Synthesis and Avidin Binding of Ruthenium Complexes Functionalized with a Light-Cleavable Free Biotin Moiety

Article abstract of DOI:10.1002/ejic.201800644

In this work the synthesis, photochemistry, and streptavidin interaction of new [Ru(tpy)(bpy)(SRR′)](PF₆)₂ complexes where the R′ group contains a free biotin ligand, are described. Two different ligands SRR′ were investigated: An asymmetric ligand 1 where the Ru-bound thioether is a N-acetylmethionine moiety linked to the free biotin fragment via a triethylene glycol spacer and a symmetrical ligand 2 containing two identical biotin moieties. The coordination of these two ligands to the precursor [Ru(tpy)(bpy)Cl]Cl was studied in water at 80 °C. In such conditions the coordination of the asymmetric ligand 1 occurred under thermodynamic control. After the reaction, a mononuclear and a binuclear complex were isolated. In the mononuclear complex, the ratio of methionine- {[6](PF₆)₂} vs. biotin-bound {[7](PF₆)₂} regioisomer was 5.3 and the free biotin fragment of [6](PF₆)₂ allowed to purify it from its isomer [7](PF₆)₂ at small scales using avidin affinity chromatography. Coordination of the symmetrical ligand 2 afforded [Ru(tpy)(bpy)(2)](PF₆)₂ {[8](PF₆)₂} in synthetically useful scales (100 mg), good yield (82 %), and without traces of the binuclear impurity. In this complex, one of the biotin remains free whereas the second one is coordinated to ruthenium. Photochemical release of ligand 2 from [8](PF₆)₂ occurred upon blue light irradiation (465 nm) with a photosubstitution quantum yield of 0.011 that was independent of the binding of streptavidin to the free biotin ligand.

Full text of DOI:10.1002/ejic.201800644

A Journal of
Accepted Article
Title: Synthesis and avidin binding of ruthenium complexes
functionalized with a light-cleavable free biotin moiety
Authors: Bianka Siewert, Michiel Langerman, Andrea Pannwitz, and
Sylvestre Bonnet
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To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201800644
Link to VoR: http://dx.doi.org/10.1002/ejic.201800644

10.1002/ejic.201800644
European Journal of Inorganic Chemistry
FULL PAPER
Synthesis and avidin binding of ruthenium complexes
functionalized with a light-cleavable free biotin moiety
Bianka Siewert[a],[b], Michiel Langerman[a], Andrea Pannwitz[a], and Sylvestre Bonnet[a]*
Abstract: In this work the synthesis, photochemistry, and streptavidin
interaction of new [Ru(tpy)(bpy)(SRR’)](PF6)2 complexes where the R’
group contains a free biotin ligand, are described. Two different
ligands SRR’ were investigated: An asymmetric ligand 1 where the
Ru-bound thioether is a N-acetylmethionine moiety linked to the free
biotin fragment via a triethyleneglycol spacer and a symmetrical ligand
2 containing two identical biotin moieties. The coordination of these
two ligands to the precursor [Ru(tpy)(bpy)Cl]Cl was studied in water
at 80 °C. In such conditions the coordination of the asymmetric ligand
1 occurred under thermodynamic control. After the reaction, a
difficult to obtain efficient release of the targeting moiety.[6]
Typically, targeting the cancer cells works well, but less
cytotoxicity is observed, because the drug is not efficiently
released from its targeting group and thus trapped in the
endosomes for example.[6] To solve this issue, strategies with pre-
determined “break junctions” have been developed. For example,
the targeting fragment can be bound via linkers that are sensitive
to intracellular triggers, such as pH, proteases, or glutathione
concentration.[6] These strategies, however, lack temporal and
spatial control. Light activation would solve this issue by allowing
to release the targeting moiety by an external trigger, i.e.
photons.[7] We reported earlier on a method to cleave methionine-
and biotin-based ligands from a ruthenium(II) polypyridyl complex
using visible light.[8] Following the steps of Lo et a,l[9], Ward et
a,l[10], and others,[11] who showed that biotin-tagged metal
compounds could keep the biotin affinity for (strept)avidin, we
embarked on investigating synthetically useful methods to
functionalize photosubstitutionally active ruthenium polypyridyl
complexes such as [Ru(tpy)(bpy)(Cl)]Cl with a free biotin
fragment that can be removed by visible light, for future use in
photochemotherapy.[12],[13]
mononuclear and
a binuclear complex were isolated. In the
mononuclear complex, the ratio of methionine- ([6](PF6)2) vs biotin-
bound ([7](PF6)2) regioisomer was 5.3 and the free biotin fragment of
[6](PF6)2 allowed to purify it from its isomer [7](PF6)2 at small scales
using avidin affinity chromatography. Coordination of the symmetrical
ligand 2 afforded [Ru(tpy)(bpy)(2)](PF6)2 ([8](PF6)2) in synthetically
useful scales (100 mg), good yield (82%), and without traces of the
binuclear impurity. In this complex, one of the biotin remains free
whereas the second one is coordinated to ruthenium. Photochemical
release of ligand 2 from [8](PF6)2 occurred upon blue light irradiation
(465 nm) with a photosubstitution quantum yield of 0.011 that was
independent of the binding of streptavidin to the free biotin ligand.
Results and Discussion
Introduction
Ligand Design and Synthesis. Thioether groups such as
those found in N-acetylmethionine or biotin are excellent ligands
for ruthenium(II) polypyridyl complexes that can be cleaved by
visible light irradiation.[8c] The dissymmetric ligand 1 that contains
both sulphur donors separated by a short ethylene glycol spacer
was hence designed first (Scheme 1). Based on the higher
thermal stability of N-acetylmethionine ruthenium complexes
compared to biotin adducts,[8c] we hypothesized that coordination
of the sterically less hindered methionine fragment of 1 may be
preferred over that of biotin. In the symmetrical bis-biotinylated
ligand 2, on the contrary, one biotin should coordinate to
ruthenium, while the second, identical fragment should remain
free to interact with streptavidin.
