S1

Supporting information

Tri-serine tri-lactone scaffold for quantification of citrate in urine

Ali Akdeniz,a Mehmet Gokhan Caglayan,a,b Pavel Anzenbacher, Jr.*,a

___________________________________

a Department of Chemistry, Bowling Green State University Bowling Green, OH 43403, USA

E-mail: pavel@bgsu.edu

bDepartment of Analytical Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey

Contents

General S2

Preparation of Polymer chips S2

Synthesis S4

UV-Vis absorption titrations S10

Fluorescence titrations S14

Job`s plot experiments S19

NMR titrations S20

Sensor-Analyte complex study by Mass spectrometry S22

Qualitative analysis S23

Semi-quantitative analysis S24

References S28

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2015

S2

General

Synthesis of S1 and S2 was performed according to the literature procedures. Starting materials

are used without further purification. Standard laboratory techniques was performed in all the

synthesis. 1H- and 13C-NMR (APT) spectra were recorded using a Bruker® Avance IITM 500

MHz UltraShieldTM (Bruker Corporation, Mass., USA) Spectrometer at 25 °C. Mass

spectrometry studies were performed using Shimadzu LCMS-8030 liquid chromatograph mass

spectrometer (ESI) or Shimadzu Axima Performance MALDI-TOF mass spectrometer. All the

optical measurements were performed in acetonitrile. Acetonitrile is dried over 4Å molecular

sieves. Optically dilute solutions (0.1 A) were used for all photophysical experiments.

Fluorescence emission spectra were acquired using an Edinburgh single photon counting

spectrofluorimeter (FLSP 920). Fluorescence titrations isotherms were calculated according to

previously published methods.1

Isotherms were constructed using emission maximum of each titration. Absorption spectra were

recorded using a Hitachi U-3010 spectrophotometer. All the optical measurements were performed

using a quartz cuvette with a path length of 1 cm at room temperature. The absolute quantum yields

were measured using a Hamamatsu Quantaurus absolute quantum yield spectrometer QY-C11347.

Preparation of Polymer chips2

The multi-well 10 x 21 (sub-microliter) glass slides were fabricated by ultrasonic drilling of

microscope slides (well diameter: 1000 ± 10 μm, depth: 250 ± 10 μm). Sensor solutions in polymer

solution (4% poly (ether-urethane) in THF w/w) were prepared. In a typical array, 200 nL of

sensor-polymer solutions were pipetted into each well of the multi-well glass slides and dried.

Then, water (400 nL) was pipetted into each well and dried to form hydrated gel matrix. Finally,

S3

analytes were added as aqueous solutions into each well and the chip was dried at room

temperature for 1 hr. Images from the sensor array were recorded using a Kodak Image Station

440CF (for preliminary experiments) and a Kodak Image Station 4000MM PRO (for qualitative

and quantitative experiments). After acquiring the images, the integrated (nonzero) gray pixel

value (n) is calculated for each well in each channel. Images of the sensor chip were recorded

before (b) and after (a) the addition of an analyte. The final responses (R) were evaluated as

indicated in the following equation:

Thus obtained data for qualitative analysis were then analyzed using Linear Discriminant Analysis

(LDA). Qualitative analysis was performed using Support Vector Machine (SVM) algorithm.

S4

Synthesis

(3S, 7S, 11S)-3,7,11-tris(tritylamino)-1,5,9-trioxacyclododecane-2,6,10-trione (1)3

(2S)-methyl 3-hydroxy-2-(tritylamino)propanoate (10 g; 27.7 mmol) and 2,2-dibutyl-1,3,2-

dioxastannolane (0.18 g; 0.61 mmol) are dissolved in toluene (200 mL) and refluxed for 3 days

under N2 equipped with a Dean and Stark device loaded 4Å sieves . All the solvent is removed.

The crude product is dissolved in CH2Cl2 and purification is achieved by column chromatography.

