Initial structural studies of charged receptors that bind to inorganic phosphate anion and to an anionic phospholipid found in bacterial membranes

Article abstract of DOI:10.1021/jo102383j

ITC titration studies of a family of bis-ammonium receptors based upon a scaffold of two bis-linked phenol rings show that several of the receptors bind to both dihydrogenphosphate and phosphatidylglycerol anions in a similar binding motif. Thermodynamic

Full text of DOI:10.1021/jo102383j

NOTE
pubs.acs.org/joc
Initial Structural Studies of Charged Receptors That Bind to Inorganic
Phosphate Anion and to an Anionic Phospholipid Found in Bacterial
Membranes
Manjula B. Koralegedara, Hong W. Aw, and Dennis H. Burns*
Department of Chemistry, Wichita State University, Wichita, Kansas 67260, United States
S Supporting Information
b
ABSTRACT: ITC titration studies of a family of bis-ammonium
receptors based upon a scaffold of two bis-linked phenol rings
show that several of the receptors bind to both dihydrogenpho-
sphate and phosphatidylglycerol anions in a similar binding
motif. Thermodynamic properties determined from ITC show that anion binding is entropy driven. Job plots determined from 1H
NMR clearly demonstrate that anion-receptor binding stoichiometry is dependent on the receptor’s length of its bis-amine linkage.
ue to the emergence of many strains of multidrug-resistant
bacteria,1-4 cationic peptides and their mimics have re-
Our modeling studies showed that a receptor with the pre-
organized (ortho-ring substituted) ammonium groups found in
compound 1 (Scheme 1) would bind to the PG lipid’s phosphate
anion group via positively charged ammonium hydrogen bonds.
Furthermore, appropriate elaboration of the receptor’s phenol
ring para-positions would orient functional groups that could
then complex with the glycerol hydroxyl groups. Because both
the ligand and receptor were charged and studies were to be
conducted in polar solvents, it was expected that complex
formation would be entropy driven.13 It was hypothesized that
such a receptor would still exhibit PG selectivity, however,
because of the multiple binding domains found in the receptor.
Each ammonium group would have two hydrogens for bonding,
reducing conformational constraints in the binding pocket.
Unknown was how the oxygen atoms in the phosphate anion
portion of PG would interact with the two ammonium ions, i.e.,
would the most stable interaction be with two of the phosphate’s
oxygens or just one, or would there be an ensemble of binding
modes. The difference in possible binding motifs for the com-
plexation of PG could affect the presentation of the lipid’s
glycerol hydroxy groups to corresponding binding units on a
receptor. To examine in detail the structural requirements for a
receptor able to bind the phosphate anion portion of PG, we
prepared a family of receptors whose linkages between the two
phenolic oxygens and two benzyl amines were of different
lengths. In this way receptors could be constructed that might
support various binding motifs, and if so, potentially maximize
the entropic contribution to complexation. Isothermal titration
calorimetry (ITC) was used to probe the thermodynamic
D
cently garnered interest as potential antibiotics.5,6 Part of the
innate immune system of such diverse organisms as plants,
invertebrates, and vertebrates, cationic antimicrobial peptides
exhibit a broad spectrum of activity against Gram-positive and
Gram-negative bacteria.7-9 They are believed to bind to the
bacteria’s inner membrane anionic lipid headgroups, insert
into, and then disrupt the membrane, leading to cell death.
However, even native cationic peptides can have substantial
host effects, including toxicity.5,10-12 Therefore, an essential
requirement for any antimicrobial peptide mimic would be
that it can selectively disrupt prokaryotic membranes and not
eukaryotic membranes.
