Chemoenzymatic synthesis and high-throughput screening of an aminoglycoside-polyamine library: Identification of high-affinity displacers and DNA-binding ligands

Article abstract of DOI:10.1021/ja049437n

Chemoenzymatic parallel synthesis and high-throughput screening were employed to develop a multivalent aminoglycoside-polyamine library for use as high-affinity cation-exchange displacers and DNA-binding ligands. Regioselective lipase-catalyzed acylation,

Full text of DOI:10.1021/ja049437n

Published on Web 09/14/2004
Chemoenzymatic Synthesis and High-Throughput Screening
of an Aminoglycoside-Polyamine Library: Identification of
High-Affinity Displacers and DNA-Binding Ligands
Kaushal Rege,† Shanghui Hu,† James A. Moore,‡ Jonathan S. Dordick,*,†,§ and
Steven M. Cramer*,†
Contribution from the Departments of Chemical and Biological Engineering, Chemistry, and
Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12180
Received January 30, 2004; E-mail: crames@rpi.edu; dordick@rpi.edu
Abstract: Chemoenzymatic parallel synthesis and high-throughput screening were employed to develop
a multivalent aminoglycoside-polyamine library for use as high-affinity cation-exchange displacers and
DNA-binding ligands. Regioselective lipase-catalyzed acylation, followed by chemical aminolysis, was used
to generate vinyl carbonate and vinyl carbamate linkers, respectively, of the aminoglycosidic cores. These
were further derivatized with polyamines, leading to library generation. A parallel batch-displacement assay
was employed to identify the efficacy of the library candidates as potential displacers for protein purification.
Using this approach, low-molecular-mass displacers with affinities higher than those previously observed
have been identified. The aminoglycoside-polyamine library was also screened for DNA binding efficacy
using an ethidium bromide displacement assay. These highly cationic molecules exhibited strong DNA-
binding properties and may have potential for enhanced gene delivery.
Introduction
appropriate high-affinity and selective displacers that are ap-
plicable across a wide spectrum of bioseparation demands.
Displacement chromatography has attracted significant at-
tention as a powerful technique for the purification of biothera-
peutic proteins and oligonucleotides.1-9 In particular, low-
molecular-weight (MW < 2000) displacers have been shown
to have significant advantages for high-resolution protein
purifications. Displacement chromatography enables simulta-
neous concentration and purification in a single step, which is
significant in the purification of biopharmaceuticals. However,
the major obstacle in implementing this technique is the lack
of a sufficient diversity of appropriate displacer candidates. Low-
molecular-weight displacers employed to date possess moderate
to high affinities, yet are unable to displace highly retained
proteins on a variety of hydrophilic and hydrophobic resins.
Thus, there is an urgent need to develop a wide range of
The identification of appropriate displacer candidates has
traditionally relied on estimation of the steric mass action (SMA)
parameters10,11 of potential candidates. Shukla et al.12,13 used a
dynamic affinity analysis to assess the effectiveness of displacers
as a function of key structural characteristics, including spacing
of the charged moieties, number of charges, types of charged
functional groups, presence of aromatic groups, and overall
geometry. While these approaches yielded important information
on actual column performance of the displacer candidates, they
were time-consuming. They also involved a number of linear
gradient experiments. Furthermore, this approach does not allow
for the a priori prediction of displacer efficacy for new displacer
lead compounds.
Empirical screening of chemical compound libraries is of
growing importance for the identification of chemical leads in
the pharmaceutical industry.14-16 Combinatorial techniques and
high-throughput screening (HTS) have also been widely em-
ployed for the identification of ligands for affinity chromatog-
† Department of Chemical and Biological Engineering.
‡ Department of Chemistry.
§ Department of Biology.
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Chromatogr., A 1995, 695, 195-204.
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Aminoglycoside−Polyamine Library
A R T I C L E S
raphy from combinatorial libraries.17 Although the earliest
reports on the use of combinatorial synthesis of affinity ligands
involved screening of epitope peptide libraries,18-22 small-
molecule ligands have been identified for a variety of targets,
including kallikrien,23 IgG,24 and elastases25 from focused
libraries.
Previous work on parallel screening of commercially available
displacer candidates in our laboratory resulted in the identifica-
tion of aminoglycosides and linear polyamines as moderate- to
high-affinity displacers in cation-exchange systems.26,27 It was
therefore hypothesized that the derivatization of aminoglycosidic
core molecules with multiple copies of linear polyamines at
different positions on the core would result in multivalent
derivatives. Consequently, chemoenzymatic synthesis was used
to generate libraries based on polyamine derivatives of ami-
noglycosides. Enzymes were used to regioselectively acylate
aminoglycoside-linker derivatives, which were further modified
by linear polyamines at the selected positions. This enabled a
multivalent approach whereby linear polyamines were grafted
onto an aminoglycosidic core to generate multivalent cationic
molecules. The components of the library were screened for
their displacement efficacy using a batch displacement assay26
and for their DNA-binding efficacy using an ethidium bromide
displacement assay.35,36 The results presented in this paper
demonstrate that this approach can be successfully employed
to identify several powerful multivalent ligands for both protein
purification and DNA condensation.
