Synthesis of bisaminoacylated pdCpAs and tandemly activated transfer RNAs

Article abstract of DOI:10.1016/j.bmc.2007.03.088

Described herein is the preparation of new bisacylated tRNAs and their participation in protein synthesis. It has been reported that Thermus thermophilus phenylalanyl-tRNA synthetase can introduce two phenylalanine moieties onto the 3′-terminal adenosine of its cognate tRNA. It is also possible to prepare bisactivated tRNAs in vitro; these participate in protein synthesis [Wang, B.; Zhou, J.; Lodder, M.; Anderson, R. D.; Hecht, S. M. J. Biol. Chem. 2006, 281, 13865]. Presently, the chemical strategy used for the synthesis of the key intermediate bisacylated pdCpAs is described. Bis-S-alanyl- and bis-S-methionyl-pdCpAs were prepared initially. Further, S-threonine, S-allo-threonine, S-homoserine, and (S)-(+)-2-amino-3-hydroxy-3-methylbutyric acid were coupled with the dinucleotide to define preparative methods applicable to more complex amino acids bearing additional functionality in the form of an OH group.

Full text of DOI:10.1016/j.bmc.2007.03.088

Bioorganic & Medicinal Chemistry 15 (2007) 4629–4642
Synthesis of bisaminoacylated pdCpAs and tandemly
activated transfer RNAs
Maria Duca, David J. Maloney, Michiel Lodder, Bixun Wang and Sidney M. Hecht*
Departments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22904, USA
Received 7 March 2007; revised 28 March 2007; accepted 29 March 2007
Available online 2 April 2007
Abstract—Described herein is the preparation of new bisacylated tRNAs and their participation in protein synthesis. It has been
reported that Thermus thermophilus phenylalanyl-tRNA synthetase can introduce two phenylalanine moieties onto the 30-terminal
adenosine of its cognate tRNA. It is also possible to prepare bisactivated tRNAs in vitro; these participate in protein synthesis
[Wang, B.; Zhou, J.; Lodder, M.; Anderson, R. D.; Hecht, S. M. J. Biol. Chem. 2006, 281, 13865]. Presently, the chemical strategy
used for the synthesis of the key intermediate bisacylated pdCpAs is described. Bis-S-alanyl- and bis-S-methionyl-pdCpAs were pre-
pared initially. Further, S-threonine, S-allo-threonine, S-homoserine, and (S)-(+)-2-amino-3-hydroxy-3-methylbutyric acid were
coupled with the dinucleotide to define preparative methods applicable to more complex amino acids bearing additional function-
ality in the form of an OH group.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
T4 RNA ligase and can, therefore, be coupled efficiently
to the truncated tRNA.5–7,15
Ribosomally mediated protein synthesis employs
aminoacylated transfer RNAs for the introduction of
each of the 20 proteinogenic amino acids into pro-
teins.1–3 There is at least one transfer RNA for each of
the amino acids, as well as an activating enzyme specific
for the amino acid and its cognate tRNA(s) that medi-
ates attachment of the amino acid at the 30-end of the
tRNA in the form of an activated ester.4 General meth-
ods for the misacylation of tRNAs,5–7 and the site-spe-
cific incorporation of non-natural amino acids into
peptides and proteins have been developed.8–14 This
method for in vitro non-natural amino acid mutagenesis
is based on the expansion of the genetic code by sup-
pressing a normal stop codon with a misacylated sup-
pressor tRNA transcript.15 This misacylated tRNA is
obtained by the coupling of a protected 20(30)-O-amino-
acylated 50-O-phosphoryl-20-(deoxy)cytidylyl(30 ! 50)
adenosine (p(d)CpA, Fig. 1A) with a tRNA transcript
lacking the 30-terminal pCpA moiety.15 The 20(30)-O-
aminoacylated pdCpA derivatives are substrates for
Ribosomal protein synthesis is believed to involve a set
of (mono)aminoacylated tRNAs that can decode indi-
vidual mRNA codons during protein translation. How-
ever, Stepanov et al. have described the ability of
phenylalanyl-tRNA synthetase from Thermus thermo-
philus to introduce two phenylalanine moieties onto
the 30-terminal adenosine moiety of its cognate
tRNA.16,17 Although the ability of such tandemly acti-
vated tRNAs to participate in protein synthesis was
not explored, presumably due to the difficulty of obtain-
ing sufficient material in pure form, it seemed possible
that they might function in protein biosynthesis.
In fact, in a recent publication, our laboratory has de-
scribed the ability of tandemly activated tRNAs to par-
ticipate in protein synthesis.18 The bisaminoacylated
tRNAs employed for that study were prepared by exten-
sion of a strategy that has been used widely for the intro-
duction of unnatural amino acids into specific,
predetermined positions in proteins.10–13
Presently, we describe the synthesis of new bisaminoacy-
lated pdCpA derivatives (Fig. 1B) and their use in the
preparation of tandemly activated tRNAs. The general
strategy for the synthesis of aminoacyl-pdCpA deriva-
tives consists of the coupling of the N-protected amino
Keywords: Aminoacylation; Transfer RNAs; Protein synthesis; Enzy-
matic ligation; Hydroxylated amino acids.
*
Corresponding author. Tel.: +1 434 924 3906; fax: +1 434 924
7856; e-mail: sidhecht@virginia.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.088

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M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
enable the characterization of the way in which a newly
NH2
N
NH2
N
recognized type of activated tRNA functions in protein
synthesis. Additionally, bisaminoacylated tRNAs exhi-
bit surprising stability both as chemical species and in
in vitro protein biosynthesizing systems. As such, they
have the wherewithal to provide significantly enhanced
yields of proteins elaborated in cell free systems.
O
O
P
O-
-O
P
O-
O
-O
O
N
O
O
N
O
O
O
O
NH2
N
NH2
N
N
N
N
N
O
P
R'
O
P
R'
O
O
O
O
N
N
O-
O-
2. Results
O
R
O
OH
O
O
O
O
2.1. Synthesis of alanine and methionine analogues
NH
P
R
NH HN
R
P
P
B
A
Bisacylated transfer RNAs bearing S-alanine and S-
methionine were prepared as outlined in Schemes 1
and 2. S-methionine and S-alanine methyl esters (1a
and 1b) were protected as N-pentenoyl derivatives19,20
by treatment with pentenoic acid, N-methylmorpholine,
hydroxybenzotriazole, and N0,N0-dicyclohexylcarbodii-
mide. The products (2a and 2b) were isolated as color-
less oils in yields of 84% and 64%, respectively
(Scheme 1). Following treatment with aq LiOH (25 ꢁC,
3 h), the carboxylates (3a and 3b) were treated with chlo-
roacetonitrile and Et3N, affording cyanomethyl esters 4a
and 4b in yields of 58% and 12%, respectively. Each of
the cyanomethyl esters was admixed with the tetra-n-
butylammonium salt of pdCpA15 in DMF (Scheme
2),21 which afforded mixtures of the monoacylated
pdCpA (5a and 5b) and bisacylated pdCpA (6a and
6b) derivatives. The yield of the latter was increased by
simply extending the period of time utilized for acylation
of pdCpA. The mono- and bisacylated pdCpAs were
separable by C18 reversed phase HPLC, as illustrated
in Figure 2 for the methionine derivatives. For methio-
nine, the monoacylated derivative (5a) was isolated in
70% yield (based on limiting pdCpA) as a colorless solid,
which was a mixture of equilibrating 20- and 30-O-ami-
noacyl derivatives (Fig. 2). The desired bisaminoacylat-
ed derivative (6a) was isolated as a colorless solid in 30%
yield. The corresponding alanyl-pdCpA derivatives (5b
and 6b) were also obtained as colorless solids in yields
of 46% and 26%, respectively.
P = protecting group
R' = H, OH
Figure 1. Structures of (A) 20(30)-O-aminoacylated dinucleotide 50-O-
phosphoryl-(20-deoxy)cytidylyl(30 ! 50)adenosine and (B) 20,30-bis-O-
aminoacylated
(30 ! 50)adenosine.
dinucleotide
50-O-phosphoryl-(20-deoxy)cytidylyl
acid, activated as a cyanomethyl ester, with the dinucle-
otide pdCpA (Schemes 1 and 2 ). Subsequent incubation
in the presence of tRNACUA-COH+ T4 RNA ligase, and
deblocking of the derived N-protected aminoacyl-
tRNA, leads to the desired activated tRNA (Scheme
2). This method has proven successful for a wide variety
of unnatural amino acids. The bisaminoacylation of
tRNA has been realized in a similar manner using a
bisaminoacylated dinucleotide in the ligation reaction.18
Presently, the preparation of bisaminoacylated pdCpA
derivatives bearing S-methionine and S-alanine is re-
ported. Further, to generalize the synthetic strategy
leading to bisactivated pdCpAs, more complex amino
acids were studied, including S-threonine, S-allo-threo-
nine, S-homoserine, and S-(+)-2-amino-3-hydroxy-3-
methylbutyric acid, in which the additional OH group
must be protected. Protection–deprotection strategies
leading to the preparation of bisacylated pdCpAs and
the final bisactivated tRNAs in good yields are de-
scribed. The ability of the derived tRNAs to function
as participants in protein synthesis is also illustrated.
The bisaminoacylated tRNAs described herein are
important as they may represent a type of activated
tRNA utilized normally in protein synthesis within cer-
tain types of organisms such as thermophilic bacteria.
To the extent that this is true, the present study will
2.2. Synthesis of threonine, allo-threonine, and homoser-
ine analogues
Following the successful synthesis of bisalanyl- and
bismethionyl-pdCpA derivatives the chemical prepara-
O
O
H
pentenoic acid, DCC
LiOH
H2N
N
OCH3
OCH3
HOBt, N-methylmorpholine
R
O
R
1a, 1b
2a, 2b
O
H
O
H
ClCH2CN
N
N
OH
O
CN
O
R
O
R
4a, 4b
Scheme 1. Synthesis of N-pentenoyl-S-methionine cyanomethyl ester (4a) and N-pentenoyl-S-alanine cyanomethyl ester (4b).
3a, 3b

