Development of new calcium receptors based on oxazolidin-2-ones containing pseudopeptides

Article abstract of DOI:10.1039/b501017a

With the aim of designing a new calcium receptor, the synthesis and the conformational analysis of a small library of dipeptides having the general formula Ac-Oxx-L-Xaa-OBn [Oxx = L-Oxd, (4S,5R)-4-methyl-5-carboxyoxazolidin-2- one; D-Oxd, (4R,5S)-4-methyl

Full text of DOI:10.1039/b501017a

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A R T I C L E
Development of new calcium receptors based on oxazolidin-2-ones
containing pseudopeptides
Gianluigi Luppi,a Andrea Garelli,a Luca Prodi,a Quirinus B. Broxterman,b Bernard Kapteinb
and Claudia Tomasini*a
a Dipartimento di Chimica “G. Ciamician” - Alma Mater Studiorum, Universita` di Bologna,
Via Selmi 2, 40126, Bologna, Italy
b DSM Research, Life Science-Advanced Synthesis & Catalysis, PO Box 18, 6160 MD, Geleen,
The Netherlands
Received 21st January 2005, Accepted 3rd March 2005
First published as an Advance Article on the web 17th March 2005
With the aim of designing a new calcium receptor, the synthesis and the conformational analysis of a small library of
dipeptides having the general formula Ac–Oxx–L-Xaa–OBn [Oxx = L-Oxd, (4S,5R)-4-methyl-5-carboxyoxazolidin-2-
one; D-Oxd, (4R,5S)-4-methyl-5-carboxyoxazolidin-2-one; or D-Oxac (4R)-(2-oxo-1,3-oxazolidin-4-yl)-acetic acid] is
reported. Ac–L-Oxd–L-Ala–OBn was identified as the most promising compound by MS–ESI analysis and this
outcome was confirmed by photoluminescence spectroscopy.
protecting group is unimportant and the benzyl was chosen
only for convenience. In contrast, we noticed that the presence
of the small acetyl moiety favors the chelation: indeed, a less
Introduction
The presence of metal ions in a biological environment is of
particular interest for diagnosis and the activity of several
biologically active compounds. As an example, some calcium-
intense M + H2O peak was obtained when the acetyl moiety
was replaced with the pivaloyl group.
dependent antibiotics (CDA) have been recently isolated and
their activity has been tested: for instance, daptomycin is the first
antibiotic of this class to have received approval for clinical use.1
Thus the development of Ca2+ receptors is of general interest,
eventually also for the design of new sensors that are extremely
important in the study of biological matrixes.2
Results and discussion
In Scheme 1 the general method for the synthesis of the
fully protected dipeptides Ac-L-Oxd–Xaa–OBn 3a–e is reported,
starting from the previously described H–L-Oxd–OBn.6
We have recently reported the synthesis of a small li-
brary of tetrapeptides containing the L-Oxd [(4S,5R)-4-methyl-
5-carboxyoxazolidin-2-one], the D-Oxd [(4R,5S)-4-methyl-5-
carboxyoxazolidin-2-one] and the D-Oxac [(4R)-(2-oxo-1,3-
oxazolidin-4-yl)-acetic acid] moieties.3 The introduction of these
moieties into an oligopeptide chain preferentially induces rigid
conformations, so that some selected compounds are able to
form helixes or small b-turn secondary structures. Moreover,
using mass spectrometry we noticed that most pseudopeptides
containing the carboxyoxazolidin-2-one moieties are able to
chelate water in the gas-phase,4 probably owing to the presence
of an additional carbonyl in the oxazolidin-2-one ring. It has
been recently observed that the presence of cooperative effects
between more than one functional group is crucial for the
hydration of a polypeptide chain and it has been demons-
trated that the gas-phase proteins should be viewed as tools
to examine the interactions that are present in solution.5 From
the mass spectra analysis, the presence of a [M + H2O]+ ion was
demonstrated by the presence of a main or an abundant peak for
the molecules containing L-pGlu, L-Oxd or D-Oxd. As the same
result was obtained when the tests were repeated, avoiding the
contact with water, we gathered that the M + H2O peaks should
be ascribed to the presence of a water molecule strongly bonded
to the tetrapeptide between two or more functional groups.
