The use of aminoglycoside derivatives to study the mechanism of aminoglycoside 6′-N-acetyltransferase and the role of 6′-NH2 in antibacterial activity

Article abstract of DOI:10.1016/j.bmc.2007.02.009

Aminoglycoside antibiotics act by binding to 16S rRNA. Resistance to these antibiotics occurs via drug modifications by enzymes such as aminoglycoside 6′-N-acetyltransferases (AAC(6′)s). We report here the regioselective and efficient synthesis of N-6′-ac

Full text of DOI:10.1016/j.bmc.2007.02.009

Bioorganic & Medicinal Chemistry 15 (2007) 2944–2951
The use of aminoglycoside derivatives to study the mechanism
of aminoglycoside 60-N-acetyltransferase and the role
of 60-NH2 in antibacterial activity
Xuxu Yan, Feng Gao, Sirilata Yotphan, Parseh Bakirtzian and Karine Auclair*
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montre´al, Que., Canada H3A 2K6
Received 5 December 2006; revised 30 January 2007; accepted 8 February 2007
Available online 11 February 2007
Abstract—Aminoglycoside antibiotics act by binding to 16S rRNA. Resistance to these antibiotics occurs via drug modifications by
enzymes such as aminoglycoside 60-N-acetyltransferases (AAC(60)s). We report here the regioselective and efficient synthesis of N-60-
acylated aminoglycosides and their use as probes to study AAC(60)-Ii and aminoglycoside–RNA complexes. Our results emphasize
the central role of N-60 nucleophilicity for transformation by AAC(60)-Ii and the importance of hydrogen bonding between 60-NH2
and 16S rRNA for antibacterial activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
gio- and chemo-selective method for the direct N-60-
derivatization of unprotected aminoglycosides in high
yield.7 This facilitated the preparation of the first gener-
ation of synthetic AAC nanomolar inhibitors. Using the
same strategy we prepared a second generation of
AAC(60) inhibitors to determine structure–activity rela-
tionships.8 This also led to a derivative able to block
aminoglycoside modification in cells.
Aminoglycosides are frequently prescribed broad spec-
trum antibiotics. They act by binding to the 16S rRNA
A-site causing mistranslation and blocking protein syn-
thesis.1,2 Their use is however threatened by the rapid
spread of resistance. Aminoglycoside resistance occurs
mainly via drug modifications3–6 such as acetylation,
adenylation, or phosphorylation. Aminoglycoside
60-N-acetyltransferase (AAC(60)) is one of the most
common determinants of resistance to this family of
antibiotics.
Besides inhibition, a more direct approach to counter
aminoglycoside resistance is to design antibacterials
active against resistant strains. Such a venture however
requires a good understanding of resistance mecha-
nisms.15,17–21 We have designed a series of N-60-acylated
aminoglycoside derivatives to no longer bind to
AAC(60)-Ii but still interact with bacterial 16S rRNA.
We report their preparation using our novel synthetic
methodology, their assay with AAC(60)-Ii, and their
antibacterial activity.
One strategy to overcome aminoglycoside resistance is
to develop inhibitors of resistance-causing enzymes.7–13
Northrop and coworkers reported the first nanomolar
inhibitor of an AAC, 3-N-(2-S-CoA-acetyl)gentamicin
C1a.12 This bisubstrate was prepared enzymatically
using its target, AAC(3)-I. The chemical synthesis of
aminoglycoside-coenzyme A (CoA) bisubstrates is chal-
lenging in part because of the judicious functional pro-
tection chemistry needed with aminoglycosides and
CoA, and the water solubility of the starting materi-
als.13–16 We recently developed an effective one-pot re-
2. Results
2.1. Design and synthesis of neamine derivatives
The 60-amino group of aminoglycosides is protonated
at physiological pH and may thus interact with RNA
via electrostatic or hydrogen bond interactions. The
reported crystal structures of aminoglycoside–RNA
complexes20,22–29 reveal an important hydrogen bond
Keywords: Antibiotic; A-site; Neamine; Regioselective; Resistance;
Aminoglycoside; Acetyltransferase; Antibacterial; RNA.
*
Corresponding author. Tel.: +1 514 398 2822; fax: +1 514 398
3797; e-mail: karine.auclair@mcgill.ca
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.009

X. Yan et al. / Bioorg. Med. Chem. 15 (2007) 2944–2951
2945
between 60-NH2 and N1 of A1408. Moreover, the
antibacterial activity of aminoglycosides containing a
60-OH suggests that hydrogen bonding at the 60 posi-
tion may be more important than the electrostatic
binding to RNA.
Synthesis of the targets was achieved using our previous-
ly reported regioselective strategy.7,8 The required N-hy-
droxyl-5-norbornene-endo-2,3-decarboximide
esters
(NBD esters, 1a–k) were prepared by standard amide
coupling with 1,3-dicyclohexylcarbodiimide in high
yields (>90%). The NBD esters were used as regioselec-
tive reagents to deliver the acyl groups to the 60-amino
group of neamine and yield molecules 2a–k (73–92%).
Boc or Fmoc protected amino acids were used (with
2g–k) to allow purification on silica gel. With derivatives
2a–f however, reverse phase HPLC was necessary. Stan-
dard deprotection protocols generated target derivatives
3g–k in yields ranging from 20% to 60% (unoptimized).
To maximize the purity, large amounts of impure prod-
ucts had to be sacrificed during purification on silica gel,
thus greatly reducing the yields.
