Synthesis of stapled β3-peptides through ring-closing metathesis

Article abstract of DOI:10.1021/ol901803d

The first synthesis of carbon-stapled β³-peptides is reported. The precursor β³-peptides, with O-allyl β-serines located in an ili+3 relationship, were prepared on solid phase. We show that efficient ring-closing metathesis (RCM) of

Full text of DOI:10.1021/ol901803d

ORGANIC
LETTERS
Synthesis of Stapled ꢀ3-Peptides
through Ring-Closing Metathesis
2009
Vol. 11, No. 19
4438-4440
Ylva E. Bergman,† Mark P. Del Borgo,‡ Romila D. Gopalan,‡ Sania Jalal,† Sharon
E. Unabia,‡ Marisa Ciampini,† Daniel J. Clayton,‡ Jordan M. Fletcher,‡ Roger
J. Mulder,§ Jacqueline A. Wilce,‡ Marie-Isabel Aguilar,‡ and Patrick Perlmutter*,†
School of Chemistry, Monash UniVersity, Clayton 3800 Victoria, Australia, Department
of Biochemistry & Molecular Biology, Monash UniVersity, Clayton 3800 Victoria,
Australia, and CSIRO Molecular and Health Technologies, Bag 10 Clayton South,
Victoria 3169, Australia
patrick.perlmutter@sci.monash.edu.au
Received August 5, 2009
ABSTRACT
The first synthesis of carbon-stapled ꢀ3-peptides is reported. The precursor ꢀ3-peptides, with O-allyl ꢀ-serines located in an i/i+3 relationship,
were prepared on solid phase. We show that efficient ring-closing metathesis (RCM) of these new ꢀ3-peptides proceeds smoothly either in
solution or on an appropriate solid support. All products were generated with high selectivity for the E-isomer.
The design and application of foldamers1 is expanding and
maturing rapidly. In particular, the propensity of ꢀ-peptides
(i.e., peptides consisting entirely of cyclic and/or acyclic
ꢀ-amino acids) to form stable, helical structures2-4 in
solution has led to applications in peptidomimetics3,5-12 as
well as new materials.13,14 The 14-helix4 is particularly
important because of its almost perfect pitch (i.e., almost
exactly three residues per turn).
Strategies adopted for further stabilizing 14-helical ꢀ3-
peptides in solution mostly rely on favorable interactions
Figure 1. Summary of NOE correlations observed for the stapled 2a
peptide consistent with the formation of a 14-helix. Gray dots or
diamonds represent NOEs ambiguously assigned due to spectral
overlap.
† School of Chemistry, Monash University.
‡ Department of Biochemistry & Molecular Biology, Monash University.
§ CSIRO.
(1) Foldamers: Structure, Properties, and Applications; Hecht, S., Huc,
I., Eds.; Wiley-VCH: New York, 2007.
between side chain residues located in an i/i+3 relationship
which are in relatively close proximity to each other (∼4.8
Å).4 These include a disulfide clamp,15 one or more salt
bridges,7,16-18 and a lactam bridge.15,19 Other important
(2) Seebach, D.; Becak, A. K.; Bierbaum, D. J. Chem. BiodiVersity 2004,
1, 1111–1239.
(3) Wu, Y.-D.; Gellman, S. Acc. Chem. Res. 2008, 41, 1231–1232.
(4) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001,
101, 3219–3232.
10.1021/ol901803d CCC: $40.75
Published on Web 08/31/2009
 2009 American Chemical Society

