Synthesis of biotin-containing phosphoramidite linker with polyether spacer arm

Article abstract of DOI:10.1080/15257770.2011.587702

A phosphoramidite linker unit, based on glycerol backbone and containing a biotin residue attached through a tetraethylene glycol spacer arm, was synthesized. DMTr-Glycidol and tetraethylene glycol were used as starting materials. After conversion of one of hydroxy groups in tetraethylene glycol into an amino group, the epoxy cycle in DMTr-glycidol was opened by this amino alcohol, resulting in the corresponding ether and some quantity of secondary amine. After attaching of biotin residue to the ether followed by phosphitylation, the desirable linker was obtained. The structure of the linker was confirmed by ¹H-¹H COSY, ¹H-¹³C HSQC, ¹H-¹³C HMBC, ¹H-¹⁵N HSQC, and ¹H-¹⁵N HMBC spectra. The resulted phosphoramidite linker unit is suitable for use in common DNA synthesizers. This approach can be used for preparation of various modifiers containing reporter groups attached to the primary amino function using conventional procedures. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.587702

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Synthesis of Biotin-Containing
Phosphoramidite Linker with Polyether
Spacer Arm
Dr Alexey Kayushin a , Dr Alexandra Demekhina a , Dr Maria
Korosteleva a , Professor Anatoly Miroshnikov a & Professor Alex
Azhayev b
a Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, Moscow, Russia
b School of Pharmacy, Faculty of Health Sciences, University of
Eastern Finland, Kuopo, Finland
Version of record first published: 02 Sep 2011.
To cite this article: Dr Alexey Kayushin, Dr Alexandra Demekhina, Dr Maria Korosteleva, Professor
Anatoly Miroshnikov & Professor Alex Azhayev (2011): Synthesis of Biotin-Containing Phosphoramidite
Linker with Polyether Spacer Arm, Nucleosides, Nucleotides and Nucleic Acids, 30:7-8, 490-502
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Nucleosides, Nucleotides and Nucleic Acids, 30:490–502, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.587702
SYNTHESIS OF BIOTIN-CONTAINING PHOSPHORAMIDITE
LINKER WITH POLYETHER SPACER ARM
Alexey Kayushin,1 Alexandra Demekhina,1 Maria Korosteleva,1
Anatoly Miroshnikov,1 and Alex Azhayev2
1Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy
of Sciences, Moscow, Russia
2School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopo,
Finland
A phosphoramidite linker unit, based on glycerol backbone and containing a biotin residue at-
2
tached through a tetraethylene glycol spacer arm, was synthesized. DMTr-Glycidol and tetraethylene
glycol were used as starting materials. After conversion of one of hydroxy groups in tetraethylene
glycol into an amino group, the epoxy cycle in DMTr-glycidol was opened by this amino alcohol, re-
sulting in the corresponding ether and some quantity of secondary amine. After attaching of biotin
residue to the ether followed by phosphitylation, the desirable linker was obtained. The structure of
the linker was confirmed by 1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC, 1H-15N HSQC, and 1H-
15N HMBC spectra. The resulted phosphoramidite linker unit is suitable for use in common DNA
synthesizers. This approach can be used for preparation of various modifiers containing reporter
groups attached to the primary amino function using conventional procedures.
Keywords Biotin; phosphoramidite; linker; polyether spacer arm
INTRODUCTION
Oligonucleotide and polynucleotide probes containing biotin residues
are widely used in diagnostics as well as in scientific research. Presently a
number of various approaches for the introduction of biotin residue into
oligonucleotides were reported. Biotin may be linked to the heterocyclic
base of a nucleoside using a diethyleneglycol-based spacer arm.[1] But the
modification of the base can change the hybridization properties of a probe.
Moreover, in this construction, the biotin residue is located in the inner
Received 9 March 2011; accepted 9 May 2011.
The authors would like to thank Professor Jouko Vepsa¨la¨inen (University of Eastern Finland) and
Dr. T. Balashova (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
Sciences) for help with NMR spectra.
Address correspondence to Alexey Kayushin, Shemyakin and Ovchinnikov Institute of Bioorganic
Chemistry, Russian Academy of Sciences, Mikluho-Maklaya st, 16/10, Moscow 117997, Russia. E-mail:
kaushin@mail.ibch.ru
490

Biotin-Containing Phosphoramidite Linker
491
part of a double helix; this can prevent the formation of biotin–avidin or
biotin–streptavidin complex.
