Development of a solid-supported biotinylation reagent for efficient biotin labeling of SH groups on small molecules

Article abstract of DOI:10.1016/j.tetlet.2011.11.089

We report here the design and synthesis of a novel and selective SH-group biotinylating reagent, KSH-1 (1), for the biotinylation of small molecules using solid phase chemistry. The results demonstrate that 1 efficiently biotinylated a small molecule, cap

Full text of DOI:10.1016/j.tetlet.2011.11.089

Tetrahedron Letters 53 (2012) 535–538
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Development of a solid-supported biotinylation reagent for efficient biotin
labeling of SH groups on small molecules
Kentarou Fukumoto a,b, Kumi Adachi a, Akihiro Kajiyama a, Yuri Yamazaki a, Fumika Yakushiji a,
Yoshio Hayashi a,
⇑
a Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji 192-0392, Japan
b Kokusan Chemical Co., Ltd, Tokyo 103-0023, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here the design and synthesis of a novel and selective SH-group biotinylating reagent, KSH-1
(1), for the biotinylation of small molecules using solid phase chemistry. The results demonstrate that 1
efficiently biotinylated a small molecule, captopril, and afforded the product in high yield and purity.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 31 October 2011
Revised 15 November 2011
Accepted 16 November 2011
Available online 23 November 2011
Keywords:
Solid support
Biotinylation reagent
Npys
Protein modification
SH-specific labeling
Introduction
purification steps, biotinylation using solid phase chemistry is an
attractive method because the unreacted materials or residual
In life sciences research, the biotinylation of bioactive com-
pounds can assist in analyzing the compound’s biological proper-
ties.1 This labeling approach is broadly used to label peptides,1,2
proteins,3–5 nucleic acids,6 and antibodies.7 The affinity and specific-
ity of the binding between avidin and biotin (vitamin H) is extraor-
dinary, and the binding interaction is among the strongest known
non-covalent protein–ligand interactions (Kd = 10À15 M).8 This
interaction is exploited in a variety of labeling methods, including
purification, detection, and immobilization of biomolecules, as well
as in viral vector targeting and drug targeting systems.7 Biotinyla-
tion is unlikely to perturb the natural function of a biomolecule
because the molecular structure of biotin is small (MW 244).1 In
most cases, biotinylation can be applied rapidly and specifically to
particular functional groups of a target molecule using specific
reagents.9 Excess concentrations of the biotinylation reagent are
usually used to increase the reaction efficacy. After labeling, the
unreacted reagent or by-products produced during the reaction
can be removed using purification techniques, usually gel filtration.
The biotinylation of small molecules is more complex, and
advanced purification methods, such as reverse-phase HPLC, are
required to separate the target compound from the biotin, which
can have similar molecular weights. To avoid such laborious
product remaining on the solid-support can be easily removed by
filtration. The present study describes the design and synthesis of
a novel selective SH biotinylation reagent (Fig. 1), for use in bioti-
nylating small molecules using solid phase chemistry techniques.
We achieved the efficient biotinylation of an example small mole-
cule, captopril,10 with excellent product purity.
Results and discussion
Design of the new SH-selective solid-supported biotinylation
reagent
As shown in Figure 1, the newly designed solid-supported
biotinylation reagent comprised three parts, namely, a solid
support, an active disulfide, and a biotin unit. The following three
O
HN
S
O
H
NH
H
O2N
N
H
Linker
solid-support
Linker
S
S
N
O
active disulfide
biotin unit
⇑
Corresponding author.
E-mail address: yhayashi@toyaku.ac.jp (Y. Hayashi).
Figure 1. Design of the SH-selective solid-supported biotinylation reagent.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.11.089

536
K. Fukumoto et al. / Tetrahedron Letters 53 (2012) 535–538
principles guided this design. (1) A hydrophilic resin with a poly-
ethyleneglycol (PEG) linker (amino-PEG resin) was chosen as a so-
lid support to realize the biotinylation reaction in either aqueous or
organic solution. (2) An Npys11 (3-nitro-2-pyridinesulfenyl)-type
disulfide structure was adopted as a key functional group to realize
SH-selective labeling, because Npys was not only useful as a pro-
tective group for the SH during the peptide synthesis, but also
acted as an attractive active disulfide to enable the specific forma-
tion of a disulfide bond with free SH groups under aqueous condi-
tions. (3) The biotin unit was conjugated to a linker that distanced
the biotin from the modification site in the target molecule, which
reacted with the Npys moiety to form an active disulfide bond. In
the present Letter, an aminoethyl chain was used as an example
linker. Note that the length of the linker can be altered for a partic-
ular purpose, as needed.