strategies include the control of helix macrodipole orienta-
tion20 and the incorporation of varying numbers of cyclic
ꢀ-amino acids.21,22
were prepared on polyethylene-glycol based NovaPEG Wang
resin with a view to evaluating peptide RCMs on a rigid
solid support. Such resins have been shown to have good
swelling properties in both polar and apolar solvents.27,28
1d was prepared on standard Wang resin, and RCM was
also performed on resin. The product was then N-acetylated
and cleaved from resin.
We describe here a new, ring-closing metathesis (RCM)-
derived staple for ꢀ3-peptide 14-helices. Such staples have
found extensive use in R-peptides since Grubbs reported the
first carbon-stapled R-peptides in 1998.23-25 We were
interested in evaluating RCM-derived staples as a means of
introducing synthetically flexible functionality on or near the
surface of such helices. This should provide the opportunity
to assemble new structures, via functional group manipulation
of, in this case, alkenes, through supramolecular and/or
covalent processes. We chose to study hexa-ꢀ-peptides
1a-1d as our RCM precursor candidates (Scheme 1). In each
case, two ꢀ3-serines bearing an O-allyl side chain26 are
located in an i/(i+3) relationship.
Extensive screening of catalyst/solvent combinations typi-
cally employed in RCM29 yielded little success. However,
it was ultimately determined that a 4:1 mixture of TFE and
CH2Cl2 worked well in combination with Hoveyda-Grubbs
generation II catalyst (Scheme 1)30 both in solution and on
solid support. Conversion in all cases was >90% as evidenced
by LCMS. To obtain such high conversion, it was found
that RCMs on Novapeg resin required only 20 mol %
catalyst, whereas reaction on Wang resin required ap-
proximately twice the amount of catalyst. Lower catalyst
loadings led to incomplete conversion due, possibly, to
catalyst decomposition and/or adsorption onto the resin.
LCMS analysis of crude reaction material indicated minimal
byproduct formation had occurred.31 Optimum yields (typi-
cally 20-30% overall for on-resin peptide synthesis, RCM,
capping and cleavage) were obtained by washing the resin
prior to cleavage with DMSO:DMF (1:1) overnight (to
remove catalyst and catalyst-derived impurities) and by
performing HPLC purification at an elevated temperature of
60 °C. All the peptide RCMs gave the E-alkene product with
high selectivity. This outcome is similar to a recent report
from Grubbs.24
Scheme 1. Sequences for All New Hexa-ꢀ-peptides (2a-d) and
Their Precursors (1a-d) as well as Conditions for Ring Closure
We were also interested to establish whether or not this
chemistry would apply to longer peptides. Thus nona-ꢀ3-
peptide 1e (Scheme 2) was synthesized on Wang resin,
Scheme 2.
Synthesis of the New Stapled Nona-ꢀ3-peptide (2e)
All RCM precursor ꢀ3-peptides were prepared on resin
using standard SPPS methods (Scheme 1). Peptides 1a-c
via Solution Phase RCM of the Nona-ꢀ3-peptide (1e)
(5) Bautista, A. D.; Craig, C. J.; Harker, E. A.; Schepartz, A. Curr. Opin.
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ChemBioChem 2007, 8, 917–926.
N-acetylated, and then cleaved prior to RCM to evaluate the
efficiency of solution phase peptide RCM. Solution phase
RCM under our standard conditions, with no side chain
protection of the three aspartic acid residues, proceeded
smoothly providing stapled 2e.
(12) Murray, J. K.; Gellman, S. H. Biopolymers 2007, 88, 657–686.
(13) Pomerantz, W. C.; Yuwono, V. M.; Pizzey, C. L.; Hartgerink, J. D.;
Abbott, N. L.; Gellman, S. H. Angew. Chem., Int. Ed. 2008, 47, 1241–
1244.
NMR spectroscopy was used to determine the solution
structure of our peptides. The peptide RCM pair 1a and 2a
(14) Pizzey, C. L.; Pomerantz, W. C.; Sung, B.-J.; Yuwono, V. M.;
Gellman, S. H.; Hartgerink, J. D.; Yethiraj, A.; Abbott, N. L J. Chem. Phys.
2008, 129, 095103/1–095103/8.
(15) Rueping, M.; Jaun, B.; Seebach, D. Chem. Commun. 2000, 2267–
2268.
(19) Vaz, E.; Brunsveld, L. Org. Lett. 2006, 8, 4199–4202.
(20) Hart, S. A.; Bahadoor, A. B. F.; Matthews, E. E.; Qiu, X. J.;
Schepartz, A. J. Am. Chem. Soc. 2003, 125, 4022–4023.
(21) Vaz, E.; Pomerantz, W. C.; Geyer, M.; Gellman, S. H.; Brunsveld,
L. ChemBioChem 2008, 9, 2254–2259.
(16) Arvidsson, P. I.; Rueping, M.; Seebach, D. Chem. Commun. 2001,
649–650.
(17) Seebach, D.; Schreiber, J. V.; Arvidsson, P. I.; Frackenpohl, J. HelV.
Chim. Acta 2001, 84, 271–279.
(22) However, multiple salt bridges on a single face of the helix,
correctly oriented with respect to the helix macrodipole, are effective.
See: ref 20.
(18) Cheng, R. P.; DeGrado, W. F. J. Am. Chem. Soc. 2001, 123, 5162–
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Org. Lett., Vol. 11, No. 19, 2009
4439

