beta-cyclodextrin modification and host-guest complexation - Adelaide

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(exemplified by sodium dodecyl sulfonate)-CD complexation can be used to .... Poly(acrylic acid)s were stirred in 1-methylpyrrolidin-2-one (NMP) at 60˚ C ..... moles of AD in 10 mg of 3% βCD substituted PAA, which are 3.06, 2.97 and 2.89 mmol ...... -Manno-epoxide βcyclodextrin (1.0 g, 0.89 mmol) was dissolved in 25%.

BETA-CYCLODEXTRIN MODIFICATION AND HOST-GUEST COMPLEXATION

Duc-Truc Pham (Phạm Đức Trực)

Thesis submitted for the degree of Doctor of Philosophy in The University of Adelaide School of Chemistry and Physics

October, 2007

Duc-Truc Pham

Chapter 5

5 chapter

CHAPTER 5

MOLECULAR CONTROL OF POLYMER PROPERTIES: INTERACTIONS OF POLY(ACRYLIC ACID)S BEARING β-CYCLODEXTRIN AND ADAMANTYL SUBSTITUENT CHAINS

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5.1. Introduction 5.1.1. Molecular Control of Polymer Properties Polymer hydrogels are of increasing interest because of potential applications in drug delivery, sensor systems, electro-optical usage, functional nanodevices and tissue engineering.1-4 Therefore, there is a need to advance the understanding and control of aqueous supramolecular assembly through interaction of substituted polymers which exhibit predictable character and constitute new materials.

A polymer backbone

B

hydrophobe

interstrand association

intrastrand association

hydrophobe association

+n hydrophobe receptor

+n -n

C

+

E

hydrophobe-single hydrophobe receptor association

hydrophobe-bis hydrophobe receptor association

D

hydrophobe receptor-hydrophobe association

Figure 5.1. Various interaction types of water soluble substituted polymers

Some possible interactions of polymers are illustrated in Fig. 5.1. The solubilization of hydrophobes by substituting them onto water soluble polymers (A) is likely to result in

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hydrophobe-hydrophobe association (B) which increases viscosity of the polymer solution. The addition of water soluble hydrophobe receptors such as cyclodextrins (CDs) may produce polymer disaggregation (C) which reduces viscosity. The process from (B) to (C) is reversible by using competitive complexation interactions where free hydrophobe (exemplified by sodium dodecyl sulfonate)-CD complexation can be used to ‘unmask’ CDhydrophobe polymer complexation interactions.5 This completely or partially restores the viscosity of the solution to that of the hydrophobr polymer alone depending on the identity of the CD. For example, by utilizing the interaction of free α-cyclodextrin (αCD) with dodecyl (C12) substituent chains in poly(acrylic acid)s (PAA), systems that undergo gelto-sol and sol-to-gel transitions were successfully constructed with a remarkable change in the viscosity when adding αCD to 5.0 g dm-3 of the polymer.6 A photoresponsive hydrogel system has been prepared by a combination of simple components: α-CD, dodecyl substituted PAA, and a photoresponsive competitive guest, 4,4'-azodibenzoic acid. Gel-tosol and sol-to-gel transitions occurred repeatedly by alternating irradiations of UV and visible light.7

When a hydrophobe substituted polymer and a receptor substituted polymer are mixed in aqueous solution, there is the possibility of host-guest complexes being formed (D) and increasing the viscosity of the solution.8,9 The addition of another free hydrophobe receptor to (D) leads to competition of complexation between free host/polymer guest and polymer host/polymer guest and may reduce the viscosity of solution (C). For example, a polymerpolymer interaction of PAA carrying 3A-αCD* (P3αCD) and 6A-αCD* (P6αCD) receptors and a PAA carrying azobenzene receptors (PC12Azo) has been investigated by several techniques, including steady-shear viscosity (η) measurements and NOESY NMR.8 (*Substituted onto PAA through the αCD C3A and C6A carbon, respectively). The η values for the P3αCD/PC12Azo and P6αCD/PC12Azo mixtures (6.5 × 10-1 and 2.5 × 102 Pa.s, respectively) are larger than that for the PAA/PC12Azo mixture (8.4 × 10-2 Pa.s). The interaction of a copolymer of acrylamide and acrylic acid bearing βCD receptors with poly(acrylamide)s bearing aromatic substituent chains was investigated by viscometry to study the effect of interactions at multisites in macromolecular recognition.9 The formation of host-guest complexes at multi-sites caused a large difference in the size of interpolymer aggregates.

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Furthermore, the addition of bis(hydrophobe receptor) to hydrophobe substituted polymers may increase viscosity of the solution as the formation of polymer aggregation (E).10 The addition of a terephthalimide linked βCD dimer to the aqueous solutions of 1:20 copolymers of 1-adamantylacrylamide or 6-acryloylaminohexanoic acid 1-adamantylamide and N,N’-dimethylacrylamide increased the viscosity of the solutions dramatically. Stable gels formed within seconds.

5.1.2. Aims This research continues the previous studies carried out through collaboration of the Lincoln and Prud’homme research groups.5,11 These studies examined the complexation interactions of 3% substituted n-octadecyl (C18) PAA with free αCD, βCD, 3% substituted αCD-PAA and βCD-PAA by monitoring the viscosity change of the polymer solutions.

To further expand these studies, the first aim is to prepare a series of three 3% substituted βCD PAAs with different substituents lengths (Fig. 5.2): βCD-PAA is the poly(acrylic acid)s with 3% substituents 6A-amino-6A-deoxy-β-cyclodextrin (6βCDNH2),12 βCDhn-PAA is the poly(acrylic acid)s with 3% substituents 6A-(6-aminohexyl)amino-6Adeoxy-β-cyclodextrin (6βCDhn)13 and βCDddn-PAA is the poly(acrylic acid)s with 3% substituents 6A-(6-aminododecyl)amino-6A-deoxy-β-cyclodextrin (6βCDddn).13

The second aim is to prepare a series of three 3% substituted hydrophobic PAA with different substituents length similar to the βCD-PAAs. Adamantane (AD) is chosen for the hydrophobic substituent due to its strong complexation in the βCD cavity.14 Three hydrophobically substituted PAAs including AD-PAA which is poly(acrylic acid) with 3% substituents 1-amino-adamantane (ADNH2), ADhn-PAA is poly(acrylic acid) with 3% substituents 1-(6-aminohexyl)amino-adamantane (ADhn) and ADddn-PAA is poly(acrylic acid) with 3% substituents 1-(6-aminododecyl)amino- adamantane (ADddn) (Fig. 5.2).

The third aim is to advance the understanding and control of aqueous supramolecular assembly according to the models as discussed in Section 5.1.1 for the host-guest interactions between the βCD substituted poly(acrylic acid)s and adamantane; AD, - 191 -

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substituted poly(acrylic acid)s and βCD and linked βCD dimers; and between both βCD and AD substituted poly(acrylic acid)s. This is examined by 2D 1H NOESY NMR. (Detailed viscosity measurements will be carried out at Princeton).

5.2. Substituted Poly(acrylic acid)s The method for the substitution of PAA is similar to that described in the literature.11 Poly(acrylic acid)s were stirred in 1-methylpyrrolidin-2-one (NMP) at 60˚ C for 24 hours. The amine was added followed by dicylohexyldiimide (DCC). The mixture was then treated with sodium hydroxide solution and purified (Fig. 5.2). ADhn-PAA and ADddnPAA were prepared by Dr. Bruce May. Other substituted PAAs were prepared as in the general procedure.

1. 60 oC/NMP

H2 C

H C

COOH

+ NH2-R +

N

C

N 2. NaOH

n

DCC

H2 C

H C

COONa 97

H C

H2 C

CO HN

O C

H N

+ 3

H N

DCU

R

R= 2

3 4

d

a

b

e

1

H N

3

2

4 1

O

N H

f

c N H

H N

2

3 4

1

O

Figure 5.2. Preparation of 3% Substituted βCD and AD substituted PAA. a: βCD-PAA, b: βCDhn-PAA, c: βCDddn-PAA, d: AD-PAA, e: ADhn-PAA, f: ADddn-PAA.

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The 1H NMR (300 MHz) spectra of six polymers and PAA are shown in Figs. 5.3 and 5.4. Samples were dissolved in D2O at about 1 wt %. The degree of substitution at the PAA carboxyl groups by βCD or AD was determined according to the equations reported previously.11 The calculated substitution degrees for all substituted PAA are 3.0 ± 0.3%.

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βCD H

1

βCD H2-6

acrylate H

βCD-PAA

hn H hn N-CH2 H

βCDhn-PAA

ddn H ddn N-CH2 H

βCDddn-PAA

PAA

6.0

5.0

4.0

3.0

2.0

1.0

0.0 ppm

Figure 5.3. 1H NMR spectra of PAA and 3% substituted βCD-PAAs in D2O.

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acrylate and adamantyl H

AD-PAA

hn H

hn N-CH2 H

ADhn-PAA

ddn H ddn N-CH2 H

ADddn-PAA

PAA

6.0

5.0

4.0

3.0

2.0

1.0

ppm 0.0

Figure 5.4. 1H NMR spectra of PAA and 3% substituted AD-PAAs in D2O.

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The resonances of the main chain and substituent protons of substituted PAAs are shifted slightly upfield compared with that of PAA. The resonances of substituents the hn and ddn chain (multiple 1.5-1.3 and 1.6-1.2 ppm, respectively) and H2-4 of AD (H3 2.1 ppm, H2 1.7 ppm and H4 1.6 ppm) in the substituted PAAs broaden and overlap with that of the main chain. However, due to complexation by βCD, these resonances may sharpen and shift upfield or downfield and can be assigned in concert with the cross-peaks in the 2D 1H NOESY NMR spectra.

5.3. 2D 1H NOESY NMR Studies 2D 1H NOESY NMR studies (600 MHz, 0.3 s mixing time) were carried out at 298.2 K on 1 wt % total substituted PAA solutions (10 mg) in 1 cm3 D2O.

5.3.1. Interaction of βCD Substituted PAAs with Adamantane-1-carboxylate The βCDhn-PAA and βCDddn-PAA were firstly examined for the self-complexation of the hn and ddn chains in the βCD annulus. The 2D 1H NOESY NMR spectrum of βCDddn-PAA (Fig. 5.5) shows weak cross-peaks arising from interactions between the CH2- protons of the hn chain and those of βCD H3, H5 and H6 (although H6 cannot be clearly distinguished from H2 and H4).15 However, there are strong cross-peaks in the case of βCDddn-PAA (Fig. 5.6). This is consistent with the ddn chain occupying the βCD annulus whilst the shorter chain hn resides mainly outside the βCD annulus. The host-guest complexation behavior of the series of three βCD substituted PAAs with sodium adamantane-1-carboxylate (ADCO2Na) were carried out by 2D 1H NOESY NMR studies at equimolar concentrations of βCD substituted PAAs and sodium adamantane-1carboxylate in 1 cm3 D2O. The moles of free ADCO2Na were calculated equivalent to βCD in 10 mg of 3% βCD substituted PAA, which are 0.48, 0.47 and 0.46 mg ADCO2Na in 10 mg each of βCD-PAA, βCDhn-PAA and βCDddn-PAA, respectively. Under these conditions the ratio 1:1 of the host βCD substituent and the guest ADCO2Na is obtained in the solutions studied.