We have recently developed a batch-displacement assay as
a high-throughput screening technique for the rapid identification
of potential displacer molecules.26 The screening results indi-
cated that a variety of aminoglycosides and linear polyamines
show moderate to high affinities as displacers in cation exchange
systems. The high-throughput screening technique is a powerful
route for the rapid identification of efficient displacer molecules
in ion-exchange systems, and it has been recently extended to
identify selective and high-affinity displacers for protein
mixtures.27 These HTS results have also been used to develop
quantitative structure-efficacy relationship (QSER) models,26
and they have been employed in concert with the multicom-
ponent steric mass action (SMA) formalism for the prediction
of chromatographic behavior of selected displacer leads.28
The complexation of DNA by polyamines has been investi-
gated in great detail.29-34 The addition of multivalent cations
with a charge greater than three in each molecule induces the
condensation of DNA in aqueous solutions. Multivalent cations
effectively reduce the electrostatic repulsion between DNA
molecules by charge neutralization, ultimately leading to DNA
collapse or condensation, which is an important first step for
gene therapy applications. Measurement of the ability of a drug
to displace ethidium bromide from DNA is established as a valid
measurement of its DNA binding ability. It is hypothesized that
DNA binding induced by polyamine binding above a critical
concentration causes conformational changes within the double
helix leading, to the release of bound ethidium bromide.35,36
In this paper, we describe the development of a small-
molecule library for potential use as both high-affinity displacers
in ion-exchange chromatography and as DNA-binding ligands.
Experimental Section
Materials. Candida antarctica lipase B (CAL-B, Novozyme 435)
was obtained from Novozymes North America, (Franklinton, NC) as
a gift. Pseudomonas fluorescence lipase (PFL) was purchased from
Amano (Nagoya, Japan). Fast Flow Sepharose SP (FF Sepharose SP),
High Performance SP Sepharose (HP Sepharose SP), and Source 15S
stationary phase materials were donated by Amersham Pharmacia
(Uppsala, Sweden). (Note that while the Sepharose materials are
agarose-based, the Source resins consist of hydrophilized poly(styrene-
divinylbenzene.) The HTS experiments were carried out using 96-well
Multiscreen, 0.45-µm Durapore membrane-bottomed plates donated by
Millipore. The 2′-deoxyadenosine, glucosamine, mannosamine, kana-
mycin A & B, neomycin sulfate, spermine, calf-thymus DNA, chicken
egg lysozyme, horse heart cytochrome-C, ammonium bicarbonate,
sodium phosphate (dibasic), and sodium phosphate (monobasic) were
purchased from Sigma (Saint Louis, MO). Ethylenediamine, diethyl-
enetriamine, vinyl chloroformate, and dry THF were purchased from
Aldrich (Milwaukee WI). Acetone-O-(vinyloxy)carbonyl)oxime was
prepared according to a literature protocol.37 Glucosamine analogues
(Scheme 1, molecules 1b-1e) neamine (Figure 2, molecule 15) and
methyl neobiosamine (Figure 2, molecule 18) were prepared according
to literature procedures.38-40
The 1H and 13C NMR spectra were recorded on a Varian spectrometer
with TMS as the internal standard. Chemical shifts are reported in ppm
and the coupling constants (J) are given in Hertz (Hz). The ESI-MS
and MALDI-TOF were measured on a Varian mass spectrometer. Flash
chromatography was performed on 60-200-mesh silica gel (Sigma
MO). Product yields, purities, and spectroscopic data are provided in
the Supporting Information section. Cation-exchange chromatography
was performed on Fast Flow Sepharose SP (FF Sepharose SP) using
ammonium bicarbonate (NH4HCO3) as a mobile phase. Fluorescence
and absorbance analyses were carried out using a Perkin-Elmer plate
reader and the results were analyzed using the software HTSoft 2.0.
Procedures. I. Generation of Glucosamine/Mannosamine Deriva-
tives (Scheme 1). Synthesis of Vinyl Carbamate Linkers, 2a-2e
(Procedure 1). Vinyl chloroformate (426 µL, 5.0 mmol) was added
(17) Lowe, C. R. Curr. Opin. Chem. Biol. 2001, 5, 248-256.
(18) Geysen, H. M.; Meleon, R. H.; Barteling, S. J. Proc. Natl. Acad. Sci. U.S.A.
1984, 81, 3998-4002.
(19) Scott, J. K.; Smith, G. P. Science 1990, 249, 386-390.
(20) Devlin, J. J.; Panganiban, L. C.; Devlin, P. E. Science 1990, 249, 404-
406.
(21) Barrett, R. W.; Cwirla, S. E.; Ackerman, M. S.; Olson, A. M.; Peters, E.
A.; Dower, W. J. Anal. Biochem. 1992, 204, 357-364.
(22) Oldenburg, K. R.; Loganathan, D.; Goldstein, I. J.; Schultz, P. G.; Gallop
M. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5393-5397.
(23) Burton, N. P.; Lowe, C. R. J. Mol. Recognit. 1992, 5, 55-68.
(24) Rongxiu, L.; Victor, D.; Stewart, D. J.; Burton S, J.; Lowe C. R.; Nature
Biotechnol. 1998, 16, 190-195.
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(26) Mazza, C. B.; Rege, K.; Breneman, C. M.; Sukumar, N.; Dordick, J. S.;
Cramer, S. M. Biotechnol. Bioeng. 2002, 80, 60-73.
(27) Rege, K.; Ladiwala, A.; Tugcu, N.; Breneman, C.; Cramer, S. M. J.
Chromatogr., A 2004, 1033, 19-28.
(28) Rege, K.; Tugcu, N.; Cramer, S. M. Sep. Sci. Technol. 2003, 38, 1499-
1517.
(29) Saminathan, M.; Antony, T.; Shirahata, A.; Sigal, L H.; Thomas, T.;
Thomas, T. J. Biochemistry 1999, 38, 3821-3830.
(30) Deng, H.; Bloomfield, V. A.; Benevides, J. M.; Thomas, G. J., Jr. Nucleic
Acids Res. 2000, 28, 3379-3385.
(31) Vijayanathan, V.; Thresia, T.; Sriharata, A.; Thomas, T. J. Biochemistry
2001, 40, 13644-13651.
