Synthesis and properties of flavin ribofuranosides and flavin ribopyranosides

Article abstract of DOI:10.1002/1522-2675(20000906)83:9<2452::AID-HLCA2452>3.0.CO;2-6

Ribose-containing coenzymes like flavin mononucleotide (FMN) can be considered to be fossils of a prebiotic RNA world in which RNA encoded genetic information and catalyzed chemical reactions. To investigate the catalytic and base-pairing properties of FM

Full text of DOI:10.1002/1522-2675(20000906)83:9<2452::AID-HLCA2452>3.0.CO;2-6

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Helvetica Chimica Acta ± Vol. 83 (2000)
Synthesis and Properties of Flavin Ribofuranosides and Flavin
Ribopyranosides
by Anja Schwöglera)1), Volker Gramlichb), and Thomas Carella)1)*
a) Laboratorium für Organische Chemie, ETH-Zentrum, Universitätstrasse 16, CH-8092 Zürich
b) Laboratorium für Kristallographie, ETH-Zentrum, Sonneggstrasse 5, CH-8092 Zürich
Dedicated to Prof. Albert Eschenmoser on the occasion of his 75th birthday
Ribose-containing coenzymes like flavin mononucleotide (FMN) can be considered to be fossils of a
prebiotic RNA world in which RNA encoded genetic information and catalyzed chemical reactions. To
investigate the catalytic and base-pairing properties of FMN-containing oligonucleotides, the two cyclic flavin b-
d-ribosides 3 and 4 derived from riboflavin 2 were synthesized (Schemes 1 and 2). These are both
constitutionally strongly related to the nucleobase uridine and should be able to participate as catalytically
competent and informational nucleobases in DNA, RNA, and p-RNA. Ribofuranoside 3 was too unstable to be
isolated, but ribopyranoside 4 had the required stability, b-d-configuration, and anti-conformation of the
glycosidic bond.
1. Introduction. ± Genetic material is constructed from the standard nucleotides
adenosine, guanosine, cytidine, thymidine, and uridine 1. These building blocks are
connected via phosphodiester bonds to give single-stranded oligonucleotides. Two such
oligonucleotides form an antiparallel double strand in which the sequence of the four
canonical bases represents the genetic information [1]. The fact that all organisms on
earth use the same building blocks and the same principles for the construction of DNA
and RNA as information-storing systems has stimulated tremendous interest to
decipher the molecular origin of the genetic material [2]. Biochemical evidence
suggests that RNA is the chemical precursor of DNA. Strong support for this so-called
RNA-world hypothesis was derived from the discovery of ribozymes [3]. These are
RNA molecules able to encode genetic information and to catalyze a chemical reaction
[4]. Ribozymes fold like proteins into a defined three-dimensional structure, and they
possess genotypic (information encoding) and phenotypic (catalytic) properties. These
features connect RNA to the DNA and protein world.
The today popular RNA-world theory hence predicts that the evolution of the
genetic system passed through a phase in which RNA molecules self-instructed
catalytic competence, which was optimized along a gradient of increasing environ-
mental pressure [2b].
In recent years, the RNA-world hypothesis has fueled the desire to find (or to
select) new ribozymes with novel and interesting catalytic properties [3b]. With respect
to the question of how to establish catalytic functions in an oligonucleotide environ-
ment, RNA-containing coenzymes deserve particular attention. Coenzymes comprise a
1
)
New address: Fachbereich für Chemie, Philipps-Universität Marburg, D-35032 Marburg (tel.: int. code ‡
(6421)2821534; e-mail: carell@mailer.uni-marburg.de)

Helvetica Chimica Acta ± Vol. 83 (2000)
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small set of ubiquitously used small organic molecules, that catalyze chemical reactions
within the active site of coenzyme-dependent enzymes. The protein environment in this
interplay modulates through p-stacking, electrostatic forces, and H-bonding interac-
tions the catalytic properties of the coenzyme to enable specific transformations. Some
of the most important coenzymes like riboflavin (2) (present in flavin-adenine
dinucleotide (FAD) and flavin mononucleotide (FMN)), ribodeazaflavin (present in
F420), nicotinamide (present in nicotinamide-adenine dinucleotide (NAD)), or folate
derivatives contain a ribose-derived anchoring group. In addition, they possess H-bond
donor and acceptor groups similar to the standard nucleobases. Both features establish
a close constitutional relationship between coenzymes and the canonical nucleobases.
These properties should allow participation of coenzymes as informational nucleobases
with potential catalytic properties in an oligonucleotide environment [5].
To investigate the hypothesis that coenzymes embedded in oligonucleotides yield
DNA and RNA strands with novel and interesting pairing and catalytic properties, we
started a program aimed at the synthesis of coenzyme-derived nucleobases. Particular
attention deserves the question of whether DNA or RNA is able to fine-tune the
catalytic properties of embedded coenzymes similar to the surrounding protein in
contemporary coenzyme-dependent enzymes [6]. Coenzymes like the riboflavin-
containing FMN catalyze a variety of essential electron- and oxygen-transfer reactions
[7]. Thus, incorporation of this coenzyme into oligonucleotides should yield DNA or
RNA strands able to catalyze redox reactions. To increase the constitutional relation-
ship between riboflavin (2) and the standard nucleobases, we designed the flavin b-d-
ribofuranoside (3) and the flavin b-d-ribopyranoside (4) as the initial synthetic targets
(see Fig. 1). In this paper, we disclose the synthesis and some properties of the
riboflavin-derived nucleobases 3 and 4 [8]. Both compounds 3 and 4 are very close
homologs of the nucleobase uridine 1. They possess a cyclic ribose unit [8] that should
allow the correct insertion of the flavin moiety as a catalytically competent base-pairing
unit into DNA, RNA, or pyranosyl-RNA (p-RNA) [9].
