Synthesis, conformational studies and binding properties of acyclic receptors for N-protected amino acids and dipeptides

Article abstract of DOI:10.1002/1099-0690(200112)2001:24<4625::AID-EJOC4625>3.0.CO;2-L

Solid-phase syntheses of novel receptors featuring a 2,6-diamidopyridine "head" group and bearing sulfonamidopeptide sidearms are described. NMR conformational studies show that the "two-armed" receptors collapse into an intramolecularly folded structure through formation of a hydrogen-bonding network. In order to accommodate the guests, receptors have to unfold, breaking the intramolecular hydrogen bonds. The absolute binding constants of receptors with N-protected amino acids and dipeptides are therefore relatively weak. However, one receptor shows a high selectivity for N-Cbz-D-Ala-D-AlaOH over its enantiomer N-Cbz-L-Ala-L-AlaOH.

Full text of DOI:10.1002/1099-0690(200112)2001:24<4625::AID-EJOC4625>3.0.CO;2-L

FULL PAPER
Synthesis, Conformational Studies and Binding Properties of Acyclic Receptors
for -Protected Amino Acids and Dipeptides
Enrique Botana,[a] Sandrine Ongeri,[a] Rosa Arienzo,[b] Mariangela Demarcus,[b]
Jeremy G. Frey,[b] Umberto Piarulli,[c] Donatella Potenza,[a] Jeremy D. Kilburn,*[b] and
Cesare Gennari*[a]
Keywords: Enantioselectivity / Host-guest systems / Molecular recognition / Peptides / Receptors
Solid-phase syntheses of novel receptors featuring a 2,6-di-
ceptors have to unfold, breaking the intramolecular hydro-
amidopyridine ‘‘head’’ group and bearing sulfonamidopep- gen bonds. The absolute binding constants of receptors with
tide sidearms are described. NMR conformational studies
show that the ‘‘two-armed’’ receptors collapse into an intram-
olecularly folded structure through formation of a hydrogen-
bonding network. In order to accommodate the guests, re-
N-protected amino acids and dipeptides are therefore rela-
tively weak. However, one receptor shows a high selectivity
for N-Cbz-D-Ala-D-AlaOH over its enantiomer N-Cbz-L-Ala-
L-AlaOH.
Introduction
receptor systems the head group plays only a limited role
in the binding of the guest, the group in Southampton has
recently developed tweezer receptors that make use of a di-
amidopyridine moiety as the head group. This can specific-
ally bind to carboxylic acid functionalities, and such
tweezers, with peptidic arms, have proved to be selective
receptors for peptides with a carboxylic acid terminus.[9]
The diamidopyridine moiety also proved to be a highly effi-
cient carboxylic acid binding site (CBS) when incorporated
into macrocyclic receptors.[1b,10]
Recently, an interest in synthetic receptors based on pep-
tidosulfonamide sidearms has also emerged.[3,11Ϫ13] Over
the past five years, the group in Milano has been studying
the conformational preferences of unnatural biopolymer
scaffolds containing β-sulfonamidopeptides[14] and vinylog-
ous sulfonamidopeptides.[15] Sulfonamidopeptides display
an aspect of the covalent framework that is essential for the
formation of folded structures by intramolecular hydrogen
bonding: The repeating backbone structure contains both
hydrogen-bond donors (strong: CONH; very strong:
SO2NH) and hydrogen-bond acceptors (strong: CϭO;
weak: SO2N).[14,15] In this paper we report the synthesis and
conformational and binding studies of a novel class of
‘‘two-armed’’ synthetic receptors for N-protected amino ac-
ids and dipeptides, each containing a 2,6-diamidopyridine
binding unit bearing pendant pseudo-peptide legs con-
sisting of an amino acid (-Phe) and chiral β-aminosulfon-
amides.[16] One of these receptors in particular (5, Figure 1)
displays a high binding selectivity for the dipeptide N-Cbz-
-Ala--AlaOH (Cbz ϭ benzyloxycarbonyl) over its enan-
tiomer N-Cbz--Ala--AlaOH.[16]
Noncovalent interactions play an important role in sta-
bilising the secondary structures of natural biopolymers
(peptides, polysaccharides and oligonucleotides) and pro-
moting the binding of natural or synthetic receptors to their
targets. Synthetic receptors for biologically relevant sub-
strates such as amino acids and peptides are of increasing
importance for possible applications in molecular recogni-
tion (enantioselective binding, separation of racemates) and
as potential biosensors, therapeutics and catalysts.
In the field of synthetic receptors, several macrocyclic
hosts have been prepared and have been shown to bind am-
ino acids or peptide fragments with excellent selectivity.[1Ϫ3]
During the last decade, a few studies focused on a different
class of receptors for peptides; these were called ‘‘tweezer
receptors’’ or ‘‘two-armed’’ receptors.[4] In the structure of
tweezer receptors, the ‘‘linker’’ (or ‘‘head group’’ or
‘‘hinge’’) is typically a conformationally restricted moiety
such as a 1,2-diamine,[5] a steroidal core,[6] a guanidini-
um,[7a] a thiourea,[7b] or a dibenzofuran.[8] The ‘‘linker’’ co-
valently binds and directs two functionalised substrate-
binding arms. The two sidearms may be macrocyclic oligo-
mers[5] or simple peptides.[6Ϫ8] Whereas in many tweezer
[a]
`
Dipartimento di Chimica Organica e Industriale, Universita di
Milano, Centro CNR per lo Studio delle Sostanze Organiche
Naturali, Via G. Venezian 21, 20133 Milan, Italy
Fax: (internat.) ϩ 39-02/5835-4072
E-mail: cesare.gennari@unimi.it
[b]
[c]
Department of Chemistry, University of Southampton,
Highfield, Southampton, SO17 1BJ, United Kingdom
Fax: (internat.) ϩ 44-1703/593-781
E-mail: jdkl@soton.ac.uk
`
Results and Discussion
Dipartimento di Scienze Chimiche Fis. e Mat., Universita
dell’Insubria,
via Valleggio 11, 22100 Como, Italy
The synthesis of compounds 1؊3 was accomplished on
solid phase, starting from N-Fmoc-Gly Wang resin 6 (load-
Supporting information for this article is available on the
WWW under http://www.eurjoc.com or from the author.
Eur. J. Org. Chem. 2001, 4625Ϫ4634
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/1224Ϫ4625 $ 17.50ϩ.50/0
4625

J. D. Kilburn, C. Gennari et al.
FULL PAPER
Figure 1. Labelling of hydrogen atoms in receptor 5
ing 0.88 mmol/g) (Scheme 1) [Fmoc ϭ N-(9-fluorenylme-
thoxycarbonyl)]. After deprotection of the Fmoc-Gly Wang
resin, ‘‘hinge’’ 7[9] was coupled using a 20% excess of resin
to give 9. The unchanged amino groups on the resin were
capped with acetylimidazole (AcIm). Deprotection of the
Fmoc groups and treatment with the -valine-derived β-N-
Fmoc-aminosulfonyl chloride 8, with DMAP as catalyst
and methyl trimethylsilyl dimethylketene acetal (MTDA) as
HCl scavenger,[14,17] gave the bis(sulfonamide) 10. Two
cycles were required to ensure completion of the coupling,
until no free amino groups could be detected by two differ-
ent colour tests.[18] Cleavage of the Fmoc protecting groups,
followed by acetylation or mesylation, gave bis(sulfonam-
ide) derivatives with two different terminal groups. Release
Scheme 1. Reagents and conditions: (i) piperidine [20% in N,N-
dimethylformamide (DMF)]; (ii) 7, 1,3-diisopropylcarbodiimide
(DIC), 1-hydroxybenzotriazole hydrate (HOBT), 4-(dimethyl-
amino)pyridine (DMAP), dichloromethane (DCM); (iii) acetylim-
of the resin-bound products was accomplished either by a idazole (Aclm), DCM; (iv) 8, DMAP, methyl trimethylsilyl di-
methylketene acetal (MTDA), DCM, two cycles; (v) Et3N, MeOH,
direct basic methanolysis to provide the methyl ester deriv-
DMF; (vi) trifluoroacetic acid (TFA), H2O; (vii) 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (EDC), DMAP,
MeOH, THF; (viii) methanesulfonyl chloride (MsCl), Et3N, DCM
atives 1 and 2, or alternatively by acidic TFA/H2O cleavage
followed by esterification (two steps, but easier to purify).
