Ferrocene-biotin conjugates targeting cancer cells: Synthesis, interaction with avidin, cytotoxic properties and the crystal structure of the complex of avidin with a biotin-linker-ferrocene conjugate

Article abstract of DOI:10.1021/om4003126

Friedel-Crafts acylation of ferrocene with a biotin-derived carboxylic acid having 6-aminohexanoyl linkers attached to the biotin carboxylic group and with desthiobiotin afforded the corresponding ferrocene-biotin bioconjugates. These compounds as well as biotinyl ferrocenyl ketone exhibit high affinity for avidin. Their cytotoxicity against cancer cell lines having various levels of biotin receptors (SMVT) was measured and revealed that lines displaying high levels of SMVT (SW620) were the most susceptible. This suggests that biotin serves as a biological vector delivering cytotoxic ferrocenyl moieties to cancer cells. The crystal structure of the complex of avidin with a conjugate having two linker units between biotin and ferrocene was determined and revealed stabilization of several different conformations of the ligand within the protein binding pocket.

Full text of DOI:10.1021/om4003126

Article
pubs.acs.org/Organometallics
Ferrocene−Biotin Conjugates Targeting Cancer Cells: Synthesis,
Interaction with Avidin, Cytotoxic Properties and the Crystal
Structure of the Complex of Avidin with a Biotin−Linker−Ferrocene
Conjugate
Damian Plazuk,† Janusz Zakrzewski,*,† Michele Salmain,‡ Andrzej Błauz,§ Błazej Rychlik,§
̀
̇
̇
̇
Paweł Strzelczyk,∥ Anna Bujacz,∥ and Grzegorz Bujacz∥
†Department of Organic Chemistry, Faculty of Chemistry, University of Łod
‡Chimie ParisTech, Laboratoire Charles Friedel, and CNRS, UMR 7223, 11 rue Pierre et Marie Curie, F-75005 Paris, France
§Cytometry Laboratory, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łod
́
z, Tamka 12, 91-403 Łodz, Poland
́
́
́
́
z,
́
́
́
12/16 Banacha Street, 90-237 Łodz, Poland
∥Institute of Technical Biochemistry, Łod
́ ́
z University of Technology, Stefanowskiego 4/10, 90-924 Łodz, Poland
́ ́
S
* Supporting Information
ABSTRACT: Friedel−Crafts acylation of ferrocene with a
biotin-derived carboxylic acid having 6-aminohexanoyl linkers
attached to the biotin carboxylic group and with desthiobiotin
afforded the corresponding ferrocene−biotin bioconjugates.
These compounds as well as biotinyl ferrocenyl ketone exhibit
high affinity for avidin. Their cytotoxicity against cancer cell
lines having various levels of biotin receptors (SMVT) was
measured and revealed that lines displaying high levels of
SMVT (SW620) were the most susceptible. This suggests that
biotin serves as a biological vector delivering cytotoxic ferrocenyl moieties to cancer cells. The crystal structure of the complex of
avidin with a conjugate having two linker units between biotin and ferrocene was determined and revealed stabilization of several
different conformations of the ligand within the protein binding pocket.
Scheme 1. Synthesis of Biotin−Ferrocene Conjugates 1−4
INTRODUCTION
■
The bioorganometallic chemistry of ferrocene continues to
attract significant interest mainly due to the anticancer activity
of numerous ferrocene derivatives.1 Pioneering work in this
field was done by Jaouen and his co-workers, who
demonstrated that diverse ferrocenyl compounds structurally
related to a classical antibreast cancer drug, tamoxifen, exhibited
a broad spectrum of cytotoxic activity, with IC50 values in some
cases being in the submicromolar range.2 Another promising
and intensively developed axis of research in this field deals
with ferrocenyl compounds exhibiting antimalarial activity.3
We recently disclosed the synthesis of a novel type of
ferrocene−biotin conjugate, biotinyl ferrocenyl ketone 1, via
direct Friedel−Crafts acylation of ferrocene with biotin
(Scheme 1).4 We expected that ferrocenyl−biotin conjugates
might exhibit enhanced anticancer activityit is well
established that biotin (vitamin H) is necessary for cell growth
and many rapidly growing cancer cells have high levels of a
specific vitamin receptor (sodium-dependent multivitamin
transporter, SMVT) on their surface.5 Therefore, biotin may
serve as a biological vector for the selective delivery of cytotoxic
compounds to the cancer cells.6 In this paper we demonstrate
the applicability of this approach in the design of anticancer
ferrocenyl compounds. From this standpoint it was interesting
to compare the cytotoxic activity of 1 with that of analogues
having the bicyclic biotin and the ferrocenyl moieties separated
Special Issue: Ferrocene - Beauty and Function
Received: April 11, 2013
© XXXX American Chemical Society
A
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Scheme 2. Acylation of Ferrocene with 6-Acetamidohexanoic Acid (7)
by longer spacer arms. Furthermore, we intended to compare
the cytotoxic activity of 1 with that of its desthiobiotinyl
analogue, which is expected to have a lower affinity for the
target receptor (as evidenced for avidin).7 For this purpose we
synthesized analogues of 1 featuring one and two 6-amino-
hexanoic spacers between the biotin fragment and ferrocene
(2 and 3, respectively) and the desthiobiotin conjugate 4. We
measured the affinity of 1−4 for avidin and studied their
cytotoxic properties against selected cancer cells expressing
different levels of SMVT. Finally, to gain insight into the
interaction of biotinylated ferrocenes with avidin, we solved the
X-ray structure of the complex of avidin with compound 3.
