Site-specific protein propargylation using tissue transglutaminase

Article abstract of DOI:10.1039/c2ob25752a

Transglutaminases (TGases) catalyse the transamidation of glutamine residues with primary amines. Herein we report the first FRET-based activity assay for the direct detection of the ligation (transamidation) reaction mediated by tissue TGase (TG2). This novel assay was then used in a microtiter plate-based screen of a library of 18 potential amine substrates. From this screen it was discovered that propargyl amine serves as an excellent substrate for TG2. Subsequently, propargyl amine and 2-azidoethyl amine were validated independently as TG2 substrates with K_M values of 44 ± 4 μM, and 0.99 ± 0.06 mM, respectively. In a proof-of-principle protein labelling experiment, the protein casein was selectively functionalized with propargyl amine using TG2 and subsequently fluorescently labelled through a dipolar cycloaddition reaction with an azido-fluorescein conjugate. This application demonstrates the strong potential of using TG2 for site-specific protein modification through a combination of enzymatic and bioorthogonal chemistry. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25752a

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PAPER
Site-specific protein propargylation using tissue transglutaminase
Claudio Gnaccarini, Wajih Ben-Tahar, Amina Mulani,† Isabelle Roy, William D. Lubell, Joelle N. Pelletier
and Jeffrey W. Keillor†*
Received 18th April 2012, Accepted 15th May 2012
DOI: 10.1039/c2ob25752a
Transglutaminases (TGases) catalyse the transamidation of glutamine residues with primary amines.
Herein we report the first FRET-based activity assay for the direct detection of the ligation
(transamidation) reaction mediated by tissue TGase (TG2). This novel assay was then used in a microtiter
plate-based screen of a library of 18 potential amine substrates. From this screen it was discovered that
propargyl amine serves as an excellent substrate for TG2. Subsequently, propargyl amine and
2-azidoethyl amine were validated independently as TG2 substrates with KM values of 44 4 μM, and
0.99 0.06 mM, respectively. In a proof-of-principle protein labelling experiment, the protein casein was
selectively functionalized with propargyl amine using TG2 and subsequently fluorescently labelled
through a dipolar cycloaddition reaction with an azido–fluorescein conjugate. This application
demonstrates the strong potential of using TG2 for site-specific protein modification through a
combination of enzymatic and bioorthogonal chemistry.
Paramount for ascertaining the scope and limitations of TGase
in such applications is a fundamental understanding of its
specificity with respect to acyl-donor and acyl-acceptor sub-
Introduction
Transglutaminases (TGases) are Ca2+-dependent enzymes that
catalyse the transamidation of peptide- and protein-bound gluta-
mine residues with primary amines, typically the epsilon-amino
group of the side-chain of lysine residues. By catalysing the
formation of γ-glutamyl-ε-lysine isopeptide bonds, transglutami-
nases facilitate the cross-linking of proteins,1,2 which is critical
for physiological processes. For example, the stabilisation of
blood clots and the extracellular matrix are respectively catalyzed
by the TGases Factor XIIIa3,4 and tissue transglutaminase
(TG2).5
Proficiency in forming amide bonds using the side-chains of
Gln and Lys residues underpins the application of TGases in
site-specific protein modification, because unlike sequence
specific enzymes, such as subtiligase,6 sortase,7 and the split
intein system,8 TGases may be suitable for modifying side
chains anywhere within the protein sequence. Recently,
lipoic acid ligase has been engineered to mediate acylation of
the lysine side chains of a small peptide tag,9–11 albeit with
narrower substrate specificity than TGases for applications
such as site-specific protein labelling,12–14 glycosylation,15,16
and PEG-ylation.17
strates. The donor substrate specificity of TGase has been pre-
viously studied, confirming the selectivity for glutamine as an
acyl-donor substrate selectivity.18,19 Moreover, features for acyl-
donor substrate recognition have been assessed using short
peptides and peptide libraries from phage display, demonstrating
that certain flanking amino acids enhance recognition of the
glutamine residue in high-affinity donor sequences.20,21
Less is known about the acyl-acceptor substrates of TGase.
