Solution-phase synthesis of the fluorogenic TGase 2 acyl donor Z-Glu(HMC)-Gly-OH and its use for inhibitor and amine substrate characterisation

Article abstract of DOI:10.1016/j.ab.2020.113612

A reliable solution-phase synthesis of the water-soluble dipeptidic fluorogenic transglutaminase substrate Z-Glu(HMC)-Gly-OH is presented. The route started from Z-Glu-OH, which was converted into the corresponding cyclic anhydride. This building block was transformed into the regioisomeric α- and γ-dipeptides. The key step was the esterification of Z-Glu-Gly-OtBu with 4-methylumbelliferone. The final substrate compound was obtained in an acceptable yield and excellent purity without the need of purification by RP-HPLC. The advantage of this acyl donor substrate for the kinetic characterisation of inhibitors and amine-type acyl acceptor substrates is demonstrated by evaluating commercially available or literature-known irreversible inhibitors and the biogenic amines serotonin, histamine and dopamine, respectively.

Full text of DOI:10.1016/j.ab.2020.113612

Journal Pre-proof
Solution-phase synthesis of the fluorogenic TGase 2 acyl donor Z-Glu(HMC)-Gly-OH
and its use for inhibitor and amine substrate characterisation
Robert Wodtke, Markus Pietsch, Reik Löser
PII:
S0003-2697(19)31241-2
https://doi.org/10.1016/j.ab.2020.113612
YABIO 113612
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 12 December 2019
Revised Date: 3 February 2020
Accepted Date: 3 February 2020
Please cite this article as: R. Wodtke, M. Pietsch, R. Löser, Solution-phase synthesis of the fluorogenic
TGase 2 acyl donor Z-Glu(HMC)-Gly-OH and its use for inhibitor and amine substrate characterisation,
Analytical Biochemistry (2020), doi: https://doi.org/10.1016/j.ab.2020.113612.
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Author contribution
RW: Conceptualization, Investigation, Methodology, Formal Analysis, Writing- Reviewing and Editing,
Funding Acquisition; MP: Conceptualization, Formal Analysis, Writing- Reviewing and Editing, Funding
Acquisition, Supervision; RL: Conceptualization, Investigation, Formal Analysis, Writing- Original draft
preparation, Funding Acquisition, Supervision.

Z-Glu-OH
reliable synthesis
in solution
Transglutaminase 2
Z-Glu(HMC)-Gly-OH
H2O
or
RNH2
kinetic characterization of
amine substrates and
inhibitors enabled by
fluorogenic substrate
inhibitors

1
2
3
Solution-Phase Synthesis of the Fluorogenic TGase 2
Acyl Donor Z-Glu(HMC)-Gly-OH and its Use for
Inhibitor and Amine Substrate Characterisation
4
Robert Wodtke 1*, Markus Pietsch 2 and Reik Löser 1,3
*
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1
2
3
Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research; Bautzner
Landstrasse 400, 01328 Dresden, Germany
7
Institute II of Pharmacology, Centre of Pharmacology, Medical Faculty, University of Cologne, Gleueler
Strasse 24, 50931 Cologne, Germany
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Faculty of Chemistry and Food Chemistry, School of Science, Technische Universität Dresden,
Mommsenstraße 4, 01062 Dresden, Germany
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Abstract: A reliable solution-phase synthesis of the water-soluble dipeptidic fluorogenic
transglutaminase substrate Z-Glu(HMC)-Gly-OH is presented. The route started from Z-Glu-OH,
which was converted into the corresponding cyclic anhydride. This building block was
transformed into the regioisomeric α- and γ-dipeptides. The key step was the esterification of
Z-Glu-Gly-OtBu with 4-methylumbelliferone. The final substrate compound was obtained in an
acceptable yield and excellent purity without the need of purification by RP-HPLC. The advantage
of this acyl donor substrate for the kinetic characterisation of inhibitors and amine-type acyl
acceptor substrates is demonstrated by evaluating commercially available or literature-known
irreversible inhibitors and the biogenic amines serotonin, histamine and dopamine, respectively.
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Keywords: fluorogenic enzyme substrates; side-chain esterified peptides; aryl esters; peptide
synthesis; enzyme kinetics; biogenic amines
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1. Introduction
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Transglutaminase 2 is a multifunctional enzyme whose distinct physiological roles are
incompletely understood [1, 2]. Its eponymous and best-characterised function represents the
catalysis of the acyl transfer between glutamine residues and a variety of primary amines [3]. To
assess the significance of this enzymatic activity in cells, substrates (especially low molecular weight
polyamines) [4, 5] and inhibitors [6] were applied amongst other biochemical techniques [7, 8]. The
prerequisite for an accurate interpretation of potential effects mediated by those TGase 2-targeting
*Correspondence: r.wodtke@hzdr.de, r.loeser@hzdr.de ; Tel.: +49-351-260-3658
Abbreviations
d…doublet, DIPEA…N,N-diisopropylethylamine, EDTA…N,N’-ethylenediamine tetraacetic acid, GDH…glutamate
dehydrogenase,
hTGase 2…human
TGase 2;
HMC…7-hydroxy-4-methylcoumarin
(=4-methylumbelliferone),
HATU…(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, HPLC…high
performance liquid chromatography, Hsp…heat shock protein, LC…liquid chromatography, m…multiplet, MES…
2-(N-morpholino)ethanesulfonic acid, MOPS…(3-(N-morpholino)propanesulfonic acid), MS…mass spectrometry,
NMM…N-methylmorpholine,
NMR…nuclear
magnetic
resonance,
Np…4-nitrophenyl,
PyBOP…benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, q…quadruplet, RP…reversed phase,
s…singlet, t…triplet, TCEP… tris(2-carboxyethyl)phosphine, TLC…thin layer chromatography, UPLC…Ultra Performance
Liquid Chromatography, Z…benzyloxycarbonyl (=carbobenzoxy, Cbz); all other abbreviations are in accordance to the
recommendations of the IUPAC-IUB commission on biochemical nomenclature, see Eur. J. Biochem. 15 (1970) 203-208 and
Eur. J. Biochem. 138 (1984) 9-37

