Reversible Covalent and Supramolecular Functionalization of Water-Soluble Gold(I) Complexes

Article abstract of DOI:10.1002/chem.201700588

The ligation of gold(I) metalloamphiphiles with biomolecules is reported, using water-soluble Au^I-N-alkynyl substituted maleimide complexes. For this purpose, two different polar ligands were applied: 1) a neutral, dendritic tetraethylene glycol-functionalized phosphane and 2) a charged, sulfonated N-heterocyclic carbene (NHC). The retro Diels–Alder reaction of a furan-protected maleimide gold(I) complex, followed by cycloaddition with a diene-functionalized biotin under mild conditions leads to a novel gold(I) metalloamphiphile. The strong streptavidin–biotin binding affinity in buffered aqueous solution of the resulting biotin alkynyl gold(I) phosphane conjugate remains intact. The cytotoxicity of the biotinylated gold(I) complex against a T47D human breast cancer cell line is higher than for cisplatin.

Full text of DOI:10.1002/chem.201700588

A Journal of
Accepted Article
Title: Reversible covalent and supramolecular functionalization of
water-soluble Au(I)-complexes
Authors: Benedict Kemper, Maximilian von Gröning, Vanessa Lewe,
Daniel Spitzer, Tobias Otremba, Natascha Stergiou, Dieter
Schollmeyer, Edgar Schmitt, Bart Jan Ravoo, and Pol
Besenius
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To be cited as: Chem. Eur. J. 10.1002/chem.201700588
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10.1002/chem.201700588
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Reversible covalent and supramolecular functionalization of
water-soluble Au(I)-complexes
Benedict Kemper,[a] Maximilian von Gröning,[a] Vanessa Lewe,[a, b] Daniel Spitzer,[a] Tobias Otremba,[c]
Natascha Stergiou,[d] Dieter Schollmeyer,[a] Edgar Schmitt,[d] Bart Jan Ravoo,[c] Pol Besenius*[a, b]
Abstract: The ligation of Au(I)-metalloamphiphiles with biomolecules
is reported, using water-soluble Au(I)-N-alkynyl substituted maleimide
complexes. For this purpose, two different polar ligands were applied:
(1) a neutral, dendritic tetraethylene glycol functionalized phosphane
and (2) a charged, sulfonated N-heterocyclic carbene (NHC). The
retro Diels-Alder reaction of a furan protected maleimide-Au(I)-
complex, followed by cycloaddition with a diene functionalized biotin
under mild conditions leads to a novel Au(I)-metalloamphiphile. The
strong streptavidin-biotin binding affinity in buffered aqueous solution
of the resulting biotin Au(I)-alkynyl-phosphane conjugate remains
intact. The cytotoxicity of the biotinylated Au(I)-complex against a
T47D human breast cancer cell line is higher than for cisplatin.
functionalization, and have been applied for the postsynthetic
9g,
9h]
modification of oligonucleotides,[9c,
proteins,[9k-m]
,
9f]
glycoconjugates[9e,
as well as for the preparation of various
synthetic polymer architectures and polymer-bioconjugates.[11]
Given the growing research activities in oligopeptides decorated
with medicinally active organometallic compounds, we were
surprised to notice that orthogonal bioconjugation strategies of
water-soluble metal-complexes have received little attention.[12]
Note that within the context of peptidic Au(I)-complexes, the
Krause lab has reported biotin-[AuICl(NHC)] conjugates
containing hydrophobic ligands.[13] The Metzler-Nolte group has
reported a series of amino acid and peptide Au(I)-conjugates
based on AuI-NHC,[14], Au(I)-N,S-heterocyclic carbene (NSHC),[15]
Introduction
The chemoselective modification of biological macromolecules is
a steadily expanding field in life sciences with regard to a wide-
range of applications in imaging, diagnostics, therapy and the
elucidation of biological function.[1] In recent years, chemists have
developed numerous strategies for the conjunction with drugs,
contrast agents, fluorescent labels or affinity tags using
bioconjugation reactions such as CuI-catalyzed[2] or strain-
promoted[3] alkyne-azide cycloaddition (CuAAC or SPAAC,
respectively), native chemical[4] and Staudinger ligations,[5] metal-
catalyzed olefin cross metathesis[6] and cross-coupling
reactions,[7] direct cysteine arylation using organometallic
palladium reagents,[8] as well as Diels-Alder[9] and the inverse
electron demand Diels-Alder cycloaddition.[10] Maleimides are
commonly employed as dienophiles in normal-electron-demand
Diels-Alder reactions, due to their electron-poor character and
thus high reactivity, as well as their straight forward chemical
+
≡
[a]
