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Zwitterionic Reagents for Labeling, Cross-Linking and Improving the Performance of
Chemiluminescent Immunoassays
Anand Natrajan*, David Sharpe and David Wen
Siemens Healthcare Diagnostics
Advanced Technology and Pre-Development
333 Coney Street
East Walpole, MA 02032
Supplementary Material
Synthetic schemes for compounds 1,2, 4 and 5; HPLC chromatograms of intermediates,
final compounds and peptide labeling reactions; MALDI-TOF mass spectra of BSA
conjugates and, assay data for theophylline and TSH.
* Author to whom correspondence should be directed.
E-mail: [email protected]
Phone: 1-508-660-4582
Fax: 1-508-660-4591
Page 102 of 128Organic & Biomolecular ChemistryElectronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012
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N COOBn N COOBn+
O
F F
F
FF
-O3S
N COOH+
-O3S N+
-O3SO
1
i ii
iii
ab
c
Figure S1. Synthetic scheme for amine-reactive zwitterionic reagent 1.
Reagents: (a) 1,3-propane sultone, 2,6-di-tert-butylpyridine, dimethylformamide; (b)
hydrogen, 10% palladium on carbon, methanol; (c) pentafluorophenol, 1-ethyl-
(dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), 1:1, 0.1 M HCl/MeCN.
Page 103 of 128 Organic & Biomolecular ChemistryElectronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012
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NNH2 + N O
O
O
O
O
N
HN
O
O
N
HN
O
O+
N
O
O O
OH
O
N
O
O
-O3S NNH2
+-O3S
N
HN
+-O3S
iv
vvi
2
a
b c
d
Figure S2. Synthetic scheme for thiol-reactive, zwitterionic reagent 2.
Reagents: (a) chloroform; (b) 1,3-propane sultone, ethyl acetate; (c) hydrogen, 10%
palladium on carbon, methanol/water (d) (BOP) benzotriazol-1-yl-
oxy)tris(dimethylamino)phosphonium hexafluorophosphate, diisopropylethylamine,
dimethylsulfoxide.
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N NH2H2N
SO3-
+N N
HNH
SO3-
+
O
ON
O
O O
O N
O
O
HN
O
O N
O
O
O
O
HN
O
O
H2NN SO3
-+
HN
O
O
O
HN
O
O
NH
N SO3-+
HN
O
O
O
NH2
NH
N SO3-+ H
N
O
O
O
HN
NH
N SO3-+
O
N
O
O
H2N
O
HN
NH
N SO3-+
O
N
O
O
O
N
O
O
ON
O
O
HN
O
HN
NH
N SO3-+
O
N
O
O
OO
ON
O
O
3 4
5
iv
vii
viii
ix
x
a
b
c
d e
f
N-α -t-BOC-N- ε-CBZ-lysine-N-hydroxsuccinimide ester
Figure S3. Synthetic schemes for zwitterionic homobifunctional crosslinker 4 and
heterobifunctional crosslinker 5.
Reagents: (a) disuccinimidyl carbonate, diisopropylethylamine, dimethyl sulfoxide; (b)
dimethyl sulfoxide; (c) hydrogen, 10% palladium on carbon, methanol/water; (d) dimethyl
sulfoxide; (e) trifluoroacetic acid; (f) disuccinimidyl glutarate, dimethyl sulfoxide.
Page 105 of 128 Organic & Biomolecular ChemistryElectronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012
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Minut es
0 1 2 3 4 5 6 7 8 9 10
mA
U
0
100
200
300
400
500
600
700
800
900
1000
mA
U
0
100
200
300
400
500
600
700
800
900
1000
Figure S4. HPLC chromatogram of compound ii. HPLC conditions: Phenomenex,
Gemini C6-Phenyl, 3 micron, 4.6 x 50 mm column using a 10 minute gradient of 10 →
40% MeCN/water (with 0.05% TFA) at a flow rate of 1.0 mL/minute and UV detection at
220 nm.
