In-peptide synthesis of di-oxazolidinone and dehydroamino acid-oxazolidinone motifs as β-turn inducers

Article abstract of DOI:10.1039/c3ob40357b

Small and easy-to-do mimetics of β-turns are of great interest to interfere with protein-protein recognition events mediated by β-turn recognition motifs. We propose a straightforward procedure for constraining the conformation of tetrapeptides lacking a pre-formed scaffold. According to the stereochemistry array, N-Ts tetrapeptides including Thr or PhSer (phenylserine) at the positions 2 or 3 gave rise in a single step to the sequences Oxd ²-Oxd³ or ΔAbu²-Oxd³ (Oxd, oxazolidin-2-one; ΔAbu, 2,3-dehydro-2-aminobutyric). These pseudo-Pro residues displayed highly constrained, and χ dihedral angles, and induced clear β-turns or inverse turns of type I or II, as determined by extensive spectroscopic and computational analyses. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40357b

Organic&
BiomolecularChemistry
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Volume 11 | Number 26 | 14 July 2013 | Pages 4273–4420
ISSN 1477-0520
PAPER
Luca Gentilucci et al.
In-peptide synthesis of di-oxazolidinone and dehydroamino
acid–oxazolidinone motifs as β-turn inducers
1477-0520(2013)11:26;1-7

Organic &
Biomolecular Chemistry
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PAPER
In-peptide synthesis of di-oxazolidinone and
dehydroamino acid–oxazolidinone motifs as β-turn
inducers†
Cite this: Org. Biomol. Chem., 2013, 11,
4316
Rossella De Marco, Arianna Greco, Sebastiano Rupiani, Alessandra Tolomelli,
Claudia Tomasini, Silvia Pieraccini and Luca Gentilucci*
Small and easy-to-do mimetics of β-turns are of great interest to interfere with protein–protein recog-
nition events mediated by β-turn recognition motifs. We propose a straightforward procedure for con-
straining the conformation of tetrapeptides lacking
a pre-formed scaffold. According to the
stereochemistry array, N-Ts tetrapeptides including Thr or PhSer (phenylserine) at the positions 2 or
3 gave rise in a single step to the sequences Oxd2-Oxd3 or ΔAbu2-Oxd3 (Oxd, oxazolidin-2-one; ΔAbu,
2,3-dehydro-2-aminobutyric). These pseudo-Pro residues displayed highly constrained ϕ, ψ, and χ dihedral
angles, and induced clear β-turns or inverse turns of type I or II, as determined by extensive spectroscopic
and computational analyses.
Received 18th February 2013,
Accepted 10th April 2013
DOI: 10.1039/c3ob40357b
www.rsc.org/obc
compounds that can mimic or disrupt β-turn-mediated recog-
Introduction
nition events.3
Interactions between proteins (PPI) are important for the
However, the preparation of the scaffolds and their incor-
majority of biological functions and cellular signaling.1 Sys- poration in the sequence may require multi-step procedures,
tematic studies have revealed that there is typically a small resulting in low overall yields. Besides, most synthetic
cluster of residues at the interface of the proteins that contrib- methods lack flexibility and are therefore not suited to intro-
utes the majority of the recognition or binding affinity. Very duce diversity.3 As a consequence, the development of a high
often, these key regions adopt well defined turn structures.2 As yielding, single step procedure for the introduction of a confor-
a consequence, small molecules mimicking the 3D features of mational constraint that forces the peptide to adopt a β-turn is
the turn regions can find applications as therapeutic agents.
currently of considerable interest.
The β-turn occurs where the polypeptide backbone reverses
In this work we report a straightforward procedure for con-
direction, and consists of four amino acid residues i to i + 3 in straining the conformation of tetrapeptides lacking a pre-
which the distance between Cαi and Cαi + 3 is about 7 Å; formed scaffold. The resulting β-turn mimetics include 2-oxo-
according to the dihedral angles ϕ and ψ of the residues i + 1 1,3-oxazolidine-4-carboxylate rings (or oxazolidin-2-one, in
and i + 2, the turns are classified in different types.
short: Oxd), that we recently proposed as constrained pseudo-
Small mimetics of β-turns, such as the privileged structures Pro residues.4
benzodiazepines, Freidinger lactams, internal or external
Pro strongly impacts the structural and conformational pro-
bicyclic dipeptide mimetics, spirocyclic dipeptides, etc., have perties of peptide and protein backbones and their molecular
been extensively investigated and utilized to discover recognition.5 It does not have a hydrogen on the amide group
and therefore cannot act as a H-bond donor. The cyclic struc-
ture forces the ϕ angle to about −65°, therefore Pro is known
Department of Chemistry “G. Ciamician”, University of Bologna, via Selmi 2, 40126
as a classical breaker of both the α-helical and β-sheet struc-
Bologna, Italy. E-mail: luca.gentilucci@unibo.it; Fax: +39 0512099456;
tures in proteins and peptides, while it promotes the for-
Tel: +39 0512099570
mation of β-turns.2,6
†Electronic supplementary information (ESI) available: 1H-NMR of 2a, c, d, and
3b, c in 8 : 2 DMSO-d6–H2O; selected 1H-NMR chemical shifts of 2a, c, d, and 3b,
Besides, due to the small free enthalpy difference between
the cis and trans Xaa-Pro bond isomers of 2.0 kJ mol−1 (com-
c in different solvents; amide NH FT-IR of 2d, and 3b; NH chemical shift of 2a
pared to 10.0 kJ mol−1 for other peptide bonds), there is a rela-
and 3c in CDCl3–DMSO-d6 0–8%; ROESY cross-peaks for 2a, c, d, and 3b, c;
Structure of the intermediate anion A; ϕ and ψ angles of the residues i + 1, i + 2
tively high intrinsic probability of 30% cis conformation at r.t.7
for 2a, c, d, and 3b, c; ECD spectra of 2a, 2d, and 3b in DCM and MeOH; repro-
ductions of the 1H- and 13C-NMR of compounds 2 and 3. See DOI: 10.1039/
Interestingly, the cis–trans interconversion of X-Pro is one of
the rate-determining steps in protein folding.8
c3ob40357b
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In order to understand the relationship between peptide
bond geometry and bioactivity, many synthetic Pro analogues
have been developed that provide restrictions of the Xaa-Pro
conformation. In general, the modifications consist in ring
substitutions with alkyl and aromatic groups, incorporation of
heteroatoms, or the expansion or contraction of the five-mem-
bered ring.9
To take advantage of the conformational bias exerted by the
Oxd pseudo-Pro, we planned to synthesize diastereomeric
N-sulfonyl tetrapeptides containing two consecutive Oxd rings,
as β-turn inducers. According to the stereochemistry array of
the amino acids, the reaction of two β-hydroxy α-amino acids
(Thr or threo-phenylserine, PhSer) already present within the
sequences at the positions 2 and 3, with bis(succinimidyl)car-
bonate (DSC), afforded two consecutive Oxd or a 2,3-dehydro-
2-aminobutyric acid (ΔAbu) in 2 and a Oxd in 3. These combi-
nations resulted in highly constrained extended or folded
structures, in particular turns or inverse turns of type I and II,
and are especially interesting because they can control not
only the ϕ and ψ dihedral angles of the backbone but also the
χ angles of the side chains.
