Synthesis of an oxidation-stable analogue of cyclic pyranopterin monophosphate (cPMP)

Article abstract of DOI:10.1002/ejoc.201301784

Molybdenum cofactor (Moco) deficiency is a lethal hereditary metabolic disease. A recently developed therapy requires continuous intravenous supplementation of the biosynthetic Moco precursor cyclic pyranopterin monophosphate (cPMP). The limited stability of the latter natural product, mostly due to oxidative degradation, is problematic for oral administration. Therefore, the synthesis of more stable cPMP analogues is of great interest. In this context and for the first time, the synthesis of a cPMP analogue, in which the oxidation-labile reduced pterin unit is replaced by a pyrazine moiety, was achieved starting from the chiral pool materials D-galactose or D-arabitol. Our synthesis, 13 steps in total, includes the following key transformations: i) pyrazine lithiation, followed by acylation; ii) closure of the pyrane ring by nucleophilic aromatic substitution; and iii) introduction of phosphate.

Full text of DOI:10.1002/ejoc.201301784

FULL PAPER
DOI: 10.1002/ejoc.201301784
Synthesis of an Oxidation-Stable Analogue of Cyclic Pyranopterin
Monophosphate (cPMP)
Stefan Hilken,[a] Florian Kaletta,[a] Angela Heinsch,[a] Jörg-M. Neudörfl,[a][‡] and
Albrecht Berkessel*[a]
Keywords: Natural products / Metalloenzymes / Biological activity / Lithiation / Phosphorylation / Molybdenum
Molybdenum cofactor (Moco) deficiency is a lethal heredi- the first time, the synthesis of a cPMP analogue, in which the
tary metabolic disease. A recently developed therapy re-
quires continuous intravenous supplementation of the bio-
synthetic Moco precursor cyclic pyranopterin monophos-
phate (cPMP). The limited stability of the latter natural prod-
uct, mostly due to oxidative degradation, is problematic for
oral administration. Therefore, the synthesis of more stable
cPMP analogues is of great interest. In this context and for
oxidation-labile reduced pterin unit is replaced by a pyrazine
moiety, was achieved starting from the chiral pool materials
D-galactose or D-arabitol. Our synthesis, 13 steps in total, in-
cludes the following key transformations: i) pyrazine lithi-
ation, followed by acylation; ii) closure of the pyrane ring by
nucleophilic aromatic substitution; and iii) introduction of
phosphate.
Subsequently, two sulfur atoms are incorporated into
cPMP (2) to give MPT (3). Complexation to molybdenum
occurs after activation of the intermediate pterin MPT (3)
to give adenylated MPT (MPT-AMP, 4).[8] Compared to
Moco (5) itself, or to its other biosynthetic precursors,
cPMP (2) is the most oxygen-stable intermediate. Neverthe-
less, cPMP (2) is oxidized under aerobic conditions, with a
half-life of only a few hours, to give the biologically inac-
tive, ring-opened “compound Z” (6; Scheme 1).[1a,2,4b,7]
Any kind of blockage of the Moco biosynthesis causes
deactivation of all molybdenum-containing enzymes.[5b]
Human Moco deficiency is an autosomal recessive heredi-
tary metabolic disorder, with symptoms resulting mainly
from the lack of sulfite oxidase activity. This, in turn, leads
to elevated levels of toxic sulfite and tissue-specific lack of
sulfate.[9] Moco deficiency affects newborns immediately af-
ter birth. The symptoms are progressive neurologic damage,
usually resulting in early childhood death.[10] The frequency
of occurrence of Moco deficiency is assumed to be 1 in
100000 or less.[1a] Nevertheless, to date, over 100 cases have
been reported. Higher numbers can be assumed, however,
as many cases probably escape diagnosis.[5b,11] Based on the
location of the mutational blockage in the biosynthetic se-
quence, patients are classified into three groups. About two
thirds belong to group A, for whom the production of
cPMP (2) from GTP (1) is impeded. In type B and C
patients, the further conversion of cPMP (2) into MPT (3),
or the metal insertion are affected, respectively.[4b,11,12]
On the basis of promising results in a mouse model,[13]
five Moco-deficiency type A patients have been successfully
treated in replacement therapy, by permanent intravenous
supplementation of cPMP (2).[4b,11b] The cPMP (2) neces-
sary is currently being produced biotechnologically, using
Introduction
Molybdenum is essential for approximately two thirds of
all genetically investigated organisms.[1] Its catalytic activity
in the biological setting hinges on complexation to a spe-
cific ligand.[1b,2] With the exception of the enzyme nitroge-
nase, molybdenum is typically bound to a pterin molecule
(MPT, 3; Scheme 1), thus forming the molybdenum cofac-
tor (Moco, 5; Scheme 1), which is located at the enzymes’s
active site. Molybdenum enzymes catalyse a variety of re-
dox processes in carbon, nitrogen, and sulfur metabolism.[3]
In mammals, four different molybdenum enzymes have
been identified: i) sulfite oxidase; ii) aldehyde oxidase;
iii) xanthine oxidoreductase; and iv) mitochondrial benzo-
amidoxime reductase.[4] The synthesis of Moco (5) follows,
in all organisms studied to date, a conserved biosynthetic
pathway (Scheme 1).[5] Starting from GTP (1), the biosyn-
thesis proceeds in four steps with the involvement of at least
six enzymes.[1a,5] In the first step, cPMP (2) is formed by a
complex reaction sequence Ϫ and by a mechanism that is
not yet fully understood Ϫ from GTP (1).[1a] cPMP (2) con-
tains a reduced pyranopterin moiety and a cyclic phos-
phate. In addition, the geminal diol is a characteristic struc-
tural feature. Presumably, this hydrate form of the parent
ketone is favoured by the strongly electron-withdrawing
substitution pattern, i.e., O- and N-substitution of the
ketone’s α-positions.[1a,4b,5,6]
[a] Department of Chemistry, University of Cologne,
Greinstrasse 4, 50939 Cologne, Germany
E-mail: berkessel@uni-koeln.de
www.berkessel.de
[‡] X-ray crystallography
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301784.
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FULL PAPER
Scheme 1. Biosynthesis of Moco and structure of compound Z.[1a]
genetically modified E. coli developed by Schwarz et cofactor binding in the enzyme(s) is still in need of clarifica-
al.,[6,11b,13b,14a] followed by HPLC purification, and storage tion.[1a,15] We felt that modification of this part of the mo-
at –80 °C.[11b] Recently, the organic synthesis of labile lecule may be the best way to overcome cPMP’s oxygen la-
cPMP (2) using the Viscontini reaction as the key step was bility. As this lability can be assumed to be due to the pres-
reported.[14b,14c]
ence of the hydropterin moiety, and the N,O-acetal sub-
Even though a biotechnological source exists, and an or- structure,[22] the logical move was to replace cPMP’s piper-
ganic synthesis has been reported, the lability of cPMP (2) azine B-ring by a pyrazine moiety (grey in Figure 1). In
still remains a major roadblock en route to the highly desir- summary, we planned to synthesize compounds of type A
able oral administration of cPMP supplements. The sensi- as cPMP analogues (Figure 1).
tivity of hydrogenated pterins to oxygen, and their poor sol-
ubility in most solvents, are well known.[4b,15] Based on
work of Viscontini et al., the first chemical synthesis of the
pyranopterin skeleton was accomplished by Pfleiderer et al.
in 1990.[16] As regards synthetic approaches to Moco (5)
or Moco analogues, the work of the groups of Joule and
Garner,[17] Basu,[18] Burgmayer,[19] Goswami,[20] and
Figure 1. Retrosynthetic analysis of target cPMP analogue A
Holm[21] in particular should be mentioned.[15] All of these
(“eastern” building block in black, “western” building block in
grey).
investigations confirm that the hydropterin moiety lies at
the heart of the molecules’ susceptibility to oxidation and
concomitant ring cleavage (cf. formation of “compound Z”
from cPMP, Scheme 1). Therefore, the organic synthesis of
simplified and more stable analogues of cPMP (2) appears
to be the logical consequence.
For the synthesis of cPMP analogue A, we envisaged the
following three key steps: i) C–C coupling by ortho-lithi-
ation of the 1,4-diazine, followed by acylation with a Wein-
reb amide; ii) C–O coupling by nucleophilic aromatic sub-
stitution of a halide; and iii) introduction of the phosphate.
We expected the carbonyl group of analogue A to exist in
equilibrium with its hydrate, as is seen for the natural prod-
uct, cPMP (2).
