Ribonucleosides for an artificially expanded genetic information system

Article abstract of DOI:10.1021/jo402665d

Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a hydroboration/oxidation sequence. RiboZ is more stable against epimerization than its 2′-deoxyribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson-Crick complement, imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying grounds for using this second-generation AEGIS Z:P pair to add amino acids encoded by mRNA.

Full text of DOI:10.1021/jo402665d

Note
pubs.acs.org/joc
Ribonucleosides for an Artificially Expanded Genetic Information
System
Hyo-Joong Kim,†,‡ Nicole A. Leal,†,‡ Shuichi Hoshika,†,§ and Steven A. Benner*,†,§
†Foundation for Applied Molecular Evolution (FfAME), 720 SW Second Avenue, Suite 201, Gainesville, Florida 32601, United States
‡Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States
§The Westheimer Institute for Science and Technology (TWIST), 720 SW Second Avenue, Suite 208, Gainesville, Florida 32601,
United States
S
* Supporting Information
ABSTRACT: Rearranging hydrogen bonding groups adds
nucleobases to an artificially expanded genetic information system
(AEGIS), pairing orthogonally to standard nucleotides. We report
here a large-scale synthesis of the AEGIS nucleotide carrying 2-
amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a
hydroboration/oxidation sequence. RiboZ is more stable against
epimerization than its 2′-deoxyribo analogue. Further, T7 RNA
polymerase incorporates ZTP opposite its Watson−Crick comple-
ment, imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying
grounds for using this “second-generation” AEGIS Z:P pair to add
amino acids encoded by mRNA.
Because of their orthogonality, “first-generation” AEGIS pairs
are today used widely. In the clinic, AEGIS DNA is used to
monitor the load of viruses in the blood of patients infected
with human immunodeficiency and hepatitis C viruses,3a detect
mutations that cause cystic fibrosis,3b and detect viruses causing
respiratory diseases.3c,d,f In the laboratory, AEGIS is supported
by a developing molecular biology, including polymerases that
perform AEGIS PCR4 and procedures to sequence AEGIS
DNA.5 These have allowed AEGIS to support in vitro evolution
that generates AEGIS-containing aptamers that bind to cancer
cells3e (inter alia).
ne of many accomplishments of synthetic biology over
Othe past two decades has been the generation of DNA
systems that have additional nucleotide “letters” that form
additional nucleobase pairs.1 These include artificially expanded
genetic information systems (AEGIS),1e,2 species of DNA that
use nonstandard hydrogen bonding patterns to form up to six
mutually exclusive nucleobase pairs with standard Watson−
Crick geometry, four more than are found in natural DNA and
RNA (Figure 1). Other approaches to add nucleotides to the
genetic alphabet diverge in structure more severely.1
One “second-generation” pair of AEGIS nucleotides has
performed especially well with enzymes as part of an expanded
genetic system. The components of this pair carry the 2-
aminoimidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially named
P) and 6-amino-5-nitro-2(1H)-pyridone (trivially named Z)
heterocycles (Figure 1). Implementing (respectively) the
hydrogen bonding “acceptor−acceptor−donor” and “donor−
donor−acceptor” patterns, these replace a first-generation
AEGIS nucleobase analogue (based on a pyrazine ring
system6a) that implemented the donor−donor−acceptor
hydrogen bonding pattern but was susceptible to epimerization
via specific-acid catalysis.6b
The Z:P pair has also proven to perform well with natural
DNA polymerases.3d,4a As a hypothesis accounting for this,
both the Z and P heterocycles place electron density in the
minor groove, the first from the exocylic oxygen of the
pyrimidine analogue and the second from N3 of the purine
Figure 1. Second-generation artificially expanded genetic information
system (AEGIS) uses alternative arrangements of hydrogen bond
donor and acceptor groups to create a total of 12 nucleotides forming
six mutually exclusive nucleobase pairs.
Received: December 5, 2013
Published: March 5, 2014
© 2014 American Chemical Society
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Scheme 1. Attempted Synthesis of C-Ribonucleoside with 2,6-Dichloropyridine
Scheme 2. Synthesis of the Ribonucleoside of Z
analogue. This density may interact with hydrogen-bond-
donating amino acid side chains of polymerases.7
where a potential mRNA containing the Z:P pair has been
prepared by transcription.
Should these successes with DNA be transferred to RNA,
many applications can be envisioned. For example, AEGIS
nucleotides might increase the number of codons in the genetic
code, allowing ribosomes to synthesize proteins containing
additional amino acids.8−11 With the development of cells able
to replicate plasmids containing GACTZP DNA, RNA
polymerases able to synthesize Z- and P-RNA intracellularly
might allow for the expression of proteins with more than the
20 standard amino acids. This was shown already 20 years ago
for the first-generation AEGIS pair between isocytidine and
isoguanosine (implementing, respectively, the acceptor−accept-
or−donor and donor−donor−acceptor hydrogen bonding
patterns).8
To synthesize riboZ, we initially explored a method
frequently used to prepare C-nucleosides that involves
condensation of ribonolactone with a lithiated base. Unfortu-
nately, all of our efforts to obtain the lithiated 2-amino-3-
nitropyridin-6-one in protected form were not successful,
apparently due to incompatibility of the protecting group and
the conditions for lithiation.
We then considered condensation of 2,6-dichloropyridine
and ribonolactone as a precursor of 2-amino-3-nitropyridin-6-
one nucleoside (Scheme 1). Here, the condensation product
was obtained with good yield. However, deoxygenation of the
product with BF3·OEt2/Et3SiH16 gave mostly the α nucleoside.