Scheme 1 Different reactivity of the N-carbamate and N-sulfonyl oligo-
peptides. (a) DSC, cat. base (B:).
Peptides containing Oxd rings constitute an infrequent but
remarkable class of peptidomimetics. They have found some
applications in medicinal chemistry,10 in the construction of
foldamers11 or as self-assembling scaffolds forming nano-
structures.12 Also the compounds with the ΔAbu-Oxd sequence
are of some interest, since they contain two distinct secondary
structure-forming elements. The dehydroamino acids are well
known β-turn inducers; for instance, sequential placement of
dehydroPhe (ΔPhe) in oligomers gave repeated β-turns
forming 310 helices.13
Scheme 2 Reactions of the sulfonyl-peptides 1a–e, containing L- or D-Thr at
positions 2 and 3, and of 1f, g containing L-Thr at 2 and threo L- or D-PhSer at 3,
respectively. (a) DSC, cat. DIPEA.
Results and discussion
Synthesis of tetrapeptides containing di-Oxd and ΔAbu-Oxd
at the positions 2 and 3, and we reacted the tetrapeptides
(Scheme 2) with a moderate excess of DSC and a catalytic
Very recently, we reported the synthesis of Oxd rings directly amount of diisopropylethylamine (DIPEA).
within peptide sequences, by treatment of N-arylsulfonyl pep-
The preparation of the N-tosyl tetrapeptides 1a–g was con-
tides containing L- or D-configured Ser with DSC and a catalytic ducted by coupling the amino acids under MW irradiation,
amount of a base, in solution or in the solid phase (Scheme 1, using a microwave oven specifically designed for organic syn-
path a).4
thesis,14 with HBTU/HOBt as activating agents (Experimental
Apparently, the reaction was due to the presence of the section, Table 3). N-Tosyl (Ts)-Ala was prepared as described in
N-sulfonyl group; indeed, the reaction of the corresponding the literature;15 L- and D-threo-PhSer were obtained via optical
Fmoc- or Boc-peptides under the same conditions with car- resolution of the racemate,16 since the resolution by enzymatic
bonates or dicarbonates gave elimination to dehydro- hydrolysis17 gave poor results.
alanine (ΔAla), as reported in the literature (Scheme 1,
The reaction of Ts-Ala-Thr-Thr-PheOMe (1a) with 2.2 equiv.
of DSC in 3 : 1 DCM–DMF and in the presence of 0.1 equiv. of
path b).13
Interestingly, peptides having the sulfonyl group directly DIPEA (Scheme 2, entry 1) provided the corresponding di-Oxd-
connected to the Ser, or separated by one or two amino acids, containing Ts-Ala-Oxd-Oxd-PheOMe (2a) in excellent yield after
gave similar results (Scheme 1, Xaa = 0, 1, or 2 residues).4 This isolation by flash chromatography (Table 1).
observation prompted us to perform in a single step the simul-
On the other hand, the reaction of Ts-Ala-D-Thr-Thr-
taneous cyclization of sulfonyl-oligopeptides containing two PheOMe (1b), under the same reaction conditions, gave exclu-
consecutive ring-forming residues, to afford highly rigid sively a tetrapeptide containing ΔAbu at position 2, Ts-Ala-
structures.
ΔAbu-Oxd-PheOMe (3b), while the tetrapeptide containing two
Accordingly, we prepared a series of diastereomeric tetra- Oxd (2b) was observed only in trace amounts (Scheme 2, and
peptides containing two Thr residues, or Thr and threo-PhSer, Table 1, entry 2).
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Table 1 Synthesis of the constrained peptidomimetics 2 or 3 from the stereoisomers 1
Entry
1
Ala1
Thr2
Xaa3,
R
Phe4
Solventa
2b (%)
3b (%)
1
2
3
4
5
6
7
8
9
a
b
c
c
c
d
e
f
L
L
L
”
”
L
L
L
L
L
D
L
”
”
L
D
L
L
L
Me
Me
Me
Me
Me
Me
Me
Ph
L
L
L
”
”
D
D
L
L
DCM–DMF
DCM–DMF
DCM–DMF
DCM
87
—
67
85
5
90
tr.c
92
93
tr.
90
23
9
78
—
tr.c
—
tr.
L
D
”
”
D
L
DMF
DCM–DMF
DCM and/or DMF
DCM–DMF
DCM–DMF
L
g
D
Ph
a All compounds 1a–g were tested in different solvent mixtures (3 : 1 DCM–DMF, DCM, DMF); with the exception of entries 3–5, the reaction
outcomes were practically unaffected, therefore these results are not shown. b Determined after purification by flash chromatography over silica
gel; tr. = traces. c The rest being the reagent.
The reaction of Ts-Ala-Thr-D-Thr-PheOMe (1c) under the behaved in a similar way to the corresponding peptides 1a and
same conditions gave a 74 : 26 mixture of Ts-Ala-Oxd-D-Oxd- 1c (the formation of 3g was observed only in trace amounts in
PheOMe (2c) and Ts-Ala-ΔAbu-D-Oxd-PheOMe (3c) (entry 3). different solvents).
