Misacylated Transfer RNAs Having a Chemically Removable Protecting Group

Article abstract of DOI:10.1021/jo971692l

The 4-pentenoyl group and a number of derivatives have been studied as protecting groups for N^α of the aminoacyl moiety in misacylated tRNAs. The unsubstituted 4-pentenoyl group itself was found to function as efficiently as any of the derivatives studied. Four different N-(4-pentenoyl)aminoacyl-tRNA_CUAS were prepared and shown to undergo deprotection readily upon admixture of aqueous iodine; the derived misacylated tRNAs all functioned well as suppressors of a nonsense codon in an in vitro protein biosynthesizing system. Also prepared were four N^α-(4-pentenoyl)-aspartyl-tRNA_CUAS that were protected on the side chain carboxylate as the nitroveratryl ester. Following treatment with aqueous iodine, the misacylated suppressor tRNAs incorporated the aspartate derivatives into position 27 of dihydrofolate reductase by suppression of a UAG codon in the mRNA. The suppression yields were significantly better than those obtained when side chain protection was absent. The resulting "caged proteins" were inactive, but full catalytic potential was restored by irradiation under conditions sufficient to effect deprotection of the side chain carboxylate moiety.

Full text of DOI:10.1021/jo971692l

794
J . Org. Chem. 1998, 63, 794-803
Misa cyla ted Tr a n sfer RNAs Ha vin g a Ch em ica lly Rem ova ble
P r otectin g Gr ou p
Michiel Lodder, Serguei Golovine, Andrei L. Laikhter, Vladimir A. Karginov, and
Sidney M. Hecht*
Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901
Received September 10, 1997
The 4-pentenoyl group and a number of derivatives have been studied as protecting groups for NR
of the aminoacyl moiety in misacylated tRNAs. The unsubstituted 4-pentenoyl group itself was
found to function as efficiently as any of the derivatives studied. Four different N-(4-pentenoyl)-
aminoacyl-tRNACUAs were prepared and shown to undergo deprotection readily upon admixture of
aqueous iodine; the derived misacylated tRNAs all functioned well as suppressors of a nonsense
codon in an in vitro protein biosynthesizing system. Also prepared were four NR-(4-pentenoyl)-
aspartyl-tRNACUAs that were protected on the side chain carboxylate as the nitroveratryl ester.
Following treatment with aqueous iodine, the misacylated suppressor tRNAs incorporated the
aspartate derivatives into position 27 of dihydrofolate reductase by suppression of a UAG codon in
the mRNA. The suppression yields were significantly better than those obtained when side chain
protection was absent. The resulting “caged proteins” were inactive, but full catalytic potential
was restored by irradiation under conditions sufficient to effect deprotection of the side chain
carboxylate moiety.
Since the pioneering work of the Lipmann laboratory
occurs, the product of the ligation reaction is a full length,
deacylated tRNA.
which demonstrated that a tRNA bearing a noncognate
amino acid could be employed for the elaboration of a
modified protein,1 there has been great interest in the
elaboration of misacylated tRNAs. Initial efforts focused
on the chemical modification of tRNAs bearing a cognate
amino acid,1,2 but the Hecht laboratory described a
potentially general strategy in which tRNAs lacking the
3′-terminal dinucleotide, which is always pCpA, were
converted to misacylated tRNAs by incubation with an
aminoacylated pCpA derivative in the presence of T4
RNA ligase.3
Several improvements in the methodology have greatly
facilitated the preparation of misacylated tRNAs, includ-
ing the use of aminoacylated pdCpA derivatives4 and in
vitro RNA transcripts elaborated without the normal 3′-
terminal CA moiety.5 The greatest challenge, however,
has involved the aminoacyl moiety itself, which is not
completely stable under the conditions of the ligation
reaction; to the extent that hydrolysis of this group
Strategies for obtaining ligated tRNAs bearing the
aminoacyl moiety of interest have included the use of
unprotected aminoacyl-pCpA derivatives in a ligation
reaction of limited duration6 and the use of protecting
groups for NR of the amino acid. The latter afford
increased stability of the aminoacyl moiety toward hy-
drolysis, but their subsequent removal without concomi-
tant deacylation of the amino acid from the misacylated
tRNA has proved problematic. Until recently, the most
successful protecting groups had been the pyroglutamyl3i
and nitroveratryloxycarbonyl (NVOC)7 groups; these can
be removed from the derived misacylated tRNAs via the
agency of an enzyme (pyroglutamate aminopeptidase)
and light, respectively. Chemical deprotection strategies
have not been used extensively.8
On the basis of the work of Fraser-Reid and co-
workers9 who studied the protection of simple amines,
we have recently described the use of the N-pentenoyl
group as a suitable aminoacyl protecting group for the
preparation of misacylated tRNAs.10 This group can be
removed under very mild chemical conditions by treat-
ment with aqueous iodine, presumably via an iodolac-
tonization reaction (Scheme 1). That this reagent is
(1) Chapeville, F.; Lipmann, F.; von Ehrenstein, G.; Weisblum, B.;
Roy, W. J .; Benzer, S. Proc. Natl. Acad. Sci. U.S.A. 1962, 48, 1086.
(2) (a) Fahnestock, S.; Rich, A. Nature, New Biol. 1971, 229, 8. (b)
Fahnestock, S.; Rich, A. Science 1971, 173, 340. (c) J ohnson, A. E.;
Woodward, W. R.; Herbert, E.; Menninger, J . R. Biochemistry 1976,
15, 569.
(3) (a) Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. J . Biol.
Chem. 1978, 253, 4517. (b) Pezzuto, J . M.; Hecht, S. M. J . Biol. Chem.
1980, 255, 865. (c) Heckler, T. G.; Zama, Y.; Naka, T.; Hecht, S. M. J .
Biol. Chem. 1983, 258, 4492. (d) Heckler, T. G.; Chang, L. H.; Zama,
Y.; Naka, T.; Hecht, S. M. Tetrahedron 1984, 40, 87. (e) Heckler, T.
G.; Chang, L. H.; Zama, Y.; Naka, T.; Chorghade, M. S.; Hecht, S. M.
Biochemistry 1984, 23, 1468. (f) Roesser, J . R.; Chorghade, M. S.; Hecht,
S. M. Biochemistry 1986, 25, 6361. (g) Payne, R. C.; Nichols, B. P.;
Hecht, S. M. Biochemistry 1987, 26, 3197. (h) Heckler, T. G.; Roesser,
J . R.; Cheng, X.; Chang, P.-I.; Hecht, S. M. Biochemistry 1988, 27, 7254.
(i) Roesser, J . R.; Xu, C.; Payne, R. C.; Surratt, C. K.; Hecht, S. M.
Biochemistry 1989, 28, 5185. (j) Hecht, S. M. Acc. Chem. Res. 1992,
25, 545.
(6) Baldini, G.; Martoglio, B.; Schachenmann, A.; Zugliani, C.;
Brunner, J . Biochemistry 1988, 27, 7951.
(7) Robertson, S. A.; Ellman, J . A.; Schultz, P. G. J . Am. Chem. Soc.
1991, 113, 2722.
(8) See, however, ref 3a and (a) Noren, C. J .; Anthony-Cahill, S. J .;
Griffith, M. C.; Schultz, P. G. Science 1989, 244, 182. (b) Mendel, D.;
Ellman, J . A.; Schultz, P. G. J . Am. Chem. Soc. 1991, 113, 2758.
(9) (a) Debenham, J . S.; Madsen, R.; Roberts, C.; Fraser-Reid, B. J .
Am. Chem. Soc. 1995, 117, 3302. (b) Madsen, R.; Roberts, C.; Fraser-
Reid, B. J . Org. Chem. 1995, 60, 7920.
(10) Preliminary results on the preparation of N-(4-pentenoyl)valyl-
tRNA have been published: Lodder, M.; Golovine, S.; Hecht, S. M. J .
Org. Chem. 1997, 62, 778.
(4) Robertson, S. A.; Noren, C. J .; Anthony-Cahill, S. J .; Griffith,
M. C.; Schultz, P. G. Nucleic Acids Res. 1989, 17, 9649.
(5) 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.
(11) Letsinger, R. L.; Lunsford, W. B. J . Am. Chem. Soc. 1976, 98,
3655.
S0022-3263(97)01692-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/22/1998

Misacylated Transfer RNAs
J . Org. Chem., Vol. 63, No. 3, 1998 795
Sch em e 1. P u ta tive Mech a n ism of Deblock in g of
P en ten oyl Der iva tives via a n Iod ola cton iza tion
Rea ction
Sch em e 2. Syn th esis of N-Block ed Va lin e Ben zyl
Ester s
employed in the chemical synthesis of nucleic acids11
suggested that its use would be compatible with the
integrity of the derived misacylated tRNAs.
Presently, we describe the investigation of nine differ-
ent N-pentenoyl derivatives as potential protecting groups
for misacylated tRNAs, and provide a complete descrip-
tion of the preparation and assay of four misacylated
susppressor tRNAs. Also described is the elaboration of
four suppressor tRNAs misacylated with aspartic acid
(analogues) bearing a protecting group on the side chain
carboxylate whose removal obtains under conditions
orthogonal to those employed for removal of the pentenoyl
group. Side chain protection was demonstrated to sub-
stantially increase the suppression of nonsense codons
during translation and provided access to caged ana-
logues12 of dihydrofolate reductase (DHFR); subsequent
deblocking of the caged proteins afforded catalytically
active analogues of DHFR.
Ta ble 1. Dep r otection of P en ten oyl Der iva tives 2-10
deprotection
deprotection
Resu lts
compound
after 5 min (%)a
compound
after 5 min (%)a
2
3
4
5
6
92
70
0
0
0
7
8
9
<3
<3
<3
18b
Syn th esis a n d Dep r otection of N-P r otected Va -
lin e Ben zyl Ester s. (S)-Valine benzyl ester was used
as a model compound for investigation of protecting
groups that could be removed via an iodolactionization
reaction. The modified pentenoic acids were attached to
(S)-valine benzyl ester p-toluenesulfonate13 by N,N′-
dicyclohexylcarbodiimide (DCC) or N,N-bis(2-oxo-3-ox-
azolidinyl)phosphinic chloride (BOP-Cl)14-mediated cou-
pling (Scheme 2). The yield of N-(4-pentenoyl)-(S)-valine
benzyl ester (2) (87%) was comparable to the yield
achieved previously for the introduction of the pentenoyl
group using pentenoic anhydride.9
Removal of the protecting groups by treatment with
iodine was then studied. Thus compounds 2-10 were
dissolved in aqueous THF and treated with 3 equiv of
iodine. After 5 min the reactions were quenched with
solid Na2S2O3 and concentrated under diminished pres-
sure. After purification the formed (S)-valine benzyl
ester was converted to the p-toluenesulfonate salt and
the yield of isolated amino acid was calculated (Table 1).
