Stable right- and left-handed peptide helices containing C α-tetrasubstituted α-amino acids

Article abstract of DOI:10.1021/jo900222g

(Chemical Equation Presented) Short peptidomimetics with stable secondary structures in solution are of interest for applications in chemistry, biology, and medicine. One way to rigidify the backbone of a peptide is the use of cyclic C^α-tetrasu

Full text of DOI:10.1021/jo900222g

Stable Right- and Left-Handed Peptide Helices containing
Cr-Tetrasubstituted r-Amino Acids
Andreas A. Grauer,† Chiara Cabrele,‡ Manfred Zabel,§ and Burkhard Ko¨nig*,†
Institute for Organic Chemistry, UniVersity of Regensburg, D-93040 Regensburg, Germany, X-ray
Crystallography, UniVersity of Regensburg, D-93040 Regensburg, Germany, and Faculty of Chemistry and
Biochemistry, Ruhr-UniVersity Bochum, D-44780 Bochum, Germany
burkhard.koenig@chemie.uni-regensburg.de
ReceiVed February 3, 2009
Short peptidomimetics with stable secondary structures in solution are of interest for applications in
chemistry, biology, and medicine. One way to rigidify the backbone of a peptide is the use of cyclic
CR-tetrasubstituted R-amino acids (TAAs) like compound 14. The structures resulting from the
incorporation of this unnatural amino acid into peptides were investigated. In total, 13 different peptides
with a length of up to eight residues and alternating sequences of TAA 14 and (S)- or (R)-valine were
synthesized. Their structures were characterized by X-ray diffraction analysis and NMR and CD
measurements showing that the all-S-backbone-configured peptides 5 and 6 (SS)2-3 form right-handed
310-helices, while the all-R-configured peptides 11-13 (RR)2-4 form left-handed 310-helices in the solid
state and solution.
Introduction
Such amino acid mimetics attracted much attention in the last
years because of their potential use as peptide models, biological
probes, drug candidates, or catalysts.4 Their often more rigid
structure leads to conformational constraints that might lock the
peptide in its bioactive or catalytically active conformation.5
An additional advantage can be an increase of the peptide’s
metabolic stability after incorporation of amino acid mimetics.6
One very successful way among others7 to increase peptide
backbone rigidity is the use of CR-tetrasubstituted R-amino acids
The de novo design of peptides and peptidomimetics with a
defined and predictable three-dimensional structure1,2 has
received considerable interest from chemists and biologists in
recent years. Short peptide sequences consisting only of natural
R-amino acids typically show flexible structures. In contrast,
stable structures in solution can be accomplished by replacing
natural amino acids by nonproteinogenic analogues which may
induce a defined secondary structure even in short peptides.3
(4) (a) Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C.; Broxterman,
Q.; Kaptein, B. J. Inclusion Phenom. 2005, 51, 121–136. (b) Hruby, V. J.; Agnes,
R. S. Biopolymers 1999, 51, 391–410. (c) Tanaka, M. Chem. Pharm. Bull. 2007,
55, 349–358. (d) Etienne, M. A.; Aucoin, J. P.; Fu, Y.; McCarley, R. L.; Hammer,
R. P. J. Am. Chem. Soc. 2006, 128, 3522–3523. (e) Rossi, P.; Felluga, F.; Tecilla,
P.; Formaggio, F.; Crisma, M.; Toniolo, C.; Scrimin, P. J. Am. Chem. Soc. 1999,
121, 6948–6949. (f) Sissi, C.; Rossi, P.; Felluga, F.; Formaggio, F.; Palumbo,
M.; Tecilla, P.; Toniolo, C.; Scrimin, P. J. Am. Chem. Soc. 2001, 123, 3169–
3170.
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Wickramasinghe, W.; Begun, J.; Martin, J. L.; Fairlie, D. P. J. Am. Chem. Soc.
1996, 118, 3375–3379.
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S. Tetrahedron: Asymmetry 2005, 16, 3785–3794.
† Institute for Organic Chemistry, University of Regensburg.
‡ Faculty of Chemistry and Biochemistry, Ruhr-University Bochum.
§ X-ray Crystallography, University of Regensburg.
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J. Org. Chem. 2003, 68, 6208–6214. (b) Komarov, I. V.; Grigorenko, A. O.;
Turov, A. V.; Khilya, V. P. Russ. Chem. ReV. 2004, 73, 785–810. (c) Vass, E.;
Hollosi, M.; Besson, F.; Buchet, R. Chem. ReV. 2003, 103, 1917–1954.
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1232.
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Tetrahedron: Asymmetry 2006, 17, 3273–3281. (b) Paradisi, M. P.; Torrini, I.;
Zecchini, G. P.; Lucente, G. Tetrahedron 1995, 51, 2379–2386. (c) Jimenez,
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3718 J. Org. Chem. 2009, 74, 3718–3726
10.1021/jo900222g CCC: $40.75 2009 American Chemical Society
Published on Web 04/08/2009

Peptide Helices containing CR-Tetrasubstituted R-Amino Acids
(TAA).8 R-Aminoisobutyric acid (Aib), which also occurs in
natural peptide sequences of peptaibiomics or peptaibols,9 is
the most extensively investigated compound of this kind. The
helical tendencies of Aib containing peptides are well character-
ized, showing that short strands form a stable 310-helix while
longer helices emerge into mixed 310/R-helices or pure R-he-
lices.10 Cycloaliphatic CR-tetrasubstituted R-amino acids Acnc
(n ) 3-9)11 with varying ring sizes are also well studied.12
However, the lack of a CR-stereocenter and of a side-chain
functional group for further derivatization is a drawback of these
compounds. Therefore, the synthesis of cyclic CR-tetrasubstituted
R-amino acids which show at least one stereocenter13 and have
an additional functional group in their side chain was investi-
gated in recent years,14 but only very few examples of successful
incorporation into peptide chains have been reported.15 We have
recently introduced the CR-tetrasubstituted tetrahydrofuran amino
acid rac-14 that has two stereocenters at the quaternary CR-
and at Cꢀ-atom. The brominated arene substituent allows further
functionalization by standard Pd(II) or Cu(I) catalysis.16 When
inserted into small tripeptides, the molecule induces stable
secondary structures in solution by the formation of ꢀ-turns.17
We now extend this concept by incorporating the unnatural
amino acid into longer peptide chains to form stable helices as
structurally predictable and tunable scaffolds for peptide mi-
metics. The high steric hindrance of the CR-tetrasubstituted
FIGURE 1. TAA-containing isomeric di-, tetra-, hexa-, and octapep-
tides prepared and investigated in this study.
