synthesis and luminescence properties of new red … · synthesis and luminescence properties of...

Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is (c) The Royal Society of Chemistry 2010

1

Synthesis and luminescence properties of new red-shifted absorption lanthanide(III) chelates suitable for peptide and protein labelling Nicolas Maindron,a Séverine Poupart,a Maxime Hamon,a Jean-Baptiste Langlois,a Nelly Plé,a,c Ludovic Jean,a,b Anthony Romieu*a,b and Pierre-Yves Renard*a,b,c aN. Maindron, Dr. S. Poupart, M. Hamon, J.-B. Langlois, Pr. Nelly Plé, Dr. L. Jean, Dr. A. Romieu and Pr. P.-Y. Renard Equipe de Chimie Bio-Organique, COBRA - CNRS UMR 6014 & FR 3038, rue Lucien Tesnière, 76131 Mont-Saint-Aignan, France Fax (+33)2-35-52-29-71 E-mail: [email protected] or [email protected] Lab homepage: http://ircof.crihan.fr bDr. L. Jean, Dr. A. Romieu, Pr. P.-Y. Renard Université de Rouen, Place Emile Blondel, 76821 Mont-Saint-Aignan, France cPr. Nelly Plé Equipe de Chimie Hétérocyclique, , COBRA - CNRS UMR 6014 & FR 3038, rue Lucien Tesnière, 76131 Mont-Saint-Aignan, France dPr. P.-Y. Renard Institut Universitaire de France, 103 Boulevard Saint-Michel, 75005, Paris, France

Supporting Information


2

Experimental Section ................................................................................................................. 3 1H NMR spectrum of symmetrical bis-pyridinylpyrazine ligand 10 recorded in DMSO-d6. .... 6 13C NMR spectrum of symmetrical bis-pyridinylpyrazine ligand 10 recorded in DMSO-d6. ... 6 ESI-MS spectrum of symmetrical bis-pyridinylpyrazine ligand 10 recorded in the positive mode. .......................................................................................................................................... 7 RP-HPLC elution profile (system B) of symmetrical bis-pyridinylpyrazine ligand 10............. 7 1H NMR spectrum of protected non-symmetrical bis-pyridinylpyrazine ligand 17 recorded in CDCl3. ........................................................................................................................................ 8 13C NMR spectrum of protected non-symmetrical bis-pyridinylpyrazine ligand 17 recorded in CDCl3. ........................................................................................................................................ 8 1H NMR spectrum of non-symmetrical bis-pyridinylpyrazine ligand 18 recorded in DMSO-d6 + D2O.......................................................................................................................................... 9 13C NMR spectrum of non-symmetrical bis-pyridinylpyrazine ligand 18 recorded in DMSO-d6 + D2O. .................................................................................................................................... 9 ESI-MS spectrum of non-symmetrical bis-pyridinylpyrazine ligand 18 recorded in the positive mode. .......................................................................................................................... 10 RP-HPLC elution profile (system B) of non-symmetrical bis-pyridinylpyrazine ligand 18. .. 10 ESI-MS spectra of Eu(III) chelates 6 and 8 recorded in the negative mode. ........................... 11 Low- and high-resolution mass spectrum of Eu(III) chelate 6................................................. 12 Low- and high-resolution mass spectrum of Eu(III) chelate 8................................................. 14 RP-elution profile (system C) of Eu(III) chelate 6................................................................... 16 RP-elution profile (system C) of Eu(III) chelate 8a. ................................................................ 16 ESI-MS spectrum of Sm(III) chelate 7 recorded in the negative mode. .................................. 17 ESI-MS spectrum of Tb(III) chelate recorded in the negative mode. ...................................... 18 ESI-MS spectrum of activated Eu(III) chelate 23 recorded in the negative mode. ................. 19 ESI-MS spectrum of activated Eu(III) chelate 24 recorded in the negative mode. ................. 20 LC-MS analysis (system I) of the crude labelling mixture of hexapeptide 25 with activated Eu(III) chelate 23. .................................................................................................................... 21 ESI mass spectrum (recorded in the negative mode) for the peak at tR = 13.1 mina. .............. 21 ESI mass spectrum (recorded in the negative mode) for the peak at tR = 13.9 min, assigned to the luminescent labelled peptide 26. ........................................................................................ 22 ESI mass spectrum (recorded in the negative mode) for the peak at tR = 15.1 min assigned to the bis-labelled peptide 29........................................................................................................ 22 ESI-MS spectrum of luminescent labelled peptide 27 recorded in the negative mode............ 23


