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MOLECULAR DIODES by
Xin Zhou
B.Sc. Fudan University, 1983
A Dissertation Subm itted in P artial Fulfillment of the Requirem ents for th e Degree of
DOCTOR OF PHILOSOPHY
in the D epartm ent of Chemistry
We accept this th e sis as conforming to the required standard
Dr. T.M. Fyles, Supervisor (D epartm ent of Chemistry)
Dr. G.A. Poulton, D epartm ental M em ber (Departm ent of Chemistry)
_______________________________ Dr. C. Bohne, D epartm ental M em ber (Departm ent of Chemistry)
Dr. ^^w side Member (Depeirtment of Biochemistry and
Microbiology)
herm an, External Exam iner (University of B ritish Columbia)
© XIN ZHOU, 1997 University of Victoria
All rights reserved. D issertation m ay n o t be reproduced in whole or in part, by photocopying or other m eans, w ithout the permission of the author.
Supervisor: Dr. T hom as M. Fyles
ABSTRACT
The goals of th is project were to synthesize voltage-gated ion channels
based upon previously studied pore-formers an d to fu rth e r explore the
mechanism of ion tra n sp o rt w ith this type of pore-former.
The syntheses of bis-macrocyclic bola-am phiphiles started w ith two
different macrocycles prepared via a two-step cyclization from maleic
anhydride by reaction w ith 1,8-octanediol alone or w ith triethyleneglycol.
The macrocycles w ere th e n modified to a set of m ono-adducts and bis-adducts
by Michael addition of thiols (3-mercaptopropanol, 2-m ercaptoacetic acid, or
3-mercaptopropionic acid). The mercaptopropanol adduct was converted to a
mesylate and coupled w ith a carboxylate derivative to form a bis-macrocycle.
Repetitious gel perm eation chrom atography gave a bis-macrocycle bearing
only one head group, a carboxylate. The second head group was added via
Michael addition to give a bis-macrocyclic bola-am phiphile which could have
either the same h e a d groups or different head groups. Two sym m etrical
transporters were synthesized via another route: two macrocycles reacted
w ith 2-m ercaptoethyl sulfide to generate a bis-macrocycle, an d the same head
group was then sim ultaneously added to both ends to give a symm etrical
bola-amphiphile. T ransporters w ith different com binations of head groups
were synthesized to compare head group effects on cation tran sp o rt
properties, while different macrocycles were used in th e backbone of
their behaviors.
The second phase of this project investigated the tra n sp o rt properties
of candidates usin g pH -stat titratio n . The pH -stat titra tio n of bilayer
vesicles allowed determ ination of dynam ic transport properties: transport
rate, a p p a re n t kinetic order an d cation selectivity. Combined with
inform ation from p la n a r bilayer experim ents (done by D. Loock), it was found
th a t an asym m etrical bis-macrocyclic bola-amphiphile w ith a n acetate and a
succinate head group behaves as voltage-gated ion channel in planar
bilayers. An ion transport m echanism of the present system was proposed
which involves th e formation of active aggregates (probably dim ers or
ohgomers).
Examiners:
Dr. T.M. Fyles, Supervisor (D epartm ent of Chemistry)
Dr. G.A. Poulton, D epartm ental M em ber (Departm ent of Chem istry)
Dr. C. Bohne, D e p h it^ e n W M ember (D epartm ent of Chem istry)
Dr. J. AuSMf’O iit^ â é - M ^ b e r (D epartm ent of Biochemistry an d Microbiology)
TABLE OF CONTENTS
TITLE PAGE i
ABSTRACT ü
TABLE OF CONTENTS iv
LIST OF TABLES vü
LIST OF FIGURES vüi
LIST OF SCHEMES xü
LIST OF ABBREVIATIONS xüi
GLOSSARY OF BIOCHEMICAL TERMINOLOGY xiv
ACKNOWLEDGEMENTS xv
CHAPTER 1 INTRODUCTION 1
1.1 N atural ion channels 1
1.1.1 Ion channel proteins 1
1.1.2 Antibiotics, sim ple n a tu ra l ion channels 1
1.2 Overview of synthetic ion channels 6
1.3 The early explorations from Fyles’ group 17
1.4 The design of research ta rg e ts in this thesis 18
CHAPTER 2 SYNTHESIS 25
2 .1 Overview 25
2 .2 Syntheses of m acrocydes 25
2.3.1 Syntheses derivative of m acrocyde 8Trg 28
2.3.2 Coupling reaction to m ake bis-macrocydes 35
2.3.3 Syntheses of tran sp o rter candidates in the STrg series 44
2.4 Syntheses of transporter candidates in the 82 series 49
2.4.1 Synthesis of A82PA82A 49
2.4.2 Synthesis of Pa82PPA82P a 62
2.4.3 Syntheses of two redesigned transporter candidates
in the 82 series 72
2.5 “Second generation” in th e 8T rg series 80
2.6 Modification w ith bulky and/or hydrophilic head group 85
2.7 Sum m ary 89
CHAPTER 3 TRANSPORT MEASUREMENTS AND PROPERTIES
DISCUSSION 90
3.1 P reparation and characterization of unilam ellar vesicles 90
3.2 p H -stat titra tio n 91
3.3 Analysis of pH -stat resu lts 94
3.3.1 D ata processing and tra n sp o rt ra te 94
3.3 .2 T ransporters in the 8T rg series 95
3.3.2.1 Apparent kinetic o rder 97
3.3 2.2 T ransporter activities 100
3.3.2 3 Cation dependence 103
3.3.3 T ransporters in th e 82 series 104
3.3.3.1 A pparent kinetic order 106
3.3 3.2 C ation selectivity an d relative activity 107
3.3.3 3 Sum m ary for th e 82 series 110
3.4 The exploration of voltage-gated ion channel 110
3.4.1 The significant behaviors of S8TrgPA8TrgA in pH -stat
titra tio n 110
3.4.2 P lan a r bilayer experim ent 113
3.5 The proposed m echanism of ion tran sp o rt 118
3.6 Conclusion an d prospects 121
CHAPTER 4 EXPERIMENTAL 122
4.1 Synthesis 122
4.2 Molecular modeling 148
APPENDIX 1 VESICLE PREPARATION AND PH-STAT TITRATION 149
APPENDIX 2 SUPPLEMENTARY ^H AND « c NM R SPECTRA 153
LIST OF TABLES
T a b le 3.1 T ran sp o rt of allcali metal cation by candidates in the
STrg series 96
T a b le 3.2 The norm alized transport rate s for th e STrg series 102
T a b le 3.3 T ran sp o rt of alkali metal cation by candidates in the Sz series 105
LIST OF FIG U R ES
F ig u re 1.1 G ra m id d in A 3
F ig u re 1.2 A m photericin B 4