N COOBn+
-O3S
ii
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Minutes
0 1 2 3 4 5 6 7 8 9 10
mA
U
0
20
40
60
80
100
120
140
160
180
%
0
10
20
30
40
50
60
70
80
90
100
7.7
15
Figure S5. HPLC chromatogram of amine-reactive, zwitterionic reagent 1. HPLC
conditions: Phenomenex, Gemini C6-Phenyl, 3 micron, 4.6 x 50 mm column using a 10
minute gradient of 10 → 40% MeCN/water (with 0.05% TFA) at a flow rate of 1.0
mL/minute and UV detection at 260 nm.
O
F F
F
FF
N+
-O3SO
1
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Mi nutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mA
U
0
50
100
150
200
250
300
350
400
450
mA
U
0
50
100
150
200
250
300
350
400
450
Figure S6. HPLC chromatogram of compound v. HPLC conditions: Phenomenex
PRODIGY, C18, 10 micron, 10 x 250 mm column using a 30 minute gradient of 10 →
60% MeCN/water (with 0.05% TFA) at a flow rate of 4.0 mL/minute and UV detection at
260 nm.
N
HN
O
O+
-O3S
v
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Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
mA
U
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
mA
U
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Figure S7. HPLC chromatogram of thiol-reactive, zwitterionic reagent 2. HPLC
conditions: Phenomenex, C18, 10 micron, 3.9 x 300 mm column using a 30 minute
gradient of 0 → 20% MeCN/water (with 0.05% TFA) at a flow rate of 1.0 mL/minute and
UV detection at 220 nm.
O
N
O
O
N
HN
+-O3S
2
Page 109 of 128 Organic & Biomolecular ChemistryElectronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012
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Minutes
0 1 2 3 4 5 6 7 8 9 10
mA
U
0
200
400
600
800
1000
1200
1400
1600
mA
U
0
200
400
600
800
1000
1200
1400
1600
Figure S8. HPLC chromatogram of homobifunctional, zwitterionic, cross-linking reagent
4. HPLC conditions: Phenomenex, Luna PFP(2), 3 micron, 4.6 x 150 mm column using
a 20 minute gradient of 0 → 30% MeCN/water (with 0.05% TFA) at a flow rate of 1.0
mL/minute and UV detection at 220 nm.
N NH
NH
SO3-
+
O
ON
O
O O
O N
O
O
4
Page 110 of 128Organic & Biomolecular ChemistryElectronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012
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Minutes
0 1 2 3 4 5 6 7 8 9 10
mA
U
0
200
400
600
800
1000
1200
mA
U
0
20 0
40 0
60 0
80 0
10 00
12 00
Figure S9. HPLC chromatogram of compound vii. HPLC conditions: YMC, C18, 3
micron, 4.0 x 50 mm column using a 10 minute gradient of 10 → 90% MeCN/water (with
0.05% TFA) at a flow rate of 1.0 mL/minute and UV detection at 260 nm.
HN
O
O
O
HN
O
O
NH
N SO3-+
vii
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Minutes
0 1 2 3 4 5 6 7 8 9 10
mA
U
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
mAU
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
Figure S10. HPLC chromatogram of compound ix. HPLC conditions: YMC, C18, 3
micron, 4.0 x 50 mm column using a 10 minute gradient of 10 → 40% MeCN/water (with
0.05% TFA) at a flow rate of 1.0 mL/minute and UV detection at 220 nm.
HN
O
O
O
HN
NH
N SO3-+
O
N
O
O
ix
Page 112 of 128Organic & Biomolecular ChemistryElectronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012
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Minutes
0 1 2 3 4 5 6 7 8 9 10
mA
U
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
mA
U
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
Figure S11. HPLC chromatogram of heterobifunctional, zwitterionic, cross-linking
reagent 5. HPLC conditions: YMC, C18, 3 micron, 4.6 x 50 mm column using a 10
minute gradient of 0 → 30% MeCN/water (with 0.05% TFA) at a flow rate of 1.0
mL/minute and UV detection at 220 nm.
