All-thioamidated homo-α-peptides: Synthesis and conformation

Article abstract of DOI:10.1002/ejoc.201300050

Replacement of a peptide bond with its thioamide surrogate is a classical method for the generation of a peptidomimetic with altered spectroscopic, conformational, physicochemical, and biological properties. In this context, we synthesized short series of

Full text of DOI:10.1002/ejoc.201300050

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FULL PAPER
DOI: 10.1002/ejoc.201300050
All-Thioamidated Homo-α-Peptides: Synthesis and Conformation
Fernando Formaggio,*[a] Marco Crisma,[a] Claudio Toniolo,[a] and Cristina Peggion*[a]
Keywords: Conformation analysis / Synthetic methods / Peptides / Peptidomimetics
Replacement of a peptide bond with its thioamide surrogate
is a classical method for the generation of a peptidomimetic
with altered spectroscopic, conformational, physicochemical,
and biological properties. In this context, we synthesized
short series of terminally protected homo-α-oligopeptides
based on the α-amino acids Gly, Ala, and Nle, as well as their
corresponding fully thioamidated analogues. For the first
time, the preparation of the latter compounds was achieved
in single-step fashion through direct thionation of their oxy-
genated precursors. Using X-ray diffraction analysis and
NMR spectroscopy we were also able to confirm that the
thioamidated α-amino acid residues can easily adopt either
folded or fully extended conformations.
tures and the increasing number of naturally occurring
poly-α- and -β-peptides containing up to five consecutive
thioamide groups in their amino acid chains, that have been
isolated and sequenced.[66–68] In this work we decided to fill
this gap at least partially by synthesizing a large set of all-
thioamidated, selected peptide foldamers, by use of the
single-step direct thionation methodology, and by investi-
gating their conformational preferences by detailed X-ray
diffraction and 2D-NMR analyses.
Introduction
Thionated peptides are members of an interesting class
of backbone-modified peptidomimetics[1] that have been
utilized in a variety of fields, including medicinal chemistry,
spectroscopy, and photophysics (cis/trans thioamide isomer-
ization).[1–25] Their preferred conformations have been ex-
tensively investigated, in particular by X-ray diffrac-
tion, NMR, and electronic and energy calcula-
tions.[2,3,7,13,15,17,26–59] Most of these studies have involved
compounds with single, site-specific peptide bond modifica-
tions. These targets have typically been achieved by regiose-
lective thionation of very short peptides (e.g., terminally
protected tripeptides), followed by extension of the amino
acid chain by chemical synthesis.[2,3,30,60–65] The classical
protecting functionalities used in peptide synthesis (ureth-
anes at N termini and esters at C termini) are not modified
under the experimental conditions almost universally em-
ployed for thionation of an amide group (Lawesson rea-
gent[60] in an organic solvent of low polarity). Moreover,
additional regioselectivity between two (or three) amide
groups can frequently be achieved by taking advantage of
the differences in steric hindrance exerted by the side chains
of the pairs of surrounding amino acids.
Results and Discussion
Peptide Synthesis and Characterization
We used classical solution-phase methods to prepare
three short series (from dimer through tetramer) of ter-
minally protected homo-α-oligopeptides based on the Gly,
Ala, and Nle (norleucine) residues. These amino acids differ
in their linear, but increasingly bulkier, side chains (R),
where R = H for Gly, R = –CH3 for Ala, and R = –CH2–
(CH2)2–CH3 for Nle. The syntheses and characterizations
of these nine peptides had already been reported.[69–72]
Thionation of the Gly dipeptide with Lawesson reagent
(LR)[60] under typical conditions [anhydrous THF at room
temperature; Procedure A (see the Exp. Section)] proceeded
fairly smoothly in about 1 hour to afford the monothioami-
dated compound 2a (Scheme 1) after chromatographic pu-
rification (no special efforts were made to optimize the
yields in our thionation reactions).
In contrast, only a very limited number of articles deal-
ing with synthesis and conformation of all-thioamidated
peptides have appeared in the literature.[2,28] This observa-
tion is rather surprising in view of the great potential inter-
est of this class of peptidomimetics as new foldameric struc-
To obtain the desired Gly bis-thioamidated product 3a
and the tris-thioamidated product 4a, a further aliquot of
LR (to a total of 1.8–3.0 equiv.) was added after 24 hours
in each case. The reaction mixtures were left whilst stirring
for additional 24 hours.
The preparations of the Ala bis-thioamidated tripeptide
3b and the tris-thioamidated tetrapeptide 4b (but not that
[a] ICB, Padova Unit, CNR, Department of Chemistry, University
of Padova,
35131 Padova, Italy
E-mail: fernando.formaggio@unipd.it
cristina.peggion@unipd.it
Homepage: http://www.chimica.unipd.it/toniologroup/
index_file/Page1089.htm
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300050.
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tions, an intense maximum near 265 nm, accompanied by
an extremely weak maximum at about 330 nm, is found in
each case. These two bands, which are absent in the spectra
of the corresponding oxygenated peptides, are attributable
to the πǞπ* and nǞπ* transitions, respectively, of the
thioamide chromophore.[2,30,60] In the 250–400 nm region
of the CD spectra in the same solvents, two well-separated
Cotton effects at about 260–270 nm and 345–350 nm are
typically displayed.[2,3,11–14,16–21,24,25,74] The longer-wave-
length band is positive and weaker, whereas the shorter-
wavelength band is negative and much more intense. The
1H (and 13C) spectra in CDCl3 solution exhibit significant
downfield shifts for the thioamide and α-carbon protons
(and for the thiocarbonyl and α-carbons as well) with re-
spect to their corresponding amide analogues, consistently
with previously reported values for similar com-
Scheme 1. Amino acid sequences of the nine all-thioamidated
homo-α-peptides synthesized and studied (Z is benzyloxycarbonyl,
OMe is methoxy, and ψ[CSNH] is the widely used representation
of a thioamidated peptide surrogate[1]).
of the monothioamidated dipeptide 2b) required larger ex- pounds.[2,30,60]
cesses of LR (up to a total of 2.4–3.6 equiv.) and longer
reaction times (up to 9 d). Thionation of the Nle dipeptide
Crystal-State Conformation Analysis
proved to be much more sluggish.
