Selective modification of streptozotocin at the C3 position to improve its bioactivity as antibiotic and reduce its cytotoxicity towards insulin-producing β cells

Article abstract of DOI:10.3390/antibiotics9040182

With the increasing resistance of bacteria to current antibiotics, novel compounds are urgently needed to treat bacterial infections. Streptozotocin (STZ) is a natural product that has broad-spectrum antibiotic activity, albeit with limited use because of

Full text of DOI:10.3390/antibiotics9040182

antibiotics
Article
Selective Modification of Streptozotocin at the C3
Position to Improve Its Bioactivity as Antibiotic and
Reduce Its Cytotoxicity towards Insulin-Producing
β Cells
Ji Zhang 1,†, Liubov Yakovlieva 1,† , Bart J. de Haan 2, Paul de Vos 2 , Adriaan J. Minnaard 1,
Martin D. Witte 1 and Marthe T. C. Walvoort 1,
*
1
Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen,
The Netherlands; zhangji1988614@hotmail.com (J.Z.); l.yakovlieva@rug.nl (L.Y.);
a.j.minnaard@rug.nl (A.J.M.); m.d.witte@rug.nl (M.D.W.)
Department of Pathology and Medical Biology, University of Groningen Medical Center, Hanzeplein 1,
9700 RB Groningen, The Netherlands; b.j.de.haan@umcg.nl (B.J.d.H.); p.de.vos@umcg.nl (P.d.V.)
Correspondence: m.t.c.walvoort@rug.nl; Tel.: +31-50-3634275
2
*
†
Authors contributed equally to the manuscript.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 13 March 2020; Accepted: 10 April 2020; Published: 15 April 2020
Abstract: With the increasing resistance of bacteria to current antibiotics, novel compounds are
urgently needed to treat bacterial infections. Streptozotocin (STZ) is a natural product that has
broad-spectrum antibiotic activity, albeit with limited use because of its toxicity to pancreatic
β cells.
In an attempt to derivatize STZ through structural modification at the C3 position, we performed
the synthesis of three novel STZ analogues by making use of our recently developed regioselective
oxidation protocol. Keto-STZ (2) shows the highest inhibition of bacterial growth (minimum inhibitory
concentration (MIC) and viability assays), but is also the most cytotoxic compound. Pre-sensitizing
the bacteria with GlcNAc increased the antimicrobial effect, but did not result in complete killing.
Interestingly, allo-STZ (3) revealed moderate concentration-dependent antimicrobial activity and no
cytotoxicity towards β cells, and deoxy-STZ (4) showed no activity at all.
Keywords: antibiotics; regioselective oxidation; streptozotocin; β cells
1. Introduction
The most applied therapeutics for the management of bacterial infections are still antibiotics.
However, an increasing number of bacteria develops resistance against current treatments, and novel
substances and methods to combat bacterial infection are therefore needed. Next to developing
completely new compounds with a different mode of action, the promising strategy of modifying
currently available antibiotics has gained attention. Derivatization may overcome the existing resistance
towards the antibiotic, can reduce its toxicity, and may even result in analogues that inhibit bacterial
growth via a new mode of action. The effectiveness of this approach has been illustrated by the
synthesis of derivatives of vancomycin, a potent inhibitor of bacterial cell wall synthesis, that overcome
vancomycin-resistant enterococci infections [
1]. Major efforts have been directed towards the (semi)
synthesis of analogues of carbohydrate-based antibiotics, and in particular aminoglycosides [2,3
].
Aminoglycosides are natural products that are highly cationic in nature and show bactericidal activity, as
they bind the 30S component of the bacterial ribosome, thereby blocking protein synthesis and inhibiting
bacterial growth [4]. The adverse effects of aminoglycosides include renal toxicity, and ototoxicity
at high concentrations. In addition, there are multiple enzymes that work on aminoglycosides to
Antibiotics 2020, 9, 182; doi:10.3390/antibiotics9040182
www.mdpi.com/journal/antibiotics

Antibiotics 2020, 9, 182
2 of 11
inactivate their antibiotic activity, which limits their use in the clinic [
5
]. A large variety of structural
analogues, including amphiphilic aminoglycosides [
and glycosylated aminoglycosides [ ] have been synthesized to overcome the adverse effects [
These efforts led to the discovery of derivatives that cannot be modified by aminoglycoside-inactivating
enzymes [ 10], that have reduced toxicity [ ], and even inhibit bacterial growth via new mechanisms
of action [11,12].
6], conformationally-locked aminoglycosides,
7,8
3].