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molecules is the knowledge of their kinetic properties towards the isolated protein and also towards
the other members of the transglutaminase family. The other main function of TGase 2 is dealing
with the binding of GDP and GTP and catalysis of hydrolysis of the latter [9]. Both functions are
mutually exclusive as they are associated with distinct conformational states of the enzyme molecule
[10]. Therefore, occupation of the guanine nucleotide binding site of TGase 2 can be analysed via
detection of its acyltransferase activity [11, 12], which indicates the value of robust assay methods
that allow for the monitoring of this activity [13]. A rigorous kinetic assessment of the enzyme’s
acyltransferase activity is enabled by the dipeptidic coumarinyl ester Z-Glu(HMC)-Gly-OH as
fluorogenic substrate. In addition to its suitable kinetic and spectral properties, the compound’s
utility is owed to its propitious water solubility [14]. Favourably, this compound can be also used for
the assay of other transglutaminase isoforms [15]. The synthesis of peptides containing glutamate
coumarinyl esters was achieved by solid-phase peptide synthesis using the chlorotrityl linker for
attachment to the solid support. The ester moiety in the glutamate side-chain was installed after
deblocking the allyl ester-protected γ-carboxylic acid function prior to acidic cleavage from the resin
[14]. Although this synthesis strategy is advantageous for obtaining various peptidic substrates, the
product scale is limited by the use of the costly polymeric resin and the need of preparative
RP-HPLC for purification. Efficient solution-phase methods have been described by Chung et al. [16]
and Leblanc et al. [17] for the synthesis of the corresponding chromogenic dipeptidic substrate
Z-Glu(ONp)-Gly-OH, which bears a nitrophenyl group attached to the identical peptidic scaffold.
This pathway was adopted by us for an efficient access to Z-Glu(HMC)-Gly-OH, which will be
reported herein together with its kinetic evaluation towards TGase 2-catalysed hydrolysis. In this
context, it was envisaged to re-prove the fluorogenic substrate’s previously established value for
TGase 2 inhibitor and substrate characterisation exemplarily for commercially available irreversible
inhibitors for which robust second-order inactivation constants kinact/K have not been reported so far.
I
Furthermore, kinetic parameters for selected physiologically relevant amine substrates were
determined by taking advantage of the title compound.
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2. Materials and Methods
2.1. General
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All starting materials, reagents and solvents were commercially obtained and used without
further purification. Compound 5, N-monodansylpiperazine and N-acryloxysuccinimide were
synthesised as previously published [15]. Chromatographic separations and TLC detections were
carried out with Merck Silica Gel 60 (63–200 μm) and Merck Silica Gel 60 F254 sheets, respectively.
TLCs were visualised under UV light (λ = 254 nm or 365 nm). Analytical RP-HPLC was carried out
with a system consisting of a Merck Hitachi L7100 gradient pump combined with a Jasco DG2080
four-line degasser with UV detection with a Merck Hitachi L7450 diode array detector. The system
was operated with D-700 HSM software and use of a Merck Hitachi D7000 interface. A Luna C18 5
μm column (Phenomenex, 250×4.6 mm) served as stationary phase. A binary gradient system of
0.1% CF
3
COOH/water (solvent A) and 0.1% CF
3
COOH/CH CN (solvent B) at a flow rate of 1
3
mL/min served as the eluent. The following elution programme was run to separate the
components: 0–3 min 80% A, 3–45 min gradient to 70% B, 45–46 min gradient to 95% B, 46–50 min
95% B, 50–55 min gradient back to 80% A, 55–60 min 80% A. For purification of compound 7,
preparative HPLC were performed on a Varian Prepstar system equipped with a UV detector
(Prostar, Varian). A Microsorb C18 60−8 column (Varian Dynamax 250 mm × 21.4 mm) was used as
the stationary phase. The same binary gradient system as for the analytical RP-HPLC was applied
using an appropriate gradient from low to high percentage of B with a slope of 1% per min.
Melting points were determined on a Galen III Boetius apparatus from Cambridge Instruments.
NMR spectra were recorded on a Varian Unity 400 MHz or an Agilent DD2 600 MHz spectrometer
equipped with ProbeOne probe. Chemical shifts of the 1H and 13C spectra were reported in parts per
million (ppm) referenced to the (residual) solvent resonances relative to tetramethylsilane.

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UPLC-MS was performed on a Waters ACQUITY UPLC I-Class system including an ACQUITY
UPLC PDA eλ setector coupled to a Xevo TQ-S mass spectrometer High-resolution mass spectra
were obtained on an Agilent UHD Accurate-Mass Q-TOF LC MS G6538A mass spectrometer
operated in combination with coupled with the Agilent 1260 Infinity II HPLC system. Elemental
microanalysis was performed on a Euro EA 3000 Elemental Analyzer (Euro Vector Instruments &
Software).
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2.2. Benzyl (S)-(2,6-dioxotetrahydro-2H-pyran-3-yl)carbamate (1, N-Carbobenzoxyglutamic anhydride)
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95
96
97
Z-Glu-OH (2.5 g, 8.89 mmol) was suspended in acetic anhydride (20 mL) and the temperature
was slowly raised to 55 °C (oil bath temperature), whereupon a clear solution was formed. From the
time of complete dissolution, stirring was continued for additional 10 min. The solution was
concentrated under high vacuum (rotary evaporator equipped with hybrid vacuum pump) to obtain a
nearly colourless oil. Ether (30 mL) was added to the crude product and ethyl acetate (5-10 mL) was
added until a homogeneous solution was obtained upon heating. The solution was transferred to an
Erlenmeyer flask and cyclohexane (15-20 mL) was added until the solution became turbid. The
mixture was kept at -20 °C over night, whereupon an oil separated. Crystallisation was initiated by
scratching with a glass rod. More cyclohexane (10 mL) was added and the mixture was kept again at
-20 °C over night. The crystalline material was collected by vacuum filtration and dried in vacuo to
obtain 1 (2.1 g, 0.80 mmol, 90%) as a white powder; Mp 82-86 °C (lit 86-88 °C [18]); 1H NMR (400 MHz,
98
99
2
3
100
101
102
103
104
105
106
CDCl
2J=18.9 Hz, 3J=13.0 Hz, 3J=6.2 Hz, 1H, Cγ-HH), 3.06 (ddd, 2J=18.7 Hz, 3J=5.6 Hz, 3J=2.2 Hz, 1H, Cγ-HH),
4.38-4.52 (m, 1H, Cα-H), 5.15 (s, 2H, CH
O), 5.57 (br s, 1H, NH), 7.30-7.42 (m, 5H, Ph-H); 13C NMR (100
MHz, CDCl ), δ (ppm): 23.72 (Cβ), 29.83 (Cγ), 51.48 (Cα), 67.77 (CH O), 128.39, 128.64, 128.80, 135.80,
(Carom), 155.81, OCONH), 164.53, 166.65 (2×C=O anhydride). NMR data are in agreement to those
3
), δ (ppm): 1.94 (qd, J=13.1 Hz, J=5.6 Hz, 1H, Cβ-HH), 2.45-2.59 (m, 1H, Cβ-HH), 2.90 (ddd,
2
3
2
reported in [17]. Elemental analysis C13
58.33%, H: 5.15%, N: 5.16%.
H
13NO , calcd. C: 59.31%, H: 4.98%, N: 5.32%, found: C:
5
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2.3. (S)-4-(((Benzyloxy)carbonyl)amino)-5-((2-(tert-butoxy)-2-oxoethyl)amino)-5-oxopentanoic acid (2,
Z-Glu-Gly-OtBu)
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To a solution of compound 1 (0.5 g, 1.90 mmol) in chloroform (20 mL) was added glycine
tert-butyl ester hydrochloride (H-Gly-OtBu×HCl; 0.32 g, 1.90 mmol) as a solid followed by triethyl
amine (0.19 g, 0.26 mL, 1.90 mmol). The resulting solution was stirred for 30 min at room temperature.
The solvent was removed in vacuo and the obtained crude product mixture was adsorbed on silica gel
and loaded onto a silica column. Product 2 was obtained by elution with ether/ethyl acetate/acetic acid
1:1:0.01. As the UV absorption of both products is rather low, visualisation of compound spots during
monitoring of the chromatographic purification by TLC analysis of distinct fractions was done by
using Hanessian’s stain [19]. The product-containing fraction were combined and brought to dryness
in vacuo. The obtained oily residue was crystallised by dissolving in a minimum amount of ethyl
acetate and subsequent addition of a larger volume of cyclohexane. The oil which separated from the
solvent mixture solidified at -20 °C. The solid was collected by vacuum filtration to obtain 373 mg (50
%) of compound 2. R
[20]); 1H NMR (400 MHz, CDCl
f
0.35 (n-hexane/ethyl acetate/acetic acid 1:4:0.01); Mp 96-103 °C (lit 106- 107 °C
), δ (ppm): 1.45 (s, 9H, (CH C), 1.88 – 2.01 (m, 1H, Cβ-HH), 2.09 – 2.20
), 3.83 – 4.00 (m, 2H, GlyCα-H ), 4.41 – 4.49 (m, 1H, GluCα-H),
3
3)3
(m, 1H, Cβ-HH), 2.41 – 2.59 (m, 2H, Cγ-H
2
2
3
5.05-5.17 (m, 2H, CH
2
O), 5.88 (d, J=8.6 Hz, 1H, Glu-NH), 7.10-7.16 (br s, 1H, Gly-NH), 7.27-7.39 (m,
5H, Ph-H); 13C NMR (100 MHz, CDCl
53.85 (GluCα), 67.44 (CH
O), 82.75 ((CH
169.06, 171.93, 176.71 (3×C=O). NMR data are in agreement to those reported in [17]. Elemental
analysis C19 : calcd. C: 57.86%, H: 6.64%, N: 7.10%, found: C: 58.97%, H: 6.79%, N: 6.94%.
3
), δ (ppm): 28.16 (3×CH
3
), 28.28 (Cβ), 29.91 (Cγ) 42.25 (GlyCα),
2
3)3C), 128.20, 128.37, 128.68, 136.14 (4×Carom), 156.70 (OCONH),
H26
N
2
O7
128
2.4. N2-((Benzyloxy)carbonyl)-N5-(2-(tert-butoxy)-2-oxoethyl)-L-glutamine (2a)