17
The reduction of the product with L-Selectride/ZnCl2
This vision motivated us to undertake the synthesis of Z- and
P-ribonucleosides, one carrying the Z heterocycle, the other
carrying the P heterocycle. The synthesis of the P-ribo-
nucleoside having a β configuration by a standard sequence
coupling a preformed nucleobase to a perbenzoylated ribose
proved not to be problematic.12,13 Standard chemistry did not
yield the Z-ribonucleoside, however. While Heck coupling gave
the 2′-deoxyribonucleoside, the analogous strategy did not
generate the analogous species with a 2′-hydroxyl group.
The literature does report the synthesis of the ribonucleoside
carrying an unsubstituted 2-aminopyridin-6-one, which
presents the same donor−donor−acceptor hydrogen bonding
pattern as Z. However, this heterocycle is prone to oxidation.
We previously reported that, as deoxyribonucleosides, the
nitro-14 and cyano15-substituted aminopyridin-6-ones were
both stable to oxidation and slower to epimerize. Since the
nitro species was both more stable and better accepted as a
substrate for DNA polymerases, we chose the nitro substituent
for development as an RNA component.
followed by Mitsunobu reaction gave the α nucleoside as a
major product in low yield (35%). Further, the conversion of
dinitropyridine to 2-amino-3-nitropyridin-6-one was problem-
atic.
We then exploited the manipulation of the silyl enol ether
moiety in the Heck product, the intermediate in our synthesis
of the 2′-deoxyribonucleoside analogue.14 The literature reports
various transformations of silyl enol ethers of furanose
nucleosides, including their oxidation with osmium tetroxide18
or dimethyldioxirane19 to give the hydroxyketone, and
hydroboration/oxidation20 to make diols. In this system,
oxidation of the silyl enol ether of the furanose nucleoside (8
in Scheme 2) with OsO4 gave a mixture of diastereomers with
low yields. Also, treatment with dimethyldioxirane also did not
yield the desired products.
However, hydroboration/oxidation of compound 815 gave
the desired trans-diol 9 exclusively and in good yield (>80%)
(Scheme 2). This intermediate was suitable for further
elaboration to give the desired nucleoside analogue. Thus,
acetylation of 9 followed by desilylation gave 11. The
configuration of the 3′-OH group of 11 was then inverted by
a two-step oxidation/reduction sequence, where the stereo-
chemical course of the reduction was controlled by the 5′-
hydroxyl group. Acetyl migration gave two compounds (13a
and 13b) as a mixture.
We report here a route for the large-scale synthesis of the
ribonucleoside analogue of 2-amino-3-nitropyridin-6-one
(riboZ). We also report physical properties of the nucleoside,
including its susceptibility to epimerization. Finally, we provide
evidence that rZTP is incorporated by T7 RNA polymerase
opposite dP in a template and vice versa, the first examples
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Scheme 3. Triphosphate Synthesis of the Ribonucleoside of Z
Removal of acetate groups in both versions of 13 by
treatment with ammonia gave 14. Subsequent removal of the
nitrophenylethyl (NPE) group (DBU in CH3CN) gave
ribonucleoside 15. This was converted into its triphosphate
17 (rZTP) using the Ludwig−Eckstein procedure21 applied to
16, prepared from 13 with 5′-DMTr protection, acetylation,
and 5′-DMTr deprotection (Scheme 3).
The availability of riboZTP allowed us to explore the
molecular biology that might be enabled by the Z:P pair.
Several DNA molecules with dP downstream from a T7 RNA
polymerase promoter segment were synthesized (Table S1).
Transcription reactions using standard and P-containing
templates and T7 RNA polymerase (MC T7 RNA Pol, having
the wild-type sequence, purified in-house) with or without
rZTP were performed. Without rZTP, transcription of the
template having one dP provided full-length product without
significant pausing. This showed that the polymerase could
mistmatch a single standard ribonucleotide (presumably C)
efficiently opposite a single template dZ. However, substantial
pausing was observed in the transcription of increasing
numbers of dPs in the template. When the transcription
mixture contained rZTP, as well, however, no pausing was
observed; the transcription gave only full-length product
(Figure 2). Analogous results with oligonucleotides containing
dZ in the template and dPTP are shown in the Supporting
Information; these show less overall processivity.
The availability of the ribonucleoside of Z and its
triphosphates in substantial quantities, its stability to the acid-
catalyzed epimerization, and its ability to be incorporated
opposite its complementary purine analogue in a template by
T7 RNA polymerase advance the in vitro molecular biology for
the Z:P system. In parallel work, we described tools to
sequence DNA containing dZ and/or dP, polymerases that
PCR-amplify dZ and dP-containing DNA, and in vitro
evolution from libraries containing dZ and dP.3e In future
work, we hope to move the Z:P system into living cells, where
it can be used to encode additional amino acids and where the
power of GACTZP nucleic acids to evolve can be coupled to
the ability of cells to evolve.
The pKa of the Z-heterocycle of the ribonucleoside 15 was
estimated from a series of UV spectra collected at 380 nm (λmax
for protonated 15) and 400 nm (λmax for deprotonated 15) as
the pH of the aqueous solution was adjusted by adding dilute
aqueous solutions of HCl and NaOH. A plot of the ratio of
absorbance at 380 nm/400 nm (Supporting Information Figure
S1) gave a pKa value 7.9 0.1. This value is similar to that
measured for 2-deoxyribonucleoside of Z, implying (as
expected) that a hydroxyl group in the 2′-position does not
affect the pKa of the Z base.