As shown in the table, two of the pentenoyl derivatives
(3 and 10) were converted to valine benzyl ester to a
significant extent within 5 min upon admixture of I2, but
neither was converted more efficiently than the parent
N-pentenoyl derivative on this time scale. Most of the
derivatives were deblocked very inefficiently and ana-
10
a
Percentage of deprotection is based on the amount of isolated
valine benzyl ester after purification. Also isolated in 29% yield
was iodohydrin derivative 10a .
b
logues (4-6) could not be deblocked at all under these
conditions. Interestingly, treatment of 10 with I2 pro-
duced iodohydrin 10a in 29% yield in addition to valine
benzyl ester. The iodohydrin presumably arises from
solvolytic ring opening of the iminolactone intermediate
resulting from iodolactonization (vide supra).
Syn th esis of N-(4-P en ten oyl)a m in oa cyl-p d Cp A
Ester s. Aminoacyl-pdCpA esters 14a -d were prepared
as shown in Scheme 3. Methyl esters 11a -d were NR-
protected by N,N′-dicyclohexylcarbodiimide-mediated cou-
pling with 4-pentenoic acid, affording amides 12a -d .
Subsequent hydrolysis of the esters with LiOH in aque-
ous THF and reaction with chloroacetonitrile gave the
respective cyanomethyl esters 13a -d .15 Admixture of
the active esters with the tris(tetrabutylammonium) salt
of pdCpA4 in DMF provided N-(4-pentenoyl)aminoacyl-
pdCpA esters 14a -d in good yield. For comparative
purposes in the deblocking experiments, N-(6-nitrovera-
(12) For a review on caged compounds, see: Adams, S. R.; Tsien, R.
Y. Annu. Rev. Physiol. 1993, 55, 755.
(13) Zervas, L.; Winitz, M.; Greenstein, J . P. J . Org. Chem. 1957,
22, 1515.
(14) Tung, R. D.; Rich, D. H. J . Am. Chem. Soc. 1985, 107, 4342.
(15) Bodanszky, M.; Bodanszky, A. In The Practice of Peptide
Synthesis, 2nd ed.; Springer-Verlag: Berlin, Heidelberg, New York,
1994; p 96.

796 J . Org. Chem., Vol. 63, No. 3, 1998
Lodder et al.
Sch em e 4. Syn th esis a n d Dep r otection of
N-(4-P en ten oyl)Am in oa cyl-tRNAs
F igu r e 1. HPLC analysis of deprotection of valyl-pdCpAs. (A)
Deprotection of N-(4-pentenoyl)valyl-pdCpA by treatment with
aqueous iodine for 5 min at 25 °C. (B) Deprotection of N-(6-
nitroveratryloxycarbonyl)valyl-pdCpA by irradiation with a
500 W mercury-xenon lamp for 5 min at 2 °C. The deblocked
valyl-pdCpA eluted at 11.2 min. Co-injection of the reaction
mixtures confirmed comigration of the products obtained from
both deprotection reactions.
Sch em e 3. Syn th esis of
N-(4-P en ten oyl)Am in oa cyl-p d Cp As
The purity of the product obtained from 14a also com-
pared favorably with that accessible from (NVOC)valyl-
pdCpA.
N-(4-Pentenoyl)methionyl-pdCpA (14c) was deblocked
in the same fashion with iodine and the product was
compared with methionyl-pdCpA accessible by deblocking
of (NVOC)methionyl-pdCpA. Again, the deblocked me-
thionyl-pdCpAs comigrated when analyzed by reverse
phase HPLC (not shown).
Misa cyla t ed t R NA P r ep a r a t ion a n d Use in in
Vitr o P r otein Syn th esis. Preparation of misacylated
tRNAs was accomplished by T4 RNA ligase-mediated
coupling of 2′(3′)-O-[N-(4-pentenoyl)]aminoacyl-pdCpAs
Phe
14a -d with yeast suppressor tRNACUA
transcripts
lacking the 3′-terminal cytidine and adenosine moieties
(Scheme 4).3-5,7 The resulting N-(4-pentenoyl)aminoacyl-
tRNAs (15a -d ) were deprotected in aqueous THF con-
taining 5 mM iodine, affording misacylated suppressor
tRNAs 16a -d .
The misacylated tRNAs so prepared were then used
in an in vitro protein biosynthesizing system that em-
ployed rabbit reticulocyte lysate and an mRNA for
dihydrofolate reductase (DHFR) containing a nonsense
(UAG) codon at position 10.16 As shown in Table 2, the
synthesis of DHFR, which involved readthrough of the
UAG codon by a misacylated suppressor tRNA, proceeded
in reasonably good yield for each misacylated tRNACUA
studied. No synthesis of DHFR was observed if iodine
treatment was omitted. Direct comparison with valyl-
tRNA prepared by photodeprotection of N-(6-nitrovera-
tryloxycarbonyl)valyl-tRNA showed that the reaction
proceeded to the same extent for both deblocked valyl-
tRNAs.
tryloxycarbonyl)valyl-pdCpA and N-(6-nitroveratryloxy-
carbonyl)methionyl-pdCpA were prepared by a known
method.7
Valine derivative 14a was deprotected by treatment
with iodine in aqueous THF. HPLC analysis (Figure 1)
of the reaction mixture showed complete deprotection
within 5 min. To ensure the identity of the products, the
N-(6-nitroveratryloxycarbonyl)valyl-pdCpA, which is
known7 to be converted to valyl-pdCpA upon irradiation
with a 500 W mercury-xenon lamp, was deblocked and
the product compared with that obtained from 14a . As
shown in the Figure, the major products from both
protected valyl-pdCpA derivatives had essentially the
same retention times; co-injection confirmed that they
were indistinguishable by reverse phase HPLC analysis.
(16) 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, 8166-8176.

Misacylated Transfer RNAs
Ta ble 2. Efficien cy of Su p p r ession by
J . Org. Chem., Vol. 63, No. 3, 1998 797
Sch em e 5. Syn th esis of γ-Nitr over a tr yl
N-(4-P en ten oyl)Asp a r tyl-p d Cp As
Am in oa cyl-tRNACUA a t P osition 10 of Dih yd r ofola te
Red u cta se
suppression
amino acid
efficiency (%)a
valineb
30
25
17
20
30
valinec
methionine
alanine
phenylalanine
a
Defined as the percentage of DHFR produced via nonsense
codon suppression relative to production of DHFR from wild-type
b
mRNA. In the absence of I2 treatment of N-(4-pentenoyl)valyl-
tRNACUA, the suppression efficiency was <1%. c From N-(nitro-
veratryloxycarbonyl)-(S)-valyl-tRNA.
F igu r e 2. Effect of time of deprotection of N-(4-pentenoyl)-
valyl-tRNA on the production of DHFR. Aliquots of N-(4-
pentenoyl)valyl-tRNA taken at predetermined times after
treatment with iodine were added to in vitro protein synthesis
reactions programmed with an mRNA containing a nonsense
codon (UAG) at position 10. More than 90% of the modified
protein was obtained in the first 10 min of the reaction,
suggesting that deprotection was almost complete after 10 min.
As shown in Figure 2, the use of aliquots of N-(4-
pentenoyl)valyl-tRNA treated with iodine for varying
lengths of time prior to addition to the protein biosyn-
thesizing system afforded maximal yields of full length
protein after 5-10 min of iodine treatment. The yields
of DHFR did not diminish if longer I2 treatment was
employed. Because a substantial percentage of all of the
activated valine moieties are incorporated into protein,
this suggested that deprotection was largely complete
within 5-10 min. In addition, N-(4-pentenoyl)-(S)-valyl-
tRNA proved to be stable in cacodylate buffer, pH 7.4,
at 37 °C for at least 24 h (data not shown).
In the same fashion as described for dimethyl (S)-
aspartate (17), dimethyl threo-â-methyl-(S)-aspartate,19
dimethyl erythro-â-methyl-(S)-aspartate,19 and dimethyl
â,â-dimethyl-(S)-aspartate19 were converted to their re-
spective γ-nitroveratryl N-(4-pentenoyl)aspartyl-pdCpA
derivatives (22a -c).
Sid e-Ch a in -P r otected Asp a r tyl-tRNAs. P r ep a r a -
tion of Asp a r tyl-tRNAs a n d in Vitr o P r otein Syn -
th esis. Aspartyl-pdCpAs 21 and 22a -c were ligated
Ala
onto an abbreviated Escherichia coli tRNACUA tran-
Syn th esis of Sid e-Ch a in -P r otected N-(4-P en ten -
oyl)a sp a r tyl-p d Cp As. Dimethyl (S)-aspartate (17) was
NR protected with the 4-pentenoyl group (Scheme 5). The
resulting amide 18 was treated with NaOH in aqueous
THF and then with 6-nitroveratryl bromide17 and CsF18
to afford nitroveratryl diester 19. Selective hydrolysis
of the R-ester with LiOH and treatment with chloro-
acetonitrile gave cyanomethyl ester 20. Reaction of 20
with the tris(tetrabutylammonium) salt of pdCpA in
DMF afforded γ-nitroveratryl N-(4-pentenoyl)aspartyl-
pdCpA (21) as a pale yellow foam.
script as described for pdCpA derivatives 14a -d . The
resulting aspartyl-tRNAs were treated with iodine and
employed for the in vitro synthesis of DHFR (Figure 3).
A DHFR mRNA containing a nonsense (UAG) codon at
position 27 was used as described previously.16 Ana-
logues of DHFR containing side-chain-protected aspartyl
derivatives at position 27 were obtained in good yields
(Table 3). A 2- to 3-fold increase in suppression efficiency
was observed as compared to aspartyl-tRNACUA deriva-
tives lacking side chain protection.16
(18) Dijkstra, G.; Kruizinga, W. H.; Kellogg, R. M. J . Org. Chem.
1987, 52, 4230.
(19) Crasto, C. F.; Laikhter, A. L.; An, H.; Lodder, M.; Hecht, S. M.
Manuscript in preparation.
(17) Wilcox, M.; Viola, R. W.; J ohnson, K. W.; Billington, A. P.;
Carpenter, B. K.; McCray, J . A.; Guzikowski, A. P.; Hess, G. P. J . Org.
Chem. 1990, 55, 1585.

798 J . Org. Chem., Vol. 63, No. 3, 1998
Lodder et al.
peptides3j,21,22 and proteins.16,23 Because the most ver-
satile method for the preparation of such misacylated
tRNAs involves the T4 RNA ligase-mediated condensa-
tion of an abbreviated tRNA (tRNA-COH) with an N-
protected aminoacyl-pdCpA derivative,3-5,7 improvements
in the nature of the protecting group may contribute
importantly to further development of this technology.