9,
6
In our ongoing research program, we have been developing site-selective methods to functionalize
and modify carbohydrates without using chemical protecting groups. We reasoned that these methods
could give straightforward access to new derivatives of carbohydrate-based antibiotics. In this paper,
we describe our efforts on modifying streptozotocin (CAS No. 1883-66-4, STZ,
chemical derivatization to maintain antimicrobial activity but reduce the side effects such as toxicity
towards insulin-producing cells. STZ (2-deoxy-2-(3-methyl-3-nitrosourea)-1-d-glucopyranose) is a
1) as a starting point for
β
derivative of N-acetylglucosamine produced by Streptomyces achromogenes var. streptozoticus, and its
chemical structure is depicted in Figure 1. STZ was first discovered and characterized in the 1960s and
exists as an equal mixture of its
α and β anomer [13,14]. STZ is most stable at pH 4.5 and degrades
rapidly in alkaline solutions [15]. It was shown to exhibit broad-spectrum antibacterial activity and
possesses antitumor and mutagenic properties [16]. Although initially embraced as an antibiotic, also
because of its apparent lack of toxicity to mammalian cells, STZ turned out to be very toxic to the
insulin-producing
β cells of the pancreas [13]. As a result of this cytotoxicity, the application of STZ as
an antibiotic was terminated, and
of diabetes [16].
1 is nowadays mostly used to generate experimental animal models
Figure 1. Structures of Streptozotocin (STZ) (1), and the STZ-derivatives (2–4) synthesized and
evaluated in this project.
STZ is specifically toxic to pancreatic
β
cells that secrete insulin, because it is efficiently transported
18]. This may be explained by the
into the cells by the glucose transport protein 2 (GLUT2) [17
β
,
structural similarities to glucose, as STZ is a 2-amino-2-deoxyglucoside that appears to act as a carrier
for the N3-methyl-N3-nitrosourea group [19]. In contrast to glucose itself, STZ is not recognized by other
glucose transporters. Since GLUT2 is primarily found in the cellular membranes of insulin-producing
β
cells, STZ is especially toxic to the pancreas. It is now also approved for use in islet-cell carcinomas
and malignant carcinoid tumors in humans [20].
In bacteria, STZ is imported through the phosphoenolpyruvate-carbohydrate phosphotransferase
system (PTS) [21], and accumulates intracellularly in its phosphorylated form [22]. The antibacterial
activity arises from the release of diazomethane through the hydrolysis of STZ, leading to DNA
damage. Already at low intracellular concentrations, septum formation in the bacteria is shown to
be affected, the bacteria grow into filaments, and the surviving cells are also mutagenized [23]. Next

Antibiotics 2020, 9, 182
3 of 11
to antibacterial and diabetogenic activity, STZ has also been reported to display anti-leukemic and
mutagenic activity [16].
Because of the different biological activities observed with STZ, research has been devoted to
understand the relation between structural modifications of STZ and the resulting biological effects.
Fully acetylated streptozotocin has no antibacterial activity, but shows an increased inhibition of the
growth of L-1210 leukemia cells compared to STZ [24]. Furthermore, replacement of the methyl group
at N3 of the nitrosourea by an ethyl or n-butyl substituent results in the loss of antibacterial activity.
Inversion of the 4-hydroxyl group of streptozotocin (from gluco to galacto configuration) leads to the
loss of antibacterial activity against Proteus vulgaris. Inversion of the C-2 hydroxyl (from gluco to manno
configuration) reduced the antibacterial activity markedly as well [24]. O-alkylation of the anomeric
hydroxyl of STZ improves the antitumor activities and reduces toxicity, but leads to a loss of the
antibacterial and diabetogenic activity. [25] In addition, methylation of the anomeric hydroxyl group
of STZ eliminates antibacterial activity, and α-methyl STZ is twice as active as β-methyl STZ against
cultures of leukemia L1210. The cytotoxicity of β-methyl STZ is identical to STZ [24].
So far, in the reported modifications of STZ, the C3-OH position has not been altered (Figure 1). To
complement the reported modifications of STZ, we decided to focus our efforts on the C3-OH position.
The targeted analogues are depicted in Figure 1, and include the C3-keto produced by oxidation (
the C3-allo-OH produced by inversion ( ), and the C3-deoxy produced by reduction ( ). Ultimately,
we were aiming to retain or improve the antibacterial activity of STZ, while at the same time reducing
the cytotoxicity to cells. We hypothesized that changing the configuration at the C3 position may
reduce the affinity of the STZ-analogue to GLUT2, resulting in reduced toxicity to cells. At the same
2),
3
4
β
β
time, because bacteria express unique transport systems that are absent in mammalian cells, such as a
d-allose transporter [26] and the PTS system, the altered STZ compounds may still be imported by
bacteria, resulting in antibacterial activity. Here we present the chemical synthesis of STZ analogues
2–4, and the biological evaluation using minimum inhibitory concentration (MIC) and growth-viability
assays on E. coli K12 cells as well as cytotoxicity studies on insulin-producing β cells.
