A METHOD FOR FUNCTIONALIZATION OF AN AROMATIC AMINO ACID OR A NUCLEOBASE

Abstract

A method for functionalization of an aromatic amino acid or a nucleobase with a fluoroalkyl-containing moiety RF, wherein the aromatic amino acid is reacted in the presence of at least one reductant with at least one hypervalent iodine fluoroalkyl reagent carrying the floroalkyl-containing moiety RF is disclosed. Novel hypervalent iodine fluoroalkyl reagents is also disclosed.

Claims

1: A method for functionalization of an aromatic amino acid or a nucleobase with a fluoroalkyl-containing moiety R.sub.F, wherein the aromatic amino acid or the nucleobase is reacted in the presence of at least one reductant with at least one hypervalent iodine fluoroalkyl reagent, wherein the hypervalent iodine fluoroalkyl reagent is of general formula 1 ##STR00025## wherein R.sup.1 is selected from H, C1-C4 alkyl, Me(OCH.sub.2CH.sub.2).sub.nO, wherein n=1-10, X.sup.1 is not present or is selected from chloride, tetrafluoroborate, hexafluorophosphate, trifluoromethanesulfonate, methanesulfonate, toluenesulfonate, fluoride, bromide, (F(CF.sub.2).sub.sSO.sub.2).sub.2N.sup.−, wherein s=1 to 4, C1-C6 carboxylate, fluorinated C1-C6 carboxylate, hexafluoroantimonate; A.sup.1 is not present or is selected from carbonyl group (C═O), R.sup.2—C—R.sup.3, wherein R.sup.2 and R.sup.3 are independently selected from fluorine, chlorine, hydrogen, CF.sub.3, C.sub.1-C.sub.4 alkyl, phenyl, phenyl substituted by fluorine, chlorine and/or C.sub.1-C.sub.4 alkyl, omega-methoxy-(C.sub.1-C.sub.4)alkyl; or R.sup.2 and R.sup.3 together with the carbon atom to which they are bound form 1,1-cyclobutylene, 1,1-cyclopentylene, 1,1-cyclohexylene, 1,1-(4-oxacyclohexylene); A.sup.2 is not present or is selected from hydroxyl group (OH), oxygen (—O—), C.sub.1-4 alkoxy, OSiR.sup.4R.sup.5R.sup.6, wherein R.sup.4,R.sup.5,R.sup.6 are independently selected from methyl, ethyl, n-propyl, i-propyl and phenyl; R.sup.7N, wherein R.sup.7 is selected from C.sub.1-10 alkyl, phenyl-substituted C.sub.1-10 alkyl, 4-chlorophenyl-substituted C.sub.1-10 alkyl, and CH.sub.3(OCH.sub.2CH.sub.2).sub.n; R.sup.8CO—, wherein R.sup.8 is selected from C.sub.1-10 alkyl, phenyl-substituted C.sub.1-10 alkyl, CH.sub.3(OCH.sub.2CH.sub.2).sub.o—, wherein o=1-10, Cl.sup.−Me.sub.3N.sup.+(CH.sub.2).sub.p—, wherein p=1-10, —(CX.sub.2).sub.mCOOQ, —(CX.sub.2).sub.mSO.sub.3Q, wherein m=2-3, -phenyl-SO.sub.3Q, -phenyl-COOQ, —(CX.sub.2).sub.mCOO—, —(CX.sub.2).sub.mSO.sub.3—, wherein m=2-3, -phenyl-SO.sub.3—, -phenyl-COO—, X.sub.2═F or Cl, X.sub.3═H, CH.sub.3, F or Cl, Q is selected from lithium, sodium, potassium, rubidium, cesium, tetra (C1-C4 alkyl, phenyl, benzyl) ammonium, tri (C1-C4 alkyl, phenyl, benzyl) pyridinium; R.sup.9C(O)O—, wherein R.sup.9 is selected from C.sub.1-10 alkyl, phenyl-substituted C.sub.1-10 alkyl, CH.sub.3(OCH.sub.2CH.sub.2).sub.o—, wherein o=1-10, Cl.sup.−Me.sub.3N.sup.+(CH.sub.2).sub.p—, wherein p=1-10, —(CX.sub.2).sub.mCOOQ, —(CX.sub.2).sub.mSO.sub.3Q, wherein m=2-3, -phenyl-SO.sub.3Q, -phenyl-COOQ, —(CX.sub.2).sub.mCOO—, —(CX.sub.2).sub.mSO.sub.3—, wherein m=2-3, -phenyl-SO.sub.3—, -phenyl-COO—, X.sub.2═F or Cl, X.sub.3═H, CH.sub.3, F or Cl, Q is selected from lithium, sodium, potassium, rubidium, cesium, tetra (C1-C4 alkyl, phenyl, benzyl) ammonium, tri (C1-C4 alkyl, phenyl, benzyl) pyridinium; R.sub.F is —CF.sub.2-A.sup.3-A.sup.4, wherein A.sup.3 is not present and A.sup.4 is fluorine, or phenylsulfanyl (PhS) group, or A.sup.3 is CF.sub.2 and A.sup.4 is fluorine, C.sub.1-10 perfluoroalkyl, phenoxy, C.sub.1-10 alkoxy, phenylsulfanyl, N-imidazolyl, N-pyrazolyl, N-benzimidazolyl, N-triazoyl, N-(2-methyl)imidazolyl, CH.sub.3(OCH.sub.2CH.sub.2).sub.nO, where n=1-10, Cl.sup.−MeH.sub.2N.sup.+(CH.sub.2).sub.m wherein r=1-10, Cl.sup.−MeH.sub.2N.sup.+(CH.sub.2).sub.2-phenyloxy, C.sub.1-10 alkyl, ethenyl, ethynyl, omega-azido C.sub.1-10 alkyl, omega-amino C1-10 alkyl, omega-ethynyl C.sub.1-10 alkyl, C1-10 alkylthio, phenyl, chloro, bromo, iodo, azido, nitro, C.sub.1-10 alkoxycarbonyl-substituted phenyl, C.sub.2-10 alkenyl, C.sub.2-10 alkyl omega substituted with SO.sub.2F, N-succinimide-O—C(O)—, methyldiaziridine, trifluoromethyldiaziridine, rhodamine, fluorescein or coumarin; aryl p-substituted with N-succinimide-O—C(O)—, rhodamine, fluorescein or coumarin, phenoxy p-substituted with N-succinimide-O—C(O)—, rhodamine, fluorescein or coumarin, and 2-(aryliodanyl)-1,1,2,2-tetrafluoroethyl; in alkyl chains, two neighboring carbon atoms may be replaced by a methylamido group (—N(Me)-C(O)—) and/or by a phenyl group.

2: The method of claim 1, wherein in formula 1: A.sup.1 is present, A.sup.2 is a bivalent substituent, and X.sup.1 is not present; or A.sup.1 is present, A.sup.2 is a monovalent substituent bound to A.sup.1, and X.sup.1 is present.

3: The method of claim 1, wherein the aromatic amino acid is selected from phenylalanine, tryptophan, tyrosine, histidine.

4: The method of claim 1, wherein the aromatic amino acid is tryptophan.

5: The method of claim 1, wherein the nucleobase is cytosine.

6: The method of claim 1, wherein the reductant is selected from a group consisting of sodium ascorbate, potassium ascorbate, calcium ascorbate, magnesium ascorbate, esters of ascorbic acid with carboxylic acids of the formula R.sup.10CO.sub.2H wherein R.sup.10 is C.sub.1-18 alkyl, sodium sulphite, sodium dithionite, tetrakis(dimethylamino)ethylene, sodium phosphite, sodium hypophosphite, and sodium hydroxymethanesulfinate.

7: The method of claim 1, wherein the reductant is used in an amount of 0.5-1.0 equivalents, preferably of 0.9-1.1 equivalents, relative to the hypervalent iodine fluoroalkyl reagent.

8: The method of claim 1, wherein aromatic amino acids incorporated in a protein are reacted in the presence of at least one reductant with at least one hypervalent iodine fluoroalkyl reagent, and subsequently it is detected which amino acids are fluoroalkylated and these amino acids are then determined to be present on the solvent-exposed surface of the protein.

9: The method of claim 1, wherein the hypervalent iodine fluoroalkyl reagent is selected from the group consisting of the following reagents: ##STR00026## ##STR00027## ##STR00028## ##STR00029##

