Molecular amplification in a dynamic system by ammonium cations

Article abstract of DOI:10.1016/S0040-4020(01)01102-4

Using hyrazone chemistry, a simple dynamic of macrocyclic pseudo-peptides was prepared which contains a cyclic structure that resembles a known receptor for ammonium cations. A series of ammonium salts were tested as templates and the expected receptor wa

Full text of DOI:10.1016/S0040-4020(01)01102-4

TETRAHEDRON
Pergamon
Tetrahedron ꢀ8 52002) 771±778
Molecular ampli®cation in a dynamic system by
ammonium cations
Ricardo L. E. Furlan, Yiu-Fai Ng, Graham R. L. Cousins, James E. Redman
p
and Jeremy K. M. Sanders
Cambridge Centre for Molecular Recognition, University Chemical Laboratory, Lens®eld Road, Cambridge CB2 1EW, UK
Received 1 June 2001; revised 20 August 2001; accepted 22 August 2001
AbstractÐUsing hydrazone chemistry, a simple dynamic system of macrocyclic pseudo-peptides was prepared which contains a cyclic
structure that resembles a known receptor for ammonium cations. A series of ammonium salts were tested as templates and the expected
receptor was ampli®ed in different degrees according to the template used. Competitive non-covalent interactions with a crown ether were
1
used to demonstrate in situ that the templated system is still dynamic. Formation of host±guest complexes was proven by ESI-MS and H
ROESY experiments. Relative af®nities of the various templates for the isolated receptor correlate well with the effect they produce on the
library composition. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
We have recently developed the use of hydrazone exchange
4
f
to prepare DCLs. A range of interconverting macrocycles
can be generated from bifunctionalized building blocks
containing one hydrazide and one protected aldehyde linked
to a central unit 5Fig. 1). The presence of acid catalyses 5a)
hydrazone formation that produces the assembly of the
macrocycles, and 5b) hydrazone exchange that leads to
the interconversion of the macrocycles. Neutralization of
the reaction medium switches off the exchange converting
the library into a `static' mixture from which the individual
members can be isolated.
Dynamic combinatorial chemistry 5DCC) has attracted
increased interest over recent years as a new strategy for
the preparation and identi®cation of new host and guest
1
compounds. The approach combines the merits of
combinatorial chemistry with molecular evolution, and in
principle integrates preparation and screening of libraries in
one single process.
2
Dynamic combinatorial libraries 5DCLs) are generated by
the assembly of building blocks through reversible bonds.
Since every member in the mixture contains one or more
reversible connections, the library product distribution is
thermodynamically controlled and may be in¯uenced by
Here we report molecular ampli®cation from a very
simple dynamic mixture of macrocyclic pseudo-peptides.
The selected macrocycle binds selectively to different
ammonium salts used as templates for its ampli®cation.
The af®nities of the various templates for the macrocycle
3
template effects. Stabilization of one particular member of
the library by addition of a template molecule will shift the
equilibria leading to an increased concentration of the selected
compound at the expense of the other members. In this way, a
guest can be used as a template to select and amplify its
preferred host from a library of potential receptors 5Fig. 1).
Several DCLs have been prepared so far using either
covalent or non-covalent bonds to reversibly connect the
4
ꢀ
6
building blocks. Ampli®cation of selected receptors has
been observed in libraries in which the building blocks are
ꢀ
a,b
held together by hydrogen bonds
or metal coordina-
However, ampli®cation of covalently assembled
ꢀ
c±e
tion.
receptors seems to be more elusive and not many examples
have been reported so far.
7
Keywords: ammonium cations; host±guest complexes; dynamic system.
Corresponding author. Tel.: 144-122-333-6411; fax: 144-122-333-
p
6017; e-mail: jkms@cam.ac.uk
Figure 1. Generation and templating of a dynamic combinatorial library.
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020501)01102-4

772
R. L. E. Furlan et al. / Tetrahedron 58 (2002) 771±778
Figure 2.
are in accordance to the response that each salt exerts on the
system.
Kubik et al. have reported the synthesis and receptor
properties of the cyclic peptide 1 composed of 5l)-proline
and 3-aminobenzoic acid in an alternating sequence
Figure 3. 5a) HPLC trace of the reaction mixture 6 h after the reaction was
initiated from building block 3. 5b) HPLC trace of the reaction mixture after
3 days. 5c) HPLC trace of cyclic dimer and cyclic trimer.