2. Results
2.1. Chemical Synthesis of STZ Derivatives 2–4
The chemical synthesis of the three STZ derivatives exploited the regioselective oxidation protocol
developed in our group, as published previously [27
are treated with [(neocuproine)Pd( -OAc)]2(OTf)2 as the catalyst and benzoquinone as the oxidant to
selectively oxidize the C3-OH to the keto-functionality. Gratifyingly, this procedure could be employed
to oxidize STZ directly, yielding keto-STZ ( ) in one step and decent yield (58%) after purification
(Scheme 1A). For the synthesis of the analogues and , the N-nitroso moiety was introduced in
the final stage using N-nitrosocarbamate reagent 29 31]. As depicted in Scheme 1B, 4-nitrophenyl
N-nitroso-N-methylcarbamate
was prepared by treating 4-nitrophenyl chloroformate in THF at 0 ◦C
with a solution of methylamine in THF to afford 4-nitrophenyl methylcarbamate . Nitrosation of
with NaNO2 was carried out in a mixture of DCM and 12 M HCl in water and produced compound
d-Allosamine and d-lividosamine were prepared using reported procedures [32], and treated with
reagent to obtain allo-STZ ( , 41%) and deoxy-STZ ( , 70%) (Scheme 1C). Allo-STZ and deoxy-STZ
were isolated as a mixture of the pyranose and furanose forms, with the pyranose form being the major
component, and fully characterized. STZ and its analogues are best stored as solids at
20 ◦C and
shielded from light. Solutions of STZ are most stable at pH 4.5, but are known to slowly release NO
,28] In short, largely unprotected carbohydrates
µ
2
3
4
6
[ –
6
5
5
6.
6
3
4
−
gas at ambient temperature [33]. Since NO release cannot be prevented even at
prepared immediately before use.
−
80 ◦C, solutions are

Antibiotics 2020, 9, 182
4 of 11
Scheme 1.
(
A
) One-step preparation of keto-STZ (2); (B) Synthesis of the N-nitrosocarbamate 6;
(
C) Synthesis of analogues
3
and starting from N-acetyl-d-glucosamine using late-stage methyl
4
nitrosourea introduction to d-allosamine and d-lividosamine [32].
2.2. Biological Evaluation of STZ Compounds 1–4
Next, we started the biological evaluation of compounds
the compounds are incubated with E. coli K12 (TOP10) cells for 16 h. As depicted in Table 1 and
Figure 2, the parent STZ compound exhibited potent antimicrobial activity as expected, with an MIC
1–4 with a standard MIC assay, where
1
value of 1.5 mg/L in rich medium (lysogeny broth, LB). This corresponds with reported values, which
lie between 0.4 and 100 mg/L depending on the bacteria and even the E. coli strain [23]. Interestingly,
when the cells were grown in minimal medium with GlcNAc as the sole carbon source prior to addition
of STZ
with the same concentrations (MIC > 1.5 mg/L). When the novel STZ-analogues
a large reduction in antimicrobial activity was observed. Only minor impairment of bacterial growth
was observed for treatment with analogues for 16 h (MIC values > 200 mg/L, Table 1). However,
when the MIC studies were performed with continuous monitoring of bacterial growth during the
overnight incubation with STZ or analogues , a more detailed picture of the inhibition profile was
obtained, revealing significant differences in growth behavior. Overall, the growth rate in minimal
medium was reduced as compared to growth in rich medium. Parent STZ ( ), keto-STZ ( ) and
allo-STZ ( ) all revealed concentration-dependent inhibition that was increased after pre-sensitizing the
bacteria with GlcNAc (Figure 2 and Supplementary Figure S1). At the same time, pre-sensitizing led
to an increase in MIC value for STZ ( ), as even the highest concentration of STZ (1.5 mg/L) showed
1
in minimal medium containing glycerol as the carbon source, incomplete killing was observed
2–4
were evaluated,
2–4
1
2–4
1
2
3
1

Antibiotics 2020, 9, 182
5 of 11
outgrowth of cells after 12 h. Interestingly, pre-sensitizing with ribose (a configurational homolog
of allose) increased the susceptibility of E. coli to allo-STZ (
deoxy-STZ (4) was unaffected (Supplementary Figure S1).
3) in a similar way, while the effect of
Table 1. Results from the minimum inhibitory concentration (MIC) assays using compounds 1–4.
MIC Value (mg/L)
Compound
LB a
MM b + GlcNAc
MM b + Ribose
STZ (1)
1.5
>1.5
>200
>200
>200
n.d.
n.d.
>200
>200
keto-STZ (2)
allo-STZ (3)
deoxy-STZ (4)
>200
>200
>200
a
LB = rich medium; b MM = minimal medium.
