Synthesis of chiral, amphiphilic, and water-soluble macrocycles via urea formation

Article abstract of DOI:10.1016/j.tet.2003.08.019

A simple, efficient, and flexible procedure for the synthesis of chiral, amphiphilic, and water-soluble macrocycles is reported. Acylation of p-xylylenediamine with N^α-Fmoc-protected glycine, L-aspartic acid, L-glutamic acid, and L-arginine, fo

Full text of DOI:10.1016/j.tet.2003.08.019

Tetrahedron 59 (2003) 7921–7928
Synthesis of chiral, amphiphilic, and water-soluble macrocycles
via urea formation
*
Tapes Bhattacharyya, Anders Sundin and Ulf J. Nilsson
Organic and Bioorganic Chemistry, Lund University, POB 124, SE-221 00 Lund, Sweden
Received 13 May 2003; revised 18 July 2003; accepted 13 August 2003
Abstract—A simple, efficient, and flexible procedure for the synthesis of chiral, amphiphilic, and water-soluble macrocycles is reported.
Acylation of p-xylylenediamine with N a-Fmoc-protected glycine, L-aspartic acid, L-glutamic acid, and L-arginine, followed by removal of
Fmoc-groups, gave amino acid:p-xylylene conjugate diamines, which were converted to ten macrocycles via stepwise urea formation using
p-nitrophenyl chloroformate. L-Aspartic acid-containing macrocyles proved to be soluble in aqueous buffers and a macrocycle containing
four aspartate residues was found to recognize arginine and arginine esters with moderate affinity.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
preferably allow for easy structural diversification, i.e.
combinatorial chemistry, and for the incorporation of
chirality. We envisioned that acylation of diamines with
amino acids would provide molecules amenable for
macrocyclisation with bifunctional amine-reactive cross-
linking reagents, as well as for incorporation of chirality and
water-solubility through the amino acids. Furthermore, such
a strategy allows for diversification by variation of both the
diamine and amino acid structures.
Synthesis of macrocycles has been a rather successful
approach towards artificial receptor molecules ever since
the pioneering work on crown ethers and elegant and
efficient macrocyclic host molecules are continuously being
discovered.1–8 A majority of macrocyclic host molecules
rely on polar interactions (charge–charge, charge–dipole,
hydrogen bonding etc.) for the recognition of guest
molecules and most hosts consequently function in non-
polar solvents. However, biologically active molecules (e.g.
amino acids, peptides, carbohydrates, glycoconjugates etc.)
constitute important targets for artificial receptor molecules
and such receptors obviously have to be water-soluble, still
providing structural motifs capable of forming molecular
interactions in water. Typically, hydrophobic interactions
are, in addition to hydrogen bonds, polar and charged
interactions, important in a highly competitive aqueous
solution9 and re-organisation of high-energy water mol-
ecules in a protein binding site upon displacement by a
ligand has been suggested to be a major driving force for a
ligand:protein complex formation.10 Thus, receptor mol-
ecules displaying efficacy in polar, i.e. aqueous, environ-
ment have to be amphiphilic10 in order to provide for polar
and hydrophobic interactions and for a beneficial re-
organisation of water molecules surrounding the receptor
molecules, as well as to ensure water-solubility.
We herein present a simple system following this strategy;
synthesis of p-xylylenediamine- and amino acid-based
chiral amphiphilic macrocycles obtained via urea formation
with p-nitrophenyl chloroformate as a bifunctional amine-
reactive reagent,11 as well as their conformational analysis
and recognition of basic amino acids. Synthesis of
achiral,12–19 as well as chiral,20 macrocyclic ureas as host
molecules has been reported.
2. Results and discussion
2.1. Synthesis
p-Xylylenediamine was chosen as the diamine component
in our synthetic strategy, because it has reactive primary
amino groups, it is commercially available, and the aromatic
ring provides a site suitable for hydrophobic interactions,
cation-p interaction etc. with putative ligands. The charged
amino acids L-aspartic acid, L-glutamic acid, and L-arginine
were selected as the amino acid components, because they
are readily available, they confer water-solubility, and their
side-chains are suited for polar (charged) interactions with
putative ligands. Furthermore, the amide and urea moieties
A synthetic strategy towards water-soluble amphiphilic
macrocycles as potential biomimetic receptors should
Keywords: macrocycle; water-soluble; molecular recognition.
*
Corresponding author. Tel.: þ46-46-2228218; fax: þ46-46-2228209;
e-mail: ulf.nilsson@bioorganic.lth.se
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.08.019

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T. Bhattacharyya et al. / Tetrahedron 59 (2003) 7921–7928
can, in addition to providing sites for hydrogen bonding, be
expected to induce rigidity in the final macrocycles.
Diisopropylcarbodiimide (DIC) and 1-hydroxybenzotri-
azole (HOBt)-mediated acylation of p-xylylenediamine 1
with N a-Fmoc-protected glycine, L-(OtBu)aspartic acid,
L-(OtBu)glutamic acid, and L-(Mtr)arginine proceeded
smoothly to give compounds 2a–d, which were treated
with piperidine to afford the free amines 3a–d (Scheme 1).
Compounds 3a–d were activated with the bifunctional
amine-reactive cross-linking reagent p-nitrophenyl chloro-
formate to give the unstable carbamates 4a–d in 50–67%
yields after column chromatography. The moderate yields
were due to the instability of the carbamates 4a–d on silica
columns. Nevertheless, pure carbamates 4a–d were
obtained in acceptable yields and immediately used in
macrocyclisation reactions with the diamines 3a–d under
high dilution conditions in DMF in the presence of
4-dimethylaminopyridine (DMAP), which resulted in pre-
cipitation of the pure macrocycles 5–14 in 28–77%
(Scheme 2). The protected macrocycles 5–14 were all
rather insoluble in most solvents, which complicated their
analysis by FAB-HRMS. However, MALDI-TOF MS
confirmed their cyclic structure and solubility in dimethyl-
1
sulfoxide was sufficient to allow H NMR spectra to be
recorded. Fortunately, trifluoroacetic acid-mediated
removal of tBu and Mtr protecting groups from 6–14 to
furnish 15–23, conferred solubility of macrocycles 15–16,
18–19, and 21 in PBS buffer (pH 7.2). Compound 5 was
insoluble in aqueous buffers, while L-arginine-based
molecules 17, 20, 22–23 were soluble only under strongly
acidic conditions, thus failing the essential criteria for
biomimicry, i.e. water-solubility near neutral pH.
Scheme 2. (a) Pyridine, DMAP, DMF (28–77%). (b) TFA, H2O (71–
88%).
