Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center

Article abstract of DOI:10.1016/j.bmc.2013.01.002

Ribosomes containing modifications in three regions of 23S rRNA, all of which are in proximity to the ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 preparations for in vitro protein biosynthesis experiments. When utilized in the presence of mRNAs containing UAG codons at predetermined positions + β-alanyl-tRNA_CUA, the modified ribosomes produced enhanced levels of full length proteins via UAG codon suppression. In the present study, these earlier results have been extended by the use of substituted β-amino acids, and direct evidence for β-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in experiments employing four substituted β-amino acids, including α-methyl-β-alanine, β,β-dimethyl-β-alanine, β-phenylalanine, and β-(p-bromophenyl)alanine. The β-amino acids were incorporated into three different positions (10, 18 and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the UAG codons were compared with those of β-alanine and representative α-l-amino acids. The isolated proteins containing the modified β-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the β-amino acids had been incorporated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of β,β-dimethyl-β-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the β-amino acid.

Full text of DOI:10.1016/j.bmc.2013.01.002

Bioorganic & Medicinal Chemistry 21 (2013) 1088–1096
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Incorporation of b-amino acids into dihydrofolate reductase
by ribosomes having modifications in the peptidyltransferase center
Rumit Maini, Dan T. Nguyen, Shengxi Chen, Larisa M. Dedkova, Sandipan Roy Chowdhury,
⇑
Rafael Alcala-Torano, Sidney M. Hecht
Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Ribosomes containing modifications in three regions of 23S rRNA, all of which are in proximity to the
ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 preparations
for in vitro protein biosynthesis experiments. When utilized in the presence of mRNAs containing UAG
codons at predetermined positions + b-alanyl–tRNACUA, the modified ribosomes produced enhanced lev-
els of full length proteins via UAG codon suppression. In the present study, these earlier results have been
extended by the use of substituted b-amino acids, and direct evidence for b-amino acid incorporation is
provided. Presently, five of the clones having modified ribosomes are used in experiments employing four
Received 1 December 2012
Revised 27 December 2012
Accepted 2 January 2013
Available online 9 January 2013
Keywords:
Aminoacylation
Beta-amino acids
Modified ribosomes
Protein synthesis
substituted b-amino acids, including
a-methyl-b-alanine, b,b-dimethyl-b-alanine, b-phenylalanine, and
b-(p-bromophenyl)alanine. The b-amino acids were incorporated into three different positions (10, 18
and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the
UAG codons were compared with those of b-alanine and representative
a-L-amino acids. The isolated
proteins containing the modified b-amino acids were subjected to proteolytic digestion, and the derived
fragments were characterized by mass spectrometry, establishing that the b-amino acids had been incor-
porated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR
contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can
hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of b,b-dimethyl-b-alanine
into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorpo-
ration of the b-amino acid.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
largest b-mimetic, synthesized by Gellman and co-workers, was
only 17 amino acids long.10 Recombinant DNA techniques, in
The side chains of individual amino acid residues play impor-
tant roles in the formation and stability of secondary structures
in peptides and proteins. For example, some amino acids, such as
combination with in vitro translation, could in principle enable
the synthesis of larger polypeptides and proteins incorporating
b-amino acids, but this approach is presently limited by the strict
leucine and alanine, have enhanced propensities for stabilizing
a
-
bias of the ribosome,12,13 which efficiently accepts only
acids for protein synthesis.
a-L-amino
helices.1–3 It has been demonstrated that oligopeptides comprised
of b-amino acids can also have significant helical propensities,
some of which are not dramatically different in overall structure
Our efforts to modify the architecture of the PTC have shown
that it is possible to prepare ribosomal variants with greater adapt-
than peptides formed from
a
-amino acids.4–7 Additionally, peptide
ability in their acceptance of a-D L
14,15 and b- -amino acids.16 In a re-
analogues containing b-amino acids often show enhanced stability
to proteolytic digestion, making them excellent candidates for use
in peptidomimetic compounds.8–11 Thus, b-amino acid incorpora-
tion into peptides and proteins is of significant interest due to their
ability to create new biopolymers with unusual structural features.
Because virtually all peptide analogues incorporating b-amino
acids are currently prepared by chemical synthesis, the size of
the modified polypeptides accessible for study is limited. The
cent study, eight variants of modified ribosomes, selected for
resistance to erythromycin, were used to prepare a library contain-
ing mutations in two regions of the PTC. Screening of this library
for clones sensitive to b-puromycin lead to the identification of
ribosomal variants putatively capable of recognizing b-amino
acids, while retaining the ability to synthesize wild-type Esche-
richia coli dihydrofolate reductase (DHFR) with good fidelity.16 It
was also shown using S-30 preparations from two ribosomal
clones that suppression of UAG codons in DHFR mRNAs resulted
in enhanced full size protein synthesis in the presence of b-
⇑
Corresponding author. Tel.: +1 480 965 6625; fax: +1 480 965 0038.
E-mail address: sidney.hecht@asu.edu (S.M. Hecht).
16
alanyl–tRNACUA
.
However, this preliminary result was obtained
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.002

R. Maini et al. / Bioorg. Med. Chem. 21 (2013) 1088–1096
1089
only using b-alanine and the incorporation of b-alanine was not
verified by any direct method.
racemic form, enabling the incorporation of either enantiomer to
be detected.
