SYNTHESIS OF NOVEL GEX1A ANALOGUES: A POTENTIAL LEAD TOWARD THE

CURE OF NIEMANN-PICK TYPE C DISEASE

A Thesis

Submitted to the Department of Chemistry and Biochemistry

of the University of Notre Dame

in Partial Fulfillment of the Requirements

for the Designation of

Honors in Chemistry

by

Michael James Ahlers

________________________________

Richard E. Taylor, Advisor

University of Notre Dame

Notre Dame, IN

May 2015
 SYNTHESIS OF NOVEL GEX1A ANALOGUES: A POTENTIAL LEAD TOWARD THE
CURE OF NIEMANN-PICK TYPE C DISEASE

Abstract

Although great advancements in the understanding of Niemann-Pick Type C

(NPC) disease have been made within the last decade, there remains no viable

treatment for this lethal lysosomal storage disorder. Specifically, NPC is characterized

by mutations to either the NPC1 or NPC2 genes, which results in defective cholesterol

trafficking within the lysosome. Hydroxypropyl -cyclodextrin (HPCD) and histone

deacetylase inhibitors (HDACi) such as Vorinostat are current therapeutic candidates

for NPC; however, these therapies have drawbacks and challenges that encourage

exploration of additional therapeutic candidates.

The natural product GEX1A, a type 1 polyketide, has been identified as a

potential therapeutic candidate for NPC disease. Our laboratory has recently

demonstrated the ability of GEX1A, isolated from Streptomyces chromofuscus, to

restore cholesterol trafficking in NPC1 mutant cell lines. The observed biological activity

was of comparable levels to Vorinostat, yet GEX1A is not an HDACi. By restoring

cholesterol homeostasis, GEX1A appears to reverse the key molecular hallmark of NPC

disease faulty cholesterol trafficking within the lysosome. Thus, the synthesis of

GEX1A analogues to be tested in NPC1 and NPC2 mutant cell lines is necessary in

order to determine the minimal functionality required for activity against NPC as well as

the mechanism of action of GEX1A. Intriguingly, GEX1A shares similar structural

features and biological activity with another natural product, pladienolide B. To simplify

ii
analogue synthesis and explore the biological relationship between these two

molecules, hybrid analogues resembling GEX1A and pladienolide B have been

synthesized. This thesis presents progress towards the organic synthesis of these

GEX1A analogues.

iii
 TABLE OF CONTENTS

List of Abbreviations.v
Acknowledgementsvii
Chapter 1: Introduction1
1.1 Niemann-Pick Type C Disease and GEX1A.....1
1.2 Structural and Biological Relationship between GEX1A and Pladienolide B...4
1.3 GEX1A-Pladienolide B Hybrid Analogues.5
Chapter 2: Contributions toward the Synthesis of Fragment B.....8
2.1 The Leighton Crotylation...8
2.2 Synthesis of the Leighton Ligand9
2.3 First Generation Route toward Fragment B.10
2.4 Second Generation Route toward Fragment B...11
Chapter 3: Progress Toward Synthesis of Fragment B213
3.1 Modification of the Leighton Crotylation to an Allylation13
3.2 Early Stage Synthesis of Fragment B214
3.3 Exploration of Alternative Aldehyde for Leighton Allylation..15
3.4 Mid Stage Synthesis of Fragment B2......16
Chapter 4: Coupling of Abbreviated Analogues 3 and 418
4.1 Fragment Coupling for Synthesis of Analogue 3....18
4.2 Future Fragment Coupling for Analogue 4..18
Chapter 5: Experimental20
5.1 General Organic Synthesis Methods...20
5.2 Leighton Ligand (S, S) Synthesis.20
5.3 Aldehyde Substrates for Leighton Crotylation24
5.4 First Generation Route toward Fragment B29
5.5 Second Generation Route toward Fragment B..34
5.6 Allylation Route toward Fragment B2..37
5.7 NMR Spectra...43
5.8 Biological Evaluation of GEX1A Analogues73
References76

iv
ABBREVIATIONS

Ac acetic, acetate

Bn benzyl

Boc tert-butyloxycarbonyl

Bu butyl

DBU 1,8-diazabicycloundec-7-ene

DCM dichloromethane

DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone

DMP Dess-Martin periodinane

DMSO dimethylsulfoxide

dr diastereometric ratio

E entgegen

ee enantiomeric excess

Et ethyl

HG(II) Hoyveda-Grubbs catalyst, 2nd generation

Me methyl

NaHMDS sodium bis(trimethylsilyl)amide

NMR nuclear magnetic resonance

Ph phenyl

ppm parts per million

pyr pyridine

rt room temperature

v
TBAF tetra-n-butylammonium fluoride

TBS tert-butyldimethylsilyl

tBu tert-butyl

TEA triethylamine

Tf triflate, triflic

THF tetrahyrofuran

TLC thin layer chromatography

Ts tosyl

Z zusammen

vi
 ACKNOWLEDGEMENTS

I would like to thank all members of the Taylor Laboratory during my time as an

undergraduate researcher, including Jarred Pickering, Eve Granatosky, Ansel Nalin,

John Rieth, Andrew Gasparrini, Ian Harrier, Matthew Wilson, Dr. Cole Stevens, Laura

Woods, Danielle Ronnow, Erik Larsen, Chia Fu Chang, Patrick Lichtenberger, Kristin

Hillgamyer, Meredith Viera, and Emily Zion. Their kindness and support in teaching me

how to become a synthetic organic chemist is greatly appreciated. I would also like to

send a special thank you to Dr. Richard E. Taylor and Jarred R. E. Pickering. Dr. Taylor

welcomed me to his laboratory over two years ago, and his support for my professional

development has been outstanding. I have worked closely with Jarred Pickering on this

GEX1A analogue project. I am appreciative of his mentorship in helping me become an

independent researcher and his support as a great friend. Additionally, Jarred was

responsible for synthesis of Fragment A, isolation of GEX1A from S. chromofuscus

broth (along with Eve Granatosky), and development of the fragment coupling

methodology. I would also like to thank the University of Notre Dame College of

Science, the Dr. Norbert Wiech Endowment, the Ara Parseghian Medical Research

Foundation, and NIH Grant #T32GM075762 for funding my research efforts.

vii
 CHAPTER 1

INTRODUCTION

1.1 Niemann-Pick Type C Disease and GEX1A

Niemann-Pick Type C (NPC) disease is a rare and fatal lysosomal storage

disease that typically presents before the age of 10. Specifically, NPC is characterized

by mutations in either the NPC1 or NPC2 genes, which subsequently leads to defective

cholesterol trafficking1. Currently, the proposed mechanism for cholesterol transport

within the lysosome is that NPC2 captures cholesterol through interactions with the

hydrophobic end and subsequently passes the cholesterol to NPC1 (Figure 1.11). Then,

by an unknown mechanism, this membrane-bound protein shuttles cholesterol through

the glycocalyx to the ER membrane. If either of these proteins are poorly functioning,

cholesterol buildup is imminent and clinical signs of NPC appear. The ensuing buildup

of cholesterol within the lysosome leads to gradual neurologic deterioration and loss of

motor skills, ultimately causing death of the patient. Approximately 95% of NPC patients

have mutations in NPC1, while 5% have mutations in NPC2.

1
Figure 1.11. Proposed model of cholesterol egress from lysosome, including roles of

NPC1 and NPC2.

Despite these recent advances in the understanding of NPC etiology and several

proposed treatments2-3, there remains no current FDA approved treatment.

Hydroxypropyl beta-cyclodextrin and histone deacetylase inhibitors (HDACi) are two

potential therapies that have undergone early-stage clinical trials; however, additional

therapeutic candidates with alternative mechanisms of action will further increase

understanding of NPC and may eventually arise as a viable cure.

Interestingly, the natural product GEX1A4 (initially isolated as herboxidiene5) was

first identified to be influential in cholesterol homeostasis in 1997. Specifically, GEX1A

was shown to upregulate the expression of low-density lipoprotein (LDL) receptor6.

Despite this intriguing activity, the role of GEX1A in cholesterol homeostasis remains

largely unknown. Potentially, GEX1A could amend cholesterol trafficking deficiency in

2
NPC disease. This project has followed up on these preliminary findings to further

explore how GEX1A influences cholesterol homeostasis.