The reaction outcome changed upon varying the solvent uti-
The proposed reaction mechanism of Scheme 1, path a,
lized; in pure DCM the yield of 2c increased to 85% (entry 4), postulates that for n = 1 or 2, as occurring for the compounds
while in DMF the reaction afforded mainly 3c, with a 78% 1a–g, the electron-poor aromatic ring of the arylsulfonylamido
yield (entry 5).
group might effectively stabilize the anionic intermediate A by
The tetrapeptide Ts-Ala-Thr-D-Thr-D-PheOMe (1d) exclu- π-stacking interaction (ESI†), thus driving formation of the Oxd
sively gave Ts-Ala-Oxd-D-Oxd-D-PheOMe (2d) (entry 6), while Ts- ring. Possibly, the diastereoisomer 1b adopted in solution a
Ala-D-Thr-Thr-D-PheOMe (1e) failed to give either 2 or 3 in sig- conformation which allowed promoting the cyclization at posi-
nificant amounts (entry 7) under a variety of reaction con- tion 3, but not at 2, therefore giving elimination, as expected
ditions (not shown).
on the basis of the literature. For 1c, the different results
Finally, the reaction was repeated with the sequences Ts- observed for the entries 3 to 5 could be attributed to a prefer-
Ala-Thr-PhSer-PheOMe (1f) or Ts-Ala-Thr-D-PhSer-PheOMe ence for different conformations in the different solvents. As
(1g), which gave the di-Oxd compounds 2f and 2g in very satis- for 1e, showing alternating absolute stereochemistry, it
factory yields (entries 8 and 9). These peptides include a 2-oxo- appeared that the compound was unable to achieve cyclization
5-phenyloxazolidine-4-carboxylate residue at position 3, which either at 2 or 3; the compound did not eliminate to a signifi-
might effectively represent a constrained Phe mimetic, with cant extent either, as observed by treatment with different
fixed ϕ, ψ and χ dihedral angles (Table S7†).
bases in different solvents at r.t. or under heating.
This result suggests the opportunity to develop analogues
The mild cleavage of the arylsulfonyl group19 was discussed
of proteinogenic or unusual amino acids, carrying the side previously.4,20 The tosyl group was removed in good yield with
chain at the position 5 of the Oxd ring. Indeed, the literature iodotrimethylsilane,21 while the treatment with SmI2–pyrroli-
reports many procedures for the stereocontrolled preparation dine–water22 was less efficient.
of a variety of β-hydroxy α-amino acids.18 Nevertheless, the syn-
thesis of Oxds functionalized with different side chains is
beyond the scope of this work, which primarily addresses the
Conformational analyses of the di-Oxd and ΔAbu-Oxd peptides
synthesis of model tetrapeptides and investigation of the sec- To investigate the conformational bias exerted by the two con-
ondary structures.
secutive L- or D-configured Oxd, or ΔAbu and L- or D-Oxd resi-
Epimerization during the reactions was excluded on the dues at positions 2 and 3, on the overall structure of the
basis of the comparison of the NMR and HPLC analyses of the peptides, we analyzed the model compounds 2a, 2c, 2d, and
compounds, including the HPLC analysis on a chiral station- 3b, 3c, by FT-IR and NMR spectroscopy.
ary phase (see General methods).
FT-IR spectroscopy was utilized to analyze the amide N–H
From the comparison of the results (entries 1–9) it stretching regions; generally, non H-bonded amide protons
appeared that the stereochemistry array exerted a major bias in show a peak above 3400 cm−1, while H-bonded amide NH
determining the cyclization versus the elimination at position bonds exhibit a peak below 3400 cm−1 23
.
2. Homochiral sequences at positions 1 and 2 tended to give
The FT-IR absorption spectrum of 2d only showed a strong
exclusively or predominantly the di-Oxd compounds, as band above 3400 cm−1 (ESI, Fig. S1†). The spectra of 2a, 2c, 3b,
observed for 1a, 1c, and 1d. On the contrary, the peptide 1e and 3c (ESI, Fig. S1†) showed a major peak at about
containing the sequence L-Ala-D-Thr failed to give 2 or 3 in sig- 3400 cm−1, and a second peak at about 3340 cm−1. The latter
nificant amounts, and 1b solely afforded the ΔAbu-Oxd became predominant in 3c, suggestive of a significant popu-
sequence. The diastereomeric peptides 1f, 1g containing PhSer lation of H-bonded conformations in equilibrium with non-
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Fig. 1 Amide NH stretching regions of the IR absorption spectra for samples of
tetrapeptide 3c (3 mM in DCM, r.t.).
Fig. 2 Variation of NH proton chemical shift (ppm) of 3c as a function of
increasing percentages of DMSO-d6 to the CDCl3 solution (v/v).
Table 2 Δδ/Δt values (ppb K−1) for the amide protons of 2a, 2c, 2d, and 3b,
3c, in CDCl3 and 8 : 2 DMSO-d6–H2O; solvents: S1 = CDCl3; S2 = 8 : 2 DMSO-d6–
H2O; amino acid stereochemistry has been omitted
bonded structures. The spectrum of 3c dissolved in DCM is
reported in Fig. 1.
1
The H-NMR experiments were done at 400 MHz in CDCl3
Compd
2a
Solvent
Ala1NH
ΔAbu2NH
Phe4NH
or in 8 : 2 DMSO-d6–H2O. 2D gCOSY experiments were utilized
for the unambiguous assignment of the resonances. In each
solvent, all spectra showed a single set of sharp resonances,
consistent with conformational homogeneity or a fast equili-
brium between slightly different conformers. For most com-
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
−2.7
−6.1
−13.5
−5.0
−2.3
−6.3
−9.0
−6.5
−1.7
−6.0
—
—
−2.4
−3.9
−3.8
−4.1
−1.2
−3.8
−4.0
−4.1
−2.7
−4.2
3b
−8.0
−7.2
—
2c
1
—
pounds, the H-NMR resonances showed modest variations of
3c
−4.4
−7.0
—
the chemical shifts in the two solvents (Table S1†) suggesting
that the peptidomimetic sequences were conformationally
stable; exceptions are highlighted in Table S1.†
2d
—
It has been shown that the Oxd confers on the preceding
amide bond an exclusive trans conformation.11 The 1H-NMR
analyses of all of the compounds showed a significantly down-
field position of the Hα proton of the residues preceding the
Oxd rings (ESI, Table S1†). For instance, the resonances of
AlaHα in Ts-Ala-Thr-Thr-PheOMe (1a) and Ts-Ala-ΔAbu-L-(5′-
Me-Oxd)-PheOMe (3b) appeared at about 3.9 and 3.8 ppm
(CDCl3), respectively, while in Ts-Ala-(5′-Me-Oxd2)-(5′-Me-Oxd3)-
PheOMe (2a) AlaHα was at about 5.2 ppm. On the other hand,
in the same compound 2a, Oxd3H4 was at 4.2 ppm, while
Oxd2H4 was at about 5.3 ppm, for the deshielding effect of
Oxd3CvO. These observations were compatible with a non-
conventional, CH⋯OvC intramolecular H-bond,24 confirming
the trans conformation of the amide bond between the Oxd
and the preceding, deshielded residue.
