N- and C-terminal modifications of negamycin

Article abstract of DOI:10.1016/S0960-894X(03)00393-7

Negamycin 1 is a bactericidal antibiotic with activity against Gram-negative bacteria, and served as a template in an antibiotic discovery program. An orthogonally protected β-amino acid derivative 3a was synthesized and used in parallel synthesis of negamycin derivatives on solid support. This advanced intermediate was also used for N- and C-terminal modifications using solution-phase methodologies. The N-terminal modifications have resulted in the identification of active analogues, whereas the C-terminal modifications resulted in complete loss of antibacterial activity. The N-methyl negamycin analogue, 19a, inhibits protein synthesis (IC₅₀=2.3 μM), has antibacterial activity (Escherichia coli, MIC=16 μg/mL), and is efficacious in an E. coli murine septicemia model (ED₅₀=16.3 mg/kg).

Full text of DOI:10.1016/S0960-894X(03)00393-7

Bioorganic& Medicinal Chemistry Letters 13 (2003) 2413–2418
N- and C-Terminal Modifications of Negamyciny
B. Raju,* Kathleen Mortell, Sampathkumar Anandan, Hardwin O’Dowd, Hongwu Gao,
Marcela Gomez, Corinne Hackbarth, Charlotte Wu, Wen Wang, Zhengyu Yuan,
Richard White, Joaquim Trias and Dinesh V. Patel
Versicor Inc., 34790 Ardentech Court, Fremont, CA 94555, USA
Received 21 November 2002; accepted 7 February 2003
Abstract—Negamycin 1 is a bactericidal antibiotic with activity against Gram-negative bacteria, and served as a template in an
antibiotic discovery program. An orthogonally protected b-amino acid derivative 3a was synthesized and used in parallel synthesis
of negamycin derivatives on solid support. This advanced intermediate was also used for N- and C-terminal modifications using
solution-phase methodologies. The N-terminal modifications have resulted in the identification of active analogues, whereas the
C-terminal modifications resulted in complete loss of antibacterial activity. The N-methyl negamycin analogue, 19a, inhibits protein
synthesis (IC50=2.3 mM), has antibacterial activity (Escherichia coli, MIC=16 mg/mL), and is efficacious in an E. coli murine sep-
ticemia model (ED50=16.3 mg/kg).
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
N- and C-terminal regions of negamycin were under-
taken to assess the influence of such modifications on
antibacterial activity.
The emergence and spread of bacterial resistance1 to
clinically useful antibiotics is a major threat to the
health care system worldwide. This observation has
rekindled interest in the discovery of new antibacterial
agents, preferably with new mode of action.2 Pursuing
novel targets based on bacterial genomics3 is an elegant
approach towards this goal. An alternative approach is
to re-investigate previously discovered molecules with
antibacterial activity that remained under-explored for
various reasons.4 Negamycin, a natural product iso-
lated5 in 1970 from the culture filtrate of Streptomyces
purpeofuscus, is one such antibiotic. It is active mainly
against Gram-negative organisms with weak activity
against Staphylococcus aureus. The mechanism of action
is through the inhibition of protein synthesis and mis-
coding activity.7 Negamycin is bactericidal and shown
to be efficacious with low acute toxicity5 in animal
models. However, there are limited structure–activity
relationship (SAR) studies reported in the literature.6
This has prompted us to undertake a SAR investigation
around this template. To this end, modifications on the
Chemistry
We have identified the orthogonally protected b-amino
ester 3a (Fig. 1) as a key building block for the solution-
or solid-phase parallel synthesis of negamycin (1) and
various other C- and N-terminal derivatives. The
b-amino acid derivative 3a can be obtained from homo-
allylglycine 4 using classical organic transformations.
Thus, homoallylglycine8 4 (Scheme 1) was subjected to
iodolactonization conditions9 followed by treatment
with sodium azide to give azidolactone 5 as a mixture of
diastereomers (1:1). After base hydrolysis of azido-
lactone 5, the resulting hydroxy acid was reacted with
t-butyldimethylsilyl chloride (TBS-Cl). Hydrolysis of the
silyl ester gave acid 6 as a mixture of diastereomers. The
activated ester of acids 6 was formed with penta-
fluorophenyl trifluoroacetate in pyridine and the dia-
stereomers were separated by column chromatography
to give 3a and 3b. Azide 7a, obtained by condensing the
b-amino ester 3a with hydrazine 2b,10 was reduced to
the corresponding amine using catalytic hydrogenation.
