Kondrat eva Ligation: Diels Alder-based. Irreversible Reaction for Bioconjugation

Transcription

1 Kondrat eva Ligation: Diels Alder-based Irreversible Reaction for Bioconjugation Supporting Information Laurie-Anne Jouanno, a Arnaud Chevalier, a Nawal Sekkat, e Nicolas Perzo, d Hélène Castel, d Anthony Romieu, b, c Norbert Lange, e Cyrille Sabot, *,a Pierre-Yves Renard*,a a Normandie Univ, COBRA, UMR 6014 et FR 3038; Univ Rouen; INSA Rouen; CNRS, 1 rue Tesnières Mont-Saint-Aignan, Cedex (France) b ICMUB, UMR CNRS 6302, Université de Bourgogne 9, Avenue Alain Savary, Dijon (France) c Institut Universitaire de France 103, Boulevard Saint-Michel, Paris (France) d Inserm U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation (DC2N), Astrocyte and Vascular Niche, Institute of Research and Biomedical Innovation (IRIB), PRES Normandy University, University of Rouen, Mont-Saint- Aignan Cedex (France); North-West Cancéropole (CNO), Lille Cedex (France) e Université de Genève, Section des sciences pharmaceutiques, Technologie pharmaceutique, Quai Ernest Ansermet 30 CH-1211 Genève 4 (Switzerland)

2 List of Contents I. Study of the Kondrat eva reaction... S2 1. Study of the stability of 5-alkoxyoxazole and 3-hydroxypyridine scaffolds... S2 a) Study of the stability of 5-alkoxyoxazole 1 at ph, and 37 C for 15 h... S2 b) Study of the stability of the 3-hydroxypyridine 4a at ph and S3 2. Study of the Kondrat'eva reaction in different solvent systems... S5 a) In organic media:... S5 b) In binary aqueous media:... S6 3. Study of the (bio)orthogonality of the Kondrat'eva reaction... S7 a) Study of the Kondrat'eva reaction between 4-benzyl-5-ethoxy-2-methyloxazole 1 and diversely functionalized maleimide derivatives 2... S7 b) Study of the bioorthogonality of the Kondrat'eva reaction... S11 II. III. IV. Determination of F/P ratio of labeled BSA 17a and 17b... S14 Spectroscopic data... S15 Copies of 1 H/ 13 C NMR spectra... S24 V. RP-HPLC elution profiles... S49 VI. MS analyses... S55 S1

3 I. Study of the Kondrat eva reaction 1. Study of the stability of 5-alkoxyoxazole and 3-hydroxypyridine scaffolds a) Study of the stability of 5-alkoxyoxazole 1 at ph, and 37 C for 15 h To a solution of 4-benzyl-5-ethoxy-2-methyloxazole 1 (15 mg, mmol) in a solution of DMSO/ NaOAc buffer ph M (1.896 ml, 20:80, v/v) was left at 37 C for 15 h in a heating chamber. The solution was then quenched with sat. aq. NaHCO 3 (2 ml) and extracted with CH 2 Cl 2 (10 ml) and EtOAc (2 10 ml). The combined organic phases were dried over anhydrous MgSO 4 and concentrated under reduced pressure. 1-Phenyl-1-cyclohexene (10.92 mg, mmol) was finally added as internal standard for 1 H NMR analyses: recovery rate of 4-benzyl-5-ethoxy-2-methyloxazole 1 after 15 h of incubation at 37 C: 87% S2

4 b) Study of the stability of the 3-hydroxypyridine 4a at ph and 10.0 At ph : To a solution of 5-hydroxypyridine 4a (19.5 mg, mmol) in a solution of DMSO/acidic buffer (citric buffer/tfa, ph =, 0.1 M; ml, 20:80, v/v) was left at 37 C for 48 h in a heating chamber. The solution was then quenched with sat. aq. NaHCO 3 (2 ml) and extracted with CH 2 Cl 2 (2 10 ml). The combined organic phases were dried over anhydrous MgSO 4 and concentrated under reduced pressure. 1-Phenyl-1-cyclohexene (10.92 mg, mmol) was finally added as internal standard peak for the 1 H NMR analysis: recovery rate of the product 4a after 48 h of incubation at 37 C: 100% S3

