Design and synthesis of fluorescent activity probes for protein phosphatases

Article abstract of DOI:10.1016/bs.mie.2019.02.002

Protein phosphatases act in concert with protein kinases to regulate and maintain the phosphoproteome. However, the catalog of chemical tools to directly monitor the enzymatic activity of phosphatases has lagged behind their kinase counterparts. In this c

Full text of DOI:10.1016/bs.mie.2019.02.002

ARTICLE IN PRESS
Design and synthesis of
fluorescent activity probes
for protein phosphatases
Garrett R. Caseya, Jon R. Becka, Cliff I. Stainsa,b,
*
aDepartment of Chemistry and Nebraska Center for Integrated Biomolecular Communication, University of
Nebraska-Lincoln, Lincoln, NE, United States
bCancer Genes and Molecular Recognition Program, Fred & Pamela Buffet Cancer Center, University of
Nebraska Medical Center, Omaha, NE, United States
*Corresponding author: e-mail address: cstains2@unl.edu
Contents
1. Introduction
2
3
3
2. Synthesis of Sox-Br and Fmoc-CSox
2.1 Synthesis of Sox-bromide (Sox-Br)
2.2 Synthesis of Fmoc-Cys(Sox[TBDPS])-OH (Fmoc-CSox, 2)
3. Synthesis of Sox-based phosphatase substrates
3.1 General SPPS conditions
9
11
11
17
18
19
19
20
21
22
23
23
24
24
24
4. In vitro characterization of peptide substrates
4.1 Mg2+ KD determination
4.2 Fold fluorescence increase (FFI) determination
4.3 Determination of kinetic parameters with recombinant phosphatase
4.4 Determination of limit of detection for recombinant phosphatase
4.5 Panel assays with recombinant phosphatases
5. Validation in cell lysates
5.1 Preparation of cell lysates
5.2 Cell lysate assays
6. Conclusions
Acknowledgments
References
Abstract
Protein phosphatases act in concert with protein kinases to regulate and maintain
the phosphoproteome. However, the catalog of chemical tools to directly monitor
the enzymatic activity of phosphatases has lagged behind their kinase counterparts.
In this chapter, we provide protocols for repurposing the phosphorylation-sensitive
#
Methods in Enzymology
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2019.02.002
2019 Elsevier Inc.
All rights reserved.
1

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Garrett R. Casey et al.
sulfonamido-oxine fluorophore known as Sox to afford direct activity probes for phos-
phatases. With validated activity probes in-hand, inhibitor screens can be conducted
with recombinant enzyme and the role of phosphatases in cell signaling can be inves-
tigated in unfractionated cell lysates.
1. Introduction
Protein phosphorylation plays a critical role in cellular communication
(Vlastaridis et al., 2017). Consequently, there is a need to probe the enzy-
matic activities that maintain the integrity of the phosphoproteome in order
to understand fundamental aspects of cellular communication as well as
human disease. The key enzymes that regulate protein phosphorylation
are the protein kinases (PKs) (Manning, Whyte, Martinez, Hunter, &
Sudarsanam, 2002), that act as writers and the protein phosphatases (PPs)
(Chen, Dixon, & Manning, 2017), that act as erasers. While significant
efforts have been focused on developing chemical tools to interrogate PK
function (Bishop et al., 2000; Gonzalez-Vera, 2012; Karginov et al.,
2011; Oldach & Zhang, 2014; Ranjitkar, Brock, & Maly, 2010; Sharma,
Agnes, & Lawrence, 2007), relatively little attention has been given to
PPs (Brautigan, 2013; Casey & Stains, 2018; De Munter, Kohn, &
Bollen, 2013; Fahs, Lujan, & Kohn, 2016; Tonks, 2013). Our laboratory
has recently described an approach to afford peptide-based activity probes
for PPs (Beck, Lawrence, Tung, Harris, & Stains, 2016; Beck, Truong, &
Stains, 2016) by repurposing the phosphorylation-sensitive Sox fluorophore
(Fig. 1) (Pearce, Jotterand, Carrico, & Imperiali, 2001; Shults, Pearce, &
Imperiali, 2003). This strategy can be applied to both protein tyrosine
Fig. 1 Sox-based probes for protein phosphatase activity. The Sox fluorophore is placed
at the +2 or ꢀ2 position relative to the phosphoamino acid (Ser, Thr, or Tyr). Chelation of
magnesium enhances Sox fluorescence (ex.¼360nm, em.¼485nm). Enzymatic
removal of the phosphoryl group decreases affinity for magnesium and results in a con-
current decrease in Sox fluorescence. The rate of decrease in Sox fluorescence is pro-
portional to the enzymatic activity of the target protein phosphatase.

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Design and synthesis of fluorescent phosphatase probes
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phosphatases (PTPs) (Beck, Lawrence, et al., 2016) and protein serine/
threonine phosphatases (PSPs) (Beck, Truong, et al., 2016). Furthermore,
if sufficiently selective substrate sequences can be identified, PP activity
can be monitored in unfractionated cell lysates providing insight into the
temporal dynamics of PP signaling under relevant biological stimuli
(Beck, Truong, et al., 2016). In this chapter, we present our laboratory’s
routine procedures for design, synthesis, and validation of Sox-based fluo-
rescent activity probes for PPs.
2. Synthesis of Sox-Br and Fmoc-CSox
Peptides containing the Sox fluorophore are commercially available
through AssayQuant Technologies (http://www.assayquant.com/). Alter-
natively, Sox-based probes can be accessed via on-resin alkylation of pep-
tides using Sox-Br (1, Fig. 2) or direct incorporation of a Sox-containing
cysteine analog termed Fmoc-CSox (2, Fig. 2). Procedures for the synthesis
of Sox-Br and Fmoc-CSox have been published previously (Lukovic,
Gonzalez-Vera, & Imperiali, 2008; Pearce et al., 2001; Shults et al.,
2003). Below, we outline the synthesis of these reagents (Figs. 3 and 4). This
procedure assumes knowledge of standard organic chemistry techniques and
the availability of basic organic synthesis laboratory equipment.
2.1 Synthesis of Sox-bromide (Sox-Br)
On-resin alkylation of peptides with Sox-Br requires fewer synthetic steps
relative to procedures utilizing Fmoc-CSox. Thus, we recommend first
attempting the synthesis of Sox-based probes using on-resin alkylation with
Sox-Br. However, in certain cases where modifications in SPPS procedures
Fig. 2 Sox reagents used for alkylation (Sox-Br, 1) of or direct incorporation (Fmoc-CSox, 2)
into peptide sequences. R¼tert-butyldiphenylsilyl.