F ig u re 1.3 C urrent-voltage (I-V) curve for a la m e th id n in
diphytanoyl-phospatidyl choline (DiPhyPC) m em brane 6
F ig u re 1.4 Schem atic description of m echanism s of ion transporters 8
F ig u re 1.5 T abushi’s P-cyclodextrin ion channel 10
F ig u re 1.6 One of L ehn’s “bouquet” molecules 11
F ig u re 1.7 VoyeFs ion channel model 12
F ig u re 1.8 G hadiri’s cycHc peptide channel 13
F ig u re 1.9 Gokel’s trismacrocychc channel system 14
F ig u re 1.10 F uhrhop’s monolayer system 15
F ig u re 1.11 Kobuke’s ion p air system 15
F ig u re 1.12 Regen’s bolaphiles and bolaam phiphiles compounds 16
F ig u re 1.13 Tunnel-defined channels 18
F ig u re 1.14 C hannels formed by aggregate pores 19
F ig u re 1.15 Design proposal for pore form ation by aggregation of bola
am phiphiles 21
F ig u r e 2.1 S ynthetic strateg y for m aking tra n sp o rte r candidates 26 F ig u re 2.2 NMR (CDCb) spectra of A8TrgA (9) an d 8TrgA (10) 33 F ig u re 2.3 NMR (CDCb) spectra of A8TrgA (9) an d 8TrgA (10) 34
F igu re 2.4 The analytical GPC m easurem ents an d LSIMS spectra 38 F igu re 2.5 NMR (C D C b) of STrgPASTrgA an d STrgPASTrgAPSTrg 39 F igu re 2.6 NMR (CDCla) of STrgPASTrgA an d STrgPASTrgAPSTrg 40
F igu re 2.7 COSY NMR of STrgPASTrgA 41
F igu re 2.8 Heterocorrelation NMR of STrgPASTrgA 42 F igu re 2.9 'H NMR spectra of compound 13, 14, an d 15 47 F igu re 2.10 NMR spectra of compound 13, 14, an d 15 4S F igu re 2.11 NMR spectrum (CDCla) of ASzA (18) 52 F igu re 2.12 NMR spectrum (CDCI3) of ASzA (18) 53 F igu re 2.13 Negative LSEMS spectrum of S2PAS2A (19) 56 F igu re 2.14 NMR (CDCI3) spectrum of AS2PAS2A (20) 60 F igu re 2.15 "C NMR (CDCI3) spectrum of A82PAS2A (20) 61 F igu re 2.16 Negative LISMS spectrum of AS2PAS2A (20) 62 F igu re 2.17 NMR (CDCI3) spectra of compound 21 a n d 22 64 F igu re 2.18 NMR (CDCI3) spectra of compound 21 an d 22 65 F igu re 2.19 NMR spectrum of PaS2PPaS2P a (24) 70 F igu re 2.20 NMR spectrum of PaS2PPaS2P a (24) 71
F igu re 2.21 NMR spectrum of S2SUS2 (25) 75
F igu re 2.22 NMR spectrum of S2SUS2 (25) 76
F igu re 2.23 NMR spectra of 26 and 27 78
F igu re 2.24 ^^C NMR spectra of 26 and 27 79
F ig u r e 2.26 «C NMR (CDCI3) of SSTrgPASTrgA (28) 83 F ig u r e 2 .2 7 : N egative LSIMS of SSTrgPASTrgA (28) 84 F ig u re 3.1 Schem atic description o f p H -stat titratio n 92 F ig u r e 3.2 A typical pH -stat titra tio n curve of compound 13 93 F ig u re 3.3 F irs t order analysis for compound 13 95 F ig u re 3.4 The dependence of tra n s p o rt on the concentration of
ASTrgPA8TrgA (13) 97
F ig u re 3.5 The in itial ra te of tra n s p o rt as a function of concentration
of 13 98
F ig u re 3.6 The dependence of tra n s p o rt on the concentration of
SSTrgPASTrgA (28) 100
F ig u r e 3.7 The cation selectivity o f th e STrg series 103 F ig u re 3.8 The dependence of tra n s p o rt on the concentration o f 24 106 F ig u re 3.9 The low energy conform ations of ASzSuSzA (26) an d
AS2PAS2A (20) 108
F ig u re 3.10 C ation selectivity a n d tra n sp o rt activity in the 82 series 109 F ig u re 3.11 The cation effect of SSTrgPASTrgA (28) 112
F ig u re 3.12 Schem atic description of p la n a r bilayer experim ent 114 F ig u re 3.13 The typical conductance recording of ASTrgPASTrgA (13) 114 F ig u re 3.14 The macroscopic current-voltage response of
G8TrgPA8TrgA(15) 115
F ig u re 3.15 T he tim e-dependence recording (A) and macroscopic cu rren t
F ig u re 3.16 The proposed tra n sp o rt mechanism s of th e STrg series 119
F ig u re A.1 NMR spectrum of 82PA82A (19) 153
F ig u re A.2 NMR spectrum of 82PA82A (19) 154
F ig u re A.3 NMR spectrum of 82P P a8zPa (20) 155
LIST OF SCHEM ES
Schem e 2.1
Syntheses of macrocodes 27Schem e
2.2 Form ation of 8Trg 28Schem e
2.3 Synthesis of mono-adduct of th e m acrocode 2 29Schem e
2.4 Syntheses of new adducts 9 and 10 30Schem e
2.5 The carboxylate coupling reaction w ith mesylate 36Schem e 2.6
Syntheses of transporter candidates in 8Trg series 45Schem e
2.7 S ynthetic route of 82POMS (17) 49Schem e
2.8 Synthesis of A82A (18) 50Schem e
2.9 Syntheses of 82PA82A (19) and A82PA82A (20) 55Schem e
2.10 The proposed mechanism for intram olecular retro-M ichaeladdition 57
Schem e
2.11 Synthesis of P a82Pa (21) 63Schem e
2.12 Synthesis of P a82PPa82P a (24) 68Schem e
2.13 Retro-Micdiael addition for P a82P a (21) 69Schem e
2.14 The synthetic routes for A82SU82A (26) and N82SU82N (27) 73Schem e
2.15 Synthesis of S8TrgPA8TrgA (28) 81Schem e
2.16 Synthetic route for m aking dendrim eric type of head group 85Schem e
2.17 Synthetic route for making a 18C6 derivative head group 87LIST OF ABBREVIATIONS
DMSG dimethylsiilfoxide
DMF N,N-dim ethylformamide
THF te trah y d ro fu ran
FCCP carbonyl cyanide 4-(trifluoromethoxy)phenylliydrazone
TLC th in layer chrom atography
HPLC h ig h p ressure liquid chrom atography
GPC gel perm eation chromatography
NMR nu clear m agnetic resonance
s singlet
tri trip le t
m m ultiplet
hr broad
COSY Correlation Spectroscopy
HETCOR H ETeronuclear sh ift CORrelation spectroscopy
LSIMS h q uid secondary ionic mass spectrum
GLOSSARY OF BIOCHEMICAL TERMINOLOGY
U nilam ellar vesicles: Vesicles which are bounded by a single lamella
consisting o f two layers of Kpld molecules (a single bllayer). This Is In
contrast to m ultU am ellar vesicles which are Isolated by m any bllayers.
FCCP: Carbonyl cyanide 4-(trlfluoromethoxy)phenylhydrazone, a proton
carrier w hich facilitates th e release of protons from vesicles.
E lectroneutral proton-cation antiport: This process Is proton / cation counter
tran sp o rt In w hich th e cation concentration g rad ie n t drives cation fluxes
Inward across th e vesicle bllayer via some tra n sp o rte rs an d the pH
g radient drives proton fluxes outward from th e vesicle. The process
rem ains electroneutral throughout.