M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
4631
NH2
NH2
N
O
-O P O
O-
N
O
-O P O
O-
N
O
O
N
O
O
O
O
O
H
NH2
NH2
N
pdCpA
N
O
CN
+
N
N
N
N
O
O
N
O
R
O P O
O-
O P O
O-
N
N
4a, 4b
a R = CH2CH2SCH3
b R = CH3
O
R
O
OH
O O
O
R
O
NH
O
NH HN
R
O
O
5a, 5b
6a, 6b
NH2
NH2
N
tRNA-C
tRNA-C
O
N
O
-O P O
O-
-O P O
O-
N
O
O
N
O
O
O
O
NH2
N
NH2
N
N
N
N
O
O
tRNA-COH
T4 RNA ligase
aq. iodine
O P O
O-
O P O
O-
6a, 6b
N
N
N
O
O
O O
O
R
O
O
R
O
NH HN
R
R
NH2H2N
O
O
Scheme 2. Synthesis of bismethionyl-pdCpA (6a) and bisalanyl-pdCpA (6b), and T4 RNA ligase-mediated construction of tandemly activated
tRNAs.
tion of bisacylated pdCpA derivatives bearing chemi-
cally more complex amino acids was explored. The prep-
aration of threonine and its analogues was addressed, in
particular using S-threonine, S-allo-threonine, S-homo-
serine, and 2-amino-3-hydroxy-3-methylbutyric acid.
These compounds required the development of
strategies for protection and subsequent deprotection
of primary, secondary, and tertiary hydroxyl groups to
enable the condensation of these amino acids with
pdCpA.
tion of the secondary (9a and 9b) or primary alcohol
(9c) was achieved using TBSCl, imidazole, and DMF
to afford 10a0–10c0 in 91%, 92%, and 90% yields, respec-
tively. Saponification of the methyl ester using LiOH,
followed by treatment with chloroacetonitrile, triethyl-
amine in MeCN, gave the cyanomethyl esters 11a0–
11c0 in 85%, 87%, and 80% yields, respectively. Given
that S-(+)-2-amino-3-hydroxy-3-methylbutyric acid
(12, Scheme 4) contains a tertiary alcohol it seemed
likely treatment with thionyl chloride in MeOH would
result in dehydration. Accordingly, TMS–diazomethane
was utilized to selectively methylate the carboxyl group
of N-pentenoyl protected amino acid 13, affording 14 in
38% yield over two steps. Attempted protection of the
tertiary alcohol with TBSCl, imidazole, and DMF failed
to produce the desired O-silylated product. Therefore,
triethylsilyl triflate (TESOTf) was employed using 2,6-
lutidine as the base to give the desired product 15 in
90% yield. The remaining steps were identical to those
used for the other analogues and afforded 16 in 62%
yield over two steps.
The same synthetic sequence was used for S-threonine
(7a), S-allo-threonine (7b), and S-homoserine (7c)
(Scheme 3); however, for S-(+)-2-amino-3-hydroxy-3-
methylbutyric acid (12) a slightly modified route was
used (Scheme 4).
As shown in Scheme 3, compounds 7a–7c were treated
with thionyl chloride and MeOH to afford the respective
methyl esters (8a–8c) which were used as crude products
in the next step. N-pentenoyl protection was carried out
in a manner similar to that described for the synthesis of
2a and 2b, to give 9a–9c in 75%, 77%, and 75% yields
(over two steps), respectively. Subsequent TBS protec-
Each of the cyanomethyl esters 11a0–11c0 and 16 was ad-
mixed with the tetra-n-butylammonium salt of pdCpA15

4632
M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
NH2
N
NH2
N
O
-O P O
O-
O
-O P O
O-
N
O
N
O
O
O
NH2
N
NH2
N
N
N
N
N
O
O
O P O
O-
O P O
O-
N
N
O
O
O
O
O
OH
O O
O
O
O
O
N
H
N
H
N
H
S
S
S
Figure 2. Separation of monomethionyl-pdCpAs from bismethionyl-pdCpA on C18 reversed phase HPLC. Monomethionyl-pdCpA eluted first as a
mixture of (equilibrating) 20- and 30-O-aminoacyl derivatives (ꢀ16.0 and 16.5 min), followed by the bisacylated derivative (ꢀ24 min).
O
O
O
H
H
R
H
R
H
R
SOCl2
MeOH
pentenoic acid succinimide ester
NaHCO3, 1:1 dioxane-H2O
N
H2N
H2N
OCH3
OH
OCH3
O
8a-8c
7a-7c
9a-9c
a R = CH3CHOH
b R = CH3CHOH c R = CH2CH2OH
(
R
)
(S)
O
O
1) LiOH
H
H
H
R
H
R
TBSCl
imidazole, DMF
N
N
O
CN
OCH3
2) ClCH2CN
O
O
10a'-10c'
11a'-11c'
a' R = CH3CHOTBS b' R = CH3CHOTBS c' R = CH2CH2OTBS
(
R
)
(S)
Scheme 3. Synthesis of N-(4-pentenoyl)-O-tert-butyldimethylsilyl-S-threonine cyanomethyl ester (11a0), N-(4-pentenoyl)-O-tert-butyldimethylsilyl-S-
allo-threonine cyanomethyl ester (11b0) and N-(4-pentenoyl)-O-tert-butyldimethylsilyl-S-homoserine cyanomethyl ester (11c0).
in DMF (Scheme 5), which afforded mixtures of the
silyl-protected monoacylated pdCpA (17a0–17c0 and
18) and bisacylated pdCpA derivatives (19a0–19c0
and 20). The mono- and bisacylated pdCpA derivatives
were separated by C18 reversed phase HPLC using a gra-
dient of 0 ! 63% acetonitrile in 50 mM NH4OAc, pH
4.5. Monoaminoacylated compounds 17a0–17c0 were
obtained in yields of 65%, 55%, and 60%, respectively,

M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
4633
O
O
O
H
H
H
H
H
N
H2N
HO
N
TMSCH2Cl2
pentenoic acid succinimide ester
NaHCO3, 1:1 dioxane-H2O
OH
OH
OCH3
O
O
2:1 THF-MeOH
HO
HO
12
13
14
O
O
H
H
H
H
N
N
1) LiOH
2) ClCH2CN
TESOTf
OCH3
O
CN
O
O
2,6-lutidine
TESO
TESO
15
16
Scheme 4. S-N-((4-pentenoyl)-2-amino)-3-triethylsilyloxy-3-hydroxy-3-methylbutyric acid cyanomethyl ester (16).
NH2
N
NH2
N
O
-O P O
O-
O
-O P O
O-
N
O
O
N
O
O
O
O
NH2
NH2
pdCpA
+
N
N
N
N
O
O
N
N
11a'-11c' and 16
O P O
O-
O P O
O-
N
N
O
R
O
OH
O O
O
R
O
NH
O
NH HN
R
O
O
17a'-17c', 18
19a'-19c', 20
18,20 R = C(CH3)2OTES
c' R = CH2CH2OTBS
b' R = CH3CHOTBS
a' R = CH3CHOTBS
(
R
)
(S)
NH2
N
NH2
N
O
-O P O
O-
O
-O P O
O-
N
O
O
N
O
O
O
O
NH2
NH2
N
3:1:1
AcOH-THF-H2O
+
N
N
N
N
O
O
N
O P O
O-
O P O
O-
N
N
O
R
O
OH
O
O
O
R
O
NH
O
NH HN
R
O
O
21a-21c, 22
23a-23c, 24
22,24 R = C(CH3)2OH
c R = CH2CH2OH
b R = CH3CHOH
a R = CH3CHOH
(R
)
(S)
Scheme 5. Synthesis of bis-O-silyl-protected threonyl-pdCpAs (19a0–19c0 and 20).

