Synthesis, Characterization, and Antimicrobial Activity of Water

366 Pages·2007·8.27 MB·English

Synthesis, Characterization, and Antimicrobial Activity of
Water-soluble, Tri-carboxylato Amphiphiles

Eko Winny Sugandhi

Dissertation submitted to the Faculty of Virginia Polytechnic Institute and
State University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

in

Chemistry

Richard D. Gandour, Chair
Paul R. Carlier
Brian E. Hanson
David G. I. Kingston
James M. Tanko

January 31, 2007
Blacksburg, Virginia

Keywords: multi-tailed, multi-headed, tri-carboxylato, dendritic,
amphiphile, solubility, microorganism, biological and intrinsic activity

Copyright 2007, Eko W. Sugandhi
Synthesis, Characterization, and Antimicrobial Activity of
Water-soluble, Tri-carboxylato Amphiphiles

Eko Winny Sugandhi

ABSTRACT

Many previous studies of biological activity in a homologous series of
amphiphiles have shown a cut-off effect, where the biological activity increases with an
increase in chain length, after which the activity plateaus or weakens. One factor
suspected to cause this problem is solubility issues. We have designed several series of
very hydrophobic, water-soluble amphiphiles to overcome this problem. Three
homologous series containing mobile hydrophobic moieties and two series of epimers
containing rigid cholestane moieties have been synthesized; the hydrophobic moiety is
connected to the first-generation, Newkome-type dendron via a ureido linker. We have
demonstrated that as tris(triethanolammonium) salts, these amphiphiles show excellent
solubility in water. The solubilities in aqueous solution of the three series containing
mobile hydrophobic moieties are 19,500 to 25,700 µM depending on the formula weight
of the homolog, while those containing rigid cholestane moieties are 18,900 and 17,400
μM.
Having eliminated the solubility issue, the antimicrobial activity against a broad
spectrum of microorganisms has been screened. We have demonstrated that the
antimicrobial activity depends on the amphiphile-series, species, chain-length, or epimer
specificities, as well as hydrophobicity. The one-tailed, tri-carboxylato amphiphiles are
generally better than the other series, with two exceptions. First, the two-tailed tri-
carboxylato amphiphiles, 3CUr1(11) and 3CUr1(12) , are more active against
2 2
Cryptococcus neoformans; in fact, both amphiphiles (MICs are 6.9 and 7.2 μM,
respectively) are considered to display good antifungal activity. Second, amphiphile
3CUr-β-cholestane, whose MIC is 27 μM, is more active against Staphylococcus
aureus. Overall, these new tri-carboxylato amphiphiles only exhibit moderate activity
with two promising leads.
Furthermore, we have demonstrated the intrinsic activity (MIC ) of the one-tailed,
0
tri-carboxylato amphiphile series (3CUrn) against Mycobacterium smegmatis. All the
MIC ‘s observed are at least 8-fold lower than the corresponding CMCs. Amphiphile
0
3CUr16 is the most active; the MIC is 100-fold smaller than the CMC. With this
0
consideration, we have suggested that the mechanism of action of the antimycobacterial
activity in amphiphile 3CUr16 is not related to detergency.
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ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my research advisor, Dr.
Richard D. Gandour for his guidance, ideas, encouragement, and continual patience
throughout the duration of my graduate years. His method of gentle guidance made this
very difficult time in my life a little less stressful. I also acknowledge my committee
members⎯Dr. Paul Carlier, Dr. Brian Hanson, Dr. David Kingston, and Dr. James
Tanko⎯for their advice and assistance.
I thank Dr. Joseph Falkinham, III, and Ms. Myra Williams for their generous
assistance in doing biological assays. Beginning with no experience in handling
microorganisms, I learned a great deal in preparing the media, growing the culture, safe
handling, storing, running the assays, and finally interpreting the results.
I thank Mr. Tom Glass and Mr. William Bebout for their help and support in the
analytical services, and Mrs. Carla Slebodnick for her help in the X-ray crystallography
services. Many thanks go to Mrs. Kay Castagnoli and Mrs. Janice McGinty for their
special friendship. Special thanks also to Dr. Yang Yanyan, and current Gandour group
members, especially André William, Richard Macri, Brett Kite, Shauntrece Hardrict, for
making my stay a most enjoyable one.
Finally I would like to thank my husband Ernest for his unconditional love and
sacrifices in order to make this work a reality. Also, my parents and the rest of my
family, who are always loving and supporting me in their characteristic style, deserve
more gratitude than I can ever express.
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DEDICATION

To my parents,
my husband Ernest, and my little Stanley.

