High-affinity recognition of the human C-reactive protein independent of phosphocholine

Article abstract of DOI:10.1039/c7ob00684e

A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most frequently quantified targets in diagnostic applications and a target in drug development. With the exception of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine that depends on Ca²⁺ for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in the absence of Ca²⁺ with a dissociation constant of 760 nM, an order of magnitude lower than that of phosphocholine, the K_D of which is 5 μM. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) was designed based on the structural similarities between three hits from a set of compounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magnitude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract CRP from human serum. The development of a robust and phosphocholine-independent recognition element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under conditions where the concentration of Ca²⁺ ions is low, or where Ca²⁺ binding agents such as EDTA or heparin are needed to prevent blood coagulation. The identification from a compound library of a small organic molecule and its conjugation to a small set of polypeptides, none of which were previously known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for proteins with dissociation constants in the μM to nM range for which no small molecule ligands are known.

Full text of DOI:10.1039/c7ob00684e

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High-affinity recognition of the human C-reactive
protein independent of phosphocholine†
Cite this: DOI: 10.1039/c7ob00684e
Jie Yang,a Anna-Lena Gustavsson,b Martin Haraldsson,b Göran Karlsson,c
a
Thomas Norberg a and Lars Baltzer
*
A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition
of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most fre-
quently quantified targets in diagnostic applications and a target in drug development. With the exception
of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine
that depends on Ca2+ for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in
the absence of Ca2+ with a dissociation constant of 760 nM, an order of magnitude lower than that of
phosphocholine, the KD of which is 5 μM. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-car-
boxylic acid (DQ) was designed based on the structural similarities between three hits from a set of com-
pounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR
spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magni-
tude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract
CRP from human serum. The development of a robust and phosphocholine-independent recognition
element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under con-
ditions where the concentration of Ca2+ ions is low, or where Ca2+ binding agents such as EDTA or
heparin are needed to prevent blood coagulation. The identification from a compound library of a small
organic molecule and its conjugation to a small set of polypeptides, none of which were previously
known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for pro-
teins with dissociation constants in the μM to nM range for which no small molecule ligands are known.
Received 20th March 2017,
Accepted 5th May 2017
DOI: 10.1039/c7ob00684e
rsc.li/obc
able general inflammation biomarker and the measurement of
the so called high CRP is predictive of the risk of cardiac
Introduction
Human CRP is an acute phase protein present at high concen- infarction.3 The protein has a molecular weight of 123 kDa
tration in serum, and its circulating concentration is increased and contains five identical subunits that interact non-
dramatically at the early stages of inflammation.1 It is one of covalently to form a symmetric pentamer. Each subunit binds
the most frequently measured biomarkers measured in the phosphocholine (PCh) with 5 μM affinity4 in the PCh binding
clinic and it is considered to play a critical role in the develop- site in the presence of Ca2+. Most recognition elements with
ment of tissue damage in the inflammation process. CRP the exception of antibodies depend on the PCh group for their
recognizes the damaged tissue or the phosphocholine (PCh) affinities but the requirement for Ca2+ is not ideal in combi-
rich bacterial cell membranes, and the related complexes acti- nation with the requirement in medical devices, for example,
vate the complement system.1 While the biological function of for anticoagulants such as EDTA or heparin, as these, too, bind
CRP remains under debate,2 it is considered to be a very valu- Ca2+. Clinical imaging of CRP could provide important insights
into the inflammatory process, but the concentration of Ca2+ is
often low in compartments where CRP is activated, severely
limiting the usefulness of PCh based tracers. In addition, the
robustness of non-proteinogenic constructs is attractive, not
least in combination with test formats that would allow point of
care or home testing. The access to robust binder molecules
not based on PCh could thus enable the use of hitherto unex-
plored test formats, for example, in clinical imaging and in the
veterinary or home testing fields of application, where storage
under controlled conditions would not be available.
aDepartment of Chemistry-BMC, Uppsala University, Uppsala, Sweden.
E-mail: Lars.Baltzer@kemi.uu.se
bChemical Biology Consortium Sweden (CBCS), Science for Life Laboratory,
Department of Medical Biochemistry and Biophysics, Karolinska Institutet,
Stockholm, Sweden
cThe Swedish NMR Center at Göteborg University, Göteborg, Sweden
†Electronic supplementary information (ESI) available: 1H and 13C NMR spectra,
mass spectra (HRMS & ESI MS), MALDI-TOF MS for peptide conjugates, SPR sen-
sorgrams from competition experiments. See DOI: 10.1039/c7ob00684e
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Previously we reported a strategy for the development of tion into helix-loop-helix motifs that dimerize to form four-
polypeptide conjugates with high affinity and selectivity for helix bundles at μM concentrations7,8 whereas at nM concen-
proteins.5 Applied to the recognition of CRP we took advantage trations the dimers dissociated to form unordered monomers.
of the known ligand PCh and linked it covalently to a designed The structure of the polypeptide fragment of the polypeptide
sixteen-membered set of polypeptides.5,6 From the corres- conjugate complexed to proteins remains elusive, but the flex-
ponding conjugates one was identified that bound CRP with ible nature of the polypeptide in solution suggest that it binds
an affinity and selectivity that compared well to antibodies. to the protein surface in each case forming a conformation
The sixteen-membered set of 42-residue sequences was adapted to the structure of the protein.9 The main advantage
designed for conjugation to small organic molecules or pep- of polypeptide conjugation is that affinity and selectivity is
tides in order to provide polypeptide conjugates with enhanced by several orders of magnitude in a single conju-
enhanced affinities and selectivities compared to the small gation step, thus allowing for evaluation of small organic com-
molecule ligand due to interactions outside of, but in close pounds and peptides in biomedical applications at an early
proximity to, the binding site of the small molecule, Fig. 1. stage of development. The affinities of the polypeptides for
The set was based on two variables, charge and site of in- target proteins are low and enhance the affinities for reasons
corporation of the ligand. Reported results show that the of entropy, being covalently linked to a small molecule that
conjugation of the small molecule ligand at different sites of binds with measurable affinity.5,6
incorporation as well as charge variations give rise to an array
In view of the importance of CRP as a biomarker and the
of affinities for the target protein.5 Following this strategy poly- limitations imposed by Ca2+ dependent interactions of small
peptide conjugates were developed for a number of proteins molecule binder molecules based on PCh, the development of
using the same 16-membered set of polypeptides. The a non-PCh dependent polypeptide conjugate appears timely.
sequences were designed to have some propensity for the for- However, while previous polypeptide conjugates depended on
mation of amphiphilic helices and were shown to fold in solu- the availability of small molecule ligands no small molecule
ligand for CRP other than PCh was known to us.3,10 The identi-
fication of small molecule ligands from compound libraries
through high through-put screening campaigns based on
NMR spectroscopy is by now a well established procedure.