O
O2N
COOtBu
N
H
H
N
Boc
S
S
N
Synthesis of KSH-1
9
Figure 2. X-ray analysis of intermediate 9.
KSH-1 (1) was synthesized in 10 steps from the commercially
available 6-hydroxynicotinic acid12 2, as shown in Scheme 1.
Namely, 5-nitro-6-hydroxynicotinic acid 3 was prepared by the
nitration of 2 with fuming nitric acid, although the yield was not
excellent due to the high water-solubility of the product. The acid
3 was treated with phosphorus pentachloride in phosphorus oxy-
chloride under reflux conditions. After the removal of excess
amounts of the phosphorus compound, MeOH was added to give
methyl 5-nitro-6-chloronicotinate 4 in moderate yields. This ester
was refluxed with benzylmercaptan in the presence of Et3N,
followed by hydrolysis of the ester moiety with LiOH to give 6-ben-
zylsulfanyl-5-nitronicotinic acid 6 in good yield. The acid 6 was then
coupled to the linker, H-b-Ala-OtBuÁHCl, in the presence of
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chlo-
ride n-hydrate (DMT-MM)13,14 to obtain the corresponding amide 7.
The conversion of amide 7 to the pyridinesulfenylchloride deriva-
tive 8 was achieved using a method reported by Ueki et al., involving
chlorination with SO2Cl2.15 The resultant crude chlorosulfenyl
derivative was directly coupled with N-Boc-aminoethyl p-methoxy-
benzyl sulfide (Boc-NH-C2H4-S-PMB) to give the active disulfide 9 in
moderate yield. The chemical structure of 9, which was re-crystal-
lized from chloroform, was confirmed by X-ray crystallography, as
shown in Figure 2.16 In the final steps, the Boc group and t-Bu ester
of 9 were removed by TFA and subsequently coupled with
biotinyl-OSu to give the biotin conjugate 10 in good yield. This prod-
uct was then attached to the amino-PEG-resin (0.42 mmol/g)17,18
using the DIPCI-HOBt method19 to afford the desired solid-sup-
ported biotin derivative 1. Small amounts of the unreacted amino
groups on the solid support were capped by acetylation with Ac2O
in the presence of pyridine.
Activity of KSH-1 (1) as an SH-selective biotinylation reagent
The ability of KSH-1 (1) as a biotinylation reagent was tested
using captopril (11),20 an angiotensin converting enzyme (ACE)
inhibitor, as a model of SH-containing small molecule (Fig. 3). To
determine the reaction conditions for biotinylation with 1, a solu-
tion of 11 in DMF/H2O (1:1, corresponding to a concentration of
0.011 mol/L for 1) was mixed with different amounts of 1 (1–
4 equiv)21 in a filtration tube at room temperature. Captopril (11)
labeling in the reaction mixture was analyzed by HPLC. As shown
in Figure 4, the HPLC content of 11 decreased according to the
amount of 1, and the reactant was completely consumed in the
presence of 4 equiv of 1. Therefore, this amount was used to eval-
uate the isolated yield of biotin labeled captopril (12) in a subse-
quent study. Namely, 11 and 1 (4 equiv)21 were reacted within
COOH
O2N
HO
COOH
O2N
Cl
COOMe
O2N
Bn
COOMe
O2N
Bn
COOH
a
c
d
b
HO
N
N
N
S
N
S
N
2
3
4
5
6
O
O
O
COOtBu
O2N
Bn
COOtBu
O2N
O2N
COOtBu
g
e
f
N
H
N
H
N
H
H
N
Boc
S
S
N
S
N
Cl
S
N
7
8
9
O
O
HN
HN
O
O
O
H
H
NH
H
NH
H
j
h, i
O2N
S S
10
COOH
O2N
S S
N
N
H
N
PEG
H
N
H
N
H
H
S
S
N
N
O
O
KSH-1 (1)
Scheme 1. Reagents and conditions: (a) HNO3, fuming (1.52), 50 °C, 18 h, 29%; (b) PCl5, POCl3, 100 °C, 3 h then methanol, 0 °C, 1 h, 57%; (c) benzylmercaptan, Et3N, MeOH,
reflux, 5 h, 92%; (d) LiOHÁH2O, MeOH/H2O, 0 °C to rt 15 h, quant.; (e) H-b-Ala-OtBuÁHCl, Et3N, DMT-MM, CH2Cl2, rt, 18 h, 85%; (f) SO2Cl2, 1,2-dichloroethane, rt, 3 h; (g) Boc–
NH–C2H4–S–PMB, CH2Cl2, À30 °C, 12 h, 83% (two steps); (h) TFA, rt, 30 min; (i) Biotinyl-OSu, Et3N, DMF, rt, 17 h, 87% (two steps); (j) DIPCI, HOBtÁH2O, amino-PEG-resin
(0.42 mmol/g), DMF, rt, 16 h, then Ac2O, pyridine, DMF, rt, 0.5 h.