5 At ph 10.0: To a solution of 3-hydroxypyridine 4a (19.5 mg, mmol) in a mixture of DMSO/basic buffer (boric acid/naoh, ph = 10.0, 0.1 M; ml, 20:80, v/v) was left at 37 C for 48 h in a heating chamber. The solution was then quenched with sat. aq. NaHCO 3 (2 ml) and extracted with CH 2 Cl 2 (2 10 ml). The combined organic phases were dried over anhydrous MgSO 4 and concentrated under reduced pressure. 1-Phenyl-1-cyclohexene (10.92 mg, mmol) was finally added as internal standard peak for the 1 H NMR analysis: recovery rate of the product 4a after 48 h at 37 C: 86% S4

6 3-Hydroxypyridine 4a/(3-Hydroxypyridine+5- ethoxyoxazole 1) (%) 2. Study of the Kondrat'eva reaction in different solvent systems a) In organic media: To a solution of 4-benzyl-5-ethoxy-2-methyloxazole 1 (17 mg, 0.08 mmol, 1 equiv) in the corresponding deuterated solvent (0.54 ml; D 2 O, MeOH-d 4, CDCl 3, THF-d 8, CD 3 CN or DMSO-d 6 ) in a NMR tube, were added successively N-methylmaleimide 2a (22 mg, 0.20 mmol, 2.5 equiv) and TFA (20 L). The ratio 5-alkoxyoxazole 1:3-hydroxypyridine 4a was determined at different time by 1 H NMR analysis based on the comparative integration between the benzylic protons of 1 and 4a. No traces of the intermediate bicyclic adduct was observed under these conditions D2O MeOH-d4 CDCl3 THF-d8 CD3CN DMSO-d Hours Figure S1. Comparative kinetic study of the Kondrat eva reaction of 5-ethoxyoxazole 1 and N- methylmaleimide 2a in various organic media. S5

7 3-Hydroxypyridine 4a/(3-Hydroxypyridine+5- ethoxyoxazole 1) (%) b) In binary aqueous media: To a solution of 4-benzyl-5-ethoxy-2-methyloxazole 1 (17 mg, 0.08 mmol, 1 equiv) in the corresponding deuterated binary aqueous medium (deuterated organic solvent/d 2 O, 2:1, v/v, 0.54 ml) in a NMR tube, were added N-methylmaleimide 2a (22 mg, mmol, 2.5 equiv) and TFA (20 L). The ratio 5-alkoxyoxazole 1:3-hydroxypyridine 4a was determined by 1 H NMR analysis based on the comparative integration between the benzylic protons of 1 and 4a. No traces of the intermediate bicyclic adduct was observed under these conditions THF-d8/D2O MeOH-d4/D2O DMSO-d6/D2O Hours Figure S2. Comparative kinetic study of the Kondrat'eva reaction between 5-ethoxyoxazole 1 and N- methylmaleimide 2a in various binary aqueous media. S6