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Garrett R. Casey et al.
Fig. 3 Synthetic scheme for Sox-Br (1).
are required (such the use of acid labile resins) (Lukovic, Vogel Taylor, &
Imperiali, 2009) or on-resin alkylation proves unsuccessful (Beck, Zhou,
Casey, & Stains, 2015), Sox can be incorporated into the peptide sequence
using Fmoc-CSox.
2.1.1 8-Hydroxy-2-methyl-quinoline-5-sulfonyl chloride (4)
1. Add 8-hydroxy-2-methyl-quinoline (3, 10.0g, 1equiv.) to a
100–200mL round bottom flask equipped with a rubber septum.
2. To the powder, add chlorosulfonic acid (50mL, 12equiv.) via direct
pour from a graduated cylinder at room temperature with continuous
magnetic stirring.
Tip: This addition should be performed slowly as this reaction is
exothermic. Additionally, the rubber septum should be removed
from the flask during this time to allow for the corrosive gas to escape
into the fume hood.
3. Evacuate the flask with high vacuum and charge with N2 (ꢁ3) follow-
ing chlorosulfonic acid addition.
4. Stir at room temperature for 2–3h.
5. Transfer the reaction mixture to a 2L separatory funnel containing ice
(3/4ths volume) and dichloromethane (CH2Cl2).
6. Shake vigorously and allow the CH2Cl2 to separate. The ice/
dichloromethane slurry should display a light yellow/greenish hue.
7. Drain the bottom (CH2Cl2) layer into a bed of potassium carbonate in a
large Erlenmeyer flask to remove residual water.

Fig. 4 Synthetic scheme for Fmoc-CSox (2).

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Garrett R. Casey et al.
8. Repeat ꢁ3 with the remaining ice/water layer, supplementing with
fresh CH2Cl2 with each extraction.
9. The resulting combined organic fractions are then vacuum filtered, and
solvent is removed in vacuo to yield a crude yellow powder.
10. The crude product can be used in the next step without further
purification.
2.1.2 5-(N,N-dimethyl)sulfonamido-8-hydroxy-2-methylquinoline (5)
1. A solution of dimethylamine in tetrahydrofuran (2M, 5.5equiv.), is
added to tetrahydrofuran (150mL) in a three-necked round bottom flask
under N2 and continual stirring.
2. 8-hydroxy-2-methylquinoline-5-sulfonyl chloride (4) (1equiv.) is added
in small portions over 3h to the side-neck of the flask. After each addition,
the atmosphere is evacuated with vacuum and recharged with N2.
Tip: The solution will turn a bright-yellow color and dissipate into a
pale-yellow solution following evacuation of the flask. If residual pow-
der is observed on the neck of the flask, use a syringe of fresh THF to
wash off the neck of the round bottom flask to ensure the reactant
washes into the reaction solution prior to atmospheric evacuation.
3. Following the final addition of 4, allow the pale-yellow solution to stir
for an additional 10–15min.
4. Remove the round bottom flask from the stir plate apparatus and remove
the solvent through rotary evaporation (low-vacuum).
5. Excess dimethylamine is removed by re-dissolving the sticky solid in
CH2Cl2 followed by solvent removal using evaporation (3ꢁ 50mL).
6. Place the crude product under high-vacuum (minimum 2h). The
resulting crude product (5), a yellow powder, can be used in the next
step without further purification.
2.1.3 8-tert-butyldiphenylsilyloxy-5-(N,N-dimethyl)sulfonamido-
2-methylquinoline (6)
1. Add 5 (5.0g, crude), imidazole (1.28g, 1equiv.), dry dimethylformamide
(DMF, 28mL), and tert-butyldiphenylsilyl chloride (5mL, 1equiv.)
sequentially to a dry round bottom flask under N2.
2. Evacuate the atmosphere with vacuum and charge with N2 (ꢁ3).
3. Stir the reaction mixture under N2 atmosphere overnight at room
temperature.
4. Remove DMF from the reaction mixture using high-vacuum rotary
evaporation.

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Design and synthesis of fluorescent phosphatase probes
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5. Dilute the resulting sticky solid with ethyl acetate (150mL).
6. Wash with saturated ammonium chloride solution (50mL), brine
(2ꢁ 50mL), and dry over a bed of Na2SO4.
7. Remove the solvent with rotary evaporation (low-vacuum followed by
high-vacuum).
8. Purify the crude product (6) using flash chromatography (silica, 9:1
hexanes/ethyl acetate) to yield a white solid after removal of solvent.
Tip: The silica column should be pre-treated with acetone for
15–20min prior to loading the crude product, in order to decrease
decomposition in the column. Flush out acetone with hexanes
vigorously before loading. Load the crude product onto the silica
column with a minimal amount of CH2Cl2 using a Pasteur pipette.
2.1.4 8-tert-butyldiphenylsilyloxy-5-(N,N-dimethyl)sulfonamido-
2-formylquinoline (7)
1. To a two-neck round bottom flask, add molecular sieves (4A, 5.0g,
˚
1equiv.) activated at 180°C in high-vacuum for 5–6h or through
microwave activation (5ꢁ 10min, or until a burning plasma is observed
in the beaker containing the molecular sieves).
2. Add 6 (4.82g, 1equiv.) to the dry flask with activated molecular sieves.
3. Add dry 1,4-dioxane (70mL) to the reaction flask with continual
stirring.
4. Heat the reaction flask to 95°C with a condenser (5°C water circulation)
attached.
5. Add selenium dioxide (1.27g, 1.2equiv.) and evacuate atmosphere and
charge with N2 (ꢁ3).
6. Allow the reaction to proceed for 17–24h and then remove from heat
and allow to cool to room temperature.
7. Filter through a bed of celite to remove the black residue and molecular
sieves.
8. The celite bed and molecular sieves are then washed with 1,4-dioxane
until no more chromophore is detected by UV on TLC plates or
orange color is observed in washes.
9. The 1,4-dioxane is removed by rotary evaporation.
10. The resulting yellow oil is re-dissolved in ethyl acetate (900mL) and
washed with brine (100mL), nano-pure water (100mL), saturated
potassium carbonate (100mL), and dried over a bed of Na2SO4.
11. Remove the solvent by rotary evaporation to yield a sticky orange oil
(7) which is used in the next step without further purification.

