Transcript Utilization of a “Biological” Safety-Catch Linker for Direct

Utilization of a “Biological” Safety-Catch Linker
for Direct Screening Assays
David Oraina and Mark Bradleyb,*
a) Current adress : Novartis Pharma AG, Lichtstrasse 35, WSJ-507.703, CH-4056 Basel, Switzerland,
E-mail : [email protected]
b) University of Southampton, Department of chemistry, Highfield, Southampton SO17 1BJ, UK
www.soton.ac.uk/~bradley
Introduction
Over the last decade, solid phase synthesis and combinatorial chemistry have undergone major evolution. In the early
years, the process of split and mix revolutionized the concept of library synthesis, however, this strategy is less widely used, due in
large measure to the fact that compound mixtures appeared to be problematic in terms of screening, while the alternative approach,
single bead screening, suffers from many practical and synthesis problems not the least of these are the vast numbers of screens
necessary when screening large split and mix libraries, and compound quantity. Thus to apply split and mix methodology effectively,
efficient screening processes on single bead must be developed which allow the direct identification of the active bead and thus the
active compound. Where release is required, it would be ideal if the cleavage could take place directly in a biologically compatible
environment.
I. Linker synthesis1
For this purpose, a specific “biologically” compatible safety-catch linker has been tailored to release amine based
compounds for direct screening assays. The linker was synthesized in solution on a core based on proline and glutamic acid
derivatives. Amine derivatives were pre-loaded on the linker before its attachment on Tentagel resin. The release mechanism based
on the internal formation of a diketopiperazine was activated by reaction of the resin with TFA. Release was achieved in a phosphate
buffer at pH 8 after activation of the safety-catch linker with TFA. Primary, secondary, aromatic amines and amidines led to high
purity released material; only benzyl amines led to lower purity.
O
O
O
N
O
N
O
O
a)
+
O
H2N
Boc
O
O
b), c), d)
O
N
N H
Boc
CO2H
OH
O
O
N
N H
Boc
O
NO2
O
O
O
O
O
e), f)
O
O
H
R
N
R'
+
NH TG
+
O
HN
O
N
O
h), i)
O
NH TG
O
O
N
N H
Boc
OH
g)
O
O
R
N
O
O
R'
N
N H
Boc
O
O
R
N
O
R'
O
Conditions : a) THF/H2O (1/1), Et3N 1.2eq, RT, 48h ; b) 4-HO-C6H4-CHO, DCC, DMAP, DCM, 18h, RT; c) NaBH4, Green
bromocresol, 20% HCl/EtOH; d) 4-NO2-C6H4-OCOCl, Py, DCM, 15 min, RT; e) R'RNH (1.1 eq), DIPEA (1 or 2 eq), DMF, 2-4h,
RT; f) Pd(PPh3)4 (5% mol), pyrrolidine (1.5 eq), DCM, 10 min, RT; g) Tentagel-S-NH2, DIC, HOBt, DCM/DMF, O/N; h)
TFA/DCM (1/1) 45 min; i) Phosphate Buffer, pH 8, 0.1M, 4 hours, 1 mL per 10 mg of resin.
II. Synthesis of TR inhibitors2
This new linker was used in the synthesis of known inhibitors of Trypanothione Reductase to demonstrate its potential to
be used in a direct screening assay. This enzyme is key to the cell’s defence against oxidative stress, and is also present in the causative
agents of the African sleeping sickness, leishmaniasis (skin lesions) and Chaga’s disease which is responsible of thousands of deaths each
year on the north American continent.3 From a spermidine core loaded on the linker, three different inhibitors were synthesised by solid
phase peptide synthesis. After activation of the safety-catch linker, cleavage in phosphate buffer afforded relatively pure material.
H
N
O
mAU
TG
Phe-Phe
1750
1500
O
O
Fmoc-HN
Fmoc-HN
N
O
1250
1000
N
H
O
750
BocN
500
250
O
0
H
N
O
SPPS
6
TG
O
BocHN-AA2-AA1-HN
N
O
8
9
10
11
12
13
14
min
mAU
Phg-Trp
2500
2000
O
BocHN-AA2-AA1-HN
7
O
N
H
1500
BocN
O
1000
500
0
a) AA1: Phe AA2: Phe
b) AA1: Phg AA2: Trp
c) AA1: Arg AA2: Trp
1) TFA/DCM (1/1), 45 min. RT
6
7
8
9
10
11
12
13
14
min
mAU
2) Phosphate Buffer 100mM, pH 8.00
Arg-Trp
2000
1500
1000
H2N-AA2-AA1-HN
H2N-AA2-AA1-HN
500
NH
0
6
7
8
9
10
11
12
13
14
min
Kinetics showed that the cleavage rate was dependent on the hydrophilicity of the lateral chain of the amino acids used. More interestingly,
multiple releases could be achieved with this linker and so lead to the possibility of direct identification of the active compound without any
other encoding techniques required.
