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TitleSynthesis of Isotopically Labeled Co-Enzyme to Probe the Active Site of Tryptophan synthase/ New Synthetic Approach to Tetrahydrocannabinol Analogs

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AuthorBastin, Baback

Publication Date2015-01-01 Peer reviewed|Thesis/dissertation

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UNIVERSITY OF CALIFORNIA RIVERSIDE

Synthesis of Isotopically Labeled Co-Enzyme to Probe the Active Site of Tryptophan synthase/ New Synthetic Approach to Tetrahydrocannabinol Analogs

A Dissertation submitted in partial satisfaction of the requirements for the degree of

Doctor of Philosophy

in

Chemistry

by

Baback Bastin

August 2015

Dissertation Committee:

Dr. Michael Marsella, Chairperson Dr. Richard Hooley

Dr. Thomas Morton

 

Copyright by Baback Bastin

2015

 

The Dissertation of Baback Bastin is approved:

Committee Chairperson

University of California, Riverside

iv    

Acknowledgements

I am grateful to have been under the guidance of Dr. Michael Marsella. Without his

encouragement, wisdom, and patience, I wouldn’t have grown into the scientist I am

today.

I would also like to thank my parents, Hamid and Fahimeh, and my brother, Bardia, for

their support and love. Hamid and Fahimeh have been a true inspiration to me and always

supportive in all of my endeavors. My mom and dad sacrificed tremendously not only to

raise us but to give us the best education available and I will forever be grateful for their

sacrifices. Bardia has been my best friend and always there for me whenever I needed

him and I am grateful to have a brother as kind hearted and selfless as him.

I am truly thankful in having wonderful lab mates, Mackenzie Alvarez and Aiden

Aceves. They were always around for intellectually stimulating discussions and to help

with experiments in the lab.

Lastly, I would like to dedicate this thesis to the memory of both my grandfathers,

Hassan Bastin and Amir Houshang Aghdaei, who had passed away during my tenure at

UCR.

v    

ABSTRACT OF THE DISSERTATION

Synthesis of Isotopically Labeled Co-Enzyme to Probe the Active Site of Tryptophan synthase/ New Synthetic Approach to Tetrahydrocannabinol Analogs

by

Baback Bastin

Doctor of Philosophy, Graduate Program in Chemistry University of California, Riverside, August 2015

Prof. Michael J. Marsella, Chairperson

Identifying enzyme mechanisms at proton level resolution is the ultimate goal of

enzymology. Traditional enzyme mechanistic studies infer protonation states from x-ray

crystal structure and optical spectroscopy. This thesis reports work towards the first

synergistic combination of x-ray crystallography, computational chemistry, synthetic

organic chemistry and solid-state NMR to fully elucidate, at proton level resolution, the

full three-dimensional structure of the catalytic site for Tryptophan synthase during active

catalysis. Specifically, this thesis describes solutions to the synthetic challenges of

introducing site-specific isotopic labels inside the cofactor Pyridoxal-5’-Phosphate (PLP)

and highlights a synthetic route that is consistently more cost-effective and higher

yielding than previous efforts.

vi    

The second project presented focuses on efforts towards the synthesis of cannabinoids,

cannabidiol (CBD) and tetrahydrocannabinol (THC). Presently, cannabinoids have

emerged as compounds of interest for a variety of pharmacologic indications. Although

stereochemically simple compounds, economical syntheses of enantiopure cannabinoids

remain elusive. Strategies to address facile syntheses of THC and CBD, as well as their

analogs, will be presented.

vii    

Table of Contents

Chapter 1: An Overview of the enzyme Tryptophan synthase: New Methodology to Characterize Each Intermediate at Proton Level Resolution Throughout the Catalytic Cycle

1.1 Introduction ................................................................................................................2 1.2 Tryptophan synthase .................................................................................................2 1.3 Atomic Resolution characterization of E(Q)indoline Intermediate ...............................8 1.4 Characterization of the E(Ain) Intermediate ...........................................................12

References ......................................................................................................................15

Chapter 2: Various Synthetic Approaches Towards Pyridoxal-5’-Phosphate

2.1 Introduction ..............................................................................................................18

2.2 Synthesis Through Pyridone Intermediate ...............................................................18

2.3 Synthesis Through the Kondrat’eva Reaction .........................................................20

2.4 Vollhardt’s Synthesis Through [2+2+2] Cobalt Mediated Cycloaddition ..............21 Reference .......................................................................................................................23 Chapter 3: Current PLP Synthesis and Comparison to Limbach Synthesis 3.1 Introduction ...............................................................................................................25 3.2 Current Published Synthesis of PLP .........................................................................25 3.3 Improvement/Discussion of Synthetic Route to PLP ...............................................27

viii    

3.4 Synthesis of Isotopic Analogs of PLP ......................................................................34 3.5 Synthesis of [15N]-2-Aminophenol ...........................................................................36 3.6 Synthetic Approach to [17O]-PLP .............................................................................38 References .......................................................................................................................43 Chapter 4: Synthetic Background/ Synthetic Attempts Towards Cannabinoid Analogs 4.1 Introduction ................................................................................................................45 4.2 Cannabinoid Overview ..............................................................................................45 4.3 Previous Syntheses of Δ1-Tetrahydrocannabinol (Δ1-THC) .....................................48 4.4 Attempts Towards Synthesis of THC ........................................................................52 4.5 Conclusions ................................................................................................................57 References ........................................................................................................................59 Appendix A: Experimental Procedure and Spectroscopic Data ........................................60

Appendix B: 1H-NMR Spectra ..........................................................................................73

ix    

List of Figures

Figure 1.1 Crystal structure of Tryptophan synthase ..........................................................3

Figure 1.2 α-site reaction mechanism .................................................................................4

Figure 1.3 β site reaction mechanism .................................................................................5

Figure 1.4 Reaction to form E(Q)Indoline intermediate ..........................................................8

Figure 1.5 Solid state NMR of labeled (red) vs unlabeled (blue) E(Q)Indoline intermediate 9

Figure 1.6 E(Q)indoline complex with with all possible protonation sites circled in green .10

Figure 1.7 Equilibrium and proton exchange of the E(Q)indoline intermediate ...................12

Figure 1.8 E(Ain) intermediate .........................................................................................13

Figure 3.1 Proposed mechanism for the formation of oxazole 2 ......................................29

Figure 3.2 Mechanism of the Diels-Alder reaction that forms compound 3 ....................30

Figure 3.3 Possible intermediates of the Diels-Alder adduct ............................................31

Figure 3.4 Hemiacetal formation of oxidized Pyridoxine in acidic solution ....................32

Figure 3.5 Reaction of 2-Aminophenol (2AP) with the EA-A intermediate ......................38

Figure 3.6 Proposed mechanism for the transformation of 26 to 27 .................................41

Figure 4.1 Left structure shows the monoterpenoid numbering, while the right structure shows the natural occurring (-)-Δ1-THC. Stereochemistry at carbons 3 and 4 are R, R ...49 Figure 4.2 Biosynthetic pathways to THC, CBD and CBC ..............................................54  

x    

List of Schemes

Scheme 2.1 Conversion of Pyridoxine to PLP a)MnO2, primary amine (R= -p-OEtC6H4, -OH, -t-Butyl), b) H3PO4, P2O5 ..........................................................................................18 Scheme 2.2 Harris, Stiller, and Folkers synthesis a) EtOH/piperidine b)Ac2O, HNO3 c) PCl5, ClC6H5 d) PtO2, H2 e)Pt, H2 f) HCl g) HONO, h)48 % HBr i)H2O, AgCl .............19 Scheme 2.3 Kondrat’eva reaction synthesis to pyridoxine a) HCOOH/EtOH, b) P2O5, DCM c)Diethyl maleate d)Lithium Aluminum Hydride, Et2O .........................................20 Scheme 2.4 Vollhardt synthesis a)n-BuLi, SnMe3Cl b) CH3CN, CpCo(CO2), m-xylenes c) CuI, NaOMe d)1. 48% HBr 2. AgCl, H2O ....................................................................21 Scheme 3.1 Limbach Synthesis a)HCO2H/EtOH, b) P2O5 c) Diethyl maleate d) LAH e) KMnO4, NaHSO3, 50% H2SO4, p-Toluidine f) H3PO4/P2O5 ............................................26 Scheme 3.2 Current published PLP synthesis a) SOCl2/EtOH b) Triethylorthoformate (TEOF) c) P2O5 d) Diethyl Maleate e) LAH f) MnO2/p-Phenetidine g) H3PO4/P2O5 ......28 Scheme 3.3 [15N]-PLP synthesis a) SOCl2/EtOH b) Triethylorthoformate (TEOF) c) P2O5 d) Diethyl Maleate e) LAH f) MnO2/p-Phenetidine g) H3PO4/P2O5 .................................34

Scheme 3.4 [15N,13C]-PLP synthesis a) SOCl2/EtOH b) Triethylorthoformate (TEOF) c) P2O5 d) Diethyl maleate e) LAH f)MnO2/p-Phenetidine g) H3PO4/P2O5 .........................35 Scheme 3.5 Synthesis of [15N]-2AP a) H15NO3(10M), Tetra-n-butylammonium bromide (TBAB), Ethylene Dichloride b)10% Pd/C, NaBH4 ..........................................................36 Scheme 3.6 A new [15N,13C]-PLP synthesis a) SOCl2/EtOH b) triethylorthoformate (TEOF) c) P2O5 d) 2,5-dihydrofuran,hydroquinone, trichloroacetic acid e) MnO2/p-phenetidine f) H3PO4/P2O5 .................................................................................................37 Scheme 3.7 Synthesis of [15N]-2AP a) HN15O3(10M), tetra-n-butylammonium bromide (TBAB), ethylene dichloride b)10% Pd/C, NaBH4 ...........................................................38 Scheme 3.8 Proposed synthesis of [17O]-PLP a) 3.5 M HCl b) Triethylorthoformate/Ac2O then NaHCO3 c) Iminoacetylacetone d) H2SO4 then NaN3 e) H2SO4/EtOH f) LAH g) NaNO2, H2O17 h) MnO2 then p-Phenetidine i) H3PO4/P2O5 ..............................................39

Scheme 4.1 First synthesis of Δ1-THC ..............................................................................50 Scheme 4.2 Mechoulam synthesis .....................................................................................51

xi    

Scheme 4.3 Evans stereospecific synthesis of (S,S) Δ1-THC a) 1.Cationic bis(oxazoline)copper (II) catalyst 2.LiOBn b) MeMgBr c) Olivetol d)ZnBr2, MgSO4 .....52

Scheme 4.4 Trost synthesis a) 5 mol % [Mo(CO)3C7H8], sodium dimethyl malonate, 7.5% (R,R)-DACH-pyridyl TROST ligand b) 1. NaOH 2. HCl c) LDA, 4-iodo-2-methylbut-1-ene d) 1. (MeO)2SO2, K2CO3 2. Grubbs II catalyst e) MeLi f) NaSEt g) 1. ZnBr2, MgSO4 2. NaSEt .....................................................................................................53 Scheme 4.5 Olefin Metathesis synthetic route a) K2CO3, MeI b) n-BuLi, TMEDA, DMF c) Ph3P=CH2 d) Grubbs II Generation ...............................................................................55 Scheme 4.6 Diels-Alder route a) K2CO3, MeI b) n-BuLi, TMEDA, DMF c) NaOH, acetone d) Lewis acid catalyst ...........................................................................................56 Scheme 4.7 Biomimetic approach a) K2CO3, MeI b) n-BuLi, TMEDA, DMF c) NaI, AlCl3 d) NaOH, 6-methyl-5-hepten-2-one e) KOH, 6-methyl-5-hepten-2-one ................57 Scheme 4.8 Proposed synthetic pathways to CBD and THC a) 1-10% acid b) MeMgBr c) K-t-pentoxide/NaSEt ..........................................................................................................58  

xii    

List of Tables

Table 1.1 13C-NMR data from E(Q)indoline intermediate ....................................................11

Table 3.1 Summary of various Diels-Alder reactions .......................................................36

Table 4.1 Chemical structures of phyto-, synthetic and endo-cannabinoids ................. 47-8

1    

Chapter 1

An Overview of the enzyme Tryptophan synthase/ New Methodology to Characterize

Each Intermediate, at Proton Level Resolution, Throughout the Catalytic Cycle.

2    

1.1 Introduction

Tryptophan is one of the essential amino acids in living organisms. Human beings have

the ability to obtain this amino acid from their diets; however bacteria, yeasts, molds and

some plants use the catalytic ability of Tryptophan synthase to produce L-Tryptophan.1-4

This allows researchers to target this enzyme for the eradication of harmful bacteria or as

a herbicide. Having a clear understanding of the mechanism by which tryptophan

synthase creates L-Tryptophan (L-Trp) allows researchers to create an inhibitor that

would shut down the enzyme and, in turn, destroy the organism without harm to humans

or plants. The following sections will discuss an overview of the enzyme’s catalytic

mechanism, as well how a combination of synthetic, computational, X-ray

crystallography and solid state NMR has allowed for the chemically-detailed three-

dimensional structure of a few of the intermediates of the Tryptophan synthase catalytic

cycle.

