N-substituted 2-amino-4-pentenoic acids for amino acid protection and resolution

Article abstract of DOI:10.1016/S0040-4020(00)00826-7

N-Substituted 2-amino-4-pentenoic acid derivatives employed for the protection of racemic amino acids were shown to be capable of permitting the chromatographic separation of R and S isomers. When N(α) was disubstituted with benzyl and phenylfluorenyl groups, the racemic amino acids could be separated with facility on open silica gel columns. Further, the optically pure N-protected amino acids so derived could be used for the preparation of misacylated suppressor transfer RNAs. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00826-7

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 9421±9429
N-Substituted 2-Amino-4-pentenoic Acids for Amino Acid
Protection and Resolution
*
Michiel Lodder, Bixun Wang and Sidney M. Hecht
Departments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22901, USA
Received 24 February 2000; accepted 2 August 2000
AbstractÐN-Substituted 2-amino-4-pentenoic acid derivatives employed for the protection of racemic amino acids were shown to be
capable of permitting the chromatographic separation of R and S isomers. When Na was disubstituted with benzyl and phenyl¯uorenyl
groups, the racemic amino acids could be separated with facility on open silica gel columns. Further, the optically pure N-protected amino
acids so derived could be used for the preparation of misacylated suppressor transfer RNAs. q 2000 Elsevier Science Ltd. All rights
reserved.
Introduction
involved in peptide bond formation, it could sometimes
hinder peptide bond formation by tRNAs bearing the
respective S-isomer. Therefore, when possible it seems
advisable to utilize optically pure amino acids for the
preparation of misacylated transfer RNAs that are employed
for in vitro protein synthesis.
During the past two decades our laboratory has developed a
method for the chemical preparation of transfer RNAs
bearing a noncognate amino acid.1±10 The general strategy
of T4 RNA ligase-mediated ligation of a synthetic amino-
acylated pdCpA derivative with a truncated tRNA lacking
the 30-terminal cytidine and adenosine moieties has proven
to be successful for a wide variety of unnatural amino
acids.1±19 The resulting misacylated transfer RNAs have
been used for probing the peptidyltransferase reaction4,6
and for the in vitro preparation of proteins containing
unnatural amino acids at single, predetermined sites.11±19
Many different methods have been developed for the
synthesis of optically enriched amino acids.20 Few of
these methods, however, produce amino acids consisting
exclusively of one isomer and many require an additional
separation step to obtain compounds that can be considered
optically pure. Another approach to enantiomerically pure
compounds involves the separation of racemic mixtures of
unnatural amino acids. Over the years many methods have
been used. They have included preferential crystallization,
crystallization from optically active solvents, fractional
crystallization of diastereomeric salts and selective destruc-
tion of one enantiomer.21 Advances in chromatographic
techniques, especially HPLC, have also made it possible
to use chiral stationary phases for the separation of enantio-
mers.22 Another possibility is the use of chiral derivatizing
reagents to convert the enantiomers into diastereomers,
which in turn can be separated based on their physical
differences.23,24 The choice of the chiral reagent has a
signi®cant effect upon the degree of separation of the
derived diastereomers, as well as the detectability and
the recovery of the individual enantiomers. With many of
the currently used reagents, recovery of the optically pure
amino acids can be a problem. In most cases, a stable amide
bond is formed between the amino acids and the chiral
reagent. Recovery of the optically pure isomers is, therefore,
only possible by treatment with reagents such as strong acid.
Accordingly, the development of a resolving reagent for the
separation of racemic amino acids that can be removed
under mild conditions would be very useful.
An interesting aspect of the utilization of misacylated
transfer RNAs involves the chirality of the synthetic
amino acids attached to the tRNA. Based on the composi-
tion of ribosomally synthesized proteins, it has been
assumed that nature incorporates only the S-isomers of the
proteinogenic amino acids. Studies using misacylated
transfer RNAs bearing the R-isomer of a few different
amino acids showed that the unnatural isomers do not par-
ticipate in peptide bond formation to any reasonable
extent.8,10,14 Although not studied with precision, the use
of racemic amino acids apparently results in the formation
of peptide bonds to almost the same extent as when the
optically pure S-isomer is employed. However, it has also
been shown that tRNAs bearing the R-isomer of an amino
acid can bind to the ribosomes in a fashion similar to the
S-isomer.8 This implies that although the R-isomer is not
Keywords: amino acids and derivatives; protecting groups; resolution; opti-
cal properties.
* Corresponding author. Tel.: 1804-924-3906; fax: 1804-924-7856;
e-mail: sidhecht@virginia.edu
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00826-7

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M. Lodder et al. / Tetrahedron 56 (2000) 9421±9429
Described herein is the development of a reagent that
combines the function of separating racemic mixtures with
the function of a protecting group. Combining these func-
tions into one group limits the number of steps necessary for
the preparation and utilization of optically pure amino acids,
and also ensures that the derivatizing reagent can be
removed after synthesis of the ®nal compound is completed.
The utility of this reagent is illustrated by the resolution and
activation of an amino acid for ribosomally mediated
protein synthesis.
