Nucleotides. Part LXXVI: Synthesis and properties of new isoalloxazine (=Benzo[g]pteridine-2,4(1H,3H)-dione) derivatives as labels for oligonucleotides

Article abstract of DOI:10.1002/hlca.200900413

A series of new fluorescing 8-(6-hydroxyhexyl)isoalloxazine (=8-(6-hydroxyhexyl)benzo[g]pteridine-2,4(1H,3H)-dione) derivatives 4 - 13 were synthesized from 6-[(6-hydroxyhexyl)amino]uracil (2) with 1-chloro-4- nitrosobenzene via 8-chloro-10-(6-hydroxyhexyl)isoalloxazine (3) and subsequent substitution of the Cl-atom of 3 by various amines (Scheme). Analogously, 8-substituted 10-{3-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]propyl} isoalloxazines 19, 20, and 23 - 25 were prepared which yielded on deprotection the corresponding 10-[3-(2,3-dihydroxypropoxy)propyl]alloxazines 21, 22, and 26 - 28. Their conversion into the 3″-O-(4,4′-dimethoxytrityl) derivatives 29 - 33 and subsequent transformation into the corresponding 2″-(2-cyanoethyl N, N-diisopropylphosphoramidites) 34 - 38 led to new building blocks for oligonucleotide synthesis. A series of 21-mer oligodeoxyribonucleotides carrying the fluorescing isoalloxazine 37 in various positions of the chain were assembled in a DNA synthesizer. Combination with the complementary sequence yielded the stable duplexes 40 - 54 showing by the melting temperatures T_m that the fluorophor (F) does not harm the stability of the unmodified duplex 39 (Table).

Full text of DOI:10.1002/hlca.200900413

1038
Helvetica Chimica Acta – Vol. 93 (2010)
Nucleotides
Part LXXVI1)
Synthesis and Properties of New Isoalloxazine (¼ Benzo[g]pteridine-2,4(1H,3H)-
dione) Derivatives as Labels for Oligonucleotides
by Evgeny Kvassiouk, Ramamurthy Charubala, and Wolfgang Pfleiderer*
Fachbereich Chemie, Universitꢀt Konstanz, Postfach 5560, 78457 Konstanz, Germany
(phone: þ 49-7531-882279; fax: þ 49-7531-883138; e-mail: Wolfgang.Pfleiderer@uni-konstanz.de)
A series of new fluorescing 8-(6-hydroxyhexyl)isoalloxazine (¼ 8-(6-hydroxyhexyl)benzo[g]pter-
idine-2,4(1H,3H)-dione) derivatives 4 – 13 were synthesized from 6-[(6-hydroxyhexyl)amino]uracil (2)
with 1-chloro-4-nitrosobenzene via 8-chloro-10-(6-hydroxyhexyl)isoalloxazine (3) and subsequent
substitution of the Cl-atom of 3 by various amines (Scheme). Analogously, 8-substituted 10-{3-[(2,2-
dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}isoalloxazines 19, 20, and 23 – 25 were prepared which
yielded on deprotection the corresponding 10-[3-(2,3-dihydroxypropoxy)propyl]alloxazines 21, 22, and
26 – 28. Their conversion into the 3’’-O-(4,4’-dimethoxytrityl) derivatives 29 – 33 and subsequent
transformation into the corresponding 2’’-(2-cyanoethyl N,N-diisopropylphosphoramidites) 34 – 38 led
to new building blocks for oligonucleotide synthesis. A series of 21-mer oligodeoxyribonucleotides
carrying the fluorescing isoalloxazine 37 in various positions of the chain were assembled in a DNA
synthesizer. Combination with the complementary sequence yielded the stable duplexes 40 – 54 showing
by the melting temperatures Tm that the fluorophor (F) does not harm the stability of the unmodified
duplex 39 (Table).
Introduction. – Labeling of biomolecules can be regarded as the most common and
widely used methodology for a broad variety of bioanalytical purposes. Fluorescence
labeling [2], especially, is often the preferred choice due to the high sensitivity and easy
detection. We concentrated our interest on isoalloxazine (¼ benzo[g]pteridine-
2,4(1H,3H)-dione) derivatives which show strong fluorescence, high quantum yields,
and high extinctions in the VIS region of 460 – 480 nm. The best-known isoalloxazine
derivative is vitamin B2 which is produced technically on a large scale by the classical
method condensing N1-d-ribityl-4,5-dimethylbenzene-1,2-diamine with alloxan [3][4].
Alloxazine syntheses are more versatile and include the condensation of an o-
aminoazobenzene with a barbituric acid [5] or a 5-nitrosopyrimidine with an aromatic
amine [6], or the nitrosation of a 6-(arylamino)uracil [7]. Further possibilities are seen
in the reactions of 6-anilinouracils with diethyl azodicarboxylate [8][9] or by refluxing
1,3-dimethyluracil-6-amine with nitrosobenzenes in Ac2O [10]. Our approach to
various 10-substituted isoalloxazines was based [11] on the condensations of 6-
(alkylamino)uracils with 1-chloro-4-nitrosobenzene in DMF leading to 8-chloro-10-
alkylisoalloxazines which could further been modified at position 8 by nucleophilic
substitution reactions.
1
)
Part LXXV: [1].
ꢁ 2010 Verlag Helvetica Chimica Acta AG, Zꢂrich

Helvetica Chimica Acta – Vol. 93 (2010)
1039
Synthesis. – Starting from 6-chlorouracil (1), reaction with 6-aminohexan-1-ol gave
6-[(6-hydroxyhexyl)amino]uracil (2) which could be converted with 1-chloro-4-nitro-
sobenzene in DMF directly in high yield into 8-chloro-10-(6-hydroxyhexyl)isoallox-
azine (3) (Scheme). In a similar manner reacted N,N-dimethyl-4-nitrosobenzenamine
to the corresponding 8-(dimethylamino)-10-(6-hydroxyhexyl)isoalloxazine (4), and in
Scheme

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Helvetica Chimica Acta – Vol. 93 (2010)
a stepwise reaction, 2 was first converted with 1-chloro-4-nitrosobenzene into 3
followed by treatment with Pr2NH or cyclohexanamine to give 5 and 6, respectively. A
more convenient approach was the nucleophilic substitution of the Cl-substituent in 3
by Et2NH, benzylamine, N,N-dibenzylamine, dodecan-1-amine, adamantan-1-amine
(¼tricyclo[3.3.1.13,7]decan-1-amine), piperidine, and morpholine yielding 7 – 13.
To prepare appropriate building blocks for oligonucleotide synthesis, 1 and 6-
chloro-3-methyluracil (14) were treated with 3-[(2,2-dimethyl-1,3-dioxolan-4-yl)me-
thoxy]propan-1-amine to form 15 and 16, respectively. Condensation of 15 with 1-
chloro-4-nitrosobenzene in N,N-dimethylacetamide (DMA) yielded 8-chloro-10-{3-
[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}isoalloxazine (17) which was further
transformed with benzylamine or dodecan-1-amine into 19 and 20, respectively. The
deprotection of the side chain at N(10) of 19 and 20 was achieved in 80% AcOH to give
8-(benzylamino)- (21) and 10-[3-(2,3-dihydroxypropoxy)propyl]-8-(dodecylamino)-
isoalloxazine (22). The corresponding 3-methyl derivatives 26 and 27 as well as the 8-
(morpholin-4-yl) compound 28 resulted from a one-pot reaction starting with 16 which
was converted to 23 – 25 via 18 and, without isolation, directly deblocked to 26 – 28. The
deprotected 8-substituted 10-[3-(2,3-dihydroxypropoxy)propyl]isoalloxazines 21, 22,
and 26 – 28 were then treated with 4,4’-dimethoxytrityl chloride to give 29 – 33. The
latter were transformed with 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiami-
dite under 1H-tetrazole catalysis to the corresponding phosphoramidites 34 – 38 in
good yields (Scheme).