F igu r e 3. Elaboration of dihydrofolate reductase analogues
containing derivatives of aspartic acid with 6-nitroveratryl
protected side chain carboxylates at position 27. Protein
synthesis was carried out in the presence of 35S-methionine
using a rabbit reticulocyte lysate, mRNA containing a non-
sense codon at position 27, and no suppressor tRNA (lane 1)
or a suppressor tRNA activated with one of several amino acids
(lanes 2-5). Following incubation at 30 °C for 1 h, the reactions
were analyzed on a 20% SDS-polyacrylamide gel: lane 1, no
tRNACUA; lane 2, aspartic acid; lane 3, â,â-dimethylaspartic
acid; lane 4, erythro-â-methylaspartic acid; lane 5, threo-â-
A protecting group that can be removed by treatment
with aqueous iodine is very attractive because the
conditions under which this reaction occurs are very mild.
The deprotection reaction is believed to proceed via
iminolactone formation, followed by hydrolysis of the
formed intermediate (Scheme 1). All of the pentenoyl
derivatives tested, except compounds 4-6, could be
removed by treatment with 3 equiv of iodine in aqueous
THF. Contrary to what was expected, none of the
derivatives was removed more efficiently than the un-
modified pentenoyl group. It was anticipated, for ex-
ample, that the dimethyl pentenoyl derivative would
exhibit more facile cyclization based on the ”gem-di-
methyl” effect.24 It has been shown that gem-dialkyl
derivatives undergo faster cyclization in this type of
reaction than unsubstituted derivatives. The absence of
any change in the rate of the reaction suggests that the
rate-determining step in the deprotection reaction may
not be the cyclization per se, but rather the hydrolysis
of the formed intermediate. This conclusion is also
supported by the fact that none of the diphenyl deriva-
tives (7-10) was removed faster than the unsubstituted
pentenoyl derivative. In previous studies25 on diphenyl
derivatives of pentenoic acid, an increase in the rate of
the reaction was observed. The additional trans double
bonds in compounds 4 and 5 clearly did not have a
facilitating effect on the rate of deblocking. The fact that
methylaspartic acid; lane 6, wild-type mRNA, no tRNACUA
.
Ta ble 3. Efficien cy of Su p p r ession by
Asp a r tyl-tRNACUAs a t P osition 27 of Dih yd r ofola te
Red u cta se
amino acid
aspartic acid
â,â-dimethylaspartic acid
erythro-â-methylaspartic acid
threo-â-methylaspartic acid
suppression efficiencya
26 (11)
20 (16)
35 (7)
26 (10)
a
Yields in parentheses were obtained in the absence of side
chain protection.16
(20) Baccanari, D.; Phillips, A.; Smith, S.; Sinski, D.; Burchall, J .
Biochemistry 1975, 14, 5267.
(21) Killian, J . A.; Van Cleve, M. D.; Shayo, Y. F., Hecht, S. M.
Manuscript in preparation.
(22) (a) Bain, J . D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R.; Diala,
E. S. J . Am. Chem. Soc. 1989, 111, 8013. (b) Bain, J . D.; Wacker, D.
A.; Kuo, E. E.; Chamberlin, A. R. Tetrahedron 1991, 47, 2389. (c) Bain,
J . D.; Switzer, C.; Benner, S. A.; Chamberlin, A. R. Nature 1992, 356,
537.
(23) (a) Noren, C. J .; Anthony-Cahill, S. J .; Griffith, M. C.; Schultz,
P. G. Science 1989, 244, 182. (b) Ellman, J .; Mendel, D.; Anthony-
Cahill, S.; Noren, C. J .; Schultz, P. G. Methods Enzymol. 1991, 202,
301. (c) Ellman, J . A.; Mendel, D.; Schultz, P. G. Science 1992, 255,
197. (d) Ellman, J . A.; Volkman, B. F.; Mendel, D.; Schultz, P. G.;
Wemmer, D. E. J . Am. Chem. Soc. 1992, 114, 7959. (e) Mendel, D.;
Ellman, J . A.; Chang, Z.; Veenstra, D. L.; Kollman, P. A.; Schultz, P.
G. Science 1993, 256, 1798. (f) J udice, J . K.; Gamble, T. R.; Murphy,
E. C.; deVos, A. M.; Schultz, P. G. Science 1993, 261, 1578. (g) Chang,
H.-H.; Benson, D. R.; Schultz, P. G.. Science 1993, 259, 806. (h) Mendel,
D.; Ellman, J . A.; Schultz, P. G. J . Am. Chem. Soc. 1993, 115, 4359. (i)
Cornish, V. W.; Benson, D. R.; Altenbach, C. A.; Hideg, K.; Hubbell,
W. L.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2910. (j)
Thorson, J .; Chapman, E.; Murphy, E. C.; Schultz, P. G., J udice J . K.
J . Am. Chem. Soc. 1995, 117, 1157. (k) Cornish, V. W.; Mendel, D.;
Schultz, P. G. Angew. Chem., Int. Ed. Engl. 1995, 34, 621. (l) Nowak,
M. W.; Kearney, P. C.; Sampson, J . R.; Saks, M. E.; Labarca, C. G.;
Silverman, S. K.; Zhong, W.; Thorson, J .; Abelson, J . N.; Davidson,
N.; Schultz, P. G.; Dougherty, D. A.; Lester, H. A. Science 1995, 268,
439. (m) Mamaev, S. V.; Laikhter, A. L.; Hecht, S. M. J . Am. Chem.
Soc. 1996, 118, 7243. (n) Saks, M. E.; Sampson, J . R.; Nowak, M. W.;
Kearney, P. C.; Du, F.; Abelson, J . N.; Lester, H. A.; Dougherty, D. A.
J . Biol. Chem. 1996, 271, 23169. (o) Steward, L. E.; Collins, C. S.;
Gilmore, M. A.; Carlson, J . E.; Ross, J . B. A.; Chamberlin, A. R. J .
Am. Chem. Soc. 1997, 119, 6. (p) Arslan, T.; Mamaev, S. V.; Mamaeva,
N. V.; Hecht, S. M. J . Am. Chem. Soc. 1997, 119, 10877-10887.
(24) (a) Beesley, R. M.; Ingold, C. K.; Thorpe, J . F. J . Chem. Soc.
1915, 1080. (b) Ingold, C. K. J . Chem. Soc. 1921, 305. (c) Capon, B.;
McManus, S. P. Neighboring Group Participation; Plenum: New York,
1976; Vol. 1, pp 58-70. (d) Hill, E. A.; Link, D. C.; Donndelinger, P. J .
Org. Chem. 1981, 46, 1177.
F igu r e 4. Irradiation of side-chain-protected (inactive) ana-
logues of DHFR for 30 min yields catalytically active proteins
by removing the side chain protecting group.
Activation of Modified P r otein s. The derived DHFR
analogues containing a side chain protecting group on
Asp-27 had essentially no activity in the reduction of
dihydrofolate to tetrahydrofolate. Irradiation of the
modified proteins for 30 min with a 500 W mercury-
xenon lamp provided deblocked DHFRs (Figure 4) which
had activity comparable to those of DHFRs prepared
without side chain protection. Enzymatic activity assays
were carried out as described previously.20 The relative
catalytic efficiencies of the modified DHFRs containing
erythro-â-methylaspartic acid, threo-â-methylaspartic acid,
and â,â-dimethylaspartic acid were about 85%, 75%, and
75%, respectively, of that determined for DHFR contain-
ing Asp-27.
Discu ssion
The availability of a variety of misacylated tRNAs has
permitted the study of the nature of the peptidyltrans-
ferase reaction3c-f,h,21 and the elaboration of modified
(25) do Amaral, L.; Melo, S. C. J . Org. Chem. 1973, 38, 800.

Misacylated Transfer RNAs
J . Org. Chem., Vol. 63, No. 3, 1998 799
sensitive reactions were conducted under argon in oven-dried
glassware. 4-Pentenoic acid, 2,2-dimethyl-4-pentenoic acid,
2,4-pentadienoic acid, 2,4-hexadienoic acid, and 3-thiophene-
acetic acid were purchased from Aldrich Chemicals. 2,2-
Diphenyl-4-pentenoic acid, 3-methyl-2,2-diphenyl-4-pentenoic
acid, 4-methyl-2,2-diphenyl-4-pentenoic acid, and 9-allylfluo-
rene-9-carboxylic acid were prepared as described.27 All other
chemical reagents were purchased from Aldrich Chemicals or
Sigma Chemicals and used without further purification. Ac-
etonitrile and dichloromethane were distilled from CaH2; DMF
was distilled from CaH2 under diminished pressure. Triethyl-
amine was distilled from P2O5. Analytical thin-layer chroma-
tography was performed on 60 F254 (E. Merck) plates and
visualized using iodine. Flash chromatography was performed
using 230-400 mesh silica gel. Elemental analyses were
carried out by Atlantic Microlab, Inc., Norcross, GA. High-
resolution mass spectra were recorded at the Nebraska Center
for Mass Spectrometry.
Nuclease-treated rabbit reticulocyte lysate was purchased
from Promega Corp.; T4 RNA ligase was from New England
Biolabs. DEAE Sepharose CL-4B, NADPH, and dihydrofolic
acid were obtained from Sigma Chemicals. Kits for plasmid
isolation and for purification of proteins on Ni-NTA agarose
were purchased from QIAGEN Inc. (Chatsworth, CA). Am-
pliScribe transcription kits were from Epicenter Technologies
(Madison, WI). [35S]Methionine (1000 Ci/mmol) was obtained
from Amersham Corp.
compound 6 did not yield the free amine upon treatment
with iodine may be explained by the aromatic nature of
the thiophene ring.
The unsubstituted 4-pentenoyl group proved to be very
useful for the protection of aminoacyl-tRNAs. The pro-
tecting group could be introduced efficiently by a simple
N,N′-dicyclohexylcarbodiimide-mediated coupling. Ear-
lier studies9,10 employed 4-pentenoyl anhydride, but direct
coupling of the acid resulted in the same yields without
having to prepare the anhydride first. Removal of the
4-pentenoyl group proceeded smoothly within 5-10 min.
Deprotection of the more complex N-(4-pentenoyl)ami-
noacyl dinucleotides did not take any longer than depro-
tection of a simple amino acid. Control experiments with
N-(NVOC)aminoacyl-pdCpAs showed that both methods
yield the same product upon deprotection. The removal
of the 4-pentenoyl group of N-(4-pentenoyl)-(S)-methio-
nyl-pdCpA by treatment with iodine provides further
confirmation of the mild nature of the deprotection
conditions, because no oxidation of the sensitive amino
acid was observed under these conditions.
