Synthesis and conformational investigation of tetrapeptide analogues of the fragment B23-B26 of insulin

Article abstract of DOI:10.1016/j.tet.2004.07.014

Tetrapeptides containing one of a set of four different α,α-dialkyl glycines at the C-terminus were synthesized by conventional methods in solution and their conformational behavior investigated by ¹H NMR spectroscopy in connection with molecular mechanics calculations. The results were consistent with conformations stabilized by a γ-turn in the case of compounds with alkyl groups larger than methyl, while the corresponding Aib derivative did not exhibit intramolecular hydrogen bonding. Graphical Abstract

Full text of DOI:10.1016/j.tet.2004.07.014

Tetrahedron 60 (2004) 8929–8936
Synthesis and conformational investigation of tetrapeptide
analogues of the fragment B23-B26 of insulin
*
´ ´ ˆ
Lıgia M. Rodrigues, Jose I. Fonseca and Hernani L. S. Maia
´
Centro de Quımica, IBQF, University of Minho, 4710-057 Braga, Portugal
Received 13 May 2004; revised 2 July 2004; accepted 6 July 2004
Available online 18 August 2004
Abstract—Tetrapeptides containing one of a set of four different a,a-dialkyl glycines at the C-terminus were synthesized by conventional
methods in solution and their conformational behavior investigated by H NMR spectroscopy in connection with molecular mechanics
1
calculations. The results were consistent with conformations stabilized by a g-turn in the case of compounds with alkyl groups larger than
methyl, while the corresponding Aib derivative did not exhibit intramolecular hydrogen bonding.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
It is part of our program concerning this class of amino acids
to investigate the effect of incorporation of derivatives with
side chains larger than methyl into biologically active
compounds. Now, we present the synthesis and the results of
Peptides containing one or more residues of an a,a-dialkyl
glycine are conformationally constrained owing to restric-
tion of their conformational freedom imparted by the
a-alkyl substituents. This conformational rigidity is a
requirement to increase potency and selectivity, to improve
bioavailabitlity, and to enhance the resistance to pepti-
dases.1 In addition, the design of conformationally con-
strained sequences is one of the approaches for development
of bioactive peptides with high activity and selectivity
towards a specific receptor.2 Deltorfin and Leu-enkephalin
analogues containing a,a-dialkyl glycine residues are
examples of this strategy.3,4 Thus, a,a-dialkyl glycines are
interesting building blocks for modification of peptides as
probes to investigate biologically active conformations and
as models for conformational analysis of the peptide
backbone (formation of a- or 310-helices, or b-turns). It
has been observed by X-ray crystallography that a residue of
a-aminoisobutyric acid (Aib) stabilizes type II b-turn
conformations in peptides having 2–4 residues;5 it is also
known6–8 that when inserted in peptides Aib imparts a
310-helical conformation, and a,a-diethyl glycine and
a,a-dipropyl glycine convey fully planar C5 conformations.
Bulkier substituents (diethyl, di-n-propyl) favor both fully
extended structures and folded helical conformations in
crystal structures.9–11 However, it has been also reported
that the local sequence may influence the conformation
adopted at the disubstituted residue.12
1
an investigation by H NMR spectroscopy in connection
with molecular mechanics calculations of the conforma-
tional behavior of four tetrapeptide analogues of the
sequence B23-B26 of insulin having a,a-dialkyl glycine
residues at their C-terminus. A considerable number of
insulin analogues has been designed by substituting amino
acid residues within its B23-B30 sequence (GlyB23-Phe-
Phe-Tyr-Thr-Pro-Lys-ThrB30). These studies revealed that
B26-des-(B27-B30)-insulin-NH2 exhibits full potency, and
modification at its N-terminus, that is, at position B26, may
cause dramatic changes in its binding activity. For example,
replacement of B-26-tyrosine by D-Ala raised the receptor
affinity to 1250%. In addition, it has been proposed that binding
of insulin to its receptor needs a conformational change.13
Owing to steric hindrance related to the tetrasubstitution at
their a-carbon atom, a,a-dialkyl glycines are problematic
compounds with regard both to their synthesis and to insertion
into peptide chains. Nevertheless, we have been engaged in
developing an improved synthesis of these amino acids14 and
reports can be found in literature of incorporation of such
compounds into peptides by taking advantage of various
coupling reagents with different efficiencies.15–19
2. Results and discussion
Keywords: a,a-Dialkyl glycines; Peptide synthesis; Conformational
analysis; NMR.
2.1. Synthesis
* Corresponding author. Tel.: C351-253-604-371; fax: C351-253-678-
983; e-mail: hmaia@quimica.uminho.pt
N-tert-Butyloxycarbonyl-dimethyl and diethyl glycine, and
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.07.014

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L. M. Rodrigues et al. / Tetrahedron 60 (2004) 8929–8936
N-benzyloxycarbonyl-dipropyl and di-isobutyl glycine were
treated with DCC/HOBt followed by reaction with an
aqueous solution of ammonium hydroxide20 to give the
corresponding N-acylamino acid amides in yields of 78, 54,
63 and 75%, respectively. Quantitative cleavage of the N-
protecting groups and coupling to N-Boc-phenylalanine,
using the DCC ‘pre-mix’ procedure with formation of the
symmetrical anhydride in situ,17 gave the fully protected
dipeptides in yields of 83, 76, 83 and 50%, respectively. The
fully protected tetrapeptides Boc-Gly-Phe1-Phe2-X-NH2,
with XZa,a-dimethyl glycine (Aib), a,a-diethyl glycine
(Deg), a,a-dipropyl glycine (Dpg) and a,a-di-isobutyl
glycine (Dbg), were then obtained by a stepwise procedure
as shown in Scheme 1 in overall yields of 32, 22, 22 and
12%, respectively, from the N-protected amino acids. These
were prepared in yields varying within the range 43–68%,
with exception of diethyl glycine derivative, which was
commercially available.
means of the Karplus equation,21 JaH–NHZ6.7 cos2(fK
60)K1.3 cos(fK60)C1.5, the dihedral angles f were
calculated from the NH–CH coupling constants measured
1
in the H spectra (Fig. 1, Table 1).
Figure 1. Definitions of the dihedral angles f, j and u.
Plots of chemical shifts versus temperature were obtained
for all four compounds from spectra run at various
temperatures within the range 298–333 K. These were
linear and allowed to calculate the variation of the amide
proton chemical shifts with temperature, Dd/DT, which are
shown in Table 1. Chemical exchange of the NH2 amide
protons at the C-terminal was observed on heating.
