Synthesis and antibacterial activity of amphiphilic lysine-ligated neomycin B conjugates

Article abstract of DOI:10.1016/j.carres.2011.01.015

Amphiphilic lysine-ligated neomycin B building blocks were prepared by reductive amination of a protected C5″-modified neomycin B-based aldehyde and side chain-unprotected lysine or lysine-containing peptides. It was demonstrated that a suitably protected

Full text of DOI:10.1016/j.carres.2011.01.015

Carbohydrate Research 346 (2011) 560–568
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and antibacterial activity of amphiphilic lysine-ligated neomycin
B conjugates
Smritilekha Bera a, George G. Zhanel b, Frank Schweizer a,b,
⇑
a Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
b Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E 3P4
a r t i c l e i n f o
a b s t r a c t
Article history:
Amphiphilic lysine-ligated neomycin B building blocks were prepared by reductive amination of a pro-
tected C500-modified neomycin B-based aldehyde and side chain-unprotected lysine or lysine-containing
peptides. It was demonstrated that a suitably protected lysine-ligated neomycin B conjugate (NeoK)
serves as a building block for peptide synthesis, enabling incorporation of aminoglycoside binding sites
into peptides. Antibacterial testing of three amphiphilic lysine-ligated neomycin B conjugates against a
representative panel of Gram-positive and Gram-negative strains demonstrates that C500-modified neo-
mycin-lysine conjugate retains antibacterial activity. However, in most cases the lysine-ligated neomycin
B analogs display reduced potency against Gram-positive strains when compared to unmodified neomy-
cin B or unligated peptide. An exception is MRSA where an eightfold enhancement was observed. When
compared to unmodified neomycin B, the prepared lysine-neomycin conjugates exhibited a 4–8-fold
enhanced Gram-negative activity against Pseudomonas aeruginosa and up to 12-fold enhanced activity
was observed when compared to unligated reference peptides.
Received 1 November 2010
Received in revised form 13 January 2011
Accepted 14 January 2011
Available online 21 January 2011
Keywords:
Antimicrobial peptides
Glycopeptides
Aminoglycosides
Antibacterials
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
while this activity is abolished in peptide kW-NHBzl (see
Table 1). The reasons for this peculiar behavior are currently not
The rise in antibiotic resistance among pathogenic bacteria and
the declining rate of novel antibacterials reaching the market are a
major concern in medicine.1–5 As a result, there is a pressing need
for novel classes of antibacterial agents with new or combined
mechanisms of action and reduced likelihood to lead to the devel-
opment of resistance. Cationic antimicrobial peptides and their
mimetics are currently investigated as a new source of potential
antibacterial agents in preclinical and clinical settings.6,7 Cationic
antimicrobial peptides that contain 10–50 amino acids are
amphiphilic, and are rich in lysine or arginine and hydrophobic
amino acids.8–10 However, shorter amphiphilic peptide sequences
as short as di- or tri- or hexapeptides with antibacterial activity
are known.11–13 We were especially encouraged by a previous
understood.
Numerous studies with linear, cyclic, and diastereomeric cat-
ionic antimicrobial peptides have strongly supported the hypothe-
sis that their physicochemical properties rather than any precise
sequence are responsible for their activities.8–10 It is generally be-
lieved that the amphiphilic topology is essential for insertion into
and disruption of the cytoplasmic membrane. However, other
mode of actions including intracellular targets have also been sug-
gested.19 In particular, the ability to rapidly kill bacteria and the
relative difficulty with which bacteria develop resistance in vitro
make cationic antimicrobial peptides attractive targets for drug
development.9 Previously, we and others have shown that
aminoglycoside antibiotics-derived amphiphiles (AADAs) form a
novel class of potent antibacterial agents.14–17,31 The physico-
chemical similarities between AADAs and cationic antimicrobial
peptides suggest a membranolytic mode of action. This mechanism
is supported by (a) the observed concentration-dependent
hemolytic activity of AADAs; (b) the presence of one or more
hydrophobe(s) to induce antibacterial activity and (c) the prece-
dence of a plethora of cationic antibacterial amphiphiles with
membranolytic mode of actions. In this paper we report on the
synthesis of amphiphilic lysine-ligated neomycin B analogs in
which the C500-position of neomycin B is ligated to hydrophobic
lysine analogs or lysine-containing hydrophobic peptide sequence
report that has shown that both dipeptides H-D-Lys-Trp-OBn
(kW-OBn) and H- -Lys-Trp-OBn (KW-OBn) display potent antibac-
L
terial Gram-positive activity against Staphylococcus aureus
(S. aureus), methicillin resistant S. aureus (MRSA), and methicillin
resistant Staphylococcus epidermidis (MRSE) strains.11 Interestingly,
it was observed that the nature of the C-terminus contributes
substantially to the antimicrobial activity of these two peptides.22
For instance, dipeptide kW-OBn exhibits strong S. aureus activity
⇑
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.2011.01.015

S. Bera et al. / Carbohydrate Research 346 (2011) 560–568
561
Table 1
Antibacterial activity (MIC) in
lg/mL of various Lys-Trp dipeptides, neomycin B–lysine conjugate 5 and amphiphilic neomycin B–peptide conjugates 7 and 17 and control
compounds against various Gram-positive and Gram-negative bacterial strains.
Organisms
Genta-micin Neo-mycin B kW-NBn kW-OBn Fmoc-kW-OBn 5 (Fmoc-NeoK-OBn) 7 (Fmoc-NeoKW-OBn) 17 (H-WNeoKW-OH)
S. aureusa
1
2
1
512
512
256
512
>512
512
512
>512
512
16
512
2
32
32
64
16
512
>512
2
2
2
4
64
128
128
128
128
16
128
4
4
128
32
32
>256
>256
8
32
4
4
64
4
MRSAb
256
0.25
0.25
8
2
4
S. epidermidisc
MRSEd
0.25
32
32
4
128
4
4
8
S. pneumoniaee
E. colif
64
16
16
128
64
32
32
16
64
>128
E. coli (Gent-R)g
P. aeruginosah
512
512
P. aeruginosa (Gent-R)i 128
Representative minimal inhibitory concentrations (MIC) in
ATCC 14990; d Methicillin-resistant S. epidermidis (CAN-ICU) 61589; e ATCC 49619; f ATCC 25922; g CAN-ICU 61714; h ATCC 27853; i CAN-ICU 62308.
l
g/mL for various bacterial strains: a ATCC 29213; b Methicillin-resistant S. aureus ATCC 33592; c S. epidermidis
(Fig. 1). The single primary hydroxymethyl group at the ribose
moiety (C500-position) in neomycin B was chosen as a point of
modification due to its expected high reactivity in chemical
modifications.21 Previous studies have indicated that modified
C500-analogs of neomycin B retain high binding affinity to RNA
and antimicrobial activities.18,30–32 Most of the previous work on
C500-modified neomycin B analogs has focused on neomycin B–lipid
analogs and neomycin B–fluoroquinolone hybrid antibiotics34
while very little data exist on amphiphilic neomycin B–amino acid
or amphiphilic neomycin B–peptide conjugates that resemble short
antibacterial peptides. We were particularly interested to prepare
followed by reductive amination with a partially protected lysine
building block (Fig. 1). For that purpose, the synthesis of the
lysine-ligated neomycin B building block NeoK is outlined in
Scheme 1. At first, commercially available neomycin B sulfate 1
was converted into Boc-protected neomycin B according to the
literature procedure.20 The primary hydroxyl group of 2 was selec-
tively oxidized with trichlorocyanuric acid (TCCA) in the presence
of catalytic amounts of TEMPO at 0 °C–rt for 3 h, according to
Vasella’s procedure21h to afford aldehyde 3, which was directly
used for the reductive amination reaction. In a model study, alde-
hyde 3 was ligated to Fmoc-L-Lys-OBn to form intermediate imine
C500-modified neomycin B conjugates linked to the
e
-amino function
in the presence of molecular sieves and catalytic amount of acetic
acid in minimum volume of methanol for 4 h at rt. Subsequently,
the reaction mixture was diluted with methanol followed by addi-
tion of sodium cyanoborohydride and 3% methanol–acetic acid
maintaining pH 5–6 and stirring at room temperature for 12 h.
The reaction was monitored by TLC using methanol–CH2Cl2
(1:10) as solvent and developed in ethanolic ninhydrin solution.