N-60-Acetylation by resistance-causing AAC(60)s de-
creases the affinity of aminoglycosides for RNA. This
may be the result of steric hindrance, loss of hydrogen
bonds, and/or loss of electrostatic interactions. As ex-
plained above, disruption of a salt bridge by acetyla-
tion is not likely to be the major factor affecting the
interaction of aminoglycosides with RNA. Two
AAC(60)s have been successfully crystallized in the
presence of aminoglycoside derivatives. Blanchard
and coworkers have published a crystal structure of
the AAC(60)-Iy–ribostamycin complex.30 Some of us
in collaboration with the group of Berghuis have re-
ported the crystal structure of AAC(60)-Ii in complex
with a series of N-60-linked aminoglycoside–CoA
bisubstrates.7 The structures of AAC(60)-Ii with differ-
ent bisubstrate inhibitors provide valuable information
about the effect of extra carbons near N-60. Unexpect-
edly, the most potent bisubstrate inhibitor of
AAC(60)-Ii reported so far has two extra carbons
compared to the proposed tetrahedral intermediate
(Fig. 1). According to the X-ray structure, the methy-
lene groups of this bisubstrate labeled as C1 and C2
are surrounded mostly by hydrophobic residues. On
the other hand, the reported structures for aminogly-
cosides in complex with RNA20,22–29 reveal a much
more polar environment near 60-NH2. Such differences
are useful in the design of aminoglycosides that bind
to RNA but not to AAC(60)s.
Compound 2f was an intermediate in the preparation of
targets 3l–m. Shortly treating 2f with KOH yielded 3l
(61%). Compound 2f was also separately treated with
Na2S2 (generated by mixing Na2S with S8 in boiling
water) to afford a disulfide intermediate that was further
reduced by dithiothreitol to generate 3m in 68% yield.
2.2. Transformation of 2a–e and 3g–m by the resistance-
causing enzyme AAC(60)-Ii
The neamine derivatives 2a–e and 3g–m were first tested
for inhibition of AAC(60)-Ii. No reduction in activity
was observed. The molecules were next tested as sub-
strates and found to be insignificantly transformed
(UV detection after in situ derivatization of the CoA
produced). Compound 3g was selected for further anal-
ysis of the transformation by AAC(60)-Ii. No acetylation
products were detected (detection limit: 10ꢀ8 M) after
HPLC separation and ESI-MS analysis. As a control
experiment, the acetylation product of neamine was
clearly recognized (10ꢀ4 M). This verifies our assump-
tion that the neamine derivatives (2a–2e and 3g–3m)
are very poor or not substrates for AAC(60)-Ii. It also
confirms that N-60 is the position acetylated by this
enzyme.
Compounds 3g–3m (Scheme 1) were designed to
investigate the importance of the hydrogen bonds
of 60-NH2 and their specific location. Acylation at
60-NH2 of neamine decreases the hydrogen bonding
strength of the nitrogen, yet the presence of amino
and/or hydroxyl functionalities in the acyl group is
expected to compensate for this loss, if positioned
correctly. We also envisaged to take advantage of
p-stacking interactions. Thus, targets 2b–2c were
designed with aromatic groups near the 60 position
and expected to stack with A1408.
2.3. Antibacterial activity in cells
The RNA binding of derivatives 2a–2e and 3g–3m was
next evaluated indirectly via antibacterial cell-based
activity assays on paper discs and in solution. In the
assays, Escherichia coli strains JR66, JR67, pETAAC
wt, and pSF815A-1 were kanamycin-resistant. E. coli
strain JR88 was resistant to gentamicin. E. coli strain
MG1655 was not resistant to aminoglycosides and the
Staphylococcus aureus strain was sensitive to all usual
antibiotics. At 30 lg/disc, none of the derivatives
showed clear growth inhibition of any of the strains
JR66, JR67, JR88, pETAAC wt, S. aureus, or
pSF815A-1. Partial inhibition was observed with
MG1655. Minimum inhibitory concentrations (MICs)
against MG1655 were next determined using standard
dilution methods (Table 1). Compared to the parent
compound neamine, none of the derivatives displayed
significant activity, except 3g and 3l which showed
reduced antibacterial activity.
C2
a
b
C1
OH
O
O
OH
O
O
Me
CoA
HO
HO
H2N
CoA
N
H
NH2
S
N
H
NH2
S
H2N
HO
NH2
HO
NH2
OH
OH
Figure 1. (a) Previously reported nanomolar inhibitor of AAC(60)-Ii.7
This bisubstrate is two carbon longer than the putative enzymatic
tetrahedral intermediate (b).

2946
X. Yan et al. / Bioorg. Med. Chem. 15 (2007) 2944–2951
O
O
Neamine,
acetone/H2O
DCC, DMAP
DCM
O
O
N OH +
N O
R
HO
R
O
O
1a-k
OH
OH
O
O
HO
HO
N
R
N
H
NH2
R
H
O
O
Deprotection
H2N
NH2
H2N
O
O
HO
NH2
HO
NH2
OH
OH
3g-m
2a-k
Na2S2 then DTT
KOH
2f
3l
2f
3m
R ( in 1a-k and 2a-k):
R ( in 3g-m):
a: Me
b: Ph
c: CH2Naph
d: CH2N3
C CH
e:
f: CH2Br
g: CH2NH2
h: (S)-CH(CH3)NH2
i: CH2CH2NH2
j: (S)-CH(NH2)CH2NH2
k: (S)-CH(NH2)CH2OH
l: CH2OH
g: CH2NHFmoc
h: (S)-CH(CH3)NHFmoc
i: CH2CH2NHBoc
j: (S)-CH(NHBoc)CH2NHBoc
k: (S)-CH(NHBoc)CH2OtBu
m: CH2SH
Scheme 1. Synthesis of neamine derivatives 2a–f and 3g–m.
