Sublibrary numbering and screening layout for the three phosphopeptide libraries

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15 Νοε 2013 (πριν από 3 χρόνια και 7 μήνες)

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Supporting information:

1


2

Sublibrary numbering and s
creening layout for
the three
phosphopeptide

3

libraries

4

From each sublibrary of the pentapeptide libraries (yellow, blue) which contain a theoretical 3375
5

different peptides each, two wells of a 96
-
well microtiter plate (corresponding to ~ 4

000 beads) were
6

filled with beads. For the tetrapeptide library (red)
which contains only 225 peptides only one well was
7

used. This resulted in a screening plate of 75 wells. The sublibrary allocation within a 96
-
well plate is
8

shown in
Table S1
and
Figure S1

9

Table S1

Sublibrary numbering and R4 residues for library
3

10

Sublibr
ary

R4 amino acid

Well

No. of peptides





1

Ala

A1, B1

3375

2

Ile

A2, B2

3375

3

Val

A3, B3

3375

4

Phe

A4, B4

3375

5

Tyr

A5, B5

3375

6

Trp

A6, B6

3375

7

Pro

A7, B7

3375

8

Arg

A8, B8

3375

9

Lys

A9, B9

3375

10

Ser

A10, B10

3375

11

Thr

A11, B11

3375

12

Asp

A12, B12

3375

13

Glu

C12, D12

3375

14

Asn

C11, D11

3375

15

Gln

C10, D10

3375

16

Ala

C9, D9

3375

17

Ile

C8, D8

3375

18

Val

C7, D7

3375

19

Phe

C6, D6

3375

20

Tyr

C5, D5

3375

21

Trp

C4, D4

3375

22

Pro

C3, D3

3375

23

Arg

C2, D2

3375

24

Lys

C1, D1

3375

25

Ser

E1, F1

3375

26

Thr

E2, F2

3375

27

Asp

E3, F3

3375

28

Glu

E4, F4

3375

29

Asn

E5, F5

3375

30

Gln

E6, F6

3375

31

Ala

E7

225

32

Ile

E8

225

33

Val

E9

225

34

Phe

E10

225

35

Tyr

E11

225

36

Trp

E12

225

37

Pro

F7

225

38

Arg

F8

225

39

Lys

F9

225

40

Ser

F10

225

41

Thr

F11

225

42

Asp

F12

225

43

Glu

G12

225

44

Asn

G11

225

45

Gln

G10

225

For technical reasons each on
-
bead screening library used in the CONA screening process is
1

composed of individual sublibraries.

The screening of defined subsets and the picking from these
2

subsets facilitates the decoding of hit structures. Each sublibrary is defined by a specific branch which
3

it belongs to (yellow, blue and red) and by the identity of the last building block. E.g.
,
ibrary 3 consists
4

of 45 sublibraries
,
15 sublibraries per branch.. For on
-
bead screening
the sublibraries of the
5

phosphopeptide library were numbered as listed above (1
-
15
:
yellow branch, 16
-
30
:
blue branch, 31
-
6

45
:
red branch). The sublibrary is denoted i
n the second column. For each screen
,

two wells
of a 96
7

well plate
were filled with beads from each sublibrary from the two fi
r
st branches (yellow and blue) and
8

one
well only was filled
with beads of the red branch. The well number(s) where the specific su
blibrary
9

is found within the screening plate
are

listed in the third column. The fourth column contains the
10

number of compounds per sublibrary
.

11

Figure S1

Sublibrary allocation within a screening plate

12


13

Screening plates, used for the phosphotyrosine

library, consist of 75 wells. The numbers correspond
14

to the sublibrary numbers from
Table S1
. The color coding indicates the affiliation of individual
15

sublibraries to the three different branches
for

the

library.

16


17


18

Table S2

Number of hit beads per well an
d maximal ring intensities for Cy5
-
SAP

19

Well No.

Well

Sublibrary

No. of hits

Maximum ring
intensity [kHz]






1

A1

Ala

2

150

2

A2

Ile

0


3

A3

Val

4

150

4

A4

Phe

1

150

5

A5

Tyr

1

200

6

A6

Trp

0


7

A7

Pro

3

150

25
25
1
1
24
24
26
26
2
2
23
23
27
27
3
3
22
22
28
28
4
4
21
21
29
29
5
5
20
20
30
30
6
6
19
19
31
37
7
7
18
18
32
38
8
8
17
17
33
39
9
9
16
16
34
40
43
10
10
15
15
35
41
44
11
11
14
14
36
42
45
12
12
13
13
A
B
C
D
E
F
G
H
1
2
3
4
5
6
7
8
9
10
11
12
25
25
1
1
24
24
26
26
2
2
23
23
27
27
3
3
22
22
28
28
4
4
21
21
29
29
5
5
20
20
30
30
6
6
19
19
31
37
7
7
18
18
32
38
8
8
17
17
33
39
9
9
16
16
34
40
43
10
10
15
15
35
41
44
11
11
14
14
36
42
45
12
12
13
13
A
B
C
D
E
F
G
H
1
2
3
4
5
6
7
8
9
10
11
12
Well No.

Well

Sublibrary

No. of hits

Maximum ring
intensity [kHz]

8

A8

Arg

25

400

9

A9

Lys

9

250

10

A10

Ser

0


11

A11

Thr

0


12

A12

Asp

0


13

B12

Asp

0


14

B11

Thr

0


15

B10

Ser

0


16

B9

Lys

5

200

17

B8

Arg

25

600

18

B7

Pro

2

200

19

B6

Trp

0


20

B5

Tyr

0


21

B4

Phe

3

150

22

B3

Val

3

250

23

B2

Ile

0


24

B1

Ala

4

200

25

C1

Lys

14

400

26

C2

Arg

12

500

27

C3

Pro

6

200

28

C4

Trp

5

800

29

C5

Tyr

2

800

30

C6

Phe

3

800

31

C7

Val

4

250

32

C8

Ile

5

400

33

C9

Ala

0


34

C10

Gln

0


35

C11

Asn

1

200

36

C12

Glu

0


37

D12

Glu

2

500

38

D11

Asn

0


Well No.

