Novel peptide foldameric motifs: A step forward in our understanding of the fully-extended conformation/310-helix coexistence

Article abstract of DOI:10.1039/c2ob06863j

The fully-extended, multiple C₅, conformation or 2.0₅ helix is a very appealing peptide secondary structure, in particular for its potential use as a molecular spacer, as it is characterized by the longest elevation (as high as 3.62 A) between the α-carbon atoms of two consecutive α-amino acids. Despite this intriguing property, however, it is only poorly investigated and understood. Here, using a complete series of C^α,α-diethylglycine (Deg) homo-oligopeptide esters to the pentamer level, we exploited the properties of a fluorophore and a quencher, synthetically positioned at the N- and C-termini of the main chain, respectively, to check the applicability of the fully-extended conformation as a rigid molecular spacer. The fluorescence study was complemented by FT-IR absorption and NMR conformational investigations. The X-ray diffraction structures of selected compounds are also reported. Unfortunately, we find that, even in a solvent of low polarity, such as chloroform, in this peptide series an equilibrium does take place between the fragile fully-extended conformation and the 3₁₀-helical structure, the latter becoming more and more stable as the main chain is elongated. Since the Deg homo-peptide esters lacking any terminal aromatic group, previously investigated, are known to adopt a stable fully-extended conformation in chloroform solution, we tend to attribute the 3D-structure instability observed in this work to the presence of multiple aromatic rings in their blocking groups. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob06863j

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PAPER
Novel peptide foldameric motifs: a step forward in our understanding of the
fully-extended conformation/310-helix coexistence†‡
Fernando Formaggio,*a Marco Crisma,a Gema Ballano,a Cristina Peggion,a Mariano Venanzib
and Claudio Toniolo*a
Received 4th November 2011, Accepted 2nd February 2012
DOI: 10.1039/c2ob06863j
The fully-extended, multiple C5, conformation or 2.05 helix is a very appealing peptide secondary
structure, in particular for its potential use as a molecular spacer, as it is characterized by the longest
elevation (as high as 3.62 Å) between the α-carbon atoms of two consecutive α-amino acids. Despite this
intriguing property, however, it is only poorly investigated and understood. Here, using a complete series
of Cα,α-diethylglycine (Deg) homo-oligopeptide esters to the pentamer level, we exploited the properties
of a fluorophore and a quencher, synthetically positioned at the N- and C-termini of the main chain,
respectively, to check the applicability of the fully-extended conformation as a rigid molecular spacer.
The fluorescence study was complemented by FT-IR absorption and NMR conformational investigations.
The X-ray diffraction structures of selected compounds are also reported. Unfortunately, we find that,
even in a solvent of low polarity, such as chloroform, in this peptide series an equilibrium does take place
between the fragile fully-extended conformation and the 310-helical structure, the latter becoming more
and more stable as the main chain is elongated. Since the Deg homo-peptide esters lacking any terminal
aromatic group, previously investigated, are known to adopt a stable fully-extended conformation in
chloroform solution, we tend to attribute the 3D-structure instability observed in this work to the presence
of multiple aromatic rings in their blocking groups.
interestingly, a novel polypeptide 3D-structure termed fully-
Introduction
extended conformation or 2.05-helix8 is preferentially adopted
The incorporation of Cα,α-dialkylated glycyl residues into
peptide chains provides a mean for efficiently restricting the ϕ,ψ
backbone conformational space. A wealth of information from
solution and crystal-state studies and conformational energy
computations as well clearly demonstrated that Cα,α-dimethyl-
glycine (or α-aminoisobutyric acid, Aib), shown in Fig. 1,
strongly stabilizes 310- and α-helical structures.1–3 In contrast,
works from our laboratory and other groups (in particular that
of Tanaka/Suemune)4–19 showed that either the 310-helix20 or,
by homo-peptides from Cα,α-diethylglycine (Deg; Fig. 1). Since
this latter peptide 3D-structure appears to be rather fragile, we
are currently investigating the most appropriate chemical (e.g.,
effect of terminal groups)21 and environmental (e.g., effect of
nature of solvent)22 conditions to increase its stability.
The repeating motif of the 2.05-helix is the Ni–Hi ⋯OivCi′
intramolecularly H-bonded C5 conformation,23,24 shown in
Fig. 1 along with its 3D-structural parameters. The relative dis-
position of the two dipoles, Ni–Hi and Ci′vOi, is such that there
is obviously some interaction between them. Since these four
atoms, together with the Ciα atom, are involved in a pentagonal
“pseudo” cyclic structure, this conformation is also called the C5
structure.
In view of its relevant interest as a molecular spacer, the
goal of our present work was to exploit the fully-extended
peptide secondary structure in a fluorescence study. Indeed,
the elevation per residue of this flat helix is the longest (as
high as 3.62 Å) among those of peptide 3D-structures.8 To this
end, we incorporated an 1-pyrenylacetyl (PyrAc) group (Fig. 1)
at the backbone N-terminus as the fluorophore, and a para-nitro-
benzoxy [O-(pNO2)Bzl] group at the C-terminus as the
quencher.25,26
aICB, Padova Unit, CNR, Department of Chemistry, University of
Padova, 35131 Padova, Italy. E-mail: fernando.formaggio@unipd.it,
claudio.toniolo@unipd.it; Fax: +39-049-8275829; Tel: +39-049-
8275247
bDepartment of Chemical Sciences and Technologies, University of
Rome “Tor Vergata”, 00133 Rome, Italy
†This article is part of an Organic & Biomolecular Chemistry web
theme issue on Foldamer Chemistry.
‡Electronic supplementary information (ESI) available: Tables of
selected torsion angles and H-bond parameters, and packing figures for
the X-ray diffraction structures of PyrAc-(Deg)2-O-(pNO2)Bzl, PyrAc-
(Deg)5-O-(pNO2)Bzl, and PyrAc-(Aib)5-OtBu. CCDC 846155–846157.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c2ob06863j
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Fig. 1 (A) Average geometrical parameters for the fully-extended, intramolecularly H-bonded, C5 conformation from a statistical analysis of X-ray
diffraction structures.8 In addition, the chemical formulas of Deg, Aib, PyrAc, and O-(pNO2)Bzl are shown.
Results and discussion
Peptide synthesis and characterization
The Nα-protected Deg derivatives Tfa-Deg-OH (Tfa, trifluoro-
acetyl),27 Z-Deg-OH (Z, benzyloxycarbonyl),27,28 and Z-Deg-
OtBu (OtBu, tert-butoxy)29 are known compounds. The
synthesis and characterization of the homo-peptides Tfa-
(Deg)n-OtBu (n = 2–5) were also reported.27,28
Our strategy for the preparation of the PyrAc-(Deg)n-O-
(pNO2)Bzl (n = 1–5) peptides first involves the introduction of
the PyrAc moiety in the Nα-deprotected H-(Deg)n-OtBu peptides
Fig. 2 X-Ray diffraction structure of PyrAc-(Deg)2-O-(pNO2)Bzl with
(obtained, in turn, by Tfa removal of the corresponding Nα-Tfa
partial atom numbering. Only the major occupancy sites for the atoms
protected oligomers with NaBH4 in ethanol)12,27 using the 1-(3-
belonging to the disordered PyrAc moiety are shown. The intramolecular
H-bond is represented by a dashed line.
dimethylamino)propyl-3-ethylcarbodiimide/7-aza-1,2,3-benzo-
triazole method30,31 in anhydrous acetonitrile. The PyrAc-
protected peptide free acids, PyrAc-(Deg)n-OH, were prepared
by dissolving the corresponding tert-butyl esters in a 6 : 4
trifluoroacetic acid–dichloromethane mixture. In the last step, the
target PyrAc-(Deg)n-O-(pNO2)Bzl peptides were synthesized
from the corresponding free acids and para-nitrobenzylbromide
in the presence of triethylamine and silver oxide in acetone or in
a 95 : 5 acetonitrile–water mixture.