R-amino acids hampers the peptide synthesis.18 Therefore,
alternating structures of tetrahydrofuran amino acid rac-14 and
S- or R-valine were envisaged. The use of the two different
valine enantiomers allows the synthesis of helical peptides with
different backbone screw sense. We report here the synthesis
of isomeric di-, tetra-, and hexapeptides and of one octapeptide
shown in Figure 1. Their structures were analyzed in solid state
and in solution.
(7) Hruby, V. J.; Li, G.; Haskell-Luevano, C.; Shenderovich, M. Biopolymers
1997, 43, 219–266.
(8) Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopolymers 2001,
60, 396–419.
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(b) Degenkolb, T.; Kirschbaum, J.; Bru¨ckner, H. Chem. BiodiVersity 2007, 4,
1052–1067. (c) Duclohoer, H. Chem. BiodiVersity 2007, 4, 1023–1026.
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(b) Crisma, M.; Saviano, M.; Moretto, V.; Broxterman, Q. B.; Kaptein, B.;
Toniolo, C. J. Am. Chem. Soc. 2007, 129, 15471–15473. (c) Alema´n, C. J. Phys.
Chem. B 1997, 101, 5046–5050. (d) Di Blasio, B.; Pavone, V.; Saviano, M.;
Lombardi, A.; Nastri, F.; Pedone, C.; Benedetti, E.; Crisma, M.; Anzolin, M.;
Toniolo, C. J. Am. Chem. Soc. 1992, 114, 6273–6278. (e) Smythe, M. L.; Nakaie,
C. R.; Marshall, G. R. J. Am. Chem. Soc. 1995, 117, 10555–10562. (f) Smythe,
M. L.; Huston, S. E.; Marshall, G. R. J. Am. Chem. Soc. 1993, 115, 11594–
11595.
Results and Discussion
Synthesis. The target peptides were prepared by a combina-
tion of protecting and deprotecting steps together with standard
solution-phase peptide coupling chemistry. The synthesis is
shown exemplary for hexapeptide 6 (SS)3 in Scheme 1 and starts
from the N-terminally Boc-protected racemic tetrahydrofuran
amino acid Boc-TAA-OH rac-14.17 The compound was coupled
with either H-S-Val-OMe· HCl 15 or H-R-Val-OMe· HCl 16
using EDC/HOBt to give the dipeptides 1, 4 or 7, 10,
respectively. Two diastereomers emerged from the coupling
reaction, which were separated by column chromatography. The
isolated dipeptides 1, 4, 7, and 10 were then used separately in
the next steps to yield enantiomerically pure tetrapeptides. To
achieve this, two equimolar amounts of compound 4 were
deprotected either C-terminal with 1 M aqueous LiOH to give
the free acid 19 or N-terminal with HCl saturated diethyl ether
cleaving the Boc protecting group. Carboxylic acid 19 and amine
20 were coupled using HATU/HOAt to give tetrapeptide 5 in
56% yield. Tetrapeptides 2, 8, and 11 were obtained analogously
in comparable yields. Compound 5 was Boc deprotected and
subsequently coupled with dipeptide acid 19 to give hexapeptide
6. The same procedure was used to synthesize hexapeptides 3,
9, and 12. Octapeptide 13 was obtained by coupling the Boc-
(11) Ac3c (1-aminocyclopropanecarboxylic acid)-Ac9c (1-aminocyclononan-
ecarboxylic acid).
(12) (a) Willisch, H.; Hiller, W.; Hemmasi, B.; Bayer, E. Tetrahedron 1991,
47, 3047–3958. (b) Toniolo, C. Biopolymers 1989, 28, 247–257. (c) Crisma,
M.; Bonora, G. M.; Toniolo, C.; Barone, V.; Benedetti, E.; Di Blasio, B.; Pavone,
V.; Pedone, C.; Santini, A.; Fraternali, F.; Bavoso, A.; Lelj, F. Int. J. Biol.
Macromol. 1989, 11, 345–352. (d) Perczel, A.; Hollosi, M.; Foxman, B. M.;
Fasman, G. D. J. Am. Chem. Soc. 1991, 113, 9772–9784.
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Moye-Sherman, D.; Jin, S.; Ham, I.; Lim, D.; Scholtz, J. M.; Burgess, K. J. Am.
Chem. Soc. 1998, 120, 9435–9443. (c) Williams, R. M.; Fegley, G. J. J. Org.
Chem. 1993, 58, 6933–6935. (d) Ma, D.; Ding, K.; Tian, H.; Wang, B.; Cheng,
D. Tetrahedron 2002, 13, 961–969.
(14) (a) Maity, P.; Konig, B. Biopolymers 2008, 90, 8–27. (b) Pirrung, M. C.;
Dunlap, S. E.; Trink, S. E. HelV. Chim. Acta 1989, 72, 1301–1310. (c) Burgess,
K.; Ho, K. K. Tetrahedron Lett. 1992, 33, 5677–5680. (d) Curry, K.; Peet, M. J.;
Magnuson, D. S. K.; McLennan, H. J. Med. Chem. 1988, 31, 864–867. (e)
Mancuse, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978, 43, 2480–2482.
(f) Cativiela, C.; D´ıaz-de-Villegas, M. D. Tetrahedron: Asymmetry 1998, 9, 3517–
3599. (g) Cativiela, C.; D´ıaz-de-Villegas, M. D. Tetrahedron: Asymmetry 2000,
11, 645–732.
(15) (a) Yokum, T. S.; Gauthier, T. J.; Hammer, R. P.; McLaughlin, M. L.
