Expedient synthesis of pseudo-Pro-containing peptides: Towards constrained peptidomimetics and foldamers

Article abstract of DOI:10.1039/c2ob07172j

The reaction of sulfonyl peptides containing l- or d-configured Ser or Thr with bis(succinimidyl) carbonate in the presence of a catalytic amount of a base affords, in solution or in the solid phase, the corresponding peptides with one or two, consecutive or alternate oxazolidin-2-ones (Oxd). The Oxd ring can be regarded to as a pseudo-Pro with an exclusively trans conformation of the preceding peptide bond; homochiral Oxd-containing peptides adopt extended conformations, while the presence of a d-configured Oxd favours folded conformations. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob07172j

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PAPER
Expedient synthesis of pseudo-Pro-containing peptides: towards constrained
peptidomimetics and foldamers†
Rossella De Marco, Alessandra Tolomelli, Marilena Campitiello, Pasqualina Rubini and Luca Gentilucci*
Received 23rd December 2011, Accepted 13th January 2012
DOI: 10.1039/c2ob07172j
The reaction of sulfonyl peptides containing L- or D-configured Ser or Thr with bis(succinimidyl)
carbonate in the presence of a catalytic amount of a base affords, in solution or in the solid phase, the
corresponding peptides with one or two, consecutive or alternate oxazolidin-2-ones (Oxd). The Oxd ring
can be regarded to as a pseudo-Pro with an exclusively trans conformation of the preceding peptide bond;
homochiral Oxd-containing peptides adopt extended conformations, while the presence of a D-configured
Oxd favours folded conformations.
Introduction
Determining the receptor-bound structure of biologically active
peptides is fundamental in drug design. However, the direct
investigation of ligand–receptor complexes has met with con-
siderable practical obstacles; besides, many native peptides are
highly flexible molecules, therefore their conformational analysis
is a difficult task. As a consequence, many efforts have been
dedicated to the design of conformationally defined peptidomi-
metics as tools for investigating the key structural and confor-
mational features on which receptor recognition and binding are
based.1
Generally, peptide backbones serve as 3D scaffolds for posi-
tioning the side chains involved in ligand–receptor interactions.
The presence of Pro residues strongly impacts upon the structural
and conformational properties of the backbones. The cyclic
structure of Pro forces the ϕ angle to −65° 15°, thus preventing
the formation of an α-helix, and promoting the formation of
β-turns.2 Turns and inverse turns play an important role in the
architecture and bioactivity of native folded proteins.3 In
Fig. 1 Selected examples of pseudo-Pro structures.
addition, while the barrier to secondary amide cis/trans isomeri-
zation is ca. 10 kcal mol−1 (Fig. 1), the presence of Pro reduces
the barrier to just 2 kcal mol−1, increasing the cis conformer.4
Pip, Nip, etc., Fig. 1)6 were synthesized and utilized to design
Besides Pro itself, numerous derivatives were found in pro-
conformationally constrained peptidomimetics.1,7
teins of microbial or marine origins, in antibiotics or cytotoxic
For instance, pseudo-Pro such as thiazolidine- and oxazoli-
dine-4-carboxylic acid (Tc, Oxi) dimethylated at the 2 position
peptides,5 and many others (e.g. 2,4-MePro, Δ3Pro, Azi, Aze,
are known to be quantitative, or nearly quantitative, inducers of
the cis conformation around the preceding peptide bond,8 while
Department of Chemistry “G. Ciamician”, University of Bologna, via
1-aminocyclohexane-1-carboxylic acid (Chx) induces the trans
Selmi 2, 40126 Bologna, Italy. E-mail: luca.gentilucci@unibo.it;
Fax: +39 0512099456; Tel: +39 0512099570
conformation.9 On the other hand, 5,5-dimethylthiazolidine-4-
carboxylic acid (Dtc)10 and azetidine-2-carboxylic acid (Aze)11
(Fig. 1) favor angles in the γ-turn region, while 5-tert-butylpro-
line (5-tBuPro) stabilizes type VI β-turn conformation.12
†Electronic supplementary information (ESI) available: 1H NMR ana-
lyses in different solvents; VT-1H-NMR Δδ/Δt values in different sol-
vents; non-obvious ROESY cross-peaks in 8 : 2 DMSO-d6–H2O;
structures and energies of the intermediates estimated by semi-empirical
computations for the cyclization reaction; CD spectra. See DOI:
10.1039/c2ob07172j
Finally, pseudo-Pro have found use in the preparation of folda-
mers,13 short synthetic oligomers which have a tendency to form
This journal is © The Royal Society of Chemistry 2012
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well-defined secondary structures, stabilized by noncovalent
interactions.14
Results and discussion
Optimization of the reaction conditions
In recent years, we have been interested in the use of D-Pro,
β-Pro, pseudo-Pro and pseudo-β-Pro for the preparation of con-
strained bioactive peptidomimetics.15 In this regard, we recently
proposed the 2-oxo-1,3-oxazolidine-4-carboxylate (or oxazoli-
din-2-one, in short: Oxd) as a constrained pseudo-Pro residue.
Indeed, the carbonyl group of the cycle introduces a constraint
that enforces the pseudo-peptide bond to always have the trans
conformation (Fig. 1).
The reaction depicted in Scheme 1 represents the first synthesis
of a oxazolidin-2-one-4-carboxylate directly within a peptide
sequence. Apparently, the reaction outcome depends on the pres-
ence of the sulfonyl group (see Introduction). Subsequently, in
order to optimize the cyclization of these sulfonyl peptides, we
investigated the role of the carbonate, solvent, base, and sulfonyl
group, by reacting the model peptides 1a–d under different con-
ditions (Scheme 2).
The preparation of 1a–d and of the other sulfonyl-protected
peptides 4, 6, 8, 11 and 13, was conducted by coupling the
amino acids under normal conditions, using 1-ethyl-3-
[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC–HCl)
and 1-hydroxybenzotriazole hydrate (HOBt) as activating agents
(Table 1). N-Mesyl (Ms), tosyl (Ts), or nosyl (Ns) amino acids
were prepared according to the literature.21
The treatment of 1a (Table 2) with 1.2 equiv. of DSC and 1.2
equiv. of DIPEA in DCM–CH3CN20 gave the oxazolidinone-
peptide 2a, Ts-Ala-Oxd-PheNH2 (entry 1) in good yield,
together with traces of the corresponding elimination product
Ts-Ala-Dha-PheNH2 (3). The substitution of CH3CN with DMF
allowed an increase of the yield while reducing the reaction time
(entry 2); the use of solely DMF or DMSO (entries 3 and 4) was
not beneficial. Different bases such as DBU (entry 5) or DMAP
(entry 6) in DCM–DMF gave 2 with comparable yields.
A second set of reactions was designed to test the role of the
carbonate. The synthesis of an oxazolidin-2-one ring from an
aminoalcohol and a carbonate or a dicarbonate is well documen-
ted in the literature.22 Nevertheless, none of the procedures
attempted by us gave the oxazolidinone-peptide 2 in a significant
yield. The reaction of 1a with 1,1′-carbonyldiimidazole (CDI)
and DIPEA23 gave Ts-Ala-Dha-PheNH2 (3) as the major
product, and only traces of 2a (entry 7), the rest being the
reagent. The treatment of 1a with Boc2O and DIPEA (entry 8)
or DMAP24 (entry 9) gave a Boc-derivative of 1 (not isolated)
and traces of 2, as determined by the HPLC-MS analysis of
the reaction mixture. Triphosgene gave 2 and 3 only in traces
(entry 10), while ethylchloroformate was completely ineffective
(entry 11).17,22,25
Apart from the different applications in medicinal chemistry,16
Oxd-peptides have been the subject of much interest as folda-
mers or as self-assembling scaffolds forming nanostructures. The
oligomers of the Boc-(L-Ala-D-Oxd)n-OBn series exhibit a strong
tendency to form helices with 10-membered H-bonded rings in
solution.17 The α,β-hybrid oligomers of the Boc-(L-β3-hPhg-D-
Oxd)n-OBn series formed helices with 11-membered H-bonded
turns for n ≥ 5. The Boc-(L-β3-hPhe-D-Oxd)n-OBn series dis-
plays chain length-dependent behaviour. In the higher oligomers
(n > 2), there is a stronger tendency to form intramolecular H-
bonds, while the Boc-(L-β3-hPhe-D-Oxd)2-OBn motif forms an
anti-parallel β-sheet-like structure, where only one intermolecular
H-bond stabilizes the fibre-like material, as well as the hydro-
phobic forces between the aromatic side-chains.18
In this work we considerably expand the scope of our prelimi-
narily described preparation of peptides containing the Oxd
ring.19 The reaction proceeds by the direct cyclization of N-sul-
fonyl peptides containing a Ser residue (Scheme 1) by treatment
with bis(succinimidyl) carbonate (DSC) and diisopropylethyla-
mine (DIPEA).
