Oligonucleotides containing arylacetylene residues: Synthesis and post-synthetic modification via [3+2] cycloaddition

Article abstract of DOI:10.1007/s11172-006-0410-0

The synthesis of a phosphoramidite reagent for 5′-modification of oligonucleotides by introducing an arylacetylene residue has been described. Using the reaction with 3-(perylen-3-yl)propyl azide as an example, it was shown that the acetylene derivatives

Full text of DOI:10.1007/s11172-006-0410-0

Russian Chemical Bulletin, International Edition, Vol. 55, No. 7, pp. 1268—1274, July, 2006
1268
Oligonucleotides containing arylacetylene residues:
synthesis and postꢀsynthetic modification via
[3+2] cycloaddition
A. V. Ustinov and V. A. Korshunꢀ
M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry of the
Russian Academy of Sciences,
16/10 ul. MiklukhoꢀMaklaya, 117997 Moscow, Russian Federation,
Fax: +7 (495) 330 6738. Eꢀmail: korshun@mail.ibch.ru
The synthesis of a phosphoramidite reagent for 5´ꢀmodification of oligonucleotides by
introducing an arylacetylene residue has been described. Using the reaction with 3ꢀ(perylenꢀ3ꢀ
yl)propyl azide as an example, it was shown that the acetylene derivatives of oligonucleotides
synthesized using this reagent undergo CuIꢀcatalyzed [3+2] dipolar cycloaddition. Fluorescent
conjugates were obtained in high yields and characterized by mass spectra.
Key words: modified oligonucleotides, [3+2] cycloaddition, conjugation, acetylenes, azides,
perylene.
The [3+2] cycloaddition of organic azides to terminal
acetylenes, resulting in the formation of 1,2,3ꢀtriazoles
(first described in the 19th century and studied in detail in
the midꢀ20th),1 now attracts increasing attention of reꢀ
searchers as a method of combinatorial chemistry,2,3 in
particular, conjugation.4,5 This process being carried out
under mild conditions in aqueous or organic solutions in
the absence of any coupling agents can be accelerated by
addition of CuIꢀspecies.6,7 The orthogonality of both the
alkyne and azido group with respect to the majority of
functional groups allows a pronounced increase in the
conjugation multiplicity, i.e., in the number of reactions
carried out simultaneously and specifically in one vessel.
However, despite the large potential of this method,
only a few examples of catalyzed [3+2] cycloaddition of
modified oligonucleotides are known to date.8—13
To involve an oligonucleotide into [3+2] cycloaddiꢀ
tion, it is necessary to carry out its modification, namely,
introduction of a terminal acetylenic or azido group. An
azido group can be introduced only postꢀsynthetically,13,14
because organic azides rapidly undergo the Staudinger
reaction15 with the phosphoramidites used in standard
protocols for solidꢀphase automated oligonucleotide synꢀ
thesis (ONS). Unlike azides, acetylenes (including termiꢀ
nal ones) are fully compatible with all reagents used in the
synthetic cycle and the acetylenic group can be introꢀ
duced into oligonucleotides using modifying reagents
in ONS. Although syntheses of oligonucleotide derivatives
containing terminal acetylenes have been reported,16,17
examples of [3+2] cycloaddition involving them are still
unknown. Note that arylethynyl modifications were inꢀ
corporated into oligonucleotides only once.18
Since the method for the synthesis of oligonucleotide
conjugates using [3+2] cycloaddition has not been adꢀ
equately developed, we decided to synthesize a modifying
reagent for introducing a terminal alkyne group into oliꢀ
gonucleotides and to optimize the conjugation conditions.
Results and Discussion
Several reactions of azides with modified oligonucleoꢀ
tides containing an acetylenic fragment have been reꢀ
ported; however, all of these cases dealt with alkylethynyl
rather than arylethynyl fragment. One could expect that
the behavior of alkylꢀ and arylethynyl derivatives in the
[3+2] cycloaddition with azides would differ. Therefore,
we have synthesized phosphoramidite 5 containing an
arylethynyl fragment (Scheme 1).
The Sonogashira coupling of 4ꢀiodobenzoic acid (1)
with trimethylsilylacetylene was used to obtain 4ꢀtriꢀ
methylsilylethynylbenzoic acid (2), which reacted with
transꢀ4ꢀaminocyclohexanol hydrochloride in the presꢀ
ence of diisopropylethylamine and tris(pyrrolidino)benꢀ
zotriazolꢀ1ꢀyloxyphosphonium hexafluorophopsphate
(PyBOP) (as a coupling reagent) to give amide 3. Deꢀ
silylation of the product on treatment with tetrabutylꢀ
ammonium fluoride yielded amide 4 containing a termiꢀ
nal acetylenic fragment. The target reagent 5 was formed
upon phosphitylation of amide 4. Phosphoramidite 5 gives
only one signal in the 31P NMR spectrum. Full assignꢀ
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1220—1226, July, 2006.
