LIBRARY
Michigan State
University

 

 

 

This is to certify that the
thesis entitled

THE SYNTHESIS OF TAXANE SUBSTRATES AND
PHENYLPROPANOID COENZYME A THIOESTERS TO
PROBE THE MECHANISM OF TAXUS
ACYLTRANSFERASES

presented by

YEMANE A. MENGISTU

has been accepted towards fulfillment
of the requirements for the

Master of degree in Chemistry
Science

 

 

 

Major Professor’s Signature

/z//f/Zoc>7
/ f /

Date

MSU is an Affirmative Action/Equal Opportunity Employer

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 KIProj/Aoc8Pres/ClRC/Dateoue.indd

 

THE SYNTHESIS OF TAXANE SUBSTRATES AND
PHENYLPROPANOID COENZYME A THIOESTERS TO PROBE
THE MECHANISM OF TAX US ACYLTRANSFERASES

BY

YEMANE A. MENGISTU

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Chemistry
2009

ABSTRACT

THE SYNTHESIS OF TAXANE SUBSTRATES AND
PHENYLPROPANOID COENZYME A THIOESTERS TO PROBE THE
MECHANISM OF TAX US ACYLTRANSFERASES

By

Yemane A. Mengistu

The use of Taxus acyltransferase enzyme could minimize the several protection
and deprotection steps involved in the semisynthesis of next generation paclitaxel
analogues. N-Debenzoyl-2'—deoxypaclitaxel:N—benzoyltransferase (NDTBT) transfers a
benzoyl group to the free amine of the phenylpropanoid side chain of N-debenzoyl-2'-
deoxypaclitaxel. To investigate the substrate specificity of NDTBT; various congeners of
N-debenzoylpaclitaxel were made and incubated with several acyl CoA thioesters. It was
found that NDTBT transferred several different acyl groups to the amino group of these

substrates.

The paclitaxel pathway enzyme baccatin III:3-amino-3-phenylpropanoyltransferase
(BAPT) shown to transfer B-phenylalanine analogues to the baccatin 111 core contains a
glycine in place of a conserved histidine residue in the active site; thus, the substrate is
hypothesized to participate in the catalytic mechanism. Thioesters with modifications at

the B-carbon were synthesized and site-directed mutagenesis of BAPT was conducted.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................ vii
LIST OF FIGURES ..................................................................................................... viii
LIST OF SCHEMES ...................................................................................................... x
LIST OF SPECTRUM ................................................................................................... xi
LIST OF ABBREVIATIONS ........... xii

BROAD SUBSTRATE SPECIFICITY OF N-DEBENZOYLPACLITAXEL

ACYLTRANSFERASE (NDBAT) ................................................................................ 1
1. Introduction ................................................................................................................. 1
1.1 Paclitaxel Background and Development ............................................................. 1
1.2 Application of Biocatalysis in the Construction of Paclitaxel Analogues ..... 6
1.2. 1 Introduction .................................................................................................. 6
1.2.2. Acyltransferases on Paclitaxel Pathway ....................................................... 7
1.2.3 Synthesis of Taxane Substrates .................................................................... 9
1.3 Results ..................................................................................................................... 10
1.3.1 Synthesis of N—Debenzoyl-2'—deoxypaclitaxel ................................................. 10
1.3.2. Synthesis of 2'-deoxypaclitaxel .................................................................. 11
1.3.3 Synthesis of N-Debenzoylpaclitaxel Substrate .......................................... 12
1.3.4 Synthesis of 10-Deacetyl-N-debenzoylpaclitaxel ...................................... 15
1.3.5 Protein Harvest and Activity Assay of the NDTBT ................................... 16
1.3.6 Relative Substrate Specificity of NDTBT with Various Aroyl CoAs. ...... 17
1.4 Discussion ......................................................................................................... 22
1.4.1 Substrate Specificity .................................................................................. 22
1.4.2. Kinetics Analyses ........................................................................................ 24
1.4.3. Biocatalytic Applications ............................................................................ 26

1.5 Conclusion .......................................................................................................... 29

THE STUDY OF A PROPOSED SUBSTRATE-ASSISTED-CATALYSIS

MECHANISM FOR THE PHENYLPROPANOYLTRANSFERASE (BAPT) .............. 30
2.1 Introduction ............................................................................................................. 30
2.2 Results ..................................................................................................................... 32

2.2.1 The synthesis of R-B-phenylalanoyl CoA thioester ......................................... 32
2.2.2 The Synthesis of 3-Phenylpropanoid CoA ...................................................... 33
2.2.3 The synthesis of 3R-hydroxy-3-phenylpropanoyl CoA ................................... 34
2.2.4 Synthesis of 3-Keto-3-phenylpropanoyl CoASH thioester .............................. 36
2.2.4.1 Synthesis via a Mixed Anhydride Approach ................................................ 36
2.2.4.2 Synthesis through Meldrum’s acid ............................................................. 38
2.2.5 Synthesis of Radioactive Baccatin III .............................................................. 41
2.3 Enzyme expression and Activity assay of BAPT ............................................... 42
2.4 Discussion ...................................... ................................................................ 47
2.5 CONCLUSION .............................................................................................. 49

3. EXPERIMENTAL SECTION ...................................................................................... 50
3.1 General Methods ............................................................................................... 50
3.2 General Procedure for the synthesis of thioesters CoASH ..................................... 66
3.3 Enzyme expression, Protein Harvest, Protein Purification. .............................. 74

3.3.1 Enzyme Expression ..................................................................................... 74
3.3.2. Protein Harvest ............................................................................................ 74
3.3.3 Protein Purification .................................................................................... 75
3.3.4. Activity and Assay of BAPT ...................................................................... 75

Appendix ........................................................................................................................... 77

REFERENCES ................................................................................................................. 94

vi

LIST OF TABLES

Table 1: Relative kinetics of the benzoyl, substituted aroyl and heteroaroyl groups ....... 19

Table 2: Relative kinetics of NDTBT for alkanoyls CoA with taxane substrates ............ 21

vii

LIST OF FIGURES

Figure 1: Paclitaxel (Taxol®) .............................................................................................. 2
Figure 2: Docetaxel (Taxotere®) ......................................................................................... 2
Figure 3: Structure activity relationship of paclitaxel ......................................................... 4
Figure 4: The 1C50(nrn/L) of various modified paclitaxel analogs .................................... 5
Figure 5: Ojima-Holton coupling for the synthesis of Paclitaxel ....................................... 6
Figure 6: Promising paclitaxel analogues from a combinatorial chemistry library. ....... 27

Figure 7: Products detected in an attempt to synthesize 3-hydroxy-3-phenylpropanoyl
CoAs. ................................................................................................................................ 34

Figure 8: ESI-MS of the reaction product for the synthesis of B-keto thioesters using

Meldrum’s acid ................................................................................................................. 40
Figure 9: Confirmation of [13-3H] baccatin 111 with HPLC ............................................. 42
Figure 10: mass spectrum of product formation ............................................................... 45
Figure 11: Liquid chromatography of the extracted product ............................................ 45
Figure 12: Fragmentation pattern of N-debenzoyl-2'-deoxypaclitaxel ............................. 46
Figure 13: MS/MS of the product identified by side chain fragment ............................... 47
Figure 14: Future targets of B-amino acid CoA thioester ................................................. 48
Figure 15: 7-TES-baccatin III ........................................................................................... 51
Figure 16: (3R)—N—Boc-7-TES-2'-deoxytaxol ................................................................... 53
Figure 17: N-debenzoyl-2'-deoxypaclitaxel ...................................................................... 54
Figure 18: 2'-de_oxypaclitaxel ............................................................................................ 55
Figure 19: 2'-TBDMS—docetaxel ...................................................................................... 56

viii

Figure 20: lO-acetyl-2'-TBDMS-docetaxel. ...................................................................... 58

Figure 21: 10-acety1docetaxel ........................................................................................... 59
' Figure 22: N-Debenzoylpaclitaxel .................................................................................... 61
Figure 23: 10-Deacetyl-N-debenzoylpaclitaxel ................................................................ 62
Figure 24: 13-Oxo-7-TES-baccatin III ............................................................................. 64
Figure 25: Phenylpropanoyl CoA ..................................................................................... 67
Figure 26: (RS)-Phenylalanoyl CoA ................................................................................. 68
Figure 27: 3R-[3-phenyla1anoyl CoA ................................................................................. 70
Figure 28: (R)—3-hydroxy-3-phenylpropanoyl CoA .......................................................... 71
Figure 29: BenzoyI-2, 2-dimethyI-1, 3-dioxane-4, 6-Dione ............................................ 72

ix

LIST OF SCHEMES

Scheme 1: Proposed mechanism of NDTBT ...................................................................... 9
Scheme 2: Synthesis of 3'R-N—debenzoyl-2'-deoxypaclitaxel .......................................... 11
Scheme 3 The synthesis of 2'-deoxypaclitaxel ................................................................ 12
Scheme 4: Selective acylation of 10-deacetylbaccatin III ................................................ 12
Scheme 5: Acetylation of docetaxel with and without Lewis acid. .................................. 13
Scheme 6: Preparation of 10-acetyI-2'-0—(tert—butyldimethylsilyl) docetaxel ................. 14
Scheme 9: Use of acyltransferase enzymes to biocatalytically synthesize potent analogues
of paclitaxel ....................................................................................................................... 28
Scheme 10: Proposed catalytic mechanism of vinorine synthase .................................... 31
Scheme 11: Proposed substrate-assisted mechanism for BAPT catalysis. ....................... 31
Scheme 12: Synthesis of 3R-B-phenylalanoyl CoA thioester ........................................... 33
Scheme 13: The synthesis of phenylpropanoyl CoA ........................................................ 34

Scheme 14: Attempted Synthesis of 3-hydroxy-3-phenylpropanoyl CoA using 0-

triethylsilyl protection chemistry ...................................................................................... 35
Scheme 15 : Synthesis of 3R-hydroxyphenylpropanoyl CoA .......................................... 36
Scheme 16: Synthesis of 3-oxo-3-phenylpropanoic acid .................................................. 36
Scheme 19: Synthesis of benzoyl Meldrum’s acid ........................................................... 38
Scheme 20: Method of synthesis of B-keto esters using benzoyl Meldrum’s acid ........... 39
Scheme 21: Synthesis of [13-3H]baccatin III .................................................................... 42
Scheme 22: Assay of the natural substrate phenylalanoyl CoA and baccatin III ............ 43
Scheme 23 : Assay of unnatural 3-hydroxy-3-phenylpropanoid CoA .............................. 44

LIST OF SPECTRUM

Spectra 1: 1H and 13 C NMR for 7-TES-Baccatin III ................................................ 77
Spectra 2: 1H and '3 C NMR for l3-Oxo-7-TES-Baccatin III .................................. 78
Spectra 3: 1H and 13 C NMR for N-boc-N-debenzoyl-7-TES -2'-deoxytaxol ........... 79
Spectra 4: 1H and 13C NMR for N-debenzoyl-2'-deoxytaxol ................................... 80
Spectra 5: 1H NMR for 2'-Deoxypaclitaxel .............................................................. 81
Spectra 6: 1H and '3 C NMR for 2-TBDMS-docetaxel .............................................. 82
Spectra 7: 1H and 13C NMR for 10-acetyl—2-TBDMS-docetaxel ............................ 83
Spectra 8: 1H and 13C NMR for 10-acetyl-docetaxel ................................................ 84
Spectra 9: 1H and 13C NMR for N-debenzoylpaclitaxel ........................................... 85
Spectra 10: 1H NMR for l0-deacetyl-N-debenzoyltaxol .......................................... 86
Spectra 11: 1H NMR for N-Boc B-phenylpropanoyl CoA ........................................ 87
Spectra 12: 1H NMR for N- B-phenylpropanoyl CoA .............................................. 88
Spectra 13: 1H NMR for 3-Phenylpropanoyl CoA ................................................... 89
Spectra 14: 1H NMR for 3-Hydroxy-3-phenylpropanoyl CoA ................................. 9O
Spectra 15: ESI-MS (-) for B-phenylalanoyl CoA ................................................... 91
Spectra 16: ESI-MS ( -ve) for Phenylpropanoid CoA ............................................. 92
Spectra 17:ESI-MS (-ve) for 3-hydroxy-3-phenylpropnoyl CoA ...................... 93

xi

THF
DCM
br

s

d

t

Boc
ESI-MS
CoA
NMR
A020
TBDMSCI
NDTBT
BAPT

DBAT

LIST OF ABBREVIATIONS

Tetrahydrofuran
Dichloromethane

Broad

Singlet

Doublet

Triplet

t- Butyloxycarbonyl
Electrospray Ionization Mass Spectrometry.
Coenzyme A Salt

Nuclear Magnetic Resonance
Acetic Anhydride

t-butyldimethylsilyl chloride
N-debenzoylpaclitaxel benzoyltransferase
Baccatin III:3-aminophenylpropanoyltransferase

lO-Deacetylbaccatin III-10-0-acetyltransferase

xii

CHAPTER ONE

BROAD SUBSTRATE SPECIFICITY OF N-DEBENZOYLPACLITAXEL
ACYLTRANSFERASE (NDBAT)

1. Introduction

1.1 Paclitaxel Background and Development

The natural product paclitaxel (Figure l) is a diterpene pharmaceutical that is widely used
as an antimitotic agent1 The drug was first identified in and isolated from the Pacific yew
(Taxus brevifolia) in 1963.2 The mechanism of action involves promoting polymerization
of B-tubulin subunits, and specifically by inhibiting microtubule depolymerization during
cell division causing cell arrest and apoptosis.3‘ 4 Paclitaxel (Taxol®) and docetaxel
(Taxotere®) (Figure 2) are important anticancer agents in the treatment of a broad range
of malignancies including carcinoma of the ovary, lung, head, neck, bladder, and
esophagus. They have also demonstrated effectiveness in the initial therapy against
earlier stages of cancer, a setting in which any new therapy is likely to make its greatest
impact.5' 6 Currently, a combinations of docetaxel and other anticancer drugs have shown
good activity in advanced stage pretreated non-small cell lung cancer pretreated with
other cancer agents7and node positive breast cancer.8 These pharmaceuticals are also

receiving great attention for the treatment of Alzheimer’s and coronary restenosis.9' '0

 

Figure 2: Docetaxel (Taxotere®)

Paclitaxel stabilizes rnicrotubules against depolymerization, which is its primary
mechanism that inhibits cell proliferation. Moreover pleiotropic effects exhibited by this
drug may be responsible for its success where other chemotherapeutic agents fail.”' ‘2
This could be because paclitaxel also modulates the expression of genes that encode
enzymes for membrane assembly and those that participate in cell proliferation and
apoptosis--pr0cesses with mechanisms independent of microtubule stabilization.” '3 In
addition, anti-angiogenic activity, also independent of microtubule assembly, has been
shown at 1-10 nmol/L of paclitaxel.l4' ‘5 Although paclitaxel has led to improvement in

the duration and quality of life for some cancer patients,” the majority will develop

progressive disease after initially responding favorably to paclitaxel treatment.16 Despite

the success of paclitaxel and docetaxel in chemotherapy, there are demands to maximize
the potential benefit of paclitaxel and its analogues in order to minimize the resistance

developed against paclitaxel and docetaxel.l7

Various approaches have been undertaken to obtain better analogues of paclitaxel with
increased water solubility, specificity, and improved activity against multi-drug resistant
(MDR) tumors.18 The targets include the synthesis of next generation analogues and
prodrugs with better and enhanced blood—brain barrier permeability, bioavailability, and
cancer-target specificity. Preparations of new formulations with improved physical
properties, and cocktail therapy along with other drugs based on the combinatorial
chemistry libraries of several paclitaxel analogues are also being evaluated.
Investigations of the biological activity of these analogues revealed lead compounds with

better efficacy. ‘9'25

The structure-activity relationships of paclitaxel have been studied for several years.26
and some of the results are summarized in Figure 3. These drug congeners have provided
a foundation for the design of new generation paclitaxel analogues. Each functional
group of the C-13 isoserinyl side chain of paclitaxel is essential for the bioactivity of
paclitaxel.” 28 However, substitutions of the phenyl group of the naturally occurring
phenylisoserine with other alkyl groups improve the activity of paclitaxel. 29 As shown in
Figure 4, combinations of acetyl group substitutions at 010 and benzoyl at C-2 result in

more efficacious taxoids compared to the parent drug.” 3 l

Various modifications of the 3'-N— acyl group of paclitaxel demonstrates better activity.
Ojima and coworkers have synthesized several analogues by replacing the phenyl group
of 3'-N-benzoyl with other groups such as cyclopentyl, tertbutyloxy, cyclohexyl and other
groups which resulted in equipotent or more active analogues compared to the parent
drug.“ 24. For example, cyclohexyl in place of phenyl (Figure 4) creates a paclitaxel
analog that is 20 times more potent than paclitaxel in non small cell carcinoma“. The
ester group at C-2 and C-4, along with the oxetane and the rigid taxane are essential for
activity,26 however substituting the C-2 benzoyl with meta substituted benzoyl groups

produce a more cytotoxic drug.32

Acetyl or acetoxy
group may be removed
without significant loss

0f activity;some acyl-analogs {2161:5an May be esterified epimerized or
have Improved aCt'Vlty actfevity removed without significant loss
N-acyl group required; of activity. Some derivatives have
some acyl analogs improved activity.
have improved /\ AcO
activity 0 OH

  
 

   
 

0 Oxetene or cyclopropane

:2 OAcO 20 mg reqmred for actIVIty
32::ng 18ng changed) bRemoval of acetate reduces activity
substitul’ed hen l slightly. Replacement by other

p - y ' Free 2'- -hydroxy groups can increase activity

Some groups give 9 cup Hydroxy group
Improved aCt'V'ty helpful but not essential

Acyloxy group essential; certain

alkenyl and substituted aromatic
groups give improved activity

Figure 3: Structure activity relationship of paclitaxel

 

Figure 4: The IC50 (nm/L) of various modified paclitaxel analogs compared with
paclitaxel

A = Human breast cancer cell line; B = multidrug resistant human breast cancer cell line

Replacement of the natural acyl groups with surrogate groups at the 3'-N-, 2-0 and/or the
lO—O-positions ( Figure 4) by synthetic means has provided paclitaxel analogues that are
more efficacious than the parent drug, regarding physical, chemical and biological

24. 25

properties. This demonstrated that these sites were central to the development of next

generation paclitaxel compounds22 (Figure 3).

