Polycationic glycosides

Article abstract of DOI:10.3390/molecules16021508

Cationic lipids have long been known to serve as antibacterial and antifungal agents. Prior efforts with attachment of cationic lipids to carbohydrate-based surfaces have suggested the possibility that carbohydrate-attached cationic lipids might serve as antibacterial and antifungal pharmaceutical agents. Toward the understanding of this possibility, we have synthesized several series of cationic lipids attached to a variety of glycosides with the intent of generating antimicrobial agents that would meet the requirement for serving as a pharmaceutical agent, specifically that the agent be effective at a very low concentration as well as being biodegradable within the organism being treated. The initial results of our approach to this goal are presented.

Full text of DOI:10.3390/molecules16021508

Molecules 2011, 16, 1508-1518; doi:10.3390/molecules16021508
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Polycationic Glycosides
Robert Engel 1,*, Ishrat Ghani 1, Diego Montenegro 1, Marie Thomas 1, Barbara Klaritch-Vrana 1,
Alejandra Castaño 1, Laura Friedman 1, Jay Leb 1, Leah Rothman 1, Heidi Lee 1,
Craig Capodiferro 1, Daniel Ambinder 1, Eva Cere 1, Christopher Awad 1, Faiza Sheikh 2,
JaimeLee Rizzo 2, Lisa-Marie Nisbett 3, Erika Testani 3 and Karin Melkonian 3
1
Department of Chemistry and Biochemistry, Queens College of the City University of New York,
65-30 Kissena Boulevard, Flushing, NY 11367, USA
2
Department of Chemistry and Physical Sciences, Pace University, 1 Pace Plaza, New York, NY
10038, USA; E-Mail: jrizzo@pace.edu (J.L.R.)
3
Department of Biology, Long Island University, C.W. Post Campus, 720 Northern Boulevard,
Greenville, NY 11548, USA; E-Mail: kmelkoni@liu.edu (K.M.)
* Author to whom correspondence should be addressed; E-Mail: robert.engel@qc.cuny.edu;
Tel.: +1-718-997-4106; Fax: +1-718-997-5531.
Received: 23 December 2010; in revised form: 7 February 2011 / Accepted: 10 February 2011 /
Published: 11 February 2011
Abstract: Cationic lipids have long been known to serve as antibacterial and antifungal
agents. Prior efforts with attachment of cationic lipids to carbohydrate-based surfaces have
suggested the possibility that carbohydrate-attached cationic lipids might serve as
antibacterial and antifungal pharmaceutical agents. Toward the understanding of this
possibility, we have synthesized several series of cationic lipids attached to a variety of
glycosides with the intent of generating antimicrobial agents that would meet the
requirement for serving as a pharmaceutical agent, specifically that the agent be effective
at a very low concentration as well as being biodegradable within the organism being
treated. The initial results of our approach to this goal are presented.
Keywords: polycationic lipid; glycoside; cyclodextrin; starch; 1,4-diazabicyclo[2.2.2]-
octane; antibacterial

