Synthesis and properties of new biotin compounds containing hexyltriethylene glycol chain

Article abstract of DOI:10.2478/s11532-011-0113-8

The synthesis of a new halogenide containing hexyltriethylene glycol chain functionalized with biotin is reported. The general possibility of this linker to use as the building block for biotinylated compounds syntheses is demonstrated. Two biotinylated e

Full text of DOI:10.2478/s11532-011-0113-8

Cent. Eur. J. Chem. • 10(1) • 2012 • 113-120
DOI: 10.2478/s11532-011-0113-8
Central European Journal of Chemistry
Synthesis and properties of new
biotin compounds containing
hexyltriethylene glycol chain
Research Article
Algirdas Matijoška1, Tatjana Charkova1, Zenonas Kuodis1,
Vladislava Voiciuk2, Olegas Eicher-Lorka1*
1Institute of Chemistry of Center for Physical Sciences and Technology,
LT-02300 Vilnius, Lithuania
2Faculty of Natural Sciences, Vilnius University, LT-03101 Vilnius, Lithuania
Received 18 May 2011; Accepted 14 September 2011
Abstract:
The synthesis of a new halogenide containing hexyltriethylene glycol chain functionalized with biotin is reported. The general
possibility of this linker to use as the building block for biotinylated compounds syntheses is demonstrated. Two biotinylated esters
withdifferentpropertiesforusefulsurfacemodificationandasfluorescenceprobesforproteinsmarkingweresynthesized. Theproperties
of mentioned compounds were investigated by using surface plazmon resonance ellypsometry and fluorescence spectroscopy.
Keywords: Biotinylated esters • Self-assembled monolayers • Stilbazolium • Fluorescent probe
© Versita Sp. z o.o.
1. Introduction
compound containing hexyltriethylene glycol chain
functionalized with biotin and dimethylaminostilbazole
trietyleneoxyhexyldimethylaminostilbazolium biotin
Self-assembled monolayers (SAMs) are the popular tool
for tailoring the reactive properties of the surface. Ordered
molecular assemblies are formed spontaneously by the
adsorption of surfactant – often organosulfur derivatives
with a specific affinity of its headgroup to a substrate –
generally noble metals [1-5]. Due to their often dense
and stable structure, SAMs have been investigated for
use in electrochemical sensors technology, electronics,
nanodevices of any kind, and other areas [6-11]. Besides,
the close similarity of the SAMs with biomembranes
enables the use of them as model systems to study
different cell processes, such as molecular transport
through membranes, investigation of complex membrane
proteins, and others [12-14].
In our ongoing project of developing novel syntheses
of different molecular systems based on tailoring the
reactive properties of surfaces, we decided to synthesize
a new halogenide containing alkyltriethylene glycol
chain functionalized with biotin. We believe that further
investigations will help to bring it into laboratory practice
as a building block for many syntheses of biotinylated
compounds. To prove this, we synthesized a new
–
(BES) ester (Fig. 1). Stilbazolium derivatives have the
ability to transfer electrons as well as strong nonlinear
optical properties that could possibility produce
self-assembled chromophoric structures such as
fluorescence probes for protein marking, monitoring free
radical polymerization processes and for construction
of potentially useful electro-optic devices of any kind
[15-19]. Stilbazolium fluorophore was selected in this
work because of successful application of this dye as
a voltage-sensitive fluorescent membrane probe and
our intention to use stilbazolium group labeled proteins
for studies of interactions between the proteins and
sparsely tethered bilayer lipid membranes [20-22]. In
this work, we describe a novel synthesis strategy of the
trietyleneoxyhexylthiol biotin (BET) ester that can be
widely used to produce monolayers for different surface
modifications and biological applications.
We decided to synthesize biotinylated esters for
several reasons. Biotin is a small molecule with tight and
specific binding to various proteins such as avidin and
streptavidin. This great binding affinity makes the biotin-
* E-mail: lorka@ktl.mii.lt
113

Synthesis and properties of new biotin compounds
containing hexyltriethylene glycol chain
streptavidin couple one of the strongest non-covalent
interactions known in nature. Biotin surfaces are one
of the most known tools to immobilize antibodies onto
surfaces and are widely used for biological detection and
purification that has potential application for construction
of sensors [23-25]. Optimal biotin binding capabilities
can be obtained by using a biotin derivative that has
an extended spacer arm which reduces the steric
hindrance effect. The spacer arm also improves the
complex formation of biotin with the deep biotin-binding
site of protein. For this purpose, generally biotinylated
systems include polyethylene glycol (PEG) groups.
