A practical synthesis of archaeosine and its base

Article abstract of DOI:10.1016/j.tet.2018.08.003

A practical synthetic route to 7-formamidino-7-deazaguanosine (archaeosine), a hypermodified nucleoside observed in archaeal tRNA, has been developed, which involves the addition of hydroxylamine to the cyano group of 7-cyano-7-deazaguanosine (preQ_0<

Full text of DOI:10.1016/j.tet.2018.08.003

Accepted Manuscript
A practical synthesis of archaeosine and its base
Natsuhisa Oka, Akane Fukuta, Kaori Ando
PII:
S0040-4020(18)30941-4
10.1016/j.tet.2018.08.003
TET 29722
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 25 July 2018
Accepted Date: 6 August 2018
Please cite this article as: Oka N, Fukuta A, Ando K, A practical synthesis of archaeosine and its base,
Tetrahedron (2018), doi: 10.1016/j.tet.2018.08.003.
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ACCEPTED MANUSCRIPT
A practical synthesis of archaeosine and its base
Natsuhisa Oka,1,2,* Akane Fukuta,1 and Kaori Ando1,*
1 Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University
2 Center for Highly Advanced Integration of Nano and Life Sciences, Gifu University (G-CHAIN)
1-1 Yanagido, Gifu 501-1193, Japan
oka@gifu-u.ac.jp, ando@gifu-u.ac.jp
Abstract
A practical synthetic route to 7-formamidino-7-deazaguanosine (archaeosine), a hypermodified
nucleoside observed in archaeal tRNA, has been developed, which involves the addition of
hydroxylamine to the cyano group of 7-cyano-7-deazaguanosine (preQ0-nucleoside) and a subsequent
Pd-catalyzed hydrogenation. PreQ0-nucleoside was obtained from an optimized β-selective
glycosylation developed by Hocek et al. The corresponding archaeosine base was subsequently
synthesized in high yield from its precursor 7-cyano-7-deazaguanine (preQ0).
keywords: Archaeosine; PreQ0; Deazaguanosine; Nucleoside; Amidine; Mitsunobu reaction
1. Introduction
7-Formamidino-7-deazaguanosine (archaeosine or G+; Fig. 1) is a hypermodified nucleoside located
at position 15 of archaeal tRNA.1,2 Since its first appearance in the literature in 1982,1a this unique
nucleoside has been extensively studied, and its biosynthetic pathways and functions have been
gradually uncovered.3 However, there are still many aspects that remain to be elucidated. For example,
G+ is considered to stabilize the three-dimensional structure of tRNA, but the mechanism of
stabilization has not yet been fully clarified.3b Chemically synthesized G+ and oligonucleotides that
contain G+ should thus represent useful tools to investigate its functions in detail.
To date, three reports on the chemical synthesis of G+ have been published.1b,4 McCloskey et al. have
synthesized G+ in 31% yield from 7-cyano-7-deazaguanosine (preQ0-nucleoside) by the addition of
NH3 to the 7-cyano group in the presence of a catalytic amount of NH4Cl in liquid NH3 at ~100 °C. The
total synthesis was achieved in seven steps from 7-cyano-7-deazaadenosine (toyocamycin) to G+ with
an overall yield of 0.7%.1b Carell et al. have reported the synthesis of G+ via the addition of MeOH to
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the 7-cyano group of preQ0-nucleoside under acidic conditions and subsequent nucleophilic
substitution by methanolic ammonia (Pinner reaction5) in 30% yield (eight steps from
2,6-diaminopyrimidin-4-one; 0.8% overall yield).4a These authors have also reported another synthesis
of G+, in which a protected preQ0-nucleoside was subjected to Pinner conditions for the simultaneous
formation of the 7-formamidine group and the global deprotection to generate G+ in 40% yield (eight
steps from 2,6-diaminopyrimidin-4-one; 2.4% overall yield).4b
Despite their success, these studies also show two challenges that remain to be resolved for a more
efficient total synthesis of G+: i) the inefficient generation of the 7-formamidine group, for which
1b
neither the direct addition of NH3 nor the Pinner reaction is effective;4 ii) the low yield of the
glycosylation for the preparation of preQ0-nucleoside, which is the synthetic precursor of G+.