The goal of high membrane selectivity for a peptide mimic can
be achieved by increasing the recognition ability of the synthetic
antibiotic for unique bacterial membrane components. The outer
leaflets of prokaryotic cell membranes contain an abundant
supply of acidic (anionic) phospholipids, such as phosphatidyl-
glycerol, whereas the outer leaflets of eukaryotic cell membranes,
such as erythrocytes, are almost exclusively composed of zwitter-
ionic phospholipids.4 Antimicrobial cationic peptides utilize this
difference in lipid head structure to recognize and bind to anionic
phospholipids of the bacterial inner membrane. A receptor able
to selectively bind to the accessible phosphatidylglycerol (PG)
headgroup, via its phosphorus anion and glycerol dihidroxy
functionality, is therefore an attractive target as a modular
component of an antimicrobial therapeutic. The linkage of these
receptors to membrane disruptors could result in synthetic
antibiotics that exhibit bactericidal action similar to that of
cationic peptides but with lessened host toxicity. Herein we
present initial results toward the ultimate goal of preparing a
selective phosphatidylglycerol receptor. Specifically, this study
was undertaken to determine the necessary structural require-
ments for a receptor to bind the anionic phosphate group of PG
using ammonium ions as receptor binding units.
1
properties of the anion-receptor complexes, and H NMR
was used to determine the stoichiometry of binding.
The synthesis and structures of our initial receptor salts 4a-g
1
(PF6 counterions) are shown in Scheme 1. H NMR spectra
Received: December 9, 2010
Published: February 22, 2011
r
2011 American Chemical Society
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The Journal of Organic Chemistry
NOTE
Scheme 1. Preparation of Receptors 4a-g and Lipid Salt Structurea
a Reagents and conditions: (a) Br(CH2)zBr, 18-crown-6, K2CO3, THF; (b) (CH2)6N4, TFA, reflux; (c) (i) H2N(CH2)xNH2, EtOH, reflux,
(ii) triethylsilane, Pd-C, MeOH; (d) HCl/MeOH then NH4PF6, CH2Cl2, H2O.
Table 1. Results of Isothermal Titration Calorimetry
Receptor
z
x
Sa
K1 (M-1) [error]
ΔH, cal/mol [error]
ΔS, cal/mol/°C
anion salt
4a
4b
4c
5
5
4
4
3
6
1:1
1.06 ꢀ 105 [(9.6 ꢀ 103]
1.01 ꢀ 105 [(5.6 ꢀ 103]
6.60 ꢀ 105 [(1.7 ꢀ 105]
1.84 ꢀ 104 [(5 ꢀ 103]b
5000 [(60]
7300 [(50]
4000 [(60]
400 [(40]
39
47
40
21
TBAP
TBAP
TBAP
1:1
2:1
4d
4e
4f
4
4
3
5
4
6
mixture
1:1
TBAP
TBAP
TBAP
4.67 ꢀ 104 [(4.6 ꢀ 103]
1.38 ꢀ 105 [(3.3 ꢀ 104]
5.77 ꢀ 103 [(1.2 ꢀ 103]b
12000 [(300]
5600 [(200]
2000 [(500]
61
42
22
2:1
4g
3
5
mixture
TBAP
4b
4d
4e
5
4
4
3
5
4
1:1
4.06 ꢀ 104 [(6.4 ꢀ 103]
2.26 ꢀ 104 [(6.1 ꢀ 103]
4000 [(200]
3000 [(300]
35
29
TBA PG
TBA PG
TBA PG
mixture
1:1
a Binding stoichiometry determined from Job plots using 1H NMR. b K2 (M-1).
