Enantioselective lactate binding by chiral tripodal anion hosts derived from amino acids

Article abstract of DOI:10.1039/b817889e

Chiral, tripodal anion hosts derived from either S-phenylalanine or S-leucine bind d-lactate enantioselectively. The nature of the host-guest interaction has been probed by solution NMR methods and by DFT calculations. The calculations suggest that the d-

Full text of DOI:10.1039/b817889e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Enantioselective lactate binding by chiral tripodal anion hosts derived from
amino acids†‡
Anna Barnard,a Sara Jane Dickson,a Martin J. Paterson,b Adam M. Todda and Jonathan W. Steed*a
Received 10th October 2008, Accepted 23rd January 2009
First published as an Advance Article on the web 26th February 2009
DOI: 10.1039/b817889e
Chiral, tripodal anion hosts derived from either S-phenylalanine or S-leucine bind D-lactate
enantioselectively. The nature of the host–guest interaction has been probed by solution NMR methods
and by DFT calculations. The calculations suggest that the D-lactate may form an additional hydrogen
bond in the host–guest complex while the L-lactate complex contains an intramolecular hydrogen bond.
Anion binding is in competition with host dimerisation, as demonstrated by DOSY and 1H NMR
spectroscopy, and DFT calculations.
modular synthesis7,22 of this class of compound we sought to
Introduction
impart enantioselectivity by the use of amino acid derivatives
Anion sensing, and the binding or discriminating molecular
in order to create a homochiral anion binding pocket as in
recognition processes that accompany it, has become a highly
topical field with potential applications in pollutant capture
and detection, analytical and quality control chemistry, and in
biological transport, imaging and monitoring.1–7 Of particular
tripodal hosts 3, which were prepared as the S,S,S-enantiomers in
a reasonably straightforward manner starting from the S-amino
acid derivatives, as outlined in Scheme 1. The hosts are based on a
3-a-aminomethylpyridine anion binding motif which we have used
before in ruthenium(II) based anion hosts38,39 but not in pyridinium
species. As a control we therefore also prepared the achiral tripodal
hosts 4.
importance are carboxylate anions because of their variety of
structures, biochemical importance and high intrinsic basicity.
While carboxylates generally bind strongly to a variety of hydrogen
bond donor hosts their basicity can lead to host deprotonation and
chemical degradation.8–10 Carboxylate anions are frequently chiral
and biological anion recognition is invariably highly enantioselec-
tive and since ca. 70% of enzyme substrates are anions,11 chiral
anion binding is clearly an interesting biomimetic target. One
of the simplest chiral anions is lactate (2-hydroxy propanoate)
of which L-(+)-lactate is important in biological metabolism.
Compared to the rest of supramolecular anion coordination chem-
istry, enantioselective recognition is relatively underexplored,12–16
particularly with regard to the enantioselective binding of lactate
and its derivatives.17,18 We now report the preparation and anion
binding properties of two chiral tripodal hosts based on amino
acid derivatives displaying enantioselective lactate binding.
The anion binding ability of hosts 3 and 4 was probed using
1H NMR spectroscopic titrations in acetonitrile-d3. The binding
constants for halides, nitrate, acetate and lactate anions for both
hosts obtained from HypNMR 200640,41 and the corresponding
stoichiometry are shown in Table 1. The shape of the titration
isotherms for hosts 3 in particular immediately suggests that the
anion binding equilibria are more complex than a simple 1 : 1
stoichiometry. In compounds 3 it is only the doublet resonance
Table 1
Log bHG
Stoichiometry
Anion
(H–G)
3a
3b
4a
4b
Dimerisation
constant
Cl-
2 : 0
1.0a
2.3a
2.61(15) 2.82(4)
Results and discussion
1 : 1
1 : 2
2 : 1
1 : 1
1 : 2
2 : 1
1 : 1
1 : 2
1 : 1
1 : 2
2 : 1
1 : 1
1 : 2
1 : 1
1 : 2
1 : 1
1 : 2
4.06(11) 4.17(3) 4.51(5) 3.74(6)
6.64(11) 6.02(4)
Hexasubstituted triethylbenzene or ‘pinwheel’ derivatives19 have
proved extremely useful anion binding scaffolds because of the
host preorganisation imparted by the steric gearing about the
