Stereoselective Synthesis of Fluorescent α-Amino Acids Containing Oxine (8-Hydroxyquinoline) and Their Peptide Incorporation in Chemosensors for Divalent Zinc

Full text of DOI:10.1021/jo980563h

J . Org. Chem. 1998, 63, 6727-6731
6727
Ster eoselective Syn th esis of F lu or escen t
r-Am in o Acid s Con ta in in g Oxin e
(8-Hyd r oxyqu in olin e) a n d Th eir P ep tid e
In cor p or a tion in Ch em osen sor s for
Diva len t Zin c
Grant K. Walkup and Barbara Imperiali*
Division of Chemistry and Chemical Engineering, California
Institute of Technology, Pasadena, California 91125
Received March 26, 1998
The synthesis of 1, employing the Myers pseudoephe-
drine glycinamide alkylation10,11 in the key stereogenic
step, is outlined in Scheme 1. Starting from com-
mercially available 8-hydroxy-2-methylquinoline (3), bro-
mide 4 was prepared in two steps via protection of the
phenol as the benzenesulfonate ester, followed by free
radical bromination. The (R,R)-(-) enantiomer of pseu-
doephedrine glycinamide (5) was chosen for alkylations
in order to yield as the major diastereomer the R-amino
acid derivative 6 possessing the L-configuration.12 In
general, 2 equiv of 5 per equiv of bromide 4 were used in
order to drive the reactions to completion within 1-3 h
when conducted at 0 °C. No attempt was made to
determine the diastereoselectivity of the alkylation reac-
tion, yet isolated yields of 6 as high as 89% were achieved,
in >95% de as determined by 1H NMR. Basic hydrolysis
of 6 to free the R-amino acid from the benzenesulfonate
ester and pseudoephedrine auxiliary, followed by protec-
tion of the newly liberated R-amine as the 9-fluorenyl-
methoxycarbonyl (Fmoc) derivative, provided 1 in 90%
yield and >95% ee.13
The synthesis of 2, outlined in Scheme 2, was per-
formed using a strategy analogous to that used for the
preparation of 1. Methylquinoline 7 was prepared in two
steps from oxine using literature methods.14 Protection
(as the benzensulfonate ester) and bromination to yield
8 proceeded smoothly in quantitative and moderate
yields, respectively. As for the preparation of 6, stereo-
selective alkylations of 8 (to yield product 9) were
performed with 2 equiv of 5 to drive the reactions to
completion within 3 h at 0 °C. Finally, hydrolysis and
Fmoc protection of 9 were performed to provide 2 in
excellent yield with high optical purity (>99% ee).
To demonstrate the utility of these newly developed
residues, 1 and 2 were used for the preparation of
heptapeptides via standard Fmoc-based solid-phase pep-
tide synthesis techniques.15 Peptides P 1 and P 2 thus
contain the oxine functionality formally attached to the
The development of fluorescent chemosensors for the
detection of ionic species has been a focus for many
researchers.1 In the continuing search for new sensing
molecules, engineering ligands which exhibit selective
and avid divalent metal binding in aqueous solution is
critical.2,3 In light of these criteria, we have sought to
harness within synthetic constructs the often unsur-
passed metal-binding properties of naturally occurring
peptides or proteins.4 Highlighting the versatility of this
approach, we have prepared selective chemosensors for
both Zn(II) (based on the zinc finger domains)5,6 and for
Cu(II) (based on the amino-terminal Cu(II) and Ni(II)
binding domains of the serum albumins).7 More recently,
we have begun to convert these and other solution-based
chemosensing reagents to regenerable devices by attach-
ing them to water-solvated resins. In this context, the
relatively large size of our Zn(II) fluorosensors (25
residues) is prohibitive to the production of sensing
materials with well-defined composition. Toward the
goal of minimizing the size of the peptidyl component
required for a successful Zn(II) sensor, we have developed
new residues for solid-phase peptide synthesis that
contain the high-affinity, bidentate metal-binding func-
tionality of the well-known ligand oxine (8-hydroxyquino-
line); this compound and its derivatives have historically
found use as fluorometric indicators of Zn(II),8 as well
as several other cations.9 We now report the syntheses
of (S)-2-amino-NR-9-fluorenylmethoxycarbonyl-3-(oxine-
2-yl)propionic acid (Fmoc-2Oxn-OH, 1) and (S)-2-amino-
NR-9-fluorenylmethoxycarbonyl-3-(oxine-5-yl)propionic acid
(Fmoc-5Oxn-OH, 2), as well as the incorporation of these
residues in short peptides (7 residues) capable of report-
ing sub-micromolar levels of Zn(II).
(1) For a recent review see: de Silva, A. P.; Gunaratne, H. Q. N.;
Gunnlaugsson, T.; Huxley, A. J . M.; McCoy, C. P.; Rademacher, J . T.;
Rice, T. E. Chem. Rev. 1997, 97, 1515-1566.
(2) Czarnik, A. W. Chem. Biol. 1995, 2, 423-428.
