Syntheses and metabolic profiles of aminopolyamines in the murine leukemia L1210 cell and the pathogenic bacterium Psued...

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Title:
Syntheses and metabolic profiles of aminopolyamines in the murine leukemia L1210 cell and the pathogenic bacterium Psuedomonas Aeruginosa
Physical Description:
xv, 151 leaves : ill. ; 29 cm.
Language:
English
Creator:
Nguyen, John Van, 1973-
Publisher:
s.n.
Place of Publication:
2004

Subjects

Subjects / Keywords:
Polyamines   ( mesh )
Leukemia L1210 -- metabolism   ( mesh )
Leukemia L1210 -- enxymology   ( mesh )
Pseudomonas aeruginosa -- enzymology   ( mesh )
Pseudomonas aeruginosa -- metabolism   ( mesh )
Department of Medicinal Chemistry thesis Ph.D   ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Medicinal Chemistry -- UF   ( mesh )
Genre:
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D)--University of Florida, 2004.
Bibliography:
Bibliography: leaves 142-150.
Statement of Responsibility:
by John Van Nguyen.
General Note:
Typescript.
General Note:
Vita.

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Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 003166036
oclc - 57761587
System ID:
AA00011365:00001

Full Text











SYNTHESES AND METABOLIC PROFILES OF AMINOPOLYAMINES IN THE
MURINE LEUKEMIA L1210 CELL AND THE PATHOGENIC BACTERIUM
Pseudomonas aeruginosa













By

JOHN VAN NGUYEN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004































To my wonderful grandmother, my parents, and my beautiful fiance Christina Ngoc Cao.
To my chemistry teacher Dang Buom, who is always in loving memory.














ACKNOWLEDGMENTS

I would like to sincerely thank my adviser, Dr. Raymond J. Bergeron, for his

excellent guidance, support, and patience during my graduate studies at the University of

Florida. I thank him for always being on my side, and for spending so much time helping

me with my graduate research project and with the writing of this dissertation. I thank

him for giving me a distinguished opportunity to explore many aspects of science, from

organic synthesis to biological and analytical studies. I thank him for teaching me great

skills such as working independently and thinking critically. I am very honored and

grateful to be one of his students.

I would like to thank the members of my graduate committee (Dr. Margaret James,

Dr. Kenneth Sloan, Dr. James McManis, and Dr. Michael Katovich) for their helpful

advice in completing this dissertation.

I thank Dr. J. McManis and Dr. G. Huang for their useful advice and discussion in

the organic synthesis part of my graduate project. I thank Dr. E. Eiler-McManis for her

helpful assistance in microbial studies; and Mrs. H. Yao for her great assistant efforts in

eukaryotic studies. Special thanks also go to Dr. James McManis and Dr. E. Eiler-

McManis for their excellent editorial assistance. I also would like to express my sincere

appreciation for the help and great friendship of other members of Dr. Bergeron's

research group: Dr. W. Weimar, Mr. R. Smith, Mr. S. Algee, Dr. M. J. Della Vecchia,

Mrs. E. Nelson, Mr. T. Vinson, Mr. H. Snellen, Mr. B. McCosar, Ms. J. Wiegand, Ms. T.








Fannin, Ms. T. Lindstrom, Mrs. K. Ratliff-Thompson, Dr. M. Xin, Dr. N. Bharti, Dr. J.

Park, and Mrs. R. Smith.

My parents, Dinh and Huong Nguyen, and my grandmother, Chau Ho, have given

me great encouragement and support in my graduate studies, and also in other aspects of

life. I wish to thank them for their financial and, more importantly, spiritual supports. I

also would like to thank my brothers, Minh and Thong; my sisters, Loan and Thuy; and

my uncle and aunt. The support from my future in-laws, Minh and Le Cao, is also

greatly appreciated. Special thanks go to my good friend Alicia for her editorial

assistance.

Finally, I wish to thank my fiance Christina Ngoc Cao for her love, commitment,

and understanding during my graduate studies as well as in life. I thank her for being an

excellent listener and a true best friend. She has always been by my side through

difficulties and challenges in school and in many other aspects of life. I thank her for

many unforgettable sweet memories that we have been through, and I believe there will

be more to come. I am so grateful to have her in my journey of life.















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS.........................................................................iii

LIST OF TABLES................................................................................vii

LIST OF FIGURES................................................................................viii

LIST OF ABBREVIATIONS....................................................................xi

ABSTRACT.......................................................................................xiv

CHAPTER

I INTRODUCTION ................................................................................. 1

Polyamines and Their Roles..................................................................... 1
Polyamine Metabolism........................................................................... 4
Importance of Understanding Polyamine Metabolism....................................10
Polyamine Metabolic Pathway Serves as a Therapeutic Target......................... 11

2 POLYAMINE CONTENTS AND POLYAMINE METABOLISM IN
PSEUDOMONADS: LITERATURE REVIEW.........................................23

Pseudomonas sp Strain Kim.................................................................24
Pseudomonas acidovorans....................................................................25
Pseudomonas thermocarboxydovorans.....................................................................26
Burkholderia cepacia...........................................................................27
Pseudomonas aeruginosa.................................................................... 27

3 PROCESSING OF 2-HYDROXYPUTRESCINE AND 7-HYDROXYSPERMIDINE
IN Pseudomonas aeruginosa..................................................................................... 35

Impact on Bacterial Growth..................................................................35
Processing of Hydroxypolyamines and Impact on Bacterial Polyamine
contents........................................................................................ 35









4 SYNTHESES OF AMINOPOLYAMINES..................................................44

Design Concept.................................................................................44
Synthesis........................................................................................44

5 BIOLOGICAL ACTIVITIES OF AMINOPOLYAMINES IN Pseudomonas
aeruginosa AND L1210 CELLS: RESULTS AND DISCUSSION....................57

Microbial Studies...............................................................................57
Eukaryotic Studies.............................................................................64

6 EXPERIMENTAL METHODS: BIOLOGICAL STUDIES, ANALYTICAL
DEVELOPMENTS AND SYNTHESES OF AMINOPOLYAMINES................95

Cellular Studies.................................................................................95
Microbial Studies..............................................................................101
Analytical Development..................................................................... 103
Syntheses.......................................................................................105

7 SUMMARY AND CONCLUSION..........................................................138

LIST OF REFERENCES.........................................................................142

BIOGRAPHICAL SKETCH.....................................................................151

























vi















LIST OF TABLES


Table Page

2.1 Polyamines contents of selected pseudomonads...................................34

5.1 Total intracellular amine equivalence in P. aeruginosa after exposure to
aminopolyamines........................................................................88

5.2 L1210 growth inhibition and transport of various aminopolyamines..............89

5.3 Impact of 7-aminopolyamines on polyamine metabolic enzymes of L1210
cells..................................................... ...................................90

5.4 Impact of 7-aminospermidine on polyamine pools of L 1210 cells............... 91

5.5 Impact of N,NA'-diethyl-7-aminospermidine on polyamine pools of L1210
cells......................................................................................... 92

5.6 Impact of N',N 2-diethyl-6-aminospermine on polyamine pools of L 1210
cells .................................... ......... ........... ................ ......... .. ... 93

5.7 Total intracellular amine equivalence in L1210 cells after exposure to
am inopolyam ines.......................................................................... 94

6.1 HPLC method for polyamine analysis ..............................................135

6.2 Approximate retention time of natural polyamines and their analogues........136















LIST OF FIGURES


Figure Page

1.1 Naturally occurring polyamines.......................................................12

1.2 Fundamental polyamine metabolism pathway in eukaryotes..................... 13

1.3 Polyamine metabolism in plants and bacteria...................................... 15

1.4 Formation of the Schiff base between L-ornithine and PLP.....................16

1.5 Proposed mechanism of omithine decarboxylation.................................17

1.6 Proposed mechanism of AdoMetDC activity....................................... 18

1.7 Metabolic breakdown from spermine to spermidine by SSAT/PAO........... 19

1.8 Biosynthesis of FAD from riboflavin................................................20

1.9 Different oxidation states of FAD....................................................21

1.10 Structures of some polyamine metabolic enzyme inhibitors.....................22

2.1 Polyamine metabolism in P. aeruginosa............................................32

2.2 Biosynthesis of 7-hydroxyspermidine observed in P. acidovorans and P.
thermocarboxydovorans................................................................ 33

3.1 Effect of 2-OH-PUT on the growth of P. aeruginosa.............................38

3.2 Effect of 7-OH-SPD and 2-OH-SPD on the growth of P. aeruginosa...........39

3.3 Effect of 2-OH-PUT on polyamine contents of P. aeruginosa....................40

3.4 Formation of hydroxypolyamines in P. aeruginosa upon exposure to 2- OH-
PU T .......................................................................................41

3.5 HPLC chromatograph of polyamine contents of P. aeruginosa (untreated)......42








3.6 HPLC chromatograph of polyamine contents of P. aeruginosa grown in the
presence of 2-OH-PUT................................................................ 43

4.1 A known synthetic route of racemic 2-NH2-PUT..................................50

4.2 A known synthetic route of (S)-2-NH2-PUT........................................51

4.3 Synthesis of 2-aminoputrescine (2-NH2-PUT).......................................52

4.4 Synthesis of 7-aminospermidine......................................................53

4.5 Synthesis of N'I,N-diethyl-7-aminospermidine....................................54

4.6 Synthesis of 6-aminospermine..........................................................55

5.1 Effects of aminopolyamines and hydroxypolyamines on P. aeruginosa
grow th..................................................................................... 72

5.2 Effects of 7-NH2-SPD on P. aeruginosa growth................................... 73

5.3 Effects of 7-NH2-DESPD on P. aeruginosa growth...............................74

5.4 Effects of 6-NH2-SPM on P. aeruginosa growth...................................75

5.5 Effects of various aminopolyamines on P. aeruginosa colony forming units....76

5.6 Effects of various aminopolyamines on P. aeruginosa growth.................. 77

5.7 Effects of 7-NH2-SPD on P. aeruginosa colony forming units.................. 78

5.8 A comparison of polyamine levels in P. aeruginosa harvested from different
growth phases...........................................................................79

5.9 Effect of 7-NH2-SPD on polyamine contents of P. aeruginosa.................. 80

5.10 Polyamine levels in P. aeruginosa after different exposure duration............81

5.11 Metabolic breakdown of N',NI2-diethyl-6-aminospermine and N',NA -diethyl-
7-aminospermidine in P.aeruginosa.................................................82

5.12 HPLC chromatograph of polyamine contents of L 1210 cells (control, no
drug) ...................................................................................... 83

5.13 HPLC chromatograph of polyamine contents of L 1210 cells exposed to
7-N H 2-SPD .............................................................................. 84








5.14 HPLC chromatograph of polyamine contents of L1210 cells exposed to
7- NH2-SPD.............................................................................85

5.15 Metabolic breakdown of N',NA-diethyl-7-aminospermidine in murine leukemia
L 1210 cells................................................................................... 86

5.16 Metabolic breakdown of N1 ,N2-diethyl-6-aminospermine in murine leukemia
L 1210 cells................................................................................87

6.1 Dansylation reaction of 7-aminospermidine.......................................119

6.2 HPLC calibration curve for PUT and DHX........................................120

6.3 HPLC calibration curve for SPD and SPM.........................................121

6.4 HPLC calibration curve for 2-OH-PUT............................................122

6.5 HPLC calibration curve for 6-OH-SPD and 7-OH-SPD.......................... 123

6.6 HPLC calibration curve for 7-NH2-DESPD and 6-NH2-DESPM..................124

6.7 HPLC calibration curve for 2-NH2-PUT and 6-NH2-SPM...................... 125

6.8 HPLC calibration curve for 7-NH2-MESPD and 7-NH2-SPD...................126

6.9 ICso0 (48 and 96 h) graphs of 2-NH2-PUT (aminoPUT)...........................127

6.10 ICso0 (48 and 96 h) graphs of 7-NH2-SPD (aminoSPD)............................128

6.11 IC50o (48 and 96 h) graphs of 7-NH2-DESPD........................................129

6.12 IC5o (48 and 96 h) graphs of 6-NH2-SPM...........................................130

6.13 Ki graph of 2-NH2-PUT................................................................131

6.14 Ki graph of 7-NH2-SPD................................................................132

6.15 Ki graph of 7-NH2-DESPD..............................................................133

6.16 Ki graph of 6-NH2-SPM................................................................134
















ACN

AcOH

ADC

AdoMetDC

ATP

C

CHCl3

CNS

DAP

DCHA

DEHSPM

DENSPM

DFMA

DFMO

DMF

DNA

e.g.