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The 2D 1H NOESY NMR spectra of adamantane-1-carboxylate with βCD-PAA (Fig. 5.7), βCDhn-PAA (Fig. 5.8) and βCDddn-PAA (Fig. 5.9) show strong cross-peaks arising from interactions between the H2, H3 and H4 protons of the AD group and those of βCD H3, H5 and H6. This indicates that the AD group occupies the βCD annulus in all cases. Interestingly, it is also seen from the 2D 1H NOESY NMR spectrum of adamantane-1carboxylate with βCDddn-PAA (Fig. 5.9) that strong cross-peaks arise from interactions between the -CH2- protons of the ddn chain and those of βCD H3, H5 and H6. As the 2D 1H NOESY NMR spectrum shows a time averaged effect, this is consistent with the ddn chain complexing in the βCD annulus in competition with the AD group as shown in Fig. 5.9.

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CO2CO2O HN

hn CH2

βCD H1

PAA H

HOD βCD H2-6

hn N-CH2

CO2- N H

A

Figure 5.5. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt %

βCDhn-PAA in D2O. The green rectangle drawn on the spectrum, A, contains the weak cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the -CH2- protons of the hn chain. Approximate structures are shown above.

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CO2CO2O HN

βCD H2-6

βCD H1

PAA H

ddn CH2

HOD

N H

ddn N-CH2

CO2-

A

Figure 5.6. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt %

βCDddn-PAA in D2O. The green rectangle drawn on the spectrum, A, contains the crosspeaks arising from the NOE interactions between the annular H3, H5 and H6 protons of

βCD and the -CH2- protons of the ddn chain. Approximate structures are shown above.

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CO2CO2O

CO2-

HN

HOD

AD H2-4

PAA H

CO2-

βCD H2-6 βCD H1

A

Figure 5.7. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt %

βCD-PAA and adamantane-1-carboxylate in D2O. The green rectangle drawn on the spectrum, A, contains the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3 and H4 protons of the AD group. Approximate structures are shown above.

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CO2CO2O CO2-

HN

AD H2-4 hn CH2

hn N-CH2

HOD

βCD H1

PAA H

CO2- N H

βCD H2-6

A

B

Figure 5.8. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt %

βCDhn-PAA and adamantane-1-carboxylate in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3 and H4 protons of the AD group and the -CH2- protons of ddn chain, respectively. Approximate structures are shown above.

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CO2-

CO2-

CO2O

CO2O

CO2-

HN

βCD H1

βCD H2-6

PAA H A

AD H2-4

HOD

N H

ddn CH2

CO2-

HN

ddn N-CH2

CO2-

CO2-

HN

B

Figure 5.9. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt %

βCDddn-PAA and adamantane-1-carboxylate in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3 and H4 protons of the AD group and the -CH2- protons of the ddn chain, respectively. Approximate structures are shown above.

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5.3.2. Interaction of Adamantyl Substituted PAAs with βCD and Linked βCD Dimers The complexation behavior of the series of three AD substituted PAAs with free βCD and linked βCD dimers was studied by 2D 1H NOESY NMR at equimolar concentrations of AD substituted PAAs and βCD in 1 cm3 D2O. The moles of free βCD and linked βCD dimers (66βCD2ur and 66βCD2su, Chapter 2) were calculated equal to the number of moles of AD in 10 mg of 3% βCD substituted PAA, which are 3.06, 2.97 and 2.89 mmol AD in 10 mg each of AD-PAA, ADhn-PAA and ADddn-PAA, respectively. Under these conditions the ratio 1:1 of host βCD and guest is AD obtained in the solutions. No cross-peaks between the βCD and the acrylate protons are seen in the 2D 1H NOESY NMR spectrum of equimolar βCD and PAA (Fig. 5.10). The spectra of AD-PAA with βCD, 66βCD2ur and 66βCD2su (Figs. 5.11, 5.12 and 5.13, respectively) show strong cross-peaks arising from interactions between the H2, H3, H4 protons of the AD substituent (H3 2.1 ppm, H2 1.7 ppm and H4 1.6 ppm) and those of βCD H3, H5 and H6. This indicates that the AD group occupies the βCD annulus in all cases. There are two resonances for AD protons H4 seen in these cases which reflect the different effects of environment in the βCD annulus on equatorial and axial AD H4. The spectra of ADhn-PAA with βCD, 66βCD2ur and 66βCD2su (Figs. 5.14, 5.15 and 5.16, respectively) show weak cross-peaks arising from interactions between the -CH2protons of hn chain and those of βCD H3, H5 and H6. However, there are strong crosspeaks arising from interactions between AD H2, H3, H4 and H4' protons and the βCD protons H3, H5 and H6. This is consistent with the AD group occupying the βCD annulus whilst the hn chain mainly resides outside the βCD annulus. In contrast, the spectra of ADddn-PAA with βCD, 66βCD2ur and 66βCD2su (Figs. 5.17, 5.18 and 5.19, respectively) show strong cross-peaks arising from interactions between the -CH2- protons of the ddn chain and βCD H3, H5 and H6. Strong cross-peaks also arise from interactions between AD H2, H3 and H4 protons and the βCD H3, H5 and H6 protons. This

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indicates that an equilibrium exists between the ddn chain and the AD group occupying the

βCD H2-6 βCD H1

PAA H

βCD annulus as shown in Figs. 5.17-5.19.

Figure 5.10. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % PAA and free βCD in D2O.

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CO2-

CO2-

CO2O

CO2O

HN

HN

CO2-

βCD H2-6 βCD H

1

AD H2,3,4,4'

PAA H

CO2-

A

Figure 5.11. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % AD-PAA and free βCD in D2O. The green rectangle drawn on the spectrum, A, contains the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, H4 and H4' protons of the AD group. Approximate structures are shown above.

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CO2CO2O

HN

NH

O2C

-

O2C O

HN

βCD H1

HOD

O2C

PAA H

-

-

βCD H2-6

AD H2,3,4,4'

HN CO2

-

O

A

Figure 5.12. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % AD-PAA and 66βCD2ur in D2O. The green rectangle drawn on the spectrum, A, contains the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, H4 and H4' protons of the AD group. Approximate structures are shown above.

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CO2-

-

O2C O

HN -

O

HOD βCD H2-6 βCD H1

O2C

AD H2,3,4,4'

CO2-

O2C

H N

N H

su linker CH2

HN

O

PAA H

CO2O

-

A

Figure 5.13. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % AD-PAA and 66βCD2su in D2O. The green rectangle drawn on the spectrum, A, contains the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, H4 and H4' protons of the AD group. Approximate structures are shown above. (su linker refers to the succinamide linker of the linked βCD dimer).

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CO2-

CO2-

CO2O

CO2O

HN

HN

βCD H1 HOD

O

A

hn CH2

βCD H2-6

HN

PAA H

CO2-

O

AD H2-4

HN

hn N-CH2

CO2-

B

Figure 5.14. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % ADhn-PAA and free βCD in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group and the -CH2- protons of hn chain, respectively. Approximate structures are shown above.

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-

CO2-

-

O

CO2O

HN

NH

O 2C O

HN NH

HN

O2C

A

AD H2-4

PAA H

HOD βCD H2-6

hn N-CH2

βCD H1

-

O

O

hn CH2

HN CO2-

O2C

B

Figure 5.15. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % ADhn-PAA and 66βCD2ur in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group and the -CH2- protons of hn chain, respectively. Approximate structures are shown above.

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CO2-

-

CO2O

-

O 2C O

H N

HN

HN NH

O

βCD H1

PAA H

HOD βCD H2-6

O

hn N-CH2 su linker CH2

O

N H

A

-

O 2C

AD H2-4 hn CH2

O

HN CO2-

O2C

B

Figure 5.16. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % ADhn-PAA and 66βCD2su in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group and the -CH2- protons of hn chain, respectively. Approximate structures are shown above. (su refers to the succinamide linker of the linked βCD dimer, 66βCD2su).

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CO2-

HN

O

CO2-

βCD H2-6

βCD H1

ddn N-CH2

HOD

HN O

PAA H

CO2-

A

ddn CH2

HN

CO2O

H N

AD H2-4

CO2O

CO2-

B

Figure 5.17. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % ADddn-PAA and free βCD in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group and the -CH2- protons of ddn chain, respectively. Approximate structures are shown above.

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O NH

HN

CO2O

H N

HN

-

O2C

-

O2C O

H N O

HN

O

CO2-

-

O

CO2CO2O HN

NH

O2C

-

O2C O

HN -

O

2-6

βCD H1

A

AD H2-4

HOD βCD H

O2C

PAA H

O

ddn N-CH2

HN CO2-

-

NH

HN

O2C

ddn CH2

CO2-

B

Figure 5.18. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % ADddn-PAA and 66βCD2ur in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group and the -CH2protons of ddn chain, respectively. Approximate structures are shown above.

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CO2-

H N

HN

O

H N

N H

O

CO2-

-

O NH

-

O2C

-

O2C O

HN -

O

HOD βCD H2-6 βCD H1

PAA H

O

ddn N-CH2 su linker CH2

CO2

HN

H N

-

A

O2C

O2C

2-4

HN

N H

O2C O

HN

O

O

O

-

H N

CO2CO2O

O2C

AD H ddn CH2

CO2 O

-

-

B

Figure 5.19. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 1 wt % ADddn-PAA and 66βCD2su in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group and the -CH2protons of ddn chain, respectively. Approximate structures are shown above. (su refers to the succinamide linker of the linked βCD dimer, 66βCD2su). - 213 -

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5.3.3. Interaction of βCD Substituted PAAs with Adamantyl Substituted PAAs The host-guest complexation behavior of the series of three βCD substituted PAAs with the series of three AD substituted PAAs was studied by 2D 1H NOESY NMR in 1 cm3 D2O of 0.5 wt % of each βCD substituted PAAs with 0.5 wt % of each AD substituted PAA. Under these conditions a 1:1 ratio of host βCD and guest was AD obtained. The 2D 1H NOESY NMR spectrum of βCD-PAA with AD-PAA (Fig. 5.20) show no cross-peak arising from interactions between the AD group protons H2-4 and the βCD protons H3, H5 and H6 indicating that the AD groups were distant from the βCD annuli, which may be due to the short substituent chain and too much steric crowding of the PAA main chain. The spectra of βCD-PAA with ADhn-PAA and ADddn-PAA (5.21 and 5.22, respectively) show cross-peaks arising from interactions between the AD group protons H2-4 and the βCD protons H3, H5 and H6. The substituent chains are also complexed to a similar extent (Fig. 5.22). The 2D 1H NOESY NMR spectrum of the βCDhn-PAA with the ADhn-PAA (Fig. 5.24) shows cross-peaks arising from interactions between the AD group protons H2-4, and the βCD protons H3, H5 and H6 consistent with the AD group occupying the βCD annulus plus a small interaction of the hn chain. However, no cross-peaks appear in the spectrum of equimolar βCDhn-PAA and AD-PAA (Fig. 5.23). Weak cross-peaks appear in the spectrum of equimolar βCDhn-PAA and ADddn-PAA (Fig. 5.25) indicating that weak complexation of the AD group and the ddn chain in the βCD annulus occurs. The spectra of βCDddn-PAA with the series of three AD substituted PAAs (Figs. 5.26, 5.27 and 5.28) show strong cross-peaks arising from interactions between the -CH2protons of ddn chain and those of βCD H3, H5 and H6 but no significant cross-peaks arising from interactions between the AD group H2-4 protons and the βCD protons H3, H5 and H6. This indicates that the ddn chain dominantly occupies the βCD annulus and limits the complexation of the AD group.

