Nicotinic Acid Adenine Dinucleotide Phosphate ...

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Nicotinic Acid Adenine Dinucleotide Phosphate Analogues. Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on ReceptorMediated Ca. 2+.
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Nicotinic Acid Adenine Dinucleotide Phosphate Analogues Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on ReceptorMediated Ca2+ Release Christopher J. Trabbic,‡ Fan Zhang,§ Timothy F. Walseth,*,§and James T. Slama,*,‡ ‡

Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, 3000 Arlington Avenue, Toledo Ohio 43614, USA §

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA Table of Contents

Figure S1. HPLC analysis of NAADP (1) and NAADP derivatives 25-35. These analyses were performed immediately before biological testing………………………………………………..S4 Figure S2. UV absorption spectra of NAADP (1) and NAADP derivatives 25-35……………..S5 Figure S3. Elemental analysis results for synthetic derivatives described in the Experimental section……………………………………………………………………………………………S6 1

H NMR spectrum of 5-(2-Phenylethynyl)-3-pyridinecarboxylic acid ethyl ester……………...S7

1

H NMR spectrum of 5-(2-Phenylethyl)-3-pyridinecarboxylic acid ethyl ester………………...S8

1

H NMR spectrum of 5-(Acetamido)nicotinic acid (2c)………………………………………...S9

1

H NMR spectrum of 5-Thiomethoxynicotinic acid (2d)………………………………………S10

1

H NMR spectrum of 5-Bromonicotinic acid ethyl ester (2e)………………………………….S11

1

H NMR spectrum of 5-(2-Phenylethyl)-3-pyridinecarboxylic acid (2f)………………………S12

1

H NMR spectrum of 3-Acetamidoprop-1-yne (3)……………………………………………..S13

1

H NMR spectrum of 5-(3-Acetamidoprop-1-yne)nicotinic acid ethyl ester (4)………………S14

1

H NMR spectrum of 5-(3-Acetamidopropyl)nicotinic acid ethyl ester (5)…...……………….S15

S1   

1

H NMR spectrum of 5-(3-Acetamidopropyl)nicotinic acid (6)…...…………………………..S16

1

H NMR spectrum of tert-Butyl-prop-2-ynylcarbamate (7)……………………………………S17

1

H NMR spectrum of 5-(t-Butyl-prop-2-ynylcarbamate)nicotinic acid ethyl ester (8)………...S18

1

H NMR spectrum of 5-(3-t-Boc-Aminopropyl)nicotinic acid ethyl ester (9)………………....S19

1

H NMR spectrum of 5-(3-Aminopropyl)nicotinic acid ethyl ester (10)………………………S20

1

H NMR spectrum of 5-(3-t-Boc-Aminopropyl)nicotinic acid (11)……………………………S21

1

H NMR spectrum of 5-(3-Aminopropyl)nicotinic acid (12)…………………….…………….S22

1

H NMR spectrum of 5-(3-Hydroxy-1-propynyl)nicotinic acid ethyl ester (13)…..….……….S23

1

H NMR spectrum of 5-(3-Hydroxypropyl)nicotinic acid ethyl ester (14)..….…………….….S24

1

H NMR spectrum of 5-(3-Hydroxypropyl)nicotinic acid (15)..….……………………..….….S25

1

H NMR spectrum of 5-[3-(p-Methylbenzenesulfonyl)propyl]nicotinic acid ethyl ester (16)....S26