However, small molecules identified from such libraries typi-
cally bind to traditional drug targets including enzyme active
sites, ion channels and other receptors.11 We therefore set out
to explore the possibility of identifying small organic mole-
cules with some affinity and selectivity for CRP, targeting
shallow depressions or crevices on protein surfaces rather than
pockets. If successful, such an approach would considerably
broaden the applicability of the polypeptide conjugation
technology. Here we wish to report on a polypeptide conjugate
that binds CRP with nM affinity and is independent of PCh
using the set of polypeptides described above and a small
molecule ligand designed based on a fragment based NMR
screening experiment.
Results
Small molecule design and synthesis
The development of a polypeptide conjugate for the reco-
gnition and binding of CRP required the availability of a small
organic molecule with at least modest affinity for the protein
and with a structure that would allow conjugation to poly-
peptides with relative ease. From the building block collection at
Fig. 1 Illustration of the concept. A. The crystal structure of CRP (pdb
the Chemical Biology Consortium Sweden, used for drug devel-
1b09) complexed to phosphocholine reveals a symmetric pentameric
structure where each subunit binds phosphocholine in the presence of opment purposes, 18 fragments were picked based on struc-
Ca2+ ions. B. The identification of a small molecule ligand that does not
ture and solubility. The compounds were selected based on
the criteria to include 5,6- or 6,6-fused bi-cyclic ring systems
with heteroatoms and contain at least two functionalities as
depend on Ca2+ ions for its ability to bind CRP allows the development
of a polypeptide conjugate, where the interactions between the protein
and the small molecule are supplemented by peptide–protein
hydrogen accepting or donation groups, and selected after
manual inspection. The crystal structure of CRP does not
interactions. C. The small molecule developed here is conjugated to the
side chain of a lysin residue via an aliphatic spacer.
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suggest the availability of binding pockets but only, at best,
crevices or shallow surface depressions.
The selected candidates were bicyclic because small organic
molecules capable of binding to crevices on protein surfaces
could be assumed to be aromatic and bicyclic. They were also
required to be soluble enough to allow measurements by NMR
spectroscopy at μM concentrations in aqueous solution, Fig. 2.
The compounds were screened for binding to CRP using
the waterLOGSY experiment.12,13 For waterLOGSY NMR spectra
see ESI.†
Three hits were found, 1, 2 and 3, that showed a similar
spatial arrangement with two carbonyl groups flanking an
aromatic cyclic nitrogen atom in a bicyclic aromatic scaffold.
The other bicyclic aromatic compounds from the selected set
of 18 did not bind. Due to the structural similarities between
the three compounds we hypothesized that the common
structural features of the three hits were largely responsible
for the binding interactions with the CRP protein and that
they could serve as the starting point for a the design of the
small molecule ligand. The 1,2-dihydroquinoline-scaffold, 4,
was selected because it was bicyclic and aromatic and based
on the expectation that the introduction of a spacer should
not offer severe synthetic complications. Further credibility to
the selection of scaffold was obtained from the spatial
overlap between the three hits and 4 as shown in Fig. 3. An
aliphatic linker with a terminal carboxylic acid group was
incorporated in the position para- to the cyclic nitrogen in
the target molecule 5. In this spatial arrangement, the linker
was assumed not to interrupt the potential H-bond inter-
actions between the key functional groups of 5 and the
protein. Therefore, the overall binding capacity of 5 was
hypothesized to be approximately the same as that of 4. The
pentafluorophenyl ester group was chosen to allow conju-
gation of 5 to a lysine side chain of the peptide. An eight-
carbon linker was considered sufficient to allow the small
molecule ligand and the polypeptide to bind their respective
binding sites simultaneously.5 In the synthesis of 5,
Scheme 1, the zinc reagent 10 to be reacted with 8 was pre-
pared from 8-bromooctanoic acid by conversion into the
methyl ester 9 (94.5% yield), followed by treatment with LiCl
and zinc dust in THF under N2. The zinc reagent 10 was too
reactive to be characterized by TLC or 1H NMR, and was
therefore directly used, without any purification or quantifi-
cation in a Negishi-type coupling with 8 to produce 11 (98.0%
yield). Cyclization by treatment with sulfuric acid produced
monoester 13 (12.0%). The reason for the low yield is
unclear, and it is noticeable that the monoester (negative ion
Fig. 2 Subset of 18 compounds from the building block collection
used for drug development, selected based on structure and solubility
and screened by NMR spectroscopy for affinity for the C-reactive
protein. (A) Three hits, 1, 2 and 3, were obtained with structural simi-
larities, all three were bicyclic compounds where a cyclic nitrogen was
1
ES-MS m/z = 344.2, only one methyl signal in H NMR), was
the only observable product, not the expected diester. The
most likely reason for this is that partial saponification
occurred during the workup procedure. The location of the flanked by two carbonyl groups. (B) Based on these three hits 4 was
designed and synthesized. In order to allow conjugation to polypeptides
a linker was introduced with a terminal pentafluorophenyl ester, to
form 5.
