Ionic Pyridinium-Oxazole Dyads: Design, Synthesis, and their

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Ionic Pyridinium-Oxazole Dyads: Design, Synthesis, and their Application in Mitochondrial Imaging Aslam C. Shaikh, Mokshada E. Verma, Ravindra D. Mule, Somsuvra Banerjee, Prasad Padmakar Kulkarni, and Nitin T. Patil J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02528 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

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The Journal of Organic Chemistry

Ionic Pyridinium-Oxazole Dyads: Design, Synthesis, and their Application in Mitochondrial Imaging Aslam C. Shaikh,a,b Mokshada E. Varma,c,d Ravindra D. Mule,a,b Somsuvra Banerjee,a,b Prasad P. Kulkarni,d* and Nitin T. Patile* Division of Organic Chemistry CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road,

a

Pune - 411 008, India. bAcademy

of Scientific and Innovative Research (AcSIR), Ghaziabad − 201 002, India

cSavitribai

Phule Pune University, Ganeshkhind Road, Pune − 411 007, India.

dBioprospecting

eDepartment

Group, Agharkar Research Institute, G. G. Agarkar Road, Pune - 411 004, India.

of Chemistry, Indian Institute of Science Education and Research (IISER)-Bhopal,

Bhopal − 462 066, India.

ABSTRACT: Pyridinium-Oxazole Dyads (PODs): Novel Mitochondria Trackers

t

N

BuO R'

1.0 equiv Cu(OTf)2 R

2.0 equiv PhI(OAc)2 ACN, 60 °C, 3 h

O

N

OTf

R'

R

 Ionic fluorophore  Low molecular weight  Good quantum yield  High photostability  Access to library  Scalable route  Low cytotoxicity

We recently developed an oxidative intramolecular 1,2-amino-oxygenation reaction, combining gold(I)/gold(III) catalysis, for accessing structurally unique ionic pyridinium-oxazole

1 ACS Paragon Plus Environment

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dyads (PODs)with tunable emission wavelengths. On further investigation,these fluorophores turned out to be potential biomarkers; specially the one containing -NMe2functionality (NMe2POD) was highly selective for mitochondrial imaging. Of note, because of mitochondria’s involvement in early stage apoptosis and degenerative conditions, tracking the dynamics of mitochondrial morphology with such imaging technology has attracted much interest. Along this line, we wanted to build a library of such PODs which are potential mitochondria trackers.However, Au/Selecfluor, our first-generation catalyst system, suffers from undesired fluorination of electronically-rich PODs resulting in an inseparable mixture (1:1) of the PODs and its fluorinated derivatives. In our attempt to search for a better alternative to circumvent this issue, we developed second-generation approach for the synthesis of PODs by employing Cu(II)/PhI(OAC)2-mediated oxidative 1,2-amino-oxygenation of alkynes.This newly synthesised PODs exhibit tunable emissions as well as excellent quantum efficiency upto 0.96. Further, this powerful process gives rapid access to a library of NMe2-PODs which are potential mitochondrial imaging agent. Out of the library, the randomly chosen POD-3g was studied for cell imaging experiments which showed high mitochondrial specificity, superior photostability, and appreciable tolerance to microenvironment change with respect to commercially available MitoTracker green (MTG). INRODUCTION Since mitochondria plays a vital role in the celluar life and death,1mitochondrial dysfunction is greatly involved in many types of human diseases.2Therefore, selective targeting of the mitochondria has emerged as a new strategy for improved therapeutic efficiency.3 One of the best studied strategies is to covalently attach therapeutic agents to a mitochondria-targeting carrier.3c Amongst other conventional methods, fluorescent 2 ACS Paragon Plus Environment

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probes are deemed as most efficient for selective mitochondria imaging and have been generated either by conjugation of a targeting moiety or using targeting fluorophores.4 In this regard, ionic fluorophores5 which contain positively charged groups such as triphenylphosphonium (type 1) or quaternized pyridine moieties (type 2) are most commonly used because of their ability to permeate the potential barrier (−180 mV) of inner mitochondrial membranes (Figure 1).4a,6 Likewise, conjugated lipophilic cationic compounds such as rhodamine (type 3) and cyanine (type 4), if attached to appropriate receptors, can also become productive mitochondria trackers (Figure 1).7 Reports on the use of protein or peptide-based probes,8 non-ionic fluorophores9 and metal complexes10 for mitochondrial imaging are also existing. Nevertheless, most of these conventional probes suffer from the problems of photostability, photobleaching, and low quantum yields, low penetration ability in cells, higher molecular weight and solubility.11 Moreover, the reported designs of mitochondria trackers relies heavily on mere attachment of positively charged species with the existing fluorophores.4a To address these limitations and facilitate advancement in this field, design and synthesis of such probes in facile manner for selective detection of cell organelle are highly warranted.

N

P

R

R N

O

R N

R N

Probe

Probe

Receptor

Receptor

Type - 1

Type - 2

Type - 3

Type - 4

Figure 1.Design of mitochondria trackers

To this end, we recently introduced a new design of ionic PODs (A) by oxidative intramolecular 1,2-amino-oxygenationreaction of pyridino-alkynes (P), combining gold(I)/gold(III) catalysisvia Au/Selectfluor catalyst system (Scheme 1a).12 Live cell3 ACS Paragon Plus Environment


