Progress Report – October 2004 to February 2006
Synthetic receptors for inositol phosphates: a novel diagnostic tool for the
oculocerebrorenal syndrome of Lowe
Investigators in the original grant: Ramon Vilar and Rudiger Woscholski
PhD student funded with this grant: Marianna Mirabelli
Imperial College London
Introduction
The symptoms of patients suffering from the inborn error “oculocerebrorenal syndrome of
Lowe” (OCRL) are due to the loss of the phosphatidylinositol 4,5-bisphosphate (PIP2) 5phosphatase activity encoded by the OCRL1 gene, which is missing or truncated in OCRL
patients. Consequently, high levels of PIP2 are an indication of the disease, which together
with reduced phosphatase levels form the basis for the currently employed biochemical
diagnosis of OCRL. However, the biochemical OCRL diagnosis is cumbersome and
restricted to a few places worldwide, which is hindering any progress in diagnosis and
clinical research. Thus, it would be extremely useful to have an easy, robust and sensitive
method to detect the amount of PIP2 in the cell and/or tissue sample.
Aim and design
The initial aim of this project as stated in our original proposal, is to develop a chemical PIP2
receptor capable of selectively binding to the head-group of this substrate (inositol
trisphosphate; IP3) in a selective fashion. Such a receptor (when properly coupled to a
luminophore or chromophore) would be able to selectively recognise PIP2 and/or IP3 and
hence, it could be used as a diagnostic tool for detecting OCRL. The development of such a
receptor is based on a modular approach in which specific receptors for each one of the
different components of the PIP2 and IP3 (i.e. the phosphate groups and the diols) are
developed (see Figure 1 below).
Figure 1 – Schematic representation of the binding expected between the synthetic receptors and IP3.
As will be discussed in this report, over the past two years we have developed and optimised
a series of bis-phosphoester binding modules. Their binding abilities have been evaluated
showing a good degree of selectivity for the targeted guests. Over the past year, we have also
developed a methodology to link the bis-phosphoester and diol modules together using
different spacers and ancillary groups so that libraries of receptors can be efficiently prepared
(and eventually evaluated in parallel). Although the final aim has not yet been achieved, we
have made important advances towards the development of the final receptor.
Timescale and milestones as indicated in the initial proposal
The timescale with milestones that was provided in the initial proposal is included herein for
reference. In the following sections, the specific milestone achieved (or under current
investigation) will be indicated so that it is possible to evaluate the progress of the project.
Milestones: M1: Synthesis of individual phosphate- and diol-receptor modules; M2:
Coupling of receptor modules to generate libraries of IP3/PIP2-receptor; M3: Evaluation of
binding abilities of IP3/PIP2-receptor libraries; M4: Synthesis of luminophore/chromophore;
M5: Incorporation of luminophore/chromophore to selected PIP2-receptor and competition
assays; M6: Synthesis of improved receptor modules; M7: Synthesis of improved IP3/PIP2receptors; M8: Evaluation of M7; M9: Write up. (NOTE: *M4 and M5 will only be targeted
if the rest has been achieved by month 30th).
Month ►
Milestone ▼
M1, M6
M2, M7
M3, M8
M4, M5*
M9
6
12
M1
M1
M2
18
24
30
36
M6
M2
M3
M3
M7
M8
M4
M5
M9
Summary of results obtained to date
During the first year of the project, we concentrated in developing the module capable of
detecting the two neighbouring phosphates on the PIP2/IP3 species (milestone M1). This led
to the preparation of a series of compounds with various degrees of selectivity for this part of
the target molecule. After analysing several of these species, some of them were found to
have a considerable degree of selectivity for the bis-phosphoester component (i.e. it is able to
discriminate between mono-phosphoester and bis-phosphoester). With this information, it
was then possible to design and develop a second generation of phosphoester-binding motifs
with a higher degree of selectivity (milestone M6). Examples of two of these receptors are
shown in Figure 2 (a list of all the species prepared and evaluated for bis-phosphoester
binding is included in Appendix 1 at the end of this report):
Figure 2 – a) Selected examples of potential receptors prepared in the first stage of the project; b)
Di-phosphoester employed to evaluate the ability of the potential receptors to bind substrates with
neighbouring phosphates
The binding constants between the receptors and the diphosphorylated species shown in
Figure 1 were determine by 1H NMR spectroscopy (milestone M3). For some of the
receptors the constants were found to be in the order of 103 in a mixture of H2O/DMSO.