Table 1. Results of antimicrobial activity assay using the dilution
method
sufficient to increase the MIC to a clinically ineffective
level. We report here the efficient synthesis of N-60-acyl-
ated aminoglycosides and their use to probe the mecha-
nism of resistance-causing AAC(60)-Ii and the role of the
N-60 of aminoglycosides in antibacterial activity. Using
the available crystal structures of aminoglycosides com-
plexed to either RNA22 or AAC(60)s,7,30 we designed a
series of derivatives expected to be poor substrates of
AAC(60)s yet maintain binding to RNA. Such targets
also have the potential to become lead compounds in
the development of antibacterials active against resistant
strains. We expected that acylation at N-60 would pre-
vent transformation by AAC(60)-Ii yet the addition of
an amino and/or alcohol groups in the acyl group would
rescue the hydrogen bonding ability necessary for RNA
affinity.
Compound
MICa (lg mLꢀ1
)
2b
2c
N/Ab
N/A
N/A
N/A
<50
2d
2e
3g
3h
N/A
N/A
N/A
N/A
<25
3i
3j
3k
3l
3m
N/A
<6.25
Neamine
a Minimum inhibitory concentration.
b Below detection.
We used our previously reported methodology to
regioselectively modify aminoglycosides, and effi-
ciently synthesized a series of neamine derivatives
acylated at N-60 (2a–f and 3g–m). Regular protec-
tion–deprotection strategies would have required at
least four more steps for each target and greatly re-
duced the overall yield. For example, compound 2f
was previously prepared in an overall yield of
8.8%,31,32 whereas our methodology allows its synthe-
sis in 68% overall yield.
3. Discussion
With the advent of antibiotics and the spread of their
use in the late 1960s, the battle against bacterial infec-
tions was considered won. The increasing incidence of
antibiotic resistance is alarming and threatens to turn
back the clock. Investigations of resistance mechanisms
and searches for new antimicrobials are vital. The anti-
bacterial activity of aminoglycosides is due to their high
affinity for the prokaryotic A site of 16S rRNA. Acety-
lation of aminoglycosides at the 60-amino group is
As predicted, none of derivatives 2a–e or 3g–m were
significantly transformed by AAC(60)-Ii. Based on

X. Yan et al. / Bioorg. Med. Chem. 15 (2007) 2944–2951
2947
the crystal structures of bisubstrates–AAC(60)-Ii com-
plexes,7 steric hindrance likely explains the reduced
affinity observed when large acyl groups are added
to neamine (2b, 2f, 3h, 3i, 3j, and 3k). On the other
hand, the results obtained with shorter acyl groups
(2a, 2d, 2e, 3g, 3 l, and 3m) combined with our
previous studies with bisubstrate inhibitors7 suggest
that N-60-acylation affects AAC(60)-Ii catalysis by
reducing the nucleophilicity of N-60 as opposed to
steric hindrance. Indeed, bisubstrates with 4-carbon
linkers at N-60 were potent inhibitors of AAC(60)-Ii,
and those with 3-carbon linkers near N-60 were more
potent inhibitors than 2-carbon linkers. This is
surprising since the latter more closely resemble the
tetrahedral enzymatic intermediate (1-carbon). The
better binding of the bisubstrate containing a 3-carbon
linker indicates that enough space is available to
accommodate extra carbons near N-60 such as in com-
pounds 2a, 2d, 2e, 3g, 3l, and 3m. Therefore, the neg-
ative effect of aminoglycoside acylation with chains of
three atoms or less on AAC(60)-Ii-catalysis cannot be
attributed to steric hindrance to binding but rather
to a reduction in the nucleophilicity of N-60. This
supports the earlier suggestion by Wright and cowork-
ers that AAC(60)-Ii catalysis uses mainly a proximity
effect.33 In light of our results we can also conclude
that the lower nucleophilicity of alcohols compared
to that of free amines is responsible for the absence
of AAC(60)-Ii-catalysis with aminoglycosides contain-
ing a 60-OH.
In conclusion, our results not only demonstrate once
again the utility of our N-60 regioselective synthetic
methodology to derivatize aminoglycosides, but also
show the central role of N-60 nucleophilicity for trans-
formation by AAC(60)-Ii, and the importance of hydro-
gen bonding between 60-NH2 and RNA for antibacterial
activity.
4. Experimental
4.1. General
Unless mentioned otherwise, all reagents were pur-
chased from Sigma–Aldrich Canada Ltd. (Oakville,
Ontario). Reagents and solvents were used without fur-
ther purification. Flash chromatography and TLC anal-
˚
ysis (F-254) were performed with 60 A silica gel from
Silicycle (Que., Canada). Compounds 2a–2f, 3l, and
3m were purified by reversed-phase HPLC using an Agi-
lent 1100 modular system equipped with an autosam-
pler, a quaternary pump system, a photodiode array
detector, a thermostatted column compartment, and a
ChemStation (for LC 3D A.09.03) data system. The
column used was a semi-preparative Zorbax SB-CN of
4.6 · 250 mm and 5 lm (Agilent, Palo Alto, CA). Sam-
ples were eluted at a flow rate of 3 mL/min, using a com-
bination of mobile phase A (0.05% TFA in water at pH
ꢁ3.5) and mobile phase B (acetonitrile containing 0.05%
TFA). After 2 min at 1% B, the eluent was brought to
40% B in a linear gradient over 20 min. The detector
was set to 214 nm. HRMS spectra were obtained by
direct infusion electrospray ionization from a solution
of 50 mM formic acid/methanol 90:10 at 2 lL/min in
an IonSpec 7 T FTICR instrument. The resolving power
was approximately 80,000. LRMS was performed using
a Finnigan LCQDUO mass spectrometer with ESI with-
out fragmentation. 1H and 13C NMR spectra were
recorded using Varian mercury 400 or 300 spectrome-
Based on their similarity to currently used aminoglyco-
side antibiotics, we expected our derivatives to penetrate
cells. The antibacterial activity measured was thus expect-
ed to correlate with RNA affinity or translation interfer-
ence. Further studies would be required to settle this
issue and confirm cell permeability. As with AAC(60)-Ii,
the reduced hydrogen bonding ability at N-60 was expect-
ed to decrease the affinity of the aminoglycosides for
RNA. To compensate, compounds 2b and 2c were
designed to take advantage of possible p-stacking with
A1408. The absence of antibacterial activity observed
with these derivatives suggests that the aryl groups could
not reach far enough to stack with any bases.
