Synthesis of alanyl nucleobase amino acids and their incorporation into proteins

Article abstract of DOI:10.1016/j.bmc.2016.07.008

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, so

Full text of DOI:10.1016/j.bmc.2016.07.008

Accepted Manuscript
Synthesis of alanyl nucleobase amino acids and their incorporation into proteins
Poulami Talukder, Larisa M. Dedkova, Andrew D. Ellington, Petro Yakovchuk,
Jaebum Lim, Eric V. Anslyn, Sidney M. Hecht
PII:
S0968-0896(16)30500-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.07.008
BMC 13121
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 April 2016
2 July 2016
5 July 2016
Please cite this article as: Talukder, P., Dedkova, L.M., Ellington, A.D., Yakovchuk, P., Lim, J., Anslyn, E.V.,
Hecht, S.M., Synthesis of alanyl nucleobase amino acids and their incorporation into proteins, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.07.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of alanyl nucleobase amino acids and their incorporation into
proteins
Poulami Talukdera, Larisa M. Dedkovaa, Andrew D. Ellingtonb, Petro Yakovchuka,
Jaebum Limb, Eric V. Anslynb, Sidney M. Hechta,*
aBiodesign Center for BioEnergetics and School of Molecular Sciences, Arizona State
University, Tempe, AZ 85287, USA
bDepartment of Chemistry, University of Texas at Austin, Austin, TX 78712, USA
* Corresponding author. Tel: +1 480 965 6625; fax: +1 480 965 0038.
E-mail address: Sidney.Hecht@asu.edu
Keywords: Nucleobases; DNA_protein interaction; N-Boc-L-serine β-lactone; DNA polymerase I

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and
exceptionally important. Such proteins employ a variety of strategies for recognition of the
relevant structural elements in their nucleic acid substrates, some of which have been shown to
involve rather subtle interactions which might have been difficult to design from first principles.
In the present study, we have explored the preparation of proteins containing unnatural amino
acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino
acids into the nucleic acid binding domain of a protein should enable these modified proteins to
interact with their nucleic acid substrates using Watson-Crick base pairing interactions. We
describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled
their attachment to a suppressor tRNA, and their incorporation into each of two proteins with
acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and
guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was
chosen as one model protein to enable direct comparison of the facility of incorporation of the
nucleobase amino acids with numerous other unnatural amino acids studied previously. The
Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein
whose mode of action has been studied in detail.
- 2 -

1. Introduction
DNA_protein interaction plays an important role in the regulation of multiple aspects of DNA
function including DNA replication, repair, recombination and transcription.1 DNAs can assume
a wide variety of secondary and tertiary structures, and individual DNA-binding proteins
recognize specific DNA structures.2-4 In many cases, a DNA-binding domain of protein involves
a specific structured motif such as an α-helix or a β-sheet, and amino acids in the protein
structure make specific contacts to the DNA backbone and nucleobases.5 Notably, hydrogen
bonding interactions between the amino acid residues in a protein and the nucleobases in a DNA
can be important to increase the binding affinity and specificity of the protein for its DNA
substrate.5 Analogous interactions operate to mediate the binding of RNA binding proteins to
their substrates.6
The appreciation that interactions between nucleobases are important for controlling nucleic
acid structure and function, and that base-base recognition can be highly selective, led to the
preparation and characterization of peptide nucleic acids (PNAs).7 These oligomers contain
nucleobases attached at defined positions to amide-linked backbones, and have been shown to
bind surprisingly tightly and specifically to a variety of nucleic acid structures using Watson-
Crick base pairing.7 In spite of the presence of amide bonds in the PNA backbone, PNAs do not
form protein-like tertiary structures. To obtain molecules capable of recognizing nucleobases in
nucleic acids, but also capable of forming protein-like structures, peptides containing L-α-amino
acids with a nucleobase side chain were designed by the Mihara laboratory.8-10 They reported the
use of L-amino-γ-nucleobase-butyric acids for the construction of a number of RNA binding
peptides.
- 3 -

The specific nucleobase amino acids of interest in the present study are the “nucleo alanine”
derivatives first described by the Diederichsen laboratory for the elaboration of alanyl-PNA
chimeras.11 As shown in Figure 1, alanyl nucleobase amino acids contain a single methylene
group connecting the -carbon atom and nucleobase, more closely analogous to the positioning
of (hetero)cyclic functional groups in proteinogenic amino acids such as histidine, tryptophan,
phenylalanine and tyrosine. The asymmetric syntheses of alanyl nucleobase amino acids (A– E)
NH2
N
O
N
O
N
NH2
N
O
N
N
HN
N
N
N
N
HN
N
HN
H2N
O
O
O
O
O
O
CO2H
NH2
CO2H
NH2
OH
OH
OH
NH2
NH2
NH2
B
A
C
D
E
Figure 1. Series of alanyl nucleobase amino acids (A – E) synthesized for site-directed
incorporation into DHFR and the Klenow fragment of E. coli polymerase I.
were achieved by nucleophilic ring opening of N-Boc-L-serine β-lactone. A site specific
mutagenesis technique, which permits the insertion of unnatural amino acids into any
predetermined position of a protein by the suppression of TAG or four base codons with
misacylated-tRNAs, had not been used before for incorporation of amino acids having
nucleobase side chains. Therefore, this strategy was attempted for the incorporation of alanyl
nucleobase amino acids A– E into E. coli dihydrofolate reductase (DHFR) and also into the
Klenow fragment of E. coli DNA polymerase I, a DNA-binding protein. In order to synthesize
the requisite aminoacylated tRNAs for protein synthesis, the pdCpA derivatives of alanyl
nucleobase amino acids were prepared.
2. Results
2.1 Synthesis of nucleobase amino acids and tRNA activation
- 4 -

The synthesis of the pdCpA derivative of amino acid A is outlined in Scheme 1. The synthesis
of the aminoacylated pdCpA derivative of amino acid A (Figure 1) was accomplished starting
from commercially available cytosine (Scheme 1). CBz protection of cytosine afforded
compound 1 in 74% yield.12 Compound 1 underwent coupling with N-Boc-L-serine β-lactone in
the presence of DBU to afford N-protected cytosyl amino acid derivative 2 in 56% yield.11
O
BocHN
O
NHCBz
NH2
NHCBz
CBzCl, NAH, anh DMF
0 oC to rt, overnight
SOCl2, MeOH
0 oC to rt, 2 h
DBU, DMSO,
rt, 2 h
N
N
N
56%
O
N
O
74%
O
65%
O
N
H
N
H
OH
NHBoc
cytosine
1
2
1. TFA, DCM, rt, 3 h
2. NVOC-Cl, K2CO3
NH2
N
NHCBz
NH2
dioxane/water
1:1, rt, overnight
N
N
Pd/C, H2, MeOH, overnight
N
O
O
O
N
O
O
N
O
85%
35% (two steps)
OMe
OMe
OMe
NHBoc
NHNVOC
NHBoc
3
4
5
NH2
N
NH2
1. LiOH, THF/MeOH/H2O
3:1:1, rt, 2 h
N
N
TBA-pdCpA
O
O
anh DMF/Et3N, sonication
rt, 5 h
2. NaHCO3, ClCH2CN
anh DMF, rt, overnight
O
N
O
OCH2CN
NHNVOC
OpdCpA
NHNVOC
34% (two steps)
25%
6
7
Scheme 1. Synthetic route employed for the preparation of protected amino acid A and its
(NVOC-protected) aminoacyl-pdCpA derivative.
Compound 2 was treated with SOCl2 in methanol to form methyl ester 3 and then the CBz
protecting group was removed by hydrogenolysis over Pd/C to afford 4. Boc deprotection
(CF3COOH in CH2Cl2) and subsequent NVOC protection of compound 4 afforded 5 in 35%
overall yield for two steps.11,13 N-protected methyl ester 5 was converted to the free acid by
- 5 -

