PONy Dyes: Direct Addition of P(III) Nucleophiles to Organic Fluorophores

Article abstract of DOI:10.1021/acs.orglett.8b00270

Nucleophilic addition of phosphinic acid, phosphites, sodium dialkyl phosphites, phosphoramidites, phosphinites, and phosphonites to highly polarized or cationic fluorophores, followed by oxidation, results in new "PONy" dyes with auxochromic phosphinate, phosphonate, or phosphonamidate groups. The reaction was applied to a wide variety of coumarins, (thio)pyronins, and N-alkylacridinium and 5,6-dihydrobenzo[c]xanthen-12-ium salts as well as a meso-chlorinated BODIPY to provide compact dyes with red-shifted absorption and emission bands and Stokes shifts up to 8200 cm^-1.

Full text of DOI:10.1021/acs.orglett.8b00270

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
PONy Dyes: Direct Addition of P(III) Nucleophiles to Organic
Fluorophores
,
†
‡,†
Alexey N. Butkevich,* Maksim V. Sednev, Heydar Shojaei, Vladimir N. Belov,* and Stefan W. Hell
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Go
̈
ttingen, Germany
*
S Supporting Information
ABSTRACT: Nucleophilic addition of phosphinic acid, phosphites,
sodium dialkyl phosphites, phosphoramidites, phosphinites, and
phosphonites to highly polarized or cationic fluorophores, followed
by oxidation, results in new “PONy” dyes with auxochromic
phosphinate, phosphonate, or phosphonamidate groups. The reaction
was applied to a wide variety of coumarins, (thio)pyronins, and N-
alkylacridinium and 5,6-dihydrobenzo[c]xanthen-12-ium salts as well
as a meso-chlorinated BODIPY to provide compact dyes with red-
shifted absorption and emission bands and Stokes shifts up to 8200
−
1
cm .
hile searching for new fluorophores suitable for
nanoscale imaging of intracellular targets, we noticed
infrared emission. Due to their compact structures and the
presence of phosphorus (P), oxygen (O), and nitrogen (N)
atoms in close proximity, we nicknamed these compounds
“PONy dyes”. These dyes can be prepared in cationic or
zwitterionic forms, making them promising candidates for
future development of cell-permeant fluorescent markers for
living cells. In this paper, we highlight the diversity and easy
synthetic accessibility of PONy dyes.
1
W
that various organic dyes with electrophilic (cationic or highly
polarized neutral) conjugated systems reacted with nucleophilic
phosphorus(III) reagents such as phosphinic acid (H PO ),
3
2
phosphinites [ROPR′ ], phosphonites [(RO) PR′], phosphites
2
2
[
(RO) P], or phosphoramidites [(RO) PNR′ ] (Scheme 1) to
3
2
2
2
form phosphonylated leuco bases. Upon oxidation, these
intermediates provided new fluorophores with red-shifted
absorption and emission maxima and increased Stokes shifts
as compared to the precursor dyes. The phosphonylated
fluorophores were deemed particularly attractive due to their
low molecular mass (typically MW < 500) and orange to near-
In terms of the Pearson acid−base theory, a qualitative
interpretation of the orbital overlap concept, nucleophilic P(III)
3
reagents, such as trimethyl phosphite, are typical soft bases. On
the other hand, the pyronin-type cationic dyes (pyronins, thio-,
carbo-, and Si-pyronins) as well as acridinium salts (Figure 1b−
e) and also electron-poor coumarins and dipyrromethenes
(Figure 1, a,f) behave as soft acids due to high polarization and
extended delocalization of the positive charge. In our case, the
direct addition of P(III) nucleophiles (see Figure S1) to a
variety of cationic or highly polarized fluorophores (Figure S2)
Scheme 1. Addition of Neutral or Anionic P(III)
Nucleophiles to Cationic or Highly Polarized Dyes (Dye 1)
4
was followed by a Michaelis−Arbuzov rearrangement and
produced leuco-forms of phosphonylated dyes (Scheme 1).
The latter could be isolated and used in further transformations
or oxidized to the products demonstrating red-shifted
absorption and emission bands and increased Stokes shifts
(
dye 2, Scheme 1).
