<<

A Thesis

Entitled

The Attempted Synthesis of Carba-Nicotinic Acid Mononucleotide

Methyl Ester using the Zincke Reaction

By

Peng Zhao

Submitted to the Graduate Faculty as partial fulfillment of the requirement for

The Master of Science Degree in Medicinal Chemistry

James T. Slama, Ph.D., Committee Chair

L.M. Viranga Tillekeratne, D.Phil., Committee Member

Isaac T. Schiefer, Ph.D., Committee Member

Amanda Bryant-Friedrich, Ph.D. rer. Nat., Dean College of Graduate Studies

The University of Toledo

May 2017 Copyright 2017, Peng Zhao

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An abstract of

The Attempted Synthesis of Carba-Nicotinic-Acid Mononucleotide Methyl Ester

using the Zincke Reation

Peng Zhao

Submitted to the Graduate Faculty as partial fulfillment of the requirement for

The Master of Science Degree in Medicinal Chemistry

The University of Toledo

May 2017

According to the retrosynthetic analysis, the carba-nicotinic acid mononucleotide methyl ester can be synthesized from a nucleoside made using a primary and a

Zincke salt by the Zincke reaction. The primary amine which was the cyclopentane analog of 1-aminoribose was synthesized following the procedure described by

Cermak and Vince in 1981 starting from the Vince lactam. The pyridinium part was synthesized as a Zincke salt. However carba-nicotinic acid mononucleotide methyl ester cannot be synthesized using this procedure due to the unanticipated high reactivity of the nicotinic acid esters with primary amine during the Zincke reaction.

We found that pyridinium-3-carboxylic esters reacted rapidly with resulting in the formation of amids. A series of Zincke reaction were performed to study the

Zincke reaction. Even the most sterically hindered protecting group tert-butyl ester could not prevent the formation of nicotinamide.

iii Acknowledgments

I am deeply grateful to my venerable advisor Dr. James T. Slama, for his valuable academic guidance, continued encouragement and considerate understanding. Without his enlightening instruction, impressive kindness and patience, I could not have completed my researches and my degree. His knowledge and guidance enlightens me not only in this period of study but also in my future study and my future life. I also would like to thank him for his help and support in many other ways during my study at Toledo.

I would also like to thank all my teachers and all professors in the College of

Pharmacy and Pharmaceutical Sciences who have helped me to develop the fundamental and essential academic competence.

Last but not least, I'd like to thank my families and all my friends for their encouragement and support.

Peng Zhao

The University of Toledo

May 2017

iv Table of Contents

Abstract …………………….………………………………………………………...iii

Acknowledgments.………………………………………………………………….iv

Table of Contents……………………………………………………………………v

List of Figures .....……………….……………………………………………….vi

List of Abbreviations………………….…………………………………………….vii

I. Introduction….……………..…………….………………………………………….1

II. Results and discussion………………….…………………………………………10

III. Experimental section……………..………………………………………………16

References…………………..………..……………………………………………29

Appendices: 1H-NMR and 13C-NMR spectra of compounds in Experimental

Section………………………………………………………………………...…...…32

v List of Figures

Figure 1. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP).…...…………..1

Figure 2. Nicotinamide Adenine Dinucleotide Phosphate (NADP)…..……...…...…..2

Figure 3. Synthesis of NAADP analogs using the enzyme catalyzed

base-exchange reaction…………….………….……….….…...……….…..5

Figure 4. Preparation of NAADP and NAADP derivatives using NAD kinase and

cyclic ADP-ribose synthetase……………………………….…...... ……....6

Figure 5. Carba-Nicotinc Acid Adenine Dinucleotide Phosphate (NAADP)…...... …..6

Figure 6. Retrosynthetic analysis of carba-nicotinic acid mononucleotide methyl

ester...... 7

Figure 7. Zincke reaction……...………………………...... …..….……..……..8

Figure 8. Propose synthesis of 6 from the Vince lactam (8)...... 8

Figure 9. Propose thesis of 4 by Zincke reaction followed by a selective

phosphorylation………………………………………………….…...…….9

Figure 10. Zincke salt synthesis……...…………………………………...………….11

Figure 11. Zincke reaction using reagents 6 and 7…………………….….…...…….12

Figure 12. Zincke reaction to prove the activity of nicotinate……….……..………..13

Figure 13. Total synthesis of 17…………...………………………….….…………..16

Figure 14. The study of the Zincke reaction………………………..………………..17

vi List of Abbreviations cADPR…………Cyclic adenosine diphosphate ribose DI water………..Deionized water DMAP………….Dimethylamino pyridine DMF……………N,N-dimethylformamide DMP……………2,2-Dimethoxypropane DMSO………….Dimethyl sulfoxide ER……………...Endoplasmic reticulum IP3……………...Inositol 1,4,5-trisphosphate NAADP………...Nicotinic acid adenine dinucleotide phosphate NADP…………..Nicotinamide adenine dinucleotide phosphate NMN……………Nicotinamide mononucleotide NMO……………N-Methylmorpholine N-oxide OsO4……………Osmium tetroxide PTSA…………...p-Toluene sulfonic acid monohydrate SR………………Sarcoplasmic reticulum TFA…………….Trifuoroacetic acid THF…………….Tetrahydrofuran TLC…………….Thin-layer chromatography

vii Chapter One

Introduction

Nicotinic acid adenine dinucleotide phosphate (NAADP, 1) (Figure 1) is an intracellular second messenger for calcium ion release. NAADP was first identified as a compound that has a potent intracellular calcium ion mobilizing effect in sea urchin eggs (Lee & Aarhus, 1995), and later NAADP was shown to have the same effect in mammals (Cancela et al., 1999).

3 nicotinic acid mononucleotide half

adenosine-2′,5′-diphosphate half

2′

1

Figure 1. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP, 1) There are three major calcium ion mobilizing messengers (Bootman, et al., 2002).