For example, TG2 has exhibited selectivity for certain lysine
residues in protein sequences, and low specificity for small
primary amine substrates.22 The relevance of flanking amino
acids and the global three-dimensional conformation containing
the lysine residue remain matters of debate;23 however, compari-
sons of amino acid sequences from known protein substrates
have led to the design of short peptide substrates, some exhibi-
ting higher affinity than others.24
For site-specific protein modification, TG2 has been relatively
underexplored, yet exhibits promise, because of its native amino
acid acyl-acceptor substrate specificity.25 Employing a new
FRET-based activity assay, which is specific to the transamida-
tion reaction, to screen a proof-of-concept library of amines,
several new substrates were identified in this work and applied in
TG2-mediated protein labelling. In particular, propargyl amine
has been validated as an effective TG2 substrate and employed
in a proof-of-principle experiment featuring selective propar-
gylation and dipolar cycloaddition with an azido–fluorescein
conjugate to yield a fluorescently labelled protein.
Département de Chimie, Université de Montreal, Succursale centre-
ville, C.P. 6128, Montréal, Québec H3C 3J7, Canada
†Current address: Department of Chemistry, University of Ottawa,
10 Marie-Curie, Ottawa, Ontario K1N 6N5 Canada.
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emission was more practical to follow than the relative increase
in fluorescein emission.
Results and discussion
Assay development
To the best of our knowledge, this is the first report of a
FRET-based activity assay for direct detection of TG2-mediated
ligation. Moreover, it is readily amenable to microtiter plate-
based applications because as little as 4 mU of purified
TG2 gives a detectable decrease in fluorescence at 465 nm.
To begin to explore the potential for this assay to detect TG2
activity in a high-throughput fashion under a wide variety of
conditions, the FRET-based activity assay was initially employed
to screen a proof-of-concept library of potential acyl-acceptor
substrates.
A TGase-mediated ligation assay was recently reported based on
the use of a fluorescent acyl-donor substrate and a biotinylated
acyl-acceptor substrate.26 Seeking a ligation assay that was more
amenable to high-throughput screening, direct detection of the
ligation reaction has now been achieved without the need for
pull down products (Scheme 1). Fluorescein-tagged peptide 1
was employed as acyl-donor substrate.26 Acyl-acceptor substrate
2, bearing a primary amine and a complementary fluorophore,
was designed to provide a transamidation product exhibiting
intramolecular FRET in a continuous fashion, over the course of
the reaction when cross-linked to 1 by TGase.
Substrate screening
Fluorescent amine 2 was synthesized from cadaverine, which
was selectively converted to N-(Boc)-1,2-diaminopentane (4,
Scheme 2). In parallel, coumarin 3 was made from 2,4-dihydroxy-
benzaldehyde and Meldrum’s acid.27 Coupling of fluorescent
acid 3 and amine 4 was accomplished using EDC and HOAt to
give carbamate 5, which was treated with trifluoroacetic acid to
remove the Boc group and ion exchanged from trifluoroacetate
to chloride, to furnish amine 2.
The excitation and emission spectra of substrates 1 and 2 were
next measured in assay buffer to establish the feasibility of the
FRET-based ligation assay (Fig. 1) The significant overlap
between the bands for the emission of donor coumarin 2 and the
excitation of acceptor fluorescein 1 indicated efficient intramole-
cular FRET would be achievable under the reaction assay
conditions.
As we have shown previously,26 one of the most straightforward
applications of an activity assay towards a screening effort is
through the design of a simple competition experiment. To that
end, 18 different amines were evaluated against amine 2 as acyl-
acceptor substrates (Fig. 3). In a microtiter plate, 30 μM of sub-
strates 1 and 2 were initially combined with a different amine
candidate in each well at a final concentration of 10 mM and
subsequently treated with 60 mU of purified TG2. Amines
capable of serving as an effective acceptor substrate competed
with amine 2 in the coupling to glutamine donor 1, and dimin-
ished the rate of the decrease of fluorescence at 465 nm, by inhi-
bition of the assay reaction rate. Screens were performed in
triplicate and repeated at lower concentrations of competing
amine (5 mM final) in triplicate to finally provide a global
profile of the relative efficiencies of amines 6–23 as potential
acceptor substrates (Fig. 4).
Addition of purified tissue transglutaminase to a solution con-
taining substrates 1 and 2, caused a proportional change in the
intensity of the fluorescence emission bands in a time-dependent
manner: the coumarin emission band at 465 nm decreased as the
fluorescein emission band at 518 nm increased (Fig. 2). Under
the reaction conditions, the relative decrease in coumarin
Certain trends in substrate affinity may be inferred from the
activities (Fig. 4). For example, unhindered primary amines,
such as lysine (6), propargyl amine (13) and benzyl amine (17),
all competed effectively with substrate 2. Adjacent carboxylate
Scheme 1
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Scheme 2
Fig. 1 Excitation (dashed) and emission (solid) bands of equimolar
solutions of substrates 2 (400/450 nm) and 1 (490/520 nm).
