Preparation of FRET reporters to support chemical probe development

Article abstract of DOI:10.1039/c0ob00322k

In high throughput screening (HTS) campaigns, the quality and cost of commercial reagents suitable for pilot studies often create obstacles upon scale-up to a full screen. We faced such challenges in our efforts to implement an HTS for inhibitors of the phosphopantetheinyl transferase Sfp using an assay that had been validated using commercially available reagents. Here we demonstrate a facile route to the synthetic preparation of reactive tetraethylrhodamine and quencher probes, and their application to economically produce fluorescent and quencher-modified substrates. These probes were prepared on a scale that would allow a full, quantitative HTS of more than 350,000 compounds.

Full text of DOI:10.1039/c0ob00322k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Preparation of FRET reporters to support chemical probe development†‡
Timothy L. Foley,a,b Adam Yasgar,c Christopher J. Garcia,a Ajit Jadhav,c Anton Simeonovc and
Michael D. Burkart*a
Received 25th June 2010, Accepted 21st July 2010
DOI: 10.1039/c0ob00322k
In high throughput screening (HTS) campaigns, the quality and cost of commercial reagents suitable
for pilot studies often create obstacles upon scale-up to a full screen. We faced such challenges in our
efforts to implement HTS for inhibitors of the phosphopantetheinyl transferase Sfp using an assay that
had been validated using commercially available reagents. Here we demonstrate a facile route to the
synthetic preparation of reactive tetraethylrhodamine and quencher probes, and their application to
economically produce fluorescent and quencher-modified substrates. These probes were prepared on a
scale that would allow a full, quantitative HTS of more than 350,000 compounds.
Introduction
Phosphopantetheinylation is an important posttranslational mod-
ification representing an obligatory step that activates enzymes
producing fatty acid, polyketide and nonribosomal peptide
compounds.1 Compounds from these classes are essential to
Fig. 1 Phosphopantetheinyl transferase assay format. Action of PPTase
with rhodamine-CoA 1 on quencher-modified YbbR acceptor peptide 2,
assembles a FRET pair 3 and decreases the rhodamine-CoA fluorescence
signal.
bacterial cell viability, and many have been identified as small
molecule virulence factors.2 The central role of this posttrans-
lational modification to cellular maintenance and pathogenicity
has not gone unnoticed as chemical actuation of this process has
potential to produce antimicrobial therapeutics with a novel mode
of action.3
The phosphopantetheine functionality is installed onto syn-
thase enzymes by action of phosphopantetheinyl transferase, in
conjunction with a coenzyme A (CoA) cosubstrate.1 To further
our development of a method to selectively isolate and identify
synthase enzymes, we have sought to identify inhibitors of Sfp, a
canonical representative of this enzyme class.4 To this end, we have
designed a novel FRET assay platform and miniaturized it into
a high-throughput screen (HTS) protocol.5 In this system, action
of the enzyme assembles fluorescent tetramethylrhodamine-CoA
1 and quencher-modified acceptor peptide 2 into a non-emitting
“dark” product 3 (Fig. 1).
This miniaturization study identified the first known inhibitors
of this enzyme, and we now seek to further unveil new active scaf-
folds through the screening of large chemical libraries. Previously,
pilot quantities of reagents were prepared from commercially avail-
able TAMRA maleimide 4 and Black Hole Quencher-2 (BHQ-2)
carboxylic acid 5 (Fig. 2). However, in turning to interrogate large
Fig. 2 Commercially available tetramethylrhodamine maleimide 4 and
Black Hole Quencher-2 carboxylic acid 5 were used to prepare pilot
reagents 1 and 2.
compound collections with this screen, we found the high cost
of 4 and 5 prohibitive toward our ability to supply reagents. In
this report, we detail our economical preparation of structurally
and spectroscopically similar rhodamine and quencher probes to
support large-scale HTS campaigns.
aDepartment of Chemistry and Biochemistry, University of California-
San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358. E-mail:
mburkart@ucsd.edu; Tel: +1 858-534-5673
bPresent address: NIH Chemical Genomics Center, 9800 Medical Center
Drive, Bethesda, MD 200892-3370, USA
cNIH Chemical Genomics Center, 9800 Medical Center Drive, Bethesda,
MD 200892-3370, USA
Results and discussion
† This paper is part of an Organic & Biomolecular Chemistry web theme
issue on chemical biology.