They are inositol 1,4,5-trisphosphate (IP3), cyclic adenosine diphosphate ribose

(cADPR) and NAADP. IP3 was discovered 1983 and it was shown to be capable of releasing calcium ion from non-mitochondrial stores in pancreatic acinar cells (Streb, et al., 1983). IP3 mediates calcium ion release by binding to IP3 receptor on a ligand-gated calcium channel on endoplasmic reticulum (ER) which releases calcium 1 ion into the cytoplasm (Barrett, et al., 2009). Studies in sea urchin eggs showed that the pyridine nucleotides, NAD and NADP (2) could mediate calcium ion release from

Figure 2. Nicotinamide Adenine Dinucleotide Phosphate (NADP, 2) membranous stores which are different from IP3 (Clapper, et al., 1987). Further studies showed that the effect was caused by the NAD metabolite: cyclic adenosine diphosphate ribose (cADPR) (Lee, et al., 1989) and the NADP metabolite nicotinic acid dinucleotide phosphate (NAADP) (Lee & Aarhus, 1995). IP3 and cADPR were shown to act on the two know ER calcium ion release channels, IP3Rs (IP3 receptors) and RyRs (ryanodine receptors) (Galione, et al., 1991) respectively.

NAADP was initially isolated and identified as a contaminant in NADP, and was shown to release calcium ion from intracellular vesicles from sea urchin eggs

(Clapper, et al., 1987). The difference between NAADP and NADP is that the nicotinamide base of the NADP is replaced by nicotinic acid in NAADP. This slight difference in molecular structure is essential for calcium ion releasing activity and

2 produces a high degree of discrimination in biological activities. NAADP was found to release calcium ion by a pharmacologically distinct mechanism from different subcellular fractions of egg homogenate, but the precise target for NAADP is unknown. Among these three major calcium mobilizing messengers, NAADP is the most potent, active at picomolar or low-nanomolar concentrations (Lee, 1997), and releasing calcium ion from reserve granules in the sea urchin egg and from lysosome like acidic vesicles in mammalian cells. NAADP mobilizes calcium ion from acidic stores. NAADP induces alkalinization of acidic stores in sea urchin eggs (Morgan &

Galione, 2007). The concentration response relationship for NAADP mediated calcium ion release from mammalian cells is a bell shaped (Pitt & Funnell, 2010), indicating that the receptor might possess high affinity stimulatory and low affinity inhibitory binding sites. NAADP could therefore inactivate its response at a high concentrations.

The carboxyl group at 3-position of the pyridine ring, the amino group of the adenine ring and the 2′-phosphate are all important for the biological activity of

NAADP (Lee, 1997). The first important site is the 2′-phosphate. Lee (1997) claimed that the removal of the 2′-phosphate resulted in the loss of agonist activity of NAADP.

Attaching a caging group to the 2′-phosphate similarly produces an inactive analog that can regenerate NAADP on irradiation with ultra violet light and cause a large calcium ion concentration change in many cells which provides additional evidence that the NAADP is a messenger for calcium ion mobilization. Therefore, the

2′-phosphate is essential for receptor recognition. However, the 2′-phosphate could be modified without completely losing its activity. If the 2′-phosphate was changed to

3′-phosphate or cyclically linked to both positions as a 2′,3′-cyclic phosphate the molecule will retain activity with a lower potency (Lee and Aarhus, 1997). The

3 second important site is the 3-carboxylate on pyridine ring. Billington et al. (2005) suggested that the high selectivity of the NAADP binding site for NAADP is based upon an interaction between the protein and a negative charge at the 3-position of the pyridine ring. The protein could tolerant variations in the size of the charged group but it has to be a monovalent anion. The carboxylate has to be directly linked to the pyridine ring. Even adding one carbon in between, the NAADP derivative will significantly reduce its potency relative to parent NAADP. Lee and Aarhus (1997) stated that if the carboxylate group was moved to the 4-position, the analog was found to be completely inactive. Introduction of a substituent at the 4-position of the nicotinic acid was shown to result in the loss of agonist potency (Jain et al., 2010).

However, substitution at the 5-position on the pyridine ring was tolerated and resulted in the production of agonists with high potency (Trabbic, et al., 2012). Therefore, the

5-position on pyridine ring was shown to be suitable for future modification. The third important site is the amino group at the 6-position on the adenine ring. Modification of the 6-amino group was found to reduce the potency. For example, Lee and Aarhus

(1997) performed an experiment which replaced the 6-amino group of adenosine with a –OH group. The effectiveness of NAADP derivative was reduced more than

1000-fold. Conversion of the 6-amino group to an oxygen resulted in production of a compound 1000-fold less potent and much less effective compound compare to the

NAADP (Lee and Aarhus, 1997). However, the 8-position on adenine ring can be modified without loss of potency. Trabbic et al. (2015) found that if the –H on

8-position of adenine ring was changed to an azido or bromo group, the NAADP analogues did not lose much potency and the EC50 was increased only slightly. So in order to retain the potency of NAADP analogues, the 2′-phosphate, the pyridinium-3-carboxylate, and the 6-amino group on adenine ring should not be

4 modified, but the 5-position on nicotinic acid ring and 8-position on adenine ring can be modified to produce agonists that bind with a low disassociation constant.

NAADP can be synthesized enzymatically by using NAD glycohydrolase (CD38) or Aplysia california cADPR synthetase through a base-exchange reaction from

NADP. The co-product will be nicotinamide which came from NADP (Zatman et al.,

1953) (Figure 3).

NADP and NADP analogues can be synthesized by an enzymatic phosphorylation of an NAD or an NAD analog using NAD kinase. Then NAADP and

NAADP derivatives can be synthesized by pyridine base exchange from NADP or

NADP analogues (Figure 4).

Figure 3. Synthesis of NAADP analogs using the enzyme catalyzed pyridine base-exchange reaction.

5 Figure 4. Preparation of NAADP and NAADP derivatives using NAD kinase and cyclic ADP-ribose synthetase.

The goal of this study was the synthesis of carba-NAADP (3). However due to the results obtained in the course of the study showed that we would not be able to

3 Figure 5. Carba-Nicotinc Acid Adenine Dinucleotide Phosphate (NAADP, 3) synthesize carba-nicotinic acid mononucleotide methyl ester (4) by our proposed route. A retrosynthetic analysis of the carba-NAADP suggests that 3 can be made by coupling a nicotinic acid mononucleotide half with an adenosine-2′,5′-diphosphate half (Figure 1). The adenosine-2′,5′-diphosphate half is commercially available, but it is not an ideal intermediate for our proposed synthesis because it could couple with 6 NMN in two ways. However, a recent study of the synthesis of NADP produced an

adenosine 2′,5′-bisphosphate in which the 2′-phosphate was fully protected (Dowden

et al. 2004). If we can get carba-nicotinic acid mononucleotide methyl ester (4), the

nicotinic acid mononucleotide half could be coupled to the

adenosine-2′,5′-diphosphate half to produce NAADP and NAADP analogues.

