Discovery of linear receptors for multiple dihydrogen phosphate ions using dynamic combinatorial chemistry

Article abstract of DOI:10.1021/ja200130h

We describe the use of dynamic combinatorial chemistry to discover a new series of linear hydrazone-based receptors that bind multiple dihydrogen phosphate ions. Through the use of a template-driven, selection-based approach to receptor synthesis, dynamic

Full text of DOI:10.1021/ja200130h

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pubs.acs.org/JACS
Discovery of Linear Receptors for Multiple Dihydrogen Phosphate
Ions Using Dynamic Combinatorial Chemistry
Sophie R. Beeren and Jeremy K. M. Sanders*
University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
S Supporting Information
b
ABSTRACT: We describe the use of dynamic combinator-
ial chemistry to discover a new series of linear hydrazone-
based receptors that bind multiple dihydrogen phosphate
ions. Through the use of a template-driven, selection-based
approach to receptor synthesis, dynamic combinatorial
chemistry allows for the identification of unexpected host
structures and binding motifs. Notably, we observed the
unprecedented selection of these linear receptors in pre-
ference to competing macrocyclic hosts. Furthermore,
linear receptors containing up to nine building blocks and
three different building blocks were amplified in the dy-
namic combinatorial library. The receptors were formed
-
Figure 1. Helical linear hydrazone-based receptor for H2PO4 that
using a dihydrazide building block based on an amino acid-
disubstituted ferrocene scaffold. A detailed study of the
linear pentamer revealed that it forms a helical ditopic
receptor that employs four acylhydrazone hydrogen-bond
donor motifs to cooperatively bind two dihydrogen phos-
phate ions.
binds cooperatively in a 2:1 fashion.
mimics,5,6 represent an interesting class of compounds for incor-
poration into DCLs. The small barrier to rotation7 and molecular
hinge-like character8 of the ferrocene introduces a flexible turn into
the building blocks that is conducive to the production of macro-
cycles of varying sizes. The inter-ring separation of ferrocene is 3.32
Å, which is close to the N O separation in hydrogen-bonded
3 3 3
β-sheets.9 Strong and well-defined hydrogen bonds are formed
between the arms of amino acid-disubstituted ferrocenes in both
the solid and solution states.5 For ferrocene conjugates of the
general form Fc-[CO-(AA1)-OR]2 or Fc-[CO-(AA1)-
(AA2)-OR]2 [where AA1 = Val, Phe, Ala, Gly, His, Cys(Me), or
Cys(Bz)], a helical “Herrick” conformation has been ob-
served.9-11 The angle between the cyclopentadienyl (Cp) substit-
uents is ∼72°, and the trans amides are essentially parallel to the Cp
rings with the carbonyl groups pointing away from one another,
allowing two interstrand hydrogen bonds to form between the NH
groups and the carbonyls of AA1. The helicity is dependent upon the
chirality of AA1: L chirality induces P helicity and D chirality induces
M helicity, which may be detected by, respectively, strong positive
and negative circular dichroism (CD) absorbances at ∼485 nm.11b
We focused on a valine-disubstituted ferrocene dihydrazide building
block, Fc-[CO-Val-NHNH2]2 (V), which was synthesized in
two steps from ferrocene dicarboxylic acid (Figure 2).
A DCL of hydrazone-linked, ferrocene-based macrocycles and
linear oligomers was generated by reacting V (0.5 mM), iso-
phthalaldehyde (I) (0.5 mM), and 4-methylbenzhydrazide (H)
(1 mM) in the presence of 1-naphthoic acid (20 mM) in 96:4
CHCl3/MeOH. Identification of the species present in the
solution after 10 days was achieved using LC-MS (Figure 3).
In the library were detected the macrocycles (VI), (VI)2, (VI)3,
and (VI)4 and the linear oligomers HIH, HIVIH, HIVIVIH, and
e report here the discovery of a series of structurally
W
unprecedented linear receptors that bind multiple dihy-
drogen phosphate ions; these receptors have been amplified and
identified in a hydrazone dynamic combinatorial library (DCL)
of ferrocene-based cyclic and linear oligomers. The linear tetra-
hydrazone oligomer shown in Figure 1, for example, forms a
-
helical receptor that binds two H2PO4 ions with positive
cooperativity.
Dynamic combinatorial chemistry1 provides a means of effi-
ciently synthesizing libraries of receptors whose individual
molecular recognition capabilities may be explored through the
library’s response to the influences of an added template.2 A DCL
is generated when building blocks combine by way of reversible
covalent reactions to form a mixture of interconverting library
members under thermodynamic control. Stabilization of a parti-
cular library member through noncovalent interactions with an
added template alters the positions of the equilibria governing
the system, ideally resulting in increased production or amplifica-
tion of the most effective hosts. Here we employed reversible
hydrazone formation to link building blocks to form a DCL of
amino acid-functionalized ferrocene macrocycles and linear
oligomers.2c,3 Ferrocene-based receptors have previously been
explored for electrochemical sensing of anions.4
Amino acid-functionalized ferrocenes, which have been re-
cently investigated as scaffolds for inducing β-sheets in peptide
Received: January 6, 2011
Published: February 28, 2011
r
2011 American Chemical Society
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Figure 3. HPLC chromatogram (290 nm) showing the equilibrium
distribution of components in a DCL formed from V (0.5 mM), I (0.5
mM), and H (1 mM) in 96:4 CHCl3/MeOH solution containing
1-naphthoic acid (20 mM) in the absence (dashed black curve) and
presence (solid red curve) of Bu4NH2PO4 (0.25 mM).
