New synthetic amino acids for the design and synthesis of peptide-based metal ion sensors

Article abstract of DOI:10.1021/jo961466w

The syntheses of two new nonstandard amino acids, Flu (6) and XBp (20), and a new synthesis of Dmd (12) are reported. These residues exhibit fluorescence, metal-coordination, and fluorescence-quenching properties, respectively. These building blocks have been incorporated into peptides via solid phase peptide synthesis to afford the prototype for a photoinduced electron transfer-based metal ion chemosensor. The fluorescence of the peptides is modulated upon metal binding. This results from a metal ion-induced conformational change that brings the side chains of the Flu and Dmd amino acids into proximity, thereby favoring photoinduced electron transfer (PET) fluorescence quenching.

Full text of DOI:10.1021/jo961466w

8940
J . Org. Chem. 1996, 61, 8940-8948
New Syn th etic Am in o Acid s for th e Design a n d Syn th esis of
P ep tid e-Ba sed Meta l Ion Sen sor s
Alicia Torrado and Barbara Imperiali*
Division of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena, California 91125
Received J uly 31, 1996X
The syntheses of two new nonstandard amino acids, Flu (6) and XBp (20), and a new synthesis of
Dmd (12) are reported. These residues exhibit fluorescence, metal-coordination, and fluorescence-
quenching properties, respectively. These building blocks have been incorporated into peptides
via solid phase peptide synthesis to afford the prototype for a photoinduced electron transfer-based
metal ion chemosensor. The fluorescence of the peptides is modulated upon metal binding. This
results from a metal ion-induced conformational change that brings the side chains of the Flu and
Dmd amino acids into proximity, thereby favoring photoinduced electron transfer (PET) fluorescence
quenching.
The development of chemosensors for the detection of
fluorescence resonance energy transfer (FRET) has also
been described.18
metal ions in aqueous and nonaqueous media continues
to be the focus of research in a number of laboratories.1
In particular, sensors based on fluorescence measure-
ments are desired due to their high sensitivity, with
working detection limits typically below 0.1 µM analyte
concentrations. Remarkable progress has been achieved
in this regard for the determination of intracellular
calcium2-5 and proton6,7 concentrations. While sensors
Herein we report the preparation and characterization
of peptides that exhibit metal-dependent fluorescence
properties resulting from photoinduced electron transfer
(PET). The application of PET in a fluorosensor design
requires a donor (fluorescence quencher) capable of
transferring a single electron to a fluorophore (acceptor)
in its excited state to transiently form the radical anion
of the fluorophore. Commonly, the electronic properties
of the donor are modulated by the analyte to be tested.
The lone pair electrons of oxygen- and nitrogen-contain-
ing molecules are suitable donors. Indeed, this effect has
been exploited in the design of cyclic and noncyclic
polyethers, crown ethers,19-23 cryptands,24-26 and cali-
xarenes27-31 for the detection of alkali metals. Similarly,
chemosensors incorporating cyclam, thiacyclam rings,32,33
or dioxotetraaza units34,35 have been developed for the
detection of transition metal ions. The binding of cations
for other analytes, including Na+,8 K+,8 Cl-,9,10 Mg2+ 11
,
and cAMP,12 have also been introduced, the need for new
strategies for the design of selective fluorosensors con-
tinues to exist.13
In light of the selectivity and avidity with which
proteins bind metal ions,14 we have focused on the use of
polypeptide motifs for the recognition and sensing of
divalent metal cations. We have demonstrated that the
judicious placement of appropriate ligands in simple
polypeptides affords species that fold into more compact
structures upon metal complexation.15,16 Furthermore,
we have exploited metal-dependent structure modulation
as the basis for trace analyte detection in the develop-
ment of a peptidyl fluorescent chemosensor for divalent
zinc.17 Recently, a polypeptide-based sensor employing
(17) Walkup, G. K.; Imperiali, B. J . Am. Chem. Soc. 1996, 118,
3053-3054.
(18) Godwin, H. A.; Berg, J . M. J . Am. Chem. Soc. 1996, 118, 6514-
6515.
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Lett. 1991, 32, 421-424.
X Abstract published in Advance ACS Abstracts, November 15, 1996.
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S0022-3263(96)01466-1 CCC: $12.00 © 1996 American Chemical Society

Peptide-Based Metal Ion Sensors
J . Org. Chem., Vol. 61, No. 25, 1996 8941
Sch em e 1
F igu r e 1.
to these macrocycles diminishes or eliminates their
ability to quench the donor. The efficiency of electron
transfer (fluorescence quenching) is strongly influenced
by the distance between the donor and acceptor, as well
as the structure of the molecular link. Other factors
affecting PET include conformational changes, local
polarity modulations, and hydrogen-bonding effects.36
Patterned after previous PET-based sensor designs, the
system described herein utilizes the cyanoanthracene
fluorophore. This moiety exhibits strong and well-
characterized emission properties, chemical stability, and
sensitivity to electron transfer from chromophores such
as the dimethoxybenzyl group.20 The cyanoanthracene
and dimethoxybenzyl groups have been integrated into
R-amino acid constructs to allow the facile assembly of
these units into functional polypeptides. Additionally,
for the sensor design, we have formulated and imple-
mented the ω-bipyridine amino acid illustrated in Figure
1. The bidentate bipyridinyl group of this ω-amino acid
residue is well suited for this particular application, since
the rigid uncomplexed amino acid maintains the polypep-
tide in an extended conformation that minimizes chro-
mophore interactions in the absence of metal ions. In
contrast, in the presence of a complexing metal analyte,
the peptide scaffold undergoes a conformational change
to alter the PET transfer properties, thereby generating
fluorescence changes that can be monitored. The sensor
design is illustrated schematically in Figure 1.
cence. J udicious incorporation of these three amino acid
building blocks into different peptides, via solid phase
peptide synthesis (SPPS), affords the prototype for a
PET-based chemosensor. This sensor is designed to
exhibit a metal ion-induced conformational change which
modulates the fluorescence properties of the peptide.
The synthesis of (S)-2-amino-NR-(9-fluorenylmethyl-
oxycarbonyl)-3-(9-cyanoanthracen-10-yl)propanoic acid
(6; Fmoc-Flu-OH) begins from the commercially available
10-formyl-9-methylanthracene (1) as outlined in Scheme
1. The key step of this synthesis involves a stereoselec-
tive alkylation of N-(diphenylmethylene)glycine tert-butyl
ester (4) with the bromide 3 using the phase transfer
catalyst (PTC) (8S,9R)-(-)-N-benzylcinchonidinium chlo-
ride according to the method of O’Donnell et al.41 The
alkylation proceeded with modest asymmetric induction
to afford the alkylated product (S)-5 in 53% ee. Enan-
tiomerically pure material (>99% ee) was obtained after
fractional crystallization (hot hexane/EtOAc 4:1) of the
racemate from the optically enriched material. Due to
the aqueous acid lability of the nitrile group in 5,
deprotection of the imine and ester functionality was
effected using anhydrous HCl(g) in dry acetone. Since
the amino acid was intended for incorporation into
polypeptide sequences using solid phase peptide synthe-
sis methods, the crude amino acid was directly converted
to the corresponding 9-fluorenylmethoxycarbonyl (Fmoc)
derivative 6 through treatment with (9-fluorenylmethoxy-
carbonyl)succinimidyl carbonate (Fmoc-OSu) in dioxane
and 10% sodium carbonate.