Voltage-gated Ion channels: Ion channels are opened above some value of
applied voltage an d are closed a t potentials below th e threshold or a t
ACKNOWLEDGMENTS
I would like to express my t h a n k s to th e supervisor, Dr. Tom Fyles for
his help, guidance an d patience throughout th is project. T hanks to Daniela
Loock for allowing me use some of her experim ental d a ta in this thesis, and
for the great collaboration and many helpful discussions. M any thanks to
Wilma van S traaten-N ijenhuis, Pedro M ontoya-Pelaez, Binqi Zeng, Lynn
Cameron, AUana P ry h itak a, and David Lycon, for th e ir help and thoughtful
discussions, and th a n k s to Dave Robertson for h is endeavors to m ake pH -stat
titration have more fun. I would like to acknowledge th e assistance of the
technical staff in D epartm ent of Chemistry, in particulEur Mrs. Christine
Greenwood and Dr. D avid McGillivary. T h a n k s for th e financial assistance
from D epartm ent of C hem istry and University of Victoria. And fin a lly , I am grateful to my wife for h e r support throughout th is long course.
1.1 N atu ral iop nhannftlg 1.1.1 Ion ch a n n el p rotein s
The flow of ions and molecules between a ceU and its environm ent is
precisely regulated by specific transport systems. These system will regulate
ceU volume and m a in ta in the intracellular pH and ionic composition to provide a favorable environment for enzyme activity, will extract and concentrate
metabobc fuels and building blocks firom the environment and œ tru d e toxic
substances, and will generate ionic gradients which are essential for the
exdtabibty of nerve and muscled All known naturally occurring transport
systems are channels^ These conduits, such as the Na+-K+ pump, play an
im portant role in intercellular communication and transfer of metabohtes.
1.1.2 A n tib iotics, sim ple n atu ral io n ch a n n els
Channel proteins are very comphcated^* 3. A typical channel protein has
a molecular weight of ca. 250,000 Da, and its conformation often changes either
in different solvents or by itself. Thus the complete three-dimensional
structures of these multi-subunit membrane proteins are unlikely to be easily
obtained. A less difficult approach which has been successful in elucidating
some features of the structure and function of transport proteins is to study
small molecule transport antibiotics produced by microorganisms. Because
these antibiotics, mainly channel forming peptides, have molecular weights of
materials. D uring the p ast decade, advances in th e analysis of the biophysical
properties of channel forming peptides have perm itted unprecedented insight
into structure an d hm ction a t a molecular level, which in many cases can be
related to structure and function in full-size proteins. Three of the best
examples are gramicidin, alam ethidn, and am photericin B, of which the first
two are generally considered as models of ion channel proteins. AU three
display characteristic ion channel behaviors to différent degrees: ion selectivity,
voltage dependence, subconductance states an d blocking, and modulation
properties in Upid
membranes'*-G ram iddin is a linear peptide produced by Bacillus brevis, which
consists of 15 alternating l- and o-amino a d d residues®. In organic solvents,
such as methanol, gram iddin exists as a m ixture of dimeric forms in
equilibrium w ith monomers’. The most ab u n d an t spedes is an anüparaUel,
left-handed P double helical dimer®. However, a series of experiments
indicated th a t gram iddin forms a head-to-head single-heUx dimer channel to
span a lipid bilayer m em brane (both N-termini are a t the bilayer mid plane)®.
The channel is about 4 Â diameter and is lined by polar peptide carbonyl
groups®. The hydrophobic side-chains are on th e periphery of the channel in
contact with hydrocarbon chains of the Upid mem brane. The hydrophiUc
carbonyl groups form a n aqueous pore and tran sien tly coordinate to the cation
as it passes down the axis of the channel* G ram iddin channels conduct
as Ca^+ or Ba^+ Side-chain modifications to produce related peptides can
change the conductance properties of gramicidin-based channels due to
conformational changes or to electrostatic effects^.
(HCO)HN-LVal-Gly- LAla-oLeu- LAla-oVal- LVal- dVal- lTi p-dLc u- L T ip -D L e u -L T rp -D L e u -L T rp -
COCNHCHjCH^OH)
Figure 1.1: Gramicidin A
Amphotericin B is the prototype of m em brane ion channels thought to be
formed firom barrel-like aggregates of am phipathic structures^ ^ It is one of a
large group (>2 0 0) of non-peptide, polyene macrolide antibiotics produced by
Streptomyces sp. Amphotericin B contains a rigid non polar heptaene u n it and a more flexible polyol region fused together in a macrolactone ring. A
mycosamine group and a carboxyl group a t one end of th e molecule generate a
zwitterion in n eu tral aqueous solutions. The overall length of the molecule is
nearly 25 Â which is about h a lf the thickness of a phosphohpid büayer. Based
on the result of th e conductance across büayer vs. amphotericin B
channel structure as shown in Figure 1.2. The non polar hydrophobic polyene
would interact w ith the alkyl chains of the phosphohpids to stabilize the
aggregate in bilayers, while the polar polyol segments in an aggregate of
amphotericin B might self-associate within the m em brane to form a hydrated
pore. The zwitterionic head groups of the aggregate would be expected to align
favorably w ith the phosphohpid head groups. It is proposed th a t th e number of
“staves” would be 8 ~ 12 amphotericin molecules, and th a t the conducting pore
has an internal radius of about 4 Â. Other experiments, such as p lanar bilayer
studies, and spectral studies of the amphotericin B complex w ith steroids,
suggest the channels come in several distinct forms” . The actual structures of
the different channels are still unknown and aw ait fu rth e r exploration.
h y d ro p h ilic HO. h y d ro p h o b ic exterior p o la r h e a d g ro u p : H HO OH CO2H Figure 1.2: Amphotericin B
Alam ethidn, which is generated hy the fungus Trichoderma viride, is a
transport antibiotic th at has been studied extensively. It is composed of 19
amino ad d s and 1 amino alcohol: Ac-Aih-Pro-Aih-Ala-Aih-Ala-Gln-Aih-Val-Aih-
Gly-Leu-Aib-Pro-Val-Aib-Aib-Glx-Gln-Plieol (Aib refers to a-aminoisobutyric
a d d or a-methylalanine). The crystal structure of alam ethidn shows th a t it
exists in a-hehces in contrast to the gram iddin p-hehces®. A lam ethidn is
monomeric in most common organic solvents, h u t in aqueous solutions
aggregates have been found to occur. In Upid hilayer membranes, alam eth id n
aggregates to form a heUcal bundle, also called a “harrel-stave” model, w ith a
central lumen through which ions can flow®. Unlike gram iddin, alam eth id n
does not show saturation of either current or voltage a t high ionic
concentration^^. This suggests th a t the alam ethidn charmel aggregates can
expand in size in response to transm em hrane conditions.
Although alam ethidn seems have no selectivity for cations, th e most
significant feature is its voltage dependence. When it is added to planar
bllayers, alam ethidn induces a macroscopic current which is strongly voltage
dependent as shown in Figure 1.3^®. Currently there are several different
models to try to explain the mechanism of alam ethidn channel formation, b ut
the details of the structure of the alam ethidn channel are less well
characterized when compared to gram iddin. Since a-heUces are commonly
found in proteins, it is beUeved th a t alam ethidn may in fact more closely mimic
1000 800 < 3 - 600 2 § 400 ü 200 -200 -150 -100 -50 0 50 100 150 Voltage (mV)
Figure 1.3: Current-voltage (I-V) curve for alam eth id n in
diphytanoylphosphatidyl choline (DiPhyPC) membrane^®
1.2 O verview o f sv n th etic io n ch a n n els
Although structural inform ation is emerging from molecular biology^
most of the molecular detail about natural ion channels comes from low
molecular weight compounds such as gramicidin, amphotericin, or alam etbicin
as discussed above'*’ These structural details provide a range of molecular
mechanisms for the different n a tu ra l ion transporters, as shown in Figure 1.4.