in 7 and is in line with recent work disclosing triflic acid promoted
Friedel−Crafts-type reactions with amides and ureas.11
Encouraged by these results, we performed the reaction of
ferrocene with acids 5 and 6 under the same conditions. We
isolated the expected biotinylated ferrocenes 2 and 3, albeit in
significantly lower yields (28% and 16%, respectively). This
result shows that, in principle, acids containing 6-amino-
hexanoic linkers may be used as acylating agents in Friedel−
Crafts reactions, but in this case the acid/TFAA/TfOH system
works less efficiently. Fortunately, the isolation procedure for 2
and 3 is relatively easy (see the Experimental Section). Finally,
the reaction of ferrocene with desthiobiotin afforded compound
4 in a 34% yield (Scheme 1).
Interactions of 1−4 with Avidin. Relative Binding
Affinity of 1−4 to Avidin. The association of avidin with
biotin and its derivatives has attracted a great amount of
interest because of the exceptional strength of these species
(Ka ≈ 1015 M−1 for the parent biotin) and the wide range of
applications.14 In a previous paper4 we had applied a solid-
phase competitive enzymatic assay to determine the relative
binding affinity (RBA) of 1 to avidin and its ruthenocenyl and
pyrenyl analogues. In this paper we applied this method to
determine the IC50’s together with the RBA’s of complexes 2−4.
The results are shown in Table 1.
RESULTS AND DISCUSSION
■
Synthesis of 2−4. The synthesis of 1 was reported in our
earlier work.4 This compound was formed in 55% yield from
the Friedel−Crafts reaction of ferrocene with biotin in the
presence of trifluoroacetic anhydride (TFAA) and trifluor-
omethanesulfonic (triflic) acid (TfOH). Ruthenocene, pyrene,4
and several other arenes8 react similarly. Compounds obtained
in this way constitute a novel class of biotin derivatives, having a
C−C bond linking the carbonyl group in the biotin valeryl
chain with a biotinylated moiety. Their advantage over the
more common biotin conjugates having amide bonds between
biotin and the biotinylated entity is improved resistance to
biotinidase,9 an enzyme present in biological samples that
cleaves the C−N bond in biotin amides.
Table 1. Values of IC50 and the Relative Binding Affinities
(RBA’s) of Biotin, Desthiobiotin, and Conjugates 2−4 for
Avidin
In a continuation of our research program, which is focused
on the use of functionalized and biologically relevant acids in
Friedel−Crafts-type reactions,4,10 we became interested in the
possibility of synthesis of biotin−ferrocene conjugates having
aliphatic 6-aminohexanoic linkers between the biotin and
ferrocene entities directly from acids 5 and 6 and ferrocene.
Since we were aware of the instability of the amide bond and
the acylating ability of amides in strongly acidic media used in
Friedel−Crafts reactions,11 we decided to start by studying the
reaction of ferrocene with 6-acetamidohexanoic acid (7),
chosen as a model acid containing an amide bond, similar to
those present in 5 and 6. We found that this reaction,
performed in the presence of TFAA and TfOH in dichloro-
methane at room temperature, afforded ketone 8 in a
satisfactory yield (60%) along with a small amount (5%) of
acetylferrocene (Scheme 2).
In the literature there are only a few reports on the use of
acid derivatives containing amide groups as arene acylating
reagents.12 They are all limited to some N-acylated α-amino
acid chlorides or esters. β-Amidoacylation of ferrocene was
achieved using strained N-acylated azetidinones in a superacidic
medium in a reaction involving cleavage of the C(O)−N
bond in the acylating agent.13 Therefore, the synthesis of 8
provides evidence that a carboxylic acid containing a remote
amide functionality may be used, under the appropriate
experimental conditions, as an efficient acylating reagent in a
Friedel−Crafts reaction. The formation of acetylferrocene
reveals a weak acylating ability of the acetamido moiety present
compd
biotin
IC50 (nM)
RBA (%)
100
9.0 2.1
33 2.0
a
1
73
2
20.3 1.8
18 2.0
44
50
4
5
3
desthiobiotin
21.6 6.5
25.4 4.8
42 10
35
4
5
a
Reference 4. IC50(biotin) = 24 7 nM.
The RBA of 1 was found to be equal to 73% in our earlier
work.4 This means that the introduction of one or two 6-
aminohexanoic acid linkers did not increase the affinity of
bioconjugates 2 and 3 for avidin, although a conjugate with a
longer linker (3) has a slightly higher affinity than 2. As reported
in the literature,7 the affinity of desthiobiotin for avidin is
significantly lower than that of biotin. Interestingly, in this case
appending a ferrocenyl substituent practically did not influence the
RBA value.
Cytotoxicity of Ferrocene Conjugates. Biotin is a growth
promoter at the cellular level, and its content in tumors is
substantially higher than that in normal tissues. As mammals
are not able to biosynthesize biotin, they utilize the two main
sources of this vitamin: food and symbiotic bacteria inhabiting
the colon. Biotin is taken up from the intestinal content via the
sodium-dependent multivitamin transporter (SMVT),5
a
product of the Slc5a6 gene that is expressed in the apical
B
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region of the epithelial cells. We thought that it would be
interesting to study whether the biotin−ferrocene conjugates
had a cytotoxic effect on cancer cell lines and also to check
whether the response of these cancer cells to the biotin−
ferrocene conjugates correlates with the level of SMVT (or
Slc5a6). For this purpose, we selected three different human
intestine derived tumor cell lines: COLO-205 (colon
adenocarcinoma), HCT116 (colon carcinoma), and SW620
(colon adenocarcinoma).