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CO2CO2O

-

O2C

-

O2C O

HN HN

CO2-

O2C

PAA, AD H

-

HOD βCD H2-6 βCD H1

Figure 5.20. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 0.5 wt % of βCD-PAA and AD-PAA in D2O. No cross-peak arising from NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group are observed. Approximate structures are shown above.

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-

CO2CO2O

O2C

O2C O

HN NH

-

O

O2C

HN

hn CH2

AD H2-4

hn N-CH2

HOD

βCD H1

PAA H

CO2-

βCD H2-6

A

B

Figure 5.21. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 0.5 wt % of βCD-PAA and ADhn-PAA in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group and the -CH2protons of the hn chain, respectively. Approximate structures are shown above.

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Chapter 5

H N

HN

O

HN

-

CO2O

-

O 2C O 2C O

HN

O2C

NH

ddn N-CH2

HOD

HN -

CO2-

CO2-

βCD H1

-

O

O 2C

2-4

CO2O

O2C O

CO2-

AD H ddn CH2

-

O2C

PAA H

-

CO2-

βCD H2-6

A

B

Figure 5.22. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 0.5 wt % of βCD-PAA and ADddn-PAA in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from the NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group and the -CH2protons of the ddn chain, respectively. Approximate structures are shown above.

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Chapter 5

CO2-

-

CO2O

-

O2C O2C O

HN

HN

-

HOD

βCD H1

βCD H2-6

PAA, AD H

hn CH2

O2C

N H

hn N-CH2

CO2-

Figure 5.23. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 0.5 wt % of βCDhn-PAA and AD-PAA in D2O. No cross-peak arising from NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group are observed. Approximate structures are shown above.

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Chapter 5

-

CO2-

-

CO2O

βCD H1

PAA H

βCD hn N-CH2

O2C

AD H2-4 hn CH2

-

O

AD hn N-

HOD

O2C O

HN NH

HN CO2- N H

O2C

βCD H2-6

A

B

Figure 5.24. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 0.5 wt % of βCDhn-PAA and ADhn-PAA in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from NOE interactions between the annular H3, H5 and H6 protons of βCD and the H2, H3, and H4 protons of the AD group and the -CH2protons of the hn chain, respectively. Approximate structures are shown above.

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Chapter 5

CO2-

O 2C

CO2O

H N

O 2C O

O 2C

-

O 2C O

HN HN

O

-

CO2O

-

HN CO2- N H

CO2-

-

CO2- N H

-

O 2C

NH

HN -

βCD H1

βCD H2-6

A

O 2C

AD H2-4 hn, ddn CH2

PAA H

βCD hn N-CH2

HOD

AD ddn N-

O

B

Figure 5.25. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 0.5 wt % of βCDhn-PAA and ADddn-PAA in D2O. The green rectangles drawn on the spectrum, A and B, contain the cross-peaks arising from NOE interactions between the annular H3, H5 and H6 protons of βCD and the H3 and H4 protons of the AD group and the -CH2- protons of the ddn chain, respectively. Approximate structures are shown above.

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Chapter 5

-

O2C

-

O2C O

CO2CO2O

HN -

O2C

HN

HOD

βCD H1

βCD H2-6

PAA, AD H

ddn CH2

N H

ddn N-CH2

CO2-

A

Figure 5.26. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 0.5 wt % of βCDddn-PAA and AD-PAA in D2O. The green rectangle drawn on the spectrum, A, contains the cross-peaks arising from NOE interactions between the annular H3, H5 and H6 protons of βCD and the -CH2- protons of the ddn chain. Approximate structures are shown above.

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Chapter 5

-

O2C

-

O 2C O

CO2-

HN NH

CO2O

O

-

O2C

HN

HOD

βCD H1

PAA, AD H

βCD H2-6

hn, ddn CH2

N H

hn N-CH2

CO2-

A

Figure 5.27. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 0.5 wt % of βCDddn-PAA and ADhn-PAA in D2O. The green rectangle drawn on the spectrum, A, contains the cross-peaks arising from NOE interactions between the annular H3, H5 and H6 protons of βCD and the -CH2- protons of the ddn chain. Approximate structures are shown above.

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Chapter 5

-

O 2C

-

O 2C O

H N

CO2O

HN -

CO2-

O 2C

O HN CO2-

βCD H

1

βCD H2-6

PAA, AD H

ddn CH2

HOD

ddn N-CH2

N H

A

Figure 5.28. 2D 1H NOESY NMR (600 MHz, 0.3 s mixing time) spectrum of 0.5 wt % of βCDddn-PAA and ADddn-PAA in D2O. The green rectangle drawn on the spectrum, A, contains the cross-peaks arising from NOE interactions between the annular H3, H5 and H6 protons of βCD and the -CH2- protons of the ddn chain. Approximate structures are shown above.

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Chapter 5

5.4. Discussion and Summary 1. It can be seen that there are strong host-guest interactions between the series of AD substituted PAA and free βCD. Therefore, it is possible for using free βCD to displace hydrophobe-hydrophobe association in the AD substituted PAAs, which may reduce viscosity. 2. Strong host-guest interaction of urea and succinamide linked βCD dimers with the series of AD substituted PAAs suggest that is a strong potential for using the linked βCD dimers to obtain cross-linked structures for the polymers and increasing viscosity. 3. Adamantane substituted PAAs and βCD substituted PAAs mixtures show host-guest interactions between the AD and βCD substituents. This produces cross linked substituted PAA structure which should increase viscosity. Generally, it is seen that weak 2D 1H NOESY NMR cross-peaks occur with short substituents. This probably reflects steric crowding with the PAA chain. At the other extreme, the long substituent C12 chain competes with the AD group for complexation in the βCD annulus. The C6 length substituent chain optimises complexation by the βCD annulus. 4. The host-guest interaction of βCD substituted PAA with adamantane-1-carboxylate is stronger than with AD substituted PAA.

To confirm the effects of the above discus host-guest interaction on viscosity, it is necessary to complete the rheological studies at Princeton.

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Chapter 5

5.5. References 1

M. Annaka and T. Tanaka, Nature, 1992, 355, 430.

2

B. D. Ratner, A. S. Hoffman, F. J. Schoen, and J. C. Lemons, 'Biomaterials Science: An Introduction to Materials and Medecine', New York, Academic Press, 1996.

3

K. Y. Lee and D. J. Mooney, Chem. Rev., 2001, 101, 1869.

4

N. M. Sangeetha and U. Maitra, Chem. Soc. Rev., 2005, 34, 821.

5

X. Guo, A. A. Abdala, B. L. May, S. F. Lincoln, S. A. Khan, and R. K. Prud'homme, Polymer, 2006, 47, 2976.

6

I. Tomatsu, A. Hashidzume, and A. Harada, Macromol. Rapid Commun. , 2005, 26, 825.

7

I. Tomatsu, A. Hashidzume, and A. Harada, Macromol., 2005, 38, 5223.

8

I. Tomatsu, A. Hashidzume, and A. Harada, J. Am. Chem. Soc., 2006, 128, 2226.

9

A. Hashidzume, F. Ito, I. Tomatsu, and A. Harada, Macromol. Rapid Commun., 2005, 26, 1151.

10

O. Kretschmann, S. W. Choi, M. Miyauchi, I. Tomatsu, A. Harada, and H. Ritter, Angew. Chem. Int. Ed. Engl., 2006, 45, 4361.

11

X. Guo, A. A. Abdala, B. L. May, S. F. Lincoln, S. A. Khan, and R. K. Prud'homme, Macromol., 2005, 38, 3037.

12

S. E. Brown, J. H. Coates, D. R. Coghlan, C. J. Easton, S. J. v. Eyk, W. Sanowski, A. Lepore, S. F. Lincoln, Y. Luo, B. L. May, D. S. Scheisser, P. Wang, and M. L. Williams, Aust. J. Chem., 1993, 46, 953.

13

B. L. May, S. D. Ken, C. J. Easton and S. F. Lincoln, J. Chem. Soc., Perkin Trans, 1997, 1, 3157.

14

M. V. Rekharsky and Y. Inoue, Chem. Rev., 1998, 98, 1875.

15

H. Schneider, F. Hacket, V. Rudiger, and H. Ikeda, Chem. Rev., 1998, 98, 1755. - 225 -

Duc-Truc Pham

Chapter 6 - Experimental

CHAPTER 6

EXPERIMENTAL

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Chapter 6 - Experimental

6.1. General 6.1.1. Measurements 1

H and 13C NMR spectra were recorded using a Varian Gemini ACP-300 spectrometer

operating at 300.145 MHz (1H) or 75.4 MHz (13C), unless otherwise stated. A Varian Gemini 200 spectrometer operating at 199.953 MHz (1H) and 50.4 MHz (13C) was also used. The abbreviations singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m) refer to the multiplicity of the NMR resonances. Compounds were dissolved in either CDCl3, D2O or d6-DMSO, and resonances were referenced against either tetramethylsilane in CDCl3, the residual solvent multiplet (δH = 2.49, δC = 39.5) in d6-DMSO or an external standard, trimethylsilylpropiosulfonic acid, in D2O. The 2D 1H ROESY and NOESY NMR spectra were recorded on a Varian Inova 600 Spectrometer operating at 599.957 MHz, using a standard sequence with a mixing time of 0.3 seconds.

Elemental analyses were performed by the Microanalytical Service of the Chemistry Department, University of Otago, Dunedin, New Zealand. As cyclodextrin derivatives, have water molecules associated with them, were characterised by adding whole numbers of water molecules to the molecular formula to give the best fit to the microanalytical data. LC-Q Mass spectrometry was carried on a Finnigan LCQ instrument. Samples were dissolved in HPLC grade methanol, Milli-Q water or a mixture of the two at a concentration of 0.5 mg cm-3.