Oligonucleotides. – To test the new isoalloxazine derivatives as fluorescence
markers, a series of oligodeoxyribonucleotides with the fluorophor in various positions
of the chain were synthesized by well-known procedures in an ABI synthesizer A392.
As solid support was used LCAMA-CPG 500 ꢃ [12 – 14] to which 5’-O-(4,4’-
dimethoxytrityl)-3’-O-succinylthymidine was attached first. The chosen 21-mer se-
quence was 5’-d(GTG TGG AAA ATC TCT AGC AGT)-3’ which showed with its
complementary sequence 3’-d(CAC ACC TTT TAG AGA TCG TCA)-5’ a melting
temperature of 52.78 (see 39 in the Table). The fluorescing phosphoramidite 36 (cf. F)
was built into the chain from the 5’-end successively in positions 1 – 6, 8, 10, 11, 13, 15,
and 19 (Table) providing the duplexes 40 – 54 with melting temperatures which were
higher in all cases than that of the unmodified duplex 39 (Table). In duplexes 49 – 52,
position 13 in the complementary chain was also modified by the four natural bases
showing, however, no special effect on the melting temperature.
The monomeric building blocks for the oligodeoxyribonucleotide synthesis were
the 2’-deoxy-5’-O-(4,4’-dimethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}ade-
nosine, 2’-deoxy-5’-O-(4,4’-dimethoxytrityl)-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}-
cytidine, and 2’-deoxy-5’-O-(dimethoxytrityl)-N2-{[2-(4-nitrophenyl)ethoxy]carbonyl}-
O6-[2-(4-nitrophenyl)ethyl]guanosine 3’-(2-cyanoethyl N,N-diisopropylphosphorami-
dite) [15]. Deprotection at the nucleobases was achieved by DBU (1,8-diazabicy-
clo[5.4.0]undec-7-ene) treatment, a washing process, and finally cleavage from the
support by ammonia [14]. The oligomers were of high purity as seen from HPLC
analysis on reversed phase RP-18 material.

Helvetica Chimica Acta – Vol. 93 (2010)
1041
Table. Melting Temperatures and Fluorescence Intensities of Modified Oligonucleotides 40 – 54a)
Sequence
Tm [8]
Fluorescence intensity at
258
65 – 758
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
5’-d (GTG TGG AAA ATC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (FTG TGG AAA ATC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GFG TGG AAA ATC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTF TGG AAA ATC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTG FGG AAA ATC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTG TFG AAA ATC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTG TGF AAA ATC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTG TGG AFA ATC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTG TGG AAA FTC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTG TGG AAA AFC TCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTG TGG AAA ATC FCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTG TGG AAA ATC FCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG CGA TCG TCA)-5’
5’-d (GTG TGG AAA ATC FCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG GGA TCG TCA)-5’
5’-d (GTG TGG AAA ATC FCT AGC AGT)-3’
3’-d (CAC ACC TTT TAG TGA TCG TCA)-5’
5’-d (GTG TGG AAA ATC TCF AGC AGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
5’-d (GTG TGG AAA ATC TCT AGC FGT)-3’
3’-d (CAC ACC TTT TAG AGA TCG TCA)-5’
52.7
62.5
59.5
57.3
56.5
55.3
53.5
54.8
53.5
54.2
55.8
58.9
59.0
57.8
57.2
59.3
17
22
10.5
12
1.6
1.5
1.4
3.4
5.7
10.6
21.5
14
1.0
4.8
7.4
16.4
28.8
11
23.5
31
9.5
7.5
13
8.5
a) Measured at 260 nm in Na2HPO4/NaH2PO4 buffer, pH 7, [Naþ] ¼ 0.15.
Experimental Part
General. Products were dried under high vacuum. All solvents used were of anh. grade. TLC:
precoated silica gel (SiO2) thin-layer sheets 60 F 254 (Merck). Flash chromatography (FC): SiO2 (30 –
60 mm; Baker); 0.2 – 0.3 bar. Column chromatography (CC): SiO2 60, Merck. HPLC: pump L 6200,
autosampler AS 4000, UV detector L 4000 (Merck-Hitachi); column RP-18, Lichrospher 100 (125 ꢀ
4 mm, 5 mm; Merck); elution: A ¼ 0.1m AcO(NHEt3); B ¼ A þ MeCN 1:1; A/B 95 :5 (0 – 2 min), A/B
60 :40 (30 min), and B (10 min); flow rate 1 ml/min; detection wavelength 260 nm. M.p.: DNA
Synthesizer from Applied Biosystems, model 392. UV/VIS: Perkin-Elmer Lambda 5; lmax in nm (log e).
1H-NMR: Bruker AC 250; d in ppm rel. to Me4Si or CDCl3 ((D6)DMSO) as internal standard, J in Hz.
31P-NMR: JEOL JMN-GX400. EI-MS: Finnigan MAT 8200; in m/z (rel. %). Microanalyses: Vario
Macro Cube, Elementar Analysensysteme GmbH.

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Helvetica Chimica Acta – Vol. 93 (2010)
1. 6-[(6-Hydroxyhexyl)amino]uracil (¼6-[(6-Hydroxyhexyl)amino]pyrimidine-2,4-(1H,3H)-dione;
2). A mixture of 6-chlorouracil (1) [16] (2.1 g, 14.3 mmol) and 6-aminohexan-1-ol (3.5 g, 30 mmol) was
stirred at 1608 for 10 min. After cooling, the precipitate was recrystallized from 80% EtOH in presence of
a few drops of AcOH: 2.67 g (82%) of 2. Colorless crystals. M.p. 208 – 2098. UV (MeOH): 203 (4.07), 264
(4.37). 1H-NMR ((D6)DMSO): 10.14 (s, HꢁN(1)); 9.80 (s, HꢁN(3)); 6.04 (t, HNꢁC(6)); 4.37 (s,
HꢁC(5)); 4.34 (t, OH); (m, CH2OH); 3.37 (m, CH2NH); 1.40 – 1.28 (m, (CH2)4). Anal. calc. for
C10H17N3O3 (227.3): C 52.85, H 7.53, N 18.48; found: C 52.63, H 7.57, N 28.21.
2. 8-Chloro-10-(6-hydroxyhexyl)benzo[g]pteridine-2,4(1H,3H)-dione (3). A mixture of 2 (1.1 g,
5 mmol) and 1-chloro-4-nitrosobenzene (1.4 g, 10 mmol) in DMF (20 ml) was stirred at 1508 for 30 min
and then evaporated. The residue was treated with EtOH, and after cooling, the precipitate was
collected: 1.09 g (64%) of 3. Yellowish crystalline powder. M.p. 255 – 2578 (dec.). UV (MeOH): 223
(4.54), 267 (4.50), 335 (3.86), 430 (4.07). 1H-NMR ((D6)DMSO): 11.43 (s, HꢁN(3)); 8.12 (d, HꢁC(7));
8.09 (s, HꢁC(9)); 7.66 (d, HꢁC(6)); 4.53 (m, CH2N); 4.35 (t, OH); 3.38 (m, CH2OH); 1.67 (m,
CH2CH2N); 1.42 (m, (CH2)3). Anal. calc. for C16H17ClN4O3 (348.8): C 55.09, H 4.91, N 16.06; found: C
54.96, H 4.84, N 15.85.
