Angewandte
Chemie
DOI: 10.1002/anie.200802224
Helical Structures
Investigation of pH-Dependent Collagen Triple-Helix Formation**
Song-Gil Lee, Jee Yeon Lee, and Jean Chmielewski*
Collagen is a ubiquitous biomaterial that forms the support-
ing structures in skin, bone, tendons, cartilage, and blood
vessels. Numerous types of collagen have been identified, and
the tertiary structure of each shares the common structural
motif of the collagen triple helix (CTH).[1] The CTH motif is
composed of three chains, each of which adopts a left-handed
type-II polyproline helix, that come together to form a right-
handed superhelix.[2] Repeating units of GlyXaaYaa are
common within the different types of collagen, and a
tendency for X to be proline and Y to be hydroxyproline
(Hyp, O) has been observed.[3] Minimal peptide sequences
based on this idealized sequence have provided a wealth of
information concerning the structural and sequence require-
ments for triple-helix stability.[4] The ability to control triple-
helix formation fundamentally would be useful for a range of
collagen-based biomaterial applications, such as tissue engi-
neering and drug delivery.[5] Herein we disclose modifications
to collagen peptides that lead to the formation of triple helices
on demand through environmental control.
Our plan for the design of a pH-responsive CTH was to
include carboxylate moieties along the collagen peptide.[6]
Under neutral conditions, interstrand electrostatic repulsion
would disfavor triple-helix formation. However, when the
carboxylate groups are protonated under acidic conditions, a
Figure 1. a) Model of PPEG-1 showing the incorporation of a single
carboxy-modified (pink) hydroxyproline residue, PE, into each strand of
a collagen triple helix. b) Structures of the peptides used in this study.
stable triple helix should form, as long as steric repulsion from
the appended groups is not a factor. This design is comple-
mentary to the use of electrostatic interactions to promote the
stabilization of CTHs.[7] As we wished to minimize alteration
to the (POG)n helical structure, our design strategy was to
incorporate a carboxylate functionality within the Hyp
residue by O-alkylation[8] (to give a PE residue; Figure 1).
Molecular modeling of a CTH with the inclusion of PE
suggested that this nonnatural amino acid should be accom-
modated well with minimal interstrand steric interactions
(Figure 1a).
Five peptides, therefore, formed the basis of this study:
two control peptides containing seven repeating units of POG
or PEG (POG-7 and PEG-7, respectively), two host–guest
peptides containing one or three central PPEG units (PPEG-1
and PPEG-3, respectively), and PPEG-7, which contains seven
repeating units of PPEG (Figure 1b). The host–guest peptide
PPEG-1 would enable us to evaluate the destabilizing effect of
a single PE residue per strand on a CTH by comparison with
the results of previous host–guest studies.[4c,d] We envisioned
that the inclusion of increasing numbers of PE residues in
place of Hyp residues, as in PPEG-3 and PPEG-7, would
enable us to determine the degree of modification necessary
for environmental control.
A protected version of PE, Fmoc-PE(tBu)-OH, was
synthesized
from
N-(carbobenzyloxy)hydroxyproline
(Scheme 1). After benzyl protection of the carboxylic acid
to yield 2, a DMAP-promoted Michael addition to tert-butyl
propiolate gave compound 3. The treatment of 3 with
hydrogen over Pd/C led to the removal of the Cbz and
[*] S.-G. Lee,[+] J. Y. Lee,[+] Prof. J. Chmielewski
Department of Chemistry, Purdue University
560 Oval Drive, West Lafayette, IN 47907 (USA)
Fax: (+1)765-494-0239
E-mail: chml@purdue.edu
[+] These authors contributed equally to this work.
Scheme 1. Synthesis of the protected nonnatural amino acid: a) BnBr,
Cs2CO3, DMF; b) tert-butyl propiolate, DMAP, CH2Cl2; c) H2, Pd/C,
MeOH; d) Fmoc-Cl, Na2CO3, water/dioxane. Bn=benzyl, Cbz=carbo-
benzyloxy, DMAP=4-dimethylaminopyridine, DMF=N,N-
[**] We are grateful to the NSF (0078923-CHE) for support of this
research.
Supporting information for this article is available on the WWW
dimethylformamide, Fmoc=9-fluorenylmethoxycarbonyl.
Angew. Chem. Int. Ed. 2008, 47, 8429 –8432
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8429