The properties of PEG such as chemical stability, water
solubility, flexibility and low cytotoxity help to avoid the
non-specific adsorption of proteins onto the surface and
provide possibility for appropriate orientation of end
group for interaction with solution species [26]. Among
various biotinylated derivatives reported in literature
the most popular are biotin alkyl thiols (BATs) (Fig. 1)
[27-28]. We decided to synthesize a similar BET ester
(Fig. 1). Like BATs structures, it consists of three main
parts: biotin, PEG, and alkyl chain. However, in the BET
ester, the ether bond connects the hexyl chain and the
triethylene glycol group. In BATs there are two peptide
bonds instead of ether and ester bonds. Our purpose
in this synthesis was to produce a derivative without
peptide bounds to avoid possible hydrogen binding
between biotinylated system and protein. We wanted to
prove that synthesized biofunctionalized halogenide can
be used for the syntheses of biotinylated compounds
with different properties for useful surface modifications
and biological applications.
2. Experimental procedure
2.1. General procedures
Chemicals of commercial grade were used without
further purification, except dry solvents. 1H and 13C NMR
spectra were recorded in CDCl3, CD3OD and DMSO-
d6 on Varian Unity Inova NMR spectrometer (δ in ppm,
1
J in Hz) at H operating frequencies of 299.75 MHz
and 75.37 MHz for 13C; spectra were referenced using
the solvent signal as internal standard. The IR spectra
were recorded on the Perkin-Elmer Spectrum GX FT-
IR spectrometer. Elemental analyses were carried out
on a Perkin-Elmer 2400-B microanalyser. APCI mass
spectra were recorded on an Agilent 1100 ion trap mass
spectrometer in positive mode.
2.2. Synthesis of compounds 1-8
2.2.1. 2-(2-(2-(6-Chlorohexyloxy)ethoxy)ethoxy)ethanol
(1)
Commercial triethylene glycol (150 g, 1000 mmol) was
dissolved in benzene (250 mL) and refluxed in a three-
neck round bottom flask equipped with a Dean-Stark
receiver for 2 hours. The sodium (4.6 g, 200 mmol) was
added and refluxed with a Dean-Stark receiver until
the reaction finished. Next, 1,6-dichlorohexane (62 g,
400 mmol) was added and the reaction mixture refluxed
for4hours.Afterstandingovernightatroomtemperature,
it was taken up in hexane (500 mL) and decanted. The
residue was dissolved in benzene (600 mL) and washed
with water (2×200 mL). The combined benzene extracts
were dried (MgSO4), filtered, and evaporated to give
36.6 g (68%) of the corresponding compound 1.
O
HN
NH
H
H
N
O
N
SH
O
O
S
O
O
O
BAT
HN
NH
O
O
SH
O
O
S
O
BET
O
HN
NH
Cl-
O
N+
O
O
O
S
N
O
BES
Figure 1. Biotinylated structures.
114

A. Matijoška et al.
1H NMR (CD3OD, δ ppm): 1.33 – 1.51 (m, 4H, 71.02(OCH2CH2OCH2CH2),71.28(OCH2CH2OCH2CH2),
ClCH2CH2CH2CH2); 1.59 (q, J = 6.72; 2H, OCH2CH2); 71.44 (CH2CH2O)3, 72.02 (OCH2CH2CH2), 73.57
1.77 (q, J = 6.61; 2H, ClCH2CH2); 3.47 (t, J = 6.51; 2H, (HOCH2CH2). Found, %: C, 47.06; H, 8.65; Cl, 8.27; N,
OCH2CH2); 3.53 – 3.67 (m, 14H, HO(CH2CH2O)3 and 6.35; S, 7.49. Calculated for C17H37ClN2O6S (433.00): C,
ClCH2). 13C (CD3OD, δ ppm): 26.47 (OCH2CH2CH2), 47.15; H, 8.61; Cl, 8.18; N, 6.47; S, 7.40.
