Stereochemistry of fully acetylated tetrahydropterins and tetrahydroquinoxalines

Article abstract of DOI:10.3987/COM-05-10544

Completely acetylated derivatives of naturally occurring tetrahydro-D-monapterin, (6R)-2-acetamido-6-[(1R,2R)-1,2,3-triacetoxypropyl]-5,8-diacetyl-5,6,7,8-tetrahydropteridin-4(3H)-one, show plus and minus CD signs at λ 280 and 240 nm, respectively. Simila

Full text of DOI:10.3987/COM-05-10544

HETEROCYCLES, Vol. 65, No. 12, 2005
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Received, 24th August, 2005, Accepted, 7th October, 2005, Published online, 7th October, 2005
STEREOCHEMISTRY OF FULLY ACETYLATED
TETRAHYDROPTERINS AND TETRAHYDROQUINOXALINES
Ning Chen,b) Kazuhisa Ikemoto,c) Shingo Komatsu,d) and Shizuaki Murata*a)
a)Graduate School of Environmental Studies, Nagoya University, Chikusa
Nagoya 464-8601 Japan, murata@urban.env.nagoya-u.ac.jp. b)Department of
Physics, Kyoto University, Sakyo, Kyoto 606-8502 Japan. c)School of Medicine,
Fujita Health University, Toyoake 470-1192 Japan. d)Institute for Molecular
Science, Okazaki 444-8787 Japan.
Abstract – Completely acetylated derivatives of naturally occurring
tetrahydro-D-monapterin, (6R)-2-acetamido-6-[(1R,2R)-1,2,3-triacetoxypropyl]-
5,8-diacetyl-5,6,7,8-tetrahydropteridin-4(3H)-one, show plus and minus CD signs
at λ 280 and 240 nm, respectively. Similar CD spectra are obtained on
(2S)-1,4-diacetyl-1,2,3,4-tetrahydro-2-isopropylquinoxaline
and
(2S)-1,4-
diacetyl-1,2,3,4-tetrahydro-2-isobutylquinoxaline but not on (2S)-1,4-
diacetyl-1,2,3,4-tetrahydro-2-methylquinoxaline nor on the 2-ethyl derivative.
The similarity of their CD spectra is represented the same stereogenic structure
based on the boat conformation confirmed by X-Ray crystallographic analyses.
INTRODUCTION
Tetrahydropteridines are biologically important nitrogen heterocycles, since some of them are operating
in various metabolic systems. For example, biopterin cofactor, i.e. (6R)-tetrahydrobiopterin, is known to
work in biosynthesis of nitrogen oxide (NO) as well as metabolic transformation of aromatic amino acids
2
to cathecholamine.1,
Folic acids, like (6R)-tetrahydrofolate, belonging to the other series of
biologically active pteridines play essential roles in biosyntheses of nucleosides and methionine.1
Recently different (6R)-tetrahydropterin (1a and 1b) have been isolated from microorganisms known not
to produce biopterin cofactor and are supposed to replace biopterin cofactor in the enzymatic reactions.3, 4
The importance of such tetrahydropteridine derivatives and the pteridine-depending metabolisms for
controlling homeostasis would increase in chemical biology.
Since such biologically active
tetrahydropteridines generally adopt the 6R configuration, the stereochemistry of synthetic as well as

