ala-(S)-Ala-OMe
(0.105 g, 0.5 mmol) and HOBt (0.068 g, 0.5 mmol) in a
mixture of THF (2 ml) and CH2Cl2 (2 ml), a solution of
TEA (0.052 g, 0.5 mmol) in CH2Cl2 (1 ml) was added,
followed by a solution of DCC (0.065 g, 0.3 mmol) in
CH2Cl2 (1 ml). The reaction mixture was magnetically stir-
red at room temperature overnight and then evaporated in
vacuo. The residue was treated as described above for Boc-
Bip-(S)-Ala-OMe to give, after repeated chromatography on
silica gel with eluent (C), 0.100 g (74%) of pure title tri-
peptide as a solid. Mp 1138C. Found: C, 68.26; H, 6.09; N,
7.66%; C31H33N3O6 (543.598) requires C, 68.49; H, 6.12; N,
Boc-(S)-Ala-Bip-(S)-Ala-OMe. A solution of Z-Bip-(S)-
Ala-OMe (0.397 g, 0.8 mmol) in 33% HBr/AcOH (7 ml)
and Et2O (diethyl ether) (7 ml) was stirred at room tempera-
ture for 2 h, then diluted with EtOAc (100 ml) and made
basic by addition of an excess of 5% NaHCO3. The
extracted organic phase was washed with water, dried
over MgSO4, ®ltered and evaporated in vacuo. The obtained
crude H-Bip-(S)-Ala-OMe (0.269 g, 97%) was dissolved in
CH3CN (4 ml) and CH2Cl2 (10 ml). The solution was cooled
to 258C and added to a cold solution of [Boc-(S)-Ala]2O,
just previously prepared by stirring a solution of Boc-(S)-
Ala-OH (0.825 g, 4.4 mmol) and EDC (0.417 g, 2.2 mmol)
in CH3CN (6 ml) at 258C for 1 h. The reaction mixture was
magnetically stirred from 258C to room temperature over-
night and then evaporated in vacuo. The residue was treated
as described above for Z-(S)-Ala-Bip-(S)-Ala-OMe to give,
after repeated chromatography on silica gel with eluent (B),
0.176 g (43%) of pure tripeptide as a solid. Crystallization
from MeOH gave needles (0.150 g). Mp 2358C. Found: C,
65.65; H, 6.96; N, 8.14%; C28H35N3O6 (509.584) requires C,
25
25
578
25
546
25
436
7.73%. [a] ˆ231; [a] ˆ232; [a] ˆ237; [a]
ˆ
589
25
365
266; [a] ˆ2110 (c 0.5; MeOH). Rfˆ0.75 (C). 1H
NMR: 7.42±7.17 (m, 13H, ArH); 7.03 (broad d, 1H, NH
Ala); 6.77 (d, 1H, NH Ala, Jˆ7.2 Hz); 5.23 (s, 1H, NH Bip);
5.13 (s, 2H, CH2 Z); 4.53 (m, 2H, Ha Ala1 and Ala2); 3.73
(s, 3H, OMe); 3.27 (broad d, 1H, Hb Bip); 3.15 (broad d,
1H, Hb Bip); 2.66 (broad m, 2H, Hb Bip); 1.41 (d, 6H, Hb
Ala1 and Ala2, Jˆ7.0 Hz). 1H NMR (213 K; two diastereo-
isomers D1 ,55% and D2 ,45%, are observed): 7.66 and
7.54 (two broad d, NH Ala); 7.03 and 6.88 (two broad d, NH
Ala); 5.50 and 5.46 (two s, NH Bip); 5.16±4.99 and 5.13±
4.99 (two m, CH2 Z); 4.62 (m, Ha Ala); 4.46 and 3.67 (two
m, Ha Ala); 3.75 and 3.67 (two s, ,55/45, OMe); 1H NMR
(333 K): 6.90 (broad d, 1H, NH Ala); 6.80 (d, 1H, NH Ala,
Jˆ7.3 Hz); 5.21 (s, 1H, NH Bip); 5.14 (s, 2H, CH2 Z); 4.53
(m, 2H, Ha Ala1 and Ala2); 3.73 (s, 3H, OMe); 3.25 (d, 1H,
Hb Bip); 3.18 (d, 1H, Hb Bip); 2.71 (broad d, 1H, Hb Bip);
2.66 (broad d, 1H, Hb Bip); 1.41 (d, 3H, Hb Ala,
Jˆ7.0 Hz); 1.39 (d, 3H, Hb Ala, Jˆ7.0 Hz). 13C NMR:
173.0, 171.8 (CvO Ala1, Ala2 and Bip), 155.4 (CvO Z),
140.6±127.7 (CAr), 69.8 (Ca Bip), 67.2 (CH2 Z), 52.3
(OMe), 49.1, 48.2 (Ca Ala1, Ala2), ,40 (broad, Cbb0
Bip), 17.8 (Cb Ala1, Ala2).
25
589
25
578
65.99; H, 6.92; N, 8.25%. [a] ˆ2323; [a] ˆ2332;
25
546
25
436
25
365
[a] ˆ2358; [a] ˆ2527; [a] ˆ2837 (c 0.2;
MeOH). Rfˆ0.35 (B). H NMR (Boc-Ala1-Bip-Ala2-OMe)
(333 K): 7.45±7.21 (m, 9H, ArH and masked NH Ala2);
6.47 (s, 1H, NH Bip); 4.86 (d, 1H, NH Ala1, Jˆ6.9 Hz);
4.59 (dq, 1H, Ha Ala2, Jˆ7.3; 7.2 Hz); 4.07 (dq, 1H, Ha
Ala1, Jˆ6.9; 7.0 Hz); 3.75 (s, 3H, OMe); 3.30 (d, 1H, Hb
Bip); 3.24 (d, 1H, Hb Bip); 2.83 (broad d, 1H, Hb Bip); 2.73
(broad d, 1H, Hb Bip); 1.44 (d, 3H, Hb Ala2, Jˆ7.2 Hz);
1.38 (d, 3H, Hb Ala1, Jˆ7.0 Hz); 1.41 (s, 9H, Boc).