central aromatic ring.17,20–32 While a range of neutral derivatives
have proved effective anions hosts, we have shown that cationic
pyridinium species are also highly selective and versatile anion
binding and sensing platforms.33–37 By taking advantage of the
8.14(3) 7.87(15)
Br-
I-
3.80(8) 3.92(1) 3.90(2) 3.61(3)
6.29(8) 5.98(2)
7.51(4) 7.38(6)
3.27(6) 3.02(2)
5.41(5) 5.85(5)
3.19(4)
5.44(4)
—
—
–
NO3
—
3.12(4) 3.20(4)
5.69(13) 6.18(11)
–
CH3CO2
2.86(9) 3.23(1)
5.32(4) 4.86(1)
2.67(1) 2.82(3)
4.57(1) 4.95(1)
2.75(4) 3.25(1)
5.02(1) 5.12(4)
—
—
—
3.54(3)
6.12(2)
—
aDepartment of Chemistry, Durham University, South Road, Durham, UK
DH1 3LE. E-mail: jon.steed@durham.ac.uk; Fax: +44 191 384 4737;
Tel: +44 191 334 2085
–
L-CH3(OH)CO2
bSchool of Engineering and Physical Sciences, Heriot-Watt University,
Edinburgh, UK EH14 4AS
–
DL-CH3(OH)CO2
—
† Dedicated to Prof. Seiji Shinkai on the occasion of his 65th birthday.
‡ Electronic supplementary information (ESI) available: Plots and spectra
for 3a, 3b, 4a and 4b. See DOI: 10.1039/b817889e
a Manual fit from concentration dependence data.
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assigned to the pyridinium proton para to the nitrogen atom that
undergoes a significant chemical shift change, with Dd values up
to ca. 0.9 ppm. All titration plots exhibit a marked sigmoidal
shape (e.g. Fig. 1 which shows the isotherm obtained for Cl-
binding by 3a) possibly suggesting cooperative binding or multiple
equilibria. The anion binding stoichiometry was confirmed by
Job plot for 3a and acetate anion which indicated a 1 : 2 host–
guest stoichiometry (see ESI‡). This model was then applied to
fitting the titration data for hosts 3 with all of the anions studied.
However, generally this model gave a very poor fit to the data
particularly in the low anion concentration region. Accordingly
we looked for additional possible equilibria. Examination of
1
the concentration dependence of the H NMR spectra of both
3a and 3b showed a small but consistent concentration effect
(Fig. 2) allowing us to fit dimerisation constants, log Kdim = 1.0
and 2.3, respectively. A dimerisation process offers a convincing
explanation for the apparent cooperativity in binding the two
Fig. 2 Concentration dependence of the 1H NMR spectrum of 3a. Points
are experimental data, the solid line is the calculated fit for log.
equivalents of anion guest with binding of the first anion in
competition with dimer formation, while the 1 : 1 host–guest
complex can more readily bind to a second anion. While we
were unable to observe dimer formation by mass spectrometry, we
sought confirmation of the anion-induced dissociation of a host
dimer by DOSY NMR spectroscopy on 3a.42 The DOSY spectra
indicated considerably faster diffusion (10 ¥ 10-10 m2 s-1) for 3a
in the presence of 3 equivalents of racemic tetrabutylammonium
lactate than where the predominant species is expected to be a
monomeric lactate-bound host, than for the hexafluorophosphate
salt which is expected to be a dimer, diffusion coefficient 7 ¥
10-10 m2 s-1 (see ESI for DOSY spectra). Dimer formation has
also been observed in related pyrene-based hosts.43
Adoption of a stoichiometry model incorporating a dimer of
known, fixed dimerisation constant along with 1 : 1 and 1 : 2
Fig. 1 Sigmoidal Cl- binding isotherm for host 3a.
Scheme 1 (a) Synthesis of chiral tripods 3, (b) structures of the lactate anion and of compounds 4.
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host–guest complexes allowed a good fit to the titration data,
giving the anion binding constants shown in Table 1 for 3a and
3b. The selectivity pattern for halide and nitrate anions is normal
for this type of host,44 with affinity decreasing modestly in the
order Cl- > Br- > I- > NO3 more or less in accordance with the
-
anions’ charge density. Surprisingly, however, acetate and lactate
are bound relatively weakly. The strong basicity of acetate usually
results in very strong binding by hydrogen bond donor hosts in
solvents such as acetontrile.1,39,45 The comparative weakness of
the carboxylate binding is also evidenced in the carboxylate Dd
values which are uncharacteristically slightly lower than for Cl-.