8 3. Study of the (bio)orthogonality of the Kondrat'eva reaction a) Study of the Kondrat'eva reaction between 4-benzyl-5-ethoxy-2-methyloxazole 1 and diversely functionalized maleimide derivatives 2 Entry R Pyridine Yield a (ph 1) Yield b (ph ) 1 Me (2a) 4a Me (2a) 4a - 50 c 3 Ph (2b) 4b (CH 2 ) 5 CO 2 H (2c) 4c d (2d) 6 (CH 2 ) 5 CONHCH 2 COCH 3 (2e) 4e (CH 2 ) 5 CONHCH 2 CCH (2f) 4f a Conditions A (ph 1): 5-alkoxyoxazole 1 (0.46 mmol), maleimide 2 (0.60 mmol, 1.3 equiv.), THF/aq. TFA (1 % v/v) 2:1, RT, 5 h; isolated yields. b Conditions B (ph ): 5-alkoxyoxazole 1 (0.07 mmol), maleimide 2 (0.09 mmol, 1.3 equiv.), DMSO/NaOAc buffer ph ( M) 1:5, 37 C, 5 h; yield determined by 1 H NMR of the crude reaction mixture relative to the internal standard 1-phenyl-1-cyclohexene. c 5-alkoxyoxazole (0.46 mmol), L-cysteine (0.46 mmol, 1 equiv.), N-methylmaleimide (0.60 mmol, 1.3 equiv.). Conditions B (ph = ) for the preparation of 3-hydroxypyridines 4a-4f: To a solution of the 5- alkoxyoxazole 1a (0.07 mmol, 1 equiv.) in a mixture of DMSO/NaOAc buffer ( M, ph, 21:79, v/v, ml) was added the corresponding maleimide (0.09 mmol, 1.3 equiv.). The resulting reaction mixture was left at 37 C for 5 h in a heating chamber Thereafter, the reaction was quenched by adding sat. aq. NaHCO 3 (2 ml) and extracted with CH 2 Cl 2 (10 ml) and EtOAc (2 10 ml). The combined organic phases were dried over anhydrous MgSO 4 and concentrated under reduced pressure. 1-Phenyl-1-cyclohexene (10.92 mg, 0.07 mmol) was finally added to the sample as internal standard peak for 1 H NMR analyses. S7

9 S

10 S

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12 b) Study of the bioorthogonality of the Kondrat'eva reaction Entry α-amino acid mg (equiv.) Yield a 1 L-Tyrosine L-Tryptophane L-Histidine (1) 19 (1) 10.71(1) 95 2 L-Proline L-Serine L-Asparagine 7.94 (1) 7.25 (1) (1) 3 L-Cysteine 8.36 (1) 50 a Determined by 1 H NMR analysis of the crude reaction mixture relative to the internal standard 1-phenyl-1- cyclohexene. 91 To a solution of 4-benzyl-5-ethoxy-2-methyloxazole 1 (15 mg, 0.07 mmol, 1 equiv.) in a solution of DMSO/NaOAc buffer ph, M (1.896 ml, 20:80, v/v) were added N-methylmaleimide 2a (9.97 mg, mmol, 1.3 equiv.) and the corresponding α-amino acid(s) (0.069 mmol, 1 equiv.). The reaction was left at 37 C for 5 h in a heating chamber. The reaction was then quenched by adding sat. aq. NaHCO 3 (2 ml) and the aqueous layer was extracted with CH 2 Cl 2 (10 ml) and EtOAc (2 10 ml). The combined organic phases were dried over anhydrous MgSO 4 and concentrated under reduced pressure. 1-Phenyl-1-cyclohexene (10.92 mg, mmol) was finally added as internal standard peak for 1 H NMR analyses. S11

13 S

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15 Absorbance Intensity (u.a.) Normalized Intensity (u.a.) II. Determination of F/P ratio of labeled BSA 17a and 17b The absorbance of 17a/17b was measured from a stock solution of 1 L of R6G-BSA conjugate 17a/17b ( 15 nmol) in PBS buffer (1 L) diluted in 500 L aq. SDS 2.5% (w/v) Wavelength (nm) Figure S3. Normalized absorption spectra of unlabeled and labeled protein 17a and 17b (black trace for BSA, dashed trace for F/P = 9.6 and dotted trace for F/P = 5.7) at 25 C in PBS ph Wavelenght (nm) Figure S4. Absorption spectra of unlabeled and labeled protein 17a and 17b (black trace for BSA, dashed trace for F/P = 9.6 and dotted trace for F/P = 5.7) at 25 C in deionized water with 2.5% of SDS (same concentration of BSA for each sample). S14