4634
M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
O
O
H
O
H
H
H
H
N
H2N
H2N
HO
1) SOCl2, MeOH
HCl, CH3COOH
OCH3
CH3
OH
OH
CH3
O
2) pentenoic acid succinimide ester
NaHCO3, 1:1 dioxane-H2O
CH3COCl
AcO
AcO
CH3
H
H
7a
25
26
O
H
H
H
1) LiOH
pdCpA
N
O
CN
2) ClCH2CN
O
AcO
CH3
27
NH2
N
NH2
O
O
N
-O P O
O-
-O P O
O-
N
O
O
N
O
O
O
O
NH2
NH2
N
+
N
N
N
N
O
O
N
O P O
O-
O P O
O-
N
N
O
O
O
O
OH
O
O
O
AcO
H
H3C
OAc
H
CH3
H
H
H
AcO
H
NH
NH
HN
CH3
O
O
29
28
54% HBF4
in diethyl ether
54% HBF4
in diethyl ether
NH2
NH2
N
O
-O P O
O-
N
O
-O P O
O-
N
O
O
N
O
O
O
NH2
N
NH2
+
N
N
N
N
O
O
O P O
O-
O P O
O-
N
N
N
O
O
O
O
O
OH
O
O
O
HO
H
H3C
OH
H
CH3
H
H
H
HO
H
NH
NH
HN
CH3
O
O
23a
21a
Scheme 6. Synthesis of bis[O-acetyl-N-(4-pentenoyl)-S-threonyl]-pdCpA (29).
while 18 was obtained in 50% yield. As expected, the bis-
acylated derivatives (19a0–19c0 and 20) were obtained in
lower yields, about 10% in all cases. In the belief that
steric hindrance due to the trialkylsilyl groups probably
inhibited the formation of the bisacylated derivative, the
less hindered acetyl group was tested as an alternative
for the protection step. In this case, the acetyl protected
S-threonine analogue (25) (Scheme 6) was prepared
using hydrochloric acid, glacial acetic acid, and acetyl
chloride.22 The remaining steps, from 25 ! 26 !
27 ! 28 + 29, were the same as those described above
for the silyl-protected analogues. However, this route
afforded monoacylated derivative 28 in 38% yield, while
bisacylated 29 was obtained in 40% yield after 3 days of
stirring at room temperature. The acetyl protecting
group thus appears to be more suitable for the prepara-

M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
4635
tion of hydroxylated amino acid derivatives such as the
threonine derivatives studied here.
initiation of protein synthesis from methionine codons
downstream from the UAG codon and initiation codon
at position 1. While this affords bands in addition to full
length DHFR fusion protein, the construct is especially
valuable for judging the efficiency of the suppression
reaction, since protein synthesis can proceed even in
the absence of suppression.
In order to conjugate the monoacylated and bisacylated
derivatives so obtained with tRNA-COH and subse-
quently use them in protein synthesis, the deprotection
of both silyl-protected and acetyl-protected derivatives
was carried out. Deprotection of TBS and TES groups
was achieved by treatment of compounds 17a0–17c0,
18, 19a0–19c0, and 20 (Scheme 5) with a mixture 3:1:1
AcOH/THF/H2O, according to a reported procedure.21
The deprotected derivatives were obtained in 50–70%
yields. The acetyl groups were removed from com-
pounds 28 and 29 using a 54% solution of HBF4 in
diethyl ether,23 leading to deprotected derivatives 21a
and 23a in 81% and 43% yields, respectively (Scheme 6).
3. Discussion
In vitro protein mutagenesis has been shown to be a
powerful tool for modifying proteins in a site-specific
manner, and thereby permitting the detailed study of
their structure, function, and interactions. The introduc-
tion of both natural and non-natural amino acids at pre-
determined positions permits the analysis of these
parameters at high resolution. The finding that bisami-
noacylated tRNAs function well in cell free protein
synthesizing systems, exhibit better stability in those
systems, and can afford greater amounts of proteins
under conditions limiting for the misacylated tRNA18
has prompted us to explore methods for the efficient
preparation of bisaminoacylated tRNAs. The synthesis
of bisaminoacylated pdCpA derivatives, which are
precursors of the tandemly activated tRNAs, was the
objective of the present study.
2.3. Synthesis of bisactivated tRNAs
The HPLC-purified N-protected bisaminoacylated
pdCpA derivatives (6a and 6b) were each ligated to
abbreviated suppressor tRNACUA transcripts lacking
the cytidine and adenosine moieties normally present
at the 30-terminus of all tRNAs.15,24 The tRNA tran-
script was obtained by in vitro run-off transcription of
a Fok1-linearized plasmid encoding the abbreviated
25
tRNACUA
.
The ligation reaction was catalyzed by T4
RNA ligase (Scheme 2).5,7,8 The completion of the liga-
tion reaction was verified by polyacrylamide gel electro-
phoresis at pH 4.5.26 The formed N-pentenoyl
bisacylated tRNAs were deprotected by treatment with
aqueous iodine as described.19,20
Initially the synthesis of bismethionyl- and bisalanyl-
pdCpAs (6a and 6b) was investigated. These two amino
acids do not contain reactive side chains with functional
groups, such that the synthesis of the bisacylated ana-
logues did not pose major obstacles, except for the rela-
tively low yields and long reaction times. HPLC
purification is also required, but pure products can be
obtained readily (Schemes 1 and 2). The study of more
complex amino acids such as threonine analogues was
then addressed. The compounds employed (7a–7c and
12) contain an additional side-chain hydroxyl group that
can be tertiary (12), secondary (7a and 7b) or primary
(7c). This hydroxyl group requires differential protec-
tion, or else one may expect participation of the OH
group during the pdCpA coupling reaction. The protect-
ing group must also be removed under conditions
compatible with stability of the formed bisaminoacyl-
pdCpA derivatives. Schultz and co-workers reported
that TBS ethers could be removed successfully from
aminoacylated pdCpA derivatives using 3:1:1 AcOH/
THF/H2O at 37 ꢁC.27 Given this report, it seemed
appropriate to use this protecting group for the O-pro-
tection of threonine and its analogues. Initial N-protec-
tion was accomplished using the pentenoyl group which
had been shown previously to be removed easily using I2
in aqueous THF.19,20S-threonine and its analogues were
converted to the silylated cyanomethyl esters in 5-step
sequences as shown in Scheme 3. Analogue 16, however,
was synthesized in a slightly different manner. The ter-
tiary hydroxyl group presented two problems, namely
(i) acidic treatment to form the methyl esters may result
in dehydration of the tertiary alcohol and (ii) the tertiary
hydroxyl group could not be successfully protected
readily using standard conditions for TBS ethers given
its hindered nature. Accordingly, the methyl ester was
The ability of the bisacylated tRNAs to participate in
protein synthesis was investigated in a prokaryotic pro-
tein biosynthesizing system programmed with the
mRNA for a dihydrofolate reductase construct in which
a nonapeptide was fused at the N-terminus of wild-type
DHFR. A stop codon (UAG) was introduced within the
fused nonapeptide, one codon upstream from the origi-
nal initiation codon (AUG) in wild-type mRNA. As
shown in Figure 3, in the presence of the deprotected
bismethionyl- and bisalanyl-tRNACUAs, full length fu-
sion protein was obtained by suppression of the UAG
codon at the position immediately upstream from the
initiator AUG codon of wild-type dihydrofolate reduc-
tase. The suppression yields for bismethionyl- and bisal-
anyl-tRNAs were 12% and 17%, respectively. In
contrast, the bisaminoacylated tRNAs containing the
N-pentenoyl protecting groups were not able to effect
suppression of the UAG codon. As is clear from Figure
3, in addition to full length protein, there was also some
Figure 3. Introduction of alanine and methionine into E. coli dihy-
drofolate reductase by suppression of a UAG codon. The UAG codon
was introduced within an N-terminal fusion peptide, one codon
upstream from the initiator (AUG) codon present in wild-type mRNA.

