Supporting Information. Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms

Transcription

1 Supporting Information Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms Marius M. Haugland, Stefan Borsley, Dominic F. Cairns-Gibson, Alex Elmi and Scott L. Cockroft* EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh EH9 3FJ, United Kingdom * S1

2 Contents 1 Methionine residues in -hemolysin Single-molecule nanopore experiments General experimental considerations Single-channel recordings General procedures for single-molecule experiments Methionine modification of protein nanopore CuAAC reaction of alkyne-modified protein nanopore CuAAC reaction of azide-modified protein nanopore SPAAC reaction of azide-modified nanopore Nanopore current traces and analysis methionine modification Pore modification with Pore modification with 1 high-resolution data Pore modification with Pore modification with 2 single modification Pore modification with Pore modification with Nanopore current traces in situ click chemistry SPAAC reaction with CuAAC reaction with CuAAC reaction with CuAAC reaction with CuAAC reaction with CuAAC reaction with Nanopore current traces Cu(I) and Cu(II) interactions and control experiments Cu(II) and Cu(I) in a non-modified pore Cu(II) in an alkyne-modified pore Cu(II) and Cu(I) in an azide-modified pore Cu(II) and Cu(I) in a pore modified with an internal alkyne Nanopore current traces voltage dependence and kinetic analysis of Cu events Kinetic analysis Analysis of intermediate populations S2

3 2.8.1 All-points histogram analysis Graphical determination of equilibrium constants Modelling speciation concentrations Excel solver determination of equilibrium constants Other reactants for single-molecule click reactions Unsuccessful azides and cyclooctynes for CuAAC and SPAAC reactions Full structure of azido-dna (1) for nanopore CuAAC modification Synthesis of reagents General experimental procedures N-Ethyl-3-phenyl-oxaziridine-2-carboxamide (1) Prop-2-ynylurea (S8) Phenyl-N-prop-2-ynyl-oxaziridine-2-carboxamide (2) Azidopropynylurea (S1) N-(3-Azidopropyl)-3-phenyl-oxaziridine-2-carboxamide (3) But-2-ynylisoindoline-1,3-dione (S12) But-2-ynylammonium chloride (S13) But-2-ynylurea (S14) N-But-2-ynyl-3-phenyl-oxaziridine-2-carboxamide (4) References... S3

4 1 Methionine residues in -hemolysin A B C Figure S1: (A) Bottom, (B) side and (C) top view of surface-accessible methionine residues (yellow) in -hemolysin. PDB ID 7AHL. 1 2 Single-molecule nanopore experiments 2.1 General experimental considerations α-hemolysin (α-hl) was purchased from Sigma Aldrich UK, and stored at C as an ice pellet in molecular biology grade water (5 Prime Germany; approx. concentration =.2% w/v). The lipid (1,2- diphytanoyl-sn-glycero-3-phosphocholine) was purchased form Avanti Polar Lipids. HPLC grade water (Fisher UK) was used throughout for making buffers. 2.2 Single-channel recordings Single-channel experiments were performed in a custom built cell. The cell (Figure S2) was formed of two Teflon blocks, each with a machine-drilled well (approx. 1 mm diameter and 1 ml volume). Each well contained a side opening, such that when the blocks were bolted together the adjacent side openings connected the two wells. Additionally, each well contained two access channels (approx. 2 mm diameter), drilled at a 45 angle, such that the channels joined the bottom of the main well. An Figure S2: Schematic representation of the cell used in all single channel experiments. S4

5 trans cis Figure S3: Schematic representation of the experimental setup employed in all single-channel experiments. aperture (approx. 1 µm diameter) was created in a Teflon sheet (Goodfellow, 25 µm thick) using a 3 kv spark gap generator (Ealing Spark Source). The Teflon sheet containing the aperture was clamped and sealed with silicone glue (31 RTV coating, Dow Corning) between the two blocks, such that the aperture was positioned in the central lower half of the side opening between the wells. The cell was placed within a custom built Faraday cage with acoustic damping to isolate the experiment from external electrical and mechanical noise. A small hanging drop (~5 µl) of 1% (v/v) solution of hexadecane in n-pentane was touched on each side of the Teflon sheet. 6 µl of aqueous buffer (1 M KCl, 3 mm potassium phosphate, ph 8.) was added to the well on each side of the Teflon sheet. 6 1 µl of 1 µg/µl solution of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in n-pentane was dispensed using a syringe on each side of the Teflon sheet. The buffer solution was then aspirated and dispensed into each well multiple times using a Hamilton syringe to paint a phospholipid bilayer across the aperture (Figure S3). Ag/AgCl electrodes (Warner) connected to a patch clamp amplifier (Axopatch 2B, Molecular Devices) were placed on either side of the Teflon sheet and a ±1 mv pulse applied at 1333 Hz to determine when a bilayer was obtained (capacitance >3 pf). A gel loading tip fitted to a 2 µl pipette was introduced into an aqueous solution of α-hemolysin (~25 µm), such that a tiny amount (<1 µl) of the solution remained in the tip. Under an applied voltage of +1 mv the pipette tip was then submerged ~5 mm from the aperture in the Teflon sheet and plunged, before being retracted and waiting for at least 6 seconds. The process was repeated until a current corresponding to a single channel arose (~1 pa under the conditions given). The nanopore was characterised by at least four I/V sweeps (1 mv steps spanning 1 to +1 mv, Figure S4), showing the characteristic asymmetry at positive vs negative voltages. All data were collected on an Axopatch 2B patch clamp and digitised using an Axon Instruments Digidata 1332A digitiser (Molecular Devices, LLC), using the Clampex software (version 1.4). Unless otherwise indicated, all single-channel nanopore current traces were recorded with a sampling frequency of 1 khz and using a 2 khz lowpass Bessel filter, at an applied voltage of +1 mv. Postacquisition processing (Clampfit, version 1.4) involved baseline correction and additional digital S5