N-phenyl¯uorenyl-(S)-2-amino-4-pentenoic acid (2) in
84% yield. Compound 2 was conjugated to racemic valine
methyl ester (3) by a DCC-mediated coupling. The resulting
diastereomers 5 and 6 were obtained in a 1:1 ratio in 92%
yield. A similar reaction of 2 with racemic phenylalanine
methyl ester (4) gave a 1:1 mixture of diastereomers 7 and 8
(Scheme 1). HPLC analysis, using a normal phase silica gel
column, showed reasonable separation of the diastereomers
in both mixtures.31 The degree of separation did not seem to
depend on the side chain moiety of the amino acids
involved. Treatment of S-valine benzyl ester conjugated to
N-phenyl¯uorenyl-(S)-2-amino-4-pentenoic acid with
iodine in aqueous THF led to the recovery of the free
amine in 71% yield.31 These results showed that a substi-
tuted 2-amino-4-pentenoyl derivative could function as an
effective derivatizing reagent for the separation of racemic
amino acids and could be removed under the same condi-
tions as the unsubstituted 4-pentenoyl group. While
compound 2 may be useful for analytical purposes, the
necessity of using an HPLC column for the separation of
the derived diastereomers of a racemic amino acid
diminishes its attractiveness for the preparative synthesis
of optically pure derivatives.
Results and Discussion
Recently we investigated substituted 4-pentenoyl groups for
their ability to serve as amine protecting groups during the
synthesis of misacylated transfer RNAs.25,26 During this
investigation 2-amino-4-pentenoic acid (allylglycine) was
identi®ed as a possible core structure for the preparation
of a chiral derivatizing reagent useful for the separation of
racemic amino acids. The amino group provides a suitable
functional group for introducing bulky substituents poten-
tially capable of differentiating the optical antipodes of
conjugated amino acids. Further, based on an earlier
study,26 it seemed likely that Na substituents would not
seriously affect the iodolactonization reaction (vide infra)
necessary for the removal of the amino acid protecting
group.
In an effort to improve the facility of separation of the
derived diastereomers, a substituted 2-amino-4-pentenoic
acid derivative was prepared that contained an additional
N-benzyl group (Scheme 2). Reversible protection of the
carboxylate moiety proved to be more complicated than
originally envisioned. A methyl ester was used at ®rst, but
after introduction of the bulky substituents on the amine, it
was not possible to remove the methyl group. Many
different methods for the removal of methyl esters were
used,32 but none afforded the desired free acid. Apparently,
the steric bulk of the N-substituent shielded the carboxylate,
thereby hindering the hydrolysis of the methyl ester.33 It was
decided to use a cyanoethyl protecting group instead, which
is not removed by direct hydrolysis, but rather by base
induced b-elimination.34,35 Thus, commercially available
In a ®rst attempt to develop a novel derivatizing reagent, a
9-phenyl¯uorenyl27±29 group was attached to the amine.
This bulky substituent has been reported to be 6000 times
more stable to acid than the trityl group.30 Further, the rigid
planar ring system may improve the separation properties of
the substituted pentenoyl derivative. The 9-phenyl¯uorenyl
group was introduced as described (Scheme 1).29 In situ
protection of the carboxylate moiety with trimethylsilyl
chloride, followed by alkylation of the amine with
9-bromo-9-phenyl¯uorene afforded, after acidic workup,
Scheme 1.

M. Lodder et al. / Tetrahedron 56 (2000) 9421±9429
9423
Scheme 2.
(S)-2-amino-4-pentenoic acid was treated with Boc
anhydride, followed by a DCC-mediated coupling of
3-hydroxypropionitrile34 to afford protected amino acid 9
in 67% yield. The Boc group was removed by treatment
with HCl in dioxane, affording hydrochloride salt 10. The
benzyl group was introduced by reductive benzylation of
compound 10 with benzaldehyde and sodium cyanoboro-
hydride.36 The use of the hydrochloride salt instead of the
free amine proved very effective for providing the correct
pH for the reductive benzylation. Compound 11 was treated
with 9-bromo-9-phenyl¯uorene resulting in bulky
compound 12 in 83% yield. Removal of the cyanoethyl
group by treatment with K2CO3 afforded desired free acid
13 in good yield.
Substituted pentenoyl derivative 13 was conjugated to
racemic valine methyl ester (3) via DCC-mediated coupling
(Scheme 3). Interestingly, the addition of HOBt to the
reaction mixture gave an unreactive benzotriazole ester of
13. Therefore, the coupling was conducted in a mixture of
CH2Cl2 and pyridine without HOBt. The resulting dia-
stereomers 14 and 15 could be separated easily by the use
Scheme 3.

9424
M. Lodder et al. / Tetrahedron 56 (2000) 9421±9429
stereomers was much improved as compared to the separa-
tion obtained by using compound 2 as a derivatizing
reagent. Comparison of the products with diastereomers
that were derived from the optically pure isomers of the
respective amino acids showed that the diastereomers
obtained from the R-isomers eluted before those obtained
from the S-isomers. Interestingly, in case of both valine and
phenylalanine the ratio of diastereomers formed was
approximately 1:2 in favor of the (S,S) derivatives over
the (S,R) derivatives. Based on the racemic starting
materials a 1:1 ratio would have been expected, but
apparently bulky 13 reacts preferentially with the S-isomers
of the amino acids.
To show that substituted pentenoyl derivative 13 could also
be used as a protecting group for subsequent transforma-
tions, (S)-valine derivative 15 was used for the preparation
of the corresponding misacylated transfer RNA (Scheme 4).
Treatment of optically pure 15 with LiOH, followed by
reaction with chloroacetonitrile afforded cyanomethyl
ester 18 in good yield. Attachment to the dinucleotide as
described37 gave pdCpA derivative 19. Enzymatic ligation
of the aminoacyl-pdCpA derivative to an abbreviated
suppressor tRNA, i.e. a tRNA lacking the 30-terminal CA
moiety, provided full length protected aminoacyl-tRNA I.
Subsequent treatment with iodine should result in the
Figure 1. Separation of diastereomic 16 (28.6 min) and 17 (34.5 min) by
normal phase HPLC.
of an open silica gel column. Similar results were obtained
when compound 13 was conjugated to racemic phenyl-
alanine methyl ester (4). HPLC analysis of diastereomers
16 and 17 (Fig. 1) showed that the separation of the dia-
Scheme 4.

