Synthesis of constrained peptidomimetics containing 2-oxo-1,3-oxazolidine- 4-carboxylic acids

Article abstract of DOI:10.1002/ejoc.201100346

Sulfonyl peptides containing a serine or threonine residue undergo cyclization with bis(succinimidyl) carbonate (DSC) and diisopropylethylamine (DIPEA) to give peptides with a 2-oxo-1,3-oxazolidine-4-carboxylate (Oxd) group, either in solution or in the solid phase. The position of the serine or threonine residue in the sequence is relatively unimportant. Under the same conditions, the corresponding Fmoc- or Boc-peptides gave dehydration products, in agreement to previous studies. The protocol constitutes a valuable approach to the preparation of oxazolidinone-containing peptides, which are a recently emerging class of constrained peptidomimetics. As a representative example, an Oxd analogue of the endogenous opioid peptide endomorphin-1, characterized by an all-trans conformation, was readily prepared.

Full text of DOI:10.1002/ejoc.201100346

FULL PAPER
DOI: 10.1002/ejoc.201100346
Synthesis of Constrained Peptidomimetics Containing 2-Oxo-1,3-oxazolidine-4-
carboxylic Acids
Luca Gentilucci,*[a] Alessandra Tolomelli,[a] Rossella De Marco,[a] Claudia Tomasini,[a] and
Sören Feddersen[a]
Keywords: Peptides / Peptidomimetics / Protecting groups / Protein modifications / Cyclization
Sulfonyl peptides containing a serine or threonine residue
undergo cyclization with bis(succinimidyl) carbonate (DSC)
and diisopropylethylamine (DIPEA) to give peptides with a
2-oxo-1,3-oxazolidine-4-carboxylate (Oxd) group, either in
solution or in the solid phase. The position of the serine or
threonine residue in the sequence is relatively unimportant.
Under the same conditions, the corresponding Fmoc- or Boc-
peptides gave dehydration products, in agreement to pre-
vious studies. The protocol constitutes a valuable approach
to the preparation of oxazolidinone-containing peptides,
which are a recently emerging class of constrained peptido-
mimetics. As a representative example, an Oxd analogue of
the endogenous opioid peptide endomorphin-1, charac-
terized by an all-trans conformation, was readily prepared.
Introduction
In principle, many naturally occurring, biologically active
peptides can be regarded as potential pharmaceutical
agents. However, there are intrinsic weaknesses that hinder
their clinical use, in particular, their rapid metabolism in
vivo, which can lead to short half-life stability, and their
poor permeation across membrane barriers. These inherent
limitations can be bypassed by adopting the peptidomi-
metic strategy, which involves altering the structure of a
peptide. Peptidomimetics very often contain non-amino
acidic elements, but still bear identifiable similarity to the
parent peptides, and either imitate or inhibit their biochem-
ical effects.[1]
During the course of our studies on biologically active
mimetics of naturally occurring opioid peptides,[2] we be-
came interested in the preparation of dehydroalanine
(DHA) containing peptides[3] as intermediates for the syn-
thesis of sequences including substituted (S)- or (R)-trypto-
phans.[4] We obtained the DHA residue (80–90%) by dehy-
dration of Boc- or Fmoc-protected serine-containing pept-
ides with bis(succinimidyl) carbonate (DSC), according to
a procedure reported in the literature (Scheme 1).[5] Inter-
estingly, we observed that, on submitting the corresponding
sulfonyl peptides to the same reaction conditions, the serine
residue underwent cyclization to form the 2-oxo-1,3-oxazol-
idine-4-carboxylate (Oxd; Scheme 1).
Scheme 1. Different reactivities of Fmoc-, Boc-, or sulfonyl-
peptides containing serine.
Peptides containing an oxazolidinone constitute a rela-
tively uncommon class of peptidomimetics.[6] In some cases,
they have been utilized as prodrugs[7] or as intermediates for
the preparation of other peptidomimetics.[8] In medicinal
chemistry, the oxazolidinone ring is present in some bioac-
tive peptides or mimetics, such as the GPIIb/IIIa integrin
antagonists, which are based on the (oxo-oxazolidinyl)-
methyl scaffold,[9] or the HIV-1 protease inhibitors, which
incorporate an oxo-N-phenyloxazolidine-5-carboxylic acid
moiety, that are designed to improve the cellular antiviral
potency.[10] Very recently, oxazolidin-2-one-containing pept-
ides have found applications in the field of foldamers, which
are short synthetic oligomers that tend to assume ordered
conformations.[11] Short Oxd-peptides were shown to form
well-defined 3D structures, such as the dimeric Boc-(Phe-d-
Oxd)2-OBn, which forms nanofibers with an antiparallel β-
sheet structure.[12]
The synthesis of the oxazolidin-2-one ring from an
amino alcohol and a carbonate or a dicarbonate (CDI, di-
ethyl or diphenyl carbonate, Boc2O, etc.) is well documen-
ted.[13] However, this work describes a new, highly efficient
[a] Department of Chemistry “G. Ciamician”, University of
Bologna,
Via Selmi 2, 40126 Bologna, Italy
Fax: +39-051-2099456
E-mail: luca.gentilucci@unibo.it
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L. Gentilucci et al.
