Hybridization of an Aβ-specific antibody fragment with aminopyrazole-based β-sheet ligands displays striking enhancement of target affinity

Article abstract of DOI:10.1039/c4ob02411g

Determining Aβ levels in body fluids remains a powerful tool in the diagnostics of Alzheimer's disease. This report delineates a new supramolecular strategy which increases the affinity of antibodies towards Aβ to make diagnostic procedures more sensitive

Full text of DOI:10.1039/c4ob02411g

Organic &
Biomolecular Chemistry
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PAPER
Hybridization of an Aβ-specific antibody fragment
with aminopyrazole-based β-sheet ligands displays
striking enhancement of target affinity†
Cite this: Org. Biomol. Chem., 2015,
13, 2974
Marco Hellmert,a Andreas Müller-Schiffmann,b Max Sena Peters,a Carsten Korth*b
and Thomas Schrader*a
Determining Aβ levels in body fluids remains a powerful tool in the diagnostics of Alzheimer’s disease.
This report delineates a new supramolecular strategy which increases the affinity of antibodies towards Aβ
to make diagnostic procedures more sensitive. A monoclonal antibody IC16 was generated to an N-term-
inal epitope of Aβ and the variable regions of the heavy and light chains were cloned as a recombinant
protein (scFv). A 6 × histidine tag was fused to the C-terminus of IC16-scFv allowing hybridization with a
small organic β-sheet binder via Ni-NTA complexation. On the other hand, a multivalent nitrilotriacetic
acid (NTA)-equipped trimeric aminopyrazole (AP) derivative was synthesized based on a cyclam platform;
and experimental evidence was obtained for efficient Ni2+-mediated complex formation with the histi-
dine-tagged antibody species. In a proof of principle experiment the hybrid molecule showed a strong
increase in affinity towards Aβ. Thus, the specific binding power of recombinant antibody fragments to
their β-sheet rich targets can be conveniently enhanced by non-covalent hybridization with small organic
β-sheet binders.
Received 15th November 2014,
Accepted 13th January 2015
DOI: 10.1039/c4ob02411g
www.rsc.org/obc
amyloid fibrils. Similarly, the tau protein self-assembles, after
hyperphosphorylation, into highly toxic neurofibrillary
Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative tangles.4 Although other proteins are discussed as the major
disease, that is neuropathologically characterized by extracellu- cause for related protein misfolding diseases, Aβ(1-42) also
lar deposits of amyloid plaques consisting of amyloid-beta (Aβ) seems to play a certain role in Parkinson’s disease and Hun-
peptides and intracellular accumulation of hyperphosphory- tington’s chorea5 as well as in the context of diabetes mellitus
lated microtubule associated protein tau. The aggregation of type 2 and Down syndrome.6,7
Aβ and tau leads to neuronal loss and ultimately to severe
Whereas monomeric Aβ is present at a physiologically
dementia and death. To date no cure is available and recently normal metabolism and supposedly has an antimicrobial
several promising treatment options failed, most likely function,8,9 its oligomeric forms are highly neurotoxic and
because drug administration comes too late. Therefore, early considered one of the major factors in the development of
diagnosis of AD is not only important for medication of initial AD.2 Initially, levels of the aggregation prone Aβ(1-42) peptide
symptoms but will also be required for potential future thera- are elevated either by increased production or decreased clear-
peutic interventions.
ance, which leads to formation of synaptotoxic low-n oligo-
Aβ refers to peptides of 39-43 amino acids emerging from meric Aβ species, like dimers and trimers.
the amyloid precursor protein (APP) through proteolytic clea-
These oligomers adopt a β-sheet-rich tertiary structure and
vage.1 One of these, i.e., Aβ(1-42), plays a central role in the form insoluble fibrillar aggregates, which deposit outside
pathogenesis of Alzheimer’s disease (AD)2,3 since it aggregates neurons as amyloid plaques.10 Increase of β-sheet rich small
into highly neurotoxic oligomers, which ultimately form soluble Aβ oligomers and insoluble Aβ is strongly correlated to
the progress of disease and therefore displays a prime target
for diagnostic agents. However, early detection of low pico-
aInstitute for Organic Chemistry, University of Duisburg-Essen, Universitätsstr. 7,
45117 Essen, Germany. E-mail: thomas.schrader@uni-due.de
molar concentrations of Aβ oligomers in body fluids (blood
serum, cerebrospinal fluid) is challenging, and reaches the
limit of antibody sensitivities. Therefore it is highly desirable
to enhance the affinity of Aβ specific antibodies.