The reaction mixture was neutralized with 2% NaOH and then
extracted with EtOAc to afford a crude reaction mixture contain-
of lysine thereby producing novel polycationic lysine mimetics,
which may find utility in solid phase/solution phase antibacterial
peptide synthesis. Moreover, suitably-protected lysine-ligated
neomycin B building blocks may find application for incorporation
of RNA-binding sites into peptides and in addition may serve as
polyfunctional lysine surrogates in antimicrobial peptides.
2. Results and discussion
ing both neomycin–lysine conjugate
4 (53%) and alcohol 2
(47%).20 Separation was achieved by flash column chromatogra-
phy using methanol–CH2Cl2 (1:10) as the eluent. The product
was confirmed by mass as well as NMR spectroscopic data. In
the 1H NMR spectrum shows the appearance of aromatic protons
related to the Fmoc- and benzyl-group between d = 7.80–
6.41 ppm indicating the presence of lysine unit while signals at
d = 5.76–1.40 indicate the neomycin B moiety. Global deprotec-
tion of all Boc-groups afforded the desired conjugate 5 as TFA
salt (Scheme 1).
Once we had demonstrated that the synthetic strategy is appli-
cable to lysine we then developed an interest to study the reduc-
tive amination of neomycin B to the ultrashort lysine-containing
antimicrobial dipeptide KW-OBn.11 For this purpose aldehyde 3
was conjugated to side chain unprotected dipeptide Fmoc-KW-
OBn using the same protocol as previously applied to afford
The chemical manipulation of aminoglycoside antibiotics pro-
vides great opportunities for medicinal chemistry due to the
unique biological properties of this class of compounds.27–31,33
However, the polyfunctional nature of aminoglycoside antibiotic
scaffold frequently requires multi-synthetic protection–deprotec-
tion steps to perform selective chemical modifications. This is often
perceived as a serious limitation to drug development making it
difficult and expensive to access aminoglycoside analogs. We
describe herein, the preparation of lysine-ligated neomycin B
analogs by taking advantage of the presence of a single primary
hydroxy group at the C500-position in neomycin B. We were inter-
ested to develop a synthetic method that would permit regioselec-
tive ligation of the C500-position of neomycin B to the side chain
amino function of lysine. We envisaged that this could be achieved
by regioselective oxidation of the primary alcohol into an aldehyde
NHBoc
O
FmocHN
H2N
NHBoc
NH2
CO2R
FmocHN
AcO
AcO
BocHN
O
NH2
O
AcO
AcO
NHBoc
p
e
p
t
HO
HO
BocHN
NHBoc
O
O
NHBoc
O
CO2R
O
O
OHC
Boc
NHBoc
H2N
OAc
H2N
O
N
BocHN
OAc
O
NH2
OAc
O
O
BocHN
OAC
NH
H2N
i
OH
OAc
O
d
e
O
OAc
O
OH
OAc
(peptide synthesis)
O
OAc
(reductive amination)
BocHN
H2N
OH
O
BocHN
O
OH
R2OC
Neomycin B
NeoK
Figure 1. Synthesis of lysine-ligated neomycin B building block (NeoK) and use in peptide synthesis.

562
S. Bera et al. / Carbohydrate Research 346 (2011) 560–568
NH2
O
NHR
O
NHR
HO
HO
O
HO
HO
HO
HO
H2N
RHN
RHN
H2N
RHN
O
RHN
O
NH2
a
NHR
O
NHR
O
O
OHC
O
HO
NH2
HO
RHN
OH
O
OH
O
OH
O
b
RHN
OH
OH
OH
O
OH
O
OH
OH
O
O
OH
O
OH
O
OH
H2N
RHN
RHN
R = Boc
3
1
2
R = Boc
NHR
NH2
O
NHFmoc
HO
NHFmoc
HO
O
BnOOC
BnOOC
RHN
HO
HO
H2N
RHN
H2N
c
NHR
O
NH2
O
O
O
NH
OH
NH
NH2
O
OH
O
NHFmoc
NHR
d
OH
OH
BnOOC
7 x CF3COOH
OH
O
O
OH
O
OH
O
HO2CF3C x H2N
RHN
H2N HO
5
R = Boc
4
Scheme 1. Reagents and conditions: (a) (Boc)2O, Et3N, MeOH, 55 °C, overnight; (b) TCCA, EtOAc, 0 °C to rt, 3 h; (c) Lysine salt, NaCNBH3, AcOH, MeOH, molecular sieves
powder, rt, 10 h; (d) TFA, 0 °C, 3 min.
neomycin B–peptide conjugate 6 in 48% isolated yield which upon
treatment with TFA produced deblocked 7 as TFA salt (Scheme 2).
The structure of neomycin–KW-OBn conjugate was confirmed by
spectroscopic analysis.
in the presence of molecular sieves powder to give the correspond-
ing aldehyde which was directly used for ligation to Fmoc-KW-OBn
using our previously applied standard protocol to afford conjugate
11 (59%) together with the corresponding primary alcohol 10
(41%). Both compounds could be separated by flash column chro-
matography (Scheme 3).
2.1. Synthesis of AG-AMP using a stepwise approach
We also explored the synthesis of hydroxyl protected lysine-li-
gated neomycin B analog 11 using a stepwise approach (Scheme 3).
Protected lysine–neomycin B conjugate 11 contains six acetate es-
ter protecting groups which are expected to facilitate peptide elon-
gation by preventing potential side reactions involving the
unprotected neomycin B-derived polyol scaffold. Compound 11
was synthesized in a five-step synthesis. At first, the primary hy-
droxyl group was selectively protected as trityl ether 8 using trityl
chloride in pyridine at room temperature in 89% yield. Peracetyla-
tion of 8 using acetic anhydride and DMAP in pyridine afforded 9.
The trityl group in 9 was selectively removed with p-TSA in meth-
anol to afford alcohol 10. Subsequently, the primary hydroxyl
group in 10 was oxidized with pyridinium chlorochromate (PCC)
2.2. Use of lysine–neomycin B conjugate 13 as building block in
peptide chain elongation
In order to use compound 11 as a building block in peptide syn-
thesis, protection of the C500-amino function is necessary. To facil-
itate global deprotection using acidic conditions at a later stage, we
protected the secondary amine as tert-butyl carbamate. This was
achieved by exposure of 11 to excess (Boc)2O and triethylamine
in acetonitrile for 6 h to yield fully protected lysine–neomycin B
conjugate 12 in 73% yield. To explore peptide chain elongation in
solution phase we selected fully protected lysine–neomycin B con-
jugate 12 for N- and C-terminal elongation. Exposure of 12 to
piperidine in DMF produced amine 13 which was coupled to
NHR
H
O
N
HO
RHN
HO
NHR
RHN
O
NHR
O
O
O
HO
HO
O
RHN
OHC
N
H
O
OH
BnO
RHN
c
NHR
O
RHN
O
OH
FmocHN
NH
O
OH
OH
O
O
RHN
HO
RHN
RHN
OH
O
OH
R = Boc
O
OH
R = Boc
3
6
d
H
N
NH
2
O
O
O
HO
HO
HN
FmocHN
BnO
NH
2
H N
2
O
NH
2
O
NH
NH
O
OH
2
OH
7 x TFA
O
OH
O
H N
2
HO
7
Scheme 2. Reagents and conditions: (a) TBTU, DIPEA, DMF, rt, 2 h; (b) 50% TFA, CH2Cl2, rt, 2 h; (c) NaCNBH3, AcOH, MeOH, rt, 4 h; (d) TFA, 0 °C.

S. Bera et al. / Carbohydrate Research 346 (2011) 560–568
563
NHR
O
NHR
O
NHR
HO
HO
O
AcO
AcO
HO
HO
RHN
RHN
NHR
O
NHR
O
RHN
O
NHR
OH
R = Boc
HO
NHR
Ph CO
3
Ph CO
3
O
NHR
NHR
a
O
O
O
b
OH
O
O
RHN
OAc
RHN
RHN
HO
HO
AcO
OH
O
O
R = Boc
R = Boc
O OH
O
OAc
O
O
OAc
OH
RHN
OH
RHN
RHN
2
8
9
NHR
NHR
O
CO Bn
2
O
AcO
AcO
AcO
AcO
FmocHN
RHN
RHN
O
c
NHR
O
NHR
O
H
N
d
e
HO
NHR
NHR
O
O
O
OAc
OAc
RHN
RHN
AcO
AcO
OAc
O
O OAc
O
O
OAc
OAc
R = Boc
RHN
R = Boc
RHN
11
10
Scheme 3. Reagents and conditions: (a) TrCl, Pyridine, rt; (b) Ac2O, Py, DMAP, rt, overnight; (c) p-TSA, MeOH, rt; (d) PCC, CH2Cl2, molecular sieves powder, rt; (e) NaCNBH3,
AcOH–MeOH.