Results and Discussion
Structural Design of a Simplified and Oxygen Resistant
cPMP Analogue
Synthesis of the Eastern Building Block (as a Weinreb
Amide)
Bearing in mind the biosynthetic conversion of cPMP (2)
into Moco (5), conservation of the “eastern part” (black in
Figure 1), consisting of the hydropyran ring and the cyclic
Weinreb amide 9 could be synthesized in five steps start-
phosphate, was considered to be crucial for biological ac- ing from d-arabitol or d-galactose (Scheme 2). To this end,
tivity. With regard to the “western part” of cPMP (2; the known benzylidene protection[23] and periodate cleav-
Scheme 1), the exact role of the pterin moiety for substrate/ age[23c,24] of the starting carbohydrates was followed by oxi-
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Oxidation-Stable Analogue of Cyclic Pyranopterin Monophosphate
Scheme 2. Synthesis of the eastern building block, i.e., Weinreb amide 9.
dation of the intermediate aldehyde to give methyl ester estingly, using methyl ester 8 instead of the Weinreb amide
7,[25] by treatment with bromine in methanol/water (9:1), in resulted in an even better yield of up to 77%. Cleavage of
analogy to a procedure reported by Williams et al.[26] The the TBS group was effected with hydrogen fluoride in pyr-
free hydroxy group of methyl ester 7 was smoothly pro- idine to give ketones 12a and 12b (Scheme 3).
tected as its TBS (tert-butyldimethylsilyl) ether, and the re-
For the planned ring-closing nucleophilic aromatic sub-
sulting methyl ester (i.e., 8) was converted into Weinreb stitution, various bases (NaH, KOtBu, Ag2O, tetrameth-
amide 9 in 80% yield following a procedure by Williams et ylguanidine, NEt3, K2CO3) as well as acyl-transfer catalysts
al.[27] (Scheme 2).
(such as 4-pyrrolidinopyridine, PPY) were explored. In
model studies, the latter gave quite satisfactory results in
intermolecular substitutions of fluoro-substituted 1,4-diaz-
ines with alcohols. Unfortunately, in the case of the desired
intramolecular substitution, decomposition of the β-hy-
ortho-Acylation of 2-Halopyrazines, and Attempted
Cyclization by Nucleophilic Aromatic Substitution
The ortho-lithiation of 2-chloro- and 2-fluoropyrazines droxy ketones (i.e., 12a and 12b) was observed exclusively
10a and 10b with lithium tetramethylpiperidide (LTMP) ac- with PPY and with all of the bases listed above. Simple
cording to Quéguiner et al.,[28] and reaction with Weinreb heating of the starting material, without added reagents, led
amide 9 gave acylated pyrazines 11a and 11b in yields of 38 to the same result. A possible explanation for this unexpec-
and 43%, respectively (Scheme 3). In our hands, the genera- ted failure of the ring closure was revealed by the X-ray
tion of the lithiated halopyrazines in the presence of the crystal structures of β-hydroxy ketone 12a (Figure 2) and
Weinreb amide (“in situ quench” conditions) proved supe- of TBS-protected β-hydroxy ketone 11b (see Supporting In-
rior to the addition of the electrophile after lithiation of the formation). For example, the diffraction analysis of 12a re-
pyrazine. In contrast to pyrazines 10a and 10b, the corre- vealed that in the conformer present in the crystal, the hy-
sponding quinoxalines turned out to be poor substrates for droxy group and the pyrazine C–F bond are not in close
this coupling approach by ortho-lithiation/acylation. Inter- proximity, but are pointing away from one another. DFT
Scheme 3. C–C coupling by ortho-acylation of 2-halopyrazines 10a and 10b [LTMP = lithium-2,2,6,6-tetramethylpiperidide, PPY = 4-(1-
pyrrolidinyl)pyridine].
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FULL PAPER
calculations (see Supporting Information) indicated that ro- fected the desired ring-closing nucleophilic aromatic substi-
tation around the Cpyrazine–Ccarbonyl bond is possible, but tution to give compound 16, and similar treatment of 15a
that the unreactive conformation as found in the crystal is and 15b resulted in the analogous formation of compound
energetically strongly favoured. The X-ray crystal structure 17 (Scheme 4). Despite extensive testing of various bases,
shown in Figure 2 furthermore shows that the hydroxy acyl-transfer catalysts, and temperatures, the cyclization
group and the acidic proton in the ketone’s α-position have yields for either epimer could not be raised above the ones
an almost perfectly antiperiplanar diaxial orientation. Un- given in Scheme 4. The best results were obtained using po-
der the basic reaction conditions, ketones 12a and 12b may tassium tert-butoxide. High-dilution conditions led to only
therefore easily undergo undesired alternative transforma- slightly improved yields.
tions e.g., retro-aldol reaction, or elimination.
Figure 3. X-ray crystal structures of epimeric diols 14a (left) and
15a (right).
Figure 2. X-ray crystal structure of β-hydroxy ketone 12a.
Before the introduction of the phosphate, for which
benzylidene cleavage was required, the reoxidation of the
secondary hydroxy groups of cyclized epimers 16 and 17
was performed. This transformation could be achieved
smoothly using Dess–Martin periodinane in yields of 95
and 74%, respectively (Scheme 4). In the resulting tricyclic
As a consequence of the above analysis, we decided to
temporarily deactivate the keto functionality by protection.
Acetalization required unacceptably harsh reaction condi-
tions (e.g., TfOH in neopentyl glycol, 140 °C, 4 Å MS), and
ketone (i.e., 13), the carbon skeleton of cPMP analogue A
so we considered reducing the carbonyl group to a second-
ary alcohol. As shown in Scheme 4, treatment of β-hydroxy
ketones 12a and 12b with sodium borohydride gave epi-
meric diols 14a and 15a, and 14b and 15b, respectively, in
good yields and with diastereomeric ratios of ca. 1:1. The
epimeric pairs of diols could be separated chromatographi-
cally, and the configurations of the newly formed ste-
reocentres were assigned by X-ray crystallography (see Fig-
ure 3 for the X-ray crystal structures of 14a and 15a). To
our delight, treatment of diols 14a and 14b with base ef- Figure 4. X-ray crystal structure of tricyclic ketone 13.
Scheme 4. Ring-closing C–O coupling by nucleophilic aromatic substitution (DMP = Dess–Martin periodinane).
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Oxidation-Stable Analogue of Cyclic Pyranopterin Monophosphate
Scheme 5. Synthesis of cPMP analogue 21 by phosphate introduction.
is fully assembled. This central intermediate of our synthe-
sis could also be crystallized, and its X-ray structure is
shown in Figure 4. The crystal structure nicely demon-
strates the bent, non-planar shape of the tricyclic core, re-
sulting from the cis junction of the dihydropyranone and
1,3-dioxane rings.
Introduction of the Phosphate
The removal of the benzylidene protecting group from
ketone 13 was effected under iodine catalysis[29] in a mixture
of methanol and chloroform. These conditions, which re-
sulted from extensive optimization, at the same time ef-
fected the conversion of the carbonyl group into its di-
methyl acetal to give 18 (Scheme 5). In view of the difficult-
ies encountered in the acetalization of TBS-protected hy-
droxy ketones 11a and 11b (see above), this result was sur-
prising, and was attributed to a reduced steric crowding
after removal of the benzylidene group, together with a po-
tential electrophilic activation of the ketone by intramolecu-
lar hydrogen bonding.[30] Treatment of diol 18 with phos-
phorus oxychloride gave, after flash chromatography, phos-
phoryl chloride 19 as a single diastereomer in 71% yield.
The hydrolysis of the latter compound was accomplished in
a mixture of D2O and [D6]acetone (to enable monitoring
by NMR spectroscopy) to give phosphoric acid 20 in 71%
yield (Scheme 5). The synthesis of cPMP analogue A was
completed by cleaving the dimethyl acetal of compound 20
by heating in the presence of an excess of deuterium chlor-
ide solution. Due to slow accompanying phosphate hydrol-
ysis, purification was carried out by preparative reverse-
phase HPLC after treatment with sodium hydrogen carb-
onate. According to its NMR spectroscopic data, the prod-
uct was obtained as its ketone hydrate (i.e., 21), and 31P
NMR spectroscopy indicated the presence of small
amounts of inorganic phosphate. Both the relative and the
absolute configuration of the cPMP analogue were con-
firmed by the X-ray crystal structure of the sodium salt of
dimethyl acetal 20, as shown in Figure 5. Upon exposure to
air, cPMP analogue 21 and its precursors did not show any
tendency towards oxidative degradation.
Figure 5. X-ray crystal structures of phosphoryl chloride 19 (left),
the sodium salt of dimethyl acetal 20 (right), and cPMP analogue
21 (the sodium salt crystallizes with one equivalent of methanol).
Conclusions
We report the first synthesis and structural characteriza-
tion of an analogue of cyclic pyranopterin monophosphate
(cPMP, 2), namely cyclic phosphate 21. As a key feature,
the reduced pterin moiety of the natural product (i.e., 2) is
replaced by a pyrazine in 21. Unlike the natural product
(i.e., 2), its analogue 21 does not show any tendency
towards oxidative degradation. Key steps of the synthesis of
21 are: i) C–C coupling of the eastern and western building
blocks by ortho-lithiation/acylation of a 2-halopyrazine;
ii) ring-closing C–O bond formation by nucleophilic aro-
matic substitution, requiring temporary reduction of the
eastern building block’s keto functionality to an alcohol;
and iii) introduction of the phosphoric diester. Tests for
biological activity are underway.
