Content uploaded by Daniel W. Armstrong
Author content
All content in this area was uploaded by Daniel W. Armstrong on Jan 14, 2017
Content may be subject to copyright.
Enantiomeric Separation of Fused
Polycycles by HPLC with Cyclodextrin
and Macrocyclic Glycopeptide Chiral
Stationary Phases
Xinxin Han, Qinhua Huang, Jie Ding, Richard C. Larock,
and Daniel W. Armstrong
Department of Chemistry, Iowa State University, Ames, IA, USA
Abstract: The enantiomeric separation of a series of 13 new chiral polycycles has been
examined on both cyclodextrin-based and macrocyclic glycopeptide chiral stationary
phases (CSPs) using HPLC in the normal phase, reversed phase, and polar organic
modes. The most effective chiral selectors for the enantiomeric separation of these
analytes are the 2,3-dimethyl-
b
-cyclodextrin (Cyclobond I-2000 DM) and hydroxy-
propyl-
b
-cyclodextrin (Cyclobond I-2000 RSP). The other Cyclobond-type and
Chirobiotic (macrocyclic glycopeptide) CSPs only show enantioselectivity for a few
of the racemic polycycles. The effects of mobile phase composition and analyte
structure on chiral recognition and separation are considered.
Keywords: Fused polycycles, enantiomeric separation, chiral stationary phase,
cyclodextrin, macrocyclic glycopeptide
INTRODUCTION
Fused polycycles exist widely in the natural world. Two pentacyclic proapor-
phine alkaloids, (2)-misramine (1) and (2)-labrandine (2), have been found
in the Egyptian and Turkish flowering plant, Roemeria hybrida, respectively.
A complex fused polycycle, dipuupehetriol (3), has been isolated from a
Verongid sponge. From the Caribbean sponge Smenospongia aurea, aureol
Received 2 February 2005, Accepted 25 July 2005
Address correspondence to Daniel W. Armstrong, Department of Chemistry, Iowa
State University, Ames, IA 50011, USA. E-mail: sec4dwa@iastate.edu
Separation Science and Technology, 40: 2745–2759, 2005
Copyright #Taylor & Francis, Inc.
ISSN 0149-6395 print/1520-5754 online
DOI: 10.1080/01496390500290535
2745
and its derivatives have been obtained (4). Esmeraldin A and B, derivatives of
diphenazine, have been found in Streptomyces antibioticus, strain Tu
¨2706 (5).
Many fused polycycles are known to possess beneficial therapeutic acti-
vities. Dipuupehetriol has shown selectivity against the human lung cancer
cell line A549 and the CV-1 cell line (3). Two analogs of aureol inhibit the
growth of some gram-positive and gram-negative bacteria (4). Strong
antitumor activities of hexacyclic derivatives of camptothecin have been
reported (6). Some other tetracyclic compounds are inhibitors of kynure-
nine-3-hydroxylase (7) and poly(ADP-ribose)polymerase (8, 9).
Huang, Larock, and co-workers have recently prepared a set of new chiral
fused polycycles (Fig. 1) (10), which includes 8 chromene derivatives,
2 quinoline derivatives, 2 isochromene derivatives, and 1 polycyclic diester.
These compounds are obtained through palladium-catalyzed alkyl to aryl
palladium migration, followed by intramolecular arylation. Since different
enantiomers of a chiral compound can have different biological properties
(11), separation of these new chiral polycycles and evaluation of their proper-
ties are desirable.
Cyclodextrin-based (12 – 23) and macrocyclic glycopeptide (24– 35) chiral
stationary phases (Fig. 2) are well known for their high enantioselectivities for
separation of a variety of different chiral molecules. In this work, the enantio-
meric selectivity of 8 cyclodextrin and 4 macrocyclic glycopeptide chiral
stationary phases for 13 recently synthesized racemic fused polycycles have
been investigated in the reversed phase, polar organic, and normal phase modes.
EXPERIMENTAL
Materials
Cyclobond I, II, III, DM, AC, RSP, DMP, SN; as well as the Chirobiotic V, R,
T, and TAG CSPs (Fig. 2) were obtained from Advanced Separation
Figure 1. General structure and ring numbering conventions for the chiral poly-
cycles. Structure 1is a chromene (X ¼O) or quinoline (X ¼NSO
2
CF
3
) derivative.
Structure 2is an isochromene derivative. Structure 3is a polycyclic diester. The carbon
marked with an asterisk is the stereogenic center.
