From the Probiotic Bacterium Lactobacillus Acidophilus NCFM

SUPPORTING INFORMATION

An NAD+–dependent sirtuin depropionylase and deacetylase (Sir2La) from the probiotic bacterium Lactobacillus acidophilus NCFM

Sita V. Olesen,1 Nima Rajabi,2,3 Birte Svensson,1 Christian A. Olsen,2,3 Andreas S. Madsen

1Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark; 2Center for Biopharmaceuticals, Faculty of Health and Medicinal Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark; 3Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen, Denmark.

Table of content

Full amino acid sequences of the produced full-length proteins ...... S-2 Figure S1—Substrate screening of Sir2La and LBA1649 ...... S-2 Figure S2—Bisubstrate kinetic measurements of Sir2La-dependent deacylation of LGKpro (2c) ...... S-3 Figure S3—Dose-response curves from end-point assays ...... S-4 Scheme S1—Synthesis of substrates ...... S-5 Scheme S2—Synthesis of inhibitor 7 ...... S-5 Table S1—Proteins discussed in the text with organism and UniProt-codes ...... S-6 Table S2—Identity and similarity between selected sirtuins ...... S-7 Table S3—Proteins used to construct the phylogenetic tree in Fig. 1a...... S-8 Table S4—BLAST search results from the NCBI database ...... S-13 Table S5—Substrate synthesis references ...... S-16 Synthesis of substrates ...... S-17 Synthesis of inhibitor 7 ...... S-23 Supplemental references ...... S-24 1H and 13C spectra of novel compounds...... S-25

S-1 Full amino acid sequences of the produced full-length proteins

Sir2La: MGSSHHHHHHSSGLVPRGSHMRVNIMKPNDITELQKDIDQAKHITFLTGAGVSTHSGIPDYRSKN GIYNGIKESPETILSEATLFHRPELFYNFVMENMYFPSAQPNLIHKKIAQLCSQKGDLITQNIDGLD TKAGNTHVTEFHGNLYNIYCTKCHQQVSYSEYANGYLHKNCGGIIRPGIVLYGEAINPEVLNVSI NNMQKSDLIIISGTSFVVYPFAQLLAYKKGSAKIYSINKTAIPAPQVTQIIGDALDVFKQLN

LBA1649: MGSSHHHHHHSSGLVPRGSHMTDLTKANEWLKDADAVIVTAGNGMAKEEGLDILSEENFDNQF GEIAEKYDVHTIGDALDKEFDSWEEKWTFWSKLINEYSLQYEKSENMKALEKLLEDKEYFIATS TFAHFFENAGFNENRIFDAFGDWTKMQCSSGINHGQKDNREVVKQFLQGKGELPKCEDCGSPM ELHMPLNAHFFPDTDANTRFRWFLTGNEEKNVVVLELGVDATSPQLLDPMVNLVQQFSNWHYI AADLANDNLPDDLKKRAIGFGSDTHETLMNLTK

Figure S1. Substrate screening of Sir2La and LBA1649. Bar graphs of Sir2La and LBA1649-dependent deacylase activity based on endpoint assays. All reactions were incubated for 1 h at 25 °C before development and fluorescence analysis.

S-2 Figure S2. Bisubstrate kinetic measurements of Sir2La-dependent deacylation of LGKpro (2c). Global fit of steady-state rates to the sequential ordered rapid equilibrium equation and the corresponding secondary plots of (kcat,obs/kcat) and (αKMkcat,obs/KM,obskcat) as functions of substrate concentrations. Best fit is shown as lines. All reactions were monitored for at least 1 h at 25 °C.

S-3

Figure S3. Dose-response curves from end-point assays.

S-4

Scheme S1. Synthesis of substrates.

Scheme S2. Synthesis of inhibitor 7.

S-5 Table S1. Proteins discussed in the text with organism and UniProt-codes.

Protein Organism UniProt code Aligned residues Sir2La (LBA0117) L. acidophilus Q5FMQ6 12–239 LBA1649 L. acidophilus Q5FIL3 1–266

hSIRT1 Homo sapiens Q96EB6 244–498 hSIRT2 Homo sapiens Q8IXJ6 65–340 hSIRT3 Homo sapiens Q9NTG7 126–382 hSIRT4 Homo sapiens Q9Y6E7 45–314 hSIRT5 Homo sapiens Q9NXA8 41–309 hSIRT6 Homo sapiens Q8N6T7 35–274 hSIRT7 Homo sapiens Q9NRC8 90–331

Sir2Tm Thermotoga maritima Q9WYW0 3–246 Sir2Af2 Archaeoglobus fulgidus O30124 – SrtN Bacillus subtilis O07595 – F0THZ6 (lam) L. amylovorus F0THZ6 7–234 A0A133PBX8 (lga) L. gasseri A0A133PBX8 8–237 Q74LU0 (ljo) L. johnsonii Q74LU0 8–237 G9ZKM4 (lpa) L. parafarraginis G9ZKM4 4–238 CobB (lpl) L. plantarum Q88ZA0 5–234

spySirTM Streptococcus pyogenes P0DN71 14–293 sauSirTM Staphylococcus aureus A0A0H3JQ59 20–315 lpaSirTM L. parafarraginis G9ZLW5 18–290 F0TGP4 (lam) L. amylovorus F0TGP4 5–266 D1YLJ9 (lga) L. gasseri D1YLJ9 5–269 A0A137PK42 (ljo) L. johnsonii A0A137PK42 5–266 – L. plantarum not identified in UniProt –

S-6 Table S2. Identity and similarity between selected sirtuins. Percentage of identical (top–right half) and similar (lower–left half) residues, calculated at http://imed.med.ucm.es/Tools/sias.html (similarity groups: FYW, RKH, DE, ST, NQ, and VILMA). Class affiliation is indicated by circles using same colors as in

Figure 1a. sauSirTM spySirTM LBA1649 Sir2Tm Sir2La hSirt1 hSirt2 hSirt3 hSirt4 hSirt5 hSirt6 hSirt7

[ [ [ [126 - 382] [ [244 - 498] [ [ [ [ [ [

90 - 331] 35 - 274] 41 - 309] 45 - 314] 65 - 340] 14 - 293] 20 - 315] 12 - 239] 3 1 - 246] - 266]

hSirt1 44 41 26 28 23 23 34 30 17 17 15 hSirt2 59 54 24 25 27 24 33 29 16 16 16 hSirt3 59 68 26 27 31 26 36 31 17 16 15 hSirt4 42 38 39 26 28 28 32 26 14 16 13 hSirt5 44 39 41 45 22 26 29 31 18 15 13 hSirt6 43 40 44 45 36 43 36 25 15 18 14 hSirt7 37 41 40 43 39 54 33 27 14 16 12 Sir2Tm 48 50 52 49 47 50 44 32 17 17 16 Sir2La 43 43 46 46 43 40 41 49 18 16 14 sauSirTM 35 30 36 27 28 33 29 32 31 61 24 spySirTM 33 29 31 26 26 31 30 30 29 73 24 LBA1649 29 29 32 27 26 27 29 35 30 43 41

S-7 Table S3. Proteins used to construct the phylogenetic tree in Figure 1a. Eukaryota UniProtKB [residues aligned] Chordata Danio rerio (zebra fish) A1L2B7[89-346], A8E5H0[35-274], A9JRZ2[87-295], E7F8W3[180-434], Q5XJ86[41-310], Q6DHI5[37-305], Q7ZVK3[63-337] Gallus gallus (chicken) A0A1D5NWN5[55-329], A0A1D5NYI2[73-329], A0A1D5P2E2[52-306], A0A1D5PHZ8[35-274], E1BRE2[40-308], F1NB70[46-315], F1NC39[91-332] Heterocephalus glaber (naked mole rat) A0A0A7HV95[169-423], A0A0P6JFX2[61-330], G5AMA2[65-340], G5AUC0[32-288], G5BJ83[90-331], G5BN47[41-309], G5BZB2[35-274] Homo sapiens (human) Q8IXJ6[65-340], Q8N6T7[35-274], Q96EB6[244-498], Q9NRC8[90-331], Q9NTG7[126-382], Q9NXA8[41-309], Q9Y6E7[45-314] Monodelphis domestica F6PLF5[91-332], F6S8W1[35-274], F6TKW2[224-478], F6U2S4[66-259], (gray short-tailed opossum) F6Z195[24-215], F7D4X9[44-314], H9H7W7[200-456] Mus musculus (mouse) P59941[35-274], Q8BKJ9[91-332], Q8K2C6[41-309], Q8R104[74-334], Q8R216[42-317], Q8VDQ8[65-340], Q923E4[236-490] Nothobranchius furzeri A0A1A7ZCL0[63-337], A0A1A7ZFW6[43-317], A0A1A7ZLH8[103-360], (turquoise killifish) A0A1A7ZR70[78-334], A0A1A7ZYE7[36-304], A0A1A8A1K5[35-303], A0A1A8A751[94-335], A0A1A8UCK7[35-274], B3TLA8[214-467] Ornithorhynchus anatinus F6T4P8[1-111], F7A153[35-310], F7D8E9[41-310], F7G8M3[1-202] (duckbill platypus) Tetraodon nigroviridis H3BZZ4[14-284], H3D3L1[2-259], H3D6D6[101-354], H3D971[88-329], (spotted green pufferfish) H3DD00[35-275], H3DQV1[63-115], Q4RXF6[33-302] Xenopus laevis (african clawed frog) A0A1L8FJP8[232-486], A0A1L8GDN0[91-347], A0A1L8I0D6[44-319], Q4V7G8[78-335], Q5HZN8[40-308], Q5U250[93-367], Q5XG15[85-326], Q68F47[40-308], Q6GPW5[35-274] Xenopus tropicalis (western clawed frog) B1H3J8[178-432], F6RAU2[14-270], F6U8M9[56-330], F7D2Y2[91-332], F7DKV7[40-308], Q07FY7[115-372], Q28CB4[44-319], Q6P340[35-274] Branchiostoma floridae (lancelet) C3Y212[35-272], C3Y909[60-301], C3YCU3[34-302], C3YKX2[61-312], C3Z1Z2[2-262], C3Z3T3[37-310] Ciona intestinalis (sea squirt) F6SLD6[179-432], F7AUS4[112-352], H2XW94[86-346], H2XWF2[17-278], Q1RL34[35-308], Q1RL52[27-264], Q1RPU9[53-320] Ecdysozoa Anopheles darlingi (mosquito) W5J661[35-273], W5J6U2[156-428], W5J6V3[98-339], W5JBK9[35-313] Anopheles gambiae (mosquito) F5HJ98[36-315], F5HMW1[107-348], Q7PXL0[35-273], Q7QD39[162-434], Q7QDH0[887-1141] Apis mellifera (honey bee) A0A088A4P0[42-313], A0A088AIW3[92-333], A0A088AIY8[35-273], A0A088AM80[5-265], A0A088AN54[194-465], A0A088ARH5[67-342] Daphnia pulex (water flea) E9G957[35-307], E9GD30[12-274], E9GFX0[185-454], E9H1P2[35-273], E9H391[69-342] Drosophila melanogaster (fruit fly) D3PFE9[1-240], Q8IRR5[36-311], Q9I7I7[27-308], Q9VAQ1[114-355], Q9VH08[35-275], Q9VK34[212-485] Nasonia vitripennis (parasitic wasp) K7IQA4[13-281], K7IS10[465-736], K7IUV9[72-344], K7IVH1[35-273], K7IWQ7[101-342], K7IZZ5[195-466] Tribolium castaneum (red flour beetle) A0A139WG94[34-302], D6W7G2[10-273], D6W9J7[35-272], D6WFJ9[202-478], D6WU58[66-340], D7EHQ9[110-351] Brugia malayi (roundworm) A0A0H5SA91[43-282], A0A0H5SD54[35-272], A0A0J9XZ03[68-342], A0A1I9GD51[134-386] Caenorhabditis briggsae (roundworm) A8WVR7[50-308], A8X439[18-284], A8X440[17-289], Q60L58[127-376] Caenorhabditis elegans (roundworm) Q20480[18-287], Q20481[18-287], Q21921[136-385], Q95Q89[35-276] Lophotrochozoa Helobdella robusta (californian leech) T1EG11[33-304], T1EG16[64-338], T1EG71[19-260], T1FM78[17-279], T1FMY8[35-272], T1FYQ0[1-198], T1FYS5[60-305] Crassostrea gigas (pacific oyster) K1P7P4[91-350], K1PW50[109-371], K1QDT0[1001-1239], K1R299[39-310], K1R7D0[107-348], K1RHF7[163-417], K1RT25[52-306] Lottia gigantea (giant owl limpet) V3ZNN2[66-320], V3ZQA2[29-290], V3ZQZ0[70-314], V4ALP5[30-239], V4AXY6[6-266], V4B5Z2[54-329], V4BM64[35-272], V4CA59[1-126]