3

Experimental Section General: Column chromatography purifications were performed on Geduran® Si 60 silica gel (40-63 μm) from Merck. Reversed-phase column flash-chromatographies were performed on octadecyl-functionalised silica gel (mean pore size 60 Å, 37-74 μm) from Aldrich. TLC were carried out on Merck DC Kieselgel 60 F-254 aluminium sheets. The spots were visualised by illumination with UV lamp (λ = 254 nm or 365 nm), by immersion in ninhydrin solution or potassium permanganate solution. Small scale syntheses (i.e., bioconjugation experiments) were performed in standard single-use microtubes (0.5 or 1.7 mL). All solvents were dried following standard procedures (CH3CN: distillation over CaH2, CH2Cl2: distillation over P2O5, DMF: distillation over BaO, THF: distillation over Na°/benzophenone). Triethylamine (TEA) was distilled from CaH2 and stored over BaO. Fmoc-Lys(Boc)-OH was purchased from Iris Biotech. Di-tert-butyl iminodiacetate 9 was synthesised following a literature procedure.1 The HPLC-gradient grade acetonitrile (CH3CN) was obtained from Acros or Fisher Scientific. Buffers and aq. mobile-phases for HPLC were prepared using water purified with a Milli-Q system (purified to 18.2 MΩ.cm). Triethylammonium acetate (TEAA, 2.0 M) and triethylammonium bicarbonate (TEAB, 1.0 M) buffers were prepared from distilled triethylamine and glacial acetic acid or CO2 gas. 1H and 13C NMR spectra were recorded on a Bruker DPX 300 spectrometer (Bruker, Wissembourg, France). Chemical shifts are expressed in parts per million (ppm) and relative to tetramethylsilane from CDCl3 (δH = 7.26, δC = 77.16) or DMSO-d6 (δH = 2.50, δC = 39.52).2 Analytical HPLC was performed on a Thermo Scientific Surveyor Plus instrument equipped with a PDA detector. Semi-preparative HPLC was performed on a Thermo Scientific SPECTRASYSTEM liquid chromatography system (P4000) equipped with a UV-visible 2000 detector. Low-resolution mass spectra were obtained with a Finnigan LCQ Advantage MAX (ion trap) apparatus equipped with an electrospray source. High-resolution mass spectra were recorded on a LCT Premier XE benchtop orthogonal acceleration time-of-flight (oa-TOF) mass spectrometer (Waters Micromass) equipped with an electrospray source and in the negative mode (ESI-). UV-visible spectra were obtained on a Varian Cary 50 scan spectrophotometer. Fluorescence spectroscopic studies were performed with a Varian Cary Eclipse spectrophotometer. Peptide synthesis: The synthesis of hexapeptide 25 was carried out on an Applied Biosystems 433A peptide synthesizer using the standard Fmoc/tBu chemistry3 and the Wang resin (Iris Biotech, loading 0.9 mmol/g) on a scale of 0.1 mmol. Coupling reactions were performed with commercial Fmoc-protected amino acids from Iris Biotech (10 equiv.) and HBTU (10 equiv.), HOBt (10 equiv.) and DIEA (30 equiv.) in peptide-grade DMF. The resin loading with the first Fmoc-protected amino acid (10 equiv.) was performed with N,N’-diisopropylcarbodiimide (DIC, 10 equiv.) and DMAP (0.036 equiv.) in a mixture of CH2Cl2-NMP (1 : 1, v/v). Final acetylation of the free amino group from the N-terminal side of hexapeptide was achieved by treatment with Ac2O (10 equiv.), DIEA (10 equiv.) and DMAP (0.1 equiv.) in NMP for 90 min. The completion of reaction was also checked by the Kaiser ninhydrin test (negative after this treatment) and the resin was washed with NMP (3 × 2 mL), MeOH (3 × 2 mL) and CH2Cl2 (3 × 2 mL). A pre-cooled TFA solution (4 mL for 250 mg of resin) containing triisopropylsilane (TIPS, 2.5%, v/v) and deionised water (2.5%, v/v) was

1 WO Pat., 2001052898, 2001. 2 H. E. Gottlieb, V. Kotlyar and A. Nudelman, J. Org. Chem., 1997, 62, 7512. 3 G. B. Fields and R. L. Noble, Int. J. Pept. Protein Res., 1990, 35, 161.


4

added to the resin. After 3 h stirring, the resin was filtered and washed with TFA. The filtrate was evaporated to dryness and cold Et2O was added to the oily residue. The resulting white precipitate was isolated by centrifugation, lyophilised and purified by semi-preparative RP-HPLC (system A). The product-containing fractions were lyophilised to give the TFA salt of hexapeptide 25 as a white amorphous powder (54 mg, yield 75%). MS (ESI-): m/z 608.40 [M - H]- and 722.07 [M + TFA - H]-, calcd for C26H39N7O10: 609.63; HPLC (system A): tR = 9.8 min; λmax(recorded during the HPLC analysis)/nm 229 and 275. Synthesis of compound 16: The N-protected amino acid Fmoc-L-Lys(Boc)-OH was esterified with tert-butanol under non-standard conditions involving Boc2O-DMAP4, followed by the Fmoc removal. The resulting primary amine was reacted with tert-butylbromoacetate in the presence of a large excess of K2CO3 in dry CH3CN to give compound 16 in a good 45% overall yield for the three steps.