1

H NMR spectrum of 5-(3-Azidopropyl)nicotinic acid ethyl ester (17)………………………..S27

1

H NMR spectrum of 5-(3-Azidopropyl)nicotinic acid (18)…………………………………...S28

1

H NMR spectrum of 8-Hydrazinoadenosine (19)……………………………………………..S29

1

H NMR spectrum of 8-Azidoadenosine (20)………………………………………………….S30

1

H NMR spectrum of 8-Azidoadenosine-5'-monophosphate (21)…………………………......S31

1

H NMR spectrum of 8-Azido-NAD (22)……………………………………………………...S32

1

H NMR spectrum of 8-Azido-NADP (23)…………………………………………………….S33

1

H NMR spectrum of 8-Bromo-NADP (24)……………………………………………………S34

1

H NMR spectrum of 5-(Acetamido)-NAADP (25)……………………………………………S35

1

H NMR spectrum of 5-Hydroxy-NAADP (26)………………………………………………..S36

1

H NMR spectrum of 5-(3-Acetamidopropyl)-NAADP (27)…………………………………..S37

1

H NMR spectrum of 5-(3-t-Boc-Aminopropyl)-NAADP (28)………………………………..S38

1

H NMR spectrum of 5-(3-Aminopropyl)-NAADP (29)………………………………………S39

1

H NMR spectrum of 5-(3-Hydroxypropyl)-NAADP (30)…………………………………….S40 S2

1

H NMR spectrum of 5-(3-Azidopropyl)-NAADP (31)………………………………………..S41

1

H NMR spectrum of 5-Thiomethyl-NAADP (32)…………………………………………….S42

1

H NMR spectrum of 8-Bromo-NAADP (33)………………………………………………….S43

1

H NMR spectrum of 8-Azido-NAADP (34)…………………………………………………..S44

1

H NMR spectrum of 8-Bromo-5-azido-NAADP (35)………………………………………...S45

S3

NAADP (1)

5-hydroxy-NAADP (26)

1.0 0.5

0

5

10

15

20

25

30

1.0

0.5

0.0

35

2.0

Absorbance, 260nm

1.5

0.0

0

5

10

Minutes

0.0

35

0.2 0.1

0

5

10

15

20

25

30

1.5 1.0 0.5 0.0

35

0

5

10

15

20

25

30

0.5

15

20

25

30

0

5

10

8-bromo-NAADP (33)

15

20

25

30

5

10

15

20

Minutes

5

10

25

30

35

15

20

25

30

35

1.0 0.5

0

5

10

20

25

30

35

5-azido-8-bromo-NAADP (35) Absorbance, 260nm

1.5

0.5

0.0

15

Minutes

8-azido-NAADP (34) Absorbance, 260nm

0

0

1.5

0.0

35

1.0

0.5

35

0.5

Minutes

1.0

30

5-(3-acetamidopropyl)-NAADP (27)

1

Minutes

1.5

25

2.0

2

0

35

20

Time (Minutes)

Absorbance, 260nm

1.0

15

1.0

0.0

35

5-aminopropyl-NAADP (29) Absorbance, 260nm

1.5

10

10

5-propylazido-NAADP (31)

3

5

5

Minutes

5-(3-t-Boc-aminopropyl)-NAADP (28)

0

0

Minutes

Absorbance, 260nm

Absorbance, 260nm

Absorbance, 260nm

0.3

2.0

Absorbance, 260nm

30

0.5

1.5

Minutes

Absorbance, 260nm

25

2.0

0.4

0.0

20

1.0

5-(3-hydroxypropyl)-NAADP (30)

5-thiomethyl-NAADP (32)

0.0

15

1.5

Minutes

0.5

0.0

5-acetamido-NAADP (25)

1.5

Absorbance, 260nm

Absorbance, 260nm

2.0

0

5

10

15

20

Minutes

25

30

35

1.0

0.5

0.0

0

5

10

15

20

25

30

35

Minutes

Figure S1. HPLC analysis of NAADP (1), and NAADP Derivatives 25-35 using an analytical column of AG MP-1 anion exchange resin (Bio-Rad, Hercules, CA) developed using gradients formed between water (solvent A) and 150 mM aqueous trifluoroacetic acid (solvent B) at a flow rate of 1 mL/min. The chromatography was described by Alexson et al., Anal. Biochem. 1981 116, 357-360. The above chromatograms were generated immediately before biological evaluation.

