Synthesis and antibacterial properties of carbohydrate-templated lysine surfactants

Article abstract of DOI:10.1016/j.carres.2011.01.025

The synthesis of four dicationic glucose-templated d-lysine-derived surfactants and their two unmodified d-lysine-analogs is described. Replacement of d-lysine by d-glucose-templated-d-lysine provides a general tool to introduce chemical diversity into th

Full text of DOI:10.1016/j.carres.2011.01.025

Carbohydrate Research 346 (2011) 588–594
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and antibacterial properties of carbohydrate-templated
lysine surfactants
Dhananjoy Mondal a, George G. Zhanel b, Frank Schweizer a,b,
⇑
a Department of Chemistry, University of Manitoba, R3T 2N2 Winnipeg, Manitoba, Canada
b Department of Medical Microbiology, University of Manitoba, R3E 3P4, Winnipeg, Manitoba, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of four dicationic glucose-templated
-lysine-analogs is described. Replacement of -lysine by
eral tool to introduce chemical diversity into the side chain of lysine. The presence of the polyfunctional
-gluco-configured polyol scaffold provides rich opportunities to study structure–activity relationships in
lysine–lipid conjugates. All cationic lipids were tested for inhibition of bacterial growth using a panel of
clinically relevant Gram-positive and Gram-negative strains. Our results show that substitution of
D
-lysine-derived surfactants and their two unmodified
Received 29 October 2010
Received in revised form 14 January 2011
Accepted 21 January 2011
Available online 28 January 2011
D
D
D
-glucose-templated- -lysine provides a gen-
D
D
Keywords:
Antibacterials
Lipids
D
-lysine by
D
-glucose-templated
D-lysine surfactants retains, but does not improve, the antibacterial
activity. Similarly, conversion of the
D
-gluco-based polyol scaffold into a hydrophobically enhanced -
tri-O-phenylcarbamate scaffold does not further enhance the antibacterial activity of the cationic lipid.
However, improvements in the antibacterial activity were observed by guanidinylation of the two
lysine-based amino groups.
Sugar amino acids
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
extracellular molecules.5a,10 After transit through the outer mem-
brane OAAs contact the anionic surface of the cytoplasmic mem-
Multidrug-resistant bacteria are becoming more prevalent in
both the hospitals and community settings. This has resulted in
an emerging crisis where an increasing number of antibiotics cease
to be of clinical usefulness.1 As a result, there is a pressing need for
novel classes of antibacterial agents with new or combined mech-
anisms of action and reduced likelihood for the development of
resistance.
Oligocationic amphiphilic antibacterials (OAAs) containing
multiple positively charged amino or other cationic groups define
a structurally diverse class of antibacterials with broad-spectrum
activity and different modes of action.2–4 This class of antibacterial
agents are comprised of the naturally occurring cationic antimicro-
bial peptides,5 synthetic mimics of antimicrobial peptides
(SMAMPs),6 synthetic oligocationic lipopeptides,7 oligocationic lip-
ids,8 and polymers.9 The cationic charges of the OAAs ensure accu-
mulation at polyanionic microbial cell surfaces that contain acidic
polymers, such as lipopolysaccharides and wall-associated teichoic
acids in Gram-negative and Gram-positive bacteria, respectively.5a
Antimicrobial peptides (polymyxin B, defensins, gramicidin S vari-
ants, and others) transit the outer membrane by interacting at sites
at which divalent cations crossbridge adjacent polyanionic poly-
mers. This causes a destabilization of the outer membrane that is
proposed to lead to self-promoted uptake of the OAAs and/or other
brane. Here they can insert themselves into the cytoplasmic
membrane thereby either disrupting the physical integrity of the
bilayer, via membrane thinning, transient poration and/or disrup-
tion of the barrier function or translocation across the membrane
and act on internal targets.5a This mode of action has been shown
to limit the risk of cross resistance.5,11
Another class of OAAs are cationic lipids, which have been
developed as antibacterial agents, antiseptics, and disinfectants
but also as drug delivery systems against diseases14,15 and gene
transfection.16 For instance, cationic lipids including dodine,17 ben-
zalkonium chlorides,18 sphingosine19, and fatty amines,20 chloroh-
exidine,21 cationic polymers22 exhibit broad spectrum antibacterial
activities (Fig. 1). Many of these are in clinical use as antiseptics,
disinfectants for several decades with little or no occurrence of
resistance.12,13 It has been suggested that the antibacterial mode
of action of cationic lipids is similar to antibacterial peptides and
involves disruption of the bacterial envelope induced by the re-
moval (substitution) of divalent positively charged counter ions
by ammonium ions.12
In this paper we describe the synthesis and antibacterial prop-
erties of a novel class of dicationic lysine- and arginine-related
amphiphiles. Previous studies have shown that dicationic, argi-
nine-based cationic lipids prepared by C-terminal amidation of
arginine to alkyl amines bearing long alkyl chains (C-10, C-12
and C-14) exhibit broad spectrum antibacterial activity with little
to no cytotoxicity.23 However, dicationic lipids of this type do not
⇑
Corresponding author. Tel.: +1 204 474 7012; fax: +1 204 474 7608.
