3-Aza-8,10-dioxa-bicyclo[5.2.1]decane (9-exo BTKa) carboxylic acid as a new reverse turn inducer: Synthesis and conformational analysis of a model peptide

Article abstract of DOI:10.1016/j.tet.2005.11.008

Dipeptide isostere 5, belonging to the class of 9-exo BTKa, was synthesised starting from R,R-tartaric acid and 4-nitro-1-(3-nitrophenyl)butan-1-one. The nine-membered lactam showed interesting structural features and was inserted in a 5-residue model pep

Full text of DOI:10.1016/j.tet.2005.11.008

Tetrahedron 62 (2006) 1575–1582
3-Aza-8,10-dioxa-bicyclo[5.2.1]decane (9-exo BTKa) carboxylic
acid as a new reverse turn inducer: synthesis and conformational
analysis of a model peptide
*
Dina Scarpi, Daniela Stranges, Andrea Trabocchi and Antonio Guarna
`
Dipartimento di Chimica Organica ‘U. Schiff’, Universita di Firenze, Via della Lastruccia 13, 50019 Sesto Fiorentino, Italy
Received 27 July 2005; revised 13 October 2005; accepted 3 November 2005
Available online 28 November 2005
Abstract—Dipeptide isostere 5, belonging to the class of 9-exo BTKa, was synthesised starting from R,R-tartaric acid and 4-nitro-1-(3-
nitrophenyl)butan-1-one. The nine-membered lactam showed interesting structural features and was inserted in a 5-residue model peptide.
The conformational properties of this modified peptide have been studied by NMR and molecular modelling, indicating that compound 5
acted as a reverse turn inducer.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
bicyclic proline mimetics have been explored as reverse
turn inducers in model peptides.10
Reverse turns are structural motifs commonly found in
proteins and bioactive peptides that play a central role as
molecular recognition elements for many biological
processes, by virtue of presenting up to four side chains in
a well defined spatial arrangement.1 In particular, b-turns
consist of a tetrapeptide sequence (defined as i–iC1–iC2–
iC3) in a non-helical region in which the peptide chain
direction is reversed. These turns are often stabilised by an
intramolecular hydrogen bond between the carbonyl oxygen
of the first residue (i) and the amide proton of the fourth one
(iC3)2 thus being a key template for the design of the so-
called ‘turn-mimetics’ in the drug discovery area.3 During
the last decade many efforts have been dedicated to
synthesise new reverse turn inducers and to study the
conformational preferences of turn-analogues within pro-
tein secondary structure models.4 In recent years, we have
been developing a new class of aza-dioxa[3.2.1]bicyclic
compounds named BTAa5 or BTKa,6 the synthesis of which
is based on the combination of a tartaric acid derivative and
either a-amino aldehydes or a-amino ketones.7 We have
previously described the applications of these scaffolds as
dipeptide isosteres when inserted in both cyclic8 and linear9
peptide sequences, acting as mimetics of iC1–iC2 central
dipeptidic sequence of a typical b-turn motif. Moreover,
In a recent paper,11 we described the synthesis of two
classes of enantiopure molecular scaffolds, whose lactam
structure formally derives from the coupling between
tartaric acid and b- or g-ketoamines. By analogy with the
previously reported 7-exo BTKa,7c we named these
compounds as 8-exo and 9-exo BTKa, indicating the lactam
size (eight- and nine-membered ring, respectively). The
general structure is reported in Figure 1.
R
7-exo BTKa n = 0
8-exo BTKa n = 1
9-exo BTKa n = 2
( )n
N
O
O
H
CO2Me
1
O
Figure 1. General structure of BTKa.
The ring enlargement of the rigid 7-exo BTKa scaffolds
afforded, as expected, more flexible compounds that
represent a new class of dipeptide isosteres, prone to take
different conformations and potentially useful as turn
inducers. Preliminary molecular modelling calculations11
explained the experimental upfield shift of the carbo-
methoxy group observed in the 1H NMR when passing from
Keywords: Conformational analysis; Peptidomimetic; Turn inducer;
Tartaric acid.
*
Corresponding author. Tel.: C39 055 4573481; fax: C39 055 4573569;
e-mail: antonio.guarna@unifi.it
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.11.008

1576
D. Scarpi et al. / Tetrahedron 62 (2006) 1575–1582
the 7-exo BTKa to the 8- and 9-exo ones12 on the basis of the
change of the average distance between this group and
the aromatic ring on the bridgehead carbon, that decreases
2. Results and discussion
2.1. Synthesis
˚
from 3.8 to 3.0 A as the ring enlarges. This interesting
structural characteristic prompted us to further investigate
these molecules and their application as peptide turn
inducers. In particular, we concentrated our attention on
the 9-exo BTKa class (Fig. 1, nZ2), that, as a consequence
The synthesis of the target 9-exo BTKa (Scheme 1) started
from g-nitroketone 1, that was obtained from commercially
available 30-nitroacetophenone in 29% yield over three
steps. Compounds 2 and 3 were straightforwardly obtained
in quantitative and 62% yield, respectively, following the
previously reported experimental procedures.11 Reduction
of the nitro groups was first performed by the reported
method, that is, hydrogenation on Raney-Ni in methanol at
room temperature. By analogy to the synthesis of other
9-exo BTKa, a high yield of the reaction was not expected;
however, reduction took place but neither lactam 5 nor
diamine 4 were recovered. After a few more experiments,
lactam 5 was obtained by hydrogenation with ammonium
˚
of the above-mentioned shortest distance of 3.0 A between
two easily functionalisable groups, could force two peptidic
fragments to face each other, allowing the formation of a
hydrogen bond network between them. We envisaged the
ideal candidate for peptide modification in the compound
bearing a m-NH2 substituent on the aromatic ring. The 9-exo
BTKa was inserted in the model sequence Ac-VA-BTKa-
GLV-OMe and its effect on the peptide conformation is
reported in detail in the next section.