Compound 3 was synthesized starting from compound 9 by
Fmoc deprotection followed by acetylation and cleavage the signal of the amidopyridine NH1 protons appeared at
from the resin (basic methanolysis).
unexpectedly low field (δ ϭ 9.41, see Table 1, standard
The conformations of compounds 1؊3 in solution were values δ ϭ 8.00Ϫ8.50[1b][10a]), whilst the sulfonamide pro-
studied by NMR spectroscopy. In the H NMR spectrum tons NH2 were also strongly deshielded (δ ϭ 6.32 compared
1
of 1 in CDCl3, the peaks were rather broad, indicating a to their standard values δ ϭ 4.50Ϫ4.74).[14] The acetamide
slow conformational equilibrium at room temperature (on NH3 proton signals were located closer to their traditional
the NMR timescale). Assignment of all the proton signals chemical shifts (δ ϭ 6.22 compared to 6.00Ϫ6.20).
of receptor 1 could, however, be made unambiguously by
The temperature dependence of protons H1 to H3 was
means of COSY and TOCSY experiments (Figure 2). In studied between 298 and 313 K, at a concentration of
CD3COCD3 or CD3SOCD3, in contrast, the spectra were 1.5 m in CDCl3. Unfortunately, the dependence at low
well resolved, showing single sets of resonances and proving temperatures could not be studied because of the limited
the absence of diastereomeric mixtures (also proved by the solubility of compound 1 in CDCl3 and in CD2Cl2. Calcu-
13C NMR spectrum). The NϪH protons, H1 to H3, dis- lated values of ∆δ/∆T for H1, H2, H3 are Ϫ9.8, Ϫ3.8, and
played a fairly small concentration dependence in CDCl3 Ϫ5.8 ppb/K, respectively, over the range of temperatures
(or in CD2Cl2), which suggests that compound 1 essentially studied (see associated electronic Supporting Informa-
does not aggregate in the concentration range under consid- tion).[19] These temperature coefficients indicate various de-
eration (1.25Ϫ20 m, see associated electronic Supporting grees of intramolecular hydrogen bonding and equilibria
Information).[19] At a concentration of 8.5 m in CDCl3, with non-hydrogen-bonded states.[14,15,20]
4626
Eur. J. Org. Chem. 2001, 4625Ϫ4634

Acyclic Receptors for N-Protected Amino Acids and Dipeptides
FULL PAPER
reogenic centre (H5) gave cross-peaks with protons CH2SO2
(H7), and with the isopropyl protons CH3 (H10) and the
isopropyl proton CH (H11). From the NOE data, and the
1
relatively downfield position of the NH signals in the H
NMR, we can conclude that ‘‘two-armed’’ receptor 1 col-
lapses into an intramolecularly folded structure through
formation of a hydrogen-bonding network involving the
amidopyridine moiety, the sulfonamide NH and the ter-
minal acetamide.
Analogous studies were conducted on the ‘‘two-armed’’
receptor 2 and gave similar results. In summary: (a) the
1
peaks were rather broad in the H NMR spectrum of 2 in
CDCl3, indicating a slow conformational equilibrium at
room temperature (on the NMR timescale); (b) protons H1,
H2 and H3 displayed a fairly small concentration depend-
ence in CDCl3, which suggests that compound 2 essentially
does not aggregate in the concentration range considered
(1.0Ϫ22.5 m, see associated electronic Supporting In-
formation);[19] (c) the signal of the amidopyridine NH1 pro-
tons appeared at low field (δ ϭ 9.07; see Table 1), whilst the
sulfonamide NH2 and NH3 proton signals appeared at δ ϭ
6.34 and 5.13, indicating hydrogen bonding; (d) ROESY
experiments in CDCl3 (concentration: 5 m) showed the
presence of several cross-peaks indicative of an intramolec-
ularly collapsed structure similar to 1. In particular, the
cross-peaks between mesyl CH3 (H9) and isopropyl CH3
(H10), mesyl CH3 (H9) and CH of the valine stereocentre
(H8) were indicative of a cisoid conformation of the ter-
minal mesyl group. It was therefore concluded that an intra-
molecularly folded structure was also highly populated in
CDCl3 in this case, and that the presence of a poor hydro-
gen-bond acceptor such as the methanesulfonamide
group[14,15] in the terminal position was compensated for
by the very strong hydrogen-bond-donating ability of the
methanesulfonamide NH group.[14,15]
Figure 2. Labelling of hydrogen atoms in receptors 1 and 2
Table 1. Chemical shifts of NH protons H1 to H3 for compounds
1؊5 (8.5 m in CDCl3)
Proton
1
2
3
4
5
NϪH1 9.41[a] 9.07[a]
8.83[a] 8.87/8.75[a] 9.32[a]
NϪH2 6.32[b] 5.13 (6.34)[b] [c] 6.56[d] 5.98[b]
5.78[b]
6.38[b]
6.22[d]
NϪH3 6.22[d] 6.34 (5.13)[b] [c]
NϪH4
5.81[d]
In comparison, the spectrum of the control compound 3
(Scheme 1) in CDCl3 was perfectly resolved, and the amido-
pyridine NH1 signal was found at δ ϭ 8.83 (for a concentra-
[a]
Reference value for the amidopyridine protons: δ ϭ 8.00Ϫ8.50,
see refs.[1b,10a]
δ ϭ 4.50Ϫ4.74, see ref.[14]
unequivocally attributed. Ϫ Reference value for the carboxyam-
ide protons: δ ϭ 6.00Ϫ6.20.
Ϫ
Reference value for the sulfonamide protons:
[b]
[c]
Ϫ
NϪH2 and NϪH3 could not be tion of 8.5 m, see Table 1), whereas the acetamide NϪH
[d]
proton was found at δ ϭ 6.56. Protons H1 and H2 displayed
a fairly small concentration dependence in CDCl3, sug-
gesting that compound 3 essentially does not aggregate in
NOESY and ROESY experiments were performed for the concentration range under consideration (see associated
compound 1 at two different concentrations (5 and 10 m electronic Supporting Information).[19] ROESY experi-
in CDCl3) and showed the same contact pattern. The CH3 ments in CDCl3 (concentration: 10 m) showed cross-peaks
acetyl hydrogen atoms (H9) gave cross-peaks with the pro- consistent with the presence of trans-amide rotamers (a mi-
ton of the valine stereogenic centre (H8) and with the NH nor contact between CH3 of the terminal acetamide and
of the terminal acetamide (H3). While the cross-peak be- CH of the phenylalanine stereocentre was also detected, in-
tween H9 and H3 is typical of a trans-amide rotamer, the dicating the presence of a small percentage of cis-amide rot-
cross-peak between H9 and H8 is indicative of the presence amer).[22,23]
of a cis-amide rotamer,[21,22] which is usually present as a
In order to obtain a simplified view of the system, and
small percentage in secondary amides (ca. 3% for N-methyl- to study the behaviour of the single legs of receptor 1, we
acetamide in CHCl3).[23] The acetyl CH3 hydrogen atoms decided to prepare the differently substituted scaffold 4,
(H9) also gave cross-peaks with the aromatic protons of the starting from the unsymmetrically protected ‘‘hinge’’ 11
phenylalanine ring and with the benzylic protons (H6). (Scheme 2). As shown in Scheme 2, 11 was deprotected
Other interesting contacts are: The isopropyl CH3 protons (25% TFA in DCM), and treated with the -valine-derived
(H10) gave cross-peaks with the aromatic protons of the β-N-Boc-aminosulfonyl chloride 12, with DMAP as cata-
phenylalanine ring; the proton at the phenylalanine ste- lyst and methyl trimethylsilyl dimethylketene acetal
Eur. J. Org. Chem. 2001, 4625Ϫ4634
4627

J. D. Kilburn, C. Gennari et al.
FULL PAPER
(MTDA) as HCl scavenger,[14,17] to give the monosulfonam-
ide 13. Removal of the Boc group (25% TFA in DCM) and
acetylation with acetylimidazole yielded receptor 4. 1H
NMR studies on receptor 4 (8.5 m in CDCl3) showed a
single set of well-resolved signals (see Table 1): The amidop-
yridine NϪH proton signals (two different signals at δ ϭ
8.75 and 8.87) were shifted substantially upfield compared
to that of H1 of receptor 1 (δ ϭ 9.41), and the same effect
could be seen for the sulfonamide NϪH proton (δ ϭ 5.98
vs. 6.32 for 1) and the acetamide NϪH proton (δ ϭ 5.81
vs. 6.22 for 1). From these data we concluded that the hy-
drogen-bonding network in 1 and 2 was strengthened by
the cooperative effect of both legs. In the ROESY spectrum
(8.3 m in CDCl3) the acetamide methyl group gave cross-
peaks with the isopropyl CH3, the CH of the valine ste-
reocentre, and the acetamide NH. These contacts are typ-
ical of the presence both of a trans-amide rotamer and of a
cis-amide rotamer,[22,23] usually present in small percentages
in secondary amides (see the above discussion for 1).