E-mail address: schweize@cc.umanitoba.ca (F. Schweizer).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.01.025

D. Mondal et al. / Carbohydrate Research 346 (2011) 588–594
589
HN
R1
R3
HN
NH
NH
2
R2
O
dodine
H2N
NH2
H2N
O
N
HO
R
O
Cl
R = n-C H , ... n-C
gentamicins
O
H
18 37
HO
8
17
R , R , R = H, Me
HN
OH
1
2
3
benzalkonium chloride
NH2
H N
2
OH
OH
O
HO
HO
H2N
H2N
O
NH2
O
sphingosine
HO
HO
O
H2N
OH
OH
O
NH
2
O
OH
myristylamine
H2N
neomycin B
NH
NH
HN
HN
NH
NH
HN
HN
HN
HN
chlorohexidine
Cl
Cl
Figure 1. Structures of antibacterial cationic lipids and detergents. Dodine, benzalkonium chlorides, sphingosine, and chlorohexidine are currently used in commercial
antiseptics and disinfectants. The structures of the positive controls gentamicin and neomycin B are also provided for general reference.
permit additional manipulation as both the dicationic headgroup
and the hydrophobic tail form the amphiphilic pharmacophore of
antibacterial cationic lipids. We have previously outlined a strat-
egy termed carbohydrate-templated amino acid synthesis (CTAAs)
that permits the incorporation of polyfunctional sugar scaffolds
into naturally occurring amino acids, such as ornithine,24 argi-
nine,24 lysine,25 proline26–30, and GABA.31 The presence of the car-
bohydrate-scaffold introduces chemical diversity into amino acid
side chains and may mimic artificial post-translational modifica-
tions, such as hydroxylation and glycosylation. Moreover, chemical
derivatization of the auxophoric polyol scaffold can be used to tai-
lor the pharmacodynamic and pharmacokinetic properties of cat-
ionic lipids. We have now applied this strategy to prepare novel
carbohydrate-templated lysine–lipid conjugates. We were particu-
larly interested to study how incorporation of an auxophoric glu-
cose-based polyol scaffold into the cationic head group of a
lysine surfactant influences its antibacterial properties.
in glycosurfactant 3. This was achieved by reaction of 3 with
N,N0-di-tert-butoxycarbonyl-N0-triflylguanidine (NHBoc)2C@NTf)32
using Et3N in dichloromethane at ambient temperature for 12 h
to generate tert-butoxycarbonyl-protected homo-
rived glycolipid 4. Deblocking was achieved by exposure to
trifluoroacetic acid in dichloromethane to give unprotected -glu-
co-templated -homoarginine-derived glycolipid 5 (Scheme 1).
Both glycolipids 3 and 5 were used for antibacterial testing.
To explore how chemical modifications on the -gluco-config-
ured polyol scaffold influence the antibacterial properties we
decided to prepare polyol-modified -lysine-based triphenylcarba-
mate and polyol-modified homo- -arginine-based trip-
D-arginine-de-
D
D
D
D
7
D
henylcarbamte 9 (Scheme 2). Phenylcarbamoylation was selected
as a method of derivatization due to the high reactivity of phenyl
isocyanate with sugar hydroxyl groups and their antibacterial
properties.34 Compound 7 was prepared by reaction of glycoplipid
2 with phenyl isocyanate in anhydrous pyridine at room tempera-
ture for 12 h to afford Boc-protected lysine analog 6 in 88% yield.
Exposure of 6 to TFA in dichloromethane produced oligoamphi-
philic lysine analog 7 as a TFA salt in quantitative yield. The two
deblocked amino functions were guanidinylated with N,N0-di-
tert-butoxycarbonyl-N0-triflylguanidine (NHBoc)2C@NTf)32 to yield
protected homoarginine-derived lipid 8 in 83% isolated yield.
Deblocking of the Boc-protecting groups in dichloromethane pro-
duced polyamphiphilic homoarginine-derived lipid 9 (Scheme 2).
2. Results and discussion
Our synthesis began with partially protected
D-gluco-templated
D
-lysine analog 1 previously prepared from commercially available
tetra-O-benzyl gluconolactone.25 Acid 1 was coupled to dodecyl-
amine using of 2-(1H-benzotriazole-1yl)-1,1,3,3-tetra-methyluro-
nium tetrafluoroborate (TBTU) as coupling reagent and Hünig’s
base (N,N-diisopropylethylamine) in N,N0-dimethylformamide as
solvent to afford glycolipid 2 in 92% yield. Exposure of glycolipid
2 to trifluoroacetic acid in dichloromethane at room temperature
We also prepared the
D
-lysine-based cationic lipid 12 and
D-homo-
arginine-derived lipid 14 from commercially available
building block 10 as reference compounds (Scheme 3).
D-lysine
With amphiphilic carbohydrate-templated lysine-modified
lipids 3, 5, 7, and 9 and lysine-based surfactants 12 and 14 lipids
in hand, we determined the antibacterial minimum inhibitory con-
for 3 h produced deblocked
fluoroacetate salt. We also prepared deblocked homo-
derived glycolipid 5 by guanidinylation of the two amino functions
D
-lysine-based glycosurfactant 3 as tri-
D-arginine-

590
D. Mondal et al. / Carbohydrate Research 346 (2011) 588–594
BocHN
BocHN
HO
NHBoc
BnO
BnO
BnO
NHBoc
OH
C
H NH
12 25 2
Ref 25
H
N
O
O
HO
HO
O
( )10
CH
HO
TBTU, DIPEA, DMF
rt, 92%
3
O
OH
OH
O
O
OBn
1
2
NBoc
NH
NBoc
BocHN
NH x CF COOH
HOOCF C x H N
2
3
3
2
(NHBoc) C=NTf
2
NHBoc
H
H
NH
O
OH
3
HO
( )10
CH
N
TFA
O
TEA, DCM
rt, 91%
HO
HO
HO
( )10
N
3
DCM, rt, quant.