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2.1.5 8-tert-butyldiphenylsilyloxy-5-(N,N-dimethyl)sulfonamido-2-
(hydroxymethyl)quinolone (8)
1. Sodium borohydride (456mg, 1equiv.) is dissolved in absolute
ethanol (98mL) and cooled to 0°C in a round bottom flask. Evacuate
the atmosphere and charge with N2 (ꢁ3) under constant stirring at room
temperature.
2. Crude 7 from the previous reaction (6.26g) is dissolved in dry
dichloromethane (203mL) and ethanol (27mL) in a separate round
bottom flask under N2, with constant stirring at room temperature. This
mixture is then subjected to atmospheric evacuation and charged with
N2 (ꢁ3).
3. Add the solution of 7 drop-wise using an addition funnel, to the sodium
borohydride solution.
Tip: The dark orange solution of 7 should be added slowly and take
1–3h for completion.
4. Following the final addition of 7, allow the reaction mixture to stir for
15min.
5. Dilute the reaction mixture with diethyl ether (800mL).
6. Wash thesolutionwith saturatedammonium chloride (200mL), nanopore
H2O(2ꢁ 100mL), brine(100mL),anddryoverabedofNa2SO4 in a large
Erlenmeyer flask.
7. Remove the solvent by rotary evaporation to yield a pale-yellow
sticky solid.
2.1.6 2-bromomethyl-8-tert-butyldiphenylsilyloxy-5-(N,N-dimethyl)
sulfonamidoquinoline (Sox-Br, 1)
1. The resulting crude alcohol (8) from the previous reaction (4.53g,
1equiv.) is dissolved in CH2Cl2 under N2 and solid carbon tetrabromide,
(CBr4, 1equiv.) is added.
Tip: Use 50mL CH2Cl2 per 0.02mol of 8. In this example, 30mL of
CH2Cl2 was used to dissolve 8 and CBr4, and 20mL of CH2Cl2 was
used to dissolve PPh3 in step 3.
2. Cool the clear reaction mixture to 0°C in an ice bath while stirring.
3. Triphenylphosphine (PPh3, 1.5equiv.) is then dissolved in CH2Cl2 and
slowly added drop-wise. The resulting dark yellow-orange reaction
mixture is stirred at 0°C for 3h.
4. Saturated NaHCO3 (equal volume) is then added to the reaction mixture
and the organic layer is separated, washed with brine (100mL), dried over
Na2SO4, and evaporated under reduced pressure.

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Design and synthesis of fluorescent phosphatase probes
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5. The resulting oil is then purified by flash chromatography (Florisil,
100–200 mesh, 9:1 hexanes/ethyl acetate) to yield a clear oil (548mg)
after removal of solvent.
6. Product identity and purity should be assessed by MS and NMR,
referring to previously published spectra (Shults et al., 2003).
2.2 Synthesis of Fmoc-Cys(Sox[TBDPS])-OH (Fmoc-CSox, 2)
Procedures for alkylation of peptides with Sox-Br can be found in
Section 3.1.6. For certain sequences alkylation with Sox-Br has proven
difficult (Beck, Zhou, et al., 2015), possibly due to inaccessibility of the
nucleophilic cysteine. Moreover, modification of SPPS procedures may
prohibit the use of acid labile protecting groups, which are needed for
selective alkylation with Sox-Br (Lukovic et al., 2009). If these situations
are encountered, Fmoc-Sox can be prepared using Sox-Br. The resulting
Fmoc-protected amino acid can be used to incorporate Sox into peptides
without the need for alkylation.
2.2.1 Fmoc-Cys-OAllyl (11)
1. To an oven-dried round-bottomed flask, equipped with a stir bar and
under positive N2 pressure, add Fmoc-Cys(Mmt)-OH (9, 1equiv.)
followed by 50mL CH2Cl2 and 50mL methanol (MeOH).
2. Allow the colorless solution to stir for 5min at room temperature.
3. To this solution add cesium carbonate (Cs2CO3, 0.5equiv.) and allow
the mixture to stir at room temperature under N2 (evacuate atmosphere
with vacuum and charge ꢁ3) for 45min.
4. Remove the solvent by rotary evaporation and dissolve the resulting
white, fluffy solid in DMF (100mL, anhydrous).
5. Immediately add allyl bromide (3equiv.) to the resulting solution.
6. The reaction mixture is then stirred at room temperature under positive
N2 pressure for 5h. During this time, the reaction is monitored by TLC
(Rf¼0.23, 20% EtOAc in hexanes).
7. Following completion, the reaction mixture is diluted with EtOAc
(1L), washed with 2% NaHCO3 (200mL), nanopore H2O (250mL
ꢁ3), brine (250mL ꢁ3), dried (Na2SO4), and concentrated in vacuo.
8. The crude product (10) is then dissolved in degassed, anhydrous
CH2Cl2 (90mL).
9. Triisopropyl silane (TIPS, 5mL) and trifluoroacetic acid (TFA, 5mL)
are then added to the slightly yellow solution.