In the present study of modified ribosomes selected using b-
puromycin, five modified ribosomes have been employed to
characterize protein synthesis with four b2 and b3-substituted b-
alanine derivatives, all of which have been incorporated into DHFR
(Fig. 1). Three different positions in DHFR were utilized for incor-
poration of the modified amino acids with S-30 preparations from
the five ribosomal clones, demonstrating unique patterns of b-ami-
no acid incorporation by individual S-30 preparations.
Selected DHFRs were analyzed by mass spectrometry following
trypsin digestion to verify the incorporation of the modified amino
acids solely into the intended positions. It was also shown that
incorporation of a b-alanine derivative into position 18 of DHFR
blocked proteolytic digestion at the C-terminus of Glu-17 by Glu-
C endoproteinase. It is anticipated that the ability to incorporate
b-amino acids into specific positions in proteins may facilitate pro-
tein folding and stability through the intrinsic conformational
biases associated with such amino acids, and may also confer sta-
bility against proteolysis at specific amino acid residues.
Activation of suppressor tRNACUA with each of the five b-amino
acids was carried out as described previously for b-alanyl–tRNA-
16
.
As illustrated in Scheme 1 for b-(p-bromophenyl)alanyl–
CUA
tRNACUA, N-protection of the commercially available amino acid
with a pentenoyl protecting group was achieved by treatment with
the N-hydroxysuccinimide derivative of pentenoic acid.17,18 The N-
protected amino acid was then activated as the cyanomethyl ester
6;19 the overall yield for the two steps was 38%. Amino acid deriv-
ative 6 was then employed for the aminoacylation of pdCpA (7),20
which involved sonication in DMF solution in the presence of tri-
ethylamine, affording N-pentenoyl-b-S-(p-bromophenyl)alanyl-
pdCpA (8) in quantitative yield.
Activated pdCpA derivative 8 was ligated to an abbreviated sup-
pressor tRNACUA transcript using T4 RNA ligase as described previ-
ously, affording N-pentenoyl-b-(p-bromophenyl)alanyl–tRNACUA
(9).16 The Nb protecting group was removed by treatment with
aqueous iodine immediately prior to use in protein synthesis.17,18
Also prepared analogously were activated pdCpA derivatives
N-pentenoyl-3-amino-(R,S)-2-methylpropionyl-pdCpA (8a), N-
pentenoyl-3-amino-3,3-dimethylpropionyl-pdCpA (8b) and N-
pentenoyl-b-S-phenylalanyl-pdCpA (8c).
2. Results and discussion
2.1. Preparation of suppressor tRNACUAs activated with b-amino
acids
2.2. In vitro translation using b-amino acids with wild-type
ribosomes
Five b-amino acids were chosen for study (Fig. 1). These in-
cluded b-alanine (1) and four C-substituted b-alanine derivatives.
The latter included a-methyl-b-alanine (2), b,b-dimethyl-b-alanine
(3), b-phenylalanine (4) and b-(p-bromophenyl)alanine (5). This
permitted an initial assessment of the ability of the several ribo-
somal clones to tolerate b-alanine derivatives substituted on the
It was anticipated that wild-type E. coli ribosomes would be
ineffective in incorporating b-amino acids into proteins. Nonethe-
less, it seemed important to verify this experimentally before
studying the modified ribosomes. Accordingly, all five b-amino-
acyl–tRNACUAs were compared with
-threonyl–tRNACUA for their ability to suppress a UAG codon at
position 49 of DHFR mRNA using an S-30 preparation derived from
a-phenylalanyl–tRNACUA and
a
a
and b carbon atoms, and having either aliphatic or aromatic sub-
stituents. b-Alanine derivative 2 was purposefully prepared in
H2N
OH
H2N
OH
O
O
O
O
O
H2N
OH
H2N
OH
H2N
OH
1
2
3
Br
4
5
COH
(1) pdCpA-beta-
beta-amino acid
A
C
C
amino acid,
T4 RNA ligase
(
2
)
deprotection
Modified
DHFR
S-30 with
modified ribosome
CUA
in vitro
translation
beta-amino
acid
AUC
UAG
mRNA
in vitro
pET28b(+)-DHFR(TAG)
Figure 1. Structures of b-amino acids studied, and the strategy employed for their incorporation into DHFR using modified ribosomes.

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R. Maini et al. / Bioorg. Med. Chem. 21 (2013) 1088–1096
NH2
O
O
N
N
H
H2N
OH
N
O
CN
O
O
P
O
O
N
O
NH2
O
O
O
1) Na2CO3, dioxane-water
O
O
O
N
N
N
2) ClCH2CN, Et3N, CH3CN
+
O
P
O
N
Br
O
O
Br
O
5
6
OH OH
DMF-Et3N
sonication
7
NH2
N
NH2
N
NH2
NH2
N
O
P
O
O
O
O
N
N
tRNA-C
O
O
O
P
O
O
N
N
N
O
O
N
N
N
N
O
P
O
O
P
O
O
O
T4 RNA ligase
O
O
O
O
ATP, tRNA-COH
O
O
OH
O
O
OH
H
H
N
N
O
O
Br
Br
8
9
Scheme 1. Synthesis of b-(p-bromophenyl)alanyl–tRNACUA
.