Resu lts a n d Discu ssion
Herein we describe the synthesis of two new unnatural
amino acids, Fmoc-Flu (6) and Fmoc-XBp (20). These
residues incorporate a cyanoanthracene fluorophore and
a metal-coordinating bipyridine derivative. We also
describe a new synthesis for L-3,4-dimethoxy-DOPA
(12).37-40 This residue is intended to act as the “electron
donor” or “quencher” of the cyanoanthracene fluores-
Commercially available 3,4-dimethoxybenzyl alcohol
(7) was used in the first step of the synthesis of (S)-2-
amino-NR-(9-fluorenylmethyloxycarbonyl)-3-(3,4-dime-
thoxybenz-1-yl)propanoic acid (12; Fmoc-L-Dmd-OH)
(Scheme 2). The procedure follows the same steps as the
synthesis of 6, except that crystallization to isolate the
enantiomerically pure S derivative from the product
mixture was not successful. Therefore, the optically pure
(35) De Santis, G.; Fabbrizzi, L.; Manotti-Lanfredi, A. M.; Pallavi-
cini, P.; Perotti, A.; Ugozzoli, F.; Zema, M. Inorg. Chem. 1995, 34,
4529-4535.
(36) Kavarnos, G. J .; Turro, N. J . Chem. Rev. 1986, 86, 401-449.
(37) Schrecker, A. W.; Hartwell, J . L. J . Am. Chem. Soc. 1957, 79,
3827-3831.
(39) Belokon, Y. N.; Bakhmutov, V. I.; Chernoglazova, N. I.; Kochet-
kov, K. A.; Vitt, S. V.; Garbalinskaya, N. S.; Belikov, V. M. J . Chem.
Soc., Perkin Trans. 1 1988, 305-312.
(40) Belokon, Y. J anssen Chim. Acta 1992, 10, 4-12.
(41) O’Donnell, M. J .; Bennett, W. D.; Wu, S. D. J . Am. Chem. Soc.
1989, 111, 2353-2355.
(38) Belokon, Y. N.; Zel’tzer, I. E.; Bakhmutov, V. I.; Saporovskaya,
M. B.; Ryzhov, M. G.; Yanovsky, A. I.; Struchkov, Y. T.; Belikov, V. M.
J . Am. Chem. Soc. 1983, 105, 2010-2017.

8942 J . Org. Chem., Vol. 61, No. 25, 1996
Torrado and Imperiali
Sch em e 2
Sch em e 3
material was obtained via the enantioselective hydrolysis
of the corresponding methyl ester 11 using commercially
available alkaline protease.42 The enzymatic hydrolysis
proceeded smoothly at room temperature and was com-
plete within 5 h. Separation and quantitative recovery
of the two enantiomers was accomplished through simple
extraction of the D-amino acid ester 11 with chloroform
followed by lyophilization of the remaining aqueous layer
to afford the L-amino acid 10 (99% ee). The crude
L-amino acid 10 was treated with Fmoc-OSu to afford the
Fmoc-protected amino acid derivative 12.
Scheme 3 outlines the synthesis of the metal ligating
NR-(9-fluorenylmethyloxycarbonyl)-glycyl-5′-amino-2,2′-
bipyridine-5-carboxylic acid (20, Fmoc-XBp). The prepa-
ration of 20 involved a Stille coupling of the two pyridine
derivatives, 14 and 16. Methyl 2-(trifluoromethylsulfo-
nyl)pyridine-5-carboxylate 14 was synthesized by me-
thylation of the commercially available 2-hydroxypyridine-
5-carboxylic acid (13) with (trimethylsilyl)diazomethane
and subsequent treatment with N-phenyltrifluoromethane-
sulfonamide. Initial attempts to convert the commer-
cially available 2-bromo-5-nitropyridine (15) into the tin
derivative 16 by treatment with n-BuLi followed by
trimethylsilyl chloride failed, probably due to the electron-
withdrawing effect of the nitro group at the 5-position of
the pyridine ring. Ultimately, 16 was obtained by
treating 15 with hexamethylditin in benzene at reflux
for 6 h.43,44 The Stille coupling reaction between the
triflate 14 and the tin derivative 16 using standard
conditions (LiCl, Pd(PPh3)4, dioxane at reflux)45 afforded
17. The nitro compound 17 was subsequently reduced
to the amine 18 with H2/Pd-C in EtOH.46 Due to the
poor reactivity of the aromatic amine functionality in 18,
attempts to protect it as the Fmoc amino acid derivative
were singularly unsuccessful with low yields even under
forcing conditions. Further, the limited reactivity of this
amino acid was deemed unsuitable for performing solid
phase coupling reactions. Therefore, the ω-amino acid
residue was incorporated into a pseudo-dipeptide “cas-
sette” 19, wherein Boc-glycine fluoride, generated in
situ,47-49 was coupled to the nonreactive aryl amine of
the bipyridyl ω-amino acid methyl ester 18. Removal of
the Boc protecting group and hydrolysis of the methyl
ester were achieved by treatment with 0.1 N NaOH,
followed by TFA. The free amine was subsequently
protected with the Fmoc group to afford 20, ready for
incorporation into peptides via SPPS. This reaction
sequence was necessitated due to lability of the Fmoc
group under the conditions required for ester hydrolysis.
The efficient solid phase synthesis of peptides F XR01,
F XR02, and F XR03 (Chart 1), incorporating the three
nonstandard residues (Flu, Dmd, and XBp) was achieved
using macropin technology.50,51 The successful prepara-
tion of these peptides highlights the compatibility of these
residues with solid phase methodology. The peptides
were prepared with an N-terminal acetyl group and a
C-terminal carboxamide to avoid interactions between
the free amine or carboxylic acid and divalent metal ions.
These peptides were designed to adopt a random coil
(46) Long, G. V.; Boyd, S. E.; Harding, M. M.; Buys, I. E.; Hambley,
T. W. J . Chem. Soc., Dalton Trans. 1993, 3175-3180.
(47) Carpino, L. A.; Sadat-Aalaee, D.; Guang Chao, H.; DeSelms,
R. H. J . Am. Chem. Soc. 1990, 112, 9651-9652.
(48) Carpino, L. A.; El-Faham, A. J . Am. Chem. Soc. 1995, 117,
5401-5402.
(42) Chen, S. T.; Wang, K. T.; Wong, C. H. J . Chem. Soc., Chem.
Commun. 1986, 1514-1516.
(43) Azizian, H.; Eaborn, C.; Pidcock, A. J . Organomet. Chem. 1981,
215, 49-58.
(44) Kashin, A. N.; Bumagina, I. G.; Bumagin, N. A.; Bakunin, V.
N.; Beletskaya, I. P. Zh. Org. Khim. 1981, 17, 905-911.
(45) Echavarren, A. M.; Stille, J . K. J . Am. Chem. Soc. 1987, 109,
5478-5486.
(49) Dourtoglou, V.; Gross, B.; Lambropoulou, V.; Zioudrou, C.
Synthesis 1984, 572-574.
(50) Geysen, H. M.; Meloen, R. H.; Barteling, S. J . Proc. Natl. Acad.
Sci. U.S.A. 1984, 81, 3998-4002.
(51) Geysen, H. M.; Rodda, S. J .; Mason, T. J .; Tribbick, G.; Schoofs,
P. G. J . Immunol. Methods 1987, 102, 259-274.

Peptide-Based Metal Ion Sensors
J . Org. Chem., Vol. 61, No. 25, 1996 8943
Ch a r t 1
conformation with the Flu and Dmd amino acids flanking
the two XBp residues. In the current designs, the metal-
coordinating residues are linked by three R-amino acids.
In the absence of metal ions, the peptides are expected
to exhibit strong fluorescence due to the side-chain
cyanoanthracene fluorophore of the R-amino acid Flu.
Furthermore, it is anticipated that, upon coordination of
divalent metal cations to the XBp residues, the chro-
mophores on the Flu and Dmd side chains would move
into proximity and allow the PET process to occur. This
conformational change would result in a decrease in
fluorescence from the anthracene moiety.