Binding studies with receptor 1 in CDCl3 were carried
out with a series of substrates, by a standard titration ex-
periment, by monitoring the shift of the NH and CH sig-
nals on addition of successive aliquots of guest and analys-
ing the resulting binding curves by nonlinear regression
analysis.[24] In agreement with the above results, and as a
Scheme 2. Reagents and conditions: (i) TFA (25% in DCM); (ii)
K2CO3 (10% in H2O); (iii) 12, DMAP, MTDA, DCM; (iv) Aclm,
DCM
further proof of strong intramolecular self-association, change to the acetamide NH3 signal. Saturation was re-
compound 1 proved to be a rather poor receptor for N- ached upon addition of an excess of N-protected amino
protected (N-Boc and N-Cbz) amino acids and dipeptides acid as guest (20Ϫ30 equiv.). In more polar solvents such as
(see Table 2), even in a noncompetitive solvent such as chlo- [D6]acetone, CD3CN or [D6]DMSO, the spectra were well
roform. A slightly higher affinity for N-Cbz-Ala-OH (either resolved, but the chemical shifts of the host signals did not
 or ) in preference to N-Boc--Ala-OH was observed, change upon addition of an excess of N-protected amino
with binding constants of 207 (N-Cbz--Ala-OH), 270 (N- acid.
Cbz--Ala-OH) and 119 (N-Boc--Ala-OH), but little en-
Binding of dipeptide guests gave only slightly higher
antioselective binding was observed with the N-Cbz-amino binding constants, and titration of 1 with, for example, N-
acids. Binding of all three amino acid substrates gave rise Cbz--Ala--Ala-OH gave a binding constant of 245 Ϫ1
to significant downfield shifts in the amidopyridine NH1 and resulted in downfield shifts both of the amidopyridine
signal (Ն 0.5 ppm), but whereas binding of the  enanti- and of the sulfonamide NH signals (NH1: ∆δ ϭ 0.25; NH2:
omers produced a small upfield shift in the acetamide NH3 ∆δ ϭ 0.30) and an upfield shift of acetamide NH3 (∆δ ϭ
signal (ca. 0.1) and little change to the sulfonamide NH2 0.12). A change in the N-protecting group from Cbz to Boc
signal, the binding of N-Cbz--AlaOH produced a down- had only a small effect on the binding (or on the shifts of
field shift of the sulfonamide NH2 signal (ca. 0.4) and little the NH signals), while a change in the C-terminal amino
Table 2. Binding constants Kass (Ϫ1) for the 1:1 complexes formed between receptors 1 and 5 and various amino acids and dipeptides
derivatives, calculated from the chemical shifts of various proton signals of 1 and 5, in CDCl3 at 25 °C
Guest
Host 1
(Data[a]
)
Host 5
(Data[a]
)
Kass [Ϫ1
]
Kass [Ϫ1
]
N-Boc--Ala-OH
N-Cbz--Ala-OH
N-Cbz--Ala-OH
N-Boc--Ala--Ala-OH
N-Cbz--Ala--Ala-OH
N-Cbz--Ala--Ala-OH
N-Boc--Ala--Phe-OH
N-Cbz--Ala--Phe-OH
N-Boc--Ala--Trp-OH
119
207
270
361
245
242
297
292
377
(NH1, NH3)
Ϫ[b]
32
(NH1, NH3, CH72SO2)
(NH1, CH6H6ЈPh, CH6H6ЈPh)
(NH1, NH2)
(NH1)
Ϫ[b]
Ϫ[b]
107
(NH1, NH2)
(NH1, CH2)
(NH2, NH3)
2404[c]
109
(NH1, NH3, CH7H7ЈPh, CH7H7ЈPh, CH6,14
)
(NH1, NH2)
(NH1)
(NH1, NH2, NH3, CH6H6ЈPh)
(NH1, NH2)
Ϫ[b]
ND[d]
[a] Data from signals used in the binding calculation. Ϫ [b] Experiment not performed. Ϫ [c] Error was estimated as Ͻ 5%, with data from
[d]
five different proton signals. Ϫ Changes in the spectrum were too small to allow reliable estimation of the binding constant.
4628
Eur. J. Org. Chem. 2001, 4625Ϫ4634

Acyclic Receptors for N-Protected Amino Acids and Dipeptides
FULL PAPER
acid from alanine to phenylalanine or tryptophan similarly
had only a limited effect. Titration with N-Cbz--Ala--
Ala-OH resulted in little change to the amidopyridine NH1
signal (∆δ Յ 0.03), but analysis of the downfield shift of
the sulfonamide NH2 signal (∆δ ϭ 0.28) and of the upfield
shift of the acetamide NH3 signal (∆δ ϭ 0.19) gave a bind-
ing constant of 242 Ϫ1, indicating that 1 also shows little
enantioselectivity for dipeptides [Ka(N-Cbz--Ala--
AlaOH) ϭ 245 Ϫ1; Table 2]. Binding of amino acids and
dipeptides was also accompanied by a small shift of several
CH signals in host 1 (Յ 0.12). The 1:1 stoichiometry of the
complexes between host 1 and the amino acidic and pep-
tidic guests was established by Scatchard plots,[25] and by
the good fit of the experimental data to the 1:1 model.[26]
In the binding experiments with host 1, we also mon-
itored the chemical shifts of the N-protected amino acid
and dipeptide guests. The carbamate NHa signals were
shifted downfield on binding of both amino acids (∆δ ϭ
0.08Ϫ0.14) and dipeptides (∆δ ϭ 0.10Ϫ0.35).[27] The amide
NHb signals on binding the dipeptides were also shifted
downfield with a ∆δ value similar to that of the NHa signals
(∆δ ϭ 0.10Ϫ0.35).[27]
Thus, receptor 1 does appear to bind dipeptides and
simple amino acid derivatives, but the absolute binding con-
stants are rather low in comparison to those observed in
other diamidopyridine-based receptors, and the shifts of the TFA, H2O; (v) EDC, DMAP, MeOH, THF
various NH signals on binding are also rather small in com-
Scheme 3. Reagents and conditions: (i) piperidine (20% in DMF);
(ii) 8, DMAP, MTDA, DCM, two cycles; (iii) Aclm, DCM; (iv)
parison to those seen in other related receptor systems.[1b,10] values[14]), suggesting intramolecular hydrogen bonding. In
In order to study the influence of the length of the legs contrast, the signal of the acetamide protons NH4 was
and possibly to increase the affinity of our ‘‘two-armed’’ closer to the conventional value (δ ϭ 6.22 compared to δ ϭ
receptors for N-protected amino acids and dipeptides, we 6.00Ϫ6.20 for reference values).
decided to test the new receptor 5, with longer pseudo-pep-
The temperature dependence of protons H1 to H4 was
tide legs (Scheme 3). Compound 5 was synthesized from the studied between 300 and 320 K, at a concentration of 1 m
bis(sulfonamide) 10 (Scheme 1) by removal of the Fmoc in CDCl3. Calculated values of ∆δ/∆T for H1, H2, H3, H4
groups, treatment with the -valine-derived β-N-Fmoc-ami- are Ϫ 2.5, Ϫ 4.8, Ϫ 4.5, and Ϫ 4.5 ppb/K, respectively, over
nosulfonyl chloride 8, and subsequent deprotection, the range of temperatures studied (see associated electronic
acetylation and cleavage (Scheme 3).
Supporting Information).[19] These temperature coefficients
In the 1H NMR spectrum of 5 in CDCl3, the peaks were revealed a small temperature dependence in protons H1 to
rather broad, indicating a slow conformational equilibrium H4, indicating various degrees of intramolecular hydrogen
at room temperature, as in the case of compound 1. Assign- bonding.
ment of all the proton signals of receptor 5 could, however,
NOESY experiments were performed at a concentration
be accomplished by means of COSY and TOCSY experi- of 12 m in CDCl3, and showed some interesting contacts.