CH
3
O
OH
O
4
NH
NH
NH
HOOCF C x H N
3
2
NH x CF COOH
2
3
NH
TFA
H
O
HO
( )10
N
HO
DCM, rt, quant.
CH
3
OH
O
5
Scheme 1. Synthesis of
D-gluco-templated
D-lysine surfactant 3 and
D
-gluco-templated homo-
D-arginine-based surfactant 5.
BocHN
HO
BocHN
NHBoc
O
NHBoc
PhNCO
TFA
H
H
O
O
PhHNOCO
PhHNOCO
( )10
( )
10
N
N
o
HO
DCM, 0 C-rt,
quant.
CH
CH
3
3
pyridine
rt, 88%
PhNHOCO
OH
O
6
2
NBoc
NH
NBoc
NHBoc
NH
NH x CF COOH
HOOCF C x H N
2
3
(NHBoc) C=NTf
3
2
2
BocHN
H
O
PhHNOCO
PhHNOCO
( )
N
10
TEA, 1,4-dioxane:
H O (3:1), rt, 83%
2
H
CH
O
PhHNOCO
PhHNOCO
3
( )
N
PhNHOCO
10
O
CH
3
PhNHOCO
O
7
8
NH
NH
NH
TFA
HOOCF C x H N
3
2
o
NH x CF COOH
2
3
NH
DCM, 0 C-rt,
H
quant.
O
PhHNOCO
PhHNOCO
( )
N
10
CH
3
PhNHOCO
O
9
Scheme 2. Synthesis of polyol-modified carbohydrate-templated D-lysine- (7) and carbohydrate-templated homo-D-arginine-based (9) lipids.
centration (MIC) in
l
g/mL of these analogs against American Type
12 because of changes in molecular weight. However, similar
potency was observed for MRSA while a fourfold deduction in anti-
bacterial potency was observed against S. pneumoniae. A significant
fourfold reduction in antibacterial activity of 3 when compared to
12 was found against a Gentamicin-susceptible Gram-negative
E. coli strain. Interestingly, phenylcarbamoylation of the cationic
polyol-based headgroup in compounds 3 and 5 has little to no ef-
fect on the antibacterial activity once the changes in molecular
weight are considered. This suggests that polyol-based substitu-
tions aimed to enhance the hydrophobicity of the polar headgroup
do not provide a tool to enhance the antibacterial properties. Very
likely the enhanced hydrophobicity of the polar headgroup in cat-
ionic lipids 7 and 9 disturbs the monoamphiphilic nature (single
polar head and single hydrophobic tail) of classic cationic deter-
Culture Collection (ATCC) reference strains as well as clinical iso-
lates of Gram-positive strains including Staphylococcus aureus,
MRSA, Staphylococcus epidermidis, methicillin-resistant S. epidermi-
dis (MRSE) and Streptococcus pneumoniae as well as Gram-negative
strains Escherichia coli, gentamicin-resistant E. coli, and Pseudomo-
nas aeruginosa (Table 1). We also included three resistant strains
obtained from a national surveillance study assessing antimicro-
bial resistance in Canadian intensive care units CAN-ICU (Table
1).33 Gentamicin and neomycin B served as positive controls.
Our results show that substitution of
D-lysine in cationic lipid
12 by -gluco-templated -lysine results in similar antibacterial
D
D
activity. In most cases, an expected slight increase in MICs for
Gram-positive organisms was observed for 3 when compared to

D. Mondal et al. / Carbohydrate Research 346 (2011) 588–594
591
NHBoc
1. C
DIPEA, DMF, rt
H NH , TBTU
12 25 2
NHBoc
OH
H
TFA, DCM
( )
10
N
H N
2
o
CH
FmocHN
3
0 C-rt, quant.
2. Piperidine, DMF, rt
87%, over two steps
O
O
11
10
NBoc
(NHBoc) C=NTf
2
NHBoc
H
NH x CF COOH
NH
2
3
NBoc
H
TEA, DCM
rt, 96%
( )
10
N
( )
N
10
CH
HOOCF C x H N
N
H
BocHN
3
2
CH
3
3
O
O
13
12
NH x CF COOH
3
TFA, DCM
NH
2
NH
NH
H
o
0 C-rt, quant.
( )
10
N
N
HOOCF C x H N
3
2
CH
3
H
O
14
Scheme 3. Synthesis of lysine- (12) and guanidinylated lysine-based (14) reference lipids.
Table 1
Antibacterial activity (MIC) in
l
g/mL of carbohydrate-templated lysine-based lipids 3, 5, 7 and 9 and reference lipids 12 and 14 against various Gram-positive and Gram-negative
bacterial strains
Control organism
Gentamicin
Neomycin B
3
5
7
9
12
32
64
8
16
32
14
S. aureusa
MRSAb
1
2
1
256
0.25
0.5
32
64
64
16
32
16
16
8
16
64
128
128
256
128
589.4
64
32
16
32
32
32
32
64
128
64
128
128
128
946.8
4
4
2
8
32
128
128
64
S. epidermidisc
MRSEd
0.25
32
4
1
128
8
S. pneumoniaee
E. colif
128
256
256
>256
256
505.4
>128
>256
>256
>256
>256
862.7
4
8
32
E. colig
128
128
128
415.3
P. aeruginosah
P. aeruginosai
MWj
512
512
908.9
128
128
499.4
k
a
ATCC 29213.
b
Methicillin-resistant S. aureus ATCC 33592.
S. epidermidis ATCC 14990.
Methicillin-resistant S. epidermidis (CAN-ICU) 61589.
