Facile and E-selective intramolecular ring-closing metathesis reactions in 310-helical peptides: A 3D structural study

Article abstract of DOI:10.1021/ja071148m

The ring-closing metathesis reaction can be used to cross-link allylated serine residues situated at the i and i + 3 positions in 3₁₀-helical peptides containing the helicogenic amino acid, ∞-aminoisobutyric acid (Aib). An octapeptide with the sequence Boc-Aib-Aib-Aib-Ser(Al)-Aib-Aib-Ser(Al)-Aib-OMe was found to undergo a facile and >20:1 E-selective ring-closing metathesis (RCM) reaction catalyzed by the Grubbs second-generation catalyst to yield an 18-membered macrocycle. The formation of this cross-link does not significantly disturb the peptide's native 3₁₀-helicity, as judged by an X-ray diffraction study of the acyclic diene, the E-olefin RCM product, and its hydrogenated derivative. A heptapeptide system with the sequence Boc-Val-Ser(Al)-Leu-Aib-Ser(Al)-Val-Leu-OMe also underwent an efficient RCM reaction, albeit with diminished E-selectivity. It is apparent from these studies that a minimal, RCM-derived, macrocyclic constraint can be readily incorporated into 3₁₀-helical peptides. Copyright

Full text of DOI:10.1021/ja071148m

Published on Web 05/11/2007
Facile and E-Selective Intramolecular Ring-Closing Metathesis Reactions in
310-Helical Peptides: A 3D Structural Study
†
‡
‡
‡
†
Amie K. Boal, Ivan Guryanov, Alessandro Moretto, Marco Crisma, Erica L. Lanni,
‡
§
,†
Claudio Toniolo, Robert H. Grubbs, and Daniel J. O’Leary*
Department of Chemistry, Pomona College, Claremont, California 91711, Institute of Biomolecular Chemistry,
PadoVa Unit, CNR, Department of Chemistry, UniVersity of PadoVa, 35131 PadoVa, Italy, and Arnold and Mabel
Beckman Laboratories of Chemical Synthesis, DiVision of Chemistry and Chemical Engineering, California Institute
of Technology, Pasadena, California 91125
Received February 16, 2007; E-mail: doleary@pomona.edu
The intramolecular ring-closing metathesis reaction (RCM) is a
useful method for altering the conformational and metabolic stability
of R-helical peptides.1 Prior RCM investigations have utilized
tethers spanning i and i + 4 or i + 7 amino acid residues, a linkage
that encompasses approximately one or two turns of an R-helical
backbone and places the reactive side chains on the same side of
the helix (Figure 1). This strategy has built upon earlier work with
-8
9
10
R-helices containing tethers employing salt bridges, lactams,
disulfide bridges,11 hydrophobic effects, and metal ligation.
12
13
Herein, we report the development of a minimal RCM constraint
for the 310-helix, which is a relatively common structural motif in
R
proteins and peptides containing C -tetrasubstituted R-amino
Figure 1. Molecular models for (L-Ala)n R- and 310-helices. Intramolecular
hydrogen bonds are indicated with dashed lines.
acids.1
4-16
The stereochemistry of the 310-helix suggests that its
17
regularity can be affected by i, i + 3 cross-links (Figure 1). This
aspect has been investigated for the case of salt18 and lactam19 side-
Scheme 1. Synthesis of RCM Macrocyclized Peptides (E)-2 and 3
chain bridges. A recent theoretical study suggested that a minimal
RCM constraint for a 310-helix would require two five-atom i, i +
3
olefinic side chains, thus producing an 18-atom macrocycle upon
20
ring closure.
To study this proposition in greater detail, an octapeptide with
the sequence Boc-Aib-Aib-Aib-L-Ser(Al)-Aib-Aib-L-Ser(Al)-Aib-
OMe (Boc, tert-butoxy; Aib, R-aminoisobutyric acid; Al, allyl;
OMe, methoxy) (1) was prepared using solution-phase methods.21
We chose this sequence because short oligopeptides containing Aib
residues largely populate 310-helices.14,16,22 When treated with the
second-generation ruthenium catalyst 4 (7 mol % of 4, 5 mM in 1,
between peptides 1 and 2 and 0.624 Å between peptides 1 and 3.