show that upon acidification of receptor 3 the amine nitrogen
protons move downfield to around 8.5 ppm (3’s NH protons are
not observed) and integrate to four protons, consistent with the
structure of receptor 4 (an X-ray crystal structure of bis-hexa-
fluorophosphate bis-ammonium receptor 4c is provided in the
Supporting Information). The receptors were prepared without
functional groups that bind to glycerol hydroxyl groups for two
reasons: (1) these receptors would be needed anyway as control
receptors for those PG receptors fully functionalized to bind both
the lipid’s phosphate and hydroxyl groups and (2) receptors that
contain several binding units make the determination of binding
stoichiometry for inorganic phosphate anion in the bis-ammo-
nium pocket problematic (i.e., two or more anions could bind to
the receptor via both binding domains). The latter would prove
troublesome because our initial strategy was to probe the
receptor-H2PO4 anion binding motif, including binding stoi-
chiometry, in solution. Our working hypothesis was that the
structure of the binding pocket found effective for binding
dihydrogenphosphate anion would translate to a receptor
pocket that would also bind the anionic phosphate group of
the tetrabutylammonium distearoyl-phosphatidylglycerol salt
(TBAPG).14 Those receptors that bound to inorganic phosphate
anion in a well-behaved manner with 1:1 stoichiometry would be
selected from among the receptor family for binding studies with
TBAPG. While ultimately a functional receptor for antibiotic
use must bind to a membrane bound PG, it was believed that
the use of solution NMR spectroscopy would most directly
illustrate binding motifs, and thereby allows for the rational,
iterative synthesis of a PG receptor’s binding units.
The stoichiometry of phosphate anion binding for receptors
1
4a-g was determined from Job plots in DMSO-d6, using H
NMR15,16 (Table 1). Downfield movement of the ammonium
1
hydrogen H NMR resonances upon addition of the anion
showed that the phosphate anion was hydrogen bonding to the
positively charged hydrogens (Supporting Information). The
results showed that the binding stoichiometry for TBAP anion
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The Journal of Organic Chemistry
NOTE
was determined by the length of the bis-amine used to construct
the receptor’s link. Binding ratios of 1:1 were found only for the
complexes made from receptors 4a, 4b, or 4e, where x = 3 or 4,
while binding ratios of 2:1 (anion: receptor) were found for
receptors 4c and 4f, where x = 6 (an X-ray structure of the
receptor-anion complex of 4f bound to two dihydrogenphos-
phate anions is provided in the Supporting Information).
Receptors 4d and 4g, where x = 5, showed no distinct stoichi-
ometry of binding. Additionally, the ability to synthesize the
shorter bis-amine link was dependent on the length of the bis-
phenolic oxygen link, whereby x = 3 could only be prepared when
z = 5. Notably, Job plots showed receptors 4b and 4e also bound
PG in a 1:1 ratio as they did with dihydrogenphosphate anion
(Table 1). Interestingly, receptor 4d, which bound the dihydro-
genphosphate anion in mixed ratios, also bound the TBAPG in
the same not well-behaved manner. The TBAPG salt, and
perhaps the receptor-PG complex, were not completely soluble
in DMSO, and therefore the stoichiometric studies were con-
ducted in 95% DMF-d7/5% CDCl3 with 1H NMR.16
Isothermal titration calorimetry (MicroCal iTC200) was used
to determine the thermodynamic properties of receptor-anion
complexation. Receptors 4a-c, 4e, and 4f (with 1:1 or 2:1 anion:
receptor binding stoichiometries) were titrated with TBAP salt in
DMSO. Not surprisingly, the ITC data show that the binding
process for receptors 4a-c, 4e, and 4f titrated with TBAP is
entropy driven (Table 1). The ITC experiments were conducted
first by adding the guest anion to receptor solution and then
second by adding the receptor to a guest anion solution via an
inverse titration. The thermodynamic properties of both addi-
tions were very similar, showing internal consistencies (i.e., state
functions are not affected by pathway).17
chemical nature of the binding pocket is very similar in either
solvent. As in the phosphate anion titration, downfield move-
ment of the ammonium hydrogen 1H NMR resonances occurred
upon titration of the receptors with the PG anion. The receptor’s
binding constants determined for dihydrogenphosphate anion
and the PG anion are very similar, only 2 times greater for the
inorganic phosphate anion than for the PG anion. The 1H NMR
data along with the small difference in binding constants
observed in the association of receptor and the two anions
garnered from the ITC experiments suggest that, while structu-
rally very different, the two phosphate anions are likely interact-
ing with the receptor pocket in a similar binding motif,
supporting our original hypothesis.