2.2. Binding studies and conformational analysis
With the water-soluble amphiphilic macrocycles 15–16,
18–19, and 21 at hand, screening for recognition of a panel
of commercially available p-nitrophenyl glycosides (a-D-
gal, b-D-gal, a-D-glc, b-D-glc, a-D-man, b-D-man, a-L-fuc,
b-L-fuc, b-lac), amino acids, and amino acid esters was
discouraging. However, the addition of esters of L-arginine
and L-lysine clearly induced small, but significant, chemical
1
shift changes in the H NMR spectra of 15 and, to a lesser
extent, of 21, while no ligand-induced chemical shifts were
observed in the 1H NMR spectra of the symmetrical
glutamate-based macrocycle 16, nor of the unsymmetrical
glycine containing macrocycles 18 and 19. The most
1
notable effect on the H NMR spectra of 15 and 21 was a
shift of the b-protons of aspartic acid side-chains. The
induced shift changes were very small, but nonetheless
significant and reproducible. Small chemical shift changes
were also observed for aromatic, benzylic, and a-protons.
The presence of small shift changes suggested weak
interactions. This was confirmed by subsequent titration
experiments of the symmetrical aspartic acid-based macro-
cycle 15 and the unsymmetrical macrocycle 21 with
Scheme 1. (a) N a-Fmoc amino acid, DIC, HOBt, THF (70–95%).
(b) Piperidine, DMF (80–95%). (c) p-Nitrophenyl chloroformate, pyridine,
DMF (50–67%).

T. Bhattacharyya et al. / Tetrahedron 59 (2003) 7921–7928
7923
L-arginine and the methyl ester of L-arginine, which
provided binding curves fitting well to a 1:1 binding
isotherm, suggesting the formation of 1:1 complexes
(Fig. 1). Dilution experiments showed that the macrocycles
15 and 21 did not self-aggregate under conditions used.
and 21 carry four negative charges each, thus forming
repulsive interactions with the carboxylate of L-arginine.
The recognition of arginine esters was not enantioselective,
because 15 showed identical Ka-values for the methyl esters
of D-arginine and L-arginine.
Scheme 3. (a) TFA (80%). (b) Ac2O, MeOH (75%).
Figure 1. 1H NMR-titration experiments of 15 and 21 with L-arginine and
L-arginine methyl ester.
The Ka values of 15 for L-arginine methyl ester and
L-arginine were calculated to be 19 and 10 M21, respect-
ively. The unsymmetrical macrocycle 21, carrying two
aspartate and two glutamate side-chains, appeared a some-
what less efficient receptor for L-arginine methyl ester and
L-arginine (Ka 15 and 8 M21, respectively). Compounds 15,
16, 18, 19, and 21 all present sites for cation recognition, i.e.
carboxylates capable of forming salt-bridges and aromatic
rings capable of forming cation-p interactions with
ammonium and guanidinium cations. However, only the
spectra of 15 and 21 were affected by the presence of
L-arginine and the methyl ester of L-arginine, suggesting the
involvement of salt-bridges with the side-chain carboxy-
lates. Guanidinium ions are well-known to interact with
carboxylates and receptors for guanidium ions, including
arginine, typically rely on interaction with carboxylates21 or
sulfonates.22,23 Furthermore, the receptor carrying four
carboxylates close to the macrocycle core, the symmetrical
aspartic acid-based macrocycle 15, displayed the highest
affinity for L-arginine and the methyl ester of L-arginine,
clearly suggesting that the positions of the side-chain
carboxylates are important.
Figure 2. 1H NMR-titration experiments of 15 and 25 with and L-arginine
methyl ester.
The 14 lowest energy conformations obtained from Monte–
Carlo search of 15 were within 10 kJ/mol, which suggests
that 15 is relatively flexible. Analysis of the fourteen low
energy conformations showed that they could be divided
into two groups based on similarity. Furthermore, the two
groups of conformers share the common features of a
collapsed macrocycle with the two aromatic rings stacked in
an edge-face fashion. These collapsed conformations
suggest that the interactions with arginine and arginine
methyl ester are of the ‘nesting’ type, which is also
supported by the small shift changes observed upon
complexation.24 Two aspartic acid side-chain carboxylates
separated by a urea bond and a xylylene unit are placed 10–
˚
12 A apart on the rim of a shallow hydrophobic pocket made
up by edge-face stacked xylylene moieties (Fig. 3). A
˚
distance of 10–12 A between the carboxylates allow for
salt-bridge formations involving both the ammonium and
guanidinium functionalities of L-arginine methyl ester,
while the methyl group could fit into the shallow
hydrophobic pocket (Fig. 3). Finally, it cannot be ruled
out that cation-p interactions are involved in the binding of
L-arginine and L-arginine methyl ester by 15 and 21, nor that
the close proximity of one or both xylylene moieties to the
one or more side-chain carboxylates is critical in the sense
that this results in enhanced strength of salt-bridges. It has
been demonstrated in other systems, and hypothesized to be
important in protein-ligand interactions, that the positioning
of a carboxylate close to an aromatic ring strengthens the
The open structure 25 corresponding to ‘half’ 15, prepared
by ester hydrolysis of 3b to give 24, followed by
N-acetylation (Scheme 3), was included in the experiments
as a control compound. The flexible and less organised
open-chain 25 did not show any 1H NMR chemically
induced shifts in the presence of L-arginine or the methyl
ester of L-arginine (Fig. 2), further indicating the importance
of the organisation of aspartate side-chain carboxylates on
the rim of the less flexible macrocyclic core. The enhanced
affinity for the methyl ester of L-arginine as compared to
L-arginine can be explained by the fact that the receptors 15

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T. Bhattacharyya et al. / Tetrahedron 59 (2003) 7921–7928
and 500 instruments; chemical shifts are reported in ppm
downfield from TMS using residual solvent peaks as
internal standards (CHCl3, 7.26 ppm; CHD2OD, 3.31 ppm,
1
d5-DMSO, 2.50 ppm; HDO, 4.70 ppm) at 295 K. H NMR
spectral assignments were made by COSY experiments.
PBS (0.15 M, pH 7.2) was prepared by dissolving NaCl
(24 g), KCl (0.6 g), Na2HPO.4H2O (3.46 g), and KH2PO4
(0.6 g) in water (3 L) and adjusting pH to 7.2. Deuterated
PBS (d-PBS) was prepared by lyophilization of 100 mL
PBS, followed by repeated lyophilization from D2O, and
finally desolvation of the deuterated salt residue in D2O
(100 mL). MALDI-TOF-MS spectra were recorded on a
Bruker Biflex III instrument and FAB-HRMS on a JEOL
MS-SX 102 instrument. Optical rotations were recorded on
a Perkin–Elmer 341 polarimeter. Concentrations were
made using rotary evaporation with a bath temperature at
or below 408C. TLC were performed on silica gel 60 F254
Figure 3. A representative collapsed low-energy minimum conformation of
15.
