Synthesis of new trisulfonated calix[4]arenes functionalized at the upper rim, and their complexation with the trimethyllysine epigenetic mark

Article abstract of DOI:10.1021/ol300243b

A synthetic route to produce a new family of trisulfonated calix[4]arenes bearing a single group, selectively introduced, that lines the binding pocket is reported. Ten examples, including new sulfonamide and biphenyl-substituted hosts, each with additional binding elements, demonstrate the tuning of guest affinities and selectivities. NMR titrations in phosphate-buffered water show that one of the new hosts binds to the modified amino acid trimethyllysine with the highest affinity and selectivity observed to date.

Full text of DOI:10.1021/ol300243b

ORGANIC
LETTERS
2012
Vol. 14, No. 6
1512–1515
Synthesis of New Trisulfonated
Calix[4]arenes Functionalized at the Upper
Rim, and Their Complexation with the
Trimethyllysine Epigenetic Mark
Kevin D. Daze, Manuel C. F. Ma, Florent Pineux, and Fraser Hof*
Department of Chemistry, University of Victoria, P.O. Box 3065, STN CSC, Victoria,
BC, Canada
fhof@uvic.ca
Received January 30, 2012
ABSTRACT
A synthetic route to produce a new family of trisulfonated calix[4]arenes bearing a single group, selectively introduced, that lines the binding
pocket is reported. Ten examples, including new sulfonamide and biphenyl-substituted hosts, each with additional binding elements, demonstrate
the tuning of guest affinities and selectivities. NMR titrations in phosphate-buffered water show that one of the new hosts binds to the modified
amino acid trimethyllysine with the highest affinity and selectivity observed to date.
p-Sulfonatocalix[4]arene (PSC, 1) is a macrocyclic host
that has received much attention due to its ability to
complex with biologically important guests in aqueous
solution.1À6 Recently we have shown that 1 strongly binds
the biologically important post-translationally modified
amino acid, trimethyllysine.7 The trimethyllysine mark is
an important signaling site that triggers proteinÀprotein
interactions and a variety of downstream cellular events
related to gene regulation.8À10
Our goal at the outset of this work was to modify the
calix[4]arene skeleton to provide a functional handle for
synthetic tuning of the affinities for selected biological
partners. Previous work in our laboratory (unpublished)
indicated to us that synthetic modifications made to
the lower rim of 111,12 offer little ability to control the
binding of biological partners due to the distance of these
modifications from the binding pocket. Despite the domi-
nant position of sulfonated calixarenes among water-
soluble supramolecular hosts,1 we found that examples
bearing modifications on the upper rim such as the mono-
substituted, trisulfonated calix[4]arenes exemplified by 2
(Figure1) havenot beenpreparedsyntheticallyorexplored
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ChemBioChem 2010, 11, 63–66.
r
10.1021/ol300243b
Published on Web 03/07/2012
2012 American Chemical Society

as supramolecular hosts. Recent work has highlighted the
importance of selective functionalization of a variety of
other macrocylic systems, such as calixpyrroles and cyclo-
dextrins, in the tuning of binding properties and develop-
ment of new technologies.13,14
nickel under a H2 atmosphere supplied amino-intermediate
8 in quantitative yield and high purity after filtration and
lyophilization of the reaction.
With 6 and 8 in hand we proceeded to develop synthetic
conditions to provide additional functionality for contact-
ing our intended guest, trimethyllysine. From 8 we envi-
sioned reaction with sulfonyl chlorides to supply a new set
of sulfonamide-containing hosts. Initial coupling condi-
tions consisted of reacting calix[4]arene 8 with an appro-
priate sulfonyl chloride in DMF and pyridine.
Scheme 1. Synthesis of Trisulfonated Intermediates
Figure 1. (A) Host, 1, and exemplary trisulfonate, 2. (B)
Schematic showing potential for additional interactions be-
tween host and guest in generic structures such as 2. (C)
Trimethyllysine.
Previous work toward these desymmetrized synthetic
modifications to the upper rim of a calix[4]arene scaffold
led to compounds 315,16 and 417 (Scheme 1). Compounds 3
and 4 are both accessed from tribenzoylester calix[4]arene,
5. Nitration of 5 using nitronium tetrafluoroborate and
nitric acid was explored, and both supplied 3 in moderate
yields. Bromination of 5 proceeded smoothly using Br2
under literature conditions17 yielding compound 4. Our
next step was to explore sulfonation conditions starting
from the reported calix[4]arenes 3 and 4. Following litera-
ture procedures18,19 to chlorosulfonate 3 with chloro-
sulfonic acid followed by hydrolysis to the sulfonate led
to an inseparable mixture of decomposition products.
