Enantioseparation on Riboflavin Derivatives Chemically Bonded to Silica Gel as Chiral Stationary Phases for HPLC

Article abstract of DOI:10.1002/chir.22452

Acetylated and/or 3,5-dimethylphenylcarbamated riboflavins were prepared and the resulting riboflavin derivatives as well as natural riboflavin were regioselectively immobilized on silica gel through chemical bonding at the 5'-O- or 3-N-position of the riboflavin to develop novel chiral stationary phases (CSPs) for enantioseparation by high-performance liquid chromatography (HPLC). The chiral recognition abilities of the obtained CSPs were significantly dependent on the structures of the riboflavin derivatives, the position of the chemical bonding on the silica gel, and the structures of the racemic compounds. The CSPs bonded at the 5'-O-position on the silica gel tended to well separate helicene derivatives, while the CSPs bonded at the 3-N-position composed of acetylated and 3,5-dimethylphenylcarbamated riboflavins showed a better resolving ability toward helicene derivatives and bulky aromatic racemic alcohols, respectively, and some of them were completely separated into the enantiomers. The observed difference in the chiral recognition abilities of these riboflavin-based CSPs is discussed based on the difference in their structures, including the substituents of riboflavin and the positions immobilized on the silica gel. Chirality 27:507-517, 2015.

Full text of DOI:10.1002/chir.22452

CHIRALITY 27:507–517 (2015)
Special Issue Article
Enantioseparation on Riboflavin Derivatives Chemically Bonded to
Silica Gel as Chiral Stationary Phases for HPLC
1
DAISUKE KUMANO,1 SOICHIRO IWAHANA,1 HIROKI IIDA,1 CHENGSHUO SHEN,1,2 JEANNE CRASSOUS,2 AND EIJI YASHIMA
*
1Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Japan
2Sciences Chimiques de Rennes, UMR 6226, Campus de Beaulieu, CNRS-Université de Rennes 1, Rennes, France
ABSTRACT
Acetylated and/or 3,5-dimethylphenylcarbamated riboflavins were prepared and
the resulting riboflavin derivatives as well as natural riboflavin were regioselectively immobilized
on silica gel through chemical bonding at the 5’-O- or 3-N-position of the riboflavin to develop
novel chiral stationary phases (CSPs) for enantioseparation by high-performance liquid chroma-
tography (HPLC). The chiral recognition abilities of the obtained CSPs were significantly depen-
dent on the structures of the riboflavin derivatives, the position of the chemical bonding on the
silica gel, and the structures of the racemic compounds. The CSPs bonded at the 5’-O-position on
the silica gel tended to well separate helicene derivatives, while the CSPs bonded at the 3-N-
position composed of acetylated and 3,5-dimethylphenylcarbamated riboflavins showed a better
resolving ability toward helicene derivatives and bulky aromatic racemic alcohols, respectively,
and some of them were completely separated into the enantiomers. The observed difference in
the chiral recognition abilities of these riboflavin-based CSPs is discussed based on the differ-
ence in their structures, including the substituents of riboflavin and the positions immobilized
on the silica gel. Chirality 27:507–517, 2015. © 2015 Wiley Periodicals, Inc.
KEY WORDS: riboflavin; chiral recognition; chiral stationary phase; helicene
The separation of enantiomers by high-performance liquid
chromatography (HPLC) has been recognized as one of the
most useful techniques not only for obtaining both enantio-
mers on a large scale, but also for the precise determination
of the enantiomeric excess of the chiral analytes including
chiral drugs.1–4 The development of a chiral stationary phase
(CSP) showing an efficient chiral recognition for a variety of
enantiomers is of key importance for this purpose. Therefore,
a number of chiral stationary phases (CSPs) for HPLC has
been prepared in the past few decades, and more than 100
CSPs have been commercialized,5 which mostly consist of
synthetic or polysaccharide-derived helical polymers6–11 or
are derived from optically active synthetic or naturally occur-
ring small molecules including amino acids, crown ethers,
and cinchona alkaloids.12–15
Riboflavin (vitamin B2) is an important unit of cofactors of
biologically active flavoenzymes16 and provides a variety of
functions, such as catalytic and redox activities and a
photoluminescent ability. Hence, the readily available natural
riboflavin consisting of a diverse functional heterocyclic isoal-
loxazine ring together with an optically active ribityl group
and its derivatives have been considered as a promising class
of novel asymmetric organocatalysts17,18 and chiral materials
for sensing19 and separating enantiomers, but successful ex-
amples for use as a CSP are still rare except for one precedent
by Gil-Av and colleagues;20 they found that natural riboflavin
had a high chiral recognition ability toward carbohelicenes
([7] to [14]helicene) when coated on silica gel as a CSP for
HPLC using a mixture of n-hexane and CH2Cl2 as the eluent.