Experimental Section
General Remarks: 2,2,6,6-Tetramethylpiperidine, 2-chloropyrazine
(10a), 2-fluoropyrazine (10b), and phosphorus oxychloride were
distilled before use and stored over molecular sieves. 1,3-O-Benzyl-
idene-d-arabitol was prepared according to a literature pro-
cedure.[23a,23c,23d] The concentration of nBuLi solutions was deter-
mined by titration against N-benzylbenzamide.[31] All other chemi-
cals were purchased from commercial suppliers, and were used
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S. Hilken, F. Kaletta, A. Heinsch, J.-M. Neudörfl, A. Berkessel
FULL PAPER
without further purification. N,N-Dimethylformamide was pur-
chased from Acros (99.8%, extra dry, over MS). Solvents were
dried before use according to standard procedures. Aluminium-
backed TLC plates coated with 0.2 mm silica containing a fluores-
cent indicator (ALUGRAM® SIL G/UV254) were purchased from
Macherey–Nagel. Flash chromatography was carried out on silica
8.30 mmol) and imidazole (940 mg, 13.8 mmol) were added to a
solution of 7 (1.65 g, 6.90 mmol) in anhydrous DMF (4.4 mL) at
0 °C. The mixture was stirred at room temperature for 19 h, then
it was diluted with EtOAc (100 mL), and washed with brine
(50 mL) and water (70 mL). The organic layer was dried with
MgSO4, filtered, and evaporated under reduced pressure. The crude
gel (Acros, 0.035–0.070 mm, 60 Å). NMR spectra were recorded product was purified by column chromatography (silica gel, cyclo-
with Bruker DPX 200, DPX 300, AV 300, DRX 500, and AV 600
spectrometers at room temperature, unless otherwise noted, and
were referenced to the solvent used. Melting points were measured
hexane/EtOAc, 20:1 to 2:1) to give the TBS-protected alcohol 8
(2.36 g, 6.70 mmol, 97%) as a colourless solid. Rf = 0.20 (cyclohex-
ane/EtOAc, 8:1), m.p. 41–44 °C. [α]2D0 = –52.3 (c = 1.1, CH2Cl2).
with a B-545 Büchi apparatus. Optical rotations were measured 1H NMR (300 MHz, CDCl3): δ = 0.06 (s, 3 H, SiCH3), 0.13 (s, 3
with a Perkin–Elmer 343plus polarimeter. HRMS data were re-
corded with a Finnigan MAT 900 S instrument. Infrared (IR) spec-
tra were recorded with a Shimadzu IR-Affinity-1 spectrometer.
CHN analysis was carried out with an Elementar Analysensysteme
GmbH Vario EL instrument. Preparative reverse-phase HPLC was
performed using an EC-Nucleodur 100–16 C-18 column
H, SiCH3), 0.92 [s, 9 H, SiC(CH3)3], 3.78 (s, 3 H, OCH3), 4.00–
4.22 (m, 3 H), 4.57 (d, J = 1.7 Hz, 1 H), 5.55 (s, 1 H), 7.30–7.42
(m, Ar-H), 7.49–7.59 (m, 2 H, Ar-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = –5.1 (SiCH3), –4.4 (SiCH3), 18.0 [s, SiC(CH3)3], 25.6
[SiC(CH3)3], 52.1 (OCH3), 65.4 [C(H)OTBS], 72.0 (CH2), 79.3
(CH), 101.3 [C(H)Ph], 126.6 (Ar-CH), 128.3 (Ar-CH), 129.2 (Ar-
(50 mmϫ250 mm, Macherey–Nagel) with a NovaPrep 200 pump, CH), 137.6 (Ar-C), 168.8 (C=O) ppm. IR (ATR): ν = 2928, 2855,
˜
a LaChrom L-7400 (Merck Hitachi) UV detector, and a K-2401
(Knauer) refractive index detector. X-ray structural data were col-
lected with a Nonius Kappa CCD diffractometer at the tempera-
tures stated. Structures were solved using SHELXS97 and refined
with SHELXL97 using the full-matrix least-squares method on |F2|.
1767, 1738, 1439, 1362, 1292, 1250, 1210, 1163 (s), 1096 (s), 1016
(s), 935 (s), 833 (s), 808, 775 (s), 758, 694 (s) cm–1. HRMS (EI):
calcd. for C18H29O5Si [M
+
H]+ 351.163; found 351.163.
C18H28O5Si (352.50): calcd. C 61.33, H 8.01; found C 61.23, H
8.02. For X-ray data of compound 8, see Supporting Information.
Methyl (2S,4S,5R)-5-Hydroxy-2-phenyl-1,3-dioxane-4-carboxylate
(7): 1,3-O-Benzylidene-d-arabitol (6.35 g, 26.4 mmol) was dissolved
in CH2Cl2 (70 mL), and saturated aqueous NaHCO3 (13 mL) was
added. Sodium periodate (11.3 g, 52.8 mmol) was added to the
(2S,4S,5R)-5-(tert-Butyldimethylsilyloxy)-N-methoxy-N-methyl-2-
phenyl-1,3-dioxane-4-carboxamide (9): N,O-Dimethylhydroxyl-
amine hydrochloride (304 mg, 3.10 mmol) was added to a stirred
solution of ester 8 (705 mg, 2.00 mmol) in anhydrous THF (4 mL)
stirred mixture at 0 °C over 10 min. The mixture was stirred for 2 h at –25 °C, and then isopropylmagnesium chloride (2.0 m in THF;
at room temperature, then it was filtered, and the insoluble residue
was washed with CH2Cl2 (2ϫ 20 mL). The combined organic ex-
tracts were washed with brine (30 mL), dried with MgSO4, and
evaporated under reduced pressure to give the intermediate alde-
hyde (5.55 g) as a colourless foam that was used in the next step
without further purification.
3.00 mL, 6.00 mmol) was added dropwise over 20 min. The mixture
was stirred for a further 20 min at –25 °C, then it was quenched
with saturated aqueous NH4Cl (1.5 mL) and water (10 mL), and
the mixture was extracted with CH2Cl2 (2ϫ 12 mL). The combined
organic extracts were dried with MgSO4, filtered, and evaporated
under reduced pressure. The crude product was purified by column
chromatography (silica gel, cyclohexane/EtOAc, 5:2) to give Wein-
reb amide 9 (609 mg, 1.60 mmol, 80%) as a colourless solid. Rf =
0.20 (cyclohexane/EtOAc, 5:2), m.p. 134 °C. [α]2D0 = –49.7 (c = 1.31,
CH2Cl2). 1H NMR (200 MHz, [D8]toluene, 75 °C): δ = 0.08 (s, 3
H, SiCH3), 0.10 (s, 3 H, SiCH3), 0.95 [s, 9 H, SiC(CH3)3], 3.07 (s,
3 H, NCH3), 3.34 (s, 3 H, OCH3), 3.63 (dd, J = 12.5, 2.0 Hz, 1 H),
3.89–4.05 (m, 2 H), 4.42 (d, J = 1.6 Hz, 1 H), 5.31 (s, 1 H), 7.04–
7.19 (m, 3 H, Ar-H), 7.47–7.58 (m, 2 H, Ar-H) ppm. 13C NMR
(50 MHz, [D8]toluene, 75 °C): δ = –5.0 (SiCH3), –4.8 (SiCH3), 17.9
[s, SiC(CH3)3], 25.6 [SiC(CH3)3], 33.9 (NCH3), 60.3 (OCH3), 65.7
[C(H)OTBS], 72.1 (CH2), 80.6 (CH), 101.3 [C(H)Ph], 126.4 (Ar-
CH), 127.7 (Ar-CH), 128.4 (Ar-CH), 137.0 (Ar-C), 167.2 (C=O)
NaHCO3 (44.3 g, 528 mmol) was added to a solution of the crude
aldehyde in methanol and water (9:1), and then a solution of Br2
(16.9 g, 106 mmol) in methanol/water (9:1; 55 mL) was added over
15 min in a dropwise manner. The resulting suspension was stirred
for 3 h at room temperature. The mixture was cooled to 0 °C, then
solid Na2S2O3 was added until it turned colourless. Water (100 mL)
was added, and the mixture was extracted with CH2Cl2 (5ϫ
100 mL). The combined organic extracts were dried with MgSO4,
filtered, and evaporated under reduced pressure. The crude product
was purified by column chromatography (silica gel, cyclohexane/
EtOAc, 1:1) to give ester 7 (3.42 g, 14.4 mmol, 55%) as a colourless
solid. Rf = 0.28 (cyclohexane/EtOAc, 1:1), m.p. 120–122 °C (ref.[25]
124–125 °C). [α]2D0 = –48.5 (c = 1.0, CHCl3); ref.[25] –56.6 (c = 0.22,
ppm. IR (ATR): ν = 2936, 2859, 1674 (s), 1389, 1317, 1246, 1165,
˜
1099 (s), 1078 (s), 1024 (s), 988, 959, 849, 829 (s), 808 (s), 777 (s),
1
acetone). H NMR (300 MHz, CDCl3): δ = 3.06 (d, J = 11.4 Hz,
752 (s), 702 (s), 656 cm–1. HRMS (EI): calcd. for C19H31NO5Si-
1 H, OH), 3.82 (s, 3 H, OCH3), 4.00 (dd, J = 11.4, 1.4 Hz, 1 H,
CHOH), 4.11 (dd, J = 12.1, 0.8 Hz, 1 H, CH2), 4.24 (dd, J = 12.1,
1.7 Hz, 1 H, CH2), 4.61 (d, J = 1.1 Hz, 1 H), 5.59 (s, 1 H), 7.32–
7.44 (m, 3 H, Ar-H), 7.48–7.59 (m, 2 H, Ar-H) ppm. 13C NMR
CH3 [M + H]+ 366.174; found 366.174. C19H31NO5Si-CH3
:
+·
+·
calcd. C 59.81, H 8.19, N 3.67; found C 59.74, H 8.19, N 3.65. For
X-ray data of compound 9, see Supporting Information.