10: A hypervalent iodine fluoroalkyl reagent of general formula 1-1 ##STR00030## wherein R.sup.1 is selected from H, C1-C4 alkyl, Me(OCH.sub.2CH.sub.2).sub.nO, wherein n=1-10, X.sup.1 is not present or is selected from chloride, tetrafluoroborate, hexafluorophosphate, trifluoromethanesulfonate, methanesulfonate, toluenesulfonate, fluoride, bromide, (F(CF.sub.2).sub.sSO.sub.2).sub.2N.sup.−, wherein s=1 to 4, C1-C6 carboxylate, fluorinated C1-C6 carboxylate, hexafluoroantimonate; A.sup.1 is not present or is selected from carbonyl group (C═O), R.sup.2—C—R.sup.3, where R.sup.2 and R.sup.3 are independently selected from fluorine, chlorine, hydrogen, CF.sub.3, C.sub.1-C.sub.4 alkyl, phenyl, phenyl substituted by fluorine, chlorine and/or C.sub.1-C.sub.4 alkyl, omega-methoxy-(C.sub.1-C.sub.4)alkyl; or R.sup.2 and R.sup.3 together with the carbon atom to which they are bound form 1,1-cyclobutylene, 1,1-cyclopentylene, 1,1-cyclohexylene, 1,1-(4-oxacyclohexylene); A.sup.2 is not present or is selected from hydroxyl group (OH), oxygen (—O—), C.sub.1-4 alkoxy, OSiR.sup.4R.sup.5R.sup.6, wherein R.sup.4,R.sup.5,R.sup.6 are independently selected from methyl, ethyl, n-propyl, i-propyl and phenyl; R.sup.7N, wherein R.sup.7 is selected from C.sub.1-10 alkyl, phenyl-substituted C.sub.1-10 alkyl, 4-chlorophenyl-substituted C.sub.1-10 alkyl, and CH.sub.3(OCH.sub.2CH.sub.2).sub.n; R.sup.8CO—, wherein R.sup.8 is selected from C.sub.1-10 alkyl, phenyl-substituted C.sub.1-10 alkyl, CH.sub.3(OCH.sub.2CH.sub.2).sub.o—, wherein o=1-10, Cl.sup.−Me.sub.3N.sup.+(CH.sub.2).sub.p—, wherein p=1-10, —(CX.sub.2).sub.mCOOQ, —(CX.sub.2).sub.mSO.sub.3Q, wherein m=2-3, -phenyl-SO.sub.3Q, -phenyl-COOQ, —(CX.sub.2).sub.mCOO—, —(CX.sub.2).sub.mSO.sub.3—, wherein m=2-3, -phenyl-SO.sub.3—, -phenyl-COO—, X.sub.2═F or Cl, X.sub.3═H, CH.sub.3, F or Cl, Q is selected from lithium, sodium, potassium, rubidium, cesium, tetra (C1-C4 alkyl, phenyl, benzyl) ammonium, tri (C1-C4 alkyl, phenyl, benzyl) pyridinium; R.sup.9C(O)O—, wherein R.sup.9 is selected from C.sub.1-10 alkyl, phenyl-substituted C.sub.1-10 alkyl, CH.sub.3(OCH.sub.2CH.sub.2).sub.o—, wherein o=1-10, Cl.sup.−Me.sub.3N.sup.+(CH.sub.2).sub.p—, wherein p=1-10, —(CX.sub.2).sub.mCOOQ, —(CX.sub.2).sub.mSO.sub.3Q, wherein m=2-3, -phenyl-SO.sub.3Q, -phenyl-COOQ, —(CX.sub.2).sub.mCOO—, —(CX.sub.2).sub.mSO.sub.3—, wherein m=2-3, -phenyl-SO.sub.3—, -phenyl-COO—, X.sub.2═F or Cl, X.sub.3═H, CH.sub.3, F or Cl, Q is selected from lithium, sodium, potassium, rubidium, cesium, tetra (C1-C4 alkyl, phenyl, benzyl) ammonium, tri (C1-C4 alkyl, phenyl, benzyl) pyridinium; R.sub.F is —CF.sub.2-A.sup.3-A.sup.4, wherein A.sup.3 is not present and A.sup.4 is fluorine, or phenylsulfanyl (PhS) group, or A.sup.3 is CF.sub.2 and A.sup.4 is fluorine, C.sub.1-10 perfluoroalkyl, phenoxy, C.sub.1-10 alkoxy, phenylsulfanyl, N-imidazolyl, N-pyrazolyl, N-benzimidazolyl, N-triazoyl, N-(2-methyl)imidazolyl, CH.sub.3(OCH.sub.2CH.sub.2).sub.nO, where n=1-10, Cl.sup.−MeH.sub.2N.sup.+(CH.sub.2).sub.r, wherein r=1-10, Cl.sup.−MeH.sub.2N.sup.+(CH.sub.2).sub.2-phenyloxy, C.sub.1-10 alkyl, ethenyl, ethynyl, omega-azido C.sub.1-10 alkyl, omega-amino C.sub.1-10 alkyl, omega-ethynyl C.sub.1-10 alkyl, C.sub.1-10 alkylthio, phenyl, chloro, bromo, iodo, azido, nitro, C.sub.1-10 alkoxycarbonyl-substituted phenyl, C.sub.2-10 alkenyl, C.sub.2-10 alkyl omega substituted with SO.sub.2F, N-succinimide-O—C(O)—, methyldiaziridine, trifluoromethyldiaziridine, rhodamine, fluorescein or coumarin; aryl p-substituted with N-succinimide-O—C(O)—, rhodamine, fluorescein or coumarin, phenoxy p-substituted with N-succinimide-O—C(O)—, rhodamine, fluorescein or coumarin, and 2-(aryliodanyl)-1,1,2,2-tetrafluoroethyl; in alkyl chains, two neighboring carbon atoms may be replaced by a methylamido group (—N(Me)-C(O)—) and/or by a phenyl group, provided that: A2 is not —O— or —OH, when: A1 is C═O or R.sup.2—C—R.sup.3, wherein R.sup.2 and R.sup.3 are methyls; A2 is not —O—C(═O)CH.sub.3, when: R.sub.F is CF.sub.3 and A1 is C═O or R.sup.2—C—R.sup.3, wherein R.sup.2 and R.sup.3 are methyls; A2 is not —O— or —OH, when: X.sup.1 is not present, R.sub.F is CF.sub.3 and A1 is R.sup.2—C—R.sup.3, wherein R.sup.2 and R.sup.3 are selected from alkyl and CF.sub.3 or wherein R.sup.2 is methyl and R.sup.3 is phenyl or R.sup.2 and R.sup.3 together form 1,1-cyclohexylene; A2 is not —O— or —OH, when: X.sup.1 is not present, R.sub.F is selected from perfluoroethyl, perfluoropropyl, perfluorobutyl, perfluorohexyl or perfluorooctyl, A1 is R.sup.2—C—R.sup.3, wherein R.sup.2 is methyl and R.sup.3 is phenyl or isopropyl; A2 is not R.sup.7N, wherein R.sup.7 is selected from C.sub.1-10 alkyl, phenyl-substituted C.sub.1-10 alkyl, when: X.sup.1 is not present, R.sub.F is CF.sub.3 and A1 is C═O.

11: A hypervalent iodine fluoroalkyl reagent selected from the following compounds ##STR00031## ##STR00032##

Description

BRIEF DESCRIPTION OF DRAWINGS

[0111] FIG. 1: Fluoroalkylation of peptide AFRIPLYWGRI.

[0112] FIG. 2: Fluoroalkylation of peptide bradykinin

[0113] FIG. 3: Fluoroalkylation of peptide somatostatin

[0114] FIG. 4: Fluoroalkylation of peptide bombesin

[0115] FIG. 5: Identification of hCA I tryptic peptide modified by CF.sub.3 radicals using reagent 1a. Based on the fragment spectra tryptophane (A), tyrosine (B), phenylalanine (C) and histidine (D) were determined as residues susceptible to radical fluoroalkylation.

[0116] FIG. 6: Red spots represent Trp123, Tyr88, Phe84 and His67 residues on the surface accessible area of hCA I structural model (1hcb).

[0117] FIG. 7: MS/MS fragmentation patterns of monofluoroalkylated ss-DNA proving fluoroalkylation on cytosine nucleobase.

EXAMPLES

[0118] The subject-matter of present invention is further illustrated by the following examples which should not be construed as limiting the scope of the invention.

[0119] Scheme 1 generally shows the reaction scheme for preparation of individual reagents as described in the following examples.

##STR00008##

Example 1: Synthesis of Reagent 1h

[0120] ##STR00009##

[0121] Step 1: CsF (0.45 mmol, 68 mg) and 3-oxo-1λ.sup.3-benzo[d][1,2]iodaoxol-1(3H)-yl acetate (3 mmol, 918 mg) were dissolved in dry DMF (2.5 ml) under argon atmosphere. To the well stirred suspension, solution of tert-butyl methyl(4-(1,1,2,2-tetrafluoro-2-(trimethylsilyl)ethoxy)-phenethyl)carbamate (1.5 mmol, 635 mg) in dry DMF (5 ml) was added dropwise. After 2 hours, the reaction mixture was diluted with EtOAc (50 ml), washed with water (10 ml), 1 M NaHCO.sub.3 (2×10 ml), 1 M LiCl (2×10 ml), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude product was purified by filtration through a pad of alumina (20 g). The impurities were washed away with Et.sub.2O (150 ml) and the alumina-adsorbed title product was completely eluted using MeOH (75 ml). The pure intermediate was obtained after concentration under reduced pressure as a colorless oil. Yield: 604 mg (69%); .sup.1H NMR (401.00 MHz, CDCl.sub.3): δ 1.34-1.39 (bs, 9H, C(17)H.sub.3), 2.76-2.83 (bm, 5H, C(12)H.sub.2 and C(14)H.sub.3), 3.41 (t, .sup.3J.sub.HH=7.2 Hz, 2H, C(13)H2), 7.12 (d, .sup.3J.sub.HH=8.2 Hz, 2H, C(9)H or C(10)H), 7.16-7.25 (bm, 2H, C(9)H or C(10)H), 7.66-7.80 (m, 2H, C(4)H and C(5)H), 7.90 (d, .sup.3J.sub.HH=8.1 Hz, 1H, C(6)H), 8.43 (dd, .sup.3J.sub.HH=7.3 Hz, .sup.4J.sub.HH=2.1 Hz, 1H, C(3)H); .sup.19F NMR (377.28 MHz, CDCl.sub.3): δ −89.5 (bs, 2F, CF.sub.2), −84.4 (bs, 2F, CF.sub.2); .sup.13C {.sup.1H} NMR (100.84 MHz, CDCl.sub.3): δ 28.2 (s, 1C, C(17)H.sub.3), 33.3 and 33.7 (s, 1C, C(12)H.sub.2), 34.1 and 34.6 (s, 1C, C(14)H.sub.3), 49.9 and 50.4 (s, 1C, C(13)H2), 79.3 (s, 1C, C(16)), 110.5 (tt, .sup.1J.sub.CF=335.4 Hz, .sup.2J.sub.CF=40.0 Hz, 1C, CF.sub.2), 114.8 (s, 1C, C(7)), 117.2 (tt, .sup.1J.sub.CF=277.9 Hz, .sup.2J.sub.CF=25.6 Hz, 1C, CF.sub.2), 121.4 (bs, 2C, C(9)H or C(10)H), 128.1 (t, .sup.4J.sub.CF=5.7 Hz, 1C, C(6)H), 130.3 (s, 2C, C(9)H or C(10)H), 131.5 (s, 1C, C(2)), 132.3 (s, 1C, C(4)), 133.7 (s, 1C, C(3)H), 135.2 (s, 1C, C(5)), 138.8 (m, 1C, C(11)), 146.4 (s, 1C, C(8)), 155.4 (s, 1C, C(15)), 165.9 (s, 1C, C(1)); HRMS (m/z, ESI.sup.+): [M+Na].sup.+ calc. for C.sub.23H.sub.24F.sub.4INO.sub.5Na, 620.0528, found, 620.0530.