Recently, Papadimitrakopoulos and coworkers have also
demonstrated that the flavin mononucleotide (FMN), a
phosphorylated analog of riboflavin, self-assembled and
wrapped around single-walled carbon nanotubes (SWCNTs)
in a chirality and handedness selective way and enriched
the left-handed helical SWCNTs.21 These results suggest that
riboflavin and its derivatives may have potential as promising
CSPs for separation of polyaromatic racemic compounds
through Π–Π interactions including charge-transfer complex-
ation between the analytes and isoalloxazine ring of the ribo-
flavin20 when they are chemically bonded to silica gel.22–25
Riboflavin has reactive hydroxy and amino groups, and mod-
ification with various substituents and further chemical bond-
ing to silica gel are possible.
In this study, we prepared five new riboflavin-based CSPs
composed of natural riboflavin and its acetylated and/or
3,5-dimethylphenylcarbamated derivatives by regioselective
immobilization at the 5’-O- or 3-N-position of the riboflavin
on silica gel (Fig. 1), and their chiral recognition abilities
were evaluated. Among the possible derivatization methods
of the hydroxy groups of the ribityl unit, we employed
3,5-dimethylphenylcarbamoylation because the CSPs com-
posed of the tris(3,5-dimethylphenylcarbamate)s of cellulose
*Correspondence to: Eiji Yashima, Department of Molecular Design and Engi-
neering, Graduate School of Engineering, Nagoya University, Chikusa-ku,
Nagoya 464-8603, Japan. E-mail: yashima@apchem.nagoya-u.ac.jp
Present address for H. Iida: Department of Chemistry, Interdisciplinary Graduate
School of Science and Engineering, Shimane University, 1060 Nishikawatsu,
Matsue 690-8504, Japan.
Received for publication 6 February 2015; Accepted 20 March 2015
DOI: 10.1002/chir.22452
Published online 28 April 2015 in Wiley Online Library
(wileyonlinelibrary.com).
© 2015 Wiley Periodicals, Inc.

508
KUMANO ET AL.
Fig. 1. Structures of riboflavin-based chiral stationary phases.
and amylose developed by Okamoto et al. exhibit an excellent
chiral resolving ability for a wide range of racemic com-
pounds and are recognized as the most frequently used
CSPs.4–7,9–11
Synthesis
Synthesis of compound 1. The synthesis of 1 was carried out accord-
ing to Scheme 1. To a solution of RF (1.08 g, 2.87 mmol) in anhydrous
DMF (400 mL) were added 3-(triethoxysilyl)propyl isocyanate (710 μL,
2.88 mmol) and triethylamine (400 μL, 2.87 mmol) under nitrogen. The
reaction mixture was stirred at 80 °C for 24 h, and then the solvent was
evaporated to give 1 (1.71 g). The residue was used for the next reaction
without further purification (see below for immobilization of the com-
pound 1 on silica gel, Scheme 8). Among four possible carbamate
MATERIALS AND METHODS
Instruments
The NMR spectra were measured using a Varian VXR-500S spectrom-
eter (Varian, Palo Alto, CA) operating at 500 MHz for 1H and 125 MHz
for 13C using tetramethylsilane (TMS) for CDCl3 as the internal standard.
The IR spectra were recorded on a Jasco FT/IR-680 spectrometer (Jasco,
Tokyo, Japan). The chromatographic separations of enantiomers were
performed using a Jasco PU-2080 Plus liquid chromatograph equipped
with Multi UV-Vis (Jasco MD-2010 Plus or MD-2018 Plus) and CD detec-
tors (Jasco CD-1595 or CD-2095 Plus) at ~25 °C. A solution of racemate
was injected into the chromatographic system using a Rheodyne Model
7725i injector (20 μL loop). The thermogravimetric (TG) analyses were
conducted on a Seiko EXSTAR6000 TG/DTA 6200 (Seiko Instruments,
Chiba, Japan) under a heating rate of 10 °C/min in a nitrogen flow of
200 mL/min.
Chemicals and Reagents
Anhydrous dimethylformamide (DMF), dimethyl sulfoxide (DMSO),
chloroform (CHCl3), pyridine, and tetrahydrofuran (THF) (water
content <0.005%), methanol (MeOH), ethanol (EtOH), acetone,
n-hexane, N,N-dimethyl-4-aminopyridine (DMAP), and 1,8-diazabicyclo
[5.4.0]-7-undecene (DBU) were purchased from Wako (Osaka, Japan).
Riboflavin (RF), 3-(triethoxysilyl)propyl isocyanate, 11-bromo-1-undecene
(5), p-toluenesulfonic acid monohydrate, 2,2’-azobis(isobutyronitrile)
(AIBN), and 3,5-dimethylphenyl isocyanate were purchased from Tokyo
Kasei (TCI, Tokyo, Japan). Hydrogen chloride in diethyl ether (1.0 M)
was obtained from Aldrich (Milwaukee, WI). Triethylamine and acetic an-
hydride were obtained from Kishida (Osaka, Japan). Tetraacetylriboflavin
(TARF),26 (3-mercaptopropyl)triethoxy silanized silica gel (M-silica,
8.3 wt%) with a mean particle size of 7 μm and a mean pore diameter of
12 nm,27 and 5’-O-trityl riboflavin (TrRF)28 were prepared according to
previously reported methods. The solvents used in the chromatographic
experiments were of HPLC grade. The racemates were commercially
available or were prepared by the usual or reported methods.29–31 The
synthesis of new [6]helicene derivatives (26–28) will be reported else-
where. Porous spherical silica gel (Daiso gel SP-120-7P, N-silica) with a
mean particle size of 7 μm and a mean pore diameter of 12 nm was kindly
supplied from Daicel (Tokyo, Japan).
