peptide conjugation and [ 18f]-radiolabelling via unusual ... · synthesis and reactivity of a...

Synthesis and Reactivity of a Bis-Sultone Cross-Linker for Peptide Conjugation and [18F]-Radiolabelling via Unusual “Double Click” Approach Thomas Priem,a,b Cédric Bouteiller,*a David Camporese,a Anthony Romieu,b,c and Pierre-Yves Renardb,c,d aAdvanced Accelerator Applications, 20 Rue Diesel, 01630 Saint-Genis-Pouilly, France Fax: +33-4-50-99-30-71 Phone: +33-4-50-99-30-70 E-mail: [email protected] Web site: http://www.adacap.com bEquipe de Chimie Bio-Organique, COBRA-CNRS UMR 6014 & FR 3038, rue Lucien Tesnière, 76131 Mont-Saint-Aignan, France Lab homepage: http://ircof.crihan.fr cUniversité de Rouen, Place Emile Blondel, 76821 Mont-Saint-Aignan, France dInstitut Universitaire de France, 103 Boulevard Saint-Michel, 75005, Paris, France

SUPPORTING INFORMATION Abbreviations ........................................................................................................................................ S1

Experimental procedures ....................................................................................................................... S1

RP-HPLC elution profile (system A) of 1-phenylacyl-1,3-propanesultone 1. ...................................... S4

RP-HPLC elution profile (system A) of 1-benzyl-1,3-propanesultone 2. ............................................. S4 1H NMR spectrum of bis-propanesultone 3 recorded in acetone-d6 at 300 MHz. ................................. S5 13C NMR spectrum of bis-propanesultone 3 recorded in acetone-d6 at 75.5 MHz................................ S5

ESI mass spectrum of bis-sultone 3 recorded in the negative mode.a ................................................... S6

RP-HPLC elution profile (system A) of bis-sultone 3.a ........................................................................ S6

RP-HPLC elution profile (system A) of mono-fluoro-propanesultone 4.a ............................................ S7

RP-HPLC elution profile (system A) of mono-fluoro-Boc-L-lysine-NH2 conjugate 5.a ...................... S7

RP-HPLC elution profile (system A) of mono-fluoro-peptide conjugate 6.a ........................................ S8

RP-HPLC elution profile (system A) of mono-iodo-propanesultone.a ................................................. S8

RP-HPLC elution profile (system A) of 1-oxo-1-phenyl-4-(propylamino)butane-2-sulfonic acid. ...... S9

RP-HPLC elution profile (system A) of 4-fluoro-1-oxo-1-phenylbutane-2-sulfonic acid. ................... S9

RP-HPLC elution profile (system A) of 4-chloro-1-oxo-1-phenylbutane-2-sulfonic acid.................. S10

RP-HPLC elution profile (system A) of 4-bromo-1-oxo-1-phenylbutane-2-sulfonic acid. ................ S10

RP-HPLC elution profile (system A) of 4-iodo-1-oxo-1-phenylbutane-2-sulfonic acid. .................... S11

Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2011

S1

Abbreviations The following abbreviations are used throughout the text of the ESI file: ESI, electrospray

ionisation; RP-HPLC, reversed-phase high performance liquid chromatography; rt, room

temperature; TFA, trifluoroacetic acid.

Experimental procedures

Mono-iodo-propanesultone

O

O

OS

OOI

SO3H

Bis-propanesultone 3 (52.4 mg, 0.14 mmol, 1 equiv.) was dissolved in dry CH3CN (5

mL). NaI (21 mg, 0.14 mmol, 1 equiv.) in solution in dry CH3CN (8 mL) was then slowly

added. The resulting reaction mixture was stirred at rt and the reaction was checked for

completion by RP-HPLC (system A). After 6 h, the reaction was stopped to avoid

significant formation of the non-desired bis-iodo derivative. The crude product was then

dissolved with aq. TFA and purified by RP-HPLC (system B, 1 injection, tR = 35.0-41.5

min). The product-containing fractions were lyophilised to give the mono-iodo-

propanesultone as an orange solid (33.1 mg, yield 49%, mixture of two racemic

diastereomers). δH (300 MHz, CD3OD) 8.29 (d, J 8.7, 2H), 8.23 (d, J 8.3, 2H), 5.65 (ddd,

J 1.9, 6.8, 8.5, 1H), 5.21-5.16 (m, 1H), 4.68-4.54 (m, 2H), 3.25-3.15 (m, 2H), 2.83-2.74

(m, 2H), 2.72-2.60 (m, 2H); δC (75.5 MHz, CD3OD) 195.5, 189.5, 142.2, 139.9, 130.7,

129.9, 69.8, 68.1, 61.5, 34.1, 28.0, 3.4; MS (ESI-): m/z 500.93 [M - H]-, calcd for

C14H15IO8S2 501.93; HPLC (system A): tR = 22.8 and 23.0 min (two racemic

diastereomers, purity 90%); λmax(recorded during the HPLC analysis)/nm 265.

