Stereoselective recognition of tripeptides guided by encoded library screening: Construction of chiral macrocyclic tetraamide ruthenium receptor for peptide sensing

Article abstract of DOI:10.1021/jo048368s

(Chemical Equation Presented) Molecule sensor 1 is devised by incorporating the reporting unit of ruthenium(II) complex and two recognition motifs of chiral cyclotetraamides on the sidearms. The target binding tripeptides for sensor 1 were readily identified by using an encoded library screening method. This solid-phase screening indicated a preferable binding of molecule 1 with D-alanine over the L-isomer. The optical and NMR studies for the binding events of 1 with tripeptides Ac-Ala-Gly-Ala-NHC₁₂H₂₅ in the solution phase showed a consistent trend for the stereoselective recognition of the DD-isomer over the LD-, DL-, and LL-isomers.

Full text of DOI:10.1021/jo048368s

Stereoselective Recognition of Tripeptides Guided by Encoded
Library Screening: Construction of Chiral Macrocyclic
Tetraamide Ruthenium Receptor for Peptide Sensing
Kuei-Hua Chang,† Jen-Hai Liao,† Chao-Tsen Chen,*,† Barun K. Mehta,† Pi-Tai Chou,* and
Jim-Min Fang*,†,‡
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan, and
Genomic Research Center, Academia Sinica, Taipei 115, Taiwan
jmfang@ntu.edu.tw
Received September 15, 2004
Molecule sensor 1 is devised by incorporating the reporting unit of ruthenium(II) complex and two
recognition motifs of chiral cyclotetraamides on the sidearms. The target binding tripeptides for
sensor 1 were readily identified by using an encoded library screening method. This solid-phase
screening indicated a preferable binding of molecule 1 with D-alanine over the L-isomer. The optical
and NMR studies for the binding events of 1 with tripeptides Ac-Ala-Gly-Ala-NHC12H25 in the
solution phase showed a consistent trend for the stereoselective recognition of the DD-isomer over
the LD-, DL-, and LL-isomers.
Introduction
An efficient peptide receptor must exhibit a spacious
recognition site in order to accommodate the relatively
large guest molecule. For this purpose, acyclic and
macrocyclic structures are often applied as the recogni-
tion motifs, to which crown ethers can be incorporated
to enhance the interaction with the terminal ammonium
group of the peptide.3 Acyclic peptide receptors are
generally constructed by implanting two peptide or
multiple-amide chains on a structurally defined back-
bone, such as steroid,4,5 calixarene,6,7 diketopiperazine,8
and dibenzofuran.9 Conversely, a macrocyclic tetraamide
receptor has been prepared by using five building blocks
Fundamental studies of peptide recognition provide a
way to investigate the more complicated protein inter-
actions, which are often addressed in chemical biology
and drug discovery research. The main binding forces
between a peptide and its host receptor include hydrogen
bonding, electrostatic, and hydrophobic interactions.1 For
example, vancomycin is a potent antibacterial agent,
which displays quadruple hydrogen bindings with the
D-Ala-D-Ala fragment that is essential for formation of
bacterial cell wall.2 The new strain of bacteria by muta-
tion of the D-Ala-D-Ala dipeptide into D-Ala-D-lactate can
resist the attack of vancomycin, presumably due to
elimination of a crucial hydrogen bonding and the
increase of repulsive electronic interactions between the
lactate portion and vancomycin.2
(3) (a) Tsubaki, K.; Kusumoto, T.; Hayashi, N.; Nuruzzaman, M.;
Fuji, K. Org. Lett. 2002, 4, 2313. (b) Tsubaki, K.; Nuruzzaman, M.;
Kusumoto, T.; Hayashi, N.; Wang, B.-G.; Fuji, K. Org. Lett. 2001, 3,
4071.
(4) (a) Cheng, Y.; Suenaga, T.; Still, W. C. J. Am. Chem. Soc. 1996,
118, 1813. (b) Boyce, R.; Li, G.; Nestler, H. P.; Suenaga, T.; Still, W.
C. J. Am. Chem. Soc. 1994, 116, 7955.
† National Taiwan University.
(5) Muynck, H. D.; Madder, A.; Farcy, N.; Clercq, P. J. D.; Pe´rez-
Paya´n, M. N.; O¨ hberg, L. M.; Davis, N. P. Angew. Chem., Int. Ed. 2000,
39, 145.
‡ Academia Sinica.
(1) Stites, W. E. Chem. Rev. 1997, 97, 1233.
(2) Healy, V. L.; Lessard, I. A.; Roper, D. I.; Knox, J. R.; Walsh, C.
T. Chem. Biol. 2000, 7, R109.
(6) van Wageningen, A. M. A.; Liskamp, R. M. J. Tetrahedron Lett.
1999, 40, 9347.
