Supplementary Materials for - Science Advances...Le u-BF3 1 8 -Ma r-2 0 1 5 m/z 1 5 2 1 5 3 1 5 4 1...

www.advances.sciencemag.org/cgi/content/full/1/8/e1500694/DC1

Supplementary Materials for

Boramino acid as a marker for amino acid transporters

Zhibo Liu, Haojun Chen, Kai Chen, Yihan Shao, Dale O. Kiesewetter, Gang Niu, Xiaoyuan Chen

Published 11 September 2015, Sci. Adv. 1, e1500694 (2015)

DOI: 10.1126/sciadv.1500694

This PDF file includes:

Scheme S1. General synthetic routes of -amino boronic acid/ester derivatives

with high enantiopurity.

Scheme S2. BAAs are converted from BAA/ester in quantitative yield.

Scheme S3. 18F-BAAs are radiosynthesized via one-step 18F-19F isotope exchange

reaction.

Fig. S1. Radioactive HPLC chromatogram for the purification of Leu-BF3.

Fig. S2. The LC-HRMS spectrum of HPLC-purified Leu-BF3.

Fig. S3. 19F NMR spectrum of HPLC-purified Leu-BF3 ( = −148.95 ppm).

Fig. S4. 1H NMR spectrum of HPLC-purified Leu-BF3.

Fig. S5. Radioactive HPLC chromatography of preparing Phe-BF3.

Fig. S6. The LC-HRMS spectrum of HPLC-purified Phe-BF3.

Fig. S7. 19F NMR spectrum of HPLC-purified Phe-BF3 ( = −151.96 ppm).

Fig. S8. 1H NMR spectrum of HPLC-purified Phe-BF3.

Fig. S9. Radioactive HPLC chromatography of preparing Ala-BF3.

Fig. S10. The LC-HRMS spectrum of HPLC-purified Ala-BF3.

Fig. S11. 19F NMR spectrum of HPLC-purified Ala-BF3 ( = −149.11 ppm).

Fig. S12. 1H NMR spectrum of HPLC-purified Ala-BF3.

Fig. S13. Radioactive HPLC chromatography of preparing Pro-BF3.

Fig. S14. The LC-HRMS spectrum of HPLC-purified Pro-BF3.

Fig. S15. 19F NMR spectrum of HPLC-purified Pro-BF3 ( = −149.02 ppm).

Fig. S16. 1H NMR spectrum of HPLC-purified Pro-BF3.

Fig. S17. Radioactive HPLC chromatography of Sep-Pak purified Leu-BF3, Phe-

BF3, Ala-BF3, and Pro-BF3, respectively (from top to bottom).

Fig. S18. In vitro stability assay of 18F-Leu-BF3.

Fig. S19. In vitro stability assay of 18F-Phe-BF3.

Fig. S20. In vitro stability assay of 18F-Ala-BF3.

Fig. S21. In vitro stability assay of 18F-Pro-BF3.

Fig. S22. LAT-1–AdiC alignment.

Fig. S23. Schematic representation of the binding site of LAT-1.

Fig. S24. Predicted structure of the LAT-1–Phe complex.

Fig. S25. Predicted structure of the LAT-1–Phe-BF3 complex.

Fig. S26. Predicted binding modes for LAT-1 ligands: Phe and Phe-BF3.

Fig. S27. Predicted structure of the LAT-1–Leu complex.

Fig. S28. Predicted structure of the LAT-1–Leu-BF3 complex.

Fig. S29. Predicted binding modes for LAT-1 ligands.

Fig. S30. The 18F-Leu-BF3 uptake in the presence of different concentrations of

nonradioactive Leu-BF3.

Fig. S31. The 18F-Phe-BF3 uptake was measured at 0.3, 1, 3, 10, 30, 100, and 300

M in PBS and plotted against the concentration of Phe-BF3.

Fig. S32. The 18F-Ala-BF3 uptake was measured at 1, 3, 10, 30, 100, 300, and

1000 M in PBS and plotted against the concentration of Ala-BF3.

Fig. S33. The 18F-Pro-BF3 uptake was measured at 10, 30, 100, 400, 800, 1500,

2000, 3000, 6000, and 10,000 M in PBS and plotted against the concentration of

Pro-BF3.

Fig. S34. The biodistribution of 18F-Phe-BF3 with U87MG-bearing mice.