NH

OH

O

HN

O

O

O

O

Fmoc-L-Lys(Boc)-OH

NH

O

O

HN O

O

16

O

O

a-c

Scheme S1 Reagents and conditions: a) Boc2O (2.0 equiv.), DMAP (0.3 equiv.), tBuOH, 35 °C, 22 h, 67%; b) Et2NH (15 equiv.), CH2Cl2, 0 °C to rt, overnight, 94%; c) tert-Butylbromoacetate (1.2 equiv.), K2CO3 (5.0 equiv.), CH3CN, reflux 5 h, then rt overnight, 72%. (a) Fmoc-L-Lys(Boc)-OtBu: Fmoc-L-Lys(Boc)-OH (1.0 g, 2.13 mmol) was dissolved in tert-butanol (8 mL) and DMAP (78 mg, 0.64 mmol) and Boc2O (932 mg, 4.27 mmol) were sequentially added. The resulting reaction mixture was stirred at 35 °C under an argon atmosphere for 22 h. Thereafter, the reaction mixture was then diluted in AcOEt (30 mL) and the organic phase was washed with a 1.0 M aq. KHSO4 (2 × 10 mL) and a 5% aq. NaHCO3 (2 × 10 mL). The aq. phases were collected and re-extracted with CH2Cl2 (2 × 30 mL). The combined organic phases were washed with brine, dried over MgSO4, filtered and evaporated to dryness. The resulting yellow foam was purified by flash-chromatography on a silica gel column with a mixture cyclohexane-AcOEt (8 : 2, v/v). The desired full-protected lysine derivative was obtained as a white solid (750 mg, yield 67%). Rf (cyclohexane-AcOEt, 8 : 2, v/v) 0.24; δH (300 MHz, CDCl3) 7.77 (2H, d, J 7.4), 7.61 (2H, d, J 7.4), 7.40 (2H, t, J 7.4), 7.32 (2H, t, J 7.4), 5.36 (1H, bs), 4.55 (1H, bs), 4.41-4.35 (2H, m), 4.28-4.19 (2H, m), 3.15-3.05 (2H, m), 1.90-1.30 (24H, m); δC (75.4 MHz, CDCl3) 171.7, 156.1, 156.0, 144.1, 144.0, 141.4, 127.8, 127.2, 125.3, 120.1, 82.3, 67.0, 54.3, 47.3, 40.3, 32.6, 29.7, 28.5, 28.1, 22.4; MS (ESI+): m/z 525.00 [M + H]+, 541.87 [M + H2O]+• (water cluster formed during the ionisation process) and 547.20 [M +

4 K. Takeda, A. Akiyama, H. Nakamura, S.-i. Takizawa, Y. Mizuno, H. Takayanagi and Y. Harigaya, Synthesis, 1994, 1063.


5

Na]+, calcd for C30H40N2O6: 524.65. (b) H-L-Lys(Boc)-OtBu: Fmoc-L-Lys(Boc)-OtBu (744 mg, 1.42 mmol) was dissolved in CH2Cl2 (12 mL) and cooled to 0 °C. Diethylamine (2.2 mL, 21.3 mmol) was added and the reaction mixture was stirred at room temperature overnight. After solvent removal under reduced pressure, purification by flash-chromatography on a silica gel column with cyclohexane/AcOEt (8 : 2, v/v) followed by CH2Cl2/MeOH 95 : 5, v/v) yielded the compound H-L-Lys(Boc)-OtBu as a pale brown oil (404 mg, yield 94%). Rf (CH2Cl2-MeOH, 95 : 5, v/v) 0.28; δH (300 MHz, CDCl3) 4.54 (1H, bs), 3.29 (1H, dd, J 7.2 and 5.4), 3.16-3.06 (2H, m), 1.76-1.30 (24H, m); δC (75.4 MHz, CDCl3) 175.6, 156.1, 81.1, 55.0, 40.5, 34.7, 30.0, 28.6, 28.2, 23.0; MS (ESI+): m/z 303.12 [M + H]+ , calcd for C15H30N2O4: 302.41. (c) Alkylation: H-L-Lys(Boc)-OH (400 mg, 1.32 mmol) and anhydrous K2CO3 (914 mg, 6.61 mmol) were dissolved in dry CH3CN (8 mL) and tert-butylbromoacetate (195 μL, 1.32 mmol) was added. After heating under reflux for 5 h, a further amount of tert-butylbromoacetate (40 μL, 0.26 mmol) was added. The reaction mixture was stirred at room temperature overnight. The suspension was filtered and the filtrate was evaporated. The resulting residue was purified by flash-chromatography on a silica gel column with a step gradient of AcOEt (10-20%) in cyclohexane. The desired secondary amine 16 was obtained as a colourless oil (400 mg, yield 72%). Rf (cyclohexane-AcOEt, 7 : 3, v/v) 0.33;δH (300 MHz, CDCl3) 4.55 (1H, bs), 3.32 (1H, d, J 17.0), 3.21 (1H, d, J 17.0), 3.15-3.06 (3H, m), 1.70-1.55 (2H, m), 1.55-1.35 (31H, m); δC (75.4 MHz, CDCl3) 174.0, 171.1, 156.0, 81.3, 61.2, 50.0, 40.4, 33.0, 29.9, 28.5, 28.2, 23.0; MS (ESI+): m/z 417.13 [M + H]+, calcd for C21H40N2O6: 416.55.


6

1H NMR spectrum of symmetrical bis-pyridinylpyrazine ligand 10 recorded in DMSO-d6.

ppm (t1)5.010.0

9.56

0

8.49

58.

470

8.08

68.

060

8.03

4

7.70

97.

685

4.17

0

3.63

8

2.51

22.

506

2.50

02.

494

2.48

8

4.00

1.88

2.05

2.10

2.07

8.48

13C NMR spectrum of symmetrical bis-pyridinylpyrazine ligand 10 recorded in DMSO-d6.

ppm (t1)50100150

172.