Flatworm

S-8 Schistosoma mansoni (blood fluke) A6XDL2[145-459], T1VXA1[28-309], T1VXV7[35-298], T1VYA5[11-482], T1VZ88[19-305] Radiata Hydra vulgaris (fresh-water polyp) T2M2B7[46-320], T2M7Y9[64-318] Nematostella vectensis A7RLD5[1-244], A7RMK8[16-273], A7RNW2[31-304], A7SK95[51-323], (starlet sea anemone) A7SMP0[14-255], A7SUT9[35-272], A7SX90[60-306] Trichoplax adhaerens (placozoa) B3RLQ6[1-250], B3RPH8[92-333], B3RR50[35-269], B3RVB2[1-246], B3RZX6[33-315], B3S4A5[24-295] Echinodermata Strongylocentrotus purpuratus W4XEF9[35-203], W4XLT3[87-357], W4XPP2[187-448], W4XRJ0[6-285], (purple sea urchin) W4Y6W2[96-374], W4YLQ0[62-336], W4YSD6[137-392], W4YZB8[14-226] Choanozoa Monosiga brevicollis (choanoflagellate) A9UP70[2-246], A9UVV1[36-308], A9V275[82-331], A9V9C1[1-249], A9V9J9[172-425] Fungi Schizosaccharomyces pombe O94640[147-432], Q9UR39[48-339], Q9USN7[16-271] (fission yeast) Emericella nidulans (fungi) A0A1U8QFB7[17-313], C8V3W5[166-461], P0CS88[24-281], Q5AR98[749-1049], Q5AW69[24-278], Q5BE04[115-410], Q71SB1[32-383] Aspergillus terreus (fungi) Q0C868[9-263], Q0CE82[32-383], Q0CHQ4[24-278], Q0CI01[12-320], Q0CR31[167-479], Q0CUC7[324-613], Q0D1D9[84-377] Gibberella zeae A0A0E0RU38[15-591], A0A0E0SF53[165-455], A0A1C3YLX8[38-323], (wheat head blight fungus) I1RAD3[121-416], I1RL32[320-606], I1RND2[32-375], I1RXZ1[35-260] Fusarium oxysporum N4TRF3[212-577], N4TWZ4[118-405], N4UFC7[160-426], N4UJE6[314-597], (fusarium vascular wilt) N4UN44[25-263], N4UPD9[32-313], W9IQ88[23-279], W9IRF6[36-319] Neurospora crassa (red bread mold) A0A0B0DNL3[112-407], A0A0B0DU72[15-529], A0A0B0DYE3[23-277], Q6MFN0[197-517], Q7RXM3[40-394], Q7SB01[1-216], V5IM98[9-328] Candida albicans (yeast) A0A1D8PHV4[293-564], O59923[229-488], Q5A1W9[23-328], Q5A985[9-264], Q5AI90[6-306], Q5AQ47[290-585] Eremothecium gossypii (yeast) Q750H1[10-272], Q750R0[121-414], Q757M7[231-516], Q758J1[46-354], Q75DM1[9-319] Saccharomyces cerevisiae P06700[245-529], P53685[191-470], P53686[13-286], P53687[42-349], (baker's yeast) P53688[77-370] Cryptococcus neoformans (fungi) J9VDW4[26-323], J9VN91[33-357], J9VPM7[30-348], J9VSG8[61-347], J9W002[147-450] Ustilago maydis (corn smut fungus) A0A0D1CD62[181-480], A0A0D1CIL7[6-293], A0A0D1DNZ2[37-337], A0A0D1DSY4[117-563], A0A0D1E6Y0[26-373] Encephalitozoon cuniculi (fungi) M1JKD1[74-358] Plants Chlamydomonas reinhardtii (green alga) D9I2J2[43-280], D9IPD0[73-396] Ostreococcus lucimarinus (green alga) A4S2L7[31-308] Amborella trichopoda (understory shrub) W1PDN6[35-269], W1PNP1[30-319] Arabidopsis thaliana (mouse-ear cress) F4KCI5[1-269], Q94AQ6[83-371], Q9FE17[35-269] Oryza sativa (rice) A2XBC4[245-501], B8ARK7[35-270], B8BNG4[102-390] Triticum aestivum (wheat) A0A1D6D8U6[105-393], Q0Z9W4[35-269] Zea mays (maize) A0A1D6IU93[88-348], A0A1D6J8P6[35-288], A0A1D6P1A4[235-463], A0A1D6P725[102-265] Physcomitrella patens (moss) A9RKY5[69-358], A9SDL4[38-323], A9T098[1-225], A9TNF3[36-271], A9TX70[137-405] Single cell eucharyotes Plasmodium falciparum Q8IE47[1-273], Q8IKW2[36-433] (protozoan parasite) Plasmodium yoelii yoelii Q7RP35[36-418], Q7RPA5[13-278] (protozoan parasite) Entamoeba dispar (amoeba) B0E5U4[123-383], B0EBU9[18-265], B0ETW4[326-610] Dictyostelium discoideum (slime mold) Q54GV7[291-542], Q54LF0[477-778], Q54P49[169-445], Q54QE6[239-505], Q55DB0[35-302] Giardia intestinalis (parasite) E1F2G4[49-353], E1F5B1[154-507], E1F5B2[10-308], E1F694[59-373], E1F7W5[115-432]