At one extreme, a carrier m echanism can be envisioned in which a carrier-
the ionophore antibiotic valinomycin is a typical carrier. At the other extreme,
ion channels could be formed by a complete membrane-spanning transporter,
such as gramiddin. The channel provides a solvation pathw ay for ions as they
pass through the Hpid barrier. Ion channels formed hy aggregate pores, hke
amphotericin and alam ethidn, would provide a loosely structured environment
containing some w ater a t the core of the aggregate to facilitate stabilization of
ions in transit. Lastly, mem brane disrupting agents, such as m ehttin (a
peptide from bee venom) or sim ple detergents, could cause defect structures
w ithin bilayers. These might be deep aqueous Qords w ithin th e bilayer and
represent an extremely primitive type of ion “channel” capable of transport.
In order to eluddate the m echanism of ion transport, and to understand
how to achieve ion selectivity and channel gating, several research groups have
devoted their efforts to the synthesis of simple artifrdal ion channels*^. In
comparison with natural ion channels, chemists hope th a t these devised model
1 00
ï«î“ wI
I
fI
s .I
\\\v
w
\\\v
\
ww
c P o o o
I
%Carrier
Defined
tunnel
Aggregate
pote
Disrupting
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Channel
I
00To study ion channel proteins, one approach is to synthesize simphhed
peptides and compare their activity w ith natural channels. Two examples are
noted here. Mutter, Montai and coworkers*® designed template-assembled
synthetic proteins (TASPs) to adopt globular, four-hehx bundle structures
which form ion channels in hpid bilayers. Their studies suggested the protein
molecules aggregated to form heterogeneous conductive ohgomers. DeGrado
and co-workers synthesized a 21 residue peptide, HzN-(Leu-Ser-Ser-Leu-Leu-
Ser-Leu)3-CONH2, to resemble th e acetylcholine receptor which is one of the
most studied ion channel proteins*®. They found the peptide formed single
channels in planar bilayers w ith well defined ion perm eability and lifetime.
Perhaps due to the complexity of the synthesis and structure modification,
there has been no further development firom those explorations.
The earhest pioneering work w ith non-peptide models was designed by
Tabushi and coworkers^®. They used P-cydodextrin as the backbone of the
target which is shown in Figure 1.5. Its fourteen secondary hydroxyl groups
provide the polarity to contact the aqueous surface, and th e four hydrophobic
drains attached to the prim ary h y d ro ^ l groups stabilize th e amphiphile in
büayers. It was described as a “h alf drarmel”. The chaimel transported copper
and cobalt, and the transport rate was much faster th a n a specific carrier,
NH
NH
NH
Figure 1.5: Tabushi’s P-cydodextrin ion channel
In order to mimic gramicidin, a unimolecular structure w ith tunnel-like
geometry m u st be designed to span a bilayer. One example is the so called
“chundle^i”, (later changed to “bouquet” molecules) reported by Lehn and
coworkers22. An 18-crown-6 or a cydodextrin derivative was used as a rigid collar to which poly(ethylene oxide) chains or polyallqrl chains, and carboxylate
head groups were attached (Figure 1.6). Vesides containing LiCl were
prepared from egg PC (phosphatidyl choline) and DPPC (dipalmitoyl
phosphatidyl choline). Opposing gradients of Li+ (inside) and Na+ (outside of
vesides) were created and the transport of Li+ and Na+ down their
concentration gradients was monitored directly by ~’\A and ^ N a NMR. It was
found these “bouquet” molecules caused a one-for-one exchange of Na+ for Li+
via a channel mechanism. However, the tran sp o rt rates for the “bouquet”
molecules were relatively slow, their rate constants were 5 - 23 x 10"® s*i for the
NaO. .O NH N aO ^ O O ONa
X .
j
o^y V ^ °O^r^JLyO
<V*UL^O
0/ ° v ° ° \A
\
N a O ^ O N a C K ^ O o ^ O N a O ^ O N aFigure 1.6: One of Lehn’s “bouquet” molecules
Another rigid tunnel-hke model was reported by Voyer and RobitaiHe^.
They synthesized a 21 amino add peptide composed of fifteen L-leudnes and six
2 l-crown-7-L-phenylalanines. The peptide backbone formed a n a-hehcal chain,
so by placing the crown ethers on every fourth residue, the crown ethers would
pH-sta t titration method indicated th a t the peptide spanned büayer vesicles to
form a channel, although there was no monovalent cation selectivity.
= R
Figure 1.7: VoyeFs ion channel model
Structurally sophisticated aggregate channels were synthesized by
Ghadiri’s groupé'*. The system is a eight amino acid cychc polypeptide,
cydo[(Trp-D-Leu)3Gln-D-Leu-l (Figure 1.8). The amino a d d side chainm were
placed roughly along the equator of the cyde and directed away from the
center. When the peptide is incorporated into bilayer membranes, a channel is
formed through hydrophobic side chain - hpid interactions and hydrogen
bonding between cydic peptides similar to P-sheet formation. The system is
active in both veside and planar bilayers, and shows typical single channel
conductance behavior. The pore formed by stacking the cydic peptide is 0.5 nm
HN N H HN N H H N N H % ^ N H
Figure 1.8: Ghadiri’s cyclic peptide channel
A flexible tris-macrocychc channel model w as developed by Gokel’s
groupes. Two m acrocydes are used as head group anchors, and a th ird one in
the middle is a central relay. Diaza-18-crown-6 w as chosen as th e macrocycle
in the system due to its cation selectivity, an d alkyl chains connect th e three
macrocydes to reach to th e desired length (see Figure 1.9). In phosphohpid
büayer veside m em branes, cation flux through th is channel system was
assessed by a fluorescence technique using pyranine as a indicator and ^N a
NMR spectroscopy. The cation conduction for th e channel in Figure 1.9 was as
much as 40 % of th e activity of gramicidin u n d er th e sam e conditions. Their
studies also indicate th a t the ionophore really does not require a tunnel-hke
c
c
c
Figure 1.9: Grokel’s tris-macrocyclic channel system
Puhrhop’s group was the first to use th e term “bolaam phipbiles” to
nam e am phiphilic molecules w ith polar h ead groups a t both ends of a
hydrophobic core^®. They also reported a num ber of bolaam phipbiles capable of
forming th in m onolayer hpid m em branes, presum ably via aggregate
structures. O ne of th e ir typical bolaam phipbiles is shown a t Figure 1.10. They
found m onensin pyrom elhtate which h as both hydrophobic and hydrophihc
sides could perforate the thin monolayer vesicles m ade fi:um electronegative or
electroneutral bola am phipbiles and m ake th e mem brane perm eable to Li+
ions.
A nother flexible channel system is an ion p air model dem onstrated by
Kobuke and co-workers^^. As shown in Figure 1.11, the phosphate m onoester
or carboxylate term inated tetraCl, 4 - butyleneglycol) m onobutyl ether
combined w ith dioctadecyldimethylam m onium cation to form an ion pair.
When the ion p a ir w as incorporated into p la n ar hpid bilayers, a single channel
recording showed stable and constant currents. They also observed an
However, th ere w as no direct evidence to support the aggregation in bilayers,
and there w as no cation selectivity for these ion channels.