B. Kemper, M. von Gröning, V. Lewe, D. Spitzer, Dr. D.
Schollmeyer, Prof. Dr. P. Besenius
Institute of Organic Chemistry
Johannes Gutenberg-University Mainz
Duesbergweg 10–14, 55128 Mainz (Germany)
E-mail: besenius@uni-mainz.de
[b]
[c]
V. Lewe, Prof. Dr. P. Besenius
Graduate School Materials Science in Mainz
Staudingerweg 9, 55128 Mainz (Germany)
T. Otremba, Prof. Dr. B. J. Ravoo
Figure 1. Water-soluble maleimidic Au(I)-alkynyl complexes based on a neutral,
dendritic tetraethylene glycol (TEG) functionalized phosphane 1 and a charged,
sulfonated NHC 2 as well as the corresponding nonpolar derivative with PPh3
Organic Chemistry Institute
as neutral ligand
3 (top); Diels-Alder ligation of 1 with biotin (middle);
Westfälische Wilhelms-University Münster
Corrensstrasse 40, 48149 Münster (Germany)
N. Stergiou, Prof. Dr. E. Schmitt
Institute of Immunology,
University Medical Center, Johannes Gutenberg-University Mainz,
Langenbeckstrasse 1, Geb. 708, 55101 Mainz (Germany)
supramolecular complexation of the resulting metalloamphiphilic biotin Au(I)-
alkynyl-phosphane conjugate 4 (bottom) with streptavidin in aqueous solution.
Image from the RCSB PDB (www.rcsb.org) of PDB ID 2Y3E (C. E. Chivers, A.
L. Koner, E. D. Lowe, M. Howarth, Biochem. J. 2011, 435, 55-63).[18]
[d]
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.XXXXXXXX.
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Au(I)-triazolyl-phosphane complexes.[16] Laguna and Concepción
Gimeno have reported the synthesis of peptidic thiolate Au(I)-
phosphane complexes,[17] whereas Amorín and Granja described
cyclic peptides linked via hydrophobic alkynyl Au(I)-
phosphanes.[19] Water-soluble molecular Au(I)-complexes in
general[20] and those bearing alkynyl ligands in particular have
received increasingly large interest in applications for
luminescence,[21] catalysis,[22] and medicinal chemistry.[12, 23]
Most recently, we have reported a synthetic route for the
glycol monomethyl ether took place to yield the precursor ligand
16 (see SI). In situ reduction following by addition of [AuICl(tht)]
led to the desired product 1 in a moderate overall yield of 45% in
four steps (Scheme S1). The oxidation/reduction steps on the
phosphorous limit the feasibility of this synthetic route. Instead of
a late introduction of the phosphane ligand into the Au(I)-complex,
we decided to exchange the neutral ligand in the first step before
demethylation was performed. Quantitative yields were achieved
when tri(4-methoxyphenyl)phosphane was reacted with
preparation of a self-assembling peptidic Au(I)-metalloamphiphile, [AuICl(tht)]. Next, deprotection of the aromatic methyl ethers of 18
via coupling of an propargylamine-functionalized Au(I)-
phosphane with a N-hydroxysuccinimide activated peptide.[24] In
order to expand the synthetic toolbox of Au(I)-bioconjugation
strategies for the construction of more complex metallo-
amphiphile-based nanomaterials and protein conjugates, we
hereby report the synthesis of Au(I)-σ-alkynyl complexes
containing Diels-Alder protected maleimide, equipped with an
oligo(ethylene glycol) derived phosphane and a sulfonated NHC
ligand (Figure 1). The high stability of these complexes is
demonstrated. After deprotection, the former Au(I)-phosphane
with BBr3 followed by hydrolysis gave the phenolic Au(I)-
phosphane complex 19 in 99% yield. Note that the Au(I)-
phosphane complex remains fully stable in the presence of the
strong Lewis acid BBr3, and furthermore no alkylation of the
phosphane was observed during the demethylation reaction.
Finally, etherification with tetraethylene glycol monomethyl ether
tosylate led to the water-soluble Au(I)-phoshane complex 1 with
an improved overall yield of 83% in three steps, on gram scale.
For the functionalization of biomacromolecules with the water-
soluble Au(I)-phosphane and NHC complexes 1 and 2, we
decided to link them with maleimides. Maleimide bioconjugation
chemistry is widely explored and can be applied on various
molecules of biological interest either by Michael-type or Diels-
Alder click chemistry. Starting from cheap maleic anhydride and
N-propargyl amine, N-alkynyl-functionalized maleimide 24 was
synthesized, as reported in literature[27] (see SI). We decided to
protect the maleimide via reversible Diels-Alder reaction in order
to enable a long-term storage of stable Au(I)-building blocks and
further prevent undesired side reactions during the anionic ligand
exchange of Au(I)-chlorido complexes. Cycloadditions of
electron-rich dienes such as furans with maleimide derivatives
have been intensively studied. It is well-known that the unblocking
reactions of endo protected maleimides proceed at much lower
temperatures compared with the corresponding exo isomers.[28]
By reacting furan with N-propargyl maleimide 24 for one week at
room temperature, the desired, kinetically favored endo isomer 5
was obtained as the major product in 59% yield (see SI). We then
proceeded with the anionic ligand exchange of the Au(I)-chlorido
complexes 6-8 using terminal alkyne of 5 (Scheme 1). Treatment
of stoichiometric amounts of the alkyne and the water-soluble
Au(I)-complexes 6 and 7, with potassium hydroxide in degassed
methanol led to the formation of the corresponding σ-complexes
9 and 10. In the case of complex 10, a temperature increase to
35 °C was required to reach full conversion. 31P{1H} NMR analysis
of the reaction mixture of 9 indicated, that the anionic ligand
exchange proceeded selectively and without any formation of
byproducts.
complex is conjugated with
a diene-functionalized biotin
derivative. The resulting biotinylated Au(I)-metalloamphiphile is
able to self-assemble into larger tetrameric superstructures via
the supramolecular biotin-streptavidin complexation (Figure 1).
