Substrate-derived Sortase A inhibitors: targeting an essential virulence factor of Gram-positive pathogenic bacteria

The bacterial transpeptidase Sortase A (SrtA) is a surface enzyme of Gram-positive pathogenic bacteria. It has been shown to be an essential virulence factor for the establishment of various bacterial infections, including septic arthritis. However, the development of potent Sortase A inhibitors remains an unmet challenge. Sortase A relies on a five amino acid sorting signal (LPXTG), by which it recognizes its natural target. We report the synthesis of a series of peptidomimetic inhibitors of Sortase A based on the sorting signal, supported by computational binding analysis. By employing a FRET-compatible substrate, our inhibitors were assayed in vitro. Among our panel, we identified several promising inhibitors with IC50 values below 200 μM, with our strongest inhibitor – LPRDSar – having an IC50 of 18.9 μM. Furthermore, it was discovered that three of our compounds show an effect on growth and biofilm inhibition of pathogenic Staphylococcus aureus, with the inclusion of a phenyl ring seemingly key to this effect. The most promising compound in our panel, BzLPRDSar, could inhibit biofilm formation at concentrations as low as 32 μg mL−1, manifesting it as a potential future drug lead. This could lead to treatments for MRSA infections in clinics and diseases such as septic arthritis, which has been directly linked with SrtA.


Materials and Methods
All amino acid derivatives were purchased from Iris Biotech GmbH and used without further purification. AmphiSpheres™, Novabiochem®, and TentaGel® rink amide resin served as the solid support for the synthesis of carboxyl-terminal peptide amide. Novabiochem® wang resin served as the solid support for the synthesis of carboxyl group at the C-terminal. All the resins were obtained from Merck. All solvents and reagents were purchased from commercial sources and used as received. For the bioassays: The substrate Abz-LPETGK(Dnp)-NH 2 (0.2784 g, 928 g/mole) was purchased from Bachem AG, Switzerland as a 100 mM stock solution in 3 ml DMSO. H-Gly-Gly-Gly-OH (189.17 g/mol) was purchased from Bachem AG, Switzerland as 50 g powder. The reference compound 5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-amine was prepared as 10 µL of 10 mM stock concentration by Ivana Uzelac at the department of chemistry and molecular biology, Gothenburg University.
Analytical TLC was performed using silica gel plates with C18 functionality and visualized under UV light at (254 nm).
The E. Coli SrtA gene used for expression codes for a protein product of 156 amino acids with an N-terminal His6-tag. The Ampicillin-resistant host strain E. coli BL21 transfected with pET21b plasmid was prepared lab at the chemistry and molecular biology department of Gothenburg University (Medicinaregatan 5c, 41390 Göteborg). Luria broth (LB) medium (Tryptone 10 g/L, Yeast extract 6 g/L, NaCl 5 g/L, and MQ-H 2 O) was prepared to perform the expression of SrtA. Tryptone, Yeast extract, and NaCl were purchased from Sigma-Aldrich®.
DiluPhotometer was used to measure the (OD 600nm ) which is the optical density at the wavelength of 600 nm.
SDS-PAGE analysis: Mini-protein® Tris-Tricine precast Gels were used for electrophoretic analysis with running buffer containing (100mM tris, 100mM tricine plus 0.1% SDS). All samples were incubated at 90 °C for 5 min in the presence of 4 μl of 4X LDS sample buffer. The gel was done at 100 v for ca. 100 min. The gel was stained with Coomassie blue stain and de-stained in 10% acetic acid aq.solution before scanning. Precision plus protein dual Xtra standards TM was used as a protein ladder. For the reducing gel, B-Mercaptoethanol was added to the sample buffer as 5% v/v.
Fast protein liquid chromatography (FPLC): HiLoad_16/60_Supperdex_75_prep_grade size exclusion column was used as a stationary phase for the protein purification. The column was equilibrated with tris buffer and isocratic elution of this buffer was used with 1.0 ml/min flow rate and 7.9 ph. The column temperature was set to 4 °C and max pressure to 0.5 MPa. UVdetection was measured at 280 nm wavelength. The sample pump was used for loading and the machine was equipped with automatic round fraction collector F9-R.

