Novel preloaded resins for solid-phase biotinylation of carboxylic acids

Article abstract of DOI:10.1016/j.tetlet.2011.08.115

Use of solid-phase synthesis for the derivatization of carboxylic acids with biotinylated spacers consisting of ethylenoxy units is described. An aminomethylated resin provided with an acid-labile aldehyde linker is used as the polymer support and three different systems with a reactive amino group are introduced. Acylation of each system was tested with a set of model carboxylic acids and afforded crude products of excellent purity. The preloaded resins are similar to the Biotin-PEG-NovaTagTM resin but offer several advantages including simple elongation of the spacer arm. The protocols described represent a very efficient way of modifying compounds to obtain ligands for affinity chromatography studies.

Full text of DOI:10.1016/j.tetlet.2011.08.115

Tetrahedron Letters 52 (2011) 5782–5788
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Novel preloaded resins for solid-phase biotinylation of carboxylic acids
⇑
Nadezda Cankarova, Petr Funk, Jan Hlavac, Miroslav Soural
Department of Organic Chemistry, Institute of Molecular and Translational Medicine, Faculty of Science, University of Palacky, 17. listopadu 12, 771 46 Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Use of solid-phase synthesis for the derivatization of carboxylic acids with biotinylated spacers consisting
of ethylenoxy units is described. An aminomethylated resin provided with an acid-labile aldehyde linker
is used as the polymer support and three different systems with a reactive amino group are introduced.
Acylation of each system was tested with a set of model carboxylic acids and afforded crude products of
excellent purity. The preloaded resins are similar to the Biotin-PEG-NovaTagTM resin but offer several
advantages including simple elongation of the spacer arm. The protocols described represent a very effi-
cient way of modifying compounds to obtain ligands for affinity chromatography studies.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 29 May 2011
Revised 9 August 2011
Accepted 19 August 2011
Available online 26 August 2011
Keywords:
Solid-phase synthesis
Biotine
PEG linker
Acylation
Affinity chromatography
Biotinylation of organic compounds is a very frequent approach
for detailed studies of biochemical properties of biologically active
substances. Biotinylated agents are widely used to immobilize
compounds for affinity chromatography,1,2 labeling of peptides,3,4
proteins,5–7 nucleic acids, antibodies,8 etc. When the molecule to
be studied is suitably derivatized with biotin, it can be simply
immobilized on a chromatography column by using its highly spe-
cific and tight non-covalent interaction with avidin or streptavi-
din.9 When the selected compound is modified for an affinity
chromatography assay, the key task is incorporation of the spacer
between the molecule to be studied and biotin.10–12 The spacer
must meet the following basic criteria: (i) it has to be long enough
to avoid steric repulsion between avidin and the target biomole-
cule which interacts with the immobilized compound, (ii) it has
to be inert to non-specific interactions with biomolecules that do
not interact with the immobilized compound, (iii) it has to avoid
decreasing the biotinylated system’s solubility in water. Aliphatic
spacers (for instance, a spacer consisting of the caproic acid skele-
ton) have been used for a long time. However, more recently they
have been successfully replaced by ethylenoxy units—containing
ligands (ethylene glycols—EGs) which fulfill the last two men-
tioned criteria. For applications in which a longer spacer is required
(e.g., more than 12 atoms) EGs, in contrast to aliphatic chains,
avoid hydrophobic interactions resulting in deformation of the
spacer.