1.2 Application of Biocatalysis in the Construction of Paclitaxel
Analogues

1.2.1 Introduction

The tremendous demand for paclitaxel in basic research and cancer chemotherapy forced
researchers to secure better ways of resourcing paclitaxel. The total synthesis of
paclitaxel was a major achievement that involved greater than 90 steps, and thus was
never considered to address commercial supply.26' 33’” A significant source of paclitaxel
is from Taxus cells, which are grown in a suitable media and later extracted to acquire the
pharmaceutical.” This biological production circumvents the several steps of purification
needed to fraction plant chlorophylls, lignins and other non-essential plant products from
the desired taxane products when using the natural resources, the Taxus brevifolia tree; 37
Bristol-meyers Squibb adopted and optimize this method for the current supply of

Taxol®. 38' 39

 

 

   

TIPSO Ph

HO

O OH AC0 0 ores 3_ LIL Ph

0
1.Ac20,CeCI3: O > T I

; ; O 2. TES-Cl 4. Deprotection axo

H0 582 OAc
1 O-Deacetylbaccatin Ill 7-TES-baccatin Ill

Figure 5: Ojima-Holton coupling for the synthesis of Paclitaxel

An alternative method of semisynthesizing paclitaxel and its analogues40 was developed
wherein the N-benzoylisoserinyl side chain was coupled to baccatin III that is derived
synthetically from an abundant naturally occurring precursor of paclitaxel, 10-
deacetylbaccatin 111, found in the needles of Taxus baccata.41 This semisynthetic method,
however, involves silyl protection at the C7-hydroxyl, acetylation of lO-deacetylbaccatin
III at C10 to afford 7-0-protected baccatin III, synthetic attachment of the N—Bz
phenylisoserine side chain at the C-13 hydroxyl, and, finally, deprotection to yield
paclitaxel (see Figure. 5); implementing biosynthetic (i.e. enzymatic) methods to
construct paclitaxel and its analogues would circumvent these protecting group
manipulations. The demand for enantiopure pharmaceuticals and fine chemicals and
corporate awareness of the impact of their environmental footprint has prompted
initiatives to employ environmentally friendly synthesis as an alternative to chemical
synthesis laden with petroleum based solvents. Biocatalysis is being implemented into
methods that facilitate the production of rare pharmaceuticals and precursor compounds,

particularly for those that require many synthetic steps.42

1.2.2. Acyltransferases on Paclitaxel Pathway

Acyltransferases are a key class of enzymes in the biosynthesis of important natural
products, and catalyze regioselective acylation reactions during the assembly of

compounds such as paclitaxel, daunorubicin, lovastatin and penicillin.43

The gene encoding the lO-deacetylbaccatin III-lO-O-acetyltransferase (DBAT), taxane-
Zu-benzoyltransferase (TBT), taxadiene-Sq-O-acetyltransferase (TAT), N—debenzoyl-Z-
deoxypaclitaxel:N—benzoyltransferase(NDTBT), and baccatin III:3-amino—3phenylpro-
panoyltransferase(BAPT) have been isolated, expressed, and the corresponding enzymes
characterized.44

A00 0
OH BAPT, 3-amino—3
-phenylpropanoyl CoA

: TAXOL

O NDTBT, benzoyl CoA

 
 
 
   

DBAT ._
ACGIYI COA HOV.

 

 

   

H0 682 OAc

Figure 6: Biocatalytical route for the synthesis of paclitaxel

Even though paclitaxel and its precursors like baccatin III and lO-deacetylbaccatin III are
important taxoids,45 there are many naturally occurring taxoids such as cephalomanine,
taxol C, taxuspinanane J, taxuspine N, lO-deacetylcephalomanine, and other compounds
that have different acylation patterns and a variety of acyl and aroyl combinations. The
functional groups include esters of acetate, propionate, butyrate, butyrate, (including
hydroxylated and branched derivatives), tigloate, benzoate and phenylpropionate , as well
as occasional glycosyl groups.” 47 The variety of different acyl group types at a specific
regiocenter acyltransferase suggests that the acyltransferases catalyzing the acyl group
attachment could have broad substrate specificity that generates the taxane variants at a

particular hydroxyl or amino group.

1.2.3 Synthesis of Taxane Substrates

The paclitaxel biosyntheticpathway in Taxus spp. contains five acyltransferases that
transfer acyl/aroyl groups to different taxane structures. The N-debenzoyl-2'-
deoxypaclitaxel: N—benzoyltransferase (NDTBT) catalyzes the last step of paclitaxel
biosynthesis by transferring the benzoyl group to the free amine position to form a
benzamide functional group.(Scheme 1).48 NDTBT belongs to a large superfamily of

49‘ 50 acyltransferase which was named according to

acyltransferases, designated BAHD,
the first letter of each of the first four enzymes first characterized in this family
(benzylalcohol O-acetyltransferase (BEAT),gnthocyanin O-hydroxycinnam-
oyltransferase, (AHCT) N-hydroxycinnamoyl/benzoyltransferase (HCBT), and

geacetylvindoline 4-0-acetyltransferase (DAT)).51

 

 

 

Scheme 1: Proposed mechanism of NDTBT

With the aim of biocatalytically synthesizing several N-acyI-N-debenzoylpaclitaxel
variants and to test the broad specificity of NDTBT, it is imperative to synthesize various

N-debenzoyl taxane substrates.

Thus, N-debenzoylpaclitaxel, and N—debenzoyl-2'-deoxypaclitaxel were synthesized to
assess enzyme selectivity for the diterpene co-substrate. In particular, we wanted to
evaluate the dependence of 2'-hydroxylation on the N—acylation and to determine whether
N-acylation precedes hydroxylation. The other taxane 10-deacetyl-N-debenzoylpaclitaxel,
would aid in dissecting whether the acetyl group at 010 would affect N—acylation by
NDTBT catalysis. The relevant substrates were synthesized from baccatin III and
docetaxel, which are commercially available. The synthesis of these substrates discussed

in the next section.

1.3 Results
1.3.1 Synthesis of N-Debenzoyl-Z'-deoxypaclitaxel

N—Debenzoyl(3'R)-2'-deoxypac1itaxel was synthesized first by selectively protecting the
7-hydroxy1 of baccatin III (1), which is the most reactive hydroxyl group of baccatin-
III,52 to give 2 (Scheme 2). The protected baccatin III was coupled to N-Boc-R-B-
phenylalanine using 0,0-di(2-pyridyl) thiocarbonate (DPTC) in the presence of a
catalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP).53'54 to form ester 3 (N-Boc-
N-debenzoyl-2'-deoxypaclitaxel), followed by deprotection to give 4 (3'R-N-debenzoyl-

2'-deoxypaclitaxe155) as a single diasteromer.

10

A00 0 ores

TESCpr
25 °C.18h, rt, 82%

 

   

DMAP, DPTC.
N-(R)-Boc-B-phenylalanine
Toluene, 70 °C, 24h,

84 %

 

88 % Formic acid

 

rt. 65%

   

Scheme 2: Synthesis of 3'R-N—debenzoyI-2'-deoxypaclitaxel

1.3.2. Synthesis of 2'-deoxypaclitaxel

The 2'-deoxypaclitaxel was synthesized by the Schotten-Baumann method56 where 3'R-
N-debenzoyl-2'-deoxypaclitaxel (4) was acylated by treatment with benzoyl chloride
under basic conditions (Scheme 3). The final product was used as a product standard for

comparison against the biosynthetically derived product 5 (2’-deoxpaclitaxel).

ll

 

   

Scheme 3 The synthesis of 2‘-deoxypaclitaxel

1.3.3 Synthesis of N-Debenzoylpaclitaxel Substrate

Previous studies on the acetylation of IO-deacetylbaccatin III (6) using acetic anhydride
and pyridine resulted in a mixture of 7-acetylbaccatin 111,571O-deacetyl-7-acetylbaccatin
III, and baccatin III (1) (i.e.,ClO acetylation exclusively).52 Therefore, we initially
attempted to acetylate the C10 hydroxyl of docetaxel (Scheme 5) using acetic anhydride,
hoping to acquire a mixture of acetylated regioisomers, from which the desired product
would be isolated. Regrettably, 7, 10, 2'-triacetylated docetaxel (8) was formed

exclusively.

m

o Cecl3. THF, 4 h, 95%

¥
7

 

HO

 

OBz OAc
6 1

Scheme 4: Selective acylation of lO-deacetylbaccatin III

12

 

 

 

 

Scheme 5: Acetylation of docetaxel with and without Lewis acid.

As an alternative, the synthesis of N-debenzoylpaclitaxel started by chemoselectively
protecting the 2'-hydroxyl position of commercially available docetaxel(7)58 using tert-
butyldimethylsilyl chloride, (TBDMS-Cl) (Scheme 6) followed by regioselective
acetylation using cerium chloride as a Lewis acid to give lO-acetyI-2'-0-(tert-
butyldimethylsilyl) docetaxel (11) at more than 95% yield.59 The method was adopted
from selective acetylation of lO—deacetylbaccatin III (IO-DAB) to produce baccatin

III”(Soheme 4).

13

HO 0H0 O

i i TBDMSCIIImidazoIe >L )L O
0 NH
0 NH 0 ‘s. o DMF.rt.18h.99% ©/\/Il\ .
o
I

c : Fl 1’
5 0 H0 (382 er
0” —Si-

     

 

 

Ac20, CeCI3, THF
23 “C. 1.5 hr. 93%

 

Scheme 6: Preparation of 10-acety1-2'-0-(tert-butyldimethylsilyl) docetaxel

AcO 0 0H AcO O OH
HF/Pyridine

—-——-——>
THF.rt,4h.89%

   

HCOOH.4h.r1,70%

 

Scheme 7: Synthesis of N-debenzoylpaclitaxel

The resulting product (11) was deprotected with HF/pyridine to give lO-acetylated
docetaxel (12). The lO-acetyl-docetaxel was further deprotected by formic acid treatment
to give N—debenzoylpaclitaxel (13) (Scheme 7), which was to be used as a substrate for

NDTBT enzyme.60

1.3.4 Synthesis of 10-Deacetyl-N-debenzoylpaclitaxel

Various analogues of paclitaxel with the 3'N-benzoyl of paclitaxel replaced with other

1 and the acyl group at C10 replaced with various alkyl group are largely

acyl groups6
provided by synthetic means.62 These synthetic methods require several protecting group
manipulation steps that ultimately affect the optimal yield of the final target compound.
Alternatively the use of chemo-and regioselective acyltransferases could reduce the
number of protecting group steps. 10-Deacetyl-N—debenzoylpaclitaxel (14) is a model
substrate to test whether NDTBT can bioscatalytically N-acylate. paclitaxels either

acylated or deacylated at C-lO. To obtain 14, docetaxel (7) was deprotected with formic

acid as shown below (Scheme 8).

HCOOH,Toluene

r

rt, 4h, 60%

 

   

Scheme 8: Synthesis of lO-deacetyI-N-debenzoyl paclitaxel

15

1.3.5 Protein Harvest and Activity Assay of the N DTBT

With the taxanes substrates in-hand, the NDTBT enzyme needed to be overexpressed in
E. coli, isolated, and tested for activity. Recombinant ndtbt cDNA was expressed and
harvested following standard procedures.48 Briefly, the ndtbt cDNA was subcloned into
expression vector pET28a, and the resulting plasmid transformed into E.coli cells. The
bacteria was grown to log phase, then induced with 0.1 mM IPTG at 16°C for 16 h. The
cells were harvested by centrifugation and the cell pellet was resuspended in lysis buffer.
The soluble protein fraction was purified and the resulting enzyme was assayed by the

following procedure.

The presumed natural substrate N-debenzoyl-2’-deoxytaxol and other N-
debenzoylpaclitaxel substrates (10-deacetyl-N-debenzoylpaclitaxel and N-debenzoyl-
paclitaxel) were incubated with NDTBT in the presence of various CoA donors including
benzoyl; orth0-, meta-, and para-substituted benzoyl; various heteroyls, alkanoyls; and
butenoyl CoAs. The choice of aroyl, alkanoyl and heteroyl CoA donors was based on the
biological activity of various non-natural aroyl, and alkanoyl N—acylated taxanes made

synthetically by replacing the benzoyl group with these unnatural groups.” 63

To assess whether a de novo product was formed, the assays were extracted, and the
product mixture was analyzed by either UV-HPLC in mixed substrate assays to calculate
the relative specificity constants, or the mixture was separated by HPLC with the effluent
directed toward a tandem mass spectrometer (MS/MS) for selected molecular ion

fragmentation analysis. The UV and mass spectrometric data revealed that several

16

different types of N-acylated paclitaxel analogues were biosynthesized by NDTBT

catalysis.

1.3.6 Relative Substrate Specificity of NDTBT with Various Aroyl CoAs.

The relative substrate specificity of NDTBT for various aroyl CoA and N—debenzoyI-2'-
deoxypaclitaxel was calculated by competitive assay method where benzoyl CoA was
incubated in pair with each aroyl CoA used herein. The specificity constant (Vmax/KM) of
NDTBT for each aroyl CoA was estimated from the amount of N—aroylated taxane made
from the corresponding thioester in a competitive substrate reaction under typical assay

conditions. The relative specificity constants were calculated based on that of NDTBT for

benzoyl CoA (Vmax/KM = 1.6 nmoI-min"°mM'l) (Table 1; Entry 1A).

The relative velocity, instead of the specificity constants, of NDTBT with N-
debenzoylpaclitaxel and 10~deacetyl-N-debenzoylpaclitaxel substrates was calculated in
order to conserve the dearth amount of these substrates. Thus, accordingly, each was
incubated separately at a single concentration done (1 mM) with each of the 16 aroyl
CoAs (at 1 mM) for 2 h, in duplicate runs. Each sample was analyzed by ESI-MS/MS to
verify N—aroylated product identity and to quantify the relative rate at which the
biosynthesized products were formed. In these assays, the kinetic parameters of NDTBT
were unknown, regarding whether the non-natural aroyl CoAs were first-order or at

saturation at 1 mM; however, to provide a rough approximation of the relative velocities

17

(vrel) of NDTBT (100 pg) for each CoA thioester (Table 1 and 2, entries B and C), the

rates were estimated to be at steady state and first-order.

The non-aromatic CoA thioesters (acetyl CoA, butyryl CoA, butenoyl CoA, and hexanoyl
CoA) were assayed with the three N-debenzoyl taxoids. The relative velocity was
determined by using LC/MS/MS to increase detection and the velocity was compared

with the benzoyl CoA.