1066ꢀ5285/06/5507ꢀ1268 © 2006 Springer Science+Business Media, Inc.

Acetylenic oligonucleotides in [3+2] cycloaddition
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 7, July, 2006
1269
Scheme 1
Reagents and conditions: i. Me3SiC≡CH, Pd(PPh3)4, CuI, Et3N, DMF, 12 ч; HCl; ii. tris(pyrrolidino)benzotriazolꢀ1ꢀyloxyꢀ
phosphonium hexafluorophosphate, EtNPri2, 20 min; transꢀ4ꢀhydroxycyclohexylammonium chloride, EtNPri2,
iii. NBu4F, THF, 10 min; iv. (Pri2N)2PO(CH2)2CN, diisopropylammonium tetrazolide, CH2Cl2, 12 h.
4 h;
ment of the 13C NMR signals of compound 5 was done
ester 7. Its hydrogenation over palladium resulted unexꢀ
1
using 2D heteronuclear H—13C NMR spectra (HMQC
pectedly in unusual product 8 containing a partially hydroꢀ
genated perylene nucleus. Unlike the corresponding
perylene derivatives, compound 8 is readily soluble in
organic solvents, which allows, in particular, its reducꢀ
tion with lithium aluminum hydride to alcohol 9. The
structures of compounds 8 and 9 were confirmed by
and HMBC).
The reagent 5 was used for the synthesis of oligonucleoꢀ
tides ON1 and ON2 (Table 1). The structure of oligoꢀ
nucleotides was confirmed by MALDIꢀTOF mass spectra.
It seemed more challenging to attempt to conjugate
acetyleneꢀcontaining oligonucleotides with nonpolar
azides. We chose peryleneꢀderived azide as the model
compound having a high fluorescence quantum yield, high
chemical stability and photostability. We expected the
conjugates to be easily detected during the polyacrylamide
gel electrophoresis due to bright fluorescence, while the
presence of a nonpolar perylene fragment was expected to
substantially increase the HPLC retention time on a reꢀ
versedꢀphase column.
1
2D heteronuclear H—13C NMR spectra (HMQC and
HMBC). However, treatment of compound 9 with a stanꢀ
dard dehydrogenation reagent, viz., 2,3ꢀdichloroꢀ5,6ꢀ
dicyanoꢀ1,4ꢀbenzoquinone (DDQ), gave aldehyde 10 in
19% yield together with the target product 11 (yield 9%)
and a number of unidentified compounds. Dehydrogenaꢀ
tion of 9 with a milder reagent, Ph3COH and (CF3CO)2O,
in trifluoroacetic acid,19 afforded the desired product in
16% yield. 3ꢀ(Perylenꢀ3ꢀyl)propanol 11 was converted
into the target azide 12 via the corresponding methaneꢀ
sulfonate.
Upon the reaction with stabilized ylide, 3ꢀformylꢀ
perylene 6 (Scheme 2) was converted into unsaturated
Azide 12 was coupled with acetyleneꢀcontaining oliꢀ
gonucleotides ON1 and ON2 (Scheme 3, for ON1). The
reaction was carried out in a triethylammonium acetate
buffer solution in 30% aqueous DMSO (pH 7.0). Copꢀ
per(I) species in a concentration of 1 mmol L–1 generated
in situ by treatment of copper(II) sulfate with ascorbic acid
or tris(2ꢀcarboxyethyl)phosphine were used as the cataꢀ
lysts. Note that in the absence of a catalyst, no even traces
of the conjugate were formed after 24 h at room temperaꢀ
ture or at 60 °C. Meanwhile, in the presence of a catalyst,
the reaction proceeds to completion in 24 h at room temꢀ
perature. According to HPLC, half of the starting oligoꢀ
nucleotide is consumed under the conditions described in
approximately 4 h after addition of the catalyst.
Table 1. Modified oligonucleotides synthesized using reagent 5
(X is the residue 4ꢀp)
Oligoꢀ
nicleoꢀ
tide
The sequence (5´→3´)
(5´→3´)
M,a
found
calculated
τ
.
/minb
ON1
ON2
XꢀCATTACATCCAGAC
XꢀTCTGGATGTAATGG
4495.68
4495.84
4636.78
4636.84
20.0
21.0
a MALDIꢀTOF mass spectrum; M is the mass; the values for the
monoisotopic peak are given.
b Conditions of chromatography: 5% MeCN in 0.1 M aqueous
NH4OAc for 5 min; gradient from 5 to 35% in 30 min and from
35 to 80% in 30 min; τ is the retention time.