The distance between the sulfonyl and the Ser in the sequence
is relatively unimportant. Indeed, the reaction of peptides having
the sulfonyl group directly connected to the Ser, or separated by
one or two amino acids (Scheme 1, Xaa = 0, 1, or 2 residues)
proceeded with similar results. The reaction of the corresponding
Fmoc- or Boc-peptides under the same conditions gave elimin-
ation to dehydroalanine (Dha), in agreement with the literature
(Scheme 1).20
Furthermore, we observed that the reaction of 1a and DSC in
the absence of a base did not furnish 2a (entry 12). However, the
reaction was possible with a catalytic amount of DIPEA (entry
13) or DMAP (entry 14), giving 2a in excellent yield and in
reasonable time in a DCM–DMF mixture, but not in DCM alone
(entry 15).
By using DSC and a catalytic amount of DIPEA, Ts-Ala-D-
Ser-PheNH2 (1b) was converted into Ts-Ala-D-Oxd-PheNH2
(2b) with the same yield of 2a (entry 16 compared to entry 13).
Scheme 1 Different reactivity of N-carbamate and N-tosyl (Ts) or
nosyl (Ns) oligopeptides.
Scheme 2 Reactions of the sulfonyl-peptides 2a, c, d, containing L-Ser, and of 2b, containing D-Ser, under the conditions reported in Table 2.
2308 | Org. Biomol. Chem., 2012, 10, 2307–2317 This journal is © The Royal Society of Chemistry 2012

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In order to gain more information about the role of the sulfo-
nyl group in the cyclization, we compared the reaction of the Ts-
peptide 1a to that of the peptides 1c and 1d carrying the nosyl
and mesyl group, respectively.
Under the same conditions as entry 13, Ns-Ala-Ser-PheNH2
(1c) gave the corresponding Oxd-peptide Ns-AlaOxd-Phe-NH2
(2c) in very good yield (entry 17).
led to the proposal of the plausible reaction pathway b, which
accounts for the experimental observations reported in Table 2.
Upon treatment with :B, the Ts- or Ns-intermediate A would
form the anionic intermediate B, which in turn gives rise to the
five-membered anionic intermediate C. The loss of 2,5-dioxo-
pyrrolidin-1-olate from C leads to the Oxd-peptide D; the proto-
nation of the anionic leaving group by HB+ would regenerate the
free base, which can be utilized in catalytic amount.
The optimized conformations of A–D and the estimated ΔE
are shown in the ESI, Scheme S1.† The most stable confor-
mation of the anionic intermediate B shows the aromatic ring of
the sulfonyl group stacked below the delocalized anion at a dis-
tance of 3.5 Å; this distance is perfectly compatible with the
values reported in the literature for π-stacking interactions.26 The
ability of arylsulfonamido groups to form sandwich structures by
π-stacking interactions is well described in the literature.27
Besides, electron-poor aromatic rings effectively promote the
reactivity of delocalized anions such as enolates by means of a
donor–acceptor π-stacking stabilization of the transition states.28
In a similar way, it is plausible that sulfonamido groups such as
tosyl and nosyl, but not mesyl, might stabilize the intermediate
anion B as well as the plausible transition state BC (Scheme 3).
This effect would be impossible for Boc- or Fmoc-peptides; con-
sequently, under the same conditions, these peptides undergo
elimination to Dha.
On the contrary, the mesyl group failed to promote the cycli-
zation. The treatment of Ms-Ala-Ser-PheNH2 (1d) with DSC in
the presence of a catalytic amount of DIPEA did not give rise to
the formation of the Oxd-peptide (entry 18); the use of 1.2
equiv. of DIPEA (entry 19) or DMAP (entry 20) afforded the
product only in traces.
Taken together, the results reported in Table 2 give several
clues on the mechanism of the cyclization process. While an
accurate investigation of the reaction mechanism is beyond the
scope of this work, we compared the elimination of Boc- or
Fmoc-peptides containing a Ser with a plausible mechanism of
cyclization of the corresponding tosyl peptides (Scheme 3). The
two reactions proceed via a common Ser-O-carbonate intermedi-
ate A. The elimination of the Boc- or Fmoc-intermediate A
induced by the base (:B) gives Dha, as sketched in pathway a.
The alternative cyclization to Oxd was investigated with the
aid of theoretical computations performed employing ab initio
molecular orbital (MO) theory at the HF/6-31G** level. These
Interestingly, the calculated structures of C and D clearly
revealed an AlaCHα⋯−1O–C interaction, and a non-convention-
al AlaCHα⋯OvC(Oxd) hydrogen bond, respectively. The latter
interaction was also confirmed by NMR analysis and modeling
of the Oxd-peptides (see next paragraph).
Table 1 RP-HPLC and ES-MS analyses of the linear peptides
ES-MS m/z
[M + 1] vs.
ES-MS m/z
[M + 1] vs.
Compd Calcd
Puritya
(%)
Puritya
(%)
Compd calcd
Starting from the calculated structures A–D, the dioxopyrroli-
din-1-olyl group was replaced by other leaving groups resulting
from CDI, Boc2O, chloroformate, or triphosgene (see Table 2),
and these new structures were optimized (not shown). The com-
parison revealed that the anionic intermediate C carrying the
dioxopyrrolidin-1-olyl is significantly lower in energy with
respect to the analogues carrying the other leaving groups. This
comparatively higher stability might also reflect the transition
state BC, accounting for the efficacy of DSC in the cyclization,
1a
1c
4
8
11b
477.2/477.1
508.2/508.2
565.1/565.2
361.1/361.1
374.1/374.1
89
86
85
87
88
1b
1d
6
477.1/477.1
401.1/401.1
346.0/346.1
374.1/374.1
86
87
88
90
11a
13b
—
—
c
c
a Determined by analytical RP-HPLC, see General methods. b Ts-Ser-
Phe-Ser-Phe-Wang; the following cyclization was performed in solid
phase. c Not determined.
Scheme 3 Plausible reaction pathways for the elimination of carbamate-peptides (pathway a) and alternatives for the cyclization of arylsulfonyl pep-
tides (pathway b).
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Table 2 Reagents and conditions tested for the synthesis of R-SO2-Ala-Oxd-PheNH2 (2) from R-SO2-Ala-Ser-PheNH2 (1)
Entry
Compd
Carbonate (equiv.)
Base (equiv.)