As shown in table (I and 2), NDTBT displayed a range of catalytic efficiencies and

relative velocities with various CoAs with each of the N-debenzoylpaclitaxel substrates.

18

 

Table 1: Relative kinetics of the benzoyl, substituted aroyl and heteroaroyl groups

 

 

 

 

 

 

 

 

 

 

 

 

R1
. A V /K B V 1 C V 1
Derived from CoA ( “.1213? 11).! re re
(nmolmm 'mM ) R2 = OH, R3 = Ac R2 = OH, R3 = H
R2 = H, R3 = Ac
0
d9 I 1.6 100 100
O
et/ 2
Not detectable Not detectable 13%
O
U“ 3
0.27 27% 33%
0
r2)” 4
1.7 33% Not detectable
F O
o9 5
0.85 8% 38%
O
Frf/ 6
0.34 11% 19%
O
O/K/ 7 0.97 <11% 68%
F
O
“CUK/ 8 0.15 < 9% 17%
O
0.38 85% 11%
O
0.37 < 1% 8%

 

 

 

 

 

19

 

Table 1: Continued Relative kinetics of NDTBT for heteroaroyl CoA groups

 

R1 A (Vmax/KM) B Vrel C Vrel

Derived from CoA
R2=H,R3=Ac R2=OH,R3=AC R2=Ol-I,R3=H

 

11 1.1 <1% 40%

O
0*”
CI
0
N/
o 12 0.31 200% 36%
O
O \
M/ 13 0.15 19% 43%

 

 

 

 

 

o
\
[fly 14 0.39 19% 5%
o
S \ /
L 15 0.37 22% 3%
o

REE/UV 16 0.16 22% 29%

 

 

 

 

 

 

 

Vmax/KM values are listed as nmol-min'l-mM'l

A = N-debenzoyI-2'-deoxypaclitaxel, B = N—debenzoylpaclitaxel, C: 10-deacety1-N-
debenzoylpaclitaxel

20

Table 2: Relative kinetics of NDTBT for alkanoyls CoA with taxane substrates

 

 

 

 

 

 

 

R1 Vrel Vrel Vrel
R2 = H, R3 = Ac R2 = OH, R3=Ac R2 = 0H.
R3=H
O
/
©Jk 17 100% 13% 12%
i
/
18 47% 85% 35%
/\i
/
19 3% 100% 92%
Ajk
\
/ 20 2% 2% 3%
/\/\)Ok
/
21 36% 68% 100%

 

 

 

 

 

 

21

1.4 Discussion

Broad substrate specificity of enzymes on secondary metabolic pathways has been
suggested as important for the evolution of metabolite diversity.64 The results of this
study demonstrated the extraordinarily broad substrate specificity of the recombinantly
expressed NDTBT enzyme in purified form when incubated with several acyl-CoA donor
substrates and three N—debenzoylpaclitaxel derivatives, N-debenzoyl-2'-deoxypaclitaxel,

N-debenzoylpaclitaxel, and l0-deacety1-N-debenzoylpaclitaxe1.

1.4.1 Substrate Specificity

Previous work with the intent to dissect the paclitaxel biosynthetic pathway has shown
that only benzoyl CoA was transferred when NDTBT was incubated with a single taxane
substrate, N-debenzoyl-2'-deoxypaclitaxe1, while acetyl CoA and phenylacetyl CoA were
not productive.48 In a more recent biosynthetic investigation,65the assembly of the
isoserinoyl side chain of paclitaxel was investigated. The result showed that benzoyl CoA
was transferred superior on to N—debenzoylpaclitaxel compared to N-debenzoyl-2'-
deoxypaclitaxel supporting an earlier claim that 2'-hydroxy1ation precedes N-
benzoylation.66 This prior study also concluded that NDTBT did not transfer other
naturally occurring short chain alkanoyl/alkenoyl groups to the amino group of the N-

debenzoylpaclitaxel substrate.65

22

The hypothesis developed herein was that NDTBT could feasibly transfer non-natural
aroyl moieties in addition to transferring a benzoyl to the amino functional group of
various derivatives of N—debenzoylpaclitaxel. To evaluate this theory, the paclitaxel
pathway N—benzoyltransferase was examined for its utility to N-aroylate analogues of
advanced taxane metabolites. NDTBT was shown to indiscriminately transfer aroyl
groups, including heteroles, 2- and 3-substituted benzoyls, alkanoyl, and alkenoyl (cf.

Tables 1) to either of the N—debenzoylated taxanes used as a co-substrate.

These results indicate that the N-benzoyltransferase is not limited to N-debenzoyl-2'-
deoxypaclitaxel as a substrate that contains the B-phenylalanine side chain. The diverse
specificity for N-aroylation was largely unaffected by the presence of the vicinal
hydroxyl group at C2' of the phenylisoserinoyl diterpenes, and thus supports the
observation described in a previous biosynthetic study.65 In addition, the lack of a C10-
acetyl group on the taxane substrate did not affect the function of the enzyme, and
therefore, conceivably, C10-acetylation could occur as a last step in the biosynthesis of
paclitaxel. Furthermore, ortho-, para- or meta-substitution on the benzoyl group
transferred from CoA generally did not affect NDTBT activity, although the para-

regioisomers within a homologous series were typically transferred faster.
The application of a selective and more sensitive HPLC electrospray ionization tandem

mass spectrometric analysis enabled the detection of the array of biosynthetic products

made by NDTBT catalysis, described herein; moreover, this mode of analysis enabled

23

categorical identification of the fragment ion of the intact side chain for each of the

biosynthetically acquired N-acyl derivatives.

1.4.2. Kinetics Analyses

The catalytic efficiency Vmax/KM for benzoyl CoA was calculated with respect to the N-

debenzoyl-2'-deoxytaxol whereas the specificity constant (Vmax/KM) of NDTBT for each

aroyl CoA was estimated from the amount of N-aroylated taxane made from the
corresponding thioester in a competitive substrate reaction under the same assay

conditions.

p-Methylbenzoyl CoA is best transferred by the NDTBT (1.7 nmol-min'l-mM'l)
compared to the natural substrate when using the presumed taxane substrate (N-
debenzoyl-2'-deoxypaclitaxel) whereas the 2-methylbenzoyl CoA was not productive. In
the case of halogen-substituted benzoyl CoA, p-chlorobenzoyl CoA with catalytic
specificity of (1.1 nmol-min‘l'mM'l' entry 11A) is the best among halogen substituted

benzoyl CoA.

The relative velocity (Vrel) for various aroyl and non-aroyl CoAs were calculated by

setting the rate for benzoyl CoA at 100% relative to the rates of NDTBT with various
aroyl CoAs and the two taxane substrates, N—debenzoylpaclitaxel and lO-deacetyl N-
debenzoylpaclitaxel (Table 1, Columns B and C). The data shows that the velocities are

distinctly variable with respect to the N-debenzoylpaclitaxel substrates.

24

For the halogen substituted such as 2- and 3-chloro benzoyl CoA had relative velocities
of less than 1%. (See Table 1, Entries 10 B and 11 B), while the rest of acyl CoA

substrates had relative velocities between 19 to 85% of the velocity for benzoyl CoA.

The catalytic efficiencies for heteroaroyl CoAs (Table 1, entries 12A, 14A, 15A) is only
around one fourth of that of benzoyl CoA (Table 1, entry 1A). Intriguingly the relative
velocity for 2-furoyl CoA was shown to be surprisingly doubled (200%) compared to
benzoyl CoA whereas, the rate for 3-furoyl was only 19% when these CoAs were

incubated with N—debenzoylpaclitaxel.

For the alkanoyl CoAs (Tablel, Entries 18, 19, 20, 21) each incubated separately with the
three taxanes and NDTBT; the relative velocities were compared with of benzoyl CoA. In
this study, acetyl CoA was converted with greater rate than other alkanoyl CoA with N-

debenzoyl-2'-deoxpaclitaxel (column A, Table 2) as the substrate. In contrast, butenoyl

CoA showed the maximum relative velocity (Vrel =100%) with NDTBT and N-

debenzoylpaclitaxel (column B, Table 2) while the natural substrate benzoyl CoA was

lower (Vrel = 13%). For lO-deacetyl-N-debenzoyltaxol (column C, Table 2), hexanoyl

CoA showed the maximum conversion set at Vrel = 100% while benzoyl CoA was

shown to have a VH3]: 12 %.

25

1.4.3. Biocatalytic Applications

These results demonstrate that NDTBT can catalyze the conversion of several N-
debenzoylpaclitaxel analogues to various acylated derivatives of paclitaxel regardless of
the hydroxyl group at the 2'-position and acetyl group at the C10-hydroxyl. Most
interestingly, some of the biocatalytically derived compounds are the next generation
paclitaxel compounds that have been derived by synthetic means, such as 3'-N—furanoyl-

N—debenzoyl taxane derivative.

1.4.3.1 Paclitaxels Modified at 3'N and C10

Figure 5 shows promising paclitaxel analogues (16, 17, and 18) that have greater
cytotoxicity against cancer cells and microtubule assembly compared to paclitaxel. It has
been reported that aliphatic and hetero-aromatic N-acyl analogues (18) with a pr0panoyl
for acetyl replacement at C10 was more cytotoxic and have better microtubule assembly
that paclitaxel.52‘ 67 Notably, when the N-benzoyl was replaced with N—(2-furanoyl), the
efficacy of the analogue was decreased relative to that of paclitaxel; however, when the
N-(2-furanoyl) replacement was coupled with a propanoyl for acetyl replacement at C10,
the cytotoxicity of the analog for human ovarian cancer increased 20-fold while the

microtubule assembly action increased slightly (Figure 6).22

26

 

17
A = <0.001 [0.02] A = 14.6 [0.02] A =0.004[0.02]
B = 0.71 [0.44] B = 2.0 [0.44] B =o.49 [0.44]

Figure 6: Promising paclitaxel analogues derived from a combinatorial chemistry
library.

A is the IC50 (pg/mL) cytotoxicity for human ovarian cancer and B is the 150 (11M) for
microtubule assembly. Paclitaxel values in brackets.

1.4.3.2 Semisynthetic Approaches to Modified Taxanes

The semisynthesis of arylamide analogs of paclitaxel includes selective hydroxyl group
protection at C7, selective acylation at the C13 hydroxyl with either an N, O-acetal-N-
aroylphenylisoserine, activated as a mixed anhydride,68 or an N—aroyllactam precursor of

l and finally deprotection to construct the target product.63 A

N-aroylphenylisoserine,2
typical example is the synthesis of the unnatural taxane containing a 3'-N-(p-
flurobenzoyl)-3'-debenzoylpaclitaxel which would require a number of steps to install in
place of the naturally occurring 3’-benzamide in the molecule.69 Currently, analog (18) is

made by Holton-Ojima B-lactam synthon method using modified baccatin III analogue

and needs more than 13 steps for semisynthesis of 18.59' 70 The application of biocatalytic

27

acylation in the described semi-synthetic methods could potentially reduce the number of
protection steps in the assembly of these second generation compounds.71 Conceivably,
Taxus acyltransferases NDTBT and the moderately promiscuous DBAT (1013—0-
acetyltransferase) can facilitate the assembly of analogues such as 18. As noted, NDTBT
can transfer 2-furanoyl to the 3'-amino group of the paclitaxel side chain, while DBAT
catalyze the transfer of propionyl and butyryl from the corresponding acyl CoA to 10-
deacetylbaccatin III to form unnatural analogues of baccatin 111.72 Therefore, analog 18 is

a reachable target via biocatalytic production as proposed in Scheme 9.

      

0
HO 0 0 \JLO
OH 0
___i§Co_A. 0“
DBAT _
HO : 3 0
OBzOAc
5 1a

 

Scheme 9: Use of acyltransferase enzymes to biocatalytically synthesizes potent
analogues of paclitaxel.

DBAT:lO-deacetylbaccatinlll:lO-O-acetyltransferase, NDTBT=N-debenzoylpaclitaxel-N-
benzoyltransferase BAPT=Baccatin 111:3 amino 3 J ,‘r r _,' "

28

1.5 Conclusion

The data described herein shows that the wild type taxus N-benzoyltransferase(NDTBT)
functions as a general acyltransferase. The broad substrate specificity of NDTBT for a
variety of acyl CoA thioesters provides momentum for the eventual application of this

biocatalyst toward the production of modified paclitaxel compounds.

Furthermore, it was anticipated that NDTBT, a benzoyltransferase, would primarily
aroylate the N—debenzoyl substrates; thus, it was intriguing to see alkanoyl and butenoyl
groups transferred by the catalyst to the acceptor substrate. Moreover, the presence of a
2'-hydroxyl group on the phenylpropanoyl side chain of the taxane substrate increased the
rate of N—alkanoylation/alkenoylation over N-benzoylation. At the early stages of
defining the scope of NDTBT specificity, the underlying effect of the 2'-hydroxyl on
alkanoyl CoA binding remains a curiosity. Conceivably, when structural data becomes
available for NDTBT valuable insight into the mechanism of substrate specificity can be
dissected, and directed mutational analysis can be employed to potentially produce new
catalyst derivatives that are able to transfer a greater or refined scope of novel acyl
groups to the taxane core or other diterpene scaffolds. The production of efficacious
paclitaxel analogues through biocatalytic means in vitro or in vivo in a suitable host
system will facilitate semi-biosynthetic methods that interface synthetic chemistry and

molecular biology.

29

CHAPTER 2

THE STUDY OF A PROPOSED SUBSTRATE-ASSISTED-CATALYSIS
MECHANISM FOR THE PHENYLPROPANOYLTRANSFERASE (BAPT)

2.1 Introduction

The acyltransferases involved in paclitaxel biosynthesis all belong to a superfamily
designated BAHD. Enzymes in this family have played a principal role in the evolution

. 4 .
73 7 The five acyltransferases Involved

of secondary metabolism in several plant species.
in paclitaxel biosynthesis are taxadien-S-ol-O-acetyltransfease (TAT), taxane-2-0-
benzoyltransferase (TBT), lO-deacetylbaccatinIII:lO-O-acetyltransferase (DBAT), N-
debenzoylpaclitaxel-N-benzoyltransferase (NDTBT), and baccatin III:3-amino—3-
phenylpropanoyltransferase (BAPT).49 The latter enzyme transfers a B-phenylalanoyl
group to a hydroxytaxane acceptor, and the derived product is an intermediate on

paclitaxel biosynthetic of paclitaxel and its analogues.55 Dissecting the mechanism of the

acyl group transfer catalyzed by BAPT will be central topic of this chapter.

The amino acid sequences of plant-derived acyltransferases in the BAHD family,
including the acyltransferases on the paclitaxel biosynthetic pathway, reveals an HXXXD
motif that is highly conserved in this family of catalysts and is presumed to reside in the
active site.49 The amino acid identity among the taxus acyltransferase is approximately
56% (with functionally equivalent residues at about 73% similarity). However, the BAPT
sequence is unique in that the proposed catalytic motif contains a natural G for H

mutation (i.e., GXXXD). Previously, a study was carried out with the BAHD

30

acetyltransferase, vinorine synthase, wherein a site—directed mutation changed the
catalytic Hi5160 to a catalytically inert alanine. The operationally expressed mutant was
rendered functionally inactive.76 This suggested that Hisrw was indispensable for
acetyltransferase activity.” 78 Hisrw is proposed to serve solely as a general base in the
mechanism of the vinorine synthase catalysis (Scheme 10), whereas the Asp164 in the
HXXXD motif is considered to serve a role in maintaining enzyme structure but not

necessary for enzyme function.

 

 

 

 

His His '
H's CoASH
HN \ HN \ CoA 1481*»
E, CoAS 8) END g 8 —N
Y 9 H ”X " o
x / CH3 0 CH3 /U\
‘H ' 0 CH3
\0 R1 I
l R‘
R1

R1OH = 17-deacetylvinorine
Scheme 10: Proposed catalytic mechanism of vinorine synthase involves His160 as a

general base.

('0 (5)
Wm We... a.
O

.S? ( SCoA
2NH NH; O

 

 

H' \Rz
RZOH = baccatin |||

Scheme 11: Proposed substrate-assisted mechanism for BAPT catalysis.
The mutagenesis study with vinorine synthase revealed the importance of the Hisrw in

the HXXXD motif in vinorine synthase catalysis.”78 Imaginably, this conserved histidine

31

residue can be considered essential among members of the BAHD family. However, the
Taxus derived BAPT is functional despite having a H-—>G mutation in this motif. It is
therefore proposed that the mechanism of BAPT utilizes the free amine of the B-amino-3-
phenylpropanoyl CoA thioester.55 To test this hypothesis, several different
phenylpropanoid CoA thioesters bearing various replacement groups at the C3 of the
phenylpropanoid in place of the amine were synthesizedss‘ 79 The CoA thioesters of
phenylpropionate, 3-keto-phenylpropionate, 3-hydroxy-3-phenylpropionate, and [3-

phenylalanyl were separately incubated with radiolabeled baccatin III.

