Dendritic receptors designed to bind polyanions in both organic and aqueous media

Article abstract of DOI:10.1039/b618063a

This paper reports the synthesis of dendrons containing a spermine unit at their focal point. The dendritic branching is based on l-lysine building blocks, and has terminal oligo(ethyleneglycol) units on the surface. As a consequence of the solubilising s

Full text of DOI:10.1039/b618063a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Dendritic receptors designed to bind polyanions in both organic
and aqueous media
John G. Hardy,a Ian Ashworth,b Colin Brennanb and David K. Smith*a
Received 11th December 2006, Accepted 26th January 2007
First published as an Advance Article on the web 7th February 2007
DOI: 10.1039/b618063a
This paper reports the synthesis of dendrons containing a spermine unit at their focal point. The
dendritic branching is based on L-lysine building blocks, and has terminal oligo(ethyleneglycol) units
on the surface. As a consequence of the solubilising surface groups, these dendrons have high solubility
in solvents with widely different polarities (e.g., dichloromethane and water). The protonated
spermine unit at the focal point is an effective anion binding fragment and, as such, these dendrons are
able to bind to polyanions. This paper demonstrates that polyanions can be bound in both
dichloromethane (using a dye solubilisation assay) and in water (competitive ATP binding assay). In
organic media the dendritic branching appears to have a pro-active effect on the solubilisation of the
dye, with more dye being solubilised by higher generations of dendron. On the other hand, in water the
degree of branching has no impact on the anion binding process. We propose that in this case, the
spermine unit is effectively solvated by the bulk solvent and the dendritic branching does not need to
play an active role in assisting solubility. Dendritic effects on anion binding have therefore been
elucidated in different solvents. The dendritic branching plays a pro-active role in providing the anion
binding unit with good solubility in apolar solvent media.
surfaces have been investigated by the groups of Astruc and
Introduction
Kaifer.9 These multivalent anion receptors were demonstrated to
be capable of binding multiple anions in organic media, and ex-
hibited dendritic amplification of their ability to electrochemically
sense the anionic target. We used a similar anion binding strategy
within a dendrimer, only located the metallocene unit at the core,
rather than on the surface. We demonstrated that in this case, the
branching inhibited the magnitude of electrochemical response
to halide anions.10 Vo¨gtle and co-workers prepared polyvalent
dendritic ureas, which were able to extract multiple oxo-anions
from water into an organic phase.11 The groups of van Koten and
Stoddart prepared dendrimers with internal quaternised amines,
which were able to bind anions in organic media.12 They used
anionic dyes to demonstrate that binding had taken place. There
have also been a number of studies of dendrimers containing
multiple protonated amines which have been shown to effectively
internalise anionic dyes.13
As part of a wider program investigating supramolecular
dendrimer chemistry,14 we recently employed spermine groups
on the surface of dendrons, in order to achieve ultra high-
affinity DNA binding in water.15 Spermine is a simple polyamine
used extensively by biological systems as a nucleic acid binder.16
Protonated polyamines such as spermine, although good anion
receptors in aqueous media, are usually ineffective in apolar media
due to their poor solubility. We became interested in employing
spermine at the focal point of a dendron. We reasoned that
with an appropriate choice of dendritic framework, we could
generate highly soluble spermine derivatives, which would operate
in solvent media with widely different polarities—for example,
both water and organic media. This paper reports the results of
our initial investigations into this anion binding strategy.
Dendrimers and dendrons make a vital contribution to the field
of nanochemistry as a consequence of their unique structural
features.1 In particular, the inherent branched structures of
dendritic molecules can be exploited in the field of molecular
recognition to achieve intriguing binding phenomena.2 The mul-
tiple surface groups of dendritic molecules are able to amplify
binding strengths by multivalency phenomena.3 On the other
hand, binding at the encapsulated core (or focal point) of a
dendritic structure takes place in a unique micro-environment,
which can have a direct impact on the binding event.4 This mimics
theway inwhichthethree-dimensionalpeptidic architecture places
the active site of an enzyme in a well-defined local environment.5
Over recent years, anion binding has developed into a key
area of supramolecular chemistry,6 as a consequence of the wide-
ranging importance of anions in environmental and biological
processes. Whilst binding anions in organic solvents is relatively
widespread, anion binding in water is less common and requires
specific solutions. The most successful strategies involve the
use of transition metal based receptors,7 which bind anions
through dative bond formation, or protonated polyamines,8 which
bind anions using a combination of electrostatics and hydrogen
bonding.