1
Boc-(S)-Ala-(S)-Ala-Bip-(S)-Ala-OMe. To a solution of
Boc-(S)-Ala-Bip-(S)-Ala-OMe (0.150 g, 0.29 mmol) in
CH2Cl2 (5 ml) TFA (5 ml) was added. The solution was
stirred at room temperature for 3 h and evaporated in
vacuo. The residue was dissolved in EtOAc (50 ml). The
solution was extracted with 5% NaHCO3 (50 ml), dried over
MgSO4, ®ltered and evaporated in vacuo. The residue was
chromatographed on a 2.5£47 cm column of silica gel with
eluent (C) to give 0.063 g (52%) of H-(S)-Ala-Bip-(S)-Ala-
OMe which was immediately used in the next coupling step.
A solution of a mixture of this compound (0.063 g,
0.15 mmol), Boc-(S)-Ala-OH (0.044 g, 0.23 mmol) and
HOBt (0.042 g, 0.31 mmol) in THF (5 ml) and CH2Cl2
(2.5 ml) was stirred at 258C. Then, a solution of EDC
(0.044 g, 0.23 mmol) in CH2Cl2 (2.5 ml) was added. The
reaction mixture was magnetically stirred from 258C to
room temperature overnight and evaporated in vacuo. The
residue was treated as described above for Z-(S)-Ala-Bip-
(S)-Ala-OMe to give, after chromatography on a 1.6£38 cm
column of silica gel with eluent (C), 0.077 g (78%) of pure
title tetrapeptide as a solid. Mp 2308C. Found: C, 63.12; H,
7.01; N, 9.43%; C31H40N4O´70.5 H2O (589.670) requires
C, 63.14; H, 7.01; N, 9.50%. ESI1 MS m/z (relative
intensity): 581 (M,H)1 (33); 603 (M,Na)1 (100); 619
Z-Bip-(S)-Ala-OMe. To
a suspension of Z-Bip-OH
(0.387 g, 1 mmol), HCl´H-(S)-Ala-OMe (0.279 g, 2 mmol)
and HOBt (0.270 g, 2 mmol) in a mixture of THF (10 ml)
and CH2Cl2 (5 ml), a solution of TEA (0.202 g, 2 mmol) in
CH2Cl2 (2.5 ml) was added, followed by a solution of EDC
(0.230 g, 1.2 mmol) in CH2Cl2 (2.5 ml). The reaction
mixture was magnetically stirred at room temperature over-
night and then evaporated in vacuo. The residue was
dissolved in a mixture of EtOAc (100 ml) and 0.5 M HCl
(100 ml). The organic phase was extracted with 0.5 M HCl
(100 ml), followed by H2O (100 ml), 5% NaHCO3
(2£100 ml), H2O (2£100 ml), dried over MgSO4, ®ltered
and evaporated in vacuo. The crude product was chromato-
graphed on a 2.3£60 cm column of silica gel with eluent (A)
to give 0.396 g of pure title dipeptide as a solid. Mp 1558C.
Found: C, 71.06; H, 6.12; N, 6.08%; C28H28N2O5 (472.520)
25
requires C, 71.17; H, 5.97; N, 5.93%. [a] ˆ2112;
589
25
25
25
25
[a]578ˆ2115; [a]546ˆ2133; [a]436ˆ2256; [a] ˆ2482
365
1
(c 0.5; MeOH). Rfˆ0.60 (A). H NMR (333 K): 7.45±7.24
(m, 13H, ArH); 6.97 (broad d, 1H, NH Ala, Jˆ7.3 Hz); 5.16
(s, 2H, CH2 Z); 5.06 (s, 1H, NH Bip); 4.60 (dq, 1H, Ha Ala,
Jˆ7.3; 7.2 Hz); 3.75 (s, 3H, OMe); 3.28 (d, 1H, Hb Bip);
25
589
25
578
25
546
(M,K)1 (8). [a] ˆ2146; [a] ˆ2157; [a] ˆ2182;

8730
F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
25
436
25
365
[a] ˆ2341; [a] ˆ2628 (c 0.2; MeOH). Rfˆ0.45 (C).
0.55 mmol) in CH2Cl2 (2 ml). The reaction mixture was
magnetically stirred at room temperature overnight and
then evaporated in vacuo. The residue was treated as
described above for Z-(S)-Ala-Bip-(S)-Ala-OMe to give,
after repeated chromatography on silica gel with eluent
(B) followed by crystallization from a hexane/CH2Cl2 solu-
tion, 0.185 g (70%) of pure title tripeptide as crystals. Mp
1958C. Found: C, 67.33; H, 5.82; N, 7.71%; C30H31N3O´60.3
H2O (534.977) requires C, 67.35; H, 5.95; N, 7.85%.
1H NMR (Boc-Ala1-Ala2-Bip-Ala3-OMe) (333 K): 7.45±
7.22 (m, 9H, ArH and masked NH Ala3); 6.62 (s, 1H, NH
Bip); 6.56 (d, 1H, NH Ala2, Jˆ6.0 Hz); 4.85 (d, 1H, NH
Ala1, Jˆ6.7 Hz); 4.57 (dq, 1H, Ha Ala3, Jˆ7.2; 7.1 Hz);
4.30 (dq, 1H, Ha Ala2, Jˆ6.0; 6.7 Hz); 4.08 (dq, 1H, Ha
Ala1, Jˆ6.7; 7.1 Hz); 3.75 (s, 3H, OMe); 3.26 (d, 1H, Hb
Bip); 3.24 (d, 1H, Hb Bip); 2.85 (broad d, 1H, Hb Bip); 2.79
(broad d, 1H, Hb Bip); 1.43 (d, 3H, Hb Ala3, Jˆ7.1 Hz);
1.41 (s, 9H, Boc); 1.40 (masked d, 3H, Hb Ala2); 1.33 (d,
3H, Hb Ala1, Jˆ7.1 Hz). 13C NMR: 173.5, 173.2, 171.8,
171.5 (CvO Ala1, Ala2, Ala3 and Bip), 155.7 (CvO
Boc), 140.6±127.5 (CAr), 80.7 (Boc), 70.2 (Ca Bip), 52.2
(OMe), 50.5, 50.0, 48.3 (Ca Ala1, Ala2, Ala3), ,40 (broad,
Cbb0 Bip), 17.9, 17.7, 17.2 (Cb Ala1, Ala2, Ala3).
