Synthesis of pdCpAs and transfer RNAs activated with thiothreonine and derivatives

Article abstract of DOI:10.1016/j.bmc.2012.02.024

N,S-diprotected l-thiothreonine and l-allo-thiothreonine derivatives were synthesized using a novel chemical strategy, and used for esterification of the dinucleotide pdCpA. The aminoacylated dinucleotides were then employed for the preparation of activat

Full text of DOI:10.1016/j.bmc.2012.02.024

Bioorganic & Medicinal Chemistry 20 (2012) 2679–2689
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of pdCpAs and transfer RNAs activated with thiothreonine
and derivatives
⇑
Shengxi Chen, Nour Eddine Fahmi, Ryan C. Nangreave, Youcef Mehellou, Sidney M. Hecht
Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
N,S-diprotected L-thiothreonine and L-allo-thiothreonine derivatives were synthesized using a novel
Received 28 December 2011
Revised 5 February 2012
Accepted 8 February 2012
Available online 15 February 2012
chemical strategy, and used for esterification of the dinucleotide pdCpA. The aminoacylated dinucleotides
were then employed for the preparation of activated suppressor tRNACUA transcripts. Thiothreonine and
allo-thiothreonine were incorporated into a predetermined position of a catalytically competent dihydro-
folate reductase (DHFR) analogue lacking cysteine, and the elaborated proteins were derivatized site-spe-
cifically at the thiothreonine residue with a fluorophore.
Keywords:
Sulfur-containing amino acids
Aminoacylation
Ó 2012 Elsevier Ltd. All rights reserved.
Protein synthesis
Fluorescence labeling
1. Introduction
the synthesis of b-methylcysteine (thiothreonine). Thus, the syn-
theses of L-thiothreonine and L-allo-thiothreonine (Fig. 1) have
Nonproteinogenic amino acids having functionalized side
chains are constituents of biologically important peptides and pep-
tidomimetics. As a result, significant effort has been expended for
the development of synthetic routes useful for preparing such
compounds.1 One example is b-methylcysteine (thiothreonine)
which has attracted considerable attention based on its presence
as a constituent of peptides called lantibiotics.2 Peptides of this
class are of current interest due to their potent antimicrobial activ-
ity and their novel mode of action.3 Lantibiotics are ribosomally
synthesized, posttranslationally modified peptide antibiotics pro-
duced by Gram-positive bacteria. They are characterized by lanthi-
onine and methyl lanthionine residues that are connected via
thioether bridges to create ring structures critical for their bioac-
tivity.2,3 One such peptide, microbisporicin, is currently undergo-
ing preclinical development as a consequence of its superior
efficacy in animal models of multidrug resistant infections com-
pared with the antibiotics of last resort, linezolid and vancomycin.4
Further, of the proteinogenic amino acids, those containing key
side chain functional groups are especially interesting for study
since they often have functional roles in proteins. For example, cys-
teine appears in the active sites of enzymes such as cysteine prote-
ases,5 and can be critical structurally in proteins via its
participation in disulfide bridges.6 As part of our ongoing efforts
to enable the synthesis of modified proteins containing mechanis-
tically interesting non-proteinogenic amino acids,7 we focused on
been undertaken utilizing a novel strategy.
The use of reagents that react with specific amino acid side
chains is a common way to realize post-translational protein mod-
ification. The introduction of a limited number of non-natural ami-
no acids into predetermined positions in proteins can be an
effective method to target specific sites for modification. In partic-
ular, a non-natural amino acid having a single thiol functional
group can be introduced into a target protein and subsequently
transformed by reagents that react with S atoms. The thiothreo-
nines prepared in this study were incorporated into a modified
DHFR protein lacking cysteine residues (csDHFR), facilitating site-
specific protein modification at introduced thiothreonine and
allo-thiothreonine residues via treatment with a thiol specific fluo-
rophore, 7-diethylamino-3-(40-maleimidophenyl)-4-methylcoum-
arin (CPM). The fluorescent prosthetic group was introduced at
the level of pdCpA, suppressor tRNA, or protein to define the
O
SH O
SH
OH
OH
NH
NH
2
2
L-thiothreonine
L-allo-thiothreonine
(2R,3R)-β-methylcysteine
(2R,3S)-β-methylcysteine
⇑
Corresponding author. Tel.: +1 480 965 6625; fax: +1 480 965 0038.
E-mail address: sidney.hecht@asu.edu (S.M. Hecht).
Figure 1. Structures of the b-methylcysteine (thiothreonine) stereoisomers
synthesized.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.024

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S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
optimal methodology for introducing specific functionalities into
proteins.
bicarbonate and NVOCCl was added. Upon stirring for 17 h at room
temperature, NVOC-protected -allo-threonine (3) was obtained
L
following work-up in 60% yield. Although serine lactones can be
easily prepared from N-protected serines through a Mitsunobu
reaction utilizing diisopropyl azodicarboxylate and triphenylphos-
phine, the corresponding threonine counterparts are much more
difficult to prepare under the same conditions, resulting in decarb-
oxylative elimination.14,16 Therefore, lactone formation through
carboxyl group activation using BOP reagent or HBTU was envis-
aged.17 Thus, compound 3, dissolved in dichloromethane, was trea-
ted with triethylamine and HBTU and the reaction mixture was
allowed to stir at room temperature under argon for 20 h. Extrac-
tive work-up followed by silica gel column chromatography gave
the desired lactone 4 in 60% yield. Next, the ring opening of the lac-
tone with thioacetate anion was studied, constituting the crucial
2. Results and discussion
2.1. Chemistry
Several methods have been reported for the preparation of b-
methylcysteine derivatives, including displacement of an O-tosy-
lated threonine derivative with potassium thioacetate,8 conjugate
addition of a thiol to an oxazolone derivative of an a,b-dehydroa-
mino acid to yield an S-benzyl-b-alkylcysteine upon hydrolysis9
and acid hydrolysis of a threonine-derived thiazolidine.10 How-
ever, while the first method gave a single stereoisomeric b-methyl-
cysteine derivative, the latter two methods required laborious
separations to access the individual diastereoisomers. The stereo-
specific ring opening of an aziridine with thiobenzoic acid in the
presence of a Lewis acid for the synthesis of threo-b-methylcys-
step in the synthesis of protected L-thiothreonine. Gratifyingly,
treatment of 4 with potassium thioacetate in DMF at room temper-
ature for 23 h afforded thioacetate 5 regio- and stereospecifically in
85% yield after purification. Since the acetate group used for the
protection of the thiol is not easily removed under conditions com-
patible with the stability of aminoacyl-tRNAs, we sought to replace
it with a protecting group such as the butyldisulfide or 4,40-dime-
thoxytrityl (DMT) group, both of which can be cleaved under neu-
tral conditions. The first attempt to remove the S-acetyl group
using potassium carbonate, followed by reprotection of the SH
group as a mixed disulfide via the agency of butyl 1-thiobutane-
1-sulfinate,18 gave the desired butyldisulfide 1a, albeit in low yield.
The use of 1 equiv of sodium methoxide in methanol to remove the
acetate group followed by mixed disulfide formation afforded
teine from
D-threonine was originally reported by Wakamiya
et al.11 It involved a double inversion at the b-carbon but suffered
from the formation of O-acylated by-product in addition to the de-
sired S-acyl product. An optimized variant of this strategy was used
in the synthesis of orthogonally protected b-methyllanthionine by
ring opening of aziridines with several thiol nucleophiles (Fig. 2).12
The introduction of sulfur at the 2-position of threonine by ring
opening of cyclic threonine sulfamidate with trityl thiol and Boc-
protected cysteine was recently reported for the preparation of
stereochemically pure b-methylcysteine and methyllanthionine
derivatives.13 Presently, we report the preparation of
nine and -allo-thiothreonine stereoisomers utilizing a novel ring
L-thiothreo-
orthogonally protected L-thiothreonine 1a in 72% yield. The intro-
L
duction of the DMT group was performed via a two step process.