Mono-fluoro-oxyamine-peptide conjugate. The same protocol (synthesis and

purification) than described for peptide conjugate 6 was used. An aminooxy-

dodecapeptide (its sequence is confidential and not disclosed within this article but it

contains an aminooxyacetic acid residue) was used as starting material. MS (ESI+): m/z

885.33 [M + 2H]2+, 1769.53 [M + H]+, calcd for C73FH117N22O24S2 1768.80; HPLC


S2

(system A): tR = 20.9 min (four diastereomers, purity 68%); λmax(recorded during the

HPLC analysis)/nm 269.

General procedure for the nucleophilic ring-opening of monosultones 1 and 2. In a round

bottom flask were introduced one equivalent of the nucleophile (halide, amine, alcohol, thiol

or amino-acid derivatives) dissolved in CH3CN (with a small amount of deionised water or

DMF if complete solubility in CH3CN was not obtained). The mixture was stirred at room

temperature and one equiv. of monosultone (1 or 2) in solution in CH3CN was then added.

The reaction was checked for completion by RP-HPLC (system A), quenched by dilution with

aq. TFA and CH3CN and purified by RP-HPLC (system B, 1 injection).

- For potassium halide salts, 1.1 equivalent of Kryptofix[K222] was added,

- For amino acid hydrochloride derivatives, 1.1 equivalent of K2CO3 (or Cs2CO3) was added.

1-Oxo-1-phenyl-4-(propylamino)butane-2-sulfonic acid

SO3H

NHO

δH (300 MHz, CD3OD) 7.73-7.63 (m, 5H), 5.07 (t, J 8.6, 1H), 4.63-4.56 (m, 1H), 4.37-

4.29 (m, 1H), 3.90-3.84 (m, 2H), 2.87-2.77 (m, 2H), 1.93-1.84 (m, 2H), 0.96 (t, J 7.3,

3H); δC (75.5 MHz, CD3OD) 184.4, 134.6, 130.2, 130.1, 128.4, 73.3, 60.6, 54.6, 25.2,

21.8, 11.1; MS (ESI-): m/z 284.20 [M - H]-, calcd for C13H19NO4S 285.10; HPLC (system

A): tR = 12.1 min (purity 94%); λmax(recorded during the HPLC analysis)/nm 254.

4-Fluoro-1-oxo-1-phenylbutane-2-sulfonic acid

SO3H

FO

δH (300 MHz, CD3OD) 8.10 (d, J 7.3, 2H), 7.62 (t, J 7.5, 1H), 7.51 (t, J 7.2, 2H), 5.14 (dd,

J 4.1, 9.4, 1H), 4.70-4.28 (m, 2H), 2.76-2.44 (m, 2H); δC (75.5 MHz, D2O) 196.8, 139.1,

134.4, 129.6, 128.8, 72.0, 64.7, 55.1; MS (ESI-): m/z 245.20 [M - H]-, calcd for

C10H11FO4S 246.04; HPLC (system A): tR = 14.1 min (purity 94%); λmax(recorded during

the HPLC analysis)/nm 254.


S3

4-Chloro-1-oxo-1-phenylbutane-2-sulfonic acid

SO3H

ClO


J 4.5, 8.9, 1H), 3.76-3.47 (m, 2H), 2.74-2.66 (m, 1H), 2.66-2.51 (m, 1H); δC (75.5 MHz,

CD3OD) 196.5, 139.0, 134.5, 130.3, 129.6, 64.2, 43.7, 33.4; MS (ESI-): m/z 261.07 [M -

H]-, calcd for C10H11ClO4S 262.01; HPLC (system A): tR = 17.3 min (purity 93%);

λmax(recorded during the HPLC analysis)/nm 254.

4-Bromo-1-oxo-1-phenylbutane-2-sulfonic acid

SO3H

BrO


J 4.5, 8.9, 1H), 3.64-3.57 (m, 1H), 3.44-3.36 (m, 1H), 2.84-2.74 (m, 1H), 2.70-2.60 (m,

1H); δC (75.5 MHz, CD3OD) 196.1, 139.0, 134.4, 130.3, 129.5, 65.3, 33.4, 31.9; MS (ESI-

): m/z 304.93 [M - H]-, 418.80 [M + TFA - H]-, calcd for C10H11BrO4S 305.96; HPLC

(system A): tR = 17.8 min (purity 84%); λmax(recorded during the HPLC analysis)/nm 254.

4-Iodo-1-oxo-1-phenylbutane-2-sulfonic acid

SO3H

IO


J 4.5, 8.7, 1H), 3.40-3.14 (m, 2H), 2.82-2.63 (m, 2H); δC (75.5 MHz, CD3OD) 196.6,

139.0, 134.5, 130.5, 129.6, 67.3, 34.4, 3.4; MS (ESI-): m/z 352.87 [M - H]-, calcd for

C10H11IO4S 353.94; HPLC (system A): tR = 19.7 min (purity 98%); λmax(recorded during

the HPLC analysis)/nm 254.