4 5

8 6 7

Figure 6. Retrosynthetic analysis of carba-nicotinic acid mononucleotide 5 methyl ester (4) The goal of this study was the synthesis of the carba-nicotinic acid

mononucleotide methyl ester (4). A retrosynthetic analysis of 4 is shown in Figure 6.

Nucleoside analog 5 when treated with phosphoryl chloride and trimethyl phosphate

will be selectively phosphorylated on the 5′-position of the ribose analog to afford 4

(Yoshikawa et al., 1969). Next we proposed to use the Zincke reaction in which we

treat 6 with 7 to afford compound 5 (Zincke et al., 1904). The Zincke reaction is a

well-known reaction in which a pyridine is transformed into a pyridinium salt by

7 reaction with 2,4-dinitro-chlorobenzene and then reacted with a primary amine which will replace the 2,4-dinitroaniline (Zincke, et al., 1904) (Kunuqi, et al., 1976) (Figure

7).

Figure 7. Zincke reaction

8 9 10

or

11 12

6

Figure 8. Propose synthesis of 6 from the Vince lactam (8)

8 Synthesis of 6 starting from the Vince lactam (8) has been described by Cermak and Vince in 1981. We followed Cermak and Vince’s published procedure to synthesize compound 6 from the Vince lactam 8 (Figure 8). First, the Vince lactam was hydroxylated using osmium tetroxide (OsO4) to introduce two hydroxyl groups.

According to Figure 8 compound 9, the two cis-hydroxyl groups were introduced with exo-stereochemistry. The olefin starting materials were not attacked from the endo-position because that position is sterically more-hindered (Kam and

Oppenheimer, 1981). Second, the two cis-hydroxyl groups were protected by acetonide to afford 10, and the amine was protected by the tert-butyloxycarbonyl protecting group to afford 11. Third, the lactam was reductively opened to afford 12 which was performed according to the conditions described by Barbier, et al., 1996.

Forth, we removed the protecting groups to get a primary amine 6.

Finally, we proposed to use a new variation on the Zincke reaction (Figure 9) to convert 6 to 5 which can be used to make 4 by selective phosphorylation on the

5′-position.

6 4 5 Figure 9. Proposed synthesis of 4 by Zincke reaction followed by a selective phosphorylation

9 Chapter Two

Results and Discussion

Carba-nicotinic acid mononucleotide methyl ester is an analogue of nicotinic acid mononucleotide, and therefore it will be used as an intermediate in the synthesis of carba-NAADP. Carba-NAADP is an analogue of NAADP. The difference between carba-nicotinic acid mononucleotide and nicotinic acid mononucleotide or carba-NAADP and NAADP is the resistance to cleavage of the pyridinium-sugar bond. This resistance can provide a long half-life of the compound in cells. The ribofuranose oxygen on the ribose sugar was modified to a methylene group. Due to the small modification of the sugar ring, the carba-nicotinic acid mononucleotide will likely be recognized by the nicotinic acid mononucleotide specific binding site. Thus we could synthesize a more stable NAADP analog. The carba-nicotinic acid mononucleotide might also be active as an inhibitor of enzymes that synthesize or degrade NAADP due to its resistance to enzymatic cleavage and specific binding activity. According to the observation made during this study, the synthesis from 8 to

6 can be duplicated. However, when removing the acetonide protecting group (from

12 to 6 Figure 8), the tert-butyloxycarbonyl protecting group was removed with the acetonide protecting group. The Zinke reactions did not proceed as we proposed. The carboxylic group ester on nicotinate was very active. It discovered that it would react rapidly with the primary amine even when the ester was protected as a tert-butyl ester.

Therefore, this study cannot be finished due to one of the most important chain reactions cannot be performed.

Synthesis of the carbocyclic 1-aminoribofuranoce analog 6:

First, we started with the Vince lactam 8, and using osmium tetroxide introduced the two hydroxyl groups in the exo-position. The product was purified by column

10 chromatography to afford the yellow solid 9 in 54.9% yield. Second, the acetonide protecting group was introduced to the two hydroxyl groups by using

2,2-dimethoxypropane and p-toluenesulfonic acid. The compound was purified by recrystallized from hexane-ethyl acetate to afford 10 in 78.6% yield. Third, we introduced the t-boc protecting group to the amine on the lactam ring using di-tert-butyl dicarbonate to afford yellow solid 11 in 90.6% yield. Fourth, sodium borohydride was used to open the lactam ring to afford 12 in 76% yield. Last, the protecting groups were removed by using concentrated aqueous HCl to afford 6 in

76% yield. Our result in the deprotection was different from the result reported in the literature, in which they removed each protecting group individually. However, the two protecting group always were removed at the same time during my study. The

1H-NMR of compound 6 agreed closely with the data reported in the literature. In addition, the 1H-NMRs of 9, 10, 11, 12, and 6 were the same as that described in the literature. Thus, we could conclude that compound 6 can be synthesized.

Zincke salt synthesis:

13 14

15

11 15 7

Figure 10. Zincke salt synthesis The nicotinic acid was reacted with methanol and hydrochloride to afford 13. The methyl ester Zincke salt 15 was synthesized from methyl nicotinate 13 and

1-chloro-2,4-dinitrobenzene 14 and purified by extraction to afford yellow sticky oily product 15 in 53.5% yield. The chloride anion of 15 was changed to the tetrafluoroborate anion producing the yellow solid 7 which was easily purified by crystallization. The tetrafluoroborate salt is not hygroscopic and it is soluble in acetonitrile and methanol.

6 7 17

Figure 11. Zincke reaction between reagents 6 and 7

The reaction of compounds 6 and 7 was proposed to result in the synthesis of 5.

Compound 6 was dissolved in acetonitrile and a solution of 7 and triethylamine in acetonitrile was added in dropwise. After the addition was complete, the solution was 12 stirred for 2 hours. During addition the solution turns dark purple, and then orange.