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naturally occurring pteridine derivatives is one of most fundamental subjects in biochemical and
pharmaceutical studies.5-10 A facile CD spectroscopic technique has been employed for determination of
the absolute configuration, and, indeed, it is easy to distinguish the (6R)-tetrahydropteridine from the
(6S)-isomer in comparison with their CD signs at λmax 265 nm in acidic solutions, e.g. the 6R and 6S
isomers afford the minus and plus signs, respectively.11 The CD spectroscopic rule has been
rationalized by the molecular distortion based on a stable twisted-chair conformation of the
tetrahydropyrazine part.
On the contrary, the configuration of completely acetylated
(6R)-tetrahydropterins (2a and 2b) isolated from cell extracts of a protozoa and bacteria has not been
discussed based on the regularity of CD spectra due to different CD patterns.3, 4 Complete acetylation is
only one reliable technique to isolate and analyze a small amount of tetrahydropterin derivatives in
complex mixtures of biological samples. In order to understand the stereochemistry of such acetylated
tetrahydropteridine derivatives, CD spectra and X-Ray crystallographic analyses were investigated on
completely acylated and optically active tetrahydroquinoxaline derivatives which have been considered to
be model compounds of 2a and 2b.
COCH3
O
N
O
N
H
N
H
H
(CH3CO)2O
R
N
R
HN
H2N
HN
N
H
CH3CONH
N
COCH3
OH
OCOCH3
CH2OCOCH3
CH2OH
2a: R =
2b: R =
tetrahydro-D-monapterin (1a): R =
tetrahydro-L-monapterin (1b): R =
OH
OH
OCOCH3
OCOCH3
CH2OCOCH3
CH2OH
OH
OCOCH3
Scheme 1. Biologically Active Tetrahydropterins and Peracetylated Derivatives
RESULTS AND DISCUSSION
CD Spectra. The (6R)-hexaacetyltetrahydro-D-monapterin (2a) and (6R)-hexaacetyltetrahydro-L-
monapterin (2b) afforded the similar CD spectra illustrated in Figure 1, where + (λmax 322 nm), – (248
nm), + (223 nm), and – (216 nm) signs are recommended.
The similarity of the +/–/+/– figures
clearly indicated that the CD spectra were affected not by the absolute configuration of stereogenic
centers on the side chain but by the 6R chirality of the heterocyclic structure. Therefore, optically active
2-alkylated (2S)- and (2R)-tetrahydroquinoxaline derivatives, (S)- and (R)- 3 – 7, illustrated in Scheme 2

HETEROCYCLES, Vol. 65, No. 12, 2005
2919
were employable to CD studies as the model compounds, the 2S configuration of which means the same
stereogenic structure as (6R)-tetrahydropterin derivatives. CD spectra of methyl and ethyl substituted
derivatives, (R)-3 and (R)-4 in Figure 2, did not show a significant deflection in the whole wave length (λ
= 200 – 400 nm). Although their absolute configurations are same, the CD spectra look like those of an
enantiomeric pair. On the contrary, 5 and 6 with rather bulky groups (R = iPr and 2-methylpropyl,
respectively) indicated obvious CD curves illustrated in Figure 3 and 4, and the CD spectra of (S)-5 and
a
b
b
a
Figure 1. CD spectra of 2a (a) and 2b (b)
in CH3CN (C = 1.95 x 10-5 M).
H
N
H
H
H
O
F
R
O
N
R
NaHCO3
H2, 5% Pd-C
R
+
OH
COOH
NO2
N
H
NO2
NH2
CH3CO
H
H
H
N
R
LiAlH4
(CH3CO)2O-Py
N
R
N
N
H
COCH3
3: R = CH3
4: R = CH2CH3
5: R = CH(CH3)2
6: R = CH2CH(CH3)2
N
HCO
H
N COH
N
CH2CH(CH3)2
N
CHO
7
Scheme 2. Preparation of Optically Active Peracetylated Tetrahydroquinoxaline ((S)-3 – 7).

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(S)-6 (Figure 3 (b) and Figure 4, respectively) changing the +/–/+/– fashion at λ = 320 – 200 nm are
similar in each other. The similarity of the CD spectra of the (6R)-pterin (Figure 1) and
(2S)-quinoxaline derivatives (Figure 3 (a)) suggested the conformational similarity in these compounds.
Armarego and coworkers have reported based on a 1H NMR spectroscopic study that
(6R)-tetrahydrobiopterin tetraacetate preferred a half-chair conformation in which the bulky side chain
1
occupied an axial geometry.8 However, H and 13C NMR spectra of the tetrahydroquinoxaline
derivatives indicating peak broadening and missing due to conformational flexibility were not useful to
discuss their conformation. For example, only 7 signals were detected in a 13C NMR spectrum of (S)-5
at room temperature, while all 15 signals were recommended at –30 ºC.
a
b
a
b
a
b
Figure 2. CD spectra of (R)-3 (a) and
(R)-4 (b) in CH3CN (C =
1.95 x 10-5 M).
Figure 3. CD spectra of (R)- 5 (a) and
(S)-5(b) in CH3CN (C =
1.95 x 10-5 M).
Figure 4. CD spectra of (S)- 6 in CH3CN
(C = 1.95 x 10-5 M).
Figure 5. CD spectra of (S)-7 in CH3CN
(C = 1.95 x 10-5 M).

