electronic supplementary information - royal society of chemistry · 2011-02-24 · s6 figure s5:...

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Electronic Supplementary Information

An approach to enzyme inhibition employing reversible boronate ester

formation

Ivanhoe K. H. Leung, Tom Brown Jr, Christopher J. Schofield, Timothy D. W. Claridge

Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12

Mansfield Road, Oxford OX1 3TA, United Kingdom.

Table of contents Page Number

Figure S1: Effect of pH on the equilibrium between boronic acid and boronate ester

S2

Figure S2: Structures of the sugars used in this study S3

Figure S3: pKa of boronic acids 1, 2 and 3 S4

Figure S4: Monitoring ternary enzyme-boronic acid-sugar complex formation by 11B NMR

S5

Figure S5: The propensity of boronate esters to form in solution.

S6

Figure S6: 11B NMR of ternary αCT-boronic acid-sorbose formation with 1 and D- and L-sorbose

S8

Figure S7: 11B NMR of ternary αCT-boronic acid-sorbose formation with 1 and D- and L-fructose

S9

Figure S8: pH dependency for boronate ester formation S10

Figure S9: waterLOGSY between 1, 2, αCT and D-fructose S11

Figure S10: waterLOGSY between 1, 2, αCT and L-fructose S13

Supplemental Reference S14

Supplementary Material (ESI) for Medicinal Chemistry CommunicationsThis journal is (c) The Royal Society of Chemistry 2011

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Figure S1 - 11B NMR spectra showing the effect of pH on the equilibrium between 1-D-

fructose boronate ester (~8 ppm) and free boronic acid 1 (~26 ppm) (and D-fructose) in

which the acid and ester undergo a slow exchange equilibrium (on the NMR timescale).

In the absence of the sugar, a single fast-exchange averaged peak with a pH dependent

chemical shift is observed. The concentration of 1 is 10 mM and D-fructose is 100 mM.


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Figure S2: The structures of the sugars used in this study. Only the cis-diol geometry is

shown.


S4

Figure S3: The effect of pH on the chemical shift of the boron resonance (δB), as used

to determine the pKa values of the boronic acids 1, 2 and 3 in D2O. The pD was

calculated from the observed pH using the formula pD = pH + 0.4.1

11B chemical shift vs. pH

0

5

10

15

20

25

30

35

0 2 4 6 8 10 12 14 16

pD

Ch

emic

al s

hif

t / p

pm

BA6 pKa ~7.44

BA16 pKa ~8.88

BA6 Oxygen Analogue pKa ~7.30

● 3: pKa ~8.88

S

B

OH

OH

▲ 1: pKa ~7.44

♦ 2: pKa ~7.30


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Figure S4. Monitoring ternary enzyme-boronic acid-sugar complex formation by 11B

NMR: (a) 5 mM boronic acid 1 alone; (b) 5 mM boronic acid 1 + 100 mM D-fructose;

(c) 1 mM boronic acid 1 + 33 mg/ml (~1.3 mM) αCT; (d) 1 mM boronic acid 1 + 33

mg/ml (~1 mM) αCT + 2 mM D-fructose. The resonances correspond to 1-

benzothiophen-2-ylboronic acid 1 (peak A), the 1-D-fructose boronate ester (peak B),

the αCT-1-D-fructose ternary complex (peak C) and the αCT-1 binary complex (peak

D).

30 25 20 15 10 5 ppm

A(a)

(b)

(c)

(d)

B

C

D

A: B:

C: D:


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Figure S5: The propensity of boronate esters to form in solution as assayed by 1H

NMR. Binding curves for D- and L-fructose with 1 (KA ~ 52 M); D- and L-glucose with 1

(KA < 2 M); and D- and L-sorbose with 1 (KA ~ 49 M) are shown (Concentration of 1 is

1 mM).

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250 300

[sugar] / mM

Bo

ron

ate

este

r / m

M

1:D-fructose

1:L-fructose1:D-glucose

1:L-glucose

1-D-fructose boronate ester

1-L-fructose boronate ester

1-D-glucose boronate ester

1-L-glucose boronate ester

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200

[sugar] / mM

Bo

ron

ate

este

r / m

M

1:D-sorbose1:L-sorbose1:D-sorbose1:L-sorbose


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Binding curves for D-fructose and L-fructose with 1 and 2 (KA ~ 52 M); and for D-

glucose and L-glucose with 1 and 2 (KA < 2 M) are shown (Concentrations of 1 and 2

are 1 mM).