(SiO2; CH2Cl2 only). Compound 1 is obtained as a white powder (6.085 g; 6.1 mmol; 66%).

F: > 250 °C. 1H NMR (500 MHz, CDCl3) δ 7.57 – 7.04 (m, 45H), 4.09 (t, J = 10.9 Hz, 3H), 3.59

– 3.38 (m, 6H), 2.66 (d, J = 9.0 Hz, 3H). 13C- NMR (126 MHz, CDCl3) δ 172.39, 145.29, 128.67,

128.06, 126.66, 71.36, 66.71, 54.40 ppm. MS (ESI) m/z: 1010.74 [M+Na]+.

(3S,7S,11S)-3,7,11-triamino-1,5,9-trioxa-cyclododecane-2,6,10-trione, trishydrochloride (2)3

Tri-serine tri-lactone (1) (4.85 g; 4.9 mM) is suspended in dry ethanol (80 mL). 2 mL of Acetyl

S5

chloride is added slowly into 10 mL of Ethanol over 10 min (exothermic). Acid solution is added

into slurry solution of 1 over 5 min. Reaction mixture is refluxed for 2 h. Resulting solution is

concentrated to 25 mL under vacuum. Precipitates are collected by vacuum filtration and washed

with CHCl3 (50 mL), EtOH (50 mL), and Et2O (50 mL). Product is a white powder. (1.75 g; 4.7

mmol; 96%). F: > 250 °C. 1H NMR (500 MHz, DMSO) δ 9.36 (s, 9H), 5.10 (dd, J = 12.4, 1.8 Hz,

3H), 4.59 (s, 3H), 4.31 (dd, J = 12.4, 2.6 Hz, 3H). 13C-ATP NMR (126 MHz, DMSO) δ 165.40,

63.20, 53.01 ppm. MS (ESI) m/z: 262.03 [M+H -3Cl]+.

(3S,7S,11S)-2,6,10-trioxo-1,5,9-trioxacyclododecane-3,7,11-triyl)tris(3-(4- dimethylamino)naphthalen-1-yl)thiourea (3)

O

OO

O

O

O

-Cl+H3N

NH3+Cl-

NH3+Cl-

NC

S

N

O

O O

O

O

O

NHC

S

N

HN

NH

HN

HN

C

SN

HNC

S

N

TEA

DCM

(3S,7S,11S)-3,7,11-triamino-1,5,9-trioxa-cyclododecane-2,6,10-trione, trishydrochloride (2) (50

mg; 0.13 mmol) is suspended into CH2Cl2 with 4Å sieves. 120 mg of 4-Dimethylamino-1-naphthyl

isothiocyanate is added as CH2Cl2 solution. Reaction flask is stirred for 3 h at R.T. Resulting

solution is evaporated until dryness. Purification is achieved by column chromatography. (SiO2;

CH2Cl2 : MeOH : Acetonitirile (10 : 0.25 : 1)). Compound 1 is obtained as a white powder (35 mg;

0.04 mmol; 30.7 %). M.P. = 153 °C. 1H NMR (500 MHz, CD3CN) δ 8.41 (s, 1H), 8.18 (d, J = 8.3

S6

Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.54 – 7.45 (m, 2H), 7.13 (d, J = 7.3 Hz, 1H), 6.97 (d, J = 7.8

Hz, 1H), 6.35 (s, 1H), 5.25 (d, J = 4.8 Hz, 1H), 4.22 (d, J = 9.0 Hz, 1H), 4.15 – 4.05 (m, 1H), 2.78

(s, 7H). 13C NMR (126 MHz, CD3CN) δ 168.99, 151.51, 131.21, 129.28, 126.86, 126.00, 125.80,

124.69, 122.91, 117.35, 113.63, 64.94, 56.42, 44.37, 26.61. MS (ESI) m/z: 946.38 [M]+.

