Diketopiperazine receptors: A novel class of highly selective receptors for binding small peptides

Article abstract of DOI:10.1002/1521-3765(20010803)7:15<3342::AID-CHEM3342>3.0.CO;2-7

A novel class of receptors consisting of a rigid diketopiperazine backbone and peptidic side chains has been developed with the use of combinatorial chemistry. These diketopiperazine receptors interact with peptidic substrates with high specificity as sho

Full text of DOI:10.1002/1521-3765(20010803)7:15<3342::AID-CHEM3342>3.0.CO;2-7

FULL PAPER
Diketopiperazine Receptors:
A Novel Class of Highly Selective Receptors for Binding Small Peptides
Helma Wennemers,* Matteo Conza, Matthias Nold, and Philipp Krattiger[a]
Abstract: A novel class of receptors
consisting of a rigid diketopiperazine
backbone and peptidic side chains has
been developed with the use of combi-
natorial chemistry. These diketopipera-
zine receptors interact with peptidic
substrates with high specificity as shown
in combinatorial on-bead assays. The
central diketopiperazine moiety can be
easily obtained from natural 4-hydroxy-
proline and serves as a rigidifying tem-
plate for the peptidic modules which
allow for structural as well as functional
variations. Screenings of several dye-
marked receptor prototypes against an
encoded tripeptide library demonstrat-
ed not only the high binding specificities
of the diketopiperazine receptors to-
wards peptides but also revealed that
small structural changes induce signifi-
cant changes in their binding properties.
Keywords: combinatorial chemistry
´ diketopiperazine ´ molecular rec-
ognition ´ peptides ´ receptors
related receptor prototypes to different peptides within a
tripeptide library.
Introduction
The increasing need for therapeutics and sensors asks for the
development of synthetic receptors recognizing small pep-
tides selectively. Due to the many degrees of freedom of even
simple di- or tripeptides the rational design of receptors for
specific peptide sequences is an extremely challenging task.
During the last decade, combinatorial chemistry has become a
powerful tool for the search of suitable substrates for a given
receptor and vice versa.[1] Using this stochastic approach,
subtle differences in host ± guest interactions have been
disclosed which could have not been predicted by molecular
modelling or found by conventional trial and error. Con-
sequently, receptors capable of binding peptides have been
established.[2±4] In particular, two-armed receptors consisting
of a template with two modules have proven efficient in
binding peptidic substrates.[3, 4] Yet, most of the examined
receptors exhibit drawbacks such as either poor binding
selectivities, rather laborious syntheses or limited possibilities
for tuning the receptor structures. Thus, the need for receptors
with easily variable structures that allow for the selection of a
specific receptor for any desired peptide remains.
A receptor class containing members capable of binding
any desired peptide selectively should consist of a rigid,
structure-directing backbone as well as functional groups that
allow for the formation of non-covalent interactions such as
hydrogen bonds, ionic and hydrophobic interactions. Further-
more, the receptor structure should offer opportunities for
combinatorial structural and functional variations and should
be accessible by a simple synthesis both in solution and on
solid supports to ultimately permit the generation of a
combinatorial receptor library using the split-and-mix proto-
col.[5]
The novel diketopiperazine receptors depicted in Figure 1
fulfil all of the above-mentioned requirements for a versatile
class of receptors. Herein, the diketopiperazine derived from
R3
O
R1
H
N
H
N
R4
N
H
N
H
O
O
R2
R6
O
O
H
O
N
H
O
Here we introduce a novel class of two-armed receptors and
report the highly selective binding of several structurally
N
H
N
H
N
R8
N
H
N
H
R7
O
R5
[a] Dr. H. Wennemers, Dipl.-Chem. M. Conza,
Dipl.-Chem. M. Nold, Dipl.-Chem. P. Krattiger
Institute of Organic Chemistry, University of Basel
St. Johanns Ring 19, 4056 Basel (Switzerland)
Fax : (‡41)61-267-1105
Figure 1. Two-armed diketopiperazine receptors.
4-hydroxyproline provides the rigid backbone and anchor for
the peptidic side chains, that is ªthe armsº. Natural l-, as well
as d-amino acids, can be employed as building blocks for the
E-mail: helma.wennemers@unibas.ch
Supporting information for this article is available on the WWW under
http://www.wiley-vch.de/home/chemistry/ or from the author.
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Chem. Eur. J. 2001, 7, No. 15

3342±3347
receptor arms as they offer structural and functional variety.
Standard peptide synthesis can be used to assemble these
diketopiperazine receptors which could be easily attached to
solid supports through side chain functional groups of the
amino acids.
N3
N3
CO2C6F5
NBoc
N3
a
b
N
CO2Me
NBoc
CO2Me
NBoc
c
2
O
H
1
N3
CO2Me
. TFA
NH2
N3
Results and Discussion
4
3
Since the diketopiperazine receptors are a novel class of
receptors with unknown binding properties, we prepared
several receptor prototypes, marked with dyes, and identical
arms (R1 ˆ R5, R2 ˆ R6, R3 ˆ R7, R3 ˆ R7 ˆ Ac) in order to
examine their binding properties in on-bead screenings
against a peptide library. As a dye we employed the red
azo-dye Disperse Red 1 since it can be easily attached to the
side chain functionality of an amino acid (such as tyrosine)
and does not bind to peptides itself.[6]
The synthesis of the diketopiperazine template starts from
N-Boc-4-trans-azido-l-proline methyl ester (1) which is read-
ily obtained from commercially available N-Boc-4-cis-hy-
droxyproline methyl ester by a SN2 substitution with NaN3 of
the corresponding mesylate.[7] One portion of the N-Boc-4-
trans-azido-l-proline methyl ester (1) is then hydrolyzed and
transformed into the pentafluorophenyl (Pfp) ester 2 and the
other portion is transformed into the trifluoroacetic acid
(TFA) salt 3. Mixing of 2 and 3 in the presence of Hünigꢁs base
yields the cyclization precursor 4. N-Boc deprotection with
TFA and addition of Hünigꢁs base leads to the diketopiper-
azine 5. The reduction of the azide functionalities with
palladium on carbon in the presence of di-tert-butyl-dicar-
bonate (Boc2O) yields the well storable N-Boc-protected
diketopiperazine 6.
N3
NHBoc
H
N
H
N
d
e
O
O
O
O
H
N
H
N
N3
NHBoc
5
6
Scheme 1. Synthesis of the diketopiperazine template 6: a) i) NaOH,
MeOH, THF, H2O; ii) C6F5OH, EDC, CH2Cl2, 85%; b) TFA, CH2Cl2,
quant.; c) iPr2NEt, CH2Cl2, 89%; d) i) TFA, CH2Cl2; ii) iPr2NEt, CH2Cl2,
79%; e) 10% Pd/C, H2, MeOH, Boc2O, 91%.
H3CO2C
BocHN
C6F5O2C
BocHN
a, b
dye
OH
O
8a
7a
N
NO2
Et
dye-OH = Disperse Red 1:
N
N
HO
Scheme 2. Synthesis of N-Boc-l-Tyr(dye)-OC6F5 8a: a) Disperse Red 1,
PPh3, DEAD, toluene, 72%; b) i) NaOH, MeOH, THF, H2O; ii) C6F5OH,
EDC, CH2Cl2, 95%.
The red azo-dye Disperse Red 1 is attached to the phenolic
hydroxy group of N-Boc-l- or d-tyrosine methyl ester 7a or
7b, respectively, by a Mitsunobu reaction followed by
conversion of the methyl esters into the Pfp esters to yield
the dye-marked tyrosine derivatives 8a and 8b (Scheme 2).
For the assembly of the receptors, the tert-butyl groups of 6
are removed and the resulting diamine is coupled with the
dye-marked Pfp esters of N-Boc-d- or l-tyrosine 8a or 8b,
respectively. After N-Boc deprotection with HCl the remain-
ing amino acids of the arms are assembled by standard
couplings of N-a-Fmoc-protected amino acids using 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDC) as the coupling reagent and tris(2-aminoethyl)amine
(TAEA) as the Fmoc deprotection reagent.[8] Acetylation
with acetic anhydride in the presence of triethylamine after
the final Fmoc deprotection leads to the diketopiperazine
receptors 9 ± 13 (Scheme 3).