To obtain the monothioamidated compound 2c, 9 days
under reflux were found to be necessary with multiple ad-
ditions of LR to a total of 3 equiv. Thionation of the Nle
tri- and tetrapeptides under comparable conditions could
barely be achieved, however. Eventually, syntheses of the
bis-thioamidated product 3c and the tris-thioamidated
product 4c were accomplished by use of Procedure B, which
Out of the all-thioamidated homo-α-peptides synthesized
in this work, we were able to grow single crystals from Z-
Gly-ψ[CSNH]-Gly-ψ[CSNH]-Gly-OMe (3a). The X-ray dif-
fraction structure is illustrated in Figure 1. Selected torsion
angles are listed in Table 1, intra- and intermolecular H-
bond parameters are reported in Table 2, and crystal data
and structure refinement parameters are given in Table 3.
The achiral thiopeptide crystallizes in a centrosymmetric
space group, so molecules of both handedness are present
in the unit cell. In Table 1 and in the following discussion,
reference is made to the molecule characterized by a nega-
tive value of the φ1 torsion angle, arbitrarily chosen as the
asymmetric unit.
[64]
involves treatment of the oxygenated peptides with P2S5
in anhydrous tetrahydrofuran under ultrasound irradiation
conditions for 3–5 hours, with addition of a total of 5–
10 equiv. of the sulfur donor reagent in three to six aliquots.
Selective thionations of the peptide functionalities were
achieved in all cases, because urethane and ester groups do
not react under all conditions tested.[2,3,60–62,64] The various
synthetic steps ultimately leading to the all-thioamidated
tri- and tetrapeptides (in particular those affording the in-
termediate monothionated tripeptides and mono- and bis-
thionated tetrapeptides) were not investigated in detail be-
cause our goal was solely to obtain sufficient amounts of
the final materials for use in our subsequent conformation
studies. As already reported in the literature for partially
and fully thionated α- and β-peptides, peptide solubilities
were observed to have increased markedly relative to those
of their oxygenated counterparts.[2] In our study, this effect
is best demonstrated by the sparingly soluble Z-(Gly)4-OMe
tetrapeptide. Indeed, we found that its suspension in tetra-
hydrofuran became a clear solution as the thionation reac-
tion proceeded to afford 4a.
After chromatographic purifications, the nine all-thio-
amidated peptides synthesized were spectroscopically char-
acterized by IR absorption, UV absorption, circular dichro-
1
ism (chiral compounds only), and H and 13C NMR tech-
niques. FTIR absorption analysis in CDCl3 solution
showed the peptide C=O stretching band near 1650 cm–1 to
be missing[2,3,60] in each case (only bands at wavenumbers
higher than 1690 cm–1, associated with the ester and ure-
thane C=O stretching modes, are seen).[73] In the UV ab-
Figure 1. X-ray diffraction structure of Z-Gly-ψ[CSNH]-Gly-
ψ[CSNH]-Gly-OMe (3a) with atom numbering. The two intramo-
sorption spectra in methanol or 2,2,2-trifluoroethanol solu- lecular H-bonds are indicated by dashed lines.
2
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All-Thioamidated Homo-α-Peptides: Synthesis and Conformation
Table 1. Selected torsion angles [°] for Z-Gly-ψ[CSNH]-Gly- amino[75] and methyl ester groups[76] and the thioamide
linkage.[27] Specifically, the C1=S1 and C2=S2 bond lengths
ψ[CSNH]-Gly-OMe (3a).
are 1.659(2) Å and 1.6623(19) Å, respectively, and the C1–
N2 and C2–N3 bond lengths are 1.306(2) Å and 1.315(2) Å,
respectively. The CЈ=S bonds are significantly longer that
the corresponding CЈ=O bond length in peptides
(1.20 Å),[26,27] whereas the CЈ–N bonds are slightly shorter
than in the peptide unit (1.34 Å). This latter finding sug-
gests a more pronounced double bond character for CЈ–N
bonds in thioamides than for those in peptides.[26,27,41,51,52]
Both thioamides, as well as the urethane and ester units,
are in the common trans disposition, with deviations from
trans planarity [|Δω|] not exceeding 6°.
The conformation adopted by the N-terminal Gly(1)
residue, characterized by φ1 = –86.3(2)° and ψ1 = –3.4(3)°,
falls in the region of the Ramachandran map between the
helical and extended conformations, termed the “bridge”
region.[77] Conversely, the Gly(2) and Gly(3) residues, with
φ2,ψ2 = 168.95(18)°, –159.90(16)° and φ3,ψ3 = –159.38(17)°,
167.61(18)°, are essentially fully extended.[78] As a result,
two intramolecularly H-bonded forms of the C5 type[79] are
observed at the level of the Gly(2) and Gly(3) residues, in-
volving the N2–H and N3–H groups, respectively, as the
donors, and the (thioamide) S2 and (ester) O3 atoms,
respectively, as the acceptors. Interestingly, the τ (N–Cα–CЈ)
bond angle for Gly(1) is 114.86(16)°, but those for the
Gly(2) and Gly(3) residues involved in the C5 structures are
110.05(15)° and 109.99(15)°, respectively. For comparison,
the values of the τ bond angle of Gly averaged from small-
molecule[80] and protein[81] X-ray diffraction structures are
112.5° and 112.1°, respectively. The expansion of τ above
the standard tetrahedral value to about 114° for amino ac-
ids in the “bridge” region of the conformation map was
noted by Karplus[81] in his statistical survey of protein
structures. This author ascribed it to the existence of a short
contact between the nitrogen atom of residue i and the
amide H atom of the subsequent (i + 1) residue in the pept-
ide chain.