X. Han et al.2746
Technologies (Whippany, NJ, USA). All the stationary phases consist of chiral
selectors bonded to 5 mm spherical porous silica gel (14, 15, 24). The chiral
selectors are the native
a
-,
b
-, and
g
-cyclodextrins, various derivatives of
b
-cyclodextrin, vancomycin, ristocetin A, teicoplanin, and teicoplanin
aglycone (Fig. 2). The dimensions of the columns are 250 4.6 mm. HPLC
grade methanol, acetonitrile, ethanol, and heptane were obtained from
Fisher (Fairlawn, NJ, USA). The triethylamine and acetic acid used were
ACS certified grade from Fisher. Water was deionized and filtered through
active charcoal and a 5 mm filter. All chiral polycycles were prepared as
previously reported via palladium-catalyzed alkyl to aryl migrations and
cyclization (10).
Figure 2. General structure of the (a) Cyclobond and (b) Chirobiotic CSPs (there can
be 1–3 linkages for each cyclodextrin or macrocyclic glycopeptide molecule). R ¼H
for Cyclobond I (
b
-cyclodextrin), II (
g
-cyclodextrin), III (
a
-cyclodextrin). All deriva-
tized cyclodextrin CSPs are made from
b
-cyclodextrin.
Enantiomeric Separation of Fused Polycycles by HPLC 2747
Equipment
Chromatographic separations were carried out using an HP 1050 HPLC
system with a UV VWD detector, an auto sampler, and computer-controlled
Chem-station data processing software (Agilent Technologies, Palo Alto,
CA, USA). The mobile phases were degassed by ultra-sonication under
vacuum. UV detection was carried out at 254 nm for all of the compounds.
All separations were carried out at room temperature (238C) and the flow
rate of the mobile phase was 1.0 mL min
21
.
Column Evaluation
The performance of all stationary phases was evaluated in the reversed phase
mode using acetonitrile/water and methanol/water mobile phases. Cyclobond
I, II, III, AC, RSP, SN, and DMP and all Chirobiotic CSPs were evaluated in
the polar organic mode using acetonitrile as mobile phase. Cyclobond SN
and DMP and all Chirobiotic CSPs were evaluated in the normal phase
mode using an ethanol/heptane mobile phase. Over the course of 1000 injec-
tions, no degradation of these columns was observed. When using a new
mobile phase, 10 column volumes of solution were pumped through the
column prior to injection of the analytes.
Calculations
Thedeadtime(t
0
) was estimated using the peak resulting from the change in
refractive index from the injection solvent on each chiral stationary phase.
The retention factor (k) was calculated using the equation k¼(t
r
2t
0
)/t
0
.
The enantioselectivity (
a
) was calculated using
a
¼k
2
/k
1
. The resolution
factor (R
S
) was calculated using the equation R
S
¼2(t
r2
2t
r1
)/
(w
1
þw
2
), where t
r2
and t
r1
are the retention times of the second and first enan-
tiomers, respectively, and w
1
and w
2
are the corresponding base peak widths.
The efficiency (number of theoretical plates, N) was calculated using
N¼16(t
r
/w)
2
.
RESULTS AND DISCUSSION
Performance of the Chiral Stationary Phases
The chromatographic parameters for successful and unsuccessful sepa-
rations are given in Tables 1– 4. For the Cyclobond CSPs, enantiomeric sep-
arations were only observed in the reversed phase mode. No enantiomeric
separations were achieved on these CSPs in the normal phase mode or the
polar organic mode. Chirobiotic CSPs showed enantioselectivities for
X. Han et al.2748