O
O
MeO OMe
NO2
NO2
b
a
(100%)
(29%)
2
1
NO2
(62%)
NO2
NO2
c
MeO2C
MeO2C
CO2Me
CO2Me
NH2
O
O
O
O
d
NH2
NO2
O
O
(40%)
N
H
CO2Me
O
4
5
NH2
3
NO2
(35%) e
O
O
NHFmoc
NHFmoc
N
H
N
H
f
(63%)
O
O
O
O
N
N
H
CO2H
H
CO2Me
O
O
6
7
SPPS
(15%)
O
H
N
N
H
O
O
N
H
O
H
N
O
H
O
O
N
N
H
N
OMe
O
H
O
O
8
Scheme 1. (a) (i) (HCHO)n, Me2NH$HCl, EtOH, HC, reflux, 2 h; (ii) NaOH aq; (iii) CH3NO2, TRITON B, reflux, 2 h; (b) HC(OMe)3, p-TsOH cat., MeOH,
reflux, 16 h; (c) BF3$Et2O, EtOAc, 0 8C, 4 h; (d) NH4HCO2, Pd/C 10%, MeOH, reflux, 16 h; (e) Fmoc-Ala-OH (1 equiv), PyBROP (1 equiv), DIEA (1 equiv),
CHCl3, rt, 24 h; (f) LiOH aq (1.0 equiv), 1,4-dioxane/H2O 1:1, 0 8C, 300.

D. Scarpi et al. / Tetrahedron 62 (2006) 1575–1582
Table 1. Temperature dependence of amide proton chemical shifts for 8
1577
NH
CDCl3
Dd/DT
CD3CN
Dd/DT
DMSO-d6
Dd/DT
d
d
d
Val-1
Ala
NH-30
Gly
Leu
7.27
6.92
9.54
7.10
7.12
7.95
K6.83
K1.48
K5.64
K2.90
K5.64
K5.64
6.82
7.08
8.81
6.99
6.69
7.08
K5.54
K6.07
K3.44
K2.27
K3.26
K4.00
7.95
8.25
9.99
7.78
8.01
8.19
K4.61
K6.38
K5.17
K4.45
K4.64
K7.21
Val-2
d are expressed in ppm and Dd/DT values in ppb/K.
formate over 10% Pd/C in refluxing methanol for 16 h (40%
yield after purification). Compound 5 was then coupled with
Fmoc-Ala-OH using PyBrop† as the activating agent and the
resultant ester 6 was hydrolysed by LiOH in 1,4-dioxane/
water at 0 8C to afford the Fmoc-amino acid 7.
deshielded (Table 1). A temperature dependent NMR
experiment indicated the presence of an equilibrium
between hydrogen bonded and non-hydrogen bonded states,
as many amide protons showed large Dd/DT values. The low
Dd/DT coefficient of Ala NH, compared with the other
amide protons, indicated the presence of a hydrogen bonded
environment for this amide proton in CDCl3 solutions. The
glycine amide coefficient also showed a relatively tempera-
ture stable chemical shift. In this case, the low coefficient
may be due to the anti O-8 orientation of carbonyl group at
C-9 of the scaffold rather than to the existence of a hydrogen
bond. The high chemical shift of Val-2 NH in conjunction
with the corresponding Dd/DT value suggested an equili-
brium of conformers in which this amide proton experi-
enced both non-hydrogen bonded states and hydrogen bonds
with different carbonyl groups.
Peptide Ac-Val-Ala-BTKa-Gly-Leu-Val-OMe (8) was pre-
pared by means of solid-phase techniques using Fmoc
protocol and a HMBA-AM polystyrene resin, that afforded
the title peptide with the C-terminus protected as methyl
ester by a nucleophilic cleavage. Fmoc-Ala-BTKa 7 was
incorporated into the growing peptide in the third coupling
step. All amide couplings were monitored with bromo-
phenol blue as internal colorimetric indicator. Nucleophilic
cleavage from the resin was achieved by trans-esterification,
heating a suspension of the resin at 50 8C overnight in a 9:1
MeOH/triethylamine mixture. The crude peptide was
purified by semi-preparative HPLC, giving pure 8 in 15%
yield.
2.3. NMR studies in CD3CN
Experiments carried out in CD3CN showed marked
differences, suggesting that a more competitive solvent
induces peptide 8 to become more organised. As a
consequence of the greater solvating effect of CD3CN,
though still remaining non-competitive relatively to
hydrogen bonding, the chemical shift values of the amide
protons experienced significant changes, except for glycine
and alanine, that appeared slightly upfield and downfield
relative to signals recorded in CDCl3, respectively. More-
over, the same trend of temperature coefficients as observed
for CDCl3 solutions was followed, with the glycine amide
proton showing the lowest Dd/DT. In contrast the alanine
NH signal increased significantly, confirming the poor
hydrogen bonding character of this proton in CD3CN, which
is itself a moderate hydrogen bond acceptor. The amide NH-
30 coefficient also suggested the presence of an equilibrium
between hydrogen bonded and non-hydrogen bonded
structures. NOE experiments suggested the existence of
b-strand organisation of the main chain, as all the amino
acids showed strong a,N(i, iC1) sequential peaks. More-
over, ROESY spectra of 8 showed some cross-peaks
between protons on non-adjacent residues. These NOE’s
were indicative of an equilibrium between more equivalent
conformations (Fig. 2). A strong cross-strand ROESY peak
between glycine NH and H-20 clearly demonstrated the
reverse turn inducing role of the scaffold. Moreover, other
cross-strand ROESY peaks between Ala NH and Leu a-H,
and between Val-2 NH and both Val-1 b- and g-H
confirmed the existence of a well-ordered reverse turn
structure (Fig. 2, left). Interestingly, the presence of cross-
strand ROESY peaks experienced by Ala a-H with Leu b-
and g-H indicated the existence of a second conformer
2.2. NMR studies in CDCl3
Conformational studies on peptide 8 were performed by
NMR, using firstly a relatively non-polar solvent (i.e.,
CDCl3), and successively more competitive solvents such as
CD3CN and DMSO-d6, to investigate the solvent effect on
the conformational preference of 8. Solutions (4.2 mM) of 8
were used to achieve sufficient dilution to prevent
aggregation. TOCSY and ROESY spectra were recorded
to assign proton resonances and investigate both sequential
and long-range NOE’s that provide evidences of preferred
conformations and give insight into stable reverse turn and
sheet conformations.13 Temperature dependence experi-
ments were carried out, since the amide proton chemical
shifts are sensitive to temperature and dilution variations,
thus giving further insight into the conformational pre-
ferences of peptides.14 The combination of chemical shift
and Dd/DT coefficient of the amide protons provides
information on the extent of hydrogen bonding.2
1H NMR analysis of compound 8 in CDCl3 solution was
complicated by the flexibility and size of the peptidomi-
metic, and the attempted structural determination of 8
turned out to be complex, as spectral data of sufficient
quality for structure determination were not obtained in
CDCl3. Moreover, signals in the ROESY spectrum
indicated slow to intermediate exchange rates between
several conformers. All amide values except NH-30 were
found between 7 and 8 ppm, with Val-2 NH being the most
† Bromotripyrrolidinophosphonium hexafluorophosphate.