The reaction mixture was pound onto 50 mL of DI water, and the water washed 3 times with 15 mL portions of methylene chloride to remove 2,4-dinitroaniline and unreacted 6. The aqueous solution was evaporated to afford a sticky oily product in

55% yield. The TLC indicated that there are two spots, one major spot and one minor spot. These two had very similar Rfs. These two were separated by preparative thin layer chromatography. The minor spot was not isolated because the amount was too small. The 1H-NMR of the major spot indicated that it was a pure compound containing two cyclopentane rings derived from 6. The nicotinate methyl ester must therefore have reacted with 6 which is a primary amine. To prove the nicotinate is more reactive than proposed, the following reactions shown in Figure 12 were performed.

18

23

19

Methyl ester 7 R1 = -H R2 = -CH3

Ethyl ester 20 R1 = -H R2 = -CH2-CH3

Isopropyl ester 21 R1 = -OH R2 = -CH-(CH3)2

tert-Butyl ester 22 R1 = -H R2 = -C-(CH3)3

Figure 12. Zincke reaction to prove the activity of nicotinate 13 To better understand the reactivity of Zincke esters, we studied the reaction of simple branched amines (isopropyl amine 18, R1 = H and alaninol 18, R1 = OH) with Zincke reagents containing different ester substituents (20-22). Zincke reagents 20-22 were synthesized from the nicotinic acid esters and 1-chloro-2,4-dinitrobenzene following the procedure we described for 7. The reactions between the amines 18 and the Zincke reagents 19 were performed using 1:1, 1:1.5 and 1:2 ratio of starting materials. We found that the ratio did not change the product distribution. It only influenced the percent yield. Only 23 was isolated. Even using 1:1 ratio of the reagents, the primary amine still reacted with carboxylic ester group resulting in amide formation.

Discussion:

During this study, the method of synthesis 6 was reproduced and duplicated and hundred milligram quantities of 6 were produced. However, due to the unanticipated high reactivity of the nicotinic acid esters and primary amine, the reaction between 6 and 7 did not resulted in the formation of the predicted products. Because of this unanticipated result, carba-nicotinic acid mononucleotide (4) could not be synthesized using our proposed method. Even the most sterically hindered protecting group tert-butyl ester could not prevent this reaction from happening.

Carba-NAADP is still desired, since it should be an NAADP analog which is predicted to be a high-potency agonist and yet be to resistant to the cleavage of the bond between the sugar and pyridinium ring due to the substitution of the bridging ribofuranose oxygen by a methylene group. If the 2′-phosphate was similarly modified to an analog which was resistant to phosphatase cleavage, the result would be a carba-NAADP which would be resistant to the metabolism. Such a compound would be expected to have interesting and informative pharmacological properties and

14 would therefore enhance our knowledge of NAADP and NAADP mediated Ca2+ signaling.

15 Chapter Three

Experimental Section

or

Figure 13. Total synthesis of 17

16 17 18 +

+

Figure 14. The study of the Zincke reaction

General Procedure

TLC was performed using silica gel as stationary phase. The TLC plates we used

19 were silica gel 60 containing fluorescent indicator on aluminum sheets which bought from Analtech, Inc.. Compounds were visualized by UV absorption or using potassium permanganate stain. Potassium permanganate stain was made of 1.5 g of

KMnO4, 10 g K2CO3, and 1.25 mL 10 % NaOH in 200 mL water. This visualizes compounds which are sensitive to oxidation. After development, the TLC plate, immersed in the staining solution and heated, the spots appeared as yellow or light brown on a light purple or pink background.

The purification was performed using a flash chromatography apparatus called

Combiflash Purification Systems which was made by Teledyne Isco. The column was packed using silica gel 60. The purification was performed at room temperature.

All of the NMR spectra were acquired at a magnetic field of 9.39798 Tesla (400

MHz for 1H and 100 MHz for 13C) or at 14.0954 Tesla (600 MHz for 1H and 151 MHz for 13C) on Varian/Inova instruments, or using a Brucker Avance 600 MHz instrument equipped with a Cryoprobe.

Combustion analysis was performed by Atlantic Microlab, Norcross, Georgia.

High-resolution mass spectrometric (HRMS) data was acquired using Waters

Synapt high definition mass spectrometer (HDMS) equipped with nano-ESI source.

Ionization mode is nano-ESI, capillary voltage is 3 kV, flow rate is 500 nL/min, and acquisition range is m/z 180-600.

2,3-Dihydroxy-4-hydroxymethyl-1-aminocyclopentane hydrochloride (6):

Compound 12 (50 mg, 0.17 mmol) was added to 4 mL of DI water, and 1 mL of 1 M aq HCl was added to the solution. The mixture was stirred for 2 hrs. The reaction was monitored using TLC (silica gel; hexane-ethyl acetate 1:1). The was removed in vacuo. Portions of acetonitrile (3X10 mL) were added and evaporated, to afford an

1 amorphous solid 23.8 mg (0.13 mmol 76%). H-NMR (600 MHz; D2O): δ1.29 (dt, 9.5 20 Hz and 13.2, 1 H), 2.11-2.15 (m, 1 H), 2.29 (dt, 8.4 Hz and 13.2 Hz, 1H), 3.50 (q, 8.4

Hz, 1 H), 3.53-3.60 (m, 2 H), 3.87 (dd, 4.2 Hz and 5.4 Hz, 1 H), 3.94 (dd, 7.8 Hz and

6 Hz, 1 H).

1-(2,4-Dinitro)-phenyl nicotinic acid methyl ester tetrafluoroborate (7):

Nicotinic acid methyl ester (13) (1.74 g, 12.7 mmol) and 1-chloro-2,4-dinitrobenzene

(14) (1 g, 6.25 mmol) were added to a round bottom flask with a stirring bar and a condenser. Methanol (15 mL) was added, and the resulting solution heated at reflux

(75 ℃) for 48 hrs. The progress of the reaction was monitored using TLC (silica gel, hexane-ethyl acetate 1:1) and the formation of a new substance beside the starting material was observed. The color of solution turned red. The solvent was removed in vacuo, and the residue was partitioned between 50 mL of chloroform and 50 mL of DI water. Then the organic layer was washed 2 times with 25 mL of DI water. The water layer was collected and evaporated in vacuo, to afford sticky yellow oily product. This material was dissolved in 10 mL of DI water and a solution of 1 g of sodium tetrafluoroborate in 10 mL of DI water was added. A precipitate consisting of the tetrafluoroborate salt (7) was formed. The white solid was collected by filtration, washed with cold water and dried: 1.33 g (3.4, mmol 53.5%) Rf is 0.354 (silica gel; hexane-ethyl acetate 1:1); mp 197-200 ℃. Anal. calcd. for C13H9BF4N3O6: C, 40.03;

1 H, 2.33; N, 10.77. Found: C, 39.92; H, 2.58; N, 10.75. H-NMR (400 MHz; D2O): δ

4.03 (s, 3 H), 8.21 (d, 8.8 Hz, 1 H), 8.47 (t, 14.4 Hz, 1 H), 8.91 (dd, 2.4 Hz, and 8.8

Hz, 1 H), 9.34-9.39 (m, 3 H), 9.79 (s, 1H).