Table 1. Retention factor of the first peak (k
1
), enantioselectivity (
a
), and enantio-
meric resolution (R
S
) of all chiral polycycles on the Cyclobond RSP and DM CSPs
in the reversed phase mode
# Structure CSP k
1
a
R
S
Mobile phase (v/v)
1
RSP 7.34 1.09 1.0 CH
3
OH/H
2
O¼35/65
5.43 1.10 1.4 CH
3
CN/H
2
O¼20/80
DM 10.52 1.04 0.4 CH
3
OH/H
2
O¼30/70
9.45 1.05 0.7 CH
3
CN/H
2
O¼15/85
2
RSP 5.59 1.17 1.8 CH
3
OH/H
2
O¼40/60
6.02 1.11 1.5 CH
3
CN/H
2
O¼20/80
DM 6.95 1.14 1.4 CH
3
OH/H
2
O¼30/70
5.95 1.09 1.0 CH
3
CN/H
2
O¼15/85
3
RSP 6.34 1.12 1.5 CH
3
OH/H
2
O¼40/60
7.03 1.14 1.8 CH
3
CN/H
2
O¼20/80
DM 9.30 1.10 1.2 CH
3
OH/H
2
O¼30/70
9.65 1.07 0.9 CH
3
CN/H
2
O¼15/85
4
RSP 4.76 1.17 1.7 CH
3
OH/H
2
O¼50/50
4.97 1.15 2.1 CH
3
CN/H
2
O¼25/75
DM 4.95 1.18 1.2 CH
3
OH/H
2
O¼35/65
13.69 1.13 1.4 CH
3
CN/H
2
O¼20/80
5
RSP 6.55 1.10 1.3 CH
3
OH/H
2
O¼50/50
7.46 1.10 1.5 CH
3
CN/H
2
O¼25/75
DM 7.70 1.08 0.5 CH
3
OH/H
2
O¼35/65
8.24 1.06 0.8 CH
3
CN/H
2
O¼25/75
6
RSP 10.32 1 0 CH
3
OH/H
2
O¼50/50
9.34 1 0 CH
3
CN/H
2
O¼25/75
DM 7.12 1.32 3.4 CH
3
OH/H
2
O¼50/50
11.65 1.37 4.2 CH
3
CN/H
2
O¼25/75
7
RSP 7.57 1.11 1.3 CH
3
OH/H
2
O¼50/50
7.21 1.12 1.5 CH
3
CN/H
2
O¼25/75
DM 8.53 1.10 0.6 CH
3
OH/H
2
O¼35/65
8.29 1.10 1.3 CH
3
CN/H
2
O¼25/75
(continued)
Enantiomeric Separation of Fused Polycycles by HPLC 2749
several of these compounds in the reversed phase mode, but no enantiomeric
separations were observed in the polar organic mode. Only separations for
compounds 3and 4were observed for Chirobiotic CSPs in the normal phase
mode. For all of the CSPs, enantiomeric separations (Rs .0.3) of all the
13 analytes and baseline separations for 11 of them were achieved. The per-
formance of all of the CSPs is summarized in Fig. 3. Obviously, the
Cyclobond I-2000 RSP and DM CSPs are the most effective for the enantio-
meric separation of these chiral polycycles. Eleven enantiomeric and 8
baseline separations were obtained with the Cyclobond I-2000 RSP CSP
Table 1.Continued
# Structure CSP k
1
a
R
S
Mobile phase (v/v)
8
RSP 5.48 1.23 2.5 CH
3
OH/H
2
O¼40/60
5.00 1.24 2.6 CH
3
CN/H
2
O¼20/80
DM 8.81 1.18 1.8 CH
3
OH/H
2
O¼35/65
5.82 1.13 1.5 CH
3
CN/H
2
O¼20/80
9
RSP 12.77 1.03 0.3 CH
3
OH/H
2
O¼40/60
9.80 1.05 0.6 CH
3
CN/H
2
O¼20/80
DM 3.82 1.18 1.9 CH
3
OH/H
2
O¼50/50
4.96 1.12 1.5 CH
3
CN/H
2
O¼25/75
10
RSP 11.62 1.13 1.5 CH
3
OH/H
2
O¼50/50
11.58 1.14 1.8 CH
3
CN/H
2
O¼25/75
DM 9.04 1 0 CH
3
OH/H
2
O¼35/65
9.52 1.03 0.3 CH
3
CN/H
2
O¼25/75
11
RSP 5.23 1.07 0.8 CH
3
OH/H
2
O¼35/65
3.68 1.07 1.0 CH
3
CN/H
2
O¼20/80
DM 5.18 1 0 CH
3
OH/H
2
O¼35/65
7.34 1 0 CH
3
CN/H
2
O¼15/85
12
RSP 5.12 1.10 1.3 CH
3
OH/H
2
O¼40/60
5.06 1.11 1.5 CH
3
CN/H
2
O¼20/80
DM 7.78 1.17 2.0 CH
3
OH/H
2
O¼35/65
5.04 1.12 1.6 CH
3
CN/H
2
O¼20/80
13
RSP 2.28 1 0 CH
3
OH/H
2
O¼40/60
3.17 1 0 CH
3
CN/H
2
O¼20/80
DM 5.48 1.46 4.0 CH
3
OH/H
2
O¼35/65
6.16 1.26 2.5 CH
3
CN/H
2
O¼20/80
X. Han et al.2750
alone. The Cyclobond I-2000 DM CSP was able to separate 12 analytes, with 5
baseline separations. The other Cyclobond and Chirobiotic CSPs were not as
effective as the former two CSPs. Only a few analytes were resolved on
these other CSPs. For the separation of these neutral chiral fused-ring poly-
cycles, the Cyclobond CSPs are superior to the Chirobiotic CSPs. However,
for compounds 3and 4, high enantioselectivities and resolutions were
observed on Chirobiotic T and Tag columns.