2. Results and Discussion. ± 2.1. Synthesis and Properties of the Flavin Ribopyrano-
side 4. To the best of our knowledge, the only other report about the synthesis of flavin
ribosides has been published by Kuhn and Ströbele in 1937 [8]. Neither the constitution
nor the configuration of the flavin sugars obtained could be clarified. These open
questions are now addressed in this investigation.
Towards the synthesis of the flavin b-d-ribopyranoside (4), d-ribose (5) was heated
with a large excess of 3,4-dimethyl-2-nitroaniline (ˆ nitroxylidine; 6) in anhydrous
EtOH (Scheme 1). The intensively orange-colored nitroxylidine riboside 7 was
obtained after plug filtration through Celite as a 1:1 mixture of the a- and b-d-
anomer. This mixture was subsequently esterified with Ac2O in pyridine and furnished,
surprisingly, only a single anomer 8, which was isolated after column chromatography
in 44% yield. Analysis of the crystal structure of the new compound 8 (Fig. 2,a2))
established its a-d-configuration. The X-ray structure reveals a bifurcated H-bond
2
)
The data were deposited at the Cambridge Crystallographic Data Centre (CCDC, 12 Union Road,
Cambridge CB21EZ; e-mail: deposit@ccdc.cam.ac.uk). Deposition numbers: 4, CCDC-130252; 8, CCDC-
130253; 18, CCDC-44113.

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Fig. 1. Structure of uridine (1) and of the constitutionally related flavin ribosides 3 and 4, as well as of the
riboflavin coenzyme 2
Scheme 1. Synthesis of the Flavin b-d-Ribopyranoside (4)
a) Me2(NO2)C6H2NH2 (6), EtOH, NH4Cl, reflux; 61%. b) Ac2O, pyridine; 44%. c) PtO2, H2, Et3N. d) Alloxan,
boric acid, AcOH; 50%. e) NH3, MeOH, r.t.; 48%.

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Fig. 2. X-Ray crystal structures of a) 8, b) 4, and c) 18. ORTEP Plot of the molecular structure. Arbitrary
numberings. Displacement ellipsoids are shown at the 50% probability level.

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between HN(1) as the H-bond donor and the acceptor centers O(4) and O(9). We
believe that formation of this H-bond furnishes the driving force for the exclusive
formation of the a-d-anomer 8. Subsequent hydrogenolytic reduction of the nitro
group in 8 and filtration of the solution containing the diamine 9 directly into a
suspension of alloxan (ˆ pyrimidine-2,4,5-6(1H,3H)-tetrone) and boric acid in AcOH
furnished a new flavin riboside in 50% yield, whose structure was established by NMR
investigations as flavin b-d-ribopyranoside 10. During the flavin synthesis, the
configuration at the anomeric center apparently switches from a-d in 8 to b-d in 10.
The AcOH medium probably allows ring opening of the N,O-acetal of intermediate 9
to the corresponding imine. Condensation of 9 with alloxan to give 10 seems to proceed
preferentially with the b-d-configurated educt 9. Compound 10 contains the large
tricyclic flavin moiety and two of the three AcO groups in the thermodynamically
favored equatorial position. Finally, cleavage of the three acetyl protecting groups of 10
with NH3 in anhydrous methanol gave 4 as a stable yellow compound in 48% isolated
yield (6.5% overall yield) without further purification.
To elucidate the conformation of the glycosidic bond and to get an initial
insight into the potential of 4 to form a catalytically active base-pairing unit in
p-RNA [9a], an X-ray crystal structure of 4 was determined (Fig. 2,b2)). Thus,
compound 4 has a b-d-ribopyranoside structure, exists in the expected chair
conformation, and has a torsion angle of the glycosidic bond of À 122.18 (anti). This
anti-conformation is strictly required if 4 is intended to mimic a standard nucleobase in
a p-RNA double strand [9]. NOE Experiments (performed with 10 in CDCl3) showed
that the essential anti-conformation of the flavin b-d-ribopyranoside (4) could
dominate also in solution (irradiation at the HÀC(9) signal ! strong NOE at the
signals of the axially oriented HÀC(2') and HÀC(4'), but no NOE at the HÀC(1')
signal). These NOE experiments, together with the X-ray crystal structure, strongly
suggest that the flavin b-d-ribopyranoside (4) possesses a) the correct pyran-
oside constitution, b) the required b-d-configuration at the anomeric center, and c),
even the desired anti-conformation of the glycosidic bond. All three properties
should enable the participation of 4 as an informational nucleobase in a p-RNA double
helix [9].
2.2. Synthesis and Properties of the Flavin Ribofuranoside 3. For the preparation of
the flavin b-d-ribofuranoside (3), a ribose precursor fixed as a furanosidic constitutional
isomer was condensed with nitroxylidene 6 (Scheme 2). To obtain this furanose pre-
cursor, d-ribose (5) was first converted to the O-acetyl-protected ribofuranose 13 via 11
and 12 by standard procedures [10]. The ribofuranose 13 was subsequently heated with
a large excess of nitroxylidine 6 to give the O-acetyl-protected nitroxylidine
ribofuranoside 14 in 55% yield. The NMR data of 14 again pointed to the a-d-
configuration at the anomeric center. Unfortunately, all attempts to crystallize 14 to
establish the formation of a bifurcated H-bond failed.