We have further assessed the cytotoxicity of the biotinylated
Au(I)-complex against a human breast cancer cell line (T47D
cells). Our synthetic strategy will facilitate the development of
more sophisticated multifunctional Au(I)-bioconjugates.
Results and Discussion
In order to show the versatility of the synthetic route presented
herein, we decided to use water-soluble Au(I)-complexes bearing
two very different kind of ligands: (1) a neutral, tetraethylene
glycol functionalized phosphane[25] and (2) a charged, sulfonated
NHC (sIMes)[26] ligand (Schemes 1 and S1). For the preparation
of the dendritic Au(I)-phosphane complex 1, we first exploited a
strategy, which was based on a neutral ligand exchange of the
corresponding phosphane with tetrahydrothiophene (tht) in
[AuICl(tht)].[25c] Due to its tendency to oxidize very quickly, the
starting tri(4-methoxyphenyl)phosphane was first fully oxidized,
followed by deprotection of the methyl ether, before etherification
of the phenolic hydroxyl groups with the tosylate of tetraethylene
In view of potential biomedical applications for hybrid bioinorganic
metal-complexes, very high purity is mandatory and we decided
to show that the metalloamphiphilic Au(I)-complexes 9 and 10
could be purified by preparative RP-HPLC. The chromatograms
and full characterization using high-resolution mass spectrometry,
1H, 13C and 31P{1H} NMR spectroscopy are provided in the
supporting information, and confirm the successful synthesis of 9
and 10. Due to their high amphiphilicity we were unable to grow
crystals, and we turned to an apolar model complex Au(I)-alkynyl-
triphenylphosphane 11 (Scheme 1 and Figure S1). Instead of
Scheme 1. Synthesis of endo-Diels-Alder protected building blocks 9-11 based
on water-soluble Au(I)-phosphane
6 and Au(I)-NHC 7 complexes or
[AuICl(PPh3)] (8) via anionic ligand exchange: 9: 6 (1.0 eq.), 5 (1.0 eq.), KOH
(1.6 eq.), MeOH, rt, 16 h; 10: 7 (1.0 eq.), 5 (1.0 eq.), KOH (1.5 eq.), MeOH,
35 °C, 16 h; 11: 8 (1.0 eq.), 5 (1.0 eq.), KOtBu (1.5 eq.), EtOH, rt, 18 h.
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hybridization found in Au(I)-alkynyl complexes. Note that the
structure is slightly disordered, showing 70% of the endo isomer
11 and 30% of the corresponding exo isomer (see SI). Filling
almost the same space, the exo derivative does not seem to affect
the packing of the crystal (Figure S2). Most likely, high summer
temperatures during crystal growth are responsible for the
observed partial endo to exo transformation in solution. This is
1
supported by H NMR analysis before and after crystal growth,
displaying the pure endo isomer of the Au(I)-complex 11 before
and 30% of the exo isomer after crystallization (Figure S1).
Importantly, the X-ray diffraction analysis confirms the
σ-coordinating nature of the hydrophobic Au(I)-complex and the
absence of π-activating Au(I)-species, which would cause side
reactions with nucleophiles in further bioconjugation steps.
Figure 2. Perspective drawing of the crystal structure of complex 11 with atomic
numbering scheme. CCDC reference number XXXXXXX.
The purified and storable Au(I)-building blocks 9-11 could be
transformed into the corresponding reactive maleimide
complexes 1-3, as required. The retro Diels-Alder reaction of the
endo protected maleimide complexes was performed at slightly
increased temperatures. Full conversion was accomplished by
heating the protected complexes at 70 °C in non-nucleophilic
solvents like dry toluene (9 and 11) or dry DMF (10) for four to
seven hours (Scheme S1). Except for furan, which could be
removed under reduced pressure, no other byproduct was
detected in the formation of the desired N-propargyl maleimide
Au(I)-alkynyl complexes 9-11. In this regard, direct maleimide
Diels-Alder ligation with biomacromolecules could potentially be
using potassium hydroxide, potassium tert-butoxide in ethanol
was applied in the substitution of the chorido ligand in
[AuICl(PPh3)] (8) with the alkynyl ligand 5. Isolation and washing
of the precipitate afforded the pure, hydrophobic Au(I)-complex
11 in 67% yield. Vapor diffusion of n-hexane into a solution of 11
in benzene resulted in colorless crystals. The Au(I)-complex
crystallized in the monoclinic space group P 21/c. A perspective
drawing of the single-crystal X-ray diffraction analysis is shown in
Figure 2. The P–Au (2.2697(9) Å), C–Au (1.993(4) Å) and C≡C
(1.193(5) Å) bond lengths are comparable to those observed in
other Au(I)-alkynyl-phosphane complexes.[29] The P-Au-C angle
of 179.73(13)° represents the typical linear geometry and sp-
applied
in
situ
and
without
further
purification.
Figure 3. Diels-Alder ligation of diene-functionalized biotin 12 with the metalloamphiphilic Au(I)-alkynyl complex 1 as the malemidic dienophile component in CD2Cl2.
(A): Reaction scheme; (B) Progress of the conjugation followed by 1H NMR (left) and 31P NMR (right) at t = 1 d at rt (top), t = 6 d at 35 °C (middle) and t = 10 d at
50 °C.