˚
salt-bridges it forms,25 presumably due to a lower cost of
desolvation of the carboxylate.
(Merck). Flash chromatography employed silica gel 60 A
(35–70 mm) with distilled solvents. Solid-phase extractions
were performed on reversed phase SepPak C18 (Waters) and
on ion exchange PRS (IST) cartridges. Monte Carlo
simulations were performed with the MMFF/water force
field implemented in MacroModel. 5000 Conformers were
collected within 50 kJ/mol.
3. Conclusions
In conclusion, we have developed a short and high yielding
synthetic procedure towards chiral, amphiphilic, and water-
soluble macrocycles, based on the acylation of an aromatic
diamine, p-xylylenediamine, with amino acids, followed by
stepwise macrocyclic urea fomation with p-nitrophenyl
chloroformate. Despite the short synthesis and structural
simplicity, a symmetrical macrocycle 15, containing four
aspartic acid residues, and an unsymmetrical macrocycle
21, containing two aspartic acid and two glutamic acid
residues, were demonstrated to interact with arginine and
arginine methyl ester. Although the interactions were weak,
they are still noteworthy because of the synthetic and
structural simplicity of the macrocycles and of the highly
competitive media (PBS buffer, pH 7.2). Furthermore,
the binding of L-arginine methyl ester was sensitive to
macrocycle structure, because an open-chain analog (25)
of 15 did not bind, nor did the symmetrical glutamate-
based macrocycle 16 and the unsymmetrical glycine
containing macrocycles 18 and 19, suggesting that a salt-
bridge with the ammonium and/or guanidinium groups
alone is not responsible for the interaction. Instead, the
relative positioning of the carboxylates close to the
macrocycle core and the xylylene moieties seem to be of
importance.
4.2. General procedure for the acylation of p-xylylene
diamine with Fmoc-protected amino acids (!2a–d)
To a solution of 1-hydroxybenzotriazole (292 mg,
2.16 mmol) and Fmoc protected amino acid (2.21 mmol)
in dry THF (20 mL), was added diisopropylcarbodiimide
(294 mg, 02.16 mmol). The solution was stirred for 45 min.
The N,N -diisopropyl urea precipitate was removed by
filtration and the filtrate was transferred to a solution of
p-xylylenediamine (100 mg, 0.73 mmol) in THF at room
temperature. After 2 h, the product precipitated (except 2b,
which precipitated after concentration) as a white solid,
which was filtered off and washed with cold THF:CH2Cl2
mixture (60:40). The products were sufficiently pure
(.95%) to be used directly in the next step.
4.2.1. Bis-N,N0-(Fmoc-glycyl)-p-xylylenediamine (2a).
Fmoc-Gly-OH (0.62 g, 2.21 mmol) gave 2a (0.484 g,
95%). 1H NMR (400 MHz, d6-DMSO) d 8.30 (t, 2H,
J¼5.8 Hz, ArCH2NH), 7.86 (d, 4H, J¼7.3 Hz, ArH), 7.69
(d, 4H, J¼7.2 Hz, ArH), 7.53 (t, 2H, J¼6.0 Hz, COCH2-
NH), 7.39 (t, 4H, J¼7.4 Hz, ArH), 7.30 (t, 4H, J¼7.2 Hz,
ArH), 7.16 (s, 4H, ArH), 4.21–4.26 (m, 10H, OCH2, CCH,
ArCH2N), 3.62 (d, 4H, J¼5.9 Hz, CH2CO). FAB-HRMS
m/z calcd for C42H39N4O6 (MþH): 695.2870, found:
695.2891.
The synthesis is highly flexible in terms of structural
modifications. Hence, further structural diversification via
the aromatic diamine component and/or the amino acid
components may very well lead to improved recognition of
biomolecules in aqueous environment. In particular,
modification of the diamine component allow for diversi-
fication of macrocycle size and of conformational
flexibility.
4.2.2. Bis-N,N00-[Fmoc-L-aspartyl(OtBu)]-p-xylylenedia-
mine (2b). Fmoc-Asp(OtBu)-OH (0.91 g, 2.21 mmol) gave
1
2b (0.64 g, 93%). H NMR (400 MHz, d6-DMSO) d 7.77
(dd, 4H, J¼3.0, 7.6 Hz, ArH), 7.57 (d, 4H, J¼7.1 Hz, ArH),
7.40 (m,4H ArCH), 7.3 (m, 4H, ArH), 7.19 (s, 4H, ArH),
6.78 (bs, 2H, CH2NH), 5.97 (d, 2H, J¼7.9 Hz, CHNH), 4.55
(bs, 4H, COCH), 4.43 (m, 8H, NCH2, OCH2), 4.20 (t, 2H,
J¼6.7 Hz, CCH), 2.97 (d, 2H, J¼13.7 Hz, CHCH2), 2.62 (d,
2H, J¼10.7 Hz, CHCH2), 1.45 (s, 18H, CH3). FAB-HRMS
m/z calcd for C54H59N4O10 (MþH): 923.4231, found:
923.4218.
4. Experimental
4.1. General
1H NMR spectra were obtained on Bruker DRX 300, 400,

T. Bhattacharyya et al. / Tetrahedron 59 (2003) 7921–7928
7925
4.2.3. Bis-N,N0-[Fmoc-L-glutamyl(OtBu)]-p-xylylenedia-
mine (2c). Fmoc-Glu(OtBu)-OH (0.92 g, 2.21 mmol) gave
2c (0.60 g, 85%). 1H NMR (400 MHz, d6-DMSO) d 8.35 (t,
2H J¼4.6 Hz, CH2NH), 7.87 (d, 4H, J¼7.4 Hz, ArH), 7.86
(d, 2H, J¼7.4 Hz, CHNH), 7.70 (dd, 4H, J¼3.7, 6.7 Hz,
ArH), 7.50 (d, 2H, J¼8.0 Hz, ArH), 7.44 (t, 4H, J¼7.6 Hz,
ArH), 7.30 (t, 4H, J¼7.4 Hz, ArH), 7.15 (s, 4H, ArH), 4.20–
4.28 (m, 10H, NCH2, OCH2, CCH), 4.00 (m, 2H, COCH),
2.20 (t, 4H, J¼8.1 Hz, COCH2), 1.87, 1.74 (2m, 4H,
CHCH2), 1.36 (s, 18H, CH3). FAB-HRMS m/z, calcd for
C56H63O10N4 (MþH): 951.4544, found: 951.4556.