(3) Czarnik, A. W. Supramolecular Chemistry, Fluorescence, and
Sensing. In Fluorescent Chemosensors for Ion and Molecule Recogni-
tion; Czarnik, A. W., Ed.; American Chemical Society: Washington,
DC, 1993; pp 1-9.
(4) Glusker, J . P. Adv. Protein Chem. 1991, 42, 1-76.
(5) Walkup, G. K.; Imperiali, B. J . Am. Chem. Soc. 1996, 118, 3053-
3054.
(6) Walkup, G. K.; Imperiali, B. J . Am. Chem. Soc. 1997, 119, 3443-
3450.
(10) Myers, A. G.; Gleason, J . L.; Yoon, T. Y. J . Am. Chem. Soc. 1995,
117, 8488-8489.
(11) Myers, A. G.; Gleason, J . L.; Yoon, T.; Kung, D. W. J . Am. Chem.
Soc. 1997, 119, 656-673.
(12) The stereochemical outcome of the Myers alkylation has proven
to be predictable for a wide variety of electrophiles. See ref 11 and
references therein.
(7) Torrado, A.; Walkup, G. K.; Imperiali, B. J . Am. Chem. Soc. 1998,
120, 6609-6610.
(8) Vogel, A. I. Spectrofluorimetry. In Vogel’s Textbook of Quantative
Chemical Analysis; Wiley: New York, 1989; Chapter 18, pp 731-740.
(9) Ions that form fluorescent complexes with oxine include Mg(II),
Ca(II), Pb(II), and Sr(II), among others. For a leading reference, see:
Seitz, W. R. Crit. Rev. Anal. Chem. 1980, 8, 367-405.
(13) The optical integrity of compounds 1 and 2 were verified by
chiral HPLC analysis after removal of the NR-Fmoc protecting group.
See the Experimental Section.
(14) Burckhalter, J . H.; Leib, R. I. J . Org. Chem. 1961, 26, 4078-
4083.
(15) Fields, G. B.; Noble, R. L. Int. J . Pept. Protein Res. 1990, 35,
161-214.
S0022-3263(98)00563-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/28/1998

6728 J . Org. Chem., Vol. 63, No. 19, 1998
Notes
Sch em e 1
F igu r e 1. Fluorescence emission spectra of 10 µM solutions
of 3 (panel a) and P 1 (panel b) taken after the addition of the
indicated concentration of ZnCl2 (in µM) with excitation at 350
nm. Spectra were taken at room temperature in 50 mM
HEPES, pH 7.0, 150 mM NaCl and have been baseline
subtracted against a sample containing buffer only.
selectivity of P 1 and P 2 for Zn(II), cysteine was incorpo-
rated as the additional intended ligand, which by virtue
of its soft character may be expected to favor Zn(II)
binding,19 particularly in comparison to the hard ions
such as Mg(II) and Ca(II) which also form fluorescent
complexes with oxine.
Sch em e 2
Peptide P 2 was found to suffer from rapid oxidation
to form the disulfide dimer under normal handling
conditions (pH 7.0, rt, and exposure to air), thereby
complicating the metal-binding analysis for this com-
pound. However, P 1 exhibited avid Zn(II) complexation
to form a fluorescent complex with 1:1 stoichiometry.
Metal-binding titrations monitoring both UV-vis and
fluorescence emission were performed at room temper-
ature in pH 7.0 buffer (50 mM HEPES/150 mM NaCl) to
determine the apparent dissociation constant of the P 1-
Zn(II) complex (100 nM) and that of the relevant model
complex 3-Zn(II) (17 µM, data not shown). This 170-
fold enhancement in Zn(II) binding affinity suggests that
intramolecular participation of the cysteine residue of P 1
assists in metal binding. This effect is highlighted by a
comparison of the fluorescence response of 10 µM solu-
tions of 3 and P 1 to Zn(II) (Figure 1). Furthermore, at
lower concentrations of chemosensor, sub-micromolar
concentrations of Zn(II) are clearly distinguishable, with
the limit of detection less than 250 nM (Figure 2).
The response of peptide P 1 has also been studied in
the presence of a number of competing metal ion species.
At 10 µM P 1, measurements made in the presence of 1
equiv of Zn(II) and 1 equiv of a competing metal ion
reveal no significant competition from Mg(II), Ca(II), Mn-
(II), Fe(III), Co(II), or Ni(II) and only minor competition
(approximately 10% binding) from Fe(II) and Cd(II). The
only metal ion which shows competition is Cu(II); ap-
proximately 40% Cu(II) binding is observed at equivalent
concentrations of the two metal ions, suggesting that the
binding of P 1 to Zn(II) is slightly better compared to Cu-
(II).