Molecules 2011, 16
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1. Introduction
Prior efforts of this laboratory have been concerned with the preparation of surfaces that serve as
antibacterial and antifungal barriers for the prevention of disease transmission [1-6]. Of particular note
has been the modification of carbohydrate-based surfaces (cotton, wood, paper) for the attachment of
cationic lipid units that would serve as permanent disruptors of microbial cell walls as they would
impinge on the modified surface [1,4]. In this manner surfaces that are permanently prophylactic
regarding the transmission of disease causing microbes have been generated, based on simple
carbohydrate chemistry.
While highly effective surfaces that are prophylactic toward disease transmission may be so
constructed, such macro-surfaces are not applicable for the treatment of disease in infected patients.
For such pharmaceutical applications, based on the concept of cationic lipid disruption of microbial
cell walls, several structural factors need to be incorporated into the agent. First, the pharmaceutical
agent must be capable of serving as a micro-surface bearing several cationic lipid sites on each defined
molecule in order to provide a sufficient localized attack on the microbial cell wall. Further, the
pharmaceutical agent must be structurally susceptible either to relatively rapid removal of itself from
the treated organism after serving as an antimicrobial agent, or it must be structurally susceptible to
relatively rapid degradation generating inactive species that themselves are either removed from the
treated organism without deleterious effect or, alternatively, those degradation products may be usable
in the ordinary metabolism of the treated organism.
Glycosides of varying sorts can be viewed as meeting these requirements for service as scaffolding
for the antimicrobially active component of the potential pharmaceutical, the cationic lipid. Several
types of glycosides are used in the current effort as model systems for such agents, and as potential
pharmaceutical agents themselves. With regard to model systems, glycosidic derivatives of simple
monosaccharides to which polycationic lipid components have been attached have been synthesized
and investigated. In addition, the disaccharide sucrose has been used to serve as a scaffolding for the
cationic lipid components, bearing three primary hydroxyl sites at which attachment can be
accomplished within a single molecule.
More complex systems which in themselves constitute micro-surfaces provide the capability for cell
wall destruction by incorporating several polycationic lipid components within a relatively small
region of space. The difficulty with simple cationic lipids serving as antimicrobial agents stems from
the unfavorable entropy factor involved, the requirement that a relatively large number of such units be
organized for interaction with the cell wall. The incorporation of numerous polycationic lipid sites
within a single molecular unit overcomes this unfavorable entropy factor by providing the capability
for more intense local disruption of the microbial cell wall. Both linear (soluble starch) and cyclic
(cyclodextrin) polyglycosides bearing numerous polycationic lipid components have been prepared
and investigated in this effort. The cyclodextrin derivatives bear either six or seven polycationic lipid
components with each molecule, providing a highly localized effect of a bacterial cell wall with which
it impinges. In the instance of soluble starch modified in an analogous manner, a ribbon of significant
length bearing the polycationic lipid units, capable of interacting with a bacterial surface over a
large region.

Molecules 2011, 16
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Our goal in the present effort has been to determine the capability of glycosides modified by the
attachment of cationic lipid components to serve as antibacterial agents that would be subject to
chemical degradation in vivo in a relatively short period of time. With a practical view for the
generation of such materials, it need be kept in mind that such agents would need to be prepared in
significant quantity and with relatively low cost. Thus, the most readily available carbohydrate
components are to be considered as the most highly desirable scaffoldings for attachment of the
cationic lipid adjuncts. Efforts have thereby concentrated on the use of glucose derivatives (both
monosaccharide glycosides, cyclic oligomers, and polymers), including the disaccharide sucrose.
2. Results and Discussion
Polycationic lipid derivatives of simple monosaccharide glycosides have been prepared from the
simple glycoside in a two-step process. In the initial step, the available primary hydroxyl group(s) of
the monosaccharide glycoside have been activated for a displacement reaction by selective tosylation
using procedures we have previously described [1]. While the standard laboratory procedure for such
tosylation generally uses pyridine as the solvent, our efforts demonstrate that an aqueous sodium
bicarbonate medium also serves well, particularly for the large-scale preparations.
In the second step of the process, displacement of the primary tosylate functionality is accomplished
by treatment with a monoalkylated derivative of 1,4-diazabicyclo[2.2.2]octane (DABCO). Such
derivatives of DABCO have been generated in our laboratory with selective alkylation being
accomplished through the judicious choice of solvent media [7-10]. Of particular interest in the present
efforts have been those bearing alkyl chains of sixteen and twelve carbon atoms, i.e. 1-hexadecyl-1-
azonia-4-azabicyclo[2.2.2]octane halide and the twelve-carbon analogue, 1-dodecyl-1-azonia-4-
azabicyclo[2.2.2]octane halide, although other chain lengths have also been of specialized interest.
This two-step process is illustrated below in Scheme 1.
Scheme 1. Reaction for preparation of polycationic glycosides.

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Our interest in these model monosaccharide glycosides has focused particularly on D-glucose
modified by the attachment of monoalkylated DABCO species (in this report we focus on the use of
monoalkylated DABCO units; other types of amines used for attachment to carbohydrate glycoside
scaffolding will be described elsewhere). The structures of the products and the precursor
monoalkylated DABCO materials are provided in Table 1, along with the yields in the particular
preparations. Structural verification data (1H- and 13C-NMR spectra and quantitative elemental
analyses) for these materials are provided in Table 2. Toward the construction of molecular
antimicrobial micro-surfaces, of those discrete molecules bearing several cationic lipid components
each, particular focus has been given to the cyclodextrins.
Table 1. Structure of polycationic glycosides and their amine precursors.
Product
Number
Product Structure
Precursor Tertiary Amine
Yield (%)
1
94
2
3
4
95
98
99
5
6
99
99