Next, we attempted treatment with neat sulfuric acid20
followed by recrystallization from brine21 but were unable
to isolate product from the solution. One commonly
observed problem was ipso-sulfonation which displaced
the newly installed nitro group to supply symmetric 1 as
the major product isolated by HPLC. To address these
problems we sought a sulfonation condition that could be
applied to both 3 and 4 and that would avoid ipso-
sulfonation, be amenable to larger scale reactions, and
would not require purification. These requirements were
met by sulfonation using neat sulfuric acid added to
dichloromethane heated at 60 °C; treatment for 1 h for 3
and 3 h for 4 provided clean products that precipitated
directly out of the reaction mixtures and were isolated by
centrifugation. Subsequent treatment of 7 with Raney
While these conditions did provide small amounts of
product (isolable by HPLC), useful quantities were not
obtained. The low nucleophilicity of the anilino nitrogen
atom in 8 led to frequent recovery of unreacted starting
materialundera large variety of typical reactionconditions
used for making sulfonamides. We found that conducting
the sulfonamide formation step in 1 M sodium phosphate
buffer (pH 8) supplied 9 and 10 in reasonable yields after
purification by HPLC (Scheme 2).
Scheme 2. Synthesis of Sulfonamide Calix[4]arenes, 9 and 10
(13) Khan, A. R.; Forgo, P. Chem. Rev. 1998, 98, 1977–1996.
(14) Zhou, C.; Liu, K. J. Chem. Res. 2006, 2006, 398–401.
(15) Verboom, W.; Durie, A. J. Org. Chem. 2002, 57, 1313–1316.
(16) Kye Chun Nma, D. S. K. Bull. Korean Chem. Soc. 1994, 15, 284–
286.
(17) Vezina, M.; Gagnon, J. Organometallics 2000, 20, 273–281.
(18) Coquiere, D.; Cadeau, H. J. Org. Chem. 2006, 71, 4059–4065.
(19) Morzherin, Y.; Rudkevich, D. M. J. Org. Chem. 1993, 58, 7602–
7605.
Aryl bromide 6 was intended to be a precursor for
Suzuki couplings to furnish a variety of biphenyl-functio-
nalized sulfonated calix[4]arene hosts. Numerous stan-
dard Suzuki reaction conditions were attempted with
PhB(OH)2, including using H2O mixed with DME,
(20) Da Silva, E.; Coleman, A. W. Tetrahedron 2003, 59, 7357–7364.
(21) Atwood, J. L.; Orr, G. W. Inorg. Chem. 1992, 31, 603–606.
Org. Lett., Vol. 14, No. 6, 2012
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THF, or dioxane as the solvent; Pd(PPh3)4 or Pd(OAc)2
in the presence of added phosphine ligands (including
“Buchwald” ligands such as S-phos) as the catalyst; Na2CO3,
K2CO3, or K3PO4 as the base; and varied temperatures
and times of reaction. All such conditions provided little or
no detectable product, with yields <5%. Finally, wefound
Pd(OAc)2, PhB(OH)2, and Na2CO3 in water under micro-
wave irradiation supplied 11 in 50% yield after only
5 min22 (Scheme 3).
These reaction conditions also worked for a variety of
aryl boronic acids (compounds 12À15), with yields rang-
ing from 38 to 50% after HPLC purification. The reac-
tion of 4-cyanophenylboronic acid under these conditions
generated two products, the desired cyano product (12),
and its partially hydrolyzed primary amide analog (13),
which wereisolatedfrom a single HPLC purification run in
29% and 26% yields, respectively.