Ch a r t 1
side chain of an N-terminal alanine residue through the
2-position (2Oxn) and 5-position (5Oxn) of the hetero-
cycle, respectively (Chart 1). Furthermore, the sequences
of these peptides were designed to contain the internal
sequence -Val-Pro-DSer-Phe- which has been demon-
strated in prior studies to promote reverse-turn formation
in aqueous solution.16 The side chains of the flanking
metal-binding residues are therefore placed in proximity
(underlined in Chart 1); this arrangement has been
observed to result in enhanced metal-binding proper-
ties.17,18 To simultaneously enhance the metal-binding
In conclusion, this paper describes the stereoselective
synthesis and peptide incorporation of two novel R-amino
acids that contain the bidentate metal-binding function-
ality of oxine (8-hydroxyquinoline). These early results
demonstrate that sensitive and selective reporting of Zn-
(16) Imperiali, B.; Fisher, S. L.; Moats, R. A.; Prins, T. J . J . Am.
Chem. Soc. 1992, 114, 3182-3188.
(17) Imperiali, B.; Kapoor, T. M. Tetrahedron 1993, 49, 3501-3510.
(18) Cheng, R. P.; Fisher, S. L.; Imperiali, B. J . Am. Chem. Soc.
1996, 118, 11349-11356.
(19) Frau´sto da Silva, J . J . R.; Willams, R. J . P. The Biological
Chemistry of the Elements: The Inorganic Chemistry of Life; Clarendon
Press: New York, 1993; p 35.

Notes
J . Org. Chem., Vol. 63, No. 19, 1998 6729
0.1 M diisopropylethylamine (DIEA) for 2 h. Following acety-
lation, an additional piperidine deblock cycle was performed to
deprotect any aryl acetate ester that formed during the acety-
lation procedure. Final peptide deprotection was achieved with
5% v/v triisopropylsilane in TFA. The crude peptide was
precipitated into 1:1 diethyl ether/hexanes, triturated using the
same solvent (3 × 5 mL), and purified to homogeneity by reverse-
phase (C18) HPLC.
8-Ben zen esu lfon yloxy-2-m eth ylqu in olin e. 8-Hydroxy-2-
methylquinoline (3) (5.0 g, 31.4 mmol, 1 equiv) was dissolved in
DCM (30 mL) and chilled to 0 °C with constant stirring.
Triethylamine (5.29 mL, 37.7 mmol, 1.2 equiv) was added
followed by the addition of benzenesulfonyl chloride (4.41 mL,
34.5 mmol, 1.1 equiv) dropwise over 15 min. After 0.5 h, the
clear solution had become a white slurry, and the ice bath was
removed. The reaction was allowed to warm to room temper-
ature and was stirred for an additional 1.5 h. Water (10 mL)
was added, and the biphasic reaction mixture was stirred
vigorously an additional 0.5 h to quench any remaining benze-
nesulfonyl chloride. The slurry was transferred to a separatory
funnel and the volume of DCM was increased to 100 mL. The
organic layer was washed with 100 mL of saturated aqueous
K2CO3. The layers were separated, and the aqueous layer was
extracted with DCM (2 × 50 mL). The organic layers were
combined, dried (K2CO3), and concentrated to afford the desired
product as a white solid (9.33 g, 99%): mp 109-110 °C; Rf )
F igu r e 2. Fluorescence emission spectra of a 1 µM solution
of P 1 taken after the addition of the indicated concentration
of ZnCl2 (in µM). Spectra were acquired using the same buffer
and conditions described for Figure 1.
(II) is feasible within short peptides (7 residues). The
new residues 1 and 2 will be useful tools in the continuing
development of other peptidyl fluorescent chemosensors,
a task which we are currently pursuing and will report
on in due course.
1
0.18 (75:25 hexanes/EtOAc), Rf ) 0.33 (100:1 DCM/EtOAc); H
NMR δ 7.96 (m, 3H), 7.68 (dd, J ) 1.2, 8.1 Hz, 1H), 7.64 (dd, J
) 1.2, 7.8 Hz, 1H), 7.56 (tt, J ) 2.1, 7.5 Hz, 1H), 7.35-7.45 (m,
3H), 7.19 (d, J ) 8.4 Hz, 1H), 2.50 (s, 3H); 13C NMR δ 25.1, 122.6,
123.0, 125.0, 126.7, 127.8, 128.4, 128.8, 133.6, 135.6, 136.7, 140.8,
145.0, 159.5; IR (thin film, cm-1) 3064, 1605; HRMS (FAB) calcd
for C16H14NO3S [M + H]+ 300.0694, found 300.0691.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Unless otherwise
noted, all reagents and solvents obtained from commercial
suppliers were of the highest possible purity and used without
further purification. Tetrahydrofuran and diethyl ether were
distilled from sodium benzophenone ketyl. Diisopropylamine,
dichloromethane (DCM), 2-butanol, and toluene were distilled
from calcium hydride. Lithium chloride was dried under
vacuum at 150 °C for g12 h and flame dried immediately prior
to use. n-Butyllithium was titrated with an analytically pre-
pared solution of 2-butanol in toluene using 2,2′-bipyridyl in
diethyl ether as indicator.11,20 Anhydrous reactions were per-
formed in oven-dried glassware under a positive pressure of
nitrogen. Thin-layer chromatography was performed using
plates obtained from EM (SiO2 60, F-254). Flash column
chromatography was carried out according to the procedure of
Still,21 using 230-400 mesh silica gel. 1H and 13C NMR spectra
were recorded at 300 and 75 MHz, respectively, in CDCl3, unless
otherwise noted. All melting points reported are uncorrected.