Cleavage of silyl ether by treatment with tetra-
butylammonium fluoride followed by acidic treatment
yPart of this work was presented at 42nd ICAAC. Annual meeting of
the American Society for Microbiology. September 27–30, 2002, San
Diego, CA, USA.
*Corresponding author. Fax: +1-510-739-3003; e-mail: raju@
versicor.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00393-7

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B. Raju et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2413–2418
of the benzyl protecting group via catalytic hydrogena-
tion gave the acid, which was then immobilized on Sas-
rin resin under Mitsunobu reaction conditions to give
resin bound hydrazine derivative 10. The unreacted
hydroxyl groups on the resin were capped with acetyl
groups. The Fmocgroup was removed and resin bound
hydrazine 10 was reacted with activated b-amino acid
3a to give resin bound azide 11. The residual amino
groups on the resin were capped with acetyl groups. The
azide was reduced to an amine using triphenylphos-
phine, then acylated with various acids under standard
coupling conditions to give the resin bound amides.
Cleavage of the amides from the resin with 10% TFA
followed by treatment with 4 M hydrochloric acid in
dioxane resulted in global deprotection to furnish
amides 12 in a library format.
Figure 1. Retrosyntheticanalysis.
Reduction of azide 7a (Scheme 3) with triphenylphos-
phine gave amine 13. Reaction of amine 13 with tri-
methylsilylisocyanate and removal of the protecting
groups furnished urea analogue 14. Reaction of amine
13 with 2-methyl-2-thiopseudourea sulfate followed by
removal of the protecting groups under acidic condi-
tions gave guanidine analogue 15. Further, amine 13
was also reacted with benzyl bromide and 3,4-diacetoxy-
benzyl bromide11 and treated with acid to generate
monoalkylated analogues 16a and 16b, respectively.
An alternative approach to the synthesis of N-alkyl
negamycin derivatives based on Fukuyama chemistry12
is briefly summarized in Scheme 4. Sulfonamide 17,
obtained by treating amine 13 with 2-nitrobenzene-
sulfonyl chloride, was reacted with a set of alkyl halides
using cesium carbonate as a base to produce alkylated
sulfonamides 18a–c. The alkylated analogue 18d was
obtained by treatment of sulfonamide 17 with 4-methyl-
1-pentanol under Mitsunobu reaction conditions.
Removal of the 2-nitrobenzenesulfonamide protecting
group in 18a–d by treatment with thiophenol in the
presence of potassium carbonate followed by global
deprotection under acidic conditions gave the desired
N-alkylated negamycins 19a–d.
Scheme 1. Negamycin and epinegamycin. Reagents and conditions:
(a) I2, KI, NaHCO3, H2O, DCM, rt, 2.5 h; (b) NaN3, DMF, 55 ꢀC, 4 h
(62% for two steps); (c) LiOH, H2O, MeOH, rt, 2 h (89%); (d) TBS-
Cl, imidazole, DCM, rt, 18 h; (e) K2CO3, MeOH, H2O, rt, 2 h (80%);
(f) CF3CO2C6F5, pyridine, DCM, rt, 24 h (79%, for three steps); (g)
2b, DMF, rt, 3 h (96%); (h) TBAF, THF, rt, 1.25 h (70%); (i) H2, Pd/
C, EtOAc, rt, 3 h (90%); (j) 4 M HCl, dioxane, H2O, rt, 9 h (78%); (k)
benzyl 2-bromoacetate or t-butyl 2-bromoacetate, Et3N, DCM, 0 ꢀC
to rt, 2 h (99%).
furnished negamycin (1). Similar transformations on
b-amino ester 3b gave 5-epinegamycin (8).
Protection of the amine group in 2a (Scheme 2) with a
fluorenylmethoxycarbonyl group (Fmoc) and removal
To access N,N-dialkylated analogues, the iodolactone
(Scheme 5) obtained from homoallylglycine 4 as descri-
Scheme 2. Solid-phase parallel synthesis of N-terminal amides.
