Functionalized oligoanthranilamides: Modular and conformationally controlled scaffolds

Article abstract of DOI:10.1016/S0968-0896(01)00161-4

This paper describes the use of functionalize oligoanthranilamides as conformationally controlle scaffolds for molecular recognition. Oligomers of anthranilamides are stabilize by the formation of intramolecular six-membere hydrogen bonds in a linear strand conformation. Onto alternate anthranilic aci units, we have attached di- or tripeptide recognition units with the potential to form intramolecular hydrogen bonds to an intercalated peptide strand . Using ¹H NMR dilution experiments in CDCl₃, we have observe chemical shift changes that are consistent with the formation of an extended hydrogen bondes sheet dimer. We also demonstrate that the bis-alanine functionalize strands are able to form discrete hydrogen bonded complexes with dipeptide substrates an to bind hexanoyl alanylalanine selectively over its benzyl ester. In the presence of excess hydrogen bon donors and acceptors, the oligoanthranilamide strand retained its linear conformation, pointing to the potential of this modular design as a useful and stable scaffold for molecular recognition studies. Copyright

Full text of DOI:10.1016/S0968-0896(01)00161-4

Bioorganic & Medicinal Chemistry 9 (2001) 2355–2363
Functionalized Oligoanthranilamides: Modular and
Conformationally Controlled Scaffolds
Yoshitomo Hamuroa and Andrew D. Hamiltonb,*
aDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA
bDepartment of Chemistry, Yale University, New Haven, CT, 06511, USA
Received 15 March 2001; accepted 27 April 2001
Abstract—This paper describes the use of functionalized oligoanthranilamides as conformationally controlled scaffolds for mole-
cular recognition. Oligomers of anthranilamides are stabilized by the formation of intramolecular six-membered hydrogen bonds in
a linear strand conformation. Onto alternate anthranilic acid units, we have attached di- or tripeptide recognition units with the
potential to form intramolecular hydrogen bonds to an intercalated peptide strand. Using 1H NMR dilution experiments in CDCl3,
we have observed chemical shift changes that are consistent with the formation of an extended hydrogen bonded sheet dimer. We
also demonstrate that the bis-alanine functionalized strands are able to form discrete hydrogen bonded complexes with dipeptide
substrates and to bind hexanoyl alanylalanine selectively over its benzyl ester. In the presence of excess hydrogen bond donors and
acceptors, the oligoanthranilamide strand retained its linear conformation, pointing to the potential of this modular design as a
useful and stable scaffold for molecular recognition studies. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
In the present paper, we extend the application of oli-
goanthranilamides with the preparation of a series of
functionalized derivatives that are used to project di-
and tripeptide functionality from the linear strand. We
further describe preliminary results that show that these
parallel positioned peptides can dimerize through inter-
molecular hydrogen bonds to form a sheet-like aggre-
gate. Intramolecular b-sheet formation is one of the key
elements in protein folding and many groups have
attempted to template formation of this secondary
structure,10 including Feigel,11 Kemp,12 Kelly,13 Now-
ick,14 and Gellman.15 The design and synthesis of inter-
molecular b-sheet models are particularly challenging
due to the entropically unfavorable process of bringing
two or more strands together. Elegant examples in this
regard are the stacked cyclic hydrogen bonded peptide
sheets of Ghadiri16 and the interdigitated dibenzofuran
peptides of Kelly.17
In recent years the preparation of oligomeric materials
that fold into well defined shapes and structures has
received considerable attention. The principal goal is to
develop a modular design using non-peptidic and vari-
able monomers that can potentially be used as a scaffold
for recognition and catalysis.1À3 One attractive
approach is to mimic proteins and exploit intramole-
cular hydrogen bonding to control the spatial arrange-
ment of the monomeric units.4À7 We have previously
described the use of different oligoanthranilamide deri-
vatives to form well-defined strand, turn, and helical
secondary structures in chloroform solution and in the
solid state. In particular, we have established that an
oligomer of anthranilamides forms a linear strand con-
formation stabilized by six-membered intramolecular
hydrogen bonding between adjacent amides, and the trans
preference of the secondary amide bond (Scheme 1).8 Oli-
goanthranilamide scaffolds can have several advantages
over simple monomeric scaffolds because of their mod-
ular design, their iterative synthesis and their ease of
modification.9
Preparation of Functionalized Strands
Functionalized oligoanthranilamides can be easily syn-
thesized from monomeric 2-nitrobenzoic acid deriva-
tives. For example, the synthesis of scaffold 2 started
with reduction of of 2-nitrobenzoic acid n-hexyl ester to
the amine followed by coupling with the acid chloride of
nitrophthalic acid mono methyl ester to give the dimeric
*Corresponding author. Tel.:+1-203-432-5570; fax: +1-203-432-
3221; e-mail: andrew.hamilton@yale.edu
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00161-4

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Y. Hamuro, A. D. Hamilton / Bioorg. Med. Chem. 9 (2001) 2355–2363
precursor (Scheme 2).18 The dimer was reduced, cou-
pled with 2-nitrobenzoic acid to give the trimeric deri-
vative and one further reduction and coupling sequence
with the acid chloride of nitrophthalic acid mono
methyl ester gave the tetrameric compound. The tetra-
mer was reduced and capped with hexanoyl chloride to
give protected scaffold 1. Alkaline hydrolysis using
LiOH solution gave unprotected scaffold 2. Later deri-
vatives of scaffold 2 were found to have poor solubility
in organic solvents. A simple modification of the above
synthetic route allowed the preparation of more soluble
versions, such as scaffolds 3 and 4 (Scheme 1).18 In an
attempt to project peptide chains from the scaffold in a
parallel relationship we reacted the free carboxylic acid
groups in scaffolds 2 and 4 with bis- and tris-alanine
derivatives 5 and 6, respectively, which in turn were
prepared by sequential peptide coupling methods using
CDI. From scaffolds 2 and 4 and two oligo-alanine
derivatives 5 and 6, we could prepare four functiona-
lized strands. The molecules resulting from a combina-
tion of 2 and 6 had poor solubility properties and no
further investigations were carried out. The three other
combinations (7, 8, and 9) were synthesized by acid
chloride coupling in good yields (Scheme 3). This simple
synthetic strategy should also allow the attachment of
oligopeptide sidechains to the monomeric anthanila-
mide precursors prior to strand formation, raising the
potential of attaching different peptides onto the scaf-
fold.