J. Am. Chem. Soc. 1997, 119, 1167–1168. (b) Obrecht, D.; Altorfer, M.; Bohdal,
U.; Daly, J.; Huber, W.; Labhardt, A.; Lehmann, C.; Mueller, K.; Ruffieux, R.;
Schoenholzer, P. Biopolymers 1997, 42, 575–626. (c) Gershonov, E.; Granoth,
R.; Tzehoval, E.; Gaoni, Y.; Fridkin, M. J. Med. Chem. 1996, 39, 4833–4843.
(d) Burgess, K.; Ho, K. K. J. Am. Chem. Soc. 1994, 116, 799–800.
(16) (a) Zhang, H.; Cai, Q.; Ma, D. J. Org. Chem. 2005, 70, 5164–5173. (b)
Kuwabe, S. I.; Torraca, K. E.; Buchwald, S. L. J. Am. Chem. Soc. 2001, 123,
12202–12206. (c) Vorogushin, A. V.; Huang, X.; Buchwald, S. L. J. Am. Chem.
Soc. 2005, 127, 8146–8149.
(18) Toniolo, C.; Formaggio, F.; Kaptein, B.; Broxterman, Q. Synlett 2006,
9, 1295–1310.
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J. Org. Chem. Vol. 74, No. 10, 2009 3719

Grauer et al.
SCHEME 1. Synthesis of Hexapeptide 6a
a Key: (a) DIPEA, EDC, HOBt, dry DMF, 24 h, rt; (b) LiOH, water/THF, 24 h, rt; (c) HCl satd diethyl ether, DCM, 24 h, rt; (d) DIPEA, HATU, HOAt,
dry DMF, 24 h, rt.
deprotected hexapeptide 29 with dipeptide acid 25.19 All
synthetic procedures and characterization data are provided in
the Supporting Information.
Structure Analysis. The structure of the peptides was
determined in the solid state by X-ray crystallography and in
solution by 2D-NMR, temperature-dependent 1H NMR, and CD
measurements.
Solid-State Structure Analysis. From all four dipeptides
crystal structures were obtained (Figure 2) showing that the pairs
1, 7 and 4, 10 are enantiomeric in configuration and structure.
The structures indicate that molecules 4 (SS) and 10 (RR) with
either two S-configured amino acids or two R-configured amino
acids in their amide backbone show a turn-like structure,
whereas the other two compounds 1 (RS) and 7 (SR) do not
have this structural feature. This correlation becomes even more
pronounced in the structures of tetrapeptides 5 (SS)2 and 11
(RR)2, which were again determined by X-ray crystal structure
analysis (Figure 3): The homochiral tetrapeptides form a
characteristic 310-helix in the solid state.20 The helix is right-
handed for the all-S-configured peptide 5 and left-handed for
the all-R-configured enantiomer 11.
For both peptides 5 (SS)2 and 11 (RR)2 the average backbone
torsion angles ꢁ and ψ of all four amino acids were determined
(Table 1). The ꢁ-angles are between the values of an ideal 310-
and an R-helix. The ψ-angles are in good agreement with those
of an ideal 310-helix.21 One ψ-angle in compound 5 is slightly
smaller than expected, while another in compound 11 is larger.
This indicates the still high flexibility of the tetrapeptides that
form just one turn of a 310-helix. The average iri+3 CdO· · ·N
intramolecular hydrogen bond angles were found to be 133.9°
(molecule 5) and 131.9° (molecule 11): both values are slightly
higher than those reported for other 310-helices but still
significantly smaller than those of an R-helix. The pitch (axial
translation) for the two peptides being 6.61 Å (5) for the right-
handed and 6.42 Å (11) for the left-handed helix is larger than
expected for a 310-helix. A slightly smaller number of residues
per turn (3.16 and -3.20) leads to a higher axial translation
per residue for the new tetrapeptides than for an ideal 310-helix.
The extension from tetra- to hexa- and octapeptides results in
smaller torsion angles ꢁ and ψ of compounds 6 (SS)3 (-60.2°,
-26.7°) and 13 (RR)4 (59.8°, 26.3°) and less variation in
comparison to their smaller homologues 5 and 11, respectively.
This indicates a more stable structure which can be ascribed to
the increased number of intramolecular hydrogen bonds. The
average iri+3 CdO· · ·N intramolecular hydrogen bond angles,
(19) During the synthesis of compound 13, racemization of the CR-carbon
of the last amino acid (valine) in the sequence occurred. This was observed
when analyzing the crystal structure of 13. However, this has no influence on
the helical structure of the molecule in solid state or in solution. For detailed
information about all reactions performed, see the Supporting Information.
(20) Similar properties have been reported for NR-blocked tripeptides: Toniolo,
C.; Benedetti, E. ISI Atlas Sci. Biochem. 1988, 1, 225–230.
(21) (a) Crisma, M.; Formaggio, F.; Moretto, A.; Toniolo, C. Biopolymers
2006, 84, 3–12. (b) Donohue, J. Proc. Natl. Acad. Sci. U.S.A. 1953, 39, 470–
478.
3720 J. Org. Chem. Vol. 74, No. 10, 2009

Peptide Helices containing CR-Tetrasubstituted R-Amino Acids
FIGURE 2. Crystal structures of dipeptides 1 (RS), 7 (SR), 4 (SS), and 10 (RR).
133.8° for compound 6 and 132.9° for compound 13, are in the
same range as those determined for the tetrapeptides.
All iri+3 CdO· · ·H-N intramolecular H-bonds are of
typical length with N · · ·OdC distances between 2.81 and 3.04
Å. This corresponds well to values of type 2 (amide-amide)
hydrogen bonds, which are of an expected N · · ·OdC distance
between 2.8 and 3.0 Å.22
The determined parameters and their comparison to examples
from the literature assign a 310-helical geometry to all four
crystal structures. However, the higher CdO· · ·N intramolecular
hydrogen bond angles, the reduced number of residues per turn,
and the higher pitch result in helices that are a little more
stretched than the ideal 310-helix.