A special feature of C-nucleosides is their susceptibility to
epimerization. This phenomenon was described for pseudo-
uridine several decades ago22 and for other C-nucleosides
since.23 2′-Deoxyribosides where pyrazine heterocycles present
a donor−donor−acceptor hydrogen bonding pattern also
epimerize, via specific-acid catalysis, to give mixtures of
furanose and pyranose isomers.6 However, electron-with-
drawing groups (nitro and cyano) placed at the 3-position of
the 2-aminopyridin-6-one system (analogous to the 5-position
of a pyrimidine) slow epimerization.14
These results prompted us to ask whether the presence of
the 2′-hydroxyl group in riboZ also slowed the epimerization.
Epimerization is indeed slower in the ribo species than in the
2′-deoxyribo species (Table 1). While about half of a sample of
Table 1. Remaining Amount of dZ and rZ Nucleosides at pH
2 and 7
a
EXPERIMENTAL SECTION
■
N,N-Bis[(tert-butoxy)carbonyl]-5-[3′,5′-di-O-(tert-butyldi-
phenylsilyl)-β-D-2,5-dihydrofuranosyl]-3-nitro-6-[2-(4-nitro-
phenyl)ethoxy]-2-pyridinamine (8). To a mixture of compound 7
(183 g, 279 mmol) and imidazole (34.4 g, 505 mmol) in
dichloromethane (2 L) was added TBDPSCl (72.2 mL, 278 mmol).
The mixture was stirred at room temperature for 1 h and washed with
H2O, dried over Na2SO4, and filtered. The solution was concentrated
by rotary evaporation, and the residue was resolved by flash
chromatography (silica, ethyl acetate/hexanes = 1:3 to 1:2). The
fractions containing the product were combined, concentrated, and
dried. The residue was then redissolved in dichloromethane (2L), to
which were then added DMAP (1.5 g), triethylamine (130 mL), and
di-tert-butyl dicarbonate (120 g). The mixture was stirred at room
temperature overnight and washed with brine, dried over Na2SO4, and
filtered. The solution was concentrated by rotary evaporation, and the
residue was resolved by flash chromatography (silica, ethyl acetate/
hexanes = 1:4 to 1:3) to give 8 as a pale yellow solid (235 g, 77%): mp
56−58 °C; 1H NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 8.01 (d, 2H, J
= 8.7 Hz), 7.62−7.76 (m, 8H), 7.26−7.49 (m, 12H), 7.19 (d, 2H, J =
8.4 Hz), 5.59 (d, 1H, J = 3.0 Hz), 4.83 (m, 1H), 4.35−4.58 (m, 3H),
3.85−4.10 (m, 2H), 2.85−3.05 (m, 2H), 1.38 (s, 18H), 1.04 (s, 9H),
0.98 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 160.6, 151.5, 150.1,
0 h
0.5 h
1 h
2 h
4 h
6 h
pH 7, 90 °C
pH 2, 20 °C
dZ
rZ
dZ
rZ
100
100
100
100
90
99.8
94.7
100
81.7
98.4
89.9
99.9
70.4
96.2
84.8
99.9
58.5
94.2
74.0
99.6
52.5
91.2
68.3
99.4
a
The loss of the nucleoside was caused by epimerization at the given
pH and temperature.
dZ was epimerized at pH 7 and 90 °C in 6 h, the epimerization
of riboZ under these conditions was less than 10%. At pH 2 and
20 °C, dZ showed significant epimerization (∼30%) in 6 h,
while epimerization of riboZ was less than 1% in this condition.
This is consistent with the specific acid-catalyzed mechanism
observed for epimerization for an analogous systems, a
mechanism requiring initial protonation of the ribose ring
oxygen. The electron-withdrawing nature of the 2′-hydroxyl
group is expected to lower the pKa of the protonated ribose
ring oxygen, making this mechanism less effective at pH values
approaching neutrality.
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mL), and DMAP (0.2 g) in dichloromethane (1200 mL) was stirred at
room temperature for 2 h. The mixture was washed with 0.5 N HCl
solution and brine. The organic layer was concentrated by rotary
evaporation. The residue was purified by flash chromatography (silica,
ethyl acetate/hexanes = 1:4) to give a light yellow solid (128 g, 96%):
1
mp 74−76 °C; H NMR (300 MHz, CDCl3) δ 8.70 (s, 1H), 8.15 (d,
2H, J = 8.7 Hz), 7.55−7.75 (m, 4H), 7.15−7.45 (m, 18H), 4.99 (s,
1H), 4.86 (s, 1H), 4.45−4.65 (m, 2H), 4.05−4.2 (m, 3H), 3.81 (dd,
1H, J = 10.2, 3.3 Hz), 3.05 (t, 2H, J = 6.9 Hz), 1.90 (s, 3H), 1.41 (s,
18H), 1.07 (s, 9H), 0.79 (s, 9H); 13C NMR (75 MHz, CDCl3) δ
168.7, 160.2, 150.2, 147.1, 145.6, 143.7, 136.6, 136.1, 135.9, 135.8,
134.7, 133.6, 133.4, 132.7, 132.0, 130.1, 130.0, 129.9, 129.8, 127.9,
124.2, 124.0, 84.4, 84.1, 81.6, 79.4, 77.1, 67.3, 63.3, 34.8, 28.1, 27.1,
27.0, 21.0, 19.4, 19.2; HRMS (TOF-ESI) m/z calcd for
C62H74N4O14Si2Na (M + Na)+ 1177.4632, found 1177.4620.
