Design, Synthesis, and Biological Evaluation of Light-Activated Antibiotics

Inga S. Shchelik, Andrea Tomio, and Karl Gademann*

Article

Design, Synthesis, and Biological Evaluation of Light-Activated

Antibiotics

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ABSTRACT: The spatial and temporal control of bioactivity of

small molecules by light (photopharmacology) constitutes a

promising approach for study of biological processes and ultimately

for the treatment of diseases. In this study, we investigated two

diﬀerent “caged” antibiotic classes that can undergo remote

activation with UV-light at λ = 365 nm, via the conjugation of

deactivating and photocleavable units through a short synthetic

sequence. The two widely used antibiotics vancomycin and

cephalosporin were thus enhanced in their performance by

rendering them photoresponsive and thereby suppressing un-

desired oﬀ-site activity. The antimicrobial activity against Bacillus

subtilis ATCC 6633, Staphylococcus aureus ATCC 29213, S. aureus

ATCC 43300 (MRSA), Escherichia coli ATCC 25922, and

Pseudomonas aeruginosa ATCC 27853 could be spatiotemporally controlled with light. Both molecular series displayed a good

activity window. The vancomycin derivative displayed excellent values against Gram-positive strains after uncaging, and the next-

generation caged cephalosporin derivative achieved good and broad activity against both Gram-positive and Gram-negative strains

after photorelease.

KEYWORDS: antibacterial agents, photopharmacology, photocaging, vancomycin, cephalosporin

4−36

harmacotherapy often remains the treatment of choice for

many diseases via suitable medication. However, this

transmitters,

or peptides. Related to antibiotics, there

have been many examples of photoswitchable groups attached

approach is often associated with issues related to environ-

mental toxicity, poor drug selectivity causing side-eﬀects, and

to antibiotics, which have been demonstrated to successfully

12,14−16,19,20,22,38

inhibit bacterial growth by irradiation.

How-

the emergence of resistance in certain disease areas such as

infectious diseases.

systems have been developed to overcome these issues,

including either endogenous stimuli (such as enzyme, pH,

redox reactions) or exogenous stimuli (such as light, ionizing

irradiation, magnetic ﬁelds).

approaches, photopharmacology has demonstrated excellent

performance in achieving control of time, area, and dosage of

therapeutics by light.

includes incorporation of photoswitchable groups into the

molecular structure of bioactive compounds,

of functional groups for light-triggered drug self-destruction,

or in general “caging” the activity of compounds.

respect, caged compounds include photoactivatable probes

such as photoprotecting groups, photocleavable linkers, or

ever, thermodynamic equilibration of the photoswitches

invariably leads to a decrease of antibiotic activity over time,

often during the application. In order to prevent bacterial

regrowth, constant and longer irradiation needs to be

employed, which consequently might lead to side-eﬀects due

to undesired prolonged UV irradiation.

In contrast, photocaging of antibiotics presents the

complementary and unique strategy of releasing the active

compounds ad infinitum. Advantages of this strategy include

−6

So far, several stimuli-responsive

In terms of exogenous

−11

The development of such strategies

(

1) short exposure to UV light, (2) release of maximum

12−22

introduction

concentration within a short time frame, and (3) prolonged

activity of the antibacterial agents. Interestingly, there are only

24−27

In this

few reports on photocaged antibiotics, used for the study of

39,40

protein translation,

hydrogel modiﬁcation for antibacterial

28,29

photodegradable peptides,

and these compounds remain

Received: January 12, 2021

Published: March 3, 2021

biologically or functionally inert prior to uncaging. Photo-

activation of caged compounds enables the spatiotemporal

regulation of the activity of the drugs of interest, which has

been successfully applied as powerful tools in biological

studies. There are many examples of successful utilization of

30−33

photocaging handles directly on antitumor drugs,

neuro-

2021 American Chemical Society

ACS Infect. Dis. 2021, 7, 681−692

Article

Scheme 1. Synthesis of UV-Light Regulated Vancomycin Derivatives 1 and 5

Reagents and conditions: (a) MeO-PEG -amine, HATU, DIPEA, DMF, 2 h, rt, 72% for 8a. (b) MeOH, H SO , 50 °C, overnight, 97% for 8b.

(c) 20% piperidine, DMF, 1−2 h, rt, 83% for 9a, 95% for 9b. (d) Vancomycin hydrochloride, PyBop, HOBt, DMF, 2 h, rt, 28% for 1 from 9a, 30%

for 5 from 9b.

wound dressings₄, blocking the group responsible for

approach for steric blocking, PEGylation referring to the

addition of oligo or poly ethylene glycol units. We

hypothesized that the introduction of a relatively long PEG

chain to vancomycin will suppress its activity and prevent

binding with the terminal amino acid residues of the nascent

peptide chain during cell wall synthesis. In case of

cephalosporin, we expected to either avert the insertion of

the drug into penicillin-binding protein (PBP) or prevent

transport issues by, e.g., bacterial pumps by this approach. As

consequence for the design, compounds 1 and 2 were chosen

as the target caged antibiotics of our study, which should

2,43

antibiotic activity,

or living organism functionalization.

However, to the best of our knowledge, examples remain very

scarce and the important classes of vancomycin and

cephalosporin antibiotics have not been addressed so far. In

this study, we report the control of activity of two widely used

antibiotics vancomycin and cephalosporin, where the caging

functionality was appended to the pharmacophore. We

demonstrate that UV-light exposure at λ = 365 nm uncages

the precursor antibiotics and thereby releases antibacterial

activity in the presence of bacteria.

Vancomycin and cephalosporin are members of the class of

antibiotics that inhibit the cell wall biosynthesis in bacteria.

Both drugs remain on the World Health Organization’s List of

Essential Medicines. Vancomycin is active against Gram-

positive bacteria and widely used in clinics worldwide,

especially for the treatment of methicillin-resistant Staph-

ylococcus aureus (S. aureus, MRSA). We evaluated members

of the fourth generation of cephalosporins, which feature

inhibitory eﬀects against various Gram-negative bacteria,

including Pseudomonas aeruginosa (P. aeruginosa).

evolution of microbial resistance to vancomycin and

cephalosporins is an emerging problem that renders those

particularly interesting candidates for photopharmacology.

The development of photoresponsive analogues and control of

their activity could reduce undesirable bacterial interactions

with the active drug form, thus limiting the progress of

bacterial resistance.

release active compounds 3 and 4. The good antibacterial

activity of similar pyridinium cephalosporin derivatives was

52,53

shown earlier in several studies

and patents. Compounds

and 6 serve as control compounds to evaluate the role of the

PEG blocking group. Vancomycin has been modiﬁed

according to a previously reported strategy at the carboxylic

44,55

acid position,

and the cephalosporin modiﬁcation was

extending the C-3′ position of the cepham ring system with a

nitrobenzyl caging group sterically modiﬁed by ethylene glycol

units.