electron-rich 8 have identical binding profiles for lysine
and trimethyllysine, demonstrating that the π-electro-
statics of the newly substituted calixarene ring have no
influence on the binding of these cationic partners under
these conditions. The bromo-substituted 6 has 2-fold high-
er affinities for both amino acids, which we hypothesize
arises from improved dispersive interactions and hydro-
phobic contributions of the bromo substituent relative to
amino or nitro. The tosyl sulfonamide host 9 showed the
weakest binding for trimethyllysine (570 MÀ1) with some
affinity recovered when the para-methyl group is replaced
by a charged carboxylate in host 10 (2900 MÀ1 for
trimethyllysine). It is likely that the flexibility of the
sulfonamide linkage either (a) produces host conforma-
tions that do not provide additional contacts with guests or
(b) allows hydrophobic collapse of the newly introduced
aryl substituent that is detrimental to binding. This type of
collapse is impossible for the rigidly linked Suzuki pro-
ducts 11À15. Phenyl-substituted host 11 showed the high-
est affinity for trimethyllysine (64000 MÀ1) that we have
observed to date, as well as the highest selectivity over
unmethylated lysine (150-fold) among this class of hosts
including the parent compound 1. Isothermal titration
calorimetry of host 11 and trimethyllysine (see Supporting
Scheme 3. Synthesis of Extended Biphenyl Calix[4]arenes,
11À15a
Information) provided a similar Kassoc of 79700 MÀ1
.
These affinities for trimethyllysine are similar to those of
the naturally evolved proteins that bind trimethyllysine-
modified proteins and peptides.8À10 Introduction of
cyano, amido, carboxy, and amino substituents (hosts
12À15) to this host skeleton all proved to negatively
affect binding.
Table 1. Affinities and Selectivities for Trimethyllysine
a These two products arise from a single reaction. See text.
Kassoc (MÀ1 a
)
Kassoc (MÀ1 a
)
selectivity for
host
trimethyllysine
lysine
trimethyllysine
1b
6
37000 ( 18000
3900 ( 100
1700 ( 90
1600 ( 200
570 ( 180
520 ( 200
440 ( 110
150 ( 20
180 ( 4
30 ( 5
120 ( 8
420 ( 50
n.d.
70
9
NMR titrations were carried out to determine the
affinities of the new family of functionalized trisulfonated
calix[4]arene hosts for trimethyllysine, as well as their
selectivity over unmethylated lysine. Solutions were pre-
pared by dissolving the amino acid in pure, buffered D2O
(40 mM Na2HPO4/NaH2PO4, pD 7.0 = pH 7.423) to
create the receiving phase and adding solid calixarene to
a portion of the same solution to create a titrant solution
that was perfectly matched in terms of buffer, pH, and
amino acid concentrations tothe receiving phase. Addition
of the titrant solutions caused changes in the chemical
shifts of amino acid guests that were fit to 1:1 binding
isotherms in order to provide Kassoc values for each hostÀ
guest pair (Table 1).
7
11
8
8
9
17
24
150
10
11
11
12
13
14
15
2900 ( 80
64000 ( 13000
79700 ( 6300c
2100 ( 220
5900 ( 1600
1700 ( 270
5200 ( 1300
210 ( 30
140 ( 10
110 ( 20
200 ( 40
10
42
16
26
a Determined by 1H NMR spectroscopy (500 MHz) at 298 K in D2O
(40 mM Na2HPO4/NaH2PO4, pD 7.0 = pH 7.4) by titration of host
(20À50 mM) into a solution of amino acid (2À3 mM). The Kassoc values
arise from 2 to 4 trackable NMR signals from 2 replicate titrations per
guest. Errors reported are standard deviations. b Values previously
reported.7 c Value determined by ITC. See Supporting Information.
The hosts 6, 7, and 8, which have had one sulfonate
removed and replaced by a neutral heteroatom, display
significantly worsened affinities and selectivities for tri-
methyllsine relative to the parent compound PSC. The
nitro-substituted, electron-poor 7 and amino-substituted,
We sought structural clues to help explain the structure/
function relations observed. All hostÀtrimethyllysine
complexes show maximum upfield shifts for the N-methyl
and CH2-ε protons of trimethyllysine, indicating that
all form complexes with the methylated ammonium
(22) Leadbeater, N. E.; Marco, M. J. Org. Chem. 2002, 68, 888–892.
(23) Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188–190.
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Org. Lett., Vol. 14, No. 6, 2012

functional group buried deep in the calixarene’s highly
shielding pocket (Table 2).7 This is unlike the binding of
unmethylated lysine, which previous work has shown to
form a side-on complex with parent host 1.7,24 We also
considered chemical shiftsof protons at the oppositeend of
trimethyllysine, with the CH-R resonances of trimethylly-
sine providing the most easily observed set of diagnostic
peaks. We found that shifts of the trimethyllysine CH-R
protons do not occur for hosts 1, 6, 7, 8, as expected; they
have no additional functionality able to engage these distal
guest protons. Aryl sulfonamide-functionalized hosts 9
and 10 also do not produce shifts of CH-R on trimethylly-
sine, offering support to our hypothesis that these ap-
pended elements do not make significant additional
contacts with the full length of the amino acid guests.