Thin layer chromatography (TLC) was carried out on Merck Kieselgel 60 F254 on aluminium-backed sheets. For analysis of cyclodextrin derivatives, plates were developed with 7:7:5:4 v/v ethyl acetate/propan-2-ol/ammonium hydroxide/water and the compounds were visualised by drying the plate and then dipping it into a 1% sulphuric acid in ethanol solution and heating it with a heat gun. To visualise amino bearing cyclodextrins plates were dried prior to dipping into 0.5% ninhydrin in ethanol and heated with a heat-gun,

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Chapter 6 - Experimental

before dipping in 1% sulphuric acid in ethanol. For the preparations described below Rc represents the Rf of a substituted cyclodextrin relative to the Rf of the parent cyclodextrin. Melting points were measured using a Kofler hot-stage apparatus viewed through a Reichert microscope and are uncorrected. As cyclodextrin derivatives decompose without melting above 180 ºC, melting points were not determined for such compounds.

UV absorbance measurements were recorded at 0.25 nm intervals over the range 220350 nm on a UV-VIS-NIR Cary/Varian 5000 spectrophotometer. Fluorescence spectra were recorded at 0.5 nm intervals over the range 350-550 nm with a Cary/Varian Eclipse fluorimeter. In both cases the sample cuvette, 1 cm3, was thermostated at 298.2 K using a Cary/Varian temperature controller. All solutions were freshly prepared in aqueous phosphate buffer (pH 7.0, I = 0.10 mol dm-3) immediately prior to measurement. The TNSand BNS- solutions were kept to a minimum exposure to light by wrapping the containers in aluminium foil. All sample solutions were diluted by weight from stock solutions using Gilson Pipetmans P20, P100, P200, P1000 and P5000.

6.1.2. Data Analysis Equation 6.1 describes the observed absorbance when a single (host).(guest) complex is present in a solution, where A represents the total absorbance, εH, εG and εH.G represent the molar absorbances of the host, guest and (host).(guest) complex, respectively. When an additional (host)2.(guest) complex is present, the observed absorbance in given by Eqn. 6.2, where εH2.G represent the molar absorbances of the (host)2.(guest) complex. Similar relationships exist for fluorescence titrations. A = εH[host] + εG[guest] + εH.G[(host).(guest)]

(6.1)1,2

A = εH[host] + εG[guest] + εH.G[(host).(guest)] + εH2.G[(host)2.(guest)] (6.2)1,2

For the UV and fluorescence monitored titrations, K1 (stability constant for 1:1 host:guest complexes) and K2 (stability constant for 2:1 host:guest complexes) were determined by a fit to either equation 6.1 or 6.2 over a range of wavelengths where significant spectral change occurred. A non-linear least-squares regression routine based

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Chapter 6 - Experimental

on Method 5 of Pitha and Jones3 using the Specfit2 program through the Matlab4 protocol was employed. The data were not weighted.

The polynomials in Eqns. 6.3 and 6.4 were derived from the mass balance relationships associated with Eqns. 6.1 and 6.2, respectively.1,2 The initial concentrations of host and guest are denoted by [host]0 and [guest]0, and K1 and K2 represent the stepwise stability constants for the (host).(guest) and (host)2.(guest), respectively. K1[guest]2 + (K1{[host]0 – [guest]0} +1)[guest] – [guest]0 = 0

(6.3)1,2

K1K2[guest]3 + K1(K2{2[host]0 – [guest]0} +1)[guest]2 + + (K1{[host]0 – [guest]0} +1)[guest] – [guest]0 = 0

(6.4)1,2

The appropriate algorithms were iteratively fitted to the 1D 1H NMR titrations to determine Kd (dimerisation constant) and K12 (stability constant of 1:2 host:guest complexes) using the HypNMR 20035 (Protonic Software6). For the potentiometric titrations, values of K (Fe2+ complexation constants) and pKa (acid dissociation constant) were determined using the program Hyperquad 20037 (Protonic Software6) and speciation plots were calculated using Hyss 20068 (Protonic Software6).

6.1.3. Materials

N-methylpyrrolidin-2-one (NMP) was dried by distillation from calcium hydride. Acetone, ethanol, methanol and dichloromethane were dried by distillation. N,NDimethylformamide (DMF) was obtained form APS, pyridine from Ajax and diethylether from Chem Supply.

Flash column chromatography was carried out using Merck Kieselgel 60 (230-400 mesh ASTM) silica. Squat column chromatography was carried out using Merck Kieselgel 60 F254 thin layer chromatography silica. Bio-Rex 70 resin was purchased from Bio-Rad Laboratories, Inc, CA and converted to the acid form using 3 mol dm-3 hydrochloric acid. Diaion HP-20 resin was from purchased from Supelco, PA.

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Chapter 6 - Experimental

Desalting was carried out by the use of AG 501-X8 mixed bed ion-exchanged resin (Bio-Rad Laboratories, Inc., CA). The drying of products was carried out under reduced pressure (approximately 0.1 torr) over phosphorus pentoxide at room temperature. Deionised water was prepared using a Milli-Q-Reagent system to give a resistivity of >15 MΩ cm. An aqueous phosphate buffer (pH 7.0, I = 0.10 mol dm-3) was prepared from Na2PO4 (BDH) and KH2PO4 (Ajax) as described in the literature.9 β-Cyclodextrin (βCD) was donated by Nihon Shokuhin Kako Co. Hydrogen peroxide was obtained from Chem-Supply, sodium hydroxide was obtained from Ajax, ferrous sulphate and sodium carbonate were obtained from BDH; 1,2-diamino ethane (en), 1,6diamino hexane (hn), 1,12-diamino dodecane (ddn), 2,2’,2’’-triaminotriethylamine (tren) were obtained from Strem Chemicals; D2O, d6-DMSO, CDCl3 were obtained from Cambridge Isotope Laboratory. Amino acids and pheniramine maleate were obtained from Sigma. Other reagents used were obtained from Aldrich and were not further purified before use unless otherwise stated.

6.1.3. Preparation of Compounds Unless otherwise stated, 6A-O-(4-methylbenzenesulfonyl)-β-cyclodextrin (6βCDtos),10 6A-amino-6A-deoxy-β-cyclodextrin (6βCDNH2),11 6A-(2-aminoethyl)amino-6A-deoxy-βcyclodextrin (6βCDen),12 6A-(6-aminohexyl)amino-6A-deoxy-β-cyclodextrin (6βCDhn),12 6A-(6-aminododecyl)amino-6A-deoxy-β-cyclodextrin (6βCDddn),12 6A-azido-6A-deoxy-βcyclodextrin

(6βCDN3)13

and

sodium

tricarbonatocobaltate(III)

trihidrate,

Na3[Co(CO3)3].3H2O,14 were prepared by literature methods. 1-Amino-adamantane, ADNH2, was obtained by neutralizing ADNH2.HCl (Aldrich) with NaOH followed by extraction into dichloromethane. The substituted cyclodextrins were dried to a constant weight over P2O5 and stored in the dark under refrigeration.

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Chapter 6 - Experimental

6.2. Experimental for Chapter 2 6.2.1. Preparation of Compounds 2A-O-(4-Methylbenzenesulfonyl)-β-cyclodextrin, 2βCDtos

O O

O

S O (Glu)6

O

OTs

HO OH O

Compounds were prepared by a slight modification of the method described by Murakami15. A mixture of βCD (45.4 g, 0.04 mol) and dibutyltin oxide (25 g, 0.1 mol) in anhydrous DMF (150 cm3) was stirred at 100 oC for 2 hrs under nitrogen. The mixture was cooled to 0 oC, stirred vigorously and to this was added triethylamine (12.2 g, 0.12 mol), followed by a dropwise addition of p-toluenesulfonylchloride (tosylchloride) (19.2 g, 0.1 mol) in DMF (50 cm3). After 2 hrs, another portion of p-toluenesulfonylchloride (9.6 g, 0.05 mol) in DMF (20 cm3) was added dropwise. The resultant solution was stirred overnight at room temperature. The solvent was removed and the residual 10 cm3 of a yellow syrup was added to 1 dm3 of acetone and vigorously stirred for 30 min. The precipitate formed was collected by filtration, washed with acetone and diethylether and dried under vacuum. The crude solid was loaded onto a Diaion HP-20 column (5 × 20cm) which was flushed with water (~2.4 dm3) until no unreacted βCD was detected in the eluate by TLC. The column was then eluted with a water methanol solvent gradient (~400 cm3 fraction). Remaining unreacted βCD was eluted with water and 10-20% aqueous methanol and the title product was eluted with 30-50% aqueous methanol. The solvent was removed and the product was dried under vacuum to give the title compound as a white solid.

Yield: 4.64g (9%) TLC: Rc = 1.93

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Duc-Truc Pham

1

Chapter 6 - Experimental

H NMR: δH(D2O) 7.95, 7.55 (ABq, J

= 8.4 Hz, 4H, ArH); 5.04 (s, 7H, H1); 3.9-3.4 (m,

42H, H2-H6); 2.43 (2, 3H, Ar-CH3) 13

C NMR: δC(D2O) 149.7, 149.2, 132.5, 130.8 (Ar-C); 104.5 (C1B-G), 100.8 (C1A); 83.6

(C4A-G); 82.2 (C2A); 76.0-74.5 (C2B-G, C3B-G, C5A-G); 72.4 (C3A) 62.7 (C6A-G); 23.7 (ArCH3). Mano-2A,3A epoxide β-cyclodextrin16

O O (Glu)6

O

HO

O

2A-O-(4-Methylbenzenesulfonyl)-β-cyclodextrin, 2βCDtos (3.0 g, 2.3 mmol) was dissolved in aqueous 10% ammonium bicarbonate solution (100 cm3) and stirred at 60 oC for 3 hrs. The resulting solution was evaporated to dryness under vacuum. The residue was dissolved in water and the solution evaporated under reduced pressure to remove excess ammonium bicarbonate (this procedure was repeated several times). The residue was dissolved in water and added to vigorously stirring acetone. The precipitate formed was collected by filtration, washed with acetone and diethylether and dried under vacuum. It was then dissolved in water (40 cm3) and loaded onto a Diaion HP-20 column (2 × 30 cm). The column was washed with water (300 cm3) and 10-20% aqueous methanol to obtain the title compound (unreacted 2βCDtos was eluted with 30-40% aqueous methanol). The solvent was removed and the title compound was obtained as a white solid after drying under vacuum.

Yield: 2.4 g (92.3%). TLC: Rc = 1.11 1

H NMR: δH(D2O) 5.05 (m, 7H, H1); 3.9-3.4 (m, 42H, H2-H6)

13

C NMR: δC(D2O) 104.5-103.7 (C1), 83.7-83.0 (C4), 75.7-72.1 (C2B-G, C3B-G, C5), 63.6

(C6B-G), 62.9 (C6A), 56.7 (C2A), 51.8 (C3A) LCQ-MS: (M + H+) 1117 - 232 -

Duc-Truc Pham

Chapter 6 - Experimental

(2AS,3AS)-3A-Amino-3A -deoxy-β-cyclodextrin, 3βCDNH2-17

NOTE: This image is included on page 233 of the print copy of the thesis held in the University of Adelaide Library.