1578
D. Scarpi et al. / Tetrahedron 62 (2006) 1575–1582
CD3CN and DMSO-d6 were used as solvents. ROESY
H
H
1
3
N
experiments in DMSO-d6 confirmed the b-strand organi-
sation of the reverse turn peptide, as suggested by strong
a,N(i, iC1) sequential peaks experienced by all the amino
acids. Moreover, cross-strand ROESY peaks between Val-2
NH and both Val-1 b- and g-H were also maintained in this
solvent, confirming the turn structure.
N
O
O
O
O
7
O
O
O
9
O
NH
O
3'
NH
NH
O
HN
O
O
HN
2.5. Molecular modelling
HN
HN
NH
O
O
Molecular modelling using AMBER* as a force field15 was
carried out to gain further insight into the conformational
preferences of peptide 8. Full unconstrained Monte Carlo
conformational search16 using CHCl3 as explicit solvent
resulted in all the conformers having a marked tendency of
adopting a reverse turn conformation, in which the scaffold
occupies the central turn position.
O
O
O
O
NH
NH
HN
NH
O
O
O
O
8
8
Conformer I
Conformer II
The distance d and the virtual torsion angle b were
computed to investigate the turn propensity The distance d
between the C-a of the first and fourth residue of a b-turn is
diagnostic of the presence of a reverse turn when its value is
Figure 2. Peptide 8: the arrows indicate significant cross-strand ROESY
correlations in CD3CN.
(Fig. 2, right), in which the N-terminal half was found to be
flipped, thus orienting Ala NH or Ala a-H inside the turn
structure, respectively. These NOE interactions demon-
strated that the scaffold 5 acts as a mimetic of the central
dipeptidic unit of a b-turn, thus generating an unusual
reverse turn. The existence of these two highly ordered
structures was in agreement with Dd/DT data, indicating the
presence of equilibrating weak hydrogen bonds. The
absence of stable hydrogen bonds characteristic of reverse
turn structures indicated that the principal driving force for
reverse turn formation is a consequence of the structure of
the scaffold. Further stabilisation is provided by intra-
molecular hydrogen bonding and hydrophobic interactions
that contribute to the overall organisation of the peptide into
b-strands conformations.
lower than 7 A,1a and the virtual torsion angle b is indicative
˚
of a reverse turn when it assumes a value within the range of
0G308.17 For peptide 8, the chain reversing property of this
unusual turn structure was assessed computing parameter d
as the distance between Gly C-a and C-30, and considering b
as the dihedral angle formed by C-30–C-5–C-9-Gly C-a. In
all the conformers, d and b values fell within the diagnostic
range for a turn structure, confirming the hypothesis of the
scaffold acting as a nucleator of tight reverse turns. All the
conformers produced by the Monte Carlo calculation
showed that the turn structure of 8 was stabilised by two
or more hydrogen bonds in a different fashion, producing
two main groups of conformers, in agreement with the NMR
data (Fig. 3). The first group of conformers included
structures A, C and D, all having in common the same
orientation of the two peptidic halves. The second group,
represented by structure B, showed the N-terminal main
chain flipped with respect to the former one. Specifically,
the first group of conformers encompassing the global
minimum conformer (EZK545.2 kJ/mol) were found in
8% abundance, and showed two hydrogen bonds, between
Val-2 NH and Ala CO, and between Ala NH and Val-2 CO,
thus giving a bent turn structure stabilised by a distorted
b-sheet structure (Fig. 3, structure A). An additional set of
structures belonging to this group were found in 36%
abundance (Fig. 3, structure C), and displayed a twisted turn
conformation stabilised by Ala NH and Gly CO, and Val-2
NH and Ala CO hydrogen bonds. A higher energy
conformer was present in 15% abundance (Fig. 3, structure
D), and showed a distorted b-strand structure of peptide
halves stabilised by three hydrogen bonds formed by Gly
NH and Ala CO, Ala NH and Gly CO, and between Val-2
NH and acetyl CO.
2.4. NMR studies in DMSO-d6
Experiments carried out in DMSO-d6 showed all the amide
protons deshielded with respect to CDCl3 solutions, as
expected. Nevertheless, Val-2 NH only showed a small
chemical shift variation, changing from 7.96 to 8.19. Thus,
the high chemical shift observed in CDCl3 solution, in
conjunction with low influence of solvent composition on
chemical shift, indicated the Val-2 amide bond to be
engaged in different hydrogen bonds, although its high
temperature coefficients in both solvents were indicative of
the presence of non-hydrogen bonded states, and of the
absence of a specific hydrogen bond of significant strength.