Synthesis and purification of peptides
Peptides were chain assembled using Rink amide resin either automated or manually. For automated synthesis, an Intavis MultiPep CF was used. Amino acid couplings were carried out with the molar ratio of (4):(4):(8) of (Fmoc-protected amino acid):(HBTU):(NMM) at room temperature for 60 minutes and deprotection was achieved in 20% (v/v) piperidine in DMF for 15 minutes at room temperature. After coupling, peptides were capped with 5% acetic anhydride in DMF (v/v) for 5 minutes. Manual amino acid couplings were carried out with the molar ratio of (3):(3):(4.5) of (Fmoc-protected amino acid):(HATU):(DIPEA) at room temperature for 60 minutes and deprotection was achieved in 20% (v/v) piperidine in DMF for 30 minutes at room temperature. N-terminal acetylation was achieved by reacting with acetic anhydride (10 eq.) and TEA (10 eq.), benzoylation with benzoyl chloride (10 eq.) and TEA (10 eq.), cyclohexanoylation with cyclohexanecarbonyl chloride (10 eq.) and TEA (10 eq.), naphtoylation with 2-naphtoyl chloride (10 eq.) and TEA (10 eq.) in DCM for 1 hour. Incorporation of the Cbz group was achieved by reacting with benzyl chloroformate (10 eq.) and DIPEA (10 eq.) for 3 hours at 0 °C. To generate the triazoles on resin, the synthetically prepared (2S)-2-azido-propanoic acid (3 eq.) was coupled twice with HATU (2.9 eq.) and DIPEA (4.5 eq.) for 1 hour. Subsequently, the resin was split into two portions and the transsubstituted triazole was synthesized by reacting with prepared Fmoc-Leu-Alkyne (4 eq.), CuSO 4 ·5H 2 0 (20mol%), sodium ascorbate (32 eq.) and DIPEA (6 eq.) in DMF for 5 hours at 60 °C under microwave irradiation using a Biotage Initiator+ microwave synthesizer (Biotage Sweden AB, Uppsala, Sweden), followed by washing with 0.5% sodium diethyldithiocarbamate in DMF, DCM and DMF, and subsequent Fmoc deprotection. The cis-substituted triazole was synthesized by reacting with prepared Fmoc-Leu-Alkyne (4eq.) and Cp*RuCl(cod) (20mol%) in DMF for 5 hours at 60 °C under microwave irradiation, followed by washing with 0.5% sodium diethyldithiocarbamate in DMF, DCm and DMF, and subsequent Fmoc deprotection. Peptides proceeded to standard cleavage from resin using a mixture of TFA (95%), H 2 O (2.5%) and TIPS (2,5%) for 3 hours at room temperature, while for cysteine-containing peptides EDT (2.5%) was added to the cleavage cocktail additionally. TFA was removed using N 2 and the resultant residue suspended in ice-cold diethyl ether. The mixture was then centrifuged (5 min, 4600 RPM) after which the supernatant was decanted into the waste. The remaining solid was washed twice by ice-cold diethyl ether and subjected to purification. For the purification and characterization of peptides, two eluent systems were used. Mobile phase A was 0.1% TFA in MQ-H 2 O, mobile phase B was 0.1% TFA in ACN and detection was done at 214 nm. The crude peptide was dissolved in a mixture of ACN in H 2 O and purified by semi-preparative HPLC using a Waters 600 system (Waters, Milford, MA, USA) equipped with a C18 column (MultoKrom 100 -5 C18, 5 µm particle size, 100 Å pore size, 250 x 20 mm, CS Chromatographie Service, Langerwehe, Germany) and a gradient of mobile phase A and mobile phase B from 5-10% B to 20-50% B over 45 min at 8 mL/min, depending on the specific peptide. Analytical RP-HPLC was carried out on a Waters XC e2695 system (Waters, Milford, MA, USA) employing a Waters PDA 2998 diode array detector equipped with a ISAspher 100-3 C18 (C18, 3.0 µm particle size, 100 Å pore size, 50×4.6 mm, Isera GmbH, Düren, Germany) at a flow rate of 2 mL/min using various gradients. The molecular weight of the purified peptides was confirmed by ESI mass on a Waters Synapt G2-Si ESI mass spectrometer equipped with a Waters Acquity UPLC system using a Xela C18 column (C18, 1.7 µm particle size, 80 Å pore size, 50×3.0 mm, Isera GmbH, Düren, Germany).