FULL PAPER
synthesis of some representative examples of Oxd-peptides readily obtained by standard solid-phase peptide synthesis
directly from a peptide sequence, either in solution or in the (SPPS) on a Rink amide resin with Fmoc-protected amino
solid phase.
acids, with N,NЈ-dicyclohexyl carbodiimide (DCC) and
Furthermore, to demonstrate the utility of the procedure HOBt as coupling agents. After cleavage with trifluoro-
in medicinal chemistry, a constrained analogue of the en- acetic acid (TFA) in the presence of scavengers, the peptides
dogenous opioid peptide endomorphin-1 (EM1),[14] H-Tyr- were isolated by flash chromatography on silica gel and an-
Pro-Trp-PheNH2, with the 2-oxo-oxazolidine-4-carboxylate alyzed by RP-HPLC and ES-MS (see Experimental Sec-
in position 2 was prepared as a proline mimetic; the com- tion, Table 2).
pound was characterized by a trans conformation of the
preceding peptide bond.
Alternatively, the Ts-peptide 1h was prepared on Wang
resin by using Fmoc-protected amino acids, DCC, and
HOBt; Ts-Ala-Ser-Phe-Wang was then utilized for the fol-
lowing cyclization reaction on a solid phase prior to cleav-
age.
Results and Discussion
The cyclization reaction of sulfonyl-peptides 1a–g and 1i
was performed in solution (Scheme 2). The corresponding
Oxd-peptide amides 2 were isolated by flash chromatog-
raphy on silica gel (purity higher than 95% by RP-HPLC
analysis), whereas the Oxd-peptide acid 2h was obtained
after cleavage from the resin (see below). The Oxd-peptides
were characterized by IR, NMR, RP-HPLC, ES-MS, and
HRMS (FAB) analyses. 1H NMR resonances were assigned
by using 2D gCOSY techniques.
Peptides with DHA were obtained by treating a mixture
of the corresponding serine-containing N-Boc- or Fmoc-
protected peptides and DSC in acetonitrile with a solution
of DIPEA in dichloromethane at room temperature
(Scheme 1). However, when the same conditions were ap-
plied to N-sulfonyl di-, tri-, or tetrapeptides 1 having the
serine (or threonine) residue in different positions, the reac-
tion gave the N-sulfonyl peptides 2, containing a 2-oxo-ox-
azolidine-4-carboxylate ring, without significant formation
of either DHA or aziridine[15] (Scheme 2).
Optimization of the reaction conditions led to the adop-
tion of some minor changes with respect to the conditions
described in the literature, such as the replacement of aceto-
nitrile with N,N-dimethylformamide (DMF); the modifica-
tions resulted in excellent yields (Table 1) and reduced reac-
tion times.
Table 1. Yields of the Oxd-peptides 2 prepared as described in
Scheme 2.
1
R
Xaa
Yaa
R1
2
3
Scheme 2. Synthesis of Oxd-containing peptides 2.
[%][a] [%][a]
Only the reaction of dipeptide Ts-Pro-Ser-OMe (1a),
containing a serine methyl ester, gave the dehydration prod-
uct Ts-Pro-DHA-OMe (3a) as the major product (82%,
Table 1). Apparently, in this case, the comparatively high
acidity of the α-H atom of the serine methyl ester with re-
spect to that of the serine amides (1b–i)[18] promoted the
elimination of the intermediate Ser-O-succinimidyl carbon-
ate.
a
b
c
d
e
f
4-MeC6H4
4-MeC6H4
4-MeC6H4
4-MeC6H4
4-MeC6H4
4-O2NC6H4
Pro
–
Pro
Ala
Ala
Ala
OMe
PheNH2
PheNH2
PheNH2
PheNH2
PheNH2
PheNH2
PheOH[c]
Trp-PheNH2
H
H
H
H
Me
H
H
H
H
trace
80
85
88
85
82
trace
trace
–
–
–
–
–
–
90
g
4-MeC6H4 Ala-Ala
95
h[b] 4-MeC6H4
i
Ala
Tyr
77[d]
85
4-MeC6H4
[a] Determined after isolation by flash chromatography on silica
gel. [b] Ts-Ala-Ser-Phe-Wang. [c] Reaction performed on a solid
phase (Wang resin), followed by peptide cleavage. [d] Determined
after isolation by semipreparative RP-HPLC.
In contrast, all of the other Ts or Ns oligopeptides con-
taining Ser or Thr amides gave the corresponding Oxd-
peptides, as reported in Table 1. This is a significant result,
because, provided the sulfonyl group is present, these condi-
Tosyl (Ts) protected amino acids were prepared by al- tions allow the introduction of the Oxd group directly into
lowing the amino acids, TsCl, and NaHCO3 in H2O/di- oligopeptides. Indeed, when the protecting group is a carba-
oxane to react according to the literature;[16] the synthesis mate, such as Boc, Fmoc, or Cbz, the reaction of peptides
of 4-O2NC6H4SO2-(Nosyl or Ns)-Ala-OH was performed containing a Ser amide gives rise to the formation of DHA,
with Ns-Cl in H2O at pH = 8.[17]
as previously reported.[5a,5e] For confirmation, we submit-
The dipeptide ester Ts-Pro-Ser-OMe (1a) was obtained ted the corresponding Fmoc- or Boc-protected versions of
in solution from Ts-Pro-OH and H-Ser-OMe·HCl under 1d (and 1f), 1g, and 1h to the same conditions described
standard conditions by using 1-[3-(dimethylamino)propyl]- above; as expected, in all cases, the reactions gave dehy-
3-ethylcarbodiimide hydrochloride (EDC·HCl) and 1-hy- dration products in variable yields, without any trace of
droxybenzotriazole hydrate (HOBt) as activating agents. Oxd (data not shown).