Self-aggregation Properties of the Peptide Functiona-
lized Strands
The self-aggregation properties of compound 8 were
1
investigated by following the changes of H NMR che-
mical shifts as a function of concentration in CDCl3 at
room temperature. One possibility was that this com-
pound would dimerize as depicted in 10, by six inter-
molecular hydrogen bonds in a sheet arrangement. The
results of the dilution experiments are shown in Figure 1.
Significant downfield shifts of all the alanine NH pro-
tons upon increasing concentration are seen, consistent
with the formation of intermolecular hydrogen bonds
(Fig. 1a). It was not possible to unequivocally distin-
guish between NHe and NHh or NHf and NHi in 10.
However, in Figure 1a one of NHe or NHh shifts 0.6
ppm and the other 0.4 ppm and one of NHf and NHi
shifts 0.8 ppm and the other shifts 0.5 ppm, upon
increasing concentration from 0.5 to 200 mM. This
Scheme 1. Modular design of oligoanthranilamide.
Scheme 2. Synthesis of scaffold 2 from monomers.

Y. Hamuro, A. D. Hamilton / Bioorg. Med. Chem. 9 (2001) 2355–2363
2357
Scheme 3. Synthesis of functionalized strands 7, 8 and 9 from scaffolds 2 and 4.
observation is consistent with the predicted dimeriza-
derived from curve fitting analysis of the four NH pro-
.
anthranilamide scaffold also showed two distinct con-
centration dependencies (Fig. 1b). The phenyl 3-protons
on rings A and ring C moved upfield (Ád=À0.09 and
tion, shown in 10 where two NHe protons and two NHi
protons are used for hydrogen bonding but only one
NHf and one NHh proton of each pair hydrogen bond
tons was 10Æ2 MÀ1 19
The phenyl protons on the
upon dimerization. The dimerization constant (Kdim
)

2358
Y. Hamuro, A. D. Hamilton / Bioorg. Med. Chem. 9 (2001) 2355–2363
À0.15 ppm, respectively) while those on rings B and D
did not shift. These upfield shifts are presumably due to
the inductive effect of forming hydrogen bonds to the A
and C ring carboxamides on dimerization. Figure 2
shows the chemical shift changes of alanine a-CH pro-
tons as a function of concentration. Protons CHe and
CHh moved downfield by 0.2 ppm over the course of the
dilution, while protons CHf and CHi shifted slightly
upfield. Neither pair separated into distinct resonances.
No significant changes occurred in the anthranilamide
NH protons indicating that intramolecular hydrogen
bonding within the scaffold was sustained through the
experiment.
In these room temperature experiments, the low Kdim
values and the lack of saturation at high concentration
and thus of sigmoidal shapes in the dilution curves sug-
gested that a full population of the aggregated state had
not been achieved. In order to investigate the oligomer-
ization further, a low temperature 1H NMR dilution
experiment of compound 8 was performed in CDCl3 at
À20 ꢀC. At high concentrations, severe peak broadening
was observed, presumably due to higher aggregation.
This broadening prevented the measurement of the
chemical shifts of the alanine NH resonances, however
the alanine C–H resonances could be followed. Figure 2
shows the chemical shifts of alanine a-protons at room
temperature and À20 ꢀC. At À20 ꢀC, the dilution curves
Figure 1. Dilution experiment of 8 at room temperature. (a) Chemical
shifts of alanine NH protons. *, NHh (or NHe), &, NHe (or NHh),
^, NHf (or NHi), ~, NHi (or NHe). (b) Chemical shifts of the phenyl
3-protons. *, ring B; &, ring C; ^, ring D; ~, ring A.
Figure 2. The comparison of two dilution experiments of 8, at room
temperature (open) and at À20 ꢀC (closed). Chemical shifts of alanine
a-protons. *, CHe and CHh; &, CHf and CHi.

Y. Hamuro, A. D. Hamilton / Bioorg. Med. Chem. 9 (2001) 2355–2363
2359
shift to the left indicating that transition from monomer
to oligomer occurs at lower concentrations and that the
aggregated state has reached saturation. The dimeriza-
tion constants at À20 ꢀC were calculated from the CHe
We anticipated that the incorporation of an additional
amino acid into the side chains of 9 would lead to the
more extensive hydrogen bonding interaction in the
dimer (shown in 11) involving 10 hydrogen bonds
1
resonances to be 330Æ220 MÀ1 19
.