(3S,7S,11S)-2,6,10-trioxo-1,5,9-trioxacyclododecane-3,7,11-triyl)tris(5-(dimethylamino)naphthalene-1-sulfonamide (4)

(3S,7S,11S)-3,7,11-triamino-1,5,9-trioxa-cyclododecane-2,6,10-trione, trishydrochloride (2) (50

mg; 0.13 mmol) is suspended into CH2Cl2 with 4Å sieves. It is dissolved as 0.2 mL of

trimethylamine is added. 120 mg of 5-(Dimethylamino)naphthalene-1-sulfonyl chloride is added

as CH2Cl2 solution. Reaction flask is stirred for 3 h at R.T. Resulting solution is evaporated until

dryness. Purification is achieved by column chromatography. (SiO2; CH2Cl2 : MeOH (10 : 0.6 )).

M.P. = 155 °C 1H NMR (500 MHz, CDCl3) δ 8.56 (d, J = 8.3 Hz, 1H), 8.35 (d, J = 5.8 Hz, 1H),

8.24 (d, J = 6.9 Hz, 1H), 7.48 (dd, J = 10.8, 4.7 Hz, 2H), 7.12 (d, J = 7.3 Hz, 1H), 6.90 (s, 1H),

4.44 (dd, J = 11.3, 3.0 Hz, 1H), 4.13 (d, J = 9.6 Hz, 1H), 3.48 (s, 1H), 2.89 (s, 6H). 13C NMR (126

S7

MHz, CDCl3) δ 166.94, 133.97, 131.24, 130.06, 129.75, 129.38, 128.86, 123.24, 115.49, 77.29,

77.04, 76.78, 66.01, 55.46, 45.47, 26.92. MS (ESI) m/z: 961.31 [M]+.

1H and 13C NMR Spectra of 1

3.09

6.36

3.00

53.8

7

2.40

2.65

2.67

3.44

3.45

3.46

3.47

3.51

4.07

4.09

4.11

5.32

7.27

7.27

7.27

7.28

7.29

7.29

7.30

7.30

7.30

7.32

7.33

7.34

7.34

7.35

7.47

7.49

7.49

S8

1H and 13C NMR Spectra of 2

1H and 13C NMR Spectra of 3

19.4

5

2.89

2.97

2.90

2.48

3.00

2.91

6.23

3.01

3.00

2.88

1.47

1.97

1.97

1.98

2.21

2.78

2.95

4.10

4.11

4.12

4.22

4.23

5.25

5.26

6.35

6.96

6.97

7.12

7.13

7.48

7.49

7.51

7.78

7.80

8.17

8.19

8.41

S9

1H and 13C NMR Spectra of 4

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)

0

500

1000

1500

2000

2500AA121

102030405060708090100110120130140150160170180f1 (ppm)

-20000

-10000

0

10000

20000

30000AA121

S10

UV-Vis absorption titrations of S1

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

Ab

sorb

ance

Wavelength, nm

20M S1 + Acetate

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Abs

orb

anc

e

Wavelength, nm

20M S1 + Benzoate

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Abs

orba

nce

Wavelength, nm

20M S1 + Chloride

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

3.0

20M S1 + Citrate

Abs

orb

anc

e

Wavelength, nm

200 250 300 350 400 450

0

1

2

3

Abs

orba

nce

Wavelength (nm)

20 µM S1+ glutarate

200 250 300 350 400 450

0

1

2

3

Ab

sorb

anc

e

Wavelength (nm)