Following this lead, GEX1A was isolated from the bacterium Streptomyces

chromofuscus and its potential as a novel NPC therapeutic candidate was explored

using a filipin staining assay conducted by the Holly V. Goodson laboratory (Figure

1.2). The filipin staining assay imaged unesterified cholesterol within the cell and

indicated that GEX1A restores cholesterol trafficking in NPC1 mutant cell lines. The

fluorescing cholesterol is condensed within the cell in the left image, but after treatment

with GEX1A, the light intensity from the fluorescence has clearly dissipated. These

results encouraged our laboratory to continue investigation of GEX1A as a therapy for

NPC disease through the synthesis of GEX1A analogues.

Control (NPC1 cells) Treated (GEX1A, 63nM)

Figure 1.2. Filipin staining assay. Treatment of NPC1 mutant cell line with GEX1A

restores cholesterol trafficking.

Although the filipin staining assay in Figure 1.2 suggests that GEX1A restores

cholesterol trafficking, the biological mechanism by which GEX1A restores cholesterol

trafficking remains poorly understood. By generating analogues of GEX1A and

3
evaluating their biological activity through filipin staining assays such as the one above,

the requisite functional groups in GEX1A for cholesterol trafficking and homeostasis

could be determined.

1.2 Structural and Biological Relationship between GEX1A and Pladienolide B

Intriguingly, novel anti-cancer natural product pladienolide B shares both

structural and biosynthetic7 similarities with GEX1A (Figure 1.3).

Figure 1.3. Structure of the natural products GEX1A and pladienolide B. Both natural

products possess a ring on their western half and a linear side chain laced with similar

functionality and stereo-specificity on their eastern half.

GEX1A and pladienolide B have both been shown to influence function of the

spliceosome, thereby modulating the splicing of pre-mRNA8. Specifically, GEX1A

inhibits SAP155 of splicing factor 3b, while pladienolide B interacts with SAP130 9. This

process leads to cell cycle arrest and is the primary reason that these natural products

4
have been investigated for their antitumor activity10. Potentially, a relationship exists

between the cholesterol transport activity of GEX1A and the pre-mRNA splicing activity

of pladienolide B. With the synthesis of GEX1A analogues, biological studies will gleam

further insight into the relationship between these two intriguing natural products and

their mechanisms of action.

1.3 GEX1A-Pladienolide B Hybrid Analogues

In order to evaluate the biological activity of these two natural products and

greatly simplify the synthetic routes to analogues resembling them, GEX1A-pladienolide

B hybrid analogues (GEX1A analogues) became the target molecules of this project

(Figure 1.4). Of note, the tertiary epoxide of GEX1A and the macrocyclic lactone of

pladienolide B pose significant synthetic challenges. The installation of such challenging

functional groups has been avoided entirely through rational design of a hybrid

analogue, which incorporates the eastern fragment of pladienolide B along with the

western fragment of GEX1A.

Figure 1.4. Rationally designed GEX1A-pladienolide B hybrid analogues. Incorporation

5
of the GEX1A pyran ring and the linear side chain of pladienolide B simplified the

synthetic route towards Analogue 1. This analogue has been accessed from the

coupling of Fragment A and Fragment B.

In the process of synthesizing Analogue 1, the synthesis of additional analogues

was explored through simplifications of Fragment B (Figure 1.5). Analogue 2 arises

from the demethylation of Analogue 1 at C16. Analogues 3 and 4 required the

coupling of a Fragment B precursor with Fragment A. With the synthesis of Fragment

B nearing optimization, these abbreviated fragments could be rapidly accessed using

similar experimental methodology.

Figure 1.5. Additional GEX1A analogues (1-4) accessed from simplified adducts of

Fragment B. These synthetic targets were designed for their accessibility and potential

6
to contribute to the determination of minimum functionality required for cholesterol

trafficking activity.

In the subsequent chapters of this thesis, the synthetic routes explored in order

to access Fragment B and its precursors are described.

7
 CHAPTER 2

CONTRIBUTIONS TOWARD THE SYNTHESIS OF FRAGMENT B

2.1 The Leighton Crotylation

During the planning stages of developing a route to Fragment B, it was realized

that through a crotylation reaction, an alcohol and an adjacent methyl group could be

installed in an early stage fragment through delivery of a crotyl moiety to an aldehyde

(Scheme 2.1).

The synthesis of Fragment B was highlighted by novel Leighton crotylation

chemistry11 in order to set requisite stereochemistry of the hydroxyl and methyl moieties

(Scheme 2.2). The Leighton crotylation chemistry was subsequently modified to an

allylation, providing the necessary intermediate for Fragment B2.

8
 The Leighton crotylation selectively delivered the silane crotyl group by first

binding it with the chiral ligand. Conveniently, this crotylation is a one-pot procedure that

can be completed in less than 8 hours. Moreover, the reaction is highly efficient (80%)

and the ligand is readily recovered (75%). Removal of the three acidic protons on the

Leighton ligand by DBU permitted binding of the silane. Subsequent transfer of the

crotyl group to an aldehyde in step 2 gave S, S stereochemistry of the hydroxyl and

methyl moieties with excellent enantioselectivity (86% ee).

2.2 Synthesis of the Leighton Ligand

In order to leverage the utility of the Leighton crotylation, it was necessary to first

synthesize the ligand from readily available starting materials according to the

procedure outlined in Leighton et. al.11 (S, S) diamino cyclohexane 2.1 was isolated

from its racemic mixture to provide the necessary stereochemistry in the ligand for the

installment of the requisite stereochemistry in the GEX1A analogues. The diamine was

then monoprotected by a Boc group (2.2) with overnight stirring at room temperature.

Separately, the necessary aldehyde (2.4) was generated after reflux for five hours

according to Scheme 2.3.

9
 Primary amine 2.2 and benzyl aldehyde 2.4 were then coupled to give the

resulting imine (2.5) after stirring for 3 hours. Subsequent reduction by LAH gave the (S,

S) Leighton ligand as white-brown crystals (2.6).

2.3 First Generation Route toward Fragment B

With the Leighton Ligand in hand and crotyl silane 2.10 obtained commercially, it

was necessary to generate an aldehyde to be used in the Leighton crotylation. Cis diol

2.7 was protected with previously synthesized PMB-imidate to give diprotected

precursor 2.8. Subsequent cleavage of the alkene by ozonolysis gave aldehyde

substrate 2.9, which was used in the first generation route toward Fragment B.

Deprotonation of 2.6 followed by addition of the cis butyl silane and aldehyde 2.9

delivered crotylation adduct 2.11 in high selectivity (Scheme 2.6). TBS protection of the

secondary alcohol followed by PMB deprotection of the primary hydroxyl group led to

free alcohol 2.13 in good yield. Tosylation of 2.13 followed by treatment with methyl

10
Grignard led to synthetic intermediate 2.15, completing the C14 through C19 carbon

framework of Fragment B.

Although isolated, complete conversion of the tosylated precursor to 2.15 failed

despite the exploration of several conditions. After overnight reflux of the tosylated

precursor failed to provide a complete conversion, an alternate route was developed in

order to access 2.15 in large quantities with improved efficiency (Scheme 2.8).

2.4 Second Generation Route toward Fragment B

To avoid the inconsistencies and difficulties in scaling up the synthesis toward

2.15, a new route was developed using an aldehyde substrate with one additional

carbon during the Leighton crotylation.

Diol 2.16 was monoprotected with PMB imidate using 5-7 additional fold of diol to

give 2.17 in good yield and minimize formation of diprotected product (Scheme 2.7).

11
The isolated monoprotected diol (2.17) was subsequently oxidized to requisite aldehyde

2.18 for the crotylation reaction using a Swern oxidation in excellent yield. Alternatively,

the moiety conversion from alcohol to aldehyde was also explored using sulfur trioxide

pyridine. Although it was a simpler reaction to set up, this route was later abandoned

due to higher yields and better consistency obtained with the Swern oxidation.

With the requisite aldehyde synthesized, the second generation route toward

Fragment B undertaken (Scheme 2.8). Many similarities exist between the two routes,

which allowed rapid development of this pathway. Of note, TBS protection of the

crotylation alcohol was altered from using TBSOTf to TBSCl. Although the reaction with

TBSCl required additional time, it was a gentler reagent than TBSOTf, which improved

both yield and consistency for the reaction. If the reaction with TBSOTf was not carefully

monitored, the presence of triflic acid could lead to degradation.