The 5ꢁ-terminus of oligonucleotide can be modified with primary
aliphatic amine followed by attaching biotin residue.[2] Using this method,
only 5ꢁ-labeled oligonucleotides can be obtained and the additional postsyn-
thesis step of biotinylation of an oligonucleotide is required.
An alternative approach to the synthesis of biotinylated oligonucleotides
is incorporation of modifiers directly into the oligonucleotide chain. Cur-
rently, the modifiers based on 1,2-diol[3] and 1,3-diol[4] structures are most
popular. Those modifiers allow inclusion of several biotin residues into any
location of an oligonucleotide chain, increasing the sensitivity of hybridiza-
tion analysis, and providing a possibility of 3ꢁ-terminal labeling. It should be
noted that incorporation of 1,2-diol residues into an oligonucleotide chain
destabilizes a duplex;[5] thus, from a practical point of view the quantity of
biotin residues that can be incorporated using this approach is restricted.
Glen Research Corporation offers a product named BiotinTEG Phos-
phoramidite (Cat. no. 10-1955-10-1955-xx). This modifier is based on the
1,2-diol structure and was successfully used for various purposes (see, for
instance, Ebhardt & Unrau; Bai et al.; Gronewold et al.[6–8]). Herewith, we
describe some features of synthesis of BiotinTEG Phosphoramidite (8) and
suggest a simple, inexpensive, and convenient approach to prepare this com-
pound. It should be noted that this approach can be used for synthesis of
modifiers containing any reporter groups (for instance, fluorescent and spin
labeled), which can be attached to the primary amino group.
RESULTS AND DISCUSSION
The scheme for preparing 8 is shown in Scheme 1.
It is known that the epoxy cycle can be opened by primary alcohols in the
presence of bases yielding ethers of primary alcohols (see, for instance, Chit-
wood & Freure[9]). So we have used 1-dimethoxytrityl-2,3-epoxypropanol
(DMTr-glycidol) (2) and tetraethylene glycol as starting materials. DMTr-
Glycidol has been synthesized in accordance with Zhang et al.[10] with some
modifications, allowing an increase in the total yield of the product.
Using a monomethyl ether of ethylene glycol as a model, we have found
that the epoxy cycle is not opened in the presence of K2CO3, sodium tert-
butylate, or sodium. But in the presence of NaOH and under heating at
80◦C in dioxane the reaction proceeds well. We have used these conditions
in the further synthesis.
Azido alcohol 1 has been synthesized by reaction of tetraethylene gly-
col with methanesulfonylchloride followed by treatment with sodium azide.
In general, we used conditions described in Bertozzi and Bednarski,[11]
with some enhancements, which allow a decrease in the quantity of a side

492
A. Kayushin et al.
SCHEME 1 The synthesis of biotin-containing phosphoramidite linker with a polyether spacer arm.
product—diazido derivative. After purification by chromatography on silica
gel, 1 was obtained with a 72% yield.
Our next plan was to prepare compound 4 from 1 and 2. After standard
reduction of azido function to amino function, we expected to obtain a
synthon suitable for the attachment of biotin, resulting in compound 7.
However, we have found that azide 1 is decomposed upon heating with
NaOH in dioxane, leading to some unknown substances. Therefore, azide
1 has been converted into amine 3 using PPh3 in aqueous dioxane (yield
95%).
Any attempts to protect the amino group in 3 before reaction with
2 were unsuccessful. We could not use protective groups [such as tert-
butyloxycarbonyl (BOC)] that require acidic conditions for removal due
to the presence of an acid labile protective group in 5. We have tested a
number of acyl protective groups (residues of acetic, monochloroacetic,
dichloroacetic, 2,4-dichlorophenoxyacetic, benzoic, and 4-chlorobenzoic

Biotin-Containing Phosphoramidite Linker
493
acids), and all of them proved to be unstable under heating with NaOH
in dioxane. So we decided to perform the reaction of 2 and 3 without pro-
tection of an amino group.