48,49

The

The synthesis of target vancomycin derivative 1 started with

the linker preparation (Scheme 1). The acid functionality of

the Fmoc-Photo-Linker 7 was used for incorporation of the

PEG chain via amide bond formation (→ 8a), followed by

Fmoc deprotection leading to the desired linker 9a with 60%

yield over two steps. The last step included coupling the

obtained linker with vancomycin hydrochloride in the presence

of PyBop and HOBt as coupling agents to give target

compound 1. Analogously, the control compound 5 without

the PEG chain was obtained via intermediates 8b and 9b in

similar yields. The synthesis of target cephalosporin derivative

50,51

RESULTS AND DISCUSSION

■

We chose to employ a dual strategy featuring both a

photocleavable group for caging combined with a PEGylation

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Scheme 2. Synthesis of Cephalosporin Derivatives 2, 4, and 6

Reagents and conditions: (a) 1-Bromo-2-(2-methoxyethoxy)ethane, K CO , DMF, 90 °C, overnight, 98% for 11a. (b) MOMCl, DIPEA, DCM, 0

3 2

9% for 13a, 55% for 13b. (e) NaI, acetone, 1 h, rt, then 13a or 4-(aminomethyl)pyridine or 13b, acetone, 3−5 h, rt, then anisole, TFA, DCM, 2−6

C to rt, 1.5h, 85% for 11b. (c) 4-(Aminomethyl)pyridine, NaBH(OAc) , AcOH, DCE, 6 h, rt, 63% for 12a, 48% for 12b. (d) Boc O, THF, 3 h, rt,

h, rt, 2% for 2, 10% for 4, 1% for 6.

Table 1. MIC Values (in μg/mL) of Vancomycin and Cephalosporin Derivatives

Red background denotes caged precursors, and green background denotes active uncaged antibiotics.

started with an S 2 reaction between the phenolate of 2-

that the release of vancomycin amide 3 takes place rapidly

during the UV-irradiation (λ = 365 nm) of a vancomycin

nitro-5-hydroxybenzaldehyde 10 and 1-bromo-2-(2-

methoxyethoxy)ethane (Scheme 2) to give 11a. Next,

reductive amination to 12a with 4-(aminomethyl)pyridine

was carried out. Carrying out this reaction with stepwise

addition of the reducing agent could improve the performance

of the reaction, and correspondingly, the yield. For the key

coupling with the cephalosporin core, the secondary N atom

on the intermediate amine had to be blocked. The attempt to

attach the linker directly to the cephalosporin core 14 led to

the formation of undesired products. A Boc protecting group

was chosen to block the reactivity of secondary amine on the

linker, and after the screening of several conditions, the use of

THF as a solvent was crucial for the reaction, in order to

achieve full conversion and to avoid decomposition. The

desired linker 13a was obtained via this route in 23% yield over

derivative with photocleavable linker and reaches a maximum

of 70% after 5 min. Next, the photocleavage eﬃcacy of

cephalosporin derivative 5 was investigated. It was shown that

the cephalosporin with pyridyl-methylamine moiety 4 as a

product was released after UV-irradiation (λ = 365 nm, Figure

S1). Moreover, the same tendency in eﬃcacy for the

photocleavage of the linker was observed compared to the

vancomycin derivative. The maximum conversion of roughly

70% was observed after only 6 min of irradiation (Figure S2).

Importantly, no by-products except for the cleaved linker could

be detected after the photocleavage by UHPLC-MS (Figure

S4).

The antimicrobial activity studies of all obtained compounds

were investigated by performing a broth dilution method

steps. The key coupling reaction with cephalosporin 14

according to the EUCAST standard protocol. The minimum

inhibitory concentration (MIC) of the target compounds 1

and 2, compounds released after UV-irradiation 3 and 4, as

well as control compounds 5 and 6 were determined against

two Gram-negative strains E. coli ATCC 25922 and

P. aeruginosa ATCC 27853, and three Gram-positive strains

B. subtilis ATCC 6633, S. aureus ATCC 29213 (VSSA),

S. aureus ATCC 43300 (MRSA) (Table 1). As was expected,

PEG containing vancomycin derivative 1 did not display

signiﬁcant activity against Gram-positive strains with a MIC

value more than 64 μg/mL. In contrast, the compound 3

lacking a PEG group displayed excellent activity with MIC

values of 0.125 μg/mL and 1 μg/mL against B. subtilis and

S. aureus, respectively, similar to vancomycin itself. Moreover,

included three straightforward steps without the isolation of

intermediates. Moreover, both Boc and PMB protection

groups could be removed in one step in the presence of

triﬂuoroacetic acid (TFA) and anisole, to give the target

compound, albeit in poor yield. Along the same lines, the

preparation of control compound 6 was achieved by using

transient MOM protection via intermediates 11b, 12b, and

3b, and the active compound 4 was obtained by direct

reaction of core 14 with 4-(aminomethyl)-pyridine.

The general photochemistry of compounds 1 and 2 was

studied next. In an earlier study, we investigated the

photoproperties of a similar vancomycin analog with the

same nitrobenzyl photogroup attached. It was demonstrated

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Figure 1. Bacterial growth curves of Gram-positive bacteria at decreasing concentrations of the vancomycin derivative 1. (A) Sample after

irradiation at time 0 min with UV light at λ = 365 nm for 5 min in the presence of B. subtilis ATCC 6633. (B) Non-irradiated samples in the

presence of B. subtilis ATCC 6633. (C) Sample after irradiation at time 0 min with UV light at λ = 365 nm for 5 min in the presence of S. aureus

ATCC 29213. (D) Non-irradiated samples in the presence of S. aureus ATCC 29213. (E) Sample after irradiation at time 0 min with UV light at λ

365 nm for 5 min in the presence of S. aureus ATCC 43300 (MRSA). (F) Non-irradiated samples in the presence of S. aureus ATCC 43300

(MRSA). All the solutions were irradiated before inoculation. Data points represent mean value ± SD (n = 3).

released vancomycin amide 3 featured the same MIC values

64 or 32 μg/mL of corresponding antibiotic was carried out

in one-half of a 96-wells plate. The solutions were UV-

irradiated for 5 min at λ = 365 nm. Next, the dilution step was

repeated in the second half of the same 96-wells plate followed

by the bacteria inoculation at optical density OD₆₀₀= 0.1. The

bacterial growth curves were recorded at 37 °C by a plate

reader, measuring the OD₆₀₀values every 20 min during 18 h.

Vancomycin derivative 1 was ﬁrst tested against the Gram-

positive strain B. subtilis. The desired inhibition was observed

for the solutions containing compound 1 starting from

compared to vancomycin. These experimental observations

corroborate the hypothesis that the presence of long PEG

chains is responsible for low antibacterial activity of

vancomycin derivatives.

Concerning cephalosporin derivatives, an insertion of PEG

linker lowered the antibiotic activity against all the tested

strains, as shown in Table 1. The released cephalosporin

derivative 4 exhibited especially high activity against Gram-

negative strains with a MIC value of 2 μg/mL; however, it

turned out to be less active against Gram-positive strains,

especially S. aureus. This can be explained by the fact that

cephalosporins possessing a thiadiazole side chain and

zwitterionic properties in their core exhibit low β-lactamase

hydrolysis and higher penetration rate through the outer

membrane, what renders them especially active against Gram-

μg/mL and above, after the UV-irradiation. In contrast,

non-irradiated solutions did not impact on B. subtilis growth at

all tested concentrations. No diﬀerence between “non-

activated” and “activated” forms of vancomycin derivative 1

was observed at the concentration 0.5 μg/mL (Figure 1A,B,

Figure S5), which is fully compliant with MIC data for both

compound 1, released form 3, and photocleavage eﬃcacy of

introduced linker.

Next, the vancomycin derivative 1 was tested against diﬃcult

to treat strains of S. aureus, including MRSA strains. We were

pleased to observe the eﬀective inhibition of bacterial growth

at the range of 4 μg/mL and above after an 5 min UV-

irradiation at λ = 365 nm of the antibiotic 1 (Figure 1C, 1E,

Figure S6 −S7). However, the normal bacterial growth

remained for both vancomycin sensitive S. aureus (VSSA)

and MRSA in the presence of deactivated vancomycin

derivative 1 at concentration 1 μg/mL and higher (Figure

1D, 1F, Figure S6 −S7).

57,58

negative bacteria.