8
Fig. 2). Macrocycle 1 is able to bind ammonium
5
cations in chloroform with stability constants between
2
1 000 and 42 000 M . If the peptide bonds that connect
1
1
then represents around 88% of the peptide material of the
mixture 5Fig. 3b).
9
the N-terminus of 3-aminobenzoic acid with the C-terminus
of proline in 1 are replaced by hydrazones, a similar macro-
cycle 2 is obtained, which contains three additional CvN
imine bonds. Pseudo-peptide 2 could be a member of a DCL
prepared by acid cyclization of building block 3. Our expec-
tation was that ammonium salts would bind the cyclic trimer
To ascertain that the reaction mixture has reached the equi-
librium, the library was also generated from pure cyclic
trimer, and from pure cyclic dimer. In both experiments,
the ®nal distribution of products was the same as in the
library prepared from 3 5Fig. 3b and c).
2 strongly enough to template its formation leading to
molecular ampli®cation.
Having a dynamic system in which the cyclic trimer is
kinetically and thermodynamically accessible, we tested a
series of tetra alkyl ammonium salts for their templating
ability 5Fig. 4). Four equivalents of each salt were
2. Results
Building block 3 was prepared by standard EDC coupling of
l-proline methyl ester hydrochloride with 3-carboxybenzal-
dehyde dimethoxyacetal, followed by hydrazinolysis of the
methyl ester to generate the necessary hydrazide function-
ality 5Scheme 1).
Cyclization of 3 in chloroform/TFA leads initially to a series
of macrocyclic N-acyl hydrazones. Six hours after the reac-
tion is initiated, 3 has been consumed completely and
macrocycles from dimer to 1ꢀmer are detected by electro-
spray mass spectrometry 5ESI-MS). At this time, HPLC
analysis shows that the cyclic trimer constitutes the major
product of the mixture 5Fig. 3a). However, after a period of
three days, the initial distribution of macrocycles drifts
almost exclusively in favour of the cyclic dimer, which
Figure 4. Relative amounts of cyclic products in absence 5control) and in
presence of different templates.
Scheme 1. 5a) TEA, CH Cl , EDC, DMAP; 5b) NH
2 2
2
NH
´H O, MeOH.
2 2

R. L. E. Furlan et al. / Tetrahedron 58 (2002) 771±778
773
introduced into the system and the reaction was monitored
by HPLC, observing different responses from the library.
Salts containing three butyl or ethyl groups on the nitrogen
produced weak non-speci®c ampli®cation of higher
oligomers. However, when trimethyl ammonium salts or
N-methyl quinuclidinium iodide were used as templates,
the cyclic dimer was consumed to generate more cyclic
trimer. The ®nal concentration of trimer depends on the
template used and varied between 10 and 90% of the peptide
material in the mixture.
If the observed ampli®cation is due to the stabilization of the
cyclic trimer via complexation with the ammonium salts,
any change in the extent of binding should result in a
corresponding change in the library composition. When
the amount of added NMQ was varied in the range
1±7 equiv. the proportion of peptide material present as 2
similarly ranged from 12±81%.
Electrospray mass spectrometry 5ESI-MS) has proved to be
a powerful technique for the identi®cation of supramolecu-
1
0
lar complexes. ESI-MS analysis of the reacting mixtures
in presence of the templates allowed the detection of
complexes between the cyclic trimer and the corresponding
effective templates. Cyclization reactions were carried out
in presence of ꢀ equiv. of the different effective templates,
and ESI mass spectra were recorded while the system was
on its way towards the equilibrium. In all the experiments,
peaks corresponding to the complex 3mer-template were
observed at 817.ꢀ 5for 3mer: EtMe N ), 879.ꢀ 5for 3mer:
Figure 5. ESI mass spectra of the reaction mixture in presence of: 5a)
ꢀ equiv. of EtMe NI, and 5b) ꢀ equiv. of NMQ.
1
3
3
1
1
BnMe N ), 8ꢀꢀ.ꢀ 5for 3mer: NMQ ), and 87ꢀ.6 5for 3mer:
3
1
ACh ). No such adducts were observed for the cations with
any of the other library members 5Fig. ꢀ).
sponding to the complex between [18]crown-6 and the
protonated hydrazide monomer accompanied by small
peaks for the protonated cyclic dimer 5487.3), and trimer
5730.4), as well as for the complex of the protonated linear
dimer with [18]crown-6 5769.4) and for the complex of the
cyclic trimer with NMQ 58ꢀꢀ.ꢀ) 5Fig. 6).