PreQ0-nucleoside has been synthesized in low to modest yields by Lewis-acid- or
Brønsted-base-promoted glycosylations between the corresponding ribose moiety and preQ0 or some
other 7-deazaguanine derivatives with appropriate protecting groups.4,6 Although the glycosylation step
can be circumvented by using toyocamycin as the starting material, the latter is an expensive natural
product and its conversion to preQ0-nucleoside requires multiple steps.1b,7
We anticipated that the conversion of the 7-cyano group into the 7-formamidine group could
possibly be carried out more smoothly via an N-hydroxyformamidino intermediate, which can be
obtained from the addition of hydroxylamine. This strategy has previously been applied to the synthesis
of 7-formamidino-7-deazaadenosine and 7-formamidino-7-deazainosine,8 but not to the synthesis of
G+.9 We also considered that the synthesis of preQ0-nucleoside should be achieved more efficiently by
applying a modified Mitsunobu reaction, which has recently been reported by Hocek et al.10 This
reaction is completely β-selective and proceeds under nearly neutral conditions so that any potential
undesired acid- or base-promoted side reactions can be suppressed.
Fig. 1. Chemical structure of archaeosine.
2. Results and discussion
2.1. Synthesis of preQ0-nucleoside and archaeosine
Initially, we investigated the synthesis of preQ0-nucleoside. The corresponding glycosyl donor and
acceptor for the glycosylation were synthesized according to Schemes 1 and 2, respectively. The
primary hydroxy group of ribose 1 was selectively protected with a monomethoxytrityl (MMTr) group
to give 2 (Scheme 1).10 PreQ0 411 was synthesized from 2,6-diaminopyrimidin-4-one and converted into
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an appropriately protected derivative (6)10a according to a literature procedure4a (Scheme 2).
Scheme 1. Synthesis of glycosyl donor 2.
Scheme 2. Synthesis of glycosyl acceptor 6.
Then, we studied the glycosylation between 2 and 6 (Table 1). A direct application of the reaction
conditions reported by Hocek at al. using DBU, DIAD, and Bu3P10a afforded the desired product (7) in
50% yield (entry 1). Upon screening phosphines and tertiary amines, we discovered that the yield of 7
significantly improved (89%) when DBU was replaced with N,N-diisopropylethylamine (entry 2).
Reducing the amount of 2 from 1.5 equiv to 1.2 equiv decreased the yield of 7 to 78% (entry 3). Bases
weaker than N,N-diisopropylethylamine, such as triethylamine and 2,6-lutidine, furnished 7 in lower
yield (76%–85%, entries 4 and 5). More electron-deficient phosphines than Bu3P also resulted in the
less efficient formation of 7 (51%–66%, entries 6–8).
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Table 1. Synthesis of 7 via a Mitsunobu reaction.
entry
base
DBU
R3P
Bu3P
yield (%)
1
2
3a
50
89
78
85
76
66
51
55
i-Pr2NEt
i-Pr2NEt
Et3N
Bu3P
Bu3P
4
Bu3P
5
2,6-lutidine
i-Pr2NEt
i-Pr2NEt
i-Pr2NEt
Bu3P
6
MePPh2
Ph2PCH2CH2PPh2
Ph3P
b
7
8
a 2 (1.2 equiv), DIAD (1.5 equiv), and Bu3P (1.5 equiv).
b 1 equiv.
As the protected preQ0-nucleoside 7 was obtained in high yield, we attempted to deprotect 7 to
obtain preQ0-nucleoside. For that purpose, we tried to hydrolyze the 6-chloro group by applying the
conditions of a DABCO-promoted4a,12 hydrolysis (Scheme 3). However, the desired product (8) was
not produced, presumably due to the robustness of the 6-chloro group.
Scheme 3. Attempted DABCO-promoted hydrolytic deprotection of 7.
Therefore, we synthesized the preQ0 derivative 10, which contains more labile protecting groups,
from preQ0 4 via 2,9-diacetyl-preQ0 96b (Scheme 4).
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Scheme 4. Synthesis of 2-N-acetyl-6-O-(N,N-diphenylcarbamoyl)preQ0 10.
