cyclodextrin scaffolds

7/26/2019 Cyclodextrin Scaffolds

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CYCLODEXTRIN SCAFFOLDS DESIGNED FOR THE M OLECULAR

RECOGNITION OF ANIONS, CATIONS OR ORGANIC SUBSTRATES

by

Suhash Chandur Harwani

A thesis submitted in partial fulfillment

of the requirements for the Doctor of

Philosophy degree in C hemistry

in the Graduate College of

The University of Iowa

May 2008

Thesis Supervisor: Assistan t Professor Jason R. Telford


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UMI Number: 3323427

Copyright 2008 by

Harwani, Suhash Chandur

All rights reserved.

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Copyright by

SUHASH CHANDUR HARWANI

2008

All Rights Reserved


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To my grandparents and parents

for all their sacrifices and hard work


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Action expresses priorities.

Mohandas Ghandi

To My Friends:

If you live to be hundred, I want to live to be a hundred minus one day, so I

never have to live without yo u.

Winnie The Pooh

In everyone's life, at some tim e, our inner fire goes out. It is then burst into flame by an

encounter with another human being. We should all be thankful for those people who

rekindle the inner spirit.

Albert Schweitzer

i i i


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ACKNOWLEDGMENTS

First and foremost, I would like to thank my family for their unwavering support.

No matter how stubborn I can be, I thank my parents and brother for their continued

support. Professor Jason Telford has m eant just as much to me in addition to allowing

me to do research in his group, he made sure that we did not lose sight of the important

things in life and always treated us like a part of his own family. I canno t imagine a mo re

understanding advisorno matter what happened, he was there to support us in our

pursuits. Both Professors Telford and Franklin provided a nurturing environment in

which I was taught to continue to strive and set higher goals. W ithout both of them I

would n ot be where I am today or the scientist that I am.

I have made numerous friends throughout graduate school, all of which have had

a profound impact in my life. Mary Daly who m ade sure I did not lose sight that there

was a world outside of the lab. Ram on Cuellar, a friend wh o was always there no matter

what time or how much he had on his plate. Nathan Lien whom mad e the days in lab

(and sometimes the yard ) more light-hearted. Samantha Soebbing-Nolting ('my

partner in crime'don't fret the t-shirt is coming) was always there when I had a

questionno matter what pain I went through, we went through it together Koushik

Banerjee for being there w hen I needed help w ith organic synthesis, making sure I got out

of the lab and could cook Asif Rahaman always there to talk to and relax withhe w as

not here during the early years of my graduate career, but he made sure I remembered

those days only to be younger again (old age hits hard and fast you know ). Sai

Kumar Ramadugu and Harsha Vardhan Reddy Annapureddy for making sure that I

remembered one of the most vital things, making sure we do not lose sight of ourselves

no matter how much there is to accomplishthe right balance of everything always

survives all odds.

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I would also like to thank the remaining members of the Franklin and Telford

groups who made lab life much more enjoyable: Dr. Yurndong (David) Jahng, Anthony

Prudden, Heaweon Park, Kinesha Harris, Sui Wah Wong-Deyrup, Oksana Chernyshev,

Chris Lim, and Sarah Brookhart Sh ields. I would also like to thank Dr. Lynn Teesch for

allowing me to be a research assistant in the HRMSF and, along with Vic Parcell, taught

me m ore than I would have otherwise learned about m ass spectrometry.

In addition to fellow classmates, there have been many professors and support

staff that have taught me and helped through those rough moments of graduate school.

One of the most inspiring is my undergraduate advisor, Dr. Kenneth W . Olsen. No

matter what mistakes I made, he guided my early scientific mind and allowed me to

realize the potential enjoyment that comes from good science. Professor Ned Bow den

who taught me numerous techniques, the process of setting up a lab and always made me

feel like I had imm easurable poten tial. Professor Louis Messerle , who was always there

when I needed chatno matter how frustrated I was, he provided a soothing voice of

reason. Professors Pienta, Hansen, Pigge, Kohen, Meade, and, of course, Telford w ho

have helped mold my ability to teach students and ensure that I constantly improved at

teachin g. Lastly, I wo uld like to thank all support staffM ichele Gerot for dealing with

all my teaching related concerns; Janet Kugley and Sharon Robertson who always

provided reminders/updates and for dealing with important administrative tasks;

Santhana Vellupillai and Rob Brown for their help with NMR experiments and analysis;

Frank Turner for all the favors fixing equipment or crafting things; Gene Hauge and Tim

Koon for their help whenever I needed to order something or could not find something;

and Nelson R eyes-Burgos for those late night chats.

Lastly, I would like to thank my past and present comm ittee m embers (Professors

Eyman, Jensen,Goff, Kay, Messerle, Quinn, and Telford) for their constructive criticism

and not beating me over my head for all my idiosyncrasies when it came to noun and

verb agreement.

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Without all of these people I would not be the person that I have become today.

Their influences will last with me a nd always guide me on m y path to strive to be a better

scientist and, m ore impo rtantly, a better pe rson.

V I


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ABSTRACT

The overarching theme reflected herein is the molecular recognition of organic

substrates or environm entally relevant anions and cations. The synthesis of modified

cyclodextrins as molecular scaffolds for the inner- or outer-sphere coordination of Ln

m

cations or tetrahedral anions, respectively, is outlined. Further studies on the

complexation of organic substrates with (3-cyclodextrin and a per-6-modified (3-

cyclodextrin are also reported.

The outer-sphere coordination of tetrahedral anions (specifically perchlorate,

phosphate, and sulfate) has been targeted. Over the past two decades num erous studies

utilizing ESI-M S for the determination of binding affinities ha ve been reported. The

relatively soft-ionization by ESI-MS provides numerous advantages. Most importantly

are its abilities to model the solution phase closely and provide the unambiguous

stoichiometry of the complex(s) formed. Binding studies of a-cyclodextrin (1) and per-6-

(2-aminoethylamino)-per-6-deoxy-a-cyclodextrin (6) with perchlorate, phosphate or

sulfate were examined using ESI-MS. The lack of specific binding of a-cyclodextrin is

evidenced with the inclusion of multiple perchlorate ions and competition for binding

with the cations (Li

+

, M g

2+

, K

+

) of the particular tetrahedral an ion. Contrary to this, per-

6-(2-aminoethylamino)-per-6-deoxy-a-cyclodextrin (6) shows a preference for binding

anions from solution with modest log K

a

values (~ 3.6 - 4.0), and predominantly a 1 : 1

(6 : Tetrahedral Anion) binding stoichiometry.

Further studies report the synthesis of carboxylic acid-functionalized per-6- and

mono-6-modified (3-cyclodextrins for the inner-sphere coordination of the lanthanides

and actinides. Synthe sis of per-6-5'-(thiosalicylic acid)-per-6-d eoxy-P -cyclodex trin

exhibited solubility issues below a pH ~ 5. Conse quently, potentiom etric studies with

this cyclodextrin could not be performed. Additional studies of carboxylic acid mono-6-

modified P-cyclodextrins were pursued . The synthesis of mono -6-(2-(diethy lenetriam ine

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pentaacetic acid-amino)ethylamino)-mono-6-deoxy-P-cyclodextrin is reported, along

with analysis of binding w ith europium, terbium, and gadolinium using fluorescence and

ESI-MS.

The last body of this work details host and guest complexes formed betw een three

small organic molecules with native P-cyclodextrin (2) or per-6-(2-aminoethylamino)-

per-6-deoxy-(3-cyclodextrin (12 ). Determ ination of binding affinities o f these three guest

molecules with these cyclodextrins are reported using UV-V is spectroscopy.