We surmise that this halide selectivity arises from steric effects
-
that prevent the acetate and lactate CH3 groups from entering
the tripodal cavity, and indeed the slightly bulkier lactate is bound
more weakly than acetate.
Fig. 4 Absorption spectra of 3a in CH3CN upon addition of up to 17.5
-
equivalents of NBu4+CH3CO2 resulting in an orange colouration.
Despite the fact that lactate binding is relatively weak with
affinity more than an order of magnitude less than Cl-, both
hosts 3a and 3b show a significant and interesting preference
for the lactate D-enantiomer, Fig. 3. We were unable to obtain
a sample of D-lactic acid and hence titrations were carried out by
transfer state involving the pyridinium acceptor.37 This assignment
is supported by TD-DFT calculations on 3b which give a broad
band around 400 nm (red shifts are quite common in TD-DFT
calculations) that corresponds to several excited states, most of
which have some amount of lactate–pyridinium p* charge-transfer
character. Indeed the first three LUMOs are nearly degenerate and
are pyridinium p* character (see ESI‡).
We sought to address both the molecular basis for the
enantioselective lactate discrimination and the competing
dimerisation process using DFT molecular models. We have found
in the past that notionally gas phase DFT models can give excellent
insights into possible conformational effects and binding modes
in these kinds of systems when coupled with detailed solution
phase information from titration and variable temperature NMR
spectroscopic data.35–37 The DFT calculated structure of the dimer
(3b)2 is shown in Fig. 5. In these flexible systems it is impossible
to guarantee full sampling of conformational space and hence
the structure in Fig. 5 represents one of a number of possible
conformational minima which may well be in equilibrium in
solution. It does, however, show the well-known alternating
conformation around the hexasubstituted aromatic rings and a
well-defined 10-membered ring hydrogen bonding motif involving
+
comparing a commercial sample of the L-enantiomer (as its NBu4
salt) with a racemic mixture. Fig. 3 shows that there is a marked
difference in the response of the hosts to the two samples and
this difference is reflected in the binding constants with both hosts
proving selective by a factor of 1.5–3 comparing the observed b11
and b12 values. Given that we are dealing with a racemic mixture,
this corresponds to a D/L selectivity of ca. 3–6, or an ee of up to ca.
70%. While not yet of the kind of selectivity required for resolution
of chiral materials in a single step, this is a surprisingly positive
result for such a flexible host system and augurs well for future
refinement of the design. It is also interesting that the hosts are able
to discriminate between lactate enantiomers even though they are
halide-selective; indeed strong binding and effective discrimination
are often mutually antagonistic.44
Fig. 3 Comparison of DL- and L-lactate binding by 3a.
A final interesting feature of both hosts 3a and 3b upon
carboxylate binding was the observation of a visual colour change
from colourless or orange/red upon addition of an excess (up to
ca. 17.5 equivalents) of acetate or lactate. The UV-Vis absorption
spectrum of 3a upon titration with acetate in acetonitrile solution
is shown in Fig. 4. The red colour comes from the growth of
a broad shoulder at around 350 nm extending into the visible
region. This band is tentatively assigned to an anion–p* charge
Fig. 5 DFT calculated structure of the proposed dimer (3b)2 (host CH
hydrogen atoms omitted for clarity).
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the amine NH donor and ester oxygen atom acceptors. A CSD
search46,47 confirms that such a motif is common for esters
joined to amide NH atoms (161 hits) and is also precedented
for rarer amine esters (such as compounds 3) in CSD refcode
TICXIM (1,1¢-bis(p-chlorophenyl)-6,7a-dimethyl-4,7a,1¢,4¢,5¢,6¢-
hexahydro-pyrrolo(4,5-a)(1,2,4)triazole-5-spiro-6¢-(1,2,4)triazine-
3,3¢-dicarboxylate).48 The calculated structure also shows that it
is feasible for the dimer to form in the absence of added anion
by inclusion of one of the amino acid residue substituents within
the dimer cavity. Such a structure is consistent with the observed
dissociation of the dimer upon addition of anions to give 1 : 1 and
1 : 2 host–guest complexes since exposure of the hydrogen bonding
functionality is a necessary feature of anion binding. Fig. 6 shows
the DFT calculated structures of 3b binding L- and D-lactate,
respectively. The model of the D-lactate complex has an additional
lactate NH ◊ ◊ ◊ O interaction with the host compared to the
L-lactate complex, in which the additional host–guest interaction
is replaced by an intramolecular host NH ◊ ◊ ◊ O interaction. This
additional interaction is very much consistent with the stronger
binding of the D-enantiomer observed in solution. However, these
gas phase models must be treated tentatively since in solution
solvation of the lactate OH and carboxylate groups in particular
and compensating entropic effects49 may well be highly significant.