Hydrogenolytic reduction of the nitro group in 14 and filtration of the product
mixture directly into a solution of alloxan and boric acid in AcOH afforded the new O-
acetyl-protected flavin ribofuranoside 15. The yield of this step varied strongly between
5 and 20% depending on the hydrogenation time. After a minimum of 1 ± 3 min at 2 bar
H2, and after medium-pressure chromatography, 15 could be obtained in yields up to
20% as a brilliant yellow powder. Longer reduction times, however, reduced the yield

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Scheme 2. Synthesis of the Flavin b-d-Ribofuranoside 15 and Attempts to Deprotect 15 to Give 3
a) Benzyl alcohol, 1% HCl soln.; 30%. b) Ac2O, pyridine; quant. c) H2, Pd/C, MeOH; quant. d)
Me2(NO2)C6H2NH2 (6), EtOH, NH4Cl, reflux; 55%. e) PtO2, H2, Et3N. f) Alloxan, boric acid, AcOH; max.
20%.
to ca. 5% only. Under all circumstances, an additional flavin ribofuranoside was
obtained in yields of 2 ± 5%.
The b-d-configuration at the anomeric center and the syn-conformation of the
glycosidic bond of the main product 15 were established by NMR spectroscopy (strong
NOE for HÀC(1')/HÀC(4') and HÀC(9)/HÀC(1'), very weak NOE for HÀC(9)/
HÀC(2') and HÀC(3')). This syn-conformation of the glycosidic bond would
compromise the ability of the flavin b-d-ribofuranoside (3) to participate as a base-
pairing unit in a standard DNA or RNA double strand. Cleavage of the O-acetyl groups
of 15 to give the flavin ribofuranoside 3 was subsequently attempted under mild basic
conditions. However, in sharp contrast to the formation of the isomeric pyranoside 4,
from 10, we observed under all circumstances rapid decomposition of ribofuranoside 3,
which was accompanied by the formation of dimethyl-alloxazin and accelerated in the
presence of light. Although stepwise cleavage of all O-acetyl protecting groups,
including AcOÀC(2'), could be followed by TLC, isolation of the target 3 was not
possible (even under exclusion of light) due to its surprisingly high instability.

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To circumvent the possibly harmful basic saponification step 15 ! 3, we alter-
natively reacted the acid-labile isopropylidene-protected ribofuranose 16 with nitro-
xylidene 6 (Scheme 3), which furnished nitroxylidine riboside 17 in 66% yield. The
latter was immediately acetylated with Ac2O in pyridine, giving 18 in 98% yield as a
yellow powder. Crystals of compound 18 suitable for X-ray analysis were obtained after
recrystallization from EtOH. The X-ray crystal structure (Fig. 2,c2)) established the
existence of a bifurcated H-bond between the H-bond donor HN(1) and the acceptor
centers O(2) and O(2') as well as the a-d-configuration at the anomeric center.
Reduction of the nitro group of 18 furnished the diamino compound 19, which was
immediately reacted with alloxan in boric acid to give the isopropylidene-protected
flavin ribofuranoside 20 in 22% yield in a clean and reproducible reaction. All attempts,
however, to cleave the isopropylidene group, now under acidic conditions, failed again.
Treatment of 20 with various acids caused always rapid decomposition. The detection
of alloxazin as one of the products indicated that the rapid decomposition was again
caused by the cleavage of the glycosidic bond.
Scheme 3. Synthesis of the Flavin b-d-Ribofuranoside 19 and Attempts to Deprotect 19 to Give 3
a) Me2(NO2)C6H2NH2 (6), EtOH, NH4Cl, reflux; 66%. b) Ac2O, pyridine; 98%. c) Pd/C, H2, MeOH. d)
Alloxan, boric acid, AcOH; 22%.
The rapid decomposition of compound 3 under basic and acidic conditions is
surprising. The observed instability of the N(10)-linked flavin ribofuranoside 3 [11] is
particularly interesting when we take into account that all known N(1)-linked alloxazin
ribofuranosides, prepared by the Pfleiderer group and others [12], are stable
compounds. These alloxazin ribofuranosides could be chemically manipulated and
were even incorporated into DNA single and double strands as novel fluorescent
nucleobases. Our target compound 3, however, contains an N(10)-linked flavin
chromophore that is able to participate in a single-electron- and hydride-transfer
reaction. In contrast to the N(1)-linked alloxazin compounds, the N(10)-linked flavin
systems possess the special reactivity of the flavin coenzyme. This reactivity is, in our
opinion, responsible for the observed instability. Based on the known properties of the

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flavin heterocycle to abstract H-atoms from organic substrates, we can speculate that
such a H-abstraction process from the ribose moiety (preferentially HÀC(2')) could be
responsible for the observed rapid cleavage of the glycosidic bond. Surprisingly, the
more stable pyranoside form of the ribose sugar in the flavin b-d-ribopyranoside 4
seems to protect this constitutional isomer from the rapid decomposition process.
3. Conclusions. ± The hitherto unknown configurations and constitutions of the
flavin ribosides, obtained upon treatment of pyranosidic and furanosidic ribose
precursors with nitroxylidine by Kuhn and Ströbele in 1937, could be elucidated.
The observed large stability differences between the flavin ribopyranoside 4 and the
isomeric flavin ribofuranoside 3 is an unexpected result, which sets, however, the frame
for the potential of both flavin ribosides to function as informational nucleobases in
DNA/RNA or in a p-RNA environment [9].
The flavin b-d-ribopyranoside (4) clearly possesses the stability and the correct anti-
conformation of the glycosidic bond required for an informational nucleobase with
catalytic properties in p-RNA double-strand environment and, hence, for the
construction of catalytically active flavin-containing p-RNA strands. It now awaits its
incorporation into p-RNA single and double strands [13].
The instability and the preferred syn-conformation of the isomeric flavin b-d-
ribofuranoside (3), however, strongly compromises the potential of 3 to participate as
an informational base-pairing unit in DNA or RNA. DNA and RNA strands containing
3 should suffer from rapid strand cleavage, in particular under aerobic conditions and in
the presence of light [11][14]3).