The reaction of 2 and 3 under the conditions mentioned above (NaOH,
dioxane, 80◦C, 10 hours) resulted in a mixture of two compounds that can
be separated by chromatography on silica gel. Both compounds had molec-
1
ular masses expected for 5 and very similar H NMR spectra. It appeared
obvious that both products were resulted from two competing reactions of
epoxide 2 with the hydroxy group or amino group in 3, resulting in 5 or 6,
respectively. In order to unambiguously assign structures to isomeric 5 and
6, some chemical methods have been used.
First, both compounds were exhaustively acetylated. A mass spectrum of
an acetyl derivative of 5 contained signals with m/z 676.37 and 692.33, corre-
sponding to the [M + Na]+ and [M + K]+ of diacetyl derivative of 5 (676.31
and 692.28). A mass spectrum of acetyl derivative of 6 contained signals
with m/z 718.41 and 734.39, corresponding to [M + Na]+ and [M + K]+
of triacetyl derivative of 6 (718.32 and 734.29). This confirms the expected
structure of 5 and 6 (primary amine and primary alcohol, respectively).
Second, 5 (in contrast to 6) reacts with salicylic aldehyde under conditions
described in Khomutov et al.[12] The latter reaction is specific for primary
amino groups and additionally confirms the structure of 5. The ratio of 5 to
6 was nearly 6:1.
Compound 7 was synthesized by reaction of 5 and N-oxysuccinimide
ester of biotin (Biotin-NHS) in the presence of Et3N in dimethylformamide
(DMF) at room temperature. We have modified the traditional approach to
the synthesis of Biotin-NHS[13] using conditions of another reaction and an-
other method of recrystallization. As a result, we obtained a purer substance
(m.p. 206◦C–207◦C in contrast with 196◦C–200◦C in Becker et al.[13]).
It should be noted that mobilities of Biotin-NHS and 7 on silica gel are
very similar; thus, we added a little bit of NH4OH to the reaction mixture
after completing of the reaction to destroy an excess of Biotin-NHS.
1
1
The structure of 7 was proved by H-1H COSY, H-13C HSQC, and
1H-13C HMBC spectra. On the basis of these spectra, structure of the
studied molecule was unambiguously proved to be 7 because in the H
1
spectrum there are three amide protons (–CONH–) present. As follows
1
from the H-13C HMBC spectrum, two of these amide protons are in-
corporated into the biotin ring and the third one is located between
the biotin side chain carbonyl moiety and the polyethylene glycol chain
(–OCH2CH2NHCOCH2CH2–).
Finally, the target phosphoramidite 8 has been synthesized using a
bis(diisopropylamino)-2-cyanoethoxyphosphine as a phosphitylating agent
and tetrazole as an activator. Compound 8 was isolated by reversed-phase
column chromatography as a white foam with a yield of 68%. The struc-
1
ture of 8 was fully characterized and confirmed by various H-X correlated

494
A. Kayushin et al.
FIGURE 1 1H-15N HSQC spectrum of 8 (DMSO-d6).
NMR spectra. The most important details of those spectra are described
below.
1
In Figure 1, the H-15N HSQC spectrum of 8 is shown. Only three
crosspeaks are observed. Two of them (6.38/79.98 and 6.32/88.77) corre-
spond to NH groups of biotin. As can be seen from Figure 2 (1H-15N HMBC
spectrum of 8), N3 of biotin (δ 79.98) correlates with H3 (δ 6.38) and H4
of biotin (δ 3.09), whereas N1 (δ 88.77) correlates with H1 (δ 6.32) and H6
FIGURE 2 1H-15N HMBC spectrum of 8 (DMSO-d6).

Biotin-Containing Phosphoramidite Linker
495
FIGURE 3 1H-1H COSY spectrum of 8 (DMSO-d6).
(δ 2.81 and 2.57). All this confirms that 8 does contain the unsubstituted
biotin ring system.
The third crosspeak (7.78/112.47) corresponds to the NH-group
between the carbonyl group of the biotin side chain and polyether
chain (−OCH2CH2NHC(O)CH2CH2CH2−). This nitrogen correlates
with protons in CH2 group in polyether chain (δ 3.38 and 3.17).