We estimated the minimum reagent need for the quantitative
HTS6 of the NIH Molecular Libraries Small Molecule Repository
(311,260 compounds)7 to be 120 mg of rhodamine CoA and
‡ Electronic supplementary information (ESI) available: Full 1H-
and 13C-NMR spectra for compound characterization. See DOI:
10.1039/c0ob00322k
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Scheme 1 Rhodamine WT (i) C18-silica, MeOH/0.003% H3PO4, 20%. (ii) (BOC)2O, DCM, 87%. (iii) 6, NaHCO3, THF, 58%. (iv) TFA, DCM quant.
(v) EtiPr2N, HBTU, DMF, 76%. (vi) Coenzyme A, NaH2PO4, quant.
Scheme 2 (i) 3 M HCl, NaNO2; HBF4. (ii) 2,5-dimethoxyaniline, DMF, 69%. (iii) HSO3NO, H2SO4; HBF4. (iv) N-methyl-N-(2-hydroxyethyl)-aniline,
DMF, 47% (2 steps). (v) p-NO2PhCOCl, DIPEA, quant. (vi) H2N-Ahx-DSKLEFIASKLA-O-[PEG]-PS resin, DIPEA. (vii) 96 : 2 : 2 TFA : TIPS : H2O.
400 mg of quencher-peptide. While the former was prepared
from 4, no suitably inexpensive tetramethyl- precursors could be
identified. We chose to evaluate a tetraethyl- analogue, Rhodamine
WT 6 (Scheme 1), that is marketed for water-tracing applications
and recently adapted for use by McCafferty & coworkers.8 We
acquired a sample of this material (60 g) as a mixture of isomers
from Pylam Dyes; available for ~$170/lb (Pylam Dyes, Tempe,
AZ, USA). This mixture may be resolved by reversed phase
flash chromatography9 using methanol : 0.003% phosphoric acid
as a mobile phase10 in modest yield (Scheme 1). We chose to
carry forward isomer-II 7, as it more closely resembles TAMRA
maleimide 4 with respect to substitution about the benzoyl ring,
given that FRET characteristics depend heavily on alignment
of molecular dipoles.11 The maleimide linker was assembled
by mono-BOC protection of ethylene diamine 8,12 followed by
conversion to maleimide 10 with N-(methoxycarbonyl)-maleimide
9 (Scheme 1).13 After purification, the BOC-group was removed
with TFA/CH2Cl2 to provide TFA ammonium salt 11.
(Scheme 1). Conversion of maleimide 12 to the Coenzyme A
analogue 13 proceeded quantitatively in phosphate buffer and the
latter was biochemically indistinguishable from 1 (vide infra).
Finding no preparative literature concerning BHQ-2 carboxylic
acid 5, we developed a facile route based on published patents
(Scheme 2).14 This sequence began with diazotization of aniline
14 in 3 M HCl. Dilution with hydrofluoroboric acid yielded
the expected diazonium tetrafluoroborate salt. Conversion to
diazoaniline 16 was accomplished by slow addition to 15 in
DMF. Moving forward, the aqueous insolubility of 16 required
conversion to the diazonium salt in concentrated sulfuric acid with
nitrosylsulfuric acid as the oxidant. Subsequent recovery of the
tetrafluoroborate salt and reaction with aniline 17 cleanly afforded
alcohol 18.
To activate alcohol 18 for peptide coupling, we had initially
planned for oxidation to the corresponding carboxylic acid. How-
ever, the compound proved unreactive or prone to decomposition
with common oxidants. As such, we investigated the use of an
activated carbonate to install the alcohol onto the peptide. In this
respect, we found that 18 could be converted to p-nitrophenyl
carbonate 19 with ease (Scheme 2).15
From here, we evaluated several activation schemes for 7. These
experiments identified the HOBt ester to react readily with amine
11 in anhydrous DMF, and provided 12 in very good yield
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We prepared the final product by assembling the
aminocaproate-terminated YbbR peptide (sequence: NH2-
Ahx-DSKLEFIASKLA–CO2H)16 using FMOC-based solid
phase peptide synthesis protocols on a 0.3 mmol scale.17
Treatment of the resin with 2 molar equivalents (630 mg) of 19
overnight gave clean conversion to peptide 20. HPLC purification
of this material afforded 228 mg of product at >98% purity
(Scheme 2). The process was iterated three times to satisfy the
projected amount required for HTS (>500 mg).