As a consequence, we undertook a systematic analysis of some
small dipeptides containing an oxazolidin-2-one moiety and an
a-amino acid, having the general formula Ac–Oxx–L-Xaa–OBn
3–5 (where Oxx is L-Oxd, D-Oxd or D-Oxac). In order to analyze
the behavior of these molecules both common a-amino acids,
such as L-alanine (L-Ala) or L-valine (L-Val), and unusual a-
amino acids, such as aminoisobutyric acid (Aib), L-isovaline
(L-Iva), L-a-methyl–valine [L-(aMe)Val] or L-a-methyl–norvaline
[L-(aMe)Nva], have been used. Moreover, we chose as protecting
groups the acetyl for the NH moiety and the benzyl for the acid
moiety. Preliminary results showed that the nature of the acid
Scheme 1 Reagents and conditions: (i) AcCl (1 eq.), DIEA (3 eq.),
DMAP (0.5 eq.), dry DMF, rt, 16 h; (ii) Pd/C (5%), MeOH, rt, 2 h;
(iii) HBTU (1 eq.), H–L-Ala–OBn·HCl (1 eq.), TEA (3 eq.), acetonitrile,
rt, 40 min.
A similar approach was used to synthesize Ac–D-Oxd–L-Ala–
OBn 4 and Ac–D-Oxac–L-Val–OBn 5 (Fig. 1).
Fig. 1 Dipeptides containing the D-Oxd or the D-Oxac moiety.
1 5 2 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 0 – 1 5 2 4
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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All these molecules have been analyzed by 1H NMR, 13C
NMR and IR spectroscopy, and by MS–ESI spectrometry in
the absence of calcium ions. The IR spectroscopy furnished
information on the formation of NH · · · OC hydrogen bonds.
The IR absorption spectra were obtained using 3 mM solutions
in methylene chloride, as at this concentration the intramolec-
ular aggregation is usually unimportant. The absorption bands
between 3500 and 3250 cm−1 of compounds 3a–e, 4 and 5 are
reported in Fig. 2 in order to check the presence of non-hydrogen
bonded amide protons bands (above 3400 cm−1) or hydrogen
bonded amide proton bands (below 3400 cm−1).7
compounds 3a and 3c was consistent with the IR outcome but
did not furnish any additional information. For these molecules
we can foresee the conformations reported in Fig. 3.
Fig. 3 Preferential conformation of 3a and of 3c suggested by IR and
1H NMR data. Significant NOE enhancements of 3a and 3c obtained by
performing the NOESY-1D experiments on 10 mM solutions in CDCl3
(600 MHz) and using a mixing time of 1 s.
The preferential conformations of 3a and 3c have been further
detected by investigation of the DMSO-d6 dependence of NH
proton chemical shift.8 This solvent has a strong hydrogen
bonding acceptor character and, if it binds to a free NH
proton, it will be expected to dramatically move its chemical
shift downfield. The evaluation of inaccessible NH groups by
1H NMR was performed by adding increasing amounts of
DMSO-d6 to 1 mM tetramer solutions in CDCl3. The results
for Ac–L-Oxd–L-Ala–OBn 3a and Ac–L-Oxd–L-Iva–OBn 3c are
reported in Fig. 4 and show that the NH of 3a is very sensitive to
DMSO (Dd = 1.80 ppm), while the NH of 3c undergoes a lower
variation of chemical shift (Dd = 1.06 ppm), thus suggesting that
this proton is at least partly hydrogen bonded: this result is in
agreement with the IR outcome.
Fig.
4 Variation of NH proton chemical shift (ppm) of
Ac–L-Oxd–L-Ala–OBn 3a and Ac–L-Oxd–L-Iva–OBn 3c as a function
of increasing percentages of DMSO-d6 to the CDCl3 solution (v/v)
(concentration: 1 mM).
As far as calcium complexation is concerned, we expected that
a chelation in solution would be better obtained with molecules
like 3a, where the Xaa carbonyls are already displaced in the
right position. Indeed, the chelation “skill” of 3a–e, 4 and 5
has been evaluated by analysis of solutions containing protected
dipeptides and calcium ions with a mass spectrometer equipped
Fig. 2 IR spectra and significant NH stretching bands for compounds
3a–e, 4 and 5.