1
Rfˆ0.20 (B). H NMR (293 K): 7.48 (m t-like, 1H, NH
Gly); 7.42±7.21 (m, 13H, ArH); 6.96 (t, 1H, NH Gly,
Jˆ6.0 Hz); 5.31 (s, 1H, NH Bip); 5.12 (s, 2H, CH2 Z);
4.19 (q, 2H, OEt, Jˆ7.1 Hz); 4.07 (d, 2H, Ha Gly,
Jˆ6.0 Hz); 4.05 (d, 2H, Ha Gly, Jˆ5.8 Hz); 3.26 (broad
d, 2H, Hb Bip); 2.63 (broad m, 2H, Hb Bip); 1.27 (t, 3H,
OEt, Jˆ7.1 Hz). 1H NMR (333 K): 7.45±7.08 (m, 14H, ArH
and masked NH Gly); 6.87 (broad t, 1H, NH Gly); 5.18 (s,
1H, NH Bip); 5.15 (s, 2H, CH2 Z); 4.21 (q, 2H, OEt,
Jˆ7.1 Hz); 4.04 (d, 2H, Ha Gly, Jˆ6.0 Hz); 4.03 (d, 2H,
Ha Gly, Jˆ5.6 Hz); 3.26 (d, 2H, Hb Bip); 2.67 (d, 2H, Hb
Bip); 1.27 (t, 3H, OEt, Jˆ7.1 Hz). 1H NMR (223 K): 7.99 (t,
1H, NH Gly, J,5.9 Hz); 7.50±7.07 (m, 13H, ArH); 6.87
(partly masked t, 1H, NH Gly); 5.61 (s, 1H, NH Bip);
5.06 (m, 2H, CH2 Z); 4.25±3.92 (m, 4H, four Ha Gly);
4.14 (q, 2H, OEt, Jˆ7.1 Hz); 3.53 (d, 1H, Hb Bip); 3.13
(d, 1H, Hb Bip); 2.74 (d, 1H, Hb Bip); 2.34 (d, 1H, Hb Bip);
1.25 (t, 3H, OEt, Jˆ7.1 Hz). 13C NMR: 172.7, 170.0, 169.7
(CvO Gly1, Gly2 and Bip), 156.0 (CvO Z), 140.5±127.8
(CAr), 69.9 (Ca Bip), 67.4 (CH2 Z), 61.3 (OEt), 43.1, 41.0
(Ca Gly1, Gly2), ,40 (broad, Cbb0 Bip), 14.1 (OEt).
Z-Bip-(S)-Ala-(S)-Ala-Bip-(S)-Ala-OMe. To a solution of
Boc-(S)-Ala-(S)-Ala-Bip-(S)-Ala-OMe (0.056 g, 0.1 mmol)
in CH2Cl2 (5 ml) TFA (5 ml) was added. The solution was
stirred at room temperature for 3 h and evaporated in vacuo.
The residue was dissolved in EtOAc (50 ml). The solution
was extracted with 5% NaHCO3 (50 ml), dried over MgSO4,
®ltered and evaporated in vacuo, to give 0.042 g (91%) of
crude H-(S)-Ala-(S)-Ala-Bip-Ala-OMe which was immedi-
ately used in the next coupling step. A solution of a mixture
of this compound (0.042 g, 0.09 mmol), Z-Bip-OH
(0.035 g, 0.09 mmol), HOBt (0.018 g, 0.13 mmol) and
EDC (0.020 g, 0.11 mmol) in CH2Cl2 (10 ml) and THF
(10 ml) was stirred at room temperature overnight and
evaporated in vacuo. The residue was treated as described
above for Z-(S)-Ala-Bip-(S)-Ala-OMe and puri®ed by
preparative TLC on silica gel with eluent (C) to give
0.039 g (52%) of pure title pentapeptide as a solid. Mp
1188C. Found: C, 69.75; H, 6.21; N, 8.01%;
C50H51N5O´80.5 H2O (858.956) requires C, 69.91; H, 6.10;
Z-Gly-Bip-OtBu. To a solution of Z-Gly-OH (0.418 g,
2 mmol) in CH3CN (10 ml) DCC (0.206 g, 1 mmol) was
added. The mixture was stirred at room temperature for
1 h, ®ltered through glass wool and added to a solution of
H-Bip-OtBu8b (0.160 g, 0.52 mmol) in CH3CN (5 ml). The
resulting solution was stirred at room temperature for 20 h
and evaporated in vacuo. The residue was treated as
described above for Z-(S)-Ala-Bip-(S)-Ala-OMe and puri-
®ed by column chromatography on silica gel with eluent (C)
to give 0.250 g (96%) of pure title dipeptide as a solid.
Crystallization from a hexane/CH2Cl2 solution gave crystals
(0.170 g). Mp 1818C. Found: C, 71.91; H, 6.35; N, 5.46%;
C30H32N2O5 (500.572) requires C, 71.98; H, 6.44; N, 5.60%.
25
25
589
25
25
578
N, 8.15%. [a] ˆ118; [a] ˆ119; [a]546ˆ122;
25
[a] ˆ146; [a] ˆ194 (c 0.2; MeOH). Rfˆ0.40 (C).
436
365
1H NMR (Z-Bip1-Ala2-Ala3-Bip4-Ala5-OMe) (333 K):
7.45±6.99 (m, 23H, ArH and masked NH Ala5, NH Ala3);
6.88 (s, 1H, NH Bip4); 6.48 (d, 1H, NH Ala2, Jˆ5.4 Hz);
5.27 (s, 1H, NH Bip1); 5.13 (m, 2H, CH2 Z); 4.56 (dq, 1H,
Ha Ala5, Jˆ7.0; 7.2 Hz); 4.29 (dq, 1H, Ha Ala3, Jˆ6.8;
7.2 Hz); 4.25 (dq, 1H, Ha Ala2, Jˆ5.4; 7.2 Hz); 3.70 (s,
3H, OMe); 3.23 (d, 1H, Hb Bip); 3.19 (d, 1H, Hb Bip);
3.09 (broad d, 2H, Hb Bip); 2.95 (d, 1H, Hb Bip); 2.87
(broad d, 1H, Hb Bip); 2.55 (d, 1H, Hb Bip); 2.43 (broad
d, 1H, Hb Bip); 1.50 (d, 3H, Hb Ala3, Jˆ7.2 Hz); 1.46 (d,
3H, Hb Ala5, Jˆ7.2 Hz); 1.42 (d, 3H, Hb Ala2, Jˆ7.2 Hz).