treatment with aq LiOH, and the latter was treated with chloroacetonitrile to afford the desired
cyanomethyl ester 6 in 34% yield for two steps.13,14 The key intermediate cyanomethyl ester was
coupled with tris(tetrabutylammonium) salt of pdCpA15 to give a pdCpA ester 7 in 25% yield.
In order to introduce A as an amino acid constituent of proteins, the pdCpA derivative of this
amino acid was used to activate a suppressor tRNACUA (Scheme 2). Accordingly, aminoacylated
dinucleotide 7 was ligated to an abbreviated tRNACUA-COH transcript via the agency of T4 RNA
ligase in the presence of ATP to afford NVOC-aminoacyl-tRNA.16 The activated tRNA was
deprotected by UV irradiation to afford the free aminoacyl-tRNA.13
NH2
NH2
N
O
N
N
tRNA-CC
O
O P O
O
N
T RNA ligase
1.
N
4
N
O
P O
O
O
O
tRNA-COH
O
NH2
N
O
hv
2.
N
N
O
OH
O
O
N
O P O
O
N
N
NH2
O
O
H2N
O
OH
NO2
O
N
O
N
HN
O
H2N
MeO
OMe
7
Scheme 2. Preparation of suppressor tRNACUA activated with amino acid A.
The synthesis of the aminoacylated pdCpA derivative of alanyl nucleobase amino acid B was
accomplished starting from commercially available uracil (Scheme 3) which underwent coupling
with N-Boc-L-serine β-lactone in the presence of DBU to afford compound 8 in 41% yield. Free
carboxylic acid 8 was treated with SOCl2 in methanol to form the methyl ester 9 in 75% yield.
Boc deprotection of methyl ester 9 and subsequent NVOC protection afforded NVOC-carbamate
- 6 -

O
1. TFA, rt, 2 h
O
N
O
N
BocHN
O
2. NVOC-Cl, K2CO3
dioxane/water
O
SOCl2, MeOH
0 oC to rt, 2 h
DBU, DMSO,
rt, 2 h
HN
HN
1:1, rt, overnight
HN
O
O
O
54% (two steps)
41%
75%
O
N
H
CO2H
NHBoc
OMe
NHBoc
uracil
8
9
1. LiOH, THF/MeOH/H2O
3:1:1, rt, 2 h
O
N
O
O
N
TBA-pdCpA
HN
HN
2. NaHCO3, ClCH2CN
anh DMF, rt, 13 h
DMF/Et3N, sonication
rt, 5 h
HN
O
O
O
N
O
O
O
46%
30% (two steps)
OpdCpA
NHNVOC
OCH2CN
NHNVOC
OMe
NHNVOC
10
12
11
Scheme 3. Synthesis of nucleobase amino acid B and its corresponding aminoacyl-pdCpA
derivative.
10 in 54% yield over for steps. Methyl ester 10 was subjected to saponification in the presence of
aqueous LiOH to produce the free acid, which was activated as cyanomethyl ester 11 in 30%
overall yield. Treatment of cyanomethyl ester 11 with the tris(tetrabutylammonium) salt of
pdCpA14 in anhydrous DMF afforded pdCpA ester 12 in 46% yield.
The synthesis of the aminoacylated pdCpA derivative of amino acid C (Figure 1) followed the
same procedure as for amino acid B, and is outlined in Scheme 4. The synthesis of the
aminoacylated pdCpA derivative of nucleobase amino acid D was accomplished starting from
commercially available adenine (Scheme 5). N-Pentenoyl protection of adenine afforded
compound 18, the latter of which underwent coupling with N-Boc-L-serine β-lactone to afford
compound 19 in 43% yield.11,12 Boc deprotection of N and subsequent reprotection using 4-
pentenoic acid succinimide ester in the presence of K2CO3 afforded N-dipentenoyl amide 20 in
41% yield for two steps.14 Free carboxylic acid 20 was activated as cyanomethyl ester 21 in 31%
yield. The pdCpA derivative of D (22) was then prepared from cyanomethyl ester 21 in 39%
yield.
- 7 -

O
1. TFA, rt, 2 h
O
N
O
N
BocHN
O
2. NVOC-Cl, K2CO3
dioxane/water
O
SOCl2, MeOH
0 oC to rt, 2 h
DBU, DMSO,
rt, 2 h
HN
HN
HN
1:1, rt, overnight
O
O
O
33%
65%
47% (two steps)
O
N
H
CO2H
OMe
NHBoc
thymine
NHBoc
13
14
1. LiOH, THF/MeOH/H2O
3:1:1, rt, 2 h
O
O
O
N
TBA-pdCpA
HN
2. NaHCO3, ClCH2CN
anh DMF, rt, 13 h
HN
HN
DMF/Et3N, sonication, 5 h
O
N
O
O
N
O
O
O
35% (two steps)
45%
OpdCpA
NHNVOC
OCH2CN
NHNVOC
16
OMe
NHNVOC
15
17
Scheme 4. Synthesis of nucleobase amino acid C and its corresponding aminoacyl-pdCpA
derivative.
O
O
O
N
1. TFA, rt, 2 h
BocHN
O
2. pentenoyl-NHS
K2CO3
dioxane/water
1:1, rt, 4 h
HN
pentenoyl-NHS
NH
N
NH2
N
NaH, DMF
rt, 16 h
N
DBU, DMSO,
rt, 15 h
N
N
N
N
N
N
H
N
41% (two steps)
55%
43%
CO2H
N
H
18
19
adenine
NHBoc
O
TBA-pdCpA
DMF/Et3N
sonication, 5 h
O
N
O
NH
N
NaHCO3, ClCH2CN
anh DMF, rt, overnight
NH
N
NH
N
N
39%
N
N
N
N
31%
N
N
N
CO2pdCpA
CO2H
CO2CH2CN
HN
HN
HN
22
21
O
20
O
O
Scheme 5. Synthesis of nucleobase amino acid D and its corresponding aminoacyl-pdCpA
derivative.
The synthesis of the aminoacylated pdCpA derivative of amino acid E (Figure 1) was
accomplished starting from commercially available 2-amino-6-chloropurine which underwent
coupling with N-Boc-L-serine β-lactone to afford compound 23 in 80% yield (Scheme 6).17
- 8 -

Solvolysis of the purinyl chloride under acidic conditions and subsequent protection using 4-
pentenoic acid succinimide ester in the presence of 1 N NaOH afforded N-protected acid 24 in
O
TFA, H2O, rt, 2 days
pentenoyl-NHS
1.
Cl
BocHN
O
Cl
2.
N
1 N NaOH/dioxane/water
1:2:1, rt
DBU, DMSO,rt, 3 h
80%
N
N
N
N
overnight
N
H
H2N
N
O
H2N
N
24% (two steps)
OH
NHBoc
2-amino-6-chloropurine
23
O
O
O
TBA-pdCpA
DMF/Et3N
sonication, 5 h
N
N
NaHCO3, ClCH2CN
anh DMF, rt, 12 h
HN
H2N
N
N
N
N
HN
H2N
HN
H2N
O
O
N
O
O
O
53%
OH
N
N
70%
OpdCpA
OCH2CN
HN
HN
HN
O
24
25
26
Scheme 6. Synthesis of nucleobase amino acid E and its corresponding aminoacyl-pdCpA
derivative.
24% yield for two steps.17 Compound 24 was activated as cyanomethyl ester 25 in 53% yield.
The pdCpA derivative of E (26) was then prepared from cyanomethyl ester 25 in 70% yield.
The individual N-NVOC and N-pentenoyl protected aminoacylated pdCpA derivatives were
ligated to a suppressor tRNACUA lacking its 3′-terminal cytidine and adenosine residues
(tRNACUA-COH)16 via the agency of T4 RNA ligase, as illustrated in Scheme 2 for nucleobase
amino acid A. The success of the ligation reaction was verified by analysis on a polyacrylamide
gel under acidic conditions (Figure 2). The NVOC protecting groups were then removed by
1
2
3
4
5
6
Figure 2. 8% polyacrylamide−7 M urea gel electrophoresis of aminoacyl-tRNA samples. Lane
1, abbreviated tRNACUA-COH (74 nucleotides); lanes 2-6, ligation products of abbreviated
- 9 -

