Synthesis of novel biotin anchors

Article abstract of DOI:10.1016/S0040-4020(01)01003-1

Details are reported of the synthesis of biotinylated thiol and cholesteryl derivatives. The biotin derivative has been used to form multilayer films on gold through interaction with streptavidin.

Full text of DOI:10.1016/S0040-4020(01)01003-1

TETRAHEDRON
Pergamon
Tetrahedron 57 52001) 9859±9866
Synthesis of novel biotin anchors
Claire Booth, Richard J. Bushby,p Yaling Cheng, Stephen D. Evans, Quanying Liu
and Haoli Zhang
Centre for Self-Organising Molecular Systems, University of Leeds, Leeds LS2 9JT, UK
Received 27 April 2001; revised 13 September 2001; accepted 4 October 2001
AbstractÐDetails are reported of the synthesis of biotinylated thiol and cholesteryl derivatives. The biotin derivative has been used to form
multilayer ®lms on gold through interaction with streptavidin. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
covalently binds to the gold surface.4,5 Extending our
research interests in this ®eld, we have now introduced a
speci®c streptavidin/biotin interaction into our system to
form multilayer ®lms.
The bio-functionalisation of solid surfaces is one of the most
challenging tasks of multidisciplinary research involving
biology, chemistry, and physics. It has attracted a great
deal of attention in recent years because it provides a bridge
between man-made functional materials or devices and the
supramolecular assemblies found in biology.1 Potential
applications range from the biocompatibilisation of
implants for surgery, to novel biosensors.2 The most impor-
tant and commonly used solid surface is gold as it readily
reacts with thiols, or disul®des to form a stable covalent
bond.3 We have reported a simple approach to the attach-
ment of lipid bilayers to such gold surfaces. This employs
self-assembled monolayers 5SAMs) comprised of a mixture
in which the anchoring component is an ethylenoxy
oligomer, which is terminated with a cholesteryl group or
phospholipid residue at one end and a thiol or disul®de
group at the other. The cholesteryl moiety or the alkyl
chain of the phospholipid penetrates into the hydrophobic
region of the inner lea¯et of the bilayer and acts as a `hook'.
The ethylenoxy part provides a hydrophilic space between
the gold and the bilayer and the thiol or disul®de group
Streptavidin is an exceptionally stable tetrameric protein,
each of the four subunits binds biotin with a free energy
of binding which is comparable to that of a covalent
bond.6 Biotin can be attached to proteins and saccharides,
as well other compounds and streptavidin then used as a
molecular linker between the biotin-derivatized units.7
Hence, biotinylated thiols have been synthesised and
SAMs of these biotinylated thiols on gold used to bind
streptavidin.8±11 A particularly interesting point is that the
four biotin binding sites have been shown to be arranged in
two opposing pairs in each molecule such that two sites per
streptavidin molecule are bound to the biotin on the surface
and the remaining two sites are unoccupied. The bound
streptavidin thus presents a matrix of well-ordered binding
sites to the adjacent subphase, which can be employed as a
template for further functionalisation. In this paper we
report the synthesis of biotinylated thiol 3, SAMs of
which can be used to bind streptavidin to gold and
Keywords: biotin cholesteryl; biomembranes; surfaces.
Corresponding author. Tel.: 144-113-2336509; fax: 144-113-2336452; e-mail: r.j.bushby@chem.leeds.ac.uk
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020501)01003-1

9860
C.Booth et al./ Tetrahedron 57 &2001) 9859±9866
cholesteryl derivatives 1 and 2 which can then be used to
react with the free sites to bind biomembranes on top of this.
obtained facilitates the calculation of the three-dimensional
protein structure.12
Among various biotinylated thiols reported in the literature,
the derivative 3 has been widely used.10,11 However, this
kind of compound is dif®cult to make. In the original report
the analogue of 3 511-mercaptododecanoic-58-biotinoyl-
amido-3,6-dioxaoctyl) amide) was made in poor yield and
no characterisation data was given.8 This restricts access to
the ®eld. A new synthesis route is reported here and the
compounds involved are fully characterised. Biotinylated
cholesteryl derivatives 1 and 2 have similar structures to
the thiol cholesteryls we reported previously,4 but the thiol
group is replaced by biotin. They should prove useful for
binding biomembranes to streptavidin immobilised on gold
surfaces. Further more, it is known that streptavidin can
crystallise in two-dimensional arrays on biotinylated phos-
pholipids monolayers at an air/water interface. This is
important for structural biology since the helical symmetry
2. Results and discussion
2.1. Preparation of compounds 1, 2, and 3
The route for the synthesis of 1 started with the com-
mercially available amine 4 as shown in Scheme 1. Choles-
teryl chloroformate was reacted with an excess of the amine
4 in tetrahydrofuran using potassium carbonate as a base.
