Asymmetric synthesis of a new 8-hydroxyquinoline-derived α-amino acid and its incorporation in a peptidylsensor for divalent zinc

Full text of DOI:10.1021/jo001681j

3224
J . Org. Chem. 2001, 66, 3224-3228
Asym m etr ic Syn th esis of a New
8-Hyd r oxyqu in olin e-Der ived r-Am in o Acid
a n d Its In cor p or a tion in a P ep tid ylsen sor
for Diva len t Zin c
Nathalie J otterand, Dierdre A. Pearce, and
Barbara Imperiali*
Department of Chemistry, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
imper@mit.edu
into a short model peptide (7 residues).6 The synthesis
is outlined in Scheme 1 and proceeds with two key
processes starting from the commercially available 8-hy-
droxy-2-methylquinoline. The first process involves the
attachment of a phenyl ring to the 5 position of the
hydroxyquinoline via a Suzuki-type coupling; the second
involves the construction of the R-amino acid strereo-
center via an asymmetric alkylation.
8-Hydroxy-2-methyl-quinoline 3 was protected as the
tert-butyldimethylsilyl ether and regioselectively bromi-
nated at the 5 position by treatment with bromine. The
resulting bromide 4 was coupled with benzene boronic
acid in the presence of Pd(PPh3)4 to afford the corre-
sponding phenyl derivative 5. Attempts to convert 5
directly to the bromomethyl 6 under free radical condi-
tions (NBS, CCl4)2 led mainly to bromination of the
phenyl ring. Additionally, replacement of the silyl ether
by a benzenesulfonate ester7 (HCl, 1 M, then Et3N,
PhSO2Cl) led to the same observations. Finally, an
alternative was found using a three-step procedure that
included the oxidation of 5 by selenium dioxide, which
provided the corresponding aldehyde, reduction with
NaBH4, and smooth conversion to the bromomethyl
derivative, 6, by treatment with a mixture of NBS and
methyl sulfide. The overall yield is 67%, and only one
chromatographic purification is necessary.
Received November 30, 2000
The need for sensors capable of monitoring real-time
concentrations of free Zn(II) in a complex aqueous
medium remains a technological challenge. Toward this
goal, strategies for the development of sensitive fluores-
cent chemosensors with ideal photophysical properties
are still needed. The approach taken by this group
involves adapting the modular architecture of the polypep-
tide backbone for the construction of new and useful
sensing molecules. The first generation of designs were
based on the zinc finger domain.1 These constructs
demonstrated excellent selectivity toward Zn(II); how-
ever, the chemosensor molecules were large (>20 amino
acid residues in length). Second generation designs
provided smaller peptidyl fluorescent sensors (7 residues)
in which the reporter functionality was a residue bearing
the bidentate ligand 8-hydroxy-quinoline2 (oxine) 1, a
chromophore known to produce a chelation enhanced
fluorescence (CHEF) upon metal binding.3 Although
these last sensors have shown good selectivity for Zn(II),
their use coupled with fiber optic devices is restricted
because of the excitation wavelength of the oxine reporter
at 355 nm, which is too close to the limit where the
transmission of light through the optic fiber starts to
become efficient.4 Therefore, the focus has been to
redesign the oxine fluorophore to shift the excitation
wavelength of the quinoline reporter to longer, more
practical wavelengths. In many conjugated systems this
“red shift” can be achieved by extending the aromatic
system, thereby reducing the π to π* gap.5 Consequently,
emphasis has been made to substitute the quinoline
moiety with different aromatic groups that include a
more extended chromophore.
The asymmetric alkylation of commercially available
N-(diphenylmethylene)glycine tert-butylester 7 with 6
was carried out using the Corey modification8 of the
O’Donnell method,9 using the phase transfer catalyst
(8S,9R)-O-(9)-allyl-N-9-anthracenyl methylcinchonidium
bromide to favor the formation of the adduct possessing
the L configuration.10 Using a slight excess of bromide 6
(6) The asymmetric synthesis of (S)-2-Amino-NR-(9-fluorenylmethyl-
oxycarbonyl)-3-(8-hydroxyquinoline-2-yl) propanoic acid has been pre-
viously reported (see ref 2). However, the presence of a supplementary
aromatic ring on the oxine moiety required the development of a new
synthetic procedure.