4636
M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
formed using TMS–diazomethane.28 TESOTf was then
used to protect the tertiary alcohol. The coupling of
the fully protected cyanomethyl esters (11a0–11c0 and
16) with pdCpA led to the formation of monoacylated
derivatives (17a0–17c0 and 18, respectively) as well as
the bisacylated compounds (19a0–19c0 and 20, respec-
tively). However, the yield of the bisaminoacylated ana-
logues was only about 10% in all cases. Since this was
probably due to the presence of the sterically hindered
silyl protecting groups, an O-acetyl group was explored
as a possible alternative. Accordingly, N-(4-pentenoyl)-
O-acetyl-S-threonine cyanomethyl ester (27) was ob-
tained in 5 steps, as shown in Scheme 6, and coupled
with pdCpA. In this case the yield of the desired bisami-
noacylated pdCpA was much higher (40%), indicating
that the O-acetyl protecting group is better suited to
the preparation of the desired hydroxylated amino acid
derivatives. While the purpose of the present study was
the preparation of bisaminoacyl-tRNAs, the findings
made concerning protection of threonine with an acetyl
group should also find utility in the synthesis of misacy-
lated monoaminoacylated tRNAs.
Analytical TLC was performed using 0.25 mm EM silica
gel 60 F250 plates that were visualized by irradiation
(254 nm) or by staining with Hanessian’s stain (cerium
1
molybdate). H and 13C NMR spectra were obtained
using a 300 MHz Varian instrument. Chemical shifts
are reported in parts per million (ppm, d) referenced
1
to the residual H resonance of the solvent (CDCl3, d
7.26; CD3OD, d 3.31). 13C spectra were referenced to
the residual 13C resonance of the solvent (CDCl3, d
77.3; DMSO-d6, d 39.5). Splitting patterns are desig-
nated as follows: s, singlet; br, broad; d, doublet; dd,
doublet of doublets; t, triplet; q, quartet; m, multiplet.
High resolution mass spectra were obtained at the Mich-
igan State University—NIH Mass Spectrometry Facil-
ity. Phosphoroimager analysis was performed using a
Molecular Dynamics 300E Phosphorimager equipped
with Image Quant software. HPLC was performed using
a Varian 9012 pump coupled with a Varian 2050 UV
detector and an Alltech Alltima RPC18 column
(250 · 10 mm, 5 lm).
4.1.1. N-4-Pentenoyl-S-methionyl-pdCpA (5a) and bis(N-
4-pentenoyl-S-methionyl)-pdCpA (6a). To a conical vial
containing 4.10 mg (15.1 lmol) of N-4-pentenoyl-S-
methionine cyanomethyl ester (4a)20 was added a
solution of 4.10 mg (3.00 lmol) of the tris(tetrabutylam-
monium) salt of pdCpA15 in 50 lL of DMF. The reac-
tion mixture was stirred at room temperature. After
3 h the reaction mixture was diluted with 1:2 CH3CN/
50 mM NH4OAc, pH 4.5, to a total volume of 400 lL
and purified using a semi-preparative C18 reversed phase
column (250 · 10 mm). The column was washed with
1 ! 63% CH3CN in 50 mM NH4OAc, pH 4.5, over a
period of 45 min at a flow rate of 3.5 mL/min (monitor-
ing at 260 nm). After lyophilization of the appropriate
fractions both compounds were obtained as colorless
solids: N-4-pentenoyl-S-methionyl-pdCpA (5a) (reten-
tion times 16.0 and 16.5 min, for the two positional
(20,30) isomers): yield 1.8 mg (70%); mass spectrum
(FAB), m/z 850.200 (M+H)+ (C29H42N9O15P2S requires
850.200). Bis(N-4-pentenoyl-S-methionyl)-pdCpA (6a)
(retention time 24 min): yield 1 mg (30%); mass spec-
trum (FAB), m/z 1063.280 (M+H)+ (C39H57N10O17P2S2
requires m/z 1063.280).
As described recently,18 bisaminoacylated tRNAs have
significant advantages for use in cell free protein synthe-
sizing systems. These include the ability of the bisacylat-
ed tRNAs to incorporate both appended amino acids
into protein in two rounds of protein synthesis, resulting
in higher yields of proteins when the activated tRNAs
are used at limiting concentrations. It has also been
shown that bisacylated tRNAs have much greater stabil-
ity in cell free protein synthesizing systems than the
respective monoacylated species.18 For example, valyl-
pdCpA was 78% hydrolyzed to pdCpA + valine when
maintained at pH 7.8 and 37 ꢁC for 1 h. In contrast,
20-O-alanyl-30-O-valyl-pdCpA was not hydrolyzed to a
detectable extent under the same conditions. An analo-
gous finding was made for mono- and bisaminoacylated
tRNAs when maintained in an in vitro protein synthe-
sizing system for 30 min in the absence of messenger
RNA.18 This suggests that the bisaminoacylated tRNAs
may find utility in continuous cell free protein synthesiz-
ing systems. In addition, the reports that at least one
bisaminoacylated tRNA occurs naturally raise the possi-
bility that such tRNAs are normal participants in pro-
tein biosynthesis in some organisms. If this proves to
be correct, the present study will enable the study of
the participation of bisaminoacylated tRNAs as constit-
uents of protein synthesizing systems.
4.1.2. N-4-Pentenoyl-S-alanyl-pdCpA (5b) and bis(N-4-
pentenoyl-S-alanyl)-pdCpA (6b). To a conical vial
containing 3.50 mg (16.6 lmol) of N-(4-pentenoyl)-S-
alanine cyanomethyl ester (4b)20 was added a solution
of 4.50 mg (3.30 lmol) of the tris(tetrabutylammonium)
salt of pdCpA15 in 50 lL of DMF. The reaction mixture
was stirred at room temperature. After 3 h the reaction
mixture was diluted with 1:2 CH3CN/50 mM NH4OAc,
pH 4.5 to a total volume of 400 lL and purified using a
semi-preparative C18 reversed phase column (250 ·
10 mm). The column was washed with 1 ! 63% CH3CN
in 50 mM NH4OAc, pH 4.5, over a period of 35 min at a
flow rate of 3.5 mL/min (monitoring at 260 nm). After
lyophilization of the appropriate fractions both com-
pounds were obtained as colorless solids: N-(4-pente-
noyl)-S-alanyl-pdCpA (5b) (retention times 12.8 and
13.3 min, for the two positional (20,30) isomers) yield
1.20 mg (46%); mass spectrum (FAB), m/z 790.1932
4. Experimental
4.1. Chemistry
Reagents and solvents were purchased from Aldrich
Chemical Co. or Sigma Chemical Co. and used without
further purification. Anhydrous grade methylene chlo-
ride, THF, DMF, and acetonitrile were purchased from
VWR Scientific. All reactions involving air- or moisture-
sensitive reagents or intermediates were performed
under an argon atmosphere. Flash chromatography
was performed using Silicycle 40–60 mesh silica gel.

M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
4637
(M+H)+ (C27H38N9O15P2 requires m/z 790.1962). Bis(N-
4-pentenoyl-S-alanyl)-pdCpA (6b) (retention time
21 min) yield 0.8 mg (26%); mass spectrum (FAB), m/z
943.2777 (M+H)+ (C35H49N10O17P2 requires m/z
943.2752).
ture and monitored by TLC; after 3 h the starting
material had been consumed, so the reaction mixture
was diluted with 60 mL of ethyl acetate and washed with
25 mL of 1 N NaHSO4, dried (MgSO4), and concen-
trated under diminished pressure. The crude residue
was dissolved in anhydrous acetonitrile and 480 mg
(6.35 mmol) of chloroacetonitrile was added followed
by 640 mg (6.35 mmol) of triethylamine. The reaction
mixture was stirred at room temperature overnight, then
diluted with 50 mL of ethyl acetate and washed with
25 mL of 1 N NaHSO4, dried (MgSO4), and concen-
trated under diminished pressure. Purification by flash
chromatography on a silica gel column (15 · 2 cm), elu-
tion with 2:1 hexanes/ethyl acetate, gave 11a0 as a color-
less oil: yield 383 mg (85%); 1H NMR (CDCl3) d 0.02 (s,
6H), 0.84 (s, 9H), 1.18 (d, 3H, J = 6.3 Hz), 2.41 (m, 4H),
4.43 (dq, 1H, J = 14.4 and 1.8 Hz), 4.60 (dd, 1H, J = 9.3
and 2.1 Hz), 4.73 (m, 2H), 5.06 (m, 2H), 5.83 (m, 1H)
and 6.14 (d, 1H, J = 9.3 Hz); 13C NMR (CDCl3) d
ꢁ5.24, ꢁ4.28, 17.81, 20.92, 25.61, 29.31, 35.53, 48.54,
48.94, 57.56, 68.42, 115.89, 136.69, 169.45, and 172.76;
mass spectrum (FAB), m/z 355.2052 (M+H)+
(C17H31N2O4Si requires m/z 355.2053).
4.1.3. N-(4-Pentenoyl)-S-threonine methyl ester (9a). To
a solution containing 500 mg (4.20 mmol) of S-threo-
nine (7a) in 4 mL of MeOH was added 0.30 mL of thio-
nyl chloride. The reaction mixture was heated to reflux
for 1 h, then concentrated to dryness. The residue was
redissolved in 4 mL of MeOH and again treated with
0.3 mL of thionyl chloride, heated to reflux for 1 h,
and then concentrated. A 320-mg (1.89 mmol) portion
of this crude material was dissolved in 5 mL of H2O
and 320 mg (1.89 mmol) of NaHCO3 was added. To this
stirred solution was added 410 mg (2.08 mmol) of 4-
pentenoic acid succinimide ester29 in 5 mL of dioxane.
After stirring at room temperature overnight the reac-
tion mixture was diluted with 10 mL of ethyl acetate
and 10 mL of 1 N NaHSO4, then extracted with three
20-mL portions of ethyl acetate. The combined organic
extract was dried (MgSO4) and concentrated under
diminished pressure. The crude product was purified
by flash chromatography on a silica gel column
(21 · 3 cm); elution with 2:1 ethyl acetate/hexane gave
4.1.6. N-(4-Pentenoyl)-S-allo-threonine methyl ester (9b).
To a solution containing 250 mg (2.10 mmol) of S-allo-
threonine (7b) in 2 mL of MeOH was added 0.15 mL of
thionyl chloride. The reaction mixture was heated to re-
flux for 1 h, then concentrated to dryness. The residue
was redissolved in 2 mL of MeOH and again treated
with 0.15 mL of thionyl chloride, heated to reflux for
1 h, and then dried. A 350-mg (2.07 mmol) portion of
this crude material was dissolved in 5 mL of H2O and
347 mg (4.14 mmol) of NaHCO3 was added. To this stir-
red solution was added 447 mg (2.27 mmol) of 4-pente-
noic acid succinimide ester29 in 5 mL of dioxane. After
stirring at room temperature overnight the reaction mix-
ture was diluted with 10 mL of ethyl acetate and 10 mL
of 1 N NaHSO4, then extracted with three 20-mL por-
tions of ethyl acetate. The combined organic extract
was dried (MgSO4) and concentrated under diminished
pressure. The crude product was purified by flash chro-
matography on a silica gel column (21 · 3 cm); elution
with 2:1 ethyl acetate/hexane gave 9b as a colorless solid:
1
9a as a colorless solid: yield 305 mg (75%); H NMR
(CDCl3) d 1.15 (d, 3H, J = 6.6 Hz), 2.36 (m, 4H), 3.72
(br s, 1H), 4.29 (s, 3H), 4.29 (dq, 1H, J = 6.3 and
1.8 Hz), 4.54 (dd, 1H, J = 9.0 and 1.8 Hz), 5.00 (m,
2H), 5.80 (m, 1H), and 6.60 (d, 1H, J = 9.0 Hz); 13C
NMR (CDCl3) d 19.88, 29.42, 35.49, 52.44, 57.21,
67.72, 115.60, 136.69, 171.52, and 173.20; mass spectrum
(FAB), m/z 216.1235 (M+H)+ (C10H18NO4 requires m/z
216.1236).
4.1.4. N-(4-Pentenoyl)-O-tert-butyldimethylsilyl-S-threo-
nine methyl ester (10a0). To a solution containing 300 mg
(1.40 mmol) of 9a in 5 mL of anhydrous DMF were
added 420 mg (2.79 mmol) of O-tertbutyldimethylsilyl
chloride (TBSCl) and 210 mg (3.08 mmol) of imidazole.
The reaction mixture was stirred at room temperature
under argon overnight, then diluted with 50 mL of brine
and extracted with two 60-mL portions of ethyl acetate.
The combined organic layer was washed with 100 mL of
H2O, dried (MgSO4), and concentrated under dimin-
ished pressure. Purification by flash chromatography
on a silica gel column (21 · 2 cm) using 3:1 hexanes/
ethyl acetate as eluant gave 10a0 as a colorless oil: yield
420 mg (91%); 1H NMR (CDCl3) d 0.02 (s, 6H), 0.83 (s,
9H), 1.12 (d, 3H, J = 6.0 Hz), 2.42 (m, 4H), 3.67 (s, 3H),
4.40 (m, 1H), 4.56 (d, 1H, J = 8.1 Hz), 5.02 (m, 2H), 5.82
(m, 1H), and 6.12 (d, 1H, J = 9.3 Hz); 13C NMR
(CDCl3) d ꢁ5.39, ꢁ4.48, 17.76, 20.84, 25.56, 29.39,
35.65, 52.14, 57.55, 68.75, 115.59, 136.84, 171.06, and
172.52; mass spectrum (FAB), m/z 330.2099 (M+H)+
(C16H32NO4Si requires m/z 330.2101).
1
yield 342 mg (77%); H NMR (CDCl3) d 1.14 (d, 3H,
J = 6.6 Hz), 2.40 (m, 4H), 3.46 (br s, 1H), 3.70 (s, 3H),
4.13 (m, 1H), 4.66 (dd, 1H, J = 7.2 and 3.3 Hz), 5.02
(m, 2H), 5.80 (m, 1H), and 6.56 (d, 1H, J = 6.0 Hz);
13C NMR (CDCl3) d 18.73, 29.35, 35.41, 52.58, 58.19,
68.93, 115.80, 136.59, 170.68, and 173.35; mass spectrum
(FAB), m/z 216.1234 (M+H)+ (C10H18NO4 requires m/z
216.1236).
4.1.7. N-(4-Pentenoyl)-O-tert-butyldimethylsilyl-S-allo-
threonine methyl ester (10b0). To a solution containing
342 mg (1.60 mmol) of 9b in 5 mL of anhydrous DMF
were added 0.48 g (3.20 mmol) of TBSCl and 270 mg
(4.00 mmol) of imidazole. The reaction mixture was stir-
red at room temperature under argon overnight, then di-
luted with 50 mL of brine and extracted with two 60-mL
portions of ethyl acetate. The combined organic layer
was washed with 100 mL of H2O, dried (MgSO4), and
concentrated under diminished pressure. Purification
4.1.5. N-(4-Pentenoyl)-O-tert-butyldimethylsilyl-S-threo-
nine cyanomethyl ester (11a0). To a suspension contain-
ing 420 mg (1.27 mmol) of 10a0 in 5 mL of 1:1 THF/
H2O was added 160 mg (3.81 mmol) of LiOH. The reac-
tion mixture was stirred under argon at room tempera-

