Solution-phase synthesis and evaluation of tetraproline chiral stationary phases

Article abstract of DOI:10.1002/chir.22001

A protocol was developed for the solution-phase synthesis of multigram amounts of two 9-fluorenylmethoxycarbonyl (Fmoc)-protected tetraproline peptides. These tetraproline peptides were then attached to amino derivatized silica gel. The replacement of the Fmoc group with the trimethylacetyl group lead to two tetraproline chiral stationary phases (CSPs). A comparison of the chromatographic behavior of these two solution-phase-synthesized tetraproline CSPs with that prepared by stepwise solid-phase synthesis revealed that all three had similar chromatographic performance for resolving 53 model analytes. This suggests that the solution-phase synthesis of oligoprolines, which allows for the specific benefits of good batch reproducibility, selector homogeneity, and possibly low cost, is a feasible alternative to the solid-phase synthesis of oligoproline CSPs. Copyright

Full text of DOI:10.1002/chir.22001

CHIRALITY 24:329–338 (2012)
Solution-Phase Synthesis and Evaluation of Tetraproline Chiral
Stationary Phases
*
ZHI DAI, GUOZHONG YE, CHARLES U. PITTMAN JR., AND TINGYU LI
Department of Chemistry, Mississippi State University, Starkville, Mississippi
ABSTRACT
A protocol was developed for the solution-phase synthesis of multigram
amounts of two 9-fluorenylmethoxycarbonyl (Fmoc)-protected tetraproline peptides. These tet-
raproline peptides were then attached to amino derivatized silica gel. The replacement of the
Fmoc group with the trimethylacetyl group lead to two tetraproline chiral stationary phases
(CSPs). A comparison of the chromatographic behavior of these two solution-phase-synthesized
tetraproline CSPs with that prepared by stepwise solid-phase synthesis revealed that all three
had similar chromatographic performance for resolving 53 model analytes. This suggests that
the solution-phase synthesis of oligoprolines, which allows for the specific benefits
of good batch reproducibility, selector homogeneity, and possibly low cost, is a feasible alterna-
C
VV
tive to the solid-phase synthesis of oligoproline CSPs. Chirality 24:329–338, 2012.
2012 Wiley
Periodicals, Inc.
KEY WORDS: solution-phase synthesis; oligoproline; proline chiral stationary phase; peptide
stationary phase
INTRODUCTION
tion-phase synthesis of two tetraproline derivatives by using
N-9-fluorenylmethoxycarbonyl (N-Fmoc) and O-t-butyl pro-
tection on a multigram scale and their subsequent applica-
tions in chiral chromatography. Potential advantages of the
solution approach include good batch reproducibility, selec-
tor homogeneity, and possibly low cost at a large scale.
Chiral stationary phases (CSPs) are of fundamental impor-
tance in liquid chromatography for the resolution of race-
mates into their pure enantiomers.1 In the past few decades,
the design and development of CSPs has attracted much
attention with more than a hundred CSPs reported.2,3
Because of their ready availability, peptide CSPs have been
investigated by a number of groups.4–6 For example, poly(N-
benzyl-L-glutamine)-coated silica was studied for the chiral
separation of hydantoins.7 CSPs with different lengths of L-
phenylalanine peptides were developed to resolve warfarin.8
A new class of promising CSPs based on proline (Pro) pep-
tides has been developed in our group.9–12
In our previous papers, these oligoproline CSPs were pre-
pared by stepwise proline solid-phase coupling directly to
aminopropyl or 3-(N-methyl)aminopropyl silica gel.11 Solid-
phase peptide synthesis (SPPS) first reported by Merrifield
in 1963 is now routinely used for peptide synthesis including
preparations on the industrial scale.13,14 Advantages of SPPS
are numerous and include possible automation and parallel
library synthesis. However, some drawbacks remain. During
SPPS of CSPs, the chiral selector is not cleaved from silica
gel. Therefore, the chiral selector cannot be purified. This
could lead to a variety of peptides present in the chiral selec-
tor bound to the support (selector inhomogeneity). For
example, after eight solid phase proline coupling cycles,
high-performance liquid chromatography (HPLC) analysis of
the product after cleavage from the support revealed that a
mixture of three peptides was obtained, corresponding to
eight, seven, and six monomer units in a relative 63/30/7
mole ratio, respectively.6 Batch to batch reproducibility can
also be a problem in solid-phase synthesis, as it involves a
biphasic reaction. In addition, the excessive amounts of
reagents often used in solid phase synthesis could drive up
the cost for solid phase synthesis.
MATERIALS AND METHODS
Materials and Equipment
Amino acid derivatives were purchased from NovaBiochem (San
Diego, CA). All other chemicals and solvents were purchased from
Sigma-Aldrich (Milwaukee, WI), Fluka (Ronkonkoma, NY), or Fisher
Scientific (Pittsburgh, PA). Hexanes (from Fisher scientific) were a mix-
ture of 86.1% n-hexane, 9.7% methylcyclopentane, and 4.2% various meth-
ylpentanes. The 1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker AMX-300 300 MHz spectrometer operating
at 300 MHz for proton and 75 MHz for carbon. CDCl3 was used as the
NMR sample’s solvent and tetramethylsilane (TMS) served as the inter-
nal standard. Mass spectra were obtained by electrospray ionization
(ESI) on an Agilent 6100 liquid chromatography–mass spectrometry
(LC/MS) system. HPLC-grade Kromasil silica gel (particle size 5 lm,
2
˚
pore size 100 A, and surface area 298 m /g) was purchased from Akzo
Nobel (EKA Chemicals, Bohus, Sweden). A modular column system (50
3 4.6 mm2) was purchased from Isolation Technologies (Hopedale,
MA). A packing pump P-1624CP from Chrom Tech (Apple Valley, MN)
Abbreviations:: APS, 3-aminopropyl silica gel; DCC, dicyclohexylcarbodii-
mide; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine; DMAP, 4-
dimethylaminopyridine; DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylme-
thoxycarbonyl; Fmoc-Pro-OH, 6-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-proline;
HATU, O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluoro-
phosphate; MAPS, 3-(N-methyl)aminopropyl silica gel; Pfp, pentafluorophe-
nol; Pro, proline; TMA, trimethylacetyl (also known as 2,2-dimethylpropa-
noyl); TFA, trifluoroacetic acid.
Contract grant sponsor: NIH; Contract grant number: R15 GM 83326-01
*Correspondence to: Tingyu Li, 14 Paramus Ct, North Potomac, MD 20878.
E-mail: li1640@gmail.com
Received for publication 18 July 2011; Accepted 31 October 2011
DOI: 10.1002/chir.22001
For these reasons, we decided to investigate the prepara-
tion of tetraproline CSPs using the solution-phase synthesis
of the tetraproline peptide. In this article, we report the solu-
Published online 17 February 2012 in Wiley Online Library
(wileyonlinelibrary.com).
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2012 Wiley Periodicals, Inc.