6 Figure S4: Typical single-channel I/V sweep (1 M KCl, 3 mm potassium phosphate, ph 8.) of a non-modified wild-type -hemolysin nanopore. filtering (lowpass Bessel filter). Analysis such as event detection by threshold or single-channel search were performed in Clampfit, with additional data processing performed in Microsoft Excel General procedures for single-molecule experiments Methionine modification of protein nanopore A fresh solution of oxaziridine reagent 1 4 was made by dissolving the reagent in DMSO to a concentration of 1 mm. Under an applied voltage (+1 mv), oxaziridine reagent solution (1 µl) was added to either the cis or trans well of a single-channel experiment (see Figure S3; final well concentration of reagent ~.17 mm). The contents of the well were briefly mixed by aspirating and plunging with a 5 µl pipette 3 times. Upon observing a ~3% drop in the pore ion current, indicating a single methionine modification, a solution of methionine (25 mm, 8 µl, 2 equiv.) was immediately added to the same well, aiming the micropipette tip towards the location of the aperture. The contents of the well was mixed by micropipette aspirating and plunging (5 5 µl). If no further quantised drops in the ion current (indicating further methionine modifications) were observed after 5 min, the singly modified pore was characterised by at least four I/V sweeps (1 mv steps at 1 to +1 mv). S6

7 2.3.2 CuAAC reaction of alkyne-modified protein nanopore Under an applied voltage (+1 mv), a solution of CuSO 4 (1 µl, 1 M, final concentration ~1.7 mm) was added to the trans well of a single-channel experiment with an alkyne-modified protein nanopore. For continuous recordings at negative potentials the CuSO 4 solution was instead added to the cis well. A freshly made solution of sodium ascorbate (2 µl, 1 M, final concentration ~3.4 mm) was added to the well containing the CuSO 4, and the contents of the well mixed by micropipette aspirating and plunging (1 5 µl). Complete reduction to Cu(I) was evident by disappearance of the blue colour of the buffer. When reversible formation of a copper acetylide became apparent in the current trace, a solution of azide (1 2 µl, 1 M) was added to the trans well (if carrying a positive charge at ph 8) or the cis well (if carrying a negative charge at ph 8). The contents of the well were carefully mixed by micropipette aspirating and plunging (1 2 5 µl), or the azide was left to diffuse across the well. The experiment was left until a permanent drop in the pore current and cessation of reversible events indicated a CuAAC reaction had taken place. The modified pore was characterised by at least four I/V sweeps (1 mv steps spanning 1 to +1 mv) CuAAC reaction of azide-modified protein nanopore Under an applied voltage (+1 mv), a solution of CuSO 4 (1 µl, 1 M, final concentration ~1.7 mm) was added to the trans well (if using an alkyne reagent carrying a positive charge at ph 8) or the cis well (if using an alkyne carrying a negative charge at ph 8) of a single-channel experiment with an azide-modified protein nanopore. A freshly made solution of sodium ascorbate (2 µl, 1 M, final concentration ~3.4 mm) was added to the same well, and the contents of the well was mixed by micropipette aspirating and plunging (1 5 µl). Complete reduction to Cu(I) was evident by disappearance of the blue colour of the buffer. A solution of alkyne reagent (2 µl, 1 M) was added to the same well, and the contents of the well was carefully mixed by micropipette aspirating and plunging (1 2 5 µl). The experiment was left until a permanent drop in the pore current indicated a CuAAC reaction had taken place. The modified pore was characterised by at least four I/V sweeps (1 mv steps spanning 1 to +1 mv) SPAAC reaction of azide-modified nanopore Under an applied voltage (+1 mv), a solution of cyclooctyne 5 (1 µl, 1 mm, final concentration ~1.7 mm) was added to both wells of a single-channel experiment with an azide-modified protein nanopore. The contents of the well was mixed by micropipette aspirating and plunging (5 5 µl). The experiment was left until a permanent drop in the pore current indicated a SPAAC reaction had taken place. The modified pore was characterised by at least four I/V sweeps (1 mv steps spanning 1 to +1 mv). S7

8 2.4 Nanopore current traces and analysis methionine modification Pore modification with * Figure S5: Single-channel current trace of nanopore modification with 1 (+1 mv; 4.1 mm 1; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of 1. Mixing in well. New current level (reaction at methionine). S8

9 Figure S6: Frequency histogram generated across multiple experiments showing the ratio of modified pore current (I b ) and non-modified pore current (I o ) for nanopores modified once (red), twice (orange), thrice (yellow) and four times (green) with oxaziridine 1. A Gaussian curve has been fitted to the single ethyl modification (blue line), giving a mean I b /I o of.948 and a standard deviation of Figure S7: I/V sweep of an -HL nanopore before (grey) and after (yellow) modification of a single methionine residue with oxaziridine 1. S9

10 2.4.2 Pore modification with 1 high-resolution data Digital filtering None Time (ms) Sweep:1 Visible:1 of 1 5 khz Time (ms) Sweep:1 Visible:1 of 1 4 khz Time (ms) Sweep:1 Visible:1 of 1 3 khz Time (ms) Sweep:1 Visible:1 of 1 2 khz Time (ms) Sweep:1 Visible:1 of 1 1 khz Time (ms) Sweep:1 Visible:1 of 1 Figure S8: High-resolution (5 khz sampling, 1 khz filter) single-channel current trace of nanopore modification with 1 (+1 mv;.17 mm 1; 1 M KCl, 3 mm potassium phosphate, ph 8.). S1

11 2.4.3 Pore modification with 2 1 * Figure S9: Single-channel current trace of nanopore modification with 2 (+1 mv; 1.7 mm 2; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of 2. Mixing in well. New current level (reaction at methionine). S11

12 Figure S1: I/V sweep of -HL nanopore in Figure S9 before (grey) and after (violet) modification of seven methionine residues with oxaziridine 2. Figure S11: Frequency histogram generated across multiple experiments showing the ratio of modified pore current (I b ) and non-modified pore current (I o ) for nanopores modified once (red), twice (orange), thrice (yellow), four-fold (green), fivefold (blue), six-fold (indigo) or seven-fold (violet) with oxaziridine 2. A Gaussian curve has been fitted to the single alkynyl modification (black line), giving a mean I b /I o of.968 and a standard deviation of S12

13 2.4.4 Pore modification with 2 single modification * Figure S12: Representative single-channel current trace of a nanopore modification with 2 (+1 mv;.17 mm 2; 3.4 mm methionine; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 5 Hz). * Introduction of 2. Mixing in well. New current level (reaction at methionine). Quenching with methionine (2 equiv.). Reversible pore gate. S13