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We decided to illustrate the facile bioconjugation of a functional,
metalloamphiphilic Au(I)-alkynyl complex by using the established
biotin-streptavidin system. Biotin is a co-factor that plays a role in
multiple eukaryotic biological processes, and the host-guest
interaction between streptavidin and biotin is the strongest known
non-covalent interaction of a protein-ligand pair. Whitesides[30]
and Ward[31] have reported biotinylated rhodium and ruthenium
complexes in the development of artificial metalloenzymes.
As an appropriate biotin derivative carrying a diene moiety, we
prepared substrate 12 (Figure 3A), by facile esterification of
commercially available (E,E)-2,4-hexadien-1-ol with biotin (see
SI). With the diene 12 and the maleimidic dienophile embedded
in the metalloamphiphilic Au(I)-alkynyl-phosphane complex 1 in
hand, we studied the Diels-Alder ligation to form the target
molecule 4 (Figure 3). In order to follow the progress of the
reaction by NMR analysis, we mixed stoichiometric amounts of 12
and 1 in deuterated dichloromethane. In the 31P{1H} NMR, the
consumption of the dienophilic Au(I)-complex 1 (δ = 38.1 ppm) as
evaporation of CD2Cl2, facilitated full conversion of the Au(I)-
complex 1. In principle, the reaction time and temperature could
further be lowered by installation of a furan derivative as the
activated diene component[32] or water as
a
solvent.[33]
Concerning the stereochemistry of the cycloaddition, four
diastereoisomers are conceivable. Supported by the literature,[9l]
our results suggest that the cycloaddition exhibits no face-
selectivity and therefore two endo isomers are formed.
To demonstrate the strong affinity of the synthesized biotin Au(I)-
alkynyl-phosphane conjugate
4
towards streptavidin, ITC
measurements were performed (Figure 4 and Figure S3). Despite
the high water-solubility of the precursor complex 1, the ITC
experiments with the biotinylated Au(I)-complex 4 had to be
performed in an aqueous buffer containing 10% of DMSO, since
the incorporation of the hydrophobic biotin moiety impaired the
water solubility. Compared to titration experiments of streptavidin
with non-functionalized biotin performed in the same aqueous
buffer, we were not able to observe any reduction of the binding
constant and could confirm the high affinity Ka > 106 M-1 for the
biotinylated metalloamphiphilic Au(I)-alkynyl-phosphane complex
4.
We further assessed the toxicity of the Au(I)-complex 4 against a
human breast cancer cell line. The T47D cells were cultured for
24 h, 48 h and 72 h in the presence of different concentrations of
4 (Figure 5). The cytotoxicity of complex 4 (IC50 = 26±2 µM after
24 h, 38±2 µM after 48 h and 41±2 µM after 72 h) is higher than
for the positive control Cisplatin. This result is consistent with
previously disclosed studies for water soluble Au(I)-phosphane
complexes, which reported IC50 values between 1 µM and
100 µM, depending on the cell line, incubation time, ligand type
and lipophilicity of the Au(I) complexes.[12a, 14-17, 20, 34]
well as formation of the desired cycloaddition product
4
(δ = 38.0 ppm) could be easily monitored (Figure 3B, right).
These observations are supported by monitoring selected 1H
NMR signals (Figure 3B, left). Herein, a significant shift of the
diene methyl group as well as of the methylene group of the
propargyl moiety is visible (dotted lines, Figure 3B). The
disappearance of the signals belonging to the conjugated double
bonds of the diene (δ = 6.30–5.56 ppm) and those corresponding
to the maleimide double bond (δ = 6.69 ppm) indicate
a
successful ligation. We found that a stepwise temperature
increase from room temperature up to 50 °C accompanied with a
slow concentration increase concentration due to partial
Figure 5. Toxicity assessment of Au(I)-complex 4 against human breast cancer
cells. Human breast cancer cells (T47D cells) were cultured for 24h, 48h and
72h in the presence of different concentrations of Au(I)-complex 4. As positive
control the cells were treated with 30 µM Cisplatin. As negative control the cells
were treated with 2 vol% DMSO in PBS buffer, as was used within the highest
concentration of the Au(I)-complex 4. T47D cells were stained with a fixable
viability dye and cell viability was determined by Flow Cytometry. The value of
the untreated cells is considered as 100%. The other values are expressed as
percentiles. The mean of three independent experiments (±SD) is shown. A two-
tailed unpaired t-test was used to determine the significant effect of the Au(I)-
complex on human breast cancer cells in comparison to the negative control.
*≤P 0.05; **≤P 0.01; ****≤P 0.0001.
Figure 4. Isothermal titration calorimetry (ITC) of streptavidin (15 µM) with the
biotinylated metalloamphiphilic Au(I)-alkynyl-phosphane complex 4 (150 µM) at
pH 7.4 (20 mM phosphate buffer, including 10% DMSO). Top row: Change of
the heat rate during the titration; Bottom row: Integration of the measured heat.
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Due to its exceptionally high binding affinity for avidin and
streptavidin, biotin has been widely used as a recognition unit for
bioassays,[35] and therapeutics[36], for example in the development
of supramolecular multicomponent protein based delivery
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
1200
1000
800
600
400
200
0
systems[37], antibody-toxin[38], or antibody-siRNA conjugates[39]
,
and
cell-surface
engineering
via
oligosaccharide-toxin
conjugates.[40] Our synthetic strategy for biotinylated Au(I)-
complexes will thus facilitate the preparation of more
sophisticated Au(I)-bioconjugates for the development of
multifunctional targeted metallodrugs and therapeutics.