The CЈ=S bond is significantly longer than a CЈ=O
bond, so the geometric parameters of the C5 structure at
the level of Gly(2) differ from the corresponding average
values reported for N–H···O=C intra-residue H-bonds of
the C5 type.[79] In the case of the C-terminal C5 structure,
the N–H···O angle is narrower and the N···O and H···O
distances are longer than the average in C5 structures, as a
result of the deviation of the φ,ψ backbone torsion angles
from the ideal values (180°, 180°) of the fully extended con-
formation. In addition, and at variance with the observa-
tion that the N–H and C=O groups involved in regular C5
structures do not take part in any intermolecular hydrogen
bonding, in this structure the N3–H and C3=O3 groups are
also intermolecularly H-bonded to each other (see below).
In the packing mode (not shown), both N–H···S=C[41]
and N–H···O=C intermolecular H-bonds are observed.
Specifically, the N1–H group is H-bonded to an (x, ½ – y,
½ + z) symmetry equivalent of the C1=S1 group, connect-
C02–C01–C07–OU
C06–C01–C07–OU
C01–C07–OU–C0
C07–OU–C0–N1
OU–C0–N1–C1A
C0–N1–C1A–C1
N1–C1A–C1–N2
N1–C1A–C1–S1
C1A–C1–N2–C2A
C1–N2–C2A–C2
N2–C2A–C2–N3
C2A–C2–N3–C3A
C2–N3–C3A–C3
N3–C3A–C3–OT
C3A–C3–OT–CT
–69.2(3)
113.5(2)
174.5(2)
178.8(2)
–173.98(16)
–86.3(2)
–3.4(3)
176.63(15)
179.38(18)
168.95(18)
–159.90(16)
176.40(17)
–159.38(17)
167.61(18)
–179.4(3)
Table 2. Intra- and intermolecular H-bond parameters for Z-Gly-
ψ[CSNH]-Gly-ψ[CSNH]-Gly-OMe (3a).
Donor (D–H)
Ac-
Dis-
Dis-
tance
[Å]
Angle[°] Symmetry
D–H···A equivalence
of A
ceptor tance
(A)
[Å]
D···A
H···A
N2–H2
N3–H3
N1–H1
S2
O3
S1
2.9348(16) 2.44
2.715(2) 2.39
3.4183(18) 2.62
118
103
155
x, y, z
x, y, z
x, ½-y, ½
+ z
2-x, –y, –z
N3–H3
O3
3.039(2)
2.19
169
Table 3. Crystal data and structure refinement for Z-Gly-ψ[CSNH]-
Gly-ψ[CSNH]-Gly-OMe (3a).
Parameter
Data
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C15H19N3O4S2
369.45
293(2) K
0.71069 Å
monoclinic
P21/c
Unit cell dimensions
a = 9.1152(3) Å
b = 21.7511(6) Å
c = 9.8888(3) Å
1863.03(10) Å3
4
α = 90°
β = 108.153(3)°
γ = 90°
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
θ Range for data collection
Iindex ranges
1.317 Mgm–3
0.309 mm–1
776
0.50ϫ0.20ϫ0.20 mm3
2.35 to 28.27°.
–12 Յ h Յ 12, –29 Յ k Յ 29,
–13 Յ l Յ 13
Reflections collected
Independent reflections
Completeness to θ = 28.27° 100.0%
Absorption correction
Max. and min. transmission 1.00000 and 0.83404
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [IϾ2σ(I)]
R indices (all data)
33002
4627 [R(int) = 0.0246]
semiempirical from equivalents
full-matrix, least-squares on F2
4627/0/205
1.042
R1 = 0.0480, wR2 = 0.1194
R1 = 0.0610, wR2 = 0.1300
0.453 and –0.498 eÅ–3
Largest diff. peak and hole
Interatomic distances and angles are in general agree- ing molecules in a zigzag motif along the c direction. A
ment with literature values for the benzyloxycarbonyl- second intermolecular H-bond is found between the N3–
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H3 group and the (2 – x, –y, –z) equiv. of the C3=O3 group, NH(i)ǞNH(i + 1) connectivities described above, repre-
thus generating a doubly H-bonded, centrosymmetric pair sents a clear indication of the presence of fully extended
of molecules. In addition, a C–H···S contact with a H···S structures.[89] In summary, in the conformational equilib-
distance of 2.79 Å (less than the sum of the van der Waals rium mixtures of the all-thioamidated homo-oligopeptides
radii of S and H, 2.90 Å)[82,83] is observed between the CT- studied in this work, fully extended and folded residues oc-
HT1 group and the (2 – x, –y, –z) symmetry equivalent cur concomitantly, with the latter prevailing near the N ter-
of S1. Packing is then completed through van der Waals minus of the chain. Interestingly, this conclusion from our
interactions.
solution conformation analysis fits nicely with the results of
the X-ray diffraction study on peptide 3a in the crystalline
state discussed above.