F igu r e 2. (a) Uncorrected fluorescence emission spectra of a
∼1.2 µM soution of F XR01 in MeOH upon addition of increas-
ing amounts of aqueous ZnCl2: 0, 1.7, 3.4, 5.0, and 6.8 µM
ZnCl2, respectively. (b) Recovery of the fluorescence after
adding EDTA. (c) Uncorrected fluorescence emission spectra
of a ∼1.6 µM solution of F XR01 in MeOH upon addition of
increasing amounts of aqueous CoCl2: 0, 1, 2, 3, 7, and 14 µM
CoCl2 respectively. (d) Recovery of the fluorescence after
adding EDTA. Excitation was performed at 388 nm.
The first design, F XR01, incorporated three glycine
residues between the two bipyridine ω-amino acids. The
glycine spacer affords flexibility to the peptide chain,
allowing it to fold in the presence of divalent metal ions.
Unfortunately, a detailed analysis of this peptide in
aqueous solution was precluded by its insolubility in
water. However, preliminary studies with crude peptide
in methanol were promising. Fluorescence measure-
ments for this and all later samples were obtained by
excitation at 388 nm, followed by observation of the
dominant emission, which is centered between 440 and
470 nm. Notably, fluorescence quenching was observed
upon titration with Zn2+ and Co2+ (Figure 2).
The sequences for peptides F XR02 and F XR03 were
selected to enhance the aqueous solubility of the peptidyl
template while maintaining backbone flexibility. Peptide
F XR02 incorporates three glycines between the two
bipyridine ω-amino acids similar to peptide F XR01;
however, two hydrophilic residues (Thr8 and Arg9) were
also included to improve solubility. Peptide F XR03 was
designed with even more hydrophilic residues (Arg1, Ser5,
Ser6, Thr8, and Arg9) to further increase aqueous solubil-
ity.
The fluorescence of peptide F XR02 was measured
using a 2.63 µM solution of peptide in 0.01 M PIPES (pH
7.0). The emission properties of F XR02 are sensitive to
the presence of micromolar concentrations of Zn2+. The
fluorescence emission of F XR02 is quenched upon com-
plexation of Zn2+ cations (Figure 3a), which may be due
to the PET effect. The reversibility of complex formation
was assessed with different chelating agents including
1,10-phenanthroline and ethylenediaminetetraacetic acid
(EDTA). Upon addition of 1,10-phenanthroline to the
F XR02-Zn2+ complex, the system exhibited total recov-
ery of fluorescence emission (Figure 3b). In contrast,
F igu r e 3. (a) Uncorrected fluorescence emission spectra of a
2.63 µM solution of F XR02 in 0.01 M PIPES (pH 7.0) upon
addition of increasing amounts of aqueous ZnCl2: 0, 1.2, 2.3,
3.9, 5.5, and 24.2 µM ZnCl2, respectively. (b) Recovery of the
fluorescence emission after adding 1,10-phenanthroline. Ex-
citation was performed at 388 nm.
addition of EDTA to the peptide-Zn2+ system did not
result in the recovery of fluorescence emission in aqueous
solution. Interestingly, when identical titration studies

8944 J . Org. Chem., Vol. 61, No. 25, 1996
Torrado and Imperiali
F igu r e 4. (a) Uncorrected fluorescence emission spectra of a
1.34 µM solution of F XR02 in MeOH upon addition of
increasing amounts of aqueous ZnCl2: 0, 0.8, 1.4, 2.6, and 3.7
µM ZnCl2, respectively. (b) Recovery of the fluorescence
emission after adding EDTA. Excitation was performed at 388
nm.
were performed in MeOH (Figure 4) or in 50% MeOH/
0.01 M PIPES (pH 7.0) (Figure 5), addition of EDTA
completely recovered the fluorescence emission. Fur-
thermore, in 50% MeOH/0.01M PIPES (pH 7.0), although
fluorescence was completely recovered (Figure 5b), it was
subsequently quenched over a period of 1.5 h (Figure 5c),
suggesting that the EDTA quenching effect could be
solvent-dependent.
From studies involving different fluorophores, it is
known that EDTA acts as an electron donor in aqueous
photoinduced electron transfer reactions. For those
systems it has been shown that electron transfer from
the lone pair of the amine to the excited state of the
fluorophore is responsible for fluorescence quenching.52-54
A similar effect may be responsible for the quenching
observed in our system as presented in Scheme 4.
Fluorescence studies with peptide F XR03 provided
results similar to peptide F XR02 (Figure 5). Fluores-
cence was measured using a 3.75 µM solution of peptide
F XR03 in 0.01 M PIPES (pH 7.0). The fluorescence
emission properties of peptide F XR03 are more sensitive
to the presence of Zn2+ compared to peptide F XR02. A
more significant change in fluorescence is observed with
peptide F XR03 upon addition of metal (Figure 6a). The
lower apparent sensitivity of peptide F XR02 may be due
to its lower solubility in aqueous media. Furthermore,
the reversibility of the metal complexation was demon-
F igu r e 5. (a) Uncorrected fluorescence emission spectra of a
1.24 µM solution of F XR02 in 50% MeOH/0.01 M PIPES (pH
7.0) upon addition of increasing amounts of aqueous ZnCl2:
0, 0.6, 1.2, 2.3, 3.5, 4.7, and 5.8 µM ZnCl2, respectively. (b)
Recovery of the fluorescence emission after adding EDTA. (c)
Quenching of the fluorescence with time after being recovered
with EDTA: 0, 30, 60, and 90 min, respectively. Between scans
the sample was left in the dark. Excitation was performed at
388 nm.
Sch em e 4
strated by the addition of 1,10-phenanthroline (Figure
6b). The failure to recover fluorescence with EDTA again
suggested that this chelating agent participates in an
electron transfer quenching reaction. A parallel set of
experiments was also carried out in phosphate buffer at
pH 7. These studies reveal that there is some intrinsic
(52) Aono, S.; Nemoto, S.; Ohtaka, A.; Okura, I. J . Mol. Catal. A
1995, 95, 193-196.
(53) Isoda, S.; Maeda, M.; Miyasaka, H.; Mataga, N. Chem. Phys.
Lett. 1991, 182, 379-383.
(54) Karmakar, A.; Chaudhuri, R.; Bera, S. C.; Rohatgi-Mukherjee,
K. K. Ind. J . Chem. 1995, 34A, 83-93.

Peptide-Based Metal Ion Sensors
J . Org. Chem., Vol. 61, No. 25, 1996 8945
F igu r e 6. (a) Uncorrected fluorescence emission spectra of a
3.75 µM solution of F XR03 in 0.01 M PIPES (pH 7.0) upon
addition of increasing amounts of aqueous ZnCl2: 0, 2.1, 4.1,
7.2, 10.3, and 14 µM ZnCl2, respectively. (b) Recovery of the
fluorescence emission after adding 1,10-phenanthroline. Ex-
citation was performed at 388 nm.
F igu r e 7. (a) Uncorrected fluorescence emission spectra of a
9.09 µM solution of F XR03 in 0.01 M phosphate buffer (pH
7.0) upon addition of increasing amounts of aqueous ZnCl2:
0, 9.7, 16.0, 24.0, 35.8, and 47.7 µM ZnCl2, respectively. (b)
Recovery of the fluorescence emission after adding 1,10-
phenanthroline. Excitation was performed at 388 nm.
matography was carried out on hard TLC plates with fluores-
cent indicator from EM Reagents (SiO2 60, F-254). TLC plates
were visualized by UV or 0.2% ninhydrin solution in ethanol
followed by heat. Flash column chromatography was per-
formed according to the procedure of Still55 using J . T. Baker
flash silica gel (∼40 µm). 10-Formyl-9-cyanoanthracene, 3,4-
dimethoxybenzyl alcohol, 2-hydroxypyridine-5-carboxylic acid,
2-bromo-5-nitropyridine, N-(diphenylmethylene)glycine tert-
butyl ester, and (8S,9R)-(-)-N-benzylcinchonidinium chloride
were purchased from Aldrich Chemical Co. Tetramethylfluo-
roformamidinium hexafluorophosphate (TFFH) was prepared
by following the method developed by Carpino.47-49
fluorescence quenching from the PIPES buffer relative
to the phosphate buffer; however, the relative change of
metal ion-induced fluoresence and the reversibility with
1,10-phenanthroline is comparable (see Figure 7a and b,
respectively). To confirm that the electron transfer was
indeed induced by a metal ion-dependent modulation of
the peptide structure, rather than direct metal ion or
metal ion complex quenching of the cyanoanthracene
excited state, the relative fluorescence of a sample of the
free cyanoanthracene amino acid (Flu) was examined on
treatment with Zn2+, Co2+, and Co2+ in the presence of
2,2′-bipyridine. In all cases, even large excesses of the
added species failed to effect any significant change in
the fluoresence emission properties (data not shown).