After the reaction, inorganic salts were removed by ®ltration
and the monocarbamate 5 separated by column chromato-
graphy. To form the amide from biotin and the monocarba-
mate 5, the acid group of biotin was ®rst activated by
reacting with N,N0-disuccinimidyl carbonate 5DSC)13 in
DMF. This gave the biotin N-hydroxysuccinimide ester
which was reacted directly with 5 without puri®cation to
Scheme 1.

C.Booth et al./ Tetrahedron 57 &2001) 9859±9866
9861
Scheme 2.
give 1. To simplify the puri®cation procedure of 1, a slight
excess of biotin was used over the DSC to avoid the DSC
reacting with 5. The excess biotin can be easily removed in
the ®nal column chromatography since it has poor solubility
in most solvents. However, the by-product N-hydroxy-
succinimide is very dif®cult to remove and extensive
column chromatography was needed. Compound 2 was
made in a similar manner to 1.
philic reagents are used or produced during these reactions
5amine, N-hydroxysuccinimide), we argued that carrying a
bromo group throughout would certainly reduce the yields
and complicate the separation of products. Following the
introduction of benzylthiol, 12-benzylthiododecanoic
N-hydroxysuccinimide ester 7 was made using DSC
reagent. The ester 7 was reacted with excess 2,20-5ethylene-
dioxy)diethylamine to give monoamide 8 which reacted
with biotin N-hydroxysuccinimide ester to generate 9. The
benzyl protecting group of 9 was readily removed using
sodium in liquid ammonia to give the ®nal product 3.4
Compound 3 was made as shown in Scheme 2. The benzyl-
protected thiol group was introduced in the ®rst step in
excellent yield. In the original report of synthesis of this
kind of compound,8 the bromo group was retained through-
out reaction sequence until the ®nal step where it was
converted into the corresponding Bunte salt and subjected
to acid hydrolysis to give the thiol. Considering that nucleo-
2.2. Surface characterisation of a biotinylated self-
assembled monolayer
X-Ray photoelectron spectroscopy 5XPS) was used to

9862
C.Booth et al./ Tetrahedron 57 &2001) 9859±9866
Figure 1. Detailed XPS scans of the nitrogen 1s peak for SAMs of: 5a) 100% 11-mercaptoundecanol, 5b) the mixed SAM used for streptavidin adsorption and
5c) 100% biotin thiol 3.
con®rm the presence of biotin thiol 3 in a mixed monolayer
used for streptavidin adsorption below. Spectra were
recorded for SAMs of 100% 11-mercaptoundecanol,
100% biotin thiol 3 and a mixed SAM formed from a
solution containing 20% biotin thiol 3 and 80% 11-mer-
captoundecanol. All spectra were recorded at a take off
angle of 908 and showed the presence of peaks correspond-
ing to gold, carbon, oxygen and sulphur for all three SAMs.
In addition, the SAMs containing biotin thiol 3 also had a
nitrogen peak. Fig. 1 shows detailed scans of the nitrogen
peak at 908 for each of the SAMs studied.
It is clear that there is virtually no nitrogen detected in the
100% 11-mercaptoundecanol SAM, as expected. For the
100% biotin thiol 3 SAM there is a relatively large nitrogen
peak due to nitrogen in the urea and amide groups of the
molecule. In the spectrum of the mixed monolayer, the
nitrogen peak is reduced in intensity as there is a lower
Figure 2. Spectrum 1: IR spectrum of a thin ®lm of biotin thiol 3 on a calcium ¯uoride plate. Spectrum 2: IRAS of 100% biotin thiol 3 SAM. Spectrum 3: IRAS
of the mixed SAM used for streptavidin adsorption.