Scheme 1. Synthesis of 1.
Chirality DOI 10.1002/chir

ENANTIOSEPARATION ON RIBOFLAVIN DERIVATIVES
509
derivatives, the main-product was found to be the 5’-carbamated riboflavin
(Scheme 1) produced by the reaction at the primary hydroxy group at the
5’-O-position and its regioselectivity was about 80% estimated from its 1H
NMR spectrum (see Spectrum S1 in the Supporting Information (SI)).
Synthesis of compound 4. The synthesis of 4 was carried out accord-
ing to Scheme 4. To a solution of 3 (502 mg, 1.00 mmol) in anhydrous
DMF (10 mL) were added 3-(triethoxysilyl)propyl isocyanate (737 μL,
2.98 mmol) and triethylamine (413 μL, 2.98 mmol) under nitrogen. After
the reaction mixture was stirred at 75 °C for 24 h, the solvents were evap-
orated. The residue was washed with n-hexane (200 mL) and purified by
column chromatography (SiO2, EtOAc–n-hexane = 0:10 to 5:5, 9:1 (v/v))
and then subjected to SEC fractionation to give 4 as an orange solid
(280 mg, 38% yield).
Synthesis of compound 2. The synthesis of 2 was carried out accord-
ing to Scheme 2. To a solution of TrRF (1.10 g, 2.78 mmol), which had
been prepared according to the reported method,28 and DMAP
(261 mg, 2.09 mmol) in CHCl3 (93 mL) were added anhydrous pyridine
(13 mL) and acetic anhydride (71 mL) under nitrogen. After the reaction
mixture was stirred at room temperature for 24 h, to this was further
added acetic anhydride (20 mL) and anhydrous pyridine (3.8 mL). After
stirring at room temperature for 24 h, the solvents were evaporated.
The residue was dissolved in EtOAc (80 mL) and the solution was washed
with brine (50 mL x 3) and then dried over anhydrous MgSO4. After filtra-
tion, the solvent was evaporated to dryness. The residue was purified by
column chromatography (SiO2, EtOAc–n-hexane = 0:10 to 5:5, 9:1 (v/v))
and then subjected to SEC fractionation to give 2 as an orange solid
(3.2 g, 88% yield).
Spectroscopic data of 4: Mp: 206.7 °C (dec); IR (KBr, cm–1): 3173,
3019, 2810, 1742, 1663, 1579, 1542, 1226, 104; 1H NMR (500 MHz, CDCl3,
25 °C): δ 8.50 (s, CONH, 1H), 8.03 (s, ArH, 1H), 7.57 (s, ArH, 1H), 5.72-
5.62 (m, NCH2CH, 1H), 5.48-5.38 (m, NCH2CHCH, NCH2CHCHCH,
2H), 4.46-4.21 (m, OCOCH2, 2H), 3.86-3.78 (q, J = 7.1 Hz, SiOCH2, 6H),
3.35-3.25 (t, J = 6.8 Hz, OCONHCH2, 2H), 2.57 (s, ArCH3, 3H), 2.45 (s,
ArCH3, 3H), 2.29 (s, CH3COO, 3H), 2.22 (s, CH3COO, 3H), 2.08
s, CH3COO, 3H), 1.74-1.71 (m, OCONHCH2CH2, 2H), 1.31-1.20
(t, SiOCH2CH3, 9H), 0.70-0.65 (m, SiCH2, 2H); 13C NMR (125 MHz,
CDCl3, 25 °C): 170.77, 170.44 170.03, 169.88, 159.42, 154.49, 150.87,
148.28, 137.16, 136.20, 134.79, 133.14, 131.38, 115.67, 70.63, 69.59, 69.16,
62.03, 58.62, 45.54, 45.18, 25.26, 21.61, 21.19, 20.94, 20.47, 19.60, 18.42,
7.70; HRMS (ESI+): m/z calcd for C33H47N5O13Si (M + Na+) 772.2837;
found 772.2803.
1
Spectroscopic data of 2: H NMR (500 MHz, CDCl3, 25 °C): δ 8.34 (s,
CONH, 1H), 8.04 (s, ArH, 1H), 7.56 (s, ArH, 1H), 7.42-7.38 (m, OCArH,
9H), 7.24-7.13 (m, OCArH, 6H), 5.69-5.59 (m, NCH2CH, NCH2CHCH,
2H), 5.38-5.34 (m, NCH2CHCHCH, 1H), 3.46-3.16 (m, OCOCH2, 2H),
2.53 (s, ArCH3, 3H), 2.44 (s, ArCH3, 3H), 2.37 (s, CH3COO, 3H), 1.90
(s, CH3COO, 3H), 1.68 (s, CH3COO, 3H).