It was found that conjugation should be carried out in
an inert atmosphere. When the reaction is carried out in

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Russ.Chem.Bull., Int.Ed., Vol. 55, No. 7, July, 2006
Ustinov and Korshun
Scheme 2
Reagents and conditions: i. ButOCOCH=PPh3, EtOAc, 4 h, ∆; ii. H2, Pd/C, EtOAc, 2 days; iii. LiAlH4, Et2O/THF; iv. 2,3ꢀdichloroꢀ
5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone, PhMe, 100 °C, 30 min; v. Ph3COH, (CF3CO2)O, CF3CO2H, ∆, 12 h; vi. CH3SO2Cl, Et3N, CH2Cl2,
1 h; NaN3, DMF, 12 h.
Scheme 3

Acetylenic oligonucleotides in [3+2] cycloaddition
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 7, July, 2006
1271
Table 2. Properties of the perylene conjugates synthesized by
[3+2] cycloaddition of azide 12 to oligonucleotides containing
an ethynyl group
air, the product yield substantially decreases according to
HPLC, while the starting oligonucleotide is consumed
almost completely (Fig. 1). This may be attributable to
the known20 oxidative degradation of oligonucleotides meꢀ
diated by the CuII/CuI redox pair.
b
Conjuꢀ
gate
Starting
M,a
found
τ
Yc
oligonucleotide
.
/min
(%)
calculated
MeCN (%)
ON3
ON4
ON1
ON2
4833.07
4829.98
4973.98
4971.98
41.5
40.5
68 (28)
71 (30)
70
a
a MALDIꢀTOF mass spectrum; M is the mass; the values for the
monoisotopic peak are given.
60
b Conditions of chromatography are given in the notes to Table 1.
c HPLCꢀbased yields of products. The value in parentheses is
the yield of the product isolated by gel electrophoresis; its amount
was determined by spectrophotometry. The typical loss during
gel electrophoresis isolation of oligonucleotides is about 50%
(see Ref. 21) but the loss can be even higher when operating with
small amounts of oligonucleotides.
50
40
30
20
Fluorescent perylene conjugates were isolated by deꢀ
naturing polyacrylamide gel electrophoresis. The properꢀ
ties and the yields of the products are presented in Table 2.
According to HPLC, the conjugate was the only compoꢀ
nent of the reaction mixture after completion of the proꢀ
cess. Therefore, it is obvious that the low isolated yields of
the oligonucleotides presented in the Table are mainly
due to losses upon gel electrophoresis.
Thus, a modifying reagent for introduction of an ethyꢀ
nyl group into oligonucleotides was synthesized and the
possibility of conjugation of acetyleneꢀcontaining oligoꢀ
nucleotides with hydrophobic aliphatic azides by cataꢀ
lyzed [3+2] cycloaddition was demonstrated.
10
0
10
20
30
τ/min
MeCN (%)
70
b
60
50
40
30
20
Experimental
Tetrakis(triphenylphosphine)palladium,22 3ꢀformylperyꢀ
lene,23 diisopropylammonium tetrazolide,24 and cyanoethoxyꢀ
bis(diisopropylamino)phosphine25 were synthesized by previously
described procedures. Commercially available reagents were
used: transꢀ4ꢀaminocyclohexanol hydrochloride, tetrabutylꢀ
ammonium fluoride trihydrate, 4ꢀiodobenzoic acid, trimethylꢀ
silylacetylene, triphenylmethanol, trifluoroacetic anhydride,
tertꢀbutoxycarbonylmethylenetriphenylphosphorane, 2,3ꢀdiꢀ
chloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone,
tris(2ꢀcarboxyꢀ
ethyl)phosphine, and tris(pyrrolidino)benzotriazolꢀ1ꢀyloxyꢀ
phosphonium hexafluorophosphate (Aldrich, Fluka, Lancaster,
and Avocado). Extra pure grade solvents were distilled prior to
use. The reactions were monitored by TLC on Kieselgel 60 F254
plates (Merck); the spots were visualized in the UV light at
254 nm. Column chromatography was carried out on silica gel
Kieselgel 60 (Merck), particle size 40—63 µm. The solutions
were concentrated on a rotary evaporator in a water jet pump
vacuum at a bath temperature of 30—50 °C. NMR spectra were
recorded on a Bruker ACꢀ500 spectrometer at 500 MHz (1H),
10
0
10
20
30
τ/min
Fig. 1. HPLC of cycloaddition products obtained in air (a) and
in an inert atmosphere (b). The arrow marks the peak of the
starting oligonucleotide. The relative sensitivity is the same in
both cases.