(1R)-(-)-2-azabicyclo[2.2.1]hept-5-en-3one (Vince lactam, 8) The starting compound Vince lactam (8) was purchased from Carbosynth Limited (Berkshire, UK).

1 it is structure was verified by NMR. H-NMR (400 MHz; D2O): δ 2.22 (d, 5.2 Hz, 1

H), 2.35 (d, 5.2 Hz, 1 H), 3.24 (s, 1 H), 4.43 (s, 1 H), 6.71-6.73 (m, 1 H), 6.92 (dd, 1.6 21 Hz and 3.6 Hz, 1H).

5,6-Exo-dihydroxy-2-azabicyclo[2.2.1]heptan-3-one (9): The Vince lactam

(8) 1.25 g (11.45 mmol) was dissolved in 10 mL of acetone. The solution was heated while adding a solution of osmium tetroxide (11.75 mL of a 1 % w/v solution in tert-butyl alcohol) in a single portion by pipet. N-Methylmorpholine N-oxide 1.475 g

(12.6 mmol) was dissolved in DI water (10 mL) and the solution was added dropwise to the refluxing mixture. The reflux was continued for 1.5 hrs at 87 ℃. The reaction was monitored using TLC (silica gel; isopropanol-ethyl acetate 3:7) using potassium permanganate stain. The result showed that the starting material was consumed and the product was formed. Heating was stopped, sodium dithionite (0.94 g 5.38 mmol) was added and the mixture was stirred for 45 min while it cooled to room temperature.

The solvent was removed in vacuo at 40 ℃. The residue was absorbed on silica gel, and the product was purified by chromatography. Column chromatography was performed using a 12 gram flash column cartridge. The mobile phase was 30% isopropanol in ethyl acetate. All the eluate was collected and the solvent removed in vacuo. The product was light yellow solid weighing 10.9 g (6.29 mmol) 54.9 %: Rf

(isopropanol-ethyl acetate 3:7) 0.27; mp 165-168 ℃ (lit. Kam and Oppenheimer

1 1981 reported 169-170 ℃). H-NMR (600 MHz; D2O): 2.04 (br s, 2 H), 2.59 (m, 1

H), 3.75 (m, 1 H), 3.99 (d, 6 Hz, 1 H), 4.03 (d, 5.4 Hz, 1 H). 1H-NMR (600 MHz;

DMSO): δ1.71 (d, 9.6 Hz, 1H), 1.89 (d, 9.6 Hz, 1 H), 2.25 (s, 1 H), 3.42 (br s, 1 H),

3.69 (t, 5.4 Hz, 1 H), 3.76 (t, 5.4 Hz, 1 H), 4.97 (d, 5.4 Hz, 1 H), 5.03 (d, 4.8 Hz, 1 H),

7.55 (br s, 1 H).13C-NMR (600 MHz, DMSO): δ 35.57, 51.18, 58.65, 67.67, 71.02,

181.36.

(-)-5,6-Exo-dimethylmethylenedioxy-2-azabicyclo[2.2.1]heptan-3-one

22 (10): A solution of compound 9 (1.43 g, 10 mmol), 2,2-dimethoxypropane (2 mL,

1.694 g, 16.27 mmol) and p-toluenesulfonic acid monohydrate (95 mg, 0.5 mmol) in acetone (5 mL) was allowed to stand and stir at room temperature for 3.5 hrs. The reaction was monitored by TLC (silica gel; isopropanol-ethyl acetate 3:7) and the starting materials were observed to be consumed after 3 hrs. Sodium bicarbonate (42 mg, 0.5 mmol) was added to the mixture, and the solvent was removed in vacuo. The residue was extracted into chloroform, and filtered. The filtrate was concentrated in vacuo to afford a crystalline residue weighing 1.44 g (78.6 %, 7.86 mmol). The product was recrystallized from hexane-ethyl acetate to give acetonide (3): Rf 0.35;

1 mp 165-168 ℃. H-NMR (400 MHz; CDCl3): δ 1.36 (s, 3 H), 1.48 (s, 3 H), 2.05-2.07

(m, 1 H), 2.13-2.15 (m, 1 H), 2.74 (s, 1 H), 3.80 (s, 1 H), 4.42 (d, 5.4 Hz, 1 H), 4.55 (d,

5.4 Hz, 1 H), 6.09 (br s, 1 H).

(-)-t-Boc-4-amino-5,6-exo-dimethylmethylenedioxy-2-azabicyclo[2.2.1]heptan

-3-one (11): Compound 10 (0.5 g, 2.73 mmol) and dimethylamino pyridine (DMAP)

(24.4 mg, 0.2 mmol) were dissolved in 5 mL of THF. The mixture was heated under reflux at 76 ℃ while a solution of di-tert-butyl dicarbonate (0.774 g, 2.54 mmol) in

5 mL of THF was added dropwise from an addition funnel over 10 min, and then the mixture was allowed to reflux overnight. The mixture was concentrated in vacuo, and the residue was dissolved in chloroform. The solvent was removed in vacuo. The residue was partitioned between 10 mL of DI water and 30 mL of ethyl acetate. The ethyl acetate layer was washed with 25 mL of 0.5 M sodium bicarbonate twice, then washed with 25 mL of brine (saturated sodium chloride). The solvent was removed in vacuo to afford a light yellow solid weighing 0.7 g (2.47mmol, 90.6%): Rf 0.27 (silica

1 gel; hexane-ethyl acetate 1:1). H-NMR (600 MHz; CDCl3): δ1.36 (s, 3 H), 1.49 (s, 3

H), 1.51 (s, 9 H), 1.98-2.01 (m, 1 H), 2.08-2.11 (m, 1 H), 2.89 (br s, 1 H), 4.43 (br s, 1 23 H), 4.48 (d, 4.2 Hz, 1 H), 4.60 (d, 4.2 Hz, 1 H).