Protein expression and purification
The E. coli strain transfected with the recombinant plasmids was grown overnight in LB medium containing 100 μg/mL Ampicillin at 37 °C with constant shaking at 177 rpm. The bacterial culture was propagated into 1L LB at 37 °C until the OD 600 reached 0.46 and then incubated with 0.5 mM IPTG (isopropyl β-d-thiogalactoside) at 37 °C for 3 h to induce sortase expression. After the completion of incubation, the cells were harvested by centrifugation at 8000 × g for 15 min and stored at −80 °C until use. For protein purification, the cells were suspended in 35 mL lysis buffer (50 mM Tris·HCl, pH 8 at 21 °C, 150 mM NaCl, and 25 mM imidazole), lysed by high-pressure homogenization and then centrifuged at 25000 × g for 30 min at 10 °C. A LM20 Microfluidizer was used as a homogenizer for cell lysis with 25 kPsi air pressure at 4 °C. The lysate supernatant was then loaded into a gravity-flow column filled with 2 ml Ni Sepharose 6 Fast Flow resin and incubated for 30 min at 400 rpm and 4 °C. Then, the Sepharose was washed with four column volumes of lysis buffer to remove intracellular contaminating proteins. The Sortase A protein containing N-terminal His-tag was eluted with five column volumes of elution buffer (50 mM Tris·HCl, pH 8 at 21 °C, 150 mM NaCl, and 500 mM imidazole). The protein was concentrated via centrifugal filtration using Vivaspin with a molecular weight cutoff of 10 kDa. The supernatant fluid, flow through and the SrtA protein were analyzed by SDS-PAGE. Following gel analysis, the protein was desalted using a PD-10 column containing 8.

Kinetic analysis of the Sortase A activity
The kinetic assay protocol was performed based on the assay published by Kruger et al. 3 The biochemical assay was carried out in 210 µL reaction mixtures at pH 7.5 containing Tris buffer (300 mM Tris, 150 mM NaCl and 5 mM CaCl 2 ), Gly 3 (2 mM), SrtA (200 nM), and varying concentrations of Abz-LPETGK(Dnp)-NH 2 (from 30 to 900 µM). The reaction mixture was incubated for 6 hrs at 37 °C. Injections of 20 μl were taken each hour automatically and analyzed using RP-HPLC. The peptides were separated using a 30 to 50% linear gradient of eluent B over 5 min. The substrate peaks were detected by absorbance at 355 nm, and the extent of transpeptidation was calculated from the decrease of the area of the substrate peak over time. The peak area was converted to concentration units using a calibration curve of Abz-LPETGK(Dnp)-NH 2 as the standard (Figure S32b). Duplicate measurements were taken for each data point. Microsoft Origin 2019 was used to calculate the kinetic constants Km, Vmax, and kcat from the curve fit for the Michaelis-Menten equation. The data were reported as mean ± SE%.