eIF-5A

EtOAc

EtOH


LIST OF ABBREVIATIONS

acetonitrile

acetic acid

arginine decarboxylase

S-adenosylmethionine decarboxylase

adenosine triphosphate

carbon

chloroform

central nervous system

1,3-diaminopropane

dicyclohexyl ammonium sulfate

diethylhomospermine

diethylnorspermine

difluoromethylarginine

difluoromethylomithine

dimethylformamide

deoxyribonucleic acid

for example

eukaryotic translation initiation factor 5A

ethyl acetate

ethanol








Expt

FAD

HBr

HCI

HEPES


HPLC

Hex

HSPD

KOAc

MDL 72527

MFMO

MGBG

MOPS

Mts

N

7-NH2-DESPD

6-NH2-DESPM

2-NH2-PUT

7-NH2-SPD

6-NH2-SPM

NMR

ODC

2-OH-PUT


experiment

flavin adenine dinucleotide

hydrobromic acid

hydrochloric acid

N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic
acid]

high performance liquid chromatography

hexane

homospermidine

potassium acetate

N,N'-bis(2,3-butadienyl)-1,4-butanediamine

monofluoromethylornithine

methylglyoxylbis(guanylhydrazone)

3-[N-morpholino]propanesulfonic acid

mesitylenesulfonyl

nitrogen

N',N8-diethyl-7-aminospermidine

N ,N12-diethyl-6-aminospermine

2-aminoputrescine

7-aminospermidine

6-aminospermine

nuclear magnetic resonance

omithine decarboxylase

2-hydroxyputrescine









6-OH-SPD

7-OH-SPD

PAO

Pd/C

PUT

dcSAM

SAM

SPD

SPM

SSAT

THF

TLC

t-RNA


6-hydroxyspermidine

7-hydroxyspermidine

polyamine oxidase

palladium on activated carbon

putrescine or 1,4-diaminobutane

decarboxylated S-adenosylmethionine

S-adenosylmethionine

spermidine

spermine

spermidine/spermine N1-acetyltransferase

tetrahydrofuran

thin-layer chromatography

transfer ribonucleic acid














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

SYNTHESES AND METABOLIC PROFILES OF AMINOPOLYAMINES IN THE
MURINE LEUKEMIA L1210 CELL AND THE PATHOGENIC BACTERIUM
PSEUDOMONAS AER UGINOSA

By

John Van Nguyen

December 2004

Chair: Raymond J. Bergeron
Major Department: Medicinal Chemistry

The aim of our study was to extend our understanding of polyamine metabolism

by investigating how eukaryotes and prokaryotes metabolically process exogenous

polyamines. A new series of polyamines, the aminopolyamines, were first assembled.

Their metabolic profiles and impact on cellular and bacterial growth were measured.

These newly synthesized aminopolyamines were screened in both eukaryotic and

prokaryotic models. In the eukaryotic model, the murine leukemia L1210 cells were

exposed to each aminopolyamine, and the 48-h and 96-h IC50 values were measured (in

addition to their effect on polyamine metabolism). In the prokaryotic model,

P. aeruginosa was used to study the impact of the synthetic aminopolyamines on its

growth and polyamine metabolism.

Studies in L1210 cells revealed that 2-aminoputrescine (2-NH2-PUT), and 6-

aminospermine (6-NH2-SPM) had no significant impact on cellular growth with 48-h

IC50 values > 100 uLM; while 7-aminospermidine (7-NH2-SPD) had a very moderate effect








with a 48-h ICso0 of 60 uLM and a 96-h ICso0 of 9 uM. The aminopolyamine N',N-diethyl-

7-aminospermidine (7-NH2-DESPD) also showed a mild effect on cellular growth with a

48-h IC50 of 100 pM and a 96-h ICso0 of 6 pM. At a concentration of 1 pM, 7-NH2-SPD

and 7-NH2-DESPD had a slight impact on ODC by reducing the enzymatic activity to

72% and 79% of control, respectively. The aminopolyamine 6-NH2-SPM was more

active by reducing ODC activity to 52% of control. In addition, 6-NH2-SPM reduced

AdoMetDC activity to 70% of control. While 7-NH2-SPD and 6-NH2-SPM did not

significantly stimulate spermidine/spermine-N'-acetyltransferase (SSAT), 7-NH2-DESPD

showed a moderate SSAT stimulation by upregulating SSAT to 303% of control. L1210

cells were able to catabolically convert 7-NH2-SPD to 2-NH2-PUT and biosynthesize

6-NH2-SPM from 7-NH2-SPD. Besides, when exposed to 6-NH2-SPM, these cells

converted this aminopolyamine to 7-NH2-SPD, and then to 2-NH2-PUT. When exposed

to 7-NH2-DESPD or 6-NH2-DESPM, deethylation of these diethylated aminopolyamines

also occurred inside the L1210 cell, possibly via the SSAT/PAO pathway.

In P. aeruginosa, 2-OH-PUT and 7-OH-SPD are not produced endogenously.

However, when exposed to 2-OH-PUT, the bacteria elaborated 2-OH-PUT into 7-OH-

SPD. A similar scenario occurred in which 2-NH2-PUT was converted to 7-NH2-SPD by

P. aeruginosa. This bacterium was incapable of producing 6-NH2-SPM from 7-NH2-

SPD. However, when exposed to 6-NH2-SPM, it was able to metabolize 6-NH2-SPM to

7-NH2-SPD and 2-NH2-PUT. Our results in this dissertation significantly add to the

understanding of how various polyamine analogues are metabolically processed in both

eukaryotes and prokaryotes.













CHAPTER 1
INTRODUCTION

Polyamines are essential for the growth and development of prokaryotes and

eukaryotes. Life without polyamines is not known, with the exception of a few

microorganisms, such as those in the order Halobacteriales. Many research studies,

including those in our laboratory (Bergeron, 1994, 1995a, 1995b, 1997, 1999, 2000,

2001 la, 2001 b), show that disruption of the polyamine metabolic pathway is a promising

target for anticancer and antibacterial strategies. Polyamine analogues that can block

biosyntheses or interfere with the function of naturally occurring polyamines have

considerable potential as therapeutic agents. Insights into polyamine metabolism are

important in understanding how these drugs work. This chapter focuses on the

metabolism of polyamines in prokaryotes and eukaryotes.

Polyamines and their roles

Polyamines are small aliphatic molecules containing two or more amino moieties

separated by carbon chains. For instance, spermidine (SPD) is a triamine in which the

three amino groups are separated by a group of three methylenes and a group of four

methylenes (Fig. 1-1). It is designated as a (3-4) triamine. Similarly, putrescine (PUT) is

denoted as a (4) diamine, and spermine (SPM) as a (3-4-3) tetraamine. Thus, the number

of methylene groups is used to denote various polyamine structures.

The principal naturally occurring polyamines in eukaryotes are PUT, SPD, and

SPM (Fig. 1-1); although 1,3-diaminopropane (DAP) and hydroxypolyamines (including

2-hydroxyputrescine (2-OH-PUT) and 7-hydroxyspermidine (7-OH-SPD)) also exist in








some prokaryotes. Polyamines are essential for the growth and development of both

eukaryotes and prokaryotes. While the three most common polyamines (PUT, SPD and

SPM) have been found in eukaryotes, SPM is less common in prokaryotes. In some

eukaryotes and prokaryotes, cationic polyamines interact with anionic macromolecules

such as nucleic acids, phospholipids, and certain proteins (Igarashi and Kashiwagi, 2000).

However, understanding the precise roles of polyamines in cellular physiology is still a

challenge (Casero and Woster, 2001). Without polyamines, cell growth is not observed,

with the exception of two bacterial orders: Methobacteriales and Halobacteriales

(Hamana and Matsuzaki, 1992, 1993). Studies show that cellular and bacterial growths

are slowed by the inhibition of polyamine biosyntheses (Bergeron, 2000 and Bitonti,

1982). Because of the positively charged nature at physiological pH, polyamines are

believed to be involved in stabilizing DNA and initiating DNA synthesis (Matthew,

1993). The DNA undergoes condensation, conformational changes, aggregation and

resolubilization in the presence of positively charged polyamines (Vijayanathan, 2001).

In vitro, calf thymus DNA formed a diffused, planar structure, with entrapped bubbles;

but when exposed to SPD and SPM, it assumed a characteristic fingerprint structure with

hexagonal order (Saminathan, 2002). Polyamines are also involved in RNA synthesis and

metabolism (Morgan, 1999). For instance, SPM was shown to stabilize the conformation

of the anticodon loop yeast t-RNA (Tabor and Tabor, 1984; Cohen and McCormick,

1979). Polyamines are involved in stimulating RNA processing through the spermidine-

regulated protein, elF-5A, and also in stimulating Ile-tRNA formation (Igarashi and

Kashiwagi, 2000). Polyamines also play an important role in protein biosynthesis,








including both initiation (Yoshida, 2001) and elongation processes (Scalabrino and

Ferioli, 1982).

Roles of Polyamines in DNA Stabilization and Interactions

In the early 1960s, Tabor discovered that polyamines could stabilize double-

stranded DNA (Tabor, 1962). For instance, PUT, SPD and SPM can increase the melting

temperature of DNA in vitro by as much as 40 C. In addition to electrostatic interaction

with DNA, polyamines interact with nucleic-acid bases, and enter the minor or major

grooves (Feuerstein, 1991). Polyamines, present in eukaryotic cells at millimolar

concentration, are believed to have significant roles in stabilizing DNA structure.

Observed accumulation of polyamines on the chromosomes of mitotic cells further

indicates that polyamines have important interactions with DNA.

Polyamines in Cellular Growth and Development

Polyamines are required during each phase of the cell cycle. The cell cycle

generally consists of four phases: Gl, the first growth phase; S, the DNA synthetic

phase; G2, the second growth phase; and M, the mitotic phase. Increased polyamine

levels have been observed during cell-cycle progression. Early studies indicate elevated

levels of polyamines in GI and S phases (Heby, 1973). These studies also showed that

depletion of polyamines inhibited the cellular transition from G phase to S phase. In

Chinese hamster ovary cells, spermidine was increased during the entire cell cycle, while

putrescine was increased during S and G2 phases, and spermine was increased mainly

during GI and S phase. Inhibition of polyamine biosynthesis in these cells also retarded

S-phase progression (Fredlund, 1997). These results clearly show that polyamines have








critical roles in cell growth and the cell cycle. Polyamines are believed to interact with

cellular proteins that regulate the cell cycle (Thomas, 2001).

Polyamine Metabolism

The polyamine metabolic network in both eukaryotes and prokaryotes is tightly

regulated, to maintain the balance of intracellular polyamine pools. Cells and bacteria

regulate their intracellular polyamine levels by mechanisms that control biosynthesis,

degradation, uptake, and excretion.

Polyamine Biosynthesis

Most cells have the ability to synthesize PUT and SPD. Some prokaryotes (such as

Pseudomonas aeruginosa) lack the ability to synthesize SPM. Some studies also

reported a low steady-state concentration of SPM in P. aeruginosa (Weaver and Herbst,

1958). The polyamine content in both prokaryotes and eukaryotes is elaborately

regulated by biosynthesis, degradation, uptake, and excretion. Interconversions among

the fundamental polyamines PUT, SPD and SPM occur to maintain the balance of these

polyamines. Biosynthesis and uptake of polyamines are stimulated during cellular

responses to growth or proliferative stimuli. On the other hand, when polyamine contents

are elevated, degradation and excretion are induced, as is homeostatic inhibition of

uptake and biosynthesis.

The fundamental polyamine pathway in most eukaryotes is a complex process

(Fig. 1-2). It first involves the conversion of arginine by arginase to ornithine (Luk and

Casero, 1987). Omithine is then decarboxylated by omithine decarboxylase (ODC) to

form PUT. Spermine is synthesized from PUT via consecutive aminopropylations of its

amino groups. The first aminopropylation occurs with the participation of the

co-substrate, decarboxylated S-adenosylmethionine (dcSAM) and spermidine synthase.






5

As a result, SPD is formed and participates in the next aminopropylation reaction. The

second aminopropylation occurs selectively on the aminobutyl end, again using dcSAM

as an aminopropyl source. However, this time a different enzyme (spermine synthase) is

involved. To produce dcSAM (the aminopropyl group donor), methionine first reacts

with ATP to form S-adenosylmethionine, which is then decarboxylated by AdoMetDC

(Seiler, 1990).

Biosynthesis of putrescine

Mammalian cells and fungi have only one pathway for PUT synthesis. According

to this biosynthetic pathway, PUT is formed from ornithine via an enzyme-catalyzed

decarboxylation. Ornithine decarboxylase (ODC) directly removes the carboxylic

(-COOH) functional group from L-ornithine to form PUT. L-Omithine is biosynthesized

from arginine, and is present in human plasma at a concentration of about 85 gM, some

of which is obtained from the diet (Morgan, 1999). Ornithine is also a product of the

urea cycle. Some from this source may participate in the biosynthesis of PUT.

In addition to the ODC pathway to PUT in eukaryotes, many microorganisms and

higher plants possess a second pathway via agmatine (Tabor and Tabor, 1985).

Decarboxylation of arginine by arginine decarboxylase produces agmatine. Agmatine is

then hydrolyzed by agmatinase (agmatine amidinohydrolase) to form PUT and urea

(Fig. 1-3). An additional biosynthetic route to PUT has also been observed in plants via

agmatine (Smith, 1985). Agmatine deiminase converts agmatine to

N-carbamoylputrescine, which is then hydrolyzed by N-carbamoylputrescine amidase to

form PUT, ammonia and carbon dioxide (Fig. 1-3).