Synthesis of compound 6. Compound 6 was prepared according to
Scheme 5. To a solution of 5 (4.4 mL, 20 mmol) and TARF (2.20 g,
4.04 mmol)26 in anhydrous DMF (190 mL) was added DBU (1.2 mL,
8.0 mmol) under nitrogen. After the reaction mixture was stirred at 50 °
C for 20 h, the solvent was evaporated, and the residue was washed with
n-hexane (100 mL x 2). The residue was then dissolved in CHCl3
(250 mL) and the solution was washed with water (300 mL x 4), and then
dried over anhydrous MgSO4. After filtration, the solvent was evaporated
to dryness, and the residue was purified by column chromatography
(SiO2, CHCl3–n-hexane = 1:1 to 1:0 (v/v) then MeOH/CHCl3 = 0/100 to
5/95 (v/v)) and MeOH–CHCl3 = 0:10 to 1:9 (v/v)) to give 6 as an orange
solid (2.1 g, 73% yield).
Synthesis of compound 3. Compound 3 was prepared according to
Scheme 3. Compound 2 (2.19 g, 2.94 mmol) was dissolved in anhydrous
CHCl3 (34 mL) under nitrogen and the solution was stirred at 0 °C for
30 min. To this was added diethyl ether containing HCl (1.0 M) (11 mL)
and the reaction mixture was stirred at 0 °C for 45 min so as to remove
the trityl group. The solution was then neutralized with aqueous NaHCO3
(50 mL) and extracted with CHCl3 (20 mL). The organic layer was
washed with water (50 mL) and brine (50 mL), and then dried over
anhydrous MgSO4. After filtration, the solvent was evaporated to dry-
ness. The residue was purified by column chromatography (SiO2,
MeOH–CHCl3 = 0:10 to 1:9 (v/v)) to give 3 as a yellow solid (1.11 g,
75% yield).
Spectroscopic data of 6: Mp: 67.5–69.3 °C; IR (KBr, cm–1): 2927,
2855, 1751, 1663, 1588, 1551, 1221, 1050; 1H NMR (500 MHz, CDCl3,
25 °C): δ 8.03 (s, ArH, 1H), 7.53 (s, ArH, 1H), 5.86-5.76 (ddt, J = 17.1,
10.3, 6.7 Hz, CH2CH = CH2, 1H), 5.72-5.62 (m, NCH2CH, 1H), 5.50-
5.44 (m, NCH2CHCH, 1H), 5.44-5.37 (m, NCH2CHCHCH, 1H), 5.02-
4.95 (m, CH = CHZHE, 1H), 4.94-4.89 (m, CH = CHEHZ, 1H), 4.47-4.21
(m, OCOCH2, 2H), 4.09-4.12 (t, J = 7.6 Hz, NCH2C8H16CH = CH2, 2H),
Spectroscopic data of 3: 1H NMR (500 MHz, CDCl3, rt.): δ 8.08 (s, ArH,
1H), 7.76 (s, ArH, 1H), 5.73-5.65 (m, NCH2CH, 1H), 5.30-5.24 (m,
NCH2CHCH, 1H), 4.30-4.25 (m, OHCH2, 2H), 3.64 (s, NCH2CHCHCH,
1H), 2.58 (s, ArCH3, 3H), 2.47 (s, ArCH3, 3H), 2.12 (s, CH3COO, 3H),
2.09 (s, CH3COO, 3H), 1.97 (s, CH3COO, 3H).
Scheme 2. Synthesis of 2.
Scheme 4. Synthesis of 4.
Scheme 3. Synthesis of 3.
Scheme 5. Synthesis of 6.
Chirality DOI 10.1002/chir

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KUMANO ET AL.
2.55 (s, ArCH3 , 3H), 2.44 (s, ArCH3, 3H), 2.30 (s, CH3COO, 3H), 2.22
s, CH3COO, 3H), 2.08 (s, CH3COO, 3H), 2.05-2.00 (m, CH2CH = CH2,
2H), 1.73 (s, CH3COO, 3H), 1.74-1.66 (m, NCH2C7H14, 2H), 1.40-1.26
(m, NCH2C7H14, 12H); 13C NMR (125 MHz, CDCl3, 25 °C): δ 170.76,
170.45, 170.02, 169.79, 159.75, 155.14, 149.26, 147.45, 139.43, 136.54,
135.97, 134.78, 133.08, 131.29, 115.45, 114.20, 70.54, 69.57, 69.13,
62.03, 44.55, 42.26, 33.96, 29.61, 29.57, 29.49, 29.26, 29.08, 27.90,
27.14, 21.53, 21.21, 20.95, 20.84, 20.47, 19.56; HRMS (ESI+): m/z calcd
for C36H48N4O10 (M + Na+) 719.3268; found 719.3286.
Synthesis of compound 7. The synthesis of 7 was carried out accord-
ing to Scheme 6. The compound
6 (7.97 g, 11.4 mmol) and
p-toluenesulfonic acid monohydrate (6.61 g, 34.7 mmol) were dissolved
in EtOH (820 mL) and the solution was refluxed for 16 h under nitrogen.