330
DAI ET AL.
was used. Solvents were purchased from Fisher (Springfield, NJ) or
Sigma-Aldrich. An Agilent 1100 HPLC system (Agilent, Wilmington, DE)
consisting of a vacuum degasser, a quaternary pump, an autosampler,
and a multiple wavelength detector was used to evaluate the columns.
ultraviolet spectroscopy (UV) spectra for measuring Fmoc loading were
obtained with a Shimadzu UV 2550 spectrometer using a 10 3 610 mm2
cell.
Fmoc-Pro-Pro 6
Trifluoroacetic acid (TFA; 7 g) was added to a solution of 4 (9.8 g,
20.0 mmol) in CH2Cl2 (15 ml) at room temperature, and the mixture was
stirred for 6 h before being quenched with an excess of methanol. Evap-
oration of the solvents in vacuo yielded Fmoc-Pro-Pro-OH 6 as a color-
less solid (8.58 g, 98.9%). 1H NMR (CDCl3): d 5 1.71–2.20 (m, 8H; CH2),
3.41–3.74 (m, 4H; CH2), 4.13–4.27 (m, 2H; CH), 4.35–4.60 (m, 3H; CH,
CH2), 7.29–7.78 ppm (m, 8H; aromatic); 13C NMR (CDCl3): d 5 24.25,
24.76, 24.93, 25.42, (ꢀꢀCH2ꢀꢀ) 28.36, 29.13, 29.89, (ꢀꢀCH2ꢀꢀCH¼) 46.36,
46.76, 47.44, (NꢀꢀCH2ꢀꢀ) 57.60, 58.19, 59.01, 59.32, (NꢀꢀCH¼) 67.66,
(OꢀꢀCH2CH¼) 119.81, 120.02, 125.20, 125.36, 127.14, 127.77, 141.26,
143.78, 144.12, (aromatic) 154.46, 155.12, 158.07, 171.55, 172.00, 171.39,
(CO) ppm; ESI-MS: m/z: 435.2 [M1H1].
(9H-Fluoren-9-yl)methyl tert-butyl
pyrrolidine-1,2-dicarboxylate 2
4-Dimethylaminopyridine (DMAP; 1.22 g, 10.00 mmol) was added to a
solution of 6-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-proline (Fmoc-Pro-
OH) 1 (10.34 g, 30.60 mmol) in dry CH2Cl2 (80 ml) at 08C. After 10 min,
dicyclohexylcarbodiimide (DCC; 6.30 g, 30.60 mmol) was added to the
solution. t-Butanol (2.22 g, 30.00 mmol) in CH2Cl2 (10 ml) was then
added dropwise at 08C. The resulting mixture was stirred for 14 h [thin
layer chromatography (TLC) monitoring] at room temperature. Then
the mixture solution was filtered and liquor was collected. After succes-
sive washing of the filtered liquor with aqueous NaHCO3 and brine, the
separated organic phase was dried over MgSO4. After evaporation of the
solvent in vacuo, the product was purified by column chromatography
(ethyl acetate/dichloromethane [DCM] 1:20) to afford Fmoc-Pro-O-t-Bu
2 (10.98 g, 95%). 1H NMR (CDCl3): d 5 1.45–1.48, (ds, 9H; (CH3)3C),
1.89–2.08 (m, 4H; CH2), 3.53–3.68 (m, 2H; CH2), 4.12–4.24 (m, 1H; CH),
4.27–4.45 (m, 3H; CH, CH2), 7.30–7.77 ppm (m, 8H; aromatic); 13C NMR
(CDCl3): d 5 23.34, 24.29, (ꢀꢀCH2ꢀꢀ) 28.02, (OꢀꢀC(CH3)3) 29.94, 31.01
(ꢀꢀCH2ꢀꢀCH¼) 47.05, 47.37, (NꢀꢀCH2ꢀꢀ) 58.65, 59.95, (NꢀꢀCH¼) 67.37,
67.65 (OꢀꢀCH2CH¼) 81.32, 81.46 (OꢀꢀC(CH3)3) 119.04, 125.16, 125.25,
127.03, 127.12, 127.70, 141.28, 143.98, 144.40, (aromatic) 154.55, 154.81,
171.83, 171.98, (CO) ppm; ESI-MS: m/z: 416.1 [M1Na1].
Fmoc-Pro-Pro-O-pentafluorophenyl ester 7
Compound 6 (8.58 g, 19.8 mmol) and pentafluorophenol (Pfp) (5.52 g,
30 mmol) were dissolved in CH2Cl2 (150 ml) and stirred for 10 min.
DCC (6.46 g, 29.1 mmol) was then added at 08C. The mixture was
stirred for 14 h (TLC monitoring) at room temperature before being
washed with saturated NaHCO3 and brine, successively. After the or-
ganic phase had been dried over MgSO4, purification by column chro-
matography (ethyl acetate/DCM 1:3) afforded Fmoc-Pro-Pro-O-Pfp 7 as
colorless crystals (7.01 g, 59%). 1H NMR (CDCl3): d 5 1.80–2.24 (m, 8H;
CH2), 3.42–3.92 (m, 4H; CH2), 4.26–4.42 (m, 2H; CH), 4.57–4.94 (m, 3H;
CH, CH2), 7.30–7.79 ppm (m, 8H; aromatic); 13C NMR (CDCl3): d 5
24.34, 25.04, 25.21, (ꢀꢀCH2ꢀꢀ) 28.79, 28.98, 29.18, (ꢀꢀCH2ꢀꢀCH¼) 46.69,
46.86, 47.15, (NꢀꢀCH2ꢀꢀ) 58.09, 58.48, (NꢀꢀCH¼) 67.55, (OꢀꢀCH2CH¼)
120.01, 124.88, 125.19, 127.13, 127.74, 141.29, 143.85, 144.15, 144.49, (ar-
omatic) 154.23, 155.12, 168.46, 170.87, 171.22, (CO) ppm; ESI-MS: m/z:
601.1 [M1H1].
Fmoc-Pro-Pro-Pro-Pro-O-t-butyl ester 8
Pro-O-t-Bu 3
Compounds 7 (2.84 g, 4.72 mmol) and 5 (3.2g) were added to DCM
(40 ml). The resulting mixture was stirred for 14 h (TLC monitoring) at
room temperature. After evaporation of the solvent in vacuo, the mixture
purified by column chromatography (methanol/DCM 1:15) afforded
Fmoc-Pro-Pro-Pro-Pro-O-t-Bu 8 (3.10 g, 96%). 1H NMR (CDCl3): d 5 1.41
(s, 9H; (CH3)3C), 1.77–2.18 (m, 16H; CH2), 3.38–3.78 (m, 8H; CH2),
4.13–4.44 (m, 4H; CH), 4.57 (m, H; CH), 4.74 (m, 2H; CH2), 7.27–7.74
ppm (m, 8H; aromatic); 13C NMR (CDCl3): d 5 24.44, 24.64, 24.89,
25.09, 25.39, (ꢀꢀCH2ꢀꢀ) 27.73, 27.94, (OꢀꢀC(CH3)3) 28.89, 28.90, 28.93,
29.06, (ꢀꢀCH2ꢀꢀCH¼) 45.25, 46.52, 46.91, 47.13, (NꢀꢀCH2ꢀꢀ) 54.87,
58.04, 58.29, 59.39, 59.95, (NꢀꢀCH¼) 67.42, (OꢀꢀCH2CH¼) 81.24, 81.62,
(OꢀꢀC(CH3)3) 119.82, 119.98, 125.20, 125.32, 127.07, 127.66, 141.22,
141.24, 143.93, 144.16, (aromatic) 155.00, 155.63, 170.22, 170.69, 171.41,
(CO) ppm; ESI-MS: m/z: 685.4 [M1H1].