M. Lodder et al. / Tetrahedron 56 (2000) 9421±9429
9425
spectrometer. Chemical shifts are expressed relative to
CHCl3 (7.26 ppm). Moisture sensitive reactions were
conducted under argon in oven-dried glassware. All chemi-
cal reagents were purchased from Aldrich Chemicals or
Sigma Chemicals and used without further puri®cation.
Acetonitrile and dichloromethane were distilled from
CaH2; DMF was distilled from CaH2 under diminished
pressure. Analytical thin layer chromatography 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 resolu-
tion mass spectra were recorded at the Nebraska Center for
Mass Spectrometry.
Figure 2. Elaboration of DHFR by suppression of an UAG codon at posi-
tion 10 using suppressor tRNA I. Protein synthesis was carried out in the
presence of 35S-methionine using a rabbit reticulocyte lysate, mRNA
containing UAG at codon position 10 and deblocked valyl-tRNACUA
derived from I. Following incubation at 308C for 1.5 h, the reaction mixture
was analyzed on a SDS-polyacrylamide gel; lane 1, wild type, no suppres-
sor tRNA; lane 2, suppressor tRNA I before iodine treatment; lane 3,
suppressor tRNA I after iodine treatment. The minor band migrating
ahead of the full length protein was due to initiation of DHFR synthesis
at an internal methionine codon.
[35S] Methionine (1000 Ci/mmol, 10 mCi/mL) was
purchased from Amersham Corporation. Nuclease-treated
rabbit reticulocyte lysate system was obtained from
Promega Corporation. Restriction endonucleases and T4
RNA ligase were obtained from New England Biolabs.
Kits for plasmid isolation were purchased from PGC
Scienti®c. AmpliScribe transcription kits were purchased
from Epicentre Technologies. Acrylamide, N,N-methylene-
bisacrylamide, urea and Tris base were obtained from
Sigma Chemicals.
removal of the pentenoyl derivative (presumably via the
imidolactone intermediate shown in the Scheme), affording
unprotected valyl-tRNA II. To show that the bulky
pentenoyl group could indeed be removed from the amino-
acyl-tRNA by treatment with iodine, protected valyl-tRNA
I and an iodine-treated sample were used in an in vitro
protein synthesizing system. The protein synthesizing
system employed rabbit reticulocyte lysate and an mRNA
for dihydrofolate reductase (DHFR) containing a nonsense
(UAG) codon at position 10.38 The results are shown in
Fig. 2. Before treatment with iodine, use of aminoacyl-
tRNA I did not result in the synthesis of full length DHFR
(lane 2). As shown in lane 3, after treatment with iodine a
product was obtained with the same molecular weight as
wild type DHFR. This indicated that the bulky pentenoyl
derivative had been removed from the aminoacyl-tRNA and
that valine was incorporated into the protein. Comparison
with an incubation mixture containing valyl-tRNA derived
from N-(4-pentenoyl)-(S)-valyl-tRNA showed that the same
product was obtained in both.
Plasmid DNAs were isolated using a JetStar plasmid midi
kit (PGC Scienti®c) according to the protocol provided.
Sodium dodecyl sulfate±polyacrylamide gel electrophoresis
was carried out using the standard Laemmli procedure.39
Gels were visualized and quanti®ed utilizing a Molecular
Dynamics 400E PhosphorImager equipped with Image-
Quant version 5.0 software. UV spectral measurements
were made using a Perkin±Elmer Lambda 20 UV/VIS
spectrometer. Run-off transcription of abbreviated
suppressor tRNACUA (-CA) and DHFR mRNAs was carried
out as described.38 All procedures involving water
employed distilled, deionized water from a Milli-Q
system.
N-Phenyl¯uorenyl-(S)-2-amino-4-pentenoic acid (2). To
a suspension of 300 mg (2.61 mmol) of (S)-2-amino-4-
pentenoic acid (1) in 5 mL of CH2Cl2 was added 364 mL
(2.87 mmol) of trimethylsilyl chloride. After stirring at
room temperature for 2 h, 0.8 mL (5.74 mmol) of Et3N
was added. The reaction mixture was stirred for 15 min
and 563 mg (1.70 mmol) of Pb(NO3)2 was added, followed
by 1.05 g (3.26 mmol) of 9-bromo-9-phenyl¯uorene. After
stirring at room temperature for 2 days, the reaction mixture
was ®ltered through Celite and concentrated under
diminished pressure. The residue was dissolved in 40 mL
of CH2Cl2 and washed with two 40-mL portions of 5% citric
acid and with 40 mL of brine. The organic phase was dried
(MgSO4) and concentrated. The crude product was applied
to a silica gel column (25£2 cm); elution with 2% MeOH
in CH2Cl2 provided N-phenyl¯uorenyl-(S)-2-amino-4-
pentenoic acid (2) as a colorless solid: yield 775 mg
(84%); mp 63±648C; silica gel TLC Rf 0.30 (3% MeOH
in CH2Cl2); 1H NMR (CDCl3) d 1.94±2.03 (m, 1H),
2.40±2.49 (m, 1H), 2.70 (t, 1H, Jˆ5.4 Hz), 5.12±5.23 (m,
2H), 5.43±5.54 (m, 1H) and 7.19±7.76 (m, 13H); 13C NMR
(CDCl3) d 38.5, 55.6, 73.3, 120.2, 120.8, 125.5, 126.2,
126.4, 128.2, 128.6, 128.7,129.1, 129.5, 129.6, 133.6,
Conclusion
Substituted 2-amino-4-pentenoic acid derivatives 2 and 13
are useful tools for the protection and resolution of racemic
amino acids. Although the examples shown here involved
naturally occurring amino acids, the method should be
readily applicable to unnatural amino acids, since the side
chain moiety appears to have little effect on the separation
of the derived diastereomers. The ability to remove the
derivatizing reagent under mild conditions after the separa-
tion makes these compounds especially attractive for the
large scale synthesis of optically pure unnatural amino
acids and the synthesis of misacylated transfer RNAs
activated with optically pure amino acids.
