University of Groningen Selective oxidation of glycosides ... · aminoglycosides from fermentation and semisynthetic compounds. A common motif shared by all aminoglycosides is the

University of Groningen

Selective oxidation of glycosidesJäger, Manuel

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2015

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Jäger, M. (2015). Selective oxidation of glycosides. [Groningen]: University of Groningen.

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 21-05-2020

https://www.rug.nl/research/portal/en/publications/selective-oxidation-of-glycosides(ad906721-0aae-4968-b6ac-258481841d9b).html

https://www.rug.nl/research/portal/en/publications/selective-oxidation-of-glycosides(ad906721-0aae-4968-b6ac-258481841d9b).html

CHAPTER 4

Discrimination among even more equals: Aminoglycosides form an important class of

antibiotics and are in clinical use since decades. Here we describe the regioselective oxidation of

neomycin B, kanamycin A, amikacin and 3-azido-neomycin B at the 3’-position. The resulting keto-

aminoglycosides can be isolated in moderate yields as single oxidation products. Subsequent

selective reduction to the 3’-axial aminoglycoside is possible with excellent selectivity and in

excellent isolated yields. The strategy described here allows the efficient modification of

aminoglycoside antibiotics and is therefore a new, valuable tool for the development of novel

aminoglycoside antibiotics that might circumvent the bacterial resistance that has developed

against the aforementioned antibiotics.

Selective Oxidation and

Modification of Aminoglycoside

Antibiotics

Chapter 4

84

INTRODUCTION In 1944 Waksman et al.[1] discovered streptomycin as the first

representative of the aminoglycoside family and the first effective

antibiotic against tuberculosis.[2] Since then, aminoglycosides have

developed to an important class of antibiotics against both Gram positive

and Gram negative bacterial infectious diseases. They are especially

important in intensive care medication to treat a range of severe bacterial

infections like endocarditis, sepsis or (resistant) mycobacterial infections

and are used alone as well as in combination with other antibiotics.[3,4]

Structure of aminoglycosides

The family of aminoglycosides is structurally very diverse, and includes

aminoglycosides from fermentation and semisynthetic compounds. A

common motif shared by all aminoglycosides is the presence of

2-desoxystrepamin (2-DOS) as depicted below. Note that this is not a

carbohydrate but a cyclohexane derivative, like the inositols.

Figure 1 General nomenclature of aminoglycosides shown for Neomycin B and

Kanamycin A

Depending on the linkage of one or several amino sugars to 2-

desoxystrepamin, aminoglycosides are divided into three subclasses. The

first subclass comprizes the 4-mono-substituted aminoglycosides like

neamine and paromamine which are not used as drugs, in contrast to

apramycin, which has a bicyclic ring I. The second subclass comprises the

Selective Oxidation and Modification of Aminoglycoside Antibiotics

85

4,5-disubstituted aminoglycosides and contains the tricyclic ribostamycin

and butirosin, the tetracyclic neomycin and paromomycin, and the

pentacyclic lividomycin.

Figure 2 General structure of 4,5-disubstituted aminoglycosides

Table 1 An overview of the mono and 4,5-disubstituted aminoglycosides

Aminoglycoside R1 R2 R3

mono-

substituted

Neamine NH2 OH H

Paromamine OH OH H

Apramycin - - -

4,5 disubstituted

Butirosin A OH OH AHB

Butirosin B NH2 OH AHB

Ribostamycin NH2 OH H

Neomycin B NH2 OH H

Paromomycin OH OH H

Lividomycin A OH H H

The third subclass comprises the 4,6-disubstituted aminoglycosides.

Kanamycin, tobramycin and the semi-synthetic amikacin are

representatives of this class. Although the list above is far from complete;

it illustrates the structural similarities and diversity of the

aminoglycosides.

Chapter 4

86

Figure 3 General structure of 4,6-disubstituted aminoglycosides

Table 2 4,6-disubstituted aminoglycosides

Aminoglycoside R1 R2 R3 R4

4,6-

disubstituted

Kanamycin A OH OH OH H

Kanamycin B NH2 OH OH H

Amikacin OH OH OH AHB

Tobramycin NH2 H OH H

Mode of action of aminoglycoside antibiotics

In the late 1980’s it was discovered, that the target of the aminoglycosides

is the 16S rRNA of the 30S bacterial ribosome.[5]

Modelling studies and crystal structures of aminoglycosides complexed

with A-site oligonucleotides revealed a specific interaction of the

aminoglycosides with three non-complementary adenines. The

interaction forces them into a “flipped out” conformation (Figure 4). This

reduces the fidelity of the translation process and increases the number of

errors in the peptide synthesis of the ribosome, which leads to cell death.

From the crystal structures it became clear that ring I and II are highly

important for the recognition. Furthermore, the binding of the

aminoglycosides to the A-site of the ribosome is sequence specific and all

contacts of the aminoglycosides to the A-site are conserved throughout

the structural variety of the aminoglycosides.[6] Responsible for the

binding of the aminoglycosides are the electrostatic interactions of the, at

physiological pH, protonated amine groups and the negatively charged

backbone of the RNA.[7]


87

Figure 4 "Flipped out" conformation (yellow) caused by aminoglycoside coordination

to the ribosomal decoding site of the bacterial ribosome. Figure is reproduced from

the literature.[8]

Bacterial resistance against aminoglycoside

antibiotics

A major issue in the use of aminoglycoside antibiotics is the development

of bacterial resistance, first described by Umezawa et al. only eight years

after the discovery of kanamycin in the late 1960s.[9,10] Due to the

spreading of the resistance genes, there are no clinical important bacteria

nowadays, which are not somehow connected to aminoglycoside

resistance.[11] Resistance can occur based on three main concepts; 1)

mutation of the rRNA and the ribosomal-protein targets 2) modification

of aminoglycoside transport (import and efflux) and 3) the synthesis of

aminoglycoside modifying enzymes (AME).[11] The most clinically

important resistance mechanism is the latter; the modification of

aminoglycosides by enzymes. Here, three classes of enzymes can be

distinguished based on the reaction they catalyze: 1) N-acetyltransferases

(AACs), 2) O-nucleotidylyltransferases (ANTs) and 3) O-

phosphotransferases (APHs). The antibiotic activity is respectively lost

Chapter 4

88

due to acetylation, adenylylation or phosphorylation of specific amino or

hydroxyl groups in the aminoglycoside, which are crucial for the

recognition by the 30S ribosomal subunit. The nomenclature for these

enzymes is as follows: APH, AAC or ANT for the type of modification; 1,

3, 3’ etcetera for the specific position in the aminoglycoside; I, II, III

etcetera for the class, which is dependent on the substrate profile and a,

b, c etcetera for a specific gene. The enzyme APH(3’)-Va for example

phosphorylates (APH) the 3’-position of either neomycin, paromomycin

and ribostamycin (class V) and is translated from the aph(3’)-Va gene.

As said, an important class of aminoglycoside modifying enzymes

comprizes the aminoglycoside phosphotransferases (APHs), which use

adenosine triphosphate (ATP) to phosphorylate specific hydroxyl groups

in the aminoglycoside. The most important class of APHs phosphorylates

the 3’-position in an aminoglycoside, which results in a loss of

antibacterial activity.

Scheme 1 Phosphorylation of aminoglycosides by AHP(3')

Table 3 shows the seven AHP(3’)-subclasses that are distinguished based

on the substrate profile of the enzymes. Also it can be seen from Table 3

that APH(3’)s have been identified in numerous clinical isolates by

genetic screening.[12] APH(3’)s have been identified for example in

bacteria that cause tuberculosis (Mycobacterium tuberculosis), cholera

(Vibrio cholera) and nosocomial infections (Staphylococcus aureus and

Pseudomonas aeruginosa).


89

Table 3 Substrate profiles of APH(3’)s and clinical isolates containing APH(3’) genes

APH

(3’)

Substrates clinical isolates

I kanamycin, neomycin,

lividomycin, paromomycin,

ribostamycin

Klebsiella pneumonia,[13]

Salmonella enterica serovar

Typhimurium,[14] Salmonella

enterica,[15] Proteus vulgaris,[16]

Vibrio cholera,[17,18]

Campylobacter jejuni,[19]

Campylobacter like organism,[20]

Corynebacterium striatum[21]

II kanamycin, neomycin,

paromomycin, ribostamycin,

butirosin

Pseudomonas aeruginosa,[22,23]

and Achromobacter

xylosoxydans[24]

III kanamycin, neomycin,

lividomycin, paromomycin,

ribostamycin, butirosin, amikacin,

isepamicin

Staphylococcus aureus,[25][26]

Staphylococcus epidermidis,[26]

Campylobacter coli,[27]

Streptococcus faecalis,[28]

Enterococcus faecalis,[29]

Enterococcus faecium,[29]

Campylobacter spp.,[30]

Mycobacterium tuberculosis[31]

IV kanamycin, neomycin,


butirosin

only in aminoglycoside

producing bacteria

V neomycin, paromomycin,

ribostamycin

only in aminoglycoside

producing bacteria

VI kanamycin, neomycin,


butirosin, amikacin, isepamicin

Acinetobacter baumannii,[32]

amikacin resistant strains of

Acinetobacter species,[12,33]

Pseudomonas aeruginosa[34]

VII kanamycin, neomycin Campylobacter jejuni,[35]

Campylobacter coli[36]

Chapter 4

90

Aminoglycosides as RNA binders

In recent years, aminoglycosides have gained interest as universal RNA-

binders. Aminoglycosides-RNA interactions have been shown, among

others, for the hammerhead ribosome[37] and the human hepatitis δ

virus.[38] Inhibition of the binding of the HIV-1 Rev protein to the Rev

response element (RRE, its RNA target) by aminoglycosides has been

observed.[39] Even inhibition of the production of the HI-virus has been

demonstrated.[39] It is however unlikely that the native aminoglycosides

can be used in the treatment of HIV, because they lack the necessary

selectivity.[40] Nevertheless, aminoglycosides can be a starting point for

the development of more potent and more selective RNA binders. So,

apart from the antibiotic activity of aminoglycosides, they may be useful

in the future for the treatment of viral infections as well.

Total synthesis of aminoglycosides

Total synthesis of derivatives of aminoglycosides focused in the recent

past mainly on two strategies, either on selective modification of

commercially available aminoglycosides (see below) or on the

modification of the neamine core and if necessary further build-up of the

aminoglycoside by glycosylation.[41–43] The modification of the neamine

core is quite popular because it is readily available by acid hydrolysis of

e.g., neomycin B[44] and reduces the amount of protection and

deprotection steps compared to the modification of common

aminoglycosides.


91

Scheme 2 Hydrolysis of neomycin B to neamine

Some literature is available on the synthesis of derivatives of neamine and

kanamycin by conventional carbohydrate chemistry, which of course is a

tedious task. Appropriate glycosyl donors and acceptors often need 10 or

more synthetic steps.[45–49]

(Selective)-modification of aminoglycosides

The selective modification of aminoglycosides has been a vivid field of

research since the rise of enzyme-mediated bacterial resistance, already

described by Umezawa et al. in the late 1960s.[9,10] Due to the variety of

different hydroxyl- and amino-groups in aminoglycosides, selective

modification, however, is rather difficult. These transformations have

been reviewed extensively,[11,50–53] and some will be discussed shortly to

illustrate the basic challenges in the field.

Selective protection of aminoglycosides

Selective protection of aminoglycosides can be facilitated by forming

metal chelates prior to the protection. The selective protection of

kanamycin A at the 6’-, 1,3,6’- or 3,6’ position is therefore possible by just

varying the reaction conditions and/or the chelating metal.[54,55] Based on

the difference in basicity of the amino groups, azide transfer (to the N-3-

position) of a range of aminoglycosides has been shown by Bastian et

al..[56]

Chapter 4

92

Selective azide reduction

Selective Staudinger reduction of fully azide protected aminoglycosides

opens another way to obtain liberated amines for further modification.

Selective Staudinger reduction has been shown for some neamine

derivatives at the 2’-position[57] or the 1-position[58,59] depending on the

protecting groups on the 5- and 6-position. An example for the selective

Staudinger reduction of a commercially available active aminoglycoside

(neomycin B, kanamycin A) is however lacking, which is limiting the use

of this approach.

(Highly)-regioselective modifications

The single primary alcohol function in neomycin B (5’’) and kanamycin

(6’), for example, is relatively easy to modify. Therefore, a range of

modifications is known including dimerization of aminoglycosides,[60]

aminoglycoside-acridine conjugates,[61,62] carbohydrate-aminoglycoside

conjugates,[63] guanidinylated aminoglycosides[64] and aminoglycoside-

dinucleotide conjugates.[65] Although modification of the primary alcohol

is relatively straightforward, 5 to 9 synthetic steps are required for these

abovementioned aminoglycoside derivatives.

A huge range of 2’’-ether analogs of paromomycin has been prepared by

Hanessian et al., by a regioselective allylation of the 2”-hydroxy group.

Three protection steps are still required before the site selective allylation

can be employed, generating a 10 steps synthesis route for each of the 2’’-

ether analogs.[66]

Modification of neomycin B via a Mitsunobu dehydration, either

selectively to form the epoxide on ring IV or in combination with the

formation of an aziridine on ring II, has been described by Houston et al.

(Scheme 3).[67] Both rings allow subsequent modification via azide-

mediated ring-opening and subsequent click chemistry towards a range

of neomycin B analogs.


93

Scheme 3 Mitsunobu dehydration of neomycin B as described by Houston et al.[67]

Bastian et al. described a spectacular supramolecular aptameric protecting

group which allows acetylation of unprotected neomycin B and

paromomycin at ring IV in a single step.[68] Due to the high price of the

oligonucleotide, this method is currently suitable for screening purposes

only, and not for preparative scale synthesis.