Scheme 4 shows two
instead of six in 10. A H NMR dilution experiment
possible dimeric structures which could account for the
observations above. These two aggregates are inter-
convertible since the intercalation dimer transforms into
the stacked dimer by breaking the hydrogen bonds
between A2 and B1. At this point it is impossible to
distinguish between the two forms, however, it is likely
that the stacked dimer would more readily form higher
aggregates.
with 9 in CDCl3 at room temperature showed very little
change in the chemical shifts of the alanine NH reso-
nances over a 10À4–10À2 M concentration range (Fig.
3). Significantly the NHj or NHg resonances were
already downfield shifted (8.3 and 7.9 ppm, respectively)
at the lowest concentration (10À3 M) compared with
those of the terminal residues in 8 (6.5 and 6.7 ppm at
10À3 M). These downfield shifts in 9 showed no change
with concentration and are most likely due to intra-
molecular hydrogen bonding between two tris-alanine
strands. However, aggregation of a less regular nature
can occur with 9. Concentrations of 9 above 10À2 M were
able to effectively gel CHCl3 at room temperature.20
Binding Properties of the Functionalized Strands
The rigid separation of the side chains provided by the
anthranilamide strand prompted us to investigate the
ability of 7 to act as a host for small molecule guests. In
particular, the position of the amide bonds in the
flanking bis-alanine chains suggested that dipeptide
carboxylate or ester derivatives, such as 12 and 13,
might be recognized through formation of a hydrogen
bonded b-sheet, as in 14. We had earlier discussed the
use of peptide strands within a rigid framework as
recognition units for peptide carboxylates, in direct ana-
logy to the antibiotic vancomycin.21 This macrobicyclic
Figure 3. Dilution experiment of 9 at room temperature. Chemical
shifts of alanine NH’s. *, NHj (or NHg); &, NHg (or NHj); ^, NHe
(or NHh); ~, NHh (or NHe); *, NHi (or NHf); &, NHf (or NHi).
Scheme 4. Schematic representation of possible dimers, intercalated and stacked.

2360
Y. Hamuro, A. D. Hamilton / Bioorg. Med. Chem. 9 (2001) 2355–2363
forming further hydrogen bonds to dipeptide substrates
in higher order (2:1 or 3:1) complexes. This may
account for the observation that even at 10 equivalents
of dipeptide, clear saturation in the shifts of the NHe
and CHe,h resonances has not been reached. That a dis-
crete interaction is taking place between 7 and 12 is seen
in the behavior of benzyl ester 13. In contrast to 12,
when 13 is titrated into a CDCl3 solution of 7 no sig-
nificant shifts in the amide NH or a-CH resonances of
the functionalized strand are seen, even at high con-
centrations of the dipeptide ester (Fig. 4b). The absence
of a strong interaction between 7 and 13 is likely due to
the bulkiness of the benzyl groupwhich, if the four
hydrogen bonds shown in 14 were formed, would
encounter unfavorable steric interactions with the oli-
goanthranilamide strand. During both titrations, the
anthranilamide proton resonances moved little indicat-
ing that the conformation of the scaffolds remained
intact even in the presence of excess dipeptide substrate.
In summary, we have described the synthesis of a new
class of functionalized oligomers in which parallel di-
and tripeptide strands are displayed from one face of an
oligoanthranilamide scaffold. The self-aggregation
properties of the bis-alanine functionalized strands were
analyzed by 1H NMR dilution experiments which
showed chemical shift changes that were consistent with
the formation of an extended hydrogen bonded sheet
dimer. In contrast, the tris-alanine derivatives did not
self-associate suggesting that intramolecular hydrogen
bonding blocked the intermolecular binding sites. The
bis-alanine strands were also able to form discrete
hydrogen bonded complexes with dipeptide substrates.
1H NMR experiments were consistent with the forma-
tion of an intermolecular sheet structure that was avail-
able to hexanoylalanylalanine carboxylic acid but not
its benzyl ester. During both dilution and titration
experiments, the anthranilamide CH or NH protons
barely shifted suggesting that the intramolecularly
hydrogen bonded conformation of the scaffolds was
retained. These results demonstrate both the stability
and usefulness of linear oligoanthralinamides as
conformationally well-controlled scaffolds.
Figure 4. Chemical shifts of 7 upon the titration of 7 with 12 and 13
(The concentration of 7 was kept constant at 10À3 M). (a) NH protons
of 7. *, NHe; &, NHh; ^, NHf; ~, NHi; with 12. *, NHe and NHh;
&, NHf; ~;, NHi; with 13 (NHe and NHh were inseparable in this
titration). (b) *, CHe and CHh of 7 with 12; *, CHe and CHh of 7
with 13.
heptapeptide derivative binds to acyl-d-alanine-d-ala-
nine carboxylate derivatives through several hydrogen
bonds that mimic one strand of a b-sheet.22 Our initial
studies with the functionalized anthranilamides involved
acyl-l-alanine-l-alanine carboxylic acid and ester deri-
vatives 12 and 13.23
The complexation properties of compound 7 were mea-
1
sured by H NMR titration experiments in CDCl3 at
room temperature. We had earlier determined that the
dimerization constant for 7 was 55Æ10 MÀ1 indicating
that at 10À3 M (the concentration of 7 in the titration
experiments) self-aggregation should be only minor.19
On titration of 7 with bis-alanine carboxylic acid 12,
significant shifts in several of the NH and CH protons
were observed (Fig. 4a). Analysis of the titration curve
using non-linear regression analysis gave a Ka value of
Experimental
General methods
CH2Cl2 was obtained from Fisher and distilled from
P2O5. Et2O and THF were obtained from Fisher and
distilled from sodium benzophenone ketyl. All other
reagents, unless otherwise noted, were obtained from
the Aldrich Chemical Company and used without fur-
350Æ120 MÀ1 24
.
