Protein-mediated dethreading of a biotin-functionalised pseudorotaxane

Article abstract of DOI:10.1039/c3ob41612g

In this article, we describe the synthesis of new biotin-functionalised naphthalene derivatives 3 and 4 and their complexation behaviour with avidin and neutravidin using a range of analytical techniques. We have shown using 2-(4′-hydroxyazobenzene)benzoic acid displacement and ITC experiments, that compounds 3 and 4 have the propensity to form reasonably high-affinity bioconjugates with avidin and neutravidin. We have also demonstrated using ¹H NMR, UV-vis and fluorescence spectroscopy that the naphthalene moiety of 3 and 4 facilitates the formation of pseudorotaxane-like structures with 1 in water. We have then investigated the ability of avidin and neutravidin to modulate the complexation between 1 and 3 or 4. UV-vis and fluorescence spectroscopy has shown that in both cases the addition of the protein disrupts complexation between the naphthalene moieties of 3 and 4 with 1.

Full text of DOI:10.1039/c3ob41612g

Organic &
Biomolecular Chemistry
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PAPER
Protein-mediated dethreading of a
biotin-functionalised pseudorotaxane†
Stuart T. Caldwell,a Catherine Maclean,a Mathis Riehle,b Alan Cooper,a
Margaret Nutley,a Gouher Rabani,a Brian Fitzpatrick,a Vincent M. Rotello,c
Brian O. Smith,b Belal Khaled,d Patrice Woisel*d and Graeme Cooke*a
Cite this: Org. Biomol. Chem., 2014,
12, 511
In this article, we describe the synthesis of new biotin-functionalised naphthalene derivatives 3 and 4 and
their complexation behaviour with avidin and neutravidin using a range of analytical techniques. We have
shown using 2-(4’-hydroxyazobenzene)benzoic acid displacement and ITC experiments, that compounds
3 and 4 have the propensity to form reasonably high-affinity bioconjugates with avidin and neutravidin.
1
We have also demonstrated using H NMR, UV-vis and fluorescence spectroscopy that the naphthalene
Received 7th August 2013,
Accepted 14th November 2013
moiety of 3 and 4 facilitates the formation of pseudorotaxane-like structures with 1 in water. We have
then investigated the ability of avidin and neutravidin to modulate the complexation between 1 and 3 or 4.
UV-vis and fluorescence spectroscopy has shown that in both cases the addition of the protein disrupts
complexation between the naphthalene moieties of 3 and 4 with 1.
DOI: 10.1039/c3ob41612g
www.rsc.org/obc
Pseudorotaxane-based systems engineered from macro-
cyclic host units and appropriately functionalised guests offer
Introduction
Avidin1 and the related biotin-binding proteins streptavidin2 exciting possibilities to reversibly modulate protein structure
and neutravidin,3 due to their ability to strongly bind to up to and function.9 In this context, the host cyclobis(paraquat-
four units of biotin, have become important systems for devel- p-phenylene) (CBPQT4+, 1)10 has the propensity to become an
oping diagnostics,4 immunoassays,5 protein purification tech- important system for the development of novel protein conju-
niques,6 and surface-protein conjugates.7 Chicken egg avidin gates with appropriately functionalised electron rich guest
together with bacterial-based streptavidin, possess a very high- units.11 In particular, the ability to: synthesise this macrocycle
affinity for biotin (Ka ≈ 1015
M
−1). The two proteins have using reliable and high yielding protocols;12 modulate its
similar tetrameric quaternary structures and amino acid recognition processes by chemical and electrochemical redox
arrangement in their binding sites, however, their differing processes;13 form reasonably strong complexes in aqueous
degrees of glycosylation plays an important role in controlling solvents14 and conveniently monitor complexation properties
their physical properties. Avidin due to its glycosylated outer using UV-vis15,16 and fluorescence spectroscopy facilitates the
surface has a pI = 10, whereas the deglycosylated streptavidin development of novel stimuli-responsive bioconjugates.