Herein we report the asymmetric synthesis of (S)-2-
amino-NR-9-fluorenylmethoxycarbonyl-3-(5-phenyl-8-hy-
droxyquinoline-2-yl) propionic acid 2 and its incorporation
(7) The silyl ether was hydrolyzed at 40 °C in dioxane/aqueous HCl,
1 M (1: 1). The mixture was neutralized (NaOH, 1 M) and extracted
(CH2Cl2). The organic phase was dried (K2CO3) and evaporated. The
residue was taken in dry THF, and the protection of the alcohol was
carried out according to ref 2 to afford 8-benzenesulfonyloxy-2-methyl-
5-phenylquinoline with an overall yield of 90%. Rf ) 0.31 (EtOAc/
hexane 1:3); 1H NMR (CDCl3) 8.06 (d, J ) 8.5, 1H), 8.05 (d, J ) 8.5,
1H), 8.02 (d, J ) 8.8, 1H), 7.67 (d, J ) 8.0, 1H), 7.61 (bt, J ) 7.5, 1H),
7.52-7.39 (m, 8H), 7.15 (d, J ) 8.5, 1H), 2.52 (s, 3H); 13C NMR (CDCl3)
160.0, 144.9, 141.6, 140.4, 139.4, 137.4, 135.2, 134.5, 130.6, 129.6, 129.3,
(1) (a) Walkup, G. K.; Imperiali, B. J . Am. Chem. Soc. 1996, 118,
3053-3054. (b) Walkup, G. K.; Imperiali, B. J . Am. Chem. Soc. 1997,
119, 3443-3450. A sensor incorporating a zinc finger structure has
been independently reported: Godwin, H. A.; Berg, J . M. J . Am. Chem.
Soc. 1996, 118, 6514-6515.
(2) Walkup, G. K.; Imperiali, B. J . Org. Chem. 1998, 63, 6727-6731.
(3) CHEF effect of oxine in the presence of Zn(II): Vogel, A. I. In
Vogel’s Textbook of Quantitative Chemical Analysis; Wiley: New York,
1989; Chapter 18, pp 731-740. CHEF effect of oxine in the presence
of other metal ions such as Mg(II), Ca(II), Pb(II): see ref 5.
(4) (a) Thompson, R. B. IEEE Circuit Device 1994, 10, 14-21. (b)
Signal attenuation in optic fibers results from scattering caused by
defects in the fibers and becomes significant below 360 nm. The value
of a fiber’s attenuation is typically specified during manufacture.
(5) Seitz, W. R. Critical Reviews in Analytical Chemistry; CRC
Press: Boca Raton, FL, 1980; pp 367-404.
129.2, 128.6, 126.8, 126.5, 123.3, 25.8; HRMS (ES) calcd for C22H18
-
NO3S [M + H]+ 376.1007, found: 376.1054.
(8) (a) Corey, E. J .; Xu, F.; Noe, M. C. J . Am. Chem. Soc. 1997, 119,
12414-12415. (b) Corey, E. J .; Noe, M. C.; Xu. F. Tetrahedron Lett.
1998, 39, 5347-5350.
(9) O’Donnell, M. J .; Bennett, W. D.; Wu, S. J . Am. Chem. Soc. 1989,
111, 2353-2355.
10.1021/jo001681j CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/29/2001

Notes
J . Org. Chem., Vol. 66, No. 9, 2001 3225
Sch em e 1
F igu r e 1. UV-visible absorption spectra and steady-state
emission spectra of P 1 (10 µM) on addition of ZnCl2 (to 100
µM). (a) UV-visible absorption spectra of P 1 in the presence
of 0, 5, 10, and 100 µM ZnCl2. (b) Comparison of fluorescence
excitation and emission spectra of P 1 (solid line spectra) and
P 2 (dashed line spectra). Baseline spectra were acquired in
the absence of ZnCl2; other spectra were acquired on addition
of 100 µM ZnCl2. Samples were prepared in 50 mM HEPES
(pH 7.01), containing 150 mM NaCl. Spectra were acquired
at 21 °C, and baseline were corrected using a sample of the
buffer solution.
fully incorporated into the heptapeptide P 1,15 a sequence
that contains the reverse turn promoting fragment -Val-
Pro-DSer-Phe- and an additional “soft” thiolate-donor
ligand (from cysteine) to improve the affinity of the ligand
for Zn(II),16 compared with the parent 2. The major
product, containing the L-amino acid was purified from
the minor product, containing the D-amino acid, by
HPLC.17 For the purposes of comparison an analogous
peptide P 2, containing the parent oxine residue 1, was
also prepared.
(1.2 equiv), the alkylation adduct 8 was obtained with
82% yield. Simultaneous deprotection of the imine, ester,
and phenol functionalities was carried out under acid
hydrolysis (HCl, 6 M, reflux) to afford the corresponding
amino acid as the hydrochloride salt form 9. Derivatiza-
tion of 9 with Marfey’s reagent11 (1-fluoro-2,4-dinitro-
phenyl-5-L-alanineamide) followed by HPLC analysis of
the adducts revealed a 91% ee in favor of the L-amino
acid.12
To incorporate 9 into a polypeptide sequence via solid-
phase synthesis techniques,13 the amino acid was con-
verted to the corresponding 9-fluorenyl-methoxycarbonyl
(Fmoc) derivative 2 by treatment with (9-fluorenyl-
methoxycarbonyl)succinimidyl carbonate (Fmoc-Su) (88%
yield from 7).14 The amino acid derivative 2 was success-
Addition of Zn(II) to P 1 was monitored by both UV-
visible and steady-state fluorescence emission spectros-
copy. Changes in the UV-visible absorption spectra of
P 1 on addition of Zn(II) are summarized in Figure 1a.
