Data for the synthesis of oligo-γ-glutamylglutamines as model compounds for γ-glutamyltransferases (GGTs) and for normalization of activities of different GGTs

γ-Glutamyltransferases (GGTs) are widespread, conserved enzymes that catalyze the transfer of the γ-glutamyl moiety from a donor substrate to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a γ-glutamyl enzyme intermediate. Although the vast majority of the known GGTs has a short sequence called lid-loop covering the glutamate binding site, Bacillus subtilis GGT and some other enzymes from Bacillus spp. lack the lid loop. In order to assess the possible role of the lid loop of GGTs in substrate selection, synthetic oligo-γ-glutamylglutamines containing up to three γ-glutamyl residues were used as model substrates. The activities of the enzymes under investigation were standardized with respect to a common reaction to ensure comparable results. The activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from Escherichia coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT (Calvio et al., 2018) [1]. Here we report the experimental procedures for the synthesis of model substrates γ-glutamylglutamines through the method of the N-phtaloyl-L-glutamic acid anhydride and the spectral data of the synthetized compounds. The data obtained in the normalization procedure of the activities of the three enzymes are also reported.

method of the N-phtaloyl-L-glutamic acid anhydride and the spectral data of the synthetized compounds. The data obtained in the normalization procedure of the activities of the three enzymes are also reported

Value of the data
The synthesis of oligo-γ-glutamylglutamines is described. Spectroscopic data of the synthetized compounds are reported. The procedure for normalization of activities of different GGT enzymes is described.

Data
The general procedure for the preparation of the model compounds: with the synthetic plan (Scheme 1) and the spectroscopic data of the compounds 1-3 are reported. The standard test used for measure the activity of GGTs is summarized in Scheme 2. Normalization of enzyme's activities, required to obtain comparable data when evaluating different GGT enzymes towards the same substrate [1], is reported in Fig. 1.

Experimental design, materials and methods
2.1. Synthesis of the oligomers of γ-glutamylglutamines 1-3

General
All reagents were from Sigma Aldrich and were used as received. DMF was distilled prior to use by standard methods. N-phtaloyl-L-glutamic acid anhydride was synthesized starting from N-phtaloyl-L-glutamic acid as previously reported [2].
TLC was carried out on silica gel F 254 aluminum sheets using nBuOH-AcOH-water 4: 1: 1 as the eluent. TLC plates were inspected under an UV lamp (254 nm) and stained with 0.5% ninhydrin solution in ethanol and heating at 150°C ca. Dowex 1 Â 8 ion exchange resin 200-400 mesh (Aldrich) in the acetate form was used for ion exchange column chromatography. 1 H and 13 C NMR spectra were acquired at 400.13 and 100.61 MHz, respectively, using a Bruker Avance 400 spectrometer interfaced with a workstation running Windows Operating System equipped with TopSpin Software package. Chemical shifts are given in ppm (δ) and are referenced to the signals of 3-(trimethylsilyl)propionic acid-2,2,3,3-d4 as external standard. Signal multiplicities in the 13 C spectra were assigned on the basis of APT (attached proton test) experiments. Spectra analyses were carried out using inmr (www.inmr.net). Mass spectra were acquired with a ThermoFinnigan LCQ Advantage mass spectrometer.
2.1.2. General procedure for the synthesis of the oligomers of γ-glutamylglutamines 1-3 1.05 eq of the compound to be γ-glutamylated was suspended in dry DMF (2 mL/mmol of starting material) in a two-necked round-bottom flask equipped with magnetic stirrer under a stream of nitrogen. N-phtaloyl-L-glutamic acid anhydride was added in one portion. The mixture was stirred at 21°C until disappearance of the starting material (24-72 h), monitoring the progress of the reaction by TLC. The reaction mixture was diluted with water (10% of the amount of DMF) and 3-6 eq. hydrazine hydrate, depending on the number of carboxyl groups in the product molecule, was added under vigorous stirring. Within 3 h a white, sticky solid precipitated. Ethanol was then added (2 mL/mmol of expected product) and stirring was continued at 0°C for further 30 min in order to favor complete precipitation. The solid was collected by filtration on a sintered glass septum and washed in succession with cold ethanol, ethyl acetate and diethyl ether. The solid was then dried in a desiccator under reduced pressure without removing it from the septum.
The hydrazinium salt of the product was dissolved with small portions of water, leaving on the septum most of the phtalylhydrazide. The pH of the aqueous solution was adjusted to ca 9.5 with 1 M NaOH and charged onto a column of Dowex 1 Â 8 ion exchange resin in the acetate form. The column was eluted with water until disappearance of hydrazine in the eluate (tested with 2% 4dimethylaminobenzaldehyde in 10% HCl solution) and then with a scalar gradient of acetic acid (0.5, 1.0, 2.0, 3.0 M, three column volumes each). Eluate was collected in fractions; fractions containing the product were combined on the basis of TLC analysis and freeze-dried.

γ-glutamylglutamine 1
Obtained from glutamine and N-phtaloyl-L-glutamic acid anhydride in 23% yield after 72 h reaction time as a white, amorphous solid.

Enzymes standardization
Enzyme activity assays were carried out using γ-glutamyl-p-nitroanilide 0.25 mM and glycylglycine 2 mM in 0.1 M NaHCO 3 solution, pH 8.2. Reactions were initiated by adding 20 μL of an arbitrarily diluted enzyme solution. Final volume was 2 mL. Absorbance was measured at λ ¼ 410 nm for 10 min, recording data every min. On the basis of the measured activities (Fig. 1A), stock enzymes' solutions were differentially diluted in order to have the same amount of enzyme units and activities were again verified through the same assay (Fig. 1B).