0
10
20
30
40
50
0
10
20
30
40
50
Elution time (min)
Elution time (min)
Figure 6. RP-HPLC chromatograms of 9 monitored at λ = 210 nm: a)
RP-HPLC separation on a semi-preparative VariTide RPC column (flow:
18.9 mL/min), b) RP-HPLC chromatogram (baseline corrected) of the
isolated product 9 on an analytical VariTide RPC column (flow: 1.0 mL/min).
Details about the used gradient (dashed line) are provided in the SI.
Conclusions
1H NMR (600 MHz, MeOD-d4, 294 K): δ/ppm = 7.38 (dd, JPH = 12.0 Hz,
J = 8.6 Hz, 6H, PCCH), 7.04 (dd, J = 8.6 Hz, 1.5 Hz, 6H, CHCqO aryl), 6.40
(t, J = 0.9 Hz 2H, CH alkene), 5.27 (ddd, J = 3.6 Hz, 1.6 Hz, 0.9 Hz, 2H,
CH furan), 4.19–4.14 (m, 6H, CqOCH2 TEG), 4.06 (s, 2H, NCH2), 3.85–3.82
(m, 6H, CH2 TEG), 3.70–3.65 (m, 6H, CH2 TEG), 3.65–3.57 (m, 24H, CH2 TEG),
3.53 (dd, J = 3.6 Hz, 1.7 Hz, 2H, CH maleimide), 3.51–3.48 (m, 6H, CH2 TEG),
3.32 (s, 9H, CH3). 13C NMR (151 MHz, MeOD-d4, 296 K): δ/ppm =175.9
(CO), 163.0 (d, JPC = 2.3 Hz, CqO aryl), 136.8 (d, JPC = 15.4 Hz, PCC),
135.6 (C alkene), 122.7 (d, JPC = 61.6 Hz, PC), 116.6 (d, JPC = 12.3 Hz,
CCqO aryl), 110.9 (d, JPC = 154.9 Hz, CCAu), 96.8 (d, CCAu), 80.7 (C furan),
73.0 (CH2), 71.8 (CH2), 71.60 (CH2), 71.58 (CH2), 71.5 (CH2), 71.4 (CH2),
70.6 (CH2), 68.9 (CH2), 59.2 (CH3), 47.3 (C maleimide), 28.4 (d, NCH2).
31P{1H} NMR (162 MHz, MeOD-d4, 295 K): δ/ppm = 37.5 (s). ESI-HRMS
(MeOH) (m/z): Calculated for [C56H77AuNO18PNa]+: 1302.4436, found:
1302.4410.
In summary, we present a synthetic route for the bioconjugation
of metalloamphiphilic Au(I)-alkynyl complexes containing water-
soluble phosphane ligands via Diels-Alder ligation. In this context,
we improved the protocol for the preparation of polar Au(I)-
phosphane complexes bearing tetraethylene glycol moieties,
relying on the very strong Au-P bond which was stable under
demethylation conditions with boron tribromide, etherification
reactions and reversible Diels-Alder chemistry. We were able to
prepare σ-coordinated Au(I)-alkynyl metalloamphiphiles by
anionic ligand exchange of Au(I)-chlorido-phosphane and NHC
complexes with endo furan protected N-propargyl maleimide.
After RP-HPLC purification, the selective activation of these
storable Au(I)-alkynyl complexes via retro Diels-Alder reaction at
relatively low temperatures yields reactive N-propargyl maleimide
Au(I)-alkynyl building blocks. In order to demonstrate the
feasibility for bioconjugation, the maleimidic Au(I)-alkynyl-
phosphane complex 1 was reacted with diene-functionalized
biotin by Diels-Alder cycloaddition. The supramolecular function
of the resulting biotinylated amphiphilic Au(I)-alkynyl complex
remains intact as shown by ITC titration experiments with
streptavidin. The cytotoxicity of the biotinylated Au(I)-complex
against a T47D human breast cancer cell line was determined and
was higher than for cisplatin. The maleimidic and biotinylated
Au(I)-complexes will facilitate the preparation of more
sophisticated Au(I)-conjugated biomolecules, which could find
utilization in the development of multifunctional and targeted
therapeutics.
Au(I)-alkynyl-NHC complex 10: To a solution of KOH (6.00 mg, 107 µmol,
1.5 eq.) and 5 (14.1 mg, 69.4 µmol, 1.0 eq.) in dry, degassed MeOH
(2 mL), Cl-Au-sIMes (51.6 mg, 69.6 µmol, 1.0 eq.) was added under argon
in the dark. The reaction mixture was stirred at 35°C for 16 h in the dark
and concentrated in vacuo. Purification via RP-HPLC afforded a mixture
of the desired product 10 and 2 in the ratio of 1:0.32 (15.6 mg, 17.1 µmol,
25%) as white solid. Partial retro Diels-Alder reaction seemed to occur
during concentration of the HPLC fractions on the rotavap.