Figure 2. Growth curves of E. coli to determine the MIC value for STZ
right) in rich medium, and for STZ bottom left) and keto-STZ bottom right) in minimal medium
+ GlcNAc. Experiments are performed in triplicate.
1 (top left) and keto-STZ 2 (top
1
(
2 (
An alternative way to asses antimicrobial activity is to identify the percentage of viable bacteria
after a short treatment with the antibiotic (2 h). In this so-called growth-based viability assay, originally
developed by Haynes and co-workers for the toxicity screening of nanomaterials [34], the percentage of
viable cells is determined by correlating the growth (OD600) of the treated samples to control samples
with a known number of cells that are grown under the same conditions. The advantage of using
relative viabilities is that it allows straightforward comparison of viability, even when the experiments
have been performed under different growth conditions. The results of the growth-based viability
assay are presented in Table 2. In rich-medium conditions, STZ (1) showed the largest effect on bacterial

Antibiotics 2020, 9, 182
6 of 11
growth, with a viability of 0.0035% after treatment with 50 mg/L. Of the STZ-derivatives, keto-STZ
) was the most active inhibitor of growth (8% of viability with 200 mg/L), whereas allo-STZ (
showed a remaining viability of 29%. Deoxy-STZ ( ) showed no significant delay in growth, resulting
(2
3)
4
at a calculated viability of 127%, even at the highest concentration tested (200 mg/L). These results
corroborate those of the MIC assays. When the viability assays were performed on cells pre-sensitized
with GlcNAc, a similar trend was observed as compared to the MIC assays (Figure 3). The activity of
STZ (
Keto-STZ (
non-presensitized cells (0.1% viable cells with 200 mg/L). In contrast, the antibacterial activity of
allo-STZ ( ) remained the same, and deoxy-STZ ( ) showed no inhibitory effect on bacterial growth,
1
) was increased two-fold, with a resulting viability of 0.0019% after treatment with 50 mg/L.
2) also showed higher activity in the same concentration range compared to its effect on
3
4
also after pre-sensitizing with ribose (Supplementary Figure S2).
Table 2. Results from the growth-based viability assays using compounds 1–4.
Viability
Compound
LB a
MM b + GlcNAc
MM b + Ribose
STZ (1) (50 mg/L)
keto-STZ (2) (200 mg/L)
allo-STZ (3) (200 mg/L)
0.0035%
8%
29%
0.0019%
0.1%
29%
n.d.
n.d.
21%
202%
deoxy-STZ (4) (200 mg/L)
127%
170%
a
LB = rich medium; b MM = minimal medium.
Figure 3. Growth-based viability curves of E. coli for STZ
medium, and for STZ bottom left) and keto-STZ bottom right) in minimal medium + GlcNAc.
Experiments are performed in triplicate.
1 (top left) and keto-STZ 2 (top right) in rich
1
(
2 (

Antibiotics 2020, 9, 182
7 of 11
From the antimicrobial assays above it is clear that the parent STZ (
inhibiting bacterial growth, while keto-STZ ( ) and allo-STZ (
an important goal of this project is to understand the structural motifs important for antimicrobial and
cytotoxic activities, we explored the toxicity of compounds on insulin-producing cells. Using
the WST-1 dye as a colorimetric read-out of cell proliferation and mitochondrial activity, the effect of
compounds was measured, as depicted in Figure 4. Compared to the negative inhibition control
(incubation with citrate), STZ (1) shows a large reduction in β cell viability. Unfortunately, this result
is mimicked by keto-STZ ( ), which shows a similarly high cytotoxicity. In contrast, allo-STZ ( ) and
deoxy-STZ (4) show a negligible amount of cytotoxicity in this assay.
1
) remains the most active in
2
3) showed moderate to poor activity. As
1–
4
β
1–4
2
3
Figure 4. Effect of compounds
1–4 (5 mM) on viability of β cells. Significant differences compared to
the control were indicated by * p < 0.05 or **** p < 0.0001.