Significantly, we found many different results which account
for different conformations, although the tested molecules are
very similar. Indeed, the L-Ala and L-Val containing dipeptides
(3a, 4 and 5) show no aptitude to form NH · · · OC hydrogen
bonds, while 3b, 3c, 3d and 3e, which contain a,a-disubstituted
amino acids, tend to form NH · · · OC hydrogen bonds more
easily.
with an electrospray detector (MS–ESI). We prepared 10−3
M
solutions of 3a–e, 4 and 5 each in acetonitrile in the presence of
5eq. ofcalcium tetrafluoborate andanalyzed them with MS–ESI
apparatus. Table 1 shows the results obtained from the MS–ESI
analysis of 10−3 M solution of dipeptides 3a–e, 4 and 5 (left) and
10−3 M solution of dipeptides 3a–e, 4 and 5 with 5 eq. of calcium
salt (right).
1
In contrast, the H NMR spectra of 3a–e, 4 and 5 did not
furnish clear-cut results as they show a very deshielded signal
for the methyl group of the acetyl moiety, which always resonates
at about 2.5 ppm (compared with 2.02 ppm of N-methyl
acetamide). As we already observed,3 this outcome should be
attributed to the presence of the heterocycle carbonyl of the
oxazolidin-2-one ring, which strongly deshields the hydrogen
nearby, thus confirming that the imide bond always assumes
a trans-conformation even in the presence of a very small side
chain, like the acetyl group.
For this reason we choose to have a deeper insight into the
conformation of Ac–L-Oxd–L-Ala–OBn 3a (mostly non chelated
NH) and of Ac–L-Oxd–L-Iva–OBn 3c (mostly chelated NH). An
evaluation of the NOESY-1D spectra (CDCl3, 600 MHz) of the
As Table 1 shows, all the spectra on the left side (pure
compounds) contain peaks corresponding to M, M + 1, M +
Na (M + 23) and M + H2O (M + 18) in different proportions.
The spectra on the right hand side (dipeptide + calcium ions)
show the additional presence of a M + 20 peak, which can be
attributed to an ion having the formula (2M + Ca)2+, suggesting
that two molecules of dipeptide can chelate only one calcium ion,
although the calcium is in a large excess in the solution. Ions with
the formula (M + Ca), producing signals of m/z (M + 40)/2,
are totally absent.
The same results have been obtained utilizing 10−3 M solutions
of 3a–e, 4 and 5 in a 1 : 1 mixture of H2O and acetonitrile. The
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 0 – 1 5 2 4
1 5 2 1

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Table 1 Fragmentation peaks for the MS–ESI analysis of 10−3
M
solutions of dipeptides 3a–e,4 and 5 and of mixtures of 3a–e, 4, 5 and
calcium fluoborate. Only the area of the molecular weight is shown
Fig. 5 Hypothetical structure of the complex 3a + Ca2+, which is in
agreement with the IR, 1H NMR and MS–ESI outcome.
A similar result was obtained with Ac–D-Oxd–L-Ala–OBn 4
and in this case also a M + 20 peak was observed, although
less intense. This outcome shows that the L-Ala moiety is
very important for the receptor design, possibly because more
hindered side chains do not favour the useful conformation for
the formation of the Ca2+ complex.
To prove this hypothesis, a mixture of 3a and calcium
fluoborate in CD3CN [10−3 M solution of 3a, with increasing
amounts of Ca(BF4)2] was performed by 1H NMR and IR
spectroscopy, but no significant variations have been noticed in
both sets of spectra. This outcome is not surprising, bearing
in mind the results reported on the NMR analysis of the
calcium binding effects of lipopetide antibiotic daptomycin.11
The authors show that no variation of the chemical shifts in
1
the H NMR spectra between daptomycin and a mixture of
daptomycin and Ca2+ has been obtained, but only a broadening
of the resonance lines was observed. So, calcium binding induces
aggregation of daptomycin but does not noticeably alter the
conformation of the peptide.
Interestingly, the complexation process of 3a with Ca2+ can
be also monitored by photoluminescence spectroscopy. Fig. 6
shows that a new large and unstructured bond appears in
the emission spectrum of Ac–L-Oxd–L-Ala–OBn 3a between
400 and 600 nm when 0.5 eq. of Ca2+ was introduced in the
10−3 M solution of 3a in acetonitrile. On the other hand, further
additions of Ca2+ did not induce noticeable changes in the
emission spectrum. This result corroborates the hypothesis of
formation of a complex where the calcium ion is hexacoordinate
with two molecules of 3a. Further studies aiming to insert a
suitable flourophore for Ca2+ detection in vivo are in progress in
our laboratory.12
peak at (M + 18) highlights the presence of water, which does
not prevent some selected molecules from forming (2M + Ca)/2
ions.