coated stir bar, and all non-aqueous reactions were performed
under a balloon of dry argon in septum-sealed, oven-dried
glassware. When required, compounds were purified via flash
chromatography20 on 230–400 mesh Silica Gel 60 (EMD Chemi-
cals, Gibbstown, NJ, USA). Analytical TLC was performed using
250 mm silica layers on glass plates (Silica Gel 60 F254, EMD
Chemicals, Gibbstown, NJ, USA) and separated compounds
visualized by illumination with UV light. Analytical reversed
phase TLC (rp-TLC) was performed using 200 mm layers of
R
Evaluation of the above-prepared reagents 13 and 20 demon-
strated that they performed indistinguishably from their commer-
cial counterparts, providing a stable and highly reproducible dose–
response screen of the LOPAC1280 (Library of Pharmacologically
Active Compounds, Sigma-Aldrich), performed in triplicate using
a fully-automated robotic screening system (Fig. 3).18 As seen in
Fig. 3A, the robotic screen was associated with a consistently high
Partisilꢀ KC18 on glass plates (Cat. No. 4801-425, Whatman
Inc, GE Healthcare, Piscataway, NJ, USA). High pressure liquid
chromatography (HPLC) separations were performed with an
Agilent 1100 series instrument (Agilent Technologies, Santa Clara,
CA, USA) equipped with a preparative scale autosampler (fitted
with a 900 mL injection loop) and a diode array detector. Analytical
R
separations were performed on a 4.6 ¥ 150 mm Ultrasphere ODSꢀ
19
Z¢-factor and near-constant IC50 values for an inhibitor dose–
column (Cat. No. 235330, Beckman Coulter, Brea, CA, USA)
with injection volumes ranging from 50–100 mL. Semipreparative
separations were performed on a 10 ¥ 250 mm Biotage KP-
C18-HS 35/70u column (Cat. No. S1L0-1119-95050, Biotage,
Charlotte, NC, USA) using an injection volume of 500–900
mL. Preparative reversed phase separations were performed on
a Waters instrument (Waters, Milford, MA, USA) comprised of
a model 680 gradient controller, model 510 pumps fitted with
high flow volume (225 mL) heads, model 2487 dual wavelength
absorbance detector, Hewlett Packard 3396-series II integrator
(Agilent Technologies, Santa Clara, CA, USA), Pharmacia Frac-
100 fraction collector (GE Healthcare, Piscataway, NJ, USA) and a
22 ¥ 250 mm Econosphere C18 (10 mm) column (cat. no. 50195422,
W.R. Grace & Co., Deerfield, IL, USA). Prior to separation,
samples were prepared using Waters SepPak C18 (Waters, Milford,
MA, USA) solid phase extraction columns to remove salts
and nonpolar contaminants that irreversibly adsorb to reverse
phase media. Characterization data and yields correspond to
homogeneous materials. NMR data were collected on a 300 MHz
Varian Mercury or 400 MHz Varian Mercury Plus spectrometers
response series included on every screening plate. Furthermore,
two previously noted screening hits,5 PD 404,182 and calmida-
zolium chloride, displayed uniform concentration–response curves
which overlapped those previously obtained in with the initial
reagent set (Fig. 3B). These results confirmed that the assay
retained its sensitivity to inhibitors with the new reagents.
1
operating at 300.077 MHz or 399.913 MHz for H-NMR and
75.462 MHz or 100.567 MHz for 13C-NMR, respectively, at the
UCSD Department of Chemistry and Biochemistry NMR facility.
FID files were processed using MestReNova software version 6.1.0
(MestreLab Research, Escondido, CA, USA). Chemical shifts
were calibrated using the residual solvent resonance:21 D6-DMSO
(d 2.50, pentet, H-NMR) and (d 39.52, heptet, 13C-NMR), D1-
1
1
chloroform (d 7.26, singlet, H-NMR) and (d 77.16, triplet, 13C-
Fig. 3 Utilization of the new reagents in a triplicate robotic screen of the
LOPAC1280 library. (A) Excellent screening assay performance as evidenced
by the consistently high Z¢ factor and the unchanging IC50 for a control
inhibitor SCH-202676. (B) Switching to the new reagents did not change
the assay’s sensitivity to inhibitors as evidenced by the nearly identical dose
responses obtained for two inhibitors of different potency, PD 404,182
(average IC50 3.2 mM) and calmidazolium chloride (average IC50 20 mM)
when reagents 1 and 2 (PD 404,182, ᭺; calmidazolium chloride, ꢀ) or 13
and 20 (PD 404,12: ᭜, ᭹, and *; calmidazolium chloride: ꢁ, ꢀ, and ᭢)
were used.