Well

Sublibrary

No. of hits

Maximum ring
intensity [kHz]

39

D10

Gln

0


40

D9

Ala

1

200

41

D8

Ile

7

500

42

D7

Val

6

300

43

D6

Phe

6

700

44

D5

Tyr

4

400

45

D4

Trp

8

400

46

D3

Pro

7

500

47

D2

Arg

20

1000

48

D1

Lys

10

1000

49

E1

Ser

0


50

E2

Thr

3

200

51

E3

Asp

0


52

E4

Glu

0


53

E5

Asn

2

200

54

E6

Gln

4

300

55

E7

Ala

0


56

E8

Ile

0


57

E9

Val

1

300

58

E10

Phe

5

400

59

E11

Tyr

0


60

E12

Trp

3

200

61

F12

Asp

0


62

F11

Thr

0


63

F10

Ser

0


64

F9

Lys

0


65

F8

Arg

10

300

66

F7

Pro

0


67

F6

Gln

3

250

68

F5

Asn

5

400

69

F4

Glu

0


Well No.

Well

Sublibrary

No. of hits

Maximum ring
intensity [kHz]

70

F3

Asp

0


71

F2

Thr

5

500

72

F1

Ser

2

200

73

G1

Glu

0


74

G2

Asn

0


75

G3

Gln

0


Beads in total
150 000 No. of
hits: 253 hit rate: 0.0017

All beads with fluorescent ring intensities above 200 kHz were counted as hits. The number of hit
1

beads is listed for each well of the
screening plate.

2


3


4

Figure S3:
Picked SAP hit
-
beads and their respective
fl.
ring intensities

(RI)

5


6

Pick label

RI

Pick label

RI

Pick label

RI

A8
-
021

234

C2
-
186

357

C1
-
060

359

A8
-
026

282

C2
-
298

426

C1
-
082

395

A8
-
048

420

C2
-
293

185

C1
-
092

406

A8
-
091

536

C5
-
027

461

C1
-
144

105

A8
-
109

224

C5
-
054

417

C1
-
148

124

A8
-
124

302

C5
-
279

147

C1
-
159

204

A8
-
209

394

C4
-
036

268

C1
-
167

138

A8
-
282

351

C4
-
236

154

C2
-
048

316

A8
-
357

312

D1
-
027

405

D6
-
392

236

A8
-
415

337

D1
-
028

1451

D6
-
395

477

A8
-
517

335

D1
-
094

515

D6
-
413

506

A8
-
210

331

D2
-
069

594

D8
-
057

441

B8
-
517

1003

D2
-
177

500

D8
-
237

325

C1
-
047

386

D3
-
355

494

D8
-
401

404

C1
-
034

420

D5
-
062

446

E10
-
078

225

C1
-
052

555

D6
-
277

486

E10
-
588

383

Figure S2
2D
-
FIDA anisotropy data for SAP hit compounds

1


2

Single
-
point
2D
-
FIDA anisotropy
solution measurements () were performed with samples derived from
3

all picked hit beads from the Cy5
-
SAP screen. The cleaved compound fractions were diluted 1:500,
4

incubated with SAP in a 384
-
well microtiter plate and measured on the PS02

(assay volume 7

µl,
5

12x12

sec measurement time).
A literature described α
-
peptidic SAP binder, used in HPLC
-
purified
6

form, served as positive control (denoted pos c).

7


8


9

Bead #
TMR
pos c
A8-021
A8-026
A8-048
A8-095
A8-109
A8-124
A8-142
A8-209
A8-282
A8-357
A8-415
A8-517
A8-210
B8-517
C1-12
C1-47
Fluorescence Anisotropy
0.02
0.04
0.06
0.08
0.1
Cpd only
SAP (16 µM)
SAP (16 µM)
Off-bead confirmation SAP - part 1
Bead #
TMR
pos c
C1-34
C1-052
C1-060
C1-082
C1-092
C1-144
C1-148
C1-159
C1-167
C2-048
C2-186
C2-298
C2-293
C5-27
C4-036
C4-236
C5-054
Fluorescence Anisotropy
0.02
0.04
0.06
0.08
0.1
Cpd only
SAP (16 µM)
SAP (16 µM)
Off-bead confirmation SAP - part 2
Bead #
TMR
pos c
C5-279
D1-027
D1-028
D1-094
D2-069
D2-177
D3-355
D5-062
D6-277
D6-392
D6-395
D6-413
D8-057
D8-237
D8-401
E10-078
E10-588
Fluorescence Anisotropy
0.02
0.04
0.06
0.08
0.1
Cpd only
SAP (16 µM)
SAP (16 µM)
Off-bead confirmation SAP - part 3
Bead #
TMR
pos c
A8-021
A8-026
A8-048
A8-095
A8-109
A8-124
A8-142
A8-209
A8-282
A8-357
A8-415
A8-517
A8-210
B8-517
C1-12
C1-47
Fluorescence Anisotropy
0.02
0.04
0.06
0.08
0.1
Cpd only
SAP (16 µM)
SAP (16 µM)
Off-bead confirmation SAP - part 1
Bead #
TMR
pos c
C1-34
C1-052
C1-060
C1-082
C1-092
C1-144
C1-148
C1-159
C1-167
C2-048
C2-186
C2-298
C2-293
C5-27
C4-036
C4-236
C5-054
Fluorescence Anisotropy
0.02
0.04
0.06
0.08
0.1
Cpd only
SAP (16 µM)
SAP (16 µM)
Off-bead confirmation SAP - part 2
Bead #
TMR
pos c
C5-279
D1-027
D1-028
D1-094
D2-069
D2-177
D3-355
D5-062
D6-277
D6-392
D6-395
D6-413
D8-057
D8-237
D8-401
E10-078
E10-588
Fluorescence Anisotropy
0.02
0.04
0.06
0.08
0.1
Cpd only
SAP (16 µM)
SAP (16 µM)
Off-bead confirmation SAP - part 3
Kd determination and quenching analysis

1

In the simplest case of a ligand
-

receptor interaction with a 1:1 stoichiometry the dissociation constant
2

K
d

is defined as

3








RL
L
R
K
d









[ES
-
1]

4

[R], [L]: free receptor and ligand concentrations; [RL]: complex concentration at equilibrium.

5

For a (PS/PS
-
labeled) sample containing the labeled ligand and a percentage of (non
-
binding
6

competent) impurities
f
i
, the mass balances are

7





RL
R
R


0








[ES
-
2]

8





0
0
L
fi
RL
L
L










[ES
-
3]

9

R
0
, L
0
: total receptor and ligand
concentrations.