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Garrett R. Casey et al.
10. The resulting red solution is then allowed to react at room temperature
under N2 for 3h and the progress is monitored by TLC (Rf¼0.28, 20%
EtOAc in hexanes). The reaction mixture turns a clear yellow color
upon completion.
11. The reaction mixture is then the diluted in CH2Cl2 (300mL), washed
with 5% NaHCO3 (250mL ꢁ2), nanopore H2O (250mL), brine
(300mL), dried (Na2SO4), and concentrated in vacuo.
12. Perform flash column chromatography (SiO2) by loading crude prod-
uct in CH2Cl2, elute with 20% EtOAc in hexanes to yield the product
(11) as a fluffy white solid after removal of solvent.
2.2.2 Fmoc-Cys(Sox[TBDPS])-OAllyl (12)
1. To an oven-dried, round-bottomed flask equipped with a stir bar and
under positive N2 pressure, add Fmoc-Cys-OAllyl (1equiv.) dissolved
in anhydrous CH2Cl2 (70mL).
2. To this colorless solution, add Sox-Br (1, 1equiv.) followed by anhy-
drous N,N-Diisopropylethylamine, (DIPEA, 1.5equiv.). The reaction
(pale yellow in color) is then stirred at room temperature under positive
N2 pressure overnight.
3. Thecrudereaction mixture isthendilutedwith CH2Cl2 (400mL), washed
with saturated ammonium chloride (NH4Cl, 2ꢁ 100mL), nanopore H2O
(100mL), brine (100mL), dried (Na2SO4), and concentrated in vacuo.
4. The resulting crude product is then purified by flash column chromato-
graphy (SiO2 equilibrated with 10% EtOAc in hexanes). Load the crude
product in CH2Cl2 and eluent with increasing amounts of EtOAc
(10, 20, and 30%) in hexanes, yielding the product (12) as a white solid
after solvent removal.
Tip: Load the crude product onto the column in a minimal amount
of CH2Cl2. Flush with pure hexanes to elute the dichloromethane
before addition of ethyl acetate.
2.2.3 Fmoc-Cys(Sox[TBDPS])-OH (2)
1. To an oven-dried round-bottomed flask equipped with a stir bar under
positive N2 pressure, add a solution of Fmoc-Cys(Sox[TBPDS])-OAllyl
(12, 1equiv.) dissolved in anhydrous, degassed (ꢁ3) CH2Cl2 (100mL).
2. To the resulting pale red solution, add phenylsilane (25equiv.) followed
by tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4, 0.04equiv.).
After 10–20min, the reaction should turn a deep red color.

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Design and synthesis of fluorescent phosphatase probes
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3. The resulting mixture is then stirred at room temperature under positive
N2 pressure for 3h. Reaction progress is monitored by TLC (Rf¼0.36,
10% MeOH in CH2Cl2).
4. Upon completion of the reaction, the solvent is removed in vacuo.
5. The crude mixture is then passed through a short flash chromatography
column (SiO2). Load the crude product in CH2Cl2 and eluent with
1, 2, 3, 4, 5, 10, 15% MeOH in CH2Cl2 to yield the product (2).
The resulting Fmoc amino acid was used in SPPS without further
purification.
6. Product purity and identity should be characterized by reverse-phase
HPLC, MS, and NMR using previously published spectra (Lukovic
et al., 2008).
3. Synthesis of Sox-based phosphatase substrates
While several small molecule probes for PPs have been described,
increased selectivity can often be achieved utilizing peptide-based sensors
(Casey & Stains, 2018). In this section we provide general procedures for
the synthesis of peptide-based probes containing Sox using solid-phase
peptide synthesis (SPPS). This procedure assumes a general familiarity with
SPPS and access to standard SPPS equipment.
3.1 General SPPS conditions
Our laboratory typically employs Fmoc-PAL-PEG-PS resin (Applied
Biosystems, 0.18mmol/g) for SPPS. For alkylation of peptides with
Sox-Br, Fmoc-Cys-(Mmt)-OH (Chem-Impex International, 03699) is
used since the Mmt protecting group can be selectively removed prior to
reaction with Sox-Br (Section 3.1.6). The Sox-fluorophore is generally
placed at the +2 or ꢀ2 position relative to the site of dephosphorylation
(Fig. 1) (Beck, Lawrence, et al., 2016; Beck, Truong, et al., 2016;
Lukovic et al., 2008). In certain cases, addition of Sox to a peptide can alter
the efficiency of that substrate for a target enzyme. Thus, our laboratory syn-
thesizes both analogs (+2 and ꢀ2) and evaluates their efficiency using
recombinant enzyme in order to choose an optimal substrate for cell lysates
studies (Section 4). Lastly, our laboratory uses a low-cost setup for SPPS
consisting of Bio-Rad Poly-Prep chromatography columns (9cm height),
Vac-Man Laboratory Vacuum Manifolds from Promega (A7231), and
solvent resistant three-way stopcocks (Qosina, 99166, Fig. 5). Procedures

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Fig. 5 Peptide synthesis apparatus. (A) A coupling reaction bubbling under nitrogen
(1) in a column (2) with a (3) three-way stopcock. (B) A resin bed (4) after solvent removal
by vacuum (5).
given below are tailored to this setup. In addition, it should be noted that
peptides are synthesized in the C- to N-terminal direction via SPPS
(Fig. 6), which is opposite to the standard convention for writing peptide
sequences (N- to C-terminus).
3.1.1 Swelling
1. Weigh out 50–200mg of resin into the column.
2. Swell the resin by allowing it to incubate in CH2Cl2 for 5–15min.
3. Drain CH2Cl2 using vacuum.
4. Wash (ꢁ5) with the appropriate reaction solvent (Table 1).
3.1.2 Deprotection
1. Add 2mL of deprotection solution (20% 4-methylpiperidine (v/v) in the
appropriate solvent, Table 1) and bubble nitrogen through the column
for 5min.
2. Remove the solvent using vacuum.
3. Repeat steps 1–2 (ꢁ3).
4. After the final deprotection, wash the resin (ꢁ5) with 2–5mL of the
appropriate solvent (Table 1) followed by 2–5mL of CH2Cl2.
5. Using a spatula, remove a small sample of resin and place in an Eppendorf
tube.
6. Perform the TNBS (Habeeb, 1966), chloranil (Vojkovsky, 1995), or
Kaiser (Kaiser, Colescott, Bossinger, & Cook, 1970) test for free amines.
Tip: For proline residues, use the chloranil test for secondary amines.