NMR), or D4-methanol (d 3.31, pentet, 1H-NMR) and (d 49.00,
heptet, 13C-NMR). Resonance multiplicities are reported as s =
singlet, d = doublet, t = triplet, q = quartet, p = pentet, dd =
doublet of doublets, m = multiplet. 1H-NMR data are reported as
follows: Chemical shift (number of protons, multiplicity, coupling
constants). Mass spectrometric data for all compounds except
13 and 20 were collected by Dr Yongxuan Su at the UCSD
Department of Chemistry and Biochemistry Small Molecule
Mass Spectrometry facility on Finnigan LCQDECA and Thermo
Finnigan MAT900XL spectrometers. Mass spectrometric data for
compounds 13 and 20 were collected by Dr William Leister at the
NIH Chemical Genomics Center using an Agilent 6210 time-of-
flight mass spectrometer fitted with a 1200 series HPLC system
for sample introduction (Agilent Technologies, Santa Clara, CA,
USA).
Experimental
All reagents and chemical compounds were used as purchased
from commercial sources unless noted. Pyridine was distilled
from KOH. Stirring was accomplished magnetically with a teflon-
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Separation of Rhodamine WT isomers10
were combined, washed successively with water (1 ¥ 100 mL) and
brine (1 ¥ 100 mL). The organic phase was dried over Na2SO4
and evaporated to dryness. The resulting solid was recrystallized
from EtOAc/iPr2O to give methyl carbamate 9 (6.211 g, 40 mmol,
78%). NB: DIPEA is not an acceptable substitute for NMM.
dH (400 MHz, CDCl3) 6.84 (2 H, s, J 4.6), 3.94 (2 H, s, J 0.4).
dC (101 MHz, CDCl3) 165.89, 148.32, 135.53, 54.52. MS (ESI)
m/z 156.04 ([M + H]+, 100%); HRMS (ESI-FT) m/z calcd for
C6H6NO4 156.0291, found 156.0293.
Rhodamine WT 6 (1 g) was dissolved in methanol (200 mL).
Dichloromethane was added (400 mL) and the solution extracted
with 1 M HCl (400 mL). The organic phase was concentrated
in vacuo to give a burgundy solid. The solid was dissolved in
methanol (50 mL) and diluted with 0.003% phosphoric acid
(70 mL). This solution was loaded on a preparative C18-silica
column (7 cm ¥ 20 cm bed dimensions) equilibrated in 35 : 65
methanol : 0.003% phosphoric acid. The isomers were resolved
by a step gradient from 35 to 60% methanol that increased in
increments of 5% methanol. Fractions containing the separated
isomers were pooled, diluted with an equal volume 1 M HCl and
extracted with dichloromethane (3 ¥ total volume). the pooled
organic phases were dried over Na2SO4 and evaporated to give
rhodamine WT isomer I (355 mg, 35%) and rhodamine WT isomer
II 7 (162 mg, 16%) in a ratio of approximately 2 : 1 (isomer I :
isomer II).
Isomer I. dH (400 MHz, CD3OD) 8.44–8.33 (2 H, m), 7.98 (1 H,
d, J 1.1), 7.12 (2 H, d, J 9.5), 7.03 (2 H, dd, J 9.5, 2.4), 6.98 (2 H,
d, J 2.4), 3.67 (8 H, q, J 7.1), 1.30 (12 H, t, J 7.1).
dC (101 MHz, DMSO) 166.59, 166.53, 157.53, 155.37, 135.11,
131.56, 131.49, 131.06, 114.82, 113.10, 96.62, 45.91, 13.12. MS
(ESI) m/z 487.40 ([M+]+, 100%); HRMS (ESI-FT) m/z calcd for
C29H31N2O5 487.2227, found 487.2229.