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Table of Contents
List of Figures……………………………………………………………………………ix
List of Schemes…………………………………………………………………………..xi
List of Tables…………………………………………………………………………….xii
Chapter I Background of Amphiphiles and Their Antimicrobial Activity....................1
I.1 Introduction to Amphiphiles................................................................................1
I.2 Designing Amphiphiles as Antimicrobial Agents...............................................2
I.2.1 Detergency.....................................................................................................3
I.2.2 Hydrophilic Moiety of an Amphiphile..........................................................4
I.2.3 Hydrophobic Moiety of an Amphiphile.........................................................5
I.2.4 Amphiphiles of Interest..................................................................................6
I.3 Antimicrobial Properties of Amphiphiles............................................................7
I.3.1 Overview on Previous Studies in Gandour’s Laboratory..............................7
I.3.2 The Cut-off Effect........................................................................................10
I.4 Literature Review in Designing Amphiphiles of Interest..................................11
I.4.1 Multi-headed Amphiphiles..........................................................................12
I.4.2 Newkome Dendrons.....................................................................................13
I.4.3 Ureido Linker...............................................................................................15
I.4.4 One-tailed Tri-carboxylato Amphiphile......................................................18
I.4.5 Two-tailed Tri-carboxylato Amphiphile......................................................19
I.5 Cholestane-based Polyanionic Compounds Derived from Natural Product......22
I.6 Tri-carboxylato Amphiphile Containing Rigid Cholestane-based Hydrophobic
Moiety................................................................................................................26
I.7 Microorganisms in Antimicrobial Screening.....................................................26
I.7.1 Bacteria........................................................................................................27
I.7.2 Fungi............................................................................................................28
I.8 Summary............................................................................................................29
References for Chapter 1..................................................................................................29
Chapter II Synthesis of One-Tailed, Tri-headed Amphiphiles (3CUrn)......................39
II.1 Preparation of WeisocyanateTM (3)....................................................................40
II.2 Preparation of Alkan-1-amines..........................................................................41
II.2.1 Literature Review on Syntheses of Long-chain Alkan-1-amines (4)..........41
II.2.2 Literature Review on Syntheses of 1-Azidoalkanes (8)..............................43
II.2.3 Literature Review on Syntheses of 1-Methanesulfonyloxyalkanes (9).......46
II.2.4 Literature Review on Syntheses of 1-Bromoalkanes (11)...........................46
II.2.5 Preferred Starting Material⎯Alkan-1-ols (12) or 1-Bromoalkanes (11)....47
II.2.6 Synthesis of Long-chain Alkan-1-amines (4)..............................................47
II.3 Synthesis of the 3EUrn Series (2a−e)...............................................................50
II.4 Synthesis of the 3CUrn Series (1a−e)...............................................................51
II.5 X-ray Crystal Structure of Tri-tert-butyl Ester 3EUr16....................................51
II.6 CMCs (Critical Micelle Concentration) of Amphiphiles 3CUrn......................53
II.7 Experimental Procedures...................................................................................55
References for Chapter II..................................................................................................65
Chapter III Synthesis of Two-Tailed, Tri-headed Amphiphiles (3CUr(n) and
2
3CUr1(n) )..................................................................................................73
2
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III.1 The 3CUr(n) Homologous Series....................................................................74
2
III.1.1 Preparation of Secondary Amines...............................................................75
III.1.1.1 Literature Review on Syntheses of N-Alkylalkan-1-amines (4a, 4c, and
4e).......................................................................................................75
III.1.1.2 Literature Review on Syntheses of N-Alkylalkanamides (5).............77
III.1.2 Synthesis of N-Alkylalkan-1-amines (4a, 4c, and 4e).................................78
III.1.3 Synthesis of Two-tailed, Tri-headed 3CUr(n) Homologous Series..........79
2
III.2 The 3CUr1(n) Homologous Series..................................................................81
2
III.2.1 Preparation of Primary Amines (10a−f)......................................................82
III.2.1.1 Literature Review on Syntheses of 1-Alkylalkan-1-amines (10a−f)..82
III.2.1.2 Literature Review on Syntheses of Azidoalkanes..............................85
III.2.1.3 Literature Review on Syntheses of Methanesulfonyloxyalkanes.......86
III.2.1.4 Literature Review on Syntheses of Secondary Alkanols....................87
III.2.2 Synthesis of Amines (10a−f).......................................................................89
III.2.3 Synthesis of Two-tailed, Tri-headed 3CUr1(n) Homologous Series.........91
2
III.3 Comparison of Trends in Melting Temperatures (Tri-tert-butyl Esters vs Tri-
carboxylic Acids)...............................................................................................93
III.4 Comparison of NMR Spectra of 3EUr(n) vs 3EUr1(n) .................................94
2 2
III.5 Experimental Procedures...................................................................................97
References for Chapter III..............................................................................................115
Chapter IV Synthesis of Tri-headed Amphiphiles Containing Cholestane-based
Structure on the Hydrophobic Moiety.......................................................122
IV.1 The 3CUr-z-cholestane Series........................................................................123