1.1 Tryptophan synthase

Tryptophan synthase is a α2β2 bi-enzyme tetramer complex that catalyzes the

transformation of 3-indole-D-glycerol 3’-phosphate (IGP) to L-Trp in bacteria, yeasts,

molds and plants (Figure 1.1).1 The S. typhimurium bi-enzyme complex has been

extensively studied using various techniques1-6 and has been chosen as a model for the

better understanding of substrate channeling and the catalytic mechanism.

3    

Figure 1.1 Crystal structure of Tryptophan synthase1

The enzyme is found in bacteria, yeasts, molds and plants. In higher animals, Tryptophan

synthase is non-existent and has become a potential drug target for herbicides and

infectious diseases, i.e. Chlamydia trachomatis and Mycobacterium tuberculosis. 7,8 The

full understanding of the enzymatic catalysis and substrate channeling could prove

helpful in designing potent inhibitors to battle infectious disease and to create herbicides.

The catalytic pathway consists of an α-reaction and a β-reaction. The α-site reaction is

the conversion of 3-indole-D-glycerol 3’-phosphate (IGP) to D-glyceraldehyde 3-

phosphate (G3P) and indole (Figure 1.2). 1-4 For the α-site reaction, the glutamic acid

residue (αGlu49) aids in the retro aldol condensation while the aspartic acid residue

(αAsp60) hydrogen bonds with the amine to further deactivate the ring and allow for

protonation at the β position relative to the amine in the indole ring.

4    

Figure 1.2 α-site reaction mechanism2

Once indole has been made, it is channeled down a 25 Å tunnel to the β site where, with

the addition of L-serine (L-Ser), Pyridoxal 5’-Phosphate (PLP), and a monovalent cation

produces the amino acid L-Tryptophan (L-Trp) (Figure 1.3).1-4

5    

Figure 1.3 β site reaction mechanism9

Initially, PLP forms an imine with the lysine residue (βLys87) to form an intermediate

internal aldimine, E(Ain). Next, the amino group of L-Ser nucleophilically attacks the

imine in E(Ain), initiating the transamination to form the gem-diamine intermediate,

E(GD1), which then releases the βLys87 residue and forms an external aldimine imine

complex E(Aex1). At this point, the βLys87 deprotonates the α proton and the quinonoid

substrate, E(Q1), is formed. Once the electrons from the pyridine nitrogen resonate back

up and promote elimination of water, the α-aminoacrylate, E(A-A), is formed and Stage 1

of the β site reaction is complete. At this point the α site reaction has been completed and

indole has been shuttled down to react with the E(A-A) intermediate to begin Stage 2 of

the β site reaction. Indole reacts with the E(A-A) through a Michael addition where the

6    

enamine moiety of the indole ring attaches to the aforementioned intermediate to form

the new quinonoid structure E(Q2/3). The βLys87 residue then returns and protonates at

the α position giving the E(Aex2). At this point, the uncharged side-chain of the βLys87

residue becomes nucleophilic again and attacks the imine, initiating the second

transamination and giving the second gem-diamine intermediate E(GD2). The last step is

reforming the initial βLys87-PLP imine and freeing L-Trp from the active site.

The overall goal of this project is to define clearly enzymatic catalysis at a

chemical/structural level through a synergistic combination of X-ray crystallography,

computational chemistry, solid state NMR (SSNMR), and synthetic organic chemistry.

In the past, the relationship between protein structure and biological function has been

achieved with the determination of protein structures at a near atomic resolution, genetic

modification of proteins, and bioorganic mechanism experimentation. This has reduced

enzymatic catalysis to a simple set of sequential organic chemical reactions such as

Lewis acid catalysis, Brønsted acid-base catalysis, and nucleophilic/electrophilic

catalysis. This does not account, however, for the enormous rate accelerations achieved

by enzyme active sites. The introduction of computational chemistry has been

indispensible in enzymatic mechanism studies. Most of the current studies have

prioritized the electrostatic field effects at enzyme active sites to stabilizing higher energy

complexes.10-16 In order for a clearer, proton level resolution of enzyme catalysis to

occur, a better insight of the electrostatic microenvironment at the active site is necessary.

7    

Though each technique used in the field of enzymology provides insight into enzyme

catalysis, a combination of all the techniques should provide the most detailed

mechanism. For example, a high resolution X-ray crystal structure provides details of the

interaction between enzyme side chains and substrate but at 1.5-2.5 Å resolution it

doesn’t allow for determining protonation states of the acidic/basic functional groups at

the active site. Typically, protonation states are inferred based on solution pKa and

hydrogen bonding pattern intuition. SSNMR allows for proton level resolution under the

same conditions used to solve X-Ray crystal structure and can be used for large

biomolecules, such as the 144 kDa Tryptophan synthase. Work done by McDowell et

al.17 showed that when labeled 13C-Serine was added to the enzyme, SSNMR could easily

identify the E(A-A) intermediate in the crystalline state. Through a combination of

SSNMR, X-Ray crystallography, computational chemistry and synthetic organic

chemistry each individual intermediate of the Tryptophan synthase catalytic cycle can be

resolved at the proton level.

8    

1.3 Atomic resolution characterization of the E(Q)indoline Intermediate

The first intermediate fully characterized was a chemical and structural equivalent of the

E(Q2/3) intermediate, E(Q)indoline.5 Through inhibition of the α-reaction (while supplying

indoline instead of indole) the E(A-A) intermediate reacts with indoline to form the

quasi-stable intermediate (Figure 1.4).5

Figure 1.4 Reaction to form E(Q)Indoline intermediate5

The E(Q)indoline intermediate was characterized through solid-state NMR (Figure 1.6), and

distinct 13C resonances are highlighted. With the addition of isotopically labeled serine,

sharp and distinct peaks are present in the spectra (Figure 1.5).5

9    

Figure 1.5 Solid state NMR of labeled (red) vs unlabeled (blue) E(Q)Indoline intermediate5

To accurately determine the protonation state of each molecule, a model of the β-subunit

7.0 Å from the substrate was constructed, and the substrate chemical shifts were

calculated. Figure 1.6 shows the possible sites of protonation and several candidate

structures were considered with different protonation sites. Not all the sites can be

simultaneously protonated, so a potential of 28 structures were calculated and compared

with experimental data.5

10    

N

O

N

O

N O

O

HPO

OO

EnzNH2

Figure 1.6 E(Q)indoline complex with with all possible protonation sites circled green

According to the experimentally derived and computationally derived chemical shifts,

(Table 1.1), the previously reported protonated Schiff’s base hydrogen bonding to the

phenolic oxygen was found to be incorrect, and a more accurate depiction is fast

exchange between the protonated Schiff’s base and the carboxylic acid in a ratio of 34:66

(but the three-site equilibrium among the protonated Schiff base, the carboxylate, and the

phenolic form in a ratio of 33:58:9 cannot be ruled out) (Figure 1.7). The NMR data

suggest the predominent form is the protonated carboxylic acid hydrogen bonding with

the Schiff’s base, and the X-ray crystal structure also confirms the structure with

distances of the imine nitrogen in hydrogen bonding proximity to the carboxylic acid

proton. Calculations also show a build up of negative charge at Cα in the protonated

carboxylic acid structure, as well. This makes the free lysine from the enzyme more rigid

11    

in this structure, as opposed to the free enzyme. The build up of negative charge at Cα not

only helps direct the proton from the lysine residue to Cα position, which is the next step

of the mechanism, but also lowers the energy barrier along the reaction coordinate

through charge stabilization.9 Once Cα is protonated, the lysine residue becomes

nucleophilic, the proton then jumps to the Schiff base making the imine more

electrophilic catalyzing the next step in the catalytic cycle: the transamination reaction.

Exp. Calculated PSB Form

Calculated Acid Form

Two-Site Eq.

Calc. Phenolic

Form

Three-Site Eq.

Cα 103.6 106 101.3 102.9 124.7 104.8 Cβ 54.1 54.0 49.6 51.1 52.7 51.4 C’ 173.0 172.3 169.3 170.3 175.6 170.9 N 296.5 215.1 337.2 295.7 321.7 295.7 C2 50.5 50.1 51.5 51.1 50.2 51.0 C3 28.5 31.9 32.5 32.3 31.8 32.2 N1 83.5 85.0 91.1 89.0 84.5 88.5

Table 1.15 13C-NMR data from E(Q)indoline intermediate

12    

Figure 1.7 Equilibrium and proton exchange of the E(Q)indoline intermediate

1.4 Characterization of the E(Ain) Intermediate

By using 15N, 13C, and 31P NMR chemical shift measurements, UV/Vis spectroscopy, X-

ray crystallography and strategic labeling of atoms in the PLP coenzyme the full

protonation state of the E(Ain) was solved. (Figure 1.8).18

N

NN

H

O

O

O

H

OP

O

OO

ENZ

NH3

N

NN

O

O

OO

PO

OO

ENZ

NH3

N

NN

O

OH

O

H

OP

O

OO

ENZ

NH3

N

NN

O

OH

OO

PO

OO

ENZ

NH3

H

13    

Figure 1.8 E(Ain) intermediate18

In Figure 1.8, the possible sites of protonation are circled in grey, the side chain residues

are colored blue and the experimentally derived charged states are in red. As

hypothesized, 19 the imine formed with Lys87 and the PLP coenzyme at the C4’ position

should be protonated. The UV/Vis spectroscopy, along with 15N/13C NMR studies,

confirms the protonated Schiff’s base hypothesis. The protonated Schiff’s base (PSB)

makes a more reactive center and allows for a nucleophilic attack at the C4’, as opposed

to the non-protonated tautomer.19-21 Protonation at the imine nitrogen initiates catalysis

and allows for the free serine residue to nucleophilically attack forming the gem-diamine

intermediate while the protonation states of both the phenolic oxygen and the pyridyl

nitrogen serve only in establishing the specificity of the reaction pathway.22-25 Typically,

a absorption maximum between 420-430 nm indicates the E(Ain) complex in PLP

14    

dependent enzymes and the 412 nm λmax corresponds to a Schiff base linkage which is in

conjugation with the coenzyme pi-system.26 By viewing the X-ray crystal structure of the

Tryptophan synthase E(Ain) complex, the distance between the Schiff base nitrogen and

the phenolic oxygen is measured around 2.6 Å which correlates to a N−H--O hydrogen

bond. Although UV/Vis and X-ray crystal structure suggest a protonated Schiff base, it

was adamant to probe the active site using solid state NMR to confirm these findings.

These experiments were able to conclude that the phenolic oxygen was deprotonated and

hydrogen bound to the PSB, the pyridine N was deprotonated and hydrogen bound to the

hydroxyl moiety of Ser 377 residue and the phosphoryl group is in the dianionic state.

Having the pyridine nitrogen protonated aids the proton transfer from phenolic oxygen to

the PSB,27,28 which is required for the PSB hypothesis of rate enhancement. The X-ray

crystal structure does show a network of water molecules that keeps a consistent

hydrogen bonding interaction with the phenolic oxygen and this suggests that the

hydrogen bound water is satisfactory for initiating catalysis through a proton transfer to

the Schiff base nitrogen. The measuring of the chemical shifts within the enzyme active

site has allowed a full, proton level, characterization of the E(Ain) intermediate of the

Tryptophan synthase catalytic cycle.

15    

REFERENCES:

1. Raboni, S.; Bettati, S.; Mozzarelli., Cell. Mol. Life Sci. 2009, 66, 2391-2403. 2. Dunn, F., M.; Niks, D.; Ngo, H.; Barends, T,R,M.; Schlichting, I,; Trends in Biochem. Sci. 2008, 33, 254-264. 3. Casino, P,. Niks, D., Ngo, H., Pan, P., Brzovic, P., Blumenstein, L., Barens, T.R., Schlichting, I., Dunn, M.F., Biochemistry, 2007, 47, 7713-7727. 5. Weyand, M., Schlichting, I., Biochemistry, 1999, 38, 16469-16480. 6. Pan, P., Woehl, E., Dunn, M.F., Trends in Biochem. Sci. 1997,22, 22-27. 7. Schpatzidis, A., Dealwis, C., Lubetsky, J.B., Liang, P.H., Anderson, K.S., Lolis, E. Biochemistry, 1999, 38, 12665-12674. 8. Finn, J., Langevine, C., Birk, I., Birk, J., Nickerson, K., Rodaway, S., Bioorg. & Med. Chem. Lett. 1999, 9, 2297-2302. 9. Lai, J., Niks, D., Wang, Y., Domratcheva, T., Barends, T. R.M., Schwarz, F., Olsen, R.A., Elliot, D.W., Fatmi, M.Q., Chang, C.A., Schlichtin, I., Dunn, M.F., Mueller, L.J., J. Am. Chem. Soc. 2011, 133, 4-7. 10. Hur, S., Bruice, T.C., Proc. Natl. Acad. Sci. USA. 2003, 100, 12015-12020. 11. Benkovic, S.J., Hammes-Schiffer, S., Science. 2003, 301, 1196-1202. 12. Claeyssens, F.,Harvey, J.N., Manby, F.R., Mata, R.A., Mulholland, A.J., Ranaghan, K.E., Schutz,M., Thiel, S., Thiel, W., Werner, H.J., Angew. Chem. Int. Ed. 2006, 45, 6856-6859. 13. Warshel, A., Sharma, P.K., Kato, M., Xiang, Y., Liu, H., Olsson, M.H., Chem Rev. 2006, 106, 3210-3235. 14. Sigala, P.A., Fafarman, A.T., Bogard, P.E., Boxer, S.G., Herschiag, D., J. Am. Chem. Soc. 2007, 129, 12104-12105. 15. Webb, L.J., Boxer, S.G., Biochem. 2008, 47, 1588-1598. 16. Childs, W., Boxer, S.G., J. Am Chem. Soc. 2010, 132, 6474-6480. 17. McDowell, L.M., Lee, M.S., Schaefer, J., Anderson, K.S. J. Am. Chem. Soc. 1995, 117, 12352-12353.