H
N
Abstract in German: Mit Hilfe von kombinatorischer Chemie
wurde eine neue Klasse von Rezeptoren entwickelt, die aus
einem rigiden Diketopiperazin und peptidischen Seitenketten
bestehen. Diese Diketopiperazin Rezeptoren zeigen in kombi-
natorischen on-bead Screenings, dass sie mit peptidischen
Substraten mit hoher Spezifität wechselwirken. Das zentrale
Diketopiperazin ist aus 4-Hydroxyprolin synthetisch leicht
zugänglich und dient als strukturgebendes Templat für die
peptidischen Seitenketten, die die Einführung von struktureller
als auch funktioneller Vielfalt ermöglichen. Die Screenings
einiger farbstoffmarkierter Rezeptorprototypen gegen eine
kodierte Tripeptidbibliothek zeigten nicht nur, dass die Dike-
topiperazin Rezeptoren Peptide mit hoher Selektivität erken-
nen, sondern auch, dass kleine strukturelle Unterschiede zu
signifikanten ¾nderungen in den Rezeptorbindungseigen-
schaften führen.
Ac
AA3
AA2
AA1
N
H
a - f
6
O
O
H
N
Ac
AA3
AA2
AA1
N
H
receptors 9 - 13
Scheme 3. Synthesis of the diketopiperazine receptors 9 ± 13: a) i) TFA,
CH2Cl2; ii) iPr2NEt, 7, CH2Cl2; b) HCl, MeOH, dioxane; c) iPr2NEt, N-a-
Fmoc-amino acid, EDC, CH2Cl2; d) TAEA, CH2Cl2; e) repetition of c) and
d) ; f) Ac2O, NEt3.
Using this synthesis route five receptor prototypes with
closely related yet distinct structures were prepared. The two
identical arms of each receptor consist of dye-marked l- or d-
Tyr as initial amino acids followed by l-Phe and either l-Asn
Chem. Eur. J. 2001, 7, No. 15
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3343

H. Wennemers et al.
FULL PAPER
Table 1. Binding specificities of the receptor prototypes 9 ± 13 for tripep-
tides within the library Ac-AA3-AA2-AA1-NH(CH2)6CONH-PS.
or l-Gln. Thus, the structural differences of the five proto-
types (Figure 2) are as subtle as the opposite stereochemistry
of tyrosine (e.g. 9 and 10) or a single methylene group (e.g. 9
and 12 as well as 11 and 13).
AA3
AA2
AA1
freq.
freq.
found [%][a] exp. [%][a]
9 d-Val/d-Ala
10 d-Ala/d-Val
l-Asn
11 d-Ala/d-Val
d-Hph
d-Hph[b]
l-Asn/l-Gln l-Ala/Gly
d-His
100
43
57
0.04
0.03
0.02
0.08
0.04
0.08
0.04
0.04
0.04
0.06
d-Pro
l-Hph
l-Ala
l-Hph
d-Hph
l-Gln
d-Hph
l-Hph
l-Ser/l-Thr 46
l-Gln/l-Asn 23
l-Ala
d-His
d-Hph
d-Val/d-Leu 20
l-Asn 100
AA3
AA2
AA1
9
L-Phe-L-Asn(Trt)-L-Tyr(dye)
H
N
AA3
AA1
Ac
Ac
AA2
d-Hph
17
34
37
10 L-Phe-L-Asn(Trt)-D-Tyr(dye)
11 L-Asn(Trt)-L-Phe-D-Tyr(dye)
12 L-Phe-L-Gln(Trt)-L-Tyr(dye)
13 L-Gln(Trt)-L-Phe-D-Tyr(dye)
12 d-Ala/d-Val
l-Ala/l-Leu
d-Gln
N
H
O
O
H
N
13 d-Ala/d-Val/d-Leu l-Hph
AA3
AA2
AA1
N
[a] The column frequency found lists the percentage of beads selected in
the receptor binding assay for the indicated peptide sequence. The column
frequency expected lists the expected frequency for the particular tripep-
tide sequence if the beads were picked randomly. The comparison between
the percentage of ªfrequency foundº and ªfrequency expectedº is a
measure for the selectivity level of the receptor. [b] Hph ˆ hydrophobic
amino acid can be either Gly, Ala, Val, Leu or Phe.
H
Figure 2. Diketopiperazine receptor prototypes 9 ± 13.
The dye-marked receptor prototypes were tested for their
peptide binding affinities by screening them against an
encoded resin-bound tripeptide library with the general
structure Ac-AA3-AA2-AA1-NH(CH2)6-CONH-PS. The li-
brary had been synthesized on polystyrene resin by encoded
split synthesis[9] employing 29 l- and d-amino acids at each
position.[10] Thus, the library contained maximally 293 ˆ 24389
different acylated tripeptides. In order to ensure a represen-
tative screening result, an amount corresponding to at least
five theoretical copies of the library were used per assay.[11]
Upon mixing the library with dilute solutions of the
receptors (ꢀ20 ± 40 mm) in chloroform and equilibration for
at least 24 hours, several beads picked up the red color of the
receptor in all five assays. The assays of receptor 9 and 13
indicated a particularly high level of binding specificity since
only about 25 beads showed the red color of the receptor cor-
responding to a selectivity of one selected bead out of 5000.
Isolation of the colored beads and analysis of the encoding
electrophoric tag molecules by gas chromatography using
electron capture detection[9] revealed remarkable specifici-
ties: Each of the five structurally similar receptor prototypes
selects for different tripeptides within the library (Table 1).
While receptor 9 exclusively selects peptides containing a
d-His following two hydrophobic d-amino acids, receptor 13
solely chooses peptides with an l-Asn following a hydro-
phobic l- and a hydrophobic d-amino acid. Receptor 10
differing from 9 in the configuration of the tyrosine residue,
selects for sequences with an l-Asn in the middle or
N-terminal position being flanked by a combination of a d-
and l-hydrophobic amino acid. More interestingly, receptors
12 and 11 which differ from 9 and 13 only by a single
methylene group (l-Asn is exchanged by l-Gln and vice
versa) is not only selective for the tripeptides bound by 9 and
13 but also for two different peptide motifs. Thus, the novel
diketopiperazine receptors are not only able to bind to
peptides but small structural changes alter their ligand pattern
to a significant extent.
which has a strong preference for the peptide Ac-d-Val-d-Val-
d-His, shows an affinity of Ka ˆ 1420 Æ 200m 1 (DG ˆ 4.3 Æ
1
0.1 kcalmol ), receptor 12, which also recognizes other
peptides, binds to Ac-d-Val-d-Val-d-His less tightly Ka ˆ
1
260 Æ 40m 1 (DG ˆ -3.3 Æ 0.1 kcalmol ). Small modifications
of the ligand peptide such as inverting the stereochemistry of
the central d-Val lead to a considerably decreased binding
1
1
affinity of Ka ꢁ15m
(DG ꢁ1.5 kcalmol ). Thus, our
diketopiperazine receptors are not only highly selective for
certain tripeptides but also show a considerably higher
binding affinity towards the selected peptides as compared
to the non-selected peptides.
To verify that the two-armed motif is the minimal structure
necessary for binding peptides, truncated receptors with only
two amino acids attached to the diketopiperazine 14, a
ªsingle-armº receptor 15 as well as an ªisolated armº 16 were
synthesized and screened against the tripeptide library. Up to
fragment concentrations of 500 mm none of the library beads
turned red, thus, confirming that two arms consisting of three
amino acid residues are crucial for the interactions between
the ligand peptide and the receptor.
H
N
H
N
Ac-L-Asn(Trt)-D-Tyr(dye)
O
Ac-L-Phe-L-Asn(Trt)-D-Tyr(dye)
N
H
N
H
O
O
O
H
N
H
N
Ac-L-Asn(Trt)-D-Tyr(dye)
N
H
N3
15
14
16
Ac-L-Phe-L-Asn(Trt)-D-Tyr(dye)-OCH3
Figure 3. Receptor fragments 14 ± 16.