tRNACUA-COH incubated with pdCpA derivatives activated with amino acids A, B, C, D and E,
respectively (76 nucleotides).
exposure to high intensity UV light at 4 oC.13 The N-pentenoyl protecting group was removed by
treatment with aqueous iodine.14 Removal of the protecting group was done immediately prior to
the use of the misacylated tRNAs in protein synthesis.
2.2 Incorporation of nucleobase amino acids into proteins
The five aminoacyl-tRNACUAs obtained as described above were employed in an in vitro cell
free transcription-translation system, which was programmed with one of two DNA plasmids.
The first contained a TAG codons at the position corresponding to residue Val10 of DHFR. The
incorporation of all the alanyl nucleobase amino acids into position 10 was evaluated via
denaturing PAGE analysis and it was found that alanyl nucleobase amino acids (A – E) could
suppress the UAG codon at position 10 of DHFR mRNA, leading to full length DHFR synthesis
with suppression yields ranging from 6 to 18% as compared to wild-type DHFR (Figure 3A and
Table 1).
Table 1. Expression Yields of Full Length DHFRs and Klenow Fragments of DNA Polymerase
I, Modified at Positions 10 and 484, Respectively.
Amino acids
Yields of modified proteins relative to wild-type, %
DHFR (position 10)
17 ± 1
Klenow fragment (position 484)
15 ± 3
14 ± 3
11 ± 2
6 ± 3
A
B
C
D
E
18 ± 2
13 ± 3
6 ± 2
8 ± 1
7 ± 2
- 10 -

Phe (positive control)
30 ± 9
27 ± 8
− (negative control)
0.2 ±0.1
0.3 ± 0.1
A
B
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
Figure 3. (A) Analysis of products of in vitro translation of DHFR from wild-type (lane 1) and
modified mRNA (UAG codon in protein position 10) (lanes 2-8) in the absence (lane 2) and in
the presence of nucleobase aminoacyl-tRNACUAs (A, B, C, D and E in lanes 3-7, respectively),
and phenylalanyl-tRNACUA (lane 8) by 15% SDS-polyacrylamide gel electrophoresis. (B)
Analysis of products of in vitro translation of the Klenow fragment of DNA polymerase I from
wild-type (lane 1) and modified mRNA (UAG codon in protein position 484) (lanes 2-8) in the
absence (lane 2) and in the presence of nucleobase aminoacyl-tRNACUAs (A, B, C, D and E in
lanes 3-7, respectively), and phenylalanyl-tRNACUA (lane 8) by 10% SDS-polyacrylamide gel
electrophoresis. The arrows denote full length protein bands; the lower bands are truncation
products resulting from failure to suppress the UAG codon. The absence of lower bands in panel
A reflects the small size of the truncated product (UAG codon at position 10).
The same aminoacyl-tRNAs were further employed in an in vitro cell free transcription-
translation system programmed with a plasmid containing the gene for the Klenow fragment of
- 11 -

DNA polymerase I; this gene contained a TAG codon at the position corresponding to position
484 of the Klenow fragment. As shown in Figure 3B and summarized in Table 1, the suppression
efficiencies, relative to the Klenow fragment synthesized from the wild-type gene, were 6 to
15%.
The suppression yields obtained in replicate experiments are summarized in Table 1. For both
proteins studied, incorporation of the pyrimidine amino acids (A, B and C) were found to be in
the range 11 – 18%, while the purine amino acids (D and E) exhibited suppression yields from 6
– 8%. For any given nucleobase amino acid, the suppression yields were quite similar for the two
proteins studied. Relative suppression yields tend to be quite reproducible from one set of
experiments to another, such that it should be possible to introduce amino acids A – E into many
other proteins in workable yields. In comparison, absolute protein yields can vary significantly
from one set of experiments to another, especially if the several constituents of the protein
biosynthesizing reaction are from different preparations. In this context it may be noted that the
suppression yield obtained with phenylalanyl-tRNACUA (~30%) was toward the lower end of the
range that we have observed over a period of time. The most likely cause of this is the specific S-
30 preparation employed for the current experiments. If this proves to be correct, higher
incorporation yields for A – E can probably be achieved with other S-30 preparations.
3. Discussion
The nucleobase amino acid derivatives A – E were all prepared by an initial nucleophilic ring
opening of N-Boc-L-serine β-lactone with a pyrimidine or purine derivative in the presence of
DBU, in analogy with the work of Roviello et al.11 In the case of uracil and thymine, the
unprotected nucleobases were employed, while in the cases of cytosine and adenine, simple
protection of the exocyclic amino groups afforded compounds which provided the amino acid
- 12 -

derivatives. In the case of guanine, the synthesis began with the use of 2-amino-6-chloropurine,
and the initial amino acid derivative (23, Scheme 6) was converted to the respective guanine
derivative by solvolysis under strongly acidic conditions. The activation of suppressor tRNAs
with unnatural amino acids is achieved routinely using NVOC18 or pentenoyl19,20 protection of
N of the aminoacyl moiety, either of which can be removed under conditions which does not
cause concomitant deacylation of the amino acid attached to the tRNA as an activated ester. In
the present study the NVOC protecting group was employed for the three pyrimidine amino
acids. However, it was found that the syntheses of the two purine amino acids could be carried
out more conveniently using the N-pentenoyl protecting group. Further, the activation of the
adenine amino acid D as its pdCpA ester required protection of the exocyclic N group of
adenine; this was accomplished using a second pentenoyl protecting group, enabling both groups
to be removed simultaneously by treatment with aqueous iodine following ligation to the
abbreviated suppressor tRNA-COH. A few attempts were made to utilize tRNAs with nucleobase
amino acids containing protecting groups on the nucleobase; this was done in an effort to
increase the suppression yields. In no case was this strategy successful (data not shown).
As documented in Figure 3 and Table 1, all five nucleobase amino acid analogues were
incorporated into the two proteins studied. Based on earlier studies from our laboratory utilizing
DHFR16,21-25 and the Klenow fragment of DNA polymerase I,26 the present suppression yields
should be entirely sufficient to allow routine biochemical studies to be carried out on the
modified enzymes. While the suppression yields obtained with any unnatural amino acid cannot
be predicted with certainty, in general small lipophilic amino acids are incorporated with greater
facility than large or polar species. In the present case, all five modified nucleobase amino acids
are reasonably polar so the suppression yields observed were quite reasonable. Given the effect
- 13 -