10

and the fraction of bound ligand
a
, derived from the algebraic solution for the binding equation, is

11

0
0
0
0
0
2
2
)
(
4
)
(
L
L
f
R
L
R
b
b
a
im










[ES
-
4]

12

with

13

)
(
0
0
0
L
f
K
R
L
b
im
d










[ES
-
5]

14

<<why the use of minuses here, compared to the text…>>Any measur
ed average steady state readout
15

parameter
y

(i.e. anisotropy or translational diffusion time) is related to the degree of complex
16

formation by

17

a
Q
a
Q
y








)
1
(
1
)
min)
(max
(min





[ES
-
6]

18

with

19

free
bound
q
q
Q









[ES
-
7]

20

min, max: starting and end values of
the average steady state parameter; Q
: quenching factor;
qbound,
21

qfree: molecular brightnesses of the labeled ligand in the bound and free state.
For

FIDA measurements,
22

molecular brightnesses are obtained for each polarization channel and the molecular bri
ghtness values q
23

can be calculated directly by q = q
||

+ 2x q

.
For

FCS measurements, a possible quenching factor can be
24

derived from the total intensities.

25


26

Equilibrium dissociation constants (
K
d

values) were obtained by performing a nonlinear least squar
e
27

regression fit of the fluctuation data sets
, based on equation [ES
-
4 and ES
-
6
] with the software
28

package
GraFit 5.0.

Equation [ES
-
6] contains two fit parameters: the end
-
value
max
and the
29

dissociation constant
K
d
.

30


31

Decoding and re
-
synthesis of SAP
-
hits

32

Table S4 summarizes the off
-
bead confirmation and decoding results from the Cy5
-
SAP screen. All
33

six samples, for which a K
d

in solution had been obtained, were analyzed by MALDI
-
MS and MS/MS
34

measurements. Peaks in the range of the diagnostic Y
-
fragments (m
/z: 856 to 2

000 Da) were
35

compared to the theoretical fragmentation pattern.

36

With the exception of compound C4
-
236, all samples proved to be decodable and corresponding
1

candidate structures were found. Compound C4
-
236 produced an MS
-
spectrum of very low i
ntensity,
2

which
was
not be interpret
able
.

3

Table S4

Decoding table for Cy5
-
SAP

4


5

List of candidate structures for titrated hits from the Cy5
-
SAP screen. The fragment composition is
6

drawn from the C
-

to the N
-
-
amino acids are denoted in one
-
lette
r code as b3
-
-
amino
7

-
Phosphotyrosine and Ac stands for N
-
acetyl.

8

The MS
-
decoding resulted in a total of 12 candidate structures. Due to the low affinity of compound
9

A8
-
124 for SAP, no follow up experiments wer
e carried out with this compound. All other nine
10

candidate structures were re
-
synthesized on TentaGel beads with the identical chemical setup as used
11

for the screening library.

12

Table S5

List of
re
-
synthesized

hit compounds from Cy5
-
SAP

13


14

List of re
-
synthe
sized structures from the Cy5
-
SAP screen. The peptide sequence is drawn from the
15

C
-

to the N
-
terminus.
ß
³
-
amino acids are denoted in one
-
letter code as b3
-
X,
α
-
amino acids are written
16

in full names, pY means
α
-
Phosphotyrosine and Ac stands for N
-
acetyl.

17

Ta
ble S6

Scores obtained for hit series

vs native SLAM peptide

18

Ligand

Szybki S
ingle Point Energy

(kcal/mol)

Binding Affinity (Kd)

SAP
-
02

-
6.82

17 ± 1
μ
M

SAP
-
03

1.40

31 ± 11 μM

SAP
-
06

-
12.80

13 ± 1 μM

SAP
-
08

0.12

30 ± 3 μM

SAP
-
09

2.96

68 ± 10 μM

SLAM peptide unphosphorylated

-
41.98

300 nM

SLAM peptide phosphorylated

-
49.13

100 nM

Sample #
Name
Branch
Sub
Kd (µM)
Label/spacer
R1
R2
a
R3
pY
R4
Ac
1
D3-355
Blue
Pro
11
PEG-TMR
b3F
b3S
Isoleucine
pY
b3P
Ac
PEG-TMR
b3S
b3F
Isoleucine
pY
b3P
Ac
2
C5-054
Blue
Tyr
8
PEG-TMR
b3V
b3V
Phenylalanine
pY
b3Y
Ac
PEG-TMR
b3V
b3F
Valine
pY
b3Y
Ac
3
D6-392
Blue
Phe
35
PEG-TMR
b3D
b3F
Tryptophan
pY
b3F
Ac
PEG-TMR
b3F
b3D
Tryptophan
pY
b3F
Ac
PEG-TMR
b3V
b3Y
Tryptophan
pY
b3F
Ac
PEG-TMR
b3Y
b3V
Tryptophan
pY
b3F
Ac
4
C5-279
Blue
Tyr
70
PEG-TMR
b3R
b3R
Tyrosine
pY
b3Y
Ac
5
A8-124
Blue
Arg
100
PEG-TMR
b3W
b3Y
Tyrosine
pY
b3R
Ac
PEG-TMR
b3Y
b3Y
Tyrosine
pY
b3R
Ac
PEG-TMR
b3Y
b3W
Tyrosine
pY
b3R
Ac
6
C4-236
Blue
Trp
120
no interpretable MS-spectrum was obtained
Cpd
Name
Label/spacer
R1
R2
a
R3
pY
R4
Ac
NVP-VAY076
SAP-01
PEG-TMR
b3F
b3D
Tryptophan
pY
b3F
Ac
NVP-VAY079
SAP-02
PEG-TMR
b3F
b3S
Isoleucine
pY
b3P
Ac
NVP-VAY080
SAP-03
PEG-TMR
b3V
b3F
Valine
pY
b3Y
Ac
NVP-VAY081
SAP-04
PEG-TMR
b3V
b3Y
Tryptophan
pY
b3F
Ac
NVP-VAY082
SAP-05
PEG-TMR
b3V
b3V
Phenylalanine
pY
b3Y
Ac
NVP-VAY083
SAP-06
PEG-TMR
b3S
b3F
Isoleucine
pY
b3P
Ac
NVP-VAY084
SAP-07
PEG-TMR
b3Y
b3V
Tryptophan
pY
b3F
Ac
NVP-VAY093
SAP-08
PEG-TMR
b3D
b3F
Tryptophan
pY
b3F
Ac
NVP-VAY094
SAP-09
PEG-TMR
b3R
b3R
Tyrosine
pY
b3Y
Ac
Scores for native peptide (unphosphorylated and phosphorylated
) were directly calculated from PDB
1

complexes (1D4T, 1D4W). SAP
-
02, 03, 06, 08, 09 Scores were calculated from top ranked complexes
2

generated within RosettaLigand prioritized by Interface Delta.

