recent developments in the isolation biological function biosynthesis and synthesis of phenazine natural products

Review articleRecent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products a Institute of Organic Chemistry, Graz University of Te

Review article

Recent developments in the isolation, biological function, biosynthesis,

and synthesis of phenazine natural products

a Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria

b

Institute of Chemistry-Analytical Chemistry, University of Graz, Universitaetsplatz 1, 8010 Graz, Austria

c

Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstr 7, 38124 Braunschweig, Germany

d

Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr 7, 38106 Braunschweig, Germany

a r t i c l e i n f o

Article history:

Received 11 November 2016

Revised 29 December 2016

Accepted 4 January 2017

Available online xxxx

Keywords:

Antibiotics

Anticancer

Biofilm

Biosynthesis

Natural product

Phenazine

a b s t r a c t Phenazines are natural products which are produced by bacteria or by archaeal Methanosarcina species The tricyclic ring system enables redox processes, which producing organisms use for oxidation of NADH

or for the generation of reactive oxygen species (ROS), giving them advantages over other microorgan-isms In this review we summarize the progress in the field since 2005 regarding the isolation of new phe-nazine natural products, new insights in their biological function, and particularly the now almost completely understood biosynthesis The review is complemented by a description of new synthetic methods and total syntheses of phenazines

Ó 2017 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Contents

1 Introduction 00

2 Biological activity 00

2.1 Modes of action 00

2.2 Phenazines and glutathione (GSH) 00

2.3 Anticancer activity 00

2.4 Phenazines as antibiotics and their role to host defence 00

2.5 Phenazines and biofilms 00

2.6 Isolation of new phenazines 00

2.7 Synthetic phenazines with exceptional biological activity 00

3 Biosynthesis 00

3.1 Early studies 00

3.2 Anthranilate synthase genes 00

3.3 Current understanding 00

4 Synthesis 00

4.1 Classic methods 00

4.2 Cu- and Pd-catalyzed coupling reactions 00

4.3 Transition metal-catalyzed C-H functionalization 00

4.4 One-pot procedures and multicomponent reactions (MCRs) 00

4.5 Miscellaneous 00

4.6 Summary of modern strategies 00

4.7 Biomimetic synthesis of phenazine-1,6-dicarboxylic acid (PDC) 00

4.8 Total syntheses of streptophenazine A (51) 00

http://dx.doi.org/10.1016/j.bmc.2017.01.002

0968-0896/Ó 2017 The Authors Published by Elsevier Ltd.

This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

⇑ Corresponding author.

E-mail address: breinbauer@tugraz.at (R Breinbauer).

Contents lists available atScienceDirect Bioorganic & Medicinal Chemistry

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / b m c

4.9 Total syntheses of dermacozines A (61), B (64) and C (65) 00

5 Conclusion 00

Acknowledgments 00

References and notes 00

1 Introduction

Phenazines are a large class of redox-active secondary

metabo-lites produced by many Gram-positive (e.g Streptomyces) and

Gram-negative bacteria (e.g Pseudomonas), or by archaeal

Methanosarcina species (Fig 1).1The core structure of phenazines

is a pyrazine ring (1,4-diazabenzene) exhibiting two annulated

benzenes Their early discovery in the mid-19th century has been

facilitated by the fact that phenazines are intensively colored

compounds.2In 1859 Fordos described the extraction of a blue

pig-ment, which is responsible for the coloration of the ‘‘blue pus”,

observed in patients suffering from severe purulent wounds, and

named the blue pigment ‘‘pyocyanine” (nowadays more common

as pyocyanin (PYO)) from the Greek words for ‘‘pus” and ”blue”.3,4

Since then >180 phenazine natural products have been described

in the literature.5It has been shown that some phenazines exhibit

antibiotic, antifungal, insecticidal, antitumor, cancer

chemopre-ventive, antiplasmodial, antimalarial, and antiparasitic activities

Phenazines are produced at late growth stages at high cell

densi-ties and it has been demonstrated that phenazine-producing

organisms exhibit a longer lifespan in the natural environment

than their non phenazine-producing counterparts.1In addition, a

mouse model with PYO-deficient strains of P aeruginosa showed

that PYO is important for their hosts in lung infections.6Several

modes of actions of phenazines have been identified, which include

the reduction of molecular oxygen to reactive oxygen species

(ROS),7 the facilitation of energy generation,8–10 involvement in

iron homeostasis via Fe(III) reduction,11,12participation as signal

molecules via the activation of the Fe-containing transcription

fac-tor SoxR,13–15DNAp–pinteraction and intercalation,16and biofilm

morphogenesis17through influencing the intracellular redox state

In this review we aim to describe the current state of phenazine natural product chemistry with a special focus on the literature since 2005, when the last seminal reviews have been published

by Nielsen1and Beifuss.18

2 Biological activity This section highlights some newly isolated as well as synthetic phenazines, exhibiting exceptional structures or showing promis-ing anticancer, antibiotic or biofilm-eradicatpromis-ing activities Some important modes of action are discussed and a closer look is taken

at the connection between glutathione (GSH) and phenazines, as new findings indicate a possible mode of action in intracellular GSH level depletion

