The rapid analysis of trace metal cations for environmental

and biomedical applications is particularly demanding since it

requires the specific recognition of a particular element in the

presence of numerous closely related species. While remarkable

progress has been achieved for inorganic analytes, such as Ca^(2+), there is still a significant need for the genesis of new fluorosensors. In light of the selectivity and avidity with which proteins bind divalent metal cations, we have chosen to use a polypeptide architecture as the framework for metal ion recognition.In addition, our ability to manipulate synthetic polypeptides allows the coordinated integration of fluorescent reporters for signal transduction within a metal-binding peptidyl construct. Herein we report the design, synthesis, and evaluation of a selective fluorescent chemosensor, sensitive to nanomolar

concentrations of Zn^(2+)

Imperiali, Barbara

Walkup, Grant K.

Caltech Authors - Main

J. Am. Chem. Soc., 1996, 118(12), 3053-3054, DOI:10.1021/ja9538501
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Copyright © 1996 American Chemical Society
(01996 American Chemical Society J. Am. Chem. Soc. V119- Page305-3 -Walkup Supplemental Page I-
~YA~-8 so/- 3.3"Y'A%- j 19 6,53
pRlVU.._EGED DOuETRECEIVED
M. REVIEW pURpOSES ONLY SUPPLEMENTARY MATERIAL JOURNAL OF THE AMERICANfor the manuscript entitled CTEMICAL SOCy
Design and Evaluation of a Peptidyl Fluorescent Chemosensor for Divalent Zinc
Grant K. Walkup and Barbara Imperiali*
General Information
Reagents used were purchased from Aldrich Chemical Co. or Fluka. Protected amino
acid derivatives and reagents were obtained from Milligen (Perseptive) Biosearch, except Fmoc-
L-Baa(alloc)-OH which was synthesized as outlined below. Fluorescence experiments were
performed on a SLM-Aminco SPF 500c spectrofluorometer at ambient temperature. UV-Vis
spectra were obtained on a Shimadzu UV160U, or a Beckman DU 7500 recording
spectrophotometer.
All solutions and buffers employed in assays were composed of high-purity (18 Q cm)
water obtained from a Milli-Q (Millipore) deionization purification system, and were handled in
acid-washed HDPE containers. To prevent metal catalyzed- and auto-oxidation of ZNS- 1, buffer
solutions were sparged with argon for one half hour then hydrogen for fifteen minutes
immediately prior to use.
Metal titrations were performed by the addition of aliquots of concentrated stock
solutions. The concentration of these stocks were determined by complexometric titrations
performed in triplicate with standard EDTA solutions (Aldrich) and an appropriate colorometric
indicator.I
Synthesis of Fmoc-L-Baa(alloc)-OH
The amino acid derivative (4) was synthesized from the precursor Na-Boc-L-jI-amino alanine 2, 3
via the route outlined in Scheme I.
allychlorformate oo
NH2  DIEA NH TFA NH
0 COOH T)_o N COOH CNH NOOH
1 2 3
I' Fmoc-ONSu
NH DIEA
FmocN COOH THF/HP
H
4
Scheme I. Strategy for the synthesis of Fmoc-Baa(alloc)-OH.
1
(01996 American Chemical Society J. Am. Chem. Soc. VI 18 Page3053 Walkup Supplemental Page 2- - -
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Boc-L-Baa(alloc)-OH (2) A solution of the Boc-amine (1) was prepared by dissolving
4.33 g (21.2 mmol) in 100 mL of 50% v/v THF in deionized water, assisted by sonication at 50
*C. Sodium carbonate (2.46 g, 23.2 mmol) in 7 mL of water was added, and the resultant slurry
was cooled to 0 *C with constant stirring. Allyl chloroformate (2.25 mL, 21.2 mmol) and
diisopropylethylamine (3.66 mL, 21.2 mmol) were added in alternating portions over a period of
20 minutes, after which the ice bath was removed and the reaction vessel let stir an additional 1.5
hr. The organic solvent was removed under reduced pressure, and additional deionized water
added to increase the volume to 250 mL. Citric acid was added until pH 3.2, and the solution