Clearly, however, 1 : 1 binding of both lactate enantiomers is
feasible in these systems and competition between intramolecular
and host–guest hydrogen bonding interactions may be a factor in
the observed enantioselectivity.
two pyridinium substituents occupy the opposite face of the
hexasubstituted core to the other one.34 The remainder of the
spectrum becomes complex but is consistent with the presence of
a number of different species or conformers and may well reflect
a distribution of the sample into dimer and monomer, the latter
existing as a ‘2-up, 1-down’ conformer as well as a ‘3-up’ isomer,
as observed for related 3-aminopyridinium compounds,34 Fig. 7.
Re-recording the VT spectra in the presence of one equivalent
of lactate resulted in modest qualitative changes to the chemical
shifts of some resonances but a similar mixture of species was
As a further insight into the hosts’ solution conformational
1
behaviour we used variable temperature H NMR spectroscopy
in acetone-d6 solution. At room temperature, in both acetone
and acetonitrile solutions, hosts 3 display at least time-averaged
C3 symmetry. Lowering the temperature resulted in significant
broadening of many of the host resonances, including, for example,
the resonance at d = 1.0 ppm assigned to the CH3 groups of the
core aromatic ring ethyl substituents. At -70 ◦C this resonance
splits into a major component at 1.3 ppm and a much smaller
peak at 0.5 ppm. This latter upfield peak is indicative of a CH3
group in the shielding region of one of the pyridinium rings
and is consistent with some of the sample (but by no means
all) being present in the ‘2-up, 1-down’ conformation in which
Fig. 7 Variable temperature 1H NMR spectra of 3a at (a) 20 ◦C and
(b) -90 ◦C.
Fig. 6 DFT calculated structures of the 1 : 1 complexes of 3b with (a) L-lactate and (b) D-lactate (lactate atoms shown as spheres, host CH hydrogen
atoms omitted for clarity).
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again observed at low temperature. Examination of the speciation
plot for 3a + L-lactate (see ESI‡) confirms that a mixture of all of
the four possible species (1 : 0, 2 : 0, 1 : 1 and 1 : 2) is expected at
this concentration.
complex nature of the equilibria and the fact that only limited
data is available in the case of Cl- due to precipitation at higher
host–anion ratios.
Given the complex dimerisation and multiple anion binding
behaviour of hosts 3, we sought further insights into their con-
formational characteristics from the simplified model compounds
4, which contain the 3-(aminomethyl)pyridinium binding group
but without the chiral amino acid residues. The 1H NMR titration
isotherms for one of these compounds (4b) is shown in Fig. 8. Once
again, the data exhibits significant sigmoidality, although the effect
is far less pronounced than in compounds 3. Also, interestingly,
acetate appears to behave differently to the other anions with
very significant chemical shift changes well beyond the addition
of one equivalent of anion, while the plots for the other anions
start to level out at around one equivalent, with a marked change
in gradient in the case of Cl- binding. The acetate data fitted
well with the same model adopted for compounds 3, with both a
slightly enhanced acetate affinity and dimerisation constant, log
Kdim = 2.82 (Table 1). Surprisingly, however, this model gave a very
bad fit for all of the other anions. A simple 1 : 1 stoichiometry was
also inappropriate given the sigmoidal nature of the isotherms and
the fact that if dimerisation is present during the acetate titration,
it must also be a competing process in all of the other experiments.
