Versatile C3-symmetric scaffolds and their use for covalent stabilization of the foldon trimer

Article abstract of DOI:10.1039/c3ob42251h

C₃-Symmetric trimesic acid scaffolds, functionalized with bromoacetyl, aminooxyacetyl and azidoacetyl moieties, respectively, were synthesized and compared regarding their utility for the trivalent presentation of peptides using three different

Full text of DOI:10.1039/c3ob42251h

Organic &
Biomolecular Chemistry
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Versatile C3-symmetric scaffolds and their use for
covalent stabilization of the foldon trimer†
Cite this: Org. Biomol. Chem., 2014,
12, 2606
Arne Berthelmann,a Johannes Lach,a Melissa A. Gräwert,b,c Michael Grollb and
Jutta Eichler*a
C3-Symmetric trimesic acid scaffolds, functionalized with bromoacetyl, aminooxyacetyl and azidoacetyl
moieties, respectively, were synthesized and compared regarding their utility for the trivalent presentation
of peptides using three different chemoselective ligation reactions, i.e. thioether and oxime formation, as
well as the “click” reaction. The latter ligation method was then used to covalently stabilize the trimer of
foldon, a 27 amino acid trimerization domain of bacteriophage T4 fibritin, by linking the three foldon
monomers to the triazido-functionalized trimesic acid scaffold. This reaction dramatically enhanced the
thermal stability of the trimer, while maintaining the correct fold, as demonstrated by CD spectroscopy
and X-ray crystal structure analysis, respectively, of the foldon–scaffold conjugates.
Received 12th November 2013,
Accepted 18th February 2014
DOI: 10.1039/c3ob42251h
www.rsc.org/obc
trivalent peptides. Addressing this challenge, we have syn-
thesized three differently functionalized trivalent C3-symmetric
Introduction
Multivalent interactions play an important role in biological scaffolds to which peptides can be covalently linked via
systems,1 as exemplified by receptor oligomers.2 Trimeric different chemoselective ligation strategies. For this, we
ligands, such as the tumor necrosis factor (TNF) superfamily,3 explored three different ligation reactions: thioether formation
are often presented in the shape of C3-symmetric molecules. from an α-haloalkyl and a thiol group,8 the generation of 1,2,3-
C3-Symmetric trimeric proteins are also involved in the triazoles through 1,3-dipolar cycloaddition of an azide to an
complex interplay between pathogens, such as the human alkyne,9 as well as oxime formation from an aldehyde and a
immunodeficiency virus (HIV-1), and their host cells.4 Inter- hydroxylamine.10
actions of the HIV-1 envelope protein (Env), which is presented
Typically, the purpose of multivalent presentation is to
as a C3-symmetric trimer on the virus surface,5 with cellular bring into spatial proximity molecules that normally would
receptors are involved in a cascade of binding events that not self-associate into an oligomer. An alternative strategy is
result in virus entry into the cell.6 Synthetic peptides have covalent linkage of monomers of a pre-organized oligomer.
proven excellent molecular tools to explore the chemical and Such covalently stabilized oligomers could be useful in experi-
structural determinants of protein–protein interactions.7 ments involving low, i.e. nano- and picomolar concentrations,
Accordingly, peptides to be used for studying molecular inter- where the native, non-covalent oligomer may no longer be
actions involving trimeric proteins should also be presented as sufficiently stable. Such a beneficial effect of covalent stabiliz-
trimers, which requires ready and fast synthetic access to such ation has been shown for peptides that present parts of the
N-peptide region of HIV-1 gp41 fused to a trimeric coiled
coil.11 Covalent stabilization of this trimer by introducing
multiple inter-chain disulfide bridges dramatically enhanced
aDepartment of Chemistry and Pharmacy, University of Erlangen-Nurnberg, Schuhstr.
19, 91052 Erlangen, Germany. E-mail: jutta.eichler@fau.de;
the HIV-1 inhibitory activity of these trimeric peptides, most
likely due to increased thermodynamic stability of the trimer.12
The C-terminal domain of fibritin, a structural protein of
bacteriophage T4, has been shown to be essential for fibritin
trimerization and folding.13 This domain, termed foldon,
assembles into a β-propeller-like trimeric structure in which
each subunit consists of a single β-hairpin.14 Using a peptide
presenting only the 27 amino acid foldon sequence, this
characteristic fold has been shown to be independent of
the structural context of the fibritin protein.15 Furthermore,
the trimeric structure of the foldon is largely unaffected by
Fax: +49-9131-852-2587; Tel: +49-9131-852-4117
bCenter for Integrated Protein Science at the Department of Chemistry, Chair of
Biochemistry, Technical University of Munich, Lichtenbergstr. 4, 85747 Munich,
Germany. E-mail: michael.groll@ch.tum.de; Fax: +49-89-289-13363;
Tel: +49-89-289-13361
cEuropean Molecular Biology Laboratory, Hamburg Unit, EMBL c/o DESY,
Notkestraße 85, 22603 Hamburg, Germany. E-mail: graewert@embl-hamburg.de;
Fax: +49-40-89902-149; Tel: +49-40-89902-115
†Electronic supplementary information (ESI) available: NMR spectra of
3
through 8; LC-MS data/MALDI spectra of 9 through 17; X-ray data collection and
refinement statistics of foldon–scaffold conjugates; thermal unfolding of the
non-covalent foldon trimer (15), as well as the covalently stabilized trimers 18
and 19, in a buffer without detergent. See DOI: 10.1039/c3ob42251h
2606 | Org. Biomol. Chem., 2014, 12, 2606–2614
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Fig. 1 (A) Covalent stabilization of the foldon trimer by N- or C-termin-
ally linking the three monomers to a C3-symmetric scaffold. (B) Location
of N-and C-termini, respectively, of the three monomers within the
foldon NMR structure (PDB ID: 1RFO).15
Scheme 2 Functionalization of the amino scaffold 4 for different lig-
ation reactions. (i) Bromoacetic acid/DIC; (ii) azidoacetic acid/EDCI; (iii)
(a) AOA/EDCI, (b) TFA/DCM; X: –(CH2)2–O–(CH2)2–O–(CH2)2–.