Table 2. Retention factor of the first peak (k
1
), enantioselectivity (
a
), and enantio-
meric resolution (R
S
) of chiral polycycles separated on the Cyclobond AC, I, DMP,
and II CSPs in the reversed phase mode
Compound # CSP k
1
a
R
S
Mobile phase (v/v)
2AC 2.52 1.30 2.0 CH
3
OH/H
2
O¼40/60
8AC 5.15 1.07 0.6 CH
3
OH/H
2
O¼30/70
10 AC 8.27 1.11 1.1 CH
3
OH/H
2
O¼40/60
2I 1.95 1.35 1.1 CH
3
OH/H
2
O¼30/70
4I 2.10 1.40 0.7 CH
3
OH/H
2
O¼40/60
1DMP 3.74 1.04 0.6 CH
3
OH/H
2
O¼60/40
2DMP 4.81 1.04 0.4 CH
3
OH/H
2
O¼60/40
4DMP 8.66 1.04 0.4 CH
3
OH/H
2
O¼60/40
5DMP 11.25 1.02 0.3 CH
3
OH/H
2
O¼60/40
6DMP 9.81 1.10 1.5 CH
3
OH/H
2
O¼70/30
9DMP 5.89 1.13 1.8 CH
3
OH/H
2
O¼60/40
3II 2.16 1.08 0.8 CH
3
OH/H
2
O¼30/70
13 II 4.35 1.42 2.1 CH
3
OH/H
2
O¼30/70
Table 3. Retention factor of the first peak (k
1
), enantioselectivity (
a
), and enantio-
resolution (R
S
) of chiral polycycles separated on the Chirobiotic V, R, T, and Tag
CSPs in the reversed phase mode
Compound # CSP k
1
a
R
S
Mobile phase (v/v)
2V 3.04 1.20 1.7 CH
3
OH/H
2
O¼30/70
4V 6.62 1.03 0.3 CH
3
OH/H
2
O¼30/70
13 V 3.42 1.08 0.5 CH
3
OH/H
2
O¼30/70
2R 6.89 1.22 1.3 CH
3
OH/H
2
O¼20/80
9R 3.81 1.14 0.9 CH
3
OH/H
2
O¼30/70
11 R 2.59 1.09 0.4 CH
3
OH/H
2
O¼30/70
12 R 3.40 1.08 0.5 CH
3
OH/H
2
O¼30/70
3T 6.58 1.66 4.9 CH
3
OH/H
2
O¼40/60
4T 11.9 1.13 1.4 CH
3
OH/H
2
O¼40/60
3Tag 6.39 1.86 3.6 CH
3
OH/H
2
O¼50/50
4Tag 3.44 1.87 3.4 CH
3
OH/H
2
O¼60/40
Enantiomeric Separation of Fused Polycycles by HPLC 2751
Effect of Mobile Phase Composition
Based on studies reported in our previous publications, the pH of the reversed
phase mobile phase has little effect on the enantiomeric separation of
hydrophobic compounds that lack ionizable groups (21 – 23, 35). Two
organic modifiers, acetonitrile, and methanol were examined for separation
of all of the analytes on all CSPs. In most cases, the organic modifiers have
only small effects on the enantioselectivity, but they do affect resolution
to some extent (Table 1). For example, Cyclobond I-2000 RSP and DM
CSPs showed similar enantioselectivities for compound 1when using a
methanol/water or acetonitrile/water mobile phase. However, the enantio-
meric resolution was better when using an acetonitrile/water mobile phase
Table 4. Retention factor of the first peak (k
1
), enantioselectivity (
a
), and enantio-
meric resolution (R
S
) of chiral polycycles separated on the Chirobiotic V, R, T, and
Tag CSPs in the normal phase mode
Compound # CSP k
1
a
R
S
Mobile phase (v/v)
3V 8.00 1.04 0.8 HEP/EtOH ¼99/1
4V 8.32 1.06 0.9 HEP/EtOH ¼99/1
3R 7.40 1.04 0.6 HEP/EtOH ¼99/1
4R 7.99 1.04 0.4 HEP/EtOH ¼99/1
3T 4.94 1.44 3.2 HEP/EtOH ¼98/2
4T 5.14 1.30 2.3 HEP/EtOH ¼98/2
3Tag 2.08 3.29 3.1 HEP/EtOH ¼80/20
4Tag 1.61 3.50 2.7 HEP/EtOH ¼80/20
Figure 3. Summary of the number of baseline and partial separations obtained on
different Cyclobond and Chirobiotic CSPs.
X. Han et al.2752
due to an increase in the efficiency (Fig. 4a and 4b). The theoretical plate
number of the first peak, N
1
, is 3200, when methanol was used as the
organic modifier, while N
1
is 4300, when acetonitrile was used. Similar
trends were observed for the separation of compounds 3–5,7, and 9–12 on
the Cyclobond I-2000 RSP column and compounds 4–7on the Cyclobond
I-2000 DM column. The resolution usually increases when using acetonitrile
as the organic modifier due to an increase in the efficiency of the column.