Chromatographic assay of the peptide's stability
The cleavage assay set-up and protocol were modified from a previously published assay. 3 The assay was performed in 200 μl reaction mixtures. The cleavage of the synthetic peptides was done by incubating 90 µL of SrtA (270 nM final concentration) with 90 µL of compound solution (270 μM) in Tris buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , pH 7.5) for 4 h at RT. Gly 3 solution in Tris buffer (20 µL, 2 mM) was added to the mixture to accomplish the cleavage. Peptide substrate solution in Tris buffer (33.75 μM Abz-LPETGK(Dnp)-NH 2 ) was used in the experiment as a positive control and tested for cleavage in the same protocol. Samples of 50 μl were taken from the reaction mixture each hour and quenched by the addition of 25 μl 1M HCl to stop the enzymatic activity. The samples were analyzed by RP-HPLC and separated using a linear gradient from 0% to 40% of eluent B over 10 min. Dnp-containing peaks in the substrate were detected by measuring the absorbance at 355 nm while the synthetic peptides peaks were detected at 214 nm. The extent of cleavage was monitored over 4 hrs by measuring the product/substrate peaks ratio through integrating the areas under the HPLC trace. If cleavage occurred, the detected peaks were checked by ESI-MS. GK(Dnp)-NH 2 and Abz-LPETGK(Dnp)-NH 2 were detected by absorbance at 355 nm with retention times of 7.19 minutes and 9.97 minutes, respectively.

Fluorescence resonance energy transfer assay
For the FRET assay, the compound 5-((4-nitrobenzyl) thio)-1,3,4-thiadiazol-2-amine was used as a reference and prepared by dissolving 3 µL of the available stock (10 mM) in 96 µL HEPES and 1% DMSO to afford a 300 µM stock concentration for the assay. This reference compound has a reported IC 50 value of 26 ± 0.7 µM. 4 The fluorometric assay protocol has been previously used for screening of SrtA inhibitors. 5 The FRET assay was performed with an internally quenched fluorogenic (IQF) substrate to determine the inhibitory activity of the synthesized peptidomimetics. The assay was performed in a 30 μl reaction mixture in HEPES buffer (50 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , 0.05% Tween20, pH 7.5). SrtA solution (200 nM) was preincubated with compounds (200 μM) for 15 min at 22 °C followed by the addition of the substrate solution (25 μM Abz-LPETGK(Dnp)-NH 2 , 5 mM H-Gly 3 -OH, final assay concentration) in the same buffer. The cleavage of the Abz/Dnp-substrate was quantified by an increase in the fluorescence signal. The fluorescence signal was monitored each minute for 45 minutes and plotted to obtain the fluorescence rate as a line curve. The assay was performed in 384 well Corning NBS microplates (flat bottom, no lid, low flange, non-binding surface, non-sterile, white polystyrene). Fluorescence intensity was measured at emission and excitation wavelengths of 320 and 420 nm, respectively using a Spectramax iD5 plate reader (Molecular Devices, San Jose, CA, USA). Triplicate measurements were taken for each data point to ensure the reproducibility of the results. The average value of the fluorescence signal readouts was plotted against time over 60 min for each compound to produce a line curve using Origin. The reference compound was used in the experiment to act as a positive control. The slope of each compound curve (S x ) and the slope of the substrate (S 0 ) were extracted using the linear fit function in Microsoft Origin 2019. The percent inhibition was calculated according to the following equation: inhibitory rate = (1 -S x /S 0 ) × 100%. For IC 50 values, S x values at different concentrations were plotted against log transformed concentration values and fitted with a sigmodial fit using the DoseResp function using Origin. The bottom asymptote of the dose-response curve was fixed at 0. The data were reported as mean ± SE%.