Finally, the two control compounds PyrAc-(Aib)5-O-(pNO2)
Bzl and Ac-O-(pNO2)Bzl (Ac, acetyl) were synthesized as
described above for the PyrAc-(Deg)n-O-(pNO2)Bzl peptides
starting from Z-(Aib)5-OtBu32,33 and Ac–OH, respectively.
The final peptides and their synthetic intermediates, all of
them of high chromatographic purity, were characterized by
melting point determination (where appropriate), thin-layer
chromatography (TLC) in three solvent systems, solid-state
IR absorption, and 1H NMR data. In addition, electrospray
ionization mass spectra (ESI-MS) were collected for all of the
–O-(pNO2)Bzl ester peptides.
Fig. 3 X-Ray diffraction structure of PyrAc-(Deg)5-O-(pNO2)Bzl with
partial atom numbering. The three intramolecular H-bonds are rep-
resented by dashed lines.
Conformational analysis in the crystal state
We were able to grow single crystals useful for X-ray diffraction
investigations from three of the PyrAc-containing peptides syn-
thesized in this work. They are: PyrAc-(Deg)n-O-(pNO2)Bzl
(n = 2 and 5) and PyrAc-(Aib)5-OtBu (Fig. 2–4, respectively).
Lists of the most significant conformational and H-bond par-
ameters are reported in the ESI (Tables S1 to S6‡).
In the three peptides, only one τ (N–Cα–C′) bond angle8,34
[104.22(11)°] is well below 109.5°, the value expected for a
regular tetrahedral sp3 carbon atom. This angle pertains to
residue 1 of the Deg dipeptide. This property is typically taken
as a preliminary evidence for the occurrence of the strain
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Fig. 4 X-Ray diffraction structure of PyrAc-(Aib)5-OtBu with partial
atom numbering. The three intramolecular H-bonds are represented by
dashed lines.
characterizing the intramolecularly H-bonded C5(pentagonal)-
pseudoring conformation.8
Fig. 5 FT-IR absorption spectra in the 3500–3200 cm−1 region of (A)
PyrAc-(Deg)n-O-(pNO2)Bzl (n = 1–5) and (B) PyrAc-(Deg)n-OtBu (n =
2–5). Spectra obtained by subtracting that of PyrAc-(Deg)2-O-(pNO2)
Bzl from those of PyrAc-(Deg)n-O-(pNO2)Bzl (n = 3–5) (C), and that of
PyrAc-(Deg)2-OtBu from those of PyrAc-(Deg)n-OtBu (n = 3–5) (D) are
also shown. Peptide concentration (CDCl3): 1 mM.
In the Deg homo-dipeptide crystal structure (Fig. 2) residue 1
shows values for the backbone torsion angles close to 180°,
180° [ϕ1 = 171.3(1)°, ψ1 = 169.5(1)°, since molecules of both
handedness simultaneously occur in its centrosymmetric space
group, P21/n] indicative of the fully-extended C5 conformation.8
The intramolecular N1⋯O1 distance is 2.567(2) Å. However,
the second residue is folded in a helical conformation, with ϕ2 =
43.0(2)°, “ψ2” = 45.3(2)°.
conformations of the Deg and Aib homo-pentapeptides in the
crystal state are quite similar.
The Deg homo-pentapeptide, although not containing any
asymmetric carbon atom, crystallizes in a non-centrosymmetric
space group (P21). As a consequence, only molecules of the
same handedness occur in each crystal. This is a rather uncom-
mon phenomenon, termed spontaneous resolution.35,36 We
assigned the right-handed enantiomorph to the crystal selected
for the analysis since the value of the Flack parameter37,38 [0.0
(2)] indicates that it is the most probable one. Nevertheless, in
the absence of strong anomalous scatterers, this choice does not
imply that we have determined the absolute configuration of the
helical molecular species of the Deg homo-pentapeptide. In
the crystal structure (Fig. 3), the peptide backbone adopts a
310-helical conformation generated by three consecutive, intra-
molecularly H-bonded, C10 forms. The set of backbone torsion
angles is ϕ1 = −53.8(3)°, ψ1 = −41.7(3)°; ϕ2 = −55.0(4)°, ψ2 =
−31.5(3)°; ϕ3 = −53.4(3)°, ψ3 = −34.8(3)°; ϕ4 = −59.2(3)°, ψ4 =
−31.5(3)°; ϕ5 = 49.9(3)°, “ψ5” = 54.8(3)°. The range of N⋯O
distances is normal, 2.862(3) Å–3.218(3) Å,39 as it is that of the
N–H⋯O angles: 143°–174°. For all five helical Deg residues the
value of the τ bond angle is between 109.8(2)° and 112.0(2)°.
As expected for an Aib homo-peptide,2,3 in the crystal struc-
ture of PyrAc-(Aib)5-OtBu the molecules are folded in a
310-helical structure (Fig. 4) with three CvO⋯H–N intramole-
cular H-bonds. In this case, the achiral molecules crystallize
The crystallographic results reported in this work, together
with those reported in the literature,2,3,5,7–9,12 strongly suggest
that the Cα,α-diethylated Deg residue can adopt either the fully-
extended C5 conformation or the helical structure under these
conditions, whereas the Cα,α-dimethylated Aib residue overwhel-
mingly prefers to fold in a helix. Indeed, the homo-pentapeptide
Tfa-(Deg)5-OtBu is 2.05-helical (fully-extended) in the crystal
state,5 but the 3D-structures of Tfa-(Deg)5-OEt (OEt, ethoxy)12
and PyrAc-(Deg)5-O-(pNO2)Bzl, the latter described here, are
310-helical. It remains to be seen whether crystal packing, in par-
ticular originated from different crystallization solvents and/or
different blocking (protecting) groups, has played any role in
governing peptide conformation in Deg homo-peptides. For
example, in our PyrAc-/-O-(pNO2)Bzl homo-di and pentapep-
tides, dyads of pyrenyl and para-nitrophenyl moieties of differ-
ent molecules, separated by an intermolecular distance of 3.5 Å,
interact each other via π⋯π stacking (Fig. S1 and S2 in the
ESI‡).
Conformational analysis in chloroform solution
The conformational preferences of the PyrAc/O-(pNO2)Bzl
blocked Deg homo-peptides to the pentamer level were exam-
ined in CDCl3, a solvent of low polarity, by use of FT-IR absorp-
tion and 1H NMR.