S-9 Leishmania infantum (parasite) A4I0H7[5-320], A4IAM7[1-241], Q8I6E4[3-373] Leishmania major (parasite) Q25337[3-373], Q4Q2Y6[1-241], Q4QB33[11-308] Trypanosoma brucei (parasite) Q57V41[1-349], Q57YZ9[3-303], Q584D5[1-244] Phaeodactylum tricornutum (diatom) B5Y434[35-269], B7FQB1[2-219], B7FZ44[4-297], B7G335[74-366], B7G8W4[61-382], B7GE06[3-263] Archaea Aeropyrum pernix Q9YB13[5-245] Hyperthermus butylicus A2BMS1[12-256] Sulfolobus solfataricus Q97VX5[4-246] Caldivirga maquilingensis A8MBU4[11-254] Pyrobaculum aerophilum Q8ZT00[1-242], Q8ZU41[11-254] Pyrobaculum neutrophilum B1YAY2[9-250], B1YDE6[4-247] Archaeoglobus fulgidus O28597[1-245], O30124[6-251] Haloarcula marismortui Q5V4Q5[14-260] Methanobrevibacter smithii D2ZPE3[2-240] Methanopyrus kandleri Q8TWG0[4-250] Methanococcoides burtonii Q12Y78[2-245] Pyrococcus abyssi Q9UZE7[1-250] Pyrococcus furiosus Q8U1Q1[1-250] Pyrococcus horikoshii O58669[3-249] Bacteria Firmicutes Bacillus anthracis Q81NT6[5-242], Q9RMW1[19-298], Q9X2Y4[23-287] Bacillus cereus Q817S6[22-267], Q81C38[30-248] Bacillus halodurans Q9KEE5[1-237] Bacillus subtilis O07595[1-247] Geobacillus kaustophilus Q5KZE8[2-247], Q5L014[1-242] Listeria monocytogenes Q71W25[2-229], Q8Y3U2[2-229] Oceanobacillus iheyensis Q8ELR0[1-236] Staphylococcus aureus A0A0E0VLQ6[36-331], A0A0E0VSF3[9-246], A0A0E1VND0[19-314], A0A0E1VQS6[9-246], A0A0H3JQ59[20-315], A0A0H3JSS8[22-315], A0A1D4GWV9[1-163], P66815[6-243], Q2G148[19-314], Q53700[6-243], Q5HE07[6-243], Q6G7B7[6-243], Q6GEN2[6-243], T1Y5W4[20-315], T1YBN1[9-246] Clostridium acetobutylicum Q97MB4[5-245] Clostridium bolteae R0ANA7[5-248], R0BDL4[3-250], R0BP84[6-240], R0C4P1[5-248], R5F057[31-320], R5FLT4[3-250] Clostridium botulinum A0A0L7DSG2[5-246], A0A0M0ACZ2[4-245], B1ID22[1-247] Clostridioides difficile A0A1R5BSA7[1-245], Q187T0[1-359], T4BUW4[8-248], T4JSW1[7-241], T4NMA7[1-241] Clostridium perfringens A0A140GRJ0[35-273], Q8XNS6[3-244] Aerococcus urinae F2I5H1[2-243], F2I857[19-290] Enterococcus faecalis Q839C6[7-237], S4D5X5[8-281] Lactobacillus acidophilus Q5FIL3[1-266], Q5FMQ6[12-239] Lactobacillus amylovorus F0TGP4[5-266], F0THZ6[7-234] Lactobacillus gasseri A0A133PBX8[8-237], D1YKN2[8-237], D1YLJ9[5-269] Lactobacillus johnsonii A0A137PK42[5-266], Q74LU0[8-237] Lactobacillus parafarraginis G9ZKM4[4-238], G9ZLW5[18-290] Lactobacillus plantarum Q88ZA0[5-234] Oenococcus oeni Q04D36[4-233] Pediococcus pentosaceus Q9F3X3[19-260] Streptococcus agalactiae A0A0H1V4N3[16-292], A0A1S7DZ71[16-291], Q8DX62[12-299] Streptococcus mutans Q8DU02[23-286] Streptococcus pyogenes P0DN71[14-293]

S-10 Selenomonas sputigena C9LSM9[12-255], C9LYS2[4-287], F4EXI0[12-293] Caldanaerobacter subterraneus Q8R984[11-250], Q8R9N6[4-242] Veillonella parvula D1BM16[11-290] Actinobacteria Bifidobacterium longum A0A075NJD7[1-251] Corynebacterium efficiens Q8FPM9[2-246], Q8FRV5[4-254], Q8FUC8[1-281] Corynebacterium glutamicum A0A1B4WH63[3-281], Q8NSM4[11-258] Mycobacterium avium A0A0E2WKW5[1-188], A0QC96[1-233], A0QDH4[1-281] Mycobacterium paratuberculosis Q73WM7[1-237], Q73XP9[1-281] Mycobacterium tuberculosis A0A051TWE9[1-281], A0A0T9LWP9[1-211], A0A0T9M5P2[32-301], A0A1K3EE52[36-280], P9WGG2[1-237] Rubrobacter xylanophilus Q1AWC3[1-233] Streptomyces coelicolor Q8CJM9[1-241], Q9RL35[20-294] Thermobifida fusca Q47Q95[8-256] Proteobacteria Alteromonas macleodii A0A0E0Y7W5[1-371], A0A126Q0Q4[1-240] Shewanella piezotolerans B8CQZ1[1-236] Bdellovibrio bacteriovorus Q6MH60[135-372], Q6MJJ2[1-235] Bordetella bronchiseptica A0A0H3LPW0[19-256], Q7WLE5[1-274] Bordetella pertussis Q7VX46[1-274] Cupriavidus metallidurans A0A132H9S4[1-277], A0A132HNH3[7-244] Cupriavidus necator A0A1K0IGB6[3-287], A0A1K0J7Q2[14-255] Ralstonia solanacearum A0A1L4B3K1[6-272], A0A1L4B4X7[11-405], Q8Y015[12-246] Campylobacter jejuni Q5HU51[1-233], Q9JN05[1-233] Helicobacter pylori A0A083YGC1[1-234], A0A0B2ES39[1-229], A0A0J8GZ38[1-229], A0A1A9HF85[1-229], A0A1Q2R7Z2[1-219], A0A1Q2RQM4[1-244], I9P5H8[1-219], M3LLA4[11-163], M3PL00[11-237], M3R0D2[11-286], M3T1K6[11-163], M4ZQT1[1-229], M5Y9H8[1-222], O25849[1-229], Q9ZJW8[1-234] Caulobacter vibrioides Q9A2S6[1-238] Pelobacter carbinolicus Q3A6W7[23-272] Escherichia coli E1IUR3[1-365], P75960[1-274], Q8FIM4[1-273] Photorhabdus luminescens A0A1B8YE18[32-275], Q7MZG5[10-288], Q7N3L1[21-281] Salmonella typhimurium P0A2F2[1-273] Yersinia pestis Q8ZFR1[1-272] Chromobacterium violaceum Q7NZ63[32-282] Aggregatibacter actinomycetemcomitans Q9ZAB8[1-208] Actinobacillus pleuropneumoniae A3N1I5[17-255], E0F2K4[4-291], E0F2K8[1-239] Haemophilus influenzae A0A0E1SNA4[6-274], E1X7D6[1-234], E1X7E1[3-281] Azotobacter vinelandii C1DGE0[6-282], C1DPP8[6-243], C1DPQ2[2-254] Pseudomonas aeruginosa A0A0F6UA54[10-371], A0A0G6AI48[1-268], Q9I4E1[6-256], Q9I4L0[2-250], V6AKG1[1-365] Pseudomonas syringae A0A0D0LFF6[4-250], A0A0D0LN38[8-282], A0A0D0M0R2[1-247], A0A0N0G7Q9[1-257], A0A0N0G822[1-235], A0A0N0X3S7[8-281], A0A0N0X849[7-254], A0A0P9URV7[14-345], Q87W79[2-252], Q87XB5[182-453], Q882K4[7-281], Q885X7[1-248], Q88A13[1-228] Bradyrhizobium diazoefficiens Q89EA6[1-273], Q89LY4[13-254] Rhodopseudomonas palustris Q21D23[194-415], Q6N6U0[11-253], Q6NBK9[67-290] Sinorhizobium fredii P55383[21-252] Sinorhizobium meliloti A0A0E0UN59[2-272], Q7ANP5[2-272] Dechloromonas aromatica Q47EY8[15-265], Q47K66[5-271] Magnetospirillum magnetotacticum A0A0C2YPJ4[1-234] Vibrio vulnificus Q8D9J9[1-239] Xanthomonas campestris A0A109HGG3[1-87], B0RM75[1-285], B0RX96[46-430], Q4UZX0[1-285] Other bacteria

S-11 Petrotoga mobilis A0A101HSV0[6-254] Thermotoga maritima Q9WYW0[3-246] Thermotoga neapolitana B9KBG1[1-244] Aquifex aeolicus O67919[1-236] Bacteroides fragilis R5RZ55[57-300], R5RZA3[171-484] Bacteroides thetaiotaomicron Q8A3H9[1-234] Cytophaga hutchinsonii Q11SL1[2-222] Rhodothermus marinus D0MEH4[10-254], D0MJ81[4-278] Chloroflexus aurantiacus A9WFY1[5-250] Deinococcus radiodurans Q9RYD4[4-246] Thermus thermophilus Q5SIH7[2-249] Fusobacterium nucleatum F7KWU8[40-327], F9EMH9[16-239], Q8REC3[10-245] Rhodopirellula baltica Q7UFQ9[1-239] Leptospira interrogans Q72RR0[1-246], Q8F3Z6[1-246] Treponema denticola Q73KE1[9-251], Q73PX0[4-285]

S-12 Table S4. BLAST search results from the NCBI database. Species with proteins similar to Sir2La (Class U) and LBA1649 (Class M). Species and grouping included are based primarily on BLAST results and (1,2). Class M Class M Class U Class U

NQR NQR NEX NEX XFP XFP Group Species Sir2 Group Species Sir2

floricola L. floricola L. futsaii    amylophilus L. amylophilus L. ginsenosidimutans  L. amylotrophicus  L. heilongjiangensis     delbrueckii L. acetotolerans   L. kimchiensis

L. acidophilus   L. kimchii L. amylolyticus   L. mindensis  L. amylovorus   L. nantensis    L. apis   L. nodensis   L. bombicola   L. paralimentarius L. crispatus   L. tucceti   L. delbrueckii  L. versmoldensis   L. equicursoris  dextrinicus L. concavus  L. fornicalis L. dextrinicus  L. gallinarum    composti L. composti  L. gasseri     perolens L. harbinensis  L. gigeriorum   L. perolens  L. hamsteri     L. shenzhenensis  L. helsingborgensis   casei L. brantae  L. helveticus     L. camelliae  L. hominis   L. casei  L. iners L. manihotivorans  L. intestinalis   L. nasuensis  L. jensenii   L. pantheris  L. johnsonii   L. paracasei  L. kalixensis   L. porcinae L. kefiranofaciens    L. rhamnosus  L. kimbladii   L. saniviri  L. kitasatonis   L. sharpeae  L. kullabergensis  L. thailandensis  L. melliventris   L. zeae  L. pasteurii   selangorensis L. selangorensis  L. psittaci   sakei L. curvatus   L. taiwanensis   L. fuchuensis     L. ultunensis L. graminis   mellifer L. mellifer  L. sakei L. mellis  coryniformis L. backii  alimentarius L. alimentarius   L. bifermentans  L. allii  L. coryniformis   L. bobalius L. rennini  L. crustorum  algidus L. algidus   L. farciminis salivarius L. acidipiscis 