27.71 (ClCH2CH2CH2), 30.53 (OCH2CH2), 33.74
(ClCH2CH2), 45.69 (ClCH2), 62.20 (HOCH2), 71.10 2.2.4. 21-Mercapto-3,6,9,12,15-pentaoxahenicosan-1-
(OCH2CH2OCH2CH2), 71.37 (OCH2CH2OCH2CH2),
ol (4)
71.54 (HOCH2CH2OCH2CH2), 71.59 (HOCH2CH2OCH2), The compound 3 (6 g, 14 mmol) was dissolved in water
72.12 (OCH2CH2CH2), 73.65 (HOCH2CH2). Found, %: (50 mL). The sodium hydroxide (0.64 g, 16 mmol) water
C, 53.45; H, 9.45; Cl, 13.28. Calculated for C12H25ClO4 (15 mL) solution was added and the reaction mixture
(268.78): C, 53.62; H, 9.37; Cl, 13.19.
was stirred for 5 hours at 60ºC under argon. After the
mixture was allowed to cool to room temperature, the
2.2.2. 21-Chloro-3,6,9,12,15-pentaoxahenicosan- chloroform (150 mL) and hydrochloric acid solution
(0.5 ml HCl/2.5 ml H2O) were added. The combined
1-ol(2)
The compound 2 was prepared by the same way as chloroform extracts were washed with water
compound 1 from commercial pentaethylene glycol (3×50 mL), dried (MgSO4), and solvents were
(119 g, 500 mmol), 1,6-dichlorohexane (31 g, 200 mmol), evaporated. The residue was dried under reduced
and sodium (2.3 g, 100 mmol). Yield 19 g (53%).
pressure, yielding 2.8 g (56%) of compound 4.
1H NMR (CD3OD, δ ppm): 1.33 – 1.51 (m, 4H,
1H NMR (CDCl3, δ ppm): 1.34 (t, J = 7.74; 1H, SH);
ClCH2CH2CH2CH2); 1.58 (q, J = 6.78; 2H, OCH2CH2); 1.33 – 1.45 (m, 4H, HSCH2CH2CH2CH2); 1.54 – 1.70 (m,
1.76 (q, J = 6.68; 2H, ClCH2CH2); 3.47 (t, J = 6.51; 2H, 4H, HSCH2CH2CH2CH2CH2); 2.48 – 2.56 (td, J = 7.29 and
OCH2CH2); 3.53 – 3.67 (m, 22H, HO(CH2CH2O)5 and 7.07; 2H, HSCH2); 3.45 (t, J = 6.63; 2H, OCH2CH2CH2);
ClCH2). 13C (CD3OD, δ ppm): 26.43 (OCH2CH2CH2), 3.52 – 3.73 (m, 10H, (CH2CH2O)5. 13C (CDCl3, δ
27.67 (ClCH2CH2CH2), 30.47 (OCH2CH2), 33.68 ppm): 24.33 (CH2SH), 25.35 (OCH2CH2CH2), 27.94
(ClCH2CH2), 45.74 (ClCH2), 62.14 (HOCH2), 71.08 (HSCH2CH2CH2), 29.26 (OCH2CH2), 33.72 (HSCH2CH2),
(OCH2CH2OCH2CH2),71.30(OCH2CH2OCH2CH2),71.46 61.42 (HOCH2), 69.83 (OCH2CH2OCH2CH2), 70.09
(CH2CH2O)3, 72.09 (OCH2CH2CH2), 73.59 (HOCH2CH2). (OCH2CH2OCH2CH2), 70.36 (CH2CH2O)3, 71.06
Found, %: C, 53.75; H, 9.41; Cl, 9.98. Calculated for (OCH2CH2CH2), 72.37(HOCH2CH2). Found, %:C, 54.10;
C16H33ClO6 (356.88): C, 53.84; H, 9.31; Cl, 9.93.
H, 8.05; Cl, 9.78; S, 9.19. Calculated for C16H34O6S
(354.50): C, 54.21; H, 7.93; Cl, 9.66; S, 9.04.
2.2.3. 2-(1-Hydroxy-3,6,9,12,15-pentaoxahenicosan-
21-yl)isothiuronium chloride (3)
The compound 2 (17.8 g, 500 mmol) and thiourea
2.2.5.
(2-(2-(6-Chlorohexyloxy)ethoxy)ethoxy)ethyl
5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanoate (5)
(3.8 g, 50 mmol) were dissolved in n-butanol
(100 mL). The mixture was refluxed using an oil bath for Biotin (1 g, 4 mmol) was dissolved in dimethylformamide
5 hours. After standing overnight at room temperature, (20 mL) at 65ºC under argon. After the temperature was
the precipitate was filtered, and solvent was removed decreased to 40ºC, 3-((ethylimino)methyleneamino)-
under reduced pressure. The residue was washed N,N-dimethylpropan-1-aminium chloride – EDC HCl
with acetone (3×50 mL), refluxed in acetonitrile (1.12 g, 5.8 mmol) in dry dichloromethane (50 mL),
(100 mL) for 10 min, and hot filtered. Next, acetone N,N-dimethylpyridin-4-amine
–
DMAP (0.165 g,
(100 mL) was added and the solution was allowed 0.353 mmol) and compound 1 (1.07 g, 4 mmol) were
to cool in a refrigerator until oil product formed. The added. The mixture was stirred for 20 hours at 60ºC,
product was washed with acetone (3×50 mL), decanted, and the solvent was evaporated. The residue
and allowed to dry at room temperature yielding 11 g was
infused
with
NaHCO3
(25
mL)
(51%) of compound 3.