S4

RP-HPLC elution profile (system A) of 1-phenylacyl-1,3-propanesultone 1.

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

19,4

27 0

,373

19,9

28 0

,013

22,0

38 0

,324

22,6

52 0

,071

22,9

42 0

,103

24,2

37 9

8,97

0 29,0

42 0

,145

PDA-254nmRetention TimeArea Percent

RP-HPLC elution profile (system A) of 1-benzyl-1,3-propanesultone 2.

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

mAU

-250

0

250

500

750

1000

1250

1500

1750

2000

2250

2500

mAU

-250

0

250

500

750

1000

1250

1500

1750

2000

2250

2500

20,7

47 0

,245

21,8

20 0

,336

23,5

65 9

9,41

9



S5

1H NMR spectrum of bis-propanesultone 3 recorded in acetone-d6 at 300 MHz.

13C NMR spectrum of bis-propanesultone 3 recorded in acetone-d6 at 75.5 MHz.


S6

ESI mass spectrum of bis-sultone 3 recorded in the negative mode.a

ahydration of sultone moieties was occurred during the ionisation process.

RP-HPLC elution profile (system A) of bis-sultone 3.a

Minutes0,0 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 42,5 45,0

mAU

0

200

400

600

800

1000

1200

mAU

0

200

400

600

800

1000

1200

14,5

67 1

,015

19,0

67 0

,173

20,8

53 1

,651

21,4

37 0

,093

22,5

33 0

,127

23,1

25 0

,076

24,3

67 0

,272

26,6

65 9

6,59

2


aA doublet peak was observed because compound 3 is a mixture of two racemic diastereomers.


S7

RP-HPLC elution profile (system A) of mono-fluoro-propanesultone 4.a

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

mAU

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mAU

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

17,7

10 1

,399

18,1

02 2

,282

18,3

62 2

,016

18,8

17 4

5,97

319

,065

46,

128 19

,588

1,3

9119

,773

0,8

11


aA doublet peak was observed because compound 4 is a mixture of two racemic diastereomers.

RP-HPLC elution profile (system A) of mono-fluoro-Boc-L-lysine-NH2 conjugate 5.a

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

mAU

-20

0

20

40

60

80

100

120

140

160

180

200

mAU

-20

0

20

40

60

80

100

120

140

160

180

200

14,8

87 1

,123

15,3

58 5

8,83

6

15,8

63 4

0,04

1


aA split peak was observed because this conjugate is a mixture of four diastereomers.


S8

RP-HPLC elution profile (system A) of mono-fluoro-peptide conjugate 6.a

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

11,9

23 0

,320

12,3

15 0

,001

20,0

92 6

,844

21,0

95 9

2,83

6


aThis conjugate is a mixture of four diastereomers.

RP-HPLC elution profile (system A) of mono-iodo-propanesultone.a

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

mAU

0

200

400

600

800

1000

1200

1400

mAU

0

200

400

600

800

1000

1200

1400

13,2

10 0

,678

13,5

55 6

,718

13,9

95 0

,815

14,3

80 0

,837

22,2

45 0

,674

22,8

40 4

2,36

822

,982

47,

910


aA doublet peak was observed because this compound is a mixture of two racemic diastereomers.


S9

RP-HPLC elution profile (system A) of 1-oxo-1-phenyl-4-(propylamino)butane-2-sulfonic acid.

Minutes

0 5 10 15 20 25 30 35

mA

U

0

500

1000

1500

2000

mA

U

0

500

1000

1500

2000

12,1

50 9

3,79

1

13,6

50 5

,968

14,4

67 0

,241

PDA-Scan-280 nmRetention TimeArea Percent

RP-HPLC elution profile (system A) of 4-fluoro-1-oxo-1-phenylbutane-2-sulfonic acid.

Minutes0 2 4 6 8 10 12 14 16 18 20 22

mAU

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mAU

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

12,3

72 1

,150

13,3

67 0

,714

14,1

45 9

3,41

2 15,1

33 0

,766

15,6

90 0

,161

16,2

25 0

,470

16,4

53 0

,352

16,8

60 0

,861

17,1

60 0

,579

17,8

20 0

,029

19,0

00 1

,506



S10

RP-HPLC elution profile (system A) of 4-chloro-1-oxo-1-phenylbutane-2-sulfonic acid.

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

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

14,7

07 0

,221

15,4

97 0

,152

15,7

30 0

,137

17,3

27 9

3,18

3 18,3

10 0

,039

22,7

03 5

,075

27,3

28 1

,193


RP-HPLC elution profile (system A) of 4-bromo-1-oxo-1-phenylbutane-2-sulfonic acid.

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

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

7,21

2 1

2,81

5

14,1

90 1

,472

15,0

65 0

,633

17,0

92 0

,540

17,8

48 8

4,54

0



S11

RP-HPLC elution profile (system A) of 4-iodo-1-oxo-1-phenylbutane-2-sulfonic acid.

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

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

mAU

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

8,55

5 1

,020

15,6

33 1

,289

19,6

98 9

7,69

1



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