One of the strongest known non-covalent interactions in nature is
the one between (strept)avidin and biotin (kd = 10-13 to 10 -15 L ×
mol-1).[1] Once the avidin-biotin-complex (ABC) is formed only
extremely harsh conditions (6 M guanidine-HCl, pH = 1.5) will lead
to disassembly.[2] This phenomenon, discovered 48 years ago,
led to the emergence of the ABC field in biotechnology.[1,
3]
(Strept)avidin systems are utilized for bioconjugates, for
theranostic purposes, but also as tumor-targeting strategy,[4] for
which the high affinity of biotin for avidin is critical.[3] Recently, e.g.
an almost ten-fold increase of cellular uptake of microspheres was
reached by employing the ABC technique.[5] However, for drug-
delivery strong binding also represent the major drawback, as it is
The first step in the synthesis of 1 (Scheme 1) involved the
activation of biotin with disuccinimidyl carbonate to obtain the
N-hydroxysuccinimidyl ester of biotin, biotin-NHS (3).[14] In a
second step, compound 3 was reacted with 5 eq. of the linker 2,2’-
(ethylenedioxy)bis(ethylamine) in DMF at RT, which afforded the
mono-biotin (4) ligand. The final reaction of 4 with N-acetyl-L-
methionine (HAmet) afforded 1 as colorless, viscous oil in 72%
yield. Full characterization of ligand 1 was performed by NMR,
ESI-MS, and HRMS measurements (see ESI). Ligand 2 was
easily obtained from the bisamine spacer used for 1 and two
equivalents of NHS-biotin 3 (see Scheme 1 and Experimental
Part).
[a] Leiden Institute of Chemistry, Leiden University, Einsteinweg 55,
233CC Leiden, The Netherlands
E-mail: bonnet@chem.leidenuniv.nl;
https://www.universiteitleiden.nl/en/staffmembers/sylvestre-bonnet#tab-1
[b] Current address: Institute of Pharmacy, Pharmacognosy, University of
Innsbruck, CCB, Innrain 80-82, 6020 Innsbruck, Austria
Supporting information and ORCID(s) from the author(s) for this article
are available on the WWW under https://doi.org/
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10.1002/ejic.201800644
European Journal of Inorganic Chemistry
FULL PAPER
Scheme 1. Overview of the synthetic routes used to obtain bis-thioether ligands 1 and 2.
Coordination of 1 and 2 to ruthenium. For the synthesis of
[Ru(tpy)(bpy)(1)](Cl)2 ([6]Cl2, Scheme 2), [Ru(tpy)(bpy)Cl]Cl was
reacted with 1 in water at 80 °C over three days. Under these
conditions, the chloride precursor hydrolyses quickly[8c] to the
aqua complex [Ru(tpy)(bpy)(OH2)]2+, which reacts with one or the
other thioether moiety. After workup and column chromatography
on silica, two different fractions were obtained. The 1H NMR
(Figure S1) of the more polar fraction (Rf = 0.1, acetone/H2O/1 M
HCl [16:4:1]) showed two signals with the same integral between
d = 9.50 – 10.00 ppm, indicating two downfield-shifted A6 protons
on the bipyridine ligand instead of the expected unique doublet
(see Scheme 2).[8c] Furthermore, the sum of integrals of the
aromatic part was twice as high as that of the aliphatic part.
Additionally, all methyl and methylene groups in close proximity
to the sulfur-atoms showed a significant downfield shift. This
characteristic observation emerges from the shielding cone of the
polypyridyl ligands when a monodentate ligand is coordinated to
the ruthenium polypyridyl moiety.[15] Taken together, NMR
analysis indicated that this fraction contained a binuclear
ruthenium complex [5]Cl4 (Scheme 2). This hypothesis was
confirmed by mass spectrometry; a peak at m/z = 528.1 was
the expected peak at m/z = 519.2 for the mononuclear ruthenium
complex [Ru(tpy)(bpy)(1)]2+ (calc. m/z = 519.12). According to 1H-
NMR spectroscopy, the integral ratio between the characteristic
signals of the aromatic part and that of the aliphatic part was close
1
to one. Further analysis of the H NMR revealed significant up-
field shifts for the methyl signal of the methionine fragment (e.g.
Dd = -0.74 ppm for M1), which proved the formation of the desired
mononuclear ruthenium complex [6]Cl2 coordinated via the sulfur
of the N-Acetylmethionine methionine as major isomer (Scheme
2). However, next to the characteristic A6 signal at d = 9.75 ppm
for [6]2+, a smaller A6 doublet at d = 9.85 ppm (³J = 5.5 Hz) was
observed (Figure S2), suggesting a mixture of two isomers. The
integral ratio between these two A6 peaks showed that [6]2+ was
83% pure. The second, minor isomer is the biotin-coordinated
complex [7]2+, as shown by the identical mass spectrum but
shifted chemical resonances of the biotin signals. Silica column
chromatography enriched [6]2+ to some extent, however, a pure
compound (>99% purity) could not be obtained under these
conditions.
In a next step, the synthetic conditions were varied to evaluate
whether [6]2+ could be obtained regioselectively. By increasing the
ligand-to-Ru ratio, we successfully suppressed the formation of
the binuclear complex [5]4+. Changing the reaction times and
using higher reaction temperatures showed an enrichment of the
found,
which
fits
the
calculated
m/z
value
for
[{Ru(tpy)(bpy)}2(1)+H2O+Cl]3+ (528.1). For the less polar fraction
(Rf = 0.3, acetone/H2O/1 M HCl [16:4:1]), mass analysis showed
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10.1002/ejic.201800644
European Journal of Inorganic Chemistry
FULL PAPER
desired isomer [6]2+ and thus suggested that the selectivity of the
reaction towards [6]2+ was not driven by kinetics, but the result of
thermodynamic control. The equilibration process between 1,
[Ru(tpy)(bpy)(OH2)]2+, [6]2+, and [7]2+ was hence followed in time
by 1H NMR at 80 °C, as the reaction between the aqua complex
and the sulphur donor is not observed below 50 °C. After the first
45 minutes [6]2+ and [7]2+ had formed in a ratio of 51:49, which
showed that there was no measurable kinetic preference for one
or the other isomer. The amount of [6]2+ in the reaction mixture
(NOESY, COSY, HMBC, HSQC), ESI-mass spectrometric
analysis, and elemental analysis. Based on these results (see
ESI), the 1,8-diamino-3,6-dioxaoctane linker has no significant
effect on the coordination mode of biotin to ruthenium, compared
to
the
known
crystal
structure
of
the
adduct
[Ru(tpy)(bpy)(biotinmethylester)]2+.[8c]
quickly increased thereafter, to finally reach
a
plateau
characterized by a [6]2+:[7]2+ ratio of 84:16 after 24 h (Figure 1).