[Tetrahydro-6-(hydroxymethyl)-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-yl]-,

1,1-dimethylethyl ester (12): Compound 11 (100 mg, 0.35 mmol) was added to 15 mL of methanol and the mixture was warmed to 35 ℃ using an oil bath for 5 min to completely form a solution. Sodium borohydride (530 mg, 14 mmol) was added to the solution in portions, and stirring was continued for 2.5 hrs at room temperature. The reaction was monitored using TLC (silica gel; hexane-ethyl acetate 1:1) using the potassium permanganate stain. When the starting material 11 was consumed, the mixture was neutralized by adding a 1:1 mixture of acetic acid and methanol. The solvent was removed in vacuo, and the residue was dissolved in chloroform and filtered. The solvent was evaporated to afford a white solid 177 mg (0.62 mmol 76 %):

1 Rf 0.31 (silica gel; hexane-ethyl acetate 1:1). H-NMR (600 MHz; CDCl3): δ1.29 (s, 3

H), 1.46 (s, 9 H), 1.50 (s, 3 H), 2.07 (s, 1 H), 2.25-2.3 (m, 1 H), 2.41-2.46 (m, 1 H),

3.63-3.70 (m, 1 H), 3.77-3.82 (m, 1 H), 3.99 (br s, 1 H), 4.39 (br s, 1 H), 4.54 (br s, 1

H), 5.55 (br s, 1 H).

The reaction between Zincke Reagent 7 and carbocyclic ribosyl amine analog 6 which afford 17: A solution of 6 (100 mg, 0.68 mmol) was dissolved in 5 mL of acetonitrile in round bottom flask. The solution was stirred with a magnate stirring bar at room temperature. Make a solution of 7 (512 mg, 1.36 mmol) and triethylamine (69 mg, 0.68 mmol) in 5 mL of acetonitrile. The solution was added dropwise to the stirring solution of 6 using a pressure equalized dropping funnel over a period of 15 mins. After the addition was complete, continue stirring for 2 hours. During the addition the solution turns dark purple, then turn orange. The reaction mixture was pound onto 50 mL of DI water, and the water were washed 3 times with 15 mL portions of methylene chloride to remove 2,4-dinitroaniline and unreacted 6. The 24 aqueous solution was evaporated to afford a sticky oily product. The TLC indicated there were two spots which were very close to each other. The preparative thin layer chromatography was performed to separate the two compounds. The minor one was too small to be analyzed. The major spot was an oily product 68 mg (0.177 mmol,

1 52 %). Rf is 0.32 (5:2:3 butanol: acetic acid: water). H-NMR (600 MHz; D2O): δ1.31

(m, 2 H), 2.13-2.15 (m, 2 H), 2.31 (dt, 8.4 Hz and 13.2 Hz, 2 H), 3.51 (m, 2 H), 3.58

(t, 6 Hz, 2 H), 3.88-3.90 (m, 2 H), 3.94-3.96 (m, 2 H), 4.03 (br s, 2 H), 8.47 (dt, 6.6

Hz and 7.8 Hz, 1 H), 8.91 (d, 7.8 Hz, 1 H), 9.37 (d, 6.6 Hz, 1 H), 9.79 (s, 1 H).

Compound 7 (reaction g), 20 (reaction h), 21 (reaction i), 22 (reaction j) were synthesized using the same procedure as that which was used for 7.

1-(2,4-dinitro)-phenyl nicotinic acid ethyl ester tetrafluoroborate (20):

Nicotinic acid ethyl ester (24) (1.92 g, 12.7 mmol) and 1-chloro-2,4-dinitrobenzene

(14) (1 g, 6.25 mmol) were reacted according to the procedure used for the formation of 7 to afford a white solid 1.29 g (3.18 mmol, 51 %). Rf is 0.35 (silica gel;

1 hexane-ethyl acetate 1:1); H-NMR (600 MHz; D2O): δ 1.35 (t, 7.2 Hz, 3 H), 4.48 (q,

7.2 Hz, 2 H), 8.20 (d, 9 Hz, 1 H), 8.45 (t, 6 Hz, 1 H), 8.89 (dd, 1.8 Hz and 8.4 Hz, 1

H), 9.32-9.37 (m, 3 H), 9.75 (s, 1 H).

1-(2,4-dinitro)-phenyl nicotinic acid isopropyl ester tetrafluoroborate (21):

Nicotinic acid isopropyl ester (25) (2.10 g, 12.7 mmol) and

1-chloro-2,4-dinitrobenzene (14) (1 g, 6.25 mmol) were reacted according to the procedure used for the formation of 7 to afford a white solid 1.26 g (3.00 mmol,

1 48 %). Rf is 0.36 (silica gel; hexane-ethyl acetate 1:1); H-NMR (400 MHz; D2O): δ

1.40 (d, 6.6 Hz, 6 H), 5.29 (m, 1 H), 8.21 (d, 13.2 Hz, 1 H), 8.47 (t, 9.6 Hz, 1 H), 8.90

(dd, 3.6 Hz, and 13.2 Hz, 1 H), 9.33-9.38 (m, 3 H), 9.80 (s, 1 H).

1-(2,4-dinitro)-phenyl nicotinic acid tert-butyl ester tetrafluoroborate (22):

25 Nicotinic acid tert-butyl ester (26) (2.28 g, 12.7 mmol) and

1-chloro-2,4-dinitrobenzene (14) (1 g, 6.25 1.93mmol) were reacted according to the procedure used for the formation of 7 to afford a glass 835 mg (1.93 mmol, 31 %). Rf

1 is 0.36 (silica gel; hexane-ethyl acetate 1:1); H-NMR (400 MHz; D2O): δ 1.60 (s, 9

H), 8.22 (d, 13.2 Hz, 1 H), 8.44 (t, 10.8 Hz, 1 H), 8.91 (dd, 3.6 Hz and 13.2 Hz, 1 H),

9.30-9.36 (m, 3 H), 9.64 (s, 1 H).