For the control compound 6 having only

a nitrobenzyl group, the MIC value decreased only for

P. aeruginosa. The signiﬁcant diﬀerence in activity after the

incorporation of photolinker was not observed against other

strains, which necessitates the need of the PEG chain in the

structure. From these results we decided to focus on

exploration cephalosporin activity against Gram-negative

strains and vancomycin activity against Gram-positive bacteria

for the next experiments on in situ cleavage.

A time-resolved growth analysis in 96-well format was

performed, in order to investigate the dynamic eﬀect of target

antibiotics 1 and 2 on the bacterial growth before and after

irradiation. A series of 2-fold dilutions starting from

ACS Infect. Dis. 2021, 7, 681−692

Data points represent mean value ± SD (n = 3).

Article

Figure 2. Bacterial growth curves of E. coli ATCC 25922 at decreasing concentrations of the cephalosporin derivative 2. (A) Sample after

irradiation at time 0 min with UV light at λ = 365 nm for 5 min. (B) Non-irradiated samples. All the solutions were irradiated before inoculation.

Figure 3. Bacterial growth curves of P. aeruginosa ATCC 27853 at decreasing concentrations of the cephalosporin derivative 2. (A) Nonirradiated

samples. (B) Sample after irradiation at time 0 min with UV light at λ = 365 nm for 5 min. All the solutions were irradiated before inoculation. Data

points represent mean value ± SD (n = 3).

In the cephalosporin series, the antibacterial activity of

compound 2 with diﬀerent concentrations was ﬁrst tested

against Gram-negative strains E. coli and P. aeruginosa. From

MIC results, we could identify a small window in activity

between “non-activated” and “activated” forms of cephalospor-

in derivative 2 against E. coli. Corroborating these hypotheses,

bacterial growth curve demonstrated that a concentration of 4

μg/mL of compound 2 delivered the expected diﬀerence in

antibacterial activity before and after UV-irradiation (Figure

μg/mL for the compound 2 before the UV-irradiation (Figure

3A), which corresponds to the earlier obtained MIC results.

From the obtained result, we have concluded that the starting

concentration 32 μg/mL of target cephalosporin derivative 2 is

optimal to induce the expected diﬀerence between “activated”

and “non-activated” forms of antibiotic.

Unfortunately, the testing of cephalosporin derivative 2

against Gram-positive strains yielded disappointing results,

with no diﬀerence in growth evident before and after UV-

irradiation. However, this lack of diﬀerence might be due to a

small or nonexistent gap in activity between “non-active” and

“active” forms of cephalosporin derivative against B. subtilis and

S. aureus.

A). In addition, the sample at the concentration 2 μg/mL

resulted in slight inhibition of bacterial growth after the release

of the cephalosporin active form. Unfortunately, starting from

μg/mL and higher, compound 2 implied inhibitory activity

even without UV-irradiation (Figure 2B, Figure S8).

Next, to show the absence of activity coming from the

released by-product after the UV-irradiation of designed

antibiotics 1 and 2, a control experiment was carried out.

The linkers 9a and 12a in the highest concentration of 64 μg/

mL were UV-irradiated at λ = 365 nm for 5 min and inoculated

with bacteria. The time-resolved bacterial growth analysis was

repeated during 18 h at 37 °C. Gratifyingly, no inhibitory eﬀect

was observed for both linkers 9a and 12a against Gram-

positive and Gram-negative strains, respectively (Figures S9 −

S10 ).

Next, the activity of cephalosporin derivative 2 at the

diﬀerent concentrations was tested against P. aeruginosa. After

the release of active compound 4 signiﬁcant inhibition of

bacterial growth at concentration 32 μg/mL was observed

(Figure 3A). The lower concentration of compound 2 (16 μg/

mL) after UV exposure resulted in partial inhibition of

P. aeruginosa growth and an extended lag phase of 12 h.

Moreover, the further decrease of cephalosporin derivative

concentration to 8 μg/mL correlated with reduced culture OD

in the plateau phase. Finally, subsequent lowering of antibiotic

In order to demonstrate the beneﬁt of our designed

antibiotics 1 and 2, the study of their eﬀect after UV-

irradiation was carried out in the exponential phase of bacterial

loading did not impact on bacterial growth anymore. The

small inhibition eﬀect was observed only at concentration 64

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Figure 4. Bacterial growth in the presence of modiﬁed antibiotics with UV-irradiation step after 7 h of bacterial growth for 5 min at 365 nm. (A)

B. subtilis ATCC 6633 mixed with vancomycin derivative 1 (2 μg/mL). (B) S. aureus ATCC 29213 mixed with vancomycin derivative 1 (4 μg/

mL). (C) S. aureus ATCC 43300 mixed with vancomycin derivative 1 (4 μg/mL). (D) E. coli mixed with cephalosporin derivative 2 (4 μg/mL).

(E) P. aeruginosa ATCC 27853 mixed with cephalosporin derivative 2 (32 μg/mL). Data points represent mean values ± SD (n = 3).

growth. The bacteria were incubated with compounds 1 or 2

for 7 h, whereafter the solutions were exposed to UV light at

λ=365 nm for 5 min. The dynamic growth analysis was

recorded during 18 h in total. As shown in Figure 4, the clear

inhibition of the bacterial growth was observed for Gram-

positive B. subtilis (A) and VSSA (B) after the release of

vancomycin amide 3, and Gram-negative E. coli (D) in the

presence of cephalosporin derivative 4 compare to the negative

control experiments. The concentration of the compound 1

used to inhibit B. subtilis growth was 2 times higher (2 μg/mL)

compared to the experiment with irradiation at t = 0 min,

which was not the case for the other strains. This can be

explained by the insuﬃcient amount of antibiotic released at a

lower loading concentration to kill a large number of bacteria

formed after 7 h of incubation. Concerning MRSA and

P. aeruginosa, a slowdown in bacterial growth has been

detected after UV-irradiation of antibiotics 1 and 2,

respectively (Figure 4C and 4E). It was also demonstrated

that the bacteria was not aﬀected by the UV-irradiation step as

the control experiment did not exhibit any deviations in

growth.

with the starting amount of bacteria. This tendency was

observed for antibiotic 1 tested against S. aureus, when 2 μg/

mL of vancomycin derivative 1 was not suﬃcient to inhibit

bacterial growth after the release of the active drug 3, as the

bacterial density used for the experiment was 200 times higher

compared to MIC test. Additionally, the prolonged log phase

in case of P. aeruginosa in the presence of 16 μg/mL of

cephalosporin 2 reveal the importance to use the higher

concentration of antibiotic to observe full inhibitory eﬀect.

This delay in bacterial growth in the presence of insuﬃcient

amount of antibiotic was earlier showed by the group of Bunge

studying Enterococcus faecium. Another advantage of the

system reported herein is that by small changes in initial

antibiotic structure, we retained the biological activity against a

broader spectrum of bacterial strains. The small diﬀerence in

activity was observed for cephalosporin derivative; however, it

could be readily improved by the introduction of longer PEG

chains. Moreover, our system could be applicable for the

treatment of skin or wound infections, as UV-light (340−400

nm) has been shown to be eﬀective against several skin

60,61

diseases and does not trigger serious side eﬀects.

The use of photoswitching groups in antibiotics remains

challenging for retaining the desired biological eﬀect for a long

time. Thermal isomerization of these antibiotics over time led

addition, linkers cleavable by near IR radiation will be explored

in our laboratory. Introduction of diﬀerent photocleavable

groups might not aﬀect the approach described herein, as PEG

mainly contributed to antibiotics deactivation.