1
1
P
h
e
P
h
e
0. 8
0. 8
0. 6
0. 6
A
r
g
T
r
p
0. 4
P
h
g
T
r
p
0. 4
P
h
g
T
r
p
A
r
g
T
r
p
0
0
RelativHPLCAbsornc
P
h
e
P
h
e
0. 2
RelativHPLCAbsornc
0. 2
10
2
0
0
3
0
00 1 2 3 4
R
u
n
T
i
m
e
(
m
i
n
)
R
e
l
e
a
s
e
o
f
i
n
h
i
b
i
t
o
r
s
w
i
t
h
r
u
n
R
e
l
e
a
s
e
o
f
i
n
h
i
b
i
t
o
r
s
w
i
t
h
t
i
m
e
III Direct enzymatic screening
To determine the ability of this system to allow direct screening, a small library of 9 compounds was synthesized and
cleaved individually. The crude solutions of inhibitors were tested directly on the enzymatic system. The inhibition rates were followed
by consumption of NADPH at 340 nm or by generation of 2-nitrobenzothiol from the Ellman reagent at 415 nm. A good correlation was
obtained between the relative inhibition rates obtained in this study and previous works.4
NO2
NO2
S S
TSST
415 nm
NADPH
340 nm
Enzyme
NADP +
2 TSH
SH
NO2
100
Arg
AA1
75
50
Asp
100
Arg
AA1
75
50
Asp
25
Lys
25
Lys
0
0
Phe
Se r
Trp
AA2
% Inhibition recorded at 415 nm
Phe
Ser
Trp
AA2
% Inhibition recorded at 340 nm
Direct bead screening was then investigated on high-loading Tentagel beads. From Tentagel beads with a loading of
800 pmol/bead, a dendrimer was added to reach a level of loading around 2 nmol/bead. On these high-loading beads, the linkerspermidine scaffold was loaded and then the synthesis of the most active inhibitor from the previous screening was carried out. After
activation of safety-catch linker with TFA, the beads were washed carefully before being spatially addressed in wells.
H2N
TG
O
H2N
1) (BocNH-CH2CH2CH2)3CH-NCO,
DIPEA, DCM
N
H
H2N
2) TFA/DCM
TG
2 nmol/bead
H2N
800 pmol/bead
N
H
1) Linker + spermidine scaffold
2) SPPS
O
BocHN-Phe-Arg(Pmc)-HN
N
Linker
HN
N
H
BocHN-Phe-Arg(Pmc)-HN
3
TFA activation
Direct bead screening
N
H
TG
100
80
340 nm
60
40
415 nm
20
Different buffers were studied and best results were
obtained when cleavage was carried out in a sodium
tetraborate buffer 100 mM pH 8.0. Inhibition rates were
measured at 340 nm and 415 nm, and the first results
showed that 35% and 21% of inhibition were obtained
on a single bead respectively.
0
3
2
1
Be ads
% Inhibition for a direct bead screening
Conclusion
A new “biological” compatible safety-catch linker has been synthesized allowing a wide range of amines to be anchored.
This linker has then been used in the synthesis of Trypanothione Reductase inhibitors. Direct screening assays with crude cleavage solution
were then carried out and the inhibition rates were similar to previous works. On high-loading beads, direct one bead screening was realized
and relatively good inhibition was achieved. Other dendrimers are being studied to reach higher bead capacity to allow a more efficient one
bead screening library.
Acknowledgements
We thank the BBSRC (51/BO9512) for funding.
References
1
2
3
4
Orain, D.; Bradley, M. Mol. Div. 2000, 5(1), 25.
Orain, D.; Bradley, M. Tetrahedron Lett. 2001, 42, 515.
Schirmer, R. H.; Muller, J. G.; Krauth-Siegel, R. L. Angew. Chem. Int. Ed. Engl. 1995, 34, 141.
Smith, H. R.; Bradley, M. J. Comb. Chem. 1999, 1, 326.