With 10 in hand, we used the previously established optimized glycosylation conditions and 2, which
generated the desired nucleoside 11 in only 58% yield (Table 2, entry 1). Therefore, we further
optimized the reaction conditions and discovered that 11 was obtained in higher yield when the reaction
was conducted in CH2Cl2, THF, or acetone (entries 2–4). The reaction in pyridine furnished 11 in
merely 9% yield (entry 5). Changing the order of addition of the reagents (i-Pr2NEt, DIAD, and Bu3P)
did not improve the result (entry 6). However, cooling the reaction mixture at the initial stage of the
reaction is important, as many byproducts were observed by TLC analysis and the desired product (11)
could not be isolated when the reaction was carried out at RT in MeCN (entry 7). The reaction in
CH2Cl2 or THF at RT afforded 11 in 66% yield (entries 8 and 9), which is also lower than that obtained
under initial cooling (entries 2 and 3). Nevertheless, the reaction should be accelerated by warming to
RT; the yield of 11 was a somewhat lower when the reaction was conducted at 0 °C, even when the
reaction time was extended (entry 10). Under the optimized conditions, the reaction was carried out on
a larger scale to give 11 in 70% yield (entry 11).
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Table 2. Synthesis of 11 using a Mitsunobu reaction.
entrya,b solvent temperature and time
yield (%)
1
2
MeCN
CH2Cl2
THF
0 °C then RT, 24 h
0 °C then RT, 24 h
0 °C then RT, 24 h
0 °C then RT, 24 h
0 °C then RT, 24 h
0 °C then RT, 24 h
RT, 24 h
58
75
72
72
9
3
4
acetone
pyridine
CH2Cl2
MeCN
CH2Cl2
THF
5
6c
70
–
7
8
RT, 24 h
66
66
68
70
9
RT, 24 h
10
11d
CH2Cl2
0 °C, 48 h
CH2Cl2
0 °C then RT, 24 h
a 0.1 mmol of 10 was used.
b Reagents were added in the order i-Pr2NEt, DIAD, Bu3P.
c Reagents were added in the order DIAD, Bu3P, i-Pr2NEt.
d1 mmol of 10 was used.
The deprotection of the nucleobase and sugar moieties of 11 with methanolic ammonia and acetic
acid, respectively, proceeded smoothly to give preQ0-nucleoside 12 in 94% yield (Scheme 5). Finally,
the treatment of 12 with NH2OH followed by a Pd-catalyzed hydrogenation of the resulting 13 afforded
G+ 14 efficiently. The addition of NH2OH to the 7-cyano group of 12 was completed within 6 h at
80 °C in H2O. G+ 14 was isolated in its acetate form, as it has been reported that the free base form
partially hydrolyzes into 12.4
6

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Scheme 5. Synthesis of preQ0-nucleoside 12 and archaeosine 14.
2.2. Synthesis of archaeosine base
We applied the same synthetic strategy to the generation of archaeosine base. To the best of our
knowledge, there is only one report on the chemical synthesis of archaeosine base,13 which delivers the
target in 34% yield from the addition of chloro(methyl)aluminum amide to the 7-cyano group of preQ0
4. We discovered that archaeosine base 16 can be synthesized more efficiently by the addition of
NH2OH to the 7-cyano group of 4 followed by a Pd-catalyzed hydrogenation of the resulting
N-OH-archaeosine base 15 (Scheme 6), similar to the aforementioned case of G+. However, there is one
difference: the addition of NH2OH to the 7-cyano group of 4 did not proceed in H2O, not even after 8 h
at 80 °C. In contrast, the reaction was complete within 6 h in DMSO. This result may be rationalized in
terms of the low solubility of 4 in H2O or in terms of the preferential deprotonation of the free N–H at
position 9 in 4, which decreases the electrophilicity of the 7-cyano group.
HO
NH2
O
O
N
NC
NH2OH (4 equiv)
DMSO, 80 °C, 6 h
NH
NH2
NH
NH2
N
N
N
N
H
H
4
15
98%
NH2
+
O
AcO H2N
H2 (3 atm), Pd/C (10 mol%)
AcOH (2 equiv)
NH
NH2
EtOH H2O (1:1, v/v)
80 °C, 1 h
N
H
N
16
83%
Scheme 6. Synthesis of archaeosine base 16.