Solvent
Timea (h)
2b (%)
3 (%)
1
2
3
4
5
6
7
8
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
b
c
DSC (1.5)
DSC (1.5)
DSC (1.5)
DSC (1.5)
DSC (1.5)
DSC (1.5)
CDI (1.2)
Boc2O (2.1)
Boc2O (2.1)
Cl3COCOOCCl3 (0.5)
ClCOOMe
DSC (1.2)
DSC (1.2)
DSC (1.2)
DSC (1.2)
DSC (1.2)
DSC (1.2)
DSC (1.2)
DSC (1.5)
DSC (1.5)
DIPEA (1.2)
DIPEA (1.2)
DIPEA (1.2)
DIPEA (1.2)
DBU (1.2)
DMAP (1.2)
DIPEA (1.2)
DIPEA (1.2)
DMAP (1.2)
DIPEA (3.0)
DIPEA (1.2)
—
DCM–CH3CN 3 : 1
DCM–DMF 3 : 1
DMF
2
1
1
1
2
3
24
24
2
24
24
24
3
6
24
3
74
88
80
79
tracesc,d
—
—
—
DMSO
DCM–DMF 3 : 1
DCM–DMF 3 : 1
DCM
CH3CN
DCM–DMF 3 : 1
DCM–DMF
DCM–DMF 3 : 1
DCM–DMF 3 : 1
DCM–DMF 3 : 1
DCM–DMF 3 : 1
DCM
DCM–DMF 3 : 1
DCM–DMF 3 : 1
DCM–DMF 3 : 1
DCM–DMF 3 : 1
DCM–DMF 3 : 1
85
—
—
87
Tracesc,d
—
30d,e
d,e,f
—
—
d,e,f
9
—
—
—
—
92
90
10
11
12
13
14
15
16
17
18
19
20
tracesc,d
d,e,g
—
—
—
—
—
—
—
—
DIPEA (0.1)h
DMAP (0.3)h
DIPEA (0.1)h
DIPEA (0.1)h
DIPEA (0.1)h
DIPEA (0.1)h
DIPEA (1.2)
DMAP (1.2)
Tracesc,d,e
92
3
90
—
d
d
d
24
24
24
Tracesc,d,e
Tracesc,d,e
Tracesc,d,e
Tracesc,d,e
a The reaction was stopped at the disappearance of the reagent, as determined by the t.l.c. analysis of the reaction, or after 24 h. b After purification by
f
flash chromatography. c <5%. d Not isolated, determined by the HPLC-MS analysis. e The rest being the reagent 1. Boc-1 was the mayor by-product.
g MeOCO-1 was the major by-product. h The lower amount tested.
Synthesis of di-Oxd-peptides
compared to the other carbonates or dicarbonates, etc., discussed
in Table 2, entries 8–11.
Previous results19 demonstrated that the position of a Ser or Thr
residue in the sequence of the sulfonyl-peptide is practically
unimportant for the cyclization to Oxd-peptide (see above). As a
consequence, we envisaged the opportunity to perform a single-
step cyclization of sequences containing two Ser, or a Ser and a
Thr, consecutive or alternating with other amino acids.
The reaction of Ts-Ser-SerNH2 (6) with 2.5 equiv. of DSC
in 3 : 1 DCM–DMF and in the presence of a catalytic amount
of DIPEA (0.1 equiv.) gave the corresponding di-Oxd-amide
7 in excellent yield (Scheme 5) after isolation by flash
chromatography.
Finally, it is plausible that the π-stacking is functional also at
stabilizing the anionic intermediate of type B for peptides having
the arylsulfonamido group and Ser separated by two amino
acids19 (not calculated).
Epimerization during the cyclization process was excluded on
the basis of the comparison of the NMR and HPLC analyses of
2a vs. 2b, including the HPLC analysis on a chiral stationary
phase (see General methods).
The mild cleavage of the arylsulfonyl groups29 was discussed
previously.19 The tosyl group was removed in sufficient yield
with iodotrimethylsilane,30 while the treatment with SmI2–pyrro-
lidine–water31 was much less efficient. The cleavage of the Ns
group was easily performed with K2CO3–PhSH,32 giving the
deprotected peptide in good yield.
In summary, the comparison of the results listed in Table 2 led
us to conclude that the conditions of entry 13 were the best for
the cyclization reaction. Consequently, the Oxd-tetrapeptide
Ns-Ala-D-Oxd-Phe-GlyNH2 (5), useful for the investigation of
the conformational features of the constrained peptides, was
smoothly prepared from Ns-Ala-D-Ser-Phe-GlyNH2 (4) by treat-
ment with DSC and 0.1 equiv. of DIPEA in 3 : 1 DCM–DMF
(Scheme 4).
Scheme 5 Synthesis of the di-Oxd peptide amide 7.
On the other hand, the reaction of the dipeptide ester Ts-Ser-
SerOMe (8) under the same reaction conditions gave a ca.
1 : 1 mixture of the expected di-Oxd peptide 9 and the dipeptide
containing the Dha methyl ester 10 (Scheme 6).
Possibly, this result reflects the comparatively higher acidity of
the Hα of serine methyl ester with respect to that of the serine
amides,33 which promotes the elimination of the intermediate
Ser-O-succinimidyl carbonate.
Interestingly, the reaction outcome was dependent on the
solvent utilized (Scheme 6). Indeed, performing the reaction in
DCM gave Ts-Oxd-Oxd-OMe (9) and Ts-Oxd-Dha-OMe (10,
Dha = dehydroalanine) in 92 : 8 ratio (88% overall yield), while
in DMF the situation was completely reversed, giving a 95 : 5
ratio in favor of 10 (86% overall yield).
Scheme 4 Synthesis of 5 and Δδ/Δt VT-NMR values of the amide
protons in 8 : 2 DMSO-d6–H2O.
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Scheme 8 Solid-phase synthesis of 14.
Scheme 6 Solvent effect in the alternative formation of the di-Oxd
peptide 9 and Oxd-Dha peptide 10.
technique is intrinsically a low-resolution method; however, it
can furnish qualitative information on the presence of ordered
secondary structures,35 although not too many examples on short
peptides are reported.36 ECD measurements of compounds 2a,
2b, 5, 10, 12a, 12b and 14 were performed at room temperature
in TFE37 and methanol; 10 and 12a also in chloroform (ESI,
Fig. S2†). These analyses gave some information on the eventual
occurrence of secondary structures. Spectra of 2b, 5, 10 and 12b
show a negative nπ* band at ca. 235 nm, compatible with γ-turn
structures.38 Spectra of structurally correlated 2a 2b, and of 12a
12b show marked differences. CD intensity changes when
moving from methanol (and chloroform for 10) to TFE, indicat-
ing that the nature of the solvent may influence the turn popu-
lation. Different chiroptical properties are associated to the other
compounds 2a, 12a, 14 in the same spectral region. For instance,
by comparing ECD spectra of 12a with those of 12b, a specular
relationship emerges in the nπ* region, possibly reflecting the
inversion of configuration of Oxd1. CD spectra of 12a present a
positive band centered at ca. 230 nm accompanied by a weak
positive shoulder at lower energy, while a negative ππ* band is
observed at around 200 nm. In both cases the magnitude of CD
grows when moving from TFE to methanol and chloroform.
These spectral features suggest that for 12a, a different second-
ary structure becomes predominant with respect to the one char-
acterizing 12b, and its stability is increased in chloroform.
The Oxd-peptides were analyzed by NMR experiments using
standard techniques at 400 MHz in CDCl3, CH3OH, and in the
biomimetic medium 8 : 2 DMSO-d6–H2O.39 For most com-
pounds, the 1H NMR resonances of the compounds showed
modest variations of the chemical shifts in the different solvents,
suggesting conformational stability (exceptions are discussed
throughout).
Scheme 7 Synthesis of the di-Oxd peptide esters 12a, b.
This dipeptide Ts-Oxd-Dha-OMe is of some interest, since it
contains two distinct secondary structure-inducing residues, the
Oxd and a dehydroamino acid. Indeed, dehydroamino acids are
well known inducers of β-turn structures; for instance, sequential
placement of dehydroPhe (ΔPhe) in oligomers gives repeated
β-turns forming 310 helices.20,34
In a similar way as for the preparation of 9, the cyclization of
the peptide esters 11a, b containing L-Ser, Thr, and D-Ser, Thr,
respectively, gave in DCM the di-Oxd 12a, b in high yield
(Scheme 7), with only traces of the elimination products.
The trans configuration of the 5-methyl-oxazolidin-2-one-4-
1
carboxylate was deduced by the H NMR coupling constant of
the H4–H5 (J ca. 5.0 Hz), thus retaining the (2S,3R) stereo-
chemistry of Thr. The comparison of the NMR and HPLC ana-
lyses of 12a vs. 12b, including the HPLC analysis on a chiral
stationary phase (see General methods) led us to consider that
epimerization was negligible.