The NH resonances from amides E,
and I showed the largest downfield shifts (0.35–0.5 ppm)
consistant with their direct participation in a hydrogen
bond to carbonyl groups of the dipeptide substrate (seen
in 14). Amide NHs from residues F and H shifted
downfield by a lesser amount (ꢁ0.2 ppm) as would be
expected from an involvement of the residue CO in
hydrogen bonding to acidic protons in the substrate. In
the case of the amide NH from residue H this may
involve the carboxylic acid proton. One complication in
this analysis is the presence of additional amide NH and
CO groups on the exterior of the sheet in 14 capable of
ther purification. H NMR and 13C NMR spectra were
1
recorded on a Bruker AM-300 (300 MHz). NMR che-
mical shifts are reported in ppm downfield from internal
TMS. Mass spectra were determined at the Department
of Chemistry, University of Pittsburgh, USA. EI and
FAB mass spectra (MS) were obtained using a Varian
MAT CH-5 or VG 7070 mass spectrometer under the
direction of Dr. Kasi V. Somayajula. Melting points
were determined using an Electrothermal capillary

Y. Hamuro, A. D. Hamilton / Bioorg. Med. Chem. 9 (2001) 2355–2363
2361
melting point apparatus and are uncorrected. Elemental
analysis was carried out by Atlantic Microlab, Inc.,
Norcross, GA, USA. Analytical thin layer chromato-
graphy (TLC) was conducted using PolyGram 0.25 mm
silica gel precoated plates with fluorescent indicator
UV254. Silica gel 60 (particle size 0.063–0.200 mm, 70–
230 mesh ASTM; EM Science) was used for flash
chromatography.
was stirred for 1 h in the ice bath. The reaction mixture
was evaporated in vacuo to afford the TFA salt of the
desired compound. The salt was treated with CH2Cl2
(120 mL) and washed with aqueous NaHCO3 (100 mL)
and brine (100 mL). The organic layer was dried over
MgSO4 and evaporated in vacuo to give the free amine
of the desired compound. The free amine was dissolved
in hexane and HCl gas was bubbled through. The solu-
tion was evaporated in vacuo to give the titled com-
1
N-tert-Butoxycarbonyl-L-alanyl-L-alanine benzyl ester. A
solution of N-tert-butoxycarbonyl-l-alanine (3.78 g,
20 mmol) and CDI (3.41 g, 21 mmol) in dry CH2Cl2
(40 mL) was stirred at room temperature for 25 min. l-
Alanine benzyl ester hydrochloride (4.31 g, 20 mmol)
and Et3N (2.02 g, 20 mmol) were added to the solution
and the reaction mixture was stirred at room tempera-
ture overnight. CH2Cl2 (250 mL) was added to the mix-
ture which was then washed with 1 N HCl (200 mL),
aqueous NaHCO3 (200 mL) and brine (200 mL). The
organic layer was dried over MgSO4 and evaporated in
vacuo to give the desired product as a white wax (6.43 g,
pound as a clear oil (2.54 g, 91%): H NMR (300 MHz,
CDCl3) d 8.36 (broad d, J=5.4 Hz, 1H), 8.18 (broad s,
2H), 4.48 (m, 2H), 4.09 (m, 2H), 1.62 (m, 5H), 1.46 (m,
3H), 1.29 (m, 6H), 0.88 (t, J=6.6 Hz, 3H).
N-tert-Butoxycarbonyl-L-alanyl-L-alanyl-L-alanine hexyl
ester. A solution of N-tert-butoxycarbonyl-l-alanine
(1.14 g, 6.0 mmol) and CDI (1.07 g, 6.6 mmol) in dry
CH2Cl2 (20 mL) was stirred at room temperature for 15
.
min. A solution of HCl 51 (1.40 g, 5.0 mmol) and Et3N
(0.61 g, 6.0 mmol) in dry CH2Cl2 (40 mL) was added to
the CDI solution and the reaction mixture was stirred at
room temperature overnight. CH2Cl2 (100 mL) was
added to the mixture which was then washed with 1 N
HCl (100 mL), saturated aqueous NaHCO3 (100 mL)
and brine (100 mL). The organic layer was dried over
MgSO4 and evaporated in vacuo to give the desired
product as a white solid (1.95 g, 94%): mp 148–150ꢀ; 1H
NMR (300 MHz, CDCl3) d 6.71 (broad, 2H), 5.31
(broad, 1H), 4.51 (m, 2H), 4.14 (m, 3H), 1.63 (m, 2H),
1.38 (m, 24H), 0.89 (t, J=5.9 Hz, 3H); 13C NMR
(75 MHz, CDCl3) d 172.8, 172.7, 171.9, 155.4, 79.8,
65.5, 50.0, 48.7, 48.1, 31.3, 28.4, 28.3, 25.4, 22.5, 18.8,
18.1, 13.9; HR-MS m/e calcd for C16H28N3O5 (M+–
C4H9O) 342.2034, found 342.2029.
1
92%): H NMR (300 MHz, CDCl3) d 7.37 (s, 5H), 6.69
(broad d, J=6.0 Hz, 1H), 5.17 (m, 2H), 5.06 (s, 1H),
4.61 (m, 1H), 4.18 (broad m, 1H), 1.44 (m, 15H).