Solution Conformation Analysis
In our 2D-NMR analysis, specific attention was given to
Our 2D-NMR study was performed on tripeptides 3a, the TOCSY experiments. These were conducted on the six
3b, and 3c and tetrapeptides 4a, 4b, and 4c in CDCl3 solu- all-thioamidated tri- and tetrapeptides and also, for com-
tion. The assignments of all of the resonances for these parison, on the oxygenated Z-(Gly)3-OMe and Z-
compounds were achieved by DQF-COSY,[84] TOCSY,[85,86] (Gly)4-OMe. An unanticipated behavior pattern was noted,
and HMQC and HMBC[87] experiments.
As examples, Figure 2 shows that, for each of the three ated Gly peptides 3a and 4a.
but was limited to the TOCSY spectra of the all-thioamid-
tripeptides, both NH(i)ǞNH(i + 1) sequential connectivi-
The TOCSY method gives a total correlation for all pro-
ties, characteristic of helical conformations,[88] can be seen tons of a peptide chain with one another. Rotating frame
in the NOESY spectra. However, the NH(1)ǞNH(2) cross- experiments produce coherence transfer through J coupling
peak is weak and the NH(2)ǞNH(3) cross-peak is hardly and through cross-relaxation simultaneously. This phenom-
even visible. Moreover, an analysis of the relative intensities enon can lead to undesired interference and ambiguities be-
of the intraresidue αCH(i)ǞNH(i) and interresidue tween TOCSY and ROESY effects. The pulse program with
αCH(i – 1)ǞNH(i) connectivities in the NOESY spectra of MLEV17 used in our experiments provides clean TOCSY
all six tripeptides and tetrapeptides investigated provided a spectra, in which contributions from cross-relaxation are
useful hint of the conformations adopted. Firstly, we did suppressed. This procedure guarantees that the spin system
observe all of the expected intra- and interresidue cross- will evolve solely under the influence of the scalar coupling.
peaks in the spectrum of each compound. Secondly, in Accordingly, no peaks of negative sign due to cross-relax-
some cases the interresidue cross-peak is more intense than ation are present in our spectra. However, quite surpris-
the intraresidue cross-peak. This informative observation, ingly, cross-peaks (although rather weak) attributable to in-
combined with the general weakness of the sequential terresidue signals were detected. It is well known that, dur-
Figure 2. NHǞNH and αCHǞNH regions of the NOESY spectra of the all-thioamidated tripeptides 3a (left), 3b (center), and 3c (right)
in CDCl3 solution. The intraresidue αCHǞNH cross-peaks are not labeled.
4
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All-Thioamidated Homo-α-Peptides: Synthesis and Conformation
ing the mixing time in a TOCSY experiment with a peptide Two additional, anomalous cross-peaks (not shown) occur
substrate, magnetization, occurring through J coupling, in the αCH2ǞαCH2 region of the TOCSY spectrum of this
normally does not pass through different residues (it is same tripeptide 3a. Analogous cross-peaks were also found
“stopped” by the amide bonds).[88] Surprisingly, in our all- in the TOCSY spectra of the all-thioamidated Gly tetra-
thioamidated Gly tripeptide 3a and tetrapeptide 4a, cou- peptide 4a (not shown). From our TOCSY experiments, it
plings between αCH2(i)ǞNH(i + 1), αCH2(i)ǞαCH2(i + seems safe to conclude that this seemingly unprecedented
1), and αCH2(i)ǞNH(i – 1) are seen. With use of increasing phenomenon is correlated with the presence of thioamid-
mixing times (45, 64, 70, 100, and 140 msec) to record the ated bonds in the peptide sequence. However, the lack of
spectra, the intensities of the anomalous cross-peaks in- this observation in the all-thioamidated Ala and Nle homo-
crease correspondingly. Strictly comparable experiments peptides needs further experiments on related peptide sub-
performed on the all-thioamidated tri- and tetrapeptides de- strates before a definitive conclusion. These words of cau-
rived from Ala (3b and 4b) and Nle (3c and 4c), and also tion are justified by the weakness of the observed abnormal
on the corresponding oxygenated Z-Gly3–OMe and Z- interresidue signals and by the fact that all protein amino
Gly4–OMe, did not reveal any abnormal cross-peak even at acids but Gly are characterized by one αCH proton (only
the longest mixing time considered. The αCH2ǞNH region Gly has two). In other words, this effect might also occur
in the TOCSY spectra of the all-thioamidated Gly tripep- in all thioamidated Ala and Nle homopeptides, but might
tide 3a (with 70 and 140 msec mixing times), with most of simply be below the level of a possible observation.
the anomalous cross-peaks highlighted, is shown in Fig-
A tentative explanation of this phenomenon might reside
ure 3 (this figure also shows the corresponding TOCSY in the different electronic properties of carboxylic thio-
spectra for the non-thioamidated tripeptide Z-Gly3–OMe). amides in relation to amides.[26,27,41,51,52] The well-known
Figure 3. αCH2ǞNH region of the TOCSY spectra for: i) the all-thioamidated Gly tripeptide 3a at A) 70 msec, and B) 140 msec mixing
times, and ii) the corresponding oxygenated Z-(Gly)3-OMe at C) 70 msec, and D) 140 msec mixing times.