In order to obtain a measure for the strength of the
observed intermolecular association we determined the bind-
ing affinities of receptors 9 and 12 towards the peptide
selected in the on-bead assay and several non-selected
peptides by solid-phase binding assays.[4] While receptor 9,
Conclusion
We have introduced a novel class of receptors which combine
a particularly simple structure with remarkably high binding
selectivities that are very responsive to subtle structural
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Chem. Eur. J. 2001, 7, No. 15

Diketopiperazine Receptors
3342±3347
white solid. 1H NMR (300 MHz, CDCl3, 258C): d ˆ 9.0 ± 7.0 (broad, 2H;
NH), 4.47 (m, 2H; Ha, Hg), 3.82 (s, 3H; CH3), 3.77 (dd, J ˆ 12.6, 6.0 Hz,
1H; Hd), 3.38 (dd, J ˆ 12.6, 2.9 Hz, 1H; Hd'), 2.45 (m, 2H, Hb, Hb');
13C NMR (75.4 MHz, CDCl3, 258C): d ˆ 168.3, 59.3, 58.0, 53.6, 50.3; IR
(KBr): nÄ ˆ 3006, 2112, 1748, 1674; elemental analysis calcd (%) for
C9H11F3N4O4 (284.2): C 33.81, H 3.90, N 19.71; found: C 33.76, H 3.90, N
19.53.
modifications. The novel diketopiperazine derived from
natural 4-hydroxyproline has proven an ideal scaffold for
guiding the receptor arms into a conformation providing for
intermolecular binding. The simplicity of the structureÐa
simple octapeptide with the structure-directing central dike-
topiperazineÐallows for an easy synthesis and for structural
and functional variations. Additional diversity can be intro-
duced into the receptor structure by using a diketopiperazine
template with differently functionalized amino groups, there-
fore, allowing for the generation of two different arms. Thus,
the novel diketopiperazine receptors present an ideal scaffold
for the development of selective receptors for a given
substrate that could be used for any application where
selective molecular recognition is a prerequisite for example
for the development of diagnostic sensors, therapeutics or
catalysts.
N-Boc-(trans-4-Azido-l-Pro)2-OCH3 (4): The Pfp-ester
2
(3.12 g,
7.40 mmol) was added to the solution of the TFA-salt 3 (2.10 g, 7.40 mmol)
in CH2Cl2 (10 mL) and Hünigꢁs base (2.68 mL, 14.80 mmol). After stirring
at room temperature for 16 h the mixture was extracted with 0.5m HCl
(100 mL) and EtOAc (150 mL). The aqueous layer was extracted with
EtOAc (2 Â 100 mL) and the organic layers were washed with brine
(50 mL) and dried over MgSO4. Filtration and evaporation of the solvent at
reduced pressure yielded the crude product which was purified by flash
chromatography on silica gel (gradient of CH2Cl2/MeOH from 100:0 to
100:5) to yield the dipeptide 4 (2.69 g, 6.59 mmol, 89%) as a slowly
solidifying oil. (1H and 13C NMR- spectra show a double set of peaks (ꢀ2:1)
due to the s-cis and s-trans conformers around the tertiary amide and
carbamate.) 1H NMR (400 MHz, CDCl3, 258C): d ˆ 4.59 ± 4.14 (m, 4H;
Ha, Hg), 3.67/3.66 (2s (2:1), 3H; CH3), 3.79 ± 3.39 (m, 4H; Hd), 2.28 ± 1.93
(m, 4H; Hb), 1.37/1.34 (2s (2:1), 9H; tBu); 13C NMR (100.5 MHz, CDCl3,
258C): d ˆ 171.8, 171.6, 170.8, 170.7, 153.9, 153.1, 80.5, 80.4, 59.6, 59.3, 59.2,
58.7, 57.5, 57.5, 56.3, 56.0, 52.3, 58.3, 51.5, 51.4, 51.3, 51.2, 35.4, 34.7, 34.0, 33.9,
28.1, 28.0; IR (KBr): nÄ ˆ 2977, 2105, 1748, 1698, 1665; ESI-MS: m/z: calcd
Experimental Section
General methods: Materials and reagents were of the highest commercially
available grade and used without further purification. Reactions were
monitored by thin-layer chromatography using Merck silica gel 60 F254
covered aluminum plates. Compounds were visualized by UV, ceric
ammonium molybdate (CAM) and ninhydrin. Flash chromatography was
performed using Merck silica gel 60, particle size 40 ± 63 mm. Gel filtrations
were performed on Sephadex LH20 resin purchased from Sigma. 1H and
13C NMR spectra were recorded on a Varian Gemini300, a Bruker
DPX400 or a Bruker DPX500 spectrometer. Chemical shifts are reported
in ppm using CDCl3 as a reference. Infrared spectra were obtained on a
Perkin ± Elmer 1600 series; peaks are reported in cm 1. Finnigan MAT
LCQ and TSQ 700 instruments were used for electrospray ionization (ESI)
mass spectrometry. HPLC analysis were carried out on a Nucleosil 100 5
(250 Â 4.6 mm) from Macherey Nagel. The solid-phase binding assays were
carried out with CHCl3 freshly filtered through aluminum oxide.
‡
for C16H24N8O5Na: 431 [M‡Na] ; found: 431; elemental analysis calcd (%)
for C16H24N8O5 (408.4): C 47.05, H 5.92, N 27.44, O 19.59; found: C 47.18, H
5.90, N 27.07, O 19.33.
Diketopiperazine (5): Dipeptide 4 (2.15 g, 5.26 mmol) was dissolved in
TFA/CH2Cl2 1:3 (20 mL) and allowed to stir at room temperature for 1.5 h.
After removal of all volatiles at reduced pressure the oily residue was
triturated with Et2O (20 mL) to yield a white solid which was isolated by
decantation followed by removal of all residual volatiles in vacuo. The
residue was dissolved in THF (50 mL), Hünigꢁs base (1.83 mL, 10.53 mmol)
was added and the mixture was stirred at room temperature for 16 h. After
removal of all volatiles at reduced pressure, flash chromatography on silica
gel (gradient of CH2Cl2/MeOH from 100:0 to 100:6) afforded the
diketopiperazine 5 (1.15 g, 4.16 mmol, 79%) as a white solid. 1H NMR
(400 MHz, CDCl3, 258C): d ˆ 4.45 (dd, J ˆ 10.2 Hz, 6.7 Hz, 2H; Ha), 4.34
(ydt, J ˆ 5.1 Hz, 1.4 Hz, 2H; Hg), 3.70 (dd, J ˆ 12.9 Hz, 5.1 Hz, 2H; Hd),
3.61 (dd, J ˆ 12.9 Hz, 1.3 Hz, 2H; Hd'), 2.43 (ddd, J ˆ 15.4 Hz, 6.6 Hz,
1.5 Hz, 2H; Hb), 2.29 (ddd, J ˆ 15.4 Hz, 10.4 Hz, 5.1 Hz, 2H; Hb');
13C NMR (100.5 MHz, CDCl3, 258C): d ˆ 166.5, 58.8, 58.7, 50.7, 33.9; IR
(KBr): nÄ ˆ 2948, 2124, 1661, 1437, 1274; elemental analysis calcd (%) for
C10H12N8O2 (276.3): C 43.48, H 4.38, O 11.58; found: C 43.26, H 4.50, O
11.64.
N-Boc-trans-4-Azido-l-proline pentafluorophenyl ester (2): N-Boc-trans-
4-Azido-l-proline methyl ester (1)[7] (5.56 g, 20.57 mmol) was dissolved in
THF/MeOH 1:1 (50 mL). After the addition of NaOH (1.23 g, 30.86 mmol)
dissolved in water (5 mL) the mixture was stirred for 1.5 h at room
temperature and then carefully acidified with 1m HCl to pH 4. EtOAc
(200 mL) and water (100 mL) were added and the mixture was extracted
with additional 1m HCl (50 mL). The aqueous layer was extracted with
EtOAc (100 mL), the combined organic layers were washed with brine and
dried over MgSO4. Filtration and evaporation of the solvent at reduced
pressure yielded a white powder which was suspended in CH2Cl2 (10 mL).