12
 The second generation route has been optimized (Scheme 2.9) and proven to be

an efficient route toward 2.15, the necessary precursor to Fragment B. Using this

scheme, Fragment B was obtained with the assistance of graduate student mentor

Jarred Pickering from 2.15 onward.

13
 CHAPTER 3

PROGRESS TOWARD SYNTHESIS OF FRAGMENT B2

3.1 Modification of the Leighton Crotylation to an Allylation

As the project expanded toward synthesis of Analogue 2, the use of a general

delivery method of a carbon fragment to the aldehyde substrate permitted an easy

transition from the crotylation methodology to an allylation. As shown below, the

allylation mimics the crotylation delivery, with the distinction being that an allyl group is

added into the aldehyde rather than a crotyl group. Fortunately, the utility of the

Leighton ligand permitted this transition, with the only difference between the crotylation

and allylation reactions being the use of trichloroallylsilane (3.1) rather than

trichlorocrotylsilane (2.10).

3.2 Early Stage Synthesis of Fragment B2

Using the same methodology as the optimized crotylation route, 3.6 was

accessed rapidly and efficiently. Following TBS protection of the allylation adduct (3.2),

14
subsequent DDQ deprotection of the PMB group and toslyation of the resulting free

alcohol created a good leaving group on 3.5. Reduction by LAH gave compound 3.6,

which is also the abbreviation of Fragment B required for synthesis of Analogue 4.

3.3 Exploration of Alternative Aldehyde for Leighton Allylation

Simultaneous with the buildup of 3.6 through Scheme 3.2, it was explored

whether an aldehyde containing the requisite leaving group for 3.5 could be

incorporated into the allylation step (Scheme 3.3). If this were possible, the need for the

PMB group could be eliminated, removing two steps from the synthesis.

Diol 2.16 was first monoprotected with tosyl chloride in excellent yield, followed

by a Swern oxidation with DMP to give 3.8. Although partial conversion of alcohol 3.7 to

aldehyde 3.8 could be observed by TLC, full conversion was difficult to achieve and

isolation of aldehyde 3.8 before decomposition proved unsuccessful (Scheme 3.4).

15
 The challenge of isolating tosylated aldehyde 3.8 in larger quantities, combined

with uncertainty as to the success of a Leighton allylation with 3.8 led to the

abandonment of this route. None of the six aldehydes successfully used by Leighton et.

al11. (Figure 3.1) contain a tosyl group, which further discouraged the pursuit of this

abbreviated route. With Scheme 3.2 toward 3.6 successfully optimized, mid stage

synthesis toward Fragment B2 was undertaken.

3.4 Mid Stage Synthesis of Fragment B2

The coupling of alkene 3.6 and diacetate 3.9 through Grubbs metathesis gave

an excess of alcohol 3.10 with 10:1 equivalents of 3.9 to 3.6 and strong preference for

the E isomer. Subsequent iodination in the presence of triphenyl phosphine gave 3.11.

16
 Subsequent use of chiral auxiliary 3.12 gave the proper stereochemistry of the

methyl group at C12 (3.13). Reduction by LAH then cleaved the chiral auxiliary, giving

late stage alcohol 3.14. With the addition of the terminal alkene and epoxide through

chemistry previously worked out for the synthesis of Fragment B, the requisite fragment

for Analogue 2 can be readily obtained as a future direction for this work (Scheme 3.6).

17
 CHAPTER 4

COUPLING OF ABBREVIATED ANALOGUES 3 AND 4

4.1 Fragment Coupling for Synthesis of Analogue 3

The completed fragments were coupled via cross-metathesis chemistry and the

use of Grubbs second-generation catalyst (HG II) (4.1), providing the desired

regiochemistry of the double bond in excess (5:1 E: Z). Subsequent removal of the TBS

protecting group by acid treatment followed by trimethyltin hydroxide (Me 3SnOH) and

1,2-Dichloroethene (DCE) gave GEX1A Analogue 3 and its Z isomer (Scheme 4.1).

4.2 Future Fragment Coupling for Analogue 4

Using the same methodology as above in Chapter 4.1, Analogue 4 can be

readily accessed from the coupling of Fragment A with 3.6.

18
19
 CHAPTER 5

EXPERIMENTAL

5.1 General Organic Synthesis Methods

All organic reactions were performed in oven or flame-dried glassware. These

reactions were magnetically stirred under open atmosphere, unless the reaction

required an atmosphere of argon or nitrogen as specified. Reaction progress was

monitored by thin-layer chromatography (TLC) on Merck pre-coated silica gel plates

with F254 indicator. TLC was evaluated by fluorescence under ultra-violet light and by

subsequent staining in either potassium permanganate or phosphomolybdic acid

solution. Products were purified by column chromatography unless stated otherwise.

Nuclear magnetic resonance (NMR) spectra confirming product isolation were recorded

on Bruker 500 MHz or Bruker 400 MHz instruments. Optical rotation was recorded by a

PerkinElmer Model 343 Polarimeter.

5.2 Leighton Ligand (S, S) Synthesis

(1S, 2S)-cyclohexane-1, 2-diamine (2.1): A 2 liter beaker was charged with D-(-

)-tartaric acid (150 g, .99 mol) and distilled water (400 mL). After stirring at room

temperature until complete dissolution occurred, a mixture of cis and trans 1, 2-diamino

cyclohexane (240 mL, 1.94 mol) was added a rate such that the reaction temperature

remained below 70C. During addition, a thick white solid was observed, followed by a

20
dark brown solution. Next, glacial acetic acid was added (100 mL, 1.75 mol) at a rate

such that the reaction temperature remained below 90C. The mixture was vigorously

stirred and cooled to 0C over 2 hours. The resulting yellow precipitate was collected by

vacuum filtration and washed with cold distilled water (1 x 100 mL) and cold methanol (5

x 100 mL), affording a white solid.

The diamine salt (150 g) was dissolved in aqueous NaOH (250 mL, 4M). 2.1 was

extracted with DCM (5 x 50 mL). The aqueous solution was then saturated with NaCl

and extracted with DCM (10 x 50 mL) until minimal 2.1 could be extracted. The organic

extracts were combined, dried with MgSO4, and concentrated under reduced pressure

to give a dark brown oil (16.97 g, 148.6 mmol)

tert-butyl ((1S,2S)-2-aminocyclohexyl)carbamate (2.2): 2.1 (16.97 g, 148.6

mmol) was added to a 0C solution of concentrated HCl (12.3 mL, 148.6 mmol) in

MeOH (47.6 mL). The ice bath was subsequently removed and dH20 was added to the

solution after 15 minutes. After 30 minutes, a solution of Boc2O (32.40 g, 148.6 mmol) in

MeOH (15.82 mL) was added dropwise. The reaction was then allowed to stir for 12

hours at room temperature. The brownish-red mixture was concentrated and suspended

in Et2O, after which a white solid was collected by filtration and washed with Et 2O

rinses. The white solid residue was treated with 3M NaOH (108 mL) and extracted with

DCM (5 x 75 mL). The combined organic portions were dried with magnesium sulfate,

21
filtered, and concentrated under reduced pressure to give a beige solid (16.74 g, 77.98

mmol, 72%).

3-(tert-butyl)-2-hydroxybenzaldehyde (2.4): To an oven dried 2 liter round

bottom flask was added acetonitrile (400 mL). Subsequently 2-tert-butyl phenol (30.6

mL 200 mmol) was added followed by paraformaldehyde (40.6 g, 1.350 mol) and then

magnesium chloride (28.6 g, 300 mmol). Following addition of the previous compounds,

triethylamine (104.4 mL, 748 mmol) was added, turning the white solution to yellow).

The solution was then brought to a gentle reflux (90C) by sand bath for five hours, at

which time the solution appeared bright red. The solution was quenched with 5% HCl

(200 mL). The solution was transferred to a separatory funnel and extracted with DCM

(5 x 100 mL). The combined organic portions were concentrated and the residual

material was partitioned in diethyl ether (250 mL) and deionized water (250 mL). The

ether layer was retained and washed with brine (1 x 100 mL). The organic portion was

dried with magnesium sulfate, filtered, and concentrated under reduced pressure (24.95

g, 139.8 mmol, 69%).1H NMR (400 MHz, CDCl3) 11.81 (s, 1H), 9.90 (s, 1H), 7.56 (dd,

J = 7.7, 1.6 Hz, 1H), 7.43 (dd, J = 7.7, 1.7 Hz, 1H), 6.97 (t, J = 7.7 Hz, 1H), 1.45 (s, 9H).