We were, however, able to fit the data convincingly (see ESI‡)
using a model involving the binding of a single anion by both
monomer and dimer; i.e. our model incorporates four species of
H–G stoichiometry 1 : 0, 2 : 0, 1 : 1 and 2 : 1. We rationalise
this different stoichiometry by postulating that the host dimer for
these less sterically hindered compounds can encapsulate halides
and nitrate anions that do not have a pendant methyl substituent.
Similar behaviour has been observed in related pyrene-derived
compounds.43 Such a postulate is consistent with the self-inclusion
observed in the DFT model of 3b (Fig. 5) which could disfavour
the formation of such 2 : 1 complexes by that compound.
Conclusion
In conclusion, we have designed a chiral tripodal anion host
system displaying modest D-lactate selectivity amid the lactate
enantiomers. The solution behaviour of this class of host system
proved highly complex, however, with chloride binding being sig-
nificantly favoured over all of the carboxylates studied. Moreover,
anion binding is complicated by the formation of both 1 : 1 and
1 : 2 complexes, apparently in competition with a host dimerisation
process as well as conformational equilibria. The combination of
detailed NMR titration and variable temperature work coupled
with DFT calculations and precedents from our previous work
on related compounds allows some insight into the behaviour of
the system, suggesting that the D-enantiomer is able to form more
host–guest hydrogen bonding interactions. However, the solution
speciation of these very flexible, sterically crowded systems does
not allow for facile rationalisation of their behaviour. The apparent
dimer formation in this case contrasts with the anion-induced
‘zipping-up’ of a unimolecular capsule that we have studied in a
related compound.36
Experimental
All reagents were purchased from commercial sources and were
used without further purification. 1,3,5-Tris(bromomethyl)-2,4,6-
triethylbenzene was prepared as previously reported.19 Tetrabuty-
lammonium L-lactate was obtained from commercial sources. The
D-enantiomer was not available. Racemic tetrabutylammonium
lactate was prepared from the reaction of racemic lactic acid
with one molar equivalent of tetrabutylammonium hydroxide
in methanol in a Schlenk apparatus. After stirring for 2 hours,
the methanol and the water were removed via a trap-to-trap
vacuum distillation. H, 13C, H-1H COSY, H-13C HSQC and
1H-13C HMBC spectra were obtained from a Varian INOVA 500
1
1
1
1
spectrometer at a frequency of 500 MHz for H and 125 MHz
for 13C or from a Bruker Avance 400 at a frequency of 400 MHz
1
1
for H and 100 MHz for 13C. H NMR spectroscopic titrations
were performed on a Varian Mercury 400 BB spectrometer at a
frequency of 400 MHz. Mass spectrometry data were obtained on
a Thermo Finnigan LTQ spectrometer in ES+ and EI mode. C, H
and N elemental analysis was performed on an Excitor Analytical
Inc CE440 elemental analyser. Bromide analysis was performed on
a DIONEX DX120 ion chromatograph. IR spectroscopy data was
obtained from a Perkin Elmer Spectrum 100 FT-IR spectrometer.
UV-Vis spectrophotometric titrations were performed on a UNI-
CAM UV-Vis spectrometer (UV2-100) operated under PC-control
using Vision software and the scan range was set to 220–450 nm.
Geometry optimizations were performed using the B3LYP
functional in conjunction with the following basis: the 4-31G set on
all carbon, nitrogen and hydrogen atoms, and the 6-31+G(d) set on
all oxygen atoms. The basis was further augmented with two sets
of diffuse sp functions on peripheral hydrogen-bonding hydrogen
atoms. Minima were confirmed by analytical computation of
the Hessian and noting positive definiteness. The time-dependent
calculations were performed with an enlarged basis of 6-31G(d)
Fig. 8 Anion binding by 4b.
Another surprising observation is the fact that the nitro
compound 4b actually binds anions more weakly than its phenyl
analogue 4a. This is in direct contrast to the usual effect of electron
withdrawing substituents which tend to enhance amine/amide
hydrogen bond donor ability and hence anion affinity.15,39,50 In
this case, the nitro substituents appear to enhance the dimerisation
constant but it is 4a that exhibits the highest chloride anion affinity.