Addition of pentafluorophenol (3.98 g, 21.60 mmol) and EDC (5.91 g,
30.85 mmol) yielded a solution which was stirred for 1 h at room temper-
ature and then extracted with water (100 mL) and EtOAc (200 mL). The
aqueous layer was extracted again with EtOAc (100 mL) and the organic
layers were washed with brine and dried over MgSO4. Filtration and
removal of all volatiles at reduced pressure yielded the pentafluorophenyl
ester 2 (7.38 g, 17.49 mmol, 85%) as a white solid. (1H and 13C NMR spectra
show a double set of peaks (ꢀ2:1) due to the s-cis and s-trans conformers
around the tertiary carbamate.) 1H NMR (300 MHz, CDCl3, 258C): d ˆ
4.71 (m, 1H; Ha), 4.26 (m, 1H; Hg), 3.77 ± 3.55 (m, 2H; Hd), 2.61 ± 2.35 (m,
2H; Hb), 1.49/1.46 (2s (2:1), 9H; tBu); 13C NMR (75.4 MHz, CDCl3,
258C): d ˆ 168.5, 168.2, 153.8, 153.2, 142.3, 139.7, 81.8, 81.4, 60.4, 59.3, 58.6,
57.3, 57.2, 51.4, 51.3, 36.7, 35.4, 28.2, 28.0; IR (KBr): nÄ ˆ 3388, 2991, 2100,
1821, 1715, 1519, 1169; ESI-MS: m/z: calcd for C16H25F5N4O4Na: 445
Diketopiperazine (6): Palladium on carbon (10%, 50 mg) was added to the
solution of the diazide
5 (0.99 g, 3.59 mmol) and Boc2O (2.35 g,
10.76 mmol) in MeOH (20 mL). The black suspension was evacuated,
flushed with hydrogen and allowed to stir for 2 h at room temperature.
After filtration over celite and removal of the solvent at reduced pressure,
the residue was washed with Et2O (50 mL) and purified by flash
chromatography over silica gel (gradient of CH2Cl2/MeOH from 100:0 to
100:5) to afford the diketopiperazine 6 (1.38 g, 3.26 mmol, 91%) as a white
solid. 1H NMR (300 MHz, CDCl3, 258C): d ˆ 5.95 (s, 2H; NH), 4.44 (yt,
J ˆ 8.5 Hz, 2H; Ha), 4.22 (m, 2H; Hg), 3.74 (dd, J ˆ 12.8, 6.0 Hz, 2H; Hd),
3.46 (yd, J ˆ 12.8 Hz, 2H; Hd'), 2.30 (m, J ˆ 8.5 Hz, 4H; Hb, Hb'), 1.46 (s,
18H; tBu); 13C NMR (75.4 MHz, CDCl3, 258C): d ˆ 166.5, 155.5, 58.6, 51.6,
33.8, 28.1; IR (KBr): nÄ ˆ 3348, 2978, 1706, 1676, 1650, 1528, 1164; ESI-MS:
‡
m/z: calcd for C20H32N4O5Na: 447 [M‡Na] ; found: 447; elemental analysis
calcd (%) for C20H32N4O6 ´ H2O (442.5): C 54.28, H 7.74, N 12.66; found: C
54.42, H 7.70, N 12.57.
N-Boc-l-Tyr(O-Disperse Red)-OPfp (8a): (The d-tyrosine Pfp-ester 8b
was prepared analogously.) N-Boc-l-Tyrosine methyl ester 7a (1.90 g,
6.44 mmol), Disperse Red (2.02 g, 6.44 mmol) and triphenylphosphine
(1.69 g, 6.44 mmol) were dissolved in toluene (130 mL). Diethyl azodicar-
boxylate (1.17 mL, 6.44 mmol) was added dropwise over 15 min and the
mixture was allowed to stir at room temperature for 16 h. After removal of
the solvent at reduced pressure flash chromatography over silica gel
(CH2Cl2 then CH2Cl2/acetone 10:1) afforded N-Boc-l-Tyr(O-Disperse
Red) methyl ester (2.75 g, 4.65 mmol, 72%) as a red solid. 1H NMR
‡
[M‡Na] ; found: 445; elemental analysis calcd (%) for C16H25F5N4O4
(422.3): C 45.51, H 3.58, N 13.27; found: C 45.34, H 3.65, N 12.94.
TFA ´ trans-4-Azido-l-proline methyl ester (3): The N-Boc protected
methyl ester 1 (2.00 g, 7.40 mmol) was dissolved in TFA/CH2Cl2 1:3
(20 mL) and allowed to stir at room temperature for 1.5 h. After removal of
all volatiles at reduced pressure the oily residue was triturated with Et2O
(20 mL) and isolated by decantation followed by removal of all residual
volatiles in vacuo to yield the TFA-salt 3 (2.10 g, 7.40 mmol, quant.) as a
Chem. Eur. J. 2001, 7, No. 15
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0715-3345 $ 17.50+.50/0
3345

H. Wennemers et al.
FULL PAPER
(300 MHz, CDCl3, 258C): d ˆ 8.27 (d, J ˆ 9.1 Hz, 2H; arom.), 7.88 (d, J ˆ
9.1 Hz, 4H; arom.), 7.03 (d, J ˆ 8.5 Hz, 2H; arom.), 6.82 (d, J ˆ 8.5 Hz, 2H;
arom.), 6.79 (d, J ˆ 9.2 Hz, 2H; arom.), 5.01 (brd, J ˆ 7.7 Hz, 1H; NH), 4.53
(m, 1H; Ha), 4.15 (t, J ˆ 5.6 Hz, 2H; CH2), 3.83 (t, J ˆ 5.6 Hz, 2H; CH2),
3.70 (s, 3H; CH3), 3.59 (q, J ˆ 7.0 Hz, 2H; CH2), 3.02 (m, 2H; CH2), 1.41 (s,
9H; tBu), 1.27 (t, J ˆ 7.0 Hz, 3H; CH3); 13C NMR (75.4 MHz, CDCl3,
258C): d ˆ 172.2, 157.4, 156.6, 154.9, 151.2, 147.2, 143.6, 130.3, 128.4, 126.1,
124.5, 122.5, 114.3, 111.3, 79.7, 65.2, 54.4, 52.0, 49.7, 46.0, 37.3, 28.2, 12.2;
6.6 Hz, 2H; Asn-Hb), 2.47 (dd, J ˆ 14.0 Hz, 9.6 Hz, 2H; Phe-Hb'), 2.37 (dd,
J ˆ 15.2 Hz, 6.5 Hz, 2H; Asn-Hb'), 2.10 (m, 2H; Pro-Hb), 1.85 (m, 2H;
Pro-Hb'), 1.68 (s, 6H; COCH3), 1.10 (t, J ˆ 7.0 Hz, 6H; CH2CH3); 13C NMR
(125.6 MHz, 5% CD3OD in CDCl3, 258C): d ˆ 172.4, 171.7, 170.8, 170.6,
169.7, 166.4, 157.2, 156.6, 151.2, 147.0, 143.8, 143.4, 136.0, 130.0, 129.0, 128.7,
128.4, 128.3, 127.6, 126.7, 126.0, 124.4, 122.3, 114.1, 111.2, 70.2, 65.0, 58.3,
54.9, 54.4, 50.8, 49.8, 49.2, 47.2, 45.8, 37.1, 37.0, 35.6, 32.4, 22.0, 11.8; HRMS
(ESI): m/z: calcd for
C
128H128N20O18
:
1117.4931 [M‡2H]2‡
; found:
‡
ESI-MS: m/z: calcd for C31H37N5O7Na: 614 [M‡Na] ; found 614.
1117.4927.