Gly NH showed a small temperature coefficient compared to
the other protons, confirming the hypothesis that the role of
the scaffold was to control the position of the adjacent
species. Moreover, Val-1 NH proved to lower its coefficient
on moving to a more competitive solvent, probably due to
the presence of more structured conformations in the more
highly solvating system. The existence of a large
temperature coefficient did not prevent the hypothesis of
multiple hydrogen bonds, and was in agreement with the
presence of two or more equilibrating structures (due in
particular to flipping of the N-terminal chain), which in turn
generated different patterns of hydrogen bonds. This
conformational equilibrium was particularly evident when
The second group of structures (18%) was represented by
the second conformer in order of increasing energy
(EZK542.5 kJ/mol), which showed a well-organised
sheet structure stabilised by three hydrogen bonds: NH-30
and Gly CO, Val-2 NH and Val-1 CO, Val-1 NH and Val-2
CO (Fig. 3, structure B). In this structure, NH-30 was found
to be engaged in a hydrogen bond with Gly CO, in agreement

D. Scarpi et al. / Tetrahedron 62 (2006) 1575–1582
1579
data. The population of hydrogen bonded structures was
found at a lower percentage with respect to Monte Carlo
calculation, confirming the high flexibility of peptide 8 and
agreeing with the presence of more structures, though d and
b values lowered only to about 60%, demonstrating the
strong reverse turn propensity of model peptide 8.
3. Conclusions
In this work, we realised the synthesis of a new dipeptide
isostere belonging to the class of 9-exo BTKa, starting from
a suitable aromatic g-nitroketone. The title compound (5)
was inserted in a linear peptide chain and the structural
effects on the conformation of the model were studied.
We verified that compound 5 generated a tight reverse turn
stabilised by its rigid structure and by the preferred anti
orientation of carbonyl group at C-9 position. Moreover, the
ring size allowed the aromatic ring to bend towards the
carbonyl at C-9, thus producing an unusual reverse turn
inducer. Conformational analysis by NMR in different
solvent systems showed the existence of equilibrating
structures, stabilised by different patterns of hydrogen
bonds or simply by hydrophobic interactions. Sequential
and cross-strand ROESY peaks revealed well-ordered turn
structures, especially in more competitive solvents such as
CD3CN and DMSO-d6. Moreover, when moving to a more
competitive solvent, although the labile hydrogen bonds
were disrupted, the b-sheet organisation of the reverse turn
peptide was further stabilised, as was particularly evident in
ROESY spectra in CD3CN. The absence of stable hydrogen
bonds, characteristic of reverse turn structures, indicated
that the principal driving force for reverse turn formation is
due to the particular structural form of the scaffold. Further
stabilisation is provided by weak intramolecular hydrogen
bonding and hydrophobic interactions that contribute to the
overall organisation of peptide into b-strand conformations.
Molecular modelling confirmed the existence of equilibrat-
ing structures, specifically due to a flip of N-terminal chain,
which allowed the arrangement of the amide proton NH-30
to the inside or outside of the turn. Finally, molecular
dynamic calculations confirmed the low stability of
hydrogen bond networks present in the low energy
conformers, in accordance with NMR data. Introduction
of chemical functionalities on amide at position 3 of the
scaffold and on the aromatic ring might allow side chains
isosteres to be appended at the iC1 and iC2 positions of the
turn.
Figure 3. Low energy conformers obtained by Monte Carlo calculation.
with the lower Dd/DT coefficient in CD3CN, confirming the
hypothesis that such a structure was particularly relevant in
this solvent (see also ROESY correlations as in Fig. 2, right).
All the low energy conformers displayed a b-sheet structure
with the scaffold acting as external reverse turn inducer. The
presence of different hydrogen bonds experienced by Ala and
Val-2 amide protons were in agreement with NMR data. In
particular, the low Dd/DT value found in CDCl3 for Ala NH
was compatible with its hydrogen bonding character in a
non-competitive solvent like chloroform. In the case of Val-2
NH, however, the high chemical shift and Dd/DT value of
Val-2 amide proton in CDCl3 and the slight chemical shift
variation from CDCl3 to DMSO-d6 suggested an equilibrium
between hydrogen bonded and non-hydrogen bonded states
in all the solvents considered.
4. Experimental
4.1. General
Molecular dynamic calculations over 1 ns were carried out
on all of the four structures found by Monte Carlo
calculations to gain information on the vicinity of local
minima of found structures, and to verify the flexibility of
the structures found in the conformational search. The
global minimum conformer showed poor stability under
MD calculations, as the hydrogen bond percentages dropped
to 4.5%, giving an open turn structure, in agreement with the
hypothesis of equilibrating structures, as suggested by NMR
Melting points are uncorrected. Chromatographic separa-
tions were performed under pressure on silica gel by flash-
column techniques; Rf values refer to TLC carried out on
25-mm silica gel plates (Merck F254), with the same eluent
as indicated for the column chromatography. IR spectra
were recorded with a Perkin-Elmer 881 spectrophotometer
in CDCl3 solution.

1580
D. Scarpi et al. / Tetrahedron 62 (2006) 1575–1582
Apart from peptide 8, H NMR (200 MHz) and 13C NMR
(50.33 MHz) spectra were recorded with a Varian XL 200
instrument in CDCl3 solution. NMR spectra of peptide 8
were performed on a Varian MercuryPlus 400 spectrometer
operating at 400 MHz for 1H. The spectra were obtained in
4.2 mM CDCl3 or CD3CN solution where aggregation was
not significant. One-dimensional 1H NMR spectra for
determining temperature coefficients were obtained at
298–328 K with increments of 5 K. Sample temperatures
were controlled with the variable-temperature unit of the
instrument. Complete proton resonance assignments were
made with the aid of gCOSY, TOCSY, HSQC and ROESY
experiments.