In conclusion, we have designed and synthesized a new
family of peptide-based metal ion sensors that exhibit a
metal ion-modulated PET effect in aqueous solution.
These peptides incorporate three nonstandard amino
acids. These residues are fully compatible with solid
phase peptide synthesis methodology and may be applied
in the future toward the design of additional metal ion
sensors with complementary cation selectivities. Fur-
thermore, the modular nature of this approach will allow
the application of combinatorial methods for signal
intensity optimization.
10-Meth yl-9-cya n oa n th r a cen e (2). To 1 (1 g, 4.54 mmol)
suspended in 24 mL of 95% EtOH was added hydroxylamine
hydrochloride (356 mg, 5.13 mmol, neutralized with Na2CO3)
in 3.5 mL of warm (75 °C) water. The mixture was heated on
a steam bath for 15 min, cooled, and diluted with water. The
solid was collected and heated in 10% NaOH for 10 min. The
solution was cooled and diluted with water. The solid was
collected, and acetic anhydride (20 mL) was added. The
resulting solution was refluxed for 15 min and then filtered
while still hot. Upon cooling 791 mg (80%) of 2 as yellow
crystals (mp 207-208 °C (lit.56 mp 208-210 °C) appear in the
filtrate: Rf ) 0.51 (hexane/EtOAc, 8:2); 1H NMR (CDCl3, 300.2
MHz) δ 2.93 (s, 3H), 7.48 (t, 2H, J ) 7.5 Hz), 7.57 (t, 2H, J )
7.5 Hz), 8.13 (d, 2H, J ) 8.5 Hz), 8.22 (d, 2H, J ) 8.5 Hz); 13
C
NMR (CDCl3, 75.5 MHz) δ 14.6, 103.8, 117.5, 124.9, 125.3,
125.5, 125.81, 125.83, 125.9, 126.0, 128.0, 128.2, 128.9, 132.4,
137.8; MS m/z 217 (M+, 100), 216 (43), 189 (13), 165 (1).
9-(r-Br om om eth yl)cya n oa n th r a cen e (3). A suspension
of 2 (500 mg, 2.3 mmol), N-bromosuccinimide (451 mg, 2.5
mmol), and benzoyl peroxide (11 mg, 0.046 mmol) in CCl4/CH2-
Cl2 (5:1, 8 mL) was degassed four times by the freeze-pump-
thaw method and heated to 80 °C in a constant-temperature
Exp er im en ta l Section
Gen er al In for m ation . Nuclear magnetic resonance (NMR)
spectra were recorded at 300 MHz for proton frequency and
75 MHz for carbon frequency. Chemical shifts are reported
in δ units (ppm) relative to the solvent used. Optical rotations
were recorded at room temperature. Mass spectra were
obtained using fast atom bombardment (FAB), electron impact
(EI) or plasma desorption (PD) ionization. Thin-layer chro-
(55) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923-
2925.
(56) Wawzonek, S.; Hallum, J . V. J . Org. Chem. 1959, 24, 364-
366.

8946 J . Org. Chem., Vol. 61, No. 25, 1996
Torrado and Imperiali
bath for 4 h. The solvent was then removed in vacuo, and the
crude product was recrystallized from hexanes/CHCl3 to afford
320 mg (67%) of 3 as yellow crystals (mp 201-202 °C):57 Rf )
0.33 (hexane/EtOAc, 8:2); 1H NMR (CDCl3, 300.2 MHz) δ 5.48
(s, 2H), 7.6-7.8 (m, 4H), 8.3-8.4 (m, 2H), 8.4-8.5 (m, 2H);
13C NMR (CDCl3, 75.5 MHz) δ 14.6, 103.9, 117.6, 125.2, 125.7,
126.0, 128.1, 129.0, 132.5, 137.9; MS m/z 297 (M+, 3), 295 (5),
216 (100), 189 (13), 163 (4), 108 (4), 94 (8), 87 (4), 74 (6), 63
(5), 50 (6).
trated to afford 95 mg of 6 (50%): Rf ) 0.41 (CHCl3/MeOH,
1
8:2); [R]20 ) -148° (0.54, DMF, 0.5 dm path length cell); H
D
NMR (CDCl3, 300.2 MHz) δ 3.9-4.1 (m, 3H), 4.33 (dd, 1H, J
) 14.7, 9.6 Hz), 4.42 (dd, 1H, J ) 14.4, 5.0 Hz), 4.69 (m, 1H),
7.23 (td, 1H, J ) 7.5, 0.9 Hz), 7.30 (td, 1H, J ) 7.6, 1.0 Hz),
7.40 (dd, 1H, J ) 7.5, 4.0 Hz), 7.42 (dd, 1H, J ) 76.7, 4.0 Hz),
7.55 (dd, 2H, J ) 16.8, 7.5 Hz), 7.81 (td, 2H, J ) 6.9, 0.9 Hz),
7.89 (d, 4H, J ) 8.1 Hz), 8.40 (d, 2H, J ) 8.4 Hz), 8.80 (d, 2H,
J ) 9 Hz); 13C NMR (CDCl3, 75.5 MHz) δ 47.5, 56.7, 67.0,
117.8, 120.7, 125.9, 126.0, 126.1, 126.7, 127.7, 128.3, 129.9,
130.5, 133.4, 140.4, 141.7, 144.6, 144.7, 156.9, 162.9, 173.4.
IR (KBr) 3315 (w, NsH), 3072 (w, aromatic CsH), 2205 (m,
CtN), 1694 (s, CdO and amide band), 1445 cm-1 (CtN); MS
m/z 513 (MH+, 2), 391 (3), 242 (2), 179 (10); HRMS calculated
for C33H24O4N2 513.181 433, observed 513.183 600.
N-(Diph en ylm eth ylen e)-2-a m in o-3-(9-cya n oa n th r a cen -
10-yl)p r op a n oic Acid ter t-Bu tyl Ester (5). N-(diphenyl-
methylene)glycine tert-butyl ester (9; 248 mg, 0.841 mmol) and
(8S,9R)-(-)-N-benzylcinchonidinium chloride (35 mg, 0.084
mmol) were dissolved in CH2Cl2 (15 mL). To this was added
1.2 mL of 50% aqueous NaOH and solid 9-(R-bromomethyl)-
anthracene (8; 311 mg, 1.051 mmol). The reaction mixture
was then stirred at room temperature for 24 h. At this time
the organic phase was separated and the remaining aqueous
phase washed with CH2Cl2. The combined organic phases
were concentrated under reduced pressure and purified by
flash chromatography (eluent hexane/EtOAc 9:1) to afford 387
mg (72% yield) of pure product 5. The optical purity of the
product was determined to be 53%. Recrystallization of the
L/D mixture in hexane/EtOAc (4:1) afforded 165 mg of pure L
3,4-Dim eth oxyben zyl Br om id e (8). To a solution of 3,4-
dimethoxybenzyl alcohol (7; 5 g, 29.76 mmol) in CH2Cl2 (125
mL) was added PPh3 (9.4 g, 35.71 mmol) and N-bromosuccin-
imide (5.8 g, 32.74 mmol), and the resulting mixture was
stirred for 1 h at room temperature. The reaction volume was
then reduced by evaporation and directly passed through a
silica gel column (eluent, CH2Cl2) to afford 5.3 g (77%) of 8 as
white crystals (mp 50-51 °C, (lit.58 mp 47-50 °C)); Rf ) 0.83
1
(hexane/EtOAc, 4:3); H NMR (CDCl3, 300.2 MHz) δ 3.86 (s,
product (mp 162-163 °C): [R]20 ) -341° (0.5, CHCl3, 1 dm
D
3H), 3.88 (s, 3H), 4.49 (s, 2H), 6.80 (d, 1H, J ) 8.1 Hz), 6.90
(d, 1H, J ) 2.1 Hz), 6.94 (dd, 1H, J ) 8.1, 2.1 Hz); 13C NMR
(CDCl3, 75.5 MHz) δ 34.3, 55.7, 55.8, 110.8, 111.9, 121.4, 130.0,
148.9, 149.0; MS m/z 230 (M+, 2), 151 (100), 137 (10), 107 (10),
94 (29), 80 (39), 65 (12). HRMS [M+] calculated for C9H11BrO2
229.994 241, observed 229.993 400.