and the aqueous layer was extracted with
1H NMR (CD3OD, δ ppm): 1.35 – 1.53 (m, 4H, dichloromethane (3×25 mL). The organic phase
SCH2CH2CH2CH2); 1.58 (q, J = 6.75; 2H, OCH2CH2CH2); was washed with water (3×50 mL), dried (MgSO4),
1.72 (q, J = 7.16; 2H, SCH2CH2CH2); 3.17 (t, J = 7.16; and evaporated. The residue was recrystallized from
2H, SCH2); 3.47 (t, J = 6.40; 2H, OCH2CH2); 3.54 – 3.68 dichloromethane and hexane mixture (1:20), washed
(m, 22H, HO(CH2CH2O)5). 13C (CD3OD, δ ppm): 26.53 with hexane (50 mL), and dried under reduced pressure,
(OCH2CH2CH2), 29.14 (SCH2), 29.57 (SCH2CH2CH2), yielding 1.2 g (60.5%) of compound 5.
30.35 (OCH2CH2), 31.78 (SCH2CH2), 62.10 (HOCH2),
115

Synthesis and properties of new biotin compounds
containing hexyltriethylene glycol chain
1H NMR (CDCl3, δ ppm): 1.34 – 1.82 (m, 14H, 61.06 (NHCHCHS), 63.09 (COOCH2CH2O), 68.33
ClCH2(CH2)4CH2O and CH2CH2CH2CH2COO); 2.38 (COOCH2CH2O), 69.50 (COOCH2CH2OCH2), 69.78
(t, J = 7.40; 2H, CH2CH2COO); 2.73 – 2.94 (m, 2H, (OCH2CH2OCH2CH2OCH2CH2), 69.82 (CH2OCH2CH2),
CHCH2S); 3.12 – 3.18 (m, 1H, CHCHS); 3.46 (t, J = 6.53; 70.21 (OCH2CH2), 162.76 (NHCONH), 169.96 (SCNH2),
2H, OCH2CH2CH2); 3.54 (t, J = 6.78; 2H, ClCH2CH2); 172.86 (COO). Found, %: C, 48.26; H, 7.65; Cl, 5.67; N,
3.58 – 3.66 (m, 8H, COOCH2CH2OCH2CH2OCH2CH2O); 9.71; S, 11.29. Calculated for C23H43ClN4O6S2 (571.19):
3.70 (t, J = 4.91; 2H, COOCH2CH2O); 4.21 – 4.24 (m, C, 48.36; H, 7.58; Cl, 5.61; N, 9.80; S, 11.22.
2H, COOCH2CH2O); 4.28 – 4.33 (m, 1H, NHCHCHS);
4.48 – 4.52 (m, 1H, NHCHCH2S); 5.61 (m, 1H, 2.2.7.
NHCHCHS); 6.18 (m, 1H, NHCHCH2S). 13C (CDCl3,
δ ppm): 24.65 (CHCH2CH2), 25.33 (OCH2CH2CH2),
2-(2-(2-(6-Mercaptohexyloxy)ethoxy)ethoxy)
ethyl 5-(2-oxohexahydro-1H-thieno[3,4-d]
imidazol-4-yl)pentanoate (7)
26.60 (ClCH2CH2CH2), 28.14 (CHCH2CH2), 28.24 The compound 6 (0.27 g, 0.473 mmol) was dissolved
(CHCH2CH2CH2), 29.36 (OCH2CH2), 32.45 (ClCH2CH2), in water (10 mL) and chloroform (30 mL) mixture.