Fig. S35. 3D projection PET images of 18F-FDG (left) and 18F-Phe-BF3 (right) in

mice bearing U87MG xenografts and inflammation (h, heart; t, tumor; i,

inflammation; b, bladder; gb, gallbladder).

Fig. S36. Representative coronal 18F-FDG (left) and 18F-Phe-BF3 (right) PET

images in mice bearing U87MG xenografts and inflammation (h, heart; t, tumor;

i, inflammation; b, bladder; gb, gallbladder).

Fig. S37. 18F-Phe-BF3 is stable at in vivo conditions.

Table S1. A brief summary of 18F-AAs and proposed 18F-BAAs.

Table S2. Summary of the half-lives of BAAs.

Table S3. Structure of BAAs studied in this work and their counterpart AAs.

Table S4. Structure of AAs and their predicted Gbinding and Ki with LAT-1.

Schemes

Scheme S1. General synthetic routes of α-amino boronic acid/ester derivatives with high

enantiopurity.

Scheme S2. BAAs are converted from boramino acid/ester in quantitative yield.

Scheme S3. 18F-BAAs are radiosynthesized via one-step 18F-19F isotope exchange

reaction.

R1 MgBr R1 B

OMe

OMe

R1 B

O

OHO

HO

B(OMe)3

B

O

O

R1

Cl

B

O

O

R1

+H3N1. (Me3Si)2N-Li+

2. HCl

CHCl2-Li+

B

O

O

R

+H3N3.0 equiv. KF, 2.5 equiv. HCl

Trace amont of 18F-fluoride R

+H3N

BF3-

18F 19F

18F-19F IEXNH3

+

R

B-F

F 18F

NH3+

R

-O

O

NH3+

R

B-F

F 19F

Experimental data

Figure S1. Radioactive HPLC chromatogram for the purification of Leu-BF3. Under

this condition, free 18F-fluoride elutes firstly at 4.1 min, whereas the desired product Leu-

BF3 elutes from HPLC at 12.9 min. The product peak is collected and lyophilized to give

the chemically pure Leu-BF3.

Figure S2. The LC-HRMS spectrum of HPLC-purified Leu-BF3. (Top) LC spectrum

of HPLC purified Leu-BF3. The value of y-axis is defined as total ionization content

(TIC). There is only one peak being found, which is Leu-BF3 by confirming with the

following HRMS. (Bottom) High-resolution mass spectrum (HRMS) of Leu-BF3.

15:13:2518-Mar-2015Leu-BF3

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

15:13:2518-Mar-2015Leu-BF3

m/z152 153 154 155

%

1

154.0719

153.0787

155.0821154.9809

Figure S3. 19F NMR spectrum of HPLC-purified BF3-leucine (δ = −148.95 ppm).

NH

3+

- F3B

Figure S4. 1H NMR spectrum of HPLC-purified Leu-BF3.

NH

3+

- F3B

Figure S5. Radioactive HPLC chromatography of preparing Phe-BF3. Under this

condition, free 18F-fluoride should elute firstly at 4.1 min, whereas the desired product

Phe-BF3 elutes from HPLC at 15.5 min. The product peak is collected and lyophilized to

give the chemically pure Phe-BF3.

Figure S6. The LC-HRMS spectrum of HPLC-purified Phe-BF3. (Top) LC spectrum

of HPLC purified Phe-BF3. The value of y-axis is defined as total ionization content

(TIC). There is only one peak being found, which is Phe-BF3 by confirming with the

following HRMS. (Bottom) High-resolution mass spectrum (HRMS) of Phe-BF3.

15:24:1318-Mar-2015Phe-BF3

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

15:24:1318-Mar-2015Phe-BF3

m/z186 187 188 189 190

%

0

100188.0574

187.0609

189.0565

Figure S7. 19F NMR spectrum of HPLC-purified Phe-BF3 (δ = −151.96 ppm).

NH

3+

- F3B

Figure S8. 1H NMR spectrum of HPLC-purified Phe-BF3.

NH

3+

- F3B

Figure S9. Radioactive HPLC chromatography of preparing Ala-BF3. Under this


Ala-BF3 elutes from HPLC at 10.5 min. The product peak is collected and lyophilized to

give the chemically pure Ala-BF3.

Figure S10. The LC-HRMS spectrum of HPLC-purified Ala-BF3. (A) LC spectrum

of HPLC purified Ala-BF3. The value of y-axis is defined as total ionization content

(TIC). There is only one peak being found, which is Ala-BF3 by confirming with the

following HRMS. (B) High-resolution mass spectrum (HRMS) of Ala-BF3.