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with a Leitz Laborlux 12 apparatus. The solid-state (KBr disk) and
solution (CDCl3) IR absorption spectra were recorded with a Per-
kin–Elmer model 1720X FTIR spectrophotometer. TLC was per-
formed with Merck Kieselgel 60F254 pre-coated plates and the fol-
lowing solvent systems: 1) chloroform/ethanol 9:1, 2) butanol/
acetic acid/water 3:1:1; and 3) toluene/ethanol 7:1. The chromato-
grams were visualized by UV fluorescence or developed by
chlorine/starch/potassium iodide or ninhydrin chromatic reaction
as appropriate. All compounds were obtained in a chromatographi-
cally homogeneous state. Flash chromatography was carried out
restricted rotation around the C(=O)–NH bond in an amide
is associated with the partial double bond character be-
tween the Cα and N atoms. In a thioamide, the rotation
barrier (more than 21 kcalmol–1 in amides) requires about
5 kcalmol–1 more than in an amide. This property is in turn
related to a greater contribution of the dipolar resonance
structure C(–S–)=N+H in thioamides, which enhances their
double bond character. The presence of these successive (al-
though partial) C=N double bonds might be responsible for
the transfer of magnetization taking place through protons with a Merck silica gel 60 (40–63 μm mesh) stationary phase. MS
(ESI mode) spectra were measured with a Perseptive Biosystems
(Mariner model) ESI-ToF instrument. The UV absorption spectra
were recorded with a Shimadzu model UV-2501 spectrophotome-
ter. The CD spectra were measured with a Jasco model J-715 spec-
tropolarimeter equipped with a Haake thermostat.
belonging to different residues in the amino acid sequence.
Conclusions
The Z-(Gly)n-OMe,[69] Boc-(Ala)n-OMe,[70,71] and Boc-(Nle)n-
OMe[72] peptides are known compounds.
The replacement of an amide bond in a peptide by a
thioamide bond is one of the most valuable backbone
modifications for the generation of new and useful com-
pounds in bioorganic stereochemistry and biology. In most
of the examples in the literature the polypeptide chain is
selectively modified at the level of a single amide function.
Recently, however, interest in multiply thioamidated or even
all-thioamidated compounds has been steadily increas-
ing.[2,28,66–68]
General Procedures for Thionation of Peptides
Procedure A: LR (0.6–1.0 mmol for a dipeptide, 1.8–2.4 mmol for
a tripeptide, 3.0–3.6 mmol for a tetrapeptide) was added at room
temperature under nitrogen to a solution of the peptide (1 mmol)
in anhydrous tetrahydrofuran. The course of the reaction (with stir-
ring at room temperature) was monitored by TLC. After 1–3 d,
further LR (0.6–1.0 mmol) was added. After completion of the re-
action and removal of the solvent under reduced pressure, the crude
In this work, with the aim of determining the conforma-
tion effects induced in peptides by complete thionation of product was directly purified by flash chromatography.
their –C(=O)–NH– bonds, we synthesized a set of nine all-
thioamidated homo-oligopeptides, based on three α-amino
acids with side chains of enhanced steric hindrance. For the
Procedure B: P2S5 (2.4 mmol) was added at room temperature un-
der nitrogen to a solution of the peptide (1 mmol) in anhydrous
tetrahydrofuran. The reaction mixture was subjected to ultrasound
first time, we succeeded in preparing bis- and tris-thioamid- and stirred for 3–5 h, during which the bath temperature was in-
creased gradually to 45 °C. The course of the reaction was moni-
tored by TLC. Further additions of P2S5 (total amount of 5–
10 mmol) were performed over 3–5 h. After completion of the reac-
tion and removal of the solvent under reduced pressure, the crude
product was directly purified by flash chromatography.
ated tri- and tetrapeptides, respectively, in single-step fash-
ion, starting from their corresponding oxygenated ana-
logues. Complete thionation was observed to become in-
creasingly more difficult as the peptide is elongated and as
the amino acid side chain becomes bulkier. To thionate the
most resistant substrates fully, we were forced to replace the
classical LR[60] with the more efficient procedure based on
the P2S5 reagent assisted by ultrasonication.[64]
A literature search on the conformational propensities of
thionated (usually monothionated) peptides revealed that,
despite the well-established, individual geometric properties
of the thiopeptide unit (in particular of thiocarbonyl vs.
carbonyl bond length), both folded and fully extended con-
Z-Gly-ψ[CSNH]-Gly-OMe: Flash chromatography eluent: EtOAc
(ethyl acetate)/PE (petroleum ether) 1:1, yield 35%, m.p. 93–95 °C
[from EtOAc/PE]. Rf1 = 0.90; Rf2 = 0.95; Rf3 = 0.50. 1H NMR
(200 MHz, CDCl3): δ = 8.44 (br., 1 H, CSNH Gly2), 7.36 (m, Z
CH), 5.45 (br., 1 H, NH Gly1), 5.14 (s, 2 H, Z CH2), 4.40 (d, 2 H,
Gly CH2), 4.29 (d, 2 H, Gly CH2), 3.81 (s, 3 H, OCH3) ppm. 13C
NMR (200 MHz, CDCl3): δ = 200.01 (CS), 168.93 (COOMe), 156
(Z CO), 135.86–128.59–128.36–128.17 (Z), 67.56 (Z CH2), 52.71
(OCH ), 52.16 (Gly CH ), 46.81 (Gly CH ) ppm. IR (KBr): ν =
˜
3
2
2
formations are stereochemically favorable and commonly 3396, 3364, 3295, 1748, 1717, 1706, 1548 cm–1. MS (ESI+): m/z =
297.09 [M + H]+.
found for the thionated residues.[2,3,7,13,15,17,26–59] In this ar-
ticle we have extended these conclusions to all-thioamidated
α-peptides both in the crystalline state and in solution, as
Z-Gly-ψ[CSNH]-Gly-ψ[CSNH]-Gly-OMe: Flash chromatography
eluent: CH2Cl2/EtOH (ethanol) 98:2, yield 57%, m.p. 101–103 °C
assessed by detailed X-ray diffraction and 2D-NMR analy- [from EtOAc/PE]. Rf1 = 0.90; Rf2 = 0.95; Rf3 = 0.50. 1H NMR
(200 MHz, CDCl3): δ = 9.01 (br., 1 H, CSNH Gly2), 8.19 (br., 1
ses, respectively. These results pave the way for the exploi-
H, CSNH Gly3), 7.36 (m, Z CH), 5.46 (br., 1 H, NH Gly1), 5.16
tation of the all-thioamidated peptidomimetics as a new
(s, 2 H, Z CH2), 4.65 (br. d, 2 H, Gly2 CH2), 4.40 (d, J = 3.48 Hz,
class of foldameric compounds and functional modifica-
tions of bioactive peptides.