33.68(CH2CH2COO),40.46(SCH2),44.90(ClCH2),55.49 The sodium metabisulfite
–
Na2S2O5 (0.162 g,
(SCHCH2), 60.03 (NHCHCH2S), 61.85 (NHCHCHS), 0.851 mmol) was added and reaction mixture was
63.33 (COOCH2CH2O), 69.06 (COOCH2CH2O), 69.99 refluxed under argon for 5 hours. Then it was allowed to
(COOCH2CH2OCH2), 70.43 (COOCH2CH2OCH2CH2O), cool to room temperature and extracted with chloroform
70.48(OCH2CH2OCH2CH2),70.55(OCH2CH2OCH2CH2), (30 mL). The combined chloroform extracts were
71.14 (OCH2CH2OCH2CH2), 163.75 (NHCONH), 173.59 washed with water (3×50 mL), dried (MgSO4), and
(COO). MS m/z (relative intensity): 492.5 (100), 224.8 solvents removed under reduced pressure. The residue
(15). IR (film) 3247, 2937, 2863, 1730, 1702, 1459, was dried under reduced pressure, yielding 0.11 g (47%)
1118 cm-1. Found, %: C, 53.26; H, 8.05; Cl, 7.27; N, of compound 7.
5.71; S, 6.39. Calculated for C22H39ClN2O6S (495.07): C,
53.37; H, 7.93; Cl, 7.16; N, 5.65; S, 6.47.
1H NMR (CD OD, δ ppm): 1.36 – 1.73 (m, 14H,
3
ClCH2(CH2)4CH2O and CH2CH2CH2CH2COO); 2.37 (t, J
= 7.22; 2H, CH2CH2COO); 2.47 – 2.52 (dt, J = 7.11; 2H,
HSCH2); 2.68 – 2.96 (m, 2H, CHCH2S); 3.17 – 3.24 (m,
1H, CH2CHS); 3.47 (t, J = 6.47; 2H, OCH2CH2CH2); 3.56
– 3.64 (m, 8H, COCH2CH2OCH2CH2OCH2CH2O); 3.70
2.2.6.
2-(5-Oxo-1-(2-oxohexahydro-1H-thieno[3,4-d]
imidazol-4-yl)-6,9,12,15-tetraoxahenicosan-21-
yl)isothiuronium chloride (6)
The compound 5 (0.4 g, 0.81 mmol) and thiourea (t, J = 4.77; 2H, COOCH2CH2O); 4.21 (t, 2H, J = 4.49;
(0.074 g, 0.97 mmol) were dissolved in n-butanol COOCH2CH2O); 4.28 – 4.33 (dd, J = 4.45 and 3.70;
(20 mL). The mixture was refluxed under argon 1H, NHCHCHS); 4.47 – 4.41 (m, 1H, NHCHCH2S);
for 8 hours. Then it was evaporated, residue was 4.87 (m, 2H, NHCONH). 13C (CD OD, δ ppm): 24.88
3
dissolved in acetone and methanol mixture (2:1) and (HSCH2), 24.92 (CHCH2CH2), 25.92 (OCH2CH2CH2),
taken up in dry diethyl ether (100 mL). The mixture was 29.19 (HSCH2CH2CH2), 29.48 (CHCH2CH2CH2), 30.62
allowed to cool in refrigerator until the oil product was (OCH2CH2), 34.57 (CH2CH2COO), 35.17 (HSCH2CH2),
formed, and solvents were decanted. The residue was 41.06 (SCH2CH), 56.99 (SCHCH2), 61.62 (NHCHCH2S),
washed with dry diethyl ether (50 mL) and dried under 63.37 (NHCHCHS), 64.62 (COOCH2CH2O), 70.15
reduced pressure to give 0.27 g (58.5%) of compound (COOCH2CH2O),71.18(COOCH2CH2OCH2CH2O),71.55
6.
(COOCH2CH2OCH2CH2O), 71.59 (OCH2CH2OCH2CH2),
1H NMR (DMSO-d6, δ ppm): 1.30 – 1.61 (m, 14H, 71.61 (CH2OCH2CH2), 72.24 (OCH2CH2), 166.71
ClCH2(CH2)4CH2O and CH2CH2CH2CH2COO); 2.31 (t, J (NHCONH), 175.24 (COO). MS m/z (relative intensity):
= 7.34; 2H, CH2CH2COO); 2.57 – 2.86 (m, 2H, CHCH2S); 491.2 (100), 447.3 (23), 359.3 (60), 225.5 (80). IR
3.07 – 3.11 (m, 1H, CHCHS); 3.15 (t, J = 7.19; 2H, (film) 3247, 2929, 2859, 2560, 1732, 1704, 1460,
NH2SCH2CH2); 3.37 (t, J = 6.52; 2H, OCH2CH2CH2); 1118 cm-1. Found, %: C, 53.56; H, 8.25; N, 5.61; S,
3.40 – 3.52 (m, 8H, COOCH2CH2OCH2CH2OCH2CH2O); 13.12. Calculated for C22H40N2O6S2 (492.69): C, 53.63;
3.60 (t, J = 4.72; 2H, COOCH2CH2O); 4.11 – 4.16 (m; H, 8.18; N, 5.68; S, 13.01.