The most intense peak at (2M + Ca)/2 was obtained in
the MS–ESI spectrum of 3a + Ca2+. This outcome is in
agreement with what we foresaw from the study of the IR
and 1H NMR spectra. Fig. 5 shows a possible chelation model
(an hexacoordinate complex) which is in agreement with the
experimental data. Indeed, this is perhaps the most common
coordination number and the six ligands almost invariably lie at
the vertices of an octahedron or a distorted octahedron.9
Moreover, this hypothesis is in agreement with the reported
cyclo-(Pro–Gly)3, that forms a 2 : 1 complex with Ca2+ in which
the cation is sandwiched between the two peptide molecules.10
The Gly carbonyls from each of the peptides are octahedrally
coordinated to the cation with an average calcium oxygen
Fig. 6 Flourescence emission of (a) 1 mM Ac–L-Oxd–L-Ala–OBn 3a in
acetonitrile; (b) 1 mM Ac–L-Oxd–L-Ala–OBn 3a + 0.5 eq. of Ca(BF4)2
in acetonitrile; (c) 1 mM Ac–L-Oxd–L-Ala–OBn 3a + 1 eq. of Ca(BF4)2 in
acetonitrile; (d) 1 mM Ac–L-Oxd–L-Ala–OBn 3a + 2.5 eq. of Ca(BF4)2
in acetonitrile. The excitation was at 285 nm.
Conclusions
In conclusion, among a small library of dipeptides of the
general formula Ac–Oxx–L-Xaa–OBn, Ac–L-Oxd–L-Ala OBn
3a and Ac–D-Oxd–L-Ala–OBn 4 have the aptitude to chelate
˚
coordination distance of 2.26 A.
1 5 2 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 0 – 1 5 2 4

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calcium ions in acetonitrile solutions; probably as the presence
of the carboxyoxazolidin-2-one moiety facilitates its chelation
propensity due to the tendency of the imidic bond to always
adopt the trans-conformation, which in turn forces the carbonyl
group in the right direction. Indeed, as shown by 1H NMR study
on daptomicin,11 the dipeptide conformation before calcium
addition is very important.
Further studies on the preparation of oxazolidin-2-ones
containing pseudopeptides and their use in the chelation of
bivalent metal ions are currently ongoing. Above all, we will
focus our attention on the development of pseudopeptides that
are selective for similarly sized cations.
(20 mL) was added 10% palladium on charcoal (0.10 g) and the
mixture was stirred in a Parr apparatus under 3 atm of hydrogen
for 1 h. Then, the catalyst was filtered on a celite pad and the mix-
ture was concentrated. The carboxylic acid (2) was obtained pure
in quantitative yield (0.75 g) without any further purification.
◦
Mp = 146–148 C; [a]2D0 −16.2 (c 1.0, acetone); IR (nujol):
m = 3154, 1761, 1742, 1717 cm−1; 1H NMR (CDCl3, 300 MHz):
d = 1.58 (d, 3H, J = 6.3 Hz, Me–Oxd), 2.58 (s, 3H, CH3CO),
4.50 (d, 1H, J = 4.5 Hz, CHN), 4.67 (dq, J = 4.5, 6.3 Hz,
CHN), 4.80 (dq, J = 4.4, 6.4 Hz, CHO), 5.19 (AB, J = 12.4 Hz,
OCH2Ph), 6.55 (d, 1H, J = 6.5 Hz, NH), 7.80–7.98 (bs, 1H,
OH); 13C NMR (CDCl3, 100 MHz): d = 21.2, 23.1, 61.4, 73.1,
152.0, 170.8, 172.2. Elemental analysis for C17H20N2O6 (348.4):
calcd C 58.61, H 5.79, N 8.04%; found C 58.64, H 5.82, N 8.01%.
Experimental
General method for the synthesis of ligands 3a–3e
Routine NMR spectra were recorded with spectrometers at 400,
300 or 200 MHz (1H NMR) and at 100, 75 or 50 MHz (13C
NMR). Chemical shifts are reported in d values relative to
the solvent peak of CHCl3, set at 7.27 ppm. Infrared spectra
were recorded with an FT-IR spectrometer. Melting points were
determined in open capillaries and are uncorrected.