14 Figure S13: I/V sweeps of -HL nanopore in Figure S12 before (grey) and after (red) modification of a single methionine residue with oxaziridine 2 under conditions in which CuSO 4 and the reagents were added to most typical situation A) the trans-side and B) to the cis-side of the membrane (for experiments performed at negative potentials). S14

15 2.4.5 Pore modification with 3 * Figure S14: Representative single-channel current trace of a nanopore modification with 3 (+1 mv;.17 mm 3; 3.4 mm methionine; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 5 Hz). * Introduction of 3. Mixing in well. New current level (reaction at methionine). Quenching with methionine (2 equiv.). Reversible pore gate. S15

16 Figure S15: I/V sweep of -HL nanopore in Figure S14 before (grey) and after (orange) modification of a single methionine residue with oxaziridine 3. S16

17 2.4.6 Pore modification with 4 * Figure S16: Representative single-channel current trace of a nanopore modification with 4 (+1 mv;.17 mm 4; 3.4 mm methionine; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of 4. Mixing in well. New current level (reaction at methionine). Quenching with methionine (2 equiv.). S17

18 Figure S17: I/V sweep of -HL nanopore in Figure S16 before (grey) and after (green) modification of a single methionine residue with oxaziridine 4. S18

19 2.5 Nanopore current traces in situ click chemistry SPAAC reaction with Figure S18: Single-channel current trace of SPAAC reaction in an azide-bearing nanopore (+1 mv; 2.3 mm cyclooctyne 5; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). Introduction of 5. Mixing in well. Permanent current change (SPAAC reaction). Reversible pore gate. S19

20 Figure S19: I/V sweep of -HL nanopore in Figure S18 before modification (grey), after introduction of an azide (orange) and after single-molecule SPAAC reaction with cyclooctyne 5 (violet). S2

21 Figure S2: Single-channel current trace of SPAAC reaction in an azide-bearing nanopore (+1 mv; 1.6 mm cyclooctyne 5; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). Introduction of 5. Mixing in well. Permanent current change (SPAAC reaction). Reversible pore gate. S21

22 2.5.2 CuAAC reaction with 6 * Figure S21: Single-channel current trace of CuAAC reaction in an azide-bearing nanopore (+1 mv; 3.4 mm CuSO 4, 6.8 mm sodium ascorbate; 3.4 µm alkyne 6; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 6. Permanent current change (CuAAC reaction). Reversible pore gate. S22

23 Figure S22: I/V sweep of -HL nanopore in Figure S21 before modification (grey), after introduction of an azide (orange) and after single-molecule CuAAC reaction with alkyne 6 (violet). S23

24 * Figure S23: Single-channel current trace of CuAAC reaction in a nanopore bearing two azides (+1 mv; 3.4 mm CuSO 4, 6.8 mm sodium ascorbate; 6.8 mm alkyne 6; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 6. Permanent current change (CuAAC reaction). Reversible pore gate. The second CuAAC reaction took place during a reversible pore gate (t = s). S24

25 2.5.3 CuAAC reaction with 7 * Figure S24: Single-channel current trace of CuAAC reaction in an alkyne-bearing nanopore (+1 mv; 1.7 mm CuSO 4, 3.4 mm sodium ascorbate; 1.7 mm azide 7; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 7. Permanent current change (CuAAC reaction). Reversible pore gate. S25

26 Figure S25: I/V sweep of -HL nanopore in Figure S24 before modification (grey), after introduction of an alkyne (red) and after single-molecule CuAAC reaction with azide 7 (indigo). S26

27 1 * Figure S26: Single-channel current trace of CuAAC reaction in an alkyne-bearing nanopore (+1 mv; 1.7 mm CuSO 4, 3.4 mm sodium ascorbate; 3.4 mm azide 7; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 7. Permanent current change (CuAAC reaction). Reversible pore gate. S27

28 * Figure S27: Single-channel current trace of CuAAC reaction in a nanopore bearing two alkynes (+1 mv; 1.7 mm CuSO 4, 3.4 mm sodium ascorbate; 1.7 mm azide 7; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 7. Permanent current change (CuAAC reaction). Reversible pore gate. S28

29 1 2 * Figure S28: Single-channel current trace of CuAAC reaction in a nanopore bearing two alkynes (+1 mv; 1.7 mm CuSO 4, 3.4 mm sodium ascorbate; 1.7 mm azide 7; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 7. Permanent current change (CuAAC reaction). Reversible pore gate. The second CuAAC reaction took place during a reversible pore gate (t = s). S29

30 * Figure S29: Single-channel current trace of CuAAC reaction in a nanopore bearing two alkynes (+1 mv; 1.7 mm CuSO 4, 3.4 mm sodium ascorbate; 1.7 mm azide 7; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 7. Permanent current change (CuAAC reaction). Reversible pore gate. The pore became irreversibly blocked before the second CuAAC reaction could take place. S3

31 1 * Figure S3: Single-channel current trace of CuAAC reaction in a nanopore bearing two alkynes (+1 mv; 1.7 mm CuSO 4, 3.4 mm sodium ascorbate; 1.7 mm azide 7; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 7. Permanent current change (CuAAC reaction). Reversible pore gate. S31

32 2.5.4 CuAAC reaction with 8 * * Figure S31: Single-channel current trace of CuAAC reaction in an alkyne-bearing nanopore (+1 mv; 5. mm CuSO 4, 1 mm sodium ascorbate; 3.4 mm azide 8; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 8. Permanent current change (CuAAC reaction). Reversible pore gate. S32

33 Figure S32: I/V sweep of -HL nanopore in Figure S31 before modification (grey), after introduction of an alkyne (red) and after single-molecule CuAAC reaction with azide 8 (indigo). S33

34 1 * Figure S33: Single-channel current trace of CuAAC reaction in an alkyne-bearing nanopore (+1 mv; 11.3 mm CuSO 4, 23 mm sodium ascorbate; 19 mm azide 8; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 8. Permanent current change (CuAAC reaction). Reversible pore gate. S34

35 * Figure S34: Single-channel current trace of CuAAC reaction in a nanopore bearing two alkynes (+1 mv; 3.4 mm CuSO 4, 6.8 mm sodium ascorbate; 6.8 mm azide 8; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 8. Permanent current change (CuAAC reaction). Reversible pore gate. S35