K. Fukumoto et al. / Tetrahedron Letters 53 (2012) 535–538
537
remove the solid support, and the solvent was removed under re-
duced pressure. The resultant product was analyzed using HPLC
(Fig. 5C). This result demonstrated that the desired product 12
was obtained in a 96% yield and the purity was 87%.
O
KSH-1 (1)
HN
S
H
NH
H
HS
O
H
In summary, we report here the design and synthesis of a novel
and selective SH biotinylation reagent, KSH-1 (1), for the purpose
of biotinylating small molecules using solid phase chemistry. This
reagent expeditiously biotinylated the small molecule captopril,
yielding the product in good purity. This solid phase biotinylation
strategy would be applicable to peptides and proteins as well. Fur-
thermore, other tag-based molecular labeling approaches that rely
on the preparation of a key Npys-type active disulfide intermediate
using solid support approaches are in progress in our laboratory
and will be reported in the near future.
N
N
COOH
S
S
O
O
O2N
captopril (11)
N
O
DMF/H2O (1:1)
PEG
N
H
N
H
filtration
O
HN
H
NH
H
H
N
General procedure
S
S
O
S
N
O
biotin-labeled captopril (12)
Figure 3. Biotinylation of captopril (11) using KSH-1 (1).
3-[(6-Chlorosulfanyl-5-nitropyridine-3-carbonyl)amino]prop-
ionic acid tert-butyl ester (8)
COOH
To a mixture of 7 (664 mg, 1.59 mmol) and pyridine (20
lL) in
1,2-dichloroethane (5 mL) was added SO2Cl2 (283 L, 3.50 mmol).
l
After stirring for 3 h, excess amount of reagent and solvent were
removed in vacuo. Then, co-evaporated with hexane repeatedly
three times, to obtain a yellow color precipitate 8. This compound
was used for the next step without any further purification.
3-{[6-(2-tert-Butoxycarbonylaminoethyldisulfanyl)-5-nitro-py-
ridine-3-carbonyl]amino}propionic acid tert-butyl ester (9)
Boc-NH-C2H4-S-PMB (synthesized from Boc-NH-C2H4-SH and p-
methoxybenzylchloride, 473 mg, 1.59 mmol) was dissolved in dry
CH2Cl2 (10 mL) under an Ar atmosphere, followed by cooling to
À30 °C in an acetone bath. To this solution was added 8 in dry
CH2Cl2 (52 mL) dropwise over 30 min. The solution was stirred
for 12 h under the same conditions then warmed to room temper-
ature. The solution was sequentially washed with 10% aqueous cit-
ric acid, water, and brine. The organic layer was dried over
anhydrous Na2SO4, followed by concentration in vacuo. The resul-
tant residue was purified by column chromatography over silica
gel using hexane/AcOEt (3:1), and elution gave 9 as a yellow oil
(660 mg, 83% (2 steps)), which was recrystallized in hexane/CHCl3
as a yellow needle; 1H NMR (400 MHz, CDCl3) d 1.46 (s, 9H), 1.47 (s,
9H), 2.59 (t, J = 5.9 Hz, 2H), 3.01 (t, J = 5.7 Hz, 2H), 3.40 (br m, 2H),
3.73 (dt, J = 5.8, 12 Hz, 2H), 5.65 (br s, 1H), 8.87 (br s, 1H), 9.23 (br s,
1H); HRMS (ES+): m/z 503.1628 (M+H+) (calcd for C20H31N4O7S2:
503.1634).