extracted with CH2CI2 (5 x 200 mL). The organic layers were combined, dried over MgSO4,
and the solvent removed under reduced pressure to give a clear oil.. Traces of contaminating
lactam were separated by chromatography on silica gel (95:4:1, CHCl3/MeOH/AcOH) to afford
4.60 g (75% yield) of a clear, colorless oil. RF (85:15:3, CHCl3/MeOH/AcOH) = 0.47. RF
(95:5:1, CH2Cl2/MeOH/AcOH) = 0.20. IR (NaC1, neat) cm- 1 1164.0 (m), 1253.6 (m), 1517.4
(m), 1532.4 (m), 1694.5 (s), 1711.4 (s), 1728.4 (s), 2977.5 (m). 1H NMR (300 MHz, DMSO-d6)
8 1.40 (s, 9H), 3.98 (m, 1H), 4.42 (d, 2H), 5.27 (d, 1H), 5.92 (m, 1H), 6.92 (d, 1H), 7.2 (t, 1H).
13C NMR (DMSO-d6 ) 8 28.7, 43.2, 64.9, 72.9, 78.8, 117.4, 134.1, 162.8, 171.8, 172.7.
L-Baa(alloc)-OH * TFA (3) A 100-mL round bottom flask was charged with a stir bar,
Boc-L-Baa(alloc)-OH (2, 4.55 g, 15.8 mmol), and CH2 Cl2 (15 mL), then chilled to 0 *C with
constant stirring. Trifluoroacetic acid (TFA, 10 mL, 130 mmol) was added drop wise over 15
minutes. After an additional 15 minutes the reaction was allowed to come up to room
temperature and let stir for 2 hours. Excess TFA was removed under a stream of nitrogen, then
the solvent was removed in vacuo to give a clear gum. Lyophilization of this residue from
benzene gave 3 as a clear oil (4.63g, 97% yield). RF (1:1:1:1, n-butanol/AcOH/H20/EtOAc) =
0.54. RF (1:1:1, n-butanol/AcOH/H 2 0) = 0.4. 1H NMR (300 MHz, CD 30D) 8 3.58 (d, 1H),
3.73 (d, 1H), 4.1 (m, 1H) 4.53 (d, 2H), 5.18 (d, 1H), 5.28 (d, 1H), 5.88 (m, 1H). 13C NMR
(D20) 8 43.6, 66.6, 73.6, 118.0, 133.0, 165.3, 170.5.
Fmoc-L-Baa(alloc)-OH (4) The carbamate amine 3 (370 mg, 1.21 mmol) was dissolved
in 2:1 THF/H2 0 (12 mL), requiring the assistance of vigorous stirring and sonication.
Diisopropylethylamine (DIEA, 465 pL, 2.66 mmol) was added until the solution was slightly
basic to litmus. Under vigorous stirring, N-(9-Fluorenylmethoxycarbonyl)succinin-ide (Fmoc-
ONSu, 816 mg, 2.42 mmol) was added in alternating portions with DIEA (420 AL, 2.42 mmol)
over 30 minutes. The reaction was allowed to stir an additional four hours. The organic phase
was removed in vacuo, and the remaining aqueous solution was increased in volume to -100 mL,
and acidified with citric acid to pH 3. This was extracted with diethyl ether (2 x 50 mL), and the
organic phase discarded. The aqueous phase was further extracted with CH2Cl2 (4 x 100 mL),
the organic phases combined, dried over anhydrous Na2SO4, and concentrated under reduced
pressure to afford a clear oil. Purification by silica gel chromatography (95:5:3 CHC3 / MeOH /
2
(01996 American Chemical Society J. Am. Chem. Soc. V118 Page3053 Walkup Supplemental Page 3
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AcOH), followed by lyophilization from benzene gave 4 (465 mg, 96% yield) as a white powder.
RF (CH2Cl2 / MeOH / AcOH, 95:5:3) = 0.24. RF (CHCl3 / MeOH / AcOH, 85:15:3) = 0.51. IR
(CH2Cl2 , NaC1) cm-1 1407.1 (in), 1450.1 (m), 1523.8 (s), 1712.9 (s), 3044.3 (w), 3066.4 (w). 1H
NMR (300 MHz, DMSO-d6) 8 3.25-3.45 (m, 3H), 4.0-4.4 (m, 3H), 4.44 (d, 2H), 5.14 (d, 2H),
5.25 (d, 2H), 5.85 (m, 1H), 7.2-7.6 (m, 4H), 7.70 (d, 2H), 7.88 (d, 2H), 12.7 (s, 1H). 13C NMR
(D20) 6 42.8, 47.2, 54.9, 67.4, 100.2, 118.0, 120.2, 125.5, 127.4, 128.0, 132.9, 144.0, 141.5,
157.3, 163.9, 172.8.
Peptide synthesis
General. Peptides were synthesized on a Milligen 9050 peptide synthesizer at a 0.125
mmol scale. The support used was in all cases PAL-PEG-PS (Millipore) thus providing carboxy-
terminal primary amides. Couplings were performed at a concentration of 0.3 M acylating
reagent and HOBT, in a volume sufficient to achieve a three-fold excess of amino acid to resin-
bound amine. Pentafluorophenyl ester / 1-hydroxybenzotriazole (Pfp/HOBT) chemistry was
employed for all residues except Fmoc-L-Baa(alloc)-OH which was coupled by in situ active
ester generation using HOBT / NN'-diisopropylcarbodiimide (HOBT / DIPCDI) activation.