S-13 Class M Class M Class U Class U

NQR NQR NEX NEX XFP XFP Group Species Sir2 Group Species Sir2

L. agilis  vaccinostercus L. hokkaidonensis  L. animalis L. oligofermentans  L. apodemi  L. suebicus  L. aquaticus  L. vaccinostercus  L. aviarius  reuteri L. alvi L. cacaonum L. antri   L. capillatus  L. coleohominis  L. ceti  L. equigenerosi  L. equi    L. fermentum   L. ghanensis  L. frumenti    L. hayakitensis L. gastricus  L. hordei L. gorillae  L. mali L. ingluviei  L. murinus L. mucosae  L. nagelii  L. oris  L. oeni  L. panis   L. pobuzihii  L. pontis   L. ruminis  L. reuteri   L. saerimneri L. secaliphilus  L. salivarius  L. vaginalis L. satsumensis  L. wasatchensis   L. sucicola collinoides L. collinoides  L. uvarum  L. kimchicus  L. vini  L. malefermentans  Pediococcus P. acidilactici L. mixtipabuli  P. argentinicus  L. odoratitofui  P. cellicola L. oryzae  P. claussenii  L. paracollinoides  P. damnosus L. pentosiphilus  P. ethanolidurans L. silagei  P. inopinatus L. silagincola  P. lolii L. similis 

P. parvulus brevis L. acidifarinae  P. pentosaceus L. brevis    P. siamensis L. hammesii 

P. stilesii L. koreensis  plantarum L. fabifermentans  L. namurensis   L. herbarum  L. parabrevis  L. paraplantarum L. paucivorans  L. pentosus  L. senmaizukei  L. plantarum   L. spicheri  L. xiangfangensis  L. zymae  rossiae L. rossiae  kunkeei L. apinorum  L. siliginis  L. kunkeei 

S-14 Class M Class U

NQR NEX XFP Group Species Sir2

L. ozensis  fructivorans L. florum  L. fructivorans  L. homohiochii L. lindneri  L. sanfranciscensis  buchneri L. buchneri  L. curieae  L. diolivorans  L. farraginis  L. hilgardii L. kefiri  L. kisonensis  L. otakiensis  L. parabuchneri   L. parafarraginis   L. parakefiri  L. rapi  L. senioris  L. sunkii  

S-15 Table S5. Substrate synthesis references. Compound Reference 1a) Ac-Kfor-AMC This work 1b) Ac-Kac-AMC – 1c) Ac-Kpro-AMC This work 1d) Ac-Kbut-AMC This work 1h) Ac-Ki-but-AMC This work 1i) Ac-Ki-val-AMC This work 1k) Ac-Kcr-AMC This work 1o) Ac-Kbio-AMC This work 1p) Ac-Klip-AMC This work 1q) Ac-Khib-AMC This work 1r) Ac-K(R)-3-hbut-AMC This work 1s) Ac-K(S)-3-hbut-AMC This work 2b) Ac-LGKac-AMC (3,4) 2c) Ac-LGKpro-AMC This work 2d) Ac-LGKbut-AMC This work 2h) Ac-LGKi-but-AMC (5) 2i) Ac-LGKi-val-AMC (5) 2k) Ac-LGKcr-AMC (4) 2o) Ac-LGKbio-AMC (5) 2v) Ac-LGKsuc-AMC (4) 2w) Ac-LGKglu-AMC (6) 2x) Ac-LGKmg-AMC (6) 2y) Ac-LGKhmg-AMC (6) 2z) Ac-LGKtfa-AMC (4,7) 3b) Ac-ETDKac-AMC (8) 3g) Ac-ETDKmyr-AMC (8) 3h) Ac-ETDKi-but-AMC (5) 3i) Ac-ETDKi-val-AMC (8) 3j) Ac-ETDK(S)-mebut-AMC (8) 3k) Ac-ETDKcr-AMC (8) 3l) Ac-ETDKmeacr-AMC (5) 3m) Ac-ETDK(3-mecr)-AMC (5) 3n) Ac-ETDK(2-mecr)-AMC (5) 3t) Ac-ETDK(R/S)-3-hbut-AMC (8) 3u) Ac-ETDKacac-AMC (8) 4a) Ac-TARKfor-AMC (9) 4b) Ac-TARKac-AMC (4,9) 4c) Ac-TARKpro-AMC (9) 4d) Ac-TARKbut-AMC (9) 4e) Ac-TARKhex-AMC (9) 4f) Ac-TARKoct-AMC (9) 4g) Ac-TARKmyr-AMC (9) 4k) Ac-TARKcr-AMC (9) 5a) Ac-QPKKfor-AMC (9) 5b) Ac-QPKKac-AMC Fluor de lys-SIRT2 substrate (Enzo KI179) and (9) 5c) Ac-QPKKpro-AMC (9) 5d) Ac-QPKKbut-AMC (9) 5e) Ac-QPKKhex-AMC (9) 5f) Ac-QPKKoct-AMC (9) 5g) Ac-QPKKmyr-AMC (9) 5h) Ac-QPKKi-but-AMC (9) 5i) Ac-QPKKi-val-AMC (9) 5k) Ac-QPKKcr-AMC (9)

S-16 Synthesis of substrates

Ac-Lys(formyl)-AMC (1a); (S)-2-Acetamido-6-formamido-N-(4-methyl-2- oxo-2H-chromen-7-yl)hexanamide. Ammonium trifluoroacetate S1 (4) (99 mg, 215 µmol) was suspended in ethyl formate (8.8 mL) under Ar, iPr2NEt (45 µL) was added, affording a milky white suspension, which was heated at reflux for 23 h. The resulting suspension was evaporated to dryness, and the resulting residue was purified by prep-HPLC, to afford desired amide 1a (69 mg, 96%) 1 as a white fluffy material. H NMR (DMSO-d6) δ 10.49 (s, 1H, NHAMC), 8.21 (d, J = 7.6, 1H, NHα), 8.03–7.94 (m, 2H, NHε, HCO), 7.77 (d, J = 2.0, 1H, H8AMC), 7.70 (d, J = 8.6, 1H, H5AMC), 7.47 (dd, J = 8.7, 2.0, 1H, H6AMC), 6.25 (d, J = 1.4, 1H, H3AMC), 4.35 (td, J = 8.0, 5.2, 1H, Hα), 3.05 (q, J = 6.4, 2H, Hε), 2.38 (d, J = 1.3, 3H, 4AMC-CH3), 1.86 (s, 3H, CH3CO), 13 1.75–1.50 (m, 2H, Hβ), 1.50–1.18 (m, 4H, Hγ, Hδ). C NMR (DMSO-d6) δ 171.8 (CONHAMC), 169.5 (CH3CO), 160.9 (HCO), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 126.0 (C5AMC), 115.3 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 53.6 (Cα), 36.9 (Cε), 31.5 (Cβ), 28.8 (Cδ), + + 23.0 (Cγ), 22.4 (CH3CO), 18.0 (4AMC-CH3). HRMS m/z 374.1702 ([M+H] ,C19H24N3O5 Calcd 374.1710).

Ac-Lys(propanoyl)-AMC (1c); (S)-2-Acetamido-N-(4-methyl-2-oxo-2H- chromen-7-yl)-6-propanamidohexanamide. Ammonium trifluoroacetate S1 (4) (102 mg, 222 µmol) was suspended in anh CH2Cl2 (2.5 mL) and iPr2NEt (95 µL) at 0 °C, then propanoic anhydride (33 µL, 0.261 mmol) was added, resulting in a clear solution within 2 min. After stirring for 10 min, MeOH (2.0 mL) was added and the reaction mixture evaporated to dryness and purified by DCVC (0–6 % MeOH in CH2Cl2). The resulting residue was taken up in 10% MeOH in CH2Cl2 (35 mL, v/v) and washed with aq. HCl (1 M, 2 × 5 mL) and sat. aq. NaHCO3 (5 mL), each time back-extracting with 10% MeOH in CH2Cl2 (3 ×5 mL, v/v). The combined organic phase was dried over MgSO4, then evaporated to dryness to afford desired amide 1c (76 1 mg, 95%) as a white solid. TLC (10% MeOH in CH2Cl2): Rf = 0.3. H NMR (DMSO-d6) δ 10.49 (s, 1H, NHAMC), 8.21 (d, J = 7.5, 1H, NHα), 7.78 (d, J = 2.1, 1H, H8AMC), 7.68–7.77 (m, 2H, NHε, H5AMC), 7.49 (dd, J = 8.7, 2.1, 1H, H6AMC), 6.26 (d, J = 1.1, 1H, H3AMC), 4.36 (td, J = 8.1, 5.5, 1H, Hα), 3.01 (q, J = 6.4, 2H, Hε), 2.36–2.45 (d, J = 1.1, 3H, 4AMC-CH3), 2.02 (q, J = 7.6, 2H, NHεCOCH2), 1.87 (s, 3H, CH3CONH), 13 1.50–1.75 (m, 2H, Hβ), 1.19–1.49 (m, 4H, Hγ,Hδ), 0.96 (t, J = 7.6, 3H, NHεCOCH2CH3). C NMR (DMSO- d6) δ 172.7 (CONHε), 171.9 (CONHAMC), 169.5 (CH3CO), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 53.6 (Cα), 38.2 (Cε), 31.6 (Cβ), 28.9 (Cδ), 28.5 (CH2CONHε), 23.0 (Cγ), 22.4 (CH3CONH), 18.0 (4AMC-CH3), 10.0 + + (CH2CH3). UPLC-MS tR 1.13 min, m/z 402.4 ([M+H] , C21H28N3O5 Calcd 402.2); HRMS m/z 402.2021 + + ([M+H] , C21H28N3O5 Calcd 402.2023).