3. 8-(Dimethylamino)-10-(6-hydroxyhexyl)benzo[g]pteridine-2,4(1H,3H)-dione (4). A mixture of 2
(0.23 g, 1 mmol) and N,N-dimethyl-4-nitrosobenzenamine (0.6 g, 4 mmol) in DMF (10 ml) was stirred at
1608 for 3 h and, after cooling, concentrated. The residue was dissolved in CH2Cl2 and precipitated by
hexane. The dark powder was purified by CC (SiO2 (3.5 ꢀ 12 cm); AcOEt, AcOEt/MeOH 9 :1, then
CHCl3 and CHCl3/MeOH 9 :1): 0.3 g (83%) of 4. Yellow crystalline powder. M.p. 280 – 2838. UV
(MeOH): 257 (4.72), 312 (3.90), 492 (4.66). 1H-NMR ((D6)DMSO): 10.99 (s, HꢁN(3)); 7.82 (d,
HꢁC(6)); 7.22 (d, HꢁC(7)); 6.51 (s, HꢁC(9)); 4.54 (m, CH2N); 4.37 (t, OH); 3.36 (m, CH2OH); 3.24 (s,
2 Me); 1.70 (m, CH2CH2N); 1.40 (m, (CH2)3). Anal. calc. for C18H23N5O3 · 0.5 H2O (366.4): C 59.00, H
6.60, N 19.11; found: C 58.92, H 6.51, N 18.83.
4. 8-(Dipropylamino)-10-(6-hydroxyhexyl)benzo[g]pteridine-2,4(1H,3H)-dione (5). A mixture of 2
(50 mg, 0.22 mmol) and 1-chloro-4-nitrosobenzene (0.14 g, 1 mmol) in DMF (2 ml) was stirred at 1408
for 2 h. Pr2NH (0.2 ml) was added, and heating at 1408 was continued for 2 h. After evaporation, the
residue was purified by CC (SiO2 (2.5 ꢀ 10 cm); AcOEt/MeOH 20 :1 ! 4 :1). The main fraction was
recrystallized from EtOH: 40 mg (44%) 5. Yellow crystals. M.p. 219 – 2208. UV (MeOH): 258 (4.70), 314
(3.92), 498 (4.66). 1H-NMR ((D6)DMSO): 10.96 (s, HꢁN(3)); 7.79 (d, HꢁC(6)); 7.23 (d, HꢁC(7)); 6.43
(s, HꢁC(9)); 4.52 (m, CH2N); 4.36 (t, OH); 3.54 (m, N(MeCH2CH2)2); 3.37 (m, CH2OH); 1.65 (m,
3 CH2CH2N); 1.39 (m, (CH2)3); 0.94 (t, 2 Me). Anal. calc. for C22H31N5O3 (413.5): C 63.90, H 7.55, N
16.93; found: C 63.87, H 7.50, N 16.93.
5. 8-(Cyclohexylamino)-10-(6-hydroxyhexyl)benzo[g]pteridine-2,4(1H,3H)-dione (6). A mixture of
2 (55 mg, 0.25 mmol) and 1-chloro-4-nitrosobenzene (0.14 g, 1 mmol) in AcNMe2 (DMA; 1 ml) was
stirred at 1408 for 1 h. Cyclohexylamine (1 ml) was added, and heating at 1208 was continued for 1 h.
After evaporation, the residue was purified by CC (SiO2 (2.5 ꢀ 15 cm); AcOEt/MeOH 20 :1 ! 9 :1). The
main fraction was recrystallized from EtOH/H2O 1:1: 50 mg (50%) of 6. Yellow crystals. M.p. 332 – 3368
(dec.). UV (MeOH): 255 (4.62), 312 (3.83), 487 (4.58). 1H-NMR ((D6)DMSO): 10.93 (s, HꢁN(3)); 7.86
(d, HNꢁC(8)); 7.72 (d, HꢁC(6)); 7.05 (d, HꢁC(7)); 6.48 (s, HꢁC(9)); 4.48 (t, CH2N); 4.37 (t, OH); 3.63
(m, CHNH); 3.36 (m, CH2OH); 1.98 – 1.22 (m, (CH2)9). Anal. calc. for C22H29N5O3 · 2 H2O (447.5): C
59.04, H 7.43, N 15.64; found: C 59.06, H 6.98, N 15.78.
6. 8-(Diethylamino)-10-(6-hydroxyhexyl)benzo[g]pteridine-2,4(1H,3H)-dione (7). A mixture of 3
(50 mg, 0.14 mmol) and Et2NH (0.3 ml) in DMA (2 ml) was stirred at 1008 for 1 h. After evaporation, the
residue was purified by CC (SiO2 (2.5 ꢀ 10 cm); AcOEt, then AcOEt/MeOH 20 :1). The main fraction
was dried: 33 mg (66%) of 7. Yellow solid. M.p. 260 – 2628. UV (MeOH): 216 (4.08), 258 (4.71), 315
(3.92), 497 (4.67). 1H-NMR ((D6)DMSO): 10.96 (s, HꢁN(3)); 7.78 (d, HꢁC(6)); 7.20 (d, HꢁC(7)); 6.45
(s, HꢁC(9)); 4.53 (t, CH2N); 4.36 (t, OH); 3.63 (q, MeCH2N); 3.37 (m, CH2OH); 1.69 (m, CH2CH2N);
1.39 (m, (CH2)3); 1.20 (t, 2 MeCH2). Anal. calc. for C20H27N5O3 · 0.5 H2O (394.5): C 60.89, H 7.15, N 17.75;
found: C 60.85, H 7.25, N 17.79.
7. 10-(6-Hydroxyhexyl)-8-[(phenylmethyl)amino]benzo[g]pteridine-2,4(1H,3H)-dione (8). A mix-
ture of 3 (0.27 g, 0.77 mmol) and benzylamine (3 ml) was stirred at 1208 for 1 h and then evaporated. The
residue was purified by CC (SiO2 (2.5 ꢀ 15 cm); CHCl3, then CHCl3/MeOH 50 :1 ! 9 :1). The main

Helvetica Chimica Acta – Vol. 93 (2010)
1043
fraction was recrystallized from EtOH/CHCl3 2 :1: 0.11 g (34%) of 8. Yellow crystals. M.p. 295 – 3008
(dec.). UV (MeOH): 254 (4.70), 308 (3.86), 480 (4.67). 1H-NMR ((D6)DMSO): 10.95 (s, HꢁN(3)); 8.49
(t, HNꢁC(8)); 7.74 (d, HꢁC(6)); 7.36 (m, Ph); 7.10 (d, HꢁC(7)); 6.42 (s, HꢁC(9)); 4.59 (d, PhCH2); 4.48
(t, CH2CH2N); 4.37 (t, OH); 3.37 (m, CH2OH); 1.40 – 1.27 (m, (CH2)4). Anal. calc. for C23H25N5O3
(419.5): C 65.85, H 6.00, N 16.69; found: C 65.42, H 6.01, N 16.51.