0 11:45:51005-May-2015Ala-BF3

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

%

0

100

11:45:5105-May-2015Ala-BF3

m/z107 108 109 110 111 112 113 114 115 116 117 118

%

2

Ying-05-05-2015-01 26 (0.948) 1: TOF MS ES- 774112.0433

111.0525

113.0542

Figure S11. 19F NMR spectrum of HPLC-purified Ala-BF3 (δ = −149.11 ppm).

NH

3+

- F3B

Figure S12. 1H NMR spectrum of HPLC purified Ala-BF3.

NH

3+

- F3B

Figure S13. Radioactive HPLC chromatography of preparing Pro-BF3. Under this


Pro-BF3 elutes from HPLC at 14.5 min. The product peak is collected and lyophilized to

give the chemically pure Pro-BF3.

Figure 14. The LC-HRMS spectrum of HPLC-purified Pro-BF3. (A) LC spectrum of

HPLC purified Pro-BF3. The value of y-axis is defined as total ionization content (TIC).

There is only one peak being found, which is Pro-BF3 by confirming with the following

HRMS. (B) High-resolution mass spectrum (HRMS) of Pro-BF3.

22:18:3122-Mar-2015Pro-BF3-1

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

22:18:3122-Mar-2015Pro-BF3-1

m/z137 138 139

%

0

100138.0486

137.0480

139.0529

Figure S15. 19F NMR spectrum of HPLC-purified Pro-BF3 (δ = −149.02 ppm).

Tetrafluoroborate (BF4-, δ = −152.10 ppm) is literally added as an inner standard to adjust

chemical shift.

+H

2N

- F3B

Figure S16. 1H NMR spectrum of HPLC-purified Pro-BF3.

+H2N

-F3B

Figure S17. Radioactive HPLC chromatography of Sep-Pak purified Leu-BF3, Phe-

BF3, Ala-BF3, and Pro-BF3, respectively (from top to bottom). There is only one

major peak, and it corroborates with the elution time of the previous preparation.

Figure S18. In vitro stability assay of 18F-Leu-BF3. Radioactive HPLC

chromatography of Leu-BF3 after incubation in serum at 37 °C for 0, 60 and 120 min,

respectively. As shown, almost no defluorination was observed within 120 min,

suggesting strong serum stability for 18F-Leu-BF3.

Figure S19. In vitro stability assay of 18F-Phe-BF3. Radioactive HPLC chromatography

of 18F-Phe-BF3 after incubation in serum at 37 °C for 0, 60 and 120 min, respectively. As

shown, almost no defluorination was observed within 120 min, suggesting strong serum

stability for 18F-Phe-BF3.

Figure S20. In vitro stability assay of 18F-Ala-BF3. Radioactive HPLC chromatography

of 18F-Ala-BF3 after incubation in serum at 37 °C for 0, 60 and 120 min, respectively. As


stability for 18F-Ala-BF3.

Figure S21. In vitro stability assay of 18F-Pro-BF3. Radioactive HPLC chromatography

of 18F-Pro-BF3 after incubation in serum at 37 °C for 0, 60 and 120 min, respectively. As


stability for 18F-Pro-BF3.

Figure S22. LAT-1–AdiC alignment. The X-ray structure of the arginine/agmatine

transporter AdiC from E. coli (Protein Data Bank ID code 3L1L) was used as a template.

Figure S23. Schematic representation of the binding site of LAT-1. (A) LAT-1 (grey)

is in solid ribbon representation. Amino acid residues (Leu87, Ile96, Val97, Ala98, Leu99,

Val128, Leu131, and Val273) in binding site are in stick representation. (B) Schematic

representation of the binding pocket of LAT-1. The figure is prepared with DSViewPro

(BIOVIA).

!

A

B

Figure S24. Predicted structure of the LAT-1–Phe complex. LAT-1 (grey) is in solid

ribbon representation. Phenylalanine (Phe) and the residues in the binding site of LAT-1

are in stick representation. Hydrogen bonds between Phe and LAT-1 (involving residues

Leu87, Val97, Ala98, and Leu99) are shown as dotted green lines. The figure is prepared

with DSViewPro (BIOVIA).