[0122] Step 2: The iodane intermediate (3.4 mmol, 2 g) was dissolved in 1,2-dichloroethane (68 ml) in a round bottom flask. HCl (4 M in dioxane, 34 mmol, 8.5 ml) was added and the resulting mixture was stirred for 1 hour at 60° C. The solvent was evaporated, leading to formation of white particles which were subsequently suspended in Et.sub.2O. The mother liquor was decanted and pure 1h was obtained as a white solid. Yield: 1.46 g (75%); m.p. 120-123° C.; .sup.1H NMR (401.00 MHz, DMSO-d.sub.6): δ 2.54 (t, .sup.3J.sub.HH=5.3 Hz, 3H, C(14)H3), 2.98-3.01 (bm, 2H, C(12)H.sub.2, 3.06-3.21 (bm, 2H, C(13)H2), 7.25 (d, .sup.3J.sub.HH=8.0 Hz, 2H, C(9)H), 7.37 (d, .sup.3J.sub.HH=8.0 Hz, 2H, C(10)H), 7.70 (t, .sup.3J.sub.HH=7.6 Hz, 1H, C(5)H), 7.82 (t, .sup.3J.sub.HH=7.5 Hz, 1H, C(4)H), 8.24 (d, .sup.3J.sub.HH=7.6 Hz, 1H, C(6)H), 8.49 (d, .sup.3J.sub.HH=7.6 Hz, 1H, C(3)H), 9.17 (s, 2H, NH.sub.2); .sup.19F NMR (377.28 MHz, DMSO-d.sub.6): δ −88.5 (bs, 2F, CF.sub.2), −82.9 (t, .sup.3J.sub.FF=6.6 Hz, 2F, CF.sub.2); .sup.13C {.sup.1H} NMR (100.84 MHz, DMSO-d.sub.6): δ 31.1 (s, 1C, C(12)H2), 32.8 (s, 1C, C(14)H3), 49.3 (s, 1C, C(13)H.sub.2), 112.2 (tt, .sup.1J.sub.CF=341.8 Hz, .sup.2J.sub.CF=41.2 Hz, 1C, CF.sub.2), 116.9 (tt, .sup.1J.sub.CF=275.5 Hz, .sup.2J.sub.CF=26.9 Hz, 1C, CF.sub.2), 119.7 (s, 1C, C(7)), 121.5 (bs, 2C, C(9)H), 130.8 (s, 2C, C(10)H), 131.1 (s, 1C, C(2)), 132.2 (s, 1C, C(6)H), 133.1 (s, 1C, C(4)H), 135.5 (s, 1C, C(5)H), 137.0 (m, C(8)), 140.0 (s, C(3)H), 147.1 (s, C(11)), 165.5 (s, C(1)); HRMS (m/z, ESI.sup.+): [M+H].sup.+ calc. for C.sub.18H.sub.17F.sub.4INO.sub.3, 498.0184, found, 498.0183.

Example 2: Synthesis of Reagent 1i

[0123] ##STR00010##

[0124] 1a (0.5 mmol, 165 mg) was dissolved in dry DCM (1 ml) and acetyl chloride (0.75 mmol, 0.06 ml) was added in one portion. The mixture was stirred 15 minutes at laboratory temperature. After that volatiles were removed under reduced pressure and the obtained particles were washed with pentane to give pure 1i as a white solid. Yield: 198 mg (97%); .sup.1H NMR (401.00 MHz, CDCl.sub.3): δ 2.05 (s, 6H, C(8)H3), 2.19 (s, 3H, C(10)H.sub.3), 7.34 (ddd, .sup.3J.sub.HH=7.9 Hz, .sup.3J.sub.HH=6.5 Hz, .sup.4J.sub.HH=2.6 Hz, 1H, C(3)H), 7.60-7.73 (m, 2H, C(2)H and C(4)H), 8.38 (dd, .sup.3J.sub.HH=7.8 Hz, .sup.4J.sub.HH=1.1 Hz, 1H, C(1)H); .sup.19F NMR (377.28 MHz, CDCl.sub.3): δ −32.7 (s, 3F, CF.sub.3); .sup.13C {.sup.1H} NMR (100.84 MHz, CDCl.sub.3): δ 22.9 (s, 1C, C(10)H.sub.3), 28.6 (s, 2C, C(8)H.sub.3), 82.4 (s, 1C, C(7)), 107.8 (q, .sup.1J.sub.CF=388.6 Hz, 1C, CF.sub.3), 116.3 (s, 1C, C(6)), 129.1 (s, 1C, C(4)H), 131.1 (s, 1C, C(3)H), 133.2 (s, 1C, C(2)H), 141.7 (s, 1C, C(1)), 145.9 (s, 1C, C(5)), 169.5 (s, 1C, C(9)); HRMS (m/z, ESI.sup.+): [M+Na].sup.+ calc. for C.sub.12H.sub.13O.sub.2ClF.sub.3INa, 430.9493, found, 430.9489.

Example 3: Synthesis of Reagent 1j and 1k

[0125] ##STR00011##

[0126] 1-Fluoro-3,3-dimethyl-1,3-dihydro-1λ.sup.3-benzo[d][1,2]iodaoxole (1.83 g, 6.41 mmol, 1.3 equiv.) was dissolved in MeCN (15 ml) and to the resulting solution was added TBAT (133 mg, 0.247 mmol, 0.05 equiv.) The reaction mixture was cooled to −20° C. and a solution of 4-azido-1-trimethylsilyl-1,1,2,2-tetrafluorobutane (1.5 g of 80% purity, 4.93 mmol, 1 equiv.) in MeCN (10 ml) was slowly introduced to the reaction mixture within 50 minutes. After the addition was complete, the reaction mixture was gradually warmed to room temperature within 80 minutes. The resulting brownish solution was evaporated to dryness under reduced pressure and the resulting viscous oil was redissolved in cyclohexane (35 ml). The solution was filtered through a pad of alumina (activated by heatgun drying in vacuo) and evaporated to dryness under reduced pressure. The resulting liquid was dissolved in a mixture of Et.sub.2O (3 ml) and pentane (7 ml), the solution was cooled to 0° C. and HCl in Et.sub.2O (3.3 ml of 3M solution, 9.86 mmol, 2 equiv) was slowly added. The resulting white solid was filtered off, washed with pentane and dried in vacuo. Yield: 0.97 g (49%). .sup.1H NMR (401 MHz, CDCl.sub.3): δ 1.72 (s, 6H, CH.sub.3), 2.30-2.43 (m, 2H, CH.sub.2CF.sub.2), 3.59 (t, J=7.0 Hz, 2H, CH.sub.2N3), 4.56 (br s, 1H, OH), 7.25-7.29 (m, 1H, C.sub.ArH), 7.59-7.66 (m, 2H, C.sub.ArH), 8.13 (d, J=8.1 Hz, 1H, C.sub.ArH); .sup.19F NMR (377 MHz, CDCl.sub.3) δ −80.6 (s, 2F, CF.sub.2), −106.4 (t, J=18.2 Hz, 2F, CF.sub.2); .sup.13C NMR (100.8 Hz, CDCl.sub.3) δ 29.6 (t, .sup.2J.sub.CF=22.4 Hz, CH.sub.2CF.sub.2), 31.9 (CH.sub.3), 43.4 (t, .sup.3J.sub.CF=3.8 Hz, CH.sub.2N.sub.3), 74.2 (C—OH), 112.8 (C-I), 112.0-119.3 (m, CF.sub.2), 129.8, 130.1, 132.8, 139.2, 147.3; HRMS (m/z, ESI.sup.+): [M].sup.+ calc. for C.sub.13H.sub.15ON.sub.3F.sub.4I, 432.01904, found, 432.01924.

Example 4: Synthesis of Reagent 1l

[0127] ##STR00012##

[0128] CsF (120 mg, 0.822 mmol, 0.1 equiv.) was dried with a heat gun under vacuum and then suspended under Ar together with 1-acetoxy-1λ.sup.3-benzo[d][1,2]iodaoxol-3(1H)-one (5.03 g, 16.44 mmol, 2 equiv.) in DMF (30 ml). The reaction mixture was cooled to 15° C. and a solution of 4-azido-1-trimethylsilyl-1,1,2,2-tetrafluorobutane (2.5 g in 80% purity, 8.22 mmol, 1 equiv.) in dry DMF (10 ml) was gradually added over the course of 35 minutes. After that, the resulting mixture was left to react while reaching ambient temperature (2 h). Then it was poured into cold dilute solution of NaHCO.sub.3 (250 ml, 5% w/w) and stirred for 10 min to bring about complete hydrolysis of the acetoxyiodane precursor to the insoluble hydroxy derivative. EtOAc (300 ml) was added, then the biphasic mixture was stirred and subsequently filtered through a pad of Celite. The organic phase was separated and the aqueous phase was extracted with additional EtOAc. The combined organic phases were washed twice with 5% solution of LiCl (2×100 ml). The organic phase was dried over Na.sub.2SO.sub.4 and concentrated to near dryness. The precipitated product was cooled to 0° C., suspended in cold Et.sub.2O (3 ml), filtered off, washed twice with cold Et.sub.2O (2×5 ml) and finally dried in high vacuum. Yield: 0.63 g (23%); .sup.1H NMR (300 MHz, CDCl.sub.3, 25° C.) 2.65-2.37 (m, 2H), 3.70 (t, J=7.0 Hz, 2H), 7.77 (dd, J=11.0, 5.8 Hz, 3H), δ 8.54-8.40 (m, 1H); .sup.19F NMR (282 MHz, CDCl.sub.3) δ −85.70 (s), −105.85 (t, J=18.3 Hz).