8. 8-[Bis(phenylmethyl)amino]-10-(6-hydroxyhexyl)benzo[g]pteridine-2,4(1H,3H)-dione (9). A
mixture of 3 (0.1 g, 0.28 mmol) and dibenzylamine (0.5 g, 2.88 mmol) in DMF (1 ml) was stirred at
1208 for 5 h. After evaporation, the residue was purified by CC (SiO2 (2.5 ꢀ 15 cm); CHCl3, then CHCl3/
MeOH 20 :1 ! 9 :1): 50 mg (34%) of 9. Yellow amorphous solid. UV (MeOH): 258 (4.53), 310 (3.86),
488 (4.41). 1H-NMR ((D6)DMSO): 11.02 (s, HꢁN(3)); 7.81 (d, HꢁC(6)); 7.33 (m, HꢁC(7), 2 Ph); 6.47
(s, HꢁC(9)); 5.08 (s, 2 PhCH2); 4.37 (t, OH); 4.35 (t, CH2CH2N); 3.36 (m, CH2OH); 1.30 – 1.13 (m,
.
(CH2)4). EI-MS: 509 (Mþ ).
9. 8-(Dodecylamino)-10-(6-hydroxyhexyl)benzo[g]pteridine-2,4(1H,3H)-dione (10). A mixture of 3
(0.1 g, 0.28 mmol) and dodecan-1-amine (0.212 g, 1.14 mmol) in DMA (4 ml) was stirred at 1508 for 0.5 h.
After evaporation, the residue was purified by CC (SiO2 (2.5 ꢀ 25 cm); CHCl3, then CHCl3/MeOH
20 :1). The main fraction was recrystallized from MeOH: 120 mg (84%) of 10. Yellow crystals. M.p. 313 –
3158 (dec.). UV (MeOH): 255 (4.70), 311 (3.85), 484 (4.66). 1H-NMR ((D6)DMSO): 10.92 (s, HꢁN(3));
7.94 (t, HNꢁC(8)); 7.71 (d, HꢁC(6)); 7.02 (d, HꢁC(7)); 6.45 (s, HꢁC(9)); 4.48 (m, CH2N(10)); 4.35 (t,
OH); 3.37 (m, CH2OH); 3.29 (m, CH2NH); 1.60 – 1.20 (m, 12 CH2); 0.83 (t, Me). Anal. calc. for
C28H43N5O3 (498.7): C 67.57, H 8.70, N 14.07; found: C 67.19, H 8.48, N 14.18.
10. 10-(6-Hydroxyhexyl)-8-(tricyclo[3.3.1.13,7]dec-1-ylamino)benzo[g]pteridine-2,4(1H,3H)-dione
(11). A mixture of 3 (0.1 g, 0.28 mmol) and adamantan-1-amine (0.42 g, 2.8 mmol) in DMA (4 ml)
was stirred at 1608 for 9 h. After evaporation, the residue was purified by CC (SiO2 (2.5 ꢀ 25 cm);
AcOEt/MeOH 20 :1, then CHCl3/MeOH 20 :1). The main fraction was recrystallized from MeOH:
80 mg (60%) of 11. Yellow crystals. M.p. > 3308. UV (MeOH): 255 (4.70), 311 (3.90), 489 (4.68).
1H-NMR ((D6)DMSO): 10.95 (s, HꢁN(3)); 7.70 (d, HꢁC(6)); 7.55 (s, HNꢁC(8)); 7.17 (d, HꢁC(7));
6.64 (s, HꢁC(9)); 4.45 (m, CH2N(10)); 4.37 (t, OH); 3.37 (m, CH2OH); 2.13 – 1.40 (m, 3 CH, 10 CH2).
.
EI-MS: 463 (Mþ ). Anal. calc. for C26H33N5O3 (463.6): C 67.36, H 7.17, N 15.08; found: C 66.90, H 7.17, N
15.08.
11. 10-(6-Hydroxyhexyl)-8-(piperidin-1-yl)benzo[g]pteridine-2,4(1H,3H)-dione (12). A mixture of
2 (0.1 g, 0.44 mmol) and 1-chloro-4-nitrosobenzene (0.28 g, 2 mmol) in DMF (4 ml) was stirred at 1408
for 2 h. Piperidine (4 ml) was added and heating continued for 1 h. After evaporation, the residue was
purified by CC (SiO2 (2.5 ꢀ 20 cm); CHCl3 and CHCl3/MeOH 20 :1). The main fraction was
recrystallized from EtOH: 104 mg (60%) of 12. Yellow crystals. M.p. 263 – 2658 (dec.). UV (MeOH):
259 (4.64), 316 (3.83), 497 (4.58). 1H-NMR ((D6)DMSO): 11.00 (s, HꢁN(3)); 7.78 (d, HꢁC(6)); 7.41 (d,
HꢁC(7)); 6.47 (s, HꢁC(9)); 4.54 (t, CH2N); 4.37 (t, OH); 3.68 (s, 2 CH2NꢁC(8)); 3.37 (m, CH2OH);
.
1.66 – 1.39 (m, 7 CH2). EI-MS: 397 (Mþ ). Anal. calc. for C21H27N5O3 · 0.5 H2O (406.5): C 62.05, H 6.94, N
17.22; found: C 62.23, H 6.77, N 17.15.
12. 10-(6-Hydroxyhexyl)-8-(morpholin-4-yl)benzo[g]pteridine-2,4(1H,3H)-dione (13). A mixture of
3 (0.14 g, 0.4 mmol) and morpholine (3 ml) in xylene (3 ml) was stirred at 1408 for 1 h. After
evaporation, the residue was purified by CC (SiO2 (2.5 ꢀ 20 cm); CHCl3 and CHCl3/MeOH 20 :1). The
main fraction was recrystallized from MeOH/CHCl3 3 :1: 0.12 g (75%) of 13. Yellow crystals. M.p. 277 –
2798 (dec.). UV (MeOH): 257 (4.56), 310 (3.85), 484 (4.50). 1H-NMR ((D6)DMSO): 11.07 (s, HꢁN(3));
7.86 (d, HꢁC(6)); 7.40 (d, HꢁC(7)); 6.83 (s, HꢁC(9)); 4.56 (m, CH2N(10)); 4.36 (t, OH); 3.78 – 3.61 (m,
.
CH2OCH2); 3.37 (m, CH2OH); 1.69 – 1.39 (m, 6 CH2). EI-MS: 399 (Mþ ). Anal. calc. for C20H25N5O4 ·
H2O (417.5): C 57.54, H 6.52, N 16.77; found: C 57.80, H 6.53, N 16.33.
13. 6-{{3-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}amino}uracil (¼6-{{3-[(2,2-Dimethyl-
1,3-dioxolan-4-yl)methoxy]propyl}amino}pyrimidine-2,4(1H,3H)-dione; 15). A mixture of 3-[(2,2-
dimethyl-1,3-dioxolan-4-yl)methoxy]propan-1-amine (0.75 g, 4 mmol) and 6-chlorouracil (1) [16]
(0.3 g, 2 mmol) was stirred at 1208 for 20 min. After cooling, the mixture was treated with EtOH and
the solid collected, washed with Et2O, and dried: 0.4 g (65%) of 15. M.p. 215 – 2168. UV (MeOH): 264
(4.36). 1H-NMR ((D6)DMSO): 10.15 (s, HꢁN(1)); 9.91 (s, HꢁN(3)); 6.06 (t, CH2NH); 4.38 (s,
HꢁC(5)); 4.16 (m, CH); 3.97 (dd, CH); 3.59 (dd, CH); 3.45 (t, CH2); 3.38 (dd, CH2); 3.04 (m,

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Helvetica Chimica Acta – Vol. 93 (2010)
.