Compound 2d (0.20 g, 0.22 mmol) gave 3d (0.1 g, 80%).
[a]2D5¼þ4.18 (c 1.4, MeOH). 1H NMR (400 MHz, CD3OD)
d 7.25 (s, 4H, ArH), 6.65 (s, 2H, ArH), 4.37 (d, 2H,
J¼14.9 Hz, ArCH2), 4.29 (d, 2H, J¼14.9 Hz, ArCH2), 3.3
(m, 2H, COCH), 3.14 (bs, CH2NH), 2.66 (s, 6H, CH3), 2.60
(s, 6H, CH3), 2.11 (s, 6H, CH3), 1.64 (m, 4H, CHCH2), 1.54
(m, 4H, CH2CH2CH2). FAB-HRMS m/z calcd for
C40H61N10O8S2 for (MþH): 873.4115, found: 873.4110.
4.4. General procedure for the synthesis of p-nitrophenyl
carbamates (3a–d!4a–d)
4.2.4. Bis-N,N0-[Fmoc-L-arginyl(Mtr)]-p-xylylenedia-
mine (2d). Fmoc-Arg(Mtr)-OH (1.31 g, 2.21 mmol) gave
2d (0.67 g, 70%). 1H NMR (400 MHz, d6-DMSO) d 7.87 (d,
4H, J¼7.5 Hz, ArH), 7.69 (d, 4H, J¼7.4 Hz, ArH), 7.61 (d,
2H, J¼8.0 Hz, CHNH), 7.39 (t, 4H, J¼7.3 Hz, ArH), 7.30
(t, 4 h, J¼7.5 Hz, ArH), 7.27 (s, 4H, ArH), 6.65 (bs, 2H,
ArH), 4.2–4.26 (m, 10H, NCH2, OCH2, CCH), 3.87 (m, 2H
COCH), 3.75 (s, 6H, CH3), 3.00 (d, 4H, J¼5.5 Hz, CH2NH),
2.68 (s, 6H, CH3), 2.60 (s, 6H, CH3), 2.0 (s, 6H, CH3). FAB-
HRMS m/z calcd for C70H81N10O12S2 (MþH): 1317.5477,
found: 1317.5486.
p-Nitrophenyl chloroformate (6 equiv.) was added to 3a–d
and pyridine (6 equiv.) in dry DMF at 08C, and the solution
was stirred for 1 h. The solution was then brought to room
temperature and allowed for stirring for further 10 h.
Concentration of the reaction mixture and flash chromato-
graphy (SiO2, CH2Cl2/MeOH 50:1) gave 4a–d. The
compounds were unstable and used immediately in the
next step. Satisfactory FAB-HRMS nor elemental analysis
data could be recorded due to the instability of the
compounds.
4.4.1. Bis-N,N0-(p-nitrophenoxycarbonyl-glycyl)-p-xyly-
lenediamine (4a). Compound 3a (0.17 g, 0.68 mmol)
4.3. General procedure for the removal Fmoc
(2a–d!3a–d)
1
gave 4a (0.20 g, 50%). H NMR (300 MHz, d6-DMSO) d
8.55 (t, 2H, J¼5.6 Hz, ArCH2NH), 8.27 (d, 4H, J¼9.0 Hz,
ArH), 7.42 (d, 4H, J¼9.0 Hz, ArH), 7.22 (s, 4H, ArH),
4.27 (d, 4H, J¼5.5 Hz, ArCH2), 3.75 (d, 4H, J¼5.8 Hz,
CH2CO).
A solution of Fmoc protected amino acid derivative (2a–d)
in piperidine/DMF (20% in 20 mL) was stirred for 2 h, then
concentrated. The residue was dissolved in water and
applied onto a SepPak C18 cartridge. The cartridge was
washed with water, then eluted with MeOH/water (60:40).
Concentration gave the pure compounds (3a–d).
4.4.2. Bis-N,N0-[p-nitrophenoxycarbonyl-L-aspartyl
(OtBu)]-p-xylylenediamine (4b). Compound 3b (0.10 g,
1
0.20 mmol) gave 4b (0.11, g, 67%). H NMR (400 MHz,
4.3.1. Bis-N,N0-glycyl-p-xylylenediamine (3a). Compound
2a (1.0 g, 1.44 mmol) gave 3a (0.35 g, 95%). 1H NMR
(300 MHz, CD3OD) d 7.27 (s, 4H, ArH), 4.40 (bs, 4H,
ArCH2), (d, 4H, J¼1.6 Hz, CH2CO). MALDI-TOF-MS m/z
calcd for C12H18N4NaO2 (MþNa): 273.1, found: 274.0.
FAB-HRMS m/z calcd for C12H19N4O2 (MþH): 251.1508,
found: 251.1459.
CD3OD) d 8.65 (t, 2H, J¼5.4 Hz, CH2NH), 8.26 (d, 4H,
J¼9.2 Hz, ArH), 7.37 (d, 4H, J¼9.2 Hz, ArH), 7.26 (s, 4H,
ArH), 4.57 (m, 2H, CHCO), 4.40 (m, 4H, ArCH2), 2.87 (dd,
2H, J¼5.6, 16.2 Hz, CH2CO), 2.68 (dd, 2H, J¼8.1, 16.2 Hz,
CH2CO), 1.46 (s, 18H, CH3).
4.4.3. Bis-N,N0-[p-nitrophenoxycarbonyl-L-glutamyl
(OtBu)]-p-xylylenediamine (4c). Compound 3c (0.10 g,
4.3.2. Bis-N,N0-[L-aspartyl(OtBu)]-p-xylylenediamine
(3b). Compound 2b (0.50 g, 0.54 mmol) gave 3b (0.24 g,
0.197 mmol) gave 4c (90 mg, 55%). H NMR (300 MHz,
1
CD3OD) d 8.63 (t, 2H, J¼5.0 Hz, CH2NH), 8.25 (d, 4H,
J¼8.4 Hz, ArH), 7.40 (d, 4H, J¼9.0 Hz, ArH), 7.26 (s, 4H,
ArH), 4.40 (d, 4H, J¼5.4 Hz, ArCH2), 4.20 (dd, 2H, J¼5.2,
9.0 Hz, COCH), 2.40 (t, 4H, J¼7.4 Hz, CHCH2), 1.97–2.15
(2m, 4H, CH2CO), 1.46 (s, 18H, CH3).
