Extended diethylglycine homopeptides formed by desulfurization of their tetrahydrothiopyran analogues

Article abstract of DOI:10.1021/acs.orglett.9b00501

Diethylglycine (Deg) homopeptides adopt the rare 2.0₅-helical conformation, the longest three-dimensional structure that a peptide of a given sequence can adopt. Despite this unique conformational feature, Deg is rarely used in peptide design b

Full text of DOI:10.1021/acs.orglett.9b00501

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Extended Diethylglycine Homopeptides Formed by Desulfurization
of Their Tetrahydrothiopyran Analogues
Marta De Zotti*,† and Jonathan Clayden*,‡
†Department of Chemistry, University of Padova, Via Marzolo 1, 35131 Padova, Italy
‡School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, U.K.
S
* Supporting Information
ABSTRACT: Diethylglycine (Deg) homopeptides adopt the rare 2.05-helical conformation, the longest three-dimensional
structure that a peptide of a given sequence can adopt. Despite this unique conformational feature, Deg is rarely used in peptide
design because of its poor reactivity. In this paper, we show that reductive desulfurization of oligomers formed from more
reactive tetrahydrothiopyran-containing precursors provides a practical way to build the longest Deg homopeptides so far made,
and we detail some conformational studies of the Deg oligomers and their heterocyclic precursors.
omopeptides 2 of diethylglycine (Deg) 1 (Figure 1a) have
peculiar structural features, as they are able to adopt the
which hampers its coupling reactions. The longest Deg
homopeptide synthesized so far is the hexapeptide Tfa-
(Deg)6-OEt.5
H
In this paper, we describe a new, generally applicable synthetic
approach to Deg homopeptides that exploits the more readily
coupled tetrahydrothiopyran-derived Cα-tetrasubstituted resi-
due Thp (4-aminotetrahydrothiopyran-4-carboxylic acid, 3) as a
precursor to Deg, which may be revealed through desulfuriza-
tion of 3. Thp is a mimic of Met and has been employed in the
design of Met-containing peptide analogues with improved
biological activity/enzymatic stability.6 Cyclic quaternary amino
acids tend to be significantly more reactive than their acyclic
homologues,7 and the strategy of masking alkyl groups as rings
by linking them through a sulfur atom is one that proved
successful in classical diastereoselective aldol reactions. A
temporary sulfur-containing ring was, for example, crucial to
Woodward’s seminal synthesis of erythromycin.8
A supply of the α-amino acid Thp 3 was prepared using a
modified Strecker reaction9 (for details, see the Supporting
Information). Thp 3 was protected as its Fmoc derivative and
activated toward coupling by conversion to its acid fluoride
derivative Fmoc-Thp-F (Figure 2).10
The methyl ester of 3 was successively coupled to Fmoc-Thp-
F in solution, allowing the synthesis of Thp homopeptides 4 with
high yields (76−89%) for each coupling step (Figure 2). By fine
tuning the excess of the acylating agent and reaction times, we
could considerably improve both yield and purity even for the
longer peptide sequences (yield >80% even for Fmoc-(Thp)n-
OMe, n = 7,8). Each coupling between Fmoc-Thp-F and H-
(Thp)n-OMe was achieved by stirring for 12 h in anhydrous
Figure 1. Diethylglycine (Deg) and its oligomers.
rare, fully extended conformation or 2.05-helix (Figure 1b). This
motif is the longest 3D structure that a peptide of a given
sequence can adopt, with torsion angles φ = ψ = ω = 180°,1 and
is extremely rare in natural peptides. One of the few examples
known is the (Gly)4 sequence of the enzyme His-tRNA-
synthetase.2 The 2.05-helix is sensitive to external conditions and
reversibly interconverts with the much shorter 310-helix on
changing solvent polarity.3 Deg peptides, being able to undergo
such a conformational switch, may find application as molecular
springs in peptide-based devices.4
Despite these unique features, Deg peptides are not widely
exploited in peptide conformational design, mainly because of
the low reactivity of even the most activated Deg derivatives,
Received: February 7, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b00501
Org. Lett. XXXX, XXX, XXX−XXX

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sequences (n < 4). The onset of a 310-helix for Fmoc-(Thp)n-
OMe (n = 4−8) peptides is confirmed by the negligible dilution
effect (Figure S2, Supporting Information), the strong, red-
shifted amide I band centered at about 1666 cm−1, and the
occurrence of the amide II band at around 1520 cm−1 (Figure
S3, Supporting Information). More detailed information on the
secondary structure of the Thp homooligopepeptides was
provided by both FTIR absorption analysis and NMR studies of
Ac-(Thp)n-OMe 5, obtained by deprotection and acetylation of
4 (Figure 2). The results pointed to a well developed 310-helical
conformation13 for all Ac-(Thp)n-OMe (see the Supporting
Information).