7

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3. Conclusion
In conclusion, we have discovered that both archaeosine (G+) and its base can be efficiently
synthesized from preQ0-nucleoside and preQ0, respectively, by the addition of NH2OH to their 7-cyano
groups followed by the Pd-catalyzed hydrogenation. PreQ0-nucleoside can be effectively synthesized
by optimizing the Mitsunobu reaction developed by Hocek et al.10 Under the optimized conditions, the
overall yield for the total synthesis of G+ is 29% (seven steps from 2,6-diaminopyrimidin-4-one), which
is a significant improvement relative to the literature values (0.7%–2.4%).1b,4 This study thus provides a
practical method for the chemical synthesis of G+ and its base, both of which are useful tools to study
the biological functions of this unique hypermodified nucleoside and its base in detail.
4. Experimental section
4.1. General information
Commercially available reagents were used without purification. Dry organic solvents were prepared
by appropriate procedures prior to use. The other organic solvents were reagent grade and used as
received. All reactions in dry solvents were carried out under argon. Analytical thin-layer
chromatography (TLC) was performed on Merck TLC plates (No. 5715) precoated with silica gel 60
F254. Column chromatography on silica gel was carried out using Kanto silica gel 60N (spherical,
neutral, 40–50 or 63–210 µm). Preparative TLC (PTLC) was performed on silica gel 60 F254 PLC glass
1
plates (20 × 20 cm, 1 mm thickness). The H (400 MHz) and 13C NMR (100 MHz) spectra were
recorded on a JNM-ECS-400 spectrometer (JEOL). Tetramethylsilane (TMS; 0.0 ppm in CDCl3) and
1
DMSO-d5 (2.50 ppm in DMSO-d6) were used as the internal standards for H NMR measurements.
CDCl3 (77.0 ppm in CDCl3) and DMSO-d6 (39.5 ppm in DMSO-d6) were used as the internal standards
1
for 13C NMR measurements. H NMR data are reported as follows: chemical shift (multiplicity,
coupling constants, integration). Multiplicity is indicated as follows: s (singlet); d (doublet); dd
(doublet of doublets); t (triplet); dt (doublet of triplets); q (quartet); m (multiplet); br (broad).
High-resolution mass spectra were recorded on a Waters Xevo Q-Tof mass spectrometer (ESI-TOF).
4.2. 5-O-MMTr-
D-ribofuranose 2
D-Ribose (4.50 g, 30.0 mmol) was dried by repeated co-evaporation with dry pyridine (3 × 10 mL)
and dissolved in dry pyridine (120 mL). MMTrCl (9.26 g, 30.0 mmol) was added, and the resulting
solution was stirred for 16 h at RT. The mixture was then poured into a saturated aqueous solution of
NaHCO3 (300 mL) and extracted twice with CH2Cl2 (200 mL, 100 mL). The organic layers were
combined, dried over Na2SO4, filtered, concentrated, and co-evaporated with toluene (2 × 20 mL)
under reduced pressure. Finally, the residue was purified by column chromatography on silica gel
(silica gel 60N, spherical, neutral, 63–210 µm, CH2Cl2/MeOH = 100:0 → 96:4, v/v) to afford 2 (8.12 g,
19.2 mmol, 64%) as a white foam. The obtained 1H NMR data matched the literature values.10a
4.3. Protected preQ0-nucleoside 7
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2 (63.4 mg, 0.150 mmol) and 6 (27.8 mg, 0.100 mmol) were dried by repeated co-evaporation with
