Supporting Information

Interrogating Target-Specificity by Parallel Screening of a DNA-Encoded Chemical Library Against Closely Related ProteinsRaphael M. Franzini, Angela Nauer, Jörg Scheuermann, and Dario Neri*

Institute of Pharmaceutical Sciences, ETH Zürich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland.dario.neri@pharma.ethz.ch; Tel.: +41 44 633 74 01

Table of Contents

Experimental ProceduresMaterials and Instrumentation S2Affinity screening of protein ligands from DECL S2Synthesis of fluorescein-conjugates of hit compounds S3Measuring dissociation constants by fluorescence polarization S3Chemical protein biotinylation S3

EquationsEquation S1 Normalization of sequence counts. S4Equation S2 Fitting formula for Kd values. S4Equation S3 Formula for additive sequence counts S4Equation S4 Definition of NSC selectivity. S4Equation S5 Calculation of pseudo-color hue values. S5

Supporting Figures and TablesFigure S1 Structure of DAL-100K DNA-encoded chemical library S6Figure S2 DAL-100K screening results for HSA S6Figure S3 General structure of fluorescein conjugate S6Figure S4 Enlarged specificity-sequence count scatter plot S7Table S1 RSA hit compounds S8Table S2 BSA hit compounds S9Table S3 HSA hit compounds S10Table S4 General serum albumin hit compounds S11Table S5 Characterization of resynthesized fluorescein conjugates by LC-MS S12

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

Experimental Procedures

Materials and instrumentation. Standard reagents and solvents were acquired from commercial sources and used without further purification. Carboxylic acid building blocks were purchased from commercial suppliers including ABCR (Karlsruhe, Germany), ChemBridge (San Diego, CA), Sigma-Aldrich (St. Louis, MO), TCI Europe (Zwijndrecht, Belgium), Alfa Aesar (Ward Hill, MA), Matrix Scientific (Columbia, SC), and Acros Organics (Geel, Belgium). HATU, Biotin-LC-NHS and Fmoc-Dap(Mtt)-OH were acquired from ChemPep (Wellington, FL) and all DNA-synthesis reagents from Glen Research (Sterling, VA).Serum albumins were obtained from Sigma Aldrich (HSA: order number A3782, lyophilized, fatty acid free, globulin free; BSA: order number A7511, lyophilized powder, essentially fatty acid free, 1 x crystallized; RSA: order number A6414, lyophilized powder, essentially fatty acid free, essentially globulin free).Purification of fluorescein-conjugates were performed by reverse phase HPLC (Waters, Milford, CT) using a C18-XTerra column (5 μm, 10 on 10 x 150 mm, Waters, Milford, CT) and analyzed by LC-MS on an Agilent 6130 single quadrupole LC/MS system (Agilent Technologies, Basel, Switzerland) with electrospray ionization source. Affinity selections were performed robotically on a KingFisher Magnetic Particle Processor (Thermo Fisher, Waltham, MA) using T-1 or M-270 Dynabeads magnetic beads (Life Technologies). Fluorescence polarization experiments were performed using a SpectraMass paradigm multi-mode microplate reader (Molecular Devices, Sunnyvale, CA) in 384-well plates (Greiner, Frickenhausen, Germany). Instant JChem (ChemAxon) was used for structure and data management (http://www.chemaxon.com).

Affinity screening of protein ligands from DECL. Streptavidin-coated magnetic beads (Dynabeads M 270, 10 μL of suspension) were suspended in buffer (100 mM NaCl, 50 mM NaPi, pH 7.4) containing 0.25 % Tween-20. Using a KingFisher magnetic particle processor, the magnetic beads were transferred to a solution of biotinylated proteins (40 pmol in 100 μL) and incubated for 20 min with continuous gentle mixing. The beads were washed (3 ×) with biotin-containing buffer (100 μM) to block free streptavidin binding sites. The protein-loaded beads were transferred to a solution of the DECL (5 nM total concentration in 100 μL) in buffer containing 0.25 % Tween-20 and the suspension incubated for 1 h with continuous gentle mixing. The beads were removed and washed with buffer (5 × 30 s) and incubated with elution buffer (Buffer EB; Qiagen). DNA conjugates were released by heat denaturation of the proteins (96 °C for 5 min).The coding DNAs of the oligonucleotide conjugates were amplified by PCR after selection experiments and submitted to the Functional Genomics Center Zurich for high-throughput DNA sequencing on an Illumina HiSeq 2000/2500.Sequence counts were normalized to the total number of sequences obtained for each selection (Equation S1).