H–L-Iva–OH, H–L-(aMe)Nva–OH and H–L-(aMe)Val–OH
were prepared according to ref. 13, the others amino acids are
commercially available. H–Xaa–OBn·CF3CO2H were obtained
by deprotection of the corresponding fully protected Boc–Xaa–
OBn with trifluoroacetic acid in dichloromethane, following
standard procedures.
High quality infrared spectra (64 scans) were obtained at
2 cm−1 resolution using a 1 mm NaCl solution cell and a
Nicolet 210 FT-infrared spectrometer. All spectra were obtained
in 3 mM solutions in dry CH2Cl2 at 297 K. All compounds were
dried in vacuo and all the sample preparations were performed
in a nitrogen atmosphere.
To a stirred solution of Ac–L-Oxd–OH 2 (1 mmol, 187 mg) and
HBTU (1 mmol, 0.38 g) in dry acetonitrile (10 mL) was added a
mixture of H–Xaa–OBn·CF3CO2H (1 mmol) and Et3N (3 mmol,
0.44 mL) in dry acetonitrile (10 mL) at rt. The solution was
stirred 40 min, then acetonitrile was removed under a reduced
pressure and was replaced with ethyl acetate. The mixture was
washed with brine, 1 N aqueous HCl (3 × 30 mL) and with
5% aqueous NaHCO3 (1 × 30 mL), dried over Na2SO4 and
concentrated in vacuo. The product was obtained pure after
silica gel chromatography (cyclohexane–ethyl acetate, 9 : 1 to
7 : 3 as eluent) in quantitative yield.
◦
Ac–L-Oxd–L-Ala–OBn 3a. Mp = 139–141 C; [a]2D0 −12.0
(c 1.0, CH2Cl2); IR (CH2Cl2, 3 mM): m = 3410, 3341, 1786,
1739, 1699 cm−1; 1H NMR (CDCl3, 400 MHz): d = 1.42 (d, 3H,
J = 7.2 Hz, Me–Ala), 1.45 (d, 3H, J = 6.4 Hz, Me–Oxd), 2.54
(s, 3H, CH3CO), 4.30 (d, 1H, J = 4.4 Hz, CHN), 4.62 (dq, J =
6.5, 7.2 Hz, CHN–Ala), 4.80 (dq, J = 4.4, 6.4 Hz, CHO), 5.19
(AB, J = 12.4 Hz, OCH2Ph), 6.55 (d, 1H, J = 6.5 Hz, NH),
7.35–7.41 (m, 5H, Ph); 13C NMR (CDCl3, 100 MHz): d = 17.7,
20.5, 23.3, 48.6, 62.7, 67.3, 73.5, 128.1, 128.5, 128.6, 135.0, 152.4,
167.0, 171.1, 172.1. Elemental analysis for C17H20N2O6 (348.4):
calcd C 58.61, H 5.79, N 8.04%; found C 58.64, H 5.82, N 8.01%.
High quality 1H NMR spectra (600 MHz) were recorded
with a Varian Inova 600. Measurements were carried out in
CDCl3 and in DMSO-d6 using tetramethylsilane as an internal
standard. Proton signals were assigned by COSY spectra. Data
for conformational analysis are obtained with NOESY-1D
spectra with typical mixing times of 1.0 sec.
Emission and excitation spectra were obtained with a Perkin
Elmer LS 55 spectrofluorimeter. Corrections were made as
previously reported.14
Ac–L-Oxd–Aib–OBn 3b. Mp = 95–96 ◦C; [a]D20 −11.0 (c 0.8,
CH2Cl2); IR (CH2Cl2, 3 mM): m = 3424, 3389, 1792, 1739,
1706 cm−1; 1H NMR (CDCl3, 400 MHz): d = 1.38 (d, 3H, J =
6.4 Hz, Me–Oxd), 1.53 (s, 3H, Me–Aib), 1.57 (s, 3H, Me–Aib),
2.48 (s, 3H, CH3CO), 4.28 (d, 1H, J = 4.4 Hz, CHN), 4.60 (dq,
J = 4.4, 6.4 Hz, CHO), 5.12 (AB, J = 12.0 Hz, OCH2Ph), 6.93 (s,
1H, NH), 7.30–7.39 (m, 5H, Ph); 13C NMR (CDCl3, 100 MHz):
d = 20.5, 23.3, 24.1, 24.7, 56.9, 62.7, 67.4, 73.3, 128.1, 128.3,
128.5, 135.2, 152.5, 166.5, 170.9, 173.6. Elemental analysis for
C18H22N2O6 (362.4): calcd C 59.66, H 6.12, N 7.73%; found C
59.63, H 6.14, N 7.77%.