C.Booth et al./ Tetrahedron 57 &2001) 9859±9866
9863
proportion of biotin thiol 3 on the surface, but it con®rms the
presence of the biotinylated molecule in the monolayer.
Detailed scans of the oxygen peaks also provide useful
information on the composition of the SAMs. For the 11-
mercaptoundecanol SAM the oxygen peak is composed of
only one component centred at 533.2 eV, whereas for both
the pure biotin thiol SAM and the mixed SAM, the oxygen
peaks can be resolved into two components. The component
at around 533 eV corresponds to the presence of oxygen±
carbon single bonds, and the second component at 532 eV
con®rms the presence of oxygen±carbon double bonds in
the monolayer.
Spectrum 2 shows the IRA spectrum of a SAM of biotin
thiol 3 on gold. The amide I vibration of the urea group and
the amide II vibration of the secondary amides in the chain
are present in roughly the same positions as for the thin solid
®lm, as expected. However, there is a signi®cant difference
here in that the amide I vibration of the amides in the chain
of the molecule is no longer apparent. Since only vibrations
which produce a change in dipole moment perpendicular to
the substrate surface are active in the IRA spectrum, the
absence of this band suggests that the CvO bonds in
the chain are aligned parallel to the surface. In addition,
the relatively high intensity of the amide II peak indicates
that the dipole of the N±H bond oscillates nearly perpen-
dicular to the substrate plane.18 This orientation is consistent
with the presence of lateral hydrogen bonding interactions
between the amide containing chains of these molecules in
the SAM.20
Infrared re¯ection absorption spectroscopy 5IRAS) was also
used to study the SAMs. Fig. 2 shows the IR spectra in the
amide region of a thin ®lm of biotin thiol 3 on a calcium
¯uoride plate 5spectrum 1), a SAM of biotin thiol 3 on gold
5spectrum 2), and the mixed SAM formed from a solution
containing 20% biotin thiol 3 and 80% 11-mercapto-
undecanol 5spectrum 3).
The IRA spectrum of the mixed SAM has two major bands
in the amide region that are broad and less well de®ned,
perhaps re¯ecting a lower degree of order in the mixed
SAM as compared to the one component SAM. However,
the position of the band at around 1706 cm21 does suggest
that it contains a contribution from the amide I vibration of
the urea group, once again showing that the biotin thiol 3
molecule is present in the SAM.
In the IR spectrum of the thin solid ®lm of biotin thiol 3,
there are clearly three bands in the amide region. The band
at 1706 cm21 is assigned to the amide I vibration of the urea
group,16 which is predominantly the CvO stretching mode.
The equivalent mode for the amide groups in the chain of
the molecule occurs at 1640 cm21 17,18
The weaker band at
.
1560 cm21 is attributed to the amide II mode of these groups
5i.e. the out of phase combination of N±H in plane bending
and C±N stretching vibrations).17,18 The position of these
two peaks at 1640 and 1560 cm21 is consistent with inter-
molecular hydrogen bonding interactions.19
2.3. Adsorption of streptavidin to a biotinylated self-
assembled monolayers
The interaction of SAMs of biotin thiol 3 on gold surfaces
and subsequent adsorption of streptavidin was investigated
(a)
(b)
Figure 3. Atomic force microscopic images of biotinylated SAM: 5a) before and 5b) after protein adsorption. The images 5scan size of 500 nm) were taken in
tapping mode and under ambient conditions with a Z range of 10 nm.

9864
C.Booth et al./ Tetrahedron 57 &2001) 9859±9866
by AFM. Fig. 3 shows AFM images of a biotinylated SAM,
formed from a mixture of biotin thiol 3 and 11-mercapto-
undecanol, and the result of exposure to streptavidin. The
use of a mixed SAM, instead of a single component biotin
thiol 3 SAM, reduces steric hindrance to protein binding.11
The image for biotinylated SAMs before protein adsorption
shows a smooth surface similar to that of unfunctionalised
gold 5Fig. 3). However, the image for biotinylated SAMs
after exposure to streptavidin displays a layer of uniformly
adsorbed material. The height of the features in this image
rinsed with solvent and dried under a stream of N2 before
immersion in solutions of 11-mercaptoundecanol, biotin
thiol 3 and a mixed solution containing 80% 11-mercapto-
undecanol with 20% biotin thiol 3 in ethanol 5with total thiol
concentration of 1024 M) for overnight. On removal from
solution, SAMs were rinsed with ethanol, milli-Q water and
dried in a stream of N2.