4638
M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
by flash chromatography on a silica gel column
(15 · 2 cm) using 3:1 hexanes/ethyl acetate gave 10b0 as
a colorless oil: yield 480 mg (92%); H NMR (CDCl3)
3.54 (s, 3H), 4.00–4.06 (q, 1H, J = 9.2 and 9.0 Hz),
4.26 (t, 1H, J = 9 Hz), 4.46 (q, 1H, J = 9.2 and
9.0 Hz), 4.84–4.91 (m, 2H), 5.34–5.44 (m, 1H), and
7.09 (br s,1H); 13C NMR (CDCl3) d 25.5, 49.1, 66.4,
67.1, 115.6, 115.9, 136.7, 173.3, 174.4, and 177.3; mass
spectrum (CI), m/z 238.0 (M+Na+) (theoretical m/z
238.1); mass spectrum (FAB), m/z 216.1235 (M+H)+
(C10H18NO4 requires m/z 216.1236).
1
d 0.04 (s, 6H), 0.86 (s, 9H), 1.27 (d, 3H, J = 6.6 Hz),
2.37 (m, 4H), 3.75 (s, 3H), 4.56 (dd, 1H, J = 7.8 and
3.3 Hz), 4.58 (m, 1H), 5.10 (m, 2H), 5.86 (m, 1H), and
6.27 (d, 1H, J = 7.8 Hz); 13C NMR (CDCl3) d ꢁ5.06,
ꢁ4.51, 17.84, 20.70, 25.58, 29.33, 35.68, 52.05, 58.24,
70.00, 115.64, 136.83, 170.45, and 171.59; mass spectrum
(FAB), m/z 330.2099 (M+H)+ (C16H32NO4Si requires
m/z 330.2101).
4.1.10.
N-(4-Pentenoyl)-O-tert-butyldimethylsilyl-S-
homoserine methyl ester (10c0). To a solution containing
400 mg (1.86 mmol) of 9c in 5 mL of anhydrous DMF
were added 420 mg (2.79 mmol) of TBSCl and 210 mg
(3.08 mmol) of imidazole. The reaction mixture was stir-
red at room temperature under argon overnight, then di-
luted with 50 mL of brine and extracted with two 60-mL
portions of ethyl acetate. The combined organic phase
was washed with 100 mL of water, dried over MgSO4,
and concentrated under diminished pressure. Purifica-
tion by flash chromatography on a silica gel column
(20 · 3 cm) using methylene chloride–methanol as the
eluant gave 10c0 as a colorless oil: yield 560 mg (90%);
1H NMR (CDCl3) d 0.09 (s, 6H), 0.99 (s, 9H), 2.25–
2.40 (m, 6H), 3.69 (s, 3H), 4.18–4.26 (m, 1H), 4.42 (t,
1H, J = 9 Hz), 4.51–4.60 (m, 1H), 4.90–5.08 (m, 2H),
5.70–5.86 (m, 1H), and 7.08 (br s, 1H); 13C NMR
(CDCl3) d ꢁ5.6, ꢁ4.8, 16.9, 24.8, 50.1, 65.0, 67.1,
114.6, 119.6, 135.6, 161.6, 173.0, and 174.5; mass spec-
trum (CI), m/z 330.0 (M+H)+ (theoretical m/z 329.2);
mass spectrum (FAB), m/z 330.2099 (M+H)+
(C16H32NO4Si requires m/z 330.2101).
4.1.8. N-(4-Pentenoyl)-O-tert-butyldimethylsilyl-S-allo-
threonine cyanomethyl ester (11b0). To a suspension con-
taining 480 mg (1.47 mmol) of 10b0 in 5 mL of 1:1 THF/
H2O was added 180 mg (4.42 mmol) of LiOH. The reac-
tion mixture was stirred under argon at room tempera-
ture and monitored by TLC; after 3 h the starting
material had been consumed, so the reaction mixture
was diluted with 60 mL of ethyl acetate and washed with
25 mL of 1 N NaHSO4, dried (MgSO4), and concen-
trated under diminished pressure. The crude residue
was dissolved in anhydrous acetonitrile and 550 mg
(7.35 mmol) of chloroacetonitrile was added followed
by 740 mg (7.35 mmol) of triethylamine. The reaction
mixture was stirred at room temperature overnight, then
diluted with 50 mL of ethyl acetate and washed with
25 mL of 1 N NaHSO4, dried (MgSO4), and concen-
trated under diminished pressure. Purification by flash
chromatography on a silica gel column (15 · 2 cm), elu-
tion with 2:1 hexanes/ethyl acetate, gave 11b0 as a color-
less oil: yield 450 mg (87%); 1H NMR (CDCl3) d 0.05 (s,
6H), 0.86 (s, 9H), 1.27 (d, 3H, J = 6.6 Hz), 2.38 (m, 4H),
4.08 (m, 1H), 4.60 (dd, 1H, J = 7.8 and 3.3 Hz), 4.74 (m,
2H), 5.04 (m, 2H), 5.80 (m, 1H) and 6.20 (d, 1H,
J = 7.8 Hz); 13C NMR (CDCl3) d ꢁ5.10, ꢁ4.45, 17.82,
20.68, 25.55, 29.20, 35.47, 48.63, 58.09, 69.77, 113.75,
115.82, 136.60, 168.60, and 171.85; mass spectrum
(FAB), m/z 355.2051 (M+H)+ (C17H31N2O4Si requires
m/z 355.2053).
4.1.11. N-(4-Pentenoyl)-O-tert-butyldimethylsilyl-S-homo-
serine cyanomethyl ester (11c0). To a suspension contain-
ing 560 mg (1.76 mmol) of 10c0 in 5 mL of 1:1 THF/water
was added 222 mg (5.28 mmol) of LiOH. The reaction
mixture was stirred under argon at room temperature
for 3 h, then diluted with 60 mL of ethyl acetate and
washed with 25 mL of 1 N NaHSO4, dried over MgSO4,
and concentrated under diminished pressure. The crude
residue was dissolved in anhydrous acetonitrile and
370 mg (8.8 mmol) of chloroacetonitrile was added, fol-
lowed by 1.23 mL (8.8 mmol) of triethylamine. The reac-
tion mixture was stirred at room temperature overnight,
then diluted with 50 mL of ethyl acetate and washed with
25 mL of 1 N NaHSO4, dried over MgSO4, and concen-
trated under diminished pressure. Purification by flash
chromatography on a silica gel column (15 · 2 cm), elu-
tion with 9:1 dichloromethane/methanol, gave 11c0 as a
4.1.9. N-(4-Pentenoyl)-S-homoserine methyl ester (9c).
To a solution containing 300 mg (2.52 mmol) of S-
homoserine (7c) in 4 mL of MeOH was added 0.15 mL
of thionyl chloride. The reaction mixture was heated
to reflux for 1 h, then concentrated to dryness. The res-
idue was redissolved in 4 mL of MeOH and again trea-
ted with 0.15 mL of thionyl chloride, heated to reflux for
1 h, and then concentrated under diminished pressure.
The crude material obtained was then dissolved in
5 mL of water and 160 mg (1.89 mmol) of NaHCO3
was added. To this stirred solution was added 200 mg
of 4-pentenoic acid succinimide ester29 in 5 mL of diox-
ane. After stirring at room temperature overnight the
reaction mixture was diluted with 10 mL of ethyl acetate
and 10 mL of 1 N NaHSO4, then extracted with three
20-mL portions of ethyl acetate. The combined organic
extract was dried over MgSO4, and concentrated under
diminished pressure. The crude product was purified by
flash chromatography on a silica gel column (15 · 2 cm);
elution with 9:1 methylene chloride/methanol gave 9c as
1
colorless oil: yield 480 mg (80%); H NMR (CDCl3) d
0.09 (s, 6H), 0.92 (s, 9H), 2.05–2.15 (m, 2H), 2.25–2.40
(m, 6H), 2.40–2.50 (m, 2H), 3.64–3.80 (m, 1H), 4.90–
5.08 (m, 2H), 5.70–5.86 (m, 1H), and 7.14 (br s); mass
spectrum (FAB), m/z 355.2051 (M+H)+ (C17H31N2O4Si
requires m/z 355.2053).
4.1.12. N-((4-Pentenoyl)-S-2-amino)-3-hydroxy-3-meth-
ylbutyric acid methyl ester (14). To a solution containing
200 mg (1.50 mmol) of S-(+)-2-amino-3-hydroxy-3-
methylbutyric acid (12) in 3 mL of H2O was added
320 mg (3.76 mmol) of NaHCO3 followed by 410 mg
(2.07 mmol) of 4-pentenoic acid succinimide ester in
3 mL of dioxane. The reaction mixture was allowed to
1
a colorless oil: yield 400 mg (75%); H NMR (CDCl3) d
2.00–2.14 (m, 4H), 2.40–2.48 (m, 2H), 2.70 (br s, 1H),