From these data, we concluded that the regioselectivity of the
coordination to ruthenium of the methionine fragment in 1 vs that
of biotin, is due to the higher thermodynamic stability of the
methionine-coordinated isomer [6]2+, compared to [7]2+, rather
than to a slower reaction of biotin. According to our data, the
binding constant of [6]2+ is ~5 times higher than that of [7]2+, which
is too low to drive the formation of analytically pure complex [6]2+.
However, it was possible to purify [6]2+ from [7]2+ in small scales
using avidin chromatography (see below).
Analytically pure samples of a biotin-functionalized ruthenium
complex were obtained in preparative scale using the symmetrical
Figure 1. A) Evolution of the 1H NMR spectra (300 MHz, D2O) during the
coordination of ligand 1 to [Ru(tpy)(bpy)(OH2)](PF6)2 in D2O. B) The [6]2+:[7]2+
ratio obtained from integration of the A6 proton peaks in the 10.0 - 9.4 ppm
range, plotted vs. reaction time. Conditions: [Ru]0 = 4.2 mM, [1] = 42 mM, T =
353 K, in the dark. u = [Ru(tpy)(bpy)(OH2)]2+ (d = 9.54 ppm), = [6]2+(d = 9.76
ppm), = [7]2+(d = 9.86 ppm).
ligand 2. Thermal reaction of an excess of
2
to
[Ru(tpy)(bpy)(OH2)](PF6)2 in water at 80 °C, followed by silica gel
chromatography, afforded the mononuclear adduct [8](PF6)2
(Scheme 3) in ~100 mg scale and 81% yield. Coordination of only
one of the two biotin moieties to ruthenium and structural
information about the stereochemical orientation of the biotin
ligand in [8]2+ was obtained by a combination of 2D NMR studies
Scheme 2. Coordination of 1 eq. of 1 with [Ru(tpy)(bpy)Cl]Cl in water at 80 °C over three days yielded a mixture of a binuclear complex [5]Cl4 and two
mononuclear isomer complexes [6]Cl2 and [7]Cl2.
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10.1002/ejic.201800644
European Journal of Inorganic Chemistry
FULL PAPER
(λ = 465 nm, Δλ1/2 = 25 nm) yielded the same spectroscopic
changes as described in the previous section (Figure 2B). This
observation meant that [8]2+ is furthermore a promising candidate
for ABC-based strategies, as the linker in 2 is long enough, so that
the free biotin in complex [8]2+ still reaches the avidin binding
pocket. In addition, the observed behaviour of [8]2+ in the
presence of streptavidin (Figure 2A) shows that the complex is
kinetically stable under such conditions.
Scheme 3. Coordination of 5 eq. of 2 with [Ru(tpy)(bpy)Cl]Cl in water at 80 °C
yielded [8](PF6)2.
Photochemical Investigation. Ruthenium polypyridyl
complexes are known for their rich photochemistry.[16] Excitation
into the metal-to-ligand charge transfer (MLCT) absorption band
leads to either emission, singlet oxygen production, or ligand
photosubstitution. The concrete outcome depends on the ligand
field splitting of the complex.[16] For complexes of the
[Ru(tpy)(bpy)(L)]2+ family, emission and singlet oxygen production
are generally negligible as dissociative, metal-centered triplet
excited states (3MC) lie energetically close to the photochemically
generated 3MLCT states.[17] The photochemistry of compound
[8](PF6)2
was
studied
under
blue
light
irradiation
(λ = 465 nm, Δλ1/2 = 25 nm, photon flux 1.29×10-8 mol×s-1) in PBS
(pH = 7.03, I = 50 mM, [Ru]0 = 7.6×10-5 M) for 4 h at 298 K using
a setup described earlier.[8c] An isosbestic point was observed at
445 nm, indicating that a single photochemical reaction occurred;
the
photostationary
state
was
reached
after
80 minutes (Figure 1). Mass spectrometry measurements after
irradiation confirmed that photosubstitution of ligand 2 by water
had occurred, as the initial peak at m/z = 545.4 for [8]2+ was
replaced by
a peak at m/z = 508.1 characteristic for
[Ru(tpy)(bpy)(OH)]+ (calc m/z = 508.1). The photosubstitution rate
constant kΦ = 7.1×10-4 s-1 was obtained from the slope of a plot of
ln([RuS]/[Ru]0) versus the irradiation time, where [RuS]
represents the concentration of [8]2+ at time t, and [Ru]0 the total
ruthenium concentration. The corresponding photosubstitution
quantum yield was derived from the slope of a plot of the number
of moles of thioether complex nRuS vs the number of moles of
photons Q absorbed since t = 0, according to the methods
described in the experimental part (see also Figure S3). A
photosubstitution quantum yield of 0.011 was found, which is
higher, but about the same order of magnitude as the one found
for the substitution of [Ru(typ)(bpy)(SChol)]2+ by water (f =
0.0078).[18]
Binding of [8]2+ to Streptavidin and Photosubstitution. In
order to check the suitability of [8]2+ for the ABC technology, a
HABA (2-(4-hydroxyphenylazo)benzoic acid) displacement
titration was applied to test the binding behaviour of [8]2+ to
streptavidin. HABA exhibits a characteristic absorption band with
λmax at 506 nm when incorporated within streptavidin. Upon
addition of four equivalents of biotin per streptavidin tetramer, the
HABA is displaced leading to disappearance of the absorption
band at 506 nm.[19] Upon addition of more than four equivalents
of biotin, the absorbance at 506 nm is constant. This behaviour
was also observed for [8]2+ and indicates an equally strong
binding as biotin (Figure 2A). A slight increase in absorbance
upon addition of more than four equivalents is due to the
absorbance of [8]2+ itself. Irradiation of [8]2+ in absence and in
presence of one equivalent tetrameric streptavidin in NaH2PO4
buffer (3.00 mL, 20 mM, pH 7.0) with blue light
Figure 2. A) Displacement titration of HABA within streptavidin by [8]2+ and
biotin in NaH2PO4 buffer (2.7 mL, 20 mM, pH 7.0). B) Evolution of the
absorbance at 475 nm upon irradiation at λ = 465 nm (Δλ1/2 = 25 nm) of 6.0
×10-5 M [8]2+ in absence and in presence of one equivalent tetrameric
streptavidin in NaH2PO4 buffer (3.00 mL, 20 mM, pH 7.0).