Biosynthesis of SPD

Spermidine is biosynthesized from PUT via an enzyme-catalyzed addition of an

aminopropyl group. As discussed earlier, spermidine synthase, also named putrescine

aminopropyltransferase, is the enzyme that catalyzes the transfer of an aminopropyl

group from SAM to putrescine. Spermidine synthase has been isolated and characterized

from different sources such as mammalian cells, plants, and bacteria. Regardless of

sources, spermidine synthase (which consists of two identical subunits) accepts PUT as

its best subtrate.

Biosynthesis of SPM

The aminopropylation that occurs specifically on the aminobutyl end of SPD gives

rise to SPM. This time a different aminopropyltransferase named spermine synthase is

involved in the biosynthesis of spermine. Like spermidine synthase, spermine synthase

also contains two subunits of equal size; and the enzyme also uses an aminopropyl

moiety from dcSAM. However, spermine synthase accepts SPD as its best substrate. It

seems that SPM synthesis is confined to eukaryotic systems because of the lack of

spermine synthase in prokaryotes.

Ornithine decarboxylase

Omithine decarboxylase (ODC) is generally a dimer consisting of two identical

subunits. Pyridoxal 5'-phosphate (PLP) is derived from pyridoxine (vitamin B6). It is a

necessary electrophilic component for ODC activity. The PLP is always found

covalently bound to a lysine residue in the active site of ODC via a Schiff base (imine)

linkage (Silverman, 1999). The L-Omithine first forms a Schiff base with PLP (Fig. 1-4),

and undergoes decarboxylation via a mechanism similar to the decarboxylation of P-keto

acids (Fig. 1-5).








Mammalian species show more than 90% homology in amino-acid sequences of

ODC (Morgan, 1999). The similarity in ODC genetic makeup suggests that the structural

and enzymatic properties of ODC among mammalian species are very similar. Little

similarity exists in amino acid sequences between prokaryotes (e.g., Escherichia coli) and

eukaryotes (e.g., mammalian cells).

S-Adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase (AdoMetDC) plays an important role in

spermidine and spermine biosyntheses. Decarboxylation of S-adenosylmethionine

(SAM) by AdoMetDC produces decarboxylated SAM (dcSAM), which serves as an

aminopropyl donor for spermidine and spermine biosyntheses.

AdoMetDC contains two unequal subunits (a and 3), both of which are necessary

for the catalytic activity. Instead of PLP, all known forms of AdoMetDC use a pyruvoyl

group which binds covalently to the enzyme via an amide linkage (Poelje, 1990). The

decarboxylation activity of this enzyme is similar to that of ODC. SAM first connects

with the pyruvoyl group via an imine linkage to form a Schiff base (Fig. 1-6). The Schiff

base intermediate then undergoes decarboxylation. In the last step, the dcSAM-pyruvoyl-

enzyme complex is hydrolyzed to release dcSAM and the enzyme.

Aminopropyltransferases

Aminopropyltransferase catalyzes the biosynthesis of SPD and SPM by transferring

an aminopropyl group from dcSAM to PUT and SPD, respectively (Morgan, 1999).

Aminopropyltransferases that catalyze SPD and SPM biosyntheses differ significantly.

In mammalian cells, spermidine biosynthesis is catalyzed by spermidine synthase; a

putrescine aminopropyltransferase that is highly specific for PUT, and does not use SPD








as an aminopropyl acceptor (Pegg et al., 1981). Spermine biosynthesis is catalyzed by an

entirely distinct enzyme spermine synthase; a spermidine aminopropyltransferase that

only utilizes SPD as an aminopropyl acceptor (Pajula et al., 1979). Bacterial spermidine

synthase also uses SPD to produce SPM in vitro, although the aminopropylation reaction

is much slower. This explains why some bacteria (such as E. coli) do not produce SPM

endogenously; and why some pseudomonads produce trace amounts of SPM.

Polyamine Catabolism

Polyamine degradation is an important process in the polyamine metabolic

network. Elevation of certain polyamines within a cell can be toxic (Thomas and

Thomas, 2001). Thus, any excess polyamine (such as SPD, SPM or other exogenous

polyamines imported by the organism) must be degraded to maintain the balance of

intracellular polyamine pools. The retrograde pathway progresses from SPM to PUT.

Spermine is first N'-acetylated at the aminopropyl fragment by spermidine/spermine

N'-acetyltransferase (SSAT) using acetyl-CoA as an acetyl source (Casero and Pegg,

1993). The resulting N'-acetylspermine is then oxidized to imine by polyamine oxidase

(PAO). This imine is then hydrolyzed, resulting in 3-acetamidopropanal and SPD (Fig.

1-7) (Binda, 2001). The PAO is a flavin adenine dinucleotide (FAD)-containing enzyme

that catalyzes the oxidation of the secondary amino groups of SPM, SPD, and their

acetylated derivatives (Binda et al., 2002). In eukaryotes, PAO preferentially oxidizes

Nl-acetylspermidine and N'-acetylspermine into imines that are then hydrolyzed to PUT

and SPD, respectively (Morgan, 1999). Spermidine is acetylated at the N-l position of

the aminopropyl end, again by SSAT. Oxidation of N'-acetylspermidine by PAO,

followed by hydrolysis of the resulting imine, results in PUT.








Spermidine/spermine N'-acetyltransferase

Spermidine/spermine N'-acetyltransferase (SSAT) initiates the polyamine

degradation process. This enzyme catalyzes the transfer of an acetyl group from acetyl-

CoA to the aminopropyl moiety of spermine or spermidine. SSAT is known to

specifically acetylate the aminopropyl end of spermidine, forming only N'-

acetylspermidine. The rate of PAO activity is usually fast; thus, SSAT-catalyzed

acetylation is a rate-determining step in polyamine catabolism. SSAT has been purified

from rat, hamster, chicken and human tissues (Casero and Pegg, 1993). Regardless of

species, purified SSAT is unstable and particularly sensitive to heat.

Polyamine oxidase

Copper-containing amine oxidases. The bovine plasma diamine oxidase is an

example of a copper-dependent amine oxidase. Copper-containing amine oxidases act on

spermidine or spermine to produce an aminomonoaldehyde [N-(4-aminobutyl)-3-

aminopropanal] or a dialdehyde (4,9-diaza-dodecanedialdehyde), respectively, ammonia

and hydrogen peroxide (Morgan, 1999). These enzymes directly oxidize polyamines

containing primary amino groups, the aminopropyl moiety being more favored.

Flavin adenine dinucleotide-containing amine oxidases. Peroxisomal flavin-

dependent PAO is known to oxidize N'-acetylspermine and Nl-acetylspermidine to imine

forms. The PAO is present in most organisms, at levels that significantly exceed those of

SSAT (Pegg, 1986). Therefore, any acetylated polyamines formed will be quickly

further processed by PAO, making acetylated polyamines usually undetectable. FAD, an

important coenzyme of PAO, is derived from riboflavin via an intermediate flavin

mononucleotide (FMN) (Fig. 1-8). FAD is a good electron sink (Fig. 1-9) which is

reduced with concomitant oxidation of N'-acetylspermidine and N'-acetylspermine








(Silverman, 2000).

Importance of Understanding Polyamine Metabolism

Polyamine metabolism has been shown to be a potential target for

antiproliferative therapy (Marton and Pegg, 1995). Extensive research efforts have

focused on the design and synthesis of inhibitors of ODC, AdoMetDC, and PAO.

Ornithine decarboxylase and AdoMetDC are two of the most important enzymes in

polyamine assembly; and PAO is a key enzyme in polyamine catabolism. Despite little

success in clinical applications with difluoromethylornithine (DFMO), an ODC inhibitor;

methylglyoxylbis(guanylhydrazone) (MGBG), an AdoMetDC inhibitor; and

N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), a PAO inhibitor (Pegg,

1988), these inhibitors (Fig. 1-10) have been used successfully for studying polyamine

metabolism. DFMO blocks ODC activity in a dose- and time-dependent manner;

completely depletes PUT and SPD; and significantly depletes SPM, in some rabbit-

corneal cell types (Du, 2003), indicating that the biosyntheses of PUT by ODC and of

SPD from PUT are major routes. MGBG, a potent inhibitor of human AdoMetDC, has

no significant impact on AdoMetDC of the human pathogen Trypanosoma cruzi,

suggesting that this pathogen contains a genetically unique AdoMetDC as an enzyme in

the polyamine biosynthetic network (Persson et al., 1998).

Understanding polyamine metabolism could help explaining abnormal

concentrations of polyamines in some pathological conditions. For example, rats induced

with central nervous system (CNS) injury showed an abnormal increase in PUT level,

and a decrease in SPM and SPD levels, at the injured site. The high PUT level was due

to the elevated activity of PAO. The increased PUT was reduced significantly, and








N'-acetylspermidine increased after treating with MDL 72527, a PAO inhibitor. These

observations confirmed that the abnormal increase in PUT after CNS injury was mediated

by the SSAT/PAO pathway (Rao, 2000).

Polyamine Metabolic Pathway Serves as a Therapeutic Target

Polyamines are homeostatically regulated by multiple metabolic pathways such as

biosynthesis, uptake, degradation, and excretion. Polyamines have an important role in

the proliferation of normal and malignant cells (Davidson, 1999). Elevation of

polyamine concentration is not restricted to malignant conditions, because this

phenomenon has also been found in body fluids of patients with CF, and during

pregnancy (Russel, 1978; Wallace, 2003). However, many studies show a clear link

between increased polyamine content and cancer-cell activities. High concentrations of

polyamines were also found in tissues with bacterial (Maita, 1990) and parasitic

infections (Bacchi, 1999).

The abnormal increase in polyamine biosynthesis in neoplastic tissues has made

the polyamine metabolic network a potential target in anticancer (Marton, 1995) and

antibacterial strategies (Wang, 1991).









Diaminopropane (DAP)


H2N ,NH2



H
H2N2




H
H2N-^ N N, NH2
H


1 3
H2N 2NH2
OH




8 76 H 2
H24-N -NH2
OH


Putrescine (PUT)





Spermidine (SPD)






Spermine (SPM)


2-Hydroxyputrescine (2-OH-PUT)






7-Hydroxyspermidine (7-OH-SPD)


Figure 1-1. Naturally occurring polyamines. Numbers in the structures of 2-OH-PUT
and 7-OH-SPD indicate the positions of carbon molecules.


H22N' NH2








+
NH2 0
H2Nl N OH
H NH2


0
H2N- -" OH
NH2


H2N -sNH2
Putrescine


^c


5'-Methyl-
thioadenine


Decarboxylated
S-adenosylmethionine


Spermidine


H

0 0


f

ON 'N NH2
H H
" N -Acetylspermidine


0 0


H H

H 0


NI-Acetylspermine


H2NN '-N- N- NH2
H
Spermine
Figure 1-2. Fundamental polyamine metabolism pathway in eukaryotes.
(a) Arginase, (b) Ornithine decarboxylase, (c) Spermidine synthase, (d) Spermine
synthase, (e) Spermidine/Spermine N -acetyltransferase, (f) Polyamine oxidase,
(g) S-adenosylmethionine synthase, (h) S-adenosylmethionine decarboxylase.


Arginine


Ornithine


5'-Methyl-
thioadenine


c


H2N--N-,-NH2










Methionine


ATP




ADP + Pi


NH2
N- N COOH
N- N IS NH2
O +

HO H



h


NH2
N N
N N


S-Adenosylmethionine











Decarboxylated
S-adenosylmethionine


Figure 1-2. Continued


COOH
S/^ NH2








+
NH2 0
H2N N- -OH
H NH2

Arginine




0 NH2

H2N OH H2N ^^ NH2
NH2
Ornithine Urea Agmatine

SNH3



H2N NH2 N-carbamoyl-
putrescine
Putrescine


I NH3 + CO2

H2 N NH2
H

Spermidine




H
H2N" NN N2
H
Spermine


Figure 1-3. Polyamine metabolism in plants and bacteria








ODC
Lys B: B:

\ TH COO
\C J L-or
2P-O3PO-H- OH H2N R
N+H
PLP N
H


1-1


nithine, R= NH2


Lys ^B+ B:
HN :_I
V^ H
-NH .,,,Coo-
+ "" R


Lys B: NH2 B
NH2


Figure 1-4. Formation of the Schiff base between L-ornithine and PLP (PLP: coenzyme
of ODC). B: a base in ODC active site










Lys B+ B:
HN H

2-O3PO ,s OH

N
H






Lys B: i T
NH2 H
R-C


H.Mcoo-
H2' R


+ L-omithine, R= NH2













NH2


CO2


Lys B: B:
NH,


R
CH2


Lys B B:
HN H

2-O3PO OH
N
H

+ RCH2NH2 (PUT)


Figure 1-5. Proposed mechanism of omithine decarboxylation








NH2
NN COOH
N 'N S NH2
0 +

HO H

S-Adenosylmethionine
(SAM)


AdoMetDC
HN- 0 active-site
'0 I pyruvoyl group


SH20


AdoMetDC
NH HN


N N2

HO OH

CO2
AdoMetDC
NH2
N HN HNO



HO OH


AdoMetDC

HN 0O
0X'


AdoMetDC
HN


+ H20


dcSAM


Figure 1-6. Proposed mechanism of AdoMetDC activity


NH2








H
H2N N' N NH2
H

SSAT Acetyl-CoA


H H
H2N "-' N -, N
H O

FADH H
PAO
FAD 1


H
H2N- N N N
H 0


Spermine


NV-Acetylspermine



202


/2 02 + H20


H20


H2NNNH2
H
Spermidine


H

H 0

3-Acetamidopropanal


Figure 1-7. Metabolic breakdown from spermine to spermidine
by SSAT/PAO. SSAT: spermidine/spermine N -acetyltransferase.
PAO: polyamine oxidase. FAD: flavin adenine dinucleotide.
