tert-Butyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-
1-yl)ethylcarbamate 1013
A solution of tert-butyl 2-aminoethylcarbamate (1.13 g, 7.1 mmol)
in saturated sodium bicarbonate (35 mL) was prepared, the
resulting solids filtered, and cooled to 0 ◦C. Finely ground N-
methoxycarbonyl maleimide 9 (1.1 g, 7.1 mmol) was added and the
reaction stirred for 15 min at room temperature. THF (55 mL) was
added and the reaction stirred 45 min. Water (50 mL) was added
and the solution washed with ethyl acetate (3 ¥ 75 mL). The organic
washes were pooled, dried over Na2SO4, and concentrated to give
an oil that solidified upon standing. The residue was purified by
chromatography on SiO2 with a step gradient of hexane : ethyl
acetate to give the title compound 10 as a white solid (1.0922 g,
4.55 mmol, 58%). dH (400 MHz, CDCl3) 6.68 (2 H, s), 3.67–
3.55 (2 H, m), 3.29 (2 H, dd, J 11.1, 5.8), 1.36 (9 H, s). dC
(101 MHz, CDCl3) 171.04, 134.37, 79.67, 39.53, 38.19, 28.51. MS
(ESI) m/z 263.04 ([M + Na]+, 100%); HRMS (ESI-FT) m/z calcd
for C11H16N2O4Na 263.1002, found 263.1008.
Isomer II 7. dH (400 MHz, CD3OD) 8.90 (1 H, d, J 1.4), 8.41
(1 H, dd, J 7.9, 1.5), 7.53 (1 H, d, J 7.9), 7.10 (2 H, d, J 9.5),
7.03 (2 H, dd, J 9.5, 2.2), 6.97 (2 H, d, J 2.2), 3.67 (8 H, q, J
7.0), 1.30 (12 H, t, J 7.0) d (75 MHz, CDCl3) 170.72, 170.24,
C
162.60, 161.96, 159.85, 142.10, 137.20, 137.06, 136.98, 136.28,
136.11, 135.98, 135.04, 134.90, 118.31, 117.26, 100.13, 100.02,
49.65, 15.75, 15.60. MS (ESI) m/z 487.42 ([M+]+, 100%); HRMS
(ESI-FT) m/z calcd for C29H31N2O5 487.2227, found 487.2233.
2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethanammonium
trifluoroacetate 11
tert-Butyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylcarba-
mate 10 (1.000 g, 4.15 mmol) was dissolved in dichloromethane
(10 mL) and cooled on ice. Trifluoroacetic acid (2 mL) was added
and the solution stirred overnight. The reaction was diluted into
diethyl ether (38 mL), cooled on ice 1 h, and filtered to provide
the ammonium salt 11 (1.051 g, 4.14 mmol, 99%) as a white
crystalline solid. dH (300 MHz, CD3OD) 6.89 (2 H, s), 3.90–3.73
(2 H, t, J 5.6), 3.24–3.09 (2 H, t, J 5.6). dC (75 MHz, CD3OD)
171.19, 162.47, 162.01, 161.55, 161.09, 134.56, 38.65, 35.05. MS
(ESI) m/z 141.09 ([M + H]+, 100%); HRMS (ESI-FT) m/z calcd
for C6H9N2O2 141.0658, found 141.0659.
tert-Butyl 2-aminoethylcarbamate 2212
A solution of 1,2-diaminoethane 8 (7.7 mL, 114 mmol) in
chloroform (450 mL) was prepared and cooled to 0 ◦C in an
ice bath with stirring. A solution of di-tert-butyl dicarbonate
(5.26 mL, 22.9 mmol) in chloroform (46 mL) was cooled to 0 ◦C
and added dropwise via a pressure equalizing addition funnel over
2 h. The ice bath was removed and the reaction stirred overnight
at room temperature to give a heterogeneous solution. Solids
were filtered and the filtrate evaporated. The resulting oil was
dissolved in EtOAc (100 mL), washed with half-saturated brine (3
¥ 50 mL), dried over Na2SO4, and concentrated in vacuo to give
the monoamine product (3.220 g, 20.1 mmol, 87%). dH (400 MHz,
CDCl3) 3.15 (2 H, dd, J 11.5, 5.7), 2.77 (2 H, t, J 5.9), 1.42 (9 H, s,
J 9.4). dC (101 MHz, CDCl3) 177.14, 156.47, 79.28, 43.30, 41.63,
28.44. MS (ESI) m/z 160.95 ([M + H]+, 100%); HRMS (ESI-FT)
m/z calcd for C7H17N2O2 161.1285, found 161.1286.