COONa
F uhrhop’s bolaam phiphile
COOH H 00(
COOH
M onensin pyrom eH itate
Figure 1.10: Fuhrhop’s m onolayer system
CH
or
Regen and co-workers reported two series of compounds: one called
bolaphiles^® and the oth er called a bola am phiphile system ^. The form er were
diesters synthesized fi*om an homologous series of lin e ar saturated, olefinic,
and acelylenic a,o)-dicarfoo2yhc ad d s w ith hexaethylene ^ycol (see examples in
Figure 1 .1 2 ). Bilayer veside experiments indicated t h i s series behaved as
m em brane-disrupting agents and the most active bolaphiles w ere three times
more active th an the detergent Triton X -1 0 0 The bola am phiphile was a
sterol-polyether conjugated 5-androstene derivative (Figure 1 .1 2 ) designed to
mimic the antibiotic am photericin B. Using a sim ilar m ethod to Lehn^, Na+
transport w as monitored by ^N a NMR. liC l w as incorporated inside of egg PC
veside bilayers. Li+ exit (antiport) and/or Cl" en try (symport) m a in t a in s
electroneutrahty on both sides of the membrane, and perm its the collapse of a
Na+ concentration grad ien t (the transm em brane potential drives the ion
transport). The ^ p e rim e n ta l d ata indicated ion channels w ere formed through
aggregation a t high concentrations of bola-amphiphiles^s.
HOlCHaCHaOlellCHalnSocCHaCHaOleH
.OR
1.3 The ftarlv ftigplorationa firom Fvles* group
Combining inform ation firom n atiiral and artificial ion transporters leads
to the following criteria for d e s ig n in g m em brane active transporters: The transporter should be an am phiphile, and have suitable colum nar shape which
combines w ith its hydrophobic p art to incorporate into bilayers. It should span
a 4 nm thick bilayer eith er as a monomer or as a n aggregate, and it should
sim ultaneously surround an ion of a t least 0 .3 n m diam eter. The hydrophihc
p art of the tran sp o rter should provide a potential p ath for th e ions or should
provide ionophUic sites deep w ith in the hpid core of the bilayer. D uring the
tran sit of ions across th e bilayer, the transporter m u st accommodate th e ion
solvation requirem ent either by partial replacem ent of th e ion solvation sphere
w ith transporter-cation interactions or by introduction of w ater w ith in the hpid
core of the büayer. A final requirem ent for th e tran sp o rter is th a t a feasible
and efficient convergent synthesis should be possible.
In our group’s early explorations th ere w ere two different types of ion
channels m ade. One system is U lustrated in Figure 1.13, and was m ainly
synthesized by Dr. T. Jam es^. Ah transporters are unim olecular structures: an
18-crown-6 derivative is used as a central core, and m acrocydes are linked to
both sides of th e crown eth er to ahow the tran sp o rter to span th e büayer, then
polar head groups are capped on the m acrocydes to create th e amphiphUic
character needed to contact th e aqueous phases. T ransporters in this system
are fairly rigid due to th e specific conformation of th e crown ether core. They
aggregation formed^i.
W
- W
&
Figure 1.13: Tunnel-defined channels
The other system is illustrated in Figure 1.14, and m ainly synthesized
by Dr. M. Zojsgi^^ The m ain différence finm th e first system is th a t either
propyl ta rtaric ester or m eta-xylyl was used as central lin k er to form a more
flexible structure. T he kinetic m easurem ents indicated th a t th e transporters in
this system form active aggregates in bilayer vesicles.
To dimtingiTisb these two systems, th e form er is called “channel” due to
its unim olecular tunnel-like conformation, and th e la tte r one is n a m e d of “pore-
g ^ o o X o O o
L in k er HO^ 0 “O%
Figure 1.14: Channels formed by aggregate pores
0 OH
■“Y ï ° '
1.4 The d esign o f re sea r ch ta rgets in th is th e sis
The em phasis of th is thesis is to develop a new series of bolaamphipbiles
to achieve artificial voltage-gated ion channels^ based on m odular subunits
developed in earher w ork^. In essence we are looking for an artificial version
of alam ethidn w ith potential ion selectivity.
Ion selectivity of aggregated channels is d if f e r e n t firom th a t of carrier
type of compounds, such as crown ethers, or valinom ycin. In these cases the
structure relates to carrier size, charge, and binding site. The aggregated
there are a lot of unansw ered questions about th e ir detailed structures, forming
mechanism, and stru ctu rally determ ined factors. These rem aining puzzles
need to he solved g radually in th e exploration of artificial channels.
In our tran sp o rter system , to achieve alkali-m etal cation selectivity for
aggregate bola-am phiphiles, th e size of the aggregate needs to be lim ited. Thus
we are interested in active dim ers or trim ers which are closely sim ilar to hpids
in both cross-sectional area and chain length.
While bolaam phipbiles span the büayers, th e central linker should be
able not only to facilitate ionic passage th r o u ^ th e channel b u t also to stabilize
the transporter w ithin th e büayer. This m eans th a t th e central linker h as to be
a weU balanced m ixture of hydrophobic and hydrophilic character. The design
proposal is ü lu strated on Figure 1.15. Compared w ith the central linkers in
early work^^, it is envisaged th a t the new linker should be less buUqr and have
fewer functional groups. A t th e same time, the effect of central linker length on
transport activity w ill be examined.
In order to achieve the goal of a voltage-gated channel, the other
im portant component, th e head group, has to be closely considered. W hen th e
polarity of a voltage apphed to the büayer is switched, the bola-am phiphüe
should respond by a reorientation in the büayer. In other words, the head
groups on two ends of th e bola-am phiphüe should be different to create a large
molecular dipole. Therefore, our initial strategy w as to synthesize bola-
am phiphües bearing one head group having a negative charge and the other
control bolaam phipbiles should be synthesized to com pare th e relative
activities of symmetrical and n eu tral analogs. L ater in th is th esis w ork, one
m ore head group carrying -2 charge (pH = 7) w as added to th is se t o f targ ets.
As described in the early work®** macrocychc tetraesters w ere used as
building blocks in the syntheses. Two series of bola-am phiphiles w ere created
to te st th e importance of the effects of hydrophobidty and h y d ro p h id lity on
transport.
o g o S ^ o O Q
“1 C entral linker ° = C y = A STrgPA STrgNFigure 1.15: Design proposal for pore form ation by aggregation of
To get suitable structures for potential voltage-gated ion channels,
m olecular modebng w ith CAChe scientific computing program was used to
assist th e design. Since th e p resen t com puter program cannot sim ulate
behaviors of larger aggregates of molecules in Hpid bilayer m em branes, th e
possible global m iniTrm m conform ations of candidates in gas phase were
investigated to provide indirect evidence for th e ir possible molecular shapes.
The extended shapes are the potential structures expected and molecules
w hich fold are less interesting as candidate pore-formers.