Scheme 6. Synthesis of 7.
Scheme 7. Synthesis of 8.
Scheme 8. Immobilization of 1, 4, 6, 7, and 8 on silica gel.
Chirality DOI 10.1002/chir

ENANTIOSEPARATION ON RIBOFLAVIN DERIVATIVES
511
The reaction mixture was then cooled to –20 °C. The precipitated solid
was collected by filtration and washed with EtOH (60 mL) to give 7 as
an orange solid (3.94 g, 65% yield).
Synthesis of compound 8. Compound 8 was prepared according to
Scheme 7. To solution of (1.17 g, 2.22 mmol) in anhydrous
a
7
DMF (100 mL) were added anhydrous pyridine (60 mL) and
3,5-dimethylphenyl isocyanate (2.0 mL, 14 mmol) under nitrogen. The re-
action mixture was stirred at 80 °C for 20 h, and then the solvents were
evaporated. The residue was dissolved in CHCl3 (100 mL) and the solu-
tion was washed with water (150 mL x 3), and then dried over Na2S2O4.
After filtration, the solvent was evaporated to dryness. The residue was
purified by column chromatography (SiO2, EtOAc–n-hexane = 1:9 to 4:6
(v/v)) and then subjected to SEC fractionation to give 8 as a yellow solid
(1.3 g, 54% yield).
Spectroscopic data of 7: Mp: 164.0–167.9 °C; IR (KBr, cm–1): 3400,
2925, 1546; 1H NMR (500 MHz, DMSO-d6, 25 °C): δ 7.94 (s, ArH, 1H),
7.93 (s, ArH, 1H), 5.84-5.72 (ddt, J = 17.3, 10.1, 6.9 Hz, CH2CH = CH2,
1H), 5.18-5.05 (s, OH, 1H), 5.02-4.95 (m, CH = CHZHE, 1H), 4.94-4.89
(m, CH = CHEHZ, 1H), 4.89-4.80 (m, NCH2CH, OH, 2H), 4.79-4.77
(m, OH, 1H), 4.64-4.60 (m, NCH2CHCH, 1H), 4.51-4.49 (m, OH, 1H),
4.30-4.21 (m, NCH2CHCHCH, 1H), 3.90-3.83 (t, J = 7.6 Hz, NCH2C8
H16CH = CH2, 2H), 3.70-3.42 (m, NCH2, OCOCH2, 4H), 2.48 (s, ArCH3,
3H), 2.40 (s, ArCH3, 3H), 2.05-1.95 (m, CH2CH = CH2, 2H), 1.62-1.18 (m,
NCH2C7H14, 14H); 13C NMR (125 MHz, DMSO-d6, 25 °C): 159.41,
154.65, 149.42, 146.12, 138.82, 135.86, 135.79, 134.28, 132.05, 130.68,
117.41, 114.62, 114.60, 73.55, 72.78, 68.79, 63.41, 47.07, 33.16, 28.90,
28.81, 28.77, 28.50, 28.26, 27.34, 26.45, 20.77, 18.76; HRMS (ESI+): m/z
calcd for C28H40N4O6 (M + Na+) 551.2846; found 551.2863.
Spectroscopic data of 8: Mp: 217.8–219.0 °C; IR (KBr, cm–1): 3313,
2924, 1735, 1650, 1585, 1549, 1216; 1H NMR (500 MHz, CDCl3, 25 °
C): δ 7.99 (s, ArH, 1H), 7.63 (s, ArH, 1H), 7.21 (s, OCONHArH, 2H),
7.05 (s, OCONHArH, 2H), 6.85 (s, OCONHArH, 2H), 6.70 (s,
OCONHArH, 1H), 6.67 (s, OCONHArH, 1H), 6.65 (s, OCONHArH,
1H), 6.60 (s, OCONHArH, 2H), 6.48 (s, OCONHArH, 2H), 6.00-5.91
Chart 1. Racemates used for resolution on riboflavin-based CSPs (9–31).
Chirality DOI 10.1002/chir

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KUMANO ET AL.
(br, NCH2CH, 1H), 5.86-5.77 (ddt, J = 17.1, 10.5, 6.7 Hz, CH2CH = CH2,
1H), 5.61-5.44 (m, NCH2CHCH, NCH2CHCHCH, 2H), 5.02-4.95
(m, CH = CHZHE, 1H), 4.94-4.89 (m, CH = CHEHZ, 1H), 4.76-4.38
(m, OCOCH2, 2H), 4.52-4.48 (br, NCH2, 2H), 4.11-3.88 (m, NCH2C8
H16CH = CH2, 2H), 2.40 (s, ArCH3, 3H), 2.30 (s, ArCH3, 3H), 2.29-2.24
(m, OCONHArCH3, 12H), 2.21-2.12 (m, OCONHArCH3, 12H), 2.04-
1.98 (m, CH2CH = CH2, 2H), 1.69-1.24 (m, NCH2C7H14, 14H); 13C
NMR (125 MHz, CDCl3, 25 °C): 159.91, 156.48, 153.07, 152.76, 152.72,
152.60, 148.88, 147.68, 139.42, 139.40, 138.98, 138.82, 138.63, 138.61,
137.87, 137.28, 136.66, 136.38, 135.87, 135.21, 132.76, 131.66, 126.14,
125.81, 125.28, 125.06, 122.10, 117.10, 117.01, 116.65, 116.18, 115.52,
114.22, 114.20, 107.07, 71.71, 71.24, 63.56, 46.07, 42.28, 33.96, 29.65,
29.60, 29.49, 29.26, 29.08, 27.91, 27.24, 21.56, 21.50, 21.44, 21.35,
19.41; HRMS (ESI+): m/z calcd for C64H76N8O10 (M + Na+) 1139.5582;
found 1139.5577.