(32) Pelta, J.; Livolant, F.; Sikorav, J. L. J. Biol. Chem. 1996, 271, 5656-
5662.
(33) Basu, H. S. and Marton, L. J. Biochem. J. 1987, 244, 243-246.
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77, 1547-1555.
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6986.
(38) Shafizadeh, F.; Meshreki, M. H.; Susott, R. A. J. Org. Chem. 1973, 38,
1190-1194.
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P. J. Am. Chem. Soc. 1996, 82, 3938-3946.
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Am. Chem. Soc. 2001, 123, 5878-5891.
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A R T I C L E S
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Scheme 1. (a) Chemoenzymatic Synthesis of Glucosamine-Based Derivatives and (b) Mannosamine Derivatives
dropwise to a phosphate buffer solution (40 mL, pH 8.0, 50 mM) of
compounds (1a-1e) (2.0 mmol) for 1 h at 0 °C. The reaction mixture
was vigorously stirred and the solution was maintained at pH 8.0 by
continuously adding 1.0 N NaOH. After 5 h, compounds 2c and 2d
were directly precipitated from the buffer solution, then they were
washed by distilled H2O and dried under vacuum with >95% purity.
For the other compounds, the reaction mixture was lyophilized for 24
h to afford crude product, which was purified by silica gel flash
chromatography using EtOAc/MeOH (5:1 and 2:1). The above proce-
dure resulted in moderate to good yields: 2a, 88%; 2b, 33%; 2c, 42%;
2d, 67%, 2e, 80%.
Generation of Spermine Derivatives of Glucosamine and Man-
nosamine, 4a-4e (Procedure 3). A solution of vinyloxy linkers (3a-
3e, Scheme 1) (0.2-0.4 mmol) and spermine (3 equiv relative to the
free amine group) in ethanol (10 mL) was shaken at 45 °C and 250
rpm for 96 h. After removal of ethanol under pressure, the residue was
neutralized with 1.0 N HCl at 0 °C. After lyophilization, the powder
was purified using cation-exchange chromatography (resin: FF Sepharose
SP, eluent: 0.1-0.5 M NH4HCO3 solution) as described subsequently.
The fraction collection was monitored by TLC and detected by
ninhydrin reagent solution after heating at 100 °C. The NH4HCO3
solution was removed by lyophilization to yield desired products (4a-
4e) with purities in the range 85 ∼ 95% based on NMR and MS
analyses (Scheme 1) and moderate isolated yields in the range 10-
25% after cation-exchange chromatography (described below). It was
observed that the formation of isomers was usually lower than 20-
40%, presumably due to stronger steric effects of the secondary amino
than the primary amino groups in spermine. The mannosamine-spermine
derivative (4e) was synthesized in a similar manner with a 23% overall
yield.
Lipase-Catalyzed Regioselective Synthesis of 6-Vinyl Carbonate
Linkers, 3a-3e (Procedure 2). A mixture of 2a-e (0.6 mmol), acetone
O-(vinyloxy) carbonyl)oxime (1.8 mmol), and Candida antarctica lipase
B (CAL) in 15 mL of dry THF were shaken at 200 rpm at 45 °C for
24-96 h. The reaction was monitored by TLC. After evaporation of
the solvent, the residue was purified by flash chromatography using
hexane/EtOAc (2:1, 1:2) to afford 3a-3e (Scheme 1). The yields were
generally good (3a, 91%; 3b, 73%; 3c, 83%; 3d, 48%; 3e, 82%) and
the 6-vinyloxy derivatives were the sole products in most cases.
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Aminoglycoside−Polyamine Library
A R T I C L E S
Figure 1. Structures of the aminoglycosidic leads used for derivatization
with polyamines. (a) Neomycin, neamine, neobiosamine, and potential
polyamine library using CO linkers. (b) Kanamycin A and B and potential
polyamine library using CO linkers. (c) Glucosamine- and mannosamine-
based dispermine derivatives.
II. Generation of 2′-Deoxyadenosine Derivatives (Schemes 2 and
3). Monospermine Derivatives of 2′-Deoxyadenosine (Scheme 2a,b).
The vinyl linker 6 was readily formed using CAL-B catalyzed acylation
with high yields (87%).40 Aminolysis with excess spermine gave the
desired product, 7. A solution of vinyloxy derivative 6 (0.3 mmol) and
spermine (0.9 mmol) in THF (20 mL) was shaken at 30 °C and 250
rpm for 24 h. After removal of THF, the residue was purified by silica
gel flash chromatography using MeOH/1.0 M NaCl (10:1) to yield the
pure product 7 (69% yield after flash chromatography, Scheme 2a).
Pseudomonas fluorescence lipase (PFL) was used to catalyze the
acylation of 2′-deoxyadenosine; the vinyl carbonate linker was intro-
duced at 3′-hydroxyl as described above, with high regioselectivity
(>10:1, 3′-OH versus 6′-OH group) to yield the intermediate product,
8 in reasonable yield (28%).40 The desired monospermine derivative,
9, was obtained upon aminolysis with spermine (50% yield, 95%
purity).
Trispermine Derivatives of 2′-Deoxyadenosine. Vinyl chlorofor-
mate (307µL, 5.4 mmol) was added dropwise to 5 (Scheme 2a) (377
mg, 1.5 mmol) in pyridine (5 mL) and DMAP (122 mg, 1.0 mmol) at
0 °C. The reaction mixture was stirred at room temperature for 24 h,
after which the reaction mixture was diluted with EtOAc (20 mL) and
the mixture was washed with 1.0 N HCl, saturated NaHCO3, and then
saturated NaCl. The organic phase was dried over MgSO4. After
Figure 2. Neomycin-, neamine-, and neobiosamine-based polyamine
library.
evaporation of solvent the residue was purified by flash chromatography
(hexane/EtOAc, 1:1 and 1:5) to afford the desired product 10 (yield,
28%). The trispermine derivative, 11, was synthesized in a manner
similar to that described in Procedure 3 (yield 32%, purity, 85%).