Protein adsorption. The binding of protein streptavidin was
proceeded in a phosphate saline solution 5pH 7.4) of strep-
tavidin at a concentration of 30 mg ml21. One hour was
allowed for binding to complete. The substrates were then
removed, washed with milli-Q water and dried with a steam
of N2.
Ê
are in the range 50±60 A, which is consistent with the value
Ê
of 45±50 A obtained for two-dimensional crystals of
streptavidin at the air/water interface.14 Therefore, the
AFM results indicate the binding of streptavidin on the
biotinylated surfaces. It should be noted that no signi®cant
streptavidin adsorption was found in control experiments on
SAMs formed from 100% 11-mercaptoundecanol, which is
also in agreement with previous reports.10,11 The observed
adsorption is hence due to a speci®c interaction between
biotin and streptavidin.
XPS was performed using a Vacuum Generators ML 500
instrument with an Al Ka source 51486.6 eV). All spectra
were recorded using a constant pass energy of 30 eV and the
residual pressure in the chamber remained in the order of
1029 Torr. The peaks were resolved into a mixture of
Gaussian and Lorentzian curves to ®t the spectra.
Infrared re¯ection absorption spectra of SAMs were
recorded on a Bruker IFS 48 system equipped with a grazing
angle accessory 5808). Spectra were recorded using 2000
3. Conclusions
A biotinylated thiol has been made by an ef®cient new
method. The SAMs of this biotinylated thiol and their inter-
action with steptavidin have been characterised by XPS,
IRAS and AFM. Two biotin cholesteryls with different link-
ing chains have also been made and ongoing work on
construction of multilayer ®lms using these compounds
will be reported in due course.
scans and 2 cm21 resolution in the range 400±4000 cm21
,
whilst being purged with dry air at room temperature. The
transmission IR spectrum of a thin ®lm of biotin thiol 3 on a
calcium ¯uoride plate was recorded using the same
instrument.
For characterisation of binding of streptavidin onto bio-
tinylated surfaces an atomic force microscope 5Nanoscope
IIIa, Digital Instruments) was used. The images were
collected in tapping mode using a standard silicon tip
under ambient conditions.
4. Experimental
4.1. General procedures and instrumentation
4.1.1. 13-Amino-4,7,10-trioxatridecaneamine cholesteryl
carbamate +5). Cholesteryl chloroformate 52.04 g,
4.55 mmol) in dichloromethane 520 ml) was added drop-
wise to 4,7,10-trioxa-1,13-tridecanediamine 54) 55.00 g,
Nuclear magnetic resonance spectra were recorded on a
General Electric QE 300 spectrometer or a Bruker AM
400 spectrometer. Chemical shifts are expressed in parts per
1
million 5ppm) down®eld of tetramethylsilane for H reso-
22.73 mmol)
and
potassium
carbonate
53.14 g,
nances. Mass spectra were recorded on a VG Autospec mass
spectrometer. Microanalyses were carried out at Leeds Univer-
sity Microanalytical Laboratory. All C, H, N, and S, analytical
®gures are percentage values. Some cholesteryl conjugated
compounds were analysed as the corresponding hydrates.
22.73 mmol) solution in tetrahydrofuran 5100 ml). The
reaction mixture was stirred at room temperature for 4 h.