However, it should be noted that in a few special cases, better resolution
was observed when methanol was used as the organic modifier, because of
higher enantioselectivity. One typical example is the separation of
compound 13 on the Cyclobond I-2000 DM CSP. Higher enantioselectivity,
which resulted in better resolution, was observed when using a methanol/
water mobile phase as opposed to an acetonitrile/water mobile phase
(Fig. 4c and 4d).
Effects of the Structure of the Analyte
Although all analytes have similar molecular skeletons, as well as stereogenic
centers, a small difference in the structure of these analytes away from the
stereogenic center produces large effects on these enantiomeric separations.
These effects are illustrated using the following examples.
Figure 4. Chromatograms showing the difference in the separation when using two
different organic modifiers in the reversed phase mode. Chromatograms (a) and (b)
were obtained using the Cyclobond I-2000 RSP CSP. Chromatograms (c) and (d)
were obtained using the Cyclobond I-2000 DM CSP. The mobile phase composition
(volume ratio) in each case was as follows: (a) and (c) CH
3
OH/H
2
O¼35/65,
(b) and (d) CH
3
CN/H
2
O¼20/80. Enantioselectivity: (a)
a
¼1.09, (b)
a
¼1.10,
(c)
a
¼1.46, (d)
a
¼1.26. Number of theoretical plates of the first peak: (a)
N
1
¼3200, (b) N
1
¼4300.
Enantiomeric Separation of Fused Polycycles by HPLC 2753
Both the Cyclobond I-2000 RSP and DM columns displayed higher
enantioselectivities for compound 2, which has a methyl ester substituent at
the 6 position, than compound 1without such a group, when a methanol/
water mobile phase was used. Therefore, the resolution for compound 2is
higher than compound 1on these two columns. Compound 2also can be
easily separated on the Cyclobond I-2000 AC, I, Chirobiotic V, and R
columns, while no enantioselectivity was observed for compound 1on these
CSPs. Another example is the separation of compounds 11 and 12. The methy-
lenedioxy group at the 4 and 5 positions of the polycycle enhanced the enan-
tiomeric resolution. Baseline separation of compound 12 was achieved on the
Cyclobond DM CSP, while no selectivity for compound 11 was found on this
column due to the lack of substituents. In general, Cyclobond CSPs showed
higher enantioselectivities for the racemic polycycles with substituents than
the analogous compounds without substituents. A substituent on any chiral
compound can provide steric interactions that adjust the geometry of the
inclusion complexation, thereby providing a more or less favorable enantio-
selective binding site. Obviously, in these specific cases, the substituent on
the polycycle resulted in an inclusion complex that enhanced the enantio-
meric recognition between the racemic analytes and the derivatized cyclo-
dextrin, thereby improving the separations.
Another interesting example is the separation of chromene derivatives
5–7. These three compounds have similar structures, except for differing
substituents in the 5 position of the polycycle. Compound 5has a proton,
while compounds 6and 7have nitro and methoxy groups, respectively.
The methoxy group has a small effect on the enantiomeric separation on the
Cyclobond I-2000 RSP and DM CSPs. Both CSPs showed similar enantio-
selectivities for compounds 5and 7(Fig. 5). Conversely, the nitro group
affects enantiomeric separation greatly. Although Cyclobond I-2000 RSP
CSP was not able to separate the enantiomers of compound 6, the enantio-
meric separation was improved for this compound on Cyclobond I-2000
DM CSP compared with compounds 5and 7(Fig. 5).
A comparison of the separation of the structural isomers 8and 9is also
interesting. A change in the position of the methylenedioxy substituent
resulted in different enantioselectivities for these two compounds on
the Cyclobond I-2000 RSP column. Using the same mobile phase on the
Cyclobond I-2000 RSP CSP, compound 8(with the methylenedioxy substitu-
ent at the 7 and 8 positions) showed lower retention, but higher enantioselec-
tivity, than compound 9(with the same group at the 8 and 9 positions).
Clearly, the location of the same substituents on the polycycles also
affected the enantiomeric separations of these compounds.