To simplify n a m in g and give some stan d ard inform ation on th e num ber
of com binations possible, a structure-based sem i-system atic n a m in g system is
described as follows and equated on Figure 1.15: 1) Each synthon w as
assigned one or two sim ple le tte r or n u m b e r n a m e: A = 2-mercaptoacetic a d d or
2-mercaptoacetic carboxylate, G = l-mercapto-^-D-glucose, P a =
3-mercaptopropionic a d d or 3-mercatopropionic carbojQrlate, 8 = firom 1,8-
octanediol, T t^ = from triethylene glycol, P = 3-mercaptopropyl, S u = 2-
m ercaptoethyl sulfide, etc. 2) Each interm ediate is named as a com bination of
its synthon abbreviations w ith th e exception th a t the m aleate esters of th e w all
u n its are impHed: ST rg = th e m acrocydic te tra ester derived firom 1 mole of 1,8-
octanediol and 1 mole of triethylene glycol (and 2 moles of m aleic anhydride), 82
= th e macrocychc tetraester from 2 moles of 1,8-octanediol (and 2 moles of
m aleic anhydride), etc. 3) The f in a l stru ctu res are named firom one head group
head group 2. A s shown in Figure 1.15, A 8T i^P A 8T rgN represents: a
mercaptoacetic a d d head group + a te tra e ste r w all u n it m ade from 1,8-
octanediol and triethylene ^ycol + a central linker derived from 3-
m ercaptopropanol and 2-mercaptoacetic ad d + th e sam e w all u n it + an N,N-
dim ethylam inoethanethiol head group.
It can be envisaged th a t membrane spanning bola am phiphile units in
which th e m acroqrde STrg is used as w all u n it will orient to create an
aggregate in w hich th e polar part of the STrg is tow ard th e inside of the
aggregates and th e non polar segment tow ard th e hpid w hen transporters are
incorporated into büayers. However, when m acrocyde 82 is used as wall unit,
the possibihty for form ing an aggregate will be greatly decreased due to the loss
of m ost of its hydrophidhty.
The tran sp o rt properties were investigated through pH -stat titration of
vesides and w ith a p lan ar büayer experiment. The pH -stat titratio n m easured
transport properties via a cation-proton antiportP^ across veside büayers^^-
Through d ata processing, the kinetic behaviors of transporters can be dosely
investigated. T ransport rates which are norm alized to the sam e concentration
can be used to compare the transporter’s relative activities and cation
selectivities. The apparent kinetic order also can be obtained to look a t
aggregation of th e transporter in büayers^i* 32. However, the pH -stat technique
only m easures th e cation dependence of in itiatio n of a tran sp o rt process which
rapidly equüibrates each veside w ithin th e tim e of a single opening. T hat
and it only exam ines th e average behaviors of transporters.
The ion translocation process can be directly observed using the planar
bilayer experiment?®. This experim ent m easures the tim e dependence of the
current carried across a p lan ar bilayer formed on a sm all hole in a hydrophobic
support barrier. It can detect th e behavior of a single channel®®. This
technique provides unam biguous dem onstration of a channel mechanism and
can provide m olecular details of the ion translocation process. More
im portantly it is a n excellent tool to m easure the voltage-gated property of an
ion channel. Except for some very weU defined cases, th e p lan ar bilayer
^ p érim en t provides relatively httle inform ation about th e in itiatio n of channel
openings. U sing a com bination of the two different techniques (planar bilayer
and vesicle), th e tran sp o rt properties of a ion channel can be explored firom
different angles. Hopefully enough inform ation can be obtained to lead to a
composite p o rtrait of the channel mechanism in m olecular d etail not only for a
CHAPTER 2 SYNTHESIS
2 .1 O v erv iew
Following earlier w ork in th e groupé ^ , a convergent synthesis w as used in
this thesis to construct tran sp o rte r candidates based on m odular subunits as
previously explored (Figure 2.1). Commercial reagents w ere used whenever
possible to sim plify and optim ize th e synthesis. The synthesis sta rted from two
different m acrocydes, STrg an d 82, which were subsequently modified to
different derivatives. The two macrocydic derivatives, m esylate and
carbojQ^late, th en w ere coupled to hold one polar head group. Finally, the
second polar head group w as added to give a bolaam phiphile, pore-former
candidate. From different com binations of w all u n it, linker, an d head group, it
is hoped th a t the regulation of ion transport across büayer m em branes can be
detected and controlled.
2.2 S y n th e se s o f m fl<»m cycles
Syntheses of te tra e ste r diene macrocydes followed th e procedures
reported previously^' ^ w ith m inor changes to improve hanHling and
separation. 1,8-Octanediol reacted w ith two equivalent of m aleic anhydride to
give a d iad d (1) w hich w as esterified w ith either triethylene glycol or 1,8-
octanediol to form th e m acrocydes 8Trg (2) or 82 (3) ^ (Scheme 2.1). The
purification of m acrocydes w as the m ajor challenge of th is step of the
syntheses. M acrocyde 82 is relatively easy to separate; th e crude oüy product of
oily solid, followed by several recrystaHizations brom etbyl acetate to give pure
82 (3). The total yield of th e two steps w as 14%.
Transporter candidate
Wall u n it (macroc^cle)
Central linker
Polar head group
Triethylene glycol / CH3SO3H / 6.3 Benzene, quantitative HO. OH 1,8-octanediol 14 % / CH3SO3H STrg (2) 8 2 (3)
Scheme 2.1: Syntheses of macrocycles
The isolation of 8Trg is more arduous. The m ixture o f five compounds
and polymer by-products (Scheme 2.2) w as chromatographed on sfiica gel via
pre-absorption to remove compounds 3, 6, and some of th e polym er product.
Kugelrhor distillation w as th en used to ô*actionate the product-containing
chrom atography firactions. Compounds 4, 5, and the rest of the polymer
product were removed, and pure 8Trg (2) w as obtained. Due to th e various by
products and the losses in the process, the yield of 2 w as only 6.3%.
The syntheses of the two m acrocydes were revisited a t le ast four tim es
each during the course of th is project. Based on th e previous work done by
attem pted. The purification of maerocyde 2 and 3 w as im proved over previous
reports and by-products 3 and 6 were isolated in pure form firom the
purification process of m acrocyde 2. However, th e yield of macrocyde couldn’t
be fiirth er improved, and th e yields shown on Scheme 2.1 are unchanged firom
earher reports. J CH3SO3H Q / Benzene Dean-Stark condensation OH + 1 y + Polymer 4
Scheme 2.2: Form ation of STrg
2.3 S y n th eses o f tran sp o rter eandiHates in th e STrg se r ie s 2.3.1 S y n th eses o f d e r iv a tiv e s o f m a crocyd e STrg
Following prior work®’- the tetraester diene m acrocyde STrg (2) was
converted to the mono-alcohol 7 via Michael addition of 3-mercaptopropanol
(Scheme 2.3). Three compounds were isolated: unreacted macrocyde STrg 2,
the mono-adduct 7, and th e double-adduct (not show n in Scheme 2.3). In a
chrom atography could separate th is m ixture to give pure m ono-adduct 7 and
recovered macrocyde 2. Mono-alcohol 7 reacted w ith m ethanesulphonyl
chloride to yield th e m esylate 8 The yield of th e two steps was 36%,
unchanged firom previous reports.
/ 2,2,6,6-tetramethylpiperidine
STrgPOH (7)
STrgPOMs (8)
Scheme 2.3: Synthesis of mono-adduct of th e m acrocyde 2
New compounds, th e d iad d ASTrgA (9) and th e mono a d d STrgA (10),
were obtained through M ichael addition of 2-mercaptoacetic a d d catalyzed by
2,2,6,6-tetram etbylpiperidine (Scheme 2.4). The product m ixture of th is
reaction was dependent on th e reaction tim e. The double M ichael addition
could be completed w ith longer reaction tim es, typically m ore th a n 24 h, and 9
could be obtained through a sim ple work up. However, after a sh o rter reaction
adduct 1 0, plus the unreacted macrocycle 2. 2 HS-^COgH / 2,2,6,6-tetramethylpiperidine 9 10
Scheme 2.4: Syntheses of new adducts 9 and 10
Through a fortuitous accident, the separation of th e m ixture of 9 and 10
w as found to be possible by silica column chromatography using a solvent
gradient of hexanes and ethyl acetate. There was some degree of product loss
on th e süica column, b u t th e separation was achieved cleanly. When the
m ixture of reactants w as refluxed for 6 h, the yield for 1 0 w as 12% and the
yield for 9 was 69% after th e purification.