When working with dynamic systems, it is important to
show that the ®nal product distribution in presence of a
template represents the equilibrium and is not the result of
the introduction of a kinetic trap. It should be noted that the
ammonium salts added into the DCL are able to undergo
anion exchange with the TFA used as catalyst. This
exchange could render the reaction conditions insuf®ciently
acidic to permit hydrazone exchange.
In these experiments, the product distribution of the
dynamic system is controlled by two simultaneous supra-
molecular effects: 51) towards linear monomer and linear
dimer by virtue of crown ether recognition, and 52) towards
cyclic trimer by ammonium cation binding; both effects act
against the intrinsic thermodynamic preference towards the
cyclic dimer. This intricate interplay of supramolecular
If the macrocycles are interconverting, an equal number of
linear hydrazinium intermediates generated during the ring
opening/closure process must be present in the mixture. We
have previously reported that the hydrazinium terminus of
1
1
these transient species can be complexed by [18]crown-6.
This complexation leads to ampli®cation of the linear
species, which can therefore be detected by ESI-MS or
HPLC demonstrating that the system is still dynamic.
Solutions of 3 were hence established under thermodynamic
cyclization conditions with 4 equiv. of N-methyl quinu-
clidinium and variable quantities of TFA, such that the
series of experiments progresses from an excess of template
to an excess of TFA. The equilibria were maintained for
three days before [18]crown-6 was added to the solutions.
For all the experiments, after the addition of the crown
ether, a new signal was observed in the HPLC at 2.ꢀ min.
The UV spectrum recorded using the diode array of the
HPLC facility shows the expected band at 2ꢀ7 nm
characteristic of aromatic aldehydes. ESI mass spectra of
the solutions are dominated by a major peak at ꢀ26.4 corre-
Figure 6. ESI mass spectrum of the dynamic system in presence of two
competing templates: NMQ and [18]crown-6.

774
R. L. E. Furlan et al. / Tetrahedron 58 (2002) 771±778
recognition and stabilization, through a more or less
complex network of equilibria highlights the capabilities
of DCLs.
interconverting is clear from the NOESY spectrum, which
shows exchange peaks between all the a-proton signals.
This mixture of conformers and cis/trans isomers can be
envisaged as a dynamic conformational or con®gurational
1
The H NMR spectrum of pure trimer 2 in a 9ꢀ:ꢀ chloro-
12
library in itself.
form/methanol mixtureÐto ensure complete solubilityÐ
shows the presence of different conformational isomers in
slow exchange on the NMR chemical shift time scale
Addition of different templates to the mixture of inter-
converting conformers of 2 dramatically changes its H
1
5
Fig. 7a). Inspection of the a-proton region of the spectrum
reveals a range of a-proton signals between 4.ꢀ and
.0 ppm. That all of these conformers for the trimer are
NMR spectrum. The presence of 1equiv. of acetylcholine
leads to the simpli®cation of the whole spectrum of 2, which
6
is now dominated by a single C symmetric conformer
3
1
Fig. 7b). All the signals previously observed in the a
3
5
proton region collapse into one multiplet at 4.74 ppm. Simi-
0
the aromatic Ha are observed at 7.97 and 8.23 ppm where
larly two sharp singlets corresponding to the imine He and
0
several broad signals were previously present.
The binding to the chiral receptor 2 also affects the reso-
nances of all the guest protons. The N-methyl proton signal
is shifted down®eld by 0.1ꢀ ppm while the signal corre-
sponding to the acetyl group is shifted up®eld by
0.23 ppm as compared to the unbound guest. In addition
the ±CH a± and ±CH b± protons of acetylcholine become
2
2
inequivalent and split into two pairs of signals. The appear-
ance of the signals for ±CH a± and ±CH b± and the
2
2
observed gem, cis and trans coupling constants for
CH b± 51ꢀ.7, 2.9 and 7.ꢀ Hz, respectively), led to the
interpretation that acetylcholine is bound in the trans
±
2
1
4
conformation 5Fig. 7c).