10.1021/jo048368s CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/10/2005
2026
J. Org. Chem. 2005, 70, 2026-2032

Stereoselective Recognition of Tripeptides
NH of the amido group and CdO of the vicinal amido
group form an ideal hydrogen donor/acceptor pair suit-
able for bonding with R-amino acid derivatives.15,16 This
chiral receptor is expected to exert different complexation
strengths with respect to D- and L-amino acid derivatives.
Previous HPLC studies15,16d have revealed that chiral
columns using A2B2 derivatives as a stationary phase are
applicable to the separation of derivatized isomers of
R-amino acids and tripeptides. In this regard, the cage
compound with an A4B6 motif also serves as a receptor
for dipeptide and tripeptide derivatives.11, 17
As for the signal transduction, the molecular receptors
of A2B2 and A4B4 scaffolds are elaborated to detect peptide
derivatives by attaching suitable fluorophore/quencher
pairs, e.g., dansyl and dabcyl, via the fluorescence
resonance energy transfer (FRET) mechanism.18 In the
absence of guest molecule, the free receptor shows rather
weak fluorescence due to the efficient FRET, followed by
the dominant nonradiative deactivation pathways. Upon
complexation with the target peptide, an enhanced
fluorescence is observed as the fluorophore and quencher
are pushed apart.
On the basis of the protocol of two-armed receptors
devised by Still’s group,11,16,17 we demonstrate herein a
novel molecule sensor 1 that is used to detect tripeptides
with a high sequence- and stereoselectivity. Compound
1 is designed to incorporate two A2B2 motifs as the
recognition units16a,d and a tris(bipyridine) ruthenium-
(II) complex as the signal transduction unit (Figure 2).
The two macrocyclic A2B2 units are linked to a bipyridine
moiety via two amide bonds. Thus, the multiple-amide
unit provides a desirable semirigid structure and suf-
ficient hydrogen bonding sites for peptide recognition.
The appropriate disposition of two A2B2 motifs in mol-
ecule 1 may also enhance the binding with the target
peptides in a cooperative manner.16 The Ru(II) center is
sensitive to the binding event so that the changes of
luminescence properties and redox potential can be
readily monitored and correlated to the binding strength.19
FIGURE 1. A2B2 motif formed by two units of isophthalic acid
(A) and two units of vicinal diamine (B) in recognition of
R-amino acid derivatives via double hydrogen bondings.
consisting of 2,6-diaminopyridine, phenylalanine, succinic
acid, (4-aminomethyl)phenol, and (4′-bromomethylbiphen-
yl)acetic acid.10 This receptor displays a selective binding
to dipeptide N-Cbz-(â-Ala)-Ala. A similar macrocyclic
hexaamide receptor is derived from a core structure of
1,3,5-trimercaptobenzene to form a confined deep-basket
shape that well accommodates the guest molecule of
tripeptide cPr-Ala-Pro-Ala-NHC12H25.11 A podant iono-
phore12 having a three-dimensional framework also
shows a sequence-selective recognition of L-Arg-L-Phe-D-
Asp. Cyclodextrins13 and Kemp’s acid amide derivatives14
are also suitable motifs for peptide recognition. The
receptor molecules constructed by incorporation of such
motifs exhibit high affinity toward linear and cyclic
peptides. A multiloop receptor has been built by extension
of four hydrophilic cyclic peptide loops, Gly-Asp-Gly-Asp,
on a hydrophobic calix[4]arene core.7 This molecular
receptor thus provides multiple electrostatic and hydro-
gen bonding interactions to bind with the surface peptide
of cytochrome c that contains abundant lysine residues.7
In view of sensing peptides in an enantioselective
fashion, of special interest are the molecular receptors
with chiral recognition sites, e.g., the A2B2 motif of an
octadecacyclotetramide structure (Figure 1). The A2B2
motif is readily constructed by two units of isophthalic
acid and two units of (1R,2R)-1,2-diphenylethane-
diamine15 [or (1R,2R)-1,2-cyclohexanediamine],16 in which
Results and Discussion
Synthesis. Molecular sensor 1 was constructed by five
building blocks: isophthalic acid bis-chloride (2), (R,R)-
cyclohexane-1,2-diamine (3), 5-(azidomethyl)isophthalic
acid (4), 2,2′-bipyridine-4,4′-dicarboxylic acid (5), and cis-
(bpy)2RuCl2 (6). When diamine 3 was treated with Boc2O,
a mixture of mono-Boc and di-Boc derivatives were
obtained. To avoid this complication, an indirect method
was applied to prepare the mono-Boc derivative 7 (Scheme
1).20 Thus, diamine 3 was first converted to the N,N′-bis-
Cbz derivative, which was then reacted selectively with
(7) (a) Peczuh, M. W.; Hamilton, A. D. Chem. Rev. 2000, 100, 2479.
(b) Hamuro, Y.; Calama, M. C.; Park, H. S.; Hamilton, A. D. Angew.
Chem., Int. Ed. Engl. 1997, 36, 2680.