The level of expression of the Slc5a6 gene was determined
for the three selected cell lines. Slc5a6 mRNA was the most
abundant in SW620 cells (10.4 4.4 copies per 1000 copies of
the reference gene, n = 3), while COLO-205 and HCT116
exhibited the same level of expression (3.6 1.1 and 3.6 1.8
copies per 1000 copies of the reference gene, n = 3, respectively).
The cytotoxicity of the ferrocene−biotin conjugates was tested
within the range of 3 nM to 30 μM (we did not use higher
concentrations, as they would be highly above a realistic assumption
of concentration achievable in any compartment of a living
organism). The results are gathered in Table 2 and Figure 1.
approximately 6 times weaker (IC50 = 78.5 1.9 μM) than that of
1. It is comparable to that of compound 8 (IC50 = 70.1 1.8 μM),
which is an analogue of 2, having an acetyl group in the place of the
biotin moiety. The above data clearly confirm the crucial role of the
biotin moiety in the cytotoxic activity of the compounds under
study. In contrast, the presence of the desthiobiotin moiety in
compound 4 did not seem to enhance its cytotoxic activity.
The idea of using biotin as a vector to deliver a cytotoxic agent to
cancer cells overexpressing biotin receptors (SMVT) was applied
earlier by Ojima6 to improve the cytotoxic properties of taxoids. In
this context, it should be emphasized that many cancer cells
overexpress biotin receptors more than they do folate and/or
vitamin B12 receptors, which until now were more widely used as
biomarkers in targeted chemotherapy.5 Other reports on highly
cytotoxic biotin conjugates of natural products have also appeared
in the literature.15 The results obtained in this work clearly indicate
that biotin can also be used for tumor-targeted delivery of a
cytotoxic ferrocene moiety. Therefore, cytotoxic biotin−ferrocene
conjugates are promising candidates for anticancer drugs.
X-ray Structure of the Complex of Avidin with Compound
3. We attempted to obtain crystals of complexes of avidin with
compounds 1−4 suitable for X-ray diffraction studies. However,
so far we succeeded only in the case of the complex of avidin
with the conjugate 3 (hereafter denoted as avidin-3). Crystals
of this complex were obtained by the hanging drop vapor-
diffusion technique. Its X-ray structure reveals interesting
features related to protein−organometallic ligand interactions.
The model consists of residues 2−125 of monomer A and
2−124 of monomer B, which are quite visible on the electron
density map. The overall fold of the protein and its tetrameric
arrangement are similar to those described earlier.16 Each monomer
is constructed of eight antiparallel strands, which form a β-barrel
with the binding site for biotin or a derivative located at its wider
part (Figure 2). The monomer is stabilized by one disulfide bridge,
Figure 1. Viability of various tumor cell lines in the presence of
biotin−ferrocenyl conjugate 1 (for compounds 2−4 and 8 see the
Supporting Information).
As can be seen from Table 2, SW620 cells are the most
susceptible to conjugates 1, 2, 4, and 8. The COLO-205 and
Table 2. Susceptibility of Colon Cancer Cells to Biotin−
a
Ferrocene Conjugates
IC50 (μM)
compd
COLO-205
HCT116
SW620
1
2
3
4
8
≫100
≫100
≫100
≫100
≫100
NA
13.0 3.6
26.2 1.4
≫100
NA
87.5 1.8
≫100
NA
78.5 1.9
70.1 1.8
a
IC50 values are given in μM. NA denotes that the data did not allow
calculation of the IC50 parameter. Values given as ≫100 denote that
the calculated IC50 parameter value is highly above 100 μM.
HTT116 cells are resistant to these compounds, with one
exception (HCT116 cells and compound 3). Therefore, the
correlation between the susceptibility of cells to the compounds
under study and Slc5a6 expression is clear. Conjugate 1 was the
most toxic to the SW620 cells (IC50 = 13.0 3.6 μM), being
approximately twice as potent as 2 (IC50 = 26.2 1.4 μM),
which in turn was much more potent than 3. This means that 6-
aminohexanoyl spacers exert a deleterious effect on the cytotoxic
activity of the investigated conjugates. The cytotoxic activity of the
desthiobiotin−ferrocene conjugate 4 against the SW620 cells is
Figure 2. Crystal structure of avidin-3.
three intermolecular salt bridges, and intramolecular hydrogen
bonds.17
In the orthorhombic crystalline form, one molecular dyad is
coincident with the crystallographic 2-fold axis; thus, an avidin
C
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dimer is located in the asymmetric unit. Therefore, the
quaternary structure of avidin has been described as a dimer
of dimers.