The occurrence of intramolecular H-bonds in 2a, 2c, 2d, 3b,
and 3c, was evaluated by analyzing the variation of the NH
proton chemical shifts upon addition of increasing percen-
tages of DMSO-d6 to 2 mM solutions of the compounds in
CDCl3. As representative example, the titration curves of 3c are
shown in Fig. 2. The titration of 2a gave no evidence of
H-bonds involving PheNH nor AlaNH, since these signals exhibi-
ted a considerable downfield shift;25 2c, 2d gave similar results
(ESI†). On the contrary, the titration of 3c (Fig. 2) and 3b (ESI†)
revealed that PheNH, but not AlaNH nor ΔAbuNH, was much
less sensitive, therefore less accessible, accounting for a
H-bonded structure.
of PheNH with respect to AlaNH (and ΔAbuNH) were sugges-
tive of a moderate preference for conformations having PheNH
involved in a H-bond (|Δδ/Δt| < or close to 2.5 ppb K−1).23,26
The only exception was observed for 2d in CDCl3, since the Δδ/
Δt (ppb K−1) of AlaNH was lower than that of PheNH (−1.7
and −2.7, respectively).
From the comparison of the results above discussed for the
Oxd–Oxd compounds (2), it appeared that the possible exist-
ence or not of H-bonds was differently supported by the
methods employed. Reasonably, the inconsistencies reflected
the existence of equilibria between H-bonded and non-H-
bonded structures. As for the ΔAbu-Oxd compounds (3), all of
the evidence was highly coherent in support of a H-bonded
PheNH.
To determine the preferred conformations in solution and
their dynamic behaviours, the compounds were analyzed by
2D-ROESY in 8 : 2 DMSO-d6–H2O and molecular dynamics
(MD) simulations. DMSO-d6 or DMSO-d6–H2O mixtures have
been recommended by several authors as biomimetic media.27
Structures consistent with the spectroscopic analyses were
obtained by MD using the distances derived from ROESY as
constraints. The intensities of the ROESY cross-peaks were
ranked to infer the distances (Tables S2–6, ESI†). The ω bonds
were set at 180°, since the absence of Hαi-Hα(i + 1) cross-peaks
excluded cis peptide bonds. Simulations were conducted in a
box of explicit 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.
Variable temperature (VT)-1H-NMR experiments in CDCl3
and 8 : 2 DMSO-d6–H2O were also utilized to deduce the pres-
ence of intramolecular H-bonds (Table 2). For almost all com-
pounds, the comparatively lower VT-1H-NMR Δδ/Δt parameters
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Finally, the system was gradually cooled, and the structures
To investigate the dynamic behaviour of 2a (A) and (B),
were minimized with the AMBER force field.28 The results unrestrained MD were performed for 10 ns in a box of stan-
were clustered by the RMSD analysis of the backbone atoms.
dard TIP3P water molecules. The simulations showed the con-
For Ts-Ala-(5′-Me-Oxd)-(5′-Me-Oxd)-PheOMe (2a), the analy- version of one conformation into the other. The analyses of
sis gave two clusters comprising altogether more than 90% the trajectories of the conformer (A) revealed very few struc-
of the structures. For each cluster, the representative geome- tures compatible with a γ-turn centered on Oxd3 with an expli-
tries A and B with the lowest internal energy were selected and cit H-bond between PheNH and Oxd2CvO, while (B) gave no
analyzed (Fig. 3). The occurrence of these two different evidence of secondary structures.
structures, which differ almost exclusively by the opposite
For Ts-Ala-(5′-Me-Oxd)-D-(5′-Me-Oxd)-PheOMe (2c) and Ts-
orientation of the Phe residue, reflected the observation Ala-(5′-Me-Oxd)-(5′-Me-Oxd)-D-PheOMe (2d), the analyses gave
of contradictory cross-peaks: the cross-peaks PheNH-Oxd3H4 one major cluster each comprising about 80% of the struc-
and PheNH-Oxd3H5 accounted for the conformation A, tures. For each compound’s cluster, the representative geome-
while the cross peak PheNH-Oxd2H5 was coherent with tries with the lowest internal energy are shown in Fig. 3. These
B. Possibly, the two structures represented conformers in fast structures 2c (C) and 2d are compatible with a well-defined
equilibrium.
type II β-turn secondary structure (Table S7†). The structures
2c (C) and 2d were analyzed by unrestrained MD; besides
the different random conformations, the analysis of the
trajectories of 2c (C), but not of 2d, revealed an explicit H-
bond between PheNH and the AlaCvO, as shown in 2c (D),
Fig. 3.
The conformational analyses of the peptides 3b, 3c contain-
ing in their sequences ΔAbu2 and L- or D-Oxd3, respectively,
gave one cluster each, comprising the large majority of the
structures; the representative geometries are shown in Fig. 4.
Fig. 3 Top. Representative low-energy structures of 2a (A and B), 2c (C), and
2d consistent with ROESY analysis, calculated by restrained MD; low energy
structure of 2c (D) calculated by unrestrained MD. All structures determined in a
30 × 30 × 30 Å box of standard TIP3P water molecules. Backbones and Oxd are
rendered in balls and cylinders, the rest in sticks. The peptide bonds connecting
Oxd2-Oxd3 are shown horizontally and perpendicularly with respect to the
viewer. Bottom. Sketches of the extended structure of 2a (A), and of the folded
2c (D) and 2d.
Fig. 4 Top. Representative low-energy structures of 3b and 3c (E) consistent
with ROESY analysis, calculated by restrained MD; low energy structure of 3c (F)
calculated by unrestrained MD. All structures determined in a 30 × 30 × 30 Å
box of standard TIP3P water molecules. Backbones and Oxd are rendered in
balls and cylinders, the rest in sticks. The peptide bonds connecting ΔAbu2-Oxd3
are shown horizontally and perpendicularly with respect to the viewer. Bottom.
Sketches of the folded structures 3b and 3c (E).