Although there is no significant difference for the separations of two
somewhat similar quinoline derivatives 3and 4on the Cyclobond DM and
RSP CSPs, the Chirobiotic T and TAG CSPs showed different enantio-
selectivity for these two analytes. In the reversed phase mode, the Chirobiotic
T column showed much higher enantioselectivity for compound 3than
X. Han et al.2754
compound 4and the enantiomeric resolution of compound 3is about 3.5 times
that of compound 4. However, on Chirobiotic TAG column, the enantioselec-
tivity of both compounds 3and 4increased (Fig. 6). Although higher enantios-
electivity was observed for compound 3on the Chirobiotic TAG than the
Chirobiotic T column, the resolution was worse on the Chirobiotic TAG
column due to the low efficiency (N1 is 1400 on Chirobiotic TAG CSP and
2600 on Chirobiotic T CSP). The enantiomeric resolution for compound 4
was significantly greater on the Chirobiotic TAG column than on the Chiro-
biotic T column, because of the increase in the enantiomeric selectivity. In
the normal phase mode, high enantiomeric resolutions of compounds 3and
4were observed on both Chirobiotic T and TAG CSPs. The Chirobiotic
TAG column showed much higher enantioselectivities (more than twice) for
these two compounds compared to the Chirobiotic T column. However, no
great increase in separation was observed due to the poor efficiency of the
Chirobiotic TAG column. The results in the normal phase (Table 4)
indicated that the steric effect of the bulky sugar groups on the teicoplanin
decreased the chiral recognition of these two compounds. On the contrary,
these repulsive steric interactions of the Chirobiotic T column decreased the
retention and increased the efficiency greatly compared with the Chirobiotic
TAG column. In addition, compounds 3and 4are the only compounds,
which can be separated in normal phase mode on all Chirobiotic CSPs.
Figure 5. The effects of different analyte substituents on the enantiomeric separ-
ation. Chromatograms (a), (b), and (c) were obtained using the Cyclobond I-2000
RSP CSP. Chromatograms (d), (e), and (f) were obtained using the Cyclobond
I-2000 DM CSP. The mobile phase composition (volume ratio) in each case was
as follows: (a), (b), (c), and (e) CH
3
OH/H
2
O¼50/50, (d) and (f) CH
3
OH/
H
2
O¼35/65. Enantioselectivity
a
: (a)
a
¼1.10, (c)
a
¼1.11, (d)
a
¼1.08,
(e)
a
¼1.32, (f)
a
¼1.10.
Enantiomeric Separation of Fused Polycycles by HPLC 2755
CONCLUSIONS
All of the 13 chiral fused polycycles examined were separated on Cyclobond
and Chirobiotic CSPs and 11 of them were baseline separations. Cyclobond
I-2000 DM and RSP CSPs are the most broadly applicable CSPs for the sep-
aration of these chiral compounds. Although Chirobiotic CSPs are not as
effective as Cyclobond CSPs for these analytes, high enantioselectivities
and resolutions for two analytes were observed on the Chirobiotic T and
TAG columns in the reversed phase and normal phase modes. The reversed
phase mode is the best mobile phase for these separations. Enantiomeric
separations of only two analytes were observed in the normal phase mode
on Chirobiotic CSPs and no enantioselectivity was found in the polar
organic mode on any CSP. Similar enantioselectivities were found for
analytes when either acetonitrile or methanol were used in the reversed
phase mode. Generally, the acetonitrile/water mobile phases showed higher
efficiencies than methanol/water mobile phases. For some special cases, the
enantioselectivity in the methanol/water mobile phase was higher than with
the acetonitrile/water mobile phase. The structure of the individual analytes
Figure 6. Comparison of the separations of compounds 3and 4on Chirobiotic T and
TAG CSPs in reversed phase mode. Chromatograms (a) and (b) were obtained using
the Chirobiotic T CSP. Chromatograms (c) and (d) were obtained using the Chirobiotic
TAG CSP. The mobile phase composition (volume ratio) in each case was as follows:
(a) and (b) CH
3
OH/H
2
O¼40/60, (c) CH
3
OH/H
2
O¼50/50, (d) CH
3
OH/
H
2
O¼60/40. Enantioselectivity
a
: (a)
a
¼1.66, (b)
a
¼1.13, (c)
a
¼1.86,
(d)
a
¼1.87. Number of theoretical plates of the first peak N
1
: (a) N
1
¼2600,
(c) N
1
¼1400.
X. Han et al.2756
greatly affected the enantiomeric separation. Chiral analytes with substituents
generally were better separated than their unsubstituted parent compounds.
ACKNOWLEDGMENTS
We gratefully acknowledge the support of this work by the National Institutes
of Health, NIH RO1 GM53825-08.
REFERENCES
1. Kende, A.S. and Johnson, S. (1985) (2)-Misramine: an unusual proaporphine
alkaloid. J. Org. Chem., 50 (5): 729– 730.
2. Go
¨zler, B. (1990) Labrandine: a new pentacyclic proaporphine alkaloid from
roemeria hybrida.Heterocycles, 31 (1): 149 – 152.
3. Hamann, M.T. and Scheuer, P.J. (1993) Biogenetically diverse, bioactive constitu-
ents of a sponge, order verongida: bromotyramines and sesquiterpene-shikimate
derived metabolites. J. Org. Chem., 58 (24): 6565 – 6569.
