Angewandte
Chemie
DOI: 10.1002/anie.200805420
Pyrrolysine
A Pyrrolysine Analogue for Protein Click Chemistry**
Tomasz Fekner, Xin Li, Marianne M. Lee, and Michael K. Chan*
[
7]
The discovery of pyrrolysine (1, Figure 1), the 22nd genet-
parent pyrrolysine analogue 2. We subsequently postulated
that this framework could be used as a starting point for the
design of other functionalizable pyrrolysine analogues.
Herein, we report on the synthesis of the pyrrolysine
analogue 3, bearing a terminal alkyne functionality, that not
only reads through the UAG codon in E. coli overexpressing
the Methanosarcina mazei PylS and PylT, but also allows for
site-specific modification of the resulting protein through the
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC)
[
1,2]
ically encoded amino acid,
and its subsequent incorpora-
[
8]
reaction. The taggable pyrrolysine analogue 3 was prepared
from the known tetrahydrofuran 4 by a seven-step sequence
Scheme 1). Thus, desilylation of 4 with TBAF/AcOH in
[
9]
Figure 1. Pyrrolysine (1) and its THF analogues 2 and 3.
(
[
3]
tion into recombinant proteins in E. coli have laid the
foundation for the future development of novel biotechnol-
ogies and tactics in protein research. In principle, incorpo-
ration of the genes encoding the pyrrolysine tRNA synthase
(
PylS) and its cognate tRNA (PylT) could enable both
[
4,5]
[6]
bacteria
and eukaryotes to use the UAG codon for the
production of proteins containing pyrrolysine (1) and its
analogues. Herein, we describe the synthesis of a new
pyrrolysine congener that can be incorporated into recombi-
nant protein and selectively modified with azide-containing
dyes by copper(I) click chemistry. We demonstrate the
usefulness of this technology for monitoring conformational
changes of calmodulin (CaM) by Fꢀrster resonance energy
transfer (FRET) measurements.
Scheme 1. Synthesis of taggable pyrrolysine analogue 3: a) TBAF,
AcOH, THF, room temperature, 14 h, 76% or AcCl (25 mol%),
MeOH, room temperature, 10 h, 89%; b) H IO , CrO (4 mol%),
5
6
3
MeCN, H O, 08C, 90 min; c) Boc-Lys-OtBu, BOP, NMM, CH Cl , room
2
2
2
To harness the unique UAG-codon–pyrrolysine system
for biochemical applications, we decided to search for
functionalized pyrrolysine surrogates that would be both
more stable and amenable for further post-translational
modification (i.e., tagging). Our primary focus centered on
derivatives of the THF-containing amino acid 2 because of
the steric and electronic similarity between the THF ring and
the 3,4-dihydro-2H-pyrrole ring present in 1. Recently, in
collaboration with the Krzycki group, we demonstrated that
Methanosarcina barkeri PylS could charge PylT with the
temperature, 48 h, 68% from 5; d) OsO (2 mol%), NMO, Me CO,
4
2
H O, room temperature, 48 h, 93%; e) NaIO , THF, H O, room
2
4
2
temperature, 30 min, 94%; f) AcC(N
)P(O)(OMe)
(10), K CO ,
2 3
2
2
MeOH, room temperature, 3 h, 70%; g) TFA, room temperature, 1 h,
ca. 100%. TBDPS: tert-butyldiphenylsilyl, TBAF: tetrabutylammonium
fluoride, BOP: (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate, NMM: N-methylmorpholine, NMO: N-methyl-
morpholine N-oxide; Boc: tert-butoxycarbonyl; Ac: acetyl; TFA: tri-
fluoroacetic acid.
[
10]
[11]
THF or AcCl in MeOH gave alcohol 5 in 76% or 89%
yield, respectively. Its subsequent oxidation with H IO in the
[
+]
[+]
[
*] Dr. T. Fekner, X. Li, M. M. Lee, Prof. M. K. Chan
Departments of Chemistry and Biochemistry, The Ohio State
University
5
6
[12]
presence of a catalytic amount of CrO3
provided acid 6 that,
in turn, was coupled (BOP/NMM) with Boc-Lys-OtBu to give
amide 7 in 68% yield over the two steps. NMO/OsO4-
mediated dihydroxylation of 7 furnished an approximately
484 West 12th Avenue, Columbus, OH 43210 (USA)
Fax: (+1)614-292-6773
E-mail: chan@chemistry.ohio-state.edu
Homepage: http://www.chemistry.ohio-state.edu/~chan/
] These authors contributed equally to this work.
1
:1 mixture of the corresponding diastereomeric diols 8
93%) that were oxidatively cleaved with NaIO4 to give
aldehyde 9 in 94% yield. Subsequent Ohira–Bestmann
(
+
[
[
**] This research was supported by a grant from the US National
Institutes of Health (GM061796) and an American Heart Associ-
ation Great Rivers Affiliate Predoctoral Fellowship (0815449D) to
X.L. We thank Liwen Zhang (CCIC, OSU) for protein mass-
spectrometry analysis.
[13,14]
[15]
alkynylation
with diazoketophosphonate 10
in the
presence of K CO led to the protected target 11 (70%). Its
2
3
final treatment with neat TFA cleanly freed the desired
pyrrolysine analogue 3.
To test if 3 could be recognized by a pyrrolysyl-tRNA
synthetase and incorporated into recombinant protein in vivo,
Angew. Chem. Int. Ed. 2009, 48, 1633 –1635
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1633