Receptor (10): 1H NMR (500 MHz, 5% CD3OD in CDCl3, 258C): d ˆ 8.27
(d, J ˆ 9.1 Hz, 4H; dye), 7.87 (d, J ˆ 9.1 Hz, 4H; dye), 7.85 (d, J ˆ 9.1 Hz,
4H; dye), 7.24 ± 7.04 (m, 44H; trityl, Phe, Tyr-2H), 6.75 (d, J ˆ 9.2 Hz, 4H;
dye), 6.71 (d, J ˆ 8.6 Hz, 4H; Tyr), 4.49 (dd, J ˆ 10.3 Hz, 4.0 Hz, 2H; Tyr-
Ha), 4.32 (dd, J ˆ 8.1 Hz, 5.4 Hz, 2H; Phe-Ha), 4.16 ± 4.07 (m, 6H; Pro-
Ha, Pro-Hg, Asn-Ha), 4.06 (t, J ˆ 5.7 Hz, 4H; OCH2CH2N), 3.77 (t, J ˆ
5.7 Hz, 4H; OCH2CH2N), 3.60 (dd, J ˆ 12.6 Hz, 6.4 Hz, 2H; Pro-Hd), 3.54
(q, J ˆ 7.0 Hz, 4H; CH2CH3), 3.23 (dd, J ˆ 14.3 Hz, 4.0 Hz, 2H; Tyr-Hb),
3.01 (brd, J ˆ 12.6 Hz, 2H; Pro-Hd'), 2.93 (dd, J ˆ 14.0 Hz, 5.4 Hz, 2H;
Phe-Hb), 2.81 (dd, J ˆ 14.1 Hz, 8.2 Hz, 2H; Phe-Hb'), 2.78 ± 2.69 (m, 6H;
Tyr-Hb', Asn-Hb, Asn-Hb'), 2.18 ± 2.11 (m, 4H; Pro-Hb, Pro-Hb'), 1.84 (s,
6H; COCH3), 1.22 (t, J ˆ 7.0 Hz, 6H; CH2CH3); 13C NMR (125.6 MHz, 5%
CD3OD in CDCl3, 258C): d ˆ 172.8, 172.1, 171.4, 170.8, 169.9, 166.2, 157.2,
156.8, 151.4, 147.3, 144.1, 143.6, 136.0, 130.2, 130.0, 129.2, 128.6, 128.5, 127.9,
127.1, 127.0, 126.3, 124.7, 114.3, 111.4, 70.1, 65.2, 58.6, 55.2, 54.7, 50.7, 50.7,
49.8, 47.6, 46.1, 37.2, 37.0, 35.6, 33.1, 22.3, 12.2; HRMS (ESI): m/z: calcd for
N-Boc-l-Tyr(O-Disperse Red)-OCH3 (2.20 g, 3.72 mmol) was converted
into the Pfp-ester 8a (2.63 g, 3.53 mmol, 95%) following the procedure
decribed for the formation of 2. 1H NMR (300 MHz, CDCl3, 258C): d ˆ
8.31 (d, J ˆ 9.2 Hz, 2H; arom.), 7.92 (d, J ˆ 9.2 Hz, 2H; arom.), 7.89 (d, J ˆ
9.1 Hz, 2H; arom.), 7.14 (d, J ˆ 8.7 Hz, 2H; arom.), 6.87 (d, J ˆ 8.6 Hz, 2H;
arom.), 6.81 (d, J ˆ 9.1 Hz, 2H; arom.), 4.95 (brd, J ˆ 8.8 Hz, 1H; NH), 4.83
(m, 1H; Ha), 4.18 (t, J ˆ 5.8 Hz, 2H; CH2), 3.85 (t, J ˆ 5.8 Hz, 2H; CH2),
3.61 (q, J ˆ 7.0 Hz, 2H; CH2), 3.20 (m, 2H; CH2), 1.43 (s, 9H; tBu), 1.29 (t,
J ˆ 7.0 Hz, 3H; CH3); 13C NMR (75.4 MHz, CDCl3, 258C): d ˆ 168.3, 157.9,
156.8, 155.0, 151.3, 147.4, 143.8, 130.5, 127.4, 126.3, 124.7, 122.6, 114.8, 111.4,
80.7, 65.4, 54.5, 49.9, 46.2, 37.0, 28.2, 12.3; ESI-MS: m/z: calcd for
‡
C36H35F5N5O7: 744 [M‡H] ; found: 744.
Synthesis of the receptors (9 ± 13)
Coupling of 7: The tert-butyl-protecting groups of bis-N-Boc-protected
diketopiperazine 6 (50 mg, 0.12 mmol) were removed with TFA/CH2Cl2 1:3
(4 mL) as described for the formation of the TFA-salt 3. The residue was
dissolved in a solution of Hünigꢁs base (90 mL, 0.47 mmol) in CH2Cl2
(2 mL), the pentafluorophenyl ester 8a or 8b (193 mg, 0.25 mmol) was
added and the mixture was allowed to stir at room temperature for 16 h.
After a filtration over silica gel (gradient of CH2Cl2/MeOH from 100:0 to
100:5) the diketopiperazine with the attached N-Boc protected tyrosine
was isolated. The red solid was dissolved in MeOH (1 mL) and 4m HCl in
dioxane (2 mL) and allowed to react for 0.5 h. The corresponding HCl salt
was isolated as described for 3.
C
128H128N20O18: 1117.4931 [M‡2H]2‡; found: 1117.4935.
Receptor (11): 1H NMR (500 MHz, 5% CD3OD in CDCl3, 258C): d ˆ 8.32
(d, J ˆ 9.1 Hz, 4H; dye), 7.91 (d, J ˆ 9.2 Hz, 4H; dye), 7.89 (d, J ˆ 9.3 Hz,
4H; dye), 7.28 ± 7.14 (m, 36H; trityl, Phe-6H), 7.04 (d, J ˆ 6.9 Hz, 4H; Phe),
6.81 (d, J ˆ 9.3 Hz, 4H; dye), 6.73 (d, J ˆ 8.8 Hz, 4H; Tyr), 6.70 (d, J ˆ
8.9 Hz, 4H; Tyr), 4.37 (m, 4H; Asn-Ha, Pro-Ha), 4.21 (m, 4H; Tyr-Ha,
Pro-Hg), 4.12 (t, J ˆ 5.8 Hz, 4H; OCH2CH2N), 3.98 (yt, J ˆ 7.1 Hz, 2H;
Phe-Ha), 3.83 (t, J ˆ 5.7 Hz, 4H; OCH2CH2N), 3.60 (q, J ˆ 7.0 Hz, 4H;
CH2CH3), 3.45 (dd, J ˆ 12.4 Hz, 4.5 Hz, 2H; Pro-Hd), 3.18 (m, 2H; Pro-
Hd'), 2.91 (dd, J ˆ 13.7 Hz, 7.1 Hz, 2H; Phe-Hb), 2.84 (m, 4H; Tyr-Hb, Asn-
Hb), 2.77 (dd, J ˆ 13.7 Hz, 7.1 Hz, 2H; Phe-Hb'), 2.71 (m, 2H; Tyr-Hb'),
2.61 (dd, J ˆ 15.4 Hz, 5.3 Hz, 2H; Asn-Hb'), 2.35 (m, 2H; Pro-Hb), 2.06 (m,
2H; Pro-Hb'), 1.86 (s, 6H; COCH3), 1.27 (t, J ˆ 7.0 Hz, 6H; CH2CH3);
13C NMR (125.6 MHz, 5% CD3OD in CDCl3, 258C): d ˆ 171.8, 171.5,
170.9, 170.9, 170.3, 166.4, 157.2, 156.7, 151.2, 147.3, 144.2, 143.6, 136.3, 130.3,
129.1, 128.7, 128.5, 127.7, 127.0, 126.9, 126.2, 124.6, 122.5, 114.3, 111.3, 70.5,
65.2, 58.8, 56.4, 54.0, 50.7, 50.6, 49.8, 48.1, 46.1, 37.1, 36.6, 35.1, 33.1, 22.7,
Coupling of the N-a-Fmoc protected amino acids: The desired N-a-Fmoc
protected amino acid (0.48 mmol) was added along with EDC (92 mg,
0.48 mmol) to the solution of the diamino receptor precursor in CH2Cl2
(max. 1 mL) and the mixture was stirred at room temperature for 30 min.
The mixture was extracted with EtOAc (15 mL) and 0.5m HCl (5 mL), the
aqueous layer was extracted with EtOAc (20 mL) and the organic layers
were washed with brine and dried over Na2SO4. Filtration, removal of the
solvent at reduced pressure and a filtration over silica gel (gradient of
CH2Cl2/MeOH) afforded the N-Fmoc-protected receptor precursor which
was used without further purification.
12.2; HRMS (ESI): m/z: calcd for C128H128N20O18: 1117.4931 [M‡2H]2‡
;
found: 1117.4945.