The sulfonyl di-, tri-, and tetrapeptide-amides of general se-
The reaction of the dipeptide Ts-Ser-Phe-NH2 (1b) gave
quence RSO2-Xaa-Ser/Thr-Yaa-NH2 (1b–g and 1i) were Ts-Oxd-Phe-NH2 (2b) in good yield and only traces of the
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Constrained Peptidomimetics
dehydration product (3b). The tripeptides Ts-Pro-Ser-Phe-
Finally, to demonstrate the usefulness of Oxd-peptides
NH2 (1c) and Ts-Ala-Ser-Phe-NH2 (1d), containing a tosyl as conformationally defined peptidomimetics containing a
group that was not directly appended to the Ser residue, pseudo-proline,[6] an analogue of the endogenous opioid
gave the corresponding Oxd-peptides 2c and 2d, respec- peptide endomophin-1 (EM1), H-Tyr-Pro-Trp-PheNH2 was
tively, in high yields.
prepared. Among the different natural or synthetic opioid
In a similar way, Ts-Ala-Thr-Phe-NH2 (1e), which con- peptides,[23] EM1 is unique for high receptor affinity and
tains a Thr residue, gave the corresponding tripeptide with selectivity towards the μ-opioid receptor (MOR). However,
a trans-5-methyl-2-oxo-oxazolidine-4-carboxylate (2e). In despite many structural investigations performed on EM1
this case, the (2S,3R) stereochemistry of the threonine resi- and its analogues, the identification of the bioactive confor-
due was retained, as determined from the H NMR cou- mation remains elusive.[24] Extensive NMR studies revealed
1
pling constant of the 4-H–5-H doublet (J = 4.8 Hz).
that, in solution, EM1 exists as an approximate 1:3 cis/trans
The reaction could also be performed with a different mixture of conformers with respect to the Tyr1–Pro2 pept-
sulfonyl group; thus, the Nosyl-peptide Ns-Ala-Ser-Phe- ide bond.
NH2 (1f), gave the expected product Ns-Ala-Oxd-Phe-NH2
The tetrapeptide Ts-Tyr-Ser-Trp-Phe-NH2 (1i) gave Ts-
(2f) with comparable yield. Furthermore, the tetrapeptide Tyr-Oxd-Trp-Phe-NH2 (2i) in satisfactory yield. In solution,
Ts-Ala-Ala-Ser-Phe-NH2 (1g), having the Ser residue in po- the Oxd-tetrapeptide 2i adopts an all-trans conformation of
sition 3 of the sequence, underwent cyclization to the corre- the peptide bonds, as revealed by the NMR spectrum of the
sponding Oxd-tetrapeptides Ts-Ala-Ala-Oxd-Phe-NH2 (2g) compound, which shows a single set of sharp resonances
in almost quantitative yield.
(Figure 1).
To expand the synthetic scope of the reaction, the Oxd-
tripeptide acid Ts-Ala-Oxd-Phe-OH (2h) was synthesized
entirely on a solid phase (Table 1). The resin-bound precur-
sor Ts-Ala-Ser-Phe-Wang (1h) was prepared under standard
conditions, and then subjected to cyclization by treatment
with a moderate excess of DSC and DIPEA in dichloro-
methane/DMF (4:1) for 2 h. The cleavage of the Oxd-pept-
ide was performed by treatment with two consecutive por-
tions of 10% TFA in dichloromethane and scavengers.
Analysis of the crude reaction mixture by RP-HPLC and
ES-MS analysis revealed the presence of the peptide Ts-
Ala-Ser-Phe-OH (ca. 5%). The peptide 2h was isolated by
semipreparative RP-HPLC with satisfactory yield (77%,
based on an average resin loading of 0.6 mmol/g).
The feasibility of sulfonyl group removal under reason-
ably mild conditions was then investigated (Scheme 3). The
tripeptide Ts-Ala-Oxd-PheNH2 (2d) was treated with iodo-
trimethylsilane[19] to give the deprotected product H-Ala-
Oxd-PheNH2 (4) in moderate yield, together with a mixture
of by-products. As an alternative, treatment of 2d with
SmI2/pyrrolidine/water,[20] was attempted, but this method
gave 4 in only low yield.
Figure 1. 1H NMR spectrum of 2i, showing the amide and aro-
matic region.