Ac-Lys(butanoyl)-AMC (1d); (S)-2-Acetamido-6-butanamido-N-(4- methyl-2-oxo-2H-chromen-7-yl)hexanamide. Ammonium trifluoroacetate S1 (4) (103 mg, 224 µmol) was suspended in anh CH2Cl2 (2.5 mL) and iPr2NEt (95 µL) at 0 °C, then butyric anhydride (43 µL, 0.261 mmol) was added, resulting in a clear solution within 2 min. After stirring for 30 min, MeOH (200 µL) was added and the reaction mixture evaporated to dryness and purified by DCVC (0–6 % MeOH in CH2Cl2) to afford desired amide 1d (88 mg, >99%) 1 as a white solid. TLC (10% MeOH in CH2Cl2): Rf = 0.3. H NMR (DMSO-d6) δ 10.50 (s, 1H, NHAMC), 8.21 (d, J = 7.5, 1H, NHα), 7.79 (d, J = 2.0, 1H, H8AMC), 7.68–7.77 (m, 2H, NHε, H5AMC), 7.49 (dd, J = 8.7, 2.0, 1H, H6AMC), 6.26 (d, J = 1.4, 1H, H3AMC), 4.36 (td, J = 8.1, 5.5, 1H, Hα), 3.01 (q, J = 6.3, 2H, Hε), 2.39 (d, J = 1.4, 3H, 4AMC-CH3), 1.98 (t, J = 7.3, 2H, CH2CONHε), 1.87 (s, 3H, CH3CONH), 1.52–1.76 (m, 2H, Hβ), 1.20–1.52 (m, 6H, Hγ, Hδ, CH2CH3), 0.80 (t, J = 7.4, 3H, CH2CH3). 13 C NMR (DMSO-d6) δ 171.84 (CONHAMC/CONHε), 171.76 (CONHAMC/CONHε), 169.5 (CH3CO), 160.0

S-17 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.0 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 53.6 (Cα), 38.1 (Cε), 37.4 (CH2CONHε), 31.6 (Cβ), 28.9 (Cδ), 22.9 (Cγ), 22.4 (CH3CONH), 18.7 (CH2CH3), 18.0 (4AMC-CH3), 13.6 (CH2CH3). UPLC-MS tR 1.21 min, m/z 416.4 + + + + ([M+H] , C22H30N3O5 Calcd 416.2); HRMS m/z 416.2181 ([M+H] ,C22H30N3O5 Calcd 416.2180).

Ac-Lys(isobutyryl)-AMC (1h); (S)-2-Acetamido-N-(4-methyl-2-oxo-2H- chromen-7-yl)-6-(2-methylpropanamido)hexanamide. Ammonium trifluoroacetate S1 (4) (101 mg, 220 µmol) was suspended in anh CH2Cl2 (3.0 mL) and iPr2NEt (117 µL) at 0 °C, then isobutyric anhydride (66 µL, 0.330 mmol) was added, resulting in a clear solution within 2 min. After stirring for 45 min, the reaction mixture was evaporated to dryness, then redissolved in DMF (1.5 mL) and purified by prep-HPLC, to afford desired amide 1h (63 mg, 1 77%) as a white fluffy material. H NMR (DMSO-d6) δ 10.48 (s, 1H, NHAMC), 8.20 (d, J = 7.5, 1H, NHα), 7.78 (d, J = 2.0, 1H, H8AMC), 7.71 (d, J = 8.7, 1H, H5AMC), 7.67 (t, J = 5.7, 1H, NHε), 7.49 (dd, J = 8.7, 2.0, 1H, H6AMC), 6.26 (d, J = 1.3, 1H, H3AMC), 4.36 (td, J = 8.2, 5.5, 1H, Hα), 3.01 (q, J = 6.4, 2H, Hε), 2.39 (d, J = 1.3, 3H, 4AMC-CH3), 2.29 (hept, J = 6.9, 1H, COCH), 1.87 (s, 3H, CH3CONH), 1.50–1.76 (m, 2H, Hβ), 1.19–1.50 (m, 4H, Hγ, Hδ) , 0.96 (d, J = 6.9, 3H, CHCH3,A), 0.95 (d, J 13 = 6.9, 3H, CHCH3,B). C NMR (DMSO-d6) δ 175.9 (CONHε), 171.9 (CONHAMC), 169.5 (CH3CO), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.0 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 53.6 (Cα), 38.1 (Cε), 34.0 (CH(CH3)2), 31.6 (Cβ), 28.9 (Cδ), 22.9 (Cγ), 22.4 + + (CH3CONH), 19.6 (CH(CH3)2), 18.0 (4AMC-CH3). UPLC-MS tR 1.22 min, m/z 416.4 ([M+H] , C22H30N3O5 + + Calcd 416.2); HRMS m/z 416.2181 ([M+H] , C22H30N3O5 Calcd 416.2180).

Ac-Lys(isovaleryl)-AMC (1i); (S)-2-Acetamido-6-(3-methylbutanamido)- N-(4-methyl-2-oxo-2H-chromen-7-yl)hexanamide. Ammonium trifluoroacetate S1 (4) (101 mg, 220 µmol) was suspended in MeCN (3.0 mL) and iPr2NEt (117 µL) under Ar at 0 °C. Isovaleric acid (31 µL, 0.330 mmol) and HATU (125 mg, 0.329 mmol) was premixed for 10 min in MeCN (1 mL), then added to the amine solution. After stirring for 45 min, the reaction mixture was evaporated to dryness. Approx. half of the resulting residue was dissolved in DMF (1.5 mL) and purified by prep-HPLC, to afford desired amide 1i (31 1 mg, 37%) as a white fluffy material. H NMR (DMSO-d6) δ 10.48 (s, 1H, NHAMC), 8.19 (d, J = 7.6, 1H, NHα), 7.78 (d, J = 2.0, 1H, H8AMC), 7.66–7.76 (m, 2H, NHε, H5AMC), 7.49 (dd, J = 8.7, 2.0, 1H, H6AMC), 6.26 (d, J = 1.4, 1H, H3AMC), 4.36 (td, J = 8.1, 5.6, 1H, Hα), 3.02 (q, J = 6.3, 2H, Hε), 2.39 (d, J = 1.3, 3H, 4AMC-CH3), 1.83–1.96 (m, 6H, CH3CONH, COCH2CH), 1.52–1.76 (m, 2H, Hβ), 1.19–1.47 (m, 4H, Hγ, Hδ), 13 0.82 (d, J = 5.9, 6H, CHCH3). C NMR (DMSO-d6) δ 171.8 (CONHAMC), 171.3 (CONHε), 169.5 (CH3CO), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.0 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 53.6 (Cα), 44.8 (COCH2), 38.1 (Cε), 31.6 (Cβ), 29.0 (Cδ), 25.5 (CH(CH3)2), 22.9 (Cγ), 22.4 (CH3CONH), 22.3 (CH(CH3)2), 18.0 (4AMC-CH3). UPLC-MS tR 1.31 min, m/z + + + + 430.4 ([M+H] , C23H32N3O5 Calcd 430.2); HRMS m/z 430.2336 ([M+H] , C23H32N3O5 Calcd 430.2336).

Ac-Lys(crotonyl)-AMC (1k); (S)-2-Acetamido-N-(4-methyl-2-oxo-2H- chromen-7-yl)-6-(E-but-2-emamido)heaxanamide. Ammonium trifluoroacetate S1 (4) (103 mg, 224 µmol) was suspended in anh CH2Cl2 (2.5 mL) and iPr2NEt (95 µL), then crotonic anhydride (39 µL) was added. After stirring for 15 min, MeOH (2 mL) was added, then the reaction mixture adsorbed directly on silica and purified by DCVC (0–6% MeOH in CH2Cl2, v/v). The resulting residue was taken up in 10% MeOH in CH2Cl2 (35 mL, v/v) and washed with aq. HCl (1 M, 2 × 5 mL) and sat. aq. NaHCO3 (5 mL), each time back-extracting with 10% MeOH in CH2Cl2 (3 ×5 mL, v/v). The combined organic phase was dried over MgSO4, then evaporated to dryness to afford desired amide 1k (97 mg, >99%) as a white residue. TLC

S-18 1 (10% MeOH in CH2Cl2): Rf = 0.3. H NMR (DMSO-d6) δ 10.49 (s, 1H, NHAMC), 8.21 (d, J = 7.5, 1H, NHα), 7.86 (t, J = 5.6, 1H, NHε), 7.78 (d, J = 2.0, 1H, H8AMC), 7.71 (d, J = 8.7, 1H, H5AMC), 7.49 (dd, J = 8.7, 2.1, 1H, H6AMC), 6.56 (dq, J = 15.4, 6.8, 1H, CH=CHCH3), 6.26 (d, J = 1.4, 1H, H3AMC), 5.86 (dq, J = 15.1, 1.6, 1H, CH=CHCH3), 4.30–4.41 (m, 1H, Hα), 3.08 (q, J = 6.4, 2H, Hε), 2.39 (d, J = 1.4, 3H, 4AMC-CH3), 1.87 (s, 3H, CH3CO), 1.75 (dd, J = 6.9, 1.7, 3H, CH=CHCH3), 1.54–1.72 (m, 2H, Hβ), 1.19–1.50 (m, 4H, Hγ, 13 Hδ). C NMR (DMSO-d6) δ 171.9 (CONHAMC), 169.5 (CH3CO), 164.8 (CONHε), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 137.3 (CH=CHCH3), 126.0 (C5AMC/CH=CHCH3), 125.9 (C5AMC/CH=CHCH3), 115.3 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 53.6 (Cα), 38.2 (Cε), 31.5 (Cβ), 28.9 (Cδ), 23.0 (Cγ), 22.4 (CH3CO), 18.0 (4AMC-CH3), 17.3 (CH=CHCH3). UPLC-MS tR 1.38 + + + + min, m/z 414.1 ([M+H] , C22H28N3O5 Calcd 414.2); HRMS m/z 414.2026 ([M+H] ,C22H28N3O5 Calcd 414.2023).