Example 5: Synthesis of Reagents 1m and 1n

[0129] ##STR00013##

[0130] 2,1-Benzoxathiol-3-one-1,1-dioxide (184 mg, 1 mmol) was dissolved in dry DCM (5 ml) under argon atmosphere and 1a (330 mg, 1 mmol) was added in one portion. The mixture was stirred 20 minutes at room temperature. After that, the volatiles were removed under reduced pressure and the obtained particles were washed with Et.sub.2O (5 ml) to give pure 1m as a white solid. Yield: 383 mg (74%); .sup.1H NMR (600.13 MHz, DMSO-d.sub.6): δ 1.99 (bs, 6H, C(8)H.sub.3), 7.38 (dd, .sup.3J.sub.HH=7.2 Hz, .sup.4J.sub.HH=1.8 Hz, 1H, C(15)H), 7.53-7.58 (m, 3H, C(3)H, C(13)H, and C(14)H), 7.73 (dd, .sup.3J.sub.HH=7.2 Hz, .sup.4J.sub.HH=1.9 Hz, 1H, C(12)H), 7.85 (td, .sup.3J.sub.HH=7.6 Hz, .sup.4J.sub.HH=1.3 Hz, 1H, C(2)H), 7.95 (dd, .sup.3J.sub.HH=8.0 Hz, .sup.4J.sub.HH=1.7 Hz, 1H, C(4)H), 8.75 (dd, .sup.3J.sub.HH=7.9 Hz, .sup.4J.sub.HH=1.3 Hz, 1H, C(1)H); .sup.19F NMR (377.28 MHz, DMSO-d.sub.6): δ −28.6 (s, 3F, CF.sub.3); .sup.13C CHI NMR (150.92 MHz, DMSO-d.sub.6): δ 31.0 (only in HSQC, 2C, C(8)H.sub.3), 81.7 (s, 1C, C(7)), 100.1 (q, .sup.1J.sub.CF=367.8 Hz, 1C, CF.sub.3), 115.2 (s, 1C, C(6)), 126.6 (s, 1C, C(15)H), 127.2 (s, 1C, C(12)H), 129.2 (s, 1C, C(4)H), 129.8 (s, 1C, C(13)H or C(14)H), 130.1 (s, 1C, C(13)H or C(14)H), 130.7 (s, 1C, C(11)), 131.2 (s, 1C, C(3)H), 133.5 (s, 1C, C(2)H), 140.7 (s, 1C, C(10)), 141.7 (s, 1C, C(1)), 144.5 (s, 1C, C(5)), 166.4 (s, 1C, C(9)); HRMS (m/z, ESI.sup.+): [M+Na].sup.+ calc. for C.sub.17H.sub.14 O.sub.5SF.sub.3INa 536.9451, found, 536.9445.

[0131] In NMR tube, 1m (10 mg, 0.02 mmol) was dissolved in DMSO-d.sub.6 (0.4 ml). To the solution was added LiCl (5.8 mg, 0.12 mmol) and after 30 minutes, .sup.1H and .sup.19F NMR spectra were measured. .sup.1H NMR (401.00 MHz, DMSO-d.sub.6): δ 2.06 (bs, 6H, C(8)H.sub.3), 7.32-7.35 (m, 2H, C(3)H and C(15)H), 7.38-7.49 (m, 2H, C(13)H and C(14)H), 7.67 (t, .sup.3J.sub.HH=7.6 Hz, 1H, C(2)H), 7.75 (d, .sup.3J.sub.HH=7.5 Hz, 1H, C(12)H), 7.92 (d, .sup.3J.sub.HH=8.0 Hz, 1H, C(4)H), 8.40 (d, .sup.3J.sub.HH=7.7 Hz, 1H, C(1)H); .sup.19F NMR (377.28 MHz, DMSO-d.sub.6): δ −33.4 (s, 3F, CF.sub.3); .sup.13C {.sup.1H} NMR (100.84 MHz, DMSO-d.sub.6): δ 29.1 (only in HSQC, 2C, C(8)H3), 83.6 (s, 1C, C(7)), 121.3 (s, 1C, C(6)), 127.3 (s, 1C, C(15)H), 127.9 (s, 1C, C(12)H), 129.2 (s, 1C, C(4)H), 129.2 (s, 1C, C(13)H or C(14)H), 129.5 (s, 1C, C(13)H or C(14)H), 130.5 (s, 1C, C(3)H), 132.3 (s, 1C, C(11)H), 132.5 (s, 1C, C(2)H), 141.3 (s, 1C, C(1)), 144.8 (s, 1C, C(10)), 146.4 (s, 1C, C(5)), 167.3 (s, 1C, C(9)).

Example 6: Synthesis of Reagents 1o and 1p

[0132] ##STR00014##

[0133] 1c (227 mg, 0.5 mmol) was dissolved in dry DCM (2.5 ml) under argon atmosphere and 2,1-benzoxathiol-3-one-1,1-dioxide (92 mg, 0.5 mmol) was added in one portion. The mixture was stirred 15 minutes at laboratory temperature. After that volatiles were removed under reduced pressure and crude product was precipitated from DCM/Et.sub.2O mixture to obtain pure 1o as white particles. Yield: 282 mg (88%); .sup.1H NMR (401.00 MHz, CDCl.sub.3): δ 2.08 (s, 6H, C(8)H.sub.3), 7.19-7.29 (m, 3H, C(15)H and C(17)H), 7.34 (t, .sup.3J.sub.HH=7.4 Hz, 1H, C (19)H), 7.38-7.52 (m, 5H, C(3)H, C(13)H, C(14)H, and C(18)H), 7.80 (t, .sup.3J.sub.HH=7.6 Hz, 1H, C (2)H), 7.94 (dd, .sup.3J.sub.HH=8.0 Hz, .sup.4J.sub.HH=1.7 Hz 1H, C (4)H), 7.99 (d, .sup.3J.sub.HH=7.7 Hz, 1H, C(12)H), 8.29 (d, .sup.3J.sub.HH=8.0 Hz, 1H, C(1)H); .sup.1H NMR (401.00 MHz, DMSO-d.sub.6): δ 2.04 (s, 6H, C(8)H.sub.3), 7.31-7.43 (m, 4H, C(15)H, C(17)H and C(19)), 7.49-7.59 (m, 5H, C(3)H, C(13)H, C(14)H, and C(18)H), 7.76 (dd, .sup.3J.sub.HH=7.1 Hz, .sup.4J.sub.HH=2.0 Hz, 1H, C(12)H), 7.85 (td, .sup.3J.sub.HH=7.7 Hz, .sup.4J.sub.HH=1.3 Hz, 1H, C (2)H), 7.97 (dd, .sup.3J.sub.HH=8.0 Hz, .sup.4J.sub.HH, 1.7 Hz, 1H, C(4)H), 8.59 (d, .sup.3J.sub.HH=8.0 Hz, 1H, C(1)H); .sup.1H NMR (401.00 MHz, CD.sub.3CN): δ 2.07 (s, 6H, C(8)H.sub.3), 7.27-7.44 (m, 4H, C(15)H, C(17)H and C(19)), 7.44-7.62 (m, 5H, C(3)H, C(13)H, C(14)H, and C(18)H), 7.78-7.83 (m, 1H, C(12)H), 7.90 (td, .sup.3J.sub.HH=7.7 Hz, .sup.4J.sub.HH=1.3 Hz, 1H, C (2)H), 8.03 (dd, .sup.3J.sub.HH=8.0 Hz, .sup.4J.sub.HH=1.8 Hz, 1H, C(4)H), 8.51 (dd, .sup.3J.sub.HH=8.0 Hz, .sup.4J.sub.HH=1.3 Hz, 1H, C(1)H); .sup.19F NMR (377.28 MHz, CDCl.sub.3): δ −84.9 (t, .sup.3J.sub.FF=8.2 Hz, 2F, CF.sub.2O), −81.5 (s, 2F, CF.sub.2I); .sup.19F NMR (377.28 MHz, DMSO-d.sub.6): δ −80.5 (t, .sup.3J.sub.FF=6.5 Hz, 2F, CF.sub.2O), −78.7 (bs, 2F, CF.sub.2I); .sup.19F NMR (377.28 MHz, CD.sub.3CN): δ −84.1 (t, .sup.3J.sub.FF=7.4 Hz, 2F, CF.sub.2O), −80.4 (t, .sup.3J.sub.FF=7.4 Hz, 2F, CF.sub.2I); .sup.13C {.sup.1H} NMR (100.84 MHz, CDCl.sub.3): δ 28.5 (s, 2C, C(8)H3), 81.2 (s, 1C, C(7)), 108.5 tt, .sup.1J.sub.CF=279.1 Hz, .sup.2J.sub.CF=25.8 Hz, 1C, ICF.sub.2), 109.7 (s, 1C, C(6)), 115.6 (tt, .sup.1J.sub.CF=337.6 Hz, .sup.2J.sub.CF=41.1 Hz, 1C, OCF.sub.2), 121.3 (s, 2C, C(17)H), 126.4 (s, 1C, C(15)H), 127.7 (s, 1C, C(19)H), 127.8 (s, 1C, C(12)H), 130.0 (s, 1C, C(4)H), 130.0 (s, 1C, C(13)H or C(14)H), 130.2 (s, 2C, C(18)H), 130.3 (s, 1C, C(11)H), 131.1 (s, 1C, C(13)H or C(14)H), 131.6 (s, 1C, C(3)H), 134.3 (s, 1C, C(2)H), 140.0 (s, 1C, C(10)), 141.8 (s, 1C, C(1)), 146.0 (s, 1C, C(5)), 147.8 ((s, 1C, C(16)), 167.3 (s, 1C, C(9)); HRMS (m/z, ESI.sup.+): [M+Na].sup.+ calc. for C.sub.24H.sub.19 O.sub.6SF.sub.4INa 660.9775, found, 660.9768.