with carboxy group-containing compounds, (ii) acylation of an
amino group-containing compound with the biotin-EGs-COOH
system. In this Letter we describe an efficient method of biotinyla-
tion of carboxylic acids using the first mentioned method. So far,
biotinylation of appropriate compounds via EG linkers has been
commonly performed using solution-phase synthesis and/or
commercially available biotin-EG-NH2 systems. However, solution-
phase modification of compounds is usually complicated by diffi-
cult and time-consuming isolation of products from reaction
mixtures and also complicated purification of intermediates as well
as of the final products. Such problems can be eliminated when the
concept of solid-phase synthesis is applied. There are two alterna-
tives to the use of solid-phase synthesis for biotinylation of organic
compounds: (i) the substrate which undergoes biotinylation is pre-
immobilized (or directly synthesized) on an insoluble polymer
support.13 (ii) the biotin-EG-NH2 system is immobilized and the
resulting preloaded resin is used for an acylation reaction with
an appropriate carboxy group-containing compound. Quite re-
cently, Novabiochem has introduced the first preloaded resin of
this kind: Biotin-PEG-NovaTag™ resin, which has already been
successfully used in practice in several cases.14–16 In this Letter,
we describe preparation and use of alternative systems which offer
O
O
O
The most frequent way to attach the compound to be studied to
the biotin-EG system is: (i) acylation of the biotin-EG-NH2 system
HO
O
O
O HOBt, DIC
H
NH2
N
L
3O
O
DCM/DMF (1:1)
rt, overnight
O
⇑
Corresponding author. Tel.: +420 585634418; fax: +420 585634465.
E-mail addresses: souralm@seznam.cz, soural@orgchem.upol.cz (M. Soural).
Scheme 1. Attachment of an acid-labile linker to the aminomethylated resin.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.08.115

N. Cankarova et al. / Tetrahedron Letters 52 (2011) 5782–5788
5783
O2
O
N
S
O2N
H2
N
N
O
H
FAEEAA
O
S
H
10% AcOH/DMF
rt, on
HOBt, DIC
N
L
N
H
DCM/DMF (1:1)
rt, overnight
O
L
O
then NaBH(OAc)3
rt, 6 h
A1
O2N
O2N
O
S
O
N
O
H
L
N
S
O
N
L
N
biotin
H
O
50% pip/DMF
rt, 15 min
HOBt, DIC
O
O
DMF
rt, overnight
O
A2
A3
O
O
NH
Fmoc
NH2
O2N
O
S
H
NH2
L
N
SH(CH2)2OH
DBU
N
L
N
O
O
A
O
O
DMF
O
rt, 5 min
O
O
O
NH
S
O
NH
S
HN
A4
HN
NH
4
NH
4
O
O
Scheme 2. Preparation of system A.
NH2(CH2)2O(CH2)2OH
Fmoc-OSu
DCM
rt, overnight
10% AcOH/DMF
rt, on
O
OH
N
H
L
L
O
N
then NaBH(OAc)3
rt, 6 h
B1
biotin
O
S
HOBt, DIC
DIEA, DMF
O
OH
L
O
NH
O
L
N
Fmoc
4
Fmoc
HN
rt, overnight
repeated twice
O
B2
B3
1. FAEEAA
O
S
HOBt, DIC
DCM/DMF (1:1)
rt, overnight
20% pip/DMF
rt, 30 min
O
NH
O
L
N
H
4
HN
2. 20% pip/DMF
rt, 30 min
O
B4
O
S
O
NH
O
L
N
4
HN
O
O
O
H2N
B
O
Scheme 3. Preparation of system B.
several advantages including simple elongation of the spacer arm,
and can be synthesized simply with the help of developed proce-
dures involving commercially available synthons.
commercially available [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic
acid (FAEEAA hereafter).
Polystyrene resin was chosen as the polymer support because of
the very easy isolation of reaction products it allows. This consists
of only a simple filtration after each synthetic step. All the systems
were prepared by using an acid-labile linker attached to the ami-
nomethylated resin (Scheme 1).
Three different systems with various lengths and structures
were prepared and tested. The first step of the entire reaction se-
quence was the reductive amination of the benzaldehyde linker
with N-(3-aminopropyl)-2-nitrobenzenesulfonamide (for systems
A) or 2-(2-aminoethoxy)ethanol (for systems B and C). The key
building block for the preparation of each system was the
(i) System A: Immobilized N-(3-aminopropyl)-2-nitrobenzene-
sulfonamide A1 was acylated with FAEEAA, the Fmoc-pro-
tective group of the intermediate A2 was cleaved and the
resulting aminoderivative A3 was acylated with biotin.