Experimental
General methods
Melting points were taken on a Thomas Hoover melting
point apparatus and are not corrected. H NMR and 13C
NMR spectra were recorded on a General Electric QE-300
1

9426
M. Lodder et al. / Tetrahedron 56 (2000) 9421±9429
141.0, 141.4, 143.9, 147.6, 148.9 and 177.1; mass spectrum
(CI, methane) m/z 356 (M1H)1. Anal. Calcd for
C24H21NO2: C, 81.10; H, 5.96. Found: C, 80.84; H, 6.35.
combined organic extract was dried (MgSO4) and concen-
trated. The residue was dissolved in 30 mL of CH2Cl2 and
0.75 mL (10.8 mmol) of 3-hydroxypropionitrile and 122 mg
(1.0 mmol) of 4,4-(dimethylamino)pyridine (DMAP) were
added. The mixture was cooled in an ice bath and 1.97 g
(9.54 mmol) of DCC was added. The reaction was stirred
for 20 h and ®ltered. The ®ltrate was diluted with 25 mL of
CH2Cl2 and washed successively with two 50-mL portions
of 0.5N 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 residue was applied to a silica gel column (15£3 cm):
elution with 2% MeOH in CH2Cl2 afforded 2-cyanoethyl
N-(N-Phenyl¯uorenyl-(S)-2-amino-4-pentenoyl)-(R,S)-
valine methyl ester (5 and 6). To a cooled (0±58C) solution
containing 100 mg (0.28 mmol) of N-phenyl¯uorenyl-(S)-2-
amino-4-pentenoic acid (2), 47 mg (0.28 mmol) of (R,S)-
valine methyl ester hydrochloride (3) and 62 mL
(0.56 mmol) of N-methylmorpholine in 5 mL of CH2Cl2
was added 42 mg (0.31 mmol) of hydroxybenzotriazole,
followed by 64 mg (0.31 mmol) of DCC. After stirring at
room temperature for 16 h, the reaction mixture was ®ltered,
diluted with 25 mL of CH2Cl2 and washed successively with
two 25-mL portions of 0.5N HCl, two 25-mL portions of
saturated aqueous NaHCO3 and 25 mL of brine. The
organic phase was dried (MgSO4) and concentrated
under diminished pressure. The crude product was applied
to a silica gel column (25£2 cm); elution with 2% MeOH
in CH2Cl2 gave N-(N-phenyl¯uorenyl-(S)-2-amino-4-
pentenoyl)-(R,S)-valine methyl ester as a colorless oil (1:1
mixture of diastereomers 5 and 6): yield 121 mg (92%); 1H
NMR d 0.92±0.98 (m, 6H), 1.78±1.95 (m, 1H), 2.11±2.20
(m, 1H), 2.57±2.65 (m, 2H), 3.77 (s, 1.5H), 3.81 (s, 1.5H),
4.32±4.41 (m, 1H), 5.06±5.14 (m, 2H), 5.38±5.54 (m, 1H)
and 7.03±7.73 (m, 13H); mass spectrum (FAB) m/z
469.2472 (M1H)1 (C30H33N2O3 requires 469.2491).
N-butyloxycarbonyl-(S)-2-amino-4-pentenoate (9) as
colorless oil that solidi®ed upon standing: yield 1.55 mg
a
1
(67%); mp 58±598C; H NMR (CDCl3) d 1.45 (s, 9H),
2.48±2.57 (m, 2H), 2.72 (t, 2H, Jˆ6 Hz), 4.23±4.38 (m,
3H), 4.95 (br, 1H), 5.15±5.21 (m, 2H) and 5.65±5.76 (m,
1H); 13C NMR (CDCl3) d 18.4, 28.7, 29.3, 53.4, 59.9, 80.5,
117.1, 119.9, 132.5, 155.6 and 172.1; mass spectrum (CI,
methane) m/z 269 (M1H)1. Anal. Calcd for C13H20N2O4: C,
58.19; H, 7.51. Found: C, 58.29; H, 7.55.
2-Cyanoethyl (S)-2-amino-4-pentenoate (10). To a ¯ask
containing 1.47 g (5.48 mmol) of 2-cyanoethyl N-butyloxy-
carbonyl-(S)-2-amino-4-pentenoate (9) was added 14 mL of
a solution of 4N HCl in dioxane. After stirring at room
temperature for 2 h the solvent was removed under
diminished pressure. The residue was crystallized from
ethanol±ether to afford 2-cyanoethyl (S)-2-amino-4-
pentenoate (10) as the hydrochloride: yield 947 mg (84%);
1H NMR (CDCl3±MeOD) d 2.63±2.72 (m, 2H), 2.77 (t, 2H,
Jˆ6 Hz), 4.06 (t, 1H, Jˆ6 Hz), 4.31±4.43 (m, 2H), 5.21±
5.28 (m, 2H) and 5.63±5.76 (m, 1H); 13C NMR (CDCl3) d
17.5, 34.7, 52.6, 61.3, 117.7, 121.0, 130.6 and 168.8. Anal.
Calcd for C8H13N2O2Cl: C, 46.95; H, 6.40. Found: C, 47.05;
H, 6.35.