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Russ.Chem.Bull., Int.Ed., Vol. 55, No. 7, July, 2006
Ustinov and Korshun
125.7 MHz (13C), and 202.4 MHz (31P). The spectra were caliꢀ
brated using the residual signals of the DMSOꢀd6 protons
(δH 2.50 and δC 39.7) or CDCl3 (δH 7.25 for 1H and δC 77.0); the
chemical shifts were referred to SiMe4 (1H and 13C) or 85%
20% MeOH—CHCl3 system. Yield 427 mg (99%). Colorꢀ
less crystals, m.p. 220—221 °C (hexane—CHCl3). Rf 0.29
(10% MeOH—CHCl3). 1H NMR (DMSOꢀd6), δ: 1.18—1.28
(m, 2 H); 1.31—1.41 (m, 2 H), (Hax(2), Hax(3), Hax(5), Hax(6));
1.76—1.88 (m, 4 H, Heq(2), Heq(3), Heq(5), Heq(6)); 3.39 (m,
1 H, H(4)); 3.70 (m, 1 H, H(1)); 4.33 (s, 1 H, ≡CH); 4.53 (d,
1 H, OH, J = 4.2 Hz); 7.54 (d, 2 H, H(3´), H(5´), J = 8.3 Hz);
7.83 (d, 2 H, H(2´), H(6´), J = 8.3 Hz); 8.23 (d, 1 H, NH,
J = 7.9 Hz).
transꢀ1ꢀ[(N,NꢀDiisopropylamino)ꢀ(2ꢀcyanoethoxy)phosphinꢀ
oxy]ꢀ4ꢀ(4ꢀethynylbenzoylamino)cyclohexane (5). Diisopropylꢀ
ammonium tetrazolide (240 mg, 1.400 mmol) and bis(diꢀ
isopropylamino)cyanoethoxyphosphine (422 mg, 1.400 mmol)
were added under argon to a stirred suspension of transꢀ4ꢀ
(4ꢀethynylbenzoylamino)cyclohexanol (4) (227 mg, 0.933 mmol)
in dry CH2Cl2 (30 mL). The mixture was kept at room temperaꢀ
ture under argon for 16 h, diluted with CH2Cl2 (50 mL), and
poured in a water (50 mL)—CH2Cl2 (50 mL) mixture. The
organic layer was separated, washed with water (50 mL), a satuꢀ
rated aqueous solution of NaHCO3 (2×50 mL), and again with
water (50 mL), dried with Na2SO4, and concentrated. The resiꢀ
due was chromatographed on silica gel in 10% Me2CO and
1.5% Et3N in PhMe. Yield 369 mg (89%). Colorless crystals,
t.dec. >110 °C. Rf 0.47 (10% Me2CO in PhMe). 1H NMR
(DMSOꢀd6), δ: 1.14 (d, 12 H, CH3, J = 6.7 Hz); 1.42 (m, 4 H,
Hax(2), Hax(3), Hax(5), Hax(6)); 1.80—2.05 (m, 4 H, Heq(2),
Heq(3), Heq(5), Heq(6)); 2.80 (t, 2 H, CH2CN, J = 6.0 Hz);
3.53—3.62 (m, 2 H, Me2CHN); 3.64—3.80 (m, 4 H, H(1),
H(4), CH2CH2CN); 4.33 (s, 1 H, ≡CH); 7.58 (d, 2 H, H(3´),
H(5´), J = 8.3 Hz); 7.87 (d, 2 H, H(2´), H(6´), J = 8.3 Hz); 8.25
(d, 1 H, NH, J = 7.7 Hz). 13C NMR (DMSOꢀd6), δ: 19.89 (d,
H3PO4 (
31P). 2D heteronuclear 1H—13C gradient selected
NMR spectra, HMQC and HMBC, were obtained by using
2048(t2)×256(t1) complex point data sets, zero filled to
2048(F2)×1024(F1). The spectral window widths were 13 and
200 ppm for 1H and 13C, respectively. The HMBC spectra were
recorded with a 50 ms delay for evolution of longꢀrange couꢀ
plings. The melting points were determined on a Boetius hot
stage (not corrected). The oligonucleotide synthesis was carried
out on a BiosSet ASMꢀ700 instrument on a 200 nmol scale by
standard procedures. MALDIꢀTOF mass spectra were measured
on a Bruker Ultraflex spectrometer. A Beckman 153 chromatoꢀ
graph and a Supelco Discovery C18 column (250×4 mm) were
used for HPLC.
(4ꢀTrimethylsilylethynyl)benzoic acid (2). A solution of
4ꢀiodobenzoic acid (2.480 g, 10 mmol) in DMF (15 mL) was
degassed three times by evacuating the flask and then filling it
with argon. In an argon flow, trimethylsilylacetylene (1.83 mL,
13 mmol), triethylamine (4.17 mL, 30 mmol), tetrakis(triꢀ
phenylphosphine)palladium (577 mg, 0.5 mmol), and copper(I)
iodide (190 mg, 1 mmol) were added successively. The mixture
was kept under argon at room temperature for 16 h and partiꢀ
tioned between ethyl acetate (100 mL) and 10% HCl (100 mL).
The organic layer was separated, washed with water (3×100 mL)
and brine (100 mL), dried with Na2SO4, and concentrated.