path length cell); Rf ) 0.36 (hexane/EtOAc, 8:2); 1H NMR
(CDCl3, 300.2 MHz) δ 1.49 (s, 9H), 4.17 (dd, 1H, J ) 14.0, 3.1
Hz), 4.33 (t, 1H, J ) 12.0 Hz), 4.46 (dd, 1H, J ) 9.9, 3.2 Hz),
5.8-5.9 (s br, 2H), 6.83 (t, 2H, J ) 7.5 Hz), 7.02 (t, 1H, J )
7.5 Hz), 7.13 (t, 2H, J ) 7.5 Hz), 7.25 (d, 3H, J ) 7.9 Hz), 7.46
(t, 2H, J ) 7.6 Hz), 7.61 (t, 2H, J ) 7.6 Hz), 8.36 (d, 4H, J )
8.6 Hz); 13C NMR (CDCl3, 75.5 MHz) δ 28.0, 31.5, 66.5, 81.7,
104.9, 117.6, 125.7, 126.1, 126.7, 127.5, 127.8, 128.3, 128.5,
129.7, 130.1, 132.7, 135.0, 138.3, 138.5, 170.4, 170.6; IR
(CHCl3) 3060-3012 (m, aromatic CsH), 2978-2912 (m,
aliphatic CsH), 2214 (s, CtN), 1725 (s, CdO), 1619 (m, ring
CdC), 1443 cm-1 (m, CtN); MS m/z 512 (39), 511 (MH+, 100),
510 (M+, 6), 426 (6), 340 (7), 296 (23), 238 (22), 235 (18), 217
(6), 200 (17), 183 (21), 182 (16), 163 (3), 105 (4); HRMS [MH+]
calculated for C35H31N2O2 511.239500, observed 511.238554.
Deter m in a tion of En a n tiom er ic Excess. The enantio-
meric excess of the pure amino acid obtained by hydrolysis of
5 was evaluated by derivatization with 1-fluoro-2,4-(dinitro-
phenyl)-5-L-alanineamide (FDAA, Marfey’s reagent) followed
by reversed phase HPLC analysis of the adducts. Ester 5 (2
mg) was heated in 6 N HCl at 100 °C for 1 h. The solution
was then neutralized with Na2CO3 and lyophilized to dryness.
The residue was dissolved in 345 µL of 0.1 M NaHCO3 (10
mM solution of amino acid). The resulting solution was added
to 100 µL of freshly prepared solution of 1.4 mg of Marfey’s
reagent in acetone and kept at 40 °C for 1 h with frequent
mixing. After cooling, 2 N HCl was added to make the solution
slightly acidic. The mixture was the centrifuged and 10 µL of
the supernatant was analyzed by HPLC (C18 column; solvent
A, H2O/0.1% TFA; solvent B, MeCN/0.1% TFA). Retention
times: S derivative 24.06 min; R derivative 26.7 min.
N-(Diph en ylm eth ylen e)-2-am in o-3-(3,4-dim eth oxyben z-
1-yl) p r op a n oic Acid ter t-Bu tyl Ester (9). N-(Diphenyl-
methylene)glycine tert-butyl ester (4; 1.3 g, 4.33 mmol) and
(8S,9R)-(-)-N-benzylcinchonidinium chloride (364 mg, 0.86
mmol) were dissolved in CH2Cl2 (80 mL). To this was added
6.5 mL of 50% aqueous NaOH and a solution of 8 (1 g, 4.33
mmol). The reaction mixture was then stirred at room
temperature for 36 h. At this time the organic phase was
separated and the remaining aqueous phase washed with CH2-
Cl2. The combined organic phases were concentrated under
reduced pressure and purified by flash chromatography (eluent
hexane/EtOAc/Et3N, 9:0.75:0.25) to afford 1.290 g (67% yield)
of pure product 9. The optical purity of the product was
determined to be 50% ee using a chiral Crownpak CR(+)
column (Daicel): flow rate 1 mL/min, 25 °C, 280 nm, eluent
10% MeOH/70% HClO4, t-Bu ester D 5.79 min, L 7.70 min; Rf
1
) 0.33 (hexane/EtOAc, 8:2); H NMR (CDCl3, 300.2 MHz) δ
1.43 (s, 9H), 3.0-3.1 (m, 2H), 3.61 (s, 3H), 3.81 (s, 3H), 4.06
(dd, 1H, J ) 8.6, 4.8 Hz), 6.5-6.6 (m, 4H), 6.67 (d, 1H, J ) 8.1
Hz), 7.2-7.3 (m, 6H), 7.55 (dd, 2H, J ) 8.2, 1.3 Hz). 13C NMR
(CDCl3, 75.5 MHz) δ 28.0, 39.0, 55.4, 55.8, 67.9, 110.7, 112.8,
121.7, 127.6, 127.7, 127.8, 127.9, 128.0, 128.1, 128.6, 130.1,
130.8, 136.3, 139.4, 148.3, 170.1, 170.8. IR (CHCl3) 3008-
2936 (m, aromatic CsH), 2837 (w, aliphatic CsH), 1725 (s,
CdO), 1464 (m, CdN), 1260 (s, CsOsC), 1154 (s), 1140 (s),
1028 cm-1 (m, CsOsC); MS m/z 446 (MH+, 49), 390 (97), 344
(29), 294 (11), 238 (100), 193 (44), 182 (18), 165 (40), 151 (86),
137 (11), 107 (11); HRMS [MH+] calculated for C28H32NO4
446.233 134, observed 446.233 500.
(S)-2-Am in o-Nr-(9-flu or en ylm eth yloxyca r bon yl)-3-(9-
cya n oa n th r a cen -10-yl) p r op a n oic a cid (6). To a suspen-
sion of 5 (190 mg, 0.372 mmol) in dry acetone (2 mL) was added
HCl(g) at 0 °C. The resulting solution was stirred for 15 min
at 0 °C and 15 min more at room temperature. The solvent
was removed under a stream of argon, and the resulting
residue was washed with dry acetone and dried under argon.