In summary, we have developed a new and eﬃcient

PEGylation approach for the caging of antibiotic activity. In

this report, we applied photo modiﬁcations for UV light-

stimulated control of the activity of two broadly used

antibiotics vancomycin and cephalosporin. The modiﬁed

antibiotics could be irreversibly turned into an active form

using an external stimulus. The release experiments performed

in the presence of Gram-negative and Gram-positive strains

showed strong inhibition of bacterial growth after UV-

to a loss in activity, as shown by group of Feringa. For long-

term maintenance of the therapeutic eﬀect, irreversible

antibiotic release thereby constitutes an advantage. We could

successfully demonstrate the remaining antibacterial eﬀect after

the activation of the designed compounds over a period of 18

h. Moreover, the loading concentration of target antibiotics

required to observe the desired inhibition after UV-irradiation

remained low and can be predicted accurately from the MIC

results and photocleavage eﬃcacy. However, the initial

concentration of the “caged” derivative has to be correlated

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best LC-MS quality. H and ¹³C NMR spectra were recorded

on Avance II or III-500 (500 MHz with Cryo-BBO, TXI, BBI

or BBO probes). Chemical shifts are given in parts per million

(ppm) on the delta (δ) scale and coupling constants (J) were

reported in Hz. Chemical shifts were calibrated according to

irradiation (λ=365 nm, 5 min) in both lag and exponential

growth phases. In principle, the developed approach could be

applied to any antibiotic possessing active groups for the

modiﬁcation, opening the ﬁeld of photopharmacology without

the need for dramatic changes in a drug structure.

the used solvents.

METHODS

(9H-Fluoren-9-yl)methyl(1-(5-methoxy-2-nitro-4-((75-

oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,-

9,62,65,68,71-tetracosaoxa-74-azaoctaheptacontan-78-yl)

oxy)phenyl)ethyl)carbamate (8a). To a solution of Fmoc-

photolinker 7 (17.4 mg, 0.034 mmol) in anhydrous DMF

0.350 mL) at 0 °C, distilled N,N-diisopropylethylamine

0.017 mL, 0.1 mmol) and HATU (25.4 mg, 0.067 mmol)

■

Chemistry. General Methods. Reactions were carried out

under inert gas (N or Ar) in oven-dried (120 °C) glass

equipment and monitored for completion by TLC or

UHPLC−MS (ESI). Solvents for reactions and analyses were

of analytical grade. Fmoc-photolinker 7, NH C H PEG OMe

(

m-PEG -amine), 5-Hydroxy-2-nitrobenzaldehyde 10, and 1-

were added. The reaction mixture turned dark brown. m-

PEG24-amine (40 mg, 0.037 mmol) was added after 10 min.

The reaction was stirred at 0 °C for 1 h and then at rt for 1 h.

The solvents were evaporated followed by puriﬁcation of crude

product by ﬂash silica column chromatography (DCM/MeOH

bromo-2-(2-methoxyethoxy)ethane were purchased from Iris

Biotech, BroadPharm, FluoroChem, and Sigma-Aldrich,

respectively. The synthesis of compounds 11b and 14 have

62,63

already been reported.

Analytical thin-layer chromatog-

raphy (TLC) was run on Merck TLC plates silica gel 60 F254

on glass plates with the indicated solvent system; the spots

were visualized by UV light (365 nm), and stained by

00:5) to obtain the product 8a (38 mg, 0.033 mmol, 72%) as

a slightly yellow oil. R = 0.29 (DCM/MeOH 100:5); H NMR

(

500 MHz, Chloroform-d) δ 7.82−7.69 (m, 2H), 7.58−7.54

m, 3H), 7.45−7.34 (m, 3H), 7.33−7.27 (m, 2H), 6.88 (s,

anisaldehyde, ninhydrin, or KMnO stain. Silica gel column

chromatography was performed using silica gel 60 (230−400

Mesh) purchased from Sigma-Aldrich with the solvent mixture

indicated. The SPE columns used were DSC-18 (Supelco,

Sigma). Preparative HPLC separations were performed on a

Shimadzu HPLC system (LC-20AP dual pump, CBM-20A

Communication Bus Module, SPP-20, A UV/vis Detector,

FRC-10A Fraction collector) using reverse-phase (RP)

columns Gemini-NX C18 (250 mm × 21.2 mm; 10 μm, 110

Å) or Synergi Hydro-RP (250 mm × 21.2 mm; 10 μm, 80 Å).

Ultrahigh-performance liquid chromatography (UHPLC)

coupled to mass spectrometer (MS) experiments were

performed on an Ultimate 3000 LC system (HPG-3400 RS

pump, WPS-3000 TRS autosampler, TCC-3000 RS column

oven, Vanquish DAD detector from Thermo Scientiﬁc)

coupled to a triple quadrupole (TSQ Quantum Ultra from

Thermo Scientiﬁc). The separation was performed using a RP

column (Kinetex EVO C18; 50 × 2.1 mm; 1.7 μm; 100 Å,

Phenomenex), a ﬂow of 0.4 mL/min, a solvent system

composed of A (H O + 0.1% HCO H) and B (MeCN +

H), 6.49−6.45 (m, 1H), 5.60−5.49 (m, 1H), 5.40−5.36 (m,

H), 4.45−4.32 (m, 1H), 4.17 (s, 1H), 4.13−4.08 (m, 2H),

.88 (s, 3H), 3.74−3.57 (m, 121H), 3.57−3.51 (m, 6H),

.48−3.42 (m, 4H), 3.37 (s, 3H), 2.45−2.35 (m, 3H), 2.22−

.15 (m, 2H), 2.07−1.82 (m, 6H), 1.60−1.39 (m, 4H).

NMR (126 MHz, CDCl ) δ 172.23, 155.57, 153.96, 147.18,

43.98, 141.43, 140.56, 134.43, 127.81, 127.13, 125.03, 120.09,

09.99, 72.06, 70.69, 70.30, 70.05, 68.72, 66.65, 59.16, 56.47,

⁸^.⁶⁰^,⁴⁷^.³⁵^,³⁹^.³⁸^,³²^.⁶⁵^,²⁵^.⁰³^,²¹^.⁸¹^.^I^R⁽^ﬁ^l^m⁾^vmax = 2872,

1719, 1648, 1519, 1452, 1349, 1272, 1247, 1217, 1182, 1096,

−

948, 836, 762, 742 cm ; ESI-HRMS calcd for

C H O N Na [M + Na] , m/z = 1612.83457, found

77 127 31 3

1612.83540.

Methyl 4-(4-(1-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)-

amino)ethyl)-2-methoxy-5-nitrophenoxy)butanoate (8b).

Fmoc-photolinker 7 (50 mg, 0.096 mmol) was dissolved in

MeOH (0.9 mL). Sulfuric acid (3 drops) was added to the

reaction mixture and the mixture was stirred overnight at 50

°C. The solvent was removed under reduced pressure and

DCM was added to the reaction mixture. The formed

precipitate was ﬁltered oﬀ and the solution was concentrated

.1% HCO H) and an elution gradient starting with 5% B,

increasing from 5−95% B in 3.5 min, from 95−100% B in 0.05

min, and washing the column with 100% B for 1.25 min. In

UHPLC-MS measurements after the photolysis experiments,

the fragments ions were monitored by SIM mode focusing on

the m/z 505.7 and 656.8 Da. IR-spectroscopy was performed

on a Varian 800 FT-IR ATR Spectrometer. Lyophilization was

performed on a Christ Freeze-dryer ALPHA 1−4 LD plus.