13C NMR: 173.3 (broad), 172.7 (broad), 172.2, 172.1 (CvO
Bip1, Bip4, Ala2, Ala3 and Ala5), 156.2 (CvO Z), 140.5±
127.1 (CAr), 70.0, 69.4 (Ca Bip1 and Bip4), 67.2 (CH2 Z),
52.1 (OMe), 51.1, 50.4, 48.3 (Ca Ala2, Ala3 and Ala5),
,41.5, ,35.5 (broad, Cbb0 Bip1 and Bip4), 17.8, 16.9,
16.6 (Cb Ala2, Ala3 and Ala5).
1
Rfˆ0.75 (C). H NMR: 7.47±7.20 (m, 13H, ArH); 6.64 (s,
1H, NH Bip); 5.70 (m t-like, 1H, NH Gly); 5.29 (s, 2H, CH2
Z); 3.84 (d, 2H, Ha Gly, Jˆ5.5 Hz); 3.12 (broad d, 2H, Hb
Bip); 2.7 (broad m, 2H, Hb Bip); 1.46 (s, 9H, OtBu). 13C
NMR: 170.8, 168.4 (CvO Gly and Bip), 156.6 (CvO Z),
140.9±126.9 (CAr), 81.7 (OtBu), 69.3 (Ca Bip), 67.0 (CH2
Z), 44.5 (Ca Gly), 40.9 (broad, Cb Bip), 38.2 (broad, Cb0
Bip), 14.1 (OtBu).
Z-Gly-Bip-Gly-OEt. To a solution of Z-Gly-Bip-OtBu
(0.240 g, 0.48 mmol) in CH2Cl2 (5 ml) TFA (5 ml) was
added. The solution was stirred at room temperature for
3 h and evaporated in vacuo. Dissolution of the residue in
CH2Cl2 and evaporation in vacuo were repeated several
times in order to remove the excess of TFA. Then, the
residue was precipitated from a concentrated CH2Cl2 solu-
tion by addition of hexane. The precipitate was collected
and dried in vacuo to give 0.191 g (90%) of crude Z-Gly-
Bip-OH as a powder (mp 1758C), which was used in the
Synthesis of Bip/Gly peptides
Z-Bip-Gly-Gly-OEt. To
a suspension of Z-Bip-OH
(0.193 g, 0.5 mmol), HCl´H-Gly-Gly-OEt (0.196 g,
1 mmol) and HOBt (0.135 g, 1 mmol) in a mixture of
THF (5 ml) and CH2Cl2 (2 ml), a solution of NMM
(N-methylmorpholine) (0.101 g, 1 mmol) in CH2Cl2 (1 ml)
was added, followed by a solution of EDC (0.106 g,

F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
8731
next coupling step without further puri®cation. To a suspen-
sion of a mixture of this compound (0.177 g, 0.40 mmol),
HCl´H-Gly-OEt (0.111 g, 0.80 mmol) and HOBt (0.108 g,
0.80 mmol) in THF (5 ml) and CH2Cl2 (2 ml), a solution of
NMM (0.084 g, 0.80 mmol) in CH2Cl2 (1 ml) was added,
followed by a solution of EDC (0.092 g, 0.48 mmol) in
CH2Cl2 (2 ml). The reaction mixture was magnetically stir-
red at room temperature overnight and then evaporated in
vacuo. The residue was treated as described above for Z-(S)-
Ala-Bip-(S)-Ala-OMe to give, after column chromato-
graphy on silica gel with eluent (B), 0.173 g (82%) of
pure title tripeptide. Crystallization from a hexane/CH2Cl2
solution gave crystals (0.141 g). Mp 1528C. Found: C,
67.56; H, 5.77; N, 7.84%; C30H31N3O6 (529.572) requires
C, 68.04; H, 5.90; N, 7.93%. Rfˆ0.40 (B). 1H NMR
(Z-Gly1-Bip-Gly2-OEt) (293 K): 7.45±7.27 (m, 13H,
ArH); 7.25 (partly masked t, 1H, NH Gly2); 6.45 (s, 1H,
NH Bip); 5.45 (broad t, 1H, NH Gly1); 5.11 (s, 2H, CH2
Z); 4.21 (q, 2H, OEt, Jˆ7.1 Hz); 4.02 (broad d, 2H, Ha
Gly2, J,5.0 Hz); 3.83 (d, 2H, Ha Gly1, Jˆ5.7 Hz); 3.26
(broad m, 2H, Hb Bip); 2.68 (broad m, 2H, Hb Bip); 1.28
69.7, 69.2 (Ca Bip1, Bip2), 69.9 (CH2 Z), ,40 (very
broad, Cbb0 Bip1, Bip2), 27.8 (OtBu).
Z-Bip-Bip-OH. To a solution of Z-Bip-Bip-OtBu (1.091 g,
1.61 mmol) in CH2Cl2 (15 ml) TFA (15 ml) was added. The
solution was stirred at room temperature for 3 h and evapo-
rated in vacuo. The residue was dissolved in EtOAc
(150 ml). The solution was extracted with H2O
(2£100 ml), dried over MgSO4, ®ltered and evaporated in
vacuo. The residue was dissolved in MeOH, and the solution
was ®ltered and evaporated in vacuo to give 0.929 g (93%)
of crude title dipeptide as a solid, which was used in the next
step without further puri®cation. Mp 2608C. Found: C,
74.64; H, 5.67; N, 4.24%; C40H34N2O´5H2O (640.704)
requires C, 74.98; H, 5.66; N, 4.37%.
5(4H)-Oxazolone from Z-Bip-Bip-OH. A suspension of
Z-Bip-Bip-OH (0.903 g, 1.45 mmol) in Ac2O (25 ml) was
stirred at 115±1208C for 40 min. Then, the resulting solu-
tion was evaporated in vacuo. The residue was repeatedly
dissolved in toluene and the solution evaporated in vacuo to
give 0.877 g (quantitative yield) of crude title oxazolone,
which was used in the next step without further puri®cation.
This next coupling reaction being uncomplete (see Z-Bip-
Bip-Bip-OtBu), separation by column chromatography
gave a small sample of title oxazolone, obtained as a
powder. Mp 1688C. Found: C, 79.31; H, 5.45; N, 4.65%;
C40H32N2O4 (604.672) requires C, 79.45; H, 5.33; N, 4.63%.