From the 1H-1H COSY spectrum (see Figure 3), it is clear that
protons with δ 7.78, 3.17, and 3.38 form an isolated spin sys-
1
tem. In accordance with the H-13C HMBC spectrum (see Figure 4),
the carbon atom of the carbonyl group (δ 172.53) correlates with the fol-
lowing protons: −OCH2CH2NHC(O)CH2CH2CH2− (δ 1.50), −OCH2CH2
NHC(O)CH2CH2CH2− (δ 2.06), −OCH2CH2NHC(O)CH2CH2CH2−
(δ 7.78), and −OCH2CH2NHC(O)CH2CH2CH2− (δ 3.17). All this confirms
the correctness of attaching of a biotin moiety to the polyether chain.
The presence of the (diisopropyl)phosphoroamidite system in 8 is con-
1
firmed by the H-15N HMBC spectrum (see Figure 2). Crosspeaks between
CH3 protons (δ 1.15, 1.12, and 1.01) and 15N (δ 98.43 and 97.45) are ob-
served. Two signals of 15N are caused by the presence of a chiral P-atom.
Those results confirm the expected structure of this part of molecule.

496
A. Kayushin et al.
FIGURE 4 Part of 1H-13C HMBC spectrum of 8 (DMSO-d6).
CONCLUSION
We developed a preparative method of synthesis of the phosphoramidite
unit, which is based on glycerol backbone and contains a biotin residue
attached through a tetraethylene glycol spacer arm. It was found that the
epoxy cycle in DMTr-glycidol can be opened by amino alcohol, resulting in
a mixture of both ether and secondary amine. Using this generic approach,
various modifiers containing reporter groups, attached via primary amino
function, can be prepared. Those modifiers can be used for incorporation
of several reporter groups into the oligonucleotide chain without affecting
the nucleic base structure.
EXPERIMENTAL
General Materials and Methods
Unless otherwise noted, the materials were obtained from com-
mercial suppliers and used without any purification. Tetraethylene gly-
col, dimethoxytritylchloride, methanesulfonylchloride, sodium azide, N,Nꢁ-
dicyclohexylcarbodiimide, 1H-tetrazole, and triphenylphosphine were pur-
chased from Fluka (Buchs, Switzerland). N-oxysuccinimide was obtained
from Fluka and recrystallized from ethyl acetate. Glycidol and biotin were
purchased from Aldrich (St. Louis, Mo, USA). Bis(diisopropylamino)-2-
cyanoethoxyphosphine was obtained from Metkinen Chemistry (Turku,
Finland). Triethylamine and acetonitrile were distilled over calcium hy-
dride. Diethyl ether and dioxane were distilled over sodium. Thin layer
chromatography (TLC) was performed on silica gel plates (TLC Sil-
ica gel 60 F254, Merck, Darmstadt, Germany). Column chromatogra-
phy was performed on Silica gel 60 (Fluka) or on Separon SGX C18

Biotin-Containing Phosphoramidite Linker
497
60 (Tessek Ltd., Prague, Czech Republic). High performance liquid
chromatography (HPLC) was performed on the Waters system (Waters
1525, Waters 2489, Breeze 2) using column Nova Pack C18, 4.6 ×
150 mm. NMR spectra were recorded on Bruker AMX-400 and
Bruker Avance II 700 spectrometers (Bruker BioSpin, Rheinstetten,
Germany) in DMSO-d6 or CDCl3 at 30◦C. Chemical shifts in ppm
(δ) are measured relative to the residual solvent signals as internal
standards (CDCl3 7.26, DMSO-d6 2.50). Coupling constants (J ) are mea-
sured in Hz. Mass spectra MALDI-TOF were recorded on Ultraflex
TOF/TOF (Bruker Daltonics, Bremen, Germany) using THAP as a matrix.
Mass spectra [electrospray ionization (ESI)] were recorded on Esquire3000
Plus (Bruker Daltonics, Germany). Infrared (IR) spectra were recorded on
Hitachi 270-30 Infrared Spectrophotometer (Hitachi, Tokyo, Japan).
Syntheses
1-Azido-11-Hydroxy-3,6,9-Trioxaundecane (1)
Tetraethylene glycol (16.8 mL, 103 mmol) and anhydrous triethylamine
(20 mL, 252 mmol) were dissolved in 100 mL of diethyl ether and cooled in
an ice bath. A solution of methanesulfonylchloride (4.0 mL, 52 mmol) in 16
mL of diethyl ether was added dropwise under cooling during 2 hours. The
reaction mixture was allowed to warm up to the room temperature, the sol-
vent was evaporated, and the residue was coevaporated with ethanol. The re-
sulting oil was dissolved in 120 mL of ethanol, 7.210 g (111 mmol) of sodium
azide was added, and the suspension was heated at reflux for 10 hours. The
reaction progress was controlled by TLC in chloroform–methanol, 9:1.