2.1 Modes of action Although a detailed description of the modes of action of phenazines would be far beyond this review, some aspects that have already reviewed elsewhere1,19–22are indispensable to impart

a better understanding and will be therefore recapitulated shortly Phenazines are able to both donate and to accept electrons, dependent on its relative redox potential to other electron transfer molecules.19Reactive oxygen species (ROS) formation is a major mode of action of phenazines and can be beneficial to the host, e.g via an inhibition of pathogenic organisms or detrimental by

an interference with normal cell functions.19 PYO production by

P aeruginosa has shown to play a major role in lung infection via ROS generation.6,19,23–25 PYO is able to supply toxic superoxide (O2 
) and hydrogen peroxide (H2O2) via cellular redox cycling of

O

N N

O

N N

pyocyanin (PYO) (P aer uginosa)

CO2H

N N

endophenazine A (S cinnamomensis)

N

methanophenazine (M sarcina Gö1)

derm acozine A (Der macoccus aby ssi)

phenazinolin A (R=OH) phenazinolin B (R=H) (Streptomyces sp.)

N N CONH 2

CONH2

N N

CO 2 Me

CO2Me HO

streptophenazine A (Streptomy ces sp HB202)

N N

O

N

N H

H

R

N N

CO2H

OH OH HO SCH3

izuminoside B (Streptomy ces sp.)

Figure 1 Selected naturally-occurring phenazine derivatives.

molecular oxygen and various reducing agents such as NADH and

endothelial cells with PYO, hydrogen peroxide was formed

accom-panied with depletion of the glutathione (GSH) level.27In addition,

intracellular NADPH levels increased O’Malley et al found that

PYO depletes GSH level in airway epithelial cells and

overexpres-sion of catalase could not fully prevent the decline in cellular

GSH.28 Counterintuitively, GSH can have a detrimental effect on

lung cells as it can reduce PYO with concomitant formation of

O2
.22 Hydroxyl radical formation from the interaction of

pro-tease-cleaved Fe-transferrin with redox cycling of pyocyanin has

been associated with endothelial cell injury.29 Lactoperoxidase

and related dual oxidases (Duox) produce mild oxidants harmful

for several airway pathogens like Staphylococcus aureus,

Burkholde-ria cepacia, and Pseudomonas aeruginosa.30The expression of PYO

leads to oxidative stress via a consumption of NADPH and

superox-ide formation leading to a competitive inhibition of epithelial Duox

activity.30Bacterial nitric oxide synthases (bNOS) catalyze the

for-mation of NO from arginine and NO has shown to increase the

resistance of bacteria to various antibiotics via chemical

modifica-tion of toxic compounds and mitigamodifica-tion of oxidative stress.31PYO

was found to inhibit the growth of the B subtilis nos-deletion strain

significantly more compared to the wild type.31 As a protection

from oxidative stress, P aeruginosa contains cytoplasmic

superox-ide dismutases (SODs).22,32

Results obtained in the Newman lab9,10have shown that the

intracellular redox state in P aeruginosa can be influenced by

PYO in the absence of other electron acceptors via reoxidation of

under anaerobic conditions via electron-shuttling

Another role performed by phenazines is the ability to

reduc-tively dissolve ferrihydrite and hematite in the pH range of 5–8

thus making iron more bioavailable as shown for

electrochemi-cally-reduced PYO, phenazine-1-carboxylic acid (PCA),

phena-zine-1-carboxamide and 1-hydroxyphenazine by Wang and

can compete with Fe(III) as the final oxidant The authors

specu-lated that the different phenazines may accomplish different

func-tions dependent on oxygen and iron availability

Further work in the group of Newman13,14has indicated that

phenazines can act as signalling molecules via the activation of

the Fe-containing transcription factor SoxR PYO was found to

trig-ger the upregulation of transport genes and downregulates genes

involved in ferric iron acquisition

PYO was lately linked to an aberrant entrapment and killing by

neutrophil extracellular trap (NET) release leading to a damage of

host tissues, found in cystic fibrosis (CF).33The role of PYO in P

aeruginosa infection has been reviewed,23,25 and a more general

discussion of phenazines in Pseudomonas spp was recently given.34

2.2 Phenazines and glutathione (GSH) GSH, an important antioxidant ubiquitous in mammalian cells,

is important for detoxification (e.g of carcinogens) and can protect against DNA damage that is caused by ROS.35In addition, GSH is a regulator of the thiol-redox status and plays an important role in many disease states and in critical cell signalling pathways.35 GSH homeostasis is of utmost importance as for example a low intracellular GSH level decreases the antioxidant capacity, whereas higher GSH levels are observed in cancer cells and lead to increased chemo-resistance.35PYO has been known to reduce the intracellu-lar GSH level with accompanied formation of mixed disulfides.27,28 Exogenous GSH is protective against PYO toxicity,36and it has been hypothesized that this is due to the formation of a cell-impermeant GSH-PYO conjugate.37