1
ters. For all H NMR spectra of target molecules, pre-
saturation (presat.) was used to suppress the water
peak. The chemical shifts (d) were reported in parts
per million (ppm) and are referenced to either the inter-
nal standard TMS (when CDCl3 is used) or the deuter-
ated solvent used. The peak patterns are indicated as
follows: s, singlet; d, doublet; t, triplet; dt, doublet of
triplet; ddd, doublet of doublet of doublet; td, triplet
of doublet; m, multiplet; q, quartet; p, pentet; and br
s, broad singlet. The coupling constants, J, are reported
in Hz. Reactions with air or moisture sensitive reagents
were carried out under an atmosphere of argon.
As an alternative strategy to offset the amidation of
N-60, amino-hydroxyl-, and/or thio-groups were added
on the acyl group (3g–m). The use of a poor hydrogen
bond acceptor/donor such as a thiol (3m) abolished
activity. When the acyl group was more than three
atoms long (3h, 3i, 3j, and 3k), the antibacterial activity
decreased below the detection limit, likely due to steric
hindrance (based on crystal structures). With shorter
acyl groups however (3g and 3l), some activity was
detected, but it was reduced by ꢁ4-fold compared to
that of the parent compound neamine. Since the addi-
tion of acyl chains of less than three atoms should not
cause steric clashes, this confirms our initial hypothesis
that acetylation affects RNA affinity by reducing the
hydrogen bonding ability at N-60. The ꢁ4-fold decrease
in antibacterial activity observed for 3g and 3l suggests
that the newly added hydrogen bond donors/acceptors
may not be positioned properly.
4.2. Synthesis
4.2.1. General procedure for the synthesis of compounds
1a–1k from carboxylic acids. endo-N-Hydroxy-5-nor-
bornene-2,3-dicarboximide (NBD) (0.538 g, 3 mmol)
and the desired carboxylic acid (3 mmol, protected at
other functional groups if necessary) were dissolved in
dichloromethane (20 mL). When necessary a small
amount of THF (ca. 5 mL) was added to dissolve the
carboxylic acid. DCC (0.619 g, 3 mmol) was added to
the mixture, followed by a catalytic amount of DMAP

2948
X. Yan et al. / Bioorg. Med. Chem. 15 (2007) 2944–2951
(5 mg). A few minutes after addition of DCC, a white
solid (DCU) precipitated out. The reaction was stirred
under argon at room temperature overnight. The pres-
ence of the desired NBD ester (1a–1k) was monitored
by TLC. The solid DCU was removed by filtration
and the filtrate was evaporated to dryness. EtOAc
(100 mL) was added to dissolve the product. The solu-
tion was extracted several times with 10% Na2CO3
(15 mL). The organic layer was dried over anhydrous
MgSO4 and filtered. The filtrate was evaporated to near
dryness and the NBD ester was dried under vacuum.
When higher purity was needed, a silica gel chromatog-
raphy column was used for the purification.
13C NMR (CDCl3, 75 MHz): d 170.0, 166.4, 150.2,
138.2, 134.9, 127.2, 125.4, 125.0, 124.8, 124.8, 123.8,
122.5, 113.3, 51.2, 44.7, 43.2, 37.8; MS for C21H17NO4
(M+H) calcd 348.1, found: 348.1.
Compound 1d. TLC Rf = 0.35 (Hex/EtOAc 1:1); yield:
1
80%; H NMR (CDCl3, 400 MHz): d 6.20 (br s, 2H),
4.17 (s, 2H), 3.46 (s, 2H), 3.35 (br s, 2H), 2.25 (s, 3H),
1.80 (d, J = 8.8, 1H), 1.55 (d, J = 8.8, 1H); 13C NMR
(CDCl3, 75 MHz) d 169.6, 135.0, 51.6, 48.2, 45.1, 43.7;
MS for C11H10N4O4(M+H) calcd 263.1, found: 263.2.
Compound 1e. TLC Rf = 0.47 (Hex/EtOAc 1:1); yield:
1
94%; H NMR (CDCl3, 400 MHz): d 6.17 (br s, 2H),
4.2.2. General procedure for the preparation of com-
pounds 2a–2k. The general procedure can be exemplified
with the preparation of compound 2f. Neamine (32 mg,
0.1 mmol) was dissolved in water (2 mL) in a vial. endo-
N-Hydroxy-5-norbornene-2,3-dicarboximide bromoace-
tate (NBD ester 1f, 24 mg, 0.08 mmol) was dissolved in
acetone (2 mL) in another vial. The two solutions were
mixed, sonicated for 1 min, and stirred for one hour be-
fore quenching with formic acid to pH 4. Purification by
reverse phase HPLC afforded the desired product 2f as a
fluffy white powder (24 mg, 70%). Compounds 2a–2f
were purified by reverse phase HPLC and compounds
2g–2k, however, were purified by flash chromatography.