The tertiary and quaternary structures of avidin-3 are
generally similar to those of other biotin−avidin complexes
(PDB ID: 2AVI and 1AVD).16 However, several noteworthy
differences are evident. The set of hydrogen-bond interactions
is almost identical with that of the avidin−biotin complex with
respect to the bicyclic ring system. The ligand is stabilized in
the binding pocket through hydrogen bonds (Table 3; the atom
Table 3. Hydrogen Bond Protein−Ligand Interactions in
avidin-3
3
monomer A
distance (Å)
monomer B
distance (Å)
avidin
atom
atom
Ser16 OH
Tyr33 OH
Asn12 NH2
Thr35 OH
Asn118 O
Thr77 OH
Ala39 NH
Thr38 OH
Ser75 OH
Ser73 OH
O1
O1
O1
N2
N1
S1
2.98
2.88
3.12
2.98
2.94
3.01
2.57
2.89
3.08
O1
O1
O1
O4
N1
S1
2.86
2.84
3.11
3.02
2.95
3.07
a
Figure 4. Structural superimposition of avidin monomers A (violet)
and B (light blue) showing different conformations of loops L3-4 and
L6-7.
O2
O2
N3
a
a
O2
O2
3.44
3.47
Trp110 from the symmetry-related monomer. In the first part
of the linker chain, O2 interacts with the nitrogens of Ala39 and
Thr38 from the loop L3-4, whereas N3 interacts via the
hydrogen bond with OH of Ser75 (Table 3). The second part
of the ligand aliphatic chain is stabilized in the protein-binding
site by a close interaction of O3 with the ligand from the
symmetry-related monomer. Moreover, N4 is stabilized by a
water hydrogen network including two water molecules and the
main chain atoms of Leu99, Ser101, and Lys111 (distance <3 Å).
The keto oxygen bound to the ferrocene moiety (O4) is stabilized
by van der Waals contacts with loop 3-4 from the symmetry-
related molecule.
a
Conformation II of 3.
numbering scheme is shown in Figure 3), van der Waals
contacts, and hydrophobic interactions.
Figure 3. Polar atom numbering scheme for 3.
In monomer B two different conformations of 3 were
observed, owing to the different positioning of loop L3-4
(residues 35−45). The first conformation (I) is located parallel
to the axis of the β-barrel, and the second (II) is turned into the
direction of loop L3-4 and the sugar moiety attached to Asn17
(Figure 6). The bicyclic biotin moiety in both conformations
interacts by hydrogen bonds with five polar residues, as
presented in Table 3. The nitrogen atom in the ureido fragment
(N2). similarly to the case for the avidin−homobiotin
complex,17 does not display a hydrogen-bond interaction with
Thr35, as a result of a conformational change of loop L3-4. The
remaining hydrogen bonds are maintained (Table 3). The
tetrahydrothiophene ring is stabilized similarly as in monomer
A through interactions with the aromatic residues. The keto
oxygen (O2) from the aliphatic chain in conformation II of the
ligand interacts by hydrogen bonds with Ser75 and Ser73, as in
the avidin−biotin complex (PDB ID: 1AVD), and additionally
by the water-mediated hydrogen bond with Ser101. The keto
oxygen O2 in conformation I is stabilized only by water-
mediated hydrogen interactions with Ser75 and Ser101. Most
interestingly, ligand 3 in conformation II makes π-electron
contacts between both the cyclopentadienyl rings of ferrocene
and two residues: Ala36 from loop L3-4 of monomer B and
Trp110 from the symmetry-related monomer. Moreover, the
The overall structure of monomer A differs from that of
monomer B in the areas of loops L3-4 (residues 35−45) and
L6-7 (residues 85−90) (Figure 4). As a result, in the crystal
structure of avidin-3, ligand 3 adopts a single conformation in
monomer A, whereas in monomer B the ligand adopts two
different conformations (Figure 5).
In monomer A ligand 3 is stabilized in the binding pocket by
nine direct hydrogen bonds and four van der Waals contacts. In
the deepest part of the cavity several polar residues are available for
hydrogen bonding to atoms of the ureido fragment. Nitrogen N1
interacts with the oxygen of Asn118, N2 interacts with the
hydroxyl group of Thr35, and O1 acts as an acceptor for three
donor groups: OH of Ser16, OH of Tyr33, and NH2 of Asn12.
On the other side of the pocket the sulfur atom (S1) in a
tetrahydrothiophene ring interacts with the hydroxyl group of
Thr77. The set of hydrogen-bond interactions for the bicyclic
moiety of biotin is generally similar to that of the avidin−norbiotin
complex (PDB ID: 1LDO).18 When that complex is compared to
the avidin−biotin complex (PDB ID: 1AVD), the only difference
is the weak interaction of Thr77 with S1 (distance 3.75 Å).16a
Additionally, the tetrahydrothiophene ring of 3 is stabilized
by the aromatic residues Phe79, Trp97, and Trp70 and also
D
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Figure 5. Ligand 3 (yellow) in the binding pocket of monomers A and B of avidin (violet). In monomer B two conformations of the ligand
(I and II) are shown.
1
solvent signals: CDCl3, δ 7.27 ppm for H and δ 77.00 ppm for 13C;
keto oxygen O4 of the ligand acts as an acceptor to the
hydroxyl group of Thr35.
Generally, the interactions of 3 with avidin appear to be
different in the two monomers. The biotin bicyclic moiety is
located in the deepest part of the binding pocket in both
monomers, and most interactions found for the ligand in its
complex with avidin are maintained. Due to the high flexibility
of the linker, the interaction of the ferrocene moiety differs in
CD3OD, δ 3.31 ppm for 1H and δ 49.15 ppm for 13C; DMSO-d6, δ 2.50
for H and δ 39.51 for 13C. Spectra were recorded at room temperature
1
(291 K); chemical shifts are in ppm and coupling constants in Hz. EI and
HR-EI analyses were performed in the positive mode at 75 eV. Thin-layer
chromatography (TLC) was performed on aluminum sheets precoated
with Merck 5735 Kieselgel 60F254. Column chromatography was carried
out on silica gel 60 (0.040−0.063 mm, 230−400 mesh, Fluka).