In recent years our group has developed trimeric amino-
pyrazole (AP) based β-sheet ligands by rational design. These
bDepartment of Neuropathology, Heinrich Heine University, Moorenstr. 5, 40225
Düsseldorf, Germany. E-mail: ckorth@uni-duesseldorf.de
†Electronic supplementary information (ESI) available: Copies of the 1H NMR
and 13C NMR spectra for all key intermediates and final products. See DOI:
10.1039/c4ob02411g
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Fig. 1 Hydrogen-bond interactions between the trimeric aminopyra-
zole ligand (DAD)3 with the peptide backbone of a β-strand (ADA)n.
Fig. 2 Schematic representation of the Ni2+-mediated His6-antibody/
NTA-aminopyrazole hybrid targeting two different recognition sites on
the Aβ monomer (aa 1-8 and the KLVFF sequence 16-20).
contain a DAD order of hydrogen-bond donors and acceptors
which perfectly matches the β-sheet structure formed by Aβ
oligomers (Fig. 1). Most likely these agents bind to the KLVFF
sequence of mono- and oligomeric Aβ.11–13
concept may be generally applicable to other proteins involved
in neurodegenerative diseases.
Meanwhile, we have synthesized a large variety of trimeric
aminopyrazole derivatives with different C-terminal extensions
and identified the most potent β-sheet breakers; their affinities
towards Aβ, however remained moderate – in the low micro-
molar range.14 In order to enhance in vivo efficiency we joined
forces with the Willbold group and developed hybrid mole-
cules consisting of the established β-sheet breaking AP-trimer
covalently connected to polypeptides identified by phage
display as effective selectors for Aβ oligomers.15 These new
hybrids displayed striking synergistic effects in cells where
they completely suppressed Aβ oligomer formation as well as
restored long term potentiation (LTP). As a logical extension,
we decided to connect the aminopyrazole unit to Aβ-specific
antibodies.
Much effort has lately been devoted to AD immunother-
apy.16,17 Most recently instead of large immunoglobulins,
single-chain variable fragments (scFvs) have been utilized for
Aβ-recognition, with the advantage of enhanced permeability
through the blood-brain barrier accompanied by decreased
in vivo side effects. Here, we employed a scFv-construct derived
from an Aβ binding immunoglobulin (IC16) which targets the
N-terminal domain (aa 2-8) of monomeric and aggregated Aβ18
and combined it with the aminopyrazole trimer by non-
covalent complexation. To this end the antibody species was
equipped with a histidine tag at its C-terminus while the
aminopyrazole was covalently linked to a trivalent nitriloacetic
acid (NTA) moiety, allowing a highly stable Ni2+-mediated com-
plexation of the scFv with the aminopyrazole (Fig. 2). Although
the 1 : 1 complex depicted in Fig. 2 agrees with modeling
studies, one cannot exclude a priori the alternative formation
of an overlapping 2 : 1 complex, in the form of (1-8 amyloid-1)
– (IC16-scFv : AP-Ni-NTA-cyclam) – (16-20 amyloid-2).