038

152.

582

149.

038

142.

166

138.

377

124.

296

119.

965

58.9

90

54.3

80

40.3

5340

.075

39.7

9839

.520

39.2

4238

.964

38.6

86


7

ESI-MS spectrum of symmetrical bis-pyridinylpyrazine ligand 10 recorded in the positive mode.

300 350 400 450 500 550 600 650 700 750m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

525.27

547.20 578.07

600.13556.13364.53 481.33410.20 589.33466.27 604.73445.20391.93 503.07309.00252.27 561.27 625.87 743.40352.00 643.53 686.07 728.00283.13 667.00329.80 699.07

[M + H]+

525.27

300 350 400 450 500 550 600 650 700 750m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

525.27

547.20 578.07

600.13556.13364.53 481.33410.20 589.33466.27 604.73445.20391.93 503.07309.00252.27 561.27 625.87 743.40352.00 643.53 686.07 728.00283.13 667.00329.80 699.07

[M + H]+

525.27

RP-HPLC elution profile (system B) of symmetrical bis-pyridinylpyrazine ligand 10.

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24

mA

U

-25

0

25

50

75

100

125

150

175

200

225

250

275

mA

U

-25

0

25

50

75

100

125

150

175

200

225

250

275

12,6

67 1

,939

13,3

50 1

,527

13,7

67 9

6,53

4

Spectrum Max PlotRetention TimeArea Percent

* *

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24

mA

U

-25

0

25

50

75

100

125

150

175

200

225

250

275

mA

U

-25

0

25

50

75

100

125

150

175

200

225

250

275

12,6

67 1

,939

13,3

50 1

,527

13,7

67 9

6,53

4


* *

*peaks found in aq. HPLC mobile phase.


8

1H NMR spectrum of protected non-symmetrical bis-pyridinylpyrazine ligand 17 recorded in CDCl3.

ppm (t1)5.010.0

9.65

19.

624

8.48

98.

463

8.40

28.

377

7.94

77.

921

7.89

27.

866

7.81

27.

787

7.70

07.

674

7.26

0

4.65

8

4.41

64.

212

4.16

1

4.11

9

4.06

8

3.81

53.

526

3.39

33.

368

3.34

4

3.06

8

1.70

31.

493

1.46

4

1.41

1

2.000.920.95

0.901.00

2.20

1.79

1.76

3.77

0.881.80

1.91

1.98

49.98

0.75

13C NMR spectrum of protected non-symmetrical bis-pyridinylpyrazine ligand 17 recorded in CDCl3.

ppm (t1)50100150

172.

276

170.

859

168.

630

159.

529

156.

128

155.

702

153.

673

152.

979

149.

817

149.

355

142.

692

142.

340

138.

458

138.

037

125.

103

124.

277

120.

884

120.

107

82.7

91

81.7

2981

.277

79.1

03

64.3

7758

.820

58.1

71

55.0

54

53.5

46

40.3

93

29.7

2029

.655

28.5

1528

.382

28.1

74

23.4

97


9

1H NMR spectrum of non-symmetrical bis-pyridinylpyrazine ligand 18 recorded in DMSO-d6 + D2O.

ppm (t1)12.03.04.05.06.07.08.09.0

9.54

59.

533

8.47

88.

454

8.43

0

8.08

08.

060

8.05

48.

034

8.02

88.

009

7.74

17.

715

7.69

27.

667

4.19

2

4.15

2

4.10

0

4.04

6

3.99

5

3.67

33.

553

3.41

13.

386

3.36

3

2.72

62.

506

2.50

02.

494

1.67

7

1.50

4

1.37

01.

341

2.00

2.24

2.38

2.54

2.393.92

6.312.751.79

2.61

2.483.871.38

*

*

ppm (t1)12.03.04.05.06.07.08.09.0

9.54

59.

533

8.47

88.

454

8.43

0

8.08

08.

060

8.05

48.

034

8.02

88.

009

7.74

17.

715

7.69

27.

667

4.19

2

4.15

2

4.10

0

4.04

6

3.99

5

3.67

33.

553

3.41

13.

386

3.36

3

2.72

62.

506

2.50

02.

494

1.67

7

1.50

4

1.37

01.

341

2.00

2.24

2.38

2.54

2.393.92

6.312.751.79

2.61

2.483.871.38

*

*

13C NMR spectrum of non-symmetrical bis-pyridinylpyrazine ligand 18 recorded in DMSO-d6 + D2O.

ppm (t1)50100150

174.

138

172.

733

172.

071

160.

150

159.

755

158.

460

158.

038

152.

597

152.

513

149.

097

149.

066

142.

107

138.

318

138.

196

138.

061

124.

264

123.

925

119.

907

119.

782

63.2

4959

.029

57.4

29

54.4

4652

.567

45.6

9239

.520

29.0

1926

.773

22.7

53

8.57

5

*

*

ppm (t1)50100150

174.

138

172.

733

172.

071

160.

150

159.

755

158.

460

158.

038

152.

597

152.

513

149.

097

149.

066

142.

107

138.

318

138.

196

138.

061

124.

264

123.

925

119.

907

119.

782

63.2

4959

.029

57.4

29

54.4

4652

.567

45.6

9239

.520

29.0

1926

.773

22.7

53

8.57

5

*

*

*peaks assigned to triethylammonium salts recovered during lyophilisation.