16 The degree of labeling of the BSA protein, i.e. the fluorophore:protein (F/P) molar ratios, were estimated from the relative intensities of protein and dye absorption, according to the following equation: 1 The molar absorption coefficient of the BSA protein P,280 was determined to be M -1 cm -1 at 280 nm in aq. SDS 2.5% (w/v) at 25 C. The molar absorption coefficient of azaphthalimide-r6g F,539 was determined to be M -1 cm -1 at 539 nm in aq. SDS 2.5% (w/v) at 25 C. The correction factor ƒ equal to the ratio of the fluorophore absorbances at 280 and 539 nm was calculated to be Table 1: Determination of the labelling density F/P for 17a and 17b Entry Number of equiv of oxazole- R6G 15 A 280 A 539 F/P , (17b) , (17a) III. Spectroscopic data Table 2: Quantum yield Entry Compound Solvents abs (nm) a em (nm) a Stokes' shift (nm) e F 1 R6G H 2 O b PBS SDS Ethoxyoxazole 15 PBS c DMSO SDS d Azaphthalimide-R6G PBS DMSO SDS Tocopherol conjugate 16 PBS 517/ DMSO SDS Labeled BSA 17a (F/P = 9.6) PBS 508/ SDS Labeled BSA 17b (F/P = 5.7) SDS a Maxima of the main absorption ( abs ) and emission ( em ) bands. b Deionized water. c 10 mm + 15 mm NaCl, ph 7.4; d Aq. SDS 2.5% (w/v); e R6G was used as standard (94% in EtOH) 2, excitation at 500 nm. 1 J. Pauli, K. Licha, J. Berkemeyer, M. Grabolle, M. Spieles, N. Wegner, P. Welker, U. Resch-Genger, Bioconjugate Chem. 2013, 24, A. M. Brouwer, Pure Appl. Chem. 2011, 83, S15

17 Normalized Intensity (u.a.) Normalized Intensity (u.a.) 5-Ethoxyoxazole Wavelength (nm) Figure S5. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 557 nm) spectra for the 5-ethoxyoxazole 15 in PBS at 25 C Wavelength (nm) Figure S6. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 565 nm) spectra for the 5-ethoxyoxazole 15 in DMSO at 25 C. S16

18 Normalized Intensity (u.a.) Normalized Intensity (u.a.) Wavelength (nm) Figure S7. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 559 nm) spectra for the 5-ethoxyoxazole 15 in aq. SDS 2.5% (w/v) at 25 C. Azaphthalimide-R6G Wavelength (nm) Figure S8. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 558 nm) spectra for the azaphthalimide-r6g in PBS at 25 C. S17

19 Normalized Intensity (u.a.) Normalized Intensity (u.a.) Wavelength (nm) Figure S9. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 564 nm) spectra for the azaphthalimide-r6g in DMSO at 25 C Wavelength (nm) Figure S10. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 559 nm) spectra for the azaphthalimide-r6g at 25 C in aq. SDS 2.5% (w/v) S18

20 Normalized Intensity (u.a.) Normalized Intensity (u.a.) Tocopherol conjugate Wavelength (nm) Figure S11. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 557 nm) spectra for the tocopherol conjugate 16 at 25 C in PBS buffer Wavelength (nm) Figure S12. Absorption (black trace), emission (dashed trace, ex = 500 nm) and excitation (dashed trace, em = 565 nm) spectra for the tocopherol conjugate 16 at 25 C in DMSO. S19

21 Normalized Intensity (u.a.) Noramalized Intensity (u.a.) Wavelength (nm) Figure S13. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 560 nm) spectra for the tocopherol conjugate 16 at 25 C in aq. SDS 2.5% (w/v). Labeled BSA 17a/17b Wavelength (nm) Figure S14. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 560 nm) spectra for the labeled BSA 17a (F/P = 9.6) at 25 C in aq. SDS 2.5% (w/v). S20

22 Normalized Intensity (u.a.) Normalized Intensity (u.a.) Wavelength (nm) Figure S15. Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 557 nm) spectra for the labeled BSA 17b (F/P = 5.7) at 25 C in PBS buffer Wavelength (nm) Figure S16: Absorption (black trace), emission (dotted trace, ex = 500 nm) and excitation (dashed trace, em = 559 nm) spectra for the labeled BSA 17b (F/P = 9.6) at 25 C in aq. SDS 2.5% (w/v). S21

23 Noramalized Intensity (u.a.) Noramalized Intensity (u.a.) Labeled peptide Wavelength (nm) Figure S17. Absorption (solid trace), emission (dotted trace, ex = 595 nm) and excitation (dashed trace, em = 720 nm) spectra of Cy-labeled peptide 19 in PBS at 25 C Wavelength (nm) Figure S18. Absorption (solid trace), emission (dotted trace, ex = 595 nm) and excitation (dashed trace, em = 720 nm) spectra of Cy labeled peptide 19-D in PBS at 25 C. S22