III. Generation of Polyamine Derivatives of Neomycin (12),
Neamine (15), and Methyl-Neobiosamine (18) (Figure 2). Synthesis
of Vinyl Carbamate Linkers (13, 16, 19). The vinyl carbamate linkers
of neomycin, neamine, and methyl-neobiosamine were synthesized as
described in Procedure 1 in moderate to good yields (13, 71%; 16,
66%; 19, 48%, respectively).
Ethylenediamine-Based Derivatives (Procedure 4). A solution of
vinyloxy linkers 13 and 16, (0.2-0.4 mmol) and ethylenediamine (2
9
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A R T I C L E S
Rege et al.
Scheme 2. (a) Chemoenzymatic Synthesis of 2′-Deoxyadenosine Derivative Containing a Spermine Chain at the 6′-Hydroxyl Group and (b)
Chemoenzymatic Synthesis of 2′-Deoxyadenosine Derivative Containing a Spermine Chain at the 3′-Hydroxyl Group
mL) were shaken at 35 °C and 250 rpm for 24-48 h. The excess
ethylenediamine was evaporated under pressure and the residue was
precipitated by MeOH/EtOAC; it was then washed with EtOAc to yield
pure products 14a and 17a, respectively.
Diethylenetriamine-Based Derivatives (Procedure 5). A solution
of vinyloxy linkers 13 and 19 (0.3 mmol) and diethylenetriamine (4
equiv relative to the free amine group) in ethanol (10 mL) was shaken
at 45 °C and 250 rpm for 96 h. After removal of ethanol under vacuum,
the residue was precipitated and washed as described in procedure 4
to afford 14b and 20a, respectively.
Spermine-Based Derivatives. The spermine derivatives of neamine
and methyl-neobiosamine, 17b and 20b, respectively, were synthesized
according to Procedure 3 above and were obtained in moderate to good
yields.
IV. Generation of Polyamine Derivatives of Kanamycin (Figure
3). Synthesis of Vinyl Carbamate Linkers (22a and 22b). The
carbamate linkers for kanamycin A and B were synthesized according
to Procedure 1 and were obtained in moderate to good yields: 22a,
yield 37%, 22b, yield 84%.
Ethylenediamine-Based Derivatives. A solution of 22a or 22b
(0.2-0.4 mmol) and ethylenediamine (2 mL) was shaken at 35 °C and
250 rpm for 24-48 h. The excess ethylenediamine was evaporated
under pressure, and the residue was precipitated by MeOH/EtOAC and
then washed with EtOAc to yield pure products 23a and 24,
respectively.
Diethylenetriamine-Based Derivatives. A solution of 22a (0.3
mmol) and diethylenetriamine (4 equiv relative to free amine group)
in ethanol (10 mL) was shaken at 45 °C and 250 rpm for 96 h. After
removal of ethanol in vacuo the residue was precipitated and washed
as procedure A to afford 23b (76% yield).
Purification of Aminoglycoside-Polyamine Library. The purifica-
tion of the components of the aminoglycoside-polyamine library was
carried out as follows. In the case of ethylenediamine, the desired
aminoglycoside polyamines were produced in a high yield and good
purity after the evaporation of excess ethylenediamine in vacuo and
simple precipitation with a mixture of ethyl acetate and methanol (for
example, 14a and 17a, >99% yield, >95% purity).
For the purification of diethylenetriamine and spermine derivatives,
a parallel batch screen using resin packed in a membrane-bottomed
96-well microtiter plate was used to identify the correct resin-mobile
phase combination for the subsequent chromatography steps. Cation-
exchange chromatography using Fast Flow Sulfopropyl Sepharose (FF
Sepharose SP) with a multistep gradient of ammonium bicarbonate were
identified as the best candidates for the stationary phase and the mobile
phase conditions, respectively (screening data not shown). The fact that
ammonium bicarbonate could be removed from the final product by
freezedrying was also a significant factor in its selection as the mobile
phase.
Parallel Screening of Displacers.26 Protein Adsorption. The bulk
stationary phase (HP Sepharose SP or Source 15S) was first washed
once with deionized water and then three times with the buffer, (50
mM phosphate, pH 6.0) and was allowed to equilibrate for 2 h. After
gravity settling of the stationary phase, the supernatant was removed
and 3.0 mL of the remaining stationary phase slurry was equilibrated
with 36 mL containing 3 mg/mL of the protein (horse-heart cyto-
chrome-C or chicken egg lysozyme) in 50 mM phosphate buffer, pH
6.0, at 20 °C. The protein was equilibrated with the resin for 5 h to
attain complete equilibration, during which the stationary phase was
allowed to gravity settle. Upon settling, the supernatant was removed
and the protein content in the supernatant was determined using
absorbance detection at 280 or 405 nm, using a plate reader. The mass
of the protein adsorbed on the stationary phase was determined by mass
balance.
Spermine-Based Derivatives. The spermine derivatives 23c and 25
1
(structure deduction based on ESI-MS, H and 13C NMR data) were
synthesized according to Procedure 3 above and were obtained in
moderate to good yields (7% and 27%, respectively) with purities >95%
after cation exchange chromatography.