(8) (a) Wennemers, H.; Conza, M.; Nold, M.; Krattiger, P. Chem.
Eur. J. 2001, 7, 3342. (b) Conza, M.; Wennermers, H. J. Org. Chem.
2002, 67, 2696.
(9) LaBrenz, S. R.; Kelly, J. W. J. Am. Chem. Soc. 1995, 117, 1655.
(10) Dowden, J.; Edwards, P. D.; Flack, S. S.; Kilburn, J. D. Chem.
Eur. J. 1999, 5, 79.
(11) (a) Borchardt, A.; Still, W. C. J. Am. Chem. Soc. 1994, 116, 373.
(b) Borchardt, A.; Still, W. C. J. Am. Chem. Soc. 1994, 116, 7467.
(12) Burger, M. T.; Still, W. C. J. Org. Chem. 1997, 62, 4785.
(13) (a) Breslow, R.; Yang, Z.-W.; Ching, R.; Trojandt, G.; Odobel,
F. J. Am. Chem. Soc. 1998, 120, 3536. (b) Breslow, R.; Zhang, B. J.
Am. Chem. Soc. 1992, 114, 5882. (c) Breslow, R.; Greenspoon, N.; Guo,
T.; Zarzycki, R. J. Am. Chem. Soc. 1989, 111, 8296. (d) Zhang, B.;
Breslow, R. J. Am. Chem. Soc. 1993, 115, 9353. (e) Maletic, M.;
Wennemers, H.; McDonald, D. Q.; Breslow, R.; Still, W. C. Angew.
Chem., Int. Ed. Engl. 1996, 35, 1490.
(17) (a) Yoon, S. S.; Still, W. C. J. Am. Chem. Soc. 1993, 115, 823.
(b) Yoon, S. S.; Still, W. C. Tetrahedron 1995, 51, 567.
(18) (a) Chen, C.-T.; Wagner, H.; Still, W. C. Science 1998, 279, 851.
(b) Rothman, J. H.; Still, W. C. Bioorg. Med. Chem. Lett. 1999, 9, 509.
(19) Sauvage, J.-P.; Collin, J.-P.; Chambron, J.-C.; Guillerez, S.;
Coudret, C.; Baltani, V.; Barigelletti, F.; De Cola, L.; Famigni, L. Chem.
Rev. 1994, 94, 993. (a) Szemes, F.; Hesek, D.; Chen, Z.; Dent, S. W.;
Drew, M. G. B.; Goulden, A. J.; Graydon, A. R.; Grieve, A.; Mortimer,
R. J.; Wear, T.; Weightman, J. S.; Beer, P. D. Inorg. Chem. 1996, 35,
5868. (b) Prasanna de Silva, A.; Nimal Guaratne, H. Q.; Gunnlaugsson,
T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem.
Rev. 1997, 97, 1515. (c) Keefe, M. H.; Benkstein, K. D.; Hupp, J. T.
Coord. Chem. Rev. 2000, 205, 201.
(14) Jeong, K.-S.; Muehldorf, A. V.; Rebek, J., Jr. J. Am. Chem. Soc.
1990, 112, 6144.
(15) Gasparrini, F.; Misiti, D.; Pierini, M.; Villani, C. Org. Lett. 2002,
4, 3993.
(16) (a) Wennemers, H.; Yoon, S. S.; Still, W. C. J. Org. Chem. 1995,
60, 1108. (b) Still, W. C. Acc. Chem. Res. 1996, 29, 155. (b) Hu, K.;
Bradshaw, J. S.; Dalley, N. K.; Krakowiak, K. E.; Wu, N.; Lee, M. L.
J. Heterocycl. Chem. 1999, 36, 381. (d) Gasparrini, F.; Misiti, D.; Still,
W. C.; Villani, C.; Wennemers, H. J. Org. Chem. 1997, 62, 8221.
(20) Kim, Y. Y.; Lee, S. J.; Ahn, K. H. J. Org. Chem. 2000, 65, 7807.
J. Org. Chem, Vol. 70, No. 6, 2005 2027

Chang et al.
FIGURE 2. Construction of molecular sensor 1 by five building blocks: isophthalic acid bis-chloride (2, two units), (1R,2R)-
cyclohexane-1,2-diamine (3, four units), 5-(azidomethyl)isophthalic acid (4, two units), 2,2′-bipyridine-4,4′-dicarboxylic acid (5,
one unit), and cis-(bpy)2RuCl2 (one unit).