ester function on the aromatic ring was evidenced by the
presence of an aromatic ester methyl group singlet
(3.99 ppm) and the absence of a 3.66 ppm aliphatic methyl
group singlet. Partial esterification (pentafluorophenol/pyri-
dine/DIC) of diacid 14 gave 5 as the only monoester product
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Polypeptide conjugate synthesis
From the sixteen-membered set of designed polypeptides four
were selected based on the results from the previous investi-
gation,6 where the most positively charged polypeptides gave
the highest affinities, Fig. 5. The theoretical PI value of CRP is
5.45 and the previous evaluation demonstrated that at physio-
logical pH the electrostatic interactions between CRP and poly-
peptide scaffold improved the overall affinity. Even though it
was possible, and even likely, that 2-oxo-1,2-dihydroquinoline-
8-carboxylic acid 4 (DQ) would bind at a different site than
PCh, and therefore that the electrostatic potential at that site
could be different from the one surrounding the PCh binding
site, it was decided to evaluate peptides from the library with
overall positive charge first. The polypeptides were synthesized
on the solid phase using an automated peptide synthesizer,
following standard Fmoc chemistry protocols. Before cleavage
of the peptides from the solid support, modifications were per-
formed including acetylation of the N-terminal amino group
and deprotection of the alloc-protected lysine side chain fol-
lowed by incorporation of 7-methoxycoumarin-3-carboxylic
acid, included for identification and titration purposes. The
remaining protected amino acid residues, including a Boc-
protected lysine, were deprotected during cleavage in TFA solu-
tion. The crude peptides were purified by HPLC and identified
by MALDI-TOF mass spectrometry. The active ester 5 was
reacted with the purified peptide in DMSO solution at the only
available free lysine side chain in the sequence. Precipitation
of the crude polypeptide conjugates from DMSO solution gave
the desired products in approximately 50% yields, after purifi-
Fig. 3 Structural analysis of the three hit compounds 1, 2 and 3 shows
similarities in the orientation of functional groups in the bicyclic scaffold
(left). Also shown is the structure of the designed small molecule ligand
4 (right).
(38.0% yield from 13). The structure of monoester 5 was
determined by comparing 5 with 13 and a model compound
pentafluorophenyl 8-bromo-octanoate, Fig. 4. The location of
the carboxyl function on the aromatic ring was shown by the
very small chemical shift difference between the benzylic
methylene protons of compounds 13 and 5 (2.68 and
2.74 ppm), whereas the methylene protons adjacent to the ali-
phatic carboxylic acid showed the expected substantial shift
difference (2.35 to 2.66 ppm). The model compound penta-
fluorophenyl 8-bromo-octanoate showed a similar change in
chemical shift for these protons, when compared to the
corresponding acid (AIST spectra database). The synthesis of
molecule 4, Scheme 2, followed the same synthetic routes as
that of 5, but concentrated sulfuric acid was replaced by con-
centrated aqueous hydrochloric acid for the 15 to 16 cycliza-
tion step (40% yield).
Scheme 1 Synthesis route to active ester 5. Reagents and conditions: a, ice bath for 2 hours, then r.t. overnight, evaporated oxalyl chloride excess
then residue was refluxed at 120 °C for 30 min; b, thionyl chloride, DCM, then evaporated, DCM and filled with MeOH as solvent, r.t. overnight; c,
NBS, DCM, reflux 2 h; d, thionyl chloride, DCM, then evaporated DCM and filled with MeOH as solvent, r.t. for 1 h; e, Zn, anhydrous LiCl, tertbutyl-
silyl chloride, 1,2-dibromoethane,anhydours THF, 50 °C overnight; f, 8, 3% mol Pd(oAc)2, 6% mol S-phos, anhydrous THF; g, 6, pyridine, DCM; h, 95%
H2SO4, r.t. overnight; i, tert-ButOH : H2O 1 : 1, reflux 1 h; j, DCI, pentafluorophenol, pyridine, r.t. overnight.
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cation by RP-HPLC. The identities of the purified polypeptides
and polypeptide conjugates were confirmed by MALDI-TOF
mass spectrometry.
Measurements of affinity
A
preliminary evaluation of binder affinity was carried
out using a three-step fluorescence titration assay, Fig. 6.
The environmentally sensitive fluorescent probe 7-methoxy-
coumarin was incorporated in each polypeptide at a lysine
residue side chain, in close proximity to the small organic mole-
cule. The fluorescence emission intensity of the fluorophore of
the polypeptide conjugate complexed to CRP was expected to be
different from that of the free peptide in buffer solution. The
Fig. 4 Expansions of 1H NMR spectra showing chemical shifts of complex formation was monitored by measuring the change in
methylene protons of the model compound (pentafluorophenyl
fluorescence emission intensity as a function of protein con-
8-bromo-octanoate) (top spectrum), 13 (middle spectrum) and
5
centration. It should be noted that in principle, the dilution of
polypeptide conjugates can give rise to changes in fluorescence
emission intensity without interactions with proteins, because
(bottom spectrum) used for spectral assignment and structural determi-
nation. Pentafluorophenol esterifiction shifted the methylene protons
adjacent to the aliphatic acid downfield from 2.35 ppm to 2.66 ppm (in
parallel with the chemical shift of model compound) whereas the of the monomer-dimer equilibrium. For this reason the fluo-
benzylic protons of 5 were only slightly shifted from those of the methyl
ester 13 (2.76 ppm to 2.68 ppm).
rescence titration experiment is only used in preliminary evalu-
ations, to be supplemented by e.g. SPR measurements. To
perform the fluorescence assay, 500 nM of each polypeptide
conjugate was incubated with 500 nM, 1000 nM and 1500 nM
CRP in the wells of a microtitre plate. The coumarin group was
excited at 350 nm, and the emission intensity of the fluo-
rescent light was measured in the range of 380–500 nm. The
expected behavior of a high affinity binder would be to show a
change in emission intensity upon addition of 500 nM of CRP,
but to show no further change upon further addition of
protein. In contrast, a less strong interaction would be
expected to result in a change in emission intensity upon
addition of 500 nM CRP, and further increases in intensity
upon further additions of protein. Whether the addition of
CRP increases or decreases the emission intensity is not
Scheme 2 Synthesis route of 4. Reagents and conditions: k, pyridine,
DCM, over night; l, 37% HCl, r.t. overnight; m, 10% NaOH, r.t., 2 h.
Fig. 6 Screening of polypeptide conjugates for affinity towards CRP
using fluorescence titration. The concentration of conjugates was 500
Fig. 5 Polypeptide sequences selected to form polypeptide conjugates.
DQ was covalently linked to the side chains of Lys residues shown in
boldface. Coumarin was linked to the side chains of Lys residues shown
underscored. After peptide cleavage, Cys-24 remained Acm protected,
Lys-41 remained TFA protected and the amino terminal was acetylated
in all sequences effectively preventing conjugation to the wrong amino
acid side chain.
nM and the concentration of CRP was 0, 500 nM, 1000 nM and 1500
nM. Titration of 4-C25L22-DQ shows the behaviour of strong binding. In
comparison to the fluorescence emission of free polypeptide conjugate,
the lowest trace, the addition of CRP increased the intensity, and further
additions of CRP did not increase intensity further. In contrast, the emis-
sion intensity of 4-C37L34-DQ increases gradually with increased con-
centration of CRP, indicative of weak binding.