M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
4639
stir overnight, then acidified carefully with 1 N NaHSO4
to pH 4 and extracted with two 20-mL portions of ethyl
acetate. The combined organic layer was washed with
75 mL of brine, dried (MgSO4), and concentrated under
diminished pressure. The residue (crude 13) was dis-
solved in 2 mL of THF and 1 mL of MeOH, the solution
was cooled to 0 ꢁC in an ice bath, and 2.0 mL of a 2.0 M
solution of TMS–diazomethane23 was added dropwise.
Upon addition the evolution of N2 occurred immedi-
ately; after N2 evolution ceased, the reaction was
quenched by dropwise addition of AcOH. The reaction
mixture was diluted with 25 mL of brine and extracted
with two 25-mL portions of ethyl acetate. The combined
organic layer was dried (MgSO4) and concentrated un-
der diminished pressure. Purification by flash chroma-
tography on a silica gel column (20 · 2 cm), eluting
with 2:1 ethyl acetate/hexanes, gave 14 as a colorless
diluted with 50 mL of ethyl acetate and washed with
25 mL of satd aq NH4Cl, dried (MgSO4), and concen-
trated under diminished pressure. Purification by flash
chromatography on a silica gel column (15 · 2 cm), elu-
tion with 2:1 hexanes/ethyl acetate, gave 16 as a colorless
oil: yield 85 mg (62%); 1H NMR (CDCl3) d 0.57 (q, 6H,
J = 7.8 Hz), 0.93 (t, 9H, J = 7.8 Hz), 1.28 (s, 3H), 1.36
(s, 3H), 2.35 (m, 4H), 4.42 (d, 1H, J = 8.7 Hz), 4.68 (d,
2H, J = 15.6 Hz), 4.80 (d, 1H, J = 15.6 Hz), 5.04 (m,
2H), 5.80 (m, 1H), and 6.25 (d, 1H, J = 8.4 Hz); 13C
NMR (CDCl3) d 6.37, 6.85, 27.60, 27.97, 29.21, 35.45,
48.42, 60.98, 74.32, 113.85, 115.74, 136.58, 168.94, and
172.19; mass spectrum (FAB), m/z 369.2209 (M+H)+
(C18H33N2O4Si requires m/z 369.2210).
4.1.15. N-4-Pentenoyl-O-tert-trialkylsilyl-S-aminoacyl-
pdCpAs (17a0–17c0 and 18) and bis(N-4-pentenoyl-O-
tert-trialkylsilyl-S-aminoacyl)-pdCpAs (19a0–19c0 and
20). To a conical vial containing 20 mg (54–56 lmol)
1
oil: yield 130 mg (38%, 2 steps); H NMR (CDCl3) d
1.18 (s, 3H), 1.22 (s, 3H), 2.32 (m, 4H), 3.08 (br s,
1H), 3.69 (s, 3H), 4.47 (d, 1H, J = 8.7 Hz), 4.97 (m,
2H), 5.75 (m, 1H), and 6.64 (d, 1H, J = 8.7 Hz); 13C
NMR (CDCl3) d 26.62, 26.66, 29.29, 35.32, 52.06,
59.66, 71.58, 115.50, 136.65, 171.82, and 172.61; mass
spectrum (FAB), m/z 230.1391 (M+H)+ (C11H20NO4
requires m/z 230.1392).
of
N-(4-pentenoyl)-O-silyl-protected-S-amino
acid
cyanomethyl esters (11a0–11c0 and 16) was added a solu-
tion of the tetrabutylammonium salt of 5.0 mg
(3.64 lmol) of pdCpA15 in 100 lL of anhydrous DMF,
˚
followed by 20 lL of triethylamine (over 4 A molecular
sieves). The reaction mixture was stirred at 25 ꢁC and
monitored by HPLC. A 5-lL aliquot of the reaction
mixture was removed after 24 h and diluted with
45 lL of 1:2 CH3CN/50 mM NH4OAc, pH 4.5. Twenty
microliters of the diluted aliquot was analyzed by HPLC
on a C18 reversed phase column (250 · 10 mm). The col-
umn was washed with 1 ! 63% CH3CN in 50 mM
NH4OAc, pH 4.5, over a period of 35 min at a flow rate
of 3.5 mL/min (monitoring at 260 nm). The majority of
the pdCpA salt had been consumed at this time so the
reaction mixture was diluted to a total volume of
500 lL of 1:1 CH3CN/50 mM NH4OAc, pH 4.5, and
purified using the same semi-preparative C18 reversed
phase column. After lyophilization of the appropriate
fractions, N-(4-pentenoyl)-O-tert-butyldimethylsilyl-S-
threonyl-pdCpA (17a0) (retention time 28.4 min) was
obtained as a colorless solid: yield 2.22 mg (65%); mass
spectrum (MALDI-TOF), m/z 932.2766 (MꢁH)ꢁ
(C34H52N9O16P2Si requires m/z 932.2777); N-(4-pente-
noyl)-O-tert-butyldimethylsilyl-S-allo-threonyl-pdCpA
(17b0) (retention time 28.2 min) was obtained as a
colorless solid: yield 1.88 mg (55%); mass spectrum (MAL-
DI-TOF), m/z 933.84 (M+) (theoretical m/z 933.28); N-(4-
pentenoyl)-O-tert-butyldimethylsilyl-(S)-homoseryl-pdCpA
(17c0) (retention time 27.9 min) was obtained as a color-
less solid: yield 2.05 mg (60%); mass spectrum (MAL-
DI-TOF), m/z 931.2671 (Mꢁ2H)2ꢁ (C34H53N9O16P2Si
requires m/z 931.2698); N-((4-pentenoyl)-2-amino)-3-tri-
ethylsilyloxy-3-methylbutyryl-pdCpA (18) (retention
time 28.4 min) was obtained as a colorless solid: yield
1.74 mg (50%); mass spectrum (MALDI-TOF), m/z
947.47 (M+) (theoretical m/z 947.30); bis-N-(4-pent-
enoyl)-O-tert-butyldimethylsilyl-S-threonyl)-pdCpA (19a0)
(retention time 33.8 min) was obtained as a colorless so-
lid: yield 0.45 mg (10%); mass spectrum (MALDI-
TOF), m/z 1253.41 (M+Na)+ (theoretical m/z 1253.45),
bis-N-(4-pentenoyl)-O-tert-butyldimethylsilyl-S-allo-thre-
onyl)-pdCpA (19b0) (retention time 34.0 min) was ob-
tained as a colorless solid: yield 0.45 mg (10%); mass
4.1.13.
N-((4-Pentenoyl)-S-2-amino)-3-O-triethylsilyl-
oxy-3-methylbutyric acid methyl ester (15). To a solution
containing 95 mg (0.41 mmol) of 14 in 2 mL of anhy-
˚
drous CH2Cl2 with 4 A molecular sieves were added
220 mg (0.83 mmol) of triethylsilyl triflate (TESOTf)
and 110 mg (1.03 mmol) of 2,6-lutidine. The reaction
mixture was stirred at room temperature under argon
for 1 h, then diluted with 50 mL of brine and extracted
with two 30-mL portions of ethyl acetate. The combined
organic phase was washed with 75 mL of H2O, dried
(MgSO4), and concentrated under diminished pressure.
Purification by flash chromatography on a silica gel col-
umn (15 · 2 cm) eluting with 2:1 hexanes/ethyl acetate
1
gave 15 as a colorless oil: yield 130 g (90%); H NMR
(CDCl3) d 0.52 (q, 6H, J = 7.8 Hz), 0.89 (t, 9H,
J = 7.8 Hz), 1.21 (s, 3H), 1.31 (s, 3H), 2.33 (m, 4H),
3.66 (s, 3H), 4.40 (d, 1H, J = 9.0 Hz), 4.98 (m, 2H),
5.78 (m, 1H), and 6.23 (d, 1H, J = 9.0 Hz); 13C NMR
(CDCl3) d 6.39, 6.79, 27.66, 27.86, 29.31, 35.65, 51.64,
60.89, 74.49, 115.48, 136.75, 170.63, and 171.79; mass
spectrum (FAB), m/z 344.2255 (M+H)+ (C17H34NO4Si
requires m/z 344.2257).
4.1.14. N-((4-Pentenoyl)-S-2-amino))-3-triethylsilyloxy-
3-methylbutyric acid cyanomethyl ester (16). To a suspen-
sion containing 130 mg (0.37 mmol) of 15 in 3 mL of 3:1:1
THF/H2O/MeOH was added 46 mg (1.11 mmol) of
LiOH. The reaction mixture was stirred under argon at
room temperature and monitored by TLC; after 3 h the
starting material had been consumed, so the reaction mix-
ture was diluted with 60 mL of ethyl acetate and washed
with 25 mL of 1 N NaHSO4, dried (MgSO4), and concen-
trated under diminished pressure. The crude residue was
dissolved in anhydrous acetonitrile and 140 mg
(1.85 mmol) of chloroacetonitrile was added, followed
by 186 mg (1.85 mmol) of triethylamine. The reaction
mixture was stirred at room temperature overnight, then