Reagents and conditions: (a) FmocOSu, Na2CO3, dioxane, 0 ꢀC to rt,
18 h (80%); (b) H2, Pd/C, EtOAc, rt, 18 h, (100%); (c) sasrin resin,
PPh3, DIAD, THF, 0 ꢀC to rt, 16 h; (d) Ac2O, pyridine, rt, 3 h; (e) 20%
piperidine in DMF, 30 min; (f) 3a, Et3N, DMF, rt, 24 h; (g) Ac2O,
pyridine, rt, 3 h; (h) PPh3, THF, H2O, 28 h; (i) RCO2H, HATU,
DIEA, DMF, rt, 16 h; (j) 10% TFA in DCM, rt, 1 h; (k) 4 M HCl in
dioxane, rt, 1 h.
Scheme 3. N-terminal modifications of negamycin. Reagents and
conditions: (a) PPh3, THF, H2O, 14 h (80%); (b) trimethylsilyl isocy-
anate, DCM, rt, 18 h (60%); (c) 4 M HCl in dioxane, H2O, rt, 2 h
(96%); (d) 2-methyl-2-thiopseudourea sulfate, NaOAc, i-PrOH, reflux,
8 h (40%); (e) ArCH2Br, DIEA, DMF, rt, 16 h (67–77%, for two steps).

B. Raju et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2413–2418
2415
Scheme 4. Mono N-alkylated negamycin analogues. Reagents and
conditions: (a) 2-nitrobenzenesulfonyl chloride, collidine, DCM, rt, 8 h
(90–97%); (b) ROH, PPh3, DIAD, DCM, 4 ꢀC to rt, 3 h (80%); (c)
CsCO3, RI, DMF, rt, 2 h (94%); (d) PhSH, K2CO3, DMF, rt, 12 h
(76%); (e) 4 M HCl in dioxane, H2O, rt, 2 h (96%).
Scheme 6. Synthesis of homonegamycin. Reagents and conditions: (a)
i-butyl chloroformate, NMM, THF, ꢁ15 ꢀC, 45 min; then ethereal
solution of CH2N2; (b) C6H5CO2Ag, Et3N, MeOH, (79%, for two
steps); (c) LiOH, H2O, THF, 0 ꢀC to rt, 1.5 h (89%); (d) i-butyl chlor-
oformate, NMM, THF, ꢁ15 ꢀC, 45 min; then NaBH4 followed by
H2O (68%); (e) DMSO, oxalyl chloride, TEA, ꢁ60 to ꢁ40 ꢀC (93%);
(f) BrCH2CO2Et, Et2Zn, RhCl(PPh3)3, THF (57%); (g)TBS-Cl, imi-
dazole, THF, rt, 18 h (81%); (h) LiOH, H2O, MeOH, rt, 20 h (93%);
(i) CH3SO2Cl, Et3N, DCM, 0 ꢀC to rt, 2 h; (j) NaN3, DMF, 70 ꢀC, 2 h
(69%); (k) 2 M KOH, MeOH, rt, 16 h, (89%); (l) 2b, HATU, DMF,
0 ꢀC to rt, 14 h (62%); (m) H2, Pd/C, EtOAc, rt, 2.5 h (92%); (n) 4 M
HCl, dioxane, H2O (100%).
anhydride14 procedure. Hydroxy derivative 26a was
further transformed to the corresponding azido acid 27a
in three classical steps: mesylation, nucleophilic dis-
placement using sodium azide then hydrolysis of the
t-butyl ester under basicconditions. b-Amino acid 27a
was then coupled with hydrazine 2b, using HATU as a
coupling reagent. Reduction of the azide to the corre-
sponding amine by catalytic hydrogenation and removal
of the protecting groups under acidic conditions gave
homonegamycin 28a. Similar transformations on 25b
gave homonegamycin analogue 28b, a diasteromer of
28a.
Scheme 5. N,N-dialkylated negamycin analogues. Reagents and con-
ditions: (a) I2, KI, NaHCO3, H2O, DCM, rt, 2.5 h (88%); (b)
R1NHR2, DMF, 70 ꢀC, 17 h (38–65%); (c) LiOH, H2O, MeOH, 0 ꢀC
to rt, 24 h; (d) TBS-Cl, imidazole, DCM, 0 ꢀC to rt, 24 h; (e) K2CO3,
MeOH, H2O, rt, 3 h; (f) HATU, DIEA, DMF, 0 ꢀC to rt, 17 h (35–
47%, last four steps); (g) 4 M HCl in dioxane, H2O, rt, 7 h (quantita-
tive).
bed above (Scheme 1), was alkylated with various cyclic
amines to obtain lactones 20a–d. Opening of lactones
20a–d under basic conditions followed by protection of
the secondary hydroxyl group as a TBS ether then base
hydrolysis of the silyl ester furnished amino acids 21a–d
as lithium salts on lyophilization. Coupling of lithium
salts 21a–d with hydrazine 2b using HATU as a cou-
pling agent followed by acidic treatment gave the
desired analogues 22a–d as a mixture of diastereomers.