Determination of DC50. For the screening experiments, 300 µL of
different initial concentrations (ranging from 0.3 to 5 mM) of a displacer
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Aminoglycoside−Polyamine Library
A R T I C L E S
as an alternative synthetic approach.41-43 We used Candida
antarctica lipase B (CAL-B) to incorporate the carbonate linker
at the 6-OH using acetone O-(vinyloxy)carbonyl)oxime as the
acyl donor in THF.37 Based on this strategy, we envisioned that
a series of different oligosaccharide-based branched polyamines
with different orientations could be prepared, each with different
efficacies as displacers in ion-exchange chromatography. To that
end, the acylated derivatives prepared above were further
derivatized with spermine, as shown in Scheme 1. The purifica-
tion of the spermine-conjugated molecules was performed using
cation-exchange chromatography with FF Sepharose SP as the
stationary phase and a linear gradient of NH4HCO3 as the mobile
phase. The combination of the stationary and mobile phases
was based on a microtiter-plate screen for a variety of resins
and eluents. Although other eluents also showed comparable
performance, NH4HCO3 was chosen as the mobile phase,
particularly because it could be lyophilized from the final
product.
solution was added to 25-µL aliquots of the stationary phase slurry
with bound protein. (Note that a different displacer molecule and
concentration were employed for each vial to enable parallel screening.)
The system was equilibrated for 5 h. After equilibrium was achieved,
the supernatant was removed and the protein content was determined
by absorbance detection at 280 or 405 nm, using a plate reader. This
entire procedure was carried out in parallel in 96-well membrane-
bottomed plates to enable rapid screening of potential displacer
candidates. The percent protein displaced was calculated for each
aliquot based on protein mass balance and the data were plotted as a
function of the initial displacer concentration. The resulting plots were
then employed to determine the initial displacer concentration required
to displace 50% of the adsorbed protein (i.e., the DC50).
Identification of DNA-Binding Ligands. The components of the
aminoglycoside-polyamine library were screened for their DNA-
binding efficacy using an ethidium bromide displacement assay.35,36
Parallel screening of DNA-binding activity was carried out in the
following manner: 3 mL of 6 µg/mL double-stranded calf-thymus DNA
were equilibrated with 15 µL of 0.5 mg/mL ethidium bromide. After
equilibration, 25 µL of each potential DNA-binding compound (0.3
mM) were added to the DNA-ethidium bromide mixture and equili-
brated for 20 min. A portion (75 µL) of the resulting solution was
transferred into a 96-well microtiter plate and the percent fluorescence
decrease was monitored using excitation at 260 nm and emission at
595 nm. All solutions were prepared in 20 mM Tris buffer, pH 8.0.
The synthesis of monospermine compounds based on 2′-
deoxyadenosine is described in Scheme 2. The monospermine
derivative of 2′-deoxyadenosine, 7 (spermine incorporated on
the primary hydroxyl group of 2′-deoxyadenosine), was syn-
thesized chemoenzymatically; the vinyl linker, 6, was readily
formed using CAL-B-catalyzed acylation.40 Aminolysis with
excess spermine resulted in desired product, 7. For the synthesis
of the regioisomer 9, the regioselective generation of the vinyl
carbonate linker at the 3′-hydroxyl group is challenging.
Nevertheless, P fluorescence lipase (PFL) did catalyze the 3′-
acylation with high regioselectivity (>10:1, 3′-OH versus 5′-
OH group) to afford 8 in reasonable yield (28%).40 After
aminolysis of 8 with spermine similar to that described above,
the desired product, 9, was obtained in 50% yield and ∼95%
purity resulting in a 14% overall yield of 9. Finally, the
trispermine derivative of 2′-deoxy-adenosine, 11, was synthe-
sized via chemical acylation of 2′-deoxyadenosine with vinyl
chloroformate, followed by the aminolysis with spermine in a
9% overall yield for the two steps (Scheme 3).
Results and Discussion
In this paper, we describe the parallel synthesis and high-
throughput evaluation of a multivalent cationic library designed
as high efficacy displacers for protein purification and high-
affinity DNA-binding ligands. Previous work on parallel screen-
ing of commercially available displacer candidates in our
laboratory resulted in the identification of aminoglycosides and
linear polyamines as moderate- to high-affinity displacers in
cation-exchange systems.26,27 Furthermore, structure-efficacy
relationship modeling results in concert with the screening data
indicated that charge content and spacing, inclusion of aromatic
moieties in the molecule, and molecular flexibility resulted in
increased affinity of displacer candidate molecules.26,27 Based
on these results, it was determined that the generation of a
multivalent library based on the derivatization of aminoglyco-
sides by linear polyamines would result in candidates with
increased displacement affinities. Further, the availability of a
large number of reactive sites for functionalization makes
aminoglycosides (Figure 1) promising lead candidates for
chemoenzymatic syntheses of these libraries. As will be shown
later in this paper, it turns out that the high cationic content of
these library candidates also make them promising candidates
for DNA binding/complexation. Accordingly, the library was
screened for both displacer efficacy and DNA-binding affinity.
The yields, purities, and spectroscopic data of the library
candidates are described in the Supporting Information.
Chemoenzymatic Synthesis of the Aminoglycoside-
Polyamine Library. The structural and functional diversity of
aminoglycosides was expanded using chemoenzymatic synthe-
ses. Selective incorporation of a vinyl carbamate linker on amino
groups in a molecule containing multiple hydroxyl groups was
readily achieved through chemical means in yields in the range
35-90%. A vinyl carbamate linker was initially introduced on
the 2R-amino group of glucosamine (1a) and its analogues, 1b-
1d (Scheme 1) using vinyl chloroformate as the acyl donor.