300-373 K). Tetrapeptide 5 showed a high temperature
dependency for two NH-proton resonances (N1H and N2H;
-5.40 and -5.72 ppb/K, respectively), indicating that these two
protons are not involved in intramolecular hydrogen bonds. In
contrast, the N3H and N4H protons are part of intramolecular
hydrogen bonds, as supported by their much smaller temperature
coefficients (-1.12 ppb/K for N3H and -1.72 ppb/K for N4H).
These results correlate to the X-ray diffraction structure of 5,
in which protons N1H and N2H are not involved in intramo-
lecular hydrogen bonds, while the other two (N3H and N4H)
form iri+3 CdO· · ·H-N intramolecular hydrogen bonds.
Similar results were found for the variable-temperature NMR
of peptide 6, where again two proton resonances for N1H and
N2H were strongly temperature dependent (-4.63 and -8.25
ppb/K), while the other four NH-protons involved in hydrogen
bonds showed only slight resonance shifts (N3H -2.21 ppb/K,
N4H -1.31 ppb/K, N5H -0.97 ppb/K, N6H -1.36 ppb/K).
Also for octapeptide 13, N1H and N2H exhibit large temperature
coefficients (-4.56 and -7.92 ppb/K, respectively), whereas
NMR Measurements. One compound of each length (tetra-,
hexa-, and octamer) was investigated by NMR spectroscopy to
gain information about its structure in solution. With peptides
5 (SS)2, 6 (SS)3, and 13 (RR)4, temperature-dependent 1H NMR
studies were performed in DMSO-d6 solution (temperature range
(22) Go¨rbitz, C. H. Acta Crystallogr. B 1989, 45, 390–395.
J. Org. Chem. Vol. 74, No. 10, 2009 3721

Grauer et al.
FIGURE 3. Crystal structures of the helical peptides 5, 6, 11, and 13. Top: the right-handed and left-handed helices 5 and 11 (view from the side
on the left; view perpendicular to the helical axis on the right). Middle: the structure of the right-handed hexapeptide 6. Bottom: the left-handed
helix forming octapeptide 13. Hydrogen atoms except NH-protons were omitted for clarity.
3722 J. Org. Chem. Vol. 74, No. 10, 2009

Peptide Helices containing CR-Tetrasubstituted R-Amino Acids
TABLE 1. Structural Parameters Derived from the Crystal
Structures of Compounds 5, 6, 11, and 13a
preference for the formation of a 310-helix over an R-helix. For
compound 13, no structural insights could be obtained by its
ROESY spectrum, due to strong overlap of the CR-proton
resonances.24
The NMR measurements strongly indicate that the investi-
gated peptides based on alternating natural and CR-tetrasubsti-
tuted unnatural R-amino acids form stable helices in solution,
as they do in the solid state. The hexamers prefer a 310-helix
structure in deuterated DMSO.
parameter
ꢁ (deg)
ψ (deg)
N · · ·OdC hydrogen
bond angle (deg)
5 (SS)2 11 (RR)2 6 (SS)3 13 (RR)4 310-helix R-helix
-61.6
-28.4
133.9
61.7
30.9
131.9
-60.2
-26.7
133.8
59.8
26.3
132.9
-57
-30
128
-63
-42
156
N · · ·OdC hydrogen
3.00
2.99
111.1
2.99
111.6
3.12
3.04
111.6
3.10
2.81
111
3.24
2.86
99
3.63
bond distance (Å)
rotation (per residue) 111.5
(deg)
residues per turn
axial translation
(per residue) (Å)
3.16 -3.20
2.09
CD Measurements. The conformation of all synthesized
peptides was further investigated in methanol by CD spectroscopy.
Figure 5 shows the effect of the peptide chain length
elongation on the dichroic properties of the oligopeptides.
Peptides 1 (RS), 2 (RS)2, and 3 (RS)3, which have no helix
character based on the NMR investigation, show two opposite
CD bands near 210 nm (positive) and 222 nm (negative), with
a crossover close to 215 nm. The intensity of the CD spectra
decreases by increasing the peptide-chain length. Similarly,
peptides 8 (SR)2 and 9 (SR)3 (no helix character based on NMR)
are characterized by much less intense CD curves than dipeptide
7 (SR): they still show a broad minimum near 210 nm, whereas
the positive shoulder of 7 at 230 nm disappears until it becomes
a weak negative shoulder for hexapeptide 9. Upon elongation
of dipeptide 4 (SS) to tetrapeptide 5 (SS)2 and hexapeptide 6
(SS)3 (right-handed 310-helix in the crystal), the minimum near
205 nm is slightly red-shifted and increases in intensity, whereas
the positive CD contribution above 215 nm is substituted by a
negative maximum near 222 nm, followed by a minimum near
234 nm.
The CD spectra of peptides 11-13 (RR)2-4 (left-handed 310-
helix in the crystal) are reminiscent of the one of dipeptide 10
(RR) but shifted toward positive ellipticity values, which leads
to the appearance of a maximum near 210 nm and a positive
valley near 222 nm. Moreover, a new positive band is detected
near 233 nm, whose intensity increases when going from
tetrapeptide 11 up to octapeptide 13. The corresponding
enantiomers 5 (SS)2 and 6 (SS)3 also show this band, obviously
opposite in sign, whereas the tetra- and hexapeptides from the
(RS) and (SR) series do not (Figure 6): as only the peptides
from the (RR) and (SS) series adopt a 310-helical structure in
the crystal and show NOEs characteristic of helical structures,
this band near 233 nm, together with the increased CD intensity
in the range of 210-230 nm, should reflect the presence of an
ordered structure that becomes better defined in the larger
oligopeptides (CD intensity over 210-230 nm: 6 > 5, and 13
> 12 > 11).
2.01
2.10
2.10
1.94
1.56
axial translation
(per helical turn) (Å)
6.61
6.42
6.55
6.51
6.29
5.67
a The literature data for an ideal right-handed 310- and an R-helix9a,21
are reported for comparison.
FIGURE 4. Temperature-dependent NMR of the octapeptide helix 13
in deuterated DMSO as solvent recorded in a temperature range of
300-373 K.
all other proton resonances have a much smaller temperature
dependency (N3H -1.68 ppb/K, N4H -0.82 ppb/K, N5H -1.00
ppb/K, N6H -1.20 ppb/K, N7H -0.77, N8H -1.99) (Figure
4). These results clearly show that all peptides adopt a stable
C-terminal helical structure in solution.