20 µM S1 + Maleate

S11

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

Ab

sorb

ance

Wavelength, nm

20M S1 + Malonate

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.520M S1 + Oxalate

Ab

sorb

ance

Wavelength, nm

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Abs

orba

nce

Wavelength, nm

20M S1 + Tricarballylate

S12

UV-Vis absorption titrations of S2

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.520M S2 + Acetate

Abs

orb

anc

e

Wavelength, nm

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5 20M S2 + Benzoate

Ab

sorb

ance

Wavelength, nm

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

Abs

orba

nce

Wavelength, nm

20M S2 + Chloride

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.520M S2 + Citrate

Abs

orb

anc

e

Wavelength, nm

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Abs

orb

ance

Wavelength, nm

20M S2 + glutarate

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

sorb

ance

Wavelength, nm

20M S2 + Maleate

S13

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.520M S2 + Malonate

Abs

orb

anc

e

Wavelength, nm

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.520M S2 + Oxalate

Ab

sorb

ance

Wavelength, nm

200 250 300 350 400 450

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

sorb

ance

Wavelength, nm

20M S2 + Tricarballylate

S14

Fluorescence titrations of S1

360 400 440 480 520 560 6000

2

4

6

8

10

12

0 20 40 60 80 100 120 140

0

2

4

6

8

10

12

1:31:2

(I-I

0)/(I

i-I0)

[Acetate] (M)

1:1

I / I 0

Wavelength (nm)

Acetate

360 400 440 480 520 560 6000

1

2

3

4

5

0 20 40 60 80 100 120 140

0

2

4

1:31:21:1

(I-I

0)/

(Ii-I

0)

[Benzoate] (M)I / I 0

Wavelength (nm)

Benzoate

360 400 440 480 520 560 6000

1

2

3

4

0 5 10 15 20 25 30

0.0

0.2

0.4

0.6

0.8

1.0

Ka = 8.32 x 104 M-1

(I-I

0)/

(Ii-I

0)

[Citrate] (M)

I / I 0

Wavelength (nm)

Citrate

360 400 440 480 520 560 600

0

1

2

0.00000 0.00002 0.00004 0.00006

0.0

0.2

0.4

0.6

0.8

1.0

Ka = 3.72 x 105 M-1

(I-I

0)/

(Ii-I

0)

[Glutarate] (M)

Glutarate

I / I 0

Wavelength (nm)

360 400 440 480 520 560 6000.0

0.5

1.0

1.5

0.00000 0.00002 0.00004 0.00006 0.00008

0.0

0.2

0.4

0.6

0.8

1.0

Ka = 2.9 x 105 M-1

(I-I

0)/(I

i-I0)

[Maleate] (M)

I /

I 0

Maleate

360 400 440 480 520 560 6000

1

2

3

0 10 20 30

0.0

0.2

0.4

0.6

0.8

1.0

Ka = 2.25 x 105 M-1

(I-I

0)/

(Ii-I

0)

[Malonate] (M)I / I 0

Wavelength (nm)

Malonate

S15

360 400 440 480 520 560 6000

1

2

0 5 10 15 20 25 30

0.0

0.2

0.4

0.6

0.8

1.0

Ka = 2.08 x 105 M-1

(I-I

0)/(I

i-I0)

[Oxalate] (M)I / I 0

Wavelength (nm)

Oxalate

360 400 440 480 520 560 6000

1

2

3

4

0 20 40 60 80

0.0

0.2

0.4

0.6

0.8

1.0

[Tricarballylate] (M)

(I-I

0)/

(Ii-I

0)

I / I 0

Wavelength (nm)

Tricarballylate

Ka = 7.44 x 104 M-1

360 400 440 480 520 560 600

0.0

0.5

1.0

I /

I 0

Wavelength (nm)

Chloride

S16

Fluorescence titrations of S2

400 440 480 520 560 600 640 680

0.0

0.2

0.4

0.6

0.8

1.0

1.2

I /

I 0

Wavelength (nm)

Acetate

400 440 480 520 560 600 640 680

0.0

0.2

0.4

0.6

0.8

1.0

1.2

I / I 0

Wavelength (nm)

Benzoate

400 440 480 520 560 600 640

0.0

0.2

0.4

0.6

0.8

1.0

I /

I 0

Wavelength (nm)

