Quantifying end-to-end conformational communication of chirality through an achiral peptide chain

Article abstract of DOI:10.1002/anie.200901892

Successful communication: Two diastereotopic protons more than 60 bonds from the nearest chiral center appear as an AB system, showing that the intervening structure is a well-ordered helix. Decay of anisochronicity quantifies the linear persistence of a

Full text of DOI:10.1002/anie.200901892

Communications
DOI: 10.1002/anie.200901892
Chirality Transfer
Quantifying End-to-End Conformational Communication of Chirality
through an Achiral Peptide Chain**
Jonathan Clayden,* Alejandro Castellanos, Jordi Solꢀ, and Gareth A. Morris
Helicity is a widespread characteristic of the secondary
structure of peptides: those built of l-amino acids typically
adopt right-handed helical structures, even when most of the
chain is achiral.[1] Helical peptide motifs may be stabilized by
the incorporation of quaternary amino acids such as Aib
(aminoisobutyric acid),[2] and oligomers of Aib adopt racemic
helical conformations.[3,4] Favored adoption of a left- or right-
handed helix may be controlled by judicious distribution of
chiral monomers along any polymer chain.[5] A bias towards
one absolute sense of helicity may also arise from a terminal
chiral residue bound covalently[6,7] or noncovalently[8] to an
otherwise achiral peptide chain. At the limit, absolute helicity
(that is, a left- or right-handed helical preference) may be
induced in an otherwise configurationally achiral oligomer by
a single terminal amino acid. The work of Toniolo et al. (for
short Aibn oligomers)[6] and of Inai et al. (for oligomers of
Aib-DZPhe (DZPhe = (Z)-didehydrophenylalanine)[7,8] has
shown that covalent or noncovalent attachment of a terminal
chiral residue leads to a helicity preference[9] in at least part of
the peptide structure, detectable by circular dichroism.
the fidelity with which each achiral amino acid transmits a
helical preference along the chain.
The method relies on the observation by NMR spectros-
copy of anisochronicity between two 1H nuclei which are
indistinguishable unless they find themselves in a chiral
environment. In a helix built entirely of achiral monomers,
and inverting rapidly on the NMR timescale, the two
“reporter” nuclei are in fast exchange and must be isochro-
nous.[11] If a remote chiral influence succeeds in inducing
preferentially one absolute helicity in the oligomer, the
symmetry of the local environment of the nuclei will be
broken, rendering the diastereotopic nuclei anisochronous.
Provided the chiral controller is located sufficiently far away
to avoid direct interaction with the reporter nuclei, the degree
of anisochronicity reflects a weighted average of two pseu-
doenantiomeric environments and therefore reflects the local
excess of one absolute helicity over the other. Quantifying the
anisochronicity as a chemical-shift difference between these
nuclei, and observing its decay with increasing helix length,
may thus provide an empirical measure of the distance over
which a chiral influence can persist in a peptide.
What we now establish is how far the asymmetric
influence of a terminal chiral residue can persist through a
single isolated helical structure—in other words the fidelity
with which a helical peptide built of achiral monomers can
carry information about a terminal residue over ever increas-
ing distances. Previous studies have used CD to detect helicity
in the oligomer as a whole, but no information on asymmetry
localized at the helix terminus can be gathered by this
method.[10] As a helix of achiral monomers is lengthened,
every achiral residue must carry a finite chance of helix
inversion, leading to erosion of the asymmetric environment
at the terminus of the growing oligomer. Using a simple
spectroscopic technique, we have now evaluated the local
We chose 1-aminoisobutyric acid (Aib (1)) as the achiral
monomeric amino acid from which to build the helix since
oligomers incorporating Aib are well-known to form 310-
helical structures[2–4] which undergo helix inversion rapidly on
the NMR timescale at room temperature.[4] The generalized
structure of our targets is shown in Scheme 1: an N-terminal
1
asymmetry of a pair of geminal “reporter” H nuclei at the
C terminus of a peptide containing a single N-terminal chiral
residue, and hence have quantified both the distance over
which oligomers retain a helical preference in solution and
Scheme 1. General structure of the target molecules studied.