Scheme 1. Synthesis of HIVIHa
Figure 2. Building blocks Fc-[CO-Val-NHNH2]2 (V), isophthalal-
dehyde (I), and 4-methylbenzhydrazide (H). Reagents and conditions
for the synthesis of V: (i) H-Val-OMe, HOBt, EDC, Et3N, DMF,
overnight, 72%; (ii) N2H2 H2O, MeOH, 2 days, 77%.
3
HIVIVIVIH. To test whether the equilibrium was under ther-
modynamic control, an isolated sample of macrocycle (VI)2
(0.25 mM) was combined with 4-methylbenzhydrazide (H)
(1 mM) and 1-naphthoic acid (20 mM) in 96:4 CHCl3/MeOH;
after 10 days, a fully re-equilibrated library was established.
When the DCL was allowed to equilibrate in the presence of
Bu4NH2PO4 (0.25 mM), the concentrations of HIVIH, HIVI-
VIH, and HIVIVIVIH as well as (VI) and (VI)3 increased at the
expense of macrocycle (VI)2, indicating that Bu4NH2PO4 inter-
acts favorably with these oligomers (Figure 3). The amplification
of complex linear oligomers at the expense of macrocycles, and of
library members formed from as many as seven and nine building
blocks, is unprecedented; the latter is associated with a significant
entropic cost to the system.12
The linear oligomer HIVIH was prepared in two steps from its
three components (Scheme 1).
a Reagents and conditions: (i) CH3COOH, CHCl3, 3 Å molecular
sieves; (ii) CH3COOH, CHCl3, MeOH, 3 Å molecular sieves, 55%.
The UV-vis titration of HIVIH with Bu4NH2PO4 generated a
binding isotherm suggestive of multiple cooperative binding.13 A
Job plot showed a maximum at 0.45, which does not indicate a
clear 2:1 binding stoichiometry but suggests a binding mode other
than 1:1 (Figures S3 and S4 in the Supporting Information).
The NMR spectrum of HIVIH in 96:4 CHCl3/CD3OD revealed
the presence of two conformational isomers in slow exchange on the
chemical shift time scale. One isomer (HIVIHA) exhibited C2
symmetry, as deduced from the presence of only one set of
resonances for the two substituted Cp ligands of the molecule,
while the other isomer (HIVIHB) showed C1 symmetry. When the
solution of HIVIH was titrated with Bu4NH2PO4, a striking change
in the relative concentrations of the two isomers was observed over
the course of the titration, with HIVIHB gradually being converted
to HIVIHA (Figure 4). After the addition of 5 equiv of Bu4NH2PO4,
over 95% of the material was present as HIVIHA, which is evidence
of its superior binding.
In order to determine the association constants for binding of
-
H2PO4 by the two isomers of HIVIH, we used the program
DCLfit, which is designed to calculate binding constants directly
from measurements of the concentrations of the various species
in a DCL in the presence of different quantities of template.14,15
The NMR titration solution containing a mixture of isomers
HIVIHA and HIVIHB may be considered as a two-membered
DCL. The concentrations of each isomer in the presence of
increasing amounts of Bu4NH2PO4 were calculated by integra-
tion of the H3 and R-proton resonances of HIVIHA and
HIVIHB, respectively, during the titration. The measured
changes in isomer concentration fit poorly to a 1:1 binding
model but very well to a 2:1 binding mode with positive
cooperativity (Figure 5). The fitting indicated that both
isomers bound two H2PO4- anions strongly and cooperatively,
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Figure 6. CD spectra of Fc-[CO-Val-OMe]2 (1), V, and HIVIH
(2 mM in 96:4 CHCl3/MeOH).
effect spectroscopy (NOESY) experiment was performed on a
solution of HIVIH (4 mM) and Bu4NH2PO4 (24 mM) to help
elucidate the specific conformation of the hydrazone moieties in
the receptor when bound to H2PO4-. The spectrum showed
NOE cross-peaks from H2 to H3 and from H8 to H9, indicating
that the imines were both in an E conformation; additional NOE
cross-peaks from H9 to H10 were seen, suggesting that the outer
hydrazone amides were in a trans conformation.