A

A

2 ,3 -Manno-epoxide β−cyclodextrin (1.0 g, 0.89 mmol) was dissolved in 25% 3

o

aqueous ammonia (30 cm ) and stirred at 60 C for 3 hrs. The resulting solution was 3

evaporated down and the solid was dissolved in 15 cm of concentrated aqueous ammonia and added to vigorously stirred acetone. The precipitate formed was collected by filtration, washed with acetone and diethylether and dried under vacuum. +

An aqueous solution was loaded onto a BioRex 70 (H form) cation exchange column 3

(4.5 × 4.5 cm). The column was washed with water (~400 cm ) and the product was -3

3

eluted with 0.5 mol dm aqueous ammonia solution (~250 cm ). Water was removed 3

under reduced pressure and the residue was redissolved in water (10 cm ) and died under reduced pressure to remove ammonia (3 times). The residue was freeze-dried to give the title product as a white solid. Yield: 0.6 g (60%) TLC: Rc = 0.71 1

1

2

6

H NMR: δH(D2O) 5.05 (m, 7H, H ); 3.9-3.4 (m, 42H, H -H )

13

1

4

2B-G

C NMR: δC(D2O) 104.5-103.7 (C ), 83.7-83.0 (C ), 75.7-72.1 (C 62.9 6B-G

C

6A

3B-G

,C

5

, C ),

3A

), 62.3 (C ), 54.8 (C ) +

LCQ-MS: (M+H ) 1134 Elemental analysis: Cal. C42H71NO34.7H2O: C, 40.03; H, 6.80; N, 1.1. Found: C, 40.0; H, 6.52; N, 0.95.

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Chapter 6 - Experimental

Bis-4-nitrophenyl succinate17

NOTE: This image is included on page 234of the print copy of the thesis held in the University of Adelaide Library.

Succinic acid (1.0 g, 8.5 mmol) and N,N’-dicyclohexyl carbodiimide (DCC) (3.5 g, 17 3

mmol) were dissolved in dichloromethane (50 cm ) and stirred at room temperature. 4Nitrophenol (2.36 g, 17 mmol) was added to the reaction solution which was left to stir overnight. The mixture was filtered and run through a squat column after which it was mixed with aqueous sodium bicarbonate (5%) and extracted with dichloromethane. The solvent was removed and title compound was obtained as a pale yellow powder after drying under vacuum. (The chromatographic materials in the squat column were silica gel 60 (5-14 µm), Scharlau GE0033). Yield: 800 mg (25%) TLC: Rf = 0.50 (5% o

1

hexanes/dichloromethane, Rf (nitrophenol) = 0.21) m.p.: 153-154 C H NMR: δH(CDCl3) 8.30 13

(d, J = 9.3 Hz, 4H, ArH); 7.32 (d, J = 9.3 Hz, 4H, ArH); 3.06 (s, 4H, succinyl CH2) C NMR: δC(CDCl3) 169.77 (ester C=O); 155-122.2 (ArC); 29.07 (succinyl CH2) 17

General procedure for the preparation of the succinamide linked βCD dimers A

A

A

A

A

A

Either (2 S,3 S)-3 -amino-3 -deoxy-β-cyclodextrin or 6 -amino-6 -deoxy-β-cyclo3

dextrin (~ 0.88 mmol) was dissolved in pyridine (20 cm ) and stirred at room temperature. Bis-4-nitrophenyl succinate (0.4 equivalents, 0.36 mmol) was added to this solution in two or more portions over a period of 1 hr. and the reaction mixture was stirred for 48 hrs 3

at room temperature before being added dropwise to diethylether (200 cm ) with vigorous stirring. The resultant precipitate was collected by centrifugation, washed with acetone and diethylether and dried under vacuum. The product was dissolved in H2O and run +

A

A

A

A

down a BioRex 70 (H ) column to remove either excess (2 S,3 S)-3 -amino-3 -deoxy-βA

A

cyclodextrin or 6 -amino-6 -deoxy-β-cyclodextrin. The white solid product was obtained by freeze drying followed by further drying over phosphorous pentoxide.

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N,N’-bis-(6A-deoxy-6A-β-cyclodextrinyl)succinamide, 66βCD2su

O H N N H O

6A-Amino-6A-deoxy-β-cyclodextrin (1.0 g, 0.88 mmol) was dissolved in pyridine (20 cm3) and stirred at room temperature. To this, bis-4-nitrophenyl succinate (129 mg, 0.36 mmol) was added in two portions over a period of 1 hr. After the general work-up and purification procedure, the title compound was obtained as a white solid.

Yield: 710 mg (84%) TLC: Rc = 0.40 1

H NMR: δH(D2O) 5.08-5.05 (m, 14H, H1); 4.0-3.38 (m, 84H, H2-H6); 2.6 (s, 4H, succinyl

CH2). 13

C NMR: δC(D2O) 177.4 (amide C=O), 104.5 (C1), 85.6-83.7 (C4), 75.7-72.8 (C2, C3, C5),

62.9 (C6B-G), 42.8 (C6A), 33.6 (succinyl CH2). LCQ-MS: (M + H+) 2351.2; (M + Na+) 2373.9 Elemental analysis: C88H144N2O70.19H2O: C, 39.25; H, 6.81; N, 1.04. Found: C, 39.3; H, 6.6; N, 1.1. 6A-(3-(4-Nitrophenoxycarbonyl)-propionamido)-6A-deoxy-β-cyclodextrin

O O2N

O

C

C O

H N βCD

Bis-4-nitrophenyl succinate (950 mg, 2.64 mmol) was dissolved in pyridine (20 cm3) and stirred at room temperature. To this, 6βCDNH2 (1.0 g, 0.88 mmol) was added in three portions over a period of 1 hr. The mixture was then left to stir for 48 hrs at room temperature. It was then added dropwise to diethylether (200 cm3) with vigorous stirring.

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The precipitate was collected by centrifugation, washed with acetone and diethylether and dried under vacuum.

Yield: 868 mg (73%) TLC: Rc = 1.54 1

H NMR: δH(DMSO) 8.30 (d, J = 8.4 Hz, 2H, ArH); 7.32 (d, J = 8.7 Hz, 2H, ArH); 5.7,

4.8, 4.5, 3.6-3.36 (m, 78H, CD; 2.77 (m, 4H, succinyl CH2) 13

C NMR: δC(DMSO) 171, 170.8 (ester C=O); 155.5-123.1 (ArC); 102.0 (C1), 83.6-81.7

(C4), 73.1-72.0 (C2, C3, C5), 59.9 (C6B-G), 41.8 (C6A), 30.6-29.3 (succinyl CH2). N-(2AS,3AS)-(3A-Deoxy-3A-β-cyclodextrinyl)-N’-(6A-deoxy-6A-β-cyclodextrinyl)succinamide, 36βCD2su

O H N N H O

(2AS,3AS)-3A-Amino-3A-deoxy-β-cyclodextrin (1.0 g, 0.88 mmol) was dissolved in pyridine (20 cm3) and stirred at room temperature. To this, 6A-deoxy-6A-(3nitrophenoxycarbonyl)-propionamido)-β-cyclodextrin (0.868 g, 0.65 mmol) was added in two portions over a period of 1 hr. The mixture was then left to stir for 48 hrs at room temperature. After the general work-up and purification procedure, the title compound was obtained as a white solid.

Yield: 1.05 g (69%) TLC: Rc = 0.47 1

H NMR: δH(D2O) 5.15-5.05 (m, 14H, H1); 4.20-3.58 (m, 84H, H2-H6); 2.59 (s, 4H,

succinyl CH2). 13

C NMR: δC(600 MHz, D2O) 174.8, 174.6 (amide C=O); 103.7-101.1 (C1); 82.8-79.9 (C4);

73.1-69.9 (C2, C3, C5); 60.2-59.5 (C6); 50.9 (C3A’); 39.9 (C6A); 31.1, 30.9 (succinyl CH2). LCQ-MS: (M + H+) 2351.1; (M + Na+) 2374.0 Elemental analysis: C88H144N2O70.18H2O: C, 39.52; H, 6.78; N, 1.05. Found: C, 39.5; H, 6.3; N, 1.0. - 236 -

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N,N’-Bis-((2AS,3AS)-3A-deoxy-3A-β-cyclodextrinyl)succinamide, 33βCD2su

O H N N H O

(2AS,3AS)-3A-Amino-3A-deoxy-β-cyclodextrin (1.0 g, 0.88 mmol) was dissolved in pyridine (20 cm3) and stirred at room temperature. To this, bis-4-nitrophenyl succinate (129 mg, 0.36 mmol) was added in two portions over a period of 1 hr. After the general work-up and purification procedure, the title compound was obtained as a white solid.

Yield: 700 mg (82%) TLC: Rc = 0.54 1

H NMR: δH(D2O) 5.13-5.03 (m, 14H, H1); 4.1-3.58 (m, 84H, H2-H6); 2.61 (s, 4H, succinyl

CH2). 13

C NMR: δC(D2O) 177.5 (amide C=O), 106.4-103.8 (C1), 84.6-82.3 (C4), 75.7-72.5 (C2,

C3, C5), 62.4-63.9 (C6), 53.6 (C3A), 33.7 (succinyl CH2). LCQ-MS: (M + H+) 2351.0; (M + Na+) 2374.0 Elemental analysis: C88H144N2O70.20H2O: C, 38.99; H, 6.84; N, 1.03. Found: C, 39.1; H, 6.5 N, 1.1. N,N’-Bis(6A-deoxy-6A-β-cyclodextrinyl)urea, 66βCD2ur18

O HN

NH

6A-Azido-6A-deoxy-β-cyclodextrin (2.85 g, 2.45 mmol) was dissolved in DMF (40 cm3) and the solution was saturated with dry CO2 and stirred at room temperature. Triphenylphosphine Ph3P (1.0 g, 3.8 mmol) in DMF (10 cm3) was added dropwise and the reaction mixture was saturated with CO2 and stirred for 27 hrs after which it was added

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dropwise to vigorously stirred acetone. The resulting precipitate was collected by centrifugation, washed acetone and diethylether, dried under vacuum and freeze dried.

Yield: 2.69 g (47%) TLC: Rc = 0.36 1

H NMR: δH(D2O) 5.05 (m, 14H, H1); 3.9-3.4 (m, 84H, H2-H6)

13

C NMR: δC(D2O) 163.1 (amide C=O), 104.5 (C1), 85.5-83.0 (C4), 76.4-73 (C2, C3, C5),

63.0 (C6B-G), 43.2 (C6A) LCQ-MS: (M + H+) 2294.2; (M + Na+) 2317.1 Elemental analysis: C85H140N2O69.19H2O: C, 38.73; H, 6.81; N, 1.06. Found: C, 38.6; H, 6.8; N, 1.08. N-((2AS,3AS)-3A-Deoxy-3A-β-cyclodextrinyl)-N’-(6A-deoxy-6A-β-cyclodextrinyl)urea, 36βCD2ur18

O HN

NH

(2AS,3AS)-3A-Amino-3 A-deoxy-β-cyclodextrin (1.24 g, 0.88 mmol) was dissolved in 10 cm3 DMF and stirred at room temperature and dry CO2 was bubbled through the solution. After 1 hr., 6A-azido-6A-deoxy-β-cyclodextrin (918 mg, 0.79 mmol) in DMF (5 cm3) was added dropwise followed by triphenylphosphine (288 mg, 1.11 mmol) in DMF (10 cm3). The solution was then stirred overnight. TLC showed that no 6A-azido-6A-deoxy-βcyclodextrin remained. The solution was reduced to 5 cm3 and added to acetone. The resulting precipitate was collected by vacuum filtration and washed with acetone and diethylether and dried under vacuum. The collected product was dissolved in H2O and run through a BioRex 70 (H+) column. Water was removed and the product was then freeze dried.