Ray et al could show that a chronic, low-level exposure to PCA, PYO and 1-hydroxyphenazine increased protein misfolding and neurotoxic phenotypes in the model organism C elegans.38It was demonstrated that these phenotypes are not directly linked to ROS production, as the addition of the anti-oxidant N-acetyl cys-teine did not prevent the formation of the phenotypes Recently, two new phenazines exhibiting a thiol ether linkage between PCA and pantetheine were identified by Heine et al.39 The new phenazines, namely panphenazine A and B, were discovered via metabolic profiling of concentrated culture extracts of the rare actinomycete Kitasatospora sp HKI 714.39 The biosynthesis gene cluster40revealed no genes that could be responsible for CAS bond formation and upon irradiation of PCA in the presence of panteth-eine, a mixture of panphenazine A and B was formed most likely via a radical mechanism (Scheme 1) These findings suggest a pos-sible mode of action for the intracellular GSH level depletion, caused by PYO.27,28In addition, the authors could demonstrate that phenazines readily form S-conjugates with different proteins exhibiting cysteine side-chains, which could explain phenazine-induced protein misfolding in C elegans.38 Further research has

to be undertaken in order to clarify the role of phenazines in protein misfolding processes

2.3 Anticancer activity Anticancer activities of phenazines have been recently summa-rized and critically evaluated by Cimmino et al.41,42It was found that an implementation of phenazines as anticancer agents is problematic because of nonselective DNA intercalation leading to general toxicity.41 However, selectivity can be enhanced using derivatives to overcome the ‘‘flatland structure” of phenazines.41 Potent structures have been found e.g amongst dimeric or those bearing a pendant protonatable group.41 The so called ‘‘prodrug approach” using phenazine-5,10-dioxides rather than phenazines

N N

CO2H

S

H N O

H N O OH OH

panphenazine A

N N

CO2H

S H O H O HO

OH

panphenazine B

N N

CO2H

PCA

HS H

O H

O

OH OH

hv (370 nm)

or AIBN, Et3N

+

Scheme 1 Formation of panphenazines via a non-enzymatic reaction 39

Table 1

Several newly discovered phenazines exhibiting extraordinary structures or activities.

N

N

O

N

N H

H

R

phenazinolin A (1, R= OH)

phenazinolin B (2, R= H)

N

N H

H HO

phenazinolin C (3)

O

O

N

N H H

OH H

HO

phenazinolin D (4)

N

N

HO2C

OH

O

N

N H H OH H

phenazinolin E (5)

N N

HO2C

HO

OH

Phenazinolin A-C (1–3) exhibited in vitro cytotoxicity against P388, GLC, H460, and XWLC human cancer cell lines with IC 50 values between 14–40lM and antibiotic activity against Bacillus subtilis, Staphylococcus aureus, Aspergillus niger, and Botrytis cinerea MIC values were between 12–27lM

Streptomyces sp.

64

N

N

O

R 1

OMe

N

pontemazine A R1=R2= NH2, 6

pontemazine B R1= OH, R2= NH 2, 7

Neuronal cell protective effect on glutamate-induced mouse hippocampal HT-22 cell damage

Streptomyces sp.

82

N

N

O

O

8

Cytotoxic to murine P388 leukemia cells at 50lM (only tested

at this concentration)-proliferation inhibition by 78%

Bacillus sp 57

N

N

CO2H

CH3

5-methyl

phenazine-1-carboxylic acid betaine

9

IC 50 = 489 nM (A549 lung cancer cell line) P putida 69

IC 50 = 459 nM (MDA MB-231 breast cancer cell line) Growth-free inhibition zones (diameter) ranged from 11 to

23 mm towards pathogenic bacteria tested at this concentration)- proliferation inhibition by 78%

N

N

O

Br

N N O Br

IC 50 of TNF-a-induced NFjB activity (10: 4.1lM, 11: 24.2lM) Streptomyces

sp.

58

IC 50 of LPS-induced nitric oxide production (10: >48.6lM, 11:

15.1lM)

IC 50 of PGE 2 production (10: 7.5lM, 11: 0.89lM)

Table 1 (continued)

N

N

CO2H

OH OH HO

N N

CO2H

OH OH HO

izuminoside A, 12

SCH3

N

N

CO2H

O

O

O HO

HO

OH

CH 3

izuminoside B, 13

izuminoside C, 14

N

N

O

N N

CO2H H

H OH OH HO

izumiphenazine A, 15

N

N

CO2H

CO2H

S

N

H 3 CO

O

CH3 O

N

H O

Compound 13 (10lM) displayed a 22% decrease in cell viability (AGS cell line) in the presence of TRAIL (100 ng mL
1 ), compared to the cell viability where no TRAIL is present

Streptomyces sp.

59– 62

Compound 14 (60lM): 19% decrease Compound 15 (40lM): 19% decrease Compound 17 (12.5lM): 35% decrease Izuminoside A (12) and yorophenazine (16) showed no decrease in cell viability in the presence of TRAIL

N

N

CONH2

O

O

HO

HO

O

N N

O

OH HO HO

OH

N N

O

OH HO O

OH

N

N

CO2H

O

OH

HO

HO

O

N N

O

OH HO HO

N N

CO2H

O

OH

compound Bacillus subtilis Escherichia coli

Inhibition zone (mm)

18 19 20 21 22 23

8 8 14 8 15 8

7 12 9 0 7 0

Kitasatospora sp.