Ch ir a l HP LC An a lysis. Analyses were performed on ali-
quots of the intermediate R-amino acids taken from the hydroly-
ses of 6 and 9, as well as of samples resulting from deprotection
of the NR-Fmoc-amino acids 1 and 2 by treatment with 20%
piperidine in DMF. Optical purity was assessed using a Crown-
Pak CR (+) analytical column (Daicel) [eluent 0.1 N aqueous
HClO4, flow rate 0.5 mL/min, UV detection (320 nm)]. Retention
times: 2Oxn, 14.0 min (D), 15.0 min (L); 5Oxn, 10.1 min (D), 12.7
min (L).
P ep tid e Syn th esis. Peptides were assembled on macro
crown supports (Chiron, 6 µmol/crown) using standard Fmoc
chemistry batch synthesis techniques. Iterative removal of NR-
Fmoc protecting groups was performed with piperidine (20% v/v
in DMF) for 10 min, followed by washing with DMF (2 × 5 min)
and MeOH (1 × 2 min, 2 × 5 min), allowing the support to dry
for 5 min, and then reswelling the support in DMF (5 min).
Coupling reactions were carried out using diisopropylcarbodi-
imide/1-hydroxybenzotriazole (DIPCDI/HOBT) activation chem-
istry using 0.1 M active ester in sufficient volume of DMF to
provide a 6-fold excess of coupling reagent to support-bound
amine for a minimum of 4 h. At the completion of the synthesis,
the final Fmoc protecting group was removed, and the N-
terminal R-amine acetylated with 1 mL of 0.1 M acetic anhydride/
8-Ben zen esu lfon yloxy-2-b r om om et h ylq u in olin e
(4).
8-Benzenesulfonyloxy-2-methylquinoline (5.0 g, 16.6 mmol, 1.0
equiv) was placed in a round-bottom flask with deoxygenated
CCl4 (40 mL), the flask was fitted with a reflux condenser, and
the mixture was heated to reflux to dissolve. N-Bromosuccin-
imide (4.14 g, 23.2 mmol, 1.4 equiv) was added, followed by 2,2′-
azobis(2-methylpropiononitrile) (AIBN, 295 mg, 1.55 mmol, 0.1
equiv), and reflux was continued. Additional aliquots of AIBN
(295 mg) were added at 0.5 h intervals until a total of 5 additions
had been made. At 0.5 h after the final addition, the reaction
flask was allowed to cool to room temperature, and the solvent
was removed under reduced pressure. The resulting residue was
suspended in EtOAc (400 mL), transferred to a separatory
funnel, and washed with 1:1 saturated aqueous Na2CO3/Na2-
SSO3 (300 mL). The layers were separated, and the aqueous
layer was extracted with EtOAc (75 mL). The organic layers
were combined, dried (K2CO3), and concentrated. The resulting
residue was dissolved in a minimal quantity of chloroform and
purified by silica flash chromatography (eluent 75:25 hexanes/
EtOAc, Rf ) 0.22). Both starting material (1.7 g) and the product
were collected by concentration of the appropriate fractions.
NMR analysis of the product revealed the presence of a
contaminant (singlet at 1.73 ppm, CDCl3) which was assigned
to the radical termination product 2,3-dicyano-2,3-dimethylbu-
tane. Recrystallization from hot EtOAc/hexanes provided the
pure bromide 4 (3.25 g, 71% based on recovery of starting
material) as a crystalline white solid: mp 138-139 °C; Rf ) 0.40
(65:35 hexanes/EtOAc) Rf ) 0.60 (100:1 DCM/EtOAc); 1H NMR
δ 8.12 (d, J ) 8.4 Hz, 1H), 7.98 (dd, J ) 8.4, 1.2 Hz, 2H), 7.74
(d, J ) 8.1 Hz, 2H), 7.61 (m, 1H), 7.45-7.6 (m, 4H), 4.44 (s, 2H);
13C NMR δ 33.9, 122.0, 123.8, 126.6, 126.7, 128.5, 128.7, 128.8,
134.0, 137.0, 137.1, 140.2, 145.3, 157.3; IR (thin film, cm-1) 3605,
1600; HRMS (FAB) calcd for C16H13NO3SBr [M + H]+ 377.9800,
found 377.9797.
8-Ben zen esu lfon yloxy-5-m eth ylqu in olin e. 8-Hydroxy-5-
methylquinoline14 (7) (6.23 g, 39.1 mmol, 1.0 equiv) was dissolved
in DCM (90 mL) and chilled to 0 °C with constant stirring.