2.2 Results

2.2.1 The synthesis of R-B-phenylalanoyl CoA thioester

The conventional method of synthesizing CoA thioesters typically proceeds via a mixed
anhydride, but the presence of functional groups (hydroxyl, amine, or B-keto) required
protecting group manipulations to manage the reactivity of the competing functional
group. Consequently, different approaches were accessed to synthesize the proposed CoA

thioesters as follows.

To synthesize (R)-B-phenylalanyl CoA, commercially available N-Boc-3-amino-
phenylpropanate acid was mixed with ethyl chloroformate to form the mixed anhydride,
and the mixed anhydride which was subsequently coupled to CoASH under basic
conditions. After quenching the reaction with HCl, the product was isolated at 65 %

which was significantly lower than the typical>90% product yield obtained when

32

coupfing

aroyl or aliphatic carbonyl anhydrides with the CoASH under similar

conditions.60 The Boc deprotection step with 88% formic acid was slow, and after 17 h,

65% of the starting material was converted to product (21) (Scheme 12).

Bocx

NH O ? Jk
. OH CICOZEt, Et3N ¥ ©/\/U\O O/\
THF,rt, 1h

 

 

 

 

19 L _
HSCOA in 0.4M NaHCO3,
t-BUOH, I‘l, 0.511
65%,
II
NH O BOC\
; 2 NH O

 

HCOOH, rt, ?
SCOA 4f SCOA
67 °/o

21 20

Scheme 12: synthesis of 3R-B-phenylalanoyl CoA thioester

2.2.2 The Synthesis of 3-Phenylpropanoid CoA

The 3-phenylpropanoid CoA was prepared to test whether the absence of the amine

functional group at C3 (cf. Scheme 13) of the substrate would affect the substrate

selectivity of BAPT, and provide preliminary evidence supporting a substrate-assisted

catalysis

(SAC)—-based mechanism. The mixed anhydride procedure described

previously55 was used to make (22). The yield was 75%, which was comparable to other

phenylpropanoids containing functional group at C3.

33

O

O
HSCoA In NaHCO
OH cucozet NEt3> OJLOM - 3 WWW“
THF.rt, 1 h 75% 23

22

 

Scheme 13: The synthesis of phenylpropanoyl CoA

2.2.3 The synthesis of 3R-hydroxy-3-phenylpropanoyl CoA

In the process of synthesizing 3R-hydroxy-3-phenylpropanoy1 CoA using the mixed
anhydride method, as explained previously, no product was formed, but LC-MS analysis
showed the formation of polymerized anhydride products that were consistent with the

putative structures.

OH O
OH O O 0
9H 0 o ? 0 9H 0 9H
: O -‘ O ’.
we“ “o “o
m/z: 314.12 rn/z: 462.17

Figure 7: Products detected in an attempt to synthesize 3-hydroxy-3-phenylpropanoyl
CoAs.

To eliminate the formation of polymerization product, the C3-hydroxyl group was
protected with triethylsilyl (TES), and the thioesterification with CoASH was attempted
by methods previously described (Scheme 14). Still, no thioester product was detected by

LC-MS analysis of the reaction mixture.

34

{/—

 

 

' Si
OH O Im1dazole,DMAP \
? TESCI _ f 9 0
OH r OH
CHZCIZ, 25 °C
24 25
CICOOEt, NEt3
THF,rt,1h
[-Si/ — (S/ _
‘0 O |\
? HSCoA. 0.4 NaHCO3 /— 9 0 i
SCoA < X 0 O’\
t-BuOH,rt, 0.5 h
26 _ _

 

 

Scheme 14: Attempted Synthesis of 3-hydroxy-3-phenylpropanoyl CoA using 0-
triethylsilyl protection chemistry

The presence of the TES group, because of its steric effect may prevent the coupling of
the CoA to the phenylpropanoate. Therefore, we re-explored the direct conversion of the
3-hydroxyphenylpropanoid to its thioester without a protecting group. The solvent system
was changed from 100% THF to the combination THFzDCM (5:2 v/v) and the reactions
were diluted 3-fold compared to previous attempt described earlier; the latter dilution was
made to reduce the effect of polymerization. In this instance, the reaction mixture
contained predominantly the desired mixed anhydride (Scheme 15), as assessed by mass
spectrometry and 1H-NMR analysis. The thioesterification of the mixed anhydride of 24
resulted in product 27 at 25% yield (Scheme 15). The yield was judged suitable to

commence with our preliminary enzyme assays.

35

OHO QHO

? HSCoA In NaHCO ’
OH ClCOzEt, NEt3> 0,3100“ W 3 SCoA
DCM,THF.rt,1 h 25% 27

24

 

Scheme 15: Synthesis of 3R—hydroxyphenylpropanoyl CoA

2.2.4 Synthesis of 3-Keto-3-phenylpropanoyl CoASH thioester

2.2.4.1 Synthesis via a Mixed Anhydride Approach

The synthesis of 3-keto-3-phenylpropionyl CoA was started with the synthesis of 3-oxo-
3-phenylpropionic acid by hydrolyzing the ethyl ester of 3-oxo-3-phenyl propionate to
obtain 3-oxo-3-pheny1propionic acid80(Scheme 16).
O O NaOH, H20,rt,12 h 0 O
o/\ > OH
28 H2804, H20,rt, 60% 29

Scheme 16: Synthesis of 3-oxo-3-pheny1propanoic acid

 

The preparation of the mixed anhydride in the case of [El-keto acids was designed to
eliminate or minimize the condensation reaction that could partially occur as a result of
the acidic u—proton of the starting material.“ The B-keto acid (29) was dissolved in THF
and added dropwise to a mixture of ethyl chloroformate and 1 equivalent of

triethylamine. The resulting keto—enol mixture of the anhydride was verified by 1H NMR.

36

Unfortunately, displacement of the ethyl carbonate by CoASH under basic conditions did
not yield the desired acyl CoA. One of the reasons for the failure of the reaction was
probably due to the facile formation of the keto-enol in the presence of base or acid. The
resonance effect of the tautomer likely stabilizes the (II-carbon (Scheme 17) and thus

reduces the strength of the nucleophilic attack by CoASH (Scheme 18).

o o o
©/U\/U\OJKO/\

NaHCO3
e e
o o) o o o o o o 0
GAO“ / GAGA \ GAGA
@J H

Scheme 17: The effect of base on tautomerization of (ethyl carbonic)-3-oxo-phenyl

propanoic anhydride

 

o o 0 0 0 CoASH in 0 o
OH__2__C'CO Et’ 5‘3”... DADA 0.4M NaHCO3 SCoA
THF,rt, 1h X >
t-BuOH
29 25 °c, 0.5h

Scheme 18: An attempt to synthesize 3-Oxo-3-phenylpropanoyl CoA

37

2.2.4.2 Synthesis through Meldrum’s acid

The failure to synthesize B-keto thioesters using the proven mixed anhydride method
promoted an alternative method. In this next approach, Meldrum’s acid was used to
activate the carbonyl group of the carboxylate for nucleophilic attack by CoASH.
Meldrum’s acid derivatives have been used successfully in previous investigations for the
synthesis of B-ketoamides and B—thioesters.82 As an example the synthesis of B-keto-B-
phenylpropanoamides, proceeded from benzoyl Meldrum’s acids wherein the derivative
was refluxed with an alcohol, amine, or thiol nucleophile to give the corresponding
ester.82 Herein, typical reaction conditions for coupling an acyl Meldrum’s acid with a

nucleophile were modified as follows.

The benzoyl Meldrum’s acid was prepared according to a literature procedure 82(Scheme
19), and the resulting derivative was dissolved in THF, and the CoASH was added slowly
as a solution in NaHC03. The reaction was heated at 40 °C overnight instead reflux
temperature (66 °C for THF) to minimize possible decomposition of the CoA reactant
and/or the resultant CoA thioester The progress of the reaction was checked by ESI-MS

(negative mode) but no product formation was observed.

0 O O 0

(>4. + A W0
O O 7 0 0K

CH20I2, rt, 12 h, 60%
30 31 32

 

Scheme 19: Synthesis of benzoyl Meldrum’s acid

38

O O

OO

O
W0 (21%?“ We
00% OO

32 33

Scheme 20: Method of synthesis of B-keto esters using benzoyl Meldrum’s acid

After promoting no reaction at 40 °C, the stability of CoASH and benzoyl Meldrum’s
acid was empirically evaluated in refluxing THF. After 3h the benzoyl Meldrum's acid
was opened up (MW = 145) but not totally degraded as determined by ESI-MS while the
CoASH remained intact (Figure 8). But no thioesterification product, B-keto thioester,
was detected. It was presumed that the thioester might be unstable at that high

temperature or not formed at all.

39

Relative abundance

 

 

 

I
I '518.1
247.0 I

I I

247.4 i.
I I
I 248.0
I
E 315.0 787.1
I ' ' I
I 1 771.2 I
I 14:50 1' 500-1 ) 788.2
I I 1 383.0 585.1 I
I L I ITI AL 11 L i

 

041. . 1_
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

Figure 8: ESI-MS of the reaction product for the synthesis of B-keto thioesters using
Meldrum’s acid

Shown unreacted CoA salt = [M + Na - H]'l= 787.1, benzoyl Meldrum's acid, [M-H]'l =
247.4, [2M + Na - H1" = 517

To avoid long reflux time and thus minimize decomposition, a microwave synthesizer
was used to keep the temperature at 40 °C for five minute while keeping all other
reagents/reactants the same as in previous attempts. The reaction mixture was analyzed
by ESI-MS (negative mode) and benzoyl Meldrum's acid, 3-oxo-3-phenylpropanoic acid
B-ketoacid, and a significant amount of CoASH were detected. Increasing the reaction
time for an additional 5 min showed an increase in the hydrolysis of the benzoyl
Meldrum's acid and the decomposition of CoASH. The challenge to provide sufficient

energy for the coupling reaction employing the benzoyl Meldrum's acid and CoASH

4O

resulted in overshooting a fine balance to overcome the energy burner for nucleophilic
attack of the CoASH on the acid and to prevent CoASH from decomposing. The
obligatory high temperature generally needed to activate by ring opening Meldrum's

acids 82made the synthesis of the B-keto thioester challenging.

2.2.5 Synthesis of Radioactive Baccatin 111

To increase the sensitivity of the detection of biosynthetic products derived from baccatin
III, a tritium labeled baccatin III substrate was synthesized from 7-TES-baccatin III (2)
according to 8 described procedure. Briefly, 2 was oxidized followed by reduction with
tritium labeled sodium borohydride and deprotection (Scheme 21) provided the [13-
3H]baccatin III which had the same Rf (retention factor) as authentic baccatin III on silica
gel thin layer chromatography and the same retention time as authentic baccatin III on

radio-HPLC.

41

H0 0 ores

””02/CH2C'2’” _ 1. NaBT4, THF . 0 °c _

02.HF-Py,THF,0°C T : - .
HO H0 (33'; OAc
2 34 35

 

 

  

90%

   

Scheme 21: Synthesis of [13-3H]baccatin 111

 

 

 

 

 

 

 

350
a 250:
IE
‘8'
.9 20°“
‘0
(U
a: 150
,
.8 ,
E :
50 SE
04 .........................................................................
o 2 4 6 8 10
Tina (Mn)
— Raddabeled Winlll
------- Authenticbaccatinlll

 

 

 

Figure 9: Confirmation of [13-3H] baccatin 111 with HPLC

2.3 Enzyme expression and Activity assay of BAPT

Standard microbial and recombinant techniques used throughout this work are described

by Sambrook.83 The full length bapt cDNA (accession AY082804) was subcloned from

42

its original expression vector pSBET55 into pET28a (Novagen), which incorporated an N-
terrninal Hisé-tag epitope for immunoblot analysis of the expressed protein and
purification by HIS-SelectTM Nickel Affinity Gel (Sigma, St. Louis, MO). The resultant
plasmid containing the bapt cDNA in pET28a was transferred into an expression vector;
E. coli BL21-CodonPlus® (DE3), the bacteria was grown, the recombinant protein was
over expressed by S-isopropyl B-D-l-thiogalactopyranoside, the protein was harvested

and purified.

The operationally soluble BAPT was verified as functional by incubating the enzyme
with R-B-phenylalanine CoA and [13-3H]baccatin 111. After 2 h,.the assay mixture was
extracted with ethyl acetate, the solvent was evaporated, and the remaining residue was
analyzed by radio HPLC and ESI-MS. UV-absorbance profiles of the biosynthetic
product isolated from the assay containing crude enzyme extracts from cells transformed
with empty vector showed no de novo radioactive peak corresponding to the biosynthetic

product.

won + T ‘9

H6 H0 OBHz DAc

   

BAPT

 

 

Scheme 22: Assay of the natural substrate phenylalanoyl CoA and baccatin III with
BAPT

43

   

BAPT

H

 

Scheme 23 : Assay of unnatural 3-hydroxy-3-phenylpropanoid CoA

AcO O OH

    

Scheme 24: Assay of unnatural 3-phenylpropanoyl CoA

The products were purified by HPLC, and the corresponding de novo UV-absorbance
peak were collected and further analyzed by a direct injection-electrospray ionization
mass spectrometry, in positive ion mode. Further diagnostics by LC-ESI-MS/MS of
selected ion m/z 734[M+H]+ revealed (Figure 10) typical diagnostic fragment ions of m/z
509, 569, and the B-phenylalanyl side chain fragment ion (m/z 166, ordinarily the base
peak) that confirmed the identity of the product as N—debenzoyl-2'-deoxypaclitaxel when
compare to authentic sample analyzed by similar methods (figure 13). The only
productive acyl CoA substrate was B-phenylalanyl CoA, and UV-absorbance profiles of
the biosynthetic product isolated from the assay containing crude enzyme extracts from
cells transformed with empty vector showed no de novo radioactive peak corresponding

to the biosynthetic product.

°/o Relative Abundance

734.3

 

 

 

 

 

 

 

100 a
80 -
60 —
40 a
20 ~
0 T' I I 1; MI r —I
100 200 300 400 500 600 700 800
m/z
Figure 10: mass spectrum of product formation
120 -
100 4 Product
Substrate
8 80 ~ H
C
(U
'0
S
a 60 <
<1:
‘3
:5 40 .
Q)
n:
o\° 20 .
0 "‘ M W W

 

 

I I I I I I

0 2 4 6 8 10 12 14 16

1

Retention Time (min)

Figure 11: Liquid chromatography of the extracted product

45

The results show the enzyme is very productive based on the approximately 50%
conversion of substrate to product, after 1h incubation with BAPT (100 11g). Several [3-
amino acid CoA thioesters will be synthesized to assess the scope of the substrate

specificity of BAPT.