triethoxy silanized silica gel in DMF in the presence of
AIBN,27 producing the riboflavin-based CSPs regioselectively
immobilized on the silica gel (Scheme 8 and Fig. 1). Com-
plete regioselective modifications except for 1 (~80%) were
1
confirmed by the H and 13C NMR spectra of the precursors
(4, 6–8) before the immobilization on silica gel (see Experi-
mental Procedures and Supporting Information). The CSPs
were then packed into stainless-steel columns (25 cm x
0.20 cm (i.d.)) by the conventional high-pressure slurry
packing procedure.29
Enantioseparation on Riboflavin-Based CSPs.
We first performed the chromatographic enantioseparation
of 10 standard racemic compounds with different structures
and functionalities (9–18) (Chart 1)6,10,24 using the five
riboflavin-based CSPs with n-hexane–2-propanol (90:10, v/v)
and n-hexane–CH2Cl2 (90:10, v/v) as the eluent to get an in-
sight into their specific chiral recognition abilities toward
such a variety of racemates, and the results are summarized
in Tables S1 and S2, respectively. The chromatographic
parameters, the retention factor k1 [= (t1 – t0)/t0], the
separation factor α [=(t2 – t0)/(t1 – t0)], and the resolution fac-
tor RS [=2(t2 – t1)/(w1 + w2)] were used to evaluate the
resolution results, where t0, t1, and t2 are the dead time and
the retention times of the first- and second-eluted
enantiomers and w1 + w2 are the peak widths at the baseline,
Immobilizations of compounds 1, 4, 6, 7, and 8 on silica gel were car-
ried out according to Scheme 8.
Immobilization of compounds 1 and 4 on silica gel. A typical exper-
imental procedure is described below. N-Silica (901 mg) was dispersed in
a solution of 1 just after the preparation (~2.8 mmol) in anhydrous DMSO
(4.0 mL) and pyridine (1.5 mL) under nitrogen and the mixture was
heated at 70 °C. After 25 h, anhydrous DMF (60 mL) was added to this,
and the resulting silica gel was collected by filtration, washed with
DMF (20 mL), MeOH (60 mL), acetone (80 mL), and n-hexane (60 mL),
and dried in vacuo at 120 °C overnight, yielding the CSPA (1.14 g). The
content of 1 chemically bonded to silica gel was estimated by TG analysis
and was 13.4 wt%. In the same way, the compound 4 was chemically
bonded to N-silica (CSPB), and its content was estimated to be 13.2 wt%.
respectively.
A
chromatogram for the resolution of
trans-cyclopropanedicarboxylic acid dianilide (18) on CSPE
using n-hexane–2-propanol (90:10, v/v) as the eluent is
shown in Fig. 2. The peaks were detected by a UV detector
and identified by a CD detector. The (–)- and (+)-18
enantiomers eluted at the retention times of t1 and t2, showed
almost baseline separation, and the retention factor k1, the
separation factor α, and the resolution factor RS were
estimated to be 0.78, 1.38 and 1.28, respectively.
Immobilization of compounds 6, 7, and 8 on silica gel. A typical ex-
perimental procedure is described below. M-silica (1.00 g, 8.3 wt%)27
was dispersed in a solution of 7 (444 mg, 0.638 mmol) in THF (30 mL)
under nitrogen, and to this was added AIBN (35.7 mg, 0.217 mmol).
The reaction mixture was refluxed for 24 h, and AIBN (35.5 mg,
0.216 mmol) was further added. After refluxing for 15 h, the resulting
silica gel was collected by filtration, washed with THF (50 mL x 2)
and n-hexane (50 mL), and dried in vacuo at 120 °C overnight. The con-
tent of 7 chemically bonded to silica gel was estimated by TG analysis
and was 11.8 wt%. In order to improve the amount of 7 bonded to silica
gel, the immobilization procedure was repeated two times, giving CSPC
(0.92 g); the content of 7 was 18.4 wt% thus estimated by TG analysis. In
the same way, the compounds 6 and 8 were chemically bonded to
M-silica, and their contents were estimated to be 15.9 (CSPD) and
24.7 wt% (CSPE), respectively.