Compound 28 (reaction k), 29 (reaction l), and 32 (reaction n) were synthesized using the same procedure as that was which was used for 28 (reaction k). Compound

28 (reaction k), 29 (reaction l), and 32 (reaction n) were using isopropylamine as the primary amine to perform the Zincke reaction. Compound 31 (reaction m) was using

D-alaninol (2-amino-1-propanol) as the primary amine to perform the Zincke reaction.

3-Isopropylcarbamoyl-1-isopropyl-pyridinium tetrafluoroborate (28, reaction k): A solution of isopropylamine 27 (313.3 mg, 5.30 mmol) was dissolved in 5 mL of acetonitrile in round bottom flask. The solution was stirred with a magnetic stirring bar at room temperature. Make a solution of the 1-(2,4-dinitrophenyl)-3-3 methoxycarbonyl pyridinium tetrafluoroborate 7 (1 g, 2.65 mmol) in 5 mL of acetonitrile was prepared. The solution was added dropwise to the stirring solution of the isopropylamine using a pressure equalized dropping funnel over a period of 15 mins. After the addition was complete, stirring was continued for 2 hrs. During addition the solution turns dark purple, then it turns orange. The reaction mixture was pounded onto 50 mL of DI water, and the water washed 3 times with 15 mL portions of methylene chloride to remove 2,4-dinitroaniline and unreacted isopropylamine.

The aqueous solution was evaporated to afford a sticky oily product 608 mg (2.07

1 mmol, 78.1 % yield). Rf is 0.41 (5:2:3 butanol: acetic acid: water). H-NMR (600

26 MHz; D2O): δ 1.18 (d, 6.6 Hz, 6 H), 1.59 (d, 6.7 Hz, 6 H), 3.38 (m, 1 H), 4.94 (m, 1

H), 7.99 (dd, 6.54 Hz and 7.56 Hz, 1 H), 8.72 (d, 7.98 Hz, 1H), 8.86 (d, 6.18 Hz, 1 H),

13 9.11 (s, 1 H). C-NMR (600 MHz; D2O): δ 19.67 ppm, 22.04 ppm, 43.95 ppm, 65.41 ppm, 127.99 ppm, 137.15 ppm, 143.03 ppm, 143.38 ppm, 145.05 ppm, 167.82 ppm.

+ HRMS calcd for C12H19N2O : 207.149. Found m/z: 207.150 (M+).

3-Isopropylcarbamoyl-1-isopropyl-pyridinium tetrafluoroborate (29, reaction l): A solution of isopropylamine 27 (292 mg, 4.94 mmol) and 20 (1 g, 2.47 mmol) were reacted according to the procedure used for the formation of 28 to afford a sticky oily product weighing 515 mg (1.75 mmol, 71 % yield). Rf is 0.41 (5:2:3 butanol:

1 acetic acid: water). H-NMR (600 MHz; D2O): δ 1.18 (d, 6.6 Hz, 6 H), 1.59 (d, 6.7 Hz,

6 H), 3.38 (m, 1 H), 4.94 (m, 1 H), 7.99 (dd, 6.54 Hz and 7.56 Hz, 1 H), 8.72 (d, 7.98

13 Hz, 1H), 8.86 (d, 6.18 Hz, 1 H), 9.11 (s, 1 H). C-NMR (600 MHz; D2O): δ 19.67 ppm, 22.04 ppm, 43.95 ppm, 65.41 ppm, 127.99 ppm, 137.15 ppm, 143.03 ppm,

143.38 ppm, 145.05 ppm, 167.82 ppm.

3-Isopropan-1-ol-carbamoyl-1-isopropan-1-ol-pyridinium tetrafluoroborate

(31, reaction m): A solution of D-alaninol which was purchased from Oakwood

Chemical (South Carolina, USA) 30 (375 mg, 4.76 mmol) and 22 (1 g, 2.38 mmol) were reacted according to the procedure used for the formation of 28 to afford a sticky oily product 489 mg (1.50 mmol, 63 %). Rf is 0.36 (5:2:3 butanol: acetic acid: water).

1 H-NMR (400 MHz; D2O): δ 1.23 (d, 7.2 Hz, 3 H), 1.64 (d, 6.6 Hz, 3 H), 3.34 (d, 7.2

Hz, 2 H), 3.48 (m, 1 H), 3.65 (d, 6.6 Hz, 2 H) 5.01 (m, 1 H), 8.04 (dd, 6.6 Hz and 7.2

Hz), 8.77 (d, 7.8 Hz, 1 H), 8.91 (d, 6 Hz, 1 H), 9.16 (s, 1 H).

3-Isopropylcarbamoyl-1-isopropyl-pyridinium tetrafluoroborate (32, reaction n): A solution of isopropylamine 27 (66 mg, 1.12 mmol) and 22 (242 mg, 0.56 mmol) were reacted according to the procedure used for the formation of 28 to afford a glass

27 product 68 mg (0.23 mmol, 41 % yield). Rf is 0.41 (5:2:3 butanol: acetic acid: water).

1 H-NMR (600 MHz; D2O): δ 1.18 (d, 6.6 Hz, 6 H), 1.59 (d, 6.7 Hz, 6 H), 3.38 (m, 1

H), 4.94 (m, 1 H), 7.99 (dd, 6.54 Hz and 7.56 Hz, 1 H), 8.72 (d, 7.98 Hz, 1H), 8.86 (d,

13 6.18 Hz, 1 H), 9.11 (s, 1 H). C-NMR (600 MHz; D2O): δ 19.67 ppm, 22.04 ppm,

43.95 ppm, 65.41 ppm, 127.99 ppm, 137.15 ppm, 143.03 ppm, 143.38 ppm, 145.05 ppm, 167.82 ppm.

Compound 28 (reaction k), 29 (reaction l), and 32 (reaction n) were the same compound. They have the identical NMR spectra and identical mass spectrometric data.