ATCC 49619.
ATCC 25922.
CAN-ICU 61714.
ATCC 27853.
CAN-ICU 62308.
c
d
e
f
g
h
i
j
Molecular weight as TFA salt for compounds 3, 5, 7, 9, 12 and 14, trisulfate (Neomycin B).
Mixture of homologues average MW ꢀ 600.
k
gents such as 12 and 14 resulting in the formation of polyamphi-
philic surfaces. In addition, our results suggest that the enhanced
hydrophobicity of amphiphiles 7 and 9 very likely must be com-
pensated by changes in the ionization state or number of cationic
charges in the headgroup in order to enhance antibacterial activity.
Moreover, guanidinylation of 12, 3, and 7 enhances in most cases
antibacterial activity. The increase in potency is more obvious for
Gram-positive strains in lipids 12 and 3 resulting in a eight to two-
fold decrease in MIC with relative little changes for Gram-negative
organisms. Overall it appears that carbohydrate-templated lysine–
lipid conjugates exhibit higher MICs when compared to their
unmodified lysine–lipid conjugates. In particular, cationic lipid
14 exhibits impressive Gram-positive activity when compared to
the aminoglycoside antibiotics gentamicin and neomycin B.
In summary, we have synthesized four carbohydrate-templated
lysine-derived dicationic lipids as well as two unmodified lysine-
derived dicationic reference lipids and explored their antibacterial
properties against a panel of clinically relevant Gram-positive and
Gram-negative bacterial strains. Our results show that substitution
of D-lysine by carbohydrate-templated D-lysine analogs retains, but
does not improve, the antibacterial potency of the corresponding
cationic lipid.
3. Experimental
3.1. General methods
NMR spectra were recorded on a Brucker Avance 300 spectrom-
eter (300 MHz for 1H NMR, 75 MHz for 13C) and on a AMX 500
spectrometer (500 MHz for 1H NMR). Optical rotation was mea-
sured at a concentration of g/100 mL, with a Perkin–Elmer polar-
imeter (accuracy 0.002°). GC–MS analyses were performed on a
Perkin–Elmer Turbomass-Autosystem XL. Analytical thin-layer
chromatography was performed on pre-coated silica gel plates.

592
D. Mondal et al. / Carbohydrate Research 346 (2011) 588–594
Visualization was performed by ultraviolet light and/or by staining
with ninhydrin solution in ethanol. Chromatographic separations
were performed on a silica gel column by flash chromatography
(Kiesel gel 40, 0.040–0.063 mm; Merck). Yields are given after
purification, unless differently stated. When reactions were
performed under anhydrous conditions, the mixtures were main-
tained under nitrogen. Compounds were named following IUPAC
rules as applied by Beilstein-Institute AutoNom (version 2.1) soft-
ware for systematic names in organic chemistry. The purity of all
final compounds used in biological testing was assessed by ele-
mental analysis confirming >95% purity. 13C NMR spectra of com-
pounds in the form of TFA salts usually showed the characteristic
carbon peaks for TFA at d = 164.4 (q) and 116.5 (q) in D2O when
using long acquisition times.
2H), 3.48 (dd, 1H, J = 6.8, 9.0 Hz), 3.57 (dd, 1H, J = 3.3, 9.4 Hz),
3.65 (dd, 1H, J = 2.7, 14.4 Hz), 3.80 (dd, 1H, J = 4.4, 14.4 Hz),4.83
(d, 1H, J = 4.0 Hz); 13C NMR (75 MHz, MeOH-d4): d 14.5, 23.7,
28.0, 28.4 (ꢂ6), 28.5 (ꢂ3), 28.6 (ꢂ3), 30.4, 30.5, 30.6, 30.8 (ꢂ4),
33.1, 40.6, 42.9, 57.0, 72.0, 72.5, 78.6, 79.8, 80.5, 80.6, 80.9, 84.6,
84.7, 154.0 (ꢂ2), 157.1, 158.3, 164.1, 164.3, 170.4; MS (ES+): m/z
910.14 [M+Na]+; Anal. Calcd for C42H77N7O13: C, 56.80; H, 8.74;
N, 11.04. Found: C, 57.11; H, 9.02; N, 10.83.
3.5. N0-Dodecyl-[[(2R)-2-guanidinyl-2-]-[6-guanidinyl-6-deoxy)-
b-D
-glucopyranosyl]ethanamide ꢂ 2 trifluoroacetic acid (5)
A solution of TFA–CH2Cl2 (1:3, 4.0 mL) was added to compound
4 (80 mg, 0.09 mmol) at room temperature. After 3 h, the solution
was diluted with toluene (3 ꢂ 5 mL), concentrated in vacuo, quan-
titatively provided guanidine substance 5 (64.0 mg, quant.) as a tri-
3.2. N0-Dodecyl-[(2R)-2-(tert-butoxycarbonylamino)-2-[6-
deoxy-6-(t-butoxycarbonylamino)-b-
D
-glucopyranosyl]
fluroacetate salt. ½a D25
ꢁ
¼ ꢃ15:0 (c 0.60, MeOH); 1H NMR (300 MHz,
ethanamide (2)
D2O): d 0.81 (t, 3H, J = 6.6 Hz), 1.21 (s, 18H), 1.44 (br s, 2H), 3.01–
3.31 (m, 4H), 3.40–3.55 (m, 3H), 3.58 (br d, 1H, J = 13.8 Hz), 3.73 (br
d, 1H, J = 6.9 Hz), 4.50 (s, 1H); 13C NMR (75 MHz, D2O): d 14.0, 22.9,
27.1, 29.0, 29.5, 29.7, 29.9, 30.0 (ꢂ3), 32.2, 40.2, 42.8, 56.2, 70.3,
70.9, 77.3, 78.4, 79.6, 157.1, 157.8, 168.1; MS (ES+): m/z 488.01
[M+H]+; Anal. Calcd for C26H47F6N7O9: C, 43.63; H, 6.62; N, 13.70.