1
ments (Figure 1). The H NMR spectra at 320 K in CDCl3 The CH3 acetyl hydrogen atoms (H15) gave a cross-peak
and at 300 K in CD3COCD3 and in CD3CN were well re- with the NH of the acetyl group (H4), which is typical of a
solved and showed single sets of resonances, proving the trans-amide rotamer [a minor contact between CH3 of the
absence of diastereomeric mixtures (also proved by the 13C terminal acetamide (H15) and H14 at the vicinal stereocentre
NMR spectra). The signals of NϪH protons H1, H2, H3 was also detected, indicating the presence of a small per-
and H4 displayed a fairly small concentration dependence centage of a cis-amide rotamer].[22,23] The CH3 acetyl hy-
in CDCl3 (slightly higher for H3 compared to H1, H2 and drogen atoms (H15) also gave distinct cross-peaks with the
H4), suggesting that compound 5 essentially does not ag- isopropyl CH3 hydrogen atoms of two different valine res-
gregate in the concentration range under consideration idues (H11 and H13). The NH proton of the amidopyridine
(0.47Ϫ20 m, see associated electronic Supporting In- (H1) gave cross-peaks with the proton of the valine ste-
formation).[19] At a concentration of 8.5 m in CDCl3, the reogenic centre (H9) and with the isopropyl CH proton
signal of the amidopyridine NH1 protons appeared at low (H10). The aromatic protons of the phenylalanine ring gave
field (δ ϭ 9.32 compared to δ ϭ 8.00Ϫ8.50 for reference cross-peaks with the proton of the valine stereogenic centre
values,[1b][10a] see Table 1). The signals of the sulfonamide (H9), with CH2SO2 (H8), with the isopropyl CH3 (H11), and
protons NH2 and NH3 appeared at low field as well (δ ϭ with the isopropyl CH proton (H10). The benzylic protons
5.78 and 6.38 compared to δ ϭ 4.50Ϫ4.74 for reference (H7) gave cross-peaks with the isopropyl CH3 (H11), and
Eur. J. Org. Chem. 2001, 4625Ϫ4634
4629

J. D. Kilburn, C. Gennari et al.
FULL PAPER
with the isopropyl CH proton (H10). The proton of the class of synthetic ‘‘two-armed’’ receptors containing a 2,6-
phenylalanine stereogenic centre (H6) also gave cross-peaks diamidopyridine binding unit bearing pendant pseudopep-
with the isopropyl CH3 (H11) and with the isopropyl CH tide legs consisting of an amino acid (-Phe) and various
proton (H10). All these NOE contacts suggested an intram- chiral β-aminosulfonamides. These receptors collapse into
olecularly folded structure for the ‘‘two-armed’’ receptor 5, intramolecularly folded structures in CDCl3 solution,
produced by formation of a hydrogen-bonding network in- through formation of a network of intramolecular hydrogen
volving the amidopyridine moiety and the different sulfona- bonds. Addition of excesses of N-protected amino acids and
mide and acetyl groups of the two legs.
dipeptides to receptors 1 or 5 involves unfolding of the re-
As further evidence of the strong self-association, com- ceptor and breaking of intramolecular hydrogen bonds
pound 5 initially proved to be a poorer receptor than com- (with an associated energy cost) to allow interaction with
pound 1 for N-protected amino acids and N-protected di- the guest, resulting in rather low binding constants and only
peptides (see Table 2). Compound 5 binds the amino acid small changes of the signals for the hydrogen-bonding NH
N-Cbz--Ala-OH approximately one order of magnitude protons. Only a very qualitative picture of the conforma-
less tightly than compound 1 does (Ka ϭ 32 vs. 207 Ϫ1). tional properties of the hosts and of the structure of their
The NH1 chemical shift was shifted downfield, but satura- complexes with protected amino acids and dipeptides could
tion could not be reached even after addition of 20 equiv. be established; this is unfortunately also true for the inter-
of guest.
esting enantioselectivity shown by host 5 in the recognition
Receptor 5 associates less strongly than receptor 1 with of N-Cbz--Ala--Ala-OH.[28] In any event, the significant
N-Cbz--Ala--Ala-OH and with N-Boc--Ala--Phe-OH shifts of several CH and NH signals throughout the spec-
(Ka ϭ 107 and 109 vs. 245 and 297 Ϫ1, see Table 2). The trum of 5 (in particular the very large downfield shift for
NH1 chemical shift was shifted downfield (ca. 0.3) but sat- the sulfonamide NH3) suggest that the structure of the com-
uration could not be reached even after addition of 15Ϫ20 plex of N-Cbz--Ala--Ala-OH is very different from that
equiv. of dipeptide. The 1:1 stoichiometry of the complexes of the enantiomeric guest N-Cbz--Ala--Ala-OH. Such
between host 5 and the amino acidic and peptidic guests high enantioselectivity (Ͼ 20:1, effectively discriminating
was established by Scatchard plots[25] and by the good fit between methyl groups and hydrogen atoms) has seldom
of the experimental data to the 1:1 model.[26]
been observed in synthetic receptors,[1Ϫ3][10b] and is particu-
Titration of 5 with N-Cbz--Ala--Ala-OH gave a bind- larly noteworthy in such a structurally simple acyclic recep-
ing constant of 107 Ϫ1 and a downfield shift of δ ϭ 0.3 tor, which appears to lack much, if any, preorganisation for
for the amidopyridine NH1, but only small changes to the binding. Work to develop a combinatorial approach to the
sulfonamide and acetamide NH protons (NH2, NH3, NH4; binding of dipeptides is in progress, either by the screening
∆δ Յ 0.07). In contrast, titration of 5 with N-Cbz--Ala-- of a library of receptors towards single substrates of inter-
Ala-OH gave a binding constant of 2404 Ϫ1, with a par- est,[29] or by the screening of a library of dipeptides towards
ticularly large downfield shift for the sulfonamide NH3 sig- a particular receptor.[30]
nal (∆δ ϭ 0.72) but an upfield shift for the sulfonamide
NH2 (∆δ ϭ 0.38) and even a small upfield shift for the ami-
dopyridine NH1 (∆δ ϭ 0.05)! Binding of N-Cbz--Ala--
Ala-OH was also accompanied by significant shifts of sev-
eral CH signals in the host (up to δ ϭ 0.4 vs. Յ 0.08 in
the case of N-Cbz--Ala--Ala-OH). We also monitored the
chemical shifts of the N-protected amino acid and dipeptide
guests. The carbamate NHa signal of the amino acid sub-
strate was shifted downfield very modestly (ca. 0.03) on
binding with 5.[27] The carbamate NHa and amide NHb sig-
nals of the dipeptide substrates were more significantly
shifted downfield, most notably in the case of N-Cbz--Ala-
-Ala-OH in which the NHa signal shifted by δ ϭ 0.50 and
the NHb signal shifted by δ ϭ 0.42 on binding with 5 (in
the case of N-Cbz--Ala--Ala-OH, the NHa signal shifted
by δ ϭ 0.11 and the NHb signal shifted by δ ϭ 0.10 on
binding with 5).[27] Thus, receptor 5 displays remarkably
good enantioselectivity for the biologically significant Ala-
Ala-OH dipeptide sequence, albeit in a noncompetitive
solvent.
Experimental Section
General: Manipulations involving air-sensitive compounds were
carried out under argon. Anhydrous solvents were obtained from
sodium benzophenone ketyl (THF), or by refluxing over CaH2 for
at least 4 h prior to use. Reagents were used as received, without
any further purification, and were generally purchased from Ald-
rich and Fluka AG. Reactions in solution were monitored by ana-
lytical thin layer chromatography (TLC) by using Merck 60 F254
silica gel glass plates. The chromatograms were viewed under UV
light and by staining with a cerium reagent followed by heating.
Flash chromatography was performed with 60 silica gel (230Ϫ400
Mesh) purchased from MachereyϪNagel. NMR spectra were re-
corded with Bruker instruments (AC 200, AC 300 and AVANCE
400). The spectra are reported in ppm relative to tetramethylsilane.
General Procedure for Fmoc Removal from Resin-Bound Fmoc De-
rivatives: N-Fmoc-protected resins were treated with a 20% piperid-
ine/DMF solution (7 mL, 3 ϫ 15 min). Between each cycle the so-
lution was drained and the resin was washed with DMF (1ϫ). At
the end of the third cycle, the resin was drained, washed with DMF
(3ϫ), MeOH (3ϫ) and DCM (4ϫ), and dried in vacuo.
Conclusions
2,6-Bis( -Fmoc-
L-Phe-NH)pyridine-4-OCH2CO-Gly-O-Wang-
In conclusion, we have described the synthesis, the con-
Merrifield Resin (9): A solid-phase reactor was charged with N-
formational behaviour and the binding properties of a novel Fmoc-Gly-Wang-Merrifield resin 6 (0.260 mmol, 294 mg). Fmoc
4630 Eur. J. Org. Chem. 2001, 4625Ϫ4634

Acyclic Receptors for N-Protected Amino Acids and Dipeptides
FULL PAPER
removal was accomplished according to the general procedure.
night at room temperature. The solution was then filtered and the
Acid 7 (0.217 mmol, 200 mg), HOBt (0.868 mmol, 117 mg), DIC resin was washed with DMF (3ϫ), MeOH (3ϫ) and DCM (4ϫ)
(0.868 mmol, 0.134 mL) and DMAP (0.108 mmol, 13 mg) were
then added. DCM (8 mL) was added and the resulting mixture was
shaken at room temperature for 20 h. The resin was drained,
washed with DMF (3 ϫ 8 mL), MeOH (3 ϫ 8 mL) and DCM (4
ϫ 8 mL), and dried in vacuo. Yield: 426 mg, 98%. Acetylimidazole
(2.170 mmol, 239 mg) and DCM (8 mL) were then added and the
mixture was shaken at room temperature for 2 h. The solution was
then filtered and the resin was washed with DMF (3 ϫ 8 mL),
MeOH (3 ϫ 8 mL) and DCM (4 ϫ 8 mL), and dried in vacuo.
and dried in vacuo.