Modifications to circumvent the action of APH(3’)

Modification of the 3’-position in aminoglycosides as a strategy to

overcome bacterial resistance caused by the aminoglycoside modifying

enzyme APH(3’) is a rather logic approach and has been described as

early as in 1970. A huge contribution was made by Umezawa et al. with

the synthesis of 3’-deoxykanamycin A, first by total synthesis[69] and later

starting from kanamycin A in 10 steps.[70] Furthermore, Umezawa

described the synthesis of 3’,4’-dideoxykanamycin[71] and several

fluorinated derivatives.[43,49] Fluorinated derivatives should resemble

kanamycin more closely than the deoxy-derivatives since the difference

in electronegativity is not as pronounced (fluorine vs. hydroxyl compared

to hydrogen vs. hydroxyl) and obviously cannot be phosphorylated by

APH(3’). In all cases antibacterial activity against kanamycin-resistant

strains was shown. In one case a decrease in the toxicity for fluorinated

Chapter 4

94

kanamycin and tobramycin compared with the deoxy-derivative was

presented.[72]

A “self-regenerating” aminoglycoside was described by Mobashery et al..

3’-Keto-kanamycin A (1), which was synthesized in 11 steps from

kanamycin A, is in equilibrium with its hydrated form 2. 2 is still a

substrate for APH(3’), but is inherently unstable after phosphorylation.

Elimination of the phosphate regenerates 1 and therefore the antibiotic

activity. Although the minimum inhibition concentration (MIC) values

for non-resistant strains were higher compared to kanamycin A, the MIC

values for the resistant strains were 4-8 fold lower than for Kanamycin

A.[73]

Scheme 4 A self regenerating form of kanamycin A as decribed by Mobashery[73]

Although over a period of 40 years considerable progress has been made

in the modification of aminoglycosides, and the accumulated knowledge

is massive, the field is still dominated by (extremely) long synthetic

routes. This hampers, if not blocks, a thorough study on the efficacy of the

prepared derivatives, let alone their scale up for clinical studies. It means

that there is a necessity for efficient regioselective methods for the

modification of unprotected or partly protected aminoglycosides in order

to dramatically shorten the synthesis of these derivatives and allow the

preparation of new ones.


95

GOAL The goal of this project was to develop a method for the selective

oxidation of aminoglycoside antibiotics. We envisioned that the catalytic

system we developed for the oxidation of glycosides (Chapter 2, mono

and disaccharides) might be applicable to selectively oxidize

aminoglycosides at the 3’-position. A comparable catalytic method for the

oxidation of aminoglycosides or even oligosaccharides is unprecedented

and would be highly desirable since it allows selective modification of

already known and commercially available antibiotics.

A possible way to circumvent bacterial resistance caused by

aminoglycoside modifying enzymes can be the modification of the

aminoglycosides in such a way that inactivation by the enzyme is no

longer possible.[74,75] We envisioned that an axial hydroxyl group at the 3’-

position of aminoglycosides, instead of the equatorial hydroxyl group in

the parent compounds, would not be phosphorylated as the 1,3-di-axial

interaction of the phosphate with the axial substituent on the anomeric

center would preclude this. This would lead to an active antibiotic against

bacteria containing APH(3’)s. Based on our previous results (Chapter 2)

selective reduction to the axial hydroxyl group should be feasible.

RESULTS AND DISCUSSION We selected several commercially available aminoglycosides based on the

presence of a gluco-configuration and a hydroxyl group at the 3’-position

of ring I. This led to the selection of neomycin B, kanamycin A, amikacin,

paromomycin and apramycin.

Chapter 4

96

Figure 5 Selection of aminoglycoside substrates for regioselective oxidation

Global amine protection of aminoglycosides

We began our project with the synthesis of the suitable precursors for the

selective oxidation. A global amine protection of the aminoglycosides had

to be done since the catalyst for the oxidation is incompatible with free

amines. Cbz- and Boc-protection were chosen, Cbz protection because of

its orthogonal removal, Boc protection because it was not entire certain

whether the oxidation would be entirely orthogonal to the

benzyloxycarbonyl protection.

Neomycin B could be protected by Boc-groups using di-tert-

butyldicarbonate[60] in 80% yield after purification by column

chromatography. Paromomycin was protected with Cbz- and Boc-groups

as well and the products were isolated in 47% and 17% yield, respectively,

after column chromatography. The Boc-protection of paromomycin was


97

performed only once, therefore the low isolated yield of 12 is probably

due to unoptimized purification and reaction conditions.

Scheme 5 Protection of the amino groups in neomycin B and paromomycin

The protection of kanamycin A and amikacin with Cbz groups resulted

in both cases in highly insoluble material, which made further

purification difficult. Therefore, kanamycin A and amikacin were

protected by the tert-butyl-derivative of the Cbz-group. The increased

solubility now allowed purification by column chromatography or

recrystallization, and 13 and 15 were isolated in 65% and 69%

respectively. Boc-protection of kanamycin and amikacin gave the

protected aminoglycosides in 56% and 73% yield.

Scheme 6 Protection of the amino groups in kanamycin A and amikacin

Chapter 4

98

Unfortunately, the protection of apramycin with Cbz- or Boc-groups

failed, due to an incomplete reaction and complex crude reaction

mixtures.

Oxidation of the amine-protected

aminoglycosides

We started to test our hypothesis, that the catalyst would preferably

oxidize at the 3-position of a glucopyranose, by treatment of Cbz-

protected neomycin B with 2.5 mol% of [(neocuproine)PdOAc]2(OTf)2 and

3 eq of benzoquinone as the oxidant. Although the reaction stopped after

about 40% conversion, a single oxidation product was obtained after

separation by column chromatography.

Scheme 7 Regioselective oxidation of amine-protected neomycin B

Thorough NMR analysis using 1H-, 13C- HSQC-, COSY- and TOCSY-NMR

revealed oxidation of the 3’-position, as we had predicted. Optimization

of the reaction conditions by the addition of a second batch of 2.5 mol%

of catalyst after 1 h, improved the isolated yield to 41% (from 29% in the

first reaction). Also the oxidation of Boc-protected neomycin B did not go

to completion according to HPLC-analysis. Furthermore its purification

by silica column chromatography was low yielding and did not give pure

product.

The reason for the incomplete conversion was not pinned down. The

initial rate of the reaction appears to be pretty high, but decreases quickly

as well, and a second batch of catalyst leads to some increase but not to a


99

doubling of the yield. A reasonable explanation for the incomplete

conversion could be that the benzylic hydrogens of the Cbz groups are

somewhat sensitive to oxidation by palladium. This side reaction would

lead to deprotection and subsequent inhibition of the catalyst by the

liberated amine. This reasoning is contradicted by the observation that

also Boc-protected neomycin B is not fully oxidized. At the other hand, it

is not impossible that the Lewis acidic palladium catalyst leads to some

Boc-removal, again liberating a free amine. Nevertheless, also other

explanations are possible such as an inhibition of the catalyst by

complexation to the substrate in an unproductive way. A further study is

warranted here, and mass spectrometry seems a preferred choice.

Scheme 8 Regioselective oxidation of amine-protected kanamycin A

A spectacular result was obtained with kanamycin A (Boc protected, 14)

which was fully and selectively oxidized (again on the 3’-postion) using

only 2 mol% of catalyst within 30 min. Straightforward precipitation

afforded the pure product without the necessity of column

chromatography, with an isolated yield of 59%. Further precipitation

afforded only impure product. Cbz- and (tert-butyl)-Cbz protected

kanamycin A, however, gave no conversion even after prolonged reaction

times and an increase of the catalyst loading up to 10 mol%. The presence

of residual halide or residual free amines might in this case be blamed; in

the preceding protection step it is often difficult to judge whether

complete reaction of all four amine groups has been achieved.

Chapter 4

100

Scheme 9 Regioselective oxidation of amine-protected amikacin

Tert-butyl-Cbz amikacin (15) was fully oxidized using 2.5 mol% of

[(neocuproine)PdOAc]2(OTf)2 within 24 h, but the reaction appeared to be

not fully selective. HPLC-MS analysis showed several mono-oxidation

products next to the major product oxidized at the 3’-position.

Fortunately, 3’-keto-tert-butyl-Cbz amikacin could be isolated after

purification by trituration and recrystallization from THF and dioxane,

respectively, in 28% yield.

Finally, the oxidation of both Cbz- and Boc-protected paromomycin

turned out to be problematic. In both cases the reaction was not selective

and separation of the products using column chromatography did not

succeed. The research on paronomycin was therefore terminated.

Table 4 Overview of the regioselective oxidation of several amine-protected

aminoglycosides

Entry Aminoglycoside Protecting

Group

Isolated

yield Remarks

1 Neomycin B Cbz 41% incomplete

reaction

2 Kanamycin A Boc 59% complete

reaction

3 Amikacin Tert-butyl-Cbz 28% complete

reaction

4 Paromomycin Cbz nd not

selective


101

3’-Keto-aminoglycosides as antibiotics

As shown above, the 3-keto aminoglycosides could be used as self-

regenerating antibiotics as described by the group of Mobashery.[73] To

obtain the 3-keto aminoglycosides, two methods for the deprotection

were studied; deprotection using palladium on charcoal and palladium

hydroxide. Whereas for deprotection using palladium on charcoal a

hydrogen pressure of about 70 bar was necessary in order to obtain

complete deprotection, palladium hydroxide cleanly cleaved the Cbz

groups overnight at atmospheric hydrogen pressure (in deoxygenated

solvents). Both methods however, did not give the desired product 20,

instead cleavage of the ring I (21) was observed by HSQC-NMR. The

cleavage follows probably a retro-Michael or an E1cB elimination

mechanism. Due to the instability of 20, this approach was, for the time

being, discontinued.

Scheme 10 Deprotection of 3’-keto-neomycin B cleaves ring I.

Selective modification to 3’-epi-aminoglycosides

In Chapter 2 we observed, that treatment of 3-keto-glycosides, bearing an

axial substituent on the anomeric center, with sodium borohydride,

selectively provides the 3-axial alcohol.[76,77] We desired to study this also

for the aminoglycosides, because this would lead to the 3’-epimer of a

parent aminoglycoside in only 4 steps.

Chapter 4

102

Scheme 11 Epimerization of aminoglycosides by selective oxidation and reduction

As a starting point, 17 was treated with 5 eq of sodium borohydride in

methanol for 24 h and were pleased to isolate 22 as single product in 92%

yield.

Scheme 12 Selective reduction of 17

After this success, it was surprising (and disappointing) that treatment of

18 with sodium borohydride gave 23 in just 60% isolated yield, and as a

mixture of the axial and equatorial 3’-alcohol 23 in a ratio of 3.5 : 1. This

became evident after removal of the Boc-groups followed by comparison

of the NMR-spectra with those of kanamycin A (reduction of keto-

kanamycin A to the equatorial alcohol obviously gives the starting


103

compound in the sequence). The reason for this lack of selectivity remains

unknown.


Reduction of 19 by sodium borohydride for 4 h, on the other hand, gave

again cleanly 24 as single product in quantitative yield. Given the

seemingly futile differences between 18 and 19, when it comes to

reduction at the 3’-position, the lower yield and selectivity in the

reduction of 18 is hard to understand.


Deprotection and final purification

Deprotection of Cbz-protected neomycin B and tert-butyl-Cbz-protected

amikacin was carried out by hydrogenolysis using palladium hydroxide

on carbon in a mixture of methanol, water and acetic acid (36/3/1), for 3-5

days at 1 atm of hydrogen.[78] Fortunately, 3’epi-neomycin B and 3’-epi-

amikacin were isolated in quantitative yield as their tetra-acetic acid salts.

Chapter 4

104

Scheme 15 Deprotection of 22 and 24 to afford 3’epi-neomycin B (25) and 3’-epi-

amikacin (26)

Boc-protected 3’-epi-kanamycin A 23 was deprotected in a 1:1 mixture of

trifluoroacetic acid and dichloromethane (using thiophenol as cation

scavenger) for 1 h at room temperature, and could also be isolated in

quantitative yield as its tetra-trifluoroacetic acid salt.

Scheme 16 Deprotection of 23 to afford 3’-epi-kanamycin A (27)

In order to determine MIC values of these modified aminoglycosides in

bacterial strains, we had to reduce the amount of residual palladium from

either the oxidation or deprotection to a minimum. Palladium is toxic and

would blur any study on antibiotic activity. In an earlier project we had

noticed that aminoglycosides, after hydrogenolysis by palladium

hydroxide, contained up to 200 ppm of palladium according to ICP-AES

(inductively coupled plasma-atomic emission spectroscopy) analysis. By


105

column chromatography, the amount of palladium could be reduced to

16 ppm. The detection limit of this technique is below 1 ppm and the usual

error margin not more than 10 ppm, indicating the reduction of the

palladium content is significant. As eluents a mixture of dichloromethane

or chloroform with methanol and aq. 25% ammonia (2:2:1 or 2:1:1 (the

upper layer of the biphasic system)) was used. Unfortunately, the eluents

also dissolved some of the silica gel that ended up in the product.

Attempts to dissolve the aminoglycoside in a small of amount water,

followed by filtration of the residual solids were only partially successful.

As the determination of MIC-values, and activity assays in general,

requires accurate determination of the amount of compound, we turned

to quantitative NMR-spectroscopy (qNMR).[79–81] A more detailed

description of the qNMR can be found in the experimental section.

Table 5 Overview of purity and isolated yield of various 3’-epi aminoglycosides

according to qNMR

Aminoglycoside Purity (%)

by

q-NMR

axial/equatorial Isolated yield

(recalculated)

3’-epi-neomycin B

(25) 20% >10:1 20%

3’-epi-amikacin

(26) 23% >10:1 29%

3’-epi-kanamycin A

(27) 77% 3.5:1 54%

Table 5 shows the results of the quantitative NMR analysis of 25, 26 and

27. To our disappointment, the yield and the purity for the 3’-epi

derivatives of neomycin B (25) and amikacin (27) were rather low. A

purity of 20% and 23% and an isolated yield of 23% and 29% for 25 and

26 was calculated, respectively. The 3’-epi derivative of kanamycin A (27)

was isolated as TFA salt in 54% yield and a purity of 77%.