Absorbance bands at 251 and 357 nm in spectra of P 1
are replaced by bands at 269 and 391 nm on addition of
Zn(II).18 The absorbance bands at 357 and 391 nm are
red shifted compared with the corresponding absorbance
bands of spectra of free and Zn(II)-bound P 2 (308 and
(10) The stereochemical outcome of the Corey alkylation has proven
to be predictable for a wide range of electrophiles. See ref 8 and
references therein.
(11) Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M.; Harada,
K.-I. Anal. Chem. 1997, 69, 3346-3352.
(12) The general and unambiguous applicability of the Marfey
method has been well described. Thus, determination of the absolute
configuration of an amino acid by HPLC analysis is feasible: the
L-derivative elutes faster than the parent D-derivative. See ref 10 and
references therein.
(13) Fields, G. B.; Noble, R. L. Int. J . Pept. Protein Res. 1990, 35,
161-214.
(14) The product 2 was stable on storage in the dark, at 5 °C. The
internal, unprotected base is not strong enough to cause loss of the
N-Fmoc protecting group under these conditions.
(15) The peptide containing the (D) enantiomer of 9 was readily
separated by reversed phase HPLC purification.
(16) (a) Imperiali, B.; Fisher, S. L.; Moats, R. A.; Prins, T. J . J . Am.
Chem. Soc. 1992, 114, 3182-3188. (b) Imperiali, B.; Kapoor, T. M.
Tetrahedron 1993, 49, 3501-3510. (c) Cheng, R. P.; Fisher, S. L.;
Imperiali, B. J . Am. Chem. Soc. 1996, 118, 11349-11356.
(17) In the final synthetic cycle, the completed sequences were acetyl
capped. A subsequent piperidine de-block cycle was performed. HPLC
analysis of the completed peptides showed no trace of byproduct
peptides, corresponding to esterification of the quinoline OH.

3226 J . Org. Chem., Vol. 66, No. 9, 2001
Notes
recorded at 300 MHz for proton frequency and 75 MHz for carbon
frequency. Chemical shifts are reported in δ units (ppm) relative
to tetramethylsilane (external standard). Optical rotations were
recorded at room temperature.
372 nm respectively, data not shown). Fluorescence
spectra from Zn(II)-bound P 1 was compared with those
from the analogous Zn(II)-bound P 2. Neither species is
fluorescent at pH 7.0 (50 mM HEPES, 150 mM NaCl) in
the absence of the metal ion, and on addition of Zn(II)
the fluorescence emission spectra of the two species
almost overlap. Spectra of Zn(II)-bound P 1 and Zn(II)-
bound P 2 exhibit emission maxima of 546 and 544 nm,
respectively. However, while the emission from Zn(II)-
bound P 2 reaches a maximum at λex ) 372 nm, emission
from Zn(II)-bound P 1 reaches a maxima at λex ) 396 nm
in the corresponding excitation spectra, representing a
red shift of some 24 nm, Figure 1b. The quantum yield19
of Zn(II)-bound P 1 was found to be 0.005, a value
consistent with the modest quantum yields of metal
complexes of other oxine derivatives.20 A method for the
systematic increase in quantum yield of Zn(II) oxine
complexes has been recently reported21 and will be
investigated as means to improve the luminescence
properties of derivatives such as 2.
In conclusion, this note describes the asymmetric
synthesis, peptide incorporation, and characterization of
a new R-amino acid containing the bidentate ligand
8-hydroxyquinoline to which a phenyl ring has been
attached to the 5 position. This modification results in
significant red shifts in the excitation maxima of the
ligand when complexed to Zn(II). Fluorescent sensors
designed to be compatible with fiber optic technology
must take into consideration the loss of transmitted light
due to Rayleigh scattering and inhomogeneities in the
fiber that are particularly significant in the UV region
of the spectrum below 360 nm; beyond this point signal
transmittance increases almost linearly to about 500
nm.4a The simple modification reported here produces a
red shift in the excitation maxima of the derivative to
almost 400 nm and represents a prototype for the
systematic design of new sensors for the selective and
sensitive analysis of trace Zn(II) through fiber optic
devices. Further studies, to increase the quantum yield
and spectral red shifts of such residues, are in progress.
P ep tid e Syn th esis. The peptides (0.0333 mmol) were syn-
thesized using standard Fmoc amino acid protection chemistry
on Fmoc-PAL-PEG-PS resin (0.170 mmol equiv). Residues 1-6
were synthesized on a Milligen series 9050 PepSynthesizer with
a 4-fold excess of activated amino acid ester (prepared in situ
using N-hydroxybenzotriazole/2-(benzotriazole-1-yl)-1,1,3,3,-tet-
ramethyluronium hexafluorophosphate (HOBT/HBTU)). The
final residue was added manually, using benzotriazole-1-yl-oxy-
tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) and
diisopropylethylamine (DIEA) to generate the activated ester.