1H NMR (600 MHz, MeOD-d4, 296 K): δ/ppm = 7.47–7.45 (m, 3.3H, CH im),
7.17–7.14 (m, 3.3H, CH aryl), 6.73 (s, 0.6H, 2-CH alkene), 6.22 (s, 2H, 10-
CH alkene), 5.23–5.21 (m, 2H, CH furan), 4.07 (s, 0.6H, 2-CH2), 3.86 (s, 2H,
10-CH2), 3.46 (dd, J = 3.7, 1.7 Hz, 2H, CH maleimide), 2.75–2.71 (m, 9.8H,
CH3), 2.46–2.43 (m, 9.8H, CH3), 2.15–2.12 (m, 9.8H, CH3). 13C NMR
(151 MHz, MeOD-d4, 296 K): δ = 189.7 (N2CAu), 175.9 (10-CO maleimide),
171.4 (2-CO maleimide), 150.87 (C aryl), 143.23 (C aryl), 140.08 (C aryl), 137.93
(C aryl), 137.67 (C aryl), 137.64 (C aryl), 137.61 (C aryl), 136.07 (C aryl), 135.51
(2-CH alkene), 135.47 (10-CH alkene), 133.20 (CH im), 124.67 (CH aryl), 121.81
(10-CCAu), 98.17 (2-CCAu), 97.18 (10-CCAu), 80.72 (CH furan), 47.18
(CH maleimide), 29.26 (10-NCH2), 28.28 (2-NCH2), 23.74 (CH3), 23.72 (CH3),
18.31 (CH3), 18.29 (CH3), 16.95 (CH3), 16.94 (CH3). ESI-HRMS (MeOH)
(m/z): Calculated for [C32H30AuN3O9S2Na]-: 884.0987, found: 884.0986.
Experimental Section
Au(I)-alkynyl-triphenylphosphane complex 11: To a solution of KOtBu
(17.0 mg, 152 µmol, 1.5 eq.) and 5 (20.7 mg, 102 µmol, 1.0 eq.) in
degassed EtOH (11 mL), 8 (50.2 mg, 101 µmol, 1.0 eq.) was added under
argon. The resulting suspension was stirred at room temperature for 18 h.
The precipitate was isolated via centrifugation, followed by washing with
EtOH (15 mL). After drying, the desired product 11 (44.8 mg, 67.7 µmol,
67%) was obtained as a colorless solid.
Detailed synthetic procedures for the remaining compounds, their
characterization and full details about the instrumentation and methods, as
well as additional experimental data (Figures S1-S3) can be found in the
Supporting Information (SI).
Anionic ligand exchange protocol
1H NMR (400 MHz, CDCl3, 296 K): δ/ppm = 7.55–7.39 (m, 15H, CH aryl),
6.44 (t, J = 0.9 Hz, 2H, CH alkene), 5.32 (ddd, J = 3.5 Hz, 1.7 Hz, 0.9 Hz, 2H,
CH furan), 4.21 (d, JPH = 1.6 Hz, 2H, CH2), 3.52 (dd, J = 3.6 Hz, 1.7 Hz, 2H,
CH maleimide). 13C NMR (101 MHz, CDCl3, 296 K): δ/ppm = 173.9 (CO),
134.6 (C alkene), 134.4 (d, JPC = 13.8 Hz, Cortho), 131.7 (d, JPC = 2.5 Hz,
Cpara), 129.8 (d, JPC = 56.0 Hz, Cipso), 129.3 (d, JPC = 11.2 Hz, Cmeta), 124.5
(d, JPC = 142.4 Hz, CCAu), 95.3 (d, JPC = 27.1 Hz, CCAu), 79.7 (C furan),
Au(I)-alkynyl-phosphane complex 9: To a solution of KOH (21.7 mg,
387 µmol, 1.6 eq.) and 5 (49.5 mg, 244 µmol, 1.0 eq.) in dry, degassed
MeOH (8 mL), a solution of 6 (267 mg, 240 µmol, 1.0 eq.) in dry, degassed
MeOH (6 mL) was added under argon in the dark. The reaction mixture
was stirred at room temperature for 16 h in the dark and concentrated in
vacuo. Purification via RP-HPLC afforded the desired product 9 (56.6 mg,
44.2 µmol, 18%) as a yellowish oil.
This article is protected by copyright. All rights reserved.

10.1002/chem.201700588
Chemistry - A European Journal
FULL PAPER
46.2 (C maleimide), 28.9 (d, JPC = 2.6 Hz, CH2). 31P{1H} NMR (162 MHz,
CDCl3, 296 K): δ/ppm = 41.8 (s). ESI-HRMS (CH2Cl2:MeOH = 1:1) (m/z):
Calculated for [C29H23AuNO3PNa]+: 684.0973, found: 684.0967.
Retro Diels-Alder reaction
Au(I)-alkynyl-phosphane complex 1:
A solution of 9 (26.9 mg,
21.0 µmol) in dry toluene (5.0 mL) was stirred at 70 °C for 4 h under argon.
Concentration in vacuo and drying afforded the desired product 1 (25.3 mg,
20.9 µmol, 99%) as a yellowish oil.
Figure 7. RP-HPLC chromatogram of 4 monitored at λ = 254 nm on a
semi-preparative Luna RP-C18(2) column (flow: 20.0 mL/min). Details
about the used gradient (dashed line) are provided in the SI.