[*] Prof. J. Clayden, Dr. A. Castellanos, Dr. J. Solꢀ, Prof. G. A. Morris
School of Chemistry, University of Manchester
Oxford Road, Manchester M139PL (UK)
Fax: (+44)161-275-4939
chiral amino acid (in most cases, Cbz-l-Phe[12]) provides a
stereochemical influence which we hope will propagate
through a helix of achiral amino acids (Aib or glycine).
Remote from the chiral controller, a pair of diastereotopic
protons provide a spectroscopic probe,[13] reporting on the
local asymmetry at the C terminus.
The achiral Aib oligopeptides were built up from a C-
terminal protected ester 3a or alcohol 3b by the iterative
method[14] illustrated in Scheme 2: acylation with 1-azidoiso-
butyryl chloride (AzibCl (2)) to give 4a or 4b followed by
E-mail: clayden@man.ac.uk
Homepage: http://clor2.ch.man.ac.uk/home.htm
[**] We are grateful to the EPSRC and to the Leverhulme Trust for
financial support of this work, and to the Departament d’Innovaciꢁ
Universitats i Empresa de la Generalitat de Catalunya for a
postdoctoral fellowship to J.S.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200901892.
5962
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2009, 48, 5962 –5965

Angewandte
Chemie
Scheme 2. Oligopeptide synthesis: a) AzibCl (2), Et3N, CH2Cl2; b) H2,
Pd/C, MeOH; c) Cbz-l-Phe, PyBOP, iPr2NEt, CH2Cl2; d) CbzGly, EDC,
HOBt, CH2Cl2; e) HCl, MeOH. PyBOP: benzotriazol-1-yl-oxytripyrrolidi-
nophosphonium hexafluorophosphate; EDC: 3-(3-dimethylamino-
propyl)-1-ethylcarbodiimide; HOBt: 1-hydroxybenzotriazole.
hydrogenation of the azide gave the chain-extended dipep-
tides 5a or 5b. Repetition of the procedure two and three
times gave achiral tetrapeptides 6a or 6b and pentapeptide 7,
respectively. These oligopeptides were capped at the N ter-
minus with Cbz-l-Phe to yield 8 and (after desilylation) 11, or
with CbzGly to yield 9 and 10.
From these pentapeptide building blocks we constructed
polypeptides of the general structure shown at the top of
Figure 1. Direct coupling between Aib oligomers was difficult
to achieve using standard coupling methods,[14] but the Gly
linkers allowed us to build decapeptide 12 (by deprotection
and coupling of pentapeptides 8 and 10), pentadadecapeptide
13 (similarly from 8 + 9 + 10), and icosapeptide 14 (similarly
from 8 + 9 + 9 + 10) as shown in Figure 1. 1H NMR spectra of
these four peptides Cbz-l-PheAib4[GlyAib4]nAibCH2OH
(n = 0–3) were acquired in deuterated methanol, and the
form of the CH2OH AB signal for each is shown in Figure 1.
Each of the peptides 11–14 contains a single chiral residue,
the terminal l-Phe, with a stereogenic center located respec-
tively 16, 31, 46, and 61 bonds from the C-terminal reporter
nuclei. Nonetheless, all displayed 1H NMR spectra which
indicated that the diastereotopic reporter group finds itself in
a locally chiral environment which is independent of concen-
tration.[15] Lengthening the peptide by five residues decreased
the anisochronicity Dd of the signals by approximately a
factor of two, but the AB system persisted even with nineteen
achiral amino acids—61 bonds—intervening between the
single stereogenic center and the C-terminal diastereotopic
pair. Anisochronicity was also evident in the methylene
groups of the glycine linkers of 12, 13, and 14.