Fig. 6 General scheme for solid-phase peptide synthesis. Peptides are synthesized in the C- to N-terminal direction through a repetitive series
of deprotection and coupling steps. Cleavage provides the final peptide sequence. R represents amino acid side chains and PG represents
protecting groups.

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Table 1 SPPS coupling cocktails utilized by our laboratory.
Coupling Coupling
Situation
reagent
additive
Solvent Base
Basic coupling
PyBOP or HOBt
HBTU
DMF DIPEA
Repetitive Asp and Glu residues COMU Oxyma
Fmoc-CSox/difficult couplings PyAOP HOAt
NMP TMP
DMF Collidine
7. If the free amine test is negative (minimal or no color), repeat steps 1–6.
8. If the test for free amines is positive (orange-TNBS, orange—chloranil
primary amine, green—chloranil secondary amine, blue—Kaiser), pro-
ceed directly to the next section.
3.1.3 Activation and coupling
1. Weigh out 6equiv. of Fmoc-amino acid, coupling reagent, and cou-
pling additive (Table 1) into a 1.5mL Eppendorf tube.
Tip: Equivalents of Fmoc-amino acid, coupling reagent, coupling
additive, and base should be calculated based on the loading capacity
of the selected resin.
2. Dissolve the reagents in a minimal volume of the appropriate solvent
(Table 1).
3. Add 12equiv. of the appropriate base (Table 1) and incubate for 1–2min.
4. Add the contents of the Eppendorf tube directly to the resin. Add
additional solvent to bring total reaction volume to ꢂ5mL.
5. Bubble the reaction mixture with nitrogen for 45min–1h.
Tip: Most amino acid couplings are complete within 1h. However,
certain residues, such as arginine, may require longer coupling times.
6. Following completion of the reaction remove the coupling cocktail by
vacuum.
7. Wash the resin with the solvent used for the reaction (ꢁ5) followed by
CH2Cl2 (ꢁ5).
8. Perform a free-amine test. Successful coupling should result in no color
change (negative result).
9. In the case of partial or incomplete coupling, repeat steps 1–8 with fresh
reagents.
Tip: For particularly difficult sequences, free amines can be capped as
in Section 3.1.5.
10. In the case of a complete reaction (negative free amine test), continue
with successive couplings by repeating steps 1–8 above.

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3.1.4 Pausing SPPS
1. Following a successful coupling reaction, wash the resin with the solvent
used for the reaction (ꢁ5), CH2Cl2 (ꢁ5), and MeOH (ꢁ5).
2. Resin-bound peptides can be stored in the reaction column at 4°C
for weeks.
3. Restart the synthesis by swelling the resin as in Section 3.1.1, step 2.
4. Proceed with deprotection, activation, and coupling steps until peptide
sequence is complete.
3.1.5 Capping
1. Following completion of the amino acid sequence, deprotect the termi-
nal Fmoc amino acid using 20% 4-methylpiperidine in the appropriate
solvent.
2. Wash the resin with solvent (ꢁ5).
3. Test for free-amines, if the test is negative repeat the deprotection.
4. If the test is positive, add 20equiv. of pyridine and acetic anhydride
(Ac2O) to the resin and fill to 5mL with solvent.
5. Bubble under nitrogen for 30–45min.
6. Remove the capping cocktail via vacuum and wash with solvent (ꢁ5)
followed by CH2Cl2 (ꢁ5).
7. Test for free-amines, the test should be negative, if not repeat the
capping procedure.
3.1.6 Selective side-chain deprotection and Sox-Br alkylation
1. Conduct a test cleavage (Section 3.1.7) and characterization by MS
(Section 3.1.8) to ensure that the desired product is present before
alkylation with Sox-Br.
2. Prepare 100mL of deprotection solution containing TFA (1% v/v) and
triisopropylsilane (TIPS, 5% v/v) in CH2Cl2.
3. Swell the resin with DMF (5min). Remove the solution by vacuum
and incubate with CH2Cl2 (45min).
4. Remove the solution by vacuum and wash with CH2Cl2 (ꢁ5).
5. Add 3–4mL of the deprotection solution to the peptide column and
bubble with nitrogen for 20min.
6. Remove the deprotection solution using vacuum and repeat step 5
until the bright yellow color, corresponding to the liberated Mmt
protecting group, completely disappears.
7. To ensure compete Mmt removal, perform an additional round of
deprotection following the loss of yellow color.