C-Terminal analogues were obtained by coupling of
b-amino acid intermediate with various derivatives.
Alkylation of N-hydroxyphthalimide with t-butyl
2-bromoacetate 29a or with t-butyl 2-bromopropionate
29b (Scheme 7) followed by removal of the phthalimide
protecting group using hydrazine gave the desired
O-alkyl hydroxylamines 31a and 31b, respectively.
Trans esterification of activated ester 3a with methanol
followed by reduction of the azide group using catalytic
hydrogenation gave the primary amine. The amine was
then protected with a Boc group and the ester group
was hydrolyzed to give desired b-amino acid 32. Cou-
pling of b-amino acid 32 with 31b using HATU as a
coupling reagent and global deprotection gave hydro-
xamate 34. Condensation of b-amino ester 3a with
O-alkyl hydroxylamine 31a gave the fully protected
hydroxamate 35. Reduction of azide 35 to the corre-
sponding amine using triphenylphosphine followed by
treatment with 30% trifluoroacetic acid in dichloro-
methane resulted in removal of the Bocand t-butyl
protecting groups, with the TBS group intact. Removal
of the TBS group was accomplished using HF in pyri-
dine which was buffered with excess pyridine to generate
desired hydroxamate 36.
An analogue in which the terminal amine was extended
by one methylene unit was desired to investigate the role
of chain length in the b-amino acid portion of the
molecule. Homologation of aspartic acid 23 (Scheme 6)
under Arndt–Eistert13 reaction conditions, followed by
ester hydrolysis gave desired acid 24. The alcohol
obtained by reduction of acid 24 under mixed anhy-
dride14 reaction conditions was oxidized to the corre-
sponding aldehyde then reacted with ethyl
bromoacetate under modified Reformatsky15 reaction
conditions to furnish a mixture of diastereomeric alco-
hols 25a and 25b, separated by column chromato-
graphy. After protecting the secondary hydroxyl group
in 25a as its TBS ether, the ethyl ester was selectively
hydrolyzed and reduced to alcohol 26a by the mixed

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B. Raju et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2413–2418
Scheme 9. Acid surrogate building blocks. Reagents and conditions:
(a) (Boc)2O, NaHCO3, EtOAc, 0 ꢀC to rt, 3 h (100%); (b) Cbz-Cl,
NaHCO3, EtOAc, 0 ꢀC to rt, 1 h (60%); (c) 4 M HCl in dioxane, rt, 4 h
(100%); (d) ClCH2CN, DMF, rt, 14 h (87%); (e) Bu3SnN3, 110 ꢀC,
72 h (83%); (f) HBr, AcOH, rt, 1 h (64%); (g) diethyl (trifluoromethyl-
sulfonylmethyl)phosphonate, DIEA, toluene, rt, 4 h (47%); (h) Pd/C,
EtOAc, rt, 1 h (92%); (i) diethyl methylphosphonate, HCHO, THF, rt,
1 h, reflux, 1 h (51%).
Scheme 7. Hydroxamate ethers. Reagents and conditions: (a) NaH,
DMF, 0 ꢀC to rt (85%); (b) NH2NH2, EtOH, rt (50%); (c) MeOH,
Et3N, rt, 8 h (quantitative); (d) H2, 5% Pd/C, MeOH, 4 h; (e) (Boc)2O,
THF, Et3N, rt; (f) KOH, MeOH, rt, 14 h (75%, three steps); (g) 31b,
HATU, DIEA, DMF; (h) 4 M HCl in dioxane, H2O, rt (30%, for two
steps); (i) 31a, DMF, rt, 24 h (68%); (j) PPh3, THF, H2O, rt, 16 h
(93%); (k) 30% TFA in DCM, rt, 4 h; (l) HF, pyridine, rt, 2.5 h (67%,
for two steps).
ethyl (1-hydroxymethyl)phosphonate18 was used to N-
alkylate hydrazine 40 and the benzyloxycarbonyl group
was removed under catalytic hydrogenation conditions.