Figure 1. Construction of peptides Cbz-l-PheAib4[GlyAib4]nAibCH2OH
(n=0–3; 11–14) and controls 15–17 (syntheses involve sequential
deprotections and couplings: see the Supporting Information), and
form of their CH2OH signals and the chemical-shift separation Dd of
the AB system (1H NMR in CD3OD, 238C).
experiments were carried out to confirm that the anisochro-
nicity arises from induction of chirality through the helix
under thermodynamic control rather than by any form of
direct inter- or intramolecular interaction between the
terminal chiral residue and the reporter group: a) The achiral
Gly-terminated decapeptide 15 displayed no anisochronicity
in its terminal reporter group (nor its glycine methylene
groups) at temperatures between À20 and + 208C, confirming
that helix inversion in peptides constructed of Aib oligomers
is fast on the NMR timescale,[16] and that racemic helicity
alone cannot give rise to anisochronicity in these systems;
b) decapeptide 16, in which the two Aib4 segments are
insulated from one another[17] by a conformationally flexible
We interpret the remote effect of the N-terminal Phe
residue as being the result of perturbation of the equilibrium
between left- and right-handed helices. The observed local
asymmetry at the C terminus is the consequence of this
thermodynamically controlled imbalance. Several further
Angew. Chem. Int. Ed. 2009, 48, 5962 –5965
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5963

Communications
5-aminopentanoyl residue, displayed almost (though interest-
at a Gly residue must consequently be of the order of 17.5%
(Figure 2, green line).
ingly not quite) zero anisochronicity (Dd < 5 ppb) in its
CH2OH group; c) the NMR spectrum of (Æ )-12 is identical
in all respects—including the anisochronicity of the CH2OH
group—with that of (À)-12; d) an equimolar mixture of 12
and 15 shows a superimposed singlet and AB system, each
identical with that of the pure components. These last three
results, along with the concentration independence of Dd in
CD3OD,[15] indicate that anisochronicity is the result of
intramolecular communication only, and not intermolecular
interactions.
The observed decay of anisochronicity with increasing
chain length presumably results from equalization of the
populations of local M and P helicity at the C terminus as
random helix “errors” accumulate in longer chains. The decay
is in fact consistent with a simple model in which there is a
constant probability that a helix fault (inversion) will occur at
any given residue, leading to an exponential erosion of helix
preference with chain length. On this model (see the
Supporting Information), the average fault rate in the
(GlyAib4)n sections of 11–14 (Figure 2, blue squares) is of
the order of 6% per residue (blue line). However, given the
Peptides rich in Aib typically form 310 helices, and NMR
experiments (see the Supporting Information) allowed us to
ascertain that only two of the NH groups of 12 were not
involved in intramolecular hydrogen bonds in CDCl3, a result
consistent with the formation of a 310 helix (rather than an
a helix, in which three NH groups are excluded from intra-
molecular hydrogen-bonding). CD experiments[18] (see the
Supporting Information) indicated a right-handed (P) helix.
The adoption of a P-310-helical conformation with a helix rise
per residue of 1.94 ꢀ[19] by icosapeptide 14 generates a
structure of 3.9 nm length, and future work will seek to exploit
the nanoscale organizational properties of these, and related,
synthetic peptidic structures. Biological systems make exten-
sive use of conformational changes to mediate signal trans-
duction, and we propose to employ helical peptides related to
14 (which is longer than the typical thickness of a cell
membrane) to mediate communication on a similar scale in
artificial systems.[20]
Received: April 8, 2009
Published online: June 30, 2009
Keywords: anisochronicity · conformation analysis · helicity ·
.
NMR spectroscopy · peptides
[1] a) L. Pauling, R. B. Corey, H. R. Branson, Proc. Natl. Acad. Sci
USA 1951, 37, 205; b) S. Aravinda, N. Shamala, P. Balaram,
Chem. Biodiversity 2008, 5, 1238.
[2] a) V. Moretto, M. Crisma, G. M. Bonora, C. Toniolo, H.
Balaram, P. Balaram, Macromolecules 1989, 22, 2939; b) I. L.
Karle, P. Balaram, Biochemistry 1990, 29, 6747; c) J. D. Aug-
spurger, V. A. Bindra, H. A. Scheraga, A. Kuki, Biochemistry
1995, 34, 2566; d) J. R. Kumita, C. J. Weston, L.-P. Choo-Smith,
G. A. Woolley, O. S. Smart, Biochemistry 2003, 42, 4492; e) J.
Venkatraman, S. C. Shankaramma, P. Balaram, Chem. Rev. 2001,
101, 3131.