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8. Wash the resin with CH2Cl2 (ꢁ5) and DMF (ꢁ5).
9. Add 200μL of anhydrous DMF (per 50mg of resin) and 6equiv. of
freshly distilled tetramethylguanidine (TMG). Allow this solution to
incubate for 3–5min.
10. Add 3equiv. of Sox-Br (dissolved in anhydrous DMF) and agitate the
suspension on a mechanical rocker overnight.
11. Following the reaction, remove the solution and wash with DMF (ꢁ5)
followed by CH2Cl2 (ꢁ5).
12. Perform a test cleavage on a small sample of resin (Section 3.1.7) and
characterize using HPLC and MS analysis (Section 3.1.8).
13. If minimal alkylation product is observed, repeat steps 1–11.
3.1.7 Complete deprotection and cleavage
The following procedure outlines how to perform a complete side-chain
deprotection and cleavage from the resin. To perform a test cleavage, scale
the reaction back to 1mL of cleavage cocktail Reagent K* (King, Fields, &
Fields, 1990) and use a minimal amount of resin.
1. Prepare a 10mL solution of cleavage cocktail Reagent K* (TFA/
phenol/water/thioanisol/1,2-ethanedithiol/TIPS;80:5:5:5:2.5:2.5v/v).
2. Transfer the resin to a 15mL conical tube using a spatula.
3. Add the Reagent K* cocktail to the resin.
4. Rotate at room temperature for 3h.
5. Evaporate off most of the solution under a gentle stream of N2.
6. Precipitate the peptide out of the remaining solution by addition
of ice-cold diethyl ether (Et2O). For large-scale cleavages, a white
precipitate should be observed.
7. Pellet the resin and precipitated peptide by centrifugation at 13.3 rcf
for 5min.
8. Remove the supernatant using a Pasture pipette.
9. Add 5mL of fresh, ice-cold Et2O and vortex.
10. Pellet and repeat steps 7–9 (ꢁ2).
11. Following the final removal of the supernatant, dry the pellet by setting
the open tube in a fume hood for 5–20min.
12. Dissolve the white precipitate in nano-pure water (2–3mL).
13. Filter the dissolved peptide using a 10mL syringe, equipped with a filter
(0.2μm, Waters), into a fresh 15mL conical tube.
14. Wash the conical tube containing the original cleavage reaction with an
additional 2–3mL of nano-pure water and filter this solution into the
tube containing the peptide.
15. Purify the peptide by semi-preparative HPLC (Section 3.1.8).

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Design and synthesis of fluorescent phosphatase probes
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3.1.8 Purification and characterization of peptides
1. Our laboratory utilizes semi-preparative revere-phase HPLC (solvent A:
acetonitrile with 0.1% TFA; solvent B: water with 0.1% TFA) and
monitors UV absorption at 228nm (amide bond) and 316nm (Sox)
(Lukovic et al., 2008, 2009). Typical gradients consist of 5–95% solvent
A over 30min on a C18 YMC-PACK Pro (5μm, 250ꢁ20mm, flow
rate¼15mL/min) column. Gradients should be optimized based on
the purity of each sample.
Tip: Before semi-preparative HPLC, an analytical HPLC run can be
performed with 40μL of sample to determine purity. For test cleav-
ages conducted prior to incorporation of Sox, monitor absorbance at
228nm (amide bond) and 280nm (Fmoc, Trp, Tyr, and Phe).
2. Collect fractions in 15mL conical tubes and freeze at ꢀ80°C. Multiple
runs may be required for large-scale synthesis.
3. Lyophilize each fraction to remove solvent. Fractions containing large
amounts of acetonitrile should be placed on a rotary evaporator prior
to freezing and lyophilization.
4. After lyophilization, a white powder will generally be visible in the tube.
Dissolve this in 1–2mL of nano-pure water.
5. Verify the identity of the peptide using MS and assess purity using
analytical HPLC. This procedure should result in samples with >95%
purity. If significant contamination is observed, gradient conditions
for semi-preparative HPLC can be modified in order to obtain pure
samples.
6. Determine the concentration of the purified peptide by UV–Vis
spectrometry using the extinction coefficient of the Sox fluorophore,
ε¼8247Mꢀ1 cmꢀ1 at 355nm in 0.1M NaOH with 1mM Na2EDTA
(Shults et al., 2003).
7. Stock solutions can generally be stored at 4°C for at least 6 months
or ꢀ20°C for longer periods.
4. In vitro characterization of peptide substrates
Following probe synthesis, it is essential to determine optimal assay
conditions for each new sensor prior to conducting lysate assays. These
experiments involve determining the affinity for Mg2+, the optimal concen-
tration of Mg2+, and the efficiency and selectivity of a substrate for a target
enzyme. With these optimized assay conditions in-hand, the utility of the
probe for monitoring PP activity in cell lysates can be assessed (Section 5).

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Garrett R. Casey et al.
Materials
Fluorescence plate reader
384-well flat-bottom plates (Corning, 3824, 40μL assay volume)
Nano-pure water
Sodium chloride (NaCl)
MgCl2
10ꢁ Assay Buffer: 500mM Tris–HCl (pH 7.5 at 22°C), 10mM DTT,
20mM EGTA (not used in PSP assays), 0.1% Brij-35P (v/v)
Enzyme dilution buffer: Tris–HCl 50mM (pH 7.5 at 22°C), 2mM
EGTA (not used in PSP assays), 1mM DTT, 0.01% Brij-35P, 0.1%
glycerol (v/v)
Tris–HCl Buffer: 50mM, pH 7.5 at 22°C
4.1 Mg2+ KD determination
1. Determine the fluorescence of Sox peptides (1μM) in the presence of
Tris–HCl (50mM, pH 7.5 at 22°C), NaCl (150mM), and varying con-
centrations of MgCl2 (0–250mM) in a total volume of 40μL. Excite
samples at 360nm and measure emission from 400 to 650nm.
2. The fluorescence of each peptide should be measured in triplicate for
each concentration of MgCl2 and averaged.
Tip: Most peptides will display an emission maximum at 485nm,
although we have observed that certain sequences can shift the
emission maximum of Sox. This can be readily observed using the
emission scan above and the maximum emission wavelength can then
be empirically determined.
3. Subtract the fluorescence of the sample with no MgCl2 (background)
from the fluorescence of samples containing varying concentrations of
MgCl2.
4. Plot the background corrected fluorescence data versus the concentra-
tion of Mg2+ and fit the curve to a one-site binding isotherm:
Bmax ½Mg2 +
ꢃ
AFU ¼
KD + ½Mg2 +
ꢃ
where AFU is arbitrary fluorescence units, Bmax is the maximal
fluorescence at saturating concentrations of Mg2+, KD is the equilibrium
dissociation constant, and [Mg2+] is the concentration of Mg2+.
Tip: In order to discriminate between the phosphorylation state of the
peptide, the KD of the phosphopeptide for Mg2+ should be tighter than