A three component condensation19 between hydrazine
40, formaldehyde and diethyl methylphosphonate fol-
lowed by removal of the benzyloxycarbonyl group by
catalytic hydrogenation resulted in phosphinate 43.
Conformationally restricted C-terminal analogues were
generated from d- and l-aminoproline derivatives.
Aminoprolines 37a and 37b (Scheme 8) were treated
with diazomethane to produce the corresponding
methyl esters, which were treated with trifluoroacetic
acid in DCM to remove the Boc group. The resultant
amines were coupled with b-amino acid 32 using HATU
as coupling reagent. Removal of the protecting groups
in 38a and 38b gave analogues 39a and 39b, respec-
tively.
Condensation of hydrazine 41 (Scheme 10) with acti-
vated ester 3a followed by reduction of the azide to the
corresponding amine and removal of the protecting
groups under acidic conditions gave tetrazole analogue
44. Hydrazine 42 was condensed with activated ester 3a
and the TBS protecting group was removed using acetic
acid and water containing a catalytic amount of hydro-
fluoricaicd to give the hydroxy azide intermediate.
Reduction of the azide via hydrogenation followed by
treatment with trimethylsilyl iodide, then with hydro-
chloric acid in dioxane, furnished phosphonic acid 45.
Similarly, condensation of activated ester 3a with
hydrazine 43 and removal of the silyl-protecting group
gave a hydroxy azide intermediate. The azide was
reduced to the amine by catalytic hydrogenation, the
phosphinate ester was hydrolyzed to the corresponding
phoshinic acid under basic conditions and the Boc
group was cleaved in acidic conditions to generate
phosphinicacid 46.
Obtaining analogues with C-terminal acid surrogates
required the synthesis of several building blocks. Selec-
tive protection of the more reactive secondary amine16
in N-methylhydrazine 9 (Scheme 9) with a Bocgroup
followed by treatment with benzyl chloroformate and
removal of the Boc group under acidic conditions gave
hydrazine 40. N-Alkylation of hydrazine 40 using
chloroacetonitrile followed by cycloaddition with azi-
dotributyltin17 and then treatment with hydrobromic
acid furnished hydrazine 41. For the preparation of
phosphonate analogue 42, the triflate derived from di-
Scheme 10. Negamycin analogues with C-terminal acid surrogates.
Reagents and conditions: (a) 41, Et3N, DMF, rt, 24 h (31%); (b) PPh3,
THF, H2O, rt 16 h or H2, Pd/C, EtOAc, rt, 4 h (60–87%); (c) 4 M HCl
in dioxane, H2O; (d) 42, Et3N, DMF, rt, 4 h (78%); (e) AcOH, HF,
THF, H2O, rt, 11 h (72%); (f) TMSI, CH3CN, 0 ꢀC to rt, 18 h (9%);
(g) 43, Et3N, DMF; (h) LiOH, H2O, EtOH, 18 h (12% for two steps).
Scheme 8. Conformationally restricted C-terminal analogues.
Reagents and conditions: (a) CH2N2, Et2O, THF, 0 ꢀC; (b) 20% 4 N
HCl in dioxane, 30 min, rt (98%) ; (c) 32, HATU, DIEA, DMF, 0 ꢀC
to rt, 8 h (44%); (d) 1 N NaOH, MeOH, rt, 3 h; (e) 4 M HCl in diox-
ane, H2O, rt, 4 h (90%, two steps).

B. Raju et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2413–2418
Table 1. MICs21 and protein synthesis inhibition data (TC/TL)23
2417
Organisims/Compd
MIC (mg/mL)
1
8
12a
14
1516a
>256
>256
128
>256
>256
>256
>256
ND
16b
19a
128
32
8
32
128
>128
128
>128
19b
19c
19d
E. coli ATCC 25922
E. coli MG1655
E. coli MG1655 tolC
Klebsiella pneumoniae ATCC 113882
Enterobacter cloacae ATCC35030
Pseudomonas aeruginosa PA013
S. aureus ATCC 25293
4
4
2
2
8
32
32
32
128
128
32
16
16
8
32
16
64
>256
>256
>256
>256
>256
>256
>256
ND
64
32
16
32
64
16
8
4
16
8
4
8
16
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
64
4
128
>512
512
ND
16
128
128
256
>128
>128
128
128
>256
ND
256
ND
Streptococcus pneumoniae VSPN1005
TC/TL IC50 (mM)
1.0
23.4
3.5
>80
>80
0.8
0.6
2.3
4.5
20.3
>80
ND, not determined. Analogues 22a–d, 28a–b, 34, 36, 39a–b, 44, 45 and 46 are inactive (MIC >256 mg/mL and IC50 >80 mM).