The inorganic salts were removed by ®ltration. The solution
was concentrated and the product was separated by column
chromatography on silica gel 5ethyl acetate changed to ethyl
acetate/methanol 9:1) to give the product 51.73 g,
2.73 mmol, 60%) as a pale yellow viscous oil. dH
5300 MHz, CDCl3) 5.37±5.32 52H, m, vinyl cholesteryl,
and NH), 4.50±4.46 51H, m, CHO of cholesteryl), 3.64±
3.49 512H, m, 6£CH2O), 3.31±3.25 52H, m, CH2NHCO),
2.85±2.81 52H, m, CH2NH2), 2.36±2.25 52H, m,
cholesteryl). 2.01±1.72 510H, m, NH2, CH2CH2NH2,
CH2CH2NHCO, 4H of cholesteryl), 1.65±0.88 5m, 34H,
cholesteryl), 0.67 53H, s, cholesteryl). MS 5EI) m/z 633
5M1, 100%), 369 547%), 265 510%), 247 57%), 221
534%), 161 534%) 147 548%), 109 529%), 95 548%), 81
556%). Calcd for C38H68N2O5: C, 72.11; H, 10.83; N,
4.43. Found: C, 72.15; H, 11.10; N, 4.30%.
Thin layer chromatography was carried out using precoated
plastic-backed silica plates which were visualised using
ultraviolet light, and permanganate stain. Flash chromato-
graphy signi®es column chromatography on Merck silica
gel 5230±400). Petroleum ether refers to petroleum ether
5bp 40±608C) unless otherwise stated.
Preparation of pure and mixed monolayers of biotin thiol 3
and 11-mercaptoundecanol for XPS, IRAS and AFM analy-
sis. Gold substrates were prepared by evaporation of
approximately 10 nm chromium followed by 200 nm gold
onto clean silicon wafer 5for XPS) and glass microscope
slides 5for IRAS), using an Edwards 306 evaporator. For
AFM studies SAMs were formed on uniformly ¯at gold
substrates, prepared using the method described by Stamou
et al.15 The substrates were cleaned in an argon plasma,
4.1.2. 13-Biotinoylamido-4,7,10-trioxatridecaneamine
cholesteryl carbamate +1). Biotin 5112 mg, 0.46 mmol),

C.Booth et al./ Tetrahedron 57 &2001) 9859±9866
9865
N,N0-disuccinimidyl carbonate 597 mg, 0.38 mmol), and
triethylamine 5192 mg, 1.90 mmol) in DMF 515 ml) were
stirred at room temperature for 6 h. 13-Amino-4,7,10-tri-
oxatridecaneamine cholesteryl carbamate 55) 5240 mg,
0.38 mmol) was added and the reaction mixture was stirred
overnight. Solvents were removed under vacuum and the
product was separated by column chromatography on silica
gel using ethyl acetate ®rst then dichloromethane/methanol
59:1) as eluents to give the product 5215 mg, 0.25 mmol,
66%) as a pale yellow solid. dH 5300 MHz, CDCl3) 6.61
51H, br. NH of biotin), 6.10 51H, br. NH of biotin), 5.38
51H, m, vinyl cholesteryl), 5.29 51H, br. NH), 5.26 51H, br.
NH), 4.53±4.48 52H, m, CHO of cholesteryl and CHCH2S
of biotin), 4.34±4.30 51H, m, CHCHS of biotin), 3.67±3.55
512H, m, 6£CH2O), 3.42±3.34 52H, m, CH2N), 3.29±3.21
52H, m, CH2N), 3.18±3.12 51H, m, CHS of biotin), 2.92
51H, dd, Jˆ12.8, 4.9 Hz, one of CH2S of biotin), 2.74
51H, dd, Jˆ12.8, 3.1 Hz, one of CH2S of biotin),
2.35±2.21 52H, m, cholesteryl), 2.20 52H, t, Jˆ7.4 Hz,
CH2CON), 2.01±1.92 51H, m, cholesteryl), 1.87±0.88
5.29 51H, br. NH), 5.27 51H, br. NH), 4.53-4.48 52H, m,
CHO of cholesteryl and CHCH2S of biotin), 4.34±4.30 51H,
m, CHCHS of biotin), 3.67±3.55 58H, m, 4£CH2O), 3.59±
3.46 52H, m, CH2N), 3.44±3.37 52H, m, CH2N), 3.18±3.12
51H, m, CHS of biotin), 2.92 51H, dd, Jˆ12.8, 4.9 Hz, one
of CH2S of biotin), 2.74 51H, dd, Jˆ12.8, 3.1 Hz, one of
CH2S of biotin), 2.35±2.21 52H, m, cholesteryl), 2.24 52H, t,
Jˆ7.4 Hz, CH2CON), 2.01±1.92 51H, m, cholesteryl),
1.87±0.88 543H, m, cholesteryl and CH2CH2CH2CH2CON
of biotin), 0.67 53H, s, cholesteryl). MS 5ES) m/z 809
5M11Na, 62%), 322 58%), 249 58%). Acc. Mass: Calcd
for C44H74N4O6SNa: 809.5227. Found. 809.5208.