2 H, Gly3 CH2), 4.31 (d, J = 5.68 Hz, 2 H, Gly1 CH2), 3.82 (s, 3
H, OCH3) ppm. 13C NMR (200 MHz, CDCl3): δ = 199.40 (CS
Gly1), 197.32 (CS Gly2), 168.77 (COOMe), 156.95 (Z CO), 135.87–
128.60–128.35–128.15 (Z), 67.64 (Z CH2), 54.03 (Gly2 CH2), 52.88
(OCH ), 52.32 (Gly1 CH ), 47.06 (Gly3 CH ) ppm. IR (KBr): ν =
Experimental Section
˜
3
2
2
3294, 1718, 1541 cm–1. MS (ESI+): m/z = 370.09 [M + H]+.
Synthesis and Characterization: Materials and reagents were of the
highest commercially available grade and were used without further
purification. Melting points were determined in open capillaries
Z-Gly-ψ(CSNH)-Gly-ψ[CSNH]-Gly-ψ[CSNH]-Gly-OMe: Flash
chromatography eluent: CHCl3/MeOH (methanol) 97:3, yield 56%,
6
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O30050
/KAP1
Date: 26-03-13 17:50:56
Pages: 10
All-Thioamidated Homo-α-Peptides: Synthesis and Conformation
m.p. 56–58 °C [from EtOAc/PE]. Rf1 = 0.90; Rf2 = 0.95; Rf3 =
0.40. 1H NMR (200 MHz, CDCl3): δ = 8.93 (br., 2 H, CSNH Gly2
CSNH Gly3), 8.06 (br., 1 H, CSNH Gly4), 7.37 (m, Z CH), 5.55
(br., 1 H, NH Gly1), 5.17 (s, 2 H, Z CH2), 4.72 (d, 2 H, Gly2 CH2),
4.65 (d, J = 3.56 Hz, 2 H, Gly3 CH2), 4.41 (d, J = 4.64 Hz, 2 H,
Gly4 CH2), 4.33 (d, J = 6.00 Hz, 2 H, Gly1 CH2), 3.82 (s, 3 H,
OCH3) ppm. 13C NMR (200 MHz, CDCl3): δ = 200.13 (CS),
196.95 (CS), 196.78 (CS), 168.90 (COOMe), 156.95 (Z CO),
135.77–128.62–128.41–128.15 (Z), 67.72 (Z CH2), 54.51 (Gly2 Gly3
CH2), 52.86 (OCH3), 52.39 (Gly1 CH2), 46.92 (Gly4 CH2) ppm. IR
(Nle γ-CH2), 22.53 (2ϫNle δ-CH2), 13.93 (2ϫCH3 Nle) ppm. IR
(KBr): ν = 3274, 1748, 1691 cm–1. MS (ESI+): m/z = 375.24 [M +
˜
H]+.
Boc-Nle-ψ[CSNH]-Nle-ψ[CSNH]-Nle-OMe: Flash chromatog-
raphy eluent: EtOAc/PE (2:9), yield 27%, oil. Rf1 = 0.95; Rf2 =
1
0.95; Rf3 = 0.90. H NMR (400 MHz, CDCl3): δ = 8.54 (br. d, J
= 7.04 Hz, 1 H, CSNH), 8.02 (br. d, J = 7.56 Hz, 1 H, CSNH),
5.27 (m, 1 H, α-CH), 5.10 (m, 1 H, α-CH), 5.10 (br., 1 H, NH
Nle1), 4.29 (m, 1 H, α-CH), 3.78 (s, 3 H, OCH3), 2.01 (m, 2 H,
Nle2 β-CH2), 2.00 (m, 2 H, Nle3 β-CH2), 1.93 (m, 2 H, Nle1 β-
CH2), 1.94 (m, 2 H, Nle2 γ-CH2), 1.84 (m, 2 H, Nle3 γ-CH2), 1.71
(m, 2 H, Nle1 γ-CH2), 1.44 (s, 9 H, 3ϫCH3Boc), 1.34 (m, 2 H,
Nle2 δ-CH2), 1.32 (m, 4 H, Nle1 Nle3 δ-CH2), 0.89 (m, 9 H,
3ϫCH3 Nle) ppm. 13C NMR (400 MHz, CDCl3): δ = 204.01 (CS
(KBr): ν = 3358, 1735, 1696, 1585, 1561 cm–1. MS (ESI+): m/z =
˜
443.08 [M + H]+.
Boc-Ala-ψ[CSNH]-Ala-OMe: Flash chromatography eluent:
EtOAc/PE (1:3), yield 67%, oil. Rf1 = 0.90; Rf2 = 0.95; Rf3 = 0.80.