Thus, the acetate group was hydrolyzed using lithium hydroxide
and the resulting free thiol was treated with 4,40-dimethoxytrityl
chloride in the presence of triethylamine to give the orthogonally
protected L-thiothreonine 1b in 51% yield. The successful imple-
mentation of the regio- and stereoselective ring opening reaction
promoted by the thioacetate anion prompted us to extend this
opening reaction involving threonine-derived lactones. The ring
opening of such lactones is well documented but the required at-
tack of the nucleophilic sulfur atom at the hindered b-position
was expected to be difficult, as reported by Pansare and Vederas
who studied the nucleophilic ring opening of sulfonamide deriva-
tives of the lactone (Fig. 2).14 Lactone ring opening using LiSH
and NaSH as nucleophiles gave products derived exclusively from
attack at the carbonyl carbon; an exception was thiourea, which
attacked the b-carbon atom affording the b-isothiouronium salt,
a plausible precursor to a b-methylcysteine derivative. We applied
this strategy for the synthesis of NVOC protected b-methylcysteine
using potassium thioacetate as the nucleophile for the key ring
opening of the b-lactone at the b-carbon atom (Scheme 1). For
the protected stereoisomers of thiothreonine studied (Fig. 3), b-
ring opening proceeded in yields of 85% and 82%, respectively.
methodology to the synthesis of protected
L-allo-thiothreonine
(Scheme 2). -Threonine was employed as starting material and
L
was treated initially with NVOCCl, leading to N-protection and
affording compound 6 in 85% yield. Treatment with HBTU in the
presence of triethylamine in dichloromethane for 20 h gave b-lac-
tone derivative 7 in 65% yield. Subsequent treatment of compound
7 with potassium thioacetate in DMF resulted in opening of the b-
lactone ring and the formation of thioacetate derivative 8, which
was obtained in 82% yield. Deprotection of the acetate group of
compound 8 was achieved using sodium methoxide in methanol,
followed by treatment with butyl 1-thiobutane-1-sulfinate in the
same reaction flask. Following work-up and column chromato-
graphic purification, compound 2a was obtained in 90% yield.
The synthesis of protected
menced with the preparation of NVOC-
treatment of -allo-threonine with 6-nitroveratryl chloroformate
(NVOCCl), the latter of which was prepared according to the proce-
dure of Katritzky et al.15 Thus,
-allo-threonine was dissolved in a
L-thiothreonines (1a and 1b) com-
L
-allo-threonine (3) by
L
L
The DMT protected
L-allo-thiothreonine 2b was obtained after
1:1 mixture of dioxane and water in the presence of sodium
NH
O
H2N
S
H2N
H2N
β-lactone
aziridine
S
OH
NHSO2Ph
COOMe
PHN
COOMe
SR
RSH
O
PN
BF3.OEt2
PhSO2HN
O
OH
O
MSH
M = Na or Li
P = benzyloxycarbonyl or nosyl
R = Bn, p-methoxybenzyl, trityl
SH
NHSO2Ph
Figure 2. Strategies reported for the synthesis of b-methylcysteine derivatives.

S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
2681
OH
OH
O
COOH
NVOC-Cl
HBTU
COOH
NaHCO3
H2O-dioxane
60%
CH2Cl2
Et3N
60%
NH2
NHNVOC
NVOCHN
O
L-allo-threonine
3
4
S
S
1- NaOMe, MeOH
COOH
2- butyl 1-thiobutane
1-sulfinate
NHNVOC
SAc
KSAc
72%
1a
COOH
DMF
85%
NHNVOC
DMTS
1- LiOH, MeOH-H2O
COOH
2- DMT-Cl, Et3N
51%
5
NHNVOC
1b
Scheme 1. Synthesis of N,S-diprotected
L-thiothreonine derivatives 1a and 1b.
O
SR
O
SR
Optimal results with 1b and 2b were achieved by treatment with
chloroacetonitrile in dry acetonitrile in the presence of triethyl-
amine. Cyanomethyl esters 9 and 11 were obtained in 67 and
85% yields, respectively (Scheme 3). The treatment of the active es-
ters 9 and 11 with the tris-(tetrabutylammonium) salt of pdCpA in
anhydrous DMF gave the corresponding aminoacylated pdCpAs 10
and 12 in 21 and 47% yields, respectively. The removal of the DMT
OH
NHNVOC
OH
NHNVOC
1a R = SBu
2a R = SBu
2b
R = DMT
1b
R = DMT
Figure 3. N,S-diprotected derivatives of
nine (2).
L-thiothreonine (1) and L-allo-thiothreo-
group at the pdCpA level was performed on protected L-allo-thioth-
reonyl pdCpA ester 12 using silver nitrate in aqueous acetonitrile
followed by treatment with dithiothreitol (Scheme 4). The detrity-
lation was complete within 30 min and NVOC-protected L-allo-thi-
deacetylation using lithium hydroxide in aqueous methanol fol-
lowed by trapping the resulting free thiol with 4,40-dimethoxytrityl
chloride. Compound 2b was obtained in 84% yield after purifica-
tion. The conversion of protected compounds 1 and 2 to the corre-
sponding cyanomethyl esters was attempted using 1a and 1b, as
well as 2a and 2b. The conversions were found to proceed signifi-
cantly more cleanly with the S-tritylated precursors (1b and 2b).
othreonyl pdCpA ester 13 was obtained in 82% yield. The
dinucleotide was derivatized on the sulfur atom with a coumarin
fluorophore [7-diethylamino-3-(40-maleimidophenyl)-4-methyl-
coumarin] in acetonitrile/water and the fluorescent dinucleotide
14 was obtained in 64% yield after purification by HPLC (Fig. S1,
Supplementary data).
OH
OH
O
NVOC-Cl
HBTU
COOH
NH2
L-threonine
COOH
NaHCO3
H2O-dioxane
85%
CH2Cl2
Et3N
65%
NHNVOC
NVOCHN
O
7
6
S
S
1- NaOMe, MeOH
COOH
2- butyl 1-thiobutane
1-sulfinate
NHNVOC
SAc
KSAc
90%
2a
COOH
DMF
82%
NHNVOC
DMTS
1- LiOH, MeOH-H2O
COOH
8
2- DMT-Cl, Et3N
84%
NHNVOC
2b
Scheme 2. Synthesis of N,S-diprotected L-allo-thiothreonine derivatives 2a and 2b.

2682
S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
DMTS
DMTS
DMTS
O
DMTS
DMTS
O
ClCH2CN
pdCpA
COOH
O
CN
CN
OpdCpA
Et3N, CH3CN
67%
DMF
21%
NHNVOC
NHNVOC
NHNVOC
10
1b
9
DMTS
O
O
ClCH2CN
pdCpA
COOH
O
OpdCpA
Et3N, CH3CN
85%
DMF
47%
NHNVOC
NHNVOC
NHNVOC
2b
12
11
Scheme 3. Synthesis of N,S-diprotected
L
-thiothreonine and L-allo-thiothreonine derivatives of pdCpA.
DMTS
HS
O
ν
h
ACC-tRNA
ACC-tRNA
O
O
CUA
DMTS
O
DMTS
O
NH2
O
tRNA-COH
ACC-tRNA
O
pdCpA
O
CUA
T4 RNA ligase
NHNVOC
NHNVOC
10
1- AgNO3
CUA
ν
2- h
NH2
DMTS
SH
O
ν
h
ACC-tRNA
ACC-tRNA
O
CUA
DMTS
O
DMTS
O
NH2
O
tRNA-COH
ACC-tRNA
pdCpA
O
O
NHNVOC
12
CUA
T4 RNA ligase
NHNVOC
1- AgNO3
O
CUA
ν
2- h
NH2
1- AgNO3
2- DTT
CPMS
O
CPMS
O
HS
O
CPMS
O
CPM
tRNA-COH
ν
h
pdCpA
O
NHNVOC
14
ACC-tRNA
CUA
pdCpA
O
ACC-tRNA
O
CUA
O
CH3CN-H2O
T4 RNA ligase
NHNVOC
NHNVOC
13
NH2
O
N
O
CPM
=
O
O
N
Scheme 4. Synthesis of thiothreonyl- and allo-thiothreonyl-tRNACUAs.
2.2. Synthesis of tRNACUAs activated with
coupling with CPM
L-thiothreonines and
Suppressor tRNACUAs activated either with
L-thiothreonine or
1
2
3
allo- -thiothreonine were prepared as outlined in Scheme 4. The
L
aminoacylated pdCpAs were ligated to an abbreviated suppressor
tRNACUA transcript (tRNACUA-COH, lacking the terminal cytidine
and adenosine moieties present at the 30-terminus of all tRNAs)
via a T4 RNA ligase mediated ligation reaction to obtain the N
and S-protected thiothreonyl-tRNACUAs. The ligation efficiencies
were evaluated via denaturing PAGE analysis as shown in Fig-
ure 4.19 The N-protected aminoacylated tRNAs were then irradi-
ated with UV light at 0 °C for 5 min to afford the activated tRNAs
ligated
unligated
Figure 4. Ligation of thiothreonyl-pdCpA derivatives to tRNACUA-COH. The efficiency
of ligation was monitored by 8% denaturing PAGE, pH 5.2, and the gel was stained
with methylene blue. Lane 1, abbreviated tRNACUA (1
ligation of DMT-thiothreonyl-pdCpA with tRNACUA (1
allo-thiothreonyl-pdCpA with tRNACUA (1 g).
l
l
g) without pdCpA; lane 2,
g); lane 3, ligation of DMT-
having amino acids with free a-amines. Evaluation of the deprotec-
l

S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
2683
tion efficiency of the DMT group was investigated prior to photol-
ysis. A portion of the fully protected tRNAs were subjected to treat-
ment with silver nitrate, followed by treatment with dithiothreitol,
affording thiothreonyl-tRNACUAs having free sulfhydryl groups. Re-
moval of the DMT group was verified via coupling with the couma-
rin fluorophore, and visualized by irradiating with 365 nm
wavelength light (Fig. 5).
with 365 nm wavelength light (Fig. 7). As is clear from Figure 7,
the fluorophore could be introduced readily into the nascent
csDHFRs containing thiothreonine and allo-thiothreonine. As antic-
ipated, the csDHFRs elaborated with DMT-protected thiothreonine
and allo-thiothreonine did not react detectably with the coumarin
fluorophore.