The residue was chromatographed on silica gel in a 20%
Me2CO—PhMe system, and the reaction product was recrystalꢀ
lized from hexane. Yield 1.293 g (59%). Colorless crystals, m.p.
153—156 °C (hexane), Rf 0.46 (20% Me2CO—PhMe). 1H NMR
(CDCl3), δ: 0.26 (s, 9 H, CH3); 7.54, 8.03 (both d, 2 H each,
CHAr, J = 8.2 Hz).
3
3
CH2CN, JP,C = 6.9 Hz); 24.31 (d, 2 C, Me, JP,C = 6.9 Hz);
3
24.43 (d, 2 C, Me, JP,C = 6.9 Hz); 29.91, 29.97 (C(3), C(5));
32.71 (d, 3JP,C = 3.4 Hz); 32.80 (d, 3JP,C = 3.4 Hz) (C(2), C(6));
2
transꢀ4ꢀ(4ꢀTrimethylsilylethynylbenzoylamino)cyclohexanol
(3). Tris(pyrrolidino)benzotriazolꢀ1ꢀyloxyphosphonium hexaꢀ
fluorophosphate (1.147 g, 2.205 mmol) and diisopropylethylꢀ
amine (0.78 mL, 4.5 mmol) was added to a solution of 4ꢀ(triꢀ
methylsilylethynyl)benzoic acid (491 mg, 2.25 mmol) in DMF
(5 mL). The mixture was stirred for 20 min at ~20 °C, then a
solution of transꢀ4ꢀaminocyclohexanol hydrochloride (319 mg,
2.25 mmol) and diisopropylethylamine (0.78 mL, 4.5 mmol) in
DMF (5 mL) was added. After 4 h, the reaction mixture was
poured in a mixture of water (50 mL) and EtOAc (120 mL). The
organic layer was separated and washed with water (2×50 mL),
a saturated solution of NaHCO3 (50 mL), again water (50 mL),
and brine (50 mL), dried with Na2SO4, and concentrated. The
residue was recrystallized from PhMe—MeOH. Yield 576 mg
(81%). Colorless crystals, m.p. 228—229 °C (PhMe—MeOH).
42.54 (d, PNCH, JP,C = 12.6 Hz); 47.55 (C(4)); 58.01 (d,
2
2
POCH2, JP,C = 17.8 Hz); 71.90 (d, C(1), JP,C = 17.7 Hz);
82.68 (≡CH); 82.96 (C≡CH); 119.05 (CN); 124.25 (C(4´));
127.59 (C(2´), C(6′)); 131.54 (C(3´), C(5´)); 134.84 (C(1´));
164.78 (CO). 31P NMR (DMSOꢀd6), δ: 146.21.
tertꢀButyl transꢀ3ꢀ(perylenꢀ3ꢀyl)acrylate (7). Solid (tertꢀbutꢀ
oxycarbonylmethylene)triphenylphosphorane
(361
mg,
0.96 mmol) was added to a suspension of 3ꢀformylperylene
(192 mg, 0.8 mmol) in EtOAc (75 mL), the mixture was
stirred for 12 h, an additional portion of (tertꢀbutoxycarbonylꢀ
methylene)triphenylphosphorane (90 mg, 0.24 mmol) was added,
and the mixture was refluxed for 4 h. The solution was cooled,
concentrated, and then toluene (100 mL) was added to, and
distilled from the residue, and the residue was chromatographed
on silica gel in CHCl3 and recrystallized from a CHCl3—MeOH
system. Yield 227 mg (75%). Bright red crystals, m.p.
223—225 °C (MeOH—CHCl3). Rf 0.56 (CHCl3). 1H NMR
1
Rf 0.28 (10% Me2CO—CHCl3). H (CDCl3), δ: 0.25 (s, 9 H,
CH3); 1.24—1.34 (m, 2 H); 1.40—1.51 (m, 3 H) (Hax(2), Hax(3),
Hax(5), Hax(6), OH); 2.02, 2.11 (both m, 2 H each) (Heq(2),
Heq(3), Heq(5), Heq(6)); 3.64 (m, 1 H, H(4)); 3.94 (m, 1 H,
H(1)); 5.86 (d, 1 H, NH, J = 7.4 Hz); 7.49, 7.66 (both d,
2 H each, H(2´), H(3´), H(5´), H(6´), J = 8.2 Hz).
transꢀ4ꢀ(4ꢀEthynylbenzoylamino)cyclohexanol (4). A solution
of tetrabutylammonium fluoride (555 mg, 2.121 mmol) in THF
(5 mL) was added to a solution of compound 3 (539 mg,
1.767 mmol) in THF (15 mL). The mixture was stirred for
10 min at ~20 °C, diluted with PhMe (50 mL), and concenꢀ
trated, and the residue was chromatographed on silica gel in a
(CDCl3), δ: 1.58 (s, 9 H, CH3); 6.47 (d, 1 H, H(2´), J2´,3´
=
15.7 Hz); 7.49 (m, 2 H, H(8), H(11)); 7.56 (m, 1 H, H(5)); 7.70
(m, 2 H, H(9), H(10)); 7.74 (d, 1 H, H(2), J = 8.0 Hz); 8.05 (d,
1 H, H(4), J = 8.5 Hz); 8.14—8.26 (m, 4 H, H(1), H(6), H(7),
H(12)); 8.36 (d, 1 H, H(3´), J2´,3´ = 15.7 Hz).