Finally, the di-Oxd-tetrapeptide acid Ts-Oxd-Phe-Oxd-Phe-
OH (14) was entirely synthesized on solid-phase (Scheme 8).
The resin-bound precursor Ts-Ser-Phe-Ser-Phe-Wang (13) was
prepared under standard conditions, and was subjected to cycli-
zation by treatment with a 5 equiv. excess of DSC and catalytic
DIPEA (0.3 equiv.) prior to the cleavage from the resin.
The cleavage of the di-Oxd-peptide was performed by two
consecutive treatments with 10% TFA in DCM in the presence
of scavengers. The peptide 14 was isolated by semi-preparative
RP-HPLC with a very satisfactory yield.
It has been demonstrated that the Oxd confers the preceding
amide bond a well defined trans conformation.17,18 Accordingly,
the 1H NMR analyses of all of the Oxd-peptides showed a single
set of sharp resonances in each solvent, indicating conformation-
al homogeneity or a fast equilibrium between conformers; gener-
ally, linear peptides containing a Pro show two sets of
resonances, for the cis and trans conformers (see Introduction).
Conformational aspects of the Oxd peptides
1
The H NMR analyses of all of the compounds showed a sig-
nificantly downfield position of the Hα proton of the residue pre-
ceding the Oxd. For instance, for the AlaHα in Ts-Ala-Oxd-
PheNH2 (2a) it is at ca. 5.1 ppm, while in Ts-Ala-Ser-PheNH2
(1a) it is at ca. 3.5 ppm; in Ts-Oxd1-Oxd2-OMe (9), the Oxd1H4
is at ca. 6.0 ppm, while Oxd2H4 is at ca. 5.0 ppm. This accounts
for a strong deshielding effect exerted by Oxd(CvO),17,18 com-
patible with a non-conventional CH⋯OvC intramolecular
In order to analyze the conformational bias exerted by the Oxd
on the overall structure of the peptides, we performed the confor-
mational analyses of the representative examples 2a, 2b, 5, 10,
containing one Oxd, and of 12a, 12b, 14, which contain two
consecutive or alternate Oxd rings.
Electronic Circular Dichroism (ECD) is a widely used tech-
nique for studying protein and peptide conformations. This
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hydrogen bond,40 and confirms the trans conformation of the
amide bond between the Oxd and the preceding, deshielded
residue.
Variable temperature (VT)-1H-NMR experiments were utilized
to deduce the presence of H-bonds (Tables S1–3, ESI†). The
homochiral Oxd-peptide Ts-Ala-Oxd-PheNH2 (2a) did not mani-
fest the presence of any H-bond: the Δδ/Δt values of AlaNH,
PheNH in 8 : 2 DMSO-d6–H2O were −6.8, and −4.3 ppb K−1
respectively.
,
On the other hand, for Ts-Ala-D-Oxd-PheNH2 (2b) the Δδ/Δt
(ppb K−1, 8 : 2 DMSO-d6–H2O) values of AlaNH, PheNH were
−4.2, and −2.6, and for Ts-Ala-D-Oxd-Phe-GlyNH2 (5) the Δδ/
Δt (ppb K−1, 8 : 2 DMSO-d6–H2O) values of AlaNH, PheNH,
and GlyNH were −4.8, −2.7, and −7.4. The comparatively
lower VT-1H-NMR Δδ/Δt parameters of PheNH for 2b and 5 are
suggestive of the occurrence of a significant amount of folded
conformations having PheNH involved in a H-bond (|Δδ/Δt| ≲
2.5 ppb K−1).32,41 The same trends can be observed also in the
other solvents (Table S1, ESI†).
Molecular backbone conformations were investigated by 2D
ROESY analysis in 8 : 2 DMSO-d6–H2O at 400 MHz, and the
intensities of the resulting cross-peaks were ranked to infer
plausible interproton distances (Tables S4–10, ESI†). 2D
gCOSY experiments were utilized for the unambiguous assign-
ment of all of the resonances.
Structures consistent with the spectroscopic analyses were
obtained by restrained MD simulations, using the distances
derived from ROESY as constraints, and minimized with the
AMBER force field. The ω bonds were set at 180°, as the
absence of Hαi–Hα(i + 1) cross-peaks excluded cis peptide
bonds. Simulations were conducted in a box of explicit, equili-
brated TIP3P water molecules. Random structures were gener-
ated by unrestrained high-temperature MD; the structures were
subjected to high-temperature restrained MD with a scaled force
field, followed by a simulation with full restraints. Finally, the
system was gradually cooled, and the structures were minimized.
The results were clustered by the RMSD analysis of the back-
bone atoms.
For Ts-Ala-Oxd-PheNH2 (2a) and Ts-Ala-D-Oxd-PheNH2
(2b), the procedure gave one major cluster each, comprising ca.
80% of the structures. For each compound cluster, the represen-
tative geometries with the lowest internal energy were selected
and analyzed (Fig. 2). The comparison of the two structures
shows a clear difference; while 2a adopts an extended confor-
mation, the peptide containing the D-Oxd shows a preference for
a folded conformation, compatible with a γ-turn centered on D-
Oxd.
To investigate the dynamic behavior of 2a and 2b, the struc-
tures were analyzed by unrestrained MD for 10 ns in a box of
explicit water molecules. Besides the different random confor-
mations, the analysis of the trajectories of 2b, but not of 2a,
revealed a well-defined γ-turn secondary structure stabilized by
an explicit H-bond between PheNH and the Ala(CvO), in
agreement with the VT-NMR parameters.
Fig. 2 Representative low-energy structures of 2a, 2b, 5 (A, B), 10,
12a, 12b, 14 consistent with ROESY analysis, calculated by restrained
MD in a 30 × 30 × 30 box of standard TIP3P water molecules. Back-
bones and Oxd are rendered in balls and cylinders, the rest in sticks.
For Ns-Ala-D-Oxd-PheGly-NH2 (5), the conformational analy-
sis gave two clusters comprising altogether more than 85% of
the structures. For each cluster, the representative geometries 5A
and 5B with the lowest internal energy were selected and ana-
lyzed (Fig. 2). They differ almost exclusively by the opposite
orientation of the D-Oxd-Phe peptide bond. Possibly, the two
structures represent conformers in fast equilibrium. The structure
5A shows a clear inverse γ-turn centered on D-Oxd, and is
2312 | Org. Biomol. Chem., 2012, 10, 2307–2317
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ca. 1.4 kcal mol−1 lower in energy than 5B; the latter shows
some violations of the distance constraint involving PheNH.
To investigate the dynamic behavior of 5, the two conformers
A and B were analyzed by unrestrained MD for 10 ns in a box
of standard TIP3P water molecules. The simulation showed the
conversion of one conformation into the other. The analysis of
the trajectories of 5A revealed the occurrence of well-defined
secondary structures stabilized by an explicit H-bond between
Ala(CvO) and PheNH, in agreement to the VT-NMR par-
ameters. On the other hand, the analysis of the trajectories of 5b
revealed that occasionally the structure adopts an inverse β-turn
type I conformation, stabilized by a H-bond between GlyNH and
the Ala(CvO), but this observation is not supported by the
VT-NMR analysis.
The VT-NMR analysis in 9 : 1 CDCl3–DMSO-d6 indicated
some preference for a pseudo-γ-turn on Oxd1 stabilized by a H-
bond between Oxd1(SvO) and Phe2NH (Phe2NH Δδ/Δt = −2.9
ppb K−1). However, as observed for 10, this conformation is not
stable in more polar environments (for instance, in 8 : 2 DMSO-
d6–H2O Phe2NH Δδ/Δt = −5.2 ppb K−1), and it is not observed
during the unrestrained MD simulations in explicit water.
Conclusions
In this work we discuss our methodology for the straightforward
preparation of Oxd-peptides starting from arylsulfonyl peptides
containing L- or D-configured Ser or Thr by treatment with DSC
and a base.