Keeping in mind the IR absorption properties in the amide A
region of the Tfa/OtBu protected, fully-extended (Deg)n pep-
tides6 and the 310-helical (Aib)n homo-peptides,40 we analyzed
the spectra of the PyrAc-(Deg)n-O-(pNO2)Bzl (n = 1–5) pep-
tides (Fig. 5A and C). In particular, the difference spectra of
the shortest peptides reveal a strong band at about 3360 cm−1
ˉ
normally in a centrosymmetric space group (P1). The set of
backbone torsion angles is: ϕ1 = 55.4(2)°, ψ1 = 31.4(2)°; ϕ2 =
55.0(2)°, ψ2 = 30.4(2)°; ϕ3 = 52.2(2)°, ψ3 = 35.8(2)°; ϕ4 =
57.9(2)°, ψ4 = 37.6(2)°; ϕ5 = −/+49.3(2)°, “ψ5” = −/+43.2
(2)°. The range of the intramolecular H-bonding N⋯O separ-
ations is 2.921(1)–3.141(1) Å and that of the N–H⋯O angles
is 150°–162°. The range of the τ bond angles for the five
Aib residues is 109.9(1)°–111.1(1)°. Overall, the molecular
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and a very weak band near 3440 cm−1, characteristic of the
C5 conformation.6,23 However, the strong band moves to lower
frequencies for the longest oligomers, as expected for 310-helical
peptides.40 More specifically, the band of the homo-pentamer
attributed to intramolecularly H-bonded conformers appears at
3340 cm−1, close to that of the 310-helical PyrAc-(Aib)5-O-
(pNO2)Bzl (3344 cm−1) (not shown). In conclusion, our view is
that the conformational equilibrium mixtures of PyrAc/O-
(pNO2)Bzl Deg homo-peptides tend to evolve from the multiple
C5 conformation to the predominant 310-helix as the peptide
length is enhanced from the homo-trimer to the pentamer.
Furthermore, we checked the role of the terminal groups
on peptide conformation. In the spectrum of the tetrapeptide
Tfa-(Deg)4-O-(pNO2)Bzl (not shown), three bands are seen:
3448 cm−1 (very weak), 3406 cm−1 (weak), and at about
3360 cm−1 (intense and broad). Overall, the spectrum is reason-
ably close to that of a peptide in the fully-extended confor-
mation. Also, the spectra, and the difference spectra as well, of
the PyrAc-(Deg)n-OtBu (n = 2–5) peptide series (Fig. 5B and D)
resemble those of fully-extended peptides (in particular, the
occurrence of a very weak band at 3447–3450 cm−1 and a main
band at 3360 cm−1 in the difference spectra is significant). From
these data, we tend to conclude that it is the combination of the
two large aromatic moieties [PyrAc and (pNO2)Bzl] that is
somewhat deleterious to the stabilization of the fully-extended
conformation in the Deg homo-peptides.
Fig. 6 FT-IR absorption spectra in the 1750–1450 cm−1 region of: (A)
Tfa-(Deg)n-OtBu (n = 2–5), (B) PyrAc-(Deg)n-O-(pNO2)Bzl (n = 3–5)
[these are difference spectra obtained by subtracting that of PyrAc-
(Deg)2-O-(pNO2)Bzl from those of its higher homologs] and PyrAc-
(Aib)5-O-(pNO2)Bzl (Aib), (C) Tfa-(Deg)4-O-(pNO2)Bzl, and (D)
PyrAc-(Deg)n-OtBu (n = 3–5) [these are difference spectra obtained by
subtracting that of PyrAc-(Deg)2-OtBu from those of its higher homo-
logs]. Peptide concentration (CDCl3): 1 mM.
Additional information on the secondary 3D-structural pro-
pensities of the PyrAc/O-(pNO2)Bzl blocked Deg homo-
oligopeptides were extracted from an NMR investigation.
Typical titrations in CDCl3 solution as a function of addition of
the strong H-bonding acceptor solvent dimethylsulfoxide
(DMSO):42–44 (i) For the 310-helix forming (Aib)5 homo-peptide
(Fig. 7A), they induce a shift to higher δ values only for the
N-terminal NH(1) and NH(2) protons (much more pronounced
for the former), not intramolecularly H-bonded with any CvO
group of the peptide. (ii) For the fully-extended Tfa-(Deg)4-
OtBu peptide (Fig. 7 B and ref. 6), they do not produce any
sizable shift in the signals of the four NH protons, which
suggests that these protons are solvent protected. In addition, the
close positions of all four NH proton signals at low field (above
7.0 ppm), similar to those of the three C-terminal NH proton
signals of (Aib)5, definitely confirm their participation in H-
bonds. When the C-terminal alkyl ester (OtBu) is replaced by an
arylalkyl ester, as in Tfa-(Deg)4-O-(pNO2)Bzl, only one NH
proton chemical shift becomes sensitive (moving to higher δ
values) to the addition of DMSO (Fig. 7C). We assign this signal
to the NH(4) proton, near the aromatic, C-terminal protecting
group (which also produces a downfield shift by about 0.30 ppm
in the position of this proton in CDCl3 as compared to that of
the corresponding proton in the OtBu ester). Finally, the CDCl3–
DMSO titration curve for the PyrAc/O-(pNO2)Bzl Deg homo-
tetramer (Fig. 7D) is clearly anomalous and difficult to interpret,
in the sense that it is quite distinct from those characteristic of
either the fully-extended or the 310-helical peptides. In the curve
of this tetrapeptide, all NH proton chemical shifts move some-
what (0.30–0.70 ppm) from their original positions in CDCl3.
Two of them, NH(1) and NH(4), are downfield shifted, whereas
the other two, NH(3) and particularly NH(2), are unusually
upfield shifted. Furthermore, the signals of all these NH protons
We also expanded our FT-IR absorption analysis to the
1750–1450 cm−1 region. Typically, 310-helical oligopeptides
exhibit a strong band near 1665 cm−1 (amide I), followed by a
less intense band at approximately 1515 cm−1 (amide II).41 In
the shortest peptides, this latter absorption occurs as two bands
(free and H-bonded N–H groups, respectively), with that of H-
bonded N-Hs prevailing for longer peptides. The band above
1700 cm−1 is assigned to the CvO stretching mode of the ester
chromophore. In contrast, in the case of the fully-extended pep-
tides Tfa-(Deg)n-OtBu (n = 2–5) the amide II band is signifi-
cantly more intense than that of the amide I (Fig. 6A). This latter
band is seen at about 1665 cm−1 as a double peak with a separ-
ation as large as 25 cm−1 for the pentapeptide. The amide II
signal appears as a single band, which is shifted to lower wave-
numbers as compared to that of 310-helical peptides and moves
further down as the peptide chain is elongated.
The spectral behavior of the PyrAc/O-(pNO2)Bzl blocked
Deg homo-oligomers is somewhat intermediate between those of
the 310-helical and fully-extended peptides. In particular, in the
difference spectra (Fig. 6B) the amide I absorption shows a
double peak typical of the latter conformation, but the absorption
of the pentapeptide resembles more that of the (Aib)5 homo-oli-
gomer (also shown in Fig. 6B). Also in the amide II region (at
least above 1500 cm−1) there is a tendency for the spectrum of
the (Deg)5 peptide to approach that of (Aib)5. Further infor-
mation was obtained from observation of the spectra of Tfa-
(Deg)4-O-(pNO2)Bzl (Fig. 6C) and the difference spectra of the
PyrAc-(Deg)n-OtBu (n = 3–5) (Fig. 6D), which are all quite
close to that expected for peptides in the fully-extended confor-
mation. To summarize, our solution conformational analysis
based on the amide I/II regions of the FT-IR absorption spectra
of the peptides examined nicely confirm the conclusions pre-
sented above from the amide A data.