Mesylation of 2,6-Bis[( )- -FmocNHCH( Pr)CH2SO2-L-Phe-
NH]pyridine-4-OCH2CO-Gly-O-Wang-Merrifield Resin (10):
A
solid-phase reactor was charged with resin 10 (0.054 mmol), and
Fmoc removal was performed according to the general procedure.
Mesyl chloride (10 equiv.), TEA (12 equiv.) and DCM (4 mL) were
added and the mixture was shaken at room temperature overnight.
The resin was drained, washed with a 10% solution of TEA in
DCM (3ϫ), DMF (3ϫ), MeOH (3ϫ) and DCM (4ϫ), and dried
in vacuo.
2,6-Bis[( )- -FmocNHCH( Pr)CH2SO2-L-Phe-NH]pyridine-4-
OCH2CO-Gly-O-Wang-Merrifield Resin (10): A solid-phase re-
actor was charged with 2,6-bis(Fmoc--Phe-NH)pyridine-4-
OCH2CO-gly-O-Wang-Merrifield resin 9 (0.163 mmol, 320 mg),
and Fmoc removal was performed according to the general proced-
Cleavage of the 2,6-Diamidopyridine Derivatives from the Resin
General Procedure A (Basic Cleavage):[33] The appropriate resin was
treated with an anhydrous TEA/MeOH/DMF solution (4.7 mL,
1.9:1.9:0.9, v/v/v) under argon. The mixture was magnetically
stirred at 50° C for 24 h. The suspension was then filtered and the
resin was washed with DCM (3ϫ). The eluates were pooled and
the solvents were evaporated under reduced pressure to afford a
residue that was subsequently purified.
ure. Sulfonyl chloride
8
(0.652 mmol, 267 mg), DMAP
(0.130 mmol, 16 mg) and DCM (8 mL) were then added and the
mixture was shaken for 5 min; MTDA (1.300 mmol, 0.264 mL) was
added and the mixture was shaken at room temperature overnight.
The suspension was then filtered and the resin was washed with
DMF (3 ϫ 8 mL), MeOH (3 ϫ 8 mL) and DCM (4 ϫ 8 mL), and
dried in vacuo. Yield: 334 mg, 92%. The free amino content was
checked by two different tests: The TNBS test was negative,[31]
while the NF31 test,[32] a more sensitive assay, revealed the presence
of approximately 2% free amino content (slightly pink-red beads).
A basic washing was performed by addition of a solution of 5%
DIPEA in DMF (4 mL). This washing was done twice and the
resin was washed with DMF (3 ϫ 8 mL), MeOH (3 ϫ 8 mL) and
DCM (4 ϫ 8 mL), and dried in vacuo. A second coupling cycle was
then performed under the same conditions: 8 (0.163 mmol, 66 mg),
DMAP (0.032 mmol, 4 mg), MTDA (0.326 mmol, 66 µL), and
DCM (4 mL). Yield: 338 mg, 93%. After washing, both tests were
clearly negative (colourless beads).
General Procedure B (Acidic Cleavage): The appropriate resin was
treated with TFA/H2O solution (95:5 v/v) (5 mL). The mixture was
shaken for 4 min. The solution was then drained and the resin was
washed with DCM (3ϫ). A second cycle was performed. The elu-
ates were pooled and the solvents were evaporated under reduced
pressure to afford a residue that was treated with dry toluene and
sonicated for 1 or 2 min. The solvent was evaporated (the treatment
was repeated three more times). The resulting white solid was dried
in vacuo, to yield the acid derivative in 85Ϫ90% overall yield. The
acid was mixed with EDC (4 equiv.), DMAP (0.5 equiv.) and dry
THF (4.5 mL/0.043 mmol of acid), and the mixture was stirred for
5 min. Dry MeOH (6Ϫ9 equiv.) was then added and the reaction
mixture was stirred at room temperature overnight. Evaporation of
the solvent under reduced pressure afforded a residue, which was
subsequently purified.
2,6-Bis[( )- -FmocNHCH( Pr)CH2SO2-( )-NHCH( Pr)CH2SO2-
L
-Phe-NH]pyridine-4-OCH2CO-Gly-O-Wang-Merrifield
Resin
(14): A solid-phase reactor was charged with 2,6-bis[(S)-N-Fmoc-
NHCH(iPr)CH2SO2--Phe-NH]pyridine-4-OCH2CO-gly-O-Wang-
Merrifield resin 10 (0.095 mmol, 169 mg), and Fmoc removal was
performed according to the general procedure. Sulfonyl chloride 8
(0.380 mmol, 155 mg), DMAP (0.076 mmol, 9 mg) and DCM
(6 mL) were then added and the mixture was shaken for 5 min;
MTDA (0.760 mmol, 0.154 mL) was added and the mixture was
shaken at room temperature overnight. The suspension was then
filtered and the resin was washed with DMF (3 ϫ 8 mL), MeOH
(3 ϫ 8 mL) and DCM (4 ϫ 8 mL), and dried in vacuo. Yield:
163 mg, 84%. The free amino content was checked by two different
tests: The TNBS test was negative,[31] while the NF31 test[32] re-
vealed the presence of free amino content (slightly pink-red beads).
A basic washing was performed by addition of a solution of 5%
DIPEA in DMF (4 mL). This washing was performed twice and
the resin was washed with DMF (3 ϫ 8 mL), MeOH (3 ϫ 8 mL)
and DCM (4 ϫ 8 mL), and dried in vacuo. A second coupling cycle
was then performed under the same conditions: 8 (0.190 mmol,
78 mg), DMAP (0.032 mmol, 4 mg), MTDA (0.380 mmol, 77 µL)
and DCM (4 mL). Yield: 179 mg, 92%. After washing, both tests
were clearly negative (colourless beads).
2,6-Bis[( )- -AcNHCH( Pr)CH2SO2-L-Phe-NH]pyridine-4-OCH2-
CO-Gly-OMe (1): By general procedure A. The crude residue was
purified by flash column chromatography on silica gel, with DCM/
acetone (5%) and DCM/MeOH (1Ϫ5%) as eluents. The yellow
solid obtained was precipitated from DCM/diethyl ether to give a
white solid in 30% overall yield. By general procedure B. The crude
residue was purified by flash column chromatography on silica gel,
with DCM/acetone (5%) and DCM/MeOH (2Ϫ6%) as eluents. The
white solid obtained was precipitated from DCM/diethyl ether to
give a white solid in 35% overall yield. 1: 1H NMR (200 MHz,
5 m in [D6]DMSO, 307 K): δ ϭ 10.27 (s, 2 H, Py-NHCO), 8.74
(t, J ϭ 5.0 Hz, 1 H, NH-Gly), 7.85 (br. s, 2 H, NHSO2), 7.70 (d,
J ϭ 10.0 Hz, 2 H, NHAc), 7.62 (s, 2 H, CH-Py), 7.50Ϫ7.32 (m, 10
H, aromatic), 4.73 (s, 2 H, O-CH2-Gly), 4.50Ϫ4.48 (m, 2 H, CH-
Phe), 4.12Ϫ4.08 (m, 2 H, CH-iPr), 4.02 (d, J ϭ 5.0 Hz, 2 H, CH2-
Gly), 3.74 (s, 3 H, OMe), 3.30Ϫ2.65 (m, 4 H, CH2Ph), 2.60Ϫ2.30
(m, 4 H, CH2SO2), 1.88 (s, 6 H, CH3CO), 1.70Ϫ1.68 (m, 2 H,
CHMe2), 0.77 (d, 12 H, J ϭ 7.0 Hz, CH3). 1H NMR (400 MHz,
3.8 m in CDCl3, 300 K): δ ϭ 9.30 (br. s, 2 H, Py-NHCO), 7.58
(br. s, 1 H, NH-Gly), 7.37Ϫ7.28 (m, 12 H, aromatic), 6.27 (br. s, 2
H, NHSO2), 6.05 (br. s, 2 H, NHAc), 4.50 (br. s, 4 H, OϪCH2-Gly
and CH-Phe), 4.17Ϫ4.12 (m, 4 H, CH-iPr and CH2-Gly), 3.77 (s,
General Procedure for the Acetylation of Resin-Bound Fmoc Deriv-
atives: A solid-phase reactor was charged with Fmoc-protected 3 H, OMe), 3.37Ϫ3.35 (m, 2 H, CH2Ph), 3.03Ϫ2.92 (m, 4 H,
resins (9 or 10) and Fmoc removal was performed according to the CH2SO2), 2.82Ϫ2.79 (m, 2 H, CH2Ph), 1.97 (s, 6 H, CH3CO),
general procedure. Acetylimidazole (10 equiv.) and DCM (2 mL 1.76Ϫ1.74 (m, 2 H, CHMe2), 0.80 (d, J ϭ 7.0 Hz, 6 H, CH3), 0.79
per 0.054 mmol) were then added and the mixture was shaken over-
(d, J ϭ 7.0 Hz, 6 H, CH3). 13C NMR (50.3 MHz, 5 m, [D6] acet-
Eur. J. Org. Chem. 2001, 4625Ϫ4634
4631

J. D. Kilburn, C. Gennari et al.
FULL PAPER
one, 300 K): δ ϭ171.4, 170.5, 167.8, 167.4 (CO), 151.8, 138.0 (C, (33 mg, 0.044 mmol) was dissolved in a solution of 25% (v/v) TFA
arom.), 130.5, 129.1, 127.6 (CH, arom.), 96.5 (CH, Py), 67.3 (CH2, in dry DCM (1.5 mL). The solution was stirred at room temper-
OCH2CO), 59.9 (CH, CH-Phe), 55.3 (CH2, CH2SO2), 52.0 (CH3, ature for 1.5 h, diluted with DCM (100 mL), and washed with a
OCH3), 50.4 (CH, CH-iPr), 40.9 (CH2, CH2-Gly), 39.0 (CH2, 10% aqueous solution of K2CO3 and brine. The organic layer was
CH2Ph), 32.5 (CH, CHMe2), 22.9 (CH3, CH3CO), 19.1 (CH3), 17.5
dried with Na2SO4 and filtered, and the solvents were evaporated.