Desulfurization of the tetrahydrothiopyran ring of Thp reveals
two ethyl groups, allowing the synthesis of peptides containing
the otherwise synthetically challenging diethylglycine residue.
Treatment of Ac-(Thp)n-OMe peptides (n = 6−8) with Raney
Ni14 converted the Thp-homooligopeptides in a straightforward
manner to the corresponding Deg-peptides Ac-(Deg)n-OMe (n
= 6−8) in good yield and purity (Figure 4).
Figure 2. Synthesis of oligomers of Thp. Conditions: (a) 20% v/v
Et2NH in anhyd CH2Cl2, rt, 1 h; (b) Fmoc-Thp-F (1.1 equiv) in anhyd
CH2Cl2, rt overnight; (c) 30% Ac2O in anhyd CH2Cl2, rt, 1 h.
CH2Cl2. Simple purification by flash chromatography returned
pure Thp-homopeptides 4.
Thp homopeptides have never been synthesized before, so we
carried out a detailed study of their conformational character-
istics. It has been proposed6 that Thp may induce the elusive γ-
turn.11 FTIR analysis in deuterochloroform (Figure 3) provided
Figure 4. Synthesis of Ac-(Deg)n-OMe by Raney Ni desulfurization of
Ac-(Thp)n-OMe.
As a result of the differences in their molecular dipole
moments, Deg homopeptides undergo reversible conforma-
tional transitions between the 2.05- and 310-helix in response to a
change in solvent polarity (Figure 1b,c). FTIR clearly
distinguishes between a 2.05- and a 310-helix,3 so in order to
investigate this feature for these unprecedentedly long
homopeptides, we acquired IR spectra in three different solvents
(CDCl3, cyclohexane-d12, and CD3CN).
FTIR analysis on all peptides at both 0.1 and 1 mM
concentrations revealed negligible spectral differences and thus
ruled out peptide aggregation. Ac-(Deg)6-OMe shows the
typical IR absorption features of the fully extended conforma-
tion, namely a split amide I band (Δν = 20 cm−1). The amide II
shifts to a frequency lower than 1500 cm−1 (about 1490 cm−1)
and is more intense than the amide I band (Figure 5).15
Additionally, a less intense band at 1525 cm−1 may be tentatively
ascribed to a small contribution from the 310-helical con-
formation arising from the relative destabilization of the 2.05-
helix in longer oligomers of Ac-(Deg)n-OMe.
The amide I and II bands of Ac-(Deg)7-OMe in CDCl3
solution (Figure 5) reveal the population of both 310- and 2.05-
helical structures, with a broad amide I band and two equally
intense amide II bands at the position typical of 310- (1525
cm−1) and 2.05-helices (1490 cm−1). This observation is further
supported by the position and relative intensities of the bands in
the amide A region, (Figure S6, Supporting Information). In a
continuation of this trend, the IR absorption spectrum of Ac-
(Deg)8-OMe (Figure 5a) shows amide I and amide II bands
characteristic only of a 310-helical structure. In particular, the
Figure 3. (a) Portion of the FTIR absorption spectra (NH stretching
bands) of Fmoc-(Thp)n-OMe (n = 2−4) in CDCl3 (1 mM). (b) Amide
A region of the FTIR absorption spectra of the Fmoc-(Thp)n-OMe
series (n = 4−8) in CDCl3 (1 mM).
evidence that a γ-turn is indeed present in the shorter Thp
homologues. Figure 3a shows a band centered at about 3290
cm−1 for Fmoc-(Thp)3-OMe and 3315 cm−1 for Fmoc-(Thp)2-
OMe. A negligible dilution effect (see SI) shows that this band is
due to an intramolecularly H-bonded NH, arising from a γ-turn
C7 pseudocycle between the NH of the second Thp residue and
the Fmoc CO. At the same time, the third Thp residue in
Fmoc-(Thp)3-OMe makes possible the formation of a β-turn (a
sequence of which produces a 310 helix). An i ← i + 3 H-bond
between the NH of Thp3 and the urethane CO of the Fmoc
protecting group is confirmed by a band centered at 3370 cm−1.
This rare γ-turn could not be detected for longer
homopeptides, for which a 310-helical structure is preferably
adopted (Figure 3b), indicating that the γ-turn (a sequence of
which generates the 2.27-helix12) is stable only in short peptide
B
DOI: 10.1021/acs.orglett.9b00501
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Figure 5. IR absorption spectra (amide I and II region) of (a) Ac-
(Deg)n-OMe (n = 6−8) in CDCl3 (peptide concentration: 1 mM) and
(b) Ac-(Deg)7-OMe in cyclohexane-d6 (peptide concentration: 0.5
mM).