In this case, coupling was carried out with a 2-fold excess of
amino acid using the following conditions: removal of Fmoc
protecting group (20% piperidine in DMF, 2 × 8 min), wash
(DMF 2 × 2 min, MeOH 1 × 2 min, DMF 2 × 2 min); amino
acid coupling (amino acid/PyBOP/DIEA 2:2:2), wash (DMF 5 ×
2 min). The N-terminal Fmoc was removed as previously, and
the N-terminal R-amine was acetylated with 2 mL (3 × 10 min)
of a mixture consisting of DMF/CH2Cl2/Ac2O/HOBT (90 mL:10
mL:2.5 mL:48 g). Any aryl ester formed was cleaved (20%
piperidine in DMF, 2 × 8 min), and the resin washed (as above).
After a final wash with MeOH, the resin was dried under
vacuum and the peptide was cleaved from the resin (TFA, 5%
v/v triisopropylsilane, 90 min). The resulting solution was
concentrated, and the crude peptide was precipitated by addition
of an ether/hexane 1:1 solution. The suspension was triturated
using the same solution (3 × 5 mL). The pellet containing the
peptide was dissolved in water, lyophilized from the aqueous
eluent overnight, and purified to homogeneity (as evidenced by
the appearance of a single peak in HPLC traces on re-injection
of the purified sample) by reversed phase high-pressure liquid
chromatography (HPLC, C18).
8-ter t-Bu tyld im eth ylsilyloxy-2-m eth ylqu in olin e. A 100-
mL flask was successively loaded with dry CH2Cl2 (50 mL),
8-hydroxy-2-methylquinoline (5.1 g, 32.037 mmol), imidazole
(2.29 g, 33.639 mmol, 1.05 equiv), and tert-butyldimethylsilyl
chloride (5.31 g, 35.24 mmol, 1.1 equiv). The solution was stirred
at room temperature for 10 h, diluted with Et2O (500 mL),
washed with aqueous HCl (0.1 M, 50 mL), brine (100 mL), water
(100 mL), dried over K2CO3 and evaporated under reduced
pressure to afford 8.41 g (96%) of protected product as a colorless
oil. Rf ) 0.58 (EtOAc/hexane 5:95); Rf ) 0.35 (Et2O/hexane 2:98);
1H NMR (CDCl3) δ 7.98 (d, J ) 8.5, 1H), 7.35 (ap. t, J ) 8.3,
1H), 7.34 (d, J ) 8.3, 1H), 7.24 (d, J ) 8.3, 1H), 7.15 (bd, J )
8.5, 1H), 2.76 (s, 3H), 1.08 (s, 9H), 0.29 (s, 6H); 13C NMR (CDCl3)
δ 157.3, 152.4, 141.8, 136.0, 128.0, 126.1, 122.1, 120.3, 118.3,
26.3, 25.4, 19.3, -3.6; IR (thin film) 2927, 2888, 2855, 1599, 1566,
1504, 1470, 1432, 1326, 1276, 1247, 1237, 1104, 926, 833, 783,
616; HRMS (ES) calcd for C16H24NOSi [M + H]+ 274.1629, found
274.1645.
5-Br om o-8-ter t-b u t yld im et h ylsilyloxy-2-m et h yl-q u in o-
lin e (4). A solution of bromine (1.16 mL, 22.67 mmol, 1 equiv)
in CH2Cl2 (20 mL) was added dropwise to a vigorously stirred
solution of the silyl ether (6.20 g, 22.67 mmol) in CH2Cl2 (150
mL). The completion of the reaction was checked by TLC (UV
detection). If necessary, more bromine solution was added. The
solution was diluted with CH2Cl2 (1 L), washed with aqueous
saturated Na2S2O4 (50 mL), aqueous saturated NaHCO3 (100
mL), brine (50 mL), and water (100 mL), dried over K2CO3, and
evaporated under reduced pressure to afford 7.42 g (93%) of
bromide 3 as a yellowish oil. An analytical sample was obtained
by flash chromatography (UV detection, hexane/Et2O 98:2). Rf
) 0.57 (Et2O/hexane 2:98); Rf ) 0.67 (EtAc/hexane 10:90); 1H
NMR (CDCl3) δ 8.32 (d, J ) 8.8, 1H), 7.59 (d, J ) 8.3, 1H), 7.33
(d, J ) 8.5, 1H), 7.03 (d, J ) 8.3, 1H), 2.75 (s, 3H), 1.07 (s, 9H),
0.27 (s, 6H); 13C NMR (CDCl3) δ 158.0, 152.4, 142.3, 135.6, 129.6,
126.9, 123.3, 118.8, 112.5, 26.3, 25.1, 19.3, -3.5; IR (thin film)
2954, 2927, 2855, 1590, 1466, 1321, 1255, 1077, 840, 616; HRMS
(ES) calcd for C16H23NOSiBr [M + H]+ 352.0733, found 352.0727.