Larger NH temperature gradients were observed for Phe2
(Dd/DTZK6.5 to 8.2 ppb KK1) as compared to those for
the other residues. As in DMSO-d6 solutions of peptides
Dd/DT values greater than K4 ppb KK1 are indicative of
NH participation in intermolecular hydrogen bonds with
solvent,22 we assume that the Phe2 amide group has an
external orientation. In contrast, the low temperature
coefficient, Dd/DT, observed for the a,a-dialkyl glycine
NH proton in compounds 2–4 is in agreement with its
participation in intramolecular hydrogen bonding; this
effect is more pronounced in the case of compounds 3 and
4 (Dd/DTZK1.8 ppb KK1), which have the largest side
chains at their C-terminal residue. Compound 1 behaves
differently; the NH proton of its Aib residue is sensitive
to temperature (Table 1, Dd/DTZK5.8 ppb KK1) and
may be solvent exposed, and its glycine amide proton
(Dd/DTZK3.8 ppb KK1) may be involved in a weak
intramolecular hydrogen bond. Based on the NMR data
presented above, we propose that peptides 2–4 assume a
conformation stabilized by an intramolecular hydrogen
bond involving the NH proton of the a,a-dialkyl glycine
residue and the carbonyl oxygen atom of Phe1 to give rise to
a seven-membered ring structure (Fig. 2). This does not
seem to apply to compound 1, for which our NMR data is
inconclusive.
Scheme 1. i—DCC, HOBT, NH4OH (method A); ii—TFA (method B);
iii—HBr/HOAc (method C); iv—DCC (method D); v—DCC, HOBt
(method E). BocZterc-butyloxycarbonyl; ZZbenzyloxycarbonyl. XZ
dimethyl (1); diethyl (2); dipropyl (3); di-isobutyl (4).
2.2. Conformational analysis
Diluted solutions (5 mM) of the fully protected tetrapeptides
1–4 (Scheme 1, Table 1) in DMSO-d6 were prepared for
conformational 1H NMR spectroscopy experiments. The
room temperature spectra exhibited the number of signals
that would fit either a single dominant conformation or
several conformations under fast exchange. Full proton
assignment was obtained by double resonance, HMQC and
HMBC techniques in addition to peak splitting patterns. By
Table 1. Temperature coefficients of NH protons, JaH–NH coupling and calculated dihedral angles (f) in DMSO-d6 for compounds 1–4
Compound
Residue
Gly Phe1
Phe2
X
Gly
Phe1
Phe2
KDd/DT (ppb/K)
JaH–NH (Hz)
f (8)
JaH–NH (Hz)
f (8)
JaH–NH (Hz)
f (8)
1
2
3
4
3.8
5.0
5.5
6.0
4.8
6.2
6.5
6.7
6.5
8.2
8.1
8.0
5.8
2.4
1.8
1.8
5.7
6.0
6.0
6.3
34; 87;K75; K166 8.1
83; 37; K77; K163 8.7
83; 37; K77; K163 8.4
42; 78; K79; K161 8.7
K146; K154
K101; K139
K97; K143
K101; K139
6.9
7.2
7.5
8.1
60; K84;K156
K86; K154
K89; K151
K146; K154
XZdimethyl (1); diethyl (2); dipropyl (3); di-isobutyl (4).

L. M. Rodrigues et al. / Tetrahedron 60 (2004) 8929–8936
8931
NiC1H was observed and only small NOEs between CiaCH
and NiC1H were detected. Compound 2 showed NOE
values of 0.8 and 1% for CaHPhe1–NHPhe2 and CaHPhe2–
NHDeg, respectively. In the spectra for compounds 3 and 4,
values of 0.6 and 3% were observed for CaHPhe2–NHDpg/
Dbg and CH2Gly–NHPhe1, respectively. The intra-residue
NOEs (CiaCH–NiH) were very weak or absent in the case of
compound 2. In the spectrum of compound 3, values of 5
and 0.8% were observed for NH–CH2Gly and NHPhe2–
CaHPhe2, respectively. Compound 4 showed values of 5
and 2% for NH–CH2Gly and NHPhe1–CaHPhe1, respec-
tively. In the case of compound 1 the following NOEs were
detected: NHGly–NHPhe1, 2%; CH2Gly–NHPhe1, 2%;
CaHPhe1–NHPhe2, 1%; CaHPhe2–NHAib, 3%; NH–
CH2Gly, 1%; NHPhe1–CaHPhe1, 4%; NHPhe2–CaHPhe2,
0.7%. The NOE data confirms that our compounds do not
adopt extended conformations. In fact, for extended
Figure 2. Representation of the conformation for compounds 2–4.
A preference for a g-turn has been reported for peptides
containing an a,a-dialkylglycine, namely 1-aminocyclo-
propane carboxylic acid;23 this has been related to the
polarity of solvents,22 namely DMSO as it is our case.
structures small NiH–NiC1H and large CiaCH–NiC1
H
NOEs are to be expected, with CiaCH–NiC1H NOEs larger
than those for CiaCH–NiH, which does not agree with the
results reported above.12
No information concerning the involvement of carbonyl
groups in hydrogen bonds could be obtained from the 13C
NMR data. The chemical shifts of the carbonyl groups of
glycine (ca. 169 ppm), Boc (ca. 156 ppm) and Phe1 (ca.
171.5 ppm) were almost invariant in all four compounds.
The corresponding chemical shifts for the a,a-dialkyl
glycine residues varied within the range 174.4–176.2 ppm
but do not show any coherent relationship with structure.
The downfield shift observed for the carbonyl carbon atom
of Phe2 in compound 1 (0.8–1.1 ppm) as compared to
compounds 2–4 suggests deshielding of this atom.
A systematic conformational search of each of the
tetrapeptides was performed using the Hyperchem 7.0
molecular modeling software and the AMBER force field by
varying the dihedral angles of the glycine and phenylalanine
residues; the set of minimum energy conformations
obtained was then re-optimized with the same force field.
The resulting conformations were examined and those
consistent with JaH–NH coupling constants and hydrogen
bond patterns derived from NMR data were selected as the
most probable conformations in solution (Fig. 3). In the case
of compounds 2–4, a hydrogen bond between NH proton of
the a,a-dialkyl glycine residue and the carbonyl oxygen
atom of Phe1 was found, the dihedral angles calculated
being close to the experimental values. The conformations
NOE difference spectra were run for the four tetrapeptides.
In the case of compounds 2–4, no NOE between NiH and
Figure 3. Conformations calculated for compounds 1–4. Double tipped arrows indicate the observed NOE values.

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L. M. Rodrigues et al. / Tetrahedron 60 (2004) 8929–8936
calculated for these three compounds are similar and show a
bend in agreement with the proposed structure that may be
due to repulsive interaction of the two aromatic rings with
each other. For compound 1 no intramolecular hydrogen
bonding was found, but a bent conformation was also
obtained. In this conformation, the values found for the
dihedral angles were close to the experimental ones; in
addition, the NOEs observed were consistent with the
calculated structure, as the distances between the interacting
atoms for which NOEs were observed varied from 2.1 to
dissolved in a mixture of DCM (40 mL) and DMF (10 mL);
HOBt (10.1 mmol) and DCC (10.05 mmol) were added and
the mixture was stirred at room temperature for 30 min. An
aqueous solution of 25% ammonium hydroxide (13 mmol)
was then added and the reaction mixture was stirred
overnight. The insoluble materials were filtered off and
the resulting solution was diluted with DCM (50 mL). The
organic layer was washed successively with 5% NaHCO3,
water, 5% citric acid and water. After drying over MgSO4
and filtration, the solvent was removed under reduced
pressure and the residue thus obtained was crystallized from
an appropriate solvent as indicated below.