(d), 120.9 (d), 113.0 (s), 77.7 (d), 76.5 (d), 74.9 (t), 53.0 (q),
52.7 (q), 36.9 (t), 21.2 (t). MS m/z (%): 399 (MCC1, 1), 310
(MCK(CH2)3NO2, 100). Anal. Calcd for C16H18N2O10: C,
48.25; H, 4.55; N, 7.03. Found: C, 48.36; H, 4.52; N, 6.96.
1
4.1.3. (1R,7R,9R)7-(3-Aminophenyl)-2-oxo-8,10-dioxa-3-
azabicyclo[5.2.1]decane-9-carboxylic acid methyl ester
(5). Ammonium formate (1.58 g, 25.1 mmol) and Pd/C 10%
(150 mg) were added to a solution of 3 (500 mg, 1.26 mmol)
in MeOH (300 mL). The mixture was heated under reflux
for 16 h and, after cooling, filtered through a Celite layer
and finally evaporated to give crude 5, that was purified by
chromatography (eluent: EtOAc/petroleum ether 0.1%
Et3N, 3:1, RfZ0.17) affording pure 5 (154 mg, 40%) as a
white solid.
Mass spectra were carried out by EI at 70 eV, unless
otherwise stated, on 5790A-5970A Hewlett-Packard and
QMD 1000 Carlo Erba instruments. Microanalyses were
carried out with a Perkin-Elmer 2400/2 elemental analyser.
Optical rotations were determined with a JASCO DIP-370
instrument.
Compound 5. [a]2D5 K12.3 (c 1.0, CHCl3). 1H NMR d (ppm):
7.11 (t, JZ8.0 Hz, 1H), 6.87 (s, 1H), 6.82 (d, JZ2.2 Hz, 1H),
6.61 (dd, JZ8.0, 1.4 Hz, 1H), 6.52 (br s, 1H), 5.30 (d,
JZ1.8 Hz, 1H), 4.94 (d, JZ1.8 Hz, 1H), 4.15–4.05 (m, 1H),
3.46 (s, 3H), 3.40–3.20 (m, 1H), 2.21–2.13 (m, 1H), 2.07–1.84
(m, 3H). 13C NMR d (ppm): 174.0 (s), 169.4 (s), 146.0 (s),
142.5 (s), 128.9 (d), 115.5 (d), 115.0 (d), 114.9 (s), 112.0 (d),
79.5 (d), 78.7 (d), 52.4 (q), 42.1 (t), 36.2 (t), 25.6 (t). MS m/z
(%): 306 (MC, 30), 247 (MCKCO2CH3, 9), 137 (82), 120
(100). Anal. Calcd for C15H18N2O5$2H2O: C, 52.63; H, 6.48;
N, 8.18. Found: C, 52.87; H, 6.50; N, 8.29.
All the solid-phase reactions were carried out on a shaker,
using solvents of HPLC quality. HPLC purification was
performed with an HPLC system equipped with semi-
preparative C-18 10 mm, 250!10 mm, reverse-phase
column using H2O–CH3CN eluent buffered with 0.1%
TFA. Peptide 8 was characterised by ESI-MS, 2D-NMR and
HPLC system equipped with an analytical C-18 10 mm,
250!4.6 mm, reverse-phase column.
4.1.4. (1R,2S,7R,9R)7-{3-[2-(9H-Fluoren-9-ylmethoxy-
carbonylamino)propionylamino]phenyl}-2-oxo-8,10-
4-Nitro-1-(3-nitrophenyl)butan-1-one (1) was synthesised
dioxa-3-azabicyclo[5.2.1]decane-9-carboxylic
acid
as already reported.18
methyl ester (6). Fmoc-Ala-OH (312 mg, 1.08 mmol),
PyBrop (468 mg, 1.08 mmol) and DIEA (344 mL,
2.16 mmol) were added to a solution of 5 (300 mg,
1.08 mmol) in anhydrous CHCl3 (5 mL). The mixture was
left at room temperature, under stirring and nitrogen
atmosphere. After 24 h AcOEt (20 mL) was added and the
organic phase was washed with water (2!10 mL), satd
NaHCO3 (2!10 mL), and dried over Na2SO4. After
filtration and evaporation of the solvent, the obtained
crude 6 was purified by flash chromatography (eluent:
AcOEt/petroleum ether, 3:1; RfZ0.22), affording pure 6
(230 mg, 36%) as a yellowish solid.
4.1.1. 1-(1,1-Dimethoxy-4-nitrobutyl)-3-nitrobenzene
(2). Synthesised using the procedure previously reported
for closely related compounds,11 starting from 1 (1.56 g,
6.55 mmol) an refluxing for 16 h. After filtration and
evaporation of the solvent, crude 2 was obtained in
quantitative yield and used in the next step without further
purification.
1
Compound 2. Yellow oil. H NMR d (ppm): 8.36 (s, 1H),
8.20 (d, JZ8.0 Hz, 1H), 7.79 (d, JZ7.8 Hz, 1H), 7.57 (t,
JZ7.8 Hz, 1H), 4.25 (t, JZ6.6 Hz, 2H), 3.17 (s, 6H), 2.05–
1.97 (m, 2H), 1.74–1.57 (m, 2H). 13C NMR d (ppm): 148.4
(s), 142.5 (s), 132.9 (d), 129.4 (d), 123.2 (d), 122.2 (d),
102.1 (s), 74.8 (t), 48.9 (q, 2C), 33.6 (t), 21.5 (t). MS m/z
(%): 284 (MC, 1).