Yield: 1.2 g (66%) TLC: Rc = 0.31

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Chapter 6 - Experimental

H NMR: δH(D2O) 5.14-5.04 (m, 14H, H1); 3.9-3.4 (m, 84H, H2-H6)

13

C NMR: δC(D2O) 162.6 (amide C=O), 106.3-103.7 (C1), 85.5-82.4 (C4), 75.7-73.3 (C2,

C3, C5), 62.9-62.3 (C6), 54.3 (C3A), 43.2 (C6A) LCQ-MS: (M + H+) 2295.3; (M + Na+) 2318.6 Elemental analysis: C85H140N2O69.17H2O: C, 39.26; H, 6.74; N, 1.08. Found: C, 39.2; H, 6.5 N, 0.97.

Attempted

synthesis

of

N,N’-bis((2AS,3AS)-3A-deoxy-3A-β-cyclodextrinyl)urea,

33βCD2ur

Method 1: An attempt was made to prepare 33βCD2ur by a method similar to that used to prepare 66βCD2ur.13 Preparation of the required (2AS,3AS)-3A-azido-3A-deoxy-β-cyclodextrin was attempted by the addition of NaN3 (0.85 g, 13 mmol) to a solution of 2A,3A-epoxide βcyclodextrin (1.5 g, 1.3 mmol) in DMF (10 cm3). The reaction mixture was stirred at 65 oC for 24 hrs. It was then cooled to room temperature and desalted using Bio-Rad AG 501-x8 (D) (15 g) in H2O (15 cm3). The reaction products were precipitated by addition of acetone. TLC and 1H and

13

C-NMR spectra showed complex mixture of products to be

present and this method of preparation was not further pursued.

Method 2: Diphenyl carbonate (30 mg, 0.14 mmol) was added to a solution of (2AS,3AS)-3Aamino-3A-deoxy-β-cyclodextrin (500 mg, 0.44 mmol) in pyridine/H2O (10 cm3, 6:4) and left to stir at 100 oC, under similar conditions to those described in the literature.17 The reaction was monitored by TLC but no linked βCD dimer was detected.

6.2.2. Preparation of Solutions for 2D 1H ROESY NMR Studies A D2O phosphate buffer (pD 7.0, I = 0.10 mol dm-3) was prepared from Na2PO4 (BDH) and KH2PO4 (Ajax) as described in the literature.9 A stock solution of 6.0 cm3 of TNS- (1.5 mmol dm-3) in D2O phosphate buffer at pD 7.0 was prepared. Aliquots of 1 cm3 were added to test tubes contained the appropriate amount - 239 -

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of either βCD (1.7 mg); 66βCD2su (3.5 mg); 36βCD2su (3.5 mg); 33βCD2su (3.5 mg); 66βCD2ur (3.4 mg) or 36βCD2ur (3.4 mg). When dissolution was complete, portions of each solutions were transferred to 5 mm NMR tubes.

6.2.3. Preparation of Solutions for UV Titrations TNS- guest stock solutions (3.0 × 10-4 mol dm-3)

Sodium 6-(p-toluidino)-2-naphthalenesulfonate, NaTNS, (26.4 mg) was dissolved in aqueous phosphate buffer at (pH 7.0, I = 0.10 mol dm-3) to give an accurate volume of 250 cm3.

Host stock solutions Concentrations of host stock solutions were, βCD (1.0 × 10-3 mol dm-3); 66βCD2su (4.0 × 10-4 mol dm-3); 36βCD2su (2.5 × 10-4 mol dm-3); 33βCD2su (2.5 × 10-4 mol dm-3); 66βCD2ur (4.0 × 10-4 mol dm-3); 36βCD2ur (2.5 × 10-4 mol dm-3). The volume of each stock solution prepared in aqueous phosphate at (pH 7.0, I = 0.10 mol dm-3) buffer was 100 cm3. The weights of either βCD and the linked βCD dimers were: βCD (113.5 mg); 66βCD2su (94.0 mg); 36βCD2su (58.7 mg); 33βCD2su (58.7 mg); 66βCD2ur (91.7 mg) or 36βCD2ur (57.4 mg).

Sample preparation The complexation of TNS- by βCD and the linked βCD dimers in aqueous phosphate buffer (pH 7.0, I = 0.10 mol dm-3) at 298.2 K was studied by monitoring the absorbance change of 24 sample solutions containing TNS- (3.0 × 10-5 mol dm-3) with increasing concentrations of either βCD (0-5.0 × 10-4 mol dm-3); 66βCD2su (0-2.0 × 10-4 mol dm-3); 36βCD2su (0-1.25 × 10-4 mol dm-3); 33βCD2su (0-1.25 × 10-4 mol dm-3); 66βCD2ur (0-2.0 × 10-4 mol dm-3) or 36βCD2ur (0-1.25 × 10-4 mol dm-3). Twenty four reference solutions of each system containing the same concentrations of either βCD or a linked βCD dimer as in corresponding sample solutions were prepared.

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Each sample solution was prepared by mixing TNS- stock solution (1.0 cm3) and the appropriate amount of either βCD or a linked βCD dimer stock solution (0-5.0 cm3). A total volume of 10 cm3 was achieved by addition of aqueous phosphate buffer. The reference solutions were prepared in a similar manner with TNS- omitted.

6.2.4. Preparation of Solutions for Fluorimetric Titrations TNS- stock solutions (1.0 × 10-5 mol dm-3) The solution was prepared by diluted 10 cm3 of 3.0 × 10-4 mol dm-3 TNS stock solution to a total volume of 300 cm3 by further addition of the aqueous phosphate buffer at (pH 7.0, I = 0.10 mol dm-3).

Host stock solutions Concentrations of host stock solutions were, βCD (1.5 × 10-2 mol dm-3); 66βCD2su (1.0 × 10-3 mol dm-3); 36βCD2su (1.0 × 10-3 mol dm-3); 33βCD2su (1.0 × 10-3 mol dm-3); 66βCD2ur (4.0 × 10-4 mol dm-3); 36βCD2ur (4.0 × 10-4 mol dm-3). The volume of each stock solution prepared in the aqueous phosphate buffer at (pH 7.0, I = 0.10 mol dm-3) was 50 cm3. The weights of either βCD or a linked βCD dimer was: βCD (851.3 mg); 66βCD2su (117.5 mg); 36βCD2su (117.5 mg); 33βCD2su (117.5 mg); 66βCD2ur (45.9 mg) or 36βCD2ur (45.9 mg).

Sample preparation The complexation of TNS- by either βCD or a linked βCD dimer in aqueous phosphate buffer (pH 7.0, I = 0.10 mol dm-3) at 298.2 K was monitored through changes in the fluorescence of 30 sample solutions containing TNS- (1.0 × 10-6 mol dm-3) with increasing concentrations of βCD (0-7.5 × 10-3 mol dm-3); 66βCD2su (0-5.0 × 10-4 mol dm-3); 36βCD2su (0-5.0 × 10-4 mol dm-3); 33βCD2su (0-5.0 × 10-4 mol dm-3); 66βCD2ur (0-2.0 × 10-4 mol dm-3) or 36βCD2ur (0-2.0 × 10-4 mol dm-3).

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Each sample solution was prepared by adding TNS- stock solution (1.0 cm3) and the appropriate amount of either βCD or a linked βCD dimer stock solutions (0-5 cm3). A total volume of 10 cm3 was achieved by addition of aqueous phosphate buffer at (pH 7.0, I = 0.10 mol dm-3).

6.3. Experimental for Chapter 3

6.3.1. Preparation of Compounds Sodium 6-(4’-tert-butylanilino)-2-naphtalene sulphonate, BNS-19,20

H N NaO3S

The preparation was carried out according to literature methods.19,20 A solution of 2amino-6-naphtalenesulfonic acid (1.116 g, 5 mmol) (AmNS) and p-tert-butyl-aniline (2.238 g, 15 mmol) in 30 cm3 aqueous solution of Na2S2O5 (9.9 g) was refluxed for 3 days. The precipitate was filtered off at room temperature and washed with chloroform (to remove aniline) and water. The product was recrystallised three times from aqueous NaHSO3 (1.4 mol dm-3)/Na2SO3 (0.08 mol dm-3) solution and the title compound was obtained as a light pink solid.

Yield: 668 mg (36%) TLC: Rf = 0.49 (methanol:benzene 40:60) (Rf (AmNS) = 0.32) 1

H NMR: δH(D2O) 8.08-6.9 (m, 10H, ArH); 1.11 (s, 9H, -CH3)

13

C NMR: δC(DMSO) 135.4-100.1 (ArC); 31.2 (-CH3).

LCQ-MS: (M + H+) 355, (M + Na+) 377. Elemental analysis: Cal. C20H20NO3SNa.H2O: C, 60.74; H, 5.60; N, 3.54. Found: C, 60.79; H, 5.57; N, 3.50.

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6.3.2. Preparation of Solutions for 1H 2D ROESY NMR Studies A D2O phosphate buffer (pD 7.0, I = 0.10 mol dm-3) was prepared from Na2PO4 (BDH) and KH2PO4 (Ajax) as described in the literature.9 A stock solution of 6.0 cm3 of BNS- (2.0 mmol dm-3) in D2O phosphate buffer at pD 7.0 was prepared. Aliquots of 1 cm3 were added to test tubes contained the appropriate amount of either βCD (2.3 mg); 66βCD2su (4.7 mg); 36βCD2su (4.7 mg); 33βCD2su (4.7 mg); 66βCD2ur (4.6 mg) or 36βCD2ur (4.6 mg). When dissolution was complete, portions of each solution were transferred to 5 mm NMR tubes.

6.3.3. Preparation of Solutions for UV Titrations BNS- stock solutions (1.0 × 10-4 mol dm-3)

Sodium 6-(4-tert-butylanilino)-2-naphthalenesulfonate (9.4 mg) was dissolved in aqueous phosphate buffer( pH 7, 0 I = 0.10 mol dm-3) to give a total volume of 250 cm3.

Host stock solutions The concentrations of the host stock solutions were, βCD (4.0 × 10-4 mol dm-3); 66βCD2su (2.0 × 10-4 mol dm-3); 36βCD2su (4.0 × 10-5 mol dm-3); 33βCD2su (1.2 × 10-4 mol dm-3); 66βCD2ur (4.0 × 10-5 mol dm-3); 36βCD2ur (1.2 × 10-4 mol dm-3). Solutions of each of βCD and the linked βCD dimers were prepared from βCD (45.4 mg); 66βCD2su (47.0 mg); 36βCD2su (9.4 mg); 33βCD2su (28.2 mg); 66βCD2ur (9.2 mg) or 36βCD2ur (27.5 mg), respectively, in aqueous phosphate buffer (pH 7.0, I = 0.10 mol dm-3) to give total volumes of 100 cm3 in each case.