L-Alanine hexyl ester. l-Alanine (13.4 g, 150 mmol) was
suspended in 1-hexanol (100 mL) and benzene (80 mL).
HCl gas was bubbled into the mixture which was then
refluxed for 2 h. The mixture was cooled to room tem-
perature and washed with aqueous NaHCO3
(2Â100 mL) and brine (100 mL). The organic layer was
dried over MgSO4 and the drying reagent was filtered
off. The filtrate was evaporated in vacuo and purified by
column chromatography (SiO2, AcOEt) to give the
titled product as a pale yellow oil (12.2 g, 47%): 1H
NMR (300 MHz, CDCl3) d 4.11 (m, 2H), 3.54 (q,
J=5.4 Hz, 1H), 1.64 (m, 2H), 1.54 (broad, 2H), 1.32 (m,
9H), 0.89 (t, 6.7. 3H).
L-Alanyl-L-alanyl-L-alanine hexyl ester (6). To a solu-
tion of N-tert-butoxycarbonyl-l-alanyl-l-alanyl-l-ala-
nine hexyl ester (1.25 g, 3.0 mmol) in dry CH2Cl2
(10 mL) in an ice bath was added TFA (5 mL) and the
mixture was stirred for 1 h in the ice bath. The reaction
mixture was evaporated in vacuo to afford the TFA salt
of the desired compound. The salt was treated with
CH2Cl2 (100 mL) and washed with aqueous NaHCO3
(100 mL) and brine (100 mL). The organic layer was
dried over MgSO4 and evaporated in vacuo. The eva-
porated residue was solidified from hexane to give the
N-tert-Butoxycarbonyl-L-alanyl-L-alanine hexyl ester. A
solution of N-tert-butoxycarbonyl-l-alanine (3.78 g,
20 mmol) and CDI (3.41 g, 21 mmol) in dry CH2Cl2
(40 mL) was stirred at room temperature for 10 min. A
solution of l-alanine hexyl ester (3.47 g, 20 mmol) and
Et3N (2.02 g, 20 mmol) in dry CH2Cl2 (20 mL) was
added to the CDI solution and the reaction mixture was
stirred at room temperature overnight. CH2Cl2
(100 mL) was added to the mixture which was then
washed with 1N HCl (100 mL), saturated aqueous
NaHCO3 (100 mL) and brine (100 mL). The organic
layer was dried over MgSO4 and evaporated in vacuo to
1
titled compound as a white powder (496 mg, 47%): H
NMR (300 MHz, CDCl3) d 7.74 (broad, 1H), 6.72
(broad, 1H), 4.49 (m, 2H), 4.14 (m, 2H), 3.57 (broad,
1H), 1.81 (broad, 2H), 1.64 (m, 2H), 1.39 (m, 15H), 0.89
(t, J=6.7 Hz, 3H).
1
give the desired product as a clear oil (6.65 g, 97%): H
NMR (300 MHz, CDCl3) d 6.71 (broad, 1H), 5.11
(broad, 1H), 4.53 (m, 1H), 4.14 (m, 3H), 1.63 (m, 2H),
1.40 (m, 15H), 0.89 (t, 6.6H); 13C NMR (75 MHz,
CDCl3) d 172.8, 172.4, 155.4, 79.9, 65.5, 49.8, 48.0, 31.3,
28.4, 28.3, 25.4, 22.5, 18.5, 18.2, 13.9; HR-MS m/e calcd
for C17H32N2O5 344.2314, found 344.2311.
Hx-Ant(Ala-Ala-OHx)-Ant-Ant(Ala-Ala-OHx)-Ant-OHx
(7). A solution of 218 (75 mg, 0.10 mmol) and oxalyl
chloride (75 mg, 0.60 mmol) in dry THF (20 mL) was
stirred at room temperature. DMF (0.025 mL) was
added through a silica gel filter and the mixture was
stirred at room temperature for 25 min. The reaction
mixture was evaporated in vacuo to obtain the diacid
.
L-Alanyl-L-alanine hexyl ester hydrochloride (HCl 5). To
.
chloride (102 mg). To a solution of HCl 5 (max. 82%,
a solution of N-tert-butoxycarbonyl-l-alanyl-l-alanine
hexyl ester (3.45 g, 10 mmol) in dry CH2Cl2 (20 mL) in
an ice bath was added TFA (10 mL) and the mixture
273 mg, max. 0.80 mmol) and Et3N (506 mg, 5.0 mmol)
in dry THF (5 mL) was added a solution of the diacid
chloride in dry THF (5 mL) and the mixture was stirred

2362
Y. Hamuro, A. D. Hamilton / Bioorg. Med. Chem. 9 (2001) 2355–2363
at room temperature for 40 min. CH2Cl2 (50 mL) was
added to the mixture which was then washed with 1 N
HCl (50 mL), aqueous NaHCO3 (50 mL) and brine
(50 mL). The organic layer was dried over MgSO4, eva-
porated in vacuo and solidified from CHCl3 (16 mL)
and AcOEt (80 mL) to obtain the desired product as
(0.025 mL) was added through a silica gel filter and the
mixture was stirred at room temperature for 30 min.