16    

18. Caulkins, B.G., Bastin, B., Yang, C., Neubauer, T.J., Young, R.P., Hilario, E., Huang, Y.M., Chang, C.A., Fan, L., Dunn, M.F., Marsella, M.J., Mueller, L.J., J. Am. Chem. Soc, 2014, 136, 12824-12827. 19. Cordes, E.H., Jencks. W.P., Biochemistry. 1962, 1, 773. 20. Heinert, D., Martell, A.E., J. Am. Chem. Soc. 1963, 85, 188. 21. Metzler, D.E., J. Am. Chem. Soc. 1957, 79, 485. 22. Toney, M.D. Biochim. Biophys. Acta. 2011, 1814, 1407. 23. Crugeiras, J.; Rios, A.; Riveiros, E.; Richard, J. P. J. Am. Chem. Soc. 2011, 133, 3173. 24. Major, D. T.; Gao, J. L. J. Am. Chem. Soc. 2006, 128, 16345.

25. Major, D. T.; Nam, K.; Gao, J. L. J. Am. Chem. Soc. 2006, 128, 8114.

26. Peracchi, A., Bettati, S., Mozzarelli, A., Rossi, G.L., Miles, E.W., Dunn, M.F., Biochemistry. 1996, 35, 1872-1880. 27 Limbach, H. H.; Chan-Huot, M.; Sharif, S.; Tolstoy, P. M.; Shenderovich, I. G.; Denisov, G. S.; Toney, M. D. Biochim. Biophys. Acta. 2011, 1814, 1426. 28 Sharif, S.; Powell, D. R.; Schagen, D.; Steiner, T.; Toney, M. D.; Fogle, E.; Limbach, H. H. Acta Crystallogr., Sect. B 2006, 62, 480.

17    

Chapter 2

Various Synthetic Approaches Towards Pyridoxal-5’-Phosphate (PLP)

18    

2.1 Introduction Pyridoxine, precursor to PLP (Scheme 2.1), has been synthesized through various

different routes using a variety of different starting materials and synthetic strategies .1-4

The bioactive form of vitamin B6, PLP, was resolved and synthesized in 1944 by

Gunsalus et al.5 Conversion of pyridoxine to the bioactive form, PLP, occurs through two

steps; an oxidation/protection and a phosphorylation/removal of protecting group.

Scheme 2.1 Conversion of Pyridoxine to PLP a) MnO2, primary amine (R= -p-OEtC6H4, -OH, -t-Butyl), b) H3PO4, P2O5

Though numerous syntheses exist, none of them were cost effective, high yielding or able

to directly label any atom throughout the PLP molecule. Herein is a brief overview of the

various syntheses of pyridoxine.

2.2 Synthesis Through Pyridone Intermediate

In 1939, Stiller, Keresztesy and Stevens fully elucidated the strucutre pyridoxine.6 The

complete syntheis of Pyridoxine was accomplished in 1939 (by Harris, Stiller, and

Folkers), and was completely identical, and biologically equivalent in activity, to the

natural vitamin Scheme 2.2.7

N

HOOH

N

HO

NOH

R

N

HO

O

OPO3H2

Pyridoxine Pyridoxal-5'Phosphate

HO

a b

19    

Scheme 2.27 Harris, Stiller, and Folkers synthesis a) EtOH/piperidine b)Ac2O, HNO3 c) PCl5, ClC6H5 d) PtO2, H2 e)Pt, H2 f) HCl g) HONO, h)48 % HBr i)H2O, AgCl

The synthesis begins through a condensation between cyanoacetamide and ethyl

acetonoxoalate. The resulting pyridone is nitrated with fuming nitric acid, then converted

to the pyridyl chloride with PCl5. Once isolated, the nitro group is reduced with PtO2 and

H2 to the amine, reductive dechlorination removes the chloride group, and reduces the

cyano group to the primary amine. Refluxing in concentrated hydrochloric acid

protonated both primary amines to make the dihydrochloride. The dihydrochloride was

heated in H2SO4(aq) and to the reaction aqueous solution of sodium nitrite was slowly

added resulting in the 4-ethoxy analog of pyridoxine. The 4-ethoxy analog was refluxed

in 48 % HBr to afford the dibromide species, then refluxed in water and AgCl to yield

O OO

H2N

OCN

NH

CNCH2OC2H5

O

a b

NH

CNCH2OC2H5

O

O2N c

N

CNCH2OC2H5

Cl

O2N

d

N

CNCH2OC2H5

Cl

H2Ne

N

CH2NH2

CH2OC2H5H2Nf

N

CH2NH2

CH2OC2H5ClHH2Ng

N

CH2OC2H5HO

OH

N

CH2BrHO

Br

N

HOOH

HO

h

i

HCl

20    

pyridoxine. The overall yield for the reaction was not only very low, but also none of the

starting materials were commercially available isotopically labeled.

2.3 Synthesis Through the Kondrat’eva Reaction

Most of the recent pyridoxine syntheses are centered around the Kondrat’eva approach.8,9

This reaction is an inverse-demand [4+2] cycloaddition between 4-methyl-5-ethoxy

oxazole and a variety of dienophiles and has been shown to form cinchomeronic acids .10

The idea was to place all the necessary functional groups onto the oxazole in order to go

directly to a pyridoxine analog. This new way has shortened the pyridoxine synthesis

tremendously from previous approaches, and is now the most commonly used synthetic

stragey employed. Scheme 2.3 shows the most current synthesis for pyridoxine.

Scheme 2.311 Kondrat’eva reaction synthesis to pyridoxine a) HCOOH/EtOH, b) P2O5, DCM c) Diethyl maleate d) Lithium Aluminum Hydride, Et2O

HO2C NH2

ab

NH

EtO2C

O

N

O OEt

N

HOCO2Et

CO2Et

N

HOOH

c

d

HO

21    

The tandem N-formylation/esterfication is performed in a autoclave at high

temperature/pressure with formic acid and ethanol. This ester is then cyclized to the

oxazole The Diels-Alder reaction occurs with diethyl maleate but has been shown to react

with a variety of other dienophiles. The product is reduced with lithium aluminum

hydride to give pyridoxine.

2.4 Vollhardt’s Synthesis Through [2+2+2] Cobalt Mediated Cycloaddition

This particular synthesis uses a [2 + 2 + 2] cycloaddition between α,ω-diynes with nitriles

and α,ω-cyanoalkynes with alkynes Scheme 2.4.4

Scheme 2.4 Vollhardt synthesis a) n-BuLi, SnMe3Cl b) CH3CN, CpCo(CO2), m-xylenes

c) I2, CHCl3 d) CuI, NaOMe e) 1. 48% HBr 2. AgCl, H2O

The first step was the synthesis of the diyne compound by lithiating the bis-2-propynyl

ether, and then reacting with trimethylstannyl chloride. Next, a regioselective cyclization

catalyzed by η5-cyclopentadienyl dicarbonyl cobalt (CpCo(CO2)) was employed with the

OSnMe3

SnMe3 N

OSnMe3

N

OOMe

N

HO

OHHO

Oa

c

e

b

N

OId

22    

diyne and acetonitrile refluxed in m-xylenes. The product monodestannylated, upon

chormatographic purfication giving the monostanyl pyridyl compound. Cleavage of the

aryl-tin bond was first initiated with a metal-metal exchange with CuI then, the

methoxide anion was able to nucleophilically displace the resulting aryl-halide bond. The

inner ether was then cleaved by refluxing in HBr and the methoxy group was deprotected

with AgCl.

23    

References:

1. Ichiba, A., Emoto, S., Sci. Papers of Inst. Phys. And Chem. Research, 1941, 38, 317-352.

2. Harris, S.A., Folkers, K., J. Am. Chem. Soc., 1939, 61, 3307-3310. 3. Blackwood, R.K., Hess, G.B., Larrabee, C.E., Pilgrim, F.J., J. Am. Chem. Soc. 1958,

80, 6244-6249. 4. Parnell, C.,A., Vollhardt, K.P.C., Tetrahedron, 1985, 41, 5791-5796. 5. Gunsalus, I.C., Bellamy, W.D., Umbreit, W.W., J. Biol. Chem, 1944, 155, 685-686. 6. Stiller, E.T., Keresztesy, J.C., Stevens, J.R., J. Am. Chem. Soc, 1939, 61, 1237-1242. 7. Harris, S.A., Stiller, E.T., Folkers, K., J. Am. Chem. Soc. 1939, 61, 1242-1244. 8. Kondratjeva, G. Y, Khim. Nauka I Prom., 1957, 2, 666. 9. Sharif, S., Schagen, D., Toney, M.D., Limbach, H.H., J. Am. Chem. Soc., 2007, 129,

4440-4455. 10. Firestone, R.A., Harris, E.E., Reuter,W., Tetrahedron, 1967, 23, 943-955. 11. Chan-Huot, M., Niether, C., Sharif, S., Tolstoy, P.M., Toney, M.D., Limbach, H.H., J. Molec. Struc. 2010, 976, 282-289.

24    

Chapter 3

Current PLP Synthesis and Comparison to Limbach Synthesis

25    

3.1 Introduction

The current synthesis of PLP was published by the Limbach group1, but there were

several issues with their approach. There are several stipulations that were put forth

before we landed on our specific approach. First, we had to design a synthesis that

allowed us to use commercially available isotopically labeled starting material. Second,

the synthesis had to be high yielding and able to recover starting material after each step.

Third, cost analysis played a crucial role in designing the synthesis and this synthesis is

the cheapest route to our desired product. Lastly the synthesis had to be easily

reproducible so that anybody could follow the directions and easily makes isotopic

variants of PLP. We understood there was no one single synthesis to produce all

isotopically labeled PLP analogs, and after reviewing all of the literature available we

determined a modified Kondrat’eva approach satisfied all of our needs for the synthesis

of PLP.

26    

3.2 Current Published Synthesis of PLP

Scheme 3.1 details the current published synthesis and this section will detail some of the

issues with the synthesis.

Scheme 3.11 Limbach Synthesis a)HCO2H/EtOH, b) P2O5 c) Diethyl maleate d) LAH e) KMnO4, NaHSO3, 50% H2SO4, p-Toluidine f) H3PO4/P2O5

The first step is a combination of a N-formylation with an esterification of alanine. This

reaction is placed inside of a high-pressure autoclave and heated at 200 °C for 24 hours

yielding 50% of Ethyl-N-formyl-alaninate 1. Next, 1 is cyclized through a 5-exo-trig ring

cyclization which is thermally allowed by Baldwin’s rules,2-4 by dissolving the ethyl-N-

formyl-alaninate in dichloromethane (DCM) then slowly adding P2O5. This reaction yield

was roughly 50% and made 4-methyl-5-ethoxyoxazole 2. Once the oxazole has been

purified it is placed into a small vial, alongside with Diethyl maleate, sealed, and heated

H2N CO2H

a)NH

CO2Et

Ob)

N

O OEt

c)

N

HOCO2Et

CO2Etd)

N

HO

OH

N

HO

N

e)f)

N

HO

O

OPO3H2

OH

OH

1 2

34

56

27    

for 3 days. After workup 4,5-ethoxycarbonyl-3-hydroxy-2-methyl-pyridine 3 is obtained

in roughly 60 % yield. Product 3 is reduced to pyridoxine 4 with lithium Aluminum

Hydride (LAH) at 90% turnover. Pyridoxine 4, is then oxidized by an in situ formation of

MnO2 then scavenged from the aqueous mixture with p-Toluidine to give N-

(pyridoxlidene)-tolylamine hydrochloride 5 in 44 % yield. Lastly, the imine free hydroxyl

group is monophosphorylated and the imine is deprotected with a H3PO4/P2O5 mixture,

and the resulting crude product is placed on top of an ion exchange resin and eluted with

water giving 24 % yield of our desired product pyridoxal-5’-phosphate (6). Overall, the

six-step synthesis gives a 1.4 % yield, which corresponds to $9500 for 1 g of 15N-PLP

and ~$93,000 for 1 g of [15N,13C] PLP. This specific synthesis was extremely costly and

some of the steps were questionable in their reproducibility. For step a, placing the

simultaneous N-formylation/esterification returned mainly starting material regardless of

how dry the formic acid and ethanol were. In step c, regardless of how pure the oxazole,

was the yield was not consistent with the literature value and several side products

formed. For step e the allylic oxidation/imine formation did not work at all and the MnO2

was never confirmed to be formed in situ. The aromatic primary amine was too water-

soluble and wouldn’t crystallize out product (5). The following section will discuss the

new method and some changes made to make the synthesis more reproducible, higher

yielding, cheaper and the same number of steps.