24 Noramalized Intensity (u.a.) Noramalized Intensity (u.a.) Wavelength (nm) Figure S19. Absorption (solid trace), emission (dotted trace, ex = 595 nm) and excitation (dashed trace, em = 720 nm) spectra of Cy labeled peptide 20 in PBS at 25 C Wavelength (nm) Figure S20. Absorption (solid trace), emission (dotted trace, ex = 595 nm) and excitation (dashed trace, em = 720 nm) spectra of Cy labeled peptide 20-D in PBS at 25 C. S23

25 IV. Copies of 1 H/ 13 C NMR spectra S24

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50 0,200 0,002 mau V. RP-HPLC elution profiles RP-HPLC elution profiles (system A) of oxazole-based BHQ-3 22, peptides H-Ser-Gly-Arg-Ser-Ala- Asn-Ala-Lys(Dde)-NH 2 (L- and D-amino acids), 18, 18-D, 19, 19-D, 20 and 20-D (detection in "Max Plot" mode) ,467 1, ,100 2,920 23,633 94,177 24,617 1, ,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 Minutes S49

51 mau mau 19,350 96,307 Peptide H-Ser-Gly-Arg-Ser-Ala-Asn-Ala-Lys(Dde)-NH 2 (L-amino acids) mau 19,050 99, ,933 3,018 23,917 0, ,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 Minutes Peptide H-Ser-Gly-Arg-Ser-Ala-Asn-Ala-Lys(Dde)-NH 2 (D-amino acids) ,767 0, ,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 Minutes S50

52 mau 20,867 98,809 Cy-labeled peptide mau 21,167 96, ,567 30,917 1,111 0, Minutes Cy-labeled peptide 19-D ,017 3,859 24,817 0, Minutes S51

53 mau Cy-labeled maleimide-peptide ,917 99, ,983 0, ,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 Minutes S52

54 mau Cy-labeled maleimide-peptide 20-D ,083 0,805 28,067 0,256 mau 27,400 98,035 22,750 98, Minutes FRET-based upa-sensitive probe ,767 1, ,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 Minutes S53

55 mau 27,267 99,616 FRET-based upa-sensitive probe 18-D ,883 0, Minutes S54

56 Relative Abundance VI. MS analyses LRMS ESI spectrum of 19 recorded in the positive mode AC302-QC #2 RT: 0.03 AV: 1 NL: 6.95E6 T: + p ESI Full ms [ ] [M+H] m/z HRMS ESI spectrum of 19 recorded in the negative mode S55

57 Relative Abundance LRMS ESI spectrum of 19-D recorded in the positive mode AC302-QC #3 RT: 0.06 AV: 1 NL: 7.13E6 T: + p ESI Full ms [ ] [M+H] m/z S56

58 Relative Abundance LRMS ESI spectrum of 20 recorded in the negative mode AC518 QCb3 #2 RT: 0.05 AV: 1 NL: 5.65E4 T: - p ESI Full ms [ ] [M-H] m/z HRMS ESI spectrum of 20 recorded in the negative mode S57

59 Relative Abundance LRMS ESI spectrum of 20-D recorded in the negative mode AC518 QCb3 #6 RT: 0.22 AV: 1 NL: 7.15E4 T: - p ESI Full ms [ ] [M-H] m/z S58

60 Relative Abundance LRMS ESI spectrum of 18 recorded in the positive mode AC522bis2 #2 RT: 0.04 AV: 1 NL: 1.65E7 T: + p ESI Full ms [ ] [M+3H] [M+4H] 4+ [M+2H] m/z HRMS ESI spectrum of 18 recorded in the negative mode S59

61 Relative Abundance LRMS ESI spectrum of 18-D recorded in the positive mode AC522bis #5 RT: 0.11 AV: 1 NL: 2.74E7 T: + p ESI Full ms [ ] [M+3H] [M+4H] 4+ [M+2H] m/z S60