The preference for the folded structures shown by 2b and 5
makes sense. It is well known that linear oligopeptides including
a heterochiral Pro show higher propensity to adopt stable inverse
γ- or β-turns, the Pro occupying the position i + 1, compared to
the peptides composed of all L-amino acids.42 Besides, 10- and
7-membered H-bonded rings form competitively to each other,
depending on the solvent,43 on the prevalence of a trans over cis
conformation of the amide bond preceding Pro,44 and/or on the
nature and steric hindrance of the amino acids preceding and fol-
lowing Pro.45
The conformational analysis of (10) gave one cluster compris-
ing the large majority of the structures. The representative geo-
metry with the lowest internal energy is shown in Fig. 2. The
calculated geometry of the Dha residue perfectly matches the
structures reported in the literature.46
The mechanism was investigated by varying the reaction con-
ditions, and the results were rationalized with the aid of theoreti-
cal computations. The experiments highlighted the role of the
arylsulfonylamido group and DSC. Indeed, while tosyl and
nosyl gave the ring closure, the mesyl group was ineffective; and
CDI, Boc2O, triphosgene and ClCOOMe failed to afford the
Oxd-peptides in significant yield. The reaction was performed
by using catalytic amounts of DIPEA, DBU, or DMAP.
Essentially, computations suggested that the electron-poor
arylsulfonylamido group might effectively stabilize the anionic
intermediate which leads to the Oxd ring by a π-stacking inter-
action. Computations also allowed the rationalization of the
effectiveness of DSC compared to other carbonates or
dicarbonates.
We expanded the scope of the methodology by preparing in a
single step di-Oxd-peptides from peptides containing two con-
secutive Ser, or Ser and Thr, or two Ser separated by other
amino acids. The peptide amide gave the corresponding di-Oxd-
amide, as expected, while peptide methyl ester gave the di-Oxd-
peptide or the peptide Oxd-Dha, depending on the solvent
selected for the reaction. The synthesis of the linear precursors
and the cyclization reaction was performed either in solution or
in the solid phase, making the entire process a convenient
method for the preparation of constrained peptides or foldamers.
These Oxd residues can be regarded to as suitable constrained
pseudo-Pro. The peptides containing the Oxd in place of Pro
show an all-trans conformation instead of mixtures of cis and
trans conformers. Homochiral sequences tend to adopt extended
conformations, while the presence of a D-Oxd ring induces
folded conformations, with an inverse γ-turn centered on D-Oxd,
stabilized by an explicit H-bond.
The conformational preference of 10 showed some depen-
dence on the solvent. The H NMR resonances of the H4 and
H5 protons of Oxd in CDCl3 showed significant differences with
respect to 8 : 2 DMSO-d6–H2O and CH3OH.
1
The VT-NMR analysis in CDCl3 gave DhaNH Δδ/Δt = −1.8
ppb K−1, and in 8 : 2 DMSO-d6–H2O Δδ/Δt = −4.6 ppb K−1
.
Apparently, in the less competitive CDCl3 the DhaNH is
involved in a H-bond with one of the SvO, conferring on 10 a
pseudo-γ-turn structure, which is not observed in 8 : 2 DMSO-
d6–H2O. Accordingly, the unrestrained molecular dynamics
simulations for 10 ns in explicit water did not show this pseudo-
γ-turn conformation.
The ROESY-restrained MD simulations of Ts-Oxd1-(5-Me-
Oxd2)-OMe (12a) and Ts-D-Oxd1-(5-Me-Oxd2)-OMe (12b)
gave, after clustering, one cluster each, comprising almost the
totality of the structures; the representative ones are shown in
Fig. 2. The two oxazolidin-2-ones are nearly orthogonal to
each other. The unrestrained MD confirmed the rigidity of
the conformations, since the rotation of the two Oxd rings
one with respect to the other was not observed during the
simulations.
Experimental
General methods
The calculated conformation of the Ts-Oxd1-Phe2-Oxd3-Phe4-
OH (14), determined as described above, presents an extended
conformation (Fig. 2), confirming that homochiral peptides do
not tend to fold.
Unless stated otherwise, standard chemicals were obtained from
commercial sources and used without further purification. Flash
chromatography was performed on silica gel (230–400 mesh),
using mixtures of distilled solvents. Analytical RP-HPLC was
performed on an ODS column (4.6 μm particle size, 100 Å pore
diameter, 250 μm, DAD 210 nm, from a 9 : 1 H2O–CH3CN to a
1
The H NMR resonances of the compound significantly vary
in the different environments; for instance, the δ of Phe4Hα in
9 : 1 CDCl3–DMSO-d6 (this compound is not soluble in pure
chloroform), 8 : 2 DMSO-d6–H2O, and CH3OH is 4.83, 4.53,
4.22, respectively, and the δ of Phe2Hα is 5.78, 5.57, 5.68.
2 : 8 H2O–CH3CN in 20 min) at a flow rate of 1.0 mL min−1
,
followed by 10 min at the same composition. Semi-preparative
RP-HPLC was performed on a C18 column (7 μm particle size,
This journal is © The Royal Society of Chemistry 2012
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21.2 mm × 150 mm, from 8 : 2 H2O–CH3CN to 100% CH3CN
in 10 min) at a flow rate of 12 mL min−1. Purities were assessed
by analytical RP-HPLC under the above reported conditions and
elemental analysis. Chiral HPLC analysis was performed on a
CHIRALPAK IC column (0.46 cm × 25 cm), n-hexane–2-propa-
nol 1 : 1, at 0.8 mL min−1. Semi-preparative and analytical
RP-HPLC of the peptide acid 14 was performed as reported
above, with the addition of 0.1% TFA in the mobile phase.
Elemental analyses were performer using a Thermo Flash 2000
CHNS/O analyzer. NMR spectra were recorded on a Varian
instrument. Circular Dichroism (CD) spectra were recorded on a
Jasco J-710 spectropolarimeter.
ES-MS m/z 503.2 [M + 1], calcd 503.2; Elem. Anal. for
C23H26N4O7S, calcd: C 54.97, H 5.21, N 11.15, S 6.38; found:
C 54.92, H 5.19, N 11.10, S 6.36.
Ts-Ala-D-Oxd-Phe-NH2 (2b). ). The reaction of Ts-Ala-D-Ser-
Phe-NH2 (1b, 0.20 g, 0.42 mmol) under the same conditions as
described for 1a gave 2b (0.190 g, 90%, 96% pure by analytical
RP-HPLC). IR (nujol) ν 1770, 1717, 1651, 1528 cm−1 1H
;
NMR (CDCl3) δ 1.25 (d, J = 7.2 Hz, 3H, AlaMe), 2.38 (s, 3H,
TsMe), 2.85 (dd, J = 9.2, 14.0 Hz, 1H, PheHβ), 3.25 (dd, J =
4.8, 14.0 Hz, 1H, PheHβ), 3.75 (dd, J = 3.0, 8.4 Hz, 1H,
OxdH5), 4.28 (t, J = 9.2 Hz, 1H, OxdH5), 4.66 (q, J = 7.4 Hz,
1H, PheHα), 4.72 (dd, J = 3.6, 9.2 Hz, 1H, OxdH4), 5.09 (quint,
J = 7.8 Hz, 1H, AlaHα), 6.00 (br.s, 1H, CONH2), 6.62 (d, J =
9.2 Hz, 1H, AlaNH), 6.70 (br.s, 1H, CONH2), 7.02–7.29 (m,
7H, ArH), 7.65 (d, J = 8.4 Hz, 2H, ArH), 8.23 (d, J = 8.4 Hz,
1H, PheNH); 13C NMR (2 : 1 CDCl3–DMSO-d6) δ 19.0, 21.1,
40.1, 47.0, 50.2, 55.6, 55.8, 127.3, 127.5, 129.1, 129.5, 130.1,
136.7, 139.9, 143.1, 153.4, 168.1, 173.2, 173.5; RP-HPLC (see
General methods) 7.01 min; ES-MS m/z 503.2 [M + 1], calcd
503.2; Elem. Anal. for C23H26N4O7S, calcd: C 54.97, H 5.21, N
11.15, S 6.38; found: C 54.94, H 5.24, N 11.09, S 6.34.