1
(t, 3H, OEt, Jˆ7.1 Hz). H NMR (333 K): 7.45±7.27 (m,
13H, ArH); 7.19 (broad t, 1H, NH Gly2); 6.32 (s, 1H, NH
Bip); 5.30 (broad t, 1H, NH Gly1); 5.13 (s, 2H, CH2 Z); 4.23
(q, 2H, OEt, Jˆ7.1 Hz); 4.03 (d, 2H, Ha Gly2, Jˆ5.3 Hz);
3.83 (d, 2H, Ha Gly1, Jˆ5.8 Hz); 3.30 (d, 2H, Hb Bip); 2.76
1
(broad d, 2H, Hb Bip); 1.29 (t, 3H, OEt, Jˆ7.1 Hz). H
NMR (223 K): 7.59 (broad t, 1H, NH Gly2); 7.45±7.05
(m, 13H, ArH); 6.74 (s, 1H, NH Bip); 6.00 (broad t, 1H,
NH Gly1); 5.00 (m, 2H, CH2 Z); 4.04 (broad q, 2H, OEt);
4.00 (m, 2H, Ha Gly2); 3.75 (broad d, 2H, Ha Gly1); 3.18
(d, 2H, Hb Bip); 2.38 (d, 1H, Hb Bip); 3.07 (d, 1H, Hb Bip);
1.23 (t, 3H, OEt, Jˆ7.1 Hz). 13C NMR: 172.3, 170.0, 169.4
(CvO Gly1, Gly2 and Bip), 156.8 (CvO Z), 140.6±127.8
(CAr), 70.2 (Ca Bip), 67.5 (CH2 Z), 61.4 (OEt), 45.4, 41.6
(Ca Gly1, Gly2), ,40 (broad, Cbb0 Bip), 14.1 (OEt).
1
Rfˆ0.45 (D). H NMR (oxazolone from Z-Bip1-Bip2-OH):
7.45±7.22 (m, 21H, ArH); 5.19 (s, 2H, CH2 Z); 5.14 (s,
1H, NH Bip1); 3.4±2.4 (very broad m, 8H, Hbb0 Bip1,
Bip2). 13C NMR: 179.0 (O±CvO Bip2), 163.2 (O±CvN
Bip1), 154.6 (CvO Z), 140.7±127.7 (CAr), 78.0, 65.0 (Ca
Bip1, Bip2), 67.1 (CH2 Z), ,40.5, ,38.8 (very broad, Cbb0
Bip1, Bip2).
Z-Bip-Bip-Bip-OtBu. A solution of the 5(4H)-oxazolone
from Z-Bip-Bip-OH (0.518 g, 0.86 mmol) and H-Bip-
OtBu (0.300 g, 0.97 mmol) in CH3CN (25 ml) was re¯uxed
for 14 d and evaporated in vacuo. The residue was dissolved
in EtOAc (150 ml). The solution was extracted with 5%
citric acid (2£50 ml), H2O (2£100 ml), dried over MgSO4,
®ltered and evaporated in vacuo to give 0.652 g of crude
neutral part. The acidic aqueous phase was made basic by
addition of an excess of 1 M NaOH and then extracted with
EtOAc. The organic phase was washed with H2O
(2£100 ml), dried over MgSO4, ®ltered and evaporated in
vacuo to give 0.080 g (26%) of crude recovered H-Bip-
OtBu. The crude neutral part was chromatographed on a
2.3£55 cm column of silica gel with eluent (D), then (A),
to give 0.115 g (22%) of recovered pure oxazolone and
0.559 g (59%) of pure title tripeptide as a powder. Mp
2048C. Found: C, 77.35; H, 6.25; N, 4.34%;
C60H55N3O´6H2O (932.080) requires C, 77.31; H, 6.16; N,
Synthesis of Bip homo-peptides
Z-Bip-Bip-OtBu. To a solution of Z-Bip-OH (0.388 g,
1 mmol) and TEA (0.101 g, 1 mmol) in CH2Cl2 (0.5 ml)
and toluene (1 ml) cooled to 258C, a solution of Piv-Cl
(Piv, pivaloyl) (0.121 g, 1 mmol) in toluene (1 ml) was
added. The resulting suspension was stirred at 258C for
2 h, then at room temperature for 1.5 h, and ®nally ®ltered
through glass wool. The solution was evaporated in vacuo at
308C and to the crude solid Z-Bip-OPiv a solution of H-Bip-
OtBu (0.341 g, 1.1 mmol) in toluene (5 ml) was added. The
reaction mixture was stirred for 3 h at 608C and then evapo-
rated in vacuo. The residue was dissolved in EtOAc
(150 ml) and the solution was extracted with 0.5 M HCl
(2£100 ml), H2O (100 ml), 5% NaHCO3 (100 ml), H2O
(2£100 ml), dried over MgSO4, ®ltered and evaporated in
vacuo. The crude product was chromatographed on a
2.3£50 cm column of silica gel with eluent (A) to give
0.414 g (61%) of pure title dipeptide as a foam. Mp
1168C. Found: C, 77.44; H, 6.41; N, 4.06%; C44H42N2O5
(678.792) requires C, 77.85; H, 6.24; N, 4.13%. Rfˆ0.75
(A). 1H NMR (Z-Bip1-Bip2-OtBu): 7.49±7.27 (m, 21H,
ArH); 7.12 (broad s, 1H, NH Bip2); 5.09 (s, 3H, CH2 Z
and NH Bip1); 3.5±2.2 (very broad m, 8H, Hbb0 Bip1,
Bip2); 1.44 (s, 9H, OtBu). 13C NMR: 171.1 (CvO Bip1,
Bip2), 156.3 (CvO Z), 140.7±127.4 (CAr), 81.3 (OtBu),
1
4.51%. Rfˆ0.80 (A). H NMR (Z-Bip1-Bip2-Bip3-OtBu):
7.66 (s, 1H, NH Bip); 7.49±7.14 (m, 29H, ArH); 6.62
(broad s, 1H, NH Bip); 5.09 (s, 1H, NH Bip1); 4.90 (broad
m, 2H, CH2 Z); 3.6±2.3 (very broad m, 12H, Hbb0 Bip1,
Bip2, Bip3); 1.49 (s, 9H, OtBu). 13C NMR: 171.7, 171.1,
170.8 (CvO Bip1, Bip2, Bip3), 155.6 (CvO Z), 140.8±
127.2 (CAr), 81.2 (OtBu), 70.0, 69.8, 69.7 (Ca Bip1, Bip2,
Bip3), 67.3 (CH2 Z), ,40 (very broad, Cbb0 Bip1, Bip2,
Bip3), 27.9 (OtBu).