Chapter 4

106

The analysis by qNMR illustrates two problems: the purification by silica

column chromatography is low yielding and the samples contain a huge

amount of silica, ammonia- and/or formate-salts. In the future a (second)

purification of the aminoglycosides after deprotection has to be

developed. A possible solution would be the use of ion exchange

chromatography[78] or size exclusion chromatography.[43,72,82]

Targeting two mechanisms of bacterial resistance

against aminoglycoside antibiotics

A general strategy against enzyme-mediated bacterial resistance is the

modification of the positions in an aminoglycoside that are targeted by

the enzyme, in such a way that the enzyme’s action is prohibited.[74] This

probably leads to a diminished activity of the antibiotic as well, due to

less efficient binding to the target, but to some extent this is acceptable.

We envisioned that the combination of our regioselective

oxidation/reduction sequence with the selective azide transfer[56]

developed in the Herrmann group could lead to an active antibiotic

against bacteria carrying APH(3’)s and AAC(3)s.

Scheme 17 Strategy to overcome two types of bacterial resistance against neomycin B

Synthetic plan

A synthetic strategy was designed that starts with the regioselective azide

transfer of neomycin B, described by Bastian et al.[56] and followed by a

global amine protection to give 29. 29 can be oxidized selectively using

the standard conditions and subsequent reduction of the ketone with


107

sodium borohydride should give the axial hydroxyl group on the 3’-

position. Further reduction of the azide by a Staudinger reduction would

give amine 31 which, as it has only one free amino group in the molecule,

can be transformed by acylation (e.g., with amino acid derivatives),

methylation or, better, dimethylation. Deprotection would give a double

modified neomycin B (33) in just 6 to 7 synthetic steps.

Scheme 18 Synthetic strategy toward a double modified neomycin B

The synthesis of 28 was performed by azide transfer using imidazole-1-

sulfonyl azide at pH = 6.6 in 82% yield.[56] Subsequent Cbz-protection was

achieved by treatment of 28 with N-(benzyloxycarbonyloxy)-succinimide

which led to 29 in 63% yield after purification by column

chromatography.

Chapter 4

108

Scheme 19 Regioselective azide-transfer and Cbz-protection leading to 29

Oxidation of 29 was achieved in 50% isolated yield using 2.5 mol%

[(neocuproine)PdOAc]2(OTf)2 and 3 eq of benzoquinone after purification

by column chromatography. Fortunately, the azide is stable during the

reaction conditions (4 h at rt). Prolonged reaction times (>12 h), however,

showed a complex mixture of products, pointing to an involvement of the

azide.

Scheme 20 Regioselective oxidation of 29

Sodium borohydride reduction of 30 in MeOH at room temperature for

24 h gave a mixture of 34 and 31 as also partial reduction of the azide took

place. Surprisingly however, longer reaction times and/or more

equivalents of sodium borohydride did not lead to a complete reduction

to amine 31. 34 and 31 could be isolated in 17% (azide) and 46% (amine),

respectively, after separation by column chromatography. Attempts to

selectively reduce 30 to 34, by lowering the temperature to -10 °C,

shortening the reaction time to 2 h and also quenching the reaction

at -10 °C were successful on a small scale, but gave a 4:1 mixture of 34 and

31 on a bigger scale. Reduction of azides by sodium borohydride is rare,


109

although in combination with other metal salts it has been reported.[83,84]

Residual palladium from the oxidation could participate in the reduction

of the azide. Although the partial reduction of the azide function was

unexpected, it was no problem for our synthetic strategy since the next

step was the Staudinger reduction.

Scheme 21 Reduction of 30 with sodium borohydride

Therefore we decided to use the mixture of 34 and 31 without separation

by column chromatography, which enabled an excellent isolated yield of

97%.

The mixture of 31 and 34 could be completely reduced by Staudinger

reduction in 91% crude yield and was used without further purification.

Dimethylation by reductive amination with formaldehyde and sodium

cyanoborohydride gave 35 in 56% yield after purification by column

chromatography.

Scheme 22 Staudinger reduction and reductive amination leading to 35

Final deprotection by hydrogenolysis using palladium hydroxide in a

mixture of methanol, water and acetic acid gave 36 after purification by

column chromatography (silica gel, dichloromethane/methanol/25%

ammonia). The purity was 16% according to qNMR, again containing

Chapter 4

110

NMR-silent impurities, like silica gel and ammonium salts, which

resulted in a rather disappointing isolated yield of 6.5%. Nevertheless, 36

was fully characterized and the route allows to produce sufficient

material for biological studies.

Scheme 23 Deprotection of 35 to produce double modified neomycin B (36)

Apart from dimethylation, another option can be pursued in the future.

The modification by acylation with amino acid derivatives (35) would

also not change the overall positive charge, but the remote position of the

new amino group makes modification by ACC-3 hopefully impossible.

Scheme 24 Future plans for the modification of 31

CONCLUSIONS AND FUTURE PERSPECTIVES To conclude, we have developed a method for the regioselective

oxidation of aminoglycosides. The 3’-keto-derivatives of neomycin B,

kanamycin A, amikacin and the 3-azido-derivative of neomycin B have

been prepared and isolated in moderate to good yields after protection of

the amino groups. The 3’-keto-function has been stereoselectively

reduced with sodium borohydride to the 3’-axial-hydroxyl group in good


111

to excellent selectivities and isolated yields. By combining our selective

oxidation method with the selective azide transfer described by Bastian et

al.,[56] we were able to develop a strategy to selectively modify two

positions in neomycin B. Both positions are well-known targets for the

AME of resistant bacteria.[12] In the near future it will be determined

whether the modified aminoglycosides still are active antibiotics, and

moreover, whether the compounds are active against resistant bacterial

strains.

The final isolation and purification of the modified aminoglycosides

remains a critical point. It is an absolute requirement for reliable antibiotic

activity studies to remove residual palladium from the compounds.

Chromatography on silica, however, turned out to be far from ideal as the

high polarity of the products requires a mobile phase that dissolves the

silica, as shown by qNMR. Different methods for the isolation and

purification should be investigated. Possible solutions could be the

application of ion-exchange or size-exclusion column chromatography. In

addition, resins are available that selectively scavenge palladium, not

surprisingly as the contamination of active pharmaceutical ingredients

with heavy metals is a known problem.

The selective oxidation described here, despite the problems with

purification and isolation, is a powerful new tool for the modification of

aminoglycoside antibiotics. Despite the fact that the resulting keto-

aminoglucosides, though not necessarily very stable, might act as

antibiotics themselves subsequent modification is probably required. We

showed already that hydride reduction leads in most cases selectively to

the epimeric alcohol. Several other selective modifications should be

feasible as well: 1) Deoxygenation of the keto-group, for example via

reduction of the corresponding tosylhydrazone[85] could be a route to 3’-

deoxy-aminoglycosides (38) in just 5 steps (the only reported route in the

literature requires 10 steps in the case of kanamycin A)[70] 2) Via reductive

amination, a range of different aminoglycoside derivatives (39) should be

Chapter 4

112

readily accessible. This can be approached by formation of the hydrazone

with hydrazine followed by reduction towards the 3-deoxy-3-amine-

aminoglycoside[86,87] or by reductive amination with amines that form e.g.,

dimers[60,62,88,89] and acridine-[61,62] or pyrene-aminoglycoside-conjugates[90].

These conjugates have shown to increase the affinity for RNA. 3)

Transformation to the 3’,3’-difluoro derivatives (40) might be feasible via

treatment of the (hydroxyl protected) 3’-keto-aminoglycoside with DAST

or related reagents.[72] Earlier reports have shown that the toxicity of

aminoglycosides is dependent on the basicity of the amino groups.[72,91,92]

A higher pKa of the amino groups results in an increased toxicity, and a

possible remedy seems to be the fluorination of aminoglycosides. By

mono- and di-fluorination of the 5-position of dibekacin

(3’,4’deoxykanamycin B) and tobramycin (3’deoxykanamycin B) the

toxicity of the aminoglycoside could be markedly decreased, probably

due to the decreased pKa of the amino groups at the 1- and 3-position,

with retention or improvement of antibacterial activity.[72]

Figure 6 Possible modifications of 3’-keto aminoglycosides

EXPERIMENTAL SECTION

General Information

Solvents and Reagents

All solvents used for extraction, filtration and chromatography were of

commercial grade, and used without further purification. Reagents were

purchased from Sigma-Aldrich, Acros, ABCR, and Carbosynth and were used

without further purification. For purification via column chromatography silica


113

gel from either Silicycle (Sila Flash 40-63 µm, 230-400 mesh) or from Sigma

Aldrich (Silica Amorphus, precipitated, Davisil grade 62, pore size 150 Å, 60-

200 mesh) was used. [(neocuproine)PdOAc]2OTf2 and 3-azido neomycin B were

prepared according to the literature procedures.[56,93]

Analysis

TLC was performed on Merck silica gel 60, 0.25 mm plates and visualization was

done by UV and staining with Seebach’s reagent (a mixture of phosphomolybdic

acid (25 g), cerium (IV) sulfate (7.5 g), H2O (500 mL) and H2SO4 (25 mL)) and

potassium permanganate stain (a mixture of KMnO4 (3g), K2CO3 (10g), water

(300mL)).

1H-, 13C-, APT-, COSY-, HSQC-, NOESY were recorded on a Varian AMX400 (400,

100.59 MHz, respectively) using DMSO-d6, MeOD-d4 or D2O as solvent. Chemical

shift values are reported in ppm with the solvent resonance as the internal

standard (DMSO-d6: 2.50 for 1H, δ 39.51 for 13C; MeOD-d4: δ3.31 for 1H, δ 49.15 for

13C; D2O: δ 4.80 for 1H; acetonitrile-d3: δ 1.94 for 1H, δ 118 for 13C). Data are

reported as follows: chemical shifts (δ), multiplicity (s = singlet, d = doublet, t =

triplet, q =quartet, br = broad, m = multiplet), coupling constants J (Hz), and

integration.

The purity of the aminoglycosides was measured by quantitative NMR (qNMR).

The basic steps described in the literature were followed.[79,80] The 90 deg pulse

was calibrated, the relaxation time (T1) was measured and a relaxation delay (d1)

of >5T1 for the longest T1 was chosen. Carbon decoupling was done using the

GARP decoupling sequence. Calcium formate or maleic acid (Standard for

quantitative NMR, TraceCERT® from Sigma Aldrich) were used as internal

standard in D2O.

Optical rotations were measured on a Schmidt+Haensch polarimeter (Polartronic

MH8) with a 10 cm cell (c given in g/100 mL). High Resolution Mass

measurements were performed using a ThermoScientific LTQ OribitrapXL

spectrometer.

Chapter 4

114

(Cbz)6-Neomycin B(9)

(Cbz)6-Neomycin B was kindly provided by an

industrial partner and was purified by column

chromatography (silica gel, DCM/MeOH 20:1).[94] 1H

NMR (400 MHz, DMSO/drop of D2O): δ = 7.42 – 7.21

(m, 30H), 5.11 – 4.94 (m, 13H), 4.90 (d, J = 12.3 Hz, 1H),

4.78 (appears as s, 1H), 3.98 (appears as s (br), 1H), 3.94

(appears as t (br), J = 5.3 Hz, 1H), 3.85 (m (br), 1H), 3.79 (m (br), 1H), 3.75 (m (br),

1H), 3.70 (appears as d (br), J = 7.9 Hz, 1H), 3.66 – 3.48 (m, 6H), 3.44 (appears as

m, partial overlaps with HDO, 5H), 3.22 (m (br), 3H), 3.07 (m (br), 2H), 1.71

(appears as d (br), J = 11.3 Hz, 1H), 1.31 (q, J = 12.9 Hz, 1H). 13C NMR (100 MHz,

DMSO/drop of D2O): δ = 156.6, 156.5, 156.2, 156.2, 156.2, 156.1, 137.3, 137.3, 137.3,

137.2, 137.1, 137.0, 128.6, 128.5, 128.5, 128.5, 128.4, 128.0, 127.9, 127.9 127. 8, 127.7,

127.6, 127.5, 127.4, 109.8, 98.8, 98.4, 85.9, 82.1, 76.7, 73.7, 72.7, 71.1, 70.0, 69.8, 67.3,

65.7, 65.6, 65.4, 65.4, 62.2, 56.5, 52.5, 50.9, 42.4, 41.1, 30.8. HRMS (ESI) calculated

for C71H83N6O25 ([M+H]+): 1419.54, found: 1419.54 [α]D20 +24 (c 1.0, MeOH). For a

1H-NMR-spectrum in CDCl3 and IR-data see literature.[95,96]

(Boc)6-Neomycin B (10)

Neomycin B trisulfate salt hydrate (1.3 g, 1.4 mmol,

1 eq) and triethylamine (4.55 mL, 32.6 mmol, 23 eq)

were dissolved in water (6.5 mL) and MeOH (6.5 mL).