   

OBI OAc
m/z: 509.22

Figure 12: Fragmentation pattern of N—debenzoyl-2'-deoxypaclitaxel

46

00 _ 166.0

 

5692

 

 

 

 

 

m/z: 569.24
m/z: 509.22
3272
387.2 734 3
0 L I A l Lllrl A ‘ 1T A r l l. I
100 200 300 400 500 600 700 800
m/z

Figure 13: MS/MS of the product identified by side chain fragment

2.4 Discussion

The result of the assay showed that optimization of the expression of BAPT enzyme by
introducing N-terrninal His6- tag epitope and the use of high expression vector enable us
to get enough quantities of active enzyme activity of enzyme so that the biosynthetic
products could be easily detected with HPLC even without the use of radioactive baccatin
III. In this experiment three phenylpropanoid CoA cosubstrates were synthesized to probe
the role of free amine in proposed substrate assisted catalysis mechanism. In a previous
study, a diasteromeric mixture of (R,S)-[3—phenylalanoyl CoA was utilized in the seminal
assays with BAPT, while in this experiment enantiopure R-B-phenylalanoyl CoA was
used. The use of enantiopure CoA could increase the possibility of product formation as

there is no competitive binding from the racemic mixture towards the binding site. In

47

addition new phenylpropanoid CoA thioesters (3R-hydroxy-3~phenylpropanoyl CoA, and
3-phenylpropanoyl CoA) were used and these thioester CoAs were incubated individually
with the other co—substrate baccatin III in the presence of BAPT. Among these the only
productive substrate was the B-phenylalanoyl CoA and the product was identified by LC-
MS and LC-MS/MS. The other phenylpropanoid thioester CoAs have similar carbon
skeletons, except they have different functional groups at C3-i.e., OH or H instead of free
amine. The synthesis of 3-oxo-phenylpropanoid CoA as a substrate failed and thus will
be a future goal of the project. Conclusively, these results support the role of the free
amine to potentially aid BAPT catalysis, particularly considering that the BAPT enzyme
naturally lacks the lone conserved histidine residue purportedly required for catalysis of
the superfamily in which BAPT belongs. Further investigation to examine the substrate

specificity of BAPT for other derivatives of B-aminopropanoid CoA thioester is

imminent.
NH2 0
NH O ?
8H2 0 ? 2 SCOA
S Co A SCoA F3C
H3C
NH2 0
NH2 0 kHz 0 =
i : SCoA
| \ SCoA SCoA
NH2 0 NH2 0

NH2 0 NH2 0 o
i ' SCoA
fiscm SCOA MSCOA \ I

Figure 14: Future targets of B-amino acid CoA thioester

2.5 CONCLUSION

The BAPT acyltransferase enzyme couples the phenylpropanoid to the baccatin 111 core.
This work has shown preliminary data that likely suggests that the BAPT enzyme
involves substrate assisted catalysis mechanism to transfer the phenylpropanoid CoA to
the Baccatin III. The presence of an amine group at the fi-position of the substrate is
seemingly crucial. As the scope of the substrate specificity examination is expanded we
might reveal that BAPT also promotes substrate assisted catalysis of unnatural B-
aminopropanoids. Conceivably BAPT could be utilized in biocatalytic process that

reduce the number of steps to synthesize these paclitaxel analogs.

49

3. EXPERIMENTAL SECTION

3.1 General Methods:

CoASH as a trilithium salt was purchased from ARC (St. Louis, MO). (R,S)-3-(tert-
butoxycarbonylamino)-3-phenylpropanoic acid (R)-3-(tert-butoxycarbonylamino)-3-
phenylpropanoic acid, (R)—3—hydroxy-3~phenylpropanoic acid were purchased. from Alfa
Aesar (Ward Hill, MA). N-tert-Butoxycarbonyl-(3R)-phenylalanine was obtained from
Acros Organics (Morris Plains, NJ), docetaxel was obtained from OChem (Des Plaines,
IL), baccatin III was purchased from Natland (Research Triangle Park, NC). C-18
(carbon 11%) reversed phase silica gel was purchased from Silicycle, (QC, Canada).
Tetrahydrofuran and dichloromethane were obtained from dry still packed with activated
alumina that was pressurized with nitrogen gas. Silica gel (230-400 Mesh) and
aluminium backed silica gel 60 Thin Layer Chromatography (TLC) plates, embedded
with A254 chromophores were purchased from EMDTM chemicals Inc (Gibbstown,
NJ).Ethyl chloroformate, ethyl benzoylacetate, and all other reagents were purchased

from Sigma-Aldrich and used without purification unless noted otherwise.

50

All reported yields are for isolated materials. 1H and 13C NMR spectra were acquired on a
Varian Inova-3OO or a Varian Unity plus 500 spectrometer and were referenced to
residual solvent signals either at 7.24 ppm or at 4.67 ppm for CDC13 and D20
respectively. All apparent coupling constants (J values) were measured at the indicated

field strengths and expressed in hertz (Hz).

A Q—ToF Ultima Global electrospray ionization tandem mass spectrometer (ESI-MS,
ESI-MS/MS, Waters, Milford, MA) with a waters CapLC capillary HPLC was used for
mass spectral analysis at the Mass Spectrometry Facility Research Training and Support
Facility, Michigan State University. An Agilent 1100 HPLC system equipped with a UV
detector was used for liquid chromatography and absorbance (A228) monitoring of

effluent.

Synthesis of 7-TES-baccatin III (2):

 

Figure 15: 7—TES-baccatin 111

To a solution of baccatin III (165 mg, 0.28 mmol) in pyridine (10 mL) was added

chlorotriethylsilane (1.42 mL, 8.45 mmol) dropwise. The solution was stirred at 25 °C for

51

24 h. After dilution with ethyl acetate (100 ml), the solution was washed with aqueous
CuSO4 (3 x 20 mL) and brine (20 mL). The organic extract was dried (MgSO4),
concentrated, and purified by flash chromatography using silica gel as stationary‘phase
and 35% ethyl acetate in hexane as a mobile phase to give 7-TES-baccatin 111(160 mg,
82.0%). Rf = 0.50 (35:65 ethyl acetate in hexane), ESI-MS (positive ion mode)
701.3[M+H]+, 723.28 [ M+Na]+. 1H NMR (CDC13, 300 MHz) 5: 8.07 (dd, 0-Ph,), 7.60 (t,
J = 1.4, J = 7.4, p-Ph), 7.49 (m, Ph) , 6.59 (s, H—10), 5.69 (d, J = 6.9 Hz, H-2), 4.92 (d, J =
8.2 Hz, H-5), 4.8 (t, H-13) 4.48 (dd, J = 6.9 Hz, H-7), 4.33 (d, J = 8.0 Hz, H-ZOa), 4.12(

d, J = 8.0 Hz, H-20b), 3.9l(d, J = 6.9 Hz, H-3), 2.96 (d, J = 20 Hz, H-l4a), 2.65 (d, J =

20. Hz, H-20b), 2.50 (m, H-601), 2.23 (s, C(O)OCH3 at C4), 2.19 (s, C-10 C(O)OCH3 and
H-l8), 1.67, 1.28, 1.19 (s, H-16, H-17 and H-l9), 0.90 (dd. 9H, Si-CHZ CH3). 0.5(m, Si-

CH2 CH3.)

52

Synthesis of (3R)-N-Boc-7-TES-2'-deoxytaxol (3):

ACO O

  
   

OTES

i L O
082 OAC

   

HO

Figure 16: (3R)-N-Boc-7-TES-2'-deoxytaxol

Dipyridyl thiocarbonate (0.305 g, 1.30 mmol) was added to a solution of 3-(R)-N—Boc-B-
phenylalanine (0.344 g, 1.30 mmol), N,N—dimethylaminopyridine (0.167 g, 1.3 mmol)
and 7-TES-baccatin 111 (0.150 g, 0.22 mmol) in toluene (24 mL). The reaction was stirred
at 70 °C for 24 h. The solvent was evaporated under reduced pressure and the crude
residue was purified by flash ‘column chromatography using silica gel and 35% ethyl
acetate in hexane as the mobile phase. Fractions containing the product were combined
and solvent evaporated under reduced pressure to obtain the desired product as pale
yellowish solid (175 mg, 85%). Rr = 0.80 (35: 65 ethyl acetate in hexane), ESI-MS
(positive ion mode): 970.4 [M+Na] +. IH-NMR (500 MHz , CDC13 ) 6: 8.07 (dd, O-Ph),
7.60 (t, J = 1.4, J = 7.4, p-Ph), 7.49 (m, Ph) , 6.59( s, H-10), 5.69( d, J = 6.9 Hz, H-2), 5.2
( s, H-3'), 4.92 (d, J = 8.2 Hz, H-5), 4.48 (dd, J = 6.9 Hz, H-7), 4.33 (d, J = 8.0 Hz, H-

20a), 4.12 (d, J = 8.0 Hz, H-20b), 3.91 (d, J = 6.9 Hz, H-3), 3.1 (dd, H-2') ,2.96 (d, J =

20 Hz, H-14a), 2.65 (d, J = 20. Hz, H-20b), 2.50 (m, H-6a), 2.23 (s, C(O)CH3 at C-4),

53

2.19 (s, C(O)CH3 at C-10 and H-18), 1.67, 1.28, 1.19 (s, H-l6, H-17 and H-19), 1.5 (s,,

C(CH3)3), 0.9 (dd, SiCHzCH3). 0.6( m, SICHZCH3)

Synthesis of N-debenzoyl-2'-deoxypaclitaxel (4)

 

i 2 O
H0 082 OAc

Figure 17: N-debenzoyl-2'-deoxypaclitaxel

Formic acid (0.5 ml, 88% aqueous solution) was added to a solution of N-Boc-7-TES-2'-
deoxypaclitaxel (100 mg, 106 umol) in 2 mL of dichloromethane and stirred at room
temp. The completion of the reaction was monitored by TLC and the residual formic acid
was removed under reduced pressure. The reaction mixture was then diluted with ethyl
acetate and the solution was washed with 5% NaHCO3 (2 x 10 mL), water (2 x 10 mL),
brine (2 x 10 mL) successively. The organic extract was dried over Na2S04 and
concentrated. The residue was purified by PTLC to get N-debenzoyl-2'-deoxypaclitaxel
(52 mg, 65%). Rr = 0.375 (10: 90 methanol: ethyl acetate), ESI-MS (positive ion mode):
734.2 [M+H]+. 1H-NMR (500 MHz , CDC13 ) 8: 8.07 (dd, O-Ph), 7.60 (t, J = 1.4, J = 7.4,

p-Ph), 7.49 (m, Ph) , 6.40 (s, H-10), 6.00 (t, J = 7.2 Hz, H-l3), 5.75 (br,NH) 5.69 (d, J =

54

6.9 Hz, H-2) 5.2 (s, H-3'), 4.92 (d, J = 8.2 Hz, H—5), 4.48 (dd, J = 6.9 Hz, H-7), 4.33 (d, J
= 8.0 Hz, H-20a), 4.12(d, J = 8.0 Hz, H-20b), 3.91 (d, J = 6.9 Hz, H-3), 3.1 (dd, H-2')

2.96 (d, J = 20 Hz, H—l4a), 2.65 (d, J = 20. Hz, H-20b), 2.50 (m, H—601), 2.23 (s,
C(O)CH3 at 04), 2.19 (s, C(O)CH3 at C-10 and H-l8), l.87-l.8 (m,H-6B) 1.67, 1.28,

1.19 (s, H-16, H-17 and H-19)

Synthesis of 2'-deoxypaclitaxel (5):

 

Figure 18: 2'-deoxypaclitaxel

In a 10-mL tube N-debenzoyl-2'—deoxypaclitaxel (8.3 mg, 0.01 mmol) was dissolved in
ethyl acetate and saturated with NaHCO3 (1 mL) to maintain the pH at 9.00. Benzoyl

chloride (40 11L, 0.01 mmol) was added and the mixture was vigorously vortexed for 30

minute and the reaction proceeds at 25 °C. The reaction was diluted with ethyl acetate
after 24 h, washed with water and brine successively, dried over MgSO4 and

concentrated and purified using PTLC using (methanol: ethyl acetate) to get a pale white

55

solid 2'-deoxypaclitaxe1 (8.51 mg, 90%). Rf = 0.875 (10:90, methanol: ethyl acetate);

ESI-MS (positive ion mode): 861 [M+Na]+ 1H-NMR, (300 MHz , CDC13 ) 6: 8.02 (,d, J =
7, O-Ph), 7.60—7.26 (m, , phenyl), 6.36 ( s, H-10), 6.1 (d, J = 6.3, 0.9 Hz, H-13), 5.66 (b,
NH) 5.62 (d, J = 6.9 Hz, H-2), 5.18 (b, H-3') , 4.90 (dd, J = 5.4, 2.1, H-5), 4.4 (dd, J =
6.9 Hz, H-7), 4.24 (d, J = 8.0 Hz, H-20a), 4.12 (d, J = 8.0 Hz, H-20b), 3.72 (d, J = 6.6,
1H, H-3), 3.05 (dd, J = 10.5, 9.9 Hz, H-2'), 2.96 (d, J =20 Hz, H—14a), 2.65 (d, J = 20 Hz,

H-20b), 2.50 (m, H-601), 2.23 (s, C(O)CH3 at C-4), 2.19 (s, C(O)CH3 at C-10 and H-l8)

1.67, 1.28, 1.19 (s, H—l6, H-l7 and H-19).

Preparation of 2'-TBDMS-docetaxel (7 )

 

Figure 19: 2'-TBDMS-docetaxel

To a solution of docetaxel (220 mg, 0.27 mmol) dissolved in DMF (2.4 mL) were added

imidazole (92 mg, 1.32 mmol, 5 equiv) and tert-butyldimethylsilyl chloride (TBDMS-Cl)

56

(204 mg, 1.32 mmol, 5 equiv). The solution was stirred at 23 °C, and after 18 h, the
mixture was diluted with ethyl acetate and washed with water (2 x 30 mL) and brine (1 x
30 mL). The organic layer was dried (MgSO4), concentrated in vacuo, and purified by
silica gel column chromatography (1:2 (v/v), EtOAc/hexane to yield 2’-TBDMS-
docetaxel as a white powder (246 mg, 0.267 mmol, 99%) yield at 99% purity as judged
by TLC and UV absorbance visualization. ESI-MS (positive ion mode): 944.6 [M+Na]+.
lH-NMR ( 300 MHz, CDCI3) (3: 8.09 (d, J = 7.5 Hz, o-ph at C2 benzoyl), 7.56 (t, J: 7.5,
7.2 Hz, p-ArH of C2- benzoyl), 7.46 (t, J = 7.8, 7.2 Hz, m-ph of C2- benzoyl), 7.36 (m,
C3' phenyl), 7.28 (m, ArH of C3' phenyl), 6.31 (t, J = 8.7 Hz, H-13), 5.66 (d, J = 7.2 Hz,
H-2), 5.43 (br (1, J = 9.3 Hz, H-3’), 5.30 (br, (1, J = 9.0 Hz, NH), 5.18 (s, H-lO), 4.95 (d,

J = 8.1 Hz, H-S), 4.5 (d, J = 2.6 Hz, H-2'), 4.32-4.17 (m, H-7), 4.17 (dd, J = 7.2 Hz, H-
20), 3.93 (d, J = 6.6 Hz, H-3), 2.61-2.58 (m, H-6), 2.53 (s, C(O)OCH3 at C4), 2.38-2.39
(m, H- 601, H-14), 2.02 (s, H-18, H-6) 1.73 (s, H-l9), 1.28 (s, N—Boc), 1.28 (s, H—l7), 0.72

(s, SiC(CH3)3), -0.13 (s, SiCH3). -0.34 (s, SiCH3).

57

Synthesis of 10-acetyl-2’-TBDMS-docetaxel. (8)

 

Figure 20: 10-acetyl—2'-TBDMS-docetaxel.

To a solution of 2'-TBDMS-docetaxel (240 mg, 0.26 mmol) and anhydrous cerium
chloride (6.3 mg, 0.1 equiv) dissolved in THF (5 mL) was added excess acetic anhydride
(10 mL, dropwise). The reaction progress was monitored by TLC for the formation of

product. After 1 h, the reaction mixture was diluted with EtOAc (100 mL) and washed

with saturated aqueous NaHCO3 solution (3 x 40 mL) and then brine (20 mL). The

organic layer was dried (NaZSO4) and concentrated under reduced pressure. The residue

was purified by silica gel flash column chromatography (1:1 EtOAc/Hexane, v/v), and
the isolated product was concentrated in vacuo to give 10- acetyl-2'-TBDMS-docetaxel
(231 mg, 0.24 mmol, 93% yield at 95% purity as determined by lH-NMR). ESI-MS
(positive ion mode): 986.4 [M + Na]+ 'H-NMR ( 300 MHz, CDC13) a: 8.09 (d, J = 7.5
Hz, o-ph at C2 benzoyl), 7.56 (t, J: 7.5, 7.2 Hz, p-ArH of C2- benzoyl), 7.46 (t, 2H, J =

7.8, 7.2 Hz, m-ph of C2- benzoyl), 7.36 (m, C3’ phenyl), 7.28 (m, 1H, ArH of C3'

58

phenyl), 6.30 (s and br, t, H-10, H-13), 5.66 (d, J = 7.2 Hz, H-2), 5.44 (br (1, J = 9.6 Hz,
H-3'), 5.30 (br, (1, J = 9.0 Hz, NH), 4.95 (d, J = 7.8 Hz, H-S), 4.5 (d, J = 1.8 Hz, H-2'),

4.42 (dd, J = 6.6 , H-7), 4.30 (dd, J = 8.4 Hz, H-20b), 4.17 (d, J: 8.1 Hz, H-20a) 3.81 (d,

J = 7.2 Hz, H-3), 2.53 (s, C(O)OCH3 at C4), 2.41-2.33 (m, H- 601, H-14), 2.20 (s,

C(O)OCH3 at C10), 2.04 (s, H-l8, H-6), 1.83 (s, H-19), 1.28 (s, H-l6, N-Boc), 1.13 (s,

H-17), 0.72 (s, SiC(CH3)3), -0.14 (s, SiCH3). -0.32 (s, SiCH3).