Compound 2 (10.98 g, 29.1 mmol) was added to a solution of piperi-
dine/CH3CN (1:4, 90 ml). The resulting mixture was stirred for 1 h at
room temperature. After evaporation of the solvent in vacuo, the crude
Pro-O-t-Bu 3 (solid product) was obtained. It was used without further
purification for the synthesis of 4.
Fmoc-Pro-Pro-O-t-butyl ester 4
1-Hydroxybenzotriazole hydrate (HOBt) (3.55 g, 29.1 mmol) was
added to a solution of Fomc-Pro-OH (9.84 g, 29.1 mmol) in dry CH2Cl2
(120 ml) at 08C. After 10 min, DCC (6.46 g, 29.1 mmol) was added to the
solution. A solution of compound 3 (29.1 mmol) in CH2Cl2 (20 ml) was
then added dropwise. The resulting mixture was warmed to room tem-
perature and stirred for 3–4 h (TLC monitoring). Then the mixture solu-
tion was filtered and liquor was collected. After successive washing of
the filtered liquor with aqueous NaHCO3 and brine, the separated or-
ganic phase was dried over MgSO4. After evaporation of the solvent in
vacuo, the product was purified by column chromatography (ethyl ace-
tate/DCM, 1:3) to afford the colorless solid Fmoc-Pro-Pro-O-t-Bu 4 (9.84
g, 69%). 1H NMR (CDCl3): d 5 1.41–1.42, (ds, 9H; (CH3)3C), 1.89–2.17
(m, 8H; CH2), 3.43–3.76 (m, 4H; CH2), 4.07–4.24 (m, 2H; CH), 4.27–4.51
(m, 3H; CH, CH2), 7.26–7.74 ppm (m, 8H; aromatic); 13C NMR (CDCl3):
d 5 23.09, 24.30, 24.93, 25.80, (ꢀꢀCH2ꢀꢀ) 27.97, (OꢀꢀC(CH3)3) 28.74,
28.88, 29.10, (ꢀꢀCH2ꢀꢀCH¼) 46.61, 46.82, 47.18, (NꢀꢀCH2ꢀꢀ) 58.25,
58.57, 58.62, (NꢀꢀCH¼) 67.04, (OꢀꢀCH2CH¼) 81.24, (OꢀꢀC(CH3)3)
119.80, 119.96, 125.18, 125.34, 127.08, 127.51, 127.68, 141.23, 143.90,
144.21, (aromatic) 154.33, 154.93, 170.29, 170.40, 171.48, (CO) ppm; ESI-
MS: m/z: 491.2 [M1H1].
Fmoc-Pro-Pro-Pro-Pro 9
TFA (5 g) was added to a solution of 8 (3.1 g 4.54mmol) in CH2Cl2
(10 ml) at room temperature, and the mixture was stirred for 6 h before
being quenched with an excess of methanol. Evaporation of the solvents
in vacuo yielded the Fmoc-Pro-Pro-Pro-Pro-OH 9 as a colorless solid
1
(3.28 g, 97%). H NMR (CDCl3): d 5 1.67–2.27 (m, 16H; CH2), 3.43–3.80
(m, 8H; CH2), 4.21–4.36 (m, 4H; CH), 4.53–4.72 (m, 3H; CH, CH2), 7.27–
7.76 ppm (m, 8H; aromatic); 13C NMR (CDCl3): d 5 24.64, 24.74, 24.83,
24.98, 25.14, (ꢀꢀCH2ꢀꢀ) 27.71, 28.01, 28.41, 28.78, (ꢀꢀCH2ꢀꢀCH¼) 45.42,
46.95, 47.06, 47.39, (NꢀꢀCH2ꢀꢀ) 57.58, 58.26, 58.32, 58.86, 60.69,
(NꢀꢀCH¼) 67.73, (OꢀꢀCH2CH¼) 119.82, 120.03, 125.20, 125.29, 127.12,
127.77, 141.17, 141.24, 144.02, 144.05, (aromatic) 155.12, 166.59, 171.24,
171.57, 172.13, (CO) ppm; ESI-MS: m/z: 629.4 [M1H1].
4-(Fmoc-N-Methylamido)butanoic acid 11
Pro-Pro-O-t-butyl ester 5
To a stirred solution of 4-(methylamino)butyric acid hydrochloride (20
mmol, 3.07 g) in 100 ml of water, N-(9-fluorenylmethoxycarbonyloxy)
succinimide (20 mmol, 6.75 g) in 100 ml of tetrahydrofuran (THF) was
added gradually. After stirring at room temperature for 8 h, the reaction
mixture was acidified to pH 2 with concentrated hydrochloric acid. The
solution was extracted with ethyl acetate (33 100 ml), and the organic
Compound 4 (4.00 mmol) was added to a solution of piperidine/
CH3CN (1:4, 20 ml). The resulting mixture was stirred for 1 h at room
temperature. After evaporation of the solvent in vacuo, the crude Pro-
Pro-O-t-butyl 5 (solid product) was obtained and was used further with-
out purification.
Chirality DOI 10.1002/chir

SOLUTION-PHASE SYNTHESIS OF TETRAPROLINE CSPs
331
extracts were washed with water and then dried over anhydrous sodium
4-(Fmoc-Pro-Pro-N-Methylamido)butanoate
t-butyl ester 16
sulfate. Evaporation of the solvent yielded the crude product that was
purified by flash column chromatography on silica gel (methanol/DCM
1:15) to give the desired 11 (5.49 g, 81% yield). 1H NMR (CDCl3): d 5
1.56–2.37 (m, 4H; CH2), 2.85, 2.88 (ds, 3H; NꢀꢀCH3), 3.03–3.36 (m, 2H;
HOBt (1.33 g, 10.9 mmol) was added to a solution of Fmoc-Pro-OH
(3.68 g, 10.9 mmol) in dry CH2Cl2 (80 ml) at 08C. After 10 min, DCC
(2.42 g, 10.9 mmol) was added to the solution. A solution of 15 (5.38 g,
10.9 mmol) in CH2Cl2 (20 ml) was then added dropwise. The resulting
mixture was warmed to room temperature and stirred for 3–4 h (TLC
monitoring). Then the mixture solution was filtered and liquor was col-
lected. After successive washing of the filtered liquor with aqueous
NaHCO3 and brine, the separated organic phase was dried over MgSO4.