1
2
3
4
5
6
7
8
9
← full length DHFR
← truncated at position 48
100
0.7
40
0.5
50
0.4
2.2
0.5
0.7
% suppression
Figure 2. Translation of DHFR from wild-type (lane 1) and modified (lanes 2–9) (TAG codon in DHFR position corresponding to Ser49) genes in the presence of different
suppressor tRNACUAs. Lane 2, nonacylated tRNACUA; lane 3, tRNACUA activated with phenylalanine; lane 4, tRNACUA activated with b-phenylalanine (4); lane 5, tRNACUA
activated with threonine; lane 6, tRNACUA activated with b-(p-bromophenyl)alanine (5); lane 7, tRNACUA activated with b,b-dimethyl-b-alanine (3); lane 8, tRNACUA activated
with a-methyl-b-alanine (2); lane 9, tRNACUA activated with b-alanine (1). The suppression efficiency relative to wild type is shown below each lane.
wild-type ribosomes (Fig. 2). The suppression efficiency, expressed
as a percentage of full length DHFR synthesized from the wild-type
2.3. In vitro translation using b-amino acids with modified
ribosomes
gene, was 40% for
a-phenylalanyl–tRNACUA, 50% for a-threonyl–
tRNACUA and <2% above background for all of the b-amino acids,
that is, much less than that of threonine and phenylalanine. Essen-
tially the same result was obtained using DHFR mRNA containing a
UAG codon at position 18 (Supplementary data, Fig. 1). These
data demonstrated the inability of the wild-type ribosomes to
incorporate these b-amino acids into protein with reasonable
efficiency.
Five S-30 systems were prepared using different modified ribo-
some clones16 (Table 1) after b-puromycin selection and were used
for in vitro translation experiments. Two types of modified DHFR
constructs, having a TAG codon in position Val10 (pETDH10 plas-
mid) or Asn18 (pETDH18 plasmid) were used initially for the at-
tempted incorporation of the b-amino acids from their respective
activated suppressor tRNACUAs. Because the efficiency of (unmodi-

R. Maini et al. / Bioorg. Med. Chem. 21 (2013) 1088–1096
1091
Table 1
Table 3
23S rRNA sequence modifications in PTC for clones used for S-30 preparations
Incorporation of b-alanine analogues 2, 4 and 5 into E. coli DHFR (position 18) by the
use of S-30 systems having different modifications in the PTC
Clone
Sequence in 23S rRNA of modified ribosome
Amino acids
Suppression efficiency in different S-30 systems, having
modified ribosomes (%)
region 1
region 2
0403x2
0403x4
040321
040329
040217
Wild type
2057AGCGUGA2063
2057AGCGUGA2063
2057AGCGUGA2063
2057AGCGUGA2063
2057AGCGUGA2063
2057GAAAGAC2063
2502UUACCG2507
2502AGCCAG2507
2502AGAUAA2507
2502UGGCAG2507
2496AUAGUU2501
2496CACCUC2501
2502GAUGUC2507
040329
0403x2
0403x4
040321
040217
Phe
—
2
4
5
100
100
100
100
100
1.5a 0.7
6.0 0.5
18.4 2.7
13.5 1.5
1.2 0.4
1.5 1.0
5.8 1.3
5.3 0.9
2.4 1.0
6.0 2.2
13.2 1.1
15.5 2.0
0.8 0.4
1.3 0.7
6.0 1.8
7.6 2.7
1.0 0.4
0.73 0.2
7.8 0.1
9.2 0.4
a
Each number represents the average of three independent experiments SD.
fied) DHFR synthesis from wild-type and each modified ribosomal
preparation was different (Supplementary data, Table 1), the sup-
pression efficiencies were expressed relative to the suppression
clone 040217, which has the same sequence as 0403x4 and
040329 in the first randomized region (2057–2063) of 23S rRNA,
but a totally different second mutated region (2496–2501 vs
2502–2507) (Table 1). No significant difference was observed be-
tween the efficiency of incorporation of b-amino acids 1, 3, 4 or
5. This S-30 preparation was not tested with b-amino acid 2 in
the case of plasmid pETDH10, but in the case of pETDH18 essen-
tially no full length DHFR synthesis was observed. Clones 0403x2
and 040321 have little homology with 0403x4 and 040329 in re-
gion 2 of the 23S rRNA (Table 1), and they did not demonstrate rea-
sonable full length DHFR synthesis for b-amino acid 2 (1.5–3.8%)
but for the other four b-amino acids the suppression efficiencies
were moderate (5.3–7.9%).
obtained using
a-threonyl–tRNACUA (Table 2) or a-phenylalanyl–
tRNACUA (Table 3). DHFR synthesis obtained in the presence of non-
acylated-tRNACUA was used as the control for non-specific incorpo-
ration at the UAG codon. Quantification of DHFR synthesis was
achieved by the use of a phosphorimager, which monitored the
incorporation of 35S-methionine.
As shown in Table 2 for incorporation into position 10 of DHFR,
all five tested S-30 preparations gave only low levels (0.5–2.2%) of
non-specific readthrough of the UAG codon in the absence of any
activated suppressor tRNA. In comparison, UAG codon suppression
in the presence of b-aminoacyl–tRNACUAs bearing b-amino acids 1,
2 and 3 afforded b-amino acid incorporation in yields up to ꢀ11%.
Figure 2 (Supplementary data) illustrates the formation of full
length DHFR using an S-30 system prepared from clone 040329
in the presence of b-amino acids 1, 2 and 3. Only slight variations
in suppression efficiency (ꢀ2-fold) were observed for the individ-
ual b-amino acids using the five S-30 preparations having the mod-
ified ribosomes. The best DHFR yields were obtained using the S-30
preparations from clones 040329 and 040217.