N-(N-Phenyl¯uorenyl-(S)-2-amino-4-pentenoyl)-(R,S)-
phenylalanine methyl ester (7 and 8). In the same fashion
as described for compounds 5 and 6, compounds 7 and 8
were prepared as a 1:1 mixture (obtained as a colorless oil):
yield 139 mg (96%); 1H NMR d 1.82±1.89 (m, 1H), 2.34±
2.60 (m, 2H), 3.00±3.18 (m, 2H), 3.73 (s, 1.5H), 3.77 (s,
1.5H), 4.51±4.56 (m, 0.5 H), 4.71±4.77 (m, 0.5H), 5.01±
5.14 (m, 2H), 5.23±5.31 (m, 0.5H), 5.49±5.55 (m, 0.5H)
and 6.90±7.73 (m, 18H); mass spectrum (FAB) m/z
517.2502 (M1H)1 (C34H33N2O3 requires 517.2491).
2-Cyanoethyl N-benzyl-(S)-2-amino-4-pentenoate (11).
To
a
solution containing 780 mg (3.81 mmol) of
HPLC analysis of diastereomers 5 and 6 and
diastereomers 7 and 8
2-cyanoethyl (S)-2-amino-4-pentenoate hydrochloride (10)
in 20 mL of freshly distilled MeOH was added 360 mg
(5.72 mmol) of NaBH3CN, followed by 0.39 mL
(3.81 mmol) of benzaldehyde. After stirring at room
temperature for 19 h the mixture was cooled in an ice
bath, acidi®ed with conc. HCl and stirred for an additional
hour. The solvent was removed under diminished pressure.
The residue was dissolved in 20 mL of H2O and the pH
adjusted to 10 with saturated Na2CO3. The aqueous solution
was extracted with four 25-mL portions of CH2Cl2 and the
combined extract was dried (MgSO4) and concentrated. The
residue was applied to a silica gel column (35£2 cm):
elution with 3% MeOH in CH2Cl2 gave 2-cyanoethyl
N-benzyl-(S)-2-amino-4-pentenoate (11) as a colorless oil:
The mixture of diastereomers 5 and 6 was dissolved in 4 mL
of 4:1 hexanes±ethyl acetate and analyzed by HPLC. A
10-mL aliquot was diluted with 70 mL of 4:1 hexanes±
ethyl acetate and injected on a Partisil silica column
(250£10 mm). Elution was performed with a gradient of
10±50% ethyl acetate in hexanes over a period of 45 min
at a ¯ow rate of 3.5 mL/min. Detection was at 275 nm. Two
major peaks were found at 19.2 and 20.3 min in approxi-
mately 1:1 ratio. Analysis of the mixture of diastereomers 7
and 8 under the same conditions resulted in two major peaks
at 27.1 and 28.0 min in a 1:1 ratio.