Compound 6. Mp 154–155 8C. [a]2D5 K44.3 (c 0.25,
CH3OH). H NMR d (ppm): 8.46 (s, 1H), 7.78–7.74 (m,
1
12H), 6.58 (br s, 1H), 5.47 (br s, 1H), 5.39 (s, 1H), 4.96 (s,
1H), 4.46–4.40 (m, 3H), 4.22 (t, JZ7.0 Hz, 1H), 4.19–4.00
(m, 1H), 3.39 (s, 3H), 3.20–3.09 (m, 1H), 2.17–2.06 (m,
1H), 1.95–1.83 (m, 3H), 1.48 (d, JZ7.0 Hz, 3H). 13C NMR
d (ppm): 173.9 (s), 170.7 (s), 169.2 (s), 156.4 (s), 143.6 (s,
2C), 142.5 (s), 141.3 (s, 2C), 137.5 (d), 128.7 (d), 127.8 (d,
2C), 127.1 (d, 2C), 125.0 (d, 2C), 121.2 (d), 120.0 (d, 2C),
119.9 (d), 116.8 (d), 114.5 (s), 79.6 (d), 78.8 (d), 67.3 (t),
52.3 (q), 51.2 (d), 47.0 (t), 42.1 (d), 36.9 (t), 29.7 (t), 25.7
(q). MS m/z (%): 599 (MC, 20). Anal. Calcd for
C33H33N3O8$2H2O: C, 62.35; H, 5.87; N, 6.61. Found: C,
62.62; H, 5.86; N, 6.69.
4.1.2.
(4R,5R)2-(3-Nitrophenyl)-2-(3-nitropropyl)-
[1,3]dioxolane-4,5-dicarboxylic acid dimethyl ester (3).
Synthesised using the procedure previously reported for
closely related compounds,11 starting from 2 (1.8 g,
6.33 mmol). After purification by chromatography (eluent:
EtOAc/petroleum ether, 1:4, RfZ0.25), pure 3 was obtained
as pale yellow oil (1.56 g, 62%).
Compound 3. [a]D25 C81.9 (c 1.0, CHCl3). 1H NMR d
(ppm): 8.36 (s, 1H), 8.20 (d, JZ8.0 Hz, 1H), 7.83 (d, JZ
8.0 Hz, 1H), 7.55 (t, JZ8.0 Hz, 1H), 4.81 (AB system,
JABZ5.6 Hz, 2H) 4.48 (t, JZ6.6 Hz, 2H), 3.86 (s, 3H), 3.58
(s, 3H), 2.24–2.04 (m, 4H). 13C NMR d (ppm): 168.6 (s),
168.5 (s), 148.1 (s), 142.6 (d), 132.0 (d), 129.4 (d), 123.8
4.1.5. (1R,2S,7R,9R)7-{3-[2-(9H-Fluoren-9-ylmethoxy-
carbonylamino)propionylamino]phenyl}-2-oxo-8,10-
dioxa-3-azabicyclo[5.2.1]decane-9-carboxylic acid (7). A
0.40 M solution of LiOH in H2O (0.25 mL) was added

D. Scarpi et al. / Tetrahedron 62 (2006) 1575–1582
1581
dropwise to a solution of 6 (62 mg, 0.10 mmol) in 1,4-
dioxane (0.5 mL) and water (0.5 mL) cooled at 0 8C. The
resulting mixture was stirred at 0 8C for 30 min and then 5%
KHSO4 was added reducing the pH to 5. After concentration
to a small volume, the product was extracted with CHCl3
(4!10 mL), adjusting the pH to 5 after every extraction.
The combined organic phases were dried over Na2SO4.
After filtration and evaporation of the solvent, pure 7
(38 mg, 63%) was obtained as a yellowish solid.
with Ac2O in DMF using catalytic 4-dimethylamino-
pyridine. All amide couplings were monitored with
bromophenol blue as an internal colorimetric indicator.19
Nucleophilic cleavage from the resin was achieved by trans-
esterification, heating at 50 8C overnight a suspension of the
resin in a 9:1 MeOH/triethylamine mixture. Crude peptide
was purified by semi-preparative HPLC using 10–90%
ACN/55 min as gradient, giving pure 8 as a white solid
(9.5 mg, 15%).
Compound 7. [a]D25 K48.8 (c 0.3, CHCl3). 1H NMR d
(ppm): 9.13 (s, 1H), 7.76–7.67 (m, 2H), 7.57–7.40 (m, 3H),
7.40–7.14 (m, 7H), 6.25 (m, 1H), 5.23 (s, 1H), 4.89 (s, 1H),
4.42 (m, 1H), 4.29–4.11 (m, 4H), 3.17–2.74 (m, 1H), 2.03–
1.45 (m, 4H), 1.37 (m, 3H). 13C NMR d (ppm): 175.1 (s),
172.0 (s), 171.7 (s), 156.4 (s), 143.8 (s), 143.5 (s, 2C), 141.2
(s, 2C), 137.7 (s), 128.7 (d), 127.7 (d, 2C), 127.0 (d, 2C),
125.1 (d, 2C), 121.2 (d), 120.1 (d), 119.9 (d, 2C), 117.0 (d),
114.5 (s), 79.2 (d), 78.7 (d), 67.0 (t), 51.3 (d), 46.9 (t), 41.4
(d), 35.7 (t), 29.6 (t), 24.8 (q). MS m/z (%): 586 (MC, 10).
Anal. Calcd for C32H31N3O8$2H2O: C, 61.83; H, 5.67; N,
6.76. Found: C, 61.98; H, 5.72; N, 6.58.
Compound 8. tRZ20.8 min (91% HPLC purity) using 0%
ACN/5 min, 0–10% ACN/5 min, then 10–90% ACN/
20 min as gradient. ESI-MS m/z (%): 788.27 (MCC1,
35), 810.46 (MCCNa, 100), 826.40 (MCCK, 45). 1H and
13C NMR data are shown in Table 2.