Figure 4. Time course of captopril biotinylation in the presence of 1 (1–4 equiv). An
aliquot of the reaction mixture (5 L) at each time point during the reaction was
injected into an HPLC, and the captopril 11 content was calculated according to the
l
corresponding peak areas and positions.
(A)
(B)
(C)
24.2 min
24.2 min
3-[(5-Nitro-6-{2-[5-(2-oxo-hexahydrothieno[3,4-d]imidazol-6-
yl)pentanoylamino]ethyldisulfanyl}pyridine-3-carbonyl)ami-
no]propionic acid (10)
16.8 min
Compound 9 (516 mg, 1.03 mmol) was dissolved in TFA (10 mL)
and stirred for 30 min at room temperature. The solution was
concentrated and co-evaporated with hexane three times. A yellow
precipitate was obtained. The precipitate was dissolved in dry DMF
Figure 5. HPLC charts of captopril biotinylation with 1. Charts of (A) captopril in
DMF/H2O (1:1); (B) the reaction mixture after 30 min; and (C) the final compound
after removal of the solvent, resolved in 15% aqueous MeOH. HPLC conditions:
(25 mL), to which was added Et3N (660 lL, 5.14 mmol). The
YMC-pack ODS-AM (4.6 Â 150 mm) with
a linear gradient of 0.1% TFA-MeCN
solution color changed from yellow to brown, and the biotin-
succinimidyl ester (292 mg, 0.856 mmol) was added. After stirring
for 17 h, the solvent was removed in vacuo, and AcOEt (20 mL)
was added. The obtained yellow precipitate was filtered and washed
with AcOEt (10 mL) three times, followed by washing with Et2O
(10 mL) three times. The obtained compound was dried under
(100:0–60:40 over 30 min) at a flow rate of 0.9 mL minÀ1, detection at 230 nm. A
peak with a retention time of 24.2 min in (B and C) corresponded to the biotin-
labeled captopril 12.
DMF/H2O (1:1, 7 mL, corresponding to
a concentration of
0.021 mol/L of 1) for 30 min. As shown in Figure 5B, the peak for
captopril (11, rt = 16.8 min, Fig. 5A) completely disappeared, and
the biotin-labeled captopril 12 appeared as a new major peak at
a retention time of 24.2 min. The reaction mixture was filtered to
reduced pressure to give 10 as a yellow powder (425 mg, 87%). 1
H
NMR spectrum: (400 MHz, CD3OD) d 1.28–1.85 (m, 6H), 2.22
(t, J = 7.2 Hz, 2H), 2.58–2.77 (m, 4H), 2.80–3.10 (m, 2H), 3.15–3.27

538
K. Fukumoto et al. / Tetrahedron Letters 53 (2012) 535–538
(m, 1H), 3.40–3.59 (m, 2H), 3.67 (t, J = 6.8 Hz, 2H), 4.26–4.35
(m, 1H), 4.46–4.54 (m, 1H), 8.96 (d, J = 2.0 Hz, 1H), 9.23
(d, J = 2.0 Hz, 2H); HRMS (ES+): m/z 573.1292 (M+H+) (calcd for
this article can be found, in the online version, at doi:10.1016/
j.tetlet.2011.11.089.
C21H29N6O7S3: 573.1260).
References and notes
1. (a) Hofmann, K.; Finn, F. M.; Kiso, Y. J. Am. Chem. Soc. 1978, 100, 3585–3590; (b)
Hofmann, K.; Kiso, Y. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3516–3518; As a more
recent reference: (c) Fabry, M.; Brandenburg, D. BioMethods 1996, 7, 65–81.