After ligation of the N-(4-pentenoyl)aminoacyl-pdCpAs
to tRNA transcripts lacking the 3′-terminal pCpA moiety,
the protecting group could be removed under the same
conditions that had been used for the pdCpA derivatives.
Under these conditions, the N-pentenoyl group was
removed within 5-10 min, as judged by the yield of the
derived proteins. A major advantage of the strategy
described herein is the wherewithal to effect differential
protection of the NR and side chain functional group of
the aminoacyl moiety. In the present case this was
accomplished for four aspartyl-pdCpA analogues by using
the 4-pentenoyl group for NR protection and a photo-
chemically removable nitroveratryl ester for side chain
protection.
Previously, it has been noted that misacylated susp-
pressor tRNAs bearing polar amino acids tend to function
poorly in readthrough of nonsense codons.16,23k The
combination of NR and side chain protecting groups
employed here for the aspartyl-tRNACUAs permitted side
chain protection to be retained during protein synthesis
and thereby resulted in a significant increase in the
yields of DHFR produced in the cell-free protein synthe-
sizing system by nonsense codon suppression.
In addition to the increased yields of proteins accessible
by in vitro synthesis, another interesting aspect of the
orthogonal protection strategy is the possibility of creat-
ing caged proteins.12 Before removal of the side chain
protecting group, a full length protein is obtained which,
in the case of DHFR, lacks any catalytic activity. Ir-
radiation affords a fully functional protein with the same
enzymatic activity as obtained before.16 A related ap-
proach to caged proteins has been described before,
employing a BPOC group for the protection of the
R-amino group and a nitrobenzyl group for the protection
of the side chain carboxylate of aspartic acid.26 The
BPOC group, however, is labile under acidic conditions
and had to be removed before ligation of the aminoacy-
lated dinucleotide to the truncated tRNA, thereby greatly
diminishing the effectiveness of the protection strategy.
Phosphorimager analysis was performed using a Molecular
Dynamics 300E Phosphorimager equipped with Image-Quant
software.
N-(4-P en ten oyl)-(S)-va lin e Ben zyl Ester (2). To a cooled
(0-5 °C) solution containing 200 mg (0.53 mmol) of (S)-valine
benzyl ester p-toluenesulfonate, 60 µL (58 mg, 0.58 mmol) of
4-pentenoic acid, 58 µL (54 mg, 0.53 mmol) of N-methylmor-
pholine, and 78 mg (0.58 mmol) of hydroxybenzotriazole in 6
mL of CH2Cl2 was added 120 mg (0.58 mmol) of N,N′-
dicyclohexylcarbodiimide. The reaction was allowed to warm
to room temperature and was then stirred for 18 h. The
reaction mixture was filtered, diluted with 15 mL of CH2Cl2,
and washed successively with two 25-mL portions of 0.5 N HCl,
two 25-mL portions of saturated aqueous NaHCO3, and 25 mL
of brine. The organic phase was dried (MgSO4) and concen-
trated under diminished pressure. The crude product was
applied to a silica gel column (2 × 25 cm); elution with 2%
MeOH in CH2Cl2 afforded N-(4-pentenoyl)-(S)-valine benzyl
ester (2) as a colorless oil: yield 134 mg (87%); silica gel TLC
Rf 0.57 (4% MeOH in CH2Cl2); 1H NMR (CDCl3) δ 0.86 (d, 3H,
J ) 7 Hz), 0.91 (d, 3H, J ) 7 Hz), 2.12-2.23 (m, 1H), 2.31-
2.42 (m, 4H), 4.62-4.66 (m, 1H), 4.99-5.10 (m, 2H), 5.14 (d,
1H, J ) 12 Hz), 5.20 (d, 1H, J ) 12 Hz), 5.76-5.88 (m, 1H),
5.93 (d, 1H, J ) 8.5 Hz), and 7.36 (s, 5H); 13C NMR (CDCl3) δ
18.2, 19.5, 30.0, 31.8, 36.2, 57.4, 67.5, 116.2, 128.9, 129.0, 129.1,
135.8, 137.5, 172.6, and 172.8; mass spectrum (chemical
ionization, methane) m/z 290 (M + H)+. Anal. Calcd for
C
17H23NO3: C, 70.56; H, 8.01. Found: C, 70.48; H, 7.99.
N-(2,2-Dim eth yl-4-p en ten oyl)-(S)-va lin e Ben zyl Ester
(3). To a cooled (0-5 °C) solution containing 200 mg (0.53
mmol) of (S)-valine benzyl ester p-toluenesulfonate, 75 µL (74
mg, 0.58 mmol) of 2,2-dimethyl-4-pentenoic acid, 58 µL (54 mg,
0.53 mmol) of N-methylmorpholine, and 78 mg (0.58 mmol) of
hydroxybenzotriazole in 6 mL of CH2Cl2 was added 120 mg
(0.58 mmol) of N,N′-dicyclohexylcarbodiimide. The reaction
was allowed to warm to room temperature and was then
stirred for 18 h. Workup and purification as described for
compound 2 afforded 2,2-dimethyl-N-(4-pentenoyl)-(S)-valine
benzyl ester (3) as a colorless oil: yield 128 mg (76%); silica
gel TLC Rf 0.70 (4% MeOH in CH2Cl2); 1H NMR (CDCl3) δ
0.85 (d, 3H, J ) 7 Hz), 0.91 (d, 3H, J ) 7 Hz), 1.20 (s, 6H),
2.15-2.20 (m, 1H), 2.22-2.30 (m, 2H), 4.59-4.63 (m, 1H),
5.04-5.09 (m, 2H), 5.12 (d, 1H, J ) 12 Hz), 5.22 (d, 1H, J )
12 Hz), 5.68-5.82 (m, 1H), 6.12 (d, 1H, J ) 8.5 Hz), and 7.36
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were taken on a capil-
lary melting point apparatus and are not corrected. Moisture-
(27) (a) Arnold, R. T.; Searles, S. J . Am. Chem. Soc. 1949, 71, 1150.
(b) Arnold, R. T.; Parham, W. E.; Dodson, R. M. J . Am. Chem. Soc.
1949, 71, 2439.
(26) Mendel, D.; Ellman, J . A.; Schultz, P. G. J . Am. Chem. Soc.
1991, 113, 2758.

800 J . Org. Chem., Vol. 63, No. 3, 1998
Lodder et al.
(s, 5H); 13C NMR (CDCl3) δ 18.2, 19.5, 25.6, 25.7, 31.8, 42.7,
45.6, 57.4, 67.5, 118.6, 128.8, 128.9, 129.1, 134.8, 135.9, 172.5,
and 177.6; mass spectrum (chemical ionization, methane) m/z
318 (M + H)+. Anal. Calcd for C19H27NO3: C, 71.89; H, 8.57.
Found: C, 71.65; H, 8.59.
3H, J ) 7 Hz), 2.08-2.14 (m, 1H), 3.20-3.25 (m, 2H), 4.57-
4.61 (m, 1H), 4.91-5.00 (m, 2H), 5.08 (d, 1H, J ) 12 Hz), 5.16
(d, 1H, J ) 12 Hz), 5.68-5.77 (m, 1H), 5.98 (d, 1H, J ) 8 Hz),
and 7.23-7.34 (m, 15H); 13C NMR (CDCl3) δ 17.8, 19.5, 31.5,
43.8, 57.9, 61.3, 67.5, 118.3, 127.6, 128.8, 128.85, 129.0, 129.1,
129.5, 129.7, 135.7, 135.9, 143.0, 143.7, 172.0, and 174.5; mass
spectrum (chemical ionization, methane) m/z 442 (M + H)+.
Anal. Calcd for C29H31NO3: C, 78.88; H, 7.08. Found: C,
78.79; H, 7.07.
N-(2,4-P en ta d ien oyl)-(S)-va lin e Ben zyl Ester (4). To a
cooled (0-5 °C) solution containing 200 mg (0.53 mmol) of (S)-
valine benzyl ester p-toluenesulfonate, 65 mg (0.66 mmol) of
2,4-pentadienoic acid, and 222 µL (161 mg, 1.59 mmol) of Et3N
in 10 mL of CH2Cl2 was added 168 mg (0.66 mmol) of BOP-
Cl. The reaction was allowed to warm to room temperature
and was then stirred for 18 h. Workup and purification as
described for compound 2 afforded N-(2,4-pentadienoyl)-(S)-
valine benzyl ester (4) as a colorless oil: yield 85 mg (56%);
silica gel TLC Rf 0.60 (4% MeOH in CH2Cl2); 1H NMR (CDCl3)
δ 0.88 (d, 3H, J ) 7 Hz), 0.93 (d, 3H, J ) 7 Hz), 2.16-2.25 (m,
1H), 4.71-4.75 (m, 1H), 5.15 (d, 1H, J ) 12 Hz), 5.20 (d, 1H,
J ) 12 Hz), 5.45 (d, 1H, J ) 10 Hz), 5.58 (d, 1H, J ) 17 Hz),
5.95 (d, 1H, J ) 15 Hz), 6.02 (d, 1H, J ) 8.5 Hz), 6.38-6.50
(m, 1H), 7.18-7.29 (m, 1H), and 7.36 (s, 5H); 13C NMR (CDCl3)
δ 18.2, 19.5, 32.1, 57.6, 67.6, 124.7, 125.2, 128.9, 129.0, 129.1,
135.2, 135.8, 142.3, 166.2, and 172.6; mass spectrum (chemical
ionization, methane) m/z 288 (M + H)+. Anal. Calcd for
N-(2,2-Dip h en yl-3-m eth yl-4-p en ten oyl)-(S)-va lin e Ben -
zyl Ester (8). To a cooled (0-5 °C) solution containing 200
mg (0.53 mmol) of (S)-valine methyl ester p-toluenesulfonate,
176 mg (0.66 mmol) of 2,2-diphenyl-3-methyl-4-pentenoic acid,
and 222 µL (161 mg, 1.59 mmol) of Et3N in 10 mL of CH2Cl2
was added 168 mg (0.66 mmol) of BOP-Cl. The reaction was
allowed to warm to room temperature and was then stirred
for 18 h. Workup and purification as described for compound
2 afforded N-(2,2-diphenyl-3-methyl-4-pentenoyl)-(S)-valine
benzyl ester (8) as a colorless oil (1:1 mixture of diastereo-
mers): yield 171 mg (71%); silica gel TLC Rf 0.61 (1% MeOH
1
in CH2Cl2); H NMR (CDCl3) δ 0.52 (d, 1.5H, J ) 7 Hz), 0.58
(d, 1.5H, J ) 7 Hz), 0.59 (d, 1.5H, J ) 7 Hz), 0.63 (d, 1.5H, J
) 7 Hz), 0.88 (d, 1.5H, J ) 6.5 Hz), 0.92 (d, 1.5H, J ) 6.5 Hz),
2.02-2.06 (m, 1H), 3.93-3.98 (m, 1H), 4.52-4.58 (m, 1H),
4.92-4.98 (m, 2H), 5.04-5.18 (m, 2H), 5.65-5.82 (m, 1H), 5.97
(d, 0.5H, J ) 8 Hz), 6.06 (d, 0.5H, J ) 8 Hz), and 7.22-7.44
(m, 15H); 13C NMR (CDCl3) δ 16.7, 17.4, 17.6, 17.8, 19.4, 31.7,
31.8, 41.2, 57.8, 65.8, 65.9, 67.4, 116.1, 116.5, 127.4, 127.6,
128.1, 128.35, 128.4, 128.6, 128.7, 128.9, 129.1, 129.6, 129.7,
130.5, 131.15, 131.2, 135.9, 139.9, 140.2, 140.7, 141.2, 141.9,
142.3, 172.1, and 174.0; mass spectrum (chemical ionization,
methane) m/z 456 (M + H)+. Anal. Calcd for C30H33NO3: C,
79.09; H, 7.30. Found: C, 79.03; H, 7.29.