The trans conformation of 2i was confirmed by compari-
son of the NMR spectroscopic data with that of Oxd-pept-
ides described in the literature.[11,12] In Oxd-peptides, the
carbonyl group of the cycle introduces a constraint that
forces the pseudo-peptide bond to always have the trans
conformation. This feature should be of help to investigate
the bioactive conformation of EM1, which is an aspect that
is under investigation.
Conclusions
In this paper we have illustrated the ready synthesis of
some representative peptides incorporating a 2-oxo-oxazol-
idine-4-carboxylate starting from sulfonyl peptides contain-
Scheme 3. Removal of Ts from 2d and Ns from 2f affording the
deprotected Oxd-containing peptide 4.
In contrast, cleavage of the Ns group was much easier ing Ser or Thr. Interestingly, the outcome of the reaction
(Scheme 3), and treatment of Ns-Ala-Oxd-PheNH2 (2f) was practically independent of the distance between the Ser
with K2CO3/PhSH[21] gave 4 in good yield. The superior or Thr residues and the sulfonyl group. Tosyl- or Nosyl-
performance of Ns compared with Ts is not unexpected.[22] protected peptides gave equally excellent results; these sulf-
Based on this result, the Ns group can be regarded as the onyl groups can be removed under a variety of mild condi-
best choice for the preparation of deprotected Oxd-pept- tions, in particular, the removal of Nosyl gave the depro-
ides.
tected peptide in good yields. The cyclization reaction was
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L. Gentilucci et al.
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triturated and collected by using a centrifuge. The crude solid was
purified by flash chromatography on silica gel (eluent: EtOAc; col-
umn size: 10 cmϫ1.5 cm), giving 1 (70–80%). The products were
analyzed by RP-HPLC and by MS, and the results are summarized
in Table 2.
performed either in solution or on a solid phase, making
the entire process a promising approach to the preparation
of this class of constrained peptidomimetics and foldamers.
As a representative example, an all-trans Oxd analogue of
EM1 was prepared.
Solution-Phase Synthesis of Oxd-peptides 2b–g and 2i: DSC
(0.45 mmol) was added to a stirred solution of 1 (0.3 mmol) in
CH2Cl2/DMF (4:1) (3 mL) followed by DIPEA (0.45 mmol) at
room temp., under an inert gas. After 1 h, the solvent was removed
under reduced pressure, the residue was diluted with 0.1 m HCl
(5 mL), and the mixture was extracted with CH2Cl2 (3ϫ 5 mL).
The combined organic layers were dried with Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash chromatography on silica gel (hexane/EtOAc, 1:1; column
size: 15 cmϫ1.0 cm) to give 2 (80–95%; purity higher than 95%
as determined by analytical RP-HPLC). Under the same reaction
conditions and workup protocol, Ts-Pro-Ser-OMe (1a) gave Ts-
Pro-DHA-OMe (3a) (82%; 95% pure by analytical RP-HPLC).
Experimental Section
General Methods: Unless stated otherwise, standard chemicals were
obtained from commercial sources and used without further purifi-
cation. Flash chromatography of 1 was performed on silica gel
(230–400 mesh) by using mixtures of distilled solvents. Compounds
1 were analyzed by RP-HPLC and ES-MS. RP-HPLC was per-
formed on an ODS column [4.6 μm particle size, 100 Å pore dia-
meter, 250 μm, DAD 210 nm, from H2O/CH3CN (9:1) to H2O/
CH3CN (2:8) in 20 min] at a flow rate of 1.0 mL/min, followed by
10 min at the same composition. Semipreparative RP-HPLC purifi-
cation of compounds 2 were performed on a C18 column [7 μm
particle size, 21.2 mmϫ150 mm, from H2O/CH3CN (8:2) to 100%
CH3CN in 10 min] at a flow rate of 10 mL/min. The purity of 2
(more than 95%) was assessed by analytical RP-HPLC under the
above conditions. Semipreparative and analytical RP-HPLC of
compound 4 were performed under the same conditions as re-
ported for 2, but with the addition of 0.1% TFA in the mobile
phase. Compounds 2 and 4 were analyzed by RP-HPLC, ES-MS,
and HRMS (FAB). 1H NMR spectra were recorded at 400 MHz
at room temperature in CDCl3/[D6]DMSO (2:1) by using 5 mm
tubes; 13C NMR spectra were recorded at 75 MHz. Chemical shifts
are reported as δ values relative to the CDCl3 signal.
Solid-Phase Synthesis of 2h: Wang resin pre-loaded with Fmoc-
Phe (0.5 g, 0.4–0.8 mmol/g, resin particle size: 100–200 mesh) was
introduced into a reactor of an automated synthesizer apparatus.
