Modular Synthetic Approach to Silicon-Rhodamine Homologues and Analogues via Bis-aryllanthanum Reagents

Letter

Modular Synthetic Approach to Silicon-Rhodamine Homologues

and Analogues via Bis-aryllanthanum Reagents

Alexey N. Butkevich *

Cite This: Org. Lett. 2021, 23, 2604 −2609

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sı Supporting Information

ABSTRACT: A modular synthetic approach toward diverse analogues of the far-red ﬂuorophore silicon-rhodamine (SiR), based on

a regioselective double nucleophilic addition of aryllanthanum reagents to esters, anhydrides, and lactones, is proposed. The reaction

has improved functional group tolerance and represents a uniﬁed strategy toward cell-permeant, spontaneously blinking, and

photoactivatable SiR ﬂuorescent labels. In tandem with Pd-catalyzed hydroxy- or aminocarbonylation, it serves a streamlined

synthetic pathway to a series of validated live-cell-compatible ﬂuorescent dyes.

ith the recent developments in molecular-size super-

resolution ﬂuorescence microscopy techniques, the

Scheme 1. Synthetic Approaches to 5H-Spiro[furan-2,9′-

xanthenes] (Y = O) and Their Analogues (Y = CMe , SiMe ,

problem of design and development of suitable small-molecule

ﬂuorescent probes is becoming central to achieving practicality

and ﬁdelity in nanoscale ﬂuorescence imaging. In particular,

there is a deﬁciency in the range and diversity of photo-

activatable or photoconvertible small-molecule ﬂuorophores

etc.)

that also exhibit high emission brightness and photostability.

Additional requirements of cell membrane permeability and

live-cell compatibility are posed whenever superresolution

imaging in live cells or tissues is required. Among the successful

developments of the past decade, numerous triarylmethane

ﬂuorophores, in particular the rhodamine analogues with the

oxygen bridge replaced with CMe , SiMe , GeMe , and P(O)R

groups to achieve bathochromic shifts of absorption and

emission, have been reported. However, the latest disclosures

b−e

interval between −78 °C and rt.

We have concluded that

transmetalation with a more Lewis acidic metal salt should

mitigate the undesired reactivity of ArLi or ArMgX species

stemming from single-electron transfer pathways or their highly

show that the demand for chemical diversity of newly designed

labels exceeds the scope of established synthetic chemistry of

triarylmethane dyes, and the synthetic methods oﬀering

practicable yields are often lacking.

basic nature. Indeed, it is long known that alkyl- and

Recently, a reaction of bis-aryllithium or bis-arylmagnesium

Received: February 11, 2021

Published: March 15, 2021

reagents has been proposed as an alternative synthetic strategy

toward O-, Si-, and P-rhodamines and Si-ﬂuoresceins (Scheme

). While this method did provide access to an expanded range

of triarylmethane ﬂuorophores, in our hands for more

demanding examples we found it operationally complex, as it

required slow additions and temperature optimization across the

2021 The Author. Published by

American Chemical Society

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Org. Lett. 2021, 23, 2604−2609

Organic Letters

Letter

Scheme 2. Reaction of Aryllanthanum Reagents Generated from 1a−f with (a) Anhydrides 2a−j and Esters 2g′,k,l, and (b)

Lactones 4a−l

Reaction yield with bis-aryllithium reagent without addition of LaCl . Reaction with dimethyl tetraﬂuorophthalate (2g′) instead of

tetraﬂuorophthalic anhydride (2g). Reaction with methyl cinnamate (2k). Reaction with ethyl phenylpropiolate (2l). Reaction run on 2 mmol

scale. Dehalogenated product (5a) formed instead of expected 5g.