of amino acid side chain size on incorporation efficiency, it was to be expected that A – C were
incorporated with greater efficiency than D or E.
The prospects for productive use of the alanyl nucleobase amino acids described here may be
judged by the experience of the Mihara laboratory. These workers have prepared nucleobase
amino acids having one more bridging methylene group in the side chains than do compounds A
– E, and have incorporated them into peptides by chemical synthesis to enable RNA binding
studies.8-10,28,29 For example, when three nucleobase amino acid residues were included within
arginine-rich -helical peptides targeted to a hairpin RNA derived from P22 phage, the affinities
varied with the nature and location of the nucleobases employed, thus illustrating the potential
for structure dependent RNA targeting.10 In a second study, the synthetic peptides were used to
screen T7 phage libraries. A number of nucleic acid-related proteins were identified in this
fashion. Interestingly, other types of proteins were identified as well.27 Additionally, introduction
of the nucleobase amino acids within the dimerization domain of HIV-1 protease enhanced
affinity of the individual subunits, but lowered the catalytic activity of the formed dimer to some
extent.28 In the aggregate, these studies suggest numerous applications which may be anticipated
for proteins containing nucleobase amino acids. While the preparation of peptides containing
nucleobase amino acids can be addressed using existing methods of chemical synthesis, the
routine preparation of proteins by chemical synthesis represents a significantly greater challenge.
In such cases, the use of ribosomal synthesis, carried out in vitro or in vivo, represents a more
realistic alternative.
While the design of peptides having unique affinities for specific nucleic acid targets is
difficult enough, the complexity of protein−nucleic acid interactions reflected in myriad X-ray
crystal structures29-31 underscores the extraordinary challenge of attempting to alter the
- 14 -

selectivity of such interactions by any simple predictive alteration of protein structure. In
comparison, the detailed information provided by such structures might provide a useful starting
point for introducing nucleobase amino acids into proteins at sites potentially capable of forming
new H-bonds with specific nucleosides in target DNA and RNA structures. Enhanced selectivity
of gene expression and nucleic acid maturation, for example, might be more readily achieved by
this approach. Clearly, numerous other applications can be envisioned, including directed
protein-protein interactions and the assembly of supramolecular arrays.
4. Conclusions
Five alanyl nucleobase amino acids have been synthesized and their aminoacyl-tRNA
derivatives were used for incorporation of each of the five analogues into a predetermined of two
proteins by suppression of a UAG codon. The protein sites modified were position 10 of E. coli
DHFR and 484 of the Klenow fragment of E. coli DNA polymerase I. Pyrimidine-based alanyl
nucleobase amino acids (A – C) exhibited better suppression efficiencies than did purine-based
alanyl nucleobase amino acids (D and E). These findings enable the elaboration of proteins
containing nucleobase amino acids as protein constituents at predetermined positions. It is
anticipated that such “nucleoproteins” whose normal function includes DNA or RNA binding
can be targeted selectively to particular regions of nucleic acids via nucleobase interactions
presently associated with nucleic acids.
5. Experimental section
5.1 General experimental procedures
All experiments requiring anhydrous conditions were conducted in flame-dried glassware fitted
with rubber septa under a positive pressure of dry nitrogen. Reactions were performed at room
temperature unless otherwise indicated. Analytical thin layer chromatography (TLC) was
- 15 -

performed using glass plates pre-coated with silica gel (0.25 mm, 60 Å pore size, 230-400 mesh,
Silicycle) impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by
exposure to ultraviolet light (UV). Flash column chromatography was performed employing
silica gel (60 Å pore size, 40-63 μm, standard grade, Silicycle). An acetone cooling bath was
adjusted to the appropriate temperature by the addition of small portions of dry ice.
1H NMR and 13C NMR spectra were recorded on Varian INOVA 400 (400 MHz) or Varian
INOVA 500 (500 MHz) spectrometers at 25 °C. Proton chemical shifts are expressed in parts per
million (ppm, δ scale) and are referenced to residual protium in the NMR solvent (CDCl3,
DMSO-d6 or CD3OD). Splitting patterns are designated as follows: s, singlet; br s, broad singlet;
d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multiplet. High resolution mass
spectra were obtained at the Arizona State University CLAS High Resolution Mass Spectrometry
Facility or the Michigan State University Mass Spectrometry Facility. HPLC purification was
performed with a Waters 600 pump coupled with a Varian ProStar 340 detector and a Grace
Econosil C18 column (250 × 10 mm, 5 μm). The tetra-n-butylammonium (TBA) salt of pdCpAX
was prepared using Dowex 50W×8, 200-400 mesh, activated in its TBA form.
The chemicals used for synthesis were purchased from Aldrich Chemical Co., Sigma Chemical
Co. or Combi Blocks. THF was distilled under argon from sodium-benzophenone ketyl and
CH2Cl2 was distilled under argon from calcium hydride. Ni-NTA agarose was obtained from
Qiagen Inc. DNA oligonucleotides were purchased from Integrated DNA Technologies. DEAE-
Sepharose, ammonium persulfate, acrylamide, N, N´-methylene-bis-acrylamide, acetic acid,
potassium glutamate, ammonium acetate, dithiothreitol, magnesium acetate,
phospho(enol)pyruvate, Escherichia coli tRNA, isopropyl -D-thiogalactopyranoside (IPTG),
ATP, GTP, CTP, UTP, cAMP, amino acids, rifampicin, and formamide were obtained from
- 16 -

Sigma-Aldrich. Tris and SDS were purchased from Bio-Rad Laboratories (Hercules, CA). [35S]-
methionine (1000 Ci/mmol, 10 Ci/L) was purchased from PerkinElmer Inc. Protease inhibitor
(complete, EDTA-free) was obtained from Boehringer Mannheim Corp. T4 RNA ligase and T4
polynucleotide kinase were purchased from New England Biolabs Inc.
Phosphorimager analysis was performed using an Amersham Biosciences Storm 820 equipped
with ImageQuant version 5.2 software from Molecular Dynamics.
5.2. Synthesis of alanyl nucleobase aminoacyl-pdCpA derivatives
5.2.1. Methyl (S)-3-(4-(Benzyloxycarbonylamino)-2-oxopyrimidin-1(2H)-yl)-2-(tert
butoxycarbonylamino)propionate (3).
To a stirred suspension of 1.44 g (36.0 mmol) of NaH (60% in mineral oil) in 50 ml of DMF at
0 °C was added 1.00 g (9.01 mmol) of cytosine. The reaction mixture was stirred at 0 °C for 1 h,
then 1.35 mL (9.50 mmol) of benzyl chloroformate was added. The reaction mixture was stirred
at room temperature for 14 h, then diluted with 100 mL of H2O and ice. After neutralization with
5 N HCl, a colorless precipitate formed, and was filtered, washed with five 50-mL portions of
H2O and dried to give 1 as a colorless powder: yield 1.71 g (74%); mass spectrum (APCI), m/z
246.0890 (M+H)+ (C12H12N3O3 requires m/z 246.0879). To a stirred suspension containing 250
mg (1.02 mmol) of compound 1 in 5 mL of DMSO was added 0.18 mL (183 mg, 1.20 mmol) of
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Within 15 min, a solution containing 220 mg (1.20
mmol) of N-(tert-butoxycarbonyl)-L-serine β-lactone11 in 5 mL of DMSO was added. The
reaction mixture was stirred at room temperature for 2 h under an argon atmosphere, then diluted
with 10 mL of 0.5 N HCl and extracted with two 20-mL portions of EtOAc. The combined
organic phase was dried (MgSO4) and concentrated under diminished pressure. The residue was
purified by chromatography on a silica gel column (10 × 2 cm). Elution with 5:1 ethyl acetate–
- 17 -