The reaction mixture was evaporated in vacuo to obtain
the diacid chloride. To a solution of 6 (88 mg,
0.25 mmol) and pyridine (79 mg, 1.0 mmol) in dry
CH2Cl2 (2.5 mL) was added a solution of the diacid
chloride in dry CH2Cl2 (2.5 mL) and the mixture was
stirred at room temperature for 4 h. CH2Cl2 (30 mL)
was added to the mixture and which was then washed
with 1N HCl (30 mL), aqueous NaHCO3 (30 mL) and
brine (30 mL). The organic layer was dried over MgSO4
and evaporated in vacuo to obtain the crude product
(161 mg). The crude product was purified by a column
chromatography (SiO2, 1/1=THF/hexane) to give the
desired product as a yellow foam (45 mg, 50%): mp
225–226 ; H NMR (300 MHz, CDCl3) d 12.97 (s, 1H),
12.94 (s, 1H), 12.50 (s, 1H), 11.85 (s, 1H), 9.62 (s, 1H),
9.61 (s, 1H), 9.45 (s, 1H), 9.22 (s, 1H), 8.33 (m, 3H), 8.17
(m, 2H), 8.10 (d, J=8.4 Hz, 1H), 7.98 (m, 2H), 7.87 (m,
2H), 7.33 (m, 2H), 6.69 (d, J=7.2 Hz, 1H), 6.56 (d,
J=7.8 Hz, 1H), 5.40 (m, 3H), 5.26 (m, 1H), 5.08 (m,
1H), 4.79 (m, 1H), 4.64 (m, 1H), 4.50 (m, 2H), 4.17 (m,
4H), 2.5 (t, J=7.5 Hz, 2H), 1.83–1.25 (m, 58H), 1.00–
0.89 (m, 45H); 13C NMR (125 MHz, CDCl3) d 174.5,
172.9, 172.8, 172.6, 171.5, 171.2, 167.7, 167.4, 167.0,
166.8, 165.2, 164.5, 141.1, 140.8, 140.7, 140.3, 138.4,
137.8, 136.1, 135.2, 131.1, 128.2, 127.6, 124.6, 124.4,
123.5, 123.0, 122.5, 121.9, 119.6, 119.1, 74.1, 73.2, 72.9,
66.2, 65.7, 49.2, 49.0, 48.9, 48.7, 48.2, 47.8, 44.1, 44.0,
43.8, 38.6, 31.4, 28.5, 25.2, 24.9, 24.8, 23.3, 23.1, 22.6,
20.1, 19.9, 19.4, 18.7, 18.4, 17.8, 14.0; HR-MS (FAB, 3-
NBA) m/e calcd for C95H140N10O20Na (M+Na+)
1764.0143, found 1764.0074.
a pale yellow solid (62 mg, 52%): m.p. 255–256ꢀ; H
1
NMR (300 MHz, CDCl3) d 12.46 (s, 1H), 12.39 (s, 1H),
12.35 (s, 1H), 11.19 (s, 1H), 9.14 (s, 1H), 9.05 (d,
J=1.2 Hz, 1H), 8.79 (d, J=8.4 Hz, 1H), 8.69 (d,
J=8.1 Hz, 1H), 8.11 (dd, J=8.6, 1.1 Hz, 1H), 7.94 (m,
3H), 7.66 (d, J=7.5 Hz, 2H), 7.59 (m, 2H), 7.28 (m,
3H), 6.93 (m, 2H), 4.83 (t, J=7.2 Hz, 1H), 4.75 (t,
J=6.9 Hz, 1H), 4.58 (m, 2H), 4.35 (t, J=6.6 Hz, 2H),
4.14 (m, 4H), 2.41 (t, J=7.5 Hz, 2H), 1.82–1.25 (m,
46H), 0.8 (m, 12H); 13C NMR (125 MHz, CDCl3) d
172.8, 172.7, 172.3, 171.7, 171.6, 168.6, 167.7, 167.0,
166.9, 166.4, 165.8, 140.7, 140.4, 140.0, 139.9, 138.2,
137.6, 134.7, 133.3, 131.1, 127.9, 127.7, 127.5, 124.1,
123.7, 123.4, 122.7, 122.6, 122.2, 121.7, 120.63, 120.56,
119.5, 116.1, 66.1, 65.7, 65.6, 49.4, 49.3, 48.4, 48.3, 38.4,
31.4, 29.7, 28.5, 25.7, 25.5, 25.1, 22.5, 22.4, 18.7, 18.24,
18.19, 17.8, 14.0; LR-MS (FAB, MNBA) m/e calcd for
C66H88N6O14Na (M+Na+) 1239.6, found 1240.
ꢀ
1
Hx-Ant(Ala-Ala-OHx)-Ant(ODmh)-Ant(Ala-Ala-OHx)-
Ant(ODmh)-ODmh (8). A solution of 418 (58 mg,
0.050 mmol) and oxalyl chloride (135 mg, 0.12 mmol) in
dry THF (5 mL) was stirred at room temperature. DMF
(0.025 mL) was added through a silica gel filter and the
mixture was stirred at room temperature for 30 min.