2. Heuzenroeder, M. W.; Barton, M. D.; Vanniasinkam, T.; Phumoonna, T. Methods
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3. Ingvarsson, J.; Larsson, A.; Sjoholm, A. G.; Truedsson, L.; Jansson, B.; Borrebaeck,
C. A. K.; Wingren, C. J. Proteome. Res. 2007, 6, 3527–3536.
4. Wingren, Ch.; Borrebaeck, C. A. K. Curr. Opin. Biotech. 2008, 155–161.
5. Wingren, C.; Ingvarsson, J.; Dexlin, L.; Szul, D.; Borrebaeck, C. A. K. Proteomics
2007, 7, 3055–3065.
KSH-1 (1)
To a solution of 10 (250 mg, 0.436 mmol) in DMF (4 mL) was
added, sequentially, DIPCI (67.5 L, 0.436 mmol), H2N-PEG-resin
l
(346 mg, 0.145 mmol), and HOBtÁH2O (73.4 mg, 0.480 mmol). After
stirring for 16 h, the solution was filtered and washed with DMF
five times. The resin was processed using the Kaiser test, and the
reaction was assumed to have reached completion. To cap the
small quantities of unreacted –NH2 groups on the resin, Ac2O
6. Pavlickova, P.; Hug, H. Methods Mol. Biol. 2004, 264, 73–83.
7. Barat, B.; Wu, A. M. Biomol Eng. 2007, 24, 283–291.
8. (a) Green, N. M. Biochem. J. 1963, 89, 585–591; (b) Swack, J. A.; Zander, G. L.;
Utter, M. F. Anal. Biochem. 1978, 87, 114–126.
(275 lL, 2.91 mmol) and pyridine (236 lL, 2.91 mmol) were added.
After 30 min, the solution was filtered. The resin was then sequen-
tially washed with MeOH (2 mL, six times) and Et2O (2 mL, four
times). The resin was dried in vacuo to give KSH-1 (1) (410 mg,
0.145 mmol, the total molar quantity of KSH-1 was preserved
during the process).
9. (a) Flanders, K. C.; Mar, D. H.; Folz, R. J.; England, R. D.; Coolican, S. A.; Harris, D.
E.; Floyd, A. D.; Gurd, R. S. Biochemistry 1982, 21, 4244–4251; (b) Nunomura,
W.; Takakuwa, Y.; Parra, M.; Conboy, J. G.; Mohandas, N. J. Biol. Chem. 2000, 275,
6360–6367; (c) Muroi, M.; Ohnishi, T.; Tanamoto, K. J. Biol. Chem. 2002, 277,
42372–42379; (d) Prior, I. A.; Clague, M. J. Biochimica et Biophysica Acta 2000,
1475, 281–286; (e) Sutoh, K.; Yamamoto, K.; Wakabayashi, T. J. Mol. Biol. 1984,
178, 323–339; (f) Hayashi, Y.; Ezawa, K. JP Patent A-246382, 1988.; (g)
Ghebrehiwet, B.; Bossone, S.; Erdei, A.; Reid, K. B. M. J. Immnol. Methods 1988,
110, 251–260.
General procedure used for the biotinylation of captopril
10. (a) Murthy, V. S.; Waldron, T. L.; Goldberg, M. E.; Vollmer, R. R. Eur. J. Pharmacol.
1977, 46, 207–212; (b) Shimazaki, M.; Hasegawa, J.; Kan, K.; Nomura, K.; Nose,
Y.; Kondo, H.; Ohashi, T.; Watanabe, K. Chem. Pharm. Bull. 1982, 30, 3139–3146;
Kim, H. M.; Shin, D. R.; Yoo, O. J.; Lee, H.; Lee, J. FEBS Lett. 2003, 538, 65–70.
11. (a) Caldwell, T. W.; Kornfeld, E. C. J. Am. Chem. Soc. 1942, 64, 1695–1698; (b)
Saikachi, H. Yakugaku Zasshi 1944, 64, 201–202; (c) Matsueda, R.; Aiba, K. Chem.
Lett. 1978, 7, 951–952; (d) Matsueda, R.; Kimura, T.; Kaiser, E. T.; Matsueda, G.
R. Chem. Lett. 1981, 10, 737–740; (e) Vlahov, I. R.; Santhapuram, H. K. R.; Wang,
Y.; Kleindl, P. J.; You, F.; Howard, S. J.; Westrick, E.; Reddy, J. A.; Leamon, C. P. J.