Further information on the host±guest complex between the
cyclic trimer and acetylcholine was provided by H ROESY
1
studies. Analysis of a 1:1 mixture of acetylcholine and 2 in
CDCl /CH OD 59ꢀ:ꢀ) revealed nOes between the N-methyl
3
3
0
of proline in the receptor 5Fig. 8a). NOes were also observed
protons of the guest and the aromatic Ha and the C-a proton
0
acetylcholine. These intermolecular nOes suggest that
between Ha and the ±CH a± and ±CH b± protons of
2
2
when it is bound to the guest, receptor 2 adopts a conforma-
tion in which Ha and the C-a proton of proline are exposed
0
into the cavity. This could be achieved if the peptide bond is
0
slightly out of the plane of the aromatic ring. This confor-
in the trans conformation and the carbonyl is anti to Ha and
0
mation places one Hd of proline in close proximity to Ha on
the aromatic ring and is supported by the intramolecular
nOe observed between these two protons 5Fig. 8b).
The 1:1 complex stoichiometry for the complex between 2
and acetylcholine was con®rmed by using Job's method of
continuous variations.
1
ꢀ
1
Figure 7. 5a) H NMR spectrum of trimer 2 and expansion of the
1
H
NOESY spectrum showing the exchange of a proton signals for different
1
conformers. 5b) H NMR spectrum of trimer 2 in presence of 1equiv. of
1
ACh. 5c) Expansion of H NMR spectrum of acetylcholine in presence of
1equiv. of 2 showing CH
from the observed coupling constants.
2
a and CH
2
b resonances. Conformation derived
Figure 8. 5a) Intermolecular nOes observed in a 1:1 mixture of ACh and
receptor 2. 5b) Intramolecular nOes observed for bound receptor.

R. L. E. Furlan et al. / Tetrahedron 58 (2002) 771±778
77ꢀ
reaction for DCL generation. The libraries that result from
hydrazone exchange have been proven to be under thermo-
dynamic control, which is of huge signi®cance if templating
is to be realized in DCLs. This characteristic, in combina-
tion with the possibility of switching on and off of the
exchange by acid catalysis and neutralization, has allowed
the guest induced ampli®cation and isolation of receptor 2.
In the dynamic system studied, a signi®cant increase in the
cyclic trimer concentration was observed when four of the
ammonium cations tested were used as templates. The ®rst
evidence that these observations are the result of a speci®c
template effect is ESI mass spectrometry analysis, which
reveals the in situ formation of complexes only between
the templates and the ampli®ed cyclic trimer.
Figure 9. Observed intermolecular nOes between receptor 2 and 5a)
EtMe NI, 5b) BnMe NI, and 5c) NMQ.
The interaction between each effective template and the
ampli®ed receptor was proven by the observation of inter-
molecular nOes between the receptor and protons of the
3
3
1
Control experiments of 1:1 mixtures of the acetylcholine
with cyclic dimer afforded H NMR spectra, which show
templates in the H ROESY experiments. The binding
constants for the complexes are in accordance to the
response they exert on the system: Acetylcholine
1
none of the changes in chemical shifts or splitting of CH2
signals observed with cyclic trimer.
2
1
21
5Kˆ230 M ) is the best template, NMQ 5Kˆ1ꢀ0 M )
21
ampli®es the trimer to a similar extent as EtMe NI
3
1
The simpli®cation of the H NMR spectrum of 2 observed in
5Kˆ140 M ), and BnMe NI possesses the smallest binding
3
2
1
presence of acetylcholine was also produced by addition of
EtMe NI, BnMe NI, and NMQ. The similarity between the
constant 5Kˆ80 M ) and produces the weakest response
from the system.
3
3
spectra of receptor 2 in presence of the different cations
suggests that the same conformer is selected and ®xed by
all the guests. The effect of complexation on the proton
resonances of the different guests follows a similar trend:
5a) small down®eld shift in the ±NMe resonances, and 5b)
3
splitting of the signals corresponding to protons on the CH2
Molecular recognition with [18]crown-6 was used to check
that hydrazone exchange is not switched off by the excess of
template necessary to shift the equilibrium towards cyclic
trimer 2. This simple experiment represents a useful test to
con®rm that hydrazone exchange based mixtures are
dynamic at any moment.
a to the ammonium centre.
1
H ROESY experiments of 1:1 mixtures of 2 with NMQ,
EtMe NI, and BnMe NI showed similar intermolecular
The objective of this work was to generate a dynamic
system which is simple enough to permit the use of conven-
tional analytical techniques for its characterization, and still
contains one interesting member for recognition based in a
known receptor. The simplicity of the system facilitates
carrying out the necessary control experiments in a proof
of principle step; however, its low diversity decreases the
chances of discovering new interesting receptors.