C
17H21NO3: C, 71.06; H, 7.37. Found: C, 70.79; H, 7.50.
N-(2,4-Hexa d ien oyl)-(S)-va lin e Ben zyl Ester (5). To a
cooled (0-5 °C) solution containing 200 mg (0.53 mmol) of (S)-
valine benzyl ester p-toluenesulfonate, 74 mg (0.66 mmol) of
2,4-hexadienoic acid (5), and 222 µL (161 mg, 1.59 mmol) of
Et3N in 10 mL of CH2Cl2 was added 168 mg (0.66 mmol) of
BOP-Cl. The reaction was allowed to warm to room temper-
ature and was then stirred for 18 h. Workup and purification
as described for compound 2 afforded N-(2,4-hexadienoyl)-(S)-
valine benzyl ester (5) as a colorless solid: yield 102 mg (64%);
mp 59-61 °C; silica gel TLC Rf 0.62 (4% MeOH in CH2Cl2);
1H NMR (CDCl3) δ 0.87 (d, 3H, J ) 7 Hz), 0.93 (d, 3H, J ) 7
Hz), 1.84 (d, 3H, J ) 5 Hz), 2.15-2.26 (m, 1H), 4.71-4.75 (m,
1H), 5.14 (d, 1H, J ) 12 Hz), 5.20 (d, 1H, J ) 12 Hz), 5.81 (d,
1H, J ) 15 Hz), 5.94 (d, 1H, J ) 8 Hz), 6.04-6.21 (m, 2H),
7.16-7.25 (m, 1H), and 7.36 (s, 5H); 13C NMR (CDCl3) δ 18.2,
19.1, 19.5, 32.1, 57.5, 67.6, 121.5, 128.9, 129.0, 129.1, 130.1,
135.8, 138.7, 142.5, 166.7, and 172.6; mass spectrum (chemical
ionization, methane) m/z 302 (M + H)+; mass spectrum
(electron impact) m/z 301.167 (M+) (C18H23NO3 requires
301.168).
N-(2,2-Dip h en yl-4-m eth yl-4-p en ten oyl)-(S)-va lin e Ben -
zyl Ester (9). To a cooled (0-5 °C) solution containing 200
mg (0.53 mmol) of (S)-valine benzyl ester p-toluenesulfonate,
176 mg (0.66 mmol) of 2,2-diphenyl-4-methyl-4-pentenoic acid,
and 222 µL (161 mg, 1.59 mmol) of Et3N in 10 mL of CH2Cl2
was added 168 mg (0.66 mmol) of BOP-Cl. The reaction was
allowed to warm to room temperature and was then stirred
for 18 h. Workup and purification as described for compound
2 afforded N-(2,2-diphenyl-4-methyl-4-pentenoyl)-(S)-valine
benzyl ester (9) as a colorless oil: yield 176 mg (73%); silica
gel TLC Rf 0.61 (1% MeOH in CH2Cl2); 1H NMR (CDCl3) δ
0.62 (d, 3H, J ) 7 Hz), 0.72 (d, 3H, J ) 7 Hz), 1.40 (s, 3H),
2.04-2.13 (m, 1H), 3.14 (d, 1H, J ) 14 Hz), 3.28 (d, 1H, J )
14 Hz) 4.46 (s, 1H), 4.58-4.61 (m, 1H), 4.68 (s, 1H), 5.09 (d,
1H, J ) 12 Hz), 5.16 (d, 1H, J ) 12 Hz), 6.12 (d, 1H, J ) 8
Hz), and 7.21-7.41 (m, 15H); 13C NMR (CDCl3) δ 17.8, 19.5,
25.3, 31.6, 46.5, 58.0, 62.0, 67.4, 116.1, 127.5, 128.6, 128.62,
128.88, 128.9, 129.1, 129.6, 130.0, 135.9, 143.0, 144.0, 172.1,
and 174.1; mass spectrum (chemical ionization, methane) m/z
456 (M + H)+. Anal. Calcd for C30H33NO2: C, 79.09; H, 7.30.
Found: C, 79.10; H, 7.35.
N-(3-Th iop h en ea cetyl)-(S)-va lin e Ben zyl Ester (6). To
a cooled (0-5 °C) solution containing 200 mg (0.53 mmol) of
(S)-valine benzyl ester p-toluenesulfonate, 82 mg (0.58 mmol)
of 3-thiopheneacetic acid, 58 µL (54 mg, 0.53 mmol) of
N-methylmorpholine, and 78 mg (0.58 mmol) of hydroxyben-
zotriazole in 6 mL of CH2Cl2 was added 120 mg (0.58 mmol)
of N,N′-dicyclohexylcarbodiimide. The reaction was allowed
to warm to room temperature and was then stirred for 18 h.
Workup and purification as described for compound 2 afforded
N-(3-thiopheneacetyl)-(S)-valine benzyl ester (6) as a colorless
solid: yield 140 mg (80%); mp 69-70 °C; silica gel TLC Rf 0.62
1
N-(9-Allylflu or en yl-9-ca r bon yl)-(S)-va lin e Ben zyl Es-
ter (10). To a cooled (0-5 °C) solution containing 200 mg (0.53
mmol) of (S)-valine benzyl ester p-toluenesulfonate, 146 mg
(0.58 mmol) of 9-allylfluorenyl-9-carboxylic acid, 58 µL (54 mg,
0.53 mmol) of N-methylmorpholine, and 78 mg (0.58 mmol) of
hydroxybenzotriazole in 6 mL of CH2Cl2 was added 120 mg
(0.58 mmol) of N,N′-dicyclohexylcarbodiimide. The reaction
was allowed to warm to room temperature and was then
stirred for 18 h. Workup and purification as described for
compound 2 afforded N-(9-allylfluorenyl-9-carbonyl)-(S)-valine
benzyl ester (10) as a colorless oil: yield 221 mg (94%); silica
gel TLC Rf 0.64 (1% MeOH in CH2Cl2); 1H NMR (CDCl3) δ
0.50 (d, 3H, J ) 7 Hz), 0.66 (d, 3H, J ) 7 Hz), 1.93-1.99 (m,
1H), 3.09-3.12 (m, 2H), 4.43-4.48 (m, 1H), 4.75-4.88 (m, 2H),
4.98 (d, 1H, J ) 12 Hz), 5.06 (d, 1H, J ) 12 Hz), 5.20-5.34
(4% MeOH in CH2Cl2); H NMR (CDCl3) δ 0.74 (d, 3H, J ) 7
Hz), 0.85 (d, 3H, J ) 7 Hz), 2.08-2.19 (m, 1H), 3.65 (s, 2H),
4.57-4.62 (m, 1H), 5.09 (d, 1H, J ) 12 Hz), 5.17 (d, 1H, J )
12 Hz), 5.91 (d, 1H, J ) 8.5 Hz), 7.01 (d, 1H, J ) 5 Hz), 7.16
(s, 1H), and 7.30-7.39 (m, 6H); 13C NMR (CDCl3) δ 18.0, 19.5,
31.7, 38.6, 57.5, 67.5, 123.9, 127.2, 128.9, 129.0, 129.1, 135.1,
135.8, 170.9, and 172.1; mass spectrum (chemical ionization,
methane) m/z 332 (M + H)+. Anal. Calcd for C18H21NO3: C,
65.23; H, 6.39. Found: C, 65.34; H, 6.46.
N-(2,2-Dip h en yl-4-p en ten oyl)-(S)-va lin e Ben zyl Ester
(7). To a cooled (0-5 °C) solution containing 200 mg (0.53
mmol) of (S)-valine benzyl ester p-toluenesulfonate, 167 mg
(0.66 mmol) of 2,2-diphenyl-4-pentenoic acid, and 222 µL (161
mg, 1.59 mmol) of Et3N in 10 mL of CH2Cl2 was added 168
mg (0.66 mmol) of BOP-Cl. The reaction was allowed to warm
to room temperature and was then stirred for 18 h. Workup
and purification as described for compound 2 afforded 2,2-
diphenyl-N-(4-pentenoyl)-(S)-valine benzyl ester (7) as a color-
less oil: yield 153 mg (65%); silica gel TLC Rf 0.61 (1% MeOH
in CH2Cl2); 1H NMR (CDCl3) δ 0.60 (d, 3H, J ) 7 Hz), 0.74 (d,
(m, 1H), 5.68 (d, 1H, J ) 8.5 Hz), and 7.19-7.77 (m, 13H); 13
C
NMR (CDCl3) δ 17.8, 19.4, 31.7, 41.4, 57.6, 62.8, 67.4, 118.9,
120.8, 120.9, 125.1, 125.4, 128.2, 128.4, 128.8, 128.9, 129.0,
129.1, 133.7, 135.8, 141.2, 141.5, 145.5, 146.1, 171.6, and 172.8;
mass spectrum (chemical ionization, methane) m/z 440 (M +

Misacylated Transfer RNAs
J . Org. Chem., Vol. 63, No. 3, 1998 801
H)+. Anal. Calcd for C29H29NO3: C, 79.24; H, 6.65. Found:
C, 79.02; H, 6.69.
methane) m/z 246 (M + H)+. Anal. Calcd for C11H19NO3S:
C, 53.85; H, 7.81. Found: C, 53.71; H, 7.82.