28    

3.3 Improvements/Discussion of Synthetic Route to PLP

Scheme 3.2 illustrates the modified synthetic route to PLP.

Scheme 3.2 Current published PLP synthesis a) SOCl2/EtOH b) triethylorthoformate (TEOF) c) P2O5 d) diethyl maleate e) LAH f) MnO2/p-phenitidine g) H3PO4/P2O5

The first step of the reaction is an esterification of L-alanine to the ethyl ester5. A solution

of alanine in absolute ethanol is mixed with 4 equivalents of thionyl chloride and heated

gently. The key to this reaction is crystallizing out the alanine ethyl ester 7 through a

layered recrystallization. A pressure recrystallization works by dissolving 7 in a small

amount of absolute ethanol then by gently pipetting an excess of diethyl ether, without

disturbing the ethanol layer, until the heterogeneous solution becomes cloudy. This

mixture is then set aside at room temperature until the formation of crystals begin at

which point the flask is transferred to the freezer and allowed to complete the

H2N CO2H

a)

CO2EtH2Nb)

NH

CO2Et

O

N

O OEt

c)

d)

N

HO

OH

OH

N

HO

N

OEt

OH

e)

f)

N

HO

O

OPO3H2

71

2

4

86

29    

crystallizing process overnight. The yield of this reaction was ~100 % and the product 7

continued to the next step without any further purification.

The next step is the N-formylation of compound 76. The solid is reacted with 3 molar

equivalents of triethylorthoformate. Once all of the excess solvent is evaporated, the

crude is sufficiently pure and used in the next step of the synthesis and yielded 95% of

product 1.

The next step of the reaction is the formation of the 4-methyl-5-ethoxy oxazole via a 5-

exo-trig ring cyclization.1 The excess of the dehydrating agent, P2O5, forces the loss of

water instead of the better leaving group, ethanol, to give the desired oxazole 2 in 60 %

yield, Figure 3.1.

Figure 3.1. Proposed mechanism for the formation of oxazole 2, where LA stands for the Lewis acid P2O5

In Figure 3.1, the first step of the proposed mechanism is the 5-exo-trig cyclization,

which is thermally allowed according to Baldwin’s rules.2-4 Next, the oxyanion formed

chelates to the P2O5 and deprotonation at the nitrogen occurs. The phosphorous-oxy

complex is then eliminated through anchimeric assistance (since the non bonding pi

30    

electrons on the oxygen are in an anti orientation from the leaving group) and

deprotonation and rearmotization occurs to form the oxazole.

Once the oxazole has been formed, the next step is a Diels-Alder reaction. The electron-

poor dienophile diethyl maleate was used and Figure 3.2 gives a proposed mechanism

according to Firestone, Harris and Reuter.7 In Figure 3.3, the researchers were puzzled by

the mechanism of forming exclusively the 3-hydroxy derivative. The mechanism

suggests unfavorable cis elimination in order to turn the oxo bridgehead into a

hemiacetal. Figure 3.3 demonstrates the 3 possible intermediates formed with the Diels-

Alder reaction of 4-methyl-5-ethoxy oxazole.

Figure 3.2 Mechanism of the Diels-Alder reaction that forms compound 3

31    

Figure 3.3 Possible intermediates of the Diels-Alder adduct

Looking at Figure 3.3, intermediate A possesses a carbenium carbon atom at the bridge-

head of the small bicylic. The carbenium ion at the bridgehead is an extremely high-

energy form and therefore was eliminated as a possible intermediate. Intermediate C, as

shown in the mechanism, has a cis-elimination, which is unfavorable. Intermediate B

seemed the likely intermediate. It was proposed that, 7 from the workup, would attack the

carbocation in intermediate B to form a hemiacetal and elimination would occur to form

the aromatic pyridine ring. Unfortunately, if intermediate B were the correct choice, the

3-alkoxy intermediate should have been isolated but never was. Studies also with 18O-

water also never showed any incorporation of the label at the 3-position. As stated earlier,

the 3-hydroxypyridine derivatives was the exclusive product for every dienophile

reaction with the oxazole. The researchers came to two conclusions, either intermediate C

was correct where the oxo-bridgehead spontaneously opens to give a carbocation at the 6

position in the ring, or the cis-elimination is exceptionally easy.

Once 3 has been isolated, the reduction with lithium aluminum hydride afford Pyridoxine

41. Pyridoxine is dissolved into an acidic aqueous solution, with powdered MnO2 added,

32    

and stirred at room temperature to initiate the selective allylic oxidation/protection.8 At

this point, it is important to make the p-phenetidine soluble in water by stirring with

water and adding concentrated HCl until a homogenous solution is formed. Once the

reaction has completed, the crude reaction mixture is filtered and the p-phenetidine

solution, alongside with a solution of NaOAc, is added to the filtrate. After allowing the

mixture to sit in the fridge overnight a yellow solid 9, crystallizes out from the aqueous

solution. After filtering and washing with cold water product 9 is isolated, in 95% yield,

and is used through to the next step without any further purification needed. The allylic

oxidation with MnO2 is a well studied reaction9, but here it was necessary to convert the

transformed aldehyde into an imine to protect from hemiacetal formation by the adjacent

primary alcohol Figure 4.3. The hemiacetal is known to form in acidic aqueous solutions

rapidly.

Figure 3.4 Hemi acetal formation of oxidized yridoxine in acidic solution

The final step of the synthesis is the monophosphorylation at the free primary hydroxyl

group and deprotection of the imine group to form pure PLP in its biologically active

N

HO

OOH

N

HOO

HO

H

33    

form.10 First, a mixture of 85 % ortho-phosphoric acid and P2O5 are stirred together under

nitrogen gas. Once the exothermic dissolution subsides, 9 is added and the reaction

mixture is gently heated. After heating between 4-5 hours, 0.1 M HCl is added and the

reaction mixture is heated for a few minutes to deprotect the imine and form the

aldehyde.

While the reaction is heating, the cation exchange resin needs to be prepared accordingly.

The resin that works the best is the Dowex® 50WX4 hydrogen form with a 200-400 mesh

size. The resin is then poured into a column and first washed with distilled water,

absolute alcohol and lastly distilled water once more. Next, 1 M HCl is passed through,

followed by distilled water, 1 M NaOH, distilled water, 1 M HCl, and lastly distilled

water until the eluted water is no longer acidic.11

Once the column has been prepared, the crude reaction mixture is cooled to room

temperature and then added to the top of the column and the product is eluted with

distilled water. Typically 1-2 L, sometimes more, of distilled water is passed through to

collect all of the PLP. The water is then evaporated with the rotary evaporator with gentle

heating, never allowing the water bath to exceed 45 °C. All but 5.0 mL of the solution is

evaporated and the aqueous solution contacting PLP is suitable for biological studies.

34    

3.4 Synthesis of Isotopic Analogs of PLP

The synthesis discussed in Section 3.3 was utilized to make two isotopic analogs of PLP.

Scheme 3.3 and Scheme 3.4 illustrate synthetic schemes for the synthesis of two

isotopically labeled PLP analogs.

Scheme 3.3 [15N]-PLP synthesis a) SOCl2/EtOH b) triethylorthoformate (TEOF) c) P2O5 d) diethyl maleate e) LAH f) MnO2/p-phenetidine g) H3PO4/P2O5

H215N CO2H

a)

CO2EtH215Nb) CO2Et

15N

O OEt

c)

d)

15N

HO

OH

OH

15N

HO

N

OEt

OH

e)

g)

15N

HO

O

OPO3H2

10 11

1214

15 16

15NH

O

15N

HOCO2Et

CO2Et

13

f)

35    

Scheme 3.4 [15N,13C]-PLP synthesis a) SOCl2/EtOH b) Triethylorthoformate (TEOF) c) P2O5 d) diethyl maleate e) LAH f)MnO2/p-phenetidine g) H3PO4/P2O5

The overall yield for the modified PLP synthesis comes out to ~19 % which is a 11-fold

improvement over the most current published synthesis. This correlates to a $12,000

savings per 1 gram of the [15N]-PLP and $123,000 savings per [13C,15N]-PLP.

One issue with the synthesis shown in Scheme 3.3 and 3.4 is the Diels-Alder reaction.

The cycloaddition with oxazole and diethyl maleate had severe yield/reproducibility

issues. Scheme 3.5 illustrates the various Diels-Alder reactions tested in order to find a

reliable, high yielding reaction.

H215N13C13C

H313C H313C13C13C

15NH2

15N13C

13CO

13CH3

OEt13C13C15NH313C

HOCO2Et

CO2Et

13C13C15NH313C

HO

N

OEt

OH13C13C15NH313C

HO

O

OPO3H2

13CH3

13C13C15NOO

OH

O

O

O

Oa) b) c)

d)e)

g)

17 18

19

20 21

23

13C13C15NH313C

HO

HO

OH f)

22

H HH

H

36    

Scheme 3.5 Various Diels-Alder reactions attempted

Table 3.1 gives an exhaustive overview of the various reactions attempted and the yields

of pyridoxine isolated.

Table 3.1 Summary of various Diels-Alder reactions

N

O OR

R = -Et, -CO2Et

R1 R1

R1= -CO2Et, -CO2Me, -CH2OH, CH2OAc

OR2 R2

R2= -H, -(O)

O O

R3

R3 = -n-propyl, i-propyl

N

HO

OH

OH

Oxazole Dienophile Catalyst Yield of Pyridoxine

(%) -Et -CO2Et7 None/CaO/Rad.

Inhibitor 27

-CO2Me7 None 30

-CH2OH7 CaO, Hydroquinone < 5

-CH2OAc12 CaO < 5

-H7 Trichloroacetic Acid 60

-C(O)7 None < 15

-n-propyl13 CaO <5

-i-propyl13 CaO <5

-CO2Et -CO2Et14 None 30

37    

It became evident that the one dienophile, 2,5-dihydrofuran, was the best choice. Scheme

3.6 now illustrates the new synthetic scheme, which corresponds to a 23 % overall yield.

Scheme 3.6 A new [15N,13C]-PLP synthesis a) SOCl2/EtOH b) triethylorthoformate (TEOF) c) P2O5 d) 2,5-dihydrofuran,hydroquinone, trichloroacetic acid e) MnO2/p-

phenetidine f) H3PO4/P2O5

The synthesis in Scheme 3.6 is essentially the same as the previously reported synthesis

in Schemes 3.3 and 3.4 except for the new Diels-Alder reaction. The new dienophile, 2,5-

dihydrofuran, is used in 20 fold molar excess in relation to the oxazole. It was shown that

a 25 mol % of recrystallized trichloroacetic acid a catalytic amount of hydroquinone

added to the reaction improved yields drastically. The reaction is run in a steel autoclave

heated at 190 °C. for 5 hours.

3.5 Synthesis of [15N]-2-Aminophenol

The inhibitor, 2-Aminophenol (2-AP), works by binding to the E(A-A) intermediate

Figure 3.5.

H215N13C13C

H313C H313C13C13C

15NH2

15N13C

13CO

13CH3

OEt

13C13C15NH313C

HO

N

OEt

OH13C13C15NH313C

HO

O

OPO3H2

13CH3

13C13C15NOO

OH

O

O

O

Oa b c

d f

1718

19

2123

13C13C15NH313C

HO

HO

OH e

22

H HH

H

38    

Figure 3.5 Reaction of 2-Aminophenol (2AP) with the EA-A intermediate

This addition was being investigated, and at the time it wasn’t evident if the amine was

attacking the E(A-A) or if it was an electrophilic aromatic substitution reaction. To

determine which case it was, the [15N]-2AP isotope was synthesized in two steps

according to Scheme 3.7.

Scheme 3.7 Synthesis of [15N]-2AP a) HN15O3(10M), tetra-n-butylammonium bromide (TBAB), ethylene dichloride b)10% Pd/C, NaBH4

The nitration of phenol was carried out using [15N]-Nitric Acid and the phase transfer

catalyst TBAB.15 This prohibits the para nitration and limits it to only a mono ortho-

nitrophenol product. The second step is just the reduction of the nitro group to the amine

N

COO

N

HO

H2O3PO

NH2

OH

H

N

COO

N

HO

H2O3PO

H

NH

OH

E(A-A) E(Q)

OH OH15NO2

OH15NH2

a) b)

24 25

39    

using Pd/C and sodium borohydride.16 Hydrogen gas is generated, in situ, by adding

concentrated HCl. The overall yield was low, but [15N]-nitric acid is relatively cheap.