1
90%). [a]2D5¼23.18 (c 1.5, MeOH). H NMR (300 MHz,
CD3OD) d 7.26 (s, 4H, ArH), 4.37 (d, 4H, J¼4.4 Hz,
ArCH2), 3.64 (t, 2H, J¼6.0 Hz, CHCO), 2.67 (dd, 2H,
J¼5.7, 16.3 Hz, CH2CO), 2.55 (dd, 2H, J¼6.8, 16.3 Hz,
CH2CO), 1.45 (s, 18H, CH3). MALDI-TOF-MS m/z calcd
for C24H38N4NaO6 (MþNa): 501.3, found: 501.7. FAB-
HRMS m/z calcd for C24H39N4O6 (MþH): 479.2870, found:
479.2874.
4.4.4. Bis-N,N0-[p-nitrophenoxycarbonyl-L-arginyl(Mtr)]-
p-xylylenediamine (4d). Compound 3d (75 mg, 86 mmol)
1
gave 4d (52 mg, 51%). H NMR (300 MHz, d6-DMSO) d
8.15 (d, 4H, J¼9.2 Hz, ArH), 7.30 (d, 4H, J¼9.2 Hz, ArH),
7.20 (s, 4H, ArH), 6.60 (s, 2H, ArH), 4.32 (d, 4H, J¼6.4 Hz,
ArCH2), 4.12 (dd, 2H, J¼5.5, 8.9 Hz, COCH), 3.75 (s,
6H, OCH3), 3.20 (bs, 4H, CH2NH), 2.63 (s, 6H CH3), 2.55
(s, 6H, CH3), 2.05 (s, 6H, CH3), 1.55–1.75 (2m, 8H,
CHCH2CH2).
4.3.3. Bis-N,N0-[L-glutamyl(OtBu)]-p-xylylenediamine
(3c). Compound 2c (0.40 g, 0.42 mmol) gave 3c (0.20 g,
1
92%). [a]2D5¼þ8.38 (c 0.4, MeOH). H NMR (400 MHz,
CD3OD) d 7.32 (s, ArH, 4H), 4.42 (dd, 4H, J¼7.4, 14.4 Hz,
ArCH2), 3.96 (t, 2H, J¼6.2 Hz, COCH), 2.15 (m, 4H,
CH2CO), 1.71–1.81 (2m, 4H, CHCH2), 1.47 (s, 18H, CH3).
FAB-HRMS m/z calcd for C26H43N4O6 (MþH): 507.3183,
found: 507.3181.
4.5. General procedure for macrocyclization
(314!5–14)
4.3.4. Bis-N,N0-[L-arginyl(Mtr)]-p-xylylenediamine (3d).
A solution of 4 (20–210 mmol) in DMF (20–70 mL) was

7926
T. Bhattacharyya et al. / Tetrahedron 59 (2003) 7921–7928
added to a vigorously stirred solution of 3 (20–210 mmol,
1 equiv.), pyridine (2–16 ml), and DMAP (0.5–2 mg) in
DMF (20–40 mL). After 12 h, the white precipitate
formed was isolated by centrifugation (400 rpm, 5 min),
washed with DMF and MeOH and centrifuged repeatedly to
give 5–14.
MS m/z calcd for C32H41N8O10 (M22BuþH): 697.3, found:
696.7.
4.5.7. Macrocycle 11. Compounds 3d (20 mg, 34 mmol)
1
and 4a (30 mg, 34 mmol) gave 11 (30 mg, 75%). H NMR
(400 MHz, d6-DMSO) d 8.40 (bs, 2H, ArCH2NH), 8.30 (bs,
2H, ArCH2NH), 7.93 (s, 8H, ArH), 6.65 (bs, 2H, ArH), 6.38
(d, 2H, J¼7.6 Hz, CHNH), 6.26 (d, 2H, J¼5.3 Hz, CH2NH),
4.50, 3.97 (2m, 4H, ArCH2), 4.22, 4.05 (2m, 4H, ArCH2),
4.10 (m, 2H, COCH), 3.93, 3.60 (2m, 4H, CH2NH), 3.00 (s,
4H, CH2NH), 2.65 (s, 6H, CH3), 2.55 (s, 6H, CH3), 2.02 (s,
6H, CH3), 1.55 (s, 4H, CHCH2), 1.36 (s, 4H, CH2CH2CH2).
MALDI-TOF-MS m/z calcd for C54H75O12N14S2 (MþH):
1175.5, found: 1176.0.
4.5.1. Macrocycle 5. Compounds 3a (27.5 mg, 110 mmol)
and 4a (63.8 mg, 110 mmol) gave 5 (23 mg, 38%). 1H NMR
(400 MHz, d6-DMSO) d 8.49 and 8.42 (2 s, 2H each,
ArCH2NH), 7.29 (s, 8H, ArH), 6.60 (bs, 4H, COCH2NH),
4.35 (bs, 8H, ArCH2), 3.77 (d, 8H, J¼15.8 Hz, CH2CO).
MALDI-TOF-MS m/z calcd for C26H32N8NaO6 (MþNa):
575.2, found: 575.5.
4.5.2. Macrocycle 6. Compounds 3b (53 mg, 111 mmol)
1
and 4b (90 mg, 111 mmol) gave 6 (63 mg, 56%). H NMR
4.5.8. Macrocycle 12. Compounds 3b (57 mg, 119 mmol)
and 4c (100 mg, 119 mmol) gave 12 (35 mg, 29%).
Alternatively, compounds 3c (32 mg, 62 mmol) and 4b
(50 mg, 62 mmol) gave 12 (27 mg, 42%). 1H NMR
(400 MHz, d6-DMSO), d 8.25 (t, 2H, J¼6.6 Hz, ArCH2-
NH), 8.17 (t, 2H, J¼7.9 Hz, ArCH2NH), 7.17 (s, 4H, ArH),
7.13 (s, 4H, ArH), 6.51 (d, 2H, J¼7.1 Hz, CHNH), 6.30 (d,
2H, J¼8.1 Hz, CHNH), 4.44, 3.99 (2m, 8H, ArCH2), 4.30–
4.10 (2m, 4H, COCH), 2.60 (m, 4H, CHCH2CO), 2.30 (m,
4H, CH2CH2CO), 1.70–1.68 (m, 4H, CH2CH2CO), 1.40,
1.38 (2 s, 36H, CH3). MALDI-TOF-MS m/z calcd for
C52H76N8O14Na (MþNa): 1059.5, found: 1060.2.
(400 MHz, d6-DMSO) d 8.15 (bs, 4H, ArCH2NH), 7.12 (bs,
8H, ArH), 6.54 (bs, 4H, CHNH), 4.24 (bs, 8H, ArCH2), 4.10
(bs, 4H, J¼5.9 Hz, COCH), 1.35 (s, 36H, CH3). MALDI-
TOF-MS m/z calcd for C50H73N8O14 (MþH): 1009.5,
found: 1009.4.
