Glyconanobiotics: Novel carbohydrated nanoparticle antibiotics for MRSA and Bacillus anthracis

Article abstract of DOI:10.1016/j.bmc.2007.11.052

This report describes the synthesis and evaluation of glycosylated polyacrylate nanoparticles that have covalently-bound antibiotics within their framework. The requisite glycosylated drug monomers were prepared from one of three known antibiotics, an N-sec-butylthio β-lactam, ciprofloxacin, and a penicillin, by acylation with 3-O-acryloyl-1,2-O-isopropylidene-5,6 bis((chlorosuccinyl)oxy)-d-glucofuranose (7) or 6-O-acetyl-3-O-acryloyl-1,2-O-isopropylidene-5-(chlorosuccinyl)oxy-α-d-glucofuranose (10). These acrylated monomers were subjected to emulsion polymerization in a 7:3 (w:w) mixture of butyl acrylate-styrene in the presence of sodium dodecyl sulfate as surfactant (3 weight %) and potassium persulfate as a radical initiator (1 weight %). The resulting nanoparticle emulsions were characterized by dynamic light scattering and found to have similar diameters (～40 nm) and size distributions to those of our previously studied systems. Microbiological testing showed that the N-sec-butylthio β-lactam and ciprofloxacin nanoparticles both have powerful in vitro activities against methicillin-resistant Staphylococcus aureus and Bacillus anthracis, while the penicillin-bound nanoparticles have no antimicrobial activity. This indicates the need for matching a suitable antibiotic with the nanoparticle carrier. Overall, the study shows that even relatively large, polar acrylate monomers (MW > 1000 amu) can be efficiently incorporated into the nanoparticle matrix by emulsion polymerization, providing opportunities for further advances in nanomedicine.

Full text of DOI:10.1016/j.bmc.2007.11.052

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2412–2418
Glyconanobiotics: Novel carbohydrated nanoparticle
antibiotics for MRSA and Bacillus anthracis
Sampath C. Abeylath,a Edward Turos,a,* Sonja Dickeyb and Daniel V. Limb
aCenter for Molecular Diversity in Drug Design, Discovery, and Delivery, Department of Chemistry,
CHE 205, 4202 East Fowler Avenue, University of South Florida, Tampa, FL 33620, USA
bDepartment of Biology, 4202 East Fowler Avenue, University of South Florida, Tampa, FL 33620, USA
Received 20 October 2007; revised 21 November 2007; accepted 21 November 2007
Available online 28 November 2007
Abstract—This report describes the synthesis and evaluation of glycosylated polyacrylate nanoparticles that have covalently-bound
antibiotics within their framework. The requisite glycosylated drug monomers were prepared from one of three known antibiotics,
an N-sec-butylthio b-lactam, ciprofloxacin, and a penicillin, by acylation with 3-O-acryloyl-1,2-O-isopropylidene-5,6 bis((chloro-
succinyl)oxy)-D-glucofuranose (7) or 6-O-acetyl-3-O-acryloyl-1,2-O-isopropylidene-5-(chlorosuccinyl)oxy-a-D-glucofuranose (10).
These acrylated monomers were subjected to emulsion polymerization in a 7:3 (w:w) mixture of butyl acrylate–styrene in the pres-
ence of sodium dodecyl sulfate as surfactant (3 weight %) and potassium persulfate as a radical initiator (1 weight %). The resulting
nanoparticle emulsions were characterized by dynamic light scattering and found to have similar diameters (ꢀ40 nm) and size dis-
tributions to those of our previously studied systems. Microbiological testing showed that the N-sec-butylthio b-lactam and cipro-
floxacin nanoparticles both have powerful in vitro activities against methicillin-resistant Staphylococcus aureus and Bacillus
anthracis, while the penicillin-bound nanoparticles have no antimicrobial activity. This indicates the need for matching a suitable
antibiotic with the nanoparticle carrier. Overall, the study shows that even relatively large, polar acrylate monomers
(MW > 1000 amu) can be efficiently incorporated into the nanoparticle matrix by emulsion polymerization, providing opportunities
for further advances in nanomedicine.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
These nanoparticles can be prepared easily in water by
radical-induced emulsion polymerization using an acry-
lated derivative of the antibiotic as a co-monomer in a
warmed mixture of butyl acrylate and styrene. Sodium
dodecyl sulfate (3 weight %) and sodium persulfate (1
weight %) are used as surfactant and radical initiator,
respectively. The resulting emulsions are stable indefi-
nitely up to at least 80 ꢁC. Also by extending this emulsion
polymerization procedure, we established that penicillin-
containing polyacrylate nanoparticles can be easily pre-
pared and are effective at rejuvenating the in vitro antibac-
terial activity of penicillin drugs against b-lactamase-
producing microbes such as methicillin-resistant Staphy-
lococcus aureus (MRSA).4 More recently, we described
the application of this polymerization methodology to
the synthesis of carbohydrated poly(ethyl acrylate) nano-
particles which contain a variety of O-protected and even
unprotected monosaccharides within the polymeric
framework.5 These carbohydrated nanoparticles appear
to be the first examples of polymeric glyconanoparticles
reported in the literature, and required that relatively
bulky, water-soluble glycosylated acrylates be used as
monomers in the emulsion polymerization process.