Triethylamine (6.58 mL, 46.9 mmol, 1.2 equiv) was added,
followed by the dropwise addition of benzenesulfonyl chloride
(5.48 mL, 43.0 mmol, 1.1 equiv) over 15 min. After 0.5 h, the
clear solution had become a white slurry, and stirring was
continued overnight (10 h). Water (20 mL) was added, and the
biphasic mixture was stirred vigorously for 1 h to ensure
consumption of the remaining benzenesulfonyl chloride. The
(20) Watson, S. C.; Eastham, J . F. J . Organomet. Chem. 1967, 9,
165-168.
(21) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923-
2925.

6730 J . Org. Chem., Vol. 63, No. 19, 1998
Notes
organics were removed under reduced pressure, and the aqueous
phase was transferred to a separatory funnel. Saturated aque-
ous Na2CO3 (100 mL) was added, and the mixture was extracted
with EtOAc (2 × 75 mL). The organic layers were combined,
dried (K2CO3), and concentrated to afford a white solid. The
solid was dissolved in a minimal quantity of CHCl3 and loaded
onto a silica flash column (eluent 65:35 hexanes/EtOAc, Rf )
0.21) to afford the desired product (10.95 g, 94%): mp 106-108
Hz, 0.33H), 3.28* (dd, J ) 8.1, 14.7 Hz, 0.33H), 3.09 (dd, J )
5.1, 14.7 Hz, 0.67H), 3.04 (s, 2H), 2.96* (s, 1H), 2.93 (dd, J )
8.4, 14.7 Hz, 0.67H), 1.5-2.5 (broad s, 3H), 1.04* (d, J ) 6.6
Hz, 1H), 0.99 (d, J ) 6.9 Hz, 2H); 13C NMR δ 14.1, 15.5, 27.2,
31.3, 43.9, 51.2, 57.0, 57.7, 75.1, 75.4, 121.5, 122.0, 123.3, 123.5,
125.0, 125.1, 126.5, 126.6, 126.8, 127.1, 127.4, 127.8, 128.1, 128.2,
128.3, 128.4, 128.8, 128.9, 133.8, 135.8, 136.4, 140.9, 141.6, 142.0,
145.0, 159.9, 160.4, 174.8, 175.9; IR (thin film, cm-1) 3360
(broad), 3062, 2982, 1625; HRMS (FAB) calcd for
C28H30N3O5S [M + H]+ 520.1906, found 520.1909; [R]20D ) +61°
(c ) 1.0, CHCl3).
1
°C; Rf ) 0.4 (1:1 hexanes/EtOAc); H NMR δ 8.77 (dd, J ) 1.5,
4.2 Hz, 1H), 8.26 (dd, J ) 1.5, 8.7 Hz, 1H), 7.98 (dd, J ) 0.6, 7.2
Hz, 2H), 7.59 (tt, J ) 1.2, 7.5 Hz, 1H), 7.4-7.5 (m, 3H), 7.4 (dd,
J ) 4.2, 8.7 Hz, 1H), 7.32 (dd, J ) 0.9, 7.8 Hz, 1H), 2.65 (s, 3H);
13C NMR δ 18.3, 121.3, 122.1, 122.5, 126.1, 128.7, 132.3, 133.7,
134.1, 136.3, 138.6, 141.5, 143.9, 150.1; IR (thin film, cm-1) 3062,
1598; HRMS (FAB) calcd for C16H14NO3S [M + H]+ 300.0694,
found 300.0697
[(R,R)-2S]-N-(2-Hydr oxy-1-m eth yl-2-ph en yleth yl)-N-m eth -
y l-2-a m i n o -3-[5-(8-b e n z e n e s u lfo n y lo x y )q u i n o li n y l]-
p r op ion a m id e (9). A solution of n-butyllithium in hexanes
(2.40 M, 1.63 mL, 3.90 mmol, 3.9 equiv) was added to a solution
of diisopropylamine (560 µL, 4.0 mmol, 4.0 equiv) in deoxygen-
ated THF (2 mL) at 0 °C. After 15 min, the resulting solution
of lithium diisopropylamide (+ 2 mL wash) was transferred via
cannula over 5 min to a stirred slurry of anhydrous (R,R)-(-)-
pseudoephedrine glycinamide (454 mg, 2.0 mmol, 2.0 equiv) and
flame-dried lithium chloride (509 mg, 12 mmol, 12 equiv) in
deoxygenated THF (5 mL) at 0 °C. After 30 min of stirring at
0 °C, a solution of 8 (378 mg, 1.0 mmol, 1.0 equiv) in THF (3
mL + 2 mL wash) at 0 °C was added slowly to the yellow enolate
solution. The mixture was stirred for 2 h at 0 °C. Water (30
mL) was added, the resulting biphasic mixture was warmed to
room temperature, and the THF was removed under reduced
pressure. The pH of the solution was made basic by the addition
of NaOH (2 mL, 2 N), and the resulting aqueous phase was
extracted with chloroform (3 × 25 mL). The combined organic
layers were dried over anhydrous K2CO3, filtered, and concen-
trated under reduced pressure. The residue was purified by
chromatography on silica gel by gradient elution with DCM/
MeOH/Et3N (96.5:1.5:2 f 92:4:4). The C-alkylated diastereo-
mers were separable under these conditions, and only those
fractions containing the pure major diastereomer were collected.