2H; COOCH2CH2O and 1H; NHCHCHS); 4.29 – 4.34
(m, 1H, NHCHCH2S); 6.41 (m, 1H, NHCHCHS); 6.47 2.2.8.
(m, 1H, NHCHCH2S). 13C (DMSO-d6, δ ppm): 24.52
(CHCH2CH2), 25.10 (OCH2CH2CH2), 27.67 (SCH2),
28.01 (CHCH2CH2CH2), 28.40 (SCH2CH2CH2), 29.04
(E)-4-(4-(dimethylamino)styryl)-1-(5-oxo-1-(2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
6,9,12,15-tetraoxahenicosan-21-yl)pyridinium
chloride (8)
(OCH2CH2), 29.98 (SCH2CH2), 33.27 (CH2CH2COO), (E)-N,N-dimethyl-4-(2-(pyridin-4-yl)vinyl)aniline (0.2 g,
39.88 (SCH2CH), 55.39 (SCHCH2), 59.22 (NHCHCH2S), 0.892 mmol), compound 5 (0.44 g, 0.892 mmol) and
116

A. Matijoška et al.
tetraethylammonium iodide (0.002 g, 0.00781 mmol) the SOPRA program Winelli. Mixed SAM was prepared
were dissolved in toluene (10 mL). The mixture was by injecting into the cell the 0.2 mM ethanolic solution
refluxed in a domestic microwave for 2.5 hours. Then, of 5:95 mol% ratio mixtures of compounds 7 and 4.
itwascooledtoroomtemperature,decanted,andwashed After incubation for 24 hours, the cell was washed with
with benzene (3×50 mL). The residue was dissolved ethanol, and water was injected. 1 μM streptavidin
in dry methanol (3 mL), taken up in dry diethyl ether (ProSpec-Tany TechnoGene, Israel) water solution
(100 mL), decanted, and dried under reduced pressure. was injected to initiate the adsorption to biotin exposed
The product was chromatographed on a column of silica at the surface of the SAM. After the steady signal was
gel using dichloromethane and methanol (5:1) solvents observed (~ 10 min), the cell was washed with water.
as eluent. Yield 0.11 g (47%).
1H NMR (CDCl3, δ ppm): 1.33 – 1.78 (m, 12H, 2.3.2. Fluorescence measurements
ClCH2CH2CH2CH2CH2CH2N and CH2CH2CH2CH2COO); Fluorescence
spectra
were
recorded
using
1.91 -2.02 (m, 2H, NCH2CH2); 2.35 (t, J = 7.05; the Fluorescence Spectrometer (LS 55, PerkinElmer,
2H, CH2CH2COO); 2.83 – 2.94 (m, 2H, CHCH2S); USA). 15 μM streptavidin and 200 μM compound 8 water
3.06 (s, 6H, N(CH3)2); 3.11 – 3.18 (m, 1H, CHCHS); solution was dialyzed using the 12.000-14.000 MWCO
3.43 (t, J = 6.65; 2H, OCH2CH2CH2); 3.55 – 3.65 (m, dialysis membranes (ROTH ZelluTrans, Germany) for
8H, OCH2CH2OCH2CH2O); 3.70 (t, J = 4.59; 2H, 7 days. The size of membrane’s pores and dialysis
COOCH2CH2O); 4.21 – 4.24 (m, COOCH2CH2O); duration were tested to ensure complete elimination of
4.29 – 4.33 (m, 1H, NHCHCHS); 4.52 – 4.56 (m, 1H, free chromophore molecules from the solution.