Results and discussion
In initial attempts with only one NTA head fused to the amino-
pyrazole trimer the formation of the desired AP-NTA-Ni2+-His6-
antibody complex could not be confirmed. Consequently we
aimed for an analogous multivalent complex with several NTA
moieties placed on a suitable platform and connected to the
aminopyrazole. In the ideal case, all six histidine moieties of
the C-terminal tag should be addressed simultaneously. In
their seminal work, Piehler et al. developed and optimized
different scaffolds and indeed achieved greatly improved
affinities towards histidine tags.19 The most promising candi-
date consisted of a central cyclam unit equipped with three
NTA groups leaving one free amine for further functionali-
zation; this was in our case used as the attachment point for
the aminopyrazole trimer. Compared to monovalent NTA
units, the tris-NTA cyclam moiety displayed an increase in
thermodynamic stability of 3 orders of magnitude, from 10 μM
to 10 nM affinity, accompanied with extremely slow dis-
sociation kinetics. Fig. 3 shows our application for antibody-
aminopyrazole hybridization and a proposed model of the
octahedral Ni2+-mediated NTA-histidine complexation.
We observed that this hybridization of two Aβ ligands
greatly improved their affinity towards Aβ-peptides, providing
the basis for new sensitive tools for diagnostic issues. Our
results constitute a proof of principle for the general concept,
that molecular recognition of β-sheet rich proteins by specific
recombinant antibody fragments may be greatly enhanced
Fig. 3 Left: schematic representation of the proposed very tight
complex between the His6-tagged antibody and the AP-cyclam-tris-
(NTA) moiety, mediated by Ni2+ ions. Right: proposed model of the
through combination with small organic β-sheet binders. This NTA-Ni2+-histidine complexation.
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Synthesis
conditions followed by benzyl ester cleavage via Pd-catalyzed
hydrogenolysis to afford protected NTA-propanoic acid (3) in
63% yield over both steps (Fig. 4).
The cyclam-tris(NTA) scaffold was synthesized similarly as
described.19 In the first step H-Glu(OBzl)-OtBu hydrochloride
(1) was treated with excess t-butyl bromoacetate (2) under basic
Next the cyclam platform (4) was equipped with three
equivalents of tris(NTA)-propanoic acid (3) by standard peptide
coupling (TBTU). TFA cleavage of the t-butyl esters and neutral-
ization with sodium hydroxide afforded gave cyclam-tris(NTA)
sodium salt (6) which was used as a reference in the ELISA
binding experiments with IC16-scFv (Fig. 5). The free second-
ary amine of intermediate (5) was elongated with N-benzyloxy-
carbonyl-6-aminohexanoic acid, followed by hydrogenolytic
release of its primary amine to yield 7.20
Finally NTA-cyclam platform (7) was attached at the tip of
its aminohexanoyl spacer to the PMB-protected aminopyrazole
trimer (8) (HCTU/Cl-HOBt).13 Simultaneous cleavage of all t-butyl
Fig. 4 Synthesis of the cyclam-tris(NTA)-propanoic acid (3). (a) DIEA,
DMF, 55 °C, 12 h; 85%; (b) H2, Pd/C, MeOH; 74%.
Fig. 5 Synthesis of cyclam-tris(NTA) sodium salt (6) and aminopyrazol-trimer-cyclam-tris(NTA) sodium salt (9). (a) TBTU, DIEA, CH2Cl2, rt, 24 h,
46%; (b) CH2Cl2/TFA 7 : 1, rt, 24 h, 88%; (c) 1 M NaOH, rt, 10 min, 99%; (d) Cbz-6-aminohexanoic acid, TBTU, DIEA, CH2Cl2, rt, 24 h, 85%; (e) H2, Pd/
C, MeOH, 90%; (f) HCTU, Cl-HOBt, DIEA, CH2Cl2/DMF 2 : 1, 0 °C→rt, 48 h, 33%; (g) TFA, 70 °C, 5 h, 85%; (h) NaOH, Et2O, MeOH, 75%.
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ester and PMB groups was effected in hot trifluoroacetic acid,
and subsequent treatment with sodium hydroxide furnished the
desired aminopyrazole-cyclam-tris(NTA) sodium salt (9).