3


4


5

Figure S3

Plot of Autoligand results indicating total ener
gy per volume (kcal/molÅ3)
6

versus volume (Å3).
7


8

The legend indicates the number of fill points used to find the optimal fill volume.

9


10

Figure S4 Ligand and receptor
-
based pharmacophore generated with vROCS for
11

SLAM binding pocket of SH2 domain.

12


13

1,2

indicate features that mapped co
-
crystallised waters of PDB entries:1D4T, 1D4W, which
14

were subseqeuently removed from the pharmacophore.

15


16


17

-
0.21
-
0.2
-
0.19
-
0.18
-
0.17
-
0.16
-
0.15
-
0.14
-
0.13
-
0.12
0
500
1000
1500
2000
Total Energy per Volume [kcal/mol
A**3]
Volume [A**3]
Fill_10
Fill_25
Fill_50
Fill_75
Fill_100
Fill_150
Fill_200
Fill_300
Fill_400
Fill_500
Fill_600
Fill_700
Figure
S3. P
lot
of
AutoLigand
results of total energy per volume (kcal/molÅ
3
)
versus
volume (Å
3
).
The legend indicates the number of fill points used to find the optimal fill volume.

1


2

Figure S5 Binding site residues and critical interactions observed between SLAM
3

phosphopeptide and SAP SH2 binding

domain
.

4


5


6

Materials and Methods

7

1.
General methods

8

HPLC
-
instrumentation

9

Analytical HPLC:

10

HPLC analysis was performed on an Agilent 1100 series HPLC system, consisting of a quaternary
11

pump (G1311A), a degasser (G1322A), an FLD detector (G1321A) and a DAD
detector; column:
12

Vydac peptide C8, 4.6 mm x 150 mm, 5 µm particle diameter size.

13

Analyses were performed using a linear gradient of A: H
2
O containing 5% MeCN and 0.1% TFA and
14

B: MeCN containing 5% H
2
O and 0.1% TFA with a flow rate of 0.8 ml/min with FAD
detection at:
15

absorption 555 nm and emission 575 nm. Retention times (t
R
) are denoted in minutes.

16

Standard gradient: from 5% to 45% ACN in 20 min, then to 95% in 5 min.

17

Preparativ HPLC:

18

Purification of mg quantities of peptides and dyes was carried out usi
ng a preparative HPLC system
19

(Agilent 1100 prep
-
HPLC system), equipped with a preparative autosampler (G2260A), preparative
20

scale pumps (G1361A), a fraction collector (G1364B
-
prep) and a multiwavelength UV detector
21

(G13658 MWD with preparative flow cell).
The crude material was dissolved in a DMF/water mixture
22

(~ 2:1) and separated at a flow rate of 20 ml/min on an
Agilent RP
-
C
-
18
column (21.2 x 150 mm, 10
23

µm particle size), using a water/acetonitrile gradient and a detection wavelength of 210 nm.

24

Solvent A
: water, 5% acetonitrile, 0.1% TFA; solvent B: acetonitrile, 5% water, 0.1% TFA. Gradient:
25

5% to 40% B in 20 minutes, 40% B to 95 % B in 5 minutes.

26

Figure
S5.
Binding site residues and critical interactions observed between SLAM
phosphopeptide
and SAP SH2 binding domain.
ESI
-
MS instrumentation

1

The mass spectra were recorded on a Waters

MS
-
70.4000 micromass
spectrometer, fragment ions are
2

given in
m/z
.

3

2. Protein expression and fluorescence labeling of the SH2
-
domain of SAP

4

The recombinant protein was cloned, expressed and purified as N
-
terminal thioredoxin (trx)
-
tag fusion
5

construct. The purification and sol
ubility tag was cleaved by PreScission Protease (Amersham
6

Biosciences) in the last step of the purification, leaving a sequence of 4 residual amino acids GPGS
7

and the 103 amino acids from the SH2
-
domain of SAP:

8

SAP
-
SH2 sequence:

9

GPGSDAVAVYHGKISRETGEKLLLAT
GLDGSYLLRDSESVPGVYCLCVLYHGYIYTYRVSQT
10

ETGSWSAETAPGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEK

11

This sequence contains 107 aa (1 Trp, 9 Tyr, 7 Lys, 2 Cys), amounting to a molecular weight of
12

11854.5 Da. The theoretical extinction coefficient
according to [ES
-
9] therefor
e

=

19035 [l
13

mol
-
1

cm
-
1
].

14

Reducing SDS polyacrylamide gel electrophoresis of the purified protein showed one single band in
15

the expected mass range (
Figure
, lanes 2 and 3).

16

The
recombinant protein

was randomly labeled
on

lysines with the activated dye Cy5
-
N
-
17

hydroxysuccinimidyl ester (Amersham Biosciences, Cy5
-
Mono NHS ester: MW = 754.9)
using a
15
18

mM NaHCO
3

buffer at pH 8.4 and following the
manufacturer`s protocol for Cy5 labeling.

19

The
final
concentration of labeled protein

was determined by recording a UV absorption spectrum on
20

an Agilent 8453 spectrophotometer in the single beam mode, using 50

µl UV
-
silica cells (Ultra
-
Micro
21

Cuvettes, Agilent) and the sample buffer as reference solution. The buffer subtracted spectra were
22

c
orrected for Raleigh scattered light according to Heirwegh, Meuwissen and Lontie.
1

This correction
23

assumes that the contribution of Raleigh scattering to the over
-
4

and
24

t
hat any absorption of the protein solution in the range of 300 to 330 nm is exclusively due to
25

scattering. Then the scattering corrected absorption is given by

26


27

330
95
.
1
280
280
300
2
280
280
E
E
A

or

E
E
A







[E
S
-
8
]

with
A
280
: protein absorption at 280 nm and
E
280
,

E
300

and
E
330
:

measured
28

absorption signal at the respective wavelength.