8732
F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
Z-Bip-Bip-Bip-OH.
Tripeptide
Z-Bip-Bip-Bip-OtBu
puri®cation. This next coupling reaction being uncomplete
(0.657 g, 0.72 mmol) was C-deprotected in TFA (10 ml)
and CH2Cl2 (10 ml), as reported above for Z-Bip-Bip-OH,
to give 0.586 g (95%) of crude title tripeptide as a solid,
which was used in the next step without further puri®cation.
Mp 2198C. Found: C, 75.05; H, 5.78; N, 4.58%;
C56H47N3O´62 H2O (893.992) requires C, 75.23; H, 5.75;
N, 4.70%.
(see Z-Bip-Bip-Bip-Bip-Bip-OtBu below), separation by
column chromatography gave a small sample of title oxa-
zolone, obtained as a powder. Mp 1758C. Found: C, 77.86;
H, 5.76; N, 4.91%; C72H58N4O´62 H2O (1111.248) requires
1
C, 77.81; H, 5.62; N, 5.04%. Rfˆ0.10 (D); 0.80 (A). H
NMR (oxazolone from Z-Bip1-Bip2-Bip3-Bip4-OH): 7.75
(s, 1H, NH Bip); 7.45±7.25 (m, 37H, ArH); 6.33 (s, 1H,
NH Bip); 5.10 (s, 1H, NH Bip1); 4.90 (m, 2H, CH2 Z); 3.6±
2.3 (very broad m, 16H, Hbb0 Bip1, Bip2, Bip3, Bip4). 13C
NMR: 180.0 (O±CvO Bip4), 171.0, 170.6 (CvO Bip1,
Bip2), 163.2 (O±CvN Bip3), 155.7 (CvO Z), 140.3±
127.1 (CAr), 78.1, 70.0, 65.3 (Ca Bip1, Bip2, Bip3, Bip4),
67.4 (CH2 Z), ,41.8, ,39.8, ,38.5, ,37.0 (very broad,
Cbb0 Bip1, Bip2, Bip3, Bip4).
5(4H)-Oxazolone from Z-Bip-Bip-Bip-OH. Tripeptide
Z-Bip-Bip-Bip-OH (0.566 g, 0.66 mmol) was treated as
described above for the oxazolone from Z-Bip-Bip-OH to
give 0.554 g (quantitative yield) of crude title oxazolone,
which was used in the next step without further puri®cation.
This next coupling reaction being uncomplete (see Z-Bip-
Bip-Bip-Bip-OtBu below), separation by column chromato-
graphy gave a small sample of title oxazolone, obtained as a
powder. Mp 1758C. Found: C, 78.18; H, 5.62; N, 4.81%;
C56H45N3O´5H2O (857.960) requires C, 78.39; H, 5.52; N,
Z-Bip-Bip-Bip-Bip-Bip-OtBu. A solution of the 5(4H)-
oxazolone from Z-Bip-Bip-Bip-Bip-OH (0.363 g, 0.34
mmol) and H-Bip-OtBu (0.209 g, 0.67 mmol) in CH3CN
(25 ml) was re¯uxed for 18 d and treated as described
above for Z-Bip-Bip-Bip-OtBu. Chromatography of the
crude product on silica gel with gradient eluent from (D)
to (A), gave 0.026 g (7%) of recovered pure oxazolone
and 0.268 g (57%) of pure title pentapeptide as a solid.
Mp 2348C. Found: C, 78.73; H, 6.04; N, 4.87%;
C92H81N5O´8H2O (1402.624) requires C, 78.77; H, 5.96; N,
4.99%. ESI1 MS m/z (relative intensity): single peak 1385
1
4.89%. Rfˆ0.30 (D); 0.80 (A). H NMR (oxazolone from
Z-Bip1-Bip2-Bip3-OH): 7.51±7.30 (m, 29H, ArH); 7.02 (s,
1H, NH Bip2); 5.15 (broad s, 2H, CH2 Z); 5.12 (s, 1H, NH
Bip1); 3.5±2.3 (very broad m, 12H, Hbb0 Bip1, Bip2, Bip3).
13C NMR: 179.3 (O±CvO Bip3), 171.5 (CvO Bip1), 163.0
(O±CvN Bip2), 155.2 (CvO Z), 140.8±127.5 (CAr), 78.0,
76.6, 64.7 (Ca Bip1, Bip2, Bip3), 67.1 (CH2 Z), ,40.2,
,39.1, ,37.5 (very broad, Cbb0 Bip1, Bip2, Bip3).
1
(M,H)1 (100). Rfˆ0.75 (A). H NMR (Z-Bip1-Bip2-Bip3-
Z-Bip-Bip-Bip-Bip-OtBu. A solution of the 5(4H)-oxa-
zolone from Z-Bip-Bip-Bip-OH (0.533 g, 0.63 mmol) and
H-Bip-OtBu (0.200 g, 0.65 mmol) in CH3CN (25 ml) was
re¯uxed for 17 d and treated as described above for Z-Bip-
Bip-Bip-OtBu. The crude product was chromatographed on
a 2.3£52 cm column of silica gel with eluent (D), then (A),
to give 0.078 g (15%) of recovered pure oxazolone
and 0.459 g (63%) of pure title tetrapeptide as a powder.
Mp 2158C. Found: C, 78.15; H, 6.01; N, 4.87%;
C76H68N4O´7H2O (1167.352) requires C, 78.19; H, 6.04; N,
4.80%. Rfˆ0.05 (D); 0.75 (A). 1H NMR (Z-Bip1-Bip2-Bip3-
Bip4-OtBu): 7.84 (s, 1H, NH Bip); 7.57±7.02 (m, 38H, ArH
and masked NH Bip); 6.66 (s, 1H, NH Bip); 5.17 (s, 1H, NH
Bip1); 5.11 (broad m, 2H, CH2 Z); 3.7±2.2 (very broad m,
16H, Hbb0 Bip1, Bip2, Bip3, Bip4); 1.58 (s, 9H, OtBu). 13C
NMR: 172.0 (broad), 171.6 (broad), 171.4, 170.7 (broad)
(CvO Bip1, Bip2, Bip3, Bip4), 155.5 (CvO Z), 140.7±
127.1 (CAr), 80.9 (OtBu), 70.2, 69.8, 69.6, 69.4 (Ca Bip1,
Bip2, Bip3, Bip4), 67.4 (CH2 Z), ,40 (very broad, Cbb0
Bip1, Bip2, Bip3, Bip4), 27.9 (OtBu).