Perhaps surprisingly, dendritic receptors for anions remain rel-
atively unexplored. Dendrimers with metallocene-functionalised
aDepartment of Chemistry, University of York, York, UK YO10
5DD. E-mail: dks3@york.ac.uk; Fax: +44 (0)1904 432 516; Tel: +44
(0)1904 434 181
bTechnology and Projects Department, Syngenta, Huddersfield, UK HD2
1FF
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ammonium formate to give compound 5. Coupling compound
5 with acid 2 gave compound G2-Boc, which subsequently had
the Boc protecting groups removed using HCl in methanol. This
sequence gave receptor G2 in a good overall yield of 27% (six steps)
from commercially available starting materials. The compounds
were characterised using all the usual spectroscopic methods, and
full data can be found in the Experimental section.
Results and discussion
Synthesis
We targeted first and second generation L-lysine based dendritic
structures G1 and G2, and model system G0, each of which has an
anion binding spermine unit at the focal point and oligo ethylene
oxide surface group(s) (Scheme 1). We chose these surface groups
because they provide solubility across a broad range of solvents
and furthermore, do not have any innate affinity for anions. It
is worth noting that the dendrons are wholly constructed from
biocompatible building blocks.
The synthesis of these dendrons was achieved using a diver-
gent strategy (Scheme 1). To synthesise non-dendritic control
receptor G0, tri-Boc protected spermine 1 was made according
to a methodology published by the Blagbrough group,17 and
coupled to 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (2) using
DCC and HOBt to yield G0-Boc. The Boc protecting groups were
subsequently removed from the spermine unit using HCl gas in
methanol to provide G0. To synthesise G1, Z-protected lysine 3
was coupled with protected spermine 1 using DCC methodology
to yield compound Z-4. Removal of the Z protecting groups from
the product with palladium hydroxide on carbon and ammonium
formate gave key intermediate 4. Coupling of 4 with acid 2 gave
compound G1-Boc, and subsequent removal of the Boc groups
with HCl gas in methanol provided receptor G1 in a good overall
yield of 41% (four steps). To synthesise G2, intermediate 4 was
coupled with Z-protected lysine 3 to give compound Z-5. The
product had the Z protecting groups removed using Pd(OH)2 and
Anion binding in organic media
We developed assays to probe the potential of these receptors in
both organic and aqueous media. In common with other studies
of anion binding in organic media which use dendritic hosts,12 we
decided to use a dye solubilisation assay to probe the affinity of
these hosts for anions.
We chose this strategy in preference to NMR titration methods
as the N+–H protons were not readily observed and the other
protons in the receptor did not give rise to significant shifts. This is
partly a consequence of the fact that the protonated spermine unit
in these receptors already has chloride counteranions associated
with it, and consequently all anion binding experiments are
effectively competition experiments. We therefore decided to use
a highly charged anionic guest in order to maximise the binding
interaction. However, as highly charged anions are not soluble in
organic media, it was necessary to employ a solubilisation assay
approach.
Aurin tricarboxylic acid is a powerful inhibitor of cellular pro-
cesses that are dependent on the formation of protein nucleic acid
Scheme 1 Synthesis of dendritic anion binders G0, G1 and G2. (a) DCC, HOBt, Et3N, DCM; (b) HCl(g), MeOH; (c) DCC, HOBt, Et3N, DCM;
(d) Pd(OH)2/C, HCOONH4, EtOH.
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complexes,18 and is commercially available as the tri-carboxylate
anion (Fig. 1). This polyanion is soluble in water, but not in organic
media such as dichloromethane (DCM). Our receptors have good
solubility in DCM as a consequence of the oligo-ether surface
groups. We therefore decided to probe the ability of our receptors
to solubilise aurin tricarboxylate into this apolar solvent.
We have previously demonstrated that interactions between
protonated amines and carboxylate anions play an essential role in
the solubilisation of aurin-based dyes by dendritic systems.19b We
propose that in this case, the dendritic effect is a consequence of
the dendritic branching, which is compatible with organic solvents
such as DCM. Indeed, without the presence of this dendritic
branching, the highly charged spermine unit would not be soluble
in the solvent (DCM) in the first place, as protonated spermine is
not compatible with low-polarity media. Consequently, the ability
of the dendritic branching to interact favourably with the solvent
enhances the solubility of the overall complex and thus higher
generation systems are better able to solubilise the dye.