(CH3). MS (FAB): m/z ϭ 931 [M ϩ H]ϩ; 953 [M ϩ Na]ϩ. HRMS The sulfonyl chloride 12 (25 mg, 0.088 mmol), DMAP (8 mg,
(FAB) calcd. for C42H59N8O12S2 [M ϩ H]ϩ: 931.36884, found m/
z: 931.36869.
0.066 mmol), dry DCM (2 mL) and MTDA (36 µL) were then ad-
ded to the free amine (28 mg, 0.044 mmol). The mixture was stirred
at room temperature for 2 h 30. DCM was removed and the residue
was dissolved in EtOAc (75 mL). The organic layer was washed
with a 10% aqueous solution of citric acid (3 ϫ 10 mL) and brine
(10 mL), dried with Na2SO4 and filtered. The solvents were evapor-
ated to give the N-Boc-protected compound 13 (39 mg, 99% yield).
Compound 13 (39 mg, 0.043 mmol) was dissolved in a solution of
25% (v/v) TFA in DCM (1.0 mL). The solution was stirred at room
temperature for 1 h and the DCM was evaporated. Dry toluene (2
mL) was added to the residue. The mixture was sonicated and the
toluene was evaporated (3 cycles). The residue was dissolved in
DCM (2 mL) and Aclm was added (34 mg, 0.305 mmol). The mix-
ture was stirred at room temperature overnight. DCM was evapor-
ated and the residue was dissolved in EtOAc (75 mL). The organic
layer was washed with a 10% aqueous solution of citric acid (3 ϫ
10 mL), and then with a 10% aqueous solution of NaHCO3 (3 ϫ
10 mL) and brine (10 mL), dried with Na2SO4 and filtered. The
solvents were evaporated to give 33 mg (90%) of compound 4,
which could not be further purified. 4: 1H NMR (400 MHz, 8.5 m
in CDCl3, 300 K): δ ϭ 8.87 (s, 1 H, Py-NHCO), 8.75 (s, 1 H, Py-
NHCO), 7.41Ϫ7.23 (m, 17 H, aromatic), 5.98 (br. s, 1 H, NH-SO2),
5.87Ϫ5.79 (m, 2 H, CHϭC and NH-Ac), 5.74 (br. s, 1 H, NH-
Alloc), 5.28 (s, 2 H, OϪCH2-Ph), 5.23Ϫ5.14 (m, 2 H, CH2ϭC),
4.67 (s, 2 H, OϪCH2-CO), 4.57Ϫ4.55 (m, 1 H, CH-Phe), 4.53Ϫ4.51
(m, 2 H, CH2-Alloc), 4.47 (br. s, 1 H, CH-Phe), 4.24Ϫ4.20 (m, 1
H, CH-iPr), 3.34Ϫ3.24 (m, 2 H, CH2Ph), 3.08Ϫ2.82 (m, 4 H,
CH2Ph and CH2SO2), 1.95 (s, 3 H, CH3CO), 1.72Ϫ1.70 (m, 1 H,
CHMe2), 0.82 (d, J ϭ 7.0 Hz, 6 H, CH3). MS (FAB): m/z ϭ 843
2,6-Bis[( )- -MsNHCH( Pr)CH2SO2-L-Phe-NH]pyridine-4-
OCH2CO-Gly-OMe (2): This compound was prepared by general
procedure A. The crude residue was purified by flash column chro-
matography on silica gel, with DCM/acetone (5%) and DCM/
MeOH (3%) as eluents. The yellow solid obtained was precipitated
from DCM/diethyl ether to give a white solid in 30% overall yield.
1
2: H NMR (200 MHz, 5 m in [D6]DMSO, 300 K): δ ϭ 10.77 (s,
2 H, Py-NHCO), 8.78Ϫ8.76 (m, 1 H, NH-Gly), 8.13Ϫ8.11 (m, 2
H, NHSO2), 7.61 (s, 2 H, CH-Py), 7.50Ϫ7.32 (m, 12 H, aromatic,
NHSO2), 4.73 (s, 2 H, OϪCH2-Gly), 4.40Ϫ4.60 (m, 2 H, CH-Phe),
4.02 (d, J ϭ 5.0 Hz, 2 H, CH2-Gly), 3.73 (s, 3 H, OMe), 3.63Ϫ3.59
(m, 2 H, CH-iPr), 3.30Ϫ2.65 (m, 4 H, CH2Ph), 2.98 (s, 6 H,
CH3SO2), 2.60Ϫ2.30 (m, 4 H, CH2SO2), 1.88Ϫ1.74 (m, 2 H,
CHMe2), 0.76 (d, J ϭ 7.0 Hz, 6 H, CH3), 0.75 (d, J ϭ 7.0 Hz, 6
1
H, CH3). H NMR (400 MHz, 5 m in CDCl3, 300 K): δ ϭ 9.07
(br. s, 2 H, Py-NHCO), 7.65 (br. s, 1 H, NH-Gly), 7.38Ϫ7.28 (m,
12 H, aromatic), 6.34 (br. s, 2 H, NHSO2), 5.13 (br. s, 2 H,
NHSO2), 4.54 (s, 2 H, OϪCH2-Gly), 4.41 (br. s, 2 H, CH-Phe),
4.20 (br. s, 2 H, CH2-Gly), 3.79 (s, 3 H, OMe), 3.70Ϫ3.60 (m, 2 H,
CH-iPr), 3.47Ϫ3.16 (m, 2 H, CH2Ph), 3.14Ϫ2.91 (m, 8 H, CH2Ph
and CH3SO2), 2.80Ϫ2.55 (m, 2 H, CH2SO2), 2.50Ϫ2.33 (m, 2 H,
CH2SO2), 2.02Ϫ1.90 (m, 2 H, CHMe2), 0.86 (br. s, 12 H, CH3).
13C NMR (50.3 MHz, 10 m, CDCl3, 300 K): δ ϭ 170.2, 167.6,
166.1 (CO), 150.2, 136.4 (C, arom.), 129.7, 128.0, 127.4 (CH,
arom.), 96.3 (CH, Py), 66.7 (CH2, OCH2CO), 59.8 (CH, CH-Phe),
55.0 (CH3, OCH3), 53.5 (CH2, CH2SO2), 52.3 (CH, CH-iPr), 41.8
(CH3, CH3SO2), 40.7 (CH2, CH2-Gly), 38.6 (CH2, CH2Ph), 32.5
(CH, CHMe2), 17.9 (CH3), 17.6 (CH3). MS (FAB): m/z ϭ 1003 [M
ϩ H]ϩ; 1025 [M ϩ Na]ϩ. HRMS (FAB) calcd. for C40H59N8O14S4
[M ϩ H]ϩ: 1003.30281, found m/z: 1003.30330.
[M
ϩ ϩ
H]ϩ; 865 [M Na]ϩ. HRMS (FAB) calcd. for
C43H51N6O10S [M ϩ H]ϩ: 843.33819, found m/z: 843.33657.
