Fluoroalkylated hypervalent iodine reagents and their use in

Fluoroalkylated hypervalent iodine reagents and their use in thiol bioconjugation Iveta Klimánková| 6. 6. 2019

Hypervalent iodine reagents 2 1) Oxidation reagents: Hypervalent λ 3 - iodanes • T-shape • 3 cb bond • Nonclasical σ-hole 2) Transfer reagents: a) IH 3; b) IF 3 • H. P. D. E. Magalhães, O. Sala, H. P. Lüthi, 2018, pp. 1– 28. D. P. Hari, P. Caramenti, J. Waser, Acc. Chem. Res. 2018, 51, 3212– 3225. Cyclic structure – enhanced the stability

3 Fluoroalkylated hypervalent iodine regents and their reactivity No reaction with amines and carboxylates Reaction with alcohols – harsh conditions Charpentier, J. ; Früh, N. ; Togni, A. , Chem. Rev. 2015, 115 (2), 650 -682. Matoušek, V. ; Václavík, J. ; Hájek, P. ; Charpentier, J. ; Blastik, Z. E. ; Pietrasiak, E. ; Budinská, A. ; Togni, A. ; Beier, P. , Chem. Eur. J. 2016, 22 (1), 417 -424.

Reaction with thiols I. Kieltsch, P. Eisenberger, A. Togni, Angew. Chemie Int. Ed. 2007, 46, 754– 757. V. Matoušek, J. Václavík, P. Hájek, J. Charpentier, Z. E. Blastik, E. Pietrasiak, A. Budinská, A. Togni, P. Beier, Chem. - A Eur. J. 2016, 22, 417– 424. S. Capone, I. Kieltsch, O. Flögel, G. Lelais, A. Togni, D. Seebach, Helv. Chim. Acta 2008, 91, 2035– 2056. 4

Late stage fluoroalkylation of thiols by hypervalent iodine reagents M. A. Danielson, J. J. Falke, Annu Rev Biophys Biomol Struct. 1996, 25, 163– 195. E. N. G. Marsh, Acc. Chem. Res. 2014, 47, 2878– 2886. L. K. Charkoudian, C. W. Liu, S. Capone, S. Kapur, D. E. Cane, A. Togni, D. Seebach, C. Khosla, Protein Sci. 2011, 20, 1244– 1255. J. Václavík, R. Zschoche, I. Klimánková, V. Matoušek, P. Beier, D. Hilvert, A. Togni, Chem. - A Eur. J. 2017, 23, 6490– 6494. 5

Labeling of Retro-aldolase RA 95. 5 -8 S 25 C K 210 M (RA-CM) Lys 83 in hydrophobic pocket • responsible for catalytic activity of the enzyme Cys 25 J. Václavík, R. Zschoche, I. Klimánková, V. Matoušek, P. Beier, D. Hilvert, A. Togni, Chem. - A Eur. J. 2017, 23, 6490– 6494. 6

Reagents used for labeling RA-CM J. Václavík, R. Zschoche, I. Klimánková, V. Matoušek, P. Beier, D. Hilvert, A. Togni, Chem. - A Eur. J. 2017, 23, 6490– 6494. 7

Labeling of RA-CM 8 + J. Václavík, R. Zschoche, I. Klimánková, V. Matoušek, P. Beier, D. Hilvert, A. Togni, Chem. - A Eur. J. 2017, 23, 6490– 6494.

MALDI spectra after trypsin digestion of labeled RA-CM J. Václavík, R. Zschoche, I. Klimánková, V. Matoušek, P. Beier, D. Hilvert, A. Togni, Chem. - A Eur. J. 2017, 23, 6490– 6494. 9

10 Fluorolkylation of cysteine Reagent Precipitation p. H co-so Ph 1 Ph 2 Ph 1 -Ac. Cl NMT 1 NMT 2 Yes Yes Yes No No 4 4 8 4 7 DMSO DMSO CH 3 CN 19 F 1 [%] 9 6 20 18 28 98 91 13 94 NMR yield to internal CF 3 COOK or CF 3 CH 2 OH 2 [%] 8 6 2 3 2 9 4 8 3