Figure S25. Predicted structure of the LAT-1–Phe-BF3 complex. (A) LAT-1 (grey) is

in solid ribbon representation. Phe-BF3 and the residues in the binding site of LAT-1 are

in stick representation. Hydrogen bonds between phenylalanine and LAT-1 (involving

residues Leu87, Val97, Ala98, and Leu99) are shown as dotted green lines (B). The figure is

prepared with DSViewPro (BIOVIA).

A

B

Figure S26. Predicted binding modes for LAT-1 ligands: Phe and Phe-BF3. LAT-1

(grey) is in solid ribbon representation. Phe, Phe-BF3, and the residues in the binding site

of LAT-1 are in stick representation. Carbon atoms in Phe are colored in dark cyan, and

carbon atoms in Phe-BF3 are colored in light cyan. The figure is prepared with

DSViewPro (BIOVIA).

Figure. S27. Predicted structure of the LAT-1–Leu complex. LAT-1 (grey) is in solid

ribbon representation. Leu and the residues in the binding site of LAT-1 are in stick

representation. Hydrogen bonds between leucine and LAT-1 (involving residues Leu87,

Val97, Ala98, and Leu99) are shown as dotted green lines. The figure is prepared with

DSViewPro (BIOVIA).

Figure S28. Predicted structure of the LAT-1–Leu-BF3 complex. LAT-1 (grey) is in

solid ribbon representation. Leu-BF3 and the residues in the binding site of LAT-1 are in

stick representation. Hydrogen bonds between Leu-BF3 and LAT-1 (involving residues

Leu87 and Leu99) are shown as dotted green lines. The figure is prepared with DSViewPro

(BIOVIA).

Figure S29. Predicted binding modes for LAT-1 ligands. Leucine (Leu) and Leu-BF3.

LAT-1 (grey) is in solid ribbon representation. Leu, Leu-BF3, and the residues in the

binding site of LAT-1 are in stick representation. Carbon atoms in Leu are colored in

dark cyan, and carbon atoms in Leu-BF3 are colored in light cyan. The figure is prepared

with DSViewPro (BIOVIA).

Figure S30. The 18F-Leu-BF3 uptake in the presence of different concentrations of

nonradioactive Leu-BF3. As a result, the uptake-concentration correlation fits well with

Michaelis-Menten kinetics, giving the KM of 18F-Leu-BF3 to be 25.9±2.3 µM, versus KM

of Leucine to be 19.7±2.4 µM.

Figure S31. The 18F-Phe-BF3 uptake was measured at 0.3, 1, 3, 10, 30, 100, and 300

µM in PBS and plotted against the concentration of Phe-BF3. As a result, the uptake-

concentration correlation fits well with Michaelis-Menten kinetics, giving the KM of 18F-

Phe-BF3 to be 17.6 ± 4.2 µM, versus KM of phenylalanine to be 14.2 ± 4.1 µM.

Figure S32. The 18F-Ala-BF3 uptake was measured at 1, 3, 10, 30, 100, 300, and 1000

µM in PBS and plotted against the concentration of Ala-BF3. As a result, the uptake-

concentration correlation (Fig. 3B) fits well with Michaelis-Menten kinetics, giving the

KM of 18F-Ala-BF3 to be 467.6±87.2 µM, versus KM of alanine to be 370 µM.

Figure S33. The 18F-Pro-BF3 uptake was measured at 10, 30, 100, 400, 800, 1500,

2000, 3000, 6000, and 10,000 µM in PBS and plotted against the concentration of

Pro-BF3. There are two types of proline transporters, hence the curve fitting follows the

equation of double-reciprocal function. As a result, the uptake-concentration correlation

fits well with Michaelis-Menten kinetics, giving the KM of 18F-Pro-BF3 to be 65.6±24.5

µM and 7158±1760 µM, versus KM of proline to be 67.0±11.5 µM and 3300±1350 µM,

respectively (23).

Figure S34. The biodistribution of 18F-Phe-BF3 with U87MG-bearing mice. The

biodistribution of 18F-Phe-BF3 in selected organs at 120 min after injection displayed

higher uptake in the tumor in comparison with all normal organs/tissues.

Musc

le

Bone

Tumor

Bra

in

Lung

Inte

stin

Liver

Kid

ney

Pan

crea

se

Gal

l bla

dder0.0

2.5

5.0

7.5

10.0

%ID

/g

Figure S35. 3D projection PET images of 18F-FDG (left) and 18F-Phe-BF3 (right) in

mice bearing with U87MG xenografts and inflammation (h, heart; t, tumor; i,

inflammation; b, bladder; gb, gallbladder).