ATP ADP a N~ : -NH

FMN



ATP



PPi


NH2


P-O-P-O-, N
o O-
HO HO OH
OH
HO




ND
0

FAD


Figure 1-8. Biosynthesis of FAD from riboflavin


Riboflavin








R
N N 0

NONH
0
FAD




Ie + le





R
I
N NO
N
0-

[FAD']-




le + le




R


JUaN NH
H 0

[FADH]

Figure 1-9. Different oxidation states of FAD









H2N COOH
H2N C

DFMO, an irreversible inhibitor of ODC








NH H

H2N N N N- N NH2
H r NH


MGBG, an AdoMetDC inhibitor








H c CCH2

H2C H


MDL 72527, a PAO inhibitor

Figure 1-10. Structures of some polyamine metabolic enzyme inhibitors













CHAPTER 2
POLYAMINE CONTENTS AND POLYAMINE METABOLISM IN
PSEUDOMONADS: LITERATURE REVIEW

Polyamine metabolism in some prokaryotes is somewhat different from that in

animal cells. In addition to the metabolic pathway described above, some

microorganisms (e.g., P. aeruginosa, also possess a second pathway via agmatine)

(Fig. 2-1). In P. aeruginosa, PUT can be biosynthesized either directly from ornithine by

ODC or indirectly from arginine via arginine decarboxylase (ADC). Arginine is

decarboxylated by ADC to form agmatine. Agmatine is then hydrolyzed by agmatinase

to form PUT, with the elimination of urea (Morgan, 1999). The genes of two enzymes

ODC and ADC of wild type P. aeruginosa strain PAO1 were identified as speC and speA

(Nakada and Itoh, 2003). It was also reported that the activities of these two

decarboxylases were similar, and each individual decarboxylase appeared to direct

sufficient polyamine biosynthesis for normal growth.

In rapidly growing organisms including cancer cells and bacteria, the demand for

polyamines is high. The polyamine pathway has been elucidated for a relatively small

number of organisms, and there is significant interspecies variation (Casero and Woster,

2001). For example, in P. acidovorans, 2-OH-PUT, PUT, SPD, and 7-OH-SPD were

identified but in P. aeruginosa, only PUT, SPD, and trace amounts of DAP and SPM

were found (Table 2-1) (Bitonti et al., 1982; Kallio et al., 1981; Weaver and Herbst,

1958). Additional polyamines present in selected pseudomonads are also listed in Table

2-1.








Pseudomonads are allocated to three different subclasses ofproteobacteria, a, P,

and y, by their differences in polyamine distribution patterns. PUT and homospermidine

(HSPD) make up the a subclass. PUT, 2-OH-PUT, and SPD are in the P3 subclass and

PUT, DAP and SPD belong to the y subclass (Busse and Auling, 1988). Burkholderia

cepacia, DSM 50181, is a common pathogenic bacterial strain that produces a large

amount of 2-OH-PUT and a small amount of HSPD in addition to PUT and SPD. P.

aeruginosa belongs to the y subclass, which contains considerable amount of PUT, SPD,

and a small amount of DAP. Spermine is also present in P. aeruginosa at a low steady

state concentration. To date, there is no evidence of hydroxypolyamines (i.e., 2-OH-PUT

or 7-OH-SPD) in bacteria of the y subclass.

Pseudomonas sp Strain Kim

In P. sp strain Kim, a member of the P subclass (Table 2-1), PUT, 2-OH-PUT and

SPD were identified (Rosano et al., 1989), but whether 7-OH-SPD was present in these

bacteria was not reported. The steady state level of SPD was low because of the efficient

conversion of SPD to PUT and 2-OH-PUT that ultimately prevented accumulation of

SPD in the bacteria. However, whether 2-OH-PUT was derived from SPD directly or via

PUT was unclear. Incubation of 14C-SPD (12.5 p.M) with growing log phase bacteria for

6 h resulted in the formation of radioactive PUT and 2-OH-PUT. These results suggested

that P. sp strain Kim contains N'-SPD acetyltransferase and PAO, the enzymes needed to

convert SPD to PUT. When exposed to MGBG (an AdoMetDC inhibitor) and '14C-SPD

(12.5 uM) simultaneously, the amounts of radioactive PUT and 2-OH-PUT found in

these same bacteria increased 5-7 fold. Note that in the presence of MGBG, the

biosynthesis of endogenous SPD was reduced, causing a decrease in the conversion from








SPD to PUT and 2-OH-PUT. Therefore, these bacteria increased the processing of 14C-

SPD to form radiolabeled polyamines PUT and 2-OH-PUT to stabilize the polyamine

pools.

In a separate experiment, 14C-SPD was added to growing log phase Pseudomonas

sp strain Kim with and without MDL 72527, a PAO inhibitor, added to the bacteria

nutrient broth. MDL 72527 only reduced the conversion of radioactive SPD to

radioactive PUT and radioactive 2-OH-PUT by 40%, and it had no significant impact on

total intracellular levels of PUT and 2-OH-PUT. Rosano et al. suggested that 40% of

radiolabeled SPD must have been quickly converted to radioactive PUT and 2-OH-PUT

before the inhibition of PAO by MDL 72527 took effect. It was interesting to note that

inhibitor MDL 72527 did not significantly affect the levels of non-radioactive PUT and

2-OH-PUT. The authors suggested that PUT and 2-OH-PUT are important in the

bacterial growth, and these polyamines could have been synthesized under homeostatic

control (Rosano et al., 1989).

Pseudomonas acidovorans

In addition to 2-OH-PUT, PUT and SPD, 7-OH-SPD (1-3 Ig/wet wt cells) was

identified in P. acidovorans, a member of the 03 subclass (Rosano et al., 1978) (Table

2-1). It was demonstrated that 7-OH-SPD could be synthesized enzymatically from

2-OH-PUT by cell-free preparations from E. coli or P. acidovorans, which contain an

aminopropyltransferase called SPD synthase. The presence of 7-OH-SPD, but not 6-OH-

SPD, as a naturally occurring polyamine suggested that SPD synthase reacts

preferentially with the amine distal to the hydroxyl group (Fig. 2-2). The SPD synthase

from P. acidovorans was found to be capable of synthesizing both SPD and 7-OH-SPD








from their respective substrates. Recall that PUT serves as a substrate for SPD synthase

and 2-OH-PUT is a derivative of PUT with a hydroxyl substituent at the C-2 position.

This finding is consistent with the observation that in addition to PUT, spermidine

synthase can utilize PUT derivatives with a variety of substituents at the C-2 position

including hydroxyl and methyl groups (Sarhan et al., 1987).

Pseudomonas thermocarboxydovorans

Pseudomonas thermocarboxydovorans is also a member of the P3 subclass.

However, the polyamine content of these bacteria differs slightly from P. acidovorans. It

also contains DAP (Hamana and Matsuzaki, 1990). It was suggested that, like

P. acidovorans, 7-OH-SPD is synthesized from 2-OH-PUT as a result of preferential

action of the aminopropyltransferase with the amine distal to the hydroxyl group (Fig.

2-2).

Aminopropyltransferase catalyzes the biosynthesis of SPD and SPM by

transferring an aminopropyl group from dcSAM to PUT and SPD, respectively (Morgan,

1999). There appear to be significant differences in the aminopropyltransferases that

catalyze SPD and SPM biosyntheses. In mammalian cells, SPD biosynthesis is catalyzed

by spermidine synthase, a putrescine aminopropyltransferase, which is highly specific for

PUT and does not use SPD as an aminopropyl acceptor (Pegg et al., 1981). Spermine

biosynthesis is catalyzed by an entirely distinct enzyme spermine synthase, a spermidine

aminopropyltransferase, which only utilizes SPD as an aminopropyl acceptor (Pajula et

al., 1979). Spermine can be biosynthesized in some bacteria despite the absence of

spermine synthase since bacterial spermidine synthase can also utilize spermidine as a

substrate. However, the spermine biosynthetic reaction catalyzed by spermidine synthase








is much slower than by spermine synthase. Some bacteria, such as E. coli, do not

produce SPM endogenously, yet some pseudomonads produce trace amounts of SPM.

Burkholderia cepacia

In a clinical setting, B. cepacia (formerly classified as P. cepacia) often infects

patients with cystic fibrosis after a lengthy colonization by P. aeruginosa. Patients with

severe respiratory impairment, who are considered for lung transplantation, are also at a

high risk for infection. Acquisition of B. cepacia after lung transplantation was markedly

associated with morbidity and mortality (Steinbach et al., 1994). B. cepacia is also a

member of the 3 subclass (Table 2-1) and contains large amounts of OH-PUT (24.7-30.5

pmol/g dry weight) and PUT (43.3-58.2 uimol/g dry weight) (Busse and Auling, 1988).

The pathogen also contains cadaverine (8.2-14.6 p.mol/g dry weight) and a small amount

of SPD (0.3 umol/g dry weight). Whether 7-OH-SPD was measured or not was unclear

from these available studies. In the presence of abundant endogenous 2-OH-PUT in B.

cepacia, 7-OH-SPD was expected to be biosynthesized by spermidine synthase. Perhaps

the efficient conversion from 7-OH-SPD to 2-OH-PUT resulted in an undetectable

steady-state level of 7-OH-SPD.

Pseudomonas aeruginosa

Biology

P. aeruginosa is one of the best known species among pseudomonads. It grows

well in all of the common culture media at a temperature range of 30-37 oC. Although

aerobic conditions are required, some strains of P. aeruginosa can grow in anaerobic

conditions with the presence of nitrate. In these cases, the oxygen in nitrate serves as the








electron acceptor. In common with other pseudomonads, P. aeruginosa can utilize a

large number of organic substrates as carbon sources.

P. aeruginosa is usually a straight bacillus. The bacterium is motile by a single

polar flagellum. The flagellum is also important in bacterial virulence and biofilm

(bacterial protective matrix) formation. Bacterial biofilm is a large, flexible, but sturdy

matrix. It contains water channels for the intake of nutrients and export of wastes. Each

bacterium is between 1.5 to 4.0 pm long and about 0.5 gm in diameter. Pseudomonas.

aeruginosa produces fluorescein (a green fluorescent pigment) and pyocyanin (a

phenazine blue pigment). Fluorescein is found in several pseudomonads while pyocyanin

is uniquely found in P. aeruginosa.

P. aeruginosa is first formed in the biofilm layer that protects the bacteria from

extreme physical, chemical, and biological conditions. Upon maturing, the planktonic

bacteria are released from the biofilm and if survived, they will proliferated quickly

(doubling time = 40 min), cause acute infections, and develop new biofilms. Bacteria in

biofilm are 50-5000 times more resistant to anti-pseudomonal drugs compared to the

planktonic bacteria.

Pathogenicity

Pseudomonas aeruginosa was chosen for this study because infections caused by

this bacterium are of major concern in medical facilities, particularly in intensive care and

bum units. Pseudomonas aeruginosa is a Gram-negative bacterium which possesses a

complex and sturdy cellular membrane structure (Wilson and Dowling, 1998). Sites of

infection include the CNS, bones, joints, skin, eyes, ears, gastrointestinal tract, urinary

tract and lungs. Pseudomonas aeruginosa is an opportunistic organism, but is relatively








harmless to healthy humans. However, immunocompromised patients who have had

surgery, transplant, cancer, HIV, and cystic fibrosis are at high risk for infection (Fick,

1993). Pseudomonas aeruginosa is capable of utilizing a wide variety of carbon sources,

thus contributing to its virulence (Delden and Iglewski, 1998). Once infection is

established, P. aeruginosa produces toxic proteins that cause extensive tissue damage and

also interfere with human defense mechanisms. These proteins range from potent toxins

that enter and kill host cells to degradative enzymes that permanently disrupt the cell

membranes and connective tissues in various organs (Todar, 2002). In patients with

cystic fibrosis (CF), P. aeruginosa successfully colonize the respiratory tract since it is

able to produce a highly protective capsule made of the mucoid polysaccharide alginate.

This allows the bacteria to adhere better to the lining of the lungs and to resist

phagocytosis by immune cells. Antibiotics do not effectively eradicate P. aeruginosa

from the lungs in these patients (Wilson and Tsang, 1994).

Current treatment of P. aeruginosa includes the usage of many classes of

antibiotics including cephalosporins, aminoglycosides and carbapenems (Jones and

Varnam, 2002; Korvick and Yu, 1991). Development of resistance to antibiotics is a

major problem in fighting bacterial infection. Some pseudomonad strains can inactivate

the drugs that threaten them by using certain enzymes to modify the drug (Poole, 2002),

while other strains can use their active efflux systems to export various antibiotics (Levy,

2002).