22
 tert-butyl ((1S,2S)-2-(((E)-3-(tert-butyl)-2-hydroxybenzylidene)amino)cyclo

hexyl)carbamate (2.5): A solution of the protected diamine (19.7 g, 91.77 mmol) in

EtOH (870 mL) was added to the aldehyde (16.38 g 91.77 mmol). The mix was heated

to reflux (85C) and stirred for 3 hours. The reaction was then allowed to cool to room

temperature and concentrated under reduced pressure. The residue was recrystallized

from minimum boiling EtOH to give the resulting imine as long yellow crystals (23.0 g,

61.27 mmol, 67%)

tert-butyl ((1S,2S)-2-((3-(tert-butyl)-2-hydroxybenzyl)amino)cyclohexyl)

carbamate (2.6): To an oven dried flask charged with LAH (10.63 g, 280.5 mmol) was

added THF (500 mL) via addition funnel. The solution was then cooled to 0C, at which

time the imine (35.1 g, 93.5 mmol) dissolved in THF (250 mL) was added dropwise via

addition funnel along with a THF rinse. The reaction was then allowed to warm to room

temperature and stirred for two hours. The solution was then refluxed for 12 hours. The

reaction was then cooled to 0C and quenched slowly and carefully according to the

Fieser workup. Deionized water (10.63 mL) followed by 15% NaOH (10.63 mL) and

23
finally another addition of deionized water (31.89 mL). The solution was allowed to stir

for ca. one hour, at which time a white salt had precipitated. The solution was then

filtered through a plug of celite with copious amounts of ether rinses. The combined

organic filtrate was dried with magnesium sulfate, filtered, and concentrated. The

resulting Leighton ligand was purified by either recrystallization from minimum boiling

hexanes or column chromatography (9:1 DCM:MeOH, 0.5% ammonium hydroxide),

(24.84 g, 85.53 mmol, 90%). 1H NMR (500 MHz, CDCl3) 7.16 (d, J = 7.6 Hz, 1H), 6.86

(d, J = 7.0 Hz, 1H), 6.69 (t, J = 7.5 Hz, 1H), 4.01 (d, J = 13.3 Hz, 1H), 3.83 (d, J = 13.3

Hz, 1H), 2.39 (s, 3H), 2.16 (d, J = 10.4 Hz, 4H), 1.80 1.64 (m, 2H), 1.40 (s, 9H), 1.32-

1.12 (m, 3H), 1.02-0.87 (m, 1H).

5.3 Aldehyde Substrates for Leighton Crotylation

4-methoxybenzyl 2,2,2-trichloroacetimidate (PMB-imidate):To an oven dried

250 mL round bottom under N2 was added sodium hydride (0.768 g, 80% in mineral oil)

suspended in Et2O (40 mL). Then, 4-methoxyalcohol (12 mL) was added over 20

minutes and the solution was cooled to 0C with an ice bath. Trichloroacetonitrile (11.6

mL) was then added dropwise. The solution was stirred for ca. 2 hours, gradually

allowing the reaction to approach room temperature. The solution was then

concentrated to a dark red oil. To the residue was added 2% methanol in pentanes (200

mL), which was stirred at room temperature for 30 minutes leaving behind a dark brown

24
insoluble. The solution was then filtered through a plug of celite and washed with

copious amounts of pentanes. The organic filtrate was concentrated under reduced

pressure to afford a yellow oil (25.8 g crude). 1H NMR (500 MHz, CDCl3) 8.35 (s, 1H),

7.42 7.32 (m, 2H), 6.95 6.85 (m, 2H), 5.27 (s, 2H), 3.81 (s, 3H).

(Z)-1,4-bis((4-methoxybenzyl)oxy)but-2-ene (2.8): To a flame dried 500 mL

round bottom under an atmosphere of N2 was added PMB-imidate (crude, 13.0 g, 46.8

mmol) and dry DCM (200 mL), followed by cis-butene-1,4 diol (1.52 mL, 18.6 mmol).

Subsequently, catalytic camphor sulfonic acid (0.432 g, 1.86 mmol) was added and the

reaction was allowed to stir for 12 hours at room temperature. White solids appeared

indicating that the reaction was nearing completion. The reaction was concentrated

under reduced pressure to white solids and a yellow oil. The residue was then taken up

in hexanes, filtered through a plug of celite, and concentrated. The product was purified

by column chromatography (4:1 EtOAc: hexanes, Rf = 0.6) giving a yellow oil (4.23 g,

12.88 mmol, 70%). 1H NMR (500 MHz, CDCl3) 7.25 (d, J = 8.7 Hz, 4H), 6.87 (d, J =

8.7 Hz, 4H), 5.77 (td, J = 3.7, 1.8 Hz, 2H), 4.42 (s, 4H), 4.03 (d, J = 4.8 Hz, 2H), 3.80 (s,

6H).

25
 2-((4-methoxybenzyl)oxy)acetaldehyde (2.9): DCM/MeOH (120/7 mL) was

added to a 250 mL round bottom with 2.8 (3.83 g, 9.53 mmol) and cooled to -78C via

dry ice and acetone bath. An ozonolysis machine was then used to feed ozone into

reaction at a steady bubbling pace. The reaction was bubbled in ozone for ca. 30

minutes until the solution turned a slight blue. Dimethyl sulfide (3.5 mL, 43.8 mmol) was

then added to the reaction, causing the solution to become clear. The reaction was

maintained at -78C and under ozone until a persistent blue color tinted the solution (ca.

3 hours). The flask was then placed under N2 and allowed to warm to room temperature

as it stirred for 12 hours. The reaction was then concentrated under reduced pressure

and purified by column chromatography (3:2 ether: hexanes, Rf = 0.2). 1H NMR (500

MHz, CDCl3) 9.71 (s, 1H), 7.29 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.6 Hz, 2H), 4.56 (s,

2H), 4.07 (d, J = 0.8 Hz, 2H), 3.81 (s, 3H).

3-((4-methoxybenzyl)oxy)propan-1-ol (2.17): To a flame-dried round bottom

was added 1,3-propanediol (40 mL, 615 mmol) to PMB imidate (28.5 g, 103 mmol) and

dry DCM (400 mL). Catalytic CSA (2.4 g, 10.3 mmol) was subsequently added. The

reaction was monitored by TLC until all PMB-imidate had been consumed (4 hours).

The solution was then concentrated under reduced pressure, leaving a residue that was

taken up in hexanes and filtered through a plug of celite with copious washes of

hexanes. The combined organic portions were dried with MgSO 4, filtered, and

concentrated under reduced pressure. The product (9.49 g, 48.37 mmol, 60%) was

subsequently purified via column chromatography (4:1 EtOAc:Hexanes, Rf =0.3). 1H

26
NMR (400 MHz, CDCl3) 7.25 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 4.45 (s, 2H),

3.81 (s, 3H), 3.78 (dd, J = 11.2, 5.5 Hz, 2H), 3.64 (t, 2H), 2.04 (s, 1H), 1.85 (dt, J = 11.4,

5.7 Hz, 2H).

3-((4-methoxybenzyl)oxy)propanal (2.18): To a flame dried 250 mL round

bottom a solution of oxalyl chloride (2M, 12.7 mL, 25.3 mmol) in dry DCM (30.1 mL) was

cooled to -78C under N2. The temperature of the solution was closely monitored during

the addition of DMSO (6.4 mL, 42.15 mmol) in DCM (4.1 mL) such that the reaction

temperature remained below -65C. After stirring for 5 minutes, a solution of 2.17 in

DCM (6.5 mL) was added dropwise. The resulting mixture was stirred for 15 minutes,

followed by slow addition of TEA (14.7 mL, 105.37 mmol). After stirring the reaction for

10 minutes at -70C, the dry ice/acetone bath was removed and the reaction was

allowed to warm to room temperature (ca. 1 hour). The reaction was quenched with

water and allowed to stir for 15 minutes before being transferred to a separatory funnel.

The organic portion was washed successively with 5% HCl (1 x 15 mL), saturated

aqueous NaHCO3 (1 x 15 mL), and brine (1 x 15 mL). The organic layer was

subsequently dried with MgSO4, filtered, and concentrated under reduced pressure.