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X-Ray Crystallographic Analysis. Although Viscontini and coworkers determined the 6R absolute
configuration of biopterin cofactor based on the X-Ray crystallographic analysis of its peracetylated
derivative, they did not mention about the conformation and CD spectra.6, 7 Based on 2D NMR study
and X-Ray crystallographic analysis, one of us proved that the preferable conformation of 2a was the boat
form.12 If the stable boat conformation is recommended in 5 and 6, the similarity of their CD spectra
could be rationalized by the conformational similarity. The X-Ray crystallographic analysis was carried
out on (S)-5,13 and the molecular structure is illustrated in Figure 6. Here, it is obvious that the
tetrahydropyrazine moiety of (S)-5 adopts the typical boat conformation in which the N(1) acetyl group
(CH3CON(1)) and the isopropyl group are located in an anti geometry to avoid steric repulsion. Three
typical structures (8 – 10) of (S)-5 are illustrated in Figure 7. In the half-chair conformation (8) with the
equatorial isopropyl group (R), the existence of major steric interactions between R and CH3CON(1) can
be recommended. The half-chair conformation (9) is the similar structure as that was mentioned by
Armarego,8 and the quasi 1,3-diaxial relationship of R and CH3CON(4) is significant in this structure.
On the contrary, no significant interaction caused by R is recommended in the boat structure (10).
However the stability is mainly depending on the relative bulkiness of R and the acetyl group in 10.
Because of general disadvantages of the boat form 10 might become less favorable than the half-chair
forms, when the steric repulsion is smaller. The fact that CD spectra of (S)-7 (Figure 5) did not obey the
similarity to N-acetyl derivatives is rationalized by good stability of the half-chair conformation like 9 due
to the small N-formyl group. In the cases of tetrahydroquinoxaline derivatives with smaller R (3: CH3,
4: C2H5), the repulsion in 8 would be reduced very much, and the small interaction allowed the
CH3CON(1)
Figure 6. ORTEP drawing of (S)-5.