Recent advances in biotechnology have accelerated the
discovery of new drug molecules. However, many of
these compounds suffer from sensitivity to degradative
processes in the blood, or have limited water solubility
that provides for minimal dissolution in the gastrointes-
tinal tract and low bioavailability. There has been a re-
newed effort to find strategies for protecting and
delivering such drugs, including the use of liposomes
and various nanoparticles. These drug delivery vehicles
improve bioavailability, efficacy, and specificity of some
pharmaceutical compounds.1,2 Recently, our laboratory
has investigated the properties of polyacrylate emulsions
that contain nanoparticles measuring 30–50 nm in diam-
eter, and onto which an antibiotic molecule can be cova-
lently attached.3
Keywords: Carbohydrates; Emulsion polymerization; Nanobiotics;
Nanoparticle; MRSA; Anthrax; Drug delivery.
*
Corresponding author. Tel.: +1 813 974 7312; fax: +1 813 974
1733; e-mail: eturos@cas.usf.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.052

S. C. Abeylath et al. / Bioorg. Med. Chem. 16 (2008) 2412–2418
2413
Glycosylated nanoparticles have been employed to study
carbohydrate interactions in the field of chemical glycobi-
ology, but their potential in drug delivery is relatively
unexplored particularly in the context of antibacterials re-
search.6 Consequently, a number of issues regarding this
capability need to be better investigated, and the first
thing we wanted to explore was whether the glycosylated
nanoparticles could be made to have antibacterial activ-
ity. Thus, the goal of the present work was to adapt our
previous studies on antibiotic-conjugated nanoparticles
and carbohydrated nanoparticles to construct an amal-
gamated system we refer to as glyconanoparticle antibiot-
ics, or glyconanobiotics for short, and to evaluate their
in vitro antibacterial capabilities against two dangerous
pathogenic microbes, MRSA and B. anthracis. A graphic
of what this nanoparticle is comprised of is depicted in
Scheme 1. Secondly, we also were interested in whether
the relatively large monomeric acrylates required for the
formation of these nanoparticles could even be success-
fully employed, since this would then signal the potential
use of other surrogates needed for biomedical applica-
tions. At the outset of this study, it was not obvious that
this could in fact be achieved, since until now only small
(MW < 450) lipophilic monomers have been successfully
introduced as polyacrylates by emulsion polymerization.
Therefore, our study is focused on three representative
antibiotic agents having entirely different biological
modes of action, an N-thiolated b-lactam, a fluoroquino-
lone, and a penicillin. To prepare the nanoparticles (des-
ignated NP1–3), it was necessary to first convert each of
the antibiotics to their glycosylated monomers 1–3, where
the critical acrylate moiety is located at the 30 center of the
furanose ring (Fig. 1). Acrylate monomer 1 contains two
appended N-thiolated b-lactams, while monomers 2 and 3
Antibiotic
O
O
Antibiotic
O
Antibiotic
O
O
Antibiotic
O
O
O
O
glycosylated antibiotic acrylate
O
O
+
O
O
O
Antibiotic
emulsion polymerization
O
O
Antibiotic
O
O
water, 70 o
C
O
O
O
O
+
O
O
O
O
O
Antibiotic
Antibiotic
O
O
Antibiotic
Scheme 1. Synthesis of glyconanobiotics by emulsion polymerization.
S
O
N
O
O
N
O
O
O
HOOC
F
Cl
O
O
O
N
O
Cl
O
O
O
N
O
O
O
O
O
O
O
O
N
O
O
O
S
O
O
2
1
O
O
O
O
O
H
O
N
S
O
O
N
O
O
HOOC
O
3
Figure 1. Glycosylated antibiotic acrylates used for the formation of glyconanobiotics: bis-N-sec-butylthio b-lactam acrylate (1), ciprofloxacin
acrylate (2), 6-aminopenicillanic acid acrylate (3).