Gen er a l P r oced u r e for Dep r ot ect ion of P en t en oyl
Der iva tives. To a solution of 0.20 mmol of protected (S)-
valine benzyl ester in 2 mL of 1:1 THF-H2O was added 3 equiv
of I2. The reaction mixture was stirred at room temperature
for 5 min, quenched with solid Na2S2O3, and concentrated
under diminished pressure. The residue was applied to a silica
gel column (1 × 20 cm); elution with 5% MeOH in CH2Cl2 gave
(S)-valine benzyl ester as a colorless oil. The deprotected
product was then converted to the p-toluenesulfonic acid salt.
1H NMR data and melting points were the same as those of
the authentic valine benzyl ester p-toluenesulfonate salt.
During the treatment of compound 10 with iodine the
formation of iodohydrin 10a was observed. Compound 10a :
1H NMR (CDCl3) δ 0.44 (d, 1.5 H, J ) 7 Hz), 0.51 (d, 1.5 H, J
) 7 Hz), 0.59 (d, 1.5 H, J ) 7 Hz), 0.70 (d, 1.5 H, J ) 7 Hz),
1.94-2.12 (m, 2 H), 2.88-2.97 (m, 1 H), 3.06-3.20 (m, 2 H),
3.38-3.51 (m, 1 H), 4.41-4.46 (m, 1 H), 4.92-5.09 (m, 2 H),
5.61 (t, 1 H, J ) 8 Hz) and 7.17-7.81 (m, 13 H); mass spectrum
(FAB) m/z 584.3 (M + H)+.
N-(4-P en ten oyl)-(S)-va lin e Meth yl Ester (12a ). To a
cooled (0-5 °C) solution containing 500 mg (2.98 mmol) of (S)-
valine methyl ester hydrochloride (11a ), 335 µL (328 mg, 3.28
mmol) of pentenoic acid, 328 µL (301 mg, 2.98 mmol) of
N-methylmorpholine, and 443 mg (3.28 mmol) of hydroxyben-
zotriazole in 20 mL of CH2Cl2 was added 677 mg (3.28 mmol)
of N,N′-dicyclohexylcarbodiimide. The reaction was allowed
to warm to room temperature and was then stirred for 18 h.
The reaction mixture was filtered, diluted with 30 mL of CH2-
Cl2, and washed successively with two 50-mL portions of 0.5
N HCl, two 50-mL portions of saturated aqueous NaHCO3, and
50 mL of brine. The organic phase was dried (MgSO4) and
concentrated under diminished pressure. The crude product
was applied to a silica gel column (2 × 40 cm); elution with
1-2% MeOH in CH2Cl2 afforded N-(4-pentenoyl)-(S)-valine
methyl ester (12a ) as a colorless oil: yield 575 mg (90%); silica
gel TLC Rf 0.36 (4% MeOH in CH2Cl2); 1H NMR (CDCl3) δ
0.90 (d, 3H, J ) 7 Hz), 0.93 (d, 3H, J ) 7 Hz), 2.10-2.18 (m,
1H), 2.31-2.42 (m, 4H), 3.74 (s, 3H), 4.56-4.61 (m, 1H), 5.01-
5.12 (m, 2H), 5.77-5.85 (m, 1H), and 5.93 (d, 1H, J ) 8.5 Hz);
mass spectrum (electron impact) m/z 213.136 (C11H9NO3
requires 213.136).
N-(4-P en ten oyl)-(S)-a la n in e Meth yl Ester (12d ). To a
cooled (0-5 °C) solution containing 500 mg (3.58 mmol) of (S)-
alanine methyl ester hydrochloride (11d ), 402 µL (394 mg, 3.94
mmol) of pentenoic acid, 3.94 µL (362 mg, 3.58 mmol) of
N-methylmorpholine, and 532 mg (3.94 mmol) of hydroxyben-
zotriazole in 20 mL of CH2Cl2 was added 813 mg (3.94 mmol)
of N,N′-dicyclohexylcarbodiimide. The reaction was allowed
to warm to room temperature and was then stirred for 18 h.
Workup and purification as described for compound 12a
afforded N-(4-pentenoyl)-(S)-alanine methyl ester (12d ) as a
colorless oil: yield 422 mg (64%); silica gel TLC Rf 0.34 (4%
MeOH in CH2Cl2); 1H NMR (CDCl3) δ 1.40 (d, 3H, J ) 7 Hz),
2.17-2.45 (m, 4H), 3.76 (s, 3H), 4.56-4.66 (m, 1H), 5.00-5.11
(m, 2H), 5.77-5.90 (m, 1H), and 6.02 (br, 1H); mass spectrum
(chemical ionization, methane) m/z 186 (M + H)+. Anal. Calcd
for C9H15NO3: C, 58.36; H, 8.16. Found: C, 58.42; H, 8.13.
N-(4-P en ten oyl)-(S)-va lin e Cya n om eth yl Ester (13a ).
To a solution containing 570 mg (2.67 mmol) of compound 12a
in 15 mL of THF was added 336 mg (8.02 mmol) of LiOH in
10 mL of H2O. The reaction mixture was stirred at room
temperature for 3 h, and 50 mL of ether and 50 mL of
saturated aqueous NaHCO3 were then added. The layers were
separated, and the ether layer was washed with 25 mL of
saturated aqueous NaHCO3. The combined aqueous layer was
acidified with concentrated HCl and extracted with three 25-
mL portions of CH2Cl2. The organic phase was dried (MgSO4)
and concentrated under diminished pressure. The residue was
dissolved in 10 mL of CH3CN, and 1.59 mL (1.16 g, 11.42
mmol) of Et3N and 433 µL (516 mg, 6.84 mmol) of chloro-
acetonitrile were added. The reaction mixture was stirred at
room temperature for 20 h and concentrated. The residue was
dissolved in 50 mL of CH2Cl2 and washed with two 50-mL
portions of 1 N NaHSO4 and then with 50 mL of brine. The
organic phase was dried (MgSO4) and concentrated under
diminished pressure. The crude product was applied to a silica
gel column (2 × 40 cm); elution with 1:1 ethyl acetate-hexanes
afforded N-(4-pentenoyl)-(S)-valine cyanomethyl ester (13a ) as
a colorless solid: yield 490 mg (77%); mp 45-46 °C; silica gel
1
TLC Rf 0.43 (1:1 ethyl acetate-hexanes); H NMR (CDCl3) δ
0.95 (d, 3H, J ) 7 Hz), 0.98 (d, 3H, J ) 7 Hz), 2.16-2.23 (m,
1H), 2.33-2.42 (m, 4H), 4.59-4.64 (m, 1H), 4.69 (d, 1H, J )
15 Hz), 4.85 (d, 1H, J ) 15 Hz), 5.03-5.13 (m, 2H), and 5.77-
5.85 (m, 2H); 13C NMR (CDCl3) δ 18.4, 19.4, 29.9, 31.4, 35.9,
49.2, 57.4, 114.6, 116.2, 137.3, 171.3 and 173.2; mass spectrum
(chemical ionization, methane) m/z 239 (M + H)+. Anal. Calcd
for C12H18N2O2: C, 60.49; H, 7.61. Found: C, 60.23; H, 7.53.
N-(4-P en ten oyl)-(S)-p h en yla la n in e Meth yl Ester (12b).
To a cooled (0-5 °C) solution containing 500 mg (2.32 mmol)
of (S)-phenylalanine methyl ester hydrochloride (11b), 260 µL
(255 mg, 2.55 mmol) of pentenoic acid, 255 µL (235 mg, 2.32
mmol) of N-methylmorpholine, and 345 mg (2.55 mmol) of
hydroxybenzotriazole in 20 mL of CH2Cl2 was added 526 mg
(2.55 mmol) of N,N′-dicyclohexylcarbodiimide. The reaction
was allowed to warm to room temperature and was then
stirred for 18 h. Workup and purification as described for 12a
afforded N-(4-pentenoyl)-(S)-phenylalanine methyl ester (12b)
as a colorless solid: yield 505 mg (83%); mp 40-41 °C; silica
gel TLC Rf 0.46 (4% MeOH in CH2Cl2); 1H NMR (CDCl3) δ
2.24-2.36 (m, 4H), 3.05-3.19 (m, 2H), 3.73 (s, 3H), 4.87-4.97
(m, 1H), 5.00-5.06 (m, 2H), 5.71-5.83 (m, 1H), 5.91 (d, 1H, J
) 8.5 Hz), and 7.07-7.32 (m, 5H); mass spectrum (chemical
ionization, methane) m/z 262 (M + H)+. Anal. Calcd for
N-(4-P en ten oyl)-(S)-p h en yla la n in e Cya n om eth yl Ester
(13b). Treatment of compound 12b (460 mg, 1.76 mmol) as
described for 12a gave N-(4-pentenoyl)-(S)-phenylalanine cya-
nomethyl ester (13b) as a colorless solid: yield 380 mg (75%);
mp 57-58 °C; silica gel TLC Rf 0.33 (1:1 ethyl acetate-
hexanes); 1H NMR (CDCl3) δ 2.25-2.36 (m, 4H), 3.13-3.16
(m, 2H), 4.68 (d, 1H, J ) 15 Hz), 4.80 (d, 1H, J ) 15 Hz), 4.90-
5.06 (m, 3H), 5.70-5.82 (m, 2H), and 7.12-7.36 (m, 5H); 13C
NMR (CDCl3) δ 29.7, 35.8, 38.0, 49.4, 53.5, 114.5, 116.3, 128.0,
129.4, 129.7, 135.7, 137.2, 171.0, and 172.8; mass spectrum
(chemical ionization, methane) m/z 287 (M + H)+. Anal. Calcd
for C16H18N2O2: C, 67.12; H, 6.34. Found: C, 67.18; H, 6.36.
C
15H19NO3: C, 68.94; H, 7.33. Found: C, 68.81; H, 7.34.
N-(4-P en ten oyl)-(S)-m eth ion in e Meth yl Ester (12c). To
N-(4-P en ten oyl)-(S)-m eth ion in e Cya n om eth yl Ester
(13c). Treatment of compound 12c (405 mg, 1.65 mmol) as
described for 12a gave N-(4-pentenoyl)-(S)-methionine cya-
nomethyl ester (13c) as a colorless solid: yield 257 mg (58%);
mp 58-59 °C; silica gel TLC Rf 0.29 (1:1 ethyl acetate-
hexanes); 1H NMR (CDCl3) δ 1.99-2.09 (m, 1H), 2.11 (s, 3H),
2.15-2.26 (m, 1H), 2.32-2.43 (m, 4H), 2.55 (t, 2H, J ) 7 Hz),
4.73 (d, 1H, J ) 16 Hz), 4.77-4.88 (m, 1H), 4.84 (d, 1H, J )
16 Hz), 5.02-5.13 (m, 2H), 5.77-5.88 (m, 1H), and 6.15 (d,
1H, J ) 8.5 Hz); 13C NMR (CDCl3) δ 15.9, 29.8, 30.4, 31.1,
35.8, 49.6, 51.6, 114.6, 116.3, 137.2, 171.3, and 173.2; mass
spectrum (chemical ionization, methane) m/z 271 (M + H)+.