Sample preparation The complexation of BNS- by βCD and the linked βCD dimers in aqueous phosphate buffer (pH 7.0, I = 0.10 mol dm-3) at 298.2 K was studied by monitoring the absorbance change of 24 sample solutions containing BNS- (1.0 × 10-5 mol dm-3) with increasing - 243 -

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concentrations of βCD (0-2.0 × 10-4 mol dm-3); 66βCD2su (0-1.0 × 10-4 mol dm-3); 36βCD2su (0-2.0 × 10-5 mol dm-3); 33βCD2su (0-6.0 × 10-5 mol dm-3); 66βCD2ur (0-2.0 × 10-5 mol dm-3) or 36βCD2ur (0-6.0 × 10-5 mol dm-3). Twenty four reference solutions of each system were prepared at the same concentration of either βCD or the linked βCD dimers as in the corresponding sample solutions. Each sample solution was prepared by adding TNS- stock solution (1.0 cm3) and the appropriate amount of either βCD or a linked βCD dimer stock solution (0-5 cm3). A total volume of 10 cm3 was achieved by further addition of the aqueous phosphate buffer (pH 7.0, I = 0.10 mol dm-3). The reference solutions were similarly prepared with BNSomitted. BNS- dimerisation study Ten solutions of BNS- were prepared in the aqueous phosphate buffer with increasing concentration from 5.0 × 10-6 to 5.0 × 10-5 mol dm-3 by diluted the above described stock solution. A total volume of 10 cm3 was achieved by addition of the aqueous phosphate buffer.

6.3.4. Preparation of Solutions for Fluorimetric Titrations BNS- stock solution (1.0 × 10-5 mol dm-3) The solution was prepared by diluting 25 cm3 of the 1.0 × 10-4 mol dm-3 BNS- stock solution described above to a total volume of 250 cm3 with aqueous phosphate buffer (pH 7.0 I = 0.10 mol dm-3).

Host stock solutions Concentrations of stock solutions were, βCD (2.0 × 10-4 mol dm-3); 66βCD2su (2.0 × 10-5 mol dm-3); 36βCD2su (2.0 × 10-5 mol dm-3); 33βCD2su (1.0 × 10-4 mol dm-3); 66βCD2ur (1.0 × 10-5 mol dm-3) and 36βCD2ur (1.0 × 10-4 mol dm-3). A total volume of 50 cm3 of βCD and linked βCD dimers were prepared by diluting stock solutions of βCD and - 244 -

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the linked βCD dimers prepared for UV studies described above with aqueous phosphate buffer (pH 7.0 I = 0.10 mol dm-3).

Sample preparation The complexation of BNS- by βCD and linked βCD dimers in the aqueous phosphate buffer (pH 7.0, I = 0.10 mol dm-3) at 298.2 K was studied by monitoring the fluorescence intensity change of 30 sample solutions containing BNS- (1.0 × 10-6 mol dm-3) with increasing concentrations of βCD (0-1.0 × 10-4 mol dm-3); 66βCD2su (0-1.0 × 10-5 mol dm-3); 36βCD2su (0-1.0 × 10-5 mol dm-3); 33βCD2su (0-5.0 × 10-5 mol dm-3); 66βCD2ur (05.0 × 10-6 mol dm-3) or 36βCD2ur (0-5.0 × 10-5 mol dm-3). Each sample solution was prepared by adding BNS- stock solution (1.0 cm3) and an appropriate volume of either βCD or a linked βCD dimer stock solution (0-5 cm3). A total volume of 10 cm3 was achieved by the addition of aqueous phosphate buffer (pH 7.0 I = 0.10 mol dm-3).

6.3.5. Preparation of Solutions for NMR Titrations 1

H NMR titration spectra were recorded using a Varian Gemini ACP-300 spectrometer

operating at 300.145 MHz at 298.2 K. Samples were prepared in D2O phosphate buffer (pD 7.0, I = 0.10 mol dm-3), and 1H NMR resonances were referenced against an external standard of trimethylsilylpropiosulfonic acid in D2O. BNS- stock solutions (2.0 × 10-2 mol dm-3)

Sodium 6-(4-tertbutylanilino)-2-naphthalenesulfonate (12.1 mg) was dissolved in 1.6 cm3 D2O phosphate buffer (pD 7.0, I = 0.10 mol dm-3).

Host stock solutions Stock solutions were 1.0 × 10-2 mol dm-3 in either βCD or a linked βCD dimer. Stock solutions were prepared by dissolving βCD (30.7 mg); 66βCD2su (63.5 mg); 36βCD2su

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(63.5 mg); 33βCD2su (63.5 mg); 66βCD2ur (61.9 mg); 36βCD2ur (61.9 mg)in 2.7 cm3 of D2O phosphate buffer (pD 7.0, I = 0.10 mol dm-3).

Sample preparation The complexation of BNS- by βCD and linked βCD dimers in the D2O phosphate buffer (pD 7.0, I = 0.10 mol dm-3) at 298.2 K was monitoring through the variation of the chemical shift of the t-butyl 1H resonance in 15 sample solutions containing BNS- (2.0 × 10-3 mol dm-3) with increasing concentrations of βCD and linked βCD dimers (0-5.0 × 10-3 mol dm-3). Each sample solution was prepared by adding BNS- stock solution (0.10 cm3) and the appropriate volume of either βCD or linked βCD dimer stock solutions (0-0.5 cm3). The total volume of each sample was made up to 1.0 cm3 with D2O phosphate buffer (pD 7.0 I = 0.10 mol dm-3). BNS- dimerisation study Seven solutions of BNS- were prepared in 1.0 cm3 D2O phosphate buffer (pD 7.0, I = 0.10 mol dm-3) with increasing concentration: 2.0 × 10-4; 4.0 × 10-4; 2.0 × 10-3; 4.0 × 10-3; 8.0 × 10-3; 1.2 × 10-2; 2.0 × 10-2 mol dm-3.

6.4. Experimental for Chapter 4 6.4.1. Preparation of Compounds General procedure for the preparation of the polycarboxymethyl-amino-βcyclodextrins The substituted βCDs were prepared by a modification of previous procedures.21,22 Solutions containing chloroacetic acid (0.5 g, 5 mmol) in 2 cm3 H2O and NaOH (0.2 g, 0.5 mmol) in 2 cm3 H2O were cooled in an ice bath and then combined. The mixture was added to an aqueous solution of either (2AS,3AS)-3A-amino-3A-deoxy-β-cyclodextrin, 6Aamino-6A-deoxy-β-cyclodextrin (0.6 mmol) or 6A-(2-aminoethyl)amino-6A-deoxy-βcyclodextrin in 5 cm3 H2O. After adjusting the pH to 10-11 with aqueous NaOH, the - 246 -

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reaction solution was heated at 80 oC for 24h. During this time NaOH (calculated equivalent to the HCl produced from the reaction) in water (10 cm3) was very slowly added with a perfuser. The reaction was monitored by TLC until no trace of either (2AS,3AS)-3A-amino-3A-deoxy-β-cyclodextrin, 6A-amino-6A-deoxy-β-cyclodextrin or 6A(2-aminoethyl)amino-6A-deoxy-β-cyclodextrin was detected. The reaction mixture was cooled to room temperature and poured into ethanol (200 cm3). The resulting precipitate was filtered, washed with ethanol (50cm3), dissolved in water (10 cm3), and loaded onto a Dowex 50w x2 cation exchange column (1.5 × 15 cm). The column was washed with water (500 cm3) and 1.0 mol dm-3 aqueous acetic acid (500 cm3). The eluate was collected in 20 cm3 fractions. Fractions containing the product were combined and evaporated to dryness under vacuum. The residue was freeze-dried to give the product as a white solid. (2AS,3AS)-3A-Bis(carboxylmethyl)amino-3A-deoxy-β-cyclodextrin, 3βCDidaH2 OH O

O N

OH

(2AS,3AS)-3A-Amino-3A-deoxy-β-cyclodextrin (0.68 g, 0.6 mmol) was dissolved in H2O (5 cm3) and stirred at room temperature. Ice-cooled solutions containing chloroacetic acid (0.5 g, 5 mmol) in 2 cm3 H2O and NaOH (0.2 g, 0.5 mmol) in 2 cm3 H2O were combined and added to the first solution. The general work-up and purification procedure was followed and the title compound was obtained as a white solid.

Yield: 382 mg (51%) TLC: Rc = 0.46 1

H NMR: δH(D2O) 5.08-5.01 (m, 7H, H1); 4.2 (s, 4H, CH2); 4.09-3.61 (m, 42H, H2-H6).

13

C NMR: δC(D2O) 173.05 (acid C=O), 104.5-103.2 (C1), 102.1 (C1A), 85.6-83.6 (C4), 82.1

(C4A), 78.2-73.5 (C2, C3, C5), 63.1-62.1 (C6B-G), 59.2 (C6A), 53.8 (C3A), 49.7 (CH2). LCQ-MS: (M + H+) 1249; (M + Na+) 1272.

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Elemental analysis: Cal. C46H75NO38.11H2O: C, 38.15; H, 6.75; N, 0.96. Found: C, 38.10; H, 6.66; N, 0.92. 6A-Bis(carboxylmethyl)amino-6A-deoxy-β-cyclodextrin, 6βCDidaH2

O

OH O N OH

6A-Amino-6A-deoxy-β-cyclodextrin (0.68 g, 0.6 mmol) was dissolved in H2O (5 cm3) and stirred at room temperature. Ice-cooled solutions containing chloroacetic acid (0.5 g, 5 mmol) in 2 cm3 H2O and NaOH (0.2 g, 0.5 mmol) in 2 cm3 H2O were combined and added to the first solution. The general work-up and purification procedure was followed and the title compound was obtained as a white solid.

Yield: 540 mg (72%) TLC: Rc = 0.33 1

H NMR: δH(D2O) 5.08-5.05 (m, 7H, H1); 4.2 (s, 4H, CH2); 4.0-3.35 (m, 42H, H2-H6).

13

C NMR: δC(D2O) 171.8 (acid C=O), 104.5 (C1), 102.8 (C1A), 85.6-83.7 (C4), 81.7 (C4A),

75.7-72.8 (C2, C3, C5), 63.2-62.9 (C6B-G), 58.8 (C6A), 47.6 (CH2). LCQ-MS: (M + H+) 1249; (M + Na+) 1272 Elemental analysis: Cal. C46H75NO38.6H2O: C, 40.68; H, 6.46; N, 1.03. Found: C, 40.5; H, 6.2; N, 1.0. 6A-[Tri(carboxylmethyl)(2-aminoethyl)amino]-6A-deoxy-β-cyclodextrin, 6βCDedtaH3

OH O N

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Chapter 6 - Experimental

6A-(2-Aminoethyl)amino-6A-deoxy-β-cyclodextrin (0.71 g, 0.6 mmol) was dissolved in H2O (5 cm3) and stirred at room temperature. Ice-cooled solutions containing chloroacetic acid (0.5 g, 5 mmol) in 2 cm3 H2O and NaOH (0.2 g, 0.5 mmol) in 2 cm3 H2O were combined and added. to the first solution The general work-up and purification procedure was followed and the title compound was obtained as a white solid..