Fmoc-
L
-Trp(Boc)-H using TBTU, DIPEA in DMF to produce neomy-
2.3. Antibacterial properties of lysine-ligated neomycin B
conjugates
cin B-ligated dipeptide 14 in quantitative yield. C-terminal elonga-
tion required deblocking of the benzylester moiety using catalytic
hydrogenation with Pearlman’s catalyst (Pd(OH)2–C) to produce
With amphiphilic lysine–neomycin B conjugates 5, 7, and 17 in
acid 15. Coupling of acid 15 to amine H-
L-Trp(Boc)-OBn produced
hand, we determined the antibacterial minimum inhibitory con-
the tripeptide-nomycin B conjugate 16 in 58% yield. Global depro-
tection of 16 using catalytic amounts of sodium methoxide in
methanol followed by catalytic hydrogenation and exposure to
TFA produced unprotected tripeptide–neomycin B conjugate 17
(Scheme 4).
centration (MIC) in lg/mL of these conjugates against American
Type Culture Collection (ATCC) reference strains as well as clinical
isolates of Gram-positive strains including S. aureus, MRSA,
S. epidermidis, methicillin-resistant S. epidermidis (MRSE), and
Streptococcus pneumoniae as well as Gram-negative strains
1
1
NHR
O
NHR
O
NHR
NHFmoc
AcO
AcO
AcO
O
AcO
AcO
AcO
O
1
1
FmocHN
BnOOC
R NH
1
1
R NH
1
1
O
NH
R OOC
O
RHN
NHR
NHR
N
FmocHN
BnOOC
NHR
O
1
1
1
R
NHR
NHR
NR
O
NHR
HN
NR
O
2
O
O
OAc
O
OAc
O
OAc
1
1
c
R HN
RHN
a
R HN
AcO
AcO
AcO
O OAc
O
O OAc
OAc
O
O
O
OAc
OAc
OAc
1
1
R HN
RHN
R HN
12: R2 = Fmoc ; R1 = Boc
1
14: R2 = Bn ; R = Boc
11: R = Boc
b
2
1
d
2
1
13: R = H ; R = Boc
15: R = H ; R = Boc
c
1
1
NHR
2
NHR
1
NHR
NHR
3
O
R O
3
O
1
HO
R O
O
HO
1
O
NH
1
R NH
1
O
NH
1
R NH
O
NHR
N
1
NHR
N
R
e, f, g
H OC
2
1
H
Bn OC
1
2
NHR
1
NR
O
NHR
3
NR
O
O
OR
O
O
OH
1
1
R HN
N
H
O
3
N
H
R HN
R O
1
N
HO
R
HN
3
O
OR
O
O
OH
O
3
OR
OH
1
1
R HN
R HN
1
2
3
1
16: R = Boc ; R = Fmoc ; R = Ac ;
17: R = H x TFA
Scheme 4. Reagents and conditions: (a) (Boc)2O, DMAP, Et3N, acetonitrile; (b) 25% piperidine, DMF, rt; (c) TBTU, DIPEA, DMF; (d) Pd(OH)2, EtOAc, H2; (e) Na in MeOH, 0 °C to
rt; (f) Pd(OH)2, MeOH, H2; g) TFA, 0 °C.

564
S. Bera et al. / Carbohydrate Research 346 (2011) 560–568
(accuracy 0.002°). GC–MS analyses were performed on a Perkin–
Elmer Turbomass Autosystem XL. Analytical thin-layer chromatog-
raphy was performed on precoated silica gel plates. Visualization
was performed by ultraviolet light and/or by staining with ninhy-
drin solution in ethanol. Chromatographic separations were per-
formed on a silica gel column by flash chromatography (Kiesel
Gel 40, 0.040–0.063 mm; Merck). All the aminoacids used are nat-
Escherichia coli, gentamicin-resistant E. coli, and Pseudomonas aeru-
ginosa (Table 1). We also included three resistant strains obtained
from a national surveillance study assessing antimicrobial resis-
tance in Canadian intensive care units CAN-ICU (Table 1).25 Genta-
micin and neomycin B served as positive controls while the
dipeptides kW-OBn, kW-NHBn, and Fmoc-kW-OBn served as refer-
ence peptides. Our results demonstrate that lysine-ligated neomy-
ural, that is, L-isomer. Yields are given after purification, unless dif-
cin
B
analog
7
(Fmoc-NeoKW-OBn ꢀ 7 TFA, MW = 2038.5)
ferently stated. When reactions were performed under anhydrous
conditions, the mixtures were maintained under nitrogen. Com-
pounds were named following IUPAC rules as applied by Beilstein
Institute AutoNom (version 2.1) software for systematic names in
organic chemistry.
generally exhibits reduced Gram-positive activity (2–8-fold higher
MICs) when compared to reference peptide Fmoc-KW-OBn ꢀ TFA
(MW = 758.3). However, an eightfold reduction in MIC was
observed against two Gram-negative E. coli strains including a gen-
tamicin-resistant organism. Enhancing the overall amphiphilicity
of lysine-ligated neomycin conjugates leads to an increase in anti-
bacterial potency. For instance, amphiphilic dipeptide Fmoc-Neo-
KW-OBn 7 containing a hydrophobic tryptophan (W) residue
shows stronger antibacterial activity when compared to Fmoc-
NeoK-OBn 5. This is consistent with previous findings which have
shown that cationic amphiphilicity is necessary for antibacterial
activity.7 A similar trend is seen with dipeptides H-kW-OBn and
Fmoc-kW-OBn. The enhanced antibacterial activity of Fmoc-pro-
tected cationic peptides has been reported previously and is likely
related to strong hydrophobic effect of the Fmoc-protecting
group.12 The similar antibacterial activity of tripeptide–neomycin
B conjugate 17 (H-WNeoKW-OH) when compared to 7 demon-
strates that the hydrophobicity provided by the Fmoc-group can
be substituted by addition of a tryptophan residue. The preference
for tryptophan residues in short cationic peptide antibiotics has
been previously described.23,24 Rather surprising is the low activity
of amphiphilic neomycin B conjugate 7 against MRSA resulting in a
eightfold reduction of antibacterial potency when compared to
dipeptide Fmoc-kW-OBn. The loss of activity in 7 is higher than
that what would be expected by a 2.5-fold increase in molecular
weight. Overall our biological results indicate that there is little
to no synergistic effects when neomycin B is conjugated to amphi-
philic lysine-containing residues at the C500-position in neomycin B.
In summary, we have developed a synthetic access to suitably-pro-
tected polycationic lysine surrogates that serve as building blocks
in peptide synthesis. Incorporation of these building blocks per-
mits the incorporation of RNA-binding motifs in peptides. All three
lysine–neomycin B conjugates display lower antibacterial activity
when compared to unmodified neomycin B. The only exceptions
are MRSA and two P. aeruginosa strains where modest improve-
ments (up to eightfold lower MICs) were seen. The modest activity
of neomycin B conjugates 5, 7, and 17 against resistant organisms
such as MRSE, gentamicin-resistant E. coli, and P. aeruginosa strains
could be the action of aminoglycoside-modifying enzymes,
reduced binding affinity to RNA, reduced membrane permeability
or other factors. It is currently not possible to eliminate any of
these factors. Based on previous research results obtained with
C-5’’-modified neomycin B analogs it is unlikely that reduced
RNA binding will be the major driving force. Very likely a combina-
tion of organism-specific factors will be responsible to explain the
observed results. We hypothesize that the introduced amphiphilic-
ity in neomycin B conjugates 5, 7, and 17 alters the mode of
action(s) in favor of enhanced permeability.