proteins attached to it, rendering this domain an ideal auxilli-
ary to induce or stabilize trimeric structures of peptides and
proteins. Examples of such foldon fusion proteins include
HIV-1 gp41,16 a tumor necrosis factor ligand,17 as well as a
recombinant influenza H5N1 vaccine.18
Here, we asked whether the thermodynamic stability of the
foldon trimer could be enhanced, while maintaining the
correct fold, by covalently linking the three monomers to a
C3-symmetric scaffold (Fig. 1).
organic solvents, such as dichloromethane or chloroform, in
which scaffold synthesis was carried out, as well as in aqueous
media for the subsequent ligation reaction with peptides. Fur-
thermore, PEG based spacers are flexible, non-toxic and non-
immunogenic.23
Scaffold 4 provided the basis for the generation of three
differently functionalized trimeric scaffolds, through acylation
with different acetic acid derivatives (Scheme 2). The bromo-
acetylated scaffold 5, which served as a precursor for sub-
sequent thioether ligation, was synthesized by acylating the
amino groups of 4 with bromoacetic acid. For this reaction,
EDCI turned out to be a poor coupling reagent, since it was
used as a hydrochloride, resulting in a halide exchange of the
bromo alkyl group, yielding the less reactive chloroacetyl
scaffold derivative. This reaction could be avoided by using
N,N′-diisopropylcarbodiimide (DIC) as the coupling reagent,
which yielded the desired bromoacetyl scaffold derivative 5
(Fig. S5 and S6†).
The triazido scaffold 6 was generated by two different
approaches. The first strategy involved coupling of pre-formed
azidoacetic acid 824 (Fig. S11 and S12†) in conjunction with
EDCI to yield 6 (20% isolated). The second method was based
on a pseudo halide exchange of the haloacetylated scaffold 5
with sodium azide in the presence of 18-crown-6-ether.25 Both
methods worked equally well, yielding 6 as the desired product
(Fig. S7 and S8†).
Results and discussion
Synthesis of scaffolds
Benzene-1,3,5-tricarboxylic acid (trimesic acid) 1, containing a
3-fold axis, served as a starting point for the generation of the
different scaffolds. The utility of this simple aromatic com-
pound for the synthesis of complex trimeric structures, such
as monodisperse dendrimers,19 symmetric LewisX antigens20
or trimeric integrin ligands,21 has been demonstrated before.
In order to provide
scaffold,
a
trivalent amino-functionalized
(Boc-DOOA)
N1-Boc-3,6-dioxaoctane-1,8-diamine
2 was coupled as a spacer to the three carboxylic groups of 1
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)
as a coupling reagent, yielding the Boc-protected scaffold 3
(Scheme 1, Fig. S1 and S2†). EDCI was chosen because it facili-
tates work-up of the product through simple aqueous extrac-
tion of the resulting water-soluble urea derivative.22 Removal
of the Boc groups yielded the C3-symmetric scaffold
4
The hydroxylamine functionality required for subsequent
oxime ligation was introduced by coupling bis-Boc protected
(aminooxy)acetic acid (AOA) to the triamino scaffold 4, fol-
lowed by removal of the Boc groups with trifluoroacetic acid
(TFA), yielding tris(aminooxy)-functionalized scaffold 7 quanti-
tatively (Fig. S9 and S10†).