After concentration of the appropriate fractions, the product
residue was concentrated from toluene (2 × 25 mL) and then
chloroform (2 × 25 mL) to remove residual triethylamine. The
product 9 was isolated as a pale yellow granular solid (482 mg,
92%): Rf ) 0.55 (85:15:1 CHCl3/MeOH/NH4OH), Rf ) 0.23 (90:
5:5 CHCl3/MeOH/Et3N). 1H NMR (1:2.3 rotameric forms, *
denotes minor rotamer) δ 8.78* (d, J ) 2.5 Hz, 0.3H), 8.74 (d, J
) 2.5 Hz, 0.7H), 8.57* (d, J ) 7.5 Hz, 0.3H), 8.39 (d, J ) 7.5 Hz,
0.7H), 7.95-8.0 (m, 1.3H), 7.2-7.6 (m, 11.4H), 7.04* (d, J ) 3.5
Hz, 0.3H), 4.60 (m, 0.7H), 4.43* (d, J ) 8 Hz, 0.3H), 4.35 (d, J
) 8 Hz, 0.7H), 4.2* (m, 0.3H), 4.14 (m, 0.7H), 4.01* (m, 0.3H),
3.66* (dd, J ) 5 Hz, 14 Hz, 0.3H), 3.0-3.5 (m, 3.7H), 2.91* (s,
0.9H), 2.23 (s, 2.1H), 0.92* (d, J ) 6 Hz, 0.9H), 0.46 (d, J ) 6
Hz, 2.1H); 13C NMR (contributions from both rotamers listed) δ
13.9, 14.0, 15.6, 27.2, 29.5, 37.6, 37.8, 51.8, 52.1, 55.2, 55.5, 58.0,
70.7, 71.2, 74.6, 75.2, 76.7, 121.95, 122.0, 126.7, 126.8, 127.3,
128.0, 128.3, 128.4, 128.5, 128.6, 128.7, 128.8, 132.2, 132.6, 133.2,
133.9, 134.0, 136.1, 136.2, 141.3, 141.4, 141.5, 141.8, 144.4, 144.6,
150.2, 150.5, 174.9, 186.7; IR (thin film, cm-1) 3350 (broad), 3062,
2980, 1626; HRMS (FAB) calcd for C28H30N3O5S [M + H]+
8-Ben zen esu lfon yloxy-5-b r om om et h ylq u in olin e
(8).
8-(Benzenesulfonyloxy)-5-methylquinoline (1.28 g, 4.28 mmol,
1.0 equiv) was placed in a round-bottom flask with deoxygenated
CCl4 (30 mL), the flask was fitted with a reflux condenser, and
the mixture was heated to reflux to dissolve. N-Bromosuccin-
imide (1.07 g, 6.0 mmol, 1.4 equiv) was added, followed by AIBN
(70 mg, 0.43 mmol, 0.1 equiv), and reflux was continued.
Additional aliquots of AIBN (70 mg) were added at 0.5 h
intervals until a total of 5 additions had been made. At 0.5 h
after the final addition, the reaction flask was allowed to cool
to room temperature, and the solvent was removed under
reduced pressure. The resulting residue was suspended in
EtOAc (50 mL), transferred to a separatory funnel, and washed
with 1:1 saturated Na2CO3/Na2SSO3 (50 mL). The layers were
separated, and the aqueous layer was extracted with EtOAc (50
mL). The organic layers were combined, dried (K2CO3), and
concentrated. The resulting residue was dissolved in a minimal
quantity of CHCl3 and purified by silica flash chromatography
(eluent 75:25 hexanes/EtOAc, Rf ) 0.13) to yield 8 (1.02 g, 63%)
as a white solid: mp 142-142 °C; Rf ) 0.22 (65:35 hexanes/
EtOAc), Rf ) 0.60 (95:5 DCM/Et2O); 1H NMR δ 8.83 (dd, J )
1.8, 4.2 Hz, 1H), 8.44 (dd, J ) 1.5, 8.7 Hz, 1H), 8.00 (dd, 1.2, 8.4
Hz, 2H), 7.63 (m, 1H), 7.56 (d, 4.2 Hz, 2H), 7.4-7.5 (m, 3H),
4.87 (s, 2H); 13C NMR (125 MHz) δ 29.2, 121.9, 122.0, 127.4,
127.6, 128.7, 128.9, 132.1, 133.0, 134.0, 136.0, 142.0, 146.1, 150.8;
IR (thin film, cm-1) 3063, 1598; HRMS (FAB) calcd for C16H13
-
NO3SBr [M + H]+ 377.9800, found 377.9800.