10

ESI-MS spectrum of non-symmetrical bis-pyridinylpyrazine ligand 18 recorded in the positive mode.

200 300 400 500 600 700 800 900 1000 1100 1200m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

596.27

298.60

1191.00

467.33538.07269.60

552.13333.271132.73261.27 423.33 856.67832.47 1230.73721.67160.47 687.20475.33 771.73 981.73352.27 648.93 1101.401021.27231.07 905.33

[M + H]+

596.27

[M + 2H]2+

298.60

200 300 400 500 600 700 800 900 1000 1100 1200m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

596.27

298.60

1191.00

467.33538.07269.60

552.13333.271132.73261.27 423.33 856.67832.47 1230.73721.67160.47 687.20475.33 771.73 981.73352.27 648.93 1101.401021.27231.07 905.33

[M + H]+

596.27

[M + 2H]2+

298.60

RP-HPLC elution profile (system B) of non-symmetrical bis-pyridinylpyrazine ligand 18.

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mA

U

-250

0

250

500

750

1000

1250

1500

1750

2000

2250

2500

mA

U

-250

0

250

500

750

1000

1250

1500

1750

2000

2250

2500

13,7

17 9

2,92

114

,083

7,0

79



11

ESI-MS spectra of Eu(III) chelates 6 and 8 recorded in the negative mode.

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

673.20

784.671344.93

859.40 1007.73 1856.401787.671077.27 1490.471366.73577.27 1199.33 1623.80719.93 1931.601294.53483.93 1704.67273.00 386.73204.87

[M - H]-

673.20

650 655 660 665 670 675 680 685 690 695 700m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

673.20

671.20

674.07

674.93

658.07 675.80654.60 665.00 689.80

673.20

671.20

a)

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

673.20

784.671344.93

859.40 1007.73 1856.401787.671077.27 1490.471366.73577.27 1199.33 1623.80719.93 1931.601294.53483.93 1704.67273.00 386.73204.87

[M - H]-

673.20

650 655 660 665 670 675 680 685 690 695 700m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

673.20

671.20

674.07

674.93

658.07 675.80654.60 665.00 689.80

673.20

671.20

650 655 660 665 670 675 680 685 690 695 700m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

673.20

671.20

674.07

674.93

658.07 675.80654.60 665.00 689.80

673.20

671.20

a)

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

744.13

1487.13

855.87

1115.47

1773.731602.201982.871859.401131.071041.73839.73 879.20 1748.071212.73 1355.93 1429.27465.20 567.87 656.13 972.27290.93 347.40

[M - H]-

744.13

b)

[2M - H]-

1487.13

726 728 730 732 734 736 738 740 742 744 746 748 750 752 754 756 758 760m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

744.13

742.20

745.20

746.07

746.40

747.33 759.27757.80750.20748.07 751.07

742.20

744.13

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

744.13

1487.13

855.87

1115.47

1773.731602.201982.871859.401131.071041.73839.73 879.20 1748.071212.73 1355.93 1429.27465.20 567.87 656.13 972.27290.93 347.40

[M - H]-

744.13

b)

[2M - H]-

1487.13

726 728 730 732 734 736 738 740 742 744 746 748 750 752 754 756 758 760m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

744.13

742.20

745.20

746.07

746.40

747.33 759.27757.80750.20748.07 751.07

726 728 730 732 734 736 738 740 742 744 746 748 750 752 754 756 758 760m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

744.13

742.20

745.20

746.07

746.40

747.33 759.27757.80750.20748.07 751.07

742.20

744.13

(a) ESI mass spectrum of Eu(III) chelate 6, calcd mass for [C24H20EuN6O8]-: 672.42; (b) ESI mass spectrum of Eu(III) chelate 8, calcd mass for [C28H29EuN7O8]-: 743.54 ; both recorded in the negative mode, the inset show the zoom-scan spectrum of [M - H]-.


12

Low- and high-resolution mass spectrum of Eu(III) chelate 6.

9::4::5 MN -19-121-Dec-2010

m/z400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820

%

0

1001012039 24 (0.151) Cm (4:24) 1: TOF MS ES-

3.60e4673.0583

671.0536

674.0610

Elemental Composition Report Multiple Mass Analysis: 2 mass(es) processed Tolerance = 10.0 PPM / DBE: min = -1.5, max = 30.0 Element prediction: Off Number of isotope peaks used for i-FIT = 4 Monoisotopic Mass, Even Electron Ions 4239 formula(e) evaluated with 32 results within limits (up to 50 closest results for each mass) Elements Used: C: 0-500 H: 0-110 N: 0-7 O: 0-10 151Eu: 0-1 153Eu: 0-1 Minimum: 50.00 -1.5 Maximum: 100.00 10.0 10.0 30.0 Mass RA Calc. Mass mDa PPM DBE i-FIT i-FIT (Norm) Formula 671.0536 87.45 671.0538 -0.2 -0.3 18.5 649.0 2.2 C27 H22 N2 O9 153Eu 671.0532 0.4 0.6 6.5 649.5 2.7 C20 H31 O6 151Eu 153Eu 671.0541 -0.5 -0.7 18.5 648.3 1.4 C24 H20 N6 O8 151Eu 671.0545 -0.9 -1.3 11.5 649.2 2.4 C21 H27 N4 O2 151Eu 153Eu 671.0551 -1.5 -2.2 23.5 648.9 2.1 C28 H18 N6 O5 153Eu 671.0509 2.7 4.0 26.5 650.4 3.6 C35 H20 O5 151Eu 671.0564 -2.8 -4.2 -1.5 649.5 2.6 C9 H31 N6 O9 151Eu 153Eu 671.0505 3.1 4.6 7.5 649.0 2.2 C16 H27 N6 O4 151Eu 153Eu 671.0568 -3.2 -4.8 17.5 650.7 3.8 C28 H24 O10 151Eu 671.0578 -4.2 -6.3 22.5 651.6 4.7