As anticipated from the riboflavin structure in which a chi-
ral, flexible ribityl chain is located away from the achiral
Preparation of chiral columns. Each column packing material was
packed into a stainless-steel tube (25 cm x 0.20 cm (i.d.)) by conventional
high-pressure slurry packing technique using a Chemco Slurry-Packing
Apparatus Model 124A (Chemco, Osaka, Japan).29 The plate numbers
of the columns were 1500–2200 for benzene with n-hexane–2-propanol
(90:10, v/v) as the eluent at a flow rate of 0.1 mL/min. The dead time
(t0) was estimated using 1,3,5-tri-tert-butylbenzene as the nonretained
compound.32 The CSPs are stable and maintained their chiral recognition
abilities at least for more than 1 month.
RESULTS AND DISCUSSION
Synthesis of Riboflavin Derivatives and Immobilization on
Silica Gel.
In order to regioselectively immobilize natural riboflavin
and its derivatives (1, 4, 6–8) on the silica gel (7 μm particle
size, 12 nm pore size) surface via chemical linkages, a
3-(triethoxysilyl)propylcarbamate or a 1-undecenyl residue
was introduced at the 5’-O- (1 and 4) or 3-N-position (6–8)
of the riboflavins, respectively (Scheme 8). The precursors
were then allowed to react with unmodified silica gel in
DMSO in the presence of pyridine or (3-mercaptopropyl)
Chirality DOI 10.1002/chir
Fig. 2. Chromatograms for the resolution of 18 on CSPE. Eluent: n-hex-
ane–2-propanol (90:10). Flow rate: 0.1 mL/min.

ENANTIOSEPARATION ON RIBOFLAVIN DERIVATIVES
513
heterocyclic isoalloxazine ring, the CSPs separated only two
(16 and 18) (Table S1) and three racemates (10, 12, and
16) (Table S2) with n-hexane–2-propanol (90:10, v/v) and
n-hexane–CH2Cl2 (90:10, v/v) as the eluents, respectively.
The chromatographic resolution results, however, gave use-
ful information regarding the specificity of the CSPs for the
racemates and interaction mode between the CSPs and race-
mates; in the n-hexane–2-propanol system, the fully
phenylcarbamated CSPE immobilized at the 3-N-position al-
most completely resolved a bulky aromatic racemic alcohol,
1-(9-anthryl)-2,2,2-trifluoroethanol (16) (Fig. 3A) and a
cyclic dianilide 18 (Fig. 2) with high α values (α = 1.11 and
1.38, respectively) probably through an intermolecular hydro-
gen bond formation between the polar carbamate residues in-
troduced at the ribityl group of the CSPE and the hydroxy or
amide residues of the analytes, while the CSPA, CSPB, and
CSPD only partially resolved 18 (Table S1). A similar trend
was observed in the n-hexane–CH2Cl2 system for CSPE that
partially separated the bulky aromatic alcohols 12 and 16.
In contrast to the resolutions in n-hexane–2-propanol (90:10,
v/v), CSPA and CSPB immobilized at the 5’-O-position
partially resolved the Tröger base (10) in addition to 16 in
n-hexane–CH2Cl2 (90:10, v/v) (Table S2), indicating that not
only hydrogen bonding, but also hydrophobic or Π-stacking
interactions may contribute during the chiral recognition pro-
cess in this eluent system. The CSPC bearing the free ribityl
group showed a poor chiral recognition in both eluent
systems and could not separate the 10 tested racemates. It
should be noted that the bulky aromatic racemic alcohols
with an acidic hydroxy group 13 and 16 strongly interacted
with the five CSPs in n-hexane–CH2Cl2, in particular on the
CSPC and CSPD, 16 could not elute (k1 > 100) under the
present conditions.
Based on the above results together with the previously re-
ported intriguing chiral recognition abilities of riboflavin20
and FMN21 for the racemic helicenes and SWCNTs,
Fig. 3. Chromatograms for the resolution of (A) 16 on CSPE (eluent; n-hexane–2-propanol (90:10), flow rate; 0.1 mL/min), (B) 23 on CSPC (eluent: n-hexane–2-
propanol (80:20), flow rate: 0.5 mL/min), (C) 31 on CSPD (eluent: n-hexane–CH2Cl2 (90:10), flow rate: 0.5 mL/min), and (D) 23 on CSPE (eluent: n-hexane–
CH2Cl2 (80:20), flow rate: 0.5 mL/min).
Chirality DOI 10.1002/chir

514
KUMANO ET AL.
respectively, we employed 13 racemates (19–31) (Chart 1)
including the cyclic dibenzamide (19) and dianilide (20),
bulky aromatic racemic alcohols (21–23), and helicene de-
rivatives including the [6] and [7]helicenes (24–30) and a
metal-containing [8]helicene analog (31) to further evaluate
the specific chiral recognition abilities of the riboflavin-based
CSPs in n-hexane–2-propanol and n-hexane–CH2Cl2 eluent
systems; the chromatographic resolution results showing a
more or less enantioselectivity (α > 1) on either CSP for the
racemates are summarized in Tables 1 and 2, respectively.
For comparison, some resolution results in Tables S1 and
S2 are also shown.