Citrate

400 450 500 550 600 650

0.0

0.2

0.4

0.6

0.8

1.0 I

/ I0

Wavelength (nm)

Glutarate-S2

400 450 500 550 600 650

0.0

0.2

0.4

0.6

0.8

1.0

I / I

0

Wavelength (nm)

Maleate-S2

400 450 500 550 600 650

0.0

0.2

0.4

0.6

0.8

1.0

I / I

0

Wavelength (nm)

Malonate-S2

S17

400 450 500 550 600 650

0.0

0.2

0.4

0.6

0.8

1.0 I

/ I0

Wavelength (nm)

Oxalate-S2

400 450 500 550 600 650

0.0

0.2

0.4

0.6

0.8

1.0

I / I

0

Wavelength (nm)

Tricarballylate-S2

400 450 500 550 600 650

0.0

0.2

0.4

0.6

0.8

1.0

I / I

0

Wavelength (nm)

Chloride-S2

S18

Fluorescence titrations isotherms of S2

0.00000 0.00004 0.00008 0.00012 0.00016

0.0

0.2

0.4

0.6

0.8

1.0

[Glutarate] (M)

Ka = 2.08 x 104 M-1

I-I 0/

I f-I0

0 50 100 150 200

0.0

0.2

0.4

0.6

0.8

1.0

[Tricarballylate] (M)

Ka = 4.29 x 104 M-1

(I-I

o)/(

If-I

o)

0.00000 0.00005 0.00010 0.00015

0.0

0.2

0.4

0.6

0.8

1.0

Ka = 8.28 x 103 M-1

(I-I

0)/(I

i-I0)

[Citrate] (M)

S19

Job`s plot experiments Citrate - S1 and S2 Job`s plot in Acetonitrile Result: 1:1 Binding Result: 1:1 Binding

0.0 0.2 0.4 0.6 0.8 1.0

0.0

5.0x103

1.0x104

1.5x104

2.0x104

2.5x104

3.0x104

3.5x104

4.0x104

citr

ate

Flu

ore

scen

ce

Citrate = [Citrate]/([Citrate]+[S1])

0.0 0.2 0.4 0.6 0.8 1.0

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

citr

ate

Flu

ores

cenc

e Citrate = [Citrate]/([Citrate]+[S2])

Acetate – S1 and S2 Job`s plot in Acetonitrile Result ≈ 1 :3 Binding Result ≈ 1 :2 Binding

0.0 0.2 0.4 0.6 0.8 1.0

0.0

5.0x103

1.0x104

1.5x104

2.0x104

Ace

tate

Flu

ore

sce

nce

Acetate = [Acetate]/([Acetate]+[S1])

0.0 0.2 0.4 0.6 0.8 1.0

0.0

2.0x104

4.0x104

6.0x104

8.0x104

1.0x105

1.2x105

Ace

tate

Flu

ores

cenc

e

Acetate = [Acetate]/([Acetate]+[S2])

S20

NMR titrations

Titrations with S1

Figure.1 H NMR (500 MHz) titration of S1 (2 mM) upon the addition of Citrate as tri-tetrabutylammonium salt in CD3CN.

 S1 only

0.25 eq. Citrate

0.75 eq. Citrate

1 eq. Citrate

1.25 eq. Citrate

1.50 eq. Citrate

0.50 eq. Citrate

S21

Titrations with S1

5.05.45.86.26.67.07.47.88.28.69.09.4f1 (ppm)

1

2

3

4

AA124-S2-titration 0 eq citrate4 4

AA124-S2-titration 0.5 eq citrate3 3

AA124-S2-titration 1 eq citrate2 2

AA124-S2-titration 3 eq citrate1 1

Figure. 1 H NMR (500 MHz) titration of S2 (2 mM) upon the addition of Citrate as tri-tetrabutylammonium salt in CD3CN.