13

C32 H22 O7 153Eu 671.0581 -4.5 -6.7 22.5 651.0 4.2 C29 H20 N4 O6 151Eu 671.0491 4.5 6.7 2.5 650.1 3.3 C15 H31 N2 O8 151Eu 153Eu 671.0585 -4.9 -7.3 15.5 652.6 5.7 C26 H27 N2 151Eu 153Eu 671.0482 5.4 8.0 27.5 650.4 3.6 C31 H16 N6 O3 151Eu 671.0591 -5.5 -8.2 27.5 652.0 5.2 C33 H18 N4 O3 153Eu 671.0479 5.7 8.5 27.5 651.2 4.4 C34 H18 N2 O4 153Eu 671.0473 6.3 9.4 15.5 652.3 5.5 C27 H27 O 151Eu 153Eu 671.0469 6.7 10.0 22.5 651.4 4.6 C30 H20 N2 O7 151Eu 673.0583 100.00 673.0582 0.1 0.1 17.5 546.2 0.0 C28 H24 O10 153Eu 673.0585 -0.2 -0.3 17.5 550.0 3.8 C25 H22 N4 O9 151Eu 673.0589 -0.6 -0.9 10.5 557.0 10.8 C22 H29 N2 O3 151Eu 153Eu 673.0595 -1.2 -1.8 22.5 550.9 4.7 C29 H20 N4 O6 153Eu 673.0555 2.8 4.2 18.5 551.4 5.3 C24 H20 N6 O8 153Eu 673.0549 3.4 5.1 6.5 558.6 12.4 C17 H29 N4 O5 151Eu 153Eu 673.0626 -4.3 -6.4 21.5 551.6 5.5 C30 H22 N2 O7 151Eu 673.0629 -4.6 -6.8 14.5 555.3 9.2 C27 H29 O 151Eu 153Eu 673.0536 4.7 7.0 1.5 559.2 13.1 C16 H33 O9 151Eu 153Eu 673.0636 -5.3 -7.9 26.5 555.8 9.7 C34 H20 N2 O4 153Eu 673.0527 5.6 8.3 26.5 554.4 8.3 C32 H18 N4 O4 151Eu 673.0639 -5.6 -8.3 26.5 554.2 8.1 C31 H18 N6 O3 151Eu 673.0523 6.0 8.9 26.5 556.1 9.9 C35 H20 O5 153Eu 673.0648 -6.5 -9.7 1.5 559.7 13.6 C15 H33 N2 O8 151Eu 153Eu


14

Low- and high-resolution mass spectrum of Eu(III) chelate 8.

10:08:10MN -35-121-Dec-2010

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

%

0

1001012040 184 (1.119) Cm (181:386) 1: TOF MS ES-

3.92e4744.1282

742.1263

673.0538

745.1306

10:08:10MN -35-121-Dec-2010

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

%

0

1001012040 184 (1.119) Cm (181:386) 1: TOF MS ES-

3.92e4744.1282

742.1263

673.0538

745.1306

Elemental Composition Report Multiple Mass Analysis: 2 mass(es) processed Tolerance = 10.0 PPM / DBE: min = -1.5, max = 30.0 Element prediction: Off Number of isotope peaks used for i-FIT = 4 Monoisotopic Mass, Even Electron Ions 1197 formula(e) evaluated with 13 results within limits (up to 50 closest results for each mass) Elements Used: C: 0-500 H: 0-110 N: 5-9 O: 6-10 151Eu: 0-1 153Eu: 0-1 Minimum: 50.00 -1.5 Maximum: 100.00 10.0 10.0 30.0 Mass RA Calc. Mass mDa PPM DBE i-FIT i-FIT (Norm) Formula 742.1263 84.01 742.1276 -1.3 -1.8 18.5 589.8 2.1 C28 H29 N7 O8 151Eu 742.1246 1.7 2.3 19.5 589.4 1.7 C27 H27 N9 O7 153Eu 742.1236 2.7 3.6 14.5 589.1 1.4 C23 H29 N9 O10 151Eu 742.1299 -3.6 -4.9 -1.5 589.2 1.5 C13 H40 N7 O9 151Eu 153Eu 742.1316 -5.3 -7.1 22.5 590.9 3.1 C33 H29 N5 O6 151Eu 742.1200 6.3 8.5 3.5 589.4 1.6 C15 H36 N9 O6 151Eu 153Eu 744.1282 100.00 744.1290 -0.8 -1.1 18.5 631.8 0.2 C28 H29 N7 O8 153Eu


15

744.1250 3.2 4.3 14.5 637.6 6.0 C23 H29 N9 O10 153Eu 744.1320 -3.8 -5.1 17.5 633.7 2.0 C29 H31 N5 O9 151Eu 744.1244 3.8 5.1 2.5 642.8 11.2 C16 H38 N7 O7 151Eu 153Eu 744.1330 -4.8 -6.5 22.5 637.6 6.0 C33 H29 N5 O6 153Eu 744.1221 6.1 8.2 22.5 634.9 3.3 C31 H27 N7 O6 151Eu 744.1356 -7.4 -9.9 2.5 644.3 12.7 C15 H38 N9 O6 151Eu 153Eu


16

RP-elution profile (system C) of Eu(III) chelate 6.