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200

[sugar] / mM

Bo

ron

ate

este

r / m

M

1:D-fructose1:L-fructose2:D-fructose2:L-fructose

1:D-fructose1:L-fructose

2:D-fructose2:L-fructose

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250 300

[sugar] / mM

Bo

ron

ate

este

r / m

M 1:D-glucose1:L-glucose2:D-glucose2:L-glucose

1:D-glucose

1:L-glucose2:D-glucose

2:L-glucose


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Figure S6: Formation of an αCT-1-sorbose ternary complex as monitored by 11B NMR.

More D-sorbose than L-sorbose is required to form the ternary complex. The

concentrations of 1 (1 mM) and αCT (1.3 mM).

(a) L-sorbose titration

(b) D-sorbose titration

15 10 5 0 -5 -10 ppm

15 10 5 0 -5 -10 ppm

0 mM

0.5 mM

1 mM

2 mM

4 mM

0 mM

5 mM

15 mM

30 mM

50 mM

AB


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Figure S7: The formation of a ternary enzyme–boronic acid–sugar complex on titration

of D-fructose (at the concentrations shown) into a mixture of 1 or 2 (1 mM) and αCT

(1.3 mM).

20 10 0 -10 ppm

20 10 0 -10 ppm

D-fructose titration to 1 (1 mM) + αCT (1.3 mM)

D-fructose titration to 2 (1 mM) + αCT (1.3 mM)

(a) No D-fructose

(b) 0.5 mM

(c) 1 mM

(d) 2 mM

(a) No D-fructose

(b) 0.5 mM

(c) 1 mM

(d) 3 mM

(e) 6 mM

(f) 10 mM

B A

CD


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Figure S8: The dynamics of boronate ester formation depends on the pKa of the boronic

acids (naphthalen-2-ylboronic acid (3): pKa ~8.8). At pH 5.8, a ~1:500 3/D-fructose ratio

was required to form ~50% of the 3-D-fructose boronate ester complex; at pH 7.0 this

was reduced to ~1:50, and at pH 8.0, ~1:10

0

0.2

0.4

0.6

0.8

1

0 200 400 600 800 1000

[sugar] / mM

Bo

ron

ate

este

r / m

M

pH 5.8pH 7.0pH 8.0

KA ~ 2.2 MKA ~ 23 MKA ~ 76 M


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Figure S9: 1H waterLOGSY analyses of αCT and D-fructose with 1 and/or 2. (a) and

(b): In the absence of 2, both 1 and 1-D-fructose bind to αCT; (c) and (d): In the absence

of 1, both 2 and 2-D-fructose bind to αCT; (e) and (f): In a mixture of 1 and 2, 1 and 1-

D-fructose are preferentially bound by αCT. Conditions for the experiments: 200 µM

αCT; 5 mM 1; 5 mM 2; 15 mM fructose.

(a) 1H reference

(b) waterLOGSY

(c) 1H reference

(d) waterLOGSY

(e) 1H reference

(f) waterLOGSY

8.0 7.5 7.0 ppm

C D A BC+D D

A+B

C+D

A: X = SC: X = O

B: X = SD: X = O

D

In the waterLOGSY experiment, bulk water magnetization is transferred via the

solvated protein-ligand complex to the free ligand. Differences in rotational correlation


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time between the solvated protein-ligand complex and the solvated free ligand result in

binders and non-binders displaying waterLOGSY signals with opposite signs.2

The observed waterLOGSY responses for the boronic acid and boronate ester may

originate from direct binding of each species to the enzyme, or indirectly from the

binding of either species, followed by their interconversion in solution, provided the

interconversion is fast with respect to the relaxation of the binding species.

The signals observed in waterLOGSY experiments rely on, among other factors, the

exchange kinetics of the reversibly forming protein-ligand complex; in situations of

very strong binding the bound residence time may become too long, meaning

longitudinal relaxation dominates before the ligand dissociates, leading to a nil response

(a “false negative”).


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Figure S10: waterLOGSY spectrum showing αCT preferentially binds to 1 and 1-D-

fructose boronate ester, but no selectivity is observed in the presence of L-fructose.

Conditions for the experiments: 200 µM αCT; 5 mM 1; 5 mM 2; 15 mM fructose.

(a) 1H reference

(b) waterLOGSY

(c) 1H reference

(d) waterLOGSY

8.0 7.5 7.0 ppm

C D A BC+D D

A+B C+D

A: X = SC: X = O

B: X = SD: X = O

X

B

OH

OH

X

B

O

OHO

D

With

D-fructose

With

L-fructose


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Supplemental Reference:

1. P. K. Glasoe and F. A. Long, J. Phys. Chem. 1960, 64, 188–190.

2. C. Dalvit, P. Pevarello, M. Tatò, M. Veronesi, A. Vulpetti and M. Sundström, J.

Biomol. NMR 2000, 18, 65–68.


electronic supplementary information - royal society of chemistry · 2011-02-24 · s6 figure s5:...

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