˚
2.9 A.
Method B. General procedure for deprotection of N-tert-
butyloxycarbonylamino acid amides or peptide amides. The
N-tert-butyloxycarbonyl amino acid amide or peptide amide
(10 mmol) was treated with TFA (30 mL) at room
temperature. The mixture was set aside under protection
from moisture and stirred occasionally. After one to one and
a half hours the excess reagent was removed under reduced
pressure and the solid obtained was triturated with ether,
filtered, washed with ethyl ether, dried and recrystallised
from the appropriate solvent whenever indicated.
3. Conclusion
Thetetrapeptideanaloguesofthe fragmentB23-B26ofinsulin
having an a,a-dialkyl glycine residue at the C-terminus
could be obtained by DCC promoted stepwise synthesis.
The use of the DCC ‘pre-mix’ technique proved to be
efficient in the problematic coupling of Boc-Phe to the
a,a-dialkyl glycine amide.
Both NMR data and molecular mechanics calculations for
all compounds but one were consistent with a folded g-turn
type conformation stabilized by an intramolecular hydrogen
bond; this involves the NH proton of the a,a-dialkyl glycine
residue and the carbonyl oxygen atom of the first
phenylalanine residue. The exception was the peptide
having the smallest side chains at the C-terminal residue,
that is, the Aib derivative (1), which exhibited a different
folded conformation without intramolecular hydrogen
bonding. Our results indicate that substitution of methyl at
the a,a-dialkyl C-terminal residue by larger groups changes
the conformational preferences of these compounds. Little
conformational change was observed on branching at the
b-carbon.
Method C. General procedure for deprotection of N-benzyl-
oxycarbonylamino acid amides. The N-benzyloxycarbonyl
amino acid amide (10 mmol) was treated with 40% HBr in
acetic acid (20 mL) for 1 h at room temperature and under
occasional stirring. Ethyl ether was added to precipitate the
resulting hydrobromide, which was filtered, washed with
ether, dried over KOH in a desiccator and used without
further purification.
Method D. General procedure for peptide bond formation
involving a,a-dialkyl glycine amides. The Boc- or Z-amino
acid (10 mmol) was dissolved in dichloromethane (30 mL)
containing DMF (3 mL) and treated with DCC (5 mmol)
after cooling to 0 8C. The mixture was stirred for 30 min
and the a,a-dialkyl glycine amide salt as obtained from
N-deprotection (5 mmol) was added followed by triethyl-
amine (5 mmol). After leaving the reaction mixture over-
night under stirring at room temperature, the insoluble
materials were filtered off, the solvent was removed under
reduced pressure and the residue was dissolved in ethyl
acetate. The organic layer was washed successively with 5%
NaHCO3, water, 5% citric acid, water and brine. After
drying over MgSO4 and filtration, the solvent was removed
under reduced pressure and the resulting product crystal-
lized from the appropriate solvent.
4. Experimental
Melting points were determined on a Gallenkamp melting
point apparatus and are uncorrected. Thin layer chromato-
graphy was carried out on pre-coated plates (Merck
Kieselgel 60F254) and compounds were visualized by
UV-light and exposure to vaporized iodine and to ninhydrin
reagent. H and 13C NMR data were recorded on a Varian
1
Unity Plus 300 Spectrometer in DMSO-d6; d ppm were
measured vs TMS and J values are given in Hz. Double
resonance, HMQC and HMBC experiments were carried
out for complete assignment of proton and carbon signals in
the NMR spectra. Elemental analyses were carried out on a
Leco CHNS 932 instrument. Commercially available
compounds, including Boc-Deg-OH, and solvents were
used without previous purification; light petroleum refers to
the fraction boiling at 40–60 8C; the hydrobromides of
a,a-dipropyl and a,a-di-isobutyl glycine were synthesized
by a Ugi-Passerini reaction as published elsewhere14 and
Boc-Phe-OH was synthesized by a literature method.24
Method E. General procedure for peptide bond formation.
The required Boc-amino acid (10 mmol) was dissolved in
dichloromethane (40 mL) containing DMF (5 mL) and
treated with HOBt (10 mmol). After cooling to 0 8C, DCC
(10 mmol) was added followed by the N-deprotected
peptide amide (10 mmol) and triethylamine (10 mmol).
After leaving the reaction mixture stirring overnight at room
temperature, it was worked up as above.
4.1. General methods
4.1.1. Boc-Gly-Phe-Phe-Aib-NH2 (1). Boc-Aib. 2-Amino-
isobutyric acid (10 mmol, 1.03 g) was added to a mixture of
water (20 mL), dioxane (20 mL) and NaOH solution
(10 mmol, 10 mL 1 M) with vigorous stirring. The reaction
Method A. General procedure for the synthesis of a,a-dialkyl
glycine amides. The N-protected amino acid (10 mmol) was

L. M. Rodrigues et al. / Tetrahedron 60 (2004) 8929–8936
8933
mixture was cooled to 0 8C and treated with Boc2O
(0.011 mol, 2.40 g). Stirring was continued at room
temperature for three hours and the solution was concen-
trated under reduced pressure to remove the organic solvent.
The remaining aqueous layer was then cooled to 0 8C,
acidified to pH 2 with 1 M KHSO4 and extracted with ethyl
acetate. The organic layer was washed with water and dried
over MgSO4. The solvent was removed under reduced
pressure and the residue thus obtained was crystallized from
ethyl acetate/light petroleum. The white solid was filtered
and dried to yield 61% of the required product, mp 116–
117 8C (lit.25 mp 117–118 8C). 1H NMR (DMSO-d6) d
(ppm): 1.28 (s, 6H, 2!CH3), 1.35 (s, 9H, Boc), 7.05 (s, 1H,
NH), 12.20 (br s, 1H, CO2H). Anal. calcd for C9H17NO4: C:
53.20, H: 8.43, N: 6.89; found: C: 53.38, H: 8.36, N: 6.93.
was obtained, which was pure by tlc and used without
further purification. Yield 90%, mp 229–230 8C. H NMR
1
(DMSO-d6) d (ppm): 1.30 (s, 6H, 2!CH3), 2.90 (m, 2H,
bCH2 Phe), 3.10 (m, 2H, bCH2 Phe), 4.00 (m, 1H, aCH
Phe), 4.50 (m, 1H, aCH Phe), 6.80 and 6.90 (2s, 2H, NH2),
7.20 (m, 10H, Ar), 8.00 (br s, 3H, NHC3 ), 8.10 (s, 1H, NH
Aib), 8.80 (d, 1H, JZ8.2 Hz, NH Phe).