[0134] In NMR tube, 1o (23 mg, 35 μmol) was dissolved in DMSO-d.sub.6 (0.4 ml). To solution was added LiCl (4.2 mg, 0.1 mmol) and after 30 minutes was measured .sup.1H and .sup.19F NMR. .sup.1H NMR (401.00 MHz, DMSO-d.sub.6): δ 2.12 (bs, 6H, C(8)H.sub.3), 7.31-7.54 (m, 9H, C(3)H, C(13)H, C(14)H, C(15)H, C(17)H, C(18)H) and C(19)), 7.69-7.80 (m, 2H, C (2)H and C(12)H), 8.02 (dd, .sup.3J.sub.HH=8.1 Hz, .sup.4J.sub.HH=1.7 Hz, 1H, C(4)H), 8.44 (d, .sup.3J.sub.HH=7.9 Hz, 1H, C(1)H); .sup.19F NMR (377.28 MHz, DMSO-d.sub.6): δ −86.7 (bs, 1F, CF.sub.2I), −83.8 (bs, 1F, CF.sub.2I), −81.5 (t, .sup.3J.sub.FF=6.3 Hz, 2F, CF.sub.2O); .sup.13C {.sup.1H} NMR (100.84 MHz, DMSO-d.sub.6): δ 28.5 (only in HSQC, 2C, C(8)H3), 83.1 (s, 1C, C(7)), 111.2 (tt, .sup.1J.sub.CF=344.3 Hz, .sup.2J.sub.CF=40.8 Hz, 1C, CF.sub.2), 115.2 (s, 1C, C(6)), 116.3 (tt, .sup.1J.sub.CF=275.6 Hz, .sup.2J.sub.CF=26.5 Hz, 1C, CF.sub.2), 121.8 (s, 2C, C(17)H), 126.9 (s, 1C, C(15)H), 127.4 (s, 1C, C(19)H), 127.5 (s, 1C, C(12)H), 128.7 (s, 1C, C(13)H or C(14)H), 129.1 (s, 1C, C(4)H), 129.6 (s, 1C, C(13)H or C(14)H), 130.3 (s, 2C, C(18)H), 130.3 (s, 1C, C(3)H), 131.8 (s, 1C, C(11)H), 132.8 (s, 1C, C(2)H), 141.7 (s, 1C, C(1)), 144.5 (s, 1C, C(10)), 146.7 (s, 1C, C(5)), 148.0 ((s, 1C, C(16)), 166.9 (s, 1C, C(9));

Example 7: Synthesis of Reagent 1q

[0135] ##STR00015##

[0136] 1c (227 mg, 0.5 mmol) was dissolved in dry CHCl.sub.3 (1 ml) under argon atmosphere and acetyl chloride (0.1 ml, 1.5 mmol) was added in one portion. The mixture was stirred 15 minutes at laboratory temperature. After that volatiles were removed under reduced pressure and residue was washed with Et.sub.2O to give pure 1q as white particles. Yield: 194 mg (73%); .sup.1H NMR (401.00 MHz, CDCl.sub.3): δ 2.09 (s, 6H, C(8)H.sub.3), 2.22 (s, 3H, C(10)H.sub.3), 7.20 (d, .sup.3J.sub.HH=8.1 Hz, 2H, C(17)H), 7.27-7.37 (m, 2H, C(3)H and C (19)H), 7.42 (t, .sup.3J.sub.HH=7.8 Hz, 2H, C(18)H), 7.63-7.78 (m, 2H, C(2)H and C(4)H), 8.37 (d, .sup.3J.sub.HH=8.0 Hz, 1H, C(1)H); .sup.19F NMR (377.28 MHz, CDCl.sub.3): δ −87.8 (s, 2F, CF.sub.2I), −84.3 (t, .sup.3J.sub.FF=6.8 Hz, 2F, CF.sub.2O); .sup.13C {.sup.1H} NMR (100.84 MHz, CDCl.sub.3): δ 22.9 (s, 1C, C(10)H3), 28.7 (s, 2C, C(8)H3), 82.2 (s, 1C, C(7)), 112.9 (tt, .sup.1J.sub.CF=345.1 Hz, .sup.2J.sub.CF=40.7 Hz, 1C, ICF.sub.2), 114.4 (s, 1C, C(6)), 116.5 (tt, .sup.1J.sub.CF=277.2 Hz, .sup.2J.sub.CF=25.8 Hz, 1C, OCF.sub.2), 121.5 (s, 2C, C(17)H), 127.3 (s, 1C, C(19)H), 129.6 (s, 1C, C(4)H), 130.0 (s, 2C, C(18)H), 131.0 (s, 1C, C(3)H), 133.2 (s, 1C, C(2)H), 141.8 (s, 1C, C(1)), 145.6 (s, 1C, C(5)), 148.2 (s, 1C, C(16)), 169.4 (s, 1C, C(9)); HRMS (m/z, ESI.sup.+): [M+Na].sup.+ calc. for C.sub.19H.sub.18 O.sub.3ClF.sub.4INa 554.9818, found, 554.9811.

Example 8: Synthesis of Reagent 1r

[0137] ##STR00016##

[0138] N-2-Chlorocarbonylmethyl-N,N,N-trimethylammonium chloride (56 mg, 0.33 mmol) was suspended in dry CHCl.sub.3 (1 ml) under argon atmosphere and 1a (109 mg, 0.33 mmol) was added in one portion. The suspension was stirred for 30 min under ambient temperature, then diluted with Et.sub.2O (1 ml). White particles were filtered and dried under reduced pressure. Yield: 166 mg (71%, purity 75%, 25% unreacted betaine); .sup.1H NMR (401.00 MHz, DMSO-d.sub.6): δ 2.00 (bs, 6H, C(8)H3), 3.37 (bs, 9H, C(11)H3), 4.63 (bs, 2H, C(10)H2), 7.43 (t, .sup.3J.sub.HH=7.6 Hz, 1H, C(3)H), 7.72 (t, .sup.3J.sub.HH=7.6 Hz, 1H, C(2)H), 7.81 (d, .sup.3J.sub.HH=8.0 Hz, 1H, C(4)H), 8.51 (d, .sup.3J.sub.HH=7.8 Hz, 1H, C(1)H); .sup.19F NMR (377.28 MHz, DMSO-d.sub.6): δ −34.3 (s, 3F, CF.sub.3); .sup.13C {.sup.1H} NMR (100.84 MHz, DMSO-d.sub.6): δ 27.3 (s, 2C, C(8)H.sub.3), 53.4 (s, 3C, C(11)H.sub.3), 63.8 (s, 1C, C(10)H2C1), 84.4 (s, 1C, C(7)), 108.4 (q, .sup.1J.sub.CF=391.5 Hz, 1C, CF.sub.3), 120.7 (s, 1C, C(6)), 128.6 (s, 1C, C(4)H), 131.11 (s, 1C, C(3)H), 132.3 (s, 1C, C(2)H), 141.4 (s, 1C, C(1)), 143.0 (s, 1C, C(5)), 163.5 (s, 1C, C(9)); HRMS (m/z, ESI.sup.+): [M-Cl].sup.+ calc. for C.sub.15H.sub.21O.sub.2NClF.sub.3I 466.0252, found 466.0248.

Example 9: Synthesis of Reagent 1s

[0139] ##STR00017##

[0140] In a 3-necked round-bottom flask equipped with a thermometer and under Ar atmosphere, a solution of phenylmagnesium bromide in diethyl ether (63 ml of 1.49 M solution, 93.8 mmol, 2.5 equiv.) was added followed by addition of further diethyl ether (40 ml). The resulting mixture was cooled in water bath to 15° C. and a solution of 2-iodobenzoylchloride (10 g, 37.5 mmol, 1 equiv.) in diethyl ether (10 ml) was added dropwise so that the internal temperature does not exceed 25° C. After the addition was complete, the reaction mixture was stirred for 3 h at room temperature. Afterwards, the biphasic brown reaction mixture is poured onto well-stirred cold diluted phosphoric acid (150 ml, 20%) until all magnesium salts dissolve. The organic phase was separated, the aqueous phase was extracted with ethyl acetate (2×70 ml). The pooled organic phases were washed with brine and dried over magnesium sulfate and filtered. The filtrate gave upon evaporation and drying on high vac a brown viscous oil in nearly quantitative yield (14.8 g, approx. 90% purity). The crude (2-iodophenyl)diphenylmethanol was taken directly into the next step.

[0141] The crude 2-iodophenyl)diphenylmethanol (5 g in 90% purity, 11.6 mmol, 1 equiv.) was dissolved in acetonitrile (40 ml) and heated to 70° C. In a separate flask, trichloroisocyanuric acid “TCICA” (916 mg, 3.94 mmol, 0.34 equiv.) was dissolved in acetonitrile (10 ml). To the well-stirred solution of crude 2-iodophenyl)diphenylmethanol was slowly added the solution of TCICA and the resulting mixture was refluxed for 15 min. The precipitated isocyanuric acid was filtered off, the solution was concentrated to dryness, suspended in a mixture of pentane/diethyl ether (4/1 v/v, 70 ml) and cooled to −15° C. with stirring. The yellowish solid (1-chloro-3,3-diphenyl-1,3-dihydro-1λ3-benzo[d][1,2]iodaoxole) was collected by filtration and washed with little precooled pentane and dried in high vacuum (1.83 g, 37%).