All coupling reactions were allowed to proceed for greater than 45 minutes, and were monitored
for completion with the Enhanced Monitoring Option (EMO, Milligen). A coupling judged to be
incomplete (< 97%) was double coupled for an additional 45 minutes. After successful coupling
cycles, any residual amines were acylated by a 10 min wash with 0.3 M acetic anhydride/HOBT
solution in 9:1 DMF / dichloromethane. Removal of the Fmoc group was performed with
piperidine (20% v/v in DMF) with a standard wash duration of 7 minutes. This time was
extended if an inefficient deblock was detected by EMO. After addition of the final residue, the
amino-terminus was acetyl-capped (DMF / acetic anhydride / triethylamine, 4 mL:63 gL:94 pL)
for 0.5 h then washed with DMF (5 x 10 mL) and MeOH (5 x 10 m). Residual solvent was
removed under reduced pressure.
Alloc removal. The method of Kates et al.4 was employed with some minor
modifications. A typical procedure for removal of the alloc group was as follows. Under a
blanket of nitrogen, a 20-mL plastic, stoppered vial was charged with resin from the completed
peptide synthesis (300 mg, 0.21 meq/g, 63 gmol), and 5 mL of a solvent cocktail (CHCl3 /
morpholine / AcOH, 90:5:5). The resin was allowed to swell 10 min, to which
tetralds(triphenylphosphine)palladium (200 mg, 173 Ipmol) was added under a blanket of
nitrogen. The vial was capped, shielded from light, and placed on a wrist-action shaker at low
speed for 2 h. The resin was filtered, washed with CHCl3 (5 x 10 mL), and a palladium-
chelating cocktail (DMF / diethyldithiocarbamic acid * 3H20 / triethylamine, 25 mL:225 mg:250
pL). Traces of this solution were removed with a basic wash (0.5% v/v triethylamine in DMF),
3
(01996 American Chemical Society J. Am. Chem. Soc. V118 Page3053 Walkup Supplemental Page 4
and a final wash with methanol. The resin was transferred to a clean plastic vial, and the residual
solvent removed under reduced pressure.
Dansyl coupling. Lyophilized resin taken directly from the alloc-removal procedure (280
mg, 0.21 meq/g, 59 umol), was allowed to swell in DMF (5 mL). Dansyl chloride (159 mg, 590
gmol) was added, followed by triethylamine (82 mL, 590 ttmol). The vial was capped under a
blanket of nitrogen, and placed on a wrist-action shaker at low speed for 2 h. The resin was
filtered, washed with a basic wash (5 x 10 mL, see above), washed with DMF (5 x 10 mL), then
finally washed with MeOH (5 x 10 mL). The resin was transferred to a clean 20 mL
polyethylene vial, and residual solvent was removed under reduced pressure prior to peptide
cleavage.
Peptide cleavage and purifcation. Peptides were cleaved after fluorophore coupling
using 10 mL of Reagent K5 (trifluoroacetic acid / H20 / ethanedithiol / thioanisole / phenol,
82.5:5:5:5:2.5) with a 2 h incubation period. The resin was filtered, concentrated to ca. 2 mL
volume, and precipitated with ether / hexane 2:1 at -20 'C for 30 min. The supernatant was
decanted, and the peptides triturated with ether / hexane 2:1 (5 x 50 mL). The resultant solid was
resuspended in water (20 mL), lyophilized, and then purified by reverse phase (C18 ) high-
performance liquid chromatography (HPLC), gradient elution with 0.1% v/v TFA in acetonitrile
(solvent B) added to 0.1% v/v TFA in water (solvent A). The reduced (dithiol) and oxidized
(intramolecular disulfide) forms of the peptide are separable by HPLC. Both forms of the
peptide were collected and characterized, then combined and submitted together to a reduction
protocol.
Peptide reduction.. The reduction was performed by dissolving the combined HPLC
purified peptides in 0.1 M phosphate buffer, pH 8.0, 50 mM dithiothreitol (DTT), 10mM EDTA,
6 M guanidine, and incubating at 25 'C, 1 h. The reduction mixture was loaded onto a C 18 Sep-
Pak (Millipore), and buffer salts and DTT were eluted with 10 mL water. The reduced peptide