High-resolution electrospray mass spectra (HR-ESI-MS) were

recorded on a timsTOF Pro TIMS-QTOF-MS instrument

to aﬀord the desired product 8b as a white solid (49 mg, 0.096

mmol, 97%). H NMR (500 MHz, DMSO-d

) δ 8.02 (d, J =

8.0 Hz, 1H), 7.87 (d, J = 7.2 Hz, 2H), 7.64 (d, J = 7.2 Hz, 2H),

7.49−7.47 (m, 1H), 7.42−7.38 (m, 2H), 7.32−7.26 (m, 2H),

7.25 (s, 1H), 5.21 (p, J = 7.0 Hz, 1H), 4.33−4.23 (m, 2H),

4.20−4.15 (m, 1H), 4.06 (t, J = 6.1 Hz, 3H), 3.86 (s, 3H), 3.60

(s, 3H), 2.47 (t, J = 7.2 Hz, 2H), 2.04−1.92 (m, 2H), 1.41 (d, J

(

Bruker Daltonics GmbH, Bremen, Germany). The samples

= 6.7 Hz, 3H). C NMR (126 MHz, DMSO) δ 172.88,

155.25, 153.40, 146.23, 143.89, 143.61, 140.73, 139.92, 135.47,

127.59, 126.90, 125.00, 120.10, 109.37, 108.16, 67.78, 65.21,

were dissolved in (e.g., MeOH) at a concentration of ca. 50

μg/mL and analyzed via continuous ﬂow injection (2 μL/

min). The mass spectrometer was operated in the positive (or

negative) electrospray ionization mode at 4000 V (−4000 V)

capillary voltage and −500 V (500 V) end plate oﬀset with a

56.19, 51.35, 46.68, 45.93, 29.82, 23.97, 21.89. R = 0.9

(DCM/MeOH 20:1). IR (ﬁlm) 3348, 2938, 1736, 1687, 1579,

1451, 1375, 1335, 1278, 1254, 1218, 1178, 1119, 1086, 1070,

1051, 1021, 876, 758, 738, 646. HR-ESI-MS (MeOH) calcd

N nebulizer pressure of 0.4 bar and a dry gas ﬂow of 4 L/min

at 180 °C. Mass spectra were acquired in a mass range from m/

z 50 to 2000 at ca. 20 000 resolution (m/z 622) and at 1.0 Hz

rate. The mass analyzer was calibrated between m/z 118 and

for C H O N Na [M + Na] , m/z = 557.18944, found

557.18952.

Compounds 9a and 9b: General Procedure. Compound

8a or 8b was treated with a solution of piperidine in DMF

(20% v/v, 0.01 mM solution). The reaction mixture was stirred

for 1−2 h. The solvent was evaporated and the remaining

721 using an Agilent ESI-L low concentration tuning mix

solution (Agilent, USA) at a resolution of 20 000 giving a mass

accuracy below 2 ppm. All solvents used were purchased in

ACS Infect. Dis. 2021, 7, 681−692

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Article

reaction mixture was washed with ether (2 × 10 mL) to aﬀord

the desired products. Analytical data and yields for obtained

compounds are described below.

B, 5−95% B for 3.5 min; 95−100% for 0.05 min, wash. The

product 1 was eluted at 2.02 min and was detected at 270 nm.

Vancomycin Derivative with Photo Linker (5). RP-HPLC:

Gradient 5% B for 14 min; 5−30% B for 46 min; 30−100% B

for 4 min, wash. The desired product, eluting at 34.4 min, was

collected and lyophilized to aﬀord product 5 (12.3 mg, 0.023

-(4-(1-Aminoethyl)-2-methoxy-5-nitrophenoxy)-N-

2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,-

6,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)-

(

butanamide (9a). The product was obtained as a slightly

mmol, 30%) as a slightly yellowish solid. H NMR (500 MHz,

yellow oil (24 mg, 0.020 mmol, 87%). H NMR (500 MHz,

Methanol-d

(m, 2H), 7.58−7.53 (m, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.03

d, J = 2.2 Hz, 1H), 7.02−6.98 (m, 1H), 6.95 (s, 1H), 6.86 (d,

J = 8.6 Hz, 1H), 6.42 (d, J = 2.3 Hz, 1H), 5.90 (d, J = 2.0 Hz,

) δ 8.50 (s, 1H), 7.65−7.62 (m, 1H), 7.61−7.58

DMSO-d ) δ 8.27 (s, 1H), 7.92 (t, J = 5.5 Hz, 1H), 7.49−7.42

(

m, 2H), 4.02 (t, J = 6.4 Hz, 2H), 3.91 (s, 3H), 3.66−3.61 (m,

H), 3.60−3.44 (m, 121H), 3.44−3.34 (m, 12H), 3.24 (s,

H), 5.79−5.76 (m, 1H), 5.70 (q, J = 6.9 Hz, 1H), 5.45 (d, J =

.5 Hz, 1H), 5.42 (d, J = 4.2 Hz, 1H), 5.38−5.32 (m, 1H),

.31−5.28 (m, 1H), 4.19−4.16 (m, 1H), 4.15−4.10 (m, 2H),

.87−3.83 (m, 2H), 3.80 (s, 3H), 3.77−3.71 (m, 1H), 3.70 (s,

H), 3.55−3.50 (m, 1H), 3.42−3.36 (m, 1H), 2.82 (dd, J =

H), 3.20 (q, J = 5.8 Hz, 2H), 2.24 (t, J = 7.4 Hz, 2H), 1.93 (p,

J = 6.8 Hz, 2H), 1.35 (d, J = 6.1 Hz, 3H). C NMR (126

MHz, DMSO) δ 171.50, 153.17, 146.22, 140.13, 109.75,

08.38, 71.28, 69.78, 69.72, 69.58, 69.56, 69.11, 68.26, 58.05,

6.19, 38.52, 31.47, 24.65. ESI-HRMS calcd for C H O N

118 29

2.6, 16.1 Hz, 1H), 2.57 (t, J = 7.4 Hz, 2H), 2.48 (s, 3H), 2.35−

[

M + H] , m/z = 1368.78455, found 1368.78635.

.37 (m, 1H), 2.13 (p, J = 6.6 Hz, 2H), 2.08−2.02 (m, 1H),

.92 (d, J = 13.7 Hz, 1H), 1.81−1.74 (m, 1H), 1.68−1.61 (m,

H), 1.58−1.54 (m, 1H), 1.54−1.49 (m, 3H), 1.47 (s, 3H),

Methyl 4-(4-(1-aminoethyl)-2-methoxy-5-nitrophenoxy)-

butanoate (9b). The product was obtained as a slightly

yellow amorphous solid (10 mg, 0.044 mmol, 73%). R = 0.45

1.20 (d, J = 6.4 Hz, 3H), 0.98 (d, J = 6.4 Hz, 3H), 0.95 (d, J =

(

DCM/MeOH 15:1). H NMR (500 MHz, CDCl ) δ 7.47 (s,

.4 Hz, 3H). HR-ESI-MS (water) calcd for C H O N C

80 95 29 11 l2

H), 7.31 (s, 1H), 4.79 (q, J = 6.5, 2H), 4.09 (t, J = 6.4, 2H),

.96 (s, 3H), 3.69 (s, 3H), 2.55 (t, J = 7.3 Hz, 2H), 2.21−2.14

[

M + 2H] , m/z = 871.78316, found 871.78295. The purity of

the compound was analyzed by UHPLC. Gradient starts from

% B, 5−95% B for 3.5 min; 95−100% for 0.05 min, wash. The

product 5 was eluted at 2.11 min and was detected at 270 nm.

-(2-(2-Methoxyethoxy)ethoxy)-2-nitrobenzaldehyde

11a). 5-Hydroxy-2-nitrobenzaldehyde 10 (100 mg, 0.598

(

m, 2H), 1.62 (s, 3H), 1.42 (d, J = 6.5 Hz, 3H). C NMR

126 MHz, CDCl ) δ 173.51, 153.89, 146.69, 140.85, 137.72,

09.25, 109.06, 68.34, 56.42, 51.86, 46.02, 30.52, 24.91, 24.42.