1
yield 715 mg (73%); H NMR (CDCl3) d 2.48 (t, 2H,
2-Cyanoethyl N-butyloxycarbonyl-(S)-2-amino-4-pente-
noate (9). To a cooled (0±58C) solution containing 1.0 g
(8.67 mmol) of (S)-2-amino-4-pentenoic acid (1) in 30 mL
of dioxane and 30 mL of 1 M NaHCO3 was added 2.10 g
(9.62 mmol) of di-t-butyl pyrocarbonate. The reaction
mixture was stirred for 24 h at room temperature. The
mixture was diluted with 75 mL of H2O, washed with two
60-mL portions of ether, acidi®ed to pH 1 with 1N H2SO4
and extracted with four 30-mL portions of CH2Cl2. The
Jˆ7 Hz), 2.70 (t, 2H, Jˆ7 Hz), 3.44 (t, 1H, Jˆ7 Hz), 3.72
(d, 1H, Jˆ13 Hz), 3.86 (d, 1H, Jˆ13 Hz), 4.31 (t, 2H,
Jˆ6 Hz), 5.10±5.17 (m, 2H), 5.70±5.84 (m, 1H) and
7.24±7.38 (m, 5H); 13C NMR (CDCl3) d 18.6, 38.2, 52.5,
59.3, 60.6, 117.3, 118.9, 127.7, 128.8, 129.0, 129.3, 129.4,
133.8, 140.0 and 174.6; mass spectrum (CI, methane) m/z
259 (M1H)1. Anal. Calcd for C15H18N2O2: C, 69.74; H,
7.02. Found: C, 69.69; H, 7.00.

M. Lodder et al. / Tetrahedron 56 (2000) 9421±9429
9427
2-Cyanoethyl N-benzyl-N-phenyl¯uorenyl-(S)-2-amino-
4-pentenoate (12). To a solution containing 715 mg
(2.77 mmol) of 2-cyanoethyl N-benzyl-(S)-2-amino-4-
pentenoate (11) in 15 mL of anhydrous CH3CN was added
1.11 g (3.46 mmol) of 9-bromo-9-phenyl¯uorene, followed
by 647 mg (3.05 mmol) of K3PO4 and 734 mg (2.22 mmol)
of Pb(NO3)2. The reaction mixture was stirred for 17 h at
room temperature, ®ltered through Celite and concentrated
under diminished pressure. The residue was dissolved in
30 mL of CH2Cl2 and washed with two 30-mL portions of
5% citric acid and with 30 mL of brine. The organic phase
was dried (MgSO4) and concentrated. The crude product
was applied to a silica gel column (20£2 cm): elution with
4:1 hexanes±EtOAc gave 2-cyanoethyl N-benzyl-N-phenyl-
¯uorenyl-(S)-2-amino-4-pentenoate (12) as a colorless
25 mL of brine. The organic phase was dried (MgSO4) and
concentrated. The mixture of diastereomers 14 and 15 was
separated by silica gel column chromatography (30£2 cm),
elution with 15±20% ethyl acetate in hexanes. N-(N-benzyl-
N-phenyl¯uorenyl-(S)-2-amino-4-pentenoyl)-(R)-valine
methyl ester (14) was isolated as an oil: yield 31 mg (12%);
1H NMR (CDCl3) d 0.67 (d, 3H, Jˆ7 Hz), 0.78 (d, 3H,
Jˆ7 Hz), 1.37±1.50 (m, 1H), 1.89±1.98 (m, 1H), 2.44±
2.58 (m, 1H), 2.89 (d, 1H, Jˆ10 Hz), 3.66±3.70 (m, 1H),
3.93 (d, 1H, Jˆ14 Hz), 3.95 (s, 3H), 4.27±4.34 (m, 2H),
4.56 (d, 1H, Jˆ10 Hz), 5.28±5.36 (m, 1H) and 7.11±7.82
(m, 18H); 13C NMR (CDCl3) d 18.5, 18.7, 31.6, 32.2, 51.4,
52.5, 57.6, 61.2, 80.0, 115.6, 120.6, 121.4, 125.7, 127.6,
127.8, 127.8, 127.9, 128.1, 128.3, 128.6, 129.3, 130.8,
138.0, 138.3, 140.4, 141.6, 143.5, 146.6, 148.9, 172.9 and
173.1; mass spectrum (FAB) m/z 559.2945 (M1H)1
(C37H39N2O3 requires 559.2961).
1
foam: yield 1.15 g (83%); H NMR (CDCl3) d 1.61±1.73
(m, 1H), 2.21±2.37 (m, 1H), 2.43 (t, 2H, Jˆ6 Hz), 3.26±
3.30 (m, 1H), 3.52±3.60 (m, 1H), 3.73±3.80 (m, 1H), 4.05
(d, 1H, Jˆ14 Hz), 4.40 (d, 1H, Jˆ14 Hz), 4.48 (d, 1H,
Jˆ17 Hz), 4.66 (d, 1H, Jˆ12 Hz), 5.18±5.32 (m, 1H) and
7.18±7.83 (m, 18H); 13C NMR (CDCl3) d 18.2, 34.0, 51.6,
59.0, 61.0, 80.1, 117.2, 117.7, 120.4, 121.1, 127.1, 127.6,
127.8, 128.0, 128.2, 128.2, 128.5, 129.0, 129.3, 130.0,
135.9, 140.0, 140.7, 141.3, 143.9, 147.3, 148.0 and 172.7;
mass spectrum (CI, methane) m/z 499 (M1H)1. Anal. Calcd
for C34H30N2O2: C, 81.90; H, 6.06. Found: C, 81.67; H,
6.25.
N-(N-benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-pentenoyl)-
(S)-valine methyl ester (15) was isolated as an oil: yield
1
68 mg (27%); H NMR (CDCl3) d 0.68 (d, 3H, Jˆ7 Hz),
0.73 (d, 3H, Jˆ7 Hz), 1.51±1.60 (m, 1H), 1.84±1.93 (m,
1H), 2.42±2.52 (m, 1H), 3.06 (dd, 1H, Jˆ9 Hz, Jˆ4 Hz),
3.75 (s, 3H), 4.00±4.04 (m, 1H), 4.06 (d, 1H, Jˆ15 Hz),
4.28 (d, 1H, Jˆ14 Hz), 4.49 (d, 1H, Jˆ17 Hz), 4.63 (d,
1H, Jˆ10 Hz), 5.27±5.38 (m, 1H) and 7.13±7.78 (m,
18H); 13C NMR (CDCl3) d 19.1, 19.5, 32.1, 33.8, 51.5,
52.3, 58.0, 62.6, 80.7, 115.8, 120.6, 121.2, 126.4, 127.7,
127.8, 127.9, 128.3, 128.4, 129.0, 129.2, 129.4, 130.1,
137.8, 140.0, 140.4, 141.7, 143.8, 146.6, 148.1, 173.0 and
173.7; mass spectrum (FAB) m/z 559.2949 (M1H)1
(C37H39N2O3 requires 559.2961).
N-Benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-pentenoic acid
(13). To a solution of 1.1 g (2.21 mmol) of 2-cyanoethyl
N-benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-pentenoate (12)
in 10 mL of THF and 5 mL of MeOH was added a solution
of 3.65 g (22.1 mmol) of K2CO3´1.5H2O in 15 mL of H2O.
After stirring at room temperature for 14 h the solvent was
removed under diminished pressure. The residue was
dissolved in 25 mL of CH2Cl2 and washed with two 25-
mL portions of 5% citric acid and with 25 mL of brine.
The organic phase was dried (MgSO4) and concentrated to
afford N-benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-pente-
N-(N-Benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-pentenoyl)-
(R)-phenylalanine methyl ester and N-(N-benzyl-N-
phenyl¯uorenyl-(S)-2-amino-4-pentenoyl)-(S)-phenyl-
alanine methyl ester (16 and 17). Compounds 16 and 17
were prepared in the same fashion as described for
compounds 14 and 15. N-(N-benzyl-N-phenyl¯uorenyl-
(S)-2-amino-4-pentenoyl)-(R)-phenylalanine methyl ester
1
noic acid (13) as a colorless foam: yield 0.91 g (92%); H
1
NMR (CDCl3) d 1.63±1.69 (m, 1H), 2.37±2.47 (m, 1H),
3.08±3.12 (m, 1H), 4.00 (d, 1H, Jˆ14 Hz), 4.38 (d, 1H,
Jˆ13 Hz), 4.40 (d, 1H, Jˆ19 Hz), 4.63 (d, 1H, Jˆ10 Hz),
5.29±5.40 (m, 1H) and 7.19±7.82 (m, 18H); 13C NMR
(CDCl3) d 33.4, 51.8, 61.3, 80.1, 116.8, 120.6, 121.2,
127.0, 127.6, 127.7, 128.0, 128.1, 128.4, 128.5, 128.7,
129.2, 129.4, 129.5, 130.0, 136.4, 138.9, 140.6, 141.6,
143.6, 146.6, 147.8 and 177.6; mass spectrum (FAB) m/z
446.2118 (M1H)1 (C31H28NO2 requires 446.2120).