It is known that short peptides containing CR-tetrasubstituted
R-amino acids can undergo an R/310-helix transition in solu-
tion.23 To determine the predominant helix type in DMSO,
ROESY spectra of 5, 6, and 13 were recorded in DMSO-d6.
For all three peptides, strong sequential dNN NOE cross-peaks
were observed. These cross-peaks evoke from the interaction
of the NH proton of amino acid i with the NH proton of amino
acid i+1 and are characteristic for helical structures in solution
and with that provide another proof for the C-terminal helical
structure adopted by the investigated peptides in solution. To
discriminate between the R- and 310-helix, the ROE spectra were
searched for the dRN (i, i+2) contacts characteristic for 310-
helices and the dRN (i, i+4) contacts normally observed for
R-helices. These cross signals originate from the interaction
between the CR-proton of amino acid i and the NH proton of
amino acid i+2 or i+4. In contrast to peptides consisting only
of CR-tetrasubstituted R-amino acids, in our case it was possible
to observe this kind of interaction due to the presence of valine
in the peptide chain. For the tetrapeptide 5 only the dRN (i, i+2)
(i ) 2) was found, indicating the presence of a C-terminal 310-
helix. For the hexapeptide 6, two dRN (i, i+2) (i ) 2 and 4)
cross-peaks were found, but no dRN (i, i+4) (i ) 2) NOE contact
was detected. Accordingly, these findings clearly indicate a
We assume that the dipeptides contain the highest amount
of disordered and irregular conformations. Therefore, the
increase in the ordered peptide fraction of the larger peptides
can be visualized by subtracting the CD spectrum of the
dipeptides from the CD spectra of the larger peptides. This
eliminates the CD contribution of the disordered fraction and
possible CD contributions from the aromatic group of the TAA.
The difference CD spectra for the four peptide series (SS, RR,
SR, RS) are shown in Figure 7. The difference CD spectra
obtained for the peptides (SS)2-3 and (RR)2-4 that adopt a right-
handed and left-handed 310-helix, respectively, in the crystal
are characterized by a red-shifted helix-like band pattern (panel
(24) (a) Wu¨thrich, K. H. NMR of proteins and nucleic acids; J. Wiley &
Sons: New York, 1986. (b) Clore, G. M.; Gronenborn, A. M. Crit. ReV. Biochem.
Mol. Biol. 1989, 24, 479–564. (c) Clore, G. M.; Gronenborn, A. M. Protein
Eng. 1987, 1, 275–288.
(23) Moretto, A.; Formaggio, F.; Kaptein, B.; Broxterman, Q. B.; Wu, L.;
Keiderling, T. A.; Toniolo, C. Biopolymers 2008, 90, 567–574.
J. Org. Chem. Vol. 74, No. 10, 2009 3723

Grauer et al.
FIGURE 5. CD spectra of compounds 1-3 (RS)1-3 (panel A), 4-6 (SS)1-3 (panel B), 7-9 (SR)1-3 (panel C), and 10-13 (RR)1-4 (panel D),
recorded in methanol at a peptide concentration of 1 mM.
FIGURE 6. Comparison of the CD spectra of the helical peptides 5, 6 (panel A), and 11-13 (panel B) with the nonhelical oligomers 8, 9 and 2,
3, respectively.
A of Figure 7), with two minima for (5-4) and (6-4) or two
maxima for (11-10), (12-10), and (13-10) near 210 and 230
nm.
The remarkable red shift of the longer wavelength band can
be due to the highly hydrophobic character of the peptides.25
The intensity of the spectra increases with the peptide length,
suggesting an increase in ordered structure; moreover, for the
tetrapeptides of both series the ratio between the ellipticity values
at 230 and 210 nm is <1, whereas for the larger peptides this
ratio becomes >1. This suggests that the tetrapeptides are likely
to form both a 310- and an R-helical turn in methanol, whereas
the larger peptides are able to build a stable short R-helix. The
tendency of peptide chains with eight or more amino acids in
length to form an preferred R-helical structure was previously
discribed.9a The difference CD spectra obtained for the peptides
(SR)2-3 and (RS)2-3 are completely different from those ones
3724 J. Org. Chem. Vol. 74, No. 10, 2009

Peptide Helices containing CR-Tetrasubstituted R-Amino Acids
FIGURE 7. (A) Difference CD spectra of oligopeptides (RR)2-4 minus dipeptide (RR) and of oligopeptides (SS)2-3 minus dipeptide (SS). (B)
Difference CD spectra of oligopeptides (RS)2-3 minus dipeptide (RS) and of oligopeptides (RS)2-3 minus dipeptide (RS).
DIPEA (1.99 mL, 11.7 mmol, 3 equiv), HOBt (793 mg, 5.83 mmol,
1.5 equiv), and EDC (1.03 mL, 5.83 mmol, 1.5 equiv) in order.
H-Val-OMe·HCl 15 (911 mg, 5.44 mmol, 1.4 equiv) was slowly
added in portions. The mixture was allowed to warm to room
temperature and stirred for 24 h. The reaction was quenched with
6 mL of water and 4 mL of 1 M aqueous KHSO4 and extracted
with diethyl ether (3 × 10 mL). The combined organic layers were
washed twice with brine. Afterward, the solution was dried over
MgSO4 and concentrated under reduced pressure. The crude product
was then purified by column chromatography on flash silica gel
(PE/diethyl ether 80:20) to give the product as two diasteriomers
as colorless solids with an overall yield of 76% (1.47 g, 2.94 mmol).
of the peptides (SS)2-3 and (RR)2-4, which excludes a helical
conformation.
Conclusion
In summary, we have prepared 13 new peptides based on an
alternated sequence of the S- or R-configured R-amino acid
valine and the unnatural CR-tetrasubstituted tetrahydrofuran
R-amino acid rac-14. Homo- and heterochiral stereoisomers
with up to eight residues in length were systematically synthe-
sized in good yields and high purity by solution-phase chemistry.