This work was supported by the ETH Science Foundation and the Volkswagen Stiftung. We thank Dipl.
Chem. R. Quaderer for initial synthetic studies.
Experimental Part
General. All materials were obtained from commercial suppliers and were used without further
purification. Solvents of technical quality were distilled prior to use. For reactions under an inert gas
atmosphere, N2 of standard quality was used. TLC: precoated silica-gel plates (Merck 60-F 254); staining of
sugar compounds with anisaldehyde (0.5 ml) in AcOH (50 ml) and sulfonic acid (1 ml), followed by heating at
1008. Flash chromatography (FC): silica gel (Merck; 0.040 ± 0.063 mm) and silica-gel-H (Fluka; 0.005 ±
0.040 mm). MPLC: Knauer HPLC instrument with pumps 64, Knauer variable-wavelength UV detector, and
Knauer degasser. M.p.: uncorrected; Büchi Smp 20. IR Spectra: Perkin-Elmer 1600 FT-IR, KBr pellets or CHCl3
solns.; nÄ in cmÀ1. NMR Spectra: Varian Gemini 200 (200 MHz (1H), 50 MHz (13C)), Varian Gemini 300
(300 MHz (1H), 75 MHz (13C)), and Bruker AM-500 (500 MHz (1H), 125 MHz (13C)); chemical shift d in ppm
downfield from SiMe4 (ˆ0 ppm), or alternatively, residual solvent protons as reference. MS: EI, ESI, and FAB
spectra were measured by the staff of the mass-spectrometry facilities of the ETH Zürich; Hitachi-Perkin-Elmer
VG TRIBRID, 70 eV ionization energy (EI); ZAB-2 SEQ, 3-nitrobenzyl alcohol as matrix (FAB); m/z (rel. %).
Elemental analyses were performed by the microanalysis laboratory of the ETH Zürich.
X-Ray Crystal-Structure Data of 42). Yellow needles (MeOH); C17H18N4O6 ´ 2 MeOH (Mr 438.44). Triclinic
space group P1, Dc ˆ 1.440 g/cmÀ3; Z ˆ 1; a ˆ 5.206 (3), b ˆ 8.894 (5), c ˆ 11.103 (6) Š; l ˆ 93.14 (5), b ˆ
99.71 (5), g ˆ 91.08 (5); Vˆ 505.8 (5) Š3; a 1.54178 Š radiation, 4.0 ꢀ 2Vꢁ 49.98, 1037 unique reflections, T
293 K. The crystal structure was solved by direct methods (SHELXTL PLUS) and refined by full-matrix least-
squares analysis on the basis of experimental weights (heavy atoms anisotropic; H-atoms refined isotropically).
3
)
The reduced flavin coenzyme is a strong electron donor and drives a photochemical electron transfer to
repair UV-light-induced lesions in DNA photolyases.

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Final R(F) ˆ 0.040, wR(F) ˆ 0.100 for 285 variables and 1037 observed reflections with F > 4.0s(F).
Diffractometer: Picker-Stoe.
X-Ray Crystal-Structure Data of 82). Yellow needles (H2O); C19H24N2O9 (Mr 424.40). Monoclinic space
group P2, Dc ˆ 1.354 g/cmÀ3; Z ˆ 2; a ˆ 9.141 (4), b ˆ 8.148 (5), c ˆ 14.365 (11) Š; a ˆ 90, b ˆ 103.40 (5), g ˆ 90;
Vˆ 1040.8 (11) Š3; l 0.71073 Š radiation, 2.29 ꢀ 2Vꢁ 26.548, 2474 unique reflections, T 293 K. The crystal
structure was solved by direct methods (SHELXTL PLUS) and refined by full-matrix least-squares analysis on
the basis of experimental weights (heavy atoms anisotropic; H-atoms refined isotropically). Final R(F) ˆ 0.036,
wR(F) ˆ 0.087 for 277 variables and 2330 observed reflections with F > 4.0s(F). Diffractometer: Picker-Stoe.
X-Ray Crystal-Structure Data of 182). Yellow needles (H2O); C18H24N2O7 (Mr 380.39). Monoclinic space
group P2, Dc ˆ 1.306 g/cmÀ3; Z ˆ 2; a ˆ 9.133 (11), b ˆ 9.852 (12), c ˆ 11.247 (12) Š; a ˆ 90, b ˆ 107.15 (8), g ˆ
90; Vˆ 967 (2) Š3; l 0.71073 Š radiation, 2.338 ꢀ 2Vꢁ 20.04, 1034 unique reflections, T 293 K. The crystal
structure was solved by direct methods (SHELXTL PLUS) and refined by full-matrix least-squares analysis on
the basis of experimental weights (heavy atoms anisotropic; H-atoms refined isotropically). Final R(F) ˆ 0.030,
wR(F) ˆ 0.068 for 249 variables and 972 observed reflections with F > 4.0s(F). Diffractometer: Picker-Stoe.
4,5-Dimethyl-2-nitro-N-(a/b-d-ribopyranosyl)benzenamine (7). To d-ribose (5; 0.50 g, 3.33mmol) in abs.