Wodtke et al.
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Compound 2a was obtained by further elution of the chromatographic column described above
with ether/ethyl acetate/acetic acid 1:4:0.01. The product-containing fractions were combined and
brought to dryness in vacuo. The obtained oily residue was washed with cyclohexane under
ultrasonification to remove residual acetic acid, which was followed by washing with n-pentane. The
oil was dried under very low pressure (< 0.1 mbar) to obtain 223 mg (30%) of compound 2a as a white
foam. R
9H, (CH
3.96 (m, 2H, GlyCα-H
6.77 (t, 3J=4.9 Hz, 1H, Gly-NH), 7.27 – 7.35 (m, 5H, Ph-H), 13C NMR (100 MHz, CDCl
(3×CH O), 82.91 ((CH C), 128.19, 128.31,
), 28.50 (Cβ), 32.13 (Cγ), 42.35 (GlyCα), 53.48 (GluCα), 67.24 (CH
128.65, 136.28 (4×Carom), 156.61 (OCONH), 169.39, 173.61, 174.34 (3×C=O). Elemental analysis
: calcd. C: 57.86%, H: 6.64%, N: 7.10%, found: C: 57.36%, H: 6.74%, N: 6.58%.
f
0.17 (n-hexane/ethyl acetate/acetic acid 1:4:0.01); 1H NMR (400 MHz, CDCl
C), 1.99 – 2.09 (m, 1H, Cβ-HH), 2.15 – 2.27 (m, Cβ-HH, 1H), 2.30 – 2.49 (m, 2H, Cγ-H
), 4.32 – 4.43 (m, 1H, GluCα-H), 5.08 (s, 2H, CH O), 6.02 (d, 3J=7.5 Hz, 1H, Glu-NH),
) δ (ppm): 28.12
3
) δ (ppm): 1.45 (s,
3)3
2), 3.84 –
2
2
3
3
2
3)3
C
19
H26
N
2
O7
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2.5. 4-Methyl-2-oxo-2H-chromen-7-yl
(S)-4-(((benzyloxy)carbonyl)amino)-5-((2-(tert-butoxy)-2-oxoethyl)amino)-5-oxopentanoate (3,
Z-Glu(HMC)-Gly-OtBu)
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Compound 2 (0.373 g, 0.95 mmol) and HATU (0.361 g, 0.95 mmol) were dissolved in DMF (3 mL)
under a N
0.95 mmol) in DMF (1 mL) were added to this mixture. The resulting yellow solution was stirred for 2
h at room temperature. Subsequently, the solution was diluted with CH Cl (15 mL), transferred to a
separatory funnel and washed with 1 M HCl (1×3 mL), sat. NaHCO (2×3 mL), H O (1×3 mL) and brine
(1×1 mL). The organic layer was dried over Na SO and evaporated in vacuo (the remaining DMF was
2
atmosphere. DIPEA (282 μL, 1.88 mmol) and a solution of 4-methylumbelliferone (0.168 g,
2
2
3
2
2
4
removed using a rotary evaporator equipped with hybrid pump for very low pressure) to obtain a
yellow viscous oil. The crude product was purified by column chromatography on silica gel using a
mixture of n-hexane and ethyl acetate of increasing polarity (ratio starting from 6:4 over 1:1 to 4:6; the
final ratio was adjusted 20 fractions after unconverted 4-methylumbelliferone eluted). The
product-containing fractions were collected and evaporated in vacuo to obtain 243 mg of compound 3
as an off-white solid. As the product still contained 4-methylumbelliferone as trace impurity, the
material was recrystallised by adding ethyl acetate (3.2 mL) to a refluxing suspension of the product in
cyclohexane (4 mL). Upon keeping the solution at 4 °C overnight, a precipitate was formed, which was
collected by vacuum filtration, washed with cyclohexane and n-hexane and dried in vacuo to obtain
compound 3 (134 mg, 26%) as a white granulous solid. R
°C; 1H NMR (400 MHz, CDCl
), δ (ppm): 1.47 (s, 9H, (CH
1H, Cβ-HH), 2.43 (d, 4J = 1.3 Hz, 3H, CH
f
0.16 (n-hexane/ethyl acetate 1:1); Mp 61-63
3
3)3
C), 2.02 – 2.14 (m, 1H, Cβ-HH), 2.26-2.38 (m,
3
-coumarin), 2.67-2.90 (m, 2H, Cγ-H
), 3.90 (dd, 2J=18.2 Hz, 3J=5.1
2
Hz, 1H, Cα-HH of Gly), 3.98 (dd, 2J=18.3 Hz, 3J=5.4 Hz, 1H, Cα-HH of Gly), 4.37 – 4.44 (m, 1H, Cα-H of
Glu) 5.11 (d, 2J=12.3 Hz, 1H, CHHO), 5.15 (d, 2J=12.9 Hz, 1H, CHHO), 5.58 (d, 3J=8.0 Hz, 1H, Glu-NH),
6.27 (q, 4J=1.3 Hz, 1H, H-3 of coumarin), 6.51 (br s, 1H, Gly-NH) 7.07 – 7.11 (m, 1H, H-6 of coumarin),
4
3
7.13 (d, J=1.8 Hz, 1H, H-8 of coumarin), 7.28 – 7.38 (m, 5H, Ph-H), 7.57 (d, J=8.6 Hz, 1H, H-5 of
coumarin). 13C NMR (100 MHz, CDCl
), δ (ppm): 18.88 (CH of coumarin), 28.20 (3×CH ), 28.30 (Cβ),
30.52 (Cγ), 42.18 (Cα of Gly), 53.92 (Cα of Glu), 67.43 (CH O), 82.75 ((CH C), 110.65 (C8 of coumarin),
3
3
3
2
3)3
114.76 (C3 of coumarin), 118.09, 118.27 (C4a and C6 of coumarin), 125.53 (C5 of coumarin), 128.30,
128.42, 128.73 (3×CH of phenyl), 136.18 (C1 of phenyl), 151.99, 153.10, 154.34 (C4, C7 and C8a of
coumarin), 156.37 (OCONH), 160.61 (C2 of coumarin), 168.62, 171.00, 171.54 (3×C=O). HR-MS (ESI+):
[M+NH
4
]+ calculated: 570.2451, found: 570.2445 (90%), [2M+NH
4
]+ calculated: 1122.4559, found:
1122.4565 (100%). Elemental analysis C29
62.48%, H: 5.82%, N: 4.73%.
H32
N
2
O9
: calcd. C: 63.04%, H: 5.84%, N: 5.07%, found: C:
175
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2.6. (S)-(2-(((Benzyloxy)carbonyl)amino)-5-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)-5-oxopentanoyl)glycine
(4, Z-Glu(HMC)-Gly-OH)
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178
To a solution of compound 3 (130 mg, 0.24 mmol) in CH
2
Cl (0.5 mL) was added slowly
2
trifluoroacetic acid (4 mL) under ice cooling. After stirring for 2 h under ice cooling the solvents were