3.45 (s, 2H), 3.34 (br s, 2H), 3.27 (s, 1H), 1.80 (d,
J = 8.8, 1H), 1.53 (d, J = 8.8, 1H); 13C NMR (CDCl3,
75 MHz) d 169.3, 135.0, 81.9, 70.6, 51.5, 45.1, 43.6;
MS for C12H9NO4(M+H) calcd 232.2, found: 232.3.
Compound 1f. TLC Rf = 0.47 (Hex/EtOAc 1:1); yield:
95%. The crude product was recrystallized using hexane
1
to yield earth-red crystals of mp 90–92 ꢁC; H NMR
(CDCl3, 400 MHz): d 6.20 (br s, 2H), 4.04 (s, 2H),
3.46 (br s, 2H), 3.34 (br s, 2H), 1.80 (d, J = 8.8, 1H),
1.54 (d, J = 8.8, 1H); 13C NMR (CDCl3, 75 MHz) d
169.5, 135.0, 51.6, 45.1, 43.7, 21.9; HRMS for
C11H10NO4 calcd 298.98 (100) and 300.98 (97), found:
299.00 (100) and 301.00 (97).
Deprotection of Boc, Fmoc, and tert-butyl ether groups
was performed using standard procedures.34
Compound 1g. TLC Rf = 0.66 (Hex/EtOAc 1:1). The
starting material Fmoc-Gly-OH was used as the limiting
reagent; yield: 94%; 1H NMR (300 MHz, CDCl3) d 7.75
(d, J = 7.4, 2H), 7.57 (d, J = 4.0, 2H), 7.35 (m, 4H), 6.18
(br s, 2H), 5.30 (m, 1H), 4.43 (d, J = 7.4, 2H), 4.29 (m,
3H), 3.45 (m, 2H), 3.34 (br s, 2H), 1.78 (d, J = 8.8,
1H), 1.53 (d, J = 8.8, 1H).
Methyl
azidoacetate.
Methylbromoacetate
(3 g,
20 mmol) and sodium azide (2.5 g, 20 mmol) were mixed
in acetone (30 mL). The mixture was refluxed overnight.
The mixture was evaporated to dryness and the residue
was extracted with EtOAc to give methyl azidoacetate
(2.18 g, 95%) as a yellowish liquid. IR: 2110 cmꢀ1 (azide
absorption); 1H NMR (CDCl3, 400 MHz): d 3.89 (s,
2H), 3.80 (s, 3H); 13C NMR (CDCl3, 75 MHz) d
170.2, 49.2, 52.2.
Compound 1h. TLC Rf = 0.38 (Hex/EtOAc 1:1). The
starting material Fmoc-Ala-OH was the limiting re-
1
agent; Yield: 76%; H NMR (200 MHz, CDCl3) d 7.74
(d, J = 7.1, 2H), 7.56 (d, J = 7.1, 2H), 7.32 (m, 4H),
6.18 (s, 2H), 5.26 (d, J = 7.6, 1H), 4.71 (t, J = 7.6, 1H),
4.38 (m, 2H), 4.23 (m, 1H), 3.44 (s, 2H), 3.32 (m, 2H),
1.76 (d, J = 8.8, 1H), 1.57 (d, J = 7.2, 3H), 1.51 (d,
J = 8.8, 1H).
Compound 1a. TLC Rf = 0.53 (Hex/EtOAc 1/1); yield:
90%; H NMR (CDCl3, 400 MHz): d 6.18 (br s, 2H),
3.45 (br s, 2H), 3.33 (br s, 2H), 2.25 (s, 3H), 1.79 (d,
J = 8.8, 1H), 1.54 (d, J = 8.8, 1H); 13C NMR (CDCl3,
75 MHz) d 169.5, 135.0, 51.6, 45.1, 43.7, 17.2; ESI-MS
for C11H11NO4(M+H) calcd 222.1, found: 222.0, 238.2.
1
Compound 1i. Rf = 0.38 (Hex/EtOAc, 1:1). The starting
material Boc-b-Ala-OH was the limiting reagent; yield:
1
Compound 1b. TLC Rf = 0.65 (Hex/EtOAc 1:1); yield:
84%; H NMR (200 MHz, CDCl3) d 6.155 (br s, 2H),
5.12 (br s, 1H), 3.46 (br s, 2H), 3.43 (br s, 2H), 3.31
(m, 2H), 1.75 (br d, 1H), 1.54 (br d, 1H), 1.41 (s, 9H).
1
94%; H NMR (CDCl3, 400 MHz): d 8.09 (d, J = 8.0,
2H), 7.65 (t, J = 8.0, 1H), 7.48 (t, J = 8.0, 2H), 6.28
(br s, 2H), 3.50 (br s, 2H), 3.40 (br s, 2H), 1.83 (d,
J = 8.8, 1H), 1.58 (d, J = 8.8, 1H); 13C NMR (CDCl3,
75 MHz): d 170.2, 134.9, 134.7, 130.7, 130.4, 128.9,
51.7, 45.2, 43.5; MS for C16H13NO4(M+H) calcd
284.1, found: 284.1.
Compound 1j. Rf = 0.33 (Hex/EtOAc, 2:1). The starting
material di-Boc-2,3-diaminopropionic acid was the lim-
1
iting reagent; yield 52%; H NMR (200 MHz, CDCl3) d
6.132(s, 2H), 5.71 (br, 1H), 5.39 (t, J = 7.4, 1H), 4.61 (m,
1H), 3.66 (m, 1H), 3.51 (m, 1H), 3.39 (s, 2H), 3.30 (s,
2H), 1.73 (d, J = 8.8 Hz, 1H), 1.48 (d, J = 8.8, 1H),
1.38 (s, 18H).
