Reversible Covalent End-Capping of Collagen Model Peptides

Article abstract of DOI:10.1002/chem.201903460

The combination of supramolecular aggregation of collagen model peptides with reversible covalent end-capping of the formed triple helix in a single experimental set-up yielded minicollagens, which were characterized by a single melting temperature. In spite of the numerous possible reaction intermediates, a specific synthetic collagen with a leading, middle and trailing strand is formed in a highly cooperative self-assembly process.

Full text of DOI:10.1002/chem.201903460

DOI: 10.1002/chem.201903460
Communication
&
Peptides
Reversible Covalent End-Capping of Collagen Model Peptides
Christoph Priem and Armin Geyer*[a]
Abstract: The combination of supramolecular aggregation
of collagen model peptides with reversible covalent end-
capping of the formed triple helix in a single experimental
set-up yielded minicollagens, which were characterized by
a single melting temperature. In spite of the numerous
possible reaction intermediates, a specific synthetic colla-
gen with a leading, middle and trailing strand is formed in
a highly cooperative self-assembly process.
Figure 1. Top: The hydroxylation of Pro (blue: Hyp) is the most common sta-
Collagen is a fibrous protein that occurs in different forms of
life such as glass sponges or mammalians.[1,2] Its high structural
diversity originates from a multitude of mutations and post-
translational modifications (PTMs) which bulge or kink the
triple-helical framework formed by the dominant tripeptide
motif proline–hydroxyproline–glycine (POG).[3–5] Glycosylation,
phosphorylation, hydroxylation and inter- or intra-triple-helical
crosslinks lead to a multi-facetted local structural microhetero-
geneity which make collagen a scaffold for cell adhesion, bio-
mineralization and many more physiological processes.[6–12] The
local twisting, bending, widening of the triple-helical structure
guides mobile cells such as a braille code through the connec-
tive tissue.[13] Collagens from natural sources are not suitable
for the quantification of stabilizing or destabilizing effects of a
selected type of PTM without the simultaneous interference of
others. Synthetic minicollagens were developed to investigate
one type of PTM at a specific position within the triple helix.
The deconvolution achieved in such model systems (Figure 1)
identifies stabilizing or destabilizing effects which are mea-
sured as increased or decreased melting temperatures of the
model triple helix.[6–18] Comparing different collagen model
peptides (CMP) with a reference helix such as Ac-(POG)7-NH2
identifies the effect of a selected PTM.[7,11,14] Most trimeric
CMPs are assembled from one type of collagen strand but
there are also heterotrimeric CMPs, which are stabilized by
complementary charges.[19–21] Other trimers are stabilized by
bilizing PTM of collagen. The combined influence of carbohydrates, phos-
phorylation etc. are less well understood. The yellow ring symbolizes inter-
strand covalent bonds. Below: The deconvolution of microheterogeneity in
synthetic triple-helices with a single PTM (green) on each peptide strand.
metal coordination, although its only moderate stabilizing ef-
fects found no application beyond the proof of principle.[17,18]
Furthermore, collagen single-strands were linked by biological
methods[22–24] or stabilized with polymers or dendrimers.[25,26] In
spite of the progress in synthetic collagen-like aggregates with
selected properties such as predetermined register of single
strands or amino acid building blocks that increase the melting
temperature of the triple-helix, a method for the assembly of a
natural host–guest sequence in a predetermined register as a
substrate for collagen binding proteins is still missing.[27] There-
fore, we set out to develop a chiral end-cap that can fix the
single strands in a registers of leading middle and trailing
strand in a covalent reversible manner.
Covalent cross-links that connect the three peptide strands
of a triple-helix as well as linkages between different triple-heli-
ces increase the stability of natural collagens.[9,12,28] The domi-
nant crosslinks in natural collagens are formed by condensa-
tion of hydroxylysine side chains between two triple helices as
well as by disulfide bonds that fix the register of three single
strands within a triple helix.[29] Artificially linked CMPs were in-
vestigated to better understand the influence of covalent
crosslinking in natural collagens. Bonds between all three
strands are feasible with at least one strand bearing two reac-
tive groups.[9,12] Fixing the natural register of leading, middle
and lagging strand of the triple helix by correctly placing the
disulfides was incentive for the development of other collagen
models.[12,30–33] Yet, installing covalent bonds between all three
single-strands of the collagen helix requires numerous synthet-
ic transformations and restricts the application of these tech-
niques. A simpler approach towards covalently linked triple
helices is the covalent end-capping of the CMP with an end-
cap bearing three functional groups of complementary reactiv-
[a] C. Priem, Prof. Dr. A. Geyer
Department of Chemistry, Philipps-Universitꢀt Marburg
Hans-Meerwein-Straße 4, 35032 Marburg (Germany)
E-mail: armin.geyer@staff.uni-marburg.de
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/chem.201903460.