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Fig. 8 Fluorescence spectra of the blank PyrAc–OH (0), the Deg
derivative and peptides PyrAc-(Deg)n-O-(pNO2)Bzl (n = 1–5) (1–5),
and the Aib peptide PyrAc-(Aib)5-O-(pNO2)Bzl (A5) in CHCl3 solution
(concentration: 10–7 M).
group in its molecule, reveals that: (i) No pyrene excimer emis-
sion band is seen. (ii) The trend in quenching efficiency for the
shortest members of the series, the (Deg)1–3 peptides, steadily
decreases from 0.97 (n = 1) to 0.89 (n = 2), and 0.64 (n = 3).
This finding is compatible with the conclusion that these three
peptides are all essentially fully-extended in CHCl3 solution.
However, the quenching efficiencies for our longest Deg homo-
peptides, 0.68 for n = 4 and 0.65 for n = 5, are not further
reduced. The inevitable conclusion is that the tetra- and penta-
peptides populate at least two different conformations, where the
probe dyads are located at different relative distances. Unfortu-
nately, this preliminary fluorescence investigation does not allow
us to assign the relative populations to each of the secondary
structures attained by these two oligomers. In any case, a reason-
able view is that in CHCl3 the (Deg)4 and (Deg)5 peptides tend
to adopt mixtures of fully-extended and 310-helical conformers,
with the amount of the latter increasing from the tetra- to the
pentamer level. Notably, these results fit well with those obtained
from our IR absorption and NMR studies in the same solvent of
low polarity.
We obtained further interesting information from the fluor-
escence spectrum of the 310-helical pentamer PyrAc-(Aib)5-O-
(pNO2)Bzl, also reported in Fig. 8. This relatively long oligomer
is quenched more than (Deg)2, but slightly less than the Deg
monomer (quenching efficiency: 0.94). In particular, these
findings clearly indicate that the shortest distance for the fluoro-
phore–quencher dyad experienced by the Aib pentamer molecule
is more reduced than that occurring in the Deg dimer (and much
more reduced than that present in the related Deg pentamer). It is
worth pointing out that in this phenomenon a significant role
could be played not only by the peptide backbone solution con-
formations, but by the rotamers around the two single bonds
characterizing each of the probes as well, not necessarily cor-
rectly represented by those experimentally found in the crystal
state for the (Deg)2 and (Deg)5 peptides (Fig. 2–4).
1
Fig. 7 Plots of the NH proton chemical shifts in the H NMR spectra
of (A) PyrAc-(Aib)5-O-(pNO2)Bzl, (B) Tfa-(Deg)4-OtBu, (C) Tfa-
(Deg)4-O-(pNO2)Bzl, and (D) PyrAc-(Deg)4-O-(pNO2)Bzl as a function
of increasing percentages of DMSO added to the CDCl3 solution (v/v)
(peptide concentration: 1 mM).
tend to broaden as DMSO is added, which might indicate the
concomitant occurrence of multiple conformers in the solution
equilibrium mixture. Taken together, these NMR results
reinforce our conclusion that the PyrAc/O-(pNO2)Bzl system is
not an appropriate choice for the stabilization of a fully-extended
structure in Deg homo-peptides.
Molecular spacers
We checked the applicability of the PyrAc-(Deg)n-O-(pNO2)Bzl
peptides as fully-extended molecular spacers in a static fluor-
escence study. To perform a very stringent comparison with our
IR absorption and NMR results, the solvent used was chloro-
form, despite the fact that in principle it would not be the best
choice for a fluorescence analysis, due to the presence, although
to a limited extent, of potentially quenching chlorine atoms in
solution. The spectra were recorded after excitation of the
samples at 346 nm, where the pyrenyl chromophore is known to
exhibit its strongest absorption (related to a symmetry allowed
π → π* transition) above 300 nm.25,26
In the fluorescence spectra above 350 nm, the pyrenyl chro-
mophore is characterized by two main bands located near
375 nm and 395 nm endowed with fine structure.25,26 A simple
visual inspection of the curves, shown in Fig. 8 in comparison
with that of the blank (PyrAc-OH), which lacks any quencher
Conclusions
The fully-extended conformation is by far the most stretched per
residue peptide 3D-structure. In this specific rank order it is
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followed by the β-sheet conformation, and the type-II poly-
(Pro)n, 310- and α-helices. So far, this potentially quite useful
property of the fully-extended conformation has not been
exploited at all because: (i) This 3D-structure is constitutionally
fragile.21,22 (ii) The synthesis of homo-peptides typically adopt-
ing it is difficult and the coupling yields usually obtained are not
amenable to the solid-phase methodology.45
1-butanol–acetic acid–water 3 : 1 : 1; Rf3 toluene–ethanol 7 : 1.
The chromatograms were visualized by UV fluorescence or
developed by chlorine–starch–potassium iodide or ninhydrin
chromatic reaction as appropriate. All compounds were obtained
in a chromatographically homogeneous state. The solid-state IR
absorption spectra (KBr disk technique) were recorded with a
Perkin-Elmer model 1720X FT-IR spectrophotometer.
The classical α-amino acid residue recommended for building
up this unusual peptide secondary structure is Deg.4–19 We and
others have already shown that the most stabilizing, terminal pro-
tecting (or blocking), groups of the peptide main-chain, at least
in chloroform solution, are Tfa at the N-terminus and an alkyl
ester at the C-terminus.5,6,12 In the Tfa-protected Deg homo-
peptides, the N-terminal residue is held tightly in the C5 confor-
mation by virtue of a stabilizing, multiple H-bonding of the
F⋯H(N1)⋯O1vC1 type. A C-terminal primary or secondary
amide is not tolerated because its unsatisfied –NH– function
can provide the H-bonding donor responsible for the switch of
this extremely elongated 3D-structure to the more compressed
310-helix.21 Also, to avoid destabilization of the fully-extended
conformation, the solvent of choice is chloroform. Intermolecu-
lar H-bonds arising from solvents of higher polarity22 and
packing forces occurring in crystals12 may compete with the
weak intraresidue H-bond of the C5 basic unit, thus switching
the conformation to the 310-helix.
PyrAc-Deg-OtBu. This compound was obtained from PyrAc-
OH (Aldrich, St. Louis, MO) and H-Deg-OtBu (the latter
prepared in turn by catalytic hydrogenation of Z-Deg-OtBu in
anhydrous CH2Cl2) in anhydrous CH3CN in the presence of
EDC–HOAt. Yield: 21%. Melting point: oil. TLC Rf1: 0.95;
Rf2: 0.95; Rf3: 0.85. 1H NMR (200 MHz; CDCl3): δ 8.36
(d, 1H, Pyr CH), 8.14–7.93 (m, 8H, 8 Pyr CH), 6.40 (s, 1H,
NH), 4.29 (s, 2H, PyrAc CH2), 2.43 (m, 2H, Deg β-CH2), 1.60
(m, 2H, Deg β-CH2), 1.28 (s, 9H, OtBu CH3), 0.59 (t, 6H, Deg
γ-CH3).