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Design and synthesis of fluorescent phosphatase probes
19
the KD of the corresponding non-phosphopeptide for Mg2+. If a
minimal difference in KD is observed, alternative peptide sequences
should be pursued.
4.2 Fold fluorescence increase (FFI) determination
1. Measure the fluorescence of the phosphorylated and non-phosphorylated
peptides (1μM) in solutions containing 50mM Tris–HCl, pH 7.5 at
22°C, 150mM NaCl, and varying Mg2+ concentrations based on
the KD of the phosphorylated peptide for Mg2+ (0.5, 1.0, 1.5, and
2.0ꢁ the observed KD).
2. The fold fluorescence increase is defined as the maximum emission of the
phosphorylated peptide divided by the fluorescence of the corresponding
non-phosphorylated peptide.
3. The concentration of Mg2+ that yields the highest FFI should be used for
all subsequent assays.
4.3 Determination of kinetic parameters with recombinant
phosphatase
1. Wells containing varying concentrations of phosphorylated-peptide
and non-phosphorylated peptide (1–250μM) in assay buffer (1ꢁ in well)
with the optimal MgCl2 concentration are prepared in triplicate.
2. Record the emission of the assay mixture (prior to recombinant phos-
phatase addition) at 485nm for 10min.
3. Determine the average fluorescence emission of wells containing varying
concentrations of phosphorylated and non-phosphorylated peptides
without enzyme.
4. By plotting the average fluorescence as a function of peptide concentra-
tion and fitting to a linear trend line, the slope values (AFU/μM)
for samples containing phosphorylated (fp) and non-phosphorylated
(fnp) peptide can be determined. The difference between the slopes of
these two samples (fp ꢀfnp) yields a correction factor that can be used
to calculate the concentration of the phosphorylated peptide remaining
in solution at a given time after addition of enzyme (step 6).
5. Following the initial 10min read, recombinant phosphatase (25nM
stock solution in enzyme dilution buffer) is added to a final concentration
of 2.5nM in wells containing phosphorylated peptide. Fluorescence
emission is monitored for 1h.

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Garrett R. Casey et al.
6. Plot the fluorescence change versus time for wells containing phosphor-
ylated substrate peptide at each concentration. Select time points for the
initial linear portion of the data corresponding to <10% substrate turn-
over (by comparison to fluorescence prior to enzyme addition in step 2)
and fit to a linear trend line. The resulting reaction slope (AFU/min)
is divided by the correction factor (fp ꢀfnp) obtained in step 4 above,
yielding the rate of substrate depletion (μM/min).
Tip: If reactions have consumed >10% substrate, repeat the experi-
ment using less enzyme.
7. Rates of substrate depletion are converted to rates of product formation
by multiplying by ꢀ1, plotted versus the concentration of phosphory-
lated peptide, and fit to the following equation to determine kinetic
parameters (Nath & Atkins, 2006):
d½Pꢃ
d½Sꢃ Vmax ½Sꢃ
Velocity ¼
¼ ꢀ
¼
dt
dt
½Sꢃ + KM
where d[P]/dt is the change in product concentration versus time, d[S]/dt
is the change in substrate concentration versus time, Vmax is the maximal
velocity of the reaction, KM is the Michaelis-Menten constant, and [S] is
the concentration of substrate. kcat can be determined by dividing Vmax
(μM/min) by the concentration of phosphatase (μM). The specificity
constant (kcat/KM) can be used to compare the efficiency of different
substrates for a given phosphatase. Generally, substrates displaying larger
specificity constants should be used for further assays.
4.4 Determination of limit of detection for recombinant
phosphatase
1. Add optimal concentrations of phosphorylated peptide substrate (at least
1ꢁ to 2ꢁ KM) and MgCl2 to assay buffer (1ꢁ final concentration
in 40μL total) in a series of wells. Allow extra volume for subsequent
addition of enzyme, such that the final assay volume is 40μL.
2. Monitor the fluorescence emission of each well for 10min prior to
addition of enzyme.
Tip: Comparing the fluorescence of wells before and after addition
of enzyme can be used to determine if the initial portion of the
reaction has been missed. Reaction rates can be decreased by using
less enzyme.

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Design and synthesis of fluorescent phosphatase probes
21
3. Dilute recombinant phosphatase in enzyme dilution buffer and add to
wells at varying concentrations (e.g., 2.5–0.1nM). A separate reaction
without enzyme serves as the blank.
4. Monitor the fluorescence emission immediately after addition of enzyme
for 1h.
5. Readings can be background corrected using wells without enzyme
(blank).
6. The linear region of the resulting fluorescence data is plotted versus time
for each enzyme concentration and fit to a linear trend line. For ease of
interpretation, the absolute values of these reaction slopes are used form
here on. Reaction slopes for each enzyme concentration should be
averaged.
7. Plotting the reaction slope versus enzyme concentration allows for the
determination of the limit of detection using the following equation:
3∗Standard deviation of the blank
Limit of detection ¼
Slope of the linear trend line
4.5 Panel assays with recombinant phosphatases
Defining the selectivity of phosphatase probes is a critical step in determining
the utility of a sensor for cell lysate assays. Testing a panel of homologs
phosphatases, or those known to have overlapping substrate specificity,
can highlight potential issues with off-target activity. If off-target activity
is observed, inhibitors can be used to suppress off-target signal (Beck,
Truong, et al., 2016). Alternatively, if off-target activity overwhelms target
signal, new peptide substrates can be pursued or the target enzyme can be
enriched from lysates (Beck, Lawrence, et al., 2016).
1. Add optimal concentrations of phosphorylated peptide substrate (at least
1ꢁ to 2ꢁ KM) and MgCl2 to assay buffer (1ꢁ final concentration in
40μL total) in a series of wells. Allow extra volume for subsequent addi-
tion of enzyme, such that the final assay volume is 40μL.
2. Monitor the fluorescence emission of each well for 10min prior to addi-
tion of enzyme.
3. Prepare recombinant phosphatase stocks by dilution in enzyme dilution
buffer and add to the well. A reasonable starting concentration is 5nM of
each enzyme (this concentration can be adjusted based on the observed
reaction slopes).
4. Monitor the fluorescence emission immediately after addition of enzyme
for 1h.