ꢁ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of Creative Commons Attri-
bution NonCommercial License, which permits use, distribution and repro-
duction in any medium, provided the original work is properly cited and is
not used for commercial purposes.
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ity to. Triesters were used by Goodman and Raines to efficient-
ly end-cap triple-helices.[14,15,34] The Lys2 branch also found ap-
plication.[8,35] The concurrent C- and N-terminal end-capping to
obtain a macrotricyclic ring system was a heroic effort.[16]
Spacers such as 6-aminohexanoic acid between the collagen
model peptides and the C-terminal Lys2 end-cap decouple the
triple-helix register from the potentially enhancing helicity of
the chiral end-cap.[8] We recently described CMPs tethered
with triamine caps formed by dipeptides of different stereo-
chemistries and investigated the melting temperature of the
end-capped helix and its biomimetic silicification properties.[36]
Yet, the preparation of covalently end-capped helices is time-
consuming because the separation of deletion mutants is tedi-
ous.
tion reaction (slow chemical exchange). Benzoboroxoles and
aromatic 1,2-diols such as catechols appeared as a good
choice for the bio-orthogonal linkage between collagen and
end-cap. Benzoboroxole was utilized for carbohydrate recogni-
tion by Hall, who described its reversible condensation with
diols.[37–46] The heterogenic mixture of stereo- and regioisomers
limits the NMR characterization for most carbohydrates or oli-
gosaccharides. We recently described 1,2-cis-dihydroxylated bi-
cyclic dipeptides that spontaneously form boronate esters with
high diastereoselectivity. NMR spectroscopy identified the ste-
reochemistry of the newly formed boron stereocenter within
the spiro-oligocyclic ringsystem.[47] We characterized oligomeric
supramolecular assemblies of branched amyloid-type peptides
up to 10 kDa in water.[48] The present study uses similar boro-
nate esters as reversible linkage between end-caps and CMP.
The cooperative triple-helix formation of a CMP is expected to
positively influence the threefold esterification of an end-cap.
Reversibly end-capped CMPs were not reported so far, al-
though boronate based reversible end-capping seemed very
appropriate to enforce the supramolecular assembly of the
triple-helix from three individual peptide strands. The modular
self-assembling components allow the quantification of the
melting temperature of the non-covalently assembled triple-
helical strands and of the covalently assembled triple-helices
from the same molecules by measuring the collagens without
and in the presence of the cap. Even competition experiments
between different peptides are possible. We expected that the
moderate tendency of an individual boroxole to spontaneously
form the boronate from the condensation with a diol under
physiological conditions is pushed to completion by the triva-
lent character of the collagen-helix formed in the process.
Figure 3 shows that the condensation of single benzoborox-
For the present study, we set out to reduce the synthetic
effort towards covalently end-capped CMPs significantly by
using reversible self-assembly from modular components what
appeared as a practical method for triple-helical collagen. The
self-assembly was expected to yield the thermodynamically
most stable triple-helical product as schematically shown in
Figure 2 together with the complex network of various not
fully assembled species. Quantitative spectroscopic methods in
solution are necessary to prove the completeness of the as-
sembly process with defined assembly and register.
Cooperative effects from adequately chosen reactive groups
are expected to shift the equilibrium directly from the disas-
sembled state to the covalently end-capped triple helix. A two-
state folding process requires NMR analytics because it is sensi-
tive for the secondary structure (fast conformational exchange)
and simultaneously quantifies the progress of the condensa-
Figure 3. 1H NMR (600 MHz) of 3 mm 3,4-dihydroxybenzoic acid methyl ester
(1) and 3 mm benzoboroxole (2) in 50 mm pH 7.4 phosphate buffer at 300 K
shows 80% conversion to boronate 3.
ole (1) with catechole (2) is in slow exchange on the NMR time
scale (600 MHz). Spontaneous esterification is not complete
under equimolar amounts of educts and high dilution condi-
tions. The catechol shows 80% conversion at pH 7.4. The con-
densation of boroxoles to sugars needs significant excess of
one component for completion.[40–43,47]
Figure 2. Four equilibria have to be considered for the reversible end-cap-
ping of a collagen triple-helix. (a) The formation of the CMP triple-helix with-
out condensation with the end-cap. (b) The threefold condensation without
triple-helix formation. (c) The triple-helix formation after condensation and
(d) the threefold condensation after triple-helix formation. A potential PTM
which either increases or decreases the cooperativity is shown as a green
bar. (e) All incomplete condensation reactions are shown together with the
functional groups of this study. Further details of the cooperative condensa-
tion reaction are discussed in the main text.