Molecules 2011, 16
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Table 1. Cont.
7
99
8
9
91
99
10
11
33
29
12
13
33
31
14
30

Molecules 2011, 16
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Table 1. Cont.
15
16
42
56
Table 2. Structural verification data for cationic glycosides.
Product
Number
1H-NMR (D2O)()
13C-NMR ()
Elemental Analysis
1.36 (6H) d, 2.31 (3H) s, 3.28-3.80 15.5, 20.5, 44.0, 47.8, 55.1, 60.6, 68.3,
Calcd. for C23H39BrN2O8S.8.5H2O:
1
(20H) br, 7.28-7.62 (4H) AA'BB'
69.6, 71.2, 71.6, 73.1, 99.3, 125.4, 129.5,
139.5, 142.5
C, 37.50; H, 7.66. Found: C 37.53; H, 7.37
1.01 (6H) d, 2.21 (1H) br, 2.30
(3H) s, 3.35 (3H) s, 3.33-3.87
(21H) m, 7.27-7.60 (4H) AA'BB'
20.6, 21.9, 22.9, 44.0, 51.2, 55.1, 60.6,
69.6, 71.3, 71.6, 73.1, 74.2, 99.3, 125.4,
129.6, 139.6, 142.5
Calcd. for C24H41BrN2O8S.9H2O: C,
37.94; H, 7.83. Found: C 8.12; H, 7.79
2
3
0.83 (3H) t, 1.28 (2H) m, 1.64
(2H) m, 2.29 (3H) s, 3.29-3.50
(5H) br, 3.69-3.80 (16H) br, 3.81
(3H) br, 7.26-7.58 (4H) AA'BB'
12.7, 18.9, 20.5, 43.9, 46.2, 50.7, 55.0,
60.5, 65.1, 69.5, 71.2, 71.5, 73.0, 99.2,
125.3, 129.5, 139.4, 142.5
Calcd. for C24H41BrN2O8S.5H2O: C,
41.92; H, 7.48. Found: C 41.65; H, 7.76
2.34 (3H) s, 3.36 (3H) s, 3.32-3.84 20.5, 44.1, 50.6, 55.0, 60.6, 69.0, 69.6,
Calcd. for C27H39ClN2O8S.4H2O: C, 50.54;
H, 6.99. Found: C 50.51; H, 7.00
4
5
(19H) br, 4.66 (2H) br, 7.32-7.64
(9H) br
71.3, 71.6, 73.1, 99.3, 125.4, 126.3,
129.5, 129.6, 131.4, 133.0, 139.5, 145.5
0.76 (3H) t, 1.17-1.24 (10H) br,
1.69 (2H) br, 2.29 (3H) s, 3.36
(3H) s, 3.30-3.78 (21H) br, 7.26-
7.58 (4H) AA'BB'
13.3, 20.4, 21.3, 21.9, 25.2, 28.0, 28.1,
30.9, 43.9, 50.7, 55.0, 58.1, 60.5, 65.3,
71.2, 71.5, 73.0, 99.2, 125.3, 129.4,
139.4, 142.5
Calcd. for C28H49BrN2O8S.2H2O: C,
48.76; H, 7.74. Found: C 48.77; H, 7.80
0.84 (3H) t, 0.86-1.01 (18H) br,
1.24 (2H) br, 2.19 (3H) s, 3.32
(3H) br, 3.43-3.76 (21H) br, 7.08-
7.57 (4H) AA'BB'
13.7, 20.8, 21.2, 22.5, 23.8, 28.2, 29.3,
29.4, 29.6, 29.7, 30.2, 31.8, 43.9, 46.9,
50.9, 55.0, 60.5, 69.5, 71.2, 71.2, 73.0,
99.2, 125.6, 129.1, 139.8, 141.2
Calcd. for C32H57BrN2O8S.2H2O: C,
51.53; H, 8.24. Found: C 51.46; H, 8.31
6
7
0.84 (3H) t, 0.86-1.01 (22H) br,
1.24 (2H) br, 2.19 (3H) s, 3.32
(3H) s, 3.43-3.76 (21H) br, 7.04-
7.58 (4H) AA'BB'
13.9, 21.0, 21.7, 21.9, 22.8, 26.0, 26.3,
29.1, 29.7, 29.8, 30.0, 30.1, 32.2, 44.1,
46.4, 50.8, 55.1, 60.6, 64.7, 69.6, 71.3,
71.6, 73.2, 99.3, 125.8, 129.1, 139.8,
142.1
Calcd. for C34H61BrN2O8S.2H2O: C,
52.77; H, 8.47. Found: C 52.83; H, 8.52
0.81 (3H) t, 1.19 (26H) br, 1.69
(2H) br, 2.15 (3H) s, 3.32 (3H) s,
3.44-3.87 (21H) br, 6.94-7.55 (4H) 29.8, 29.9, 32.7, 44.2, 45.2, 50.6, 55.3,
14.1, 21.2, 22.7, 24.5, 26.1, 26.9, 27.1,
28.9, 29.2, 29.5, 29.66, 29.68, 29.73,
Calcd. for C36H65ClN2O8S.5H2O: C, 46.56;
H, 9.55. Found: C 46.53; H, 9.18
8
9
AA'BB'
60.8, 69.7, 71.6, 71.7, 73.4, 99.3, 125.8,
129.1, 140.1, 141.9
0.86 (3H) t, 1.24 (30H) br, 1.69
(2H) br, 2.29 (3H) s, 3.04 (2H) br,
3.31 (3H) s, 3.19-3.85 (19H) br,
7.11-7.47 (4H) AA'BB'
13.9, 21.2, 22.0, 22.8, 24.3, 26.2, 26.9,
28.9, 29.44, 29.47, 29.50, 29.6, 29.67,
29.70, 29.75, 29.8, 29.9, 32.7, 44.6, 45.7,
50.9, 55.4, 61.0, 69.9, 71.6, 71.8, 73.7,
99.6, 125.4, 128.0, 141.0, 142.1
Calcd. for C38H69BrN2O8S5H2O: C,
51.63; H, 9.01. Found: C 51.55; H, 9.23
0.87 (18H) t, 1.24 (108H) br, 1.65
15.47, 23.37, 24.26, 24.32, 27.47, 30.46,
Calcd. for C144H276Br12N12O244H2O:
10
(12H) br, 3.32 (12H) br, 3.59-3.88 30.97, 31.39, 31.82, 31.90, 33.72, 45.91,
C, 48.17; H, 7.97. Found: C 48.08; H, 8.13
(108H) br, 5.02 (6H) d
52.94, 61.28, 66.24, 72.13, 73.84, 75.77,
83.19, 103.98