3. Discussion
To overcome the increasing resistance of bacteria to current antibiotics, novel antimicrobial
compounds are desperately needed. Using our regioselective oxidation method, we generated three
novel STZ analogues. It was exciting to see that keto-derivative
by oxidizing STZ directly. The robust way of N-nitrosourea introduction using N-nitrosocarbamate
reagent proved useful for the generation of the STZ analogues based on d-allosamine (allo-STZ,
and d-lividosamine (deoxy-STZ, ). It is clear from the MIC assays that the parent STZ compound (
2 could be accessed in good yields
1
6
3
1
)
)
4
displayed the largest antimicrobial activity on E. coli K12 (TOP10) cells, with only 1.5 mg/L needed
for complete killing. When the E. coli cells were first treated with GlcNAc before incubation with
STZ, the impact of STZ was reduced. Earlier studies already revealed that the effect of exogenous
GlcNAc on carbohydrate metabolism and bacterial growth is complex [23]. When GlcNAc and STZ
were added simultaneously, E. coli cells have been observed to import less STZ, suggesting that they
compete for the same transport machinery, presumably the PTS system [22]. In contrast, when the cells
are first pre-sensitized with GlcNAc, the upregulation of transport machinery is suggested to result in
a higher sensitivity to STZ
1 and keto-STZ 2 [35]. The latter effect is especially observed in the first 8 h
of incubation (Figure 2), where the growth rate is significantly impacted by the lower concentrations
compared to rich medium (0.1875 and 0.375 mg/L for STZ, 12.5 and 25 mg/L for keto-STZ). This may
reflect the upregulation of transport machinery in the presence of GlcNAc, which primes the bacterial

Antibiotics 2020, 9, 182
8 of 11
cells to increase the uptake of STZ, resulting in an increased sensitivity. However, after 8 h the OD600
increases again and reveals incomplete killing even with the highest concentrations of STZ (Figure 2).
1
This result may reflect a loss of the sensitization effect over time, and a reduction of the expression of
GlcNAc transport machinery. Alternatively, STZ and analogues may be slowly degrading under the
assay conditions [33]. The growth-based viability experiments produce a similar trend (Figure 3), as
the largest delay in outgrowth was observed for parent STZ (
note that pre-sensitizing with ribose increased the susceptibility of E coli cells to allo-STZ (
deoxy-STZ ( ). This may indicate the upregulation of the d-allose operon, as described before [26].
Unfortunately, the trend of antibacterial activity of compounds is mirrored in the cytotoxicity
experiment. When incubated with insulin-producing cells, compounds and show the highest
reduction in cell density (Figure 4), whereas compounds and are only marginally toxic to the cells.
We postulate that the activity of keto-STZ ( ) in both the antimicrobial activity and cytotoxicity assays
1) and keto-STZ (
2). It is interesting to
3), but not to
4
1–4
β
1
2
3
4
2
may be explained by the hydrate, which results after addition of water to the ketone functionality.
Even though the hydrate has not been observed in NMR, as hydration at keto-C3 (13C NMR~204 ppm
for the keto) would result in a significant upfield shift (13C NMR~95 ppm for the hydrate) [36] that was
not observed, a small amount of the keto may be hydrated in situ. When the hydrate is formed on the
C3 position, the resulting structure may mimic the configuration of STZ more closely. The moderate
but concentration-dependent antimicrobial activity of allo-STZ (
STZ analogue did not show significant cytotoxicity to β cells.
3) is an exciting result, because this
In conclusion, we have described a straightforward and robust method to derivatize STZ on the C3
position, and generated three novel analogues by oxidation, inversion and reduction. This late-stage
modification of largely unprotected carbohydrates, compatible with sensitive functional groups such
as a free anomeric hydroxyl group and a reactive N-nitroso functionality, allows straightforward access
to analogues that normally require full syntheses. We foresee that this late stage modification approach
can be expanded, at least to other carbohydrate-based drugs, like the many other aminoglycoside
antibiotics. In the same vein it is obvious that not only epimerization and hydroxy group removal
are possible but also conversion of the keto function into other functional groups and handles. Of
the prepared analogues, keto-STZ (
in bacterial growth, albeit significantly lower than parent STZ (
was similarly largest for keto-STZ ( ), limiting the applicability of this compound as an antibiotic.
Although the allo-STZ ( ) analogue revealed a promising selectivity for the killing of bacterial cells over
pancreatic cells, the antimicrobial activity is not competitive with STZ. This suggests that the bacterial
2) showed the highest antimicrobial activity and largest delay
1
). Moreover, the toxicity to cells
β
2
3
β
carbohydrate uptake systems only moderately tolerate the modification of the C3 position in glucose
and GlcNAc analogues. This research invites a more thorough analysis of the various carbohydrate
uptake mechanisms bacteria use. Once we know the precise binding of glucose and GlcNAc, also with
respect to the anomeric configuration, and the pyranose versus furanose form, rational design and
synthesis of STZ analogues will be possible. Alternatively, chemical modifications can be investigated
to stimulate the bacterial uptake of STZ analogues.
4. Materials and Methods
4.1. Determination of MIC Values
The minimum inhibitory concentration (MIC) of STZ derivatives
2–4, in comparison to STZ
(1), was determined according to the recommendations proposed by the Clinical and Laboratory
Standard Institute (CLSI) [37], using LB as rich medium. See the Supplementary Information for a
detailed procedure.
4.2. Growth-Based Viability Assays
To better assess the impact of the STZ derivatives
2–4 on bacterial growth and viability,
growth-based viability assays were performed [34]. The assay was performed in rich medium

Antibiotics 2020, 9, 182
9 of 11
(lysogeny broth, LB) as well as in minimal medium with N-acetylglucosamine (GlcNAc) or ribose
as additives to study the effect of these compounds on the uptake of the streptozotocin-analogues.