[3] a) C. Toniolo, M. Crisma, F. Formaggio, C. Peggion, Biopolymers
2001, 60, 396; b) C. Toniolo, M. Crisma, G. M. Bonora, E.
Benedetti, B. di Blasio, V. Pavone, C. Pedone, A. Santini,
Biopolymers 1991, 31, 129.
&
Figure 2. Experimental and modeled anisochronicity in Cbz-l-
PheAib4[GlyAib4]nAibCH2OH oligomers (length of peptide
chain=5x+5) and Cbz-l-PheAibnAibCH2OH oligomers (length of
&
[4] a) R.-P. Hummel, C. Toniolo, G. Jung, Angew. Chem. 1987, 99,
1180; Angew. Chem. Int. Ed. Engl. 1987, 26, 1150; b) M. Kubasik,
A. Blom, ChemBioChem 2005, 6, 1187.
peptide chain=y+1); modeled signal decay c (blue line) fault rate
of 6.0%; c (orange line) fault rate of 3.5%; c (green line) fault
rates of 3.5% for Aib monomers and 17.5% for Gly monomers.
[5] a) M. M. Green, M. P. Reidy, J. Am. Chem. Soc. 1989, 111, 6452;
b) M. M. Green, N. C. Peterson, T. Sato, A. Teramoto, R. Cook,
S. Lifson, Science 1995, 268, 1860.
[6] B. Pengo, F. Formaggio, M. Crisma, C. Toniolo, G. M. Bonora,
Q. B. Broxterman, J. Kamphuis, M. Savino, R. Iacovino, F. Rossi,
E. Benedetti, J. Chem. Soc. Perkin Trans. 2 1998, 1651.
[7] a) Y. Inai, Y. Kurokawa, T. Hirabashi, Biopolymers 1999, 49, 551;
b) Y. Inai, Y. Kurokawa, A. Ida, T. Hirabashi, Bull. Chem. Soc.
Jpn. 1999, 72, 55.
[8] a) Y. Inai, K. Tagawa, A. Takasu, T. Hirabayashi, T. Oshikawa,
M. Yanashita, J. Am. Chem. Soc. 2000, 122, 11731; b) Y. Inai, N.
Ousaka, T. Okabe, J. Am. Chem. Soc. 2003, 125, 8151; c) N.
Ousaka, Y. Inai, J. Org. Chem. 2009, 74, 1429.
[9] For examples of helicity controlled from one terminus of
nonpeptide structures, see: a) T. Nakano, Y. Okamoto, Chem.
Rev. 2001, 101, 4013; b) D. J. Hill, M. J. Mio, R. B. Prince, T. S.
Hughes, J. S. Moore, Chem. Rev. 2001, 101, 3993; c) H. Jiang, C.
Dolain, J.-M. Lꢁger, H. Gornitzka, I. Huc, J. Am. Chem. Soc.
difference in helix stability of Gly- and Aib-containing
peptides,[2a,b,e] we suspected that the helix faults would be
located principally at the Gly residues. We therefore made
“all-Aib” tetrapeptide 17 (Cbz-l-PheAib3AibCH2OH; Dd =
166 ppb) and, by an alternative route employing an azlactone
derivative 18 of the azidotetrapeptide precursor to 7, “all-
Aib” decapeptide 19 (Cbz-l-PheAib9AibCH2OH). Aniso-
chronicity Dd in the reporter CH2OH group of 19 was
quantified as 109 ppb (see Figure 1), significantly greater than
that observed in the Gly-containing homologue 12. From the
Dd values for 17, 11, and 19 (Figure 2, red squares) we deduce
that the apparent fault rate (probability of helix inversion) at
an Aib residue is only 3.5% (Figure 2, orange line). To give
correct values for the Gly-containing oligomers, the fault rate
5964 www.angewandte.org
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2009, 48, 5962 –5965

Angewandte
Chemie
2004, 126, 1034; d) J. Clayden, L. Lemiꢂgre, G. A. Morris, M.
Pickworth, T. J. Snape, L. H. Jones, J. Am. Chem. Soc. 2008, 130,
15193.