2.18 was purified via column chromatography (1:1 EtOAc:hexanes, Rf = 0.9) (3.481 g,

17.92 mmol, 92%). 1H NMR (500 MHz, CDCl3) 9.79 (s, 1H), 7.25 (d, J = 9.3 Hz, 2H),

6.88 (d, J = 8.6 Hz, 2H), 4.46 (s, 2H), 3.80 (s, 3H), 3.78 (t, J = 6.1 Hz, 2H), 2.68 (td, J =

6.1, 1.8 Hz, 2H).

27
 3-((4-methoxybenzyl)oxy)propanal (2.18): SO3 pyridine complex (19.5 g 122.4

mmol) was added portionwise to a stirred solution of 2.17 (8.0 g, 40.76 mmol) in DCM

(20.8 mL) under N2. DMSO (20.48 mL, 285.32 mmol) was subsequently added

dropwise followed by TEA (28.64 mL, 203.8 mmol). Inconsistencies in this reaction

ultimately led to the abandonment of this route in favor of Swern oxidation.

3-hydroxypropyl 4-methylbenzenesulfonate (3.7): 2.16 (4.7 mL, 65.8 mmol)

was dissolved in dry DCM (200 mL) and cooled to 0 C under an atmosphere of N 2.

Next, DMAP (0.8 g, 6.56 mmol) in 10 mL of DCM was added followed by TEA (3.68 mL,

26.32 mmol). Finally, a 0.5 M solution of TsCl was added (2.5g, 13.12 mmol). The

reaction was maintained at 0 C for 4 hours, at which time TLC showed complete

conversion of 2.16 to 3.7. dH2O was added to dilute the reaction solution, which was

subsequently transferred to a separatory funnel. The aqueous layer was extracted with

DCM (5 x 50 mL). The organic portions were combined , washed with brine (1 x 50 mL),

dried over MgSO4, filtered, and concentrated under reduced pressure (2.60 g, 11.29

mmol, 86%). The product was purified by column chromatography (2:3 EtOAc :

hexanes, Rf = 0.5).

28
 3-oxopropyl 4-methylbenzenesulfonate (3.8): 3.7 (108 mg, 0.434 mmol) was

dissolved in dry DCM (20 mL) and placed under an atmosphere of N2. Subsequently

NaHCO3 (54.72 mg, 0.651 mmol) was added followed by DMP (276 mg, 0.651 mmol).

Under atmospheric N2, the reaction was allowed to stir at room temperature. After 2

hours, TLC showed formation of the aldehyde but not complete conversion to 3.8. The

reaction subsequently decomposed overnight and 3.8 was not isolated (Rf = 0.8 in 3:2

EtOAc:hexanes).

5.4 First Generation Route toward Fragment B

(2R,3S)-1-((4-methoxybenzyl)oxy)-3-methylpent-4-en-2-ol (2.11): To a 25 mL

flame dried flask under N2 was added 2.6 (0.177 g, 0.61 mmol) dissolved in dry DCM (2

mL). The solution was cooled to 0C, followed by the addition of DBU (dried over

sieves). Silane 2.10 (0.127 g, 0.67 mmol) was then added dropwise. Following addition,

the reaction was allowed to warm to room temperature over ca. 1 hour. The reaction

was then cooled to 0C for the dropwise addition of aldehyde 2.9 (0.100 g, 0.56 mmol).

The reaction was maintained at 0C for 1 hour, at which time TLC confirmed

consumption of the aldehyde. The solution was concentrated under reduced pressure

and the residue was suspended in Et2O (10 mL) with vigorous stirring for 20 minutes,

29
allowing precipitation of white DBU salts. The mixture was then filtered through a plug of

celite and washed with copious amounts of Et2O. The filtrate was then treated with

TBAF (0.20 g, 0.67 mmol, 1M in THF) and stirred for two hours. Then 1 M HCl (2.78

mL) was added and the mixture was transferred to a separatory funnel. The aqueous

layer was extracted with Et2O (5 x 15 mL). The combined organics were washed with

H2O (2 x 25 mL) and saturated aqueous NaHCO3 (1 x 25 mL). The organics were then

dried with MgSO4, filtered, and concentrated under reduced pressure. The product (0.45

g, 0.201 mmol, 36%) was purified via column chromatography (2:3 Et2O:hexanes Rf =

0.45). 1H NMR (500 MHz, CDCl3) 7.29 7.22 (m, 2H), 6.90 6.87 (m, 2H), 5.73 (ddd,

J = 17.3, 10.3, 7.9 Hz, 1H), 5.07 (dd, J = 1.8, 1.2 Hz, 1H), 5.03 (dd, J = 1.8, 1.2 Hz, 1H),

4.47 (s, 2H), 3.81 (s, 3H), 3.67 3.59 (m, 1H), 3.53 (dd, J = 9.5, 3.1 Hz, 1H), 3.36 (dd, J

= 9.5, 7.9 Hz, 1H), 2.36 2.27 (m, 1H), 1.07 (d, J = 6.8 Hz, 3H).

2.0 g aldehyde scale recovery: To recover the Leighton (S, S) ligand, the

combined aqueous layers were treated with 1 M aqueous NaOH (70 mL) and extracted

with DCM (20 x 25 mL). The combined organics were washed with water (2 x 50 mL),

dried over MgSO4, filtered, and concentrated. The Leighton (S, S) ligand could be

purified by dissolving the residue in minimum hot 9:1 MeOH:H2O. The hot, saturated

solution was allowed to cool to room temperature, and deionized water was added. The

30
solution was then cooled to 0C overnight to ensure complete crystallization of the

ligand. The white solid was then collected by vacuum filtration (79% recovery).

tert-butyl(((2R,3S)-1-((4-methoxybenzyl)oxy)-3-methylpent-4-en-2-

yl)oxy)dimethylsilane (2.12): To a -78C solution of 2.11 (0.430 g, 1.918 mmol) in dry

DCM (10 mL) was added 2,6-lutidine (0.45 mL, 3.84 mmol) dropwise followed by the

dropwise addition of TBSOTf (2.20 mL g, 9.592 mmol). The reaction was maintained at

-78C for three hours and monitored by TLC until completion. The reaction was

quenched with saturated NaHCO3 and warmed to room temperature. After transferring

to a separatory funnel, the aqueous layer was extracted with DCM (3 x 25 mL). The

organic layers were combined, washed with brine (1 x 50 mL), dried with MgSO 4, and

concentrated under reduced pressure. The product (0.500 g, 1.426 mmol, 74%) was

purified via column chromatography (2:3 Et2O:hexanes Rf = 0.85). 1H NMR (500 MHz,

CDCl3) 7.25 (dd, J = 5.6, 4.7 Hz, 2H), 6.87 (d, J = 8.3 Hz, 2H), 5.82 (ddd, J = 17.3,

10.1, 7.4 Hz, 1H), 5.04 4.98 (m, 1H), 4.97 (dd, J = 10.4, 1.0 Hz, 1H), 4.42 (q, J = 11.6

Hz, 2H), 3.81 (s, 3H), 3.72 (dd, J = 10.4, 5.3 Hz, 1H), 3.42 (dd, J = 9.7, 4.9 Hz, 1H),

3.33 (dd, J = 9.7, 6.0 Hz, 1H), 2.39 (dd, J = 12.1, 6.5 Hz, 1H), 0.98 (d, J = 6.7 Hz, 3H),

0.88 (s, 9H), 0.03 (s, 6H).

31
 (2R,3S)-2-((tert-butyldimethylsilyl)oxy)-3-methylpent-4-en-1-ol (2.13): To a

DCM: pH 7 buffer (18:1, 37 mL) solution containing 2.12 (0.500 g, 1.426 mmol), DDQ

(0.973 g, 4.27 mmol) was added. The reaction was stirred at room temperature for one

hour, followed by quenching with saturated aqueous NaHCO3. The reaction mixture was

diluted with EtOAc and then filtered through a plug of celite with DCM and EtOAc rinses.