LC–MS Analysis. Acetonitrile and methanol for HPLC were
purchased by Riedel-de Hae¨n. All the samples were prepared
by diluting 1 mg in 5 mL of a 1 : 1 mixture of H2O and
acetonitrile, in pure acetonitrile or in pure methanol. The
samples were analyzed with a liquid chromatography Agilent
R
Technologies HP1100 equipped with a Zorbax Eclipseꢀ XDB-
C8 Agilent and Technologies column (flow rate 0.5 mL min−1)
and provided with a diode-array UV detector (220 and 254 nm).
The MSD1100 mass detector was utilized under the following
conditions: mass range 100–2500 uma, positive scanning, energy
of fragmentor 50 V, drying gas flow (nitrogen) 10.0 mL min−1,
nebulizer pressure 45 psig, drying gas temperature 350 ◦C,
capillary voltage 4500 V.
◦
Ac–L-Oxd–L-Iva–OBn 3c. Mp = 122–124 C; [a]2D0 +1.0
(c 0.8, CH2Cl2); IR (CH2Cl2, 3 mM): m = 3428, 3385, 1792, 1733,
1699 cm−1; 1H NMR (CDCl3, 600 MHz): d = 0.78 (t, 3H, J =
7.2 Hz, CH2–CH3), 1.48 (d, 3H, J = 6.6 Hz, Me–Oxd), 1.63 (s,
3H, Me–Iva), 1.86 (dq, 1H, J = 7.8, 13.8 Hz, CHH–CH3), 2.29
(dq, 1H, J = 7.2, 13.8 Hz, CHH–CH3), 2.57 (s, 3H, CH3CO),
4.29 (d, 1H, J = 4.2 Hz, CHN), 4.72 (dq, J = 4.8, 6.6 Hz, CHO),
5.20 (AB, J = 12.0 Hz, OCH2Ph), 6.87 (s, 1H, NH), 7.28–7.40
(m, 5H, Ph); 13C NMR (CDCl3, 100 MHz): d = 8.2, 20.7, 22.3,
23.3, 61.5, 63.1, 67.6, 73.4, 128.2, 128.5, 128.6, 135.1, 152.4,
166.2, 171.0, 173.7. Elemental analysis for C19H24N2O6 (376.4):
calcd C 60.63, H 6.43, N 7.44%; found C 60.59, H 6.48, N 7.40%.
(4R,5S)-3-Acetyl-5-methyl-2-oxo-oxazolidine-4-carboxylic acid 2
A solution of acetyl chloride (5.5 mmol, 0.4 mL) in DMF
(0.5 mL) was added dropwise to a stirred solution of oxazolidin-
2-one (1)15 (5 mmol, 1.18 g), DIEA (15 mmol, 0.6 mL) and
DMAP (2.5 mmol, 0.31 g) in dry DMF (3 mL). The mixture
was stirred 16 h under nitrogen at rt, then was diluted with ethyl
acetate (50 mL), washed with 1 N aqueous HCl (3 × 30 mL)
and 5% aqueous NaHCO3 (1 × 30 mL), dried over Na2SO4 and
concentrated in vacuo. The fully protected oxazolidin-2-one was
obtained pure in 95% yield (1.32 g) as an oil after silica gel
chromatography (cyclohexane–ethyl acetate, 8 : 2 as eluant).