Peptide synthesis
A stirred solution of the N-protected amino acid in 4 : 1
DCM–DMF (5 mL) was treated with HOBt (1.2 equiv.), at r.t.
and under inert atmosphere. After 10 min, the C-protected amino
acid (1.1 equiv.), EDCI–HCl (1.2 equiv.) and TEA (3 equiv.)
were added while stirring at r.t. under inert atmosphere. After
3 h, the mixture was concentrated at reduced pressure, and the
residue was diluted with EtOAc (25 mL). The solution was
washed with 0.1 M HCl (5 mL), and a saturated solution of
NaHCO3 (5 mL). The organic layer was dried over Na2SO4 and
the solvent was evaporated at reduced pressure. The crude pep-
tides were analyzed by HPLC-MS analysis, and were used
without further purifications (Table 1).
The intermediate N-Boc peptides were deprotected by treat-
ment with 1 : 2 TFA/DCM (5 mL), with stirring, at r.t. After
15 min, the solution was evaporated under reduced pressure, and
the treatment was repeated. The residue was suspended in Et2O
(20 mL). The peptide-TFA salts which precipitated were used for
the next couplings without further purifications.
Ns-Ala-Oxd-Phe-NH2 (2c). The reaction of Ns-Ala-Ser-Phe-
NH2 (1c, 0.20 g, 0.39 mmol) under the same conditions as
described for 1a gave 2c (0.189 g, 90%, 96% pure by analytical
RP-HPLC). IR (nujol) ν 1783, 1710, 1705, 1653, 1525 cm−1
;
1H NMR (CDCl3) δ 1.21 (d, J = 6.8 Hz, 3H, AlaMe), 1.33 (d,
J = 7.2 Hz, 3H, AlaMe), 2.43 (s, 3H, TsMe), 3.02 (dd, J = 6.4,
13.8 Hz, 1H, PheHβ), 3.06 (dd, J = 6.9, 13.8 Hz, 1H, PheHβ),
3.77 (quint, J = 7.3 Hz, 1H, AlaHα), 4.22 (dd, J = 4.3, 9.0 Hz,
1H, OxdH5), 4.44 (t, J = 9.2 Hz, 1H, OxdH5), 4.62 (q, J = 7.1
Hz, 1H, PheHα), 4.92 (dd, J = 4.3, 8.8 Hz, 1H, OxdH4), 5.27
(quint, J = 6.8 Hz, 1H, AlaHα), 5.79 (br.s, 1H, CONH2), 6.51
(br.s, 1H, CONH2), 6.61 (d, J = 8.4 Hz, 1H, AlaNH), 7.10–7.28
(m, 8H, ArH + AlaNH), 7.72 (d, J = 8.0 Hz, 2H, ArH), 7.80 (d,
J = 7.2 Hz, 1H, PheNH); 13C NMR (2 : 1 CDCl3–DMSO-d6) 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; Elem. Anal. for C22H23N5O9S, calcd: C 49.53, H
4.35, N 13.13, S 6.01; found: C 49.49, H 4.33, N 13.13, S 5.98.
Mono-Oxd-peptide synthesis
Ts-Ala-Oxd-Phe-NH2 (2a). DSC (0.13 g, 0.50 mmol) was
added to a stirred solution of the linear peptide Ts-Ala-Ser-Phe-
NH2 (1a, 0.20 g, 0.42 mmol), in 3 : 1 DCM–DMF (4 mL) fol-
lowed by DIPEA (7.4 μL, 0.04 mmol) at r.t. and under inert
atmosphere. After 3 h, the solvent was removed under reduced
pressure, the residue was diluted with 0.1 M HCl (5 mL), and
the mixture was extracted three times with DCM (5 mL). The
combined organic layers were dried over sodium sulfate, filtered,
and concentrated at reduced pressure. The residue was purified
by flash chromatography over silica-gel (eluant 1 : 1 hexane–
EtOAc, column size: 15 × 1.0 cm2) to give 2a (0.194 g, 92%,
95% pure by analytical RP-HPLC). IR (nujol) ν 1766, 1722,
Ns-Ala-D-Oxd-Phe-Gly-NH2 (5). The reaction of Ns-Ala-D-
Oxd-Phe-Gly-NH2 (4, 0.20 g, 0.35 mmol) under the same con-
ditions as described for 1a gave 5 (0.178 g, 85%, 96% pure by
analytical RP-HPLC). IR (nujol)
ν 1768, 1719, 1655,
1534 cm−1; 1H NMR (9 : 1 CDCl3–DMSO-d6) δ 1.13 (d, J = 7.0
Hz, 3H, Me), 3.01 (dd, J = 6.2, 13.8 Hz, 1H, PheHβ), 3.23 (dd,
J = 6.8, 13.8 Hz, 1H, PheHβ), 3.64 (dd, J = 3.1, 8.9 Hz, 1H,
D-OxdH5), 3.70–3.79 (m, 2H, GlyHα), 4.30 (t, J = 8.6 Hz, 1H,
D-OxdH5), 4.64 (q, J = 7.1 Hz, 1H, PheHα), 4.72 (dd, J = 2.9,
9.0 Hz, 1H, D-OxdH4), 5.13 (quint, 7.1 Hz, 1H, AlaHα), 6.62
(br.s, 1H, CONH2), 7.05 (br.s, 1H, CONH2), 7.11–7.25 (m, 5H,
ArH), 7.90 (d, J = 8.4 Hz, 2H, ArH), 8.02 (t, J = 7.9 Hz, 1H,
GlyNH), 8.29 (d, J = 8.0 Hz, 1H, PheNH), 8.35 (d, J = 8.4 Hz,
2H, ArH), 8.40 (d, J = 9.3 Hz, 1H, AlaNH); 13C NMR (DMSO-
d6) δ 18.0, 37.6, 42.2, 51.6, 56.0, 58.2, 62.4, 125.2, 125.9,
127.8, 128.8, 128.6, 136.7, 150.4, 151.3, 153.9, 169.9, 171.7,
172.3, 173.2; RP-HPLC (see General methods) 7.85 min;
ES-MS m/z 591.2 [M + 1], calcd 591.1; Elem. Anal. for
1
1649, 1530 cm−1; H NMR (CDCl3) δ 1.31 (d, J = 7.3 Hz, 3H,
AlaMe), 2.39 (s, 3H, TsMe), 3.01–3.16 (m, 2H, PheHβ), 4.26
(dd, J = 4.2, 9.3 Hz, 1H, OxdH5), 4.44 (t, J = 9.3 Hz, 1H,
OxdH5), 4.63 (q, J = 7.4 Hz, 1H, PheHα), 4.90 (dd, J = 4.2, 8.6
Hz, 1H, OxdH4), 5.12 (dq, J = 7.4, 9.3 Hz, 1H, AlaHα), 6.03
(br.s, 1H, CONH2), 6.35 (br.s, 1H, CONH2), 6.40 (d, J = 9.3 Hz,
1H, AlaNH), 7.09–7.26 (m, 7H, ArH), 7.75 (d, J = 8.4 Hz, 2H,
ArH), 7.96 (d, J = 8.0 Hz, 1H, PheNH); 13C NMR (2 : 1 CDCl3–
DMSO-d6) 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; RP-HPLC (see General methods) 7.01 min;
2314 | Org. Biomol. Chem., 2012, 10, 2307–2317
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C24H26N6O10S, calcd: C 48.81, H 4.44, N 14.23, S, 5.43; found:
C 48.77, H 4.47, N 14.19, S, 5.47.
treated as described above for the synthesis of 9, giving 12a
(0.20 g 88%, 94% pure by analytical RP-HPLC). IR (nujol) ν,
1788, 1768, 1719; H NMR (CDCl3) δ 1.63 (d, J = 6.0 Hz, 3H,
1
5-Me), 2.46 (s, 3H, TsMe), 3.82 (s, 3H, COOMe), 4.39 (dd, J =
3.2, 9.2 Hz, 1H, Oxd1H5), 4.64 (d, J = 5.0 Hz, 1H, Oxd2H4),
4.73 (br.t, 2H, Oxd1H5 + Oxd2H5), 6.04 (dd, J = 4.0, 10.0 Hz,
1H, Oxd1H4), 7.36 (d, J = 8.0 Hz, 2H, ArH), 7.99 (d, J = 8.0
Hz, 2H, ArH); 13C-NMR (9 : 1 CDCl3–DMSO-d6) δ 20.6, 21.3,
53.5, 57.6, 60.6, 65.0, 75.1, 128.9, 129.0, 133.7, 145.6, 151.2,
152.0, 167.1, 167.7; RP-HPLC (see General methods) 8.33 min;
ES-MS m/z 427.3 [M + 1], calcd 427.1; Elem. Anal. for
C17H18N2O9S, calcd: C 47.89, H 4.25, N 6.57, S 7.52; found: C
47.93, H 4.2 N 6.60, S 7.49.