X-ray crystallography, NMR, and CD measurements showed
that all homochiral peptides, even the tetrapeptides, form helical
structures in the solid state and in solution. The handedness of
the helix is determined by the use of S-amino acids for right-
handed or R-amino acids for left-handed peptide helices.
The stable and predictable secondary structure of the new
peptides makes them suitable for applications as scaffolds and
peptidomimetics. Additional moieties, e.g., dyes, can be intro-
duced by metal-catalyzed functionalization of the brominated
arene substituent.17 Comparison of the crystallographic data of
octamer 13 (RR)4 with an idealized R-helix geometry reveals
that the residues i, i+3, and i+6 of the octamer correspond to
residues i, i+3, and i+7 of the natural R-helix. These positions
are of importance for numerous protein-protein interactions,
as the side chains of these residues are located on the same
face of an amphipathic R-helical protein or peptide segment.26
1
1. Rf (PE/diethyl ether 70:30) ) 0.24. Mp: 147 °C. H NMR
3
(300 MHz, CDCl3): δ ) 0.48 (d, JH,H ) 6.9, 3 H, 18), 0.61 (d,
3
3JH,H ) 6.9, 3 H, 18), 1.47 (s, 9 H, 1), 1.86 (septet, JH,H ) 4.6,
3JH,H ) 6.8, 1 H, 17), 2.52-2.71 (m, 1 H, 7), 2.73-2.92 (m, 1 H,
3
3
7), 3.70 (s, 3 H, 16), 4.21 (dd, JH,H ) 4.4, JH,H ) 8.5, 1 H, 13),
4.27-4.45 (m, 2 H, 8), 5.42 (bs, 1 H, 10), 6.16 (bs, 1 H, NH),
6.48 (d, 3JH,H ) 8.0, 1 H, NH), 7.24 (d, 3JH,H ) 8.2, 2 H, 20), 7.41
3
(d, JH,H ) 8.5, 2 H, 21). 13C NMR (75 MHz, CDCl3): δ ) 17.3
(+, 1 C, 18), 18.3 (+, 1 C, 18), 28.4 (+, 3 C, 1), 31.0 (+, 1 C,
17), 36.1 (-, 1 C, 7), 52.2 (+, 1 C, 16), 57.3 (+, 1 C, 13), 66.6
(-, 1 C, 8), 67.5 (Cquat, 1 C, 6), 80.2 (Cquat, 1 C, 2), 80.4 (+, 1 C,
10), 121.6 (Cquat, 1 C, 22), 127.0 (+, 2 C, 20), 131.3 (+, 2 C, 21),
136.4 (Cquat, 1 C, 19), 154.2 (Cquat, 1 C, 4), 171.2 (Cquat, 1 C, 11),
172.1 (Cquat, 1 C, 14). IR (neat) (cm-1): ν˜ ) 3384, 3271, 2959,
2518, 2361, 1725, 1673, 1527, 1491, 1437, 1373, 1211, 1147, 1072,
1009, 830, 802. MS (CI, NH3): m/z ) 500.9 (100) [MH+], 518.0
(17) [MNH4+]. Anal. Calcd for C22H31BrN2O6 (499.4): C, 52.95;
H, 6.28; N, 5.59. Found: C, 53.02; H, 6.33; N, 5.50. MF:
C22H31BrN2O6. MW: 499.4.
Experimental Section
4. Rf (PE/diethyl ether ) 70:30) ) 0.20. Mp: 135 °C. 1H NMR
3
1
(300 MHz, benzene-d6): δ ) 0.68 (d, JH,H ) 6.9, 6 H, 18), 1.44
For further details regarding the H and 13C NMR assignments
of compounds 1, 2, 4, 17, and 18, see the Supporting Information.27
(2S)-Methyl 2-(2-(4-Bromophenyl)-3-(tert-butoxycarbonylami-
no)tetrahydrofuran-3-carboxamido)-3-methylbutanoate (1/4). Un-
der an atmosphere of nitrogen, compound rac-14 (1.50 g, 3.88
mmol, 1 equiv) was dissolved in 3.9 mL of DMF (1 mL/mmol)
and cooled to 0 °C in an ice bath. To the solution were added
(s, 9 H, 1), 1.70-1.91 (m, 1 H, 17), 2.22-2.42 (m, 1 H, 7),
2.59-2.81 (m, 1 H, 7), 3.15 (s, 3 H, 16), 3.75-3.94 (m, 1 H, 13),
3.92 (dd, 3JH,H ) 7.1, 3JH,H ) 16.2, 8), 4.27 (dd, 2JH,H ) 4.9, 3JH,H
) 7.9, 1 H, 8), 5.51 (bs, 1 H, 10), 6.40 (s, 1 H, NH), 6.75 (d, 3JH,H
3
3
) 5.8, 1 H, NH), 7.20 (d, JH,H ) 8.5, 2 H, 20), 7.30 (d, JH,H
)
8.2, 2 H, 21). 13C NMR (75 MHz, CDCl3): δ ) 17.9 (+, 1 C, 18),
18.7 (+, 1 C, 18), 28.4 (+, 3 C, 1), 31.8 (+, 1 C, 17), 36.1 (-, 1
C, 7), 52.1 (+, 1 C, 16), 57.2 (+, 1 C, 13), 66.5 (-, 1 C, 8), 67.6
(25) Ohmae, E.; Fukumizu, Y.; Iwakura, M.; Gekko, K. J. Biochem. 2005,
137, 643–652.
(26) Davis, J. M.; Tsou, L. K.; Hamilton, A. D. Chem. Soc. ReV. 2007, 36,
326–334.