3
3
0
of the guests as observed with ACh 5Fig. 9).
nOes between Ha in the receptor and the N-methyl protons
The stability of the complexes between 2 and the four effec-
tive templates were estimated by means of NMR titrations.
Increasing amounts of the receptor were added to solutions
of the different salts in CDCl /CH OD 59ꢀ:ꢀ) and the shifts
3
3
of the resonances of the guests' protons were followed in the
H NMR spectra. Stability constants were calculated using a
non-linear least squares ®tting method for 1:1 complexes.
The next obvious step is to expand the diversity and screen
with a range of templates. The main challenge is analytical
in nature; how can active species be identi®ed from such
diverse mixtures? Although changes in product distribution
should be indicative of lead compounds, many building
blocks will not be components of these lead compounds
and thus not all the library members will be consumed to
generate the ampli®ed species. The existence of a wide
range of untemplated species could cloud traditional
analysis such as HPLC.
1
1
6
2
1
The values obtained were: 230 M for acetylcholine,
ꢀ0 M for N-methyl quinuclidinium, 140 M for ethyl-
for benzyl-trimethyl
2
1
21
1
trimethyl ammonium, and 80 M
2
1
ammonium. These relative binding constants correlate
well with the degree of ampli®cation of 2 that each template
produces. It should be noted that the conditions under which
titrations were performed do not entirely re¯ect those of the
cyclization reaction and it is reasonable to believe that the
presence of ꢀ% methanol could give values for the binding
constants lower than would be operating in 100%
chloroform.
Fourier transform ion cyclotron electrospray mass
spectrometry 5FT-ICR-MS) has been demonstrated as a
powerful technique for the analysis of complex DCLs
4
d,17
1
8
and conventional combinatorial libraries.
Taking
advantage of the high resolution and sensitivity of
FT-ICR-MS, small increases in the concentration of the
best binders in the library may be detected. Once the best
binders have been identi®ed, the library composition can be
3
. Discussion and outlook
Hydrazone chemistry has been demonstrated as a suitable

776
R. L. E. Furlan et al. / Tetrahedron 58 (2002) 771±778
tuned by mixing only the right relative amounts of those
building blocks which feature in the species that bind to
the template.
distilled from CaH under Ar5g). N-methylquinuclidinium
2
2
1
iodide was prepared according to a literature procedure.
Column chromatography was carried out using silica gel 60
F 5Merck).
Alternatively, templates supported on polymer beads can
become a very useful tool.
4
e,19,20
Since the template is
attached to a solid support, the untemplated species will
eventually become washed from the solid support by
4.2.1. 3-Carboxybenzaldehyde dimethoxyacetal ,4). 3-
Carboxybenzaldehyde 5ꢀ.00 g, 0.033 mol) and ammonium
chloride 510 g, 0.20 mol) were re¯uxed in dry methanol
5110 ml) during 72 h under an atmosphere of argon. The
reaction mixture was ®ltered while warm and the solvent
removed under reduced pressure to give a white solid. The
product was puri®ed by recrystallization from hexane to
®
ltration leaving only `strong' binders appended to the
bead. The species attached to the solid phase may be washed
off under different solvent conditions that disrupt the
template±receptor interaction and subsequently identi®ed.
The development of selection techniques using guests
appended to polymer beads is pivotal to the management
and success of highly diverse DCLs.
1
afford compound 4 as a white solid 56.1g, 93%). H NMR
5400 MHz, CDCl ) dˆ8.22 51H, s, Ar-H), 8.08 51H, d,
3
Jˆ7.8 Hz, Ar-H), 7.715 H1 , d,
1
CH), 3.3ꢀ 56H, s, OMe); C NMR 562.ꢀ MHz, CDCl )
Jˆ7.8 Hz, Ar-H), 7.49,
5
1H, t, Jˆ7.8 Hz, Ar-H, Jˆ7.8 Hz, Ar-H), ꢀ.47 51H, s,
3
What is clear is that dynamic combinatorial chemistry is a
highly powerful technique, which by using reversible
chemistry can generate and screen highly diverse libraries
of candidate molecules for the identi®cation of host and
guest molecules with potential as catalysts or drugs.