3.2. 500-N-(Fmoc-Lys-OBn)-1,3,20,60,2000,6000-hexa-N-(tert-
butoxycarbonyl)-500-deoxy-neomycin B (4)
A 0.05 M solution of alcohol 2 (1.0 g, 0.823 mmol) in EtOAc at
ꢁ10 °C was treated with trichloroisocyanuric acid (TCCA; 0.209 g,
0.902 mmol) and a catalytic amount of TEMPO (0.006 g, 0.041
mmol) under N2 was allowed to warm to 25 °C and stirred for 1 h.
After completion the reaction mixture was basified with 1 N NaOH.
After separation of the phases, the aq layer was extracted with EtOAc
(3 ꢀ 50 mL). The combined organic layers were dried (Na2SO4) and
evaporated to obtain the aldehyde 3 (77%). This crude aldehyde
was used for next reaction.
To a solution of 3 (0.08 g, 0.065 mmol) in MeOH (10 mL) was
added Fmoc-Lys-OBn (0.060 g, 0.132 mmol) and stirred at room
temperature for 1 h and then AcOH (0.36 mL) was added and the
mixture was stirred for 30 min. NaBH3CN (0.045 g, 0.72 mmol)
was then added and the mixture was stirred at room temperature
for 4 h. The reaction mixture was neutralized with NaOH and ex-
tracted with AcOEt (3 ꢀ 50 mL). The combined organic layers were
washed with satd NaCl (3 ꢀ 30 mL), dried over Na2SO4, filtered,
and the solvents were evaporated in vacuo to give an oily residue.
Flash column chromatography afforded the desired product 4 as a
white solid. Yield = 53%; Rf 0.46 (CH2Cl2–MeOH 11:1); 1H NMR
(300 MHz, CD3OD):
d 7.80 (d, 2H, J = 9.0 Hz), 7.66 (d, 2H,
J = 7.6 Hz), 7.40 (t, 2H, J = 7.6 Hz), 7.30 (m, 7H), 5.76 (d, 1H,
J = 4.0 Hz), 5.60 (br s, 1H), 5.20–5.10 (m, 4H), 5.09 (br s, 1H), 5.00
(d, 2H, J = 8.3 Hz), 4.79 (m, 1H), 4.61 (m, 1H), 4.49 (d, 1H,
J = 5.0 Hz), 4.40–4.30 (m, 4H), 4.20–4.12 (m, 3H), 4.10 (m, 1H),
3.92 (m, 3H), 3.80 (m, 2H), 3.51 (m, 6H), 3.40 (m, 1H), 3.21 (m,
1H), 2.85 (m, 1H), 2.60 (m, 1H), 1.97 (m, 2H), 1.66 (m, 2H), 1.53
(m, 2H), 1.40 (m, 54H); 13C NMR (75 MHz, CD3OD): d 173.8 (ester–
CO), 159.2, 159.0, 158.7, 158.5, 158.4, 158.3, 157.9, (amide–CO),
145.0–120.7 (aromatic carbons), 111.6, 99.4, 98.1 (anomeric car-
bons), 80.9 (ꢀ2), 80.7 (ꢀ2), 80.3 (ꢀ4), 79.0, 76.3, 75.2, 74.3, 73.1,
72.9 (ꢀ2), 72.8, 72.6, 71.8, 71.4 (ꢀ2), 68.4, 68.0 (ꢀ2), 67.7, 56.0,
54.2, 53.1, 52.3, 50.6, 42.4, 41.4, 35.8, 32.8, 32.3, 29.0–28.7, 24.7;
þ
½
a 2D5
ꢂ
¼ þ34:0 (c 0.2, MeOH); EIMS: calcd for C81H122N8NaO28
1677.84, found: 1678.02 [M+Na]+. Anal. Calcd for C81H122N8O28: C,
58.75; H, 7.43; N, 6.77. Found: C, 59.11; H, 7.67; N, 6.91.
3.3. 1,3,20,60,2000,6000-Heptaammonium-500-N-(Fmoc-Lys-OBn)-500-
deoxy-neomycin B-hexafluoroacetate (5)
Compound 4 (0.12 g, 0.7255 mmol) was treated with 99% triflu-
oroacetic acid (2 mL) for 3 min at 0 °C. The volatiles were removed
in vacuo. The nonpolar residue was removed by washing with
ether–methanol (1%) mixture and decanted the solvent to get the
conjugates 5 as TFA salt. Yield = 88%; Rf 0.25 (CH2Cl2–MeOH–
3. Experimental
3.1. General methods—chemistry
NH4OH 7:5:2); 1H NMR (300 MHz, CD3OD):
d 7.80 (d, 2H,
NMR spectra were recorded on a Brucker Avance 300 spectrom-
eter (300 MHz for 1H NMR, 75 MHz for 13C) and AMX 500 spec-
trometer (500 MHz for 1H NMR). Optical rotation was measured
at a concentration of g/100 mL, with a Perkin–Elmer polarimeter
J = 7.3 Hz), 7.66 (t, 2H, J = 7.4 Hz), 7.40 (d, 2H, J = 7.3 Hz), 7.30 (m,
7H), 5.97 (d, 1H, J = 4.3 Hz), 5.82 (d, 1H, J = 1.8 Hz), 5.35 (t, 1H,
J = 4.3 Hz), 5.33 (br s, 1H), 5.15 (dd, 2H, J = 6.6, 10.5 Hz), 4.76 (m,

S. Bera et al. / Carbohydrate Research 346 (2011) 560–568
565
1H), 4.61 (m, 1H), 4.40 (m, 2H), 4.32 (dd, 2H, J = 6.6, 10.5 Hz), 4.25–
4.12 (m, 3H), 4.10 (m, 1H), 4.05–3.88 (m, 4H), 3.73 (q, 2H,
J = 9.0 Hz), 3.71 (m, 2H), 3.67–3.58 (m, 2H), 3.49–3.40 (m, 3H),
3.26 (m, 1H), 3.18 (m, 3H), 2.86 (t, 1H, J = 7.3 Hz), 2.49 (m, 1H),
2.22 (m, 1H), 1.86 (m, 1H), 1.66 (m, 2H), 1.43 (m, 2H); 13C NMR
(75 MHz, CD3OD): d 173.8 (ester), 158.7 (Fmoc-CO), 145.3–120.9
(aromatic carbons), 111.2, 99.4, 98.9 (anomeric carbons), 87.8,
77.9, 77.2, 75.0, 74.3, 73.6, 72.0, 71.4, 71.2, 71.0, 70.9, 69.8, 69.4,
69.2, 68.1, 62.1, 61.5, 55.4, 55.2, 52.7, 51.4, 50.4, 50.2, 42.1, 42.0,
40.5, 31.9, 28.1, 24.5; EIMS: calcd for C51H75N8O16þ 1055.52, found:
1055.60 [M+H]+. Anal. Calcd for C65H81F21N8O30: C, 42.12; H, 4.41;
F, 21.53; N, 6.05. Found: C, 42.22; H, 4.39; F, 21.5763; N, 6.11.
41.6, 31.9, 30.1, 28.9, 23.8; ½a D25
ꢂ
¼ þ29:0 (c 0.55, MeOH); EIMS:
calcd for C62H85N10O17 1241.60, found: 1241.64 [M+H]+. Anal.
Calcd for C76H91F21N10O31: C, 44.76; H, 4.50; F, 19.56; N, 6.87.
Found: C, 44.89; H, 4.55; F, 19.87; N, 6.31.
þ
3.6. 500-O-Triphenylmethyl-1,3,20,60,2000,6000-hexa-N-(tert-
butoxycarbonyl)-neomycin B (8)
A solution of 2 (1.0 g, 0.82 mmol) in dry pyridine (20 mL) was
treated with trityl chloride (0.66 g, 2.46 mmol) and stirred at rt
for 10 h and then removed the solvent under reduced pressure.
The crude reaction mixture was partitioned between H2O
(60 mL) and EtOAc (30 mL). The aqueous layer was separated and
extracted with EtOAc (2 ꢀ 40 mL). The combined organic layer
was washed with brine, dried over Na2SO4, and concentrated in va-
cuo. Flash column chromatography afforded 8 as a white solid.