After evaporation of the solvent in vacuo, the product was purified by col-
umn chromatography (ethyl acetate/DCM 1:3) to afford a colorless solid
CH2), 4.22–4.54 (m, 3H; CH, CH2), 7.30–7.77 ppm (m, 8H; aromatic); 13
C
NMR (CDCl3):
d
5
22.58, 25.38, 30.84, 31.07, (ꢀꢀCH2ꢀꢀ) 34.06,
(ꢀꢀCH2ꢀꢀCOOH) 34.63, (NꢀꢀCH3) 47.32, 47.93, (NꢀꢀCH2ꢀꢀ) 67.10, 67.50,
(OꢀꢀCH2CH¼) 119.97, 120.02, 124.77, 125.06, 127.16, 127.73, 141.36,
144.06, (aromatic) 156.44, 156.67, 178.11, (CO) ppm; ESI-MS: m/z: 362.1
[M1Na1].
1
product 16 (4.30 g, 67%). H NMR (CDCl3): d 5 1.36, (s, 9H; (CH3)3C),
4-(Fmoc-N-Methylamido)butanoate t-butyl ester 12
1.73–2.18 (m, 12H; CH2), 2.84, 2.99 (d, 3H; NꢀꢀCH3), 3.37–3.75 (m, 6H;
CH2), 4.08–4.19 (m, 2H; CH), 4.20–4.79 (m, 3H; CH, CH2), 7.19–7.67
ppm (m, 8H; aromatic); 13C NMR (CDCl3): d 5 22.45, 23.01, 23.75,
24.83, 24.91, 25.64, (ꢀꢀCH2ꢀꢀ) 28.04, (OꢀꢀC(CH3)3) 28.32, 28.90, 29.07,
29.71 (ꢀꢀCH2ꢀꢀCH¼) 33.80, (ꢀꢀCH2ꢀꢀCO) 35.11, (NꢀꢀCH3) 46.62, 47.18,
47.39, 47.54, 48.72, 48.98, (NꢀꢀCH2ꢀꢀ) 54.69, 56.36, 56.57, 57.68, 58.24,
(NꢀꢀCH¼) 67.34, (OꢀꢀCH2CH¼) 81.11, 81.57, (OꢀꢀC(CH3)3) 119.70,
119.85, 125.11, 125.21, 126.89, 127.57, 141.19, 143.92, 144.15, (aromatic)
154.26, 154.93, 170.45, 171.95, 172.53, (CO) ppm; ESI-MS: m/z: 590.3
[M1H1].
DMAP (1.22g, 10.00 mmol) was added to a solution of compound 11
(5.49 g, 16.20 mmol) in dry CH2Cl2 (80 ml) at 08C. After 10 min, DCC
(3.34 g, 16.20 mmol) was added to the solution. t-Butanol (1.48 g, 20.00
mmol) in CH2Cl2 (10 ml) was then added dropwise at 08. The resulting
mixture was stirred for 14 h (TLC monitoring) at room temperature.
Then the mixture solution was filtered and liquor was collected. After
successive washing of the filtered liquor with aqueous NaHCO3 and
brine, the separated organic phase was dried over MgSO4. After evapora-
tion of the solvent in vacuo, the product was purified by column chroma-
tography (ethyl acetate/DCM 1:20) to afford product 12 (6.08 g, 95%).
1H NMR (CDCl3): d 5 1.46 (s, 9H; (CH3)3C), 1.81–2.26 (m, 4H; CH2),
2.89, 2.92 (ds, 3H; NꢀꢀCH3), 3.07–3.45 (m, 2H; CH2), 4.43–4.51 (m, 3H;
CH, CH2), 7.30–7.79 ppm (m, 8H; aromatic); 13C NMR (CDCl3): d 5
22.94, 23.18, (ꢀꢀCH2ꢀꢀ) 28.13, (OꢀꢀC(CH3)3) 32.38, 32.58, (ꢀꢀCH2ꢀꢀ)
34.01, (ꢀꢀCH2ꢀꢀCO) 34.62, (NꢀꢀCH3) 48.07, 48.44, (NꢀꢀCH2ꢀꢀ) 67.23,
(OꢀꢀCH2CH¼) 80.35, (OꢀꢀC(CH3)3) 119.94, 124.78, 125.02, 127.02,
127.64, 141.37, 144.14, (aromatic) 156.30, 172.51, (CO) ppm; ESI-MS: m/
z: 418.2 [M1Na1].
4-(Pro-Pro-N-Methylamido)butanoate t-butyl ester 17
Compound 16 (4.30 g, 7.3 mmol) was added to a solution of piperi-
dine/CH3CN (1:4, 10 ml). The resulting mixture was stirred for 1 h at
room temperature. After evaporation of the solvent in vacuo, the crude
solid 17 was obtained and was used without further purification.
4-(Fmoc-Pro-Pro-Pro-Pro-N-Methylamido)butanoate
t-butyl ester 18
Compounds 17 (7.3 mmol) and 7 (4.39 g 7.3 mmol) were added to
DCM (80 ml). The resulting mixture was stirred for 14 h (TLC monitor-
ing) at room temperature. After evaporation of the solvent in vacuo, the
mixture was purified by column chromatography (methanol/DCM 1:15)
affording the product 18 (5.03 g, 88%). 1H NMR (CDCl3): d 5 1.41, (s,
9H; (CH3)3C), 1.62–2.21 (m, 20H; CH2), 2.89, 3.03 (d, 3H; NꢀꢀCH3),
3.45–3.77 (m, 10H; CH2), 4.17–4.56 (m, 4H; CH), 4.71–4.93 (m, 3H; CH,
CH2), 7.25–7.74 ppm (m, 8H; aromatic); 13C NMR (CDCl3): d 5 22.47,
23.30, 24.40, 24.64, 24.70, 24.96, 25.58, (ꢀꢀCH2ꢀꢀ) 27.66, 27.79,
(ꢀꢀCH2ꢀꢀCH¼) 28.04, (OꢀꢀC(CH3)3) 29.01, 29.85 (ꢀꢀCH2ꢀꢀCH¼) 33.97,
(ꢀꢀCH2ꢀꢀCO) 35.03, (NꢀꢀCH3) 46.80, 47.07, 47.19, 47.56, 48.95,
(NꢀꢀCH2ꢀꢀ) 56.75, 57.72, 57.87, 58.14, 58.27, (NꢀꢀCH¼) 67.48,
(OꢀꢀCH2CH¼) 81.33, 81.70, (OꢀꢀC(CH3)3) 119.73, 119.89, 125.10,
125.23, 127.01, 127.61, 141.25, 143.89, 144.17, (aromatic) 154.38, 155.04,
170.17, 170.84, 171.64, 171.99, 172.58, (CO) ppm; ESI-MS: m/z: 784.5
[M1H1].
4-(Methylamino)butanoate t-butyl ester 13
Compound 12 (15.4 mmol) was added to a solution of piperidine/
CH3CN (1:4, 40 ml). The resulting mixture was stirred for 1 h at room
temperature. After evaporation of the solvent in vacuo, the crude solid
13 was obtained. It was used without further purification.