IV.1.1 Preparation of 5α-Cholestan-3-amine (4a−b)...........................................125
IV.1.1.1 Literature Review on Syntheses of Amines (4a−b)..........................125
IV.1.1.2 Literature Review on Syntheses of 3-Azidocholestane (5a−b)........127
IV.1.1.3 Literature Review on Syntheses of 3-Methanesulfonyloxy-5α-
cholestane (6a–b)..............................................................................129
IV.1.1.4 Literature Review on Synthesis of 3α-Bromo-5α-cholestane (7)....129
IV.1.2 Synthesis of 5α-Cholestan-3-amines (4a−b).............................................131
IV.1.3 Synthesis of 3CUr-z-cholestane (1a−b)...................................................133
IV.1.4 X-ray Structure of 3EUr-α-cholestane (2a).............................................134
IV.2 The 3CUrnEs-z-cholestane Homologous Series............................................137
IV.2.1 Preparation of 3α- and 3β- Epimer of 5α-Cholestan-3-yl Aminoethanoate
(11a−b)......................................................................................................138
IV.2.1.1 Literature Review on the Synthesis of 5α-Cholestan-3α-yl
Aminoethanoates (11a−b)................................................................139
IV.2.1.2 Literature Review on the Synthesis of 5α-Cholestan-3α-yl
Azidoethanoate (12a−b)...................................................................140
IV.2.1.3 Literature Review on the Synthesis of 5α-Cholestan-3α-yl
Chloroethanoate (13a−b)..................................................................140
IV.2.2 Synthesis of 5α-Cholestan-3α-yl Aminoethanoate (11a−b).....................141
IV.2.3 Synthesis of 3CUr1Es-z-cholestane (9a−b)...............................................143
IV.3 Experimental Procedures.................................................................................144
References for Chapter IV..............................................................................................162
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Chapter V Antimicrobial Activity of Various Tri-headed Amphiphiles.....................168
V.1 Introduction to Biological Assays....................................................................168
V.1.1 Measurement of Antimicrobial Activity....................................................168
V.1.2 Goals..........................................................................................................169
V.2 Recent Studies of Solubility of One-tailed Tri-carboxylato Amphiphiles.......170
V.3 Results of Biological Assays...........................................................................171
V.3.1 Range of Concentration of Active Amphiphiles........................................174
V.3.2 The Most Active Amphiphiles within Each Homologous Series..............175
V.3.3 Various Specificities Patterns of Antimicrobial Activity..........................177
V.3.3.1 One- and Two-tailed Amphiphiles...................................................177
V.3.3.2 Cholestane-based Amphiphiles........................................................179
V.3.4 Comparing Hydrophobicity and Antimicrobial Activity...........................180
V.3.4.1 One- and Two-tailed Amphiphiles....................................................180
V.3.4.2 Cholestane-based Amphiphiles..........................................................182
V.4 Comparison with Prior Work...........................................................................182
V.5 Conclusions......................................................................................................184
V.6 Experimental Procedures.................................................................................185
V.6.1 Preparation of Tri-carboxylato Amphiphiles Solutions in Aqueous
Triethanolamine.........................................................................................185
V.6.2 Microbial Strains, Culture Conditions, and Preparations of Inocula for
Susceptibility Testing.................................................................................185
V.6.3 Measurement of MICs...............................................................................187
References for Chapter V................................................................................................188
Chapter VI Intrinsic Activity of the 3CUrn Homologus Series Against Mycobacterium
smegmatis...................................................................................................192
VI.1 The “inoculum effect”......................................................................................194
VI.2 Intrinsic Activity of Homologous Amphiphiles..............................................194
VI.3 Results and Discussions...................................................................................197
VI.3.1 MICs of the 3CUrn Series.........................................................................197
VI.3.2 Comparison with Previous Work...............................................................199
VI.3.3 Comparison of MICs and CMCs...............................................................199
VI.4 Conclusions......................................................................................................201
VI.5 Experimental Procedures.................................................................................201
VI.5.1 Microbial Strain, Culture Conditions, and Preparations of Inoculum for
Susceptibility Testing.................................................................................201
VI.5.2 Measurement of MICs...............................................................................202
References for Chapter VI..............................................................................................203
Chapter VII Accomplishments, Conclusions, and Future Work....................................205
VII.1 Accomplishments.............................................................................................205
VII.2 Conclusions......................................................................................................206
VII.3 Future Work.....................................................................................................207
References for Chapter VII.............................................................................................208
APPENDICES................................................................................................................209
VITA...............................................................................................................................354