Ac–L-Oxd–L-aMeNva–OBn 3d. Waxy solid; [a]2D0 +5.5
(c 1.0, CH2Cl2); IR (CH2Cl2, 3 mM): m = 3427, 3389, 1789, 1730,
1702 cm−1; 1H NMR (CDCl3, 300 MHz): d = 0.84 (t, 3H, J =
7.5 Hz, CH2–CH2–CH3), 0.93–1.08 (m, 1H, CH2–CHH–CH3),
1.18–1.34 (m, 1H, CH2–CHH–CH3), 1.44 (d, 3H, J = 6.6 Hz,
Me–Oxd), 1.61 (s, 3H, C–aMe), 1.75 (dd, 1H, J = 4.8, 13.2 Hz,
To
a solution of 3-acetyl-5-methyl-2-oxo-oxazolidine-4-
carboxylic acid benzyl ester (4 mmol, 1.11 g) in ethyl acetate
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 0 – 1 5 2 4
1 5 2 3

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CHH–CH2–CH3), 2.16 (dd, 1H, J = 4.5, 13.2 Hz, CHH–CH2–
CH3), 2.54 (s, 3H, CH3CO), 4.29 (d, 1H, J = 4.2 Hz, CHN),
4.66 (dq, J = 4.2, 6.6 Hz, CHO), 5.17 (AB, J = 12.0 Hz,
OCH2Ph), 6.91 (s, 1H, NH), 7.31–7.42 (m, 5H, Ph); 13C NMR
(CDCl3, 100 MHz): d = 13.8, 17.2, 20.7, 22.6, 23.3, 38.9, 60.9,
63.0, 67.6, 73.4, 128.2, 128.5, 128.6, 135.1, 152.4, 166.2, 171.0,
173.7. Elemental analysis for C20H26N2O6 (390.4): calcd C 61.53,
H 6.71, N 7.18%; found C 61.54, H 6.75, N 7.15%.
Acknowledgements
C. T. thanks MURST Cofin 2002 (“Sintesi di peptidi ciclici o
lineari contenenti amminoacidi inusuali analoghi di arginina,
glicina o acido aspartico (RGD) come nuovi inbitori di inte-
grine”), FIRB 2001 (“Eterocicli azotati come potenziali inbitori
enzimatici”) and 60% (Ricerca Fondamentale Orientata), and
the University of Bologna (funds for selected topics “Processi di
sintesi innovativi ed eco-compatibili di molecole bioattive”) for
financial support. G. L. thanks Consorzio C.I.N.M.P.I.S. (Bari)
for a grant.
Ac–L-Oxd–L-aMeVal–OBn 3e. Waxy solid; [a]2D0 −12.2
(c 0.9, CH2Cl2); IR (CH2Cl2, 3 mM): m = 3430, 3390, 1786,
1737, 1701, 1638 cm−1; 1H NMR (CDCl3, 400 MHz): d = 0.88
(d, 3H, J = 6.8 Hz, Me–iPr), 0.94 (d, 3H, J = 6.8 Hz, Me–iPr),
1.38 (d, 3H, J = 6.4 Hz, Me–Oxd), 1.58 (s, 3H, C–aMe), 2.15–
2.29 (m, 1H, CH–iPr), 2.54 (s, 3H, CH3CO), 4.28 (d, 1H, J =
4.0 Hz, CHN), 4.68 (dq, J = 4.8, 6.4 Hz, CHO), 5.14 (AB, J =
References
1 (a) C. Kempter, D. Kaiser, S. Haag, G. Nicholson, V. Gnau, T. Walk,
K. H. Gierling, H. Decker, H. Zaehner, G. Jung and J. W. Metzger,
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Morytko, X. Yu, Y. Zhang, J. Silverman, N. Controneo, V. Laganas,
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12.0 Hz, OCH2Ph), 6.78 (s, 1H, NH), 7.31–7.42 (m, 5H, Ph); 13
C
NMR (CDCl3, 100 MHz): d = 17.2, 18.5, 20.5, 23.4, 35.0, 62.9,
63.7, 67.3, 73.1, 128.4, 128.5, 128.6, 135.2, 152.4, 166.4, 171.3,
172.7. Elemental analysis for C20H26N2O6 (390.4): calcd C 61.53,
H 6.71, N 7.18%; found C 61.50, H 6.77, N 7.10%.
Ac–D-Oxd–L-Ala–OBn 4. To a stirred solution of Ac–D-
Oxd–OH (1 mmol, 187 mg) and HBTU (1 mmol, 0.38 g) in
dry acetonitrile (10 mL) was added a mixture of H–L-Ala–
OBn·CF3CO2H (1 mmol, 0.29 mg) and Et3N (3 mmol, 0.44 mL)
in dry acetonitrile (10 mL) at rt. The solution was stirred 40 min,
then acetonitrile was removed under a reduced pressure and was
replaced with ethyl acetate. The mixture was washed with brine,
1 N aqueous HCl (3 × 30 mL) and with 5% aqueous NaHCO3
(1 × 30 mL), dried over Na2SO4 and concentrated in vacuo.