36 2.5.5 CuAAC reaction with * Figure S35: Single-channel current trace of CuAAC reaction in an alkyne-bearing nanopore (+1 mv; 1.7 mm CuSO 4, 3.4 mm sodium ascorbate; 6.7 mm azide 9; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 9. Permanent current change (CuAAC reaction). Reversible pore gate. S36

37 Figure S36: I/V sweep of -HL nanopore in Figure S35 before modification (grey), after introduction of an alkyne (red) and after single-molecule CuAAC reaction with azide 9 (indigo). S37

38 1 * Figure S37: Single-channel current trace of CuAAC reaction in an alkyne-bearing nanopore (+1 mv; 1.7 mm CuSO 4, 3.4 mm sodium ascorbate; 13 mm azide 9; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 9. Permanent current change (CuAAC reaction). Reversible pore gate. S38

39 2.5.6 CuAAC reaction with 1 * Figure S38: Single-channel current trace of CuAAC reaction in an alkyne-bearing nanopore (+1 mv; 1.7 mm CuSO 4, 3.4 mm sodium ascorbate; 1.7 µm azide 1; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 1. Permanent current change (CuAAC reaction). Reversible pore gate. S39

40 Figure S39: I/V sweep of -HL nanopore in Figure S38 before modification (grey), after introduction of an alkyne (red) and after single-molecule CuAAC reaction with azide 1 (indigo). S

41 * Figure S: Single-channel current trace of CuAAC reaction in an alkyne-bearing nanopore (+1 mv; 3.4 mm CuSO 4, 6.8 mm sodium ascorbate; 3.4 µm azide 1; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Introduction of 1. Permanent current change (CuAAC reaction). Reversible pore gate. S41

42 2.6 Nanopore current traces Cu(I) and Cu(II) interactions and control experiments Cu(II) and Cu(I) in a non-modified pore * Figure S41: Single-channel current trace of a non-modified nanopore in the presence of Cu(II) and Cu(I) (+1 mv; 3.4 mm CuSO 4, 6.8 mm sodium ascorbate; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Reversible pore gate. S42

43 2.6.2 Cu(II) in an alkyne-modified pore * Figure S42: Single-channel current trace of an alkyne-modified nanopore in the presence of Cu(II) (+1 mv; 8.3 mm CuSO 4 ; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Reversible pore gate. S43

44 1.8 * Figure S43: Single-channel current trace of an alkyne-modified nanopore in the presence of Cu(I) (+1 mv; 6.5 mm CuSO 4, 26 mm sodium ascorbate; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. S44

45 2.6.3 Cu(II) and Cu(I) in an azide-modified pore 2 * Figure S44: Single-channel current trace of an azide-modified nanopore in the presence of Cu(II) and Cu(I) (+1 mv; 6.5 mm CuSO 4, 26 mm sodium ascorbate; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Reversible pore gate. S45

46 2.6.4 Cu(II) and Cu(I) in a pore modified with an internal alkyne * Figure S45: Single-channel current trace of a nanopore modified with an internal alkyne, in the presence of Cu(II) and Cu(I) (+1 mv; 8.1 mm CuSO 4, 16.3 mm sodium ascorbate; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). * Introduction of CuSO 4. Mixing in well. Introduction of sodium ascorbate. Reversible pore gate. S46

47 2.7 Nanopore current traces voltage dependence and kinetic analysis of Cu events Figure S46: Single-channel current trace of a nanopore modified with alkyne reagent 2 in the presence of Cu(I) (+3 mv;.8 mm CuSO 4, 8. mm sodium ascorbate; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). S47

48 Figure S47: Single-channel current trace of a nanopore modified with modified with alkyne reagent 2 in the presence of Cu(I) (+ mv;.8 mm CuSO 4, 8. mm sodium ascorbate; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). S48

49 Figure S48: Single-channel current trace of a nanopore modified with modified with alkyne reagent 2 in the presence of Cu(I) (+5 mv;.8 mm CuSO 4, 8. mm sodium ascorbate; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). S49

50 Figure S49: Single-channel current trace of a nanopore modified with modified with alkyne reagent 2 in the presence of Cu(I) (+6 mv;.8 mm CuSO 4, 8. mm sodium ascorbate; 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 2 Hz). S5

51 2.7.1 Kinetic analysis A τ on = 27 ms τ off = 52 ms B τ on = 31 ms τ off = ms C τ on = 53 ms τ off = 33 ms D τ on = 97 ms τ off = 2 ms Figure S5: Scatter plots of residual ion current (I b /I o ) against copper-alkyne event duration, histograms of distribution of residual ion current of events, and histograms of interevent (τ on ) and event (τ off ) durations, at (A) +3 mv (Figure S46), (B) + mv (Figure S47), (C) +5 mv (Figure S48), and (D) +6 mv (Figure S49). 1 M KCl, 3 mm potassium phosphate, ph 8.. τ on and τ off were determined by fitting to exponential functions. S51

52 Table S1: Calculated kinetic parameters for the formation of a copper-alkyne complex at the given voltage. The observed association constant K obs is given from the relation K obs = τ on 1 /τ off 1 = τ off /τ on. Voltage / mv τ on /s τ off /s K obs S52

53 Figure S51: Single-channel current traces of a nanopore modified with a terminal alkyne in the presence of Cu(I) at a range of negative applied potentials.8 mm CuSO 4 (cis-side only), 8. mm sodium ascorbate (cis-side only); 1 M KCl, 3 mm potassium phosphate, ph 8.; filter 5 Hz). S53

54 2.8 Analysis of intermediate populations All-points histogram analysis Data for copper binding at varying [Cu(I)] was analysed by an all-points histogram. Current histograms of the current traces were plotted with a bin width of.1 pa. The raw data (with no additional digital filtering) was used for this analysis. The current bins were converted to I b /I o values. Subsequently, the histograms were fitted to four gaussian distributions according to the equation: f(x) = (a 1 e (x b1)2 2 2c 1 ) + (a 2 e (x b2)2 2 2c 2 ) + (a 3 e (x b3)2 2 2c 3 ) + (a 4 e (x b4)2 2 2c 4 ) Where b defines the centre of the gaussian distribution while a and c define its height and width. b values were defined manually, while a and c values were fitted using the solver function in Microsoft Excel, minimising the square of the residuals between the data and the fitted model using an iterative method. Fitted parameters and the associated errors obtained using the Solverstat macro 1 and OriginPro are listed in Tables S2-S5 and displayed graphically in Figure S52. It should be noted that most of the integral fitting errors are very small and therefore underestimate the errors associated with integral determination. Thus, an error estimate (equal to the largest fitting error) of 12% was used (propagated as a 13% error for ratios 12 /12, 13 /13 and 13 /12) in subsequent analyses and error bar plots. Such an error estimate appears reasonable based on the quality of the graphical and iterative data fitting presented in section and S54