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List of Figures

Figure I.1 Common representations of an individual amphiphile...............................1
Figure I.2 Representation of amphiphile aggregates...................................................2
Figure I.3 Mono-, di-, and tri-ionic amphiphiles.........................................................5
Figure I.4 Schematic representations of proposed multi-headed amphiphiles............7
Figure I.5 Plots of log MEC (M) vs chain length in spermicidal (left) and log MIC
(M) vs chain length in antifungal (right) assays of Z-n..............................8
Figure I.6 Plots of log MEC vs chain length in spermicidal assays of 1(p) and 2(p)10
Figure I.7 Various multi-headed amphiphiles with anti-HIV properties...................12
Figure I.8 Various Newkome-type dendrons (first and second generations) associated
with ferrocene, dansyl, pyrene, and viologen...........................................14
Figure I.9 Structures of one-tailed tri-carboxylato amphiphiles having amido and
carbamato linkers......................................................................................15
Figure I.10 Various compounds containing ureido functional groups........................17
Figure I.11 General representation and structure of one-tailed tri-carboxylato
amphiphiles...............................................................................................18
Figure I.12 Various fatty acids tested for skin penetration rates and skin irritation....21
Figure I.13 General representations of two-tailed, tri-headed amphiphile series........22
Figure I.14 Polyol and polyamine dendrimers associated with cholestane moieties..24
Figure I.15 General structures of cholestane-based, tri-headed amphiphile series.....26
Figure II.1 X-ray crystal structure of tri-tert-butyl ester 3EUr16, showing the packing
diagram and the intermolecular hydrogen bonding to water and to a
neighboring molecule. Hydrogen-bond Donor(D)–Acceptor(A) distances
and approximate ∠DHA: O(8)···N(2), 2.863(3) Å and 159.1°;
O(15)···O(1), 2.718(4) Å and 170(4)°; O(15)···O(3), 2.843(4) Å and
169(4)°; N(4)···O(15), 2.832(3) Å and 153.4°. The other nitrogens on the
ureido linker are just outside the range for significant hydrogen bonding.
N(1)···O(8), 3.201(3) Å and 145.6°; N(3)···O(15), 3.074(3) Å and 141.6°
...................................................................................................................53
Figure III.1 General representation of two-tailed, tri-headed amphiphiles..................73
Figure IV.1 Structural representations of 5α- and 5β-cholestane..............................122
Figure IV.2 General structures of tri-headed amphiphiles series containing cholestane
moiety.....................................................................................................123
Figure IV.3 X-ray crystal structure of 3EUr-α-cholestane (2a), showing the packing
and the intermolecular hydrogen bonding to ethanol. Hydrogen-bond
Donor(D)–Acceptor(A) distances and approximate ∠DHA: O(16)···O(1),
2.621(7) Å and 158.1°; O(15)···O(8), 2.625(6) Å and 161.0°; O(4)···O(16),
2.865(7) Å and 155(7)°; N(2)···O(15), 2.849(8) Å and 159.6°. The other
nitrogens on the ureido linker are just outside the range for significant
hydrogen bonding. N(3)···O(16), 3.035(7) Å and 141.6°; N(1)···O(15),
3.084(8) Å and 146.0°.............................................................................135
Figure V.1 Relationship between log MIC of the 3CUrn series vs chain length for E.
coli, M. smegmatis (left) and C. albicans, C. neoformans, and S. cerevisiae
(right). Error bars (not shown for clarity) are ± 0.3. Lines connecting
points are eye guides...............................................................................175
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Figure V.2 Relationship between log MIC of the 3CUr(n) and 3CUr1(n) series vs
2 2
chain length for C. neoformans (left) and C. albicans (right). Error bars
(not shown for clarity) are ± 0.3. Lines connecting points are eye guides
.................................................................................................................176
Figure V.3 Relationship between log MIC of one- and two-tailed amphiphiles vs logD
for M. smegmatis (left) and C. albicans (right). Error bars (not shown for
clarity) are ± 0.3......................................................................................181
Figure V.4 Relationship between log MIC and logD for C. neoformans (left) and S.
cerevisiae (right). Error bars (not shown for clarity) are ± 0.3...............182
Figure VI.1 Effect of initial cell density on inhibition of growth of M. smegmatis for
the 3CUrn series. Combined data of two separate assays⎯3.2 to 3.2 × 107
CFU/mL and 500 to 5.0 × 108 CFU/mL. Error bars (not shown for clarity)
are ± 0.3...................................................................................................197
Figure VI.2 Effect of chain length on inhibition of growth of M. smegmatis of the
3CUrn series as function of the initial cell density. Error bars (not shown
for clarity) are ± 0.3.................................................................................198
Figure VI.3 Comparison of log CMC and log MIC of the 3CUrn series at the highest
and lowest initial cell densities of M. smegmatis....................................200

x