[(R,R)-2S]-N-(2-Hydr oxy-1-m eth yl-2-ph en yleth yl)-N-m eth -
y l-2-a m i n o -3-[2-(8-b e n z e n e s u lfo n y lo x y )q u i n o li n y l]-
p r op ion a m id e (6). A solution of n-butyllithium in hexanes
(2.30 M, 1.70 mL, 3.90 mmol, 3.9 equiv) was added to a solution
of diisopropylamine (560 µL, 4.0 mmol, 4.0 equiv) in deoxygen-
ated THF (2 mL) at 0 °C. After 15 min, the resulting solution
of lithium diisopropylamide (+ 2 mL wash) was transferred via
cannula over 5 min to a stirred slurry of anhydrous (R,R)-(-)-
pseudoephedrine glycinamide (454 mg, 2.0 mmol, 2.0 equiv) and
flame-dried lithium chloride (509 mg, 12.0 mmol, 12.0 equiv) in
deoxygenated THF (5 mL) at 0 °C. After 30 min at 0 °C, a
solution of 4 (378 mg, 1.0 mmol, 1.0 equiv) in tetrahydrofuran
(5 mL + 2 mL wash) at 0 °C was added slowly to the yellow
enolate solution. The reaction mixture was stirred for 3 h at 0
°C. Water (30 mL) was added, the resulting biphasic mixture
was warmed to room temperature, and the THF was removed
under reduced pressure. The pH of the solution was made basic
by the addition of aqueous NaOH (2 mL, 2 N), and the resulting
aqueous phase was extracted with chloroform (3 × 20 mL). The
combined organic layers were dried (K2CO3), filtered, and
concentrated under reduced pressure. The residue was purified
by chromatography on silica gel eluting with DCM/MeOH/Et3N
(92:4:4). The C-alkylated diastereomers were separable under
these conditions, and only those fractions containing the pure
major diastereomer were collected. After concentration of the
appropriate fractions, the product residue was concentrated from
toluene (2 × 25 mL) and then chloroform (2 × 25 mL) to remove
residual triethylamine. This afforded 6 as a pale yellow granular
solid (0.47 g, 89%): mp 67-69 °C; Rf ) 0.44 (90:5:5 CHCl3/
MeOH/TEA), Rf ) 0.28 (95:5:1 CHCl3/MeOH/NH4OH); 1H NMR
(approximately 2:1 rotamer ratio, * denotes minor rotamer) δ
8.65* (d, J ) 8.4 Hz, 0.33 H), 8.04 (d, J ) 8.4 Hz, 0.67H), 7.9-8
(m, 2H), 7.2-7.8 (m, 12H), 4.74* (d, J ) 8.1 Hz, 0.33H), 4.61 (d,
J ) 8.1 Hz, 0.67H), 4.4-4.6 (m, 2H), 3.46* (dd, J ) 3.6, 14.7
520.1906, found 520.1906; [R]20 ) +12° (c ) 1.0, CHCl3).
D
(S)-2-Am in o-Nr-9-flu or en ylm eth oxyca r bon yl-3-(oxin e-2-
yl)p r op ion ic Acid (1). Alkylated pseudoephedrine derivative
6 (100 mg, 0.192 mmol, 1.0 equiv) was dissolved in deoxygenated
dioxane (1.5 mL), deoxygenated aqueous NaOH (0.5 M, 1.54 mL,
0.768 mmol, 4 equiv) was added, and the reaction mixture was
heated to reflux. After 4 h, TLC analysis of the amber solution
(n-BuOH/AcOH/H2O/EtOAc 1:1:1:1) showed that only the depro-
tected amino acid (Rf ) 0.23) remained as a UV-, ninhydrin-,
and FeCl3-active species. The solution was cooled, and the
dioxane was removed under reduced pressure. The aqueous
solution was washed with DCM (3 × 5 mL) to remove pseu-
doephedrine. A small aliquot of the aqueous phase (20 µL) was
removed for chiral HPLC analysis, the remainder was chilled
to 0 °C, and NaHCO3 (64 mg, 0.768 mmol, 4.0 equiv) was added.