2,6-Bis[( )- -AcNHCH( Pr)CH2SO2-( )-NHCH( Pr)CH2SO2-L-
Phe-NH]pyridine-4-OCH2CO-Gly-OMe (5): By general procedure
B. The crude residue was purified by flash column chromatography
on silica gel, with DCM/EtOH (5%)/EtOAc (5%) as eluents. The
white solid obtained was precipitated from DCM/diethyl ether to
give a white solid in 20% overall yield. 5: 1H NMR (200 MHz,
20 m in CD3CN, 300 K): δ ϭ 9.18 (s, 2 H, Py-NH1CO),
7.58Ϫ7.48 (3 H, NH-Gly and CH-Py), 7.33Ϫ7.21 (m, 10 H, aro-
matic), 6.56 (d, J ϭ 6.0 Hz, 2 H, NH3-SO2), 6.38 (d, J ϭ 10.0 Hz,
2 H, NH4-CO), 5.71 (d, J ϭ 10.0 Hz, 2 H, NH2-SO2), 4.58 (s, 2 H,
OϪCH2-Gly), 4.38Ϫ4.29 (m, 4 H, CH9-iPr and CH14-iPr), 3.96 (d,
J ϭ 6.0 Hz, 2 H, CH2-Gly), 3.71Ϫ3.65 (m, 5 H, OMe and CH6-
Phe), 3.26Ϫ2.63 (m, 12 H, CH122SO2 and CH82SO2 and CH72Ph),
1.88 (s, 6 H, CH315CO), 1.82Ϫ1.73 (m, 4 H, CH10Me2 and
CH16Me2), 0.87Ϫ0.70 (m, 24 H, CH313 and CH131). 1H NMR
(400 MHz, 2 m in CDCl3, 300 K): δ ϭ 9.27 (s, 2 H, Py-NH1CO),
7.43 (br. s, 1 H, NH-Gly), 7.37Ϫ7.28 (m, 12 H, aromatic), 6.24 (br.
s, 2 H, NH3-SO2), 6.17 (br. s, 2 H, NH4-CO), 5.72 (br. s, 2 H, NH2-
SO2), 4.57 (s, 2 H, OϪCH2-Gly), 4.46 (br. s, 4 H, CH9-iPr and
CH14-iPr), 4.17 (s, 2 H, CH2-Gly), 3.79 (br. s, 5 H, OMe and CH6-
Phe), 3.40Ϫ3.37 (m, 4 H, CH122SO2 and CH82SO2), 3.20Ϫ3.12 (m,
2 H, CH122SO2), 3.03Ϫ2.97 (m, 2 H, CH82SO2), 2.77 (br. s, 2 H, CH
72Ph), 2.56 (br. s, 2 H, CH72Ph), 2.04 (br. s, 8 H, CH135CO and
CH16Me2), 1.88 (br. s, 2 H, CH10Me2), 0.89 (br. s, 12 H, CH133),
2,6-Bis( -Ac-L-Phe-NH)pyridine-4-OCH2CO-Gly-OMe (3): This
compound was prepared by general procedure A. The yellow solid
obtained was precipitated twice from DCM/diethyl ether to give a
white solid in 30% overall yield. 3: 1H NMR (200 MHz, 8.5 in
[D6]acetone, 300 K): δ ϭ 9.39 (s, 2 H, Py-NHCO), 8.07 (t, J ϭ
6.0 Hz, 1 H, NH-Gly), 7.76 (d, J ϭ 8.0 Hz, 2 H, NHAc), 7.42 (s,
2 H, CH-Py), 7.35Ϫ7.10 (m, 10 H, aromatic), 4.96Ϫ4.85 (m, 2 H,
CH-Phe), 4.53 (s, 2 H, OϪCH2-Gly), 4.01 (d, J ϭ 6.0 Hz, 2 H,
CH2-Gly), 3.63 (s, 3 H, OMe), 3.29Ϫ3.19 (m, 2 H, CH2Ph),
3.02Ϫ2.90 (m, 2 H, CH2Ph), 1.90 (s, 6 H, CH3CO). 1H NMR
(400 MHz, 8.5 in CDCl3, 300 K): δ ϭ 8.83 (s, 2 H, Py-NHCO),
7.35 (br. s, 1 H, NH-Gly), 7.33Ϫ7.23 (m, 12 H, aromatic), 6.56 (d,
J ϭ 8.0 Hz, 2 H, NHAc), 4.96Ϫ4.91 (m, 2 H, CH-Phe), 4.49 (s, 2
H, OϪCH2-Gly), 4.17Ϫ4.11 (m, 2 H, CH2-Gly), 3.78 (s, 3 H,
OMe), 3.33Ϫ3.27 (m, 2 H, CH2Ph), 3.14Ϫ3.09 (m, 2 H, CH2Ph),
2.03 (s, 6 H, CH3CO). 13C NMR (75.4 MHz, 8.5 m, CDCl3,
300 K): δ ϭ 171.4, 170.2, 167.6, 166.1 (CO), 150.6, 136.4 (C,
arom.), 129.2, 128.6, 127.0 (CH, arom.), 96.3 (CH, Py), 66.8 (CH2,
OCH2CO), 55.3 (CH, CH-Phe), 52.3 (CH3, OCH3), 40.7 (CH2,
CH2-Gly), 37.1 (CH2, CH2Ph), 23.2 (CH3, CH3CO). MS (FAB):
m/z ϭ 633 [M ϩ H]ϩ; 655 [M ϩ Na]ϩ. HRMS (FAB) calcd. for
C32H37N6O8 [M ϩ H]ϩ: 633.26674, found m/z: 633.26722.
2-[( )- -AcNHCH( Pr)CH2SO2-L-Phe-NH]-6-( -Alloc-L
-Phe- 0.77 (br. s, 12 H, CH131). 13C NMR (100.6 MHz, 20 m in CDCl3,
NH)pyridine-4-OCH2CO-Gly-OBn (4): The asymmetric scaffold 11
300 K): δ ϭ 172.3, 170.9, 168.1, 166.7 (CO), 151.0, 137.1 (C,
Eur. J. Org. Chem. 2001, 4625Ϫ4634
4632

Acyclic Receptors for N-Protected Amino Acids and Dipeptides
FULL PAPER
[18]
[19]
Test 1 (TNBS test): W. S. Hancock, J. E. Battersby, Anal. Bi-
ochem. 1976, 71, 260Ϫ264. Test 2 (NF31 test): A. Madder, N.
Farcy, N. G. C. Hosten, H. De Muynk, P. J. De Clercq, J. Barry,
A. P. Davis, Eur. J. Org. Chem. 1999, 2787Ϫ2791. See more
details in the Exp. Sect. and in refs.[31,32]
Electronic Supporting Information available (see footnote on
the first page of this article): details on concentration depend-
ence experiments, details of temperature dependence experi-
ments, NMR binding studies and chemical shift data.
arom.), 130.2, 129.2, 127.8 (CH, arom.), 97.1 (CH, Py), 67.2 (CH2,
OCH2CO), 60.2 (CH, CH-Phe), 56.1 (CH2, CH2SO2), 55.1 (CH3,
OCH3), 52.8 (CH, CH-iPr), 50.8 (CH, CH-iPr), 41.2 (CH2, CH2,
CH2-Ph), 39.2 (CH2-Gly), 33.7 (CH, CHMe2), 32.1 (CH, CHMe2),
23.8 (CH3, CH3CO), 19.6 (CH3), 18.2 (CH3). MS (FAB): m/z ϭ
1230 [M ϩ H]ϩ; 1252 [M ϩ Na]ϩ. HRMS (FAB) calcd. for
C52H81N10O16S4 [M ϩ H]ϩ: 1229.47094, found m/z: 1229.47036.
[20]
[21]
[22]
L. Belvisi, C. Gennari, A. Mielgo, D. Potenza, C. Scolastico,
Eur. J. Org. Chem. 1999, 2, 389Ϫ400.
G. J. Pernia, J. D. Kilburn, J. W. Essex, R. J. Mortishire-Smith,
M. Rowley, J. Am. Chem. Soc. 1996, 118, 10220Ϫ10227.
Acknowledgments
H. J. Dyson, P. E. Wright in 2-Dimensional NMR spectroscopy
(Eds.: W. R. Croasmun, R. M. K. Carlson), VCH-Verlagsge-
sellschaft, Weinheim, 1994, p. 672. We checked the possibility
that the observed contact between the hydrogen atom at the
amino acid stereogenic centre and the acetyl hydrogen atoms
might be an artefact due to scalar coupling-transfer through
the NH signal; this spin diffusion interference is usually caused
by the frequency of the transmitter. However, ROESY spectra
recorded with a different transmitter frequency (original trans-
mitter frequency: 5 ppm; modified transmitter frequencies: 6
ppm and 7 ppm) gave the same results. Thus, the observed
contacts are real, and account for a certain percentage of cis-
amide.
R. R. Gardner, S. L. Mc Kay, S. H. Gellman, Org. Lett. 2000,
2, 2335Ϫ2338, and references therein. No separate set of sig-
nals due to the cis rotamer could be detected in the proton
spectra of 1, and it was therefore concluded that its contribu-
tion does not exceed the standard percentage (Յ 5%).
Data were analysed by using a modified version of the algo-
rithm described by C. A. Hunter and used in his software
NMRTit HG (see: A. P. Bisson, C. A. Hunter, J. C. Morales, K.