55 Table S2: Gaussian fitting parameters corresponding to the populations of 12, 12, 13, and 13 at 1.7 mm Cu(I) at +1 mv in 1 M KCl, 3 mm potassium phosphate buffer (ph 8.). 1.7 mm Cu(I) Fitted value Standard error % Error 13' a % b1.92 c % Integral % 13 a % b2.942 c % Integral % 12' a % b3.989 c E-4 1.1% Integral % 12 a % b c E-5.3% Integral % S55

56 Table S3: Gaussian fitting parameters corresponding to the populations of 12, 12, 13, and 13 at 3.3 mm Cu(I) at +1 mv in 1 M KCl, 3 mm potassium phosphate buffer (ph 8.). 3.3 mm Cu(I) Fitted value Standard error % Error 13' a % b1.92 c % Integral % 13 a % b2 9.42E-1 c2.168.% Integral % 12' a % b3.989 c E-5.6% Integral % 12 a % b4 1.E+ c4 8.47E E-5.3% Integral % S56

57 Table S4: Gaussian fitting parameters corresponding to the populations of 12, 12, 13, and 13 at 6.6 mm Cu(I) at +1 mv in 1 M KCl, 3 mm potassium phosphate buffer (ph 8.). 6.6 mm Cu(I) Fitted value Standard error % Error 13' a % b1.915 c E-4.6% Integral % 13 a % b2.93 c E-4 1.1% Integral % 12' a % b c E-5.1% Integral % 12 a % b c E-5.1% Integral % S57

58 Table S5: Gaussian fitting parameters corresponding to the populations of 12, 12, 13, and 13 at 9.9 mm Cu(I) at +1 mv in 1 M KCl, 3 mm potassium phosphate buffer (ph 8.). 9.5 mm Cu(I) Fitted value Standard error % Error 13' a % b1.91 c E-5.3% Integral % 13 a % b2.925 c E-4 1.% Integral % 12' a % b c E-5.2% Integral % 12 a % b c E-5.3% Integral % S58

59 Figure S52: (A) to (D) All-point histogram analysis of the blue and yellow ion current levels resolved two Cu(I)-dependent Gaussian distributions of states corresponding to alkyne species 12, 12, 13 and 13. Data obtained at +1 mv in 1 M KCl, 3 mm potassium phosphate buffer (ph 8.) Graphical determination of equilibrium constants For the equilibrium shown above, the equilibrium constants K 1, K 2 and K 3 are defined by equations 1-3. S59

60 K 1 = [12 ] [12][Cu] M 1 (1) K 2 = [13] [12 ] (2) K 3 = [13 ] [13][Cu] M 1 (3) The overall equilibrium constant over the three steps, K total, is then given by equation 4 K total = K 1 K 2 K 3 = [13 ] [12][Cu] 2 M 2 (4) Equation 1 can be rearranged to give equation 5, which is structurally similar to a straight line. [Cu]K 1 = [12 ] [12] (5) A plot of [12 ] [12] versus [Cu] will therefore produce a straight line with a gradient of K 1 Figure S53: Determination of K 1 from experimental population data as the concentration of Cu(I) was varied. Data determined at +1 mv in 1 M KCl, 3 mm potassium phosphate buffer (ph 8.). Similarly, equation 3 can be rearranged to give equation 6. S6

61 [Cu]K 3 = [13 ] [13] (6) A plot of [13 ] [13] versus [Cu] will therefore produce a straight line with a gradient of K 3 Figure S54: Determination of K 3 from experimental population data as the concentration of Cu(I) was varied. Data determined at +1 mv in 1 M KCl, 3 mm potassium phosphate buffer (ph 8.). Equation 4 can be rearranged to give equation 7 [Cu] 2 K total = [13 ] [12] (7) A plot of [13 ] [12] versus [Cu]2 will therefore produces a straight line with a gradient of K total Figure S55: Determination of K total from experimental population data as the concentration of Cu(I) was varied. Data determined at +1 mv in 1 M KCl, 3 mm potassium phosphate buffer (ph 8.). Two fits are shown, both including and excluding the final data point. With known values of K 1, K 3 and K total, the value of K 2 can be calculated from equation 4. S61

62 2.8.3 Modelling speciation concentrations The concentrations of every component of the system must sum to the total population of the system. This can be defined as being equal to 1. [12] + [12 ] + [13] + [13 ] = 1 (8) Using ratios R 1, R 2 and R 3 (defined by equations 9 11) any given concentration can be expressed in terms of any other concentration. This can then be used to express equation 8 in terms of a single concentration, which can be solved for algebraically. R 1 = [12 ] [12] (9) R 2 = [13 ] [13] (1) R 3 = [13 ] [12] (11) Deriving an Expression for [12] Using the definitions of R 1, R 2, and R 3 (equations 9 11) [12 ], [13], and [13 ] can be expressed in terms of [12]. [12 ] = R 1 [12] (12) [13 ] = R 3 [12] (13) [13] = [13 ] R 2 (14) Substituting equation 13 into equation 14 then gives S62

63 [13] = R 3 R 2 [12] (15) Equations 12, 13 and 15 can be substituted into equation 8 to give equation 16. [12] + R 1 [12] + R 3 [12] + R 3 R 2 [12] = 1 (16) Dividing by a factor of [12] affords equation 17 1 [12] = 1 + R 1 + R 3 + R 3 R 2 (17) Therefore: [12] = (1 + R 1 + R 3 + R 1 3 (18) ) R 2 Deriving an Expression for [12 ] Using the definitions of R 1, R 2, and R 3 (equations 9 11) [12], [13], and [13 ] can be expressed in terms of [12 ]. [12] = [12 ] R 1 (19) [13 ] = R 3 [12] (2) Substituting equation 19 into equation 2 then gives: [13 ] = R 3[12 ] R 1 (21) S63