Yield = 89%; Rf 0.45 (CH2Cl2–MeOH 12:1); 1H NMR (300 MHz,
CD3OD): d 7.42 (d, 6H, J = 7.5 Hz), 7.29 (t, 6H, J = 7.2 Hz), 7.21 (t,
3H, J = 6.9 Hz), 6.12 (d, 1H, J = 8.7 Hz), 5.66 (br s, 1H), 5.32 (s,
1H), 5.11 (s, 1H), 4.90 (m, 2H), 4.74 (br s, 1H), 4.36 (br s, 1H),
4.11 (m, 3H), 3.92 (d, 1H, J = 8.7 Hz), 3.70 (m, 3H), 3.60 (m, 5H),
3.25 (m, 5H), 2.66 (br s, 1H), 2.39 (m, 1H), 1.46 (s, 54H); 13C
NMR (75 MHz, CD3OD): 158.9, 158.5, 158.2 (ꢀ2), 157.9, 150.1
(Boc-CO), 145.5–128.1 (anomeric carbons), 110.8, 99.6, 98.0 (ano-
meric carbons), 88.0, 87.7, 82.2, 80.8 (ꢀ2), 80.7, 80.5 (ꢀ2), 80.0,
78.1, 77.7, 75.6, 75.3, 74.2, 73.5, 73.0, 68.6, 64.2, 56.4, 53.5, 52.4,
3.4. 500-N-(Fmoc-Trp-Lys-OBn)-1,3,20,60,2000,6000-hexa-N-(tert-
butoxycarbonyl)-500-deoxy-neomycin B (6)
To a solution of 3 (0.08 g, 0.065 mmol) in MeOH (10 mL) were
added Fmoc-Lys-Trp-OBn (0.060 g, 0.132 mmol) and molecular
sieves powder and stirred at room temperature for 1 h and then
AcOH (0.36 mL) was added and the mixture was stirred for
30 min. NaBH3CN (0.045 g, 0.72 mmol) was then added and the
mixture was stirred at room temperature for 8 h. The reaction
mixture was neutralized with NaOH and extracted with AcOEt
(3 ꢀ 50 mL). The combined organic layers were washed with satd
NaCl (3 ꢀ 30 mL), dried over Na2SO4, filtered, and the solvents
were evaporated in vacuo to give an oily residue. Flash column
chromatography afforded the desired product 6 as a white solid.
Yield = 48%; Rf 0.45 (CH2Cl2–MeOH 17:1); 1H NMR (300 MHz,
CD3OD): d 8.13 (t, 1H, J = 8.3 Hz), 7.84 (t, 1H, J = 6.3 Hz), 7.82
(d, 2H, J = 6.3 Hz), 7.60 (s, 1H), 7.52 (m, 1H), 7.45–7.20 (m,
10H), 7.03 (dd, 1H, J = 2.6, 7.6 Hz), 6.98 (d, 1H, J = 5.4 Hz), 5.33
(s, 1H), 5.17 (s, 2H), 4.91 (s, 1H), 4.78 (m, 4H), 4.58 (s, 1H),
4.36 (t, 1H, J = 7.8 Hz), 4.20 (m, 2H), 3.98 (m, 1H), 3.91 (m, 3H),
3.73 (m, 4H), 3.66 (m, 1H), 3.52 (m, 9H), 3.38 (m, 2H), 3.20 (m,
3H), 2.96 (m, 1H), 2.76 (m, 1H), 2.01 (m, 2H), 1.62 (m, 2H),
1.48 (3 s, 54H), 1.37 (m, 2H); 13C NMR (75 MHz, CD3OD, HSQC):
d 127.7–119.7 (aromatic carbons), 110.9, 106.1, 99.5 (anomeric
carbons), 86.0, 85.7, 85.3, 82.2, 80.9 (ꢀ2), 80.7 (ꢀ2), 78.2, 77.6,
76.8, 74.0, 73.3, 71.5, 71.1, 70.0, 68.1, 66.5, 65.9, 55.5, 54.6,
53.9, 52.0, 51.6, 50.8, 49.6, 48.3, 46.5, 40.6, 34.3, 34.0, 27.6–
42.2, 41.2, 37.1, 36.0, 31.8, 29.3–28.9; ½a D25
ꢂ
¼ þ22:0 (c 0.55,
þ
MeOH); EIMS: calcd for
C
72H108N6NaO25
1479.74, found:
1480.04 [M+Na]+. Anal. Calcd for C72H108N6O25: C, 59.33; H, 7.47;
N, 5.77. Found: 59.11; H, 7.25; N, 5.89.
3.7. 6,30,40,200,500,4000-Hexa-O-acetyl-1,3,20,60,2000,6000-hexa-N-(tert-
butoxycarbonyl)- 500-O-triphenylmethyl neomycin B (9)
To a solution of 8 (1.36 g, 10 mmol), in pyridine (10 mL) were
added acetic anhydride (1.3 g, 13 mmol) and DMAP (1.5 g,
12 mmol). After stirring at rt for 6 h the reaction mixture was neu-
tralized by adding hydrochloric acid (1.0 N) and aqueous sodium
bicarbonate solution and then partitioned between H2O (100 mL)
and EtOAc (100 mL). The organic layer was dried (anhydrous
Na2SO4), evaporated, and purified by flash column chromatogra-
phy to afford 9 as a white solid. Yield = 89%; Rf 0.45 (CH2Cl2–MeOH
17:1); 1H NMR (300 MHz, CD3OD): d 7.50 (d, 3H, J = 7.3 Hz), 7.45 (d,
3H, J = 7.3 Hz), 7.36 (t, 4H, J = 6.6 Hz), 7.26 (m, 2H), 7.12 (d, 3H,
J = 7.6 Hz), 5.66 (s, 1H), 5.32 (s, 1H), 5.25 (s, 1H), 5.04–4.77 (m,
5H), 4.64 (m, 4H), 3.87 (m, 3H), 3.70 (m, 4H), 3.52 (t, 1H,
J = 8.8 Hz), 3.32 (m, 1H), 3.22 (m, 4H), 2.91 (m, 1H), 2.28 (m, 1H),
2.22 (s, 3H), 2.18 (s, 3H), 2.11 (s, 3H), 2.01 (s, 3H), 1.93 (s, 3H),
1.66 (s, 3H), 1.46 (s, 54H); 13C NMR (75 MHz, CDCl3): 171.1,
170.3, 169.4, 169.1 (ꢀ2), 168.4, 156.6, 155.3 (ꢀ2), 154.9 (ꢀ2),
154.6, 144.5–127.0 (aromatic carbons), 107.8, 97.6, 96.7 (anomeric
carbons), 86.4, 82.9, 80.2, 79.7 (ꢀ2), 79.4 (ꢀ2), 79.3 (ꢀ3), 78.8, 77.3,
75.7, 74.2, 73.2, 71.9, 68.9, 68.6 (ꢀ2), 66.6, 61.6, 52.4, 49.4, 49.0,
28.0, 24.3;
C
½
a 2D5
ꢂ
¼ þ43:0 (c 0.35, MeOH); EIMS: calcd for
92H132N10NaO29 1863.92, found: 1864.13 [M+Na]+. Anal. Calcd
þ
for C92H132N10O29: C, 59.99; H, 7.22; N, 7.60. Found: C, 60.11;
H, 7.31; N, 7.55.
3.5. 1,3,20,60,2000,6000-Hexaammonium-500-N-(Fmoc-Trp-Lys-OBn)-
500-deoxy-neomycin B-hexafluoroacetate (7)
Procedure same as above. Yield = 85%; Rf 0.12 (NH4OH–CH2Cl2–
MeOH 2:9:4); 1H NMR (300 MHz, CD3OD): d 7.82 (dd, 2H, J = 3.4,
7.3 Hz), 7.72 (d, 2H, J = 7.9 Hz), 7.60 (d, 2H, J = 7.3 Hz), 7.41 (t,
2H, J = 7.5 Hz), 7.35 (m, 3H), 7.22 (m, 3H), 7.16 (m, 3H), 7.03 (t,
1H, J = 7.5 Hz), 5.97 (d, 1H, J = 3.4 Hz), 5.85 (d, 1H, J = 3.6 Hz),
5.46 (m, 1H), 5.39 (d, 1H, J = 3.4 Hz), 5.33 (m, 1H), 5.29 (m, 1H),
5.18 (m, 2H), 4.97 (m, 1H), 4.58 (d, 1H, J = 5.5 Hz), 4.45 (d, 1H,
J = 14.6 Hz), 4.32 (m, 3H), 4.16 (m, 2H), 4.03 (m, 3H), 3.87 (m,
1H), 3.75 (dd, 1H, J = 4.8, 10.1 Hz), 3.69 (br d, 1H, J = 2.8 Hz), 3.64
(m, 1H), 3.59 (m, 1H), 3.44 (m, 4H), 3.28 (m, 1H), 3.24 (m, 1H),
3.20 (m, 1H), 3.17 (d, 1H, J = 4.5 Hz), 3.10 (m, 3H), 2.83 (m, 1H),
2.48 (m, 2H), 2.16 (m, 1H), 2.01 (m, 1H), 1.72 (m, 1H), 1.58 (m,
2H); 13C NMR (75 MHz, CD3OD): d 173.6 (ester–CO), 164.0–162.7
40.0, 35.8, 28.4–28.0, 21.0–20.5; ½a D25
ꢂ
¼ þ36:0 (c 0.55, MeOH);
þ
EIMS: calcd for
C
84H120N6NaO31
1731.80, found: 1731.9
[M+Na]+. Anal. Calcd for C84H120N6O31: C, 59.00; H, 7.07; N, 4.91.