However, regioselective introduction of a vinyl carbonate linker
is very challenging and led us to consider enzymatic acylation
Neomycin-, neamine-, methyl-neobiosamine- (Figure 2) and
kanamycin-based polyamine derivatives (Figure 3) were syn-
thesized using a procedure similar to that used in generating
the glucosamine/mannosamine derivatives. Incorporation of the
vinyl carbamate linker on the amino groups of these molecules
was performed chemically in yields ranging from ∼50 to 70%,
using vinyl chloroformate as the acyl donor. The acylated
derivatives were further derivatized with ethylenediamine,
diethylenetriamine, and spermine to yield 14a, 14b, 17a, 17b,
20a, 20b, 23a, 23b, 23c, 24, and 25 in yields ranging from ∼40
to 85%.
Evaluation of the Aminoglycoside-Polyamine Library.
Identification of High-Affinity Cation-Exchange Displacers.
As described in the Introduction, we have recently developed
an efficient technique for the high-throughput screening of
displacer molecules.26 In this approach, small-scale parallel batch
displacement experiments are used for rapidly screening a large
number of potential displacer candidates. The technique can be
(41) Riva, S.; Chopineau, J.; Kieboom, A. P. G.; Klibanov, A. M. J. Am. Chem.
Soc. 1988, 110, 584-589.
(42) Hennen, W. J.; Sweers, H. M.; Wang, Y.-F.; Wong, C.-H. J. Org. Chem.
1988, 53, 4939-4945.
(43) Ferreira, L.; Ramos, M. A.; Gil, M. H.; Dordick, J. S. Biotechnol. Prog.
2002, 18, 986-993.
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A R T I C L E S
Rege et al.
Scheme 3. Synthesis of a 2′-Deoxyadenosine Derivative Having
Three Spermine Chains
applied to screen a diverse library of molecules and can be used
for different modes of interactions, such as ion exchange,
hydrophobic interaction, and reversed phase, and presents a
significant advantage over previous column based tech-
niques.12,13
In the present work, we used a modified batch-displacement
assay to investigate displacer efficacy over a wide range of
concentrations. Different initial concentrations of the displacer
candidates were employed to determine the DC50 value that is
the initial displacer concentration required to displace 50% of
the initially bound protein; clearly, the lower the DC50 value,
the more efficacious the displacer. Figure 4 presents representa-
tive data for the displacement of cytochrome-C by various initial
concentrations of neamine and its derivatives on HP Sepharose
SP. The resulting plots were then employed to determine the
DC50 values of neamine (DC50, 5.6 mM), 17a (DC50, 2.1 mM),
and 17b (DC50, 0.7 mM).
The above approach was employed to determine the DC50
values of the library candidates (Table 1) for the displacement
of horse cytochrome-C on HP Sepharose SP and Source 15S
cation-exchange resins at pH 6.0. As seen in the table, while
commercially available displacer candidates such as spermine
and neomycin show moderate to high affinities as displacers
with DC50 values of 4.8 and 2.8 mM, respectively, compounds
17b (neamine tetraspermine, Figure 2) and 23c (kanamycin A
tetraspermine, Figure 3) showed submillimolar DC50 values.
Furthermore, 11 displacers from the library exhibited higher
affinities than neomycin, while the others had comparable
affinities. In previous work, spermine and neomycin were among
the highest-affinity displacers identified by our laboratory.12,13,26
These results are important in that they demonstrate that
individual displacers with moderate affinities can be conjugated
to generate a new class of high-affinity displacers. This
represents a novel approach for the design of high-affinity
displacers for biomolecule purification by displacement chro-
matography. The DC50 values of neomycin and its derivatives
14a (neomycin derivatized with ethylenediamine, Figure 2) and
14b (neomycin derivatized with diethylenetriamine, Figure 2)
Figure 3. (a) Kanamycin-polyamine library. (b) Proposed structure of
one of the major isomers of kanamycin-trispermine derivatives.
were 2.8, 1.6, and 1.1 mM, respectively, for the displacement
on HP Sepharose SP. This indicates that displacer affinity
increases as larger polyamine homologues are conjugated to
neomycin. We hypothesize that the derivatization of neomycin
with ethylenediamine (14b) serves to increase the flexibility of
the primary amines on the resulting molecule as compared to
the parent neomycin. The greater flexibility of the cationic
charge results in an increased affinity of the molecule, a result
consistent with previous observations.26,27 Neomycin derivatized
with diethylenetriamine (14a) was found to have the highest
affinity among the neomycin derivatives, the increased affinity
due to an increase in charge on the molecules as well as added
flexibility of the charged groups. It turns out that the displacer
affinity for the molecules, as well as for their derivatives,
followed the trend neomycin > neamine > neobiosamine.
Glucosamine and mannosamine derivatives 4a-e (Scheme 1)
show interesting trends in their DC50 values on HP Sepharose
SP (Table 1). The DC50 values of 4a, 4d, and 4e are similar
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12312 J. AM. CHEM. SOC. VOL. 126, NO. 39, 2004

Aminoglycoside−Polyamine Library
A R T I C L E S
paragraphs, indicate that the affinity of the resulting multivalent
displacers can be significantly improved by the conjugation of
appropriate moieties.