With the exception of the C-terminal residue 8, which is helical in
40 °C, 30 min), diene 1 underwent a rapid and E-selective (>20:
1) ring-closing reaction to yield an 18-membered macrocycle in
93% yield (Scheme 1). This result is interesting because E/Z
1
and 2 while semi-extended in 3, most of the backbone φ,ψ torsion
angle values of corresponding residues in 2 and 3 do not differ by
more than 10° if compared to 1. For 2, the largest φ,ψ deviations
are observed at Ser(4) and Ser(7) [|∆φ|,|∆ψ| ) 22°, 39° and 14°,
mixtures are normally observed in RCM reactions between side
chains in helical peptides.1 The olefin moiety in peptide 2 was
-3
2
reduced (cat. 10% Pd-C, 1 atm H , EtOH, 25 °C, 6 h) to provide
the saturated macrocycle 3 in excellent yield.
An X-ray crystallographic analysis23 (Figure 2) of peptides 1-3
provided a structural comparison at each stage of the modification.
Each of the three peptides adopts a well-developed right-handed
1
ψ
6°, respectively]. For 3, deviations within 10-16° are found for
. As commonly found,1
4,16,22
all internal Aib
2
, ψ
5
, φ
6
, and ψ
6
residues exhibit φ,ψ torsion angles typical of helical residues. In
alkene 2, the 310-helical H-bonding pattern is interrupted by the
lack of the intramolecular H-bond between N6 and O3, as each of
these two atoms is intermolecularly H-bonded to a co-crystallized
solvent molecule. In 3, the N6‚‚‚O3 separation, 3.573(4) Å, is only
slightly above the upper limit for a CdO‚‚‚H-N H-bond. To the
best of our knowledge, this is the first X-ray diffraction 3D structural
comparison of a helical peptide before and after installation of a
side-chain cross-link, RCM-derived or otherwise.
310-helical structure. Peptide 1 is 310-helical for residues 1-6 and
contains a type-I â-turn at the C-terminal residues 6 and 7 (a 310
-
helix consists of repeat type-III â-turns). This C-terminal turn
behavior is also seen in peptides 2 and 3, where the regularity of
the helix is slightly disturbed at residues 4 and 5, with a deviation
greater for alkene 2 than for the saturated macrocycle 3. Despite
these small differences, the structures are quite similar to one
another, with rms deviations for backbone atoms of 0.996 Å
We note that in methanol solution peptides 1-3 exhibited circular
2
4
dichroism (CD) spectra consistent with 310-helical structures
(
Figure 3). This helix is characterized by a strong negative
†
Pomona College.
maximum near 205 nm and a much weaker (60-75% less intense)
negative maximum at 222-232 nm.
‡
University of Padova.
§
California Institute of Technology.
6986
9
J. AM. CHEM. SOC. 2007, 129, 6986-6987
10.1021/ja071148m CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
especially in peptides with high Aib content. In an Aib-rich
octapeptide, this macrocyclization does not significantly disturb 310
-
helicity, as judged by an X-ray diffraction study of acyclic diene
1
, E-olefin RCM product 2, and its hydrogenated derivative 3. While
R
other sequences (also including C -tetrasubstituted R-amino acids
with allyl side chains) and tether lengths remain to be studied, it is
apparent from these studies that a minimal, RCM-derived, macro-
cyclic constraint can be readily incorporated into 310-helical
peptides.
Acknowledgment. D.J.O. thanks the Mellon Foundation and
Susan and David Hirsch for financial support. Work at Caltech was
supported by the National Institutes of Health. We thank Lawrence
M. Henling and Dr. Michael W. Day (Caltech) for providing the
X-ray crystallographic analysis of peptide 2, and Professor Helen
Blackwell (University of Wisconsin) for helpful discussions.