Yield: 535 mg (66%) TLC: Rc = 0.31 1

H NMR: δH(D2O) 5.08-5.06 (m, 7H, H1); 4.7, 4.2 (m, 6H, acid CH2); 4.0-3.35 (m, 42H,

H2-H6). 13

C NMR: δC(D2O) 176.7 (acid C=O), 104.5 (C1), 102.5 (C1A), 85.6-83.7 (C4), 81.7 (C4A),

75.7-71.2 (C2, C3, C5), 63.2-62.1 (C6B-G), 58.8 (C6A), 47.5 (CH2). LCQ-MS: (M + H+) 1350; (M + Na+) 1373 Elemental analysis: Cal. C50H82N2O40.11H2O: C, 38.76; H, 6.76; N, 1.80. Found: C, 38.99; H, 6.78; N, 1.62.

6.4.2. Potentiometric Titrations Potentiometric titrations were carried out using a Metrohm Dosino 800 titrimator, a Metrohm Titrando 809 potentiometer and an Orion 81-03 combination electrode that was filled with aqueous 0.10 mol dm-3 NaClO4 solution. The electrode was soaked in 0.10 mol dm-3 NaClO4 solution for at least three days prior to use. Titrations were performed in a water-jacketed 2 cm3 titration vessel held at 298.2 ± 0.1 K. During the titrations, a gentle stream of nitrogen bubbles (previously passed through aqueous 0.10 mol dm-3 KOH to remove any CO2 traces and then aqueous 0.10 mol dm-3 NaClO4) was passed through the titration solutions, which were magnetically stirred. The titration solutions were allowed to stand in the titration vessel for 15 minutes before commencement of the titration to allow the solution to equilibrate to 298.2 K and become saturated with nitrogen.

The electrode was calibrated every 24 hours by titration of a solution that was 0.01 mol dm-3 in HClO4 and 0.09 mol dm-3 in NaClO4. For each system, 0.10 NaOH was titrated against the species of interest (0.001 mol dm-3) in solutions 0.010 mol dm-3 in HClO4 and 0.09 mol dm-3 in NaClO4.

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Deionised water that had been purified with a Milli-Q-Reagent system to give a resistivity of >15 MΩ cm was subsequently boiled to remove CO2, and was used in all solution preparations.

6.4.3. Preparation of Solutions for NMR Enantioselectivity Studies The

stock

solutions

of

0.05

mol

dm-3

substituted

βCD,

europium(III)

trifluoromethanesulfonate (europium triflate), Eu(CF3SO3)3, (Aldrich) and each of the guests as either the racemate or either the D or L enantiomers (at pD 10 (NaOD) were prepared in D2O. The 1 cm3 D2O sample solutions were prepared by mixing in 0.8 cm3 of D2O 0.1 cm3 of each of the Eu(CF3SO3)3 and the racemate stock solutions, or replacing the latter with 0.08 cm3 of each of the D enantiomer and 0.02 cm3 of L enantiomer stock solutions. 1H NMR spectra were recorded using a Varian Inova 600 Spectrometer operating at 599.957 MHz at 298.2 K and resonances were referenced against an external standard, trimethylsilylpropiosulfonic acid, in D2O. Only those resonances of the guest not overlapped by cyclodextrin resonances were examined for enantioselectivity in the substituted βCDs and europium(III)-βCDs.

6.5. Experimental for Chapter 5 6.5.1. Preparation of Compounds General procedure for the preparation of the 3% substituted polyacrylic acids.23

Poly(acrylic acid) (PAA) wt. 35% solution in water (Aldrich) was diluted to wt. 10% and freeze-dried to give the product as white solid. Poly(acrylic acid) (0.9g, 12.5 mmol of –COOH groups) was dissolved in 1-methylpyrrolidin-2-one (30 cm3) at 60˚ C and stirred for 24 hours. The substituting amine (0.37 mmol) was added in 1-methylpyrrolidin-2-one (2.0 cm3) followed by dicylohexyldiimide (0.48 mmol) in 1-methylpyrrolidin-2-one (2.0 cm3) and the reaction mixture was stirred at 60˚ C for at least 48 hrs.

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The reaction mixture was cooled to room temperature and diluted with 40% w/w sodium hydroxide solution (35 cm3). This was followed by the addition of 15 cm3 of 1methylpyrrolidin-2-one at 60 oC. The mixture was centrifuged and the liquid mixture was decanted to leave the white substituted sodium polyacrylate solid behind. A second 15 cm3 of 1-methylpyrrolidin-2-one at 60 oC was added and the centrifugation and decanting was repeated. This was followed by repeated washings with 50 cm3 of methanol and decanting until the decanted methanol became clear. The crude product was twice dissolved in water (12.5 cm3) and precipitated into methanol (100 cm3). The substituted sodium polyacrylate was then dissolved in water (20 cm3) and dialysed (cutoff 3500 g/mol) against deionised water for at least 4 days until the conductivity of the water outside the dialysis tube remained constant. The final substituted sodium polyacrylate product was isolated after freeze-drying from aqueous solution. Yield 70-80%. The substitution was determined to be 3% by NMR spectroscopy. The 1D and 2D 1H NMR spectra of the substituted sodium polyacrylates (PAAs) are shown in Chapter 5.

ADhn-PAA (PAA 3% substituted with 1-(6-aminohexyl)amino-adamantane (ADhn)) and ADddn-PAA (PAA 3% substituted with 1-(6-aminododecyl)amino- adamantane (ADddn)) were prepared by Dr. Bruce May. Other substituted PAAs, AD-PAA (PAA 3% substituted with 1-amino-adamantane (ADNH2)), βCD-PAA (PAA 3% substituted with 6βCDNH2), βCDhn-PAA (PAA 3% substituted with 6βCDhn) and βCDddn-PAA (PAA 3% substituted with 6βCDddn) were prepared as described in the general procedure.

6.5.2. Preparation of Solutions for 1H 2D NOESY NMR Studies

All solutions were prepared and equilibrated in test tubes before being transferred to 5 mm NMR tubes. Solutions for complexation studies of βCD-substituted polyacrylates with adamantan-1carboxylate contained either βCD-PAA, βCDhn-PAA or βCDddn-PAA (10 mg) and ADCO2Na (0.5 mg) in 1 cm3 D2O (equimolar with the βCD substituents of the substituted polyacrylates).

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Solutions for complexation studies of adamantly substituted polyacrylates by βCD, 66βCD2su and 66βCD2ur contained either AD-PAA, ADhn-PAA, ADddn-PAA (10 mg) and either βCD (3.3 mg), or 66βCD2su (3.5 mg), 66βCD2ur (3.4 mg) in 1 cm3 D2O (such that the βCD annuli were equimolar with the adamantyl substituents of the substituted polyacrylates). Solutions for complexation studies of βCD-substituted polyacrylates with adamantly-substituted polyacrylates contained βCD-PAA, βCDhn-PAA, βCDddn-PAA (5 mg) and either AD-PAA, ADhn-PAA, ADddn-PAA (5 mg) in 1 cm3 D2O (such that the βCD annuli were equimolar with the adamantyl substituents of the substituted polyacrylates).

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6.6. References 1

C. A. Haskard, C. J. Easton, B. L. May, and S. F. Lincoln, J. Phys. Chem., 1996, 100, 14457.

2

T. Kuruscev, The University of Adelaide, 1984.

3

J. Pitha and R. N. Jones, Can. J. Chem., 1966, 44, 3031.

4

Copyright, 1984-1994, The MathWorks, Inc., Cochituate Place, 24 Prime Park Way, Natich, Mass. 01760, U.S.A.

5

C. Frassineti, S. Ghelli, P. Gans, A. Sabatini, M. S. Moruzzi, and A. Vacca, Anal. Biochem., 1995, 231 374.

6

Protonic Software, 2, Templegate Avenue, Leeds LS15 0HD, UK.

7

P. Gans, A. Sabatini, and A. Vacca, Talanta, 1996, 1739.

8

L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, and A. Vacca, Coord. Chem. Rev., 1999, 184, 311.

9

C. Long, 'Biochemist Handbook / Compiled by One Hundred and Seventy-One Contributors ', ed. E. J. King and W. M. Sperry, London, Spon, 1971.

10

B. Brady, N. Lynam, T. O'Sullivan, C. Ahern, and R. Darcy, Org. Syntheses, 77, 770.

11

S. E. Brown, J. H. Coates, D. R. Coghlan, C. J. Easton, S. J. v. Eyk, W. Sanowski, A. Lepore, S. F. Lincoln, Y. Luo, B. L. May, D. S. Scheisser, P. Wang, and M. L. Williams, Aust. J. Chem., 1993, 46, 953.

12

B. L. May, S. D. Ken, C. J. Easton and S. F. Lincoln, J. Chem. Soc., Perkin Trans, 1997, 1, 3157.

13

R. C. Petter, J. S. Salek, C. T. Sikorski, G. Kumaravel, and Fu-Tyanlin, J. Am. Chem. Soc., 1990, 112, 3867.

14

H. F. Bauer and W. C. Drinkard, J. Am. Chem. Soc., 1960, 82, 5031.

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15

T. Murakami, K. Harata, and S. Morimoto, Tet. Lett., 1987, 28, 312.

16

R. Breslow and A. W. Czarnik, J. Am. Chem. Soc., 1983, 105, 1390.

17

C. J. Easton, S. J. v. Eyk, S. F. Lincoln, B. L. May, J. Papageorgiou, and M. L. Williams, Aust. J. Chem., 1997, 50, 9.

18

M. M. Cieslinski, P. Clements, B. L. May, C. J. Easton, and S. F. Lincoln, J. Chem. Soc., Perkin Trans. 2, 2002, 5, 947.

19

R. Breslow, N. Greenspoon, T. Guo, and R. Zarycki, J. Am. Chem. Soc., 1989, 111, 8296.

20

E. M. Kosower, H. Dodiuk, K. Tanizawa, M. Ottolenghi, and N. Orbach, J. Am. Chem. Soc., 1975, 97, 2167.

21

M. Sandow, B. L. May, P. Clements, C. J. Easton, and S. F. Lincoln, Aust. J. Chem., 1999, 52, 1143.

22

S. D. Kean, C. J. Easton, S. F. Lincoln, and D. Parker, Aust. J. Chem., 2001, 54, 535.

23

X. Guo, A. A. Abdala, B. L. May, S. F. Lincoln, S. A. Khan, and R. K. Prud'homme, Marcromol., 2005, 38, 3037.

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