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Synthesis of fluorescein-conjugates of hit compounds. The fluorescein-containing linker structure was synthesized by solid-phase DNA-phosphoramidite chemistry using a fluorescein-CPG (2-(6-FAM) CPG), a spacer phosphoramidite (Spacer phosphoramidite 3 or Spacer phosphoramidite 9) and a 5’-amino-modifier (5’-amino modifier C12 or 5’-amino-modifier 5). The conjugates were synthesized on a 20 – 50 nmol scale. The Mmt group was removed from the 5’-amine using cycles of washing with 3 % TCA in DCM and DCM washes (5 ×). The solid support was treated with Fmoc-Dap(Mtt)-OH (50 mM), HATU (50 mM) and DIEA (150 mM) in NMP (0.5 mL) for 4 h. The Mtt-group was removed using cycles of washing with 3 % TCA in DCM and DCM washes (5 ×). DE-1 was derivatized by reaction with carboxylic acid building block (50 mM), HATU (50 mM), and DIEA (150 mM) in NMP (0.5 mL) for 2 × 2 h. The residue was treated with 10 % Et2NH in MeCN (2 × 5 min) followed by 20 % piperidine in DMF (2 × 5 min). DE-2 was introduced by reaction with carboxylic acid building block (50 mM), HATU (50 mM) and DIEA (150 mM) in NMP (0.5 mL) for 2 × 2 h. The conjugate was cleaved from the solid support by incubation with AMA. The AMA solution was evaporated and the residue purified by reverse-phase HPLC. Conjugates were characterized by LC-MS (Table S5) and analytical HPLC (λabs = 495 nm) and found to be > 95 % pure.

Measuring dissociation constants by fluorescence polarization. The fluorescence polarization (λex = 485 nM; λem = 515 nM) was measured of solutions containing the fluorescein-labeled conjugates (0.5 nM) and variable concentrations of the protein in buffer (100 mM NaCl, 50 mM NaPi, pH 7.4 for all other proteins). The fluorescence polarization values of triplicate measurements were plotted versus the protein concentration and fitted using Equation S2.

Chemical Protein Biotinylation. The protein was incubated with a 20-fold excess of Biotin-LC-NHS in buffer (100 mM NaCl, 50 mM NaPi, pH 7.4) containing 5 % of DMSO for 1 h at room temperature. The reaction was quenched with an excess of Tris (10-fold relative to Biotin-LC-NHS). The biotinylated protein was separated from small-molecule reagents using a disposable PD-10 desalting column (GE Healthcare Life Sciences, Piscataway, NJ). Protein-containing fractions were combined and the concentration determined by measuring the absorbance at 280 nm. Protein quality and purity was evaluated by denaturing gel electrophoresis and analytical gel-filtration chromatography. Biotinylation was verified by performing a band-shift assay involving the disappearance of the protein band in SDS-PAGE gel electrophoresis upon addition of avidin. Proteins were stored at -78 C.

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Equations

Equation S1: Normalization of sequence counts.

𝑁𝑆𝐶(𝑥,𝑦) = 𝑆𝐶(𝑥,𝑦)[ 240

∑𝑢 = 1

( 430

∑𝑣 = 1

𝑆𝐶(𝑢,𝑣))] ‒ 1 × 240 × 430

NSC(x,y): normalized sequence count; SC(x,y): sequence count (x and y define the number of the building block at DE-1 and DE-2).

Equation S2. Fitting formula for Kd measurements.