1H NMR (200 MHz, CDCl3): δ = 8.37 (br., 1 H, CSNH Ala2), 5.12 Ala1), 202.16 (CS Ala2), 171.60 (COOMe), 155.57 (COBoc), 80.21
(br., 1 H, NH Ala1), 5.09 (m, 1 H, α-CH), 4.45 (m, 1 H, α-CH),
(C4Boc), 64.18 (α-CH Nle), 63.94 (α-CH Nle), 61.59 (α-CH Nle),
3.79 (s, 3 H, OCH3), 1.54 (d, 3 H, CH3 Ala), 1.54 (d, 3 H,CH3 Ala), 52.62 (OCH3), 35.14 (Nle β-CH2), 34.43 (Nle β-CH2), 30.70 (Nle
1.45 (s, 9 H, 3ϫCH3 Boc) ppm. 13C NMR (200 MHz, CDCl3): δ
= 205.27 (CS), 172.60 (COOMe), 155.90 (COBoc), 79.50 (C4 Boc),
68.34 (Ala1 CH), 53.30 (Ala2 CH), 52.84 (OCH3), 28.44 (3ϫCH3
β-CH2), 28.34 (3ϫCH3Boc), 27.83 (Nle γ-CH2), 27.30 (2ϫNle γ-
CH2), 22.27 (3ϫNle δ-CH2), 13.90 (3ϫCH3 Nle) ppm. IR (KBr):
˜
ν = 3295, 1731, 1691 cm–1. MS (ESI+): m/z = 504.29 [M + H]+.
Boc), 21.65 (Ala1 CH3), 17.17 (Ala2 CH ) ppm. IR (KBr): ν =
˜
3
Boc-Nle-ψ(CSNH)-Nle-ψ[CSNH]-Nle-ψ[CSNH]-Nle-OMe: Flash
chromatography eluent: EtOAc/PE (2:8), yield 33%, oil. Rf1 = 0.95;
Rf2 = 0.95; Rf3 = 0.90. 1H NMR (400 MHz, CDCl3): δ = 8.64 (br.,
1 H, CSNH Nle2), 8.37 (br. d, J = 7.00 Hz, 1 H, CSNH Nle3), 8.06
(br. d, J = 5.18 Hz, 1 H, CSNH Nle4), 5.23 (m, 2 H, α-CH Nle2 α-
CH Nle3), 5.16 (br., 1 H, NH Nle1), 5.13 (m, 1 H, α-CH Nle4),
4.31 (m, 1 H, α-CH Nle1), 3.78 (s, 3 H, OCH3), 1.98 (m, 2 H, Nle2
3261, 1746, 1689 cm–1. MS (ESI+): m/z = 291.13 [M + H]+.
Boc-Ala-ψ[CSNH]-Ala-ψ[CSNH]-Ala-OMe: Flash chromatog-
raphy eluent: CHCl3/EtOH (97:3), yield 43%, m.p. 115–117 °C
[from EtOAc/PE]. Rf1 = 0.90; Rf2 = 0.95; Rf3 = 0.80. 1H NMR
(400 MHz, CDCl3): δ = 8.96 (br. d, J = 5.72 Hz, 1 H, CSNH Ala2),
8.17 (br. d, J = 5.20 Hz, 1 H, CSNH Ala3), 5.30 (m, 1 H, α-CH
Ala2), 5.14 (br., 1 H, NH Ala1), 5.06 (m, 1 H, α-CH Ala3), 4.47 β-CH2), 2.01 (m, 2 H, Nle3 β-CH2), 2.00 (m, 2 H, Nle4 β-CH2),
(m, 1 H, α-CH Ala1), 3.80 (s, 3 H, OCH3), 1.56 (d, J = 6.64 Hz, 3 1.93 (m, 2 H, Nle1 β-CH2), 1.90 (m, 2 H, Nle2 γ-CH2), 1.91 (m, 2
H, CH3 Ala2), 1.52 (d, J = 7.12 Hz, 3 H, CH3 Ala3), 1.47 (d, J = H, Nle3 γ-CH2), 1.83 (m, 2 H, Nle4 γ-CH2), 1.71 (m, 2 H, Nle1 γ-
6.96 Hz, 3 H, CH3 Ala1), 1.44 (s, 9 H, 3ϫCH3Boc) ppm. 13C NMR
CH2), 1.44 (s, 9 H, 3ϫCH3Boc), 1.31 (m, 2 H, Nle3 δ-CH2), 1.30
(400 MHz, CDCl3): δ = 203.65 (CS Ala1), 202.65 (CS Ala2), 172.21 (m, 4 H, Nle1 Nle4 γ-CH2), 1.28 (m, 2 H, Nle2 δ-CH2), 0.89–0.88
(COOMe), 155.12 (CO Boc), 80.53 (C4 Boc),59.19 (α-CH), 57.20 (m, 12 H, 4ϫCH3 Nle) ppm. 13C NMR (400 MHz, CDCl3): δ =
(α-CH), 53.41 (α-CH), 52.83 (OCH3), 28.32 (CH3 Boc), 21.61
203.86 (CS), 201.81 (CS), 201.25 (CS), 171.59 (COOMe), 155.57
(COBoc), 80.33 (C4Boc), 64.18 (α-CH Nle), 63.94 (α-CH Nle),
61.59 (α-CH Nle), 57.60 (α-CH Nle), 52.62 (OCH3), 35.07 (Nle β-
CH2), 34.41 (2ϫNle β-CH2), 30.87 (Nle β-CH2), 28.44
(3ϫCH3Boc), 27.83 (Nle γ-CH2), 27.40 (Nle γ-CH2), 27.29 (Nle γ-
CH2), 27.20 (Nle γ-CH2), 22.35 (4ϫNle δ-CH2), 13.81 (4ϫCH3
(CH Ala), 20.76 (CH Ala), 19.95 (CH Ala) ppm. IR (KBr): ν =
˜
3
3
3
3262, 1726, 1691, 1595 cm–1. MS (ESI+): m/z = 378.15 [M + H]+.