IR (ﬁlm) 2954, 1735, 1577, 1516, 1442, 1333, 1272, 1210,

174, 1052, 818, 759; HR-ESI-MS calcd for C H O N [M +

(

mmol) was dissolved in dry DMF (6 mL) and powdered

K CO (99 mg, 0.718 mmol) was added. After 5 min 1-bromo-

H] , m/z = 313.13941, found 313.13930.

Compounds 1 and 5: General Procedure. Vancomycin

hydrochloride (1 equiv), PyBoP (3 equiv) and HOBt (1

equiv) were dissolved in dry DMF (0.05 mM, based on

vancomycin). To the reaction mixture freshly distilled N,N-

diisopropylethylamine (3 equiv) was added followed by the

addition of linker 9 or 16 (1.1 equiv). The reaction mixture

was stirred for 1 h at rt and the solvent was evaporated. The

-(2-methoxyethoxy)ethane (0.090 mL, 0.658 mmol) was

added dropwise and the reaction mixture was stirred at 90 °C

overnight. Then, the mixture was cooled to rt, diluted with

H O, and extracted with DCM (3 × 30 mL). The combined

organic phases were washed with brine, dried over Na SO and

concentrated under reduced pressure. The crude was puriﬁed

by column chromatography (n-pentane:EtOAc 2:1) to aﬀord

mixture was dissolved in MeCN:H O (1:1, with 0.1%

the desire product 11a as yellow oil (158 mg, 0.598 mmol,

HCO H) and ﬁltered through a SPE column. The solvent

8%). R (n-pentane:EtOAc 2:1) = 0.3. H NMR (400 MHz,

was evaporated and the compound was puriﬁed by preparative

RP-HPLC. The puriﬁcation methods, analytical data, and

yields are shown below.

CDCl ) δ 10.47 (s, 1H), 8.15 (d, J = 9.1 Hz, 1H), 7.34 (d, J =

.9 Hz, 1H), 7.18 (dd, J = 9.1, 2.9 Hz, 1H), 4.30−4.25 (m,

H), 3.92−3.86 (m, 2H), 3.74−3.69 (m, 2H), 3.59−3.55 (m,

Vancomycin Derivative with PEG₂₄Linker (1). RP-HPLC:

Gradient 5% B for 14 min; 5−40% B for 36 min; 40−100% B

for 2 min, wash. The desired product, eluting at 28.2 min, was

collected and lyophilized to aﬀord product 1 (3.1 mg, 0.008

H), 3.38 (s, 3H). C NMR (101 MHz, CDCl ) δ 188.60,

63.44, 142.48, 134.40, 127.36, 119.26, 114.06, 72.04, 71.04,

9.42, 68.78, 59.25. IR (ﬁlm) 2881, 1695, 1583, 1516, 1485,

425, 1389, 1329, 1288, 1246, 1234, 1199, 1164, 1108, 1074,

048, 934, 886, 846, 746, 676, 631. HRMS (ESI) calcd for

mmol, 28%) as a white solid. H NMR (500 MHz, Methanol-

d ) δ 8.48 (s, 1H), 7.68−7.63 (m, 1H), 7.61 (s, 2H), 7.24 (d, J

C H O N [M + H] , m/z = 270.09721, found 270.09718.

12 16 6

8.0 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 6.96 (s, 1H), 6.86 (d,

J = 8.6 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 5.93−5.88 (m, 1H),

.69 (q, J = 6.9 Hz, 1H), 5.48−5.44 (m, 1H), 5.42 (d, J = 3.7

Hz, 1H), 5.38−5.28 (m, 1H), 4.63−4.58 (m, 1H), 4.16−4.09

m, 2H), 3.88−3.83 (m, 1H), 3.81 (s, 2H), 3.78−3.72 (m,

Compounds 12a and 12b: General Procedure. In a ﬂask

covered with aluminum foil, NaBH(OAc) (1 equiv) and

molecular sieves (3 Å) were set under argon and suspended in

,2-dichloroethane (0.2 M solution). Then, 4-(aminomethyl)-

pyridine (1.1 equiv) was added by syringe, followed by AcOH

glacial, 0.1 mL, 2 mmol). The reaction mixture was stirred at

(

H), 3.70−3.58 (m, 95H), 3.58−3.51 (m, 6H), 3.51−3.47 (m,

H), 3.39 (t, J = 5.4 Hz, 3H), 3.36 (s, 3H), 2.87−2.79 (m,

H), 2.53 (s, 3H), 2.45 (d, J = 7.6 Hz, 2H), 2.14 (p, J = 6.7 Hz,

H), 2.05 (dd, J = 4.1, 13.3 Hz, 1H), 1.93 (d, J = 13.3 Hz, 1H),

.76 (p, J = 6.7 Hz, 1H), 1.71−1.68 (m, 1H), 1.58 (q, J = 6.9

(

rt while a solution of 11a or 11b (1 equiv) in dry 1,2-

dichloroethane (0.2 M solution) was added dropwise by

syringe over 10 min. After 3 h another equivalent of

NaBH(OAc)₃was added. The reaction was stirred for

additional 2 h at rt followed by the addition of one more

Hz, 1H), 1.52 (d, J = 6.9 Hz, 3H), 1.47 (s, 3H), 1.20 (d, J =

.4 Hz, 3H), 0.98 (d, J = 6.4 Hz, 3H), 0.95 (d, J = 6.4 Hz, 3H).

equivalent of NaBH(OAc) . After 6 h in total, the reaction

HR-ESI-MS (water) calcd for C₁₂₈H₁₉₂O N Cl [M + 2H] ,

mixture was poured into sat. NaHCO solution and extracted

m/z = 1399.60573, found 1399.60396. The purity of the

compound was analyzed by UHPLC. Gradient starts from 5%

with DCM (3 × 20 mL). The combined organic phase was

washed with brine, dried over Na SO , and the solvent was

ACS Infect. Dis. 2021, 7, 681−692

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Article

removed under a vacuum. The crude product was puriﬁed by

column chromatography. The puriﬁcation methods, analytical

data, and yields are shown below.

N-(5-(2-(2-Methoxyethoxy)ethoxy)-2-nitrobenzyl)-1-(pyri-

din-4-yl)methanamine (12a). The puriﬁcation is carried out

by column chromatography (DCM/MeOH 20:1) The desired

MHz, DMSO-d ) δ 8.55−8.48 (m, 2H), 8.09 (d, J = 9.0 Hz,

1H), 7.23 (d, J = 5.6 Hz, 2H), 7.13 (dd, J = 9.0, 2.7 Hz, 1H),

6.91 (d, J = 2.7 Hz, 1H), 5.28 (s, 2H), 4.49 (s, 2H), 3.40 (s,

3H), 1.34 (s, 9H). C NMR (126 MHz, DMSO) δ 161.07,

154.98, 149.65, 147.54, 141.41, 137.19, 127.89, 127.73, 121.03,

114.58, 93.93, 79.95, 55.98, 50.01, 48.76, 27.75. IR (ﬁlm)

2976, 1696, 1581, 1517, 1482, 1455, 1413, 1338, 1276, 1241,

1206, 1156, 1068, 995, 925, 842, 755. HRMS (ESI) calcd for

product 12a was obtained as yellow oil (41 mg, 0.186 mmol,

3%). R = 0.31 (DCM/MeOH 20:1). H NMR (500 MHz,

DMSO-d ) δ 8.48 (dd, J = 4.4, 1.6, 2H), 8.02 (d, J = 9.0 Hz,

C H N O [M + H] , m/z = 404.18161 found 404.18187.

