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Puromycin analogue synthesis by aminolysin-A
In the case of the single donor/single acceptor system (Fig. 4A),
the reaction mixture (2.5 mL) was composed of 0.1 M phosphate
buffer (pH 8.0), 4.0 mM ester derivatives of amino acids as acyl
donors, 35 mM PAN as an acyl acceptor, 1.0 mg aminolysin-A, and
10% (v/v) DMSO. Enzyme reaction, quenching of the reaction,
and LC–UV–MS and LC–MS/MS analysis were conducted in a
similar manner as described above. In this experiment, UV moni-
toring at 270 nm and MS/MS monitoring with the MRM method
were adopted for the detection and quantification of the produced
X-PANs as a substitute for authentic PM. In this analysis, the cal-
ibration curve for authentic PM was used for the quantification of
each product (Section 7-2, ESI†). HRESIMS data describing the
isolated puromycin analogues were as follows. L-Phe-PAN, calcd
for C21H28N7O4 [M+H]+: 442.2197, found: 442.2190. D-Phe-PAN,
calcd for C21H28N7O4 [M+H]+: 442.2197, found: 442.2193. L-Trp-
PAN, calcd for C23H29N8O4 [M+H]+: 481.2306, found: 481.2305.
L-Tyr-PAN, calcd for C21H28N7O5 [M+H]+: 458.2146, found:
458.2139. L-Leu-PAN, calcd for C18H30N7O4 [M+H]+: 408.2354,
found: 408.2350. L-Ile-PAN, calcd for C18H30N7O4 [M+H]+:
408.2354, found: 408.2348. L-Val-PAN, calcd for C17H28N7O4
[M+H]+: 394.2197, found: 394.2201. L-Met-PAN, calcd for
C17H28N7O4S [M+H]+: 426.1918, found: 429.1910. In the case
of the multi-donor/single acceptor system (Fig.5), the reaction
mixture (1.2 mL) was composed of 0.1 M phosphate buffer
(pH 8.0), a mixture of L-Phe-OMe/D-Phe-OBn/L-Leu-OBn/L-Ile-
OBn/L-Trp-OBn/L-Tyr-OBn/L-Met-OMe/Val-OBn (0.85 mM
each), 50 mM PAN, 100 mg of aminolysin-A, and 10% (v/v)
DMSO. The enzyme reaction was conducted at 50 ◦C for 6 h,
followed by the addition of 300 ml of 10% (v/v) HCOOH
for quenching. After centrifugation, 10 mL of the supernatant
was subjected to LC–MS/MS analysis. The procedures for the
quantification of each product by the MRM method are described
above.
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Acknowledgements
Unnatural amino acids were kindly provided by Nagase and Co.,
Ltd. This research was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science
and a Technology and a Grant-in-Aid for JSPS Fellows. The
authors are grateful to the MS Laboratory of the Faculty of
Agriculture, Okayama University for assistance and the HRMS
experiments.
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