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HETEROCYCLES, Vol. 65, No. 12, 2005
H
R
R
H
H
N
H3COC
H
N
COCH3
H
H
N
R
N
H
H3COC
N
N
COCH3
H3COC
COCH3
H
H
8
9
10
Figure 7. Major conformations of diacetyltetrahydroquinoxaline.
rapid interconversion of 8 and 9. Based on the quadrant rule14 employed to the theoretical explanation
on the CD spectra of tetrahydropterins,11 the two half-chair conformations could afford opposite CD signs
in each other. The obscure CD spectra of 3 and 4 shown in Figure 2 might be caused by the
conformational flexibility.
EXPERIMENTAL
(R)-1,4-Diacetyl-1,2,3,4-tetrahydro-2-methylquinoxaline ((R)-3). A mixture of D-alanine (2.0 g, 22
mmol), o-fluoronitrobenzene (3.5 g 25 mmol), and NaHCO3 (9.0 g) in 50% aq. ethanol (180 mL) was
heated under reflux for 6 h. The mixture was concentrated on a rotary evaporater (20 mmHg, 50 °C) to
half of the volume, and the residue was washed by ether (30 mL x 2). The aqueous solution was
acidified (pH 1) with HCl, and crude N-(2-nitrophenyl)-D-alanine (3.3 g) was obtained as pale yellow
precipitates.
A mixture of the crude product (2.0 g, 9.5 mmol) and 5% Pd-C in 50% aq. ethanol (50
mL) was stirred vigorously at room temperature under atmospheric pressure of H2. Catalysts were
removed by filtration, and solvents were removed by evaporation. The resulting crude product (1.2 g)
was dissolved in anhydrous ether (100 mL), and to this was added LiAlH4 (2.5 g). The mixture was
heated under reflux for 5 h, and the reaction was quenched by addition of water (10 mL) and 3M aq.
NaOH (2.5 mL). Precipitates were filtered off, and the filtrate was concentrated. To the obtained oily
compound were added pyridine (8 mL) and acetic anhydride (24 mL), and the mixture was stirred at rt for
18 h. Then, the excess reagents were decomposed by the addition of methanol (30 mL), and solvents were
removed. Column chromatography on silica gel eluting with a mixture of toluene and ethyl acetate (7:3,
15, 16
v/v) gave pure (R)-3 (1.16 g).
Colorless needles (recrystallized from toluene), mp 138 – 140 °C;
1
TLC (silica gel, toluene:ethyl acetate = 1:5): Rf = 0.26; H NMR (CDCl3, 17 °C): δ 1.15 (d, 3H, J = 6.0
Hz, CH3), 2.18 (s, 3H, CH3CO), 2.22 (s, 3H, CH3CO), 2.80 (br s, 1H), 5.00 (br s, 2H), 7.24–7.30 (m, 4H,
Ar); 13C NMR (CDCl3, -30 ºC): δ 18.59 (CH3), 22.20 (CH3CO), 23.32 (CH3CO), 49.92 (C(3)), 52.38
(C(2)), 124.32 (Ar), 126.35 (Ar), 126.39 (Ar), 126.52 (Ar), 133.51 (Ar), 136.10 (Ar), 168.48 (CO),