Compound 1c. TLC Rf = 0.55 (Hex/EtOAc 1:1); yield:
1
99%; H NMR (CDCl3, 400 MHz): d 7.93 (d, J = 8.0,
1H), 7.86 (d, J = 8.0, 1H), 7.82 (d, J = 8.0, 1H), 7.58
(t, J = 8.0, 1H), 7.50 (t, J = 8.0, 1H), 7.45 (m, 2H),
6.15 (br s, 2H), 4.30 (s, 2H), 3.41 (br s, 2H), 3.27
(br s, 2H), 1.74 (br s, 1H), 1.47 (d, J = 8.8, 1H);
Compound 1k. Rf = 0.68 (Hex/EtOAc, 1:4). The starting
material Fmoc-Ser (tBu)-OH was the limiting reagent;
yield 89%; 1H NMR (300 MHz, CDCl3) d 8.30 (m,

X. Yan et al. / Bioorg. Med. Chem. 15 (2007) 2944–2951
2949
1
1H), 7.75 (d, J = 11, 3H), 7.50 (m, 2H), 7.30 (m, 4H),
6.58 (d, J = 2, 1H), 6.20 (s, 1H), 6.08 (s, 2H), 4.33 (m,
4H), 3.90 (m, 1H), 3.67 (m, 1H), 3.36 (m, 2H), 3.13
(m, 2H), 1.78 (m, 1H), 1.30 (s, 9H).
Compound 2f. Yield 70%; H NMR (D2O, 400 MHz,
pD = 4.0, presat.): 5.50 (d, J = 4.0, 1H), 3.92 (s, 2H),
3.70–3.61 (m, 3H), 3.43 (t, J = 8.8, 1H), 3.38–3.31 (m,
3H), 3.21–3.16 (m, 2H), 3.10 (td, J = 10, 3.8, 1H), 2.31
(td, J = 8.8, 4.0, 1H), 1.66 (q, J = 8.8, 1H); 13C NMR
(D2O, 75 MHz): d 169.6, 96.8 (C10), 79.0 (C30), 75.0
(C40), 72.4 (C50), 71.8 (C6), 70.2 (C5), 68.3 (C4), 54.0
(C20), 50.7 (–COCH2Br), 49.8 (C1), 48.2 (C3), 39.2
(C60), 28.8 (C2); ESI-MS for C14H27BrN4O7 (M+H)
calcd 442.11, 444.10, found: 442.20 (100), 444.20 (98).
1
Compound 2a. Yield 60%; H NMR (D2O, 400 MHz,
pD = 4.0, presat.): 5.50 (d, J = 4.0, 1H), 3.72–3.63 (m,
3H), 3.46 (t, J = 8.8, 1H), 3.40–3.32 (m, 3H), 3.24–3.19
(m, 2H), 3.13 (td, J = 10, 4.0, 1H), 2.32 (td, J = 12.0,
4.0, 1H), 1.82 (s, 3H), 1.67 (q, J = 12.0, 1H); 13C
NMR (D2O, 75 MHz): d 169.6, 96.8 (C10), 79.0 (C30),
75.0 (C40), 72.4 (C50), 71.8 (C6), 70.2 (C5), 68.3 (C4),
54.0 (C20), 49.8 (C1), 48.2 (C3), 39.2 (C60), 28.8 (C2),
21.9 (–COCH3); ESI–MS for C14H28N4O7 (M+Na)
calcd 387.20, found: 387.20.
Compound 2g. Rf = 0.65 (CHCl3/MeOH/NH4OH, 4:3:1);
yield 89%; ESI-MS (MeOH): (M+H) calcd 602.2, found:
602.2.
Compound 2h. Rf = 0.38 (CHCl3/MeOH/NH4OH, 4:3:1);
yield 91%; ESI-MS (MeOH): (M+H) calcd 616.2, found:
616.2.
1
Compound 2b. Yield 80%; H NMR (D2O, 400 MHz,
pD = 4.0, presat.): 7.59 (d, J = 8.0, 2H), 7.45 (t,
J = 6.8, 1H), 7.35 (t, J = 8.0, 2H), 5.55 (d, J = 4.0,
1H), 3.81–3.70 (m, 3H), 3.62 (m, 1H), 3.49 (t, 1H),
3.40–3.30 (m, 3H), 3.26 (br d, J = 13.2, 1H), 3.13
(td, J = 10.0, 4.0, 1H), 2.32 (td, J = 12.0, 4.0, 1H),
1.67 (q, J = 12, 1H); 13C NMR (D2O, 75 M) d
167.7, 135.4, 132.2, 128.7, 126.5, 97.0 (C10), 79.0
(C30), 75.2 (C40), 72.8 (C50), 72.0 (C6), 71.0 (C5),
69.0 (C4), 54.0 (C20), 50.0 (C1), 48.6 (C3), 40.0
(C60), 28.5 (C2); ESI-MS for C19H30N4O7 (M+Na)
calcd 439.21, found: 439.11.
Compound 2i. Rf = 0.42 (CHCl3/MeOH/NH4OH, 4:3:1);
yield 87%; ESI-MS (MeOH): (M+H) calcd 493.2, found:
493.2.
Compound 2j. Rf = 0.45 (CHCl3/MeOH/NH4OH, 4:3:1);
yield 92%; ESI-MS (MeOH): (M+H) calcd 609.2, found:
609.2.
Compound 2k. Rf = 0.62 (CHCl3/MeOH/NH4OH,
4:3:1); yield 79%; ESI-MS (MeOH): (M+H) calcd
688.3, found: 688.3.