has a pI = 6.8. Neutravidin like streptavidin is deglycosylated
Previously, we have reported the fabrication of tuneable bio-
and has a similar pI to streptavidin (pI = 6.3), however, it pos- conjugates from 1, biotin-functionalised axle 2 and avidin.11b
sesses the highest degree of specificity of this family of biotin- However, the poor aqueous solubility of compound 2 required
binding proteins.8
the investigations to be carried out in the presence of 30%
ethanol limiting future biological applications of these
systems. Furthermore, the ester link between the biotin and
naphthalene units may be susceptible to cleavage by avidin, as
this protein has previously been shown to possess hydrolytic
activity.17 Here, we report the synthesis of a new generation of
naphthalene-functionalised biotin systems that have improved
hydrolytic stability conferred by the amide linker group and in
the case of oligomer 4, significantly better water solubility
than compounds 2 or 3. We report the binding properties of 3
and 4 to avidin and neutravidin, and we have investigated the
role the differing proteins have in controlling complexation of
the naphthalene moieties of 3 and 4 with 1.
aGlasgow Centre for Physical Organic Chemistry, WestCHEM, School of Chemistry,
Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, UK.
E-mail: Graeme.Cooke@glasgow.ac.uk
bCollege of MVLS, University of Glasgow, Glasgow, G12 8QQ, UK
cDepartment of Chemistry, University of Massachusetts at Amherst, Amherst,
MA 01002, USA
dUnité Matériaux et Transformations (UMET), UMR-CNRS 8207, Université Lille
Nord de France, ENSCL, Université des Sciences et Technologies de Lille, Villeneuve
d’Ascq, 59655, France
†Electronic supplementary information (ESI) available: Synthesis of 3 and 4. See
DOI: 10.1039/c3ob41612g
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Table 1 Selected isothermal calorimetry data for the binding of 4 with
avidin and neutravidin at 25 °C in water. N is the binding stoichiometry
Ka (M−1
)
N
Avidin
Neutravidin
2.0 0.3 × 107
4.7 1.1 × 106
2.9 0.1
3.2 0.1
absorption peak at λ = ∼500 nm due to original avidin–HABA
complex. Similar data were obtained when experiments were
repeated with neutravidin.
The good solubility of compound 4 in water has allowed the
investigation of the binding of this compound to avidin and
neutravidin using isothermal titration microcalorimetry (ITC)
(see Table 1 and ESI†). In all cases the ITC data indicate
exothermic binding of 4 to the target proteins, with larger
observed binding affinities (Ka) for avidin compared to neutra-
vidin. The protein binding affinities for 4 (Ka of the order of
107 M−1) are much lower than previously observed for avidin
and biotin, presumably due to the inevitable constraints
imposed by the long chain attached to the biotin moiety of 4.
Bearing in mind the uncertainties regarding absolute concen-
trations and purities of the proteins, the apparent binding stoi-
chiometries (N) determined by ITC (Table 1) are consistent
with the four binding sites present in the tetrameric proteins
and suggests that 4 may only bind to three of the four possible
binding sites of these proteins.21 In line with related investi-
gations involving biotin species featuring poly(ethylene glycol)
spacer moieties, we attribute the oligomeric nature of 4 being
partly responsible for preventing this unit binding to all four
of the available binding sites.22 Thus, the data are consistent
with the HABA measurements, demonstrating that compound
4 has a good affinity for avidin and neutravidin in aqueous
environments.
