Fluorescent Activity-Based Probe To Image and Inhibit Factor XIa Activity in Human Plasma

Anticoagulation therapy is a mainstay of the treatment of thrombotic disorders; however, conventional anticoagulants trade antithrombotic benefits for bleeding risk. Factor (f) XI deficiency, known as hemophilia C, rarely causes spontaneous bleeding, suggesting that fXI plays a limited role in hemostasis. In contrast, individuals with congenital fXI deficiency display a reduced incidence of ischemic stroke and venous thromboembolism, indicating that fXI plays a role in thrombosis. For these reasons, there is intense interest in pursuing fXI/factor XIa (fXIa) as targets for achieving antithrombotic benefit with reduced bleeding risk. To obtain selective inhibitors of fXIa, we employed libraries of natural and unnatural amino acids to profile fXIa substrate preferences. We developed chemical tools for investigating fXIa activity, such as substrates, inhibitors, and activity-based probes (ABPs). Finally, we demonstrated that our ABP selectively labels fXIa in the human plasma, making this tool suitable for further studies on the role of fXIa in biological samples.


Substrate specificity profiles for APC, thrombin, fXa, and fXIa
To determine the substrate specificity of fXIa at the P1 position, we used the defined library with the general structure of Ac-Ala-Arg-Leu-P1-ACC containing 133 individual fluorogenic substrates. This tailored library of fluorogenic substrates consists of a fixed P4-P2 motif (Ala-Arg-Leu) and various amino acid residues at the P1 position. The general structure of the library was Ac-Ala-Arg-Leu-P1-ACC, in which ACC was 7-amino-4-carbamoylmethylcoumarin, Ac was an acetyl group, and P1 was an individual natural or unnatural amino acid. Figure S1. Substrate specificity profiles for APC, thrombin, fXa, and fXIa at the P1 position presented as heatmaps [1].
To determine the binding pocket preferences at the P4-P2 positions we utilized the Hybrid Combinatorial Substrate Library (HyCoSuL) approach. Since fXIa preferentially accommodated arginine in the S1 pocket, we employed the combinatorial library with arginine fixed at the P1 position. This library consisted of three tetrapeptide sublibraries (Ac-P4-Mix-Mix-Arg-ACC, Ac-Mix-P3-Mix-Arg-ACC, Ac-Mix-Mix-P2-Arg-ACC) and contained a natural and a large pool of unnatural amino acids at the investigated position (P4, P3 or P2) (135 amino acids for P2, 136 amino acids for P3 and P4) and an equimolar mixture of natural amino acids (Mix) at the remaining positions (18 natural amino acids and norleucine (cysteine and methionine were not used due to the possibility of oxidation)).  Figure S2. Substrate specificity profiles for APC, thrombin, fXa, and fXIa at the P4-P2 positions presented as heatmaps [1].

ABPs and inhibitor synthesis
The peptide sequences of the best two substrates were utilized to design irreversible ABPs (two biotinylated and one fluorescent) and one inhibitor. In the first step, 100 mg of 2-chlorotrityl resin (for each ABP) was placed in the peptide synthesis vessel and swollen in anhydrous DCM for 30 min, and then, the resin was washed once with DCM. Next, Fmoc-P2-OH (2.5 equiv.) was dissolved in anhydrous DCM, preactivated with DIPEA (3 equiv., 84 mL), added to the resin under an argon atmosphere, and stirred gently for 12 h. After that, the mixture was filtered, the resin was washed with DCM (three times), and the remaining active sites on the 2-chlorotrityl resin were deactivated with DCM/MeOH/DIPEA (%, v/v/v, 80:15:5) solution for 1 h. Next, the mixture was filtered, the resin was washed with DMF (six times), and N-terminal Fmoc-protecting group deprotection was performed using 20% piperidine in DMF (5, 5, and 25 min). After Fmoc-protecting group removal, the P3, P4 amino acids, and Fmoc-PEG(4)-OH were attached to the H2N-P2-resin with HOBt (2.5 equiv., 60 mg) and DICI (2.5 equiv., 52 mL) as coupling reagents in coupling/deprotection cycles. The biotin tag was coupled to H2N-PEG(4)-P4-P3-P2-resin using HBTU (2.5 equiv., 152 mg) and DIPEA (2.5 equiv., 70 mL) as coupling reagents in a DMF/DMSO mixture (%, v/v, 50:50). After 3 h, the resin was washed with DMF (six times), DCM (three times), and MeOH (three times) and dried over P2O5.
The organic phase was collected, dried over MgSO4, and evaporated. Finally, the side chain amino acid protecting groups were removed with a mixture of TFA/DCM/TIPS (%, v/v/v, 80:15:5). After 30 min, solvents were removed with argon flow, and the obtained product (biotin-PEG(4)-P4-P3-P2-Arg P (OPh)2) was dissolved in peptide grade DMSO, purified on HPLC, and lyophilized. The ABP purity was confirmed by analytical HPLC and analyzed using HRMS. Additionally, NMR analysis was performed. ABP was then dissolved in peptide grade DMSO to a final concentration of 10 mM and stored at -80 °C until use.