4-(Fmoc-Pro-N-Methylamido)butanoate t-butyl ester 14
HOBt (1.88 g, 15.4 mmol) was added to a solution of Fmoc-Pro-OH
(5.21 g, 15.4 mmol) in dry CH2Cl2 (80 ml) at 08C. After 10 min, DCC
(3.42 g, 15.4 mmol) was added to the solution. A solution of compound
13 (6.08 g, 15.4 mmol) in CH2Cl2 (20 ml) was then added dropwise. The
resulting mixture was warmed to room temperature and stirred for 3–4 h
(TLC monitoring). Then the mixture solution was filtered and liquor was
collected. After successive washing of the filtered liquor with aqueous
NaHCO3 and brine, the separated organic phase was dried over MgSO4.
After evaporation of the solvent in vacuo, the product was purified by col-
umn chromatography (ethyl acetate/DCM 1:3) to afford a colorless solid
14 (5.38 g, 71%). 1H NMR (CDCl3): d 5 1.37, 1.42 (ds, 9H; (CH3)3C),
1.63–2.22 (m, 8H; CH2), 2.89, 2.95 (d, 3H; NꢀꢀCH3), 3.28–3.77 (m, 4H;
CH2), 4.22–4.30 (m, 1H; CH), 4.34–4.72 (m, 3H; CH, CH2), 7.27–7.74
ppm (m, 8H; aromatic); 13C NMR (CDCl3): d 5 22.56, 23.11, 23.73,
24.45, 24.97, 25.64, (ꢀꢀCH2ꢀꢀ) 28.07, (OꢀꢀC(CH3)3) 29.54, 30.11, 30.40,
(ꢀꢀCH2ꢀꢀCH¼) 33.87, (ꢀꢀCH2ꢀꢀCO) 35.10, (NꢀꢀCH3) 46.71, 47.23, 47.51,
48.89, (NꢀꢀCH2ꢀꢀ) 56.59, 56.67, 56.98, 60.35, (NꢀꢀCH¼) 67.37,
(OꢀꢀCH2CH¼) 80.24, 81.64, (OꢀꢀC(CH3)3) 119.80, 119.88, 125.14,
125.32, 127.02, 127.61, 141.22, 141.27, 143.95, 144.27, (aromatic) 154.82,
172.05, 172.37, 172.53, (CO) ppm; ESI-MS: m/z: 515.3 [M1Na1].
4-(Fmoc-Pro-Pro-Pro-Pro-N-Methylamido)butanoic
acid 19
TFA (8 g) was added to a solution of 18 (5.03 g 6.42 mmol) in CH2Cl2
(15 ml) at room temperature, and the mixture was stirred for 6 h before
being quenched with an excess of methanol. Evaporation of the solvents
in vacuo yielded 19 (4.43 g, 95%). 1H NMR (CDCl3): d 5 1.66–2.16 (m,
20H; CH2), 2.87, 3.02 (d, 3H; NꢀꢀCH3), 3.30–3.78 (m, 10H; CH2), 4.20–
4.55 (m, 4H; CH), 4.72–4.90 (m, 3H; CH, CH2), 7.26–7.75 ppm (m, 8H;
aromatic); 13C NMR (CDCl3): d 5 22.53, 23.20, 24.40, 24.51, 24.87, 25.48,
(ꢀꢀCH2ꢀꢀ) 27.74, 27.84, 29.04, 31.32, (ꢀꢀCH2ꢀꢀCH¼) 33.58,
(ꢀꢀCH2ꢀꢀCOOH) 35.17, (NꢀꢀCH3) 46.87, 47.07, 47.23, 47.57, 49.43,
(NꢀꢀCH2ꢀꢀ) 56.67, 57.74, 58.05, 58.26, (NꢀꢀCH¼) 67.43, (OꢀꢀCH2CH¼)
119.76, 119.81, 125.14, 125.25, 127.02, 127.68, 141.23, 141.26, 143.94,
144.10, (aromatic) 154.39, 154.99, 157.66, 170.50, 170.68, 177.22, (CO)
ppm; ESI-MS: m/z: 728.3 [M1H1], 750.4 [M1Na1].
4-(Pro-N-Methylamido)butanoate t-butyl ester 15
Compound 14 (10.9 mmol) was added to a solution of piperidine/
CH3CN (1:4, 10 ml). The resulting mixture was stirred for 1 h at room
temperature. After evaporation of the solvent in vacuo, the crude solid
15 was obtained. The crude 15 was used without further purification.
Preparation of 3-(N-methyl)aminopropyl Silica Gel
3-(N-methyl)aminopropyl silica gel (APS) was prepared from Kromasil
silica gel and 3-(N-methyl)aminopropyltrimethoxysilane according to a
Chirality DOI 10.1002/chir

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DAI ET AL.
Scheme 1. Solution-phase synthesis of Fmoc-protected tetraproline 9 and its conversion to CSP-2.
previous procedure.15 The surface amino concentration is 1.07 mmol/g,
based on nitrogen elemental analysis (C, 4.81; H, 1.16; N, 1.54).
N,N-diisopropylethylamine (DIPEA; 3 equiv., 0.17 g) in 7 ml of N,N-
dimethylformamide (DMF) were added to 0.8 g of MAPS prepared as
described above (the surface methylamino concentration was 0.561
mmol/g). After agitating for 18 h, the resulting silica was filtered and
washed with DMF, methanol, and DCM 10 ml, respectively. Then any
unreacted free methylamine groups on the silica gel were end capped by
reacting with acetic anhydride and pyridine in DCM. The surface proline
concentration was determined to be 0.47 mmol/g using the Fmoc cleav-
age method on an aliquote.16 The Fmoc protecting group was then
removed by treating the silica with 100 ml of 20% (v/v) piperidine in
DMF for 3 h. The deprotected silica, H-Pro-MAPS, was collected by fil-
tration and washed with DMF, methanol, and DCM. Then three succes-
sive identical coupling steps were carried out using Fmoc-Pro-OH,
Preparation of 3-(N-Methyl)Aminopropyl Silica Gel
3-(N-methyl)aminopropyl silica gel (MAPS) was prepared from Kroma-
sil silica gel and 3-(N-methylamino)propyltrimethoxysilane according to
a procedure described for the preparation of 3-APS.15 The surface meth-
ylamino concentration is 0.561 mmol/g, based on nitrogen elemental
analysis (C, 3.09; H, 0.76; N, 0.77).
Preparation of CSP-1
Fmoc-Pro-OH (3 equiv., 0.45 g), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tet-
ramethyluronium hexafluorophosphate (HATU; 3 equiv., 0.51 g), and
Chirality DOI 10.1002/chir

SOLUTION-PHASE SYNTHESIS OF TETRAPROLINE CSPs
333
Scheme 2. Synthesis for tetraproline derivative 19 and its coupling to APS to prepare CSP-3.