HETEROCYCLES, Vol. 65, No. 12, 2005
2923
169.46 (CO); UV (CH3CN): λmax 228 (ε 27,300), 253 nm (ε 14,200). Anal. Calcd for C13H16N2O2: C,
67.22; H, 6.94; N, 12.06. Found: C, 67.08; H, 6.95; N, 11.93.
(R)-1,4-Dacetyl-2-ethyl-1,2,3,4-tetrahydroquinoxaline ((R)-4).
Pale brown oil TLC (silica gel,
1
toluene:ethyl acetate = 1:5): Rf = 0.29; H NMR (CDCl3, 17 °C): δ 0.85 (t, 3H, J = 7.6 Hz, CH3),
1.36–1.50 (m, 2H), 2.18(s, 3H, CH3CO), 2.22 (s, 3H, CH3CO), 3.02 (br s,1H), 4.93 (br s, 2H), 7.25 (br s,
13
4H, Ar); C NMR (CDCl3, -30 ºC): δ 9.78 (CH3), 22.54 (CH2), 23.13 (CH3CO), 26.27 (CH3CO), 48.95
(C(3)), 55.59 (C(2)), 124.34 (Ar), 126.35 (Ar), 126.41 (Ar), 126.54 (Ar), 133.54 (Ar), 135.81 (Ar),
168.96 (CO), 169.42 (CO).
(S)-1,4-Diacetyl-1,2,3,4-tetrahydro-2-isopropylquinoxaline ((S)-5). Colorless prisms (recrystallized
from toluene), mp 133 – 134 °C; TLC (silica gel, toluene:ethyl acetate = 1:5): Rf = 0.36; 1H NMR (CDCl3,
17 °C): δ 0.85 (d, 3H, J = 6.9 Hz, CH3), 0.87 (d, 3H, J = 6.9 Hz, CH3), 1.68 (m, 1H), 2.15 (s, 3H,
13
CH3CO), 2.21 (s, 3H, CH3CO), 3.19 (br s, 1H), 4.80 (br s, 2H), 7.27 (s, 4H, Ar); C NMR (CDCl3, -30
ºC): δ 17.04 (CH3), 19.12 (CH3), 22.52 (CH3CO), 23.04 (CH3CO), 30.70 (CH), 46.12 (C(3)), 59.26 (C(2)),
124.27 (Ar), 126.37 (Ar), 126.43 (Ar), 126.65 (Ar), 134.26 (Ar), 136.00 (Ar), 169.07 (CO), 169.60 (CO);
UV (CH3CN): λmax 228 (ε 21,200), 253 nm (ε 10,700). Anal. Calcd for C15H20N2O2: C, 69.20; H, 7.74; N,
10.76. Found: C, 69.24; H, 7.87; N, 10.62.
(R)-1,4-Diacetyl-1,2,3,4-tetrahydo-2-isopropyloquinoxaline ((R)-5). Colorless prisms (recrystalized
from toluene), mp 134 – 135 °C. Anal. Calcd for C15H20N2O2: C, 69.20; H, 7.74; N, 10.76. Found: C,
69.29; H, 7.86; N, 10.65.
(S)-1,4-Diacetyl-1,2,3,4-tetrahydro-2-(2-methylpropyl)quinoxaline ((S)-6). Colorless oil, TLC (silica
1
gel, toluene:ethyl acetate = 1:5): Rf = 0.41; H NMR (CDCl3, 17 °C): δ 0.86 (d, 3H, J = 6.8 Hz, CH3),
0.93 (d, 3H, J = 6.3 Hz, CH3), 1.06–1.23 (m, 1H), 1.29–1.36 (m, 1H), 1.46–1.56 (m, 1H), 2.16 (s, 3H,
CH3CO), 2.22 (s, 3H, CH3CO), 3.05 (br s, 1H), 4.71 (br s, 1H), 5.14 (br s, 1H), 7.21–7.29 (br s, 4H, Ar);
13C NMR (CDCl3, -30 ºC): δ 22.19 (CH3), 22.64 (CH3), 22.98 (CH3CO), 23.82 (CH3CO), 24.51 (CH2),
42.38 (CH), 49.65 (C(3)), 51.89 (C(2)), 124.32 (Ar), 126.28 (Ar), 126.49 (Ar), 126.66 (Ar), 133.16 (Ar),
135.68 (Ar), 169.05 (CO), 169.08 (CO); UV (CH3CN): λmax 228 (ε 20,800), 254 nm (ε 10,900).
(S)-1,4-Diformyl-1,2,3,4-tetrahydro-2-(2-methylpropyl)quinoxaline (7). Colorless oil, TLC (silica gel,
toluene:ethyl acetate = 1:5): Rf = 0.72; 1H NMR: δ (CDCl3, 17 °C): 0.89 (d, 3H, J = 6.8 Hz, CH3), 0.96 (d,
3H, J = 6.8 Hz, CH3), 1.16–1.33 (m, 3H), 3.25 (dd, 1H, J = 13.2 and 3.9 Hz), 4.58 (dd, 1H, J = 13.2 and
2.0 Hz), 4.98–5.04 (m, 1H), 7.18–7.35 (m, 4H), 8.78 (s, 1H, CHO), 8.96 (s, 1H, CHO); 13C NMR (CDCl3,
17 ºC) δ: 22.49 (CH3), 22.53 (CH3), 24.85 (CH2), 38.69 (CH), 41.99 (C(3)), 44.34 (C(2)), 116.52 (Ar),
118.42 (Ar), 125.49 (Ar), 125.63 (Ar), 160.15 (CHO), 160.39 (CHO).

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ACKNOWLEDGEMENTS
X-Ray crystallographic analysis was carried out at Institute for Molecular Science in Okazaki Japan.
We thank Prof. T. Suzuki (Institute for Molecular Science) and Prof. S.-i. Iwamatsu (Nagoya University)
for their help on the X-Ray analysis.
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as follows: Radiation: MoKα (0.7107 Å); Reflection measurement device: Rigaku® RAXIS-RAPID
imaging plate; Space group: P21; Cell dimension: a = 15.643 Å, b = 12.214 Å, c = 7.129 Å, α = 90 °,
β = 89.46 °, γ = 90 °; cell volume = 1362.1 Å3; Z = 4; Unique reflections: 3173; R = 0.1061.
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