(Scheme 1, Fig. S3 and S4†). The ethylene glycol nature of the
diamine spacer 2 was chosen based on its favorable chemical
and physical characteristics, which include solubility in
Ligation reactions
The utility of the three differently functionalized scaffolds 5, 6
and 7 for the respective ligation reactions was tested using the
peptide sequence AKYRP, a short trypsin inhibitor.26 This
peptide was synthesized thrice (peptides 9, 10 and 11). The
Scheme 1 Synthesis of the amino-functionalized trimesic acid scaffold 4. sequence was N-terminally extended by ε-aminohexanoic acid
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(Ahx) as a spacer, followed by an N-acetylated cysteine (peptide
9, Fig. S13†), N-acetylated propargyl glycine (peptide 10,
Fig. S14†), and 4-formylbenzoic acid (peptide 11, Fig. S15†),
respectively. For the cleavage of peptide 11 from the resin, the
scavenger composition of the TFA cocktail had to be modified
in order to avoid side reactions, in particular reduction of the
aldehyde to the alcohol. Therefore, thioanisole was omitted
from the cleavage cocktail, and formylbenzoic acid was added
as an additional scavenger. All three peptides could be syn-
thesized in good yields of 42–58%.
The functionalized scaffolds 5, 6 and 7, as well as peptides
9, 10 and 11, subsequently served as mutually reactive precur-
sors for the three ligation reactions to be studied (Scheme 3).
To analyze and compare the kinetics of these ligations, the
reaction progress was followed by LC-MS over time.
Formation of a thioether from haloacetylated and thiol
containing precursors in aqueous media (Scheme 3A) is well
established.27 This reaction, however, is not absolutely
chemoselective. Peptide dimerization through disulfide for-
mation is a prominent side reaction, which can be reversed by
reducing the disulfide using tris(2-carboxyethyl)phosphine
(TCEP).28 The ligation reaction was performed at pH 9.6 to
provide a highly nucleophilic thiolate species of 9. As shown in
Fig. 2A, the trimeric product 12 (Fig. S16†) was generated
instantaneously upon mixing of precursors 5 and 9. The
first LC-MS sample was taken after approximately 1 min at
which time the conversion rate was already 55%, indicating a
very fast reaction. Kinetic analysis by HPLC indicated 85% con-
version to the thioether trimer within 30 min. The disulfide
dimer of 9 was detected merely as a minor side product, while
the remaining side products were identified as mono- and di-
substituted scaffolds, respectively. These results demonstrate
that thioether ligation is an excellent method for the
Fig. 2 Kinetics of ligation reactions: (A) thioether ligation (generation of
12), (B) 1,3-dipolar cycloaddition (generation of 13), (C) oxime ligation
(generation of 14). Monomeric peptides were used at
3 mM and
scaffolds at 1 mM, resulting in a concentration of 1 mM for the generated
trivalent peptides.
generation of trivalent peptides. Furthermore, this method can
also be used to trimerize proteins using free cysteine residues
in their sequences as ligation points.
Copper(I)-catalyzed 1,3-dipolar cycloaddition of azides to
alkynes generates 1,4-substituted (1,2,3)-triazoles (Scheme 3B)
and is better known as “click chemistry”.29 This type of chemo-
selective reaction has been extensively used to generate diverse
bioactive molecules, including combinatorial peptidotriazole
libraries,30 cyclopeptide analogs,31 or β-turn mimics.32 The
click reaction is controlled by several parameters, most impor-
tantly the source of Cu(I). A convenient way to provide the cata-
lytic Cu(I) species is in situ reduction of Cu(II)-salts with a
suitable reductive agent, such as ascorbic acid. Another critical
factor is the solvent. While an aqueous environment is favor-
able for reactions involving unprotected peptides, protected or
Scheme 3 Ligation reactions. (A) Thioether ligation; (B) 1,3-dipolar
cycloaddition; (C) oxime ligation. (i) Carbonate buffer pH 9.6; (ii) 6 M
CuSO4, 18 M sodium ascorbate; (iii) citrate buffer pH 2.5. X: –(CH2)2–O–
(CH2)2–O–(CH2)2–; peptide: Ahx-AKIYRP.
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resin-bound peptides, as well as other, less polar compounds, biomolecular interactions, which may mask the native inter-
require a polar organic solvent. To cover both scenarios, two actions. These specifics should be taken into account when
solvent mixtures were tested, i.e. a water–isopropanol (1 : 1) selecting a ligation strategy for a specific application.
and a water–DMF (1 : 1) mixture.
Covalent stabilization of the foldon trimer
Similar to the thioether ligation, kinetic analysis (Fig. 2B)
demonstrated a fast reaction of peptide 10 with triazido
scaffold 6. In isopropanol, the reaction was almost complete
within 10 min, with a conversion rate to the trimeric product
13 (Fig. S17†) of 86% (isopropanol) and 90% (DMF), respecti-
vely, illustrating the outstanding value of click chemistry for
the chemoselective attachment of alkyne peptides to C3-sym-
metric azido scaffolds. Mono- and disubstituted scaffolds were
found as side products (11% and 9%, respectively), whereas
only traces of unreacted 10 were detectable after 30 minutes.