N-Acetyl-5-(2′-O-acetyl-β-D-xylofuranosyl)-3-nitro-6-[2-(4-
nitrophenyl)ethoxy]-2-pyridinamine (11). To a solution of
compound 10 (128 g, 110.8 mmol) in dichloromethane (1100 mL)
was added trifluoroacetic acid (33 mL) at room temperature. The
mixture was stirred for 3 h, neutralized by aqueous NaHCO3, and
mixed with water and extracted with dichloromethane. The combined
organic layers were dried over Na2SO4, filtered, and concentrated. The
residue was then resolved by flash chromatography (silica, ethyl
acetate/hexanes = 1:2) to give the NH2 (Boc removed) product as a
pale yellow solid; 1H NMR (300 MHz, CDCl3) δ 8.62 (d, 1H, J = 0.9
Hz), 8.15 (d, 2H, J = 8.7 Hz), 7.55−7.7 (m, 4H), 7.1−7.5 (m, 18H),
5.02 (s, 1H), 4.77 (s, 1H), 4.4−4.55 (m, 2H), 4.05−4.15 (m, 3H),
3.86 (dd, 1H, J = 13.8, 7.2 Hz), 3.04 (t, 2H, J = 6.8 Hz), 1.89 (s, 3H),
1.07 (s, 9H), 0.79 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 168.9,
162.3, 153.1, 147.1, 145.9, 136.2, 136.1, 135.9, 135.8, 135.4, 133.7,
133.5, 133.1, 132.1, 130.0, 129.9, 129.8, 128.0, 127.9, 127.7, 124.0,
122.8, 114.4, 84.1, 81.8, 79.4, 77.4, 66.7, 63.4, 34.9, 27.2, 26.9, 21.1,
19.4, 19.2.
The product was then dissolved in pyridine (600 mL). DMAP (4 g)
and acetic anhydride (60 mL) were added to the solution, and the
mixture was stirred for 5 h at 60 °C. Pyridine was then removed by
rotary evaporation, and the residue was dissolved in ethyl acetate. The
mixture was treated with 0.5 N HCl and stirred vigorously. The
organic phase was separated, dried, and purified by column
chromatography (silica gel, ethyl acetate/hexanes) to give a pale
yellow solid, which was a mixture of the N-acetyl and N,N-diacetyl
compounds. Spectral data for N-acetyl compound: 1H NMR (300
MHz, CDCl3) δ 10.64 (s, 1H), 8.72 (d, 1H, J = 0.9 Hz), 8.15 (d, 2H, J
= 8.7 Hz), 7.6−7.7 (m, 4H), 7.1−7.5 (m, 18H), 5.03 (s, 1H), 4.83 (s,
1H), 4.55−4.65 (m, 2H), 4.05−4.2 (m, 3H), 3.85 (dd, 1H, J = 10.2,
3.3 Hz), 3.12 (t, 2H, J = 6.9 Hz), 2.45 (s, 3H), 1.92 (s, 3H), 1.08 (s,
9H), 0.76 (s, 9H).
The mixture of N-acetyl and N,N-diacetyl compounds was mixed
with triethylamine trihydrofluoride (160 mL), triethylamine (140 mL),
and THF (1400 mL) then stirred at room temperature for 24 h. The
mixture was poured into aqueous NaHCO3 and extracted with ethyl
acetate. The combined organic layer was dried over Na2SO4, filtered,
and concentrated. The residue was purified by flash chromatography
(silica, ethyl acetate/hexanes = 3:1 to ethyl acetate 100%) to give the
product as a pale yellow solid (49 g, this is a mixture of mono- and
diacetyl compounds and the monoacetyl is major, 84%): mp 52−54
°C; 1H NMR (300 MHz, CDCl3) δ 10.52 (s, 1H), 8.73 (d, 1H, J = 0.6
Hz), 8.16 (d, 2H, J = 8.7 Hz), 7.43 (d, 2H, J = 8.7 Hz), 5.03 (dd, 1H, J
= 3.0, 1.8 Hz), 4.91 (dd, 1H, J = 2.7, 0.9 Hz), 4.6−4.8 (m, 2H), 4.3 (m,
1H), 4.05−4.2 (m, 3H), 3.9−3.95 (m, 1H), 3.22 (t, 2H, J = 6.9 Hz),
2.95−3.05 (m, 1H), 2.44 (s, 3H), 2.06 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 170.2, 169.3, 161.6, 147.1, 145.6, 145.5, 135.9, 130.0, 126.1,
124.0, 118.2, 83.5, 81.0, 78.5, 77.4, 68.1, 61.5, 34.9, 26.5, 21.1; HRMS
(TOF-ESI) m/z calcd for C22H24N4O11Na (M + Na)+ 543.1334,
found 543.1331.
Figure 2. PAGE (20%, with 7 M urea) of transcription products with
four RNA length standards (left, 42, 25, 14, 5 nt). Transcription of
“standard template” shows unreacted 32P-GTP (large band at bottom),
typical “stutter” bands, full-length transcript (30 nt), and small
amounts of very long products (unassigned). Remaining bands are
with indicated templates containing from 1 to 3 dPs (Table S1)
obtained without rZTP (left cluster) and with rZTP (right cluster).
Full-length product is made in the absence of rZTP by mis-
incorporation of a standard nucleotide (presumably rC) opposite
dP, with increasing pausing with increasing numbers of template dPs.
Pausing disappears when rZTP is added.
147.0, 145.7, 143.2, 136.5, 135.9, 135.8, 135.6, 134.4, 133.4, 133.3,
131.5, 131.4, 130.6, 129.9, 128.2, 128.1, 127.9, 127.9, 126.7, 123.9,
101.3, 84.5, 84.0, 78.1, 67.4, 65.2, 35.1, 28.0, 27.0, 26.5, 19.43, 19.39;
HRMS (TOF- ESI) m/z calcd for C60H70N4O12Si2Na (M + Na)+
1117.4421, found 1117.4405.