In the presence of a non-nucleophilic counterion, such as Cl−
−
or ClO , the formation of phosphonylated leuco dyes was
4
slow, resulting in low yields (<40%) even after prolonged
reaction times (overnight) or at 60 °C. Addition of a
stoichiometric amount of an external nucleophile (in the
+
−
form of Bu N X ) resulted in complete conversion within 0.5−
4
1
h at room temperature in CH Cl for X = I (Scheme 1, path
2 2
A; with competing cyanation for X = CN) and increased the
Received: January 25, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b00270
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Figure 1. Structural diversity of stable PONy dyes obtained according to Scheme 1. Highly polarized neutral fluorophores (green: coumarins (a), 8-
chloro-BODIPY (f)) or cationic dyes (blue: (b−e)) react regioselectively with P(III) nucleophiles according to Scheme 1; the reaction sites are
marked with red dots (see Figure S3 for the corresponding atomic LUMO coefficients). Photophysical properties (as compared to those of the
parent fluorophores) are given in Table 1 and Tables S1 and S2. For synthetic procedures and additional examples, see the Supporting Information.
preparative yield of the leuco phosphonate. The intermediate
leuco phosphonylated compounds can be easily oxidized with a
stoichiometric amount of DDQ (2,3-dichloro-5,6-dicyano-1,4-
benzoquinone). In many cases (Figure 1b−e), the oxidation
was rapid at −78 °C; however, coumarin-derived 3,4-
dihydrocoumarin-4-phosphonates required heating (the oxida-
tion was complete at 70 °C within 5 min). The most far-red-
emitting PONy dyes prepared from carbopyronins and Si-
pyronins (e.g., by oxidation of the stable leuco-SiP1 compound,
Figure 1e) or from electron-poor P(III) nucleophiles (e.g.,
Heating carbopyronins or Si-pyronins with 50% aq H PO
3 2
resulted only in their reduction to the corresponding
diarylmethanes, while pyronins and 10-alkylacridinium salts
produced the desired leuco phosphinic acids. After isolation,
these intermediates could be oxidized into zwitterionic red-
fluorescent pyronin- (P1) or orange-fluorescent acridinephos-
phinic acids (A1, Table 1). Otherwise, the leuco phosphinic
acids were treated with excess N,O-bis(TMS)acetamide (BSA)
to produce di-O-TMS-phosphonites as the reactive analogues
6
of alkylphosphonous acids, which underwent the nucleophilic
Ph POMe) rapidly hydrolyzed in protic solvents, forming
phosphorus-free products (Figure S4). Nevertheless, it was
addition reaction with Michael acceptors (e.g., N-substituted
acrylamide in Scheme 2).
2
7
possible to prepare a hydrolytically stable NIR-emitting (740−
The photophysical properties of the stable PONy dyes are
compiled in Table 1. The versatility of phosphorus addition at
the sp2 carbon, combined with the wide availability of
functionally substituted P(III) reagents, offers an extended
array of conceivable PONy dyes with broadly varying properties
(see Table S1, including the hydrolytically unstable examples).
Comparison of the UV−vis spectra indicates that PONy dyes
absorb and emit at longer wavelengths and possess larger
Stokes shifts than the parent dyes (Figure S5). The increase in
Stokes shift values is smallest for pyronins (+100 to +250
7
50 nm) PONy dye P7 from the recently reported extended
5
7
pyronin analogue H-hNR.
The reactivity of neutral coumarins and 10-alkylacridinium
salts (Figure 1a,c) toward neutral P(III) reagents was
insufficient; however, sodium dimethyl phosphite underwent
Michael addition at C-4 of coumarins at room temperature and
at C-9 of acridinium salts at 100 °C in DMF (Scheme 1, path
B). While BODIPY 505/515 proved resistant under these
conditions, its 8-chloro derivative (BP) yielded purple
nonfluorescent BP1 upon heating with excess triisopropyl
phosphite (Figure 1f). Because of its very low fluorescence
quantum yield (<0.2%) and relatively high absorbance (ε = 33
−1
−1
cm ), larger for 10-alkylacridinium salts (around +600 cm ),
and may exceed +6000 cm− for push−pull coumarins initially
1
−1
possessing large Stokes shifts (typically 2000−2500 cm ). 4-
Phosphonylated coumarins with a 2-thiazolyl (C3) or 2-
benzothiazolyl (C5,C6) substituent at the 3-position provide a
−1
−1
0
00 M cm ), the dye BP1 may be recommended as a FRET
quencher in the range between 500 and 600 nm.