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TABLE OF CONTENTS

LIST OF TABLES xi

LIST OF FIGUR ES xii

LIST OF ABBR EVIATIONS xix

CHAPTER

1 OUTER-SPHERE COORD INATION OF METAL COM PLEXES IN

BIOLOGICAL AND CYCLODEXTRIN-BASED SYSTEMS 1

1.1 Backg round 1

1.2 Natu ral Systems 2

1.2.1 Bacterial Iron Transpo rt 2

1.2.2 M amm alian An tibacterial Proteins 3

1.2.3 Blue Copp er Proteins 4

1.2.4 Recognition of M g( H

2

0)

6

2+

6

1.3 Cyclod extrin-based Systems 7

1.3.1 Host-G uest Chem istry of Me tal Com plexes 8

1.3.2 W aste rem ediation 11

1.4 Ca talysis 12

1.4.1 Horserad ish Peroxidase (HRP ) M imic 13

1.4.2 W ater-soluble Phosp hane with Cyclo dextrin 14

1.5 Future Outlook 14

Notes 17

2 ANION RECOGNITION USING A CYCLODEXTRIN-BASED

SCAFFOLD 21

2.1 Introduction 21

2.1.1 Oxoanion Recognition Chemistry 21

2.1.2 Outer-sphere Coordination Using Cyclodextrins 21

2.1.3 Tetrahedral Anions 23

2.1.4 Determ ination of Binding Affinities 23

2.2 Experimental 24

2.2.1 ESI-MS 24

2.2.2 Synthesis 25

2.2.3 Titrations 25

2.2.4 Calculation of Bindin g Affinities 26

2.2.5 pK

a

Determination 27

2.2.6 T\Inversion Recovery Experiments 27

2.3 Results and Discussion 28

2.3.1 Binding mode of Tetrahedral Anions 35

2.4 Conclusions 36

Notes 40

3 CATION RECOGNITION USING ACID-MODIFIED

CYCLO DEXTRIN-BASED SCAFFOLDS 42

3.1 Introduction 42

IX


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3.1.1 Cation Recogn ition.. 42

3.1.2 Nuclear Waste Refinement 44

3.1.3 Lanthanides and Actinid es 45

3.1.4 Supramolecular Host and Choice of Functional Group 46

3.2 Experim ental 50

3.2.1 Synthesis 50

3.2.2 Fluorescence Measurem ents 62

3.2.3 Ma ss Spectrometry 63


3.3.1 Synthesis 75

3.3.2 Fluorescence 78

3.3.3 Mass Spectrom etry 79

3.4 Conc lusions 80

3.5 Future Work 80

Notes 82

4 ORGANIC SUBSTRATES BINDING WITH NATIVE AND

MO DIFIED (3-CYCLOD EXTRINS..... 85

4.1 Introduction 85

4.2 Experimental 85

4.2.1 Synthesis 86

4.2.2 UV-V is Spectroscopy 87

4.2.3 Crystallization 88

4.2.4 ESI-MS Measurem ents .88


4.4 Conclusions 97

Notes 98

5 SUMMARY AND FUTURE WORK 99

APPENDIX A DETERMINATION OF BINDING CONSTANTS USING ESI-

MS 101

A. l Importance and Background of Binding Affinities 102

A.2 Utility of ESI-M S in Determ ining Binding Affinities 103

Notes 106

APPENDIX B SPECTRA AND RELEVANT DATA FOR THE BINDING OF

TETRAHEDRAL ANIONS WITH 1 AND 6 107

APPENDIX C SPECTRA: MOD IFIED CYLODEX TRINS 140

REFERENCES 167

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LIST OF TABLES

Table

2.1 Properties of Tetrahedral Anions Studied with Cyclodextrin 6 30

2.2 Sample Calculations for the Titration of 6 with LiC104 31

2.3 Delay Time Prior to ^ - N M R Spectrum Collection for Experiments

Performed to Determine T\Relaxation Time Perturbations 38

2.4 Experimentally Calculated T\Relaxation Times (sec) for

H-N MR Peaks of

Cyclodextrin 6 Titrated with L iC10

4

39

3.1 Ionic Radii of the Lanthan ides (A) 49

4.1 Log K

a

of Guest Molecules with Cyclodextrins 2 or 12 90

B.l ESI-MS Intensities of Host(I

H

)and Host / Guest Complex (7#

G

) for 6 Titrated

withLiC10

4

133

B.2 ESI-M S Intensities of Host (///) and Host / Guest Comp lex (I

HG

)for 6 Titrated

w i t h M g S 0

4

134

B.3 ESI-M S Intensities of Host (7#) and Host / Guest Comp lex (I

HG

)for 6 Titrated

w i t h K H

2

P 0

4

135

B.4 Relative

J

H-N MR Peak Heights for peaks of interest of

per-6-(2-

aminoethylamino)-per-6-deoxy-a-cyclodextrin(6) 136

B.5 Relative 'H-NM R Peak Heights for peaks of interest ofper-6-(2-

aminoethylamino)-per-6-deoxy-a-cyclodextrin (6) titrated with LiC10

4

(1 :

1.2) 137

B.6 Relative ^ - N M R Peak Heights for peaks of interest of

per-6-(2-

aminoethylam ino)-per-6-deoxy-a-cyclodextrin (6) titrated with LiC10

4

(1:

4) 138

B.7 Relative 'H-N MR Peak Heights for peaks of interest of

per-6-(2-

aminoethylamino)-per-6-deoxy-a-cyclodextrin (6) titrated with LiC10

4

(1 :8)....139

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LIST OF FIGURES

Figure

1.1 Diagram of(A) the export of a siderophore from bacterial cell, (B)

complexation with Fe

+

, (C) uptake of the siderophore-Fe

3+

complex by an

outer membrane cell surface receptor. (OM = outer membrane, IM = inner

membrane)

1.2 A view of the binding site of Fhu showing hydrogen bonding interactions to

the octahedral iron center (PDB Accession C ode: 1QFF)

1.3 Repre sentations of a-, |3-,and y-cyclod extrins. The top structure represents

the full drawn chemical structures of a-, P-, and y-cyclodextrins (b = 1, 2, or

3,

respectively). The lower center and right structures are shorthand

representations of the a-,

(3-,

and y-cyclodextrins (n = 6, 7 or 8, respectively).

Lastly, the lower left-most represen tation is a cartoon of toroidal sha pe. The

dispositions of the primary and secondary hydroxyls are presented. The

edges ringed with

1

and 2 hydroxyls are referred to as the lower and upper

rims,respectively 10

1.4 Representation of the inclusion of a guest molecule within the cyclodextrin

cavity. Guest molecules could include organic molecules, inorganic metal

complexes, cations, or anions 11

1.5 (a) 3-D mod el of uranyl carbonate showing itsD symmetry and available

hydrogen bond acceptors (carbon = gray, oxygen = red, uranium = yellow)

(b) a portion of the ^ - N M R spectrum of per-6-(2-aminoethylamino)-per-6-

deoxy-ot-cyclodextrin titrated with uranyl carbonate shows the clear upfield

shift of proton resonan ces near the bindin g site 13

1.6 (a) F e

m

2-hydroxy-l-naphthaldehyde thiosemicarbazone (HNT) Schiff-base

complex. This figure shows a three-coordinate Fe; however, the structure is

actually either a square pyramidal or octahedral geometry which utilizes

another HNT ligand to satisfy the additional bonding sites, (b) Representation

of the Schiff-base salicylidene-2-amino-4-phenylthiazole complex (M

n

-

(SAPTS)

2

) 15

1.7 Phosp hane ligand for complexation with a metal ion (e.g. Pd[4]3) and

inclusion into the cyclodex trin cavity 16

2.1 Synthetic transformation for a-cyclodextrin (1) to per-6-iodo-a-cyclodextrin

(5).

Rem oval of the iodides via nucleo philic attack by either of the free

terminal amines of e thylenediamine results in the f inal per-6-(2-

aminoethylam ino)-per-6-deoxy-a-cyclodextrin (6) product 22

2.2 pH-dependent speciation diagram based on estimated P values for the hexa-

protonated per-6-(2-aminoethylamino)-per-6-deoxy -a-cyclodextrin (6) 32

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2.3 Calculated T\ relaxation times in graphical format for protons of interest in

the 'H-N M R spectrum. The only significant deviation greater than 10% is

seen at 8 3.61 ppm . The yellow line indicates a perturbation greater than 10%

at 5 4.01 ppm ; however, no deviation is seen at 8 4.01 ppm with 8 equivalents

of perchlorate indicating that its relaxation time is most likely unaffected by

the perchlorate 33

2.4 Generic representation of a tetrahedral anion (TA = C 10

4

",SCV",orPCV")

binding to the upper-rim of per-6-(2-aminoethylam ino)-per-6-deoxy-a-

cyclodextrin. Depending on the tetrahedral anion binding and charge state of

the species in M S, the charge of the final species in the gas phase will vary,

therefore n would indicate the appropriate charge to get the overall charge on

the species and w ould be the charge of the each respective tetrahedral anion.

The binding mode seen in this diagram is only assumed as it provides the

mo st likely electrostatic interaction 34

3.1 Structures of 12-crown-4 and 18-crown-6 which are know n to bind cations K

+

and Na

+

, respectively 43

3.2 Rela tion betw een ionic radius and binding free energy (AG) for the trivalent

lanthanide species with the peptide from Imperiali

et.

al. The table included

provides Kd values for each trivalent lanthanide and their peptide 44

3.3 7. Representation of a calix[n]arene with 4-8 mono mers. 8. Repesentation of

carbamoylmethylphosphine oxide. 9/10. Representations of calix[4]arene

modified with a CMPO derivative and with the lowers-methyl group also

modified 46

3.4 The decrease ('lanthanide contraction') in the ionic radii of the Ln species is

apparent from the above graphical representation w here as Z increases, the

radius decreases 48

3.5 A generic represe ntation of

(a)

per-6-modified P-cyclodextrins and (b) mono-

6-modified P-cyclode xtrins. In unmo dified natural P-cyclodex trin R = -OH 48

3.6 Rea ction sequenc es for the synthesis of per-6-mo dified P-cyclodextrins (11 -

15). The arrow for the synthetic route of

12

to 15 is marked w ith an ' X '

through it to denote that the reaction did not generate the desired product 56

3.7 Synthesis of16 57

3.8 Syntheses of 17 and 18 58

3.9 Synthesis of19 59

3.10 Synthes is of 20 60

3.11 Representations of the functional groups attached to the per-6- or mono-6-

modified P -cyclodextrins. R is the location where the cyclodextrin group is

attached at C6. For the per-6-modified p-cyclodextrins each of the C6

positions is modified w ith the labeled group; wh ereas, for the mon o-6-

modified P-cyclodextrins only one of the seven C6 positions is modified with

the labeled group. Each position for a C-H bond is labeled for ease of NM R

assignments for the ^ - N M R listed in the characterization of each synthesis 61

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3.12 Titration of Eu

3+

with up to 5 equivalents of

19 .