Both functional groups are compatible with solid phase pep-
tide synthesis (SPPS) and the boronate formation is comple-
mentary to other functional groups found in a CMP. Collagen
single-strand 4 (Figure 4) was synthesized according to the op-
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Figure 4. Four different dipeptide end-caps consisting of either l-Lys (5), d-Lys (6), l-Orn (7) or d-Orn (8) were investigated for the ability to stabilize the
triple-helical assembly of peptide 4 which contains a (POG)7 repeat functionalized with a N-terminal boroxole. The boronate ester 43@5 is shown; the other
esters 43@6, 43@7, and 43@8 were obtained analogously. Arrows are drawn in the direction of N- to C-terminus of the peptides.
timized SPPS protocol described earlier.[11] The N-terminal gly-
cine served as spacer between boroxole and the heptameric
POG repeat (Further synthetic details are described in the Sup-
porting Information). The main advantage of a modular ap-
proach was the combination of peptide 4 with different end-
caps without the necessity to restart the synthesis from the
building blocks. End-caps are easily accessible in alternative
stereochemistries bearing three catechol functionalities which
are expected to condense spontaneously with the benzobor-
oxole moiety. The use of dipeptides containing lysine or orni-
thine, modified with three catechol functionalities on side
chains and N-terminus fulfilled these requirements. We investi-
gated four dipeptide end-caps 5–8 with either butylene (Lys)
or propylene spacer (Orn) and l,l or d,d stereochemistry, re-
spectively (Figure 4).
may act as chromophores in CD spectroscopy. NMR identifies
the esterification from chemical shift variations and quantifies
the triple-helical formation from the high-field shift of Pro-dH.
The shielding of an individual proton which is caused by the
triple-helix formation is a reliable proof of the temperature de-
pendence of collagen.[10,11,15] This proton characterizes the co-
operative unfolding of CMPs at elevated temperatures by its
sigmoidal decrease of intensity and quantifies the triple-helix
formation of boroxole-modified CMP 4 (Tm =48.28C). Well-re-
1
solved H NMR spectra were observed over the whole range
between triple-helix (78C) and monomer (778C) (see Support-
ing Information.). The end-caps showed a high-resolution
1H NMR and allowed for a complete signal assignment as well.
Different from the individual components however, the equi-
molar mixture of boroxole peptide 4 and cap 7 (4/7=3:1)
showed severely broadened signals, similar to the NMR spectra
observed for boroxols and carbohydrates[45] wherein the micro-
heterogeneity of numerous regio- and diastereoisomeric boro-
nates having slightly different chemical shifts prohibits the
signal assignment. A complete NMR titration was expected to
yield more data. Therefore, we titrated CMP 4 to a solution of
cap 7 at pH 7.4 in phosphate buffer and monitored the con-
The incomplete (80%) esterification of each individual bor-
oxole may either destructively multiply to a mere 0.83 =50%
overall yield or, the condensation is pushed to completion by
the cooperativity of triple-helix formation after formation of
the first ester bond. The two alternative scenarios which
depend on pH and temperature were already shown in
Figure 2. NMR spectroscopy is the method of choice for the
analytical characterization of the supramolecular assemblies in
solution because it is not influenced by aromatic rings which
1
sumption of the end-cap by H NMR (Figure 5, complete spec-
tra in the Supporting Information). Cap 7 was used because a
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Figure 5. Expansions from the H NMR (600 MHz, 300 K, D2O) titration of benzoboroxole-substituted CMP 4 and the end-cap 7. Due to the cooperative revers-
ible condensation in a 3:1 ratio (4/7) at pH 7.4 phosphate buffer, there is neither unbound end-cap (aromatic protons in blue), nor unbound CMP (aromatic
protons in red) visible, thus indicating that the boronate capping of this CMP yielding 43@7 is largely complete.
the Orn–Orn dipeptide structure yields the highest stabilization
of the triple-helix.[8,14,16,36] Substoichiometric amounts of 4 left
completely unbound end-cap in the solution until the 3:1 ratio
of peptide/cap (4/7) was reached. The detection of completely
unbound end-cap with substoichiometric amounts of peptide
is expected only for a cooperative threefold esterification.