Finally, oxime ligation33 was used to generate trivalent
peptide 14 by reacting the nucleophilic hydroxylamines of
scaffold 7 with peptide 11, which was N-terminally acylated
with 4-formylbenzoic acid, providing the aldehyde moiety for
oxime formation (Scheme 3C). Analysis of reaction kinetics for
the formation of 14 (Fig. S18†) indicated a maximal yield of
80% after 20 minutes (Fig. 2C), which could not be further
improved over time. As expected,34 addition of aniline as a
catalyst accelerated the reaction, but did not improve the final
yield (data not shown). Interestingly, the main side product
here was unreacted 11 (18%) rather than the respective mono-
and disubstituted scaffolds. It should also be noted that the
trimerization yields of parallel experiments involving an
analog of 11, in which the aldehyde moiety was replaced by a
ketone (pyruvoyl moiety), were much lower and did not exceed
23% after 10 hours.
Based on the excellent results obtained using the click reaction
to attach peptide monomers to the trimesic acid scaffold, as
well as the robustness of this reaction and ease of synthesis of
precursors, we chose the click reaction to covalently stabilize
the trimer of the 27 amino acid peptide foldon. The three
dioxaoctane spacer units of scaffold 6 were thought to be ade-
quate to cover the distances between the N-(13.7 Å) and
C- (5.15 Å) termini of the three foldon monomers within the
trimer structure. In addition to the unmodified foldon
sequence (15, Fig. S19†), we synthesized two peptides, in
which a propargylglycine residue was added either to the N- or
C-terminus of the foldon sequence (16 and 17, Fig. S20 and
S21,† see Table 1 for peptide sequences). These functionalized
peptides were then “clicked” to the triazido scaffold 6, yielding
conjugates 18 and 19 (Fig. 3 and S22 through S25†), whose
folding and thermal stabilities were subsequently analyzed by
CD spectroscopy and X-ray crystal structure analysis,
respectively.
Comparison of the CD spectra of 15, 18 and 19 indicated
that covalent attachment of the three foldon monomers to the
scaffold does not affect folding of the trimer, as all three CD
spectra are very similar (Fig. 4).
The notion of structural similarity between wt foldon (15)
and the two foldon–scaffold conjugates was corroborated by
Comparing the results of the above three ligation strategies,
it is apparent that thioether ligation and click reaction yielded
the desired trivalent peptides in excellent yields and purities,
while the results of the oxime ligation were less satisfactory.
Furthermore, all three methods have their specific strengths
and limitations. Thioether ligation yields metabolically stable
and largely non-reactive molecules that are well compatible
with biological systems. In fact, methionine, one of the
proteinogenic amino acids, contains a thioether in its side
chain. Possible side reactions associated with thioether lig-
ation include an attack of other nucleophilic moieties on the
bromoacetylated scaffold, impairing the site-selectivity of the
ligation reaction. Similarly, the carbonyl carbon of the alde-
hyde or ketone moieties involved in oxime ligation may also be
attacked by nucleophiles other than the cognate aminooxy
group of the scaffold. Furthermore, the limited choice of sca-
vengers to be used during the acidic cleavage of the aldehyde
peptide precursor from the resin may impair the purity of
longer peptides containing multiple side chain protection
groups. In addition, due to the susceptibility of oximes to
hydrolysis, this ligation reaction may not be ideal for the gene-
ration of molecules to be used in biological systems. Triazoles,
on the other hand, which are the product of the click reaction,
are thought to be fairly stable in a range of different milieus. It
should be noted, however, that the nitrogen atoms of the tri-
Table 1 Peptide sequences
Peptide
Sequence
9
Ac-CXaAKIYRP-NH2
10
11
15
16
17
Ac-Prab-XAKIYRP-NH2
Fbac-XAKIYRP-NH2
Ac-GYIPEAPRDGQAYVRKDGEWVLLSTFL-NH2
Ac-Pra-XGYIPEAPRDGQAYVRKDGEWVLLSTFL-NH2
Ac-GYIPEAPRDGQAYVRKDGEWVLLSTFLX-Pra-NH2
a X,
4-formylbenzoic acid; Ac, acetyl.
6-aminohexanoic
acid.
b Pra,
propargylglycine.
c Fba,
Fig. 3 Covalently stabilized foldon trimers, in which the three mono-
mers are linked N- (18, left) and C- (19, right) terminally respectively, via
azole ring are potentially strong hydrogen bond acceptors in triazole linkers, to a trimesic acid scaffold.
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observed at the flexible spacer region (root mean square devi-
ation of Cα < 0.3 Å) (Fig. 5a and S27†). Furthermore, the struc-
tures are largely congruent with the previously solved NMR
structure.15 The calculated size of the interface between the
three foldon monomers (approximately 3000 Å2) was
very similar in wt foldon and the covalently stabilized trimers
18 and 19. The scaffold and linker regions of 18 and
19, however, are resolved differently in the two structures. The
electron density map of 18 merely displays the amide part of
the linker that is directly attached to the foldon N-terminus
(Fig. 5b). In the structure of 19, on the other hand, the trimesic
acid scaffold is well defined and positioned at the center of the
tri-fold non-crystallographic symmetry axis atop the C-terminal
leucine residues, whereas the first three N-terminal foldon
residues, as well as the linker region composed of 6-amino-
hexanoic acid, the triazole ring and DOOA, are structurally dis-
torted (Fig. 5c). In summary, the crystallographic data confirm
that the structure of the foldon trimer is largely unaffected by
covalent linking of the three monomers to the trimesic acid
scaffold.