1H NMR (400 MHz, MeOD-d4, 296 K): δ/ppm = 7.36 (dd, JPH = 12.1 Hz,
J = 8.8 Hz, 6H, PCCH), 7.02 (dd, J = 8.6 Hz, 1.8 Hz, 6H, CHCqO aryl), 6.81
(s, 2H, CH alkene), 4.25 (s, 2H, NCH2), 4.18–4.13 (m, 6H, CqOCH2 TEG),
3.85–3.80 (m, 6H, CH2 TEG), 3.69–3.65 (m, 6H, CH2 TEG), 3.65–3.55 (m,
24H, CH2 TEG), 3.51–3.47 (m, 6H, CH2 TEG), 3.32 (s, 9H, CH3). 13C NMR
(101 MHz, MeOD-d4, 296 K): δ/ppm = 171.4 (CO), 163.0 (d, JPC = 2.2 Hz,
CqO aryl), 136.8 (d, JPC = 15.4 Hz, PCC), 135.7 (C alkene), 122.7 (d,
JPC = 61.8 Hz, PC), 116.7 (d, JPC = 12.4 Hz, CCqO aryl), 111.4 (CCAu),
97.9 (CCAu), 73.0 (CH2), 71.8 (CH2), 71.60 (CH2), 71.58 (CH2), 71.5 (CH2),
71.4 (CH2), 70.6 (CH2), 68.9 (CH2), 59.2 (CH3), 28.6 (NCH2). 31P{1H} NMR
(162 MHz, MeOD-d4, 296 K): δ/ppm = 37.4 (s). 1H NMR (400 MHz,
CD2Cl2, 296 K): δ/ppm = 7.40 (ddd, JPH = 12.1 Hz, J = 8.8 Hz, 1.7 Hz, 6H,
PCCH), 6.98 (dd, J = 8.8 Hz, 1.8 Hz, 6H, CHCqO aryl)), 6.70 (s, 2H,
CH alkene), 4.29 (d, JPH = 1.7 Hz, 2H, NCH2), 4.17–4.11 (m, 6H,
CqOCH2 TEG), 3.85–3.79 (m, 6H, CH2 TEG), 3.69–3.64 (m, 6H, CH2 TEG),
3.64–3.53 (m, 24H, CH2 TEG), 3.51–3.47 (m, 6H, CH2 TEG), 3.32 (s, 9H, CH3).
31P{1H} NMR (162 MHz, CD2Cl2, 296 K): δ/ppm = 38.1 (s). ESI-HRMS
(MeOH) (m/z): Calculated for [C52H73AuNO17PNa]+: 1234.4174, found:
1234.4153.
1H NMR (600 MHz, CD2Cl2, 294 K, mixture of isomers): δ/ppm = 7.40
(dd, JPH = 12.0 Hz, J = 8.8 Hz, 6H, PCCH), 6.98 (dd, J = 8.7 Hz, 1.5 Hz,
6H, CHCqO aryl), 5.78–5.71 (m, 2H, CH alkene), 5.15 and 5.12 (s, 1H, NH),
4.87 and 4.85 (s, 1H, NH), 4.67–4.58 (m, 1H, CO2CHACHBCH), 4.51–4.42
(m, 2H, CHCH2S, CO2CHACHBCH), 4.33–4.26 (m, 2H, CHCHS), 4.18–
4.11 (m, 8H, NCH2, CqOCH2 TEG), 3.85–3.79 (m, 6H, CH2 TEG), 3.68–3.65
(m, 6H, CH2 TEG), 3.63–3.54 (m, 24H, CH2 TEG), 3.50–3.47 (m, 6H, CH2 TEG),
3.32 (s, 9H, OCH3), 3.29–3.24 (m, 1H, CH2CHCH maleimide), 3.18–3.13 (m,
1H, CHS), 3.09–3.05 (m, 1H, CH3CHCH maleimide), 2.92–2.87 (m, 1H,
CHAHBS), 2.70–2.64 (m, 1H, CHAHBS), 2.64–2.59 (m, 1H, CO2CH2CH),
2.47–2.41 (m, 1H, CHCH3), 2.38–2.31 (m, 1H, CH2CH2CO2), 1.77–1.58
(m, 4H, CH2 alkyl), 1.48–1.39 (m, 5H, CH2 alkyl, CHCH3), 13C NMR (151 MHz,
CD2Cl2, 294 K, mixture of isomers): δ/ppm = 176.84 and 176.82
(CO maleimide), 176.64 and 176.60 (CO maleimide), 173.81 and 173.80 (CO ester),
163.4 (CO urea), 161.9 (d, JPC = 2.2 Hz, CqO aryl), 136.2 (d, JPC = 15.5 Hz,
PCC), 135.1 (C alkene), 129.32 and 129.26 (C alkene), 125.8 (d,
JPC = 143.3 Hz, CCAu), 122.1 (d, JPC = 61.4 Hz, PC), 115.7 (d,
JPC = 12.3 Hz, CCqO aryl), 95.9 (d, JPC = 27.3 Hz, CCAu), 72.4 (CH2 TEG),
71.3 (CH2 TEG), 71.1 (CH2 TEG), 71.01 (CH2 TEG), 70.99 (CH2 TEG), 70.9
(CH2 TEG), 69.9 (CH2 TEG), 68.2 (CH2 TEG), 64.75 and 64.72 (CO2CH2CH),
62.23 and 62.23 (CHCHS), 60.61 and 60.57 (CHCH2S), 59.2 (OCH3),
55.91 and 55.89 (CHS), 45.5 (CH3CHCH maleimide), 43.1 and 43.0
(CH2CHCH maleimide), 41.16 and 41.13 (CH2S), 36.21 and 36.17
(CO2CH2CH), 34.38 and 34.35 (CH2CH2CO2), 31.7 (CHCH3), 29.2 (NCH2),
28.82 and 28.80 (CH2 alkyl), 28.77 and 28.75 (CH2 alkyl), 25.38 and 25.34
(CH2 alkyl), 17.0 (CHCH3). 31P{1H} NMR (162 MHz, CD2Cl2, 296 K): δ/ppm
Au(I)-alkynyl-NHC complex 2: A solution of 10 (4.64 mg, 5.11 µmol) in
DMF (750 μL) was stirred at 70 °C for 4 h under argon. Concentration in
vacuo and drying afforded the desired product 2 (4.4 mg, 5.2 µmol, quant.)