Di-tert-butyldicarbonate (3.25 g, 14.9 mmol, 10.6 eq)

was added and the mixture was stirred overnight at

60 °C. The MeOH was evaporated and a sticky white

solid precipitated. The solid was dissolved in EtOAc and the water layer was

extracted with EtOAc. The combined organic layers were washed with brine,

dried and concentrated in vacuo to give 1.6 g of crude product. The crude

product was purified over a plug of silica (pure product was eluted with

DCM/MeOH (20:1)) and coevaporated with toluene to give (Boc)6-Neomycin B

as a white solid (1.36 g, 1.12 mmol, 80%). 1H NMR (400 MHz, CD3OD): δ = 5.29

(appears as s (br), 1H), 5.16 (s, 1H), 4.90 (appears as s (br), 1H), 4.18 (appears as s

(br), 2H), 4.04 – 3.93 (m, 1H), 3.92 – 3.79 (m, 3H), 3.79 – 3.68 (m, 3H), 3.66 (dd, J =

10.1, 9.3 Hz, 1H), 3.59 – 3.41 (m, 6H), 3.41 – 3.32 (m, 3H), 3.30 (overlaps with HOD,


115

m, 1H), 3.28 – 3.22 (m, 1H), 3.19 (dd, J = 9.7, 9.2 Hz, 1H), 1.95 (d, J = 12.8 Hz, 1H),

1.51 – 1.39 (m, 54H), 1.38 – 1.27 (m, 1H). 13C NMR (100 MHz, CD3OD): δ = 159.2,

159.1, 158.7, 158.6, 158.4, 158.0, 110.7, 100.5, 99.4, 87.8, 83.6, 80.8, 80.8, 80.6, 80.5,

79.8, 78.5, 75.8, 74.6, 73.2, 73.1, 72.5, 71.7, 69.0, 63.7, 57.3, 53.7, 52.4, 51.9, 42.7, 41.9,

36.0, 29.1, 29.0, 29.0, 29.0, 28.9. HRMS (ESI) calculated for C53H94N6O25Na

([M+Na]+): 1237.62, found: 1237.62. [α]D20 +36 (c 1.5, MeOH). Characterization

matches literature.[60]

3’-Keto-(Cbz)6-Neomycin B (17)

(Cbz)6-neomycin B (1 g, 0.7 mmol, 1 eq) and

benzoquinone (0.23 g, 2.1 mmol, 3 eq) were dissolved

in DMSO (6 mL) and a drop of water was added.

[(Neocuproine)PdOAc]2(OTf)2 (18 mg, 18 μmol,

2.5 mol%) was added and the reddish solution was

stirred for 1 h. Another batch of

[(neocuproine)PdOAc]2(OTf)2 (18 mg, 18 μmol, 2.5 mol%) was added and the

mixture was stirred overnight. The reaction was quenched by addition of water

(50 mL). The resulting white precipitate was filtered, washed with water and

transferred by dissolution in DCM (dried with Na2SO4). Concentration in vacuo

gave 970 mg crude product. Pure 3’-Keto-(Cbz)6-Neomycin B (408 mg, 0.29 mmol,

41%) was obtained by purification using automated column chromatography

(40 g silica column, DCM/MeOH gradient: 0% for 3 CV, 0-5% in 8 CV, 5% for

10 CV) as a yellow foam. 1H NMR (400 MHz, DMSO/drop of D2O): δ = 7.41 –

7.23 (m, 30H), 7.19 (d, J = 5.8 Hz, 1H), 7.11 (s (br), 1H), 6.87 (d (br), J = 8.3 Hz, 1H),

6.55 (s, 1H), 6.19 (d, J = 9.2 Hz, 1H), 5.59 (appears as s (br), 1H), 5.14 – 4.92 (m,

13H), 4.88 (appears as d (br), J = 12.2 Hz, 1H), 4.80 (appears as s (br), 1H), 4.65 (dd

(br), J = 8.4, 4.2 Hz, 1H), 4.19 (d, J = 9.8 Hz, 1H), 4.12 – 3.95 (m, 2H), 3.95 – 3.86 (m,

1H), 3.86 – 3.78 (m, 2H), 3.78 – 3.73 (m, 1H), 3.69 (d (br), J = 10.4 Hz, 1H), 3.49

(overlaps with HOD, m, 5H), 3.35 (appears as s (br), 2H), 3.31 – 3.23 (m, 2H), 3.23

– 3.13 (m, 2H), 1.72 (appears as d (br), J = 11.2 Hz, 1H), 1.32 (dd, J = 24.4, 11.9 Hz,

1H). 13C NMR (100 MHz, DMSO/drop of D2O): δ = 202.9, 156.6, 156.6, 156.3,

156.2, 156.1, 156.1, 156.0, 155.9, 155.7, 137.3, 137.2, 137.2, 137.0, 137.0, 136.8, 128.5,

128.5, 128.5, 128.5, 128.0, 127.9, 127.9, 127.9, 127.8, 127.8, 107.7, 100.3, 98.6, 84.4,

82.2, 79.0, 77.1, 73.7, 73.6, 73.4, 72.8, 72.7, 69.8, 67.3, 66.1, 65.7, 65.7, 65.6, 65.6, 65.4,

Chapter 4

116

61.6, 60.4, 52.7, 50.7, 50.3, 42.0, 41.1, 34.6. HRMS (ESI) calculated for C71H81N6O25

([M+H]+): 1417.52, found: 1417.51. [α]D20 +11 (c 3.6, MeOH).

3’-epi-(Cbz)6-Neomycin B (22)

3’-Keto-(Cbz)6-neomycin B (72 mg, 51 μmol, 1 eq) was

dissolved in dry MeOH (2 mL) and sodium

borohydride (9.6 mg, 254 μmol, 5 eq) was added. The

mixture was stirred overnight and neutralized by

addition of Amberlite (H+-form), filtered and

coevaporated with MeOH (4 x 5 mL), which gave pure

3’-epi-(Cbz)6-neomycin B (66 mg, 46 μmol, 92%). Identification of 22 is difficult

due to overlapping signals. It was done by comparison of the NMR-spectra of 9

and 22, which showed significant differences, indicating 3’-epi-(Cbz)6-neomycin

B (22) as only product. 1H NMR (400 MHz, CD3OD): δ = 7.41 – 7.20 (m, 30H), 5.30

– 4.94 (m, 14H), 4.79 (appears as s (br), 1H), 4.01 (m (br), 1H), 3.97 – 3.86 (m, 1H),

3.86 – 3.77 (m, 1H), 3.70 – 3.63 (m, 1H), 3.62 – 3.46 (m, 6H), 3.46 – 3.33 (m, 6H),

1.99 (appears as d (br), J = 12.9 Hz, 1H), 1.40 (m (br), 1H). 13C NMR (100 MHz,

CD3OD): δ = 159.4, 159.3, 159.1, 158.7, 158.4, 158.1, 138.4, 138.4, 138.4, 138.2, 137.9,

129.8, 129.8, 129.7, 129.7, 129.6, 129.6, 129.6, 129.5, 129.3, 129.2, 129.1, 129.1, 129.0,

129.0, 128.8, 109.4, 100.6, 100.1, 86.3, 83.7, 81.3, 78.3, 75.9, 75.4, 74.6, 71.9, 69.3, 69.1,

68.4, 68.2, 67.8, 67.7, 67.6, 63.1, 54.2, 53.5, 52.6, 52.2, 42.9, 42.7, 35.5. HRMS (ESI)

calculated for C71H83N6O25 ([M+H]+): 1419.54, found: 1419.54. [α]D20 +10 (c 1.2,

MeOH).

3’-epi- Neomycin B (25)

3’-Epi-(Cbz)6-neomycin B (46 mg, 32 μmol, 1 eq) was

dissolved in a MeOH-water-AcOH mixture (3.2 mL,

36:3:1) and palladium hydroxide on carbon (4.5 mg,

20 wt% Pd) was added. The mixture was hydrogenated

at 1 atm. hydrogen for 5 days. The mixture was filtered

over celite, which was subsequently washed with water

and MeOH. The mixture was concentrated in vacuo, redissolved in water and

freeze-dried to give 3’-epi-neomycin B (44 mg, quant.) as its acetic acid salt. In

order to remove residual palladium (for biological tests), the 3’-epi-neomycin B


117

was passed over a silica column (0.9 g silica, DCM/MeOH/25% ammonia 2 : 2 : 1)

and evaporated in vacuo. To remove dissolved silica the product was re-dissolved

in a small amount of water and filtered through a membrane filter (0.45 μm),

which gave 20 mg of 20% pure acc. to qNMR 3’-epi-neomycin B (6.6 μmol, 20%

pure, contaminants being NMR-silent, 20% yield) after freeze-drying. 1H NMR

(400 MHz, D2O): δ = 5.92 (d, J = 4.3 Hz, 1H), 5.47 (d, J = 2.2 Hz, 1H), 5.36 (d, J = 1.4

Hz, 1H), 4.61 (dd, J = 6.3, 5.1 Hz, 1H), 4.48 (dd, J = 4.8, 2.3 Hz, 1H), 4.40 (m (br),

1H), 4.33 (appears as t, J = 3.1 Hz, 1H), 4.31 – 4.26 (m, 2H), 4.19 – 4.09 (m, 2H), 4.02

– 3.93 (m, 2H), 3.90 – 3.86 (m, 1H), 3.86 – 3.74 (m, 4H), 3.66 (appears as s (br), 1H),

3.60 – 3.53 (m, 2H), 3.52 (appears as t, J = 3.8 Hz, 1H), 3.49 – 3.44 (m, 2H), 3.44 –

3.40 (m, 1H), 3.40 – 3.33 (m, 1H), 2.53 (appears as dt, J = 12.4, 4.0 Hz, 1H), 1.98

(appears as q, J = 13.1 Hz, 1H). 13C NMR (100 MHz, D2O): δ = 109.5, 95.2, 93.6,

84.1, 81.4, 75.3, 74.6, 73.4, 72.3, 70.1, 67.6, 67.2, 66.5, 65.9, 65.1, 60.6, 50.8, 50.0, 49.7,

48.7, 40.4, 39.9, 28.0. HRMS (ESI) calculated for C23H46N6O13Na ([M+Na]+):

637.302, found: 637.301

3-Azido-(Cbz)5-neomycin B (29)

3-Azido-neomycin B[56] (4.4 g, 6.8 mmol, 1 eq) and

potassium carbonate (3.3 g, 23.8 mmol, 3.5 eq) were

dissolved in water (60 mL). A solution of N-

(benzyloxycarbonyloxy)succinimide (15.2 g, 61.2 mmol,

9 eq) in THF (60 mL) was added and the mixture was

stirred overnight. The reaction was quenched by

addition of 3-dimethylaminopropylamine (7.6 mL) and diluted with EtOAc

(12 mL) and water (15 mL). The organic layer was separated and washed with a

mixture of 20% citric acid and brine (30 mL, 1:1) and with a mixture of sat. aq.

NaHCO3 and water (30 mL, 1:1). The organic layer was dried and concentrated

in vacuo to give 8.8 g of crude product. The crude product was dissolved in EtOAc

(25 mL) and was added dropwise to MTBE (115 mL). The precipitate was filtered,

washed with ether and dried in vacuo to give 6.37 g of product. The purity was

not sufficient for the oxidation, therefore the precipitate was further purified by

column chromatography (Grace automated column chromatography, 2x 120 g

silica column, gradient DCM/MeOH, 5% MeOH eluted the product) which gave

4.73 g of pure 3-azido penta-N-benzyloxycarbonyl-neomycin B as white solid.

Purification of the mother liquor of the precipitation by column chromatography

Chapter 4

118

gave an additional 245 mg of pure product (total yield 4.98 g, 63%). 1H NMR (400

MHz, CD3OD): δ = 7.44 – 7.19 (m, J = 10.2, 6.6, 5.1 Hz, 25H), 5.30 (s, 1H), 5.18 –

4.95 (m, 11H), 4.83 (overlap with HOD peak, 1H), 4.07 – 4.00 (m, 1H), 3.91 (s, 5H),

3.82 (s, 1H), 3.76 (d, J = 10.5 Hz, 1H), 3.71 (d, J = 7.9 Hz, 1H), 3.67 – 3.54 (m, 3H),

3.51 – 3.32 (m, 9H), 3.24 (appears as t, J = 9.4 Hz, 1H), 2.11 (appears as d, J = 12.4

Hz, 1H), 1.37 – 1.22 (m, 1H). 13C NMR (100 MHz, CD3OD): δ = 157.8, 157.7, 157.5,

157.4, 157.2, 136.9, 136.9, 136.8, 136.6, 128.2, 128.1, 128.1, 128.0, 128.0, 127.8, 127.6,

127.6, 127.6, 127.5, 127.4, 127.2, 108.9, 98.9, 98.4, 85.1, 82.1, 78.9, 76.6, 74.0, 73.1,

71.7, 71.1, 70.1, 67.7, 66.6, 66.2, 66.1, 61.6, 60.9, 56.2, 52.7, 41.7, 41.1. HRMS (ESI)


MeOH)

3-Azido-3’-keto-(Cbz)5-neomycin B (30)

3-Azido-(Cbz)5-neomycin B (1.25 g, 0.95 mmol, 1 eq)

and benzoquinone (0.31 g, 2.86 mmol, 3 eq) were

dissolved in DMSO (6.4 mL) and

[(neocuproine)PdOAc]2OTf2 (25 mg, 24 μmol,

2.5 mol%) was added. The mixture was stirred for 4 h

and was quenched by the addition of water. The

precipitate (very finely divided) was filtered over celite and washed with water.

The solid was dissolved in DCM, dried and concentrated in vacuo to give 1.3 g of

crude product. Purification by column chromatography (Grace automated

column chromatography, 40 g silica column, gradient DCM/MeOH, 5% MeOH

eluted the product) gave 623 mg (0.46 mmol, 50%) of pure 3-azido-3’-keto (Cbz)5-

Neomycin B along with starting material (206 mg) and mixed fractions (229 mg).