8-ter t-Bu t yld im et h ylsilyloxy-2-m et h yl-5-p h en ylq u in o-
lin e (5). A 50-mL Schlenk flask containing bromide 4 (369 mg,
1.0473 mmol) was successively loaded with benzene (11 mL),
aqueous Na2CO3 2 M (12.9 mL), ethanol (2.9 mL), and ben-
zeneboronic acid (140 mg, 1.152 mmol, 1.1 equiv). The emulsion
Exp er im en ta l Section
Gen er a l P r oced u r es. All reagents and solvents from com-
mercial suppliers were used without further purification. O-(9)-
Allyl-N-9-anthracenylmethylcinchonidium bromide was pre-
pared according to the method developed by Corey.8 Methylene
chloride was distilled from calcium hydride. Anhydrous reactions
were performed in oven-dried glassware under a positive pres-
sure of nitrogen. Thin-layer chromatography was carried out
using TLC plates (silica gel IB-F) obtained from J . T. Baker.
TLC plates were visualized by UV or 0.2% ninhydrin solution
in ethanol followed by heat. Flash column chromatography was
performed according to the procedure of Still22 using 230-400
mesh silica gel. Nuclear magnetic resonance (NMR) spectra were
(18) Addition of a single equivalent of Zn(II) almost saturated P 1
(10 µM); however, analysis of titration data was complicated by
nonlinear behavior due to peptide aggregation in solution. The appar-
ent dissociation constant was estimated to be 0.1-0.3 µM, using the
method described in ref 16c.
(19) Determined with reference to quinine sulfate in 0.1 M H2SO4.
Uncertainty estimated at 15%. Chen, R. F. Anal. Chem. 1967, 19, 374-
387.
(20) Prodi, L.; Bargossi, C.; Montalti, M.; Zaccheroni, N.; Su, N.;
Bradshaw, J . S.; Izatt, R. M.; Savage, P. B. J . Am. Chem. Soc. 2000,
122, 6769-6770.
(21) Pearce, D. A.; J otterand, N.; Carrico, I. S.; Imperiali, B. 2000,
submitted for publication.
(22) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923-
2925.

Notes
J . Org. Chem., Vol. 66, No. 9, 2001 3227
was degassed, and Pd(PPh3)4 (36.7 mg, 0.0317 mmol, 0.0303
equiv) was rapidly added under a stream of nitrogen. The flask
was equipped with a condenser, and the mixture was refluxed
with vigorous stirring until no more bromide 4 remained (ca.
48 h). The mixture was cooled, diluted with benzene (100 mL),
washed with water (25 mL) and brine (25 mL), dried over K2-
CO3, and evaporated under reduced pressure. The crude residue
was purified by flash column chromatography (Et2O/hexane 2:98,
UV detection) to afford 307 mg (84%) of phenyl 5 as a yellowish
oil. Rf ) 0.57 (EtOAc/hexane 5:95); Rf ) 0.41 (Et2O/hexane 2:98);
1H NMR (CDCl3) 8.07 (d, J ) 8.5, 1H), 7.47-7.35 (m, 5H), 7.28
(ap. t, J ) 7.7, 1H), 7.18 (d, J ) 8.8, 1H), 2.73 (s, 3H), 1.09 (s,
9H), 0.31 (s, 6H); 13C NMR (CDCl3) 157.0, 151.9, 141.5, 140.1,
134.4, 133.0, 130.3, 128.6, 128.5, 127.2, 126.9, 126.0, 122.0, 117.7,
26.3, 25.1, -3.4; IR (thin film) 2927, 2854, 1570, 1467, 1320,
1247, 1099, 856, 620; HRMS (ES) calcd for C22H28NOSi [M +
H]+ 350.1941, found 350.1913.
784, 615; HRMS (ES) calcd for
428.1046, found 428.1026.
C
22H27NOSiBr [M + H]+
(N-Dip h en ylm eth ylen e)-2-a m in o-3-(8-ter t-bu tyld im eth -
ylsilyloxy-5-p h en yl-qu in olin e-2-yl)-p r op a n oic Acid ter t-
Bu tyl Ester (8). A 10-mL Schlenk flask was successively loaded
with CH2Cl2 (0.5 mL), tert-butylglycinate benzophenone imine
7 (45.5 mg, 0.154 mmol, 1 equiv), O-(9)-allyl-N-9-anthracenyl-
methylcinchonidium bromide (9 mg, 0.0154 mmol, 0.1 equiv),
and rapidly with CsOH monohydrate (258 mg, 1.54 mmol, 10
equiv). The flask was filled with nitrogen and cooled to -78 °C.