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The calculated geometry of ΔAbu closely matched that of de- between the Oxd carbonyls and the Hα protons of the preced-
hydroamino acids reported in the literature.13,29
ing residues.
The structure 3b, stabilized by an explicit H-bond between
In particular, the heterochiral Oxd2-D-Oxd3 scaffold induced
AlaCvO and PheNH, was compatible with a type I β-turn. The a stable and well defined type II β-turn, albeit the H-bond
structure of 3c (E) nicely reproduced an inverse type I β-turn between the residues i − i + 3 was relatively loose. This ability
(Table S7†), and showed a H-bond between AlaCvO and to promote folded structures makes sense, and is consistent
PheNH. The unrestrained MD simulations also revealed a ten- with the observations on previously described Oxd-pep-
dency for structures characterized by a H-bond between tides.11,12 The oxazolidin-2-one ring can be regarded as a
PheCvO and AlaNH, such as the representative 3c (F), Fig. 4.
pseudo-Pro; it is well known that heterochiral linear oligopep-
To further confirm the presence of folded structures, we tides including a Pro show higher propensity to fold compared
used Electronic Circular Dichroism (CD) spectroscopy. Spectra to the homochiral sequences.23,26
of model peptides 2a, 2d, and 3b, were recorded both in
dichloromethane and in methanol.
The spectra in dichloromethane (Fig. 5) showed a negative
band centered at about 230 nm, less intense for 2a. These
Conclusions
spectral features could be indicative of a significant population In this paper we reported a straightforward, one-step pro-
of bent conformers, in particular in the case of 2d and 3b. In cedure for constraining a peptide backbone without the need
fact, a negative nπ* ECD band near 225 nm is generally for a pre-formed scaffold. The cyclization of two consecutive
observed in the presence of β-turn structures (I or II type).23,30 β-hydroxy amino acid residues (L/D-Thr or threo-L/D-PhSer)
The observed different intensities of ECD signals were in embedded in N-arylsulfonyl tetrapeptide sequences afforded
agreement with ROESY and MD data which evidenced homo- in excellent yields the peptides containing Oxd2-L/D-Oxd3 (2) or
geneous conformations for 2d and 3b.
By moving from dichloromethane to methanol, intensities the precursors.
ΔAbu2-L/D-Oxd3 (3), according to the stereochemistry array of
of the negative ECD bands slightly reduced for all compounds
Conformational analyses of the model peptides revealed
(see ESI, Fig. S3†). This spectral behavior could be ascribed to that the homochiral Oxd2-Oxd3 dipeptidomimetic induced an
a dependence of the conformer population on the polarity extended disposition of the backbone. The heterochiral
and/or the competitive nature of the solvent.
sequences exerted a strong conformational control, giving rise
Taken together, all of the experimental evidence supports to type II β-turns. The unrestrained MD simulations confirmed
that the peptides 3 containing the ΔAbu at the position 2 the rigidity of the conformations, since the rotation of the two
formed highly stable type I β-turns reinforced by H-bonds, Oxd rings with respect to each other was not observed during
normal or inverse depending on the stereochemistry of the the simulations. On the other hand, the sequence ΔAbu2-Oxd3
Oxd3. This result is particularly gratifying, since the formation induced highly stable β-turns of type I, stabilized by clear
of the ΔAbu-Oxd motif was somewhat unpredictable.
H-bonds.
On the other hand, the data of the peptides 2 collected by
For their ability to control the ϕ and ψ dihedral angles of
ROESY/MD analyses confirmed a certain flexibility of the Phe4 the backbone as well as the χ dihedral angles of side chains,
residue. This observation could explain the discrepancies in peptide mimetics based on the structures of the model pep-
the determination of H-bonds by IR, VT-NMR, and NMR titra- tides 2 and 3 might find applications in medicinal chemistry
tion experiments. Nevertheless, the results highlighted the as 3D rigid scaffolds, in the field of foldamers, or as self-
rigidity of the Ala-Oxd-Oxd portions; the rotation of the Ala assembling scaffolds to form nanostructures.
and the two Oxd rings with respect to each other was not
observed during the unrestrained MD simulations. The confor-
mational homogeneity was correlated to the strong preference
for all-trans conformations stabilized by the weak interactions
Experimental
General methods
Unless stated otherwise, standard chemicals were obtained
from commercial sources and used without further purifi-
cation. 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 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, 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
.
Fig. 5 ECD spectra recorded in dichloromethane at room temperature (2a
dotted line, 3b dashed line, 2d full line).
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reported conditions and elemental analysis. Chiral HPLC ana- organic layers were dried over sodium sulfate, filtered, and
lysis was performed on a CHIRALPAK IC column (0.46 cm × concentrated at reduced pressure. The residue was purified by
25 cm), 1 : 1 n-hexane–2-propanol, at 0.8 mL min−1. Semi-pre- semi-preparative RP-HPLC (see General methods), giving 2
parative and analytical RP-HPLC of the peptide 14 was per- (85–93%, see Table 1, 94–97% pure by analytical RP-HPLC) or
formed as reported above, with the addition of 0.1% TFA in 3 (78–90%, see Table 1, 95–98% pure by analytical RP-HPLC),
the mobile phase. Elemental analyses were performed using a as waxy solids.
Thermo Flash 2000 CHNS/O analyzer. High-quality IR were
Ts-Ala-(5′-Me-Oxd2)-(5′-Me-Oxd3)-PheOMe (2a). IR (CH2Cl2)
obtained at 2 cm−1 resolution using a FT-IR spectrometer and ν: 3406, 3335, 1789, 1736, 1703, 1670 cm−1; 1H-NMR (CDCl3) δ:
1
1 mm NaCl solution cell. H-NMR spectra were recorded on a 1.43 (d, J = 6.8 Hz, 3H, AlaMe), 1.52 (d, J = 6.3 Hz, 6H, Oxd2Me
Varian instrument at 400 MHz, 13C-NMR spectra at 100 MHz. + Oxd3Me), 2.43 (s, 3H, TsMe), 3.11 (dd, J = 5.5, 14.1 Hz, 1H,
Circular Dichroism (CD) spectra were recorded on a Jasco J-710 PheHβ), 3.15 (dd, J = 5.4, 14.1 Hz, 1H, PheHβ), 3.77 (s, 3H,
spectropolarimeter. The synthetic procedure by MW OMe), 4.26 (d, J = 3.7 Hz, 1H, Oxd3H4), 4.58 (dq, J = 2.1, 6.3 Hz,
irradiation was performed using a microwave oven (Micro- 1H, Oxd2H5), 4.75 (dq, J = 3.7, 6.3 Hz, 1H, Oxd3H5), 4.85 (q,
SYNTH Microwave Labstation for Synthesis).