The reaction mixture was evaporated in vacuo to obtain
.
the diacid chloride. A solution of HCl 5 (112 mg,
0.40 mmol) and Et3N (101 mg, 1.0 mmol) in dry CH2Cl2
(10 mL) was added to the diacid chloride and the mix-
ture was stirred at room temperature for 2 h. CH2Cl2
(50 mL) was added to the mixture and which was then
washed with 1N HCl (50 mL), saturated NaHCO3 (50
mL) and brine (50 mL). The organic layer was dried
over MgSO4 and evaporated in vacuo to obtain the
crude product. The crude product was purified by col-
umn chromatography (silica gel, 1:1=AcOEt/hexane)
to give the desired product as a yellow foam (154 mg,
Hexanoyl-L-alanyl-L-alanine benzyl ester (13). A solu-
tion of hexanoyl-l-alanine (5.62 g, 30 mmol) and CDI
(5.11 g, 32 mmol) in dry CH2Cl2 (50 ml) was stirred at
room temperature for 30 min. A solution of l-alanine
benzyl ester hydrochloride (6.80 g, 32 mmol) and Et3N
(3.19 g, 32 mmol) in dry CH2Cl2 (150 mL) was added to
the previous solution and the reaction mixture was stir-
red at room temperature overnight. CH2Cl2 (100 mL)
was added to the mixture which was then washed with
1 N HCl (150 mL), saturated NaHCO3 (150 mL) and
brine (150 mL). The organic layer was dried over
MgSO4 and evaporated in vacuo. The crude product
was purified by column chromatography (silica gel,
1:1=AcOEt/CH2Cl2) and then recrystallized from
AcOEt/hexane to give the desired prodꢀuct as a white
(300 MHz, CDCl3) d 7.36 (m, 5H), 6.56 (d, J=7.2 Hz,
1H), 6.01 (d, J=7.5 Hz, 1H), 5.18 (m, 2H), 4.59 (m,
1H), 4.49 (m, 1H), 2.19 (t, J=6.2 Hz, 2H), 1.60 (m,
2H), 1.42 (d, J=7.2 Hz, 3H), 1.36 (d, J=7.0 Hz, 3H),
1.30 (m, 4H), 0.89 (t, J=6.8 Hz, 3H); 13C NMR
(75 MHz, CDCl3) d 173.1, 172.5, 172.4, 135.4, 128.6,
128.4, 128.1, 67.0, 48.5, 48.2, 36.4, 31.4, 25.3, 22.4,
18.8, 17.7, 13.9; HS-MS m/e calcd for C19H28N2O4
348.2049, found 348.2059.
80%): m.p. 147-148ꢀ; H NMR (300 MHz, CDCl3) d
1
12.51 (s, 1H), 12.45 (s, 1H), 12.22 (s, 1H), 11.25 (s, 1H),
9.41 (s, 1H), 9.36 (s, 1H), 9.15 (s, 1H), 9.08 (s, 1H), 8.16
(d, J=8.1 Hz, 1H), 8.00 (m, 5H), 7.82 (m, 4H), 6.83 (d,
J=7.5 Hz, 1H), 6.75 (d, J=7.5 Hz, 1H), 5.38 (m, 3H),
4.74 (m, 2H), 4.57 (m, 2H), 4.13 (m, 4H), 2.44 (m, 2H),
1.78–1.25 (m, 52H), 0.95–0.80 (m, 45H); 13C NMR
(75 MHz, CDCl3) d 172.8, 172.7, 172.3, 172.0, 167.7,
167.0, 166.9, 166.8, 166.4, 165.7, 165.1, 165.0, 140.8, 140.5,
140.0, 139.9, 138.5, 138.0, 136.1, 134.9, 131.0, 127.9, 127.8,
127.7, 124.8, 124.3, 123.5, 123.3, 123.2, 122.9, 122.4, 122.1,
121.8, 121.1, 119.9, 119.5, 74.0, 73.2, 72.9, 65.6, 49.5, 48.4,
44.0, 38.4, 31.4, 28.5, 25.5, 24.8, 23.2, 22.5, 18.0, 14.0; HR-
MS (FAB, 3-NBA) m/e calcd for C89H130N6O16Na
(M+Na+) 1621.9401, found 1621.9406.
cotton (4.06 g, 39%). Mp130–131
;
1H NMR
Hx-Ant(Ala-Ala-Ala-OHx)-Ant(ODmh)-Ant(Ala-Ala-Ala-
OHx)-Ant(ODmh)-ODmh (9). A solution of 4 (115 mg,
0.10 mmol) and oxalyl chloride (64 mg, 0.50 mmol) in
dry THF (5 mL) was stirred at room temperature. DMF
Hexanoyl-L-alanyl-L-alanine (12). A solution of 13
(3.49 g, 10 mmol) and 10% Pd/C (350 mg) in dry THF
(50 mL) was prepared in a 250-mL round-bottomed

Y. Hamuro, A. D. Hamilton / Bioorg. Med. Chem. 9 (2001) 2355–2363
2363
flask provided with a magnetic stirrer. H2 was intro-
References and Notes
duced after removal of air by an aspirator and the
solution was stirred vigorously for 2 h at room tem-
perature. The catalyst was removed by filtration
through Celite. The filtrate was evaporated in vacuo to
give the crude product. The crude product was recrys-
tallized from AcOEt/hexane to give the desired product
as a white powder (1.96 g, 76%): mp 147–148 ꢀ; 1H
NMR (300 MHz, CDCl3) d 7.15 (d, J=7.1 Hz, 1H), 6.53
(d, J=7.6 Hz, 1H), 4.64 (m, 2H), 4.54 (m, 2H), 2.22 (t,
J=7.5 Hz, 2H), 1.62 (m, 2H), 1.45 (d, J=7.1 Hz, 3H),
1.37 (d, J=7.0 Hz, 3H), 1.30 (m, 4H), 0.89 (t, J=6.7 Hz,
3H); 13C NMR (75 MHz, DMSO-d6) d 174.0, 172.3,
172.0, 47.6, 47.4, 35.1, 30.9, 24.9, 21.9, 18.2, 17.2, 13.9;
MS m/e calcd for C12H22N2O4 259.1658, found
259.1665.