The mixture was then basified with 10% Na2CO3/dioxane (2
mL/5 mL), followed by a solution of Fmoc-ONSu (138 mg, 0.409
mmol) in dioxane (2 mL). The resulting yellow suspension was
stirred at room temperature for 6 h. The aqueous phase was
extracted with Et2O (3×), and the aqueous phase was acidified
to pH 4 with 2 N HCl and extracted with EtOAc (5×). The
combined ethyl acetate phases were dried over Na2SO4 and
concentrated to give a yellow solid. The residue was dissolved
in a small amount of CH2Cl2/MeOH and triturated with
hexanes. The solid was redissolved in CH2Cl2 and concen-
(D,L)-2-Am in o-3-(3,4-dim eth oxyben z-1-yl)pr opan oic Acid
(10). The t-Bu ester 9 (200 mg, 0.449 mmol) was refluxed in
6 N HCl (3 mL) for 5 h. The hydrolyzed mixture was then
cooled, extracted with EtOAc (3 × 5 mL), and concentrated to
dryness. The residual material was lyophilized from water
overnight to afford 107 mg (92%) of the isomeric mixture of
amino acid hydrochlorides: Rf ) 0.4 (AcOH/H2O/nBuOH/
1
EtOAc, 1:1:1:1); H NMR (D2O, 300.2 MHz) δ 3.13 (dd, 1H, J
) 14.7, 7.6 Hz), 3.26 (dd, 1H, J ) 14.7, 5.5 Hz), 3.81 (s, 6H),
4.25 (dd, 1H, J ) 7.4, 5.6 Hz), 6.87 (dd, 1H, J ) 8.5, 1.5 Hz),
6.92 (d, 1H, J ) 1.7 Hz), 6.99 (d, 1H, J ) 8.2 Hz); 13C NMR
(D2O, 75.5 MHz) δ 35.3, 54.3, 55.6, 112.1, 112.6, 122.2, 126.9,
147.7, 148.3, 171.7; MS m/z 226 (M+, 100), 209 (13), 180 (22),
(57) Bausch, M. J .; Guadalupe-Fasano, C.; J irka, G.; Peterson, B.;
Selmarten, D. Polycycl. Aromat. Compd. 1991, 2, 19-27.
(58) TenBrink, R. E.; McCall, J . M. J . Heterocycl. Chem. 1981, 18,
821-824.

Peptide-Based Metal Ion Sensors
J . Org. Chem., Vol. 61, No. 25, 1996 8947
163 (11), 151 (55); HRMS [M+] calculated for C11H16NO4
226.107 933, observed 226.108 000.
Met h yl 2-[(t r iflu or om et h yl)su lfon yl]p yr id in e-5-ca r -
boxyla te (14). To a suspension of the methyl ester (1g, 6.53
mmol) and N-phenyltrifluoromethanesulfonimide (2.3 g, 6.53
mmol) in CH2Cl2 (20 mL) was slowly added Et3N (0.9 mL, 7.18
mmol). The resulting solution was the stirred at room tem-
perature for 48 h. After washing the mixture with 1 N NaOH
(2×) and Na2CO3, the organic layer was dried and concen-
trated. Purification by flash chromatography on silica gel (99:1
CH2Cl2/MeOH) afforded 1.5 g (80% yield) of triflate 14:59 1H
NMR (CDCl3, 300.2 MHz) δ 3.98 (s, 3H), 7.25 (d, 1H, J ) 8.5
Hz), 8.49 (dd, 1H, J ) 8.5, 2.3 Hz), 9.01 (d, 1H, J ) 2.3 Hz);
13C NMR (CDCl3, 75.5 MHz) δ 53.1, 115.0, 127.0, 142.6, 150.7,
158.4, 164.3.
2-(Tr im eth ylsta n n yl)-5-n itr op yr id in e (16). A mixture
of Me3SnSnMe3 (1.07 g, 3.27 mmol), 2-bromo-5-nitropyridine
(15; 500 mg, 2.46 mmol), Pd(PPh3)4 (25 mg, 0.0213 mmol), and
benzene (8 mL) was heated under reflux in a N2 atmosphere
for 12 h. It was then cooled, the benzene concentrated under
reduced pressure, and the brownish residue purified by
alumina chromatography (eluent, CHCl3) to give 365 mg (52%)
of 16 as pale yellow crystals (mp 47-48 °C): Rf ) 0.62 (hexane/
EtOAc, 8:2); 1H NMR (CDCl3, 300.2 MHz) δ 0.38 (9H, s), 7.69
(1H, d, J ) 8.1 Hz), 8.24 (1H, dd, J ) 8.1, 2.6 Hz), 9.48 (1H,
d, J ) 2.4 Hz); 13C NMR (CDCl3, 75.5 MHz) δ -9.2, 107.2,
127.2, 131.1, 143.3, 144.1, 184.5; IR (CHCl3) 2987 (w), 1587
(m), 1571 (m), 1447 (w), 1352 (s), 1258 (w), 1216 (s), 1210 (m),
1018 (w), 852 cm-1 (s); MS m/z 288 (M+cluster, 7), 273 (92),
243 (79), 165 (54), 135 (100), 120 (41). HRMS [M+] calculated
for C8H12O2N2Sn 279.994 701, observed 279.993 629.
2-Am in o-3-(3,4-d im et h oxyb en z-1-yl)p r op a n oic Acid
Meth yl Ester (11). Benzene (4 mL) was added to the amino
acid hydrochloride (260 mg, 1.15 mmol, 50% ee) and lyophi-
lized. The residue was dissolved in dry MeOH (3 mL) and
cooled to 0 °C. Anhydrous hydrogen chloride was passed
through the mixture for 10 min. After standing at room
temperature overnight, the solvent was removed under re-
duced pressure. MeOH (3 × 5 mL) was added and removed.
The oily residue was the dissolved in 0.2 N NaHCO3 and
extracted with CHCl3 (3 × 10 min). The combined organic
phases were dried over Na2SO4 and concentrated in vacuo to
yield 210 mg (76%) of a racemic mixture of methyl esters 11:
1H NMR (CDCl3, 300.2 MHz) δ 2.84 (dd, 1H, J ) 13.5, 7.5 Hz),
3.06 (dd, 1H, J ) 13.5, 4.8 Hz), 3.74 (s, 3H), 3.87 (s, 6H), 6.73
(s, 1H), 6.76 (s, 1H), 6.82 (d, 1H, J ) 7.8 Hz); 13C NMR (CDCl3,
75.5 MHz) δ 40.3, 51.8, 55.5, 55.6, 111.0, 112.0, 121.1, 129.3,
147.6, 148.6, 175.1; IR (CHCl3) 3360 (w, NsH), 3025-2925
(m, aromatic CsH), 2825 (w, aliphatic C-H), 1731 (s, CdO),
1510 (s, CdC ring), 1259 (s, CsOsC), 1243 (s), 1024 cm-1 (m,
CsOsC); MS m/z 239 (M+, 8), 180 (10), 151 (100), 135 (3), 59
(3). HRMS [M+] calculated for C12H17NO4 239.115 758, ob-
served 239.115 600.
En zym a tic Resolu tion of L-3-(3,4-d im eth oxyben z-1-yl)-
p r op a n oic Acid (10). The optical purity of the underivatized
amino acid was determined by resolving the enantiomers
directly on a chiral Crownpak CR(+) column (Daicel).
A
solution of the protease type VIII, Sigma (1.7 mg) in 6.8 mL
of 0.2 M NaHCO3 was added to the racemic mixture of the
amino acid methyl esters (100 mg, 0.418 mmol). The mixture
was agitated on an orbital shaker and monitored by HPLC at
30 min intervals until the ratio of the D-amino acid methyl
ester to the L-amino acid methyl ester was >50:1 (∼5 h). At
this stage, the mixture was extracted with CHCl3 (6 × 20 mL).
The aqueous phase was reduced in volume to remove any
chloroform and then lyophilized to yield a mixture of amino
acid and carbonate salts. The combined organic layers were
dried over Na2SO4, and the solvent was evaporated. HPLC
analysis of the aqueous phase showed a 100% ee of the L-amino
acid 10, while analysis of the organic residue showed 72% ee
of the D-amino acid methyl ester 11.