2.4. Suppression of UAG codon in three different positions of the
DHFR gene by b-(p-bromophenyl)alanyl–tRNACUA
To study the ability of a single b-aminoacyl–tRNACUA to sup-
press different positions on an mRNA, three plasmids (pETDH10,
pETDH18 and pETDH49) have been tested comparatively for
in vitro translation using an S-30 system prepared from clone
040217 and b-(p-bromophenyl)alanyl–tRNACUA prepared from b-
amino acid 5 (Fig. 3). b-Amino acid 5 was chosen because it was
found to incorporate well into DHFR position 18 (Table 3), and be-
cause the bromine atom could confer an isotopic pattern in subse-
quent mass spectrometric analysis (Fig. 4). As shown in the Figure,
Next, the incorporation of aromatic b-amino acids 4 and 5 into
position 18 of DHFR was studied. b-Amino acid 2 was also included
to facilitate a comparison with the results in Table 2. The protocol
for the translation reactions was the same as in previous experi-
ments and the suppression efficiency was expressed relative to
that obtained using
a-phenylalanyl–tRNACUA (Table 3). b-Amino
acid 2 gave comparable relative suppression efficiencies when
using S-30 preparations from clones 0403x4 and 040329 (6.0% in
both cases). However, minimal suppression was observed for the
other S-30s preparations, in spite of the fact that 2 included both
stereoisomers of the amino acid. S-30 preparations from clones
0403x4 and 040329 also afforded the best yield of DHFR for both
b-amino acids with aromatic side chains 4 (13–18%) and 5 (13–
15%). These clones have the same sequence in the first mutated re-
gion (2057AGCGUGA2063 vs 2057GAAAGAC2063 for wild-type
ribosomes) and a high level of homology in the second mutated re-
gion (2502AGCCAG2507 and 2502UGGCAG2507), respectively.
Interesting results were observed for S-30 system prepared from
relative to the suppression obtained using
a-phenylalanyl–tRNA-
CUA, b-(p-bromophenyl)alanine (5) was incorporated into positions
10, 18 and 49 in yields of 11%, 7.4% and 6.3%, respectively. Thus
incorporation could be achieved in reasonable yields in a few dif-
ferent positions of DHFR.
2.5. Characterization of b-amino acids in DHFRs by MALDI
analysis of peptides resulting from ‘in-gel’ trypsin digestion
In order to provide direct evidence for the incorporation of b-
amino acids into DHFR, we prepared two modified DHFRs on a lar-
ger scale. The first employed plasmid pETDH49 and b-(p-bromo-
Table 2
Incorporation of b-alanine analogues 1, 2 and 3 into E. coli DHFR (position 10) by the
use of S-30 systems having different modifications in the PTC
1
2
3
4
5
6
7
8
9
Amino acids
Suppression efficiency in different S-30 systems, having
modified ribosomes (%)
040329
0403x2
0403x4
040321
040217
100%
11
0.01
100
7.4
1.0
100
6.3
1.1
Thr
—
1
2
3
100
100
100
100
100
0.5a 0.3
8.2 3.0
8.8 1.0
11b
2.1 1.4
6.8 3.2
3.8 1.0
7.9 1.1
1.4 0.7
8.1 3.4
5.4 1.9
7.1 3.6
0.9 0.2
5.4 1.3
Not tested
5.6 3.0
2.2 1.0
9.9 3.9
Not tested
10.9 4.4
Figure 3. Relative suppression efficiency of b-(p-bromophenyl)alanyl–tRNACUA at
three different positions (10, 18 and 49) in DHFR, using an S-30 system prepared
from clone 040217. Lanes 1, 4 and 7,
bromophenyl)alanyl–tRNACUA; lanes 3, 6 and 9, unacylated tRNACUA. The suppres-
sion efficiency relative to -phenylalanine is shown below each lane. Lanes 1–3,
modification of position 10 in DHFR; lanes 4–6, modification of position 18 in
DHFR; lanes 7–9, modification of position 49 in DHFR.
L-phenylalanyl–tRNACUA; lanes 2, 5 and 8, b-(p-
L
a
Each number represents the average of three independent experiments SD.
Tested in a single experiment (Supplementary data Fig. 2).
b

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R. Maini et al. / Bioorg. Med. Chem. 21 (2013) 1088–1096
Figure 4. MALDI-MS of tryptic fragments of wild-type (A) and modified DHFRs (B), the latter having b-amino acid 5 in position 49. Mass range 1800–2500 Da (⁄ = estimated
value in Da).