29


30

The concentration of unlabeled protein was calculated according to the Bouguer
-
Lambert
-
Beer law
31

using the corrected protein absorption at 280 nm and the molar protein extinction coefficient at
280
32

nm,

280
.

280

was calculated by an empirical formula according to Gill and von Hippel
2
:

33


34



Cystines
#

Tyr
#


Trp
#


-1
cm
-1
M
l


125
490
1
500
5
280





[E
S
-
9
]


35

The protein concentration and average labeling ratio of Cy5
-
conjugated proteins was calculated from
36

UV
-
VIS data of the purified Cy5
-
labeled protein and the free dye in the respective buffer. A correction
37

factor for Cy5 dye absorption at 280 nm was determin
ed from the spectrum of the free dye by forming
38

the ratio of the absorption at 280 nm to the absorption maximum at 649
-
650 nm.

39

The protein concentration and the labeling ratio for the labeled protein were calculated from the
40

absorbance value at the absorp
tion maximum of Cy5
-
protein, 649 nm and the signal at 280 nm,
41

corrected for contribution of Cy5 to the absorption at 280 nm. Additionally, straylight correction
42

factors, based on the absorption at 330 nm were included.

43

Calculation basis: extinction coeffi
cient of Cy5: ε = 250

000 [M
-
1
cm
-
1
].

1


2


3

0.0274)

x

E
E
corrected
-
Cy5

A
649
(
280
280



[
ES
-
10
]



0.0434)

x

E
E
corrected
-
Cy5

30
A
649
(
330
3



[
ES
-
11
]



1.929)

x

corrected
Cy

E

corrected
Cy

E
corr.
stray
&

Cy5

A




5
330
(
5
280
280

[
ES
-
12
]


4

Protein

labeling result
:

5

Cy5
-
SAP

6

A total amount of 13.5 nmol SAP
tr

w
as

used for buffer exchange to 0.15 M NaHCO
3

buffer, pH 8.4.
7

The stray
-
light corrected UV
-
quantification before labeling yielded 7.3 nmol protein, which were
8

reacted with Cy5
-
NHS ester (3 equivalents). After a reaction time of 3 hours HPLC
-
analysis indicated
9

a consumption of 56.8% of labeling reagen
t or 1.7 equ. of Cy5
-
NHS. After separation of unreacted
10

dye by size exclusion the stock solution contained 3.16 nmol protein with a concentration of 5.27 µM
11

and a protein/Cy5 labeling stoichiometry of 1/1.85.
For CONA on
-
bead screening using the PS04
12

instr
ument, a minimal concentration of of protein of 1 nM dye equivalents are needed in order to
13

obtain a high enough signal intensity for hit bead detection. Ideally, the concentration should be
14

between 5 and 50 nM dye equivalents. Based in these
specification
s
, the obtained Cy5 labeled SAP
15

protein was found suitable for on
-
bead screening and used at a concentration of 5 nM (9.25 dye
16

equivalents
).


17

Figure
S6

Absorption spectrum of unlabeled SAP

18


19


20


21

-0.05
0.15
0.35
0.55
0.75
0.95
1.15
1.35
220
270
320
370
Wavelength (nm)
Absorbance (AU)

Figure

S7

Absorption spectrum of Cy5
-
SAP

1


2


3

Figure
S8

SDS
-
PAGE
gel of Cy5
-
labeled SAP

4


5

Reducing SDS
-
PAGE gel electrophoresis of unlabeled
SAP
-
SH2 and Cy5
-
SAP
-
SH2. After labeling
6

and purification by size exclusion chromatography the sample shows an identical purity as the
7

unlabeled protein. For electrophoresis
Novex

pre
-
cast gels 4
-
12% Bis
-
Tris (Invitrogen) together with
8

the
Mark12

size standard (Invitrogen) were used. Protein bands were visualized, using the
SILVER
9

Express

staining kit (Invitrogen
)
.

10


11

Confocal Nanoscanning (CONA) hit bead ranking

12

Image analyses were c
arried out by the
BeadEval software (Perkin Elmer, former Evotec)
. It first
13

performs a bead
-
detection, based on predefined thresholds (e.g. bead diameter, ring width, minimal
14

signal increase within a fluorescent ring, etc.). For each detected bead both, ri
ng and area fluorescence
15

intensities are then determined (
Figure S9
).

16


17

-0.05
0.15
0.35
0.55
0.75
0.95
220
320
420
520
620
720
Wavelength (nm)
Absorbance (AU)

21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Mark 12
Mark 12
Mark 12
0.5
µ
g SAP
0.5
µ
g Sost
0.5
µ
g IRAK4
0.5
µ
g MK2
21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Mark 12
Mark 12
Mark 12
0.5
µ
g SAP
0.5
µ
g Sost
0.5
µ
g IRAK4
0.5
µ
g MK2
21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Mark 12
0.5
µ
g SAP
0.5
µ
g Sost
21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Mark 12
0.5
µ
g SAP
0.5
µ
g Sost
21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Mark 12
0.5
µ
g IRAK4
0.5
µ
g MK2
21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Mark 12
0.5
µ
g IRAK4
0.5
µ
g MK2
Mark 12
0.25
µ
g SAP
-
Alexa488
0.25
µ
g SAP
-
Cy5
21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Mark 12
0.25
µ
g SAP
-
Alexa488
0.25
µ
g SAP
-
Cy5
21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Mark 12
0.25
µ
g ERR
α
-
Biotin
0.25
µ
g ERR
γ
-
Biotin
21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Mark 12
0.25
µ
g ERR
α
-
Biotin
0.25
µ
g ERR
γ
-
Biotin
21.5kDa
14.5kDa
6kDa
36.5kDa
31kDa
55.4kDa
66.3kDa
3.5kDa
97.4kDa
Figure
S9

Schematic illustration of the parameters "
ring
intensity" and "
area


1

intensity"

2


3

Schematic i
llustration of the parameters “area

intensity” and “ring intensity” used for evaluation of
4

scanned beads. The
area

intensity is the average fluorescence intensity within the bead interior. The
5

ring intensity i
s the average fluorescence intensity in the outer few µm of the bead area (= ring). The
6

fluorescent ring is defined by a sharp and steep signal change in the outer regions of a bead. A
7

recorded bead scan image corresponds to the optical cross
-
section throu
gh the bead along the
8

scanning laser focus (dotted line).