2414
S. C. Abeylath et al. / Bioorg. Med. Chem. 16 (2008) 2412–2418
bear a single ciprofloxacin or 6-aminopencillinanic acid
moiety, respectively. The latter acrylate was used as a con-
trol, since our prior investigation on penicillin-bound
nanoparticles identified it as being devoid of antibacterial
activity against MRSA, with a broth minimum inhibitory
concentration of >256 lg/mL.4
tected glucofuranose 4 as shown in Scheme 2.
Subsequently, the antibiotics were attached by direct
acylation according to the procedures described below.
2.1. Synthesis of acyl chlorides 7 and 10
3-O-acryloyl-1,2-O-isopropylidene-5,6-bis((chloro-succi-
nyl)oxy)-D-glucofuranose (7) and 6-O-acetyl-3-O-acry-
loyl-1,2-O-isopropylidene-5-(chlorosuccinyl)oxy-a-D-gluco-
furanose (10) were both synthesized from 1,2:5,6-di-
O-isopropylidine-a-D-glucofuranose-3-acrylate (4) as
shown in Scheme 2. Partial deacetonation of 4 with
2. Results and discussion
To construct acrylates 1–3, it was first necessary to syn-
thesize the two acyl chlorides 7 and 10 from the pro-
O
O
HO
HO
O
O
O
O
O
80% CH3COOH
80o C, 4 hrs
O
Acetyl Chloride
O
HO
O
O
O
O
O
O
Collidine, CH2Cl2, -40o C
O
O
O
O
4
5
8
O
O
Et3N, CH2Cl2
Et3N, CH2Cl2
O
O
O
O
O
OH
O
O
O
O
O
O
O
O
O
O
HO
O
HO
O
O
O
O
O
O
O
O
O
6
9
SOCl2
SOCl2
O
O
Cl
O
O
O
O
O
O
O
O
O
Cl
O
Cl
O
O
O
O
O
O
O
O
O
10
7
Scheme 2. Synthesis of 3-O-acryloyl-1,2-O-isopropylidene-5,6 bis((chlorosuccinyl)oxy)-D-glucofuranose (7) and 6-O-acetyl-3-O-acryloyl-1,2-O-
isopropylidene-5-(chlorosuccinyl)oxy-a-D-glucofuranose (10).

S. C. Abeylath et al. / Bioorg. Med. Chem. 16 (2008) 2412–2418
2415
80% acetic acid at 80 ꢁC afforded product 5 in 62% iso-
O
N
HOOC
F
lated yield. Diol 5 was diacylated with succinic anhy-
dride in the presence of Et3N in CH2Cl2 to give
di-acid 6 in 65% yield. Thionyl chloride was used to con-
vert compound 6 to the corresponding di-acyl chloride 7
in 98% yield. Acyl chloride 10 was obtained similarly by
selective acylation7 of the primary hydroxyl group of
diol 5 with acetyl chloride in dichloromethane in the
presence of 2,4,6-collidine at ꢁ40 ꢁC to afford com-
pound 8 in 72% yield. Following succinic acid acylation
of alcohol 8 to afford carboxylic acid 9, conversion to
the mono-acyl chloride 10 with thionyl chloride pro-
ceeded in 65% yield for the two steps. A significant fea-
ture revealed in this synthesis is the stability of the endo-
cyclic acetonide of the monosaccharide during the reac-
tion of thionyl chloride (generation of HCl).
N
NH
12
10
Et3N, CH2Cl2
O
N
O
O
HOOC
F
O
N
O
O
N
O
2.2. Synthesis of antibiotic-conjugated acrylates 1–3
O
O
O
O
With the two key acyl chlorides in hand, we evaluated
their use for acylating the three selected antibiotic drugs
to prepare monomers 1–3 (Schemes 3–5). N-thiolated b-
lactams discovered in our group are a new family of
antibacterial agents active against Staphylococcus bacte-
ria, including methicillin-resistant strains of Staphylo-
coccus aureus (MRSA),8,9 and Bacillus anthracis10
which is the causative agent of anthrax infections. For
the goal of preparing glyconanoparticle variants of this
class of antibacterial agents, bis-N-thiolated b-lactam
glucose acrylate 1 was prepared. Monomer 1 was syn-
thesized in 62% yield from N-sec-butylthio b-lactam
derivative 1111 by coupling with di-acyl chloride 7 and
Et3N in CH2Cl2 (Scheme 3). As it was expected that sim-
ilar bis-ciprofloxacin and bis-penicillin glucose acrylates
would be less lipophilic due to the presence of their car-
2
(MW 730 amu)
Scheme 4. Synthesis of ciprofloxacin acrylate (2).