1
Compound 2c. Yield 85%; H NMR (D2O, 400 MHz,
presat.): d 7.84 (t, J = 8.4, 2H), 7.79 (d, J = 8.0, 1H),
7.47(m, 2H), 7.40 (t, J = 8.0, 1H), 7.36 (d, J = 8.0,
1H), 5.33 (d, J = 4.0, 1H), 3.98 (s, 2H), 3.64–3.58 (m,
3H), 3.48–3.35 (m, 3H), 3.32 (m, 1H), 3.15 (dt,
J = 11.0, 4.0, 1H), 2.88 (t, J = 9.6, 1H), 2.69 (dd,
J = 11.2, 4.0, 1H), 2.33 (td, J = 12.4, 4.8, 1H), 1.67 (q,
J = 12.8, 1H); 13C NMR (D2O, 100 MHz, presat.): d
163.3 (CO), 133.8, 131.8, 131.1, 129.1, 128.9, 128.5,
126.9, 126.4, 126.2, 123.7, 96.7 (C10), 79.0 (C30), 75.2
(C40), 72.7 (C50), 71.9 (C6), 70.0 (C5), 68.6 (C4), 53.9
(C20), 49.9 (C1), 48.7 (C3), 40.5 (CH2), 38.9 (C60), 28.6
(C2);MS (ESI) for C24H34N4O7 (M+Na) calcd 513.2,
found: 513.2.
1
Compound 3g. Yield 27%; H NMR (D2O, 400 MHz,
pD = 10, presat.): 5.03 (d, J = 4.0, 1H), 3.72 (m, 1H),
3.52–3.18 (m, 3H), 3.18 (s, 2H), 3.14–3.07 (m, 4H),
2.96 (t, J = 9.2, 1H), 2.67 (m, 2H), 1.82 (m, 1H), 1.76
(q, J = 12.0, 1H); 13C NMR (D2O, 75 MHz): d 175.8,
101.2, 88.0, 77.2, 75.8, 73.3, 71.3, 71.2, 55.1, 50.2, 49.3,
43.6, 40.0, 35.4; ESI-MS for C14H29N5O7 (M+H) calcd
requires 380.2, found: 380.1.
1
Compound 3h. Yield 17%; H NMR (D2O, 400 MHz,
pD = 10, presat.): 5.05 (d, J = 4.0, 1H), 3.71 (dt,
J = 9.2, 4.0, 1H), 3.47–3.28 (m, 4H), 3.15–2.94 (m,
4H), 2.78 (m, 3H), 1.82 (m, 1H), 1.18 (d, J = 6.8, 3H),
1.04 (q, J = 12.0, 1H); ESI-MS for C15H31N5O7
(M+H) calcd 394.2, found: 394.2.
1
Compound 2d. Yield 75%; H NMR (D2O, 400 MHz,
pD = 4.0, presat.): 5.57 (d, J = 4.0, 1H), 3.91 (s, 2H),
3.73 (dt, J = 8.8, 4.0, 1H), 3.56 (d, J = 12.4, 1H), 3.43
(t, J = 7.6, 1H), 3.35 (m, 2H), 3.14 (t, J = 9.2, 1H),
3.10 (t, J = 9.2, 1H), 3.00 (t, J = 9.2, 1H), 2.55 (dt,
J = 9.2, 3.6, 1H), 1.83 (td, J = 12.0, 4.0, 1H), 1.05 (q,
J = 12.0, 1H); 13C NMR (D2O, 75 MHz): d 163.5,
101.4, 88.2, 77.4, 75.0, 73.7, 72.9, 72.6, 55.6, 52.0, 50.2,
49.6, 40.3, 35.9; ESI-MS for C14H27N7O7 (M+H) calcd
406.2, found: 406.1.
1
Compound 3i. Yield 33%; H NMR (D2O, 400 MHz,
pD = 10, presat.): 5.05 (d, J = 4.0, 1H), 3.71 (dt, J = 9.2,
4.0, 1H), 3.47–3.28 (m, 4H), 3.15–2.94 (m, 4H), 2.82 (t,
J = 6.9, 1H), 2.68–2.50 (m, 3H), 2.33 (t, J = 6.9, 2H),
1.82 (m, 1H), 1.04 (q, J = 12.0, 1H); 13C NMR (D2O,
75 MHz): d 174.8, 101.4, 88.2, 77.5, 76.1, 73.6, 71.7,
71.5, 55.4, 50.5, 49.6, 40.3, 37.2, 37.0, 35.8; ESI-MS for
C15H31N5O7 (M+H) calcd 394.2, found: 394.2.
1
Compound 2e. Yield 77%; H NMR (D2O, 400 MHz,
1
Compound 3j. Yield 60%; H NMR (D2O, 400 MHz,
pD = 4.0, presat.): 5.57 (d, J = 4.0, 1H), 3.79–3.64 (m,
3H), 3.55–3.40 (m, 6H), 3.32–3.28 (m, 2H), 3.20 (m,
1H), 2.32 (td, J = 12.0, 4.0, 1H), 1.67 (q, J = 12.0, 1H);
13C NMR (D2O, 75 MHz): d 155.0, 97.1, 79.4, 77.3,
75.8, 75.1, 72.6, 71.6, 70.6, 68.9, 53.9, 49.8, 48.7, 39.9,
28.5; ESI-MS for C15H26N4O7 (M+H) calcd 375.2,
found: 375.1.
pD = 10, presat.): 5.50 (d, J = 4.0, 1H), 3.73–2.91 (m,
11H), 2.62–2.50 (m, 2H), 1.76 (m, 1H), 1.00 (m, 2H);
13C NMR (D2O, 75 MHz): d 173.4, 101.2, 87.0, 77.5,
76.2, 73.5, 71.8, 63.7, 61.5, 56.0, 55.0, 50.2, 49.6,45.4,
35.3; ESI-MS for C15H31N5O8 (M+H) calcd 410.2,
found: 410.2.