In the present study, we used the DMT group to protect the thiol
L-Allo-thiothreonyl-tRNACUA derivatized with the same fluoro-
groups of L-thiothreonine and L-allo-thiothreonine. The DMT group
phore was also prepared by the T4 RNA ligase-mediated coupling
of dinucleotide 14 with tRNA-COH (Scheme 4). Following irradia-
tion with UV light to remove the NVOC protecting group, the
CPM-derivatized L-allo-thiothreonyl-tRNACUA was employed in a
protein synthesizing system, as described below.
could be removed readily at the pdCpA and tRNA levels, and both
the DMT-protected and unprotected thiothreonyl- and allo-thioth-
reonyl-tRNACUAs were used to incorporate the thiolated amino
acids into position 10 of csDHFR. The nascent csDHFRs containing
unprotected thiothreonine and allo-thiothreonine reacted readily
with CPM, affording protein fluorescently labeled at a single posi-
tion. However, the DMT group could not be removed from the thi-
othreonines at the protein level due to protein precipitation under
the conditions used for deprotection. The free thiol groups of thi-
othreonine and allo-thiothreonine could be modified with the fluo-
rophore CPM at the pdCpA, tRNA and protein levels. However, the
CPM-protected allo-thiothreonyl-tRNACUA proved to be a poor sub-
strate in the peptidyltransferase reaction, such that the preferred
method for elaborating the fluorescent protein was by direct deriv-
atization of the initially formed csDHFR containing a free SH group
in the amino acid at position 10.
2.3. Participation of the activated tRNAs in protein synthesis
and post-translational modification of csDHFR
The ability of thiothreonyl-tRNACUAs to participate in protein
synthesis was evaluated in a bacterial cell free protein synthesizing
system. A plasmid encoding a modified Escherichia coli DHFR in
which the Cys85 and Cys152 codons had been replaced with serine
codons was used. This modified DHFR was found to retain essen-
tially full enzymatic activity (Fig. S2, Supplementary data). Addi-
tionally, a stop codon (TAG) was incorporated into the plasmid at
position 10 of DHFR to facilitate incorporation of the thiothreonine
derivatives from the misacylated suppressor tRNAs (Scheme 5).
3. Conclusions
Both L-thiothreonyl-tRNACUA and L-allo-thiothreonyl-tRNACUA were
found to participate in protein synthesis, affording DHFRs with 43%
and 45% suppression efficiencies, respectively, relative to the
amount of csDHFR produced from the comparable mRNA lacking
any stop codon (Fig. 6). The DMT-protected thiothreonine and
allo-thiothreonine were incorporated in yields of 28% and 26%,
respectively. In comparison, the incorporation of the fluorescently
labeled allo-thiothreonine from CPM-derivatized allo-thiothreonyl-
tRNACUA (prepared by ligation of 14 and tRNA-COH) proceeded only
in low (ꢀ5%) yield. In replicate experiments, the incorporation of
CPM-allo-thiothreonine into csDHFR ranged from 2–8%. All of the
proteins were purified successively on Ni-NTA and then DEAE-Se-
pharose columns, as illustrated for the thiothreonine-containing
DHFR (Fig. S3, Supplementary data). Coupling of the purified pro-
tein containing thiothreonine and allo-thiothreonine in position
10 with the coumarin fluorophore was visualized by irradiating
This study has addressed the synthesis of the two stereoisomers
of
step in these syntheses involved the formation of an intermediate
-threonyl-b-lactone, which was opened using thioacetate as a
nucleophile to afford the desired stereoisomers of -thiothreonine.
This synthesis provided access to -thiothreonine and -allo-thi-
othreonine cleanly and in good yields. The two isomers so prepared
were used to esterify pdCpA and then ligated to tRNACUA-COH
L-thiothreonine in five steps using a novel approach. The key
L
L
L
L
.
To illustrate specific coupling between the thiothreonine amino
acids and a thiol-reactive fluorophore, we employed a modified
DHFR having serines in lieu of the Cys85 and Cys152 in wild-type
DHFR. The mRNA used to direct the syntheses of DHFR had a stop
(UAG) codon corresponding to position 10 of the protein.
Thiothreonyl- and allo-thiothreonyl-tRNACUAs functioned well
in the cell free protein synthesizing systems employed, effecting
suppression of the UAG codon at position 10 in csDHFR in 43%
and 45% yields, respectively. The derived proteins both reacted
with the thiol-specific fluorophore, while proteins elaborated with
the S-tritylated amino acids as a control did not react with the fluo-
rophore detectably.
1
2
3
4
5
4. Experimental
4.1. General materials and methods
Reagents and solvents for chemical synthesis were purchased
from Aldrich Chemical Co. or Sigma Chemical Co. and used without
further purification. All reactions involving air- or moisture-sensi-
tive reagents or intermediates were performed under argon. Flash
chromatography was performed using Silicycle silica gel (40–
60 mesh). Analytical TLC was performed using EM silica gel 60
F254 plates (0.25 mm) and was visualized by UV irradiation
(254 nm). 1H and 13C NMR spectra were obtained using
a
400 MHz Varian NMR instrument. Chemical shifts are reported in
parts per million (ppm, d) referenced to the residual 1H resonance
of the solvent (CDCl3, d 7.26; CD3OD, d 3.31; DMSO-d6, d 2.50). 13C
NMR spectra were referenced to the residual 13C resonance of the
solvent (CDCl3, d 77.16; CD3OD, d 49.00, DMSO-d6, d 39.52).
Figure 5. Coupling of the NVOC-protected thiothreonyl-tRNA analogues with CPM
after DMT group removal. The fluorescence was monitored at ꢀ470 nm following
irradiation at 365 nm. Sample 1, CPM treated suppressor tRNA (3 lg/lL); sample 2,
CPM treated DMT-thiothreonyl-tRNA (3
nyl-tRNA (3 g/ L); sample 4, CPM treated DMT-allo-thiothreonyl-tRNA (3
sample 5, CPM coupled allo-thiothreonyl-tRNA (3 g/ L).
l
g/lL); sample 3, CPM coupled thiothreo-
l
l
lg/lL);
l
l

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S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
in vitro
transcription
mRNA
UAG
csDHFR
in vitro
translation
pET28b(+)-csDHFR(10)
SH
CH3
CPM
(1) pdCpA-aa,
T4 RNA ligase
NH2
O
tRNA-CCA
tRNACUA-COH
CH3
(2) deprotection
SH
O
CPM
NH2
csDHFR
O
tRNA-CCA
CH3
S
CPM
S
O
CPM
CH3
Scheme 5. Strategy employed for incorporation of thiothreonines into csDHFR and coupling with CPM.
1
2
3
4
5
1
2
0
3
4
5
6
7
35S
yield (%)
100
28
26
43
45
5
Figure 6. In vitro incorporation of thiothreonine derivatives into csDHFR at
position 10. The samples were analyzed by 15% SDS–PAGE at 100 V for 2 h.
Phosphorimager analysis was performed using an Amersham Biosciences Storm
820 equipped with ImageQuant version 5.2 software from Molecular Dynamics.
Lane 1, wild-type csDHFR; lane 2, incorporation in the absence of any amino acid;
lane 3, incorporation of DMT-thiothreonine; lane 4, incorporation of DMT-allo-
thiothreonine; lane 5, incorporation of thiothreonine; lane 6, incorporation of allo-
thiothreonine; lane 7, incorporation of CPM-allo-thiothreonine.
fluorescence
Figure 7. Coupling of thiothreonyl-csDHFR analogues with CPM. All of the protein
samples were purified chromatographically prior to treatment with the fluoro-
phore. The samples were analyzed by 15% SDS–PAGE at 100 V for 2 h. The 35S-
methionine labeled protein bands were obtained through phosphorimager analysis,
and the fluorescence was monitored at ꢀ470 nm following irradiation at 365 nm.
Lane 1, CPM treated wild-type csDHFR (1
nyl-csDHFR (1 g); lane 3, CPM treated DMT-allo-thiothreonyl-csDHFR (1
4, CPM treated thiothreonyl-csDHFR (1 g); lane 5, CPM treated allo-thiothreonyl-
csDHFR (1 g).
l
g); lane 2, CPM treated DMT-thiothreo-
l
lg); lane
Splitting patterns are designated as follows: s, singlet; d, doublet;
dd, doublet of doublets; t, triplet; q, quartet; quint, quintet; m,
multiplet; br, broad. High resolution mass spectra were obtained
at the Arizona State University CLAS High Resolution Mass Spec-
trometry Laboratory or at Michigan State University Mass Spec-
trometry Facility.