Figures 2.2, and 2.3 com pare the and NMR spectra of ASTrgA (9)
and STrgA (10). In th e NMR spectrum of ASTrgA (9), there was a broad
singlet a t S.75 ppm due to th e acidic protons (a). The m ethylene protons
(b)
adjacent to the carboj^l are seen between 4.27 ppm and 4.06 ppm as am ultiplet, and the m ethylene protons (d) adjacent to th e ether group formed
another m ultiplet betw een 3.69 ppm and 3.64 ppm. The two inequivalent
methylene protons (e) on th e acetic ad d side ch ain coupled w ith each other.
Since there were three possible regio-iosomers formed, a total of four protons
gave three sets of doublets of doublets firom 3.57 ppm to 3.30 p p m The
m ethine proton (c) adjacent to sulfide coupled with two inequivalent methylene
protons (f) on the ring to form a m ultiplet a t 3.89 ~ 3.82 ppm, an d those two
methylene protons (f) gave two sets of m ultiplets from 3.00 to 2.66 p p m The
methylene protons (g, h , i) on th e hydrocarbon chain of th e m acrocyde gave a
broad m ultiplet and a broad singlet a t 1.61 - 1.59 ppm and 1.31 ppm. Note
th a t there are sixteen possible enantiom ers plus diasterom ers for 9.
In comparison w ith 9, the NMR spectrum of 8TrgA (10) revealed
more details about the m acrocyde conformation. Both spectra had quite
sim ilar patterns for most protons, except for very sm all differences in chemical
shifts. The addic proton (a) of compound 1 0 moved to 8.34 ppm. Two singlets
a t 6.77 and 6.19 ppm w ere due to th e m ethine protons (b) of th e olefin. The
former was fi*om the trans conformation of the olefin, and the la tte r one was
firom the dom inant cis conformation. The m ethine proton (d) on th e r in g , and
two different inequivalent m ethylene protons (f, g) aU showed doublets of
doublets. One set of doublet of doublets firom proton f w as m uch bigger th a n
the other one. This could be due to the selective form ation of one of two
For the NMR spectrum of 9, th e carbonyls from th e carbosylic ad d
(a) showed four peaks a t 173.6,173.5,173.4, and 173.3 ppm, an d th e carbonyls
from the ester groups of th e m acrocyde (b) gave three peaks a t 170.9, 170.8,
170.0 p p m The m ethylene carbons (c) from the ether group w ere in two sets a t
70.1, 70.0, 68.7, 6 8 .6 ppm. The m ethylene carbons (d) adjacent to th e carboxyl
gave four lines a t 65.4, 64.9, 64.3, 63.7 p p m A pair of lines a t 41.7 and 41.5
ppm w as contributed from the m e t h in e carbon (e) adjacent to sulfrde. The
m ethylene carbons ( g ) on th e acetic a d d side r h a in were a p air of peaks a t 33.1,
32.8 ppm, and the m ethylenes (f) on th e ring were another p a ir a t 35.9, 35.7
ppm. The rem aining signals 28.4, 28.1, 25.2, and 25.1 ppm w ere due to the
m ethylene carbons (h, i, j) from th e hydrocarbon chain of m acrocyde.
In comparison to 9, th e NMR spectrum of 8TrgA (10) had some extra
peaks: 165.3, 165.0 ppm w ere due to th e a,P-unsaturated carbonyl carbons (c),
133.8, 133.0 ppm were contributed from th e trans, and 130.2, 130.1, 129.3,
129.1 ppm were from th e cis olefin (d). The rest of the assignm ents for
chemical shifts were quite sim ilar to compound 9 except th e relative ratios were
different. These different ratios again verified the selective contribution from
one of two possible regio-isomers indicated in the NMR spectrum analysis.
From chemical shifi: considerations, th e favored regio-isomer for 10 would be
the one shown in Figures 2 .2 and 2.3.
The successful isolation of m ono-adduct 8TrgA (10) w ill allow more and
exploration.
-S « X
CO
I
I* 7
^
g & •o S2.3.2 C oup lin g r e a ctio n to m ake b is-m aeroeyeies
The coupling of the two macrocydic derivative 8 and 9, w as carried out
using tétram éthylam m onium hydroxide pentahydrate in DMSO to form the
ester bond as th e middle linker. Once again, separation w as th e main obstacle
in the synthesis. Statistically, the products formed firom this mono-alkylation
are the desired bis-macrocycle, 8TrgPA8TrgA (11), and a by-product tris-
macrocycle 8TrgPA8TrgAP8Trg (12), plus unreacted startin g m aterials
(Scheme 2.5). Due to th eir comparable polarity, silica or alum ina column
chrom atography is not able to separate bis-macrocyde (11) firom tris-macrocyde
(12). In consideration of the molecular w eight difference betw een them , gel perm eation chrom atography, also called size exdusion chrom atography, was
used to explore th e separation. After several different types and pore-sizes of
gel were investigated, Lipophihc Sephadex LH-20 w as finally chosen as the
most suitable one, and th e mixed solvent, chloroform : 2-propanol = 4 : 3 , was
used as packing solvent and eluent. However, the m ixture could not be
separated in a single step. Therefore, after each separation, the mixed
fi*actions were combined, and carried over to a subsequent separation step.
Since TLC w as not good enough for monitoring to detect mixed firactions, HPLC
equipped w ith an analytical gel permeation column w as used to determ ine the
composition of each firaction. Usually the pure tran sp o rter precursor, bis-
macrocyde 8TrgPA8TrgA (11), could be obtained through a cyde of ten stages
I
I
I
COFigure 2.4 shows th e analytical GPC detection before and after
purification, the negative LSIMS spectrum for th e bis- macrocyde, and th e
positive LSIMS spectrum for th e tris-m acrocyde. Figures 2.5 and 2.6 give 'H
and NMR spectral com parisons for 11 and 12. Figures 2.7 and 2.8 give 2D
NMR spectra for STrgPASTrgA (11), this key precursor.