Fig. 2 Time-dependent decrease of coumarin emission (∼465 nm) and
concomitant increase in fluorescein emission (∼518 nm), due to trans-
glutaminase-mediated ligation of equivalent concentrations of substrates
1 and 2.
moieties reduced efficacy [e.g. 6-aminohexanoic acid (7),
γ-aminobutyric acid (8, GABA), alanine (10), and p-amino-
methylbenzoic acid (19)], which could be restored by esterifica-
tion as in the cases of ethyl 3-aminopropionate (9) and alanine
methyl ester (11). Anilines 20–23 were relatively less active than
the benzyl amines, perhaps owing to their decreased nucleophili-
city, and the presence of a carboxylate moiety in p-aminophenyl-
acetic acid (23) diminished activity. The observation that
propargyl amine (13) served as an efficient substrate for TG2
opened the door to site-specific protein modification by enzy-
matic propargylation followed by dipolar cycloadditions with
azides (vide infra).
TG2-mediated protein labelling
The discovery of propargyl amine (13) as an effective substrate
inspired investigation of the site-specific transamidation of
glutamine residues with TG2 followed by conjugation with an
appropriate label by azide–alkyne cycloaddition. In addition to
propargyl amine (13), 2-azidoethylamine was examined as sub-
strate for TG2 in order to develop a complementary protein
labelling process. 2-Azidoethylamine (25) was synthesized in
three steps from 2-bromoethylamine by amine protection with
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Fig. 3 Amines 6–23 screened as potential acyl-acceptor TG2 substrates.
di-tert-butyldicarbonate, displacement of bromide with sodium
azide and carbamate cleavage with HCl (Scheme 3).
Ac-PNPQLPF, which was prepared by solid-phase synthesis
(see Experimental) and had previously exhibited high affinity
for TG2 with an apparent KM of 11 1 μM in competition28
with a chromogenic assay.22 The heptapeptide was reacted with
propargyl amine for two hours in the presence of TG2 at 37 °C.
Product formation was observed as the appearance of a new
peak with corresponding mass in the LC-MS chromatogram.
Proof-of-concept site-specific labelling with propargyl amine
was then performed using dimethyl casein, a known protein
donor substrate of TG2,29 followed by cycloaddition on the
alkyne employing fluorescein azide 26. Azide 26 was prepared
by acylation of amino azide 25 with FITC (Scheme 3). Dimethyl
casein was reacted with one equivalent of propargyl amine at a
final concentration >10 times of its KM value in the presence of
TG2. After two hours, the reaction mixture was treated with
It is possible, although improbable, that a positive result from
our indirect competition assay may derive from inhibition of the
assay reaction by the competing amine, without that amine
serving as a genuine substrate. Therefore, we deemed it critical
to evaluate the most important putative amine substrates by inde-
pendent assay methods. Using a known chromogenic activity
assay,22 the kcat and KM values for propargyl amine (13) were
thus determined to be 2.39 0.04 min−1 and 44 4 μM, respec-
tively, whereas for 2-azidoethylamine (25) they were found to be
2.09 0.04 min−1 and 0.99 0.06 mM. In subsequent appli-
cations, amine 13 was employed, because of its greater affinity
(KM) and efficiency (kcat/KM). Amine 13 was furthermore
reacted with the glutamine residue of the heptapeptide
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Fig. 4 Relative efficiency of amines 6–23 (Fig. 3) in competition with fluorescent amine 2 (Scheme 1). Activity was measured at final concentrations
of 5 and 10 mM of competing amine and error bars represent standard deviations from screening results obtained in triplicate.