Ac-Lys(biotinoyl)-AMC (1o); (S)-2-acetamido-N-(4-methyl-2-oxo-2H- chromen-7-yl)-6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanamido)hexanamide. (+)-Biotinyl-N-hydroxy- succinimide (25 mg, 73 µmol) was suspended in anh. CH2Cl2 (2 mL) at 0 °C, followed by addition of iPr2NEt (18 µL, 105 µmol) and ammonium trifluoroacetate S1 (4) (32 mg, 72 µmol). The resulting suspension was stirred for 90 min while allowing to heat to r.t., then anh. CH2Cl2 (1 mL) and iPr2NEt (18 µL, 105 µmol) was added and stirring continued. After stirring for a total of 3 h, MeOH (1 mL) was added to the reaction mixture. The resulting solution was evaporated to dryness and the resulting residue taken up in DMF (1.2 mL) and MeOH (0.2 mL) and purified 1 by prep-HPLC, to afford desired amide 1o (18 mg, 43%) as a white fluffy material. H NMR (DMSO-d6) δ 10.49 (s, 1H, NHAMC), 8.20 (d, J = 7.5, 1H, NHα), 7.78 (d, J = 2.1, 1H, H8AMC), 7.75 (t, J = 5.9, 1H, NHε), 7.72 (d, J = 8.7, 1H, H5AMC), 7.49 (dd, J = 8.7, 2.1, 1H, H6AMC), 6.41 (s, 1H, H–N3biotin), 6.36 (s, 1H, H– N1biotin), 6.26 (d, J = 1.4, 1H, H3AMC), 4.36 (td, J = 8.0, 5.5, 1H, Hα), 4.30 (dd, J = 7.7, 5.1, 1H, H6abiotin), 4.11 (dd, J = 7.7, 4.4, 1H, H3abiotin), 3.07 (ddd, J = 8.5, 6.1, 4.4, 1H, H4biotin), 3.01 (q, J = 6.5, 2H, Hε), 2.81 (dd, J = 12.4, 5.1, 1H, H6pro-R,biotin), 2.57 (d, J = 12.4, 1H, H6pro-S,biotin), 2.39 (d, J = 1.3, 3H, 4AMC-CH3), 2.02 (t, J = 7.4, 2H, NHCOCH2), 1.88 (s, 3H, CH3CONH), 1.75–1.54 (m, 3H, Hβ, CH2,AC4biotin), 1.53–1.19 (m, 13 9H, Hγ, Hδ, NHCOCH2CH2CH2CH2,BC4biotin). C NMR (DMSO-d6) δ 171.9 (CONHε,CONHAMC), 169.5 (CH3CO), 162.7 (C2biotin), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 61.0 (C3abiotin), 59.2 (C6abiotin), 55.4 (C4biotin), 53.6 (Cα), 39.8 (C6biotin), 38.2 (Cε), 35.2 (NHCOCH2), 31.5 (Cβ), 28.9 (Cδ), 28.2 (NHCOCH2CH2CH2), 28.0 (CH2C4biotin), 25.3 (NHCOCH2CH2), 22.9 (Cγ), 22.4 (CH3CONH), 18.0 (4AMC- + + CH3). UPLC-MS tR 1.33 min, m/z 572.2 ([M+H] , C28H38N5O6S Calcd 572.3); HRMS m/z 572.2532 + + ([M+H] , C28H38N5O6S Calcd 572.2537).

Ac-Lys((R)-lipoyl)-AMC (1p); (S)-6-(5-((R)-1,2-dithiolan-3- yl)pentanamido)-2-acetamido-N-(4-methyl-2-oxo-2H-chromen-7- yl)hexanamide. (R)-Lipoic acid (36 mg, 175 µmol) and HOBt (26 mg, 190 µmol) was suspended in anh. CH2Cl2 (2.2 mL) at 0 °C, followed by addition of iPr2NEt (57 µL, 327 µmol) and DIC (22 µL, 142 µmol). After sturring for 10 min, ammonium trifluoroacetate S1 (4) (50 mg, 109 µmol) was added, cooling was removed, and the reaction allowed to heat at r.t. After stirring for 2 h, MeOH (1 mL) was added, the reaction mixture evaporated to dryness and the resulting residue was taken up in DMF (1.5 mL) and purified by prep-HPLC, to afford desired amide 1p 1 (27 mg, 47%) as a white fluffy material. H NMR (DMSO-d6) δ 10.48 (s, 1H, NHAMC), 8.20 (d, J = 7.6, 1H, NHα), 7.78 (d, J = 2.1, 1H, H8AMC), 7.74 (t, J = 5.7, 1H, NHε), 7.72 (d, J = 8.7, 1H, H5AMC), 7.49 (dd, J = 8.7, 2.1, 1H, H6AMC), 6.26 (d, J = 1.2, 1H, H3AMC), 4.36 (td, J = 8.1, 5.5, 1H, Hα), 3.57 (dq, J = 8.7, 6.2, 1H, CHS), 3.17 (ddd, J = 10.9, 6.9, 5.5, 1H, CH2,AS), 3.10 (dt, J = 11.0, 6.8, 1H, CH2,BS), 3.01 (q, J = 6.5, 2H, Hε), 2.44–2.34 (m, 4H, CH(S)CH2,ACH2S, 4AMC-CH3), 2.01 (t, J = 7.4, 2H, NHCOCH2), 1.87 (s, 3H,

S-19 CH3CONH), 1.82 (dt, J = 12.7, 6.8, 1H, CH(S)CH2,BCH2S), 1.74–1.54 (m, 3H, CH2CH2CH2,ACHSS, Hβ), 13 1.54–1.21 (m, 9H, Hγ, Hδ, CH2CH2CH2,BCHS). C NMR (DMSO-d6) δ 171.8 (CONHε/CONHAMC), 171.7 (CONHε/CONHAMC), 169.5 (CH3CO), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.2 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.0 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 56.1 (CHS), 53.6 (Cα), 39.9 (CH(S)CH2CH2S), 38.12 (Cε/CH2S), 38.08 (Cε/CH2S), 35.3 (NHCOCH2), 34.1 (CH2CH2CH2CHSS), 31.5 (Cβ), 28.9 (Cδ), 28.3 (NHCOCH2CH2CH2), 25.1 (NHCOCH2CH2), 22.9 (Cγ), 22.4 (CH3CONH), 18.0 (4AMC- + + CH3). UPLC-MS tR 1.77 min, m/z 534.2 ([M+H] , C26H36N3O5S2 Calcd 534.2); HRMS m/z 534.2086 + + ([M+H] , C26H36N3O5S2 Calcd 534.2091).

Ac-Lys(2-hydroxyisobutanoyl)-AMC (1q); (S)-2-Acetamido-6-(2- hydroxy-2-methylpropamido)-N-(4-methyl-2-oxo-2H-chromen-7- yl)hexanamide. 2-hydroxyisobutanoic acid (20 mg, 0.194 mmol) and HOBt (22 mg, 166 mmol) was dissolved in anh CH2Cl2 (2.2 mL) and iPr2NEt (57 µL), cooled to 0 °C and DIC (22 µL) was added. After stirring for 10 min, ammonium trifluoroacetate S1 (4) (57 mg, 123 µmol) was added, cooling was removed, and the reaction allowed to heat at r.t. After stirring for 2 h, MeOH (1 mL) was added, the reaction mixture evaporated to dryness and the resulting residue was taken up in DMF (1 mL) and purified by prep-HPLC, to afford desired amide 1q (49 mg, 92%) 1 as a white fluffy material. H NMR (DMSO-d6) δ 10.48 (s, 1H, NHAMC), 8.20 (d, J = 7.5, 1H, NHα), 7.78 (d, J = 2.0, 1H, H8AMC), 7.71 (d, J = 8.7, 1H, H5AMC), 7.62 (t, J = 6.0, 1H, NHε), 7.49 (dd, J = 8.7, 2.1, 1H, H6AMC), 6.26 (d, J = 1.4, 1H, H3AMC), 4.40–4.31 (m, 1H, Hα), 3.05 (q, J = 6.7, 2H, Hε), 2.39 (d, J = 1.4, 3H, 4AMC-CH3), 1.87 (s, 3H, CH3CO), 1.75–1.55 (m, 2H, Hβ), 1.49–1.37 (m, 2H, Hδ), 1.37–1.22 (m, 2H, Hγ), 13 1.20 (d, J = 3.7, 6H, COH(CH3)2). C NMR (DMSO-d6) δ 176.3 (CONHε), 171.9 (CONHAMC), 169.5 (CH3CO), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 71.9 (COH), 53.6 (Cα), 38.0 (Cε), 31.5 (Cβ), 29.0 (Cδ), 27.8 + (COH(CH3)2), 22.8 (Cγ), 22.4 (CH3CONH), 18.0 (4AMC-CH3). UPLC-MS tR 1.24 min, m/z 432.2 ([M+H] , + + + C22H30N3O6 Calcd 432.2); HRMS m/z 454.1947 ([M+Na] , C22H29N3O6Na Calcd 454.1949).