4.3. Computational methods
Molecular mechanics calculations were carried out on a SGI
IRIX 6.5 workstation, using MacroModel (v6.5) molecular
modelling software,20 with AMBER* as a force field15 and
the implicit chloroform GB/SA solvating system.21 Monte
Carlo conformational search16 was carried out without
imposing any constraint and including amide bonds among
all rotatable bonds. Two thousand structures were generated
4.2. Peptide synthesis
˚
Peptide 8 was prepared by means of solid-phase techniques
using a HMBA-AM polystyrene resin (100 mg, 0.08 mmol).
A five equivalent excess of Fmoc amino acids and DIPC/
HOBt carboxylic-activating mixture were used throughout
the synthesis, and DMF was used as solvent. Compound 7
was used in 2 equiv excess. Final acetylation was performed
and minimised until the gradient was less than 0.05 kJ/A/
mol using the TNCG gradient implemented in Macro-
Model.22 All the conformers having an energy of 6 kcal/mol
above the global minimum conformer were discarded.
Molecular dynamic (MD) hybrid simulation algorithm was
used to assess stability of low energy conformers. AMBER*
Table 2. Proton and carbon chemical shifts of peptide 8 (d values are expressed in ppm, and in parentheses are reported J values in Hz)
1H (DMSO-d6)
1H (CD3CN)
1H (CDCl3)
13C (CDCl3)
Ac
1.92
1.99
2.08
7.27
4.33
2.04
0.95
6.79
4.82
1.46 (d, 6.6)
5.31
6.20
4.20 and 3.19
2.13 and 1.88
2.46 and 2.13
5.01
7.62
9.49
7.26
22.9
—
58.9
31.4
19.4
—
49.8
19.3
80.0
—
40.9
25.3
22.2
79.8
118.5
—
Val-1 NH
Val-1H-a
Val-1H-b
Val-1H-g
Ala NH
7.95 (d, 8.2)
4.20 (t, 6.8)
2.00
0.90
8.25 (d, 7.0)
4.42
1.34 (d, 7.0)
4.99 (d, 2.7)
7.91
4.02, 3.09
1.60, 2.07
2.0, 2.07
4.88 (d, 2.8)
7.74
6.82 (d, 6.8)
3.99 (dd, 6.1; 6.1)
2.02
0.87 (d, 6.9)
7.08 (d, 6.2)
4.39 (dq, 7.1; 6.2)
1.31 (d, 7.1)
4.99 (d, 2.7)
6.37 (t, 7.1)
4.00 and 3.07
1.65 and 2.03
2.05
4.80 (d, 2.7)
7.71
8.81
7.19 (d, 8.0)
7.26 (t, 7.8)
7.73 (d, 8.0)
6.99 (t, 5.9)
3.54 (t, 5.9)
Ala a-H
Ala b-H
BTK H-1
BTK H-3
BTK H-4
BTK H-5
BTK H-6
BTK H-9
BTK H-20
BTK NH-30
BTK H-40
BTK H-50
BTK H-60
Gly NH
9.99
7.70 (d, 8.0)
7.33 (t, 7.8)
7.24 (d, 7.9)
7.78 (t, 5.6)
3.73 (dd, 16.6, 6.0)
3.53 (dd, 16.8, 4.9)
8.01 (d, 8.2)
4.43
1.36, 1.60
1.60
0.90
8.19 (d, 8.0)
4.14 (t, 7.8)
2.07
121.0
129.0
104.3
—
7.36 (dd, 8.0; 7.6)
8.22
7.10
Gly a-H
4.33 and 3.73
42.4
Leu NH
Leu a-H
Leu b-H
Leu g-H
Leu d-H
Val-2 NH
Val-2H-a
Val-2H-b
Val-2H-g
OMe
6.69 (d, 8.0)
4.35
1.45
7.10
4.84
1.50 and 1.35
1.58
0.89 (d, 5.9)
8.02
4.55 (dd, 8.7; 5.8)
2.10
—
52.1
42.1
24.7
22.8 and 19.1
—
57.4
31.7
18.9
1.65
0.79 (d, 7.1)
7.08 (d, 6.2)
4.19 (dd, 8.3; 6.2)
2.01
0.79 (d, 7.1)
3.58
0.91
3.64
0.89 (d, 5.9)
3.75
52.2
Experiments in CDCl3 solution have been carried out at 303 K, and at 298 K for DMSO-d6 and CD3CN solutions.

1582
D. Scarpi et al. / Tetrahedron 62 (2006) 1575–1582
6333–6338. (j) Baek, B.-H.; Lee, M.-R.; Kim, K.-Y.; Cho,
U.-I.; Boo, D.-W.; Shin, I. Org. Lett. 2003, 5, 971–974. (k)
Cristau, P.; Martin, M. T.; Tran Huu Dau, M.-E.; Vors, J.-P.;
Zhu, J. Org. Lett. 2004, 6, 3183–3186. (l) Grotenberg, G. M.;
Timmer, M. S. M.; Llamas-Saiz, A. L.; Verdoes, M.; van der
Marel, G. A.; van Raaij, M. J.; Overkleeft, H. S.; Overhand, M.
J. Am. Chem. Soc. 2004, 126, 3444–3446.
was used as force field, as implemented in Macromodel
(v6.5). A time step of 0.75 fs was used and the total
simulation was 2000 ps; samples were taken at 1 ps
intervals, yielding 2000 conformations for analysis.
Acknowledgements
5. Bicyclic compounds deriving from tartaric acid and amino
acid.
`
MIUR, Universita di Firenze, COFIN 2003-2005, COFIN
2004-2006 and FIRB project (code RBNE01RZH4_004)
contributed to the financial support of this work. Mr.
Maurizio Passaponti and Mrs. Brunella Innocenti are
acknowledged for their technical support. Ente Cassa di
Risparmio di Firenze is acknowledged for granting a
400 MHz NMR spectrometer.
6. Bicyclic compounds deriving from tartaric acid and
ketoamines.