Fmoc deprotection: The N-Fmoc-protected receptor precursor was dis-
solved in CH2Cl2 (5 mL) followed by addition of tris(2-aminoethyl)amine
(0.45 mL, 3.00 mmol) which caused the formation of a precipitate after 2 ±
5 min. The suspension was stirred at room temperature for 0.5 h and then
first extracted with brine (3 Â 10 mL) followed by phosphate buffer pH 5.5
(3 Â 10 mL). The aqueous layers were washed once with CH2Cl2 (10 mL)
the organic layers were dried over MgSO4 to yield the corresponding amine
after removal of all volatiles at reduced pressure.
Receptor (12): 1H NMR (500 MHz, 5% CD3OD in CDCl3, 258C): d ˆ 8.30
(d, J ˆ 9.1 Hz, 4H; dye), 7.87 (d, J ˆ 9.1 Hz, 4H; dye), 7.83 (d, J ˆ 9.3 Hz,
4H; dye), 7.32 ± 7.09 (m, 44H; trityl, Phe, Tyr-4H), 6.76 (d, J ˆ 8.8 Hz, 4H;
Tyr), 6.72 (d, J ˆ 9.3 Hz, 4H; dye), 4.63 (dd, J ˆ 11.8 Hz, 3.4 Hz, 2H; Tyr-
Ha), 4.58 (m, 2H; Pro-Hg), 4.52 (dd, J ˆ 10.0 Hz, 7.4 Hz, 2H; Pro-Ha), 4.12
(dd, J ˆ 10.6 Hz, 3.9 Hz, 2H; Phe-Ha), 4.04 (t, J ˆ 5.7 Hz, 4H;
OCH2CH2N), 3.92 (m, 2H; Gln-Ha), 3.73 (t, J ˆ 5.7 Hz, 4H; OCH2CH2N),
3.61 (dd, J ˆ 12.4 Hz, 5.7 Hz, 2H; Pro-Hd), 3.53 (q, J ˆ 7.1 Hz, 4 H ;
CH2CH3), 3.48 (brd, J ˆ 12.4 Hz, 2H; Pro-Hd'), 3.41 (dd, J ˆ 14.3 Hz,
3.3 Hz, 2H; Tyr-Hb), 3.07 (dd, J ˆ 14.3 Hz, 3.8 Hz, 2H; Phe-Hb), 2.79 (dd,
J ˆ 14.3 Hz, 11.8 Hz, 2H; Tyr-Hb'), 2.52 (dd, J ˆ 14.3 Hz, 10.7 Hz, 2H; Phe-
Hb'), 2.30 (m, 2H; Pro-Hb), 2.15 (m, 2H; Pro-Hb'), 1.95 (m, 2H; Gln-Hg),
1.75 (m, 4H; Gln-Hg', Gln-Hb), 1.59 (m, 2H; Gln-Hb'), 1.62 (s, 6H;
COCH3), 1.23 (t, J ˆ 7.1 Hz, 6H; CH2CH3); 13C NMR (125.6 MHz, 5%
CD3OD in CDCl3, 258C): d ˆ 173.8, 173.5, 172.6, 171.0, 170.4, 167.5, 157.1,
156.7, 151.2, 147.4, 144.2, 143.7, 136.1, 131.1, 130.4, 128.9, 128.7, 128.1, 127.5,
127.2, 126.3, 124.7, 122.6, 114.3, 111.4, 70.9, 65.5, 59.0, 56.4, 55.3, 54.6, 50.5,
49.8, 48.1, 46.1, 36.7, 36.1, 33.9, 32.9, 25.3, 22.9, 12.4; HRMS (ESI): m/z:
calcd for C130H132N20O18 1131.5087: [M‡2H]2‡; found: 1131.5085.
The coupling and Fmoc deprotection cycle was repeated until the desired
tripeptidic arms were assembled. After the final Fmoc deprotection, the
diamine was dissolved in CH2Cl2 (1 mL) followed by the addition of
triethylamine (0.067 mL, 0.48 mmol) and acetic anhydride (45 mL,
0.48 mmol). After stirring the mixture for 30 min, the acetylated receptor
was purified by flash chromatography (gradient of CH2Cl2/MeOH 100:0 to
100:10) and gel filtration (LH 20, CH2Cl2/MeOH 95:5). Receptors 9 ± 13
were isolated in amounts of 25 ± 40 mg (10 ± 15% from 6).
Receptor (9): 1H NMR (500 MHz, 5% CD3OD in CDCl3, 258C): d ˆ 8.17
(d, J ˆ 9.1 Hz, 4H; dye), 7.76 (d, J ˆ 9.1 Hz, 4H; dye), 7.73 (d, J ˆ 9.3 Hz,
4H; dye), 7.12 ± 6.94 (m, 40H; trityl, Phe), 6.89 (d, J ˆ 8.7 Hz, 4H; Tyr), 6.62
(d, J ˆ 9.3 Hz, 4H; dye), 6.60 (d, J ˆ 8.7 Hz, 4H; Tyr), 4.31 (dd, J ˆ 8.3 Hz,
5.2 Hz, 2H; Tyr-Ha), 4.12 (dd, J ˆ 9.6 Hz, 5.1 Hz, 2H; Phe-Ha), 4.17 (yt,
J ˆ 6.6 Hz, 2H; Asn-Ha), 4.07 (m, 2H; Pro-Hg), 3.91 (yt, J ˆ 8.1 Hz, 2H;
Pro-Ha), 3.85 (t, J ˆ 5.7 Hz, 4H; OCH2CH2N), 3.57 (t, J ˆ 5.7 Hz, 4H;
OCH2CH2N), 3.50 (dd, J ˆ 12.3 Hz, 2.5 Hz, 2H; Pro-Hd), 3.40 (q, J ˆ
7.1 Hz, 4H; CH2CH3), 3.02 (dd, J ˆ 12.3 Hz, 4.0 Hz, 2H; Pro-Hd'), 2.91
(dd, J ˆ 14.3 Hz, 5.1 Hz, 2H; Tyr-Hb), 2.75 (dd, J ˆ 14.3 Hz, 8.3 Hz, 2H;
Tyr-Hb'), 2.70 (dd, J ˆ 14.0 Hz, 5.0 Hz, 2H; Phe-Hb), 2.63 (dd, J ˆ 15.2 Hz,
Receptor (13): 1H NMR (500 MHz, 5% CD3OD in CDCl3, 258C): d ˆ 8.32
(d, J ˆ 9.1 Hz, 4H; dye), 7.91 (d, J ˆ 9.2 Hz, 4H; dye), 7.89 (d, J ˆ 9.2 Hz,
4H; dye), 7.26 ± 7.16 (m, 36H; trityl, Phe-6H), 6.97 (d, J ˆ 7.0 Hz, 4H; Phe),
6.82 (d, J ˆ 8.7 Hz, 4H; Tyr), 6.80 (d, J ˆ 9.2 Hz, 4H; dye), 6.71 (d, J ˆ
8.7 Hz, 4H; Tyr), 4.42 (dd, J ˆ 7.7 Hz, 5.3 Hz, 2H, Tyr-Ha), 4.38 (dd, J ˆ
10.5 Hz, 7.6 Hz, 2H, Pro-Ha), 4.28 (yt, J ˆ 5.7 Hz, 2H; Pro-Hg), 4.13 (yt,
J ˆ 7.1 Hz, 2H; Gln-Ha), 4.09 (t, J ˆ 5.8 Hz, 4H; OCH2CH2N), 3.87 (yt,
J ˆ 8.4 Hz, 2H; Phe-Ha), 3.81 (t, J ˆ 5.8 Hz, 4H; OCH2CH2N), 3.65 (dd,
3346
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0715-3346 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 15

Diketopiperazine Receptors
3342±3347
J ˆ 12.9, 5.7 Hz, 2H; Pro-Hd), 3.58 (m, J ˆ 7.1 Hz, 6H; CH2CH3, Pro-Hd'),
2.94 (dd, J ˆ 14.0, 7.7 Hz, 2H, Tyr-Hb), 2.82 ± 2.77 (m, 4H; Phe-Hb, Tyr-
Hb'), 2.72 (dd, J ˆ 13.6, 8.4 Hz, 2H; Phe-Hb'), 2.41 ± 2.35 (m, 4H; Gln-Hg,
Pro-Hb), 2.31 (m, 2H; Gln-Hg'), 2.17 (m, 2H; Pro-Hb'), 1.88 (s, 6H;
COCH3), 1.76 (m, 4H, Gln-Hb, Gln-Hb'), 1.26 (t, J ˆ 7.1 Hz, 6H; CH2CH3);
13C NMR (125.6 MHz, 5% CD3OD in CDCl3, 258C): d ˆ 173.1, 172.5,
General procedure for the determination of binding constants on the solid
support by UV: An accurately measured amount (ca. 5 mg) of resin-bound
peptide was placed in a 1 mL UV cuvette. The UV cuvette was then
charged with a solution of the receptor in CHCl3, sealed with a teflon
stopper and slightly agitated for 72 h. After this period of time the UV
absorbance did not change anymore. The beads were allowed to float to the
top of the CHCl3 solution before determining the receptor concentration at
equilibrium by UV. For the calculation of the binding constant it was
assumed that all peptides on the resin are able to participate in the binding
process.