Ac-Lys((R)-3-hydroxybutanoyl)-AMC (1r); (S)-2-Acetamido-6-((R)-3- hydroxybutanamido)-N-(4-methyl-2-oxo-2H-chromen-7-yl)hexanamide. (R)-3-Hydroxybutyric acid (32 mg, 0.307 mmol) and HOBt (36 mg, 0.268 mmol) was dissolved in anh CH2Cl2 (2.5 mL) and iPr2NEt (50 µL) at 0 °C. DIC (40 µL) was added and the reaction mixture stirred for 10 min, then added to a suspension of ammonium trifluoroacetate S1 (4) (98 mg, 213 µmol) in CH2Cl2 (2.5 mL) and iPr2NEt (50 µL). After stirring for 100 min, MeOH (1 mL) was added and the reaction mixture and evaporated to dryness. The resulting residue was dissolved in DMF (1.5 mL) and purified by prep–HPLC to afford desired amide 1r (44 mg, 53%) as a 1 white fluffy material. TLC (10% MeOH in CH2Cl2): Rf = 0.3. H NMR (DMSO-d6) δ 10.49 (s, 1H, NHAMC), 8.20 (d, J = 7.5, 1H, NHα), 7.75–7.83 (m, 2H, NHε, H8AMC), 7.72 (m, 1H d, J = 8.6, 1H, H5AMC), 7.49 (dd, J = 8.6, 1.3, 1H, H6AMC), 6.26 (d, J = 1.1, 1H, H3AMC), 4.36 (td, J = 8.2, 5.5, 1H, Hα), 3.93 (dp, J = 6.9, 6.2, 1H, CHOH), 3.02 (tt, J = 13.3, 6.9, 2H, Hε), 2.39 (d, J = 1.1, 3H, 4AMC-CH3), 2.16 (mABX,A, J = 13.8, 7.1, 1H, CH2,ACHOH), 2.06 (mABX,B, J = 13.8, 5.9, 1H, CH2,ACHOH), 1.88 (s, 3H, CH3CONH), 1.51–1.77 (m, 13 2H, Hβ), 1.20–1.49 (m, 4H, Hγ, Hδ), 1.02 (d, J = 6.2, 3H, CHOHCH3). C NMR (DMSO-d6) δ 171.9 (CONHAMC), 170.5 (CONHε), 169.5 (CH3CO), 160.0 (C2AMC), 153.7 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 63.8 (CHOH), 53.6 (Cα), 45.4 (CH2CHOH), 38.1 (Cε), 31.5 (Cβ), 28.9 (Cδ), 23.4 (CHOHCH3), 22.9 (Cγ), 22.4 + + (CH3CONH), 18.0 (4AMC-CH3). UPLC-MS tR 1.04 min, m/z 432.4 ([M+H] , C22H30N3O6 Calcd 432.2); + + HRMS m/z 432.2134 ([M+H] , C22H30N3O6 Calcd 432.2129).

S-20 Ac-Lys((S)-3-hydroxybutanoyl)-AMC (1s); (S)-2-Acetamido-6-((S)-3- hydroxybutanamido)-N-(4-methyl-2-oxo-2H-chromen-7-yl)hexanamide. (S)-3-hydroxybutyric acid (27 mg, 0.261 mmol) and HOBt (36 mg, 0.268 mmol) was dissolved in anh CH2Cl2 (2.5 mL) and iPr2NEt (45 µL) at 0 °C. DIC (40 µL) was added and the reaction mixture stirred for 10 min, then added to a suspension of ammonium trifluoroacetate S1 (4) (115 mg, 250 µmol) in CH2Cl2 (2.0 mL) and iPr2NEt (100 µL). After stirring for 2 h a second aliquot of activated acid [made by stirring S-3-hydroxybutyric acid (17 mg, 0.167 mmol), HOBt (22 mg, 0.166 mmol) and DIC (40 µL) in CH2Cl2 (2.5 mL) and iPr2NEt (50 µL) for 5 min] was added. After stirring for additional 40 min, H2O (1 mL) was added and the reaction mixture stirred for 30 min, then evaporated to dryness and coevaporated with MeCN (4 × 3 mL). The resulting residue was dissolved in DMF (1.5 mL) and purified by prep–HPLC for afford desired amide 1s (82 mg, 85%) as a 1 white fluffy material. TLC (10% MeOH in CH2Cl2): Rf = 0.2. H NMR (DMSO-d6) δ 10.49 (s, 1H, NHAMC), 8.20 (d, J = 7.5, 1H, NHα), 7.74–7.82 (m, 2H, NHε, H8AMC), 7.72 (d, J = 8.6, 1H, H5AMC), 7.49 (dd, J = 8.6, 1.3, 1H, H6AMC), 6.26 (d, J = 1.1, 1H, H3AMC), 4.36 (td, J = 8.1, 5.4, 1H, Hα), 3.92 (h, J = 6.2, 1H, CHOH), 3.02 (hept, J = 6.4, 2H, Hε), 2.39 (d, J = 1.1, 3H, 4AMC-CH3), 2.16 (mABX,A, J = 13.8, 7.1, 1H, CH2,ACHOH), 2.05 (mABX,B, J = 13.8, 5.9, 1H, CH2,ACHOH), 1.88 (s, 3H, CH3CONH), 1.53–1.77 (m, 2H, Hβ), 1.20–1.48 13 (m, 4H, Hγ, Hδ), 1.02 (d, J = 6.2, 3H, CHOHCH3). C NMR (DMSO-d6) δ 171.9 (CONHAMC), 170.5 (CONHε), 169.5 (CH3CO), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.3 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.7 (C8AMC), 63.8 (CHOH), 53.6 (Cα), 45.3 (CH2CHOH), 38.1 (Cε), 31.5 (Cβ), 28.9 (Cδ), 23.3 (CHOHCH3), 22.9 (Cγ), 22.4 (CH3CONH), 18.0 (4AMC-CH3). UPLC- + + + MS tR 1.04 min, m/z 432.4 ([M+H] , C22H30N3O6 Calcd 432.2); HRMS m/z 432.2120 ([M+H] , + C22H30N3O6 Calcd 432.2129).

Ac-Leu-Gly-Lys(propanoyl)-AMC (2c); (S)-2-(2-((S)-2- acetamido-4-methylpentanamido)acetamido)-N-(4-methyl-2- oxo-2H-chromen-7-yl)-6-propionamidohexanamide. iPr2NEt (17.3 µL, 99 µmol) and propanoic anhydride (6.2 µL, 48 µmol) was dissolved in anh CH2Cl2 (1.0 mL), followed by addition of Ac-Leu- Gly-Lys-(7-amino-4-methyl-coumarin) trifluoroacetate salt (S2 (4), 26 mg, 41 µmol). After stirring at r.t. for 40 min, MeOH (1 mL) was added, and the reaction mixture evaporated to dryness. The resulting residue was purified by prep–HPLC, affording Ac-Leu-Gly-Lys(propanoyl)-(7-amino-4-methyl- 1 coumarin) (2c, 20 mg, 86%) as a white fluffy material. H NMR (DMSO-d6) δ 10.36 (s, 1H, NHAMC), 8.31 (mABX,X, J = 6.0, 5.9 Hz, 1H, NHGly), 8.09 (d, J = 7.3 Hz, 1H, NHLeu), 8.00 (d, J = 7.7 Hz, 1H, NHα,Lys), 7.79 (d, J = 2.0 Hz, 1H, H8AMC), 7.72 (d, J = 8.7 Hz, 1H, H5AMC), 7.70 (t, J = 5.6 Hz, 1H, NHε,Lys), 7.52 (dd, J = 8.7, 2.0 Hz, 1H, H6AMC), 6.26 (d, J = 1.4 Hz, 1H, H3AMC), 4.34–4.40 (m, 1H, Hα,Lys, overlap with solvent peak), 4.19–4.25 (m, 1H, Hα,Leu, overlap with solvent peak), 3.74 (mABX,A, J = 16.7, 5.9 Hz, 1H, Hα,Gly,A), 3.71 (mABX,B, J = 16.7, 6.0 Hz, 1H, Hα,Gly,B), 2.99–3.03 (m, 2H, Hε,Lys), 2.39 (d, J = 1.3 Hz, 3H, 4AMC-CH3), 2.03 (q, J = 7.6 Hz, 2H, COCH2CH3), 1.85 (s, 3H, CH3CONHLeu), 1.69–1.77 (m, 1H, Hβ,Lys,A), 1.57–1.69 (m, 2H, Hγ,Leu, Hβ,Lys,B), 1.42–1.49 (m, 2H, Hβ,Leu), 1.36–1.42 (m, 2H, Hδ,Lys), 1.21–1.36 (m, 2H, Hγ,Lys), 0.96 13 (t, J = 7.6 Hz, 3H, COCH2CH3), 0.88 (d, J = 6.7 Hz, 3H, CH3,Leu,A), 0.84 (d, J = 6.5 Hz, 3H, CH3,Leu,B). C NMR (DMSO-d6) δ 172.9 (COLeu), 172.7 (NHε,LysCO), 171.4 (COLys), 169.7 (NHLeuCO), 169.0 (COGly), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.1 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.8 (C8AMC), 53.6 (Cα,Lys), 51.5 (Cα,Leu), 42.0 (Cα,Gly), 40.5 (Cβ,Leu), 38.2 (Cε,Lys), 31.4 (Cβ,Lys), 28.9 (Cδ,Lys), 28.5 (NHε,LysCOCH2CH3), 24.2 (Cγ,Leu), 22.9 (CH3,Leu,A), 22.8 (Cγ,Lys), 22.5 (CH3CO), 21.6 (CH3,Leu,B), 18.0 (4AMC-CH3), 10.0 (NHε,LysCOCH2CH3). HRMS m/z 594.2889 + + ([M+Na] ,C29H41N5O7Na Calcd 594.2898).