(75 MHz, CDCl3): δ = 52.4 (OCH3), 64.8 [C(H)OH], 72.1 (CH2), [(2S,4S,5R)-2-(tert-Butyldimethylsilyloxy)-2-phenyl-1,3-dioxan-4-
78.7 (CH), 101.3 [C(H)Ph], 126.1 (Ar-CH), 128.3 (Ar-CH), 129.2
yl](3-chloropyrazin-2-yl)methanone (11a):
(Ar-CH), 136.8 (Ar-C), 168.4 (C=O) ppm. IR (ATR): ν = 3505,
˜
From 9: A solution of 2,2,6,6-tetramethylpiperidine (119 μL,
98.9 mg, 700 μmol) in anhydrous THF (2 mL) was cooled to
–30 °C, and nBuLi (2.4 m in hexanes; 228 μL, 550 μmol) was added
over 5 min. The mixture was stirred at 0 °C for 30 min, then the
solution was cooled to –78 °C, and it was added over 8 min to
a solution of 2-chloropyrazine (62.5 μL, 80.2 mg, 700 μmol) and
Weinreb amide 9 (191 mg, 500 μmol) in anhydrous THF (3 mL) at
–78 °C. The mixture was stirred at this temperature for a further
2874, 1744 (s), 1734 (s), 1441, 1396, 1250 (s), 1223 (s), 1153 (s),
1090 (s), 1015 (s), 935, 822, 750 (s), 689 (s), 658 cm–1. HRMS (EI):
calcd. for C12H15O5 [M + H]+ 237.076; found 237.076. C12H14O5
(238.24): calcd. C 60.50, H 5.92; found C 60.75, H 6.12. For X-ray
data of compound 7, see Supporting Information.
Methyl (2S,4S,5R)-5-(tert-Butyldimethylsilyloxy)-2-phenyl-1,3-diox-
ane-4-carboxylate (8): tert-Butyldimethylsilyl chloride (1.25 g,
2236
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2231–2241

Oxidation-Stable Analogue of Cyclic Pyranopterin Monophosphate
1.5 h, then it was quenched with saturated aqueous NH4Cl (2 mL),
and warmed to room temperature. The organic layer was separated,
and the aqueous layer was extracted with EtOAc (3ϫ 3 mL). The
combined organic layers were dried with MgSO4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography (silica gel, cyclohexane/EtOAc, 3:1) to
give acylated chloropyrazine 11a (82 mg, 189 μmol, 38 %) as a
colourless solid.
the solution was cooled to –78 °C, and it was added over 20 min
to a solution of 2-fluoropyrazine (849 μL, 1.03 g, 10.5 mmol) and
methyl ester 8 (2.64 g, 7.50 mmol) in anhydrous THF (45 mL) at
–78 °C. The mixture was stirred at this temperature for 60 min,
then it was quenched with saturated aqueous NH4Cl (40 mL), and
warmed to room temperature. The organic layer was separated, and
the aqueous layer was extracted with MTBE (3 ϫ 50 mL). The
combined organic layers were dried with MgSO4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography (silica gel, cyclohexane/EtOAc, 3:1) to
give acylated fluoropyrazine 11b (2.43 g, 5.81 mmol, 77%) as a pale
yellow solid. Rf = 0.30 (cyclohexane/EtOAc, 5:2), m.p. 144 °C.
From 8: A solution of 2,2,6,6-tetramethylpiperidine (1.79 mL,
1.48 g, 10.5 mmol) in anhydrous THF (30 mL) was cooled to
–30 °C, and nBuLi (2.4 m in hexanes; 3.42 mL, 8.25 mmol) was
added over 5 min. The mixture was stirred at 0 °C for 30 min, then
the solution was cooled to –78 °C and added over 20 min to a solu-
tion of 2-chloropyrazine (937 μL, 1.20 g, 10.5 mmol) and methyl
ester 8 (2.64 g, 7.50 mmol) in anhydrous THF (45 mL) at –78 °C.
The mixture was stirred at this temperature for another 60 min,
then it was quenched with saturated aqueous NH4Cl (40 mL), and
warmed to room temperature. The organic layer was separated, and
the aqueous layer was extracted with MTBE (methyl tert-butyl
ether; 3ϫ 50 mL). The combined organic layers were dried with
MgSO4, filtered, and evaporated under reduced pressure. The crude
product was purified by column chromatography (silica gel, cyclo-
hexane/EtOAc, 8:1 to 4:1) to give acylated chloropyrazine 11a
(1.50 g, 3.45 mmol, 46%) as a colourless solid. Rf = 0.55 (cyclohex-
ane/EtOAc, 2:1), m.p. 147 °C. [α]2D0 = –75.1 (c = 1.10, CH2Cl2). 1H
NMR (300 MHz, CDCl3): δ = –0.17 (s, 3 H, SiCH3), 0.05 (s, 3 H,
SiCH3), 0.86 [s, 9 H, SiC(CH3)3], 4.15 (dd, J = 12.4, 1.3 Hz, 1 H,
CH2), 4.21 (dd, J = 12.4, 1.4 Hz, 1 H, CH2), 4.31–4.38 (m, 1 H,
CHOTBS), 5.51 (d, J = 1.2 Hz, 1 H), 5.68 (s, 1 H), 7.29–7.39 (m,
3 H, Ar-H), 7.46–7.57 (m, 2 H, Ar-H), 8.51 (d, J = 2.3 Hz, 1 H,
Pyr-H), 8.54 (d, J = 2.3 Hz, 1 H, Pyr-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = –5.0 (SiCH3), –4.3 (SiCH3), 18.0 [s, SiC(CH3)3], 25.7
[SiC(CH3)3], 66.6 [C(H)OTBS], 71.8 (CH2), 82.8 (CH), 101.3
[C(H)Ph], 126.4 (Ar-CH), 128.2 (Ar-CH), 129.0 (Ar-CH), 137.7
(Ar-C), 141.1 (Pyr-CH), 145.7 (Pyr-CH), 146.0 (Pyr-C), 147.4 (Pyr-
1
[α]2D0 = –95.6 (c = 1.28, CH2Cl2). H NMR (300 MHz, CDCl3): δ
= –0.30 (s, 3 H, SiCH3), 0.03 (s, 3 H, SiCH3), 0.83 [s, 9 H, SiC-
(CH3)3], 4.16–4.23 (m, 2 H, CH2), 4.35–4.41 (m, 1 H, CHOTBS),
5.64 (d, J = 1.6 Hz, 1 H), 5.70 (s, 1 H), 7.32–7.42 (m, 3 H, Ar-H),
7.53–7.63 (m, 2 H, Ar-H), 8.39–8.46 (m, 1 H, Pyr-H), 8.52–8.60
(m, 1 H, Pyr-H) ppm. 13C NMR (75 MHz, CDCl3): δ = –5.3
(SiCH3), –4.2 (SiCH3), 17.9 [s, SiC(CH3)3], 25.5 [SiC(CH3)3], 65.7
[C(H)OTBS], 71.7 (CH2), 82.5 (CH), 101.3 [C(H)Ph], 126.5 (Ar-
CH), 128.2 (Ar-CH), 129.1 (Ar-CH), 136.0 (JC,F = 19.3 Hz, Pyr-
C), 137.7 (Ar-C), 140.6 (JC,F = 5.3 Hz, Pyr-CH), 145.4 (JC,F
9.6 Hz, Pyr-CH), 158.2 (JC,F = 268.7 Hz, Pyr-C), 190.7 (JC,F
=
=
6.5 Hz, C=O) ppm. 19F NMR (282 MHz, CDCl3): δ = –71.1 ppm.
IR (ATR): ν = 2959, 2930, 2855, 1722, 1570, 1389, 1358, 1252,
˜
1155, 1096 (s), 1026 (s), 1016, 982, 932, 845, 826, 775 (s), 750 (s),
702 (s), 606 cm–1. HRMS (ESI): calcd. for C21H27FN2O4SiNa [M
+ Na]+ 441.162; found 441.162. C21H27FN2O4Si (418.54): calcd. C
60.26, H 6.50, N 6.69; found C 60.21, H 6.47, N 6.65. For X-ray
data of compound 11b, see Supporting Information.