Polyamine Contents and Metabolism

Pseudomonas aeruginosa is a member of the y subclass (Table 2-1). It contains

DAP (2.9 uimol/g dry weight), PUT (34 utmol/g dry weight), and SPD (8.6 rimol/g dry








weight). A study also reported that P. aeruginosa contains trace amount of spermine

(Weaver, 1958).

Monofluoromethylomithine (MFMO) at 2 mM can inhibit the activity of ODC in

P. aeruginosa by 98%. Difluoromethylarginine (DFMA) at 1 mM can inhibit arginine

decarboxylase (ADC) activity by 94% but cannot inhibit ODC activity (Kallio et al.,

1981). Although MFMO and DFMA were efficiently transported into bacteria from the

nutrient broth, these drugs individually could not inhibit PUT biosynthesis. In order to

inhibit PUT biosynthesis, it is necessary to use both drugs to block both ODC and ADC

activities. However, because of the rapid turnover of ODC and ADC, a high

concentration of drugs is needed to achieve significant inhibition on these enzymes.

Addition of a combination of three drugs, MFMO (2 mM), DFMA (2.5 mM) and

DCHA (dicyclohexyl ammonium sulfate, a competitive inhibitor of SPD synthase) (5

mM), to cultures of P. aeruginosa slowed the bacterial growth (Bitonti et al., 1984). The

doubling time increased from 42 min to 62 min. Intracellular concentrations of PUT and

SPD decreased significantly to 41% and 19% of control levels, respectively. The normal

growth rate was restored by the addition of 0.1 mM SPD or 5 mM PUT. Addition of

SPD resulted in the increase of intracellular PUT, and addition of SPM resulted in

increases of both PUT and SPD. On the other hand, addition of PUT only increased the

intracellular PUT but had no effect on SPD or SPM. It was clear that SPD and SPM were

more effective in reversing the growth inhibition. SPD at a low concentration (0.1 mM)

was found to be sufficient to reverse the growth inhibition while a large amount of

exogenous PUT (5 mM) was required to reverse the growth inhibition (Bitonti et al.,






31


1984). However, it was still unclear from this study whether or not SPD itself was

responsible for the growth recovery.

P. aeruginosa does not produce any 2-OH-PUT or 7-OH-SPD. However, this

bacterium can utilize these hydroxypolyamines without affecting its growth. In the next

chapter, we will describe how this microorganism incorporated the hydroxypolyamines

into its polyamine metabolic network.








NH2 0
H2N N OH
H NH2


Arginine


0
H2N OH
NH2


Ornithine


+
NH2
H2N N NH2
H
Agmatine


Putrescine



H2N N ^ NH2
H


Spermidine



11I


H2NNN NH2
H
Spermine
Figure 2-1. Polyamine metabolism in Pseudomonas aeruginosa









H
H2 N2
OH

7-Hydroxyspermidine



A


Spermidine synthase


SDistal to the OH group

H2N- NH2
OH


2-Hydroxyputrescine


Not observed


H2 N N NH2
H OH

6-Hydroxyspermidine

Figure 2-2. Biosynthesis of 7-hydroxyspermidine observed in
P. acidovoran and P. thermocarboxydovorans.










Table 2-1. Polyamine contents of selected pseudomonads


Species Subclass Naturally occurring
polyamines (pmole/g dry
weight unless specified)
P. aminovorans a PUT (122)
SPD (0.8)
HSPD (53.0)
SPM (0.8)
P. aeruginosa y DAP (2.9)
PUT(34.0)
SPD (8.6)
P. acidovorans p 2-OH-PUT (57.1)
PUT (57.8)
SPD (0.6)
7-OH-SPD (1-3 pg/g wet wt)
Burkholderia cepacia Cadaverine (8.2-14.6)
2-OH-PUT (24.7-30.5)
PUT(433-582)
SPD (03)
P. sp strain Kim PUT(133 nmol/OD unit)
2-OH-PUT (9.08 nmol/OD unit)
SPD (trace amount)
P. thermocarbaxydovorans DAP (0.100 pmnol/g wet wt)
PUT (0.500 pmol/g wet wt)
SPD (0.760 pmol/g wet wt)
2-OH-PUT (0.129 pmol/g wet wt)
7-OH-SPD (0.400 pmol/g wet wt)













CHAPTER 3
PROCESSING OF 2-HYDROXYPUTRESCINE AND 7-HYDROXYSPERMIDINE IN
PSEUDOMONAS AER UGINOSA

In the previous chapter, we discussed hydroxypolyamines in a few pseudomonads.

Unlike pseudomonads in the 03 subclass, P. aeruginosa, a member of the y subclass, does

not contain hydroxypolyamines. We would like to understand how P. aeruginosa

incorporates 2-OH-PUT and 7-OH-SPD into its polyamine biosynthetic network as well

as its catabolic machinery.

Hydroxypolyamines

Free forms of 2-OH-PUT and 6-OH-SPD are not observed in living organisms.

However, they incorporate in some naturally occurring compounds. The 2-OH-PUT is a

component of hypusine (a naturally occurring compound that is present in bovine brain

tissue) (Shiba, 1971), while 6-OH-SPD is a vital component of a cytotoxic marine

compound. Hypusine is also present in various organs (such as liver, kidney, spleen,

heart, and lung) of Wistar rats (Nakajima, 1971). Unlike 2-OH-PUT and 6-OH-SPD,

7-OH-SPD is a naturally occurring hydroxypolyamines present in some pseudomonads as

discussed in chapter 2. The three hydroxypolyamines were prepared previously in our

laboratory; and thus, the authentic samples were used as standards for analytical analyses.

Impact on Bacterial Growth

As the exposure concentrations of 2-OH-PUT increased from 0.05 to 1.0 mM, the

lag time (the time it took before the bacteria started to enter logarithmic phase) gradually

increased (Fig. 3-1). Even at high concentration, i.e. 1.0 mM for 2-OH-PUT and 1.0 mM








for 7-OH-SPD, these two hydroxypolyamines did not have any significant impact on

bacterial growth by stationary phase. When grown without these exogenous polyamines,

the bacteria usually started the logarithmic phase at t = 11 h, and reached the stationary

phase by 18 h. In the presence of each hydroxypolyamine at 1.0 mM, the bacteria started

the logarithmic phase at 15 h, and reached the stationary phase by 23 h (Fig. 3-2). In

addition, the maximum bacterial turbidity usually decreased to about 80 % of control.

Processing of Hydroxypolyamines and Impact on Bacterial Polyamine Contents

Incubation of P. aeruginosa with 2-OH-PUT resulted in decreasing intracellular

concentrations of PUT and SPD. The depletion of these bacterial natural polyamines

occurred in a dose-dependent manner. At a concentration of 1.0 mM, 2-OH-PUT

reduced PUT and SPD levels to 66% and 69% of control, respectively. It was also

noticeable that as the exposure concentration of 2-OH-PUT increased, the intracellular

level of 2-OH-PUT also increased. In addition, 2-OH-PUT was converted to both

7-OH-SPD and 6-OH-SPD (Fig. 3-3). The formation of each of the two

hydroxypolyamines, 7-OH-SPD and 6-OH-SPD, were dependent of the exposure

concentration of 2-OH-PUT (Fig. 3-4). The low steady state levels of 7-OH-SPD and

6-OH-SPD (< 1.0 nmol/OD unit) were probably due to the dynamic back conversion to

2-OH-PUT, or perhaps because these hydroxypolyamines are not important for the

growth of P. aeruginosa. Unlike P. acidovorans or P. thermocarboxydovorans, it was

surprising that 6-OH-SPD was biosynthesized from 2-OH-PUT in P. aeruginosa.

Spermidine synthase in this bacterium must have catalyzed the aminopropylation from

either amino end of 2-OH-PUT. Careful inspection of the HPLC chromatographs further

confirmed the formation of 6-OH-SPD and 7-OH-SPD in P. aeruginosa grown in the








presence of 2-OH-PUT. In the untreated bacteria, a HPLC chromatograph showed no

detectable level of any of the hydroxypolyamines (Fig. 3-5). On the other hand, in the

bacteria incubated with 1.0 mM 2-OH-PUT for 16 h (mid log phase), 6-OH-SPD and

7-OH-SPD were formed. According to a HPLC chromatograph (Fig. 3-6), 6-OH-SPD

eluted at 20.001 min (peak # 9) with an area under the curve of 203786 (- 2.1% of PUT

area) while 7-OH-SPD eluted at 19.598 min (peak # 8) with an area of 65543 (- 0.7% of

PUT area). These interesting observations have stimulated further studies on polyamine

metabolic activity in prokaryotes and eukaryotes with respect to exogenous polyamine

analogues.





















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__________________,6













CHAPTER 4
SYNTHESES OF AMINOPOLYAMINES

Design Concept

Based on the surprising observation that P. aeruginosa both incorporated and

processed 2-OH-PUT and 7-OH-SPD, we elected to further investigate the structural

boundary conditions set by this microorganism on the polyamine analogues it would

process. We elected to replace the hydroxyl oxygen with another heteroatom, nitrogen.

Thus, 2-OH-PUT would be replaced by 2-NH2-PUT and 7-OH-SPD by 7-NH2-SPD.

This modification could clearly have an important impact on the charge property of the

molecule.

Synthesis

Previous Methods

Aminoputrescine (1, 2, 4-butanetriamine, also known as 2-NH2-PUT) was

synthesized in the laboratory as early as 1921 by Windaus and coworkers (Windaus,

1921). It was prepared from histamine by acylation of the imidazole moiety using 2-

methylpropanoyl chloride as an acylating agent (Fig. 4-1). The reaction also utilizes

aqueous potassium acetate in acetonitrile. This imidazole ring opening reaction, also

known as Bamberger ring cleavage, was invented in 1893. Methanolysis of the

diformamide isomer mixture 38 yielded a single ene-triamide 39. The double bond of the

ene-triamide intermediate was reduced by hydrogenation in the presence of palladium.








The resulting 1, 2, 4-butane triamide (40) was then hydrolyzed by utilizing 6 N HCI to

furnish 2-NH2-PUT (5).

Recently, Altman and coworkers reported a different method for the Bamberger

cleavage of the imidazole ring. Histamine was first acylated at the N-4 position utilizing

2-methylpropanoyl chloride. The imidazole ring of the N4-(2-methylpropanoyl)histamine

was then cleaved in the presence of di-tert-butyl dicarbonate and aqueous potassium

acetate in acetonitrile (Altman, 1989). Reduction of the ene-triamide was improved by

utilizing Raney nickel instead of palladium (Altman, 1991).

In 1993, Altman and Ben-Ishai introduced a new method for synthesizing chiral 2-

NH2-PUT (37) (Altman, 1993) (Fig. 4-2). The starting material (S)-5-(hydroxymethyl)-

pyrrolidone (30) was prepared from (S)-pyroglutamic acid methyl ester (Silverman,

1980). The alcohol 30 was converted to (S)-5-(azidomethyl)-2-pyrrolidone (31) by a

Mitsunobu reaction utilizing hydrazoic acid and phosphine in diethylazodicarboxylate

(DEAD). Hydrogenation of the azide 31 produced the primary amine 32, which was

further hydrolyzed with 6 N HCI to afford (S)-4,5-diaminovaleric acid (33). The diamino

acid was acylated with iso-butyl chloroformate to give N4,N5-protected amino acid 34,

which was then converted to an azide by the mixed anhydride procedure and sodium

azide. Upon heating, this azide intermediate underwent a Curtius rearrangement to form

the isocyanate 35, which was then hydrolyzed by utilizing LiOH in THF and H20 to yield

(S)-N',N2-bis(iso-butoxycarbonyl)-1,2,4-butanetriamine (36). Finally, the di-protected

amine 36 was hydrolyzed with 6 N HCI to afford (S)-1,2,4-butanetriamine (37) as a

trihydrochloride salt.








In this dissertation, we report a modified procedure for the synthesis of racemic 2-

NH2-PUT. Histamine, without first being acylated at the N-4 position, was subjected to a

Bamberger ring cleavage utilizing di-tert-butyl dicarbonate. Reduction of the ene-tri-Boc

was performed at a higher temperature (55 C) to improve the yield. This was a simple

method which provided 2-NH2-PUT in high overall yield (69%). Another advantage of

this method was to avoid the azide intermediate of Fig. 4-2, compound 31, which is

known to be explosive and hazardous. The aminopolyamines 7-NH2-SPD, N'-ethyl-7-

aminospermidine (7-NH2-MESPD), 7-NH2-DESPD, and 6-NH2-SPM were synthesized

for the first time in our laboratory. The aminopolyamine 6-NH2-DESPM was

synthesized previously in our laboratory (Bergeron, R. J.; Guangfei, H., unpublished).

Synthesis of 2-NH2-PUT

The aminopolyamine 2-NH2-PUT was synthesized in our laboratory according to

our modified procedure. Histamine dihydrochloride 1 was subjected to a Bamberger ring

cleavage reaction (Bamberger and Liebigs, 1921) in the presence of di-tert-butyl

dicarbonate (Boc anhydride), aqueous potassium acetate, and acetonitrile stirring at room

temperature for 5 d to yield a mixture of N-formyl products 2 in 66% yield (Fig. 4-3).