There is also the possibility of secondary interactions between
the anionic dye and hydrogen bonding amide groups in the
dendritic branching, which will enhance the degree of uptake—
such interactions are well-known in the literature.20
Furthermore, it should be noted that the ether units in the
dendritic branches may interact with the NH4+ cation and further
enhance the solubilisation process. However, these ether units also
have the potential to interact with the protonated spermine unit at
the focal point of the dendron itself, and so we do not believe that
ether–NH4+ interactions provide the main driving force behind the
solubilisation event.
Fig. 1 Structures of anions investigated in this paper.
Anion binding in water
Solid aurin tricarboxylate (as its tri-ammonium salt) was
suspended in a 1 mM solution of the anion receptor (dissolved
in DCM) and stirred for 24 h.19 The mixture was then filtered
to remove excess dye, and the solution analysed by UV-Vis
spectrometry. Fig. 2 provides a visual assessment of the degree
of dye solubilisation in each case and a quantitative measure is
given in Table 1 (normalised with respect to the solubilisation
caused by G0). In the absence of receptor, effectively no dye was
solubilised into DCM; however, in the presence of the receptors
solubilisation occurred. The extent of solubilisation increased in
the order G0 < G1 < G2. There is, therefore, a clear dendritic
effect on the uptake of the anionic dye into organic solvent.
We then decided to determine whether our receptors would operate
in aqueous solution. We chose to probe the interaction between
our polyanionic receptors and adenosine triphosphate (ATP),
a biologically-relevant phosphate polyanion. In 1977, Nakai
and Glinsmann developed a simple and innovative competition
assay to determine the binding between protonated amines and
ATP and we applied their methodology in this study.21 Their
method involves placing a constant amount of ATP and cationic
resin (Dowex AG1-X2) in tris-chloride buffered water (pH 7.5).
Various amounts of soluble receptor are then added. The receptor
competes with the solid cationic resin for binding the ATP, and the
residual amount of ATP in the solution, which can be determined
by UV-Vis spectrometry, reflects the affinity of the receptor for
ATP. The Igarashi group have since published minor modifications
to handling the data from this procedure.22 We applied both data-
handling approaches to investigate ATP binding in water at pH 7,
and for the purposes of comparison also determined the binding
of spermine and spermidine (Fig. 3) to the target anion.
Fig. 2 Solutions resulting from the solubilisation study performed with
G0, G1 and G2 (each 1 mM) in dichloromethane solution with aurin
tricarboxylate (ammonium salt).
Table 1 Solubilisation of aurin tricarboxylate into dichloromethane as
assessed by UV-Vis spectrometry using absorption at kmax (522 nm) and
normalised relative to the uptake exhibited by compound G0
Fig. 3 Structures of spermidine and spermine.
Importantly, the binding constants for spermine and spermi-
dine, generated using the assumption of 1 : 1 binding with the
polyanion, were in good agreement with the data in the literature,
validating our use of the assay (Table 2). We then investigated the
performance of receptors G0–G2 in this assay. It is clear from the
data that receptors G0, G1 and G2 show very similar affinities for
Host
Degree of solubilisation
None (DCM alone)
0.02
1.00
1.81
3.64
G0
G1
G2
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Table 2 Log K values determined for ATP binding with the receptors at
pH 7 in tris-buffered water using competitive binding assay and assuming
a 1 : 1 binding model using methods from ref. 21 and 22
phy was performed on commercially available Merck aluminium
backed silica plates. Preparative gel permeation chromatography
was carried out using a 2 m glass column packed with Biobeads
SX-1 supplied by Biorad, or a shorter length column (0.5 m)
packed with Sephadex LH-20. Proton and carbon NMR spectra
were recorded on a Jeol 400 spectrometer (1H 400 MHz, 13C
100 MHz). Samples were recorded as solutions in CDCl3 and
chemical shifts (d) are quoted in parts per million, referenced
to residual solvent. Coupling constant values (J) are given in
Hz. DEPT experiments were used to assist in the assignment
of 13C NMR spectra. Melting points were measured on an
Electrothermal IA 9100 digital melting point apparatus and are
uncorrected. Positive ion electrospray mass spectra were recorded
on a Finnigan LCQ mass spectrometer. Positive ion fast atom
bombardment mass spectra were recorded on a Fisons Instru-
ments Autospec mass spectrometer, with 3-nitrobenzyl alcohol as
matrix. Polyethylene glycols and/or polyethylene glycol mono-
methyl ethers were used as calibrants for HRMS determinations.
The isotope distribution observed for mass spectral ions of
the larger molecules is consistent with data calculated from
isotopic abundances. Infra-red spectra were recorded using an
ATI Mattson Genesis Series FTIR spectrometer.