The increase in the

fluorescence emission at 615 nm is a result of the exclusion of water from the

first coordination sphere of the Eu

+

65

3.13 Relative fluorescence of Eu

3+

w hen titrated w ith 18 , one of the starting

materials for the synthesis of15 . Imp ortantly, there is no increase in the

fluorescence intensity at 615 nm as seen inFigure 3.12 66

3.14 Titration ofTb with up to 5 equivalents of

19 .

The increase in the

fluorescence emission at 545 nm is a result of the exclusion of water from the

first coordination sphere of the Tb

3+

67

3.15 Titration ofT b

3+

with up to 5 equivalents of

18 .

Importantly, there is no

increase in the fluorescence intensity at 545 nm as seen in

Figure 3.14

68

3.16 Binding curve generated from fluorescence emission at 615 nm ofE u

3+

titrated with 19 is provided in black. The calculated fluorescence emission

based upon fitting to equation 3.1 for determination of Kd is provided in

orange 69

3.17 Bind ing curve generated from fluorescence em ission at 545 nm of Tb

titrated with 19 is provided in black. The calculated fluorescence em ission

based upon fitting to equation 3.1 for determination ofK


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4.3 UV -Vis absorbance of 23 titrated with 2 from 265 nm - 385 nm. No m ajor

absorban ce shifts are noted 92

4.4 UV -Vis absorbance of 25 titrated with 2 from 265 nm - 385 nm. A blue shift

in the absorption maxim um at 340 nm to 332 nm is noted 93

4.5 UV -Vis absorbance of 25 titrated with 12 from 265 n m - 385 nm. A blue

shift in the absorption maximu m at 340 nm to 332 nm is noted 94

4.6 Negative ESI-MS for the analysis of binding of 25 with P-cyclodextrin (2).

At a ratio of1: 1 (25 / 2), it is evident that th ere are both the free forms of 2

and 25 predominant in solution 95

B. l ESI mass spectrum for 1, a-cyclodextrin 108

B.2 ESI ma ss spectrum for 1, a-cyclo dextrin titrated with LiC104 (1 : 3) 109

B.3 ESI mass spectrum for 1, a-cyclodextrin titrated with

MgSC>4

(1 : 3) 110

B.4 ESI mass spectrum for 1, a-cyclo dextrin titrated with KH2PO4 (1 : 7) 111

B.5 ESI mass spectrum for 6, per-6-(2-aminoethylamino)-per-6-deoxy-a-

cyclodextrin 112

B.6 ESI ma ss spectrum for titration of6, per-6-(2-aminoethylamino)-per-6-

deoxy-a-cyclodextrin withL iClO 4(10 : 1) 113

B.7 ESI m ass spectrum for titration of6, per-6-(2-aminoethylamino)-per-6-

deox y-a-cy clode xtrin with LiC104 (1 : 1) 114

B.8 ESI mass spectrum for titration of6, per-6-(2-aminoethylamino)-per-6-

deoxy-a-cyclodextrin with LiC104 (1 : 5) 115

B.9 ESI mass spectrum for titration of 6, per-6-(2-aminoethylamino)-per-6-

deox y-a-cy clodex trin with LiC104 (1 : 10) 116


deoxy-a-cyclodextrin with Mg S04 (10 : 1) 117

B.l

1

ESI mass spectrum for titration of 6, per-6-(2-am inoethylamino)-per-6-

deoxy-a-cyclodextrin with Mg S04 (1 : 1) 118


deoxy-a-cyclodextrin with M gS 04 (1 : 5) 119

B.l3 ESI mass spectrum for t i t ra t ion of 6 , per-6-(2-aminoethylamino)-per-6-

deoxy-a-cyclodex trin with M gS04 (1 : 10) 120


deoxy-a-cyclodextrinwithK H2PO 4(10 : 1) 121

B .l5 ESI mass spectrum for titration of 6, per-6-(2-aminoethylamino)-per-6-

deox y-a-cy clodex trin with KH2PO4 (1 : 1) 122

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B.16 ESI mass spectrum for titration of 6, per-6-(2-aminoethylamino)-per-6-

deox y-a-cy clodex trin with KH2PO4 (1 : 5) 123

B.17 ESI mass spectrum for titration of

6,

per-6-(2-aminoethylamino)-per-6-

deoxy-a-cyclodextrin with KH

2

P0

4

(1 : 10) 124

B.

18 Linear regression attained by inputing data from the first titration of 6 with

LiC104 to equation

A.6.

The slope of the line is then used to generate the Kd

value as outlined in

Appendix

A 125

B.19 Linear regression attained by inputing data from the second titration of

6

with

LiC104 to equation A .6. The slope of the line is then used to gen erate the Kd


Appendix

A 126

B.20 Linear regression attained by inputing data from the third titration of6with

LiC104 to equation A. 6. The slope o f the line is then used to genera te the Kd


Appendix

A 127

B.21 Linear regression attained by inputing data from the first titration of 6with

M gS0 4 to equation A . 6. The slope of the line is then used to g enerate the Kd


Appendix A

128

B.22 Linear regression attained by inputing data from the second titration of 6 with

MgSC>4

to equation A.6 . The slope of the line is then used to gene rate the Kd

value as outlined inAppendix A 129

B.23 Linear regression attained by inputing data from the first titration of 6 with

KH2PO4 to equation A.6 . The slope of the line is then used to generate the Kd

value as outlined inAppendix A 130

B.24 Linear regression attained by inputing data from the second titration of 6 with

KH2PO4 to equation A.6 . The slope of the line is then used to generate the Kd


Appendix A

131

B.25 Linear regression attained by inputing data from the third titration of 6 with

KH2PO4 to equ ation A.6 . The slope of the line is then used to generate the Kd


Appendix A

132

C.l Positive ESI-MS spectrum for 6, per-6-(2-aminoethylam ino)-per-6-deoxy-a-

cyclodextrin 141

C.2 'H-N MR spectrum for 6, per-6-(2-aminoethylamino)-per-6-deoxy-a-

cyclodextrin 142

C.3 Positive ESI-M S spectrum for 12, per-6-(2-aminoethy lamino)-per-6-deo xy-(3-

cyclodextrin 143

C.4 ^ - N M R spectrum of for 12, per-6-(2-aminoethylamino)-per-6-deoxy-P-

cyclodextrin 144

C.5 Positive ESI-MS spectrum of

14,

per-6-5'-(thiosalicylic acid)-per-6-deoxy-P-

cyclodextrin (

[1 4

+ K

+

]

+

= 2125 m/z) along with breakdown products 145

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C.6 ^ - N M R spectrum from 8 6.2 - 8.4 ppm of14 ,per-6-5'-(thiosalicylic acid)-