N,N-Bis[(tert-butoxy)carbonyl]-5-[3′,5′-di-O-(tert-butyldi-
phenylsilyl)-β-D-xylofuranosyl]-3-nitro-6-[2-(4-nitrophenyl)-
ethoxy]-2-pyridinamine (9). To a solution of the compound 8 (65
g, 59.3 mmol) in THF (700 mL) was added diborane in THF (1 M,
∼200 mL, 3.4 equiv) at 0 °C. The mixture was slowly warmed to room
temperature and stirred overnight. It was quenched with 70 mL of
H2O for 5 min and treated with sodium perborate (100 g) in water
(600 mL). The mixture was vigorously stirred at room temperature for
1 h. THF was removed by rotary evaporation, and the residue was
extracted with ethyl acetate. The organic layer was then dried over
Na2SO4 and filtered. The solution was concentrated by rotary
evaporation, and the residue was resolved by flash chromatography
(silica, ethyl acetate/hexanes = 1:4 to 1:3) to give 9 as a pale yellow
solid (61 g, 92%): mp 71−73 °C; 1H NMR (300 MHz, CDCl3) δ 8.62
(s, 1H), 8.10 (d, 2H, J = 8.7 Hz), 7.64−7.74 (m, 4H), 7.14−7.50 (m,
18H), 4.68 (s, 1H), 4.60 (t, 2H, J = 6.3 Hz), 4.00−4.28 (m, 4H), 3.64
(br s, 1H), 3.09 (t, 2H, J = 5.4 Hz), 1.42 (s, 18H), 1.09 (s, 9H), 0.81
(s, 9H); 13C NMR (75 MHz, CDCl3) δ 160.2, 150.3, 147.1, 145.6,
143.3, 136.7, 136.0, 135.9, 135.8, 134.2, 133.9, 133.7, 133.4, 132.0,
130.2, 129.95, 129.86, 128.1, 127.9, 125.0, 124.0, 84.2, 83.7, 81.9, 79.4,
67.3, 62.8, 35.0, 28.1, 27.2, 26.9, 19.5, 19.2; HRMS (TOF-ESI) m/z
calcd for C60H72N4O13Si2Cl (M + Cl)− 1147.4328, found 1147.4365;
C62H75N4O15Si2 (M + CH3COO)− 1171.4773, found 1171.4825.
N,N-Bis[(tert-butoxy)carbonyl]-5-[3′,5′-di-O-(tert-butyldi-
phenylsilyl)-2′-O-acetyl-β-D-xylofuranosyl]-3-nitro-6-[2-(4-
nitrophenyl)ethoxy]-2-pyridinamine (10). A mixture of com-
pound 9 (128 g, 115 mmol), acetic anhydride (20 mL), pyridine (30
N-Acetyl-5-(3′-deoxy-3′-oxo-2′-O-acetyl-β-D-ribofuranosyl)-
3-nitro-6-[2-(4-nitrophenyl)ethoxy]-2-pyridinamine (12). A
mixture of compound 11 (23.4 g, 45.0 mmol), trityl chloride (13.8
g, 49.5 mmol), DMAP (0.4 g) in triethylamine (12.5 mL), and
dichloromethane (350 mL) was stirred at room temperature for 2 h. It
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was washed with water, and the organic layer was dried over Na2SO4,
filtered, and concentrated. The residue was purified by flash
chromatography (silica, ethyl acetate/hexanes = 2:1) to give a light
N-Acetyl-5-[2′,3′-O-diacetyl-β-D-ribofuranosyl]-3-nitro-6-[2-
(4-nitrophenyl)ethoxy]-2-pyridinamine (16). To a solution of
compound 13 (500 mg, 0.961 mmol), triethylamine (0.27 mL), and
DMAP (10 mg) in dichloromethane (10 mL) was added DMTr
chloride (358 mg, 1.057 mmol). The mixture was stirred at room
temperature for 1 h then washed with water. The organic layer was
dried over Na2SO4, filtered, and concentrated. The residue was
purified by flash chromatography (silica, ethyl acetate) to give a light
yellow solid. This was dissolved in dichloromethane (20 mL) and
pyridine (0.5 mL) and treated with acetic anhydride (0.18 mL) and
DMAP (10 mg). The mixture was stirred at room temperature for 3 h
then washed with water. The organic layer was dried over Na2SO4,
filtered, and concentrated. The residue was then resolved by flash
chromatography (silica, ethyl acetate/hexanes = 2:1) to give product
1
yellow solid: H NMR (300 MHz, CDCl3) δ 10.59 (s, 1H), 8.73 (d,
1H, J = 0.6 Hz), 8.16 (d, 2H, J = 8.7 Hz), 7.2−7.5 (m, 17H), 5.08 (dd,
1H, J = 2.1, 0.9 Hz), 4.92 (dd, 1H, J = 2.1, 0.9 Hz), 4.6−4.8 (m, 2H),
4.15−4.3 (m, 2H), 3.65 (d, 2H, J = 4.5 Hz), 3.22 (t, 2H, J = 3.9 Hz),
3.08 (d, 1H, J = 3.0 Hz), 2.44 (s, 3H), 2.07 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 169.6, 168.9, 161.4, 147.1, 145.6, 145.5, 143.3, 135.7,
130.0, 128.7, 128.3, 127.6, 126.1, 124.0, 118.4, 87.9, 82.7, 80.9, 79.2,
79.9, 68.0, 62.2, 35.0, 26.5, 21.1.