Young, Chem. Eur. J. 1998, 4, 845Ϫ851) to allow simultaneous
evaluation of data from several signals in the NMR of the host.
The normalised sum of squares for all the experimental data,
χ2 ϭ (obsd. Ϫ calcd.)2/σ2, was minimised by floating the host
and hostϪguest chemical shifts for each proton considered and
the common binding constant by using the solver function in
a Microsoft ExcelTM spreadsheet.
We thank the Commission of the European Union (TMR Network
grant ‘‘Enantioselective separations’’ ERB FMRX-CT-98Ϫ0233)
for financial support and for postdoctoral and postgraduate fellow-
ships to E. Botana, S. Ongeri and M. Demarcus.
[1]
For a recent review on peptide receptors, see: [1a]M. W. Peczuh,
A. D. Hamilton, Chem. Rev. 2000, 100, 2479Ϫ2494. See also:
[1b]
P. D. Henley, C. P. Waymark, I. Gillies, J. D. Kilburn, J.
Chem. Soc., Perkin Trans. 1 2000, 1021Ϫ1031, and references
therein.
[23]
[24]
[2]
W. C. Still, Acc. Chem. Res. 1996, 29, 155Ϫ163.
C. Gennari, H. P. Nestler, U. Piarulli, B. Salom, Liebigs
[3]
Ann./Recueil 1997, 637Ϫ647.
For original work on tweezer receptors, see:
[4]
[4a]
C.-W. Chen,
H. W. Whitlock Jr., J. Am. Chem. Soc. 1978, 100, 4921Ϫ4922.
[4b]
S. C. Zimmerman, W. Wu, Z. Zeng, J. Am. Chem. Soc.
1991, 113, 196Ϫ201.
[5] [5a]
H. Wennemers, S. S. Yoon, W. C. Still, J. Org. Chem. 1995,
[5b]
60, 1108Ϫ1109.
Y. Shao, W. C. Still, J. Org. Chem. 1996,
61, 6086Ϫ6087. [5c] Z. Pan, W. C. Still, Tetrahedron Lett. 1996,
37, 8699Ϫ8702. [5d] M. D. Weingarten, K. Sekanina, W. C. Still,
[5e]
J. Am. Chem. Soc. 1998, 120, 9112Ϫ9113.
M. Torneiro, W.
C. Still, Tetrahedron 1997, 53, 8739Ϫ8750.
[6] [6a]
R. Boyce, G. Li, H. P. Nestler, T. Suenaga, W. C. Still, J.
Am. Chem. Soc. 1994, 116, 7955Ϫ7956. [6b] Y. Cheng; T. Suen-
aga, W. C Still, J. Am. Chem. Soc. 1996, 118, 1813Ϫ1814.
[25]
[26]
[27]
R. E. Lenkinski, J. Reuben, J. Am. Chem. Soc. 1976, 98,
3089Ϫ3094.
[7] [7a]
M. Davies, M. Bonnat, F. Guillier, J. D. Kilburn, M. Brad-
[7b]
ley, J. Org. Chem. 1998, 63, 8696Ϫ8703.
G. M. Kyne, M.
For a full set of all the binding data and curve-fitting, see ref.[10]
and associated electronic Supporting Information; see ref.[19]
E. Light, M. B. Hursthouse, J. de Mendoza, J. D. Kilburn, J.
Chem. Soc., Perkin Trans. 1 2001, 1258Ϫ1263.
Ha and Hb were named as follows: ROϪCOϪ
NHaϪCH(Me)COOH;
[8]
S. R. LaBrenz, J. Am. Chem. Soc. 1995, 117, 1655Ϫ1656.
[9] [9a]
T. Fessmann, J. D. Kilburn, Angew. Chem. Int. Ed. 1999,
ROϪCOϪNHaϪCH(Me)COϪNHbϪCH(R1)COOH.
[9b]
38, 1993Ϫ1996.
T. Braxmeier, M. Demarcus, T. Fessmann,
[28]
ROESY spectra were recorded for: (a) host 5 alone (5 m in
CDCl3); (b) host 5 (5 m in CDCl3) with Cbz--Ala--Ala-
OH (10 m in CDCl3) Ϫ i.e., guest/host ratio of 2:1 (ca. 80%
saturation); (c) host 5 (2 m in CDCl3) with Cbz--Ala--Ala-
OH (10 m in CDCl3) Ϫ i.e., guest/host ratio of 5:1 (ca. 92%
saturation). In spectrum (a) we detected the NOE contacts,
which we have already described in the text. In spectrum (b)
we essentially detected no major changes. The experiment
therefore gave no useful information regarding the binding. In
spectrum (c) very few signals could be observed and the signal-
to-noise ratio was too high.
S. McAteer, J. D. Kilburn, Chem. Eur. J. 2001, 7, 1889Ϫ1898.
[10] [10a] J. Dowden, P. D. Edwards, S. S. Flack, J. D. Kilburn, Chem.
[10b]
Eur. J. 1999, 5, 79Ϫ89.
P. D. Henley, J. D. Kilburn, Chem.
Commun. 1999, 1335Ϫ1336.
[11]
[12]
[13]
C. Gennari, H. P. Nestler, B. Salom, W. C. Still, Angew. Chem.
Int. Ed. Engl. 1995, 34, 1765Ϫ1768.
D. W. P. M. Löwik, S. J. E. Mulders, Y. Cheng, Y. Shao, R. M.
J. Liskamp, Tetrahedron Lett. 1996, 37, 8253Ϫ8256.
D. W. P. M. Löwik, M. D. Weingarten, M. Broeckema, A. J.
Brouwer, W. C. Still, R. M. J. Liskamp, Angew. Chem. Int. Ed.
1998, 37, 1846Ϫ1850.
[29]
[14]
[15]
[16]
For examples of the screening of a library of receptors with a
C. Gennari, M. Gude, D. Potenza, U. Piarulli, Chem. Eur. J.
1998, 4, 1924Ϫ1931.
[29a]
chosen substrate, see refs.[2,5d,6a,6b,9b]; see also:
H. P.
Nestler, Mol. Diversity 1996, 2, 35Ϫ40. [29b] R. Xu, G. Greiveld-
inger, L. E. Marenus, A. Coopper, J. A. Ellman, J. Am. Chem.
H. Hioki, T. Yamada, C. Fu-
jioka, M. Kodama, Tetrahedron Lett. 1999, 40, 6821Ϫ6825.
B. Salom, D. Potenza, C. Gennari, C. Longari, E. Fioravanzo,
O. Carugo, N. Sardone, Chem. Eur. J. 1996, 2, 644Ϫ655.
For a preliminary communication on part of this work, see: E.
Botana, S. Ongeri, R. Arienzo, M. Demarcus, J. G. Frey, U.
Piarulli, D. Potenza, C. Gennari, J. D. Kilburn, Chem. Com-
mun. 2001, 1358Ϫ1359.
[29c]
Soc. 1999, 121, 4898Ϫ4899.
[30]
For examples of the screening of a library of substrates towards
a particular receptor, see: H. Wennemers, M. Conza, M. Nold,
P. Krattiger, Chem. Eur. J. 2001, 7, 3342Ϫ3347, and
refs.[5a,7,11Ϫ13]
[17]
M. Gude, U. Piarulli, D. Potenza, B. Salom, C. Gennari, Tetra-
hedron Lett. 1996, 37, 8589Ϫ8592.
Eur. J. Org. Chem. 2001, 4625Ϫ4634
4633

J. D. Kilburn, C. Gennari et al.
FULL PAPER
[31]
TNBS test: A few resin beads were sampled and washed several
(nitrophenyl)azo]phenyl]amino-3-oxapentanoate}. After heat-
ing at 70 °C for 10 min, the beads were filtered and washed
with DMF (3ϫ), MeOH (3ϫ) and DCM (3ϫ). The beads were
inspected under a microscope. Beads containing free amino
functions appear as red spheres while completely coupled beads
(no free amino groups) remain colourless. For further details
see ref.[18]
times with ethanol. The sample was placed in a vial and 1Ϫ2
drops (25Ϫ50 µL) of a 10% of DIPEA solution in DMF and
1Ϫ2 drops of 1% 2,4,6-trinitrobenzenesulfonic acid (TNBS) in
DMF were added. The sample was inspected under a micro-
scope and colour changes were noted. The TNBS test is consid-
ered to be positive (presence of free amino groups) if the resin
beads turn orange or red within a minute and negative if the
beads remain colourless. For further details see ref.[18]
[33]
D. L. Holmes, E. M. Smith, J. S. Nowick, J. Am. Chem. Soc.
1997, 119, 7655Ϫ7669.
[32]
NF31 test: A few beads of resin were suspended in 100 µL
Received July 18, 2001
of a 2 m solution of NF31 {4-nitrophenyl-5-[N-ethyl-N-[4-
[O01354]
4634
Eur. J. Org. Chem. 2001, 4625Ϫ4634