Found: C, 59.23; H, 7.38; N, 5.03.
3.8. 6,30,40,200,500,4000-Hexa-O-acetyl-1,3,20,60,2000,6000-hexa-N-(tert-
butoxycarbonyl)- neomycin B (10)
1
(TFA carbons, q with JCF ꢃ34.8 Hz), 124.1–112.4 (TFA carbons, q
To compound 9 (0.76 g, 0.44 mmol) in methanol was added a
solution containing p-TSA (0.08 g) in MeOH (10 mL) at rt. After
1 h, the reaction mixture was neutralized with 5% NaHCO3 and
diluted with CH2Cl2 and extracted with CH2Cl2 (3 ꢀ 50 mL). The
organic phase was dried (anhydrous Na2SO4), filtered, and the
1
with JCF ꢃ292.0 Hz), 158.4 (ꢀ2) (amide CO), 145.5–119.7 (aro-
matic carbons), 110.4, 97.1, 96.6 (anomeric carbons), 86.4, 78.5,
76.9, 75.1, 74.3, 74.0, 73.3, 72.0, 71.8, 71.0, 69.8, 69.4, 69.2, 68.4,
58.9, 55.9, 55.2, 54.9, 52.9, 51.4, 51.2, 50.6, 50.3, 50.2, 44.3, 42.0,

566
S. Bera et al. / Carbohydrate Research 346 (2011) 560–568
solvent was removed in vacuo to afford a solid residue. Flash col-
umn chromatography afforded 10 as a white solid. Yield = 78%; Rf
0.44 (CH2Cl2–MeOH 17:1); 1H NMR (300 MHz, acetone-d6): d
6.07 (br s, 1H, NH), 5.92 (d, 1H, J = 8.7 Hz), 5.81 (d, 1H,
J = 8.7 Hz), 5.68 (s, 1H), 5.29 (m, 2H), 5.08 (t, 1H, J = 10.3 Hz),
4.74 (br s, 1H), 4.94 (m, 3H), 4.80–4.72 (m, 3H), 4.31 (t, 1H,
J = 5.8 Hz), 4.14 (t, 1H, J = 7.7 Hz), 4.08 (m, 2H), 3.97 (dt, 1H,
J = 3.6, 9.9 Hz), 3.87 (t, 1H, J = 5.8 Hz), 3.75 (m, 3H), 3.62 (m, 1H),
3.32 (m, 2H), 3.22 (m, 1H), 2.10 (4 s, 12H), 1.97 (s, 3H), 1.90 (s,
3H), 1.75 (t, 1H, J = 12.1 Hz), 1.51 (m, 1H), 1.40 (s, 54H); 13C NMR
(75 MHz, CD3OD): 171.2, 170.7, 170.4, 169.6, 169.2, 168.4 (ace-
tates), 155.9 (ꢀ2), 155.7, 155.0 (ꢀ3) (Boc-CO), 105.9, 99.1, 98.8
(anomeric carbons), 82.5, 81.9, 80.4, 79.9, 79.5 (ꢀ3), 79.4 (ꢀ3),
77.3, 76.1, 74.3, 72.9, 71.8, 70.1, 69.1, 68.9, 66.6, 61.1, 53.4, 49.2,
3.63 (m, 4H), 3.40 (m, 3H), 3.24 (m, 3H), 3.10 (m, 1H), 2.70 (m,
1H), 2.40 (t, 1H, J = 5.8 Hz), 2.28–1.90 (4 s, 18H), 1.60 (m, 2H),
1.50 (s, 9H), 1.40 (3s, 54H), 1.35 (m, 2H); 13C NMR (75 MHz,
CD3OD): 173.6–170.4 (carbonyls), 158.9–157.3 (Boc-CO), 145.3–
121.0 (aromatic carbons), 110.3, 99.1, 97.8 (anomeric carbons),
84.8, 81.3, 80.9, 80.7, 80.4, 78.9, 78.1, 77.4, 75.1, 74.3, 73.3, 70.3,
70.1, 69.7, 68.1, 68.0, 67.5, 55.4, 54.3, 52.9, 50.6, 50.1, 41.1, 40.5,
34.9, 32.3, 29.2–28.9, 24.3–20.9; ½a D25
ꢂ
¼ þ27:0 (c 0.04, MeOH);
þ
EIMS: calcd for
C
98H142N8NaO36
2030.94, found: 2029.33
[M+Na]+. Anal. Calcd for C98H142N8O36: C, 58.61; H, 7.13; N, 5.58.
Found: C, 58.95; H, 7.39; N, 5.31.
3.11. 500-N-(H-Lys-OBn)-6,30,40,200,500,4000-hexa-O-acetyl-1,3,20,60,-
500,2000,6000-hepta-N-(tert-butoxycarbonyl)- 500-deoxy-neomycin B
(13)
48.9, 41.1, 40.5, 33.9, 28.3–28.2, 21.0–20.2 (acetates);
þ
½
a 2D5
ꢂ
¼ þ28:0 (c 0.45, MeOH); EIMS: calcd for C65H106N6NaO31
1489.69, found: 1489.69 [M+Na]+. Anal. Calcd for C65H106N6O31
C, 53.20; H, 7.28; N, 5.73. Found: C, 53.41; H, 7.45; N, 5.41.