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Garrett R. Casey et al.
5. Plot the linear region of the fluorescence emission versus time and
determine the average, absolute reaction slope for each enzyme
sample.
6. Data can be background corrected using wells without enzyme.
7. Plot the average reaction slope, and corresponding standard deviation,
for each enzyme in bar graph format.
5. Validation in cell lysates
Once a selective probe is identified, validation studies in cell lysates
can be performed. Target selectivity can be probed using siRNA knock-
down of phosphatase expression (Beck, Truong, et al., 2016). A general
protocol for preparation and analysis of cell lysates is given below. Reducing
reagents are excluded from PTP assays in order to preserve oxidative
modifications to the catalytic cysteine. Catalase and superoxide dismutase
are included in lysis buffers for PTPs in order to prevent non-specific
oxidation (Beck, Lawrence, et al., 2016). PSP assays do not contain chelating
reagents to preserve PSP catalytic activity.
Materials
Fluorescence plate reader
384-well flat-bottom plates (Corning, 3824, 40μL assay volume)
Corning 150mm culture dishes (Sigma, CLS430599)
Cell scrapers (Fisher Scientific, 08-100-242)
Liquid nitrogen in Dewar flask
Phosphate-buffered saline (PBS; Life Technologies, 10010-023), ice cold
Refrigerated microcentrifuge
MgCl2
10ꢁ PTP Assay Buffer: 500mM Tris–HCl (pH 7.5 at 22°C), 20mM
EGTA, 0.1% Brij-35P (v/v)
PTP Lysis Buffer: 50mM Tris–HCl (pH 7.5 at 22°C), 150mM NaCl,
1% Triton X-100 (v/v), 1% (v/v) Protease Inhibitor Cocktail (Life Tech,
78430), 2mM EGTA, 100μg/mL catalase (Millipore, 219261), and
100μg/mL superoxide dismutase (Millipore, 574594)
10ꢁ PSP Assay Buffer: 500mM Tris–HCl (pH 7.5 at 22°C), 10mM
DTT, 0.1% Brij-35P (v/v)
PSP Lysis Buffer: 50mM Tris–HCl (pH 7.5 at 22°C), 1mM DTT,
150mM NaCl, 1% Triton X-100 (v/v), 1% (v/v) Protease Inhibitor
Cocktail (Life Tech, 78430)

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Design and synthesis of fluorescent phosphatase probes
23
5.1 Preparation of cell lysates
1. Culture cells as specified by the supplier.
2. Seed cells onto 150mm culture dishes. A control sample (no treatment) as
well as a sample for subsequent siRNA knockdown should be prepared.
3. Allow cells to grow to the desired confluency (e.g., 80%).
4. Add siRNA knockdown reagents to the treatment sample and culture
according to manufacturer recommendations.
5. After sufficient time has passed for knockdown, place culture dishes
on ice and remove the media. Wash 3ꢁ with ice cold PBS.
6. Lyse cells by adding 200μL of the appropriate lysis buffer and scrap
using a cell scrapper.
7. Incubate for 15min and collect lysate in an Eppendorf tube.
8. Centrifuge at 10,000ꢁg for 5min at 4°C.
9. Collect the supernatant and flash freeze aliquots in liquid nitrogen.
10. Use one aliquot to determine total protein concentration. Our labora-
tory routinely uses the BioRad protein assay (BioRad, 500-0006).
11. Verify knockdown of the target PP via Western blotting according
to supplier protocols.
5.2 Cell lysate assays
1. Mix optimized concentrations of phosphorylated peptide substrate and
MgCl2 with assay buffer (1ꢁ final concentration in 40μL total) in a series
of wells. Allow extra volume for subsequent addition of lysate, such that
the final assay volume is 40μL.
2. Monitor the fluorescence emission of each well for 10min prior to
addition of lysate.
3. Lysate stocks can be diluted using lysis buffer and are generally assayed
at 5–20μg total protein per well where the lysate is <5% of the total
volume of the assay. Optimal amounts of total protein may vary and
should be optimized empirically.
4. Monitor the fluorescence emission for 1h.
5. Plot the linear region of the fluorescence emission versus time and
determine absolute reaction slopes for each sample. Average the reaction
slopes for each sample and determine standard deviations.
6. Reaction slopes and corresponding standard deviations can be plotted in
bar graph form. A selective probe should yield a reduced reaction slope
upon siRNA knockdown that is proportional to the extent of target PP
knockdown.

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Garrett R. Casey et al.
6. Conclusions
Recent work has demonstrated the importance of PPs in cell signaling
as well as the potential of these enzymes as drug targets. However, chemical
tools to study the activity dynamics of these enzymes have lagged behind
those available for protein kinases (Casey & Stains, 2018). The method
described above provides an approach for the synthesis and validation of
Sox-based, fluorescent activity probes for PPs. With a validated probe
in-hand, the activity dynamics of PPs can be assessed under relevant biolog-
ical conditions (Beck, Lawrence, et al., 2016; Beck, Truong, et al., 2016).
Thus, these probes provide a new means to investigate the chemical biology
of PPs, adding to our understanding of cellular signaling.
Acknowledgments
This work was supported by the NIH (R35GM119751). The content of this work is solely
the responsibility of the authors and does not necessarily represent the official views of
the NIH.
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