boxylic acid moieties, which in turn could cause prob-
lems in the emulsion polymerization due to poor
solubility in the butyl acrylate–styrene mixture, we
decided to instead try to prepare the mono-antibiotic
NH2
S
N
O
HOOC
13
N,O-Bis(trimethylsilyl)
acetamide, CH2Cl2
O
S
HO
Cl
N
NH2
S
N
11
O
Me3SiO2C
14
7
Et3N, CH2Cl2
10
Et3N, CH2Cl2
S
O
N
O
O
O
O
Cl
O
O
O
Cl
O
O
H
O
O
O
N
O
O
O
S
O
N
O
O
O
O
N
O
S
O
O
O
HOOC
O
1
3
(MW 1010 amu)
(MW 651 amu)
Scheme 3. Synthesis of bis-N-sec-butylthio b-lactam acrylate (1).
Scheme 5. Synthesis of 6-aminopenicillinic acid acrylate (3).

2416
S. C. Abeylath et al. / Bioorg. Med. Chem. 16 (2008) 2412–2418
acrylate forms. Mono-ciprofloxacin acrylate 2 was suc-
cessfully synthesized in 56% yield from ciprofloxacin
by coupling with acyl chloride 10 and Et3N in CH2Cl2
(Scheme 4), whereas the mono-penicillin acrylate 3 was
synthesized4 in 51% yield from 6-aminopenicillanic acid
(13) via its trimethylsilyl ester 14, which was then re-
acted with acyl chloride 10 (Scheme 5). The structures
of the carbohydrated monomers 1 and 2 were deter-
icillin-bound glyconanoparticles and non-drug contain-
ing nanoparticles made of just butyl acrylate–styrene
(7:3) (controls) were completely inactive against these
microbes, with MIC values of >256 lg/mL. The bioac-
tivities are similar to or better than that of the corre-
sponding drug monomers. The finding that the
N-thiolated b-lactam and cipro nanoparticles are active,
while the penicillin variant is not, suggests that the type
of antibiotic and the comparative activity of the free
drug being released (presumably by ester or amide cleav-
age) are both critical. Our laboratories’ attempts to mea-
sure the rate of drug release from the nanoparticles have
not yet been successful, and are ongoing, as are experi-
ments to identify the mechanism of how these nanopar-
ticle antibiotics may exert their effects on bacterial cells.
1
13
mined by H and C NMR, and mass spectrometry.12
2.3. Preparation of glyconanobiotics
The carbohydrate-conjugated antibiotic monomers (1–
3) prepared from N-sec-butylthio b-lactam, ciprofloxa-
cin, and 6-aminopenicillanic acid were found to be read-
ily soluble in a warm 7:3 (w:w) mixture of butyl acrylate
and styrene, and undergo emulsion polymerization with
ease using the prescribed protocol. In this way, the
nanoparticles bearing one of the three antibiotics could
be prepared by emulsion polymerization of antibiotic
monomers 1–3 (2 weight %) in a 7:3 (w:w) blend of butyl
acrylate–styrene, upon stirring in water for 6 h at 70 ꢁC.
Sodium dodecyl sulfate (3 weight %) and potassium per-
sulfate (1 weight %) were used as surfactant and radical
initiator, respectively. Dynamic light scattering (DLS)
analysis of the final emulsion revealed that the average
diameter of the glyconanoparticles is uniformly about
40 nm with narrow size distributions spanning only
from about 30–50 nm, as has been previously reported
for similar polyacrylate nanoparticles.4
3. Experimental
Solvents and chemicals were purchased from Acros
Organics and used without further purification.1,2:5,6-
Di-O-isopropylidine-a-D-glucofuranose-3-acrylate (4)13
was prepared following the cited literature procedure.
1H NMR and 13C NMR spectra were recorded on Bru-
ker DPX-250 and Varian Inova 400 spectrometers.
Mass spectra were obtained with an ESI LC MS system
(Agilent Technologies).