Scheme 3
azide 26, copper and ascorbic acid30 and allowed to sit over-
night, prior to analysis by SDS–PAGE (Fig. 5). Both bands of
casein were efficiently fluorescently labelled by this procedure;
however, in control experiments without TG2 no labelling was
observed.
screened in competition with the fluorescent amine substrate to
provide a structure–activity profile for acyl-acceptor substrates of
TG2. From this screen, propargyl amine was discovered to be an
effective TG2 substrate, as confirmed by independent assays,
and subsequently employed in a proof-of-principle protein
labelling experiment featuring a transamidation/cycloaddition
sequence. Considering the potential of TG2 to site-specifically
propargylate diverse proteins, including those modified with
high-affinity glutamine sequences,31 our results may open many
doors for various applications, featuring modification of the pro-
pargylated proteins using a spectrum of different azide tags.
In addition, the complementary modification of azide bearing
proteins with different alkyne derivatives11,32 may be performed,
because we have also shown that TG2 can recognize
Summary
A new assay for studying TG2 transpeptidation activity has been
developed featuring ligation of fluorescently labelled substrates
and detection of intramolecular FRET. This assay is specific for
transpeptidation, sensitive, and suitable for high-throughput
applications. Using this assay, a library of 18 amines was
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dioxane–water was added over a period of 4 h to a solution of
1,5-diaminopentane (2 g, 19.6 mmol) in 60 mL of 9 : 1 dioxane–
water. The solution was stirred at room temperature overnight
and concentrated by rotary evaporation. The residue was dis-
solved in 40 mL of water. The white precipitate corresponding to
N,N′-di-Boc-1,5-diaminopentane was removed by filtration
through a fritted glass funnel, and the filtrate was extracted
with CH2Cl2 (4 × 30 mL). The combined organic extracts
were concentrated. The residue was purified by silica gel
chromatography (90 : 10 CH2Cl2–methanol). Evaporation of
the collected fractions provided N-(Boc)-1,5-diaminopentane
(4, 1.4 g, 6.95 mmol, 71%) as a dense liquid: bp 97 °C;
1H NMR (CDCl3, 300 MHz) δ 4.63 (s, 1H), 3.1 (dd, 2H, J =
6.0, J = 12.4), 2.68 (t, 2H, J = 6.7), 1.75 (s, 3H), 1.49–1.52 (br,
3H), 1.42 (s, 9H), 1.27–1.36 (br, 2H). 13C NMR (CDCl3,
75 MHz) δ 156.9, 79.9, 42.8, 41.3, 34.0, 30.7, 29.2, 24.9.
HRMS m/z (M + H+): calcd for C10H23N2O2 203.1754; found
203.1761.
Fig. 5 Protein labelling with fluorescent tag by TG2-mediated propar-
gylation of casein, followed by azide cycloaddition: in the absence of
TG2, no labelling occurs.
2-azidoethyl amine as a substrate. These protein-labelling
methods take advantage of the site-specificity and orthogonality
of TG2-mediated transamidation, as well as the chemical diver-
sity of subsequent cycloaddition reactions, rather than attempting
to use a wide variety of labelling agents as substrates for TG2.
TG2-based modifications have thus been established as effective
methods for labelling proteins.
{5-[(7-Hydroxy-2-oxo-2H-chromene-3-carbonyl)amino]pentyl}-
carbamic acid tert-butyl ester (5). A solution of acid 3 (30 mg,
0.15 mmol) in 3 mL of DMF was treated with amine 4
(31 mg, 0.15 mmol), HOAt (21 mg, 0.15 mmol), EDC·HCl
(29 mg, 0.15 mmol) and DIEA (0.027 mL, 0.15 mmol) and
stirred at room temperature for 4 h, when TLC showed the disap-
pearance of the starting amine (Rf = 0, solvent: 5% MeOH–95%
AcOEt) and appearance of a new less polar spot (Rf = 0.2,
solvent: 5% MeOH–95% AcOEt). The volatiles were removed
by rotary evaporation and the residue was treated with 5 mL of
MeOH, leading to formation of a white precipitate. This precipi-
tate was recovered by filtration onto a fritted funnel, washed with
3 × 2 mL of MeOH, and dried under vacuum overnight yielding
amide 5 (50.1 mg, 0.13 mmol, 87%) as white solid: mp
176.3 °C; 1H-NMR (DMSO d6, 400 MHz) δ 11.16 (s, 1H),
8.77 (s, 1H), 8.61 (t, 1H, J = 5.6), 7.81 (d, 1H, J = 8.6), 6.88
(dd, 1H, J = 8.6, J = 2.2), 6.8 (d, 1H, J = 2.1), 6.73 (br, 1H),
3.18 (d, 1H, J = 4.8), 2.91 (q, 2H, J = 12.8, J = 6.6), 1.51 (m,
2H), 1.36 (s, 9H), 1.28 (br, 3H). 13C-NMR (CDCl3, 100 MHz)
δ 164.9, 162.3, 156.8, 155.2, 149.1, 134.8, 130.6, 126.1, 119.5,
119.4, 117.4, 79.9, 41.3, 40.5, 30.5, 30.0, 29.2, 25.0. HRMS m/z
(M + Na+): calcd for C20H26N2O6 413.1683; found 413.1695.