11%

ID/g

0

gb

b

t

11%

ID/g

0

h

b

t

i

Figure S36. Representative coronal 18F-FDG (left) and 18F-Phe-BF3 (right) PET

images in mice bearing with U87MG xenografts and inflammation (h, heart; t,

tumor; i, inflammation; b, bladder; gb, gallbladder). As shown in Fig. S34 and S35,

compare with 18F-FDG, 18F-Phe-BF3 demonstrated equally high tumor-to-background

ratio with 18F-FDG in tumor-bearing mice. In addition, 18F-Phe-BF3 demonstrated much

lower uptake in inflammation region, highlighting the potential of using boramino acids

to distinguish the inflammation tissue from tumor lesion. The study of 18F-Phe-BF3 was

performed one day after 18F-FDG study. Therefore the U87MG xenografts is visibly

larger.

0

12%

ID/g

0

12%

ID/g

t

b

gb

t

b

h

t

i i

18F-FDG 18F-Phe-BF3

Figure S37. 18F-Phe-BF3 is stable at in vivo conditions.

As shown in Fig. S37, the stability of 18F-Phe-BF3 is tested in mice. The blood sample

and urine sample were taken out of the mice at 30 and 60 min post injection. The

radioactivity was extracted from these samples and then reinjected into analytical HPLC

system. Based on the HPLC chromatography, the degradation of 18F-Phe-BF3 is almost

negligible in both urine and blood. Table S1. A brief summary of 18F-AAs and proposed

18F-BAAs.

Urine

0 min

30 min

60 min

In Vivo Stability Assay

Blood

0 min

30 min

60 min

NH3+B-

F

F18F

NH3+B-

F

F18F

Rad

io (

mV

)R

ad

io (

mV

)

Rad

io (

mV

)R

ad

io (

mV

)R

ad

io (

mV

)

Table S1. A brief summary of 18F-AAs and proposed 18F-BAAs.

As shown, many AAs have not been radiolabeled with 18F due to the lack of proper

chemistry, while all the boramino acids can be readily synthesized and labeled with 18F

via 19F-18F exchange reaction.

Name Major

AATs 18F-AA Name

Major

AATs 18F-AA

Ala System A/

ASCT N.A. Lys

System

CAAT N.A.

Val System L N.A. Asp System

EAAT N.A.

Ile System L N.A. Glu System

EAAT

Leu System L N.A. Ser System

ASCT N.A.

Met System L N.A. Thr System

ASCT N.A.

Phe System L Asn System ATA N.A.

Tyr System L Gln System ATA .

Try System L Pro System P

Arg System

CAAT N.A. Gly System G N.A.

His System

CAAT N.A. Cys

System

ASCT N.A.

NH3+

-OOC

18F

NH3+

-OOC COOH

18F

COO-

NH3+

NH

O 18F

NH3+

-OOC

O

18F

+H2N

-OOC

18F

-OOC

NH3+

CONH2

18F

Table S2. Summary of the half-lives of BAAs.

The chemical stability of boramino acids, which is defined as its half-life (t1/2) in

phosphate buffer at pH=7.4, is calculated based on reported linear correlation (18).

Table S3. Structure of BAAs studied in this work and their counterpart AAs.

Name Major

AATs AA 18F-AA BAA

Leu System L N.A.

Phe System L

Ala System A/

ASCT N.A

Pro System P

NH3+

-OOC

+H2N

-OOC

18F

NH3+-F3B

NH3+

-F3B

NH3+

-F3B

+H2N

-F3B

NH3+-OOC

NH3+

-OOC

NH3+

-OOC

+H2N

-OOC

18F

Table S4. Structure of AAs and their predicted ∆Gbinding and Ki with LAT-1.

Name Structure ∆Gbinding

a (kcal/mol)

Kib (µM)

RMSDc (Å)

Phe

−5.98 41.49 −

Phe-BF3

−5.67 60.33 0.69

Leu

−5.01 212.40 −

Leu-BF3

−4.79 308.55 1.39

aBinding free energy. bInhibition constant (T = 298.15 K). cThe root-mean-square

deviation (RMSD) value is calculated based on the best docked conformation between

natural amino acid and boramino acid.

Supplementary Materials for - Science Advances...Le u-BF3 1 8 -Ma r-2 0 1 5 m/z 1 5 2 1 5 3 1 5 4 1...

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