1H NMR (400 MHz, CD3OD): δ = 7.44 – 7.18 (m, 25H), 5.84 (d, J = 4.1 Hz, 1H), 5.21

– 4.96 (m, 11H), 4.87 (s, J = 4.4 Hz, 1H), 4.71 (d, J = 3.8 Hz, 1H), 4.25 – 4.16 (m, 1H),

4.12 (d, J = 10.2 Hz, 1H), 4.08 (appears as s (br), 2H), 4.01 – 3.88 (m, 3H), 3.84

(appears as s (br), 1H), 3.74 – 3.64 (m, 2H), 3.61 (dd, J = 12.1, 5.0 Hz, 1H), 3.54 –

3.41 (m, 5H), 3.41 – 3.33 (m, 4H), 2.11 (appears as dt, J = 12.9, 3.9 Hz, 1H), 1.42 –

1.20 (m, 1H). 13C NMR (100 MHz, CD3OD): δ = 204.3, 159.3, 159.2, 159.1, 158.7,

158.5, 138.4, 138.4, 137.9, 129.8, 129.7, 129.7, 129.6, 129.6, 129.6, 129.4, 129.2, 129.1,

129.1, 129.0, 129.0, 128.9, 109.7, 101.8, 100.4, 85.8, 83.6, 79.9, 78.4, 75.9, 75.6, 75.3,

74.8, 71.7, 69.4, 68.5, 67.8, 67.8, 67.7, 67.7, 63.1, 61.8, 61.4, 54.3, 52.2, 43.6, 42.7, 34.0.


119

HRMS (ESI) calculated for C63H73N8O23 ([M+H]+): 1309.48, found: 1309.48. [α]D20

+26 (c 1.1, MeOH).

3-Azido-3’-epi -(Cbz)5-neomycin B (34)

Sodium borohydride (289 mg, 7.6 mmol, 5 eq) was

dissolved in dry MeOH (75 mL) and cooled to -10 °C

(internal temperature). 3-Azido-3’-keto penta-N-

benzyloxycarbonyl-neomycin B (2 g, 1.53 mmol, 1 eq)

was added dropwise over 25 min as a solution in dry

MeOH (75 mL) at -10 °C (internal temperature). The

mixture was stirred for 2 h at -10 °C and then quenched by addition of acidic ion

exchange resin (Amberlite® 120, H+-form, prewashed with MeOH) until the

mixture was slightly acidic. The ion exchange resin was filtered and washed with

MeOH. Concentration in vacuo and co-evaporation with MeOH (5 x 150 mL) gave

1.94 g (1.47 mmol, 97%) of 3-azido-3’-epi-(Cbz)5-neomycin B as an off-white foam,

that contained approximately 20% of 3-amino-3’-epi-penta-N-

benzyloxycarbonyl-neomycin B (from reduction of the azide) according to TLC

and NMR. An analytically pure sample was obtained after purification by

column chromatography (DCM/MeOH gradient 3-9%). 1H NMR (400 MHz,

CD3OD): δ = 7.48 – 7.16 (m, 25H), 5.34 (d (br), J = 2.5 Hz, 1H), 5.18 – 4.97 (m, 11H),

4.83 (overlaps with HOD, s, 1H), 4.18 (m (br), 1H), 4.04 (s (br), 1H), 4.00 – 3.87 (m,

5H), 3.83 (appears as s (br), 2H), 3.70 – 3.60 (m, 2H), 3.56 (dd, J = 12.6, 5.3 Hz, 1H),

3.52 – 3.41 (m, 5H), 3.40 – 3.33 (m, 4H), 3.31 (overlaps with MeOD, J = 3.3, 1.6 Hz,

1H), 2.14 (appears as d (br), J = 11.8 Hz, 1H), 1.41 – 1.26 (m, 1H).13C NMR (100

MHz, CD3OD): δ = 159.3, 159.3, 159.1, 158.7, 158.2, 138.4, 138.4, 138.3, 137.9, 129.8,

129.7, 129.7, 129.7, 129.6, 129.5, 129.2, 129.1, 129.1, 129.1, 129.0, 129.0, 128.9, 109.5,

100.5, 99.3, 86.0, 83.6, 79.5, 78.4, 75.5, 75.4, 74.7, 72.0, 71.7, 69.6, 69.3, 68.4, 68.0,

67.8, 67.8, 67.7, 63.1, 61.4, 54.2, 53.4, 52.2, 43.2, 42.7, 33.9. HRMS (ESI) calculated

for C63H74N8O23Na ([M+Na]+): 1333.48, found: 1333.48. [α]D20 +23 (c 1.4, MeOH).

Chapter 4

120

3-Amino-3’-epi-(Cbz)5- neomycin B (31)

To a reaction flask equipped with a reflux condenser, a

solution of 34 (50 mg, 0.038 mmol) in THF (0.7 ml) was

added and combined with 0.1 M NaOH aq (110 µL).

Then 1 M trimethylphosphine in THF (188 uL, 0.19

mmol, 5 eq) was added and the reaction mixture was

stirred at 50 °C for 4.5 h, whereupon TLC indicated

completion of the reaction (Rf = 0.56, EtOAC/MeOH). Ethyl acetate (5 mL) and

water (5 mL) were added and the organic layer was separated and dried with

MgSO4. The solution was concentrated in vacuo. The product was obtained as a

white solid (44.5 mg, 0.0346 mmol, crude yield: 91%) and was used without

further purification. An analytical pure sample was obtained after purification

by column chromatography (DCM/MeOH gradient 3-9%). 1H NMR (400 MHz,

CD3OD): δ = 7.41 – 7.20 (m, 25H), 5.22 – 4.93 (m, 12H), 4.68 (s, 1H), 4.06 – 3.91 (m,

4H), 3.90 – 3.76 (m, 4H), 3.69 – 3.55 (m, 3H), 3.52 (appears as s (br), 2H), 3.47 –

3.33 (m, 6H), 3.31 (overlaps with MeOD, m, 1H), 3.25 – 3.16 (m, 1H), 2.61 (m (br),

1H), 1.84 (d (br), J = 10.5 Hz, 1H), 1.22 – 1.07 (m, 1H). 13C NMR (100 MHz,

CD3OD): δ = 159.3, 159.2, 159.2, 158.8, 157.8, 138.6, 138.5, 138.4, 138.4, 137.6, 130.0,

129.9, 129.7, 129.7, 129.6, 129.4, 129.3, 129.2, 129.1, 129.1, 129.0, 129.0, 109.3, 100.8,

100.3, 85.4, 83.7, 77.7, 76.5, 75.0, 74.6, 71.9, 71.7, 69.8, 69.3, 68.6, 68.5, 67.9, 67.8,

67.7, 67.6, 62.9, 54.1, 53.7, 52.8, 52.1, 43.3, 42.8, 36.5. HRMS (ESI) calculated for

C63H77N6O23 ([M+H]+): 1285.50, found: 1285.51. [α]D20 +13 (c 0.85, MeOH)

3-N,N-dimethyl-3’-axial-hydroxyl-(Cbz)5-neomycin B (35)

To a solution of 31 (1.16 g, 0.9 mmol, 1 eq), in 12 mL of

acetonitrile, formaldehyde (0.85 mL) and NaBH3CN

(202 mg, 3.2 mmol, 3.6 eq) were added. The reaction

mixture was cooled to 0 °C, 0.1 mL of glacial acetic acid

was added, and the mixture was stirred for 1.5 h at rt.

Then a second portion of acetic acid was added

(0.1 mL), and stirring was continued for an additional 1.5 h. The reaction mixture

was diluted with ethyl acetate (500 mL) and washed with saturated aq. NaHCO3

(500 mL), brine (500 mL) and water (500 mL). The organic layer was separated

and dried over MgSO4, filtered and concentrated in vacuo. Purification with


121

column chromatography (EtOAc/MeOH, gradient 2-9%) yielded 878 mg of a

mixture of starting material and product, which was again subjected to column

chromatography (DCM/MeOH 20:1) to give 684 mg, (0.51 mmol, 56%) as a white

solid with a purity sufficient for the subsequent step. 1H NMR (400 MHz,

CD3OD, D2O): δ= 7.38 – 7.28 (m, 25H), 5.56 (s(br), 1H), 5.31 (s, 1H), 5.19 – 4.99 (m,

11H), 4.40 – 4.29 (m, 1H), 4.10 (s(br), 1H), 4.01 – 3.88 (m, 4H), 3.85 (s, 1H), 3.81 –

3.57 (m, 7H), 3.55 – 3.46 (m, 2H), 3.43 – 3.34 (m, 4H), 3.21 (dd, J = 14.0, 6.7 Hz, 1H),

2.56 (s (br), 1H), 2.20 (s, 6H), 1.99 – 1.91 (m, 1H), 1.09 – 0.95 (m, 1H). 13CNMR (126

MHz, CD3OD) δ =159.3, 158.9, 158.9, 158.6, 158.2, 138.4, 138.3, 138.3, 138.2, 138.0,

129.6, 129.5, 129.5, 129.5, 129.2, 129.1, 129.1, 129.0, 129.0, 117.3, 106.5, 99.8, 96.1,

83.2, 77.9, 75.9, 74.8, 72.3, 71.6, 69.7, 69.2, 68.7, 68.0, 67.7, 67.6, 67.5, 54.1. HRMS

(ESI) calculated for C65H79N6O23 ([M-H+]): 1311.53, found: 1311.52.

3-N,N-di-methyl-3’-epi-neomycin B (36)

Prior to use, all solvents were degassed by sonication for

1 h followed by bubbling with argon for 1 h. 35 (300 mg,

0.23 mmol) was dissolved in a freshly prepared mixture

of MeOH-water-AcOH (24 mL, 36 : 3 : 1) and 180 mg of

10 wt% Pd/C was added followed by degassing of the

mixture for 30 min. Subsequently, the reaction mixture

was stirred under a H2 atmosphere (ambient pressure) at room temp for 24 h. The

reaction mixture was filtered through celite, the filter cake was washed with

water, and the combined filtrate was concentrated in vacuo and freeze-dried. The

crude product was dissolved in water and purified by column chromatography,

(silica gel, eluted with a mixture of DCM/MeOH/25% ammonia, 2:2:1). The

solvent was evaporated, the product dissolved in water and filtered through

cotton and freeze-dried. The product was redissolved in water, filtered through

a syringe PTFE membrane filter (pore size 0.45 µm) and again freeze-dried. 36

was obtained as a white solid (56.3 mg, 16.5% pure acc. to qNMR, 0.014 mmol,

6.3%). 1H NMR (600 MHz, D2O): δ = 5.64 (d, J = 3.3 Hz, 1H), 5.42 (s, 1H), 4.96 (s,

1H), 4.49 (dd, J = 6.7, 5.1 Hz, 1H), 4.35 – 4.26 (m, 1H), 4.17 – 4.13 (m, 1H), 4.04 –

4.01 (m, 1H), 4.01 – 3.98 (m, 1H), 3.93 – 3.91 (m, 1H), 3.90 – 3.86 (m, 1H), 3.86 –

3.82 (m, 1H), 3.80 – 3.75 (m, 1H), 3.75 – 3.72 (m, 1H), 3.72 – 3.70 (m, J = 3.2 Hz,

1H), 3.70 – 3.68 (m, 1H), 3.65 (s (br), 1H), 3.59 (dd, J = 10.2, 2.8 Hz, 1H), 3.29 – 3.24

(m, J = 13.2, 6.1 Hz, 1H), 3.10 – 3.07 (m, 1H), 3.02 (s, 1H), 3.01 – 2.97 (m, 2H), 2.93

Chapter 4

122

– 2.90 (m, 1H), 2.82 – 2.74 (m, 1H), 2.73 – 2.65 (m, 1H), 2.36 (s, 6H), 2.03 (appears

as d, J = 12.6 Hz, 1H), 1.30 (appears as m, 1H). 13CNMR (400 MHz, D2O): δ = 110.1,

101.5, 98.9, 88.1, 83.7, 79.2, 78.1, 76.7, 76.1, 74.5, 73.4, 73.0, 72.1, 71.0, 70.1, 69.6,

64.5, 63.3, 55.1, 53.3, 53.1, 43.5, 43.4, 42.7, 26.9. HRMS (ESI) calculated for

C25H51N6O13 ( [M+H+]) 643.350, found 643.350.

(Boc)4-kanamycin A (14)

Kanamycin A (0.5 g, 1 mmol, 1 eq) was suspended in

water (2 mL). Triethylamine (0.58 mL, 0.42 g, 4 mmol,

4 eq) and di-tert-butyldicarbonate (1.8 g, 8 mmol,

8 eq) were added as solution in DMSO (12 mL). The

suspension completely dissolved within 3 h and was

stirred overnight. To the resulting white suspension, NH4OH (5 mL) was added

and the very finely divided solid was filtered over celite , washed with water and

EtOAc. The product was transferred by dissolution in MeOH. Concentration in

vacuo gave 511 mg (0.58 mmol, 56%) of pure (Boc)4-kanamycin A as a white solid.