Ni-NTA agarose was obtained from Qiagen Inc. (Valencia, CA).
DNA oligonucleotides were ordered from Integrated DNA Technol-
ogies (Coralville, IA). DEAE-Sepharose, ammonium persulfate,
acrylamide, N,N0-methylene-bis-acrylamide, acetic acid, potassium
glutamate, ammonium acetate, dithiothreitol, magnesium acetate,
l
l
polynucleotide kinase were purchased from New England Biolabs
Inc. (Ipswich, MA). HPLC analysis was carried out using a Grace
Davison Econosil C18 reversed phase column (10
l
; 250 ꢁ 10 mm).
Phosphorimager analysis was performed using an Amersham
Biosciences Storm 820 equipped with ImageQuant version 5.2 soft-
ware from Molecular Dynamics. UV spectral measurements were
made using a Perkin–Elmer Lamdba 20 UV/vis spectrometer. Fluo-
rescence was monitored through 365 nm UV light. The mutant
csDHFR analogues modified at position 10 with thiothreonine were
prepared using the general strategy shown in Scheme 5.7,20
phospho(enol)pyruvate, Escherichia coli tRNA, isopropyl b-D-thio-
galactopyranoside (IPTG), ATP, GTP, CTP, UTP, cAMP, amino acids,
rifampicin, formamide and 7-diethylamino-3-(40-maleimidophe-
nyl)-4-methylcoumarin (CPM) were obtained from Sigma-Aldrich
(St. Louis, MO). Tris and SDS were obtained from Bio-Rad Laborato-
4.2. Synthesis of L-thiothreonines and their pdCpA derivatives
ries (Hercules, CA). [35S]-methionine (1000 Ci/mmol, 10
lCi/lL)
was purchased from PerkinElmer Inc. (Boston, MA). Protease inhib-
itor (complete, EDTA-free) was obtained from Boehringer Mann-
heim Corp. (Indianapolis, IN). T4 RNA ligase and T4
4.2.1. N-(6-Nitroveratryloxycarbonyl)-
To a solution containing 0.50 g (4.19 mmol) of
in 20 mL of 1:1 dioxane/H2O was added 0.70 g (8.38 mmol) of
L
-allo-threonine (3)
L
-allo-threonine

S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
2685
NaHCO3. This was followed by the addition of 1.38 g (5.03 mmol) of
4.2.4. N-(6-Nitroveratryloxycarbonyl)-
disulfide (1a)
A solution containing 189 mg (0.45 mmol) of S-acetyl-N-(6-
nitroveratryloxycarbonyl)- -thiothreonine (5) in 10 mL of anhy-
drous MeOH was purged repeatedly by subjecting it to vacuum
and then argon. A solution of 98 L (25% w/v, 0.45 mmol) of
L-thiothreonine n-butyl
6-nitroveratryl chloroformate. The reaction mixture was stirred at
room temperature for 17 h. Twenty mL of CHCl3 was then added
to the reaction mixture, and the biphasic mixture was separated.
The chloroform layer was discarded and the aqueous layer was
acidified to pH ꢀ4 by the addition of 1 N NaHSO4. The aqueous solu-
tion was then washed with three 30-mL portions of ethyl acetate.
The combined organic layer was dried (MgSO4) and the solvent
was concentrated under diminished pressure. The crude residue
was purified on a silica gel column (15 ꢁ 4 cm); elution with
9:1:0.01 CH2Cl2/MeOH/AcOH afforded N-(6-nitroveratryloxycar-
L
l
NaOMe in MeOH was added and the reaction mixture was stirred
at room temperature under argon for 1 h. Butyl 1-thiobutane-1-
sulfinate (94 mg, 0.48 mmol) in 0.8 mL of MeOH was added and
stirring was continued for another 2 h. The solvent was concen-
trated under diminished pressure and residue was partitioned be-
tween 10 mL of EtOAc and 8 mL of 1 N aq NaHSO4. The organic
layer was washed with 10 mL of water, dried (MgSO4) and concen-
trated under diminished pressure. The crude product was purified
on a silica gel column (14 ꢁ 4 cm) using 9:1:0.01 CH2Cl2/MeOH/
bonyl)-L-allo-threonine (3) as a yellow foam: yield 0.90 g (60%); sil-
ica gel TLC Rf 0.20 (9:1:0.01 CH2Cl2/MeOH/AcOH); ½a D24
ꢃ7.1 (c 1.0,
ꢂ
MeOH); 1H NMR (CD3OD) d 1.24 (d, 3H, J = 6.4 Hz), 3.89 (s, 3H), 3.96
(s, 3H), 4.14 (m, 1H), 4.27 (d, 1H, J = 5.2 Hz), 5.38, 5.42 (ABq, 2H,
J = 15.6 Hz), 7.18 (s, 1H), and 7.71 (s, 1H); 13C NMR (CD3OD) d
19.2, 56.8, 57.0, 61.3, 64.6, 68.6, 109.2, 110.6, 129.8, 140.5, 149.4,
155.4, 158.3, and 173.6; mass spectrum (APCI), m/z 359.1083
(M+H)+ (C14H19N2O9 requires m/z 359.1091).
AcOH for elution. N-(6-Nitroveratryloxycarbonyl)-L-thiothreonine
n-butyl disulfide (1a) was obtained as a yellow syrup: yield
152 mg (72%); silica gel TLC Rf 0.55 (9:1:0.01 CH2Cl2/MeOH/AcOH);
½
a 2D4 +39.9 (c 1.0, CH2Cl2); 1H NMR (CDCl3) d 0.90 (t, 3H, J = 7.4 Hz),
ꢂ
1.38 (m, 2H), 1.42 (d, 3H, J = 7.2 Hz), 1.62 (quint, 2H, J = 7.4 Hz),
2.69 (t, 2H, J = 7.4 Hz), 3.59 (m, 1H), 3.94 (s, 3H), 3.98 (s, 3H),
4.61 (dd, 1H, J = 9.2 and 3.2 Hz), 5.52, 5.60 (ABq, 2H, J = 15.2 Hz),
5.78 (d, 1H, J = 9.2 Hz), 7.01 (s, 1H), and 7.71 (s, 1H); 13C NMR
(CDCl3) d 13.8, 18.6, 21.7, 31.1, 39.5, 47.1, 56.5, 56.6, 58.2, 64.3,
108.3, 109.5, 128.3, 139.6, 148.2, 153.9, 156.1, and 175.3; mass
spectrum (APCI), m/z 463.1212 (M+H)+ (C18H27N2O8S2 requires
m/z 463.1209).
4.2.2. N-(6-Nitroveratryloxycarbonyl)-L-allo-threonyl-b-lactone
(4)
A sample of 0.87 g (2.42 mmol) of compound 3 was dissolved in
25 mL of dichloromethane and 1.35 mL (0.98 g, 9.68 mmol) of tri-
ethylamine was added. To this solution was added 1.83 g
(4.84 mmol) of HBTU (O-benzotriazole-N,N,N0,N0-tetramethyluro-
niumhexafluorophosphate) and the reaction mixture was stirred
at room temperature for 20 h under argon. The solvent was con-
centrated under diminished pressure and the crude product was
purified by flash column chromatography on a silica gel column
(15 ꢁ 4 cm) using 1:1 hexanes/EtOAc for elution. The appropriate
fractions were collected and the solvent was concentrated under
diminished pressure to give the desired product 4 as a pale yellow
solid: yield 498 mg (60%); mp 140–142 °C; silica gel TLC Rf 0.20
4.2.5. S-(4,40-Dimethoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
L-thiothreonine (1b)
A solution containing 335 mg (0.80 mmol) of S-acetyl-N-(6-
nitroveratryloxycarbonyl)-L-thiothreonine (5) in 10 mL of MeOH
was purged repeatedly by subjecting it to vacuum and then argon.
A degassed solution of 5 mL of 1 N aq LiOH was added and the
reaction mixture was stirred at room temperature under argon
for 1 h. The reaction mixture was diluted with 10 mL of satd. aq
NaHCO3 and the methanol was removed under diminished pres-
sure. The aqueous residue was extracted with 10 mL of Et2O and
the ethereal solution was discarded. The pH of the aqueous phase
was adjusted to ꢀ 3 with 1 N HCl and then extracted with two 15-
mL portions of EtOAc. The organic layer was dried (MgSO4) and
concentrated under diminished pressure to give the crude thiol
product as a yellow syrup. The crude product was dissolved in
(1:1 hexanes/ethyl acetate); ½a D24
ꢂ
ꢃ58.5 (c 1.0, acetone); 1H NMR
(DMSO-d6) d 1.49 (d, 3H, J = 5.6 Hz), 3.87 (s, 3H), 3.90 (s, 3H),
4.77 (m, 2H), 5.37 (s, 2H), 7.19 (s, 1H), 7.70 (s, 1H), and 8.41 (d,
1H, J = 7.2 Hz); 13C NMR (DMSO-d6) d 18.2, 56.1, 56.2, 63.26,
63.29, 75.4, 108.2, 111.1, 126.7, 139.5, 147.9, 153.3, 155.0, and
168.9; mass spectrum (APCI), m/z 341.1005 (M+H)+ (C14H17N2O8
requires 341.0985).