Bacterial cells (Escherichia coli K12 TOP10) were first exposed to the streptozotocin derivatives (either
in rich or minimal medium) at room temperature and constant shaking to ensure good mixing.
Subsequently, a portion of the exposed mixture was diluted 40 by addition to fresh medium to
×
alleviate the effect of the antibiotic, and the cells were incubated in a Biotek plate reader for 16 h at
37 ◦C with optical density (OD) measurements at 600 nm taking place every 20 min preceded by 30 s
of shaking. The resulting data was processed to obtain viability values, as described in detail in the
Supplementary Information.
4.3. Cytotoxicity Assays
The MIN6 cell line (pancreatic
(ATCC, Manassas, VA, USA). MIN6 cells (passages 30–45) were cultured in DMEM High glucose
medium (Lonza, Basal, Switzerland), containing 15% fetal bovine serum (FBS, Lonza), 50 mol/L
g/mL streptomycin (all from
β cells) was purchased from American Type Culture Collection
µ
β
-mercaptoethanol, 2 mmol/L l-glutamine, 50 U/mL penicillin, and ◦50
µ
Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured at 37 C in a humidified atmosphere
containing 95% air and 5% CO2. The effect of streptozotocin (STZ) and the derivatives on
was determined by the cell proliferation reagent WST-1 (Roche, Indianapolis, USA). Briefly, MIN6 cells
(1
105 cells/well) were seeded in 96-well plates. Cells were cultured overnight and the following
day incubated with or without STZ or analogue (Sigma-Aldrich) at 5 mM for 48 and 72 h followed by
WST-1 assay. After 30 min incubation with WST-1 (10
L/well) at 37 ◦C, the absorbance was measured
β cell viability
×
µ
at 450 nm using a Bio-Rad Benchmark Plus microplate spectrophotometer reader (Bio-Rad Laboratories
B.V, Veenendaal, The Netherlands.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/4/182/s1,
Figure S1: Growth curves of E. coli TOP10 to determine the MIC value for allo-STZ
3
(top left) and deoxy-STZ
(middle right) in minimal medium +
(bottom right) in minimal medium + ribose. Experiments
are performed in triplicate; Figure S2: Growth-based viability curves of E. coli TOP10 for allo-STZ (top left)
and deoxy-STZ (top right) in rich medium, allo-STZ (middle left) and deoxy-STZ (middle right) in minimal
medium + GlcNAc, and allo-STZ (bottom left) and deoxy-STZ (bottom right) in minimal medium + ribose.
Experiments are performed in duplicate.
4
(top right) in rich medium, allo-STZ
3 (middle left) and deoxy-STZ 4
GlcNAc, and allo-STZ (bottom left) and deoxy-STZ
3
4
3
4
3
4
3
4
Author Contributions: Conceptualization of the project, J.Z., L.Y., A.J.M., M.D.W., M.T.C.W.; chemical synthesis,
J.Z.; MIC and viability assays, L.Y.; cytotoxicity assay, B.J.d.H. and P.d.V.; data interpretation, all authors; writing
the manuscript, all authors. All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by the Dutch Organization for Scientific Research (NWO VENI
722.016.006 to MTCW) and the China Scholarship Council (201408530053 to J.Z.).
Acknowledgments: We thank T.M. Wood, and N.I. Martin for sharing their expertise in the MIC studies.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Mühlberg, E.; Umstätter, F.; Kleist, C.; Domhan, C.; Mier, W.; Uhl, P. Renaissance of vancomycin: Approaches
for breaking antibiotic resistance in multidrug-resistant bacteria. Can. J. Microbiol. 2020, 66, 11–16. [CrossRef]
[PubMed]
Khan, F.; Pham, D.T.N.; Kim, Y.-M. Alternative strategies for the application of aminoglycoside antibiotics
against the biofilm-forming human pathogenic bacteria. Appl. Microbiol. Biotechnol. 2020, 104, 1955–1976.
[CrossRef] [PubMed]
Chandrika, N.T.; Garneau-Tsodikova, S. Comprehensive review of chemical strategies for the preparation
of new aminoglycosides and their biological activities. Chem. Soc. Rev. 2018, 47, 1189–1249. [CrossRef]
[PubMed]

Antibiotics 2020, 9, 182
10 of 11
4.
Kotra, L.P.; Haddad, J.; Mobashery, S. Aminoglycosides: Perspectives on Mechanisms of Action and
Resistance and Strategies to Counter Resistance. Antimicrob. Agents Chemother. 2000, 44, 3249–3256.
[CrossRef]
5.
6.