1H NMR (400 MHz, DMSO-d6/drop of D2O): δ = 4.90 (appears as d (two

overlapping d), J = 3.7 Hz, 2H), 3.80 (appears as dt, J = 10.1, 3.3 Hz, 1H), 3.57

(appears as dt, J = 9.8, 3.8 Hz, 1H), 3.53 – 3.15 (m, 14H), 3.05 (appears as t, J = 9.3

Hz, 1H), 1.79 (appears as d, J = 12.5 Hz, 1H), 1.42 – 1.31 (m, 37H). 13C NMR

(100 MHz, DMSO-d6/drop of D2O): δ = 156.5, 156.4, 155.6, 155.1, 101.2, 97.9, 84.0,

80.4, 78.1, 77.6, 75.1, 73.0, 72.7, 72.1, 70.5, 70.3, 67.4, 60.3, 55.8, 50.1, 49.1, 41.4, 34.8,

28.5, 28.4, 28.3. HRMS (ESI) calculated for C38H68N4O19Na ([M+Na]+): 907.437,

found: 907.438. [α]D20 -73 (c 1.0, DMSO). Characterization matches literature.[97]

3’-Keto-(Boc)4-kanamycin A (18)

(Boc)4-kanamycin A (300 mg, 0.34 mmol, 1 eq) and

benzoquinone (110 mg, 1 mmol, 3 eq) were

dissolved in DMSO (4.5 mL) and

[(neocuproine)PdOAc]2(OTf)2 (7 mg, 6.8 μmol,

2 mol%) was added. The mixture was stirred for 1 h

and subsequently quenched by addition of 40 mL of water. The resulting solid

was filtered over celite and transferred by dissolution in MeOH. Evaporation


123

gave 700 mg of crude product (which remained wet, also after coevaporation

with toluene). The crude product was dissolved in toluene and EtOAc and was

precipitated by addition of pentane. The solid was filtered and dried in vacuo, to

give 3’-keto-(Boc)4-kanamycin A (236 mg) containing residual DMSO (3 mol%)

and hydroquinone (0.4 mol%) according to NMR (177 mg, 0.20 mmol, 59%). 1H

NMR (400 MHz, CD3OD): δ = 5.50 (d, J = 4.1 Hz, 1H), 5.06 (d, J = 3.0 Hz, 1H), 4.42

(d, J = 4.1 Hz, 1H), 4.13 (dd, J = 10.0, 1.1 Hz, 1H), 4.09 – 4.01 (m, 1H), 3.90 (appears

as d, J = 9.4 Hz, 1H), 3.79 (dd, J = 11.6, 2.3 Hz, 1H), 3.73 – 3.62 (m, 2H), 3.62 – 3.46

(m, 5H), 3.46 – 3.34 (m, 4H), 2.03 (appears as d (br), J = 13.5 Hz, 1H), 1.54 – 1.32

(m, 37H). 13C NMR (100 MHz, DMSO-d6/drop of D2O): δ = 205.4, 156.3, 155.8,

155.3, 154.8, 103.6, 97.9, 84.3, 80.4, 77.8, 77.2, 74.6, 72.9, 72.5, 72.2, 70.3, 67.4, 60.3,

55.9, 49.9, 45.0, 41.5, 34.6, 28.3, 28.2, 28.1. HRMS (ESI) calculated for

C38H66N4O19Na ([M+Na]+): 905.421, found: 905.422. [α]D20 -42 (c 0.6, DMSO)

3’-epi-(Boc)4-kanamycin A (23)

3’-Keto-(Boc)4-kanamycin A (150 mg, 0.17 mmol,

1 eq) was dissolved in MeOH (14 mL), cooled to 0 °C

and sodium borohydride (32 mg, 0.85 mmol, 5 eq)

was added. The mixture was allowed to warm up to

rt and was stirred overnight. The mixture was

neutralized by Amberlite® (120, H+-form, prewashed with MeOH), filtered and

co-evaporated with MeOH (4 x). Trituration with diethyl ether (10 mL) gave pure

3’-epi-(Boc)4kanamycin A (90 mg, 60%). 1H NMR (400 MHz, DMSO-d6/drop of

D2O): δ = 6.87 (s, 1H), 6.51 (m, 2H), 6.32 (s, 1H), 4.95 (d, J = 3.9 Hz, 1H), 4.89 (d, J

= 3.8 Hz, 1H), 3.83 – 3.73 (m, 3H), 3.56 – 3.47 (m, 4H), 3.40 – 3.14 (m, 10H), 1.83

(appears as d, J = 13.1 Hz, 1H), 1.37 (appears as s, 28H), 1.35 (s, 9H). 13C NMR

(100 MHz, DMSO-d6/drop of D2O): δ = 156.5, 156.3, 155.5, 155.1, 100.8, 98.0, 83.4,

80.9, 78.2, 78.1, 78.0, 77.5, 74.8, 73.1, 71.2, 70.3, 68.0, 67.5, 67.1, 65.7, 60.4, 55.8, 50.0,

49.2, 41.2, 34.6, 28.5, 28.4, 28.3, 28.3. HRMS (ESI) calculated for C38H68N4O19Na

([M+Na]+): 907.437, found: 907.437. [α]D20 -39 (c 2.4, DMSO)

Chapter 4

124

3’-epi-kanamycin A (27)

3’-epi-(Boc)4-kanamycin A (200 mg, 0.23 mmol, 1 eq)

and thiophenol (70 µL, 0.68 mmol, 3 eq) were

dissolved in dry DCM (2.8 mL) and trifluoroacetic

acid (2.8 mL). The mixture was stirred for 1 h and

subsequently coevaporated with toluene. The

remaining solid was dissolved in water and freeze-dried to give 230 mg of the

product as acetic acid salt. 161 mg of this salt was subjected to column

chromatography (2.7 g silica gel, eluent: the upper layer of a biphasic system of

CHCl3/MeOH/ammonia 25% 2 : 1 : 1, the column being pretreated with this

eluent) to give 155 mg of the product containing silica gel. The product was

dissolved in 1 mL of water and the solids were filtered off by a 0.45 µm PTFE-

syringe-filter. After freeze-drying, a 3.5 : 1 mixture of 3’-epi-kanamycin and

kanamycin A was isolated as their TFA-salt with a purity of 77% (113 mg,

93 µmol, 54%, still containing silica gel and/or ammonia salts) according to

qNMR analysis as a highly hydroscopic yellowish solid. 1H NMR (400 MHz,

D2O): δ = 5.49 (d, J = 4.0 Hz, 1H), 5.10 (d, J = 3.6 Hz, 1H), 4.16 (appears as s (br),

1H), 4.05 (m (br), 1H), 3.97 – 3.77 (m, 7H), 3.77 – 3.64 (m, 2H), 3.64 – 3.34 (m, 5H),

3.19 (dd, J = 13.4, 7.9 Hz, 1H), 2.52 (appears as dt (br), J = 12.4, 4.1 Hz, 1H), 1.90

(appears as q, J = 12.4 Hz, 1H). 13C NMR (100 MHz, D2O): δ = 163.6 (appears as

d, J = 35.4 Hz), 117.0 (q, J = 291.8 Hz), 101.3, 96.4, 84.6, 77.9, 73.6, 73.4, 70.7, 68.8,

68.0, 67.5, 66.1, 64.9, 60.5, 55.7, 50.6, 48.7, 41.0, 28.3. HRMS (ESI) calculated for

C18H37N4O11 ([M+H]+): 485.245, found: 485.245.

(Boc)4-amikacin (16)

Amikacin (0.5 g, 0.85 mmol, 1 eq), triethylamine

(0.48 mL, 3.4 mmol, 4 eq) and di-tert-

butyldicarbonate (1.49 g, 6.8 mmol, 8 eq) were

dissolved in DMSO (9.6 mL) and water

(1.6 mL). The suspension turned into a clear

solution within 1 h and was stirred overnight. Ammonia (10 mL) was added to

the clear solution, which resulted in the formation of a white precipitate. The

finely divided solid was filtered over celite and washed with water. After transfer

of the solid by dissolution in MeOH and concentration in vacuo, pure (Boc)4-


125

amikacin was obtained as a white solid (611 mg, 0.62 mmol, 73%). 1H NMR (400

MHz, CD3OD): δ = 5.13 (d, J = 2.6 Hz, 1H), 5.05 (d, J = 3.4 Hz, 1H), 4.06 – 4.01 (m,

1H), 3.99 (dd, J = 9.3, 3.6 Hz, 1H), 3.88 – 3.77 (m, 2H), 3.75 – 3.56 (m, 6H), 3.55 –

3.34 (m, 6H), 3.31 (m, overlaps with MeOD, 1H), 3.25 – 3.14 (m, 3H), 2.66 (s, 13H),

2.13 (appears as d, J = 12.8 Hz, 1H), 1.95 (appears as dtd, J = 11.4, 7.6, 3.6 Hz, 1H),

1.76 (appears as td, J = 14.6, 6.3 Hz, 1H), 1.63 – 1.51 (m, 1H), 1.45 (s, J = 7.7 Hz,

27H), 1.43 (s, 9H). 13C NMR (100 MHz, CD3OD): δ = 177.3, 159.5, 159.3, 158.7,

157.8, 102.8, 100.6, 85.2, 82.1, 80.7, 80.5, 80.3, 80.1, 77.7, 74.7, 74.2, 72.5, 71.9, 71.2,

69.9, 62.6, 57.4, 51.1, 51.0, 42.0, 38.4, 35.5, 35.2, 29.0, 29.0, 29.0, 28.9. HRMS (ESI)


MeOH). Characterization matches literature.[98]

(p-Tert-butyl-Cbz)4-amikacin (15)

(p-Tert-butyl)benzyl chloroformate

p-(Tert-butyl)benzyl alcohol (3.5 g, 21.3 mmol, 1 eq) was

dissolved in dry THF (32 mL) and phosgene solution (14.6 mL,

27.7 mmol, 1.3 eq, 20 wt% in toluene) was added dropwise. The

mixture was stirred for 3 h at r.t. and concentrated in vacuo to give 4.37 g of pure

p-(tert-butyl)benzyl chloroformate (4.37 g, 19.3 mmol, 90%) as a clear oil, which

was used immediately without purification.

Amikacin (1.48 g, 2.5 mmol, 1 eq) and

potassium carbonate (1.22 g, 8.8 mmol,

3.5 eq) were dissolved in water (23 mL)

and p-(tert-butyl)benzyl chloroformate

(4.37 g, 19.3 mmol, 7.7 eq) was added as a

solution in THF (23 mL). The mixture was stirred overnight, the THF was

removed in vacuo and the precipitated white solid was triturated with water,

filtered and coevaporated with toluene. The solid was nearly dissolved in 350 mL

of hot MeOH, filtered while hot. Water was carefully added to the clear solution

until a precipitate was observed. The suspension was heated to reflux to dissolve

all solids and the solution was allowed to cool down overnight (slowly).

Filtration of the resulting precipitate, washing with MeOH, ether and EtOAc and

drying in vacuo gave pure p-(tert-butyl-Cbz)4-amikacin (2.06 g). The mother

liquor was concentrated to give 4.2 g of crude product, which was purified over

Chapter 4

126

a silica-plug (500 mL each: DCM, 20 : 1 DCM/MeOH, 10 : 1 DCM/MeOH (eluted

the product), 1:1 DCM/MeOH (eluted the product)) yielding 1 g of still impure

product. Recrystallization as described above gave 0.3 g of pure p-(tert-butyl-

Cbz)4-amikacin (in total: 2.36 g, 1.75 mmol, 69%). 1H NMR (400 MHz, DMSO-

d6/CD3OD 1:1): δ = 7.35 – 7.12 (m, 16H), 5.13 – 4.77 (m, 10H), 3.94 – 3.81 (m, 2H),

3.77 – 3.67 (m, 1H), 3.67 – 3.55 (m, 4H), 3.55 – 3.41 (m, 4H), 3.40 – 3.19 (m, 6H),

3.15 – 3.09 (m, 2H), 3.04 (appears as t, J = 9.4 Hz, 1H), 2.00 – 1.81 (m, 2H), 1.64 (td,

J = 14.5, 6.5 Hz, 1H), 1.47 (appears as q, J = 12.2 Hz, 1H), 1.29 – 1.16 (m, 36H). 13C

NMR (100 MHz, DMSO-d6/ CD3OD): δ = 176.1, 158.7, 158.4, 157.9, 157.3, 151.6,

151.6, 135.1, 135.1, 135.0, 134.9, 128.7, 128.6, 128.5, 126.0, 126.0, 102.4, 99.6, 85.4,

81.3, 76.7, 74.2, 74.0, 73.6, 72.0, 71.7, 71.5, 70.5, 68.9, 66.7, 66.6, 66.5, 61.9, 57.5, 51.0,

50.2, 42.4, 38.3, 35.0, 34.6, 31.6. HRMS (ESI) calculated for C70H100N5O21 ([M+H]+):

1346.69, found: 1346.69. [α]D20 +41 (c 1.0, DMSO)

3’-Keto-(p-tert-butyl-Cbz)4-amikacin (19)

p-(Tert-butyl-Cbz)4-amikacin (0.5 g,

0.37 mmol, 1 eq) and benzoquinone

(120 mg, 1.1 mmol, 3 eq) were dissolved in

DMSO (2.5 mL),

[(neocuproine)PdOAc]2(OTf)2 (9.7 mg,

9.8 μmol, 2.5 mol%) was added and the mixture was stirred for 24 h. The reaction

was quenched by addition of water (15 mL), the resulting solid was filtered,

washed with water (3 x 5 mL) and transferred by suspending in MeOH.