Wodtke et al.
removed under a N
5 of 17
Cl (20
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2
stream under ambient pressure. The oily residue was taken up into CH
2
2
mL) and washed with 0.1 M HCl (5 mL). As addition of HCl resulted in the formation of an emulsion,
brine (3 mL) was added, whereupon a white precipitate was formed, which was filtered off and
redissolved in ethyl acetate (15 mL). The aqueous layer was separated from the combined biphasic
mixture and the organic layer was dried over Na
2
SO
4
and evaporated in vacuo to obtain 112 mg of a
1
white solid. Since an impurity was detectable in the H NMR spectrum, the crude product was
recrystallised. To this end, the solid was dissolved in a mixture of ethyl acetate (4 mL) and acetonitrile
(2 mL) under heating. Cyclohexane (6 mL) was added and the resulting solution was kept at 4 °C for
30 min, whereupon a gelatinous precipitate formed. The material was collected by vacuum filtration,
washed with cyclohexane and n-pentane and dried in vacuo to obtain compound 4 (77 mg, 65%) as a
1
white solid. R
f
0.01 (n-hexane/ethyl acetate 2:8); Mp 167-171 °C; H NMR (400 MHz, DMSO-d
6) δ
(ppm): 1.88 – 1.98 (m, 1H, Cβ-HH), 2.02 – 2.14 (m, 1H, Cβ-HH), 2.44 (d, 4J=1.3 Hz, 3H, CH
3
-coumarin),
2
3
2
3
2.68 – 2.75 (m, 2H, Cγ-H
2
), 3.73 (dd, J=17.5, J=5.6 Hz, 1H, Cα-HH of Gly), 3.83 (dd, J=17.5, J=6.0 Hz,
1H, Cα-HH of Gly), 4.15-4.21 (m, 1H, Cα-H of Glu), 5.02 (d, 2J=12.6 Hz, 1H, CHHO), 5.07 (d, 2J=12.6 Hz,
1H, CHHO), 6.39 (d, 4J=1.3 Hz, 1H, H-3 of coumarin), 7.18 (dd, 3J=8.7, 2.3 Hz, 1H, H-6 of coumarin), 7.26
(d, 4J=2.3 Hz, 1H, H-8 of coumarin), 7.28 – 7.39 (m, 5H, Ph-H), 7.54 (d, 3J=8.3 Hz, 1H, Glu-NH), 7.81 (d,
3J=8.7 Hz, 1H, H-5 of coumarin), 8.29 (t, 3J=5.8 Hz, 1H, Gly-NH). 13C NMR (100 MHz, CDCl
18.11 (CH of coumarin), 26.98 (Cβ), 30.00 (Cγ), 40.66 (Cα of Gly), 53.49 (Cα of Glu), 65.51 (CH
3
), δ (ppm):
O),
3
2
110.04 (C8 of coumarin), 113.69 (C3 of coumarin), 117.47 (C4a of coumarin), 118.34 (C6 of coumarin),
126.32 (C5 of coumarin), 127.66, 127.74, 128.27 (3×CH of phenyl), 136.88 (C1 of phenyl), 152.82,
152.90, 153.48 (C4, C7 and C8a of coumarin), 155.92 (OCONH), 159.55 (C2 of coumarin), 170.82,
171.03, 171.51 (3×C=O). NMR data are in agreement to those reported in [14]. HR-MS (ESI+): m/z
calculated for [M+NH
1010.3313 (57.2%), [3M+NH
O calcd. C: 58.36%, H: 5.09%, N: 5.45%, found: C: 58.36%, H: 4.92%, N: 5.23%.
4
]+: 514.1825, found: 514.1821 (100%), [2M+NH
4
]+: calculated: 1010.3307, found:
]+: calculated: 1506.4789, found: 1506.4799 (80.52%). Elemental analysis:
4
C
25
H24
N
2
O9
H2
Chromatograms for UPLC-MS analysis of Z-Glu(HMC)-Gly-OH (4) are shown in Figure S2
(Supporting Information).
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208
2.7. tert-Butyl (2-(4-((5-(dimethylamino)naphthalen-1-yl)sulfonyl)piperazin-1-yl)-2-oxoethyl)carbamate (6a,
N-Boc-glycine-4-dansylpiperazide)
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226
To a solution of Boc-glycine (98.8 mg, 0.56 mmol) and DIPEA (196.5 μL, 0.12 mmol) in DMF
(5 mL) was added PyBOP (293 mg, 0.56 mmol). The solution was stirred for 5 min at room
temperature and, subsequently, N-monodansylpiperazine (90 mg, 0.28 mmol) was added. After
stirring for 3 h, the solvent of the reaction mixture was removed in vacuo and the obtained residue
was dissolved in ethyl acetate (15 mL). The organic phase was washed with sat. NaHCO
and brine (1×10 mL), dried over Na SO and evaporated to dryness. The obtained crude product was
subjected to column chromatography (silica; eluent: petroleum ether/ethyl acetate (25 ‰ 50%)) to
3
(2×10 mL)
2
4
obtain 6a (133 mg, 100%) as a light-green solid; 1H NMR (400 MHz, CDCl
Boc), 2.90 (s, 6H, 2×CH of dansyl), 3.23–3.15 (m, 4H, 2×CH of piperazine), 3.45–3.39 (m, 2H, CH
piperazine), 3.69–3.62 (m, 2H, CH
7.21 (d, 3J=7.5 Hz, 1H, HDansyl), 7.55 (dd, 3J=8.6, 7.6 Hz, 2H, 2×HDansyl), 8.20 (dd, 3J=7.3 Hz, 4J=1.2 Hz, 1H,
3
): δ=1.41 (s, 9H, 3×CH
3
of
of
3
2
2
3
2
of piperazine), 3.86 (d, J=4.5 Hz, 2H, CH Gly), 5.36 (br s, NH),
2
3
3
H
Dansyl), 8.37 (d, J=8.7 Hz, 1H, HDansyl), 8.59 (d, J=8.5 Hz, 1H, HDansyl); 13C NMR (100 MHz, CDCl
δ=28.45 (3×CH Boc), 41.68 (CH of piperazine), 42.28 (CH of Gly), 44.24 (CH of piperazine), 45.48
(CH of piperazine), 45.63 (CH of piperazine), 45.65 (2×CH dansyl), 80.03 (Cquartär of Boc), 115.67,
3
):
3
2
2
2
2
2
3
119.65, 123.45, 128.45, 130.16 (Cquart of dansyl), 130.44 (Cquart of dansyl), 131.03, 131.16, 132.30 (Cquart of
1
dansyl), 151.68 (Cquart of dansyl), 155.87 (CO of Boc), 167.07; MS (ESI+) 477.3 ([M+H]+). H and 13C
NMR spectra in agreement to published data [21].
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228
2.8. 2-Amino-1-(4-((5-(dimethylamino)naphthalen-1-yl)sulfonyl)piperazin-1-yl)ethan-1-one (6b,
Glycine-4-dansylpiperazide)