4640
M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
spectrum (MALDI-TOF), m/z 1230.39 (M+) (theoretical
m/z 1230.46); bis-N-(4-pentenoyl)-O-tert-butyldimethyl-
added, followed by 2.2 mL (15.5 mmol) of triethyl-
amine. The reaction mixture was stirred at room temper-
ature overnight under argon. The reaction mixture was
then diluted with ethyl acetate, washed with 50 mL of
NaHCO3, 100 mL of brine, dried (MgSO4), and concen-
trated under diminished pressure. Purification by flash
chromatography on a silica gel column (20 · 3 cm) using
2:1 ethyl acetate/hexanes gave 27 as a colorless oil: yield
655 mg (75%); 1H NMR (CDCl3) d 1.76 (d, 3H,
J = 6.9 Hz), 2.25–2.35 (m, 7H), 4.22–4.25 (m, 1H), 4.77
(s, 2H), 5.02 (s, 2H), 5.65–5.75 (m, 2H), and 7.14 (br
s, 1H); 13C NMR (CDCl3) d 14.9, 25.7, 28.7, 29.6,
32.9, 35.7, 48.8, 116.5, 125.4, 136.9, 138.0,163.2, and
171.8; mass spectrum (FAB), m/z 283.1295 (M+H)+
(C13H19N2O5 requires m/z 283.1294).
silyl-S-homoseryl-pdCpA
(19c0)
(retention
time
33.8 min) was obtained as a colorless solid: yield
0.45 mg (10%); mass spectrum (MALDI-TOF), m/z
1253.89 (M+Na)+ (theoretical m/z 1253.45), bis-N-((4-
pentenoyl)-2-amino)-3-triethylsilyloxy-3-methylbutyryl)-
pdCpA (20) (retention time 33.9 min) was obtained as a
colorless solid: yield 0.46 mg (10%); mass spectrum
(MALDI-TOF), m/z 1257.55 (MꢁH)ꢁ (theoretical m/z
1257.48).
4.1.16. N-(4-Pentenoyl)aminoacyl-pdCpAs (21a–21c and
22) and bis-(N-4-pentenoyl)aminoacyl-pdCpAs (23a–23c
and 24). The silyl groups were removed from com-
pounds 17a0–17c0, 18, 19a0–19c0, and 20 by treatment
with 75 lL of 3:1:1 AcOH/THF/H2O.27 After stirring
for 3 h at 25 ꢁC the reaction mixture was diluted to a to-
tal volume of 200 lL of 1:1 CH3CN/50 mM NH4OAc,
pH 4.5, and purified using a semi-preparative C18 re-
versed phase column. After lyophilization of the appro-
priate fractions, N-(4-pentenoyl)-pdCpAs (21a–21c and
22) (retention times 13.5–14.0 min) were obtained as col-
orless solids; bis-(N-(4-pentenoyl)aminoacyl)-pdCpAs
(23a–23c and 24) (retention times 15.5–18 min) were ob-
tained as colorless solids. 21a: yield 67%; mass spectrum
(MALDI-TOF), m/z 834.04 (M+H)+ (theoretical m/z
834.22); 21b: yield 57%; mass spectrum (MALDI-
TOF), m/z 834.16 (M+H)+ (theoretical m/z 834.22);
21c: yield 55%; mass spectrum (MALDI-TOF), m/z
829.50 (MꢁH)ꢁ (theoretical m/z 829.18); 22: yield
60%; mass spectrum (MALDI-TOF), m/z 848.68
(M+H)+ (theoretical m/z 848.24); 23a: yield 24%; mass
spectrum (MALDI-TOF), m/z 1038.69 (M+Na)+ (theo-
retical m/z 1038.29); 23b: yield 18%; 23c: yield 25%; mass
spectrum (MALDI-TOF), m/z 1015.76 (M+) (theoretical
m/z 1015.30); 24: yield 20%.
4.1.18. O-Acetyl-N-(4-pentenoyl)-S-threonyl-pdCpA (28)
bis-(O-acetyl-N-(4-pentenoyl)-S-threonyl)-pdCpA
and
(29). To a conical vial containing 20 mg (56 lmol) of
O-acetyl-N-(4-pentenoyl)-S-threonine cyanomethyl es-
ter (27) was added a solution of the tetrabutylammoni-
um salt of 5.0 mg (3.64 lmol) of pdCpA15 in 100 lL of
anhydrous DMF, followed by 20 lL of triethylamine
˚
(over 4 A molecular sieves). The reaction mixture was
stirred at 25 ꢁC and monitored by HPLC. A 5-lL
aliquot of the mixture was removed after 24 h and
diluted with 45 lL of 1:2 CH3CN/50 mM NH4OAc,
pH 4.5. Twenty microliters of the diluted aliquot was
analyzed by HPLC on a C18 reversed phase column
(250 · 10 mm). The column was washed with a gradient
of CH3CN (0 ! 63%) in 50 mM NH4OAc, pH 4.5, over
a period of 35 min at a flow rate of 3.5 mL/min (monitor-
ing at 260 nm). The majority of the pdCpA salt had been
consumed at this point so the reaction mixture was di-
luted to a total volume of 500 lL of 1:1 CH3CN/50 mM
NH4OAc, pH 4.5, and purified using the same semi-pre-
parative C18 reversed phase column. After lyophilization
of the appropriate fractions, 30-O-acetyl-N-(4-pente-
noyl)-S-threonyl-pdCpA (28) (retention time 15.6 min)
was obtained as a colorless solid: yield 1.2 mg (38%); mass
spectrum (MALDI-TOF), m/z 862.2125 (M+H)+
(C30H42N9O17P2 requires m/z 862.2114), and bis-(O-acet-
4.1.17. O-Acetyl-N-(4-pentenoyl)-S-threonine cyanometh-
yl ester (27). To
a solution containing 500 mg
(4.20 mmol) of S-threonine (7a) which was dissolved in
1 mL of 6 N HCl, glacial acetic acid (1 mL) was added
and the solution was cooled to 0 ꢁC in an ice bath. Acet-
yl chloride was then added slowly. The O-acetoxyamino
acid chloride precipitated within a few minutes; quanti-
tative precipitation was realized by adding 2–3 vol of
ether. The solid was filtered, washed with ether, and
dried in vacuo. Compound 25 was obtained as a color-
less oil: yield 675 mg (100%).
yl-N-(4-pentenoyl)-S-threonyl)-pdCpA(29)
(retention
time 20.3 min) was obtained as a colorless solid: yield
1.59 mg (40%); mass spectrum (MALDI-TOF), m/z
1088.21 (M+2H)2+ (theoretical m/z 1088.32).
4.1.19. N-4-Pentenoyl-S-threonyl-pdCpA (21a) and bis-
N-4-pentenoyl-S-threonyl-pdCpA (23a). To a conical vial
containing 0.5 mg of compound 28 or compound 29
(5.73 lmol or 4.55 lmol, respectively) in 150 lL of dry
methanol was added 50 lL of a 54% solution of HBF4
in diethyl ether. The reaction mixture was stirred at
room temperature for 3 h. A 10-lL aliquot of the reac-
tion mixture was diluted with 40 lL of 1:1 acetonitrile/
50 mM NH4OAc, pH 4.5, and was analyzed by HPLC
on a C18 reversed phase column (250 · 10 mm). The col-
umn was washed with 0% ! 63% acetonitrile in 50 mM
NH4OAc, pH 4.5, over a period of 35 min at a flow rate
of 3.5 mL/min (monitoring at 260 nm). The remaining
reaction mixture was diluted to a total volume of
200 lL of 1:1 acetonitrile/50 mM NH4OAc, pH 4.5,
and purified using the same C18 reversed phase column.
To 500 mg (3.10 mmol) of 25 in 5 mL of H2O was added
521 mg (6.2 mmol) of NaHCO3. To this stirred solution
was added 794 mg (4.03 mmol) of 4-pentenoic acid suc-
cinimide ester29 in 5 mL of dioxane. The reaction mix-
ture was allowed to stir at room temperature under
argon overnight, then diluted with ethyl acetate and
washed with 1 N NaHSO4. The organic layer was sepa-
rated and the aqueous layer was back-extracted with
two 25-mL portions of ethyl acetate. The combined or-
ganic phase was washed with 50 mL of brine, dried over
MgSO4, and concentrated under diminished pressure.
The crude material was dissolved in 10 mL of acetoni-
trile and 1.0 mL (15.5 mmol) of chloroacetonitrile was