Ramirez, M.S.; Tolmasky, M.E. Aminoglycoside modifying enzymes. Drug Resist. Updat. 2010, 13, 151–171.
[CrossRef]
Kato, T.; Yang, G.; Teo, Y.; Juskeviciene, R.; Perez-Fernandez, D.; Shinde, H.M.; Salian, S.; Bernet, B.;
Vasella, A.; Böttger, E.C.; et al. Synthesis and Antiribosomal Activities of 40-O-, 60-O-, 4”-O-, 40,60-O- and
4”,6”-O-Derivatives in the Kanamycin Series Indicate Differing Target Selectivity Patterns between the 4,5-
and 4,6-Series of Disubstituted 2-Deoxystreptamine Aminoglycoside Antibiotics. ACS Infect. Dis. 2015, 1,
479–486. [CrossRef]
7.
8.
9.
Chen, W.; Matsushita, T.; Shcherbakov, D.; Boukari, H.; Vasella, A.; Böttger, E.C.; Crich, D. Synthesis,
antiribosomal and antibacterial activity of 40-O-glycopyranosyl paromomycin aminoglycoside antibiotics.
MedChemComm 2014, 5, 1179–1187. [CrossRef]
Matsushita, T.; Chen, W.; Juskeviciene, R.; Teo, Y.; Shcherbakov, D.; Vasella, A.; Böttger, E.C.; Crich, D.
Influence of 40-O-Glycoside Constitution and Configuration on Ribosomal Selectivity of Paromomycin. J. Am.
Chem. Soc. 2015, 137, 7706–7717. [CrossRef]
Sati, G.C.; Sarpe, V.A.; Furukawa, T.; Mondal, S.; Mantovani, M.; Hobbie, S.N.; Vasella, A.; Böttger, E.C.;
Crich, D.; Mantovani, M. Modification at the 20-Position of the 4,5-Series of 2-Deoxystreptamine
Aminoglycoside Antibiotics To Resist Aminoglycoside Modifying Enzymes and Increase Ribosomal Target
Selectivity. ACS Infect. Dis. 2019, 5, 1718–1730. [CrossRef]
10. Sonousi, A.; Sarpe, V.A.; Brilkova, M.; Schacht, J.; Vasella, A.; Böttger, E.C.; Crich, D. Effects of
the 1-N-(4-Amino-2S-hydroxybutyryl) and 60-N-(2-Hydroxyethyl) Substituents on Ribosomal Selectivity,
Cochleotoxicity, and Antibacterial Activity in the Sisomicin Class of Aminoglycoside Antibiotics. ACS Infect.
Dis. 2018, 4, 1114–1120. [CrossRef]
11. Herzog, I.M.; Feldman, M.; Eldar-Boock, A.; Satchi-Fainaro, R.; Fridman, M. Design of membrane targeting
tobramycin-based cationic amphiphiles with reduced hemolytic activity. MedChemComm 2013, 4, 120–124.
[CrossRef]
12. Fosso, M.Y.; Zhu, H.; Green, K.D.; Garneau-Tsodikova, S.; Fredrick, K. Tobramycin variants with enhanced
ribosome-targeting activity. ChemBioChem 2015, 16, 1565–1570. [CrossRef]
13. Ogbonnaya, E.C.; Eleazu, K.; Chukwuma, S.; Essien, U.N. Review of the mechanism of cell death resulting
from streptozotocin challenge in experimental animals, its practical use and potential risk to humans.
J. Diabetes Metab. Disord. 2013, 12, 60. [CrossRef]
14. Agarwal, M. Streptozotocin: Mechanisms of action—Proceedings of a Workshop Held on 21 June 1980,
Washington, Dc. FEBS Lett. 1980, 120, 1–3. [CrossRef]
15. Reusser, F. Mode of Action of Streptozotocin. J. Bacteriol. 1971, 105, 580–588. [CrossRef]
16. King, A.J.F. The use of animal models in diabetes research. Br. J. Pharmacol. 2012, 166, 877–894. [CrossRef]
17. Wang, Z.; Gleichmann, H. GLUT2 in pancreatic islets: Crucial target molecule in diabetes induced with
multiple low doses of streptozotocin in mice. Diabetes 1998, 47, 50–56. [CrossRef]
18. Schnedl, W.J.; Ferber, S.; Johnson, J.H.; Newgard, C.B. STZ transport and cytotoxicity. Specific enhancement
in GLUT2-expressing cells. Diabetes 1994, 43, 1326–1333. [CrossRef]
19. Elsner, M.; Guldbakke, B.; Tiedge, M.; Munday, R.; Lenzen, S. Relative importance of transport and alkylation
for pancreatic beta-cell toxicity of streptozotocin. Diabetol. 2000, 43, 1528–1533. [CrossRef]
20. Moertel, C.G.; Hanley, J.A.; Johnson, L.A. Streptozocin Alone Compared with Streptozocin plus Fluorouracil
in the Treatment of Advanced Islet-Cell Carcinoma. N. Engl. J. Med. 1980, 303, 1189–1194. [CrossRef]
21. Postma, P.W.; Lengeler, J.W.; Jacobson, G.R. Phosphoenolpyruvate:carbohydrate phosphotransferase systems
of bacteria. Microbiol. Rev. 1993, 57, 543–594. [CrossRef]
22. Ammer, J.; Brennenstuhl, M.; Schindler, P.; Holtje, J.V.; Zahner, H. Phosphorylation of streptozotocin during
uptake via the phosphoenolpyruvate: Sugar phosphotransferase system in Escherichia coli. Antimicrob.