Wodtke et al.
Compound 6a (167 mg, 0.35 mmol) was dissolved in a mixture of TFA and CH
6 of 17
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2
Cl
2
(10 mL; 1:1,
v/v) and stirred for 2 h at room temperature. The volatiles were removed at ambient pressure in a
nitrogen stream. The residue was dissolved in sat. NaHCO (20 mL) and extracted with CH Cl
(5×5 mL). The organic phase was dried over Na SO and subsequently evaporated to dryness to
obtain 6b (131 mg, 100%) as a light-green solid; 1H NMR (400 MHz, DMSO-d
): δ=2.83 (s, 6H, 2×CH
of dansyl), 3.12–3.05 (m, 4H, 2×CH of piperazine), 3.53–3.20 (m, 6H, 2×CH of piperazine, CH Gly),
7.27 (d, 3J=7.5 Hz, 1H, HDansyl), 7.70–7.59 (m, 2H, 2×HDansyl), 8.13 (dd, 3J=7.4 Hz, 4J=1.2 Hz, 1H, HDansyl),
8.30 (d, 3J=8.7 Hz, 1H, HDansyl), 8.53 (d, 3J=8.5 Hz, 1H, HDansyl), signal for NH not detectable; 13C NMR
(100 MHz, DMSO-d ): δ=40.89 (CH of piperazine), 42.53 (CH Gly), 43.39 (CH of piperazine), 45.04
(2×CH of dansyl), 45.37 (2×CH of piperazine), 115.33, 118.86, 123.70, 129.20 (Cquart of dansyl), 129.62
3
2
2
2
4
6
3
2
2
2
2
6
2
2
2
3
2
(Cquart of dansyl), 130.06, 130.39, 132.48 (Cquart of dansyl), 151.42 (Cquart of dansyl), 171.55 (CO); MS
(ESI+) 377.2 ([M+H]+) 1H and 13C NMR spectra are in agreement to published data [21].
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243
2.9. N-(2-(4-((5-(Dimethylamino)naphthalen-1-yl)sulfonyl)piperazin-1-yl)-2-oxoethyl)acrylamide (7,
N-Acryloylglycine-4-dansylpiperazide)
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To a solution of compound 6b (112 mg, 0.30 mmol) and TEA (82.5 μL, 0,60 mmol) in CH
2
Cl
2
(10 mL) was added N-acryloxysuccinimide (50.3 mg, 0,30 mmol) as solid. After 1 h, the same amount
of N-acryloxysuccinimide was added again and the mixture was stirred for additional 1 h at room
temperature. The solution was washed with sat. NaHCO
Na SO and evaporated to dryness. The crude product was purified by preparative RP-HPLC to
obtain 7 (10.9 mg, 7%) as a white solid; 1H NMR (400 MHz, CDCl
): δ=3.01 (s, 6H, 2×CH of dansyl),
3
(2×10 mL), brine (1×10 mL); dried over
2
4
3
3
3.27–3.18 (m, 4H, 2×CH
2
of piperazine), 3.52–3.45 (m, 2H, CH
2
of piperazine), 3.72–3.66 (m, 2H, CH
2
3
3
2
of piperazine), 4.08 (d, J=4.2 Hz, 2H, CH
2
Gly), 5.68 (dd, J=10.2 Hz, J=1.4 Hz, 1H, CH=CHH), 8.62
(d, 3J=8.6 Hz, 1H, HDansyl), 6.15 (dd, 3J=17.0, 10.2 Hz, 1H, CH=CH
CH=CHH), 6.74 (bs, 1H, N
H of Gly), 7.31 (d, 3J=7.6 Hz, 1H, HDansyl), 7.64–7.56 (m, 2H, 2×HDansyl), 8.24
(dd, 3J=7.4 Hz, 4J=1.1 Hz, 1H, HDansyl), 8.48 (d, 3J=8.7 Hz, 1H, HDansyl), 8.62 (d, 3J=8.6 Hz, 1H, HDansyl); 13C
NMR (100 MHz, CDCl ): δ=41.38 (CH of Gly), 41.86 (CH of piperazine), 44.36 (CH of piperazine),
45.41 (CH of piperazine), 45.59 (CH of piperazine), 45.84 (2×CH
127.71 (CH=CH ), 128.30, 129.57 (Cquart of dansyl), 130.01 (CH=CH
2
), 6.28 (dd, 3J=17.0 Hz, 2J=1.4 Hz, 1H,
α
3
2
2
2
2
2
3
of dansyl), 116.22, 121.07, 124.15,
2
2
), 130.43 (Cquart of dansyl), 130.57,
1
131.26, 132.59 (Cquart of dansyl), 149.58 (Cquart of dansyl), 165.92, 166.56. H and 13C NMR spectra in
agreement to published data [21].
260
2.10. General assay procedure and analysis
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All measurements were conducted at 30 °C over 900 s (interval of 8 or 10 s) using a Cytation 5
Microplate Reader (BioTek Instruments) and black 96-well microplates with μCLEAR® bottom
(greiner bio-one). Fluorescence was detected in bottom read mode and predefined settings for
excitation (360±20 nm) and emission (450±20 nm) of 7-HMC were chosen. Measurements at pH=8.0
and pH=6.5 were conducted with a sensitivity of 45 and 50, respectively. The assay mixture (200 μL)
contained 190 μL aqueous solution and 10 μL DMSO (5% v/v). The following three buffer systems
were used: assay buffer I (100 mM MES, 3 mM CaCl
NaOH), assay buffer II (100 mM MOPS, 3 mM CaCl
NaOH) and enzyme buffer (100 mM MOPS, 3 mM CaCl
2
, 50 μM EDTA, adjusted to pH 6.5 with 1 M
, 50 μM EDTA, adjusted to pH 8.0 with 1 M
, 10 mM TCEP, 20% (v/v) glycerol). The
2
2
buffers were stored at 0 °C for periods of up to 2 weeks and freshly prepared after that period. The
concentration of the hTGase 2 stock solution was 1 mg/mL. All regression analyses were
accomplished using GraphPad Prism (version 8.2.1, August 20, 2019). To provide values of mean
and SEM, the respective regression analyses were separately accomplished for each experiment and
the obtained fit values were collected and statistically analysed. hTGase 2 (T022) and inhibitors Z006
and Z013 were purchased from Zedira (Darmstadt, Germany).
Detailed descriptions of the assay procedures and the kinetic analyses for the characterisation of
Z-Glu(HMC)-Gly-OH (4) towards enzymatic hydrolysis at pH 6.5 and 8.0 as well as for the kinetic
characterisation of amines and irreversible inhibitors are given in recent publications of our group