3.6 Synthetic Approach to [17O]-PLP

Unfortunately, selectively labeling each individual atom within the PLP molecule

couldn’t be accomplished cheaply, through the modified synthesis. The phenol oxygen

protonation state is still something that needs to be investigated fully. Using the modified

synthesis would mean inserting the 17O label in the second step, making it extremely

costly and an inefficient use of isotopic starting material. Introducing the isotope in the

beginning would cost roughly $80,000 per gram of [17O]-PLP. The goal was to redesign

the synthesis and allow for the 17O label to be inserted towards the end. Scheme 3.8

illustrates a different PLP synthesis, one of the very first, completed by Reuben G. Jones

from Eli Lilly.17

Scheme 3.8 Proposed synthesis of [17O]-PLP a) 3.5 M HCl b) triethylorthoformate/Ac2O then NaHCO3 c) Iminoacetylacetone d) H2SO4 then NaN3 e) H2SO4/EtOH f) LAH g)

NaNO2, H2O17 h) MnO2 then p-phenetidine i) H3PO4/P2O5

EtO2C

OCO2Et

Na

EtO2C

OCO2Et EtO2C

OCO2Et

OH

EtO2C

OCO2Et

NH O

N

H2NCO2H

CO2H

N

H2NCO2Et

CO2Et

N

H17O

HO

OH

N

H17O

O

OPO3H2

a b cd

e f,g h,i

27

29[17O]-Pyridoxine [17O]-PLP

30

26 28

40    

In the first step of the synthesis in Scheme 3.8, commercially available enolate of diethyl

oxaloacetate is acidified to transform the enolate anion into the keto form 26.18 Next,

refluxing 26 in triethylorthoformate and acetic anhydride gives the ethoxy analog of 27,

which can then be deprotected to the alcohol with solid sodium bicarbonate. The next

step is just a nucleophillic addition of iminoacetylacetone to 27 at room temperature in

diethyl ether.17 Product 28 is then dissolved in concentrated sulfuric acid and, after an

hour, powdered sodium azide is added slowly.18 Compound 28 initially cyclizes to

diethyl-2-methyl-3-acetyl-4,5-pyridinedicarboxylate. Then upon the addition of sodium

azide a Schmidt reaction occurs and compound 29 is isolated Figure 3.6.

41    

Figure 3.6 Proposed mechanism for the transformation of 26 to 27

The reduction of 29 with sodium borohydride and AlCl3 in diglyme has been

unsuccessful so far.18 This particular reduction was chosen to eliminate the formation of

lactones. A possible solution to this might be the esterification of 29 to the ethyl

EtO2CCO2Et

O

NH O

N

O HO CO2EtCO2Et

N

CO2EtCO2Et

O

N3

H

N

N OH CO2EtCO2Et

NN

N

N CO2EtCO2Et

NN

H

H2ON

NCO2Et

CO2Et

H

-N2

OHH

Proton transfer

N

HN

CO2EtCO2Et

-H2O

N

NCO2Et

CO2EtOHH

O

Hydrolysis

N

H2NCO2H

CO2H

42    

carboxylate, 30, then reduction using lithium aluminum hydride.19 Once a viable

reduction has been discovered and 3-amino pyridoxine has been isolated, the 17O label

can be inserted using sodium nitrite dissolved in [17O]-H2O. After the insertion of the

isotope, the last two steps are the same as the previous synthesis.

43    

References:

1. Chan-Huot, M., Niether, C., Sharif, S., Tolstoy, P.M., Toney, M.D., Limbach, H.H., J. Molec. Struc. 2010, 976, 282-289. 2. Baldwin, J.E., Thomas, R.C., Kruse, L.I., Silberman, L. J. Org. Chem. 1977, 42, 3846-3852. 3. Baldwin, J.E. J. Chem. Soc. Chem. Commun., 1976, 18, 734-736. 4. Baldwin, J.E., Cutting, J., Dupont, W., Kruse, L., Silberman, L., Thomas, R.C. J. Chem. Soc., Chem. Commun., 1976, 18, 736-738. 5. Lee, S.J., Kim, E., Seo, M.L., Do, Y., Lee, Y.A., Lee, S.S., Jung, J.H., Kogiso, M., Shimizu, T. Tetrahedron, 2008, 64, 1301-1308. 6. Dean, A., Ferlin, M.G., Brun, P., Castagliuolo, I., Badocco, D., Pastore, P., Venzo, A., Bombi, G. G., Di Marco, V.B. Dalton Trans., 2008, 7,1689-1697. 7. Firestone, R.A., Harris, E.E., Reuter,W., Tetrahedron, 1967, 23, 943-955. 8. Florentiev, V.L., Ivanov, V.I., Kapreisky, M.Ya., Methods in Enzymology, 1970, 18, 567-598. 9. G. Cahiez, M. Alami, R. J. K. Taylor, M. Reid, J. S. Foot, "Manganese Dioxide", in Paquette, Leo A., Encyclopedia of reagents for organic synthesis, New York: J. Wiley & Sons, 2004. 10. Iwanami, M., Numata, T., Murakami, M., Bull. Chem. Soc. Japan., 1968, 41, 161-165. 11. Armarego, W.L.F., Perrin, D.D., Purification of Laboratory Chemicals. Oxford: Butterworth/Heinemann, 1996. Paperback. 12. Harris, E.E., Zabriskie, J.L., Chamberlin, E.M., Crane, J.P., Peterson, E.R., Reuter, W., JOCS. 1969, 34, 1993-1996. 13. Zou, Y., Shi, X., Zhang, G., Li, Z., Jin, C., Su, W., Org. Process Res. Dev. 2013, 14, 1498-1502. 14. Murakami, M., Iwanami, M,. Bull. Chem. Soc. Jap. 1968, 41, 726-727.

44    

15. Joshi, A.V., Baidoosi, M., Mukhopadhyay, S., Sasson, Y., Org. Proc. Research and Develop., 2003, 7, 95-97. 16. Smith, C.J., Ali, A., Chen, L., Hammond, M.J., Anderson, M.S., Chen, Y., Eveland, S.S., Guo, Q., Hyland, S.A., Milot, D.P., Sparrow, C.P., Wright, S.D., Sinclair, P.J. Bioorg. Med. Chem. Lett. 2010, 20, 346-349. 17. Jones, R.G., J. Am. Chem. Soc., 1951, 73, 5244-5247. 18. Jones, R. G. 2-Methyl-3-Acetyl-4,5-Pyridine Dicarboxylic Acid, Lower Alkyl Esters Thereof and Intermediates. USPTO, assignee. Patent 2,724,714. 22 Nov. 1955. Print. 19. Jones, R.G., 2-Methyl-3-Amino-4,5-Di-Hydroxy Methylpyridine And Its Salts and The Preparation Thereof. USPTO, assignee. Patent 2,650,232. 25 Aug. 1953. Print.

45    

Chapter 4

Synthetic Background/ Synthetic Attempts Towards Cannabinoid Analogs

46    

4.1 Introduction

Cannabinoid analogs have evolved from a recreational to a promising therapeutic agent.

The first isolated cannabinoid analog, Δ1-tetrahydrocannabinol (Δ1-THC), has been used

to treat a wide range of disease (cancer, MS, etc.) along with easing nausea and

stimulating appetite during chemotherapy.1-12 Its efficacy in a wide range of symptom

and disease controls has sparked many synthetic strategies to solve this tricyclic

compound. The following sections will give a background of the previous syntheses of

some cannabinoid analogs and an attempt by our lab to devise a new synthetic route.

4.2 Cannabinoid Overview

Cannabinoids are usually segregated into three distinct classes, phyto-, synthetic, and

endogenous cannabinoids. The phytocannabinoids encompass the over 60 different

compounds that were extracted from the plant Cannabis satvia. Synthetic cannabinoids

are synthetic variants modeled after the phytocannabinoids, and the endogenous

cannabinoids represent those naturally produced within an organism (Table 4.1).

47    

Class Name Structure

Phytocannabinoid Δ1-Tetrahydrocannabinol

Cannabidiol

Cannabivarin

Cannabichromene

Synthetic HU-210

Nabilone

CP-55,940

O

OH

OH

HO

O

OH

OH

O

O

OH

OH

O

OH

O

OH

OH

OH

 

Table 4.1 Chemical structures of phyto-, synthetic and endo- cannabinoids  

48    

Endocannabinoid Anandamide

2-arachdionoyl glycerol

2-arachidonoyl glycerol

ether

Table 4.1 Chemical structures of phyto-, synthetic and endo- cannabinoids continued.

Most of these compounds act as agonists for either the Cannabinoid receptor 1 (CB1) or

Cannabinoid receptor 2 (CB2) or both. CB1 and CB2 are both G protein-coupled

receptors, with the former being found predominately in the central nervous system,

making it a prime target for CNS disorders.

Δ1-Tetrahydrocannabinol (Δ1-THC) was first isolated and fully characterized in 1964 by

Mechoulam13 and is also responsible for the psychoactive affects during recreational use.

It has been shown to have several therapeutic affects as well. Figure 4.1 illustrates the

numbering scheme of Δ1-THC and the stereochemistry of the naturally occurring

psychoactive and therapeutic analog.

NH

OH

O

O

OOH

OH

OOH

OH

49    

Figure 4.1 Left structure shows the monoterpenoid numbering the right structure shows

the natural occurring (-)-Δ1-THC. Stereochemistry at carbons 3 and 4 are R, R.

4.3 Previous Syntheses of Δ1-Tetrahydrocannabinol (Δ1-THC)

The very first synthesis of Δ1-THC was accomplished by Gaoni and Mechoulam in

1965.14 In this synthesis, the lithium derivative of the methoxy protected olivetol, 31,

condenses with citral gave the methoxy protected Cannabidiol (CBD) 32. Heating 32

with methylmagnesium idodie deprotects and converts it to a racemic mixture of CBD.

Upon heating with HCl the CBD is converted to Δ1-THC with an overall yield of 2 %.

(Scheme 4.1)

O

OH1

2

3

45

6

7

O

OHH

H

50    

Scheme 4.1 First synthesis of Δ1-THC

Roughly a year later, Taylor and coworkers were able to synthesize a racemic mixture of

Δ6-THC in one step using 10% BF3 as a catalyst in roughly 10-20 % yield.15

Unfortunately this reaction also yielded the cis-Δ1-THC along with the isocannabinoid

33. Mechoulam altered the reaction by using 1 % BF3 and was able to get the trans-Δ1-

THC in roughly 20 % yield along with the cis- Δ1-THC isomer. (Scheme 4.2)

OMe

MeO C5H11

LiOOMe

MeO C5H11

OH

HO C5H11 O

OH

C5H11

H

H

31 32

CBD

51    

Scheme 4.2 Mechoulam synthesis

Evans, in 1997, tried making Δ1-THC through the first stereospecific route.16

Unfortunately the inactive (S,S) Δ1-THC was isolated through numerous steps in 21 %

yield. The chiral catalyst, bis(oxazoline) copper (II), was used to catalyze the Diels-Alder

reaction between acrylamide, 34, and 1-acetoxy-3-methylbutadiene 35. The resulting

cycloadduct is then cleaved with LiOBn to form 36, which in turn is reacted with

methylmagnesium bromide to form the Diol 37. The Diol is reacted with olivetol and

cyclized to form (S,S) Δ1-THC. (Scheme 4.3)

(±)-cis-Δ1-THC

(±)-trans-Δ6-THC

(±)-trans-Δ1-THC + cis-Δ1-THC

OH

HO C5H11

OO

OH

C5H11

H

HOH

O C5H11

32

O

OH

C5H11

H

H

10 % BF3

1 % BF3

33

52    

Scheme 4.3 Evans stereospecific synthesis of (S,S) Δ1-THC a) 1. Cationic

bis(oxazoline)copper (II) catalyst 2. LiOBn b) MeMgBr c) Olivetol d) ZnBr2, MgSO4

Trost’s synthesis in 2007 was the first stereospecific synthesis, which led to the most

prevalent isomer in Cannabis sativa, (R,R) Δ1-THC, through 12 steps with an overall

yield of 30 %.17 This reaction uses a Molybdenum catalyzed asymmetric allylic alkylation

to set the correct stereochemistry. The first part of the synthesis converts olivetol into the

carbonate 39 in 4 steps. Next, the Mo-catalyzed allylic alkylation of 39 with sodium

dimethyl malonate yields product 40. The product is then decarboxylated, and then

alkylated followed by a ring closing metathesis to form 43. Lastly this product was

isomerized, methylated with MeLi, mono-deprotected using NaSEt, cyclized with ZnBr2,

and lastly fully deprotected, once again with NaSEt, yielding the natural occurring isomer

of Δ1-THC. (Scheme 4.4)

OAc N O

OO

3435

O

HOAc

BnO36 OH

HOH

37

H

HO

OH

C5H11

HO

H

38

O

OH

C5H11

H

H

1-THC(S,S)

ab

c d

53    

Scheme 4.4 Trost synthesis a) 5 mol % [Mo(CO)3C7H8], sodium dimethyl malonate, 7.5% (R,R)-DACH-pyridyl TROST ligand b) 1. NaOH 2. HCl c) LDA, 4-iodo-2-

methylbut-1-ene d) 1. (MeO)2SO2, K2CO3 2. Grubbs II catalyst e) MeLi f) NaSEt g) 1. ZnBr2, MgSO4 2. NaSEt

4.4 Attempts towards synthesis of THC

The goal was to design a new synthetic strategy that was shorter and used cheaper

starting material, since olivetol is quite expensive and difficult to synthesize.18 The

following synthetic attempts uses orcinol as the starting material, since we hypothesized

that the shorter carbon chain should reduce the psychotropic side affects but retain the

therapeutic affects and also bind to the CB1 receptor as well as the natural occurring

isomer. The second goal was to design a synthesis that mimicked the biosynthetic route.