The product was then dissolved in dichloromethane (350 mL) and
treated with Dess-Martin periodinane (28 g, 66 mmol) at room
temperature and stirred for 4 h. It was filtered through Celite, and the
filtrate was concentrated. The residue was purified by flash
chromatography (silica, ethyl acetate/hexanes = 1:1) to give a light
yellow solid. It was then dissolved in dichloromethane (600 mL) and
treated with trifluoroacetic acid (30 mL) and stirred at room
temperature for 3 h. The mixture was neutralized by aqueous sodium
bicarbonate and mixed with water and extracted with dichloro-
methane. The combined organic layers were dried over Na2SO4,
filtered, and concentrated. The residue was purified by flash
chromatography (silica, ethyl acetate/hexanes = 2:1) to give a light
yellow solid (15.9 g, 68%): mp 66−68 °C; 1H NMR (300 MHz,
CDCl3) δ 10.64 (s, 1H), 8.65 (s, 1H), 8.17 (d, 2H, J = 9.0 Hz), 7.47
(d, 2H, J = 8.7 Hz), 5.22 (d, 1H, J = 9.6 Hz), 5.09 (d, 1H, J = 9.3 Hz),
4.6−4.9 (m, 2H), 4.25 (t, 1H, J = 2.7 Hz), 3.98 (m, 2H), 3.26 (t, 2H, J
= 6.9 Hz), 2.44 (s, 3H), 2.07 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
207.5, 169.6, 168.4, 162.8, 147.3, 146.5, 145.2, 137.3, 130.1, 125.7,
124.1, 115.5, 81.4, 76.4, 75.0, 68.7, 61.6, 35.0, 26.6, 20.5; HRMS
(TOF-ESI) m/z calcd for C22H22N4O11Na (M + Na)+ 541.1177,
found 541.1181.
5-(2′-O-Acetyl-β-D-ribofuranosyl)-3-nitro-6-[2-(4-nitro-
phenyl)ethoxy]-2-pyridinamine (13a) and 5-(3′-O-Acetyl-β-D-
ribofuranosyl)-3-nitro-6-[2-(4-nitrophenyl)ethoxy]-2-pyridina-
mine (13b). To a solution of compound 12 (29.0 g, 51.7 mmol) in
acetic acid (200 mL) and acetonitrile (200 mL) was added sodium
triacetoxyborohydride (21.9 g, 103 mmol) at 0 °C and stirred for 1 h.
It was poured into water and extracted with ethyl acetate. The
combined organic layers were dried over Na2SO4, filtered, and
concentrated. The residue was resolved by flash chromatography
(silica, ethyl acetate/hexanes = 2:1 to 100% ethyl acetate) to give 13a/
13b as a pale yellow solid (20.5 g, 70%): mp 164−165 °C; HRMS
(TOF-ESI) m/z calcd for C22H24N4O11Na (M + Na)+ 543.1334,
found 543.1333.
1
as a light yellow solid: H NMR (300 MHz, CDCl3) δ 10.61 (s, 1H),
8.66 (s, 1H), 8.17 (d, 2H, J = 8.7 Hz), 7.15−7.5 (m, 11H), 6.81 (d,
4H, J = 8.1 Hz), 5.49 (t, 1H, J = 4.8 Hz), 5.32 (dd, 1H, J = 12.3, 3.6
Hz), 5.01 (d, 1H, J = 4.2 Hz), 4.69 (t, 2H, J = 6.6 Hz), 4.2−4.3 (m,
1H), 3.77 (s, 6H), 3.35−3.45 (m, 2H), 3.21 (t, 2H, J = 6.6 Hz), 2.44
(s, 3H), 2.06 (s, 3H), 2.04 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
169.8, 169.5, 168.7, 161.9, 158.7, 147.1, 145.9, 145.5, 144.7, 135.9,
135.8, 135.7, 130.3, 130.1, 128.3, 128.1, 127.1, 125.9, 124.0, 117.0,
113.4, 86.7, 80.2, 77.6, 74.8, 71.2, 68.1, 62.5, 60.6, 55.4, 35.0, 26.6,
20.8, 20.7.
The product was dissolved in dichloromethane (30 mL) and treated
with dichloroacetic acid (0.6 mL). The mixture was stirred at room
temperature for 1 h then washed with water. The organic layer was
dried over Na2SO4, filtered, and concentrated. The residue was
resolved by flash chromatography (silica, ethyl acetate/hexanes = 2:1
to ethyl acetate 100%) to give 16 as a light yellow solid (400 mg,
74%): mp 160−161 °C; 1H NMR (300 MHz, CDCl3) δ 10.60 (s, 1H),
8.73 (s, 1H), 8.17 (d, 2H, J = 8.7 Hz), 7.47 (d, 2H, J = 8.7 Hz), 5.2−
5.4 (m, 2H), 4.96 (d, 1H, J = 4.5 Hz), 4.74 (t, 2H, J = 6.3 Hz), 4.1−4.2
(m, 1H), 3.95−4.05 (m, 1H), 3.74−3.84 (m, 1H), 3.26 (t, 2H, J = 7.2
Hz), 2.44 (s, 3H), 2.23 (t, 1H, J = 5.7 Hz), 2.10 (s, 3H), 2.06 (s, 3H);
13C NMR (75 MHz, CDCl3) δ 170.3, 169.6, 168.7, 162.1, 147.2, 146.1,
145.4, 136.7, 130.1, 125.8, 124.1, 116.6, 81.6, 78.4, 74.7, 70.5, 68.3,
61.5, 35.0, 26.6, 20.8; HRMS (TOF-ESI) m/z calcd for
C24H26N4O12Na (M + Na)+ 585.1439, found 585.1436.