PyrAc-Deg-OH. This compound was prepared from PyrAc-
Deg-OtBu by treatment with TFA diluted with CH2Cl2. Yield:
95%. Melting point: 279–281 °C [after repeated washings with
1
diethyl ether (Et2O)]. TLC Rf1: 0.25; Rf2: 0.95; Rf3: 0.35. H
NMR (400 MHz; DMSO, d6): δ 8.42–7.98 (m, 9H, Pyr CH),
7.79 (s, 1H, NH), 4.28 (s, 2H, PyrAc CH2), 1.98 (m, 2H, Deg
β-CH2), 1.74 (m, 2H, Deg β-CH2), 0.68 (t, 6H, Deg γ-CH3).
In this work, we made an attempt to exploit the fully-extended
peptide conformation as a rigid molecular ruler using (static) flu-
orescence spectroscopy. However, this physico-chemical tech-
nique implies the introduction of aromatic moieties as a
fluorophore and a quencher. The results of our present combi-
nation of FT-IR absorption, NMR, and X-ray diffraction tech-
niques force us to conclude that not only in the crystal state, but
in chloroform as well, in the Deg homo-peptides investigated the
fully-extended conformation and the 310-helix coexist to differ-
ent extents. Clearly, terminal aromatic groups are not eligible for
the fabrication of a stable fully-extended conformation, at least
in this homo-peptide series.
PyrAc-Deg-O-(pNO2)Bzl. This compound was prepared from
PyrAc-Deg-OH and 4-nitrobenzylbromide in acetone solution in
the presence of triethylamine and Ag2O. Yield: 34%. Melting
point: 141–143 °C [from CH2Cl2–petroleum ether (PE)]. TLC
Rf1: 0.95; Rf2: 0.95; Rf3: 0.70. IR (KBr): 3385, 1738, 1649,
1
1519 cm−1. H NMR (400 MHz; CDCl3): δ 8.27–7.92 (m, 11H,
9 Pyr and 2 Bzl CH), 7.18 (d, 2H, 2 Bzl CH), 6.10 (s, 1H, NH),
5.00 (d, 2H, Bzl CH2), 4.30 (s, 2H, PyrAc CH2), 2.30 (m, 2H,
Deg β-CH2), 1.75 (m, 2H, Deg β-CH2), 0.53 (m, 6H, Deg
γ-CH3). MS (ESI-TOF): [M + H]+
509.213; [M + H]+
calcd
exptl
509.212.
We are currently extending our investigation of this confor-
mation in Deg homo-peptides in chloroform solution to an
NMR-based technique, REDOR,46 which allows the use of the
fluorine atoms of the stabilizing Tfa group at the N-terminus as a
first probe and requires an appropriate isotopically labelled Deg
residue or protecting group at the C-terminus as the second
probe.
PyrAc-(Deg)2-OtBu. This compound was prepared from
PyrAc-OH and H-(Deg)2-OtBu [the latter obtained in turn from
NaBH4 treatment of Tfa-(Deg)2-OtBu27,28 in ethanol under
reflux] in anhydrous CH3CN in the presence of EDC–HOAt.
Yield: 21%. Melting point: 131–133 °C [from ethyl acetate
(EtOAc)–PE]. TLC Rf1: 0.95; Rf2: 0.95; Rf3: 0.70. IR (KBr):
3399, 3341, 1721, 1653, 1494 cm−1 1H NMR (400 MHz;
.
CDCl3): δ 8.27 (d, 1H, 1 Pyr CH), 8.10–7.86 (m, 8H, 8 Pyr
CH), 6.82 (s, 1H, NH1), 6.61 (s, 1H, NH2), 4.29 (s, 2H, PyrAc
CH2), 2.45 (m, 2H, Deg1 β-CH2), 2.20 (m, 2H, Deg2 β-CH2),
1.60 (m, 4H, Deg β-CH2), 1.32 (s, 9H, OtBu CH3), 0.56
(m, 12H, Deg γ-CH3).
Experimental
Peptide synthesis and characterization
General methods. Melting points were determined in capillary
tubes by use of a Stuart SMP10 apparatus (Bibby Scientific,
Stone, Staffordshire, UK) and are uncorrected. ESI-MS
were obtained by using a PerSeptive Biosystem Mariner model
instrument (Framingham, MA). Flash chromatography was
carried out by use of a Merck (Darmstadt, Germany) silica
gel 60 (40–63 μm mesh) stationary phase. Analytical TLC was
performed on Kieselgel F 254 precoated plates (Merck). The
retention factors (Rf) values were determined using the following
solvent mixtures as eluants: Rf1 chloroform–ethanol 9 : 1; Rf2
PyrAc-(Deg)2-OH. This compound was prepared from PyrAc-
(Deg)2-OtBu as described above for PyrAc-Deg-OH. Yield:
94%. Melting point: 223–225 °C (after repeated washings with
Et2O). TLC Rf1: 0.25; Rf2: 0.95; Rf3: 0.30. 1H NMR (400 MHz;
DMSO, d6): δ 8.7–8.03 (m, 9H, 9 Pyr CH), 7.77 (s, 1H, NH),
7.32 (s, 1H, NH), 4.29 (s, 2H, PyrAc CH2), 2.09 (m, 2H, Deg
β-CH2), 1.85 (m, 4H, Deg β-CH2), 1.67 (m, 2H, Deg β-CH2),
0.67 (m, 6H, Deg γ-CH3), 0.58 (m, 6H, Deg γ-CH3).
2418 | Org. Biomol. Chem., 2012, 10, 2413–2421
This journal is © The Royal Society of Chemistry 2012

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(d, 2H, Bzl CH2), 7.02 (s, 1H, NH3), 6.82 (s, 1H, NH2), 6.75
(s, 1H, NH4), 6.55 (s, 1H, NH1), 5.26 (s, 2H, Bzl CH2), 4.30
(s, 2H, PyrAc CH2), 2.40–2.15 (m, 8H, Deg β-CH2), 1.91
(m, 2H, Deg β-CH2), 1.70–1.56 (m, 6H, Deg β-CH2), 0.77–0.60
PyrAc-(Deg)2-O-(pNO2)Bzl. This compound was prepared
from PyrAc-(Deg)2-OH in a 95 : 5 CH3CN–H2O mixture as
described above for PyrAc-Deg-O-(pNO2)Bzl. Yield: 50%.
Melting point: 166–168 °C (from CH2Cl2–PE). TLC Rf1: 0.95;
Rf2: 0.95; Rf3: 0.55. IR (KBr): 3340, 1736, 1646, 1520,
(m, 24H, Deg γ-CH3). MS (ESI-TOF): [M + H]+
848.471;
calcd
1
1494 cm−1. H NMR (200 MHz; CDCl3): δ 8.26 (d, 1H, 1 Pyr
[M + H]+exptl 848.447.