9


10

For calculating the fluorescence ring intensity, the intensities of the brightest pixels in the
11

neighborhood (in a ring of radius R + dr) of a detected bead are averaged. The
area

intensity of a be
ad
12

is determined by averaging all pixel intensities within a circle of diameter 0.8 x R around the bead
13

origin.

14


To set a threshold for hit
-
bead classification the average level of background in the currently reported
15

on
-
bead screen was determined and the

threshold of hit
-
bead classification was set to four
-
times the
16

average of background signal
.

17

Chemical Syntheses

18

Abbreviations
: Boc (
tert
-
butoxycarbonyl), Boc
2
O (di
-
tert
-
butyl dicarbonate), DCM (dichloromethane),
19

DIPEA (diisopropylethylamine), DMAP (4
-
(
dimethylamino)pyridine), Fmoc (9
-
20

fluorenylmethoxycarbonyl), Fmoc
-
OSu (9
-
fluorenylmethoxycarbonyl
-
N
-
succinimidyl
-
carbonate),
21

HATU (O
-
(7
-
azabenzotriazol
-
1
-
yl) N,N,N,N
-

tetramethyluronium hexafluorophosphate, HMBA (4
-
22

hydroxymethylbenzoic acid), HPLC (high pe
rformance liquid chromatography), h.v. (high vacuum
23

(0.01
-
0.1 Torr), MeIm (1
-
methylimidazole), MSNT (1
-
mesitylene
-
2
-
sulfonyl)
-
3
-
nitro
-
1H
-
1,2,4
-
24

triazole), NMM (N
-
methylmorpholine), NMR (nuclear magnetic resonance), r.t. (room temperature),
25

TFA (trifluoroace
tic acid), TIS (triisopropylsilane), TMR (carboxy
-
tetramethylrhodamin), TNBS
26

(2,4,6
-
trinitrobenzosulfonic acid), UV (ultra violet), eq. (equivalent)

27

Reagents:

The Fmoc
-
protected

a

and


3
-
homoamino acids were purchased from
Fluka, AnandChem

28

GmbH

or

Chem
-
Imp
ex

International. The
HMBA

and
Wang

resins were purchased from
Rapp
29

Polymere
. All other reagents were used as received from

Sigma
-
Aldrich.


30

General procedures
:

31

Anchoring of FmocPra
-
OH on HMBA resin (
GP 1
)
: Esterification of the Fmoc
-
protected
32

propargylglycine amino acid with the HMBA r
esin was performed as follows:

A solution of the Fmoc
33

protected amino acid (5 equiv.) in dry DCM (3 ml/mmol) was treated with MeIm (3.75 equiv.)
34

followed by MSNT (5 eq.) at r.t. After complete dissolution of the M
SNT the solution was added to
35

the pre
-
swollen resin (1 eq.). The suspension was mixed by Ar bubbling for 2h. Subsequently, the
36

resin was filtered, washed with DCM (5 mL, 5 x 1min) and dried under h.v. for 24 h.

37

Solid
-
phase ß
-
peptide synthesis on the HMBA
resin (
GP 2
)
: The Fmoc group of the

first amino acid
38

attached to the resin was removed using 20% piperidine in DMF (3 mL, 4 x 10 min). After filtration,
39

the resin was washed with DMF (2 mL, 4 x 1 min). Solid phase peptide synthesis was then continued
40

for e
ach resin part by sequential incorporation of
N
-
Fmoc
-
protected amino acids. For each coupling
41

step, the resin was treated with a solution of
N
-
Fmoc
-
protected amino acid (4 eq.), HATU (3.9 eq.),
42

DIPEA (10 eq.) in DMF (1 mL) and mixed for 45
-
60 min. Monitori
ng of the coupling reaction was
43

Ring Intensity

Scanning Laser Focus

~

130

µ

m









µ

m

oi湧 f湴敮獩ty

p捡c湩湧 䱡獥r 䙯捵c

B敡d

oi湧 f湴敮獩ty

p捡c湩湧 䱡獥r 䙯捵c

~

ㄳ〠

µ

m









µ

m

oi湧 f湴敮獩ty

p捡c湩湧 䱡獥r 䙯捵c

~

ㄳ〠

µ

m









µ

m

oi湧 f湴敮獩ty

p捡c湩湧 䱡獥r 䙯捵c

B敡d

oi湧 f湴敮獩ty

p捡c湩湧 䱡獥r
䙯捵c

~

ㄳ〠

µ

m









µ

m

Area Intnesity

performed with TNBS test. In case of a positive TNBS test (indicating incomplete coupling), the
1

suspension was filtered off and the peptide resin was treated again with the same amino acid and
2

coupling reagents
.

3


4

N
-
terminal

acetylation (
GP3
)

5

After the last amino acid, the resin was Fmoc deprotected as above and the N
-
terminus was acetylated
6

by treating the resin with a
solution of Ac
2
O (10 equ.) and DIPEA (20 equ.) in DMF. After a reaction
7

time of 1 h at r.t., the resin was
drained and again treated with the same amount of fresh reaction
8

mixture for another 1 h. Finally, the resin was washed extensively with DMF (4x3 min), DCM (4x3
9

min) and dried under h.v. for 12 h.

10


11

Side chain deprotection (
GP4)
:

12

For side chain
deprotection, each resin sample was treated with a solution containing TFA/H
2
O/Tis
13

(95:2.5:2.5) at r.t.. After a reaction time of 2 h, water (10% v/v) was added to the reaction mixture and
14

the reaction allowed to continue for further 16 h. The resin was th
en washed with DCM (4x2 min),
15

DIPEA (10% v/v) in DCM (4x2 min), DCM (4x2 min) and dried in vacuo to yield the final resin
-
16

bound peptide, which was stored at 4° C.

17


18

On
-
bead labeling and cleavage of batch synthesized peptides (
GP 5
)
:
Several hundred beads ar
e
19

suspended in a mixture of 40 µL
t
BuOH and 35 µL H
2
O and treated with 20 µL of a freshly prepared
20

solution of CuSO
4

(3 mg) and ascorbic acid (1.5 mg) in 1 mL H
2
O. 30 µL of a solution of TMR
-
azide
21

5

in MeOH

(approx. 5 mm) are then added and the reaction mixture is left to stand at r.t. for 24 h. The
22

solution is then removed and the beads are washed 5
-
times with 100 µL MeOH and 5
-
times with 50
23

µL H
2
O. Finally, the labeled compound is cleaved from the bead by
treatment of the resin with 120 µL
24

of a cold (0° C) solution of NaOH (1n)
-
dioxane 1:3 for 15 min, followed by addition of 80 µL of HCl
25

(1n). The solution is then purified by RP
-
HPLC.