methanol gave 2 as a colorless solid: yield 246 mg (56%); silica gel TLC Rf 0.31 (5:1 ethyl
acetate/methanol). To a cooled (0 °C) solution containing 246 mg (0.60 mmol) of 2 in 5 mL of
anhydrous MeOH was added dropwise 0.04 mL (67.7 mg, 0.60 mmol) of SOCl2. The reaction
mixture was allowed to warm slowly to room temperature with stirring for 2 h, then diluted with
50 mL of brine and extracted with two 50-mL portions of EtOAc. The combined organic phase
was dried (MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 2 cm). Elution with 10:1 ethyl acetate/methanol
gave 3 as a colorless solid: yield 165 mg (65%); silica gel TLC Rf 0.72 (5:1 ethyl
acetate/methanol); 1H NMR (CDCl3) δ 1.35 (s, 9H), 3.64 (s, 3H), 4.01-4.60 (m, 3H), 5.19 (s,
2H), 6.29 (br s, 1H), 7.17 (s, 1H), 7.31- 7.37 (m, 4H), 7.55 (s, 1H), 7.75 (s, 1H) and 8.70 (br s,
1H); 13C NMR (CDCl3) δ 28.2, 50.8, 52.1, 52.8, 67.9, 80.5, 94.8, 128.3, 128.58, 128.63, 135.1,
149.7, 150.5, 152.5, 155.5, 162.9 and 170.4; mass spectrum (APCI), m/z 447.1890 (M+H)+
(C21H27N4O7 requires m/z 447.1880).
5.2.2. Methyl (S)-3-(4-Amino-2-oxopyrimidin-1(2H)-yl)-2-(tert-
butoxycarbonylamino)propionate (4).
To a solution containing 164 mg (0.36 mmol) of 3 in 5 mL of MeOH was added a catalytic
amount of 10% Pd/C and the reaction was maintained under 1 atm of H2 (g) overnight. The
catalyst was removed by filtration through a pad of Celite and the filtrate was concentrated under
diminished pressure. The crude product was purified by chromatography on a silica gel column
(10 × 2 cm). Elution with 3:1 ethyl acetate–methanol gave 4 as a colorless solid: yield 98.0 mg
(85%); silica gel TLC Rf 0.11 (5:1 ethyl acetate/methanol); 1H NMR (CD3OD) δ 1.37 (s, 9H),
3.71-3.73 (m, 4H), 4.34-4.59 (m, 2H), 5.83 (d, 1H, J = 7.2 Hz) and 7.44 (d, 1H, J = 7.2 Hz); 13C
- 18 -

NMR (CD3OD) δ 28.6, 53.2, 53.6, 80.9, 95.8, 147.9, 148.2, 157.6, 158.7, 167.9 and 172.0; mass
spectrum (APCI), m/z 313.1506 (M+H)+ (C13H21N4O5 requires m/z 313.1512).
5.2.3. Methyl (S)-3-(4-Amino-2-oxopyrimidin-1(2H)-yl)-2-((4,5-dimethoxy-2-
nitrobenzyloxy)carbonylamino)propionate (5).
To a stirred solution containing 97.0 mg (0.31 mmol) of 4 in 2 mL of anhydrous CH2Cl2 was
added 0.24 mL (353 mg, 3.10 mmol) of trifluoroacetic acid. The reaction mixture was stirred at
room temperature for 2 h and then concentrated under diminished pressure. To a stirred solution
containing the Boc-deprotected product in 2 mL of 1:1 dioxane/water was added 129 mg (0.93
mmol) of K2CO3 followed by 85.5 mg (0.31 mmol) of NVOC-Cl. The reaction mixture was
stirred at room temperature for 14 h under argon, then diluted with 50 mL of brine and extracted
with two 50-mL portions of EtOAc. The combined organic phase was dried (MgSO4) and
concentrated under diminished pressure. The residue was purified by chromatography on a silica
gel column (10 × 2 cm). Elution with 5:1 ethyl acetate/methanol gave 5 as a light yellow solid:
yield 49.2 mg (35% for two steps); silica gel TLC Rf 0.25 (5:1 ethyl acetate/methanol); 1H NMR
(CD3OD) δ 3.71 (s, 3H), 3.84 (s, 3H), 3.90 (s, 3H), 4.35-4.39 (m, 1H), 4.77-4.81 (m, 2H), 5.31
(ABq, 2H, J = 10.8 Hz), 5.83 (d, 1H, J = 7.2 Hz), 7.04 (s, 1H), 7.48 (d, 2H, J = 7.2 Hz) and 7.58
(s, 1H); mass spectrum (APCI), m/z 452.1413 (M+H)+ (C18H22N5O9 requires m/z 452.1417).
5.2.4. Cyanomethyl (S)-3-(4-Amino-2-oxopyrimidin-1(2H)-yl)-2-((4,5-dimethoxy-2-
nitrobenzyloxy)carbonylamino)propionate (6).
To a stirred solution containing 14.0 mg (0.03 mmol) of compound 5 in 1 mL of 1:3:1
water/THF/methanol was added 90.0 µL (0.09 mmol) of 1 N LiOH. The reaction mixture was
stirred at room temperature for 2 h, and then concentrated under diminished pressure. The
residue was dissolved in 1 mL of anhydrous DMF under argon and 25.0 mg (0.30 mmol) of
- 19 -

NaHCO3 was added followed by 4.0 µL (5.0 mg, 0.06 mmol) of chloroacetonitrile. The reaction
mixture was stirred at 23 °C overnight, then diluted with 20 mL of brine and extracted with two
50-mL portions of EtOAc. The combined organic phase was dried (MgSO4) and concentrated
under diminished pressure. The residue was purified by chromatography on a silica gel column
(10 × 2 cm). Elution with 5:1 ethyl acetate/methanol gave 6 as a light yellow solid: yield 5.01 mg
(34% for two steps); silica gel TLC Rf 0.26 (5:1 ethyl acetate/methanol); 1H NMR (CD3OD) δ
3.89 (s, 3H), 3.94-3.96 (m, 4H), 4.36-4.41 (m, 1H), 4.71-4.75 (m, 1H), 4.92 (s, 2H), 5.38-5.48
(m, 2H), 5.84 (br s, 1H), 7.12 (s, 1H), 7.48 (d, 2H, J = 7.6 Hz) and 7.71 (s, 1H); mass spectrum
(APCI), m/z 477.1377 (M+H)+ (C19H21N6O9 requires m/z 477.1370).
5.2.5. ((S)-3-(4-Amino-2-oxopyrimidin-1(2H)-yl) 2-((4,5-dimethoxy-2-
nitrobenzyloxy)carbonylamino)propionic Acid pdCpA Ester (7).
To a solution containing 5.20 mg (4.00 μmol) of pdCpA tetrabutylammonium salt in 100 µL of
9:1 anhydrous DMF/triethylamine was added 10.0 mg (21.0 μmol) of compound 6. The reaction
mixture was sonicated for 5 h. The reaction mixture was purified by HPLC on a C18 reversed
phase column (250 × 10 mm) using a linear gradient of 99:1 → 1:99 50 mM aq ammonium
acetate, pH 4.5/acetonitrile. The retention time of the desired product was 20.1 min. The
fractions containing the product were lyophilized to afford 7 as a colorless solid: yield 1.01 mg
(25%); mass spectrum (ESI), m/z 1054.2106 (M-H)- (C36H42N13O21P2 requires m/z 1054.2093).
5.2.6. Methyl (S)-2-(tert-Butoxycarbonyl)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)propionate (9).
To a stirred suspension containing 250 mg (2.23 mmol) of uracil in 5 mL of DMSO was added
0.40 mL (407 mg, 2.68 mmol) of DBU. Within 15 min, a solution containing 491 mg (2.68
mmol) of N-(tert-butoxycarbonyl)-L-serine β-lactone in 5 mL of DMSO was added. The reaction
- 20 -