Org. Chem. 2007, 72, 5968–5972; (f) Cros, E.; Planas, M.; Bardaji, E. Lett. In Pept.
Sci. 2002, 9, 1–4.
12. 6-Hydroxynicotinic acid 2 was purchased from Wako Pure Chemical Industries
and used without further purification.
13. (a) Kunishima, M.; Kawachi, C.; Morita, J.; Terao, K.; Iwasaki, F.; Tani, S.
Tetrahedron 1999, 55, 13159–13170; (b) Kaminski, Z. J. Tetrahedron Lett. 1985,
26, 2901–2904; (c) Kaminski, Z. J. Synthesis 1987, 917–920.
KSH-1 (1) (410 mg, 0.145 mmol, 4 equiv from captopril) was
poured into a filtration tube. A solution of captopril (11) (7.89 mg,
36.3 lmol) in DMF/water = 1/1 (7 mL) was added and the solution
was stirred for 30 min. The reaction mixture was analyzed by
reverse-phase HPLC. After the disappearance of the captopril peak,
solutions were separated from the resin by filtration. The resin
was washed away using DMF, and the collected filtrate was concen-
trated in vacuo. The resultant residue was co-evaporated with water
and ethanol after drying under reduced pressure to give the biotin-
labeled captopril 12 (18.0 mg, 96%) as a colorless solid; (12 was
dissolved in 15% aqueous MeOH and analyzed by reverse-phase
HPLC. The purity of 12 was 87%.). 1H NMR (400 MHz, CD3OD) d
1.21 (d, J = 6.8 Hz, 2H), 1.28–1.81 (m, 6H), 1.98–2.14 (m, 3H),
2.16–2.41 (m, 3H), 2.66–2.76 (m, 2H), 2.76–2.89 (m, 1H), 2.89–
3.06 (m, 2H), 3.08–3.25 (m, 3H), 3.38–3.54 (m, 2H), 3.60–3.80 (m,
2H), 4.26–4.35 (m, 1H), 4.37–4.46 (m, 1H), 4.46–4.54 (m, 1H); HRMS
(ES+): m/z 519.1745 (M+H+) (calcd for C21H35N4O5S3: 519.1770).
14. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride
n-
hydrate (DMT-MM) was purchased from Kokusan Chemical Corporation and
used without further purification.
15. Ueki, M.; Honda, M.; Kazama, Y.; Katoh, T. Synthesis 1994, 21–22.
16. Crystal structure data for 9: Crystallographic data (excluding the structure
factors) for the structure of
9 have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication number CCDC
846957. Copies of the data may be obtained, free of charge, upon application to
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [Fax: (internet) +44 1223-
336003 or e-mail: deposit@ccdc.cam.ac.uk].
Acknowledgments
17. (a) Bayer, E. Angew. Chem., Int. Ed. 1991, 30, 113–129; (b) Hutchins, S. M.;
Chapman, K. T. Tetrahedron Lett. 1994, 35, 4055–4058.
18. The resin used was purchased from Watanabe Chemical Industries
(polyethyleneglycol spacer on a polystyrene bead (110 mm, 0.42 mmol/g)—
NH2-PEG-resin TG HL-NH2—cat. # A00303).
This research was supported by the Adaptable and Seamless
Technology Transfer Program (A-STEP) through target-driven
R&D, Exploratory Research (AS231Z04408F) from the Japan Science
and Technology Agency, and by a Grant-in-Aid for Scientific
Research (B) (No. 20390036) from MEXT (Ministry of Education,
Culture, Sports, Science and Technology) of Japan.
19. Westerduln, P.; Veeneman, G. H.; Pennings, Y.; van der Marel, G. A.; van Boom,
J. H. Tetrahedron Lett. 1987, 28, 1557–1560.
20. Captopril was purchased from Wako Pure Chemical Industries and purified
before use. The reagent was purified by preparative HPLC (with a linear
gradient of 0–40% CH3CN in 0.1% aqueous TFA over 40 min). The collected
fractions were lyophilized, following recrystallization from hexane/AcOEt to
give a colorless solid.
Supplementary data
21. The amount of biotin on the resin was calculated based on the original NH2–
loading content.
Supplementary data (the preparation and chemical data for
compounds 3–7, including 1H NMR and HRMS) associated with