CH2CH2N); 1.69 (m, CH2CH2N); 1.30, 1.25 (2s, 2 Me). EI-MS: 299 (Mþ ). Anal. calc. for C13H21N3O5
(299.3): C 52.16, H 7.07, N 14.04; found: C 52.04, H 6.99, N 14.14.
14. 6-{{3-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}amino}-3-methyluracil (¼6-{{3-[(2,2-Di-
methyl-1,3-dioxolan-4-yl)methoxy]propyl}amino}-3-methylpyrimidine-2,4(1H,3H)-dione; 16). A mix-
ture of 3-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]propan-1-amine (1.9 g, 10 mmol) and 6-chloro-3-
methyluracil (14) [17] (0.8 g, 5 mmol) in N-ethyldiisopropylamine (3 ml) was stirred at 1208 for 2.5 h.
After cooling, the mixture was dissolved in CHCl3 (100 ml) and washed with H2O (2 ꢀ 20 ml), the org.
phase dried (Na2SO4) and concentrated, and the residue treated with Et2O and dried: 1.2 g (77%) of 16.
Colorless amorphous powder. UV (MeOH): 264 (4.33). 1H-NMR ((D6)DMSO): 10.12 (s, HꢁN(1)); 6.08
(t, CH2NH); 4.56 (s, HꢁC(5)); 4.16 (m, CH); 3.96 (dd, CH); 3.59 (dd, CH); 3.40 (m, 2 CH2O); 3.32 (s,
.
MeꢁN(3)): 3.05 (m, CH2CH2N); 1.70 (m, CH2CH2N); 1.29, 1.25 (2s, 2 Me). EI-MS: 313 (Mþ ). Anal.
calc. for C14H23N3O5 (313.5): C 53.66, H 7.40, N 13.41; found: C 53.64, H 7.25, N 13.13.
15. 8-Chloro-10-{3-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}benzo[g]pteridine-2,4(1H,3H)-
dione (17). To a soln. of 15 (2.42 g, 8.08 mmol) in DMA (40 ml), 1-chloro-4-nitrosobenzene (2.28 g,
16.2 mmol) was added gradually, and the mixture was stirred at 110 – 1208 in an oil bath for 5 h. After
evaporation, the residue was purified by CC (SiO2 (5 ꢀ 15 cm); CHCl3/MeOH 50 :1 ! 9 :1). The main
fraction was recrystallized from CHCl3/MeOH 20 :1: 1.6 g (48%) of 17. Yellow crystals. M.p. 2718. UV
(MeOH): 262 (4.22), 313 (3.64), 428 (3.73). 1H-NMR ((D6)DMSO): 11.43 (s, HꢁN(3)); 8.13 (d,
HꢁC(6)); 8.05 (s, HꢁC(9)); 7.67 (d, HꢁC(7)); 4.59 (t, CH2N(10)); 4.17 (m, CH); 3.98 (dd, CH); 3.59 (m,
.
CH, CH2O); 3.41 (d, CH2O); 2.00 (m, CH2CH2N); 1.29, 1.25 (2s, 2 Me). EI-MS: 420 (Mþ ). Anal. calc.
for C19H21ClN4O5 (420.8): C 52.44, H 5.03, N 13.31; found: C 52.42, H 5.29, N 13.34.
16. 10-{3-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}-8-[(phenylmethyl)amino]benzo[g]-
pteridine-2,4(1H,3H)-dione (19). A mixture of 17 (1.4 g, 3.32 mmol) and benzylamine (3 ml) in DMA
(30 ml) was stirred at 120 – 1308 for 30 min and then evaporated. The solid was purified by CC (SiO2 (5 ꢀ
15 cm); CHCl3, then CHCl3/MeOH 40 :1). The main fraction was recrystallized from EtOH: 0.6 g (37%)
of 19. Yellow crystals. M.p. 297 – 2988. UV (MeOH): 254 (4.74), 308 (3.88), 481 (4.71). 1H-NMR
((D6)DMSO): 10.96 (s, HꢁN(3)); 8.45 (t, HNꢁC(8)); 7.73 (d, HꢁC(6)); 7.41 (m, Ph); 7.09 (d, HꢁC(7));
6.50 (br. s, HꢁC(9)); 4.56 (d, CH2NH); 4.48 (m, CH2N(10)); 4.13 (m, CH); 3.99 (dd, CH); 3.50 (m, CH,
.
CH2O); 3.38 (d, CH2O); 1.77 (m, CH2CH2N); 1.27, 1.21 (2s, 2 Me). EI-MS: 491 (Mþ ). Anal. calc. for
C26H29N5O5 (491.5): C 63.33, H 5.94, N 14.21; found: C 63.04, H 6.02, N 14.22.
17. 10-{3-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}-8-(dodecylamino)benzo[g]pteridine-
2,4(1H,3H)-dione (20). As described for 19, with 17 (0.5 g, 1.2 mmol), dodecan-1-amine (0.55 g,
3 mmol), and DMA (8 ml) at 1208 for 3 h. The main CC fraction gave 0.27 g (40%) of 20. Yellow
1
amorphous powder. UV (MeOH): 255 (4.68), 309 (3.85), 484 (4.58). H-NMR ((D6)DMSO): 10.93 (s,
HꢁN(3)); 7.95 (br. s, HNꢁC(8)); 7.71 (d, HꢁC(6)); 7.03 (d, HꢁC(7)); 6.49 (br. s, HꢁC(9)); 4.56 (t,
CH2N(10)); 4.15 (m, CH); 3.95 (dd, CH); 3.58 (m, CH, CH2O); 3.41 (d, CH2O); 1.96 (m, CH2CH2N);
.
1.62 (m, CH2CH2N); 1.29, 1.21 (2s, 2 Me); 1.24 (m, (CH2)9); 0.83 (t, MeCH2). EI-MS: 569 (Mþ ). Anal.
calc. for C31H47N5O5 (569.7): C 65.35, H 8.31, N 12.29; found: C 65.05, H 8.20, N 12.40.
18. 10-[3-(2,3-Dihydroxypropoxy)propyl]-8-[(phenylmethyl)amino]benzo[g]pteridine-2,4(1H,3H)-
dione (21). A soln. of 19 (0.8 g, 1.62 mmol) in 80% AcOH (50 ml) was stirred at 608 for 30 min and then
concentrated. EtOH (4 ꢀ 30 ml) was added to the residue and co-evaporated; then the residue was
dissolved in CH2Cl2/MeOH 9 :1, the soln. dropwise added into Et2O (100 ml), and the precipitate
recrystallized from EtOH: 0.45 g (61%) of 21. Yellow crystals. M.p. 276 – 2788. UV (MeOH): 254 (4.64),
308 (3.80), 481 (4.59). 1H-NMR ((D6)DMSO): 10.97 (s, HꢁN(3)); 8.46 (t, HNꢁC(8)); 7.73 (d,
HꢁC(6)); 7.41 (m, Ph); 7.08 (d, HꢁC(7)); 6.55 (br. s, HꢁC(9)); 4.76 (br. s, CH2OH); 4.55 (d, CHOH,
.