Drawing the same conclusion from a comparison of the
binding enthalpies and entropies of the two anions when
complexed to receptors 4b or 4e is problematic, however, since
the two anions have such vastly different structures. Presumably
the PG anion would have less configurational entropy because its
anion portion is more sterically hindered, consequently less able
to form a multitude of geometrical arrangements upon complex
formation. This would lead to less positive entropy afforded upon
formation of a receptor-PG complex, as is observed with both
receptor 4b and 4e. The amount of binding enthalpy and entropy
is larger for 4e than 4b upon complex formation with inorganic
phosphate anion, but larger for 4b than 4e for complex formation
with PG anion. Given that, it is interesting to note that the
binding constants for both PG anion and inorganic phosphate
anion are twice as large for receptor 4b as for receptor 4e. One
thing is clear, that the strongest interaction between both
receptor’s ammonium cation and the anion’s phosphorus-
oxygen bond(s) is found when x = 3.
The formation of receptor-H2PO4 complexes produced a
large increase in the entropy of the system, from 39 to 61 eu, with
positive enthalpies in the 4-12 kcal/mol range, resulting in
binding constants ranging from 104 to 105. Receptors 4c and 4f
exhibited slightly wider ranges (103 to 105) for both their K1 and
K2 binding constants. While the binding of dihydrogenphosphate
anion by receptors 4b or 4e in a 1:1 ratio affords the largest
entropy gains in the receptor family, complex formation with
either receptor requires the highest enthalpic costs as well. The
high positive enthalpies suggest that desolvation of these recep-
tors requires the disruption of a more stabilized solvent shell
about the ammonium cations when x = 3 or 4. The high positive
entropies additionally suggest that there may be an ensemble of
low energy structures available to the binding partners, resulting
in an increase in what has been termed configurational entropy.13
Much less positive enthalpy or entropy is produced upon binding
the second anion to receptors 4c or 4f. This is most likely due to
the fact that the two bound phosphate anions are able to
hydrogen bond with each other (see X-ray crystal structure in
the Supporting Information), thus decreasing enthalpy (via
H-bond stabilization) and entropy (via higher ordering in the
complex).
In summary, ITC titration studies of bis-ammonium receptors
4b and 4e show that they bind in a 1:1 stoichiometry to both
dihdrogenphosphate anion and phosphatidygylcerol anion with
similar binding constants, and that complexation is entropy
driven in both systems. When titrated with either anion all
the receptor’s 1H NMR spectra indicate the anion is binding to
the ammonium hydrogens, forming charged hydrogen bonds.
The stoichiometry of binding for both anions is determined by the
length of the bis-amine spacer; 1:1 binding is seen with x = 3 or 4,
not well-behaved binding is observed when x = 5, and 2:1 binding
results when x = 6. All of these data taken together suggest that
the binding motifs are similar for a complex formed between
receptor 4b or 4e and either anion. The preparation of receptors
containing functional groups that also bind to the glycerol
headgroup of PG, based on the structures of receptors 4b and
4e, is in progress.
’ EXPERIMENTAL SECTION
The following experimental procedure is provided as representative
of the preparation for all corresponding compounds 1-4:
1,5-Bis(4-methylphenoxy)pentane (1a). p-Cresol (5.17 g,
0.048 mol) was dissolved in 475 mL of dry THF under N2 and to this
mixture was added K2CO3 (6.54 g, 0.047 mol) and 18-crown-6 (2.59 g,
0.007 mol). 1,5-Dibromopentane (3.32 mL, 0.024 mol) was added and
the reaction mixture was refluxed for 20 h. The reaction was allowed to
cool to room temperature and quenched with 10% HCl. The layers were
separated and the aqueous layer was extracted with CH2Cl2 (3 ꢀ
40 mL). The combined organic layer was washed with a saturated
sodium bicarbonate solution (2 ꢀ 40 mL) and brine (40 mL) and dried
with anhydrous Na2SO4. Removal of the solvent under reduced pressure
yielded the crude product as a white solid. Column chromatography
ITC was also used to measure the thermodynamic properties
of complex formation with TBAPG and receptors 4b and 4e (two
receptors that bound TBAP with 1:1 binding stoichiometry).