Complexation of IC16-scFv with AP-Ni-NTA-cyclam
IC16-scFv. The variable domains of the heavy (VH) and light
chain (VL) of IC16 were cloned into pET22b allowing the peri-
plasmic expression with C-terminal myc- and His6-tags in
E. coli. IC16-scFv was purified via immobilized metal chelating
chromatography (IMAC) and Aβ(1-16)-GB1 affinity purification
(Fig. 6). Purified IC16-scFv binds to Aβ(2-8) with a Kd of
112 nM.18
Fig. 7 Native PAGE for verification of complex formation between
IC16-scFv (5 μM) and increasing amounts of Ni-NTA-cyclam-AP. Gel
pH = 6.7.
Complex formation between the antibody and the amino-
pyrazole trimer was confirmed by native polyacrylamid electro-
phoresis (PAGE) (Fig. 7). The His6-IC16-scFv was incubated
with increasing concentrations of the AP-tris(NTA)-cyclam (9),
that had been associated with a 60-fold excess of Ni2+ ions.
Successful binding of three negatively charged AP-tris(NTA)-
cyclam molecules to IC16-scFv led to a pI-shift of the antibody
from 5.9 to 5.4, thus changing the migration speed when sep-
arated on a native gel with a pH of 6.7. This was observed for
all concentrations higher than a 30-fold excess of (9).
After verification of the complex formation we further inves-
tigated the binding properties of the self-assembled hybrid
compound towards Aβ by use of enzyme-linked immuno-
sorbent assays (ELISA).
In order to measure the affinity enhancement of the IC16-
scFv induced by complex formation with the aminopyrazole-
cyclam-tris(NTA) moiety we applied 120 nM IC16-scFv corres-
ponding to approximately semimaximal binding (Fig. 8, top).
Addition of AP-tris(Ni-NTA)-cyclam Ni-(9) strongly enhanced
the affinity of IC16-scFv to Aβ(1-42) in a dose-dependent
manner, indicating the improved binding properties of the
hybrid (Fig. 8, bottom). Already a two-fold excess (250 nM) of
AP-tris(Ni-NTA)-cyclam led to increased 1.75-fold binding,
whereas a 20-fold excess (2.5 µM) nearly doubled the antibody
affinity to Aβ(1-42). Finally, addition of a 160-fold excess
(20 µM) of Ni-(9) raised Aβ(1-42) binding to 4-fold relative of
the level of the antibody fragment alone.
Fig. 8 ELISA-experiment for the investigation of binding properties of
the self-assembled complex between AP-Ni-NTA-cyclam and IC16-
scFv. Top: dose response chart of IC16-scFv binding to Aβ(1-42).
Bottom: 120 nM IC16-scFv was complexed with increasing amounts of
either AP-Ni-NTA-cyclam, AP-NTA-cyclam or Tris-Ni-NTA-cyclam.
Binding intensities are shown in comparison to 120 nM IC16-scFv alone
(n-fold increase).
To verify that the affinity enhancement is related to
complex formation between the IC16-scFv antibody fragment
and AP-tris(Ni-NTA)-cyclam Ni-(9), the same experiment was
carried out without Ni2+-ions, thus preventing binding of AP-
tris(Ni-NTA)-cyclam to the His-tag of IC16-scFv. Here, the
binding of IC16-scFv was not influenced, indicating that
hybrid formation is essential for affinity enhancement. In
addition application of tris(NTA)-cyclam unit (6), which lacks
the AP trimer, also did not affect the affinity of IC16-scFv to
Aβ(1-42), demonstrating the functional role of the AP moiety.
In the absence of IC16-scFv we yielded no significant signals
above background, confirming the lack of non-specific binding
of the primary and secondary antibody used for the ELISA
assay. We would like to emphasize that the two above-
described negative control experiments strongly support the
idea, that both parts of the self-assembled hybrid complex
recognize different epitopes on the Aβ(1-42) target simul-
taneously, just as proposed in Fig. 2.
Fig. 6 Schematic representation of the His6-IC16-scFv preparation
(left) and purity control after IMAC purification and Aβ-affinity polishing
(right).
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Altogether the ELISA data nicely reflect the results from with variable concentrations of Ni-(9) (60-fold molar excess of
native PAGE, because maximal binding occurred only when Ni2+). As controls IC16-scFv was either incubated with (9) in
AP-trimer-tris(Ni-NTA)-cyclam Ni-(9) was added to IC16-scFv in the absence of Ni2+ ions or with Tris(Ni-NTA)-cyclam Ni-(6)
concentrations higher than a 30-fold excess.
that lacks the AP.