Found: C, 43.43; H, 6.30; N, 13.33.
To a mixture of compound 1 (100 mg, 0.23 mmol) and TBTU
(265 mg, 0.83 mmol) in DMF (5.0 mL) were added dodecylamine
(51 mg, 0.25 mmol) and N,N-diisopropylethylamine (200 lL,
1.1 mmol) and stirred for 2 h at room temperature. The solvent
was removed in vacuo and the residue was purified by flash col-
umn chromatography (MeOH–EtOAc 1:25) to yield 2 (127 mg,
0.21 mmol, 92%) as a thick colorless liquid. Rf = 0.20 (MeOH–EtOAc
3.6. N0-Dodecyl-[(2R)-2-(t-butoxycarbonylamino)-2-[6-deoxy-6-
(tert-butoxycarbonylamino-2,3,4-tri-O-phenylcarbamoyl)-b-D-
glucopyranosyl]ethanamide (6)
1:19); ½a 2D5
ꢁ
¼ þ10:0 (c 0.30, MeOH); 1H NMR (300 MHz, MeOH-d4):
d 0.91 (t, 3H, J = 6.9 Hz), 1.30 (br s, 18H), 1.45–1.55 (m, 20H), 3.01–
3.12 (m, 2H), 3.16–3.26 (m, 3H), 3.29 (m, 1H), 3.35–3.40 (m, 2H),
3.55 (d, 1H, J = 13.5 Hz), 4.40 (s, 1H); 13C NMR (75 MHz, MeOH-
d4): d 14.5, 23.7, 28.0, 28.7 (ꢂ3), 28.9 (ꢂ3), 30.5 (ꢂ3), 30.8 (ꢂ4),
33.1, 40.4, 42.9, 56.9, 71.9, 72.9, 79.1, 80.3, 80.8, 81.0, 81.2,
157.4, 158.7, 171.9; MS (ES+): m/z 626.21 [M+Na]+; Anal. Calcd
for C30H57N3O9: C, 59.68; H, 9.52; N, 6.96. Found: C, 59.43; H,
9.90; N, 7.23.
To a solution of tert-butoxycarbonyl-protected derivative 2
(1 equiv) in dry pyridine phenyl isocyanate (4.0 equiv) was added
and stirred at room temperature for 12 h. Pyridine was removed
under reduced pressure and the crude residue was purified by flash
column chromatography, eluted by EtOAc–hexane to get pure car-
bamate derivative 6 as thick liquid in 88% yield. Rf = 0.20 (EtOAc–
Hexane 4:1); ½a 2D5
ꢁ
¼ ꢃ27:0 (c 1.3, MeOH); 1H NMR (300 MHz,
3.3. N0-Dodecyl-[[(2R)-2-ammonium-2-]-(6-ammonium-6-
deoxy)-b-D-glucopyranosyl]ethanamide bistrifluoroacetate (3)
MeOH-d4): d 0.89 (t, 3H, J = 7.0 Hz), 1.29 (s, 18H), 1.44 (2s, 18H),
1.50 (m, 2H), 3.04–3.20 (m, 2H), 3.20–3.28 (m, 1H), 3.56 (m, 1H),
3.68 (m, 1H), 4.00 (dd, 1H, J = 2.5, 10.0 Hz), 4.45 (m, 1H), 4.89 (m,
1H), 5.09 (dd, 1H, J = 9.5, 10.0 Hz), 5.32 (dd, 1H, J = 9.5, 10.0 Hz),
6.80 (br s, 1H), 6.92–7.02 (m, 3H), 7.09–7.30 (m, 8H), 7.32–7.44
(m, 4H), 7.93 br s, 1H); 13C NMR (75 MHz, MeOH-d4): d 14.5,
23.7, 28.1, 28.7 (ꢂ3), 28.8 (ꢂ3), 30.3, 30.4, 30.5, 30.8 (ꢂ4), 33.1,
40.8, 42.5, 56.8, 71.8, 72.0, 76.3, 78.9, 79.2, 80.4, 81.3, 120.2–
139.7 (aromatic carbons), 154.5, 154.9, 155.2, 157.6, 158.2,
170.5; MS (ES+): m/z 983.58 [M+Na]+; Anal. Calcd for
A solution of TFA–CH2Cl2 (1:3, 4.0 mL) was added to 2 (127 mg,
0.21 mmol) at room temperature. After 3 h, the solution was
diluted with toluene (3 ꢂ 10 mL), concentrated in vacuo, to provide
3 (133 mg, quant.) as a trifluoroacetate salt. ½a D25
¼ þ15:0 (c 0.35,
ꢁ
MeOH); 1H NMR (300 MHz, D2O): d 0.84 (t, 3H, J = 7.0 Hz), 1.24
(br s, 18H), 1.42–1.55 (m, 2H), 3.04 (m, 1H), 3.12–3.30 (m, 3H),
3.35–3.70 (m, 4H), 3.82 (m, 1H), 4.35 (s, 1H); 13C NMR (75 MHz,
D2O): d 14.1, 22.9, 27.2, 28.9, 29.4, 29.7, 29.9, 30.0 (ꢂ3), 32.2,
40.4, 41.1, 53.7, 70.0, 71.4, 76.4, 76.9, 77.3, 165.3; MS (ES+): m/z
404.17 [M+H]+; Anal. Calcd for C24H43F6N3O9: C, 45.64; H, 6.86;
N, 6.65. Found: C, 45.32; H, 6.53; N, 6.85.