Application of the selective streptavidin binding. The
observation that the free biotin in [8]2+ interacted strongly with
streptavidin suggested avidin-based column chromatography as
a potential method to separate the isomeric mixture of [6]2+ and
[7]2+ obtained above. An avidin column was hence incubated for
24 h with a mixture of [6]2+/[7]2+ in PBS. In a second step, the
unbound complex mixture was washed from the column using
buffer (PBS). Then, a biotin-loaded PBS buffer was used to
displace the non-coordinated, avidin-bound biotin moiety in [6]2+,
with native biotin. This procedure yielded a mixture of [6]2+ and
native biotin without traces of [7]2+, as shown by the unique A6
proton in the aromatic region of the 1H NMR spectrum of the
purified sample (9.6-10.0 ppm, Figure 3). The low loading
capacity and high price of commercially available avidin-based
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10.1002/ejic.201800644
European Journal of Inorganic Chemistry
FULL PAPER
purifications columns, however, was not suitable for a large scale
separation of [6]2+, but it confirmed that the dangling biotin moiety
in [6]2+ can still interact selectively with streptavidin.
Experimental Section
Material and Methods
Biotin was obtained from TCI Chemicals, N,N-diisopropylethylamine
(DIPEA) was obtained from Acros Organics. Disuccinimidyl carbonate,
1,8-diamino-3,6-dioxaoctane, N,N’-diisopropylcarbodiimide (DIC), 4-
[7]2+
[6]2+
dimethylaminopyridine
(DMAP),
Et3N
and
HABA
(2-(4-
hydroxyphenylazo)benzoic acid) were obtained from Sigma Aldrich.
DSPE-PEG-2K-Biotin [385437-57-0] was obtained from Nanocs.
[Ru(tpy)(bpy)(Cl)]Cl and [Ru(tpy)(bpy)(OH2)](PF6)2 were synthesized
following literature procedures[20] as well as wild type streptavidin.[21] DMF
was obtained from Alfa Aesar and dried over molecular sieves (3 Å). DCM
was obtained from Alfa Aesar and dried using a PureSolve 400 solvent
dispenser. Silica gel (60 Å, 230-400 mesh particle size, Ca ~0.1 %) was
used for column chromatography. Size exclusion chromatography was
done over Sephadex LH-20 prepared in MeOH. Avidin affinity column
chromatography was done with a 2 mL Pierce Monomeric Avidin Agarose
Kit from Thermo Scientific. 1H NMR and 13C NMR spectra were recorded
with a Bruker DPX 300 spectrometer or a Bruker DMX 400 spectrometer.
Chemical shifts are indicated in ppm relative to TMS. Numbering of the
compounds and NMR-spectra can be found in the supporting material.
Mass spectra were recorded on
a ThermoQuest Finnagen AQA
spectrometer. UV-Vis spectra were obtained on a Varian Cary 50 UV-
visible spectrometer. The irradiation setup for UV-vis experiments
consisted of custom made LEDs directly placed on top of a 1 cm quartz
cuvette, as described by Bahreman et al.[22]
Figure 3. Comparison of the 1H NMR spectra (D2O, 300 MHz, zoom of A6
proton peaks) of the isomeric mixture [6]2+ and [7]2+ (top) and the isolated
complex [6]2+ (bottom), before and after avidin affinity column chromatography,
respectively.
Biotin-NHS (3) Biotin (1.00 g, 4.10 mmol), disuccinimidyl carbonate (1.37
g, 5.33 mmol), and Et3N (2.30 mL, 16.4 mmol) were added to dry DMF (14
mL). The resulting suspension was stirred at room temperature under
argon for 16 hours. The solvent was removed by vacuum distillation at 323
K. The resulting solid was triturated in diethyl ether and ethyl acetate and
subsequently filtered and washed with diethyl ether over a glass filter. The
white powder was dried under air. Yield: 1.26 g (3.70 mmol, 90%). Rf =
0.68 (SiO2, BuOH/H2O/AcOH [3:1:1]); 1H NMR (300 MHz, Chloroform-d)
δ (ppm): 4.60 – 4.49 (m, 1H), 4.36 (dd, J = 7.9, 4.5 Hz, 1H), 3.17 (td, J =
7.2, 4.5 Hz, 1H), 2.94 (dd, J = 12.8, 5.0 Hz, 1H), 2.89 – 2.81 (m, 4H), 2.75
(d, 2H), 2.65 (q, J = 7.0 Hz, 2H), 1.93 – 1.65 (m, 6H), 1.57 (q, J = 7.6 Hz,
2H). 1H NMR data is in agreement with known literature.[23]
Conclusions
In summary, the two different biotin-containing thioether
ligands 1 and 2, when coordinated to [Ru(tpy)(bpy)(OH2)]2+ in
water, led to different results. The coordination of ligand 1 to
[Ru(tpy)(bpy)Cl]Cl provided one of the rare regioselectivity study
of the binding of thioethers to ruthenium(II). It showed that in the
conditions of the experiment coordination is under
thermodynamic control and that although biotin is more hindering
than N-acetylmethionine, the thermodynamic preference for [6]2+
vs [7]2+ was not strong enough to lead to the regioselective
preparation of [6]2+. Purification of [6]2+ from its regioisomer [7]2+
eventually succeeded in low scale via avidin-affinity
chromatography, proving that in [6]2+ the free biotin moiety allows
for establishing strong interactions with (strept)avidin.