Molecules 2011, 16
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Table 2. Cont.
0.86 (18H) t, 1.26 (156H) br,
1.64 (12H) br, 3.35 (12H) br,
3.55-3.90 (108H) br, 5.03 (6H) d 31.87, 31.93, 33.80, 45.99, 52.88, 60.97,
66.03, 72.19, 73.91, 75.76, 83.04, 103.94
15.33, 23.41, 24.28, 24.31, 24.35, 27.48,
28.22, 28.71, 30.45, 30.68, 30.99, 31.41,
Calcd. for C168H324Br12N12O244H2O:
11
C, 51.38; H, 8.52. Found: C 51.29; H,
8.63
0.85 (21H) t, 1.16 (126H) br,
1.60 (14H) br, 3.25 (14H) br,
3.48-3.78 (126H) br, 4.96 (7H) d 51.20, 51.86, 60.02, 65.25, 72.17, 73.61,
81.26, 102.38
13.74, 21.02, 21.72, 22.34, 25.82, 28.64,
28.76, 28.87, 28.92, 29.04, 31.48, 44.20,
Calcd. for C168H322Br14N14O283H2O:
12
13
14
C, 48.51; H, 7.95. Found: C 48.36; H,
8.18
0.86 (21H) t, 1.27 (182H) br,
1.65 (14H) br, 3.36 (14H) br,
3.45-3.81 (126H) br, 4.99 (7H) d 31.91, 31.94, 33.79, 46.01, 52.85, 60.86,
66.11, 72.23, 73.87, 75.82, 83.10, 103.96
15.37, 23.42, 24.22, 24.35, 24.39, 27.44,
28.20, 28.75, 30.41, 30.57, 30.97, 31.28,
Calcd. for C196H378Br14N14O283H2O: C,
51.72; H, 8.50. Found: C 51.66; H, 8.58
0.86 (21H) t, 1.28 (266H) br,
1.66 (14H) br, 3.37 (14H) br,
15.34, 23.41, 24.29, 24.37, 24.42, 24.58,
25.36, 27.50, 28.19, 28.83, 29.24, 30.43,
Calcd. for C238H462Br14N14O283H2O: C,
55.60; H, 9.18. Found: C 55.53; H, 9.22
3.44-3.80 (126H) br, 4.99 (7H) d 30.54, 30.61, 30.95, 31.30, 31.67, 31.93,
31.95, 33.72, 33.81, 46.03, 52.91, 60.92,
66.18, 72.27, 73. 95, 75.80, 83.17, 103.91
0.79-0.85 (9H) br, 1.23 (54H) br, 13.71, 14.22, 14.76, 19.73, 20.28, 20.35,
Calcd. for C87H151Cl3N6O17S3H2O: C,
15
1.71 (6H) br, 2.14 (9H) s, 3.40-
3.88 (56H) br, 6.93-7.53 (12H)
AA'BB'
22.57, 22.86, 23.31, 23.79, 24.00, 24.43,
24.78, 25.25, 25.63, 26.04, 26.41, 26.80,
27.07, 27.65, 27.97, 28.36, 28.72, 29.23,
29.79, 30.30, 30.56, 31.18, 31.66, 32.18,
32.67, 32.94, 33.34, 44.89, 42.62, 43.01,
43.17, 44.55, 45.31, 45.87, 46.18, 46.32,
47.72, 47.93, 48.29, 49.50, 52.22, 59.48,
60.31, 65.64, 71.75, 73.47, 83.12, 99.93,
103.38, 125.56, 128.84, 141.10, 141.96
58.91; H, 8.69. Found: C 58.78; H, 8.74
0.80-0.85 (9H) br, 1.21 (78H) br, 13.80, 14.13, 14.62, 19.98, 20.11, 20.31,
Calcd. for C99H175Cl3N6O17S3H2O: C,
16
1.70 (6H) br, 2.15 (9H) s, 3.42-
3.91 (56H) br, 6.95-7.55 (12H)
AA'BB'
20.87, 22.43, 22.75, 23.29, 23.44, 23.57,
23.82, 24.10, 24.22, 24.56, 24.90, 25.23,
25.57, 25.87, 26.02, 26.39, 26.75, 27.12,
27.27, 27.54, 27.80, 28.04, 28.23, 28.58,
28.65, 29.18, 29.70, 30.44, 30.67, 31.32,
31.65, 31.99, 32.27, 32.31, 32.54, 32.86,
33.29, 44.65, 42.71, 43.09, 43.18, 44.67,
45.27, 45.73, 46.03, 46.38, 47.52, 47.99,
48.18, 49.82, 52.34, 59.67, 60.21, 65.48,
71.47, 73.76, 82.66, 99.71, 102.84, 125.73,
129.29, 140.02, 141.80
61.23; H, 9.19. Found: C 61.17; H, 9.28
Several intriguing points are to be noted in these syntheses. First, the isolated products, being
polycationic organic salts, as with the previously noted polycationic organic salts we have investigated
[6-10] are significantly hydroscopic. In all analyses, account must be taken for the formation of solid
materials in which varying amounts of water are agglomerated.
Further, the yields for the syntheses of the model monosaccharide polycationic glycosides are
excellent, a result not unexpected with simple nucleophilic substitution reactions of this type, and in
keeping with our prior observations. However, the yields of isolated polycationic cyclodextrin
derivatives are rather low, and the isolated salts in the direct syntheses bear only halide ion and no
tosylate ion. The absence of tosylate ion was an intriguing observation from the NMR spectra of the
precipitated salts. Further investigation of the solutions from which these salts were isolated in yields
of approximately 30% indicated that the tosylate and tosylate/bromide ion conjugates remain soluble;
the salts precipitating readily under our conditions are those units bearing only bromide ion (we have
also investigated the ion exchange of these materials to generate salts with a single type of anion, but
this does not affect the ultimate results.) Apparently, under the solvent conditions used, the pure