As expected, an analogous cyclic dibenzamide (19) and
more bulky aromatic alcohols (22 and 23) were partially or
almost completely resolved on the phenylcarbamated CSPE
in n-hexane–2-propanol (Table 1). The four-membered cyclic
dianilide (20) was not resolved on CSPE, but 20 as well as
the bulky alcohols (22 or 23) were partially resolved on
CSPA, CSPB, and CSPD and the reversed elution order was
observed for 23 on CSPD. The fact that the relatively less
bulky 2,2‘-dihydroxy-6,6‘-dimethylbiphenyl (13) and 1,1‘-
bi-2-naphthol (21) were not separated at all on all of the CSPs
independent of the eluents (Tables 1 and 2) clearly indicated
the important role of the steric or bulky effect; in other words,
the molecular size for the efficient separation of aromatic ra-
cemic alcohols on the riboflavin-based CSPs. A similar molec-
ular size effect was suggested by Gil-Av et al. during the
separation of a series of achiral polyaromatic compounds on
silica gel coated with natural riboflavin.20 Interestingly, CSPC
exhibited an exceptionally high chiral recognition ability only
for the bulky racemic alcohol 23 (α = 1.54) and completely re-
solved it (Fig. 3B), although CSPC showed a poor chiral rec-
ognition for the other racemates in both eluent systems. In
addition, most of the CSPs could not resolve the polycyclic
fully aromatic [6] and [7]helicenes (24 and 30) except for
CSPA bearing mostly free hydroxy groups at the ribityl chain,
which partially separated [7]helicene (30) into enantiomers.
Interestingly, all of the eight helicene derivatives (24–31)
were efficiently recognized by the 5’-O-bonded CSPA and
CSPB except for 29 on CSPB in n-hexane–CH2Cl2, showing
not complete, but partial separations in the same elution or-
der, while interacting strongly with the CSPs as supported
by high k1 values (Table 2). However, CSPC and CSPE
immobilized at the 3-N-position could not resolve the
helicenes at all, indicating that the interaction of the original
isoalloxazine ring of CSPA and CSPB with the polyaromatic
helicenes may be the major driving force for their efficient
chiral recognition of the helicenes rather than the ribityl
moiety, since CSPA and CSPC possess the same ribityl group
except for the 5’-O-position of CSPA. Obviously, the chirality
of the ribityl chain is essential for the chiral recognition of
the present riboflavin-based CSPs. The elution order of [7]
helicene (30) on CSPA and CSPB was identical to that on
the reported riboflavin-coated silica gel CSP,20 judging from
the Cotton effect sign of the first-eluted enantiomer of 30 at
350 nm.20,33
More interestingly, most of the helicene derivatives, in par-
ticular the [6]helicene derivatives (25 and 27), [7]helicene
(30), and an [8]helicene analog (31), were better resolved
on the 3-N-bonded CSPD composed of the fully acetylated
ribityl residue, and 25 and 31 (Fig. 3C) were completely sep-
arated. It is noteworthy that the elution order of the helicene
derivatives (24–31) on CSPD was totally reversed without
Chirality DOI 10.1002/chir

ENANTIOSEPARATION ON RIBOFLAVIN DERIVATIVES
515
Chirality DOI 10.1002/chir

516
KUMANO ET AL.
exception when compared to that on CSPA and CSPB in
n-hexane–CH2Cl2 (Table 2). The difference between the acet-
ylated CSPB and CSPD is the position immobilized on the sil-
ica gel (5’-O- and 3-N-positions, respectively). Therefore, the
results reveal the critical role of the immobilization positions
of the riboflavin-based CSPs during the enantioselectivity of
the helicenes as well as their elution order.
Again, CSPE exhibited a good chiral recognition ability for
bulky aromatic racemic alcohols (22 and 23) (α = 1.26 and
1.49, respectively) and completely resolved 23 (Fig. 3D), al-
though CSPE showed poor enantioselectivities (α = ~1) to-
ward the racemic helicene derivatives. The other CSPs also
separated either 22 or 23; the latter bulky alcohol was
almost base-line resolved on CSPD with the reversed elution
order to that on CSPE. The retention factors of bulky racemic
alcohols (13, 16, 22, and 23) as well as helicene derivatives
(26–28) significantly decreased when n-hexane-CH2Cl2
(90:10 or 80/20) containing 1% 2-propanol was used as the
eluent (Table 2), while the separation factors slightly de-
creased or were improved depending on the CSPs.
based CSPs because such polymers may form a preferred-
handed helical structure18,34,35 and the research along this
line is now in progress in our laboratory.
Acknowledgments
This work was supported in part by a Grant-in-Aid for
Scientific Research (S) (to E.Y.) and a Grant-in-Aid for Young
Scientists (B) (to H.I.) from the Japan Society for the Promo-
tion of Science. C.S. expresses his thanks for the Program for
Leading Graduate Schools “Integrative Graduate Education
and Research in Green Natural Sciences,” MEXT, Japan.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article at the publisher’s web-site.
LITERATURE CITED
A comparison of the enantioseparation results on the five
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Chirality DOI 10.1002/chir