N-biotinyl-8-amino-3,6-dioxaoctane-1-amonium acetate (4) Compound 3
(560 mg, 1.64 mmol) was dissolved in DMF (6 mL). The solution was
added dropwise to a solution of 1,8-diamino-3,6-dioxaoctane (1.20 mL,
8.20 mmol) and Et3N (1.14 mL, 8.20 mmol) in DCM (24 mL). The resulting
solution was stirred at room temperature for 5 hours, after which the
solvents were removed in vacuum to give an oil. The crude product was
purified twice by silica column chromatography using butanol/H2O/acetic
acid (3:1:1) as the eluent. Excess of acetic acid was removed by co-
evaporation with toluene. Yield: 547 mg (1.26 mmol, 77%). Rf = 0.33 (SiO2,
butanol/H2O/acetic acid [3:1:1]). 1H NMR (400 MHz, Methanol-d4) δ
(ppm): 4.50 (dd, J = 7.9, 4.8 Hz, 1H, H2), 4.31 (dd, J = 7.8, 4.4 Hz, 1H,
H3), 3.70 (t, J = 4.9 Hz, 2H, H13), 3.66 (s, 4H, H11+H12), 3.56 (t, J = 5.6
Hz, 2H, H10), 3.37 (t, J = 5.6 Hz, 2H, H9), 3.21 (ddd, J = 8.9, 5.8, 4.4 Hz,
1H, H4), 3.11 (t, J = 4.7 Hz, 3H, H14), 2.93 (dd, J = 12.7, J = 4.9 Hz, 1H,
H1B), 2.71 (d, J = 12.7 Hz, 1H, H1A), 2.23 (t, J = 7.3 Hz, 2H, H8), 1.91 (s,
3H, CH3), 1.79 – 1.54 (m, 4H, H5+H7), 1.44 (p, J = 7.6 Hz, 2H, H6). 13C
NMR (75 MHz, Methanol-d4) δ (ppm): 178.9 (C=O), 176.2 (Cb), 166.1 (Ca),
71.4 + 71.3 (C11+C12), 70.7 (C10), 68.0 (C13), 63.4 (C3), 61.6 (C2), 57.0
(C4), 41.0 (C1), 40.6 (C14), 40.2 (C9), 36.7 (C8), 29.7 (C6), 29.5 (C5),
26.9 (C7), 23.1 (CH3). ESI MS m/z (calc): 375.1 (375.2, [M - AcO]+).
The symmetrical bis-biotin ligand
2 offered a more
straightforward and synthetically useful route towards the
preparation of a ruthenium complex with a light-cleavable, free
biotin group. Complex [8](PF6)2 was obtained in ~100 mg scale
with good yields (82%). Meanwhile, photochemical studies with
[8]2+ showed that, whether or not the dangling, non-coordinated
biotin moiety was bound to streptavidin, efficient photosubstitution
of the coordinated biotin ligand occurred, thus providing a proof-
of-concept that light-cleavable free biotin moieties can be installed
on photoactivated chemotherapeutic complexes to prepare
streptavidin-mediated targeted PACT systems. The synthetic
strategy presented in this work would circumvent current
problems of (strept)avidin-biotin based cancer-targeting methods,
where the biologically active drug remains covalently bound to the
biotin targeting group.
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10.1002/ejic.201800644
European Journal of Inorganic Chemistry
FULL PAPER
N’’-(acetyl-L-methionine)-N’-biotinyl-3,6-dioxaoctane-1,8-diamine (1): N-
Characterization of [6]Cl2 (contaminated with [7]Cl2 see supplementary):
Rf = 0.30 (SiO2, acetone/H2O/1 M HCl [16:4:1]); 1H NMR (300 MHz, D2O)
δ (ppm): 9.75 (d, J = 5.5 Hz, 1H, A6), 8.70 – 8.63 (m, 3H, T3’+A3), 8.49 (d,
J = 7.8 Hz, 2H, T3), 8.42 (d, J = 8.1 Hz, 1H, B3), 8.36 (d, J = 8.0 Hz, 1H,
T4’), 8.39 – 8.27 (m, 1H, A4), 8.02 (dd, J = 8.0, 7.6 Hz, 2H, T4), 8.06 –
7.92 (m, 1H, A5), 7.83 (dd, J = 7.8 Hz, 1H, B4), 7.84 – 7.71 (m, 2H, T6),
7.35 (dd, J = 7.5, 5.5 Hz, 2H, T5), 7.23 (d, J = 5.6 Hz, 1H, B6), 7.08 (dd, J
= 7.5, 5.6 Hz, 1H, B5), 4.54 (dd, J = 8.0, 4.7 Hz, 1H, H2), 4.34 (dd, J = 8.1,
4.3 Hz, 1H, H3), 4.14 (dd, J = 9.2, 4.5 Hz, 1H, M4), 3.59 – 3.54 (m, 2H,
H10), 3.51 (t, J = 5.0 Hz, 2H, H13), 3.32 (t, J = 5.1 Hz, 2H, H9), 3.27 (t, J
= 5.2 Hz, 2H, H14), 3.24 – 3.15 (m, 1H, H4), 2.88 (dd, J = 13.0, 4.8 Hz,
1H, H1a), 2.65 (d, J = 13.0 Hz, 1H, H1b), 2.15 (t, J = 7.7 Hz, 3H, H8), 2.01
– 1.98 (m, 1H, M2), 1.88 (s, 3H, M5), 1.85 – 1.45 (m, 5H, M3+H5+H7),
1.35 (s, 3H, M1), 1.31 – 1.22 (m, 1H, H6). ESI MS m/z (calc): 519.2 (519.2,
[M - 2Cl]2+).