Figure 4.1 shows the key intermediate that can be transformed into THC or CBD.

OMe

C5H11 OMe

4 stepsOMe

C5H11 OMe

OCO2CH3

OMe

C5H11 OMe

CO2MeMeO2C

OMe

C5H11 OMe

MeO2COMe

C5H11 OMe

MeO2COMe

C5H11 OMe

MeO2C

O C5H11

OMe

e,f,g

3940

43

44

4142

54    

Figure 4.2 Biosynthetic pathways to THC, CBD and CBC

Here, in Figure 4.1, one intermediate is easily converted into three distinct cannabinoids

using three distinct enzymes. The synthetic plan was to functionalize orcinol at the

position ortho to both alcohol groups, with a carbonyl which would allow for either a

Wittig reaction to make a vinylic group, which could potentially react with a terpenoid

through an olefin metathesis or an aldol condensation to create a dienophile to help create

the fused bicylic for THC or the cyclohexane ring for CBD.

CnH2n+1

OH

HOCOOH

O

OH

RCnH2n+1

O

OH

RCnH2n+1

HO

OH

CnH2n+1R

n = 3, 5CBGA

R = COOH (CBCA)R = H (CBC)- CO2

R = COOH (THCA)R = H (THC)- CO2

R = COOH (CBDA)R = H (CBD)- CO2

THCA synthase

CBDAsynthase

CBCA synthase

55    

The first synthetic attempt was to utilize the Grubbs catalysis because of its ubiquity and

stability under various conditions. Scheme 4.5 summarizes the synthetic approach.

Scheme 4.5 Olefin Metathesis synthetic route a) K2CO3, MeI b) n-BuLi, TMEDA, DMF c) CH2=PPh3 d) Grubbs II Generation

The first three steps were the same approach that Trost’s synthesis had employed, which

begins by the dimethyl protection of the hydroxyl groups on orcinol.17 Next, 45 was

formylated α to the two hydroxyl groups. Lastly, 46 was reacted with

methyltriphenylphosphonium Bromide, whereby a Wittig reaction takes place to add the

vinylic group and obtain 47. Unfortunately the olefin metathesis between 47 and linalool

would not take place, since neither was able to couple with the Ru center regardless of

which Grubbs catalyst was used.

Next we turned our attention to a slight variation in the synthesis with the key step a

Diels-Alder reaction. (Scheme 4.6)

OH

OH

OMe

OMe

OMe

OMe

O

OMe

OMe

OH

OMe

OMe

OHOH

O

OH

THC CBD

45 46 47

48

a b c

d

OH

56    

Scheme 4.6 Diels-Alder route a) K2CO3, MeI b) n-BuLi, TMEDA, DMF c) NaOH, acetone d) Lewis acid catalyst

In this variation of the synthesis steps a and b are identical to the aforementioned route.

Once 46 was obtained an aldol condensation with acetone occurs and 49 is obtained in

high yield. The Diels-Alder step was extensively studied using various dienes and Lewis

acid catalysts. Unfortunately neither of the Diels-Alder reactions gave any appreciable

amount of 50. According to Piermatti et. al.18 this reaction occurs only under extremely

high pressures, (18 kBar), which is not practical or safe, so this synthetic strategy was

deemed useless.

Scheme 4.7 depicts the most up to date attempt (and most promising) synthetic route

towards THC/CBD.

OH

CBD

OH

OH

OMe

OMe

OMe

OMe

O

OMe

OMe

O OTMS

OMe

OTMS

OH

OTHC

OMeO

OH

a b c

d

45 46 49

50

57    

Scheme 4.7 Biomimetic approach a) K2CO3, MeI b) n-BuLi, TMEDA, DMF c) NaI, AlCl3 d) KOH, 6-methyl-5-hepten-2-one e) NaOH, 6-methyl-5-hepten-2-one

Once again the first few reactions are very similar as the previous syntheses. In order to

isolate CBD the Aldol condensation of 46 with 6-methyl-5-hepten-2-one gave 52 in

relatively good yields isolated as a solid. The proposed conversion to CBD from 52 is

shown in Scheme 4.8. The monodeprotection of 46 to form 51 was accomplished with

NaI and AlCl3 and was converted to 53 through an Aldol condensation with 6-methyl-5-

hepten-2-one. The proposed conversion of 53 to THC is illustrated in Scheme 4.8 as well.

OH

OH

OH

OMe

OMe

OMe

OMe

O

OH

O

OMe

OMe

OOMe

OH

O

OMe

OH

O

OH

a b c

d e

45 4651

5253

THCCBD

58    

A      B

Scheme 4.8 Proposed synthetic pathways to CBD and THC a) 1-10% acid b) MeMgBr

c) K-t-pentoxide/NaSEt

In Scheme 4.8 reaction pathway A, 52 could potentially be cyclized using a catalytic

amount of a weak organic acid (for example trifluoroacetic acid). Next, a Grignard

reaction will form the tertiary alcohol. The last steps include a dehydration with a bulky

base and deprotection of the methoxy groups with sodium ethanethiolate, which would

hypothetically give CBD. For reaction pathway B, the tricyclic fusion will occur, since

the one methoxy group had been previously removed. With the cannabinoid skeleton

intact, once again a Grignard reaction with methylmagnesium bromide and

dehydration/deprotection with a bulky base and sodium ethanethiolate, respectively,

should produce Δ1-THC. Though this reaction is not stereospecific (and might give a

mixture of various isomers) it is a good starting point and uses relatively cheap materials.

Preliminary results for the cyclization of 53 indicate a disappearance of the benzylic

alkene in the 1H-NMR spectrum, a disappearance of the –OH functional group in the IR

spectrum, and a m/z peak value which corresponds to the project mass of the product.

OH

OH

O

OMe

OMe

O

OMe

OH

O

OH52

53THC

CBD

OMe

O

OMe

OMe

OMe

OH

OMe

O

O

OMe

O

OH

59    

4.5 Conclusion

Various cannabinoids have been previously reported to have potential therapeutic affects

ranging from anti-cancer fighting capabilities to combating various CNS disorders.

Although several syntheses do exist there is still more room for improvement before a

reliable, cheap synthetic drug could be put to market. We had tried several different

routes in order to synthesize CBD and Δ1-THC and found that a biomimetic approach

seemed the most feasible route.

60    

References:

1. Pacher, P., Batkai, S., Kunos, G., Pharmcol. Rev., 2006, 58, 389-462. 2. Guindon, J., De Léan, A., Beaulieu, P., Pain, 2006, 121, 85-93. 3. Amar, M., Journal of Ethnopharmacology, 2006, 105, 1-25. 4. Zajicek, J., Fox, P., Sanders, H., Wright, D., Vickery, J., Thompson, A., Lancet, 2003, 362, 1517-1526 5. Compston, A., Coles, A., Lancet, 2008, 372, 1502–17 6. Guzman, M., Sanchez, C., Galve-Roperh, I., J Mol Med, 2001, 78, 613-625. 7. Sanchez, C., Galve-Roperh, I., Canova, C., Brachet, P., Guazman, M., FEBS Lett, 1998, 436, 6-10 8. Sarker, K. P., Obara, S., Nakata, M., Kitajima, I., Maruyama, I., FEBS Lett, 2000, 472, 39-44 9. Chan, G. C-K., Hinds, R. T., Impey, S., Storm, D.R., J. Neurosci, 1998, 18, 5322-5332. 10. Chan, P.C., Sills, R.C., Braun, A.G., Hasaman, J.K., Bucher, J.R., Fundam Appl Toxicol, 1996, 30, 109-117. 11. Petrocellis, L.D., Melck, D., Palmisano, A., Bisogno, T., Laezza, C., Bifulco, M., Di Marzo, V., Proc Natl Acad Sci USA, 1998, 95, 8375-8380. 12. Melck, D., De Petrocellis, L., Orlando, P., Bisogno, T., Laezza, C., Bifulco, M., di Marzo, V., Endocrinology, 2000, 141, 118-126. 13. Mechoulam, R.; Gaoni, Y. J Amer Chem Soc, 1964, 86, 1646. 14. Mechoulam, R.; Gaoni, Y., J Amer Chem Soc, 1965, 87, 3273-3275. 15. Taylor, E.C., Lenard, K., Shvo, Y., J Amer Chem Soc, 1966, 88, 367. 16. Evans, D. A.; Shaughnessy, E. A.; Barnes, D. M. Tetrahedron Letters 1997, 38, 3193. 17. Trost, B. M.; Dogra, K. Organic Letters 2007, 9, 861. 18. Focella, A., Teitel, S., Brossi, A., J. Org. Chem. 1977, 42, 3456-3457.

61    

Appendix A Experimental Procedure and Spectroscopic Data

Chemicals and solvents were purchased from Sigma Aldrich and used without further

purification. Unless otherwise noted, reactions were performed using standard synthetic

organic techniques under an atmosphere of nitrogen gas. Nuclear magnetic resonance

(NMR) spectra were acquired using a Varian Inova 300 MHz spectrometer. Chemical

shifts (δ) are reported in parts per million (ppm) and are calibrated to the residual solvent

peak. Coupling constants (J) are reported in Hz. Purifications by column chromatography

were performed using silica gel (40 - 63 µm, 230 - 400 mesh) and appropriate eluent.

Analytical data acquired for all previously reported compounds were fully consistent with

existing spectral data.

Compound 10: A round bottom flask charged with L-15N-Alanine (0.231g, 2.56 mmol) ,

purchased from Sigma-Adlrich, dissolved in 5.0 mL absolute ethanol (1M) is chilled in

an ice-salt bath. Once cooled, SOCl2 (0.65 mL, 8.80 mmol) is added slowly. After the

addition is complete, the mixture is heated to 40 °C for 5 hours. After heating, the solvent

is evaporated and crystallized by dissolving in small amount of absolute ethanol and then

diethyl ether is slowly added on top of the ethanol layer, without disrupting the ethanol

layer. The mixture was then allowed to stand at room temperature until crystals started to

form and then placed into the freezer overnight to complete crystallization. The yield was

0.374 g (95 % yield). 1H NMR (D2O, 400 MHz) δ 1.31 (3H, t, J = 7.2 Hz), δ 1.57 (3H,

dd, J = 2.8, 4.4 Hz), δ 4.18 (1H, qt, J = 7.2 Hz), δ 4.31 (2H, qt, J = 7.2 Hz). 13C-NMR

62    

(D2O, 400 MHz) δ 8.40, 10.34, 44.06, 58.75, 166.11. HRMS (m/z): [M+Na] calc for

C5H11(15N)NaO2 141.0657; found 141.0646.

Compound 11: 0.36 g of 10 (2.32 mmol) is dissolved into triethylorthoformate (1.2 mL,

6.96 mmol) and heated at 80 °C for 1.5 hours. The solvent is distilled off in vacuo and 11

was obtained in quantitative yields. 1H NMR (CDCl3, 300 MHz): δ 1.30 (3H t, J = 7.2

Hz), δ 1.45 (3H, dd, J = 2.4, 4.5 Hz), δ 4.23 (2H, qt, J = 7.2 Hz), δ 4.67 (1H, quint, J =

7.2 Hz), δ 6.26 (15NH, dt, J = 92Hz, 5.7 Hz), δ 8.19 (1H, d, J = 16.2 Hz). 13C-NMR

(CDCl3, 400 MHz) δ 14.3, 18.76, 47.07 (d, J = 46 Hz), 61.91, 160.50, 172.80. HRMS

(m/z): [M+H]+ Calc for C6H1215NO3 147.0782; found 147.0800

Compound 12: In a 100 mL round bottom flask 11 (0.53 g, 3.63 mmol) is dissolved in

30 mL dichloromethane. While stirring, 2.08 g of P2O5 is added and the reaction is

refluxed for 24 hrs. The reaction mixture is cooled to room temperature and the reaction

quenched with 20 % aqueous NaOH. The aqueous layer is extracted three times with

dichlormethane, and then the organic phase is washed with water. The organic phase is

then dried with MgSO4 and the solvent removed with a rotary evaporator. The yield of

the reaction was 50 %. 1H NMR (CDCl3, 400 MHz) δ 1.35 (t, 3H, J = 7.2 Hz), δ 2.05 (d,

3H, J = 2 Hz), δ 4.15 (qt, 2H, J = 7.2 Hz), δ 7.37 (d, 1H, J = 12.8 Hz). 13CNMR (CDCl3,

400 MHz) δ 10.2 (d, J = 23.2 Hz), 15.2, 70.3, 104.99, 112.46 (dd, J = 42.8 Hz), 142.31

(d, J = 39.21 Hz). HRMS (m/z): [M+H]+ calc C6H1015NO2 129.0676; found 129.0676.