6-Amino-3-(5′-O-triphosphate-β-D-ribofuranosyl)-5-nitro-
2(1H)-pyridone (17). To a solution of compound 16 (60 mg, 0.107
mmol) in pyridine (1 mL) and dioxane (2 mL) was added a solution
of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (32.4 mg, 0.16
mmol) in dioxane (1.0 mL) at room temperature. After 15 min, a
mixture of tributylammonium pyrophosphate in DMF (0.2 M, 1.6 mL,
0.32 mmol) and tributylamine (0.17 mL) was added. After 20 min, a
solution of iodine (40.6 mg, 0.16 mmol) and water (0.063 mL) in
pyridine (3.1 mL) was added. After 30 min, the reaction was quenched
by the addition of aqueous Na2SO3 (5%, until color disappears). The
pyridine and dioxane were removed in vacuo. The residue was
dissolved in acetonitrile (10 mL) and water (10 mL) and kept at room
temperature overnight. It was purified by reverse-phase prep HPLC
(25 mM TEAA to 25 mM TEAA/CH3CN = 30:70 in 20 min, running
time 30 min), then the collected fraction was lyophilized (12.9 min in
the analytical HPLC, 50 mM TEAB to 60% CH3CN + 40% 50 mM
TEAB in 20 min). The lyophilized residue was dissolved in CH3CN (5
mL) and treated with DBU (0.1 mL). The mixture was stirred at room
temperature for 24 h. Volatiles were removed by rotary evaporation.
To the residue was added ammonium hydroxide (5 mL), and the
mixture was stirred at room temperature for 1 h. Ammonia was
removed by rotary evaporation, and the residue was diluted with water
and lyophilized. The residue was dissolved in water, filtered, and
resolved by ion exchange HPLC (water to 0.5 M ammonium
bicarbonate in 25 min). The collected fraction was lyophilized to give a
yellow solid (0.042 mmol, 39%): 1H NMR (300 MHz, D2O) δ 8.17 (s,
1H), 4.0−4.2 (m, 6H); 31P NMR (121 MHz, D2O) δ −7.9 (d, 1P, J =
20 Hz), −10.0 (d, 1P, J = 19 Hz), −21.6 (t, 1P, J = 19 Hz); HRMS
(TOF-ESI) m/z calcd for C10H15N3O16P3 (M − H)− 525.9671, found
525.9666.
5-(β-D-Ribofuranosyl)-3-nitro-6-[2-(4-nitrophenyl)ethoxy]-2-
pyridinamine (14). A mixture of compound 13 (15 g, 28.8 mmol)
and ammonium hydroxide (100 mL) in methanol (500 mL) was
stirred at room temperature overnight. The solution was then
concentrated in vacuo, and the residue was resolved by flash
chromatography (silica, ethyl acetate/methanol = 15:1) to give a
1
light yellow solid (11.3 g, 90% for two steps): mp 155−156 °C; H
NMR (300 MHz, CD3OD) δ 8.52 (d, 1H, J = 0.6 Hz), 8.18 (d, 2H, J =
9.0 Hz), 7.60 (d, 2H, J = 8.7 Hz), 4.82 (d, 2H, J = 3.6 Hz), 4.6−4.7
(m, 2H), 3.65−3.95 (m, 5H), 3.24 (t, 2H, J = 6.3 Hz); 13C NMR (75
MHz, CD3OD) δ 163.6, 154.0, 147.0, 146.8, 135.2, 130.3, 123.4, 121.7,
113.5, 83.4, 79.5, 75.9, 70.9, 67.0, 61.9, 34.7; HRMS (TOF-ESI) m/z
calcd for C18H20N4O9Na (M + Na)+ 459.1122, found 459.1118.
6-Amino-3-(β-D-ribofuranosyl)-5-nitro-2(1H)-pyridone (15).
To a solution of compound 14 (30 mg, 0.069 mmol) in acetonitrile
(5 mL) was added DBU (0.1 mL); the mixture was then stirred
overnight at room temperature. The mixture was then concentrated in
vacuo, and the residue was resolved by flash chromatography (silica,
CH2Cl2/MeOH = 5:1 to 5:2) to give a yellow solid (15 mg, 75%): mp
200−202 °C;1H NMR (300 MHz, D2O) δ 8.14 (s, 1H), 4.59 (d, 1H, J
= 4.8 Hz), 4.03 (t, 1H, J = 4.8 Hz), 3.96 (t, 1H, J = 6.0 Hz), 3.8−3.9
(m, 1H), 3.72 (dd, 1H, J = 12.6, 3.0 Hz), 3.59 (dd, 1H, J = 12.6, 4.5
Hz); 13C NMR (75 MHz, D2O) δ 163.3, 150.9, 135.4, 116.7, 115.0,
83.1, 80.3, 74.3, 70.7, 61.5; HRMS (TOF-ESI) m/z calcd for
C10H12N3O7 (M − H)− 286.0681, found 286.0681.
3198
dx.doi.org/10.1021/jo402665d | J. Org. Chem. 2014, 79, 3194−3199

The Journal of Organic Chemistry
Note
Measuring the pKa of 15. An aqueous solution (30 mL) of 15
(∼0.5 mg) was acidified to pH 4 by the addition of dilute aqueous HCl
(10 mM). The pH of the solution was then varied by addition of
various amounts of dilute aqueous NaOH (10 or 100 mM). UV scans
(220−500 nm) were taken at the various pH values. The pKa was
determined by plotting the pH versus the quotient of absorption at
two different wavelengths (400 nm/380 nm).