Host
Log K21
Log K22
Spermine
Spermidine
G0
4.06
3.08
2.80
2.74
2.82
3.76
3.22
3.28
3.28
3.37
G1
G2
ATP as spermidine. Although our receptors contain four nitrogen
atoms, they are actually more similar to spermidine than spermine,
as one of the four nitrogen atoms has been converted to an
amide and thus cannot be protonated. Therefore the similarity
in behaviour to spermidine is to be expected.
Furthermore, it is clear that receptors G0–G2 all give similar
degrees of binding, irrespective of the extent of functionalisation.
This indicates that the dendritic branching does not inhibit the
ability of the polyamine unit at the focal point to bind ATP
anions in water. Furthermore, it indicates that in water, unlike
in organic media, the dendritic branching does not assist the
anion binding process. In aqueous solution, the polar dendron
structure and the spermine binding unit will both be heavily
solvated, and we propose it is therefore unable to generate a specific
microenvironment at the core. In addition, the affinity of neutral
amides for anions is limited in polar media, and there will be
no additional anion binding within the dendritic framework. For
this reason, we argue there is no dendritic effect in water and the
binding afforded by the protonated (solvated) polyamine unit is
unaffected by dendritic functionalisation.
Compound 1 was synthesised according to the methodology
previously published by Blagbrough and co-workers.17 Com-
pounds 2 and 3 were commercially available. The full synthetic
methodology and characterisaton data for G0 can be found in the
supplementary information of our previous publication.15a
Synthesis and characterisation
Compound Z-4. Compound 1 (4.0 g, 8.75 mmol, 1.1 eq.) and
compound 3 (3.63 g, 8 mmol, 1 eq.) were dissolved in EtOAc
(100 mL). DCC (2.48 g, 12 mmol, 1.5 eq.), HOBt (1.62 g, 12 mmol,
1.5 eq.) and Et3N (1.21 g, 12 mmol, 1.5 eq.) were added and
the mixture was placed in an ice bath for 1 h and then left
to stir at rt for 2 d. Dicyclohexylurea (DCU) was removed by
filtration, and the solvents removed by rotary evaporation. The
crude product was purified by silica column chromatography (70 :
30 cyclohexane–EtOAc), a white solid was recovered (5.56 g,
Conclusions
In summary, we have demonstrated that these new dendritic
structures enable anion binding to be achieved by the same
receptor in solvent media with large difference in polarities (DCM,
e = 9.1, H2O, e = 80). The dendritic branching plays a pro-active
role in enhancing the solubility of the system in apolar media. The
dendritic structure has a direct impact on the binding in organic
media where it can help ensure the charged spermine unit and
its charged guest remain compatible with the surrounding apolar
solvent. The more extensive the dendritic branching, the more able
it is to solubilise the charged complex, and hence higher generation
systems cause enhanced uptake of the dye—a ‘dendritic effect’.23
However, there is no dendritic effect in water, where the whole
structure will be largely solvated and the branching will have less
impact on the charged binding unit. We propose that these systems,
which are compatible with a broad range of solvent systems may
have future potential as anion transport agents. Furthermore,
the incorporation of additional functionality into the dendritic
structures may enable the developmentof medically-relevantanion
complexation agents.
1
62%). H NMR (CD3OD, 400 MHz) dH 7.28–7.13 (10H, m, CH
aromatic), 6.98–6.87 (1H, br m, NH amide), 6.54–6.39 (3H, br m,
NHBOC, NHZ), 4.97 (2H, s, OCH2Ph), 4.94 (2H, s, OCH2Ph),
3.90 (1H, dd, J = 5.2, 8.9 Hz, COCH(R)NH), 3.28–3.14 (14H,
br m, CH2N), 3.11 (2H, t, J = 6.6 Hz, CH2N), 3.01 (2H, t, J =
7.3 Hz, CH2N), 1.71–1.53 (4H, br m, CH2CH2NH), 1.50–1.21
(37H, br m, (CH3)3C, CH2CH2NBOC, CH2); 13C NMR (CD3OD,
100 MHz) dC 175.12 (CONH, amide), 158.92, 158.41, 157.78
(CONBoc × 2, CONHBoc, CONHZ × 2, overlapping), 138.43,
138.15, 129.47, 129.44, 128.75 (Ar–C, overlapping), 80.92, 79.93
(C(CH3)3 × 3, overlapping), 67.67, 67.31 (CH2 benzylic), 56.01
(COCH(R)NH), 46.56, 46.37, 41.81, 40.66, 38.23, 37.61, 37.30,
30.46 (CH2, overlapping), 28.78 ((CH3)3C × 9, overlapping),
27.64, 25.41, 24.09 (CH2CH2N, overlapping); IR (KBr disc)
Experimental
mmax cm−1 3444, 3336 (NH), 1600 (C O), 1639 (C O), 1600
=
=
=
(C O), 1580, 1552 (CONH, amide 2), 1509, 1251 (OCONH
Materials and methods
carbamate); ESI-MS (m/z) calculated value for C47H74N6O11
898.5: (ES+) found 921.4 ([M + Na]+, 100%); HR-FAB calculated
value for C47H74N6O11Na 921.5313: found 921.5316; Rf 0.13
Silica column chromatography was carried out using silica gel
provided by Fluorochem Ltd. (35–70 l). Thin layer chromatogra-
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◦
(70 : 30 EtOAc–hexane, ninhydrin stain); m.p. 52–54 C; [a]D293
−5.1 (c = 1.0, CH3OH).