Synthesis of lO-acetyldocetaxel (10)

 

Figure 21: 10-acetyldocetaxel

To a solution of 10-acetyl-2’-TBDMS-docetaxel (231 mg, 0.24 mmol) dissolved in THF
(5 mL) at 0 °C were added pyridine (0.75 mL) and a solution of 70% hydrogen fluoride
dissolved in pyridine (0.75 mL). The reaction was stirred at 0 °C and then gradually

warmed to room temperature. The progress of the reaction was monitored by TLC,

59

showing 40% conversion of the starting material. Additional pyridine (0.5mL) and HF/
pyridine solution (0.5 mL) were added at 0 °C, and the mixture was warmed to room
temperature and stirred for 10 h to complete the reaction. The solution was diluted with
EtOAc (100 mL), and the organic layer was washed successively with a 5% (w/v)
solution of NaHCO3 (2 x 10 mL), a 5% (v/v) solution of HCl (2 x 10 mL), water (10
mL), and brine (15 mL). The organic fraction was dried (MgSOa) and concentrated under
vacuum, the crude mixture was purified by silica gel column chromatography (1:1(v/v)
EtOAc/ hexane), and the fractions containing product were concentrated in vacuo to
provide lO-acetyldocetaxel (193 mg, 0.22 mmol, 89% yield at 95% purity as judged by
1H NMR). ESI-MS (positive ion mode): 872.3 [M+Na]+ lH-NMR (CDC13, 500 MHz) 6:
8.12 (d, J = 7.5 Hz, o-ph of C2 benzoyl), 7.62 (t, J = 7.5, 7.0 Hz, p-ArH of C2 -benzoyl),
7.51 (t, J = 7.5 Hz, 772- ArH of C2- benzoyl), 7.40 (m, ArH of C3’ phenyl), 7.33 (m, ArH
of C3' phenyl), 6.31 (s, H-10), 6.25 (t, J = 9.0 Hz, H-l3), 5.68 (d, J = 7.0 Hz, H-2), 5.45
(br (1, J = 9.3 Hz, H-3'), 5.30 (br d, J: 9.3 Hz, NH), 4.95 (d, J: 8.0 Hz, H-S), 4.64 (d, J:
2.3 Hz, H-2'), 4.42 (dd, J = 9.0, 8 Hz, H-7), 4.30 (d, J = 8.5 Hz, H-20a), 4.17 (d, J: 8.5
Hz, H-20b), 3.81 (d, J: 7.5 Hz, H-3), 3.4 (br d, 2'-OH), 2.61-2.57 (m, H-601), 2.38 (s, 3H,

C(O)OCH3 at G4), 2.33 (m, H-14), 2.25 (s, C(O)OCH3 at C-10), 1.91-1.89 (m, H-6),

1.86 (s, H-l8), 1.70 (s, H-l9), 1.35 (s, OC(CH3)3), 1.27 (s, H-l6), 1.16 (s, H-l7).

60

 

Synthesis of N-Debenzoylpaclitaxel. (11)

 

Figure 22: N-debenzoylpaclitaxel

To a solution of 10- acetyldocetaxel (170 mg, 0.20 mmol) dissolved in acetonitrile (1
mL) was added (dropwise) 88% formic acid solution (2 mL) at 0 °C, and the reaction was

slowly warmed to room temperature. After 4 h, the reaction was judged to be 50%

complete by TLC (90:10 (v/v) chloroform/methanol, Rf = 0.3) and was quenched by
partitioning between NaHCO3 solution and chloroform. The aqueous fraction was

removed, and the organic layer was extracted with 5% (w/v) NaHCO3 solution (40 mL).

The aqueous fractions were combined and back extracted with chloroform (2 x 25 mL).

The organic fractions were combined, dried (MgSO4), and concentrated in vacuo, and the

resultant residue was purified by silica gel column chromatography (8% MeOH in
CHC13) to provide N-debenzoylpaclitaxel (11). (70% yield, 105 mg, 0.14 mmol,) as a

pale yellow solid. ESI-MS (positive ion mode): 750.2 [M + HT“, 768 [M + Na]+ iH-NMR

(CDCI3, 300 MHz) 5: 8.07-8.04 (m, o-ArH of C2 -benzoyl), 7.65 (t, J = 8.3 Hz, p- ArH

61

of C2- benzoyl), 7.51 (t, 2H, J = 8.1, 6.9 Hz, meta-ArH of C2 benzoyl), 7.38 (m, ArH of
C3' phenyl), 7.28 (m, ArH of C3’ phenyl), 6.27 (s, H-10), 6.14 (t, J = 8.0 Hz, H-l3), 5.63
(d, J = 7.2 Hz, H-2), 4.93 (d, J = 8.1 Hz, H-5), 4.42-4.34 (m, H-7), 4.32-4.27 (m, H-20a,

and H-2'), 4.13 (d, J = 8.7Hz, H-32), 3.75 (d, J = 6.9 Hz, H-3), 2.56-2.48 (m, H-6a), 2.24
(s, C(O)OCH3 at C 10 or C4), 2.23 (s, C(O)OCH3 at C10 or C4), 1.98-2.10 (m, H-14 and

H-6b), 1.88 (br s, H-l8), 1.65 (s, H-19), 1.24 (s, H-16), 1.12 (s, H-17).

Synthesis of 10-Deacetyl-N-debenzoylpaclitaxel (12)

 

Figure 23: 10-Deacetyl-N-debenzoylpaclitaxel

A solution of docetaxel (100 mg, 0.124 mmol) in concentrated formic acid (25 mL) was
stirred for 4 h at room temperature. The mixture was concentrated, and the residual acid

was removed as an azeotrope with toluene. The residue was partitioned against 5% (w/v)

NaHCO3 solution and ethyl acetate. The aqueous fraction was removed, and the organic

62

layer was again extracted with 5% (w/v) NaHCO3 solution (40 mL).The aqueous
fractions were combined and back- extracted with ethyl acetate (3 X 25 mL). The organic

layer was dried (MgSO4) and concentrated under vacuum. The resulting crude product
was purified by silica gel column chromatography (95:5 (v/v) EtOAc/MeOH) to obtain
10-deacetyl-N-debenzoylpaclitaxel as a white powder (54 mg, 0.075 mmol, 60% yield).
ESI-MS (positive ion mode): 708.2 [M+H]+ 1H-NMR (300 MHz, CDC13) 6: 8.06 (d, J =
7.8 Hz, o-ArH of C2 benzoyl), 7.62 (t, J = 7.5, 7.2 Hz, p-ArH of C2- benzoyl), 7.51 (t, J
=7.5 Hz, m-ArH of C2 benzoyl), 7.39 (m, ArH of C3’phenyl), 7.30 (m, ArH of
C3'phenyl), 6.13 (t, J = 9.0 Hz, 13-H), 5.64 (d, J = 7.2 Hz, H-2), 5.20 (s, H-10), 4.93 (d,

J = 7.8 Hz, H-S), 4.35-4.28 (m, H-2', H-3’, H-20a), 4.26 (m, H-7), 4.15 (d, J = 8.4 Hz, H-
20b), 3.88 (d, J = 7.2 Hz,H—3), 2.62-2.52 (m, H-6a), 2.25 (s, C(O)OCH3 at C-4), 2.07-

2.02 (m, H-14), 1.90 (s, H-18), 1.82 (m, H-6), 1.75 (s, H-l9), 1.21 (s, H-16), 1.11 (s, H-

17).

63

Synthesis of -13-Oxo-7-TES-baccatin III (27)

   
 

HO 5 2 OAc

   

Figure 24: 13-Oxo-7—TES—baccatin III

7-TES-baccatin III (100 mg, 0.14 mmol) was dissolved in dichloromethane (6 mL), three
batches of MnO;;, (2 g) were added with constant stirring. The reaction was followed up
using TLC, and after most of the reaction converted into product, the reaction suspension
was filtered and diluted with ethyl acetate. The organic solvent concentrated and purified
by silica gel chromatography (Ethyl acetate: Hexane 35:65, as mobile phase) to get a
white powder of l3-oxo-7-TES-baccatin III (85 mg, 86.9%).ESI- MS (positive mode):
700.3 [M+ Na]+ 1H-NMR (300 MHz , CDC13) 8: 8.03 (d, J = 7.2 Hz,o-ArH of C2
benzoyl), 7.62 (t, J = 7.5 Hz, Bz), 7.45 (t, J = 7.5 Hz, B2), 6.55 (s, lO-H), 5.67 (d, J = 6.0
Hz, 2-H), 4.88 (d, J = 7.5 Hz, 5-H), 4.44 (dd, J = 10.3, 6.9 Hz, 7-H), 4.28 (d, J = 8.7 Hz,
20-Ha), 4.10 (d, J = 8.4 Hz, 20Hb ), 3.87 (d, J = 6.6 Hz, 3-H), 2.92 (d, J = 20.1 Hz, 14-

Ha ), 2.61(d, J = 20.7 Hz, 14- HI, ), 2.50 (m, 6-H), 2.21 (s, 3H, OAC), 2.17 (s, 3 H, OAC),

2.06 (s, 18-CH3), 1.82 (m, 6-H), 1.65 (s, l9-CH3), 1.25 (s, l6—CH3), 1.17 (s, 17-CH3),

0.884 (t, J = 8.4 Hz, s1( CH2CH3)3. 0.58-0.51 (band, 6H, Si(CH2 CH3)3).

Synthesis of [l3-H3] baccatin III.(28)

l3-Oxo-7-TES-baccatin III (20 mg, 0.029 mmol) was dissolved in THF (250 11L) and
added to the original vial containing [3H]NaBH4 (50 mCi, specific activity, 150
mCi/mmol, ARC Inc.) and stirred at 0 °C in ice bath for 30 minutes and the temperature
was kept at room temperature for 2 h after which 0.1 ml of 0.01 N sodium hydroxide
solution was added and the reaction was followed by TLC and kept for additional 2 h and
the reaction quenched with water (0.5 ml). Finally, the reaction was extracted with ethyl
acetate (3 x 5ml), dried over sodium sulfate, and the solvent was evaporated under
nitrogen. The resulting residue was purified by PTLC to get [13-3H]-7-TES baccatin III
(yield = 15 mg), and verified with authentic 7-TES-baccatin III by comparison against
authentic sample on TLC. [13-H3]-7-TES baccatin III (15mg, 0.021mmol) was dissolved
in 1 mL of THF, and 0.3 mL of pyridine was added and stirred at 0 °C and HF/pyridine
(0.3 mL) added dropwise over 0.5 h, and the reaction was warmed to room temperature

and stirred for 2 h. The reaction progress was monitored by TLC, and the reaction was

diluted with EtOAc (10 mL) and washed with sat. NaHCO3 (2 x 2 mL), 10% NaOH

solution (2 mL), and finally with brine. The organic layer was separated from the aqueous
layer, and, the organic fraction was dried over sodium sulfate. The solvent was removed
under a stream nitrogen gas, and the crude product was purified by PTLC using (ethyl
acetate: hexane, 50:50) and 8 mg, (0.013mmol, 65%). The specific activity of baccatin 111
(1.01 x108 DPM, 0.0455 mCi, and specific activity 3.46 mCi/mmol) was determined

using liquid scintillation counting on a 14/4C Wallace Instruments counter (Copley, OH)

65

3.2 General Procedure for the synthesis of thioesters CoASH

Triethylamine (1.1 equiv.) was added to a solution of the corresponding carboxylic acid
(1.0 equiv.) in 5:2 (v/v )CH2C12/ THF (1.4 mL) under N2 gas. The mixture was stirred for

10 min at 23 °C, ethyl chloroformate (1.2 eq.) was added in one portion, and the reaction
was stirred for 1 h. The solvents were evaporated under reduced pressure, and the residue
was dissolved in 0.5 mL of tert-butyl alcohol. CoASH as the sodium salt (1.1 equiv.) in
0.4 M NaHCO3 was added to the solution, and the mixture was stirred for 0.5 h at room
temperature (23 °C), then quenched by dropwise addition of 1M HCl and adjusted to pH
3-5. The solvents were evaporated under vacuum, and the residue was dissolved in water
(5 ml, pH 3-5) and loaded onto a C-18 silica gel column (10 % capped with trimethyl
silyl (TMS)) that had been washed with 100% MeOH (50 mL) and pre-equilibrated with
distilled water (100 mL, pH 5). The sample was washed with water (100 mL, pH 3-5) and
then eluted with 15% MeOH in water (50 mL, pH 3-5). The fractions containing
Coenzyme A thioesters, as determined by TLC, were combined and the solvent was
evaporated, and the derived solution containing the product was lyophilized. The purity
of the product was assessed by analytical TLC, and the identities of the compounds were

then confirmed by ESI-MS and lH-NMR spectroscopy.

66

Phenylpropanoyl CoASH (17)

Figure 25: phenylpropanoyl CoA

Following the general procedure, 3-phenylpropanoic acid (9.8 mg, 65 pmol) was
dissolved in CHzClz ITHF (5:2 (v/v), 3.0 mL) and 1 M triethylamine in DCM (8 11L, 78
umol) was added and mixed for 10 min. ethyl chloroformate (11 1.1L, 97.5 umol) was
added to one portion under nitrogen. The reaction stirred for 1 h at room temperature and
the reaction progress was monitored by TLC. The resulting acid anhydride was dried
overnight under vacuum to remove traces of ethyl chlorformate. The resulting residue
was dissolved in 2.5 ml of t-BuOH. The CoA was dissolved (as the trilithium salt, 50 mg,

60 pmol) in NaHC03 (0.4 M, 2.5 mL) and added to the solution. The reaction was stirred

for 1 h at room temperature, quenched with 1M HCl, adjusted to pH 3-5 and the solvents

were evaporated under reduced pressure at room temperature. The residue was purified
by reverse-phase flash chromatography on C13 silica gel chromatography (using water

and methanol as a mobile phase). The fractions containing the product were combined

67

together, and the solvent was evaporated in vacuo, and the remaining water was removed

by lyophilization to provide the phenylpropanoyl CoASH as a white solid (69 mg, 51%).