HN
O
HN
NH
N SO3-+
O
N
O
O
OO
ON
O
O
5
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M i n u t e s
0 5 1 0 1 5 2 0 2 5
mA
U
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
1 8 0
2 0 0
mA
U
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
1 8 0
2 0 0
Figure S12. HPLC chromatograms of (a) coupling reaction of zwitterionic reagent 1 with
penta(phenylalanine) (red trace) (b) penta(phenylalanine) (blue trace). Compound
eluting at 18.5 minutes (red) is the zwitterion-labeled peptide 6. Compound eluting at
16.5 minutes (red) is pentafluorophenol that is released in the coupling reaction. HPLC
conditions: Phenomenex, 10 micron, C18, 3.9 x 300 mm column and a 30 minute gradient
of 10 → 100% MeCN/water (with 0.05% TFA) at a flow rate of 1.0 mL/minute and UV
detection 260 nm.
HN
O
NH O
HN
O
NH O
HN COOH
O
N-O3S+
6
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M i n u t e s
0 5 1 0 1 5 2 0 2 5 3 0
mA
U
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
mA
U
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
Figure S13. HPLC chromatograms of (a) coupling reaction of zwitterionic reagent 1 with
Phe-Gly-Gly-Phe (red trace) (b) Phe-Gly-Gly-Phe (blue trace). Compound eluting at 17.3
minutes (red) is the zwitterion-labeled peptide 7. Compound eluting at 25 minutes is
pentafluorophenol that is released in the coupling reaction. HPLC conditions:
Phenomenex, 10 micron, C18, 3.9 x 300 mm column and a 40 minute gradient of 10 →
60% MeCN/water (with 0.05% TFA) at a flow rate of 1.0 mL/minute and UV detection at
260 nm.
HN
O
NH O
HN
O
NH O
OH
O
N-O3S+
7
Page 115 of 128 Organic & Biomolecular ChemistryElectronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012
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Minutes
6 8 10 12 14 16 18 20 22 24 26 28 30
mA
U
0
20
40
60
80
100
120
140
160
180
200
mA
U
0
20
40
60
80
100
120
140
160
180
200
Figure S14. HPLC chromatograms of (a) coupling reaction of zwitterionic reagent 1 with
penta(leucine) (red trace) (b) crude penta(leucine) (blue trace) eluting at 23.5 minutes
with impurity at 25 minutes. Compound eluting at 27 minutes (red) is the zwitterion-
labeled peptide 8. Compound eluting at 21.5 minutes is pentafluorophenol that is
released in the coupling reaction. Unreacted peptide is shifted slightly at 24 minutes.
HPLC conditions: Phenomenex, 10 micron, C18, 3.9 x 300 mm column and a 30 minute
gradient of 10 → 70% MeCN/water (with 0.05% TFA) at a flow rate of 1.0 mL/minute and
UV detection at 220 nm.
HN
O
NH O
HN
O
NH O
HN COOH
O
N-O3S+
8
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M i n u t e s
0 5 1 0 1 5 2 0 2 5 3 0
mA
U
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
mA
U
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
Figure S15. HPLC chromatograms of (a) coupling reaction of zwitterionic cross-linking
reagent 4 with penta(phenylalanine) (blue trace) (b) further coupling of zwitterion-
modified penta(phenylalanine) intermediate xi with Phe-Gly-Gly-Phe (red trace).
Compound eluting at 17 minutes (blue) is the zwitterion-labeled penta(phenylalanine)
intermediate xi. Compound eluting at 16 min (blue) is unreacted penta(phenylalanine).
Compound eluting at 18 minutes (red) is the zwitterion cross-linked nonapeptide 9.
Compound eluting at 9 minutes (red) is excess, unreacted Phe-Gly-Gly-Phe. HPLC
conditions: Phenomenex, 10 micron, C18, 3.9 x 300 mm column and a 30 minute gradient
of 10 → 100% MeCN/water (with 0.05% TFA) at a flow rate of 1.0 mL/minute and UV
detection at 260 nm.