The crystals were filtered off, the filtrate was concentrated, and the
residue was dissolved in 30 mL of semi-saturated aqueous NaCl. The re-
sulting solution was washed by diethyl ether, and the ether layer was washed
by semi-saturated aqueous NaCl (5 × 25 mL). The aqueous layers were com-
bined and washed by chloroform (4 × 100 mL). The organic layers were
combined, dried by sodium sulfate, and concentrated to oil. The desirable
product was purified by column chromatography, column 3.5 × 31 cm,
elution by chloroform (300 mL) followed by 3% methanol in chloroform,
1
flow rate 6.8 mL/minute. Yield 8.102 g (37 mmol, 72%), viscous oil. H
NMR (CDCl3, 400 MHz): 3.72 (m, 2H, CH2 OH), 3.67–3.64 (m, 10H,
O CH2 CH2 O , CH2 CH2 N3), 3.60 (m, 2H, CH2 CH2 OH),
3.37 (t, 2H, J = 5.3, CH2 CH2 N3), 2.47 (bs, 1H, OH). MALDI (m/z):
[(M + Na)+] calculated for C8H17N3O4Na 242.11, found 241.97; [(M +
K)+] calculated for C8H17N3O4K 258.08, found 257.97.
DMTr-Glycidol (2)
Glycidol (2.4 mL, 36 mmol) and triethylamine (7.5 mL, 54 mmol) were
dissolved in 50 mL of dioxane. Dimethoxytritylchloride (10.170 g, 30 mmol)

498
A. Kayushin et al.
was dissolved in dioxane, the solution was concentrated up to the beginning
of a precipitate formation, and the resulting solution was added to a solution
of glycidol and triethylamine under argon and cooled in an ice bath. The
reaction progress was controlled by TLC in dichloromethane. In 1 hour,
1.5 mL of methanol was added to the reaction mixture. In 1 hour, the
precipitate was filtered off, the filtrate was concentrated, the residue was twice
coevaporated with chloroform, and the residue was dissolved in chloroform
and washed by 50 mL of 0.1 M triethylammonium bicarbonate (TEAB).
The aqueous layer was washed by chloroform (3 × 50 mL), and combined
organic layers were washed by 0.1 M TEAB (2 × 50 mL), dried by Na2SO4, and
concentrated. The desired product was isolated by column chromatography
(2.5 × 33 cm, elution by dichloromethane, flow rate 6.8 mL/minute). Yield
10.529 g (28 mol, 93%), viscous oil. IR (thin film): 2910, 1590, 1430, 1260,
1
1100, 800, 710 cm . 1H NMR (DMSO-d6, 400 MHz): 7.38 (m, 2H, DMTr),
7.28 (m, 7H, DMTr), 6.89 (m, 4H, DMTr), 3.74 (s, 6H, O CH3), 3.27
(m, 2H, CH2 O DMTr), 3.12 (m, 1H, CH), 2.71 (dd, 1H, J = 4.3, 5.0,
O CH2 CH ), 2.56 (dd, 1H, J = 2.7, 5.1, O CH2 CH ).
1-Amino-11-Hydroxy-3,6,9-Trioxaundecane (3)
To the solution of 1 (5.900 g, 27 mmol) in 40 mL of dioxane, 4 mL
of water and 7.770 g (27 mmol) of triphenylphosphine were added and the
reaction mixture was incubated for 10 hours at room temperature. The reac-
tion progress was controlled by TLC in chloroform–methanol, 7:1; the chro-
matograms were visualized using 0.3% solution of ninhydrin in methanol or
by heating. The reaction mixture was concentrated up to minimal volume,
and the residue was dissolved in 100 mL of water and left at 5◦C for 10 hours.
The crystals were filtered off, and the filtrate was concentrated and dried.