Rhodamine-WT maleimide 12
Rhodamine WT isomer II 7 (10 mg, 0.02 mmol) was dissolved in
DMF (2 mL). HBTU (8.56 mg, 0.02 mmol) and DIPEA (5.31 mg,
7.15 mL, 0.04) were added successively with stirring. After 30 min,
the reaction was checked by rp-TLC (CH3OH/0.003% H3PO4;
80/20) to ensure the formation of the HOBt ester (Rf 0; deep
purple). N-(2-Aminoethyl) maleimide TFA salt 11 (10.43 mg,
0.04 mmol) was added and the reaction followed to completion
by rp-TLC (product Rf 0.5). The reaction was diluted with
dichloromethane (50 mL) and washed with NaH2PO4 buffer
(50 mM, pH 7.4, 1 ¥ 50 mL), brine (1 ¥ 50 mL), dried over Na2SO4,
and evaporated. The resulting purple solid was further purified by
reversed phase HPLC to yield maleimide 12 (9.5 mg, 76%). dH
(400 MHz, CD3OD) 8.69 (1 H, d, J 1.6), 8.16 (1 H, dd, J 7.9, 1.7),
N-(Methoxycarbonyl)-maleimide 922
To a solution of maleimide (5 g, 51.5 mmol) in ethyl acetate
(250 mL) was added N-methyl morpholine (5.6 mL, 51.5 mmol)
via syringe and the solution cooled on ice for 20 min. Methyl
chloroformate (4.8 mL, 61.8 mmol) was added dropwise and the
reaction stirred 30 min. Solids were collected on a Bu¨chner funnel
and washed with ethyl acetate (100 mL). The filtrate and washes
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7.51 (1 H, d, J 7.9), 7.12 (3 H, d, J 9.5), 7.03 (3 H, dd, J 9.5, 2.3),
6.98 (3 H, d, J 2.3), 6.84 (2 H, s), 3.87–3.76 (2 H, m), 3.75–3.60
(15 H, m), 1.30 (27 H, t, J 7.2). dC (101 MHz, CDCl3) 171.37,
159.61, 157.95, 155.64, 136.66, 135.98, 134.40, 131.80, 131.65,
130.27, 130.20, 114.16, 113.69, 96.21, 46.11, 39.02, 37.69, 12.67.
MS (ESI) m/z 609.47 ([M+]+, 100%); HRMS (ESI-FT) m/z calcd
for C35H37N4O6 609.2708, found 609.2720.
dissolving NaNO2 (100 mg) in sulfuric acid (5 mL) and warming
to 50 C. After complete dissolution, the solution was cooled in
◦
an ice bath and added dropwise to diazoaniline 16. After 1 h of
stirring, ice cold 1 M hydrogen tetrafluoroborate (300 mL) was
slowly added and the solution extracted with dichloromethane (5
¥ 100 mL). The organic extract was pooled, dried over Na2SO4 and
evaporated to yield 0.661 g of the diazonium tetrafluoroborate salt
that was dissolved in THF (200 mL) and to which a solution of N-
methyl-N-(2-hydroxyethyl)-aniline 17 (0.286 g, 1.73 mmol) in THF
(20 mL) was added dropwise. The reaction was stirred for 1 h and
stripped of solvent. The residue was dissolved in dichloromethane
(300 ml) and washed with 1 M HCl (1 ¥ 150 mL) and brine (1 ¥
150 mL) and dried over Na2SO4 to give 18 as a green–purple solid
(0.748 g, 1.56 mmol, 47%). dH (400 MHz, CDCl3) 8.38 (2 H, dd, J
9.3, 2.3), 8.05 (2 H, dd, J 9.3, 2.3), 7.95 (2 H, d, J 9.1), 7.49 (2 H, d, J
13.3), 6.85 (2 H, d, J 9.1), 4.10 (3 H, s), 4.05 (3 H, s), 3.91 (2 H, t, J
5.6), 3.66 (2 H, t, J 5.7), 3.17 (3 H, s). dC (101 MHz, CDCl3)
156.57, 153.71, 152.67, 150.98, 148.50, 146.82, 144.61, 142.19,
126.34, 124.90, 123.70, 111.74, 101.19, 100.18, 59.81, 56.87, 54.70,
50.40, 39.44. MS (ESI) m/z 465.10 ([M + H]+, 100%); HRMS
(ESI-FT) m/z calcd for C23H24N6O5Na 487.1700, found 487.1702.