S-21 Ac-Leu-Gly-Lys(butanoyl)-AMC (2d); (S)-2-(2-((S)-2- acetamido-4-methylpentanamido)acetamido)-6-butyramido- N-(4-methyl-2-oxo-2H-chromen-7-yl)hexanamide. iPr2NEt (17.3 µL, 99 µmol) and butanoic anhydride (7.8 µL, 48 µmol) was dissolved in anh CH2Cl2 (1.0 mL), followed by addition of Ac-Leu- Gly-Lys-(7-amino-4-methyl-coumarin) trifluoroacetate salt (S2 (4), 25 mg, 40 µmol). After stirring at r.t. for 40 min, MeOH (1 mL) was added, and the reaction mixture evaporated to dryness. The resulting residue was purified by prep–HPLC, affording Ac-Leu-Gly-Lys(butanoyl)-(7-amino-4-methyl- 1 coumarin) (2d, 18 mg, 76%) as a white fluffy material. H NMR (DMSO-d6) δ 10.36 (s, 1H, NHAMC), 8.31 (t, J = 5.8 Hz, 1H, NHGly), 8.08 (d, J = 7.3 Hz, 1H, NHLeu), 8.00 (d, J = 7.6 Hz, 1H, NHα,Lys), 7.79 (d, J = 2.0 Hz, 1H, H8AMC), 7.72 (d, J = 8.7 Hz, 1H, H5AMC), 7.71 (t, J = 5.6 Hz, 1H, NHε,Lys), 7.52 (dd, J = 8.7, 2.0 Hz, 1H, H6AMC), 6.26 (d, J = 1.5 Hz, 1H, H3AMC), 4.33–4.40 (m, 1H, Hα,Lys), 4.18–4.25 (m, 1H, Hα,Leu), 3.73 (m, 2H, Hα,Gly, overlap with solvent signal), 3.01 (q, J = 6.6 Hz, 2H, Hε,Lys), 2.40 (d, J = 1.2 Hz, 3H, 4AMC-CH3), 1.99 (t, J = 7.3 Hz, 2H, COCH2CH2), 1.85 (s, 3H, CH3CONHleu), 1.69–1.77 (m, 1H, Hβ,Lys,A), 1.56–1.69 (m, 2H, Hγ,Leu, Hβ,Lys,B), 1.42–1.50 (m, 4H, Hβ,Leu, COCH2CH2), 1.36–1.42 (m, 2H, Hδ,Lys), 1.21– 1.36 (m, 2H, Hγ,lys), 0.88 (d, J = 6.5 Hz, 3H, CH3,Leu,A), 0.84 (d, J = 6.6 Hz, 3H, CH3,Leu,B), 0.80 (t, J = 7.4 13 Hz, 3H, CO(CH2)2CH3). C NMR (DMSO-d6) δ 172.9 (COLeu), 171.8 (NHε,LysCO), 171.4 (COLys), 169.7 (NHLeuCO), 169.0 (COGly), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.1 (C7AMC), 125.9 (C5AMC), 115.3 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.8 (C8AMC), 53.6 (Cα,lys), 51.5 (Cα,Leu), 42.0 (Cα,Gly), 40.5 (Cβ,Leu), 38.2 (Cε,Lys), 37.4 (NHε,lysCOCH2CH2), 31.4 (Cβ,Lys), 28.9 (Cδ,Lys), 24.2 (Cγ,Leu), 22.9 (CH3,Leu,A), 22.8 (Cγ,Lys), 22.5 (CH3CO), 21.6 (CH3,Leu,B), 18.7 (NHε,LysCOCH2CH2), 18.0 (4AMC-CH3), 13.6 + + (NHε,LysCO(CH2)2CH3). HRMS m/z 586.3227 ([M+H] , C30H44N5O7 Calcd 586.3235).

S-22 Synthesis of inhibitor 7

Benzyl ((9S,12S)-12-((1H-indol-3-yl)methyl)-15-methyl-10,13-dioxo-3- thioxo-2,4,11,14-tetraazahexadecan-9-yl)carbamate (7). A solution of methylamine (33% (w/w) in absolute EtOH, 21 µL, 0.16 mmol) and iPr2NEt (30 µL, 0.17 mmol) in CH2Cl2 (4.0 mL) was added dropwise over 5 minutes to a solution of bis(1-benzotriazolyl)methanethione (46 mg, 0.16 mmol) in anhydrous CH2Cl2 (2.0 mL) at 0 °C. After stirring for 10 minutes at r.t., additional methylamine (33% (w/w) in absolute EtOH, 12 µL, 0.10 mmol) was added. The reaction mixture was stirred for 20 minutes at r.t. and was then concentrated under reduced pressure. The resulting residue, Cbz-Lys-Trp-NHiPr∙TFA (10) (51 mg, 0.08 mmol), and iPr2NEt (30 µL, 0.17 mmol) were dissolved in anhydrous DMF (2.0 mL). The mixture was stirred at r.t. overnight and was then concentrated under reduced pressure. The crude residue was redissolved in EtOAc (50 mL) and washed with H2O (2 × 50 mL), sat. aq. NaHCO3 (2 × 50 mL), aq. HCl (1 M, 2 × 50 mL) and brine (2 × 50 mL). The organic phase was dried over Na2SO4 and concentrated under reduced pressure and the resulting residue was purified by HPLC, affording desired thiourea 7 (10.7 mg, 1 22%) as a colorless fluffy material. H NMR (DMSO-d6) δ 10.79 (d, J = 2.5 Hz, 1H, NHIndole), 7.85 (d, J = 8.1 Hz, 1H, NHα,Trp), 7.65 (d, J = 7.7 Hz, 1H, COα,TrpNH), 7.55 (d, J = 7.9 Hz, 1H, H4Indole), 7.45–7.24 (m, 9H, NHε,Lys, NHCH3, NHα,Lys, HAr,Cbz, H7Indole), 7.10 (d, J = 2.3 Hz, 1H, H2Indole), 7.04 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H, H6Indole), 6.98–6.93 (m, 1H, H5Indole), 5.07–4.97 (m, 2H, CH2,Cbz), 4.45 (td, J = 7.8, 6.1 Hz, 1H, Hα,Trp), 3.94 (td, J = 8.5, 5.1 Hz, 1H, Hα,Lys), 3.83–3.71 (m, 1H, CHiPr), 3.27 (br s, 2H, Hε,Lys overlap with residual water), 3.06 (mABX, J = 14.6, 6.1 Hz, 1H, Hβ,Trp,A), 2.96 (mABX, J = 14.6, 7.7 Hz, 1H, Hβ,Trp,B), 2.79 (s, 3H, NHCH3), 1.59–1.34 (m, 4H, Hβ,Lys, Hδ,Lys), 1.31–1.13 (m, 2H, Hγ,Lys), 1.00 (d, J = 6.6 Hz, 3H, 13 CH3,iPr,A), 0.91 (d, J = 6.6 Hz, 3H CH3,iPr,B). C NMR (DMSO-d6) δ 171.5 (COLys), 170.0 (COTrp), 156.0 (COCbz), 137.0 (C1Ar,Cbz), 135.9 (C7aIndole), 128.3 (C3Ar,Cbz, C5Ar,Cbz), 127.8 (C4Ar,Cbz), 127.6 (C2Ar,Cbz, C6Ar,Cbz), 127.4 (C3aIndole), 123.5 (C2Indole), 120.8 (C6Indole), 118.5 (C4Indole), 118.1 (C5Indole), 111.1 (C7Indole), 109.8 (C3Indole), 65.4 (CH2,Cbz), 54.9 (Cα,Lys), 53.3 (Cα,Trp), 43.4 (Cε,Lys), 40.4 (CHiPr), 31.6 (Cβ,Lys), 30.5 (NHCH3), 28.5 (Cδ,Lys), 27.9 (Cβ,Trp), 22.9 (Cγ,Lys), 22.2 (CH3,iPr,A), 22.1 (CH3,iPr,B). The peak for C=S was 13 13 not visible in C NMR and the peaks for Cε,Lys and NHCH3 were broad and of low intensity in C NMR, 14 probably due to fast quadrupolar relaxation via the nearby N-nuclei. UPLC-MS tR 2.09 min, m/z 581.3 + + + + ([M+H] , C30H41N6O4S Calcd 581.3); HRMS m/z 581.2911 ([M+H] , C33H45N6O6S Calcd 581.2905).

S-23 Supplemental references

1. Zheng, J., Ruan, L., Sun, M., and Gänzle, M. (2015) A genomic view of lactobacilli and pediococci demonstrates that phylogeny matches ecology and physiology. Appl. Environ. Microbiol. 81, 7233- 7243 2. Salvetti, E., Torriani, S., and Felis, G. E. (2012) The genus Lactobacillus: A taxonomic update. Probiotics Antimicrob. Prot. 4, 217-226 3. Wegener, D., Wirsching, F., Riester, D., and Schwienhorst, A. (2003) A fluorogenic histone deacetylase assay well suited for high-throughput activity screening. Chem. Biol. 10, 61-68 4. Madsen, A. S. and Olsen, C. A. (2012) Substrates for efficient fluorometric screening employing the NAD-dependent sirtuin 5 lysine deacylase (KDAC) enzyme. J. Med. Chem. 55, 5582-5590 5. Moreno-Yruela, C., Galleano, I., Madsen, A. S., and Olsen, C. A. (2017) Histone deacetylase 11 is an ε-N-myristoyllysine hydrolase. bioRxiv https://doi.org/10.1101/211839 6. Anderson, K. A., Huynh, F. K., Fisher-Wellman, K., Stuart, J. D., Peterson, B. S., Douros, J. D., Wagner, G. R., Thompson, J. W., Madsen, A. S., Green, M. F., Sivley, R. M., Ilkayeva, O. R., Stevens, R. D., Backos, D. S., Capra, J. A., Olsen, C. A., Campbell, J. E., Muoio, D. M., Grimsrud, P. A., and Hirschey, M. D. (2017) SIRT4 is a lysine deacylase that controls leucine metabolism and insulin secretion. Cell Metab. 25, 838-855 7. Bradner, J. E., West, N., Grachan, M. L., Greenberg, E. F., Haggarty, S. J., Warnow, T., and Mazitschek, R. (2010) Chemical phylogenetics of histone deacetylases. Nat. Chem. Biol. 6, 238-243 8. Galleano, I., Schiedel, M., Jung, M., Madsen, A. S., and Olsen, C. A. (2016) A continuous, fluorogenic sirtuin 2 deacylase assay: Substrate screening and inhibitor evaluation. J. Med. Chem. 59, 1021-1031 9. Madsen, A. S., Andersen, C., Daoud, M., Anderson, K. A., Laursen, J. S., Chakladar, S., Huynh, F. K., Colaço, A. R., Backos, D. S., Fristrup, P., Hirschey, M. D., and Olsen, C. A. (2016) Investigating the sensitivity of NAD+-dependent sirtuin deacylation activities to NADH. J. Biol. Chem. 291, 7128- 7141 10. Rajabi, N., Auth, M., Troelsen, K. R., Pannek, M., Bhatt, D. P., Fontenas, M., Hirschey, M. D., Steegborn, C., Madsen, A. S., and Olsen, C. A. (2017) Mechanism-based inhibitors of the human sirtuin 5 deacylase: Structure-activity relationship, biostructural, and kinetic insight. Angew. Chem. Int. Ed. 56, 14836-14841

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