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mA

U

-200

0

200

400

600

800

1000

1200

1400

1600

1800

mA

U

-200

0

200

400

600

800

1000

1200

1400

1600

1800

8,38

7 1

00,0

00

PDA-345nmRetention TimeArea Percent

RP-elution profile (system C) of Eu(III) chelate 8a.

Minutes0 2 4 6 8 10 12 14 16 18 20

mA

U

-25

0

25

50

75

100

125

150

175

200

225

250

275

mA

U

-25

0

25

50

75

100

125

150

175

200

225

250

275

4,63

8 0

,036

5,22

2 9

8,00

8

15,4

98 0

,986

16,6

23 0

,970

PDA-345nmRetention TimeArea Percent

athe broad peak can be explained by an incomplete protonation of lysine amino group of 8 under these HPLC conditions (TEAA 25 mM, pH 7.0 as aq. mobile phase). Indeed, it is not possible to analyse Ln(III) chelates with an acidic aq. mobile phase (e.g., TFA 0.1%) due to their instability under these HPLC elution conditions.


17

ESI-MS spectrum of Sm(III) chelate 7 recorded in the negative mode.

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

672.40

1343.07

1363.07581.40 945.87

565.20 1284.93786.20726.93 1492.60382.33 1433.33624.27 1192.731074.73858.27 959.00267.53 517.80312.27 462.53 908.47

[M - H]-

672.40

660 662 664 666 668 670 672 674 676 678 680 682 684m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

672.40674.20

667.33669.33

668.40

670.27

673.33675.20

671.27

664.40679.33

664.07 676.27665.20680.40663.87

684.40666.47 677.87 683.00682.33660.67

667.33668.40

669.33

670.27

672.40 674.20

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

672.40

1343.07

1363.07581.40 945.87

565.20 1284.93786.20726.93 1492.60382.33 1433.33624.27 1192.731074.73858.27 959.00267.53 517.80312.27 462.53 908.47

[M - H]-

672.40

660 662 664 666 668 670 672 674 676 678 680 682 684m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

672.40674.20

667.33669.33

668.40

670.27

673.33675.20

671.27

664.40679.33

664.07 676.27665.20680.40663.87

684.40666.47 677.87 683.00682.33660.67

667.33668.40

669.33

670.27

672.40 674.20

660 662 664 666 668 670 672 674 676 678 680 682 684m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

672.40674.20

667.33669.33

668.40

670.27

673.33675.20

671.27

664.40679.33

664.07 676.27665.20680.40663.87

684.40666.47 677.87 683.00682.33660.67

660 662 664 666 668 670 672 674 676 678 680 682 684m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

672.40674.20

667.33669.33

668.40

670.27

673.33675.20

671.27

664.40679.33

664.07 676.27665.20680.40663.87

684.40666.47 677.87 683.00682.33660.67

660 662 664 666 668 670 672 674 676 678 680 682 684m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

672.40674.20

667.33669.33

668.40

670.27

673.33675.20

671.27

664.40679.33

664.07 676.27665.20680.40663.87

684.40666.47 677.87 683.00682.33660.67

667.33668.40

669.33

670.27

672.40 674.20


18

ESI-MS spectrum of Tb(III) chelate recorded in the negative mode.

500 550 600 650 700 750 800 850 900 950 1000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

679.20

792.67

906.53724.40950.93782.47747.87673.40562.20 808.00694.67 926.67901.93850.67 967.40507.40 862.93 993.20830.87527.87 618.53 635.07586.47

[M - H]-

679.20

[M + TFA - H]-792.67

670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

679.20

680.20

681.13

682.07673.40 682.87 688.80 689.67688.20683.33674.07

679.20

680.20

500 550 600 650 700 750 800 850 900 950 1000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

679.20

792.67

906.53724.40950.93782.47747.87673.40562.20 808.00694.67 926.67901.93850.67 967.40507.40 862.93 993.20830.87527.87 618.53 635.07586.47

[M - H]-

679.20

[M + TFA - H]-792.67

670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

679.20

680.20

681.13

682.07673.40 682.87 688.80 689.67688.20683.33674.07

670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

679.20

680.20

681.13

682.07673.40 682.87 688.80 689.67688.20683.33674.07

679.20

680.20


19

ESI-MS spectrum of activated Eu(III) chelate 23 recorded in the negative mode.