mixture was stirred at room temperature for 2 h under argon, then diluted with 10 mL of 0.5 N
HCl and extracted with two 20-mL portions of EtOAc. The combined organic phase was dried
(MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 2 cm). Elution with 5:1 ethyl acetate/methanol gave
8 as a colorless solid: yield 273 mg (41%); silica gel TLC Rf 0.25 (5:1 ethyl acetate/methanol).
To a cooled (0 °C) solution containing 246 mg (0.91 mmol) of 8 in 5 mL of anhydrous MeOH
was added dropwise 0.07 mL (71.2 mg, 0.60 mmol) of SOCl2. The reaction mixture was allowed
to warm slowly to room temperature with stirring for 2 h, and was then diluted with 50 mL of
brine and extracted with two 50-mL portions of EtOAc. The combined organic phase was dried
(MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 2 cm). Elution with 10:1 ethyl acetate/methanol
gave 9 as a colorless solid: yield 214 mg (75%); silica gel TLC Rf 0.81 (5:1 ethyl
acetate/methanol); 1H NMR (CDCl3) δ 1.36 (s, 9H), 3.74 (s, 3H), 4.20-4.49 (m, 3H), 5.64 (s,
1H), 5.73 (s, 1H), 7.17 (s, 1H) and 10.10 (s, 1H); 13C NMR (CDCl3) δ 28.3, 49.7, 52.4, 53.1,
80.8, 102.2, 145.2, 151.4, 155.4, 164.2 and 170.6; mass spectrum (APCI), m/z 314.1345 (M+H)+
(C13H20N3O6 requires m/z 314.1352).
5.2.7. Methyl (S)-2-((4,5-Dimethoxy-2-nitrobenzyloxy)carbonylamino)-3-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)propionate (10).
To a stirred solution containing 94.0 mg (0.30 mmol) of 9 in 2 mL of anhydrous CH2Cl2 was
added 0.23 mL (342 mg, 3.00 mmol) of trifluoroacetic acid. The reaction mixture was stirred at
room temperature for 2 h and then concentrated under diminished pressure. To a stirred solution
containing the Boc-deprotected product in 2 mL of 1:1 dioxane/water was added 124 mg (0.90
mmol) of K2CO3 followed by 82.7 mg (0.30 mmol) of NVOC-Cl. The reaction mixture was
- 21 -

stirred at room temperature for 14 h under argon, then diluted with 50 mL of brine and extracted
with two 50-mL portions of EtOAc. The combined organic phase was dried (MgSO4) and
concentrated under diminished pressure. The residue was purified by chromatography on a silica
gel column (10 × 2 cm). Elution with 10:1 ethyl acetate/methanol gave 10 as a light yellow solid:
yield 73.3 mg (54% for two steps); silica gel TLC Rf 0.60 (10:1 ethyl acetate/methanol); 1H
NMR (DMSO-d6) δ 3.66 (s, 3H), 3.73-3.78 (m, 1H), 3.87 (s, 3H), 3.91 (s, 3H), 4.19-4.23 (m,
1H), 4.43-4.45 (m, 1H), 5.34 (ABq, 2H, J = 14.5 Hz), 5.46 (d, 1H, J = 7.5 Hz), 7.15 (s, 1H), 7.48
(d, 1H, J = 8.0 Hz), 7.70 (s, 1H) and 8.10 (d, 1H, J = 8.5 Hz); 13C NMR (DMSO-d6) δ 48.0, 52.1,
52.3, 56.0, 56.1, 62.8, 100.7, 108.1, 110.5, 127.3, 139.2, 145.8, 147.7, 150.9, 153.3, 155.5, 163.5
and 170.0; mass spectrum (APCI), m/z 453.1259 (M+H)+ (C18H21N4O10 requires m/z 453.1257).
5.2.8. Cyanomethyl (S)-2-((4,5-Dimethoxy-2-nitrobenzyloxy)carbonylamino)-3-(2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)propionate (11).
To a stirred solution containing 22.7 mg (0.05 mmol) of 10 in 1 mL of 1:3:1
water/THF/methanol was added 150 µL (0.15 mmol) of 1 N LiOH. The reaction mixture was
stirred at room temperature for 2 h, and then concentrated under diminished pressure. The
residue was redissolved in 1 mL of anhydrous DMF and 25.0 mg (0.30 mmol) of NaHCO3 was
added followed by 10.0 L (11.3 mg, 0.15 mmol) of chloroacetonitrile. The reaction mixture was
stirred at 23 °C overnight, then diluted with 20 mL of brine and extracted with two 50-mL
portions of EtOAc. The combined organic phase was dried (MgSO4) and concentrated under
diminished pressure. The residue was purified by chromatography on a silica gel column (10 × 2
cm). Elution with 10:1 ethyl acetate/methanol gave the desired product 11 as a yellow solid:
yield 7.17 mg (30% for two steps); silica gel TLC Rf 0.75 (5:1 ethyl acetate/methanol); 1H NMR
(CD3OD) δ 3.91 (s, 3H), 3.99 (s, 3H), 4.33-4.36 (m, 2H), 4.67-4.71 (m, 1H), 4.87-4.93 (m, 2H),
- 22 -

5.41-5.51 (m, 2H), 5.67 (d, 1H, J = 7.6 Hz), 7.13-7.15 (m, 1H), 7.33-7.43 (m, 1H), 7.73 (s, 1H)
and 7.97 (s, 1H); mass spectrum (APCI), m/z 478.1213 (M+H)+ (C19H20N5O10 requires m/z
478.1210).
5.2.9. ((S)-2-((4,5-Dimethoxy-2-nitrobenzyloxy)carbonylamino)-3-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)propionic Acid pdCpA Ester (12).
To a solution containing 5.20 mg (4.00 μmol) of pdCpA tetrabutylammonium salt in 100 µL of
9:1 anhydrous DMF/triethylamine was added 10.0 mg (21.0 μmol) of 11. The reaction mixture
was sonicated for 5 h. The reaction mixture was purified by HPLC on a C18 reversed phase
column (250 × 10 mm) using a linear gradient of 99:1 → 1:99 50 mM aq ammonium acetate, pH
4.5/acetonitrile. The retention time of the desired product was 18.6 min. The fractions containing
the product were lyophilized to afford 12 as a colorless solid: yield 2.02 mg (46%); mass
spectrum (ESI), m/z 1055.1943 (M-H)- (C36H41N12O22P2 requires m/z 1055.1934).
5.2.10. Methyl (S)-2-(tert-Butoxycarbonylamino)-3-(2,4-dioxo-3,4-dihydro-5-
methylpyrimidin-1(2H)-yl)propionate (14).
To a stirred suspension containing 280 mg (2.23 mmol) of thymine in 5 mL of DMSO was
added 0.40 mL (407 mg, 2.68 mmol) of DBU. Within 15 min, a solution containing 491 mg
(2.68 mmol) of N-(tert-butoxycarbonyl)-L-serine β-lactone in 5 mL of DMSO was added. The
reaction mixture was stirred at room temperature for 2 h under argon, then diluted with 10 mL of
0.5 N HCl and extracted with two 20-mL portions of EtOAc. The combined organic phase was
dried (MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 2 cm). Elution with 10:1 ethyl acetate/methanol
gave 13 as a colorless solid: yield 230 mg (33%); silica gel TLC Rf 0.30 (5:1 ethyl
acetate/methanol). To a cooled (0 °C) solution containing 230 mg (0.73 mmol) of 13 in 5 mL of
- 23 -