(27) CCDC 726669–726676 contain the supplementary crystallographic data
for the compounds 4, 1, 5, 11, 13, 6, 7, and 10 in this order. These data can be
obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
(Cquat, 1 C, 6), 80.2 (Cquat, 1 C, 2), 80.7 (+, 1 C, 10), 121.6 (Cquat
,
1 C, 22), 127.3 (+, 2 C, 20), 131.0 (+, 2 C, 21), 135.5 (Cquat, 1 C,
19), 154.3 (Cquat, 1 C, 4), 171.3 (Cquat, 1 C, 11 + 14). IR (neat)
(cm-1): ν˜ ) 3440, 3271, 2957, 2874, 2357, 1742, 1719, 1652, 1507,
1367, 1244, 1162, 1088, 1014, 987, 798. MS (CI, NH3): m/z )
500.9 (100) [MH+], 518.0 (33) [MNH4+]. Anal. Calcd for
J. Org. Chem. Vol. 74, No. 10, 2009 3725

Grauer et al.
C22H31BrN2O6 (499.4): C, 52.91; H, 6.26; N, 5.61. Found: C, 53.02;
H, 6.33; N, 5.50. MF: C22H31BrN2O6. MW: 499.4.
nitrogen, compound 17 (150 mg, 0.31 mmol, 1 equiv) was dissolved
in 2.5 mL of DMF (8 mL/mmol) and cooled to 0 °C in an ice bath.
To the solution were added DIPEA (159 µL, 0.93 mmol, 3 equiv),
HOAt (64 mg, 0.46 mmol, 1.5 equiv), and HATU (176 mg, 0.46
mmol, 1.5 equiv) in sequence. The amine 18 (148 mg, 0.34 mmol,
1.1 equiv) was then slowly added in portions. The mixture was
allowed to warm to room temperature and stirred for 24 h. The
reaction was quenched with 2 mL of water and 1 mL of 1 M
aqueous KHSO4 and extracted with diethyl ether (3 × 10 mL).
The combined organic layers were washed twice with brine.
Afterward, the solution was dried over MgSO4 and concentrated
under reduced pressure. The crude product was then purified by
column chromatography on flash silica gel (PE/diethyl ether 60:
40, Rf ) 0.14) to give the product as colorless solids with an overall
(S)-2-((2S,3R)-2-(4-Bromophenyl)-3-(tert-butoxycarbonylami-
no)tetrahydrofuran-3-carboxamido)-3-methylbutanoic Acid (17).
Compound 1 (440 mg, 0.88 mmol) was dissolved in 22 mL of a
MeCN/water mixture (4:1, 25 mL/mmol). To the solution was added
1 M aqueous LiOH (970 µL, 0.97 mmol) drop by drop. The mixture
was stirred overnight at room temperature. After acidification with
1 M aqueous KHSO4 solution, the mixture was extracted with DCM
(3 × 10 mL). The combined organic layers were dried over MgSO4
and concentrated under reduced pressure to give the product as
colorless solid 93% yield (400 mg, 0.82 mmol). Mp: 133-135 °C.
3
1H NMR (300 MHz, CDCl3): δ ) 0.44 (d, JH,H ) 5.2, 3 H, 16),
3
0.62 (d, JH,H ) 5.8, 3 H, 16), 1.42 (m, 9 H, 1), 1.78-1.95 (m, 1
1
H, 15), 2.41-2.65 (m, 1 H, 7), 2.67-2.86 (m, 1 H, 7), 4.13-4.39
(m, 3 H, 8 + 13), 5.39 (bs, 1 H, 10), 6.17 (bs, 1 H, NH), 6.47 (d,
3JH,H ) 7.7, 1 H, NH), 7.20 (d, 3JH,H ) 8.2, 2 H, 18), 7.35 (d, 3JH,H
) 8.8, 2 H, 19). 13C NMR (75 MHz, CDCl3): δ ) 17.2 (+, 1 C,
16), 18.4 (+, 1 C, 16), 28.4 (+, 3 C, 1), 30.7 (-, 1 C, 15), 36.0 (-,
1 C, 7), 57.2 (+, 1 C, 13), 66.5 (-, 1 C, 8), 67.5 (Cquat, 1 C, 6), 80.2
(+, 1 C, 10), 80.3 (Cquat, 1 C, 2), 121.6 (Cquat, 1 C, 20), 127.0 (+, 2
C, 18), 131.4 (+, 2 C, 19), 136.4 (Cquat, 1 C, 17), 154.3 (Cquat, 1 C, 4),
171.4 (Cquat, 1 C, 11), 176.3 (Cquat, 1 C, 14). IR (neat) (cm-1): ν˜ )
3397, 3334, 2967, 2891, 1734, 1710, 1667, 1492, 1256, 1164, 1067,
1044, 1010, 827, 793. MS (ES, DCM/MeOH + 10 mM NH4OAc):
m/z ) 485.1 (100) [MH+], 502.2 (57) [MNH4+], 523.1 (26) [MK+],
1007.3 (22) [2 M + K+]; 409.0 (35) [M - tBuOH - H+], 483.1 (100)
[M - H+]. MF: C21H29BrN2O6. MW: 485.37.