per-6-deoxy-P-cyclodextrin 146

C.7 ^ - N M R spectrum from 8 2.0 - 5.3 ppm of14 ,per-6-5'-(thiosalicylic acid)-

per-6-deoxy-(3-cyclodextrin 147

C.8 Positive ESI-MS spectrum for 16, mono-6-tosyl-mono-6-deoxy-P-

cyclodextrin 148

C.9 'H-N M R spectrum from 8 4.5 - 8.6 ppm of

16 ,

mono-6-tosyl-mono-6-deoxy-

P-cyclodextrin 149

C IO ^ - N M R spectrum from 8 1.5 - 4.2 ppm of16 , mono-6-tosyl-mono-6-deoxy-

P-cyclodextrin 150

C.l1Positive ESI-MS spectrum of

17 ,

mono-6-iV-(p-aminonicotinic acid)-mono-6-

deoxy-(3-cyclodextrin 151

C.12 ^ - N M R spectrum from 8 5.9 - 9.0 ppm of

17 ,

mono-6-JV-(p-aminonicotinic

acid)-mono-6-deoxy-P-cyclodextrin 152

C.13 ^ - N M R spectrum from 8 3.2 - 5.3 ppm of

17 ,

mono-6-A^-(p-aminonicotinic

acid)-mono-6-deoxy-p-cyclodextrin 153

C.14 ^ ^ H COSY of

17 ,

mono-6-JV-(p-aminonicotinic acid)-mono-6-deoxy-mono-

6-deoxy-P-cyclodextrin

154

C.15 'H - ^ C H M Q C o f H , mono-6-iV-(p-aminonicotinicacid)-mono-6-deoxy-P-

cyclodextrin 155

C.l6 ESI-MS spectrum of

18 ,

mono-6-(2-aminoethylamino)-mono-6-deoxy-P-

cyclodextrin 156

C.l7

H-NMR spectrum of

18,

mono-6-(2-aminoethylamino)-mono-6-deoxy-P-

cyclodextrin 157

C.l8 Negative ESI-MS spectrum of

19 ,

mono-6-(2-(diethylenetriamine pentaacetic

acid-amino)ethylamino)-mono-6-deoxy-P-cyclodextrin 158

C.l9 ' H - N M R o m mono- mono-6-(2-(diethylenetriamine pentaacetic acid-

amino)ethylamino)-mono-6-deoxy-P-cyclodextrin 159

C .20 ^ H C OS Y of

19 ,

mono-6-(2-(diethylenetriamine pentaacetic acid-

amino)ethylamino)-mono-6-deoxy-P-cyclodextrin. 160

C .2 1 ^ - ^ C H M Q C o f l ^ m o n o - 6- (2 - (d ie th y le n et ri am i n e p e nt aa ce ti c a c id -

amino)ethylamino)-mono-6-deoxy-P-cyclodextrin 161

C.22 P ositive ESI-MS spectrum of20, mono-6-(2-(dansylamino)ethylamino)-

mono-6-deoxy -P-cyclodextrin ; 162

C.23 ^ - N M R spectrum from 8 7.1 - 9.2 ppm of20,mono-6-(2-

(dansylamino)ethylamino)-mono-6-deoxy-P-cyclodextrin 163

xvii


20/194

C.24 ^ - N M R spectrum from 8 2.3 - 5.5 ppm of20,m ono-6-(2-

(dansylamino)ethylamino)-mono-6-deoxy-P-cyclodextrin 164

C .25 ^ H C OSY of

20,

mono-6-(2-(dansylamino)ethylamino)-mono-6-deoxy-p-

cyclodextrin 165

C.26 'H-

13

C HMQC of

20,

mono mono-6-(2-(dansylamino)ethylamino)-mono-6-

deoxy-P-cyclodextrin 166

xvm


21/194

LIST OF ABBREVIATIONS

ACN

CMPO

COSY

ddH

2

0

DEAE

DTPA

ESI

ESI-MS

Fhu

FRET

HEPES

HMQC

IROMP

/PrOH

MeOH

MS

MWCO

NGAL

NMR

PBP

PUREX

TC

TRLFS

Acetonitrile, CH

3

CN

Carbamoylmethylphosphine oxide

Correlation spectroscopy

Distilled-deionized water

2-(diethylamino)ethyl

Diethylene triamine pentaacetic acid

Electrospray ionization

Electrospray ionization m ass spectrometry

Ferric hydroxamate uptake

Fluorescence resonance energy transfer

4-(2-hyd roxyethy l)-1 -piperazineethan esulfonic acid

Heteronuclear multiple quantum coherence

Iron responsive outer-memb rane protein

Isopropanol

Methanol

Mass spectrometry

Molecular weight cut off

Neutrophil gelatinase-associated lipocalin

Nuclear magnetic resonance

Phosphate binding protein

Plutonium and uranium recovery extraction

To-contain

Time-resolved laser fluorescence spectroscopy


22/194

1

CHAPTER 1

OUTER-SPHERE COORDINATION OF METAL COMPLEXES IN

BIOLOGICAL AND CYCLODEXTRIN-BASED SYSTEMS

1.1 Background

This background text has been adapted from the following publication: "Outer-

sphere Coordination of Metal Complexes in Biological and Cyclodextrin Systems",

Harwani, S.; Telford, J. R. Chemtracts, 2 0 0 5 ,18(8 ) , 437-448.

The use of both inner- and outer-sphere coordination play fundamental roles in

metal-ion reactivity.

1

"

3

Inner-sph ere coordination (the first coord ination sphe re) refers to

the ligands directly bound to a metal; in Nature, this is most often realized by amino acids

and a few other cofactors

(i.e.

ligands ). The inner coordination sphere around meta ls

have been studied extensively over time ; although m ost instances of metal-bound systems

characterized to date fall within the realm of supra-molecular and molecular recognition

using outer-sphere coordination. Outer-sphere coordination (second coordination sphere)

specifically refers to those interactions outside the inner or primary coordination sphere.

Examples include ordered or disordered solvent and amino acid residues which utilize

hydrogen bonding or dipolar interactions with ligand atoms. In protein systems, a

receptor provides a binding site with a spatial arrangement of hydrogen bonds or

hydro phob ic patches to target a specific substrate. The interactions employ ed to bind a

ligand in natural protein systems consist mainly of (1) hydrogen bonds, (2) TC-U and other

donor-acceptor interactions, (3) electrostatic interactions, and (4) bonding from the direct

overlap of metal orbitals with outer-sphere ligands.

1

"

4

In addition to naturally occurring systems there are a number of synthetic

achievements in the form of supra-molecular scaffolds designed to bind metal com plexes.

These scaffolds have been applied to a variety of tasksmainly in the areas of enzyme

mim ics, medicinal chemistry, and environmental waste remediation. Utilizing many of


23/194

2

the same interactions as natural systems, much effort has been directed towards

mimicking biological systems using supramolecular scaffolds such as cyclodextrins.

This review focuses on some recent developments in outer-sphere coordination chem istry

with an em phasis on: (1) natural systems and (2) cyclodextrin-based systems.

1.2 Na tural System s

1.2.1 Bacterial Iron Transport

Recognition, transport, and assimilation of nutrients are essential for bacteria to

satisfy their growth requirements.

5

'

6

This is often accom plished by outer-sph ere

recog nition of the nutrient substra te. Of specific interest are sideropho res, small, iron-

binding compounds produced by bacteria, which serve as a paradigm for micro-nutrient

acquisition. Siderophore transport and internalization also provides an exam ple of outer-

sphere recognition of metal com plexes (Figure 1.1).

Siderophores play an important role in bacterial and fungal life cycles.

7

"

11

An

important step in the iron uptake pathway is the internalization of the Fe-siderophore

com plex. This is accomplished by a variety of outer mem brane transport proteins. These

transport proteins are expressed under iron limiting condition, and therefore, are

generally classified as iron responsive outer-membrane proteins (IROM P). IRO MP s are

either exceed ingly specific or generic in their recognition of substrate. Specific IRO M Ps

may recognize one or a few substrates, while generic IROMPs recognize a wide variety

of a class of siderop hores, such as any iron-tris-hydrox am ate com plex. This variation in

substrate recognition is one of several ways that bacteria compete for the multitude of

siderophore architectures.

The ferric hydroxamate uptake (Fhu) protein is an IROMP that generically

transports iron-bound hydroxamate siderophores. Recognition is effected by hydrogen

bonding contacts with the octahedral metal center (Figure 1.2) rather than contacts with

the organ ic sideroph ore back bon e. The Fhu protein is therefore used as an exam ple for


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3

the general phenomenon of iron uptake, and an interesting example of metal-centric

recog nition. It is only after the binding of Fe-sidero phore by Fh u that uptake into the cell

can occur.

Figure 1.1

Diagram of (A) the export of a siderophore from bacterial cell, (B)

complexation with Fe

3 +

, (C) uptake of the siderophore-Fe

3+

complex by an outer

membrane cell surface receptor. (OM = outer membrane, IM = inner membrane).

1.2.2 M am ma lian An tibacterial Protein s

Another example of iron-dependent recognition proteins is the lipocalin binding

proteins; however, lipocalins perform a very different function by withholding the Fe-

sideropho re com plex from the bacteria. In essenc e, lipocalins are beating bacteria at their

own game of iron acquisition. One mem ber of the lipocalin family, neutrophil

gelatinase-associated lipocalin (NGAL), has been implicated in numerous cellular

processes

13

"

15

, such as providing an Fe recognition system. Recent studies have

elucidated the x-ray crystal structure of the protein.

16

"

18

NG AL contains an unusually

shallow and broad binding pocket (calyx) lined with polar and positively charged


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4

residu es. The NG A L calyx has an affinity for small hydro phob ic mo lecules . In

particular, it has recently been sho wn that the NGA L calyx has a very high affinity for

Fe-enterobactin (a cyclotriserine-based siderophore), binding and stabilizing the

siderophore from hydrolytic degradation into its monomeric building blocks.

