A new strategy for the synthesis of bisaminoacylated tRNAs

Article abstract of DOI:10.1021/ol201993c

Tandemly activated tRNAs participate effectively in protein synthesis and exhibit superior chemical and biochemical stability compared to the more commonly used singly aminoacylated tRNAs. While several bisaminoacylated tRNAs have been prepared via the T4 RNA ligase-mediated condensation of bisaminoacylated pdCpAs and abbreviated tRNA transcripts (tRNA-C_OH), the bisaminoacylated pdCpAs are difficult to prepare when using bulky amino acids. Described herein is a new strategy for preparing bisaminoacylated tRNAs, applicable even for bulky amino acids.

Full text of DOI:10.1021/ol201993c

ORGANIC
LETTERS
2011
Vol. 13, No. 18
4906–4909
A New Strategy for the Synthesis of
Bisaminoacylated tRNAs
Ryan C. Nangreave, Larisa M. Dedkova, Shengxi Chen, and Sidney M. Hecht*
Center for BioEnergetics, The Biodesign Institute, and Department of Chemistry and
Biochemistry, Arizona State University, Tempe, Arizona 85827, United States
sidney.hecht@asu.edu
Received July 23, 2011
ABSTRACT
Tandemly activated tRNAs participate effectively in protein synthesis and exhibit superior chemical and biochemical stability compared to the
more commonly used singly aminoacylated tRNAs. While several bisaminoacylated tRNAs have been prepared via the T4 RNA ligase-mediated
condensation of bisaminoacylated pdCpAs and abbreviated tRNA transcripts (tRNA-COH), the bisaminoacylated pdCpAs are difficult to prepare
when using bulky amino acids. Described herein is a new strategy for preparing bisaminoacylated tRNAs, applicable even for bulky amino acids.
Chemically misacylated tRNAs have facilitated the in-
troduction of non-natural amino acids into proteins at
predetermined sites.1ꢀ6 This has enabled the detailed
investigation of thebiochemicaland biophysical properties
of numerous proteins.7ꢀ10 While monoaminoacylated
tRNAs are ordinarily used in protein synthesizing systems,
Lavrik and co-workers11 have described a phenylalanyl-
tRNA synthetase from Thermus thermophilus that can
incorporate more thanone molecule of phenylalanine onto
tRNAPhe, producing bis- (20,30-O-phenylalanyl)-tRNAs.
Our laboratory has shown that such bisaminoacylated
tRNAs can be prepared in vitro and employed for cell free
protein synthesis; these species have the advantages of
improved chemical and biochemical stabilities, as well as
their ability to participate in two cycles of peptide elon-
gation.12 Under protein synthesizing conditions limiting
(1) Heckler, T. G.; Zama, Y.; Naka, T.; Hecht, S. M. J. Biol. Chem.
1983, 258, 4492.
(2) Heckler, T. G.; Chang, L. H.; Zama, Y.; Naka, T.; Hecht, S. M.
Tetrahedron 1984, 40, 87.
(3) Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G.
Science 1989, 244, 182.
(4) Heckler, T. G.; Chang, L. H.; Zama, Y.; Naka, T.; Chorghade,
M. S.; Hecht, S. M. Biochemistry 1984, 23, 1468.
(5) Baldini, G.; Martoglio, B.; Schachenmann, A.; Zugliani, C.;
Brunner, J. Biochemistry 1988, 27, 7951.
(6) Hecht, S. M. In Protein Engineering; RajBhandary, U. L., Koehrer,
C., Eds.; Springer: New York, 2008; pp 251ꢀ266.
(7) Ellman, J. A.; Mendel, D.; Schultz, P. G. Science 1992, 255, 197.
(8) Karginov, V. A.; Mamaev, S. V.; An, H.; Van Cleve, M. D.;
Hecht, S. M.; Komatsoulis, G. A.; Abelson, J. N. J. Am. Chem. Soc.
1997, 119, 8166.
(11) (a) Stepanov, V. G.; Moor, N. A.; Ankilova, V. N.; Lavrik, O. I.
FEBS Lett. 1992, 311, 192. (b) Stepanov, V. G.; Moor, N. A.; Ankilova,
V. N.; Sukhanova, M. V.; Lavrik, O. I. Biochim. Biophys. Acta 1998,
1386, 1.
(12) (a) Wang, B.; Zhou, J.; Lodder, M.; Anderson, R. D.; Hecht,
S. M. J. Biol. Chem. 2006, 281, 13865. (b) Duca, M.; Maloney, D. J.;
Lodder, M.; Wang, B.; Hecht, S. M. Bioorg. Med. Chem. 2007, 15, 4629.
(c) Maloney, D. J.; Ghanem, N.; Zhou, J.; Hecht, S. M. Org. Biomol.
Chem. 2007, 5, 3135. (d) Duca, M.; Chen, S.; Hecht, S. M. Methods 2008,
44, 87. (e) Duca, M.; Chen, S.; Hecht, S. M. Org. Biomol. Chem. 2008, 6,
3292. (f) Duca, M.; Trindle, C.; Hecht, S. M. J. Am. Chem. Soc. 2011,
133, 11368.
(9) England, P. M.; Lester, H. A.; Dougherty, D. A. Biochemistry
1999, 38, 14409.
(10) Yakovleva, L.; Chen, S.; Hecht, S. M.; Shuman, S. J. Biol. Chem.
2008, 283, 16093.
r
10.1021/ol201993c
Published on Web 08/23/2011
2011 American Chemical Society