𝐹𝑃 = 𝑎 + 0.5 × 𝑏 × ((𝑐𝑝𝑟𝑜𝑡 + 𝑐𝑙𝑖𝑔 + 𝐾𝑑) ‒ ((𝑐𝑝𝑟𝑜𝑡 + 𝑐𝑙𝑖𝑔 + 𝐾𝑑)2 ‒ 4 × 𝑐𝑝𝑟𝑜𝑡 × 𝑐𝑙𝑖𝑔))FP: fluorescence polarization; cprot: protein concentration; clig: ligand concentration; Kd: dissociation constant; a, b: fitting constants.

Calculation of

NSC(RSA,BSA,HSA): Normalized sequence count for specified protein (RSA, BSA, HSA).

Max(NSC): maximal normalized sequence count for any of the three serum albumins.

Min(NSC): minimal normalized sequence count for any of the three serum albumins.

Equations S3, S4 and S5 were used to calculated for better visualization of the data in Figure 2.

Equation S3. Formula for additive sequence counts (ASC).

𝐴𝑆𝐶 = 𝑁𝑆𝐶(𝑅𝑆𝐴) + 𝑁𝑆𝐶(𝐵𝑆𝐴) + 𝑁𝑆𝐶(𝐻𝑆𝐴)

Equation S4. Definition of NSC-selectivity.

𝑃𝑆 =32

∙ (𝑀𝑎𝑥(𝑁𝑆𝐶)𝐴𝑆𝐶

‒13)

PS: protein selectivity.

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Equation S5. Calculation of pseudo-hue values.

𝐻𝑢𝑒: {𝑀𝑎𝑥(𝑁𝑆𝐶) = 𝑁𝑆𝐶(𝑅𝑆𝐴): 𝐻 = 60° ∙ (𝑁𝑆𝐶(𝐵𝑆𝐴) ‒ 𝑁𝑆𝐶(𝑅𝑆𝐴)𝑀𝑎𝑥(𝑁𝑆𝐶) ‒ 𝑀𝑖𝑛(𝑁𝑆𝐶)

𝑚𝑜𝑑6)𝑀𝑎𝑥(𝑁𝑆𝐶) = 𝑁𝑆𝐶(𝐵𝑆𝐴):𝐻 = 60° ∙ (𝑁𝑆𝐶(𝐻𝑆𝐴) ‒ 𝑁𝑆𝐶(𝑅𝑆𝐴)

𝑀𝑎𝑥(𝑁𝑆𝐶) ‒ 𝑀𝑖𝑛(𝑁𝑆𝐶)+ 2)

𝑀𝑎𝑥(𝑁𝑆𝐶) = 𝑁𝑆𝐶(𝐻𝑆𝐴):𝐻 = 60° ∙ (𝑁𝑆𝐶(𝑅𝑆𝐴) ‒ 𝑁𝑆𝐶(𝐵𝑆𝐴)𝑀𝑎𝑥(𝑁𝑆𝐶) ‒ 𝑀𝑖𝑛(𝑁𝑆𝐶)

+ 4) �

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Supporting Figures and Tables

Figure S1. Structure of DAL-100K DNA-encoded chemical library. The library contains 103,200 encoded compounds consisting of combinatorial pairs of 670 carboxylic acid building blocks (240 at DE-1 and 430 at DE-2; Reference 5 in main text).

Figure S2. DAL-100K screening results for HSA. Figure reproduced from Reference 5 in main text.

Figure S3. General structure of fluorescein-conjugates.

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Figure S4. Enlarged specificity-sequence count scatter plot. Selected hit compounds are indicated by arrows and labeled by the compounds designation. The hue of the marker indicates the albumin-preference of the corresponding compounds (RSA: red, BSA: green, HSA: blue). The entire data set is shown in Figure 2b in the main text.

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Table S1. RSA hit compounds. Structures normalized sequence counts and dissociation constants for compounds with predicted specificity for RSA.