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Org. Lett. 2021, 23, 2604−2609

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Letter

Scheme 3. Preparation of (a) Live-Cell-Compatible Fluorescent Dyes 6a−f by Pd-Catalyzed Carbonylative Hydroxylation of 3j−

m and 5e,f and (b) Live-Cell Far-Red-Fluorescent Tubulin Probe 4-SiR-CTX (8) by Pd-Catalyzed Carbonylative Amination of

aryllanthanide reagents of type RMX (M = La, Ce) retain high

nucleophilicity toward carbonyl groups and oﬀer good yields

with base-sensitive substrates such as CH-acids and Michael

The reaction of aryllanthanum reagents with phthalide and

coumarin provided the desired spirocyclic products 5a,i in

comparable yields to those obtained from nontransmetalated

aryllithium reagents (Scheme 2b). However, the aryllanthanum

nucleophiles better tolerated the presence of a base-sensitive aryl

acceptors. Replacing the MgBr additive with the commercially

available LaCl ·2LiCl complex, we were able to quickly extend

^t^rⁱ^ﬂ^a^t^e^s^u^b^s^tⁱ^t^u^eⁿ^t⁽⁵^l⁾^aⁿ^d^,^t^o^a^l^e^s^s^e^r^e^x^t^eⁿ^t^,^a^r^o^m^a^tⁱ^c^N^O2

this chemistry to a number of sensitive substrates. In this report,

we highlight the scope of this transformation and demonstrate

its application to the synthesis of established live-cell-compatible

ﬂuorescent labels.

(

5k) and CN (5h) groups. The reaction of 7-bromophthalide

with the aryllanthanum reagent allows synthetic access to the

little explored 7′-HMSiR ﬂuorophore core of 5g, providing a

new entry to its derivatives recently developed into ﬂuorogenic

The reaction between the bis-aryllanthanum reagents derived

from 1a−d and cyclic anhydrides 2a−j resulted in formation of

the expected spirocyclic products with 40−70% isolated yields

far-red-ﬂuorescent labels for Cu-free click chemistry. Treat-

ment of this substrate with the basic bis-aryllithium reagent

resulted in formation of only the corresponding proto-

dehalogenated product 5a.

(Scheme 2a). In the case of 3-substituted phthalic anhydrides

(2f,i,j), double nucleophilic addition proceeded regioselectively

The expanded functional group tolerance of the proposed

aryllanthanum addition allows the introduction of a one-step

approach to a variety of halogen-substituted triarylmethane

ﬂuorophores (3i−m, 5e−g) and their novel halogen- and

triﬂate-substituted spirochromene analogues (5l−n). The easy

access to the former prompted us to explore their potential as

substrates in carbonylative cross-coupling reactions (Scheme 3).

The previously reported syntheses of regioisomerically pure 4′-,

at the least substituted carbonyl group. This method allowed the

preparation of previously inaccessible Si-rhodamine analogues

a,b lacking the pendant o-phenylene ring and containing an

electrophilic cyclic unsaturated ester functionality, poorly

compatible with basic RLi or RMgX nucleophiles. Reactive

NO and F substituents in the aryl ring of the electrophilic

partner were also tolerated. The new procedure provided

markedly improved yields in the reactions with 3-bromophthalic

′-, or 6′-carboxyrhodamine dyes relied on the extensive use of

6a,10

(

3j; 67% vs 5%) and glutaric anhydrides (thus, the photo-

protecting groups.

We envisaged that, given the regiose-

activatable ﬂuorophore PA-SiR was isolated in 71% vs

reported 5% yield). Under identical conditions, the addition of

bis-aryllanthanum reagent was 1,2-selective with α,β-unsatu-

rated esters, producing 9-alkenyl- and 9-alkynyl-substituted Si-

lectivity of bis-aryllanthanum addition to 3-substituted phthalic

anhydrides, recently proposed 4′-carboxycarbo-, silico-, and

oxygen-bridged rhodamine ﬂuorophores, demonstrating out-

standing imaging performance in stimulated emission depletion

xanthenes 3n and 3o.

(STED) microscopy in living cells, can be accessed from the

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Org. Lett. 2021, 23, 2604−2609

Organic Letters

The Supporting Information is available free of charge at

Letter

intermediates 3j−m via a carbonylative hydroxylation or

amination. To demonstrate the versatility of the proposed

method, we have prepared the ﬂuorescent dyes 4-TMR, 4-

(I, III) at pH > 1. Typical of the carbo- and Si-rhodamines is

their propensity to close into the colorless form I under strongly

acidic conditions (pH < 2), while the rhodamine dyes (e.g., 3k)

10CP, 4-SiR, and its novel analogue 4-SiR700 by carbon-

and thiophene analogues of Si-rhodamine such as 3c remain

ylative hydroxylation with CO gas safely generated in a small

zwitterionic (II) across the entire pH 1−10 range. However,

compound 3m undergoes attack by the nucleophilic solvent

(water) and is only present in its colored and NIR-ﬂuorescent

form II between pH 1 and 2.

amount ex situ in a two-chamber reactor (Scheme 3a).