Anion receptor G1. Compound G1-Boc (0.060 g, 0.79 mmol)
was dissolved in MeOH (150 mL) and gaseous hydrogen chloride
was bubbled through for 30 seconds. The reaction was allowed to
stir for 2 h, after which time the solvent was removed under reduced
pressure, and a white solid was recovered (0.39 g, 81%). Yield
calculated for HCl salt, FW: 760. 1H NMR (CD3OD, 400 MHz)
dH 7.99–7.83 (2H, br m, NH amide), 7.06–7.02 (1H, br m, NH
amide), 4.23 (1H, dd, J = 5.3, 8.9 Hz, COCH(R)NH), 4.01–3.89
(4H, br s, OCH2CONH), 3.70–3.40 (16H, br m, OCH2), 3.35–3.30
(6H, br s, OCH3), 3.28–3.14 (10H, br m, CH2N), 3.15–2.93 (4H, br
Compound 4. Compound Z-4 (5.0 g, 5.5 mmol, 1 eq.),
ammonium formate (1.0 g, 16.5 mmol, 3 eq.) and palladium
hydroxide on carbon (20 wt%, wet) (0.35 g, 2.75 mmol, 0.5 eq.)
were refluxed in ethanol (100 mL) for 48 h. The mixture was
allowed to cool to room temperature and filtered over celite to
remove the solids, and the solvents were removed on a rotary
evaporator. The mixture was dissolved in DCM (50 mL), washed
with conc. NH4OH and brine, dried over MgSO4, and the solvents
removed on a rotary evaporator. A light yellow viscous oil was
m, CH2NH), 2.05–1.27 (14H, br m, CH2CH2N (amide), CH2); 13
C
NMR (CD3OD, 100 MHz) dC 174.25, 174.19, 173.96 (CONH ×
3), 73.07, 71.56, 71.03, (CH2O, overlapping), 59.38 (OCH3), 53.25
(COCH(R)NH), 39.34, 37.61, 37.30, 32.03, 30.32, 28.38, 24.50,
24.45 (CH2, overlapping); IR (KBr disc) mmax cm−1 3444, 3336
1
recovered (3.05 g, 88%). H NMR (CD3OD, 400 MHz) dH 6.71–
6.69 (1H, br m, NH amide), 5.62–5.60 (1H, br m, NHBOC),
3.60 (1H, dd, J = 5.2, 8.9 Hz, COCH(R)NH), 3.29–3.15 (10H,
br m, CH2N), 3.16 (2H, t, J = 6.6 Hz, CH2NH), 2.73 (2H,
t, J = 7.3 Hz, CH2NH2), 1.71–1.53 (4H, br m, CH2CH2N),
1.50–1.21 (37H, br m, (CH3)3C, CH2CH2NBoc, CH2); 13C NMR
(CD3OD, 100 MHz) dC 177.03 (CONH, amide), 157.60, 155.63
(CONBoc × 2, CONHBoc, overlapping), 79.77, 79.72, 78.70
(C(CH3)3), 54.96 (COCH(R)NH), 47.76, 47.55, 44.313, 41.04,
37.56, 35.32, 34.92, 32.30, (CH2, overlapping), 27.45 ((CH3)3C ×
9, overlapping), 25.65, 22.72 (CH2CH2N, overlapping); IR (KBr
=
(NH), 1641 (C O), 1604, 1553 (CONH, amide 2), 1366 (O–
CH3, ether); ESI-MS (m/z) calculated value for C30H62N6O9Na
673.5 (100.0%), 674.5 (33.5%): (ES+) found 673.5 (100.0%), 674.5
(30.5%); [a]2D93 −5.3 (c = 1.0, CH3OH).