A solution of bromide 5 (79.2 mg, 0.1848 mmol, 1.2 equiv) in
CH2Cl2 (0.7 mL) was added dropwise and the suspension was
vigorously stirred at -55 °C till the imine was completely
consumed (ca. 23 h). The suspension was poured into Et2O (50
mL), washed with water (2 × 10 mL) and brine (10 mL), dried
over MgSO4, and evaporated under reduced pressure. The
residue was purified by flash chromatography (EtOAc/ hexane
5:95, UV detection) to afford 82 mg (82%) of adduct 7 as a yellow
oil. Rf ) 0.15 (EtOAc/hexane 5:95); Rf ) 0.27 (Et2O/hexane 10:
8-ter t-Bu t yld im et h ylsilyloxy-5-p h en ylq u in olin e-2-ca r -
ba ld eh yd e. Molecular sieves 4 Å (200 mg) then selenium
dioxide (66 mg, 0.5952 mmol, 1.01 equiv) were added under
nitrogen to a solution of 5 (206 mg, 0.5893 mmol) in dry 1,4-
dioxane (4 mL). The mixture was stirred at 90 °C for 3 h, cooled,
and filtered over a pad of Celite. The Celite was rinsed with
dioxane (6 mL), and the solvent was removed under reduced
pressure. The residue was taken up in Et2O (100 mL), washed
with brine (25 mL) and water (25 mL), dried over K2CO3, and
evaporated under reduced pressure to afford 74 mg (81%) of the
corresponding aldehyde as a yellowish oil. An analytical sample
was obtained by flash chromatography (Et2O/ hexane 2:98, UV
detection). Rf ) 0.54 (EtOAc/hexane 5:95); Rf ) 0.38 (Et2O/
1
90); H NMR (CDCl3) δ 8.03 (d, J ) 8.5, 1H), 7.81 (d, J ) 7.7,
1H), 7.59-7.15 (m, 16H), 6.65 (bd, J ) 7.2, 1H), 4.54 (dd, J )
9.3, 4.1, 1H), 3.66 (dd, J ) 13.0, 4.1, 1H), 3.56 (dd, J ) 13.0, 9.3,
1H), 1.44 (s, 9H), 1.03 (s, 9H), 0.13 (s, 3H), 0.11 (s, 3H); 13C NMR
(CDCl3) δ 171.1, 170.8, 157.5, 152.1, 141.7, 140.0, 139.7, 137.7,
136.3, 134.1, 132.9, 132.6, 130.3, 129.1, 129.0, 128.5, 128.4, 128.1,
127.7, 127.3, 127.2, 126.4, 122.8, 113.6, 81.4, 67.2, 43.0, 28.3,
26.3, 19.2, -3.3, -3.7; IR (thin film) 2929, 2856, 1570, 1466,
1367, 1279, 1249, 1150, 838, 782, 700; HRMS (ES) calcd for
C41H47N2O3Si [M + H]+ 643.3357, found 643.3350. [R]20
-12.2° (c ) 3.33, CHCl3).
)
D
(S)-2-Am in o-3-(5-p h en yl-8-h yd r oxyqu in olin e-2-yl)p r op i-
on ic Acid Hyd r och lor id e Sa lt (9). The tert-butyl ester 8 (82
mg, 0.1275 mmol) was refluxed in HCl 6 M (2 mL) under
nitrogen until TLC analysis of the solution (nBuOH/AcOH/H2O/
EtOAc 1:1:1:1, UV and ninhydrin detection) showed complete
deprotection (ca. 5 h). The hydrolyzed mixture was cooled,
diluted with HCl 1 M (5 mL), extracted with EtOAc (2 × 10 mL),
and concentrated to dryness under reduced pressure to afford
49 mg (93%) of the double hydrochloride salt 9 as a yellow
solid: mp ) 176-177 °C; Rf ) 0.16 (CH2Cl2/MeOH/AcOH 85:
15:3); Rf ) 0.61 (nBuOH/H2O/AcOH/EtOAc 1:1:1:1); 1H NMR
(CD3OD) δ 8.76 (d, J ) 8.0, 1H), 7.89 (d, J ) 8.0, 1H), 7.67 (d,
J ) 7.8, 1H), 7.60-7.44 (m, 6H), 4.72 (dd, J ) 6.0, 7.0, 1H), 3.99
(dd, J ) 16.0, 7.0, 1H), 3.82 (dd, J ) 16.0, 6.0, 1H); 13C NMR
(CD3OD3) δ 169.0, 153.9, 148.3, 143.5, 137.6, 132.0, 131.1, 130.6,
130.0, 128.8, 128.6, 128.1, 127.2, 123.3, 115.3, 51.7, 34.4; IR (thin
film) 3388 (broad), 2934, 1732, 1633, 1594, 1542, 1514, 1380,
1314, 1222, 1072, 847, 765, 704; HRMS (ES) calcd for C18H17N2O3
1
hexane 2:98); H NMR (CDCl3) δ 10.3 (s, 1H), 8.34 (d, J ) 8.4,
1H), 7.94 (d, J ) 9.0, 1H), 7.54-7.42 (m, 6H), 7.30 (d, J ) 7.8,
1H), 1.14 (s, 9H), 0.35 (s, 6H); 13C NMR (CDCl3) δ 193.7, 153.1,
151.2, 141.9, 139.2, 135.9, 135.5, 130.8, 130.3, 129.7, 128.8, 127.8,
118.7, 117.4, 26.2, 19.2, -3.4; IR (thin film) 2928, 2856, 1712,
1595, 1567, 1467, 1362, 1332, 1320, 1307, 1256, 1092, 1071, 906,
840, 784, 759, 703. HRMS (ES) calcd for C22H26NOSi [M + H]+
364.1733, found 364.1712.