19

'

20

Experimental studies of various substrates binding to the NGAL calyx indicate that the

positive charge and polarity of the residues in the binding pocket are critical for

binding . ' This has also been revealed throug h additional studies wh ich demonstra te

that NGAL does not bind positively charged iron alone ([Fe(H20)6]

3+

), suggesting an

electrostatic contribution to the interaction between the negatively charged Fe-

enterobactin complex and the positively charged NGA L calyx. Overall, it has been

proposed that NGAL and other lipocalins are bacteriostatic agents used by the immune

system in order to fight off bacterial infections more efficiently by removing a vital

bacterial iron sourc e. Other similar mechan ism s for iron hom eostasis in the body , such as

transferrin and hepicidin-dependent uptake mechanisms (along with NGAL) are

described in a review by Kaplan.

21

1.2.3 Blue Copper Proteins

Outer-sphere influences are seen not only through the interaction of two unique

molecules (host and guest), but also intramolecularly through the influences that protein

backbones have on the coordination of metal complexes, as observed in blue copper

prote ins. C rystallograp hic studies of azurin and plastocyan in show that the back bone

of the protein is preorganized to bind the copper ion, with halo forms essentially identical

to their respective apo forms. This preorganized structural mo tif is seen with many blue

c o p p e r p r o t e i n s .

In pseudoazurin, the redox potential of the copper ion is sensitive to an extended

array of amino acid residu es. One of the first mutational studies altered the proline (P80)

adjacent to the metal center to alanine or isoleucine. These mu tations resulted in an


26/194

5

increase in reduction potential, as the adjacent histidine is not as rigidly enforced in

position nor is it as solvent exposed.

24

Recently published data with mutations on

amicyanin give similar results. Mutations were constructed in the binding loop because

these are thought to relax the ligation around Cu

1

allowing it to assume its preferred

geometry and thus, increase the reduction potential,.

25

The two mutants studied, P94A

and P94F, have a shift in reduction potential by +100 m V, from = 265 mV to 380 mV

and 415 m V, respectively

25

Figure 1.2

A view of the binding site of Fhu showing hydrogen bonding interactions to

the octahedral iron center (PDB Accession Code: 1QFF).

1


27/194

6

This interpretation is further corroborated by quantum mechanical calculations by

Li et.al. Their calculations indicate that the dominant influences on the copper reduction

potential in type I blue copper proteins are residues within 6A of the copper center(s) that

contribute H-bonding.

26

Their studies also show influences by three key factors: (1) axial

ligand interactions, (2) hydrogen bonding to S

cys

, and (3) constraint of the inner-sphere

ligand orientation viathe protein backbone. These results, along with previous studies,

indicate that the hydrogen bonding network indirectly influences the geometry around the

copper in the active-site(s) and thereby controls the physical properties of the metal. The

experimental data from mutations in the binding loop of the blue copper proteins coupled

with the quantum mechanical data provide additional support that the electronic

properties of the copper center(s) are modulated by the inner-most H-bonding array to the

copper ligands.

1.2.4 Recognition of M g(H

2

0)

6

2+

Many natural biological systems require the use of [Mg(H20)6]

2+

for stability and

or catalytic activity of enzym es. A survey of the Cambridge Structural Database (CSD)

done in 1994 by Bock et. al. revealed the stability of hexaaquamagnesium(II) in many

crystal structures.

27

Systems such as topoisom erases, ribonucleases, and bacterial

transport systems, are known to include Mg

2+

(

fli?

) as a cofactor. Magnesium has poorly

defined requirements for coordination, and it is not clear how the ion is recognized or

coordinated in biological systems. In order to probe how Mg

+

(

a?

) is coordinated, the

inert hexammine cobalt(III) complex, [Co(NH3)g]

3+

, has been utilized as a surrogate.

[Co(NH3)6]

3+

is thought to bind in proteins via outer-sphere coordination, because the

amine ligands are kinetically inert, and not readily displaced. Thus, binding to the

complex must occur through the amine ligands, rather than directly to the metal.

Analysis of proteins with [Co(NHs)6]

3+

bound to Mg

2+

(

a?

) sites suggests that


28/194

7

hexaaquamagnesium(II) also binds via an outer-sphere interaction with the aqua

ligands.

28

"

32

Even though much work has been done in this area, novel methods of studying

the outer-sphere nature of aqueous cations are still being developed. Com putational

studies, along with infrared and Raman spectroscopies, have been used to probe the

binding modes of Mg

2 +

and Ca

2+

with dimethyl phosphate anion (DMP")an anion used

to mimic the phosphate moiety. These studies indicate that the calcium cationic center

prefers at least one inner-sphere contact with a hard oxygen donor from DMP", while the

magnesium cation prefers outer-sphere binding through the aqua ligands.

33

1.3 Cyclodextrin-based Systems

Cyclodextrins (Figure 1.3) can be used in the recognition of different molecules

(or complexes) and have been proposed for a variety of possible applications.

Cyclodextrins are polymeric glucose molecules consisting of 6, 7, or 8 a-(l->4) linked

glucose moieties, known as a-, P-, and y-cyclodextrin, respectively.

34

'

35

The diameter of

the cyclod extrin cavity increases as the numbe r of glucose residues increases from a-, p \

and y-cyclodextrin, with cavity diameters of -5 .2 , ~6.6, ~8.4 A, respectively. Many areas

exploiting the utility of cyclodextrins have been targeted, including both their host-guest

chemistry and synthetic methods for functionalizing the upper and lower rims of

cyclodextrins.

Host-guest chemistry of cyclodextrins has been studied extensively for the

complexation of hydrophobic molecules for applications, such as drug delivery, chiral

recognition, enzyme mimics, and molecule detection, including secondary detection

m et h o d s s u ch a s f l u o res cen ce en h an cem en t o r q u en c h i n g . " In o rd e r t o s e l ec ti v e l y

comp lex molecules and increase the solubility of com pounds, native cyclodextrins are not

used often due to their mod est solubility. The synthesis of mod ified cyc lodex trins,

therefore, has received much deserved attention. The ability to functionalize one or all of


29/194

8

the primary hydroxyl groups on the upper rim and the secondary hydroxyl groups on the

lower rim of cyclodextrin molecules allows for the development of a wide range of

possible outer-sphere hosts or enzyme m imics, The multitude of possible hosts presents

the ability to target the com plexation of certain metal ions or substra tes. Hos t-guest

chemistry and the synthetic methods for the modification of the upper and lower rims of

cyclodextrin will not be discussed as there are many review articles in these areas.

42

"

45

Cyclodextrin-based chemistry has developed quite extensively since its discovery

in the late nineteen th and early twen tieth centurie s. Initial wo rk focusing on oute r-sphere

coordination of metal complexes with cyclodextrins was done during the middle-to-late

1980's.

46

"

52

Part of the focus in the remainin g sections will be recent progress u sing

cyclodextrins as outer-sphere host architectures.

In addition to the complexation studies, work with cyclodextrins has focused on

their use as catalys ts. Mu ch of the research in this area has centered on design of the

cyclod extrin as the catalytic center. A sum mary of this area is presen ted in review

articles by Stoddart et. al, Szejtli, et. al, and Russell, et . a/.

3

53

54

Less work has been

done exploring cyclodextrin as an architectural structure to position a catalytically active

metal site. W e will prese nt three exam ples of research that focuses on using cy clodex trin

as a scaffold on which to append a metal-binding center.

1.3.1 Host-Guest Chem istry of Metal C omplexes

The ability of cyclodextrins to incorporate molecules within its cavity

(Figure

1.4) has been studied extensively; however, analysis of these host-guest complexes

reveals only a few structures with the inclusion of a metal co mp lex. This is surprising as

the inclus ion of me tal com plex es can provide the versat i l i ty to mi mi c b io log ical

enzymes and create supramolecular assemblies .

A minimalist example of host-guest relationship is provided by Ando et. al.,

wherein they were able to study the inclusion complexes of


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9

[pentaammineruthenium(II)]

2+

and [pentacyanoferrate(II)]

4

" with a-, P-, and y-

cyclodextrin.

56

It is significant to report that it is rare for a charged (specifically,

positively charged) molecule to be included within the cyclodextrin. This was

accomplished by using the inner sphere ligands, which are hydrophobic and provide the

anchor in the core of the cyclodextrin. The steric bulk of the outer-sphere cyclodextrin

serves to stabilize the monomeric metal center.


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10

HO

\

I

.>

H O ^

OH

/

Lx

yp:y

^, OH

~lS \K

H

^ -S

\|

i O H /

AAoH ] (^ O H

\

0

\

OH-y

0

HO

b

b = 1(1), 2(2), or 3(3)

upper rim

l -OH

/v. ^A / J

/ \ / A

^

c 3 v

lower rim

2-OH

OH

\

r ^

/

[

OH

\

\

7

If

H O -

n = 6, 7 or 8

OH

V

\ ^ -

^o, \

\ \

*>A

M / l

/ n

Figure 1.3 Repre sentation s of a-, p-, and y-cyclode xtrins. The top structure represents

the full d rawn chem ical struc tures of a-, (3-, and y-cyclodex trins (b = 1, 2, or 3,

respec tively). The lower center and right structures are shorthand repre sentatio ns of the

a-, P-, and y-cyclodextrins (n = 6, 7 or 8, respec tively). Lastly, the lower left-most

represen tation is a cartoon of toroidal shap e. The dispositions of the primary and

secondary hyd roxyls are presen ted. The edges ringed with 1 and 2 hydro xyls are

referred to as the lower and upper rims, respectively.