C51H72N6O12: C, 63.73; H, 7.55; N, 8.74. Found: C, 63.51; H, 7.84;
N, 8.67.
3.7. N0-Dodecyl-[[(2R)-2-ammonium-2-]-(6-ammonium-6-
deoxy-2,3,4-tri-O-phenylcarbamoyl-)-b-D-glucopyranosyl]
ethanamide bistrifluoroacetate (7)
3.4. N0-Dodecyl-[[(2R)-2-N,N0-di-tert-butoxycarbonyl-guanidi-
nyl-2-]-[6-N,N0-di-tert-butoxycarbonyl-guanidinyl-6-deoxy)-b-
D
-glucopyranosyl]ethanamide (4)
Deblocking of the tert-butoxycarbonyl groups in derivative 6
(50.0 mg) was performed with 25% trifuoroacetic acid (4.0 mL) in
dichloromethane for 3 h at 0 °C to rt. The volatile components were
removed in vacuo under reduced pressure and the non-polar resi-
dues were removed by washing with ether and decanted the sol-
To a solution of diamine salt 3 (0.040 g, 0.006 mmol) in dichlo-
romethane (2.0 mL) was added N,N0-di-t-butoxycarbonyl-N00-tri-
flylguanidine (86 mg, 0.22 mmol, 3.5 equiv). After 5 min, NEt3
(31.0
l
L, 0.22 mmol, 3.5 equiv) was added at room temperature.
vent to get the TFA salt 7 in quantitative yield. ½a D25
ꢁ
¼ ꢃ9:0 (c
After 12 h, the dichloromethane was removed under reduced pres-
sure. The remaining residue product was purified by flash column
chromatography with EtOAc on silica gel to give guanidinylated
derivative 4 (52 mg, 0.058 mmol, 91%) as an oil. Rf = 0.20 (EtOAc);
1.7, MeOH); 1H NMR (300 MHz, MeOH-d4):
d 0.89 (t, 3H,
J = 7.0 Hz), 1.22–1.48 (m, 18H), 1.58–1.69 (m, 2H), 3.12–3.22 (m,
2H), 3.34–3.41 (m, 2H), 4.06 (m, 1H), 4.27 (m, 1H), 4.35 (br s,
1H), 4.89 (m, 1H), 5.13 (dd, 1H, J = 7.9, 8.2 Hz), 5.49 (dd, 1H,
J = 9.0, 9.5 Hz), 6.96–7.08 (m, 3H), 7.15–7.46 (m, 12H); 13C NMR
(75 MHz, MeOH-d4): d 14.5, 23.8, 28.2, 30.1, 30.5 (ꢂ2), 30.9 (ꢂ4),
33.1, 41.2, 41.7, 54.3, 71.1, 71.4, 75.0, 76.2, 77.5, 120.1–139.4 (aro-
½
a 2D5
ꢁ
¼ ꢃ55:0 (c 0.30, CHCl3); 1H NMR (300 MHz, MeOH-d4): d 0.91
(t, 3H, J = 7.0 Hz), 1.30 (br s, 19H), 1.47 (2s, 18H), 1.56 (m, 19H),
3.16 (dd, 1H, J = 8.8, 9.2 Hz), 3.25 (t, 2H, J = 7.0 Hz), 3.35–3.45 (m,

D. Mondal et al. / Carbohydrate Research 346 (2011) 588–594
593
matic carbons), 154.3, 154.8, 155.2, 165.5; MS (ES+): m/z 761.11
[M+H]+; Anal. Calcd for C45H58F6N6O12: C, 54.65; H, 5.91; N, 8.50.
Found: C, 54.86; H, 6.27; N, 8.59.
column chromatography (MeOH–EtOAc 1:20) to afford the 11
(768 mg, 87%). ½a D25
ꢁ
¼ ꢃ21:0 (c 0.5, MeOH); 1H NMR (300 MHz,
MeOH-d4): d 0.91 (t, 3H, J = 6.8 Hz), 1.24–1.40 (m, 20H), 1.44 (s,
9H), 1.46–1.60 (m, 4H), 1.60–1.74 (m, 2H), 3.06 (t, 2H, J = 6.8 Hz),
3.12–3.30 (m, 3H); 13C NMR (75 MHz, MeOH-d4): d 14.6, 23.8,
24.0, 28.1, 28.9 (ꢂ3), 30.5 (ꢂ2), 30.6, 30.7, 30.8 (ꢂ2), 30.9 (ꢂ2),
33.1, 36.3, 40.4, 41.2, 56.1, 79.8, 158.5, 177.5; MS (ES+): m/z
414.61 [M+H]+, 436.60 [M+Na]+; Anal. Calcd for C23H47N3O3: C,
66.78; H, 11.45; N, 10.16. Found: C, 66.56; H, 11.82; N, 10.41.
3.8. N0-Dodecyl-[[(2R)-2-N,N0-di-t-butoxycarbonyl-guanidinyl-2-
]-[6-N,N0-di-t-butoxycarbonyl-guanidinyl-6-deoxy-2,3,4-tri-O-
phenylcarbamoyl)-b-D-glucopyranosyl]ethanamide (8)
To a solution of diamine salt 7 (0.050 g, 0.05 mmol) in the mix-
ture of 1,4-dioxane and water (3:1, 2.0 mL) was added N,N0-di-t-
butoxycarbonyl-N00-triflylguanidine (69 mg, 0.18 mmol, 3.5 equiv).