4.5.3. Macrocycle 7. Compounds 3c (60 mg, 120 mmol)
and 4c (100 mg, 120 mmol) gave 7 (50 mg, 40%). 1H NMR
(400 MHz, d6-DMSO), d 8.12 (bs, 4H, ArCH2NH), 7.17–
7.19 (2 s, 8H, ArH), 6.26 (t, 4H, J¼7.1 Hz, CHNH), 4.24 (d,
8H, J¼5.3 Hz, ArCH2), 4.15 (t, 4H, J¼5.9 Hz, COCH), 2.27
(t, 4H, J¼7.6 Hz, CH2CO), 2.19 (t, 4H, J¼7.2 Hz, CH2CO),
1.92–1.72 (2m, 8H, CHCH2), 1.37–1.40 (2 s, 18H, CH3).
MALDI-TOF-HRMS m/z calcd for C54H80N8NaO14
(MþNa): 1087.6, found: 1086.3.
4.5.9. Macrocycle 13. Compounds 3d (50 mg, 57 mmol)
1
and 4b (46 mg, 57 mmol) gave 13 (60 mg, 75%). H NMR
(400 MHz, d6-DMSO) d 8.26 (bs, 2H, ArCH2NH), 8.14 (bs,
2H, ArCH2NH), 7.17 (s, 4H, ArH), 7.11 (s, 4H, ArH), 6.65
(s, 2H, ArH), 6.42 (d, 2H, J¼7.1 Hz, CHNH), 6.28 (d, 2H,
J¼8.8 Hz, CHNH), 4.45, 3.94 (2m, 4H ArCH2), 4.35–4.10
(2m, 4H, ArCH2), 4.20 (m, 2H, COCH), 3.92 (m, 2H,
COCH), 3.75 (s, 6H, OCH3), 3.00 (s, 4H, CH2NH), 2.65
(s, 6H, CH3), 2.20 (s, 6H, CH3), 2.00 (8 s, 6H, CH3), 1.55
(bs, 4H, CHCH2), 1.40 (bs, 4H, CH2CH2CH2), 1.37 (s,
4.5.4. Macrocycle 8. Compounds 3d (72 mg, 82 mmol) and
1
4d (100 mg, 83 mmol) gave 8 (108 mg, 72%). H NMR
(400 MHz, d6-DMSO), d 8.25 (bs, 4H ArCH2NH), 7.2 (s,
8H, ArH), 6.65 (s, 4H, ArH), 6.16 (d, 4H, J¼8.7 Hz,
CHNH), 4.45 (m, 4H, ArCH2), 3.93 (m, 4H, ArCH2), 3.85
(bs, 4H, COCH), 3.75 (s, 12H, OCH3), 3.0 (s, 8H, CH2NH),
2.67 (s, 12H, CH3), 2.00 (s, 12H, CH3), 1.54 (bs, 8H,
CHCH2), 1.40 (bs, 8H, CH2CH2CH2). MALDI-TOF-MS
m/z calcd for C82H117N20O18S4 (MþH): 1797.8, found:
1798.2.
18H,
CH3).
MALDI-TOF-MS
m/z
calcd
for
C66H94O16N14S2 (MþH): 1403.6, found: 1402.9 and m/z
calcd for C66H94O16N14S2Na (MþNa): 1425.6, found:
1424.8.
4.5.10. Macrocycle 14. Compounds 3c (10 mg, 20 mmol),
1
and 4d (25 mg, 20 mmol) gave 14 (16 mg, 56%) H NMR
4.5.5. Macrocycle 9. Compounds 3b (100 mg, 209 mmol)
and 4a (121 mg, 209 mmol) gave 9 (46 mg, 28%). 1H NMR
(400 MHz, d6-DMSO) 8.32 (bs, 4H, ArCH2NH), 7.14 (s,
4H, ArH), 7.12 (s, 4H, ArH), 6.52 (dd, 2H, J¼8.2, 14.4 Hz,
NHCOCH), 6.43 (t, 2H, J¼7.0 Hz, NHCOCH2), 4.41 (dd,
2H, J¼8.2, 14.4 Hz, CH), 4.16–4.32 (m, 8H, ArCH2), 3.78
(dd, 2H, J¼6.9, 16.7 Hz, COCH2NH), 3.44 (dd, 2H, J¼2.5,
16.7 Hz, COCH2NH), 2.59–2.63 (m, 4H, CHCH2), 1.36 (s,
18H, CH3). MALDI-TOF-MS m/z calcd for C30H37N8O10
(M22BuþH): 669.3, found: 669.8.
(400 MHz, d6-DMSO) d 8.38 (bs, 2H, ArCH2NH), 8.25 (bs,
2H, ArCH2NH), 7.16 (s, 4H, ArH), 7.14 (s, 4H, ArH), 6.65
(s, 2H, ArH), 6.20 (d, 2H, J¼7.9 Hz, CHNH), 6.07 (d, 2H,
J¼6.8 Hz, CHNH), 4.46, 4.00 (2m, 4H, ArCH2), 4.21, 4.05
(2m, 4H, ArCH2), 4.10 (m, 2H, COCH), 3.95, (m, 2H,
COCH), 3.75 (s, 6H, OCH3), 3.00 (s, 4H, CH2NH), 2.65 (s,
6H, CH3), 2.55 (s, 6H, CH3), 2.40 (m, 4H, CH2CH2), 2.00 (s,
6H, CH3), 1.85, 1.70 (2brs, 4H, CH2CH2), 1.65 (s, 4H,
CHCH2), 1.54 (s, 4H, CH2CH2CH2), 1.38 (s, 18H, CH3).
MALDI-TOF-MS m/z calcd for C68H98O16N14S2 (MþH):
1431.7, found 1431.3, and m/z calcd for C68H98O16N14S2Na
(MþNa): 1453.7, found: 1453.3.