Boc-Gly-Phe-Phe-Aib-NH2. General Method E was used
to obtain the required product in a yield of 77%, mp
116–118 8C (ethyl acetate/light petroleum). 1H NMR
(DMSO-d6) d (ppm): 1.20 (s, 6H, 2!CH3), 1.30 (s, 9H,
Boc), 2.80 (m, 2H, bCH2 Phe), 3.00 (m, 2H, bCH2 Phe),
3.40–3.60 (dABq, 2H, JZ5.7, 16.0 Hz, CH2Gly, partially
under water signal), 4.30 (m, 1H, aCH Phe2), 4.50 (m, 1H,
aCH Phe1), 6.80–6.90 (m, 3H, NH GlyCNH2), 7.20 (m,
10H, Ar), 7.82 (s, 1H, NH Aib), 7.90 (d, 1H, JZ8.1 Hz, NH
Phe1), 8.37 (d, 1H, JZ6.9 Hz, NHPhe2). 13C NMR (DMSO-
d6) d (ppm): 176.2 (CO Aib), 171.3 (CO Phe1), 170.0 (CO
Phe2), 169.2 (CO Gly), 155.7 (CO Boc), 137.6 (2!C Ar),
136.43 (2!CH Ar), 129.2 (2!CH Ar), 128.1 (2!CH Ar),
128.0 (2!CH Ar), 126.3 (2!CH Ar), 78.0 (C Boc), 55.8 (C
Aib), 54.6 (aCH Phe2), 53.6 (aCH Phe1), 43.0 (CH2 Gly),
37.5 (bCH2 Phe), 36.9 (bCH2 Phe), 28.2 (3!CH3 Boc),
25.3 (CH3 Aib), 24.3 (CH3 Aib). Anal. calcd for
C29H39N5O6: C: 62.91, H: 7.10, N: 12.65; found: C 63.03,
7.09 H, 12.61.
Boc-Aib-NH2. General Method A was used to give the
product in a yield of 78%, mp 168–170 8C (ethyl
acetate/hexane). H NMR (DMSO-d6) d (ppm): 1.30 (6H,
1
s, 2!CH3), 1.40 (9H, s, Boc), 6.70 (1H, s, NH Aib), 6.80
and 7.00 (2H, 2s, NH2). Anal. calcd for C9H18N2O3: C:
53.45, H: 8.91, N: 13.85; found: C: 53.36, H: 8.62, N: 13.83.
H-Aib-NH2, TFA. Following Method B, one-hour after the
reaction had been started ethyl ether was added and the
mixture was cooled for 2 h; the precipitated solid was
filtered off, washed with ethyl ether and dried; no further
1
purification was required. Yield 96%, mp 206–207 8C. H
NMR (DMSO-d6) d (ppm): 1.40 (s, 6H, 2!CH3), 7.50 and
4.1.2. Boc-Gly-Phe-Phe-Deg-NH2 (2). Boc-Deg-NH2.
General Method A was used to obtain the required product
in a yield of 54%, mp 155–156 8C (ethyl acetate/light
petroleum). 1H NMR (DMSO-d6) d (ppm): 0.64 (t, 6H, JZ
7.2 Hz, 2!CH3), 1.36 (s, 9H, Boc), 1.64–2.07 (m, 4H, 2!
CH2), 6.09 (s, 1H, NH Deg), 7.29 and 7.45 (2s, 2H, NH2).
Anal. calcd for C11H22 N2O3: C: 57.37, H: 9.63, N: 12.16;
found: C: 57.51, H: 9.60, N: 12.11.
7.70 (2s, 2H, NH2), 8.10 (br s, 3H, NHC3 ).
Boc-Phe-Aib-NH2. General Method D was used to prepare
this compound as a crystalline material in a yield of 83%,
1
mp 182–182.2 8C (from ethyl acetate/light petroleum). H
NMR (DMSO-d6) d (ppm): 1.24 (s, 6H, 2!CH3), 1.28 (s,
9H, Boc), 2.70–2.90 (m, 2H, bCH2 Phe), 4.10 (m, 1H, aCH
Phe), 6.90 (br s, 2H, NH2), 7.10 (d, 1H, JZ8.2 Hz, NH Phe),
7.30 (m, 5H, Ar), 8.00 (s, 1H, NHAib). Anal. calcd for
C18H27N3O4: C: 61.87, H: 7.78, N: 12.02; found: C: 61.75,
H: 8.09, N: 11.68.
H-Deg-NH2, TFA. Following general Method B, 90 min
after the reaction had been started a solid compound was
obtained in a quantitative yield, which was pure by tlc and
1
used without further purification. H NMR (DMSO-d6) d
(ppm): 0.86 (t, 6H, JZ7.1 Hz, CH3), 1.60–1.90 (m, 4H, 2!
H-Phe-Aib-NH2, TFA. Following general Method B, 90 min
after the reaction had been started a solid compound was
obtained, which was used without further purification. Yield
CH2), 7.62 and 7.73 (2s, 2H, NH2), 7.95 (s, 3H, NHC3 ).
1
96%, mp 269–270 8C dec. H NMR (DMSO-d6) d (ppm):
Boc-Phe-Deg-NH2. General Method D was used to obtain
the required product in a yield of 76%, mp 218–219 8C
(ethyl acetate/light petroleum). 1H NMR (DMSO-d6) d
(ppm): 0.56 (t, 3H, JZ7.3 Hz, CH3), 0.64 (t, 3H, JZ7.3 Hz,
CH3), 1.29 (s, 9H, Boc), 1.63 (m, 2H, CH2), 2.34 (m, 2H,
CH2), 2.74–3.00 (m, 2H, bCH2 Phe), 3.90 (m, 1H, aCH
Phe), 7.26 (m, 5H, Ar), 7.37 and 7.48 (2s, 2H, NH2), 7.49 (d,
1H, JZ7.8 Hz, NH Phe), 7.68 (s, 1H, NH Deg). Anal. calcd
for C20H31N3O4: C: 63.63, H: 8.28, N: 11.13; found: C:
63.54, H: 8.40, N: 11.05.
1.24 (s, 6H, 2!CH3), 3.00 (m, 2H, bCH2 Phe), 4.00
(apparent t, 1H, JZ7.5 Hz, aCHPhe), 6.90 and 7.00 (2s, 2H,
NH2), 7.30 (m, 5H, Ar), 8.10 (br s, 3H, NHC3 ), 8.30 (s, 1H,
NH Aib). Anal. calcd for C15H20N3O4F3: C: 49.59, H: 5.55
H, N: 11.56; found: C: 49.56, H: 5.50, N: 11.56.
Boc-Phe-Phe-Aib-NH2. General Method E was used to obtain
the required product in a yield of 77%, mp 187–188 8C
(ethyl acetate/light petroleum). 1H NMR (DMSO-d6) d
(ppm): 1.20 (s, 6H, 2!CH3), 1.30 (s, 9H, Boc), 2.85 (m, 2H,
bCH2 Phe), 3.00 (m, 2H, bCH2 Phe), 4.10 (m, 1H, aCH
Phe), 4.40 (m, 1H, aCH Phe), 6.80–6.90 (m, 3H, NH2CNH
Phe), 7.20 (m, 10H, Ar), 7.90 (s, 1H, NH Aib), 8.10 (d, 1H,
JZ8.3 Hz, NH Phe). Anal. calcd for C27H36N4O5: C: 65.30,
H: 7.31 H, N: 11.28; found: C: 65.51, H: 7.33, N: 11.24.