SCHEME 1. Synthesis of Molecular Sensor 1a
presumably due to the sterically demanding environ-
ment. After the Cbz groups were removed by catalytic
hydrogenation, the mono-Boc derivative 7 was obtained
in 67% overall yield. The coupling reaction of 7 (2 equiv)
with isophthalic acid bis-chloride (2) occurred smoothly
in the presence of Hu¨nig base to give diamide 8.16b After
removal of the Boc groups by CF3COOH, the resulting
amine (as the TFA salt) was treated with the pentafluo-
rophenyl diester of 5-(azidomethyl)isophthalic acid in the
presence of Hu¨nig base to give a cyclotetraamide 9. The
azido group of 9 was reduced by catalytic hydrogenation
or by using Ph3P/H2O (Staudinger reaction); and the
amine product (2 equiv) was then reacted with 2,2′-
bipyridine-4,4′-dicarboxylic acid (5) using EDC and HOBt
as the coupling reagents. The subsequent reaction with
cis-Ru(bpy)2Cl2, followed by exchange of Cl- with PF6
-
counterions, thus culminated in the synthesis of sensor
1 with a Ru reporter and double A2B2 recognition motifs.
Because one of bipyridyl ligands contains the chiral A2B2
substituents, the Ru(II) compound 1 likely exists in two
diastereomeric forms with ∆- or Λ-configurations at the
ruthenium center. This speculation is supported by the
NMR study (vide infra).
a Reagents and conditions: (i) i-Pr2NEt, CH2Cl2, 25 °C, 3 h, 95%;
(ii) TFA, CH2Cl2, 25 °C, 1.5 h, then diC6F5-ester of 4, i-Pr2EtN,
THF, 25 °C, 12 h, 99%; (iii) Ph3P, THF, reflux, 2 h, H2O, THF,
reflux, 14 h, 87%, or H2, 10% Pd/C, MeOH, 25 °C, 8 h, 99%; (iv)
diacid 5, EDC, HOBt, DMF, rt, 49 h, 87%; (v) 6 (hydrate), EtOH,
H2O, reflux, 6 h, NH4PF6, 83%.
The Ru(II) compound 1 (as the PF6- salt) appeared as
orange red solids. Its CH2Cl2 solution showed the char-
acteristic absorption bands at λmax ) 289 nm (ꢀ ) 73 200
M-1cm-1) for the bipyridyl ligand and at λmax ) 457 nm
(ꢀ ) 15 100 M-1cm-1) attributable to the metal to ligand
Boc2O to give exclusively the N-Boc-N,N′-Cbz derivative.
Attachment of the second Boc group was prohibited
2028 J. Org. Chem., Vol. 70, No. 6, 2005

Stereoselective Recognition of Tripeptides
FIGURE 3. Bead-bound tripeptide library with electrophoric
tags. Tripeptide AA1-AA2-AA3 represents any combination of
15 amino acids: Gly, L-Ala, D-Ala, L-Val, D-Val, L-Ser, D-Ser,
L-Pro, D-Pro, L-Asn, D-Asn, L-Gln, D-Gln, L-Lys, and D-Lys.
charge transfer (MLCT).19 In more polar media [e.g., a
mixed solution of MeOH/CHCl3 (1:9)], the absorption
maxima shifted to longer wavelengths, 299 nm (ꢀ )
71 100 M-1cm-1) and 465 nm (ꢀ ) 15 300 M-1cm-1). Upon
excitation at 457 nm, compound 1 (2 × 10-6 M) exhibited
an emission band with peak wavelengths at 595 nm (Φ
∼0.054) and 630 nm in CH2Cl2 and MeOH/CHCl3 (1:9),
respectively. The relatively long lifetime of 1.2 µs mea-
sured in aerated CH2Cl2 leads us to unambiguously
assign the 595-nm emission (or 630 nm in MeOH/CHCl3)
to a phosphorescence manifold. In degassed CH2Cl2 the
lifetime increases to 1.7 µs. Taking O2 concentration of
2.6 × 10-3 M in aerated CH2Cl2,21 an O2 quenching rate
constant of ∼9.0 × 107 M-1 s-1 was thus deduced, which
is ∼20 times smaller than 1/9 of the diffusion controlled
rate of 1.8 × 1010 M-1 s-1 calculated in CH2Cl2.22 Note
that the O2-triplet quenching rate is generally derived
from the theory of electron-exchange type of energy
transfer, in which the overall spin must be conserved
upon forming a collisional complex. Accordingly, the
possibility of each collision generating 1O2 is statistically
1/9. The relatively small O2 quenching constant of 9.0 ×
107 M-1 s-1 in 1 clearly indicates that the 3MLCT
emission is less effectively quenched by O2 due to the
introduction of two chiral cyclotetraamide motifs on the
sidearms.
Screening of Tripeptide Library. To find the pep-
tides best fitted to molecule 1, we screened a 15 × 15 ×
15 tripeptide library (3375 different combinations) that
has been established by Still and co-workers using
encoded split combinatorial chemistry (Figure 3).11,16a,23
The tripeptide-bound polystyrene beads incorporate a
photocleavable linker (o-nitrobenzyl carbonate) and var-
ied electrophoric tags (chlorinated phenoxyalkanes) for
encoding specific sequences of tripeptides.
A typical screening procedure is described as follows.