32/194

11

+ G ue s t

Figure 1.4

Repre sentation of the inclusion of a guest molecule within the cyclodextrin

cavity. Guest molecules could include organic molecules, inorganic metal complexes,

cations, or anions.

1.3.2 Wa ste reme diation

Our group has focused on the remediation of environmental metal complexes,

including the uranyl carbonate anion, [U02(C03)3]

4

"

(Figure 1.5).

Urany l carbon ate is

the predom inant form of uranium in the environment. The carbonate anion is a powerful

ligand for the Lewis acidic U

VI

center, and carbonates' ubiquitous presence drives the

formation of the tris-carbona te species in mo st aquatic system s. It has been shown that

the ligands in uranyl carbonate are not easily displacedthe uranyl tris-carbonato species

has a stability constant, Poo = 10 . " We have been able to com plex uranyl carbona te

by providing a series of complementary hydrogen bond donors via synthetic

mo difications to the upper rim of cyclode xtrin molec ules. In design ing a host structure

for uranyl carbonate, both the D symm etry and charge of the guest must be considered

as part of the design . One of the first synthetic comp ound s used as a host was per-6 -(2-

aminoethylamino)-per-6-deoxy-a-cyclodextrin.

60

This com pound ma tches the 3-fold

symmetry of [U02(C03)3]

4

" while providing a pH-dependent ring of positive charges

from protonation of the amines. NM R titrations of per-6-(2-aminoethylamino)-per-6-

deoxy-a-cyclodextrin with uranyl carbonate allowed for determination of the stability

constant (log p = 253 22 M"

1

) of the cyclodextrin-uranyl carbonate complex in an


33/194

12

aqueo us environm ent. It has been shown through complex ation induced shifts of the

ethylenediamine protons that the uranyl carbonate moiety is binding through the upper-

rim.

61,62

It wa s also shown that the complex formed wa s 1:1 betwe en the synthetic

cyclodextrin and uranyl carbonate anion.

Additional work exploring the effects of outer-sphere ligation of the uranyl

species has been published by Navaza et . al.

63

Although the uranyl hydroxide systems

generally form bimetallic complexes, N ava za's wo rk illustrates the ability of a- and p-

cyclodextrin to stabilize the formation of monomeric benzoatoOHUO2 species within

dimeric cyclodextrins. The mode of binding of the benzoato-OH UO 2 species, like the

work by Ando, is again driven by the interaction of ligand and cyclodextrin with

inclusion of the hydrophobic benzoate ligand in the cavity of the p-cyclodextrin dimer.

63

1.4 Catalysis

In catalytic processes, it is important to make the distinction between

heterogeneous and homo geneous catalysis. Typically, the main advantage of

heterogeneous catalysis is that the catalyst can be recycled more easily, and is therefore

more cost efficient; however, both cyclodextrin-mediated homogeneous and

heterog eneou s catalysis historically have not been an area of great focus. In addition to

homogeneous and heterogeneous systems, some work has been done in biphasic systems

where the cyclodextrin acts as an integral part of the catalytic system.

64

"

66

In many cases

of cyclodextrin-mediated catalysis in biphasic systems, the cyclodextrin aids by acting as

a phase transfer agent. M any experim ental results have been publishe d with the use of

cyclodextrins in biphasic systems

66

"

69

; however, the work represented here will focus on

the use of outer-sphere coordination in homo geneous systems.


34/194

13

Figure 1.5 (a) 3-D model of uranyl carbona te showing its D symmetry and available

hydrogen bond acceptors (carbon = gray, oxygen = red, uranium = yellow) (b) a portion

of the H-N MR spectrum of per-6-(2-am inoethylamino)-per-6-deox y-a-cyclodextrin

titrated with uranyl carbonate shows the clear upfield shift of proton resonances near the

binding site.

1.4.1 Horseradish Peroxidase (HRP ) Mimic

Occasionally, a significant advance can be developed from seemingly unrelated

research area s. It wa s established in the late 1970 's by Tabushi et. al. that P-cyclodextrin

molecules can be linked to create dimers (or polymeric) cyclodextrin molecules, thereby

creating a molecule with two (or more) hydrophob ic pockets. Tang et. al. first showed

the catalytic ability of a Schiff-base metal complex, such as Fe

111

2 -hydroxy- l -

naphthaldehyde thiosemicarbazone (HNT), to be used for the determination ofH2O2and

glucose concentrations. Building on these studies, Tanget. al. have recently shown that

tethering two cyclodextrins forms dual hydrophobic pockets. The Schiff-base metal

complex

(Figure 1.6)

includes as a guest within these pockets, and forms a

supramolecular mimic of HRP.

7 2

Also synthesized were a series of metal com plexes

using salicylidene-2-amino-4-phenylthiazole (SAPTS) as a ligand. These complexes


35/194

14

were then investigated as potential superoxide dismutase (SOD) mim ics. Of the metals

studied (Cu

2+

, Zn

2+

, N i

2+

, and Co

2 +

), copper was the mo st active. The ability of

polymeric-P-CD (P-CDP) to recognize the benzene rings of the M

n

-(SAPTS)2 clearly

reveals the binding mode of the complex. Their work has also provided additional

methodology for the detection of superoxide dismutase (SOD ) activity.

1.4.2 Wa ter-soluble Pho sphan e with Cyclodextrin

Caron et. al. investigated the influences of cyclodextrin on reactivity of a water

soluble Pd-phosphane complex.

73

The binding mode of

4 (Figure 1.7)

with P-

cyclodextrin was characterized using NM R. The NM R experimental evidence indicates

that the phosphorous is distant enough from the cyclodextrin molecule that the nuclei

from the two moieties do not couple. A palladium-catalyzed cleavage of allyl undecyl

carbonate was tested using [Pd(l)3] in the absence and presence of methylated-P-

cyclode xtrin. The results indicate that as the ratio of meth ylated p-cy clodextrin/4 is

increased the rate of cleavage decreased when the ratio was between - 0- 2 , constant from

- 2 - 8 , and increased for ratios from - 8- 16 . Since the catalytic activity of the palladium

complex is similar in the absence and presence of cyclodextrin this signifies that the

cyclodextrin does not electronically or sterically hinder the metal (catalytic) center.

73

These studies have illustrated that an organometallic complex in the presence of

cyclodextrin can be used to mimic functional bioenzy mes. Furthermore, with these

synthetic systems, there is no obligate restriction on biologically relevant metals, and thus

a wider variety of metal-chemistry can be explored.

1.5 Future Outlook

Outer-sphere coordination, either in natural or synthetic systems, provides a

versatile framework for recognition and control of coordination complexes.

Cyclodextrins particularly have proven to be versatile compounds since their initial use in

host-guest chemistry and have shown great potential for their employment in outer-


36/194

15

sphere comple xation . The use of cyclod extrins in host-gue st chemistry is well stud ied,

though their full potential has yet to be completely harnessed with outer-sphere metal

complexe s. Wo rk aimed toward the developmen t of enzyme mim ics, catalysts, and the

remediation of chemical wastes is ongoing.

Figure 1.6

(a) Fe 2-hydrox y-l-naphthalde hyde thiosemicarbazone (HN T) Schiff-base

com plex. This figure shows a three-co ordinate Fe ; how ever, the structure is actually

either a square pyramidal or octahedral geometry which utilizes another HNT ligand to

satisfy the additional bonding sites, (b ) Representation of the Schiff-base salicylidene-2-

amino-4-phenylthiazole complex (M -(SAPTS)2).

The use of cyclodextrin derivatives as catalysts has great potential due to the

arrangement of cyclodextrin's hydrophobic inner cavity rimmed with outer hydrophilic

functional groups . The recent wo rk by Tang el. al. producing a horseradish peroxidase

mim ic has show n the advanc eme nt of cyclod extrins in the area of catalysis . The ability

to mimic a specific enzyme has been accomplished and now it remains to be seen if

functional mode ls for other enzym es can be develo ped. The ability to modify

cyclodextrin on the upper and lower rims, and possibly attach additional scaffolds to

create an ordered system similar to the tertiary structure of proteins provides many levels

of control over a guest metal complex. The robust nature of the cyclodextrin systems

suggests that they may be suitable for use in environments where proteins are not stable.


37/194

16

Nevertheless, the ability to more precisely design supramolecular analogues of natural

systems that utilize outer-sphere coordination hinges on a deeper understanding of the

numerous biological systems still currently being studied.