Fig. 4 CD spectra of the non-covalent foldon trimer (15), as well as co-
valently stabilized trimers 18 and 19.
solving the crystal structures of 18 and 19, and comparing it
with the structure of wt foldon. Conjugates 18 and 19 were
subjected to a number of crystallization trials with various
salts at different concentrations. The conjugates spontaneously
crystallized in more than 70% of the tested buffers (approxi-
mately 500). The most promising crystals were obtained in
0.2 M ammonium sulfate, 30% PEG 4000 and 1.4 M Na/
K-phosphate, pH 5. Despite the relatively small size of the crys-
tals, high resolution data could be collected up to 1.1 Å resol-
utions. Interestingly, the X-ray data revealed a high tendency of
the foldon to crystallize in different space groups. Even crystals
harvested from the same drop showed different symmetries.
We determined the structures of conjugates 18 and 19, which
crystallized in the space groups p21 (1.1 Å) and C2 (1.3 Å). Both
proteins present a native-like fold, comprising a 310-helix at
the C-terminus, which is preceded by a β-hairpin and an
extended structure at the N-terminus (Fig. 5). Superposition of
18 and 19 with wt foldon (PDB ID 4NCU) confirmed the overall
similarity of the three structures, as local heterogeneity is only
Thermal unfolding of wt and covalently stabilized foldon
trimers was assessed by monitoring the temperature-depen-
dent changes in CD signals at 228 nm.35
As shown in Fig. 6, the stability of the trimer was greatly
enhanced by covalently attaching the three monomers to the
trimesic acid scaffold, as evidenced by a strong shift in Tm of
18 and 19, compared to 15. Even under denaturing conditions
(5 M urea), the covalently stabilized trimers (18, Tm = 77 °C
and 19, Tm = 78 °C) were substantially more stable, with Tm
approximately 30 degrees higher than that of the non-covalent
foldon (15, Tm = 48 °C) (Fig. 6). In a buffer without detergent,
on the other hand, the trimers of 18 and 19 did not start to
unfold until temperatures as high as 80 °C were reached, so
that no melting points could be determined (Fig. S26†). These
results demonstrate a strong stabilization of the foldon trimer
by covalently attaching the three monomers to a C3-symmetric
scaffold, regardless of the orientation of the foldon sequence
with respect to the scaffold (N- and C-terminal attachment,
respectively).
Fig. 5 (a) Backbone superposition of one monomer of wt foldon (red: crystal structure (PDB ID 4NCU), purple: NMR structure15) with the covalent
conjugates 18 (cyan) and 19 (gold). The structurally distorted N-termini of 19 are labelled. (b) Trimer structure of 18. The acetamide of the linker is
well defined in the electron density map. (c) Trimer structure of 19 (rotated about the x-axis by 180°). The electron density (blue) represents a 2FO–
FC-omit map contoured at 1.0 σ of the trimesic acid scaffold.
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Peptide synthesis. Peptides (see Table 1 for sequences) were
synthesized as C-terminal amides by Fmoc/t-Bu-based solid-
phase synthesis on 100 mg TentaGel S RAM resin (0.24 mmol
g−1) from Rapp Polymere, using an automated multiple
peptide synthesizer (SYRO I from MultiSynTech). In a standard
coupling cycle, five eq. of Fmoc-amino acid/DIC/HOBt in DMF
were coupled for 60 min, followed by a capping step using a
mixture of acetic anhydride–pyridine–DMF (1 : 2 : 3; 30 min).
The Fmoc group was removed using 20% piperidine–DMF
(20 min). Fmoc-propargyl glycine (peptides 10, 16 and 17) as
well as 4-formylbenzoic acid (peptide 11) were coupled over-
night using 3 and 10 eq., respectively, of activated acid. To
avoid aspartimide formation in peptides 15, 16 and 17, Fmoc-
L-Asp(OMpe)-OH was used to introduce aspartic acid residues
in these peptides. Peptide 9, 10, 15, 16 and 17 were cleaved
from the resin using a mixture of TFA–thioanisole–phenol–
water–EDT (82.5 : 5 : 5 : 5 : 2.5). Peptide 11 was cleaved with a
mixture of TFA–TIPS–4-formylbenzoic acid–water (85 : 5 : 5 : 5)
to avoid reduction of the aldehyde group. The cleaved peptides
were precipitated in a cold 1 : 1 mixture of cyclohexane and
t-butyl methyl ether (10 mL), extracted with water (10 mL), lyo-
philized twice, purified by preparative HPLC, and character-
Fig. 6 Thermal unfolding of the non-covalent foldon trimer (15),
as well as the covalently stabilized trimers 18 and 19, in the presence of
5 M urea.
Experimental section
Materials and methods
Thin-layer chromatography. Thin-layer chromatography was ized by LC-MS and high resolution ESI mass spectrometry,
performed on silica gel 60 F254 on aluminum foils (Merck). respectively.