was eluted with 30% acetonitrile in water. Acetonitrile was removed from this solution under a
stream of nitrogen until the sample could be frozen at 0 C, whence it was lyophilized. This was
resuspended in high-purity water that had been sparged with argon 0.5 h then hydrogen 0.5 h. to
delay oxidation. The resulting stock solution was periodically analyzed by HPLC and no
significant (> 2%) disulfide formation was detected.
Peptide characterization. ZNS-1: Ac-Tyr-Gln-Cys-Gln-Tyr-Cys-Glu-Lys-Arg-Baa(dns)-
Ala-Asp-Ser-Ser-Asn-Leu-Lys-Thr-His-Ile-Lys-Thr-Lys-His-Ser-NH2
tR (oxidized, linear gradient 0-70% B over 25 min) = 18.1 min.
tR (reduced, linear gradient 0-70% B over 25 min) = 18.8 min.
tR (oxidized, linear gradient 20-40% B over 25 min) = 10.6 min.
tR (reduced, linear gradient 20-40% B over 25 min) = 13.5 min.
Electrospray-MS (oxidized) MW Calcd. for C13gH212042N42S3 3227.6; Obs. 3227.2
4
.. iQ(0996.Ameriran C CMical qacieP J I A Chem. Soc. U118 R-ag43453- Walku Supplemental Page 5
13)
Electrospray-MS (reduced) MW Calcd. for C138H214042N42S3 3229.7; Obs. 3228.9
The concentration of stock solutions ZNS-1 was determined by reaction with Ellman's reagent,
2,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 6 . These assays were performed in triplicate, with
excellent agreement (< 5% error) between replicate runs. With this knowledge, the extinction
coefficient of the dansyl chromophore was determined at 333 nm for ZNS-1 to be 5.3 x 103 M- 1
cm-1 in 50 mM Hepes, pH 7.0. This value was used for all subsequent quantitations of ZNS-1.
Emission fluorescence assay. Assays were performed at pH 7.0 in 50 mM Hepes buffer,
0.5 M NaCl in a stoppered 750 ptL (1cm x 3mm) quartz fluorometer cell. The concentration of
ZNS-1 in a given assay was determined by measurement of the unique absorbance of the dansyl
chromophore (e 333 nm = 5.3 x 103). Blank scans were taken against the same buffer
immediately prior to ZNS-1 addition. Emission spectra were accumulated at 1 nm intervals with
the following parameters: excitation wavelength = 333 nm, emission wavelength = 400-750 nm,
excitation band pass = 4 mm, emission band pass = 2 mm, lamp potential = 975 V, gain = 10,
filter (time constant) = 3. To mask the effects of adventitious metal ions present prior to the
initiation of metal titration, small aliquots of EDTA were added (in increments of 0.5 PM) until
overlaying emission spectra were observed, and the wavelength of maximum emission was 560
nm. The EDTA concentration at the initiation of the metal titration was never in excess of 4 RM
(<0.001% of the Mg 2+, and <4% of the Co2+ subsequently added). With the concentrations of
Zn2+ and Co 2+ present over the course of a titration, and the relative stability constants of EDTA
for these species, at most 10% of the Zn2+ would be complexed by EDTA.7 Divalent metal ions
were added to the assay from stock solutions prepared in unbuffered water. These included a
2.83 M solution of MgCl2, a 0.0969 M solution of CoCl2, and a 100 PM solution of ZnCl2.
References
(1) Vogel's Textbook of Quantitative Inorganic Analysis; Bassett, J.; Denney, R. C.; Jeffery, G.
H. Mendham, J.; William Clowes: London, 1978; pp 223-402.
(2) Arnold, L. D.; Kalantar, T. H.; Vederas, J. C. J. Am. Chem. Soc. 1985,107,7105-9.
(3) Kucharczyk, N.; Badet, B.; Goffic, F. L. Synth. Comm. 1989, 19, 1603-1609.
(4) Kates, S. A.; Daniels, S. B.; Albericio, F. Anal. Biochem. 1993, 212, 303-3 10.
(5) King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Pept. Protein Res. 1990, 36, 255-266.
(6) Riddles, P. W.; Blakeley, R. L.; Zerner, B. Meth. Enzymol. 1983, 91, 49-60.
(7) O'Sullivan, W. J.; Smithers, G. W. Meth. Enzymol. 1979, 63, 294.
5


Design and Evaluation of a Peptidyl Fluorescent Chemosensor for Divalent Zinc

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Design and Evaluation of a Peptidyl Fluorescent Chemosensor for Divalent Zinc

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