CH), 8.21–7.87 (m, 10H, 8 Pyr and 2 Bzl CH), 7.32 (d, 2H, 2
Bzl CH), 6.58 (s, 1H, NH), 6.56 (s, 1H, NH), 5.08 (s, 2H, Bzl
CH2), 4.22 (s, 2H, PyrAc CH2), 2.35–2.16 (m, 4H, β-CH2),
1.68–1.50 (m, 4H, β-CH2), 0.55 (m, 12H, Deg γ-CH3). MS
(ESI-TOF): [M + H]+calcd 622.299; [M + H]+exptl 622.288.
PyrAc-(Deg)5-OtBu. This compound was prepared from Tfa-
(Deg)5-OtBu as described above for PyrAc-(Deg)2-OtBu. Yield:
89%. Melting point: 207–209 °C (from EtOAc–PE). TLC Rf1:
0.70; Rf2: 0.95; Rf3: 0.40. IR (KBr): 3412, 3319, 1728, 1666,
1
1527 cm−1. H NMR (400 MHz; CDCl3): δ 8.35 (m, 1H, Pyr
PyrAc-(Deg)3-OtBu. This compound was prepared from Tfa-
(Deg)3-OtBu as described above for PyrAc-(Deg)2-OtBu. Yield:
83%. Melting point: 188–190 °C (from CH2Cl2–PE). TLC Rf1:
0.90; Rf2: 0.95; Rf3: 0.70. IR (KBr): 3336, 1720, 1658,
CH), 8.19–7.95 (m, 8H, 8 Pyr CH), 7.32 (s, 1H, NH), 7.27 (s,
1H, NH), 7.18 (s, 1H, NH), 6.87 (s, 1H, NH), 6.84 (s, 1H, NH),
4.29 (s, 2H, PyrAc CH2), 2.60–2.33 (m, 10H, Deg β-CH2),
1.88–1.55 (m, 10H, Deg β-CH2), 1.48 (s, 9H, OtBu CH3),
0.85–0.60 (m, 30H, Deg γ-CH3).
1
1491 cm−1. H NMR (200 MHz; CDCl3): δ 8.41–7.87 (m, 9H,
Pyr CH), 7.21 (s, 1H, NH), 6.89 (s, 1H, NH), 6.76 (s, 1H, NH),
4.29 (s, 2H, PyrAc CH2), 2.48 (m, 6H, Deg β-CH2), 1.62 (m,
6H, Deg β-CH2), 1.47 (s, 9H, OtBu CH3), 0.78–0.58 (m, 18H,
Deg γ-CH3).
PyrAc-(Deg)5-OH. This compound was prepared from PyrAc-
(Deg)5-OtBu as described above for PyrAc-Deg-OH. Yield:
96%. Melting point: 227–229 °C (after repeated washings with
Et2O). TLC Rf1: 0.25; Rf2: 0.95; Rf3: 0.25. 1H NMR (200 MHz;
DMSO, d6): δ 8.59 (s, 1H, NH), 8.53 (m, 1H, Pyr CH), δ
8.27–8.02 (m, 8H, 8 Pyr CH), 7.38 (s, 1H, NH), 6.96 (s, 2H, 2
NH), 6.81 (s, 1H, NH), 4.38 (s, 2H, PyrAc CH2), 1.80 (m, 10H,
Deg β-CH2), 1.65 (m, 10H, Deg β-CH2), 0.88–0.54 (m, 30H,
Deg γ-CH3).
PyrAc-(Deg)3-OH. This compound was prepared from PyrAc-
(Deg)3-OtBu as described above for PyrAc-Deg-OH. Yield:
96%. Melting point: 210–212 °C (after repeated washings with
Et2O). TLC Rf1: 0.25; Rf2: 0.95; Rf3: 0.30. 1H NMR (200 MHz;
DMSO, d6): δ 8.19–8.10 (m, 10H, 9 Pyr CH and 1 NH), 7.56
(s, 1H, NH), 7.15 (s, 1H, NH), 4.28 (s, 2H, PyrAc CH2),
2.20–1.80 (m, 6H, Deg β-CH2), 1.80–1.65 (m, 6H, Deg β-CH2),
0.80–0.50 (m, 18H, Deg γ-CH3).
PyrAc-(Deg)5-O-(pNO2)Bzl. This compound was prepared
from PyrAc-(Deg)5-OH as described above for PyrAc-(Deg)2-O-
(pNO2)Bzl. Yield: 65%. Melting point: 234–236 °C (from
CH2Cl2–PE). TLC Rf1: 0.95; Rf2: 0.95; Rf3: 0.35. IR (KBr):
PyrAc-(Deg)3-O-(pNO2)Bzl. This compound was prepared
from PyrAc-(Deg)3-OH as described above for PyrAc-(Deg)2-O-
(pNO2)Bzl. Yield: 25%. Melting point: 183–184 °C (from
CH2Cl2–PE). TLC Rf1: 0.95; Rf2: 0.95; Rf3: 0.45. IR (KBr):
1
3318, 1737, 1666, 1523 cm−1. H NMR (400 MHz; CDCl3): δ
8.36–7.96 (m, 11H, 9 Pyr and 2 Bzl CH), 7.55 (d, 2H, Bzl CH2),
7.09 (s, 2H, 2 NH), 7.06 (s, 1H, NH), 6.37 (broad s, 1H, NH),
6.17 (broad s, 1H, NH), 5.24 (s, 2H, Bzl CH2), 4.33 (s, 2H,
PyrAc CH2), 2.20–1.63 (m, 20H, Deg β-CH2), 0.70–0.58
3338, 1737, 1648, 1520, 1490 cm−1 1H NMR (400 MHz;
.
CDCl3): δ 8.34–7.97 (m, 11H, 9 Pyr and 2 Bzl CH), 7.50
(d, 2H, Bzl CH2), 6.92 (s, 1H, NH2), 6.62 (s, 1H, NH3), 6.61
(s, 1H, NH1), 5.24 (s, 2H, Bzl CH2), 4.30 (s, 2H, PyrAc CH2),
2.27 (m, 6H, Deg β-CH2), 1.86 (m, 2H, Deg β-CH2), 1.59
(m, 4H, Deg β-CH2), 0.65 (m, 18H, γ-CH3). MS (ESI-TOF):
[M + H]+calcd 735.385; [M + H]+exptl 735.370.
(m, 30H, Deg γ-CH3). MS (ESI-TOF): [M + H]+
961.557;
calcd
[M + H]+exptl 961.546.
PyrAc-(Aib)5-OtBu. This compound was obtained from
PyrAc–OH and H-(Aib)5-OtBu [the latter prepared in turn from
catalytic hydrogenation of Z-(Aib)5-OtBu32,33 in methanol] as
described above for PyrAc-(Deg)2-OtBu. Yield: 26%. Melting
point: 296–298 °C (from EtOAc–PE). TLC Rf1: 0.45; Rf2: 0.95;
PyrAc-(Deg)4-OtBu. This compound was prepared from Tfa-
(Deg)4-OtBu as described above for PyrAc-(Deg)2-OtBu. Yield:
87%. Melting point: 104–106 °C (from EtOAc–PE). TLC Rf1:
0.90; Rf2: 0.95; Rf3: 0.55. IR (KBr): 3332, 1721, 1657,
1
Rf3: 0.20. IR (KBr): 3423, 3322, 1731, 1665, 1525 cm−1. H
NMR (200 MHz; CDCl3): δ 8.24–7.90 (m, 9H, Pyr CH), 7.29
(s, 1H, NH), 7.28 (s, 1H, NH), 7.12 (s, 1H, NH), 6.00 (s, 1H,
NH), 5.81 (s, 1H, NH), 4.27 (s, 2H, PyrAc CH2), 1.74 (s, 6H,
Aib β-CH3), 1.56 (s, 12H, Aib β-CH3), 1.50 (s, 12H, Aib
β-CH3), 1.43 (s, 9H, OtBu CH3).
