One‐Pot Chemoenzymatic Synthesis of Microviridin Analogs Containing Functional Tags

Abstract Microviridins are a prominent family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) featuring characteristic lactone and lactam rings. Their unusual cage‐like architecture renders them highly potent serine protease inhibitors of which individual variants specifically inhibit different types of proteases of pharmacological interest. While posttranslational modifications are key for the stability and bioactivity of RiPPs, additional attractive properties can be introduced by functional tags. To date – although highly desirable – no method has been reported to incorporate functional tags in microviridin scaffolds or the overarching class of graspetides. In this study, a chemoenzymatic in vitro platform is used to introduce functional tags in various microviridin variants yielding biotinylated, dansylated or propargylated congeners. This straightforward approach paves the way for customized protease inhibitors with built‐in functionalities that can help to unravel the still elusive ecological roles and targets of this remarkable class of compounds and to foster applications based on protease inhibition.

. Mass spectrometric analysis of the cyclization assays. Table S2. Overall theoretical yields, isolated yields and percent yields for the enzymatic conversions. Figure S1. Chemoenzymatic synthesis of C-terminally modified Mv J and Mv B derivatives. Figure S2. Results of the the repeated enzymatic conversion of MvJ_CP_N(Prop). Figure S3. MALDI-TOF MS spectrum of unmodified MvJ_CP. Figure S4. MALDI-TOF MS spectrum of bicyclic MvJ_CP. Figure S5. MALDI-TOF MS spectrum of tricyclic MvJ_CP. Figure S6. MALDI-TOF MS spectrum of unmodified MvJ_CP_N(Bio).                               Figure S38. Results of the protease inhibition assay of the microviridin J derivatives. Figure S39. Results of the protease inhibition assay of the microviridin B derivatives. Figure S40. Blank control for protease labeling assay.

Coupling of Fmoc/tBu-protected amino acids:
To 200 mg of the resin (~ 0.5 mmol/g), a 0.25 M solution of the amino acid in DMF (2.5 eq. relative to resin loading) was added. After addition of a 0.5 M solution of DIPEA in DMF (2.5 eq.) and a 0.25 M solution of TBTU in DMF (2.5 eq.), the reaction solution was mixed for 15 min. A second coupling was performed for 15 min. For couplings subsequent to the 5th amino acid, double couplings with 30 min coupling time were performed. For couplings subsequent to the 10th amino acid, a third coupling with 45 min was performed. After each coupling cycle capping with 0.5 M acetic anhydride in DMF (2 x 2.5 mL, 10 min) was performed. Finally, the resin was washed with DMF (6 x 2.5 mL). Fmoc removal: DMF/piperidine (4:1, 2.5 mL) was added to the resin and mixed for 2.5 min. The procedure was repeated 4 times. The resin was washed with DMF (6x 2.5 mL). After the final coupling cycle, the resin was washed with DCM (3 x 2 ml).

Global deprotection:
The resin was transferred to a 5 mL syringe with frit and cap. After addition of the cleavage cocktail (TFA, H 2 O, TES, DODT (3,6-dioxa-1,8-octane-dithiole) 92.5:2.5:2.5:2.5), the syringe was shaken for 3 h. The peptide was precipitated in ice cold diethyl ether and centrifuged. The supernatant was removed and the precipitate was washed with diethyl ether twice. The peptide was resolved in MeCN/H 2 O (1:4) and lyophilized. Table S1. Mass spectrometric analysis of the cyclization assays. The masses of the modified core peptides correspond to the monocyclic, bicyclic and tricyclic microviridin products due to mass shifts suggestive of the loss of one, two, or three water molecules (-H2O: mass shift of 18.01 Da) as a consequence of lactone and lactame ring forming during condensation reactions.