Figure 6. (a) Insensitivity of NH proton chemical shifts in the 1H NMR
spectra Ac-(Deg)6-OMe as a function of the amount of DMSO-d6
added to the CDCl3 solution (v/v) (peptide concentration: 1 mM). (b)
Amide region of the NOESY spectrum of Ac-(Deg)7-OMe in CD3OH
solution (peptide concentration: 1 mM) showing NH(i) → NH(i + 1)
connectivities.
(2.05-helix) amide II band at a frequency lower than 1500 cm−1
is completely absent.
This result suggests that the Deg7 heptapeptide is already too
long to adopt a fully extended 2.05 conformation, which
apparently does not gain stability by increasing cooperativity.
The same holds true for the 310-helix, which often disappears
with peptide elongation in favor of the onset of an α-helix.16 This
is the first evidence of a length-dependent change in the
conformational preferences of Deg homopeptides, with the 310-
helix preferred over the fully extended conformation for longer
7- and 8-residue homologues.
Even in nonpolar cyclohexane, which is expected to favor the
greater number of intramolecular hydrogen bonds supported by
a 2.05-helix, the amide I band of Ac-(Deg)7-OMe revealed a
mixture of 2.05- and 310-helix (Figure 5b), though with a greater
contribution from the “split” signal characteristic of the 2.05-
helix. In the polar solvent CD3CN (known to induce 310-helical
conformation in Deg-peptides), the amide II band of all Ac-
(Deg)n-OMe peptides studied (n = 6−8) falls at about 1530
cm−1, characteristic of helical structures (see Figure S7,
Supporting Information). Ac-(Deg)6-OMe thus reveals a
conformational switch between a 2.05 helix in CDCl3 and a 310
helix in CD3CN solutions.17
Further information on the secondary structure of Deg
homopeptides was gained from 1D and 2D 1H NMR
spectroscopy in CDCl3, DMSO-d6, and CD3OH. NMR analysis
on Ac-(Deg)6-OMe in CDCl3 solution in the presence of an
increasing percentage of DMSO-d618 caused no change in any of
the NH proton chemical shifts, confirming the adoption of a
fully extended conformation in CDCl3 solution for Ac-(Deg)6-
OMe (Figure 6a). The same analysis carried out on Ac-(Deg)8-
OMe showed that the two NHs are sensitive to the presence of
the H-bond donor, as expected for a 310-helical conformation
(Figure S8, Supporting Information). The 1D 1H NMR
spectrum of Ac-(Deg)7-OMe in CDCl3 solution at 23 °C was
characterized by very broad signals which sharpened when
acquired at 53 °C, presumably as a result of the exchange
between the 310- and 2.05-helical structures revealed by FTIR.
NOESY experiments in a more polar solvent CD3OH (Figure
6b) revealed all sequential NH−NH cross peaks (apart from the
one between NH6 and NH7 falling into the diagonal). Such
connectivity is possible only for 310- or α-helical peptides and is
inconsistent with a 2.05-helix.19
In summary, we report the synthesis of the homopeptide
series Fmoc-(Thp)n-OMe (n = 2−8) and Ac-(Thp)n-OMe (n =
6−8). A conformational analysis by means of FTIR absorption
spectroscopy highlighted the presence of the elusive γ-turn
conformation for short Thp homopeptides. Raney Ni reductive
desulfurization of these Thp oligomers converted them into the
corresponding Deg homopeptides Ac-(Deg)n-OMe (n = 6−
8)the longest Deg-homopeptides ever synthesizedwith
good yield and purity. This approach overcomes the usual
challenge of coupling the highly unreactive Deg oligomers and
paves the way to their synthesis on a solid support.
Ac-(Deg)n-OMe oligomers adopt stable fully extended
conformations only for n < 7 in nonpolar solvents. Such
length-dependent stability may be explained considering that
although for a single Deg residue the minimum energy
conformation corresponds to a 2.05 helix, the 310-helical
structure is less than 2 kcal/mol higher in energy.3 Moreover,
as the number of intramolecular H-bonds increases as a result of
peptide elongation, the stabilization gained by the two
additional intraresidue H-bonds in the 2.05 conformation
becomes less important. We conclude that the longest
homologue of the Ac-(Deg)n-OMe series that can adopt a
stable fully extended 2.05 conformation is the hexapeptide,
which thus represents the current limit for using peptide design
to induce this maximally extended conformation. The fact that
lengthening oligomers of Deg, and presumably also of other
similar dialkylglycines, causes them to revert to a 310 helical
secondary structure has wider implications for the use of these
residues in the design and synthesis of conformationally
controllable and switchable helical foldamer structures,20
particularly with regard to their potential membrane activity.21
The use of Thp as a precursor to Deg has potential wider utility
in peptide synthesis in solution and on solid phase and opens
C
DOI: 10.1021/acs.orglett.9b00501
Org. Lett. XXXX, XXX, XXX−XXX

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[using HOAt/EDC] was also tried for the synthesis of the short
peptides Fmoc-(Thp)n-OMe (n < 5). Fmoc-(Thp)4-OMe was prepared
several times in order to compare these two methods (see SI). Both
yield and purity were better with Fmoc-Thp-F, and the reaction times
were markedly shorter (12 h versus the 3 days needed with the activated
ester), clearly demonstrating the superiority of the amino acid fluoride
method.