Bip4-Bip5-OtBu): 7.90 (broad s, 1H, NH Bip); 7.56±7.02
(m, 46H, ArH and masked NH Bip); ,6.90 (broad s, 1H,
NH Bip); 6.66 (broad s, 1H, NH Bip); 5.15 (broad s, 1H, NH
Bip1); 5.15±4.50 (broad m, 2H, CH2 Z); 3.7±2.1 (very broad
m, 20H, Hbb0 Bip1, Bip2, Bip3, Bip4, Bip5); 1.51 (s, 9H,
1
OtBu). H NMR (333 K): 7.72 (s, 1H, NH Bip); 7.47±7.05
(m, 45H, ArH); 6.90 (s, 1H, NH Bip); 6.88 (s, 1H, NH Bip);
6.63 (s, 1H, NH Bip); 5.01 (s, 1H, NH Bip1); 4.85 (s, 2H,
CH2 Z); 3.30±2.35 (m, 20H, Hbb0 Bip1, Bip2, Bip3, Bip4,
Bip5); 1.46 (s, 9H, OtBu). 13C NMR: 172.6 (broad), 171.7
(broad), 170.3 (broad) (CvO Bip1, Bip2, Bip3, Bip4, Bip5),
155.6 (CvO Z), 140.8±126.8 (CAr), 81.0 (OtBu), 70.5,
70.4, 69.9, 69.5, 69.3 (Ca Bip1, Bip2, Bip3, Bip4, Bip5),
67.5 (CH2 Z), ,44±,34 (very broad, Cbb0 Bip1, Bip2,
Bip3, Bip4, Bip5), 28.0 (OtBu).
FT-IR absorption
The solid-state infrared absorption spectra (KBr disk tech-
nique) were recorded with a Perkin±Elmer model 580 B
spectrophotometer equipped with a Perkin±Elmer model
3600 IR data station. The solution IR absorption spectra
were recorded using a Perkin±Elmer model 1720XFT±IR
Z-Bip-Bip-Bip-Bip-OH. Tetrapeptide Z-Bip-Bip-Bip-Bip-
OtBu (0.423 g, 0.37 mmol) was C-deprotected in TFA
(10 ml) and CH2Cl2 (10 ml) as reported above for Z-Bip-
Bip-OH to give 0.395 g (98%) of crude title tetrapeptide as a
solid, which was used in the next step without further puri-
®cation. Mp 2388C. Found: C, 76.59; H, 5.87; N, 4.95%;
C72H60N4O´72 H2O (1129.264) requires C, 76.57; H, 5.71; N,
4.96%.
spectrophotometer, nitrogen-¯ushed, equipped with
a
sample-shuttle device, at 2 cm21 nominal resolution,
averaging 100 scans. Solvent (baseline) spectra were
obtained under the same conditions. Cell with path lengths
of 0.1, 1.0 and 10 mm (with CaF2 windows) were used.
Spectrograde deuterochloroform (99.8% d) was purchased
from Fluka.
5(4H)-Oxazolone from Z-Bip-Bip-Bip-Bip-OH. Tetra-
peptide Z-Bip-Bip-Bip-Bip-OH (0.382 g, 0.35 mmol) was
treated as described above for the oxazolone from Z-Bip-
Bip-OH to give 0.376 g (quantitative yield) of crude title
oxazolone, which was used in the next step without further
Nuclear magnetic resonance
1
The H NMR spectra were recorded with a Bruker model
AM 400 spectrometer. Measurements were carried out in

F. Formaggio et al. / Tetrahedron 56 (2000) 8721±8734
8733
Weinstein, B., Ed.; Dekker: New York, 1983; Vol. 7, pp. 267±
357. (b) Hruby, V. J.; Al-Obeidi, F.; Kazmierski, W. Biochem.
J. 1990, 268, 249±262. (c) Rizo, J.; Gierasch, L. M. Annu. Rev.
Biochem. 1992, 61, 387±418. (d) Liskamp, R. M. J. Recl. Trav.
Chim. Pays-Bas 1994, 113, 1±9. (e) Gante, J. Angew. Chem. Int.
Ed. Engl. 1994, 33, 1699±1720. (f) Hanessian, S.; McNaughton-
Smith, G.; Lombart, H. G.; Lubell, W. D. Tetrahedron 1997, 53,
12789±12854. (g) Toniolo, C. Int. J. Pept. Protein Res. 1990, 35,
287±300. (h) Aubry, A.; Boussard, G.; Cung, M. T.; Marraud, M.;
Vitoux, B. J. Chim. Phys. 1988, 85, 345±359.
deuterochloroform (99.96% d; Aldrich) and deuterated
DMSO (99.96% d6; Acros Organics) with tetramethylsilane
as the internal standard.
X-Ray diffraction
The a-amino carboxylic ester H-Bip-OtBu crystallises in
the non-centrosymmetric space group An (No. 9) with
Ê
Ê
unit cell parameters aˆ10.435(1) A, bˆ17.256(2) A,
11
Ê
cˆ9.991(2) A, bˆ103.44(1)8, while the Ca complex of
the Na-protected a-amino acid Boc-Bip-OH crystallises as a
water/methanol solvate in the centrosymmetric space group
2. (a) Toniolo, C.; Benedetti, E. Macromolecules 1991, 24, 4004±
4009. (b) Toniolo, C.; Crisma, M.; Formaggio, F.; Valle, G.;
Ê
Ê
Ê
Â
Cavicchioni, G.; Precigoux, G.; Aubry, A.; Kamphuis, J.
Biopolymers 1993, 33, 1061±1072.
P-1 with aˆ6.352(2) A, bˆ11.465(2) A, cˆ15.778(4) A,
aˆ76.47(2)8, bˆ79.98(2)8, gˆ86.97(2)8. For these two
structures data were collected on a Enraf±Nonius CAD-4
diffractometer with graphite monochromated CuKa
3. (a) Valle, G.; Crisma, M.; Bonora, G. M.; Toniolo, C.; Lelj, F.;
Barone, V.; Fraternali, F.; Hardy, P. M.; Langran-Goldsmith, A.;
Maia, H. L. S. J. Chem. Soc., Perkin Trans. 2 1990, 1481±1487.