H-Phe-Deg-NH2, TFA. Following general Method B,
90 min after the reaction had been started a solid compound
was obtained in a yield of 98%, which required no further
1
purification, mp 230–231 8C dec. H NMR (DMSO-d6) d
(ppm): 0.47 (t, 3H, JZ7.3 Hz, CH3), 0.61 (t, 3H, JZ7.3 Hz,
CH3), 1.63 (m, 2H, CH2), 2.20 (m, 2H, CH2), 2.80 (dd, 1H,
JZ7.3, 13.8 Hz, bCH2 Phe), 3.10 (dd, 1H, JZ7.3, 13.9 Hz,
bCH2 Phe), 4.36 (m, 1H, aCH Phe), 7.36 (s, 1H, NH2), 7.32
H-Phe-Phe-Aib-NH2, TFA. Following general Method B,
90 min after the reaction had been started a solid compound

8934
L. M. Rodrigues et al. / Tetrahedron 60 (2004) 8929–8936
1
(m, 6H, ArCNH2), 7.89 (s, 1H, NH Deg), 8.15 (br s, 3H,
NHC3 ). Anal. calcd for C17H24N3O4F3: C: 52.17, H: 6.14, N:
10.74; found: C: 51.58, H: 6.11, N: 10.62.
76–78 8C. H NMR (DMSO-d6) d (ppm): 0.80 (t, 6H, JZ
7.1 Hz, 2!CH3), 1.10 (m, 4H, 2!CH2), 1.65 (m, 4H, 2!
CH2), 5.00 (s, 2H, CH2Ar), 7.00 (s, 1H, NH Dpg), 7.30 (m,
5H, Ar), 12.60 (br s, 1H, CO2H). Anal. calcd for C16H23
NO4: C: 65.51, H: 7.90, N: 4.78; found: C: 65.24, H: 7.79,
N: 5.08.
Boc-Phe-Phe-Deg-NH2.General Method E was used to obtain
the required product in a yield of 83%, mp 113–115 8C
(ethyl acetate/light petroleum). 1H NMR (DMSO-d6) d
(ppm): 0.51 (t, 3H, JZ7.3 Hz, CH3), 0.63 (t, 3H, JZ7.3 Hz,
CH3), 1.25 (s, 9H, Boc), 1.65 (m, 2H, CH2), 2.27 (m, 2H,
CH2), 2.86 (m, 2H, bCH2 Phe), 3.07 (m, 2H, bCH2 Phe),
4.17 (m, 1H, aCH Phe), 4.40 (m, 1H, aCH Phe), 6.87 (d, 1H,
JZ8.5 Hz, NH Phe), 7.30 and 7.40 (2s, 2H, NH2), 7.27 (m,
10H, Ar), 7.60 (s, 1H, NH Deg), 8.60 (d, 1H, JZ7.6 Hz, NH
Phe). Anal. calcd for C29H40N4O5: C: 66.39, H: 7.68, N:
10.68; found: C: 66.00, H: 7.92, N: 10.37.
Z-Dpg-NH2. General Method A was used to give the product
in a yield of 62%, mp 155–156 8C (ethyl ether/light
petroleum). H NMR (DMSO-d6) d (ppm): 0.80 (t, 6H,
JZ7.2 Hz, 2!CH3), 1.10 (m, 4H, 2!CH2), 1.60 (m, 2H,
CH2), 2.00 (m, 2H, CH2), 5.00 (s, 2H, CH2Ar), 6.50 (s, 1H,
NH Dpg), 7.30 (m, 6H, ArCNH2), 7.45 (s, 1H, NH2). Anal.
calcd for C16H24N2O3: C: 65.73, H: 8.27, N: 9.58; found: C:
66.02, H: 8.49, N: 9.63.
1
H-Phe-Phe-Deg-NH2, TFA. Following general Method B,
90 min after the reaction had been started a solid compound
was obtained, which was pure by tlc and used without
H-Dpg-NH2, HBr. Following general Method C, a solid
compound was obtained, which was pure by tlc and used
without further purification. Yield 93%, mp 227–229 8C. 1H
NMR (DMSO-d6) d (ppm): 0.80 (t, 6H, JZ6.9 Hz, 2!
CH3), 1.10 (m, 2H, CH2), 1.40 (m, 2H, CH2), 1.70 (m, 4H,
CH2), 7.60 and 7.70 (2s, 2H, NH2), 7.90 (s, 3H, NHC3 ).
1
further purification. Yield 86%, mp 127–130 8C. H NMR
(DMSO-d6) d (ppm): 0.54 (t, 3H, JZ7.3 Hz, CH3), 0.65 (t,
3H, JZ7.3 Hz, CH3), 1.68 (m, 2H, CH2), 2.30 (m, 2H,
CH2), 2.80 (m, 2H, bCH2 Phe), 3.10 (m, 2H, bCH2 Phe),
3.95 (m, 1H, aCH Phe), 4.50 (m, 1H, aCH Phe), 7.30 (m,
10H, Ar), 7.40 and 7.50 (2s, 2H, NH2), 7.63 (s, 1H, NH
Deg), 8.10 (br s, 3H, NH3C), 9.13 (d, 1H, JZ7.6 Hz, NH
Phe).
Boc-Phe-Dpg-NH2. General Method D was used to obtain
the required product in a yield of 83%, mp 191–192 8C
(ethyl acetate/light petroleum). 1H NMR (DMSO-d6) d
(ppm): 0.80–0.90 (m, 8H, 2!CH3CCH2), 1.30 (sCm,
11H, BocCCH2), 1.55 (m, 2H, CH2), 2.30 (m, 2H, CH2),
2.70–2.90 (dd, 1H, JZ10.8, 13.2 Hz, bCH2 Phe), 3.00 (dd,
1H, JZ4.8, 13.7 Hz, bCH2 Phe), 3.90 (m, 1H, aCH Phe),
7.25 (m, 5H, Ar), 7.50 (dCs, 2H, NH PheCNH2), 7.40 (s,
1H, NH2), 7.65 (s, 1H, NH Dpg). Anal. calcd for
C22H35N3O4: C: 65.16, H: 8.70, N: 10.36; found: C:
65.15, H: 8.75, N: 10.24.