Compounds were detected under a UV lamp at 254 nm and
366 nm, respectively.
CD spectra. CD spectra of 15, 18 and 19 were recorded
using a Jasco J-815 instrument at 20 °C and 50 µM in phos-
Column chromatography. Column chromatography was per- phate buffer pH 7.0. Thermal unfolding transitions were
formed using Merck Silica Gel 60 (0.04–0.063 nm) as the measured by monitoring the changes in CD signals at 228 nM
stationary phase.
at peptide concentrations of 30 µM (18 and 19) and 90 µM
1H/13C-NMR spectra. 1H/13C-NMR spectra were recorded on (15), respectively, in phosphate buffer pH 7.0, as well as in the
Bruker Avance 600 (1H: 600 MHz, 13C: 151 MHz) and Bruker same buffer containing 5 M urea.
Avance 360 (1H: 360 MHz, 13C: 91 MHz) using CDCl3, CD3OD
Crystal structure analysis. The covalently stabilized folden
and CD3CN as solvents referenced to TMS (0 ppm), CHCl3 trimers 18 and 19 were dissolved in water at 8–12 mg mL−1
.
(7.26 ppm), CHD2OD (3.31 ppm) or CHD2CN (1.94 ppm). The initial crystallization conditions were determined by the
Chemical shifts are reported in parts per million (ppm). Coup- sitting drop vapor diffusion method at 20 °C. For this, equal
ling constants (J) are in hertz (Hz). The following abbreviations volumes (0.1 µL + 0.1 µL) of protein solution and precipitant
are used for the description of signals: s (singlet), d (doublet), solution were mixed (crystal screens ClassicsI, ClassicsII, JCSG,
t (triplet), q (quadruplet), m (multiplet).
PEG I and PEG II suites (Qiagen, Hilden)) using the robot from
LC-MS. Analytical HPLC was performed with online ESI Art Robbins (Phoenix), and equilibrated against the reservoir
mass spectrometry detection (LC-MS) using an Agilent HP containing 45 µL crystallization solution. In general, crystal
1100 instrument, and the following conditions: column: formation could be observed within a few hours. Suitable con-
Kinetex C18, 2.6 µm, 100 Å, 50 × 2.1 mm, flow rate: 0.4 mL ditions were further optimized using the hanging drop vapor
min−1, gradient: 5–95% ACN–water (both containing 0.1% diffusion method, resulting in the selection of 20% PEG 200,
TFA) in 15 min. Mass spectra were measured on an AB SCIEX 0.2 M ammonium sulfate, 30% PEG 4000 and 1.4 M Na/
API 2000. The recorded spectra were analyzed using the K-phosphate, pH 5. Crystals were soaked in cryobuffer contain-
Analyst software (1.4.2) by AB SCIEX.
ing 25% PEG 200 in addition to the precipitant solution, and
Preparative HPLC. The following conditions were applied: cooled in a stream of nitrogen gas at 100 K (Oxford Cryo
column: Reprosil 100 C18, 5 µm, 250 × 10 mm, flow rate: 9 mL Streams). Datasets were collected using the synchrotron radi-
min−1, gradient: 15–55% ACN–water (both containing 0.1% ation facility at the X06SA-beamline, SLS (Villigen, Switzerland)
TFA) in 60 min (12–14), and 20–60% in 80 min (15–19), (Table S1†). Datasets were processed either with PROTEUM2 or
respectively, UV detection at 220 nm.
using the program package XDS.36 For structure determi-
High resolution ESI mass spectrometry. High resolution ESI nation, molecular replacement was performed in PHASER37 by
mass spectra were recorded on a Bruker Daltonik Maxis 4G using the coordinates of a wild type foldon structure solved
using electrospray ionization in the positive mode. Peptides were by NMR spectroscopy (PDB ID: 1RFO). The models were
dissolved in ACN–H2O at 1 mg mL−1. The recorded spectra were completed using the interactive three-dimensional graphics
analysed using the open source mass spectrometry tool mMass.
program MAIN.38 Conventional crystallographic rigid body,
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positional, and temperature factor refinements were carried
Scaffold 6. Scaffold 4 (180 mg, 0.300 mmol) and DIPEA
out with REFMAC5 from the CCP4 package.39 Water molecules (0.052 mL, 0.300 mmol) were dissolved in 10 mL chloroform.
were located with the ARP/wARP solvent within the CCP4i EDCI (230 mg, 1.199 mmol) was added. To the white suspen-
GUI,40 and verified manually. Refinement with restraints sion, 2-azidoacetic acid (182 mg, 1.798 mmol) was added drop-
between bonded and non-crystallographic symmetry related wise and allowed to react at RT for 8 h. The reaction was then
atoms using anisotropic TLS parameters and REFMAC541 quenched with water (3 × 25 mL), the organic layer dried over
yielded excellent values for Rwork and Rfree as well as r.m.s.d. anhydrous Na2SO4, concentrated, and the crude product puri-
bond and angle values (Table S1†).
