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. Author manuscript; available in PMC: 2021 May 20.
Published in final edited form as: Bioconjug Chem. 2020 Apr 23;31(5):1463–1473. doi: 10.1021/acs.bioconjchem.0c00156

Table 1.

Effect of Substrate and Nucleophile on MA-SML.a

graphic file with name nihms-1591711-t0009.jpg
Entry Substrate (~GGXX) Nucleophile (GnX) % Ligation (+Ni2+) % Ligation (−Ni2+)
1 ~GGHG-OH G-OH b 5.9 ± 0.1 1.8 ± 0.1
2 ~GGHG-OH G-NH2 68 ± 0.4 43 ± 1.8
3 ~GGHG-OH GG-OH 77 ± 1.8 38 ± 1.0
4 ~GGHG-OH GG-NH2 74 ± 5.8 42 ± 0.7
5 ~GGHG-OH GGG-OH 69 ± 0.5 39 ± 0.1
6 ~GGHG-OH GGG-NH2 64 ± 1.6 41 ± 0.7
7 ~GGH-OH GG-NH2 65 ± 0.8 44 ± 0.6
8 ~GGH-NH2 GG-NH2 81 ± 1.2 45 ± 2.6
9 ~GGH-NH2 c GG-NH2 65 ± 4.4 44 ± 0.5
10 ~GGG-NH2 c GG-NH2 46 ± 0.9 46 ± 0.2
11 ~GGS-NH2 c GG-NH2 45 ± 0.1 45 ± 0.3
12 ~GGD-NH2 c GGG-NH2 44 ± 1.0 43 ± 1.1
a

Unless otherwise indicated, reaction conditions were 100 μM substrate, 100 μM nucleophile, 10 μM SrtAstaph, 0/200 μM NiSO4, sortase reaction buffer (pH 7.5), glycerol (0.2% v/v), 6 h at room temperature. Substrates and nucleophiles contained either native carboxylic acids (−OH) or primary amides (−NH2) at their C-termini. Estimates of reaction progress were obtained by comparing RP-HPLC peak areas for the unreacted substrate peptide, ligation product, and hydrolysis by-product. All data points represent three independent experiments (mean ± standard deviation).

b

Data for the G-OH nucleophile reported for the 17 h timepoint.

c

Conditions: 50 μM substrate, 50 μM nucleophile, 10 μM SrtAstaph, 0/200 μM NiSO4, sortase reaction buffer (pH 7.5), glycerol (0.2% v/v), 3 h at room temperature.