28 References:

Barbier, D.; Marazano, C.; Das, B.; and Potier, P. (1996). New chiral isoquinolinium slat derivatives from chiral primary amines via Zincke reaction. J. Org. Chem., 61, 9596-9598

Barrett, K.; Barman, S.; Boitano, S.; Brooks, H. (2009). Chapter 2. Overview of cellular physiology in medical physiology. Ganong's Review of Medical Physiology, 23e. New York: McGraw-Hill Medical. page 52-60

Billington, R.; Tron, G.; Reichenbach, S.; Sorba, G.; and Genazzani, A. (2005). Role of the nicotinic acid group in NAADP receptor selectivity. Cell Calcium 37, 81–86.

Bootman, M.; Berridge, M.; Roderick, H. (2002). Calcium signalling: more messengers, more channels, more complexity. Curr Biol, 12: R563

Cancela, J.; Churchill, G.; and Galione, A. (1999). Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells. Nature, 398, 74-76

Cermak, R.; Vince, R.(1981) (±)4β-amino-2α,3α-dihydroxy-1β-cyclopentanemethanol hydrochloride. Carbocyclic ribofuranosylamine for the synthesis of carbocyclic nucleosides. Tetrahedron Letters, 22, 2331-2332

Clapper, D.; Walseth, T.; Dargie, P.; et al. (1987). Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate. J. Biol. Chem., 262, 9561– 9568

Dowden, J.; Moreau, C.; Brown, R.; Berridge, G.; Galione, A.; and Potter, B. (2004). Chemical synthesis of the second messenger nicotinic acid adenine dinucleotide phosphate by total synthesis of nicotinamide adenine dinucleotide phosphate. Angew. Chem. Int. Ed. 43, 4637-4640

Galione, A.; Lee, H.; and Busa, W. (1991). Ca2+-induced Ca2+ release in sea urchin egg homogenates: modulation by cyclic ADP-ribose. Science, 253, 1143–1146

Galione, A.; Morgan A.; Arredouani A.; Davis L.; Rietdorf K.; Ruas M.; and Parrington J. (2010). NAADP as an intracellular messenger regulatinglysosomal calcium-release channels, Biochem. Soc. Trans. 38, 1424–1431.

Jain, P.; Slama, J.; Perez-Haddock, L.; Walseth, T. (2010) Nicotinic acid adenine dinucleotide phosphate analogues containing substituted nicotinic acid: effect of modification on Ca2+ release. J. Med. Chem. 53, 7599-7612

Kam, B.; and Oppenheimer, N. (1981) Carbocyclic sugar amines: synthesis and stereochemistry of racemic α- and β-carbocylic ribofuranosylamine, carbocyclic lyxofuranosylamine, and related compounds. J. Org. Chem. 46, 3268-3272

Kunuqi, S.; Okubo, T.; Ise, N. (1976) A study on the mechanism of the reaction of N-(2,4-dinitrophenyl)-3-carbamoylpyridinium chloride with amines and amino acids with reference to effect of polyelectrolyte addition. J Am Chem Soc. 98 (8), 2282-2287 29 Lee, H. (1997). Mechanisms of calcium signaling by cyclic ADP-ribose and NAADP. Physiol. Rev. 77, 1133–1164

Lee, H.; & Aarhus, R. (1995). A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose. J. Biol. Chem. 270, 2152–2157.

Lee, H.; and Aarhus, R. (1997). Structural determinants of nicotinic acid adenine dinucleotide phosphate important for its calcium mobilizing activity. J. Biol. Chem. 272, 20378–20383

Lee, H.; Aarhus, R.; Gee, K.; and Kestner, T. (1997). Prolonged inactivation of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release mediates a spatiotemporal Ca2+ memory. J. Biol. Chem. 272, 4172–4178

Lee, H.; Walseth, T.; Bratt, G.; et al. (1989). Structural determination of a cyclic metabolite of NAD with intracellular calcium-mobilizing activity. J. Biol. Chem., 264: 1608–1615

Morgan, A.; and Galione, A. (2007). NAADP induces pH changes in the lumen of acidic Ca2+ stores. J. Biol. Chem. 402, 301–310

Pitt, S.; and Funnell T. (2010). TPC2 Is a novel NAADP-sensitive Ca2+ release channel, operating as a dual sensor of luminal pH and Ca2+. J. Biol. Chem. 285, 35039-35046

Slama, J.; and Simmons, A. (1988). Carbanicotinamide adenine dinucleotide: synthesis and enzymological properties of a carbocyclic analog of oxidized nicotinamide adenine dinucleotide. Biochemistry, 27, 183

Streb, H.; Irvine, R.; Berridge, M.; et al. (1983). Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature, 306: 67–69

Trabbic, C.; Walseth, T.; and Slama, J. (2012). Synthesis, biochemical activity, and structure-activity relationship among nicotinic acid adenine dinucleotide phosphate (NAADP) analogs. Messenger, 1, 108-120.

Trabbic, C.; Zhang, F.; Walseth, T.; and Slama, J. (2015). Nicotinic acid adenine dinucleotide phosphate analogues substituted on the nicotinic acid and adenine ribosides. Effects on receptor mediated Ca2+ release. J. Med. Chem., 58, 3593-3610

Yoshikawa, M.; Kato, T.; and Takenishi, T. (1969) Studies of phosphorylation. III. Selective phosphorylation of unprotected nucleosides. Bulletin of the Chemical Soceity of Japan, 42, 3505-3508

Zatman, L. J.; Kaplan, N. O.; and Colowick, S. P. (1953). Inhibition of spleen diphosphopyridine nucleotidase by nicotinamide, an exchange reaction. J. Biol. Chem. 200, 197–212

Zincke, Th.; Heuser, G.; Moller, W. (1904). Ueber Dinitrophenylpyridiniumchlorid 30 und dessen Umwandlungsproducte. Justus Liebigs Annalen der Chemie 333 (2–3): 296–345.

31 Appendix

1H-NMR: Compound 6

6

32 1H-NMR: Compound 7

7

33 1H-NMR: Compound 8

8

34 1H-NMR: Compound 9

9

35 1H-NMR: Compound 10

10

36 1H-NMR: Compound 11

11

37 1H-NMR: Compound 12

or

12

38 1H-NMR: Compound 17

17

39 1H-NMR: Compound 20

20

40 1H-NMR: Compound 21

21

41 1H-NMR: Compound 22

22

42 1H-NMR: Compound 28, 29, and 32

28, 29, and 32

43 13C-NMR: Compound 28, 29, and 32

28, 29, and 32

44 1H-NMR: Compound 31

31

45