(16): yield 51 mg (19%); H NMR (CDCl3) d 1.41±1.48
(m, 1H), 2.45±2.60 (m, 1H), 2.82±3.00 (m, 3H), 3.75±
3.93 (m, 3H), 3.86 (s, 3H), 4.26±4.36 (m, 2H), 4.59 (d,
1H, Jˆ8 Hz), 5.31±5.41 (m, 1H) and 6.83±7.82 (m, 23H);
13C NMR (CDCl3) d 31.5, 38.2, 51.5, 52.7, 54.2, 61.5, 80.0,
115.6, 120.6, 121.4, 125.5, 127.1, 127.6, 127.7, 127.9,
128.1, 128.2, 129.0, 129.2, 129.4, 129.9, 130.9, 136.9,
138.0, 138.3, 140.2, 141.7, 143.6, 146.6, 148.6, 172.7 and
173.2; mass spectrum (FAB) m/z 607.2962 (M1H)1
(C41H38N2O3 requires 607.2961).
N-(N-Benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-pentenoyl)-
(R)-valine methyl ester and N-(N-benzyl-N-phenyl¯uor-
enyl-(S)-2-amino-4-pentenoyl)-(S)-valine methyl ester
(14 and 15). To a solution containing 200 mg (0.45
mmol) of N-benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-
pentenoic acid (13) in 3 mL of CH2Cl2 was added a solution
of 92 mg (0.56 mmol) of (R,S)-valine methyl ester hydro-
chloride (3) in 1 mL of pyridine, followed by 116 mg
(0.56 mmol) of DCC. After stirring at room temperature
for 18 h the solvent was removed under diminished
pressure. The residue was dissolved in 25 mL of CH2Cl2,
®ltered, and washed with two 25-mL portions of 0.5N HCl,
two 25-mL portions of saturated aqueous NaHCO3 and with
N-(N-benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-pentenoyl)-
(S)-phenylalanine methyl ester (17): yield 78 mg (29%); 1H
NMR (CDCl3) d 1.66±173 (m, 1H), 2.41±2.65 (m, 3H),
2.97 (dd, 1H, Jˆ9Hz, 2 Hz), 3.57 (s, 3H), 4.00 (d, 1H,
Jˆ13 Hz), 4.09±4.40 (m, 3H), 4.59 (d, 1H, Jˆ10 Hz),
5.26±5.37 (m, 1H) and 6.98±7.78 (m, 23H); 13C NMR
(CDCl3) d 32.9, 38.6, 51.3, 52.4, 53.9, 61.8, 80.4, 115.7,
120.6, 121.3, 125.9, 127.6, 127.9, 128.0, 128.3, 128.6,
128.9, 129.1, 129.2, 129.4, 130.4, 137.1, 137.8, 139.5,
140.3, 141.6, 143.6, 146.7, 148.4, 173.0 and 173.3; mass
spectrum (FAB) m/z 607.2933 (M1H)1 (C41H38N2O3
requires 607.2961).

9428
M. Lodder et al. / Tetrahedron 56 (2000) 9421±9429
HPLC analysis of diastereomers 14 and 15 and
diastereomers 16 and 17
to a total volume of 400 mL of 1:1 CH3CN-50 mM
NH4OAc, pH 4.5, and puri®ed using the same semi-prepara-
tive C18 reversed phase column. After lyophilization of the
appropriate fractions N-(N-benzyl-N-phenyl¯uorenyl-(S)-2-
amino-4-pentenoyl)-(S)-valine pdCpA ester (19) was
obtained as a colorless solid: yield 2.2 mg (51%); mass
spectrum (FAB) m/z 1163.3760 (M1H)1 (C55H61N10O15P2
requires 1163.3793).
A small portion of the crude reaction mixture of diastereo-
mers 14 and 15 was analyzed by HPLC. A 10-mL aliquot
was diluted with 70 mL of 4:1 hexanes±ethyl acetate and
injected on a Partisil silica column (250£10 mm). Elution
was performed with a gradient of 5±25% ethyl acetate in
hexane over a period of 30 min at a ¯ow rate of 3.5 mL/min.
Detection was at 275 nm. Two product peaks were found at
22.3 and 28.0 min in approximately 1:2.5 ratio. Analysis of
the mixture of diastereomers 16 and 17 under the same
conditions resulted in two major peaks at 28.6 and
34.5 min in a 1:2 ratio.