3.1. Synthesis
3.1.1. Synthesis of 3-O-acryloyl-1,2-O-isopropylidene-5,6
bis((chlorosuccinyl)oxy)-D-glucofuranose (7). 1,2:5,6-Di-
O-isopropylidine-a-D-glucofuranose-3-acrylate (4) (0.5 g,
1.59 mmol) was dissolved in 4 mL of 80% acetic acid
and stirred 4 h at 80 ꢁC. The solvents were removed in
vacuo and the residue was purified by column chroma-
tography (silica gel, hexane-EtOAc, 1:1) to afford com-
pound 5 (0.32 g, 62%) as a syrup. This monoacetonide
product (0.3 g, 1.09 mmol) and succinic anhydride
(0.65 g, 6.54 mmol) were stirred with Et3N (0.76 mL,
5.45 mmol) in CH2Cl2 (10 mL) at rt for 5 h. The reaction
mixture was washed with 5% KHSO4, then water. The
organic layer was dried over Na2SO4, filtered, and evap-
orated to dryness. The brown oil obtained was chro-
matographed on silica gel (EtOAc) and a solid
material was obtained that contained ammonium salt
and succinic acid, which could easily be removed by cen-
trifugation in 10 mL of CH2Cl2. After evaporation,
2.4. Biological activity
The glycosylated nanoparticle antibiotics NP1–3 were
evaluated for in vitro antibacterial activity against three
pathogenic bacteria, namely Staphylococcus aureus, a
strain of methicillin-resistant Staphylococcus aureus
(MRSA), and Bacillus anthracis. Additionally, the acry-
lated monomers 1–3 were also examined for compari-
son. Activity was assessed by determining the
minimum inhibitory concentration (MIC) values by
broth serial dilution, and is summarized in Table 1.
The concentration of antibiotic in the nanoparticle was
assumed to be equivalent to that used for the polymeri-
zation, as expressed in lg of drug per mL of undiluted
emulsion. The results show that the glyconanoparticles
containing N-thiolated b-lactam and ciprofloxacin
exhibited significant antibacterial activity, while the pen-
Table 1. Antibacterial activity of glycosylated monomers 1–3 and their nanoparticle emulsions NP1–3 against Staphylococcus aureus (ATCC 25923),
MRSA (ATCC 43300) and Bacillus anthracis, as determined by broth dilution MIC assaysa
Sample
Microbe
S. aureus (lg/mL)
MRSA (lg/mL)
B. anthracis (lg/mL)
Penicillin G (control)
Lactam monomer 1
Lactam NP1
0.012
128
64
16
n/a
64
128
64
64
16
Cipro monomer 2
Cipro NP 2
32
32
32
32
16
>256
>256
Penicillin monomer 3
Penicillin NP 3
>256
>256
>256
>256
a MIC values refer to the lowest concentration of the bound drug (lg/mL) required to completely inhibit bacterial growth for 24 h in culture.

S. C. Abeylath et al. / Bioorg. Med. Chem. 16 (2008) 2412–2418
2417
compound 6 (0.34 g, 65%) was obtained as a colorless
syrup. This di-acid (0.3 g, 0.80 mmol) was dissolved in
3 mL of thionyl chloride, stirred for 45 min. at room
temperature, and the solvents were removed in vacuo
to afford compound 7 (0.31 g, 98%) as a syrup. 1H
NMR (400 MHz, CDCl3): d 6.39 (m, 1H), 6.04 (m,
1H), 5.87 (m, 2H), 5.28 (m, 2H), 4.50 (m, 3H), 4.17
(m, 1H), 3.13 (m, 4H), 2.62 (m, 4H) 1.48 (s, 3H), 1.27
(s, 3H); 13C NMR (100 MHz, CDCl3): d 173.2, 172.9,
171.0, 170.1, 169.8, 165.7, 164.7, 132.5, 127.5, 112.8,
105.3, 83.3, 76.6, 75.2, 68.5, 63.8, 41.8, 29.3, 28.6, 26.9.
(m, 9H), 0.96 (m, 6H); 13C NMR (100 MHz, CDCl3): d
171.2, 170.3, 169.8, 169.0, 164.7, 134.6, 123.6, 130.7,
130.1, 130.0, 128.9, 127.3, 126.9, 112.7, 105.3, 83.3, 75.3,
68.0, 63.7, 63.6, 48.6, 28.5, 28.3, 26.9, 26.4, 19.2, 18.9,
11.3. ESI-MS calcd for [M+NH4]+, 1028.0; Found:
1028.3.