Methanolysis of this mixture under refluxing conditions produced a single isomer 3 in

91% yield. At room temperature, the double bond of the ene-tri-boc amine 3 was not

completely reduced by hydrogenation with the presence of 10% palladium on activated

carbon in absolute ethanol. However, when heated to 55 oC for 17 h the hydrogenation

was completed in 90% yield. Removal of the Boc groups in N,N2,N4-tri-Boc-1,2,4-

butanetriamine (4) with 20% HCl(g) in absolute ethanol furnished 2-NH2-PUT

trihydrochloride as a white hygroscopic powder (5) in 92% yield.








Synthesis of 7-NH2-SPD

The two available nitrogens of histamine dihydrochloride 1 were protected by

mesitylenesulfonyl (Mts) blocking groups using mesitylenesulfonyl chloride (2.4 equiv)

under biphasic conditions (CH2C12/1 NNaOH) in 95% yield (Fig. 4-4). The resulting

histamine bis(mesitylenesulfonamide) 6 was first treated with NaH in dry DMF and then

alkylated at the N-4 position using N-(3-bromopropyl)phthalimide to generate the fully

protected N4-(aminopropyl)histamine 7 in 45% yield. Hydrazinolysis of 7 in refluxing

ethanol resulted in the primary amine 8 in 56% yield. The Mts groups in 8 were cleanly

removed under strong reductive conditions, using 30% HBr in acetic acid and phenol in

CH2C2, to provide N4-(3-aminopropyl)histamine trihydrobromide (9) in 66% yield. This

histamine derivative was then subjected to Bamberger ring opening in the presence of di-

tert-butyl dicarbonate to afford 10 as a mixture of two isomers in 73% yield. An attempt

to cleave the Mts-protecting imidazole in 8 using the same reaction condition failed

probably due to steric effects. The formyl groups of the mixture 10 were removed by

methanolysis to obtain the ene-tetra-Boc amine 11 in 95%. Reducing the double bond of

11 was achieved successfully by hydrogenation at 55 oC in the presence of Pd on

activated carbon in absolute ethanol to give the tetra-Boc amine 12 in 95% yield. Finally,

removal of the Boc groups in 12 by utilizing 20% of HCl(g) in absolute ethanol furnished

7-NH2-SPD tetrahydrochloride (13) as a white hygroscopic solid in 98% yield.

Synthesis of N', NA-Diethyl-7-aminospermidine

A completely different method was developed to prepare 7-NH2-DESPD. As

shown in Fig. 4-5, treating the commercially available N-ethyl-1,3-diaminopropane (14)

with mesitylenesulfonyl chloride under basic conditions resulted in N, N'-








bis(mesitylenesulfonyl)-N-ethyl-1,3-diaminopropane (15) in 80% yield (Bergeron,

2001b). This disulfonamide 15 was alkylated with 4-bromo-l,2-epoxybutane in 90%

yield in the presence of NaH and DMF. The alkylation occurred selectively at the

bromide end of 4-bromo-1,2-epoxybutane (Cruickshank, 1969). The disulfonamide

epoxide 16 was next reacted with N-ethylmesitylenesulfonamide anion (prepared from

the corresponding sulfonamide in NaH and DMF), to produce the tri-mesitylene

secondary alcohol 17, in 66% yield. The epoxide opening reaction occurred selectively

at the terminal carbon, which was a less sterically hindered electrophile, of 16. The

hydroxyl group of 17 was converted to a corresponding tosylate in 75% yield by reaction

with tosyl chloride (1.2 equiv) and pyridine in CH2Cl2 at room temperature. The tosylate

18 was reacted with potassium phthalimide in DMF at 85 oC to afford the phthalimide 19

in 90% yield. It was a surprising result since phthalimide anion was a poor nucleophile

and the electrophilic carbon in the tosylate 18 was very sterically hindered. Perhaps,

relief of the steric constraint was the driving force for this displacement reaction.

Attempting to convert the N-phthalimide protecting group in 19 to free amine using

hydrazine in refluxing ethanol failed. However, using NaBH4 in 2-propanol and water at

room temperature followed by acetic acid at 80 oC (Osby, 1984) afforded the amine 20 in

55% yield. Finally, the three Mts masking groups in compound 20 were removed under

reductive conditions, using 30% HBr in acetic acid and phenol in CH2C12, and the

corresponding polyamine bromide salt was converted to NM, AN-diethyl-7-

aminospermidine tetrahydrochloride (21) in 50% yield.








Synthesis of 6-Aminospermine

The starting material N-benzyl-l,3-diaminopropane (41), also commercially

available, was prepared previously in our laboratory (Bergeron, 1984). Diamine 41 was

first treated with mesitylenesulfonyl chloride in CH2Cl2 and aqueous NaOH to form the

bis(mesitylenesulfonamide) 22 in 60% yield. N, N'-Bis(mesitylenesulfonyl)-N-benzyl-

1,3-diaminopropane (22) anion, formed in the presence of NaH and DMF, then reacted

with 4-bromo-1,2-epoxybutane (Cruickshank, 1969) to afford epoxide 23 in 79% yield

(Fig. 4-6). The alcohol 24 was formed regiospecifically, simply due to steric effects, as

the sulfonamide anion 22 reacted with epoxide 23 in 65% yield. The hydroxyl group of

24 was converted to a good leaving group, the corresponding O-tosyl, in 88% yield, by

reaction with tosyl chloride (1.2 equiv) in the presence of pyridine in CH2C2 at room

temperature. The O-tosyl in the tosylate 25 was displaced by the phthalimide anion in

DMF at 85 oC to afford protected amine 26 in 54% yield. Conversion of the N-

phthalimide protecting group in 26 to the free amine was achieved successfully in 63%

yield by utilizing NaBH4 in 2-propanol and water at room temperature, followed by

treatment with acetic acid at 80 oC (Osby, 1984). The four Mts masking groups in amine

27 were removed under reductive conditions, using HBr in acetic acid and phenol in

CH2C12 followed by concentrated HCI in ethanol to afford N', NM2-dibenzyl-6-

aminospermine (28) as a pentahydrochoride salt. The two benzyl groups in pentaamine

28 were finally removed by hydrogenation at atmospheric pressure in the presence of 1 N

HCI and 10% palladium on activated carbon. The final racemic product 6-

aminospermine (29) was generated in 60% yield from compound 27 after two synthetic

steps.










HN NH2
\=N


.2 HCI


H
R. N N-R
H HN.R


a


H
RN N-R





R-=
0

R, = CHO, R2 = H or
R1 = H, R2 = CHO



b


H
R, N- N-R
H HN"R


\d


H2N NH2
NH2

5


.3 HCI


A Known Synthetic Route of Racemic 2-NH2-PUT.
Reagents: (a) RCl/Potassium acetate/H20/CH3CN,
(b) CH30H/Reflux, (c) H2/Pd, (d) 6 N HCI


Figure 4-1.











"N 0
OH H
30







H2N~ OOH
NH2 .2HCI


N3 H


b


c


NH2 H
32


ROOCHN COOH
NHCOOR


e ROOCHN"N=C=O

NHCOOR


R = iso-butyl 34


H2N NH2
NH2
.3 HCI


Figure 4-2.


g


ROOCHNN2
NHCOOR


A known Synthesic Route of (S)-2-NH2-PUT.
Reagents: (a) PPh3-HN3-DEAD, (b) H2/Pd, (c) 6 N HC1, (d) CICOOR,
(e) CICOOR, TEA/ NaN3/ Heat, (f) THF-H20/ LiOH, (g) 6 N HC1.


a










HNNH2
HNN 2 HC1

1


H
Boc N/- N-Boc
H HN.
Boc


a


H
Boc N .N-BOC
R N.

2

RI = CHO, R2= H or
R = H, R2 = CHO




b


H
c Boc..N N-Boc
H HNBoc

3


d


H2N NH22
NH2


S3 HCI


5

Figure 4-3. Synthesis of 2-aminoputrescine (2-NH2-PUT)
Reagents: (a) (Boc)20, KOAc/H20/CH3CN, r.t., 5 d, 66%,

(b) MeOH, reflux, 4 h, 91%, (c) H2, Pd-C, 55 oC, 17 h, 90%,
(d) 20% HC1/EtOH, r.t., 12 h, 92%.










HN / NH2 a
\N 2 HCI


Mts
Mts-N NH2
N 8


H
Mts-N^ N-Mts
\sN


Mts 0

Mts-7
\-N 0


H Boc H
N NH2
HN \ N- I Nii
N 3 HBr R 20 1Bo R =CHO,R2=H or
RI = H, R2 =CHO


Boc H

BN N N N-B
H HN-Boc 12

h


H
H2N N2NH2
NH2 4 HCI

13


Boc H
g BoC N N N.Boc
H HN-Boc
11


Synthesis of 7-aminospermidine (7-NH2-SPD).
Reagents: (a) MtsCl/CH2C12, 1 NNaOH, 0 oC to rt, 95%; (b) NaH/DMF,
N-(3-bromopropyl)phthalimide, 45%; (c) Hydrazine/EtOH, reflux, 56%;
(d) 30% HBr/AcOH, phenol, CH2Cl2, 66%; (e) (Boc)20, KOAc/H20,
CH3CN, rt, 73%; (f) MeOH, reflux, 95%; (g) H2, Pd-C, EtOH, 55 0C,
12h, 95%; (h) 20% HCl/EtOH, 12h, r.t., 98%.


Figure 4-4.









a


H2N- N"-


Mts OH

Mts Mts
17


Mts OTs

Mts Mts


H NH2

H H
4 HCI


HN ~N"
Mts Mts


0

Mts Mts
16


.-,


e


Mts.OMN 0

M.ts Mts


Mts NH2

MAts Mts
20


Synthesis of NI,NV-diethyl-7-aminospermidine
Reagents: (a) MtsCl, CH2C12, 1 N NaOH, 80%; (b) NaH/DMF,
4-Bromo-1,2-epoxybutane, rt, 90%; (c) NaH/DMF/ CH3CH2NH-Mts,
70 0C, 20 h, 66%; (d) TsCl, CH2C12, Pyr, rt, 20 h, 75%; (e) Potassium
Phthalimide, DMF, 85 0C, 20 h, 90%; (f) NaBH4/2-propanol/H20,
rt, 20 h then AcOH, 80 oC, 20 h, 55%; (g) 30% HBr/AcOH, phenol,
CH2C12, 0 oC to rt, 20 h, then conc. HCl/EtOH, 50%.


Figure 4-5.








H2N-N- a HNaN"
H Mts Mts
41 22

b

0
Mts= -I \ / N N
Mts Mts
23

/C

Mts Mts OH
NN N N N -
24Mts Mts



Mts Mts OTs
NNN NN-N
Mts Mts
25
e




SMts Mts N

I I
26 Mts Mts

Figure 4-6. Synthesis of 6-aminospermine (continued on next page)












Y Mts Mts N


26 Mts Mts
f



7 Mts Mts NH2
2N7N N"'t N
27 Mts Mts k


H H NH2

28 H H 5 HCI

h



H NH2
H2N N N'NH2 5 HCI

29 H


Synthesis of 6-aminospermine (continued)
Reagents: (a) MtsCl/ NaOH/ CH2CI2, 60%; (b) NaH/ DMF/ rt, then 4-bromo-
1,2-epoxybutane, 79%; (c) 22, NaH/ DMF/ 85 OC, 65%; (d) TsCl/ Pyr/
CH2C12, 88%; (e) Potassium phthalimide/ DMF/ 90 OC, 54%; (f) NaBH4/H20/
2-propanol, then acetic acid, 80 0C, 63%; (g) HBr/ acetic acid/ Phenol/
CH2C12, then cone. HC1; (h) H2/ 10% Pd-C/ 55 C, 60% from 27.


Figure 4-6.













CHAPTER 5
BIOLOGICAL ACTIVITIES OF AMINOPOLYAMINES IN P. AERUGINOSA AND
L1210 CELLS: RESULTS AND DISCUSSION

Microbial Studies

While a great deal of information is known about the metabolism of polyamine

analogues in various eukaryotes, little is known how these analogues are managed in

prokaryotes. In this study, Pseudomonas aeruginosa was used as a prokaryotic model for

the evaluation of the impact of some aminopolyamines on the bacterial polyamine

metabolism. The impacts on bacterial polyamine contents and the processing of

aminopolyamine by the bacteria were assessed. In addition, the effects of each

aminopolyamine on bacterial growth were also measured.

Impact on Bacterial Growth

Bacterial turbidity was monitored by measuring optical density at a wavelength of

660 nm (OD660). Since OD660 only reflected the bacterial protein levels but not the

viability, colony forming units (CFU) were also determined.