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[14, 15]. In brief, the recorded time courses of type (RFU-RFU
0
)=f(t) for the enzymatic and
spontaneous conversion were analysed by nonlinear (one-phase association) and linear regressions
to the experimental data. Subsequently, initial rates v0total (in the presence of enzyme) and v0control
(spontaneous reaction in the absence of enzyme) were derived. Both data sets were globally
analysed by applying the model of total and nonspecific binding (implemented in GraphPad Prism)
to obtain the kinetic parameters for the enzymatic reaction. According to that model, the following
rule was defined:
286
vꢀꢁꢂꢁꢃꢄ(enzymatic + spontaneous) = vꢀꢅꢂꢆꢆ(enzymatic) + vꢀꢅꢂꢇꢁꢆꢂꢄ(spontaneous)
(1)
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289
where v0corr represents the rates for the enzymatic conversions (without spontaneous reaction). The
spontaneous and enzymatic conversions are mathematically defined within this model by the
following equations:
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291
vꢀꢅꢂꢇꢁꢆꢂꢄ = ꢈꢂꢉꢊ ∗ [S]
(2)
(3)
ꢋ
ꢌꢍꢎ
∗[ꢏ]
vꢀꢅꢂꢆꢆ
=
ꢐ
ꢑ[ꢏ]
ꢌ
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293
Accordingly, v0total is defined by the sum function of equations (2) and (3). Due to negligible
spontaneous reaction of 4 at pH 6.5, the above mentioned rule simplifies as follows:
294
vꢀꢁꢂꢁꢃꢄ = vꢀꢅꢂꢆꢆ
(4)
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302
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305
However, concerning the kinetic analyses the previously published procedures were slightly
adjusted, as detailed below.
Concerning the enzymatic hydrolysis of Z-Glu(HMC)-Gly-OH (4) at pH 6.5, the recorded time
courses of type (RFU-RFU )=f(t) were analysed by either nonlinear (one-phase association) or linear
0
regression over the entire measurement period of 900 s (instead of only the first 300 s) to the
experimental data depending on the shape of the curve.
Concerning the kinetic characterisation of the biogenic amines, the two sets of initial rates
(v0total=f([amine]) and v0control=f([amine])) were also globally analysed using the model of total and
nonspecific binding (instead of simple subtraction of v0control from vototal values) as conducted for the
analysis of the enzymatic hydrolyes).
306
3. Results and Discussion
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The four-step synthesis started from commercially available Z-Glu-OH and the first two steps
followed mainly the procedures published by Leblanc et al. [17] (Scheme 1). Treatment of Z-Glu-OH
with acetic anhydride, which acts both as solvent and condensation agent, at elevated temperature
led to the corresponding cyclic anhydride 1. The oily crude product obtained by concentration of the
reaction mixture could be crystallised by treatment with ether, ethyl acetate and cyclohexane (see
Material and Methods section), which resulted in an almost quantitative yield of 90%. From a
historical point of view, it is worth to mention that glutamic anhydride 1 has been used as building
block for the synthesis of glutamate-containing peptides when peptide chemistry was still in its
infancy [22]. Compound 1 was subjected to ring opening with glycine tert-butyl ester, which resulted
in quantitative consumption of the anhydride within 30 min under the formation of the
regioisomeric α- and γ-dipeptides 2 and 2a. The ratio of both isomers was determined to be 7:3 in the
favour to the desired product 2 by HPLC analysis (see Figure S1, Supporting Information). As
reported by Leblanc et al., both products were conveniently separated by chromatography on silica
gel. By-product 2a was characterised and could – in its Nα-Fmoc-protected version – be a useful
building block for solid-phase peptide synthesis as it represents an extended glutamate analogue.
Dipeptide Z-Glu-Gly-OtBu (2) was esterified with the fluorophore 4-methylumbelliferone by
applying conditions that have been elaborated by Twibanire and Grindley for the efficient acylation

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of alcohols of varying reactivity using HATU as activating agent [23], which provided compound 3.
Column chromatographic purification of the crude product yielded the product, which contained an
unknown impurity according to the 1H NMR spectrum beside trace amounts of unreacted
4-methylumbelliferone. As these impurities would be more difficult to remove after the final step
considering the higher polarity of the free carboxylic acid, the obtained material was recrystallised to
furnish highly pure compound 3. In the final step, tert-butyl ester 3 was cleaved by treatment with
trifluoroacetic acid. During work-up, excessive TFA was efficiently removed by acidic aqueous
washing to obtain the crude product in crystalline state. This material was recrystallised form ethyl
acetate/acetonitrile/cyclohexane to obtain compound 4 as monohydrate, according to results of
elementary microanalysis. Its purity has been confirmed by RP-HPLC/MS analysis.
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Scheme 1: Four-step synthesis of Z-Glu(HMC)-Gly-OH (4) starting from Z-Glu-OH. Reagents and conditions:
a) acetic anhydride, 55 °C, 10 min; b) H-Gly-OtBu×HCl, triethylamine, CHCl
3, room temperature; c) HATU,
DIPEA, 4-methylumbelliferone, DMF, room temperature; d) TFA, CH Cl , room temperature.
2
2
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342
343
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Compound 4 was investigated regarding the kinetics of hTGase 2-catalysed hydrolysis both at
pH 6.5 and pH 8.0. The results are shown in Figure 1 and Table 1 (see also Figure S19 in Supporting
Information). The obtained catalytic and Michaelis constants are in good agreement with the
recently published values [14]. The fact that the determined K values are slightly lower than those
m
recently published can be attributed to the better-defined composition of the crystalline compound
material obtained herein whereas previously obtained substrate preparations furnished amorphous
and slightly hygroscopic material after lyophilisation.

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Figure 1: hTGase 2-catalysed hydrolysis of Z-Glu(HMC)-Gly-OH (4) at pH=8.0 and pH 6.5. Plots of v0corr=f([4])
for pH 8.0 and pH 6.5 with the nonlinear regressions (―) using Equation (3) (Michaelis-Menten equation,
Materials and Methods section). Data shown are mean values ±SEM of 3 separate experiments, each performed
in duplicate. When not apparent, error bars are smaller than the symbols. Conditions: pH=8.0 or 6.5, 30 °C, 5%
DMSO, 500 μM TCEP, 3 μg/ml of hTGase 2.
353
Table 1: Kinetic parameters for the hTGase 2-catalysed hydrolyses of Z-Glu(HMC)-Gly-OH (4) at pH 6.5 and 8.0
pH value
6.5
Km (µM)
4.05 (0.23)
3.93 (0.48)
kcat (s-1)
0.30 (0.01)
0.61 (0.03)
kcat/Km (M-1s-1)
74,100
8.0
155,000
354
355
Rate constant for spontaneous hydrolysis: kobs (TCEP, pH 8.0) = 16.8 (0.5) × 10-3 min-1
For details on calculation of the kinetic parameters see Materials and Methods Section. Data shown are mean
356
357
values (±SEM) of three separate experiments, each performed in duplicate. Active concentrations of hTGase 2
(E
T=30.8 nM) were determined by active site titration as recently described [24].
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Following the hTGase 2-catalysed hydrolysis at pH 6.5, the utility of substrate 4 for the kinetic
characterisation of irreversible inhibitors was demonstrated/validated by investigating three
inhibitors which were reported in the literature. The selected compounds comprised the
commercially available peptidic inhibitors Z006 and Z013, which bear a diazomethyl ketone or an
α,β-unsaturated carboxylic ester as electrophilic warhead, respectively. Furthermore, the
nonpeptidic acrylamide 1-155 (compound 7), which was recently published by Badarau et al. [21,
25], was synthesised according to Scheme 2 in orientation to the described procedure. In contrast to
the former two compounds, a second-order inactivation constant kinact/K
I
has been reported for the
latter. The results of the inhibitor characterisation are included in Table 2.
Table 2: Second-order inactivation constants (kinact/K
I
) and K values of different literature-known irreversible
i
369
inhibitors of hTGase 2 at pH 6.5 compared to their reported inhibitory parameters
compound
structure
kinact/KI (M-1s-1)a* Ki (µM)a*
IC50 (nM)