J = 7.6 Hz, 1H, PheHα), 5.22 (dq, J = 6.8, 10.4 Hz, 1H, AlaHα),
5.26 (d, J = 2.1 Hz, 1H, Oxd2H4), 5.39 (d, J = 10.4 Hz, 1H,
AlaNH), 6.28 (d, J = 7.6 Hz, 1H, PheNH), 7.04–7.11 (m, 2H,
PheArH), 7.21–7.26 (m, 3H, PheArH), 7.31 (d, J = 8.0 Hz, 2H,
TsArH), 7.75 (d, J = 8.0 Hz, 2H, TsArH); 13C-NMR (DMSO-d6) δ:
18.5, 20.5, 21.3, 21.4, 36.9, 50.1, 52.5, 54.3, 61.0, 61.6, 74.7,
76.6, 126.0, 127.2, 128.6, 128.8, 129.6, 130.1, 137.0, 138.2,
143.5, 152.0, 153.2, 167.4, 167.7, 171.7, 172.4; ES-MS (m/z)
659.3 [M + 1], calcd 659.2; Elem. Anal. for C30H34N4O11S,
calcd: C 54.70, H 5.20, N 8.51, S 4.87; found: C 54.67, H 5.17,
N 8.45, S 4.81%.
Synthesis of the peptides 1a–g
A stirred solution of the N-protected amino acid in 4 : 1 DCM–
DMF (5 mL) was treated with HOBt (1.2 equiv.) and HBTU (1.2
equiv.), at r.t. and under inert atmosphere. After 5 min, the
C-protected amino acid (1.1 equiv.) and DIPEA (2.4 equiv.) were
added, and the mixture was stirred under inert atmosphere
and under MW irradiation (150 W). After 10 min, 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 peptides were ana-
lyzed by HPLC-MS analysis, and were used without further
purification.
The intermediate N-Boc peptides were deprotected by treat-
ment with 1 : 2 TFA–DCM (5 mL), while 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 were collected by centri-
fuge, and used for the next couplings without further purifi-
cation (80–85% pure, Table 3).
Ts-Ala-ΔAbu-(5′-Me-Oxd)-PheOMe (3b). IR (CH2Cl2) ν:
3407, 3318, 1789, 1748, 1708 cm−1 1H-NMR (CDCl3) δ: 1.23
;
(d, J = 6.8 Hz, 3H, AlaMe), 1.43 (d, J = 6.0 Hz, 3H, OxdMe),
1.79 (d, J = 7.2 Hz, 3H, ΔAbuMe), 2.43 (s, 3H, TsMe), 3.03
(dd, J = 8.2, 13.8 Hz, 1H, PheHβ), 3.23 (dd, J = 5.4, 13.8 Hz,
1H, PheHβ), 3.71 (s, 3H, OMe), 3.85 (quint, J = 7.2 Hz, 1H,
AlaHα), 4.36–4.41 (m, 2H, OxdH4,5), 4.84 (q, J = 8.2 Hz, 1H,
PheHα), 5.92 (d, J = 6.8 Hz, 1H, AlaNH), 6.04 (q, J = 7.2 Hz,
1H, ΔAbuHβ), 7.14–7.22 (m, 2H, PheArH), 7.24–7.34 (m, 5H,
PheArH + TsArH), 7.64 (d, J = 8.0 Hz, 1H, PheNH), 7.77 (d,
J = 7.6 Hz, 2H, TsArH), 8.66 (s, 1H, ΔAbuNH); 13C-NMR
(DMSO-d6) δ: 12.8, 19.4, 20.3, 37.0, 51.9, 52.4, 54.1, 62.1,
75.0, 120.5, 126.9, 127.2, 128.8, 129.6, 130.1, 130.5, 134.1,
137.1, 138.7, 143.2, 151.8, 168.6, 170.8, 170.9, 171.9; ES-MS
Synthesis of Ts-Ala-Oxd-(5′-R-Oxd)-PheOMe (2a, c, d, f, g),
Ts-Ala-ΔAbu-(5′-Me-Oxd)-PheOMe (3b, c).
(m/z) 615.4 [M
+
1], calcd 615.2; Elem. Anal. for
C29H34N4O9S, calcd: C 56.67, H 5.58, N 9.11, S 5.22; found:
C 56.78, H 5.56, N 9.08, S 5.18%.
DSC (0.73 mmol) was added to a stirred solution of 1
(0.33 mmol) in the solvent mixture of Table 1 (4 mL) followed
by DIPEA (0.07 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
Ts-Ala-(5′-Me-Oxd2)-D-(5′-Me-Oxd3)-PheOMe (2c) IR (CH2Cl2)
1
ν 3406, 3347, 1793, 1736, 1707 cm−1; H-NMR (CDCl3) δ: 1.33
(d, J = 6.6 Hz, 3H, AlaMe), 1.47 (d, J = 6.2 Hz, 3H, Oxd2Me),
1.53 (d, J = 6.4 Hz, 3H, Oxd3Me), 2.42 (s, 3H, TsMe), 3.01 (dd,
J = 6.4, 14.1 Hz, 1H, PheHβ), 3.20 (dd, J = 6.4, 14.1 Hz, 1H,
PheHβ), 3.77 (s, 3H, OMe), 4.29 (d, J = 4.8 Hz, 1H, Oxd3H4),
4.53 (dq, J = 2.4, 6.4 Hz, 1H, Oxd2H5), 4.64 (quint, 1H, J = 6.2
Hz, Oxd3H5), 4.82 (q, J = 6.4 Hz, 1H, PheHα), 5.11 (dq, J = 6.8,
13.6 Hz, 1H, AlaHα), 5.24 (d, J = 2.4 Hz, 1H, Oxd2H4), 5.44 (d,
J = 10.0 Hz, 1H, AlaNH), 6.55 (d, J = 7.6 Hz, 1H, PheNH), 7.09 (d,
J = 7.2 Hz, 2H, PheArH), 7.24–7.31 (m, 5H, PheArH + TsArH),
7.73 (d, J = 7.6 Hz, 2H, TsArH); 13C-NMR (DMSO-d6) δ: 18.5,
20.4, 20.5, 21.4, 37.4, 50.3, 52.6, 53.8, 61.3, 62.0, 69.4, 70.1,
127.2, 128.4, 128.7, 129.8, 130.1, 137.4, 138.3, 143.5, 152.3,
153.3, 166.5, 167.1, 171.7, 172.0; ES-MS (m/z) 659.4 [M + 1],
Table 3 RP-HPLC and ES-MS analyses of the linear peptides 1a–f
ES-MS m/z
[M + 1] vs. calcd
Puritya
(%)
ES-MS m/z [M + 1]
vs. calcd
Puritya
(%)
1
1
a
b
c
607.3/607.2
607.2/607.2
607.3/607.2
607.1/607.2
85
83
81
80
e
f
g
607.2/607.2
669.3/669.3
669.4/669.3
84
84
83
d
a Determined by analytical RP-HPLC, see General methods.