10 mL of DMF under argon and 241 lL (1.73 mmol) of triethyl-
4.2.3. S-Acetyl-N-(6-nitroveratryloxycarbonyl)-L-thiothreonine
(5)
amine was added. Argon was bubbled through the mixture and
then 325 mg (0.96 mmol) of 4,40-dimethoxytrityl chloride was
added. The reaction mixture was stirred at room temperature for
18 h. The solvent was concentrated and the residue was purified
on a silica gel column (22 ꢁ 3 cm) using a stepwise gradient of ace-
tone in hexanes (40 ? 70%) for elution. S-(4,40-Dimethoxytrityl)-N-
A solution containing 0.25 g (0.73 mmol) of compound 4 in
8 mL of DMF was treated with 105 mg (0.92 mmol) of potassium
thioacetate. The reaction mixture was stirred at room temperature
for 23 h under argon. Twenty-five mL of ethyl acetate was added
followed by 20 mL of 3% aq HCl. The organic layer was washed with
20 mL of 3% aq HCl. The organic layer was then washed with two
15-mL portions of 5% aq LiCl. The organic layer was dried (MgSO4)
and concentrated to dryness under diminished pressure. The crude
solid was purified by flash column chromatography on a silica gel
column (15 ꢁ 3 cm) using 9:1:0.01 CH2Cl2/MeOH/AcOH for elu-
tion. The appropriate fractions were collected and concentrated
under diminished pressure to give 5 as a yellow foam: yield
(6-nitroveratryloxycarbonyl)-L-thiothreonine (1b) was obtained as
a yellow foam: yield 277 mg (51%); 1H NMR (CDCl3) d 1.00 (d, 3H,
J = 6.8 Hz), 3.76 (s, 7H), 3.94 (s, 6H), 4.37 (m, 1H), 5.48–5.59 (m,
3H), 6.78 (d, 4H, J = 8.0 Hz), 6.98 (s, 1H), 7.17 (m, 1H), 7.26 (m,
2H), 7.37 (m, 4H), 7.46 (d, 2H, J = 7.2 Hz), and 7.71 (s, 1H); 13C
NMR (CDCl3) d 29.4, 53.8, 55.4, 56.5, 56.7, 59.3, 108.3, 109.6,
113.3, 113.4, 126.9, 127.2, 127.9, 128.0, 128.1, 129.3, 129.4,
130.8, 136.7, 139.6, 145.1, 148.2, 153.9, 158.3, and 173.6; mass
spectrum (APCI), m/z 675.2023 (MꢃH)ꢃ (C35H35N2O10S requires
m/z 675.2012).
262 mg (85%); ½a 2D4
ꢂ
+12.7 (c 1.0, MeOH); 1H NMR (CDCl3) d 1.41
(d, 3H, J = 6.8 Hz), 2.30 (s, 3H), 3.93 (s, 3H), 3.97 (s, 3H), 4.12 (m,
1H), 4.56 (dd, 1H, J = 8.8 and 4.2 Hz), 5.49, 5.55 (ABq, 2H,
J = 15.0 Hz), 5.75 (d, 1H, J = 8.8 Hz), 6.99 (s, 1H), and 7.69 (s, 1H);
13C NMR (CDCl3) d 16.6, 30.7, 41.0, 56.4, 56.7, 57.6, 64.1, 108.1,
109.7, 127.9, 139.5, 148.1, 153.7, 155.8, 173.6, and 195.2; mass
spectrum (APCI), m/z 417.0967 (M+H)+ (C16H21N2O9S requires m/
z 417.0966).
4.2.6. S-(4,40-Dimethoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
L
-thiothreonine cyanomethyl ester (9)
To a solution containing 259 mg (0.38 mmol) of S-(4,40-dime-
thoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
L-thiothreonine (1b)
in 8 mL of anhydrous CH3CN was added 267
l
L
(193 mg,

2686
S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
1.91 mmol) of Et3N followed by 242
lL (288 mg, 3.82 mmol) of
4.2.9. N-(6-Nitroveratryloxycarbonyl)-L-threonyl-b-lactone (7)
chloroacetonitrile. The reaction mixture was stirred at room tem-
perature under argon for 24 h. The solvent was concentrated and
the residue was partitioned between 15 mL of EtOAc and 10 mL
of 1 N NaHSO4. The organic layer was separated, washed with
10 mL of brine, dried (MgSO4) and concentrated under diminished
pressure. The crude product was purified on a silica gel column
(20 ꢁ 2.5 cm), eluting with 1:1 hexanes/EtOAc. N-(6-Nitroveratryl-
A sample of 1.20 g (3.34 mmol) of compound 6 was dissolved in
35 mL of dichloromethane and 1.86 mL (1.35 g, 13.4 mmol) of tri-
ethylamine was added. To this solution was added 1.90 g
(5.02 mmol) of HBTU (O-benzotriazole-N,N,N0,N0-tetramethyluro-
niumhexafluorophosphate) and the reaction mixture was stirred
at room temperature for 20 h under argon. The solvent was con-
centrated under diminished pressure and the crude product was
purified by flash column chromatography on a silica gel column
(15 ꢁ 4 cm) using 1:1 hexanes/EtOAc for elution. The appropriate
fractions were collected and the solvent was concentrated under
diminished pressure to give the desired product 7 as a pale yellow
solid: yield 741 mg (65%); mp 165–168 °C; silica gel TLC Rf 0.25
oxycarbonyl)-S-(4,40-dimethoxytrityl)-
L-thiothreonine cyanometh-
yl ester (9) was obtained as a yellow foam: yield 189 mg (67%);
silica gel TLC Rf 0.24 (3:2 hexanes/ethyl acetate); 1H NMR (CDCl3)
d 0.98 (d, 3H, J = 6.8 Hz), 2.72 (m, 1H), 3.72 (s, 6H), 3.89 (s, 3H),
3.93 (s, 3H), 4.34 (m, 1H), 4.40, 4.56 (ABq, 2H, J = 15.6 Hz), 5.47
(m, 3H), 6.75 (d, 4H, J = 9.0 Hz), 6.92 (s, 1H), 7.09–7.23 (m, 3H),
7.30 (dd, 4H, J = 9.0 and 3.0 Hz), 7.39 (d, 2H, J = 7.6 Hz), and 7.65
(s, 1H); 13C NMR (CDCl3) d 42.5, 49.3, 49.5, 55.4, 56.6, 56.7, 59.5,
64.3, 108.3, 108.4, 109.8, 110.0, 113.3, 113.4, 127.0, 127.2 127.9,
128.08, 128.13, 129.3, 129.4, 130.8, 136.5, 136.6, 139.6, 145.1,
153.9, 155.8, 158.4, 158.8, and 169.2; mass spectrum (APCI), m/z
738.2091 (M+Na)+ (C37H37NaN3O10S requires m/z 738.2097).
(1:1 hexanes/ethyl acetate); ½a D24
ꢂ
+6.4 (c 1.0, acetone); 1H NMR
(acetone-d6) d 1.48 (d, 3H, J = 6.4 Hz), 3.94 (s, 3H), 3.95 (s, 3H),
4.94 (q, 1H, J = 6.4 Hz), 5.48 (br s, 2H), 5.56 (dd, 1H, J = 9.6 and
6.0 Hz), 7.20 (s, 1H), 7.67 (d, 1H, J = 8.4 Hz), and 7.71 (s, 1H); 13C
NMR (acetone-d6) d 15.1, 56.6, 56.7, 61.4, 64.5, 75.3, 109.1, 111.4,
128.1, 140.8, 149.4, 154.8, 156.2, and 169.8; mass spectrum (APCI),
m/z 341.0992 (M+H)+ (C14H17N2O8 requires m/z 341.0985).
4.2.10. S-Acetyl-N-(6-nitroveratryloxycarbonyl)-L-allo-
thiothreonine (8)
4.2.7. S-(4,40-Dimethoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
L
-thiothreonyl pdCpA ester (10)
A solution containing 0.20 g (0.58 mmol) of compound 7 in
6 mL of DMF was treated with 84 mg (0.73 mmol) of potassium
thioacetate. The reaction mixture was stirred at room temperature
for 2 h under argon. Fifteen mL of ethyl acetate was added followed
by 20 mL of 3% aq HCl. The organic layer was washed with 20 mL of
3% aq HCl. The organic layer was then washed with two 15-mL por-
tions of 5% aq LiCl. The organic layer was dried (MgSO4) and con-
centrated to dryness under diminished pressure. The crude solid
was purified by flash column chromatography on a silica gel col-
umn (15 ꢁ 4 cm) using 9:1:0.01 CH2Cl2/MeOH/AcOH for elution.