NHCHCH2S); 4.63 (t, J = 6.95; 2H, NCH2CH2); 5.81 (m,
1H, NHCHCHS); 5.88 (m, 1H, NHCHCH2S); 6.67 – 6.70
3. Results and discussion
(m,2H,Ph-3,5);6.82–6.88(d,J=15.95;1H,CH=CHPh);
7.51 – 7.53 (m, 2H, Ph-2,6); 7.60 – 7.65 (d, J = 15.96;
1H, CH=CHPy); 7.87 – 7.89 (m, 2H, Py-2,6); 8.89 – 8.90 3.1. Synthesis
(m, 2H, Py-3,5). 13C (CDCl , δ ppm): 24.64 (CHCH2CH2), It was found in literature [29], that compound 1 can be
3
25.46 (OCH2CH2CH2), 25.74 (NCH2CH2CH2), 28.20 made by the addition of n-ethylene glycol to dihaloalkane
(CHCH2CH2CH2), 28.29 (CHCH2CH2CH2), 29.17 or terminal haloalkene.Although this alkylation by means
(OCH2CH2CH2), 33.72 (CH2CH2COO), 40.04 (CH3)2, of haloalkene is usually more effective, we decided to
40.34 (SCH2CH), 40.60 (NCH2CH2), 55.64 (SCHCH2), achieve monoalkylated product 1 with dihaloalkane for
59.99 (NCH2), 60.14 (NHCHCH2S), 61.75 (NHCHCHS), further addition of biotin. The route for the main synthesis
63.32 (COOCH2CH2O), 68.97 (COOCH2CH2O), 69.92 started with the commercially available triethylene
(COOCH2CH2OCH2), 70.40 (OCH2CH2OCH2CH2O), and pentaethylene glycols as shown in Scheme 1. In
70.44 (OCH2CH2OCH2CH2CH2), 70.49 (CH2OCH2CH2), the first step, triethylene glycol was deprotonated in
70.87 (OCH2CH2), 111.83 (Ph-3,5); 116.55 (Ph-1); anhydrous benzene by sodium, then reacted with 1,6-
122.34 (Ph-CH=); 122.79 (Py-3,5); 130.48 (Ph-2,6); dichlorohexane to yield 6-(chlorohexyl)triethylene glycol
142.61 (Py-CH=); 143.52 (Py-2,6); 152.10 (Ph-4); (1) (68% yield). Inorganic components were removed
153.87 (Py-4); 164.09 (NHCONH), 173.51 (COO). by extraction with water and benzene. The compound
MS m/z (relative intensity): 682.7 (50), 223.1 (100). IR 2 was prepared similarly to give 53% yield. Diluent
(film) 3249, 2922, 2860, 1724, 1693, 1642, 1582, 1527, compound 4 for SPRE measurements was prepared by
1162 cm-1. Found, %: C, 61.68; H, 7.75; Cl, 4.51; N, method of two steps. Firstly, thiuronium chloride 3 was
7.71; S, 4.49. Calculated for C37H55ClN4O6S (719.37): C, synthesized from compound 2 and thiourea (51% yield).
61.77; H, 7.70; Cl, 4.45; N, 7.78; S, 4.45.
Then, it was hydrolyzed with sodium hydroxide yielding
(56%) 6-(mercaptohexyl)pentaethylene glycol (4).
Next, we decided to choose the same strategy of
2.3. Measurements
2.3.1. Surface plasmon resonance ellipsometry (SPRE) biotinylated linker’s 5 synthesis through halogenides
measurements
like Hansen [30] and made it coupling 6-(chlorohexyl)
The experimental set-up consisted of a spectral triethylene glycol (1) with the carbodiimide activated
ellipsometer, SOPRA GES-5 with rotating analyzer and biotin. The resulting compound 5 was recrystallized
45° BK7 glass prism connected with gold coated glass from dichloromethane and hexane mixture in good
slide by the refraction index matching fluid. The glass yield (60%); next it was refluxed with excess thiourea
prism was mounted on the cell; the gold sensor surface in n-butanol under argon. Thiuronium salt 6 was
was placed in contact with solutions injected in the cell. isolated from methanol, acetone, and ether mixture
The experimental ellipsometric data was analyzed using (58.5% yield). The usual procedures for conversion
117

Synthesis and properties of new biotin compounds
containing hexyltriethylene glycol chain
Na, Cl(CH2)6Cl
C6H6
OH
H
OH
Cl
O
O
n
n
1 (n = 3), 2 (n =5).
+
NH2
(NH2)2CS
C4H9OH
Cl-
S
2
O
HO
HO
O
O
O
O
NH2
3
NaOH
H2O
O
SH
O
O
O
O
4
O
EDC HCl, DMAP, DMF
HN
NH
1
O
(C2H5)4NI, C6H5CH3, MW
Cl
HN NH
O
O
O
O
S
OH
O
S
N
O
N
5
O
S
HN
NH
NH
+
(NH2)2CS
C4H9OH
NH2
Cl-
S
O
O
O
O
O
NH2
O
6
O
HN
Na2S2O5
CHCl3, H2O
SH
O
O
O
S
O
O
7
HN
NH
Cl-
N
O
O
O
O
8
S
N
O
Scheme 1. Synthesis of compounds 1-8.
of the mercapto group from thiuronium salts employ for protein marking of compound 8 were proved
potassium hydroxide in ethanol. In our synthesis, with streptavidin using SPRE and fluorescence
these reaction conditions cannot be used because experiments described below.
of the presence of ester groups. So, we choose
All products, except compound 8, were purified
mild hydrolysis by sodium metabisulfite solution without column chromatography.
in chloroform to provide the corresponding thiol 7
3.2. Surface plasmon resonance ellipsometry
(47% yield) [31]. Stilbazolium salt 8 production was
attempted by conventional heating. The week-long
synthesis was unsuccessful and finished with only traces
of product. Finally, stilbazolium salt 8 was prepared (47%
yield) from compound 5 and dimethylaminostilbazole by
microwave irradiation with toluene as the solvent.