Fmoc removal and couplings with the following N-protected amino
acids were performed as described above; the quantities of the rea-
gents were calculated based on an average resin loading of
0.6 mmol/g. The resin-bound peptide was suspended in CH2Cl2/
DMF (4:1) (5 mL), and DSC (0.75 mmol) and DIPEA (0.75 mmol)
were added at room temp. under an inert gas. After 2 h, the mixture
was filtered, and the resin-bound peptide was washed three times
in sequence with CH2Cl2 (5 mL) and CH3OH (5 mL). The resin-
bound peptide was suspended in a mixture of TFA (1 mL), H2O
(0.33 mL), ethanedithiol (0.33 mL), and PhOH (0.33 mL), in
CH2Cl2 (8 mL), and mechanically shaken at room temp. After 2 h,
the mixture was filtered, the resin was washed with 5% TFA in
Et2O (2ϫ 5 mL) and Et2O (2ϫ 5 mL). The cleavage procedure was
repeated, and all of the filtrates and washes were collected; solvent
and volatiles were removed under an N2 flow at room temp. The
resulting residue was suspended in Et2O, and the crude solid that
precipitated was triturated and collected by using a centrifuge. The
Oxd-peptide acid 2i was isolated by semipreparative RP-HPLC (see
General Methods) (77% based on the average resin loading of
0.6 mmol/g; 96% pure by analytical RP-HPLC).
Synthesis of 1: A measure of Fmoc-Rink amide resin (0.5 g,
1.1 mmol/g, resin particle size: 100–200 mesh) was introduced into
a reactor of an automated synthesizer apparatus. Fmoc was re-
moved with DMF/piperidine (4:1) (5 mL) under mechanical shak-
ing. After 15 min, the suspension was filtered, the resin was washed
with CH2Cl2 (5 mL) and treated, while shaking, with a second por-
tion of DMF/piperidine (4:1). After 40 min, the suspension was
filtered, and the resin was washed three times in sequence with
CH2Cl2 (5 mL) and CH3OH (5 mL). The resin was swollen in
CH2Cl2 (5 mL), and a solution of the N-protected amino acid
(1.1 mmol) and HOBt (1.1 mmol) in DMF (4 mL) was added, fol-
lowed by DCC (1.1 mmol). The mixture was mechanically shaken,
and, after 3 h, the resin was filtered and washed three times with
the sequence CH2Cl2 (5 mL) and CH3OH (5 mL). Coupling effi-
cacy was determined by means of the Kaiser test. The resin-bound
peptide was suspended in a solution of TFA (4.8 mL), H2O
(0.20 mL), and PhOH (0.050 g), in CH2Cl2 (5 mL), and mechani-
cally shaken at room temp. After 2 h, the mixture was filtered, the
resin was washed with 10% TFA in Et2O (2ϫ 5 mL) and Et2O (2ϫ
5 mL). Filtrate and washes were collected, and solvent and volatiles
were removed under N2 flow at room temp. The resulting residue
was suspended in Et2O, and the crude solid that precipitated was
Removal of the Ts Group: To a suspension of sodium iodide
(0.045 g, 0.3 mmol) in CH3CN (3 mL), TMSCl (0.038 mL,
0.3 mmol) was added, and the mixture was stirred at 0 °C under
an inert gas for 10 min. To this stirred suspension, a solution of 2d
(0.10 g, 0.2 mmol) in CH3CN (3 mL) was added, and the reaction
mixture was heated to reflux for 3 h. The solvent was removed un-
der reduced pressure, and the residue was diluted with satd. aque-
ous Na2CO3 (5 mL) and extracted with EtOAc (4ϫ 5 mL). The
collected organic layers were dried with anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified by
semipreparative RP-HPLC (see General Methods) to afford 4
(0.045 g, 65%; 95% pure by analytical RP-HPLC).
Table 2. RP-HPLC and ES-MS analyses of 1.
Removal of the Ns Group: To a stirred solution of 2f (0.11 g,
0.2 mmol) in DMF (4 mL), PhSH (0.026 mL, 0.24 mmol) and
K2CO3 (0.83 g, 0.6 mmol) were added under an inert gas, and the
mixture was stirred at room temp. for 1 h. The solvent was removed
under reduced pressure, and compound 4 was isolated as described
above (0.063 g, 90%; 96% pure by analytical RP-HPLC).
1
1
ES-MS [M + 1] Purity
found/calcd.
1
ES-MS [M + 1] Purity
found/calcd.
[%][a]
[%][a]
a
c
e
g
371.1/371.1
503.2/503.1
491.2/491.1
548.2/548.1
85
91
93
84
b
d
f
406.1/406.2
477.2/477.1
508.2/508.2
755.3/755.2
87
89
86
90
Ts-Pro-DHA-OMe (3a): IR (nujol): ν = 3050, 1720, 1690 cm–1. H
i
˜
NMR: δ = 1.58–1.80 (m, 3 H, ProHβ, ProHγ), 2.15 (m, 1 H,
ProHβ), 2.42 (s, 3 H, TsCH3), 3.20 (m, 1 H, ProHδ), 3.46 (m, 1 H,
[a] Determined by analytical RP-HPLC; for conditions, see General
Methods.