4322 | Org. Biomol. Chem., 2013, 11, 4316–4326
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calcd 659.2; Elem. Anal. for C30H34N4O11S, calcd: C 54.70, H Oxd2Me), 2.43 (s, 3H, TsMe), 3.03 (dd, J = 7.2, 14.4 Hz, 2H,
5.20, N 8.51, S 4.87; found: C 54.60, H 5.14, N 8.44, S 4.81%. PheHβ), 3.23 (dd, J = 6.0, 14.4 Hz, 2H, PheHβ), 3.76 (s, 3H,
Ts-Ala-ΔAbu-D-(5′-Me-Oxd)-PheOMe (3c). IR (CH2Cl2) ν: OMe), 4.53–4.61 (m, 2H, Oxd2H5 + Oxd3H4), 4.88 (q, J = 7.6 Hz,
3400, 3334, 1781, 1744, 1707 cm−1; 1H-NMR (CDCl3) δ: 1.28 (d, 1H, PheHα), 5.13 (dq, J = 7.6, 10.8 Hz, 1H, AlaHα), 5.29 (d, J =
J = 7.2 Hz, 3H, AlaMe), 1.54 (d, J = 6.0 Hz, 3H, OxdMe), 1.89 (d, 2.4 Hz, 1H, Oxd2H4), 5.50–5.58 (m, 2H, AlaNH + Oxd3H5), 6.63
J = 7.2 Hz, 3H, ΔAbuMe), 2.46 (s, 3H, TsMe), 3.09 (dd, J = 9.0, (d, J = 7.7 Hz, 1H, PheNH), 7.04–7.11 (m, 2H, PheArH),
13.8 Hz, 1H, PheHβ), 3.18 (dd, J = 6.2, 13.8 Hz, 1H, PheHβ), 7.15–7.20 (m, 2H, 5′-PhArH), 7.20–7.25 (m, 3H, PheArH), 7.31
3.72 (s, 3H, OMe), 3.87 (m, 1H, AlaHα), 4.45 (d, J = 7.2 Hz, 1H, (d, J = 8.0 Hz, 2H, TsArH), 7.39–7.43 (m, 3H, 5′-PhArH), 7.76
OxdH4), 4.59 (quint, J = 6.5 Hz, 1H, OxdH5), 4.79 (q, J = 8.4 Hz, (d, J = 8.0 Hz, 2H, TsArH); 13C-NMR (CDCl3) δ: 19.3, 20.6, 21.5,
1H, PheHα), 5.66 (br.d, 1H, AlaNH), 6.27 (q, J = 6.6 Hz, 1H, 37.6, 50.7, 53.3, 53.5, 60.8, 63.4, 71.8, 79.0, 125.3, 127.4, 128.7,
ΔAbuHβ), 7.21 (d, J = 8.4, 2H, PheArH), 7.23–7.33 (m, 3H, 129.2, 129.5, 129.7, 130.1, 134.7, 135.7, 136.7, 143.6, 151.3,
PheArH), 7.35 (d, J = 7.8 Hz, 2H, TsArH), 7.80 (d, J = 7.8 Hz, 152.3, 165.4, 167.0, 171.2, 173.1; ES-MS (m/z) 721.3 [M + 1],
2H, TsArH), 7.98 (d, J = 8.0 Hz, 1H, PheNH), 8.28 (s, 1H, calcd 721.2; Elem. Anal. for C35H36N4O11S, calcd: C 58.32, H
ΔAbuNH); 13C-NMR (DMSO-d6) δ: 12.8, 16.9, 21.5, 25.7, 37.8, 5.03, N 7.77, S, 4.45; found: C 58.22, H 5.07, N 7.71, S, 4.49%.
52.8, 54.7 56.9, 62.0, 75.2, 121.9, 126.0, 127.0, 128.7, 129.9,
Conformational analysis
132.0, 134.4, 136.3, 143.0, 154.8, 168.4, 170.8, 171.6; ES-MS
(m/z) 615.4 [M + 1], calcd 615.2; Elem. Anal. for C29H34N4O9S, IR analyses. Infrared spectra were obtained at 2 cm−1 resolu-
calcd: C 56.67, H 5.58, N 9.11, S 5.22; found: C 56.60, H 5.52, tion using a 1 mm NaCl solution cell and a FT-IR spectrometer
N 9.02, S 5.15%.
(64 scans). All spectra were obtained in 3 mM solutions in dry
IR CH2Cl2 at 297 K. The compounds were dried in vacuo, and all
Ts-Ala-(5′-Me-Oxd2)-D-(5′-Me-Oxd3)-D-PheOMe
(2d).
(CH2Cl2) ν: 3409, 1776, 1747, 1707, 1608, 1420 cm−1; H-NMR the sample preparations were performed in
a
inert
1
(CDCl3) δ: 1.40 (d, J = 7.0 Hz, 3H, AlaMe), 1.46 (d, J = 6.0 Hz, atmosphere.
3H, Oxd3Me), 1.58 (d, J = 6.3 Hz, 3H, Oxd2Me), 2.44 (s, 3H,
Circular dichroism. ECD spectra were recorded from 200 to
TsMe), 3.05 (dd, J = 8.3, 13.9 Hz, 1H, PheHβ), 3.17 (dd, J = 5.3, 400 nm at 25 °C. 1 mM solutions were made up in spectral
13.9 Hz, 1H, PheHβ), 3.74 (s, 3H, OMe), 4.29–4.37 (m, 2H, grade solvents and run in a 0.1 cm quartz cell. Data are
Oxd3H4,5), 4.58 (dq, J = 3.5, 6.3 Hz, 1H, Oxd2H5), 4.88 (ddd, J = reported in molar ellipticity [θ] (deg cm2 dmol−1).