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310[15]b
14 (8.2)[26]c
4,880 (20)[15]
5.73 (0.77)[15]
5
20[27]c
Z006
191,000 (3,000)
0.096 (0.01)
Z013
51,500 (5,800)
0.45 (0,02)
1.43 (0,01)
200[28]d
12,700 (700)
4,962[21]f
6.1 (0.4)[21]e
1-155 (7)
aData shown are mean values (±SEM) of two separate experiments, each performed in duplicate.
bTGase 2-catalysed incorporation of R-I-Cad in DMC (transamidation) at pH 8.0 (5 min preincubation period
of enzyme and inhibitor). cTGase 2-catalysed incorporation of Boc-K-NH(CH
NH-Dns in DMC
2)2
(transamidation) at pH 7.4 (30 min preincubation period of enzyme and inhibitor). dIsopeptidase-assay using
the substrate Abz-NE(CAD-DNP)EQVSPLTLLK-OH (A101, Zedira). eTGase 2-catalysed incorporation of
N-(biotinyl)cadaverine in immobilised DMC (transamidation) at pH 8.5 (30 min preincubation period of
enzyme and inhibitor. fGDH-coupled assay (deamidation) at pH 7.2.
*
KI=(koff+kinact)/kon, Ki=koff/kon
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377
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381
382
383
384
385
386
387
388
389
390
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393
Schaertl et al. have determined an IC50 value of 20 nM for Z006 together with various other
compounds by employing
a
fluorescence-based transamidase-detecting assay using
N,N-dimethylcasein as acyl donor and N-monodansylcadaverin as acyl acceptor substrate,
respectively [27]. In a later study, Nε-acryloyllysine-based irreversible inhibitor 5 has been
synthesised, which was investigated in the same assay [26]. The IC50 value of 5 was 14 nM under
these conditions, which suggests that it should be equipotent to Z006. However, the kinact/K value of
I
Z006 determined herein (191 000 M-1s-1) is 37-fold greater than that of 5. Even though the applied
assay methods detect different TGase 2-catalysed reactions (transamidation in Schaertl et al. vs.
hydrolysis herein) and the pH values are different (6.5 vs. 7.4), the potencies of Z006 and 5 should
not be influenced to such extent. In agreement with the value of kinact/K determined for Z006,
I
Khosla’s group reported inactivation constants of 48 000 M-1s-1 [29] and 139 000 M-1s-1 [30] for
structurally related peptidic diazomethyl ketones, which have been determined using the
GDH-coupled assay. The higher reactivity of Z006 towards TGase 2 in comparison to 5 is reasonable,
as diazomethyl ketones are inherently more reactive towards thiols than acrylamides. Moreover, as
the peptidic structures of Z006 and Z013 are based on gliadin peptides, which are natural substrates
of TGase 2, they can probably be better accommodated by the active site, which in turn results in
stronger non-covalent interactions. This is reflected in the low K value of 96 nM for Z006. The similar
i
IC50 values of Z006 and 5 and the drastically differing inactivation constants can be rationalised
when the assay conditions are considered. The IC50 values were determined using a TGase 2
concentration of 20 nM and a preincubation time of 30 min [27]. Therefore, the IC50 limit, which
corresponds to half of the employed active enzyme concentration ([E]
reached for both compounds. In this context, it should be pointed out that the IC50 value of an
irreversible inhibitor in general will always be equal to [E] /2, provided that [inhibitor] ≥ [enzyme]/2
T
/2) [31, 32], might have been
T

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and enough incubation time is given. The other investigated peptidic inhibitor Z013 exhibits a
of 51 500 M-1s-1, which is significantly more potent than 5. As for Z006, the higher inhibitory
k
inact/K
I
potency of Z013 can be attributed to the more reactive electrophilic warhead and the peptidic
structure, which corresponds to that of Z006, apart from one amino acid side chain. An IC50 value of
200 nM has been reported for Z013; however, no information on enzyme concentration and
preincubation time were provided [28]. Again, the comparison to the kinetic parameter kinact/K
I
indicates that IC50 values are not suitable for evaluating the potency of irreversible inhibitors, unless
identical conditions and carefully adjusted preincubation times are used [33].
402
403
Scheme 2: Synthesis of inhibitor 1-155
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
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427
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430
431
432
433
434
435
436
437
438
439
440
Reagents and conditions: a) PyBOP, DIPEA, DMF, 3 h; b) TFA/CH
2
Cl
2
(1:1, v/v), 2 h; c) TEA, CH
2
Cl , 2 h.
2
The synthesis of 1-155 (compound 7) started with the amide bond coupling between
Boc-Gly-OH and N-monodansylpiperazine to 6a using PyBOP and DIPEA, in difference to the
published procedure, which used EDC, HOBt and NMM as coupling reagents (Scheme 2) [21]. After
TFA-mediated Boc cleavage the acryloyl moiety was introduced by reacting 6b with
N-acryloxysuccinimide. The last step proceeded in low yield and should better be performed with
acryloyl chloride as acylating agent. A kinact/K
of 12 700 M-1s-1 was determined for 1-155. This value is
I
approximately 2.5-fold higher than both that for the Nε-acryloyllysine 5 and that of 1-155 determined
by Badarau et al. using the GDH-coupled assay. However, taking into account that the two assay
methods work at different pH values (6.5 vs. 7.2) the results can be considered comparable. The
higher efficiency of 1-155 compared to 5 as determined in the fluorimetric assay is remarkable. This
is probably the result of well-balanced conformational flexibility/rigidity and targeting of interaction
partners in the enzymes binding site for 1-155. Considering its smaller size (30 vs. 35 non-hydrogen
atoms), binding of 1-155 might be entropically more favoured than that of 5.
In addition to the kinetic characterisation of inhibitors, a further asset of the fluorogenic
substrate 4 is the convenient investigation of amine-based acyl acceptor substrates, which are
interesting for the development of TGase 2-directed imaging agents [34]. Moreover, their substrate
properties are of general interest. Therefore, we decided to determine exemplarily the kinetic
parameters of histamine, serotonin and dopamine as common biogenic amines of the primary
arylethylamine type. These compounds are known as classical neurotransmitters and the former two
are important mediators in non-neuronal cells and tissues as well. They have been known for 60
years to be substrates of TGase 2 [35]. However, the physiological significance of TGase-mediated
aminylation as an important post-translational modification has been recognised only in the recent
decades [36-39]. In particular, the TGase 2-catalysed serotonylation of small GTPases such as RhoA
and Rab4 in thrombocytes has been shown to be critical for blood haemostasis. Worth of note,
serotonylation of Gln63 in RhoA renders its GTPase activity permanently active [40]. A similar
mechanism has been identified for the constitutive activation of Rac1 in the central nervous system
[41, 42]. In particular, activation of the Gq/11-coupled 5-HT2A receptors in rat cortical cells raises the
cytosolic Ca2+ level, which in turn activates TGase 2 for catalysing the serotonylation of Rac1 [42].
Apart from small GTPases, other proteins have been identified as substrates for TGase 2–mediated
serotonylation [43] and, most recently, the transamidation of Gln5 of histone H3 to serotonin has
been discovered as a transcription-permissive modification [44]. Similar to serotonylation, TGase
2-mediated histaminylation leads to the constitutive activation of small and heterotrimeric G
proteins [45]. Histamine transfer to extracellular proteins such as fibrinogen has been suggested as a
mechanism for attenuating the pro-inflammatory effects of this endogenous mediator [46].
Furthermore, fibronectin, another extracellular TGase 2 substrate, has been shown to undergo