26


27

Library Synthesis, g
eneral handling

28

All library syntheses were carried o
ut by manual SPPS, using filter equipped, discard able syringes (2
29

ml, 5 ml and 10 ml from Becton Dickinson, NJ, USA) and fritted filter funnels for peptide synthesis
30

(15 and 30 ml, Sigma
-
Aldrich) as reaction compartments and vacuum extraction devices (Mal
ickrodt
31

Baker, NJ, USA) for filtering and washing of resin samples within the syringes.

32

For the phosphopeptide library 1.5 g of TentaGel S HMB resin (loading: 0.26 mmol/g, custom sieved
33

to achieve a size distribution of 90 to 100 µm diameter, Rapp polymers
, Tübingen, Germany) were
34

used. Before library synthesis each resin batch was checked for autofluorescence by placing 1 mg of
35

swollen resin (PBS, 0.005% Tween20) in a 96
-
well microtiter plate and recording a CONA scan
36

images.

37

After initial resin loading wi
th Fmoc
-
propargylglycine according to

GP1
, the spacer unit, N
-
(Fmoc
-
8
-
38

amino
-
3,6
-
dioxa
-
octyl)
-
succinamic acid, was coupled to the resin and the samples were subsequently
39

subjected to Fmoc deprotection. For processing of combinatorial positions the deprotect
ed resin
40

samples were then split into sub
-
samples (one for each bulding block to be coupled), using Pasteur
41

pipettes (Marienfeld laboratory glassware, Germany). To these N
-
terminally deprotected resin samples
42

the respective amino acid building blocks were
coupled according to
GP2
. Then the sub
-
samples were
43

recombined in larger syringes and thoroughly mixed and washed. The mixed samples were again
44

subjected to Fmoc
-
deprotection and re
-
split for the next coupling step. This split
-
mix
-
split steps were
45

repeated

according to the

46

library synthesis scheme (
Fig. S5
) until the last combinatorial coupling step, after which the resins
1

were kept separated. (as sublibraries). Finally, the resin of each sub
-
library was N
-
acetylated (GP3)
2

and subjected to side chain deprot
ection (
GP4
).

3


4

Single
-
bead PS/PS
-
labeling and library quality control


5

For quality control, a random selection of beads were manually picked under a standard microscope
6

using a syringe needle and placed
-

one bead per vial
-

into autosampler

glass vials with a conical inlet
7

(8002
-
SC
-
H, Glastechnik Gräfenroda, Germany). The PS/PS
-
labeling of the single beads cleavage of
8

labeled material from solid support was carried out as described in the materials and method section in
9

the main
-
text. The cl
eaved and dried material was dissolved in 20 µl of 20% acetonitrile (in water) and
10

15 µl were subjected to HPLC
-
analysis and compound quantification.

11


12

Resynthesized hit compounds from the phosphopeptide library pYL1

13

The MALDI
-
MS analysis and decoding yield
ed a series of hit
-
structures for each screen. These
14

candidate structures were resynthesized on the TentaGel S HMB resin (loading: 0.26 mmol/g), using
15

20 mg resin per peptide (theoretical yield: 5.2 µmol peptide) and following the synthesis procedures
16

desc
ribed above. In brief: After resin loading with Fmoc
-
propargylglycine according to
GP1
, the N
-
17

(Fmoc
-
8
-
amino
-
3,6
-
dioxa
-
octyl)
-
succinamic acid (ADO) spacer unit was coupled to the resin. After
18

coupling of the spacer, the synthesis of the actual peptide seque
nces proceeded according to to the
19

standard methods of building block coupling and Fmoc
-
deprotection
GP2
, N
-
Acetylation
GP3
and
20

side chain deprotection
GP4
.

21


22

Synthesis of unlabeled peptides:

23

Unlabeled hit compounds were synthesized without the spacer unit and the propargyl
-
glycine

24

labeling site, contained in the original hit. The synthesis was performed on a polystyrene PHB Wang
25

resin (loading 1.15 mmol/g, Rapp polymere, Germany), typically us
ing 50


100 mg of dry resin per
26

peptide (theoretical yield: 58
-

115 µmol peptide). Loading of the first amino acid and peptide
27

synthesis was performed according to
GP1.
Final side chain deprotection and resin cleavage was
28

effected by treating the beads w
ith a solution of TFA/H2O/Tis (95:2.5:2.5). After a reaction time of 2
29

hours, the resin was filtered off and washed 3 times with a small amount of cleavage solution. Water
30

(10% v/v) was added to the combined filtrates and the solution agitated for another
16 hours. The
31

solvents were then removed in vacuo and the crude peptide was precipitated by addition of cold ether,
32

followed by repeated washing steps (3 times) with another aliquot of cold ether. The crude peptides
33

were then purified by preparative RP
-
HPL
C.

34


35

Detailed list of resynthesized peptides in PS/PS
-
labeled form

36

The compound identity was confirmed by ESI
-
MS, purity was assessed by RP
-
HPLC (fluorescence
37

detection
λ
ex/
λ
em 555/575nm. The purity values correspond to the crude PS/PS
-
labeled

peptides, as
38

this reflects best the actual purity on
-
bead. For solution testing, all peptides were HPLC
-
purified to
39

yield purities > 90%.

40


41

Detailed list of re
-
synthesized hit
-
compounds in PS/PS
-
labeled form

1

The compound identity was confirmed by ESI
-
MS, purity was assessed by RP
-
HPLC
2

(fluorescence detection
ex/
em 555/575nm). The purity values correspond to the crude
3

PS/PS
-
labeled peptides, as this reflects best the actual purity on
-
bead.

4

SAP
-
01

5

Sequence:

6

A
c
-

³桐桥
-
呹爨PO
3
H
2
)
-
Trp
-

³h獰
-

³桐桥
-
7

NH(CH
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
Pra(TMR)
-
OH

8

RP
-
HPLC
t
R

(%purity): 17.12 min (74%).
MS

(m/z): [C
90
H
105
N
15
O
22
P+3H]
3+

calc. 593.90,
9

found: 594.2; [C
90
H
105
N
15
O
22
P+2H]
2+

calc. 890.36, found: 890.2.