Rhodamine coenzyme A 13
Coenzyme A (76 mg, 0.10 mmol) was dissolved in 20 mM
NaH2PO4 pH 7.4 (100 mL) and cooled on ice. A solution of
rhodamine maleimide 12 (76 mg, 0.13 mmol) in methanol (20 mL)
was added in 1 mL portions. The flask was wrapped in foil and
stirred for 3 h; at which point no detectable coenzyme A was
present (determined by HPLC). The reaction was transferred
to a separatory funnel and washed with dichloromethane (5 ¥
100 mL). The resulting aqueous phase was passed over a Waters
SepPak solid phase extraction column equilibrated in 0.05% TFA,
washed with 5 column volumes 20% acetonitrile/0.05% TFA and
rhodamine CoA 13 eluted with 80% acetonitrile/0.05% TFA.
Acetonitrile was removed by rotary evaporation and provided
13 as a fine purple precipitate. HRMS (ESI-TOF) m/z calcd for
C56H74N11O22P3S 1377.3938, found 1377.3934.
2-((4-((E)-(2,5-Dimethoxy-4-((E)-(4-nitrophenyl)diazenyl)phenyl)-
diazenyl)phenyl)(methyl)amino)ethyl 4-nitrophenyl carbonate 19
(E)-2,5-Dimethoxy-4-((4-nitrophenyl)diazenyl)aniline 1614
To
a solution of diazo-hydroxyethylaniline 18 (0.800 g,
1.72 mmol) in dry dichloromethane (200 mL) was added p-
nitrophenylchloroformate (0.382 g, 1.89 mmol), followed by
pyridine (0.416 mL, 0.408 g, 5.17 mmol), and the reaction was
stirred for 2 h. The reaction was washed (1 ¥ 200 mL each)
with 1 M HCl, 50% saturated sodium carbonate, brine, and dried
over Na2SO4. Silica gel (5 g) was added and the mixture stripped
of solvent and dried under vacuum (<10 mmHg). A silica gel
column (60 g) was dry-loaded with this material and eluted with
hexanes/ethyl acetate (1 : 1) to give p-nitrophenyl carbonate 19
(1.0551 g, 1.70 mmol, 97%) as a purple solid. dH (400 MHz,
CDCl3) 8.38 (2 H, d, J 9.0), 8.25 (2 H, d, J 9.2), 8.05 (2 H, d,
J 9.1), 7.96 (2 H, d, J 9.2), 7.49 (2 H, d, J 16.5), 7.24 (2 H, s,
J 3.2), 6.85 (2 H, d, J 9.3), 4.53 (2 H, t, J 5.7), 4.10 (3 H, s),
4.06 (2 H, s), 3.87 (2 H, t, J 5.7), 3.18 (3 H, s). dC (101 MHz,
CDCl3) 177.25, 156.56, 155.47, 153.65, 152.70, 151.96, 151.17,
148.60, 146.61, 145.10, 142.47, 126.26, 125.50, 124.93, 123.75,
122.01, 111.90, 101.25, 100.29, 66.29, 56.94, 50.86, 50.65, 39.10,
29.87. MS (ESI) m/z 630.15 ([M + H]+, 100%); HRMS (ESI-FT)
m/z calcd for C30H28N7O9 630.1943, found 630.1941.
Sigma-Aldrich supplied 2,5-dimethoxyaniline 15 as pellets of a
black solid that required purification before proceeding. Aniline
15 (10 g) was dissolved in ethyl acetate (200 mL) and the persisting
solids filtered to give a black solution. Activated carbon (2 g) was
added and stirred 20 min; after which celite was added and the
solution filtered. The resulting faint yellow filtrate could not be
further decolorized by reiteration of the above treatment, and was
concentrated in vacuo to give a solid. This crude material was
recrystallized from boiling hexanes to give 15 as white crystalline
flakes.
2,5-Dimethoxyaniline 15 (0.9506 g, 6.21 mmol) was dissolved in
DMF (10 mL). A solution of p-diazoniumnitrobenzene tetraflu-
oroborate (prepared from 14, see ref. 14) in DMF (10 mL) was
added slowly and evolved a deep red color. Saturated sodium
bicarbonate was added every 5 min (6 ¥ 1 mL additions). After
1 h, the reaction was diluted with H2O (200 mL) and placed on
ice. The dark voluminous precipitate was collected by filtration,
washed with H2O, and desiccated over Ca2CO3. The resulting
solid was recrystallized from hexanes : EtOAc (3 : 1) to yield the
diazoaniline 16 (1.24 g, 4.1 mmol, 69%) as metallic green crystals.