4. Aiello, A., Fattorusso, E., and Menna, M. (1993) A new antibiotic chloro-
sesquiterpene from the Caribbean sponge Smenospongia aurea.Z. Naturforsch.,
48b (2): 209–212.
5. Van’t Land, C.W., Mocek, U., and Floss, H.G. (1993) Biosynthesis of the
phenazine antibiotics, the saphenamycins and esmeraldins, in Streptomyces anti-
bioticus.J. Org. Chem., 58 (24): 6576 – 6582.
6. Sugimori, M., Ejima, A., Ohsuki, S., Uoto, K., Mitsui, I., Matsumoto, K.,
Kawato, Y., Yasuoka, M., Sato, K., Tagawa, H., and Terasawa, H. (1994)
Antitumor agents. 7. Synthesis and antitumor activity of novel hexacyclic camp-
tothecin analogues. J. Med. Chem., 37 (19): 3033– 3039.
7. Varasi, M., Pevarello, P., Heidempergher, F., Toma, S., and Speciale, C. Prep-
aration of condensed pyrazole compounds as a kynurenine-3-hydroxylase inhibi-
tors. WO Patent 9906374, 1999.
8. Li, J.-H., Zhang, J., and Kalish, V.-J. Preparation of substituted 4,9-dihydrocyclo-
penta[lmn]-phenanthridine-5-ones and derivatives thereof as PARP inhibitors.
WO Patent 2002006240, 2002.
9. Li, J.-H., Zhang, J., and Kalish, V.-J. Preparation of substituted 4,9-dihydrocyclo-
penta[lmn]-phenanthridine-5-ones as PARP inhibitors. WO Patent 2003057145,
2003.
10. Huang, Q., Fazio, A., Dai, G., Campo, M.A., and Larock, R.C. (2004) Pd-catalyzed
alkyl to aryl migration and cyclization: An efficient synthesis of fused polycycles
via multiple C-H activation. J. Am. Chem. Soc., 126 (24): 7460 – 7461.
11. FDA’s policy statement for the development of new stereoisomeric drugs.
Chirality, 1992, 4: 338– 340.
12. Armstrong, D.W. and DeMond, W. (1984) Cyclodextrin bonded phases for the
liquid chromatographic separation of optical, geometrical, and structural
isomers. J. Chromatogr. Sci., 22 (9): 411 – 415.
13. Armstrong, D.W., Ward, T.J., Armstrong, R.D., and Beesley, T.E. (1986) Separa-
tion of drug stereoisomers by the formation of
b
-cyclodextrin inclusion complexes.
Science, 232: 1132–1135.
Enantiomeric Separation of Fused Polycycles by HPLC 2757
14. Stalcup, A.M., Chang, S.-C., and Armstrong, D.W. (1990) (S)-2-Hydroxypropyl-
b
-cyclodextrin, a new chiral stationary phase for reversed-phase liquid chromato-
graphy. J. Chromatogr., 513: 181–194.
15. Armstrong, D.W., Stalcup, A.M., Hilton, A.M., Duncan, J.D., Faulkner, J.R., Jr.,
and Chang, J.R. (1990) Derivatized cyclodextrins for normal-phase liquid chroma-
tographic separation of enantiomers. Anal. Chem., 62 (15): 1610–1615.
16. Armstrong, D.W., DeMond, W., and Czech, B.P. (1985) Separation of metallocene
enantiomers by liquid chromatography: chiral recognition via cyclodextrin bonded
phases. Anal. Chem., 57 (2): 481– 484.
17. Armstrong, D.W., Ward, T.J., Czech, A., Czech, B.P., and Bartsch, R.A. (1985)
Synthesis, rapid resolution, and determination of absolute configuration of
racemic 2,20-binaphthyldiyl crown ethers and analogs via
b
-cyclodextrin com-
plexation. J. Org. Chem., 50 (26): 5556 – 5559.
18. Camilleri, P., Reid, C.A., and Manallack, D.T. (1994) Chiral recognition of
structurally related aminoalkylphosphonic acid derivatives on an acetylated
beta-cyclodextrin bonded phase. Chromatographia, 38 (11 – 12): 771 – 775.
19. Armstrong, D.W., Chang, L.W., Chang, S.C., Wang, X., Ibrahim, H.,
Reid, G.R., III, and Beesley, T.E. (1997) Comparison of the enantioselectivity
of
b
-cyclodextrin vs. heptakis-2,3-O-dimethyl-
b
-cyclodextrin LC stationary
phases. J. Liq. Chromatogr. Rel. Technol., 20 (20): 3279 – 3295.