EtOH (15 ml) under Ar, 3,4-dimethyl-2-nitrobenzenamine (ˆ nitroxylidine, 6; 2.80 g, 16.65 mmol) and a cat.
amount of NH4Cl were added. The orange soln. was heated to reflux for 1.5 h, then allowed to cool to r.t., and
filtered. Celite was added to the soln. and the mixture evaporated in vacuo until a dry powder was obtained. The
material was added to the soln. on top of a silica-gel column, and residual 6 was eluted with CHCl3. The product
was subsequently eluted with MeOH/CHCl3 1:25: 0.60 g (61%) of 7 (a/b-d 1:1). Orange powder. M.p. > 668
(dec.). Rf (silica gel, CH2Cl2/MeOH 25 :1) 0.13 and 0.23 (a- and b-d). IR (KBr): 3386, 2922, 1630, 1570, 1506,
1439, 1411, 1328, 1239, 1194, 1150, 1078, 889. 1H-NMR (300 MHz, CDCl3; a/b-d-isomers): 2.19 (s, Me); 2.20
(s, Me); 2.28 (s, Me); 2.29 (s, Me); 3.41 ± 4.17 (m, 10 H, HÀC(2'), HÀC(3'), HÀC(4'), CH2(5')); 4.86 ± 4.90
(m, 1 H, HÀC(1') (a)); 5.10 ± 5.15 (m, 1 H, HÀC(1') (b)); 6.78 (s, 1 arom. H, (a)); 7.92 (s, 1 arom. H (a)); 6.92
(s, 1 arom. H (b)); 7.94 (s, 1 arom. H (b)); 8.14 (d, J ˆ 7.5, NH (b)); 8.86 (d, J ˆ 8.7, NH (a)). 13C-NMR
(125 MHz, (CD3)2SO, a/b-d-isomers): 18.07; 18.10; 20.14; 30.65; 61.12; 63.66; 66.89; 67.16; 68.89; 70.01; 70.36;
70.83; 80.27; 82.04; 115.64; 115.89; 125.22; 125.30; 125.36; 126.1; 129.99; 130.08; 142.09; 142.60; 146.89; 147.35.
ESI-MS (neg. mode; 5m HN4OAc): 357 (40, [M ‡ OAc]À), 333 (100), 335 (20, [M ‡ Cl]À), 297 (52, MÀ). Anal.
calc. for C3H18N2O6 (298.30): C 52.35, H 6.08, N 9.39; found: C 52.18, H 5.82, N 9.11.
4,5-Dimethyl-2-nitro-N-(2',3',5'-tri-O-acetyl-a-d-ribopyranosyl)benzenamine (8). To 7 (0.80 g, 2.68 mmol)
in pyridine (10 ml), Ac2O (3 ml) was added. The mixture was stirred for 12 h at r.t. and then heated for 30 min to
508. After evaporation, the brownish solid residue was submitted to FC (silica gel, CH2Cl2/MeOH 25 :1): 0.50 g
(44%) of 8, after recrystallization from EtOH. Yellow needles. M.p. 1498. Rf (silica gel, toluene/AcOEt 5 :1)
0.50. IR (KBr): 3378, 2941,1752, 1632, 1572, 1508, 1411, 1376, 1227, 1027, 879. 1H-NMR (300 MHz, CDCl3): 2.02
(s, Me); 2.04 (s, Me); 2.21 (s, Ac); 2.29 (s, Ac); 2.37 (s, Ac); 3.61 (dd, J ˆ 5.3, 11.2, 1 HÀC(5')); 3.90 (t, J ˆ 11.2,
1 HÀC(5')); 5.08 (qd, J ˆ 3.2, 5.2, HÀC(4')); 5.16 ± 5.30 (m, HÀC(2')); 5.45 ± 5.49 (m, HÀC(3')); 5.73 ± 5.75
(m, J ˆ 3.1, HÀC(1')); 6.90 (s, HÀC(6)); 7.98 (s, HÀC(3)); 8.93 (d, J ˆ 6.8, NH). 13C-NMR (125 MHz, CDCl3):
18.68; 20.46; 20.64; 20.68; 20.86; 55.80; 66.06; 66.17; 67.90; 76.77;115.53; 115.73; 127.04; 131.51; 141.47; 147.42;
‡
169.56; 169.57; 170.38. FAB-MS (pos. mode): 425 (100 MH ). Anal. calc. for C19H24N2O9 (424.41): C 53.77,
H 5.70, N 6.60; found: C 53.70, H 5.72, N 6.53.
7,8-Dimethyl-10-(2',3',5'-tri-O-acetyl-b-d-ribopyranosyl)benzo[g]pteridine-2,4(3H,10H)-dione (10). To a soln.
of 8 (0.10 g, 0.24 mmol) in AcOEt (3 ml), Et3N (0.17 g, 1.65 mmol) and PtO2 (0.05 g, 0.20 mmol) were added.
The resulting suspension was stirred under H2 at 2 bar in an autoclave until a colorless soln. was obtained (ca. 1 h).
The soln. containing 9 was filtered under exclusion of light into a suspension of alloxan monohydrate (0.07 g,
0.42 mmol) and boric acid (0.07 g, 1.07 mmol) in AcOH (30 ml). The colorless soln. turned immediately green
and, subsequently, upon stirring, yellow-brown. After 2 h of stirring, H2O (50 ml) was added, the mixture
extracted with CHCl3 (3Â), the combined org. phase washed with H2O (4Â), dried (Na2SO4), and evaporated.
The residue showed 3 yellow (irradiation at 366 nm) flavin spots on TLC. The main b-d-isomer 10 was isolated
by FC (silica gel-H, CH2Cl2/MeOH 10 :1). Prep. MPLC (CHCl3/MeOH 100 :0 ! 75 :25 within 1 h) allowed
removal of the minor a-d-isomer: 0.60 g (50%) of 10. M.p. 1778. Rf (silica gel, CH2Cl2/MeOH 25 :1) 0.25. IR
(neat): 3278, 2933, 1750, 1583, 1539, 1367, 1233, 1083, 1044. 1H-NMR (300 MHz, CDCl3): 1.75 (s, Ac); 2.10
(s, Ac); 2.30 (s, Ac); 2.42 (s, Me); 2.55 (s, Me); 4.13±4.24 (m, 2 HÀC(5')); 4.19 (qd, Jˆ 11, HÀC(4')); 5.80±5.85
(m, HÀC(2'), HÀC(3')); 7.69 (d, J ˆ 10.7, HÀC(1')); 7.95 (s, HÀC(6 or 9)); 7.96 (s, HÀC(6 or 9)); 8.63 (s, NH).