The combinatorial tripeptide beads (1 mg, containing
about 4 copies of each tripeptide component) were
suspended in CHCl3 (0.5 mL). A solution of sensor 1 (40
µL, 1 × 10-3 M) in MeOH/CHCl3 (1:9) was added, and
FIGURE 4. Phosphorescence titration spectra of 1 (4.0 × 10-6
M) by addition of various amounts of the tripeptide derivative
10-DD [as a 4.0 × 10-3 M solution in MeOH/CHCl3 (1:9)] at
293 K, λex ) 466 nm. Inset: A curve fitting of the data using
the nonlinear least-squares method. Ka is derived to be 2.9 ×
105 M-1
.
the mixture was agitated for 40 h to ensure equilibrium.
The 22 deep red beads that refer to the binding of 1 with
the surface tripeptides were picked out under a micro-
scope. The selected beads were then irradiated with 365-
nm light to remove the photolabile linkers, and the
released electrophoric tags were decoded by gas chroma-
tography using an electron capture detector.
Based on this approach, we quickly identified 19
tripeptides having high affinity toward the host molecule
1 (see the Supporting Information). This solid-phase
screening showed preference for three peptide se-
quences: Ac-(D-Ala)-Gly-(D-Ala), Ac-Gly-(D-Ala)-Gly and
Ac-(D-Asn)-(L-Ser-(L-Pro). The binding preference for D-
Ala over L-Ala suggests the stereoselective recognition
between 1 and peptides derived from alanine residues.
Confirmation of Stereoselective Recognition in
Solution System. It is also crucial to evaluate whether
the stereoselective recognition in the solid-phase system
can also be applied in solution. We thus synthesized four
tripeptide isomers, Ac-(D-Ala)-Gly-(D-Ala)-NHC12H25 (10-
DD), Ac-(L-Ala)-Gly-(D-Ala)-NHC12H25 (11-LD), Ac-(D-Ala)-
Gly-(L-Ala)-NHC12H25 (12-DL), and Ac-(L-Ala)-Gly-(L-Ala)-
NHC12H25 (13-LL), and examined their binding behaviors
with compound 1.
The phosphorescence titrations were undertaken to
probe the binding of receptor 1 with tripeptides. In this
experiment, to a solution of 1 (4.0 × 10-6 M) in MeOH/
CHCl3 (1:9, 2 mL) at 20 °C were added aliquots of a
specific tripeptide derivative, e.g., 10-DD (as a 4.0 × 10-3
M solution) in MeOH/CHCl3 (1:9), and the phosphores-
cence spectra (with excitation at 466 nm) were recorded
on individual titration (Figure 4). Despite negligible
changes of the absorption spectra, the phosphorescence
intensity gradually increases as the concentration of 10-
DD increases. This indicates the enhancement of phos-
phorescence yield upon complexation between 1 and the
tripeptide. Two plausible mechanisms were proposed in
an attempt to rationalize the experimental results.
Theoretically, via the rigidification effect,24 the non-
(21) Murov, S. L.; Carmichael, I.; Hug, G. L. Handbook of Photo-
chemistry, 2nd ed.; Marcel Dekker: New York, 1993.
(22) Birks, J. B. Photophysics of Aromatic Molecules; Wiley: New
York, 1970, p 313.
(23) Ohlmeyer, M. H. J.; Swanson, R. N.; Dillard, L. W.; Reader, J.
C.; Asouline, G.; Kobayashi, R.; Wigler, M.; Still, W. C. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 10922.
J. Org. Chem, Vol. 70, No. 6, 2005 2029

Chang et al.
TABLE 1. Association Constants and Free Energy Changes for 1/Tripeptide Complexation in MeOH/CHCl3 (1:9)
Solutiona
entry
peptide derivative
Kab (103 M-1
)
∆Gc (kJ mol-1
)
1
2
3
4
Ac-D-Ala-Gly-D-Ala-NHC12H25 (10-DD)
Ac-L-Ala-Gly-D-Ala-NHC12H25 (11-LD)
Ac-D-Ala-Gly-L-Ala-NHC12H25 (12-DL)
Ac-L-Ala-Gly-L-Ala-NHC12H25 (13-LL)
294 ( 33
170 ( 5
154 ( 27
58 ( 9
-30.7
-29.3
-29.1
-26.7
a Receptor 1 (4.0 × 10-6 M) was titrated with tripeptide (4.0 × 10-3 M) in MeOH/CHCl3 (1:9) at 293 K.Two or three measurements
were performed for each tripeptide substrate. b The average value derived from the phosphorescence intensity change (as the signal area
between 500 and 850 nm) of two independent titrations. The excitation wavelength λex ) 466 nm. c Calculated from ∆G ) -RT ln Ka.
radiative decay rates of free receptor 1 may be reduced
upon multiple hydrogen bonding complexation with tri-
peptides. This, along with the possible stabilization due
to the MLCT in such a supramolecular complex,19 may
rationalize the phosphorescence enhancement during the
titration. Alternatively, the complex formation may, to
certain extents, hamper the penetration of the oxygen
to proceed with the sensitization. Since the absorption
spectral feature both in peak wavelength and absorbance
remained unchanged during titration, the latter case
seems to be favorable. Though an actual mechanism is
pending for resolution, it is unambiguous that the
spectral enhancement originates from the multiple hy-
drogen bonding complexation between 1 and tripeptides.