(b) Crisma, M.; Valle, G.; Bonora, G. M.; Toniolo, C.; Lelj, F.;
Barone, V.; Fraternali, F.; Hardy, P. M.; Maia, H. L. S.
Biopolymers 1991, 31, 637±641.
Ê
(lˆ1.54184 A) radiation. The structures were solved by
using the SHELXS 86 program21a and re®ned with the
SHELXL 93 program.21b The amino H-atoms of H-Bip-
OtBu were located on the difference Fourier map and
re®ned with ®xed isotropic thermal parameters. In the two
structures all H-atoms bound to carbons were placed in ideal
positions and re®ned with a rigid model and ®xed isotropic
displacement parameters (Ux 1.2). The high temperature
factors observed for the terminal tert-butyl groups in the
two structures indicated a low accuracy in the determination
of the position of their atoms. Final R-factors are R1ˆ0.0454
and wR2ˆ0.1234 for H-Bip-OtBu, while R1ˆ0.0565 and
wR2ˆ0.1596 for the Ca11 complex of Boc-Bip-OH.
4. (a) Toniolo, C. C.R.C. Crit. Rev. Biochem. 1980, 9, 1±44.
(b) Toniolo, C.; Benedetti, E. in Molecular Conformation and
Biological Interactions; Balaram, P., Ramaseshan, S., Eds.; Indian
Academy of Sciences: Bangalore, India, 1991; pp. 511±521.
5. (a) Valle, G.; Crisma, M.; Toniolo, C.; Sudhanand; Balaji Rao,
R.; Sukumar, M.; Balaram, P. Int. J. Pept. Protein Res. 1991, 38,
511±518. (b) Benedetti, E.; Di Blasio, B.; Iacovino, R.; Menchise,
V.; Saviano, M.; Pedone, C.; Bonora, G. M.; Ettorre, A.; Graci, L.;
Formaggio, F.; Crisma, M.; Valle, G.; Toniolo, C. J. Chem. Soc.,
Perkin Trans. 2 1997, 2023±2032. (c) Toniolo, C.; Crisma, M.;
Formaggio, F.; Benedetti, E.; Santini, A.; Iacovino, R.; Saviano,
M.; Di Blasio, B.; Pedone, C.; Kamphuis, J. Biopolymers 1996, 40,
519±522.
The tripeptide Z-Gly-Bip-Gly-OEt crystallises in the centro-
symmetric space group P21/a with unit cell parameters
Ê
Ê
Ê
aˆ9.924(2) A, bˆ28.190(3) A, cˆ10.515(2) A, bˆ
106.3(1)8. Data were collected on a Philips PW 1100
diffractometer with graphite monochromated MoKa
6. (a) Venkatachalam, C. M. Biopolymers 1968, 6, 1425±1436.
(b) Rose, G. D.; Gierasch, L. M.; Smith, J. P. Adv. Protein
Chem. 1985, 37, 1±109.
Ê
(lˆ0.71073 A) radiation. The structure was solved by
using the SHELXS 86 program21a and re®ned by full-matrix
least-squares procedures on F2, using all data, with the
SHELXL 97 program.21c The C-terminal ethyl moiety is
disordered. Its methyl group was isotropically re®ned on
two sites (CT2 and CT20) with occupancies 0.70 and 0.30,
respectively. All other non-H atoms were anisotropically
re®ned. H-atoms were calculated at idealised positions
and during the re®nement they were allowed to ride on
their carrying atom, with Uiso set equal to 1.2 (or 1.5 for
methyl groups) times the Ueq of the parent atom. The re®ne-
ment converged to R1ˆ0.0466 and wR2ˆ0.1530.
7. Toniolo, C.; Benedetti, E. Trends Biochem. Sci. 1991, 16, 350±
353.
8. (a) Mazaleyrat, J.-P.; Gaucher, A.; Wakselman, M.;
Tchertanov, L.; Guilhem, J. Tetrahedron Lett. 1996, 37, 2971±
Ï
2974. (b) Mazaleyrat, J.-P.; Gaucher, A.; Savrda, J.; Wakselman,
M. Tetrahedron: Asymmetry 1997, 8, 619±631.
9. (a) Mazaleyrat, J.-P.; Gaucher, A.; Wakselman, M.; Toniolo,
C.; Crisma, M.; Formaggio, F. In Peptides 1996; Ramage, R.,
Epton, R., Eds.; May¯ower Scienti®c: Kingswinford, UK, 1998;
pp. 623±624. (b) Formaggio, F.; Crisma, M.; Toniolo, C.;
Mazaleyrat, J.-P.; Wakselman, M. In Peptides 1998; Bajusz, S.,
Â
Â
Hudecz, F., Eds.; Akademiai Kiado: Budapest, 1999; pp. 352±353.
10. Ridvan, L.; Abdallah, N.; Holakovsky, R.; Tichy, M.; Zavada,
J. Tetrahedron: Asymmetry 1996, 7, 231±236.
Acknowledgements
È
11. (a) Konig, W.; Geiger, R. Chem. Ber. 1970, 103, 788±798.
F. F., M. C. and C. T. gratefully acknowledge MURST, the
Ministry of University and Scienti®c and Technological
Research, and the National Research Council (CNR) of
Italy for their continuous support to this research. We
thank the student Yolaine Goubard for her contribution to
the synthesis.
(b) Leplawy, M. T.; Jones, D. S.; Kenner, G. W.; Sheppard, R. C.
Tetrahedron 1960, 11, 39±51. (c) Jones, D. S.; Kenner, G. W.;
Preston, J.; Sheppard, R. C. J. Chem. Soc. 1965, 6227±6239.
12. (a) Mizushima, S.; Shimanouchi, T.; Tsuboi, M.; Souda, R.
J. Am. Chem. Soc. 1952, 74, 270±271. (b) Bonora, G. M.; Mapelli,
C.; Toniolo, C.; Wilkening, R. R.; Stevens, E. S. Int. J. Biol.
Macromol. 1984, 6, 179±188. (c) Kennedy, D. F.; Crisma, M.;
Toniolo, C.; Chapman, D. Biochemistry 1991, 30, 6541±6548.
(d) Crisma, M.; Formaggio, F.; Toniolo, C.; Yoshikawa, T.;
Wakamiya, T. J. Am. Chem. Soc. 1999, 121, 3272±3278.
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