Yield 4.943 г (26 mmol, 95%), viscous oil. 1H NMR (CDCl3, 400 MHz): 3.69
(m, 2H, CH2 OH), 3.65–3.63 (m, 8H, O CH2 CH2 O), 3.58 (m, 2H,
O CH2 CH2 OH), 3.51 (t, 2H, J = 5.2, CH2 CH2 NH2), 2.84 (t,
2H, J = 5.3, CH2 NH2), 2.41 (bs, 3H, OH, NH2). MALDI (m/z): [(M
+ H)+] calculated for C8H20NO4 194.14, found 193.95; [(M + Na)+] cal-
culated for C8H19NNaO4 216.12, found 215.93; [(M + K)+] calculated for
C8H19KNO4 232.10, found 231.92.
1-Amino-14-Hydroxy-15-(O-DMTr)-3,6,9,12-Tetraoxapentadecane (5)
Compound 3 (15.456 g, 80.0 mmol) was twice coevaporated with diox-
ane up to a minimal volume; the reaction flask was filled with argon af-
ter each evaporation. Compound 2 (15.040 g, 40.0 mmol) was twice co-
evaporated with dioxane up to a minimal volume; the reaction flask was
filled with argon after each evaporation. The residue was dissolved in 100
mL of dioxane, added dropwise to the solution of 3, and 0.500 g (12.5
mmol) of powdered NaOH were added to the mixture. The reaction was

Biotin-Containing Phosphoramidite Linker
499
kept at 80◦C for 10 hours. The reaction progress was controlled by TLC
in dichloromethane and in dioxane-NH4OH (95:5); the chromatograms
were visualized using 0.3% solution of ninhydrin in methanol or by heat-
ing. The reaction mixture was concentrated; the residue was dissolved in
200 mL of dichloromethane and washed by 10% aqueous citric acid and by
semi-saturated aqueous NaHCO3. Organic layer was dried by Na2SO4 and
concentrated. The desired product was isolated by column chromatography
(3.5 × 36 cm, elution by gradient of methanol in chloroform, 10%–30%,
1600 mL, flow rate 6.8 mL/min.) Yield 6.056 g (10.6 mmol, 27%), vis-
1
cous oil. H NMR (DMSO-d6, 400 MHz): 7.42 (m, 2H, DMTr), 7.32 (m,
7H, DMTr), 6.90 (m, 4H, DMTr), 4.81 (bs, 1H, CH OH), 3.77-3.71 (m,
1H, CH OH, 6H, O CH3), 3.50–3.45 (m, 12H, O CH2 CH2 O ),
3.42–3.38 (m, 4H, O CH2 CH OH, O CH2 CH2 NH2), 2.95 (m,
2H, DMTr O CH2 CH), 2.87 (m, 2H, CH2 NH2), 1.89 (bs, 2H,
CH2 NH2). MALDI (m/z): [(M + H)+] calculated for C32H44NO8
570.31, found 570.30; [(M + Na)+] calculated for C32H43NNaO8 592.29,
found 592.28; [(M + K)+] calculated for C32H43KNO8 608.26, found
608.26.
1
Side product 6: Yield 1.024 g (1.8 mmol, 2%), viscous oil. H NMR
(DMSO-d6, 400 MHz): 7.39 (m, 2H, DMTr), 7.26 (m, 7H, DMTr), 6.88 (m,
4H, DMTr), 4.84 (br, 1H, OH), 4.51 (br, 1H, OH), 3.74 (m, 6H, O CH3),
3.71 (m, 1H, CH OH), 3.50–3.49 (m, 12H, O CH2 CH2 O ,
CH2 NH , CH2 OH), 2.91 (m, 2H, DMTr O CH2 CH ), 1.15 (m,
1H, NH). MALDI (m/z): [(M + H)+] calculated for C32H44NO8 570.31,
found 570.40; [(M + Na)+] calculated for C32H43NNaO8 592.29, found
592.37; [(M + K)+] calculated for C32H43KNO8 608.26, found 608.36.