The NMR spectrum of compound 11 is w ell resolved. The addle
proton (a) was a very broad singlet a t 7.82 ppm. Two singlets a t 6.76 and 6.17
ppm were the trans and cis m ethine protons (b) of th e olefin. The methylene
protons adjacent to carbo]cyl w hich were sixteen fi’om two macrocydes plus two
firom the middle linker (c) formed a m ultiplet fi*om 4.26 to 3.94 ppm. The
m ethylene protons (e) firom the eth er groups of the m acrocydes gave another
m ultiplet between 3.68 and 3.53 ppm. Two sets of two isolated inequivalent
m ethylene protons (f) adjacent to th e carboxylate coupled w ith each other to
form three sets of doublets of doublets firom 3.50 ~ 3.23 ppm. Three m ethine
protons (d) adjacent to sulfide h ad two sets of m ultiplets a t 3.84 -3.71 ppm and
3.68 - 3.53 ppm, and th e m ethylene protons (g) gave two sets of m ultiplets each
for one type of protons in 2.95 - 2.84 ppm and 2.72 - 2.55 ppm. The m ethylene
protons (h) firom the propyl linker adjacent to sulfide showed a m ultiplet
overlapped w ith one of th e protons g in range 2.72 - 2.55 ppm, and protons (i)
of th e middle m ethylene group gave another m ultiplet a t 1.86 ppm. Twenty
four methylene protons (j, k , 1) firom th e hydrocarbon rbain of two macrocydes
A n a ly tic a l GPC L SIM S Mixture Bis-macrocycle STrgPASTrgA (11) tiœj 400 600 800 1000 1200 1400 1600 1800 M a s s Tris-macrocycle L7DL4 M a a s STrgPASTrgAPSTrg (12) * : 2-propanol u sed as an internal reference
I
m ]9.0 27.0 STrgPASTrgA (11) d (cis) STrgPASTrgAPSTrg (8) 00 m 160 ISO 130 n o no too (ppm) ê
I f
(ppm) 1.00 2.00 3.00 4.00 5.00 6.00 7.00 (ppm) 7 .0 0 6 .0 0 5 .0 0 4.00 3 .0 0 2 .0 0 1.00(ppm) - 1.00 -2.00 -3.00 -4.00 -5 .0 0 -6.00 -7 .0 0 120 100 80 60 40 (ppm)
The assignm ent of th e NMR spectrum for 11 is also well established.
Three peaks a t 172.1, 171.9, 171.8 ppm represented th e carbonyl carbon (a) of
the carbo^H c ad d . T he other twelve peaks firom 171.2 to 169.3 ppm were firom
seven carbonyl carbons (b) firom two m acrocydes and th e m iddle linker. The
lefl»ver two a , P- u n satu rated carbonyl carbons (c) gave five peaks firom 165.2
to 164.6 p p m Signals a t 133.8 and 132.9 ppm w ere firom th e trans olefin (d),
and 130.1, 129.9, 129.1, and 128.9 ppm w ere cis olefin (d). The eight
m ethylene carbons (e) of th e ether groups gave eight peaks in the range
between 70.5 and 6 8 .6 ppm, and the nine m ethylene carbons (f) adjacent to
carboxyl (induding th e one firom the linker) form ed eleven peaks firom 65.3 to
63.6 ppntL The signals a t 41.9,41.5,41.4, and 41.2 ppm revealed three m ethine carbons (g) adjacent to th e sulfide substituent, and th ree nearby methylene
carbons (h) gave signals a t 36.3, 36.1, 35.9, and 35.7 ppm. Two methylene
carbons (i) firom th e sulfide m ethyl carboxyl group occurred a t 33.5, 33.0, 32.9
ppm. The signal a t 27.8 ppm was the m ethylene carbon (j) of propyl group
adjacent to sulfide, an d th e other methylene carbon (k), in th e middle position
of the propyl group, w as revealed a t 27.7, 27.5 ppm . All of m ethylene carbons
(1, m, n ) on the hydrocarbon chain of m acrotydes gathered in two ranges of
29.4 ~ 28.1 and 25.5 ~ 25.2 ppm.
All of th e ID NMR assignm ents for 11 w ere supported by 2D NMR
(COSY and HETCOR^?) data. In HETCOR spectrum (Figure 2.8), the
3.50 ~ 3.23 ppm which didn’t couple w ith other protons except self-coupling,
th is indicated these protons and carbons were the methylene groups betw een
sulfide and carboxyl. Com bining the d ata firom COSY and HETCOR, it was
found th a t the m ethine protons (d in Figure 2.5) gave two m ultiplets between
3.84 -3.71 ppm and 3.68 - 3.53 ppm due to th eir coupling w ith two
inequivalent protons (g) indicated in th e COSY spectrum. The COSY spectrum
also indicated th a t th e m ultiplet a t 1.86 ppm (proton i) coupled individually
w ith two m ultiplets in 4.26 - 3.94 ppm and 2.68 - 2.53 ppm (proton c and h)
w hich clearly revealed th e proton i w as firom th e middle m ethylene group in
th e propyl linker.
Since the tris-macrocycle has a sym m etrical structure, its and
NMR spectra were sim pler th a n th e spectra of his-macrocycle 11 in Figures 2.5
and 2.6. The detailed assignm ents of and NMR spectra of 12 do not
follow directly firom th e w ell established spectra of 11. The overall chemical
sh ift groups are however consistent w ith th e structure. The positive LSIMS
spectrum indicated th e signal a t 1701.4 w as th e molecular ion (M + H)+ for 12,
and th e structure proof rests principalLy on th is MS result.
2.3.3 S y n th e se s o f tr a n s p o r te r c a n d id a te s in th e 8T rg s e rie s
The last step in th e synthesis of tran sp o rter candidate w as to add the
second head group on th e precursor 8TrgPA8TrgA (11) by M ichael addition.
The reaction was catalyzed by 2,2,6,6-tetram ethylpiperidine in TEDF or DMF.
The desired product w as acidified by washing w ith IM HCl an d purified by
-m ercaptoaœtic a d d , N, N’-di-m ethyla-m inoethanethiol, or 1-thio-P-D-^ucose as
different com binations of head groups, a negatively charged (pH 7), positively
charged (pH 7), or n eu tral head group on one side of a transporter were
obtained (Scheme 2.6). o
C
o COgH 11 RSH / 2,2,6,6-tetramethylpiperidine S^COaH R = H O O C ^ 13 R = (CH3)aN^^^ 14 R = HO^ 15Scheme 2.6: Syntheses of transporter candidates in 8Trg series
The iH and NMR spectra are compared in Figures 2.9 and 2.10. The
^H NMR spectra of these three compounds are quite sim ilar except for some
specific chemical sh ifts due to the different head groups. For 13, th e broad
singlet a t 5.14 ppm w as due to the carboxylic ad d proton. Compound 14 h ad a
N,N-dim ethylam ino ethyl head group. Compound 15 gave a very broad m ultiplet
between 5.33 - 4.65 ppm caused by four hydrosyl protons of th e D-glucose head
group. Two peaks a t 1.24 and 1.22 ppm of 13 indicated a low level of
contam ination by formation a 2-propyl ester firom 2-propanol and th e ca rb o ^ h c
ad d during th e work-up.
The differences among the three compounds were also d early indicated
in th eir NMR spectra. Compound 14 h ad three distinct peaks, 43.0 ppm for
the m ethyl carbon, and 56.9, 34.1 ppm for th e two methylene carbons betw een
nitrogen and sulfur in the head group, w hich verified the structure. Compound
15 gave seven peaks a t 78.1, 72.8, 70.8, 70.7, 66.5, 66.2, and 62.1 ppm for the
D-glucose head group. Due to the two identical carb o ^lic a d d head groups of
13, th e new head group only gave some overlapping peaks in th e spectrum .
The 2-propyl ester conta m in a t ion was d early seen a t 22.6, 21.7, and 14.1 ppm, and th e negative LSIMS also verified th e analysis.
The negative LSIMS spectra of these three candidates, 13, 14 and 15,
individually gave th eir molecular ion (M - H)* a t 1261.4,1274.6 and 1365.4. All
of th e evidence indicated these three candidates in 8Trg series h ad been
successfully synthesized. In the second phase of th e program, these different
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