Non-covalent interactions (e.g. donor–acceptor interactions)
can have a profound influence on the fluorescence properties
of dialkoxynaphthalene units.14e Thus, we have investigated
the fluorescence properties of 3 and 4 both free in solution
and bound to avidin and neutravidin. In all cases, the biotin-
mediated binding of 3 or 4 to the proteins had negligible
affect on the fluorescence intensity of the naphthalene moiety,
suggesting that this unit does not undergo significant inter-
actions with these proteins (Fig. 2). This observation is in
accordance with previously reported fluorophore-appended
biotin/avidin conjugates featuring ethylene glycol linker
units.23
Results and discussion
Avidin and neutravidin binding properties of 3 and 4
The synthesis of the new naphthalene derivatives 3 and 4 is
outlined in the ESI.† Evidence to indicate that compounds 3
and 4 successfully bind to avidin and neutravidin was obtained
by performing competition experiments between the com-
plexes of 2-(4′-hydroxyazobenzene)benzoic acid (HABA) and
avidin and neutravidin. The characteristic red-coloured com-
plexes that arise when HABA binds to the biotin binding site
on avidin,18 streptavidin19 and neutravidin,20 allows its displa-
cement to be conveniently monitored using the naked eye or
UV-vis spectroscopy upon the addition of the more strongly
binding guests 3 or 4 (see Fig. 1). Indeed, the addition of 3 or
4 to a solution of the avidin–HABA complex resulted in the
formation of a new peak at λ = ∼350 nm which is characteristic
of free HABA and in the immediate disappearance of the
Complexation properties of 3 and 4 with 1 in water
We have investigated the complexation of 1 with 3 and 4 using
NMR spectroscopy in D2O. The 1H NMR of a 1 : 1 admixture of
1·3 and 1·4 were in accordance with previously reported data
for complexes of this type,12,14g and revealed significant shifts
for the Hα, Hβ and –C6H4– protons of 1 and the H(4,8) protons
of the naphthalene moiety of 3 or 4 upon complexation (Fig. 3
and ESI†). Thus the NMR data are in accordance with the
formation of pseudorotaxane-like architectures in aqueous
Fig. 1 UV-vis spectra of: (a) avidin (1 × 10−5 M) (——); (b) upon the
addition of 4 equivalents of HABA (---); (c) upon the addition of 4
equivalents of 4 (- -). Spectra were recorded in water at 21 °C.
Á
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2.6 0.1 × 105 M−1 for complex 1·4, which is approximately
two orders of magnitude lower than that observed for 4
binding to the biotin-binding proteins.
Investigation of the avidin/neutravidin-mediated complexation
between 3 or 4 with 1
With complexation verified between 3 and 4 with avidin and
neutravidin and pseudorotaxane formation demonstrated
between the naphthalene-based systems and 1 in aqueous
solution, we next investigated whether pseudorotaxane-like
bioconjugates could be fabricated. To investigate these pro-
cesses we have adopted two strategies. Firstly, we have investi-
gated the addition of avidin and neutravidin to pre-formed
pseudorotaxanes of 1 with 3 and 4. To complement this study,
we have also investigated the complexation behaviour of the
avidin/neutravidin complexes of 3 and 4 with 1.
Fig. 2 Representation of the change in the normalised fluorescence
emission intensity at 330 nm for compound 4 (∼1 × 10−5 M) (blue cylin-
der) and upon complexation with avidin (red cylinder). Spectra were
recorded in water at 21 °C. Excitation wavelength = 295 nm.
The addition of avidin to a purple coloured solution of 1·3
or 1·4 immediately resulted in the decolourisation of the solu-
tion suggesting the disruption of the pseudorotaxane architec-
ture.24 UV-vis spectroscopy experiments revealed
a total
disappearance of the absorption at 520 nm indicating that
cyclophane 1 is no longer bound to the naphthalene moiety of
3 to any significant extent (Fig. 5a). In addition, fluorescence
spectroscopy was also used to monitor changes in the emis-
sion spectrum of 1·3 upon the addition of avidin. A marked
increase in fluorescence intensity for the naphthalene moiety
was observed. When the same UV-vis experiment was repeated
Fig. 3 Partial 1H NMR spectra of 1 (top spectrum) and upon the
addition of one equivalent of 3 (bottom spectrum). Recorded in D2O
at 25 °C.