8-ter t-Bu tyldim eth ylsilyloxy-5-ph en ylqu in olin e-2-m eth a-
n ol. A solution of the previous aldehyde (214 mg, 0.5893 mmol)
in CH2Cl2 (2 mL) and absolute ethanol (2 mL) was added
dropwise under nitrogen to an ice-cooled solution of NaBH4 (22.3
mg, 0.5893 mmol, 1 equiv) in absolute ethanol (3 mL). The
solution was stirred at 0 °C for 10 min, diluted with Et2O (50
mL), washed with aqueous HCl 0.1 M (5 mL) and water (2 × 15
mL), dried over Na2SO4, and evaporated under reduced pressure
to afford 200 mg (93%) of the corresponding alcohol as a
yellowish oil. An analytical sample was obtained by flash
chromatography (EtOAc/hexane 5:95, UV detection). Rf ) 0.14
(EtOAc/hexane 5:95); Rf ) 0.12 (Et2O/hexane 2:98); 1H NMR
(CDCl3) δ 8.19 (d, J ) 8.8, 1H), 7.50-7.42 (m, 5H), 7.37 (d, J )
8.0, 1H), 7.21 (ap. t, J ) 8.0, 1H), 4.93 (s, 2H), 1.15 (s, 9H), 0.31
(s, 6H); 13C NMR (CDCl3) δ 157.3, 151.2, 140.7, 139.7, 135.5,
133.6, 130.3, 129.2, 128.7, 128.4, 127.5, 127.4, 118.5, 118.2, 64.4,
26.2, 18.9, -3.8; IR (thin film) 3386, 2928, 1711, 1471, 1254,
839, 762, 703; HRMS (ES) calcd for C22H28NO2Si [M + H]+
366.1890, found 366.1866.
[M + H]+ 309.1240, found 309.1234. [R]20 ) +2.4° (c ) 0.83,
MeOH/aqueous HCl, 1 M, 9:1).
D
Deter m in a tion of th e En a n tiom er ic Excess. The enan-
tiomeric excess of the pure amino acid was evaluated by
derivatization with 1-fluoro-2,4-(dinitrophenyl)-5-L-alanineamide
(FDAA, Marfey’s reagent) followed by reversed phase HPLC
analysis of the adducts. The hydrochloride salt (1.3 mg, 0.00422
mmol) was dissolved in 0.1 M NaHCO3 (370 µL), and the
resulting solution was added to a freshly prepared solution of
1.49 mg (0.00548 mmol, 1.3 equiv) of Marfey’s reagent in acetone
(120 µL). The mixture was kept at 40 °C for 1 h with frequent
vortexing/shaking. After cooling, the pH was adjusted to 4 by
addition of HCl 1 M. The mixture was centrifuged, and 5 µL of
the supernatant solution was analyzed by HPLC (C18 column;
solvent A ) H2O, 0.1% v/v TFA; solvent B ) MeCN, 0.1% v/v
TFA; linear gradient 30-70% over 25 min). tR: S derivative )
22.78 min, R derivative ) 25.04 min. A 91% ee was calculated
by comparison of peak areas. The compounds comprising the
peaks were confirmed by mass spectroscopy. ESMS calcd for
C27H25O8N6 [M + H]+ 561.2, found, 561.3.
2-Br om om e t h yl-8-t er t -b u t yld im e t h ylsilyloxy-5-p h e -
n ylqu in olin e (6). To a solution of N-bromosuccinimide (487 mg,
2.737 mmol, 1.5 equiv) in anhydrous CH2Cl2 (8 mL) was added
dry methyl sulfide (241 µL, 3.284 mmol, 1.8 equiv) dropwise at
0 °C under nitrogen. The yellow suspension was cooled to -15
°C, and a solution of the methyl alcohol oil (667 mg, 1.82465
mmol) freshly dried by azeotropic distillation with toluene (3 ×
10 mL) in anhydrous CH2Cl2 (2.5 mL) was added dropwise. The
reaction mixture was stirred at 0 °C for 4 h, diluted with ether
(200 mL), washed with water (50 mL) and brine (50 mL), dried
over K2CO3, and evaporated under reduced pressure. The
residue was purified by flash chromatography (Et2O/hexane 2:98,
UV detection) to afford 594 mg (89%) of bromide 6 as a colorless
oil. Rf ) 0.64 (EtOAc/hexane 5:95); Rf ) 0.50 (Et2O/hexane 2:98);
1H NMR (CDCl3) δ 8.22 (d, J ) 8.6, 1H), 7.49-7.37 (m, 7H),
(S)-2-Am in o-NR-(9-flu or en ylm eth yloxyca r bon yl)-3-(8-h y-
d r oxy-5-p h en ylqu in olin e-2-yl) P r op a n oic Acid (2). The
previous hydrochloride salt (89 mg, 0.4957 mmol) was dissolved
in aqueous Na2CO3 (1 M, 2.6 mL). (9-Fluorenylmethyl)succin-
imidyl carbonate (184 mg, 0.5453 mmol, 1.1 equiv) was dissolved
in dioxane (6.6 mL) and added dropwise to the amino acid
solution. The reaction mixture was stirred for 5 h. The dioxane
was evaporated under reduced pressure at room temperature,
and the aqueous phase was extracted with Et2O (3 × 5 mL).