A solution of 9-fluorenylmethoxychloroformate (60 mg, 0.23
mmol, 1.2 equiv) in dioxane (1.5 mL) was added, and the reaction
mixture was stirred for 8 h. The dioxane was removed under
reduced pressure, and HCl (1 N) was added dropwise until the
solution was ca. pH 4. Water was added to increase the volume
to ca. 10 mL, and the solution was extracted with DCM (3 × 10

Notes
J . Org. Chem., Vol. 63, No. 19, 1998 6731
mL). The organic layers were concentrated, and the resulting
yellow residue was concentrated from toluene (2 × 25 mL) to
remove traces of water. The residue was dissolved in CHCl3 (2
mL) and precipitated by addition to a rapidly stirred solution of
1:1 hexanes/ether (25 mL). The yellow precipitate was filtered
and dried under vacuum for 6 h to afford 81 mg (90%) of a pale
resulting pale yellow solid was dried under vacuum for 6 h to
afford 90 mg (95%) of 2: Rf ) 0.37 (85:15:3 CHCl3/MeOH/AcOH);
mp 114-116 °C; 1H NMR (MeOH-d4) δ 3.27 (dd, J ) 4.5 Hz,
14.1 Hz, 1H), 3.65 (dd, J ) 4.5 Hz, 14.1 Hz, 1H), 3.86 (d, J ) 6.9
Hz, 2H), 4.01 (t, J ) 4.5 Hz, 1H), 4.45 (m, 1H), 7.02 (d, J ) 7.8
Hz, 1H), 7.1-7.8 (m, 10H), 8.61 (d, J ) 7.8 Hz, 1H), 8.78 (d, J
) 4.2 Hz, 1H); 13C NMR (DMSO-d6) δ 32.3, 46.5, 55.0, 65.6,
114.3, 120.1, 122.0, 125.1, 125.2, 127.0, 127.1, 137.6, 127.8, 130.9,
1
yellow solid: Rf ) 0.70 (85:15:3 CHCl3/MeOH/AcOH); H NMR
(MeOH-d4) δ 3.64 (t, J ) 9.9 Hz, 1H), 3.7-4.5 (m, 3H), 4.81 (m,
1H), 7.0-8.0 (m, 12H), 8.9 (d, J ) 8.7 Hz, 1H); 13C NMR δ 38.1,
46.3, 47.0, 52.8, 67.1, 115.0, 117.3, 119.9, 122.7, 124.7, 127.0,
127.2, 127.6, 127.8, 129.4, 141.1, 141.7, 143.6, 143.8, 154.7, 156.2;
IR (thin film, cm-1) 3000 (broad), 2919, 1717, 1640, 1602; HRMS
(FAB) calcd for C27H23N2O5 [M + H]+ 455.1607, found 455.1605;
140.6, 140.8, 143.6, 143.7, 144.6, 155.9, 172.9; IR (thin film, cm-1
)
3000 (broad), 3067, 2924, 1713, 1598; HRMS (FAB) calcd for
C27H23N2O5 [M + H]+ 455.1607, found 455.1599; [R]20D ) -5° (c
) 0.5, 9:1 MeOH/1.0 N aqueous HCl).
Ac-2Oxn -Val-P r o-DSer -P h e-Cys-Ser -NH2 (P 1). HPLC (C18
;
[R]20 ) -340° (c ) 0.5, 9:1 MeOH/1.0 N aqueous HCl).
D
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) ) 18.5 min; UV
(0.1 N NaOH), λmax (nm) ) 335; MS (electrospray) calcd for
C42H56N9O11S [M + H]+ 894.3, found 894.3.
(S)-2-Am in o-Nr-9-flu or en ylm eth oxyca r bon yl-3-(oxin e-5-
yl)p r op ion ic Acid (2). Alkylated pseudoephedrine derivative
9 (100 mg, 0.192 mmol, 1.0 equiv) was dissolved in deoxygenated
dioxane (1.5 mL), deoxygenated aqueous NaOH (0.5 M, 1.54 mL,
0.768 mmol, 4 equiv) was added, and the reaction mixture was
heated to reflux. After 6 h, the solution was deep yellow, and
analysis by TLC (1:1:1:1 n-BuOH/AcOH/water/EtOAc) showed
that only the deprotected amino acid (Rf ) 0.23) remained as a
UV-, ninhydrin-, and FeCl3-active species. The solution was
cooled, and the dioxane was removed under reduced pressure.
The aqueous solution was washed with DCM (3 × 5 mL) to
remove pseudoephedrine. A small aliquot of the aqueous phase
(20 µL) was removed for chiral HPLC analysis, the remainder
was chilled to 0 °C, and NaHCO3 (64 mg, 0.768 mmol, 4.0 equiv)
was added. A solution of N-(9-fluorenylmethoxycarbonyloxy)-
succinimide (78 mg, 0.23 mmol, 1.2 equiv) in dioxane (1.5 mL)
was added, and the reaction mixture was stirred for 8 h. The
dioxane was removed under reduced pressure, and HCl (1 N)
was added dropwise until the solution was ca. pH 4. Water (5
mL) was added, and the solution was cooled to 4 °C and held at
that temperature for 1 h, during which time a yellow precipitate
formed. The solution was filtered and washed with cold water.
The solid was transferred to a round-bottom flask and concen-
trated from toluene several times to remove traces of water. The
Ac-5Oxn -Val-P r o-DSer -P h e-Cys-Ser -NH2 (P 2). 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) ) 17.9 min; UV
(0.1 N NaOH), λmax (nm) ) 366; MS (electrospray) calcd for
C42H56N9O11S [M + H]+ 894.3, found 894.3.
Ack n ow led gm en t. This research was supported by
the NSF and the Gates Foundation. The award of a NIH
predoctoral training grant in biology and chemistry to
G.K.W. (GM07616) 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 chiral HPLC traces from the analysis of the optical
purity of 1 and 2 (17 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 O980563H