fied by flash column chromatography on silica gel (DCM–
Scaffold 3. Trimesic acid (630.4 mg, 3.00 mmol) and EDCI methanol 1% → 5%) yielding 50.9 mg (0.06 mmol, 20%) of
(1898 mg, 9.90 mmol) were suspended in dry DCM (15 mL) product. 1H-NMR (CDCl3–CD3OD, 600 MHz): δ [ppm] = 8.46 (s,
under argon. After stirring for 15 min at 0 °C, Boc-DOOA 3 H), 4.64 (s, 12 H), 3.89 (s, 3 H), 3.63–3.75 (m, 12 H), 3.59 (t,
(2458 mg, 9.90 mmol) was added dropwise, and the mixture J = 5.5 Hz, 6 H), 3.41–3.50 (m, 6 H), 3.38 (s, 3 H). 13C-NMR
was stirred overnight at RT. The resulting orange solution was (CDCl3–CD3OD, 151 MHz): δ [ppm] = 167.9, 166.6, 134.6,
washed with water (3 × 25 mL), and after separation, the 128.3, 69.5, 68.9, 68.7, 51.4, 39.3, 38.5, 26.2. MS (ESI, positive):
organic layer was dried over anhydrous Na2SO4. The filtrate C33H51N15O12, [M + H]+ calc.: 849.87 found: 849.80.
was concentrated to dryness and purified by flash column
Scaffold 7. Scaffold 4 (244 mg, 0.406 mmol) was dissolved
chromatography (DCM) to yield 1539.8 mg (1.71 mmol, 57%) in 10 mL chloroform. After pre-activation of bis-Boc-amino-
1
of a slightly yellow oil. H-NMR (CDCl3, 600 MHz): δ [ppm] = oxyacetic acid (390 mg, 1.340 mmol) with EDCI (701 mg,
8.41 (s, 3 H), 7.10–7.33 (m, 3 H), 5.29 (br. s, 3 H), 3.62–3.70 3.66 mmol) in chloroform (5 mL) for 20 min at 0 °C, the result-
(m, 24 H), 3.56 (t, J = 5.3 Hz, 6 H), 3.29 (t, J = 4.7 Hz, 6 H), ing suspension was added dropwise to the amine. The mixture
1.40 (s, 27 H). 13C-NMR (CDCl3, 91 MHz): δ [ppm] = 166.0, turned slowly into a colorless solution and was stirred over-
156.2, 135.1, 128.5, 79.3, 70.3, 70.2, 69.7, 40.6, 40.1, 28.4. night at RT. The reaction was quenched with water (30 mL).
MS (ESI, positive): C42H72N6O15 [M + H]+ calc.: 901.51, After separation, the organic layer was dried with anhydrous
found: 901.41.
MgSO4, filtered and evaporated until dryness. The protected
Scaffold 4. Scaffold 3 (180 mg, 0.200 mmol) was dissolved scaffold was purified via flash column chromatography (DCM–
in 10 mL methanol. After cooling to 0 °C, concentrated HCl methanol: 1% → 5%) yielding 126.5 mg (0.154 mmol, 38%) of
(2.2 mL, 26.6 mmol) was added dropwise. The reaction was product. The Boc groups were removed with 20% TFA in DCM
stirred in the cold until TLC indicated full deprotection. After (10 mL) for two hours. After evaporating the solvent, the solid
evaporation of the solvent, the residue was re-dissolved in was re-dissolved in DCM and evaporated three times, dried
1
methanol and stepwise neutralized to pH 7 with a saturated under vacuum and used without further purification. H-NMR
solution of sodium methoxide in methanol. The generated (CDCl3–CD3OD, 600 MHz): δ [ppm] = 8.45 (br. s, 3 H), 4.03 (br.
sodium chloride was filtered off and the filtrate was dried over s, 28 H), 3.66 (br. s, 24 H), 3.60 (br. s, 6 H), 3.44 (br. s, 6 H).
anhydrous sodium sulfate. After evaporation, the free triamine 13C-NMR (CDCl3–CD3OD, 151 MHz): δ [ppm] = 167.2, 135.1,
compound was afforded quantitatively and was directly used 129.1, 70.3, 70.2, 69.8, 69.7, 69.6, 69.5, 40.1, 40.1, 38.8. MS
for further reactions. 1H-NMR (CDCl3–CD3OD, 600 MHz): (ESI, positive): C33H57N9O15, [M + H]+ calc.: 820.86 found:
δ [ppm] = 8.56 (s, 3 H), 3.68–3.77 (m, 12 H), 3.64 (s, 12 H), 3.38 820.70.