Although a different solvent system was required for binding
studies with TBAP than for TBAPG, a comparison of the binding
constants is still valid for the following two reasons: (1) the
dielectric constants of DMSO and DMF are extremely close, but
1
more importantly, (2) the H NMR spectra of the receptor in
either solvent system is nearly identical, suggesting that the
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NOTE
(silica gel) eluting with 35% ethyl acetate and 65% hexanes furnished the
pure product as a white crystalline solid (6.73 g, 0.023 mol, 99% yield): mp
58-60 °C; 1H NMR(400 MHz, CDCl3) δ1.61-1.66 (m, 2H), 1.80-1.87
(m, 4H), 2.27 (s, 6H), 3.94 (t, 4H, J = 6.7 Hz), 6.78 (d, 4H, J = 7.9 Hz), 7.06
(d, 4H, J = 8.5 Hz); 13C NMR (100 MHz, CDCl3) δ 21, 23, 29, 68, 115,
129.9, 130.1, 157; MS (ESI) m/z 307.1 (M þ Na)þ; HRMS (ESI) calcd for
C19H24O2 (M þ Na)þ 307.1675, found 307.1740.
(m, 4H), 8.49 (s, 4H); 13C NMR (100 MHz, DMSO) δ 21, 23, 25, 30, 45,
46, 69, 113, 120, 130, 132, 133, 156; MS (ESI) m/z 397.1 (M - H)þ,
199.2 (M2þ/2)þ, 543.1(M2þ PF6-)þ; HRMS (ESI) calcd for C25H37-
3
O2N2 (M - H)þ 397.2855, found 397.2864.
’ ASSOCIATED CONTENT
S
1,5-Bis(2-formyl-4-methylphenoxy)pentane (2a). Compound
1a (1.58 g, 0.0055 mol) and Duff reagent (1.82 g, 0.0129 mol) were
dissolved in 20 mL of TFA and the reaction mixture was allowed to reflux
under N2 for 24 h. The mixture was concentrated, water was added, and the
aqueous layer was extracted with 50 mL of CH2Cl2. The organic layer was
separated, and the aqueous layer was acidified with concentrated HCl and
extracted with CH2Cl2 (2 ꢀ 50 mL). The combined organic layer was
washed sequentially with 4 N HCl (2 ꢀ 50 mL), 50 mL of a saturated
Na2CO3 solution, and 50 mL of brine. Removal of the solvent under vacuum
furnished the crude product as a brown gluey mass. Column chromatogra-
phy (silica gel) eluting with hexanes furnished the pure product as a white
crystalline solid (0.75 g, 0.0022 mol, 40%): mp 76-77 °C; 1H NMR (400
MHz, CDCl3) δ1.66-1.72 (m, 2H), 1.88-1.95 (m, 4H), 2.29 (s, 6H), 4.07
(t, 4H, J = 6.2 Hz), 6.96 (d, 2H, J = 8.7 Hz), 7.32 (d, 2H, J = 8.6 Hz), 7.61 (s,
2H), 10.46 (s, 2H); 13C NMR (100 MHz, CDCl3) δ20, 23, 29, 68, 113, 125,
130, 137, 160, 190; MS (ESI) m/z 409.0 (M þ Na)þ; HRMS (ESI) calcd
for C21H24O4 (M þ Na)þ 363.1572, found 363.1584.
Supporting Information. Full characterizations for 1b,
b
1c, 2b, 2c, 3b, 3c, 3e, 3f, 4b, 4c, 4e, and 4f, HRMS, 1H NMR, and
13C NMR spectra of 1a-c, 2a-c, 3a, 3b, 3c, 3e, 3f, and 4a-g,
1H NMR spectra of TBAPG, Job plots of receptors 4a, 4b, 4c,
-
-
and 4f with TBA H2PO4 and of 4e with TBA H2PO4 or
1
TBAPG, stacked plots of H NMR titrations of 4e þ TBA
H2PO4 or TBAPG, and ITC binding isotherms of 4e þ TBA
H2PO4 or TBAPG, and X-ray crystal structures for anion-
-
-
receptor complexes of 4f þ 2H2PO4 and for 4c þ 2PF6
,
and estimated complex structure 4b þ H2PO4-. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: dennis.burns@wichita.edu.