91

represents a more promising strategy against cancer.41,43–45 A

review on fused aryl phenazine derivatives summarizes their

anti-cancer activities.46 PCA was identified to exhibit both protective

and anticancer activities against human skin melanoma cell line

SK-MEL-2.47Further efforts in testing both synthetic48–53and

nat-urally-occuring54–69 phenazines as anticancer agents have been

made recently

2.4 Phenazines as antibiotics and their role to host defence

Research into new antibiotics is particularly urgent as antibiotic

drug-resistance has emerged widely and most antibiotics rely only

on structures identified during the 1940s to 1960s, known as the

‘‘golden era” of antibiotic discovery.70Most antibiotics during this

time were identified via the screening of soil-derived

actino-mycetes.70 Phenazines could present a promising scaffold for the

development of a new class of antibiotics

In a seminal publication, Moura-Alves et al could show that the

ligand-dependent transcription factor aryl hydrocarbon receptor

(AhR) plays a crucial role to host defence against both acute and

chronical bacterial infections as AhR-deficient mice were more

vul-nerable to both P aeruginosa and M tuberculosis.71Upon phenazine

binding to AhR, several processes like the transcription of canonical

detoxifying genes or a regulation of cytokine and chemokine

production are initiated This work has now established the

sought–after mechanistic connection for phenazines in host

defence interaction

2.5 Phenazines and biofilms

Biofilm formation represents a major burden in antimicrobial

therapy The term ‘‘biofilm” has been defined as an ‘‘aggregate of

microorganisms in which cells that are frequently embedded

within a self-produced matrix of extracellular polymeric substance

(EPS) adhere to each other and/or to a surface”.72Extracellular DNA

(eDNA) stabilizes bacterial biofilms and protects against physical

and chemical stress, thereby being a promising target against

bac-terial infections.73 A biofilm offers protection against

antimicro-bials by reducing their amount to a sublethal concentration,

which in turn can lead to resistance Resistance can also emerge

via an alleviated horizontal gene transfer.74 Slow growth states

are also expected to account for the failure of antibiotic

treat-ment.74PYO was found to play a central role in P aeruginosa

addition, PYO influences the binding of eDNA to P aeruginosa

PA14 cells via intercalation.76,77Sakhtah et al recently discovered

that 5-methylphenazine-1-carboxylate (5-Me-PCA) is transported

by the efflux pump MexGHI-OpmD that controls gene expression

and biofilm development in P aeruginosa.78O’May et al showed

that iron supply in P aeruginosa biofilms is important and that iron

chelators can facilitate to prevent biofilm formation.79The

impor-tance of phenazines on biofilms has been outlined in a review.34,80

2.6 Isolation of new phenazines

A plethora of novel phenazines have been discovered in the last couple of years and some noteworthy examples are outlined in this section Important examples of newly isolated phenazines exhibit-ing exceptional structures or biological activities are listed in

Table 1 Dimeric phenazines: Dimeric phenazines are rare and some iso-lates exhibit extraordinary structures Worthwhile mentioning are phenazinolins, dimeric phenazines exhibiting uncommon azabicy-clo[3.3.1]nonadienol (1–3) and oxabicyazabicy-clo[3.3.1]nonadienol (4 and 5) ring systems These phenazines have been isolated from Strepto-myces sp and showed anticancer and antibiotic activity.64 Diasta-phenazine is a further example of dimeric Diasta-phenazines and was N