Finally, the 2-Nos-protective group of the derivative A4
was cleaved to obtain the system A (Scheme 2).
(ii) System B: Immobilized 2-(2-aminoethoxy)ethanol B1 was
N-Fmoc protected using Fmoc-OSu and the terminal
hydroxy group of the intermediate B2 was biotinylated
(the reaction had to be repeated twice for quantitative ester-
ification). Subsequently, the Fmoc-protective group was

5784
N. Cankarova et al. / Tetrahedron Letters 52 (2011) 5782–5788
NH2(CH2)2O(CH2)2OH
10% AcOH/DMF
biotin
HOBt, DIC
DMF
rt, on
O
OH
L
L
N
H
L
O
then NaBH(OAc)3
rt, 6 h
rt, overnight
B1
O
H
N
O
O
O
FAEEAA
HOBt, DIC
DIEA
OH
L
N
O
Fmoc
O
N
O
O
4
4
rt, overnight
repeated once
C1
C2
HN
S
HN
S
O
O
N
H
N
H
O
NH2
O
O
O
O
L
N
20% pip/DMF
rt, 30 min
O
4
C
HN
S
O
N
H
Scheme 4. Preparation of system C.
FAEEAA
L
1.
HOBt, DIC
DCM/DMF (1:1)
rt, overnight
L
O2N
O
S
N
O2N
N
2
O
HN
O
O
S
O
HN
O
O
O
50% pip/DMF
rt, 15 min
O
2.
O
D1
NH
H
A3
H2N
biotin
HOBt, DIC
HS(CH2)2OH
DBU
L
O2N
O
L
N
N
2
DMF, rt, overnight
O
DMF, rt
5 min
S
O
2
HN
O
O
NH2
O
O
O
O
O
O
NH
O
NH
NH
HN
NH
HN
D2
4
D
S
4
S
Scheme 5. Preparation of system D.
O
S
carboxylic acid
HOBt, DIC,
DCM/DMF
O
L
HN
O
1.
HN
NH
O
N
O n
O
R
HN
NH
n
O
HN
50% TFA/DCM,
O
2.
O
NH2
rt, 30 min
O
4
S
4
N
H
N
H
O
A: n = 1
D: n = 2
carboxylic acid
HOBt, DIC,
DCM/DMF
1.
2.
O
S
O
S
4
O
O
NH
NH
O
HN
O
O
L
N
50% TFA/DCM,
rt, 30 min
4
HN
HN
O
O
O
O
H
O
O
H2N
O
N
O
O
B
R
O
carboxylic acid
HOBt, DIC,
DCM/DMF
H
N
O
O
O
1.
2.
NH2
O
O
O
O
HN
O
O
L
N
R
50% TFA/DCM,
rt, 30 min
O
4
O
4
HN
S
HN
S
O
C
N
O
N
H
H
Scheme 6. Acylation and cleavage of final systems A, B, C, and D from the polymer support.

N. Cankarova et al. / Tetrahedron Letters 52 (2011) 5782–5788
5785
Table 1
cleaved and the intermediate B4 was acylated with FAEEAA.
Finally, the Fmoc-protective group was cleaved to give the
system B (Scheme 3).
Acids used for acylation of systems A, B, C, and D
R
System
Product
Puritya (%)
(iii) System C: 2-(2-Aminoethoxy)ethanol B1 was immobilized
and its amino group was biotinylated to give the intermedi-
ate C1. Subsequently the hydroxy group was acylated with
FAEEAA (the reaction had to be repeated once for a quantita-
tive conversion). Cleavage of the Fmoc-protective group of
intermediate C2 afforded target system C (Scheme 4).