1
Figure 4. Partial H NMR spectra (298 K) of HIVIH (4 mM, 96:4
The CD spectrum of the receptor HIVIH shows a positive
peak at 485 nm suggestive of a P-helical Herrick conformation
(Figure 6).9,10 The spectrum mimics those of the building block
V and the precursor Fc-[CO-Val-OMe]2 (1) and was not
significantly altered in the presence of 2 equiv of H2PO4-. The
suggested Herrick conformation, the structural information
deduced from the NOESY spectrum, and the location of binding
sites suggested by the NMR titration lead to the proposed helical
binding of two H2PO4- anions to HIVIH depicted in Figure 1.
In the DCL templated with H2PO4-, HIVIH, HIVIVIH, and
HIVIVIVIH were amplified with amplification factors18 of 1.6,
1.8 and 2.5, respectively. Since the amplification of oligomers
containing many building blocks is entropically unfavorable, the
amplification of HIVIVIH and HIVIVIVIH indicates either that
they have an extremely high affinity for one or two H2PO4- ions
(significantly stronger than the interaction between HIVIH and
the two ions) or that they cooperatively bind more than two
anions. HIVIVIH and HIVIVIVIH were synthesized in a kineti-
cally controlled manner similar to that for HIVIH. Attempts to
CDCl3/CD3OD) in the presence of (i) 0, (ii) 2, (iii) 4, (iv) 6, (v) 8, and
(vi) 10 mM Bu4NH2PO4. Resonances corresponding to HIVIHA are
colored in red, and assigned protons are labeled accordingly.
Figure 5. Experimentally determined concentrations of HIVIHA (2)
and HIVIHB (0) in the presence of increasing amounts of Bu4NH2PO4
along with fitted curves for a 2:1 binding model generated using
DCLfit.14
-
fully characterize the binding of H2PO4 to these longer
receptors were hampered by the complexity of their NMR
spectra resulting from the presence of multiple isomers. How-
ever, their behavior in the presence of Bu4NH2PO4, when
monitored using UV-vis titrations, Job plots, and CD spectros-
copy, was similar to that of HIVIH (see the Supporting
Information).
with K1K2(HIVIHA) = 800 000 M-2, K1K2(HIVIHB) =
100 000 M-2, and K1 , K2.
During the course of the NMR titration, significant downfield
shifts in the spectrum of HIVIHA were observed for imine
protons H3 (0.55 ppm) and H8 (0.45 ppm), Cp proton H200
(0.65 ppm), and hydrazone NH protons H2 (1.6 ppm) and H9
(0.65 ppm).16 These protons evidently act as hydrogen-bond
donors to bind the two H2PO4- ions, possibly supplemented by
additional interactions between the imine nitrogens and the
anions’ hydrogen-bond donors.
In conclusion, we have identified from a hydrazone-based
DCL a linear receptor based on a valine-functionalized ferrocene
that binds two molecules of H2PO4- cooperatively with K1K2 =
800 000 M-2. This is the first example of a DCL in which
templating has resulted in the amplification of such long and
complex linear species in preference to macrocycles. This
-
Of the many theoretically possible isomers of HIVIH resulting
from cis or trans amides and E or Z imines,3c,17 a single isomer
was selected as the best host for HIVIH. A nuclear Overhauser
remarkable affinity for H2PO4 displayed by linear receptors
illustrates how the use of dynamic combinatorial chemistry may
provide access to unexpected receptors and recognition modes.
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preorganization provided by intramolecular hydrogen bonding
combined with sufficient flexibility (due to its linearity and the
rotational freedom of the ferrocene) to adapt its shape to
optimize the intermolecular interactions between the host and
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’ ASSOCIATED CONTENT
S
Supporting Information.
Experimental procedures;
b
compound characterization; UV-vis spectra, titrations and Job
plots; NMR titrations; VT and 2D NMR spectra; and CD
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
jkms@cam.ac.uk
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2003, 42, 3821–3825. (b) Severin, K. Chem.—Eur. J. 2004,
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Chem. Soc. 2005, 127, 9390–9392. (d) Corbett, P. T.; Sanders, J. K. M.;
Otto, S. Angew. Chem., Int. Ed. 2007, 46, 8858–8861.
’ ACKNOWLEDGMENT
We are grateful to the Gates Cambridge Trust, St. John’s
College Cambridge, and the EPSRC for financial support;
Dr. Michael Pittelkow, Dr. Dan Panto-s, Dr. Emiliano Tamanini,
and Rosemary Hunt for helpful discussions; and Dr. Ana Belenguer
for maintaining the HPLC facility.
(13) Hunter, C. A.; Anderson, H. L. Angew. Chem., Int. Ed. 2009,
48, 7488–7499.
(14) Ludlow, R. F.; Liu, J.; Li, H.; Roberts, S. L.; Sanders, J. K. M.;
Otto, S. Angew. Chem., Int. Ed. 2007, 46, 5762–5764.
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(16) The amide NH protons were not observed in the protic solvent
mixture; their role in the binding of H2PO4- is therefore not known.
(17) Palla, G.; Predieri, G.; Domiano, P.; Vignali, C.; Turner, W.
Tetrahedron 1986, 42, 3649–3654.
(18) Amplification factor = ([receptor] in the templated library)/
([receptor] in the untemplated library).
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