13C-NMR (125 MHz, CDCl3): 19.45; 20.22; 20.61; 20.90; 22.09; 64.46; 65.77; 65.85; 67.99; 80.72; 117.65; 129.26;
133.30; 134.95; 135.50; 137.27; 148.05; 151.46; 154.53; 158.97; 168.97; 169.59; 170.22. FAB-MS (pos. mode): 501
‡
‡
‡
(46, MH ), 405 (22), 259 (100). FAB-HR-MS (pos. mode): 501.1629 (C23H25N4O9 ; MH ; calc. 501.1621).

Helvetica Chimica Acta ± Vol. 83 (2000)
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7,8-Dimethyl-10-(b-d-ribopyranosyl)benzo[g]pteridine-2,4(3H,10H)-dione (4).
A
soln. of 10 (55 mg,
0.11 mmol) in abs. MeOH was saturated with NH3 (50 ml) and stirred for 3 h at r.t. Evaporation gave 4
(20 mg, 48%). Yellow powder. M.p. 1878. Rf (silica gel, MeOH): 0.66. IR (neat): 3360, 2944, 1721, 1667, 1542,
1449, 1390, 1275, 1099, 104. 1H-NMR (300 MHz, (D6)DMSO): 2.35, 2.45 (2s, 2 Me); 3.68 (m, CH2(5')); 4.04 (br.,
HÀC(4'), HÀC(3')); 4.34 (br. s, HÀC(2')); 4.91 (br. s, 2 OH), 5.15 (br. s, 1 OH); 7.11 (d, HÀC(1')); 7.84, 7.95
(2s, HÀC(9), HÀC(6)). 13C-NMR (300 MHz, (D6)DMSO): 18.58; 20.76; 65.67; 65.90; 66.24; 71.16; 83.13;
118.75; 129.25; 131.25; 134.00; 135.52; 136.86; 144.95; 151.75; 155.25; 159.44. ESI-MS (MeOH): 787 (15, [2M ‡
‡
‡
‡
‡
‡
K] ), 771 (100, [2M ‡ Na] ), 413 (25, [M ‡ K] ), 397 (100, [M ‡ Na] ), 375 (58, MH ). FAB-HR-MS (pos.
‡
‡
mode): 375.1301 (C17H19N4O6 ; MH ; calc. 375.1304).
2',3',5'-Tri-O-acetyl-1'-O-benzyl-b-d-ribofuranose (12). To benzyl b-d-ribofuranoside (11; 1.00 g,
4.16 mmol) in pyridine (10 ml), Ac2O (3 ml) was added. The mixture was stirred for 8 h at r.t. and then
heated for additional 1.5 h to 508. EtOH (10 ml) and Et2O (100 ml) were added, and the mixture was extracted
with H2O (3 Â 100 ml). The combined org. phase was dried (Na2SO4) and evaporated and the residue dried
under high vacuum: 1.5 g (quant.) of 12. Colorless solid material. M.p. 60 ± 658. Rf (silica gel, CH2Cl2/MeOH
10 :1) 0.84. IR (KBr): 3356, 3022, 2900, 1747, 1433, 1381, 1220, 1083, 967, 745, 702, 626. 1H-NMR (200 MHz,
CDCl3): 2.05 (s, Ac); 2.07 (s, Ac); 2.11 (s, Ac); 4.10 ± 4.41 (m, HÀC(4')); CH2(5')); 4.53 (d, J ˆ 12, 1 H, PhCH2);
4.77 (d, J ˆ 12, 1 H, PhCH2); 5.41 (s, HÀC(1')); 5.09 ± 5.51 (m, HÀC(2'), HÀC(3')); 7.33 ± 7.38 (m, 5 arom. H).
13C-NMR (100 MHz, CDCl3): 20.53; 20.61; 20.77; 64.43; 69.42; 71.54; 74.86; 78.65; 104.24; 127.92 (2Â); 127.94;
‡
‡
128.48 (2Â); 136.82; 169.64; 169.70; 170.71. FAB-MS (pos. mode): 625 (10, [M ‡ 259] ), 389 (10, [M ‡ Na] ),
‡
367 (12, MH ), 259 (100). Anal. calc. for C18H22O8 (366.37): C 59.01, H 6.05; found: C 59.05, H 5.96.
2',3',5'-Tri-O-acetyl-a/b-d-ribofuranose (13) [10]. A soln. of 12 (1.48 g, 4.04 mmol) in AcOEt (20 ml) and
10% Pd/C (1.00 g) was stirred under H2 at 4.5 bar. The soln. was filtered through Celite and evaporated and the
residue subjected to FC (silica gel, CH2Cl2/MeOH 15 :1): 1.10 g (quant.) of 13. Colorless oil. Rf (silica gel,
CH2Cl2/MeOH 10 :1) 0.34. 1H-NMR (300 MHz, CDCl3); mixture of the a/b-d-isomers): 2.07 (s, 3 H, Ac); 2.10
(s, 3 H, Ac); 2.10 (s, 3 H, Ac); 2.11 (s, 3 H, Ac); 2.13 (s, 3 H, Ac); 2.14 (s, 3 H, Ac), 3.05 ± 3.25 (m, 2 OH); 4.13 ±
‡
‡
4.42 (m, 6 H); 5.08 ± 5.40 (m, 6 H). ESI-MS (CH2Cl2): 294 (100, [M ‡ NH4] ), 259 (24, [MH] ).