N

HO

HO

N N

Figure 2 Unprecedented glyceride phenazines showed O-acyl isomerism 81

N N

NH2 O HO

O

endophenazine E

N N

OH

endophenazine A1

N N

endophenazine F

N

endophenazine G

OH

Figure 3 Endophenazine E, 86

A1, 40

F 40 and G 40

N N OH Br

Br

24: MIC= 1.56 µM

N N O Br

Br

~2-fold increase in potency

O

CH 3

N N O Br

Br

O cyclohexyl

inactive

N N O Br

Br

O R

same activity

26: R= iPr

27: R= Ph

N N

NH 2

Br

Br

inactive

N N OH H

Br

inactive

N N OMe Br

Br

inactive

N N OH H

H

N N OH Br

H

4-fold decrease in potency

25

28 inactive

29

30 31

32 33

Figure 4 SAR of phenazine 24 against S aureus 92

weakly cytotoxic against five human tumor cell lines and showed

weak antimicrobial activity against S aureus (MIC = 64lg mL
1).63

Uncommon phenazines: Wu et al isolated unprecedented

glyc-eride O-acyl isomeric phenazines and HPLC analysis revealed its

uncommon phenazines present pontemazines A (6) and B (7),

exhibiting an unusual methyl amine linkage and showed

protec-tive effect to HT-22 neuronal cells.82The uncommon phenazine 8

exhibits anticancer activity.57Uncommon 5-methyl

phenazine-1-carboxylic acid betaine (MPCAB, 9) isolated by Kennedy et al from

P putida displayed antimicrobial as well as anticancer activities.69

Terpenoid phenazines: The terpenoid phenazines 10 and 11 have

been isolated by Kondratyuk et al from Streptomyces sp and are

potential cancer chemopreventive83 agents.58 Further examples

of recently isolated terpenoid phenazines are phenaziterpenes A

and B84isolated from Streptomyces niveus A genome

mining/isola-tion approach by Heine et al of Kitasatospora sp HKI 714 led to the

discovery of formerly unknown terpenoid endophenazine

deriva-tives, namely endophenazine A1, F and G (Fig 3) showing

antibac-terial activity.40The total synthesis of endophenazine G has been

disclosed recently.85 Via heterologous expression of the

biosyn-thetic gene cluster for endophenazines from Streptomyces anulatus

9663 in engineered host strains, derived from Streptomyces

coeli-color M145, C–isoprenylated endophenazine E was produced

Endophenazine E is a conjugate between endophenazine A, and

L-glutamine (Fig 3).86Six new phenazines, named

chromophenazi-nes, exhibiting a prenyl group attached on one of the two nitrogens

were tested for antimicrobial activity, but only one

chro-mophenazine displayed moderate activity against B subtilis,

E coli, and the fungus M miehei.87Geranylphenazinediol, isolated

from Streptomyces sp showed some activity against the enzyme

acetylcholinesterase.88

Glycosylated phenazines: Much progress has been made towards

the isolation of bioactive phenazines by the group of Ishibashi in

their screening program89for new natural products from

actino-mycetes (see compounds 12–17) Several phenazines displayed

activity in overcoming tumor necrosis factor-related apoptosis

inducing ligand (TRAIL) resistance in AGS cells.59–62,68 Some of

the new phenazines, namely izuminosides A–C (12–14), belong

to the rare class90 of glycosylated phenazines Further examples

of glycosylated phenazines have been published lately by Wu

et al.,81,91 who reported the isolation of several glycosylated

endophenasides (see compounds 18–22) Six new glycosylated

et al from Streptomyces sp.65 These phenazines exhibit one or

two rhamnose moieties and three compounds showed some

cyto-toxicity against HCT-116 cancer cells No antibacterial activity was

observed, which is uncommon for carbohydrate-containing

phenazines.1

2.7 Synthetic phenazines with exceptional biological activity

A diverse library of several phenazines was synthesized in the Huigens lab from which 24 was identified as a lead antibiotic displaying a MIC value of 1.56lM against S aureus (Fig 4).92Via systematic structural diversification, a twofold increase in potency could be realized for compound 25 Furthermore, a structure-activity relationship (SAR) was established, which will be of use for further focused libraries

Clofazimine93and derivatives94have shown remarkable in vitro activity against multidrug-resistant tuberculosis (MDR-TB) and clofazimine has been in clinical trials for the treatment of MDR-TB.95 A library of clofazimine derivatives has been estab-lished containing compounds that exhibit lower logP values com-pared to clofazimine in order to reduce undesired side effects like skin discoloration which is caused by accumulation in skin and fat tissues.96–99Some compounds displayed good in vitro activ-ity against M tuberculosis and were further tested for their acute toxicity and pharmacokinetic properties Compounds, exhibiting

a significantly reduced skin discoloration potential were selected for further evaluation in a mouse model of acute MDR-TB infection Clofazimine and two other promising candidates for the treatment

clofazimine N N

N

Cl

N N

Cl

N

34

O

N

Cl

N

35

O

IC50(Vero) (µg/m L) = 68.6 log CFU/lung* = 3.54

IC50(Vero) (µg/m L) = >64 log CFU/lung* = 4.04

IC50(Vero) (µg/m L) = 51 log CFU/lung* = 3.25

Figure 5 Active compounds against MDR-TB ⁄after 20 days of treatment in BALB/C mice infected with clinical isolated MDR-TB dosed orally at 20 mg/kg CFU = colony forming unit 98

O

N N

MIC = 2.2 µM againstM tuber culosis H37Rv and rifampicin-resistant strain ATCC 35338

Figure 6 A phenazine exhibiting an allyl-pyran group was active against M tuberculosis H37Rv and rifampicin-resistant strain ATCC 35338.

N N

I

Br OH

Cl Cl

36

MRSA MBEC= 12.5 µM MRSE MBEC= 1.56 µM

N N

I

OH

Cl Cl

37

MtB MIC= 3.13 µM

Figure 7 HPs with promising activity against persistent bacteria 102,103 MBEC: minimum biofilm eradication concentration, MRSA: methicillin-resistant S aureus, MRSE: methicillin-resistant S epidermidis, VRE:vancomycin-resistant Enterococcus.