Boc-Ala-ψ[CSNH]-Ala-ψ[CSNH]-Ala-ψ[CSNH]-Ala-OMe: Flash
chromatography eluent: CHCl3/MeOH (97:3), yield 49%, m.p. 150–
1
151 °C (from EtOAc/PE). Rf1 = 0.90; Rf2 = 0.95; Rf3 = 0.80. H
Nle) ppm. IR (KBr): ν = 3286, 1729, 1691 cm–1. MS (ESI+): m/z =
˜
NMR (400 MHz, CDCl3): δ = 8.91 (br., 1 H, CSNH Ala2), 8.70
(br. d, J = 5.72 Hz, 1 H, CSNH Ala3), 8.15 (br. d, J = 5.92 Hz, 1
633.33 [M + H]+.
H, CSNH Ala4), 5.26 (m, 2 H, α-CH Ala2 α-CH Ala3), 5.18 (br., 1H NMR Spectroscopy: All NMR experiments were performed at
1 H, NH Ala1), 5.07 (m, 1 H, α-CH Ala4), 4.48 (m, 1 H, α-CH 298 K with a Bruker AVANCE DRX-400 instrument operating at
Ala1), 3.80 (s, 3 H, OCH3), 1.58 (2ϫd, J = 6.04 Hz, 6 H, CH3 Ala2
CH3 Ala3), 1.54 (d, J = 7.16 Hz, 3 H, CH3 Ala4), 1.48 (d, J =
the frequency of 400 MHz for H, with use of the TOPSPIN soft-
ware package. The homonuclear 2D-spectra were recorded in
1
6.92 Hz, 3 H, CH3 Ala1), 1.44 (s, 9 H, 3ϫCH3 Boc) ppm. 13C CDCl3 as solvent. The DQF-COSY[84] spectra were acquired in the
NMR (400 MHz, CDCl3): δ = 203.81 (CS Ala1), 202.22 (CS Ala3), magnitude mode, and CLEAN-TOCSY[85,86] (spin lock period 45,
201.26 (CS Ala2), 172.21 (COOMe), 155.47 (COBoc), 80.75 (C4
64, 70, or 140 ms) and NOESY (mixing time 400 ms) spectra were
Boc), 59.19 (2α-CH), 57.44 (α-CH), 53.53 (α-CH), 52.84 (OCH3), recorded in the phase-sensitive mode. All homonuclear 2D-spectra
28.32 (CH3 Boc), 21.52 (CH3 Ala), 20.78 (2ϫCH3Ala), 16.97 (CH3 were acquired by collecting 400 experiments of 32 scans each, with
Ala) ppm. IR (KBr): ν = 3259, 1730, 1691, 1595 cm–1. MS (ESI+): a relaxation delay of 1–1.2 s and 2000 data points. The spectral
˜
m/z = 465.16 [M + H]+.
width was 11 ppm in each dimension. The assignments of the
chemical shifts of each residue were obtained by means of 2D
1H,13C correlation spectra. HMBC[87] experiments with selective
excitation in the CO region were performed with use of a long-
range coupling constant of 7.5 Hz and a spectral width in F1 of
60 ppm centered at δ = 190 ppm.
Boc-Nle-ψ(CSNH)-Nle-OMe: Flash chromatography eluent:
EtOAc/PE (1:9), yield 27%, oil. Rf1 = 0.95; Rf2 = 0.95; Rf3 = 0.85.
1H NMR (200 MHz, CDCl3): δ = 8.24 (br. d, J = 7.26 Hz, 1 H,
CSNH Nle2), 5.14 (m, 1 H, α-CH),5.12 (br., 1 H, NH Nle1), 4.29
(m, 1 H, α-CH), 3.77 (s, 3 H, OCH3), 1.44 (s, 9 H, 3ϫCH3 Boc),
2.04 (m, 4 H, 2ϫNle β-CH2), 1.35–1.26 (m, 8 H, 2ϫNle γ-CH2,
X-Ray Diffraction: Single crystals of Z-Gly-ψ[CS-NH]-Gly-ψ[CS-
δ-CH2), 0.89 (m, 9 H, 2ϫCH3 Nle) ppm. 13C NMR (200 MHz, NH]-Gly-OMe were grown by slow evaporation of a methanol
CDCl3): δ = 205.13 (CS), 172.60 (COOMe), 155.90 (COBoc), 80.21
(C4Boc), 61.48 (α-CH Nle), 57.62 (α-CH Nle), 52.62 (OCH3), 35.13
(2ϫNle β-CH2), 28.44 (3ϫCH3 Boc), 28.01 (Nle γ-CH2), 27.23
solution. X-ray diffraction data were collected with an Oxford Dif-
fraction Gemini E four-circle kappa diffractometer fitted with a
92 mm EOS CCD detector, with use of graphite-monochromated
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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/KAP1
Date: 26-03-13 17:50:56
Pages: 10
F. Formaggio, M. Crisma, C. Toniolo, C. Peggion
FULL PAPER
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Received: January 11, 2013
Published Online: ᭿
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Job/Unit: O30050
/KAP1
Date: 26-03-13 17:50:56
Pages: 10
F. Formaggio, M. Crisma, C. Toniolo, C. Peggion
FULL PAPER
Peptidomimetics
F. Formaggio,* M. Crisma, C. Toniolo,
C. Peggion* ..................................... 1–10
All-Thioamidated Homo-α-Peptides: Syn-
thesis and Conformation
Short series of terminally protected, fully
thioamidated, homo-α-peptides were syn-
thesized. For the first time, their prepara-
tion was achieved in single-step fashion
through direct thionation of their oxygen-
ated precursors. Conformation analysis
confirms that the thioamidated α-amino
acid residues can easily adopt either folded
or fully extended conformations.
Keywords: Conformation analysis / Syn-
thetic methods / Peptides / Peptidomimetics
10
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