20 26

H), 7.36−7.33 (m, 2H), 7.32 (d, J = 2.8 Hz, 1H), 7.03 (dd, J

9.1, 2.8 Hz, 1H), 4.25−4.21 (m, 2H), 4.00 (s, 2H), 3.79−

.76 (m, 2H), 3.75 (s, 2H), 3.61−3.58 (m, 2H), 3.47−3.44

Cephalosporin Derivatives 2, 4, and 6: General

Procedure. Under an Ar atmosphere, NaI (3 equiv) was

added to a mixture of compound 15 (1.5 equiv) in dry acetone

(0.1M). The reaction mixture was stirred at rt for 40 min. After

this time the linker 13a, 4-(aminomethyl)pyridine or 13b (1

equiv) in dry acetone (0.05 M) was added and the reaction

was stirred at rt for 3−4 h. After the reaction was ﬁnished, the

solvent was evaporated and the mixture was washed with

isopropyl ether (IPE, 2 mL). The formed precipitate was

dissolved in mixture of DCM:anisole:TFA 5:1:1 (0.1 mL). The

reaction mixture was stirred overnight and then IPE (2 mL)

was added into the reaction. The resulting suspension was

centrifuged. The supernatant was removed, and the precipitate

was washed with IPE two more times. The crude was dissolved

in mixture of MeOH/H O/CH CN, ﬁltered through SPE

18 24 5 3

H), 7.33−7.30 (m, 2H), 7.26−7.24 (d, J = 2.6, 1H), 7.02

column, and puriﬁed by RP-HPLC. The puriﬁcation methods,

analytical data, and yields are shown below.

(

ddd, J = 9.1, 2.3, 1.0 Hz, 1H), 5.25 (s, 2H), 4.09 (s, 2H), 3.87

d, J = 2.0 Hz, 2H), 3.49 (s, 3H). ¹³C NMR (126 MHz,

Cephalosporin Derivative with OEG₂Linker (2). RP-

HPLC: Gradient 5% B for 14 min; 5−45% B for 56 min;

45−100% B for 4 min, wash. The desired product, eluting at 31

min, was collected and lyophilized to aﬀord product 2 (0.700

CDCl ) δ 161.24, 149.99, 148.91, 142.68, 138.20, 127.92,

23.19, 118.24, 115.02, 94.40, 56.64, 52.15, 50.91. IR (ﬁlm)

907, 1603, 1579, 1513, 1485, 1414, 1337, 1249, 1206, 1152,

089, 1068, 993, 925, 840, 798, 755, 485. HRMS (ESI) calcd

mg, 0.043 mmol, 2%) as a slightly yellowish solid. H NMR

for C H N O [M + H] , m/z = 304.12918, found

(500 MHz, Methanol-d ) δ 9.05 (d, J = 6.6 Hz, 2H), 8.44−

04.12899.

Compounds 13a and 13b: General Procedure. Com-

pound 12a or 12b was dissolved in dry THF (0.2 M) and TEA

8.40 (m, 1H), 8.07−8.05 (m, 2H), 7.22 (d, J = 2.6 Hz, 1H),

7.00 (dd, J = 9.1, 2.6 Hz, 1H), 5.85 (d, J = 4.9 Hz, 1H), 5.67

(d, J = 13.8 Hz, 1H), 5.20−5.13 (m, 2H), 4.28−4.21 (m, 2H),

4.12 (h, J = 3.1, 2.4 Hz, 4H), 4.01 (s, 3H), 3.88−3.84 (m, 2H),

3.73−3.66 (m, 3H), 3.63−3.58 (m, 1H), 3.57−3.54 (m, 2H),

3.49 (s, 1H), 3.22 (d, J = 13.8 Hz, 2H), 3.09 (d, J = 17.8 Hz,

(

2 equiv) was added. Boc O (2 equiv) was dissolved in THF

0.1 M solution) and added to the solution. The reaction was

stirred for 3−4 h. The formation of product was observed by

UHPLC. The solvent was removed under reduced pressure.

The resulting crude mixture was puriﬁed by column

chromatography. The puriﬁcation methods, analytical data,

and yields are shown below.

1H). HRMS (ESI) calcd for C H O N S [M] , m/z =

36 10

9 2

758.20211, found 758.20314. The purity of the compound was

analyzed by UHPLC. Gradient starts from 5% B, 5−95% B for

3.5 min; 95−100% for 0.05 min, wash. The product 2 was

eluted at 2.49 min and was detected at 270 nm.

tert-Butyl (5-(2-(2-methoxyethoxy)ethoxy)-2-nitrobenzyl)-

(

pyridin-4-ylmethyl)carbamate (13a). The puriﬁcation is

Cephalosporin Derivative (4). RP-HPLC (Hydro): Gra-

dient 0% B for 14 min; 0−20% B for 16 min; 20−100% B for 2

min, wash. The desired product, eluting at 4.3 min, was

collected and lyophilized to aﬀord product 4 (3.56 mg, 0.072

carried out by column chromatography (DCM/MeOH 40:1)

The desired product 13a was obtained as a dark yellow oil (50

mg, 0.221 mmol, 49%). R = 0.30 (DCM/MeOH 40:1). H

NMR (500 MHz, DMSO-d ) δ 8.53 (s, 2H), 8.16−8.10 (m,

mmol, 10%) as a white solid. H NMR (500 MHz, MeOD) δ

(

H), 7.24 (s, 2H), 7.09 (dd, J = 9.1, 2.7 Hz, 1H), 6.76 (d, J =

.7 Hz, 1H), 4.84−4.74 (m, 1H), 4.54−4.44 (m, 1H), 4.23−

.16 (m, 2H), 4.12−4.02 (m, 1H), 3.79−3.75 (m, 2H), 3.59

9.23 (d, J = 6.7 Hz, 2H), 8.14 (d, J = 6.7 Hz, 2H), 5.87 (d, J =

4.2 Hz, 1H), 5.73 (d, J = 14.0 Hz, 1H), 5.32 (d, J = 14.0 Hz,

1H), 5.18 (d, J = 4.2 Hz, 1H), 4.52 (s, 2H), 4.02 (s, 3H),

3.74−3.66 (m, 1H), 3.24−3.19 (m, 1H). HRMS (ESI) calcd

dd, J = 5.8, 3.6 Hz, 2H), 3.46 (dd, J = 5.8, 3.6 Hz, 2H), 3.24

s, 3H), 1.33 (d, J = 17.2 Hz, 9H). ¹C NMR (126 MHz,

for C H O N S [M] , m/z = 505.10708, found 505.10666.

8 2

DMSO) δ 162.71, 154.99, 149.67, 140.71, 113.56, 71.23,

The purity of the compound was analyzed by UHPLC.

Gradient starts from 5% B, 5−95% B for 3.5 min; 95−100% for

0.05 min, wash. The product 4 was eluted at 0.34 min and was

detected at 270 nm.

9.72, 68.51, 68.07, 58.05, 27.75, 22.78. IR (ﬁlm) 2961, 1700,

579, 1516, 1462, 1414, 1337, 1279, 1110, 1071, 993, 841.