Building Blocks NSC Kd [nM]DE-1 DE-2 RSA BSA HSA RSA BSA HSA

145 108

393 7 1 7.4 93 25

228 128

234 8 1 4.5 121 45

233

424

219 4 2 4.7 6.0 9.1

228 74

210 7 1 5.3 35 46

1382

74

207 5 1 17.1 420 29

162 143203 5 2 8.5 490 48

60 174

137 6 2 26 87 44

1402

74

132 2 0 12.1 164 47

772

74

110 2 0 25 113 30

233

168

105 2 0 8.7 10.4 10.3

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Table S2. BSA hit compounds. Structures normalized sequence counts and dissociation constants for compounds with predicted specificity for BSA.

Building Blocks NSC Kd [nM]DE-1 DE-2 RSA BSA HSA RSA BSA HSA

149 11119 297 34 26 1.9 7.8

357

3

13 204 25 24 3.0 26

14811

16 164 9 33 15.1 10.9

14011

6

9 128 12 3.4 2.4 17.6

20915

2

8 124 14 40 2.3 33

14011

010 123 14 2.2 3.6 4.6

88

111

5 111 10 53 33 42

140 947 95 6 1.7 3.3 7.8

88

73

3 78 7 27 25 34

14015

2

3 53 4 22 10.3 12.3

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Table S3. HSA hit compounds. Structures normalized sequence counts and dissociation constants for compounds with predicted specificity for HSA.

Building Blocks NSC Kd [nM]DE-1 DE-2 RSA BSA HSA RSA BSA HSA

83 1342 18 151 10.9 3.0 0.4

53 158

0 1 148 -a -a -a

152 253

0 0 103 -a -a -a

7 416

2 1 93 -a -a -a

77 286

2 11 90 15 -b 0.7

94 253

0 1 61 -a -a -a

101 321

0 4 26 26 27 8.0

9934

4

0 0 23 77 300 5.4

1727

80 0 19 11.7 26 1.4

85 2150 0 17 8.9 16 5.7

a Synthesis of compound was unsuccessful or no binding was observed (A007/B416). b Broad transition unsuitable for sigmoidal curve fitting.

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Table S4. General serum albumin hit compounds. Structures normalized sequence counts and dissociation constants for compounds with predicted affinity for all three serum albumins.

Building Blocks NSC Kd [nM]DE-1 DE-2 RSA BSA HSA RSA BSA HSA

34

321

206 66 131 24 -a 2.0

17011

1

198 152 24 5.4 6.8 151

12911

1

104 150 21 5.1 2.6 16.1

2097

371 112 13 11.7 18.2 28

129 74

87 56 10 6.2 26 22

1297

3

56 69 9 5.4 72 8.5

1457

356 61 11 8.5 10.8 4.3

387

342 46 7 7.3 28 7.6

1407

3

23 42 7 4.4 24 9.2

7714

3

24 13 29 2.9 5.8 5.5

a Broad transition unsuitable for sigmoidal curve fitting.

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Table S5. Characterization of resynthesized fluorescein conjugates by LC-MS.

DE-1 DE-2 Calculated mass

Measured mass DE-1 DE-2 Calculated

massMeasured

mass145 108 1412.0 1412.0 88 73 1335.2 1334.4228 128 1344.0 1343.4 140 152 1368.8 1368.2233 424 1539.9 1539.6 83 134 1419.1 1420.6228 74 1362.0 1361.4 77 286 1383.2 1383.8138 274 1346.0 1345.3 101 321 1326.4 1325.7162 143 1384.0 1383.1 99 344 1441.1 1440.660 174 1357.1 1356.5 17 278 1436.1 1435.1

140 274 1362.0 1363.5 85 215 1357.1 1356.377 274 1342.3 1341.7 34 321 1327.3 1327.9

233 168 1484.3 1484.4 170 111 1344.9 1344.4149 111 1318.9 1318.2 129 111 1334.9 1334.335 73 1313.1 1312.5 209 73 1265.2 1265.4

148 111 1301.0 1302.4 129 74 1353.0 1352.3140 116 1374.9 1374.2 129 73 1301.1 1300.5209 152 1315.0 1314.5 145 73 1336.1 1335.4140 110 1336.9 1336.3 38 73 1285.2 1284.788 111 1369.1 1368.3 140 73 1319.0 1318.4

140 94 1338.9 1338.2 77 143 1362.2 1362.4

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