Thermodynamic control invites the template to dictate its
preferred receptor thus amplifying the concentration of the
receptor. This reversal of thinking, away from design to
choreographed molecular ampli®cation, may unlock the
doors towards the discovery of unexpected novel receptors.
3
dˆ171.94 5CvO), 138.73, 132.08, 130.28, 129.ꢀ0, 128.49
5Ar), 102.33 5CH), ꢀ2.67 5OMe).
4.2.2. ,S) N-,3-Dimethoxymethyl-benzoyl)-proline methyl
ester ,5). l-Proline methyl ester hydrochloride 50.ꢀ9 g,
3.ꢀ6 mmol) and 3-carboxybenzaldehyde dimethoxyacetal
50.70 g, 3.ꢀ7 mmol) were dissolved in dry CH Cl 520 ml)
2
2
containing dry Et N 51.0 ml, 7.17 mmol) under Ar5g) and
3
the solution was cooled to 08C on an ice bath. To this solu-
tion were added EDC 50.68 g, 3.ꢀ7 mmol) and DMAP
52ꢀ mg, 0.20 mmol) and the solution stirred at 08C for 1h
4. Experimental
before being allowed to warm to room temperature and
stirred overnight. CH Cl 5120 ml) was added and the solu-
2
2
4
.1. General
tion washed with H O 53£100 ml). The organic phase was
2
dried 5MgSO ) and the solvent removed under reduced pres-
4
HPLC analysis was performed on a Hewlett-Packard 10ꢀ0
instrument using reversed phase conditions of H O/MeCN
sure to give a yellow oil. Column chromatography 5SiO2)
[EtOAc/Hex, 8:2] yielded the titled compound 5 as a colour-
2
1
gradients with a 1ꢀ cm£4.6 mm i.d. 3 mm particle size,
less oil 50.76 g, 69%). H NMR 5400 MHz, CDCl ) dˆ7.6ꢀ
3
1
Supelco ABZ plus C16 alkylamide column. Data were
51H, s, Ar-H), 7.ꢀ3±7.33 53H, band, Ar-H), ꢀ.39 51H, s,
CH5OMe) ), 4.67±4.63 51H, m, a-H), 3.76 53H, s, OMe),
analysed using HP ChemStation. All NMR spectroscopy
was performed on Bruker DRX 400 or DPX ꢀ00 instruments
and chemical shifts are quoted in parts per million with
respect to TMS. Electrospray mass spectra were recorded
on a Micromass Quattro-LC triple quadrupole apparatus
2
3.66±3.ꢀ0 52H, band, Pro-CH ), 3.3156H, s, CH5O Me) ),
2
2
2.3ꢀ±2.24 51H, m, Pro-CH CH ), 2.03±1.99 52H, band, Pro-
a
b
1
CH ), 1.91±1.82 51H, m, Pro-CH H ); C NMR 5100 MHz,
3
2
a
b
CDCl ) dˆ174.1, 170.9 5CvO), 139.7, 137.ꢀ 5Arquat),
3
®
tted with a z-spray source. The electrospray source was
heated to 1008C and the sampling cone voltage 5V ) was
129.9, 129.6, 128.8, 127.1 5Ar-H), 104.0 5CH5OMe) ),
2
c
61.7 5a-H), 60.ꢀ 5COOMe), ꢀ4.1, ꢀ3.6 5CH5OMe) ), ꢀ1.3,
2
1
3
source without work-up with an LC pump 5Shimadzu LC-
0 V. Samples were introduced into the mass spectrometer
30.8, 26.8 5CH ); ESI-MS 51) m/zˆ308 [M1H] , 276
2
1
[M2OMe] .
2
1
9
A) at a rate of 4 ml min of MeCN/H O 51:1). Calibration
2
was performed using protonated horse myoglobin. Scanning
was performed from m/z 200 to 2200 in 6 s and several scans
were summed to obtain the ®nal spectrum, which was
processed using MassLynx V3.0 software. High-resolution
mass spectra were recorded on a Micromass Q-tof
instrument, incorporating time-of-¯ight analysis with
electrospray ionization through a standard z-spray source.
Several scans were summed to obtain the ®nal spectrum
which was processed using MassLynx V3.0 software.
Calibration was performed using erythromycin as the
standard.