N-Oxysuccinimide Ester of Biotin (Biotin-NHS)
Biotin (9.000 g, 36.9 mmol) was suspended in 300 mL of DMF. N-
oxysuccinimide (4.242 g, 36.9 mmol) and N,N’-dicyclohexylcarbodiimide
(7.590 g, 36.9 mmol) were added to the suspension, and the reaction mix-
ture was kept at room temperature under stirring. In 24 hours, 1 mL of water
was added, the precipitate was filtered off, the filtrate was concentrated up
to the beginning of precipitate formation, and the residue was recrystal-
lized from mixture of DMF and isopropanol (nearly 7:3) under heating up
to 100◦C. The crystals were washed by isopropanol and diethyl ether and
1
dried. Yield 9.879 g (28.9 mmol, 79%). m.p. 206–207◦C. H NMR (DMSO-
d6, 400 MHz): 6.38 (s, 1H, NH of biotin), 6.33 (s, 1H, NH of biotin), 4.31
(m, 1H, H-6a of biotin), 4.15 (m, 1H, H-3a of biotin), 3.10 (m, 1H, H-4 of
biotin), 2.85 (d, 1H, J = 5.1, H-6 of biotin), 2.81 (bs, 4H, CH2 of succin-
imide), 2.67 (t, 2H, J = 7.4, C(O) CH2 CH2), 2.59 (d, 1H, J = 12.4,
Hꢁ-6 of biotin), 1.69–1.38 (6H, CH2 CH2 CH2 ). MALDI (m/z): [(M +

500
A. Kayushin et al.
H)+] calculated for C14H20N3O5S 342.11, found 342.10; [(M + Na)+] calcu-
lated for C14H19NaN3O5S 364.09, found 364.10; [(M + K)+] calculated for
C14H19KN3O5S 380.07, found 381.40.
Biotin Derivative of 5 (7)
Compound 5 (10.759 g, 18.9 mmol) was dissolved in 170 mL of DMF and
triethylamine (2.9 mL, 20.8 mmol) and Biotin-NHS (7.090 g, 20.8 mmol)
were added to the solution. The reaction progress was controlled by TLC in
chloroform–methanol, 7:3. In 1 hour, 2.5 mL of aqueous 25% NH4OH was
added to the reaction mixture; in 10 minutes, the mixture was concentrated,
the residue was dissolved in chloroform, washed by water (3 × 100 mL),
dried by Na2SO4, and concentrated. The target product was isolated by
column chromatography (3.5 × 36 cm, gradient of methanol in chloroform,
0%–12%, 1100 mL, flow rate 10.2 mL/minute). Yield 10.083 g (12.6 mmol,
1
68%), white foam. H NMR (DMSO-d6, 400 MHz): 7.76 (t, 1H, J = 5.6,
CH2 NH C(O) CH2 ), 7.40 (m, 2H, DMTr), 7.25 (m, 7H, DMTr),
6.88 (m, 4H, DMTr), 6.37 (s, 1H, NH of biotin), 6.31 (s, 1H, NH of biotin),
4.80 (d, 1H, J = 5.4, CH OH), 4.30 (m, 1H, H-6a of biotin), 4.12 (m, 1H,
H-3a of biotin), 3.76–3.73 (m, 1H, CH OH, 6H, O CH3), 3.49–3.47
(m, 12H, O CH2 CH2 O ), 3.40–3.37 (m, 4H, O CH2 CH OH,
O CH2 CH2 NH ), 3.18 (m, 2H, O CH2 CH2 NH ), 3.12–2.95
(m, 3H, H-4 of biotin, DMTr O CH2 CH ), 2.81 (dd, 1H, J = 12.4,
J = 5.1, H-6 of biotin), 2.58 (d, 1H, J = 12.4, Hꢁ-6 of biotin), 2.06 (t, 2H,
J = 7.4, C(O) CH2 CH2 ), 1.69–1.21 (m, 6H, CH2 CH2 CH2 ).
MALDI (m/z): [(M + Na)+] calculated for C42H57N3NaO10S 818.37, found
818.35; [(M + K)+] calculated for C42H57KN3O10S 834.34, found 834.32.