The product was obtained pure after silica gel chromatography
(cyclohexane–ethyl acetate, 7 : 3 as eluent) in 93% yield. Mp =
86–87 ◦C; [a]D20 −17.1 (c 0.9, CH2Cl2); IR (CH2Cl2, 3 mM): m =
3397, 3345, 1790, 1743, 1697 cm−1; 1H NMR (CDCl3, 300 MHz):
d = 1.43 (d, 3H, J = 7.2 Hz, Me–Ala), 1.49 (d, 3H, J = 6.3 Hz,
Me–Oxd), 2.54 (s, 3H, CH3CO), 4.41 (d, 1H, J = 3.9 Hz, CHN),
4.60 (dq, J = 6.9, 7.2 Hz, CHN–Ala), 4.85 (dq, J = 3.9, 6.3 Hz,
CHO), 5.17 (AB, J = 12.0 Hz, OCH2Ph), 6.92 (d, 1H, J = 6.9 Hz,
NH), 7.33–7.41 (m, 5H, Ph); 13C NMR (CDCl3, 100 MHz): d =
17.5, 20.2, 23.0, 48.2, 62.2, 66.9, 73.6, 127.7, 128.1, 128.3, 134.9,
152.5, 167.3, 170.5, 172.0. Elemental analysis for C17H20N2O6
(348.4): calcd C 58.61, H 5.79, N 8.04%; found C 58.60, H 5.75,
N 8.09%.
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Ac–D-Oxac–L-Val–OBn 5. To a stirred solution of Ac–D-
Oxac–OH16 (1 mmol, 187 mg) and HBTU (1 mmol, 0.38 g)
in dry acetonitrile (10 mL) was added a mixture of H–L-Val–
OBn.CF3CO2H (1 mmol, 0.32 mg) and Et3N (3 mmol, 0.44 mL)
in dry acetonitrile (10 mL) at rt. The solution was stirred 40 min,
then acetonitrile was removed under a reduced pressure and was
replaced with ethyl acetate. The mixture was washed with brine,
1 N aqueous HCl (3 × 30 mL) and with 5% aqueous NaHCO3
(1 × 30 mL), dried over Na2SO4 and concentrated in vacuo.
The product was obtained pure after silica gel chromatography
(cyclohexane–ethyl acetate 7 : 3 as eluent) in 95% yield. Mp =
126–128 ◦C; [a]D20 +72.6 (c 1.0, CH2Cl2); IR (CH2Cl2, 3 mM): m =
3419, 1776, 1731, 1698, 1678 cm−1; 1H NMR (CDCl3, 400 MHz):
d = 0.84 (d, 3H, J = 6.8 Hz, Me–iPr), 0.89 (d, 3H, J = 6.8 Hz,
Me–iPr), 2.05–2.15 (m, 1H, CH–iPr), 2.51 (s, 3H, CH3CO), 2.55
(dd, 1H, J = 9.6, 14.8 Hz, CHHCO), 3.05 (dd, 1H, J = 3.2,
15.2 Hz, CHHCO), 4.30 (dd, 1H, J = 3.6, 9.2 Hz, CHHO),
4.46 (t, 1H, J = 8.8 Hz, CHHO), 4.53 (dd, 1H, J = 4.8, 8.8 Hz,
CHN), 4.65–4.78 (m, 1H, CHO), 5.16 (AB, 2 H, J = 12.0 Hz,
OCH2Ph), 6.33 (d, 1H, J = 8.4 Hz, NH), 7.30–7.35 (m, 5H, Ph);
13C NMR (CDCl3, 100 MHz): d = 17.9, 19.2, 24.0, 31.3, 37.8,
51.6, 57.4, 67.4, 67.9, 128.6, 128.7, 128.8, 135.4, 153.9, 169.2,
171.0, 171.9. Elemental analysis for C19H24N2O6 (376.4): calcd
C 60.63, H 6.43, N 7.44%; found C 60.67, H 6.40, N 7.42%.
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Crisma, C. Toniolo, C. Piazza, B. Kaptein, Q. B. Broxterman, I. Ruiz,
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15 For the preparation of H–L-Oxd–OBn 1, see ref. 6.
16 For the preparation of Ac–D-Oxac–OH, see ref. 3d.
1 5 2 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 0 – 1 5 2 4