/

\ -

NaCHB

f V S O J N H

4

Figure 1.7

Phosphane ligand for complexation with a metal ion (e.g. Pd[4]3) and

inclusion into the cyclodextrin cavity.


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17

Notes

(1) Karakhanov, E. E.; Maksimov, A. L.; Runova, E. A.; Kardasheva, Y. S.;

Terenina, M . V.; Buchneva, T. S.; Guchkova, A. Y.

Macromol.

Symp.2003,204,

159-173.

(2) Colquhoun, H. M.; Stoddart, J. F.; Williams, D. J.Angew . Chem., Int. Ed. Engl.

1983,2 5,487-582.

(3) Stoddart, J. F.; Zarzycki, R.Reel.Trav. Chim. Pays-Bas1988,107,515-528.

(4) Zamarev, K.New. J. Chem1994,18,3-18.

(5) Yue, W. W.; Grizot, S.; Buchanan, S. K.J.Mol. Biol.2003,332, 353-368.

(6) Telford, J. R.; Raymond, K. N . InComprehensive SupramolecularChemistry;

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CHAPTER 2

ANION RECOGNITION USING A CYCLODEXTRIN-BASED

SCAFFOLD

2.1 Introduction

2.1.1 Oxoanion Recognition Chemistry

Num erous examples of oxoanion recognition exist. A relevant example in

relation to these studies is provided by Nature with phosphate binding protein (PBP).

Medveczky et. al. have shown that PBP binds phosphate with a Kd of -0.8 uM.

1

'

2

Binding of phosphate is accomplished with 12 strong hydrogen bonds from the phosphate

binding protein backbone. Encompassed w ithin the hydrogen bonding manifold are 10

hydrogen bonds to three of the phosphate oxygens (01, 02, and 03).

2

Complementing

this are two hydrogen bonds to 04 of the phosphate, one of which is "from an NH of a

backbone peptide unit whose carbonyl oxygen is in turn the recipient of two hydrogen

bonds".

2

This set of 12 hydrogen bonds aids in understanding why phosphate binding

protein has such a high affinity for phosp hate. Other examples in Nature of oxoanion

binding proteins which exhibit a substrate specificity have also been well characterized

{e.g. sulfate).

3

"

9

2.1.2 Outer-sphere Coordination Using Cyclodextrins

The presence of literature reports for the outer-sphere coordination of metals

using cyclodextrins are lacking. Some notable examples of have been summarized in a

recent review.

10

The aim of these studies is on utilizing the oxygen atoms of tetrahedral

anions as 'ancho rs' for binding to modified cy clodextrins. The focus here remains on the

complexation of perchlorate, phosphate, and sulfate with a-cyclodextrin (1) and per-6-(2-

aminoethylamino)-per-6-deoxy-a-cyclodextrin

(6, Figure 2.1).


43/194

O

P

1

D

M

F

7

C

1

e

e

h

e

a

m

n

W

c

:

7

4

1

%

y

e

d

F

g

2

1

S

h

c

a

o

m

o

f

o

a

c

o

n

(

1

o

p

6

o

a

c

o

n

(

5

R

m

o

h

o

d

v

a

n

e

c

a

a

b

e

h

o

h

f

e

e

m

n

a

m

n

o

e

h

e

a

m

n

r

e

u

s

n

h

f

n

p

6

2

a

m

n

h

a

m

n

p

6

d

a

c

o

n

(

6

p

o

t

o

t

o


44/194

23

2.1.3 Tetrahedral Anions

Perchlorate, phosphate, and sulfate are prevalent in nature in high concentrations.

2

Since 1997 it has been shown that perchlorate levels in drinking water have been steadily

increasing ' , more-so in areas related to the testing of solid rocket propellant, companies

synthesizing perchlorates, and compan ies utilizing perchlorates. Furthermore,

experimental data has shown that both perchlorate and pertechnetate can compete with

iodide uptake by the thyroid gland in mam mals.

9

'

11

Therefore it is necess ary, that prior to

discarding waste, toxic compounds, such as perchlorate, be remediated by the companies

or agencies that produce these wastes. In addition to this, other anions (even though no t

targeted here) are of immediate con cern. For instance, the heavy use of fertilizers ha s

created a need for the rem ediation of nitrates from the environm ent. '

In order to specifically target one tetrahedral anion over another it is important to

pay attention to their size and charge/size ratio

(Table 2.1).

Here the binding these

anions is first being targeted by the size inherent to the a-cyclod extrin framew ork,

followed by the modification to the uppe r (primary hyd roxyl) rim of the cyclodextrin . It

is with these upper-rim modifications that targeting a specific charge/size ratio is being

analyzed.

2.1.4 Determ ination of Bindin g Affinities

In these studies, the binding of perchlorate has primarily been targeted; however,

it is vital to also investigate the binding of other common tetrahedral anions (e.g.

phosphate and sulfate). Even though numerous methods (NMR, UV -Vis, calorimetric,

etc.)

have previously been used for the determination of binding constants, these m ethods

were not particularly suited to the binding of tetrahedral anions with cyclodextrins.

13

'

14

A

relatively new method for determination of binding affinities using electrospray-

ionization mass spectrometry (ESI-MS) was employed.

15

Num erous studies have shown

that the determination of binding affinities can be accomplished via the intensity ratio of


45/194

24

peaks for the uncomplexed and complexed species from ESI-MS spectra.

15

"

17

Of

additional importance, studies indicate that ESI-MS, being a soft-ionization technique,

allows the speciation in the gas phase to mimic that of the solution (aqueous) phase.

1 7

Consequently the mlz ratio will unambiguously indicate the most stable complex, thereby

providing the exact stoichiometry of the host and guest complex formed.

15

'

18

"

21

Other

groups have shown that making assumptions as to the stoichiometry of the host and guest

complex can provide an erroneous binding constant.

2.2 Experimental

a-Cyclodextrin was obtained from Avocado Research Chemical Ltd (UK) and

was dried under vacuum. Ethylenediamine was obtained from Aldrich Chemical

Com pany. LiC104 and M gS0 4 were obtained from Aldrich Chemical Com pany.

Potassium phosphate (monobasic, dibasic, and tribasic) was obtained from Fisher

Scientific. These chemicals were dried under vacuum prior to use. All chemicals were

of reagent grade. NM R experiments were performed at 25 C on a Briiker DP X-30 0,

AV AN CE-300, DR X-400 or AVA NCE-600 spectrometer using deuterated solvents .

2.2.1 ESI-MS

A Thermo Finnigan LCQ Deca Spectrometer (University of Iowa, High

Resolution Mass Spectrometry Facility, spectrometer purchased with fund from NIH

grant 510 -RR 137 99- 01) was used in direct injection mode . All experiments were

conducted at a capillary temperature of 150 C, spray voltage of 4.5 V, and sample

collection flow rate of 3 uL/min. Prior to each sample, M S solvent (9 : 1 FbO /Me OH ,

0 .1%

NH4OH) was flowed through the system until the detection of relevant mlz species

wa s not seen. Initial optim ization (tuning) on the samples was done, followed by sa ving

the parameters and using this parameter set for subsequent trials. Each sample w as

allowed to equilibrate into the mach ine at 10 - 15 uL/m in for 1 - 2 min utes prior to

reducing the flow rate to 3 ul/min and collecting data. After adjusting the flow rate , data


46/194

25

was collected in positive mode for 2 - 3 minutes from a range of mlz 100 - 1500.

Generally, the initial minute of data collected was discarded, followed by averaging of

100 spectra/sam ple to obtain intensity value s for analy sis. Com plete spectra for ESI-M S

analysis of each tetrahedral anion with cyclodextrins 1 and 6 are located in Appendix B.

Intensity values (I

H

an d

IHG)

and concentrations of the host (6) and guests (tetrahedral

anions) are provided in Tables B.l - B.3 inAppend ix B.

2.2.2 Synthesis

Per-6-(2-aminoethylamino)-per-6-deoxy-a-cyclodextrin (6). The methodology

for this procedure was taken from Ashtonet. al.and Prudden et. al. ' Per-6-iodo-per-6-

deoxy-a-cyclodextrin (5) used for the synthesis of 6 was synthesized by Anthony

Prudden of the Telford group from a-cyclodextrin based upon Ashton's previous work.

22

Per-6-iodo-per-6-deoxy-a-cyclodextrin (5, 1.97 g, 1.21 mmol) was added to

ethylen ediam ine (25 mL , 0.37 mol) under positive argon press ure. After dissolving, the

reaction mixture was heated to 70 C. The reaction mixture was cooled to room

tem perature after 72 h, reduced in volum e at reduced pressu re to 1 - 2 mL and

precipitated out of -250 mL of cold ethanol. The material was dissolved and re-

precipitated two tim es. The precipitate was then isolated via Buchner funnel and vacuum

dried. Ov erall yield 1.09 g, 45 .1%. ' H NM R ( D

2

0, 400 MHz) 5 = 5.09 (6H, br s, HO,

4.01 (12H, br t, H3/H5), 3.67 (6H, br m, H

2

), 3.56 (6H, br t, H

4

), 3.21 (6H, br s, -

HNCH2CH2NH2), 2.65-3.15 (30H, br m, H

6

/ -HNCH2CH2NH2).