Boc-Gly-Phe-Phe-Deg-NH2. General Method E was used to
obtain the required product in a yield of 75%, mp 218–
1
219 8C (ethyl acetate/light petroleum). H NMR (DMSO-
d6) d (ppm): 0.55 (t, 3H, JZ7.3 Hz, CH3), 0.64 (t, 3H, JZ
7.3 Hz, CH3), 1.30 (s, 9H, Boc), 1.70 (m, 2H, CH2), 2.30 (m,
2H, CH2), 2.60–2.80 (m, 2H, bCH2 Phe), 3.10 (m, 2H, bCH2
Phe), 3.30–3.60 (dABq, 2H,, JZ5.7, 16.8 Hz, CH2 Gly,
partially under the water signal), 4.35 (m, 1H, aCH Phe2),
4.54 (m, 1H, aCH Phe1), 6.78 (t, 1H, JZ6.0 Hz, NH Gly),
7.22 (m, 10H, Ar), 7.37 and 7.44 (2s, 2H, NH2), 7.57 (s, 1H,
NH Deg), 7.92 (d, 1H, JZ8.7 Hz, NH Phe1), 8.75 (d, 1H,
JZ7.2 Hz, NH Phe2). 13C NMR (DMSO-d6) d (ppm): 174.4
(CO Deg), 171.6 (CO Phe1), 169.2 (CO Phe2), 168.8 (CO
Gly), 155.6 (CO Boc), 138.0 (C Ar), 137.9 (C Ar), 129.2
(2!CH Ar), 129.0 (2!CH Ar), 128.2 (2!CH Ar), 127.9
(2!CH Ar), 126.3 (CH Ar), 126.2 (CH Ar), 78.0 (C Boc),
64.0 (C Deg), 55.5 (aCH Phe2), 53.5 (aCH Phe1), 42.9 (CH2
Gly), 37.9 (bCH2 Phe), 37.0 (bCH2 Phe), 28.2 (3!CH3
Boc), 27.2 (CH2 Deg), 24.5 (CH2 Deg), 8.1 (CH3 Deg), 7.8
(CH3 Deg). Anal. calcd for C31H43N5O6: C: 64.00, H: 7.45,
N: 12.04; found: C: 63.84, H: 7.56, N: 11.86.
H-Phe-Dpg-NH2, TFA. Following general Method B, a solid
compound was obtained, which was pure by tlc and used
without further purification. Yield 88%, mp 232–233 8C
dec. 1H NMR (DMSO-d6) d (ppm): 0.60–0.90 (m, 8H, 2!
CH3CCH2), 1.20 (m, 2H, CH2), 1.60 (m, 2H, CH2), 2.00–
2.20 (m, 2H, CH2), 2.90 (dd, 1H, JZ7.3, 13.8 Hz, bCH2
Phe), 3.00 (dd, 1H, JZ7.3, 13.9 Hz, bCH2 Phe), 4.30 (t, 1H,
JZ7.3 Hz, aCH Phe), 7.20–7.45 (m, 7H, ArCNH2), 7.85
(s, 1H, NH Dpg), 8.15 (br s, 3H, NHC3 ). Anal. calcd for
C19H28N3O4F3: C: 54.41, H: 6.68, N: 10.02; found: C:
54.06, H: 6.42, N: 9.97.
Boc-Phe-Phe-Dpg-NH2. General Method E was used to
obtain the required product in a yield of 69%, mp 106–
1
108 8C (ethyl acetate/light petroleum). H NMR (DMSO-
d6) d (ppm): 0.75 (m, 6H, 2!CH3), 0.90 (m, 2H, CH2), 1.10
(m, 2H, CH2), 1.30 (s, 9H, Boc), 1.60 (m, 2H, CH2), 2.20
(m, 2H, CH2), 2.55–2.90 (m, 2H, bCH2 Phe), 2.90–3.10 (m,
2H, bCH2 Phe), 4.20 (m, 1H, aCH Phe), 4.40 (m, 1H, aCH
Phe), 6.85 (d, 1H, JZ8.2 Hz, NH Phe), 7.20 (m, 11H, ArC
NH2), 7.40 (s, 1H, NH2), 7.55 (s, 1H, NH Dpg), 8.50 (d, 1H,
JZ7.5 Hz, NH Phe). Anal. calcd for C31H44N4O5: C: 67.34,
H: 8.02, N: 10.14; found: C 67.08, H: 8.21, N: 9.86.
4.1.3. Boc-Gly-Phe-Phe-Dpg-NH2 (3). Z-Dpg-OH. The
amino acid hydrobromide (10.8 mmol, 2.12 g) was dis-
solved in 1 M NaOH (21.6 mL) and stirred with cooling in
an ice bath. A solution of benzyl chloroformate was added
dropwise with the pH kept at 11 by addition of 1 M NaOH
under vigorous stirring; the temperature was then raised to
40 8C and maintained as such for 4 h, while the pH was kept
at 11. The mixture was then extracted with four portions of
ethyl ether, the aqueous layer cooled and the pH adjusted to
1.5 by addition of 6 M HCl. The oil thus formed was
extracted into ethyl acetate and the solution dried over
MgSO4. The solvent was removed under reduced pressure
to give an oil that crystallized on standing. Yield 68%, mp
H-Phe-Phe-Dpg-NH2, TFA. Following general Method B,
90 min after the reaction had been started a solid compound
was obtained, which was pure by tlc and used without
1
further purification. Yield 89%, mp 126–128 8C. H NMR

L. M. Rodrigues et al. / Tetrahedron 60 (2004) 8929–8936
8935
(DMSO-d6) d (ppm): 0.75 (m, H, 2!CH3), 0.80–1.00 (m,
2H, CH2), 1.00–1.10 (m, 2H, CH2), 1.60 (m, 2H, CH2), 2.20
(m, 2H, CH2), 2.70–2.90 (m, 2H, bCH2 Phe), 3.10 (dd, 2H,
JZ5.6, 13.9 Hz, bCH2 Phe), 3.95 (m, 1H, aCH Phe), 4.50
(m, 1H, aCH Phe), 7.20–7.40 (m, 11H, ArCNH2), 7.45 (1s,
1H, NH2), 7.60 (s, 1H, NH Dpg), 8.00 (br s, 3H, NHC3 ), 9.10
(d, 1H, JZ7.9 Hz, NH Phe).
(m, 4H, 2!CH2), 2.30 (m, 2H, 2!CH), 2.70 (dd, 1H, JZ
3.4, 14.0 Hz, bCH2 Phe), 2.80–3.00 (m, 1H, bCH2 Phe),
3.90 (m, 1H, aCH Phe), 7.30 (m, 5H, Ar), 7.40 and 7.60 (2s,
2H, NH2), 7.50 (d, 1H, JZ8.1 Hz, NH Phe), 7.90 (s, 1H, NH
Dbg). Anal. calcd for C24H39N3O4: C: 66.48, H: 9.09, N:
9.69; found: C: 66.11, H: 9.02, N: 9.68.
H-Phe-Dbg-NH2, TFA. Following general Method B,
90 min after the reaction had been started a solid compound
was obtained, which was pure by tlc and used without
Boc-Gly-Phe-Phe-Dpg-NH2. General Method E was used to
obtain the required product in a yield of 86%, mp 120–
1
122 8C (ethyl acetate/light petroleum). H NMR (DMSO-
1
further purification. Yield 90%, mp 264 8C dec. H NMR
d6) d (ppm): 0.75 (m, 6H, 2!CH3), 0.80–1.00 (m, 2H,
CH2), 1.00–1.10 (m, 2H, CH2), 1.30 (s, 9H, Boc), 1.60 (m,
2H, CH2), 2.20 (m, 2H, CH2), 2.60– 2.90 (m, 2H, bCH2
Phe), 3.10 (dd, 2H, JZ5.7, 13.9 Hz, bCH2 Phe), 3.30–3.50
(dABq, 2H, JZ6.0, 16.2 Hz, CH2Gly, partially under water
signal), 4.30 (m, 1H, aCH Phe2), 4.50 (m, 1H, aCH Phe1),
6.80 (t, 1H, JZ6.0 Hz, NH Gly), 7.00–7.20 (m, 10H, Ar),
7.30 and 7.40 (2s, 2H, NH2), 7.50 (s, 1H, NH Dpg), 7.90 (d,
1H, JZ8.6 Hz, NH Phe1), 8.70 (d, 1H, JZ7.6 Hz, NH
Phe2). 13C NMR (DMSO-d6) d (ppm): 174.7 (CO Dpg),
171.5 (CO Phe1), 168.9 (CO Phe2), 168.8 (CO Gly), 155.6
(CO Boc), 137.9 (2!C Ar), 129.2 (2!CH Ar), 129.0 (2!