4.2.3. ,S) N-,3-Dimethoxymethyl-benzoyl)-proline carb-
oxylic acid hydrazide ,3). Proline ester 5 50.74 g,
2.41mmol) was treated with hydrazine monohydrate
51.2 ml, 24.7 mmol) in methanol 52ꢀ ml). The reaction
was left overnight before removal of the solvent under
vacuum to give a yellow oil which was puri®ed by ¯ash
chromatography 5SiO ) [CH Cl /MeOH, 90:10] to afford
2
2
2
1
the monomer 3 50.ꢀ7 g, 77%). H NMR 5400 MHz,
CDCl ) dˆ8.315 H1 , s, N HNH ), 7.62 51H, s, Ar-H),
3
2
7.ꢀ3±7.4H 52H, band, Ar-H), 7.40 51H, m, Ar-H), ꢀ.39
51H, s, CH5OMe) ), 4.67±4.66 51H, m, a-H), 3.94 52H,
2
bs, NHNH ), 3.ꢀ9±3.ꢀ6 51H, band, Pro-CH CH ), 3.47±
b
2
a
4
.2. Materials
3.4ꢀ 51H, band, Pro-CH CH ), 3.3156H, s, CH5O Me) ),
a b 2
2
.37 51H, m, Pro-CH CH ), 2.11±2.04 52H, band, Pro-
a b
1
3
All chemicals were purchased from Aldrich, Lancaster or
Fluka and were used without further puri®cation. All
solvents were distilled prior to use and dry solvents freshly
CH
2
), 1.82 51H, m, Pro-CH
) dˆ172.0, 170.7 5CvO), 138.ꢀ, 13ꢀ.9 5Arquat),
128.7, 128.3, 127.3, 12ꢀ.6 5Ar-H), 102.4 5CH5OMe) ),
H ); C NMR 5100 MHz,
a b
CDCl
3
2

R. L. E. Furlan et al. / Tetrahedron 58 (2002) 771±778
777
ꢀ
8.6 5a-H), ꢀ2.7 5CH5OMe) ), ꢀ0.4, 27.8, 2ꢀ.ꢀ 5CH ); ESI-
2
dˆ7.96 52H, s, CHvNR), 7.ꢀ2 52H, d, Jˆ6.1Hz, Ar-H),
7.37 52H, t, Jˆ6.7 Hz, Ar-H), 7.33 52H, d, Jˆ6.7 Hz, Ar-H),
2
1
1
MS 51) m/zˆ308 [M1H] , 276 [M2OMe] , 244
1
M22OMe] ; HRMS 5QTOF) [M1Na] C H N O Na
1
[
requires 330.1430, found 330.1446.
ꢀ.77 52H, m, a-H), 3.90±3.80 54H, m, Pro-CH ), 2.ꢀ1±2.49
2
1
ꢀ
21
3
4
1
3
52H, m, ProCH ), 2.16±2.01 56H, band, Pro-CH ); C NMR
2 2
5
100 MHz, CDCl /CD OD) dˆ173.3, 169.6 5CvO), 143.6
3
3
4.3. General procedure for cyclization and templating
experiments
5Arquat), 136.9 5Arquat), 132.4, 130.7, 129.3 5Ar-H), 121.2
5CHvNR), ꢀ7.7 5a-C), 32.1, 28.8, 24.9 5Pro-CH ); ESI-MS
5
C H N O Na requires ꢀ09.1913, found ꢀ09.1891.
2
1
1
1) m/zˆ487.3 [M1H] ; HRMS 5QTOF) [M1Na]
ꢀ
mM solutions of 5S) N-53-dimethoxymethyl-benzoyl)-
2
6
26
6
4
proline carboxylic acid hydrazide 53) in freshly distilled
chloroform containing TFA 52ꢀ mM) were stirred at room
temperature during ꢀ days. The equilibrium is reached
within 3 days. The ampli®cation observed is achieved by
addition of the ammonium salts either when the reaction is
started or after it has reached equilibrium. All templates are
solids and thus simply weighed out and added to the reac-
tion. Comparative cyclization experiments were carried out
in a 2 ml scale. HPLC or ESI-MS analysis was performed by
the removal of 100 ml aliquots for analysis from the reaction
mixture.