The influence of the stationary phase on displacer affinity
was investigated using a second strong cation-exchange resin,
Source 15S, and the resulting displacement data are also shown
in Table 1. As seen in the table, the performance of the
displacers on Source 15S (backbone matrix: crosslinked hy-
drophilized poly(styrene-divinyl benzene)) is significantly dif-
ferent from that observed on HP Sepharose SP (backbone
matrix: crosslinked agarose), with as many as eight displacers
exhibiting submillimolar affinities on the Source resin. Although
neamine-tetraspermine (17b, Figure 2) and kanamycin A-
tetraspermine (23c, Figure 3) were still among the highest-
affinity displacers, with DC50 values of 0.6 and 0.9 mM,
respectively, other displacers (including 4b, 4c, 4d, 4a, 14b,
and 20b) had comparable submillimolar affinities. Furthermore,
the affinities of the glucosamine/mannosamine derivatives (4a-
e, Scheme 1) on the Source 15S resin showed significant
differences when compared to their affinities on HP Sepharose
SP. The DC50 values of these molecules ranged between 0.8
and 1.1 mM for the Source resin. This indicated that these were
among the highest-affinity molecules on that resin. The glu-
cosamine/mannosamine derivatives (4a-e) follow the affinity
trend 4b ∼ 4c ∼ 4d > 4a > 4e (DC50 values: 0.8, 0.8, 0.8,
0.9, and 1.1 mM, respectively). It is also interesting to note
that 4b (DC50, 3.6 mM), the least-effective displacer among this
family of molecules on the HP Sepharose SP resin, was the
most effective on the Source resin (DC50, 0.8 mM). The trend
described above indicates that displacer efficacy on the Source
resin increased with the introduction of hydrophobic/aromatic
moieties in the molecule. This is probably due to the fact that
the backbone of the Source resin is based on poly(styrene-divinyl
benzene) and that displacers with hydrophobic/aromatic moieties
may be able to exploit secondary interactions with the resin
backbone.12,13 These results indicate that although this library
was designed for generic high-affinity displacers, unique
selectivities can be observed on different resins.
Figure 4. Determination of DC50 values from percentage protein displaced
data for cytochrome-C displaced on HP Sepharose SP by neamine and its
derivatives. The lines in the graph are for visualization only. DC50 values:
neamine, 5.6 mM; 17a, 2.1 mM; 17b, 0.7 mM.
Table 1. Performance of the Novel Aminoglycoside Library: DC50
(mM) Values of Horse Cytochrome C Displaced on HP Sepharose
SP and Source 15 S (Data Arranged in Increasing Order of DC50
on HP Sepharose SP)
DC50-cytochrome C on
HP Sepharose SP (mM)
DC50-cytochrome C on
source 15S (mM)
displacer
17b
23c
25
14b
20b
4c
14a
24
23a
17a
11
4e
4a
4d
neomycin
4b
23b
spermine
neamine
bekanamycin
spermidine
20a
diethylenetriamine
7
9
0.7 ( na
1.0 ( na
1.0 ( 0.02
1.1 ( 0.06
1.1 ( na
0.6 ( 0.03
0.9 ( 0.08
2.4 ( 0.13
0.9 ( 0.12
0.8 ( 0.04
0.8 ( 0.02
1.3 ( 0.09
1.9 ( 0.06
1.7 ( 0.02
2.3 ( 0.04
1.1 ( 0.14
1.1 ( 0.00
0.9 ( 0.07
0.8 ( 0.00
1.7 ( 0.02
0.8 ( 0.04
2.4 ( 0.13
4.3 ( 0.64
4.9 ( 1.67
3.1 ( 2.29
5.8 ( 0.44
4.8 ( 0.03
14.4 ( 3.67
6.2 ( 0.46
12.9 ( 1.46
17.6 ( 2.15
1.3 ( 0.08
1.6 ( 0.01
1.9 ( 0.01
2.0 ( 0.15
2.1 ( 0.06
2.3 ( na
2.7 ( 0.68
2.8 ( na
2.8 ( 0.20
2.8 ( 0.87
3.6 ( 0.25
4.1 ( 0.29
4.8 ( 1.04
5.6 ( 1.99
6.2 ( 0.37
7.7 ( 1.44
9.4 ( 2.65
10.8 ( 0.19
11.7 ( 0.10
12.3 ( 0.25
20.2 ( 2.67
To evaluate this displacer library with another protein,
experiments were carried out with chicken-egg lysozyme
adsorbed on HP Sepharose SP. Figure 5 shows a comparison
of the percent protein displaced for both lysozyme and cyto-
chrome-C at displacer concentrations of 2.5 mM; the data are
arranged in increasing order of percent lysozyme displaced. As
seen in the figure, a large number of library compounds had
significantly higher affinities than those of the previously
identified commercial compounds neomycin and spermine. In
addition, molecules such as kanamycin A tetraspermine (23b,
Figure 3) and neamine tetraspermine (17b, Figure 2) were found
to be particularly effective at displacing lysozyme with values
of 67.8 and 52.8% protein displaced, respectively. It may be
noted further that while these molecules demonstrated differ-
ences in affinities for displacing lysozyme, they resulted in
almost 100% displacement of cytochrome-C from the resin. This
is indicative of the fact that the relative efficacies of these high-
affinity displacers were strongly influenced by both the displacer
chemistry and the protein being displaced.
ethylenediamine
(∼2.7-2.8 mM). However, 4c and 4b show very different
affinities. While 4c (DC50 ) 1.3 mM) has the highest affinity
among this family of molecules, 4b (DC50 ) 3.6 mM) has the
lowest affinity. These results indicate that the introduction of
the benzyl glycoside moiety in molecule 4c increases displacer
efficacy, while the introduction of a methyl group (4b) decreases
the affinity. This may be due to greater van der Waals
interactions of the benzyl moiety of 4c with the stationary phase
resin, a phenomenon known to enhance displacer affinity in ion-
exchange systems.12,13,26 In addition, the increased flexibility
of the benzyl group as compared to the phenyl group could
contribute to the increased affinity of 4c over 4d. These results,
along with the general trends observed in the preceding
As seen in Figure 5, the displacer candidates exhibited a wide
range of efficacies for displacing lysozyme on HP Sepharose
SP. Consequently, interesting structure-efficacy information may
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J. AM. CHEM. SOC. VOL. 126, NO. 39, 2004 12313

A R T I C L E S
Rege et al.
Figure 5. Comparison of percent lysozyme and percent cytochrome-C displaced on HP Sepharose SP at input displacer concentration ) 2.5 mM. Data
increased in increasing order of percent cytochrome-C displaced.