Fmoc
A
B
C
1
2
3
4
93
95
97
94
N
H
D
Fmoc
A
B
C
5
6
7
88
95
97
N
H
A
B
C
8
9
10
95
95
98
O
To increase further the diversity of the method we subsequently
focused on possible elongation of the spacer arm. For such a mod-
ification we chose system A, which was very simply transformed to
system D consisting of twice the number of ethyleneoxy units:
HN
NH
O
A
B
C
11
12
13
96
94
97
NO2
N
H3C
(iv) System D : Polymer-supported aminoderivative A3 was
acylated with FAEEAA. Following cleavage of Fmoc-protec-
tive group intermediate D1 was biotinylated to yield deriva-
tive D2. Finally, the 2-Nos-protective group was cleaved to
access target system D (Scheme 5).
A
B
C
14
15
16
17
93
92
96
85
D
F
Cl
A
B
18
19
90
94
C
20
95
NO2
Systems A, B, C, and D were obtained in very good purity (about
a
95%, HPLC-UV traces). Their applicability for the modification of
Purity of crude product (calculated from HPLC-UV traces).
M:\rok 2010\...\np38-121B_100602182956
2.6.2010 18:29:56
RT: 0.00 - 5.00
NL:
1.39
2.47E5
200000
Total Scan
PDA
np38-
100000
0.18
121B_10060
2182956
1.78
1.24
0.21
1.02
1.84
4.84
0
NL:
1.39
2.26E5
200000
Total Scan
PDA
np38-1201bc
1.44
1.52
100000
0.17
0.85
1.14
0.92
0.23
4.79 4.86
0
NL:
0.53
2.15E5
200000
Total Scan
PDA
np38-
100000
131b_10060
0.17
2183605
0.21
0.21
0.98
0.97
1.48
1.05
0.77
1.30
1.30
4.84
0
NL:
4.33E5
400000
Total Scan
PDA
np38-171b
200000
0
0.18
0.17
0.76
1.44
0.71
4.84
NL:
0.88
8.28E4
Total Scan
PDA
50000
np38-151bi
1.45
0.21
4.79
0
NL:
1.14
2.32E5
200000
Total Scan
PDA
1.19
1.33
np38-1141b
100000
0
0.18
1.49
0.97
1.0
0.21
1.62
0.06
0.48
0.5
4.85
0.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Time (min)
Figure 1. HPLC-UV traces of crude products 1, 5, 8, 11, 14, and 18 obtained after acylation of system A.

5786
N. Cankarova et al. / Tetrahedron Letters 52 (2011) 5782–5788
M:\rok 2011\...\kity\nove\pf-KIT04-03
20.1.2011 11:29:42
RT: 0.00 - 5.00
NL:
1.57
1.45E5
Total Scan
PDA
100000
50000
pf-KIT04-03
0.17
0.22
1.53
1.39
1.32
4.76
0.40
0.25
0.65
1.35
1.19
0.11
0
NL:
1.53
1.55E5
150000
Total Scan
PDA
100000
50000
pf-kit04-04
0.16
0.21
1.63
0.10
4.79
0
NL:
1.10
2.11E5
200000
Total Scan
PDA
150000
100000
50000
0
pf-kit04-05
0.16
0.17
0.82
1.22
0.21
4.79
4.79
NL:
1.05
4.88E4
Total Scan
40000
30000
20000
10000
0
PDA
pf-kit04-01
1.20
1.29
0.77
0.22
0.29
0.50
NL:
1.20E5
Total Scan
100000
50000
0
PDA
pf-kit04-02
0.17
0.21
1.02 1.25
1.0
1.45
1.53
0.28
4.79
0.0
0.5
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Time (min)
Figure 2. HPLC-UV traces of crude products 2, 6, 12, 15, and 19 obtained after acylation of system B.
carboxy group-containing substrates was tested using a set of
model carboxylic acids. Acylation was performed with the help
of in situ generated HOBt esters of appropriate acids (Scheme 6).
After acid-mediated cleavage from the resin, the products were
obtained in excellent crude purity (Table 1). However, it must be
pointed out that the method is unsuitable for modification of
acid-labile compounds due to their possible decomposition during
the cleavage from the polymer support. In such cases, a base-labile
linker would have to be applied for the preparation of appropriate
systems.