The appropriate fractions were collected and concentrated under
diminished pressure to give 8 as a light yellow foam: yield 0.78 g
To a conical vial containing 16.0 mg (22.4
dimethoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
l
mol) of S-(4,40-
-thiothreonine
cyanomethyl ester (9) was added a solution containing 5.8 mg
(4.3
mol) of the tris-(tetrabutylammonium) salt of pdCpA21 in
90 L of anhydrous DMF followed by 10 L of triethylamine. The
reaction mixture was stirred at room temperature under an argon
atmosphere for 24 h. A 5- L aliquot of the reaction mixture was di-
luted with 55 L of 1:1 CH3CN/50 mM NH4OAc at pH 4.5 and was
L
l
l
l
l
l
analyzed by HPLC on a C18 reversed phase column (250 ꢁ 10 mm).
The column was washed with 1 ? 65% CH3CN in 50 mM NH4OAc
at pH 4.5 over a period of 45 min at a flow rate of 3.5 mL/min
(monitoring at 260 nm). The remaining reaction mixture was di-
luted to a total volume of 0.6 mL with CH3CN and purified using
the same C18 reversed-phase column. S-(4,40-Dimethoxytrityl)-N-
(82%); silica gel TLC Rf 0.43 (9:1:0.01 CH2Cl2/MeOH/AcOH); ½a D24
ꢂ
ꢃ24.2 (c 1.0, MeOH); 1H NMR (CD3OD) d 1.30 (d, 3H, J = 7.2 Hz),
2.28 (s, 3H), 3.90 (s, 3H), 3.98 (s, 3H), 4.15 (m, 1H), 4.60 (d, 1H,
J = 4.0 Hz), 5.44–5.51 (ABq, 2H, J = 15.6 Hz), 7.19 (s, 1H), and 7.73
(s, 1H); 13C NMR (CD3OD) d 16.1, 30.4, 42.1, 56.8, 57.0, 58.6, 64.6,
109.2, 110.5, 129.9, 140.5, 149.4, 155.4, 158.5, 172.7, and 196.5;
mass spectrum (APCI), m/z 417.0967 (M+H)+ (C16H21N2O9S re-
quires m/z 417.0968).
(6-nitroveratryloxycarbonyl)-L-thiothreonyl pdCpA ester (10)
(retention time 33.8 and 34.4 min) was recovered from the appro-
priate fractions as a colorless solid by lyophilization: yield 1.2 mg
(21%); mass spectrum (ESI), m/z 1295.3136 (M+H)+ (C54H61N10O22P2S
requires m/z 1295.3158).
4.2.8. N-(6-Nitroveratryloxycarbonyl)-
L
-threonine (6)
4.2.11. N-(6-Nitroveratryloxycarbonyl)-L-allo-thiothreonine n-
butyl disulfide (2a)
To a solution containing 0.50 g (4.19 mmol) of
L
-threonine in
20 mL of 1:1 dioxane/H2O was added 0.70 g (8.38 mmol) of NaH-
CO3. This was followed by the addition of 1.38 g (5.03 mmol) of
6-nitroveratryl chloroformate. The reaction mixture was stirred
at room temperature for 17 h. Twenty mL of chloroform was then
added to the reaction mixture and the biphasic mixture was sepa-
rated. The chloroform layer was discarded and the aqueous layer
was acidified to pH ꢀ4 by the addition of 1 N NaHSO4. The aqueous
solution was then washed with three 30-mL portions of ethyl ace-
tate. The combined organic layer was dried (MgSO4) and the sol-
vent was concentrated under diminished pressure. The crude
residue was purified on a silica gel column (15 ꢁ 4 cm); elution
with 9:1:0.01 CH2Cl2/MeOH/AcOH afforded N-(6-nitroveratryloxy-
A solution containing 0.49 g (1.19 mmol) of compound 8 in
25 mL of methanol was purged repeatedly by subjecting it alter-
nately to vacuum and argon. A solution of 258 lL (25% w/v,
1.19 mmol) of NaOMe in MeOH was added and the reaction mix-
ture was stirred at room temperature under argon for 1 h. Butyl-
1-thiobutane-1-sulfinate (0.24 g; 1.25 mmol) in 2 mL of MeOH
was then added to the reaction mixture, which was stirred at room
temperature for an additional 2 h. The solvent was concentrated
under diminished pressure and the crude product was partitioned
between 30 mL of ethyl acetate and 30 mL of 1 N NaHSO4. The or-
ganic layer was dried (MgSO4) and concentrated and the crude was
purified by flash column chromatography on a silica gel column
(18 ꢁ 4.5 cm), eluting with 9:1:0.01 CH2Cl2/MeOH/AcOH. The
appropriate fractions were combined and concentrated to afford
2a as a yellow syrup: yield 0.50 g (90%); silica gel TLC Rf 0.50
carbonyl)-L-threonine (6) as a solid yellow foam: yield 1.28 g
(85%); silica gel TLC Rf 0.16 (4:1 CH2Cl2/MeOH); ½a D24
ꢃ5.5 (c 1.0,
ꢂ
MeOH); 1H NMR (CD3OD) d 1.24 (d, 3H, J = 6.4 Hz), 3.89 (s, 3H),
3.96 (s, 3H), 4.18 (d, 1H, J = 2.8 Hz), 4.34 (m, 1H, J = 3.6 and
2.8 Hz), 5.41, 5.54 (ABq, 2H, J = 15.6 Hz), 7.19 (s, 1H), and 7.71 (s,
1H); 13C NMR (CD3OD) d 20.5, 56.8, 57.0, 61.1, 64.6, 68.3, 109.2,
110.5, 129.8, 140.5, 149.4, 155.4, 158.6, and 174.0; mass spectrum
(APCI), m/z 359.1083 (M+H)+ (C14H19N2O9 requires m/z 359.1091).
(9:1:0.01 CH2Cl2/MeOH/AcOH); ½a D24
ꢂ
ꢃ68.2 (c 1.0, CH2Cl2); 1H
NMR (CDCl3) d 0.89 (t, 3H, J = 7.4 Hz), 1.30 (d, 3H, J = 7.2 Hz), 1.38
(m, 2H, J = 7.4 Hz), 1.62 (quint, 2H, J = 7.4 Hz), 2.71 (t, 2H,
J = 7.4 Hz), 3.44 (m, 1H), 3.94 (s, 3H), 3.97 (s, 3H), 4.86 (dd, 1H,
J = 9.0 and 3.8 Hz), 5.55 (d, 2H, J = 7.6 Hz), 5.59 (d, 1H, J = 9.0 Hz),

S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
2687
6.99 (s, 1H), and 7.70 (s, 1H); 13C NMR (CDCl3) d 13.8, 15.4, 21.7,
31.4, 39.3, 47.2, 56.5, 56.6, 64.2, 108.2, 109.6, 128.4, 139.6, 148.1,
153.9, 155.9, and 174.9; mass spectrum (APCI), m/z 463.1217
(M+H)+ (C18H27N2O8S2 requires m/z 463.1209).
169.2; mass spectrum (APCI), m/z 738.2089 (M+Na)+ (C37H37Na-
N3O10S requires m/z 738.2097).
4.2.14. S-(4,40-Dimethoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
L
-allo-thiothreonyl pdCpA ester (12)
To a conical vial containing 16.0 mg (22.4
4.2.12. S-(4,40-Dimethoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
l
mol) of S-(4,40-
-allo-thiothre-
onine cyanomethyl ester (11) was added a solution of 5.8 mg
(4.3
mol) of the tris-(tetrabutylammonium) salt of pdCpA21 in
90 L of anhydrous DMF, followed by 10 L of triethylamine. The
reaction mixture was stirred at room temperature under an argon
atmosphere for 24 h. A 5- L aliquot of the reaction mixture was di-
luted with 55 L of 1:1 CH3CN/50 mM NH4OAc at pH 4.5 and was
L
-allo-thiothreonine (2b)
dimethoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
L
A solution containing 540 mg (1.29 mmol) of S-acetyl-N-(6-
nitroveratryloxycarbonyl)-L-allo-thiothreonine (8) in 17 mL of
l
l
MeOH was purged repeatedly by subjecting it to vacuum and then
argon. A solution of 8.5 mL (8.5 mmol) of 1 N aq LiOH was added
and the reaction mixture was stirred at room temperature for
1 h, while bubbling argon continuously through the solution. The
reaction mixture was diluted with 15 mL of satd. aq NaHCO3 and
methanol was concentrated under diminished pressure. The aque-
ous phase was extracted with 10 mL of ether and the organic layer
was discarded. The aqueous phase was acidified to pH ꢀ1 with 2 N
HCl and again extracted with two 20-mL portions of EtOAc. The or-
ganic layer was dried (MgSO4) and concentrated under diminished
pressure. The crude product was immediately dissolved in 15 mL
of anhydrous DMF and 0.39 mL (283 mg, 2.79 mmol) of triethyl-
amine was added followed by 524 mg (1.54 mmol) of dimethoxy-
trityl chloride, while bubbling argon continuously through the
reaction mixture. The reaction mixture was then stirred under an
argon atmosphere at room temperature for 18 h. DMF was concen-
trated under diminished pressure and the residue was dissolved in
25 mL of EtOAc. The organic layer was washed with 10% aq citric
acid, dried (MgSO4) and concentrated under diminished pressure.
l
l
l
analyzed by HPLC on a C18 reversed phase column (250 ꢁ 10 mm).