(3-Chloro-2-pyrazinyl)[(2S,4S,5R)-5-hydroxy-2-phenyl-1,3-dioxan-
4-yl]methanone (12a): HF·pyridine (ca. 70:30 w/w; 3.55 mL) was
added to a solution of TBS-protected alcohol 11a (1.13 g,
2.59 mmol) in anhydrous THF (70 mL) and anhydrous pyridine
(7 mL) at 0 °C. The mixture was stirred for 24 h at room temp., and
then saturated aqueous NaHCO3 was added until the evolution of
gas had ceased. The mixture was extracted with EtOAc (5 ϫ
80 mL). The combined organic extracts were dried with MgSO4,
filtered, and evaporated under reduced pressure. The crude product
was purified by column chromatography (silica gel, cyclohexane/
EtOAc, 3:2) to give alcohol 12a (726 mg, 2.26 mmol, 87%) as a
colourless solid. Rf = 0.30 (cyclohexane/EtOAc, 1:1), m.p. 140–
143 °C. [α]2D0 = –143.4 (c = 0.35, CH2Cl2). 1H NMR (300 MHz,
CDCl3): δ = 3.24 (br. s, 1 H, OH), 4.08–4.15 (m, 1 H, CHOH),
4.19–4.29 (m, 2 H, CH2), 5.61–5.67 (m, 1 H), 5.72 (s, 1 H), 7.31–
7.41 (m, 3 H, Ar-H), 7.45–7.56 (m, 2 H, Ar-H), 8.49–8.57 (m, 2 H,
Pyr-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 64.8 [C(H)OH], 72.1
(CH2), 82.5 (CH), 101.2 [C(H)Ph], 126.0 (Ar-CH), 128.3 (Ar-CH),
129.2 (Ar-CH), 136.9 (Ar-C), 140.9 (Pyr-CH), 145.6 (Pyr-C), 146.1
C), 193.0 (C=O) ppm. IR (ATR): ν = 2955, 2930, 2859, 1721, 1539,
˜
1383, 1368, 1250, 1163, 1103, 1084, 1022 (s), 1011, 982, 924, 806,
775 (s), 731 (s), 696 (s), 654 cm–1. HRMS (ESI): calcd. for
C21H27ClN2O4SiNa [M + Na]+ 457.133; found 457.132.
C21H27ClN2O4Si (434.99): calcd. C 57.98, H 6.26, N 6.44; found C
57.87, H 6.23, N 6.35.
[(2S,4S,5R)-2-(tert-Butyldimethylsilyloxy)-2-phenyl-1,3-dioxan-4-
yl](3-fluoropyrazin-2-yl)methanone (11b)
From 9: A solution of 2,2,6,6-tetramethylpiperidine (238 μL,
198 mg, 1.40 mmol) in anhydrous THF (4 mL) was cooled to
–30 °C and nBuLi (2.4 m in hexanes; 456 μL, 1.10 mmol) was added
over 5 min. The mixture was stirred at 0 °C for 30 min, then the
solution was cooled to –78 °C, and it was added over 8 min to
a solution of 2-fluoropyrazine (112 μL, 137 mg, 1.40 mmol) and
Weinreb amide 9 (382 mg, 1.00 mmol) in anhydrous THF (6 mL)
at –78 °C. The mixture was stirred at this temperature for 1.5 h,
then it was quenched with saturated aqueous NH4Cl (5 mL), and
warmed to room temperature. The mixture was extracted with
EtOAc (3ϫ 8 mL). The combined organic extracts were dried with
MgSO4, filtered, and evaporated under reduced pressure. The crude
product was purified by column chromatography (silica gel, cyclo-
hexane/EtOAc, 5:2) to give acylated fluoropyrazine 11b (178 mg,
426 μmol, 43%) as a pale yellow solid.
(Pyr-CH), 147.7 (Pyr-C), 192.7 (C=O) ppm. IR (ATR): ν = 3374,
˜
2967, 1726, 1543, 1454, 1377 (s), 1292, 1246, 1152, 1113, 1101,
1076, 1016 (s), 1001 (s), 966 (s), 916, 862, 765, 741 (s), 696 (s), 652,
602 cm–1. HRMS (ESI): calcd. for C15H13ClN2O4Na [M + Na]+
343.046; found 343.046. C15H13ClN2O4 (320.73): calcd. C 56.17, H
4.09, N 8.73; found C 56.44, H 4.13, N 8.63. For X-ray data of
compound 12a, see Supporting Information.
(3-Fluoro-2-pyrazinyl)[(2S,4S,5R)-5-hydroxy-2-phenyl-1,3-dioxan-
4-yl]methanone (12b): HF·pyridine (ca. 70:30 w/w; 200 μL) was
added to a solution of TBS-protected alcohol 11b (60.2 mg,
144 μmol) in anhydrous THF (4 mL) and anhydrous pyridine
(400 μL) at 0 °C. The mixture was stirred for 18 h at room temp.,
From 8: A solution of 2,2,6,6-tetramethylpiperidine (1.79 mL,
1.48 g, 10.5 mmol) in anhydrous THF (30 mL) was cooled to
–30 °C, and nBuLi (2.4 m in hexanes; 3.42 mL, 8.25 mmol) was and then saturated aqueous NaHCO3 (8 mL) was added until the
added over 5 min. The mixture was stirred at 0 °C for 30 min, then evolution of gas had ceased. The mixture was extracted with EtOAc
Eur. J. Org. Chem. 2014, 2231–2241
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2237

S. Hilken, F. Kaletta, A. Heinsch, J.-M. Neudörfl, A. Berkessel
FULL PAPER
(4ϫ 6 mL). The combined extracts were dried with MgSO4, fil-
tered, and evaporated under reduced pressure. The crude product
was purified by column chromatography (silica gel, cyclohexane/
EtOAc, 1:1) to give alcohol 12b (26.9 mg, 88.4 μmol, 61%) as an
orange oil. Rf = 0.40 (cyclohexane/EtOAc, 1:2), m.p. 41–44 °C. H
NMR (300 MHz, CDCl3): δ = 3.23 (br. s, 1 H, OH), 4.19–4.31 (m,
ethanol (2.3 mL) was cooled to 0 °C, and sodium borohydride
(143 mg, 3.79 mmol) was added with stirring. After 2 h at this tem-
perature, saturated aqueous NaHCO3 (15 mL) and water (10 mL)
were added. The mixture was extracted with CH2Cl2 (6ϫ 20 mL).
The combined organic extracts were dried with MgSO4, filtered,
and evaporated under reduced pressure. The crude product was
1
3 H), 5.70–5.81 (m, 2 H), 7.33–7.45 (m, 3 H, Ar-H), 7.49–7.60 (m, purified by column chromatography (silica gel, cyclohexane/
2 H, Ar-H), 8.39–8.49 (m, 1 H, Pyr-H), 8.51–8.59 (m, 1 H, Pyr-H) EtOAc, 1:2) to give alcohol 14b (417 mg, 1.36 mmol, 43%) and
ppm. 13C NMR (75 MHz, CDCl3): δ = 65.0 [C(H)OH], 72.2 (CH2), epimeric alcohol 15b (322 mg, 1.05 mmol, 33%) as colourless sol-
82.3 (CH), 101.3 [C(H)Ph], 126.1 (Ar-CH), 128.3 (Ar-CH), 129.2
ids.