5-hydroxy-NAADP (26)

1.5

1.5

1.0 0.5 0.0 220 240 260 280 300 320 340 360 380 400 420

2.0

1.0 0.5

5-thiomethyl-NAADP (32)

0.0 220 240 260 280 300 320 340 360 380 400 420

Wavelength (nm)

5-(3-hydroxypropyl)-NAADP (30)

5-propylazido-NAADP (31)

2.0

0.2 0.0 220 240 260 280 300 320 340 360 380 400 420

1.5

1.5

Absorbance

Absorbance

0.8

0.4

1.0 0.5

5-(3-t-Boc-aminopropyl)-NAADP (28)

Wavelength (nm)

5-(3-acetamidopropyl)-NAADP (27)

5-aminopropyl-NAADP (29) 1.5

2.0

Absorbance

Absorbance

Absorbance

2.0 1.0

0.5

0.5 0.0 220 240 260 280 300 320 340 360 380 400 420

0.0 220 240 260 280 300 320 340 360 380 400 420

8-bromo-NAADP (33)

Wavelength (nm)

5-azido-8-bromo-NAADP (35)

1.0

2.0

8-azido-NAADP (34) 8-azido-NAADP (34) + UV

0.5

0.0 220 240 260 280 300 320 340 360 380 400 420

Wavelength (nm)

Absorbance

Absorbance

Absorbance

Wavelength (nm)

0.5 0.0 220 240 260 280 300 320 340 360 380 400 420

1.5

0.0 220 240 260 280 300 320 340 360 380 400 420

1.0

8-azido-NAADP (34)

1.5

0.5

1.5

Wavelength (nm)

Wavelength (nm)

1.0

0.5

Wavelength (nm)

2.5

1.0

1.0

0.0 220 240 260 280 300 320 340 360 380 400 420

0.0 220 240 260 280 300 320 340 360 380 400 420

Wavelength (nm)

1.5

1.0

wavelength nm

1.0

0.6

1.5

0.5

0.0 220 240 260 280 300 320 340 360 380 400 420

Wavelength (nm)

Absorbance

5-acetamido-NAADP (25) 2.5

Absorbance

2.0

Absorbance

Absorbance

NAADP (1) 2.0

1.5

5-azido-8-bromo-NAADP (35) 5-azido-8-bromo-NAADP (35) + UV

1.0 0.5 0.0 220 240 260 280 300 320 340 360 380 400 420

Wavelength (nm)

Figure S2. Ultra violet absorption spectra of NAADP 1 and NAADP analogs 25-35, obtained during HPLC of purified dinucleotides using a diode array detector under the conditions described in Figure S1.

Compound

Formula C16H13NO2

C (calcd) 76.48

H (calcd) 5.21

N (calcd) 5.57

C (found) 76.21

H (found) 5.41

N (found) 5.54

5-(2-Phenethynyl)nicotinic acid ethyl ester 5-(2-Phenethyl)nicotinic acid ethyl ester

C16H17NO2

75.27

6.71

5.49

75.48

6.80

5.40

2f 4 8

C14H13NO2 C13H14N2O3 C16H20N2O4

73.99 63.40 61.81

5.77 5.73 6.51

6.16 11.38 8.95

74.04 63.57 61.83

5.99 5.80 6.42

6.03 11.20 8.60

53.95

6.56

8.68

53.63

6.36

8.85

64.38

5.40

6.83

64.43

5.28

6.78

·0.1CH2Cl2 11

C14H20N2O4

·0.5CH2Cl2 13

C11H11NO3

Figure S3. Elemental analysis results for synthetic derivatives described in the Experimental section.

5-phenthynylnicotinicacidethylester

12

1

1

/home/ctrabbi/avance600

10

8

6

4

2

0

[ppm]

5-phenethylnicotinicacidethylesterH2

12

1

1

/home/ctrabbi/avance600

10

8

6

4

2

0

[ppm]

"5phenethylnicotinic acid"

15

1

1

/home/ctrabbi/avance600

10

5

0

[ppm]

8-hydrazinoadenosine

12

1

1

/home/ctrabbi/avance600

10

8

6

4

2

0

[ppm]

8-azidoadenosine

12

1

1

/home/ctrabbi/avance600

10

8

6

4

2

0

[ppm]

8azidoAMP

12

1

1

/home/ctrabbi/avance600

10

8

6

4

2

0

[ppm]

8-azidoNADP

1

15

1

/home/ctrabbi/avance600

10

5

0

[ppm]

2

1

/home/yjin2/avance600

[rel]

8azidoNAADP

0

5

10

15

20

NAADP 1mM 10% D2O sample

12

10

8

6

4

2

0

- 2

[ppm]

8Bromo5AzidoNAADP

12

1

1

/home/ctrabbi/avance600

10

8

6

4

2

0

[ppm]