Growth Inhibition Assays
For the growth profiling experiments the S. aureus strain CCUG10778 was cultured overnight in sterile TSB medium and diluted 1:100 by adding 1 μL into TSB medium in a sterilized, clear polystyrene 96-well plate to a final assay volume of 100 μL. Subsequently, peptides were added to the bacteria from filter sterilized stocks in MQ-H 2 O to give 2, 8, 32 and 128 μg/ml final concentrations. Then, bacteria were incubated at 37 °C for 3, 6, 12, 24, 36 or 48 hours and their absorbance was measured at 600 nm using a Varioskan Lux microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). All individual samples were carried out in triplicate. For the fluorescence experiments S. aureus was cultured overnight in sterile TSB medium and diluted 1:250 by adding 2.5 μL bacterial culture into TSB medium in sterilized 1.5 mL Eppendorf tubes to a final volume of 500 μL. Subsequently, peptides were added to the bacteria from filter sterilized stocks in MQ-H 2 O to give 8, 32 and 128 μg/ml final concentrations. Then, bacteria were incubated at 37 °C for 24 hours and centrifuged for 3 minutes at 3000 rpm in an Eppendorf Microstar 30r centrifuge. The supernatant was discarded and the cells were washed once with sterilized PBS and centrifuged. 100 μL PBS-T (PBS + 0.1% Triton-X) was added to the cells for 15 minutes to induce cell membrane permeabilization. After removal of the PBS-T by centrifugation, 100 μL of 5 μM Sytox Green (Invitrogen, Waltham, MA, USA) solution in PBS was added and the cells were stained for 30 minutes, centrifuged and washed with PBS twice. For the fluorescence measurements, the cells were resuspended with 100 μL of PBS and added in a sterilized, white plastic polystyrene 96-well plate and their fluorescence was measured by exciting the samples at 488nm and measuring the fluorescence intensity at 533 nm in a microplate reader. All individual samples were carried out in duplicate. For the fluorescence microscopy experiments, the cells were treated in the same, but resuspended in 10 μL of PBS and 5 μL of bacterial suspension was spotted on a glass target plate dipped in a melted solution of 1.2% agarose in Tris-HCl (50 mM) to ensure fixation of the sample. The sledes were covered by a cover glass and observed on an Axio Imager Z2m fluorescence microscope (Zeiss, Jena, Germany) at 100 times magnification at the emission wavelength of 533 nm.

Biofilm inhibition assays
For the biofilm inhibition experiments the S. aureus strain CCUG10778 was cultured overnight in sterile BHI broth. A sterilized, clear polystyrene 96-well plate was prepared by coating with plasma by addition of 20% of final assay volume rabbit plasma to the wells and incubating overnight at 4 °C. Then, the cultured strain was diluted 1:100 by adding 2 μL into BHI medium on the plasma coated plate to a total volume of 200 μL. Subsequently, peptides were added to the bacteria from filter sterilized stocks in MQ-H 2 O to give 8, 32 and 128 μg/ml final concentrations and incubated for 12 hours at 37 °C without shaking. The supernatant was removed and the wells were rinsed with PBS and 100 μL of 1% crystal violet solution (diluted in PBS) was added and the biofilms were stained for 10 minutes. The wells were washed with PBS twice and 200 μL of a 95% ethanol solution in water was added to solubilize the cells for 30 minutes. 100 μL was transferred into a new 96-well plate and the absorbance measured at 595 nm using a Varioskan Lux microplate reader. All individual samples were carried out in triplicate.

Computational Studies
All computational studies and analysis steps were performed employing the YASARA molecular modeling program. 6,7 Molecular graphics were created with YASARA (www.yasara.org) and POVRay (www.povray.org).

Docking
Docking was performed using VINA 8 (default parameters) and the best scoring and/or fitting results from 25 independent docking runs were further analysed. Docking was restricted to the substrate binding pocket of SrtA. Ligands and receptor residues were kept flexible during the docking runs.

