Rational design, synthesis and biological evaluation of modular fluorogenic substrates with high affinity and selectivity for PTP1B

Article abstract of DOI:10.1002/cbic.201400033

Protein-tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type-2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B

Full text of DOI:10.1002/cbic.201400033

Supporting Information
ꢀ
Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2014
Rational Design, Synthesis and Biological Evaluation of
Modular Fluorogenic Substrates with High Affinity and
Selectivity for PTP1B
[
a]
Silvano Sanchini,* Francesca Perruccio, and Grazia Piizzi
cbic_201400033_sm_miscellaneous_information.pdf

I. Synthetic procedures and compound characterization.
O
a
)
OEt
8
a
Br
b)
O
OtBu
8b
Scheme S1. Reagents and conditions: a) ethyl isobutyrate, LDA, THF, –78 °C to rt, overnight; b) t-butyl
isobutyrate, LDA, THF, –78 °C to rt, overnight.
i
General procedure for the synthesis of compounds 8a and 8b: to a stirred solution of freshly prepared
LDA (1.1 equiv.) in THF (0.05 M), the opportune isobutyrate (1.0 equiv., ethyl isobutyrate for 8a; t-butyl
isobutyrate for 8b) was added at -78 °C and under argon atmosphere. After 2 hours 5-bromo-pent-1-ene
(1.0 equiv) was added, the mixture was allowed to warm to room temperature and stirred overnight. The
reaction was then quenched with HCl 2N and extracted with Et O. Organic layers were combined,
2
washed with brine, dried over MgSO , filtered and concentrated under reduced pressure. The residue was
4
filtered over a short pad o silica gel by eluting with heptane and used as is for the next step.
Ethyl 2,2-dimethylhept-6-enoate (8a): the spectroscopic data recorded are in agreement with those
1
reported in the reference. H NMR (400 MHz, CDCl ) δ 5.76 (ddt, J = 16.9, 10.2, 6.6 Hz, 1H), 4.98 (dd, J
3
=
17.1, 1.4 Hz, 1H), 4.93 (d, J = 10.2 Hz, 1H), 4.10 (qd, J = 7.2, 2.4 Hz, 2H), 2.05 – 1.97 (m, 2H), 1.53 –
13
1
.48 (m, 2H), 1.37 – 1.25 (m, 2H), 1.23 (t, J = 7.1 Hz, 4H), 1.14 (s, 1H) ppm; C NMR (101 MHz,
CDCl ) δ 178.0, 138.7, 114.6, 60.3, 42.2, 40.3, 34.2, 25.3, 24.4, 14.4 ppm.
3
1
tert-Butyl 2,2-dimethylhept-6-enoate (8b): H NMR (400 MHz, CDCl ) δ 5.78 (ddt, J = 17.0, 10.2, 6.7
3
Hz, 1H), 4.98 (dd, J = 17.1, 1.4 Hz, 1H), 4.93 (d, J = 10.2 Hz, 1H), 2.02 (q, J = 7.0 Hz, 2H), 1.51 – 1.44
1
3
(
m, 2H), 1.42 (s, 9H), 1.37 – 1.27 (m, 2H), 1.10 (s, 6H) ppm; C NMR (101 MHz, CDCl ) δ 177.4, 138.8,
3
1
14.6, 79.7, 42.7, 40.3, 34.3, 28.1, 25.3, 24.4 ppm.

O
a
)
I
OEt
12
Scheme S2. Reagents and conditions: a) ethyl isobutyrate, LDA, THF, -78 °C to rt, overnight.
Synthesis of ethyl 2,2-dimethylhept-6-ynoate (12): to a stirred solution of freshly prepared LDA (1.1
equiv.) in THF (0.05 M), ethyl isobutyrate (1.1 equiv.) was added at -78 °C and under argon atmosphere.
After 2 hours 5-iodo-pent-1-yne (1.0 equiv) was added, the mixture was allowed to warm to room
temperature and stirred overnight. The reaction was then quenched with HCl 2N and extracted with Et O.
2
Organic layers were combined, washed with brine, dried over MgSO , filtered and concentrated under
4
reduced pressure. The residue was filtered over a short pad o silica gel by eluting with heptane and used
1
as is for the next step. H NMR (400 MHz, CDCl ) δ 4.12 (q, J = 7.1 Hz, 2H), 2.17 (td, J = 7.0, 2.6 Hz,
3
2
6
H), 1.95 (t, J = 2.6 Hz, 1H), 1.66 – 1.59 (m, 2H), 1.52 – 1.42 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H), 1.18 (s,
1
3
H) ppm; C NMR (101 MHz, CDCl ) δ 177.8, 84.3, 68.5, 60.4, 42.1, 39.9, 25.2, 24.2, 19.0, 14.3 ppm.
3
ABBREVIATIONS USED
TMSBr, bromotrimethylsilane; DCM, dicloromethane; SiO , silica gel; MOMCl, chloromethyl methyl
2
ether;
DIPEA,
N,N-diisopropylethylamine;
DMAP,
4-dimethylaminopyridine;
DIBAL-H,
diisobutylaluminium hydride; TEA, triethylamine; LDA, lithium diisopropylamide; LiTMP, lithium
tetramethylpiperidine.