HRMS (ESI) calcd for C H O N [M + H] , m/z =

62.22348 found 462.22356.

tert-Butyl (5-(methoxymethoxy)-2-nitrobenzyl)(pyridin-4-

Cephalosporin Derivative with Photo Linker (6). RP-

HPLC: Gradient 5% B for 14 min; 5−45% B for 56 min; 45−

100% B for 4 min, wash. The desired product, eluting at 34.4

min, was collected and lyophilized to aﬀord product 6 (0.35

mg, 0.052 mmol, 1%) as a slightly yellowish solid. H NMR

(500 MHz, MeOD) δ 8.82 (d, J = 6.7 Hz, 2H), 8.62 (d, J = 6.7

ylmethyl)carbamate (13b). The puriﬁcation is carried out by

column chromatography (DCM/MeOH, 95:5) The desired

product 13b was obtained as a dark yellow oil (30 mg, 0.072

mmol, 55%). R = 0.35 (DCM/MeOH 95:5). H NMR (500

ACS Infect. Dis. 2021, 7, 681−692

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The Supporting Information is available free of charge at

Article

Hz, 1H), 8.31 (s, 1H), 8.06 (d, J = 6.6 Hz, 2H), 8.01 (d, J =

volume of 50 μL for each line of the plate. Each well containing

the antibiotic solution and the growth control wells were

inoculated with 50 μL of the bacterial suspension in

.0 Hz, 1H), 7.90−7.85 (m, 1H), 7.03 (d, J = 2.7 Hz, 1H),

.84−6.82 (m, 1H), 6.81 (dd, J = 9.0, 2.7 Hz, 1H), 5.65 (d, J =

.9 Hz, 1H), 5.61 (d, J = 14.2 Hz, 1H), 5.41 (d, J = 3.9 Hz,

H), 5.29 (d, J = 14.2 Hz, 1H), 4.60 (s, 2H), 4.12 (s, 2H), 4.10

−1

concentration 1 × 10 cfu/mL , which results in ﬁnal desired

−1

inoculum of 5 × 10 cfu/mL in a volume 100 μL. The plate

was then incubated at 37 °C for 18 h, and the cell density (600

nm) was measured every 20 min (with shaking between

measurements) in a microplate reader with the irradiation step

after 7 h of bacterial growth. All experiments were performed

in triplicates.

(

[

s, 2H), 4.05 (s, 3H). HRMS (ESI) calcd for C H O N S

M] , m/z = 656.13403, found 656.13410. The purity of the

26 26 8 9 2

compound was analyzed by UHPLC. Gradient starts from 5%

B, 5−95% B for 3.5 min; 95−100% for 0.05 min, wash. The

product 6 was eluted at 0.91 min and was detected at 270 nm.

Photolysis Experiment. Photolysis experiments were

performed using a Sina UV lamp (SI-MA-032-W; equipped

with UV lamps 4 × 9, 365 nm) at the distance of ∼5 cm. See

the Supporting Information for the detailed procedure.

Microbiological Assays. Bacterial Strains, Media,

Reagents, and Equipment. Bacillus subtilis (B. subtilis,

ATCC 6633) Staphylococcus aureus (VSSA strain ATCC

Antibacterial Activity of the Byproducts after the UV-

Irradiation. In 96-well microtiter plate, linkers 9a and 13a

(concentration 64 μg/mL) were prepared in Mueller-Hinton-

II broth (MHB) in a ﬁnal volume of 50 μL. Each well

containing the linker solution and the growth control wells

were UV-irradiated at a wavelength of 365 nm for 5 min. After

that the wells were inoculated with 50 μL of the bacterial

−1

9213), methicillin and oxacillin-resistant Staphylococcus aureus

suspension in concentration 1 × 10 cfu/mL , which results

−1

(

MRSA strain ATCC 43300), Gram-negative Escherichia coli

strain ATCC 25922), and Pseudomonas aeruginosa (strain

in ﬁnal desired inoculum of 5 × 10 cfu/mL in a volume 100

μL. The plate was then incubated at 37 °C for 18 h, and the

cell density (600 nm) was measured every 20 min (with

shaking between measurements) in a microplate. All experi-

ments were performed in triplicates.

ATCC 27853) were purchased from either the German

Collection of Microorganisms and Cell Cultures (DSMZ) or

the American Type Culture Collection (ATCC). The bacteria

culture was stored at −80 °C, and new cultures were prepared

by streaking on Luria Broth (LB) or Trypsic Soy agar plates.

The overnight culture was prepared by inoculating a single

colony into a sterile plastic tube (15 mL) containing the

bacteria medium (5 mL, LB or Trypsic Soy) and the cultures

were shaken (200 rcf/min) overnight at 37 °C. Synthesized

compounds were prepared in water at stock concentrations of

ASSOCIATED CONTENT

■

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acsinfecdis.1c00015.

H and C NMR spectra of new compounds and details

mg/mL. The microplate reader used for the experiments was

about photochemical data, microbiological assays, and

bacterial growth curves (PDF )

the Synergy H1 apparatus from BioTek. The Incubation assays

were performed using an Eppendorf Thermomixer Compact

with 1.5 mL blocks at 25 °C with a mixing speed of 700 rpm.

Optical density for bacterial suspension adjustment was

measured by Biochrom Cell Density Meter Ultrospec 10.

MICs of Tested Compounds. The minimum inhibitory

concentration (MIC) of tested compounds and control

antibiotics was determined using the broth microdilution

method according to the guidelines outlined by the European

Committee on Antimicrobial Susceptibility Testing (EU-

AUTHOR INFORMATION

■

Corresponding Author

Karl Gademann − Department of Chemistry, University of

Zurich, 8057 Zurich, Switzerland; orcid.org/0000-0003-

3053-0689; Email: karl.gademann@uzh.ch

Authors

CAST). See the Supporting Information for the detailed

procedure.

Inga S. Shchelik − Department of Chemistry, University of

Zurich, 8057 Zurich, Switzerland

Andrea Tomio − Department of Chemistry, University of

Zurich, 8057 Zurich, Switzerland

Time-Resolved Bacterial Growth Analysis. In 96-well

microtiter plate, 2-fold serial dilutions of antibiotics 1 or 2

(

ranging from 64 μg/mL to 0.125 μg/mL) were prepared in

Complete contact information is available at:

https://pubs.acs.org/10.1021/acsinfecdis.1c00015

Cation-adjusted Mueller-Hinton-II broth (MHB) in a ﬁnal

volume of 50 μL for each second line of the plate. The mixture

was UV-irradiated at a wavelength of 365 nm for 5 min. 2-fold

serial dilutions (ranging from 64 μg/mL to 0.125 μg/mL) were

repeated for the unﬁlled lines in the same 96-well microtiter

plate. Each well containing the antibiotic solution and the

growth control wells were inoculated with 50 μL of the

Author Contributions

I.S.S. and K.G. designed the study. I.S.S. carried out the

synthesis and characterization of all derivatives and biological

experiments. A.T. carried out the synthesis and optimization of

cephalosporin derivatives. I.S.S. and K.G. analyzed data and

discussed the results. I.S.S. and K.G. wrote the manuscript.

−1

bacterial suspension in concentration 1 × 10 cfu/mL , which

−1

results in ﬁnal desired inoculum of 5 × 10 cfu/mL in a

volume 100 μL. The plate was then incubated at 37 °C for 18

h, and the cell density (600 nm) was measured every 20 min

Notes

The authors declare no competing ﬁnancial interest.

(

with shaking between measurements) in a microplate reader.

All experiments were performed in triplicates.

ACKNOWLEDGMENTS

Antibacterial Activity at Exponential Phase of Bacterial

Growth. In 96-well microtiter plate, 2-fold serial dilutions of

antibiotics 1 or 2 (ranging from 64 μg/mL to 0.25 μg/mL)

were prepared in Mueller-Hinton-II broth (MHB) in a ﬁnal

■

We acknowledge the Swiss National Science Foundation

(SNSF, Grant No. 182043) and a Bundesstipendium (to

I.S.S.) for ﬁnancial support.

ACS Infect. Dis. 2021, 7, 681−692

ACS Infectious Diseases

Article

21) Pianowski, Z. L. (2019) Recent Implementations of Molecular

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