The origins of this effect are unclear and may be rooted in the
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on all carbon, nitrogen and hydrogen atoms, plus the oxygen and
peripheral hydrogen atoms as described above. Although the full
conformational space was not sampled, preliminary computations
were performed for each system to ensure the most sensible
initial host–guest binding conformation. All computations were
performed using the Gaussian03 program.51
Py C-H); 7.22 (1H, t, J = 4.9, Py C-H); 3.80 (2H, dd, Jd = 161,
Jdd = 13.3, CH2NH); 3.71 (3H, s, CO2CH3); 3.24 (1H, t, J = 7.7,
CHCO2CH3); 1.75 (1H, m, CH2CH(CH3)2); 1.44 (2H, t, J = 7.7,
CH2CH(CH3)2); 0.88 (3H, d, J = 7.0, CH2CHCH3); 0.82 (3H, d,
1
J = 6.3, CH2CHCH3). 13C{ H} NMR (CDCl3, d/ppm): 176.2,
149.7, 148.5, 135.8, 135.0, 123.4, 59.4, 51.8, 49.5, 42.7, 25.0, 22.6,
22.0. EI-MS: m/z = 259.2 [M + Na]+, 237.2 [M + H]+. IR (n/cm-1):
2954, 1732, 1579, 1468, 1426, 1368, 1269, 1196, 1149, 1026, 990,
786, 713, 631.
Synthesis of methyl 3-phenyl-2-[(pyridin-3-ylmethyl)amino]
propanoate (2a)
L-Phenylalanine methyl ester hydrochloride (10.0 g, 55.9 mmol)
and one equivalent of triethylamine (5.65 g, 55.9 mmol) were
dissolved in dry methanol (100 mL) and the mixture was stirred for
15 minutes. Two equivalents of 3-pyridinecarboxaldehyde (12.0 g,
111 mmol) were added along with magnesium sulfate. The mixture
was heated to reflux for 24 hours under an inert atmosphere.
The solution was then allowed to cool to room temperature
at which point sodium borohydride (3.13 g, 82.7 mmol) was
added slowly over a half hour period. The solution was then
stirred for 2 hours under an inert atmosphere. The reaction
was quenched to pH 3 with 1 M hydrochloric acid and then
adjusted to pH 8 with 1 M sodium hydroxide. The product
was extracted with dichloromethane and the residual solvent
removed under reduced pressure. The product was purified by
column chromatography using a 5% ethanol in dichloromethane
solvent system resulting in the pure methyl 3-phenyl-2-[pyridin-3-
Synthesis of phenylalanine-derived tripod (3a)
1,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene
(0.499
g,
2.12 mmol) and phenylalanine derivative 2a (2.00 g, 7.41 mmol)
were dissolved in dichloromethane (50 mL) and stirred at room
temperature under a nitrogen atmosphere for 20 hours. The
solvent was removed under reduced pressure to leave an orange
oil which was washed with diethyl ether yielding the bromide
salt as an orange solid (yield 1.18 g, 0.94 mmol, 44%). 1H NMR
(DMSO-d6, d/ppm, J/Hz): 9.08 (1H, s, py C-H); 8.99 (1H, d, J =
6.0, py C-H); 8.45 (1H d, J = 8.0, py C-H); 8.08, 1H, t, J = 6.8,
py C-H); 7.40 (5H, m, Ar CH); 6.03 (2H, 2,CH2py); 4.13, 3.94
(2H, AB, J = 15.6, CH2NH); 3.62 (2H, s, CO2CH3); 2.96 (2H, d,
J = 6.6, PhCH2CH3); 2.65 (2H, d, J = 6.8, CHCH2Ph); 0.88 (3H,
1
t, J = 6.6, PhCH2CH3). 13C{ H}NMR (DMSO-d6, 100 MHz,
d/ppm): EI-MS: m/z = 546.4 [M - Br]2+, 337.6 [M - 3Br]3+. The
bromide salt (1.18 g, 0.94 mmol) was dissolved in methanol and
excess ammonium hexafluorophosphate (1.53 g, 9.4 mmol) was
added and the mixture was stirred for 2 hours. The solution was
filtered leaving the isolated PF6 salt of the phenylalanine◦tripod
1
ylmethyl]amino] propanoate (yield 4.56 g, 16.9 mmol, 30%). H
NMR (DMSO-d6, d/ppm, J/Hz): 8.55 (1H, s, Py C-H); 8.53 (1H,
d, J = 6.3, Py C-H); 8.38 (1H, m, Ar C-H); 7.73 (1H, d, J = 7.7, Py
C-H); 7.56 (1H, d, J = 7.7, Ar C-H); 7.39 (1H, t, J = 3.9, Py C-H);
7.20 (2H, m, Ar C-H); 3.6 (2H, dd, Jd = 112, Jdd = 14 CH2NH);
3.38 (1H, t, J = 7.0, CHCO2Me); 3.28 (3H, s, CO2CH3); 2.85 (2H,
1
(0.63 g, 0.44 mmol, 46% yield). Mp—decomposes 130 C. H
NMR (CD3CN, d/ppm, J/Hz): 8.51 (1H, s, Py C-H); 8.32 (1H,
d, J = 8.0, Py C-H); 8.23 (1H, d, J = 5.6, Py C-H); 7.87 (1H, t,
J = 6.4, Py C-H); 7.20 (5H, m, Ar C-H); 5.74 (2H, s, CH2Py);
4.13, 3.67 (2H, AB, J = 16.0, CH2NH); 3.66 (3H, s, CO2CH3);
3.50 (1H, t, J = 6.0, CHCO2CH3); 2.95 (2H, m, PhCH2CH3); 2.52
(2H, d, J = 7.2, CHCH2Ph); 0.91 (3H, t, J = 7.2, PhCH2CH3).