The filtrate was then transferred to a separatory funnel and extracted with EtOAc (3 x 75

mL). The organic portions were combined, washed with brine (1 x 50 mL), dried with

MgSO4, filtered, and concentrated under reduced pressure. The product (0.260 g, 1.130

mmol, 79%) was then purified via column chromatography (2:3 Et2O: hexanes, Rf = 0.7).
1
H NMR (500 MHz, CDCl3) 5.89 5.69 (m, 1H), 5.08 (dd, J = 1.8, 1.3 Hz, 1H), 5.04

(dt, J = 2.8, 1.4 Hz, 1H), 3.55 (dd, J = 5.0, 1.2 Hz, 2H), 2.48 2.35 (m, 1H), 1.75 (t, J =

6.1 Hz, 1H), 1.25 (s, 1H), 1.02 (d, J = 6.9 Hz, 3H), 0.92 (s, 9H), 0.09 (s, 6H). 13C NMR

(126 MHz, CDCl3) 140.81, 115.04, 76.54, 64.60, 41.39, 26.08, 16.07, -4.19, -4.29.

(2R,3S)-2-((tert-butyldimethylsilyl)oxy)-3-methylpent-4-en-1-yl 4-

methylbenzenesulfonate (2.14): An oven dried round bottom containing 2.13 (0.242 g,

1.08 mmol) in pyridine (0.8 mL) under N2 was cooled to 0C by ice bath, after which

TsCl (0.40 g, 2.12 mmol) was added. The reaction was warmed to room temperature

32
and monitored by TLC. After 3 hours, the completed reaction was diluted with dH2O.

The solution was transferred to a separatory funnel and the aqueous layer was

extracted with DCM (3 x 50 mL). The combined organic portions were washed with

water (1 x 25 mL), dried over MgSO4, filtered, and concentrated under reduced

pressure. 2.14 (0.400 g, 1.04 mmol, 98%) was purified via column chromatography

(9.5:0.5 Et2O:hexanes). 1H NMR (500 MHz, CDCl3) 7.93 (d, J = 8.3 Hz, 2H), 7.78 (d, J

= 8.2 Hz, 2H), 5.71 (ddd, J = 12.7, 10.0, 7.4 Hz, 1H), 5.02 4.95 (m, 2H), 3.98 (dd, J =

9.9, 4.1 Hz, 1H), 3.82 (dd, J = 9.9, 6.6 Hz, 1H), 3.72 (dd, J = 10.1, 5.8 Hz, 1H), 2.45 (s,

3H), 2.36 2.24 (m, 1H), 0.94 (d, J = 6.9 Hz, 3H), 0.84 (s, 9H), 0.01 (d, J = 4.8 Hz, 6H).

tert-butyldimethyl(((3S,4S)-4-methylhex-5-en-3-yl)oxy)silane (2.15): To an

oven dried 25 mL round bottom under N2 was dissolved 2.14 (0.07 g, 0.182 mmol) in

0.9 mL of dry ether (0.2 M). The reaction was then cooled to 0C for the addition of

MeMgI (0.182 mL, 1M) dropwise. After a half hour, a second equivalent (0.182 mL, 1M)

of MeMgI was again added dropwise. The reaction was monitored by TLC, fully

converting 2.14 after a half hour. The reaction was quenched with ammonium chloride

and transferred to a separatory funnel. The aqueous layer was extracted with Et2O (3 x

15 mL). The combined organic portions were washed with brine (1 x 15 mL), dried with

MgSO4, filtered, and concentrated under reduced pressure (0.32 mg, 0.146 mmol,

80%). []20D -27.6 (c 1.00, CHCl3). Scaling the reaction to 0.400 and 0.210 resulted in

little conversion to the product, despite modifications of heating the reaction to reflux

33
and stirring for 12 hours. 1H NMR (500 MHz, CDCl3) 5.69 (ddd, J = 17.4, 10.4, 7.8 Hz,

1H), 5.14 (ddd, J = 17.3, 1.8, 1.2 Hz, 1H), 5.05 (ddd, J = 10.3, 1.7, 0.8 Hz, 1H), 3.29

(dd, J = 9.8, 5.3 Hz, 1H), 3.24 3.20 (m, 2H), 2.56 2.48 (m, 1H), 1.01 (d, J = 6.8 Hz,

3H), 0.92 (s, 9H), 0.09 (d, J = 19.2 Hz, 6H).

5.5 Second Generation Route toward Fragment B

(3S,4S)-1-((4-methoxybenzyl)oxy)-4-methylhex-5-en-3-ol (2.19): See

experimental of 2.11 for synthesis methodology, incorporating aldehyde 2.18 for 2.9.

The reaction was scaled up to using 4.00 g of 2.18 successfully. Yield: 79% Rf = 0.7

(2:3 Et2O:hexanes Rf = 0.60). 1H NMR (500 MHz, CDCl3) 7.25 (d, J = 8.7 Hz, 2H),

6.87 (d, J = 8.6 Hz, 2H), 5.86 5.69 (m, 1H), 5.07 (d, J = 1.3 Hz, 1H), 5.05 5.01 (m,

1H), 4.45 (s, 2H), 3.80 (s, 3H), 3.73 3.67 (m, 1H), 3.67 3.57 (m, 2H), 2.90 (d, J = 3.2

Hz, 1H), 2.25 (dq, J = 13.6, 6.8 Hz, 1H), 1.78 1.70 (m, 2H), 1.04 (d, J = 6.8 Hz, 3H).

tert-butyl(((3S,4S)-1-((4-methoxybenzyl)oxy)-4-methylhex-5-en-3-

yl)oxy)dimethylsilane (2.20): See experimental of 2.12 for synthesis methodology,

incorporating 2.19 for 2.11. Yield: 65%, Rf = 0.80 in 4:1 EtOAc:Hexanes. 1H NMR (400

34
MHz, CDCl3) 7.31 7.21 (m, 2H), 6.93 6.84 (m, 2H), 5.94 5.79 (m, 1H), 5.01 (dq,

J = 3.6, 1.7 Hz, 1H), 5.00 4.96 (m, 1H), 4.41 (q, J = 11.5 Hz, 1H), 3.80 (s, 2H), 3.76

3.66 (m, 1H), 3.55 3.42 (m, 1H), 2.36 2.22 (m, 1H), 1.80 1.58 (m, 1H), 0.96 (d, J =

6.9 Hz, 3H), 0.88 (s, 9H), 0.04 (d, J = 3.7 Hz, 6H).

tert-butyl(((3S,4S)-1-((4-methoxybenzyl)oxy)-4-methylhex-5-en-3-

yl)oxy)dimethylsilane (2.20): To an oven dried round bottom flask was added 2.19

(0.112 g, 0.47 mmol) and dry DMF (1 mL) to make a 0.5 M solution under an

atmosphere of N2. Solid imidazole (0.064 g, 0.94 mmol) followed by TBSCl (0.12 g, 0.80

mmol) was subsequently added. The reaction was then allowed to stir at room

temperature for 12 hours and monitored for completion by TLC (R f = 0.80 in 4:1

EtOAc:hexanes). The solution was then quenched w/ saturated aqueous NaHCO3 and

extracted with DCM (3 x 25 mL). The resulting oil was purified by column

chromatography (4:1 EtOAc:hexanes) (127 mg, 0.349 mmol, 78%).1H NMR (400 MHz,

CDCl3) 7.31 7.21 (m, 2H), 6.93 6.84 (m, 2H), 5.94 5.79 (m, 1H), 5.01 (dq, J =

3.6, 1.7 Hz, 1H), 5.00 4.96 (m, 1H), 4.41 (q, J = 11.5 Hz, 1H), 3.80 (s, 2H), 3.76 3.66

(m, 1H), 3.55 3.42 (m, 1H), 2.36 2.22 (m, 1H), 1.80 1.58 (m, 1H), 0.96 (d, J = 6.9

Hz, 3H), 0.88 (s, 9H), 0.04 (d, J = 3.7 Hz, 6H).

35
 (3S,4S)-3-((tert-butyldimethylsilyl)oxy)-4-methylhex-5-en-1-ol (2.21): See

experimental of 2.13 for synthesis methodology, incorporating 2.20 for 2.12. Yield: 65%

over two steps.

