International Journal of Peptide Research and Therapeutics
solid material, Fmoc-Gly-Pro-6-amino-d-luciferin free car-
boxylic acid, started to precipitate. At pH 6.3, this material
started to dissolve, and at pH 7.40, it dissolved completely.
Here the Fmoc-Gly-Pro-6-amino-d-luciferin formed Na-salt,
which dissolved under these conditions.
reaction to 320 pmol/min/reaction. Assay buffer for POP/
PREP and Endoproteinase Pro-C contained 25 mmol
tris(hydroxymethyl)aminomethane HCl-salt (Tris∙HCl) pH
7.4, 250 mmol NaCl, 2.5 mmol 1,4-dithiothreitol (DTT)
and the assay buffer for FAP contained 50 mmol Tris∙HCl
pH 7.4, 1 M NaCl, 1 mg/mL BSA. The N-Fmoc-GP-aLuc
substrate was applied in 1 µmol to 100 µmol in 25 µL final
reaction volume in a black plastic microtiter plate. The
effect of protease inhibition (Complete protease inhibitor
cocktail) was prepared by dissolving one tablet in 2 mL
POP/PREP buffer and used in 2.5-fold dilution in each
reaction with 10 µmol N-Fmoc-GP-aLuc and 32 pmol/min/
reaction protease activity. After 2 h incubation at 37 °C
25 µL Luminescence Detection Reagent was added to each
well. Luminescence was recorded within 5 min. The blank
wells contained each component except proteases. Pre-
sented values are blank-subtracted.
After another 20 min’ stirring at room temperature, the
organic solvent was evaporated. From the remaining aque-
ous solution, a pale yellow solid material, Fmoc-Gly-Pro-
6-amino-d-luciferin Na-salt precipitated partly. This aque-
ous mixture was extracted with 3×15 mL ethyl acetate, in
order to get rid of possible impurities. The combined organic
layers were extracted with saturated NaCl solution. Hav-
ing dropped the resulting solution on a mixture of ice and
cc HCl, a fine yellow precipitate, Fmoc-Gly-Pro-6-amino-
d-luciferin free carboxylic acid, formed. It was allowed
to settle for 10 min, filtered and washed with 2 × 5 mL
water, then air-dried to constant weight, which was 5.115 g
(7.80 mmol), yield corresponding to the crude product:
1
78%. H NMR (500 MHz, [D6]DMSO) δ 10.39 (s, 1H),
8.59 (t, J = 15.85 Hz, 1H), 8.09 (d, J = 8.98 Hz, 1H), 7.88
(d, J = 7.43 Hz, 2H), 7.71 (d, J = 7.48 Hz, 2H), 7.66–7.60
(m, 1H), 7.48 (t, J = 5.65 Hz, 1H), 7.39 (q, J1 = 7.60 Hz,
J2 = 15.29 Hz, 2H), 7.30 (q, J1 = 6.78 Hz, J2 = 13.76 Hz,
2H), 5.43 (t, J = 8.98 Hz, 1H), 4.47 (dd, J1 = 2.92 Hz,
J2=5.21 Hz, 1H), 4.29–4.25 (m, 1H), 4.21 (q, J1=6.68 Hz,
J2=14.87 Hz, 1H), 3.95–3.67 (m, 4H), 3.65–3.48 (m, 4H),
2.20–2.11 (m, 1H), 2.06–1.99 (m, 1H), 1.97–1.88 (m, 2H)
(Fig. S14). 13C NMR (125 MHz, [D6]DMSO) δ 171.15,
171.05, 167.43, 164.43, 159.04, 156.55, 148.58, 143.86,
140.70, 138.38, 136.28, 127.61, 127.08, 125.27, 124.20,
120.10, 119.64, 111.52, 78.11, 65.70, 60.47, 46.62, 45.92,
42.72, 34.78, 29.28, 24.52 (Figure S15). m/z [M+H]+ calcd
for C33H29N5O6S2 655.74 found 656.0 (Figure S16). RP-
HPLC: 70–100% B in 15 min, tR= 12.608 min. TLC: tolu-
ene/EtOH 50:30 saturated with water, Rf: 0.44.
Statistics
Statistical significance was calculated by unpaired t-test
(two-tailed, homoscedastic) between untreated and inhibi-
tor containing samples.
Conclusion
We have developed an improved route for the synthesis of
N-peptid-6-amino-d-luciferin conjugates. The method is
more reliable than the standard practice as the preparation
of one of the building blocks and two operations have been
improved, which has led to better yields and significantly
faster production time. The produced N-Fmoc-GP-aLuc was
successfully used to measure FAP and POP/PREP enzyme
activity in vitro.
Purification of Crude Fmoc-Gly-Pro-6-Amino-d-Luciferin (3)
This optimized method provides a practical and scalable
way for the preparation of other N-peptide-6-amino-d-lucif-
erin conjugates as well.
160 mg crude peptide (45% desired material content, 72 mg)
was dissolved in 1 mL N,N-dimethylformamide (DMF), then
filtered, using a 0.45∝ m nylon filter. Gradient elution was
used, 40–70% eluent B in 60 min at a 4 mL min−1 flow rate
with detection at 220 nm. Pure fractions were collected and
lyophilized to give a pale yellow material, the weight of
which was 23 mg (0.035 mmol), yield corresponding to the
isolated pure product: 32%.
Author Contributions Conceptualization: László G. Puskás, Investiga-
tion: Anita K. Kovács, Péter Hegyes, Gábor J. Szebeni, NMR analysis:
Krisztián Bogár, Writing - original draft, review & editing: Anita K.
Kovács, Supervision: Gábor K. Tóth.
Funding This work was partly supported by the following Grants:
GINOP-2.3.2-15-2016-00030 and GINOP-2.3.2-15-2016-00001 from
the National Research, Development and Innovation Office (NKFI),
Hungary. Gábor J. Szebeni was supported by János Bolyai Research
Scholarship of the Hungarian Academy of Sciences (BO/00139/17/8).
Fmoc-Gly-Pro-aLuc Assay
The following proteases were used in the assay: recom-
binant human POP/PREP, recombinant human FAP
and Endoproteinase Pro-C at equivalent protease activ-
ity in tenfold serial dilution starting from 32 fmol/min/
Compliance with Ethical Standards
Conflict of interest The authors declare no conflict of interest.
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