Phosphoramidite of 7 (8)
Compound 7 (200 mg, 0.25 mmol) was dissolved in 10 mL of ace-
tonitrile and evaporated in vacuo up to nearly 2 mL; the reaction flask
was filled with argon after evaporation. The 0.45 M solution of tetrazole
(0.17 mL, 0.08 mmol) followed by a solution of bis(diisopropylamino)-2-
cyanoethoxyphosphine in 0.1 mL acetonitrile (0.12 mL, 0.40 mmol) were
added. The reaction progress was controlled by HPLC (gradient 75%–85%
acetonitrile in 0.1 M triethylammonium acetate, pH 7.5, 15 minutes, flow
rate 0.5 mL/minute, 250 nm). In 1 hour, the reaction mixture was evapo-
rated up to dryness, the residue was dissolved in dichloromethane, and the
solution was washed by semi-saturated aqueous NaHCO3; the organic layer
was dried by Na2SO4, evaporated up to dryness, and the residue was dissolved
in 40% aqueous acetonitrile. The desired product was isolated by column
chromatography on Separon SGX C18 [8 × 210 mm, elution by a gradient
of acetonitrile in water (40%–100%) plus 0.05% Et3N, 100 mL, flow rate 3
mL/minute]. The solution containing the target product was concentrated

Biotin-Containing Phosphoramidite Linker
501
1
and dried in vacuo. Yield 169 mg (0.17 mmol, 68%), white foam. H NMR
(DMSO-d6, 700 MHz): 7.78 (t, 1H, J = 5.5, CH2 NH C(O) CH2 ),
7.41 (m, 2H, DMTr), 7.27 (m, 7H, DMTr), 6.87 (m, 4H, DMTr), 6.38 (s,
1H, NH of biotin), 6.32 (s, 1H, NH of biotin), 4.29 (m, 1H, H-6a of biotin),
4.12 (m, 1H, H-3a of biotin), 4.02 (m, 1H,
O CH2 CH CH2),
3.75–3.72 (m, 7H, O CH3, CH2 CH2 CN), 3.67 (m, 1H,
CH2 CH2 CN), 3.62 (m, 1H, O CH2 CH CH2 ), 3.60–3.49 (m, 4H,
CH CH2 O , CH3 CH CH3, CH3 CH CH3), 3.49–3.44 (m, 12H,
O CH2 CH2 O ), 3.38 (m, 2H, O CH2 CH2 NH ), 3.17 (m, 2H,
O CH2 CH2 NH ), 3.14–2.96 (m, 3H, DMTr O CH2 CH ,
H-4 of biotin), 2.81 (dd, 1H, J = 12.4, J = 5.1, H-6 of biotin),
2.76 (m, 1H,
CH2 CH2 CN), 2.64 (m, 1H,
CH2 CH2 CN),
2.57 (dd, 1H, J = 12.4, H’-6 of biotin), 2.06 (t, 2H, J = 7.4,
C(O) CH2 CH2 ), 1.64–1.22 (m, 6H,
CH2 CH2 CH2 ), 1.13
(m, 9H, CH3), 1.01 (d, 3H, J = 6.8, CH3). 13C NMR (DMSO-d6):
158.70, 145.30, 136.21, 130.08, 128.14, 127.06, 113.60, 85.63, 55.38
(DMTr); 64.14 (DMTr O CH2 ); 72.45 (DMTr O CH2 CH ),
71.77 ( CH2 CH CH2 O CH2 ), 119.16, 58.58, 43.01, 24.71,
24.52,
20.13
(diisopropylamino-2-cyanoethoxyphosphoroamidite
segment); 69.53–70.13 (polyether chain), 38.88 ( O CH2 CH2 NH
C(O) CH2 ), 172.53 ( CH2 CH2 NH C(O) CH2 CH2 ), 35.43
( NH C(O) CH2 CH2 CH2 CH2 ),
25.60
( NH C(O) CH2
CH2 CH2 CH2 ), 28.46 ( NH C(O) CH2 CH2 CH2 CH2 ),
25.66 ( NH C(O) CH2 CH2 CH2 CH2 ), 163.11 (C-2 of bi-
otin), 61.39 (C-3a of biotin), 55.70 (C-4 of biotin), 40.24 (C-6
of biotin), 59.67 (C-6a of biotin). 15N NMR (DMSO-d6): 112.47
( CH2 CH2 NH C(O) CH2 CH2 ), 98.43, 97.45 ( N(i Pr)2),
88.77 (N-3 of biotin), 79.98 (N-1 of biotin). 31P NMR (DMSO-d6): 151.41,
150.82 (relative to 80% H3PO4). ESI-MS (m/z): [(M + Na)+] calculated
for C51H74N5NaO11PS 1018.5, found 1018.2, [(M + K)+] calculated for
C51H74KN5O11PS 1034.5, found 1034.3.
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