3.11.
D-Lys-NHC12H25 ꢂ 2 trifluoroacetic acid (12)
After 5 min, NEt3 (25.0 lL, 0.18 mmol, 3.5 equiv) was added at
room temperature. After 12 h, the reaction mixture was extracted
with dichloromethane (2 ꢂ 5 mL) and dried over anhydrous
Na2SO4. Then the combined organic layer was removed under re-
duced pressure. The remaining residue product was purified by
flash column chromatography with EtOAc on silica gel to give gua-
nidinylated compound 8 (52 mg, 0.042 mmol, 83%) as an oil.
Deblocking of the tert-butoxycarbonyl groups in derivative 11
(100 mg) was performed with 25% trifluoroacetic acid (4 mL) in
dichloromethane for 3 h at 0 °C to room temperature. The volatile
components were removed in vacuo under reduced pressure and
the non-polar residues were removed by washing with ether and
the solvent was decanted to get the compound 12 as quantitative
Rf = 0.20 (EtOAc–Hexane 2:3); ½a D25
ꢁ
¼ ꢃ17:0 (c 1.1, CHCl3); 1H
as a TFA salt. ½a 2D5
ꢁ
¼ ꢃ12:0 (c 1.1, MeOH); 1H NMR (300 MHz,
NMR (300 MHz, MeOH-d4): d 0.90 (t, 3H, J = 6.9 Hz), 1.26–1.31
(m, 15H), 1.33 (br s, 8H), 1.42 (s, 9H), 1.47 (s, 9H), 1.52 (s, 9H),
1.57 (s, 9H), 3.17–3.28 (m, 2H), 3.56–3.67 (m, 1H), 3.86–3.97 (m,
2H), 4.11 (dd, 1H, J = 6.0, 10.0 Hz), 4.95–5.05 (m, 2H), 5.19 (dd,
1H, J = 9.5, 10.0 Hz), 5.37 (dd, 1H, J = 9.5, 10.0 Hz), 6.90–7.04 (m,
3H), 7.09–7.28 (m, 8H), 7.30–7.38 (m, 4H), 8.17 (t, 1H, J = 5.5 Hz);
13C NMR (75 MHz, MeOH-d4): d 14.5, 23.8, 28.1, 28.2 (ꢂ4), 28.4
(ꢂ3), 28.5 (ꢂ3), 28.6 (ꢂ3), 30.2, 30.5, 30.6, 30.8 (ꢂ3), 33.1, 40.7,
41.9, 57.0, 70.9, 72.8, 76.3, 77.2, 78.1, 80.4, 80.6, 84.0, 84.6,
120.3–139.8 (aromatic carbons), 153.2, 153.8, 154.2, 154.3, 155.1,
157.1, 158.0, 164.2, 164.4, 169.8; MS (ES+): m/z 1267.40
[M+Na]+; Anal. Calcd for C63H92N10O16: C, 60.75; H, 7.45; N,
11.25. Found: C, 60.98; H, 7.69; N, 11.63.
MeOH-d4): d 0.90 (t, 3H, J = 7.0 Hz), 1.30 (m, 18H), 1.42–1.63 (m,
4H), 1.63–1.79 (m, 2H), 1.80–2.00 (m, 2H), 2.94 (t, 2H, J = 7.5 Hz),
3.23 (t, 2H, J = 7.0 Hz), 3.85 (t, 1H, J = 6.5 Hz), 7.99 (s, 1H), 8.45 (s,
1H); 13C NMR (75 MHz, MeOH-d4): d 14.5, 23.0, 23.7, 28.0 (ꢂ2),
30.3, 30.4, 30.5, 30.7, 30.8 (ꢂ3), 32.1, 33.1, 40.3, 40.7, 54.3, 169.9;
MS (ES+): m/z 314.19 [M+H]+; Anal. Calcd for C22H41F6N3O5: C,
48.79; H, 7.63; N, 7.76. Found: C, 49.10; H, 8.00; N, 7.38.
3.12. N,N0-Di-t-Butoxycarbonyl-guanidinyl- -Lys(N,N0-di-t-
D
butoxycarbonyl-guanidinyl)-NHC12H25 (13)
To a solution of diamine 12 (95 mg, 0.175 mmol) in dichloro-
methane (2.0 mL) was added N,N0-di-t-butoxycarbonyl-N00-triflyl-
guanidine (206 mg, 0.53 mmol, 3 equiv). After 5 min, NEt3 (73 lL,
3.9. N0-Dodecyl-[[(2R)-2-guanidinyl-2-]-[6-guanidinyl-6-deoxy-
0.53 mmol, 3 equiv) was added at room temperature. After over
night, the dichloromethane was removed under reduced pressure.