tertꢀButyl 3ꢀ(4,5,6,7,8,9ꢀhexahydroperylenꢀ3ꢀyl)propionate
(8). Compound 7 (1.09 g, 3.00 mmol) and 5% Pd/C (300 mg)
were suspended in EtOAc (150 mL). The suspension was evacuꢀ
ated, flushed with hydrogen, stirred for 48 h in a hydrogen
atmosphere, and filtered through a glass fiber filter. The filtrate

Acetylenic oligonucleotides in [3+2] cycloaddition
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 7, July, 2006
1273
was concentrated and the residue was chromatographed on silica
gel in PhMe. Yield 1.01 g (87%). Yellowish crystals, m.p.
156—160 °C (MeOH—CHCl3), Rf 0.44 (PhMe). 1H NMR
(CDCl3), δ: 1.45 (s, 9 H, CH3); 2.09 (m, 4 H, H(5), H(8)); 2.57
(t, 2 H, H(2´), J2´,3´ = 8.0 Hz); 3.02—3.14 (m, 10 H, H(4),
H(6), H(7), H(9), H(3´)); 7.33 (d, 1 H, H(10), J10,11 = 7.0 Hz);
3ꢀ(Perylenꢀ3ꢀyl)propanol (11). A solution of 3ꢀ(4,5,6,7,8,9ꢀ
hexahydroperylenꢀ3ꢀyl)propanol (130 mg, 0.41 mmol), triꢀ
phenylmethanol (321 mg, 1.23 mmol), and trifluoroacetic anꢀ
hydride (0.23 mL, 1.64 mmol) in CF3CO2H (10 mL) was reꢀ
fluxed for 12 h and concentrated. The residue was dissolved in
MeOH (10 mL) and added to a solution of NaOH (100 mg).
After 1 h, the solution was acidified with AcOH (200 µL) and
concentrated. The residue was treated with PhMe (50 mL),
concentrated, and chromatographed on silica gel (10% Me2CO
in PhMe). Yield 21 mg (16%).
7.39 (d, 1 H, H(2), J1,2 = 8.5 Hz); 7.44 (dd, 1 H, H(11), J10,11
=
7.0 Hz, J11,12 = 8.5 Hz); 8.47 (d, 1 H, H(1), J1,2 = 8.5 Hz); 8.49
(d, 1 H, H(12), J11,12 = 8.5 Hz).
3ꢀ(4,5,6,7,8,9ꢀHexahydroperylenꢀ3ꢀyl)propanol (9). A soluꢀ
tion of tertꢀbutyl ester 8 (920 mg, 2.38 mmol) in an ether (10 mL)
and THF (10 mL) mixture was added dropwise to a stirred
suspension of LiAlH4 (190 mg, 5 mmol) in anhydrous ether
(5 mL) at such a rate that the mixture was maintained at boiling.
Then the mixture was kept for 10 min, propanꢀ2ꢀol (0.5 mL)
and water (5 mL) were added dropwise, and the reaction mixꢀ
ture was poured into Et2O (100 mL) and 10% HCl (50 mL). The
organic layer was washed with water (2×50 mL) and brine
(50 mL), dried with Na2SO4, and concentrated. The residue was
recrystallized from a PhMe—light petroleum mixture. Yield
620 mg (82%). Colorless crystals, m.p. 94—96 °C (PhMe—peꢀ
troleum ether), Rf 0.30 (10% Me2CO in PhMe). 1H NMR
(CDCl3), δ: 1.93 (m, 2 H, H(2´)); 2.05—2.13 (m, 4 H, H(5),
H(8)); 2.91 (t, 2 H, H(3´), J2´,3´ = 7.8 Hz); 3.03—3.13 (m, 8 H,
H(4), H(6), H(7), H(9)); 3.74 (t, 1 H, H(1´), J1´,2´ = 6.3 Hz);
3ꢀ(Perylenꢀ3ꢀyl)propyl azide (12). Methanesulfonyl chloride
(26 µL, 0.34 mmol) and triethylamine (35 µL, 0.25 mmol) were
added successively to a suspension of 3ꢀ(perylenꢀ3ꢀyl)propanol
(52 mg, 0.17 mmol) in CH2Cl2 (5 mL). The reaction mixture
was stirred for 1 h at ~20 °C, the solvent was evaporated, the
residue was dissolved in DMF (5 mL), and sodium azide was
added (82 mg, 1.3 mmol). The mixture was kept for ~16 h and
poured into an H2O (50 mL)—EtOAc (100 mL) mixture. The
organic layer was washed with H2O (2×50 mL), saturated aqueꢀ
ous NaHCO3 (2×50 mL), again H2O (50 mL), and brine
(50 mL), dried with Na2SO4, and concentrated. The residue was
chromatographed on silica gel in PhMe. Yield 49 mg (87%).