Although this might be seen as a relatively small binding constant if nanomolar quantities of
PIP2/IP3 are to be detected, it should be pointed out that this is only one of the modules of
the final receptor. The binding constants are expected to be much higher once the diolbinding module is attached to the bisphosphoester-binding moiety.
Regarding the diol-binding module, we will use boronic acids since they have been
previously shown to bind diols selectively, reversibly and with high binding constants (up to
105 when they are combined with other binding motifs – as will be our case when the
phospho- and diol-binding modules are linked together)1. More specifically, the binding
module to be used for this part of the receptor is shown in Figure 3.
Figure 3 – Reversible binding of diols by boronic acids. The presence of a methyl-amino group in
position 2 has shown to increase the binding to the diol
Once the essential characteristics of binding modules were determined, we moved into the
second part of the project which consists on linking this module with that one capable of
recognising the diol (milestone M2). It is important to mention that in order to be able to
detect selectively the targeted compound (i.e. the PIP2/IP3) it is essential that this compound
fits perfectly well in the chemical receptor (like a key in a lock). Consequently, the size,
length and flexibility of the linker that brings together the two modular components (the one
that recognises the bis-phosphate and the one that recognises the diol) has to be carefully
chosen and optimised to do the job. Consequently we decided to develop a parallel synthetic
methodology to prepare and test several compounds in fast and efficient fashion (milestones
M3 – and this will then lead to achieving milestone M7).
To develop the parallel synthetic methodology, there are several potential approaches that
can be taken. Thus, we had to spend some months investigating, evaluating and optimising
the best possible approach that would allow us to prepare from easily accessible building
blocks a wide range of potential receptors. After these investigations, the methodology
shown in Figure 4 was chosen.
Figure 4 – Schematic representation of the synthetic approach for the parallel synthesis (in solid
support) of a wide range of potential receptors.
Several structural and electronic variations can be incorporated in this approach, namely the
length, geometry and flexibility of the spacer between the two urea groups (first step) the
length and electronic properties of the spacer that links the bis-phosphorylated recognition
unit with the diol recognition moiety (in this case a boronic acid) and finally, the electronic
and steric properties of the terminal urea-group (which comes from the amine used to detach
the receptor from the solid support. An example of the type of receptor that has recently
obtained is shown in Figure 5.
Figure 5 – Example of one of the receptors prepared using the parallel synthetic approach described
in Figure 4.
Due to the di-functional nature of the building blocks (i.e. the di-isocyanate to generate ureas
and the di-amine to link the urea with the boronic acid moiety) the general methodology
shown in Figure 4 demonstrated to be more challenging than initially envisaged. Now that
the conditions have been optimized we are in the process of preparing a larger set of potential
PIP3 receptors which will then be evaluated for binding and the best candidates chosen form
this library.
Optical reporter
In order for these receptors to have real applications for detection of PIP2/IP3 a simple readout component needs to be incorporated onto the receptor molecules. Although 1H NMR
spectroscopy has allowed us to determine the biding constants between the receptor and
substrate, this would not be practical for the real detection of levels of PIP2/IP3.
Consequently, we have started investigating optical means of doing so (milestones M4 and
M5). Two options are currently being explored: a) attaching a luminophore/chromophore
directly to the receptors;2 b) displacement assays.3 Preliminary investigations with both these
options have shown the second to be potentially more useful for our purposes since it yields a
more selective means of sensing the targeted substrate. An example of the way it works is
shown in Figure 6.
Figure 6 – This assay relies in the formation of a host-guest complex between a coloured dye
(alizarine complexone) and the receptor. In the presence of the substrate (a better guest for
the optimized receptor) the dye is displaced changing colour (there are several dyes known
to change their optical –absorption or emission – properties in whether they are bound or
unbound.
Using the above assay, it has already been possible to determine optically the binding
constant between the receptor and bis-phosphorylated species shown in Figure 6. The value
of this constant is practically the same than that one determined using 1H NMR spectroscopy.