B
DOI: 10.1021/acs.orglett.8b00270
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. Photophysical Properties of the Parent Fluorophores and the Derived PONy Dyes (See Figure 1)
a
λmax (nm), Φfl Stokes shift (nm) (Δν (cm−1))
b
c
dye (molecular mass, Da)
P(III) reagent
λmax (nm)/ε (M−1cm−1
)
τ (ns)
solvent
abs
em
̅
8
coumarin 6 (350)
457/51000
468/32000
425/21000
439/25000
431/22000
439/24000
434/39000
419/22000
424/19000
546/77000
501, 0.63
555, 0.03
564, 0.10
642, 0.23
562, 0.39
651, 0.20
658, 0.29
613, 0.04
651, 0.16
569, 0.36
568, 0.38
627, 0.33
616, 0.44
635, 0.26
620, 0.50
672, 0.16
660, 0.22
663, 0.21
691, 0.26
704, 0.10
693, 0.32
638, 0.09
622, 0.36
644, 0.70
754, 0.02
737, 0.17
524, 0.30
595, 0.10
579, 0.32
587, 0.38
739, 0.04
728, 0.10
515, 0.98
44 (1922)
87 (3350)
139 (5799)
203 (7203)
131 (5408)
212 (7418)
224 (7844)
194 (7554)
227 (8224)
23 (740)
22 (709)
37 (1000)
37 (1037)
32 (836)
3.2
n.d.
0.9
2.1
2.6
2.6
2.5
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
PBS
C1 (350)
C2 (397)
C3 (480)
C4 (455)
C5 (458)
C6 (543)
C7 (430)
C8 (458)
(MeO) PONa
2
(MeO) PONa
2
(MeO) PONa
2
(MeO) PONa
2
(MeO) PONa
2
t
(Bu O) PONa
2
t
d
e
(Bu O) PONa
1.2, 0.3
2.0
2
(MeO) PONa
MeCN
PBS
2
9
pyronin Y (267)
1.8
5
46/93000
590/59000
79/67000
603/18000
88/73000
632/51000
24/62000
1.9
MeOH
PBS
P1 (330)
H PO
3
1.8
2
5
2.7
MeOH
PBS
P1-Halo (608)
P2 (375)
see Scheme 2
P(OMe)3
1.9
5
32 (878)
3.1
MeOH
PBS
40 (942)
0.8
6
36 (874)
1.6
MeOH
MeOH
MeOH
PBS
P3 (421)
P4 (480)
P5 (577)
PhP(OMe)2
P(OMe)3
629/62000
657/69000
660/38000
34 (815)
1.6
34 (749)
2.1
f
e
see the SI
44 (947)
1.7, 0.7
2.7
g
6
51/46000
603/69000
89/49000
42 (931)
MeCN
PBS
f
P6 (493)
see the SI
35 (910)
1.9
5
33 (901)
3.0
MeOH
MeOH
PBS
5
H-hNR
608/100000
36 (920)
61 (1167)
56 (1115)
29 (1118)
60 (1885)
52 (1704)
24 (726)
85 (1759)
74 (1555)
10 (384)
3.9
P7 (427)
P(OMe)3
H PO
693/29000
n.d.
1.5
6
81/40000
MeCN
MeOH
PBS
1
0
methylacridine orange (280)
495/71000
1.7
A1 (416)
535/38000
1.4
3
2
5
27/67000
2.6
MeOH
MeOH
MeOH
MeCN
CH Cl
1
1
thiopyronin [SP] (283)
SP1 (391)
563/114000
654/43000
654/64000
505/79000
574/33000
2.1
e
P(OMe)3
1.3, 0.4
0.8
i
SP2 (505)
(MeO) PNPr
2
2
1
2
BODIPY 505/515 (248)
n.d.
2
2
BP1 (412)
P(OPri)3
647, <0.002
h
73 (1966)
MeCN
a
b
c
d
Lowest energy absorption peak. Fluorescence quantum yield (absolute value). Fluorescence lifetime. Deprotection of C6 with TFA.
e
f
g
h
Biexponential (Table S2). Multiple steps, see the Supporting Information. With addition of 1% (v/v) TFA. Dark quencher (nonemissive). PBS:
phosphate-buffered saline (pH 7.4).
Scheme 2. Transformation of Commercial Dyes into PONy
Fluorophores Suitable for Bioconjugation
emitting (λmax 627 nm) zwitterionic fluorophore with bright
emission in aqueous buffers.