anhydrous MeOH was added dropwise 0.05 mL (87.4 mg, 0.73 mmol) of SOCl2. The reaction
mixture was allowed to warm slowly to room temperature with stirring for 2 h, then diluted with
50 mL of brine and extracted with two 50-mL portions of EtOAc. The combined organic phase
was dried (MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 2 cm). Elution with 10:1 ethyl acetate/methanol
gave 14 as a colorless solid: yield 156 mg (65%); silica gel TLC Rf 0.81 (5:1 ethyl
acetate/methanol); 1H NMR (CDCl3) δ 1.41 (s, 9H), 1.88 (s, 3H), 3.78 (s, 3H), 4.03-4.20 (m,
2H), 4.47-4.50 (m, 1H), 5.55 (s, 1H), 6.98 (s, 1H) and 9.34 (br s, 1H); 13C NMR (CD3OD) δ
12.4, 28.6, 50.0, 50.3, 53.1, 80.9, 110.7, 143.0, 152.6, 157.2, 166.5 and 171.4; mass spectrum
(APCI), m/z 328.1500 (M+H)+ (C14H22N3O6 requires m/z 328.1509).
5.2.11. Methyl (S)-2-((4,5-Dimethoxy-2-nitrobenzyloxy)carbonylamino)-3-(2,4-dioxo-3,4-
dihydro-5-methylpyrimidin-1(2H)-yl)propionate (15).
To a stirred solution containing 49.2 mg (0.15 mmol) of 14 in 1.5 mL of anhydrous CH2Cl2
was added 0.12 mL (171 mg, 1.50 mmol) of trifluoroacetic acid. The reaction mixture was
stirred at room temperature for 2 h and then concentrated under diminished pressure. To a stirred
solution containing the Boc-deprotected product in 2 mL of 1:1 dioxane/water was added 124 mg
(0.90 mmol) of K2CO3 followed by 41.4 mg (0.15 mmol) of NVOC-Cl. The reaction mixture
was stirred at room temperature for 14 h under argon, then diluted with 50 mL of brine and
extracted with two 50-mL portions of EtOAc. The combined organic phase was dried (MgSO4)
and concentrated under diminished pressure. The residue was purified by chromatography on a
silica gel column (10 × 2 cm). Elution with 10:1 ethyl acetate/methanol gave 15 as a light yellow
solid: yield 32.7 mg (47% for two steps); silica gel TLC Rf 0.75 (5:1 ethyl acetate/methanol); 1H
NMR (CDCl3) δ 1.88 (s, 3H), 3.78 (s, 3H), 3.95 (s, 3H), 3.99 (s, 3H), 4.13-4.15 (m, 1H), 4.34-
- 24 -

4.37 (m, 1H), 4.57-4.59 (m, 1H), 5.49 (s, 2H), 6.03 (br s, 1H), 6.99 (s, 2H), 7.69 (s, 1H) and 9.08
(br s, 1H); 13C NMR (CDCl3) δ 12.3, 49.2, 53.2, 53.4, 56.5, 56.6, 64.3, 108.3, 110.4, 111.2,
127.5, 139.8, 140.9, 148.3, 151.7, 153.7, 155.7, 164.4 and 170.0; mass spectrum (APCI), m/z
467.1411 (M+H)+ (C19H23N4O10 requires m/z 467.1414).
5.2.12. Cyanomethyl (S)-2-((4,5-Dimethoxy-2-nitrobenzyloxy)carbonylamino)-3-(2,4-dioxo-
3,4-dihydro-5-methylpyrimidin-1(2H)-yl)propionate (16).
To a stirred solution containing 23.3 mg (0.05 mmol) of 15 in 1 mL of 1:3:1
water/THF/methanol was added 150 µL (0.15 mmol) of 1 N LiOH. The reaction mixture was
stirred at room temperature for 2 h, and then concentrated under diminished pressure. The
residue was redissolved in 1 mL of anhydrous DMF and 25.0 mg (0.30 mmol) of NaHCO3 was
added followed by 10.0 L (11.3 mg, 0.15 mmol) of chloroacetonitrile. The reaction mixture was
stirred at 23 °C overnight, then diluted with 20 mL of brine and extracted with two 50-mL
portions of EtOAc. The combined organic phase was dried (MgSO4) and concentrated under
diminished pressure. The residue was purified by chromatography on a silica gel column (10 × 2
cm). Elution with 10:1 ethyl acetate/methanol gave the desired product 16 as a yellow solid:
yield 8.59 mg (35% for two steps); silica gel TLC Rf 0.77 (5:1 ethyl acetate/methanol); 1H NMR
(CD3OD) δ 1.83 (s, 3H), 3.91 (s, 3H), 3.96 (s, 3H), 4.28-4.34 (m, 2H), 4.70-4.73 (m, 1H), 4.92
(s, 2H), 5.44 (ABq, 2H, J = 14.8 Hz), 7.10 (s, 2H), 7.26 (s, 1H) and 7.70 (s, 1H); 13C NMR
(DMSO-d6) δ 11.8, 47.7, 49.8, 51.9, 56.0, 56.2, 62.9, 108.1, 108.4, 110.6, 115.5, 127.1, 139.2,
141.5, 147.7, 150.9, 153.3, 155.5, 164.1 and 168.9; mass spectrum (APCI), m/z 492.1363
(M+H)+ (C20H22N5O10 requires m/z 492.1366).
5.2.13. ((S)-2-((4,5-Dimethoxy-2-nitrobenzyloxy)carbonylamino)-3-(2,4-dioxo-3,4-dihydro-
5-methylpyrimidin-1(2H)-yl)propionic Acid pdCpA Ester (17).
- 25 -

To a solution containing 5.20 mg (4.00 μmol) of pdCpA tetrabutylammonium salt in 100 µL of
9:1 anhydrous DMF/triethylamine was added 10.3 mg (21.0 μmol) of 16. The reaction mixture
was sonicated for 5 h. The reaction mixture was purified by HPLC on a C18 reversed phase
column (250 × 10 mm) using a linear gradient of 99:1 → 1:99 50 mM aq ammonium acetate, pH
4.5/acetonitrile. The retention time of the desired product was 20.3 min. The fractions containing
the product were lyophilized to afford 17 as a colorless solid: yield 1.81 mg (45%); mass
spectrum (ESI), m/z 1069.2089 (M-H)- (C37H43N12O22P2 requires m/z 1069.2090).
5.2.14. N-(9H-Purin-6-yl)pent-4-enamide (18).
To a stirred solution containing 1.44 g (36.0 mmol) of NaH (60% in mineral oil) in 30 mL of
anhydrous DMF at 0 °C was added 1.22 g (9.00 mmol) of adenine. The reaction mixture was
stirred at 0 °C for 1 h under argon and then 1.87 g (9.50 mmol) of pentenoic acid NHS ester was
added. The reaction mixture was stirred at room temperature overnight under argon, diluted with
150 mL of brine and extracted with two 100-mL portions of EtOAc. The organic phase was dried
(MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 4 cm). Elution with 10:1 ethyl acetate/methanol
gave the desired product 18 as a yellow solid: yield 1.08 g (55%); silica gel TLC Rf 0.50 (10:1
ethyl acetate/methanol); 1H NMR (DMSO-d6) 2.47-2.49 (m, 2H), 2.64-2.28 (m, 2H), 5.00 (dd,
1H, J = 10.4 and 1.6 Hz), 5.11 (dd, 1H, J = 17.2 and 2.0 Hz), 5.86-5.96 (m, 3H), 8.39 (s, 1H) and
8.60 (s, 1H); 13C NMR (DMSO-d6) 28.5, 34.5, 112.9, 115.4, 137.1, 143.7, 145.7, 151.1, 161.9
and 172.5; mass spectrum (APCI), m/z 218.1037 (M+H)+ (C10H12N5O requires m/z 218.1042).
5.2.15. (S)-2-(tert-Butoxycarbonylamino)-3-(6-Pent-4-enamido-9H-purin-9-yl)propionic
acid (19).
- 26 -