Synthesis and deprotection of aminoacyl-tRNA I
Transfer RNA activation reactions were carried out in
100 mL (total volume) of 50 mM Na Hepes buffer, pH
7.5, containing 0.5 mM ATP, 15 mM MgCl2, 50 mg
(2 nmol) of suppressor tRNACUA transcript lacking the 30-
terminal pCpA moiety, 1.0 A260 unit (40 nmol) of pdCpA
derivative 19, 20% dimethyl sulfoxide (v/v), and 200 units
of T4 RNA ligase. After incubation at 378C for 45 min, the
reaction was quenched by the addition of 0.1 vol. of 3 M
sodium acetate, pH 5.2, and the tRNA was precipitated with
2.5 vol. of ethanol, collected by centrifugation, washed with
70% ethanol, and dried. The product was redissolved in
1 mM KOAc to a ®nal concentration of 3 mg/mL. The
ef®ciency of ligation was estimated by gel electrophoresis
(pH 5.0).41
N-(N-Benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-pente-
noyl)-(S)-valine cyanomethyl ester (18). To a solution
containing 48 mg (0.086 mmol) of N-(N-benzyl-N-phenyl-
¯uorenyl-(S)-2-amino-4-pentenoyl)-(S)-valine methyl ester
(15) in 2 mL of THF was added a solution of 18 mg
(0.43 mmol) LiOH monohydrate in 1 mL of H2O. After
stirring at room temperature for 20 h, the reaction mixture
was diluted with 20 mL of ethyl acetate and washed with
two 20-mL portions of 1N NaHSO4. The organic phase was
dried (MgSO4) and concentrated under diminished pressure.
The residue was applied to a silica gel column (20£1 cm);
elution with 4% MeOH and 1% AcOH in CH2Cl2 gave the
free acid as a colorless oil. The residue was dissolved in
1 mL of CH3CN and 60 mL (0.43 mmol) of Et3N was added,
followed by 27 mL (0.43 mmol) of chloroacetonitrile. After
stirring at room temperature for 17 h the reaction mixture
was diluted with 10 mL of ethyl acetate and washed with
two 10-mL portions of 1N NaHSO4 and then with 10 mL of
brine. The organic phase was dried (MgSO4) and concen-
trated under diminished pressure. The residue was applied
to a silica gel column (20£1 cm); elution with 2:3 ethyl
acetate±hexanes gave N-(N-benzyl-N-phenyl¯uorenyl-(S)-
2-amino-4-pentenoyl)-(S)-valine cyanomethyl ester (18) as
Deprotection was accomplished by admixture of 0.25 vol.
of 40 mM I2 in 4:1 THF±H2O. The combined solution was
maintained at 258C for 60 min, and aminoacyl-tRNA II was
recovered by centrifugation following successive additions
of 0.1 vol. of 3 M NaOAc, pH 5.2, and 2.5 vol. of cold
ethanol. The tRNA pellet was washed with 70% ethanol,
dried, and then dissolved in RNase-free water to a ®nal
concentration of 3 mg/mL and used in in vitro suppression
in a protein biosynthesis experiment immediately following
deprotection.
1
a colorless solid: yield 38 mg (76%); mp 77±78 8C; H
In vitro protein biosynthesis
NMR (CDCl3) d 0.82 (d, 3H, Jˆ6.9 Hz), 0.87 (d, 3H,
Jˆ6.9 Hz), 1.58±1.72 (m, 1H), 1.75±1.84 (m, 1H), 2.42±
2.56 (m, 1H), 3.06 (dd, 1H, Jˆ9.2 Hz, Jˆ2.5 Hz), 3.86±
3.90 (m, 1H), 4.04 (d, 1H, Jˆ13.5 Hz), 4.29 (d, 1H,
Jˆ13.5 Hz), 4.44 (d, 1H, Jˆ17.3 Hz), 4.62 (d, 1H,
Jˆ15.8 Hz), 4.63 (d, 1H, Jˆ13.0 Hz), 4.76 (d, 1H,
Jˆ15.4 Hz), 5.26±5.34 (m, 1H), 6.90 (d, 1H, Jˆ8.8 Hz)
and 7.19±7.79 (m, 18H). Anal. Calcd for C38H37N3O3: C,
78.19; H, 6.39. Found: C,77.83; H, 6.65.
In vitro protein synthesis reactions contained per 100 mL:
70 mL of methionine-depleted, nuclease-treated rabbit
reticulocyte lysate, 80 mCi of [35S]-S-methionine (1000 Ci/
mmol), 2 mL of a solution 1 mM in the 19 amino acids used
in ribosomal protein synthesis (but lacking methionine),
10 mg of the DHFR mRNA, and 25 mg (,1.0 nmol) of
protected misacylated tRNACUA I or deprotected mis-
acylated tRNACUA II. The reaction mixture was incubated
at 308C for 1.5 h and quenched by cooling to 08C. Aliquots
(typically 1 mL) were utilized for analysis by 15% SDS±
PAGE. After electrophoresis, the gel was ®xed in 40%
methanol-10% acetic acid and dried. Autoradiography of
the gels was carried out to determine the location of
35S-labeled protein; quanti®cation of the bands was carried
out using a phosphorimager.
N-(N-Benzyl-N-phenyl¯uorenyl-(S)-2-amino-4-pente-
noyl)-(S)-valine pdCpA ester (19). To a conical vial
containing 5 mg (15.7 mmol) of N-(N-benzyl-N-phenyl-
¯uorenyl-(S)-2-amino-4-pentenoyl)-(S)-valine cyanomethyl
ester (18) was added a solution of 5 mg (3.7 mmol) of the
tris(tetrabutylammonium) salt of pdCpA40 in 50 mL of
DMF. The reaction mixture was stirred at room temperature
for 2 h. A 5-mL aliquot of the mixture was diluted with
45 mL of 1:2 CH3CN-50 mM NH4OAc, pH 4.5. Ten mL
of the diluted aliquot was analyzed by HPLC on a 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 ¯ow rate of 3.5 mL/min (moni-
toring at 260 nm). After 3 h the reaction mixture was diluted
Acknowledgements
This work was supported by NIH Research Grant CA77359
awarded by the National Cancer Institute.

M. Lodder et al. / Tetrahedron 56 (2000) 9421±9429
9429
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