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related to affinity, only the change in intensity as a function of
protein concentration.
Two polypeptide conjugates, 4-C15L8-DQ and 4-C25L22-DQ
showed the behaviour expected for strong binders, Fig. 6, with
4-C25L22-DQ qualitatively the stronger of the two. Both
4-C10L17-DQ and 4-C37L34-DQ showed the behaviour
expected from more weakly binding molecules. The crystal
structure of CRP reveals that it is a non-covalently associated
symmetric pentamer, where each subunit binds phosphocho-
line. Therefore we assumed that each CRP molecule binds five
polypeptide conjugates. A lower limit to the apparent affinity
for CRP of 4-C25L22-DQ and of 4-C15L8-DQ can be estimated
based on the equation defining the dissociation constant, KD
=
[A][B]/[AB], where [A], the concentration of free peptide is [A]0
− [AB], and [B], the concentration of free protein, is [B]0 − [AB].
[AB] is the concentration of the peptide–protein complex.
When 500 nM of CRP was added to 500 nM peptide, in the
case of 4-C15L8-DQ and 4-C25L22-DQ, the peptide was found
to be essentially completely bound to CRP. Assuming an esti-
mated 10% margin of error ([A]0 − [AB]) equals 50 nM, [B]0 −
[AB] equals 2050 nM and [AB] equals 450 nM. Integrated in the
equation above, the apparent dissociation constant of
4-C25L22-DQ and 4-C15L8-DQ is estimated not to be higher
than 225 nM, although it may be lower. Assuming an error
limit of 20% would give an estimated upper limit to the
affinity of 525 nM. The affinity of 4-C25L22-DQ therefore ful-
filled the criteria for a high-affinity binder for CRP and no
further polypeptides were evaluated by SPR as DQ conjugates.
Based on the preliminary evaluation of affinity and an esti-
mated dissociation constant in the range from 200–500 nM
4-C25L22-DQ was further evaluated by SPR analysis. The
experiments were performed according to standard protocols
provided by the manufacturer with CRP immobilized on a
CM-5 chip and a series of concentrations of 4-C25L22-DQ in
running buffer used to measure the binding kinetics in real
time. In order to monitor the Ca2+ dependence two sets of
experiments were performed and an equation describing a
1 : 1 binding model was fitted to the experimental data.
BIAevaluation 4.1 (GE Healthcare Biosciences) was used to
Fig. 7 SPR biosensor analysis of interactions between immobilized CRP
and DQ, and between immobilized CRP and 4-C25L22-DQ in the pres-
ence and absence of 10 mM CaCl2. Running buffer was 3% DMSO in
HBS-P at pH 7.4. (A) DQ in the range of 64 nM–1 mM and 10 mM CaCl2,
(B) 4-C25L22-DQ in the range of 7 nM–30 μM* and 10 mM CaCl2, (C)
DQ in the range of 64 nM–1 mM, (D) 4-C25L22-DQ in the range of 5.85
nM–3 μM*. Panels E and F show fits of an equation describing a 1 : 1
equilibrium to the maximum response as a function of the concentration
of 4-C25L22-DQ with and without CaCl2 in the running buffer, (E)
4-C25L22-DQ and 10 mM CaCl2, (F) 4-C25L22-DQ. *: The sensorgrams
B/D display the same concentration range as the panels E/F.
estimate that the affinity of 4-C25L22-DQ is three orders of
magnitude higher than that of DQ.
DQ competition assay
determine the KD values from the recorded sensorgrams, for In order to further evaluate the affinity increase resulting from
the method of analysis see ESI.† In the absence of Ca2+ polypeptide conjugation and also to establish that 4-C25L22-
4-C25L22-DQ was found to have a KD of 760 198 nM, approxi- DQ binds selectively to the binding site of DQ, a competition
mately the same as that in the presence of 10 mM Ca2+, which experiment was set up. CRP was immobilized on the CM-5
was measured to be 370 160 nM. The small effect of the Ca2+ chip and pre-incubated with 0.5 μM 4-C25L22-DQ with DQ in
ions suggested that 4-C25L22-DQ binds to CRP independently the running buffer, Fig. 8. The responses recorded 5 s after
of Ca2+. The slight apparent loss of affinity may be due to a injection were normalized by setting that of 0.5 μM of
Ca2+ induced conformational change in CRP affecting the 4-C25L22-DQ without added DQ to 100. The responses were
interactions of 4-C25L22-DQ with the protein or to a salt effect. plotted versus the concentration of DQ.
To provide further understanding of the interactions between
The results show that DQ inhibits the formation of the
4-C25L22-DQ and CRP, the interactions between the small CRP-4-C25L22-DQ complex and therefore that 4-C25L22-DQ
molecule DQ and CRP were studied by SPR, in the presence as binds to the same binding site as DQ with DQ as the anchor.
well as absence of Ca2+ ions, Fig. 7. Unfortunately, the weak While this is what would be expected, it excludes the possi-
binding and the poor response made quantitative analysis bility that the polypeptide dominates binding interactions and
difficult. An inspection of the sensorgrams revealed that con- shows that the polypeptide simply enhances the DQ affinity.
centrations higher than 1 mM were required to allow obser- The affinities of the polypeptides have been shown a number
vation of CRP–DQ complex formation in agreement with an of times to be immeasurably small and thus orders of magni-
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polyethylene glycol and polypropylene glycol.6 Coating with
Pluronics F108-PDS prevents nonspecific protein adsorption to
the hydrophobic bead surface and provides a reactive group
for the coupling of molecules equipped with free SH groups.