Yellow crystals, m.p. 128—130 °C, Rf 0.39 (30% PhMe in hexꢀ
1
ane). H NMR (CDCl3), δ: 2.05 (m, 2 H, H(2´)); 3.11 (t, 2 H,
H(3´), J = 7.6 Hz); 3.39 (t, 2 H, H(1´), J = 6.5 Hz); 7.33 (d,
1 H, H(2), J1,2 = 7.8 Hz); 7.46 (m, 2 H, H(8), H(11)); 7.52 (m,
1 H, H(5)); 7.66 (m, 2 H, H(9), H(10)); 7.84 (d, 1 H, H(4),
J4,5 = 8.5 Hz); 8.11 (d, 1 H, H(1), J1,2 = 7.8 Hz); 8.15 (d, 1 H,
J = 7.3 Hz); 8.18 (d, 1 H, J = 7.6 Hz) (H(7), H(12)); 8.21 (d,
1 H, H(6), J5,6 = 7.3 Hz).
7.31 (d, 1 H, H(10), J10,11 = 7.0 Hz); 7.39 (d, 1 H, H(2), J1,2
=
8.5 Hz); 7.48 (dd, 1 H, H(11), J10,11 = 7.0 Hz, J11,12 = 8.5 Hz);
8.49 (m, 2 H, H(1), H(12)). 13C NMR (CDCl3), δ: 22.83, 23.13
(C(5), C(8)); 27.34, 27.95, 28.33 (C(4), C(6), C(7)); 29.78
(C(3´)); 31.50 (C(9)); 33.40 (C(2´)); 62.63 (C(1´)); 120.63,
120.67 (C(1), C(12)); 124.79 (C(11)); 125.29 (C(10)); 126.90
(C(2)); 128.10 (C(12b)); 128.32 (C(6b´)); 128.86 (C(3a´));
129.09 (2 C, C(6a), C(6b)); 129.61 (C(12a)); 133.39 (C(3a));
136.35 (2 C, C(3), C(9a)).
Oligonucleotide synthesis was carried out in an automated
mode using standard phosphoramidites (dABz, dCBz, dGiBu, dT)
according to manufacturer´s protocols. The condensation of the
terminal phosphoramidite 5 was carried out for 1 min (capping
and removal of the dimethoxytrityl group were omitted). Oligoꢀ
nucleotides were cleaved from the support and deprotected by
treatment with concentrated (28%) ammonia (0.75 mL). After
deprotection, the resulting solution was freezeꢀdried, the resiꢀ
due was dissolved in water (150 µL), and the oligonucleotide
was precipitated with acetone (1.5 mL), centrifuged, washed
with acetone (1 mL), dissolved in 50% aqueous formamide
(200 µL), and subjected to electrophoresis in 20% denaturing
(7 M urea) polyacrylamide gel (20 сm, 400 V, 20 mA, Broꢀ
mophenol Blue and Xylene Cyanol as leading dyes). Oligoꢀ
nucleotides were visualized in the gel by absorption, eluted with
0.5 M LiClO4 for 12 h, filtered to remove the gel, and desalted
on Sephadex Gꢀ50 columns. The oligonucleotide concentration
was determined by spectrophotometry by measuring absorption
at 260 nm. The mass spectra of the oligonucleotides were reꢀ
corded using a 1 : 1 (v/v) freshly prepared mixture of solutions of
2,6ꢀdihydroxyacetophenone (40 mg in 1 mL of methanol) and
diammonium citrate (80 mg in 1 mL of water) as the ionization
matrix for MALDIꢀTOF.
transꢀ3ꢀ(Perylenꢀ3ꢀyl)acrolein (10) and 3ꢀ(perylenꢀ3ꢀyl)proꢀ
panol (11). A solution of 3ꢀ(4,5,6,7,8,9ꢀhexahydroꢀ3ꢀperylꢀ
enyl)propanol 9 (474 mg, 1.50 mmol) and 1,2ꢀdichloroꢀ5,6ꢀ
dicyanobenzoꢀ1,4ꢀquinone (1.362 g, 6 mmol) in PhMe (15 mL)
was kept for 30 min at 100 °C. The solution was cooled, 6 M
NaOH (20 mL) was added, and the mixture was filtered through
celite. The organic layer was separated, washed with water
(2×50 mL), dried with Na2SO4, and concentrated. The residue
was chromatographed on silica gel (4→8% Me2CO in PhMe).