Dichloromethane was distilled from calcium hydride and stored over
both monomers. As a result of the this flexibility and
metallocene moiety interaction with the protein in monomer
B, a spatial rearrangement of loop L3-4 takes place. In this
context the crystal structure of avidin-3 is considered to be
important for a better understanding of the avidin−ligand
interactions. It is also worth noting that only a few crystal
structures of the complexes of proteins with ferrocene-derived
ligands have hitherto been reported.19
activated molecular sieves 4A (8−12 mesh). Reactions with ferrocene
were carried out using standard Schlenk techniques. Chemicals,
biochemicals, and solvents (HPLC grade) were purchased from Sigma-
Aldrich or AK Scientific (USA) and used as received. Avidin from egg
white was purchased from Interchim; its activity was equal to 14.8 units/
mg. A 1 mg mL−1 stock solution of avidin was prepared in PBS and kept at
4 °C. Stock solutions of biotin (2 mg mL−1), desthiobiotin (1 mg mL−1),
and conjugates (1 mg mL−1) were prepared in PBS and DMSO,
respectively.
Synthesis of Compounds. Compounds 14 and 6-biotinamido-
hexanoic acid (5)20 were prepared according to published procedures.
6-Acetamidohexanoic Acid (7). To a stirred slurry of 6-amino-
hexanoic acid (5.0 g, 38.1 mmol)) and sodium carbonate (8.2 g,
77.4 mmol) in anhydrous THF (50 mL) was added a solution of acetyl
chloride (3.29 g, 2.98 mL, 41.9 mmol) in THF (5 mL). The resulting
mixture was stirred in a water cooling bath (exothermic reaction!)
until the reaction temperature decreased to room temperature. After
4 h, distilled water (50 mL) was added and the resulting solution was
acidified with hydrochloric acid (up to pH 0−1). The product was
extracted 5 times with ethyl acetate (100 mL each portion), the
extracts were dried over sodium sulfate, and the solvent was
evaporated. Yield: 1.9 g. The crude product was washed with diethyl
ether and n-pentane and used in the next step without further
purification. Its spectroscopic data were identical with those of the
authentic sample.21 1H NMR (DMSO-d6): δ 11.95 (s, 1 H, COOH),
7.75 (br s, 1 H, NH), 2.95−3.03 (m, 2 H, H-6), 2.18 (t, J = 7.53 Hz, 2
H, H-2), 1.77 (s, 3 H, CH3), 1.44−1.52 (m, 2 H, H-3), 1.33−1.41 (m,
2 H, H-5), 1.21−1.29 (m, 2 H, H-4). 13C NMR (DMSO-d6): δ 174.3
(CO), 168.8 (CO), 38.3, 33.6, 28.8, 26.0, 24.2, 22.6. MS (EI): (M+)
341.0. HRMS (EI): calcd for C18H23FeNO2 341.10783, found
CONCLUSIONS
■
We demonstrated the feasibility of a direct Friedel−Crafts
acylation of ferrocene with biotin-derived acids featuring
aminoacyl linkers and with desthiobiotin. The resulting
conjugates along with compound 1 obtained by acylation of
ferrocene with biotin display high affinity for avidin and
cytotoxicity against SW620 cancer cell lines, thus expressing a
high level of the biotin receptor. This suggests that the biotin
moiety may serve as a biological vector for the targeted cancer
cells with cytotoxic ferrocenyl groups. Finally, an X-ray
diffraction study of avidin-3 revealed unusual protein−ligand
interactions and stabilization of different conformations of the
ligand in the protein binding pocket.
EXPERIMENTAL SECTION
■
General Considerations. 1H and 13C NMR spectra were recorded
on a Bruker ARX 600 MHz spectrometer (600 MHz for 1H and 151 MHz
for 13C). Chemical shifts in the H spectra were referenced relative to
1
E
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Figure 6. Protein−ligand interactions in avidin-3: (a) in monomer A; (b) in monomer B (ligand conformation I); (c) in monomer B (ligand
conformation II).
341.10666 (δ 1.08641 ppm). Anal. Calcd for C18H23FeNO2: C, 63.36;
H, 6.79; N, 4.10. Found: C, 63.31; H, 6.75; N, 4.10.