(3S,4S)-3-((tert-butyldimethylsilyl)oxy)-4-methylhex-5-en-1-yl 4-

methylbenzenesulfonate (2.22): See experimental of 2.14 for synthesis methodology,

incorporating 2.21 for 2.13. Yield: 65% over two steps. 1H NMR (400 MHz, CDCl3)

7.78 (d, J = 8.3 Hz, 2H), 7.34 (dd, J = 8.0, 0.6 Hz, 2H), 5.78 (ddd, J = 17.3, 10.6, 6.8 Hz,

1H), 5.01 (dd, J = 9.6, 1.4 Hz, 1H), 4.95 (t, J = 1.6 Hz, 1H), 4.18 4.01 (m, 2H), 3.69

3.50 (m, 1H), 2.45 (s, 3H), 2.25 (dt, J = 13.7, 6.9 Hz, 1H), 1.83 1.58 (m, 2H), 0.92 (d, J

= 6.9 Hz, 3H), 0.82 (s, 9H), 0.03 (dd, J = 27.7, 12.9 Hz, 6H).

tert-butyldimethyl(((3S,4S)-4-methylhex-5-en-3-yl)oxy)silane (2.15): 2.22

(0.107 g, 0.268 mmol) dissolved in 1.0 mL THF was added dropwise to precooled

solution (-78 C) of LAH (0.031 g, 0.805 mmol) in THF (2.0 mL). The solution was then

36
allowed to warm to room temperature and stir for 4 hours. The solution was then cooled

to -78 C, and the reaction was quenched according to the Fieser workup. After the

solution turned white, MgSO4 was added and the solution was filtered through a plug of

celite with copious amounts of ether. Additional MgSO4 was then added to the organic

solution, which was subsequently filtered and concentrated under reduced pressure.

The oil was purified via column chromatography (ether:hexanes 0.1:9.9) to afford 2.15

(0.052 g, 0.245 mmol, 86%).

5.6 Allylation Route toward Fragment B2

(R)-1-((4-methoxybenzyl)oxy)hex-5-en-3-ol (3.2): See experimental of 2.11 for

synthesis methodology, incorporating aldehyde 2.18 for 2.9 and silane 3.1 for 2.10.

Yield: 62%, Rf = 0.65 in EtOAc:Hexanes 3:2. 1H NMR (400 MHz, CDCl3) 7.24 (d, J =

8.3 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 5.82 (dddd, J = 11.2, 10.3, 7.5, 6.7 Hz, 1H), 5.15

5.04 (m, 2H), 4.44 (s, J = 11.2 Hz, 2H), 3.78 (s, 3H), 3.68 (dt, J = 10.6, 5.4 Hz, 1H), 3.64
13
3.57 (m, 2H), 2.96 (d, J = 2.5 Hz, 1H), 2.31 2.18 (m, 1H), 1.81 1.67 (m, 1H). C

NMR (101 MHz, CDCl3) 159.52, 135.16, 130.35, 129.53, 117.69, 114.09, 73.16,

70.56, 68.79, 55.48, 42.16, 36.12.

37
 (R)-tert-butyl((1-((4-methoxybenzyl)oxy)hex-5-en-3-yl)oxy)dimethylsilane

(3.3): See experimental of 2.20 using TBSCl for synthesis methodology, incorporating

3.2 for 2.19. Purified by column chromatography (0.5:9.5 ether:hexanes). Yield: 72%.

[]20D -16.6 (c 1.00, CHCl3). 1H NMR (400 MHz, CDCl3) 7.30 7.22 (m, 2H), 6.90

6.83 (m, 2H), 5.87 5.74 (m, 1H), 5.04 (ddq, J = 3.3, 2.2, 1.2 Hz, 1H), 5.03 4.98 (m,

1H), 4.41 (q, J = 11.5 Hz, 2H), 3.93 3.84 (m, 1H), 3.80 (s, 3H), 3.51 (t, J = 6.7 Hz, 2H),
13
2.31 2.13 (m, 2H), 1.85 1.64 (m, 2H), 0.88 (s, 9H), 0.05 (d, J = 4.3 Hz, 6H). C

NMR (101 MHz, CDCl3) 159.37, 135.21, 129.49, 117.15, 113.99, 77.56, 72.83, 69.23,

67.00, 55.51, 42.52, 36.97, 26.11, -4.11, -4.48.

(R)-3-((tert-butyldimethylsilyl)oxy)hex-5-en-1-ol (3.4): See experimental of

2.13 for synthesis methodology, incorporating 3.3 for 2.12. Yield: 65% over two steps.

Rf = 0.20 in 1:3 ether:hexanes.

(R)-3-((tert-butyldimethylsilyl)oxy)hex-5-en-1-yl 4-methylbenzenesulfonate

(3.5): See experimental of 2.14 for synthesis methodology, incorporating 3.4 for 2.13.

38
Yield: 65% over two steps. Rf = 0.70 in 1:3 ether:hexanes. 1H NMR (500 MHz, CDCl3)

7.81 7.76 (m, 2H), 7.36 7.32 (m, 2H), 5.71 (ddt, J = 17.4, 10.3, 7.2 Hz, 1H), 5.02

(dtd, J = 6.8, 2.0, 1.0 Hz, 1H), 4.99 4.98 (m, 1H), 4.10 (dd, J = 7.3, 5.8 Hz, 2H), 3.85

3.76 (m, 1H), 2.45 (s, 3H), 2.17 (ddt, J = 7.1, 5.8, 1.3 Hz, 2H), 1.85 1.75 (m, 1H), 1.69

(ddt, J = 13.8, 7.9, 5.8 Hz, 1H), 0.82 (s, 9H), -0.00 (d, J = 25.3 Hz, 6H).

(S)-tert-butyl(hex-5-en-3-yloxy)dimethylsilane (3.6): See experimental of 2.20

using LAH for synthesis methodology, incorporating 3.5 for 2.22. Yield: 86%. Rf = 0.95

in ether:hexanes 0.5:9.5. 1H NMR (500 MHz, CDCl3) 5.82 (ddt, J = 17.3, 10.2, 7.2 Hz,

1H), 5.05 (dt, J = 2.3, 1.5 Hz, 1H), 5.02 (dddd, J = 4.9, 3.3, 2.2, 1.1 Hz, 2H), 3.66 3.57

(m, 1H), 2.25 2.15 (m, 1H), 1.57 1.36 (m, 2H), 1.33 1.27 (m, 1H), 0.89 (s, 9H),
13
0.88 (t, J = 3.7 Hz, 3H), 0.05 (d, J = 0.8 Hz, 6H). C NMR (101 MHz, CDCl3) 135.79,

116.70, 73.42, 41.68, 29.70, 26.13, 9.87, -4.20, -4.31.

(S,E)-5-((tert-butyldimethylsilyl)oxy)hept-2-en-1-ol (3.10): 3.6 (0.200 g, 0.935

mmol) dissolved in dry DCM (1 mL) and 3.9 (1.61 g, 9.35 mmol) in dry DCM (3 mL) was

added to a oven-dried round bottom flask under N2. Subsequently, Grubbs catalyst

(0.029 g, 0.047 mmol) was added to the reaction, which was then heated to reflux for 12

hours. The solution was then concentrated under reduced pressure and dissolved in

39
methanol. K2CO3 (1.55 g, 11.22 mmol) was then added to the reaction which was

allowed to stir at room temperature for 30 minutes. The solution was then diluted with

dH2O and ether. The aqueous layer was extracted with ether, and the combined organic

portions were washed with brine. The organic solution was subsequently dried with

MgSO4, filtered, and concentrated under reduced pressure. The oil (0.197 g, 48.16

mmol, 82%) was purified by column chromatography (ether:hexanes 1:4). 1H NMR (400

MHz, CDCl3) 5.75 5.60 (m, 2H), 4.10 (t, J = 4.8 Hz, 2H), 3.66 3.58 (m, 1H), 2.23

2.16 (m, 2H), 1.53 1.36 (m, 1H), 1.24 (t, J = 5.6 Hz, 2H), 0.89 (s, 9H), 0.86 (t, J = 6.1
13
Hz, 3H), 0.04 (d, J = 0.8 Hz, 6H). C NMR (101 MHz, CDCl3) 131.32, 129.99, 73.41,

64.06, 39.94, 34.79, 29.77, 26.11, 9.89, -4.21, -4.29.