64 [13] = [13 ] R 2 (22) Substituting equation 21 into 22 yields [13] = R 3[12 ] R 1 R 2 (23) Equations 19, 21 and 23 can be substituted into equation 8 to give equation 24. [12 ] + [12 ] R 1 + R 3[12 ] R 1 + R 3[12 ] R 1 R 2 = 1 (24) Dividing by a factor of [12 ] affords equation [12 ] = R 3 + R 3 (25) R 1 R 1 R 3 1 R 2 Therefore: [12 ] = ( R 3 + R 1 3 (26) ) R 1 R 1 R 1 R 2 Deriving an Expression for [13] Using the definitions of R 1, R 2, and R 3 (equations 9 11) [12], [12 ], and [13 ] can be expressed in terms of [13]. [13 ] = R 2 [13] (27) [12] = [13 ] R 3 (28) Substituting equation 27 into 28 gives: S64

65 [12] = R 2[13] R 3 (29) [12 ] = R 1 [12] (3) Substituting equation 29 into 3 yields: [12 ] = R 1R 2 [13] R 3 (31) Equations 27, 29 and 31 can be substituted into equation 8 to give equation 32. [13] + R 2 [13] + R 2[13] + R 1R 2 [13] = 1 R 3 R 3 (32) Dividing by a factor of [13] affords equation [13] = 1 + R 2 + R 2 R 3 + R 1R 2 R 3 (33) Therefore: [13] = (1 + R 2 + R 2 + R 1 1R 2 (34) ) R 3 R 3 Deriving an Expression for [13 ] Using the definitions of R 1, R 2, and R 3 (equations 9 11) [12], [12 ], and [13] can be expressed in terms of [13 ]. [12] = [13 ] R 3 (35) S65

66 [13] = [13 ] R 2 (36) [12 ] = R 1 [12] (37) Substituting equation 35 into 37 gives: [12 ] = R 1[12] R 3 (38) Equations 35, 36 and 38 can be substituted into equation 8 to give equation 39 [13 ] + [13 ] + [13 ] R 3 R 2 + R 1[12] R 3 = 1 (39) Dividing by a factor of [13 ] affords equation. 1 [13 ] = R R 2 + R 1 R 3 () Therefore: [13 ] = ( R 1 1 (41) ) R 3 R 2 R Excel solver determination of equilibrium constants Establishing the models above that relate equilibrium constants to the relative populations of 12, 12, 13 and 13 as the concentration of Cu(I) was varied (as described in the previous section), meant that it was also possible to determine the equilibrium constants using iterative fitting. In this case, the sum of the root mean squared difference between the modelled concentrations of species 12, 12, 13 and 13 as the concentration of Cu(I) was varied was minimized by iteratively solving the equilibrium constants K 1, K 3, and K total (thereby also yielding K 2 ). The resulting equilibrium constants are listed in Table S6, and were found to be numerically similar in value to those that were determined graphically, with the differences between these values giving an indication of the errors associated with such an approach. Furthermore, the resulting speciation diagram in Figure S56 was similar to the graphically determined variant presented in the main text Figure 6E. S66

67 Table S6: Equilibrium constants determined for interconversion between species 12, 12, 13 and 13 using the graphical method and iterative minimization, as described in sections to Graphically determined Iterative minimization K1 / M K2 / no units K3 / M Ktotal / M 2 1,762 5,975 Figure S56: (A) to (D) Copper binding equilibria and associated minimized geometries and electrostatic potential surfaces for alkyne intermediates calculated using B3LYP/LACVP. Electrostatic potential surfaces are scaled from +2 to -2 kj mol -1 for neutral molecules and to +7 kj mol -1 for cations (red to blue). (E) Speciation diagram showing experimentally determined populations of 12, 12, 13 and 13 as the concentration of Cu(I) was varied (points) and the modelled populations (lines) determined by iterative fitting of the experimental population data (described by the fitted equilibrium constants listed in Table S6). The modelled populations are similar to those determined graphically (Section 2.8.2) and presented in Figure 6E of the main text. S67

68 3 Other reactants for single-molecule click reactions 3.1 Unsuccessful azides and cyclooctynes for CuAAC and SPAAC reactions Azides S1 and S2 were purchased from TCI and Sigma-Aldrich, respectively. Azides S3 and S4 were synthesised according to literature procedures. 2-4 Cyclooctyne S5 was purchased from Sigma-Aldrich. 3.2 Full structure of azido-dna (1) for nanopore CuAAC modification Azido-DNA 1-mer 1, purified by HPLC, was purchased through the IDT Custom DNA oligo service. S68