as off-white solid.
1H NMR (600 MHz, MeOD-d4, 296 K): δ/ppm = 7.46 (s, 2H, CH imidazole),
7.16 (s, 2H, CH aryl), 6.73 (s, 2H, CH alkene), 4.07 (s, 2H, CH2), 2.73 (s, 3H,
CH3), 2.44 (s, 3H, CH3), 2.13 (s, 3H, CH3). ESI-MS (MeOH) (m/z):
Calculated for [C28H26AuN3O8S2Na]-: 816.0725, found: 816.5405.
Au(I)-alkynyl- triphenylphosphane complex 3: A solution of 11 (14.6 mg,
22.1 µmol) in dry toluene (2.5 mL) was stirred at 70 °C for 7.5 h under
argon. Concentration in vacuo and drying afforded the desired product 3
(13.1 mg, 22.1 µmol, quant.) as a colorless solid.
=
38.0
(s).
ESI-HRMS
(MeOH)
(m/z):
Calculated
for
[C68H97AuN3O20PSNa]+: 1558.5681, found: 1558.5712.
1H NMR (400 MHz, CDCl3, 296 K): δ/ppm = 7.55–7.39 (m, 15H, CH aryl),
6.79 (s, 2H, CH alkene), 4.43 (d, JPH = 1.4 Hz, 2H, CH2). 13C NMR (101 MHz,
Acknowledgements
CDCl3, 296 K): δ/ppm
=
169.8 (CO), 134.5 (C alkene), 134.4 (d,
JPC = 13.7 Hz, Cortho), 131.7 (d, JPC = 2.2 Hz, Cpara), 129.8 (d,
JPC = 55.8 Hz, Cipso), 129.3 (d, JPC = 11.2 Hz, Cmeta), 124.3 (d,
JPC = 142.3 Hz, CCAu), 96.3 (d, JPC = 27.0 Hz, CCAu), 28.2 (d,
JPC = 2.3 Hz, CH2). 31P{1H} NMR (162 MHz, CDCl3, 296 K): δ/ppm = 41.9
We thank Nicolas Ritt (JGU Mainz) for his help in synthesizing
molecule 6. This work was supported by the DFG (SFB 858, [B.K.,
T.O.], SFB 1066 [N.S.] and GSC 266, Materials Science in Mainz
[scholarship for V.L.])
(s).
ESI-HRMS
(CH2Cl2:MeOH = 1:1)
(m/z):
Calculated
for
[C25H19AuNO2PH]+: 594.0892, found: 594.0916.
Keywords: Metalloamphiphiles • Diels-Alder ligation •
Diels-Alder ligation
biotinylation • self-assembly • click chemistry
Biotinylated amphiphilic Au(I)-alkynyl-phosphane complex 4:
A
solution of 1 (25.3 mg, 20.9 µmol, 1.1 eq.) in CD2Cl2 (0.6 mL) was treated
with 12 (6.4 mg, 19.4 µmol, 1.0 eq.) in a NMR tube under argon in the dark.
The progress of the reaction was followed by 1H and 31P{1H} NMR analysis
while stepwise heat acceleration from room temperature up to 50 °C over
a period of ten days was performed until full conversion was observed.
Concentration in vacuo and drying afforded the desired product 4 as a
yellowish oil, which contained a small amount of unreacted 12 (20%,
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Entry for the Table of Contents (Please choose one layout)
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COMMUNICATION
The ligation of Au(I)-
B. Kemper, M. von Gröning, V. Lewe, D.
Spitzer, T. Otremba, N. Stergiou, D.
Schollmeyer, E. Schmitt, B. J. Ravoo, P.
Besenius*
metalloamphiphiles with biomolecules
is reported. Two different polar ligands
were applied: a dendritic tetraethylene
glycol functionalized phosphane and a
sulfonated N-heterocyclic carbene. A
biotinylated Au(I)-alkynyl-phosphane
complex was obtained by retro Diels-
Alder reaction of a furan protected
maleimide-Au(I)-precursor complex,
followed by Diels-Alder cycloaddition
with a diene functionalized biotin
under mild conditions.
Page No. – Page No.
Reversible covalent and
supramolecular functionalization of
water-soluble Au(I)-complexes
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