Figure S10. MALDI-TOF MS spectrum of monocyclic MvJ_CP_C(Bio) with relevant adduct ions labelled.
Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1.  Table S1.  Table S1. Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1.  Table S1. Figure S18. MALDI-TOF MS spectrum of monocyclic MvB_CP with relevant adduct ions labelled. Overview of measured and calculated masses can be found in Table S1.  Table S1. Figure S20. MALDI-TOF MS spectrum of tricyclic MvB_CP with relevant adduct ions labelled. Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1.  Table S1. Figure S24. MALDI-TOF MS spectrum of tricyclic MvB_CP_N(Bio) with relevant adduct ions labelled. Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1. Figure S28. MALDI-TOF MS spectrum of monocyclic MvJ_CP_N(dansyl) with relevant adduct ions labelled. Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1. Overview of measured and calculated masses can be found in Table S1. Figure S31. MALDI-TOF MS/MS spectrum of tricyclic MvJ_CP (microviridin J core peptide) (1), which shows a fragmentation pattern diagnostic for the tricyclic architecture of microviridins. As previously reported, [1] the Nterminal lactone ring of microviridin opens during MALDI-TOF MS/MS analysis due to a rearrangement reaction, yielding a dehydrated threonine (iso-dehydrobutyrine; iso-Dhb) and the carboxylic acid moiety of aspartate instead of a lactone bond (Fig. S37). For a comprehensive list of all calculated and observed fragment ions see the supplementary Excel file.  (2), which shows a fragmentation pattern diagnostic for the tricyclic architecture of microviridins. As previously reported, [1] the N-terminal lactone ring of microviridin opens during MALDI-TOF MS/MS analysis due to a rearrangement reaction, yielding a dehydrated threonine (iso-dehydrobutyrine; iso-Dhb) and the carboxylic acid moiety of aspartate instead of a lactone bond (Fig. S37). For a comprehensive list of all calculated and observed fragment ions see the supplementary Excel file.  terminal O-propargyl-L-tyrosine) (3) , which shows a fragmentation pattern diagnostic for the tricyclic architecture of microviridins. As previously reported, [1] the N-terminal lactone ring of microviridin opens during MALDI-TOF MS/MS analysis due to a rearrangement reaction, yielding a dehydrated threonine (iso-dehydrobutyrine; iso-Dhb) and the carboxylic acid moiety of aspartate instead of a lactone bond (Fig. S37). For a comprehensive list of all calculated and observed fragment ions see the supplementary Excel file. Figure S34. MALDI-TOF MS/MS spectrum of tricyclic MvB_CP (microviridin B core peptide) (4), which shows a fragmentation pattern diagnostic for the tricyclic architecture of microviridins. As previously reported, [1] the Nterminal lactone ring of microviridin opens during MALDI-TOF MS/MS analysis due to a rearrangement reaction, yielding a dehydrated threonine (iso-dehydrobutyrine; iso-Dhb) and the carboxylic acid moiety of aspartate instead of a lactone bond (Fig. S37). For a comprehensive list of all calculated and observed fragment ions see the supplementary Excel file. Figure S35. MALDI-TOF MS/MS spectrum of tricyclic MvB_CP_N(Bio) (microviridin B core peptide with Nterminal N ε -biotinyl-L-lysine) (5), which shows a fragmentation pattern diagnostic for the tricyclic architecture of microviridins. As previously reported, [1] the N-terminal lactone ring of microviridin opens during MALDI-TOF MS/MS analysis due to a rearrangement reaction, yielding a dehydrated threonine (iso-dehydrobutyrine; iso-Dhb) and the carboxylic acid moiety of aspartate instead of a lactone bond (Fig. S37). For a comprehensive list of all calculated and observed fragment ions see the supplementary Excel file. Figure S36. MALDI-TOF MS/MS spectrum of tricyclic MvB_CP_N(dansyl) (microviridin B core peptide with a Nterminal dansyl group) (6), which shows a fragmentation pattern diagnostic for the tricyclic architecture of microviridins. As previously reported, [1] the N-terminal lactone ring of microviridin opens during MALDI-TOF MS/MS analysis due to a rearrangement reaction, yielding a dehydrated threonine (iso-dehydrobutyrine; iso-Dhb) and the carboxylic acid moiety of aspartate instead of a lactone bond (Fig. S37). For a comprehensive list of all calculated and observed fragment ions see the supplementary Excel file. Figure S37. Ring-opening reactions during MS/MS analysis. Opening of the N-terminal lactone ring of microviridin due to a rearrangement reaction yielding a dehydrated threonine (iso-dehydrobutyrine; iso-Dhb) and the carboxylic acid moiety of aspartate. This reaction is frequently observed in MALDI-TOF MS/MS analysis of microviridins and microviridin-like compounds.
[1] Figure S38. Results of the protease inhibition assay of the microviridin J derivatives. The plots were created with the Quest Graph™ IC50 Calculator. [2] Figure S39. Results of the protease inhibition assay of the microviridin B derivatives. The plots were created with the Quest Graph™ IC50 Calculator. [2] Figure S40. Blank control for protease labeling assay.