3.1.4. Synthesis of ciprofloxacin acrylate (2). To a stirred
solution of ciprofloxacin hydrochloride (0.2 g, 0.6 mmol)
and triethylamine (0.5 mL, 3.6 mmol) in CH2Cl2 (8 mL)
at 0 ꢁC was added dropwise acyl chloride 10 (0.31 g,
0.66 mmol in 0.5 mL of CH2Cl2). The solution was stir-
red at rt for 10 h, then treated with water (10 mL), and
extracted with CH2Cl2 (3 · 15 mL). The combined organ-
ic layers were washed with water (2· 50 mL), dried
(MgSO4), and evaporated under reduced pressure. The
residue was purified by column chromatography (EtOAc)
to afford compound 2 (0.23 g, 56%) as a syrup. 1H NMR
3.1.2. Synthesis of 6-O-acetyl-3-O-acryloyl-1,2-O-isopro-
-pylidene-5-(chlorosuccinyl)oxy-a-D-glucofuranose (10).
3-O-acryloyl-1,2-O-isopropylidene-a-D-glucofuranoside
(5) (0.4 g, 1.52 mmol) was dissolved in 10 mL of CH2Cl2
and cooled to ꢁ40 ꢁC. Collidine (0.36 g, 3.04 mmol) was
added to the solution, then acetyl chloride (0.14 g,
1.82 mmol), and the mixture was stirred at ꢁ40 ꢁC for
3 h and at rt for an additional hour. The mixture was
washed with water and extracted into CH2Cl2
(3 · 25 mL). The solvents were evaporated and the residue
was purified by column chromatography to afford com-
pound 8 (0.33 g, 72 %) as a syrup. This intermediate prod-
uct (0.3 g, 0.98 mmol) and succinic anhydride (0.29 g,
2.96 mmol) were stirred with Et3N (0.34 mL, 2.45 mmol)
in CH2Cl2 (10 mL) at rt for 5 h. The reaction mixture
waswashedwith5%KHSO4, thenwater.Theorganiclayer
was dried over Na2SO4, filtered, and evaporated to dry-
ness. The brown oil obtained was chromatographed on sil-
ica gel (EtOAc), and a solid material was obtained that
contained ammonium salt and succinic acid, which could
easily be removed by centrifugation in 10 mL of CH2Cl2.
Afterevaporation, compound9(0.26 g,65%)wasobtained
asa syrup. Subsequently, this acid was dissolvedin 3 mLof
thionyl chloride, stirred 45 min. at room temperature, and
the solvents were removed in vacuo to afford compound 10
(0.34 g, 98%) as a syrup. 1H NMR (400 MHz, CDCl3): d
6.35 (d, 1H, J = 17.2 Hz), 6.04 (dd, 1H, J = 10.4, 17.2
Hz), 5.87 (m, 2H), 5.33 (d, 1H, J = 2.8 Hz), 5.17 (m, 1H),
4.52 (m, 3H), 4.08 (dd, 1H, J = 5.6, 12.4 Hz), 3.10 (m,
3H), 2.57 (m, 3H), 2.00 (s, 3H), 1.49 (s, 3H), 1.27 (s, 3H);
13C NMR (100 MHz, CDCl3): d 172.8, 170.8, 169.8,
164.8, 132.7, 127.3, 112.7, 105.3, 83.3, 76.8, 75.2, 68.6,
63.2, 41.7, 29.4, 28.6, 26.3, 20.8.
(400 MHz, CDCl3):
d 8.64(s, 1H), 7.90 (d, 1H,
J = 12.4 Hz), 7.32 (d, 1H, J = 6.8 Hz), 6.39 (d, 1H,
J = 17.2 Hz), 6.05 (dd, 1H, J = 10.4, 17.2 Hz), 5.88 (d,
1H, J = 3.6 Hz), 5.86 (d, 1H, J = 10.4 Hz), 5.32 (d, 1H,
J = 2.8 Hz), 5.24(m, 1H), 4.52(m, 3H), 4.12 (dd, 1H,
J = 5.2, 12.0 Hz), 3.79(m, 2H), 3.68(m, 2H), 3.53(m,
1H), 3.33(m, 2H), 3.25(m, 2H), 2.59(m, 4H), 2.02(s,
3H), 1.49(s, 3H), 1.37(m, 2H), 1.27(s, 3H), 1.18(m, 2H);
13C NMR (100 MHz, CDCl3): d 177.1, 171.8, 170.8,
169.6, 166.9, 164.8, 155.0, 152.5, 147.6, 145.6, 145.5,
139.1, 132.3, 127.7, 120.2, 112.7, 112.4, 108.2, 105.3,
105.2, 83.3, 75.5, 68.0, 63.3, 53.6, 50.2, 49.5, 45.2, 41.5,
35.5, 29.4, 28.1, 26.9, 26.4, 20.9, 8.4. ESI-MS calcd for
[M+Na]+, 752.6; Found: 752.2.