(Ar-CH), 135.5 (JC,F = 18.6 Hz, Pyr-C), 136.9 (Ar-C), 140.6 (JC,F
14b: Rf = 0.40 (cyclohexane/EtOAc, 1:3), m.p. 116–118 °C. 1H
NMR (300 MHz, CDCl3): δ = 3.54–3.71 (m, 2 H), 3.94 (d, J =
4.0 Hz, 1 H, OH), 4.04 (d, J = 12.1 Hz, 1 H), 4.20 (d, J = 12.1 Hz,
1 H), 4.37–4.45 (m, 1 H), 5.33–5.43 (m, 1 H), 5.60 (s, 1 H), 7.29–
7.38 (m, 3 H, Ar-H), 7.39–7.49 (m, 2 H, Ar-H), 8.13–8.25 (m, 1 H,
Pyr-H), 8.42–8.54 (m, 1 H, Pyr-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 64.6 [C(H)OH], 69.4 [JC,F = 5.6 Hz, C(H)OH], 72.3
(CH2), 79.8 (CH), 101.5 [C(H)Ph], 125.9 (Ar-CH), 128.2 (Ar-CH),
129.1 (Ar-CH), 137.3 (Ar-C), 141.0 (JC,F = 4.8 Hz, Pyr-CH), 141.3
(JC,F = 8.6 Hz, Pyr-CH), 142.1 (JC,F = 28.5 Hz, Pyr-C), 158.1 (JC,F
= 255.4 Hz, Pyr-C) ppm. 19F NMR (282 MHz, CDCl3): δ =
= 5.4 Hz, Pyr-CH), 145.8 (JC,F = 9.9 Hz, Pyr-CH), 158.4 (JC,F
=
269.3 Hz, Pyr-C), 190.7 (JC,F = 6.9 Hz, C=O) ppm. IR (ATR): ν
˜
= 3417 (br. s), 2974, 2936, 2876, 1722, 1537, 1450, 1418, 1396, 1260,
1206, 1173, 1094 (s), 1078 (s), 1020 (s), 870, 735 (s), 731 (s), 698 (s)
cm–1. HRMS (ESI): calcd. for C15H13FN2O4Na [M + Na]+
327.0752; found 327.0754.
(2S,4R,5R)-4-[(Ξ)-(3-Chloropyrazin-2-yl)hydroxymethyl]-2-phenyl-
1,3-dioxan-5-ol (14a and 15a): A solution of alcohol 12a (1.70 g,
5.30 mmol) in a mixture of anhydrous THF (40 mL) and anhydrous
ethanol (4 mL) was cooled to 0 °C, and sodium borohydride
(261 mg, 6.89 mmol) was added with stirring. After 60 min at this
temperature, saturated aqueous NaHCO3 (30 mL) was added. The
mixture was extracted with CH2Cl2 (6ϫ 30 mL). The combined
organic extracts were dried with MgSO4, filtered, and evaporated
under reduced pressure. The crude product was purified by column
chromatography (silica gel, cyclohexane/EtOAc, 2:3 to 0:1) to give
alcohol 14a (633 mg, 1.96 mmol, 37%) and epimeric alcohol 15a
(831 mg, 2.57 mmol, 49%) as colourless solids.
–77.9 ppm. IR (ATR): ν = 3281 (br. s), 1451, 1400, 1360, 1277,
˜
1242, 1153, 1063 (s), 1028 (s), 853, 820, 745 (s), 702 (s) cm–1
.
HRMS (ESI): calcd. for C15H15FN2O4Na [M + Na]+ 329.0908;
found 329.0909. For X-ray data of compound 14b, see Supporting
Information.
15b: Rf = 0.30 (cyclohexane/EtOAc, 1:3), m.p. 148 °C. [α]2D0
=
1
+108.7 (c = 0.96, CH2Cl2). H NMR (300 MHz, CDCl3): δ = 3.32
(d, J = 10.9 Hz, 1 H, OH), 3.94 (d, J = 7.9 Hz, 1 H, OH), 3.98–
4.15 (m, 3 H), 4.29 (d, J = 11.9 Hz, 1 H), 5.33–5.48 (m, 2 H), 7.27–
7.38 (m, 5 H, Ar-H), 8.08–8.22 (m, 1 H, Pyr-H), 8.40–8.55 (m, 1
H, Pyr-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 63.0 [C(H)OH],
66.1 [JC,F = 5.3 Hz, C(H)OH], 72.4 (CH2), 82.0 (CH), 100.9
[C(H)Ph], 125.6 (Ar-CH), 128.1 (Ar-CH), 128.9 (Ar-CH), 137.1
(Ar-C), 140.9 (JC,F = 8.8 Hz, Pyr-CH), 141.0 (JC,F = 5.0 Hz, Pyr-
CH), 144.7 (JC,F = 30.0 Hz, Pyr-C), 158.4 (JC,F = 255.2 Hz, Pyr-
C) ppm. 19F NMR (282 MHz, CDCl3): δ = –78.0 ppm. IR (ATR):
Data for 14a: Rf = 0.40 (cyclohexane/EtOAc, 1:2), m.p. 109–111 °C.
1
[α]2D0 = +80.7 (c = 0.83, CH2Cl2). H NMR (300 MHz, CDCl3): δ
= 3.69–3.79 (m, 1 H), 3.93 (d, J = 6.1 Hz, 1 H, OH), 4.04 (d, J =
12.0 Hz, 1 H), 4.13 (d, J = 5.5 Hz, 1 H, OH), 4.22 (d, J = 12.0 Hz,
1 H), 4.41–4.49 (m, 1 H), 5.43–5.52 (m, 1 H), 5.56 (s, 1 H), 7.28–
7.36 (m, 3 H, Ar-H), 7.36–7.45 (m, 2 H, Ar-H), 8.32 (d, J = 2.1 Hz,
1 H, Pyr-H), 8.50 (d, J = 2.1 Hz, 1 H, Pyr-H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 65.2 [C(H)OH], 70.9 [C(H)OH], 72.4 (CH2),
78.8 (CH), 101.4 [C(H)Ph], 126.0 (Ar-CH), 128.2 (Ar-CH), 129.1
(Ar-CH), 137.4 (Ar-C), 141.6 (Pyr-CH), 143.6 (Pyr-CH), 148.1
ν = 3165 (br. s), 2922, 1452, 1416, 1393, 1269, 1233, 1142, 1096 (s),
˜
1020 (s), 980, 854, 754 (s), 700 (s) cm–1. HRMS (ESI): calcd. for
C
1 5 H1 5 FN2 O4 Na [M + Na]+ 329.0908; found 329.0909.
(Pyr-C), 151.5 (Pyr-C) ppm. IR (ATR): ν = 3381 (br. s), 3279 (br.
˜
C15H15FN2O4 (306.29): calcd. C 58.82, H 4.94, N 9.15; found C
58.86, H 4.97, N 9.15. For X-ray data of compound 15b, see Sup-
porting Information.
s), 2968, 2928, 1452, 1389, 1233, 1144, 1098, 1051 (s), 1028 (s),
1001 (s), 866, 760, 700, 656 cm–1. HRMS (ESI): calcd. for
C15H15ClN2O4Na [M + Na]+ 345.0613; found 345.0614. For X-ray
data of compound 14a, see Supporting Information.
(2S,4R,10S,10aR)-2-Phenyl-4,4a,10,10a-tetrahydro-2H-[1,3]di-
oxino[4Ј,5Ј:5,6]pyrano[2,3-b]pyrazin-10-ol (16)
Data for 15a: Rf = 0.25 (cyclohexane/EtOAc, 1:2), m.p. 118–120 °C.
1
[α]2D0 = +37.4 (c = 1.19, CH2Cl2). H NMR (300 MHz, CDCl3): δ
From 14a: Potassium tert-butoxide (192 mg, 1.72 mmol) was added
to a solution of diol 14a (615 mg, 1.91 mmol) in anhydrous THF
(220 mL) with stirring at –20 °C. The reaction mixture was warmed
to room temperature over 18 h, and then saturated aqueous
NaHCO3 (150 mL) was added. The organic layer was separated,
and the aqueous layer was extracted with CH2Cl2 (3ϫ 150 mL).
The combined organic layers were dried with MgSO4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography (silica gel, cyclohexane/EtOAc, 1:2 to
0:1) to give cyclized alcohol 16 (129 mg, 451 μmol, 26 %) as a
colourless oil, together with recovered starting material 14a
(133 mg, 412 μmol, 22%).
= 3.28 (d, J = 10.2 Hz, 1 H, OH), 3.81 (d, J = 8.6 Hz, 1 H, OH),
3.96–4.14 (m, 3 H), 4.29 (d, J = 12.0 Hz, 1 H), 5.41 (s, 1 H), 5.51–
5.61 (m, 1 H), 7.27–7.38 (m, 5 H, Ar-H), 8.32 (d, J = 2.4 Hz, 1 H,
Pyr-H), 8.50 (d, J = 2.4 Hz, 1 H, Pyr-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 63.1 [C(H)OH], 67.8 [C(H)OH], 72.5 (CH2), 82.5
(CH), 101.1 [C(H)Ph], 125.7 (Ar-CH), 128.2 (Ar-CH), 129.0 (Ar-
CH), 137.2 (Ar-C), 142.0 (Pyr-CH), 143.2 (Pyr-CH), 149.2 (Pyr-
C), 154.1 (Pyr-C) ppm. IR (ATR): ν = 3364 (br. s), 2957, 2924,
˜
1450, 1379, 1356, 1153, 1086 (s), 1061 (s), 1051 (s), 1001 (s), 962,
760, 746, 704 cm–1. HRMS (ESI): calcd. for C15H15ClN2O4Na [M
+ Na]+ 345.0613; found 345.0614. C15H15ClN2O4 (322.75): calcd.