1, with the resulting silica. This yielded the Fmoc-Pro-Pro-Pro-Pro-
MAPS. The surface Fmoc concentrations determined for the second
through the fourth coupling steps were 0.46, 0.44, and 0.42 mmol/g
based on the Fmoc cleavage method. After the fourth coupling step, the
Fmoc protecting group of Fmoc-Pro-Pro-Pro-Pro-MAPS was removed by
treatment with 10 ml of 20% (v/v) piperidine in DMF for 2 h. The result-
ing surface-substituted silica was end capped by reaction with trimethyla-
cetyl (TMA) chloride (0.60 g, 5 mmol) and DIPEA (0.65 g, 5 mmol) in
10 ml of dry DCM for 3 h. The desired CSP-1 CSP was collected and
washed with DMF, methanol, and DCM 10 ml each, respectively.
h. The resulting coated silica gel was end capped by reaction with TMA
chloride (0.60 g, 5 mmol) and DIPEA (0.65 g, 5 mmol) in 10 ml of dry
DCM for 3 h. The desired CSP-2 CSP was collected and washed with
DMF, methanol, and DCM 10 ml each, respectively.
Preparation of CSP-3
Compound 19 (3 equiv., 0.97 g), HATU (3 equiv., 0.51 g), and DIPEA
(3 equiv., 0.17 g) in 7 ml of DMF was added to 0.8 g of APS prepared as
described above. After agitating for 24 h, the resulting silica was filtered
and washed with DMF, methanol, and DCM 10 ml, respectively. The
surface concentration was determined to be 0.46 mmol/g based on the
Fmoc cleavage method. The Fmoc group was removed by treatment
with 10 ml of 20% (v/v) piperidine in DMF for 2 h. The resulting surface-
modified silica gel was end capped by reaction with TMA chloride (0.60
g, 5 mmol) and DIPEA (0.65 g, 5 mmol) in 10 ml of dry DCM for 3 h.
The desired CSP-3 CSP was collected and washed with DMF, methanol,
and DCM 10 ml each, respectively.
Preparation of CSP-2
Compound 9 (3 equiv., 0.84 g), HATU (3 equiv., 0.51 g), and DIPEA
(3 equiv., 0.17 g) in 7 ml of DMF were added to 0.8 g of MAPS prepared
as described above. After agitating for 24 h, the resulting silica was fil-
tered and washed with DMF, methanol, and DCM 10 ml, respectively.
The surface concentration chiral selector was determined to be 0.31
mmol/g based on the Fmoc cleavage method. The Fmoc group was
removed by treatment with 10 ml of 20% (v/v) piperidine in DMF for 2
Chirality DOI 10.1002/chir

334
DAI ET AL.
Fig. 1. Structures of analytes (1a–53a) used in this study.
equals (tr 2 t0)/t0, where tr is the retention time and t0 is the dead time.
Chromatographic Measurements
The separation factor (a) equals k2/k1, the ratio of the retention factors
of the two enantiomers. The resolution factor (Rs) was calculated using
the equation Rs 5 2 3 (tr2 2 tr1)/((w)1 1 (w)2), where (w)1 and (w)2
are the peak widths. The dead time t0 was measured with 1,3,5-tri-t-butyl-
benzene as the void volume marker. A flow rate of 1 ml/min was used.
Each CSP was packed into a 50 3 4.6 mm2 HPLC column following
the conventional high-pressure slurry packing method with ethanol as
described in the literature.17
A packing pump P-1624CP from
Chrom Tech (Apple Valley, MN) was used. The retention factor (k)
Chirality DOI 10.1002/chir

SOLUTION-PHASE SYNTHESIS OF TETRAPROLINE CSPs
335
TABLE 1. The resolution of 53 analytes on CSP-1, CSP-2, and CSP-3. CSP-1 and CSP-2 have the same linker, while CSP-3 has a
different linker
CSP-1 (solid-phase synthesis)
CSP-2 (solution-phase synthesis)
CSP-3 (solution-phase synthesis)
Analyte
a
Rs
k1
Mobile phase
a
Rs
k1
Mobile phase
a
Rs
k1
Mobile phase
1a
2a
3a
4a
5a
6a
7a
8a
1.07
1.09
1.09
1.16
1.42
1.43
1.51
1.28
1
0.15
0.35
0.33
0.78
2.67
2.14
2.37
1.94
0
0.572
6.363
4.963
6.503
6.007
6.175
4.078
11.360
9.158
2.245
4.223
4.380
4.917
7.523
7.297
4.822
5.212
4.202
4.503
3.805
4.063
8.293
8.660
3.510
2.653
3.308
1.738
5.617
5.413
5.500
2.353
5.203
9.250
10.822
10.867
13.653
4.392
6.692
7.573
6.592
5.268
5.318
5.653
7.417
8.297
12.960
4.412
4.060
6.667
3.422
12.747
19.610
7.185
1%IH
1%IH
1%IH
1.21
1.04
1.06
1.08
1.3
1.27
1.3
1.21
1
0.93
0.31
0.37
0.64
2.44
2.21
1.97
2.2
0
0.67
0
0.77
0.82
5.29
0.7
0.927
5.885
6.428
6.312
8.955
9.712
6.387
17.073
11.053
3.090
4.075
7.873
8.508
9.805
12.380
6.983
8.737
8.167
15.658
6.347
9.598
13.962
14.960
8.005
6.162
10.912
5.427
4.460
6.282
9.000
4.257
6.667
13.917
9.752
13.057
15.993
5.643
10.250
5.998
10.282
4.983
7.078
8.710
12.000
10.037
13.540
5.610
7.842
6.687
4.337
4.642
26.163
4.367
1%IH
1%IH
1%IH
1%IH
10%IH
8%IH
10%IH
8%IH
10%IH
5%IH
5%IH
3%IH
3%IH
8%IH
8%IH
8%IH
10%IH
8%IH
8%IH
8%IH
8%IH
1.27
1.03
1.05
1.12
1.43
1.36
1.43
1.22
1
0.84
0.34
0.63
0.9
2.18
2.17
2.05
1.64
0
0.63
0
0.64
0.9
1.013
8.145
4.240
6.170
8.080
9.063
5.762
17.500
9.158
3.175
4.920
7.228
8.637
11.118
10.180
6.778
5.212
10.167
6.115
7.742
7.893
16.940
18.917
6.012
4.663
10.942
5.350
6.745
7.375
7.682
3.560
5.280
11.982
14.273
14.277
17.880
6.212
10.757
9.400
10.748
5.942
10.863
13.062
15.833
11.413
15.952
6.363
13.690
9.957
4.622
12.747
20.800
7.738
1%IH
1%IH
1%IH
1%IH
10%IH
8%IH
10%IH
8%IH
10%IH
5%IH
5%IH
3%IH
3%IH