13

C N M R

2 3

( D

2

0, 150

MH z) 5 104.2 ( d ) , 86.0 (C

4

), 75.8 (C

3

), 74.3 (C

2

), 73.3 (C

5

), 53.9 (-NHCH

2

C H

2

N H

2

),

51.9 (C

6

) , 42.4 (-NHC H

2

C H

2

N H

2

) ESI-MS:(m/z) 1226 (M + H

+

)

+

, 613 (M + 2H

+

)

2+

, 409

(M + 3H

+

)

3+

, 205 (M + 6H

+

)

6+

-

2.2.3 Titrations

Conc entrated stock solutions (ranging from ~1 - 9 mM ) of cyclod extrins 1 and 6,

and the tetrahedral anions were made using volumetric glassware (TC) and then


47/194

26

immediately transferred to Corning conical vials that had been pre-rinsed with MS

solvent. 1 rtiL samp les for ESI-M S were mad e from the stock solutions, with a final

[Cyclodextrin] = 90 uM. The desired amounts of each tetrahedral anion were ad ded

ranging from [Cyclode xtrin] : [Tetrahedral An ion] from 100 : 1 to 1 : 10 and with the

remaining volume filled to 1 mL with MS solvent. Titrations with higher guest

concentrations were tested, but at such high salt concentrations the noise in the

instrumen t provides data that could not be interpreted with any reliability. It is important

to note that samples with ratios of [Cyclodextrin] : [Tetrahedral Anion] ranging from

100:1 to 1:1 generally did not provide any appreciable binding in the case of any

tetrahedral anion.

A set of example calculations for the titration of cyclodextrin 6 with LiC104 is

provided in the following sentences and

Table 2.2.

Stock solutions were made of

6

(7.96

mM) and LiC104 (7.24 raM). For the example reported here, titrations of the following

mola r ratios of [6] : [LiC104] ranging from 100 : 1, 10 : 1, 1 : 1, 1 : 5, and 1 : 1 0 we re

performed (seeTables B. l - B.3 inAppendix B for a ratios all titrations). An Eppendorf

tube for each titration point was made, followed by addition of the varying amounts of

each stock solution and MS solvent based on Table 2.2 below. The Eppendorf tubes

wer e agitated and allow ed to stand for 6 - 12 hours to allow for each mixture to reac h

equilibrium, followed by ES I-MS analysis of each sam ple.

2.2.4 Calculation of Bindin g A ffinities

The analysis of ESI-MS data was done using equations A.1 - A .7. Relevant

information for these equations is provided in

Appendix A.

Experimental values for the

intensity ratios for + 1, +2, and +3 charge states of uncomplexed and complexed

cyclode xtrins were analyzed and fit into equation A.6. Initial value s of R were

determined, followed by sampling ofR' and n values that provided the best linear fit and

conforming to the initial calculated R values. Values used for n were used to correct the


48/194

27

y-intercept determined based on the initial concentration of host. The determination of

these values was done by trying to obey trends established by the Hoffmeister with

partition coefficients. The Hoffmeister series indicates the increasing water solubility in

the order ofS0

4

2

">P0

4

3

"> > > C10

4

".

2.2.5 pK

a

Determination

Attempts to determine the pK

a

values for hexa-protonated cyclodextrin 6 using

potentiometric titrations resulted in large buffer region from which pK

a

(acid

dissociation) values could not be deconvoluted. Consequently, an estimation of the pH

speciation distribution was created using the program HySS.

24

The initial P values were

estimated beginning at 12 and working our way up to accommodate all species. The list

of

p

values were 12, 24, 33, 41 , 48, and 54 for

-AH*",

AH

2

2+

, AH

3

3+

, AH

4

4+

, AH

5

5+

, and

AHg

+

, respectively.

Figure 2.2

clearly indicates the propensity of primary amine system

to retain 2, or possibly more protons in the presence of additional species (tetrahedral

anions in this case). These data are also supported by observance of mainly + 1, +2 , and

+3 charged states in mass spectra upon binding of tetrahedral anions. This most likely

indicates a protonation state greater than two and less than six, but is highly dependent on

the tetrahedral anion due to their distinct charge states at pH ~ 10.

2.2.6 T\ Inversion Recovery Experiments

T\ inversion recovery experiments were chosen in order to clarify the binding of

the perchlora te, phosphate and sulfate with cyclodextrin 6. Only one tetrahedral anion

was chosen (perchlorate) for these studies due to the time required for each inversion

recovery experiment. Four

T\

inversion recovery experiments were performed with

molar ratios of 6 : perchlorate of

1

: 0, 1 : 1.2, 1 : 4, and 1 : 8.

Table 2.3

provides the

delay times prior to capturing spectra. Once collected, peak height data for 'H-N MR

peaks of relevance for each titration were noted and are seen in

Tables B.4

-

B.7

located

inAppendix B. Data analysis was performed by fitting peak height data to equation 2.8.


49/194

28

Final proton T\ relaxation data can be seen inTable 2.4 and Figure 2.3,which provides a

graphical version of the data.

2.3 Results and Discussion

Studies of tetrahedral anion binding with cyclodextrin 1 indicate a lack of

specificity. In the case of 1 with LiC104, spectra indicate the association of Li

+

(m/z

979),

2 L i

+

and C10

4

"(m/z 1085), 3 Li

+

and 2 C10

4

"(m/z 1191), or 4 Li

+

and 3 C10

4

"(m/z

1298). Similar studies with KH2PO4 indicate that it incorporates m ainly K

+

(m/z 1011) or

2 H

+

, 2 K

+

and PO4

3

"(m/z 1147 ). The last studies with sulfate show predom inantly the

inclusion of H

+

, M g

2+

and SO4

2

"(m/z 1093). In the former two cases, the ability of 1 to

bind the cation of the tetrahedral anion indicates a partial preference toward positively-

charged spec ies. Furtherm ore, the inclusion of mu ltiple CIO4" mo ieties indicates the

possibility of multiple association sites. In the cases of PO4

3

" and SO4 the inclusion of

more than one tetrahedral anion does occur, but to a lesser extent (less than 10% of

relative intensity for these peaks) in the ESI -M S spectrum . In the case of pho spha te,

using mono basic, dibasic, or tribasic phos phate could provid e differing resu lts. Studies

with monobasic, dibasic and tribasic phosphate were all performed providing similar

results. With the pH of each titration point betw een 10.1 - 10.5, control of the species

distribution of phosphateno matter what starting phosphate was usedwas controlled

predominantly by the 0 .1%NH4OH in the MS solvent.

In the case of cyclodextrin 6, the high propensity of the six pendant

ethylenediamine arms to be protonated provides the most likely area of electrostatic

interaction between the tetrahedral oxyanions and the host (Figure 2.4). This is an

impo r tan t d i s t inc t ion wi th 1 , wh ere the p r im ary upper - r im hyd roxy l mo ie t i es show a

partial preference for the cations of each tetrahedral anion. The estimate of protonation

states for cyclodextrin 6 was illustrated in Figure 2.2. Furthe r support for the estimated

proton ation state is provide d by the pH of the solutions being studied. As m entioned


50/194

29

previously, solution pH measurem ents indicated valued between 10.1 - 10.5. At this pH

a mixture of mono-, di-, and tri-protonated species should be present (based on pK

a

estimates earlier presented). This was further supported by the presence o f+ 1 , +2, and

+3 m/zspecies in the ESI mass spectra.

Given the clear identification of unbound and bound species via ESI-MS,

determination of binding constants for each tetrahedral anion with cyclodextrin 6 was

performed. The log K

a

values for each tetrahedral anion can be seen in Table 2.1.

Qualitative analysis of the calculated association constants indicates a small amount of

discrimination, which is almost negligible for the purpose of separating these ions in a

mixture. It also goes without saying that binding for these tetrahedral oxyanions does not

depend on their charge/size ratio in the case of cyclodextrin

6 (Table 2.1).

The analysis of binding constants for perchlorate yielded linear plots with R

2

values of 0.76, 0.91, and 0.95. The case of phosphate (either monobasic or dibasic)

yields reproducible results that produce linear plots (R

2

values of 0.98, 0.93, and 0.98).

Sulfate also produces two linear plots with good regressions (R

2

values of 0.84 and 0.92).

Overall, all three anions provide linear regressions that are within reasonable limits. This

data can be seen inAppendix B, Figures B.18 - B.25.


51/194

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cyclodextrin scaffolds

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