Di-Oxd-peptide synthesis
Ts-Oxd1-Oxd2-NH2 (7). To a stirred solution of Ts-Ser-Ser-
NH2 (6, 0.2 g, 0.58 mmol) in 3 : 1 DCM–DMF (4 mL), DSC
(0.37 g, 1.45 mmol) and DIPEA (10 μL, 0.06 mmol) were
added at r.t. under inert atmosphere. After 4 h, work up was per-
formed as described above for 2a, giving 7 (0.189 g, 82%, 95%
pure by analytical RP-HPLC). IR (nujol) ν 1776, 1770, 1725,
1
1711; H NMR (CDCl3) δ 2.43 (s, 3H, Me), 4.37 (dd, J = 2.8,
9.2 Hz, 1H, Oxd1H5), 4.48 (dd, J = 3.6, 9.6 Hz, 1H, Oxd2H5),
4.62–4.71 (m, 2H, Oxd1H5+Oxd2H5), 5.00 (dd, J = 2.8, 8.8 Hz,
1H, Oxd2H4), 5.99 (dd, J = 3.6, 9.6 Hz, 1H, Oxd1H4), 7.07 (s,
1H, CONH2), 7.35 (d, J = 8.1 Hz, 2H, ArH), 7.76 (s, 1H,
CONH2), 7.93 (d, J = 8.1 Hz, 2H, ArH); 13C NMR (9 : 1
CDCl3–DMSO-d6) δ 21.1, 50.6, 57.4, 62.9, 63.2, 128.2, 129.7,
132.7, 137.9, 151.4, 154.1, 167.9, 170.6; RP-HPLC (see
General methods) 5.7 min; ES-MS m/z 398.0 [M + 1], calcd
398.1; Elem. Anal. for C15H15N3O8S, calcd: C 45.34, H 3.80, N
10.57, S 8.07; found: C 45.28, H 3.82, N 10.60, S 8.10.
Ts-D-Oxd1-(5-Me-Oxd2)-OMe (12b). The reaction of Ts-D-
Ser-ThrOMe (11b, 0.20 g. 0.53 mmol) under the same con-
ditions as described for 9 gave 12b (0.196 g, 86%, 94% pure by
1
analytical RP-HPLC). IR (nujol) ν, 1786, 1760, 1724; H NMR
(CDCl3) δ 1.53 (d, J = 6.0 Hz, 3H, 5-Me), 2.45 (s, 3H, TsMe),
3.90 (s, 3H, COOMe), 4.27 (dd, J = 3.2, 10.0 Hz, 1H, Oxd1H5),
4.53 (d, J = 5.3 Hz, 1H, Oxd2H4), 4.72 (t, J = 9.2 Hz, 1H,
Oxd1H4), 4.74 (t, J = 6.0 Hz, 1H, Oxd2H5), 5.98 (dd, J = 3.2,
9.6 Hz, 1H, Oxd1H4), 7.33 (d, J = 8.0 Hz, 2H, ArH), 7.96 (d,
J = 8.0 Hz, 2H, ArH); 13C NMR (2 : 1 CDCl3–DMSO-d6)
δ 20.9, 21.7, 53.5, 58.2, 61.7, 65.2, 75.3, 129.3, 129.4, 134.2,
145.7, 151.3, 152.2, 167.0, 167.6; RP-HPLC (see General
methods) 8.33 min; ES-MS m/z 427.3 [M + 1], calcd 427.1;
Elem. Anal. for C17H18N2O9S, calcd: C 47.89, H 4.25, N 6.57,
S 7.52; found: C 47.93, H 4.2 N 6.60, S 7.49.
Ts-Oxd1-Oxd2-OMe (9). A stirred solution of Ts-Ser-Ser-OMe
(8, 0.2 g, 0.56 mmol) in DCM (4 mL), was treated as described
above for the synthesis of 7. The same work up gave 9 (0.187 g,
81%, 94% pure by analytical RP-HPLC) and 10 (0.016 g, 8%),
separated by flash chromatography.
1
(9). IR (nujol) ν 1779, 1769, 1721; H NMR (CDCl3) δ 2.45
(s, 3H, Me), 3.82 (s, 3H, COOMe), 4.39 (dd, J = 3.6, 9.6 Hz,
1H, Oxd1H5), 4.50 (dd, J = 2.2, 9.1 Hz, 1H, Oxd2H5), 4.74 (t,
J = 9.4 Hz, 1H, Oxd1H5), 4.76 (t, J = 9.3 Hz, 1H, Oxd2H5),
5.08 (dd, J = 1.8, 9.1 Hz, 1H, Oxd2H4), 6.00 (dd, J = 3.4,
9.6 Hz, 1H, Oxd1H4), 7.36 (d, J = 8.0 Hz, 2H, ArH), 7.98
(d, J = 8.0 Hz, 2H, ArH); 13C NMR (2 : 1 CDCl3–DMSO-d6)
δ 21.0, 51.5, 52.1, 55.7, 62.1, 62.5, 128.9, 129.3, 133.0, 137.4,
151.3, 153.3, 165.1, 166.6; RP-HPLC (see General methods)
7.81 min; ES-MS m/z 413.0 [M + 1], calcd 413.1; Elem. Anal.
for C16H16N2O9S, calcd: C 46.60, H 3.91, N 6.79, S 7.78;
found: C 46.55, H 3.88, N 6.76, S 7.75.
Di-Oxd-peptide solid-phase synthesis
Ts-Oxd1-Phe2-Oxd3-Phe4-OH (14). Wang resin pre-loaded
with Fmoc-Phe (0.5 g, 0.4–0.8 mmol g−1, resin particle size:
100–200 mesh) was introduced in one reactor of an automated
synthesizer apparatus.
Fmoc was removed with 4 : 1 DMF–piperidine (5 mL) under
mechanical shaking. After 15 min, the suspension was filtered,
the resin was washed with DCM (5 mL) and treated while
shaking with a second portion of 4 : 1 DMF–piperidine. After
40 min, the suspension was filtered, and the resin was washed
three times in sequence with DCM (5 mL) and CH3OH (5 mL).
The resin was swollen in DCM (5 mL), and a solution of the
N-protected amino acid (1.2 equiv.) and HOBt (1.2 equiv.) in
DMF (4 mL) was added, followed by DCC (1.2 equiv.). The
mixture was mechanically shaken, and after 3 h the resin was
filtered and washed three times with the sequence DCM (5 mL),
CH3OH (5 mL). Coupling efficacy was determined by means of
the Kaiser test.
The resin-bound peptide was suspended in 5 : 1 DCM–DMF
(5 mL), and DSC (5 equiv.) and DIPEA (0.3 equiv.) were added
at r.t. under inert atmosphere. After 3 h the mixture was filtered,
and the resin-bound peptide was washed three times in sequence
with DCM (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 DCM (8 mL), and mechanically shaken at r.t. After
2 h the mixture was filtered, the resin was washed twice with 5%
TFA in Et2O (5 mL), twice with Et2O (5 mL). The cleavage
Ts-Oxd-Dha-OMe (10). A stirred solution of 8 (0.20 g,
0.56 mmol) in DMF (3.0 mL) was treated as described above for
the synthesis of 7. The work up gave 10 (0.169 g, 82%, 94%
pure by analytical RP-HPLC) and 9 (9.2 mg, 4%), isolated by
flash chromatography.