ˆ
171.8, 171.3 (2 Â C O), 166.3, 157.4, 156.8, 151.3, 147.3, 144.3, 143.7, 136.6,
130.4, 129.3, 129.1, 128.7, 128.6, 127.8, 127.0, 126.3, 124.7, 122.6, 114.4, 111.4,
70.5, 65.3, 59.0, 56.6, 54.1, 52.4, 49.6, 49.1, 48.2, 46.1, 36.1, 35.2, 33.6, 32.1,
27.2, 22.5, 12.2; HRMS (ESI): m/z: calcd for C130H132N20O18: 1131.5087
[M‡2H]2‡; found 1131.5068.
Receptorfragment (14): 1H NMR (500 MHz, 5% CD3OD in CDCl3, 258C):
d ˆ 8.31 (d, J ˆ 9.1 Hz, 4H; dye), 7.91 (d, J ˆ 9.1 Hz, 4H; dye), 7.89 (d, J ˆ
9.3 Hz, 4H; dye), 7.26 ± 7.14 (m, 30H; trityl), 7.01 (d, J ˆ 8.7 Hz, 4H; Tyr),
6.80 (d, J ˆ 9.3 Hz, 4H; dye), 6.78 (d, J ˆ 8.7 Hz, 4H; Tyr), 4.47 (yt, J ˆ
6.4 Hz, 2H; Tyr-Ha), 4.44 (yt, J ˆ 5.6 Hz, 2H; Asn-Ha), 4.15 (m, 2H; Pro-
Hg), 4.13 (t, J ˆ 5.8 Hz, 4H; OCH2CH2N), 3.92 (yt, J ˆ 8.3 Hz, 2H; Pro-
Ha), 3.82 (t, J ˆ 5.8 Hz, 4H; OCH2CH2N), 3.74 (dd, J ˆ 12.4 Hz, 6.8 Hz,
2H; Pro-Hd), 3.60 (q, J ˆ 7.1 Hz, 4H; CH2CH3), 3.07 (dd, J ˆ 14.1 Hz,
6.7 Hz, 2H; Tyr-Hb), 3.03 (dd, J ˆ 12.4 Hz, 4.2 Hz, 2H; Pro-Hd'), 2.95 (m,
4H; Tyr-Hb', Asn-Hb), 2.55 (dd, J ˆ 15.8 Hz, 5.5 Hz, 2H; Asn-Hb'), 2.48
(m, 2H; Pro-Hb), 2.45 (m, 2H, Pro-Hb'), 1.83 (s, 6H; COCH3), 1.27 (t, J ˆ
7.1 Hz, 6H; CH2CH3); 13C NMR (125.6 MHz, 5% CD3OD in CDCl3,
258C): d ˆ 171.2, 171.0, 170.6, 169.9, 166.0, 157.5, 156.8, 151.3, 147.3, 144.0,
143.7, 130.5, 128.9, 128.6, 128.0, 127.1, 126.3, 124.7, 122.6, 114.5, 111.4, 70.6,
65.4, 58.3, 53.9, 50.4, 50.4, 49.8, 47.3, 46.1, 37.9, 35.6, 32.7, 22.7, 12.2; HRMS
Acknowledgements
This work was supported by the BACHEM company. We thank Dr. Mark
Brönstrup from Aventis for recording the HRMS. H.W. is grateful to the
Fonds der Chemischen Industrie for a Liebig Fellowship and an assistant
professorship sponsored by the BACHEM company.
[1] For reviews see: a) W. C. Still, Acc. Chem. Res. 1996, 29, 155 ± 163;
b) C. Gennari, H. P. Nestler, U. Piarulli, B. Salom, Liebigs Ann./
Recueil 1997, 637 ± 647; c) Y. R. de Miguel, J. K. M. Sanders, Curr.
Opin. Chem. Biol. 1998, 2, 417 ± 421; d) H. P. Nestler, R. Liu, Comb.
Chem. High Throughput Screening 1998, 1, 113 ± 126.
[2] a) A. Borchardt, W. C. Still, J. Am. Chem. Soc. 1994, 116, 373 ± 374;
b) H. Wennemers, S. S. Yoon, W. C. Still, J. Org. Chem. 1995, 60,
1108 ± 1109; c) S. S. Yoon, W. C. Still, Tetrahedron 1995, 51, 567 ± 578;
d) M. Maletic, H. Wennemers, D. Q. McDonald, R. Breslow, W. C.
Still, Angew. Chem. 1996, 108, 1594 ± 1596; Angew. Chem. Int. Ed.
1996, 35, 1490 ± 1492.
[3] a) R. C. Boyce, G. Li, H. P. Nestler, T. Suenaga, W. C. Still, J. Am.
Chem. Soc. 1994, 116, 7955 ± 7956; b) C. Gennari, H. P. Nestler, B.
Salom, W. C. Still, Angew. Chem. 1995, 107, 1894 ± 1896; Angew.
Chem. Int. Ed. 1995, 34, 1765 ± 1768; c) M. Davies, M. Bonnat, F.
Guillier, J. D. Kilburn, M. Bradley, J. Org. Chem. 1998, 63, 8696 ± 8703;
d) H. Hioki, T. Yamada, C. Fujioka, M. Kodama, Tetrahedron Lett.
1999, 40, 6821 ± 6825; e) T. Fessmann, J. D. Kilburn, Angew. Chem.
1999, 111, 2170 ± 2174; Angew. Chem. Int. Ed. 1999, 38, 1993 ± 1996;
‡
(ESI): m/z: calcd for C110H110N18O16: 1939.8420 [M‡H] ; found: 1939.8405.