[0142] In a Schlenk flask equipped with a magnetic stirring bar, potassium acetate (326 mg, 3.32 mmol, 2 equiv.) was dehydrated with heatgun under vacuum and then backfilled with Ar. To the cooled Schenk was added 1-chloro-3,3-diphenyl-1,3-dihydro-113-benzo[d][1,2]iodaoxole (700 mg, 1.66 mmol, 1 equiv.) followed by anhydrous acetonitrile (8 ml) and the resulting mixture was stirred at room temperature for 5 h. Subsequently, the reaction mixture was cooled to −15° C. and trifluoromethyl trimethylsilane (472 mg, 5 mmol, 2 equiv.) was added at once. The reaction mixture was warmed up to room temperature over the course of 5 h, then was filtered over a pad of Celite and concentrated to dryness on rotavap. The brownish oil was redissolved in a mixture of pentane/diethylether (3/1 v/v, 20 ml) and filtered through a pad of alumina. The resulting filtrate was again concentrated to dryness and subjected to silica gel chromatography (gradient elution hexane to hexane/diethylether 1:1) to give after evaporation to dryness is as a yellowish viscous oil. Yield: 196 mg (26%). HRMS (m/z, ESI+): [M+Na].sup.+ calc. for C.sub.20H.sub.14F.sub.3INaO, 476.9939, found, 476.9933.

Example 10: Synthesis of Reagent 1t

[0143] ##STR00018##

[0144] In a Schlenk flask equipped with a magnetic stirring bar, potassium acetate (326 mg, 3.32 mmol, 2 equiv.) was dehydrated with heatgun under vacuum and then backfilled with Ar. To the cooled Schenk was added 1-chloro-3,3-diphenyl-1,3-dihydro-113-benzo[d][1,2]iodaoxole (700 mg, 1.66 mmol, 1 equiv.) followed by anhydrous acetonitrile (7 ml) and the resulting mixture was stirred at room temperature for 5 h. Subsequently, the reaction mixture was cooled to −15° C. and a solution of trimethyl(1,1,2,2-tetrafluoro-2-(4-methoxyphenoxy)ethyl)silane (983 mg, 3.32 mmol, 2 equiv.) was added at once. The reaction mixture was warmed up to room temperature over the course of 5 h, then was filtered over a pad of Celite and concentrated to dryness on rotavap. The brownish oil was redissolved in a mixture of pentane/diethylether (3/1 v/v, 20 ml) and filtered through a pad of alumina. The resulting filtrate was again concentrated to dryness and subjected to silica gel chromatography (gradient elution hexane to hexane/diethylether 1:1) to give after evaporation to dryness 1t as a yellow viscous oil. Yield: 192 mg (19%). HRMS (m/z, ESI+): [M+Na].sup.+ calc. for C.sub.28H.sub.21F.sub.4INaO.sub.3, 631.0369, found, 631.0377.

Example I: Fluoroalkylation of 3-Methylindole (Scheme 11)

[0145] 3-Methylindole (66 mg, 0.5 mmol) was dissolved in MeOH (2.14 ml) and the solution was kept under argon atmosphere using the Schlenk line technique. Sodium ascorbate (50 mg, 0.25 mmol, 50 mol %) was dissolved in water (0.72 ml) and added to the solution of substrate in MeOH. 1d (264 mg, 0.6 mmol, 1.2 equiv.) was dissolved in MeOH (2.14 ml) and the resulting solution was slowly (in 5 minutes) added to the mixture of substrate and sodium ascorbate. The reaction mixture was stirred for 5 minutes at room temperature. After this period, the solvent was evaporated and the mixture was partitioned between DCM (30 ml) and water (30 ml). The organic phase was washed with water (3×15 ml), brine (3×15 ml), saturated aq. NaHCO.sub.3 solution (3×15 ml) and dried over MgSO.sub.4, followed by solvent removal under reduced pressure. Purification by flash chromatography (cyclohexane/DCM, 3:1] afforded pure product as a pale yellow oil. Yield: 140 mg (87%); R.sub.f=0.27 (cyclohexane/DCM, 3:1); .sup.1H NMR (400 MHz, CD.sub.3OD): δ 2.45-2.47 (t, J=2.2 3H, CH.sub.3), 7.07-7.14 (m, 3H, Car H), 7.20-7.26 (m, 2H, Car H), 7.31-7.38 (m, 2H, Car H), 7.41-7.44 (m, 1H, Car H), 7.59-7.62 (m, 1H, C.sub.ArH); .sup.19F NMR (376 MHz, CD.sub.3OD): δ −86.46 to −86.50 (t, .sup.3J.sub.FF=6.4, 2F, CF.sub.2), δ −110.81 to −110.86 (tt, .sup.3 J.sub.FF=6, 2F, CF.sub.2); .sup.13C H, {.sup.1H, .sup.19F} NMR (126 MHz, CD.sub.3OD): δ 9.20 (3H, CH.sub.3), 113.02, 115.05, 115.38, 119.75, 120.68, 120.79, 122.88, 123, 125.09, 127.91, 129.76, 131.11, 138.05, 150.82; HRMS (ESI) m/z Calcd for C.sub.17H.sub.13F.sub.4NO [M].sup.+ 323.0933, found 323.0932.

Example II: Fluoroalkylation of a Protected Tryptophan Derivative

[0146] ##STR00019##

[0147] Protected Trp derivative (137 mg, 0.5 mmol) was dissolved in MeOH (1.1 ml) and the solution was kept under argon atmosphere using the Schlenk line technique. Sodium ascorbate (50 mg, 0.25 mmol, 50 mol %) was dissolved in water (0.7 ml) and added to the solution of substrate in MeOH. 1cl (264 mg, 0.6 mmol, 1.2 equiv.) was dissolved in MeOH (1 ml) and the resulting solution was slowly (in 5 minutes) added to the mixture of substrate and sodium ascorbate. The reaction mixture was stirred for 5 minutes at room temperature. After this period, the solvent was evaporated and the mixture was partitioned between DCM (30 ml) and water (30 ml). The organic phase was washed with water (3×15 ml), brine (3×15 ml), saturated aq. NaHCO.sub.3 solution (3×15 ml) and dried over MgSO.sub.4, followed by solvent removal under reduced pressure. Purification by flash chromatography (cyclohexane/DCM, 6:1] afforded pure product as a pale brown oil. Yield: 95 mg (41%); R.sub.f=0.3 (cyclohexane/DCM, 6:1); .sup.1H NMR (400 MHz, CD.sub.3OD): δ 0.95-0.99 (t, J=7.1, 3H), 1.22-1.26 (t, J=7.1, 1H), 1.92 (s, 2H), 2.01 (s, 1H), 3.34-3.51 (m, 2H), 4.74-4.78 (m, 1H), 7.13-7.18 (m, C.sub.ArH, 3H), 7.24-7.29 (m, C.sub.ArH, 2H), 7.34-7.39 (m, C.sub.ArH, 2H), 7.45-7.48 (m, C.sub.ArH, 1H), 7.69-7.71 (m, C.sub.ArH, 1H); HRMS (ESI) m/z Calcd for C.sub.23H.sub.22F.sub.4N.sub.2O.sub.4 [M−H].sup.+ 466.15, found 466.1520.

Example III: Fluoroalkylation of Amino Acids

[0148] ##STR00020## ##STR00021##

Experiment IIIa—Trifluoromethylation of Individual Aromatic Amino Acids

[0149] To a solution of amino acid (0.1 mmol) in MeOH (0.2 ml) was added a solution of reagent 1b (0.12 mmol, 1.2 equiv.) in MeOH (0.65 ml). A solution of sodium ascorbate (0.05 mmol) in water or buffer of pH 5 (phosphate) or pH 9 (carbonate/acetate) (0.15 ml) was added dropwise over 2 minutes. After 2 h of stirring at 25° C. the resulting mixture was analyzed by .sup.19F NMR to determine NMR yield using sodium trifluoroacetate as an internal standard (Table 1).

TABLE-US-00001 TABLE 1 .sup.19F NMR yields of fluoroalkylation of amino acids with 1b Aminoacid pH Product yield (%) Trp — 76 Tyr — 9 9 20 Phe 5 0 9 2 His — 5 5 2 9 13

[0150] In a separate experiment, stock solutions of natural amino acid standards (Trp, Tyr, Phe, His, Gly, Ala, Ser, Pro, Val, Thr, Ile, Leu, Asn, Asp, Glu, Gln, Arg, Lys, Met (Waters, 5 mM standard solution at pH=7.5) were dissolved in an ammonium bicarbonate buffer (pH=7.5, 50 mM) to reach the final concentration 2 mM for each amino acid. Reagent 1a (10 equiv. calculated to each amino acid, 30 mM solution in DMSO) was added followed by ascorbic acid (5 equiv. calculated to each amino acid, 283 mM solution in water) were added. After stirring at 25° C. for 15 minutes, semiquantitative LCMS analysis shower reactivity order as follows: Trp>>Tyr>Phe>His. Extracted-ion chromatograms indicated that only aromatic aminoacids and cystine underwent fluoroalkylation (FIG. 2).