phenyl)alanyl–tRNACUA, putatively affording modified DHFR 1 con-
taining b-amino acid 5 at position 49 (Table 4). The second em-
ployed pETDH18 and b,b-dimethyl-b-alanyl–tRNACUA, affording
modified DHFR 2 believed to have b-amino acid 3 at position 18
(Table 4). Both modified DHFRs were purified by the use of Ni-
NTA chromatography, followed by purification on DEAE–Sephadex

R. Maini et al. / Bioorg. Med. Chem. 21 (2013) 1088–1096
1093
Table 4
MALDI analysis of tryptic digests of wild-type and modified DHFR samples
Position
Peptide sequence
MALDI-MS analysis, molecular mass, Da
Modified DHFR 1
Wild-type
Modified DHFR 2
Est MS
Est
MS
Est
MS
13–32
34–44
2304
1242
2304.1
1242.8
2304
1242
2303.6
1242.6
2289
1242
2289.9
1242.8
NTLNKPVIMGR
45–57
59–71
72–76
77–98
99–106
1506
1415
632
2028
1023
1505.8
1415.8
632.5
2029.1
1023.7
1644
1415
632
2028
1023
1645.6
1415.6
634.3
2029.7
1023.5
1506
1415
632
2028
1023
1505.9
1415.8
632.4
2030.7
1023.6
NIILSSQPGTDDR
VTWVK
VDEAIAASGDVPEIMVIGGGR
VYEQFLPK
Modified DHFR 1: b-alanine analogue 5 in position 49; modified DHFR 2: b-alanine analogue 3 in position 18. Positions 18 and 49 are indicated in red.
The values in boldface reflect the presence of the modified b -amino acids.
and then SDS–polyacrylamide gel electrophoresis. Samples of the
M
1
2
3
DHFR were treated with trypsin according to a published ‘in-gel’
protocol21and the resulting peptide fragments were subjected to
MALDI analysis. A sample of wild-type DHFR was used as a control
in MALDI analysis for both modified DHFRs. The molecular weights
of the tryptic peptides from the wild-type and modified DHFRs
were compared (Table 4). In the case of the modified DHFR 1 hav-
ing b-amino acid 5 at position 49, a tryptic fragment encompassing
amino acids 45–57 was anticipated to have a molecular mass of
1644. As shown in Table 4 and Figure 4, there was an ion peak ob-
served m/z 1644 having a characteristic bromine isotope pattern.
This ion was not present in the tryptic digest of wild-type DHFR,
but the anticipated ion at m/z 1506 (reflecting the presence of
Ser49) was readily apparent. The other six tryptic peptides encom-
passing amino acids 13–106 of DHFR were all observed in both the
wild-type and modified DHFRs, and gave ion peaks at the same
molecular masses.22 Similarly, the modified DHFR 2 having b-ami-
no acid 3 incorporated at position 18 gave an ion peak at m/z
2289.9 (estimated value 2289) corresponding to a peptide encom-
passing amino acids 13–32 (Table 4 and Supplementary data,
Fig. 3). This peak was not present in the tryptic digest of the
wild-type DHFR, but the anticipated ion peak at m/z 2304 (reflect-
ing the presence of Asn18) was present. Again, the remaining six
tryptic peptides in the wild-type and modified DHFRs were all ob-
served and gave the same ion peaks.
26 kDa →
14 kDa →
3.5 kDa →
← 5.5 kDa peptide
← 3.7 kDa peptide
← 1.85 kDa peptide
2.8 kDa →
Figure 5. GluC endoproteinase digestion of modified DHFR sample. Lane 1, non-
digested DHFR; lane 2, digested sample of wild-type DHFR; lane 3, digested sample
of DHFR having b-amino acid 3 in position 18; lane M, molecular weight marker.
weights of 3.7 and 1.85 kDa, as would be expected for the pep-
tides MISLIAALAFDRVIGME and NAMPWNLPADLAWFKRNTLNKPVI
MGRHTWE, resulting from hydrolysis at Glu17 and Glu48. In com-
parison, analogous digestion of the modified DHFR putatively hav-
ing b-amino acid 3 at position 18 afforded only a single radioactive
peptide (5.5 kDa) (lane 3). This peptide likely resulted from failed
digestion following Glu17, resulting in the appearance of the pep-
tide
instead of the two peptides produced from wild-type DHFR. This
observation provides further evidence for the presence of b-amino
acid 3 at position 18 of modified DHFR 2. It also suggests that pro-
teins containing b-amino acids at specific positions may be ex-
pected to exhibit enhanced protease resistance in proximity to
those positions, a finding of potential importance to the design of
proteins for therapeutic and other uses.25
2.6. GluC digestion probing of DHFR structure
The proteolytic stability of b-peptides has been studied in detail
by Seebach and co-workers6 through the use of a set of 15 different
proteases, including the commercially available serine proteases
trypsin (Arg and Lys in P1 position) and chymotrypsin (Tyr, Phe,
Trp and Leu in P1 position). Peptides containing b2, b2,2, b2,3, b2,2,3
and b2b3 substituted amino acids were studied and it was shown
that none of the proteases was able to degrade the b-amino acid
containing species. There is presently no direct evidence that the
presence of a b-amino acid adjacent to a protease cleavage site in
a protein would actually affect protease cleavage. However, it is
logical to anticipate that cleavage would be impaired, and we uti-
lized DHFR 2, putatively containing b-amino acid 3 in position 18,
to study this possibility. DHFR has a glutamic acid at position 17,
and we have shown previously that GluC endoproteinase (which
hydrolyzes peptide bonds at the C-terminus of glutamic acid resi-
dues23) hydrolyzes DHFR between Glu17 and Asn18.24 Shown in
Figure 5 is a polyacryalamide gel used to analyze the hydrolysis
products resulting from treatment of wild-type and modified
DHFRs with GluC endoproteinase. Although hydrolytic cleavage
was not complete under the conditions employed, a comparison
of lanes 1 and 2 shows that GluC endoproteinase produced bands
from wild-type DHFR corresponding to fragments having molecular
3. Conclusions
Four b-amino acids having quite different substitution patterns,
have been used to activate a suppressor tRNA transcript, and were
then incorporated into each of three positions in DHFR by suppres-
sion of UAG codons engineered into specific positions in DHFR
mRNA. The incorporation was mediated by S-30 preparations con-
taining ribosomes modified in the peptidyltransferase center, and
selected for their ability to recognize b-amino acids. The suppres-
sion yields, which depended on the specific modified ribosomes
employed, as well as the structures of the individual amino acids,
ranged up to 18% and were entirely sufficient to enable the prepa-
ration of modified DHFRs in amounts sufficient for routine bio-
chemical studies. The incorporation of selected b-amino acids
was verified following trypsin digestion by mass spectrometric
analysis of the derived peptide fragments, and was limited in each
case analyzed to the intended fragment. b-Amino acids have been
shown to impart specific conformational biases to peptides into
which they have been incorporated,1–3 and it is anticipated that

1094
R. Maini et al. / Bioorg. Med. Chem. 21 (2013) 1088–1096
analogous effects will be observed for proteins containing b-amino
acids. This may be of great advantage in stabilizing protein struc-
tures conformationally, especially where multiple folded confor-
mations are substantially populated. The ability of such amino
acids to stabilize proteins from proteolytic degradation was sug-
gested by the stability of a DHFR containing b,b-dimethyl-b-ala-
nine (3) at position 18 to GluC endoproteinase-mediated
cleavage following a glutamic acid residue at position 17.