Bacterial turbidity (OD0o)

Various aminopolyamines and hydroxypolyamines were tested for their impacts on

bacterial growth. In the control experiment, bacteria were grown in nutrient broth

without addition of any drug. The bacterial turbidity was determined by optical density

at 660 nm (OD660). At a high concentration of 1.0 mM, similar to 2-OH-PUT and 7-OH-

SPD, 2-NH2-PUT and 7-NH2-SPD slightly interfere with the bacterial growth by








stationary phase (22-28 h). In the control growth, the log phase started at 11 h while in

the case of 7-OH-SPD and 7-NH2-SPD, it started at 15 h (Fig. 5-1). Similar growth

retardation activities of 7-OH-SPD and 7-NH2-SPD were observed until late log phase

(t = 19 h) when the growth curves started to diverge. By stationary phase (t = 24 h),

7-OH-SPD reduced the bacterial growth to about 92% (OD660, 7-OH-SPD = 1.2 versus OD660,

control = 1.3) while 7-NH2-SPD reduced the bacterial growth to about 73% (OD660, 7-NH2-SPD

= 0.95 versus OD660, control = 1.3). Since 7-NH2-SPD showed a slightly better inhibitory

effect on bacterial growth, we decided to examine further the activity of this

aminopolyamine in terms of dose dependence. At the concentration range of 0.05-0.2

mM, 7-NH2-SPD had no significant impact on the lag time of the bacterial growth curves.

However, at the time when the bacteria reached stationary phase, 0.05 mM 7-NH2-SPD

reduced the bacterial growth to about 93%, 0.1 mM reduced the growth to 85%, and 0.2

mM reduced the growth to 80% of control. At high concentrations such as 0.5 mM and

1.0 mM, this aminopolyamine reduced the bacterial growth to as low as 70% of control.

As the exposure concentration of 7-NH2-SPD increased, the lag time (the amount of time

the bacteria took to reach logarithmic phase) also increased (Fig. 5-2). In this particular

experiment, the untreated bacteria had a lag time of 8 h while 0.5 mM 7-NH2-SPD

increased the lag time to about 11 h and 1.0 mM increased it to 14 h.

Different concentrations (0.1 mM to 1.0 mM) of each of the two diethyl

analogues, 7-NH2-DESPD and 6-NH2-DESPM, were also tested for their ability to

interfere with bacterial growth. Even at high concentrations, both of these terminally

diethylated aminopolyamines had no significant impact in reducing bacterial growth by








stationary phase (22-28 h). However, at a concentration of at least 0.5 mM, there was a

4-6 hour delay before the logarithmic phase started (Fig. 5-3, 5-4).

Colony forming unit (CFU)

Since the OD660 does not reflect the amount of viable bacteria, we would like to

look at the CFU. Colony forming units were determined during the course of growing P.

aeruginosa in the presence or absence of drugs (aminopolyamines). At each time point,

besides recording OD660, bacterial counts were also performed by serial dilution of the

bacterial culture to 10-, 10"8, and 10-9 and spreading these diluted culture solutions on

nutrient agar plates. Similar to OD660 measurements, at a high concentration of 1.0 mM,

6-NH2-SPM, 7-NH2-DESPD, and 6-NH2-DESPM individually had no significant impact

on bacterial growth (Fig. 5-5). However, at a concentration of 1.0 mM, 7-NH2-SPD was

able to reduce the CFU by two logarithmic units (Fig. 5-5). It was interesting that in the

presence of 7-NH2-SPD (1.0 mM), no significant retardation in CFU occurred until

shortly after the bacteria reached stationary phase (t = 19 h). In the corresponding OD660

growth curve (Fig. 5-6), 7-NH2-SPD (1.0 mM) also showed the greatest impact on

bacterial growth although it was a moderate effect. The result from OD660 growth studies

were consistent with CFU studies both of which indicated that 7-NH2-DESPD, 6-NH2-

SPM, and 6-NH2-DESPM had no significant impact on bacterial growth (by stationary

phase). Since 7-NH2-SPD at 1.0 mM had some significant effects on the bacterial cell

division, we further investigated the inhibitory activity of this aminopolyamine. Bacteria

were incubated with 7-NH2-SPD at various concentrations (0.01-0.5 mM) and CFU was

performed during the course of bacterial growing (Fig. 5-7). At concentrations of 0.01

mM and 0.1 mM, 7-NH2-SPD showed unnoticeable impact on bacterial CFU. However,








at 0.5 mM, a reduction of CFU by one logarithmic unit was observed. Thus, at a

concentration of at least 0.5 mM, 7-NH2-SPD significantly reduced the cell division

ability of P. aeruginosa shortly after the bacteria reached stationary phase. The impact of

each aminopolyamine on polyamine metabolism of P. aeruginosa is more interesting as

discussed in the next section.

Processing of Aminopolyamines by P. aeruginosa

As shown in Fig. 5-8, when P. aeruginosa was exposed to 7-NH2-SPD at a

concentration of 1.0 mM and harvested at early log phase (15-16 h), the intracellular level

of 7-NH2-SPD was three times that of 2-NH2-PUT. When harvested at mid log phase

(19-20 h) or stationary phase (26 h), 7-NH2-SPD level was reduced to 75% of the 2-NH2-

PUT level. However, in a separate experiment, the bacteria was grown in various

concentrations (0.05 0.5 mM) of 7-NH2-SPD and harvested at stationary phase, the

intracellular 7-NH2-SPD was observed to be 33% of the intracellular 2-NH2-PUT (Fig.

5-9). Thus, the conversion of 7-NH2-SPD to 2-NH2-PUT in P. aeruginosa exposed to

7-NH2-SPD was not completed until the bacteria reached stationary phase. In the control

untreated bacteria, PUT and SPD levels were 15-20 nmole/OD unit and 4-6 nmole/OD

unit, respectively but when exposed to 0.05 mM of 7-NH2-SPD, these natural polyamine

levels reduced to 5-7 nmole/OD unit and < 1 nmole/OD unit, respectively while the

intracellular 2-NH2-PUT and 7-NH2-SPD levels were 3-4 nmole/OD unit and < 1

nmole/OD unit, respectively. At an exposure concentration of 0.2 mM, the intracellular

levels of PUT and SPD were further reduced to < 2 nmole/OD unit. At 0.5 mM, these

natural polyamines were reduced to < 1 nmole/OD unit while 2-NH2-PUT and 7-NH2-

SPD continued to increase to 45-50 nmole/OD unit and 14-16 nmole/OD unit,








respectively. It appeared that at the exposure concentration range of 0.05- 0.5 mM,

conversion from 7-NH2-SPD to 2-NH2-PUT occurred to an extent that resulted in 2-NH2-

PUT and 7-NH2-SPD at a 3:1 molar ratio. On the other hand, it is interesting to note that

at a concentration of 1.0 mM, a large amount of 7-NH2-SPD still remained unprocessed

in the bacteria (Fig 5-8, growth phases 2 and 3 on horizontal axis). An investigation was

then carried out to study how P. aeruginosa process this aminopolyamine in a timely

manner. An experiment was performed in which the bacteria were exposed to 1.0 mM

7-NH2-SPD and analyzed for polyamine contents at different time points. At 10 h, which

is the beginning of the logarithmic phase, the intracellular levels of 7-NH2-SPD and 2-

NH2-PUT were about 65 and 25 nmole/OD unit, respectively while the levels of SPD and

PUT were reduced to 2 and 8 nmole/OD unit, respectively (Fig. 5-10). The conversion

from 7-NH2-SPD to 2-NH2-PUT continued to proceed as a function of time. As time

progressed, the levels of 2-NH2-PUT increased, while 7-NH2-SPD, PUT, and SPD

decreased. When bacteria reached mid log phase (t = 14 h), the intracellular levels of

7-NH2-SPD decreased to about 40 nmole/OD unit while 2-NH2-PUT increased to about

55 nmole/OD unit. These results clearly revealed that there was a catabolic conversion

from 7-NH2-SPD to 2-NH2-PUT, and this conversion plateaued as the bacteria progressed

through stationary phase. At 26 h (the end of the stationary phase), the intracellular

levels of 7-NH2-SPD reduced to about 34 nmole/OD unit and 2-NH2-PUT increased to

about 64 nmole/OD unit while SPD and PUT were barely detectable. Note that at high

exposure concentration, i.e., 1.0 mM of 7-NH2-SPD, the bacteria were not able to further

convert 7-NH2-SPD to 2-NH2-PUT. Besides, the intra-bacterial levels of PUT and SPD

were almost completely depleted. The reduction in CFU counts was also observed at








these time points as discussed earlier. Perhaps the complete depletion of intracellular

PUT and SPD prevented the bacteria from normal activities such as cell division. Unless

specified, P. aeruginosa in all experiments were harvested at stationary phase.

In P. aeruginosa exposed to 7-NH2-SPD, the intracellular levels of 2-NH2-PUT

and 7-NH2-SPD increased while the endogenous levels of PUT and SPD decreased in a

dose-dependent manner (Fig. 5-9). When exposed to 7- NH2-SPD at a concentration of

0.5 mM, a nearly complete depletion of PUT and SPD was observed and these bacterial

natural polyamines were replaced by 2-NH2-PUT and 7-NH2-SPD at a 3:1 molar ratio.

These results indicated that 7-NH2-SPD was subjected to the SSAT/PAO degradation

process taking place within P. aeruginosa. The polyamine catabolic enzyme SSAT must

have first transferred an acetyl group from acetylCoA to the amine of the aminopropyl

moiety of 7-NH2-SPD. The resulting N'-acetyl-7-NH2-SPD was then oxidized by PAO

and subsequently hydrolyzed to 2-NH2-PUT plus 3-acetamidopropanal. In a separate

experiment when P. aeruginosa was exposed to 2-OH-PUT, elaboration into 7-OH-SPD

occurred, although only a relatively small amount of 7-OH-SPD was measured by HPLC.

A similar scenario occurred with 2-NH2-PUT which was elaborated into a small amount

of 7-NH2-SPD in P. aeruginosa. Poor substrate affinity for spermidine synthase of

2-OH-PUT and 2-NH2-PUT may account for these observations. In other words, the

bacteria polyamine biosynthetic enzyme SPD synthase does not effectively utilize the

two substrates 2-OH-PUT and 2-NH2-PUT to biosynthesize the corresponding

hydroxyspermidine and aminospermidines. When P. aeruginosa was exposed to

7-NH2-SPD, one would anticipate the absence or a very small amount of 6-NH2-SPM in

the bacterial intracellular polyamine pool since the bacteria do not have spermine








synthase. As expected, polyamine pool analysis showed no formation of 6-NH2-SPM

from 7-NH2-SPD in P. aeruginosa. In a separate experiment when the bacteria were

grown in the presence of 6-NH2-SPM, significant levels of 7-NH2-SPD and 2-NH2-PUT

were detected. It is interesting that P. aeruginosa was not able to biosynthesize

6-NH2-SPM from 7-NH2-SPD, but it was able to utilize 6-NH2-SPM and catabolically

process this exogenous aminopolyamine, likely via the SSAT/PAO pathway, to produce

7-NH2-SPD and 2-NH2-PUT.

Expected polyamine catabolism processes such as deethylation and SSAT/PAO

pathway have occurred in P. aeruginosa when the bacteria were exposed to either

7-NH2-DESPD or 6-NH2-DESPM (Fig. 5-11). Common metabolites, including N'-ethyl-

7-aminospermidine (7-NH2-MESPD), 7-NH2-SPD, and 2-NH2-PUT, were detected by

HPLC analyses.

Impact on the Total Intracellular Charges in P. aeruginosa

Previous studies revealed that there was a conservation of charge with respect to

total amine equivalence in the cells treated with various spermidine and spermine

analogues (Bergeron, 1997). After 48 h of exposure to an aminopolyamine, the total

intracellular charge was calculated by adding the charges of the natural polyamines, the

aminopolyamine and its metabolites. For example, each equivalent of PUT is associated

with two equivalents of cationic charges, each equivalent of SPD with three, and each

equivalent of SPM with four. Each aminopolyamine has an additional amino group

compared to its parent natural polyamine. Thus, if all nitrogen atoms in amino-

polyamines are protonated at physiological pH, each equivalent of 2-NH2-PUT will be

associated with three equivalents of cationic charge, each equivalent of 7-NH2-SPD or








7-NH2-DESPD with four, and each equivalent of 6-NH2-SPM or 6-NH2-DESPM with

five.

The total intracellular charge was not conserved in P. aeruginosa when the

bacteria were exposed to each aminopolyamine. In the untreated bacteria, the total

charge was about 112.24 + 15.36 nanoequiv/OD unit while in the treated bacteria, the

total charge increased significantly (Table 5-1). The larger charge amounts (Table 5-1,

middle column) were calculated based on the assumption that all nitrogen atoms of the

aminopolyamines were protonated at physiological pH, and the smaller charge amounts

(Table 5-1, far right column) were estimated assuming that the last nitrogen of each

aminopolyamine was not protonated. Thus, when the bacteria were incubated with

7-NH2-SPD (1.0 mM) for 22-24 h (stationary phase), the total intracellular charge

increased to 169.94-272.87 nanoequivalent/OD unit (1.5-2 fold of control) (Table 5-1). It

appeared that the bacteria can tolerate a high amount of charge without disrupting their

growth. There was an exception in the case of 7-NH2-SPD (1.0 mM) in which the

bacterial growth was reduced by two logarithmic units at the stationary phase. Our result

clearly indicated that the accumulation of charge was not responsible for this growth

reduction. This rationale holds true because the bacteria survived without any significant

growth reduction when exposed to other aminopolyamines (7-NH2-DESPD and 6-NH2-

DESPM), at which the total intracellular charge was markedly elevated (Table 5-1).