63    

Compound 13: Under N2, 12 (0.681 g, 5.3 mmol) is combined with diethyl maleate (1.7

mL, 10.6 mmol) and 2.5 g SiO2 (1:1 by total mass). The reaction is heated at 120 °C for 6

hrs. Upon the completion of heating, the reaction is cooled to room temp and placed on a

filter and the crude is eluted off with diethyl ether, then dichloromethane and benzene.

The filtrate is evaporated then a small amount of 1.25 M Ethanolic-HCl is added to

dissolve the crude product. To the ethanol solution, diethyl ether is added, gently, on top

in a way to not disrupt the ethanol layer then the bilayer is placed in the freezer overnight

to crystallize out the product. Roughly 0.65 g of 13 was isolated (50 % yield). 1H NMR

(CDCl3, 300 MHz) δ 1.42 (3H, t, J = 7.2 Hz), δ 1.43 (3H, t, J = 7.2 Hz), δ 2.97 (3H, d, J =

3 Hz), δ 4.44 (2H, qt, J = 7.2 Hz), δ 4.54 (2H, qt, J = 7.2 Hz), δ 8.36 (1H, d, J = 3.3 Hz).

HRMS (m/z): [M+H]+ calc for C12H15 (15 N)O5 255.0993; found 255.1026.

Compound 14: Under a flow of N2, lithium aluminum hydride (0.44 g, 13.6 mmol) is

added, very carefully, to 90 mL of anhydrous diethyl ether while stirring at room

temperature. Next, crystalline 13 (0.43 g, 1.7 mmol) is added into the ethereal solution.

The reaction is refluxed overnight in a N2 atmosphere. Once cooled to room temperature,

35 mL of DI H2O is gently added and the mixture is filtered and washed with boiling

water (2x50 mL). The filtrate is then saturated with CO2 for an hour, and then evaporated

to dryness in vacuo. The resulting solid is then extracted with boiling methanol (2 x 40

mL) and the solvent evaporated once again to dryness. A small amount of 3 M

methanolic-HCl is added to dissolve resulting oil and then diethyl ether is gently poured

on top of the methanol layer in a way not to disrupt the layer and lastly the bilayer is

64    

placed inside the freezer overnight. Pure 14 (0.26 g) was isolated (90 % yield). 1H NMR

(D2O, 300 MHz) δ 2.50 (3H, d, J = 3.3 Hz), δ 4.68 (2H, s), δ 4.86 (2H, s), δ 7.98 (1H, d, J

= 3.3 Hz). HRMS (m/z): M*+[-H] calc for C8H10 (15N )O3 169.0636; found 169.1360.

Compound 15: Crystalline 14 (0.18 g, 1.05 mmol) was dissolved in 0.3 M H2SO4 (3.5

mL). To the acidic solution, activated MnO2 (95 mg, 1.1 mmol) was added and the

reaction is stirred at room temperature for 3.5 hrs. The reaction is filtered and the filter

cake was rinsed with 1 mL of DI water. At this point the 0.5 M p-phenetidine

hydrochloride solution needs to be made. In a small beaker with 15 mL of DI water, 1.3

mL of p-Phenetidine is added followed by the immediate addition of 1 mL of

concentrated HCl then more DI water is added to make the total volume 20 mL. This

solution is stirred until no solid is present. Once made, 2.8 mL of the p-Phenetidine

hydrochloride solution is added to the filtrate followed immediately by the addition of 4.2

mL of a 2M NaOAc solution. This mixture is placed in the fridge and 15 separates out as

a yellow solid. The solid is filtered off and washed with cold DI water. Roughly 0.29 g of

15 was isolated (95 % yield). 1H NMR (CDCl3, 300 MHz) δ 1.44 (3H, t, J = 6.9 Hz), δ

2.56 (3H, d, J = 2.4 Hz), δ 4.08 (2H, qt, J = 7.2 Hz), δ 4.87 (2H, s), δ 6.96 (2H, d, J = 8.7

Hz), δ 7.34 (2H, d, J = 8.7 Hz), δ 8.06 (1H, d, J = 10.5 Hz), δ 9.15 (1H, s). HRMS (m/z):

[M+H]+ calc for C16H19(15N)NO3 288.1361; found 288.1371.

Compound 16: In a 25 mL round bottom flask, P2O5 (0.75 g, 5.4 mmol) and 85 % o-

H3PO4 (0.41 mL, 7.0 mmol) are stirred until homogenous. Once the reaction is cooled to

65    

room temperature, solid 15 (100 mg, 0.35 mmol) is added and stirred at room

temperature until homogeneous. The reaction is then heated at 40 °C for 4 hours, cooled

to room temperature, and then 0.1 M HCl (0.22 mL) is added to the mixture and heated at

60 °C for 15 mins. Before adding the crude product to the ion-exchange resin, it needs to

be prepared as follows. After packing a column with the resin, DI water, ethanol then DI

water are passed through individually and the filtrate discarded. Then the following are

added to the resin in order; 1M HCl, DI water, 1M NaOH, DI water, 1M HCl, and then

once more DI water until eluent is no longer acidic. Once the column is prepared the

crude reaction mixture is added to the column and eluted with a large amount (1 L) of DI

water. The combined filtrate is evaporated on the rotary evaporator to about 5-10 mL. It

is imperative that the water bath temperature not exceed 40 °C. Roughly 700 mg of 16

was isolated (80 % yield). 1H NMR (DMSO-d6, 400 MHz) δ 2.44 (3H, d, J = 2.7 Hz), δ

5.20 (2H, d, J = 7.2 Hz), δ 8.13 (1H, d, J = 10.5 Hz), δ 10.4 (1H, s). HRMS (m/z):

[M+Na]+ calc for C8H10(15N)NaO6P 271.0114; found 271.0103.

Compound 17: See experimental procedure for 10. 1H NMR (DMSO-d6, 300 MHz) δ

1.23 (3H, t, J = 7.2 Hz), δ 1.4 (3H, dt, J = 130, 2.7 Hz) δ 4.02 (1H, m, J = 146 Hz), δ 4.2

(dq, 2H, J = 3.6 Hz), δ 8.50 (15NH, s, br). 13C-NMR δ 13.3, 15.22 (d, J = 136 Hz), 48.96

(ddd, J = 26, 84, 110 Hz), 170.97 (d, J = 246 Hz). HRMS (m/z): [M+Na-H] calc for

13C315NC2H11O2 143.0686; found 143.0677.

66    

Compound 18: See experimental procedure for 11. 1H NMR (CDCl3, 300 MHz) δ 1.23

(3H, t, J = 7.2 Hz), δ 1.38 (3H, m, J = 130 Hz), δ 4.15 (2H, dq, J = 3 Hz), δ 4.59 (1H, dsp,

J = 143, 6 Hz), δ 6.25 (15NH, ddd, J = 92, 7.5 Hz), δ 8.12 (1H, ddd, J = 16, 5 Hz). 13C-

NMR (CDCl3, 400 MHz) δ 14.3, 18.78 (d, J = 138 Hz), 47.07 (ddd, J = 46.8, 59, 92 Hz),

61.92, 160.56 (d, J = 53 Hz), 172.81 (d, J = 243 Hz). HRMS (m/z): [M+H]+ Calc for

13C315NC3H11O3 150.0883; found 150.0882.

Compound 19: See experimental procedure for 12. 1H NMR (CDCl3, 400 MHz) δ 1.29

(3H, t, J = 7.2 Hz), δ 1.98 (3H, dsp, J = 2.4, 128 Hz), δ 4.09 (2H, dqt, J = 3.2 Hz), δ 7.31

(1H, m, J = 1.2, 2.8, 4.5 Hz). 13C-NMR (CDCl3, 400 MHz) δ 10.17 (dt, J = 26, 224 Hz),

15.17, 70.32, 112.46 (dd, J = 165, 223 Hz), 142.39, 154.34 (dd, J = 28, 467 Hz). HRMS

(m/z): [M+H]+ calc for 13C315NC3H9O2 132.0777; found 132.0780.

Compound 21: In a steel autoclave, the oxazole 19 (g, mmol) is dissolved in 2,5-

dihydrofuran. Recrystallized trichloroacetic acid (g, mmol) and a small amount of

hydroquinone are added. The reaction is heated in an oven at 190 °C for 5 hours then

allowed to cool to room temperature over night. Excess 2,5-dihydrofuran is evaporated

and the crude is purified through column chromatography to elute with 10:1

DCM/MeOH. The intermediate is then refluxed in 48 % hydrobromic acid (mL, mmol)

for 1 hr then cooled to room temperature. Once cooled, the crude mixture dried in vacuo

and then refluxed in water. Freshly prepared AgCl is added to the refluxing aqueous

solution and the refluxing continued for an additional hour. The reaction is cooled to

67    

room temperature then filtered. The filtrate is evaporated to dryness and used without any

further purification. Approximately g of 21 was isolated (%yield). 1H NMR (DMSO-d6,

300 MHz) 13C-NMR (DMSO-d6, 400 MHz) δ 16.32 (J = 136 Hz), 53.46, 56.10, 141.96,

142.10, 142.23, 148.70 (J = 288 Hz), 152.50 (J = 284 Hz). HRMS (m/z): [M+H]+ calc for

13C315NC5H11O3 174.0883; found 174.0908.

Compound 22: See experimental procedure for 15. 1H NMR (CDCl3, 300 MHz) δ 1.38

(3H, t, J = 6.9 Hz), δ 2.51 (3H, d, J= 132 Hz) δ 4.01 (2H, qt, J = 7.2, 5.7 Hz), δ 4.81 (2H,

s), δ 6.89 (2H, d, J = 7.8 Hz), δ 7.28 (2H, d, J = 7.2 Hz), δ 7.88 (1H, m), δ 9.08 (1H, s).

HRMS (m/z): [M+H]+ calc for 13C315NC13H19NO3 291.1461; found 291.1537

Compound 23: See experimental procedure for 16. 1H NMR (D2O, 600 MHz) δ 2.61

(3H, dd, J = 3, 132 Hz), δ 5.10 (2H, d, J = 6 Hz), δ 8.19 (1H, d, J = 6 Hz), δ 10.46 (1H, s).

13C NMR (DMSO-d6, 400 MHz) δ HRMS (m/z): [M+H]+ calc for 13C315NC5H10O6P

252.0389; found 252.0439.

Compound 24: Phenol (0.75 g, 7.8 mmol) was dissolved in ethylene dichloride (16 mL)

and stirred at room temperature. To the mixture tetrabutylammonium bromide (0.25 g,

0.78 mmol), 15N-nitric acid (70 wt%, 1 g), and sufficient water to make up the nitric acid

concentration to 6 wt %. The reaction was stirred overnight at room temperature. Upon

completion of the reaction, the aqueous layer was separated from the organic layer. The

aqueous layer was extracted with pentane, combined with the other organic layer, and the

68    

combined organic layers were washed once with DI water. A silica plug eluting with 1:3

EtOAc/Hex gave 0.262 g of 24 (24 % yield). 1H NMR (CDCl3, 300 MHz): δ 7.01 (1H, dt,

J = 1.5, 5.7 Hz), δ 7.18 (1H, dd, J = 1.5, 7.2 Hz), δ 7.60 (1H, dt, J = 1.5, 5.7 Hz), δ 8.13

(1H, dd, J = 1.5, 6.9 Hz), δ 10.6 (1H, s).

Compound 25: In a flame dried 3 neck flask, affixed with reflux condenser, a positive

flow of N2, and a balloon 0.076 g of 10 % Pd/C is dissolved in 7.0 mL of DI water.

sodium borohydride (0.14 g, 3.8 mmol) is then added and the reaction stirred at room

temperature. 24 (0.27 g, 1.9 mmol) is dissolved in 2 M NaOH (10 mL) and added

dropwise. The reaction is stirred at room temperature until the yellow color disappears

and then the reaction mixture was filtered. The filtrate was acidified with 2 M HCl,

filtered once again, and then neutralized with 0.5 M NaOH. The product was extracted

with diethyl ether, and dried over anhydrous magnesium sulfate. Upon removal of the

solvent, 200 mg of 25 was isolated (96 % yield). 1H NMR (CDCl3, 300 MHz) δ 6.66-6.8

(4 H, m) HRMS (m/z): [M+] calc for C6H8NO 110.0498; found 110.0449.

Compound 26/27: To the commercially available sodium salt of oxaloacetate (126 g, 0.6

mol) was added 200 mL of 3.5 N HCl with stirring. The reaction mixture was then

extracted with ether and the organic phase dried over MgSO4. The solvent was

evaporated and the crude ethyl oxaloacetate was heated on a steam bath under vacuum

for 15 minutes. No further purification was necessary, and the crude product was carried

through to the next step. To 26 (126 g, 0.6 mol) was added triethyl orthoformate (140

69    

mL, 0.85 mol) and acetic anhydride (130 mL, 1.37 mol) and heated at 140 °C. Heating

continued for 2 hrs, and then the volatiles were distilled off under reduced pressure. To

the resulting crude product was added 150 mL of water, and 50 g of solid NaHCO3 was

gently added with stirring in 1.0 g increments. The aqueous solution was washed twice

with 50 mL portions of ether and then the aqueous solution was gently acidified with an

excess of concentrated hydrochloric acid. The resulting aqueous solution is extracted

three times with ether and then dried over anhydrous MgSO4. The solvent was evaporated

and the crude prodcut was heated on a steam bath, under vacuum, for 15 mins.