The beads were incubated overnight with Pluronics F108-PDS
and then functionalized with 4-C25L22-DQ. After washing to
remove unreacted polypeptide conjugate it was incubated in
human CRP-free serum spiked with CRP to a total concen-
tration of 10 mg L−1. The concentration was chosen because it
is the upper limit of CRP in healthy individuals, whereas infec-
tions increase the levels quite significantly to as much as
several hundred mg L−1 in the case of exposure to bacterial
agents.3 After several washes, 4-C25L22-DQ and the captured
proteins were released by reductive cleavage and analyzed by
SDS-PAGE, Fig. 9. Spiked human CRP-free serum and human
CRP-free serum were exposed to the functionalized beads. The
extracts were compared to neat human CRP and as the nega-
tive control extracts from beads that had not been functiona-
lized were used. The apparent molecular weight of neat CRP
was compatible with that of the CRP monomer, which is not
surprising in view of the fact that SDS-PAGE is run under dena-
turing conditions. The developed gel shows that CRP was
extracted from spiked human serum, while no CRP or any
protein with similar molecular weight was extracted from the
CRP-free serum nor was any band with comparable molecular
weight extracted non-specifically by the unfunctionalized
beads. The results show that the small organic molecule DQ,
obtained from a compound library and conjugated to a poly-
peptide from the designed set can bring about the selective
recognition of a native protein and extract it from the complex
mixture of proteins that make up human serum. In contrast,
the peptide 4-C15L8-DQ, which appeared to have an affinity
for CRP comparable to that of 4-C25L22-DQ based on fluo-
rescence titration, did not extract CRP from the same serum.
The polypeptide conjugate 4-C25L22-DQ thus shows a high
level of selectivity for CRP apparently emanating from the
combined interactions of DQ and the polypeptide 4-C25L22
with CRP.
Fig. 8 SPR biosensor analysis showing inhibition of the CRP
–
4-C25L22-DQ complex formation by DQ. CRP was immobilized on chip
and 4-C25L22-DQ was incubated with DQ in HBS-P running buffer, pH
7.4, prior to injection. Maximum response values are plotted versus DQ
concentration. RUs are normalized to
4-C25L22-DQ.
a level of 100 for 0.5 μM
tude weaker than those of the small organic molecule
ligands.5,6 The interaction with CRP is not non-specific in the
sense that it sticks in many positions to the protein. Although
complete inhibition is not observed due to limiting DQ solubi-
lity that prevents measurements at mM DQ concentrations, the
amount of complex formed is clearly and gradually reduced.
An estimate of the DQ inhibitory capacity can be made from
the concentration of DQ that inhibits complex formation to
around 50% at which concentration the amounts of DQ and
4-C25L22-DQ in complex with CRP are approximately the
same. The concentration ratio is then related to the ratio of
dissociation constants. At 512 μM, the highest concentration
of added DQ, with a level of inhibition that was less than 50%
the ratio of concentrations was three orders of magnitude
since the concentration of 4-C25L22-DQ was 0.5 μM. An even
higher ratio would be obtained if the concentration of DQ
required to achieve 50% inhibition would be estimated from
the extrapolation to low mM concentrations as suggested from
an inspection of the plot. The ratio is clearly an approximation,
partly because the concentrations of free DQ and free
4-C25L22-DQ are not known but the results strongly suggest
that three orders of magnitude of affinity enhancement was
obtained from the extra binding interactions between poly-
peptide scaffold and CRP.
Selectivity in human serum
PCh is known to bind CRP and the corresponding polypeptide
conjugate was expected to bind CRP with a high degree of
selectivity.6 In contrast, DQ was obtained from a compound
library and the level of selectivity of 4-C25L22-DQ towards CRP
was unknown and could not be predicted. It was investigated
by a pull-down experiment extracting CRP from human serum Fig. 9 SDS-PAGE analysis of proteins extracted in pull-down experi-
ment. Lane 1, 4-C25L22-DQ extract from CRP-free human serum. Lane
that contains thousands of proteins over a wide range of con-
centrations. The side chain of Cys-24 was deprotected and
reacted with the activated pyridyl disulfide group of Pluronics
F108-PDS (Allvivo Inc) immobilized on polystyrene beads.
2, 4-C25L22-DQ extract from CRP-free human serum spiked with CRP
to a concentration of 10 mg L−1. Lane 3, 4-C25L22-DQ extract from
HEPES buffer (5 mM Ca2+). Lane 4, human CRP. Lane 5, extract from
human serum spiked with CRP to 10 mg L−1 concentration by unfunc-
Pluronics is a functionalized block copolymer formed from tionalized beads.
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sive pharmaceutical agents that are essentially limited to extra-
cellular targets. Antibodies remain the golden standard of
in vitro diagnostics although important future developments
such as home testing require higher stability. Recombinant
proteins are competing with antibodies for the same targets in
biomedicine but have not yet reached the market to a great
extent due to limited selectivity and rapid renal clearance.
They remain too expensive for the in vitro diagnostic market
but may have found a niche in clinical diagnostics. Small
molecule drugs remain the preferred choice for a number of
indications where patients do not accept anything but oral
administration, but the success rate in small molecule drug
development is very low. There is not a single technology that
addresses all problems and new approaches are clearly of con-
siderable interest. We have previously reported on a technology
where a small set of polypeptides can be used to identify
sequences that upon covalent conjugation to these poly-
peptides enhance affinities of small molecule ligands by as much
as 3–4 orders of magnitude and selectivities between highly
homologous proteins by as much as 1–2 orders of magnitude.
The attraction of such a technology is partly that it is based on
synthesis and that batch to batch variation is neglible, partly
that it is robust and partly that one can save small organic
molecules and peptides that did not bind well enough. Until
now, however, addressing a protein for which no small mole-
cule ligand is known had not been attempted. Furthermore,
the need for binder molecules seem to be the most acute for
proteins that do not expose well-defined binding pockets or
protruding epitopes, but only shallow crevices or depressions
on the protein surface. The discovery that a bicyclic aromatic
scaffold decorated to achieve selectivity opened a route to the
recognition and binding of a protein devoid of strong struc-
tural characteristics provided some optimism for the reco-
gnition, e.g. of protein–protein interfaces and proteins with
few structural features.
CRP is an important protein in biomedicine and although
it is being measured many times every day, worldwide in hos-
pital laboratories at low cost, applications remain that are
unsuitable for hospital execution. Home testing would require
a different level of stability, because antibodies and for that
matter proteins require storage at 4 °C in order to provide
reliable data. In contrast, synthetic peptides do not denature
and are not rapidly degraded if not stored under controlled
conditions. Clinical diagnostics, i.e. clinical imaging, of CRP
would be of considerable interest, but requires a binder
technology not based on PCh, because the concentration of
free Ca2+ is not high enough in for example lung fluid or in
the small coronary arteries to allow imaging of the inflamma-
tory process as a result of lung inflammation or cardiac infarc-
tion. The latter is of considerable interest as there are reports
to suggest that the removal of CRP from blood reduces tissue
damage immediately after the onset of the infarction.15
Fig. 10 SPR biosensor analysis showing inhibition of CRP – 4-C25L22-
DQ complex formation by PCh. CRP was immobilized on chip and
4-C25L22-DQ was incubated with PCh in HBS-P running buffer, pH 7.4,
prior to injection. Maximum response values are plotted versus PCh con-
centration. RUs are normalized to a level of 100 for 0.5 μM 4-C25L22-DQ.