Supporting Information Available: Preparative procedures and
characterization data, including X-ray crystal structure coordinates and
files in CIF format. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Figure 2. X-ray crystal structures of octapeptides 1-3. Hydrogen atoms
have been omitted for clarity. Dashed lines represent intramolecular
N-H‚‚‚OdC hydrogen bonds. In 3, the co-crystallized water molecule (W)
is also shown.
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(
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Figure 3. CD spectra of peptides 1-3 (1 mM in MeOH) at 25 °C.
60, 396 and references cited within.
(17) Relative to the R-helix (φ ) -63°, ψ ) -42°, i, i + 4 hydrogen bonds),
Concerning the highly E-selective RCM reactivity of octapeptide
diene 1, we note that rapid RCM reactions and 12:1 E-selectivity
are observed in a shorter sequence, the hexapeptide Boc-Aib-L-
Ser(Al)-Aib-Aib-L-Ser(Al)-Aib-OMe (5). We have also investigated
the RCM reaction in a heptapeptide with the sequence Boc-Val-
Ser(Al)-Leu-Aib-Ser(Al)-Val-Leu-OMe (6).25 When treated with
the 3 -helix (φ ) -57°, ψ ) -30°, i, i + 3 hydrogen bonds) is more
1
0
tightly wound, contains a different pattern of intramolecular hydrogen
15
bonds, and possesses a triangular shape when viewed down the long axis.
The 3 -helix is characterized by three amino acids per turn and 10 atoms
1
0
in the pseudo-ring formed by an intramolecular i, i + 3 hydrogen bond (a
type-III â-turn).
(
18) Yokum, T. S.; Bursavich, M. G.; Gauthier, T.; Hammer, R. P.; McLaugh-
lin, M. L. Chem. Commun. 1998, 1801.
(
19) Schievano, E.; Pagano, K.; Mammi, S.; Peggion, E. Biopolymers 2005,
the second-generation ruthenium catalyst 4 (10 mol % of 4, 5 mM
in 6, 40 °C, 3 h), diene 6 formed an 18-membered macrocycle in
quantitative yield with 7:1 E/Z-selectivity. The origin of the higher
E-selectivity in the Aib-rich peptides may be due to φ/ψ confor-
80, 294 and references cited within.
(20) Saviano, M.; Benedetti, E.; Vitale, R. M.; Kaptein, B.; Broxterman, Q.
B.; Crisma, M.; Formaggio, F.; Toniolo, C. Macromolecules 2002, 35,
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(21) Full experimental details are provided in the Supporting Information.
(22) Marshall, G. R.; Hodgkin, E. E.; Langs, D. A.; Smith, G. D.; Zabrocki,
J.; Leplawy, M. T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 487.
R
mational restrictions imposed by the C -tetrasubstituted R-amino
residues. CD curves in 2,2,2-trifluoroethanol solution comparable
to those of Figure 3 have been also obtained for the RCM
macrocyclic products derived from both hexapeptide 5 and hep-
tapeptide 6 (spectra not shown).
In conclusion, we have shown that an RCM-derived 18-
membered macrocycle can be used to cross-link the side chains of
i and i + 3 amino acids in short 310-helical peptide sequences. The
intramolecular RCM reactions are efficient and highly E-selective,
(
23) Peptide 1 was crystallized from ethyl acetate, peptide 2 from slow
evaporation of a 2:1 dichloromethane/isopropanol solution, and peptide
3
from moist acetonitrile. The structures were solved by standard methods,
and the atomic coordinates have been deposited with the Cambridge
Crystallographic Data Centre.
(
24) Toniolo, C.; Polese, A.; Formaggio, F.; Crisma, M.; Kamphuis, J. J. Am.
Chem. Soc. 1996, 118, 2744.
(25) The chiral amino acids have the L configuration; this sequence is a
permutation of that used in refs 1 and 2.
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