TABLE-US-00002 Trifluoro- methylation Amino Acid detected? m/z Note Trp Yes 273.085 1 major isomer along (monofluoro- with 2 minor isomers alkylation) detected Trp Yes 341.072 2 isomers detected (bis(fluoro- alkylation)) Tyr Yes 250.069 1 major isomer (monofluoro- detected alkylation) Phe Yes 234.074 2 isomers detected His Yes 224.064 1 major isomer detected Gly, Ala, Ser, No N/A No mass corresponding Pro, Val, Thr, to trifluoromethylated Leu, Ile, Asn, amino acids could be Asp, Gln, Lys, detected Glu, Met, Arg

Example IV: Fluoroalkylation of Peptide AFRIPLYWGRI (FIG. 1)

[0151] A solution of peptide AFRIPLYWGRI (1 mg) in MeCN (0.7 ml) containing 1% formic acid and water (0.3 ml) was prepared. Solution of 1d (50 mM) in MeCN and a solution of sodium ascorbate (50 mM) in water were prepared. Solution of the peptide (45 μl) was diluted with water (0.4 ml) containing methionine (20 mM) and to 0.1 ml of this solution 1.46 μl of ascorbate solution was added. Finally, the solution of hypervalent iodine reagent (1.46 μl, 10 equiv.) was added and the mixture was vortexed for a few seconds. After 1 h at ambient temperature the mixture was analyzed by MALDI MS: m/z starting peptide calcd for C.sub.69H.sub.104N.sub.19O.sub.12 [M+H].sup.+ 1390.8112, found 1390.8065; peptide+one modification calcd for C.sub.77H.sub.108F.sub.4N.sub.19O.sub.13 [M+H].sup.+ 1582.8310, found 1582.8227; peptide+two modifications calcd for C.sub.84CH.sub.112F.sub.8N.sub.19O.sub.14 [M+H].sup.+1775.8542, found 1775.8403. MS/MS analysis confirmed that monofluoroalkylation took place only on Trp.

TABLE-US-00003 AFRIPLYWGRI AFRIPLYWGRI approx. m/z Reagent conversion Masses detected 1390.8065 1d High 1582.8227 (monofluoroalkylated) 1775.8403 (bis(fluoroalkylated))

Example V: Fluoroalkylation of Peptide Bradykinin (FIG. 2)

[0152] Bradykinin (1 equiv. as 1 mg/ml degassed solution in 20% v/v MeCN/50 mM pH 7 HEPES buffer) was mixed with sodium ascorbate (100 equiv. calculated to molar amount of aromatic residues, 20 mM solution in water) and solution of 1h was added (100 equiv. calculated to molar amount of aromatic resides, 8.7 mM in 50% v/v MeCN/H.sub.2O) The mixture was shaken for 15 min at 25° C. MALDI MS analysis indicated partial formation of fluoroalkylated bradykinin and traces of 2× fluoroalkylated bradykinin MS/MS analysis revealed that Phe residue is fluoroalkylated.

TABLE-US-00004 Bradykinin Bradykinin approx. m/z Reagent conversion Masses detected 1060.626 1h Low 1309.709 (monofluoroalkylated) 1559.802 (bis(fluoroalkylated)), trace)

Example VI: Fluoroalkylation of Peptide Somatostatin (FIG. 3)

[0153] Somatostatin (1 equiv. as 1 mg/ml degassed solution in 20% v/v MeCN/50 mM pH 7 HEPES buffer) was mixed with sodium ascorbate (100 equiv. calculated to molar amount of aromatic residues, 20 mM solution in water) and solution of 1h was added (100 equiv. calculated to molar amount of aromatic resides, 8.7 mM in 50% v/v MeCN/H.sub.2O). The mixture was shaken for 15 min at 25° C. MALDI MS analysis indicated formation of monofluoroalkylated somatostatin, bis(fluoroalkylated) somatostatin, tris(fluoroalkylated) somatostatin and traces of fluoroalkylated oxidized somatostatin. MS/MS analysis revealed that the only positions of first and second modification were on the Trp residue.

TABLE-US-00005 Somatostatin Somatostatin m/z Reagent conversion Masses detected 1637.819 1h High 1886.897 (monofluoroalkylated) 2135.979 (bis(fluoroalkylated)) 2385.073 (tris(fluoroalkylated), trace)

Example VII: Fluoroalkylation of Peptide Bombesin (FIG. 4)

[0154] Bombesin (1 equiv. as 1 mg/ml degassed solution in 20% v/v MeCN/50 mM pH 7 HEPES buffer) was mixed with sodium ascorbate (100 equiv. calculated to molar amount of aromatic residues, 20 mM solution in water) and solution of 1h was added (100 equiv. calculated to molar amount of aromatic resides, 8.7 mM in 50% v/v MeCN/H.sub.2O). The mixture was shaken for 15 min at 25° C. MALDI MS analysis indicated formation of monofluoroalkylated bombesin, bis(fluoroalkylated) bombesin and traces of tris(fluoroalkylated) bombesin and oxidized fluoroalkylated bombesin. MS/MS analysis revealed that the only positions of first and second modification were on the Trp residue.

TABLE-US-00006 Bombesin Bombesin approx. m/z Reagent conversion Masses detected 1619.944 1h High 1868.974 (monofluoroalkylated) 1884.967 (monofluoroalkylated, monooxidized, trace) 2118.047 (bis(fluoroalkylated)) 2134.038 (bis(fluoroalkylated), monooxidized, trace) 2369.139 (tris(fluoroalkylated), trace)

Example VIII: Fluoroalkylation of Insulin

[0155] ##STR00022##

[0156] Human recombinant insulin in HEPES or TRIS buffer (pH 7-9) was subjected to fluoroalkylation with reagents 1e-1h (20-100 equiv. calculated to molar amount of aromatic residues) in MeCN and sodium ascorbate (20-100 equiv. calculated to molar amount of aromatic residues) at 25° C. for 15 min. The comparison of reagents 1e and 1f revealed that the “acid-type” reagent 1f gave a deeper degree of fluoroalkylation (up to 8 modifications). To determine the site of fluoroalkylation after the reaction, the A and B chains of insulin were reductively cleaved by the treatment with DTT and the individual chains were subjected to MS/MS analysis. MS/MS analysis showed (experiment with reagents 1f at pH 8) that Phe and Tyr residues were fluoroalkylated at roughly equal proportions, while there was a minor fluoroalkylation of histidine.

[0157] Another experiments were carried out using 100 equiv. of reagents 1g or 1h (calculated to molar amount of aromatic residues), 100 equiv. of sodium ascorbate (calculated to molar amount of aromatic residues) at pH 7, 8 and 9. It was found that at pH 9 the extent of fluoroalkylation was higher than in pH 7 and 8.

TABLE-US-00007 Human Insulin recombinant approx. insulin m/z Reagent, note conversion Masses detected 5809 1e Medium 5975 (monofluoroalkylated) 20 equiv 6141 (bis(fluoroalkylated)) ascorbate, 6307 (tris(fluoroalkylated)) 100 equiv 6474 (tetrakis(fluoroalkylated), 1e used trace) 6641 (pentakis(fluoroalkylated), trace) 6810 (hexakis(fluoroalkylated), trace) 5809 1f High 5975 (monofluoroalkylated) 20 equiv 6142 (bis(fluoroalkylated)) ascorbate, 6308 (tris(fluoroalkylated)) 100 equiv 6475 (tetrakis(fluoroalkylated), 1f used trace) 6641 (pentakis(fluoroalkylated), trace) 6807 (hexakis(fluoroalkylated), trace) 6973 (heptakis(fluoroalkylated), trace) 7139 (octakis(fluoroalkylated), trace)

TABLE-US-00008 Human recombinant pH and Approx. insulin m/z Reagent buffer conversion Masses detected 5809 1g pH = 7, 21% 6057 (monofluoroalkylated) HEPES 6308 (bis(fluoroalkylated)) buffer 5809 1g pH = 8, 20% 6057 (monofluoroalkylated) HEPES 6308 (bis(fluoroalkylated)) buffer 5809 1g pH = 9, 63% 6057 (monofluoroalkylated) TRIS 6308 (bis(fluoroalkylated)) buffer 5809 1h pH = 7, 24% 6057 (monofluoroalkylated) HEPES 6308 (bis(fluoroalkylated)) buffer 5809 1h pH = 8, 25% 6057 (monofluoroalkylated) HEPES 6308 (bis(fluoroalkylated)) buffer 5809 1h pH = 9, 71% 6057 (monofluoroalkylated) TRIS 6308 (bis(fluoroalkylated)) buffer

Example IX: Fluoroalkylation of Ubiquitin—Protein Modification

[0158] ##STR00023##

[0159] A stock solution of ubiquitin from bovine erythrocytes in water was diluted in 50 mM ammonium bicarbonate buffer (pH 7.5) to reach 58 μM concentrations (0.5 mg/ml). Freshly prepared solution of ascorbic acid in water (283 mM, 100 equiv. calculated to molar amount of aromatic residues) and 1e in DMSO (10 mg/1 ml, 100 equiv. calculated to molar amount of aromatic residues) were added and the reaction mixture which was incubated for 15 min at 25° C. The mixture was desalted using Peptide Microtrac in the off-line holder (MichromBioresources) according manufacturer instruction, and eluted with 50 μl of 80% MeCN/5% AcOH. The protein was analysed using solariX XR FT-ICR mass spectrometer (Bruker Daltonics) equipped with a 15T superconducting magnet and ParaCell. The instrument was internally calibrated using Agilent tuning mix (Agilent Technologies, USA). Mass spectra were acquired in the positive mode over the m/z range 250-2500 with 2 M data points transient and 1 s ion accumulation, 8 scans were accumulated per spectrum. Data acquisition and data processing were performed using ftmsControl 2.2.0 and DataAnalysis 5.0. ESI MS spectra revealed predominantly monofluoroalkylation of the protein.