Evaporation gave 497 mg of crude product. 360 mg of the crude product was

triturated in THF (18 mL) overnight. The solid was centrifuged and the

supernatant removed. The solid was transferred by suspending in MeOH, and

evaporation gave 200 mg of still impure material. Recrystallization from dioxane

(4 mL, slowly cooled down in a hot sand bath over two days), decantation,

washing with dioxane (3 x 1 mL) and drying in vacuo gave nearly pure 3’-keto-

(tert-butyl-Cbz)-amikacin (100 mg, 74 μmol, 28%). 1H NMR (400 MHz, DMSO-

d6/ CD3OD 1:1): δ = 7.29 (appears as t, J = 6.9 Hz, 8H), 7.19 (appears as dd, J = 15.0,

8.0 Hz, 8H), 5.37 (d, J = 4.4 Hz, 1H, 1’), 5.08 – 4.78 (m, 9H, CH2Ph, 1”), 4.32 (d, J =

3.6 Hz, 1H, 2’), 4.01 (d, J = 9.9 Hz, 1H, 4’), 3.87 (appears as dd, J = 9.0, 3.6 Hz, 2H,

5”, 1’’’), 3.81 – 3.73 (m, 1H, 5’), 3.73 – 3.55 (m, 4H, 3, 4, 3”, 6”), 3.55 – 3.46 (m, 2H,

6, 6”), 3.46 – 3.36 (m, 3H, 1, 5, 6’), 3.32 (appears as dd, J = 10.9, 3.1 Hz, 2H, 6’, 2”),


127

3.27 – 3.19 (m, 1H, 4”), 3.15 – 3.10 (m, 2H, 3’’’), 2.00 – 1.80 (m, 2H, 2, 2’’’), 1.64 (dt,

J = 21.2, 7.1 Hz, 1H, 2’’’), 1.47 (appears as dd, J = 23.0, 11.3 Hz, 1H, 2), 1.26 – 1.12

(m, 36H). 13C NMR (100 MHz, DMSO-d6/ CD3OD): δ = 206.2 (3’), 176.1, 158.7,

158.2, 157.9, 157.2, 151.6, 151.6, 135.1, 135.1, 135.1, 135.0, 134.9, 128.7, 128.6, 128.5,

126.0, 126.0, 104.6 (1’), 99.7 (1”), 85.3 (5), 81.3 (4), 76.4 (6), 76.0 (2’), 74.9 (5’), 74.3

(1’’’), 73.6 (4’), 71.5 (2”), 70.5 (5”), 69.0 (4”), 66.7, 66.6, 66.5, 62.0 (6”), 57.4 (3”), 50.8

(1), 50.1 (3), 42.6 (6’), 38.3 (3’’’), 35.1 (2’’’), 34.6 (2) 31.6. HRMS (ESI) calculated for

C70H98N5O21 ([M+H]+): 1344.67, found: 1344.67. [α]D20 +33 (c 0.3, DMSO)

3’-epi-(p-tert-butyl-Cbz)4-amikacin (24)

3’-keto-(tert-butyl-Cbz)4-amikacin

(174 mg, 129 μmol, 1 eq) was dissolved in

dioxane (14 mL) and MeOH (3.6 mL),

which yielded a slightly cloudy solution.

The mixture was cooled to 0 °C and

sodium borohydride (15 mg, 388 μmol, 3 eq) was added. The mixture was

allowed to warm up to r.t. and stirred in total for 4 h. The clear solution was

neutralized by addition of Amberlite® (120 H+-form, prewashed with MeOH),

filtered (the filter was washed with MeOH) and coevaporated with MeOH to give

3’-epi-(tert-butyl-Cbz)4-amikacin (175 mg, 129 μmol, quant.) as an off-white solid.

Identification of the epi-configuration could be done by measurement of the

couplings constant at the 3’-position after final deprotection (26). Here,

comparison of NMR-spectra of 15 and 24 showed significant differences. 1H

NMR (400 MHz, DMSO-d6/ CD3OD 1:1): δ = 7.37 – 7.26 (m, 8H), 7.26 – 7.15 (m,

8H), 5.02 (appears as d (br), J = 14.5 Hz, 2H), 4.98 – 4.89 (m, 7H, CH2Ph, 1’, 1”),

4.85 (d (br), J = 12.1 Hz, 1H, CH2Ph), 3.86 (appears as dd (br), J = 8.7, 3.6 Hz, 2H,

5”, 1’’’), 3.82 (appears as s (br), 1H, 3’), 3.76 (appears as dd (br), J = 9.5, 4.6 Hz, 1H,

5’), 3.68 (m (br), 1H, 3), 3.66 – 3.54 (m, 4H, 4, 6, 3”, 6”), 3.54 – 3.45 (m, 3H, 1, 2’,

6”), 3.42 – 3.29 (m, 3H, 5, 6’, 2”), 3.23 (appears as t, J = 9.9 Hz, 3H, 4’, 6’, 4”), 3.11

(overlaps with CD3OD, m, 2H, 3’’’), 2.00 – 1.90 (m, 1H, 2), 1.90 – 1.81 (m, 1H, 2’’’),

1.63 (dt (br), J = 14.4, 6.2 Hz, 1H, 2’’’), 1.48 (dd, J = 25.0, 12.9 Hz, 1H, 2), 1.22

(appears as d, J = 2.9 Hz, 36H). 13C NMR (100 MHz, DMSO-d6/ CD3OD 1:1): δ =

175.6, 158.3, 158.0, 157.5, 157.1, 151.3, 151.3, 135.0, 135.0, 134.9, 134.8, 128.5, 128.4,

128.4, 125.8, 125.8, 101.7 (1’), 99.4 (1”), 84.5 (5), 81.5 (4), 76.3 (6), 74.1 (5”), 72.1 (3’),

71.2 (2”), 70.3 (1’’’), 69.1 (2’), 68.7 (4’, 4”), 68.2, 67.2 (5’), 66.5, 66.4, 66.3, 66.2, 61.7

Chapter 4

128

(6”), 57.2 (3”), 50.7 (1), 49.9 (3), 42.3 (6’), 38.2 (3’’’), 34.9 (2’’’), 34.3 (2), 31.5. HRMS

(ESI) calculated for C70H100N5O21 ([M+H]+): 1346.69, found: 1346.69. [α]D20 +14 (c

0.78, DMSO/MeOH 1:1)

3’-epi-amikacin (26)

3’-epi-(tert-butyl-Cbz)4-amikacin (121 mg,

90 μmol, 1 eq) and palladium hydroxide on

carbon (27.5 mg, 20 wt% Pd) were suspended in

a freshly degassed (freeze-pump-thaw)

MeOH/water/AcOH-mixture (12.2 mL, 36 : 3 : 1).

The mixture was hydrogenated for 5 days at 1 atm. of H2. The mixture was

filtered over celite and the filter was washed with water and MeOH. Evaporation,

re-dissolving in water and freeze-drying gave 83 mg of crude product.

Purification was done by column chromathography (3 g silica, eluent:

DCM/MeOH/Ammonia 25%, 2:2:1, 6 CV to wash off traces of mono-protected

product, then the upper layer of the biphasic system of DCM/MeOH/Ammonia

25% 2:1:1, 7 CV eluted the product) Product containing fractions were evaporated

and suspended in 1 mL water and filtered through a 0.45 µm PTFE-syringe-filter

followed by freeze-drying. This gave 23% pure, according to qNMR, 3’-epi-

amikacin as formate salt (87 mg, 26 µmol, 29%, still containing silica gel and/or

formate/ammonia salts) analysis as a white solid. The epi-configuration could be

assigned by measurement of the couplings constant at the 3’-position. The 3’-

position was assigned by HMQC- and COSY-NMR to the triplet at 4.14 ppm. It

shows a couplings constant of 3.1 Hz, which points to an axial hydroxyl group at

3’. 1H NMR (400 MHz, D2O/drop CD3OD): δ = 5.48 (d, J = 4.1 Hz, 1H, 1’), 5.14 (d,

J = 3.7 Hz, 1H, 1’’), 4.26 (dd, J = 9.2, 3.6 Hz, 1H, 1’’’), 4.14 (appears as t, J = 3.1 Hz,

1H, 3’), 4.12 – 4.00 (m, 3H, 5’, 1, 5’’), 3.90 – 3.79 (m, 4H, 2’, 4, 5, 6), 3.77 (d, J = 2.9

Hz, 2H, 6’’), 3.74 (dd, J = 11.1, 3.7 Hz, 1H, 2’’), 3.67 (t, J = 10.1 Hz, 1H, 4’’), 3.59 (dd,

J = 9.9, 2.9 Hz, 1H, 4’), 3.55 – 3.46 (m, 1H, 3), 3.43 (dd, J = 13.4, 3.1 Hz, 1H, 6’), 3.37

(appears as t, J = 10.7 Hz, 1H, 3’’), 3.19 (overlaps, appears as d, J = 8.1 Hz, 1H, 6’),

3.15 (t, J = 7.1 Hz, 2H, 3’’’), 2.24 – 2.08 (m, 2H, 2, 2’’’ ), 2.00 – 1.89 (m, 1H, 2’’’), 1.81

(appears as dd, J = 25.2, 12.6 Hz, 1H, 2). 13C NMR (100 MHz, D2O/drop CD3OD):

δ = 176.5, 99.1, 96.4, 81.0, 78.6, 74.3, 73.0, 71.2, 70.7, 69.1, 68.5, 68.0, 66.6, 65.4, 60.7,

56.4, 50.0, 49.4, 41.5, 38.1, 31.9, 31.1. HRMS (ESI) calculated for C22H44N5O13

([M+H]+): 586.293, found: 586.292.


129

(Cbz)5-paromomycin (11)

Paromomycin sulfate salt (1.56 g, 2.2 mmol, 1 eq) and

potassium carbonate (1.06 g, 7.7 mmol, 3.5 eq) were

dissolved in water (20 mL). A solution of

N-(benzyloxycarbonyloxy)succinimide in THF (20 mL)

was added. The mixture was stirred overnight and

quenched by addition of 3-dimethylaminopropylamine

(2.4 mL). EtOAc (4 mL) and water (5 mL) were added and the organic layer was

extracted with EtOAc (2 x 4 mL). The organic layer was first washed with a 1:1

mixture of citric acid (20%) and brine (10 mL) and then with a 1:1 mixture of sat.

aq. NaHCO3 solution and water (10 mL). The organic layer was dried and

concentrated in vacuo to give 2.9 g of crude product. Purification by automated

column chromatography (80 g silica column, DCM/MeOH gradient, 3% MeOH

for 5.5 min, 3% to 6% in 16.5 min, 6% to 7% in 4.5 min, 7% for 12 min, 7% to 8%

in 4 min, 7% MeOH eluted the product) yielded pure Cbz-protected

paromomycin (1.33 g, 1.0 mmol, 47%) as a white foam. 1H NMR (400 MHz,

CD3OD): δ = 7.41 – 7.23 (m, 20H), 7.06 (s, 1H, NH), 5.27 – 4.95 (m, 12H), 4.82

(appears as s (br), 1H), 4.09 – 3.95 (m, 2H), 3.96 – 3.87 (m, 3H), 3.82 (appears as d

(br), J = 9.7 Hz, 2H), 3.78 – 3.69 (m, 3H), 3.68 (appears as d (br), J = 5.0 Hz, 1H),

3.65 – 3.54 (m, 4H), 3.54 – 3.44 (m, 3H), 3.41 – 3.33 (m, 5H), 1.97 (appears as dd

(br), J = 9.2, 3.4 Hz, 1H), 1.41 (appears as dd (br), J = 24.6, 12.1 Hz, 1H). 13C NMR

(100 MHz, CD3OD): δ = 159.4, 159.1, 158.9, 158.7, 158.6, 138.4, 138.4, 138.3, 138.2,

129.8, 129.7, 129.7, 129.6, 129.5, 129.4, 129.2, 129.2, 129.1, 129.1, 129.0, 129.0, 128.8,

110.7, 100.6, 100.3, 87.4, 83.9, 81.3, 78.2, 76.0, 75.6, 75.0, 74.6, 72.8, 71.8, 71.7, 69.3,

68.0, 67.9, 67.8, 67.6, 63.3, 62.9, 57.9, 54.2, 52.8, 52.5, 42.7, 35.6. HRMS (ESI)

calculated for C63H75N5O24Na ([M+Na]+): 1308.47, found: 1308.47. [α]D20 +27 (c 1.3,

MeOH)

ACKNOWLEDGEMENTS Highly acknowledged is the cooperation with Dr. Andreas Bastian, Eliza

Warszawik and Prof. Dr. Andreas Herrmann on the synthesis of the

double modified aminoglycoside, who performed the synthesis of 29

from 4 and 33 from 31 (Scheme 18).

Chapter 4

130

LITERATURE [1] A. Schatz, E. Bugle, S. A. Waksman, Exp. Biol. Med. 1944, 55, 66–69.

[2] B. Singh, D. A. Mitchison, BMJ 1954, 1, 130–132.

[3] M. L. Avent, B. A. Rogers, A. C. Cheng, D. L. Paterson, Intern. Med. J. 2011, 41,

441–449.

[4] P. Poulikakos, M. E. Falagas, Expert Opin. Pharmacother. 2013, 14, 1585–1597.

[5] D. Moazed, H. F. Noller, Nature 1987, 327, 389–394.

[6] B. François, R. J. M. Russell, J. B. Murray, F. Aboul-ela, B. Masquida, Q. Vicens, E.

Westhof, Nucleic Acids Res. 2005, 33, 5677–5690.

[7] H. Wang, Y. Tor, J. Am. Chem. Soc. 1997, 119, 8734–8735.

[8] M. B. Feldman, D. S. Terry, R. B. Altman, S. C. Blanchard, Nat. Chem. Biol. 2010, 6,

54–62.

[9] H. Umezawa, M. Okanishi, S. Kondo, K. Hamana, R. Utahara, K. Maeda, S.

Mitsuhashi, Science 1967, 157, 1559–1561.

[10] O. Doi, M. Miyamoto, N. Tanaka, H. Umezawa, Appl. Microbiol. 1968, 16, 1282–

1284.

[11] D. P. Arya, Aminoglycoside Antibiotics: From Chemical Biology to Drug Discovery,

WILEY-VCH Verlag, New Jersey, 2007.

[12] K. J. Shaw, P. N. Rather, R. S. Hare, G. H. Miller, Microbiol. Rev. 1993, 57, 138–163.

[13] K. Y. Lee, J. D. Hopkins, M. Syvanen, Mol. Microbiol. 1991, 5, 2039–2046.

[14] C. J. Wrighton, P. Strike, Plasmid 1987, 17, 37–45.

[15] T. S. Frana, S. A. Carlson, R. W. Griffith, Appl. Environ. Microbiol. 2001, 67, 445–

448.

[16] T. Murata, M. Ohnishi, T. Ara, J. Kaneko, C.-G. Han, Y. F. Li, K. Takashima, H.

Nojima, K. Nakayama, A. Kaji, Y. Kamio, T. Miki, H. Mori, E. Ohtsubo, Y.