of MDR-TB are depicted inFig 5 In this regard the results from

Coelho et al should be mentioned, who tested several phenazine

derivatives for their in vitro activity against M tuberculosis H37Rv

(ATCC 27294) and the Rifampicin-resistant strain (ATCC 35338)

containing a His-526-Tir mutation in the rpoB gene and the most

active derivative showed a MIC value of 2.2lM for both strains

(Fig 6).100

A small focused library of halogenated phenazines (HPs) was

promising activity against persistent bacteria In addition, a SAR

was established Most importantly, it was demonstrated that the

HPs were selective for bacterial cells over mammalian cells

Com-pound 36 exhibits the most potent biofilm-eradicating activities to

date against several multi-resistant germs (which can be expressed

as MBEC (minimum biofilm eradication concentration)) and

com-pound 37 was identified as a potent inhibitor of the slow-growing

M tuberculosis (Fig 7)

sub-stituent have been recently synthesized by Conda-Sheridan et al

addition, two QSAR models were reported which could be of use

in the future Synthetic phenazine derivatives have shown to

exhi-bit antiplasmodial and antiprotozoal,104–107as well as insecticidal

activities.108

3 Biosynthesis

Research into phenazine biosynthesis has continued to attract

interest since early studies in the second half of the 19th century

Comprehensive reviews, dealing with phenazine biosynthesis have

been published recently and important facts are iterated.2,109–113

The focus of this section is to give a short overview of seminal

find-ings in the field of phenazine biosynthesis including the most

recent mechanistic insights

3.1 Early studies

Studies on early phenazine biosynthesis have been reviewed by

Turner and Messenger and some important milestones will be

iter-ated here.114

Initial research into phenazine biosynthesis was mainly focused

on PYO and it was found that the amount of PYO formed is

signif-icantly affected by culture conditions and bacterial origin.112Early

studies on phenazine biosynthesis turned out to be troublesome as

rich media with different bacterial isolates of poorly defined com-position had been used.109Jordan115was the first to take a more systematic approach with synthetic media and his achievements led to the development of a medium for the detection of P aerug-inosa in the clinic (e.g King’s A medium116).109P aeruginosa was found to produce a variety of additional colored compounds.112 Importantly, it was realized that trace amounts of iron and exclu-sion of air led to an increase in phenazine production in several strains of Pseudomonas117but the physiological role of phenazines

as ‘‘respiratory pigments” was demonstrated only recently by Price–Whelan et al.9In the 1950s, Blackwood and Neish118could show that glycine, alanine, leucine and isoleucine were the pre-ferred amino acid substrates for phenazine biosynthesis and that

incorpo-rated into PYO Millican119performed further studies with 14 C-labeled shikimic acid and proved some incorporation into PYO, whereas anthranilate was found not to be incorporated These results were somewhat conflictive to previous findings120where anthranilate was shown to stimulate PYO formation and phenazine biosynthesis had been proposed to originate in anthranilate.121By means of feeding experiments with14C-shikimic acid, Hollstein and McCamey122proposed two identical C-6- or C-1-N-substituted chorismic acids as precursors in the biosynthesis of the phenazine moiety

3.2 Anthranilate synthase genes Research into the biosynthesis of strain-specific phenazines had been difficult because of unstable intermediates and the discovery

of specific genes has leveraged our current knowledge of the biosynthetic pathway towards phenazines.112Essar et al identified anthranilate synthase genes in PYO-producing strains of P aerugi-nosa,123which upon inactivation caused a significant decrease in PYO levels such that they concluded that phenazine biosynthesis proceeds via anthranilate, which is in contrast to our current knowledge of phenazine biosynthesis It was later confirmed that anthranilate synthase genes are indeed responsible for the genera-tion of anthranilate but rather for the generagenera-tion of the Pseu-domonas Quinolone Signal (PQS), than being a precursor for the synthesis of phenazines.124PQS plays an important role in quorum

express specific genes, in P aeruginosa phenazine biosynthesis.126 3.3 Current understanding

Seminal results were obtained by McDonald et al.,127who found that 2-amino-2-deoxyisochorismic acid (ADIC) is completely con-verted into phenazine-1-carboxylic acid (PCA) in cell-free extracts

of E coli containing phz gene products On the contrary, anthrani-late was not converted to PCA, indicating that phenazine biosyn-thesis branches off from primary shikimate pathway at ADIC Pierson et al.128,129were the first to report genes directly involved

in phenazine biosynthesis and genes for phenazine biosynthesis have been discovered in several bacterial phenazine produc-ers.9,130–132The five enzymes responsible in phenazine synthesis, namely PhzB, PhzD, PhzE, PhzF, and PhzG are conserved among all phenazine-producing bacteria and it is assumed that all phenazines found in nature share a small number of common pre-cursors as the gene cluster has most likely spread via horizontal gene transfer.109,133,134

Chorismic acid, an intermediate in the shikimate pathway (Scheme 2) is a common precursor for many primary and sec-ondary metabolites such as vitamin K, aromatic amino acids, folate, ubiquinone or the siderophores Chorismic acid is also the first substrate in the core biosynthetic pathway for the synthesis of

OH

CO2H

chorismic acid

CO2H

OH OH HO

H

O

O

OH

OH

HO

erythrose 4-phosphate

OH

O

O

CH2

P

HO

O

OH

phosphoenolpyruvate

+

shikimic acid

O

OH OH

CO2H HO

PiO

DAHP

PhzC

H2O

- P i

Scheme 2 Chorismic acid is biosynthesized via the shikimate pathway, starting

from erythrose 4-phosphate and phosphoenolpyruvate.