1
d, J = 7.0, CH2Ph). 13C{ H} NMR (DMSO-d6, d/ppm): 174.7,
150.0, 149.7, 148.3, 139.0, 136.0, 134.7, 129.7, 128.3, 127.0, 124.0,
62.3, 53.7, 48.3, 39.6. EI-MS: m/z = 293 [M + Na]+, 271 [M +
H]+. IR (n/cm-1): 3309, 2970, 1734, 1579, 1496, 1427, 1201, 1172,
1087, 1047, 1028, 746, 700, 634.
1
13C{ H} NMR (DMSO-d6, d/ppm): EI-MS: m/z = 337.6 [M -
3PF6]3+. IR (n/cm-1): 1732, 1497, 1455, 1202, 1141, 1034, 830.
Anal. Calcd. for C63H75F18N6O6P3·2H2O: C 51.02, H 5.37, N 5.67.
Found: C 51.12, H 4.92, N 5.67%.
Synthesis of methyl 4-methyl-2-[(pyridin-3-ylmethyl)amino]
pentanoate (2b)
L-Leucine methyl ester hydrochloride (10.0 g, 69.0 mmol) and one
equivalent of triethylamine (6.97 g, 69.0 mmol) were dissolved in
dry methanol (100 mL) and the mixture was stirred for 15 minutes.
Two equivalents of 3-pyridinecarboxaldehyde (14.8 g, 138 mmol)
were then added along with magnesium sulfate. The mixture was
heated to reflux for 24 hours under an inert atmosphere. The
solution was then allowed to cool to room temperature and two
equivalents of sodium borohydride (5.24 g, 138 mmol) were added
over a half hour period. The solution was stirred for 2 hours under
an inert atmosphere. The reaction was quenched to pH 3 with 1 M
hydrochloric acid and then adjusted to pH 8 with 1 M sodium
hydroxide. The product was extracted with dichloromethane and
the residual solvent removed under reduced pressure. The product
was purified by column chromatography using a 15% ethanol
in dichloromethane solvent system resulting in the pure methyl
4-methyl-2-[(pyridin-3-ylmethyl)amino] pentanoate (yield 6.37 g,
27.0 mmol, 39%). 1H NMR (CDCl3, d/ppm, J/Hz): 8.52 (1H, s,
Py C-H); 8.47 (1H, d, J = 4.9, Py C-H); 7.66 (1H, d, J = 7.7,
Synthesis of leucine-derived tripod (3b)
1,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene (2.40 g, 5.45mmol)
and 3.5 equivalents of leucine ligand 2b (4.50 g, 19.1 mmol) were
dissolved in dichloromethane and the solution was left to stir for
20 hours under an inert atmosphere. The solvent was then removed
under reduced pressure to leave the bromide salt as an orange solid.