Compound Z-5. Compound 4 (1.25 g, 1.94 mmol, 1 eq.) and
compound 3 (1.76 g, 4.26 mmol, 2.2 eq.) were dissolved in DCM
(100 mL). DCC (0.88 g, 4.26 mmol, 2.2 eq.), HOBt (0.58 g,
4.26 mmol, 2.2 eq.) and Et3N (0.43 g, 0.59 mL, 4.26 mmol, 2.2 eq.)
were added and the mixture was placed in an ice bath for 1 h
and then left to stir at rt for 2 days. Dicyclohexylurea (DCU)
was removed by filtration, and the solvents removed by rotary
evaporation. The crude product was purified by preparative gel
permeation chromatography (Biobeads, 90 : 10 DCM–MeOH),
a tacky white solid was recovered (2.26 g, 82%). 1H NMR
(CD3OD, 400 MHz) dH 7.81–7.70 (2H, br m, NH amide), 7.28–
7.13 (20H, m, CH aromatic), 6.71–6.69 (1H, br m, NH amide
G1), 6.63–6.33 (5H, br m, NHBOC, NHZ), 5.05–4.92 (8H,
m, OCH2Ph), 4.25 (1H, dd, J = 4.5, 9.1 Hz, COCH(R)NH),
4.09 (1H, dd, J = 5.4, 8.5 Hz, COCH(R)NH), 4.00 (1H, dd,
J = 5.2, 9.1 Hz, COCH(R)NH, 1H), 3.28–3.14 (16H, br m,
CH2N), 3.01 (2H, t, J = 7.3 Hz, CH2NH), 1.86–1.04 (53H, br
m, CH2, CH3); 13C NMR (CD3OD, 100 MHz) dC 175.72, 174.78,
173.69 (CONH, amide), 159.55, 159.04, 158.04 (CONHBoc,
CONBoc × 2, CONHZ × 4 overlapping), 138.62, 138.58, 129.93,
129.37 129.23 (Ar–C, overlapping), 81.14, 80.15, (C(CH3)3 × 3,
overlapping), 67.96, 67.91, 67.47 (Ar–CH2, overlapping), 55.85,
55.77 (COCH(R)NH × 3, overlapping), 46.56, 46.37, 41.39, 39.93,
32.86, 32.52, 32.34, 30.42 (CH2N, overlapping), 28.74 ((CH3)3C ×
9, overlapping), 27.64, 25.41, 23.93 (CH2CH2N, overlapping);
disc) mmax cm−1 3443, 3336 (NH), 1638 (C O), 1553 (CONH, amide
=
2), 1509, 1251 (OCONH carbamate); ESI-MS (m/z) calculated
value for C31H62N6O7 630: (ES+) found 653 ([M + Na]+, 100%).
HR-FAB calculated value for C31H62N6O7Na 653.4578: found
653.4568.
Compound G1-Boc. Compound 4 (0.30 g, 0.6 mmol, 1 eq.)