acetyl-L-methionine
(512
mg,
2.68
mmol)
and
N,N’-
diisopropylcarbodiimde (DIC) (411 mg, 3.26 mmol) were dissolved in DMF
(15 mL) and the resulting turbid solution was added dropwise to a
suspension of compound 4 (1.12 mg, 2.57 mmol) in DMF (20 mL). DIPEA
(0.885 mL, 5.35 mmol) and DMAP (37.1 mg, 0.30 mmol) were
subsequently added. The resulting mixture was stirred under argon at
room temperature for 20 hours, after which DMF was removed by vacuum
distillation at 323 K. The crude product was dissolved in H2O and the
precipitated DIPEA was filtered off and washed with H2O. Water was
removed by rotary evaporation and the crude oil was purified by silica
column chromatography using DCM/MeOH (8:2) as eluent. Yield: 1.01 g
(1.84 mmol, 72%). Rf = 0.7 (SiO2, DCM/MeOH [8:2]). 1H NMR (300 MHz,
Methanol-d4) δ (ppm): 8.24 (d, J = 7.6 Hz, 1H, NH), 8.09 (t, J = 5.2 Hz, 1H,
NH), 8.01 (t, J = 5.5 Hz, 1H, NH), 6.57 (s, 1H, NH), 6.45 (s, 1H, NH), 4.50
(dd, J = 7.8, 4.3 Hz, 1H, H2), 4.44 (dd, J = 8.6, 5.5 Hz, 1H, M4), 4.32 (dd,
J = 7.9, 4.4 Hz, 1H, H3), 3.62 (s, 4H, H11+H12), 3.55 (t, J = 5.4 Hz, 4H,
H10+H13), 3.43 – 3.33 (m, 4H, H9+H14), 3.21 (ddd, J = 8.8, 5.8, 4.4 Hz,
1H, H4), 2.93 (dd, J = 12.8, J = 4.9 Hz, 1H, H1B), 2.71 (d, J = 12.7 Hz, 1H,
H1A), 2.63 – 2.42 (m, 2H, M2), 2.23 (t, J = 7.3 Hz, 2H, H8), 2.09 (s, 3H,
M1), 2.00 (s, 3H, M5), 2.12 – 1.81 (m, 2H, M3), 1.84 – 1.50 (m, 4H, H5+H7),
1.44 (p, J = 7.2 Hz, 2H, H6). 13C NMR (75 MHz, Methanol-d4) δ (ppm):
176.1 (Cb), 174.0 (Cc), 173.3 (Cd), 166.0 (Ca), 71.3 (C11+C12), 70.6 +
70.4 (C10+C13), 63.3 (C3), 61.6 (C2), 57.0 (C4), 54.0 (M4), 41.1 (C1),
40.3 (C9), 40.3 (C14), 36.7 (C8), 32.8 (M3), 31.1 (M2), 29.8 (C6), 29.5
(C5), 26.8 (C7), 22.6 (M5), 15.30 (M1). ESI MS m/z (calc): 570.3 (570.2
[M + Na]+). HRMS m/z (calc): 548.25717 (548.25710 [M + H]+).
Characterization of [5]Cl4: Rf = 0.10 (SiO2, acetone/H2O/1 M HCl [16:4:1]);
1H NMR (300 MHz, Methanol-d4) δ (ppm): 9.86 (d, J = 5.4 Hz, 1H, A6),
9.76 (d, J = 5.3 Hz, 1H, A6*), 8.89 – 8.79 (m, 4H, T3’), 8.81 (d, J = 8.3 Hz,
2H, A3), 8.74 – 8.58 (m, 6H, T3’’+T3+B3), 8.48 – 8.36 (m, 4H, A4+T4’),
8.18 – 8.08 (m, 4H, T4’’+T4), 8.09 – 8.04 (m, 2H, A5), 8.00 – 7.88 (m, 4H,
B4+T6’’), 7.84 – 7.74 (m, 2H, T6), 7.53 – 7.42 (m, 4H, T5’’+T5), 7.32 –
7.20 (m, 4H, B6+B5), 4.28 – 4.21 (m, 1H, M4), 4.21 – 4.12 (m, 2H, H2+H3),
3.58 (s, 4H, H11+H12), 3.52 (t, J = 5.9 Hz, 2H, H10), 3.46 (t, J = 5.9 Hz,
2H, H13), 3.40 – 3.31 (m, 2H, H9), 3.30 – 3.23 (m, 2H, H14), 2.33 (d, J =
11.7 Hz, 1H, H4), 2.05 (t, J = 7.1 Hz, 2H, H8), 1.95 (d, J = 12.2 Hz, 1H,
H1a), 1.89 (s, 3H, M5), 1.76 (dd, J = 12.2, 4.3 Hz, 1H, H1b), 1.83 – 1.65 (m,
2H, M2), 1.60 (dd, J = 10.5, 5.4 Hz, 2H, M3), 1.50 – 1.35 (m, 1H, H5b),
1.38 (s, 3H, M1), 1.28 – 1.14 (m, 2H, H7), 1.14 – 1.00 (m, 1H, H6a), 0.66
– 0.53 (m, 1H, H6b), 0.52 – 0.38 (m, 1H, H5a). 13C NMR (75 MHz,
Methanol-d4) δ (ppm): 175.7, 173.1 (d), 172.9, 159.6, 159.4, 158.7, 158.6,
158.5, 158.1, 158.1, 158.0, 154.8, 154.4, 154.3, 153.4 + 153.1 (A6), 150.8,
150.7, 140.4, 140.3, 139.8, 139.7, 139.6, 139.5, 138.6, 138.4, 129.9, 129.7,
129.2, 129.1, 128.6, 128.5, 126.7, 126.5, 126.4, 126.2, 126.0, 125.9, 125.6,
125.3, 125.2, 71.3 (C11+12), 70.5 (C10), 70.3 (C13), 60.2 (C3), 58.5 (C2),
57.5 (C4), 52.8 (M4), 40.5 (C1), 40.4 (C9), 40.2 (C14), 36.3 (C8), 31.3 (M3),
30.1 (M2), 28.0 (C6), 27.7 (C5), 26.3 (C7), 22.8 (M5), 14.2 (M1). ESI MS
m/z (calc): 528.1 (528.1, [M-3Cl+H2O]3+), 405.1 (405.3, [M-
3Cl+Na+MeOH]4+). UV-Vis: λmax (ε in L mol-1 cm-1) in pure H2O: 447 nm
(1.16×104).