Synthesis
Conclusion
General procedures. Unless otherwise noted, materials and
reagents were obtained from commercial suppliers and were
used without further purification. Cyclohexane and ethyl
acetate were freshly distilled before use. Air- and moisture-sen-
sitive reactions were performed under an argon atmosphere.
Silica gel (Kieselgel 60, Merck) was used for column chromato-
graphy. TLC was performed on Macherey-Nagel ALUGRAM and
POLYGRAM SIL G/UV254, 0.2 mm thick.
In summary we demonstrated that the non-covalent Ni2+-
mediated hybridization of IC16-scFv with the AP-trimer-tris-
(NTA)-cyclam (9) strongly increased antibody affinity towards
Aβ(1-42). Various ELISA experiments with truncated versions of
the self-assembled hybrid complex strongly suggest, that the
specific binding properties of an antibody can be combined
with the unspecific backbone recognition of a β-sheet binder
and lead to significant affinity increase.
These results may open a promising new path for the devel-
opment of enhanced antibodies applicable in amyloid diag-
nostics and possibly even in therapeutic applications to
Aβ-related neurodegenerative disorders.
1H NMR measurements were recorded on a Bruker Advance
DRX500 spectrometer. Chemical shifts are reported in parts
per million (ppm) and were referenced to CDCl3 (δ H7.25),
DMSO-d6 (δ H2.50) and D2O (δ H4.80). NMR assignments were
made using a 1D technique. Multiplicities are described using
the following abbreviations: s = singlet, d = doublet, t = triplet,
q = quartet; quint = quintet; sext = sextet; m = multiplet, br =
broad. High-resolution mass spectra (HRMS) were recorded
with a Bruker maXis 4G time of flight mass spectrometer in
positive or negative ion electrospray mode.
Experimental
Native polyacrylamid gel electrophresis
Expression and purification of scFv-IC16 has been described
before.18 AP-trimer-tris(NTA)-cyclam Ni-(9) was complexed with
Ni2+ with a 60-fold excess of NiSO4 for 30 min at rt. 5 µM of
purified IC16-scFv were incubated with increasing amounts of
Ni-(9) for 30 min at rt. Fractions of 2 µg of IC16-scFv were then
diluted in native sample buffer (5% sucrose, 100 mM sodium
phosphate, pH 6.7) and loaded on a phosphate buffered (pH
6.7) 10% polyacrylamid gel. After electrophoresis at 100 V for
1.5 h in native running buffer (100 mM sodium phosphate pH
6.7) the gel was stained with Coomassie blue.
2,2′,2″,2′″,2″″,2′″″-(((1,4,8,11-Tetraazacyclotetradecane-1,4,8-triyl)-
tris(1-carboxy-4-oxobutane-4,1-diyl))tris(azantriyl))hexa-acetic acid
(5a). 0.359 g Tris(tbutyl-NTA)cyclam (5) (0.25 mmol, 1.0 eq.)
was dissolved in 2 mL TFA and 14 mL dichloromethane and
stirred for 24 h at rt. The solvent was evaporated under
reduced pressure and the residue was redissolved in TFA. Cold
diethyl ether was added to the solution, the precipitating solid
was centrifuged and washed three times with cold diethyl
ether. The solid was dried in vacuo to afford 0.203 g
(0.22 mmol, 91%) of (5a) as a pale yellow solid. The product
was directly used to form the sodium salt.