We have designed and synthesized biofunctionalized
halogenide 5 and used it for the syntheses of two
biotinylated esters. The synthesis of BET ester (7) was
performed under an atmosphere of argon by an efficient
method of three steps. BES ester (8) was made by
microwave irradiation in two steps. The reactive surface
properties of compound 7 and chromophoric properties
(SPRE)
Fig. 2 shows the results of SAM formation on the
gold surface and subsequent streptavidin adsorption
to biotin groups of compound 7. Refractive index
dispersion of compact octadecanethiol monolayer was
used for SAM’s modeling, thus obtaining the resulting
SAM thickness of 2.6 nm which is consistent with the
length (2.7 nm) of molecule 4, the major constituent
of the SAM. This result indicates the high fill factor
and the quality of the monolayer. The estimated
thickness of adsorbed streptavidin layer assuming the
homogeneous layer at the SAM’s surface is 2.6 nm.
118

A. Matijoška et al.
distribution of compound 7 in the monolayer, resulting
in close packing of biotin groups that hinder molecular
recognition with the biotin-binding pockets of streptavidin
atthesurface.Alternatively, the5:95solutionalkylthiolate
molar ratio employed in this experiment may have
resulted in SAMs with insufficient portion of compound
7 molecules adsorbed at the gold surface. Additionally,
the used of diluent compound 4 could be unfavorable
due to its length, approximate to compound 7 molecule’s
length, as well as resulting in reduced availability of the
biotin terminus at the surface.
3.3. Fluorscence
The results of streptavidin labeling with chromophore via
biotingroupareshowninFig.3.Thefluorescencespectra
of compound 8 and streptavidin solution were recorded
before and after dialysis. The effective binding of the
chromophore is evidenced by the ratio of fluorescence
integral intensity before and after the dialysis. Initial
200 μM chromophore concentration is expected to
decrease by 3.3 fold to 60 μM considering the 15 μM
streptavidin × 4 binding sites per tetrameric molecule.
The ratio of before/after dialysis integral intensity is 2.9;
the slight variance can be explained by unfolding of
some protein structures, resulting in fewer biotin binding
sites and/or imprecise baseline subtraction.
Figure 2. SPR
ellipsometry spectra of parameter
ψ for bare
Au substrate; Au is coated with mixed compound
7/compound 4 SAM; streptavidin is adsorbed onto the
monolayer’s surface (all spectra recorded in water).
Experimental data points are shown with markers,
smooth lines represent data fits.
4. Conclusions
In conclusion, we have developed an efficient synthesis
of halogenide containing hexyltriethylene glycol chain
functionalized with biotin, which is a building block for the
synthesis of biotinylated compounds. Two biotinylated
esters were successful synthesized in several steps
and their useful properties for surface modifications
and protein marking were investigate using the surface
plasmon resonance ellypsometry and fluorescence
spectroscopy.
Acknowledgements
Figure 3. Fluorescence spectra of 15 μM streptavidin
The authors gratefully acknowledge the Lithuanian State
Science and Studies Foundation (Project B-38/2008) for
financial support. We thank Aušra Linkevičiūtė and dr.
Zigmas Balevičius from Center For Physical Sciences
and Technology for MS and SPRE spectrum recording.
The authors gratefully acknowledge the Department of
Bioelectrochemistry and Biospectroscopy at the Institute
of Biochemistry of Vilnius University for possibility to use
the FT-IR spectrometer.
and compound
8 water solution before and after
the dialysis. Concentration of the chromophore before
dialysis was 200 μM, after dialysis, considering binding
to tetrameric protein – 60 μM. Excitation wavelength
is 479 nm.
The dimensions of streptavidin tetramer molecule are
4.5×4.5×5 nm, wherefore the protein layer was filled
incompletely, resulting in about 50% of surface fill factor.
This can be explained due to inhomogeneous
119

Synthesis and properties of new biotin compounds
containing hexyltriethylene glycol chain
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