[14] M. T. Leplawy, D. S. Jones, G. W. Kenner, R. C. Sheppard,
Tetrahedron 1960, 11, 39; D. S. Jones, G. W. Kenner, J. Preston,
R. C. Sheppard, J. Chem. Soc. 1965, 6227; E. Frꢁrot, J. Coste, A.
Pantaloni, M.-N. Dufour, P. Jouin, Tetrahedron 1991, 47, 259; M.
Meldal, M. A. Juliano, A. N. Jansson, Tetrahedron Lett. 1997, 38,
2531; M. Jost, J.-C. Greie, N. Stemmer, S. D. Wilking, K.
Altendorf, N. Sewald, Angew. Chem. 2002, 114, 4438; Angew.
Chem. Int. Ed. 2002, 41, 4267.
[10] Contrasting chromophores have been used to quantify helicity in
different segments of a helical oligomer (Ref. [8c]) and the
chain-length dependence of a CD signal has been used to
estimate the degree of order in a polyisocyanate carrying a chiral
terminator: G. Y. Nath, S. Samal, S.-Y. Park, C. N. Murthy, J.-S.
Lee, Macromolecules 2006, 39, 5965.
[11] Helices containing only achiral amino acids but which invert
slowly on the NMR timescale would of course exhibit aniso-
chronous signals, see: a) N. Ousaka, T. Sato, R. Kuroda, J. Am.
Chem. Soc. 2008, 130, 463; b) V. Maurizot, C. Dolain, Y. Leydet,
J.-M. Lꢁger, P. Guionneau, I. Huc, J. Am. Chem. Soc. 2004, 126,
10049; helix preference in structures of this type has been
controlled by a single terminal residue, see: c) J.-M. Lꢁger, P.
Guionneau, I. Huc, J. Am. Chem. Soc. 2005, 127, 12943.
[12] The choice of Cbz-l-Phe as the helix controller was made after a
brief survey of amino acids, in which this terminator performed
exceptionally well, presumably because it maximizes the differ-
ence in stability between the two diastereomeric helices, and
consequently the chemical-shift difference observed in the
reporter.
[15] Spectra obtained in CD3OD remained identical over a four-fold
range of concentration. Spectra obtained in CDCl3 were broad
and concentration-dependent, suggesting aggregation in this
solvent.
[16] Between À20 and À508C, increased broadening of these singlets
suggests that the rate of helix inversion approaches the slow-
exchange regime only below À508C; see ref. [4].
[17] For a related idea, see: C. Dolain, J.-M. Lꢁger, N. Delsuc, H.
Gornitzka, I. Huc, Proc. Natl. Acad. Sci. USA 2005, 102, 16146.
[18] C. Toniolo, F. Formaggio, M. Crisma, H. E. Schoemaker, J.
Kamphuis, Tetrahedron: Asymmetry 1994, 5, 507.
[19] a) C. Toniolo, E. Benedetti, Trends Biochem. Sci. 1991, 16, 350;
b) R. Gessmann, H. Brꢃckner, K. Petratos, J. Peptide Sci. 2003, 9,
753.
[20] a) G. Krauss, Biochemistry of Signal Transduction and Regula-
tion, Wiley-VCH, Weinheim, 1999; b) J. Clayden, A. Lund, L.
Vallverdffl, M. Helliwell, Nature 2004, 431, 966; c) J. Clayden,
Chem. Soc. Rev. 2009, 38, 817. Peptides 11–14 have structural
similarity with the Aib-rich membrane-active peptaibols, in
particular cephaibol C, whose N-terminal sequence PheAib4Gly
is identical with that of 11–14; see Ref. [1b].
[13] A CH2OH group was chosen as the probe (as opposed, for
example, to the diastereotopic methyl groups of the C-terminal
Aib (see ref. [4])) since it displayed a well-separated AB system
in an uncluttered region of the spectrum. Protected CH2OR
analogues showed poor anisochronicity, perhaps due to freer
bond rotation in the absence of a C-terminal hydrogen bond.
Angew. Chem. Int. Ed. 2009, 48, 5962 –5965
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5965