(iii) The distance of the amino group from the resin’s surface is
longer (especially in systems B, C, and D) which is sterically
more favorable for acylation with bulkier and sterically more
demanding substrates.
(iv) Finally, the most significant advantage of the method
described consists of a very simple elongation of the spacer
arm. With the use of the protocol described and synthons,
the arm can simply be doubled (see transformation of sys-
tem A to system D) but also longer systems could be synthe-
sized if necessary.
The preloaded resins introduced are an alternative solution to
the use of Biotin-PEG-NovaTag™ resin. Compared to this commer-
cially available resin, the amino group which undergoes acylation
is not part of a cleavage site and this offers at least three
advantages:
In conclusion, we have introduced an efficient method of mod-
ifying carboxy group-containing compounds for affinity chroma-
tography studies. The simple solid-phase synthesis methodology
that was used for both the preparation and application of appropri-
ate biotin-EG-NH2 systems eliminates problems resulting from dif-
ficult or time-consuming isolation of products from reaction
mixtures. Small quantities of appropriately modified compounds
can be easily produced by the method. Furthermore, synthesis
and application of systems A, B, C, and D can be performed without
the need for complicated equipment (polypropylene fritted syrin-
ges were used as reaction vessels). The method was successfully
tested for set of model acids and afforded the corresponding prod-
ucts in excellent purity without need of their further purification
(see Figs. 1–4)—HPLC-UV spectra of the cleaved products) which
makes the procedure theoretically applicable for routine biotinyla-
tion of a wide range of carboxylic acid derivatives.
(i) The cleavage from the polymer support is not influenced by
the character of the substrate (resonance effects can
decrease the sensitivity of the amidic group towards proton-
ation resulting in an incomplete release from the polymer
support).
(ii) Evaluation of the reaction conversion can be done directly
from the analytical sample of the resin with the use of
HPLC-UV-(MS) traces (starting material from resins A–D is
cleavable in contrast to Biotin-PEG-NovaTag™ resin which
is uncleavable when not acylated and suitable derivatization
is needed before analysis).

N. Cankarova et al. / Tetrahedron Letters 52 (2011) 5782–5788
5787
M:\rok2011\...\kity\nove\pf-KIT04-03
RT:0.00 - 5.00
20.1.2011 11:29:42
NL:
1.45E5
1.57
Total Scan
PDA
pf-KIT04-03
100000
50000
0.17
1.53
1.39
4.76
4.79
0.22
0.40
0.25
0.65
1.35
0.11
0
NL:
1.55E5
1.53
150000
Total Scan
PDA
100000
50000
pf-kit04-04
0.16
1.32
1.63
0.21
0.10
1.19
1.10
0
NL:
2.11E5
200000
Total Scan
PDA
150000
100000
50000
0
pf-kit04-05
0.16
0.82
1.22
0.21
4.79
4.79
NL:
4.88E4
1.05
Total Scan
40000
30000
20000
10000
0
PDA
0.17
pf-kit04-01
1.20
1.29
0.77
0.22
0.29
0.50
NL:
1.20E5
Total Scan
100000
50000
0
PDA
pf-kit04-02
0.17
0.21
1.02 1.25
1.0
1.45
1.53
0.28
0.5
4.79
0.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Time (min)
Figure 3. HPLC-UV traces of crude products 3, 7, 13, 16, and 20 obtained after acylation of system C.
Q:\CR\MS_2010\np38-1211B
24.9.2010 10:32:34
2.51
RT: 0.00 - 10.00
NL:
1.57E5
Total Scan
PDA
140000
120000
100000
80000
60000
40000
20000
0
np38-
1211B
0.30
0.39
2.66
2.71
2.23
0.45
3.95
9.62
9.63
9.58
9.57
0.63
0.75
3.30
2.77
1.70
NL:
70000
60000
50000
40000
30000
20000
10000
0
7.53E4
Total Scan
PDA
0.30
np38-
1511B
1.80
2.40
2.37
2.12
0.38
0.47
1.25
1.5
0.0
0.5
1.0
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
Time (min)
Figure 4. HPLC-UV traces of crude products 4, and 17 obtained after acylation of system D.