CH Ar), 128.2 (2!CH Ar), 127.9 (2!CH Ar), 126.3 (CH
Ar), 126.2 (CH Ar), 78.0 (C Boc), 62.9 (C Dpg), 55.4 (Ca
Phe1), 53.4 (Ca Phe2), 42.8 (CH2 Gly), 37.9 (bCH2 Phe),
37.0 (bCH2Phe), 36.9 (CH2), 36.8 (CH2), 28.2 (3!CH3
BocCCH2), 16.6 (CH2), 16.2 (CH2), 14.1 (2!CH3 Dpg).
Anal. calcd for C33H47N5O6: C: 65.00, H: 7.77, N: 11.49;
found: C: 65.11, H: 7.79, N: 11.21.
(DMSO-d6) d (ppm): 0.70–0.80 (m, 12H, 4!CH3), 1.20 (m,
1H, CH), 1.55 (m, 3H, CHCCH2), 2.24 (m, 2H, CH2), 2.85
(dd, 1H, JZ6.0, 13.9 Hz, bCH2 Phe), 3.10 (m, 1H, bCH2
Phe), 4.40 (apparent t, 1H, JZ6.0 Hz, aCH Phe), 7.30 (m,
6H, ArCNH2), 7.55 (s, 1H, NH2), 8.00 (br s, 3H, NHC3 ),
8.10 (s, 1H, NH Dbg). Anal. calcd for C21H32N3O4F3: C:
56.38, H: 7.16, N: 9.40; found: C: 56.55, H: 7.13, N: 9.50.
Boc-Phe-Phe-Dbg-NH2.General Method E was used to
obtain the required product in a yield of 67%, mp 93.2–
1
95.4 8C (ethyl acetate/light petroleum). H NMR (DMSO-
d6) d (ppm): 0.70–0.80 (m, 12H, 4!CH3), 1.25 (s, 9H,
Boc), 1.50 (m, 4H, 2!CHCCH2), 2.30 (m, 2H, CH2),
2.60–2.90 (m, 2H, bCH2 Phe), 3.00–3.20 (m, 2H, bCH2
Phe), 4.20 (m, 1H, aCH Phe1), 4.40 (m, 1H, aCH Phe), 6.85
(d, 1H, JZ8.8 Hz, NH Phe), 7.20 (m, 10H, Ar), 7.40 and
7.55 (2s, 2H, NH2), 7.80 (s, 1H, NH Dbg), 8.60 (d, 1H, JZ
8.2 Hz, NH Phe). Anal. calcd for C33H48N4O5: C: 68.24, H:
8.33, N: 9.65; found: C: 68.14, H: 8.25, N: 9.64.
H-Phe-Phe-Dbg-NH2, TFA. Following general Method B,
90 min after the reaction had been started a solid compound
was obtained, which was pure by tlc and used without
4.1.4. Boc-Gly-Phe-Phe-Dbg-NH2 (4). Z-Dbg-OH. Follow-
ing the procedure described for Z-Dpg-OH above, this
compound was synthesized in a 15 mmol scale. The reaction
was carried out at 50 8C for 4 h. After working up the
reaction mixture an oil was obtained, which crystallized on
standing. Yield 43%, mp 71–72 8C. 1H NMR (DMSO-d6) d
(ppm): 0.80 (2d, 12H, JZ4.9 Hz, 4!CH3), 1.50 (m, 2H,
2!CH), 1.60 (dd, 2H, JZ6.7, 13.8 Hz, CH2), 2.00 (dd, 2H,
JZ5.5, 13.8 Hz, CH2), 5.00 (s, 2H, CH2Ar), 6.50 (s, 1H,
NH), 7.30 (m, 5H, Ar). Anal. calcd for C18H27NO4: C:
67.26, H: 8.47, N: 4.36; found: C: 67.34, H: 8.76, N: 4.49.
1
further purification. Yield 53%, mp 119–121 8C. H NMR
(DMSO-d6) d (ppm): 0.70–0.80 (m, 12H, 4!CH3), 1.30 (m,
1H, CH), 1.50 (m, 3H, CHCCH2), 2.30 (m, 2H, CH2), 2.80
(m, 2H, bCH2 Phe), 3.15 (m, 2H, bCH2 Phe), 3.95 (m, 1H,
aCH Phe), 4.50 (m, 1H, aCH Phe), 7.30 (m, 10H, Ar), 7.45
and 7.60 (2s, 2H, NH2), 7.80 (s, 1H, NH Dbg), 8.00 (br s,
3H, NHC3 ), 9.20 (d, 1H, JZ8.2 Hz, NH Phe).
Boc-Gly-Phe-Phe-Dbg-NH2.General Method E was used to
obtain the final product, requiring no further purification, in
Z-Dbg-NH2. General Method A was used to give the product
in a yield of 75%, mp 129.5–130 8C (ethyl ether/light
petroleum). H NMR (DMSO-d6) d (ppm): 0.80 (2d, 12H,
1
a yield of 98%, mp 144–146 8C. H NMR (DMSO-d6) d
1
(ppm): 0.70–0.80 (m, 12H, 4!CH3), 1.30 (m, 10H, BocC
CH), 1.50 (m, 3H, CHCCH2), 2.30 (m, 2H, CH2), 2.60–
2.80 (m, 2H, bCH2 Phe), 3.10 (m, 2H, bCH2 Phe), 3.30–
3.50 (m, 2H, CH2 Gly), 4.40 (m, 1H, aCH Phe2), 4.50 (m,
1H, aCH Phe1), 6.78 (t, 1H, JZ6.3 Hz, NH Gly), 7.20 (m,
10H, Ar), 7.48 and 7.62 (2s, 2H, NH2), 7.85 (s, 1H, NH
Dbg), 7.99 (d, 1H, JZ8.7 Hz, NH Phe1), 8.86 (d, 1H, JZ
8.1 Hz, NH Phe2). 13C NMR (DMSO-d6) d (ppm): 175.6
(CO Dbg), 171.5 (CO Phe1), 168.9 (CO Phe2), 168.7 (CO
Gly), 155.6 (CO Boc), 138.1 (C Ar), 138.0 (C Ar), 129.2
(2!CH Ar), 128.9 (2!CH Ar), 128.2 (2!CH Ar), 127.9
(2!CH Ar), 126.2 (2!CH Ar), 77.9 (C Boc), 61.9 (C
Dbg), 55.3 (Ca Phe1), 53.4 (Ca Phe2), 43.9 (2CH2 Dbg)
42.8 (CH2 Gly), 38.1 (bCH2 Phe), 36.8 (bCH2 Phe), 28.2
(3!CH3 BocCCH), 24.1 (CH3 Dbg), 24.0 (CH3 Dbg), 23.9
(CH Dbg), 23.1 (CH3 Dbg), 22.7 (CH3 Dbg). Anal. calcd for
C35H51N5O6: C: 65.91, H: 8.06, N: 10.98; found: C: 65.86,
H: 8.29, N: 10.75.