Similarly, the established spontaneously blinking ﬂuorophore

HMSiR, employed in stochastic optical reconstruction

microscopy (STORM) in live and ﬁxed cells, along with its

On the contrary, spiroether analogues and homologues 5a−h

5c,13

azetidine analogue, have been prepared from the intermedi-

ates 5e,f. By changing the catalyst and base, the same method

could also be extended to a Pd-catalyzed carbonylative

amination, allowing the installation of the carboxyl group and

the amide coupling to be performed in one step on a

regioisomerically pure aryl bromide 3j. Hydrolysis of the

crude tert-butyl ester, followed by the separation of the expected

product 7a from the minor bis-carboxylic acid impurity 7b and

of the HMSiR dye

demonstrate the second distinct

transition (II ↔ III, corresponding to pK ) within the

biologically relevant pH 4−10 range due to higher nucleophil-

icity of the reversibly formed alkoxide as compared to the

carboxylate group. Similar behavior of the spirochromenes 5i−

m is further complicated with the reversible E/Z-isomerization

(

IV ↔ V) of the xanthylium form of these dyes observed upon

the protonation of colorless VI (see Figure S3 in the Supporting

Information ). Because of free rotation of the styrene fragment,

no far-red ﬂuorescence emission characteristic of Si-pyronins is

observed in solutions containing the protonated forms IV and V

of the compounds 5i−m. Rigidizing the double bond in the (Z)-

conﬁguration (V) within the cyclopentene context of 5n results

in recovery of Si-pyronin ﬂuorescence upon its protonation.

the amide coupling with de-N-Boc-cabazitaxel (H N-CTX),

demonstrated an eﬃcient technical shortcut in the synthesis of a

far-red ﬂuorescent live-cell tubulin probe 4-SiR-CTX (8).

The selected properties of Si-rhodamine analogues (3c,f,j-m

and 5a,b,d-n), relevant for the development of ﬂuorescent

probes, are compiled in Table 1. The lactones 3a,b,h and the

spiroether 5c exist exclusively in the colorless spirolactone forms

4c,6a,15

As described previously,

the position of the zwitterion−

spirolactone (II ↔ III) equilibrium of the rhodamine dyes

strongly depends on the nature of the solvent and increases with

the increasing content of water in aprotic solvents such as 1,4-

dioxane. We have measured these changes in absorbance and

ﬂuorescence (see Figure S2 the Supporting Information)

according to the two proposed metrics: D₀_.₅(dielectric constant

of a dioxane−water mixture at which the normalized extinction

Table 1. Properties of the Selected Fluorophores 3 and 5

ε/ε of the dye is equal to one-half of the maximal value

max

observed across the entire dioxane−water gradient, range 2 to

8) and K (the spirolactone−zwitterion equilibrium constant

L‑Z

in 1:1 dioxane−water, range 0 to +∞). We conclude that only

the D metric is appropriate for evaluating the response of the

position of this equilibrium to solvent polarity for both

spirolactones 3 and spiroethers 5, since the range of the K_L_‑_Z

metric is compressed between 0 and 1 for the compounds

existing predominantly in the colorless closed forms (III, VI),

resulting in zero measured values for all spiroethers. According

to the data based on D₀_.₅(Table 1), the response of the

spiroether ﬂuorophores 5b,e-h to the changes in solvent polarity

is similar to that of HMSiR (5a) and carbo- and Si-

compd

^λmax(abs/ﬂuor), nm

pK , pK

^D0.5 ^,^KL‑Z

646/674

658/682

656/676

558/584

620/646

708/738

650/674

652/676

650/647

658/678

664/688

658/680

656/−

open

1.2, −

1.3, 2.3

open

8, 5.0

38, 0.36

48, 0.0041

20, 20

42, 0.17

39, 0.016

41, 0

36, 0

−

42, 0

41, 0

47, 0

−

1.6, −

rhodamines, while the spiroethers derived from dihydrocou-

marin or coumarin do not undergo ring opening (VI → IV, V)

under neutral conditions.