Finally, the biphenyl-containing hosts 11À15 all show
similar upfield chemical shifts for CH-R that are indicative
of significant CH-aromatic contacts between CH-R and
the appended aromatic rings
highly potent phenyl host 11? The possibilities include
CHÀπ interactions weakened by aryl electron-withdraw-
ing groups, and decreased hydrophobic contributions in
12À15 due to the increased polarity and improved solva-
tion of the hosts’ appended aryl rings relative to the more
hydrophobic 11. The functionalized aromatic ring in ques-
tion is engaging the methylenes and zwitterionic functions
of the amino acid guest, so it is possible that both the
electronic modulation of CHÀπ contacts between the host
and guest and changes in solvation play a role. Whatever
the detailed explanation, it is clear that the phenyl-
for-sulfonate substitution made between the parent com-
pound 1 and 11 more than compensates for the affinity loss
caused by removing a sulfonate and succeeds in producing
a highly specific host for the trimethyllysine epigenetic
motif that can operate in the medium that matters, pure
water.
In summary, we have developed a simple method to
access a new family of desymmetrized trisulfonated calix-
[4]arenes. We have used these novel calix[4]arenes to access
sulfonamide-functionalized and biphenyl-functionalized
reaction products providing additional contact points for
hostÀguest interactions. Sulfonated calixarenes are in-
creasingly being used in biomedical research, including as
medical agents that are active in vivo,25 as critical compo-
nents of novel enzyme assays,26 and as protein-binding
small molecules.1,27 The intermediates 6 and 8 offer re-
searchers the ability to tune potencies and selectivities of
sulfonated calix[4]arenes by specific installation of a new
functionality directly lining the binding pocket and pro-
mise to facilitate efforts to optimize molecules for each of
these diverse applications.
Table 2. Maximum Chemical Shifts for Trimethyllysine
Resonances upon Complexation by Different Hostsa
ÀΔδmax, ppm
host
N-CH3
CH2-ε
CH-R
6
2.24
2.29
2.49
2.14
2.38
2.16
2.53
2.40
2.39
2.49
1.33
1.39
1.52
1.26
1.38
1.38
1.61
1.54
1.66
1.59
0.025b
0.045b
0.08b
0c
7
8
9
10
11
12
13
14
15
0c
0.31
0.29
0.32
0.40
0.32
Acknowledgment. K.D.D. gratefully acknowledges the
support of Ride-To-Live and PCFBC Prostate Cancer
Fellowships. This work was funded by Prostate Cancer
Canada. F.H. is a Canada Research Chair and Career
Scholar of the Michael Smith Foundation for Health
Research.
a All resonances shift upfield upon binding. Averaged |Δδmax| values
obtained from the Kassoc fits (see Table 1 and Supporting Information)
are reported unless otherwise noted. b These small shifts do not fit the 1:1
binding isotherm, so maximum observed shifts are reported. c No
measurable change in signal during titration.
Supporting Information Available. Experimental pro-
cedures for synthesis and binding studies, spectral data
for new compounds, stacked NMR plots and titration
curves for all hostÀguest pairs. This material is available
free of charge via the Internet at http://pubs.acs.org.
If the geometries of the complexes of 11À15 with
trimethyllysine are generally similar (as revealed by the
similarity in |Δδmax| for all trimethyllysine protons), then
what is the basis for the weakened binding of cyano,
amido, carboxy, and amino hosts 12À15 relative to the
(26) Guo, D.-S.; Uzunova, V. D.; Su, X.; Liu , Y. Chem. Sci. 2011, 2,
1722–1734.
(27) Silva, E.; Rousseau, C. J. Inclusion Phenom. Macrocyclic Chem.
2006, 54, 53–59.
ꢀ
(24) Selkti, M.; Coleman, A. W.; Nicolis, I.; Douteau-Guevel, N.;
Villain, F.; Tomas, A.; de Rango, C. Chem. Commun. 2000, 161–162.
(25) Wang, G.-F.; Ren, X.-L.; Zhao, M.; Qiu, X.-L.; Qi, A.-D.
J. Agric. Food Chem. 2011, 59, 4294–4299.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 6, 2012
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