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Constrained Peptidomimetics
ProHδ), 3.85 (s, 3 H, COOCH3), 4.15 (dd, J = 3.2, 8.6 Hz, 1 H,
ProHα), 5.91 (s, 1 H, =CH), 6.57 (s, 1 H, =CH), 7.33 (d, J = 8.0 Hz,
55.3, 65.8, 123.7, 126.3, 127.8, 127.9, 128.9, 136.2, 146.4, 149.3,
152.3, 167.1, 172.1, 172.4 ppm. HRMS (FAB): calcd. for
2 H, ArH), 7.74 (d, J = 8.0 Hz, 2 H, ArH), 9.15 (br. s, 1 H, NH) C22H24N5O9S [M + H]+ 534.1295; found 534.1300.
ppm. 13C NMR: δ = 19.1, 22.0, 22.4, 40.2, 50.5, 56.8, 105, 125.3,
Ts-Ala-Ala-Oxd-PheNH2 (2g): IR (nujol): ν = 1783, 1710, 1705,
˜
129.5, 136.3, 138.4, 141.5, 165.3, 171.5 ppm. ES-MS: calcd. for [M
+ 1] 371.1; found 371.2.
1653, 1525 cm–1. 1H NMR: δ = 1.20 (d, J = 6.8 Hz, 3 H, AlaCH3),
1.35 (d, J = 7.2 Hz, 3 H, AlaCH3), 2.40 (s, 3 H, TsCH3), 3.03 (dd,
J = 6.4, 13.8 Hz, 1 H, PheHβ), 3.08 (dd, J = 6.9, 13.8 Hz, 1 H,
PheHβ), 3.78 (quint, J = 7.3 Hz, 1 H, AlaHα), 4.22 (dd, J = 4.3,
9.0 Hz, 1 H, 4-H), 4.43 (t, J = 9.2 Hz, 1 H, 5-H), 4.63 (q, J =
7.1 Hz, 1 H, PheHα), 4.91 (dd, J = 4.3, 8.8 Hz, 1 H, 5-H), 5.28
(quint, J = 6.8 Hz, 1 H, AlaHα), 5.80 (br. s, 1 H, CONH2), 6.50
(br. s, 1 H, CONH2), 6.60 (d, J = 8.4 Hz, 1 H, AlaNH), 7.10–7.27
(m, 8 H, ArH, AlaNH), 7.72 (d, J = 8.0 Hz, 2 H, ArH), 7.81 (d, J
= 7.2 Hz, 1 H, PheNH) ppm. 13C NMR: δ = 16.3, 18.0, 20.5, 36.7,
47.1, 51.3, 53.6, 55.2, 64.8, 125.9, 126.1, 127.4, 128.4, 128.7, 135.6,
136.0, 142.6, 151.4, 166.8, 170.6, 171.7, 171.9 ppm. HRMS (FAB):
calcd. for C26H32N5O8S [M + H]+ 574.1972; found 574.1977.
Ts-Oxd-PheNH2 (2b): IR (nujol): ν = 1777, 1710, 1651 cm–1. 1H
˜
NMR: δ = 2.43 (s, 3 H, TsCH3), 3.15–3.28 (m, 2 H, PheHβ), 4.16
(dd, J = 4.2, 8.6 Hz, 1 H, 4-H), 4.53 (t, J = 8.8 Hz, 1 H, 5-H), 4.65
(q, J = 6.8 Hz, 1 H, PheHα), 5.12 (dd, J = 4.2, 8.4 Hz, 1 H, 5-H),
6.82 (br. s, 1 H, CONH2), 7.23 (br. s, 1 H, CONH2), 7.23–7.42 (m,
7 H, ArH), 7.76 (d, J = 8.0 Hz, 2 H, ArH), 8.60 (d, J = 8.0 Hz, 1
H, PheNH) ppm. 13C NMR: δ = 19.9, 36.0, 52.7, 55.9, 65.2, 124.1,
125.7, 127.2, 127.9, 128.2, 128.3, 132.5, 133.0, 135.9, 150.2, 166.4,
171.8 ppm. HRMS (FAB): calcd. for C20H22N3O6S [M + H]+
432.1229; found 432.1232.
Ts-Pro-Oxd-PheNH2 (2c): IR (nujol): ν = 1773, 1715, 1660 cm–1.
˜
1H NMR: δ = 1.52 (m, 1 H, ProHγ), 1.72 (m, 1 H, ProHγ), 1.82
(m, 1 H, ProHβ), 1.97 (m, 1 H, ProHβ), 2.35 (s, 3 H, TsCH3), 3.00–
3.09 (m, 3 H, ProHδ, PheHβ), 3.42 (m, 1 H, ProHδ), 4.22 (dd, J =
3.6, 9.4 Hz, 1 H, 4-H), 4.51 (t, J = 9.2 Hz, 1 H, 5-H), 4.62 (q, J =
7.2 Hz, 1 H, PheHα), 5.02 (dd, J = 3.6, 9.2 Hz, 1 H, 5-H), 5.42
(dd, J = 3.8, 9.0 Hz, 1 H, ProHα), 6.50 (br. s, 1 H, CONH2), 6.73
(br. s, 1 H, CONH2), 7.08–7.22 (m, 5 H, ArH), 7.24 (d, J = 8.4 Hz,
2 H, ArH), 7.62 (d, J = 8.4 Hz, 2 H, ArH), 7.78 (d, J = 8.0 Hz, 1
H, PheNH) ppm. 13C NMR: δ = 17.9, 20.7, 23.1, 38.5, 40.7, 49.0,
51.1, 54.8, 56.4, 126.1, 127.8, 128.5, 129.4, 136.9, 139.1, 143.5,
152.6, 170.2, 173.1, 175.0 ppm. HRMS (FAB): calcd. for
C25H29N4O7S [M + H]+ 529.1757; found 529.1760.