4.5.6. Macrocycle 10. Compounds 4a (24 mg, 47 mmol)
1
and 3c (27 mg, 47 mmol) gave 10 (29 mg, 76%). H NMR
(400 MHz, d6-DMSO) d 8.33, 8.25 (2m, 4H, ArCH2NH),
7.12, 7.13 (2 s, 8H, ArH), 6.43 (d, 2H, J¼7.6 Hz,
CHNHCO), 6.24 (t, 2H, J¼7.1 Hz, CH2NHCO), 4.40,
3.90 (2m, 8H, ArCH2), 4.30, 4.10 (2m, 4H, COCH), 3.79,
3.4 (2m, 4H, COCH2N), 2.24 (m, 4H, CH2CH2CO), 1.89–
1.67 (2m, 4H, CHCH2), 1.37 (s, 18H, CH3). MALDI-TOF-
4.6. General procedure for deprotection of compounds
6–7, 9, 10, and 12 (!15–16, 18–19, and 21)
Compounds 6–7, 9–10, and 12 were stirred with TFA
(5 mL) at rt for 12 h. The solutions were concentrated and

T. Bhattacharyya et al. / Tetrahedron 59 (2003) 7921–7928
7927
co-concentrated with toluene. The resulting TFA salts were
dissolved in PBS (pH 7.2), followed by precipitation upon
acidification to pH 1, filtration and thorough washing with
water followed by methanol. The residues were dissolved in
0.1 M HCl (2 mL) applied onto an ion exchange cartridge.
Washing with water (2£5 mL), methanol (2£5 mL) and
water (2£2 mL), elution with 4 M HCl, concentration, and
co-concentration with methanol afforded pure macrocycles
15–16, 18–19, and 21. The macrocycles did not provide
satisfactory FAB-HRMS or analysis results, presumably
due to their low solubility and/or hygroscopic nature. NMR
and MALDI-TOF MS-analysis gave satisfactory results in
all cases.
m/z calcd for C30H37N8O10 (MþH): 669.2633, found:
669.2647.
4.7.5. Macrocycle 19. Compound 10 (8.0 mg, 1.0 mmol)
gave 19 (4.0 mg, 88%). 1H NMR (400 MHz, d-PBS, pD 7.2)
d 7.05 (s, 4H, ArH), 7.00 (s, 4H, ArH), 4.31 (d, 2H,
J¼19.9 Hz, ArCH2), 4.17 (ABq, 4H, J¼15.0 Hz, ArCH2),
4.06 (d, 2H, J¼19.9 Hz, ArCH2), 4.03 (dd, 2H, J¼5.0,
8.4 Hz, CHCO), 3.91 (d, 2H, J¼17.2 Hz, CH2CO), 3.62 (d,
2H, J¼17.2 Hz, CH2CO), 2.19 (bt, 4H, J¼7.4 Hz, CH2-
COOH), 1.95 and 1.82 (2m, 2H each, CHCH2). MALDI-
TOF-MS m/z calcd for C32H40O10NaN8 (MþNa): 719.3,
found: 719.0.
4.7. General procedure for deprotection of compounds 8,
11, 13–14 (!17, 20, and 22–23)
4.7.6. Macrocycle 20. Compound 11 (15 mg, 12 mmol)
gave 11 (7.0 mg, 80%). 1H NMR (500 MHz, d-PBS, pD 7.2)
d 7.10 (s, 4H, ArH), 7.05 (s, 4H, ArH), 4.23 (ABq, 4H,
J¼12.2 Hz, ArCH2), 4.13 (ABq, 4H, J¼11.6 Hz, ArCH2),
4.05 (dd, 2H, J¼4.6, 6.3 Hz, COCH), 3.90 (d, 2H,
J¼13.8 Hz, CH2CO), 3.61 (d, 2H, J¼13.8 Hz, CH2CO),
3.10 (t, 4H, J¼4.5 Hz, CH2CH2NH), 1.75 (m, 2H, CHCH2-
CH2), 1.60 (m, 6H, CHCH2CH2, CHCH2CH2). MALDI-
TOF-MS m/z calcd for C34H51O6N14 (MþH): 751.4; found:
751.4.
Compounds 8, 11, and 13–14 were stirred with TFA (5 mL)
at rt for 12 h. The solutions were concentrated and co-
concentrated with toluene. The resulting TFA salts were
dissolved in 0.1 M HCl (2 mL) applied onto an ion
exchange cartridge. Washing with water (2£5 mL), metha-
nol (2£5 mL) and water (2£2 mL), elution with 4 M HCl,
concentration, and co-concentration with methanol afforded
pure macrocycles 17, 20, and 22–23. The macrocycles did
not provide satisfactory FAB-HRMS or analysis results,
presumably due to their low solubility and/or hygroscopic
nature. NMR and MALDI-TOF MS-analysis gave satisfac-
tory results in all cases.
4.7.7. Macrocycle 21. Compound 12 (24 mg, 29 mmol)
gave 21 (15 mg, 81%). 1H NMR (400 MHz, d-PBS, pD 7.2)
d 7.17 (s, 4H, ArH), 7.14 (s, 4H, ArH), 4.25 (m, 8H,
ArCH2), 4.21 (m, 2H, COCH), 3.94 (dd, 2H, J¼8.6, 5.0 Hz,
COCH), 2.55 (dd, 2H, J¼16.1, 5.4, Hz, CHCH2CO), 2.47
(dd, 2H, J¼8.12, 15.62 Hz, CHCH2CO), 2.13 (t, 4H,
J¼7.58, Hz, CH2CH2CO), 1.94, 1.80 (2m, 4H, CHCH2-
CH2). MALDI-TOF-MS m/z calcd for C36H45O14N8
(MþH): 813.3, found: 815.2.
4.7.1. Macrocycle 15. Compound 6 (20 mg, 19 mmol) gave
1
15 (12 mg, 78%). H NMR (400 MHz, d-PBS, pD 7.2) d
7.17 (s, 8H, ArH), 4.27 (ABq, 8H, J¼15.9, 22.2 Hz,
ArCH2), 4.24 (dd, 4H, J¼4.6, 8.3 Hz, CHCH2), 2.56 (dd,
4H, J¼4.6, 16.0 Hz, CHCH2), 2.47 (dd, 4H, J¼8.3, 16.0 Hz,
CHCH2). MALDI-TOF-MS m/z calcd for C34H41N8O14
(MþH): 785.3, found: 784.7.
4.7.8. Macrocycle 22. Compound 13 (30 mg, 21.3 mmol),
1
gave 22 (15 mg, 80%). H NMR (500 MHz, d-PBS, pD
7.2) d 7.20, (2 s, 4H each, ArH), 7.19 (s, 4H, ArH), 4.25
(m, 8H, ArCH2), 4.18 (m, 2H, CHCO), 4.00 (m, 2H,
CHCO), 3.00 (m, 4H, CH2NH), 2.58 (dd, 2H, J¼9.17,
16.25 Hz, CHCH2), 2.48 (dd, 2H, J¼6.52, 16 Hz,
CHCH2), 1.75 (m, 4H, CHCH2CH2) 1.55 (m, 4H,
4.7.2. Macrocycle 16. Compound 7 (8.0 mg, 7.5 mmol)
gave 16 (4.0 mg, 78%). 1H NMR (400 MHz, d-PBS, pD 7.2)
d 7.15 (s, 8H, ArH), 4.25 (m, 8H, ArCH2), 3.95 (m, 4H,
CHCO), 2.14 (t, 8H, J¼8.5 Hz, CH2COOH), 1.97 and 1.80
(2m, 4H each, CHCH2). MALDI-TOF-MS calcd for
C38H49O14N8 (MþH): 841.3, found: 841.8.