When the helix unfolds at temperatures above its melting tem-
perature, significantly different NMR spectra are obtained be-
cause the esterification is no longer cooperative and leads to a
complex mixture of incompletely esterified regioisomers (see
Supporting Information). CD melting curves are interpreted
under the assumption of quantitative triple-helix formation at
low temperatures while NMR integrals quantify a maximum of
80% folded triple-helix for uncapped CMPs and for the end-
capped CMPs of this study, too.[11] In a solution containing an
equimolar 3:1 ratio of end-cap and triple helix, the cap quanti-
tatively formed the desired boronate and the signals of the
end-cap and the boroxole broadened close to disappearance.
The signal broadening of the end-capped triple helix is restrict-
ed to the end-cap including the boroxole moiety of the CMP
while the aliphatic signals of the POG heptamer are well re-
solved and not effected by signal broadening (Figure 5). Only
after addition of an excess of CMP 4, unbound triple helix ap-
shown in Figure 2. Aliphatic protons are well resolved und not
affected by line broadening (Pro-dH in green identifies the
triple-helical assembly). The intensity of the signals between
1.5 and 4 ppm scales with the amount of added CMP 4 but in-
tegrals was adjusted for better clarity. Temperature dependent
1H NMR spectra quantified the effect of capping on the melt-
ing temperature of the triple-helix. We used the characteristic
upfield shift of Pro-dH in triple-helical environment (green in
Figure 5) to calculate the folded fraction as reported previous-
ly.[11] The melting temperature of the CMP 4 without end-cap,
served as reference with a value of 48.28C. The N-terminal ben-
zoboroxole is a relatively bulky functionalization of the N-ter-
minus in CMPs and showed neither significant stabilizing nor
destabilizing effects on the helix. The increased melting tem-
perature compared to Ac-(POG)7-NH2 (Tm =44.68C) of about
48C is explained by the N-terminal glycine and boroxole. The
stabilizing effect is roughly a third of 10–128C for a Pro-Hyp-
Gly triplet.[11,49] To investigate of origin of this stabilization, we
synthesized Ac-G-(POG)7-NH2 (9, Tm =46.48C, see Supporting
Information) and identified an equal contribution of the gly-
cine und boroxole moiety to the increased melting tempera-
ture of CMP 4. All end-caps from Figure 3 stabilized the triple
helix from 7.2 to 10.18C. The d-Lys–d-Lys cap 8 had the lowest
influence on the triple-helical stability resulting in an increase
of the melting temperature of only 7.28C. End-capping with 5–
7 led to a stabilization of 9.2, 10.1, and 9.68C, respectively
(Figure 6). These values are in a similar range that is obtained
by coordinative reversible stabilization with metal ions (4–
108C)[17,18] but below the values measured for covalent irre-
versible C-terminal end-capping (up to 208C increase in
Tm).[18,36] Our former study on covalent irreversible end-capping
showed strong differences for Tm in the region 158C between
1
pears in the H NMR, indicated by well resolved aromatic bor-
oxole protons. The exchange broadening of these specific pro-
tons at the equal ratio of end-cap and triple-helix proves the
quantitative esterification, characteristic for a highly coopera-
tive folding of the triple-helix and the complete binding of all
three catechol moieties of the end-cap in a cooperative
manner.
The absence of aromatic signals is interpreted by the micro-
heterogeneity of the stereogenic esterification process as
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Conflict of interest
The authors declare no conflict of interest.
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Keywords: collagen model peptides · end-capping · NMR
spectroscopy · supramolecular assembly
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Manuscript received: July 29, 2019
Accepted manuscript online: September 26, 2019
Version of record online: && &&, 0000
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Chem. Eur. J. 2019, 25, 1 – 7
www.chemeurj.org
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ꢀ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communication
COMMUNICATION
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Peptides
C. Priem, A. Geyer*
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Reversible Covalent End-Capping of
Collagen Model Peptides
Supramolecular assembly of collagen
model peptides was combined with re-
versible covalent end-capping. The
quantitative formation of a single-
capped triple helix was analysed via
NMR spectroscopy. This leads to a novel
class of end-capped collagen model
peptides with less effort.
Chem. Eur. J. 2019, 25, 1 – 7
www.chemeurj.org
7
ꢀ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