other bacteria possess the phzC gene, which encodes a type-II

which catalyzes the first step of the shikimate pathway, the

reac-tion of erythrose 4-phosphate, water, and phosphoenolpyruvate

to give DAHP.112It is believed that in the case of inhibition of other

DAHP synthases, PhzC acts to ensure sufficient flow for the

phena-zine biosynthesis.112

The core biosynthetic pathway towards strain-specific

phenazi-nes (Scheme 3) commences with the conversion of chorismic acid

to ADIC, catalyzed by Mg(II)-dependent PhzE,135and the proof that

PhzE is an effective ADIC synthase was given by Li et al.135PhzE

consists of two domains In the first domain chorismate is

con-verted to ADIC, whereas in the second domain ammonia, needed

for this reaction, is generated from glutamine.135

It was found that upon binding of chorismic acid, a channel of

approximately 25 Å in length is induced in order to prevent the loss

of ammonia to the solvent.135 Fascinatingly, stereochemistry in

ADIC is induced due to the fact that the channel ends at the Si-face

at C-2 of chorismate.135An enzyme related to PhzE is anthranilate

synthase (AS),137–139exhibiting virtually an identical active site,

but in contrast to AS, pyruvate elimination takes not place in PhzE

and ADIC enters the consecutive biocatalytic cascade for the synthe-sis of strain-specific phenazines The vinyl ether functional group of ADIC is cleaved off in the next step of the biosynthetic cascade

trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA), the last stable intermediate in the biosynthesis of phenazines PhzD is an isochorismatase with major structure similarities to other structures from a subfamily of

a/b-hydrolase enzymes that includes pyrazinamidase and N-car-bamoylsarcosine amidohydrolase.140Different to related structures, PhzD does not contain a nucleophilic cysteine but rather uses aspar-tic acid to protonate the vinyl ether functionality of ADIC Further-more, PhzD catalyzes a dissimilar reaction compared to the aforementioned related structures.140The subsequent double bond isomerization is catalyzed by PhzF141,142and the underlying mech-anism is still under discussion.143,144PhzF exhibits two active sites which are occupied by sulfate ions in the available crystal struc-ture.141The enzyme-substrate complex suggests that a conserved glutamate E45 abstracts a proton from C-3 of DHHA, which is then attached to C-1 after double-bond shift to yield an enol.142Catalyzed

by PhzF144the obtained enol tautomerizes to 6-amino-5-oxocyclo-hex-2-ene-1-carboxylic acid (AOCHC) and it is suggested that a

OH

CO2H

CO 2 H

NH2

CO2H

OH

NH2

H

NH2

OH

CO2H H

NH2

O

CO 2 H H

NH2

O

CO2H H

2

H

chorismic acid

PhzB

- 2 H2O

ADIC

Gln Glu

DHHA

CO2H

N

CO2H

H

CO2H

N

CO2H

O2

H 2 O 2

PhzG

H

CO2H

N

CO2H

O2

H2O2

N H

CO2H

CO2H

O2

H2O2

N N

CO2H

CO2H

PDC

O2

H 2 O 2

N

H

CO2H

O2

H2O2

N

H

CO2H

O2

H2O2

N N

CO2H

PCA

N

H

CO2H

O2

H 2 O 2

N H

O2

H2O2

N N

phenazine

CO2

AOCHC

strain-specific phenazines THPDC

THPCA

DHPHZ

pyruvate

HHPDCa

CO2

- H2O

PhzG PhzG

THPCAa

2 2

2

Scheme 3 Current understanding of the biosynthesis towards strain-specific phenazines starting from chorismic acid 2,136