(10). IR (nujol) ν 1768, 1721, 1715; 1H NMR (CDCl3) δ 2.42
(s, 3H, Me), 3.88 (s, 3H, COOMe), 4.45 (dd, J = 4.6, 9.0 Hz,
1H, OxdH5), 4.52 (t, J = 9.2 Hz, 1H, OxdH5), 4.99 (dd, J = 4.4,
9.2 Hz, 1H, OxdH4), 6.05 (s, 1H,vCH), 6.65 (s, 1H,vCH),
7.34 (d, J = 8.4 Hz, 2H, ArH), 7.93 (d, J = 8.4 Hz, 2H, ArH),
8.54 (s, 1H, DhaNH); 13C NMR (2 : 1 CDCl3–DMSO-d6) δ
21.4, 52.6, 57.9, 66.0, 111.0, 128.7, 128.9, 129.1, 129.1, 131.0,
133.8, 145.4, 151.5, 163.6, 166.8; RP-HPLC (see General
methods) 7.47 min; ES-MS m/z 369.1 [M + 1], calcd 369.1;
Elem. Anal. for C15H16N2O7S, calcd: C 48.91, H 4.38, N 7.60,
S 8.70; found: C 48.88, H 4.35, N 7.57, S 8.68.
Ts-Oxd1-(5-Me-Oxd2)-OMe (12a). A stirred solution of Ts-
Ser-ThrOMe (11a, 0.20 g. 0.53 mmol) in DCM (4 mL), was
This journal is © The Royal Society of Chemistry 2012
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View Online
procedure was repeated, and all of the filtrates and washes were
collected; solvent and volatiles were removed under N2 flow at r.
t. The resulting residue was suspended in Et2O, and the crude
solid which precipitated was triturated and collected by centri-
fuge. The Oxd-peptide acid 14 was isolated by semipreparative
RP-HPLC (General methods) (80%, 96% pure by analytical
RP-HPLC). IR (nujol) ν 3300–2900, 1784, 1776, 1727, 1719;
1715; 1H NMR (9 : 1 CDCl3–DMSO-d6) δ 2.37 (s, 3H, Me),
2.70 (dd, J = 3.0, 14.0 Hz, 1H, Phe2Hβ), 3.17 (dd, J = 6.0, 13.7
Hz, 1H, Phe4Hβ), 3.27 (dd, J = 3.8, 14.0 Hz, 1H, Phe2Hβ), 3.41
(dd, J = 5.8, 13.7 Hz, 1H, Phe4Hβ), 3.98 (dd, J = 4.0, 8.4 Hz,
1H, Oxd3H5), 4.25 (t, J = 9.0 Hz, 1H, Oxd3H5), 4.45 (t, J = 8.9
Hz, 1H, Oxd1H5), 4.52 (dd, J = 4.1, 8.3 Hz, 1H, Oxd1H5), 4.64
(dd, J = 4.1, 7.9 Hz, 1H, Oxd3H4), 4.79 (dd, J = 3.8, 8.4 Hz,
1H, Oxd1H4), 4.83 (q, J = 6.6 Hz, 1H, Phe4Hα), 5.78 (q, J = 6.8
Hz, 1H, Phe2Hα), 6.54 (d, J = 7.6 Hz, 1H, Phe4NH), 6.67 (d,
J = 7.2 Hz, 1H, Phe2NH), 7.15–7.30 (m, 12H, Phe2ArH +
Phe4ArH + TsArH), 7.76 (d, J = 8.4 Hz, 2H, ArH); 13C NMR
(2 : 1 CDCl3–DMSO-d6) δ 23.9, 36.1, 37.8, 53.4, 54.1, 56.2,
59.2, 61.9, 62.8, 125.9, 127.7, 127.7, 128.3, 128.6, 128.8,
128.9, 129.3, 129.5, 133.7, 136.6, 136.6, 137.6, 151.9, 153.1,
171.7, 171.7, 174.7, 175.2; RP-HPLC (see General methods)
10.39 min; ES-MS m/z 693.2 [M + 1], calcd 693.2; Elem. Anal.
for C33H32N4O11S, calcd: C 57.22, H 4.66, N 8.09, S, 4.63;
found: C 57.18, H 4.69, N 8.12, S, 4.64.
sensitive mode and processed using a 90°-shifted, squared sine-
bell apodization. 2D ROESY experiments were recorded in the
biomimetic medium 8 : 2 DMSO-d6–H2O, with a 250 ms mixing
time with a proton spectral width of 3088 Hz. Peaks were cali-
brated on DMSO.
ROESY and molecular dynamics. Only ROESY-derived con-
straints were included in the restrained molecular dynamics.
Cross-peak intensities were classified as very strong, strong,
medium, and weak, and were associated with distances of 2.2,
2.6, 3.0, and 4.5 Å, respectively. Geminal couplings and other
obvious correlations were discarded. For the absence of Hα(i,
i + 1) ROESY cross peaks, all of the ω bonds were set at 180°
(force constant: 16 kcal mol−1 Å−2). The restrained MD simu-
lations were conducted using the AMBER force field48 in a 30 ×
30 × 30 Å box of standard TIP3P models of equilibrated water.49
All water molecules with atoms that come closer than 2.3 Å to a
solute atom were eliminated. A 100 ps simulation at 1200 °C
was used for generating 50 random structures that were sub-
sequently subjected to 50 ps restrained MD with a 50% scaled
force field at the same temperature, followed by 50 ps with full
restraints (distance force constant of 7 kcal mol−1 Å−2), after
which the system was cooled in 20 ps to 50 °C. H-bond inter-
actions were not included, nor were torsion angle restraints. The
resulting structures were minimized with 3000 cycles of steepest
descent and 3000 cycles of conjugated gradient (convergence of
0.01 kcal Å−1 mol−1). The backbones of the structures were
clustered by the rmsd analysis module of HyperChem.44
Theoretical computations
Unrestrained MD simulation was performed in a 30 × 30 ×
30 Å box of standard TIP3P water for 10 ns at 298 °C, at con-
stant temperature and pressure (Berendsen scheme,50 bath relax-
ation constant of 0.2). For 1–4 scale factors, van der Waals and
electrostatic interactions are scaled in AMBER to half their
nominal value. The integration time step was set to 0.1 fs.
All theoretical calculations were performed employing the
HyperChem package.47 The structures of the product D, and of
the plausible reaction intermediates A, C, and D, were calculated
employing ab initio molecular orbital (MO) theory. A systematic
conformational analysis for the structures was done at the HF/6-
31G* level. The conformers were re-optimized at the HF/6-
31G** level. Optimization was performed by conjugate gradient
algorithm, convergence at 0.001; energies are expressed in kcal
mol−1. The following molecules were included in the compu-
tations of A–D: A, DIPEA; B, DIPEAH+; C, DIPEAH+; D,
1-hydroxypyrrolidine-2,5-dione, and DIPEA.
Acknowledgements
We thank MIUR (PRIN 2008), “Fondazione del Monte di
Bologna e Ravenna”, the Italian Minister for Foreign Affairs
(bilateral proj. Italy-Mexico 2011–13), Fondazione per la
Ricerca sulla Fibrosi Cistica, Verona (FFC#11/2011) for
financial support, and EC (COST Action CM0803). We also
warmly thank Dr. Silvia Pieraccini for ECD measurements.
Conformational analysis
Circular dichroism. ECD spectra were recorded from 200 to
300 nm at 25 °C. Solutions were made up in spectral grade sol-
vents and run in a 0.01 cm quartz cell. For each sample the
absorbance value was set to 1.0 at λmax (225–260 nm); concen-
trations used were in the range 5–11 mM. Data are reported in
ellipticity (millidegree).
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