dry MeCN (5 × 1 mL). Dry MeCN (2.0 mL) was added to the residue and the resulting mixture was
cooled to 0 °C. Under stirring, dry i-Pr2NEt (17.0 µL, 0.100 mmol), DIAD (41.3 µL, 0.210 mmol), and
Bu3P (49.6 µL, 0.200 mmol) were added successively. The mixture was then allowed to warm to RT
over 3 h, where it was stirred for a further 24 h. Then, 0.5 M phosphate buffer (pH = 7; 5 mL) was
added, and the mixture was extracted with CH2Cl2 (3 × 5 mL). The organic layers were combined,
dried over Na2SO4, filtered, and concentrated under reduced pressure. Finally, the residue was purified
1
by PTLC (CH2Cl2/MeOH = 95:5, v/v) to afford 7 (60.6 mg, 88.8 µmol, 89%) as a colorless foam. H
NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 7.90 (s, 1H), 7.26 (s, 1H), 7.19–7.09 (m, 10H), 7.06 (dt, J =
8.8, 2.6 Hz, 2H), 6.74 (dt, J = 8.8, 2.4 Hz, 2H), 5.94 (d, J = 5.2 Hz, 1H), 5.08 (m, 1H), 4.53 (t, J = 2.8
Hz, 1H), 4.51 (d, J = 5.2 Hz, 1H), 3.79 (s, 3H), 3.55 (s, 1H), 3.30 (dd, J = 10.6, 2.8 Hz, 1H), 3.27 (dd, J
13
= 10.6, 2.8 Hz, 1H), 1.39 (s, 9H). C NMR (100 MHz, CDCl3) δ 177.0, 158.7, 153.4, 151.8, 149.5,
143.5, 143.2, 134.9, 134.2, 130.2, 128.0, 127.9, 127.8, 127.7, 127.1, 127.1, 113.1, 113.0, 94.4, 87.7,
87.2, 85.1, 77.3, 74.6, 64.0, 55.2, 40.6, 27.3. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
+
C37H36ClN5NaO6 704.2246; found 704.2261.
4.4. 6-O-Diphenylcarbamoyl-2-N-acetyl-preQ0 10
2,9-Diacetyl-preQ0 96b (0.778 g, 3.00 mmol) was dried by repeated co-evaporation with dry pyridine
(3 × 5 mL) and dry toluene (3 × 5 mL), before it was dissolved in dry pyridine (9.00 mL). Under
stirring, dry i-Pr2NEt (1.15 mL, 6.60 mmol) and diphenylcarbamoyl chloride (0.765 g, 3.30 mmol)
were added to the solution at RT, before stirring was continued for 2 h at RT. Then, the solvent was
removed by evaporation and repeated co-evaporation (3 × 10 mL) with toluene under reduced pressure.
The residue was then dissolved in CH2Cl2 (30 mL) and washed with 0.1 M HCl aq. (30 mL). The
aqueous layer was extracted with CH2Cl2 (4 × 30 mL). The organic layers were combined, dried over
Na2SO4, filtered, and concentrated under reduced pressure. EtOH (15.0 mL) and H2O (15.0 mL) were
added to the residue, and the mixture was cooled to 0 °C. Under stirring, 1.0 M NaOH aq. (3.00 mL)
was added dropwise and stirring was continued for 2 h at RT. The mixture was cooled at 0 °C again,
before 1.0 M HCl aq. (3.50 mL) was added dropwise until the pH of the mixture reached ~7. The
resulting precipitate was collected by suction filtration, washed with EtOH (5 × 1 mL), and dried under
vacuum. The resulting solid was suspended in EtOH (5.00 mL), and the mixture was heated to reflux
for 30 min and cooled to RT. The solid was collected by suction filtration, washed with EtOH (5 × 1
1
mL), and dried under vacuum to afford 10 (1.07 g, 2.59 mmol, 86%) as a light brown fine powder. H
NMR (400 MHz, DMSO-d6) δ 10.3 (br, 1H), 8.18 (s, 1H), 7.60–7.30 (m, 11H), 2.15 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 168.6, 160.2, 155.6, 151.1, 150.8, 143.5, 141.8, 129.4, 127.8, 126.8, 117.4,
+
106.5, 79.3, 24.3. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C22H16N6NaO3 435.1176; found
435.1179.