20 h, and the precipitated product was filtered off, washed with water, and
dried at 40 °C under vacuum (ca. 5 Pa). Yield: 198 mg. The product was
used in the next step without further purification. Its spectral data were
identical with those of the authentic sample.22 1H NMR (DMSO-d6): δ
11.94 (br s, 1 H, COOH), 7.79−7.59 (m, 2 H, 2 × NH, H-12 and H-19),
6.39 (br s, 1 H, 3-NH), 6.33 (br s, 1 H, 1-NH), 4.30 (dd, J = 5.1, 7.3 Hz, 1
H, H-6a), 4.13 (dd, J = 4.7, 7.0 Hz, 1 H, H-4a), 3.13−3.05 (m, 1 H, H-4,
CH2-alkyl chain), 3.04−2.97 (m, 4 H, CH2-alkyl chain), 2.82 (dd, J = 5.3,
12.4 Hz, 1 H, H-6), 2.58 (d, J = 12.4 Hz, 1 H, H-6), 2.18 (t, J = 7.5 Hz, 2
H, CH2-alkyl chain), 2.08−2.00 (m, 4 H, CH2-alkyl chain), 1.66−1.57
6-(6-Biotinamidohexanamido)hexanoic Acid (6). To a solution of
6-biotinamidohexanoic acid (5; 357.5 mg, 1.0 mmol) in a mixture of
DMF (10 mL) and water (2 mL) were added DIPEA (381.4 mg,
514 μL, 2.95 mmol), TSTU (380 mg, 1.26 mmol), and (after stirring at
room temperature for 20 min) 6-aminohexanoic acid (196 mg, 1.5 mmol).
After 1 h the solvents were evaporated to dryness, the solid residue was
dissolved in 50% ethanol (10 mL), and the solution was acidified with
concentrated hydrochloric acid. The resulting solution was kept at 4 °C for
F
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1 H, H-4a), 4.19 (s, 5 H, Cp), 3.25 (q, J = 6.7 Hz, 2 H, H-10), 3.18−3.11
(m, 1 H, H-4), 2.90 (dd, J = 4.5, 12.8 Hz, 1 H, H-6), 2.69−2.75 (m, 3H,
H-6 and H-17), 2.21 (t, J = 7.3 Hz, 2 H, CH2-alkyl chain), 1.80−1.61 (m,
6 H, CH2-alkyl chain), 1.56 (quin, J = 7.2 Hz, 2 H, CH2-alkyl chain),
1.48−1.36 (m, 4 H, CH2-alkyl chain). 13C NMR (CDCl3): δ 204.7 (18-
CO), 173.2 (11-CO), 164.4 (2-CO), 79.0 (Cp-ipso), 72.2 (Cp), 69.8
(Cp), 69.3 (Cp), 61.8 (C-4a), 60.2 (C-6a), 55.6 (C-4), 40.5 (C-6), 39.4
(C-17), 39.2 (C-10), 36.1 (CH2-alkyl chain), 29.4 (CH2-alkyl chain), 28.2
(CH2-alkyl chain), 28.1 (CH2-alkyl chain), 26.7 (CH2-alkyl chain), 25.8
(CH2-alkyl chain), 24.0 (CH2-alkyl chain). MS (EI): 525.3 (M+). HRMS
(EI): calcd for C26H35FeN3O3S 525.174875 found 525.17428 (δ 1.08641
ppm). Anal. Calcd for C26H35FeN3O3S: C, 59.43; H, 6.71; N, 8.00.
Found: C, 59.44; H, 6.93; N, 8.09.
6-(6-Biotinamidohexanamido)hexanoylferrocene (3). The proce-
dure was the same as for 8, except 6-(6-biotinamidohexanamido)-
hexanoic acid (6; 94 mg, 0.200 mmol), ferrocene (37 mg, 0.200 mmol),
TFAA (227 mg, 150 μL, 1.08 mmol), and TfOH (85 mg, 50 μL,
0.565 mmol) were used. Chromatography on silica (silica 25 mL, eluent
dichloromethane−MeOH gradient 0−10% MeOH) and then on a SF10-
8g column (Varian) afforded 8 as an orange solid. Yield: 21 mg (16.4%).
1H NMR (CD3OD): δ 4.83 (t, J = 1.88 Hz, 2 H, Cp), 4.59 (t, J = 1.88
Hz, 2 H, Cp), 4.49 (dd, J = 7.53, 4.89 Hz, 1 H, H-6a), 4.30 (dd, J = 7.91,
4.52 Hz, 1 H, H-4a), 4.23 (s, 5 H, Cp), 3.18−3.22 (m, 3 H, H-4 and
CH2-alkyl chain), 3.14−3.18 (m, 2 H, CH2-alkyl chain), 2.92 (dd, J =
12.80, 4.89 Hz, 1 H, H-6), 2.79 (t, J = 7.34 Hz, 2 H, H-24), 2.71 (d, J =
12.80 Hz, 1 H, H-6), 2.17−2.21 (m, 4 H, CH2-alkyl chain), 1.35−1.75
(m, 18H, CH2-alkyl chain). 13C NMR (CD3OD): δ 208.03 (25-CO),
176.1 (11- or 18-CO), 176.1 (11- or 18-CO), 166.2 (2-CO), 80.2 (Cp-
ipso), 74.0 (Cp), 71.1 (Cp), 70.8 (Cp), 63.5 (C-4a), 61.8 (C-6a), 57.1
(C-4), 41.2 (C-6), 40.6, 40.4 (2 × C), 37.2, 37.0, 30.5, 30.3, 29.9, 29.6,
27.9, 27.7, 27.1, 26.9, 25.6 (14 × CH2-alkyl chain). MS (EI): 638.4 (M+).