JZ5.2 Hz, 4!CH3), 1.40 (m, 2H, 2!CH), 1.58 (dd, 2H,
JZ5.9, 14.0 Hz, CH2), 2.10 (dd, 2H, JZ6.1, 14.3 Hz, CH2),
5.00 (s, 2H, CH2Ar), 6.50 (s, 1H, NH), 7.30 (m, 5H, Ar),
7.42 and 7.60 (2s, 2H, NH2). Anal. calcd for C18H28N2O3:
C: 67.47, H: 8.81, N: 8.74; found: C: 67.46, H: 8.85, N: 8.95.
H-Dbg-NH2, HBr. Following general Method C, a solid
compound was obtained, which was pure by tlc and used
without further purification. Yield 98%, mp 205.7–206 8C.
1H NMR (DMSO-d6) d (ppm): 0.80 (apparent t, 12H, JZ
6.4 Hz, 4!CH3), 1.40–1.80 (m, 6H, 2!CHC2!CH2),
7.60 and 7.80 (2s, 2H, NH2), 7.9 (br s, 3H, NHC3 ).
Boc-Phe-Dbg-NH2.General Method D was used to obtain
the required product in a yield of 50%, mp 169.8–171 8C
(ethyl acetate/light petroleum). 1H NMR (DMSO-d6) d
(ppm): 0.70–0.80 (m, 12H, 4!CH3), 1.30 (s, 9H, Boc), 1.50

8936
L. M. Rodrigues et al. / Tetrahedron 60 (2004) 8929–8936
Pedone, C.; Benedetti, E.; Crisma, M.; Anzolin, M.; Toniolo,
Acknowledgements
C. J. Am. Chem. Soc. 1992, 114(16), 6273–6278.
10. Crisma, M.; Valle, J.; Toniolo, C.; Rao, R.; Prasad, S.;
Balaram, P. Biopolymers 1995, 35, 1–9.
´
We wish to acknowledge Dr. Sılvia M. A. Pereira-Lima for
the preparation of amino acids H-Dpg-OH and H-Dbg-OH,
Miss Elisa Pinto for running the NMR experiments and for
the elemental analyses, and Mr. Nuno Cerqueira for help
with molecular modeling. We also wish to acknowledge the
11. Benedetti, E.; Barone, V.; Bavoso, A.; Di Blasio, B.; Lelj, F.;
Pavone, V.; Pedone, C.; Bonora, G.; Toniolo, C.; Leplawy, M.;
Kaczmarek, K.; Redlinski, A. Biopolymers 1988, 27, 357–371.
12. Kaul, R.; Banumathi, S.; Velmuragan, D.; Ravikumar, K.;
Balaji, R.; Balaram, P. J. Peptide Res. 2000, 55, 271–278.
13. Brandenburg, D. Exp. Clin. Endocrinol. Diabetes 1999,
107(Suppl. 2), S6–S11.
ˆ
Fundac¸a˜o para a Ciencia e a Tecnologia for financial
support to the Instituto de Biotecnologia e Quımica Fina
´
(University of Minho).
14. Costa, S.; Maia, H.; Pereira-Lima, S. Org. Biomol. Chem.
2003, 1, 1475–1479.
15. Spencer, J.; Antonenko, V.; Delaet, N.; Goodman, M. Int.
J. Peptide Protein Res. 1992, 40, 282–293.
References and notes
´
16. Coste, J.; Frerot, E.; Jouin, P. J. Org. Chem. 1994, 59,
2437–2446.
1. Lombard, A.; Simone, G.; Galdiero, S.; Nastri, F.; Costanzo,
L.; Makihira, K.; Yamada, T.; Pavone, V. Biopolymers 2000,
53, 150–160 and references cited therein.
17. Gross, E.; Meinhofer, J.; The Peptides, Synthesis, Analysis,
Biology, Vol. 1; Academic: London, 1979 pp 256–58.
18. Akaji, K.; Kuriyama, N.; Kiso, Y. Tetrahedron Lett. 1994,
35(20), 3315–3318.
2. Lombardi, A.; De Simone, G.; Galdiero, S.; Lombard, A.;
Simone, G.; Galdiero, S.; Nastri, F.; Costanzo, L.; Makihira,
L.; Yamada, T.; Pavone, V. Biopolymers 2000, 53, 182–188
and references cited therein.
19. Fu, Y.; Hammer, R. Org. Lett. 2002, 4(2), 237–240 and
references cited therein.
3. Benedetti, E.; Isernia, C.; Nastri, F.; Pedone, C.; Saviano, M.;
Mierke, D.; Melchiorri, P.; Negri, L.; Potenza, R.; Severini, C.;
Erspamer, V. Eur. J. Org. Chem. 1998, 2279–2287.
4. Horikawa, M.; Shigeri, Y.; Yumoto, N.; Yoshikawa, S.;
Nakajima, T.; Ohfune, Y. Bioorg. Med. Chem. Lett. 1998, 8,
2027–2032 and references cited therein.
20. Chen, S.-T.; Wu, S.-H.; Wang, K.-T. Synth. Commun. 1989,
37–38.
21. Ludvigsen, S.; Andersen, K.; Poulsen, F. J. Mol. Biol. 1991,
731–736.
22. Stachel, S.; Hu, H.; Van, Q.; Shaka, A.; Van Vranken, D.
Bioorg. Med. Chem. 1998, 6, 1439–1446.
23. Paradisi, M.; Torrini, I.; Zecchini, G.; Lucente, G.; Gavuzzo,
E.; Mazza, F.; Pochetti, G. Tetrahedron 1995, 51, 2379–2386
and references cited therein.
´
5. Halab, L.; Belec, L.; Lubell, W. Tetrahedron 2001, 57,
6439–6446 and references cited therein.
6. Karle, I.; Balaram, P. Biochemistry 1990, 29, 6747–6756.
7. Benedetti, E.; Toniolo, C.; Hardy, P.; Barone, V.; Bavoso, A.;
Grimaldi, P.; Lelj, F.; Pavone, V.; Pedone, C.; Bonora, G.;
Lingham, I. J. Am. Chem. Soc. 1984, 106, 8146–8152.
8. Benedetti, E. Biopolymers 1996, 40, 3–44.
24. Bodansky, M.; Bodansky, A. The Practice of Peptide
Synthesis; Springer: Berlin, 1984 p 20.
25. Pettit, G.; Synthetic Peptides, Vol. 3; Academic: London,
1975.
9. Blasio, B.; Pavone, V.; Saviano, M.; Lombardi, A.; Nastri, F.;