The column was washed with 1 ? 65% CH3CN in 50 mM NH4OAc
at pH 4.5 over a period of 45 min at a flow rate of 3.5 mL/min
(monitoring at 260 nm). The remaining reaction mixture was di-
luted to a total volume of 0.6 mL with CH3CN and purified using
the same C18 reversed-phase column. S-(4,40-dimethoxytrityl)-N-
(6-nitroveratryloxycarbonyl)-L-allo-thiothreonyl pdCpA ester (12)
(retention time 34.0 and 34.5 min) was recovered from the appro-
priate fractions as a colorless solid by lyophilization: yield 2.6 mg
(47%); mass spectrum (ESI), m/z 1295.3203 (M+H)+ (C54H61N10O22P2S
requires 1295.3158).
4.2.15. N-(6-nitroveratryloxycarbonyl)-L-allo-thiothreonyl
pdCpA ester (13)
The crude product was purified on
a silica gel column
To a solution containing 1.6 mg (1.23
thoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
pdCpA ester (12) in 0.60 mL of 3:1 acetonitrile/water (v/v) was
added a solution containing 3.13 mg (18.5 mol) of AgNO3 in
40 L of water. The reaction mixture was stirred at room temper-
ature for 1 h then 9.5 mg (61.6 mol) of dithiothreitol in 50 L of
water was added and stirring was continued for 90 min. The mix-
ture was centrifuged at 15,000ꢁg at 4 °C for 5 min and the super-
natant was collected and purified by HPLC on a C18 reversed phase
column (250 ꢁ 10 mm). The column was washed with 1 ? 65%
CH3CN in 50 mM NH4OAc at pH 4.5 over a period of 45 min at a
flow rate of 3.5 mL/min (monitoring at 260 nm). N-(6-Nitroverat-
l
mol) of S-(4,40-dime-
-allo-thiothreonyl
(18 ꢁ 3 cm) eluting with 7:3 hexanes/acetone and then 1:1 hex-
L
anes/acetone. S-(4,40-Dimethoxytrityl)-N-(6-nitroveratryloxycar-
bonyl)-L-allo-thiothreonine (2b) was obtained as a yellow foam:
l
yield 739 mg (84%); 1H NMR (CDCl3) d 1.06 (d, 3H, J = 7.6 Hz),
3.76 (s, 7H), 3.89 (s, 3H), 3.93 (s, 3H), 4.05 (dd, 1H, J = 9.2 and
2.8 Hz), 5.35 (d, 1H, J = 9.2 Hz), 5.53 (s, 2H), 6.79 (d, 4H,
J = 8.8 Hz), 6.97 (s, 1H), 7.17 (m, 1H), 7.26 (m, 2H), 7.37 (d, 4H,
J = 8.8 Hz), 7.45 (d, 2H, J = 7.6 Hz), and 7.71 (s, 1H); 13C NMR
(CDCl3) d 17.9, 41.7, 53.3, 56.5, 56.7, 58.4, 63.9, 67.4, 108.2,
109.2, 113.4, 126.8, 128.1, 128.9, 129.4, 130.7, 136.9, 139.3,
145.1, 148.0, 153.9, 155.8, 158.2, and 173.6; mass spectrum (APCI),
m/z 675.2002 (M-H)ꢃ (C35H35N2O10S requires m/z 675.2012).
l
l
l
ryloxycarbonyl)-L-allo-thiothreonyl pdCpA ester (13) (retention
time 22.9 and 23.3 min) was recovered from the appropriate frac-
tions as a colorless solid by lyophilization and was used immedi-
ately in the next step: yield 1.0 mg (82%); mass spectrum
(MALDI), m/z 993.4 (M+H)+ (theoretical 993.2).
4.2.13. S-(4,40-Dimethoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
L
-allo-thiothreonine cyanomethyl ester (11)
To a solution containing 600 mg (0.88 mmol) of S-(4,40-dime-
thoxytrityl)-N-(6-nitroveratryloxycarbonyl)-
(2b) in 16 mL of anhydrous CH3CN was added 613
4.39 mmol) of Et3N followed by 558 L (663 mg, 8.80 mmol) of
L
-allo-thiothreonine
lL (445 mg,
4.2.16. S-([N-(7’’-Diethylamino-4’’-methylcoumarin-3’’-yl)-4’-
phenyl]succinimid-3-yl)-N-(6-nitroveratryloxycarbonyl)-L-allo-
thiothreonyl pdCpA ester (14)
l
chloroacetonitrile. The reaction mixture was stirred at room tem-
perature under argon for 19 h. The solvent was concentrated and
the residue was dissolved in 25 mL of EtOAc and 20 mL of 1 N NaH-
SO4. The organic layer was separated, washed with 20 mL of brine,
dried (MgSO4) and concentrated under diminished pressure. The
crude product was purified on a silica gel column (16 ꢁ 3 cm), elut-
ing with 3:2 hexanes/EtOAc. S-(4,40-dimethoxytrityl)-N-(6-nitrov-
To a solution containing 1.0 mg (1.0
oxycarbonyl)- -allo-thiothreonyl pdCpA ester (13) in 375
acetonitrile/water (v/v) was added 0.8 mg (2.0 mol) of 7-diethyl-
amino-3-(40-maleimidophenyl)-4-methylcoumarin in 75
L of ace-
lmol) of N-(6-nitroveratryl-
L
lL of 2:1
l
l
tonitrile. The mixture was stirred at room temperature for 3 h then
purified by HPLC on a C18 reversed-phase column (250 ꢁ 10 mm).
The column was washed with 1 ? 65% CH3CN in 50 mM NH4OAc
at pH 4.5 over a period of 45 min at a flow rate of 3.5 mL/min (mon-
itoring at 260 nm). S-([N-(7’’-Diethylamino-4’’-methylcoumarin-
3’’-yl)-4’-phenyl]succinimid-3-yl)-N-(6-nitroveratryloxycarbonyl)-
eratryloxycarbonyl)-L-allo-thiothreonine cyanomethyl ester (11)
was obtained as a yellow foam: yield 539 mg (85%); silica gel
TLC Rf 0.23 (3:2 hexanes/ethyl acetate); 1H NMR (CDCl3) d 1.16
(d, 3H, J = 7.6 Hz), 2.86 (m, 1H), 3.78 (s, 6H), 3.92 (s, 3H), 3.94 (s,
3H), 4.57, 4.66 (ABq, 2H, J = 15.6 Hz), 5.29 (d, 1H, J = 8.8 Hz), 5.52
(q, 2H, J = 15.2 Hz), 6.81 (d, 4H, J = 8.8 Hz), 6.95 (s, 1H), 7.20 (t,
1H, J = 7.2 Hz), 7.28 (t, 2H, J = 7.2 Hz), 7.36 (m, 4H), 7.45 (m, 2H),
and 7.71 (s, 1H); 13C NMR (CDCl3) d 41.4, 49.0, 55.4, 56.5, 56.6,
58.0, 64.1, 67.7, 108.2, 109.4, 113.5, 113.8, 127.0, 128.2, 128.4,
129.3, 130.6, 136.5, 139.5, 144.9, 148.1, 153.9, 155.6, 158.4, and
L-allo-thiothreonyl pdCpA ester (14) (retention time 34–35 min)
was recovered from the appropriate fractions as a yellow solid by
lyophilization: yield 0.9 mg (64%); mass spectrum (MALDI), m/z
1395.6 (M+H)+ (theoretical 1395.3); mass spectrum (ESI), m/z
1394.3382 (M+) (C57H64N12O24P2S requires 1394.3352).