Approxamately 100 g of 27 was recovered (70 % yield). 1H NMR (CDCl3, 300 MHz) δ

1.30 (3H, t, J = 7.2 Hz), δ 1.39 (3H, t, J = 7.2 Hz), 4.26 (2H, qt, J = 6.9 Hz), 4.39 (2H, qt,

J = 6.9 Hz), 8.7 (1H, s).

Compound 28: In a round bottom flask, 27 (15.2 g, 70 mmol) was dissolved in 110 mL

diethyl ether. To this solution, iminoacetylacetone (14 g, 88 mmol) was added and the

reaction stirred overnight at room temperature. Once the reaction has commenced the

resulting precipitate was filtered and extracted with warm ethyl acetate. The filtrate was

evaporated to dryness and a 1:1 mixture of ether and petroleum ether added to crystallize

the product. About 9.16 g of 28 was isolated as a yellow crystal (44 % yield). 1H NMR

(CDCl3, 300 MHz) δ 1.29 (3H, t, J = 7.2 Hz), δ 1.36 (3H, t, J = 7.2 Hz), δ 2.19 (3H, s), δ

2.23 (3H, s), δ 4.25 (2H, qt, J = 7.2 Hz), δ 4.34 (2H, qt, J = 7.2 Hz), δ 5.7 (1H, s), δ 8.2

(2H, d, J = 13.8 Hz).

70    

Compound 29: In a 500 mL beaker, 28 g of concentrated H2SO4, and 8.0 g of 28 (27

mmol) were stirred at room temperature for 1 hr. After stirring, 1.85 g of powdered NaN3

(28 mmol) was added gently, under ice cooling. The ice bath was removed and the

reaction stirred at room temperature overnight. The solution was then poured over a large

amount of crushed ice and the aqueous solution heated at 70 °C for 10 hrs. Once cooled,

concentrated ammonium hydroxide was added to the reaction mixture until a pH of 2.0

(roughly 36 mL), and the reaction placed into the back of the hood to allow 29 to

crystallize out slowly. Roughly 2.9 g of 29 was isolated (50 % Yield). 1H NMR (DMSO-

d6, 300 MHz) δ 2.58 (3H, s), δ 8.9 (1H, s). HRMS (m/z): [M+H]+ calc for C8H9N2O4

197.0557; found 197.0557.

Compound 30: In a large round bottom flask, 29 (0.55 g, 2.6 mmol) was dissolved in 65

mL of absolute ethanol. To the ethanol solution, 3.3 mL of concentrated H2SO4 was

slowly added into the reaction flask. The reaction was refluxed for 24 hrs, cooled to room

temp., and the solvent removed by rotary evaporation. The crude product was dissolved

in ethyl acetate, washed with NH4OH(aq), and the organic layer dried over MgSO4. After

removal of the solvent, the crude is purified by column chromatography (1:1

EtOAc/DCM). The yield of the diester was 30 0.15 g (23 % yield). 1H NMR (CDCl3,

300 MHz) δ 1.37 (6H, t, 6.6 Hz), δ 2.62 (3H, s), δ 4.38 (4H, qn, J = 7.5 Hz), δ 8.22 (1H,

s).

71    

Compound 45: A mixture of orcinol (4.55 g, 36.7 mmol), K2CO3 (25 g, 184 mmol), MeI

(11.4 mL, 184 mmol), and acetone (104 mL) was refluxed for 6 hrs. Once cooled, the

reaction was filtered through celite and the solvent removed. The crude product was

passed through a small silica pad using a mixture of 2 % ethyl acetate in petroleum ether.

Upon removal of the solvent, 4.7 g of 45 was obtained (84 % yield). 1H NMR (CDCl3,

300 MHz) δ 2.32 (3H, s), δ 3.79 (6H, s), δ 6.3 (1H, s), δ 6.35 (2H, s).

Compound 46: Under N2, a mixture of 45 (4.7 g, 31 mmol) in 60 mL of anhydrous

diethyl ether cooled to 0 °C and TMEDA (7.0 mL, 46 mmol), followed by n-butyllithium

(30 mL, 46 mmol) added. The solution was refluxed for 3 hrs, then cooled back down to

0 °C. Once cooled, DMF (7 mL, 92 mmol) was added slowly. The solution is allowed to

warm back up to room temperature slowly and stirred for 2 hrs. At this point the reaction

was quenched with 25 mL of DI water and extracted with ethyl acetate (3 x 125 mL). The

combined organic extracts were dried over anhydrous MgSO4 and the solvent removed in

vacuo. The crude was purified by flash chromatography (60 % diethyl ether in petroleum

ether). Approximately 2.8 g of 46 was isolated (51 % yield). 1H NMR (CDCl3, 300 MHz)

δ 2.38 (3H, s), δ 3.89 (6H, s), δ 6.39 (2H, s), δ 10.45 (1H, s).

Compound 47: Methyl triphenylphosphonium bromide (2.5 g, 7.1 mmol) was stirred in

20 mL of dry THF. To the solution, at room temperature, was added sodium

bis(tirmethylsilyl)amide (8 mL, 8 mmol, 1 M in THF). The solution was stirred for 3 hrs

at room temperature then cooled to -78 °C. To the mixture was added 46 (0.91 g, 5.1

72    

mmol) dissolved in 20 mL of dry THF, and the reaction mixture allowed to warm to

room temperature overnight. The reaction was quenched with acetone and water,

extracted with diethyl ether, and the combined organic extracts dried over anhydrous

MgSO4. Silica gel flash chromatography eluting with 5% diethyl ether in petroleum ether

gave 0.52 g of the product (58% yield). 1H NMR (CDCl3, 300 MHz) δ 2.34 (3H, s), δ

3.83 (6H, s), δ 5.38 (1H, dd, J = 2.1, 7.0 Hz), δ 6.01 (1H, dd, J= 2.1, 11.4 Hz), δ 6.38

(2H, s), δ 6.93 (1H, dd, J = 4.2, 9.3 Hz). HRMS (m/z): [M+H]+ calc for C11H14O2

179.1067; found 179.1080.

Compound 49: A solution of 46 (0.56 g, 3.1 mmol) was dissolved in 5.0 mL of acetone

and cooled to 0 °C. To the solution 1 M aq NaOH (5 mL) was added dropwise and the

reaction stirred at room temperature for 3 hrs. At this point, the reaction was cooled once

again to 0 °C and treated with 1 M aq HCl (5 mL) dropwise. DI Water (17 mL) was

added and the mixture extracted with ethyl acetate (3 x 10 mL), and the combined

organic extracts were dried over anhydrous MgSO4. Flash chromatography eluting with

7:3 n-pentane/EtOAc afforded 0.41g of 49 (60 % yield). 1H NMR (CDCl3, 300 MHz) δ

2.37 (6H, s), δ 3.88 (6H, s), δ 6.39 (2H, s), δ 7.13 (1H, d, J = 16.5 Hz), δ 7.97 (1H, d, J =

16.5 Hz). HRMS (m/z): [M+H]+ calc for C13H17O3 221.1172; found 221.1205.

Compound 51: To a solution of 46 (5.4 g, 30 mmol) in 65 mL of acetonitrile and 31 mL

of dichloromethane was slowly added 10.0 g of AlCl3 and 11 g of NaI under ice cooling.

The reaction was stirred at room temperature for 1 hr then 20 mL water is added under

ice cooling. The mixture is extracted with dichloromethane, and the combined organic

73    

extracts dried over anhydrous MgSO4. The crude prodcut was purified by flash

chromatography on silica gel eluting with 1:5 EtOAc/Cyclohexane. The reaction gave 4.0

g of 51 (81 % yield). 1H NMR (CDCl3, 300 MHz) δ 2.33 (3H, s), δ 3.88 (3H, s), δ 6.19

(1H, s), δ 6.36 (1H, s), δ 10.26 (1H, s), δ 11.97 (1H, s). HRMS (m/z): [M+H]+ calc for

C9H11O3 167.0703; found 167.0710.

Compound 52: Sodium hydroxide (0.77 g, 19 mmol) was crushed to a powder and

dissolved in 7 mL of absolute ethanol. Once homogenous, 6-methyl-5-hepten-2-one (3.2

mL, 21.7 mmol) was added and the solution is stirred at room temperature for 10

minutes. At this point 51 (2.65 g, 16 mmol) was added and the reaction heated at 40 °C

overnight. The reaction was quenched with dilute aqueous HCl and extracted three times

with ethyl acetate. The combined organic layers were dried over anhydrous MgSO4 and

the solvent removed with rotary evaporation. The crude product was subjected to column

chromatography on silica gel using a 10:1 chloroform/methanol as an eluent. The product

could be further purified through recrystallizing with diethyl ether/hexanes. An amount of

0.36 g of 52 was isolated (28 % yield). 1H NMR (CDCl3, 300 MHz) δ 1.66 (3H, s), δ 1.7

(3H, s), δ 2.31 (3H, s), δ 2.40 (2H, m), δ 2.73 (2H, t, J = 7.8 Hz), δ 3.87 (3H, s), δ 5.18

(1H, t, J = 7.2 Hz), δ 6.30 (1H, s), δ 6.38 (1H, s), δ 7.25 (1H, d, J = 16.5), δ 8.00 (1H, d, J

= 16.5). HRMS (m/z): [M+H]+ calc for C17H23O3 275.1642; found 275.1651

Compound 53: To a 50 mL round bottom flask 1 mL of a 50 % solution of KOH was

added to 10 mL of methanol. While stirring, 6-methyl-5-hepten-2-one (0.15 mL) was

74    

added and the reaction stirred at room temperature for 10 minutes. At this point, 46 (0.18

g, 0.99 mmol) was added and the reaction stirred at room temperature for 6 hrs. The

reaction was quenched with dilute aqueous HCl and extracted three times with ethyl

acetate, and the combined organic layers were dried over anhydrous MgSO4. After

removal of the solvent through rotary evaporation, the crude is subjected to flash

chromatography on silica gel eluting with 60 % diethyl ether/petroleum ether solvent

system. Roughly 0.55 g of 53 was isolated (20 % yield). 1H NMR (CDCl3, 300 MHz) δ

1.65 (3H, s), δ 1.70 (3H, s), δ 2.27-2.47 (2H, m), δ 2.36 (3H, s), δ 2.69 (2H, t, J = 7.8

Hz), δ 3.88 (6H,s), δ 5.18 (1H, tt, J = 1.5, 7.2 Hz), δ 6.39 (2H, s), δ 7.14 (1H, d, J = 16.5

Hz), δ 8.00 (1H, d, J = 16.5 Hz). 13C NMR (CDCl3, 300 MHz) δ 17.9, 22.7, 23.6, 25.9,

40.5, 55.9, 104.9, 123.6, 128.6, 132.5, 134.1, 142.6, 160.2, 202.4. HRMS (m/z): [M+] calc

for C18H24O3 288.1725; calc 288.1714.

75    

Appendix B: 1H-NMR Spectra

H2 15N

CO2 Et

400 MH

z, D2 O

76    

H15N

CO2 Et

O

300 MH

z, CD

Cl3

77    

O

15N

OEt

300  MHz,  CDCl3  

78    

15N

HO

CO2 EtCO

2 Et

300 MH

z, CD

Cl3

79    

15N

HO

OHOH

300 MH

z, D2 O

80    

15N

HO

NOH

OEt

300 MH

z, CD

Cl3

81    

15N

HO

O

OPO

3 H2

400 MH

z, DM

SO-d6

82    

H2 15N

13CH13C

13CH3O

OEt

400 MH

z, D2 O

83    

H15N

13CH13C

13CH3O

OEt

O300 MH

z, CD

Cl3

84    

O

15N13C 13C13CH

3 OEt

400 MH

z, CD

Cl3

85    

OH

15NH2

300 MH

z, CD

Cl3

86    

EtO2 C

OCO

2 Et

OH

300 MH

z, CD

Cl3

87    

EtO2 C

OCO

2 Et

NH

O

300 MH

z, CD

Cl3

88    

N

H2 N

COOHCO

OH

300 MH

z, DM

SO-d6

89    

N

H2 N

CO2 EtCO

2 Et

300 MH

z, CD

Cl3

90    

OMe

OMe

300  MHz,  CDCl3  

91    

OMe

OMe

O

300  MHz,  CDCl3  

92    

OMe

OH

O

300  MHz,  CDCl3  

93    

OMe

OMe

300 MH

z, CD

Cl3

94    

OMe

OMe

O

300 MH

z, CD

Cl3

95    

OMe

OMe

O

300 MH

z, CD

Cl3

96    

OMe

OH

O

300 MH

z, CD

Cl3

97    

13C13C15N

H3 13C

HO

N

OH

OEt

300 MH

z, CD

Cl3

98    

13C13C

15NH3 13C

HO

OO

PO3 H

2

600 MH

z, D2 O