Bis(N-biotinyl)-3,6-dioxaoctane-1,8-diamine (2): A solution of 1,8-diamino-
3,6-dioxaoctane (110 mg, 0.74 mmol) and Et3N (152 mg, 1.50 mmol) in
dry DMF (1.0 mL) was added to a solution of compound 3 (500 mg, 1.46
mmol) in dry DMF (15 mL). The resulting solution was stirred under argon
for 26 hours at room temperature, after which the DMF was removed by
vacuum distillation at 323 K. The crude was purified twice by silica column
chromatography, using BuOH/H2O/AcOH (3:1:1) and DCM/MeOH (78:22)
as eluents, respectively. Yield: 308 mg (0.51 mmol, 69%). Rf = 0.38 (SiO2,
BuOH/H2O/AcOH [3:1:1]). 1H NMR (300 MHz, Methanol-d4) δ (ppm): 4.49
(dd, J = 7.8, 4.8 Hz, 2H, H2), 4.31 (dd, J = 7.8, 4.4 Hz, 2H, H3), 3.62 (s,
4H, H11), 3.55 (t, J = 5.5 Hz, 4H, H10), 3.37 (t, J = 5.5 Hz, 4H, H9), 3.21
(ddd, J = 8.7, 5.6, 4.5 Hz, 2H, H4), 2.93 (dd, J = 12.8, J = 4.9 Hz, 2H, H1B),
2.71 (d, J = 12.7 Hz, 2H, H1A), 2.23 (t, J = 7.3 Hz, 4H, H8), 1.81 – 1.55
(m, 8H, H5+H7), 1.44 (p, J = 7.2 Hz, 4H, H6). 13C NMR (75 MHz,
Methanol-d4) δ (ppm): 176.2 (Cb), 71.3 (C11), 70.6 (C10), 63.4 (C3), 61.6
(C2), 57.0 (C4), 41.1 (C1), 40.3 (C9), 36.8 (C8), 29.8 (C6), 29.5 (C5), 26.7
(C7). ESI MS m/z (calc): 623.4 (623.3 [M + Na]+), 320.2 (320.1 [M + H +
K]2+). HRMS m/z (calc): 601.28364 (601.28365 [M + H]+).
Optimization attempts for the synthesis of [Ru(tpy)(bpy)(1)](PF6)2
–
([6](PF6)2) A solution of ligand 1 (52 mg, 0.095 mmol) in H2O (2.9 mL) was
added to acetone (0.9 mL). The solution was deoxygenated with argon,
after which [Ru(tpy)(bpy)(OH2)](PF6)2 (15 mg, 0.019 mmol) was added and
the resulting solution was heated (different conditions (see table S1): 333
K, 353 K, or reflux) under argon for either 2 or 48 hours. The solvents were
removed by rotary evaporation at 303 K. The crude was purified by silica
column chromatography, using acetone/H2O/KPF6(sat) as the eluent,
followed by size exclusion chromatography (Sephadex LH-20) using
MeOH as the eluent in order to remove the excess KPF6 salt. Analysis of
the product composition in the different reaction conditions was performed
by 1H NMR (see Figure S1).
[{Ru(tpy)(bpy)}2(1)]Cl4 and [Ru(tpy)(bpy)(1)]Cl2 (compound [5]Cl2 and
[6]Cl2). A solution of ligand 1 (292 mg, 0.533 mmol) in H2O (15 mL) was
deoxygenated with argon and [Ru(tpy)(bpy)Cl]Cl (299 mg, 0.533 mmol)
was added. The resulting solution was stirred and heated to 353 K over 72
hours in the dark under argon. Solvent was removed by rotary evaporation
at 303
K and the crude product was purified by silica column
chromatography using acetone/H2O/1 M HCl (16:4:1) as the eluent. Two
orange fractions (Rf = 0.3 and Rf = 0.1, acetone/H2O/1 M HCl [16:4:1])
were collected. After removal of the solvent of the first fraction (Rf = 0.30,
acetone/H2O/1 M HCl [16:4:1]) by rotary evaporation at 303 K, the isomeric
mixture [6]Cl2/[7]Cl2 (83:17) was obtained as an orange powder. Yield:
488 mg (0.440 mmol, 83%). The binuclear complex [5]Cl4 was obtained as
a second fraction (Rf = 0.10, SiO2, acetone/H2O/1 M HCl [16:4:1]). The
solvent was removed by rotary evaporation and [5]Cl4 was obtained as an
orange powder. Yield: 81 mg (0.048 mmol, 9%).
[Ru(tpy)(bpy)(2)](PF6)2 ([8](PF6)2): Compound 2 (250 mg, 0.416 mmol) and
[Ru(tpy)(bpy)(OH2)](PF6)2 (66.5 mg, 83.2 µmol) were dissolved in
degassed H2O (16 mL). The resulting solution was stirred and heated to
353 K under argon for 47 hours. The solvent was removed by rotary
evaporation at 303 K and the crude product was purified by silica column
chromatography, using acetone/H2O/ KPF6,sat. (12:4:1) as the eluent.
Excess KPF6 was removed by size exclusion chromatography (Sephadex
LH-20) using MeOH as solvent. After evaporation of the solvent, an orange
solid was obtained. Yield: 94 mg (0.068 mmol, 82%). Rf = 0.62 (SiO2,
Acteon/H2O/HCl [100:10:1]). 1H NMR (400 MHz, Methanol-d4) δ (ppm):
This article is protected by copyright. All rights reserved.