ELISA
2,2′,2″,2′″,2″″,2′″″-(((1,4,8,11-Tetraazacyclo-tetradecane-1,4,8-triyl)-
tris(1-carboxylat-4-oxobutane-4,1-diyl))tris(azantriyl))-hexaacetate
Wells of a 96-well Maxisorp plate (Nunc, Thermo Scientific,
Waltham, MA, USA) were coated with 500 ng of synthetic
Aβ(1-42) (JPT, Berlin, Germany), that were diluted in 50 mM
NaHCO3, pH 8.3 over night at rt. Following washing with water,
unspecific binding sites were blocked with PBS/5% skim milk
for 2 h at rt. After removal of the blocking buffer increasing
amounts of purified IC16-scFv, diluted in PBST/0.2% milk, were
added to the wells and incubated for 2 h at rt. Subsequently,
the wells were washed three times with PBS, 0.1% Tween-20
(PBS-T) and then probed for 1 h at rt with 9E10 anti-Myc mAb,
that has been derived from hybridoma supernatant and diluted
1 : 2 in PBS-T. After three additional washing steps with PBS-T,
the antibody complex was detected with an HRP-conjugated
goat anti-mouse-IgG (Pierce, Thermo Scientific, Waltham, MA,
nona sodium salt (6). 0.050
g
Tris(NTA)cyclam (5a)
(0.053 mmol, 1.0 eq.) was dissolved in 0.481 mL 1 M NaOH
(0.481 mmol, 9.0 eq.) and stirred for 10 min at rt. Subsequently
the solution was lyophilized and furnished 0.060
g
(0.053 mmol, 99%) of (6) as a pale yellow solid. 1H NMR
(500 MHz, D2O): δ [ppm] = 1.85–2.01 (m, 10H, H-4, H-8, H-13),
2.46–2.48 (m, 6H, H-7, H-9, H-12), 2.75 (m, 2H, H-14),
2.82–2.87 (m, 2H, H-15), 3.11–3.28 (m, 15H, H-2, H-3),
3.48–3.58 (m, 13H, H-5, H-10, H-11, H-16, H-17).
Hexa-tert-butyl-2,2′,2″,2′″,2″″,2′″″-(((11-(6-(3-(3-(3-nitro-1H-pyrazol-
5-carboxamido)-1H-pyrazol-5-carboxamido)-1H-pyra-zol-5-carbox-
amido)hexanoyl)-1,4,8,11-tetraazacyclotetrade-cane-1,4,8-triyl)tris-
(1-(tert-butoxy)-1,5-dioxo-pentane-5,2-diyl))-tris(azantriyl)) hexa-
USA; 1 : 5000 diluted in PBS-T). Signals were quantified by using acetate (9a). Under argon 0.043
the OptEIA detections reagent (BD Bioscience, Franklin Lakes, carboxylic acid (8) (0.06 mmol, 1.0 eq.) was dissolved in 10 mL
NJ, USA) according to the manufacturer’s recommendations. dry dichloromethane and 5 mL dry DMF. 0.029 g HCTU
g PMB-protected trimer
In order to determine an increased affinity of IC16-scFv in (0.07 mmol, 1.2 eq.), 0.025 g Cl-HOBt (0.15 mmol, 2.5 eq.) and
complex with AP-trimer-tris(NTA)-cyclam Ni-(9), 120 nM of 0.04 mL DIEA (0.23 mol, 4.0 eq.) were added to the solution,
IC16-scFv were preincubated in PBST/0.2% milk for 30 min cooled down to 0 °C and stirred for additional 10 min. Sub-
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sequently, 0.136
g of t-butyl ester-protected amine (7) the Forschungskommission of the University of Düsseldorf
(0.09 mmol, 1.5 eq.) was added to the reaction mixture and Medical School.