(s, 3 H), 3.05–3.10 (m, 6 H), 2.76–3.04 (m, 6 H). 13C-NMR
Trivalent peptide 12. Solutions of peptide 9 (12 mM) and
(CDCl3–CD3OD, 151 MHz): δ [ppm] = 167.1, 134.5, 129.3, 69.9, scaffold 5 (4 mM, 100 µL each, both in ACN–water (1 : 1)) were
69.8, 69.5, 66.2, 39.7, 39.4. MS (ESI, positive): C27H48N6O9 mixed with 200 µL 0.1 M carbonate buffer pH 9.6, resulting in
[M + H]+ calc.: 601.71 found: 601.60.
a final concentration of 1 mM of peptide 9. After an overnight
Scaffold 5. Scaffold 4 (240 mg, 0.400 mmol) and DIPEA reaction, the product was analyzed by LC-MS, lyophilized, puri-
(500 µL, 2.86 mmol) were dissolved in 10 mL chloroform. fied by preparative HPLC, and analyzed by LC-MS.
After pre-activation of 2-bromoacetic acid (555 mg, 4.00 mmol)
Trivalent peptides 13, 18 and 19. Solutions of peptides 10,
with DIC (778 µL, 4.99 mmol) in chloroform (5 mL) at 0 °C 16 and 17, respectively (100 µL, 12 mM in isopropanol–water
for 20 min, the solution was added dropwise into the reac- (1 : 1)), were mixed with 100 µL of 4 mM scaffold 6 solution,
tion mixture and stirred at RT until no educt was evident by 100 µL of 24 mM CuSO4 in isopropanol–water (1 : 1), as well as
TLC. After evaporation of the solvent the crude solid was with 100 µL of 72 mM sodium ascorbate in isopropanol–water
purified via flash column chromatography on silica gel (DCM– (1 : 1), resulting in a final concentration of 1 mM of peptides.
methanol: 1% → 8%) yielding 50.1 mg (0.052 mmol, 13%) of a Isopropanol can be replaced with DMF. After an overnight reac-
yellow solid product. 1H-NMR (CDCl3–CD3OD, 600 MHz): tion, the product was analyzed by LC-MS, lyophilized, purified
δ [ppm] = 8.46 (s, 3 H), 4.20 (s, 9 H), 3.79 (s, 3 H), 3.59–3.70 by preparative HPLC, and analyzed by LC-MS and high resolu-
(m, 18 H), 3.55 (t, J = 5.3 Hz, 6 H), 3.38 (t, J = 5.3 Hz, 6 H). tion ESI mass spectrometry (18 and 19).
13C-NMR (CDCl3–CD3OD, 91 MHz): δ [ppm] = 168.0, 167.5,
Trivalent peptide 14. Solutions of peptide 11 (12 mM) and
135.4, 129.5, 70.6, 70.6, 70.1, 69.8, 40.3, 40.2, 28.7. MS (ESI, scaffold 8 (4 mM, 100 µL each, both in ACN–water (1 : 1)) were
positive): C33H51Br3N6O12, [M + H]+ calc.: 964.51 found: mixed with 200 µL of 0.1 M citrate buffer pH 2.5, resulting in a
964.70.
final concentration of 1 mM of peptide 14. After an overnight
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reaction, the product was analyzed by LC-MS, lyophilized, puri- structure factors of the two covalent conjugates 18 and 19,
fied by preparative HPLC, and analyzed by LC-MS.
have been deposited in the Protein Databank (PDB accession
codes 4NCV and 4NCW).
Conclusions
In conclusion, we have generated three differently functiona-
lized C3-symmetric scaffolds based on trimesic acid for the tri-
valent presentation of biomolecules. The utility of these
versatile scaffolds for trimerization reactions was demon-
strated using appropriately functionalized peptides in conjunc-
tion with three different ligation strategies, i.e. thioether
formation, click chemistry and oxime ligation, respectively.
Kinetic analysis of each ligation reaction demonstrated that all
three reactions are powerful tools for the synthesis of trivalent
peptides. Depending on the desired flexibility of the trivalent
molecules, the length and flexibility of the spacer can be
varied using other diamines. It should also be noted that the
utility of these scaffolds is not limited to the three ligation
reactions presented here, but could readily be extended to
include other ligation reactions, such as Staudinger ligation42
and native chemical ligation,43 simply by reacting the scaffold
amino groups with appropriate chemical moieties. Further-
more, covalent attachment of alkyne-modified foldon peptides
to the triazido scaffold using the click reaction distinctly
enhanced the stability of the foldon trimer, while maintaining
its correct fold. This illustrates an alternative application of
multivalent peptide presentation, in which folded, non-
covalent peptide and protein oligomers are thermodynamically
stabilized by covalently linking the monomers to an appropri-
ate multivalent scaffold.
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Abbreviations
ACN
Boc
DCM
DIC
Acetonitrile
t-Butyloxycarbonyl
Dichloromethane
N,N′-Diisopropylcarbodiimide
DIPEA N,N-Diisopropylamine
DOOA 3,6-Dioxaoctane-1,8-diamine
EDCI
EDT
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
1,2-Ethanedithiol
Fmoc 9-Fluorenylmethoxycarbonyl
HOBt 1-Hydroxybenzotriazole
TFA
Trifluoroacetic acid.
Acknowledgements
This work was supported by SFB 796 (Project A5) from the
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