Res. 2004, 37, 784. (f) Hirao, I.; Ohtsuki, T.; Fujiwara, T.; Mitsui, T.;
Yokogawa, T.; Okuni, T.; Nakayama, H.; Takie, K.; Yabuki, T.;
Kigawa, T.; Kodama, K.; Yokogawa, T.; Nishikawa, K.; Yokogawa, S.
Nat. Biotechnol. 2002, 20, 177. (g) Hirao, I.; Mitsui, T.; Kimoto, M.;
Yokoyama, S. J. Am. Chem. Soc. 2007, 129, 15549. (h) Leconte, A. M.;
Matsuda, S.; Hwang, G. T.; Romesberg, F. E. Angew. Chem., Int. Ed.
2006, 45, 4326.
Measuring the Rate of Epimerization of 15. Compound 15
was dissolved in a dilute HCl aqueous solution (pH 2.0) and solution
of 25 mM Et3N−HOAc buffer (pH 7.0). The concentration of 15 was
0.4 mM. The solutions were incubated at room temperature (pH 2) or
90 °C (pH 7). At time intervals, aliquots (10 μL) were removed,
neutralized with aqueous triethylammonium bicarbonate buffer (50
mM; pH 8; 0.1 mL), and analyzed by analytical rp-HPLC (Sunfire
C18 5 μm, 3.0 × 150 mm, eluent A = 50 mM TEAB, eluent B = 35%
CH3CN and 65% 50 mM TEAB, gradient from 100% A to 70% A,
30% B in 30 min, flow rate 0.5 mL/min).
Transcription Reaction. Transcription templates were prepared
by independently combining equimolar ratios of top strand (NLT1)
and bottom strand (NL standard, NLP1, NLP2, NLP3, or NLP4) in
1× transcription buffer (20 mM NaCl, 40 mM Tris pH 7.8, 6 mM
MgCl2, 2 mM spermidine, and 10 mM DTT), heating to 95 °C, and
then cooling to room temperature.
Transcription reactions contained a final concentration of 0.2 μM
template DNA (NLT1 and various bottom strands as indicated in
Figure 2), 1× transcription buffer (20 mM NaCl, 40 mM Tris pH 7.8,
6 mM MgCl2, 2 mM spermidine, and 10 mM DTT), 1 μCi/μL α32P-
GTP, MC T7 RNA Pol RNA polymerase (0.05 units/μL final), and
0.5 mM each rNTP in the minus experiment or 0.5 mM each rNTP
and rZTP in the plus experiment. Reactions were incubated at 37 °C
for 40 min and 2 h. Reactions were quenched with three-fold
formamide quench buffer, and samples were resolved on a 20% PAGE.
(2) Geyer, C. R.; Battersby, T. R.; Benner, S. A. Structure 2003, 11,
1485.
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Chang, C.; Horn, T.; Ahle, D.; Detmer, J.; Shen, L.-P.; Kolberg, J.;
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(b) Johnson, S. C.; Marshall, D. J.; Harms, G.; Miller, C. M.; Sherrill,
C. B.; Beaty, E. L.; Lederer, S. A.; Roesch, E. B.; Madsen, G.; Hoffman,
G. L.; Laessig, R. H.; Kopish, G. J.; Baker, M. W.; Benner, S. A.; Farrell,
P. M.; Prudent, J. R. Clin. Chem. 2004, 50, 2019−2027. (c) Arens, M.
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W.-M.; Storch, G. A. J. Clin. Microbiol. 2010, 48, 2387−2395. (d) Lee,
W. M.; Grindle, K.; Pappas, T.; Marshall, D. J.; Moser, M. J.; Beaty, E.
L.; Shult, P. A.; Prudent, J. R.; Gern, J. E. J. Clin. Microbiol. 2007, 45,
2626−2634. (e) Sefah, K.; Yang, Z.; Bradley, K. M.; Hoshika, S.;
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Natl. Acad. Sci. U.S.A. 2013, DOI: 10.1073/pnas.1311778111.
(f) Nolte, F. S.; Marshall, D. J.; Rasberry, C.; Schievelbein, S.;
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(4) (a) Yang, Z.; Chen, F.; Chamberlin, S. G.; Benner, S. A. Angew.
Chem., Int. Ed. 2010, 49, 177. (b) Sismour, A. M.; Lutz, S.; Park, J.-H.;
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ASSOCIATED CONTENT
* Supporting Information
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Nature 1992, 356, 537. (b) Switzer, C.; Moroney, S. E.; Benner, S. A. J.
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A. B.; King, D. S.; Horn, D. M.; Schultz, P. G. J. Am. Chem. Soc. 2003,
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■
S
Epimerization and pKa measurement of compound 15, and 1H,
13C, and 31P NMR spectra for compounds 8, 9, 10, 11, 12, 13,
14, 15, 16, and 17, and data on incorporation of rPTP opposite
template dZ. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: sbenner@ffame.org.
■
Notes
(10) Heinemann, I. U.; Rovner, A. J.; Aerni, H. R.; Rogulina, S.;
The authors declare the following competing financial
interest(s): Two authors are named as coinventors on patent
applications covering this technology.
Cheng, L.; Olds, W.; Fischer, J. T.; Soll, D.; Isaacs, F. J.; Rinehart, J.
FEBS Lett. 2012, 586, 3716.
̈
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Lett. 2007, 48, 907.
ACKNOWLEDGMENTS
■
We thank Dr. Matthew Carrigan for providing polymerase
(MC T7 RNA Pol). We are also indebted to HDTRA1-13-1-
0004 from the Defense Threat Reduction Agency, DARPA
under its “Foundries” program C13K11520, the Department of
Defense under W911NF-12-C-0059, and the NIAID under its
program to analyze chronic and recent HIV infections
(R01AI098616).
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