1H NMR (CDCl3, d/ppm, J/Hz): 9.77 (1H, s, Ph C-H); 9.46 (1H,
d, J = 5.6, Py C-H); 8.35 (1H, d, J = 8.4, Py C-H); 8.12 (1H,
t, J = 7.2, Py C-H), 6.19 (2H, s, CH2Py); 4.13, 4.09 (2H, AB,
J = 15.2, CH2NH); 3.67 (3H, s, CO2CH3); 3.29 (1H, t, J = 7.2,
CHCO2CH3); 2.53 (2H, q, J = 7.0, ArCH2CH3); 1.71 (1H, m,
CH2CH(CH3)2); 1.47 (2H, t, J = 7.2, CH2CH(CH3)2), 1.01 (3H,
t, J = 7.0, ArCH2CH3); 0.85 (6H, m, CH2CH(CH3)2). 13C{ H}
1
NMR (CDCl3, d/ppm): 175.5, 150.9, 144.4, 143.8, 143.1, 142.2,
129.1, 127.5, 59.8, 53.4, 51.7, 48.1, 42.4, 24.8, 22.8, 22.2, 15.9.
EI-MS: m/z = 528 [M - 2Br]2+, 304 [M - 3Br]3+. The bromide
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salt (7.40 g, 6.43 mmol) was dissolved in acetonitrile (50 mL) and
an excess of ammonium hexafluorophosphate (10.5 g, 64.3 mmol)
was then added and the mixture was heated to reflux overnight.
The solvent was then removed under reduced pressure and the
product redissolved in dichloromethane. The dichloromethane
was removed under reduced pressure to leave the pure leucine
(CD3CN, d/ppm, J/Hz): 8.57 (3H, s, Py-H); 8.51 (3H, d, J = 8.4,
Py-H); 8.37 (3H, d, J = 6.0, Py-H); 8.00 (9H, m, Ar-H and Py-H);
6.65 (6H, m, Ar-H); 6.28 (3H, t, J = 6.4, NH); 5.80 (6H, s, CH2);
4.71 (6H, d, J = 6.4, CH2); 2.50 (6H, q, J = 7.2, CH2); 0.80 (9H,
1
t, J = 7.2, CH3). 13C{ H}-NMR (CD3CN, d/ppm): 153.0; 151.0;
145.4; 142.4; 142.4; 141.6; 138.8; 128.8; 128.0; 126.2; 117.6; 112.1;
58.2; 43.6; 24.2; 14.5. ES+ MS: 1178 [M - PF6]+, 516 [M - 2PF6]2+,
296 [M - 3PF6]3+. Anal: calcd. for C51H54N6O9(PF6)3: C 46.26, H
4.08, N 9.52. Found: C 45.73, H 4.13, N 9.30%. IR: 3239 n(NH).
◦
tripod (2.63 g, 1.95 mmol, 36% yield). Mp—decomposes 95 C.
1H NMR (CD3CN, d/ppm, J/Hz): 8.67 (1H, s, Py C-H); 8.51
(1H, d, J = 8.0, Py C-H); 8.28 (1H, d, J = 6.0, Py C-H); 7.96
(1H, t, J = 6.4, Py C-H); 5.85 (2H, s, CH2Py); 4.14, 3.66 (2H,
AB, J = 15.6, CH2NH); 3.67 (3H, s, CO2CH3); 3.29 (1H, t, J =
7.6, CHCO2CH3); 2.56 (2H, q, J = 7.6, ArCH2CH3); 1.77 (1H, m,
CH2CH(CH3)2); 1.47 (2H, t, J = 7.2, CH2CH(CH3)2); 0.90 (6H,
Acknowledgements
We are grateful to the EPSRC for funding (SJD and AMT).
d, J = 6.8, CH2CH(CH3)2); 0.86 (3H, t, J = 2.8, ArCH2CH3). 13
C
NMR (DMSO-d6, 100 MHz, d/ppm): 175.1, 149.9, 145.2, 143.1,
142.3, 141.7, 128.2, 127.8, 58.7, 57.3, 51.5, 47.2, 41.7, 24.3, 23.5,
22.6, 22.0, 14.8. EI-MS: m/z = 1345 [M]+, 528 [M - 2PF6]2+, 304
[M - 3PF6]3+. IR (n/cm-1): 2960, 1728, 1456, 1260, 1198, 1017,
828. Anal. Calcd. for C54H81F18N6O6P3·3H2O: C 46.35, H 6.27,
N 6.01. Found: C 46.45, H 5.90, N 6.03%.
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