4.1.5. 12-Benzylthiododecanoic acid +6). 12-Bromodode-
canoic acid 55.0 g, 17.92 mmol), benzyl mercaptan 55.5 g,
44.8 mmol), and triethylamine 59.0 g, 89.6 mmol) in aceto-
nitrile 5150 ml) were stirred in room temperature under N2
for 48 h then dilute hydrogen chloride acid was added until
the solution become acidic 5pHˆ6). The product was
extracted with dichloromethane, and the organic layer was
dried 5MgSO4), ®ltered, and concentrated to give crude
product which was puri®ed by column chromatography on
silica gel 5petroleum spirit/ethyl acetateˆ4:1) to give the
product 55.4 g, 16.7 mmol, 93%) as a white solid. dH
5300 MHz, CDCl3) 7.32±7.28 55H, m, ArH), 3.70 52H, s,
CH2 of Bn), 2.43±2.32 54H, m, CH2S, CH2COOH), 1.65±
1.59 54H, m, CH2CH2S, CH2CH2COOH), 1.32±1.25 514H,
m, CH2). MS 5EI) m/z 322 5M1, 3%), 213 518%), 123 519%),
91 5100%). Calcd for C19H30SO2: C, 70.81; H, 9.32, S, 9.94,
Found: C, 70.70; H, 9.50, S, 10.00%.
547H,
m,
cholesteryl,
2£CH2CH2N,
and
CH2CH2CH2CH2CON of biotin), 0.67 53H, s, cholesteryl).
MS 5ES) m/z 882 5M11Na, 97%), 865 521%), 631 517%),
611 516%), 555 5100%), 517 510%), 413 518%), 322 52%),
511 53%). Calcd for C48H82N4SO7: C, 67.10; H, 9.62; N,
6.52; S, 3.73. Found: C, 66.80; H, 9.70; N, 6.25; S, 3.60%.
4.1.3. 8-Amino-3,6-dioxaoctaneamine cholesteryl car-
bamate. Cholesteryl chloroformate 52.00 g, 4.45 mmol) in
dichloromethane 520 ml) was added dropwise to 2,20-5ethyl-
enedioxy)diethylamine 53.30 g, 22.25 mmol) and potassium
carbonate 53.07 g, 22.25 mmol) in tetrahydrofuran 5100 ml).
The reaction mixture was stirred at room temperature for
4 h. The inorganic salts were removed by ®ltration. The
solution was concentrated and the product was separated
by column chromatography on silica gel 5ethyl acetate
changed to ethyl acetate/methanol 9:1) to give product
51.37 g, 2.45 mmol, 55%) as a pale yellow viscous oil. dH
5300 MHz, CDCl3) 5.36±5.34 51H, m, vinyl cholesteryl),
5.31 51H, m, NH), 4.48±4.51 51H, m, CHO of cholesteryl),
3.63 54H, s, OCH2CH2O), 3.58±3.51 54H, m,
2£OCH2CH2N), 3.40±3.37 52H, m, CH2N), 2.90 52H, t,
Jˆ5.3 Hz, CH2NH2), 2.36±2.25 52H, m, cholesteryl),
2.00±1.78 54H, m, cholesteryl), 1.67 52H, m, NH2), 1.65±
0.88 5m, 34H, cholesteryl), 0.67 53H, s, cholesteryl). MS
4.1.6. 12-Benzylthiododecanoic-+8-amino-3,6-dioxaoctyl)
amide +8). 12-Benzylthiododecanoic acid 55.25 g, 16.30
mmol), N,N0-disuccinimidyl carbonate 55.43 g, 21.20
mmol) and triethylamine 56.59 g, 65.20 mmol) in aceto-
nitrile 550 ml) were stirred at room temperature for 6 h.