II. Stability Studies
Figure S1. Spontaneous degradation of compound 4f. Compound 4f was serially diluted (10 µM, 5 µM,
2
.5 µM, 1.25 µM, 0.625 µM, 0.313 µM, 0.156 µM, 0.078 µM, 0.039 µM, 0.020 µM, 0.010 µM and 0.005
µM) in cold reaction buffer (50 mM HEPES, pH 7.1, 50 mM KCl, 1mM EDTA, 3 mM DTT, 0.05% NP-
0) for a total volume of 25 µl. Fluorescence production (mAU, y-axis) was monitored (λ = 342 nm, λ
4
ex
em
=
441 nm) every minute for one hour. The assay was run in duplicate.
O
LF
O
LF
F
O
LF
F
O
O
O
O
HO
P
O
F
O
HO
HO
HO
F
O
O
P
O
P
O
HO
O
HO
O
P
+ CO2 + LF
OH
OH
-
O O
H
HO
A
B
[ ]
C
[ ]
D
[ ]
[
]
Figure S2. Proposed decomposition mechanism for compounds 4h and 4k. [A] When unmasked, the free
phenol attacks the phosphate group to generate an unstable pentacyclic intermediate [B] which re-opens
and triggers the 1,6-elimination [C] with subsequent decomposition of the scaffolds [D].

III. Computational Studies
Table S1 – Graphical summary of the BLAST output
a
b
c
Protein
TCPTP
PTP-β
SHP1
Pdb ID
1L8K
2I4E
Score
Identities
68 %
39%
Positives
84 %
58%
449.5
197.2
186.2
182.6
115.2
ND
305X
1LAR
1YGR
ND
38 %
35 %
25 %
ND
59 %
51 %
45%
LAR
CD45
VHR*
PP2A*
ND
ND
ND
ND
ND
a) numerical value describing the overall quality of an alignment: higher numbers correspond to a higher
similarity; b) parameter indicating amino acid residues that are identical in the query (PTP1B) and in the
hit when the two are optimally aligned; c) parameter indicating residues that are similar to each other.
BLAST output highlights a very high degree of similarity between PPT1B and TCPTP. A minor degree
of homology was found in PTP-β, SHP1, LAR and CD45 whereas PP2A and VHR scored below the
threshold value. The obtained data was in complete agreement with the biological results on the potential
secondary targets for the synthesized substrates.

Arg24
Gln262
Asp181
Cys215
Arg221
Compound 4d docked into the active site of 2BGE

Arg24
Gln262
Asp181
Arg221
Cys215
Compound 4e docked into the active site of 2BGE

Arg24
Gln262
Asp181
Arg221
Cys215
Compound 4g docked into the active site of 2BGD

Arg24
Gln262
Asp181
Arg221
Cys215
Compound 4i docked into the active site of 2BGD

Arg24
Gln262
Asp181
Cys215
Arg221
Compound 4j docked into the active site of 2BGE

V. NMR spectra
1
H NMR
(400 MHz, DMSO)
4
a
13
C NMR
101 MHz, DMSO)
(
4
a

1
H NMR
400 MHz, DMSO)
(
4
b
13
C NMR
101 MHz, DMSO)
(
4
b

1
H NMR
400 MHz, DMSO)
(
4
c
13
C NMR
101 MHz, DMSO)
(
4
c

1
H NMR
400 MHz, DMSO)
(
4
d
13
C NMR
101 MHz, DMSO)
(
4
d

1
H NMR
400 MHz, DMSO)
(
4
e
13
C NMR
101 MHz, DMSO)
(
4
e

1
H NMR
400 MHz, DMSO)
(
4
f
13
C NMR
101 MHz, DMSO)
(
4
f

1
H NMR
400 MHz, DMSO)
(
4
g
13
C NMR
101 MHz, DMSO)
(
4
g

1
H NMR
600 MHz, DMSO)
(
4
h
31
P NMR
162 MHz, DMSO)
(
4
h

1
H NMR
400 MHz, DMSO)
(
4
i
13
C NMR
101 MHz, DMSO)
(
4
i

1
H NMR
400 MHz, DMSO)
(
4
j
13
C NMR
101 MHz, DMSO)
(
4
j

1
H NMR
600 MHz, DMSO)
(
4
k
31
P NMR
162 MHz, DMSO)
(
4
k

1
H NMR
400 MHz, DMSO)
(
3
a
13
C NMR
101 MHz, DMSO)
(
3a

1
H NMR
400 MHz, DMSO)
(
3
b
13
C NMR
101 MHz, DMSO)
(
3
b

1
H NMR
400 MHz, DMSO)
(
3
c
13
C NMR
101 MHz, DMSO)
(
3
c

1
H NMR
600 MHz, DMSO)
(
3
d
13
C NMR
151 MHz, DMSO)
(
3
d

1
H NMR
400 MHz, Acetone)
(
3
e
13
C NMR
101 MHz, Acetone)
(
3
e

1
H NMR
400 MHz, DMSO)
(
3
f
13
C NMR
101 MHz, DMSO)
(
3
f

1
H NMR
400 MHz, Acetone)
(
3
g
13
C NMR
101 MHz, Acetone)
(
3
g

1
H NMR
400 MHz, Acetonitrile)
(
3
h
13
C NMR
101 MHz, Acetonitrile)
(
3
h

1
H NMR
400 MHz, DMSO)
(
3
i
13
C NMR
101 MHz, DMSO)
(
3
i