1
1490 cm−1. H NMR (400 MHz; CDCl3): δ 8.35 (m, 1H, Pyr
CH), 8.18–7.93 (m, 8H, 8 Pyr CH), 7.28 (s, 1H, NH), 7.21
(s, 1H, NH), 6.91 (s, 1H, NH), 6.83 (s, 1H, NH), 4.28 (s, 2H,
PyrAc CH2), 2.50 (m, 8H, Deg β-CH2), 1.64 (m, 8H, Deg
β-CH2), 1.48 (s, 9H, OtBu CH3), 0.88–0.61 (m, 16H, Deg
γ-CH3).
PyrAc-(Aib)5-OH. This compound was prepared from PyrAc-
(Aib)5-OtBu as described above for PyrAc-Deg-OH. Yield:
94%. Melting point: 240–241 °C (after repeated washings with
Et2O). TLC Rf1: 0.25; Rf2: 0.90; Rf3: 0.10. 1H NMR (200 MHz;
DMSO, d6): δ 8.85 (s, 1H, NH), 8.45 (d, 1H, Pyr CH),
8.47–8.00 (m, 9H, 1 NH and 8 Pyr CH), 7.23 (s, 1H, NH), 7.12
(s, 1H, NH), 7.08 (s, 1H, NH), 4.28 (s, 2H, PyrAc CH2), 1.40
(s, 6H, Aib β-CH3), 1.25 (s, 6H, Aib β-CH3), 1.20 (s, 6H, Aib
β-CH3), 1.18 (s, 6H, Aib β-CH3), 0.64 (s, 6H, Aib β-CH3).
PyrAc-(Deg)4-O-(pNO2)Bzl. This compound was prepared
from PyrAc-(Deg)4-OH, obtained by acidolysis of PyrAc-
(Deg)4-OtBu, as described above for PyrAc-(Deg)2-O-(pNO2)
Bzl. Yield: 52%. Melting point: 180–182 °C (from CH2Cl2/PE).
TLC Rf1: 0.95; Rf2: 0.95; Rf3: 0.40. IR (KBr): 3411, 3327,
1
1738, 1672, 1521 cm−1. H NMR (400 MHz; CDCl3): δ 8.33
(m, 1H, Pyr CH), 8.15–7.98 (m, 10H, 8 Pyr and 2 Bzl CH), 7.52
This journal is © The Royal Society of Chemistry 2012
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were acquired in magnitude mode, while CLEAN-TOCSY49,50
(spin lock period: 70 ms) and NOESY spectra were recorded in
the phase-sensitive mode. All homonuclear 2D spectra were
acquired by collecting 400–512 experiments of 64–128 scans
each, with a relaxation delay of 1.0–1.2 s, and 2 K data points.
The spectral width was 11 ppm in each dimension.
PyrAc-(Aib)5-O-(pNO2)Bzl. This compound was obtained
from PyrAc-(Aib)5-OH as described above for PyrAc-(Deg)2-O-
(pNO2)Bzl. Yield: 87%. Melting point: 234–236 °C (from
EtOAc–PE). TLC Rf1: 0.75; Rf2: 0.95; Rf3: 0.25. IR (KBr):
1
3318, 1740, 1663, 1523 cm−1. H NMR (400 MHz; CDCl3): δ
8.35–7.98 (m, 11H, 9 Pyr and 2 Bzl CH), 7.48 (s, 1H, NH), 7.39
(d, 2H, 2 Bzl CH), 7.30 (s, 1H, NH), 7.12 (s, 1H, NH), 6.47
(s, 1H, NH), 5.81 (s, 1H, NH), 5.16 (s, 2H, Bzl CH2), 4.25
(s, 2H, PyrAc CH2), 1.50 (s, 6H, Aib β-CH3), 1.40 (s, 6H, Aib
β-CH3), 1.30 (s, 6H, Aib β-CH3), 1.28 (s, 6H, Aib β-CH3), 0.92
Fluorescence
Steady-state fluorescence spectra on the PyrAc-containing pep-
tides were recorded with a Jobin Ivon Fluoromax-2 (Longju-
meau, France) spectrofluorimeter. Cells with path length of 1 cm
were used. Spectrograde MeOH was purchased from Fluka
(Büchs, Switzerland).
(s, 6H, Aib β-CH3). MS (ESI-TOF): [M + H]+
821.397;
calcd
[M + H]+exptl 821.385.
Tfa-(Deg)4-O-(pNO2)Bzl. This compound was obtained from
Tfa-(Deg)4-OtBu via formation (without isolation) of the inter-
mediate Tfa-(Deg)4-OH. The procedures for the two steps
are those described above for PyrAc-Deg-OH and PyrAc-
(Deg)2-O-(pNO2)Bzl, respectively. Yield: 72%. Melting point:
185–187 °C (from CH2Cl2–PE). TLC Rf1: 0.95; Rf2: 0.95; Rf3:
X-Ray diffraction
Single crystals of PyrAc-(Deg)2-O-(pNO2)Bzl and PyrAc-
(Deg)5-O-(pNO2)Bzl were grown by slow evaporation from
methanol–dichloromethane mixtures, while those of PyrAc-
(Aib)5-OtBu by slow evaporation from methanol. X-ray diffrac-
tion data were collected with an Agilent Gemini E four-circle
kappa diffractometer equipped with a 92 mm EOS CCD detec-
tor, using graphite monochromated Cu Kα radiation (λ =
1.54178 Å). The structures were solved by direct methods of
the SIR 2002 program,51 and refined by full-matrix least-squares
procedures on F2, using all data, by application of the
SHELXL-97 program,52 with all non-H atoms anisotropic.
H-atoms were calculated at idealized positions and refined using
a riding model. Crystal structure data have been deposited at the
Cambridge Crystallographic Data Centre‡, and allocated the
deposition numbers CCDC 846155, CCDC 846156, and CCDC
846157 for PyrAc-(Deg)2-O-(pNO2)Bzl, PyrAc-(Deg)5-O-
(pNO2)Bzl, and PyrAc-(Aib)5-OtBu, respectively.
1
0.55. IR (KBr): 3346, 1726, 1655, 1523, 1492 cm−1. H NMR
(400 MHz; CDCl3): δ 8.25 (d, 2H, 2 Bzl CH), 7.99 (s, 1H, NH),
7.54 (d, 2H, 2 Bzl CH), 7.39 (s, 1H, NH), 7.28 (s, 1H, NH),
6.59 (s, 1H, NH), 5.37 (s, 2H, Bzl CH2), 2.73–2.38 (m, 8H, Deg
β-CH2), 1.90–1.63 (m, 8H, Deg β-CH2), 0.89–0.68 (m, 24H,
Deg γ-CH3). MS (ESI-TOF): [M
+
H]+
702.378;
calcd
[M + H]+exptl 702.349.