Solvent was removed and the product 12-benzylthiodode-
canoic acid N-hydroxysuccinimide ester 57) 56.49 g,
15.49 mmol, 95%) separated by column chromatography
on silica gel 5petroleum spirit/ethyl acetate 1:3). The acti-
vated ester 53.00 g, 7.16 mmol) in dichloromethane 550 ml)
was added dropwise to a mixture of 2,20-5ethylenedioxy)-
diethylamine 55.30 g, 35.80 mmol) and triethylamine
53.62 g, 35.8 mmol) in dichloromethane 5100 ml). The reac-
tion mixture was stirred at room temperature for 5 h.
Solvent was removed and the product was separated by
column chromatography on silica gel 5ethyl acetate change
to ethyl acetate/methanol 1:1) to give the product 52.20 g,
4.87 mmol, 68%) as a white solid. dH 5300 MHz, CDCl3)
7.32±7.23 55H, m, ArH), 6.24 51H, br. NH), 3.70 52H, s,
CH2 of Bn), 3.64±3.52 58H, m, CH2O), 3.49±3.45 52H, m,
CH2N), 2.89 52H, t, Jˆ5.2 Hz, CH2NH2), 2.40 52H, t,
Jˆ7.3 Hz, CH2CH2S), 2.17 52H, t, Jˆ7.3 Hz, CH2CON),
1.70±1.51 54H, m, CH2), 1.32±1.16 514H, m, CH2); MS
5ES) m/z 453 5M111, 100%), Calcd for C25H44N2SO3: C,
66.33; H, 9.80; N, 6.19; S, 7.08. Found: C, 66.45; H, 9.80;
N, 6.05; S, 7.20%.
5ES)
m/z
583
5M11Na,
25%),
Calcd
for
C34H60N2O4´0.5H2O: C, 71.66; H, 10.79; N, 4.92. Found:
C, 71.55; H, 10.55; N, 4.70%. Acc. Mass: Calcd for
C34H60N2O4Na: 583.4451. Found. 583.4444.
4.1.4. 8-Biotinoylamido-3,6-dioxaoctaneamine cholesteryl
carbamate +2). Biotin 50.11 g, 0.47 mmol), N,N0-disuccini-
midyl carbonate 50.12 g, 0.47 mmol), and triethylamine
50.24 g, 2.35 mmol) in DMF 515 ml) were stirred at room
temperature for 6 h. 8-Amino-3,6-dioxaoctaneamine
cholesteryl carbamate 50.26 g, 0.47 mmol) was added and
the reaction mixture was stirred over night. Solvents were
removed under vacuum and the product was separated by
column chromatography on silica gel using ethyl acetate
®rst then dichloromethane/methanol 59:1) as eluents to
give the product 50.25 g, 0.32 mmol, 67%) as a pale yellow
solid. dH 5300 MHz, CDCl3) 6.58 51H, br. NH of biotin),
6.25 51H, br. NH of biotin), 5.38 51H, m, vinyl cholesteryl),
4.1.7. 12-Benzylthiododecanoic-+8-biotinoylamido-3,6-
dioxaoctyl) amide +9). Biotin 50.36 g, 1.46 mmol), N,N0-
disuccinimidyl carbonate 50.31 g, 1.22 mmol), and triethyl-
amine 50.59 g, 5.84 mmol) in DMF 55 ml) were stirred at
room temperature for 6 h. 12-Benzylthiododecanoic-58-

9866
C.Booth et al./ Tetrahedron 57 &2001) 9859±9866
2. Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.;
Osman, P. D. J.; Raguse, B.; Wleczorek, L.; Pace, R. J. Nature
1997, 387 55), 580±583. Offenhausser, A.; Knoll, W.
Kobunshi 1995, 44, 18±22.
amino-3,6-dioxaoctyl) amide 50.46 g, 1.02 mmol) in
dichloromethane 510 ml) was added and the reaction
mixture was stirred overnight. Solvents were removed
under vacuum and the product was separated by column
chromatography on silica gel using ethyl acetate ®rst then
dichloromethane/methanol 59:1) as eluents to give the
product 50.50 g, 0.74 mmol, 72%) as a white solid. dH
5300 MHz, CDCl3) 7.32±7.23 55H, m, ArH), 6.28 51H, br.
NH), 6.27 51H, br. NH), 5.67 51H, br. NH), 4.76 51H, br.