S2 only

0.5 eq. Citrate

1 eq. Citrate

3 eq. Citrate

S22

Sensor-Analyte complex study by Mass spectrometry

Figure. (A) ESI mass spectrum of the complex of [S1+Citrate+Na+MeCN]-. Inset: Calculated

isotope pattern for [S1+Citrate+Na+MeCN]- (B) ESI mass spectrum of the complex of

S2+Citrate+TBA. Inset: Calculated isotope pattern for S2+Citrate+TBA.

S23

Qualitative analysis

Linear discriminant analysis (LDA)

Table. The jackknifed classification matrix of qualitative analysis of 8 analytes and a control by ing S1 and S2 in hydrogel matrix.

Figure. The canonical scores plot of qualitative analysis of 8 analytes and a control by using S1 and S2 in hydrogel matrix.

S24

Semi-quantitative Semi-quantitative assay for Citrate-Na in water

-20 0 20 40-40

-20

0

20

40

F2

(13 .

7%)

F1 (73.5%)

100 % Correct Classification in Water Control 10 M Citrate 20 M Citrate 30 M Citrate 40 M Citrate 50 M Citrate 60 M Citrate 70 M Citrate 80 M Citrate 90 M Citrate

Figure. Linear discriminant analysis (LDA) of Citrate-Na in hydrogel matrix. LDA shows the trend depending on increasing concentration of citrate.

0 20 40 60 80 100

0

20

40

60

80

100

Calibration data set Validation data set

RMSEC=1.26 RMSECV=1.84

RMSEP=2.14

Pre

dict

ed

[Citr

ate]

(M

)

Actual [Citrate] (M)

Prediction of Citrate in Water

Figure.The result of the linear regression using support vector machine (SVM) affords quantitative analysis of various concentration of citrate. The plots of actual vs. predicted concentration show high accuracy of prediction for multiple analyte concentrations. Two unknown samples (red squares ■) were simultaneously correctly analyzed.

S25

Table. The jackknifed classification matrix of Semi-quantitative analysis of citrate tri-sodium salt by using S1 and S2 in hydrogel matrix.

Figure. The canonical scores plot of Semi-quantitative analysis of citrate tri-sodium salt by using S1 and S2 in hydrogel matrix.

S26

Semi-quantitative assay for Citrate-Na in 25 mM HEPES at pH: 7.4

Table. The jackknifed classification matrix of Semi-quantitative analysis of citrate tri-sodium salt in buffer solution by using S1 and S2 in hydrogel matrix.

Figure. The canonical scores plot of Semi-quantitative analysis of citrate tri-sodium salt in buffer solution by using S1 and S2 in hydrogel matrix.

S27

Semi-quantitative assay for Citrate-Na in Urine

Table. The jackknifed classification matrix of Semi-quantitative analysis of citrate tri-sodium salt in urine by using S1 and S2 in hydrogel matrix.

Figure. The canonical scores plot of Semi-quantitative analysis of citrate tri-sodium salt in urine by using S1 and S2 in hydrogel matrix.

Jackknifed Classification Matrix

199.5 139.5 119.5 99.5 79.5 59.5 19.5 %correct

199.5 10 0 0 0 0 0 0 100

139.5 0 10 0 0 0 0 0 100

119.5 0 0 10 0 0 0 0 100

99.5 0 0 0 10 0 0 0 100

79.5 0 0 0 0 10 0 0 100

59.5 0 0 0 0 0 10 0 100

19.5 0 0 0 0 0 0 10 100

Total 10 10 10 10 10 10 10 100

S28

References

1 A. K. Connors Binding Constants: the Measurement of Molecular Complex Stability; Wiley: New York, 1987. 2 Y. Liu, T. Minami, R. Nishiyabu, Z. Wang, and P. Anzenbacher Jr., J. Am. Chem. Soc. 2013, 135, 7705. 3 R. J. A. Ramirez, L. Karamanukyan, S. Ortiz, and C. G. Gutierrez, Tetrahedron lett., 1997, 38, 749.