The influence of the carrier molecule on amoxicillin recognition by specific IgE in patients with immediate hypersensitivity reactions to betalactams

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1 Supporting Information The influence of the carrier molecule on amoxicillin recognition by specific IgE in patients with immediate hypersensitivity reactions to betalactams Ariza A, Mayorga C,2, Salas M 2, Doña I 2, Martín-Serrano A,3, Pérez-Inestrosa E 3,4, Pérez-Sala D 5, Guzmán AE 6, Montañez MI,3, Torres MJ 2. Research Laboratory, IBIMA Regional University Hospital of Malaga UMA, Málaga, Spain. 2 Allergy Unit, IBIMA Regional University Hospital of Malaga UMA, Málaga, Spain. 3 Andalusian Center for Nanomedicine and Biotechnology - BINAND, Málaga, Spain. 4 Department of rganic Chemistry, University of Málaga, IBIMA, Málaga, Spain. 5 Centro de Investigaciones Biológicas, CSIC, Madrid, Spain 6 Pharmacy Unit, Regional University Hospital of Malaga, Málaga, Spain

2 Figure Supplementary. RAST inhibition results (expressed as the mean + SD of percentage of inhibition) for allergic patients to AX. Solid phase: cellulose discs modified with AX-PLL. Inhibitors: AX (00-0. mm), amoxicilloic acid (00-0. mm), AX-BA (00-0. mm -) and AX-HSA ( mm of AX groups bound on HSA) % Inhibition % Inhibition Concentration of amoxicillin (mm) Concentration of amoxicilloioc acid (mm) 00 % Inhibition Concentration of AX-BA (mm)

3 Figure 2 Supplementary. H-NMR spectra of inhibitors (amoxicilloic acid and HSA-AX) in D 2. a) H-NMR spectra of amoxicilloic acid in D 2 H 7 6 NH 2 H N S N H C 2 H H H-6 H-7 H-5 J = 4.8 Hz CH-Ph H-6 H-3 J = 4.8 Hz CH 3 (β) CH 3 (α) δ (ppm) b) H-NMR spectra of AX-BA in D NH H-7 H-6 J= 7.7 Hz J= 7.7 Hz 4 9 NH 2 NH HN 0 NH S CH 3 β α CH 3 H C 2 - Na + CH(4) H-5 H-6 J= 8.6 Hz J= 8.6 Hz H-3 CH 2 (9) CH 2 (9 ) CH 3 (β) CH 3 (α) CH 2 (0,0 ) CH 2 (, ) CH 3 (2) CH 3 (2 ) δ (ppm)

4 Figure 3 Supplementary. verlapped MALDI-TF MS spectra of HSA control sample (yellow) and HSA-AX conjugate (violet). HSA control HSA-AX conjugate HSA control sample HSA-AX conjugate

5 Figure 4a Supplementary. H-NMR spectra of low molecular weight fraction <3 KDa of sera incubated with AX (lyophilized and solved in D 2 ). Signals corresponding to AX, diketopiperazine (DKP) and amoxicilloic acid (AX) and its enantiomer (C5) are identified. Figure 4b Supplementary. MALDI-TF MS spectra of low molecular weight fraction <3 KDa of sera incubated with AX.

6 Figure 4c Supplementary. HPLC- H-ESI-MS of low molecular weight fraction <3 KDa of sera incubated with AX. AX, diketopiperazine (DKP) and two enantiomers of amoxicilloic acid were detected as indicated in the following table: Compounds Amoxicillin (AX) Amoxicilloic Acid (both enantiomers) Diketopiperazine (DKP) Quantification (μg/ml) DKP AX Amoxicilloic Acid (two enantiomers 5R, 6R and 5S, 6R) Equipment: Dionex Ultimate 3000 HPLC system and rbitrap High Resolution Mass Spectrometer (Thermo Scientific). Ionization through electrospray by H-ESI-II Method: Sample was lyophilized and solved, in miliq water, for the analysis of AX and amoxicilloic acid, and in a mixture of miliq water-methanol for the analysis of DKP. Samples were injected into a Dionex Ultimate 3000 HPLC system equipped with a accucore RP-MS (50x2, mm ID, 2.6 μm) (Thermo Scientific), chromatographic column previously equilibrated with acetonitrile/amonic formiate ph:2.5 (2/98). Samples were eluted with isocratic flow 98/2 (acetonitrile/amoniac formiate ph:2.5) for 2 min, followed by gradient from 98/2 to 30/70 in min, then isocratic mobil phase 30/70 (acetonitrile/amoniac formiate ph:2.5) for 2 min, and gradient from 30/70 to 98/2 (acetonitrile/amoniac formiate ph:2.5) for 3 min. Analysis time: 9 min. Injection volume: 0 μl; Flow: 0.2 ml/min. Detection was carried out with positive polarity, in selected ion monitoring (SIM mode), selecting ions (for AX and DKP) and (for amoxicilloic acid) and MS/MS mode for analytes confirmation.