was chosen because these peptides displayed the best
dispersion in their 1D NMR spectra at 500 MHz in
d3-MeOH. NOESY experiments of 1a and stapled 2a
revealed all the expected21 i/i+2 and i/i+3 connectivities
for a 14-helix (Figure 1).
2(a)). The two water-soluble pairs, 1c/2c and 1e/2e, were
also helical in 5 mM phosphate buffer pH 7 (Figure 2(b)).
Thus, the excellent results for the peptide RCMs described
here are, very likely, due to the relatively close proximity
of the two alkenes as a result of the helical nature of each
RCM precursor in solution. In addition to the NMR evidence
cited above, CD analysis showed that all new ꢀ3-peptides,
closed as well as unclosed, exhibited spectra in TFE at
ambient temperature, consistent with a 14-helix,4 with a
minimum between 211 and 214 nm and a maximum between
195 and 198 nm.32 Indeed, 1a and 2a were consistently
helical in a range of solvents, including TFE, MeOH, and
75% acetonitrile/25% 5 mM phosphate buffer pH 7 (Figure
Figure 2. )
CD spectra of (a) ꢀ3-peptides 1a and 2a (50 µg mL-1
in TFE, MeOH, and 75% acetonitrile/25% 5 mM phosphate buffer
pH 7 and (b) ꢀ3-peptides 1c/2c and 1e/2e in 5 mM phosphate buffer
pH 7.
(23) Blackwell, H. E.; Grubbs, R. H. Angew. Chem., Int. Ed. 1998, 37,
3281–3284.
In summary, we have developed the first synthesis of
RCM-stapled ꢀ3-peptides. We have also demonstrated that
efficient RCM of suitably substituted ꢀ3-peptides is possible
either in solution or on an appropriate solid support. All
RCMs generated the E-isomer with high selectivity. Fur-
thermore we have shown that RCM-stapled ꢀ-peptides form
14-helices in both organic solvents and water. This study
provides new avenues for the design of ꢀ3-peptides where
defined three-dimensional structure is a prerequisite for
further functionalization and assembly.
(24) Boal, A. K.; Guryanov, I.; Moretto, A.; Crisma, M.; Lanni, E. L.;
Toniolo, C.; Grubbs, R. H.; O’Leary, D. J. J. Am. Chem. Soc. 2007, 129,
6986–6987.
(25) For reviews on the use of RCM-derived staples in helical R-peptides,
see: (a) Kim, Y.-W.; Verdine, G. L. Bioorg. Med. Chem. Lett. 2009, 19,
2533–2536. (b) Brik, A. AdV. Synth. Catal. 2008, 1661–1675. (c) Davis,
B. G. Chembiochem 2009, 959–969.
(26) Bergman, Y.; Ciampini, M.; Jalal, S.; Lagiakos, H.; Aguilar, M.-
I.; Perlmutter, P. Tetrahedron: Asymmetry 2008, 19, 2861–2863.
(27) Camperi, S. A.; Marani, M. M.; Iannucci, N. B.; Cote, S.; Albericio,
F.; Cascone, O. Tetrahedron Lett. 2005, 46, 1561–1564.
(28) Garcia-Martin, F.; Quintanar-Audelo, M.; Garcia-Ramos, Y.; Cruz,
L. J.; Gravel, C.; Furic, R.; Cote, S.; Tulla-Puche, J.; Albericio, F. J. Comb.
Chem. 2006, 8, 213–220.
(29) Grubbs, R. H. Handbook of Metathesis; Wiley-VCH: Weinheim,
Germany, 2003.
(30) Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H. J. Am.
Chem. Soc. 2000, 122, 8168–8179.
(31) Byproduct formation can be an issue with Grubbs catalysis, see:
Hong, S. H.; Day, M. W.; Grubbs, R. H J. Am. Chem. Soc. 2004, 126,
7414–7415.
Supporting Information Available: Experimental pro-
cedures, HPLC, MS data, CD, and 1D NMR spectra of all
peptides. TOCSY and NOESY data for 1a and 2a. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(32) See Supporting Information.
OL901803D
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Org. Lett., Vol. 11, No. 19, 2009