Delays on Milestones
As can be seen from the above results, the project has progressed well and most of the aims
initially stated have been investigated (although not fully achieved yet). There has been some
delay in achieving the milestones stated in the initial proposal, particularly in the parallel
synthetic approach and parallel evaluation of the receptors prepared (milestones M3 and M6
for the parallel synthetic approach and M5 for the incorporation of the luminophore on the
receptors). This has been due to two main factors:
a) Six months after this project had started, the PI of the project (R. Vilar) moved to
a new institution (the Institute of Chemical Research of Catalonia). Although the
student involved in the project (Marianna Mirabelli) did not stop her research
work during this move, there was obviously a small delay of approximately 2
months.
b) The optimization of the parallel synthetic approach took a longer time than
initially expected. As described above, part of the problem comes form the bifunctional nature of the building blocks employed which lead to the formation of
unwanted by-products. Although the steps employed in the synthetic methodology
are all well documented, the formation of asymmetric molecules with a relatively
high number of functionalities is not straight forward. Consequently, more time
was devoted to optimizing each of the steps. Currently, the problems initially
encountered have been solved and it is hence expected that a large number of
potential receptors will be prepared soon.
Related outputs and “cross-fertilization”
To date, we have not yet published any of the results coming out directly from this project
(although one publication is currently in preparation and it is expected that a second one will
follow). However, it should be pointed out that there have been related publications and
grants from the principal investigator’s laboratory that will have a direct effect on this
project.
During the two years the principal investigator (R. Vilar) spent in the Institute of Chemical
Research of Catalonia (ICIQ) his laboratory received extra funding to carry out research on
“Metalla-receptors for phosphorylated species” from the Ministry of Science of Spain
(105,800 euros). One of the aims of this project is to develop metal-containing receptors for
the recognition of di- and tri-phosphorylated species – such as the substrates being targeted in
the OCRL project. Hence, it is expected that interaction between the researchers involved in
the two projects will provide an advantage for the development of the receptors.
Besides the extra funding already obtained, the principal investigator’s laboratory has
recently published some papers on the general area of molecular recognition of anionic
species (with an impact on the development IP3/PIP2 receptors) which are worth pointing
out:
a)
J.A. Tovilla, R. Vilar, A.J.P. White, “A di-palladium urea complex as molecular
receptor for anions”, Chem. Comm., 2005, 4839.
b)
P. Diaz, D.M.P. Mingos, R. Vilar, A.J.P. White, D.J. Williams, “Anion templated
synthesis of metalla-cages as means for the colorimetric detection of chlorides”,
Inorg. Chem. 2004, 43, 7597.
c)
H.A. Burkill, R. Vilar, A.J.P. White, “Synthesis of a series of ruthenium and rhodium
complexes with tridentate pyridine-based ligands containing hydrogen bonding
group”, Inorg. Chim. Acta, 2006, in press.
In the first of these three papers (the most relevant to the current project) a new metalcontaining receptor capable of selectively recognizing phosphoryalted species is reported.
This provides some important clues toward the future development of more sophisticate
receptors for PIP2/IP3.
References
[1]
[2]
[3]
S.L. Wiskur, J.J. Lavigne, A. Metzger, S.L. Tobey, V. Lynch, E. V. Anslyn, Chem.
Eur. J., 2004, 10, 3792.
J.H. Liao, C.T. Chen, J.M. Fang, Org. Lett., 2002, 4, 561.
S.L. Wiskur, H. Ait-Haddou, J.L. Lavigne, E. V. Anslyn, Acc. Chem. Res., 2001, 34,
963.
Appendix 1
The following is a list of species that have been prepared and evaluated as di-phosphate
binding units.
The steric and electronic properties of the potential receptors have been systematically
changed to evaluate their impact in the phosphate-binding capabilities of the compounds. For
example, the different hydrogen bonding groups have been studied, namely ureas and
thioureas (both neutral) and thiouronium (positively charged). The length and flexibility of
the spacer that links the two hydrogen-bonding groups has also been modified to include
flexible ones (aliphatic), pre-organised systems (the bi-phenyl), etc. Finally, the terminal
groups on the hydrogen bonding moieties have also been investigated so that different steric
and electronic properties can be screened.