PCh competition assay
DQ does not mimic the structural features of PCh, except that
DQ has a negatively charged carboxylate group and hydrophobic
components whereas PCh has a negatively charged phosphate
group and a hydrophobic aliphatic chain. DQ was not expected
to bind to the same binding site as PCh because it binds also in
the absence of the Ca2+ ions required for PCh binding. In the
PCh–CRP complex the Ca2+ ions bridge the negatively charged
phosphate group and CRP carboxylate groups.14 We neverthe-
less wanted to establish unequivocally that DQ and PCh bind to
different sites on the CRP protein surface. A competition experi-
ment was set up where PCh was allowed to vary in the concen-
tration range from 0.75 μM to 50 mM at a concentration of
4-C25L22-DQ of 0.5 μM, Fig. 10. No inhibition was observed at
PCh concentrations as high as 12.5 mM but a sharp decrease in
binding was observed at 50 mM of PCh. The highest ratio of
concentrations at which no inhibition was observed, 25 000, in
combination with a KD of 5 μM for PCh would require a dis-
sociation constant for 4-C25L22-DQ of less than 0.2 nM to
explain the results if PCh is a competitive inhibitor of 4-C25L22-
DQ. A sub-nM KD for the CRP-4-C25L22-DQ complex is not in
agreement with the results from neither fluorescence titration
nor SPR analysis. We conclude that PCh and 4-C25L22-DQ do
not bind in the same binding site.4 An alternative explanation
for the observed results could be that the high concentration of
the PCh gives rise to a salt effect on CRP conformation, thus
affecting the affinity for 4-C25L22-DQ, or that aggregation
occurs. Based on the results we find it more likely that a pre-
viously unknown binding site on CRP has been discovered that
allows the binding of 4-C25L22-DQ with high nM affinity.
The availability of 4-C25L22-DQ further expands the useful-
ness of the polypeptide conjugate 4-C15L8-PC, based on PCh,
Discussion
Binder technologies for proteins remain crucial in biomedical as they form a synthetic sandwich, a common requirement for
applications. Antibodies are emerging as important but expen- in vitro bioanalytical applications. The competition experiment
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showed that they bind to different epitopes and therefore that added, and the flask was dried again with the heat gun under
they can be used to enhance the recognition in applications high vacuum. Anhydrous THF (20 mL) was added and the
where both capture and recognition are required.
mixture was activated: 1,2-dibromoethane and trimethylsilyl
chloride. The 1,2-dibromoethane (69 µL) was added first, then
the flask was heated until foaming, this procedure was repeated
one more time, then trimethylsilyl chloride (18.9 µL) was added
and the mixture was stirred for 10 minutes. After that, 9 (3.54 g,
15 mmol) and KI (2.49 g, 15 mmol) was sequentially added.
The flask was evacuated and refilled with nitrogen gas, then the
reaction mixture was stirred at 50 °C overnight. The obtained
solution of 10 was directly used in the synthesis of 11.
Conclusion
The identification of a small molecule ligand that recognizes
CRP in spite of the fact that no pockets or deep crevices are
found on the protein surface has enabled the development of
a polypeptide conjugate with nM affinity. The conjugate bound
CRP with an affinity that was three orders of magnitude higher
than that of the small molecule, at a different site from that of
PCh. The polypeptide conjugate technology thus increases the
group of tools available for the recognition and binding of pro-
teins, for which no small molecule ligands exist, and that
requires synthetic, robust high-affinity recognition elements.
Methyl 2-amino-5-(7′-methoxycarbonyheptyl)-benzoate (11).17
8 (1.8 g, 7.89 mmol), Pd(OAc)2 (53 mg, 3.0%) and S-phos
(194 mg, 6.0%) were dissolved in anhydrous THF (10 mL). The
reaction mixture was stirred for 10 minutes, then the solution
of 10 was dropwise added and stirring was continued at room
temperature for 48 hours. TLC (9 : 1 toluene/EA). After work-up
and purification, pure 11 formed (2.38 g, 98%). 1H NMR
(400 MHz): 7.66 (1H, s), 7.11 (1H, d, 8.3 Hz), 6.67 (1H, d,
8.3 Hz), 3.87 (3H, s), 3.66 (3H, s), 2.48 (2H, t, 7.8 Hz), 2.29 (2H,
t, 7.5 Hz), 1.69–1.50 (4H, m), 1.36–1.23 (6H, m). 13C NMR
(101 MHz): 174.27, 168.53, 147.44, 134.56, 131.29, 130.30,
117.26, 111.21, 51.53, 51.43, 34.83, 34.07, 31.50, 29.08, 29.05,
28.94 24.94. HRMS calcd for C17H25NNaO4 330.1676, found
330.1679 [M + Na]+.
Experimental
General methods
All reagents were purchased from commercial sources and
used without further purification unless otherwise noted.
Concentrations during small molecule synthesis were per-
formed at reduced pressure with a rotatory evaporator. Crude
peptides and peptide conjugates were purified by HPLC on a
reverse phase C-8 semi-preparative column. Gradient and
method: acetonitrile/water (30%–60%) in 30 minutes, with
0.1% TFA added, UV detection at 210 nm. The HPLC fractions
were analyzed off-line by MALDI-TOF MS using α-cyano-4-
hydroxycinnamic acid as matrix. In small molecule organic
synthesis, reaction completion was ascertained by TLC. Unless
mentioned, the work-up procedure was performed as: the reac-
tion mixture was concentrated then partitioned between two-
phase layers (organic/water). Subsequently the organic layer
was dried with anhydrous sodium sulphate then concentrated.
Purification was performed with silica gel column chromato-
graphy. ESI-MS was recorded using an Agilent 1100/Waters
Micromass ZQ instrument using a 3.0 × 50 mm C18 column
and a gradient elution with 5 : 95% acetonitrile–water (with
0.1% formic acid) to 95 : 5% acetonitrile–water (with 0.1%
formic acid). The mass detector was operated in alternating
positive and negative ion modes. Unless otherwise stated NMR
spectra were recorded at 300 or 400 MHz and 298 K using
CDCl3 as the solvent (internal CHCl3, δH 7.26, δC 77.66, 77.34,
77.02 ppm) and Varian Unity spectrometers.