and carboxylic acid 2 (0.24 g, 0.2 mL, 1.32 mmol, 2.2 eq.) were
dissolved in DCM (75 mL). DCC (0.30 g, 1.5 mmol, 2.5 eq.),
HOBt (0.202 g, 1.5 mmol, 2.5 eq.) and Et3N (0.15 g, 1.5 mmol,
2.5 eq.) were added and the mixture was placed in an ice bath for
1 h and then left to stir at rt for 2 d. Dicyclohexylurea (DCU)
was removed by filtration, and the solvents removed by rotary
evaporation. The crude product was purified by preparative gel
permeation chromatography (MeOH, Sephadex), a clear oil was
recovered (0.42 g, 0.45 mmol, 75%). 1H NMR (CD3OD, 400 MHz)
dH 7.99–7.83 (2H, br m, NH amide), 7.06–7.02 (1H, br m, NH
amide), 6.46–6.36 (1H, br m, NHBOC), 4.23 (1H, dd, J = 5.2,
8.9 Hz, COCH(R)NH), 4.01–3.89 (4H, br s, OCH2CONH), 3.80–
3.40 (16H, br m, OCH2), 3.35–3.30 (6H, br s, OCH3), 3.28–3.14
(10H, br m, CH2N), 3.13–3.09 (2H, t, J = 6.6 Hz, CH2NH),
3.06–2.95 (2H, t, J = 7.3 Hz, CH2NH), 1.71–1.53 (4H, br m,
CH2CH2N), 1.52–1.22 (37H, br m, (CH3)3C, CH2CH2N, CH2);
13C NMR (CD3OD, 100 MHz) dC 173.78, 172.60 (CONH × 3,
amide, overlapping), 158.92, 157.78 (CONBoc × 2, CONHBoc,
overlapping), 80.87, 79.93 (C(CH3)3 × 3, overlapping), 71.56,
71.37, 71.36, 71.34, 71.28 (CH2O, overlapping), 59.11 (OCH3),
55.25 (COCH(R)NH), 46.56, 46.37, 41.81, 40.66, 38.23, 37.61,
37.30, 30.08 (CH2, overlapping), 28.78 ((CH3)3C × 9, overlap-
ping), 27.64, 25.41, 24.32 (CH2CH2N, overlapping); IR (KBr
IR (KBr disc) mmax cm−1 3441, 3330 (NH), 1638 (C O), 1600,
=
1580 (aromatic), 1553 (CONH, amide 2), 1509, 1251 (OCONH,
carbamate); ESI-MS (m/z) calculated value for C75H110N10O17Na
1445.8 (100.0%), 1446.8 (83.0%), 1447.8 (40.6%): (ES+) found
1445.5 (100%), 1446.6 (70.0%), 1447.8 (30.0%); Rf 0.37 (90 : 10
DCM–MeOH, ninhydrin stain); m.p.: 138–140 ◦C; [a]D293 −9.4 (c =
1.0, CH3OH).
Compound 5. Compound Z-5 (2.0 g, 1.40 mmol, 1 eq.),
ammonium formate (0.53 g, 8.4 mmol, 6 eq.) and palladium
hydroxide on carbon (20 wt%, wet) (0.90 g, 0.71 mmol, 0.5 eq.)
were refluxed in ethanol (100 mL) for 48 h. The mixture was
allowed to cool to room temperature and filtered over celite to
remove the solids, and the solvents were removed on a rotary
evaporator. The mixture was dissolved in DCM (50 mL), washed
disc) mmax cm−1 3444, 3336 (NH), 1641 (C O), 1553 (CONH,
=
amide 2), 1509 (CONH, carbamate), 1366 (ether), 1251 (C–O,
carbamate). ESI-MS (m/z) calculated value for C45H86N6O15Na
973.6 (100.0%), 974.6 (50.2%), 975.6 (16.5%): (ES+) found 973.5
(100%), 974.5 (48.2%), 975.5 (12.4%); Rf 0.61 (70 : 30 EtOAc–
hexane, ninhydrin stain); [a]2D93 −5.2 (c = 1.0, CH3OH).
904 | Org. Biomol. Chem., 2007, 5, 900–906
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with conc. NH4OH and brine, dried over MgSO4, and the solvents
removed on a rotary evaporator. A hygroscopic waxy orange solid
was recovered (1.02 g, 82%). H NMR (CD3OD, 400 MHz) dH
(1H, br m, NH), 4.35 (1H, dd, J = 5.6, 8.7 Hz, COCH(R)NH),
4.27 (1H, dd, J = 5.2, 9.1 Hz, COCH(R)NH), 4.16 (1H, dd, J =
5.2, 9.1 Hz, COCH(R)NH), 4.10–3.80 (8H, br m, OCH2CONH),
3.80–3.40 (32H, br m, OCH2), 3.35–3.29 (12H, br s, OCH3), 3.28–
3.14 (10H, br m, CH2N), 3.13–2.95 (4H, br m, CH2NH, 8H),
2.11–1.14 (26H, br m, CH2); 13C NMR (CD3OD, 100 MHz) dC