10


11

SAP
-
02

12

Sequence:

13

Ac
-

³桐牯
-
呹爨PO
3
H
2
)
-
Ile
-

³桓敲
-

³桐桥
-
N(C
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
14

Pra(TMR)
-
OH

15

RP
-
HPLC
t
R

(%purity): 16.29 min (71%).
MS

(m/z): [C
80
H
104
N
14
O
21
P+3H]
3+

calc. 543.57,
16

found: 543.8; [C
80
H
104
N
14
O
21
P+2H]
2+

calc. 814.86, found: 814.6.

17


18

SAP
-
03

19

Sequence:

20

Ac
-

³桔yr
-
呹爨PO
3
H
2
)
-
Val
-

³桐桥
-

³桖慬
-
21

NH(CH
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
Pra(TMR)
-
OH

22

RP
-
HPLC
t
R

(%purity): 17.45 min (82%).
MS

(m/z): [C
85
H
108
N
14
O
21
P+3H]
3+

calc. 564.92,
23

found: 565.0; [C
85
H
108
N
14
O
21
P+2H]
2+

calc. 846.88, found: 846.9.

24


25

SAP
-
04

26

Sequence:

27

Ac
-

³桐桥
-
呹爨PO
3
H
2
)
-
Trp
-

³桔yr
-

³桖慬
-
28

NH(CH
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
Pra(TMR)
-
OH

29

RP
-
HPLC
t
R

(%purity): 20.29 min (46%).
MS

(m/z): [C
91
H
109
N
15
O
21
P+3H]
3+

calc. 593.929,
30

found: 594.1; [C
91
H
109
N
15
O
21
P+2H]
2+

calc. 890.38, found: 890.3.

31


32

SAP
-
05

33

Sequence:

34

Ac
-

³桔yr
-
呹爨PO
3
H
2
)
-
Phe
-

³桖慬
-

³桖慬
-
35

NH(CH
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
Pra(TMR)
-
OH

36

RP
-
HPLC
t
R

(%purity): 19.48 min (76%).
MS

(m/z): [C
85
H
108
N
14
O
21
P+3H]
3+

calc. 564.92,
37

found: 565.1; [C
85
H
108
N
14
O
21
P+2H]
2+

calc. 846.88, found: 846.6.

38


39

SAP
-
06

40

Sequence:

41

Ac
-

³桐牯
-
呹爨PO
3
H
2
)
-
Ile
-

³桐桥
-

³桓敲
-
N(C
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
1

Pra(TMR)
-
OH

2

RP
-
HPLC
t
R

(%purity): 16.75 min (79%).
MS

(m/z): [C
80
H
104
N
14
O
21
P+3H]
3+

calc. 543.57,
3

found: 544.0; [C
80
H
104
N
14
O
21
P+2H]
2+

calc 814.86, found: 814.7.

4


5

SAP
-
07

6

Sequence:

7

Ac
-

³桐桥
-
呹爨PO
3
H
2
)
-
Trp
-

³桖慬
-

³桔yr
-
8

NH(CH
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
Pra(TMR)
-
OH

9

RP
-
HPLC
t
R

(%purity): 19.61 min (65%).
MS

(m/z): [C
91
H
109
N
15
O
21
P+3H]
3+

calc. 593.92,
10

found: 594.1; [C
91
H
109
N
15
O
21
P+2H]
2+

calc 890.38, found: 890.2.

11


12

SAP
-
08

13

Sequence:

14

Ac
-

³桐桥
-
呹爨PO
3
H
2
)
-
Trp
-

³桐桥
-

³h獰
-
15

NH(CH
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
Pra(TMR)
-
OH

16

RP
-
HPLC
t
R

(%purity): 19.37 min (77%).
MS

(m/z): [C
90
H
105
N
15
O
22
P+3H]
3+

calc. 593.91,
17

found: 594.0.

18


19

SAP
-
09

20

Sequence:

21

Ac
-

³桐桥
-
呹爨PO
3
H
2
)
-
Tyr
-

³h牧
-

³h牧
-
22

NH(CH
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
Pra(TMR)
-
OH

23

RP
-
HPLC
t
R

(%purity): 14.35 min (64%).
MS

(m/z): [C
87
H
114
N
20
O
22
P+3H]
3+

calc. 608.27,
24

found: 608.6; [C
87
H
114
N
20
O
22
P+2H]
2+

calc 911.91, found: 911.7.

25


26

1.1.1

Synthesis of
a
-
灨潳p桯灥灴楤is:


27


28

Ac
-
Thr
-
Ile
-
Tyr(PO
3
H
2
)
-
Ala
-
Gln
-
Ile
-
N(CH
2
CH
2
O)
2
CH
2
CH
2
CONHCH
2
CH
2
CO
-
Pra(TMR)
-
OH


29

RP
-
HPLC

t
R

(%purity): 15.07 min (61%).
MS

(m/z): [C
78
H
108
N
16
O
23
P+2H]
2+

calc. 556.91, found:
30

556.7

31


32

1.1.2

Synthesis of unlabeled hit
-
compounds for competition experiments

33

The compound identity was confirmed by ESI
-
MS, purity was assessed by RP
-
HPLC
34

(absorption detection 220 nm). The purity values correspond to the purfied peptides, which
35

were used for the competition experiments.

36


37

SAP
-
06

38

Sequence:

39

Ac
-

³桐牯
-
呹爨PO
3
H
2
)
-
Ile
-

³桐桥
-

³桓敲
-
O

40

RP
-
HPLC
t
R

(%purity):
14.30

min (>95%).
MS

(m/z): [C
37
H
51
N
5
O
12
P+K]
+

calc. 827.29, found:
1

827.6.

2


3


4


5


6


7


8


9


10

Reference List

11


12

1.

Heirwegh, K. P. M.; Meuwissen, J. A. T. P.; Lontie, R., Selective absorption and scattering of
13

light by solutions of macromolecules and by particulate suspensions.
Journal of Biochemical and
14

Biophysical Methods
1987,

14, (6), 303
-
322.

15

2.

Gill, S. C.; von H
ippel, P. H., Calculation of protein extinction coefficients from amino acid
16

sequence data 1.
Anal.Biochem.
1989,

182, (2), 319
-
326.

17


18


19