3.1.5. Synthesis of 6-aminopenicillanic acid acrylate (3).12
6-Aminopenicillanic acid (2 g, 0.924 mmol) and N,O-
bis(trimethylsilyl)acetamide (0.21 g, 1.02 mmol) were
added to CH2Cl2 (10 mL) and stirred at rt overnight.
Triethylamine (0.19 mL, 1.38 mmol) was added and
cooled on ice. Acyl chloride 10 (0.52 g, 1.11 mmol in
0.5 mL of CH2Cl2) was added dropwise and stirred at
room temperature for 10 h. The reaction mixture was
treated with water (10 mL) and extracted into CH2Cl2
(3 · 15 mL). The combined organic layers were washed
with water (2 · 50 mL), dried (MgSO4), and evaporated
under reduced pressure. The residue was purified by col-
umn chromatography (EtOAc) to afford compound 312
1
3.1.3. Synthesis of bis-N-sec-butylthio b-lactam acrylate
(1). To a stirred solution of N-sec-butylthio b-lactam
derivative 1111 (0.2 g, 0.7 mmol) and triethylamine
(0.29 mL, 2.1 mmol) in CH2Cl2 (5 mL) at 0 ꢁC was added
dropwise diacyl chloride 7 (0.173 g, 0.42 mmol in 0.5 mL
of CH2Cl2). The solution was stirred at rt for 10 h, then
treated with water (10 mL), and extracted with CH2Cl2
(3 · 15 mL). The combined organic layers were washed
with water (2 · 50 mL), dried (MgSO4), and evaporated
under reduced pressure. The residue was purified by col-
umn chromatography (hexane-EtOAc, 2:1) to afford
compound 1 (0.22 g, 62%) as a syrup. 1H NMR
(400 MHz, CDCl3): d 7.35 (m, 8H), 6.35 (d, 1H,
J = 17.2 Hz), 6.14 (m, 2H), 6.03 (dd, 1H, J = 10.8,
17.6 Hz), 5.84 (m, 2H), 5.47 (m, 2H), 5.25 (s, 1H), 5.09
(m, 1H), 4.45 (m, 2H), 4.33 (m, 1H), 4.05 (m, 1H), 3.04
(m, 2H), 2.17 (m, 8H), 1.56 (m, 4H), 1.47 (s, 3H), 1.26
(0.23 g, 56%) as a syrup. H NMR (400 MHz, CDCl3):
d 6.55 (d, 1H, J = 8.8 Hz), 6.39 (d, 1H, J = 17.2 Hz),
6.02 (m, 1H), 5.88 (m, 2H), 5.63 (dd, 1H, J = 4.0,
8.8 Hz), 5.48 (d, 1H, J = 4.0 Hz), 5.31 (m, 1H), 5.19 (m,
1H), 4.48 (m, 3H), 4.04 (m, 1H), 2.55 (m, 4H), 2.01 (s,
3H), 1.62 (s, 3H), 1.54 (s, 3H), 1.48 (s, 3H), 1.27 (s, 3H).
3.2. Emulsion polymerization
The general procedure is as previously reported.3,4 The
drug monomer (2 weight %) was pre-dissolved in a 7:3
(w:w) mixture of butyl acrylate–styrene, and sodium
dodecyl sulfate in purified water was added to pre-emul-
sify the mixture with stirring under a nitrogen atmo-
sphere. The amounts of each component added to the
mixture are given in Table 2. The polymerized nanopar-
ticles were prepared by adding potassium persulfate to a

2418
S. C. Abeylath et al. / Bioorg. Med. Chem. 16 (2008) 2412–2418
antibiotic effects of these nanoparticles, including how,
where, and at what rate the drug is being released, and
their in vivo properties as potential treatment of life-
threatening bacterial infections.
Table 2. Formulation parameters used for emulsion polymerization
Components
Amounts
Solid content (%)
Drug monomer
Butyl acrylate
Styrene
20 mg
658 mg
282 mg
30 mg
10 mg
4 mL
2
66
28
3
Sodium dodecyl sulfate
Potassium persulfate
Water
Acknowledgments
1
We sincerely thank Gil Brubaker (University of Florida
Particle Engineering Research Center) for providing
assistance with the particle analyses, and generous finan-
cial support from the National Institutes of Health
(Grant AI05135) and National Science Foundation
(NSF 0620572).
rapidly stirred (magnetic stirrer) mixture at 70 ꢁC for
6 h, at which time heating and stirring were stopped
and the sample could be transferred to a vial for storage
or biological testing.