Figure 1. Preparation of mono and bisaminoacylated tRNACUAs by chemical aminoacylation via pdCpA.
for activated tRNA, twice as much full-length protein is
produced when employing bisaminoacylated tRNAs.12a
The synthesis of misacylated tRNAs is outlined in
Figure 1. The N-protected amino acid is activated as the
cyanomethyl ester.13 The amine must be protected such
that deprotection can be achieved under conditions com-
patible with maintenance of the labile aminoacyl moiety
that results. The nitroveratryloxycarbonyl (NVOC) group
can be removed by simple UV irradiation,13,14 while the
4-pentenoyl group can be utilized as an alternative protect-
ing group as it is removable with aqueous iodine.15
Acylation of pdCpA (1) with the activated amino acid
affords the monoaminoacylated pdCpA (2); when the
activation employs a pentenoyl-protected amino acid that
is not bulky, some of the bisaminoacylated pdCpA (3) is
alsoobtained.12 TheacylatedpdCpA intermediates2 and 3
are subsequently used in the T4 RNA ligase-catalyzed
condensation reaction with an abbreviated suppressor
tRNA (tRNA-COH) to afford the desired misacylated
tRNAs (Figure 1). However, the use of bulky amino acids
generally results in poor yields of 3 (Table 1). Thus, the
pdCpA derivatives containing two phenylalanine or bi-
phenylalanine moieties were accessible in modest yield, but
only the monoaminoacylated pdCpAs could be obtained
when dimethoxyphenylalanine or either of two regio-
isomers of naphthylalanine were employed. While bisami-
noacylated tRNAs are potentially of great utility for the
elaboration of modified proteins, the difficulty in obtain-
ing bisaminoacylated pdCpAs (3) containing bulky amino
acids has limited their use to date.
T4 RNA ligase is an ATP-dependent enzyme that catal-
yzes the formation of phosphodiester bonds from RNA
donor and acceptor substrates.16 The ligation is initiated
by adenylation of the 50-phosphate moiety of the RNA
donor to form a phosphoroanhydride intermediate (A50-
pp50N...). Finally the adenylated donor is condensed with
(13) Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc.
1991, 113, 2722.
(14) (a) Patchornik, A.; Amit, B.; Woodward, R. B. J. Am. Chem.
Soc. 1970, 92, 6333. (b) Amit, B.; Sehavi, V.; Patchornik, A. J. Org.
Chem. 1974, 39, 192.
(15) (a) Lodder, M.; Golovine, S.; Hecht, S. M. J. Org. Chem. 1997,
62, 778. (b) Lodder, M.; Golovine, S.; Laikhter, A. L.; Karginov, V. A.;
Hecht, S. M. J. Org. Chem. 1998, 63, 794.
(16) (a) Walker, G. C.; Uhlenbeck, O. C.; Bedows, E.; Gumport, R. I.
Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 122. (b) England, T. E.;
Uhlenbeck, O. C. Nature 1978, 275, 560. (c) Barrio, J. R.; Barrio,
M. C. G.; Leonard, N. J.; England, T. E.; Uhlenbeck, O. C. Biochemistry
1978, 17, 2077.
Org. Lett., Vol. 13, No. 18, 2011
4907