Experimental section
Materials
Synthesis generalities. All Fmoc-protected amino acids, resins
and coupling reagents were purchased from GL Biochem; Wang
resin was purchased from NovaBiochem. All other reagents
were obtained from Sigma-Aldrich. Reactions requiring an-
hydrous conditions were carried out under a dry nitrogen atmos-
phere employing conventional bench-top techniques. 1H- and
13C-NMR spectra were recorded on AMXR400 and AMX300
spectrometers and were referenced to the residual proton or 13C
signal of the solvent. Mass spectra were determined by FAB+
ionization on an AutoSpec Q spectrometer at the Regional Mass
Spectrometry Centre at the Université de Montréal. Reactor
tubes for solid-phase peptide synthesis were obtained from
Supelco. All resins were swollen in DMF and washing steps
were performed using CH2Cl2 and DMF (EMD Chemicals).
7-Hydroxy-2-oxo-2H-chromene-3-carboxylic acid (5-amino-pentyl)-
amide (2). A solution of carbamate 5 (20 mg, 0.06 mmol) in a
mixture of TFA–DCM (1 mL/1 mL) was stirred for 2 h. The
volatiles were removed under vacuum. A salt exchange was per-
formed by dissolving the residue in 2 mL of 1 M HCl and
freeze-drying, twice. Amine hydrochloride 2 was isolated as a
Synthesis of the FRET donor substrate
7-Hydroxycoumarin-3-carboxylic acid (3). Preparation of cou-
marin 3 was based on a literature procedure.27 A mixture of
2,4-dihydroxyl benzaldehyde (2 g, 20 mmol), Meldrum’s acid
(2.89 g, 20 mmol), piperidinium acetate (58 mg, 0.4 mmol) and
ethanol (10 mL) was stirred at room temperature for 20 min,
heated at reflux for 2 h, allowed to cool to room temperature and
chilled in an ice bath for 1 h. The crystallized product was
filtered, washed three times with ethanol, and dried in vacuo
yielding 3 (3.4 g, 83%) as an off-white powder; 1H NMR
(DMSO-d6, 400 MHz) δ 8.66 (s, 1H), 7.73 (d, 1H, J = 8.5),
6.84 (d, 1H, J = 8.4), 6.73 (s, 1H). 13C NMR (DMSO-d6,
100 MHz) δ 164.3, 164.1, 157.7, 157.1, 149.3, 132.1, 114.1,
112.7, 110.7, 101.9.
yellow gel in quantitative yield (19.3 mg, 0.06 mmol); λex
=
400 nm, λem = 448 nm. 1H NMR (CD3OD, 400 MHz) δ:
9.63 (s, 1H), 8.63 (d, 1H, J = 8.80), 7.88 (d, 1H, J = 8.80),
7.77 (s, 1H), 4.36 (t, 2H, J = 6.60), 3.94 (t, 2H, J = 7.74),
2.56–2.71 (m, 4H), 2.35–2.46 (m, 2H). HRMS m/z (M + H+):
calcd for C15H19N2O4 291.1341; found 291.1339.
Synthesis of azide substrate
tert-Butyl 2-azidoethylcarbamate (24). A solution of 2-bromo-
ethylamine hydrobromide (5 g, 24.4 mmol) in 20 mL of MeOH
was treated with 3 mL of triethylamine and 10 g (45.8 mmol) of
di-tert-butyldicarbonate, stirred at room temperature overnight,
treated with another 5 g (22.9 mmol) of di-tert-butyldicarbonate,
(5-Aminopentyl)carbamic acid tert-butyl ester (4). A solution of
di-tert-butyldicarbonate (2.13 g, 9.8 mmol) in 40 mL of 9 : 1
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and stirred for 75 min. Without isolation, the carbamate solution
was cooled to 5 °C, treated with 4.8 g (73.2 mmol) of sodium
azide and 20 mL of H2O and stirred for 4 h at 5 °C. The reaction
mixture volume was reduced by evaporation, and treated with
40 mL of water. The pH was adjusted to 4 using 1 M HCl.