As a result, the corresponding changes of the phospho-
rescence intensity can be exploited to deduce the ther-
modynamic parameters upon complexation. The phos-
phorescence intensity, taken from the signal area between
500 and 850 nm, was then monitored against the
concentration of tripeptide. The 1:1 stoichiometry and
binding constant (Ka) of complex (Table 1) were deter-
mined by curve fitting using the nonlinear least-squares
method, taking into account the dilution effect.25 The
binding strength toward receptor 1, according to the
deduced association constants (see Table 1), follows an
order of 10-DD > 11-LD ∼ 12-D; > 13-LL. This result clearly
indicated that receptor 1 favorably binds tripeptide 10-
DD which contains two D-Ala residues. The binding
affinity of 10-DD is 5- and 2-fold stronger than its antipode
13-LL and diastereomers 11-LD and 12-DL, respectively.
The resulting stereoselectivity in solution is in good
agreement with that obtained in the solid-phase library
screening.
A similar phenomenon was also observed in the titration
spectra of 1 with 13-LL (see the Supporting Information).
Due to the complex proton configuration, further calcula-
tion of the binding constants on the basis of the concen-
tration dependent chemical-shifts is impractical. Note
that in the phosphorescence titration, to simplify the
derivation, the emission yields of two diastereomeric
complexes of 1/10-DD are assumed to be the same.
In comparison, a prototype A2B2 molecule 14 was
prepared and subjected to 1H NMR titration with alanine
derivatives in CDCl3 solution. The results revealed that
molecule 14 exhibited a weak binding with 3,5-dini-
trobenzoylalanine hexylamide (15) albeit no apparent
binding with Ac-Ala-OMe ester. The association con-
stants for both 14/15-D and 14/15-L complexes at 295 K
in CDCl3 were estimated to be in a range of 50-150 M-1
,
which are 3 orders of magnitude weaker than that of
1/10-DD. Unfortunately, a similar titration experiment for
14 using 10-DD was not feasible due to the sparse
solubility of tripeptide in CDCl3, neither could the titra-
tion experiments be conducted in protic solvents, e.g.,
CD3OD/CDCl3 (1:9), due to the complicated exchange of
amido protons.
1H NMR titration experiments provided supportive
evidence to the binding modes. When receptor 1 was
titrated with tripeptide 10-DD in CH3OD/CDCl3 (1:9), the
NH on the tetraamide rings and the C3/C3′ protons on
the bipyridinediamide rings (see structure 1 in Figure 2
for numbering) showed significant changes in chemical
shifts (Figure 5). This result indicated that the bipy-
ridinediamide moiety, in addition to the A2B2 motifs, also
contributed considerable hydrogen bondings to the com-
plexation with the tripeptide substrate. The NMR analy-
sis indicated that compound 1 might exist as a mixture
of two diastereomers (∼1:1) with different configurations
(∆ or Λ) at the ruthenium center. Upon addition of more
than 22 equiv of 10-DD, the C3/C3′ protons (at ∼9.35 ppm)
shifted downfield and split into two signals, which might
account for the two diastereomeric complexes of 1/10-DD.
We also prepared the analogues of 10-DD and 11-LD by
replacing the acetyl (Ac) capping group with a bulky tert-
butyloxycarbonyl (t-Boc) group. These t-Boc analogues
turned out to have very weak affinity toward molecule
1, and no apparent change of the phosphorescence was
observed. Since the bipyridinediamide moiety in molecule
1 also participated in the molecular recognition of tri-
peptides 10-DD and 11-LD (vide supra), introducing a
bulky t-Boc group might not fit in the interior binding
pocket of 1.
Conclusion. We have demonstrated the differentia-
tion of four tripeptide isomers of Ac-Ala-Gly-Ala-NHC12H25
via a deliberately designed chiral receptor. The two-
armed receptor 1 incorporating a bipyridinediamide
moiety and two chiral cylcotetraamide motifs achieves
an effective multiple hydrogen bondings recognition with
tripeptides in a stereoselective manner. The attachment
(24) (a) Sandanayaka, K. R. A. S.; Nakashima, K.; Shinkai, S. Chem.
Commun. 1994, 1621. (b) Fang, J.-M.; Selvi, S.; Liao, J.-H.; Slanina,
Z.; Chen, C.-T.; Chou, P.-T. J. Am. Chem. Soc. 2004, 126, 3559-3566.
(25) Connors, K. A. Binding Constants; Wiley: New York, 1987.