4,5-Dimethyl-2-nitro-N-(2',3',5'-tri-O-acetyl-a-d-ribofuranosyl)benzenamine (14). Under Ar, 13 (1.00 g,
3.62 mmol) was dissolved in EtOH (15 ml). Then, 6 (3.62 g, 18.1 mmol, 5 equiv.) and a cat. amount of NH4Cl
(0.03 g) were added. The orange mixture was heated to reflux for 2.5 h, then allowed to cool to r.t., and filtered.
Celite was added to the soln., and the mixture was evaporated. The solid material was added ontop of a silica gel
column, and residual 6 was eluted with CH2Cl2. The product was then eluted with CH2Cl2/MeOH 25 :1: 0.85 g
(55%) of 14 (with traces of the corresponding b-d-isomer). Yellow oil. Rf (silica gel, CH2Cl2/MeOH 10 :1) 0.38.
IR (neat): 3372, 2934, 1747, 1627, 1572, 1506, 1433, 1372, 1232, 1058. 1H-NMR (300 MHz, CDCl3; a-d-isomer):
2.13 (s, Ac); 2.14 (s, Ac); 2.21 (s, Ac); 2.22 (s, Me); 2.29 (s, Me); 4.20 ± 4.34 (m, CH2(5'), HÀC(4')); 5.27 ± 5.31
(m, HÀC(3')); 5.39 ± 5.42 (m, HÀC(2')); 5.75 ± 5.80 (m, HÀC(1')); 6.95 (s, 1 arom. H); 7.96 (s, 1 arom. H); 8.74
(d, J ˆ 7.8, NH). 13C-NMR (125 MHz, CDCl3; a-d-isomer): 18.59; 20.36; 20.53; 20.58; 20.74; 63.48; 69.75; 71.45;
78.91; 82.05; 115.43; 126.29; 126.91; 131.42; 141.12; 147.31; 169.40; 169.97; 170.44. FAB-MS (pos. mode): 424
‡
(100, M ), 259 (87), 139 (42). Anal. calc. for C19H24N2O9 (424.40): C 53.77, H 5.70, N 6.60; found: C 53.87,
H 5.78, N 6.42.
7,8-Dimethyl-10-(2',3',5'-tri-O-acetyl-b-d-ribofuranosyl)benzo[g]pteridine-2,4(3H,10H)-dione (15). To a
soln. of 14 (0.30 g, 0.70 mmol) in AcOEt (3 ml), Et3N (0.49 g, 4.90 mmol) and PtO2 (0.06 g, 0.27 mmol) were
added. The suspension was briefly (!) stirred under H2 at 2 bar in an autoclave until a colorless soln. was
obtained. The mixture was filtered into a suspension of alloxan monohydrate (0.20 g, 1.24 mmol) and boric acid
(0.20 g, 3.17 mmol) in AcOH (10 ml) under exclusion of light. Upon stirring at r.t., the initially green suspension
changed into a green-brown soln. After 2 h, the mixture was worked up as described for 10 (CH2Cl2 instead of
CHCl3). The main b-d-isomer 15 was isolated by FC (silica gel-H (CH2Cl2/MeOH 10 :1; 3 spots on TLC).
Separation of the two minor products was achieved by MPLC (silica gel, CHCl3/MeOH 100 :0 ! 75 :25 within
1 h): 24 mg (18%) of 15. Rf (silica gel, CH2Cl2/MeOH 10 :1) 0.71. IR (KBr): 3455, 2367, 1750, 1733, 1683, 1655,
1578, 1544, 1394, 1377, 1233, 1050. 1H-NMR (300 MHz, CDCl3): 2.08 (s, Ac); 2.12 (s, Ac); 2.16 (s, Ac); 2.43
(s, Me); 2.53 (s, Me); 4.45 ± 4.56 (m, CH2(5'), HÀC(4')); 5.80 ± 5.85 (m, HÀC(3')); 5.98 (dd, J ˆ 4.0, 6.7,
HÀC(2')); 6.92 (d, J ˆ 3.8, HÀC(1')); 7.51 (s, 1 arom. H); 8.04 (s, 1 arom. H); 8.39 (s, NH). 13C-NMR
(125 MHz, CDCl3): 19.37; 20.35; 20.42; 20.80; 21.86; 62.78; 69.72; 71.83; 80.20; 90.11; 115.40; 129.89; 133.39;
134.94; 136.47; 137.12; 147.89; 153.66; 156.10; 158.84; 169.48; 170.02; 170.45. FAB-MS (pos. mode): 501 (15,
‡
‡
‡
MH ), 393 (100). FAB-HR-MS (pos. mode): 501.1629 (C23H24N4O9 , MH ; calc. 501.1622).
N-(2',3'-O-Isopropylidene-a-d-ribofuranosyl)-4,5-dimethyl-2-nitrobenzenamine (17). Under Ar, 16 (0.53 g,
2.76 mmol) was dissolved in EtOH (15 ml). Then 6 (3.30 g, 13.8 mmol, 5 equiv.) and a cat. amount of NH4Cl

2462
Helvetica Chimica Acta ± Vol. 83 (2000)
(70 mg) were added. The orange mixture was heated to reflux for 2.5 h. After 10 min, 3 Š molecular sieves were
added. The mixture was allowed to cool to r.t. and filtered. Celite was added to the soln. and the slurry
evaporated. The residue was added to the soln. on top of a silica gel column, and 6 was eluted with CH2Cl2. The
product was eluted with CH2Cl2/MeOH 15 :1: 0.59 g (66%) of 17 (a/b-d 12 :1). Yellow oil. Rf (silica gel, CH2Cl2/
MeOH 25 :1) 0.36. IR (neat): 3370, 2933, 2300, 1627, 1567, 1506, 1406, 1327, 1272, 1239, 1094, 856. 1H-NMR
(300 MHz, CDCl3; a-d-isomer): 1.38 (s, 3 H, Me2C); 1.59 (s, 3 H, Me2C); 2.20 (s, Me); 2.29 (s, Me); 3.71 ± 3.77
(m, CH2(5')); 4.33 ± 4.36 (m, HÀC(4')); 4.70 (dd, J ˆ 2.5, 5.9, HÀC(3')); 4.92 (dd, J ˆ 1.9, 6.1, HÀC(2')); 5.45
(dd, J ˆ 1.5, 9.5, HÀC(1')); 6.96 (s, 1 arom. H); 7.93 (s, 1 arom. H); 8.35 (d, J ˆ 9.1, NH). 13C-NMR (100 MHz,
CDCl3; a-d-isomer): 18.59; 20.57; 24.92; 26.13; 63.29; 79.66; 81.43; 81.43; 84.36; 114.51; 116.09; 126.29; 126.39;
‡
‡
131.32; 141.28; 146.91. FAB-MS (pos. mode): 677 (7, 2 MH ), 391 (33), 362 (28, [M ‡ Na] , 339 (100), 339
‡
(MH ).