Methyl
6-(7′-carboxyheptyl)-2-oxo-1,2-dihydroquinoline-8-
carboxylate (13). 12 (1.2 g, 3.5 mmol) was mixed with concen-
trated 95% sulfuric acid (5 ml) and the mixture was stirred
overnight at room temperature. To monitor the progress of the
reaction, an aliquot (10 µL) of the reaction mixture was mixed
with concentrated 6 M NaOH (1 mL), then extracted with EA
(500 µL). The EA layer was analyzed by TLC (20 : 1 DCM/metha-
nol). The reaction mixture was poured onto ice, neutralized
with NaOH pellets, then continued with normal work-up pro-
cedure. After purification (30 : 1 DCM/methanol and several
drops of acetic acid), pure mono-ester 13 formed (145 mg,
12%). 1H NMR (400 MHz): 11.65 (NH), 8.06 (1H, d, 2.0 Hz),
7.70 (1H, d, 9.6 Hz), 7.53 (1H, d, 2.0 Hz), 6.69 (1H, d, 9.6 Hz),
3.99 (3H, s), 2.68 (2H,t,7.7 Hz), 2.35 (2H, t, 7.5 Hz), 1.73–1.57
(4H, m), 1.42–1.30 (6H, m). 13C NMR (101 MHz) 178.60,
167.12, 162.48, 140.23, 137.58, 136.08, 133.56, 133.06, 122.48,
120.70, 113.16, 52.52, 34.96, 33.83, 31.25, 28.99, 28.87, 28.80,
24.60. ESI-MS calcd for C19H23NO5 345.1, found 368.0
[M + Na]+, 344.2 [M − H]−.
2-Oxo-6-(7′-pentafluorophenoxycarbonylheptyl)-1,2-dihydro-
quinoline 8-carboxylic acid (5). 13 (145 mg, 0.413 mmol),
LiOH (19.5 mg, 0.826 mmol) and t-butanol/water (1 : 1, 10 mL)
were mixed in a 25 ml RB flask. The reaction was stirred under
reflux for 2 hours. TLC (20 : 1 DCM/methanol). 1 M HCl was
Small molecule synthesis of 5
Synthetic steps to achieve 6, 7, 8, 9, 12, 15, 16 and 4 were routi- added to adjust the pH to 7, and the mixture was extracted
nely performed, the specific details and spectroscopy infor- with EA. The organic layer was dried with anhydrous sodium
mation were included in the ESI.†
sulphate, and concentrated to give the diacid intermediate 14
1
7-Methoxylcarbonylheptylzinc bromide lithium chloride (137 mg, 100%). A small amount was taken to obtain H NMR
complex (10).16 A flame dried flask was charged with an- spectrum. 1H NMR (400 MHz, DMSO-d6) 11.99 (NH), 8.02 (1H,
hydrous LiCl (870 mg, 21 mmol) and dried with a heat gun d, 2.0 Hz), 7.96 (1H, d, 9.7 Hz), 7.80–7.75 (1H, m), 6.57 (1H,
under high vacuum, then zinc dust (1.335 g, 21 mmol) was dd, 9.7, 2.0 Hz), 2.66 (2H, t, 7.6 Hz), 2.18 (2H, t, 7.4 Hz), 1.64–1.43
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(4H, m), 1.34–1.21 (6H, m). 13C spectrum was not recorded.
HRMS calcd for C18H20NO5 330.1346, found 330.1347 [M − H]−.
SPR. All the experiments were performed with a biacore
2000 instrument at 25 °C. Reagents were purchased either
A 25 ml R.B. flask charged with 14 (137 mg, 0.413 mmol) from GE Healthcare Biacore or from Sigma Aldrich.
and pentafluorophenol (76 mg, 0.413 mmol), was evacuated
CRP (3.0 mg mL−1) preserving buffer was switched to the
and refilled with N2 three times. Anhydrous pyridine (10 mL) coupling buffer. The protein was immobilized on a CM-5 chip
was added. The reaction was stirred for 5 minutes, and DIC according to the standard amide coupling protocol. Final
(51.7 mg, 0.413 mmol) was added dropwise. The reaction response in human CRP immobilized channel was 8051 Ru.
was left stirring overnight at room temperature. TLC (8 : 2 Details were in ESI.†
EA/toluene). After work-up and purification, pure 5 formed
In the interaction studies below, filtration with 0.2 μm PES
(80 mg, 38%). 1H NMR (400 MHz): 11.18 (NH), 8.28 (1H, d, filter was performed when the substances (DMSO, CaCl2) were
2.0 Hz), 7.75 (1H, d, 9.7 Hz), 7.70 (1H, d, 2.0 Hz), 6.73 (1H, d, added to HBS-P buffer. Detection mode was set to 2-1 with 1 as
9.6 Hz), 2.76 (2H, t, 7.8 Hz), 2.66 (2H, t, 7.4 Hz), 1.83–1.66 (4H, reference channel. After dissociation, regeneration was per-
m), 1.47–1.38 (6H, m). 13C NMR (101 MHz) 171.15, 169.48, formed by running buffer clearance for 10 minutes at a flow
162.00, 139.95, 138.67, 136.38, 135.12, 134.04, 123.21, 120.95, rate of 50 μL min−1
109.87, 34.94, 33.25, 31.23, 29.68, 28.91, 28.87, 28.69, 24.65.
.
To perform DQ, 4-C25L22-DQ kinetic assays in 10 mM Ca2+,
HRMS calcd for C24H20F5NNaO5 520.1154, found 520.1167 HBS-P containing 10 mM CaCl2 and 3% DMSO was used as
[M + Na]+.
running buffer.