cavity in dimeric PhzF could be suited for the ensuing ketone

con-densation of two molecules AOCHC for the generation of

hexahy-drophenazine-1,6-dicarboxylic acid (HHPDC).142 The head-to tail

condensation of two molecules AOCHC to give HHPDC can proceed

spontaneously in vitro, but involves PhzB, a small dimeric protein

of theD5-3-ketosteroid isomerase/nuclear transport factor family

in vivo.145It is thought that AOCHC is toxic because of possible side

reactions with other amines, e.g on proteins, thus its accumulation

has to be limited.2By means of crystallization experiments with

product and substrate analogues, it has been proposed that

dou-ble-imine formation is catalyzed through orienting two substrate

molecules and by protonation of the tetrahedral intermediate.145

Pseudomonas species contain an approximately 70% sequence

iden-tical copy of the phzB gene, namely phzA, and PhzA has shown to play

a role127in the biosynthesis of phenazines.2HHPDC is a central

inter-mediate towards the likely end products of the pathway,

5,10-dihy-dro-PDC (DHPDC) and 5,10-dihydro-PCA (DHPCA) that are central

precursors for strain-specific phenazines.136Earlier, PCA and PDC

have been claimed as final products of the pathway.136As HHPDC

is not stable, it undergoes rapid oxidative decarboxylation to

tetrahydrophenazine-1,6-carboxylic acid (THPCA) Starting from

HHPDC, two oxidative decarboxylation reactions and a spontaneous

oxidation lead to the unsubstituted phenazine.136The final steps of

the biosynthesis of DHPDC and DHPCA involve flavin-dependant

PhzG-catalyzed oxidation reactions.136,146PhzG was found to

exhi-bit close similarities to PdxH that catalyzes the final step in

pyri-doxal-50-phosphate (PLP) biosynthesis.146 PhzG is not perfectly

specific, which explains the appearance of PCA, PDC and

unsubsti-tuted phenazine and that competition between PhzG-catalyzed

oxi-dation reactions and spontaneous oxidative decarboxylations

governs the ratio of these compounds.136

Further modifications of the phenazine core involve e.g

hydrox-ylation, methylation or N-oxidation Zhao et al.147recently found

that the aromatic N-monooxogenase LaPhzNO1, which is

homolo-gous to Baeyer
Villiger flavoproteins, catalyzes in a

substrate-selective fashion phenazine N-oxidation and its possible use in

chemoenzymatic aromatic N-oxidation reactions is speculated

Chin-A-Woeng et al showed that the introduction of the gene phzH

of Pseudomonas chlororaphis can efficiently extend the range of the

biocontrol ability of bacterial strains.148

Methanophenazine, isolated from Methanosarcina mazei Gö1 is

suggested to play an important role in membrane-bound electron

transport,149 and its synthesis might proceed via a biosynthetic

pathway different to that in bacteria.2

Future research has to be directed towards

phenazine-modify-ing enzymes as there are major gaps in understandphenazine-modify-ing when it

comes to the biochemistry of modifications or the generation of

species-specific phenazine compounds.34,109

4 Synthesis

Classic synthetic strategies towards phenazines have been

reviewed1,18,150–152and an annual update153on diazines and benzo

derivatives can also be found The following section gives an

over-view of highlights in the field of phenazine synthesis since 2004, as

extensive progress both in methodology and natural product

syn-thesis has been made Established synthetic strategies towards

phenazines will be touched shortly in order to convey an integral

overview

4.1 Classic methods

An overview of classic methods for the synthesis of phenazines

is given inScheme 6 These methods are primarily based on the

construction of the central heterocyclic ring and suffer from major

disadvantages like limited substrate scope, harsh reaction condi-tions, low yields or the requirement of several synthetic steps rather than a one-step synthesis from commercially available starting materials.1,18This fact, combined with a constant interest

in differently substituted phenazines has spurred the development

of new methods for the preparation of phenazines

An old but rarely applied method, discovered by Wohl and Aue,154involves the fusion of anilines and nitrobenzenes at high temperatures under basic conditions The Bamberger-Ham155 pro-cedure comprises the reaction of two para-substituted nitrosoben-zenes under acidic conditions and suffers from various limitations The Beirut-reaction156–158can be used for the synthesis of phenazi-nes by reacting benzofurazan oxide with phenols to give 5,10-phe-nazine dioxides, which can be easily reduced to phe5,10-phe-nazines This approach offers some advantages compared to the two strategies mentioned earlier like a broader substrate scope and milder reac-tion condireac-tions Broad applicareac-tion has been found for the conden-sation of substituted 1,2-benzoquinones (can be in-situ generated

compromise either a reductive cyclization of diphenylamines with

an ortho-nitro160–163, ortho,ortho0-dinitro,163or ortho-nitro,ortho0 -fluoro164arrangement The oxidative cyclization of diphenylami-nes with an ortho,ortho0-diamino arrangement (Tomlinson oxida-tion)165,166 is also described in the literature.165,167 A very promising approach involves a sequential aniline arylation fol-lowed by aniline arylative intramolecular cyclication via a Buch-wald-Hartwig coupling reaction.167,168

4.2 Cu- and Pd-catalyzed coupling reactions Winkler et al succeeded in a homocoupling of substituted bro-moanilines via two subsequent Pd-catalyzed Buchwald-Hartwig amination,169followed by an in-situ oxidation to yield symmetrical phenazines in moderate to good yields (Scheme 8(a)) This concept was extended by Yu et al in the same year by using an environ-mentally friendly aqueous system to yield substituted phenazines

in moderate to high yields under Cu-catalysis (Scheme 8(b)).170 Laha et al achieved the synthesis of various phenazines in good

to high yields via a Pd-catalyzed coupling reaction between readily

(Scheme 8(e)).171Monoarylated products could be isolated when the reaction was stopped earlier, indicating a domino reaction pathway 1,2-Dichlorobenzenes were also tested as substrates but gave the corresponding phenazines in lower yields This approach gives access to unsymmetrical phenazines (no C2-axis) 4.3 Transition metal-catalyzed C-H functionalization

Seth et al accomplished a synchronous twofold C-N bond for-mation via an oxidative ortho-aryl C-H activation in poor to very good yields (Scheme 8(c)).172The reaction was catalyzed by a bin-ary Pd-Ag nanocluster Azoarenes were identified as sideproducts and the lowest yields were observed for a substrate containing a thioether substituent The presented protocol suffers from the need of a stoichiometric amount of Ag2CO3and only symmetrical phenazines have been synthesized Upon further assessing sub-strate scope and reaction conditions, this sub-strategy would be per-fectly suited for the establishment of a library for SAR assessment, as differently substituted anilines are commercially available

In a seminal publication, Lian et al disclosed the Rh(III)-cat-alyzed, formal [3 + 3] annulation of aromatic azides with aromatic azobenzenes to yield phenazines (Scheme 8(d)).173This strategy

phenazines are easily accessible and the strategy is also applicable for the synthesis of acridines This strategy, when applied for