The reaction mixture was extracted with 40 mL of CH2Cl2,
followed by 3 × 20 mL of CH2Cl2. The combined organic
phases were dried on MgSO4, filtered and evaporated on a rotary
evaporator to yield 1.8 g of azide 24 (9.7 mmol, 40% yield).
1H-NMR (CDCl3, 400 MHz) δ: 1.40 (s, 9H), 3.25 (m, 2H), 3.36
(t, J = 5.39 Hz, 2H) 4.84 (br s, 1H). 13C-NMR (CDCl3,
100 MHz) δ: 15.0, 29.1, 40.9, 52.0, 80.6, 156.56. HRMS m/z
(M + Na+): calcd for C7H14N4O2Na 209.1009; found: 209.1009.
sample of a few beads. Subsequently, each Fmoc protected
amino acid (1.6 mmol) was coupled to Wang resin preloaded
with the necessary carboxyl-terminal amino acid (0.63 mmol) in
DMF (5 resin volumes) using HOBt (1.6 mmol) and DIC
(1.6 mmol). Couplings were performed twice for 30 min and
verified by a negative Kaiser test on a sample of a few beads.
Final acetylation was performed by shaking the resin for 15 min
with a mixture of acetic anhydride and triethylamine in DMF
(2.5/0.75/25, 10 resin volumes). Acetylation was verified by a
negative Kaiser test on a sample of a few beads.34 The final
peptide was cleaved from the resin (1 g) by incubating with
TFA–DCM (1 : 1) over 2 h. The peptide was precipitated from
the cleavage solution using diethyl ether and hexane, and the
crude peptide was purified by preparative HPLC, using a
Synergi Polar-RP, 100 × 21.20 mm column (Phenomenex,
Torrance, CA) on a Varian (Prep Star) HPLC system.
2-Azidoethylamine (25). A solution of 200 mg (1.1 mmol) of
carbamate 24 in 3 mL MeOH was treated with 4 mL of 1 M HCl
and stirred at room temperature for 7 h. The volume was reduced
by rotary evaporation, diluted with 3 mL of water and freeze-
dried overnight, giving quantitatively 131.6 mg (1.1 mmol) of
Enzyme purification. Recombinant guinea pig liver transglu-
taminase (TG2) was expressed and purified according to a litera-
ture procedure.35 In brief, N-terminal His-tagged guinea pig liver
transglutaminase was co-expressed with the DnaK/DnaJ chaper-
one protein36 from co-transformed XL1-Blue E. coli cells,
induced overnight in the presence of 1 mM IPTG at 28 °C. After
lysis of the bacterial cells by sonication, the overexpressed His-
tagged TG2 was purified by affinity chromatography on Ni-NTA
agarose, followed by desalting.
1
25 as its hydrochloride salt. H-NMR (CD3OD, 400 MHz) δ:
3.14 (br t, 2H), 3.75 (br t, J = 5.65 Hz, 2H). 13C-NMR (CD3OD,
100 MHz) δ: 39.4, 48.9. HRMS m/z (M + H)+ calcd for
C2H7N4: 87.0665; found: 87.0662.
5-(3-(2-Azidoethyl)thioureido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-
benzoic acid (31). A solution of fluorescein isothiocyanate
(300 mg, 0.77 mmol) in 30 mL of MeOH was treated with 1 mL
of Et3N and 131 mg of 2-azidoethyl amine (25, 1.07 mmol),
stirred overnight, and evaporated to a residue that was purified
by silica gel chromatography using ethyl acetate–methanol–
acetic acid 15 : 1 : 0.1 as eluant. Evaporation of the collected
fractions gave 230 mg (0.48 mmol) of azide 26 (63% yield).
1H-NMR (CD3OD, 400 MHz) δ: 3.53 (t, J = 5.77, 2H), 3.75
(t, J = 5.77, 2H), 6.47–6.64 (m, 6H), 7.10 (d, J = 8.24, 1H), 7.70
(dd, J = 8.24, J = 1.90, 1H), 8.10 (d, J = 1.90, 1H). 13C-NMR
(CD3OD, 100 MHz) δ: 44.2, 50.6, 103.0, 110.9, 113.1, 119.7,
125.2, 128.5, 129.8, 131.6, 141.6, 149.2, 153.6, 161.0, 170.6,
174.8, 182.8. HRMS m/z (M + H+) calcd for C23H18N5O5S:
476.1016; found: 476.10232.