3.3. Dynamic light scattering analysis
Sample size and distribution of the emulsions were exam-
ined by dynamic light scattering using a UPA 150 Honey-
well MicroTrac DLS instrument equipped with a laser
beam at 780 nm. The sample was pre-diluted with de-ion-
ized distilled water (0.05 mL of emulsion in 9.95 mL of
water, polymer concentration 0.1% wt) and sonicated
for 3 min for the analysis. The value is expressed in
weight-averaged scales as unimode at a scattering angle
of 180ꢁ (backscatter) at a temperature of 25 ꢁC.
References and notes
1. Alle’mann, E.; Gurny, R.; Doelker, E. Eur. J. Pharm.
Biopharm. 1993, 39, 173–191.
2. Couvreur, P.; Dubernet, C.; Puisieux, F. Eur. J. Pharm.
Biopharm. 1995, 41, 2–13.
3. Turos, E.; Shim, Y. J.; Wang, Y.; Greenhalgh, K.; Reddy,
G. S. K.; Dickey, S.; Lim, D. V. Bioorg. Med. Chem. Lett.
2007, 17, 53–56.
4. Turos, E.; Reddy, G. S. K.; Greenhalgh, K.; Ramaraju,
P.; Abeylath, S. C.; Jang, S.; Dickey, S.; Lim, D. V.
Bioorg. Med. Chem. Lett. 2007, 17, 3468–3472.
5. Abeylath, S. C.; Turos, E. Carbohydr. Polymer 2007, 70,
32–37.
4. Conclusions
´
6. Fuente, J. M.; Penades, S. Biochim. Biophys. Acta 2006,
1760, 636–651.
7. Ishihara, K.; Kurihara, H.; Yamamoto, H. J. Org. Chem.
1993, 58, 3791–3793.
In summary, these studies have demonstrated the prep-
aration and in vitro evaluation of polyacrylate glyco-
nanoparticles that bear
a
covalently-attached
antibiotic. With this methodology, even large monomers
(MW > 1000 amu) can be efficiently incorporated into
the polyacrylate nanoparticle matrix up to 2 weight %
of the total solid content. The antibiotic-conjugated
acrylate monomers needed for the polymerization are
readily prepared by a simple one-step procedure, from
acylation of the antibiotic with the corresponding carbo-
hydrated acyl chloride. These structurally-complex acry-
late drug monomers show excellent solubility in the
butyl acrylate–styrene mixture required for radical-in-
duced emulsion polymerization in aqueous media. The
resulting nanoparticles measure around 40 nm in diam-
eter according to dynamic light scattering experiments,
with narrow size distributions (30–50 nm), and possess
equivalent or improved in vitro antibacterial activity
to that of the corresponding acrylated antibiotic mono-
mer. Further investigations in our laboratory are
attempting to fully assess the underlying reasons for
8. Turos, E.; Konaklieva, M. I.; Ren, R. X. F.; Shi, H.;
Gonzalez, J.; Dickey, S.; Lim, D. Tetrahedron 2002, 56,
5571–5578.
9. Turos, E.; Long, T. E.; Konaklieva, M. I.; Coates, C.;
Shim, J. Y.; Dickey, S.; Lim, D. V.; Cannons, A. Bioorg.
Med. Chem. Lett. 2002, 12, 2229–2231.
10. Turos, E.; Long, T. E.; Heldreth, B.; Leslie, J. M.; Reddy,
S. K.; Wang, Y.; Coates, C.; Konaklieva, M.; Dickey, S.;
Lim, D. V.; Alonso, E.; Gonzalez, J. Bioorg. Med. Chem.
Lett. 2006, 16, 2084–2090.
11. Heldreth, B.; Long, T. E.; Jang, S.; Reddy, S. K.; Turos,
E.; Dickey, S.; Lim, D. V. Bioorg. Med. Chem. 2006, 14,
3773–3784.
12. A small amount of carboxylic acid 9 mixed with penicillin
acrylate 3 showed up in the 1H NMR spectrum after
eluting with ethyl acetate, and this mixture was used for
the emulsion polymerization as a mixture.
13. Krishna, P. R.; Kannan, V.; Ilangovan, A.; Sharma, G. V.
M. Tetrahedron: Asymmetry 2001, 12, 829–837.