NaHSO4 followed by 10 mL of brine, then dried (MgSO4) and con-
centrated to dryness under diminished pressure, affording a crude
residue. The crude product was purified on a flash silica gel column
(15 ꢁ 1 cm) using 1:1 ethyl acetate/hexane for elution to obtain 6
as a colorless semi-solid: yield 140 mg (38%); Rf 0.33 (1:1 ethyl
acetate/hexane); 1H NMR (CDCl3) d 2.27–2.40 (m, 4H), 2.85–3.03
(m, 2H), 4.65 (s, 2H), 4.99–5.07 (m, 2H), 5.39 (q, 1H, J = 6.8 Hz),
5.74–5.84 (m, 1H), 6.45 (d, 1H, J = 8.0 Hz), 7.15 (d, 2H, J = 8.0 Hz),
and 7.45–7.47 (m, 2H); 13C NMR (CDCl3) d 29.4, 35.6, 39.2, 48.6,
49.0, 113.9, 115.9, 121.9, 128.1, 132.0, 136.7, 138.8, 169.3, and
171.8; mass spectrum (APCI), m/z 365.0631 (M+H)+ (C17H20NO3Br
requires m/z 365.0627).
4. Experimental
4.1. General materials and methods
4.2.2. N-4-Pentenoyl-b-S-(p-bromophenyl)alanyl-pdCpA (8)16
Reagents and solvents for chemical synthesis were purchased
from Aldrich Chemical Co. or Sigma Chemical Co. and were used
without further purification. Compounds 4 and 5 were purchased
from Peptech Corporation and were used without further purifica-
tion. All reactions involving air- or moisture-sensitive reagents or
intermediates were performed under argon. Analytical TLC was
performed using Silicycle silica gel 60 Å F254 plates (0.25 mm),
and was visualized by UV irradiation (254 nm). Flash chromatogra-
phy was performed using Silicycle silica gel (40–60 mesh). 1H and
13C NMR spectra were obtained using a Varian 400 MHz NMR spec-
trometer. Chemical shifts are reported in parts per million (ppm, d)
referenced to the residual 1H of the solvent (CDCl3, d 7.26). 13C
NMR spectra were referenced to the residual 13C resonance of
the solvent (CDCl3, d 77.16). Splitting patterns are designated as
follows: s, singlet; d, doublet; q, quartet; m, multiplet. High resolu-
tion mass spectra were obtained at the Arizona State University
CLAS High Resolution Mass Spectrometry Facility or the Michigan
State University Mass Spectrometry Facility.
To a 1.5-mL tube containing 5.3 mg (3.7
butylammonium) salt of pdCpA20 (TBA–pdCpA, 7) was added
10 mg (27 mol) of 6 dissolved in 100 L of 9:1 DMF/Et3N. After
lmol) of the tris(tetra-
l
l
2 h of sonication, the reaction mixture was purified by C18 reversed
phase HPLC using a gradient of 1%?65% acetonitrile in 50 mM
ammonium acetate, pH 4.5, over a period of 45 min. The fractions
eluting at 20.8 and 22.1 min were collected, combined and lyoph-
ilized to afford a colorless solid: yield 3.1 mg (100%); mass spec-
trum (ESI), m/z 942.1234 (M-H)- (C33H39N9O15BrP2 requires m/z
942.1230).