NH), 4.54±4.50 51H, m, CHCH2S of biotin), 4.37±4.31 51H,
m, CHCHS of biotin), 3.70 52H, s, CH2 of Bn), 3.64±3.56
58H, m, CH2O), 3.50±3.46 54H, m, CH2NHCO), 3.18±3.13
51H, m, CHS of biotin), 2.93 51H, dd, Jˆ5.1, 12.4 Hz, one
of CH2S of biotin), 2.74 51H, d, Jˆ12.4 Hz, one of CH2S of
biotin), 2.40 52H, t, Jˆ7.3 Hz, CH2CH2S), 2.27±2.17 54H,
m, CH2CON), 1.77±1.21 524H, m, CH2); MS 5ES) m/z 702
5M11Na, 100%), Calcd for C35H58N4S2O5´0.5H2O: C,
61.10; H, 8.64; N, 8.14; Found: C, 60.90; H, 8.70; N, 7.95%.
3. Garg, N.; Friedman, J. M.; Lee, T. R. Langmuir 2000, 16,
4266±4271.
4. Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.; Knowles,
P. F.; Marsh, A. Tetrahedron 1997, 53 531), 10939±10952.
Boden, N.; Bushby, R. J.; Liu, Q.; Evans, S. D.; Knowles, P. F.;
Jenkins, T. A.; Miles, R. E. Tetrahedron 1998, 54 538),
11537±11548.
5. Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.;
Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997,
13, 751±757. Cheng, Y.; Boden, N.; Bushby, R. J.; Clarkson,
S.; Evans, S. D.; Knowles, P. F.; Marsh, A.; Miles, R. E.
Langmuir 1998, 14, 839±844. Jenkins, A. T. A.; Bushby,
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4.1.8.
12-Mercaptododecanoic-+8-biotinoylamido-3,6-
6. Green, N. M. Adv.Protein Chem. 1975, 29, 85±133.
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dioxaoctyl) amide +3). About 50 ml liquid ammonia was
condensed into a 250 ml 2-neck ¯ask. Sodium 50.10 g) was
added and a dark blue colour observed. 12-Benzylthiodode-
canoic-58-biotinoylamido-3,6-dioxaoctyl) amide 50.22 g,
0.32 mmol) in 1:1 mixture of tetrahydrofuran and 1,4-diox-
ane 530 ml) was added and the reaction mixture stirred at
2788C for 1 h and re¯uxed for 2 h. Powdered ammonium
chloride 51.0 g) and tetrahydrofuran 580 ml) was added after
the reaction mixture was cooled to 2788C and the ammonia
was left to evaporate over night. The inorganic salts were
removed by ®ltration and washed with dichloromethane.
The product was separated by column chromatography on
silica gel 510% methanol in dichloromethane) to give
product 50.12 g, 0.20 mmol, 65%) as a pale yellow glass.
dH 5300 MHz, CDCl3) 6.81 51H, br. NH), 6.80 51H, br. NH),
6.50 51H, br. NH), 5.81 51H, br. NH), 4.54±4.50 51H, m,
CHCH2S of biotin), 4.37±4.31 51H, m, CHCHS of biotin),
3.64±3.56 58H, m, CH2O), 3.50±3.46 54H, m, CH2NHCO),
3.18±3.13 51H, m, CHS of biotin), 2.93 51H, dd, Jˆ5.1,
12.4 Hz, one of CH2S of biotin), 2.74 51H, d, Jˆ12.4 Hz,
one of CH2S of biotin), 2.52 52H, q, Jˆ7.2 Hz, CH2SH),
2.27±2.17 54H, m, CH2CON), 1.77±1.21 524H, m, CH2);
MS 5ES) m/z 611 5M11Na, 100%), Acc Mass Calcd for
C28H52N4NaS2O5: 611.3277; Found: 611.3276.
8. Haussling, L.; Michel, B.; Ringsdorf, H.; Rohrer, H. Angew.
1991, 30 55), 569±572. Knoll, W.;
Chem,. Int.Ed.Engl.
Schmitt, F. J.; Klein, Ch. International Patent Application
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This work was funded by a grant from EPSRC 5Grant No.
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