:
To a solution of compound 12 (0.15 g, 0.074 mmol) in DMF
(5 mL), 15% piperidine in DMF was added and stirred at rt for
3 h. The solvent was removed under reduced pressure and purified
through flash chromatography afforded the desired product as a
white solid. Yield = 78%; Rf 0.25 (CH2Cl2–MeOH 10:1); 1H NMR
(300 MHz, CD3OD): d 7.40 (m, 5H), 5.63 (t, 1H, J = 3.1 Hz), 5.27
(m, 1H), 5.21 (m, 2H), 5.17 (m, 2H), 5.05 (t, 1H, J = 9.7 Hz), 4.96
(m, 2H), 4.89 (m, 1H), 4.75 (br s, 1H), 4.64 (m, 1H), 4.12 (m, 3H),
3.96 (m, 3H) 3.88 (m, 2H), 3.74 (m, 2H), 3.67 (m, 2H), 3.63 (t, 2H,
J = 7.5 Hz), 3.53 (m, 2H), 3.48 (m, 1H), 3.44 (m, 1H), 3.27 (m, 1H),
3.21 (m, 3H), 2.28 (m, 1H), 2.18–1.90 (4 s, 18H), 1.86 (m, 1H),
3.9. 500-N-(Fmoc-Lys-OBn)-6,30,40,200,500,4000-hexa-O-acetyl-1,3,20,60,-
2000,6000-hexa-N-(tert-butoxycarbonyl)-500-deoxy-neomycin B (11)
To a solution of alcohol 10 (1.0 g, 0.823 mmol) in CH2Cl2,
pyridinium chlorochromate (0.162 g, 0.74 mmol) and molecular
sieves powder were added and stirred at rt for 3 h. After comple-
tion the reaction mixture was diluted with CH2Cl2 and filtered
through pad of Celite. The filtrate was evaporated to obtain the
aldehyde. The crude aldehyde was used for next reaction. Cou-
pling was performed with H-Lys-OBn ester as above described
procedure to get 11. Yield = 59%; Rf 0.45 (CH2Cl2–MeOH 17:1);
1H NMR (300 MHz, CD3OD): d 7.80 (d, 2H, J = 8.1 Hz), 7.65 (d,
2H, J = 7.7 Hz), 7.56 (t, 2H, J = 7.0 Hz), 7.39 (t, 2H, J = 7.2 Hz),
7.35 (m, 3H), 7.29 (m, 2H), 5.66 (d, 1H, J = 4.3 Hz), 5.22–5.13
(m, 5H), 5.08 (t, 1H, J = 9.7 Hz), 4.96 (t, 2H, J = 8.3 Hz), 4.81 (m,
2H), 4.72 (m, 2H), 4.31 (dd, 1H, J = 6.2, 10.2 Hz), 4.18 (t, 2H,
J = 6.7 Hz), 4.08 (m, 2H), 3.92 (m, 4H), 3.76–3.52 (m, 5H), 3.44
(m, 2H), 3.25 (m, 2H), 2.93 (m, 2H), 2.17–1.90 (4 s, 18H), 2.08
(m, 2H), 1.87 (m, 1H), 1.71 (m, 2H), 1.61 (m, 2H), 1.55 (m, 1H),
1.50 (3s, 54H); 13C NMR (75 MHz, CD3OD): 173.7–170.4 (carbon-
yls), 158.9–157.2 (Boc-CO), 145.3–121.0 (aromatic carbons),
110.3, 98.9, 97.8 (anomeric carbons), 84.8, 81.3, 80.9, 80.7, 80.5
(ꢀ2), 80.4 (ꢀ2), 78.9, 78.1, 77.4, 75.1, 74.3, 73.3, 70.3, 70.1,
69.7, 68.1 (ꢀ2), 68.0 (ꢀ2), 67.5, 55.4, 54.3, 52.9, 50.6, 50.1, 48.4,
1.72–1.63 (m, 2H), 1.52 (s, 9H), 1.48 (3 s, 54H); ½a D25
¼ þ32:0 (c
ꢂ
þ
0.65, MeOH); EIMS: calcd for C83H132N8NaO34 1807.88, found:
1808.12 [M+Na]+. Anal. Calcd for C83H132N8O34: C, 55.82; H, 7.45;
N, 6.27. Found: C, 55.79; H, 7.38; N, 6.55.
3.12. 500-N-(Fmoc-Trp(Boc)-Lys-OBn)-6,30,40,200,500,4000-hexa-O-
acetyl-1,3,20,60,500,2000,6000-hepta-N-(tert-butoxycarbonyl)-500-
deoxy-neomycin B (14)
To a solution of 13 (1 equiv) in dry DMF, TBTU (2 equiv), Fmoc-
Trp(Boc)-OH (1 equiv) and DIPEA (3 equiv) were added and stirred
at room temperature for 2 h. The reaction mixture was triturated
with H2O and EtOAc. The EtOAc layer was washed with H2O, brine,
dried over sodium sulfate and concentrated. The crude residue was
purified using flash silica gel by eluting with MeOH–CH2Cl2 to ob-
tain pure 14 as syrup. Yield = 74%; Rf 0.45 (CH2Cl2–MeOH 15:1); 1H
NMR (300 MHz, CD3OD): d 8.07 (d, 2H, J = 8.3 Hz), 7.75 (d, 2H,
J = 8.3 Hz), 7.60 (d, 1H, J = 7.7 Hz), 7.56 (m, 2H), 7.50 (m, 2H),
7.35 (m, 3H), 7.22 (m, 4H), 7.16 (m, 3H), 5.60 (d, 1H, J = 2.9 Hz),
5.26 (m, 2H), 5.20 (m, 2H), 5.08 (t, 1H, J = 9.8 Hz), 4.98 (m, 1H),
4.89 (m, 1H), 4.74 (m, 2H), 4.64 (br s, 1H), 4.57 (m, 2H), 4.57 (m,
1H), 4.36 (m, 1H), 4.10 (m, 4H), 4.00 (dd, 2H, J = 4.5, 10.7 Hz),
3.92 (m, 1H), 3.86 (m, 2H), 3.74 (m, 2H), 3.63 (m, 3H), 3.54 (m,
2H), 3.53 (m, 1H), 3.45 (m, 1H), 3.34 (m, 1H), 3.20 (m, 2H), 3.10
(m, 2H), 2.30–1.90 (m, 20H), 1.67 (m, 2H), 1.56 (s, 9H), 1.49 (s,
9H), 1.40 (s, 54H), 1.30 (m, 2H); 13C NMR (75 MHz, CD3OD): d
174.0 (ester–CO), 173.1, 172.9, 172.3, 171.9, 171.7, 170.7 (ace-
tates–CO), 167.4, 164.5 (peptide–amide, NHCO), 158.8, 158.4,
158.1 (ꢀ2), 157.9, 157.4 (ꢀ2), 157.2 (amide CO), 145.2–118.7 (aro-
matic carbon), 109.3, 99.7, 98.7 (anomeric carbon), 84.7, 81.7, 81.2
(ꢀ2), 81.0, 80.4 (ꢀ2), 80.3 (ꢀ3), 80.0, 79.4, 78.7, 76.9, 75.3, 74.7,
74.0, 73.3, 70.8, 69.9, 68.4 (ꢀ2), 68.1 (ꢀ2), 66.5, 55.9, 54.9, 52.9,
50.8, 50.4, 48.3, 41.6, 38.9, 37.6, 37.0, 35.2, 32.4, 31.9, 30.1, 29.9–
41.1, 40.5, 34.9, 32.3, 29.0–28.8, 24.2–20.8;
½
a 2D5
ꢂ
¼ þ29:0 (c
þ
0.93, MeOH); EIMS: calcd for C93H134N8NaO34 1929.90, found:
1929.90 [M+Na]+. Anal. Calcd for C93H134N8O34: C, 58.54; H,
7.08; N, 5.87. Found: C, 58.81; H, 7.39; N, 5.69.
3.10. 500-N -(Fmoc-Lys-OBn)-6,30,40,200,500,4000-hexa-O-acetyl-
1,3,20,60,500,2000,6000-hepta-N-(tert-butoxycarbonyl)-500-deoxy-
neomycin B (12)
To compound 11 (0.15 g, 0.078 mmol) in acetonitrile (1 mL)
were added triethylamine (0.040 mL, 0.314 mmol) and (Boc)2O
(0.685 g, 0.314 mmol) and stirred at rt for 4 h. The reaction mixture
was partitioned between EtOAc and H2O and the organic layer was
dried over Na2SO4, and filtered, the solvents were evaporated in va-
cuo to give an oily residue. Flash column chromatography afforded
the desired product 12 as a white solid. Yield = 73%; Rf 0.45
(CH2Cl2–MeOH 17:1); 1H NMR (300 MHz, CD3OD): d 7.81 (d, 2H,
J = 7.1 Hz), 7.67 (d, 2H, J = 8.3 Hz), 7.41 (t, 4H, J = 6.2 Hz), 7.31 (t,
5H, J = 6.2 Hz), 5.60 (d, 1H, J = 3.1 Hz), 5.24 (m, 5H), 5.05 (t, 1H,
J = 9.7 Hz), 4.96 (m, 2H), 4.83 (m, 2H), 4.70 (m, 2H), 4.37 (t, 1H,
J = 6.2 Hz), 4.31 (q, 2H, J = 7.1 Hz), 4.21 (t, 1H, J = 6.2 Hz), 4.01 (m,
3H), 3.99 (m, 2H) 3.85 (t, 1H, J = 7.6 Hz), 3.73 (d, 1H, J = 7.5 Hz),
28.8, 23.8–20.8; ½a D25
¼ þ36:0 (c 0.2, MeOH); EIMS: calcd for
ꢂ
þ
C
114H160N10NaO39 2316.08, found: 2316.44 [M+Na]+. Anal. Calcd
for C114H160N10O39: C, 59.67; H, 7.03; N, 6.10. Found: C, 60.11; H,
7.23; N, 6.16.

S. Bera et al. / Carbohydrate Research 346 (2011) 560–568
567
3.13. 500-N-(Fmoc-Trp(Boc)-Lys-OH)-6,30,40,200,500,4000-hexa-O-
acetyl-1,3,20,60,500,2000,6000-hepta-N-(tert-butoxycarbonyl)-500-
deoxy-neomycin B (15)
decanted to get a solid which was lyophilized to produce 17 as salt.