TABLE-US-00009 Ubiquitin Approximate m/z Reaction conditions conversion Masses detected 779.15658 10 equiv. 1e, Negligible — (11+ charge 10 equiv. ascorbate state) 779.15658 100 equiv. 1e, Medium 794.24967 (11+ charge 100 equiv. (monofluoroalkylated, state) ascorbate 11+ charge state) 809.34281 (bis(fluoroalkylated), 11+ charge state, trace) 714.30842 1k Medium 728.39401 (12+ charge (monofluoroalkylated, state) 12+ charge state) 794.52022 (monofluoroalkylated, 11+ charge state) 714.30842 1k, followed by Medium 751.40587 (12+ charge incubation with (monofluoroalkylated state) DBCO-amine and ligated with DBCO-amine, 12+ charge state) 819.62388 (monofluoroalkylated, 11+ charge state)

Example X: Fluoroalkylation of Ubiquitin Followed by Click Reaction of the Conjugate

[0160] ##STR00024##

[0161] A stock solution of ubiquitin from bovine erythrocytes in water was transferred to 50 mM ammonium bicarbonate buffer (pH 7.5) using a micro BioSpin 6 column (Biorad) and diluted in same buffer to reach 58 μM concentrations (0.5 mg/ml). Freshly prepared solution of ascorbic acid in water (50 mg/ml, 37 equiv.) and 1k in DMSO (10 mg/1 ml, 100 equiv.) were added and the reaction mixture was incubated for 1 min at 25° C. and transferred to 50 mM ammonium bicarbonate buffer pH 7.5 using a micro BioSpin 6 column in order to eliminate the effect of side products of radical reaction or unreacted ascorbic acid. In the second step, the fluoroalkylated ubiquitin solution was mixed with DBCO-amine (100 equiv.) and incubated for 1 hour at 25° C. in the dark. The mixture was desalted using Peptide Microtrap in the off-line holder (MichromBioresources) according to manufacturer's sinstructions, and eluted with 50 μl of 80% MeCN/5% AcOH. The protein was analysed using solariX XR FT-ICR mass spectrometer (Bruker Daltonics) equipped with a 15T superconducting magnet and ParaCell. The instrument was internally calibrated using Agilent tuning mix (Agilent Technologies, USA). Mass spectra were acquired in the positive mode over the m/z range 250-2500 with 2 M data points transient and 1 s ion accumulation, 8 scans were accumulated per spectrum. Data acquisition and data processing were performed using ftmsControl 2.2.0 and DataAnalysis 5.0. ESI-MS indicated that ubiquitin underwent predominantly monofluoroalkylation with trace bis(fluoroalkylation) and the mono- and bis(fluoroalkylated) ubiquitin subsequently underwent clean copper-free click reaction with a dibenzocyclooctyne-amine reagent, affording the corresponding desired conjugates.

TABLE-US-00010 Ubiquitin Reaction Approximate m/z conditions conversion Masses detected 714.30842 1k Medium 728.39401 (12+ (monofluoroalkylated, charge 12+ charge state) state) 794.52022 (monofluoroalkylated, 11+ charge state) 714.30842 1k, followed by Medium 751.40587 (12+ incubation with (monofluoroalkylated charge DBCO-amine and ligated with DBCO- state) amine, 12+ charge state) 819.62388 (monofluoroalkylated, 11+ charge state)

Example XI: Human Carbonic Anhydrase I—Aminoacid Side Chains Reactivity on Protein

[0162] For the reaction, a stock solution of carbonic anhydrase I from bovine erythrocytes (hCA I, Merck) in water was transferred to 50 mM ammonium bicarbonate buffer pH 7.5 using a micro BioSpin 6 column (Biorad) and diluted in same buffer to reach 17 μM concentration (0.5 mg/me. Freshly prepared solution of ascorbic acid in water and one of reagents (1a, 1b, 1e and 1f) in DMSO were added in 150× and 200× molar excess to protein, respectively. The reaction mixture was incubated 10 min at room temperature and mixed with 4×concentrated LDS sample buffer (Invitrogen) containing 100 mM dithiothreitol as the reducing agent in a 3:1 (v/v) ratio. Samples were loaded onto a NuPage 4-12% Bis-Tris gel (80.0×80.0×1.0 mm, 10 wells, Invitrogen). Separation was performed in MES running buffer (Invitrogen) for 35 min at 200 V. After separation, the gels were stained by Coomassie Brilliant Blue R250 and destained with ethanol, water, and acetic acid in the ratio 55:35:10. Identification of modified hCA I was performed as described previously (Rozbeský, D.; Rosůlek, M.; Kukac̆ka, Z.; Chmelík, J.; Man, P.; Novák, P. Analytical Chemistry 2018, 90 (2), 1104-1113.) Briefly, bands of the modified enzymes were excised. In gel proteolysis by trypsin was carried out for 6 hours at 37° C. (enzyme:protein ratio 1:20 w/w), dryed using SpeedVac and re-suspended in water containing 2% MeCN and 0.1% TFA.

[0163] Nano Reversed phase column (EASY-Spray column, 50 cm×75 μm ID, PepMap C18, 2 μm particles, 100 Å pore size) was used for LC/MS analysis. Mobile phase buffer A was composed of water and 0.1% formic acid. Mobile phase B was composed of acetonitrile and 0.1% formic acid. 1 μg of re-suspended samples were loaded onto the trap column (Acclaim PepMap300, C18, 5 μm, 300 Å Wide Pore, 300 μm×5 mm) at a flow rate of 15 μl/min. Loading buffer was composed of water, 2% MeCN and 0.1% TFA. Peptides were eluted with gradient of B from 4% to 35% over 60 min at a flow rate of 300 nl/min. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT-qIT, Thermo). Survey scans of peptide precursors from 350 to 1400 m/z were performed at 120 K resolution (at 200 m/z) with a 5×10.sup.5 ion count target. Tandem MS was performed by isolation at 1.5 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 10.sup.4 and the max injection time was 35 ms. Only those precursors with charge state 2-6 were selected for MS2. The dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 2 s cycles. The data were exported to mgf files and MASCOT 2.0 search engine was used to interpret the data. The precursor mass error was set to 5 ppm, the fragment tolerance was set to 10 ppm, oxidation of methionine (+15.9959 a.m.u), and modification of aromatic residues (66.9784 a.m.u. for 1a,b and 166.0174 a.m.u. for 1e,f) were included as well. Only assignments passing the 1% FDR were considered as positive hits. Such comprehensive analysis revealed thryptophane, tyrosine, phenylalanine and histidine modified by fluoroalkyl radicals in all cases (FIG. 5). The assignment showing the selectivity of fluoralkyl radical reaction is shown for 1a. According to the MS/MS analysis the Trp123, Tyr88, Phe84 and His67 residues were modified with one CF.sub.3 group. All modified residues are solvent accessible using 1hcb structural model of hCA I (FIG. 6), which clearly demonstrated that after radical fluoroalkylation the native structure of the protein was not perturbed.

Example XII: Labelling of Single Stranded DNA (TOP17)-TAATACGACTCACTATA

[0164] Single stranded DNA (from IDT company) was dissolved in 50 mM ammonium bicarbonate buffer pH 7.5 to reach 50 μM concentration. Freshly prepared solution of ascorbic acid in water (50 mg/ml) and 1f in DMSO (10 mg/l ml) were added each in 100× molar excess relative to ssDNA. The reaction mixture was incubated for 5 min at room temperature. The labeling reaction was quenched by adding AcOH to 1% final concentration. The reaction products were analyzed using solariX XR FT-ICR mass spectrometer (Bruker Daltonics) equipped with a 15T superconducting magnet and ParaCell. The instrument was internally calibrated using Agilent tuning mix (Agilent Technologies, USA). Mass spectra were acquired in the negative ion mode over the m/z range 200-3000 with 2 M data points transient and 0.5 s ion accumulation, 8 scans were accumulated per spectrum. MS/MS spectra were acquired in for −4 charge state with 2 M data points transient and 2.5 s ion accumulation, 64 scans were accumulated per spectrum. Data acquisition and data processing were performed using ftmsControl 2.2.0 and DataAnalysis 5.0, respectively. The modified ssDNA was identified using MS2links software (cit Kellersberger K A, Yu E, Kruppa G H, Young M M, Fabris D. Anal Chem. 2004 May 1; 76(9):2438-45.)

TABLE-US-00011 Oligonucleotide Approximate m/z Reagent conversion Masses detected 1711.313 (3- 1f Low 2649.387 charge state) (monofluoroalkylated, 2− charge state) 1765.946 (monofluoroalkylated, 3− charge state) 1324.214 (monofluoroalkylated, 4− charge state) CID of monofluoroalkylated product ion, 4− charge state (1324.214 m/z) List of detected ions [w2].sup.− (634.101); [a3-B].sup.− (714.126); [w6].sup.2− (926.143); [w6 + R.sub.f].sup.2− (1009.149); [w4].sup.− (1251.191); [a5-B].sup.− (1331.214); [w10].sup.2− (1523.723); [w5].sup.− (1540.253); [w10 + R.sub.f].sup.2− (1606.753); [a7-B].sup.− (1933.331); [a12-B].sup.2− (1728.287); [w5 + R.sub.f].sup.− (1706.269); [AATAC].sup.− (1725.252); [w6].sup.− (1853.272); [w12].sup.2− (1832.794); [w12 + R.sub.f].sup.2− (1925.804); [AATAC + R.sub.f].sup.− (1891.268)

[0165] The MS-spectrum showed an approximately 10% conversion to the mono-fluoroalkylated derivative. The subsequent MS/MS fragmentation revealed that all fluoroalkylations can be traced to cytosine modification (FIG. 7).

INDUSTRIAL APPLICABILITY

[0166] The disclosed method can be used for C—H functionalization of aromatic substrates, covalent modification of peptides and proteins containing aromatic amino acids and nucleotides containing cytosine with fluoroalkyl groups (bioconjugation), analytical biochemistry, protein surface mapping and fluoroalkylation, epitope mapping via surface modification and protein crosslinking. Proteins containing solvent exposed tryptophane residues and oligonucleotides with cytosine nucleobases represent the most suitable substrates.