C 55.82, H 4.68, N 8.68; found C 55.87, H 4.79, N 8.44. For X-
ray data of compound 15a, see Supporting Information.
From 14b: Potassium tert-butoxide (59.0 mg, 526 μmol) was added
to a solution of diol 14b (179 mg, 584 μmol) in anhydrous THF
(80 mL) at –20 °C. The reaction mixture was stirred at this tem-
perature for 4 h, and then saturated aqueous NaHCO3 (80 mL) was
added. The mixture was extracted with CH2Cl2 (3ϫ 80 mL), the
(2S,4R,5R)-4-[(Ξ)-(3-Fluoropyrazin-2-yl)hydroxymethyl]-2-phenyl-
1,3-dioxan-5-ol (14b and 15b): A solution of alcohol 12b (960 mg,
3.16 mmol) in a mixture of anhydrous THF (23 mL) and anhydrous
2238
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2231–2241

Oxidation-Stable Analogue of Cyclic Pyranopterin Monophosphate
combined organic layers were dried with MgSO4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography (silica gel, cyclohexane/EtOAc, 1:2) to
give cyclized alcohol 16 (42.9 mg, 150 μmol, 29%) as a colourless
oil, together with recovered starting material 14b (36.8 mg,
120 μmol, 21%). Rf = 0.30 (cyclohexane/EtOAc, 1:3). [α]2D0 = +77.6
organic layer was separated, and the aqueous layer was extracted
with CH2Cl2 (3ϫ 8 mL). The combined organic layers were dried
with MgSO4, filtered, and evaporated under reduced pressure. The
crude product was purified by column chromatography (silica gel,
EtOAc) to give ketone 13 (100 mg, 352 μmol, 95%) as a colourless
solid.
1
(c = 0.48, CH2Cl2). H NMR (300 MHz, CDCl3): δ = 4.24 (dd, J
From 17: Dess–Martin periodinane (457 mg, 1.08 mmol) was added
to a stirred solution of alcohol 17 (257 mg, 898 μmol) in anhydrous
CH2Cl2 (27 mL) at 0 °C. The reaction mixture was warmed to
room temperature over 7.5 h, and then saturated aqueous NaHCO3
(10 mL) and saturated aqueous Na2S2O3 (10 mL) were added. The
organic layer was separated, and the aqueous layer was extracted
with CH2Cl2 (3ϫ 20 mL). The combined organic layers were dried
with MgSO4, filtered, and evaporated under reduced pressure. The
crude product was purified by column chromatography (silica gel,
EtOAc) to give ketone 13 (189 mg, 666 μmol, 74%) as a colourless
solid. Rf = 0.50 (EtOAc), m.p. Ͼ160 °C (decomp.). [α]2D0 = –123.0
= 12.7, 1.0 Hz, 1 H), 4.45–4.49 (m, 1 H), 4.49–4.54 (m, 1 H,
CHOH), 4.68 (dd, J = 12.7, 1.4 Hz, 1 H), 4.83–4.93 (m, 1 H), 4.99–
5.18 (m, 1 H, OH), 5.70 (s, 1 H), 7.27–7.35 (m, 3 H, Ar-H), 7.35–
7.44 (m, 2 H, Ar-H), 8.05 (d, J = 2.5 Hz, 1 H, Pyr-H), 8.23 (d, J
= 2.5 Hz, 1 H, Pyr-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 67.0
[C(H)OH], 67.4 (CH), 68.9 (CH2), 74.3 (CH), 101.2 [C(H)Ph],
126.2 (Ar-CH), 128.2 (Ar-CH), 129.3 (Ar-CH), 137.0 (Ar-C), 137.2
(Pyr-CH), 137.7 (Pyr-C), 143.7 (Pyr-CH), 157.0 (Pyr-C) ppm. IR
(ATR): ν = 3261 (br. s), 2924, 1545, 1418, 1351, 1277, 1165, 1140,
˜
1103 (s), 1045, 995 (s), 966, 910, 806, 731, 698 cm–1. HRMS (EI):
calcd. for C15H14N2O4 [M]+ 286.095; found 286.095.
1
(c = 0.84, CH2Cl2). H NMR (300 MHz, CDCl3): δ = 4.31 (dd, J
(2S,4R,10R,10aR)-2-Phenyl-4,4a,10,10a-tetrahydro-2H-[1,3]di-
oxino[4Ј,5Ј:5,6]pyrano[2,3-b]pyrazin-10-ol (17)
= 13.1, 1.1 Hz, 1 H), 4.53–4.61 (m, 1 H), 4.68 (d, J = 1.1 Hz, 1 H),
4.73 (dd, J = 13.1, 1.5 Hz, 1 H), 5.76 (s, 1 H), 7.28–7.38 (m, 3 H,
Ar-H), 7.41–7.52 (m, 2 H, Ar-H), 8.51 (d, J = 2.0 Hz, 1 H, Pyr-
H), 8.53 (d, J = 2.0 Hz, 1 H) ppm. 13C NMR (75 MHz, CDCl3): δ
= 68.0 (CH2), 71.3 (CH), 76.6 (CH), 101.1 [C(H)Ph], 126.1 (Ar-
CH), 128.3 (Ar-CH), 129.5 (Ar-CH), 130.4 (Pyr-C), 136.4 (Ar-C),
141.1 (Pyr-CH), 148.9 (Pyr-CH), 163.0 (Py-C), 184.6 (C=O) ppm.
From 15a: Potassium tert-butoxide (357 mg, 3.18 mmol) was added
to a solution of diol 15a (1.14 g, 3.54 mmol) in anhydrous THF
(350 mL) with stirring at –20 °C. The reaction mixture was warmed
to room temperature over 18 h, and then saturated aqueous
NaHCO3 (200 mL) was added. The organic layer was separated,
and the aqueous layer was extracted with CH2Cl2 (3ϫ 200 mL).
The combined organic layers were dried with MgSO4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography (silica gel, cyclohexane/EtOAc, 1:3 to
0:1) to give cyclized alcohol 17 (286 mg, 1.00 mmol, 31%) as a pale
yellow solid, together with recovered starting material 15a (311 mg,
946 μmol, 27%).
IR (ATR): ν = 1717 (s), 1558, 1543, 1470, 1321, 1271, 1192, 1134,
˜
1096 (s), 1043, 966, 912, 733, 698 cm–1. HRMS (ESI): calcd. for
C15H12N2O4Na [M + Na]+ 307.0689; found 307.0691. For X-ray
data of compound 13, see Supporting Information.
(6R,7S)-6-(Hydroxymethyl)-8,8-dimethoxy-7,8-dihydro-6H-pyrano-
[2,3-b]pyrazin-7-ol (18): Ketone 13 (12.6 mg, 44.3 μmol) was dis-
solved in a mixture of MeOH (800 μL) and CHCl3 (400 μL), and
iodine (4.5 mg, 17.7 μmol) was added. The solution was stirred at
45 °C for 27 h with the exclusion of light. Repeated purification
by column chromatography (silica gel, CHCl3/MeOH, 20:1) gave
dimethyl acetal diol 18 (7.0 mg, 28.9 μmol, 65%) as a colourless
From 15b: Potassium tert-butoxide (83.1 mg, 740 μmol) was added
to a solution of diol 15b (252 mg, 823 μmol) in anhydrous THF
(110 mL) at –20 °C. The reaction mixture was stirred at this tem-
perature for 4 h, and then saturated aqueous NaHCO3 (100 mL)
was added. The mixture was extracted with CH2Cl2 (3ϫ 100 mL),
the combined organic layers were dried with MgSO4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography (silica gel, cyclohexane/EtOAc, 1:3) to
give cyclized alcohol 17 (47.9 mg, 167 μmol, 23%) as a colourless
solid, together with recovered starting material 15b (40.0 mg,
131 μmol, 16%). Rf = 0.20 (cyclohexane/EtOAc, 1:3), m.p. 121–
125 °C. [α]2D0 = +59.6 (c = 0.61, CH2Cl2). 1H NMR (300 MHz,
CDCl3): δ = 4.08–4.17 (m, 1 H, OH), 4.20 (dd, J = 12.6, 1.0 Hz, 1
H), 4.38–4.47 (m, 1 H), 4.60 (dd, J = 12.6, 1.6 Hz, 1 H), 4.64–4.71
(m, 1 H), 4.96–5.09 (m, 1 H, CHOH), 5.71 (s, 1 H), 7.24–7.41 (m,
5 H, Ar-H), 7.99–8.14 (m, 2 H, Pyr-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 67.3 [C(H)OH], 69.1 (CH2), 69.2 (CH), 72.8 (CH),
101.3 [C(H)Ph], 126.4 (Ar-CH), 128.2 (Ar-CH), 129.3 (Ar-CH),
136.9 (Ar-C), 137.2 (Pyr-CH), 138.4 (Pyr-C), 142.2 (Pyr-CH), 156.3
1
oil. Rf = 0.45 (CHCl3/MeOH, 9:1). H NMR (600 MHz, [D6]acet-
one): δ = 3.25 (s, 3 H, OCH3), 3.46 (s, 3 H, OCH3), 3.98–4.07 (m,
2 H, CH2), 4.32–4.37 (m, 2 H), 4.46 (d, J = 3.9 Hz, 1 H, CHOH),
4.51–4.55 (m, 1 H), 8.21 (d, J = 2.4 Hz, 1 H, Pyr-H), 8.25 (d, J =
2.4 Hz, 1 H, Pyr-H) ppm. 13C NMR (150 MHz, [D6]acetone): δ =
48.9 (OCH3), 49.9 (OCH3), 62.1 (CH2), 65.5 [C(H)OH], 78.1 (CH),
97.2 [C(OMe)2], 137.5 (Pyr-CH), 137.7 (Pyr-C), 143.6 (Pyr-CH),
158.0 (Pyr-C) ppm. IR (ATR): ν = 3360 (br. s), 2945, 1555, 1418
˜
(s), 1337, 1283, 1211, 1171, 1132, 1101 (s), 1076 (s), 1053 (s), 978,
758 cm–1. HRMS (ESI): calcd. for C10H14N2O5Na [M + Na]+
265.0795; found 265.0796.