The yield of 3ꢀ(perylenꢀ3ꢀyl)propanol (11) was 40 mg (9%).
Yellow crystals, m.p. 143—145 °C, Rf 0.20 (20% Me2CO in
1
PhMe). H NMR (DMSOꢀd6), δ: 1.80—1.87 (m, 2 H, H(2´));
3.04 (t, 2 H, H(3´), J2´,3´ = 7.6 Hz); 3.53 (t, 2 H, H(1´), J1´,2´
=
6.4 Hz); 4.56 (br.s, 1 H, OH); 7.41 (d, 1 H, H(2), J1,2 = 7.9 Hz);
7.49—7.55 (m, 2 H, H(8), H(11)); 7.58 (m, 1 H, H(5)); 7.76 (m,
2 H, H(9), H(10)); 7.96 (d, 1 H, H(4), J4,5 = 8.5 Hz); 8.27 (d,
1 H, H(1), J = 7.9 Hz); 8.30 (d, 1 H, J = 7.3 Hz), 8.34 (d, 1 H,
J = 7.6 Hz) (H(7), H(12)); 8.37 (d, 1 H, H(6), J5,6 = 7.3 Hz).
The yield of transꢀ3ꢀ(perylenꢀ3ꢀyl)acrolein (10) was 88 mg
(19%). Red crystals, m.p. 234—236 °C, Rf 0.47 (10% Me2CO in
Conjugation (general procedure). Dimethyl sulfoxide (15 µL),
an aqueous solution of triethylammonium acetate (0.2 M, pH 7,
32 µL), a solution of azide 12 in DMSO (1.19 mmol L–1
,
16.8 µL), and an aqueous solution of copper(II) sulfate
pentahydrate (7.94 mM, 12.6 µL) were mixed in a 1.5 mL disꢀ
posable plastic tube. The solution was degassed by purging with
argon for 30 s, and then an aqueous solution of ascorbic acid
(12.5 mM, 8.0 µL) and a solution of the oligonucleotide (0.1 mM,
PhMe). 1H NMR (CDCl3), δ: 6.87 (dd, 1 H, H(2´), J2´,3´
=
15.6 Hz, J1´,2´ = 7.6 Hz); 7.51 (m, 2 H, H(8), H(11)); 7.59 (m,
1 H, H(5)); 7.71 (d, 1 H, J = 8.2 Hz), 7.74 (d, 1 H, J = 8.2 Hz)
(H(9), H(10)); 7.79 (d, 1 H, H(2), J1,2 = 7.9 Hz); 8.01 (d, 1 H,
H(4), J4,5 = 8.5 Hz); 8.16 (d, 1 H, H(1), J1,2 = 7.9 Hz);
8.19—8.27 (m, 4 H, H(6), H(7), H(12), H(3´)); 9.84 (d, 1 H,
CHO, J1´,2´ = 7.6 Hz).

1274
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 7, July, 2006
Ustinov and Korshun
20 µL) were added. The tube was purged with argon once again,
closed, and kept at room temperature for 24 h. The oligonucleoꢀ
tide was precipitated with acetone (1.4 mL) and centrifuged.
The precipitate was dissolved in 50% aqueous formamide (15 µL)
and subjected to electrophoresis in 20% denaturing (7 M urea)
polyacrylamide gel (20 сm, 550 V, 15 mA, Bromophenol Blue
and Xylene Cyanol as leading dyes). The oligonucleotides were
visualized in the gel based on fluorescence, eluted with 0.5 M
LiClO4 for 12 h, filtered to remove the gel, and desalted on
Sephadex Gꢀ50 columns.
7. V. V. Rostovtsev, L. G. Freen, V. V. Fokin, and K. B.
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The authors are grateful to Z. O. Shenkarev and K. D.
Nadezhdin (Institute of Bioorganic Chemistry of the RAS
(IBCh RAS)) for recording the NMR spectra, D. G.
Alekseev (IBCh RAS) for recording the MALDIꢀTOF
mass spectra, M. V. Kvach and S. V. Gontarev (Institute
of PhysicoꢀOrganic Chemistry, National Academy of Sciꢀ
ences of the Republic of Belarus, Minsk) for the help in
oligonucleotide synthesis, M. V. Skorobogatyi and A. D.
Malakhov (IBCh RAS) for participation in experiments,
and to I. A. Stepanova (IBC RAS) for the help in manuꢀ
script preparation.
This work was supported by the Russian Foundation
for Basic Research (Projects Nos. 03ꢀ03ꢀ32196 and 06ꢀ04ꢀ
81019). The Russian Foundation for Basic Research also
supported the NMR Spectroscopy Center of the IBC RAS
(Projects No. 98ꢀ03ꢀ08).
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Received November 11, 2005;
in revised form May 29, 2006