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imaging studies revealed that Phen-POD (A, R' = 9-phen) is non-selective and localized in the cytoplasm as well as in the nucleus; whereas, NMe2-POD (A, R' = 4-(NMe2)C6H4) was found to be highly selective for mitochondrial imaging. Because the primary structural feature which distinguishes NMe2-POD from Phen-POD is the presence of 4(NMe2)C6H4/9-phen group, it was believed thatthe existence of -NMe2 group in POD is essential for mitochondrial imaging. But while working with strongly electron-rich PODs, we encountered a limitation of this methodology. Due to stoichiometric requirement ofthe oxidant Selectfluor, a highly electrophilic fluorinating source, PODs with stronger electron donating groupsended up undergoing fluorination. For instance, when pyridinoalkyneP'was treated with Au/Selectfluor conditions, the reaction led to an inseparable mixture of A'and its fluorinated derivativeA'' in 1:1 ratio (Scheme 1b).12 This prompted us to search for an alternative strategy which will not only address the current issue but also allow the concise and rapid construction of a POD library suitable for mitochondrial imaging. Scheme 1. Previous approach and associated problems: fluorination by Selectflour

(a) Our previous approach

t

cat Au(I)

N

BuO R'

R

O

2.0 equiv Selectfluor

P

BF4

N

R R' A  Ionic fluorophore  Low molecular weight

(b) Drawback of the approach: Fluorination

t

BuO

OMe

N OMe P'

cat Au(I) 2.0 equiv Selectfluor

O

N

BF4

+ O OMe

MeO

N

BF4 F

OMe MeO

A': A'' (1:1 )


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In this context, copper mediated amino-oxygenation reactions of olefins caught our attention.13 Pioneering work from the group of Chemler and co-workers disclosed a series of copper catalyzed/mediated oxidative amino-oxygenation reactions to access various nitrogen heterocycles (Scheme 2a).14 Chiba and co-workers elegantly demonstrated, in their series of papers, the Cu-catalyzed/mediated intramolecular oxidative aminooxygenation reaction of olefines to access indolines.15 In 2016, Yu and co-workers disclosed

copper-catalyzed

oxidative

intra/intermolecular

amino-oxygenation

of

unactivated alkenes (Scheme 2b).16 Later, Wang and co-workers reported the coppercatalyzed intermolecular three-component amino oxygenation of olefins using external source of nitrogen and oxygen (Scheme 2c).17The fact thatsuch oxidative copper catalyzed amino-oxygenation reactions is restricted to only alkenes, made us curious to examine the Cu/oxidant catalyst system for amino-oxygenation of alkynes.Here, we postulated that such PODs (3) could be obtained from difunctionalization of pyridinoalkynes such as 1 utilizing the sequence of syn-aminocupration, nucleophilic attack (of amide oxygen atom) and reductive elimination (Scheme 2d).18 Herein, we discloseour complete study based on the successful implementation of second generation approach to ionic PODs via Cu(II)-mediated oxidative 1,2-amino-oxygenation of alkynes. This approach allowed the concise and rapid construction of a POD library which is highly suitable for mitochondrial imaging. Further, the randomly chosen POD-3g was extensively studied for cell imaging experiments, and is found to be selective for mitochondrial imaging application as expected.



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Scheme 2. Cu-catalyzed amino-oxygenation reactions - Previous work and current hypothesis

a) Cu-catalyzed intermolecular oxidative amino-oxygenation of alkenes - Chemler's, Chiba's and co-workers R' R''O R' R' cat Cu/oxidant R' NR NHR source OR" R' = H, alkyl, aryl b) Cu-catalyzed oxidative amino-oxygenation of alkenes - Yu and co-workers OH N

H

R'

HNR'2

NR'2

N O

cat Cu/oxidant R'

R'

R'

c) Cu-catalyzed amino-oxygenation of alkenes using O-benzoylhydroxylamines -Wang and co-workers O

O cat Cu/oxidant

OH

O

BzO-NR'2

R'

NR'2

R'

d) Cu-mediated oxidative amino-oxygenation of alkynes: Current hypothesis

t

BuO

N R'

R

Cu/PhIX2

R'

1

via Cu(III) OtBu

R'

N

R

N

X

R

3

reductive elimination

syn-aminocupration R'

O

Cu(III) O

R'

N

R

Cu(III) O N

R

RESULTS AND DISCUSSION In search of a Cu-based catalyst system, we initiated our investigation with 4-((2(6-(tert-butoxy)pyridin-2-yl)phenyl)ethynyl)-N,N-dimethylaniline

(1a)

as

model

substrate. At the outset, the POD3a was obtained in 24% yield by employing 20 mol% of Cu(OTf)2 as the catalyst and 1.5 equiv. of PhI(OAc)2 as the oxidant (2) in CH3CN at 60 °C for 3 h (Table 1, entry 1).13 Screening of several copper catalysts, oxidants and equiv. of Cu(OTf)2 revealed that 1.0 equiv. of Cu(OTf)2 gives improved yield of desired product 6 ACS Paragon Plus Environment

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in 82% (Table 1, entries 2−7). Further enhancement in yield upto 90% was observed when 2.0 equiv. of PhI(OAc)2 was employed (Table 1, entry 8). We surmised that the presence of OTf-containing additives would make the reaction catalytic with respect to Cu(OTf)2. With this expectation, various additives such as NaOTf, Zn(OTf)2, Sc(OTf)3, TfOH were examined. However, they were found not to be satisfactory (Table 1, entries 9-12). In absence of either Cu(OTf)2 or PhI(OAc)2, reaction failed to yield desired product (Table 1, entries 13-14). Table 1. Optimization Studies

t

BuO

N

Cu oxidant 2

O

N

X

CH3ACN, 60 °C 3h Me2N

entry

1a Me2N

[Cu]

Additives (1.0 equiv.)

3a, X = OTf or OAc

Oxidant

Yield (%) 3a 1 Cu(OTf)2 PhI(OAc)2 24 2 Cu(OTf)2 PhI(CF3CO2)2 12 3 Cu(OTf)2 K2S2O8

Ionic Pyridinium-Oxazole Dyads: Design, Synthesis, and their

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