7.24 (d, J ) 7.8, 1H), 4.75 (s, 2H), 1.10 (s, 9H), 0.31 (s, 6H); 13
C
NMR (CDCl3) δ 155.0, 152.2, 141.3, 139.7, 135.8, 133.1, 130.3,
128.6, 128.4, 127.5, 127.0, 121.3, 118.4, 34.6, 26.3, 19.3, -3.5;
IR (thin film) 2926, 2854, 1594, 1469, 1319, 1248, 1093, 839,

3228 J . Org. Chem., Vol. 66, No. 9, 2001
Notes
The aqueous phase was acidified to pH 3 with HCl (1 M) and
extracted with EtOAc (4 × 20 mL). The combined EtOAc phases
were dried over Na2SO4 and concentrated under reduced pres-
sure. The residue was taken in CH2Cl2 and crystallized by slow
addition of chilled hexane to afford 276 mg (95%) of N-protected
amino acid 2 as a yellow powder: mp 105-106 °C, Rf ) 0.64
(CHCl3/MeOH 4:1); Rf ) 0.10 (EtAc); 1H NMR (CDCl3) δ 8.41
(d, J ) 8.9, 1H), 7.72 (bd, J ) 7.3, 2H), 7.59-7.20 (m, 14H),
6.49 (d, J ) 6.1, 1H), 4.73 (bs, 1H), 4.30 (d, J ) 7.0, 2H), 4.14 (t,
J ) 7.0, 1H), 3.70 (bm, 2H); 13C NMR (CDCl3) δ 176.0, 173.9,
156.9, 155.6, 150.3, 144.5, 144.4, 141.9, 141.0, 138.8, 138.0, 133.7,
131.5, 130.7, 130.5, 129.4, 128.9, 128.4, 127.8, 126.9, 126.6, 125.9,
123.5, 120.6, 115.2, 67.8, 53.7, 47.7, 38.8; IR (thin film) 3332,
3058, 2924, 1702, 1596, 1509, 1449, 1380, 1220, 1079, 846, 761,
737, 703; HPLC (C18; solvent A ) water, 0.1% v/v TFA; solvent
B ) MeCN, 0.1% v/v TFA), tR (linear gradient 0-70% B over 25
min) ) 29.45 min; HRMS (ES) calcd for C33H28N2O5 [M + H]+
531.1920, found 531.1925; [R]20D ) -32.3° (c ) 1.5, MeOH/CH2-
Cl2/aqueous HCl 1 M 10:5:1).
Ac-L-2Oxn (5′-ph en yl)-Val-P r o-DSer -P h e-Cys-Gly-NH2 (P 1).
Yield, following HPLC purification, ∼20% from 16 chemical
steps. HPLC (C18; solvent A ) water, 0.1% v/v TFA; solvent B
) MeCN, 0.1% v/v TFA), tR (linear gradient 0-70% B over 25
min) ) 27.36 min; UV (0.1 M NaOH), λmax ) 369 nm. HRMS
(ES) calcd for C47H58O10N9S [M + H]+ 940.4027, found 940.4022.23
Ac-L-2Oxn -Val-P r o-DSer -P h e-Cys-Gly-NH2 (P 2). The Fmoc-
Oxn-OH amino acid, 1, was prepared according to the literature
method.2 Yield of P 2, following HPLC purification, ∼20% from
16 chemical steps. HPLC (C18; solvent A ) water, 0.1% v/v TFA;
solvent B ) MeCN, 0.1% v/v TFA), tR (linear gradient 0-70% B
over 25 min) ) 25.77 min; UV (0.1 M NaOH), λmax ) 335 nm.
MS (ES) calcd for C41H53O10N9S [M + H]+ 864.3714, found
864.3717.23
Ack n ow led gm en t. This research was supported by
grants from the NSF (CHE-9996335) in the initial
phases of this project and continued support from the
NIH (GM-58716). A postdoctoral fellowship to N.J . from
the Swiss National Science Foundation is also gratefully
acknowledged.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra and relevant HPLC traces of 2, Marfey derivatives of
9, and peptides P 1 and P 2. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O001681J
(23) Peptides P 1 and P 2 were confirmed to contain the free thiol
(> 95%) by quantitative Ellman test. (a) Ellman, G. L. Arch. Biochem.
Biophys. 1959, 82, 70-77. (b) Riddles, P. W.; Blakeley, R. L.; Zerner,
B. Methods Enzymol. 1983, 91, 49-60.