5.3, 8.3, 8.5 Hz, 1H, PheHα), 5.16 (dq, J = 7.0, 10.8 Hz, 1H,
NMR analyses. 1H-NMR spectra were recorded at 400 MHz
AlaHα), 5.42 (d, J = 3.5 Hz, 1H, Oxd2H4), 5.61 (d, J = 10.8 Hz, in 5 mm tubes, using 0.01 M peptide at room temperature.
1H, AlaNH), 6.63 (d, J = 8.5 Hz, 1H, PheNH), 7.14 (d, J = 6.6 Hz, Solvent suppression was performed by the solvent presatura-
2H, PheArH), 7.22–7.37 (m, 5H, PheArH + TsArH), 7.76 (d, J = tion procedure implemented in Varian (PRESAT). 13C-NMR
8.0 Hz, 2H, TsArH); 13C-NMR (CDCl3) δ: 18.9, 20.6, 20.9, 21.5, spectra were recorded at 100 MHz. Chemical shifts are
37.9, 51.1, 52.6, 53.4, 61.1, 62.4, 74.2, 76.0, 127.2, 127.4, 128.6, reported as
δ values. The unambiguous assignment of
129.3, 129.7, 135.6, 136.9, 143.8, 151.1, 152.0, 165.7, 168.1, 1H-NMR resonances was performed by 2D gCOSY, HMBC, and
171.4, 173.8; ES-MS (m/z) 659.3 [M + 1], calcd 659.2; Elem. HSQC. gCOSY experiments were conducted with a proton spec-
Anal. for C30H34N4O11S, calcd: C 54.70, H 5.20, N 8.51, S 4.87; tral width of 3103 Hz. VT-1H-NMR experiments were per-
found: C 54.65, H 5.26, N 8.50, S 4.82%.
formed over the range of 298–348 °K. 2D spectra were recorded
Ts-Ala-(5′-Me-Oxd2)-(5′-Ph-Oxd3)-PheOMe (2f). IR (CH2Cl2) in the phase sensitive mode and processed using a 90°-shifted,
ν: 3407, 3350, 1790, 1740, 1699, 1660 cm−1; 1H-NMR (CDCl3) δ: squared sine-bell apodization. 2D ROESY experiments were
1.41 (d, J = 7.2 Hz, 3H, AlaMe), 1.55 (d, J = 6.2 Hz, 6H, recorded in 8 : 2 DMSO-d6–H2O, with a 250 ms mixing time
Oxd2Me), 2.43 (s, 3H, TsMe), 3.12 (d, J = 5.6 Hz, 2H, PheHβ), with a proton spectral width of 3088 Hz. Peaks were calibrated
3.78 (s, 3H, OMe), 4.53 (d, J = 4.4 Hz, 1H, Oxd3H4), 4.66 (dq, J = on DMSO.
2.0, 6.2 Hz, 1H, Oxd2H5), 4.90 (q, J = 7.6 Hz, 1H, PheHα), 5.24
ROESY and molecular dynamics. Only ROESY-derived con-
(dq, J = 7.6, 10.8 Hz, 1H, AlaHα), 5.35 (d, J = 2.0 Hz, 1H, straints were included in the restrained molecular dynamics.
Oxd2H4), 5.41 (d, J = 10.8 Hz, 1H, AlaNH), 5.57 (d, J = 4.4 Hz, Cross-peak intensities were classified very strong, strong,
1H, Oxd3H5), 6.31 (d, J = 7.7 Hz, 1H, PheNH), 7.04–7.12 (m, medium, and weak, and were associated with distances of 2.2,
2H, PheArH), 7.20–7.30 (m, 5H, PheArH + 5′-PhArH), 7.31 (d, 2.6, 3.0, and 4.5 Å, respectively. Geminal couplings and other
J = 8.0 Hz, 2H, TsArH), 7.39–7.44 (m, 3H, 5′-PhArH), 7.77 (d, J = obvious correlations were discarded. For the absence of Hα(i, i
8.0 Hz, 2H, TsArH); 13C-NMR (CDCl3) δ: 19.5, 21.1, 21.5, 37.2, + 1) ROESY cross peaks, all of the ω bonds were set at 180°
50.6, 52.7, 53.7, 61.2, 65.8, 73.8, 79.0, 125.2, 127.3, 127.6, (force constant: 16 kcal mol−1 Å−2). The restrained MD simu-
129.1, 129.4, 129.5, 129.9, 130.1, 135.9, 136.6, 143.8, 151.1, lations were conducted using the AMBER force field in a 30 ×
152.3, 166.1, 167.4, 173.1, 173.4; ES-MS (m/z) 721.2 [M + 1], 30 × 30 Å box of standard TIP3P models of equilibrated
calcd 721.2; Elem. Anal. for C35H36N4O11S, calcd: C 58.32, water.31 All water molecules with atoms that come closer than
H 5.03, N 7.77, S, 4.45; found: C 58.29, H 5.09, N 7.80, S, 4.40%. 2.3 Å to a solute atom were eliminated. A 100 ps simulation at
Ts-Ala-(5′-Me-Oxd2)-D-(5′-Ph-Oxd3)-PheOMe (2g). IR (CH2Cl2) 1200 °K was used for generating 50 random structures that
ν: 3409, 3345, 1779, 1730, 1700, 1656 cm−1; 1H-NMR (CDCl3) δ: were subsequently subjected to a 50 ps restrained MD with a
1.30 (d, J = 7.3 Hz, 3H, AlaMe), 1.52 (d, J = 6.4 Hz, 3H, 50% scaled force field at the same temperature, followed by
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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 °
K. H-bond interactions 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.32 Unrestrained MD simulation was performed
in a 30 × 30 × 30 Å box of standard TIP3P water for 10 ns at
298 °K, at constant temperature and pressure (Berendsen
scheme,33 bath relaxation 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.
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Acknowledgements
We thank Fondazione Umberto Veronesi, Milano/Roma, MIUR
(PRIN 2010), the Italian Minister for Foreign Affairs (bilateral
proj. Italy-Mexico 2011–13), Fondazione per la Ricerca sulla
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