4.5. Protected preQ0-nucleoside 11
2 (0.634 g, 1.50 mmol) and 10 (0.412 g, 1.00 mmol) were dried by repeated co-evaporation with dry
9

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pyridine (3 × 5 mL) and dry toluene (3 × 5 mL). Dry CH2Cl2 (20.0 mL) was added to the residue and
the resulting mixture was cooled to 0 °C. Under stirring, dry i-Pr2NEt (0.174 mL, 1.00 mmol), DIAD
(0.413 mL, 2.10 mmol), and Bu3P (0.496 mL, 2.00 mmol) were added successively. The mixture was
then allowed to warm to RT over 3 h, where it was further stirred for 24 h. The mixture was then
concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (5 mL) and reprecipitated
from hexane (200 mL). The precipitate was collected by suction filtration, washed with hexane (5 × 10
mL), and re-dissolved in CH2Cl2 (30 mL). The resulting solution was concentrated under reduced
pressure, and the residue was purified by column chromatography on silica gel (silica gel 60N,
spherical, neutral, 40–50 µm, hexane/AcOEt = 50:50 → 25:75, v/v) to afford 11 (0.572 g, 0.700 mol,
1
70%) as a pale yellow foam. H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 7.85 (s, 1H), 7.57–7.06 (m,
23H), 6.75 (d, J = 9.2 Hz, 2H), 5.90 (d, J = 5.2 Hz, 1H), 4.91 (m, 1H), 4.50 (m, 1H), 4.39 (d, J = 5.6 Hz,
1H), 3.73 (s, 3H), 3.47 (s, 1H), 3.32 (dd, J = 10.8, 3.2 Hz, 1H), 3.22 (dd, J = 10.8, 3.2 Hz, 1H), 2.21 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 169.3, 158.5, 157.7, 151.9, 151.8, 150.4, 143.4, 141.4, 141.0,
134.2, 134.0, 130.1, 129.4, 129.0, 127.9, 127.8, 127.6, 127.6, 126.9, 126.1, 113.6, 113.0, 105.8, 92.9,
86.9, 86.8, 83.9, 76.6, 73.7, 63.7, 55.0, 24.4. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
+
C47H40ClN6NaO8 839.2800; found 839.2827.
4.6. PreQ0-nucleoside 12
11 (0.409 g, 0.501 mmol) was treated with a 2 M solution of ammonia in MeOH (15.0 mL) at 50 °C
for 3 h. The mixture was cooled to RT and concentrated under reduced pressure. The residue was
treated with an 80% aqueous solution of AcOH (15.0 mL) at RT for 2 h, before the mixture was
concentrated under reduced pressure. Residual AcOH was removed by repeated co-evaporation with
H2O. The residue was extracted with hot water (300 mL). The aqueous solution was cooled, transferred
to a separation funnel, washed with CH2Cl2 (3 × 200 mL), and lyophilized to afford 12 (0.145 g, 0.472
mmol, 94%) as a fluffy white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.8 (br, 1H), 7.93 (s, 1H), 6.57
(br, 2H), 5.86 (d, J = 5.8 Hz, 1H), 5.37 (d, J = 5.8 Hz, 1H), 5.12 (d, J = 4.4 Hz, 1H), 5.03 (t, J = 5.2 Hz,
1H), 4.24 (q, J = 5.8 Hz, 1H), 4.05 (dd, J = 8.0, 4.4 Hz, 1H), 3.85 (dd, J = 8.0, 3.6 Hz, 1H), 3.63–3.49
13
(m, 2H). C NMR (100 MHz, DMSO-d6) δ 157.3, 154.0, 151.5, 127.6, 115.4, 98.7, 86.5, 86.4, 85.1,
+
74.1, 70.3, 61.3. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C12H14N5O5 308.0989; found 308.0963.
4.7. N-OH-archaeosine 13
A mixture of 12 (0.123 g, 0.400 mmol), H2O (1.60 mL), and a 50% aqueous solution of NH2OH
(98.0 ꢀL, 1.60 mmol) was stirred for 2 h at 80 °C, before the mixture was lyophilized. The thus
obtained residue was triturated with i-PrOH (2.0 mL), and the resulting solid was collected by suction
filtration, washed with i-PrOH (3 × 0.50 mL), and dried in vacuo. Residual i-PrOH was removed by
repeated lyophilization with H2O. 13 (0.116 g, 0.341 mmol, 85%) was obtained as a fluffy off-white
solid. 1H NMR (400 MHz, DMSO-d6) δ 8.98 (br, 1H), 7.27 (s, 1H), 6.68 (br, 2H), 6.41 (br, 2H), 5.92 (d,
J = 6.4 Hz, 1H), 5.28 (d, J = 6.4 Hz, 1H), 5.06 (d, J = 4.4 Hz, 1H), 4.97 (t, J = 5.0 Hz, 1H), 4.23 (q, J =
6.4 Hz, 1H), 4.01 (dd, J = 7.0, 4.4 Hz, 1H), 3.82 (dd, J = 7.0, 3.2 Hz, 1H), 3.52 (m, 2H). 13C NMR (100
10

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MHz, DMSO-d6) δ 159.8, 152.4, 151.9, 147.8, 115.3, 112.5, 97.0, 85.6, 84.8, 73.8, 70.7, 61.6. HRMS
+
(ESI-TOF) m/z: [M + H]+ calcd for C12H17N6O6 341.1204; found 341.1177.