4.2.3. Preparation and characterization of the pdCpA esters of
amino acids 2, 3 and 4
The pdCpA esters of the remaining three amino acids (2, 3 and
4) were prepared in the same fashion as the synthesis of 8 de-
scribed above. Each was purified by C18 reversed phased HPLC
(Supplementary data, Fig. 4) and afforded satisfactory high resolu-
tion mass spectrometric data as follows:
Tris, acrylamide, bis-acrylamide, urea, ammonium persulfate,
N,N,N0,N0-tetramethylenediamine (TEMED), dihydrofolic acid, glyc-
erol, ampicillin, pyruvate kinase, lysozyme, erythromycin, isopro-
4.2.3.1. N-4-Pentenoyl-3-amino-(R,S)-2-methylpropionyl-pdCpA
(8a). Mass spectrum (ESI), m/z 802.1977 (MꢂH)ꢂ (C28H38N9O15P2
requires m/z 802.1968).
pyl-b-
mercaptoethanol were purchased from Sigma Chemicals (St. Louis,
MO). 35S-Methionine (10
Ci/ L) was obtained from Amersham
D-thiogalactopyranoside (IPTG), dithiothreitol (DTT) and 2-
l
l
4.2.3.2. N-4-Pentenoyl-3-amino-3,3-dimethylpropionyl-pdCpA
(Pitscataway, NJ). BL-21 (DE-3) competent cells and T4 RNA ligase
were from Promega (Madison, WI). Plasmid MaxiKit (Life Science
Products, Inc., Frederick, CO) and GenEluteTMHP plasmid miniprep
kit (Sigma) were used for plasmid purification.
Phosphorimager analysis was performed using a Molecular
Dynamics 400E PhosphorImager equipped with ImageQuant ver-
sion 3.2 software. Ultraviolet and visible spectral measurements
were made using a Perkin-Elmer lambda 20 spectrophotometer.
(8b).
Mass spectrum (ESI), m/z 816.2130 (MꢂH)ꢂ
(C29H40N9O15P2 requires m/z 816.2125).
4.2.3.3. N-4-Pentenoyl-b-S-phenylalanyl-pdCpA (8c).
Mass
spectrum (ESI), m/z 864.2126 (MꢂH)ꢂ (C33H40N9O15P2 requires
m/z 864.2125).
4.3. b-Aminoacyl–tRNACUAs
4.2. Synthesis and characterization of aminoacylated pdCpA
derivatives
The activation of suppressor tRNACUAs was carried out as de-
scribed previously.16 Briefly, 100-
lL reaction mixtures of 100 mM
Na Hepes, pH 7.5, contained 1.0 mM ATP, 15 mM MgCl2, 100
l
g
4.2.1. N-4-Pentenoyl-b-S-(p-bromophenyl)alanine cyanomethyl
ester (6)16
of suppressor tRNACUA-COH, 0.5 A260 unit of N-pentenoyl-protected
b-aminoacyl-pdCpA, 15% DMSO, and 100 units of T4 RNA ligase.
The reaction mixture was incubated at 37 °C for 1 h, then quenched
by the addition of 0.1 vol of 3 M NaOAc, pH 5.2. The N-protected
aminoacylated tRNA was precipitated with 3 vol of cold ethanol.
The efficiency of ligation was estimated by 8% polyarylamide–
7 M urea gel electrophoresis (pH 5.0).26
The N-pentenoyl-protected aminoacyl–tRNACUAs were depro-
tected by treatment with 5 mM aqueous I2 at 25 °C for 15 min.
The solution was centrifuged, and the supernatant was adjusted
to 0.3 M NaOAc and treated with 3 vol of cold ethanol to precip-
itate the aminoacylated tRNA. The tRNA pellet was washed with
To a solution containing 100 mg (0.41 mmol) of 5 and 70.0 mg
(0.82 mmol) of NaHCO3 in 4 mL of 1:1 dioxane/H2O was added
90 mg (0.45 mmol) of 4-pentenoyloxysuccinimide. The reaction
mixture was stirred at room temperature for 24 h under argon.
The reaction was quenched by the addition of 15 mL of 1 N aq NaH-
SO4 and the aqueous layer was extracted with two 25-mL portions
of ethyl acetate. The combined organic extract was dried (MgSO4)
and concentrated to dryness under diminished pressure. The crude
product was then dissolved in 4 mL of acetonitrile. To this solution
were added 300 lL (2.10 mmol) of triethylamine and 130 lL
(2.10 mmol) of chloroacetonitrile. The reaction mixture was stirred
at room temperature for 24 h at which time 20 mL of ethyl acetate
was added. The organic layer was washed with 10 mL of 1 N aq
70% aq EtOH, air dried and dissolved in 50
water.
lL of RNase free

R. Maini et al. / Bioorg. Med. Chem. 21 (2013) 1088–1096
1095
4.4. Preparation of S-30 extracts from cells having modified
ribosomes
of the same buffer. DHFR was eluted with a NaCl step gradient (0.1;
0.2; 0.3; 0.4; 0.5 M) in 10 mM Na phosphate, pH 7.4. The fractions
were analyzed on 12% polyacrylamide gel, stained using Coomassie
R-250, then destained in 20% ethanol/7% acetic acid. The fractions
containing DHFR were combined, dialyzed against 50 mM Na
phosphate, pH 7.4, and concentrated by ultrafiltration using a
YM-10 filter (Amicon Ultra, Millipore Corp, Billerica, MA).