(11) (a) Paglialunga Paradisi, M.; Torrini, I.; Pagani Zecchini, G.;
Lucente, G.; Gavuzzo, E.; Mazza, F.; Pochetti, G. Tetrahedron 1995, 51,
2379. (b) Torrini, I.; Pagani Zecchini, G.; Paglialunga Paradisi, M.;
Lucente, G.; Mastropietro, G.; Gavuzzo, E.; Mazza, F.; Pochetti, G.;
Spisani, S.; Traniello, S. Biopolymers 1996, 39, 327.
opportunities for wider use of Deg as a conformational control
element in the design of peptidomimetics.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.9b00501.
Experimental and spectroscopic data for all new
compounds. Further spectroscopic data and conforma-
tional analysis for Thp and Deg peptides (PDF)
(12) Crisma, M.; De Zotti, M.; Moretto, A.; Peggion, C.; Drouillat, B.;
Wright, K.; Couty, F.; Toniolo, C.; Formaggio, F. New J. Chem. 2015,
39, 3208.
(13) Homooligopeptides of cyclic quaternary residues often adopt
310-helical structures. For examples, see: (a) Cho, J.-il; Tanaka, M.;
Sato, S.; Kinbara, K.; Aida, T. J. Am. Chem. Soc. 2010, 132, 13176.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: marta.dezotti@unipd.it.
*E-mail: j.clayden@bristol.ac.uk.
̈
(b) Maity, P.; Konig, B. Biopolymers 2008, 90, 8.
(14) Rentner, J.; Kljajic, M.; Offner, L.; Breinbauer, R. Tetrahedron
2014, 70, 8983.
ORCID
Marta De Zotti: 0000-0002-3302-6499
Jonathan Clayden: 0000-0001-5080-9535
Notes
(15) A fully extended conformation (2.05-helix) exhibits a broad band
at about 3360 cm−1 in the Amide A region because all of its NHs are
intramolecularly (intraresidue) H-bonded. 310-helical peptides exhibit a
strong band at about 1666 cm−1 (amide I): (a) Polese, A.; Formaggio,
F.; Crisma, M.; Valle, G.; Toniolo, C.; Bonora, G. M.; Broxterman, Q.
B.; Kamphuis, J. Chem. - Eur. J. 1996, 2, 1104. The band is followed by a
less intense band at about 1515−1530 cm−1 (amide II). In contrast, in
2.05-helical peptides, the amide I absorption at 1660 cm−1 is split into
two components: (b) Toniolo, C.; Bonora, G. M.; Bavoso, A.;
Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Barone, V.; Lelj, F.;
Leplawy, M. T.; Kaczmarek, K.; Redlinski, A. Biopolymers 1988, 27, 373.
The amide II band, significantly more intense than the amide I, is found
at wavenumbers lower than 1500 cm−1: (c) Formaggio, F.; Crisma, M.;
Ballano, G.; Peggion, C.; Venanzi, M.; Toniolo, C. Org. Biomol. Chem.
2012, 10, 2413. For α-helical structures, the amide I band falls typically
at about 1659 cm−1, while amide II is at about 1548 cm−1: (d) Nevskaya,
N. A.; Chirgadze, Y. N. Biopolymers 1976, 15, 637.
(16) Longo, E.; Moretto, A.; Formaggio, F.; Toniolo, C. Chirality
2011, 23, 756.
(17) Peggion, C.; Crisma, M.; Toniolo, C.; Formaggio, F. Tetrahedron
2012, 68, 4429.
(18) (a) Martin, R.; Hauthal, G. Dimethyl Sulfoxide; Van Nostrand-
Reinhold: Wokingham, U.K., 1975. (b) Pitner, T. P.; Urry, D. W. J. Am.
Chem. Soc. 1972, 94, 1399.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge MIUR (Futuro in Ricerca 2013, Grant No.
RBFR13RQXM) and the ERC (Advanced Grant ROCOCO)
for support.
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D
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