10,21-Dimethyldibenzo-[h,r]-1,7-dioxa-13,18-diaza-2,3,
4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo-
nonadecene (3a). Compound 2a (0.06 g, 0.176 mmol) was
suspended in 22 mL of dry ethanol and purged with dry N2 for 30
min, then the reaction was brought to reflux. Butane-1,4-diamine
(0.0224 mL, 0.222 mol) in 9 mL of dry ethanol was added dropwise to
the reaction mixture. The reaction was allowed to reflux for 20 h, and
the solvent was then removed under reduced pressure yielding the bis-
imine as a yellow solid, which was subjected to reduction without
further purification. The crude bis-imine was redissolved in 2 mL of
anhydrous methanol under N2. Pd-C (10%, 0.1 g) was added and the
reaction mixture was allowed to stir. Triethylsilane (1.0 mL, 0.006
mol) was added dropwise into the methanolic bis-imine solution with
an addition funnel, the top of which was attached to a rubber balloon.
After the addition was complete, the mixture was stirred for an
additional 6 h at room temperature. The reaction mixture was filtered
through Celite, and removal of the solvent under vacuum gave the
crude product as a yellow oil. Column chromatography (silica gel)
eluting with 10% CHCl3, 4% isopropylamine, and 86% diethyl ether
furnished compound 3a as an off-white solid (0.060 g, 0.151 mmol,
86% after both steps): mp 95-96 °C; 1H NMR (400 MHz, DMSO) δ
1.39-1.42 (m, 4H), 1.62-1.67 (m, 2H), 1.77-1.84 (m, 4H, J = 6.87
Hz), 2.21 (s, 6H), 2.47 (t, 4H, J = 5.6 Hz), 3.61 (s, 4H), 3.96 (t, 4H, J =
5.9 Hz), 6.85 (d, 2H, J = 8.2 Hz), 6.99 (d, 2H, J = 8.8 Hz), 7.04 (s, 2H);
13C NMR (100 MHz, DMSO) δ 21, 25, 27, 30, 48.3, 48.5, 69, 113,
128.7, 128.9, 129.4, 131, 156; MS (ESI) m/z 397.1 (M þ H)þ; HRMS
(ESI) calcd for C25H36O2N2 (M þ H)þ 397.2855, found 397.2852.
10,21-Dimethyldibenzo-[h,r]-1,7-dioxa-13,18-diaza-2,3,
4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo-
nonadecenediium bis(hexafluorophosphate (4a). Bis-amine
3a (15 mg, 0.038 mmol) was dissolved in 3 mL of methanol, 6 N HCl
(2 mL) was added, and the reaction mixture was allowed to stir for 3 h at
room temperature. The solvent was removed under vacuumand the crude
reaction mixture was redissolved in 3 mL of H2O. Ammonium hexa-
fluorophosphate (>30 equiv) dissolved in CH2Cl2 was added and the
reaction mixture was stirred overnight during which time 4a precipitated
from the solvent mixture as an off-white solid (24 mg, 0.034 mmol, 92%):
mp >165 °C dec; 1H NMR (400 MHz, DMSO) δ 1.71 (s, broad, 6H),
1.82-1.87 (m, 4H, J = 5.9 Hz), 2.26 (s, 6H), 2.92 (s, broad, 4H), 4.04
(t, 4H, J = 5.4 Hz), 4.08 (s, 4H), 7.01 (d, 2H, J = 8.7 Hz), 7.21-7.23
’ ACKNOWLEDGMENT
The authors thank Professor David M. Eichhorn and John C.
Bullinger for the acquisition of the X-ray structures. The authors
thank Professor Franz P. Schmidtchen for helpful discussions
regarding the use of ITC.
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