The purity (98%) was assessed by analytical TLC, Rf = 0.46 (n-butanol/HZO/ACOH,

5:3:2 v/v/v) and visualization under UV on the TLC plate. The compound was confirmed
by ESI—MS and lH-NMR. ESI-MS: calculated 899, experimental [M-H]' 898, [M-2H]'2
448.5. 1H-NMR (500 MHz, D20) see figure for numbering 5 (in ppm): 0.67 ( s, H-lO'),

0.81 (s, H-1 1'), 2.26 (t, J = 6, H-4'), 2.44 (dd, J = 5, H-2), 2.84 (1H, m, H-l'), 3.16 (dd,

J = 5.5, H-2'), 3.25 (m, H-3), 3.31 (dd, J = 6.5, H—5'), 3.48 (m, Ha-S"), 3.75 (m, Hb-S"),
3.94 (s, H-7' ), 4.14 (s, H-9', 2H), 4.5 (br,H-4"), 4.68 (D20, H-2", H-3"), 6.05 ((1, 1:5, H-

1"), 7.08-7.26 (5H, m, phenyl proton), 8.12 (s, CH-adenine), 8.45(s, CH-adenine)

Synthesis of (RS)-Phenylalanoyl CoA (20)

NH2
or 111 <':l| N//le
C CH3 (1)1
H033 0‘13 “0’13 (3‘0
”a 9' OH 0H4 .. 3"0 2'
8 GIN 9
321 S\1'/2\N/“\/IH 5' 0 :3 00
NH2 0

Figure 26: (RS-Phenylalanoyl CoA

68

 

(R,S)-N-Boc-B-3-Phenylalanine CoA was prepared similarly to the procedures described
previously except (R,S)-N-boc-B-3-Phenylalanine (17 mg, 65 nmol) was dissolved in 5:2
(v/v) CH2C12/ THF (1.4 mL ) and 1 M triethylamine in DCM (8 ILL, 78 umol) was added
and mixed for 10 min. Ethyl chloroformate (11 (IL, 97.5 pmol) was then added on to the
reaction solution under nitrogen. The reaction was stirred for 1 h at room temperature,
and the progress of the reaction was monitored by analytical TLC. The resulting acid
anhydride was dried over night under vacuum to remove traces of ethyl chlorformate. To

the resulting residue was added in 2.5 ml of t-BuOH. CoA (as the trilithium salt, 50 mg,

60 umol) was dissolved NaHCO3 (0.4 M, 2.5ml) and added to the solution containing the

mixed anhydride. The mixture was stirred for 2 h at room temperature, the reaction was
quenched with 1M HCI, adjusted to pH 3—5 and the solvents were evaporated under

reduced pressure at room temperature. The residue was purified by reverse phase flash

chromatography on C13 silica gel chromatography, using water and methanol as a

gradient mobile phase. The product comes with about 10% methanol in water and the
solvent of these fractions were combined removed under high vaccum. The residual
water was lyophilization to yield (RS )-N-Boc-B-phenylalnine CoA as a white solid (46
mg, 45 nmol, 69%).The purity (98%) was assessed by analytical TLC (Rf = 0.47 (n-
butanol: acetic acid: water, (522:3)as mobile phase).The derived product was confirmed
by ESI-MS and lH-NMR. ESI-MS: analysis calculated mw 1013. The Boc protected
product was dissolved in 1 mL of water and the Boc group was removed by 88% formic

acid solution. The reaction progress was followed up by TLC. At completing the reaction

69

was diluted with water and concentrated under vacuum. The dilution/concentration
process was repeated two more times, and the sample was lyophilized to obtain RS-

phenylalanyl CoA (22 mg, 24 umol, 53%) ESI-MS (negative ion mode) 913 [M-H]'l

1H-NMR (500 MHz, D20) 5: 0.66 (s, H-10'), 0.78 ( s, H-1 1'), 2.16 (2H, t, H-l') 2.8 (2H,

m, H-l'), 3.1 (t, H-2’), 3.26-3.28 (m, H—2 & H-5'), 3.44 (dd, J: 4.8 and 9.6 Hz, Ha-S"),
3.66 (dd, J = 4.8 and 9.6 Hz, Hb-S"), 3.86 (s, H-7') , 4.06 (s, H-9'), 4.43 (ddd, J = 2.7 and
5.3 Hz, H-4”), 4.56- 4.66 (m, H-2" and H-3"), 6.0- 6.1 (two doublets; one set from each
stereoisomer, J = 6.9 Hz for both, H-l"), 7.21-7.27 (m, HA phenyl protons), 8.26 (8, HC

adenine-CH), 8.33 (8, H3 adenine-CH)

Synthesis of 3(R)-|3-pheny1alanoyl CoASH (15)

NH2

10' 11' HO (1:31: He
H30 CH3 l9
HO, O‘P 'P‘O- (3‘0
HA 7 9' OH OH 4K3"O TEL"
8 67N ?
P—

321 S\1'/2\N/u\/I5H' O: I 00

O'
NH2 0

Figure 27 : 3R-B-phenylalanoyl CoA

Following the same procedure as preparation of (RS)-B-phenylalanoyl CoASH, but we

use the R enantiomer of N-Boc-R-B-phenylalnine, to get (20 mg, Zlumol, 50%) R-B-

70

 

phenylalanine CoA, verified by LC-MS. Compound then confirmed by ESI-MS( negative
mode) 913[M-H]', 1H-NMR (500 MHz, D20) , 8: 0.66 (s, H-10’) , 0.78 (s, H-ll’), 2.16
(t, H-l ’) 2.8 (m, H-1 ’), 3.1 (t, H-2’), 3.26-3.28 (m, H-2 & H-5’), 3.44 (dd, J: 4.8 and 9.6
Hz, Ha-S"), 3.66 (dd, J: 4.8 and 9.6 Hz, Hb-S"), 3.86 (5, H-7'), 4.06 (s, H-9'), 4.43 (ddd, J
= 2.7 and 5.3 Hz, H-4"), 4.56- 4.66 (m, H-2" and H-3"), 6.0- 6.1 (dd, J = 6.9 Hz , H-l"),

7.21-7.27 (111, HA phenyl protons), 8.26 (3, HC adenine-CH), 8.33 (8, H3 adenine-CH).

Synthesis of (R)-3-hydroxy-3-phenylpropanoyl CoA (20)

NH2
Ho
10' 11' <’:I N’/‘HB
H3C CH3 ll
Ho,, o-P -o— P'- o
”A 7' 9' OH OH 4" 3"0 2'
8 GIN 9
321 s\1'/2\N/”\)NH 5' 0 T; 00
OH 0

Figure 28: (R)-3-hydroxy-3-phenylpropanoyl CoA

Following the general procedure, R-3-hydroxy-3-phenylpropanoyl CoA was similarly
prepared as the previous except R-3-hydroxy-3-pheny1propanoic acid (10mg, 65 pmol)
was used with slight variation in the composition of solvent in a 5:1 CHzClz: THF (v/v,

1.8 mL) to yield (R)-3-hydroxy-3-phenylpropanoyl CoA (15 mg, 16 umol, 25%) ESI-
MS: calculated 915.17, experimental [M-H]' 914.09. ‘H-NMR ( 500 MHz, 1320) , 6 0.66

(s, H-lO') , 0.78 (s, H-1 1'), 2.39 (t, H-4'), 2.8 (m, H-l'), 3.20 (t, H-2'), 3.26-3.28 (m, H-2

71

 

& H-5'), 3.46 (dd, J = 4.8 and 9.6 Hz, Ha-S”), 3.80 (dd, J = 4.8 and 9.6 Hz, Hb-S"), 3.96
(s, H-7') , 4.06 (s, H-9’), 4.46 (dd, J = 2.7 and 5.3 Hz, H-4"), 4.60 - 4.82 (m, H-2" and H-
3"), 5.80 (m, H-3), 6.40 ( dd, J=6.9 Hz , H-l"), 7.21-7.27 (5H, m, HA phenyl protons),

8.20 (1H,s, Hc adenine-CH), 8.43 (1H,s, H3 adenine-CH)

Synthesis of benzoyl-2, 2-dimethyl-l, 3-dioxane-4, 6-dione

Figure 29: benzoyl-2, 2-dimethyl-1, 3-dioxane—4, 6-Dione

A solution of 2,2—dimethyl-1,3-dioxane-4,6-dione (1.2 g, 8.32 mmol) and 4
(dimethylamino)pyridine (DMAP, 2.4g, 10.9 mmol) in DCM (25 ml) was cooled in an
ice-salt bath to —10 °C, and a solution of benzoyl chloride (1.62 g, 9.25 mmol) in
dichloromethane (10 mL) was added dropwise over 2 h. The resulting yellow solution
was kept in the cooling bath for 4 h and allowed to stir at room temperature overnight.

The Orange-yellow solution was then cooled in an ice-water bath and washed with 3 x 5
mL of a l M aqueous HCl solution and water. The organic layer was dried over MgSO4

and filtered, and the filtrate was evaporated to dryness. The residue was triturated with

hexanes (10 ml), and dried under vacuum to give the benzoyl-2,2—dimethyl-l,3-dioxane-

72

 

4,6—dione as a yellow powder. A suspension of 1g of the (4 mmol) was made in 40 ml of
toluene, was stirred at room temperature for 45 min and then filtered to remove insoluble
material. The toluene solution was then treated with neat isopropyl amine (300 11L, 0.14
g, and 3.5 mmol) added drop wise through a syringe. The resulting precipitate was
isolated by filtered and washed with toluene (3 x 5 ml) and hexanes (2 x 5 ml) to afford a

salt as a yellow solid. The salt was then suspended in dichloromethane and washed with l
M aqueous HCl solution. The organic layer was separated, dried (MgSO4), filtered, and

concentrated to give a yellow solid.

1H-NMR (300 MHz, CDC13): 5 15.5 (br, s, 1H, H-S), 7.7 (d, J = 8.1, 2H, o-Ph), 7.64 (dd,

J = 7.5, 1H, p-Ph), 7.05 (dd, J = 7.2, 2H, m-Ph), 1.88 (s, 6H, CH3).

73

 

3.3 Enzyme expression, Protein Harvest, Protein Purification.

3.3.1 Enzyme Expression.

Standard microbial and recombinant techniques used throughout this work are described
by Sambrook. The full length bapt cDNA (accession AY082804) was subcloned from its
original expression vector pSBET55 into pET28a (Novagen), which incorporated an N-
terminal Hisé-tag epitope for immunoblot analysis of the expressed protein and
purification by HIS-SelectTM Nickel Affinity Gel (Sigma, St. Louis, MO). To The
resultant plasmid containing the bapt cDNA in pET28a was transformed into expression

vector, E. coli BL21-CodonPlus® (DE3).

3.3.2. Protein Harvest

Recombinant bapt was expressed in the described bacterial expression system and
harvested according to a previously reported protocol48 with slight modifications.
Cultures were grown overnight at 37°C in 5 mL Luria-Bertani medium supplemented
with 50 ug/mL kanamycin. Bacteria transformed with empty vector were processed
analogously. The 5-mL inoculum was added to and grown at 37°C to OD600 = 0.7 in 1 L
Luria—Bertani medium supplemented with the kanamycin (50 mg/L) antibiotic, and then
gene expression was induced with 100 MM isopropyl-B-D—l-thiogalactopyranoside, and
the culture was incubated at 18°C. After 18 h, the cells were harvested by centrifugation

at 2000g for 20 min at 4°C, the supernatant discarded, and the pellet was resuspended in

74

Lysis Buffer (25 mM 2-(N-morpholino) ethanesulfonic acid, pH 7.0 at 5 mL/g cells ) at
4°C. The cells were lysed at 4°C by sonication (6 x 20 s bursts at 60% power at l min
intervals) with a Misonix XL-2020 sonicator (Misonix Inc., Farmingdale, NY), and the

cell-lysate was clarified by ultracentrifugation at 45000g for l h at 4°C.

3.3.3 Protein Purification

The supernatant of the lysed bacterial cells was incubated with HIS-SelectTM Nickel
Affinity Gel in batch mode at 4°C. After 2 h, the mixture was poured into an Econo-
column (BioRad, 20 cm x 2.5 cm), the Lysis Buffer was drained. The resin was washed
with five column volumes of Wash Buffer (50 mM sodium phosphate and 300 mM
sodium chloride, pH 8.0) containing 20 mM imidazole, and the bound protein was eluted
with one column volume of Wash Buffer containing 250 mM imidazole. The imidazole
was removed from the eluent by consecutive concentration by ultracentrifugation (30000
MWCO, YM30 membrane, Millipore, Billerica, MA) and dilution in Assay Buffer (50

mM sodium phosphate, 5% glycerol, pH 8.0)

3.3.4. Activity and Assay of BAPT

To verify functional expression of the BAPT, 100 11g of the purified soluble protein was
withdrawn and assayed with three acyl CoAs 100 nmol of R-B-phenylalanine CoA and
[13-3H]baccatin III (70 umol, 0.5/1C1) at 31°C. The assay mixture was quenched with
saturated sodium bicarbonate and extracted with ethyl acetate (2 x 1 mL), the organic

fractions were combined, the solvent was evaporated, and the residue was redissolved in

75

50 uL acetonitrile. A 25-111. aliquot was loaded onto a reverse-phase column (Allsphere

ODS2-5um, 250 x 4.6 mm, Alltech, Mentor, OH) and eluted isocratically with 50:50

(v/v) solvent Azsolvent B [solvent A: 97.99% H20 with 2% CH3CN and 0.01% H3PO4

(v/v); solvent B: 99.99% CH3CN with 0.01% H3PO4 (v/v); flow rate of 1 mL/min; A228

monitoring of the effluent for 20 min] on an Agilent 1100 HPLC system (Agilent
Technologies, Wilmington, DE) connected to a UV detector. UV-absorbance profiles of
the biosynthetic product isolated from the assay containing crude enzyme extract of bapt-
expressing cells were compared to absorbance profiles of the products identically isolated

from control assays containing extracts of cells transformed with empty vector

76

‘

Appendix
Spectra 1: 1H and 13 C NMR for 7-TES—Baccatin III

 

 

 

 

 

 

 

77

Spectra 2: 1H and '3 c NMR for 13-Oxo-7-TES—Baccatin 111

 

 

 

 

 

 

 

 

 

 

l l w 1111111

F m r 1 1 1 1 1 1 1 1 T 1 1 1 1 fi

 

200 150 100 50

78

Spectra 3: l H and I 3 C NMR for N-boc-N-debenzoyl-7-TES -2'-deoxytaxol

 

 

 

£1133.”le

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

 

 

79

Spectra 4: 1H and 13C NMR for N -debenzoyl-2'-deoxytaxol

 

 

 

 

 

 

 

 

fi—fI—ilrfi j—f T—T—r—l—‘T'Tjhfr TTfl iffifij-rf’flgr—T—j—r—r—TfijfiT—T_
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0
1 .111 1 nil" 1 l 111 ii 1i 1““ iii
7 7 * #7 150 100 50 7 —71—

80

 

Spectra 5: 1H NMR for 2'-Deoxypaclitaxel

 

 

 

 

 

WWW

I I I 1" I I I I T I I I I I I I I I I I l I | I I j fir I I I I I I I I T
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

 

81

Spectra 6: 1H and 13C NMR for 2-TBDMS-docetaxel

 

 

 

 

 

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

82

Spectra 7: 1H and 13C NMR for 10-acetyl-2-TBDMS-docetaxel

O

' : ': O
0 H0 032 OAc

 

 

Maw M

~v—r——x—r—v—k+—rfi——v—v fi—I—r

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

 

 

1.1 -. 1.1M - IJIIH. i- ll 1 I‘lltliflllifinfilu

'7'" 7' ,. "'I'IT . - . . ‘11

 

150 100 50

83

 

Spectra 8: 1H and 13C NMR for lO-acetyl-docetaxel

 

 

 

 

 

8.0 7.0 6.0 5.0 4.0 3.0

.1- . . H1 1 1 lJ‘ll ”I.

2.0

111113 I 11 .' 1 1111“;

 

 

r fi 1? 1

200 150 100

84

2',

50

2.,

1,

I

v

Spectra 9: 1H and 13 C NMR for N-debenzoylpaclitaxel

 

 

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

 

 

PDT" ("I

85

Spectra 10: I H NMR for 10-deacetyl-N-debenzoyltaxol

 

8.0 7.0 6.0 5.0 4.0

86

 

 

 

 

Spectra I I: 1H NMR for N—Boc B-phenylpropanoyl CoA

 

 

 

 

 

I

l

 

T‘V’T‘ftlTIII‘I’IrIIIIIII

9.0

8.0

7.0

6.0

llllllIlllIlrrIITIIIlIIl

5.0

87

4.0

3.0

2.0

1.0

Spectra 12: 1H NMR for N- B-phenylpropanoyl CoA

 

 

8.0 7.0 6.0 5.0 4.0

88

3.0

2.0

Spectra 13: 1H NMR for 3-Phenylpropanoyl CoA

 

 

 

 

 

 

 

89

 

 

Spectra 14: 1H NMR for 3-Hydroxy-3-phenylpropanoyl CoA

 

 

 

8.0 7.0 6.0 5.0 4.0 3.0

90

2.0

1.0

Electro spray Mass spectrum for CoA thioesters

Spectra 15: ESI-MS (-) for B—phenylalanoyl CoA

 

 

 

 

100 913
1
1 1
1 1
1 1
1 1
I 1
3‘ 1
.5; I
t:
8 456
.S
Q)
.2
E5
d)
a:
1
1
1 , 506 915
1 1
01. L 724 l :Ail .2 3 L l .L vb ‘ ' ‘
200 400 600 800 1000 1200 1400 M/z

ESI-MS of R-B-phenylalanoyl CoA [M-H] ‘= 913, [M-2H1'2 = 456.0

91

Spectra 16: ESI-MS ( -ve) for Phenylpropanoid CoA

449

Relative intensitv

1
149 1

’J 255

 

 

 

‘ ' .
T1 L 4‘ Y ‘1 4L '1 T LII A“ 111‘ A .x L i

898

 

l

 

044.... H44...
100 150 200 250 300 350 400 450 500 550 600 650 700

ESI-MS (-) 3-phenylpropanoyl CoA [M-2H]'2= 448.5

92

1. .V . L
750 800 850 900 m/z

[M—H] ' =898,

Spectra l7: ESI-MS (-ve) for 3-hydroxy-3—phenylpropnoyl CoA

IUU: 382.5

 

456.5
5° .
5' 1 |
E i
3 1
.E 1
o l
.2
E 1
Q) .
°‘ 1
‘ _ 766
' 1- . 111111-311-

 

 

 

 

.7

01. ' i . 1 i * A T ‘ ‘
100 150 200 250 300 350 400 450 500 550 600 650 700 750

M/z

ESI-spectra of 3R-hydroxy -3-phenyl propanoyl CoA, the result is dominated by the CoA
salt, 456.5 [M-2H]’2. 382.5 [HSCoA-ZH]

93

(1)
(2)

(3)
(4)
(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

REFERENCES

Ojima, 1.; Das, M., J. Nat. Prod. 2009, 72, 554-565.