N-(5'-O-Acetyl-2',3'-O-isopropylidene-a-d-ribofuranosyl)-4,5-dimethyl-2-nitrobenzenamine (18). To
a
soln. of 17 (0.86 g, 2.53 mmol) in pyridine (10 ml), Ac2O (3 ml) was added and the mixture stirred for 3 h at
r.t. and then heated for 8 h at 508. EtOH (10 ml) and Et2O (100 ml) were added, and the mixture was washed
with H2O (3 Â 100 ml). The org. phase was dried (Na2SO4) and evaporated and the residue (yellow powder)
dried under high vacuum for 2 days: 0.95 g (98%) of 18. An anal. sample of 18 was obtained by recrystallization
from EtOH. Yellow needles. M.p. 157 ± 1618. Rf (silica gel, CH2Cl2/MeOH 10 :1) 0.73. IR (neat): 3367, 2933,
1742, 1627, 1567, 1508, 1433, 1372, 1239, 1094, 1033, 861. 1H-NMR (300 MHz, CDCl3): 1.44 (s, 3 H, Me2C); 1.68
(s, 3 H, Me2C); 1.14 (s, Ac); 2.20 (s, Me); 2.29 (s, Me); 4.13 ± 4.16 (m, 1 HÀC(5')); 4.31 ± 4.40 (m, HÀC(4'),
1 HÀC(5')); 4.73 (dd, J ˆ 1.8, 6.6, HÀC(3')); 4.89 (dd, J ˆ 3.3, 4.9, HÀC(2')); 5.49 (dd, J ˆ 4.8, 7.6, HÀC(1'));
6.93 (s, 1 arom. H); 7.95 (s, 1 arom. H); 8.77 (d, J ˆ 7.5, NH). 13C-NMR (100 MHz, CDCl3): 18.64; 20.53; 20.96;
25.16; 26.19; 63.87; 79.27; 79.49; 81.57; 84.29; 114.79; 116.18; 126.33; 126.59; 131.50; 141.08; 146.99; 170.58. FAB-
‡
MS (pos. mode): 381 (34, MH ), 289 (41). Anal. calc. for C18H24N2O7 (380.40): C 56.83, H 6.36, N 7.36; found:
C 56.86, H 6.36, N 7.36.
10-(5'-O-Acetyl-2',3'-O-isopropylidene-b-d-ribofuranosyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-
dione (20). To a soln. of 18 (0.30 g, 0.79 mmol) in AcOEt (9 ml) and Et3N (0.59 g, 5.53 mmol), PtO2 (0.07 g,
0.31 mmol) was added and the mixture stirred under H2 at 2 bar in an autoclave until a colorless soln. was
obtained (1 h). The mixture containing 19 was filtered under exclusion of light into a suspension of alloxan
monohydrate (0.22 g, 1.40 mmol) and boric acid (0.22 g, 3.58 mmol) in AcOH (30 ml). The mixture turned
immediately green and, upon stirring at r.t., yellow-brown. After 2 h, the mixture was worked up as described for
10 (2 Â CHCl3). The main b-d-isomer 20 was isolated by FC (silica gel H, CH2Cl2/MeOH 10 :1; 3 spots on TLC).
Separation of the two minor products was achieved by MPLC (silica gel, CHCl3/MeOH 100 :0 ! 75 :25 within
1 h): 80 mg (22%) of 20. M.p. 1428. Rf (silica gel, CH2Cl2/MeOH 10 :1) 0.74. IR (neat): 3178, 2933, 2811, 1717,
1657, 1578, 1542, 1395, 1256, 1156, 1078. 1H-NMR (300 MHz, CDCl3): 1.38 (s, 3 H, Me2C); 1.63 (s, 3 H, Me2C);
2.04 (s, Ac); 2.44 (s, Me); 2.55 (s, Me); 4.47 ± 4.57 (m, CH2(5'), HÀC(4')); 5.41 (dd, J ˆ 4.0, 6.3, HÀC(3')); 5.57
(dd, J ˆ 1.2, 6.5, HÀC(2')); 6.68 (s, HÀC(1')); 7.66 (s, 1 arom. H); 8.05 (s, 1 arom. H); 8.35 (s, NH). 13C-NMR
(125 MHz, CDCl3): 19.40; 20.81; 21.64; 25.27; 27.25; 64.60; 82.77; 84.24; 88.35; 92.83; 114.79; 114.97; 130.75;
‡
133.08; 135.03; 136.72; 137.18; 148.42; 149.03; 153.40; 158.87; 170.48. ESI-MS: 456 (10, M ), 243 (100). FAB-
‡
‡
HR-MS (pos. mode): 457.1727 (C22H25N4O7 , MH ; calc. 457.1723).
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Received May 12, 2000