Molecules 2011, 16
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bromide salts precipitate readily while the salts bearing one or more tosylate ions, possibly held within
the cyclodextrin cavity, remain in solution. We have proceeded in the investigations of the
antimicrobial characteristics using the bromide salts.
Antibacterial and antifungal activity of the synthesized salts were measured using the procedure
described in the Experimental section. As a general reference for antibacterial activity, the effect
against S. aureus was noted. As may be noted in Table 3, the MIC for each of the simple
monosaccharide glycosides with S. aureus is relatively high, a consequence of a significant entropy
factor involved with bringing an effective concentration of cationic lipid to impinge on the bacterial
cell wall. However, with the modified cyclodextrins, much lower values for MIC are to be noted, a
consequence of multiple impingement of cationic lipid units (six or seven) with the bacterial cell wall
within a small surface area of that bacterial cell wall.
Table 3. Activity of synthesized materials toward S. aureus and other organisms (MIC; mg/mL).
Compound
S. aureus
>5.8
P. aeruginosa
A. niger
C. albicans
1
-
-
-
2
>5.9
-
-
-
3
>5.9
-
-
-
4
3.11
-
-
-
5
>6.5
-
-
-
6
>6.6
1.99 × 10−1
12.5
-
-
7
-
-
-
8
>7.2
-
4.9 × 10−2
2.4 × 10−2
9
>7.9
-
-
-
10
4.6 × 10−2
2.3 × 10−2
2.3 × 10−2
2.3 × 10−2
2.4 × 10−2
1.3
-
-
-
11
-
-
-
12
3.9 × 10−1
2.0 × 10−1
4.9 × 10−2
13
1.56
4.9 × 10−2
2.4 × 10−2
14
-
-
-
-
-
-
-
-
-
15
16
2.4
modified soluble
starch
1.3 × 10−7
-
-
-
In addition to the Gram-positive bacterium studied, S. aureus, for selected systems activity against a
Gram-negative bacterium, Pseudomonas aeruginosa, and two fungi, Candida albicans and Aspergillus
niger, have been investigated. These results (MIC values) are also provided in Table 3.
The construction of a polycationic glycoside from potato starch (Aldrich, ACS Reagent starch,
soluble, partially hydrolyzed) was performed in the same manner as it was used to accomplish
functionalization of both the monosaccharide derived glycosides and the cyclodextrin derivatives.
With a system such as soluble starch, a long chain bearing regularly spaced pendant alkyl-substituted
DABCO units could be constructed that would have the capability of impinging on a bacterial cell wall
with a very large number of cationic lipids. As noted in Table 3, such a system exhibits a very
significantly lower MIC toward S. aureus (a factor of 5 × 104 more efficient than the cyclodextrin
derivatives) and holds the greatest potential to serve as a potential antimicrobial pharmaceutical agent.