2688
S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
4.3. Ligation of suppressor tRNACUA-COH with thiothreonine
analogues and deprotection of DMT and NVOC groups
4.6. In vitro translation of csDHFR analogues
The wild-type csDHFR plasmid was obtained by site-directed
mutagenesis as described previously25 using the wild-type DHFR
plasmid as the template. The DNA primer for the mutation at posi-
tion 85 was 50-GATGAAGCCATCGCGGCGT CTGGTGACGTACCA-
GAAATC-30; and the primer for the mutation at position 152 was
50-CAGAACTCTCACAGCTATAGCTTTGAGATTCTGGAGC-30. The mu-
tant csDHFR (TAG at position 10) was obtained by the same site-di-
rected mutation using the wild-type csDHFR plasmid as the
template and the DNA sequence 50-GTCTGATTGCGGCGTTAGCGT
AGGATCGCGTTATCGGCATG-30 as the primer.
The yeast suppressor tRNAPhe
transcript was prepared as
CUA
previously reported.7i Activation of the suppressor tRNACUA was
carried out in 100 L (total volume) of 100 mM Hepes buffer, pH
7.5, containing 2.0 mM ATP, 15 mM MgCl2, 100 g of suppressor
l
l
tRNA-COH, 2.0 A260 units of protected aminoacyl-pdCpA (5–10 fold
molar excess), 15% DMSO and 200 units of T4 RNA ligase. After
incubation at 37 °C for 1 h, the reaction was quenched by the addi-
tion of 10 lL of 3 M NaOAc, pH 6.3, followed by 300 lL of ethanol.
The reaction mixture was incubated at –20 °C for 30 min, then cen-
trifuged at 15,000ꢁg at 4 °C for 30 min. The supernatant was care-
The in vitro expression mixture (300
l
L total volume) contained
fully decanted and the tRNA pellet was washed with 100
lL of 70%
30
l
g of mutant DHFR (TAG at position 10) plasmid DNA, 120 l
L of
ethanol and dissolved in 100
l
L of RNase free H2O. The efficiency of
premix (35 mM Tris-acetate, pH 7.0, 190 mM potassium glutamate,
30 mM ammonium acetate, 2.0 mM dithiothreitol, 11 mM maga-
nesium acetate, 20 mM phospho(enol)pyruvate, 0.8 mg/mL of
E. coli tRNA, 0.8 mM IPTG, 20 mM ATP and GTP, 5 mM CTP and
ligation was estimated by 8% denaturing PAGE (pH 5.2).19 The DMT
protecting group of N-(6-nitroveratryloxycarbonyl)-thiothreonyl-
tRNA or N-(6-nitroveratryloxycarbonyl)-allo-thiothreonyl-tRNA
was removed by treatment with 50 mM AgNO3 at room tempera-
ture for 30 min, followed by treatment with 60 mM DTT at room
temperature for 30 min.22 The reaction mixture was centrifuged
at 15,000ꢁg at 4 °C for 10 min, and then the supernatant was care-
UTP and 4 mM cAMP), 100
30 -methionine, 10
Ci of [35S]-
tected misacylated tRNACUA and 90
l
M of each of the 20 amino acids,
g/ L rifampicin, 90 g of depro-
L of S-30 extract from E. coli
l
L
l
l
l
l
strain BL21(DE3). The reaction mixture was incubated at 37 °C
for 45 min. Plasmid DNA containing the gene for wild-type csDHFR
was used as the positive control, and an abbreviated tRNA (tRNA-
fully decanted. The precipitated pellet was washed with 100
0.3 M NaOAc, pH 6.3. The combined aqueous supernatant was trea-
ted with 600 L of ethanol to precipitate the tRNA. The tRNA pellet
was washed with 100 L of 70% ethanol and then dissolved in
30 L of RNase free H2O. The NVOC-protected aminoacyl-tRNA
lL of
l
C
OH) lacking any amino acid was used as the negative control. An
aliquot containing 2 L of reaction mixture was removed, treated
with 2 L of loading buffer and heated at 90 °C for 2 min. This
l
l
l
l
was cooled to 2 °C and irradiated with a 500 W mercury–xenon
lamp for 5 min.23,24 After irradiation, the deblocked aminoacylated
suppressor tRNAs were used in in vitro suppression experiments
without further purification.
was analyzed by 15% SDS–PAGE at 100 V for 2 h.
4.7. Purification of analogues of csDHFRs
Also prepared were samples of the thiothreonyl- and allo-thi-
othreonyl-tRNAs in which the DMT group was not removed prior
to photolysis of the NVOC protecting group.
The analogues of csDHFR containing an N-terminal hexahisti-
dine fusion peptide were purified by Ni-NTA chromatography.26
The in vitro translation reaction mixture (300
900 L of 50 mM Tris–HCl, pH 8.0, containing 300 mM NaCl and
10 mM imidazole, and mixed gently with 100 L of a 50% slurry
of Ni-NTA resin at 4 °C for 2 h. Then the mixture was loaded on a
column and washed with 600 L of 50 mM Tris–HCl, pH 8.0, con-
taining 300 mM NaCl and 20 mM imidazole. Finally, the csDHFR
analogue was washed three times with 200 L of 50 mM Tris–
lL) was diluted with
l
l
4.4. Coupling of NVOC-thiothreonyl-tRNA analogues with CPM
l
The NVOC-thiothreonyl-tRNA coupling was carried out in 50
lL
(total volume) of 100 mM NaOAc, pH 6.3, containing 90 g of thi-
l
l
othreonyl-tRNA analogue and 1 mM CPM. The reaction mixture
was incubated at room temperature for 1 h and was quenched by
the addition of 3 lL of 3 M NaOAc, pH 6.3, followed by 150 lL of
ethanol. The reaction mixture was incubated at –20 °C for
30 min, then centrifuged at 15,000ꢁg at 4 °C for 30 min. The super-
natant was carefully decanted and the tRNA pellet was washed
HCl, pH 8.0, containing 30 mM NaCl and 150 mM imidazole. The fi-
nal three elutions from the Ni-NTA column were combined and
loaded on a 200
washed with 300
100 mM NaCl, 300
200 mM NaCl, and then three 300-
l
l
L DEAE-Sepharose column. The column was
L of 50 mM Tris–HCl, pH 8.0, containing
L of 50 mM Tris–HCl, pH 8.0, containing
L portions of 50 mM Tris–
l
l
with 50 lL of 70% ethanol and dissolved in 30 lL of RNase free
HCl, pH 8.0, containing 300 mM NaCl. Aliquots of each fraction
were analyzed by 15% SDS–PAGE.
H2O. The fluorescence was monitored at ꢀ470 nm following irradi-
ation at 365 nm.
4.8. Testing the activities of wild-type DHFR and csDHFRs at pH
7.0
4.5. Ligation of suppressor tRNA-COH with CPM-allo-
thiothreonyl-pdCpA and deprotection of NVOC group
The enzymatic activities of wild-type DHFR and csDHFR were
measured in 1 mL of MTEN buffer (containing 50 mM MES,
25 mM trizma base, 25 mM ethanolamine, 100 mM NaCl, 0.1 mM
EDTA and 10 mM b-mercaptoethanol, pH 7.0). MTEN buffer
Suppressor tRNA aminoacylation was carried out in 100
tal volume) of 100 mM Hepes buffer, pH 7.5, containing 2.0 mM
ATP, 15 mM MgCl2, 100 g of suppressor tRNA-COH, 2.0 A260 units
of NVOC-protected CPM-allo-thiothreonyl-pdCpA, 15% DMSO and
200 units of T4 RNA ligase. After incubation at 37 °C for 1 h, the
lL (to-
l
(0.97 mL) was mixed with 10
protein. The mixture was incubated at 37 °C for 3 min. Then
20 L of 5 mM dihydrofolate in MTEN buffer, pH 7.0, was added.
lL of 10 mM NADPH and 100 ng of
reaction was quenched by the addition of 10
lL of 3 M NaOAc,
l
pH 5.2, followed by 300 L of ethanol. The reaction mixture was
l
The OD value at 340 nm was monitored over a period of 6 min.
incubated at –20 °C for 30 min, then centrifuged at 15,000ꢁg at
4 °C for 30 min. The supernatant was carefully decanted and the
4.9. Coupling thiothreonyl-csDHFR analogues with CPM
tRNA pellet was washed with 100
lL of 70% ethanol, and dissolved
in 30 L of RNase free H2O. The NVOC-protected CPM-allo-thioth-
reonyl-tRNA was cooled to 2 °C and irradiated with a 500 W mer-
cury–xenon lamp for 5 min.
l
The thiothreonyl-csDHFR coupling was carried out in 30
tal volume) of 50 mM Tris–HCl, pH 7.0, containing 100 mM NaCl,
of thiothreonyl-csDHFR analogue and 1 mM CPM.
lL (to-
3 lg

S. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2679–2689
2689
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The reaction was incubated at room temperature for 5 min and
was quenched by extraction with two 30- L portions of ethyl ace-
tate. The reaction mixtures were analyzed by 15% SDS–PAGE. The
fluorescence was monitored at ꢀ470 nm following excitation at
365 nm.
l
Acknowledgement
This study was supported by Research Grant GM 092946 from
the National Institutes of Health.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.02.024.
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