8%IH
8%IH
8%IH
10%IH
8%IH
8%IH
8%IH
8%IH
1%IH
10%IH
10%IH
10%IH
10%IH
10%IH
5%IH
5%IH
5%IH
5%IH
8%IH
10%IH
10%IH
10%IH
10%IH
10%IH
10%IH
10%IH
10%IH
10%IH
5%IH
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
27a
28a
29a
30a
31a
32a
33a
34a
35a
36a
37a
38a
39a
40a
41a
42a
43a
44a
45a
46a
47a
48a
49a
50a
51a
52a
53a
1.12
1
1.1
0.7
0
1.11
1
1.12
1
0.81
1.02
5.04
0.82
1.8
1.07
1.06
1.72
1.06
1.12
1
1.04
1.41
1.06
1.04
1.07
1.07
1.06
1.05
1.04
1
1.08
1.08
2.02
1.08
1.15
1
1.03
1.91
1.1
1.06
1.12
1.12
1.07
1.07
1.05
1
1.1
2.12
1.09
1.17
1
1.04
2.14
1.1
1.07
1.12
1.13
1.09
1.08
1.05
1
6.22
0.45
1.98
0
1.6
0
0
0.23
2.68
0.85
0.45
1.11
1.35
0.91
0.62
0.35
0
0
1.91
2
2.19
1.49
1.9
0.35
1.75
0.75
0.38
0.9
1.06
0.69
0.48
0.37
0
0.15
3.70
0.81
0.49
1.12
1.31
0.62
0.54
0.39
0
8%IH
8%IH
5%IH
5%IH
5%IH
1%IH
8%IH
8%IH
5%IH
5%IH
5%IH
1%IH
5%IH
5%IH
5%IH
1
1.5
30%IH
30%IH
30%IH
30%IH
30%IH
30%IH
30%IH
30%IH
30%IH
30%IH
30%IH
60%IH
30%IH
50%IH
30%IH
30%IH
30%IH
30%IH
20%EH
60%IH
60%EH
60%IH
30%IH
50%EH
20%ID
10%ID
1
0
30%IH
20%IH
20%IH
30%IH
30%IH
20%IH
20%IH
20%IH
20%IH
20%IH
20%IH
50%IH
20%IH
50%IH
20%IH
20%IH
20%IH
30%IH
20%EH
50%IH
40%EH
50%IH
30%IH
50%EH
20%ID
10%ID
1
0
30%IH
20%IH
20%IH
30%IH
30%IH
20%IH
20%IH
20%IH
20%IH
20%IH
20%IH
50%IH
20%IH
50%IH
20%IH
20%IH
20%IH
30%IH
20%EH
50%IH
40%EH
50%IH
30%IH
50%EH
20%ID
10%ID
1.34
1.26
1.42
1.24
1.26
1.15
1.15
1.14
1.17
1.1
1.28
1.15
1.61
1.2
1.24
1.38
1
2.03
1.87
2.68
1.58
1.96
1.43
1.59
1.74
1.57
0.6
1.56
1.19
3.82
1.46
1.5
1.51
1.31
1.63
1.29
1.28
1.2
2.82
1.92
2.56
1.54
1.89
1.72
1.73
1.67
1.76
1.01
1.81
1.06
5.32
1.41
1.98
2.06
0
1.51
2.31
1.2
2.39
1.28
2.31
0.83
1.32
1.36
1.66
1.37
1.33
1.23
1.23
1.2
1.27
1
1.38
1.09
2
1.22
1.49
1.38
1
1.22
1.59
1.19
1.55
1.21
1.25
1.15
1.26
1.7
1.74
1.51
2.07
0
1.73
0.25
3.96
1.42
1.86
1.7
1.2
1.18
1.24
1.11
1.35
1.11
1.97
1.19
1.35
1.34
1.0
1.16
1.53
1.12
1.52
1.18
1.43
1.12
1.21
1.91
0
0
1.71
1.73
1.31
2
1.21
1.71
0.85
1.41
1.09
1.36
1.12
1.32
1.15
1.3
1.31
1.74
1.12
1.62
1.50
1.82
0.46
1.48
1.03
1.24
a is the separation factor. Rs is the resolution factor. The mobile phase constituents are designate by I for isopropanol, H for mixed hexanes, D for dichlorome-
thane, E for ethanol, 1%IH for 1% isopropanol in mixed hexanes. Column dimensions, 50 3 4.6 mm2 ID. Flow rate, 1.0 ml/min; UV diode array detector.
available Fmoc-proline 1 and t-butyl alcohol in the presence
of DCC and DMAP in DCM afforded the Fmoc-Pro-O-t-butyl
ester, 2. Fmoc was removed with 20% piperidine in acetoni-
trile yielding the Pro-O-t-butyl ester, 3. Coupling Fmoc-pro-
line, 1, with Pro-O-t-butyl, 3, in the presence of DCC and
RESULTS AND DISCUSSION
The solution-phase route used to prepare Fmoc-protected
tetraproline 9 is depicted in Scheme 1, using Fmoc-proline 1
as the starting material. Reaction between commercially
Chirality DOI 10.1002/chir

336
DAI ET AL.
TABLE 2. Summary of the chromatographic behavior
of CSP-1, CSP-2, and CSP-3
O-Pfp, 7. The tetrameric ester, Fmoc-Pro-Pro-Pro-Pro-O-t-
butyl, 8 was prepared by directly coupling 7 with 5 in
DCM at room temperature. Ester 8 was then hydrolyzed
with TFA in DCM to give the desired Fmoc-protected tet-
rapeptide Fmoc-Pro-Pro-Pro-Pro-OH, 9. Overall this pro-
cess gave a 36% yield of 9 on a multigram scale from 1.
Another tetraproline derivative with a four carbon linking
unit (Fmoc-Pro-Pro-Pro-Pro-N(CH3)CH2CH2CH2COOH, 19)
was prepared by a similar solution-phase procedure (Scheme
2). All compounds 2–19 shown in both Schemes 1 and 2
were characterized as analytically pure by 1H, 13C NMR spec-
troscopy and ESI-MS.
Average
Number of
analytes
baseline-
separated
Rs > 1.5
values
from all 53
analytes
Surface
concentration
(mmol/g)
Number of
analytes
resolved
a
Rs
CSP-1
CSP-2
CSP-3
0.42
0.31
0.46
46
47
47
23
23
24
1.26
1.17
1.24
1.31
1.27
1.44
Fmoc-Pro-Pro-Pro-Pro-OH 9 was attached to 3-N-methyla-
minopropyl silica gel (MAPS) in the presence of the coupling
reagent HATU and DIPEA in DMF to afford the silica deriva-
tive Fmoc-Pro-Pro-Pro-Pro-MAPS (Scheme 1). The surface
chiral selector concentration of this stationary phase was
determined by measuring the Fmoc concentration, after
Fmoc cleavage by piperidine in DMF from an aliquote, as
described previously.16 This selector’s surface concentration
HOBt in DCM, afforded Fmoc-Pro-Pro-O-t-butyl, 4. Deprotec-
tion of 4 with 20% piperidine in acetonitrile yielded the Pro-
Pro-O-t-butyl ester, 5. t-Butyl group removal from Fmoc-
Pro-Pro-O-t-butyl ester 4 with TFA in DCM freed the car-
boxylic acid, giving Fmoc-Pro-Pro-OH, 6, which was then
treated with Pfp in the presence of DCC in DCM. This
generated the active pentaflurophenyl ester Fmoc-Pro-Pro-
Fig. 2. Resolution of analyte 14a (mobile phase 8% isopropanol/hexanes) on CSP-1 (a), CSP-2 (b), and CSP-3 (c) and resolution of analyte 41a (mobile
phase 50% isopropanol/hexanes) on CSP-1 (d), CSP-2 (e), and CSP-3 (f).