To a stirred suspension containing 217 mg (1.00 mmol) of compound 18 in 5 mL of DMSO
was added 0.18 mL (183 mg, 1.20 mmol) of DBU. Within 15 min, a solution containing 220 mg
(1.20 mmol) of N-(tert-butoxycarbonyl)-L-serine β-lactone in 5 mL of DMSO was added. The
reaction mixture was stirred at room temperature for 12 h under argon, then diluted with 10 mL
of 0.5 N HCl and extracted with two 20-mL portions of EtOAc. The combined organic phase
was dried (MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 2 cm). Elution with 5:1 ethyl acetate/methanol gave
19 as a colorless solid: yield 174 mg (43%); silica gel TLC Rf 0.20 (9:1:0.05
EtOAc/MeOH/HOAc); 1H NMR (3:1 CD3OD/CDCl3) 1.30 (s, 9H), 2.47-2.49 (m, 2H), 2.64-
2.28 (m, 2H), 2.64-2.28 (m, 2H), 4.51-4.57 (m, 2H), 5.00 (dd, 1H, J = 10.4 and 1.6 Hz), 5.11
(dd, 1H, J = 17.2 and 2.0 Hz), 5.90-5.95 (m, 2H), 8.25 (s, 1H) and 8.64 (s, 1H); 13C NMR (3:1
CD3OD/CDCl3) 28.5, 29.9, 37.4, 46.1, 54.8, 80.7, 116.0, 123.1, 129.2, 129.8, 137.9, 145.7,
150.2, 152.9, 157.2 and 173.6; mass spectrum (APCI), m/z 405.1880 (M+H)+ (C18H25N6O5
requires m/z 405.1886).
5.2.16. Cyanomethyl (S)-2-Pent-4-enamido-3-(6-pent-4-enamido-9H-purin-9-yl)propionate
(21).
To a stirred solution containing 125 mg (0.31 mmol) of 19 in 2 mL of anhydrous CH2Cl2 was
added 0.24 mL (353 mg, 3.10 mmol) of trifluoroacetic acid. The reaction mixture was stirred at
room temperature for 2 h and then concentrated under diminished pressure. To a stirred solution
containing the Boc-deprotected product in 2 mL of 1:1 dioxane/water was added 129 mg (0.93
mmol) of K2CO3 followed by 61.1 mg (0.31 mmol) of pentenoic acid NHS ester. The reaction
mixture was stirred at room temperature for 14 h under argon, then diluted with 20 mL of 0.5 N
HCl and extracted with two 50-mL portions of EtOAc. The combined organic phase was dried
- 27 -

(MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 2 cm). Elution with 5:1 ethyl acetate/methanol gave
20 as a light yellow solid: yield 49.1 mg (41% for two steps); silica gel TLC Rf 0.10 (9:1:0.05
EtOAc/MeOH/HOAc); mass spectrum (APCI), m/z 387.1781 (M+H)+ (C18H23N6O4 requires m/z
387.1781). To a stirred solution containing 54.0 mg (0.14 mmol) of 20 in 1 mL of anhydrous
DMF was added 44.0 mg (0.42 mmol) of NaHCO3 followed by 26.0 µL (32.0 mg, 0.42 mmol) of
chloroacetonitrile. The reaction mixture was stirred at 23 °C for 16 h, then diluted with 20 mL of
satd aq NaHCO3 and extracted with two 50-mL portions of EtOAc. The combined organic phase
was dried (MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 2 cm). Elution with ethyl acetate gave 21 as a light
yellow solid: yield 18.4 mg (31%); silica gel TLC Rf 0.6 (1:10 methanol/ethyl acetate); 1H NMR
(CD3OD)  2.19-2.21 (m, 4H), 2.48-2.50 (m, 2H), 2.73-2.76 (m, 2H), 3.77 (s, 2H), 4.64-4.75
(m, 2H), 4.99-5.03 (m, 2H), 5.10-5.15 (m, 4H), 5.68-5.69 (m, 1H), 5.90-5.97 (m, 1H), 8.26 (s,
1H) and 8.63 (s, 1H); 13C NMR (CD3OD) 30.2, 30.5, 35.9, 37.5, 45.0, 45.4, 50.7, 53.2, 116.0,
123.7, 138.0, 138.2, 145.9, 150.5, 153.2, 153.7, 169.6, 171.0, 173.8 and 175.4; mass spectrum
(APCI), m/z 426.1881 (M+H)+ (C20H24N7O4 requires m/z 426.1890).
5.2.17. (S)-2-Pent-4-enamido-3-(6-pent-4-enamido-9H-purin-9-yl)propionic Acid pdCpA
Ester (22).
To a solution containing 5.20 mg (4.00 μmol) of pdCpA tetrabutylammonium salt in 100 µL of
9:1 anhydrous DMF/triethylamine was added 8.76 mg (21.0 μmol) of 21. The reaction mixture
was sonicated for 5 h. The reaction mixture was purified by HPLC on a C18 reversed phase
column (250 × 10 mm) using a linear gradient of 99:1 → 1:99 50 mM aq ammonium acetate, pH
4.5/acetonitrile. The retention time of the desired product was 20.2 min. The fractions containing
- 28 -

the product were lyophilized to afford 22 as a colorless solid: yield 1.50 mg (39%); mass
spectrum (MALDI), m/z 1003.22 (M-H)- (theoretical m/z 1003.26).
5.2.18. (S)-3-(2-Amino-6-chloro-9H-purin-9-yl)-2-(tert-butoxycarbonylamino)propionic
acid (23).
To a suspension containing 2.05 g (12.1 mmol) of 2-amino-6-chloropurine in 5 mL of DMSO
was added 1.50 mL (1.56 g, 10.2 mmol) of DBU over a period of 15 min. Within 15 min, a
solution containing 1.74 g (9.30 mol) of N-(tert-butoxycarbonyl)-L-serine β-lactone dissolved in
5 mL of DMSO was added dropwise and the reaction mixture was stirred for additional 3 h. The
reaction was stopped by adding HOAc (585 µL, 10.2 mmol) and the solvent was removed under
diminished pressure. Purification of the crude product was done by flash chromatography on a
silica gel column (10 × 2 cm) using a gradient of 9:1 EtOAc/MeOH to 9:1:0.05
EtOAc/MeOH/HOAc. After co-evaporation with portions of toluene, the product was obtained
as a white solid: yield 2.60 g (80%); Rf 0.40 (9:1:0.05 EtOAc/MeOH/HOAc); 1H NMR (DMSO-
d6) 1.24 (s, 9H), 4.15-4.45 (m, 3H), 6.69 (s, 1H), 6.89 (s, 2H), 7.89 (s, 1H) and 8.04 (s, 1H);
13C NMR (DMSO-d6)27.9, 43,6, 52.6, 78.3, 123.2, 143.3, 149.1, 154.1, 155.1, 159.7 and
171.2; mass spectrum (APCI), m/z 357.1083 (M+H)+ (C13H18N6O4Cl requires m/z 357.1078).
5.2.19. Cyanomethyl (S)-3-(2-Amino-6-oxo-1,6-dihydropurin-9-yl)-2-pent-4-
enamidopropionate (25).
(S)-3-(2-Amino-6-chloro-9H-purin-9-yl)-2-(tert-butoxycarbonyl)propionic acid (23) (386 mg,
0.75 mmol) was dissolved in 10 mL of 3:1 TFA/H2O and the reaction mixture was stirred at
room temperature for 48 h. Toluene (~50 mL) was added and the solvent was concentrated. After
drying in vacuo a white solid was obtained in quantitative yield; Rf 0.25 (70:30:3:0.3
CHCl3/MeOH/H2OHOAc). The crude solid residue was dissolved in 20 mL of 1:1:2 H2O/1 N
- 29 -