dH (400 MHz, CDCl3) 8.30 (2 H, d, J 9.1), 7.90 (2 H, d, J 9.0),
7.40 (2 H, s), 6.35 (2 H, s), 4.61 (2 H, s), 3.98 (3 H, s), 3.90 (3 H,
s). dC (101 MHz, CDCl3) 157.56, 156.74, 146.87, 145.79, 142.18,
133.64, 124.79, 122.59, 97.69, 97.00, 56.62, 55.86, 50.08. MS (ESI)
m/z 303.06 ([M + H]+, 100%); HRMS (ESI-FT) m/z calcd for
C14H15N4O4 303.1088, found 303.1090.
Quencher-apo-YbbR peptide 20
YbbR peptide appended with
a 6-aminohexanoyl residue
appended to the N-terminus (sequence Fmoc-N-Ahx-
DSLEFIASKLA-OH) was synthesized with a Pioneer automated
peptide synthesizer (Applied Biosystems, Foster City, CA, USA)
on a 0.3 mmol scale using standard 9-fluorenylmethyloxycarbonyl-
(Fmoc) conditions, including 2-(1H-7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU)
activation and extended coupling times (1 h per residue) in the
presence of 4-fold molar excess of Fmoc-L-amino acid relative to
resin capacity. After completion, the N-terminal Fmoc protecting
group was left intact and the resin collected from the column after
2-((4-((E)-(2,5-Dimethoxy-4-((E)-(4-nitrophenyl)diazenyl)phenyl)-
diazenyl)phenyl)(methyl)amino)ethanol 18
Diazoaniline 16 was dissolved in sulfuric acid (concentrated,
100 mL) at room temperature to give a thick, deep purple solution
that was cooled on ice (20 min). Nitrosyl sulfate was prepared by
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Org. Biomol. Chem., 2010, 8, 4601–4606 | 4605
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washing with dichloromethane. The resin was stored at room
temperature in the dark until further use.
Su (UCSD Small Molecule Mass Spectrometry Facility) and
William Leister (NIH Chemical Genomics Center) for performing
mass spectrometric analyses; and Elizabeth Komives (UCSD) for
assistance with peptide synthesis.
For quencher labeling, 1.35
g of the peptide resin
(0.19 mmol/gram substitution) was swollen in an Econo-Pac
disposable column (product number 732-1010, Bio-Rad, Hercules,
CA, USA). A solution of piperidine was added (20% vol/vol,
10 mL) and the resin shaken gently at room temperature. After 1
h, the resin bed was allowed to settle and the piperidine solution
drained. The resin was washed with copious amounts of DMF
(~250 mL) and transferred to a 50 mL disposable falcon tube. To
this was added a solution of p-nitrophenyl carbonate 18 (0.400 g,
0.63 mmol, 2 eq.) and diisopropylethylamine (51 mL, 0.3 mmol,
1 eq.) in DMF (30 mL) and the reaction shaken gently overnight.
In the morning, the resin was collected in an Econo-Pac column
and washed with DMF until the effluent was no longer purple.
The resin was transferred to a 50 mL falcon tube and 35 mL
of cleavage cocktail added (2% H2O, 2% triisopropylsilane, 96%
trifluoroacetic acid) (N.B.: reaction turns true blue). The tube was
shaken intermittently for 2 h, after which the resin was filtered
and washed with cleavage cocktail (15 mL). The pooled filtrate
and wash was transferred to a falcon tube, evaporated to ~5 mL
under a dry stream of nitrogen, and the peptide precipitated by
the addition of 40 mL of cold diethyl ether. After incubation on
dry ice (1 h), the solids were collected by centrifugation (20 min ¥
1000 g). The pellet was titrated in cold diethyl ether (3 ¥ 30 mL)
and dried under vacuum.
Notes and references
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Conclusion
The methods presented here for the general preparation of
tetraethylrhodamine and quencher reporters is economical and
executable at a preparative scale. Preparation of these probes
enabled the initiation of an HTS campaign where the cost and
availability of commercial probes had previously limited access.
These procedures should be found applicable not only for the
preparation of labeled peptides as above, but also as a direct
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This work was funded by NIH R01GM086225, R03MH83266,
and R21AI090213. The authors wish to thank Drs. Yongxuan
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4606 | Org. Biomol. Chem., 2010, 8, 4601–4606
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The Royal Society of Chemistry 2010
©