same monoaminoacylated tRNAs (I) and bisaminoacylated
tRNAs (II), accessible by ligation of tRNA-COH þ amino-
acylated pdCpAs. The suppressor tRNACUA-CCOH was ob-
tained by in vitro RNA transcription from a DNA template
that had been linearized with restriction enzyme FokI. The
Table 1. Mono and Bisaminoacylation of pdCpA and AMP
suppressor tRNA was structurally related to yeast tRNAPhe 8
.
Table 2. Mono and Bisacylated AppAs Prepared from Ami-
noacylated Adenosine 50-Monophosphates
the 30 terminal OH group of the RNA acceptor (e.g.,
tRNA-COH) to form a new 30f50 phosphodiester link-
age.17 Hecht and co-workers first described a “chemical
aminoacylation” procedure in which a chemically synthe-
sized aminoacylated AppA, identical with the putative
intermediate in the T4 RNA ligase reaction, was utilized
as a donor substrate for the enzyme, thus producing a
misacylated tRNA (5 f 5a f I; Figure 2).18
It seemed possible that the bisaminoacylation of 50-
AMP with bulky amino acids might proceed more readily
than of pdCpA and that the resulting bisaminoacylated
AMP could still be converted to the respective AppA
derivative with reasonable efficiency. As documented in
Table 1, treatment of 50-AMP with each of five bulky
amino acids (N-pentenoyl protected derivatives, activated
as the cyanomethyl esters) afforded both mono and bis-
aminoacylated derivatives, with the latter isolated in yields
of 54ꢀ77%. All of the 50-AMP derivatives were purified by
C18 reversed phase HPLC using a gradient of 0 f 65%
acetonitrile in 50mM NH4OAc, pH 4.5. Treatment of each
monoaminoacylated AMP (5) and bisaminoacylated
AMP (6) with imidazolium AMP then afforded the re-
spective aminoacylated AppA derivative in yields ranging
from 81 to 94%, after purification by HPLC (Table 2).
Interestingly, the coupling efficiencies did not differ dra-
matically for the mono and bisaminoacylated AMPs.
These AppA derivatives were then ligated to tRNA-CCOH
via the agency of T4 RNA ligase (Figure 2), affording the
The ability of the mono- and bisaminoacylated tRNAs
prepared via aminoacylated AppA intermediates to parti-
cipate in protein synthesis was investigated in vitro in an
E. coli S30 coupled transcription-translation system pro-
grammed with DHFR mRNA having a UAG codon at
the position corresponding to Val10. Each of the five
N-pentenoyl protected monoaminoacylated tRNAs (I)
and the five N-pentenoyl protected bisaminoacylated
tRNAs (II) prepared via aminoacylated AppA intermedi-
ates (Table 2) was deprotected by treatment with aqueous
iodine as described previously15 before introduction into
the protein synthesis reaction. The synthesis of full length
DHFRs in the presence of the monoaminoacylated and
bisaminoacylated tRNAs is illustrated in Figures 3 and 4,
respectively. Both the mono- and bisaminoacylated-tRNAs
were shown to suppress the UAG codon effectively, result-
ing in the production of DHFR with reasonable efficiencies.
Some variations were observed while employing mono- vs
bisaminoacylated-tRNAs for incorporating the same amino
(17) (a) Ohtsuka, E.; Nishikawa, S.; Sugiura, M.; Ikehara, M. Nucleic
Acids Res. 1976, 3, 1613. (b) Bryant, F. R.; Benkovic, S. J. J. Am. Chem.
Soc. 1981, 103, 697. (c) Hoffmann, P. U.; McLaughlin, L. W. Nucleic
Acids Res. 1987, 15, 5289.
(18) Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. J. Biol.
Chem. 1978, 253, 4517.
4908
Org. Lett., Vol. 13, No. 18, 2011

Figure 2. Preparation of mono and bisaminoacylated tRNACUAs by chemical aminoacylation via AMP.
Figure 4. In vitro synthesis of DHFR utilizing bisaminoacylated
Figure 3. In vitro synthesis of DHFR utilizing monoacylated
tRNACUAs to suppress a UAG codon at position 10 of DHFR
mRNA. Lane 1, wild-type mRNA; Lane 2, no tRNACUA; lane 3,
L-phenylalanyl-tRNACUA (prepared from pdCpA); lane 4, L-
phenylalanyl-tRNACUA; lane 5, 3,4-dimethoxy-L-phenylalanyl-
tRNACUA; lane 6, L-1-naphthylalanyl-tRNACUA; lane 7, L-2-naph-
tRNACUAs to suppress a UAG codon at position 10 of DHFR
mRNA. Lane 1, wild-type mRNA; lane 2, no tRNACUA; lane 3,
L-phenylalanyl-tRNACUA (prepared from pdCpA); lane 4, bis-
L-phenylalanyl-tRNACUA; lane 5, bis-L-3,4-dimethoxyphenyl-
alanyl-tRNACUA; lane 6, bis-L-1-naphthylalanyl-tRNACUA
lane 7, bis-L-2-naphthylalanyl-tRNACUA; lane 8, bis-L-4,40-
biphenylalanyl-tRNACUA
;
thylalanyl-tRNACUA; lane 8, L-4,40-biphenylalanyl-tRNACUA
.
.
acid. In the case of L-1-naphthylalanine, L-2-naphthylalanine
and L-biphenylalanine, the use of the bisaminoacyl-tRNAs
resulted in approximately 2ꢀ3 times more DHFR than
when the respective monoaminoacylated tRNAs were em-
ployed. In contrast, the yields of DHFRs were somewhat
greater when monophenylalanyl-tRNACUA was employed,
regardless of whether the phenylalanyl-tRNAs were pre-
pared by the method outlined in Figure 1 or 2. This
presumably reflects differences in the relative stabilities of
the individual mono and bisaminoacylated tRNAs in the
in vitro synthesizing system,12 and plausibly also differences
in the facility of utilization of individual bisaminoacylated
tRNAs by the ribosome.
In summary, by the modification of a procedure for the
misacylation of tRNAs, first described by Hecht et al.,18 it
is now possible to prepare bisaminoacylated tRNAs in
good yield even where bulky amino acids are involved
Supporting Information Available. Experimental de-
tails for the preparation and characterization of all
aminoacylated nucleotides. This material is available free
of charge via the Internet at http://pubs.acs.org.
Org. Lett., Vol. 13, No. 18, 2011
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