M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
4641
Dinucleotide derivative 21a (retention time ꢀ14.0 min)
was recovered from the appropriate fractions by lyoph-
ilization as a colorless solid: yield 0.4 mg (81%); mass
spectrum (MALDI-TOF) m/z 828.70 (Mꢁ2H)ꢁ (theo-
retical 828.17). Dinucleotide derivative 23a (retention
time 18.2 min) was recovered from the appropriate frac-
tions by lyophilization as a colorless solid: yield 0.2 mg
(43%); mass spectrum (MALDI-TOF) m/z 1012.14
(Mꢁ2H)ꢁ (theoretical 1012.27).
amino acids lacking methionine, 50 lM methionine,
40 lCi of [35S]-S-methionine, and 30 lL of S-30 extract
that had been heat treated at 42 ꢁC for 6 min.31Suppres-
sion reaction mixtures (100 lL) contained 25 lg of
deprotected misacylated tRNACUA and were incubated
at 37 ꢁC. Aliquots from in vitro translation reactions
were removed for analysis by 15% SDS–PAGE.32 Auto-
radiography of the gels was carried out to determine the
location of 35S-labeled protein. Quantification of the
bands was carried out using a phosphorimager. Sup-
pression efficiency was calculated as the percentage of
the protein produced via nonsense codon suppression
relative to the production of wild-type protein.
4.2. Preparation of bisaminoacyl-tRNACUAs
Bisaminoacyl-tRNAs were prepared by a T4 RNA ligase-
mediated ligation of the chemically synthesized pdCpA
derivatives (6a and 6b) with the abbreviated suppressor
.
15,24,25 Ligation reactions utilized 100 lL (to-
Acknowledgment
tRNA-COH
tal volume) of 50 mM Na Hepes, pH 7.5, containing
0.5 mM ATP, 15 mM MgCl2, 50 lg of tRNACUA-COH
This work was supported by NIH Research Grant CA
77359, awarded by the National Cancer Institute.
,
1.0 A260 unit of N-pentenoyl-protected bisaminoacyl-
pdCpA derivatives (5- to 10-fold molar excess), 15%
DMSO, and 200 U of T4 RNA ligase. After incubation
at 37 ꢁC for 45 min, the reaction was quenched by adding
0.1 vol of cold EtOH, and the product was collected by
centrifugation, washed with 70% EtOH, and dried. The
tRNA was dissolved in 1 mM KOAc to a final concentra-
tion of 5 lg/lL. The ligation efficiency was estimated by
gel electrophoresis at pH 4.5.26 Deprotection of pente-
noyl-containing aminoacyl-tRNAs utilized 100 lL of
reaction mixtures containing 5 lg/lL of N-pentenoylami-
noacyl-tRNA, 15% THF, and 8 mM I2 (incubation at
25 ꢁC for 50 min). The deprotected bisaminoacyl-tRNAs
were precipitated by addition of 0.1 vol of 3 M NaOAc,
pH 5.2, and then 2.5 vol of cold EtOH. The samples were
collected by centrifugation. The tRNA pellets were
washed with 70% EtOH, centrifuged, and dried. The
deprotected bisaminoacyl-tRNAs were dissolved in
1 mM KOAc to a final concentration of 5 lg/lL and
stored in aliquots at ꢁ80 ꢁC.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at Supplementary data
associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2007.03.088.
References and notes
1. Kozak, M. Microbiol. Rev. 1983, 47, 1.
2. Merrick, W. C. Microbiol. Rev. 1992, 56, 291.
3. Kozak, M. Gene 1999, 234, 187.
4. Hecht, S. M. Acc. Chem. Res. 1992, 25, 545.
5. Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S.
J. Biol. Chem. 1978, 253, 4517.
6. Heckler, T. G.; Chang, L. H.; Zama, Y.; Naka, T.;
Chorghade, M. S.; Hecht, S. M. Tetrahedron 1984, 40, 87.
7. Heckler, T. G.; Chang, L. H.; Zama, Y.; Naka, T.;
Chorghade, M. S.; Hecht, S. M. Biochemistry 1984, 23,
1468.
4.3. Synthesis of DHFR mRNA by in vitro transcription
Plasmid pTHD(ꢁ1) was linearized with BamHI and
transcribed using AmpliScribe T7 transcription kit.25
The transcribed mRNA solution was extracted succes-
sively with phenol and chloroform, then precipitated
by the addition of 2.5 vol of EtOH, washed with 70%
EtOH, and dried. The mRNA was dissolved in water
and stored at ꢁ80 ꢁC.
8. Heckler, T. G.; Zama, Y.; Naka, T.; Hecht, S. M. J. Biol.
Chem. 1983, 258, 4492.
9. Roesser, J. R.; Xu, C.; Payne, R. C.; Surratt, C. K.; Hecht,
S. M. Biochemistry 1989, 28, 5185.
10. Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.;
Schultz, P. G. Science 1989, 244, 182.
11. Xie, J.; Schultz, P. G. Methods 2005, 36, 227.
12. Xie, J.; Schultz, P. G. Curr. Opin. Chem. Biol. 2005, 9, 548.
13. Tan, Z.; Blacklow, S. C.; Cornish, V. W.; Forster, A. C.
Methods 2005, 36, 279.
4.4. In vitro protein translation reactions
14. Josephson, K.; Hartman, M. C. T.; Szostak, J. W. J. Am.
Chem. Soc. 2005, 127, 11727.
15. Robertson, S. A.; Noren, C. J.; Anthony-Cahill, S. J.;
Griffith, M. C.; Schultz, P. G. Nucleic Acid Res. 1989, 17,
9649.
16. Stepanov, V. G.; Moor, N. A.; Ankilova, V. N.; Lavrik,
O. I. FEBS Lett. 1992, 311, 192.
17. Stepanov, V. G.; Moor, N. A.; Ankilova, V. N.; Vasil’eva,
I. A.; Sukhanova, M. V.; Lavrik, O. I. Biochim. Biophys.
Acta 1998, 1386, 1.
Suppression of a UAG stop codon at position ꢁ1 of
DHFR was performed in an Escherichia coli S-30 sys-
tem.18 Translation reactions were carried out in 10–
2000 lL reaction mixtures that contained the following
per 100 lL: 10 lg of plasmid DNA dissolved in diethyl
pyrocarbonate-treated water, 40 lL of premix [35 mM
Tris–acetate (pH 7.0), 190 mM potassium glutamate,
30 mM ammonium acetate, 2 mM dithiothreitol,
11 mM magnesium acetate, 20 mM phosphoenolpyr-
uvate, 0.8 mg/mL E. coli tRNA, 0.8 mM isopropyl b-
D-thiogalactopyranoside, 20 mM ATP and GTP,
5 mM CTP and UTP, and 10 mM cAMP],30 100 lM
18. Wang, B.; Zhou, J.; Lodder, M.; Anderson, R. D.; Hecht,
S. M. J. Biol. Chem. 2006, 281, 13865.
19. Lodder, M.; Golovine, S.; Hecht, S. M. J. Org. Chem.
1997, 62, 778.

4642
M. Duca et al. / Bioorg. Med. Chem. 15 (2007) 4629–4642
20. Lodder, M.; Golovine, S.; Laikhter, A. L.; Karginov, V.
A.; Hecht, S. M.. J. Org. Chem. 1998, 63, 794.
21. Robertson, A. A.; Ellman, J. A.; Schultz, P. G. J. Am.
Chem. Soc. 1991, 11, 2722.
22. Wilchek, M.; Patchornik, A. J. Org. Chem 1964, 29, 1629.
23. Fahmi, N. E.; Golovine, S.; Wang, B.; Hecht, S. M.
Carbohydr. Res. 2001, 330, 149.
24. Noren, C. J.; Anthony-Cahill, S. J.; Suich, D. J.; Noren,
K. A.; Griffith, M. C.; Schultz, P. G. Nucleic Acids Res.
1990, 18, 83.
25. 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.
26. Varshney, V.; Lee, C. P.; RajBhandary, U. L. J. Biol.
Chem. 1991, 266, 24712.
27. Chung, H. H.; Benson, D. R.; Cornish, V. W.; Schultz, P.
G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10145.
28. Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm.
Bull. 1981, 29, 1475.
29. Lodder, M.; Wang, B.; Hecht, S. M. Methods 2005, 36,
245.
30. Pratt, J. M. Transcription and Translation: A Practical
Approach; IRL Press: Oxford, 1984, pp 179–209.
31. Short, G. F., III; Golovine, S. Y.; Hecht, S. M. Biochem-
istry 1999, 38, 8808.
32. Laemmli, U. K. Nature 1970, 227, 680.