Methods
Ligation activity assay and competition screen. In a 96-well
microtiter plate, each well was charged with 10 μL of 0.6 mM
fluorescein amide substrate 1, 10 μL of 0.6 mM coumarin amine
substrate 2, and 80 μL buffer (200 mM Tris HCl (pH 8), 10 mM
CaCl2, 1.2 mM EDTA), and treated subsequently with either
10 or 20 μL of a 100 mM stock solution of the amine candidate
(6–23), followed by 90 or 80 μL of water to give final concen-
trations of 5 or 10 mM, respectively, in a final volume at 200 μL.
In control runs, 100 μL water was added instead of the amine
solution. Reactions were initiated by adding 20 μL of a
3 U mL−1 stock solution of purified TG2. In control experi-
ments, the enzyme storage buffer [25 mM Tris acetate (pH 7)]
was used instead of TG2 solution. The time-dependent decrease
in fluorescence was followed using a 465 nm emission filter.
Synthesis of peptide substrate
Ac-PNPQLPF. The peptide Ac-PNPQLPF was synthesized
using standard solid-phase Fmoc chemistry.33 Briefly, the first
Fmoc protected amino acid (5 mmol) was coupled to Wang resin
(1.1 mmol) using DIC (5 mmol) and DMAP (0.1 mmol) in
DMF (5 resin volumes). The level of loading of the amino acid
on the resin after the first coupling step was determined by
spectroscopic measurement of the UV absorbance of the piper-
idine dibenzofulvalene adduct formed during Fmoc deprotection.
This loading level was used as the resin loading capacity for all
subsequent steps. The remaining free hydroxyl functionalities
were capped by shaking the resin for 2 h in a mixture of acetic
anhydride–pyridine (2 : 3). After washing with DMF (three times
with 10 resin volumes), DCM (three times with 10 resin
volumes), and ether (three times with 10 resin volumes), the
Fmoc group was removed by incubating with piperidine in DMF
(20% v/v; 10 resin volumes) for 1 h, followed by washing with
DMF (three times with 10 resin volumes), DCM (three times
with 10 resin volumes), and ether (three times with 10 resin
volumes) in preparation for the next amide coupling. Deprotec-
tion was qualitatively verified by a positive Kaiser test on a
Peptide propargylation. A 1.5 mL Eppendorf tube was
charged with 10 μL of a 66 U mL−1 TG2 solution, 5 μL of a pro-
pargyl amine stock solution (100 mM in buffer), 50 μL of a solu-
tion of peptide Ac-PNPQLPF (11.7 mM in DMF) and 935 μL of
buffer (200 mM MOPS (pH 7), 0.1 M CaCl2, 20 mM EDTA).
After 2 h at 37 °C, to the mixture was centrifuged over an
Amicon Centrifugal Filter Unit (30 kDa MWCO) at 4 °C for
30 min at 1962g. The filtrate was collected and characterized by
LC-MS, whereby a new peak was detected, whose mass was
892.4, corresponding to the expected product [Ac-PNPQ(N-pro-
pargyl)LPF]+.
Protein labelling. To 1 mL of a 12 mg mL−1 (0.5 mM) solu-
tion of dimethyl casein in buffer (100 mM Tris HCl (pH 8),
5 mM CaCl2), 5 μL of 100 mM propargyl amine and 40 μL of
18 mg mL−1 purified TG2 were added and the reaction was
5264 | Org. Biomol. Chem., 2012, 10, 5258–5265
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14 M. Jäger, E. Nir and S. Weiss, Protein Sci., 2006, 15, 640–646.
mixed gently for 2 h at room temperature. As a negative control
the same reaction was prepared without adding TG2. The TG2
and control reaction mixtures were treated with 4 μL of 50 mM
CuCl2, 20 μL of 50 mM ascorbic acid, 10 μL of 50 mM FITC-
azide 26, and 12 μL of 50 mM 1,2-phenylene diamine, and
mixed gently overnight at 4 °C. The reaction products were sub-
sequently analysed by 10% SDS–PAGE, separating the two
casein proteins present in the commercial product. The fluore-
scence of these bands was recorded prior to Coomassie staining.
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