2950
X. Yan et al. / Bioorg. Med. Chem. 15 (2007) 2944–2951
1
Compound 3k. Yield 22%; H NMR (D2O, 400 MHz,
pD = 10, presat.): 5.05 (d, J = 4.0, 1H), 3.73 (m, 1H),
3.47–3.08 (m, 8H), 2.78 (m, 5H), 1.82 (m, 1H), 1.04 (q,
J = 12.0, 1H); 13C NMR (D2O, 75 MHz): d 175.2,
101.2, 87.6, 77.5, 76.1, 73.6, 71.4, 71.3, 56.6, 55.4, 50.6,
49.6, 45.2, 40.1, 35.7; ESI-MS for C15H32N6O7 (M+H)
calcd 409.2, found: 409.2.
4.5. Enzymatic transformation and detection of the
acetylated neamine derivatives
The reaction mixtures (total volume of 2 mL) consisted
of EDTA (1 mM), AcCoA (0.7 mM), the molecule test-
ed (4 mM), and AAC(60)-Ii (0.4 mg) in Hepes (25 mM,
pH 7.5). It was incubated at room temperature with
orbital shaking at 200 rpm. Additional portions of
enzyme (0.1 mg) were added every 2 h for 10 h, and
the mixture was left stirring at room temperature
overnight. The enzyme was separated from the reaction
mixture using an Amicon ultrafiltration system (10K
MWCO membrane, filter code: YM10), and the filtrate
was concentrated by rotary evaporation. The residue
was suspended in water (1 mL) and applied to HPLC
for separation. The column used was a semi-preparative
Zorbax SB-CN of 4.6 · 250 mm and 5 lm (Agilent, Palo
Alto, CA). The sample was eluted at a flow rate of 2 mL/
min, using a combination of mobile phase A (0.05%
TFA in water at pH ꢁ3.5) and mobile phase B (acetoni-
trile containing 0.05% TFA). After 10 min at 0% B, the
eluent was brought to 40% B in a linear gradient over
30 min. The detector was set to 214 nm. After collection,
the fractions were analyzed by ESI-MS. A control
reaction was performed with neamine.
Compound 3l. Compound 2f (TFA salt, 36 mg,
0.05 mmol) was dissolved in H2O (3 mL), KOH
(120 mg, 2 mmol) was added, and the mixture was
refluxed overnight. The mixture was run through a short
column of neutralized Amberlite IRA-67 and the
collected crude product was purified by reverse phase
HPLC. Yield: 61%; 1H NMR (D2O, 400 MHz,
pD = 4.0, presat.): 5.50 (d, J = 4.0, 1H), 3.88–3.58 (m,
5H), 3.55–3.28 (m, 5H), 3.19 (m, 2H), 2.38 (td,
J = 12.0, 4.0, 1H), 1.76 (q, J = 12.0, 1H); 13C NMR
(D2O, 75 MHz): d 166.7, 96.4, 78.1, 75.4, 72.7, 70.8,
69.5, 68.9, 53.6, 49.9, 48.7, 40.3, 28.5; ESI-MS for
C14H28N4O8 (M+Na) calcd 403.2, found: 403.2.
Compound 3m. The Na2S2 solution was prepared by
mixing Na2S (39 mg, 0.5 mmol) and S8 (16 mg,
0.5 mmol) in H2O (2 mL) in a glass tube, and gently
boiled using a heat gun for a few minutes until the
solution turns clear yellow. Compound 2f (free base,
23 mg, 0.05 mmol) was dissolved in H2O (3 mL), the
Na2S2 solution (0.2 mL, 0.05 mmol) was added, and
the mixture was stirred for 3 h at room temperature.
Dithiothreitol (8 mg, 0.05 mmol) was added and the
mixture was stirred overnight at room temperature.
After acidification to pH 3 using 20% aqueous TFA,
the product was purified by HPLC to afford com-
pound 3m as a white fluffy powder. Yield: 68%; 1H
NMR (D2O, 400 MHz, pD = 4.0, presat.): 5.55 (d,
J = 4.0, 1H), 3.83-3.79 (m, 3H), 3.52–3.10 (m, 10H),
2.34 (td, J = 12.0, 4.0, 1H), 1.70 (q, J = 12.0, 1H);
13C NMR (D2O, 75 MHz): d 172.3, 96.9, 78.9, 75.2,
72.7, 71.8, 70.8, 69.4, 68.5, 54.0, 49.9, 48.8, 40.0,
28.5; ESI-MS for C14H28N4O7S (M+H) calcd 397.2,
found: 397.1.
4.6. Anti-microbial activity test
Escherichia coli strains JR66, JR67, pETAACWT, and
pSF815A-1 were kanamycin-resistant. E. coli strain
JR88 was resistant to gentamicin. The S. aureus strain
was sensitive to all usual antibiotics. The tests on solid
medium were carried out at 30 lg/disc on LB. Standard
dilution assays were performed in overnight cultures of
MG1655 in LB (3 mL) diluted 100 times. The readings
were OD at 600 nm.
Acknowledgments
The authors thank Karen Lu and Prof. Julian E. Davies
for performing the antimicrobial activity assays. This
work was supported by the Natural Science and Engi-
neering Research Council of Canada (NSERC).
4.3. AAC(60)-Ii inhibition assays
AAC(60)-Ii was expressed and purified as previously de-
scribed elsewhere.7 Enzyme activity was monitored
using a procedure reported earlier.35
References and notes
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(25 mM, pH 7.5) containing EDTA (1 mM), 4,40-dithi-
odipyridine (DTDP, 400 lM), AcCoA (100 lM), and
various concentrations of the molecule tested as a poten-
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