Anal. Calcd for C12H18N2O3S: C, 53.31; H, 6.71. Found: C,
53.23; H, 6.67.
a cooled (0-5 °C) solution containing 500 mg (2.50 mmol) of
(S)-methionine methyl ester hydrochloride (11c), 281 µL (275
mg, 2.75 mmol) of pentenoic acid, 275 µL (253 mg, 2.50 mmol)
of N-methylmorpholine, and 372 mg (2.75 mmol) of hydroxy-
benzotriazole in 20 mL of CH2Cl2 was added 567 mg (2.75
mmol) of N,N′-dicyclohexylcarbodiimide. The reaction was
allowed to warm to room temperature and was then stirred
for 18 h. Workup and purification as described for 12a
afforded N-(4-pentenoyl)-(S)-methionine methyl ester (12c) as
a colorless oil: yield 514 mg (84%); silica gel TLC Rf 0.38 (4%
MeOH in CH2Cl2); 1H NMR (CDCl3) δ 1.94-2.04 (m, 1H) 2.10
(s, 3H), 2.12-2.23 (m, 1H), 2.31-2.60 (m, 6H), 3.76 (s, 3H),
4.70-4.77 (m, 1H), 5.01-5.12 (m, 2H), 5.76-5.88 (m, 1H), and
6.15 (d, 1H, J ) 7 Hz); mass spectrum (chemical ionization,

802 J . Org. Chem., Vol. 63, No. 3, 1998
Lodder et al.
N-(4-P en ten oyl)-(S)-a la n in e Cya n om eth yl Ester (13d ).
Treatment of compound 12d (370 mg, 2.0 mmol) as described
for 12a gave N-(4-pentenoyl)-(S)-alanine cyanomethyl ester
(13d ) as a colorless solid: yield 50 mg (12%); mp 48-49 °C;
analyzed by C18 reverse phase HPLC using the same conditions
as described above for compound 14a . Deblocked (S)-methio-
nyl-pdCpA had the same retention time (10.3 min) as a sample
of (S)-methionyl-pdCpA derived from N-(6-nitroveratryloxy-
carbonyl)-(S)-methionyl-pdCpA.28
1
silica gel TLC Rf 0.24 (1:1 ethyl acetate-hexanes); H NMR
(CDCl3) δ 1.46 (d, 3H, J ) 7.5 Hz), 2.30-2.44 (m, 4H), 4.61-
4.68 (m, 1H), 4.72 (d, 1H, J ) 16 Hz), 4.84 (d, 1H, J ) 16 Hz),
5.01-5.13 (m, 2H), and 5.77-5.88 (m, 2H); 13C NMR (CDCl3)
δ 18.2, 29.8, 35.8, 48.2, 49.5, 114.5, 116.3, 137.2, 172.2, and
172.7; mass spectrum (chemical ionization, methane) m/z 211
(M + H)+. Anal. Calcd for C10H14N2O3: C, 57.13; H, 6.71.
Found: C, 57.22; H, 6.67.
Dim eth yl N-(4-P en ten oyl)-(S)-aspar tate (18). To a cooled
(0-5 °C) solution containing 1.0 g (5.06 mmol) of dimethyl (S)-
aspartate hydrochloride (17), 0.57 mL (558 mg, 5.57 mmol) of
pentenoic acid, 0.56 mL (512 mg, 5.06 mmol) of N-methylmor-
pholine, and 0.75 g (5.57 mmol) of hydroxybenzotriazole in 20
mL of CH2Cl2 was added 1.15 g (5.57 mmol) of N,N′-dicyclo-
hexylcarbodiimide. The reaction was allowed to warm to room
temperature and was then stirred for 18 h. The reaction
mixture was filtered, diluted with 30 mL of CH2Cl2, and
washed successively with two 50-mL portions of 0.5 N HCl,
two 50-mL portions of saturated aqueous NaHCO3, and 50 mL
of brine. The organic phase was dried (MgSO4) and concen-
trated under diminished pressure. The crude product was
applied to a silica gel column (2 × 40 cm); elution with 1:1
ethyl acetate-hexanes afforded dimethyl N-(4-pentenoyl)-(S)-
aspartate (18) as a colorless oil: yield 1.10 g (89%); silica gel
N-(4-P en ten oyl)-(S)-va lin e p d Cp A Ester (14a ). To a
conical vial containing 4.1 mg (19 µmol) of N-4-pentenoyl-(S)-
valine cyanomethyl ester (13a ) was added a solution of 4.8
mg (3.5 µmol) of the tris(tetrabutylammonium) salt of pdCpA
in 50 µL of DMF. The reaction mixture was stirred at room
temperature for 2 h. A 5-µL aliquot of the mixture was diluted
with 45 µL of 1:2 CH3CN-50 mM NH4OAc, pH 4.5. Ten
microliters of the diluted aliquot was analyzed by HPLC on a
C18 reverse phase column (10 × 250 mm). The column was
washed with 1 f 63% CH3CN in 50 mM NH4OAc, pH 4.5, over
a period of 45 min at a flow rate of 3.5 mL/min (monitoring at
260 nm). After 3 h the reaction mixture was diluted to a total
volume of 400 µL of 1:2 CH3CN-50 mM NH4OAc, pH 4.5, and
purified using the same semipreparative C18 reverse phase
column [retention times 15.3 and 15.7 min, for the two
positional (2′,3′) isomers]. After lyophilization of the appropri-
ate fractions, N-(4-pentenoyl)-(S)-valyl-pdCpA (14a ) was ob-
tained as a colorless solid: yield 2.7 mg (94%); λmax 260 nm
(pH 4.5); mass spectrum (FAB) m/z 840.209 (M+Na)+
(C29H41N9O15P2Na requires 840.209).
N-(4-P en ten oyl)-(S)-p h en yla la n yl-p d Cp A (14b). To a
conical vial containing 4.5 mg (15.7 µmol) of N-(4-pentenoyl)-
(S)-phenylalanine cyanomethyl ester (13b) was added a solu-
tion of 3.8 mg (2.8 µmol) of the tris(tetrabutylammonium) salt
of pdCpA in 50 µL of DMF. The reaction mixture was stirred
at room temperature for 3 h. HPLC purification (retention
times 18.9 and 19.3 min) as described for 14a afforded N-(4-
pentenoyl)-(S)-phenylalanyl-pdCpA (14b) as a colorless solid:
yield 1.4 mg (58%); mass spectrum (FAB) m/z 866.230 (M +
H)+ (C33H42N9O15P2 requires 866.228).
N-(4-P en ten oyl)-(S)-m eth ion yl-p d Cp A (14c). To a coni-
cal vial containing 4.1 mg (15.1 µmol) of N-(4-pentenoyl)-(S)-
methionine cyanomethyl ester (13c) was added a solution of
4.1 mg (3.0 µmol) of the tris(tetrabutylammonium) salt of
pdCpA in 50 µL of DMF. The reaction mixture was stirred at
room temperature for 3 h. HPLC purification (retention times
16.0 and 16.5 min) as described for 14a afforded N-(4-
pentenoyl)-(S)-methionyl-pdCpA (14c) as a colorless solid:
yield 2.1 mg (82%); mass spectrum (FAB) m/z 850.200 (M +
H)+ (C29H42N9O15P2S requires 850.200).
N-4-P en ten oyl-(S)-a la n yl-p d Cp A (14d ). To a conical vial
containing 3.5 mg (16.6 µmol) of N-4-pentenoyl-(S)-alanine
cyanomethyl ester (13d ) was added a solution of 4.5 mg (3.3
µmol) of the tris(tetrabutylammonium) salt of pdCpA in 50 µL
of DMF. The reaction mixture was stirred at room tempera-
ture for 3 h. HPLC purification (retention times 12.8 and 13.3
min) as described for 14a afforded N-4-pentenoyl-(S)-alanyl-
pdCpA (14d ) as a colorless solid: yield 1.2 mg (46%); mass
spectrum (FAB) m/z 790.193 (M + H)+ (C27H38N9O15P2 requires
790.196).
1
TLC Rf 0.33 (1:1 ethyl acetate-hexanes); H NMR (CDCl3) δ
2.30-2.41 (m, 4H), 2.84 (dd, 1H, J ) 17.5, 4 Hz), 3.04 (dd,
1H, J ) 17.5, 4 Hz), 3.69 (s, 3H), 3.76 (s, 3H), 4.84-4.89 (m,
1H), 4.99-5.10 (m, 2H), 5.75-5.86 (m, 1H), and 6.51 (d, 1H, J
) 9 Hz); 13C NMR (CDCl3) δ 29.8, 35.9, 36.5, 48.8, 52.4, 53.2,
116.0, 137.2, 171.7, 172.0, and 172.6; mass spectrum (chemical
ionization, methane) m/z 244 (M + H)+. Anal. Calcd for
C
11H17NO5: C, 54.31; H, 7.04. Found: C, 54.15; H, 7.14.