S6
In the case fluorescently labeled probe, to the amine group of H2N-PEG(4)-P4-P3-P2-resin, the BODIPY FL fluorophore was coupled. The amount of 2-chlorotrityl resin was reduced to 50 mg. BODIPY FL (0.3 equiv., 9 mg) was dissolved in DMF, preactivated with DIPEA (1.2 equiv., 17 mL), and stirred with the resin overnight. Next, the resin was washed with DMF (six times), DCM (three times), and MeOH (three times) and dried over P2O5. The remaining steps were analogous to the synthesis of the biotin-labeled ABPs. Scheme S2. Synthesis of the fluorescently labeled ABP.
of AcOH, 5 equiv. of HBTU, and 5 equiv. of DIPEA in DMF. After 1 h, the solution was filtered, and the resin was washed with DMF (six times), DCM (three times), and MeOH (three times) and dried over P2O5. The remaining steps were analogous to the synthesis of the biotinylated ABPs.
Synthesis of the inhibitor. Table S1. Purity and MS analysis of fXIa compounds.

S36
The chromatograms of some substrates are not sharp but appear as two peaks. This was the case with the two sets of substrates listed below in the Table S2. What they have in common is the fact that they all have the amino acid Glu(Bzl) at the P2 position. A series of the same substrates differing only at the P2 position (His(Bzl) instead of Glu(Bzl)) no longer give such a false double peak. We believe that this is due to their structure, namely the interactions of large hydrophobic amino acids at positions P4 and P2 with the stationary phase.
To more closely explain the fact that the double peak does not affect the purity, we have presented the close-up of spectra and the MS result from the beginning and the end of the peak of two exemplary substrates from two series -SMXI6 and SMXI13 (the situation for the rest of the substrates was the same). It can be noticed that the masses of both peaks are the same, which means that the compound is pure, and only its separation on the column gives two separate -although essentially the same peaks. Proper masses were also confirmed by the HRMS analysis.

S48
To perform the NMR analysis, we used the Bruker Avance Neo spectrometer 600 MHz.
P-SMXI51, P-SMXI52, and I-SMXI5 were dissolved in deuterated DMSO. 1 H NMR was performed at 600.58 MHz, and 13 C NMR was performed at 151.02 MHz. Since the P-SMXI52 sample was already dissolved in DMSO (not deuterated), we could only make 1 H NMR analysis by performing selective quenching of methyl groups from the solvent. The spectra provided are: 1 H NMR, 13 C NMR for P-SMXI51, I-SMXI5, and 1 H NMR for P-SMXI52. Variable Temperature NMR (VT NMR) experiments for P-SMXI51, P-SMXI52, and I-SMXI5 were performed at 600.58 MHz and the 298-338 K range every 10 K. Analyses were calibrated for the residual signals from DMSO.  P-SMXI52 anti-fXI merged