A novel synthesis of peptide-6-amino-D-luciferin conjugates for detection of peptidase activities
A. Kovács
a,b,*, P. Hegyes
b, B. Ózsvári
b, L. G. Puskás
b,c, G. K. Tóth
aa
University of Szeged, Department of Medical Chemistry, Szeged, Hungary
b
AVIDIN Biotechnology Ltd., Szeged, Hungary
c
AVICOR Ltd., Szeged, Hungary
*kovacs.anita@med.u-szeged.hu
min
10 15 20 25 30
mAU
0 200 400 600 800 1000
VWD1 A, Wavelength=220 nm (A:\PL000151.D)
Biological application
In vivo bioluminescence imaging has become a cornerstone technology for preclinical molecular imaging. This imaging method is based on light-emitting enzymes, luciferases, which require specific substrates for light production. When linked to a specific biological process in an animal model of human biology or disease, the enzyme-substrate interactions become biological indicators that can be studied noninvasively in living animals [1].
Aminoluciferin (aLuc) is american firefly (Photinus pyralis) luciferin with its 6-position hydroxyl group substitued with an amino group. This modification allows aLuc to form amide bond with a peptide, while at the same time retaining the transport and bioluminescent properties of luciferin, resulting in a molecule called peptide-aminoluciferin. Many, particially protected peptide-aminoluciferin (e.g. Z-Asp-Glu-Val-Asp-aLuc, Z-Leu-Glu-His-Asp-aLuc, Suc-Leu-Leu-Val-Tyr-aLuc) are good substrates for bioluminescence assays, for example in the detection of protease actvity. Proteases represent important pharmaceutical targets because of their involvement in numerous disease processes [2]. The above mentioned conjugates generally offer significant advantages, such as increased sensitivity, ease of use, and high throughput screening capacity. Luciferase-based assays are typically 10- to 100-fold more sensitive than comparable fluorescent assays (Rhodamine 110, AMC and AFC) [3].
Light emission of the peptide-aminoluciferin Synthetic problems
The synthesis of different type peptide-6-amino-D-luciferin conjugates and their precursors have been published [2] and some of them are commercially available. However, because of their high price the in vivo application of these conjugates is limited. To solve this problem we successfully worked out a new, easier and more convenient and economical method for the preparing these derivatives starting from 2-chloro-benzothiazole. Moreover this products have excellent purity (>99%) and adequate yield (82-93%).
ka_110906 #52-64RT:1.28-1.58AV:13NL:6.42E3 T:+ c ESI m s [ 149.98-1499.97]
500 600 700 800 900 1000 1100 1200 1300 1400
m /z 0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Relative Abundance
870.3
871.1
889.4
532.8 670.5
514.4 653.9 896.8
1425.6
464.5 536.2
1321.81387.8
1058.0 1171.2
772.9
699.5 991.2
587.0 733.6 845.3 909.2 1132.7 1232.81305.31325.1
1008.5 1231.8
833.2
S N
N S
NH COOH
Peptide
S N
N S H2N COOH
luciferase ATP Mg2+
O2
* protease *
Peptide +
S N
N S N O
H2
+ AMP + CO 2 + PP i + Light
m in
0 5 10 15 20 25 30
m AU
0 500 1000 1500 2000
VWD1 A, Wavelength=220 nm (A:\PL000291.D)
References
[1] Biochemistry, 2006, 45, 11103-11112.; [2] J. of Biomol. Sc., 2005, 10(2), 137-148.
[3] BioTechniques, 2011, 51(2), 105-110.
Synthesis
1H-NMR spectra of 2-chloro-6-nitro-benzothiazole
RP-HPLC-chromatogram of the purified Fmoc-Asp(OtBu)-6-amino-2-cyano-benzothiazole
tR = 25.50 min
RP-HPLC-chromatogram of the crude Z-Asp-Glu-Val-Asp-6-amino-luciferin tR = 19.20 min
ESI-mass spectra of the Z-Asp-Glu-Val-Asp-6-amino-luciferin 870.3 = [M + H]+
Acknowledgement
We are grateful for financial support from the grants of GOP-1.3.1-11/B-2011-0002,
GOP-1.3.1-11/C-2011-0027, GOP-1.1.1-11- 2012-0060 and TÁMOP 4.2.2/B-10/1-2012-0012.
Biological tests Caspase-3/7 activity (24 h)
Luminescence
control treated with toxin
In vivo:
Immunosupressed SCID mice injected with A375luc melanoma cells + antitumour drug + Z-DEVD-aLuc
→the measured luminescence indicate the increased caspase activity
In vitro:
Primary rat retinal cell culture + toxin (thapsigargin)
→measurement of caspase activity N
S Cl
N S
Cl O2N
N S
Cl N
H2
cc. H2SO4 / KNO3 0-10°C
aq. NH4Cl / Fe-powder EtOAc / reflux
KCN / KI / DMF / 120° C / 10 h
KCN / 18-crown- 6 / DMF / 120 ° C / 8 h
N S
CN N
H2
Fmoc-AA / TCFH / DCM / rt / overnight Fmoc-AA / TFFH / DCM / rt / overnight
N S
CN N
H
O NH Fmoc
R
*
D-Cys / MeOH rt / 25 min
N N S
H
O NH Fmoc
R
N S
COOH
*
* N
N S H
O NH Fmoc
R
N S
O O resin
* 2-Cl-Trt resin *
N N S
H
O NH
R
N S
O O resin
peptide Fmoc
*
*
DIPEA / DMF / rt / 3 h
20% piperidine / DMF Fmoc-AA / DCC / HOBt / DMF
20% piperidine / DMF Z-AA / DCC / HOBt / DMF
N N S
H
O NH
R
N S
O O
peptide Z
resin
*
*
N N S
H
O NH
R
N S
O OH
peptide Z
*
* 50% TFA / 40% AcN / 10% H2O
45 min / rt Fmoc-AA / isobutyl-chloroformate