(2R,4aR,10aS)-2-Chloro-10,10-dimethoxy-4,4a,10,10a-tetrahydro-
1,3,2-dioxaphosphinino[4Ј,5Ј:5,6]pyrano[2,3-b]pyrazine 2-Oxide (19):
A solution of anhydrous triethylamine (32.6 μL, 23.7 mg,
234 μmol) and phosphorus oxychloride (10.9 μL, 17.9 mg,
117 μmol) in anhydrous CH2Cl2 (4 mL) was added at 0 °C to a
solution of dimethyl acetal diol 18 (27.0 mg, 111 μmol) in anhy-
drous CH2Cl2 (4 mL) over 5 min. The mixture was stirred for 3 h,
then the volatile components were evaporated under reduced pres-
sure. The crude product was purified by column chromatography
(silica gel, EtOAc) to give diastereomerically pure chlorophosphate
19 (25.6 mg, 79.3 μmol, 71 %) as a colourless solid. Rf = 0.30
(Pyr-C) ppm. IR (ATR): ν = 3273 (br. s), 2914, 1545, 1458, 1416
˜
(s), 1366, 1337, 1281, 1157, 1099 (s), 1038, 1024, 1011, 964, 912,
829, 731 (s), 698 cm–1. HRMS (EI): calcd. for C15H14N2O4 [M]+
286.095; found 286.095.
(6R,7S)-7-Hydroxy-6-(hydroxymethyl)-6,7-dihydro-8H-pyrano-
[2,3-b]pyrazin-8-one (13)
From 16: Dess–Martin periodinane (189 mg, 444 μmol) was added
to a stirred solution of alcohol 16 (106 mg, 370 μmol) in anhydrous
1
(EtOAc), m.p. Ͼ130 °C (decomp.). H NMR (500 MHz, CDCl3):
CH2Cl2 (11 mL) at 0 °C. The reaction mixture was warmed to δ = 3.27 (s, 3 H, OCH3), 3.52 (s, 3 H, OCH3), 4.72–4.77 (m, 1 H),
room temperature over 7.5 h, and then saturated aqueous NaHCO3
(4 mL) and saturated aqueous Na2S2O3 (4 mL) were added. The
4.78–4.81 (m, 1 H), 4.89–5.00 (m, 1 H), 5.01–5.05 (m, 1 H), 8.33
(d, J = 2.4 Hz, 1 H, Pyr-H), 8.41 (d, J = 2.4 Hz, 1 H, Pyr-H) ppm.
Eur. J. Org. Chem. 2014, 2231–2241
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2239

S. Hilken, F. Kaletta, A. Heinsch, J.-M. Neudörfl, A. Berkessel
FULL PAPER
13C NMR (125 MHz, CDCl3): δ = 49.4 (OCH3), 50.0 (OCH3), 68.0 paper. These data can be obtained free of charge from The Cam-
(JC,P = 6.2 Hz, CH), 71.7 (JC,P = 8.8 Hz, CH2), 73.8 (JC,P = 7.6 Hz, bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
CH), 94.1 [JC,P = 11.4 Hz, C(OMe)2], 133.5 (Pyr-C), 138.7 (Pyr- data_request/cif
CH), 144.1 (Pyr-CH), 155.4 (Pyr-C) ppm. 31P NMR (203 MHz,
Supporting Information (see footnote on the first page of this arti-
CDCl ): δ = –5.6 ppm. IR (ATR): ν = 2974, 2947, 1555, 1412 (s),
˜
3
cle): DFT calculations on the conformations of compound 12b;
NMR spectra (1H, 13C, 31P) of compounds 12b, 13, 14a, 15a, 14b,
15b, 16, 17, 18, 19, 20, and 21; X-ray data of compounds 7, 8, 9,
11b, 12a, 13, 14a, 15a, 15b, 19, and 20.
1337, 1315, 1289, 1269, 1171, 1128, 1096, 1061 (s), 988, 874 cm–1.
HRMS (ESI): calcd. for C10H12ClN2O6PNa [M + Na]+ 345.0014;
found 345.0014. For X-ray data of compound 19, see Supporting
Information.
(4aR,10aS)-10,10-Dimethoxy-2-oxo-4,4a,10,10a-tetrahydro-
1,3,2λ5-dioxaphosphinino[4Ј,5Ј:5,6]pyrano[2,3-b]pyrazin-2-(2H)ol
Mono(2H)hydrochloride (20): In an NMR tube, chlorophosphate 19
(23.1 mg, 71.6 μmol) was dissolved in [D6]acetone (500 μL), and
then D2O (50 μL) was added dropwise. After mixing, the solution
was kept at room temperature for 24 h. The solution was then
transferred to a round-bottomed flask and cooled to –50 °C, and
the solvent was evaporated under vacuum (ca. 10–3 mbar) with slow
warming to room temperature. D2O (1.5 mL) was added to the
resulting residue, the mixture was filtered, and the filtrate was
freeze dried to give acid 20 (17.3 mg, 50.5 μmol, 71%) as a pale
Acknowledgments
F. K. and A. H. gratefully acknowledge doctoral fellowships from
the Fonds der Chemischen Industrie, Frankfurt, Germany. The au-
thors thank Silvia Elfert for the DFT calculations on the confor-
mational space of compounds 12a and 12b.
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yellow solid, m.p. Ͼ75 °C (decomp.). H NMR (300 MHz, D2O):
δ = 3.19 (s, 3 H, OCH3), 3.50 (s, 3 H, OCH3), 4.50–4.65 (m, 2 H,
CH2), 4.73–4.78 (m, 1 H), 4.97–5.05 (m, 1 H), 8.32 (d, J = 2.6 Hz,
1 H, Pyr-H), 8.34 (d, J = 2.6 Hz, 1 H, Pyr-H) ppm. 13C NMR
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=
5.8 Hz, CH2), 70.1 (JC,P = 4.0 Hz, CH), 70.6 (JC,P = 4.4 Hz, CH),
94.6 [JC,P = 9.8 Hz, C(OMe)2], 133.6 (Pyr-C), 137.3 (Pyr-CH),
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= –5.2 ppm. IR (ATR): ν = 2947, 1555, 1458, 1414, 1300, 1269,
˜
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Sodium (4aR,10aS)-10,10-Dihydroxy-2-oxo-4,4a,10,10a-tetrahydro-
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= 0.1 ppm). Assuming a purity of 70 %, a yield of 8.1 mg
(27.2 μmol, ca. 60%) was calculated. 1H NMR (600 MHz, D2O): δ
= 4.49–4.58 (m, 2 H, CH2), 4.62–4.64 (m, 1 H), 4.75–4.77 (m, 1
H), 8.32 (d, J = 2.6 Hz, 1 H, Pyr-H), 8.34 (d, J = 2.6 Hz, 1 H, Pyr-
H) (OH-Protons not detectable) ppm. 13C NMR (150 MHz, D2O):
δ = 68.1 (JC,P = 6.0 Hz, CH2), 70.1 (JC,P = 4.5 Hz, CH), 75.5 (JC,P
= 5.2 Hz, CH), 88.8 [JC,P = 10.0 Hz, C(OH)2], 137.2 (Pyr-C), 138.3
(Pyr-CH), 143.8 (Pyr-CH), 155.6 (Pyr-C) ppm. 31P NMR
(244 MHz, D2O): δ = –5.0 ppm (also a signal at 0.1 ppm). HRMS
(ESI): calcd. for C8H8N2O7P [M – Na] 275.0064; found 275.0071;
calcd. for C8H6N2O6P [M – H2O – Na] 256.9958; found 256.9966.
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2240
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2231–2241

Oxidation-Stable Analogue of Cyclic Pyranopterin Monophosphate
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