yield of 67% (180 mg, 0.21 mmol). Mp: 159-161 °C. H NMR
3
(600 MHz, COSY, CDCl3): δ ) 0.65 (d, JH,H ) 6.8, 3 H, 38),
3
3
0.67 (d, JH,H ) 7.2, 3 H, 28), 0.74 (d, JH,H ) 6.6, 3 H, 38), 0.81
(d, 3JH,H ) 6.8, 3 H, 28), 1.48 (s, 9 H, 1), 1.91-1.99 (m, 1 H, 37),
2.00-2.06 (m, 1 H, 27), 2.46-2.61 (m, 2 H, 19 + 29), 2.82-2.91
(m, 1 H, 29), 3.15 (bs, 1 H, 19), 3.59-70 (m, 4 H, 9 + 18),
3
3
3.98-4.08 (m, 1 H, 30), 4.16 (dd, JH,H ) 5.5, JH,H ) 8.8, 1 H,
15), 4.23 (dd, 3JH,H ) 8.7, 3JH,H ) 15.7, 1 H, 20), 4.33 (dt, 3JH,H
)
3
3
3
2.8, JH,H ) 8.5, 1 H, 30), 4.43 (dt, JH,H ) 5.3, JH,H ) 8.6, 1 H,
20), 5.00 (s, 1 H, 22), 5.21 (s, 1 H, 32), 5.84 (s, 1H 5), 6.32 (s, 1H,
3
3
8), 6.87 (d, JH,H ) 7.7, 1 H,14), 7.14 (d, JH,H ) 8.4, 2 H, 24),
3
3
7.17 (d, JH,H ) 8.6, 2 H, 34), 7.28 (d, JH,H ) 7.9, 2 H, 25), 7.35
(d, JH,H ) 8.4, 2 H, 35), 7.48 (s, 1 H, 11). 13C NMR (150 MHz,
3
HSQC, HMBC, CDCl3): δ ) 17.4 (+, 1 C, 28), 17.7 (+, 1 C, 38),
18.7 (+, 1 C, 38), 19.0 (+, 1 C, 28), 28.4 (+, 3 C, 1), 30.0 (+, 1
C, 27), 31.2 (+, 1 C, 37), 35.5 (-, 1 C, 29), 35.6 (-, 1 C, 19),
51.8 (+, 1 C, 18), 57.5 (+, 1 C, 15), 59.7 (+, 1 C, 9), 67.4 (-, 1
C, 30), 67.6 (-, 1 C, 20), 69.0 (Cquat, 1 C, 6), 69.6 (Cquat, 1 C, 12),
81.7 (Cquat, 1 C, 2), 83.8 (+, 1 C, 32), 84.3 (+, 1 C, 22), 121.7
(Cquat, 1 C, 36), 122.6 (Cquat, 1 C, 26), 127.7 (+, 2 C, 24), 127.9
(S)-Methyl 2-((2S,3R)-3-Amino-2-(4-bromophenyl)tetrahy-
drofuran-3-carboxamido)-3-methylbutanoate (18). Compound 1
(290 mg, 0.58 mmol) was dissolved under ice bath cooling at 0 °C
in diethyl ether. To this solution was added 4.1 mL of ice-cold
HCl saturated ether (7 mL/mmol Boc), and the mixture was stirred
overnight at room temperature. The solvent was removed under
reduced pressure to give the HCl salt of the product as a white
hygroscopic solid in quantitative yield (250 mg, 0.57 mmol). No
(+, 2 C, 34), 131.0 (+, 2 C, 35), 131.4 (+, 2 C, 25), 135.1 (Cquat
1 C, 23), 136.6 (Cquat, 1 C, 33), 155.5 (Cquat, 1 C, 4), 189.7 (Cquat
1 C, 13), 169.9 (Cquat, 1 C, 10), 171.0 (Cquat, 1 C, 7), 171.8 (Cquat
,
,
,
1
further purification was necessary. Mp > 180 °C. H NMR (300
MHz, MeOH-d4): δ ) 0.62 (d, 3JH,H ) 8.2, 3 H, 14), 0.64 (d, 3JH,H
1 C, 16). IR (neat) (cm-1): ν˜ ) 3252, 3312, 3241, 2964, 2941,
2884, 2361, 2341, 1736, 1668, 1595, 1519, 1463, 1259, 1118, 1010,
994, 829. MS (ES, DCM/MeOH + 10 mM NH4OAc): m/z ) 867.3
(100) [MH+], 884.3 (74) [MNH4+]; 865.3 (100) [M - H+], 925.3
(55) [M + OAc-]. HR-MS (PI-LSIMS, MeOH/DCM/NBA): [MH+]
calcd for C38H50Br2N4O9 865.2023, found 865.2022. MF:
C38H50Br2N4O9. MW: 866.63.
) 8.0, 3 H, 14), 1.78 (sextet, 3JH,H ) 6.7, 1 H, 13), 2.36 (ddd, 3JH,H
3
2
3
) 2.5, JH,H ) 7.3, JH,H ) 14.4, 1 H, 3), 3.00 (dt, JH,H ) 14.4,
3JH,H ) 9.3, 1 H, 1 H, 3), 3.67 (s, 3 H, 12), 3.84 (d, 3JH,H ) 6.6, 1
3
3
3
H, 9), 4.18 (dt, JH,H ) 7.2, JH,H ) 9.3, 1 H, 4), 4.54 (dt, JH,H
)
2.5, 3JH,H ) 9.1, 1 H, 4), 4.99 (s, 1 H, 6), 7.32 (d, 3JH,H ) 8.2, 2 H,
16), 7.53 (d, 3JH,H ) 8.8, 2 H, 17). 13C NMR (75 MHz, MeOH-d4):
δ ) 19.1 (+, 1 C, 14), 19.4 (+, 1 C, 14), 31.1 (+, 1 C, 13), 36.0
(-, 1 C, 3), 52.6 (+, 1 C, 12), 60.6 (+, 1 C, 9), 67.8 (-, 1 C, 4),
70.0 (Cquat, 1 C, 2), 86.8 (+, 1 C, 10), 123.8 (Cquat, 1 C, 18), 129.2
Acknowledgment. We thank the Fonds der Chemischen
Industrie, the University of Regensburg, and the DFG graduate
college 760 for support.
(+, 2 C, 16), 132.8 (+, 2 C, 17), 136.2 (Cquat, 1 C, 15), 169.2 (Cquat
,
1 C, 7), 173.3 (Cquat, 1 C, 10). IR (neat) (cm-1): ν˜ ) 3288, 2965,
2874, 2362, 2341, 1749, 1684, 1646, 1519, 1488, 1157, 1074, 1010,
841. MS (ES, DCM/MeOH + 10 mM NH4OAc): m/z ) 399.1 (100)
[MH+]. MF: C17H23BrN2O4 ·HCl. MW: 435.74.
Supporting Information Available: Experimental proce-
dures, characterization data for all new compounds, and X-ray
(CIF), NMR, and CD data. This material is available free of
charge via the Internet at http://pubs.acs.org.
(S)-Methyl 2-((2S,3R)-2-(4-Bromophenyl)-3-((S)-2-((2S,3R)-
2-(4-bromophenyl)-3-(tert-butoxycarbonylamino)tetrahydrofuran-
3-carboxamido)-3-methylbutanamido)tetrahydrofuran-3-car-
boxamido)-3-methylbutanoate (2). Under an atmosphere of
JO900222G
3726 J. Org. Chem. Vol. 74, No. 10, 2009