20. Risley, D.S. and Strege, M.A. (2000) Chiral separations of polar compounds by
hydrophilic interaction chromatography with evaporative light scattering
detection. Anal. Chem., 72 (8): 1736 – 1739.
21. Mitchell, C., Desai, M., McCulla, R., Jenks, W., and Armstrong, D. (2002) Use of
native and derivatized cyclodextrin chiral stationary phases for the enantiosepara-
tion of aromatic and aliphatic sulfoxides by high performance liquid chromato-
graphy. Chromatographia,56(3/4): 127 – 135.
22. Schumacher, D.D., Mitchell, C.R., Xiao, T.L., Rozhkov, R.V., Larock, R.C., and
Armstrong, D.W. (2003) Cyclodextrin-based liquid chromatographic enantiomeric
separation of chiral dihydrofurocoumarins, an emerging class of medicinal
compounds. J. Chromatogr. A, 1011 (1–2): 37 – 47.
23. Han, X., Yao, T., Liu, Y., Larock, R.C., and Armstrong, D.W. (2005) Separation of
chiral furan derivatives by liquid chromatography using cyclodextrin-based chiral
stationary phases. J. Chromatogr. A, 1063 (1– 2): 111 – 120.
24. Armstrong, D.W., Tang, Y., Chen, S., Zhou, Y., Bagwill, C., and Chen, J.-R.
(1994) Macrocyclic antibiotics as a new class of chiral selectors for liquid chrom-
atography. Anal. Chem., 66 (9): 1473 – 1484.
25. Armstrong, D.W. and Zhou, Y. (1994) Use of a macrocyclic antibiotic as the chiral
selector for enantiomeric separations by TLC. J. Liq. Chromatogr., 17 (8):
1695–1707.
26. Ward, T.J., Dann, C.I., and Blaylock, A. (1995) Enantiomeric resolution using the
macrocyclic antibiotics rifamycin B and rifamycin SV as chiral selectors for
capillary electrophoresis. J. Chromatogr. A, 715 (2): 337 – 344.
27. Gasper, M.P., Berthod, A., Nair, U.B., and Armstrong, D.W. (1996) Comparison
and modeling study of vancomycin, ristocetin A, and teicoplanin for CE enantio-
separations. Anal. Chem., 68 (15): 2501 – 2514.
28. Berthod, A., Liu, Y., Bagwill, C., and Armstrong, D.W. (1996) Facile LC enantio-
resolution of native amino acids and peptides using a teicoplanin chiral stationary
phase. J. Chromatogr. A, 731 (1/2): 123– 137.
X. Han et al.2758
29. Peter, A., Torok, G., Toth, G., Van den Nest, W., Tourwe, D., and
Armstrong, D.W. (1998) Enantiomeric separation of unusual secondary aromatic
amino acids. Chromatographia, 48 (1–2): 53 – 58.
30. Aboul-Enein, H.Y. and Serignese, V. (1998) Enantiomeric separation of several
cyclic imides on a macrocyclic antibiotic (vancomycin) chiral stationary phase
under normal and reversed phase conditions. Chirality, 10 (4): 358 – 361.
31. Berthod, A., Chen, X., Kullman, J.P., Armstrong, D.W., Gasparrini, F.,
D’Acquarica, I., Villani, C., and Carotti, A. (2000) Role of the carbohydrate
moieties in chiral recognition on teicoplanin-based LC stationary phases. Anal.
Chem., 72 (8): 1767 – 1780.
32. Karlsson, C., Karlsson, L., Armstrong, D.W., and Owens, P.K. (2000) Evaluation
of a vancomycin chiral stationary phase in capillary electrochromatography using
polar organic and reversed-phase modes. Anal. Chem., 72 (18): 4394 – 4401.
33. Xiao, T.L., Zhang, B., Lee, J.T., Hui, F., and Armstrong, D.W. (2001) Reversal of
enantiomeric elution order on macrocyclic glycopeptide chiral stationary phases.
J. Liq. Chromatogr. Rel. Technol., 24 (17): 2673 – 2684.
34. Berthod, A., Xiao, T.L., Liu, Y., Jenks, W.S., and Armstrong, D.W. (2002)
Separation of chiral sulfoxides by liquid chromatography using macrocyclic
glycopeptide chiral stationary phases. J. Chromatogr. A, 955 (1): 53 – 69.
35. Xiao, T.L., Rozhkov, R.V., Larock, R.C., and Armstrong, D.W. (2003) Separation
of the enantiomers of substituted dihydrofurocoumarins by HPLC using macro-
cyclic glycopeptide chiral stationary phases. Anal. Bioanal. Chem., 377 (4):
639–654.
Enantiomeric Separation of Fused Polycycles by HPLC 2759