69 4 Synthesis of reagents 4.1 General experimental procedures All reagents and synthetic starting materials were obtained from commercial sources (Alfa Aesar, Fisher UK, Fluorochem, Sigma Aldrich, TCI, VWR) and used as received. Anhydrous solvents were dried by being passed through an activated alumina column on a solvent tower under N 2 pressure. Analytical TLC was carried out on Merck aluminium sheets coated with silica gel 6F and visualized using UV light (254 nm) and/or staining with vanillin. Flash column chromatography was carried out on silica gel Geduran 6 ( 63 µm). Nuclear magnetic resonance (NMR) spectra were recorded using a MHz Bruker Avance III spectrometer, or a 5 MHz Bruker Avance III spectrometer with a 13 C cryoprobe. NMR chemical shifts (δ) are reported in parts per million (ppm) relative to trimethylsilane (δ = ppm). 1 H spectra were referenced to residual protons in CDCl 3 (δ 7.26), DMSO-d 6 (δ 2.5) or CD 3 OD (δ 3.31). Coupling constants (J) are given in Hz to an accuracy of.1 Hz. Assignment of 1 H resonances were determined on the basis of unambiguous chemical shift, coupling patterns, and/or by analysis of 2D NMR (COSY, HSQC and/or HMBC) spectra. 13 C NMR spectra ( 1 H decoupled) were recorded at ambient probe temperatures on the instruments mentioned above (1 MHz and 125 MHz, respectively), and referenced to the solvents CDCl 3 (δ 77.16), DMSO-d 6 (δ 39.52) or CD 3 OD (δ 49.). Mass spectrometry was performed by the University of Edinburgh departmental mass spectrometry service, using a ThermoElectron MAT 9 spectrometer for EI-HRMS and a Bruker microtof for ESI-HRMS. The parent ion [M] + or adduct [M+H] + are calculated to five decimal places; all values are within an error of 5 ppm. Infrared (IR) spectra were recorded as solids or thin films on a Bruker Tensor 27 FTIR instrument with a Pike Miracle ATR ZnSe module. Wavelengths of maximum absorbance (ν max ) are reported in wavenumbers (cm 1 ). Only a selection of characteristic resonances are reported. Melting points were measured in a Gallenkamp melting point apparatus. 4.2 N-Ethyl-3-phenyl-oxaziridine-2-carboxamide (1) N-Ethylurea (S6, 881 mg, 1. mmol, 1. equiv.) and benzaldehyde (1.22 ml, 12. mmol, 1.2 equiv.) were dissolved in dry THF (2 ml) under N 2. Ti(Oi-Pr) 4 (3.3 ml, 11 mmol, 1.1 equiv.) was added dropwise over stirring, and the reaction mixture was stirred for 19 hours at room temperature. The solvent was removed under reduced pressure to give an oily residue. In a separate flask, m-cpba (77% w/w, 6.72 g, 3. mmol, 3. equiv.) was dissolved in CH 2 Cl 2 (3 ml), and a solution of K 2 CO 3 (aq. sat., 3 ml) was added carefully over vigorous stirring. After stirring for 1 min, a solution of the above residue in CH 2 Cl 2 (3 ml) was added dropwise over vigorous stirring (NB! Gas evolution). The thickness of the resulting suspension was adjusted by addition of CH 2 Cl 2 (5 ml). The mixture was stirred at room temperature for 22 hours, before being diluted with H 2 O (1 ml) and extracted with CH 2 Cl 2 (3 5 ml). The organic extracts were washed with brine (2 ml) and dried (Na 2 SO 4 ). The solvent was removed, and the residue was purified by column chromatography (diethyl ether/ch 2 Cl 2 (1:99)) to give an oily mixture of 1 and benzaldehyde. The oil was dissolved in diethyl ether (5 ml) and poured into hexane (75 ml) over rapid stirring. The product was allowed to precipitate at 2 C, S69

70 collected by vacuum filtration, and dried under high vacuum to give oxaziridine 1 as a white, fluffy solid (234 mg, 1.22 mmol, 12%). R f.42 (diethyl ether/ch 2 Cl 2 (5:95)); 1 H NMR (5 MHz, CDCl 3 ) δ (5H, m, ArH), 6.5 (1H, br s, NH), 5. (1H, s, ArCH), (2H, m, CH 2 ), 1.21 (6H, t, J = 7.3 Hz, CH 3 ); 13 C NMR (126 MHz, CDCl 3 ) δ 162.2, 132.6, 131.1, 128.7, 128.1, 79.5, 35.5, Data consistent with literature values. 5 Figure S57: 1 H NMR (5 MHz, CDCl 3 ) spectrum of 1. Figure S58: 13 C NMR (126 MHz, CDCl 3 ) spectrum of Prop-2-ynylurea (S8) Propargylamine (S7, 2.4 ml, 37 mmol, 1. equiv.) was dissolved in HCl (1 M aq., 37 ml) and KOCN (12.2 g, 15 mmol, 4. equiv.) was added at room temperature. The reaction mixture was stirred at 6 C for 26 hours, until TLC analysis indicated complete conversion. The mixture was cooled to nearfreezing at 2 C, and the precipitate was collected by vacuum filtration and dried. The solid was S7

71 dissolved in methanol (15 ml) and silica gel (25 g) was added, and the mixture was stirred for 18 hours at room temperature, before being filtered through celite. The solvent was removed, and the urea S8 was isolated as a white solid after drying under high vacuum (1.93 g, 19.7 mmol, 53%). R f.5 (methanol/ch 2 Cl 2 (2:)); 1 H NMR (5 MHz, DMSO-d 6 ) δ 6.25 (1H, t, J = 5.9 Hz, NH), 5.57 (2H, s, NH 2 ), 3.75 (2H, dd, J = 5.8, 2.5 Hz, CH 2 ), 3.2 (1H, t, J = 2.5 Hz, CH); 13 C NMR (126 MHz, DMSO-d 6 ) δ 158.1, 82.5, 72.4, Data consistent with literature values. 5 Figure S59: 1 H NMR (5 MHz, DMSO-d 6 ) spectrum of S8. Figure S6: 13 C NMR (126 MHz, DMSO-d 6 ) spectrum of S Phenyl-N-prop-2-ynyl-oxaziridine-2-carboxamide (2) Urea S8 (3 mg, 3.6 mmol, 1. equiv.) and benzaldehyde (3 µl, 3.67 mmol, 1.2 equiv.) were dissolved in dry THF (12 ml) under N 2. Ti(Oi-Pr) 4 (1. ml, 3.37 mmol, 1.1 equiv.) was added dropwise S71

72 over stirring, and the reaction mixture was stirred for 2 hours at room temperature. The solvent was removed under reduced pressure to give an oily residue. In a separate flask, m-cpba (77% w/w, 2.6 g, 9.18 mmol, 3. equiv.) was dissolved in CH 2 Cl 2 (9 ml), and a solution of K 2 CO 3 (aq. sat., 9 ml) was added carefully over vigorous stirring. After stirring for 1 min, a solution of the above residue in CH 2 Cl 2 (9 ml) was added dropwise over vigorous stirring (NB! Gas evolution). The thickness of the resulting suspension was adjusted by addition of CH 2 Cl 2 (3 ml). The mixture was stirred at room temperature for 6 hours, before being diluted with H 2 O (5 ml) and extracted with CH 2 Cl 2 (3 5 ml). The organic extracts were washed with H 2 O (2 ml) and dried (Na 2 SO 4 ). The solvent was removed, and the residue was purified by column chromatography (diethyl ether/ch 2 Cl 2 (1:9)) to give an oily mixture of 2 and benzaldehyde. The oil was dissolved in diethyl ether (5 ml) and poured into hexane (75 ml) over rapid stirring. The product was allowed to precipitate at 2 C, collected by vacuum filtration, and dried under high vacuum to give oxaziridine 2 as a white, fluffy solid (172 mg,.851 mmol, 28%). R f.57 (diethyl ether/ch 2 Cl 2 (1:9)); 1 H NMR (5 MHz, CDCl 3 ) δ (5H, m, ArH), 6.27 (1H, br s, NH), 5.3 (1H, s, ArCH), 4.9 (2H, ddd, J = 5.5, 4., 2.6 Hz, CH 2 ), 2.31 (1H, t, J = 2.5 Hz, CH); 13 C NMR (126 MHz, CDCl 3 ) δ 162., 132.2, 131.3, 128.8, 128.1, 79.6, 78.4, 72.7, 3.3. Data consistent with literature values. 5 Figure S61: 1 H NMR (5 MHz, CDCl 3 ) spectrum of 2. Figure S62: 13 C NMR (126 MHz, CDCl 3 ) spectrum of 2. S72