CH2CH2NH); 3.60 (m, CHOH); 3.47 (m, 3 CH2O); 1.77 (m, CH2CH2N). EI-MS: 451 (Mþ ). Anal. calc.
for C23H25N5O5 (451.5): C 61.19, H 5.58, N 15.51; found: C 60.80, H 5.69, N 15.56.
19. 10-[3-(2,3-Dihydroxypropoxy)propyl]-8-(dodecylamino)benzo[g]pteridine-2,4(1H,3H)-dione
(22). A mixture of 17 (0.53 g, 1.33 mmol) and docecan-1-amine (0.555 g, 3 mmol) in DMA (8 ml) was
heated to 1108 for 4 h and then concentrated. The residue was dissolved in CHCl3 (50 ml), the soln.
washed with H2O (2 ꢀ 20 ml), dried (Na2SO4), and concentrated, the resulting solid treated in 80%
AcOH (50 ml) at 608 for 30 min, and the mixture again concentrated. H2O (3 ꢀ 10 ml) and EtOH (3 ꢀ
10 ml) were added to the residue and co-evaporated. The solid was purified by CC (SiO2 (5 ꢀ 12 cm);

Helvetica Chimica Acta – Vol. 93 (2010)
1045
CHCl3 and CHCl3/MeOH 40 :1 ! 9 :1). The main fraction was recrystallized from MeOH/CHCl3 3 :1:
0.55 g (78%) of 22. Yellow crystals. M.p. 255 – 2588. UV (MeOH): 255 (4.73), 311 (3.92), 485 (4.68).
1H-NMR ((D6)DMSO): 10.94 (s, HꢁN(3)); 7.94 (t, HNꢁC(8)); 7.72 (d, HꢁC(6)); 7.04 (d, HꢁC(7));
6.54 (br. s, HꢁC(9)); 4.74 (br. s, CH2OH); 4.54 (m, CHOH, CH2CH2NH); 3.56 – 3.46 (2m, CHOH,
3 CH2O); 1.95 (m, CH2CH2N); 1.61 (m, CH2CH2N); 1.21 (m, (CH2)9); 0.82 (t, MeCH2). EI-MS: 529
.
(Mþ ). Anal. calc. for C28H43N5O5 (529.7): C 63.49, H 8.18, N 13.22; found: C 63.27, H 8.04, N 13.12.
20. 10-[3-(2,3-Dihydroxypropoxy)propyl]-3-methyl-8-[(phenylmethyl)amino]benzo[g]pteridine-
2,4(1H,3H)-dione (26). A mixture of 16 (1.0 g, 3.2 mmol) and 1-chloro-4-nitrosobenzene (0.9 g,
6.4 mmol) in DMA (10 ml) was stirred at 1208 for 2 h and then concentrated. The residue was dissolved
in CHCl3, and the soln. dropwise added into hexane (100 ml). The amorphous precipitate of 18 was
collected and dissolved in DMA (10 ml), benzylamine (2 ml) was added and the mixture heated to 1208
for 2 h. Evaporation and treatment with hexane gave crude 23 which was dissolved again in 80% AcOH
(70 ml). The soln. was stirred at 608 for 1 h and concentrated, H2O (3 ꢀ 10 ml) and EtOH (3 ꢀ 20 ml)
were added to the residue and co-evaporated. The resulting solid was purified by CC (SiO2 (5 ꢀ 12 cm);
CHCl3 and CHCl3/MeOH 40 :1 ! 20 :1). The main fraction was recrystallized from MeOH: 0.5 g (34%)
of 26. Yellow crystals. M.p. 173 – 1768. UV (MeOH): 254 (4.76), 309 (3.88), 482 (4.71). 1H-NMR
((D6)DMSO): 8.50 (t, HNꢁC(8)); 7.78 (d, HꢁC(6)); 7.39 (m, Ph); 7.10 (d, HꢁC(7)); 6.54 (s, HꢁC(9));
4.73 (d, CHOH); 4.54 (m, CH2OH, CH2NH, CH2N(10)); 3.60 – 3.30 (m, CH, 3 CH2O); 3.21 (s, MeN);
.
1.78 (m, CH2CH2N). EI-MS: 465 (Mþ ). Anal. calc. for C24H27N5O5 (465.5): C 61.92, H 5.84, N 15.04;
found: C 61.87, H 5.82, N 15.16.
21. 10-[3-(2,3-Dihydroxypropoxy)propyl]-8-(dodecylamino)-3-methylbenzo[g]pteridine-2,4(1H,3H)-
dione (27). As described for 26, with 1-chloro-4-nitrosobenzene (1.9 g, 13.4 mmol), 16 (2.1 g, 6.7 mmol),
DMA (10 ml), dodecan-1-amine (2.6 g, 14 mmol), and 80% AcOH (100 ml) (via 24). CC (SiO2 (5 ꢀ
12 cm); CHCl3 and CHCl3/MeOH 20 :1) and final recrystallization from MeOH gave 1.72 g (47%) of
27. Yellow crystals. M.p. 173 – 1758. UV (MeOH): 255 (4.76), 310 (3.90), 486 (4.69). 1H-NMR
((D6)DMSO): 7.97 (m, HNꢁC(8)); 7.76 (d, HꢁC(6)); 7.05 (d, HꢁC(7)); 6.55 (s, HꢁC(9)); 4.75 (br. s,
CHOH); 4.54 (m, CH2OH, CH2CH2N); 3.60 – 3.30 (m, CHOH, 3 CH2O, CH2NH); 3.21 (s, MeN); 1.95
.
(m, CH2CH2N); 1.61 (m, CH2CH2NH); 1.21 (m, (CH2)9); 0.82 (t, MeCH2). EI-MS: 543 (Mþ ). Anal. calc.
for C29H45N5O5 (543.7): C 64.06, H 8.34, N 12.88; found: C 63.96, H 8.25, N 12.92.
22. 10-[3-(2,3-Dihydroxypropoxy)propyl]-3-methyl-8-morpholin-4-yl)benzo[g]pteridine-2,4(1H,3H)-
dione (28). As described for 26, with 1-chloro-4-nitrosobenzene (0.9 g, 6.38 mmol), 16 (1.0 g, 3.2 mmol),
DMA (5 ml), morpholine (3 ml), and 80% AcOH (70 ml) (via 25). CC (SiO2 (3 ꢀ 12 cm); CHCl3 and
CHCl3/MeOH 4 :1) and final recrystallization from EtOH gave 0.5 g (39%) of 28. Yellow crystals. M.p.
1
245 – 2498. UV (MeOH): 257 (4.76), 310 (3.89), 485 (4.57). H-NMR ((D6)DMSO): 7.91 (d, HꢁC(6));
7.40 (d, HꢁC(7)); 6.91 (s, HꢁC(9)); 4.67 (d, CHOH); 4.62 (m, CH2CH2NH); 4.54 (t, CH2OH); 3.77 –
.
3.44 (m, CHOH, 5 CH2O, 2 CH2N); 3.23 (s, MeN); 1.96 (m, CH2CH2N). EI-MS: 445 (Mþ ). Anal.
calc. for C21H27N5O6 (543.7): C 64.06, H 8.34, N 12.88; found: C 63.96, H 8.25, N 12.92.
23. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-8-[(phenylmethyl)amino]ben-
zo[g]pteridine-2,4(1H,3H)-dione (29). To a soln. of 21 (0.8 g, 1.77 mmol) in pyridine (20 ml) was added
under stirring at r.t. gradually 4,4’-dimethoxytrityl chloride (0.89 g, 2.6 mmol). After 2 h, MeOH (2 ml)
was added and the mixture stirred for 15 min, then diluted with CHCl3 (100 ml), and washed with sat.
NaHCO3 soln. (30 ml). The org. layer was dried (Na2SO4) and concentrated. Toluene (2 ꢀ 20 ml) was
added to the residue and co-evaporated. The crude material was purified by CC (SiO2 (5 ꢀ 15 cm);
AcOEt/MeOH 40 :1 ! 9 :1 containing 0.1% Et3N). The crude product was dissolved in little CH2Cl2 and
the soln. added dropwise to hexane. The solid gave, after drying, 0.4 g (30%) of 29. Amorphous orange-
red powder. UV (MeOH): 236 (4.52), 255 (4.66), 488 (4.58). 1H-NMR ((D6)DMSO): 10.97 (s, HꢁN(3));
8.41 (t, HNꢁC(8)); 7.74 (d, HꢁC(6)); 7.70 – 6.75 (m, 18 arom. H); 7.09 (d, HꢁC(7)); 6.40 (br. s,
HꢁC(9)); 5.04 (br. s, CHOH); 4.43 (d, CH2NH); 4.31 (m, CH2N(10)); 3.81 (m, CHOH); 3.63 (s,
2 MeO); 3.43 (m, 2 CH2O); 2.96 (m, CH2O); 1.75 (m, CH2CH2N). Anal. calc. for C44H43N5O7 (753.8): C
70.10, H 5.75, N 9.29; found: C 69.44, H 5.66, N 9.31.
24. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-8-(dodecylamino)benzo[g]pter-
idine-2,4(1H,3H)-dione (30). As described for 29, with 22 (0.5 g, 0.9 mmol), dimethoxytrityl chloride
(0.7 g, 2 mmol), and pyridine (10 ml). CC (SiO2 (3.5 ꢀ 12 cm); CHCl3/MeOH 100 :1 ! 50 :1) gave 0.3 g

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Helvetica Chimica Acta – Vol. 93 (2010)
(39%) of 30. Amorphous orange-red powder. UV (MeOH): 236 (4.52), 255 (4.65), 486 (4.60). 1H-NMR
((D6)DMSO): 10.95 (s, HꢁN(3)); 7.90 (t, HNꢁC(8)); 7.72 (d, HꢁC(6)); 7.40 – 6.74 (m, 13 arom. H); 7.03
(d, HꢁC(7)); 6.30 (br. s, HꢁC(9)); 5.08 (br. s, CHOH); 4.35 (m, CH2N(10)); 3.82 (m, CHOH); 3.63 (s,
2 MeO); 3.56 – 2.99 (m, CH2NH, 3 CH2O); 1.87 (m, CH2CH2N); 1.54 (m, CH2CH2NH); 1.19 (m,
(CH2)7); 0.82 (t, MeCH2). Anal. calc. for C49H61N5O7 (832.1): C 70.73, H 7.39, N 8.42; found: C 70.31, H
7.38, N 8.40.
25. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-3-methyl-8-[(phenylmethyl)-
amino]benzo[g]pteridine-2,4(1H,3H)-dione (31). As described for 29, with 26 (0.824 g, 1.77 mmol),
4,4’-dimethoxytrityl chloride (0.89 g, 2.6 mmol), and pyridine (20 ml). CC (SiO2 (3 ꢀ 12 cm); AcOEt/
MeOH 100 :1 ! 50 :1) gave 0.66 g (84%) of 31. Amorphous orange-red powder. UV (MeOH): 237
(4.50), 254 (4.72), 482 (4.66). 1H-NMR ((D6)DMSO): 8.45 (t, HNꢁC(8)); 7.78 (d, HꢁC(6)); 7.40 – 6.70
(m, 19 arom. H, HꢁC(7)); 6.41 (br. s, HꢁC(9)); 5.02 (br. s, CHOH); 4.44 (d, CH2NH); 4.34 (m,
CH2N(10)); 3.79 (m, CHOH); 3.63, 3.62 (2s, 2 MeO); 3.43 – 2.96 (m, 3 CH2O); 3.21 (s, MeN); 1.75 (m,
CH2CH2N). Anal. calc. for C45H45N5O7 (785.9): C 68.77, H 5.77, N 8.91; found: C 68.12, H 5.99, N 8.31.
26. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-8-(dodecylamino)-3-methylben-
zo[g]pteridine-2,4(1H,3H)-dione (32). As described for 29, with 27 (1.14 g, 2.1 mmol), 4,4’-dimethox-
ytrityl chloride (1.02 g, 3.1 mmol), and pyridine (20 ml). CC (SiO2 (5 ꢀ 12 cm); CHCl3/MeOH 100 :1
containing 0.1% Et3N) gave 1.2 g (68%) of 32. Amorphous orange-red solid. UV (MeOH): 237 (4.50),
1
254 (4.74), 486 (4.67). H-NMR ((D6)DMSO): 7.92 (t, HNꢁC(8)); 7.74 (d, HꢁC(6)); 7.40 – 6.70 (m, 13
arom. H); 7.04 (d, HꢁC(7)); 6.32 (br. s, HꢁC(9)); 5.05 (br. s, CHOH); 4.37 (m, CH2N(10)); 3.82 (m,
CHOH); 3.61, 3.60 (2s, 2 MeO); 3.55 – 2.95 (m, 3 CH2O, CH2NH); 3.21 (s, MeN); 1.88 (m, CH2CH2N);
1.54 (m, CH2CH2NH); 1.18 (m, (CH2)8); 0.81 (t, MeCH2). Anal. calc. for C50H63N5O7 (846.1): C 70.98, H
7.50, N 8.28; found: C 70.64, H 7.46, N 8.05.
27. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-3-methyl-8-(morpholin-4-yl)-
benzo[g]pteridine-2,4(1H,3H)-dione (33). As described for 29, with 28 (1.12 g, 2.5 mmol), 4,4’-
dimethoxytrityl chloride (1.02 g, 3.1 mmol), and pyridine (20 ml). CC (SiO2 (3 ꢀ 12 cm); CHCl3 and
CHCl3/MeOH 100 :1 containing 0.1% Et3N) gave 0.8 g (36%) of 33. Amorphous orange-red solid. UV
(MeOH): 235 (4.49), 259 (4.59), 487 (4.67). 1H-NMR ((D6)DMSO): 7.90 (d, HꢁC(6)); 7.5 – 6.75 (m, 13
arom. H); 7.39 (d, HꢁC(7)); 6.66 (br. s, HꢁC(9)); 4.97 (br. s, CHOH); 4.42 (m, CH2N(10)); 3.78 (m,
CHOH); 3.61, 3.60 (2s, 2 MeO); 3.70 – 2.97 (m, 5 CH2O, CH2NCH2); 3.23 (s, MeN); 1.88 (m,
CH2CH2CH2N). Anal. calc. for C42H45N5O8 (747.85): C 76.45, H 6.06, N 9.36; found: C 76.03, H 7.15, N
9.12.
28. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-8-[(phenylmethyl)amino]ben-
zo[g]pteridine-2,4(1H,3H)-dione 2’’-[2-Cyanoethyl N,N-Diisopropylphosphoramidite] (34). To a mix-
ture of 29 (0.35 g, 0.46 mmol) and 1H-tetrazole (16 mg, 0.23 mmol) in CH2Cl2 (4 ml) was added 2-
cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (0.21 g, 0.69 mmol) at r.t. and stirred under Ar
for 8 h. The mixture was diluted with CHCl3 (50 ml) and washed with sat. NaHCO3 soln. (2 ꢀ 30 ml), the
org. layer dried (Na2SO4) and concentrated, the residue dissolved in CH2Cl2, and the soln. added
dropwise into hexane. The amorphous powder was dried in vacuo: 0.39 g (67%) of 34. UV (MeOH): 236
(4.63), 254 (4.77), 482 (4.71). 31P-NMR (CDCl3): 150.074; 149.942; 149.397. Anal. calc. for C53H60N7O8P
(954.1): C 66.72, H 6.38, N 10.27; found: C 66.26, H 6.32, N 10.15.
29. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-8-(dodecylamino)benzo[g]pter-
idine-2,4(1H,3H)-dione 2’’-[2-Cyanoethyl N,N-Diisopropylphosphoramidite] (35). As described for 34,
with 30 (0.52 g, 0.62 mmol), 1H-tetrazole (21 mg, 0.3 mmol), 2-cyanoethyl N,N,N’,N’-tetraisopropyl-
phosphorodiamidite (0.26 g, 0.87 mmol), and CH2Cl2 (7 ml). The amorphous powder was dried in vacuo:
0.5 g (77%) of 35. UV (MeOH): 235 (4.62), 255 (4.72), 486 (4.68). 31P-NMR (CDCl3): 149.736. Anal.
calc. for C58H78N7O8P (1032.3): C 67.48, H 7.61, N 9.49; found: C 67.25, H 7.65, N 9.20.
30. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-3-methyl-8-[(phenylmethyl)-
amino]benzo[g]pteridine-2,4(1H,3H)-dione 2’’-[2-Cyanoethyl N,N-Diisopropylphosphoramidite] (36).
As described for 34, with 31 (0.4 g, 0.52 mmol), 1H-tetrazole (17 mg, 0.24 mmol), 2-cyanoethyl
N,N,N’,N’-tetraisopropylphosphorodiamidite (0.22 g, 0.73 mmol), and CH2Cl2 (5 ml). The amorphous
powder was dried in vacuo: 0.39 g (77%) of 36. UV (MeOH): 235 (4.64), 254 (4.72), 483 (4.68). 31P-

Helvetica Chimica Acta – Vol. 93 (2010)
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NMR (CDCl3): 149.915; 149.832; 149.298. Anal. calc. for C54H62N7O8P (968.1): C 66.99, H 6.45, N 10.13;
found: C 66.72, H 6.52, N 9.87.
31. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-8-(dodecylamino)-3-methylben-
zo[g]pteridine-2,4(1H,3H)-dione 2’’-[2-Cyanoethyl N,N-Diisopropylphosphoramidite] (37). As descri-
bed for 34, with 32 (1.14 g, 1.34 mmol), 1H-tetrazole (47 mg, 0.7 mmol), 2-cyanoethyl N,N,N’,N’-
tetraisopropylphosphorodiamidite (0.61 g, 2.0 mmol), and CH2Cl2 (15 ml). The amorphous powder was
dried in vacuo: 1.2 g (85%) of 37. UV (MeOH): 234 (4.65), 255 (4.69), 487 (4.69). 31P-NMR (CDCl3):
149.652. Anal. calc. for C59H80N7O8P (1045.7): C 67.76, H 7.71, N 9.37; found: C 67.21, H 7.26, N 9.52.
32. 10-{3-{3-[(4,4’-Dimethoxytrityl)oxy]-2-hydroxypropoxy}propyl}-3-methyl-8-(morpholin-4-yl)-
benzo[g]pteridine-2,4(1H,3H)-dione 2’’-[2-Cyanoethyl N,N-Diisopropylphosphoramidite] (38). As de-
scribed for 34, with 33 (0.28 g, 0.37 mmol), 1H-tetrazole (13 mg, 0.19 mmol), 2-cyanoethyl N,N,N’,N’-
tetraisopropylphosphorodiamidite (0.17 g, 0.56 mmol), and CH2Cl2 (5 ml). The amorphous powder was
dried in vacuo: 0.25 g (70%) of 38. UV (MeOH): 235 (4.65), 257 (4.64), 487 (4.55). 31P-NMR (CDCl3):
150.250; 149.574. Anal. calc. for C51H62N7O9P (948.1): C 64.61, H 6.59, N 10.34; found: C 64.50, H 6.81, N
9.56.
33. Oligodeoxyribonucleotide Syntheses. The solid-support material LCAMA-CPG 500 ꢃ was
prepared according to [12 – 14], and loading with 5’-O-4,4’-(dimethoxytrityl)-3’-O-succinylthymidine was
achieved in the usual manner [14]. The assembly of the oligodeoxyribonucleotides in an Applied-
Biosystems-392 synthesizer was performed with 2-(4-nitrophenyl)ethyl(npe)/[2-(4-nitrophenyl)ethoxy]-
carbonyl(npeoc)-protected 2’-deoxyribonucleoside 3’-(2-cyanoethyl N,N-diisopropylphosphoramidites)
[15].
REFERENCES
[1] T. Maier, W. Pfleiderer, Helv. Chim. Acta 2009, 92, 2722.
[2] M. Sameiro, T. Goncalves, Chem. Rev. 2009, 109, 190.
[3] R. Kuhn. F. Weygand, Ber. Dtsch. Chem. Ges. 1932, 68, 1282.
[4] R. Kuhn, Angew. Chem. 1936, 49, 6.
[5] M. Tishler, K. Pfister. R. D. Babson, K. Ladenburg, A. J. Fleming, J. Am. Chem. Soc. 1947, 69, 1487.
[6] P. Hemmerich, B. Prijs, H. Erlenmeyer, Helv. Chim. Acta 1959, 42, 1604.
[7] H. Goldner, G. Dietz, E. Carstens, Liebigs Ann. Chem. 1966, 694, 142.
[8] F. Yoneda, S. Fukazawa, J. Chem. Soc., Chem. Commun. 1972, 503.
[9] F. Yoneda, S. Matsumoto, Y. Sakuma, J. Chem. Soc., Perkin Trans. 1 1975, 1907.
[10] E. C. Taylor, F. Sowinski, T. Yee, F. Yoneda, J. Am. Chem. Soc. 1967, 89, 3369.
[11] F. Yoneda, Y. Sakuma, M. Ichiba, K. Shinomura, J. Am. Chem. Soc. 1976, 98, 830.
[12] K. P. Stengele, W. Pfleiderer, Nucleic Acids Res., Symp. Ser. 1989, 21, 101.
[13] K. P. Stengele, W. Pfleiderer, Tetrahedron Lett. 1990, 31, 2549.
[14] T. Wagner, W. Pfleiderer, Helv. Chim. Acta 1997, 80, 200.
[15] H. Lang, M. Gottlieb, M. Schwarz, S. Farkas, B. S. Schulz, F. Himmelsbach, R. Charubala, W.
Pfleiderer, Helv. Chim. Acta 1999, 82, 2172.
[16] G. Nꢂbel, W. Pfleiderer, Liebigs Ann. Chem. 1960, 631, 168.
[17] G. Nꢂbel, W. Pfleiderer, Chem. Ber. 1962, 95, 1605.
Received November 23, 2009