173.63, 172.59, 172.53 (CONH × 7, amides, overlapping), 72.65,
71.72, 71.37, 71.11 (CH2O, overlapping), 58.94 (OCH3), 55.23,
54.99 (COCH(R)NH, overlapping), 39.34, 37.61, 37.35, 32.03,
30.87, 29.78 28.38, 24.50, 24.45 (CH2, overlapping); IR (KBr disc)
1
7.80–7.73 (2H, br m, NH amide G2), 6.71–6.69 (1H, br m, NH
amide G1), 6.31–6.23 (1H, br m, NHBOC), 4.70–4.50 (8H, br m,
NH2), 4.29–4.04 (3H, br m, COCH(R)NH, overlapping), 3.28–
3.14 (12H, br m, CH2N), 3.01 (2H, t, J = 6.5 Hz, CH2NH), 2.65
(4H, t, J = 7.3 Hz, CH2NH2), 1.86–1.04 (53H, br m, CH2, CH3);
13C NMR (CD3OD, 100 MHz) dC 175.73, 174.88, 173.64 (CONH,
amide), 159.55, 159.03 (CONBoc × 2, CONHBoc, overlapping),
80.95, 80.91, 79.90, (C(CH3)3), 56.21, 56.18 (COCH(R)NH × 3,
overlapping), 42.32, 33.61 (CH2, overlapping), 28.81 ((CH3)3CO ×
9, overlapping), 29.53, 27.64, 25.41, 23.93 (CH2CH2N, overlap-
mmax cm−1 3444, 3336 (NH), 1641 (C O), 1602 (NH2 , NH3 ),
1553 (CONH, amide 2), 1366 (O–CH3, ether); ESI-MS (m/z)
calculated value for C56H110N10O19Na 1249.8 (100.0%), 1250.8
(62.6%), 1251.8 (25.4%): (ES+) found 1249.8 (100.0%), 1250.8
(65.5%), 1251.8 (20.5%); also found [C56H110N10O19Na2]2+, with
a peak at 637.0 (10% of the intensity of the M+ ion). [a]D293 −2.34
(c = 1.0, CH3OH).
+
+
=
ping); IR (KBr disc) mmax cm−1 3431, 3330 (NH), 1639 (C O),
=
1553 (CONH, amide 2), 1509, 1251 (OCONH carbamate); ESI-
MS (m/z) calculated value for C43H86N10O9 887: (ES+) found 910
([M + Na]+, 100%); [a]D293 −20.7 (c = 1.0, CH3OH).
Compound G2-Boc. Compound 5 (0.35 g, 0.4 mmol, 1 eq.)
and acid 2 (0.36 g, 0.3 mL, 2 mmol, 5 eq.) were dissolved in DCM
(50 mL). DCC (0.41 g, 2 mmol, 5 eq.), HOBt (0.27 g, 2 mmol,
5 eq.) and Et3N (0.2 g, 2 mmol, 5 eq.) were added and the mixture
was placed in an ice bath for 1 h and then left to stir at rt for
2 d. Dicyclohexylurea (DCU) was removed by filtration, and the
solvents removed by rotary evaporation. The crude product was
purified initially by preparative gel permeation chromatography
(MeOH, Sephadex), and subsequently by silica column chro-
matography (90 : 10, DCM–MeOH), a clear oil was recovered
(0.44 g, 0.29 mmol, 73%). 1H NMR (CD3OD, 400 MHz) dH 8.12–
7.81 (6H, br m, NH amide), 7.06–7.02 (1H, br m, NH amide),
6.55–6.36 (1H, br m, NHBOC), 4.35 (1H, dd, J = 5.6, 8.7 Hz,
COCH(R)NH), 4.27 (1H, dd, J = 5.2, 9.1 Hz, COCH(R)NH),
4.16 (1H, dd, J = 5.2, 9.1 Hz, COCH(R)NH), 4.01–3.80 (8H, br
m, OCH2CONH), 3.80–3.40 (32H, br m, OCH2), 3.35–3.30 (12H,
br s, OCH3), 3.28–3.14 (14H, br m, CH2N), 3.13–3.09 (2H, t, J =
6.6 Hz, CH2NH), 3.01 (2H, t, J = 7.3 Hz, CH2NH), 1.71–1.16
(53H, br m, CH2, CH3). 13C NMR (CD3OD, 100 MHz) dC 173.62,
172.59, 172.54 (CONH × 7, amides, overlapping), 159.32, 157.87
(CONBoc × 2, CONHBoc, overlapping), 80.91, 79.90 (C(CH3)3 ×
3, overlapping), 72.91, 71.39, 71.37, 71.34 (CH2O, overlapping),
59.17 (OCH3), 55.25, 55.02 (COCH(R)NH, overlapping), 46.58,
46.37, 41.83, 40.65, 39.68, 39.65, 38.23, 37.61, 37.30, 30.13 (CH2,
overlapping), 28.81 ((CH3)3C × 9, overlapping), 24.31 (CH2CH2N,
overlapping); IR (KBr disc) mmax cm−1 3444, 3341 (NH), 1659
Acknowledgements
We acknowledge EPSRC and Syngenta for funding the research.
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