Meth yl 5′-Nitr o-2,2′-bip yr id in e-5-ca r boxyla te (17). Tri-
flate 14 (1.17 g, 4.12 mmol), tin derivative 16 (1.30 g, 4.53
mmol), LiCl (525 mg, 12.36 mmol), and Pd(PPh3)4 (238 mg,
0.21 mmol) were heated in dioxane at 100 °C for 12 h. The
mixture was cooled and filtered to obtain 461 mg of 17 as a
pale orange solid (mp 234-235 °C), and the filtrate was
concentrated to give a dark solid. The solid was purified by
flash column chromatography (silica gel, CH2Cl2/MeOH, 98:
2) to give 317 mg more of 17 (total yield, 73%). 17 was an
insoluble crystal in most of organic and aqueous solvents: Rf
1
) 0.72 (CHCl3/MeOH, 98:2); H NMR (CDCl3, 300.2 MHz) δ
4.00 (3H, s), 8.47 (1H, dd, J ) 8.4, 2.1 Hz), 8.61 (1H, d, J )
8.4 Hz), 8.62 (1H, d, J ) 8.7 Hz), 8.73 (1H, d, J ) 8.7 Hz),
9.31 (1H, d, J ) 2.1 Hz), 9.50 (1H, d, J ) 2.7 Hz); IR (KBr)
3300-3080 (w, aromatic CsH), 1719 (s, CdO), 1520 (m, NdO),
1349 (s, NdO), 856 cm-1 (m, CsN); MS m/z 259 (M+, 100),
228 (64), 213 (25), 200 (14), 186 (12), 154 (23), 142 (3), 136 (6),
127 (12), 115 (3), 100 (6), 77 (12), 59 (11), 51 (15); HRMS [M+]
calculated for C12H10N3O4 259.059 306, observed 259.059 900.
Meth yl 5′-Am in o-2,2′-bipyr idin e-5-car boxylate (18). The
nitro compound 17 (139 mg, 0.54 mmol) was suspended in
absolute EtOH (20 mL). Pd/C catalyst was added (14 mg) and
the mixture stirred under hydrogen at atmospheric pressure
for 12 h. The obtained amine was soluble in EtOH. The
reaction mixture was filtered through Celite and the solvent
removed to afford 110 mg of 18 as a yellow solid (89%) (mp
>250 °C): Rf ) 0.52 (CHCl3/MeOH, 8:2); 1H NMR (CD3OD,
300.2 MHz) δ 3.94 (3H, s), 7.13 (1H, dd, J ) 8.6, 2.8 Hz), 8.07
(1H, dd, J ) 2.8, 0.5 Hz), 8.13 (1H, dd, J ) 8.6, 0.5 Hz), 8.21
(1H, dd, J ) 8.4, 0.8 Hz), 8.35 (1H, dd, J ) 8.4, 2.2 Hz), 9.09
(1H, dd, J ) 2.2, 0.8 Hz); 13C NMR (CD3OD, 75.5 MHz) δ 52.8,
120.3, 122.0, 124.2, 125.5, 137.2, 139.0, 144.2, 147.7, 151.1,
161.2; IR (KBr) 3430 (w, NsH), 3283-3194 (w, aromatic
CsH), 1707 (s, CdO), 1579 (s), 1289 (s), 842 (w, CsH); MS
m/z 229 (M+, 100), 198 (46), 187 (4), 170 (24), 143 (16), 116
(5), 99 (5), 93 (12), 85 (5), 70 (13), 59 (5); HRMS [M+] calculated
for C12H11O2N3 229.085 127, observed 229.085 300.
(S)-2-Am in o-Nr-(9-flu or en ylm eth yloxyca r bon yl)-3-(3,4-
d im eth oxyben z-1-yl) p r op a n oic Acid (12). The amino acid/
carbonate salt mixtures from above were dissolved in 1.4 mL
of 10% Na2CO3. (9-Fluorenylmethyl)succinimidyl carbonate
(112 mg, 0.331 mmol) was dissolved in 1.4 mL of dioxane and
added dropwise to the amino acid solution. The reaction
mixture was stirred for 1.5 h. The dioxane was evaporated in
vacuo, and the aqueous phase was extracted with Et2O (3 × 5
mL). The aqueous phase was acidified to pH 3 and extracted
with EtOAc (5 × 5 mL). The combined organic phases were
dried over Na2SO4 and concentrated to afford 115 mg (93%)
of 12 as a white powder: Rf ) 0.56 (CHCl3/MeOH, 8:2); [R]20
D
) -28° (0.485, DMF, 0.5 dm path length cell); 1H NMR (CDCl3,
300.2 MHz) δ 3.08 (dd, 1H, J ) 8.4, 5.6 Hz), 3.16 (dd, 1H, J )
14.0, 5.1 Hz), 3.79 (s, 3H), 3.83 (s, 3H),4.19 (t, 1H, J ) 6.5
Hz), 4.39 (d, 2H, J ) 6.8 Hz), 4.6-4.7 (m, 1H), 5.25 (d, 1H, J
) 7.84 Hz), 6.6-6.8 (m, 4H), 7.2-7.4 (m, 4H), 7.54 (t, 2H, J )
6.1 Hz), 7.25 (d, 2H, J ) 7.4 Hz); 13C NMR (CDCl3, 75.5 MHz)
δ 25.3, 37.3, 47.0, 55.8, 67.1, 111.2, 112.2, 120.0, 121.4, 125.0,
127.0, 127.7, 127.8, 141.2, 143.5, 143.6, 148.1, 148.8, 155.8;
IR (CHCl3) 3332 (w, NsH), 2958-2938 (w), 1687 (s, CdO),
1540 (s, CdC), 1515 (s), 1264 (s, CsOsC), 1235 cm-1 (s); MS
m/z 447 (M+, 13), 391 (4), 282 (61), 179 (66), 165 (17); HRMS
[M+] calculated for C26H25NO6 447.168 188, observed
447.167 600.
Met h yl N-Boc-glycyl-5′-a m in o-2,2′-b ip yr id in e-5-ca r -
boxyla te (19). N-Boc-glycine (344 mg, 1.96 mmol) and TFFH
(778 mg, 2.95 mmol) were dissolved in CH2Cl2 (18 mL).
Pyridine (318 mL, 3.93 mmol) was added, and the solution was
stirred at room temperature for 2 h. The resulting mixture
was added over a suspension of the amine 18 (150 mg, 0.65
Meth yl 2-h yd r oxyp yr id in e-5-ca r boxyla te. (Trimethyl-
silyl)diazomethane (5 mL, 2.0 M in hexanes, 10 mmol) was
added to a suspension of 2-hydroxypyridine-5-carboxylic acid
13 (1.07 g, 7.7 mmol) in MeOH (15 mL) and benzene (54 mL).
The resulting solution was stirred at room temperature for 2
h and concentrated in vacuo to afford 1.06 g of the methyl ester
(91% yield): 1H NMR (CDCl3, 300.2 MHz) δ 3.87 (s, 3H), 6.58
(dd, 1H, J ) 9.5, 0.5 Hz), 8.00 (dd, 1H, J ) 9.5, 2.4 Hz), 8.19
(dd, 1H, J ) 2.4, 0.5 Hz).
(59) Torrado, A.; Lo´pez, S.; Alvarez, R.; de Lera, A. R. Synthesis
1995, 285-293.
(60) Biosearch Technical Bulletin No. 9000-02.