In DQ assay, flow rate was set to 50 μL min−1. A concen-
tration series of DQ in the range of 64 nM to 1 mM by sequen-
Peptide synthesis
The polypeptides were synthesized on a peptide synthesizer tially five times dilution was injected to surface for 1 minute,
using standard Fmoc chemistry with HCTU and DIPEA as acti- then dissociation was followed by 3 minutes. In 4-C25L22-DQ
vating agents. Fmoc-deprotection of the amino terminal was assay, a concentration series in the range of 7 nM to 30 μM by
performed using 20% piperidine in NMP. The synthesis was sequentially four times dilution was used. Flow rate was set to
performed on a 0.1 mmol scale using Rink-amide resin 30 μL min−1. Injection time: 2 minutes. Dissociation time:
(0.23 mmol g−1). Before cleavage of the peptide, the alloc- 10 minutes.
protected Lys was deprotected by palladium(0). Subsequently
the fluorescent probe 7-methoxycourmarin-3-carboxylic acid was HBS-P containing 3% DMSO was used as running buffer.
coupled to the Lys via amide bond formation. Eventually the In DQ assay, the same procedure was repeated. In
peptide was liberated from resin by TFA and purified by HPLC. 4-C25L22-DQ assay, a concentration series in the range of 5.85
To perform DQ, 4-C25L22-DQ kinetic assays without Ca2+,
(Details are in the ESI.†)
nM to 3 μM by sequentially two times dilution was used. Flow
rate was set to 30 μL min−1. Injection time: 5 minutes.
Dissociation time: 10 minutes.
Conjugation procedure
The ligand 5 was conjugated to the free lysyl amino group of
the peptides by the addition of 2.5 equivalents of 5 in DMSO
DQ competition assay
stock solution (10 mg mL−1) to a solution of the polypeptide The flow rate was set to 15 μL min−1. For competition study, a
in DMSO (1 mM) in the presence of 5 equivalents of DIPEA. DQ concentration series in the range of 0.5 to 512 μM by sequen-
The reaction was monitored by analytical HPLC. The crude tially four times dilution were injected to CRP immobilized chip
conjugated polypeptide was lyophilized and then purified by for 5 minutes, dissociation was then followed by 10 minutes.
preparative HPLC (in yields 30–50%). (The mass spectra Except that 0.5 μM 4-C25L22-DQ was in the samples, both
of ligand 5 conjugated peptides were available in the ESI, running buffer and samples contained 3% DMSO.
S22–S25.†)
PCh competition assay
Affinity measurements
The same procedure in DQ competition was performed using
Fluorescence screening. Titration of binders with CRP (3 mg a PCh concentration series in the range of 0.76 to 50 000 μM
mL−1) (Millipore) was carried out by fluorescence spectroscopy by sequentially four times dilution. Except that 0.5 μM
using a GeminiXPS platereader. BRANDplates®96 pureGrade 4-C25L22-DQ was in the samples, both running buffer and
non-sterile plates were coated with Pluronic® F108NF Prill samples contained 10 mM CaCl2 and 3% DMSO.
poloxamer338 (BASF) before use. Tris Buffer (50 mM, 5 mM
Selectivity
CaCl2, 150 mM NaCl, pH 7.0) was used in the experiment.
In each well, 500 nM peptide was used together with 0, 500, Free SH groups were introduced to polystyrene beads by incu-
1000, 1500 nM of CRP. Buffer was used as blank. The 7-methoxy- bating them in 10 mg mL−1 Pluronic F108-PDS solution. Acm-
coumarin probe of each peptide was excited at 350 nm deprotected peptides were then immobilized on beads via di-
using a high pmt setting and the emission was recorded at sulfide bond formation. Incubating with human serum/
380–500 nm. All measurements were performed at 25 °C and human serum with 10 mg L−1 CRP, the beads and trapped pro-
were made in triplicates. Data was read after 30 minutes incu- teins were spun down to bottom. Following DTT cleavage, pep-
bation. (Details are in ESI.†)
tides and their trapped proteins were liberated and analysed
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by SDS-PAGE. (Preparation of Acm-deprotected peptides and
following details of selectivity study are in ESI.†)
3 G. M. H. M. B. Pepys, J. Clin. Invest., 2003, 111, 1805–1812.
4 T. Christopeit, T. Gossas and U. H. Danielson, Anal.
Biochem., 2009, 391, 39–44.
5 L. Baltzer, Anal. Bioanal. Chem., 2011, 400, 1653–1664.
6 L. T. Tegler, G. Nonglaton, F. Büttner, K. Caldwell,
T. Christopeit, U. H. Danielson, K. Fromell, T. Gossas,
A. Larsson, P. Longati, T. Norberg, R. Ramapanicker,
J. Rydberg and L. Baltzer, Angew. Chem., Int. Ed., 2011, 50,
1823–1827.
Abbreviations
Acm
CRP
DCM
DCI
Acetamidomethyl
Human C-reactive protein
Dichloromethane
N,N-Diisopropylcarbodiimide
7 L. Brive, G. T. Dolphin and L. Baltzer, J. Am. Chem. Soc.,
1997, 119, 8598–8607.
DIPEA Diisopropylethylamine
EA
Ethyl acetate
8 K. P. R. Nilsson, J. Rydberg, L. Baltzer and O. Inganäs, Proc.
Natl. Acad. Sci. U. S. A., 2003, 100, 10170–10174.
9 T. Andersson, M. Lundquist, G. T. Dolphin, K. Enander,
B.-H. Jonsson, J. W. Nilsson and L. Baltzer, Chem. Biol.,
2005, 12, 1245–1252.
10 C. Srikantiah, IJRCI, 2014, 2, 1–24.
11 B. J. Stockman and C. Dalvit, Prog. Nucl. Magn. Reson.
Spectrosc., 2002, 41, 187–231.
MQ
NMP
PCh
PE
Milli-Q
N-Methylpyrrolidone
Phosphocholine
Pentane
SPR
TFA
THF
Surface plasmon resonance
Trifluoroacetic acid
Tetrahydrofuran.
12 C. Dalvit, P. Pevarello, M. Tatò, M. Veronesi, A. Vulpetti and
M. Sundström, J. Biomol. NMR, 2000, 18, 65–68.
13 C. Dalvit, G. Fogliatto, A. Stewart, M. Veronesi and
B. Stockman, J. Biomol. NMR, 2001, 21, 349–359.
14 J. Volanakis, Mol. Immunol., 2001, 38, 189–197.
15 T. Bansal, A. Pandey, D. Deepa and A. K. Asthana, J. Clin.
Diagn. Res., 2014, 8, ZE21–ZE24.
Acknowledgements
Financial support from the Swedish Research Council, grant
number 2014-4057, to L. B. is gratefully acknowledged.
16 A. Krasovskiy, V. Malakhov, A. Gavryushin and P. Knochel,
Angew. Chem., Int. Ed., 2006, 45, 6040–6044.
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