Aliquots (5–10
harboring plasmids with a wild-type or modified rrnB gene, were
placed on LB agar supplemented with 100 g/mL of ampicillin
lL) from liquid stocks of E. coli BL-21(DE-3) cells,
l
and grown at 37 °C for 16–18 h. One colony was picked from each
agar plate and transferred into 3 mL of LB medium supplemented
with 100 lg/mL of ampicillin and 0.5 mM IPTG. The cultures were
grown at 37 °C for 3–6 h in a thermostated shaker until OD600
4.7. ‘In gel’ trypsin digestion
ꢀ0.15–0.3 was reached (about 3–5 h), diluted with LB medium
Samples to be digested in the gel were run in 3–4 lanes of a 12%
SDS–polyacrylamide gel, stained with Coomassie R-250 and de-
stained until the background was clear. That area of the gel having
the DHFR was cut from the gel and washed with 0.1 M ammonium
bicarbonate (1 h, room temperature). The solution was discarded
supplemented with 100 lg/mL ampicillin, 1 mM IPTG and 3 lg/
mL of erythromycin (for selectively enhancing the modified ribo-
some fraction) until OD600 0.01 was reached, and then grown at
37 °C for 12–18 h. The optimal concentration of the final cultures
was OD600 0.5–1.0. Cells were harvested by centrifugation
(5000ꢁg, 4 °C, 10 min), washed three times with S-30 buffer
(1 mM Tris–OAc, pH 8.2, containing 1.4 mM Mg(OAc)2, 6 mM KOAc
and 0.1 mM DTT) supplemented with b-mercaptoethanol (0.5 mL/
L) and once with S-30 buffer having 0.05 mL/L b-mercaptoethanol.
The weight of the wet pellet was estimated and 1.27 mL of S-30
buffer was added to suspend 1 g of cells. The volume of the suspen-
sion was measured and used for estimating the amount of other
components. Pre-incubation mixture (0.3 mL) (0.29 M Tris, pH
8.2, containing 9 mM Mg(OAc)2, 13 mM ATP, 84 mM phosphoenol
and 0.1–0.2 mL of 0.1 M ammonium bicarbonate and 10–30 lL of
0.045 mM DTT were added. Gel pieces were incubated at 60 °C
for 30 min, cooled to room temperature and incubated at room
temperature for 30 min in the dark after the addition of 10–
30 lL of 0.1 M iodoacetamide. Gel pieces were washed in 50% ace-
tonitrile/0.1 M ammonium bicarbonate until they became color-
less. After discarding the solution, the gel pieces were incubated
in 0.1–0.2 mL of acetonitrile (10–20 min at room temperature)
and, after removal of solvent, were re-swelled in 50–100
lL of
25 mM ammonium bicarbonate containing 0.02 g/ L trypsin.
l
l
pyruvate, 4.4 mM DTT and 5
lM amino acids mixture), 15 units of
After incubation at 37 °C for 4 h, the supernatant was removed to
a new tube and the peptides were extracted with 60% acetoni-
trile/0.1% TFA (20 min at room temperature). The combined frac-
tions were dried and reconstituted in minimum amount of 60%
acetonitrile/0.1% TFA.
pyruvate kinase and 10 g of lyzozyme were added per 1 mL of cell
l
suspension and the resulting mixture was incubated at 37 °C for
30 min. The incubation mixture was then frozen at ꢂ80 °C
(ꢀ30 min), melted (37 °C, 30 min), and again frozen and melted
at room temperature (ꢀ30 min). Ethylene glycol tetraacetic acid
(EGTA) was then added to 2.5 mM final concentration and the cells
were incubated at 37 °C for 30 min and again frozen (ꢂ80 °C,
30 min). The frozen mixture was centrifuged (15,000ꢁg, 4 °C, 1 h)
and the supernatant was stored in aliquots at ꢂ80 °C.
4.8. GluC endoproteinase digestion
DHFR samples radiolabeled by in vitro translation in the pres-
ence of 35S-methionine were purified by successive chromato-
graphic purification of Ni-NTA and DEAE-Sepharose columns. The
4.5. In vitro protein translation
samples were transferred to 50 lL of 50 mM Tris–HCl, pH 8.0, con-
taining 0.5 mM Glu–Glu, by the use of a YM-10 ultrafiltration de-
vice (Amicon Ultra, Millipore Corp, Billerica, MA). The
radioactivity in the samples was checked by liquid scintillation
counting. About 20 ng of each DHFR sample (ꢀ107cpm) was mixed
Protein translation reactions were carried out in 12–2000
incubation mixture containing 0.2–0.4 L/ of S-30 system,
100 ng/ L of plasmid, 35 mM Tris acetate, pH 7.4, 190 mM potas-
sium glutamate, 30 mM ammonium acetate, 2 mM DTT, 0.2 mg/
mL total E. coli tRNA, 3.5% PEG 6000, 20 g/mL folinic acid,
20 mM ATP and GTP, 5 mM CTP and UTP, 100 M amino acids mix-
ture, 0.5 Ci/ g/mL rifampicin. In the
L of 35S-methionine and 1
case of plasmids having a gene with a TAG codon, a suppressor
tRNA was added to a concentration of 0.3–0.5 g/ L (for -amino-
acyl-tRNAs) and 0.6–1.0 g/ L (for b-aminoacyl-tRNAs). Reactions
lL of
l
lL
l
with 2
The total reaction volume was 20
the addition of 2 L of 1.1% formic acid and the final samples (5–
lg of GluC endoproteinase and incubated at 25 °C for 16 h.
l
l
l
L. Reactions were quenched by
l
l
l
l
10 lL) were analyzed by 20% Tris–tricine polyacrylamide gel elec-
trophoresis27 followed by analysis using a phosphorimager.
l
l
a
l
l
were carried out at 37 °C for 1 h and terminated by chilling on ice.
Aliquots from in vitro translation mixtures were analyzed by SDS–
PAGE followed by quantification of the radioactive bands by phos-
phorimager analysis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.002.
4.6. Purification of DHFR
References and notes
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