7. (a) Guarna, A.; Guidi, A.; Machetti, F.; Menchi, G.; Occhiato,
E. G.; Scarpi, D.; Sisi, S.; Trabocchi, A. J. Org. Chem. 1999,
64, 7347–7364. (b) Guarna, A.; Cini, N.; Machetti, F.; Menchi,
G.; Occhiato, E. G. Eur. J. Org. Chem. 2002, 873–880. (c)
Guarna, A.; Bucelli, I.; Machetti, F.; Menchi, G.; Occhiato,
E. G.; Scarpi, D.; Trabocchi, A. Tetrahedron 2002, 58,
9865–9870. (d) Trabocchi, A.; Menchi, G.; Rolla, M.;
Machetti, F.; Bucelli, I.; Guarna, A. Tetrahedron 2003, 59,
5251–5258. (e) Trabocchi, A.; Cini, N.; Menchi, G.; Guarna,
A. Tetrahedron Lett. 2003, 44, 3489–3492.
References and notes
1. (a) Rose, G. D.; Gierasch, L. M.; Smith, J. A. Adv. Protein
Chem. 1985, 37, 1–109. (b) Stanfield, R. L.; Fieser, T. M.;
Lerner, R. A.; Wilson, I. A. Science 1990, 248, 712–719. (c)
Chou, P. Y.; Fasman, G. D. J. Mol. Biol. 1977, 115, 135–175.
(d) Wilmot, C. M.; Thorton, J. M. J. Mol. Biol. 1988, 203,
221–232. (e) Richardson, J. S. Adv. Protein Chem. 1981, 34,
167–339. (f) Mu¨ller, G. Angew. Chem., Int. Ed. 1996, 35,
2767–2769.
8. Scarpi, D.; Occhiato, E. G.; Trabocchi, A.; Leatherbarrow,
R. J.; Brauer, A. B. E.; Nievo, M.; Guarna, A. Bioorg. Med.
Chem. 2001, 9, 1625–1632.
9. Trabocchi, A.; Occhiato, E. G.; Potenza, D.; Guarna, A. J. Org.
Chem. 2002, 67, 7483–7492.
10. Trabocchi, A.; Potenza, D.; Guarna, A. Eur. J. Org. Chem.
2004, 4621–4627.
11. Scarpi, D.; Stranges, D.; Cecchi, L.; Guarna, A. Tetrahedron
2004, 60, 2583–2591.
12. 3.75, 3.70 and 3.37 ppm, respectively.
2. Belvisi, L.; Gennari, C.; Mielgo, A.; Potenza, D.; Scolastico, C.
Eur. J. Org. Chem. 1999, 389–400.
3. (a) Cho, N.; Harada, M.; Imaeda, T.; Imada, T.; Matsumoto,
H.; Hayase, Y.; Sasaki, S.; Furuya, S.; Suzuki, N.; Okubo, S.;
Ogi, K.; Endo, S.; Onda, H.; Fujino, M. J. Med. Chem. 1998,
41, 4190–4195. (b) Souers, A. J.; Virgilio, A. A.; Schu¨rer,
S. S.; Ellman, J. A.; Kogan, T. P.; West, H. E.; Ankener, W.;
Vanderslice, P. Bioorg. Med. Chem. Lett. 1998, 8, 2297–2302.
(c) Souers, A. J.; Virgilio, A. A.; Rosenquist, A.; Fenuik, W.;
Ellman, J. A. J. Am. Chem. Soc. 1999, 121, 1817–1825.
4. (a) Virgilio, A. A.; Ellman, J. A. J. Am. Chem. Soc. 1994, 116,
11580–11581. (b) Gante, J. Angew. Chem., Int. Ed. 1994, 33,
1699–1720. (c) Schneider, J. P.; Kelly, J. W. Chem. Rev. 1995,
95, 2169–2187. (d) Gardner, R. R.; Liang, G.-B.; Gellman,
S. H. J. Am. Chem. Soc. 1995, 117, 3280–3281. (e) Hanessian,
S.; McNaughton-Smith, G.; Lombart, H.-G.; Lubell, W. D.
13. Sieber, V.; Moe, G. R. Biochemistry 1996, 35, 181–188.
14. Haque, T. S.; Little, J. C.; Gellman, S. H. J. Am. Chem. Soc.
1996, 118, 6975–6985.
15. Weiner, S. J.; Kollman, P. A.; Nguyen, D. T.; Case, D. A.
J. Comput. Chem. 1986, 7, 230–252.
16. Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc. 1989,
111, 4379–4386.
17. Ball, J. B.; Hughes, R. A.; Alewood, P. F.; Andrews, P. R.
Tetrahedron 1993, 49, 3467–3478.
18. Scarpi, D.; Occhiato, E. G.; Guarna, A. Tetrahedron:
Asymmetry 2005, 16, 1479–1483.
´ ´ ´
19. Krchnak, V.; Vagner, J.; Safar, P.; Lebl, M. Collect. Czech.
Chem. Commun. 1998, 53, 2542–2548.
20. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still,
W. C. J. Comput. Chem. 1990, 11, 440–467.
¨
Tetrahedron 1997, 53, 12789–12854. (f) Krauthauser, S.;
Christianson, L. A.; Powell, D. R.; Gellman, S. H. J. Am.
Chem. Soc. 1997, 119, 11719–11720. (g) Feng, Y.;
Pattarawarapan, M.; Wang, Z.; Burgess, K. Org. Lett. 1999,
1, 121–124. (h) Soth, M. J.; Nowick, J. S. J. Org. Chem. 1999,
21. Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T.
J. Am. Chem. Soc. 1990, 112, 6127–6129.
´
64, 276–281. (i) Alonso, E.; Lopez-Ortiz, F.; Del Pozo, C.;
22. Ponder, J. W.; Richards, F. M. J. Comput. Chem. 1987, 8,
1016–1024.
´
Peralta, E.; Macıas, A.; Gonzalez, J. J. Org. Chem. 2001, 66,
´