Receptorfragment (15): 1H NMR (500 MHz, CDCl3, 258C): d ˆ 8.32 (d,
J ˆ 9.1 Hz, 2H; dye), 7.92 (d, J ˆ 9.1 Hz, 2H; dye), 7.90 (d, J ˆ 9.3 Hz, 2H;
dye), 7.33 ± 7.12 (m, 22H; trityl, Phe, Asn-NH, Phe-NH), 7.08 (d, J ˆ 8.6 Hz,
2H; Tyr), 6.87 (d, J ˆ 8.6 Hz, 1H, Pro-NH), 6.80 (m, 1H, Tyr-NH), 6.78 (d,
J ˆ 9.3 Hz, 2H; dye), 6.75 (d, J ˆ 8.6 Hz, 2H; Tyr), 6.11 (m, 1H, Asn-NH),
4.63 (dd, J ˆ 14.6 Hz, 6.3 Hz, 1H; Tyr-Ha), 4.47 (m, 1H; Phe-Ha), 4.40 (dd,
J ˆ 11.6 Hz, 5.6 Hz, 1H; Asn-Ha), 4.30 (yt, J ˆ 4.9 Hz, 1H; Pro-Hg), 4.15
(m, 2H; Pro-Ha, Pro'-Hg), 4.08 (t, J ˆ 5.8 Hz, 2H; OCH2CH2N), 3.78 (t,
J ˆ 5.7 Hz, 2H; OCH2CH2N), 3.71 (yt, J ˆ 8.4 Hz, 1H; Pro'-Ha), 3.63 (dd,
J ˆ 13.1, 5.1 Hz, 1H; Pro-Hd), 3.58 (q, J ˆ 7.0 Hz, 2H; CH2CH3), 3.51 (brd,
J ˆ 13.0 Hz, 1H; Pro-Hd'), 3.46 (m, 1H; Pro'-Hd), 3.16 (dd, J ˆ 16.5,
11.6 Hz, 1H; Asn-Hb), 3.07 (m, 3H; Phe-Hb, Tyr-Hb, Tyr-Hb'), 2.93 (dd,
J ˆ 14.2 Hz, 7.7 Hz, 1H; Phe-Hb'), 2.88 (dd, J ˆ 13.9 Hz, 3.0 Hz, 1H; Pro'-
Hd'), 2.64 (dd, J ˆ 16.5, 5.5 Hz, 1H; Asn-Hb'), 2.32 (brdd, J ˆ 13.8, 6.7 Hz,
1H; Pro-Hb), 2.22 (ddd, J ˆ 13.8, 10.6, 4.9 Hz, 1H; Pro-Hb'), 2.04 (m, 2H,
Pro'-Hb, Pro'-Hb'), 1.69 (s, 6H; COCH3), 1.26 (t, J ˆ 7.0 Hz, 6H; CH2CH3);
13C NMR (125.6 MHz, CDCl3, 258C): d ˆ 171.5, 171.2, 171.0, 170.7, 169.7,
166.0, 165.5, 157.4, 156.8, 151.3, 147.3, 144.1, 143.7, 135.8, 130.7, 129.3, 128.9,
128.6, 128.0, 127.4, 127.1, 126.3, 124.7, 122.6, 114.4, 111.4, 70.7, 65.2, 58.7, 58.5,
58.3, 54.9, 54.5, 50.6, 50.3, 50.0, 49.8, 47.8, 46.1, 37.3, 37.1, 35.9, 34.0, 33.4, 22.7,
12.3; IR (KBr): nÄ ˆ 3311, 2925, 2105, 1664, 1599, 1133; HRMS (ESI): m/z:
Â
f) H. De Muynck, A. Madder, N. Farcy, P. J. De Clercq, M. N. Perez-
Payan, L. M. Öhberg, A. P. Davis, Angew. Chem. 2000, 112, 149 ± 152;
Angew. Chem. Int. Ed. 2000, 39, 145 ± 148.
Â
[4] a) Y. Cheng, T. Suenaga, W. C. Still, J. Am. Chem. Soc. 1996, 118,
1813 ± 1814; b) D. W. P. M. Löwik, M. D. Weingarten, M. Broekema,
A. J. Brouwet, W. C. Still, R. M. J. Liskamp, Angew. Chem. 1998, 110,
1947 ± 1950; Angew. Chem. Int. Ed. 1998, 37, 1846 ± 1850.
Â
Â
Ã
[5] a) A. Furka, F. Sebestyen, M. Asgedom, G. Dibo, Int. J. Pept. Protein
Res. 1991, 37, 487 ± 493; b) K. S. Lam, S. E. Salmon, E. M. Hersh, V. J.
Hruby, W. M. Kazmierski, R. J. Knapp, Nature 1991, 354, 82 ± 84.
[6] H. Wennemers, W. C. Still, Tetrahedron Lett. 1994, 35, 6413 ± 6417.
[7] 1: CAS [121147 ± 97 ± 5], a) B. P. Gangamani, V. A. Kumar, K. N.
Ganesh, Tetrahedron 1996, 52, 15017 ± 15030; b) M. L. Petersen, R.
Vince, J. Med. Chem. 1991, 34, 2787 ± 2797; c) K. Eva Ng, L. E. Orgel,
J. Med. Chem. 1989, 32, 1754 ± 1757.
‡
calcd for C69H70N14O10: 1255.5472 [M‡H] ; found 1255.5474.
Ac-l-Phe-l-Asn(Trt)-d-Tyr(dye)-OCH3 (16): 1H NMR (500 MHz, CDCl3,
258C): d ˆ 8.32 (d, J ˆ 9.1 Hz, 2H; dye), 7.91 (d, J ˆ 9.1 Hz, 2H; dye), 7.90
(d, J ˆ 9.1 Hz, 2H; dye), 7.53 (d, J ˆ 7.7 Hz, 1H, Asn-NH), 7.27 ± 7.10 (m,
21H; trityl, Phe, Tyr-NH), 7.03 (d, J ˆ 8.7 Hz, 2H; Tyr), 7.01 (s, 1H, Asn-
NHg), 6.79 (d, J ˆ 9.3 Hz, 2H; dye), 6.77 (d, J ˆ 8.7 Hz, 2H; Tyr), 5.90 (d,
J ˆ 6.8 Hz, 1H, Phe-NH), 4.66 (ddd, J ˆ 7.6, 6.2, 3.8 Hz, 1H; Asn-Ha), 4.62
(brddd, J ˆ 7.9, 5.7, 2.0 Hz, 1H; Tyr-Ha), 4.54 (ddd, J ˆ 6.8, 5.5 Hz, 2.8 Hz,
1H; Phe-Ha), 4.11 (t, J ˆ 5.9 Hz, 2H; OCH2CH2N), 3.81 (t, J ˆ 5.9 Hz, 2H;
OCH2CH2N), 3.62 (s, 3H, OCH3), 3.59 (q, J ˆ 7.1 Hz, 2H; CH2CH3), 3.07
(dd, J ˆ 10.1, 5.5 Hz, 1H; Phe-Hb), 3.05 (dd, J ˆ 10.1 Hz, 5.6 Hz, 1H; Tyr-
Hb), 2.92 (dd, J ˆ 15.3, 3.8 Hz, 1H; Asn-Hb), 2.88 (m, 1H, Phe-Hb'), 2.86
(dd, J ˆ 10.1 Hz, 2.0 Hz, 1H; Tyr-Hb'), 2.46 (dd, J ˆ 15.3 Hz, 6.3 Hz, 1H;
Asn-Hb'), 1.80 (s, 6H; COCH3), 1.27 (t, J ˆ 7.1 Hz, 6H; CH2CH3); 13C NMR
(125.6 MHz, CDCl3, 258C): d ˆ 171.3, 171.3, 170.7, 170.2, 170.0, 157.4, 156.8,
151.3, 147.3, 144.2, 143.7, 136.1, 130.4, 129.0, 128.8, 128.7, 128.6, 127.9, 127.1,
127.0, 126.3, 124.7, 122.6, 114.4, 111.4, 70.1, 65.1, 54.8, 54.2, 52.2, 49.8, 49.7,
46.1, 37.5, 36.9, 22.9, 12.3; ESI-MS: m/z: calcd for C60H60N8O9Na: 1060
[8] a) M. Beyermann, M. Bienert, H. Niedrich, L. A. Carpino, D. Sadat-
Aalee, J. Org. Chem. 1990, 55, 721 ± 728; b) L. A. Carpino, D. Sadat-
Aalaee, M. Beyermann, J. Org. Chem. 1990, 55, 1673 ± 1675.
[9] a) M. H. J. Ohlmeyer, R. N. Swanson, L. W. Dillard, J. C. Reader, G.
Asouline, R. Kobayashi, M. H. Wigler, W. C. Still, Proc. Natl. Acad.
Sci. USA 1993, 90, 10922 ± 10926; b) H. P. Nestler, P. Bartlett, W. C.
Still, J. Org. Chem. 1994, 59, 4723 ± 4724.
[10] AA1 ± AA3 ˆ Gly, d-Ala, l-Ala, d-Val, l-Val, d-Leu, l-Leu, d-Phe,
l-Phe, d-Pro, l-Pro, d-Ser, l-Ser, d-Thr, l-Thr, d-Asp, l-Asp, d-
Glu, l-Glu, d-Asn, l-Asn, d-Gln, l-Gln, d-His, l-His, d-Lys, l-Lys,
d-Arg, l-Arg.
[11] a) K. Burgess, A. I. Liaw, N. Y. Wang, J. Med. Chem. 1994, 37, 2985 ±
2987; b) P.-L. Zhao, R. Zambias, J. A. Bolognese, D. Boulton, K. T.
Chapman, Proc. Natl. Acad. Sci. USA 1995, 92, 10212 ± 10216.
‡
[M‡Na] ; found: 1060; elemental analysis calcd (%) for C60H60N8O9
(1037.2): C 69.48, H 5.83, N 10.80, O 13.88; found: C 69.15, H 6.07, N
10.63, O 14.11.
Received: January 30, 2001 [F3053]
Chem. Eur. J. 2001, 7, No. 15
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