Din itr over a tr yl N-(4-P en ten oyl)-(S)-a sp a r ta te (19). To
a solution containing 203 mg (0.83 mmol) of dimethyl N-(4-
pentenoyl)-(S)-aspartate (18) in 2 mL of THF was added 25
mL of 0.1 N NaOH. After stirring at room temperature for 3
h the reaction mixture was acidified to pH 2 with 10% aqueous
NaHSO4 and extracted with three 30-mL portions of ethyl
acetate. The combined organic extract was dried (Na2SO4) and
concentrated under diminished pressure. The residue was
dissolved in 2 mL of DMF and 429 mg (2.82 mmol) of CsF was
added, followed by 276 mg (1.0 mmol) of nitroveratryl bro-
mide.17 The reaction mixture was stirred at room temperature
for 6 h, diluted with 30 mL of ethyl acetate, and washed with
two 10-mL portions of water. The organic layer was dried
(Na2SO4) and concentrated under diminished pressure. The
crude product was applied to a silica gel column (2 × 25 cm);
elution with 1:3 ethyl acetate-hexanes afforded dinitroveratryl
N-(4-pentenoyl)-(S)-aspartate (19) as a pale yellow solid: yield
200 mg (40%); mp 167.5-168.5 °C; silica gel TLC Rf 0.22 (1:1
ethyl acetate-hexanes); 1H NMR (CDCl3) δ 2.32-2.38 (m, 4H),
2.95 (dd, 1H, J ) 17.5, 4.5 Hz), 3.19 (dd, 1H, J ) 17.5, 4.5
Hz), 3.95 (s, 3H), 3.96 (s, 3H), 3.98 (s, 3H), 3.99 (s, 3H), 4.96-
5.08 (m, 3H), 5.44-5.59 (m, 4H), 5.72-5.83 (m, 1H), 6.57 (d,
1H, J ) 8.5 Hz), 6.96 (s, 1H), 6.98 (s, 1H) 7.69 (s, 1H), and
7.71 (s, 1H); mass spectrum (chemical ionization, methane)
m/z 606 (M + H)+. Anal. Calcd for C27H31N3O13: C, 53.55; H,
5.16. Found: C, 53.44; H, 5.18.
γ-Nit r over a t r yl N-(4-P en t en oyl)-(S)-a sp a r t a t e Cya -
n om eth yl Ester (20). To a cooled (0-5 °C) solution contain-
ing 200 mg (0.33 mmol) of dinitroveratryl N-(4-pentenoyl)-(S)-
aspartate (19) in 2 mL of THF was added a solution of 21 mg
(0.50 mmol) of LiOH in 2 mL of water. The reaction mixture
was stirred for 4 h, diluted with 5 mL of water, and extracted
with three 10-mL portions of CH2Cl2. The aqueous layer was
then acidified with 10% NaHSO4 and extracted with three 15-
mL portions of ethyl acetate. The combined organic layer was
dried (Na2SO4) and concentrated under diminished pressure.
The residue was dissolved in 2 mL of acetonitrile and 68 µL
(0.48 mmol) of Et3N was added, followed by 152 µL (2.4 mmol)
of chloroacetonitrile. The reaction mixture was stirred at room
temperature for 6 h and the solvent was concentrated under
diminished pressure. The crude product was applied to a silica
gel column (2 × 20 cm); elution with 1:1 ethyl acetate-hexanes
(S)-Va lyl-p d Cp A. To a solution containing 1 A260 unit of
N-(4-pentenoyl)-(S)-valyl-pdCpA (14a ) in 50 µL of H2O was
added 0.1 mg (∼6 equiv) of I2. After 5 min the reaction was
quenched by the addition of 50 µL of 0.1 N Na2S2O3 and
analyzed by C18 reverse phase HPLC using the same conditions
as described above for compound 14a . Deblocked (S)-valyl
pdCpA ester had the same retention time (11.2 min) as a
sample of (S)-valine pdCpA ester derived from N-(6-nitrovera-
tryloxycarbonyl)-(S)-valyl-pdCpA.
(S)-Meth ion yl-p d Cp A. To a solution containing 1 A260 unit
of N-(4-pentenoyl)-(S)-methionyl-pdCpA (14c) in 50 µL of H2O
was added 0.1 mg (∼6 equiv) of I2. After 10 min the reaction
was quenched by the addition of 50 µL of 0.1 N Na2S2O3 and
(28) Shayo, Y. F.; Hecht, S. M. Unpublished data. We thank Mr.
Yuda Shayo for the authentic sample.

Misacylated Transfer RNAs
J . Org. Chem., Vol. 63, No. 3, 1998 803
gave γ-nitroveratryl N-(4-pentenoyl)-(S)-aspartate cyano-
methyl ester (20) as a pale yellow oil: yield 68 mg (46%); Rf
0.25 (1:1 ethyl acetate-hexanes); 1H NMR (CDCl3) δ 2.34-
2.38 (m, 4H), 2.96 (dd, 1H, J ) 17.5, 4.5 Hz), 3.16 (dd, 1H, J
) 17.5, 4.5 Hz), 3.97 (s, 3H), 4.01 (s, 3H), 4.70-4.81 (m, 2H),
4.99-5.11 (m, 3H), 5.46 (d, 1H, J ) 14 Hz), 5.55 (d, 1H, J )
13.5 Hz), 5.77-5.90 (m, 1H), 6.50 (d, 1H, J ) 8.5 Hz), 6.97 (s,
1H), and 7.74 (s, 1H); mass spectrum (electron impact) m/z
449.142 (M+) (C20H23N3O9 requires 449.143).
γ-Nit r over a t r yl N-(4-P en t en oyl)-(S)-a sp a r t yl-p d Cp A
(21). A solution containing 4 mg (2.94 µmol) of the tris-
(tetrabutylammonium) salt of pdCpA and 10 mg (22.3 µmol)
of γ-nitroveratryl N-(4-pentenoyl)-(S)-aspartate cyanomethyl
ester (20) in 50 µL of DMF was stirred at room temperature
for 4 h. HPLC purification as described for compound 14a
afforded compound 21 as a pale yellow foam: yield 1.1 mg
(36%); mass spectrum (FAB) m/z 1029.238 (M + H)+
(C37H47N10O21P2 requires 1029.239).
of cold ethanol. The precipitated N-(4-pentenoyl)-aminoacyl-
tRNA (15) was collected by centrifugation. The pellet was
washed with ethanol, dried, and then dissolved in H2O to a
final concentration of 1 µg/µL.
Deprotection was accomplished by admixture of 0.25 volume
of 25 mM I2 in 1:1 THF-H2O. The combined solution was
maintained at 25 °C for 5-60 min, and the aminoacyl-tRNA
(16) was recovered by centrifugation following successive
additions of 0.1 volume of 3 M NaOAc, pH 5.3, and 2.5 volumes
of cold ethanol. The tRNA pellet was washed with ethanol,
dried, and then dissolved in H2O for the translation experi-
ments.
Syn th esis a n d Dep r otection of N-(Nitr over a tr yloxy-
ca r bon yl)-(S)-va lyl-tRNA. The protected aminoacyl-tRNA
was prepared as described above for N-(4-pentenoyl)aminoacyl-
tRNAs.
Deprotection was accomplished by irradiation of a cold (2-
2.5 °C) aqueous solution of the NVOC-valyl-tRNA (at
a
γ-Nitr over a tr yl N-(4-P en ten oyl)-th r eo-â-m eth yl-(S)-a s-
p a r tyl-p d Cp A (22a ). A solution containing 10 mg (7.35 µmol)
of the tris(tetrabutylammonium) salt of pdCpA and 25 mg (53.9
µmol) of γ-nitroveratryl N-(4-pentenoyl)-threo-γ-methyl-(S)-
aspartate cyanomethyl ester19 in 100 µL of DMF was stirred
at room temperature for 16 h. HPLC purification as described
for compound 14a afforded compound 22a as a pale yellow
solid: yield 4.5 mg (59%); mass spectrum (FAB) m/z 1043.254
(M + H)+ (C38H49N10O21P2 requires 1043.255).
concentration of 1 µg/µL) with a 500 W mercury-xenon lamp
for 5 min using Pyrex and water filters.
In Vitr o P r otein Syn th esis. In vitro protein synthesis
reactions (typically 25 µL final volume) contained 17.5 µL of
rabbit reticulocyte lysate, 1 µg of the DHFR mRNA, 40 µM
amino acids with or without 40 µM methionine, 20 µCi [35S]-
methionine (1000 Ci/mmol), and 2.5 µg of aminoacyl-tRNACUA
.
Reactions were incubated at 30 °C for 60 min and quenched
by cooling to 0 °C. One-microliter aliquots were analyzed by
20% SDS-polyacrylamide gel electrophoresis. For quantita-
tion of the suppression efficiency after electrophoresis, the gel
was fixed in 40% methanol-10% acetic acid, dried, and
analyzed on a phosphorimager.
γ-Nitr over a tr yl N-(4-P en ten oyl)-er yth r o-â-m eth yl-(S)-
a sp a r tyl-p d Cp A (22b). A solution containing 10 mg (7.35
µmol) of the tris(tetrabutylammonium) salt of pdCpA and 25
mg (53.9 µmol) of γ-nitroveratryl N-(4-pentenoyl)-erythro-â-
methyl-(S)-aspartate cyanomethyl ester19 in 100 µL of DMF
was stirred at room temperature for 6 h. HPLC purification
as described for compound 14a afforded compound 22b as a
pale yellow solid: yield 4.0 mg (52%); mass spectrum (FAB)
m/z 1043.254 (M + H)+ (C38H49N10O21P2 requires 1043.255).
γ-Nit r over a t r yl N-(4-P en t en oyl)-â,â-d im et h yl-(S)-a s-
p a r tyl-p d Cp A (22c). A solution containing 4.4 mg (3.24
µmol) of the tris(tetrabutylammonium) salt of pdCpA and 8.0
mg (16.8 µmol) of γ-nitroveratryl N-(4-pentenoyl)-â,â-dimethyl-
(S)-aspartate cyanomethyl ester19 in 100 µL of DMF was
stirred at room temperature for 6 h. HPLC purification as
described for compound 14a afforded compound 22c as a pale
yellow solid: yield 2.1 mg (61%); mass spectrum (FAB) m/z
1057.271 (M + H)+ (C39H51N10O21P2 requires 1057.271).
Syn th esis a n d Dep r otection of N-(4-p en ten oyl)a m i-
n oa cyl-tRNAs. An incubation mixture having 0.5 A260 unit
(20 nmol) of a N-(4-pentenoyl)aminoacyl-pdCpA (14) and 5 µg
(0.2 nmol) of a tRNACUA transcript lacking the 3′-terminal
pCpA moiety in 50 µL of 50 mM Hepes buffer, pH 7.5,
containing 15 mM MgCl2, 0.75 mM ATP, 10% DMSO (v/v), and
100 units of T4 RNA ligase was incubated at 37 °C for 60 min.
The incubation mixture was treated with 3 M NaOAc, pH 5.3,
to a final salt concentration of 0.3 M and then with 2.5 volumes
Qu a n tita tion of DHF R Activity. The enzymatic activity
of DHFR was determined as previously described,16 with one
modification. Before enzymatic assay, samples of purified
DHFRs containing nitroveratryl-protected derivatives of as-
partic acid were irradiated for 30 min at 2 °C with a 500 W
mercury-xenon lamp using Pyrex and water filters. No
enzymatic activity was detected without irradiation. After
irradiation the activities of the DHFRs containing modified
aspartic acid analogues were comparable to those described.16
Ack n ow led gm en t. This paper is dedicated to Pro-
fessor Dieter Seebach on the occasion of his 60th
birthday. This study was supported by grant BIO-96-
008 from Virginia’s Center for Innovative Technology.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 5, 10a and 20 (3 pages). This material is contained
in libraries on microfiche, immediately follows this article in
the microfilm version of the journal, and can be ordered from
the ACS; see any current masthead page for ordering
information.
J O971692L