Mass spectrometry

The molecular weight of synthetic probe precursors was determined by mass spectrometry (Bruker Daltonics, MA, USA). Particle size was determined by transmission electron microscopy (JEOL, Japan). Gel electrophoresis (BIO‐RAD, CA, USA), zeta potentiometer monitoring (Malvern, UK) the potential of the probe and multispectral laser imaging system (Azure Sapphire, USA) used to detect the conjugation of NPs and antibody was successful. Gd content in solution was determined by inductively coupled plasma optical emission spectrometry (Thermo Fisher Scientific, USA), and Gd content in mouse tissue was calculated. Absorption spectra were analyzed using a Multiskan Spectrum Microplate Spectrophotometer (Thermo Fisher Scientific, USA).

bLF (20% iron saturated) was kindly provided by DMV International (Veghel, The Netherlands). LF derived peptides (Table 1) were synthesized by solid phase peptide synthesis using Fmoc chemistry with a Siro II synthesizer (Biotage, Uppsala, Sweden) according to the manufacturer’s protocol. Purification by Reverse Phase-HPLC was conducted as described previously (Bolscher et al. 2009 (link)). Identity of the peptides was confirmed by mass spectrometry (Bruker Daltonik GMBH, Bremen, Germany) and molar concentrations were calculated based on their weight.Table 1

Sequences and characteristics of the peptides investigated

Peptidea	Primary structure	Charge	MBC (μM)b
			Y. e.	Y. p.
LFampin265–284	DLIWKLLSKAQEKFGKNKSR	+ 4	6.3	ND
LFcin17–30	FKCRRWQWRMKKLG	+ 6	0.8	ND
LFchimerac		+ 12	0.2	1.6
LFampin265–284 and LFcin17–30	DLIWKLLSKAQEKFGKNKSRand FKCRRWQWRMKKLG	+ 10	0.8	ND

Y. e.: Y. enterocolitica, Y. p.: Y. pseudotuberculosis

^aThe purity of the peptides was at least 95% and the authenticity of the peptides was confirmed by MALDI-TOF mass spectrometry

^bMinimal bactericidal concentration (MBC), in 1 mM phosphate buffer (from Sijbrandij et al. 2017 (link)). ND: not detected up to 50 µM peptide

^cA single C-terminal lysine amide (K) was substituted at the α- and ε-amino groups with the two peptides via the C-terminal site, while leaving the two N-termini as free ends

An X-4 melting point apparatus with a binocular microscope was used to determine the melting point (m.p.) of the target compound 6a–u. HRMS were obtained using mass spectrometry (Bruker Daltonik, Germany). Using tetramethylsilane as the internal standard, the ¹H-NMR and ¹³C-NMR data of the target compound were measured using a Bruker AV-500 nuclear magnetic resonance instrument (compounds 6a–u were dissolved in CDCl₃ or DMSO_d6). The positive fluoxetine used in the pharmacological experiment was of a high-purity standard which can be directly used in pharmacological experiments. All chemical reagents used in the chemical experiment were of analytical reagent grade or chemically pure, which were directly used in the experiment without further purification. The NMR and HRMS spectra of compounds 4, 5, and 6a–u are presented in Figures S2–S68.