Terawaki, T. Hayashi, J. Bacteriol. 2002, 184, 3194–3202.

[17] D. Böltner, C. MacMahon, J. T. Pembroke, P. Strike, A. M. Osborn, J. Bacteriol. 2002,

184, 5158–5169.

[18] B. Hochhut, Y. Lotfi, D. Mazel, S. M. Faruque, R. Woodgate, M. K. Waldor,

Antimicrob. Agents Chemother. 2001, 45, 2991–3000.

[19] J. M. Musser, Clin. Microbiol. Rev. 1995, 8, 496–514.

[20] M. Ouellette, G. Gerbaud, T. Lambert, P. Courvalin, Antimicrob. Agents Chemother.

1987, 31, 1021–1026.

[21] A. Tauch, S. Krieft, J. Kalinowski, A. Pühler, MGG - Mol. Gen. Genet. 2000, 263, 1–

11.

[22] H. Hächler, P. Santanam, F. H. Kayser, Antimicrob. Agents Chemother. 1996, 40,

1254–1256.

[23] C. K. Stover, X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey,

F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry,

E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R.

Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T.

Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, M. V Olson, Nature 2000,

406, 959–964.


131

[24] M. L. Riccio, L. Pallecchi, R. Fontana, G. M. Rossolini, Antimicrob. Agents

Chemother. 2001, 45, 1249–1253.

[25] G. S. Gray, W. M. Fitch, Mol. Biol. Evol. 1983, 1, 57–66.

[26] K. Ubukata, N. Yamashita, A. Gotoh, M. Konno, Antimicrob. Agents Chemother.

1984, 25, 754–759.

[27] D. E. Taylor, W. Yan, L.-K. Ng, E. K. Manavathu, P. Courvalin, Ann. l’Institut

Pasteur / Microbiol. 1988, 139, 665–676.

[28] P. Trieu-Cuot, P. Courvalin, Gene 1983, 23, 331–341.

[29] E. E. Udo, A. A. Dashti, Int. J. Antimicrob. Agents 2000, 13, 273–279.

[30] B. Papadopoulou, P. Courvalin, Antimicrob. Agents Chemother. 1988, 32, 945–948.

[31] J.-W. Ahn, K.-J. Kim, J. Microbiol. Biotechnol. 2013, 23, 1529–1535.

[32] P. Martin, E. Jullien, P. Courvalin, Mol. Microbiol. 1988, 2, 615–625.

[33] T. Lambert, G. Gerbaud, P. Bouvet, J. F. Vieu, P. Courvalin, Antimicrob. Agents

Chemother. 1990, 34, 1244–1248.

[34] C. Torres, Int. J. Antimicrob. Agents 2000, 15, 257–263.

[35] F. C. Tenover, T. Gilbert, P. O’Hara, Plasmid 1989, 22, 52–58.

[36] F. C. Tenover, P. M. Elvrum, Antimicrob. Agents Chemother. 1988, 32, 1170–1173.

[37] T. K. Stage, K. J. Hertel, O. C. Uhlenbeck, RNA 1995, 1, 95–101.

[38] J. Rogers, A. H. Chang, U. von Ahsen, R. Schroeder, J. Davies, J. Mol. Biol. 1996,

259, 916–925.

[39] M. L. Zapp, S. Stern, M. R. Green, Cell 1993, 74, 969–978.

[40] S. H. L. Verhelst, P. J. A. Michiels, G. A. van der Marel, C. A. A. van Boeckel, J. H.

van Boom, Chembiochem 2004, 5, 937–942.

[41] J. Haddad, L. P. Kotra, B. Llano-Sotelo, C. Kim, E. F. Azucena, M. Liu, S. B.

Vakulenko, C. S. Chow, S. Mobashery, J. Am. Chem. Soc. 2002, 124, 3229–3237.

[42] C.-H. Liang, A. Romero, D. Rabuka, P. W. M. Sgarbi, K. A. Marby, J. Duffield, S.

Yao, M. L. Cheng, Y. Ichikawa, P. Sears, C. Hu, S.-B. Hwang, Y.-K. Shue, S. J.

Sucheck, Bioorg. Med. Chem. Lett. 2005, 15, 2123–2128.

[43] Y. Takahashi, T. Tsuchiya, S. Umezawa, H. Umezawa, Carbohydr. Res. 1991, 210,

221–232.

[44] S. B. A. M. W. van den Broek, B. W. T. Gruijters, F. P. J. T. Rutjes, F. L. van Delft,

R. H. Blaauw, J. Org. Chem. 2007, 72, 3577–3580.

[45] S. Hanessian, O. M. Saavedra, M. A. Vilchis-Reyes, A. M. Llaguno-Rueda,

Medchemcomm 2014, 5, 1166.

[46] W. A. Greenberg, E. S. Priestley, P. S. Sears, P. B. Alper, C. Rosenbohm, M.

Hendrix, S.-C. Hung, C.-H. Wong, J. Am. Chem. Soc. 1999, 121, 6527–6541.

[47] G. Kavadias, P. Dextraze, R. Massé, B. Belleau, Can. J. Chem. 1978, 56, 2086–2092.

[48] M. Mizuno, H. Matsumoto, K. Goto, K. Hamasaki, Tetrahedron Lett. 2006, 47, 8831–

8835.

[49] T. Tsuchiya, Y. Takahashi, Y. Kobayashi, S. Umezawa, H. Umezawa, J. Antibiot.

(Tokyo). 1985, 38, 1287–1290.

[50] M. Chittapragada, S. Roberts, Y. W. Ham, Perspect. Medicin. Chem. 2009, 3, 21–37.

[51] M. W. Aslam, J. Chem. Soc. Pak. 2013, 35, 1571–1581.

[52] J. L. Houghton, K. D. Green, W. Chen, S. Garneau-Tsodikova, Chembiochem 2010,

11, 880–902.

Chapter 4

132

[53] J. Zhou, G. Wang, L.-H. Zhang, X.-S. Ye, Med. Res. Rev. 2007, 27, 279–316.

[54] S. H. Lee, C. S. Cheong, Tetrahedron 2001, 57, 4801–4815.

[55] A. Ricci, Ed. , Amino Group Chemistry, Wiley-VCH Verlag GmbH & Co. KGaA,

Weinheim, Germany, 2007.

[56] A. A. Bastian, E. M. Warszawik, P. Panduru, C. Arenz, A. Herrmann, Chem. Eur.

J. 2013, 19, 9151–9154.

[57] P. T. Nyffeler, C.-H. Liang, K. M. Koeller, C.-H. Wong, J. Am. Chem. Soc. 2002, 124,

10773–10778.

[58] J. Li, H.-N. Chen, H. Chang, J. Wang, C.-W. T. Chang, Org. Lett. 2005, 7, 3061–3064.

[59] R. Rai, H.-N. Chen, P. G. Czyryca, J. Li, C.-W. T. Chang, Org. Lett. 2006, 8, 887–889.

[60] K. Michael, H. Wang, Y. Tor, Bioorg. Med. Chem. 1999, 7, 1361–1371.

[61] S. R. Kirk, N. W. Luedtke, Y. Tor, J. Am. Chem. Soc. 2000, 122, 980–981.

[62] N. W. Luedtke, Q. Liu, Y. Tor, Biochemistry 2003, 42, 11391–11403.

[63] M. Fridman, V. Belakhov, S. Yaron, T. Baasov, Org. Lett. 2003, 5, 3575–3578.

[64] R. J. Fair, M. E. Hensler, W. Thienphrapa, Q. N. Dam, V. Nizet, Y. Tor,

ChemMedChem 2012, 7, 1237–1244.

[65] J. Alguacil, S. Defaus, A. Claudio, A. Trapote, M. Masides, J. Robles, Eur. J. Org.

Chem. 2010, 3102–3109.

[66] S. Hanessian, J. Szychowski, S. S. Adhikari, G. Vasquez, P. Kandasamy, E. E.

Swayze, M. T. Migawa, R. Ranken, B. François, J. Wirmer-Bartoschek, J. Kondo, E.

Westhof, J. Med. Chem. 2007, 50, 2352–2369.

[67] S. Quader, S. E. Boyd, I. D. Jenkins, T. A. Houston, J. Org. Chem. 2007, 72, 1962–

1979.

[68] A. A. Bastian, A. Marcozzi, A. Herrmann, Nat. Chem. 2012, 4, 789–793.

[69] S. Umezawa, T. Tsuchiya, R. Muto, Y. Nishimura, H. Umezwaw, J. Antibiot.

(Tokyo). 1971, 24, 274–275.

[70] T. Jikihara, T. Tsuchiya, H. Umezawa, S. Umezawa, Carbohydr. Res. 1981, 89, 91–

101.

[71] H. Umezawa, S. Umezawa, T. Tsuchiya, Y. Okazaki, J. Antibiot. (Tokyo). 1971, 24,

485–487.

[72] T. Shitara, Y. Kobayashi, T. Tsuchiya, S. Umezawa, Carbohydr. Res. 1992, 232, 273–

290.

[73] J. Haddad, S. Vakulenko, S. Mobashery, J. Am. Chem. Soc. 1999, 121, 11922–11923.

[74] J. B. Aggen, E. S. Armstrong, A. A. Goldblum, P. Dozzo, M. S. Linsell, M. J. Gliedt,

D. J. Hildebrandt, L. A. Feeney, A. Kubo, R. D. Matias, S. Lopez, M. Gomez, K. B.

Wlasichuk, R. Diokno, G. H. Miller, H. E. Moser, Antimicrob. Agents Chemother.

2010, 54, 4636–4642.

[75] G. G. Zhanel, C. D. Lawson, S. Zelenitsky, B. Findlay, F. Schweizer, H. Adam, A.

Walkty, E. Rubinstein, A. S. Gin, D. J. Hoban, J. P. Lynch, J. A. Karlowsky, Expert

Rev. Anti. Infect. Ther. 2012, 10, 459–473.

[76] M. Jäger, M. Hartmann, J. G. de Vries, A. J. Minnaard, Angew. Chem. Int. Ed. Engl.

2013, 52, 7809–7812.

[77] W. Li, K. Koike, Y. Asada, T. Yoshikawa, T. Nikaido, Carbohydr. Res. 2003, 338,

729–731.


133

[78] J. Zhang, F.-I. Chiang, L. Wu, P. G. Czyryca, D. Li, C.-W. T. Chang, J. Med. Chem.

2008, 51, 7563–7573.

[79] G. F. Pauli, B. U. Jaki, D. C. Lankin, J. Nat. Prod. 2007, 70, 589–595.

[80] G. F. Pauli, B. U. Jaki, D. C. Lankin, J. Nat. Prod. 2005, 68, 133–149.

[81] G. F. Pauli, S.-N. Chen, C. Simmler, D. C. Lankin, T. Gödecke, B. U. Jaki, J. B.

Friesen, J. B. McAlpine, J. G. Napolitano, J. Med. Chem. 2014, 57, 9220–9231.

[82] Y. Kobayashi, T. Tsuchiya, T. Ohgi, N. Taneichi, Y. Koyama, Carbohydr. Res. 1992,

230, 89–105.

[83] H. S. Prakash rao, P. Siva, Synth. Commun. 1994, 24, 549–555.

[84] F. Fringuelli, F. Pizzo, L. Vaccaro, Synthesis (Stuttg). 2000, 2000, 646–650.

[85] V. Nair, A. K. Sinhababu, J. Org. Chem. 1978, 43, 5013–5017.

[86] J. Anders, R. Buczys, E. Lampe, M. Walter, E. Yaacoub, K. Buchholz, Carbohydr.

Res. 2006, 341, 322–331.

[87] M. Pietsch, M. Walter, K. Buchholz, Carbohydr. Res. 1994, 254, 183–194.

[88] F. Agnelli, S. J. Sucheck, K. A. Marby, D. Rabuka, S.-L. Yao, P. S. Sears, F.-S. Liang,

C.-H. Wong, Angew. Chem. Int. Ed. Engl. 2004, 43, 1562–1566.

[89] J. B.-H. Tok, L. J. Dunn, R. C. Des Jean, Bioorg. Med. Chem. Lett. 2001, 11, 1127–1131.

[90] K. Hamasaki, M.-C. Woo, A. Ueno, Tetrahedron Lett. 2000, 41, 8327–8332.

[91] T. Shitara, E. Umemura, T. Tsuchiya, T. Matsuno, Carbohydr. Res. 1995, 276, 75–89.

[92] L. Chen, M. Hainrichson, D. Bourdetsky, A. Mor, S. Yaron, T. Baasov, Bioorg. Med.

Chem. 2008, 16, 8940–8951.

[93] N. R. Conley, L. A. Labios, D. M. Pearson, C. C. L. McCrory, R. M. Waymouth,

Organometallics 2007, 26, 5447–5453.

[94] P. Dozzo, A. A. Goldblum, J. B. Aggen, M. S. Linsell, Antibacterial Aminoglycoside

Analogs, 2011, U.S. Patent WO 2011/044502.

[95] J. L. Asensio, A. Hidalgo, A. Bastida, M. Torrado, F. Corzana, J. L. Chiara, E.

García-Junceda, J. Cañada, J. Jiménez-Barbero, J. Am. Chem. Soc. 2005, 127, 8278–

8279.

[96] V. Kumar, W. A. Remers, J. Org. Chem. 1981, 46, 4298–4300.

[97] M. D. Disney, O. J. Barrett, Biochemistry 2007, 46, 11223–11230.

[98] S. Bera, G. G. Zhanel, F. Schweizer, Hydrophobically Enhanced Aminoglycosides,

2011, U.S. Patent US2011178037 (A1).

Chapter 4

134

Documents

University of Groningen Selective oxidation of glycosides ... · aminoglycosides from fermentation and semisynthetic compounds. A common motif shared by all aminoglycosides is the