Figure 6. DNA-binding performance of the aminoglycoside-polyamine library: percent fluorescence decreased using ethidium bromide displacement
assay. Ligand concentration: 0.3 mM.
be observed in this case. For example, although 20b (Figure 2)
has the same number of amines as 4a (Scheme 1), the latter
showed much lower affinity for displacing lysozyme (4a: 23%,
20b: ∼41%). This suggests that the presence of an additional
1-methoxyribose unit may be responsible for the enhanced
affinity of 20b. Furthermore, an increase in the aromatic
character of the substituents on glucosamine-dispermine (Scheme
1) derivatives resulted in increased displacement affinities for
lysozyme. As seen in Figure 5, while 4c and 4d have the same
cationic content as 4a, 4b, and 4e due to the presence of the
two spermine chains on the molecules (Scheme 1), the presence
of the benzyl group on 4c and the phenyl group of 4d resulted
in high affinities of these displacers. These results are in
agreement with previous reports from our group that the
presence of terminal aromatic groups serves to enhance the
efficacies of displacer candidates in ion exchange systems.12,13,26
As seen from these screening results, the rational design of
a displacer library has resulted in the identification of several
novel high-affinity displacers for cation exchange systems. These
molecules have the potential to significantly improve the
efficiency of ion-exchange processes ranging from large-scale
preparative chromatography to microscale proteomic applica-
tions.
Evaluation of the Aminoglycoside-Polyamine Library.
Identification of High-Affinity DNA-Binding Ligands. We
hypothesized that the constituents of the aminoglycoside-
polyamine library could also be employed as high-affinity DNA-
binding ligands due to the high cationic nature of this library.
Accordingly, the ethidium bromide displacement assay35,36 was
employed to evaluate the DNA-binding affinity of this library
in a 96-well format. Using this screen, the percent fluorescence
decreased35,36 was used as a parameter to rank the DNA-binding
efficacy of the various compounds (Figure 6).
As seen in the figure, commercially available polyamines such
as spermidine, spermine, bekanamycin, and neomycin showed
low efficacies (the percent fluorescence decreased values ranging
from 0 to 15%). On the other hand, a large number of the library
constituents acted as effective DNA binding agents. Particularly
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12314 J. AM. CHEM. SOC. VOL. 126, NO. 39, 2004

Aminoglycoside−Polyamine Library
A R T I C L E S
significant are the results with 23c (Figure 3), 17b (Figure 2),
4a (Scheme 1), 25 (Figure 3), and 4b (Scheme 1), which resulted
in values of 82, 70, 66, 66, and 60% fluorescence decreased,
respectively. While the trend in Figure 6 clearly reveals that an
increase in cationic charge leads to the generation of higher
DNA-binding affinity ligands, some interesting trends can also
be observed. For example, an increase in the hydrophobic
character of the substituents on glucosamine-dispermine (Scheme
1) derivatives resulted in an overall decrease in DNA-binding
efficacy of the molecules; the results followed the trend 4a
(66%) > 4b (60%) > 4c (52%) > 4e (47%). In particular,
differences in the position of spermine (2-R versus 2-â) led to
significant differences in efficacy, as seen in case of 4a
(glucosamine-dispermine, 66% fluorescence decreased) and 4e
(mannosamine-dispermine, 47% fluorescence decreased). Clearly,
these data demonstrates that several candidates from the
aminoglycoside-polyamine library exhibited high DNA-binding
efficacies. Furthermore, differences in the chemistry and location
of the substituted moieties resulted in different DNA-binding
efficacies.
conjugated with four spermine molecules show enhanced
displacer and DNA-binding activities, due primarily to an overall
increased cationic content of the molecules. It was found that
increased charge, size, and charge spacing contributed to overall
efficacy of the molecules. These results justify the rational,
multivalent design of high-affinity displacers and DNA-binding
ligands from low- to moderate-affinity leads. Future work will
involve the use of the displacer efficacy DC50 data and the DNA-
binding data for the generation of predictive structure-property
models, which will then be employed to direct the a priori design
of a second generation of high-affinity displacers and DNA-
binding ligands. The identification of high-affinity displacers
will enable relatively low displacer concentrations to be
employed in protein separation processes, resulting in dramatic
improvements in yield and purity. This will have implications
for protein separation processes ranging from large-scale
manufacturing to microscale proteomic applications. The iden-
tification of high-affinity DNA-binding ligands will set the stage
for further evaluation of these compounds for their efficacies
as transfection agents.
Conclusions
Acknowledgment. The authors acknowledge financial support
from NSF (Grant BES-0079436) and NIH (Grant GM 47372).
The authors also thank Dr. Dmitri Zagorevski for the mass
spectrometry data and NSF (Grant CHE-0091892) for financial
support of the mass spectrometry facility, Department of
Chemistry, RPI.
In this paper, chemoenzymatic syntheses have been employed
with rapid, parallel screening techniques for the identification
of both high-affinity displacers and DNA-binding ligands.
Regioselective lipase-catalyzed acylation, followed by chemical
aminolysis, was used to generate vinyl carbonate and vinyl
carbamate linkers, respectively, of the aminoglycosidic lead
molecules. The resulting derivatives were further derivatized,
using polyamines to generate the library.
Supporting Information Available: Yields and spectroscopic
data of the aminoglycoside-polyamine library. This material
is available free of charge via the Internet at http://pubs.acs.org.
Screening results indicate that many components of the library
indeed show high affinities as cation-exchange displacers and
DNA-binding ligands. Particularly, neamine and kanamycin
JA049437N
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J. AM. CHEM. SOC. VOL. 126, NO. 39, 2004 12315