Eukaryotic Studies

The murine LI 210 cell is routinely used to study polyamine metabolism. In this

model, a great deal of information is available on the key enzymes involved in the

biosynthetic pathway as well as in the processing of polyamine analogues. The effects on








cell growth in addition to the impact on polyamine metabolism of each aminopolyamine

were measured. Results from 6-NH2-DESPM studies (Bergeron, R. J.; Guangfei H.; and

Yao, H., unpublished) were compared with the results from studies of other amino-

polyamines in this dissertation.

ICso values in L1210 Cells

As shown in Table 5-2, 2-NH2-PUT, 7-NH2-DESPD and 6-NH2-SPM had no

significant impact on the growth of L1210 cells. These three aminopolyamines had 48-h

IC50 values of 100 pM and greater. At 96 h, 7-NH2-DESPD and 6-NH2-SPM, both with

IC50 values of 6 and 10 IM, respectively, were much more active than 2-NH2-PUT

although this effect on cellular growth was still very moderate. At 48 h, 7-NH2-SPD was

almost twice as active as its diethyl analogue, 7-NH2-DESPD, but at 96 h their activities

were similar. The aminopolyamine 6-NH2-DESPM had a 48-h IC50 of 9 pM and a 96-h

ICso of 0.35 uM (Bergeron et al, unpublished). Among all aminopolyamines studied

(Table 5-2), 6-NH2-DESPM was the most active analogue in reducing the cell growth.

Competitive Uptake Determination in L1210 Cells

The ability of 2-NH2-PUT, 7-NH2-SPD, 6-NH2-SPM, and 7-NH2-DESPD to

compete with radiolabeled SPD for uptake was evaluated (Table 5-2). As predicted,

2-NH2-PUT had a Ki value of > 500 upM. The diethyl analogue 7-NH2-DESPD also had

a poor uptake affinity with a Ki value of 130 ptM while the free amine parent (7-NH2-

SPD), with a Ki of 84 jiM, was a more effective competitor. A similar trend was seen in

which 6-NH2-SPM (Ki = 21 pM) was better than 6-NH2-DESPM (Ki = 33.7 PM)

(Bergeron et al, unpublished) in competing with SPD for uptake. The general trend is

that the terminally diethylated aminopolyamines have higher Ki values than the








unethylated aminopolyamines and are thus less easily taken up by the cells. In addition,

the aminospermine analogues have lower Ki values than the aminospermidine analogues

(6-NH2-SPM versus 7-NH2-SPD; 6-NH2-DESPM versus 7-NH2-DESPD). Thus, the

aminospermines, namely 6-NH2-SPM and 6-NH2-DESPM, are more easily taken up by

the cell compared to the aminospermidines, namely 7-NH2-SPD and 7-NH2-DESPD.

Bergeron R. J. and coworkers have shown that charge is critical to cellular

recognition of the polyamine analogues at the transport level. Factors that influence the

"polyamine recognition" by the uptake apparatus are (1) the number of nitrogens in the

polyamine analogue, (2) the spacing between the nitrogens, and (3) the steric effects of

substituents in proximity to the nitrogens. Studies in Bergeron's laboratory revealed that

tetraamines, e.g., NI,N 2-diethylspermine (DESPM) and N,N'-bis(4-piperidinylmethyl)-

1,4-diaminobutane [PIP (4,4,4)], both of which form tetracations at physiological pH,

compete well with SPD for uptake (Bergeron, 1995). The Ki values for DESPM and PIP

(4,4,4) are 1.6 pM and 4.9 pM, respectively. The triamines, e.g., N',N-

dimethylspermidine (DMSPD) and homospermidine (HSPD), both of which form

trications at physiological pH, also compete well with SPD for the polyamine transport

apparatus. These two triamines have competive Ki values of 5.1 gpM (DMSPD) and 3.4

pAM (HSPD). It was concluded that as long as a polyamine analogue has at least +3

charges separated by 3 or 4 methylene groups, it will be "recognized" by the polyamine

transport apparatus. On the other hand, dicationic polyamine analogues such as NN12-

bis(2,2,2-trifluoroethyl)spermine (FDESPM) (Ki value of 285 pM), or PUT (Ki > 500

pM), competes poorly with SPD (Bergeron, 1995).








As mentioned earlier, 2-NH2-PUT (Ki > 500 jpM) had a poor affinity for the

polyamine uptake apparatus. Although it is a triamine, 2-NH2-PUT is more likely present

as a dication at physiological pH. Thus, similar to FDESPM and PUT, 2-NH2-PUT

competes poorly with SPD for the polyamine uptake apparatus. The observed increase in

Ki values when the aminopolyamines are terminally ethylated (Table 5-2) was probably

due to the steric effect of the terminal ethyl groups. The ethyl groups interfere with the

interaction between the protonated terminal nitrogen cations and the biological

counteranions in the transport apparatus, thus reducing the binding affinity of the

polyamine analogue.

Impact on ODC and AdoMetDC Activities

The effect of the polyamine analogues on ODC and AdoMetDC is fairly rapid

(Porter, 1987 and Porter, 1990). For example, the induced reduction from DESPM on

ODC activity plateaued at 4 h (Porter, 1987), and AdoMetDC at 6 h (Porter, 1990). On

the basis of these studies, the impact of aminopolyamines on ODC and AdoMetDC were

evaluated at 4 h and 6 h, respectively.

At a concentration of 1 jiM, 7-NH2-SPD reduced ODC activity to 72% of control

and the corresponding diethyl analogue, 7-NH2-DESPD, to 79% (Table 5-3). It appeared

that the parent aminospermidine and its diethyl analogue had similar impacts on ODC

activity. On the other hand, the diethyl aminospermine analogue, 6-NH2-DESPM, was

significantly more active than the corresponding parent aminospermine, 6-NH2-SPM,

with reduction to 19% versus 52% of control. Both aminospermines, 6-NH2-SPM and

6-NH2-DESPM, were more active in reducing ODC activity than the aminospermidines,

7-NH2-SPD and 7-NH2-DESPD. A similar trend in reduction of AdoMetDC activity was








observed in which the aminospermines were more active than the aminospermidines

(Table 5-3). The aminopolyamine 6-NH2-DESPM was more active than its parent

aminospermine, 6-NH2-SPM, in reducing AdoMetDC activity. At a concentration of 1

utM, 6-NH2-DESPM reduced AdoMetDC activity to 53% of control, while 6-NH2-SPM

reduced enzyme activity to 70% of control. The two aminospermidines 7-NH2-SPD

(reducing AdoMetDC activity to 81%) and 7-NH2-DESPD (reducing AdoMetDC activity

to 86%) had similar effects on the reduction of AdoMetDC activity.

Impact on SSAT Activity

At a concentration of 10 pM for 48 h, each of the following aminopolyamines

stimulated SSAT activities: 7-NH2-SPD, 189%; 7-NH2-DESPD, 303%; 6-NH2-SPM,

117%; and 6-NH2-DESPM, 668% of control. It was noticeable that 7-NH2-DESPD was

slightly more active that its free amine parent (7-NH2-SPD) in up-regulating the SSAT

while 6-NH2-DESPM was noticeably more active than its corresponding parent 6-NH2-

SPM. The general trend observed was that the terminally diethylated aminopolyamines

were more effective than their corresponding parent aminopolyamines in stimulating the

activity of SSAT.

Impact on Polyamine Pools and Metabolism of L1210 Cells

When the L1210 cells were exposed to 7-NH2-SPD, it appeared that there was

only a small amount of catabolic conversion to 2-NH2-PUT (29-35 nmol/OD unit) as a

result of SSAT/PAO activities. There was a significant new peak elutedd after spermine)

in the HPLC chromatograms of the L1210 cells which increased in a dose-dependent

manner. This suspicious peak was later shown to correspond to 6-NH2-SPM, formed by

the aminopropylation of 7-NH2-SPD. While the control cells did not have any of the








aminopolyamines (Fig. 5-12), the cells incubated with 60 itM 7-NH2-SPD for 48 h had

6-NH2-SPM with an area under the curve of about 10% of the SPM area (Fig. 5-13).

When the cells were incubated with 300 iM 7-NH2-SPD, 6-NH2-SPM achieved a

concentration of 25-30% of SPM (Fig. 5-14).

When cells were treated with 60 uM of 7-NH2-SPD for 48 h, intracellular PUT,

SPD, and SPM levels decreased to 35%, 34%, and 77% of control levels, respectively

(Table 5-4). At the same exposure concentration, the intracellular level of 7-NH2-SPD

was about 2500 pmole/106 cell, which was 2.5 times of that of SPD. This indicated an

efficient cellular uptake of 7-NH2-SPD even though 7-NH2-SPD showed a poor affinity

(Ki = 84 pM) for the polyamine transport apparatus (Table 5-2).

When LI1210 cells were exposed to 100 pM of 7-NH2-DESPD for 48 h, a

significant level of the metabolite Nl-ethyl-7-aminospermidine (1765 pmol/106 cells) was

detected; only a relatively small amount of 7-NH2-SPD (7 pmol/106 cells) was present in

the intracellular polyamine pool. At a higher exposure concentration (500 pM), the level

of Nl-ethyl-7-aminospermidine increased to 2700 pmole/106 cells (1.5 fold) and 7-NH2-

SPD to 12 pmole/106 cells. The aminopolyamine 7-NH2-DESPD must have undergone

NA-deethylation to form N'-ethyl-7-aminospermidine. A second deethylation then

occurred on Nl-ethyl-7-aminospermidine to produce 7-NH2-SPD (Fig. 5-15). At a

concentration of 100 pM, 7-NH2-DESPD reduced intracellular PUT, SPD and SPM in

treated cells to 56%, 29% and 60% of control, respectively while at 500 pM of the same

exposure analogue, PUT, SPD and SPM levels were reduced to 43%, 18% and 49%,

respectively (Table 5-5).








Incubation of L1210 cells with 10 utM or 50 pM of N', N'2-diethyl-6-

aminospermine (6-NH2-DESPM) for 48 h also resulted in the formation of two

metabolites, N'-ethyl-7-aminospermidine and 7-NH2-SPD although the intracellular

levels of these metabolites were much lower (Bergeron, R. J.; Guangfei, H.; Yao, H.,

unpublished). In the case of smaller exposure concentration, N'-ethyl-7-amino-

spermidine was detected at a level of 54 pmol/106 cells and 7-NH2-SPD at 21 pmol/106

cells. At the higher exposure concentration, these metabolites were found at similar levels

(Table 5-6). First, 6-NH2-DESPM must have undergone N'-deethylation before any

further metabolism could occur. The resulting N12-ethyl-6-aminospermine then

underwent deaminopropylation by the SSAT/PAO polyamine degradation pathway to

produce N'-ethyl-7-aminospermidine (Fig. 5-16). Formation of 7-NH2-SPD was a result

of deethylation of the intermediate metabolite N'-ethyl-7-aminospermidine. At the 48-h

IC50 concentration of 10 pM, 6-NH2-DESPM reduced intracellular PUT, SPD, and SPM

of treated cells to 39%, 16%, and 54% of control, respectively (Table 5-6) (Bergeron, R.

J.; Guangfei, H.; Yao, H., unpublished). At five times the IC5o concentration, i.e. 50 pM,

6-NH2-DESPM reduced intracellular PUT, SPD, and SPM to 26%, 7%, and 32% of

control, respectively. These results indicated that 6-NH2-DESPM had a greater impact on

the polyamine pool of the L 1210 cells than 7-NH2-SPD and the terminally ethylated

analog, 7-NH2-DESPD.

Impact on Total Cellular Charges of L1210 Cells

Our results indicated that there was a slight impact on the total cellular charges

when L 1210 cells were exposed to each aminopolyamine (Table 5-7). The total charge in

untreated cells was 12.78 x 103 picoequivalent/106 cells. When exposed to 7-NH2-SPD








(60 /M) for 48 h, the total charge increased to the range 13.43 x 103 + 16.00 x 103

picoequivalent/106 cells. As the exposure concentration of 7-NH2-SPD raised five times

to 300 ^M, the total intracellular charge slightly increased to 14.79 x 103 + 18.45 x 103

picoequivalent/106 cells (1.2 1.5 fold of control values). Similar results were recorded

when cells were exposed to 6-NH2-DESPM, which caused the total cellular charge to

increase to 1.2 1.6 fold of control. In the case of 7-NH2-DESPD (at two different

exposure concentrations, 100 pM and 500 pM, for 48 h), the total cellular charge was

conserved. In order to maintain the balance of charge, the cell processed some portions

of the natural polyamines and exported them as it incorporated the aminopolyamine. It is

interesting to note that, when exposed to 7-NH2-DESPD, cells did not incorporate this

exogenous polyamine analogue beyond the point where the total cellular charge would be

disrupted.







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