Ts-Ala-Oxd-Phe-OH (2h): IR (nujol): ν = 2800–3450, 1766, 1722,
˜
1716, 1649 cm–1. 1H NMR: δ = 1.20 (d, J = 7.2 Hz, 3 H, AlaCH3),
2.41 (s, 3 H, TsCH3), 3.05–3.17 (m, 2 H, PheHβ), 4.27 (dd, J = 4.1,
8.5 Hz, 1 H, 4-H), 4.36 (t, J = 9.2 Hz, 1 H, 5-H), 4.63 (q, J =
7.6 Hz, 1 H, PheHα), 4.92 (dd, J = 4.0, 9.2 Hz, 1 H, 5-H), 5.20 (m,
1 H, AlaHα), 6.30 (d, J = 9.0 Hz, 1 H, AlaNH), 7.10–7.25 (m, 5
H, ArH), 7.30 (d, J = 8.0 Hz, 2 H, ArH), 7.52 (d, J = 7.6 Hz, 1 H,
PheNH), 7.73 (d, J = 8.0 Hz, 2 H, ArH), 9.32 (br. s, 1 H, COOH)
ppm. 13C NMR: δ = 17.9, 20.5, 38.4, 47.9, 50.1, 54.2, 56.4, 126.8,
127.7, 128.2, 129.1, 130.2, 136.3, 139.2, 144.1, 152.4, 168.0, 173.2,
176.0 ppm. HRMS (FAB): calcd. for C23H26N3O8S [M + H]+
504.1441; found 504.1436.
Ts-Ala-Oxd-PheNH2 (2d): IR (nujol): ν = 1766, 1722, 1649,
˜
1
1530 cm–1. H NMR: δ = 1.32 (d, J = 7.3 Hz, 3 H, AlaCH3), 2.40
Ts-Tyr-Oxd-Trp-PheNH2 (2i): IR (nujol): ν = 1776, 1718, 1675,
˜
(s, 3 H, TsCH3), 3.00–3.17 (m, 2 H, PheHβ), 4.27 (dd, J = 4.2,
9.3 Hz, 1 H, 4-H), 4.45 (t, J = 9.3 Hz, 1 H, 5-H), 4.64 (q, J =
7.4 Hz, 1 H, PheHα), 4.91 (dd, J = 4.2, 8.6 Hz, 1 H, 5-H), 5.15
(dq, J = 7.4, 9.3 Hz, 1 H, AlaHα), 6.07 (br. s, 1 H, CONH2), 6.39
(br. s, 1 H, CONH2), 6.41 (d, J = 9.3 Hz, 1 H, AlaNH), 7.10–7.27
(m, 7 H, ArH), 7.75 (d, J = 8.4 Hz, 2 H, ArH), 7.98 (d, J = 8.0 Hz,
1 H, PheNH) ppm. 13C NMR: δ = 18.5, 20.8, 39.1, 47.8, 50.7, 54.9,
56.0, 127.1, 127.3, 128.7, 129.4, 129.8, 136.4, 139.5, 143.9, 152.4,
167.9, 173.5, 174.5 ppm. HRMS (FAB): calcd. for C23H27N4O7S
[M + H]+ 503.1600; found 503.1598.
1
1526 cm–1. H NMR: δ = 2.38 (s, 3 H, TsCH3), 2.42 (dd, J = 8.0,
14.0 Hz, 1 H, TyrHβ), 2.82 (dd, J = 7.6, 14.2 Hz, 1 H, TrpHβ),
2.99 (dd, J = 6.4, 14.2 Hz, 1 H, TrpHβ), 3.15 (dd, J = 3.0, 14.0 Hz,
1 H, TyrHβ), 3.22 (d, J = 7.6 Hz, 2 H, PheHβ), 4.22 (dd, J = 4.1,
9.2 Hz, 1 H, 4-H), 4.50 (t, J = 9.2 Hz, 1 H, 5-H), 4.57 (q, J =
6.8 Hz, 1 H, TrpHα), 4.92 (q, J = 7.0 Hz, 1 H, PheHα), 5.02 (dd,
J = 4.1, 8.8 Hz, 1 H, 5-H), 5.30 (quint, J = 6.8 Hz, 1 H, TyrHα),
5.72 (br. s, 1 H, CONH2), 6.05 (br. s, 1 H, CONH2), 6.75 (d, J =
8.4 Hz, 2 H, TyrArH), 6.95 (d, J = 7.8 Hz, 1 H, TyrNH), 7.02–7.15
(m, 4 H, TrpNH, TrpH-5, TyrArH), 7.19 (t, J = 7.6 Hz, 1 H, TrpH-
6), 7.25 (s, 1 H, TrpH-2), 7.30 (d, J = 8.0 Hz, 2 H, ArH), 7.32–7.45
(m, 5 H, PheArH), 7.50 (d, J = 8.0 Hz, 1 H, TrpH-7), 7.61 (s, 1 H,
TyrOH), 7.66 (d, J = 7.9 Hz, 1 H, TrpH-4), 7.68 (d, J = 8.0 Hz, 2
H, ArH), 8.55 (d, J = 8.8 Hz, 1 H, PheNH), 9.81 (br. s, 1 H, TrpH1)
ppm. 13C NMR: δ = 21.4, 31.9, 36.6, 37.3, 49.3, 52.7, 59.2, 60.1,
71.3, 111.8, 112.3, 116.6, 120.2, 121.0, 123.5, 125.0, 126.2, 127.3,
129.5, 132.1, 132.3, 136.2, 137.6, 140.1, 141.3, 155.7, 156.2, 173.2,
174.0, 174.2, 176.0 ppm. HRMS (FAB): calcd. for C40H41N6O9S
[M + H]+ 781.2656; found 781.2662.