8948 J . Org. Chem., Vol. 61, No. 25, 1996
Torrado and Imperiali
mmol) and 2,6-di-tert-butyl-4-methylpyridine (403 mg, 1.96
mmol) in CH2Cl2 (6 mL) and stirred at room temperature for
30 min (when the reaction is over by TLC). Then, H2O was
added to the suspension and the aqueous phase extracted with
CH2Cl2 (5×). The combined organic phases were dried over
Na2SO4 and concentrated. The resulting solid was purified
by flash column chromatography (silica gel, CHCl3/MeOH, 98:
2) to afford 157 mg of 19 as a yellow powder (62%): Rf ) 0.69
(CH2Cl2/MeOH, 8:2); 1H NMR (CDCl3, 300.2 MHz) δ 1.46 (9H,
s), 3.96 (3H, s), 4.00 (2H, d, J ) 5.7 Hz), 5.60 (1H, t, J ) 5.7
Hz), 8.21 (1H, dd, J ) 8.4, 0.6 Hz), 8.3-8.4 (3H, m), 8.73 (1H,
d, J ) 2.1 Hz), 9.19 (1H, d, J ) 0.6 Hz), 9.24 (1H, s, br); 13C
NMR (CDCl3, 75.5 MHz) δ 28.3, 45.6, 46.4, 52.3, 120.1, 122.1,
125.2, 127.4, 135.4, 137.9, 140.7, 150.4, 150.6, 156.7, 158.9,
165.8, 168.6; MS m/z 387 (MH+, 100), 258 (34), 230 (14), 173
(9); HRMS [M+] calculated for C19H23O5N4 387.166 845, ob-
served 387.165 300.
support by treatment with DMF/Ac2O/TEA (12 eq). The
peptides were deprotected and removed from the solid support
by treatment with reagent R: trifluoroacetic acid/thioanisole/
ethanedithiol/anisole, 90:5:3:2) for 4 h. The resulting solution
was concentrated to ∼1 mL and precipitated with ethyl ether/
hexane 1:1. The precipitated product was subsequently
triturated with ether/hexane 1:1 three times (6 mL). The
solvent was decanted and the residue redissolved in H2O and
lyophilized overnight. The peptides were visualized by re-
versed phase high-pressure liquid chromatography (HPLC)
(C18) on a dual-pump system (solvent A, 0.1% TFA/H2O; solvent
B, 0.1% TFA/MeCN), purified on appropiate gradients and
identified by mass spectroscopy.
Peptides F XR02 and F XR03 were purified by reversed
phase HPLC on a semipreparative column (C18) and identified
by electrospray mass spectroscopy (MW(F XR02) ) 1475); MW-
(F XR03) ) 1692).
Nr-(9-F lu or en ylm et h yloxyca r b on yl)-glycyl-5′-a m in o-
2,2′-bip yr id in e-5-ca r boxylic Acid (20). The dipeptide 19
(132 mg, 0.34 mmol) was dissolved in a 50% solution of 0.01
N NaOH/dioxane and stirred at room temperature for 4 h. The
reaction mixture was acidified to pH 3 and the resulting solid
collected and treated with 50% TFA/CH2Cl2 for 30 min. The
TFA was evaporated under N2, and the residue washed with
CH2Cl2 (2×), toluene (1×), and MeOH (1×). The oily residue
was dissolved in a mixture of dioxane/10% Na2CO3 (1:1, 2 mL),
and Fmoc-ONSu (89 mg, 0.26 mmol) in dioxane (0.5 mL) was
added over it. The reaction mixture was stirred for 4 h at room
temperature. The dioxane was evaporated under reduced
pressure and the aqueous phase extracted with Et2O (3×). The
aqueous phase was acidified to pH ∼3 and extracted with
EtOAc (5×). The combined organic extracts were dried (Na2-
SO4) and evaporated to afford 50 mg (46%) of the N-protected
dipeptide 20: Rf ) 0.17 (CHCl3/MeOH/AcOH, 85:15:5); 1H
NMR (DMSO-d6, 300.2 MHz) δ 3.87 (2H, d, J ) 6 Hz), 4.24
(1H, t, J ) 6 Hz), 4.30-4.33 (2H, m), 7.33 (2H, t, J ) 6.7 Hz),
7.42 (2H, t, J ) 7.2 Hz), 7.73 (2H, d, J ) 7.2 Hz), 7.89 (2H, d,
J ) 7.2 Hz), 8.23 (1H, dd, J ) 9, 2.4 Hz), 8.35 (1H, dd, J )
8.4, 2.1 Hz), 8.42 (2H, d, J ) 8.4 Hz), 8.87 (1H, d, J ) 2.1 Hz),
9.10 (1H, d, J ) 2.1 Hz); 13C NMR (DMSO-d6, 75.5 MHz) δ
44.1, 46.6, 65.7, 119.6, 120.1, 121.5, 125.2, 126.6, 127.0, 127.6,
128.3, 136.6, 138.0, 140.2, 140.7, 143.8, 148.9, 150.1, 156.6,
157.8, 166.3, 168.8. MS m/z 517 (MNa+, 4), 495 (MH+, 29),
391 (4), 242 (5), 216 (4), 189 (3), 179 (20), 176 (8), 165 (10);
HRMS [M+] calculated for C28H22O5N4 495.166 845, observed
495.169 600.
Ma cr op in P ep tid e Syn th esis. Peptides were synthesized
on Macrocrowns purchased from Chiron Mimotopes. Com-
mercially available starting materials and reagents were
purchased from Milligen Biosearch, EM Science, Nova Biose-
arch, or Aldrich Chemical Co. A typical coupling cycle involved
a 30 min soak in 20% (v/v) piperidine/DMF followed by a 5
min wash with DMF and three rinses with methanol. After
air-drying for 10 min and 5 min soaking in DMF, the pin was
submerged in the coupling mixture. Typical coupling were
performed in 2 h with Fmoc-protected amino acid pentafluo-
rophenyl esters and N-hydroxybenzotriazole (HOBt) at 80 mM
concentration each with 450 µL of activated amino acid ester
solution per pin. After coupling, the pin was washed once with
DMF and three times with methanol. The pin was then air-
dried for 10 min to complete a full cycle. Residues Flu, Dmd,
and XBp were coupled with 6 equiv of amino acid and HOBt/
DIPCDI/DIPEA chemistry in DMF. For these nonstandard
amino acids, the coupling time was longer: 5 h for Flu and
Dmd and 12 h for XBp. These cycles were repeated until the
peptide was complete. Peptides were N-acetylated on the solid
F lu or escen ce Stu d ies. A typical fluorescence spectrum
was acquired in a 1 cm path length cuvette using a SLM-
Aminco SPF-500C spectrofluorometer, with the following
parameters: HVA 975, gain 10, λex ) 388 nm, λem ) 400 nm,
filter (time constant) 3, and both emission and excitation slit
widths 4 nm. The concentrations of the peptide solutions were
determined first by quantitative amino acid analysis (QAA)
and then spectrophotometrically using the absorption band of
the chromophores (λmax ) 252 nm, ꢀ ) 98570, λmax ) 307 nm,
ꢀ ) 54 569, λmax ) 416 nm, ꢀ ) 8425, and λmax ) 437 nm, ꢀ )
8366). The methanolic and aqueous solutions of known
peptide concentrations were then titrated with standard metal
cation solutions at room temperature. Aqueous titrations were
performed in 0.01 M PIPES buffer (pH 7.0) and 0.01 M
phosphate buffer (pH 7.0). The recorded spectra were analyzed
using KaleidaGraph version 3.0 (Abelbeck Software, CA).
Abbr evia tion s
Dmd, (S)-2-amino-3-(3,4-dimethoxybenz-1-yl)propanoic
acid; DIPEA, diisopropylethylamine; DIPCDI, diisopro-
pylcarbodiimide; EDTA, ethylenediaminetetraacetic acid;
FDAA, 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide; Flu,
(S)-2-amino-3-(9-cyanoanthracen-10-yl)propanoic acid;
FRET, fluorescence resonance energy transfer; PET,
photoinduced electron transfer; HOBt, N-hydroxybenzo-
triazole; SPPS, solid phase peptide synthesis; PTC, phase
transfer catalyst; Fmoc, 9-fluorenylmethoxycarbonyl;
PIPES, 1,4-piperazinebis(ethanesulfonic acid); XBp, gly-
cyl-5′-amino-2,2′-bipyridine-5-carboxylic acid.
Ack n ow led gm en t. This research was supported by
the NSF and the Caltech President’s Fund. The award
of Postdoctoral fellowships from NATO and Xunta de
Galicia (to A.T.) is also gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra for
5, 6, 12, and 16-20 and the 13C NMR spectra for 9 (recorded
on a General Electric QE-300 spectrometer) and HPLC traces
for peptides F XR02 and F XR03 (obtained on a Beckman
reversed phase HPLC (C18 column)) (11 pages). This material
is contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
J O961466W