Yield = 33% (in 3 steps); Rf 0.10 (CH2Cl2–MeOH–NH4OH 5:6:1); 1H
NMR (300 MHz, CD3OD): d 7.62–7.40 (m, 4H), 7.33–7.00 (m, 6H),
5.28 (br s, 2H), 5.11 (br s, 2H), 4.86 (m, 2H), 4.35 (m, 5H), 4.19
(m, 2H), 3.99 (m, 1H), 3.86 (m, 2H), 3.80 (m, 2H), 3.70–3.65 (m,
5H), 3.51 (m, 2H), 3.40 (m, 3H), 3.33 (m, 1H), 3.26 (m, 3H), 3.00
(m, 2H), 2.32 (m, 2H), 2.18 (dt, 2H, J = 3.7, 12.1 Hz), 2.00 (m, 1H),
1.68 (m, 1H), 1.49 (m, 1H), 1.30 (m, 1H); 13C NMR (75 MHz, CD3OD,
HSQC): d 1214.8–114.4 (aromatic carbons), 109.0, 98.5, 98.3 (ano-
meric carbons), 84.6, 80.6, 79.4, 78.5, 73.4, 74.1, 71.5, 74.0, 72.9,
71.1, 70.0, 69.4, 66.9, 55.0, 54.6, 54.1, 52.9, 51.9, 51.0, 50.2, 49.6,
The solution of 14 (130 mg, 0.104 mmol) and 10% Pd(OH)2–C in
EtOAc (5 mL) was hydrogenated at normal temperature and
pressure for 5 h and then filtered through Celite. The filtrate was
concentrated and the residue was purified on flush column chro-
matography to afford 16 as a white solid. Yield = 87%; Rf 0.16
(CH2Cl2–MeOH 9:1); 1H NMR (300 MHz, CD3OD): d 8.07 (d, 1H,
J = 6.0 Hz), 7.73 (t, 2H, J = 7.3 Hz), 7.70 (br s, 1H), 7.56 (m, 1H),
7.48 (d, 1H, J = 7.1 Hz), 7.41 (d, 1H, J = 7.1 Hz), 7.37 (d, 2H,
J = 4.1 Hz), 7.27 (d, 1H, J = 4.2 Hz), 7.23 (s, 1H), 7.25 (t, 2H,
J = 7.5 Hz), 5.60 (s, 1H), 5.28 (m, 2H), 5.05 (t, 1H, J = 9.4 Hz), 4.95
(m, 1H), 4.73 (m, 2H), 4.64 (m, 2H), 4.39 (m, 2H), 4.17 (m, 5H),
4.00 (m, 3H), 3.97 (t, 1H, J = 4.4 Hz), 3.92 (t, 1H, J = 4.4 Hz), 3.88
(m, 2H), 3.63 (m, 5H), 3.51 (s, 1H), 3.42 (m, 2H), 3.24–3.10 (m,
5H), 2.30–1.90 (6 s, 18H), 2.10 (m, 2H), 1.89 (m, 1H), 1.68 (m,
1H), 1.57 (m, 2H), 1.50 (2 s, 18H), 1.42 (s, 54H); 13C NMR
(75 MHz, CD3OD from HSQC): d 127.0–114.5 (aromatic carbons),
106.4, 96.6, 97.8 (anomeric carbons), 80.8, 79.1, 77.8, 76.8, 75.4,
75.1, 73.4, 72.6, 72.4, 71.9, 71.2, 68.5, 68.0, 66.7, 66.0, 65.0, 63.6,
60.3, 55.0, 54.8, 54.4, 53.7, 51.9, 51.2, 48.2, 47.3, 46.8, 39.4, 33.4,
49.3, 49.0, 47.4, 41.2, 39.9, 35.1, 34.4, 33.4, 26.9–23.2;
þ
½
a 2D5
ꢂ
¼ þ21:0 (c 0.11, MeOH); EIMS: calcd for C51H79N12O16
1115.57, found: 1115.99 [M+H]+. Anal. Calcd for C67H86F24N12O32
:
C, 39.69; H, 4.28; F, 22.49; N, 8.29. Found: C, 40.11; H, 4.52; F,
22.76; N, 8.54.
3.16. Determination of the MIC values for aminoglycoside–
peptide conjugates 5, 7, and 17
Bacterial isolates were obtained from the American Type Cul-
ture Collection (ATCC). Isolates were kept frozen in skim milk at
ꢁ80 °C until minimum inhibitory concentration (MIC) testing
was carried out. Following two subcultures from frozen stock,
the in vitro activities of peptides were determined by macrobroth
dilution in accordance with the Clinical and Laboratory Standards
Institute (CLSI) 2006 guidelines.26 Stock solutions of peptides were
prepared and dilutions were made as described by CLSI. Test tubes
contained doubling antimicrobial dilutions of cation adjusted
Mueller–Hinton broth and inoculated to achieve a final concentra-
tion of approximately 5 ꢀ 105 CFU/mL then incubated in ambient
air for 24 h prior to reading. Colony counts were performed period-
ically to confirm inocula. Quality control was performed using
ATCC QC organisms.
32.8, 28.8–27.3, 22.2–18.9; ½a D25
¼ þ26:0 (c 0.2, MeOH); EIMS:
ꢂ
calcd for C107H154N10NaO39 2226.04, found: 2226.04 [M+Na]+.
Anal. Calcd for C107H154N10O39: C, 58.30; H, 7.04; N, 6.35. Found:
C, 58.77; H, 7.29; N, 6.89.
þ
3.14. 500-N-(Fmoc-Trp(Boc)-Lys-Trp(Boc)-OBn)-1,3,20,60,500,2000,6000-
hepta-N-(tert-butoxycarbonyl)-6,30,40,200,500,4000-hexa-acetyl-500-
deoxy-neomycin (16)
Coupling was performed as previously described and compound
16 was purified after column chromatography. Yield = 58%; Rf 0.35
(CH2Cl2–MeOH 15:1); 1H NMR (300 MHz, CD3OD): d 7.93 (d, 1H,
J = 8.3 Hz), 7.89 (d, 1H, J = 8.1 Hz), 7.59 (m, 2H), 7.58 (m, 2H),
7.56 (m, 1H), 7.52 (m, 1H), 7.50 (m, 2H), 7.44–7.32 (m, 3H), 7.20
(m, 4H), 7.16–6.98 (m, 6H), 5.42 (s, 1H), 5.05 (m, 2H), 5.00 (s,
1H), 4.97 (s, 1H), 4.88 (m, 2H), 4.74 (m, 2H), 4.54 (s, 1H), 4.43
(m, 1H), 4.22 (m, 2H), 3.90 (m, 5H), 3.76 (m, 4H), 3.53 (m, 1H),
3.43 (m, 4H), 3.40 (t, 2H, J = 2.3 Hz), 3.33 (t, 1H, J = 5.6 Hz), 3.22
(dd, 3H, J = 5.6 Hz), 3.18 (d, 1H, J = 11.1 Hz), 3.03 (m, 4H), 2.88
(m, 1H), 2.80 (m, 3H), 2.07–1.60 (6 s, 18H), 1.67 (m, 2H), 1.48
(m, 1H), 1.43 (m, 2H), 1.37 (s, 3H), 1.30 (s, 15H), 1.22 (s, 63H);
13C NMR (75 MHz, CD3OD): d 173.8, 173.2, 172.3–170.5 (ester
and acetate–CO), 158.4, 158.1–156.6 (amide CO), 145.1–116.2
(aromatic carbons), 109.3, 99.0, 98.4 (anomeric carbons), 85.2,
84.7, 81.9, 81.2 (ꢀ3), 81.0, 80.3 (ꢀ3), 79.8, 76.9, 75.8, 75.0, 73.8,
72.3, 71.7, 71.3, 68.4, 67.5 (ꢀ3), 69.1, 66.5, 66.1, 60.7, 56.0, 54.5,
53.8, 52.9, 51.7, 50.7, 50.3, 48.6, 46.6, 41.9, 41.2, 34.0, 33.5, 30.5,
Acknowledgments
The author thanks the Natural Sciences and Engineering
Research Council of Canada (NSERC), the Canadian Institutes for
Health Research (CIHR), and the Manitoba Health Research Council
(MHRC) for financial support.
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