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

891.07

1181.80742.27

1624.13870.07 1703.13 1916.60949.00532.80 1508.80 1821.131367.731065.93521.13 1595.20269.00 1879.60413.20 1303.13 1784.07682.67 1026.20 1208.331107.73 1944.00807.27570.87595.60

[M - H]-

891.07

880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

891.07

889.20

892.07

893.20890.27

893.40894.20 899.13

895.07

896.13

883.67 897.00881.00 887.13884.13883.40 888.27

887.53 897.67884.53881.73

889.20

891.07

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

891.07

1181.80742.27

1624.13870.07 1703.13 1916.60949.00532.80 1508.80 1821.131367.731065.93521.13 1595.20269.00 1879.60413.20 1303.13 1784.07682.67 1026.20 1208.331107.73 1944.00807.27570.87595.60

[M - H]-

891.07

880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

891.07

889.20

892.07

893.20890.27

893.40894.20 899.13

895.07

896.13

883.67 897.00881.00 887.13884.13883.40 888.27

887.53 897.67884.53881.73

880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

891.07

889.20

892.07

893.20890.27

893.40894.20 899.13

895.07

896.13

883.67 897.00881.00 887.13884.13883.40 888.27

887.53 897.67884.53881.73

889.20

891.07


20

ESI-MS spectrum of activated Eu(III) chelate 24 recorded in the negative mode.

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

1025.29

1108.95981.35928.27 1537.951165.05 1293.26 1615.861365.35 1723.81212.16 1818.86 1965.78845.33257.69 1468.44359.25 726.62602.10525.29429.19

[M - H]-

1025.29

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

1025.29

1108.95981.35928.27 1537.951165.05 1293.26 1615.861365.35 1723.81212.16 1818.86 1965.78845.33257.69 1468.44359.25 726.62602.10525.29429.19

[M - H]-

1025.29


21

LC-MS analysis (system I) of the crude labelling mixture of hexapeptide 25 with activated Eu(III) chelate 23.

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

2.16

13.09

13.902.64

33.1015.12 28.97 33.6831.86 35.5029.30 36.4928.734.04 15.37 27.164.37 25.5923.6919.98 23.1118.4117.677.41 11.034.78 6.75 9.06

1.17

NL:8.39E7TIC F: - c ESI Full ms [150.00-2000.00] MS NM-46-2-brut-4h

ESI mass spectrum (recorded in the negative mode) for the peak at tR = 13.1 mina.

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1598.25

1428.37

799.79

1513.66

212.111649.211369.23830.24

243.31 713.79 1679.33920.01 1134.311018.42455.49 1960.331281.85 1838.88587.01313.51

[M - H]-

1428.37

[M' - H]-

Activated chelatedimer

799.79

[M' - 2H]2-

1598.25

Mono-labelledcyclo-peptide 28

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1598.25

1428.37

799.79

1513.66

212.111649.211369.23830.24

243.31 713.79 1679.33920.01 1134.311018.42455.49 1960.331281.85 1838.88587.01313.51

[M - H]-

1428.37

[M' - H]-

Activated chelatedimer

799.79

[M' - 2H]2-

1598.25

Mono-labelledcyclo-peptide 28

aUnder these chromotagraphic conditions, luminescent mono-labelled cyclo-peptide 28 and activated chelate dimer are not resolved and so eluted as a single peak.


22

ESI mass spectrum (recorded in the negative mode) for the peak at tR = 13.9 min, assigned to the luminescent labelled peptide 26.

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

1464.40

1407.35212.09 1486.32 1593.28731.80 1279.291222.20796.50 1336.25 1952.651133.30 1867.181665.81255.12 569.86 956.61884.68 1782.691055.83478.17321.10 673.01

1464.40

[M - H]-

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

1464.40

1407.35212.09 1486.32 1593.28731.80 1279.291222.20796.50 1336.25 1952.651133.30 1867.181665.81255.12 569.86 956.61884.68 1782.691055.83478.17321.10 673.01

1464.40

[M - H]-

ESI mass spectrum (recorded in the negative mode) for the peak at tR = 15.1 min assigned to the bis-labelled peptide 29.

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

1159.11

212.16

1546.10891.12243.281131.13 1216.44

1605.601314.821088.401017.52 1872.97255.10 1465.34763.55 1751.10 1937.26928.81495.17 864.24593.07 664.96431.98345.04

1159.11

[M - 2H]2-

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

1159.11

212.16

1546.10891.12243.281131.13 1216.44

1605.601314.821088.401017.52 1872.97255.10 1465.34763.55 1751.10 1937.26928.81495.17 864.24593.07 664.96431.98345.04

1159.11

[M - 2H]2-


23

ESI-MS spectrum of luminescent labelled peptide 27 recorded in the negative mode.

1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

1519.67

1632.93

1655.80

1842.731259.201739.731686.13

1831.271577.27 1885.47 1916.93 1967.001324.73 1790.531501.87

1097.27 1719.471267.601212.00 1932.401409.27 1439.401345.401159.871014.73 1073.73

1365.531053.00

[M - H]-

1519.67

1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

1519.67

1632.93

1655.80

1842.731259.201739.731686.13

1831.271577.27 1885.47 1916.93 1967.001324.73 1790.531501.87

1097.27 1719.471267.601212.00 1932.401409.27 1439.401345.401159.871014.73 1073.73

1365.531053.00

[M - H]-

1519.67

synthesis and luminescence properties of new red … · synthesis and luminescence properties of...

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