CHCH2CH2).
MALDI-TOF-MS
m/z
calcd
for
C38H55O10N14 (MþH): 867.4, found: 867.4.
4.7.3. Macrocycle 17. Compound 8 (20 mg, 11 mmol) gave
1
17 (10 mg, 80%). H NMR (400 MHz, d-PBS, pD 7.2) d
4.7.9. Macrocycle 23. Compound 14 (12 mg, 8.0 mmol),
gave 23 (5.0 mg, 71%). 1H NMR (400 MHz, d-PBS, pD 7.2)
d 7.17 (bs, 8H, ArH), 4.33–4.20 (m, 8H, ArCH2), 4.10–
3.90 (m, 4H, COCH), 3.10 (s, 4H, CH2NH), 2.40 (bs, 4H,
CH2CO), 2.00–1.50 (m, 12H, CHCH2CH2). MALDI-TOF-
MS m/z calcd for C40H59O10N14 (MþH): 895.5, found:
895.0.
7.12 (s, 8H, ArH), 4.22 (bs, 8H, ArCH2), 3.90 (dd, 4H,
J¼5.0, 8.9 Hz, CHCO), 3.02 (t, 8H, J¼7.0 Hz, CH2NH),
1.60 (m, 8H, CHCH2), 1.50 (m, 8H, CHCH2CH2). MALDI-
TOF-MS m/z calcd for C42H69O6N20 (MþH): 949.6, found:
949.9.
4.7.4. Macrocycle 18. Compound 9 (25 mg, 32 mmol) gave
1
18 (17 mg, 80%). H NMR (400 MHz, d-PBS, pD 7.2) d
4.7.10. Bis-L-aspartyl-p-xylyldiamide 24. Compound 3b
(130 mg, 0.27 mmol) was stirred with TFA (2.5 mL) at rt for
6 h. The solution was concentrated and TFA was removed
by co-evaporation with toluene. The residue was applied
onto a C18 cartridge and elution with water methanol (2:1)
afforded 24 (80 mg, 81%). [a]2D5¼2248 (c 0.0026, MeOH).
1H NMR (400 MHz, D2O) d 7.19 (s, 4H, ArH), 4.30 (d,
J¼15.3 Hz, ArCH2), 4.26 (d, 2H, J¼15.1 Hz, ArCH2), 4.14
(t, 2H, J¼5.5 Hz, CHCO), 2.72 (dd, 2H, J¼17.2, 4.7 Hz,
CHCH2), 2.62 (dd, 2H, J¼18.9, 9.1 Hz, CHCH2).
7.05 (s, 4H, ArH), 7.01 (s, 4H, ArH), 4.35 (dd, 2H, J¼4.6,
8.6 Hz, CHCH2), 4.27 (d, 2H, J¼15.7 Hz, ArCH2), 4.17 (d,
2H, J¼15.5 Hz, ArCH2), 4.15 (d, 2H, J¼15.7 Hz, ArCH2),
4.10 (d, 2H, J¼15.5 Hz, ArCH2), 3.90 (d, 2H, J¼17.5 Hz,
CH2CON), 3.62 (d, 2H, J¼17.5 Hz, CH2CON), 2.58
(dd, 2H, J¼4.6, 15.7 Hz, CHCH2), 2.46 (dd, 2H, J¼8.6,
15.7 Hz, CHCH2). MALDI-TOF-MS m/z calcd for
C30H37N8O10 (MþH): 669.3, found: 669.6. FAB-HRMS

7928
T. Bhattacharyya et al. / Tetrahedron 59 (2003) 7921–7928
6. Lavigne, J. J.; Anslyn, E. V. Angew. Chem. Int. Ed. 2001, 40,
3118–3130.
FAB-HRMS m/z calcd for C16H22N4NaO6 (MþNa):
389.1437, found: 389.1462.
7. Fitzmaurice, R. J.; Kyne, G. M.; Douheret, D.; Kilburn, J. D.
J. Chem. Soc., Perkin Trans. 1 2002, 841–864.
8. Hof, F.; Craig, S. L.; Nuckolls, C.; Rebek, Jr. J. Angew. Chem.
Int. Ed. 2002, 41, 1488–1508.
4.7.11. Bis-L-(N-acetyl)-aspartyl-p-xylyldiamide 25.
Compound 24 (4.0 mg, 11 mmol) was stirred with methanol
(0.6 mL) and Ac2O (1.5 mL) for 12 h, then concentrated.
The residue was dissolved in water and applied onto a C18
cartridge. Elution (water/methanol 2:1) afforded 25 (3.8 mg,
77%). [a]2D5¼29.08 (c 0.0013, MeOH). 1H NMR (400 MHz,
D2O) d 7.30 (s, 4H, ArH), 4.57 (s, ArCH2), 4.4 (m, 2H,
CHCO), 2.55 (dd, 4H, J¼14.9, 7.4 Hz, CHCH2). FAB-
HRMS m/z calcd for C20H26N4NaO8 (MþNa): 473.1648,
found: 473.1631.
9. Davis, A. M.; Teague, S. J. Angew. Chem. Int. Ed. Engl. 1999,
38, 736–749.
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12. Kaplan, L. J.; Weisman, G. R.; Cram, D. J. J. Org. Chem.
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891–894.
4.8. NMR-titrations
NMR titration experiments26 were performed at 298K by
recording the 1H NMR spectra of a macrocyclic compound
(2 mM) in the presence of varying concentration of a ligand
in d-PBS. The change of proton chemical shifts were plotted
against the ligand concentration and the data points were
fitted to a standard 1:1 binding isoterm using the program
Kaleidagraph (Abelbeck Software Inc.), which provided the
association constants.
16. Cordier, D.; Dumaine-Bouaziz, M.; Coulet, P. R. Anal. Lett.
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Acknowledgements
This work was supported by the Swedish Research Council,
the Swedish Strategic Research Foundation, and the
program ‘Glycoconjugates in Biological Systems’ spon-
sored by the Swedish Strategic Research Foundation.
´
20. Tejeda, A.; Oliva, A. I.; Simon, L.; Grande, M.; Caballero,
´
M. C.; Moran, J. R. Tetrahedron Lett. 2000, 41, 4563–4566.
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