Chirality DOI 10.1002/chir

SOLUTION-PHASE SYNTHESIS OF TETRAPROLINE CSPs
337
was 0.31 mmol/g. Then the Fmoc group was removed to
free the amine terminal function of Pro-Pro-Pro-Pro-MAPS
bound to silica. Then this amino group was end capped by
reacting with TMA chloride to provide CPS-2, Tma-Pro-Pro-
Pro-Pro-MAPS.
Compound 19 (Scheme 2) was attached to the APS in the
presence of the coupling reagent HATU and DIPEA in DMF
to afford the silica-bound derivative. A surface chiral selector
concentration of 0.46 mmol/g was determined by Fmoc
cleavage for this system. The Fmoc group was then removed
by piperidine/DMF, followed by end capping with TMA
chloride to give CPS-3.
mmol/g surface concentration of this chiral selector on CSP-
3 was achieved. This amount is much larger than that for
CSP-2 (0.31 mmol/g) and is slightly larger than that on
CSP-1 (0.42 mmol/g). Clearly, the coupling efficiency of 19
with 3-APS is greater than that of 9 with the secondary
amine function of MAPS. The flexibility associated with the
extra four carbons in the side chain of 19 probably enhances
the carboxylic acid group’s ability to couple with the primary
amine function of 3-aminopropylsilica compared with cou-
pling the more crowded ꢀꢀCOOH function of 9 with the sec-
ondary amine of MAPS. The use of HATU, DIPEA in DMF
was identical in each case.
Both CSP-2 and CPS-3 were packed into 50 3 4.6 mm2
HPLC columns using a standard slurry packing method,17
and these columns were tested in an Agilent 1100 HPLC sys-
tem. Fifty three analytes were chosen to evaluate these two
tetraproline peptide CSPs for their chiral resolution capabil-
ities (Fig. 1). This group of analytes has been used to evalu-
ate CSPs previously in our group.9 The chromatographic per-
formance of these two tetraproline peptide systems CSP-2
and CPS-3 was studied with each of these analytes. The sep-
aration factor (a), resolution factor (Rs), and retention factor
(k) were determined for each of the 53 analytes on each CSP
(Table 1).
A previously described tetraproline CSP, CSP-1, had been
prepared by solid-phase synthesis.18 This CSP-1 has the
same structure as CSP-2 and its surface selector concentra-
tion was determined to be 0.42 mmol/g using the Fmoc
cleavage method. The chiral resolution capabilities of CPS-1,
prepared via solid-phase synthesis, was evaluated under the
same column packing and evaluation conditions that were
used for CSP-2 and CSP-3 (Table 1). The average separa-
tion and resolution factors for all 53 analytes and the number
of these analytes resolved for each of these three columns
are summarized in Table 2. Representative chromatograms
for the resolution of analytes 14a and 41a using CSP-3,
CSP-2 and CSP-1 are illustrated in Figure 2.
CSP-2 resolved 47 of the 53 analytes with an average sepa-
ration factor of 1.17 and an average resolution factor of 1.27.
Furthermore, 23 analytes were baseline separated. CSP-1,
the solid-phase synthesized CSP with the same structure as
CSP-2, resolved 46 of the 53 analytes with an average sepa-
ration factor of 1.26 and an average resolution factor of 1.31.
Also, 23 analytes were baseline-separated, the same number
as CSP-2. However, the average separation factor (1.17) of
CSP-2 is substantially smaller than that of CSP-1 (1.26).
Thus, the extent of chiral separation of CSP-2 is clearly
smaller than on CSP-1. This is possibly due to the lower se-
lector concentration on CSP-2 (0.31 mmol/g) versus
that on CSP-1 (0.42 mmol/g). The size of the
solution phase-synthesized reagent Fmoc-Pro-Pro-Pro-Pro-
OH reagent 9, used for the solution-phase attachment to
CSP-2, is larger than Fmoc-Pro-OH, 1, which is used during
each solid phase coupling step to build CSP-1. This size dif-
ference between 9 and 1 could lower the coupling efficiency
of 9 versus 1 on reaction with 3-(N-methyl)aminopropyl
silica gel.
CSP-3 resolved 47 of 53 analytes with an average separa-
tion factor of 1.24 and an average resolution factor of 1.44.
Additionally, 24 of the analytes gave baseline separation. The
average separation factor (1.24) of CSP-3 was clearly larger
than that of CSP-2 (1.17) suggesting that chiral selector sur-
face concentration is important to chiral separation. The
number of baseline separated analytes (24) slightly exceeds
that of CSP-1 (23). Perhaps, most striking is the large aver-
age resolution separation factor (1.44) of CSP-3. It is larger
than that of CSP-1 (1.31). Moreover, the average separation
factor of CSP-3 (1.24) is just slightly less than that of CSP-1
(1.26). The chiral separation achieved with CSP-3 is very
similar to that of CSP-1.
CONCLUSIONS
The solution-phase synthesis of two tetraproline peptide
chiral selectors using the N-Fmoc-protection method was car-
ried out. These two compounds were anchored to, in one
case, 3-(N-methyl)aminopropyl silica gel and in the other
case to a APS. Finally end capping was performed with the
TMA group. These routes were developed on a multigram
scale. This approach achieved the attachment of two pure tet-
raproline chiral selectors to silica gel and created effective
CSPs for HPLC enantiomer separation. The chromatographic
behavior of these two tetraproline-immobilized phases, which
had been synthesized in solution and then attached to silica,
was compared to that of a tetraproline CSP synthesized by a
solid phase route. This CSP has an identical structure to one
of the two solution-phase synthesized systems. Comparing
the chromatographic behavior revealed that both synthetic
routes gave similar CSP performance characteristics for a
selection of 53 model analytes. This supports the view that
the solution-phase synthesis of oligoprolines or other pep-
tides may offer an alternative to the solid-phase routes to con-
struct CSPs. Solution-phase syntheses allow the specific ben-
efits of good batch repetition, knowledge of exact structure
of all the attached selector molecules, and possibly low cost
at large scale.
ACKNOWLEDGMENTS
The authors appreciate the assistance and helpful discus-
sions with Professor Gerald Rowland, who also provided gen-
erous access to, and use of, HPLC instrumentation in his lab-
oratory.
Tetraproline derivative 19 was constructed to try to
increase the selector concentration on the surface of 3-APS.
The extra four carbon unit link (Fmoc-Pro-Pro-Pro-Pro-
N(CH3)CH2CH2CH2COOH 19) was designed and synthe-
sized via a solution-phase route (Scheme 2). Then 19 was
successfully attached to the APS to obtain CSP-3. A 0.46
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