4.8. Puri®cation of cyclic trimer ,2)
5S) N-53-dimethoxymethyl-benzoyl)-proline carboxylic acid
hydrazide 53) 5400 mg, 1.30 mmol) was dissolved in chloro-
form 5130 ml) to give a 10 mM solution. To this solution
was added N-methyl quinuclidinium iodide 51.3 g,
ꢀ.21mmol) and TFA 50.1ml, 1. 30 mmol)Ðas a solution
in chloroform. The reaction was left stirring at room
temperature for 2 days before washing with H O 5100 ml)
2
and subsequent washing of the aqueous layer with three
portions of chloroform 5100 ml). The organic extracts
4.4. Preparation of the dynamic system from the cyclic
dimer
were combined, dried 5MgSO ) and the solvent removed
4
under reduced pressure to give a white solid. This solid
was subjected to silica gel column chromatography using
2
ml of a 2ꢀ mM solution of TFA in freshly distilled chloro-
gradient elution [CHCl
cyclic trimer as a white solid 51ꢀ8 mg, ꢀ0%). H NMR
3
/MeOH, 98:2!90:10] to give the
2
3
1
form were added to cyclic dimer 52.4 mg, ꢀ£10 mmol)
and the resulting solution stirred for 3 days. The reaction
was followed by HPLC and ESI-MS.
5400 MHz, CDCl /CD
3
OD) dˆ8.49±7.14 51ꢀH, band, Ar-H
3
and CHˆNR), ꢀ.82±4.67 53H, band, a-H), 3.93±3.49 56H,
band, Pro-CH ), 2.43±2.0ꢀ 512H, band, Pro-CH ); ESI-MS
2
2
1
1
4.5. Preparation of the dynamic system from the cyclic
trimer ,2)
51) m/zˆ730.4 [M1H] ; HRMS 5QTOF) [M1Na]
C H N O Na requires 7ꢀ2.2921, found 7ꢀ2.2913.
39 39 9 6
4
.9. Job plot
2
ml of a 2ꢀ mM solution of TFA in freshly distilled chloro-
2
3
form were added to cyclic trimer 52.4 mg, 3.3£10 mmol)
and the resulting solution stirred for 3 days. The reaction
was followed by HPLC and ESI-MS.
Equimolar solutions 5ꢀ mM) of trimer 2 and acetylcholine
chloride in ꢀ% CD OD/CDCl were prepared and mixed in
various ratios. H NMR spectra of the resulting solutions
were recorded, and the change in chemical shift of the
3
3
1
4.6. Use of [18]crown-6 to check that the templated
system is dynamic
1
ꢀ
N-methyl proton signals of the guest was analysed.
.10. Host±guest titrations
Solutions of cyclic trimer 2 52ꢀ mM, CDCl /CD OD, 9ꢀ:ꢀ)
4
Cyclization reactions were carried out at ꢀ mM concentra-
tion of building block 3 containing 4 equiv. of NMQ 5with
respect to the amount of 3) and increasing amounts of TFA
3
3
were titrated into ꢀ mM solutions of the different guests
CDCl /CD OD, 9ꢀ:ꢀ). The chemical shifts of the guest
5
from 1to 10 equiv. with respect to the amount of 3). The
5
3
3
reaction were stirred for 3 days before [18]crown-6 was
added 5same number of equivalents as TFA). The ampli®ed
monomer was detected by HPLC analysis 5260 nm,
protons were plotted against the host concentration. The
binding constants were calculated by non-linear least-
squares ®tting method for 1:1 complexes using EQNMR
R ˆ2.ꢀ min) and by ESI-MS 5m/zˆꢀ26.4) in all the experi-
t
1
6
program. The values were estimated to have errors within
0%.
ments.
2
4
.7. Puri®cation of cyclic dimer
Acknowledgements
5
S) N-53-Dimethoxymethyl-benzoyl)-proline carboxylic
acid hydrazide 53) 50.28 g, 0.90 mmol) was dissolved in
freshly distilled chloroform 5180 ml) to afford a ꢀ mM solu-
tion. To this solution was added TFA 50.3ꢀ ml, ꢀ mmol) and
the reaction stirred at room temperature for 3 days. After
We are grateful for support from EPSRC, BBSRC, ethyl
petroleum, Consejo Nacional de Investigaciones Cient Âõ ®cas
y
T e cnicas 5Argentina) and Fundaci o n Antorchas
5Argentina).
w
this time, the solution was treated with Amberlyst A-21
beads to remove the TFA, ®ltered and the solvent removed
under reduced pressure to give a white solid. This solid was
subjected to silica gel column chromatography [CHCl3/
References
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3
3

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