stirred for 48 h at rt. The solvent was removed and the residue
was dissolved in ethyl acetate. The organic layer was washed
three times with saturated aqueous NaHCO3 as well as three
Notes and references
times with saturated aqueous NaCl, dried over magnesium
sulfate and evaporated under reduced pressure. The crude
product was purified by column chromatography (ethyl
acetate) and gave 0.043 g (0.02 mmol, 33%) of (9a) as a color-
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1
less solid. RF: 0.44 (ethyl acetate). H-NMR (500 MHz, DMSO-
d6): δ [ppm] = 1.24–1.31 (m, 2H), 1.38 (s, 54H, H-1), 1.40 (s,
27H, H-1′), 1.50–1.52 (m, 8H), 1.65–1.84 (m, 11H), 2.34–2.46
(m, 8H), 3.36–3.40 (m, 27H), 3.33–3.36 (m, 27H), 3.70–3.71 (3
s, 9H, H-35), 5.61 (s, 2H, H-30), 5.67 (s, 2H, H-30), 5.81 (s, 2H,
H-30), 6.85–6.91 (m, 7H, H-28, H-32, H-33), 7.16–7.30 (m, 7H,
H-32, H-33), 7.71 (s, 1H, H-28), 7.98 (s, 2H, H-28), 8.62 (bs, 1H,
H-25), 11.33 (s, 1H, H-36), 11.49 (s, 1H, H-36). HRMS (ESI†):
for C115H171N17O30Na: calcd: 2294.2301, found: 2294.2476, for
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C115H171N17O30Na2: calcd: 1158.6097, found: 1158.6217.
2,2′,2″,2′″,2″″,2′″″-(((11-(6-(3-(3-(3-Nitro-1H-pyrazol-5-carbox-
amido)-1H-pyrazol-5-carboxamido)-1H-pyrazol-5-carboxami-do)-
hexanoyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-triyl)tris(1-carboxy-
4-oxobutane-4,1-diyl))tris(azantriyl))hexaacetic acid (9b). Under
argon 0.028 g (0.01 mmol, 1.0 eq.) trimer-tris(tBu-NTA)cyclam
(9a) was dissolved in 4 mL dry TFA and stirred for 5 h at 70 °C
in a closed system. Cold diethyl ether was added to the cooled
reaction mixture; the precipitating solid was centrifuged and
washed three times with cold diethyl ether. The crude product
was dried in vacuo to afford 0.019 g (0.01 mmol, 99%) of (9b)
as a colorless solid, which was directly converted into the
sodium salt.
12 C. N. Kirsten and T. H. Schrader, J. Am. Chem. Soc., 1997,
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2,2′,2″,2′″,2″″,2′″″-(((11-(6-(3-(3-(3-Nitro-1H-pyrazol-5-carbox-
amido)-1H-pyrazol-5-carboxamido)-1H-pyrazol-5-carbox-amido)-
hexanoyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-triyl)tris(1-carboxy-
lat-4-oxobutane-4,1-diyl))tris(azan-triyl)) hexaacetic acid nona
sodium salt (9). 5.0 mg of the deprotected hybrid compound
(9b) (0.004 mmol, 1.0 eq.) was dissolved in 1 mL 2 M NaOH,
and 2 mL diethyl ether were added to the solution. After drop-
wise addition of methanol (up to 1 mL) a yellow solid precipi-
tated. The solution was centrifuged and the solid was washed
with cold diethyl ether. Redissolution in water and subsequent
lyophilization gave 5.2 mg (0.003 mmol, 75%) of (9) as a pale
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1
yellow solid. H-NMR (500 MHz, D2O): δ [ppm] = 1.44 (m, 2H,
H-19), 1.66 (m, 4H, H-18, H-20), 1.86–2.00 (m, 10H, H-4, H-8,
H-13), 2.45 (m, 8H, H-5, H-17), 3.12–3.63 (m, 33H, H-2, H-3,
H-7, H-9, H-10, H-11, H-12, H-14, H-15, H-16, H-21), 6.81 (s, 1H,
H-25), 6.93 (s, 1H, H-25), 7.46 (s, 1H, H-25). HPLC: 92–99%
(retention time: 2.1–3.0 min, RP 18 column, 214 nm detection,
flow rate: 1.0 mL min−1, linear gradient 95–40% eluent A in
30 min, eluent A: 0.1% TFA in water, eluent B: acetonitrile).
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Y. Cinar, M. Wördehoff, C. Korth, S. Aileen Funke and
D. Willbold, PLoS One, 2013, 8, e59820.
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Acknowledgements
M. Hellmert thanks the Bruno-Werdelmann foundation for
financial support. A. Müller-Schiffmann received a grant from
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 2974–2979 | 2979