4.8. Archaeosine 14
A mixture of 13 (85.1 mg, 0.250 mmol), EtOH/H2O (1:1, v/v) (10.0 mL), AcOH (28.6 ꢀL, 0.500
mmol), and 5% Pd/C containing 54% water (0.116 g, 25.1 ꢀmol) was stirred in an autoclave under H2
(3 atm) for 1 h at 80 °C. The mixture was then filtered through a pad of celite. The celite pad was
washed with hot EtOH/H2O (1:1, v/v, 80 °C, 10 × 1 mL). The filtrate and washings were combined,
concentrated under reduced pressure, and lyophilized to afford 14 (83.8 mg, 0.218 mmol, 87%) as a
fluffy white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.1 (br, 2H), 9.93 (br, 2H), 8.14 (s, 1H), 7.25 (br,
2H), 5.93 (d, J = 6.0 Hz, 1H), 4.23 (t, J = 6.0 Hz, 1H), 4.06 (t, J = 4.2 Hz, 1H), 3.87 (m, 1H), 3.59 (dd,
J = 12.0, 4.0 Hz, 1H), 3.52 (dd, J = 12.0, 5.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 175.4, 162.5,
159.4, 156.0, 154.0, 123.8, 107.8, 96.6, 86.1, 85.2, 73.9, 70.5, 61.8, 24.1. HRMS (ESI-TOF) m/z: [M +
+
H]+ calcd for C12H17N6O5 325.1255; found 325.1246.
4.9. N-OH-archaeosine base 15
A mixture of 4 (70.1 mg, 0.400 mmol), dry DMSO (1.60 mL), and a 50% aqueous solution of
NH2OH (98.0 ꢀL, 1.60 mmol) was stirred for 6 h at 80 °C. The mixture was cooled to RT, and H2O
(10.0 mL) was added, before the mixture was lyophilized. The residual dark brown oil was triturated
with CH2Cl2 (2.0 mL) to give a light brown powder, which was collected by suction filtration, and
washed with CH2Cl2 (5 × 2 mL). Residual DMSO was removed from the product by repeated
lyophilization with H2O (5 × 2 mL) to give 15 (81.4 mg, 0.391 mmol, 98%) as a fluffy off-white solid.
1H NMR (400 MHz, DMSO-d6) δ 11.3 (br, 1H), 10.7 (br, 1H), 8.94 (br, 1H), 6.97 (br, 1H), 6.73 (br,
13
2H), 6.22 (br, 2H). C NMR (100 MHz, DMSO-d6) δ 160.2, 152.4, 152.1, 148.7, 115.7, 111.4, 96.8.
+
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C7H9N6O2 209.0781; found 209.0774.
4.10. Archaeosine base 16
A mixture of 15 (41.6 mg, 0.200 mmol), EtOH/H2O (1:1, v/v, 12.0 mL), AcOH (22.9 µL, 0.400
mmol), and 5% Pd/C containing 54% water (92.5 mg, 20.0 µmol) was stirred in an autoclave under H2
(3 atm) for 3 h at 80 °C. The mixture was then filtered through a pad of celite. The celite pad was
washed with hot EtOH/H2O (1:1, v/v; 80 °C, 10 × 1 mL), and the filtrate and washings were combined,
concentrated under reduced pressure, and lyophilized to afford 16 (41.6 mg, 0.165 mmol, 83%) as a
fluffy white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.3 (br, 2H), 10.4 (br, 2H), 9.68 (br, 2H), 7.85 (s,
13
1H), 7.08 (br, 2H), 1.72 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 175.8, 162.0, 159.8, 155.3, 154.5,
124.6, 107.2, 96.6, 24.5. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C7H9N6O+ 193.0832; found
193.0842.
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References and notes
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