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monoaminylation, including dopaminylation [47, 48]. Despite this overwhelming body of evidence
for the biological relevance of TGase 2-catalysed protein acyl transfer to biogenic monoamines, exact
kinetic data on their substrate properties are scarce or have not been determined at all.
444
445
446
447
448
449
450
451
Figure 2. hTGase 2-catalysed incorporation of different biogenic amines into compound Z-Glu(HMC)-Gly-OH
(4). Plots of v0corr/[hTGase 2]=f([amine]) with nonlinear regressions (―) using Equation (3) (Michaelis-Menten
equation, Materials and Methods section). Data shown are mean values ±SEM of two separate experiments,
each performed in duplicate. When not apparent, error bars are smaller than the symbols. Conditions: pH=8.0
(100 mM MOPS was used, which ensures that the pH value is maintained over the entire range of amine
concentrations), 30 °C, 5% DMSO, 100 μM Z-Glu(HMC)-Gly-OH (4), 500 μM TCEP, 0.6, 2 and 3 μg/mL of
hTGase 2 for histamine, dopamine and serotonin, respectively.
452
Table 3. Kinetic parameters of different biogenic amines as acyl acceptors for hTGase 2 at pH=8.0
ꢔꢕꢕ
ꢗꢔꢘ
ꢒꢔꢓꢕꢕ (mM)
0.08 (0.01)
0.96 (0.06)
0.50 (0.17)
ꢖ
(s-1)
biogenic amine
histamine
kcat/Km (M-1s-1)
66,900
5.35 (0.58)
2.90 (0.04)
1.35 (0.04)
dopamine
3,000
serotonin
2,700
453
454
455
456
For details on calculation of the kinetic parameters see Experimental section. Data shown are mean values
(±SEM) of two separate experiments, each performed in duplicate. Active concentrations of hTGase 2
(ET=6.15 nM for histamine, 20.5 nM for dopamine and 30.8 nM for serotonin) were determined by active site
titration as recently described [24].
457
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466
The results on the kinetic characterisation of histamine, serotonin and dopamine are shown in
Figure 2 and the calculated parameters are included in Table 3 (see also Figure S20 in Supporting
Information). The substrate concentration range and the employed enzyme concentration were
adjusted for each substrate in preliminary tests. Notably, no substrate inhibition occurred for any of
the amine substrates. The formation of the corresponding secondary amide products upon TGase
2-catalysed substrate conversion was confirmed by LC-MS/MS analysis (see Figures S21-S26 in
Supporting Information). In agreement with previously published data using the GDH-coupled
assay and the gluten-derived heptapeptide Ac-PQPQLPF-NH as acyl donor, histamine is a very
2
efficient acyl acceptor substrate for TGase 2 [49]. In contrast, the second-order performance constants
for both serotonin and dopamine are approximately 20-fold lower. In the GDH-assay mentioned

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before, which employs TGase 2 at a concentration as high as 50 μg/mL (640 nM), conversion of
serotonin was not detectable. This enzyme concentration is approximately 20-fold higher than that
applied herein for the analysis of serotonin. This demonstrates the value of activated fluorogenic
acyl donors such as compound 4 as even poor acyl acceptor substrates can be analysed and the
consumption of valuable enzyme material is minimised. According to its kcat/K , dopamine is a
m
slightly more efficient amine substrate for TGase 2 than serotonin, even though the apparent
Michaelis constant is lower for serotonin. Considering that all three studied biogenic amines share
the 2-aminoethyl chain and an (hetero)aromatic core, the distinct substrate properties of histamine
are remarkable. One could argue that its imidazole ring could assist in the deacylation step of the
catalytic cycle by acting as general base in the proton transfer from the amine nitrogen, as
intramolecular hydrogen bonds have been identified in both the neutral and side-chain protonated
form of histamine [50]. Thus, the imidazole ring of the substrate would fulfil the same function
during attack of the neutral amino group on the thioester intermediate as the imidazole of active-site
His335 [51]. However, its K
app of 80 μM, which is significantly lower than the corresponding values
m
for dopamine and serotonin, suggests specific interactions within the enzyme’s active site.
Furthermore, it is surprising that serotonin is the least efficient substrate of the investigated amines
even though the most findings on physiologically relevant protein monoaminylation have been
obtained for this biogenic amine [52]. In this context, it is worth to consider that beside availability of
the particular monoamine substrate, which can vary from cell type and depends on the subcellular
localisation, the sequence in which the Gln substrate residue is embedded should also influence the
substrate specificity towards the amine-based acyl acceptor substrate. This is evidenced by
investigations on the sequence dependence of TGase 2-catalysed modification of Hsp20, which
occurs at two of its five Gln residues, Gln31 and Gln66. Interestingly, concerning the differentiation
between the acyl acceptor the outcome is different for both residues as Gln31 is exclusively
transamidated whereas Gln66 is exclusively deamidated. However, when the isolated
undecapeptides of the sequences in which both residues are embedded are considered, Gln31 is not
converted at all whereas Gln66 undergoes both transamidation and deamidation [53]. This indicates
that the results on the substrate properties of the biogenic monoamines obtained with
Z-Glu(HMC)-Gly-OH (4) as acyl donor substrate should be extrapolated with care towards large
protein substrates. Therefore, we would like to define the elucidation of the molecular mechanisms
that lead to the coupling between acyl donor and acyl acceptor substrate specificity as a future
challenge for TGase research. In this context, the influence of the acceptor nucleophile on the
mechanism of the acylation step in TGase 2 catalysis has been established very recently on the basis
of 14N/15N kinetic isotope effects [54].
502
4. Conclusions
503
504
505
506
507
508
A reliable solution phase synthesis of the title substrate compound 4 has been established,
which will support its use in transglutaminase research. Its value for the evaluation of TGase 2
inhibitors based on meaningful kinetic parameters has been demonstrated. The compound’s utility
for the characterisation of amine substrates as exemplified by the biogenic amines histamine,
serotonin and dopamine has been shown, which allows for the first time their comparison on a
robust kinetic basis.
509
510
511
Supporting Information: Supplementary data associated with this article (chromatograms for synthesis, NMR
spectra for compounds 1-4, kinetic data, identification of reaction products for biogenic amines) can be found in
the online version at doi:
512
513
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515
Acknowledgments: We highly appreciate the skilful assistance of Ms. Peggy Nehring and Mr. Kay Fischer in
substrate synthesis and that of Mr. David Bauer in that of inhibitor 1-155. Dr. Markus Laube and Ms. Karin
Landrock are kindly acknowledged for the measurement of HR-MS spectra and elemental microanalyses,
respectively.
516

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Highlights
•
Reliable and scalable solution-phase synthesis of the fluorogenic acyl donor substrate Z-
Glu(HMC)-Gly-OH was established
•
•
Substrate properties towards TGase 2-catalysed hydrolysis were confirmed
Suitability of the substrate for the kinetic characterization of irreversible inhibitors that were
recently reported in the literature is demonstrated
•
Kinetic investigation of histamine, serotonin and dopamine as acyl acceptors using Z-
Glu(HMC)-Gly-OH allows for the first time the comparison of their substrate properties
towards TGase 2