Agents Chemother. 1979, 16, 801–807. [CrossRef] [PubMed]
23. Lengeler, J. Analysis of the physiological effects of the antibiotic streptozotocin on Escherichia coli K 12 and
other sensitive bacteria. Arch. Microbiol. 1980, 128, 196–203. [CrossRef] [PubMed]
24. Bannister, B. Synthesis and Biological-Activities of some Analogs of Streptozotocin. J. Antibiot. 1972, 25,
377–386. [CrossRef]

Antibiotics 2020, 9, 182
11 of 11
25. Iwasaki, M.; Ueno, M.; Ninomiya, K.; Sekine, J.; Nagamatsu, Y.; Kimura, G. Alkyl streptozotocin analogues
with improved biological activities. J. Med. Chem. 1976, 19, 918–923. [CrossRef]
26. Kim, C.; Song, S.; Park, C. The D-allose operon of Escherichia coli K-12. J. Bacteriol. 1997, 179, 7631–7637.
[CrossRef]
27. Jäger, M.; Hartmann, M.; De Vries, J.G.; Minnaard, A.J. Catalytic Regioselective Oxidation of Glycosides.
Angew. Chem. Int. Ed. 2013, 52, 7809–7812. [CrossRef]
28. Jumde, V.R.; Eisink, N.N.H.M.; Witte, M.D.; Minnaard, A.J. C3 Epimerization of Glucose, via Regioselective
Oxidation and Reduction. J. Org. Chem. 2016, 81, 11439–11443. [CrossRef]
29. Martinez, J.; Oiry, J.; Imbach, J.L.; Winternitz, F. A Selective Synthesis of N-Alkyl N-Nitrosoureas. Eur. J. Med.
Chem. 1980, 15, 211–213.
30. Martinez, J.; Oiry, J.; Imbach, J.L.; Winternitz, F. Activated N-nitrosocarbamates for regioselective synthesis
of N-nitrosoureas. J. Med. Chem. 1982, 25, 178–182. [CrossRef]
31. Gassmann, N.; Stoos, F.; Meier, A.; Helali, S.E.; Hardegger, E. Varianten im Zuckerteil des Streptozotocins.
Helv. Chim. Acta 1975, 58, 182–185. [CrossRef] [PubMed]
32. Zhang, J.; Eisink, N.N.H.M.; Witte, M.D.; Minnaard, A.J. Regioselective Manipulation of GlcNAc Provides
Allosamine, Lividosamine, and Related Compounds. J. Org. Chem. 2018, 84, 516–525. [CrossRef] [PubMed]
33. Goud, B.J.; Dwarakanath, V.; Chikka, B.K. Streptozotocin—A diabetogenic agent in animal models. Int. J.
Pharm. Pharm. Res. 2015, 3, 253–269.
34. Qiu, T.A.; Nguyen, T.H.T.; Hudson-Smith, N.V.; Clement, P.L.; Forester, D.-C.; Frew, H.; Hang, M.N.;
Murphy, C.J.; Hamers, R.J.; Feng, Z.V.; et al. Growth-Based Bacterial Viability Assay for Interference-Free
and High-Throughput Toxicity Screening of Nanomaterials. Anal. Chem. 2017, 89, 2057–2064. [CrossRef]
35. Jacobson, G.R.; Poy, F.; Lengeler, J.W. Inhibition of Streptococcus mutans by the antibiotic streptozotocin:
Mechanisms of uptake and the selection of carbohydrate-negative mutants. Infect. Immun. 1990, 58, 543–549.
[CrossRef]
36. Liu, H.M.; Sato, Y.; Tsuda, Y. Chemistry of Oxo-Sugars.2. Regioselective and Stereoselective Synthesis of
Methyl D-Hexopyranosiduloses and Identification of their Forms Existing in Solutions. Chem. Pharm. Bull.
1993, 41, 491–501. [CrossRef]
37. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 9th ed.; Clinical
and Laboratory Standards Institute: Wayne, PA, USA, 2012.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).