Patricia Isabel Stewart, Una Lim, Alex Eddenden, Mark Nitz. “Labelling of glycans using carbohydrate acetyltransferases via simple thioesters.” ChemRxiv (). https://doi.org/10.26434/chemrxiv.15007712/v1

S-(4-methylphenyl) 2-azidoethanethioate
Identifier AzTE
NMR reporting reference CD₂Cl₂: 5.32 ppm for ¹H NMR and 53.84 ppm for ¹³C NMR
¹H NMR (400 MHz, CD₂Cl₂) δ 7.34 – 7.30 (m, 2H), 7.29 – 7.25 (m, 2H), 4.12 (s, 2H), 2.40 (s, 3H)
¹³C NMR (126 MHz, CD₂Cl₂) δ 194.7, 140.9, 135.1, 130.6, 123.0, 58.3, 21.5
DART-MS m/z [M+H]⁺ calcd. for C₉H₉N₃OS 208.05, found 208.1
SI, pp. S2–S3
S-(cyanomethyl) ethanethioate
Identifier AcTE
NMR reporting reference CDCl₃: 7.26 ppm for ¹H NMR and 77.16 ppm for ¹³C NMR
¹H NMR (500 MHz, CDCl₃) δ 3.67 (s, 1H), 2.43 (s, 2H)
¹³C NMR (126 MHz, CDCl₃) δ 191.8, 115.8, 30.1, 14.6
DART-MS m/z [M+H]⁺ calcd. for C₄H₅NOS 116.01, found 116.0
SI, pp. S2–S3
S-(cyanomethyl) pent-4-ynethioate
Identifier AlkTE
NMR reporting reference CDCl₃: 7.26 ppm for ¹H NMR and 77.16 ppm for ¹³C NMR
¹H NMR (400 MHz, CDCl₃) δ 3.71 (s, 2H), 2.88 (t, J = 7.2 Hz, 2H), 2.58 (td, J = 7.2, 2.7 Hz, 2H), 2.02 (t, J = 2.7 Hz, 1H)
¹³C NMR (101 MHz, CDCl₃) δ 193.6, 115.7, 81.2, 70.1, 42.2, 14.4
DART-MS m/z [M+H]⁺ calcd. for C₇H₇NOS 154.02, found 154.0
SI, pp. S2–S4
2-(cyanomethyl)-1,1,3,3-tetramethylisothiouronium bromide
NMR reporting reference CDCl₃: 7.26 ppm for ¹H NMR and 77.16 ppm for ¹³C NMR
¹H NMR (500 MHz, CDCl₃) δ 4.28 (s, 1H), 3.25 (s, 6H)
¹³C NMR (101 MHz, CDCl₃) δ 171.9, 117.1, 44.2, 19.4
SI, pp. S2, S4
Thiol-thioester exchange of CoASH with AcTE, AzTE, and AlkTE
NMR reporting reference D₂O: 4.79 for ¹H NMR
¹H NMR (600 MHz, D₂O) CoASH (1 mM) and each thioester (1 mM) in triethylammonium bicarbonate buffer (pH 8.0, 50 mM) for one hour at 37 °C. Disappearance of the methylene peak from CoASH (2.61 ppm) along with the corresponding appearance of triplets (AcCoA: 2.96 ppm; AzCoA: 3.05 ppm; AlkCoA: 3.02 ppm)
SI, pp. S2, S4, S15
Thiol-thioester exchange of CoASH with AcTE, AzTE, and AlkTE
¹H NMR The methylene protons adjacent to the CoA thiol appeared at 2.60 ppm as a triplet, while a triplet downfield at 2.97 has been assigned to the methylene proximal to the thioester AcCoA. The disappearance of the 2.60 ppm CoA peak was diagnostic for acylation
Main, p. 6
CoASH, AcCoA, AlkCoA, and AzCoA from thiol-thioester exchange
ESI-MS Detection of the expected [M+H]⁺ ions of AcCoA, azidoacetyl Coenzyme A (AzCoA), and alkynyl Coenzyme A (AlkCoA) respectively, as well as the [M+Li]⁺ ions which are due to the commercial CoA trilithium salt
Main, pp. 6–7
MalE-NeuO constructs
Size-exclusion chromatography, conditions Chromatograms were obtained by loading protein aliquots (1 mg/mL) in dialysis buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl) onto ENrich SEC650 column using an NGC Chromatography system (Bio-Rad)
Size-exclusion chromatography MalE-NeuO constructs exclusively forms trimers. MalE-NeuO eluted at approximately the same elution volume as IgG (150 kDa), which corresponds to that of the functional trimer (~200 kDa) as opposed to the monomer (~70 kDa)
SI, p. S18
EndoN-treated colominic acids without or with NeuO M134A
ESI-MS (negative mode), sample 2 mg/mL colominic acids, 0.5 mM AzCoA, ± 20 µg/mL NeuO M134A, in PBS for 1 h at 37 °C, followed by treatment with EndoN neuraminidase (10 µg/mL EndoN, in PBS for 2 h at 37 °C)
ESI-MS (negative mode), without NeuO M134A The major signals were consistent with sialic acid oligomers (DP 3–7)
ESI-MS (negative mode), with NeuO M134A A new set of signals appeared, corresponding to the single and double azide modifications