Results and Discussion
negamycin in both assays. The N-benzylated analogues
16a and 16b are equipotent to negamycin in cell-free
protein synthesis assay but these analogues are sig-
nificantly less active than negamycin in the whole cell
assay. These analogues are not significantly effluxed in
E. coli (Table 1) suggesting they may not be getting
transported as efficiently into the bacterial cell. All other
analogues are inactive in either the whole cell or the
protein synthesis assays (MIC >256 mg/mL; IC50 >80 mM).
A flexible method for the synthesis of negamycin and its
analogues was developed starting from commercially
available 3-R-t-butoxycarbonylaminohex-5-enoic acid8
(4). The orthogonally protected advanced intermediate
3a was used in the synthesis of N-acylated analogues on
a solid support. This method enabled us to generate a
library of 180 discrete compounds.20 In vitro evaluation
of the antimicrobial properties21 of amide analogues
against Gram-positive and Gram-negative organisms
indicated that all analogues were inactive except leu-
cylnegamycin 12a, which is 2–16 times less active than
negamycin (Table 1). Interestingly, leucylnegamycin has
been proposed as a biosyntheticprecursor of negamy-
cin.7a The transformation of the N-terminal amine in
negamycin to the corresponding urea and guanidine
derivatives resulted in analogues 14 and 15, respectively,
which are devoid of antibacterial activity. On the other
hand, the antibacterial activity of N-methyl and N-ethyl
derivatives 19a and 19b are comparable to that of
negamycin (1). The N-methyl analogue is also effica-
cious in an E. coli murine septicemia model22
(ED50=16.3 mg/kg). The analogues bearing slightly
longer alkyl groups (19c and 19d) and the cyclic-alkyl
groups (22a–d) exhibited a drasticloss in antibacterial
activity. These results dictate a stringent requirement for
a basicamino group that is either unsubstituted or has
small linear alkyl groups (methyl or ethyl) on it. An
exception to this observation are the N-benzyl deriva-
tives 16a and 16b, which are significantly less active than
negamycin (1) but retain some antibacterial activity. In
contrast, homonegamycin analogues 28a or 28b lack
antibacterial activity. These observations indicate that
not only is the basicN-terminal primary amine essential
for antibacterial activity, but its distance from the
hydroxyl group or from the internal amine is also cri-
tical for antibacterial activity. Replacement of the C-
terminal acid with the hydroxamate ether moiety, as in
analogue 34 and 36, resulted in inactive analogues.
Replacement of the acid with surrogates such as tetra-
zole, phosphonic acid, and phosphinic acid (44, 45 and
46, respectively) also resulted in complete loss of anti-
bacterial activity. In addition to a whole cell assay,21 all
the analogues were evaluated in a cell-free protein
synthesis assay.23 The N-methyl and N-ethyl analogues,
19a and 19b respectively, are slightly less active than
In conclusion, the small molecule antibacterial agent
negamycin appears to have stringent requirements with
regard to the chemical nature of the basic N-terminal
and acidic C-terminal groups and also with regard to
the spacing of the basic amino group relative to the
remainder of the functional groups on the scaffold.
These results suggest an active-transport mediated event
may account for the permeability of such a small but
highly charged molecule across the cell wall membrane of
Gram-positive and across Gram-positive organisms. Our
efforts are now focused on probing the importance of the
internal functional groups of negamycin for antibacterial
activity and will be communicated in due course.
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is an efflux pump mutant.
21. Micro dilution MICs were determined in Mueller–Hinton
broth supplemented with 50% human serum and an inoculum
size of 5 ꢂ 105 cfu/mL.
22. In vivo efficacy: Mice were infected ip with approximately
1 ꢂ 105 cfu of E. coli ATCC25922. The compound was admi-
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for 7 days and the ED50 calculated by non-linear regression.
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a
standard in this study
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23. Inhibition of protein synthesis was measured with a
cell-free transcription/translation coupled assay (E. coli S30
Extract System, Promega, Madison, WI, USA) using
pGEMbgal as a DNA template. Compounds were pre-
incubated with the reaction mixture for 10 min prior to the
addition of template. After 1 h of incubation, ONPG was
added and b-galactosidase activity was monitored at
420 nm.