HN
HN N
SO3-
HN
O
+
O
HN
O
NHO
HN
O
NHO
HO
O
NH O
HN
O
NH O
HN COOH
9
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Minutes
0 5 10 15 20 25
mA
U
0
50
100
150
200
mA
U
0
50
100
150
200
Figure S16. HPLC chromatograms of (a) coupling reaction of heterobifunctional
zwitterionic, cross-linking reagent 5 with penta(phenylalanine) (blue trace) (b) further
coupling of zwitterion-modified penta(phenylalanine) intermediate xii with Cys-Gly (red
trace). Compound eluting at 18.5 minutes (blue, earlier eluting) is the zwitterion-labeled
penta(phenylalanine) intermediate xii. Starting material penta(phenylalanine)elutes as a
later eluting shoulder peak. Compound eluting at 16.5 minutes (red) is the zwitterion
cross-linked heptapeptide 10. HPLC conditions: Phenomenex, 10 micron, C18, 3.9 x 300
mm column and a 30 minute gradient of 10 → 100% MeCN/water (with 0.05% TFA) at a
flow rate of 1.0 mL/minute and UV detection at 260 nm.
HN
O
NHO
HN
O
NHO
HNHOOC
HN
O
HN
NH
N SO3-+
O
N
O
O
OOH2N
S
O
NH O
OH
10
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A
B
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C
D
Figure S17. MALDI-TOF mass spectra of (A) Unlabeled BSA, (B) BSA labeled with
NSP-DMAE-NHS indicating a mass increase of 634 units corresponding to ~1.4
acridinium ester labels, (C) NSP-DMAE-BSA further labeled with 10 equivalents of
zwitterion label 1 indicating a mass increase of 1200 units corresponding to ~5 zwitterion
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labels and, (D) NSP-DMAE-BSA further labeled with 20 equivalents of zwitterion label 1
indicating a mass increase of 2034 units corresponding to ~8 zwitterion labels.
N
SO3-
O O
O O N
O
O
+
NSP-DMAE-NHS
O
F F
F
FF
N+
-O3SO
1
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Table S1. Theophylline assay using conjugates 11 and 12.
Theophylline Assay
Compound 11 Compound 12 Theophylline [µM]
Mean Chemiluminescence
(RLU)
B/B0
(%) Relative Precision (c.v.%)
Mean Chemiluminescence
(RLU)
B/B0 (%)
Relative Precision (c.v.%)
0 2,138,770 100 6.2 2,321,548 100 5.8 1 2,089,048 98 4.3 2,074,255 89 3.5 2 1,941,391 91 3.3 1,905,715 82 1.5 4 1,675,088 78 3.1 1,651,129 71 4.0 10 1,232,974 58 1.7 1,160,276 50 5.6 20 850,536 40 2.1 849,644 37 3.9 40 511,047 24 0.7 470,761 20 1.8
100 242,815 11 2.4 197,789 8.5 5.0 500 53,563 2.5 0.6 39,429 1.7 0.9 1000 27,932 1.3 1.3 20,006 0.9 1.9
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Table S2. TSH assay using anti-TSH conjugate of labels 13 and 14.
TSH Assay Compound 13 Compound 14 TSH [mIU/L] Mean
Chemiluminescence (RLU)
Relative Precision (c.v.%)
Mean Chemiluminescence
(RLU)
Relative Precision (c.v.%)
0 9,186 3.3 10,887 3.6 0.002 9,566 2.0 12,064 2.9 0.004 9,800 2.0 12,652 5.5 0.01 10,697 2.6 12,875 7.4
0.015 11,061 3.0 12,983 4.8 0.02 11,536 4.6 13,295 1.3
0.025 11,871 3.7 13,642 1.4 0.03 12,580 3.3 14,443 1.1 0.1 16,982 3.3 21,818 4.6 1 102,981 3.0 124,400 2.5 10 621,486 1.5 965,677 5.1
100 4,853,307 2.6 6,673,056 3.1
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