2030 J. Org. Chem., Vol. 70, No. 6, 2005

Stereoselective Recognition of Tripeptides
FIGURE 5. 1H NMR titration of compound 1 (1 × 10-4 M) by addition of various amounts of 10-DD [as 1 × 10-2 M solution in
CH3OD/CDCl3 (1:9)].
H, m); 13C NMR (DMSO-d6, 100 MHz) δ 166.7 (C), 166.3 (C),
of a Ru(II) reporter renders a real-time detection of the
binding property based on the phosphorescence spectros-
136.4 (C), 135.4 (C), 134.9 (C), 129.7 (CH), 129.4 (CH), 128.3
(CH), 126.7 (CH), 126.5 (CH), 53.8 (CH), 53.7 (CH), 53.0 (CH2),
copy. This, in combination with the encoded library
31.5 (CH2), 24.7 (CH2); FAB-MS m/z (rel intensity) 544 (M +
screening21 technique, proves to be a rapid and efficient
H+, 30); HRMS calcd for C29H34N7O4 (M + H+) 544.2672, found
method for search and sensing of target peptides.
544.2665.
Synthesis of Molecular Sensor 1. To a solution of azide
Experimental Section
9 (500 mg, 0.92 mmol) in THF (40 mL) was added solid PPh3
(362 mg, 1.38 mmol) at room temperature. The mixture was
refluxed for 2 h until no nitrogen evolved. The solution was
cooled, and a mixture of H2O (0.2 mL) and THF (9 mL) was
added. The mixture was refluxed for an additional 14 h,
concentrated, and purified by chromatography on a silica gel
column (CH3OH/CHCl3, 1:4) to give the corresponding amine
as white solids (433 mg, 87%).
Synthesis of Cyclotetraamide 9. To a solution of 5-azi-
domethylisophthalic acid (301 mg, 1.36 mmol) in CH2Cl2 (35
mL) was added a solution of pentafluorophenol (563 mg, 3.06
mmol) and EDC (586 mg, 3.06 mmol) in CH2Cl2 (10 mL). The
mixture was stirred for 3 h at room temperature, concentrated,
and purified by flash chromatography on a silica gel column
(EtOAc/hexane, 1:9) to afford 5-azidomethylisophthalic acid
dipentafluorophenyl ester as white solids (741 mg, 98%).
Diamide 816b (1.75 g, 3.1 mmol) in CH2Cl2 (56 mL) was
treated with TFA (14 mL) for 1.5 h at room temperature to
remove the Boc protecting groups. The reaction mixture was
concentrated under reduced pressure and triturated several
times with Et2O to give precipitates. The solids were collected
and dried in vacuo for at least 6 h to afford the corresponding
TFA salt (1.83 g, quantitative yield).
To a mixture of 5-azidomethyl-isophthalic acid dipentafluo-
rophenyl ester (1.53 g, 2.77 mmol) and the above prepared TFA
salt (1.60 g, 2.77 mmol) suspended in CH2Cl2 (65 mL) was
added i-Pr2NEt (2.85 g. 22 mmol) dropwise over a period of
10 min at room temperature. The mixture was stirred for 12
h, concentrated, and purified by chromatography on a silica
gel column (CH3OH/CH2Cl2, 1:19) to afford compound 9 as
white solids (1.49 g, 99%): mp > 320 °C; TLC (MeOH/CHCl3
(1:19)) Rf ) 0.2; IR (KBr) 3315, 3072, 2105, 1657 cm-1; 1H NMR
(DMSO-d6, 400 MHz) δ 8.41 (2 H, d, J ) 7.6 Hz), 8.36 (2 H, d,
J ) 7.6 Hz), 8.23 (1 H, s), 8.21 (1 H, s), 7.80 (2 H, dd, J ) 7.6,
1.6 Hz), 7.78 (2 H, s), 4.55 (2 H, s), 3.89 (4 H, m), 1.96-1.93 (4
H, m), 1.77-1.74 (4 H, m), 1.60-1.50 (4 H, m), 1.35-1.25 (4
The amine product (350 mg, 0.68 mmol) was added to a
suspension of 2,2′-bipyridyl-4,4′-dicarboxylic acid (83 mg, 0.34
mmol) and HOBt (94 mg, 0.70 mmol) in DMF (10 mL) at 0 °C,
followed by addition of EDC (142 mg, 0.74 mmol). The mixture
was stirred for 1 h at 0 °C and 49 h at room temperature to
give a pink suspension. The reaction mixture was concentrated
under reduced pressure, dissolved in CH3OH/CHCl3 (1:9), and
washed twice with H2O. The aqueous phase was extracted
three times with CH3OH/CHCl3 (1:9). The combined organic
phase was dried over anhydrous Na2SO4 and filtered, and the
filtrate was concentrated to give pale pink solids. The crude
product was purified by chromatography on a silica gel column
(CH3OH/CHCl3, 1:9) to afford the two-arm bipyridyl ligand as
white solids (367 mg, 87%).