HRMS (EI): calcd for C32H46FeN4O4S 638.25966, found 638.25984 (δ
0.43170 ppm). Anal. Calcd for C32H46FeN4O4S: C, 60.18; H, 7.26; N,
8.77. Found: C, 60.05; H, 7.03; N, 8.85.
Table 4. Crystallographic Data and Refinement Statistics for
the Complex of Avidin with 3 (PDB: 4JHQ)
a
X-ray Data Collection and Processing Statistics
radiation source
wavelength (Å)
temp (K)
BESSY BEAMLINE 14.2
0.91841
100
rotation range (deg)
space group
0.5
P21212
unit cell (Å)
a = 69.69, b = 78.66, c = 42.76
molecules in asymmetric unit
completeness (%)
redundancy
2
96.5 (82.6)
4.6 (3.9)
31.45 (6.30)
4.3 (17.9)
I/σ(I)
b
Rmarge (%)
Refinement Statistics
resolution range high (Å)
resolution range low (Å)
1.99 (1.99)
28.96 (2.05)
16.22/21.02
0.018
c
c
Rwork /Rfree (%)
RMSD bond lengths (Å)
RMSD bond angles (deg)
av B factor (Å2)
1.957
37.00
Ramachandran Plot
most favored regions (%)
allowed regions (%)
93.5
6.5
generously allowed regions (%)
disallowed regions (%)
0.0
0.0
a
b
Values in parentheses are for the last resolution shell. Rmerge = ∑h∑j|
Ihj − ⟨Ih⟩|/∑h∑jIhj, where Ihj is the intensity of observation j of
c
reflection h. R = ∑h||Fo| − |Fc||/∑h|Fo| for all reflections, where Fo and
Fc are observed and calculated structure factors, respectively. Rfree is
calculated analogously for the test reflections, randomly selected and
excluded from the refinement.
Desthiobiotinoylferrocene (4). The procedure was the same as
for 8, except D-desthiobiotin (214 mg, 1 mmol), ferrocene (187 mg,
1 mmol), TfOH (149 mg, 88 μL, 0.995 mmol), and TFAA (227 mg,
150 μL, 1.08 mmol) were used. Yield: 130 mg (34%). Orange solid.
1H NMR (CDCl3): δ 5.15 (br s, 1 H, 1-NH), 4.84 (br s, 1 H, 3-NH),
(m, 1 H, CH2-alkyl chain), 1.56−1.42 (m, 7 H, CH2-alkyl chain), 1.41−
1.33 (m, 4 H, CH2-alkyl chain), 1.32−1.13 (m, 6 H, CH2-alkyl chain). 13
C
NMR (DMSO-d6): δ 174.3 (CO), 171.7 (CO), 171.7 (CO), 162.6 (CO),
61.0 (C-4a), 59.2 (C-6a), 55.3 (C-4), 39.77 (C-6), 38.3, 38.2, 35.3, 35.2,
33.6, 28.9, 28.8, 28.1, 28.0, 26.1, 25.9, 25.3, 25.0, 24.2 (14 × CH2-alkyl
chain).
4.78 (t, J = 1.9 Hz, 2 H, Cp), 4.54−4.45 (m, 2 H, Cp), 4.19 (s, 5 H,
Cp), 3.84 (quin, J = 6.7 Hz, 1 H, H-5), 3.75−3.67 (m, 1 H, H-4), 2.71
(t, J = 7.3 Hz, 2 H, H-11), 1.72 (quin, J = 7.2 Hz, 2 H, H-10), 1.59−
1.38 (m, 5 H, CH2-alkyl chain), 1.35−1.28 (m, 1 H, CH2-alkyl chain),
1.13 (d, J = 6.4 Hz, 3 H, H-6). 13C NMR (CDCl3): δ 204.3 (12-CO),
163.5 (2-CO), 79.1 (Cp-ipso), 72.1 (Cp), 69.7 (Cp), 69.3 (Cp), 56.0
(C-4), 51.4 (C-5), 39.4 (C-11), 29.5 (CH2-alkyl chain), 29.4 (CH2-
alkyl chain), 26.4 (CH2-alkyl chain), 24.3 (C-10), 15.7 (C-6). MS
(EI): (M+) 382.1. HRMS (EI): calcd for C20H26FeN2O2 382.13440,
found 382.13399 (δ 0.97867 ppm). Anal. Calcd for C20H26FeN2O2: C,
62.84; H, 6.86; N, 7.33 Found: C, 62.81; H, 6.83; N, 7.28.
Competitive Binding Assay for Biotin and Desthiobiotin
Conjugates. Flat-bottomed polystyrene 96-well microtiter plates
(Greiner) were treated with a solution of avidin (10 μg mL−1, 100 μL
well−1) in 0.1 M carbonate buffer pH 9.5 overnight at 4 °C. The plates
were blocked by the addition of PBS containing 1% BSA (w/v)
(100 μg well−1) for 30 min at room temperature and washed three
times with PBS containing 0.05% Tween 20 (v/v) (PBS-T, 100 μL
well−1). Standard solutions of biotin (0−200 nM), desthiobiotin
(0−2400 nM), 2 and 3 (0−2000 nM), and 4 (0−5200 nM) were
applied to a series of Micronic tubes (200 μL tube−1), and HRP-biotin
solution (400 ng mL−1, 200 μL tube−1) was added to each tube. A 100 μL
amount of each solution was added in duplicate to the plates, which were
incubated 30 min at room temperature. The plates were washed four
times with PBS-T, and an OPD solution in citrate−phosphate buffer pH
5 and 0.16% H2O2 v/v (0.7 mg mL−1, 100 μL well−1) was added to the
wells. After sufficient color development, the enzymatic reaction was
stopped by H2SO4 (2.5 M, 50 μL well−1) and the optical densities at 485
and 410 nm were read with a microliter plate reader (BMG Labtech).