10.1002/ejic.201800644
European Journal of Inorganic Chemistry
FULL PAPER
9.84 (d, J = 5.1 Hz, 1H, A6), 8.84 (d, J = 8.1 Hz, 1H, A3), 8.79 (d, J = 8.3
Hz, 2H, T5’), 8.77 (d, J = 8.4 Hz, 1H, T3’), 8.66 (d, J = 8.0 Hz, 1H, T3’’),
8.62 (d, J = 8.0 Hz, 1H, T3), 8.60 (d, J = 8.1 Hz, 1H, B3), 8.41 (ddd, J =
7.9, 7.9, 1.5 Hz, 1H, A4), 8.40 (dd, J = 8.2, 8.2 Hz, 1H, T4’), 8.12 (ddd, J =
7.8, 7.8, 1.5 Hz, 1H, T4’’), 8.10 (ddd, J = 7.8, 7.8, 1.6 Hz, 1H, T4), 8.08
(ddd, J = 7.7, 5.6, 1.3 Hz, 1H, A5), 7.94 (ddd, J = 8.2, 7.4, 1.6 Hz, 1H, B4),
7.91 (dd, J = 5.5, 0.6 Hz, 1H, T6’’), 7.78 (dd, J = 5.5, 0.8 Hz, 1H, T6), 7.47
(ddd, J = 7.7, 5.5, 1.2 Hz, 1H, T5’’), 7.44 (ddd, J = 7.6, 5.6, 1.3 Hz, 1H, T5),
7.29 – 7.22 (m, 2H, B6+B5), 4.49 (dd, J = 7.8, 4.5 Hz, 1H, H2), 4.31 (dd, J
= 7.9, 4.4 Hz, 1H, H3), 4.20 (dd, J = 8.4, 4.5 Hz, 1H, H2’), 4.16 (dd, J =
8.4, 4.4 Hz, 1H, H3’), 3.63 (s, 4H, H11+H11’), 3.60 – 3.51 (m, 4H,
H10+H10’), 3.41 – 3.32 (m, 4H, H9+H9’), 3.21 (ddd, J = 8.8, 5.9, 4.4 Hz,
1H, H4), 2.92 (dd, J = 12.8, 4.9 Hz, 1H, H1b), 2.69 (d, J = 12.7 Hz, 1H,
H1A), 2.32 (ddd, J = 11.6, 4.4, 2.5 Hz, 1H, H4’), 2.21 (t, J = 7.4 Hz, 2H, H8),
2.06 (t, J = 7.1 Hz, 2H, H8’), 1.93 (d, J = 12.2 Hz, 1H, H1’a), 1.75 (dd, J =
12.2, 4.6 Hz, 1H, H1’b), 1.72 – 1.51 (m, 4H, H7+H5), 1.51 – 1.36 (m, 3H,
H5’b+H6), 1.31 – 1.20 (m, 2H, H7’), 1.15 – 1.02 (m, 1H, H6’b), 0.65 – 0.53
(m, 1H, H6’a), 0.53 – 0.43 (m, 1H, H5’a). 13C NMR (101 MHz, Methanol-d4)
δ (ppm): 176.1 (b), 175.7 (b’), 165.2 (a’), 159.5, 159.3, 158.4, 158.1, 154.7
(T6’’), 154.3 (T6), 153.1 (A6), 150.6 (B6), 140.4 (T4+T4’’), 139.8 (A4),
139.6 (B4), 138.7 (T4’), 129.8 (T5), 129.7 (T5’’), 129.1 (A5), 128.6 (B5),
126.6 (T3’’), 126.4 (B3), 126.2 (A3), 126.0 (T3’), 125.8 (T3’), 125.2 (T3),
71.2 (C11+C11’), 70.5 (C10+C10’), 63.3 (C3), 61.6 (C2), 60.2 (C3’), 58.5
(C2’), 57.4 (C4’), 57.0 (C4), 41.1 (C1), 40.5 (C1’), 40.3 (C9+C9’), 36.8 (C8),
36.3 (C8’), 29.7 (C6), 29.5 (C5), 28.0 (C6’), 27.8 (C5’), 26.9 (C7), 26.3
(C7’). ESI MS m/z (calc): 545.4 (545.7, [M]2+). Elemental analysis calcd
(%) for C51H63F12N11O6P2RuS2 + 1 H2O: C 43.78, H 4.68, N 11.01; found:
C 43.81, H 4.54, N 11.04. UV-Vis: λmax (ε in L mol-1 cm-1) in pure H2O: 448
nm (5.50×103).
or [8](PF6)2 in methanol (1.26 mM) were added and after each addition, a
spectrum was recorded.
Acknowledgements
The European Research Council is acknowledged for an ERC
starting grant to S.B. NWO is acknowledged for a VIDI grant to
S.B. A.P. gratefully acknowledges a postdoctoral fellowship from
the Swiss National Science Foundation (SNSF), Grant
P2BSP2_175003. The authors thank Thomas Ward for a
generous gift of mature streptavidin. The COST action CM1105 is
acknowledged for stimulating scientific discussions. Prof. E.
Bouwman is kindly acknowledged for support and scientific
discussions.
Keywords: streptavidin • biotin • photoactivated chemotherapy •
photosubstitution • thioether
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mg in 2.5 mL PBS solution, 1.7×10-4 M) was prepared. To investigate the
photochemical properties of complex [8](PF6)2 x mL of stock solution (x =
1.30 mL) was added to a quartz cuvette filled with 3.00-x mL PBS solution.
Final concentration of [8]2+ was 7.5×10-5 M. The sample was deoxygenated
with argon for 20 minutes. Irradiation was performed using a custom 465
nm LED (Δλ1/2 = 25 nm, 1.4 mW, 220 min, 19 J·cm-2), and the samples
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of one equivalent of streptavidin was performed in NaH2PO4 buffer (3.00
mL, 20 mM, pH 7.0) at 298 K. After deoxygenation with nitrogen irradiation
occurred at 298 K under nitrogen flow with the abovementioned 465 nm
LED at 3.30 W = 4.46·10-8 mol·s-1.
HABA displacement titration. For each experiment streptavidin was added
to a HABA solution (1.07 mM) in NaH2PO4 buffer (2.7 mL, 20 mM, pH 7.0)
in a cuvette resulting in concentration of tetrameric streptavidin of 6.26 µM
for the measurement with biotin and 7.27 µM with [8](PF6)2. The solution
equilibrated for five minutes. UV-vis spectra of this sample were recorded
using the buffer as background. Aliquots (5µL) of biotin in buffer (1.56 mM)
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10.1002/ejic.201800644
European Journal of Inorganic Chemistry
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Key Topic*
Synthesis and avidin binding of
ruthenium complexes functionalized with
a light-cleavable free biotin moiety
Page No. – Page No.
A new synthetic strategy is presented for preparing photoactivated chemotherapeutic
ruthenium compounds functionalized with a free biotin group. The free biotin fragment,
which is bound to ruthenium via a thermally stable Ru-S bond, can still bind to streptavidin.
Blue light irradiation resulted in the cleavage of the biotin-bearing protecting ligand, thus
liberating the ruthenium aqua compound.
Title
This article is protected by copyright. All rights reserved.