<1, −

2.0, 4.2

1.4, 5.1

2.6, 4.0

1.9, 4.4

1.2, 2.9

1.7, 4.7

1.8, 4.5

2.1, 4.0

2.2, 4.0

1.7, 3.5

1.7, 3.6

1.3, 2.2

In summary, we have proposed a uniﬁed synthetic approach

toward diverse analogues of cell-permeant, spontaneously

blinking, and photoactivatable versions of the silicon-rhodamine

ﬂuorophore, and we investigated their behavior in aqueous

solutions at diﬀerent pH values and with varying contents of

organic cosolvent (dioxane). We are currently applying these

insights into the behavior of spirocyclic ﬂuorophores 3 and 5 to

the design and development of switchable ﬂuorescent probes for

biologically relevant superresolution microscopy.

642/−

658/−

650/674

−

ASSOCIATED CONTENT

■

See Figures S1 and S2 in the Supporting Information for the

corresponding spectra. Apparent pK values in 20% (v/v) DMSO−

00 mM Na phosphate buﬀer. With 1% (v/v) DMSO. 3m forms a

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.orglett.1c00512.

colorless water adduct at pH > 1.5.

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Org. Lett. 2021, 23, 2604−2609

Organic Letters

K.; Piao, W.; Kusakabe, M.; Saito, N.; Terai, T.; Okabe, T.; Nagano, T.

Letter

Synthetic procedures and characterizations of the

compounds, UV−vis spectra (Figures S1 and S2), and

copies of the NMR spectra (PDF)

Development of NIR fluorescent dyes based on Si-rhodamine for in

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Johnsson, K. A near-infrared fluorophore for live-cell super-resolution

microscopy of cellular proteins. Nat. Chem. 2013, 5, 132−139.

AUTHOR INFORMATION

Corresponding Author

■

Alexey N. Butkevich − Department of Optical Nanoscopy, Max

Planck Institute for Medical Research, 69120 Heidelberg,

Germany; orcid.org/0000-0002-9885-6434;

Email: alexey.butkevich@mr.mpg.de

(

d) Grimm, J. B.; Sung, A. J.; Legant, W. R.; Hulamm, P.; Matlosz, S.

M.; Betzig, E.; Lavis, L. D. Carbofluoresceins and carborhodamines as

scaffolds for high-contrast fluorogenic probes. ACS Chem. Biol. 2013, 8

6), 1303 −1310. (e) Sednev, M. V.; Wurm, C. A.; Belov, V. N.; Hell, S.

W. Carborhodol: a new hybrid fluorophore obtained by combination of

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.orglett.1c00512

fluorescein and carbopyronine dye cores. Bioconjugate Chem. 2013, 24

(4), 690−700. (f) Fukazawa, A.; Suda, S.; Taki, M.; Yamaguchi, E.;

Grzybowski, M.; Sato, Y.; Higashiyama, T.; Yamaguchi, S. Phospha-

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Notes

The author declares no competing ﬁnancial interest.

(g) Grzybowski, M.; Taki, M.; Senda, K.; Sato, Y.; Ariyoshi, T.;

ACKNOWLEDGMENTS

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photostable near-infrared labeling agent based on a phospha-rhod-

amine for long-term and deep imaging. Angew. Chem., Int. Ed. 2018, 57,

■

We thank Dr. Richard Lincoln (Department of Optical

Nanoscopy, Max Planck Institute for Medical Research) for

the preparation of compound 1e and for proofreading the

manuscript, Dr. Sebastian Fabritz and the staﬀ of the MS core

facility (Max Planck Institute for Medical Research) for

performing numerous LC-HRMS measurements, and the

Department of Optical Nanoscopy (Prof. Stefan W. Hell, Max

Planck Institute for Medical Research) for the technical and

ﬁnancial support of this project.

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