Data sets were fitted by nonlinear regression analysis applying the four-
parameter logistic equation (1), where a and d are the upper and lower
6-Acetamidohexanoylferrocene (8). To a suspension of 7 (174
mg, 1.0 mmol) in dichloromethane (5 mL) was added TFAA (227 mg,
150 μL, 1.08 mmol), and the mixture was stirred until the acid was
completely dissolved. Then ferrocene (186 mg, 1.0 mmol) and TfOH
(149 mg, 88 μL, 0.995 mmol) were added, and stirring was continued
for 2 h. Quenching with water, extraction with dichloromethane, and
chromatography (silica 50 mL, eluent dichloromethane−MeOH
gradient 0−10% MeOH) afforded, in order of elution, acetylferrocene
(11 mg, 5%, identified by comparison with an authentic sample) and 4
1
as an orange solid (205 mg, 60%). H NMR (CDCl3): δ 5.97 (br s, 1
H, NH), 4.75 (s, 2 H, Cp), 4.48 (d, J = 1.51 Hz, 2 H, Cp), 4.17 (s, 5
H, Cp), 3.25 (dd, J = 6.40, 5.27 Hz, 1 H, H-6), 2.69 (t, J = 7.15 Hz, 2
H, H-2), 1.96 (s, 3 H, CH3), 1.70 (quin, J = 7.43 Hz, 2 H, H-3), 1.54
(quin, J = 7.15 Hz, 2 H, H-5), 1.33−1.42 (m, 2 H, H-4). 13C NMR
(CDCl3): δ 204.55 (C-1), 170.62 (C-8), 79.05 (Cp-ipso), 72.22 (Cp),
69.76 (Cp), 69.30 (Cp), 39.41 (C-2 or C-6), 39.37 (C-2 or C-6),
29.39 (C-5), 26.70 (C-4), 23.96 (C-3), 23.34 (CH3). MS (EI): (M+)
341.0. HRMS (EI): calcd for C18H23FeNO2 341.10783, found
341.10666 (δ 1.08641 ppm). Anal. Calcd for C18H23FeNO2: C,
63.36; H, 6.79; N, 4.10. Found: C, 63.31; H, 6.75; N, 4.10.
6-Biotinamidohexanoylferrocene (2). The procedure was the same
as for 8, except 6-biotinamidohexanoic acid (5; 177 mg, 0.495 mmol),
ferrocene (94 mg, 0.505 mmol), TFAA (227 mg, 150 μL, 1.08 mmol),
and TfOH (75 mg, 44 μL, 0.497 mmol) were used. Yield: 72.8 mg
1
(28%). Orange solid. H NMR (CDCl3): δ 6.52 (br s, 1 H, 1-NH),
6.32 (t, J = 4.9 Hz, 1 H, 3-NH), 5.64 (br s, 1 H, 12-NH), 4.77 (t, J =
1.9 Hz, 2 H, Cp), 4.57−4.45 (m, 3 H, Cp and H-6a), 4.31 (d, J = 4.5 Hz,
G
dx.doi.org/10.1021/om4003126 | Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
JLigand29 from the CCP4 package. Data collection and processing
statistics for all experiments are shown in Table 4. The ligand−protein
interactions were analyzed by LIGPLOT v.4.5.3.30
a − d
OD485nm = d +
1 + ([ligand]/c)b
(1)
asymptotes, respectively, c is the value of the [ligand] at the inflection
point (IC50), and b is related to the slope at the center of the sigmoid.
The relative binding affinity (RBA) is given by eq 2.
ASSOCIATED CONTENT
■
S
* Supporting Information
Figures giving 1H NMR, MS, and HRMS spectra of compounds
2−8 and viability of various tumor cell lines in the presence of
biotin−ferrocenyl conjugates of 2−4 and 8. This material is
available free of charge via the Internet at http://pubs.acs.org.
IC50(biotin)
IC50(ligand)
RBA =
× 100
(2)
AUTHOR INFORMATION
Corresponding Author
*J.Z.: e-mail, janzak@uni.lodz.pl; tel, (4842) 635 58 50.
Notes
■
Determination of IC50 for Compounds 1−4 and 8. Cell line
sensitivity to compounds 1−4 and 8 was determined by the modified
MTT-reduction assay.23 Cells suspended in 100 μL of medium were
seeded on the 96-well plates at a density of 10000/well. The cells were
allowed to attach for 24 h, and then the tested compound at the
desired concentration was administered. Stock solutions of com-
pounds 2−4 were prepared in DMSO, and care was taken to keep the
solvent concentration identical in all wells, including controls. The
final DMSO concentration did not exceed 0.01% v/v and was proven
to be nontoxic to the cells. After 70 h of incubation, MTT was added
to the medium to a final concentration of 1.1 mM. After a further 2 h
the medium was removed and the formazane crystals were dissolved in
100 μL of DMSO. Absorbance was measured at 580 nm analytical
wavelength and 720 nm reference wavelength. The results were turned
into a percentage of controls, and the IC50 values for each cell line and
substance were calculated by GraphPad Prism 4.03 software
(GraphPad Inc.), using the formula
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the Polish National Science Center for
financial support (Grant No.1856/B/H03/2011/40). The
Polish National Science Center had no impact on the study
design, on the collection, analysis and interpretation of data, on
the writing of the report, and on the decision to submit this
article for publication.
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