(S,E)-tert-butyl((7-iodohept-5-en-3-yl)oxy)dimethylsilane (3.11): 3.10 (0.185

g, 0.757 mmol) was dissolved in dry THF (5 mL) under N 2 and cooled to 0 C. After

cooling, PPh3 (0.298 g, 1.135 mmol) was added followed by imidazole (0.103 g, 1.514

mmol). Iodine (0.250 g, 0.984 mmol) was then added to the solution, which was

monitored by TLC (Rf = 0.25 in hexanes). After several hours, the reaction was diluted

with dH2O and extracted with ether (5 x 25 mL). The combined organic layers were

dried with MgSO4, filtered, and concentrated under reduced pressure. 3.11 (0.199 g,

0.562 mmol) was subsequently purified via column chromatography (1:9 ether:hexanes)

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 (R)-4-benzyl-3-((2S,7S,E)-7-((tert-butyldimethylsilyl)oxy)-2-methylnon-4-

enoyl)oxazolidin-2-one (3.13): NaHMDS (1.12 mL, 1.124 mmol) dissolved in THF (0.4

mL) was cooled to -78 C, followed by dropwise addition of 3.12 (0.262 g, 1.124 mmol).

After 30 minutes of stirring, 3.11 (0.199 g, 0.562 mmol) dissolved in THF (1.0 mL) was

added along with THF rinses (2 mL). After stirring for 3 hours, the reaction was

quenched with saturated aqueous NH4Cl and extracted with EtOAc (5 x 10 mL). The

combined organics were then washed with brine (1 x 15 mL) and dried with NaSO 4. The

solution was then filtered and concentrated under reduced pressure. The oil and

remaining starting material was purified via column chromatography (1:9

ether:hexanes). Rf = 0.5 in 1:4 ether:hexanes, Yield: 38% overall, 80% brsm. 1H NMR

(400 MHz, CDCl3) 7.32 (t, J = 7.1 Hz, 2H), 7.27 (d, J = 7.1 Hz, 2H), 7.21 (d, J = 6.8

Hz, 1H), 5.60 5.38 (m, 2H), 4.67 (tdd, J = 7.4, 5.0, 2.1 Hz, 1H), 4.22 4.10 (m, 2H),

3.85 3.74 (m, 1H), 3.63 3.53 (m, 1H), 3.28 (dd, J = 13.3, 3.3 Hz, 2H), 2.71 (td, J =

13.2, 9.7 Hz, 2H), 2.52 2.42 (m, 1H), 2.21 2.11 (m, 1H), 1.54 1.35 (m, 2H), 1.17 (t,

J = 6.6 Hz, 3H), 0.88 (s, 9H), 0.85 (t, J = 7.4 Hz, 3H), 0.03 (d, J = 1.6 Hz, 6H).13C NMR

(101 MHz, CDCl3) 176.91, 153.30, 135.64, 130.04, 129.62, 129.16, 129.00, 127.54,

73.62, 73.59, 66.22, 55.57, 55.54, 40.44, 38.37, 37.86, 37.16, 31.82, 29.62, 26.13,

16.54, 9.85, -4.20, -4.33.

41
 (2S,7S,E)-7-((tert-butyldimethylsilyl)oxy)-2-methylnon-4-en-1-ol (3.14): 3.13

(0.097 g, 0.211 mmol) in THF (0.5 mL) was added along with three 0.5 mL THF rinses

to LAH (0.024 g, 0.633 mmol) in THF (2.5 mL) precooled to -78 C. The reaction stirred

at -78 C for 2 hours and then room temperature for 1.5 hours. After TLC (Rf = 0.5 in 1:1

ether:hexanes) confirmed full conversion of 3.13, the solution was then cooled to -78 C

and quenched using the Fieser workup. After the solution turned white, MgSO 4 was

added and the solution was filtered through a plug of celite with copious amounts of

ether. Additional MgSO4 was then added to the organic solution, which was

subsequently filtered and concentrated under reduced pressure. The oil was purified via

column chromatography (ether:hexanes 1:4) to afford 3.14 (0.049 g, 0.171 mmol,

85%).1H NMR (400 MHz, CDCl3) 5.43 (td, J = 5.7, 3.7 Hz, 2H), 3.63 3.54 (m, 1H),

3.47 (dt, J = 16.5, 10.4 Hz, 2H), 2.24 2.17 (m, 1H), 2.15 (dd, J = 8.0, 4.3 Hz, 1H), 2.12

2.06 (m, 1H), 1.99 1.82 (m, 1H), 1.76 1.62 (m, 1H), 1.51 1.33 (m, 2H), 0.91 (t, J
13
= 6.7 Hz, 3H), 0.88 (s, 9H), 0.86 (t, J = 7.4 Hz, 3H), 0.03 (s, 6H). C NMR (101 MHz,

CDCl3) 130.53, 128.78, 73.75, 68.24, 40.39, 36.90, 36.21, 31.37, 29.63, 26.13, 16.63,

9.87, -4.21, -4.33.

42
5.7 NMR Spectra

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5.8 Biological Evaluation of GEX1A Analogues

We are interested in understanding how GEX1A affects cholesterol homeostasis

at both the cellular and molecular level. To probe the pharmacophore responsible for

GEX1As observed biological activity against NPC, we will conduct preliminary

biological screening of all GEX1A analogues in cell models of NPC disease. To

evaluate cholesterol trafficking in NPC cells, a filipin staining assay will be performed.

To determine whether GEX1A analogues influence the alternative splicing of the genes

directly involved with lysosomal cholesterol egress (NPC1 and NPC2), an alternative

splicing assay will be utilized.

Filipin Staining Assay

To assess whether GEX1A analogues can affect intracellular cholesterol

trafficking in NPC cells, we will conduct a microscopy-based cholesterol detection

assay. Filipin, a highly fluorescent stain specific for free cholesterol, will be used to

visualize cholesterol distribution in both treated and untreated cells. To understand the

changes in cholesterol transport induced by our compounds, we will utilize both CHO

cells exhibiting an NPC phenotype (CHO-K1 10-3)12, and human fibroblasts with a

mutation in either NPC1 (GM03123)3 or NPC2 (GM18455)13. The CHO-K1 10-3 cells

were generously provided by Prof. Laura Liscum (Tufts University), and GM03123 and

GM18455 were obtained from the Coriell Institute. Healthy human fibroblasts

(GM05659, Coriell Institute) will be used as a control in these experiments. Cells will be

maintained under standard culture conditions (Dulbeccos Modified Eagle Medium or

Hams F12 Medium supplemented with 10% FBS, at 37C and 5% CO 2) and seeded

73
into 4-well chamber slides (well volume of 1 mL) suitable for imaging as necessary. The

cells will be incubated with a purified GEX1A analogue at 100 nM for 24 hours, at which

point the cells will be fixed and stained with filipin (50 g/mL). Intracellular fluorescence,

corresponding to filipin-stained cholesterol, will then be visualized using a Nikon TE-

2000U epifluorescence microscope with UV filters at 40x magnification. Using Nikon

Elements and ImageJ software, we will analyze the collected images both qualitatively

and quantitatively. We will specifically determine the efficacy of drug treatment by

calculating both the average filipin intensity and the lysosome compartment ratio

compared to treatment with vehicle control DMSO14. Compounds that are found to

reverse cholesterol accumulation in this initial screen will be subjected to further studies

to elucidate their potential mechanism of action.

Alternative Splicing Assay

To determine whether GEX1A analogues influence the alternative splicing of

NPC1 and NPC2, we will analyze the production of NPC1 splice variants in response to

treatment with GEX1A analogues. We will compare the size and relative abundance of

the spliceoforms of NPC1 or NPC2 in RNA isolated from treated vs. untreated cells.

CHO 10-3, GM03123, or GM18455 cells will be incubated with purified GEX1A

analogue at 100 nM for 24 hours, at which point total RNA will be isolated from the cells

using the RNeasy Mini Kit (Qiagen)15. RNA will also be isolated from cells incubated

with DMSO as a control, as well as from healthy human fibroblasts (GM05659). In

collaboration with Prof. Chalfant, cDNA will be synthesized using this isolated RNA,

random hexamers, and SuperScript II Reverse Transcriptase (Invitrogen) according to

74
the manufacturers instructions. Using primers specific for either NPC1 (to analyze

cDNA corresponding to GM03123 cells) or NPC2 (for GM18455 cells), we will amplify

the gene of interest from the cDNA using standard PCR and analyze the products using

2% agarose gels16. Alternative splice variants of the gene of interest will be gel purified,

cloned into pCR-Blunt, and sequenced in order to determine the exact changes in exon

inclusion induced by drug treatment.

75
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