The remaining residue product was purified by flash column chro-
matography (hexane–EtOAc 1:9) on silica gel to give guanidinylat-
ed derivative 13 in 96% yield as an oil. Rf = 0.20 (EtOAc);
2,3,4-tri-O-phenylcarbamoyl)-b-D-glucopyranosyl]ethanamide
ꢂ 2 trifluoroacetic acid (9)
Deblocking of the tert-butoxycarbonyl groups in derivative 8
(0.050 mmol) was performed with 25% trifluoroacetic acid (4 mL)
in dichloromethane for 3 h at 0 °C to rt. The volatile components
were removed in vacuo under reduced pressure and the non-polar
residues were removed by washing with ether and the solvent was
decanted to get the guanidine derivative 9 as TFA salt quantita-
½
a 2D5
ꢁ
¼ ꢃ17:0 (c 0.6, CHCl3); 1H NMR (300 MHz, CDCl3): d 0.84 (t,
3H, J = 6.8 Hz), 1.16–1.30 (m, 18H), 1.32–1.41 (m, 4H), 1.44 (2s,
36H), 1.53–1.63 (m, 2H), 1.70 (m, 1H), 1.88 (m, 1H), 3.10–3.22
(m, 2H), 3.30–3.40 (m, 2H), 4.42 (q, 1H, J = 7.0 Hz), 6.76 (t, 1H,
J = 5.5 Hz), 8.27 (br t, 1H, J = 4.8 Hz), 8.62 (d, 1H, J = 7.3 Hz), 11.3
(br s, 2H); 13C NMR (75 MHz, CDCl3): d 14.1, 22.6, 22.8, 26.8, 28.0
(ꢂ6), 28.2 (ꢂ6), 28.7, 29.2 (ꢂ2), 29.3, 29.5, 29.6 (ꢂ3), 31.0, 31.9,
39.5, 40.6, 54.0, 79.3, 79.5, 83.1, 83.6, 152.3, 153.2, 156.0, 156.1,
162.9, 163.5, 170.8; MS (ES+): m/z 820.48 [M+Na]+; Anal. Calcd
for C40H75N7O9: C, 60.20; H, 9.47; N, 12.29. Found: C, 60.03; H,
9.19; N, 12.53.
tively. ½a 2D5
ꢁ
¼ ꢃ29:0 (c 1.0, MeOH); 1H NMR (300 MHz, MeOH-
d4): d 0.79 (t, 3H, J = 6.8 Hz), 1.20 (s, 18H), 1.40–1.50 (m, 2H),
3.04–3.19 (m, 2H), 3.30 (dd, 1H, J = 6.0, 15.0 Hz), 3.60 (dd, 1H,
J = 2.5, 15.0 Hz), 3.83 (m, 1H), 4.17 (dd, 1H, J = 2.5, 10.0 Hz), 4.54
(d, 1H, J = 2.5 Hz), 4.86 (dd, 1H, J = 9.8, 10.0 Hz), 5.10 (dd, 1H,
J = 9.8, 10.0 Hz), 5.34 (dd, 1H, J = 9.8, 10.0 Hz), 6.84–6.98 (m, 3H),
7.02–7.20 (m, 8H), 7.24–7.35 (m, 4H), 8.03 (t, 1H, J = 5.4 Hz); 13C
NMR (75 MHz, MeOH-d4): d 14.4, 23.7, 28.2, 30.3, 30.5, 30.6,
30.7, 30.8, 30.9 (ꢂ2), 33.1, 41.1, 43.2, 56.9, 71.1 (ꢂ2), 75.8, 78.2,
79.6, 120.3–139.4 (aromatic carbons), 154.6, 154.8, 155.0, 159.0,
159.5, 167.5; MS (ES+): m/z 845.13 [M+H]+; Anal. Calcd for
3.13. Guanidinyl-D-Lys(guanidinyl)-NHC12H25 (14)
Deblocking of the tert-butoxycarbonyl groups in derivative 13
(0.050 mmol) was performed with 25% trifluoroacetic acid (4 mL)
in dichloromethane for 3 h at 0 °C to room temperature. The vola-
tile components were removed in vacuo under reduced pressure
and the non-polar residues were removed by washing with ether
and the solvent was decanted to get guanidine derivative 14 as
C47H62F6N10O12: C, 52.61; H, 5.82; N, 13.05. Found: C, 52.36; H,
6.02; N, 12.87.
3.10. t-Butoxycarbonyl-NH-D-Lys-NHC12H25 (11)
TFA salt quantitatively. ½a D25
ꢁ
¼ ꢃ24:0 (c 1.5, MeOH); 1H NMR
To the mixture of compound 10 (1 g, 2.1 mmol) and TBTU (2.5 g,
7.8 mmol) in DMF (15.0 mL) were added dodecylamine (540 L,
(300 MHz, MeOH-d4): d 0.90 (t, 3H, J = 7.0 Hz), 1.20–1.38 (m,
18H), 1.39–1.58 (m, 4H), 1.59–1.68 (m, 2H), 1.78 (m, 1H), 1.89
(m, 1H), 3.15–3.26 (m, 4H), 4.12 (dd, 1H, J = 5.7, 7.5 Hz), 8.22 (t,
1H, J = 5.7 Hz); 13C NMR (75 MHz, MeOH-d4): d 14.5, 23.6, 23.7,
28.0, 29.5, 30.3, 30.4, 30.5, 30.7 (ꢂ2), 30.8 (ꢂ2), 33.1, 33.4, 40.8,
42.2, 56.5, 158.6, 158.7, 172.1; MS (ES+): m/z 398.46 [M+H]+; Anal.
l
2.31 mmol) and N,N-diisopropylethylamine (1.8 mL, 10.08 mmol)
and stirred for 2 h at room temperature. The solvent was removed
in vacuo and the residue was stirred with piperidine (5.0 mL) in
DMF (20.0 mL) for 1 h at room temperature. The solvent was
removed in vacuo and the crude product was purified by flash

594
D. Mondal et al. / Carbohydrate Research 346 (2011) 588–594
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3.14. Determination of the MIC values for cationic lipids 3, 5, 7,
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