1. Tanatani, A.; Mio, M. J.; Moore, J. S. J. Am. Chem. Soc.
2001, 123, 1792 and references therein.
2. Louis, A.; Lehn, J.-M.; Homo, J.-C.; Schmutz, M. Angew.
Chem., Int. Ed. 2000, 39, 233.
3. Gellman, S. H. Acc. Chem. Res. 1998, 31, 173.
4. For a discussion of the role of intramolecular hydrogen
bonding in a family of simple diamides, see: Dado, G. P.;
Gellman, S. H. J. Am. Chem. Soc. 1993, 115, 4228.
5. Appella, D. H.; Christianson, L. A.; Klein, D. A.;
Richards, M. R.; Powell, D. R.; Gellman, S. H. J. Am. Chem.
Soc. 1999, 121, 7574.
6. Seebach, D.; Abele, S.; Siffereln, T.; Hanggi, M.; Gruner,
S.; Seiler, P. Helv. Chem. Acta. 1998, 81, 2218.
7. Nowick, J. S.; Muna, A.; Bellamo, K. A.; Love, J. A.;
Martinez, E. J.; Noronha, G.; Smith, E. M.; Ziller, J. W. J.
Amer. Chem. Soc. 1995, 117, 89.
8. Hamuro, Y.; Geib, S. J.; Hamilton, A. D. J. Am. Chem.
Soc. 1996, 118, 7529. Hamuro, Y.; Geib, S. J.; Hamilton, A. D.
J. Am. Chem. Soc. 1997, 119, 10587.
Dilution experiment with 8
A representative dilution experiment was as follows:
compound 8 (128 mg, 0.08 mmol) was dissolved in
0.4 mL of CDCl3 to give a solution of 8 at 200 mM and
the H NMR of the solution was measured at room
temperature. Sequential dilution of the solution with
9. Liskamp, R. M. J. Angew. Chem. Int. Ed. Engl. 1994, 33, 633.
10. (a) For recent reviews, see: Nowick, J. S.; Smith, E. M.;
Pairish, M. Chem. Soc. Rev. 1996, 401. (b) Schneider, J. P.;
Kelly, J. W. Chem. Rev. 1995, 95, 2169.
11. Brandmeier, V.; Sauer, W. H. B.; Feigel, M. Helv. Chim.
Acta 1994, 77, 70.
12. Kemp, D. S.; Li, Z. Q. Tetrahedron Lett. 1995, 36, 4179.
13. Schneider, J. P.; Kelly, J. W. J. Am. Chem. Soc. 1995, 117,
2533.
14. Nowick, J. S.; Mahrus, S.; Smith, E. M.; Ziller, J. W. J.
Amer. Chem. Soc. 1996, 118, 1066.
1
CDCl3 gave solutions of 8 at 100, 50, 20, 10, 5, 2, 1,
1
0.5 mM and the H NMR of each solution was mea-
1
sured. The same procedure was repeated and H NMR
spectra were measured at À20 ꢀC. Dimerization con-
stants were calculated by Saunders and Hyne’s model19
using KaleidaGraph.
15. Gardner, R. R.; Liang, G.-B.; Gellman, S. H. J. Am.
Chem. Soc. 1995, 117, 3280.
16. Kobayashi, K.; Granja, J. R.; Ghadiri, M. R. Angew.
Chem., Int. Ed. Engl. 1995, 34, 95.
Titration Experiment of 7 with 12
17. LaBrenz, S. R.; Kelly, J. W. J. Am. Chem. Soc. 1995, 117,
1655.
A representative dilution experiment was as follows:
compound 7 (12 mg, 0.01 mmol) was dissolved in 10 mL
of CDCl3 to give a solution of 7 at 10 mM (solution A).
Compound 12 (13 mg, 0.05 mmol) was dissolved in 5 mL
of solution A (solution B, 10 mM of 7 and 100 mM of
12). After various ratios of solution A and solution
B were mixed (including 100% solution A and 100%
18. For the syntheses of monomers and scaffolds 1–4, see ref 8.
19. Determined using the method of Saunders and Hyne.
Saunders, M.; Hyne, J. B. J. Chem. Phys. 1958, 20, 1319.
20. For other examples of peptide based gelators see; Estroff,
L.; Hamilton, A. D. Angew. Chem., Int. Ed. 2000, 39, 3447 and
references therein.
1
21. Pant, N.; Hamilton, A. D. J. Am. Chem. Soc. 1988, 110,
2002.
solution B), H NMR spectra of the resulting solutions
were measured at room temperature. The resulting
binding isotherm was fit to a 1:1 binding model using
KaleidaGraph.
22. Williams, D. H. J. Am. Chem. Soc. 1981, 1037, 6580.
23. For other approches to dipeptide carboxylate recognition
through sheet hydrogen bonding see; Weiner, W. S.; Hamil-
ton, A. D. Bioorg. Med. Chem. Lett. 1998, 8, 681. Dowden, J.;
Edwards, P. D.; Kilburn, J. D. Tetrahedron Lett. 1997, 38,
1095. Pan, Z.; Still, W. C. Tetrahedron Lett. 1996, 37, 8699.
Pieters, R. J.; Diederich, F. J. Chem. Soc., Chem. Commun.
1996, 2255.
Acknowledgements
The authors thank the National Science Foundation
for financial support of this work. We also thank the
Kureha Chemical Industry for a fellowshipto Y.H.
24. Association constant analysis was carried out using
KaleidaGraph ver 2.1.