Ts-Ala-(5-Me-Oxd)-PheNH2 (2e): IR (nujol): ν = 1776, 1716,
˜
1
1633 cm–1. H NMR: δ = 1.24 (d, J = 7.2 Hz, 3 H, AlaCH3), 1.33
(d, J = 6.0 Hz, 3 H, 5-CH3), 2.36 (s, 3 H, TsCH3), 3.03 (d, J =
6.4 Hz, 2 H, PheHβ), 4.22 (d, J = 4.8 Hz, 1 H, 4-H), 4.42 (t, J =
5.2 Hz, 1 H, 5-H), 4.55 (q, J = 7.3 Hz, 1 H, PheHα), 4.98 (m, 1 H,
AlaHα), 5.70 (br. s, 1 H, CONH2), 5.81 (br. s, 1 H, CONH2), 6.17
(d, J = 7.9 Hz, 1 H, AlaNH), 7.09–7.20 (m, 5 H, ArH), 7.22 (d, J
= 7.2 Hz, 2 H, ArH), 7.38 (d, J = 8.0 Hz, 1 H, PheNH), 7.65 (d, J
= 7.2 Hz, 2 H, ArH) ppm. 13C NMR: δ = 18.5, 20.3, 21.5, 37.8,
51.0, 54.9, 75.0, 75.9, 127.1, 127.3, 128.7, 129.2, 129.8, 136.3, 136.7,
147.5, 151.8, 167.0, 173.0, 174.7 ppm. HRMS (FAB): calcd. for
C24H29N4O7S [M + H]+ 517.1757; found 517.1761.
H-Ala-Oxd-PheNH2 (4): IR (nujol): ν = 3352, 3445, 1764, 1722,
˜
1
1645 cm–1. H NMR: δ = 1.22 (d, J = 6.8 Hz, 3 H, AlaCH3), 2.96
Ns-Ala-Oxd-PheNH2 (2f): IR (nujol): ν = 1769, 1724, 1648, (dd, J = 9.8, 13.8 Hz, 1 H, PheHβ), 3.12 (dd, J = 4.6, 13.8 Hz, 1
˜
1532 cm–1. 1H NMR: δ = 1.32 (d, J = 7.0 Hz, 3 H, AlaCH3), 3.00– H, PheHβ), 3.30 (q, J = 6.8 Hz, 1 H, AlaHα), 4.08 (dd, J = 4.6,
3.15 (m, 2 H, PheHβ), 4.20 (dd, J = 4.1, 9.1 Hz, 1 H, 4-H), 4.40 8.4 Hz, 1 H, 4-H), 4.36 (dd, J = 4.6, 9.2 Hz, 1 H, 5-H), 4.43 (t, J
(t, J = 9.1 Hz, 1 H, 5-H), 4.59 (q, J = 7.3 Hz, 1 H, PheHα), 4.81
(dd, J = 4.1, 8.5 Hz, 1 H, 5-H), 5.15 (dq, J = 7.0, 8.8 Hz, 1 H,
AlaHα), 6.21 (br. s, 1 H, CONH2), 6.80 (br. s, 1 H, CONH2), 7.10–
7.30 (m, 5 H, ArH), 7.37 (d, J = 8.8 Hz, 1 H, AlaNH), 7.79 (d, J
= 8.0 Hz, 1 H, PheNH), 7.96 (d, J = 9.0 Hz, 2 H, ArH), 8.22 (d, J
= 9.0 Hz, 2 H, ArH) ppm. 13C NMR: δ = 19.6, 37.1, 50.2, 53.9,
= 8.4 Hz, 1 H, 5-H), 4.50 (m, 1 H, PheHα), 7.10 (br. s, 1 H,
CONH2), 7.15 (br. s, 1 H, CONH2), 7.16–7.30 (m, 5 H, ArH), 8.05–
8.25 (m, 3 H, PheNH, AlaNH) ppm. 13C NMR: δ = 17.3, 37.9,
47.7, 49.0, 56.1, 63.2, 126.0, 128.7, 129.2, 137.1, 153.4, 172.4, 173.8,
174.0 ppm. HRMS (FAB): calcd. for C16H21N4O5 [M + H]+
349.1512; found 349.1509.
Eur. J. Org. Chem. 2011, 4925–4930
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4929

L. Gentilucci et al.
FULL PAPER
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Received: March 11, 2011
Published Online: July 13, 2011
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