A mixture of the two-arm bipyridyl ligand (59 mg, 0.047
mmol) and cis-Ru(bpy)2Cl2 hydrate (25 mg, 0.052 mmol) in
EtOH (8 mL) and H2O (2 mL) was heated under reflux for 6
h. After removal of solvents, the residue was dissolved in CH2-
Cl2 and triturated with Et2O. The solids were collected by
filtration, washed with CH2Cl2/Et2O (1:3), and dried in vacuo
to afford the chloride salt of 1 (75 mg). The chloride salt was
J. Org. Chem, Vol. 70, No. 6, 2005 2031

Chang et al.
dissolved in H2O, and treated with saturated NH4PF6 aqueous
solution (5 mL). The mixture was stirred for 30 min at room
temperature, filtered, and washed with cold water to give the
phosphorus hexafluoride salt of Ru(II) sensor 1 as orange red
solids: mp > 330 °C dec; yield 77 mg (83%); IR (KBr) 3422,
The binding constant of complex and 1:1 stoichiometry were
determined by curve fitting using the nonlinear least-squares
method, and the association constant (Ka) is derived from the
following equation.22
F ) F0 + (∆Fmax/2C0){1/Ka + C0 + C -
1
2942, 1654, 1540 cm-1; H NMR (DMSO-d6, 500 MHz) δ 9.72
[(1/Ka+ C0 + C)2 - 4C0C]1/2
}
(2H, s), 9.24 (2H, s), 8.83 (6H, m), 8.37 (4H, m), 8.22-8.13 (8H,
m), 7.90-7.67 (16H, m), 7.52-7.45 (8H, m), 4.45 (4H, m), 3.88
(8H, m), 1.96-1.90 (8H, d), 1.92 (8H, m), 1.74 (8H, m), 1.52
(8H, m), 1.31 (8H, m); 13C NMR (DMSO-d6, 125 MHz) δ 166.6
(C), 166.5 (C), 166.4 (C), 162.9 (C), 157.1 (2 × C), 152.0 (CH),
151.6 (CH), 151.3 (CH), 141.4 (C), 139.0 (CH), 138.2 (CH),
135.1 (C), 134.9 (C), 129.6 (CH), 129.0 (CH), 128.3 (CH), 127.9
(CH), 126.7 (CH), 125.7 (CH), 125.4 (CH), 124.5 (CH), 122.0
(CH), 53.5 (CH), 42.8 (CH2), 31.5 (CH2), 24.7 (CH2); FAB-MS
m/z (rel intensity) 1802 (M + H+ - PF6), 1658 (M + H+ - 2
PF6).
Phosphorescence Titration Studies. Phosphorescence
spectra were recorded on AMINCO/Bowman Series 2 spec-
trometer. A solution of compound 1 (4.0 × 10-6 M) in MeOH/
CHCl3 (1:9, 2 mL) was placed in a quartz cuvette (1 cm width)
at 293 K. Aliquots of tripeptide derivative (4.0 × 10-3 M) in
MeOH/CHCl3 (1:9) were added in an incremental fashion (0.5,
1, 2, 3, 4, 6, 10, 15, 30, 40, and 60 equiv). The phosphorescence
spectra with 466-nm excitation were recorded for each addi-
tion. The phosphorescence intensity (as the signal area
between 500 and 850 nm) was monitored as a function of
tripeptide concentrations. Nanosecond-microsecond lifetime
studies were performed by an Edinburgh FL 900 photon-
counting system with a hydrogen-filled/or a nitrogen lamp as
the excitation source. The emission decays were analyzed by
the sum of exponential functions, which allows partial removal
of the instrument time broadening and consequently renders
a temporal resolution of ∼200 ps.
F is the emission intensity, F0 is the original emission
intensity of the free receptor, and ∆Fmax is the largest change
of emission intensity after saturation with the substrate. C is
the concentration of substrate; C0 is the initial concentration
of the receptor.
1
1H NMR Titration Studies. H NMR spectra were mea-
sured on a Bruker Avance-400 NMR spectrometer. A typical
experiment was performed as follows. A solution of compound
1 (1.0 × 10-4 M) in CD3OD/CDCl3 (1:9, 0.5 mL) was placed in
a 5-mm NMR tube. A small aliquot of tripeptide derivative
(1.0 × 10-2 M, e.g., Ac-D-Ala-Gly-D-Ala-NHC12H25) in CD3OD/
CDCl3 (1:9) was added in an incremental fashion (2, 8, 14, 22,
32, 44, and 64 equiv), and their corresponding spectra were
recorded. The chemical-shift changes of NH on the tetraamide
rings and C3/C3′-H were monitored as a function of tripeptide
concentrations.
Acknowledgment. We thank the National Science
Council for financial support.
Supporting Information Available: Synthetic proce-
dures, table of 19 tripeptides selected by encoded screening,
and absorption, phosphorescence, and NMR spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO048368S
2032 J. Org. Chem., Vol. 70, No. 6, 2005