73 4.5 3-Azidopropynylurea (S1) Azide S9 (245 µl, 2.5 mmol, 1. equiv.) was dissolved in HCl (1 M aq., 2.5 ml) and KOCN (811 mg, 1. mmol, 4. equiv.) was added at room temperature. The reaction mixture was stirred at 6 C for 18 hours. The solvent was removed, and the residue redissolved in H 2 O (2 ml). The solution was extracted with ethyl acetate (3 2 ml), the combined extracts were dried (Na 2 SO 4 ), and the solvent was removed. After drying under high vacuum, the urea S1 was isolated as a white solid (257 mg, 1.79 mmol, 72%). M.p C; IR (thin film, ν max / cm 1 ) 3431, 3366, 319, 287, 1539, 1384; 1 H NMR (5 MHz, CD 3 OD) δ 3.36 (2H, t, J = 6.8 Hz, NHCH 2 ), 3.18 (2H, t, J = 6.8 Hz, CH 2 N 3 ), 1.74 (2H, p, J = 6.8 Hz, CH 2 CH 2 CH 2 ).; 13 C NMR (126 MHz, CD 3 OD) δ 162.2, 5.1, 38.3, 3.5; HRMS (ES+) calc. for C 4 H 1 ON 5 [M+H] , found Figure S63: 1 H NMR (5 MHz, CD 3 OD) spectrum of S1. Figure S64: 13 C NMR (126 MHz, CD 3 OD) spectrum of S1. S73

74 4.6 N-(3-Azidopropyl)-3-phenyl-oxaziridine-2-carboxamide (3) Urea S1 (143 mg, 1. mmol, 1. equiv.) and benzaldehyde (122 µl, 1.2 mmol, 1.2 equiv.) were dissolved in dry THF (4 ml) under N 2. Ti(Oi-Pr) 4 (326 µl, 1.1 mmol, 1.1 equiv.) was added dropwise over stirring, and the reaction mixture was stirred for 17 hours at room temperature. The solvent was removed under reduced pressure to give an oily residue. In a separate flask, m-cpba (77% w/w, 673 mg, 3. mmol, 3. equiv.) was dissolved in CH 2 Cl 2 (3 ml), and a solution of K 2 CO 3 (aq. sat., 3 ml) was added carefully over vigorous stirring. After stirring for 1 min, a solution of the above residue in CH 2 Cl 2 (3 ml) was added dropwise over vigorous stirring (NB! Gas evolution). The thickness of the resulting suspension was adjusted by addition of CH 2 Cl 2 (1 ml). The mixture was stirred at room temperature for 6 hours, before being diluted with H 2 O (5 ml) and extracted with CH 2 Cl 2 (3 5 ml). The organic extracts were washed with H 2 O (2 ml) and dried (Na 2 SO 4 ). The solvent was removed, and the residue was purified by column chromatography (diethyl ether/ch 2 Cl 2 (2:98)) to give oxaziridine 3 as a colourless oil which turned into a white solid upon freezing (96 mg,.388 mmol, 39%). M.p C; R f. (diethyl ether/ch 2 Cl 2 (1:9)); IR (thin film, ν max / cm 1 ) 3399, 3323, 29, 297, 1695, 152, 1252; 1 H NMR (61 MHz, CDCl 3 ) δ (5H, m, ArH), 6.27 (1H, br s, NH), 5.1 (1H, s, ArCH), (4H, m, NCH 2, CH 2 N 3 ), 1.85 (2H, p, J = 6.7 Hz, CH 2 CH 2 CH 2 ).; 13 C NMR (151 MHz, CDCl 3 ) δ 162.5, 132.4, 131.2, 128.8, 128.1, 79.6, 49.3, 38.2, 28.7.; HRMS (EI+) calc. for C 11 H 13 O 2 N 5 [M] , found Data consistent with literature values. 5 Figure S65: 1 H NMR (61 MHz, CDCl 3 ) spectrum of 3. S74

75 Figure S66: 13 C NMR (151 MHz, CDCl 3 ) spectrum of But-2-ynylisoindoline-1,3-dione (S12) 1-Bromo-2-butyne (S11, 2. ml, 22.9 mmol, 1. equiv.) was carefully added to a suspension of potassium phthalimide in dry DMF under N 2. The reaction mixture was stirred at 9 C for 18 hours, before being cooled to room temperature and poured into H 2 O (5 ml). The mixture was extracted with CHCl 3 (3 5 ml) and the combined extracts were washed with NaOH (1 M aq., 2 3 ml) and dried (MgSO 4 ). The solvent was removed, and the residue was recrystallized from CHCl 3 /hexane. After drying under high vacuum, phthalimide S12 was isolated a white needles (3.64 g, 18.2 mmol, 79%). 1 H NMR (5 MHz, DMSO-d 6 ) δ (4H, m, ArH), 4.31 (2H, q, J = 2.4 Hz, CH 2 ), 1.75 (3H, t, J = 2.5 Hz, CH 3 ); 13 C NMR (126 MHz, DMSO-d 6 ) δ 166.8, 134.7, 131.4, 123.3, 78.8, 73.6, 27., 2.9. Data consistent with literature values. 6,7 Figure S67: 1 H NMR (5 MHz, DMSO-d 6 ) spectrum of S12. S75