Acknowledgments
References and notes
1. Scriba, G. K. E. Methods and Principles in Medicinal Chemistry 2004, 21, 211–241
(Molecular Biology in Medicinal Chemistry).
The authors are grateful to project CZ.1.07/2.3.00/20.0009 com-
ing from the European Social Fund and to the Student Project of the
Palacky University (PrF_2011_023). The infrastructural part of this
project (Institute of Molecular and Translational Medicine) was
supported from the Operational Program Research and Develop-
ment for Innovations (project CZ.1.05/2.1.00/01.0030).
2. Roque, A. C. A.; Lowe, Ch. R. Methods in Molecular Biology (Totowa, NJ, United
States) 2008, 421, 1–21 (Affinity Chromatography (2nd Edition)).
3. Heuzenroeder, M. W.; Barton, M. D.; Vanniasinkam, T.; Phumoonna, T. Methods
in Molecular Biology (Totowa, NJ, United States) 2009, 524, 137–144 (Epitope
Mapping Protocols (2nd Edition)).
4. Fabry, M.; Brandenburg, D. BioMethods (Basel) 1996, 7, 65–81 (Laboratory
Guide to Biotin-Labeling in Biomolecule Analysis).
5. Wingren, Ch.; Borrebaeck, C. A. K. Curr. Opin. Biotech. 2008, 1, 55–61.
6. Ingvarsson, J.; Larsson, A.; Sjoholm, A. G.; Truedsson, L.; Jansson, B.; Borrebaeck,
C. A. K.; Wingren, C. J. Proteome. Res. 2007, 6, 3527–3536.
7. Wingren, C.; Ingvarsson, J.; Dexlin, L.; Szul, D.; Borrebaeck, C. A. K. Proteomics
2007, 7, 3055–3065.
Supplementary data
Supplementary data (experimental procedures, analytical data
of syntesized compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.tetlet.2011.08.115.
8. Pavlickova, P.; Hug, H. Methods Mol. Biol. 2004, 264, 73–83 (Protein
Arrays).

5788
N. Cankarova et al. / Tetrahedron Letters 52 (2011) 5782–5788
9. Stayton, P. S.; Freitag, S.; Klumb, L. A.; Chilkoti, A.; Chu, V.; Penzotti, J. E.; To, R.;
Hyre, D.; Le Trong, I.; Lybrand, T. P.; Stenkamp, R. E. Biomol. Eng. 1999, 16, 39–
44.
10. O’Carra, P.; Barry, S.; Griffin, T. FEBS Lett. 1974, 43, 169–175.
11. Phillips, T. M. Encyclopedia of Chromatography, 2nd ed.; CRC Press: Boca Raton,
Florida, USA, 2010; Vol. 1, pp 31–32.
13. Baumeister, B.; Beythien, J.; Ryf, J.; Schneeberger, P.; White, P. D. Int. J. Pep. Res.
Ther. 2005, 11, 139–141.
14. Gilmore, B. F.; Quinn, D. J.; Duff, T.; Cathcart, G. R.; Scott, Ch. J.; Walker, B.
Bioconjug. Chem. 2009, 20, 2098–2105.
15. Dang, T. H. T.; de la Riva, L.; Fagan, R. P.; Storck, E. M.; Heal, W. P.; Janoir, C.;
Fairweather, N. F.; Tate, E. W. ACS Chem. Biol. 2010, 5, 279–285.
16. Berry, A. F. H.; Heal, W. P.; Tarafder, A. K.; Tolmachova, T.; Baron, R. A.; Seabra,
M. C.; Tate, E. W. ChemBioChem 2010, 11, 771–773.
´
12. Janson, J. C., Ryden, L., Eds.; Protein Purification, VCH Publishers: New York,
USA, 1989; pp 289–306.