Receptor structure generation
The structure of SrtA S. aureus was derived from pdb 2kid and was structurally aligned with pdb 7S51. 9, 10 Then the binding peptide Abz-LPATAG from 7S51 was transferred to 2kid keeping its original binding orientation and then transformed into LPRDSar. The resulting protein structure was geometry optimized with the YASARA YAPAC module using semiempirical, quantum chemical methods followed by an energy minimization employing the Yasara2 force field 11 and refined in a 110 ns molecular dynamic simulation. Thereby, the ligand was fixed in the binding poket through harmonic restraints (O Leu 2, NH1 Arg 197; O Pro 3, NE Arg 197; O Asp 5, N Cys 184; O Asp 5, N Thr 121) which mimic the hydrogen bonds between the ligand and the protein in 7S51. The parent structure as well as the structure of the protein at 92 ns were derived from the refinement simulation, energy minimized and used as the input receptor structure for docking. Energy minimizations and molecular dynamic simulations of the inhibitor-SrtA complex were performed as all-atom molecular dynamics simulation in explicit water (TIP3P) using the PME method 12 to describe long-range electrostatics at a cut-off distance of 8 Å at physiological conditions (0.9% NaCl, pH 7.4 13 ) at constant temperature (298 K) using a Berendsen thermostat and constant pressure (1 bar). Charged amino acids were assigned according to the predicted pKa of the amino acid side chains by Ewald summation and were neutralized by adding counter ions (NaCl). 13 In order to increase the simulation performance a multiple time step algorithm together with a simulation time step interval of 2.5 fs. 7 Simulation snapshots were saved every 100 ps. Time traces were computed from these snapshots. Energy minimizations were performed by simulated annealing including optimization of the hydrogen bond network 14 and equilibration of the water shell until system convergence was achieved (<0.05 kJ/mol/200/steps).  Figure S1. A) HPLC chromatogram (gradient from 5 to 25% ACN in water over 15 min at 1 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 1 H 2 N-LPRDA-OH. Figure S2. A) HPLC chromatogram (gradient from 5 to 25% ACN in water over 15 min at 1 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 2 LPRDA-Ado. Figure S3. A) HPLC chromatogram (gradient from 5 to 25% ACN in water over 15 min at 1 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 3 Ado-LPRDA. Figure S4. A) HPLC chromatogram (gradient from 5 to 25% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 4 LPRCA. Figure S5. A) HPLC chromatogram (gradient from 0 to 25% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 5 LPRD-Sar. Figure S6. A) HPLC chromatogram (gradient from 5 to 25% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 6 LPET-Sar. Figure S7. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 7 LPETP. Figure S8. A) HPLC chromatogram (gradient from 5 to 35% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 8 KSFLPATGGAE. Figure S9. A) HPLC chromatogram (gradient from 5 to 25% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 9 GTEPL. Figure S10. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 10 AcLPRDA. Figure S11. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 11 BzLPRDA. Figure S12. A) HPLC chromatogram (gradient from 5 to 50% ACN water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 12 ChLPRDA. Figure S13. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 13 2NapLPRDA. Figure S14. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 14 CbzLPRDA. Figure S15. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 15 AzARDA. Figure S16. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 16 LtARDA. Figure S17. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 17 LcARDA. Figure S18. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 18 BzLPRDSar. Figure S19. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 19 BzLPETP. Figure S20. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 20 LPRDP. Figure S21. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 21 BzLPRDP. Figure S22. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 22 FLPRDA. * impurity in the LC-MS system. Figure S23. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 23 BzLPRDF. Figure S24. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 24 LPRDF. Figure S25. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 25 FPRDF. Figure S26. A) HPLC chromatogram (gradient from 5 to 50% ACN in water over 10 min at 2 ml/min, detection at 214 nm) and B) high resolution mass spectrum for peptide 26 FPRDPSar.                Computational Analysis Figure S41. Optimized sortase A structures derived from pdb 2kid (SrtA*). Light-blue -energy minimized, initial SrtA structure from 2kid (lowest energy member from NMR ensemble) after ligand transfer form pdb 7S51 and transformation into ligand 5 (SrtA*), blue -SrtA* structure after 16 ns and 92 ns (red) of all-atom molecular dynamic simulation.