Fig. 4 Representation of the change in the normalised fluorescence
emission intensity at 330 nm for compound 3 (∼1 × 10−5 M) (blue cylin-
der) and upon the addition of one equivalent of 1 (red cylinder). Spectra
were recorded in water at 21 °C. Excitation wavelength = 295 nm.
conditions. In both cases, the formation of the admixture
resulted in the formation of a purple-coloured solution (λ =
520 nm) and a significant decrease in the fluorescence inten-
sity of the naphthalene moiety that are consistent with
complex formation (Fig. 4).16 ITC experiments provided a Ka of
Fig. 5 UV-vis spectra of: (a) complex formed between 3 and 1 (purple
line) (∼1 × 10−4 M) and upon the addition of one equivalent of avidin
(red line) and (b) the spectra obtained when 5 was used instead of 3
upon addition of avidin. Spectra were recorded in water at 21 °C.
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with non-biotin functionalised naphthalene derivative 5,25 no
change in the colour or intensity of the absorption at 520 nm
was observed indicating that the presence of the biotin moiety
was required to induce the avidin-mediated disruption of com-
plexation and its associated colour change (Fig. 5b). Similar
UV-vis and fluorescence data was also observed when neutra-
vidin was added to 1·3 and 1·4.
Fig. 7 Schematic diagram showing the avidin-mediated decomplexa-
tion of pseudorotaxane 1·3 or 1·4.28
avidin or neutravidin upon the addition of 1. The addition of
aliquots of 1 to a solution containing either avidin and neutra-
vidin and 4 led to a negligible change in colour, suggesting
that despite their differing pI values, binding does not occur
between these species to any significant extent under the con-
ditions investigated, and suggests that steric factors may be
responsible.27
Further evidence for the avidin-induced dethreading
process of 1·3 was obtained by adding either guest 6 or 7 after
the protein-mediated decomplexation process. Previous experi-
ments have shown that in aqueous conditions guest 6 forms a
lower affinity complex with macrocycle 1 than naphthalene-
based guests such as 3.14g Therefore compound 6 could be a
good system for investigating whether free 1 is available in
solution following the addition of the protein to 1·3. The
addition of excess 6 following the addition of avidin to 1·3
resulted in an orange/red coloured solution consistent with
the formation of 1·6. Furthermore, when experiments were
repeated with guest 7, an emerald-green solution was formed
due to an absorption in the UV-vis spectrum at ∼850 nm. This
observation is consistent with complexation between 1 and 7
(Fig. 6).26
Conclusions
In conclusion, we have shown that compound 3 and 4 have the
propensity to form reasonably high-affinity complexes with
avidin and neutravidin and moderate affinity complexes with 1
in water. Furthermore, experiments show that, in a thermodyn-
amic equilibrium situation and under similar concentration
conditions, the proteins avidin or neutravidin will complete
favourably with 1 for binding of 3 or 4, and will thus shift the
equilibrium from the pseudorotaxanes to the protein-biotin
complexes, as illustrated in Fig. 7. This study paves the way for
the elaboration of these systems into protein mediated mole-
cular machines,29 and our investigations in this area will be
reported in due course.
Finally, we have investigated whether pseudorotaxane-like
bioconjugates could be prepared from pre-complexed 4 and
Acknowledgements
G.C. and B.F. thank the EPSRC for funding. C.M. thanks
the University of Glasgow for the award of a Kelvin–Smith
Scholarship. P.W. thanks the Agence Nationale de la Recherche
(STRAPA project, ANR-12-BS08-0005). VR acknowledges CHE-
1307021 from the National Science Foundation.
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(purple line) (∼1 × 10−4 M); (b) upon the addition one equivalent of
avidin (black line) and (c) upon the addition of excess 7 to the cuvette
(green line). Spectra were recorded in water at 21 °C.
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