11 Reactivity of watersoluble reagents p. H time 4 7 8 10 min 19 F 4 [%] 94 99 97 3 F- or 3 HF [%] 52 18 17 NMR yield to internal CF 3 COOK 3 [%] 0 13 13

Fluoroalkylation of DAVACAK The reaction mixture was further analyzed by LC-MS (I - Class - SYNAPT G 2). Lower concentration (cysteine 30 m. M, DAVACAK 0. 6 – 1. 5 m. M) Higher excess of reagent (10 eq. – 30 eq. ) Identification of nonfluorinated side products 12

Fluoroalkylation of DAVACAK 13

14 Fluoroalkylation of DAVACAK 5 6 102 [AUC] p. H = 4 Blank 5 6 137 0 NMT 1 1 h 25 29 NMT 1 24 h 21 31 p. H = 8 NMT 2 1 h 25 14 NMT 2 24 h 22 15 Blank 121 0 NMT 1 1 h 39 40 NMT 1 24 h 21 36 NMT 2 1 h 24 h 14 8 136 106 c = 0. 15 m. M [AUC] 300 600 c = 1. 5 m. M [AUC] 170 3460

Fluoroalkylation of DAVACAK – Formation of oxidized side products 15 • Decomposition of 21 102 [AUC] p. H = 4 Blank 5 6 7 8 9 10 137 0 0 1 0 0 NMT 1 1 h 25 29 11 20 18 28 NMT 1 24 h 21 31 12 22 12 23 p. H = 8 NMT 2 1 h 25 14 10 35 5 28 NMT 2 24 h 22 15 12 38 5 22 Blank 121 0 0 1 0 2 NMT 1 1 h 39 40 0. 5 6 0 7 NMT 1 24 h 21 36 0. 7 8 0 6 NMT 2 1 h 14 136 0 5 NMT 2 24 h 8 106 0. 5 7 0 2

Reaction in H 2 O 18 16

Reaction in H 2 Blank 102 [AUC] 20 b 21 b 0 0 17 O 18 NMT 1 1 h 102 [AUC] 29 11 NMT 2 24 h 1 h 24 h 102 [AUC] 31 14 15 12 10 12

18 Fluoroalkylation of DAVACAK – Formation of N-acylated side products • Unselective – functional group is transfer also on Lysine, N-term… 102 [AUC] p. H = 4 Blank 5 6 11 12 13 137 0 0 NMT 1 1 h 25 29 0 0 0 NMT 1 24 h 21 31 0 0 0 p. H = 8 NMT 2 1 h 25 14 0 0 0 NMT 2 24 h 22 15 0 0 0 Blank 121 0 0 NMT 1 1 h 39 40 4 0. 5 15 NMT 1 24 h 21 36 5 1 16 NMT 2 1 h 14 136 5 0. 5 7 NMT 2 24 h 8 106 31 0. 5 10

19 Fluoroalkylation of DAVACAK – Formation of N-acylated side products Reagent Precipitation p. H co-so Ph 1 Ph 2 Ph 1 -Ac. Cl NMT 1 NMT 2 Yes Yes Yes No No 4 4 8 4 7 DMSO DMSO CH 3 CN 1 [%] 9 6 20 18 28 98 91 13 94 2 [%] 8 6 2 3 2 9 4 8 3

Reaction in H 2 O 18 20

Conclusion • Thiol fluoroalkylation by hypervalent iodine reagent is usefull: 1. In organic solvents at low temperature 2. In water at rt with water soluble reagents • The excess of reagent, lower concenration and longer reaction time are unsuitable for this reaction, due to the formation of side products 21

Radical fluoroalkylation with hypervalent iodine reagents in aqueous media • Only solvent acesible aminoacids 22

Acknowledgement 23 • Petr Beier • Jiří Václavík, Sonia Rahimi • Martin Hubálek and Michal Korecký from MS department • Václav Matoušek (CF Plus Chemicals) IOCB Prague Iveta Klimánková | 27. 2. 2019