Wani, M. C.; Taylor, H. L.; Wall, M. B.; Coggon, P.; McPhail, A. T., J. Amer.
Chem. Soc. 1971, 93, 2325-2327.

Jordan, M. A.; Wilson, L., Current Opinion in Cell Biology 1998, 10, 123-130.
Schiff, P. B.; Fant, J .; Horwitz, S. B., Nature 1979, 277, 665—667.

Mielke, S.; Sparreboom, A.; Steinberg, S. M.; Gelderblom, H.; Unger, C.;
Behringer, D.; Mross, K., Clin. Cancer Res. 2005, 11, 4843-4850.

Rowinsky, E. K., Annual Review of Medicine 1997, 48, 353-374.

Correale, P.; Miano, S. T.; Remondo, C.; Migali, C.;.Rotundo, M. S.; Macri, P.;
Tagliaferri, P.; Tassone, P.; Caraglia, M.; Gotti, G.; Francini, G., Cancer Biology
& Therapy 2009, 8, 497-502.

Hayes, D. F.; Thor, A. D.; Dressler, L. G.; Weaver, D.; Edgerton, S.; Cowan, D.;
Broadwater, G.; Goldstein, L. J .; Martino, S.; Ingle, J. N .; Henderson, 1. C.;
Norton, L.; Winer, E. P.; Hudis, C. A.; Ellis, M. J .; Berry, D. A., N. Engl. J. Med.
2007, 357, 1496-1506.

Seyb, K. I.; Ansar, 8.; Bean, J.; Michaelis, M. L., J. Mol. Neurosci. 2006, 28, 111-
124.

Weissman, N. J .; Ellis, S. G.; Grube, E.; Dawkins, K. D.; Greenberg, J. D.; Mann,
T.; Cannon, L. A.; Cambier, P. A.; Fernandez, S.; Mintz, G. S.; Mandinov, L.;
Koglin, J .; Stone, G. W., Eur. Heart J. 2007, 28, 1574-1582.

Bogdan, C.; Ding, A., Journal of Leukocyte Biology 1992, 52, 119-121.

Moos, P. J .; Fitzpatrick, F. A., Proc. Natl. Acad. Sci., U.S.A. 1998, 95, 3896-3901.

White, C. M.; Martin, B. K.; Lee, L. F.; Haskill, J. S.; Ting, J. P. Y., Cancer
Immunology Immunotherapy 1998, 46, 104-112.

Pasquier, B.; Carre, M.; Pourroy, B.; Camoin, L.; Rebai, 0.; Briand, C.; Braguer,
D., Mol. Cancer Ther. 2004, 3, 1301-1310.

Wang, J. Y.; Lou, P. P.; Lesniewski, R.; Henkin, J ., Anti-Cancer Drugs 2003, 14,
13-19.

94

(16) Thomas, H.; Rosenberg, P., Critical Reviews in Oncology/Hematology 2002, 44,
43-5 1 .

(17) Singla, A. K.; Garg, A.; Aggarwal, D., International Journal of Pharmaceutics
2002, 235, 179-192.

(18) Ganta, S.; Amiji, M., Molecular Pharmaceutics 2009, 6, 928-939.

(19) Ganesh, T.; Norris, A.; Sharma, S.; Bane, S.; Alcaraz, A. A.; Snyder, J. P.;
Kingston, D. G. I., Bioorg. Med. Chem. 2006, 14, 3447-3454.

(20) Kingston, D. G. l.; Bane, S.; Snyder, J. P., Cell Cycle 2004, 4, 279-289.

(21) Ojima, I.; Fumero-Oderda, C. L.; Kuduk, S. D.; Ma, 2.; Kirikae, F.; Kirikae, T.,
Bioorg. Med. Chem. 2003, 11, 2867-2888.

(22) Baloglu, B.; Hoch, J. M.; Chatterjee, S. K.; Ravindra, R.; Bane, S.; Kingston, D.
G. I., Bioorg. Med. Chem. 2003, 11, 1557-1568.

(23) Kingston, D. G. 1.; Jagtap, P. G.; Yuan, H.; Samala, L., The Chemistry of Taxol
and Related Taxoids. In Progress in the Chemistry of Organic Natural Products,
ed.; Herz, W.; Falk, H.; Kirby, G. W., 'Ed.""Eds.' Springer-Verlag Wien: New
York, 2002; 'Vol.‘ 84, pApp 53-225.

(24) Rob, E. J .; Song, C. E.; Kim, D.; Pae, H. 0.; Chung, H. T.; Lee, K. S.; Chai, K.;
Lee, C. 0.; Un Choi, S., Bioorg. Med. Chem. 1999, 7, 2115-2119.

(25) Xue, M.; Long, B. H.; Fairchild, C.; Johnston, K.; Rose, W. C.; Kadow, J. F.;
Vyas, D. M.; Chen, S. H., Bioorg. Med. Chem. Lett. 2000, 10, 1327-1331.

(26) Kingston, D. G., Trends Biotechnol. 1994, 12, 222-227.

(27) Ganesh, T.; Yang, Q; Norris, A.; Glass, T.; Bane, S.; Ravindra, R.; Banerjee, A.;
Metaferia, B.; Thomas, S. L.; Giannakakou, P.; Alcaraz, A. A.; Lakdawala, A. S.;
Snyder, J. P.; Kingston, D. G., J. Med. Chem. 2007, 50, 713-725.

(28) Kingston, D. G. I., Phytochemistry.Phytochemical Society of North America 2007,
68, 1844-1854.

(29) Miller, M. L.; Roller, E. E.; Zhao, R. Y.; Leece, B. A.; Ab, 0.; Baloglu, B.;
Goldmacher, V. 8.; Chari, R. V. J ., J. Med. Chem. 2004, 47, 4802-4805.

(30) Ojima, 1.; Slater, J. C.; Michaud, B.; Kuduk, S. D.; Bounaud, P. Y.; Vrignaud, P.;

Bissery, M. C.; Veith, J. M.; Pera, P.; Bernacki, R. J ., J. Med. Chem. 1996, 39,
3889-3896.

95

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

Ojima, 1.; Wang, T.; Miller, M. L.; Lin, S. N.; Borella, C. P.; Geng, X. D.; Pera,
P.; Bernacki, R. J ., Bioorg. Med. Chem. Lett. 1999, 9, 3423-3428.

Kingston, D. G. 1.; Chaudhary, A. G.; Chordia, M. D.; Gharpure, M.; Gunatilaka,
A. A. L.; Higgs, P. 1.; Rimoldi, J. M.; Samala, L.; Jagtap, P. G.; Giannakakou, P.;
Jiang, Y. Q.; Lin, C. M.; Hamel, B.; Long, B. H.; Fairchild, C. R.; Johnston, K.
A., J. Med. Chem. 1998, 41, 3715-3726.

Holton, R. A.; Somoza, C.; Kim, H. B.; Liang, F.; Biediger, R. J .; Boatman, P. D.;
Shindo, M.; Smith, C. C.; Kim, S.; et al., J. Am. Chem. Soc. 1994, 116, 1597-
1598.

Holton, R. A.; Kim, H. B.; Somoza, C.; Liang, F.; Biediger, R. J.; Boatman, P. D.;
Shindo, M.; Smith, C. C.; Kim, S.; et al., J. Am. Chem. Soc. 1994, 116, 1599-
1600.

Nicolaou, K. C.; Yang, 2.; Liu, J. J .; Ueno, H.; Nantermet, P. G.; Guy, R. K.;
Claiborne, C. F.; Renaud, J .; Couladouros, E. A.; Paulvannan, K.; Sorensen, E. J .,
Nature 1994, 367, 630-634.

Gibson, D. M.; Ketchum, R. E. B.; Vance, N. C.; Christen, A. A., Plant Cell Rep.
1993, 12, 479-482.

Witherup, K. M.; Look, S. A.; Stasko, M. W.; Ghiorzi, T. J .; Muschik, G. M.;
Cragg, G. M., J. Nat. Prod. 1990, 53, 1249-1255.

Jennewein, S.; Croteau, R., Appl. Microbiol. Biotechnol. 2001, 57, 13-19.
Ketchum, R. E. B.; Croteau, R. B., Int. Congr. Ser. 1998, 1157, 339-348.
Holton, R. A.; Biediger, R. J .; Boatman, P. D., Semisynthesis of Taxol and
Taxotere. In Taxol: Science and Applications, ed.; Suffness, M., 'Ed.""Eds.' CRC
Press: Boca Raton, FL, 1995; 'Vol.' pApp 97-121.

Magri, N. F.; Kingston, D. G. 1.; Jitrangsri, C.; Piccariello, T., J. Org. Chem.
1986, 51, 3239-3242.

Bommarus, A. S.; Polizzi, K. M., Chem. Eng. Sci. 2005, 61, 1004-1016.

Garcia-Estrada, C.; Vaca, 1.; Ullan, R. V.; van den Berg, M. A.; Bovenberg, R. A.
L.; Martin, J. F., Bmc Microbiology 2009, 9.

Zhang, R; Zhou, P. P.; Jiang, C.; Yu, H.; Yu, L. J ., Biotechnology Letters 2008,
30, 2119-2123.

Baloglu, B.; Kingston, D. G. 1., J. Nat. Prod. 1999, 62, 1068-1071.

96

 

(46)
(47)

(48)

(49)
(50)

(51)

(52)

(53)

(54)

(55)

(56)

(57)

(58)

(59)

(60)

(61)

Baloglu, B.; Kingston, D. G. 1., J. Nat. Prod. 1999, 62, 1448-1472.
Chau, M.; Jennewein, S.; Walker, K.; Croteau, R., Chem. Biol. 2004, 11, 663-672.

Walker, K.; Long, R.; Croteau, R., Proc. Natl. Acad. Sci., U.S.A. 2002, 99, 9166-
9171.

D'Auria, J. C., Curr. Opin. Plant Biol. 2006, 9, 331-340.

St-Pierre, B.; De Luca, V., Recent Adv. Phytochem. 2000, 34, 285-315.
St-Pierre, B.; Luca, V. D., Chapter Nine Evolution of acyltransferase genes:
Origin and diversification fo the BAHD superfamily of acyltransferases involved
in secondary metabolism.Recent Advances in Phytochemistry. In Evolution of
Metabolic Pathways, Volume 34 ed.; John T. Romeo, R. 1., Luc Varin and
Vincenzo De Luca, 'Ed.""Eds.' Elsevier: 2000; 'Vol.' p"pp 285-315.

Guerittevoegelein, F.; Senilh, V.; David, B.; Guenard, D.; Potier, P., Tetrahedron
1986, 42, 4451-4460.

Shiina, 1.; Suenaga, Y.; Nakano, M.; Mukaiyama, T., Bull. Chem. Soc. Jpn. 2000,
73, 2811-2818.

Saitoh, K.; Shiina, 1.; Mukaiyama, T., Chem. Lett. 1998, 679-680.

Walker, K.; Fujisaki, S.; Long, R.; Croteau, R., Proc. Natl. Acad. Sci., U.S.A.
2002, 99, 12715-12720.

Georg, G. 1.; Boge, T. C.; Cheruvallath, Z. S.; Harriman, G. C. B.; Hepperle, M.;
Park, H.; Himes, R. H., Bioorg. Med. Chem. Lett. 1994, 4, 335-338.

Magri, N. F.; Kingston, D. G. I., J. Org. Chem 1986, 51, 797.

Tilburg, E. W. V.; Franssen, E. J. F.; Hoven, v. d.; Meij, M. v. d.; Elshove, D.;
Lammertsma, A. A.; Windhorst, A. D., J Label Compd Radiopharm 2004, 47,
763-777. .

Holton, R. A.; Zhang, Z.; Clarke, P. A.; Nadizadeh, H.; Procter, D. J .,
Tetrahedron Lett. 1998, 39, 2883-2886.

Nevarez, D. M.; Mengistu, Y. A.; Nawarathne, I. N .; Walker, K. D., J. Amer.
Chem. Soc. 2009, 131, 5994-6002.

Roh, E. J .; Song, C. E.; Kim, D.; Pae, H. 0.; Chung, H. T.; Lee, K. S.; Chai, K.
B.; Lee, C. 0.; Choi, S. U., Bioorg. Med. Chem. 1999, 7, 2115-2119.

97

(62)

(63)

(64)

(65)

(66)

(67)

(68)

(69)

(70)

(71)

(72)
(73)
(74)
(75)
(76)

(77)

(78)

Baloglu, E. Synthesis and Biological Evaluation of Paclitaxel Analogs. 2001.
Kingston, D. G. 1.; Ganesh, T.; Snyder, J. P.; Lakdawala, A. S.; Bane, S.
Preparation of conformationally constrained paclitaxel analogs as anticancer and
anti-Alzheimer's agents. W02005070414, January 14, 2005.

Fim, R. D.; Jones, C. G., Nat. Prod. Rep. 2003, 20, 382-391.

Long, R. M.; Lagisetti, C.; Coates, R. M.; Croteau, R. B., Arch. Biochem.
Biophys. 2008, 477, 384-389.

Long, R. M.; Croteau, R., Biochem. Biophys. Res. Commun. 2005, 338, 410-417.

George, K. M.; Chatterjee, D.; Gunawardana, G.; Welty, D.; Hayman, J .; Lee, R.;
Small, P. L. C., Science 1999, 283, 854-857.

McChesney, J. D.; Henri, J. T.; Venkataraman, S. K.; Lamb, R. L.; Foster, J. B.;
Sumner, C. M.; Ye, S.; Yiannkouros, G. P.; Stemphoski, A. M.; Walker, D. G.
Processes for the preparation of taxane derivatives and intermediates useful
therein. WO 2007075870, December 21, 2007.

Roh, E. J .; Kim, D.; Lee, C. 0.; Choi, S. U.; Song, C. E., Bioorg. Med. Chem.
2002, 10, 3145-3151.

Ojima, 1.; Habus, 1.; Zhao, M.; Zucco, M.; Park, Y. H.; Sun, C. M.; Brigaud, T.,
Tetrahedron 1992, 48, 6985-7012.

Jennewein, S.; Wildung, M. R.; Chan, M.; Walker, K.; Croteau, R., Proc. Natl.
Acad. Sci., U.S.A. 2004, 101, 9149-9154.

Loncaric, C.; Merriweather, E.; Walker, K. D., Chem. Biol. 2006, 13, 1-9.
Walker, K.; Croteau, R., Proc. Natl. Acad. Sci., U.S.A. 2000, 97, 583-587.
Walker, K.; Schoendorf, A.; Croteau, R., Arch. Biochem. Biophys. 2000, 374,
371-380.

D'Auria, J. C., Curr. Opin. Plant Biol. 2006, 9, 331-340.

Bayer, A.; Ma, X.; Stockigt, J ., Bioorg. Med. Chem. 2004, 12, 2787-2795.

Nakayama, T.; Suzuki, H.; Nishino, T., J. Mol. Catal. B Enzym. 2003, 23, 117-
132.

Ma, X.; Koepke, J .; Panjikar, S.; Fritzsch, G.; Stockigt, J., J. Biol. Chem. 2005,
280, 13576-13583.

98

(79)

(80)

(81)

(82)

(83)

Nims, B.; Dubois, C. P.; Roberts, S. C.; Walker, E. L., Metab. Eng. 2006, 8, 385-
394.

Grayson, D. H.; Tuite, M. R. j., J. CHEM. Soc. PERKIN TRANS. 1986, I, 2137-
2142.

Bunting, J. W.; Kanter, J. P., J. Amer. Chem. Soc. 2002, 115, 11705-11715.
Hogenkamp, D. J .; Johnstone, T. B. C.; Huang, J. C.; Li, W. Y.; Tran, M.;
Whittemore, E. R.; Bagnera, R. B.; Gee, K. W., J. Med. Chem. 2007, 50, 3369-
3379.

Sambrook, J .; Fritsch, E. F.; Maniatis, T., Molecular Cloning: A laboratory
Manual. 2nd ed.; Cold Spring Harbor Laboratory Press: 1989; 'Vol.' p.

99

"I
H
III
MI
"
I”
T“

1293 03083 3782

3

 

1111111111