Ac-O-(pNO2)Bzl. This compound was prepared from acetic
acid (AcOH) as described above for PyrAc-(Deg)2-O-(pNO2)
Bzl. Yield: 71%. Melting point: 74–76 °C (from CH2Cl2–PE).
TLC Rf1: 0.95; Rf2: 0.95; Rf3: 0.85. IR (KBr): 1738, 1655,
1
1520 cm−1. H NMR (200 MHz; CDCl3): δ 8.13 (d, 2H, 2 Bzl
CH), 7.19 (d, 2H, 2 Bzl CH), 5.13 (s, 2H, Bzl CH2), 2.08 (s, 3H,
Ac CH3).
Crystal data for PyrAc-(Deg)2-O-(pNO2)Bzl: C37H39N3O6,
M = 621.71, monoclinic, space group P21/n, a = 10.68181(10)
Å, b = 11.93226(15) Å, c = 25.6913(3) Å, β = 93.4477(9)°; V =
FT-IR absorption
The FT-IR absorption spectra were recorded at 293 K using a
Perkin Elmer model 1720X FTIR spectrophotometer, nitrogen
flushed, equipped with a sample shuttle device, at 2 cm−1
nominal resolution, averaging 100 scans. Cells with path lengths
of 0.1, 1.0, and 10 mm (with CaF2 windows) were used. Spec-
trograde deuterated chloroform (99.8%, d) was purchased from
Aldrich (Milwaukee, WI). Solvent (baseline) spectra were
recorded under the same conditions.
3268.64(6) Å3, Z = 4; ρcalcd = 1.263 Mg m−3, μ = 0.697 mm−1
,
43 609 reflections measured, 6306 independent reflections (Rint
=
0.0562), θmax = 71.44°, T = 295(2) K. The N-terminal PyrAc
group shows librational disorder. It was refined on two sets of
positions (atoms C01 to C17 and C01′ to C17′, respectively)
with refined occupancies of 0.674(16) and 0.326(16). Restraints
were applied to the bond distances, planarity, and anisotropic
displacement parameters of the disordered parts. The final R1
values were 0.0539 [I > 2σ(I)] and 0.0591 (all data). The final
wR(F2) values were 0.1549 [I > 2σ(I)] and 0.1621 (all data).
Data/restraints/parameters 6306/188/570. Goodness-of-fit on
F2 = 1.032. The largest difference peak and hole were 0.276 and
−0.247 e Å−3, respectively.
Nuclear magnetic resonance
1
The H NMR spectra were recorded with Bruker AC 200 and
AM 400 spectrometers. Measurements were carried out in
CDCl3 (99.96%, d; Aldrich) and deuterated DMSO (99.96%, d6;
Acros Organics) with tetramethylsilane as the internal standard.
Splitting patterns are abbreviated as follows: (s) singlet, (d)
doublet, (t) triplet, (q) quartet, (qd) quartet of doublets, (m) mul-
tiplet. The 2D-NMR experiments were performed with a Bruker
AVANCE DRX-400 spectrometer, operating at 400 MHz,
equipped with a 5 mm probe BBI-Z grad. Processing and evalu-
ation of the experimental data were carried out using the pro-
grams TOPSPIN 1.3 and SPARKY.47 The DQF-COSY48 spectra
Crystal data for PyrAc-(Deg)5-O-(pNO2)Bzl: C55H72N6O9,
M = 961.19, monoclinic, space group P21, a = 10.0556(2) Å,
b = 23.1736(5) Å, c = 11.9038(2) Å, β = 98.010(2)°; V =
2746.81(9) Å3, Z = 2; ρcalcd = 1.162 Mg m−3, μ = 0.638 mm−1
,
38 894 reflections measured, 10 511 independent reflections (Rint
= 0.0329), θmax = 72.39°, T = 293(2) K. Although lacking chiral
carbon atoms, the molecule crystallizes in a chiral space group.
The chosen enantiomorph is that giving the lowest value for the
2420 | Org. Biomol. Chem., 2012, 10, 2413–2421
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15 C. Toniolo, M. Crisma, F. Formaggio, C. Peggion, Q. B. Broxterman and
B. Kaptein, Biopolymers, 2004, 76, 162–176.
16 M. Crisma, F. Formaggio, A. Moretto and C. Toniolo, Biopolymers,
2006, 84, 3–12.
17 M. Crisma, E. Andreetto, M. De Zotti, A. Moretto, C. Peggion,
F. Formaggio and C. Toniolo, J. Pept. Sci., 2007, 13, 190–205.
18 J. Torras, D. Zanuy, M. Crisma, C. Toniolo, O. Betran and C. Aleman,
Biopolymers, 2008, 90, 695–706.
19 A. Moretto, M. De Zotti, M. Crisma, F. Formaggio and C. Toniolo,
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Flack parameter [0.0(2)]. Restraints were applied to the anisotro-
pic displacement parameters of a number of atoms belonging to
the PyrAc moiety, the Cγ atoms of the Deg residues, and the O
atoms of the C-terminal nitro group. The final R1 values were
0.0638 [I > 2σ(I)] and 0.0705 (all data). Data/restraints/
parameters 10 511/91/631. The goodness of fit on F2 was 1.032.
The largest difference peak and hole were 0.661 and −0.206 e
Å −3, respectively.
20 C. Toniolo and E. Benedetti, Trends Biochem. Sci., 1991, 16, 350–353.
21 F. Formaggio, M. Crisma, C. Peggion, A. Moretto, M. Venanzi and
C. Toniolo, Eur. J. Org. Chem., 2012, 167–174.
Crystal data for PyrAc-(Aib)5-OtBu: C42H55N5O7, M =
ˉ
741.91, triclinic, space group P1, a = 11.5910(8) Å, b = 12.4620
(8) Å, c = 15.4573(12) Å, α = 97.958(1)°, β = 107.430(1)°, γ =
91.224(1)°; V = 2105.1(3) Å3, Z = 2; ρcalcd = 1.170 Mg m−3, μ =
0.647 mm−1, 37 968 reflections measured, 8448 independent
reflections (Rint = 0.0328), θmax = 73.34°, T = 293(2)
K. Restraints were applied to the anisotropic displacement para-
meters of a number of atoms belonging to the PyrAc moiety
and the C-terminal tBu group. The final R1 values were 0.0537
[I > 2σ(I)] and 0.0559 (all data). The final wR(F2) values were
0.1598 [I > 2σ(I)] and 0.1638 (all data). Data/restraints/
parameters 8448/66/487. The goodness of fit on F2 was 1.031.
The largest difference peak and hole were 0.266 and −0.237 e
Å−3, respectively.
22 C. Peggion, M. Crisma, C. Toniolo and F. Formaggio, Tetrahedron, 2012,
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M. C. is grateful to Prof. A. Dolmella (Department of Pharma-
ceutical Sciences, University of Padova) for granting access
to the Gemini diffractometer and for his help with X-ray data
collection and processing. Financial support for the acquisition
of the Agilent Gemini diffractometer was provided by the
University of Padova through the 2008 “Scientific Equipment
for Research” initiative.
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