Molecules 2011, 16
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3. Experimental
3.1. General
All chemicals and solvents used in these syntheses and purifications were of commercial reagent
13
quality and were used without further purification. All 1H- and C-NMR spectra were measured with
the samples in commercial deuterated solvents (D2O) using a Brüker 400-MHz DPX400 instrument.
Elemental analyses were performed by Schwarzkopf Microanalytical Services of Woodside, NY, USA.
3.2. Syntheses of cationic lipid appended glycosides
General procedure - monosaccharide glycosides and starch. The appropriate glycoside (approximately
5.0 g) was dissolved in water (100 mL) to which a small excess of NaHCO3 was added (1.1 mol
amount for monosaccharide glycosides, and 1.1 molar amount based on calculation of monomeric D-
glucose units of starch), followed by 1 molar amount of p-TsCl. The mixture was stirred vigorously for
1 h at ambient temperature to allow the formation of the appropriate 6-O-tosyl derivative. After this
time 1 molar amount (based on the tosylation performed) of the selected monoalkylated DABCO
derivative was added, and the reaction mixture was stirred under reflux for 14 h. After cooling, the
solvent was evaporated under reduced pressure leaving a viscous liquid that became solid when
residual solvent was removed under high vacuum. The impure solid was treated with 95% ethanol and
any insoluble materials were removed by suction filtration. The remaining solvent was then evaporated
under reduced pressure and the remainder was dried under high vacuum to provide the target material.
Structures, yields, and analytical data for these materials are presented in Tables 1 and 2.
General procedure - cyclodextrins. The appropriate cyclodextrin hydrate (0.003 mol) was added to a
solution of 10% NaHCO3 in water (50 mL). This was followed by the addition of a solution of
p-toluenesulfonyl chloride (0.025 mol) in 10% aqueous NaHCO3 solution (75 mL). The solution was
allowed to stir at ambient temperature overnight after which the solution was filtered with suction and
the filtrate evaporated under reduced pressure to yield the crude polytosylate as a viscous liquid. The
crude polytosylate was dissolved in acetonitrile (75 mL) and a solution of the appropriate
monoalkyated DABCO reagent (0.025 mol) dissolved in acetonitrile (50 mL), was added slowly with
stirring. The reaction mixture was heated at reflux with stirring for 6 days after which the tan powder
that had formed was collected by suction filtration, washed with portions of ethyl acetate and diethyl
ether, and dried under high vacuum. Structures, yields and analytical data for these materials are
presented in Tables 1 and 2.
3.3. Evaluation of antimicrobial activity of salts
A measured quantity of the salt to be investigated and a stock solution of bacteria (5 µL, ~5 × 104
cells, S. aureus) were added to Luria-Bertani (LB) broth (2 mL). (Corresponding additions were made
for P. aeruginosa and the fungi investigated.) The growth medium was incubated overnight at 37 °C in
a shaking water bath. The absorbance (abs, indicating turbidity of the medium) at 600 nm was
recorded. The results were noted as percent growth:

Molecules 2011, 16
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(abs of sample/abs of blank) × 100.
The results are reported in Table 3. Minimum inhibitory concentrations (MIC) were determined by
preparing serial dilutions of each salt and adding the same amount of bacteria to each dilution.
dilutions were incubated overnight at 37 °C in a shaking water bath and growth was observed in
comparison to a blank.
4. Conclusions
Cationic lipid units attached through the 6-position of monosaccharide glycosides exhibit a
moderate antibacterial effect. While relatively low in activity, these species are nonetheless more
active toward bacteria than the simple alkyl-substituted cationic lipids.
The concept of grouping several cationic lipid sites within a single molecule to increase the
antibacterial efficacy of the material has been borne out in this investigation. The use of cyclodextrin
scaffoldings through which six or seven cationic lipids are organized to impinge on a bacterial cell
wall simultaneously within a small region of surface has been found to be an effective approach to
increasing antibacterial activity for the cationic lipid species. The glycosidic linkages of the
cyclodextrin derivatives provides the capability for hydrolysis of the unit to individual fragments that
can then be disposed of by a host organism. This structural characteristic of the cyclodextrins enables
one to overcome the deleterious entropy factor involved in organizing cationic lipids for destruction of
a bacterial cell wall, and provides an effective approach for the design of non-antibiotic
antibacterial agents.
Further, it is of particular note that the use of soluble starch modified by the incorporation of
polycationic lipid units along the entire chain length provides a significantly greater antibacterial effect
even compared to the cyclodextrin derivatives. The impingement on the bacterial cell wall by such a
material will provide an extended swath of disruption to that cell wall sufficient to produce
cytotoxicity at a very low concentration.
As noted, the goal of the current effort was the determination of the potential of simple, readily
available glycosidic materials to be modified to serve as antibacterial agents, potentially for in vivo
applications. The presented results demonstrate this potential to be real. Simple glycosides, readily
available in sizable quantity, are capable of being modified by the rapid chemical attachment of
cationic lipid adjuncts to serve the desired purpose. Most significantly, the use of a simple starch as a
scaffolding for the cationic lipid adjuncts provides a significantly high efficacy as an antibacterial
agent that warrants continuing investigation toward serving this purpose. Our current goals are the
optimization of the starch/adjunct structure for this end, along with determination of the conditions and
rates for in vitro, and ultimately in vivo degradation of the agent.
Acknowledgements
We are grateful to the Magnet Program at the CUNY Graduate Center and the NYC LSAMP for
financial support to one of us (M.T.), as well as to the Howard Hughes Medical Institute for financial
support of the pre-college research experience. The Pace University Research Award (J.R.) and the
PSC-CUNY Research Award program are thanked for general financial support of this effort.

Molecules 2011, 16
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Sample Availability: Samples of the compounds are available from the corresponding author.
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