Acid hydrolysis of two tert-butyl groups off the phosphonate
ester in dye C6 led to compound C7 (Figure 1) bearing a net
negative charge in aqueous buffer. On the other hand, the
hydrolysis of dialkyl phosphonates or phosphonamidates (see
examples in the Supporting Information) allows the prepara-
tion of PONy dyes in highly water-soluble zwitterionic forms.
The extinction coefficients of the PONy dyes are comparable
with the values found for the parent fluorophores while the
fluorescence quantum yields are lower, in particular in aqueous
solutions; the effect can be explained in terms of increased
probabilities of nonradiative internal conversion (due to the
1
3a
narrowing energy gap ) and intersystem crossing (for
1
3b
thiopyronin derivatives ). The fluorescence lifetimes of
PONy dyes generally do not exceed 3 ns (with shorter
lifetimes corresponding to lower emission efficiences). The
excited state lifetimes vary, and their sensitivity to the nature of
the solvent may allow using the PONy-derived probes in
fluorescence lifetime imaging or as polarity sensors.
new entry to very large Stokes shift dyes (>7000 cm− in
1
acetonitrile), maintaining practical fluorescence quantum yield
(
≥0.2) and excited-state lifetimes (≥2 ns). The dye P1
To demonstrate the versatility of the proposed trans-
formation, we performed facile functionalizations of the
represents an extremely small (330 Da) and compact red-
C
DOI: 10.1021/acs.orglett.8b00270
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
commercial fluorophores Atto 495 (in the form of tert-butyl
ester) and Pyronin Y (Scheme 2). Using the phosphinylation/
oxidation chemistry, Atto 495 was converted into an orange-
emitting and amino-reactive A1 NHS ester (Scheme 2a) ready
for immunolabeling. Similarly, the red-emitting P1 self-labeling
(2) (a) Zheng, S.; Barlow, S.; Parker, T. C.; Marder, S. R. Tetrahedron
Lett. 2003, 44, 7989. (b) Kempf, B.; Mayr, H. Chem. - Eur. J. 2005, 11,
9
(
9
17.
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1
4
HaloTag ligand was prepared via a di-O-TMS-phosphonite
followed by the reaction with a HaloTag O2 ligand-derived
acrylamide and oxidation with DDQ (Scheme 2b). Both
transformations required only two separate synthetic steps and
provided the functional derivatives of the PONy dyes A1 and
P1 in zwitterionic form with a very short charge separation
distance (which is known to favor intact membrane
permeability in living cells).
We are currently exploring the possibilities of applying
PONy dyes as analytical reagents (e.g., in electrophoresis), cell-
permeant fluorescent probes for living cells, and dyes for optical
microscopy.
(
5) Niu, G.; Liu, W.; Zhou, B.; Xiao, H.; Zhang, H.; Wu, J.; Ge, J.;
Wang, P. J. Org. Chem. 2016, 81, 7393.
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ASSOCIATED CONTENT
Supporting Information
(13) (a) Valeur, B.; Berberan-Santos, M. N. Characteristics of
Fluorescence Emission. In Molecular Fluorescence: Principles and
Applications, 2nd ed.; Wiley-VCH: Weinheim, 2012; p 56. (b) Kryman,
M. W.; Schamerhorn, G. A.; Hill, J. E.; Calitree, B. D.; Davies, K. S.;
Linder, M. K.; Ohulchanskyy, T. Y.; Detty, M. R. Organometallics
2014, 33, 2628.
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b00270.
(14) Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.;
Synthetic procedures and characterizations of the
compounds, including Figures S1−S7 and Tables S1
and S2 (PDF)
Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.;
Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris,
G.; Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.; Wood, K. V.
ACS Chem. Biol. 2008, 3, 373.
AUTHOR INFORMATION
Corresponding Authors
■
*
E-mail: abutkev@gwdg.de.
E-mail: vbelov@gwdg.de.
*
ORCID
Stefan W. Hell: 0000-0002-9638-5077
Present Address
‡(
Universita
Wurzburg, Germany.
M.V.S.) Institut fu
t Wurzburg, Am Hubland, Geb. C1, 97074
̈
r Organische Chemie, Julius-Maximilians-
̈
̈
̈
Author Contributions
†A.N.B. and M.V.S. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the Bundesministerium fu
Forschung (BMBF, Germany) for funding in the program
KMU-innovativ: Photonik/Optische Technologien (FKZ
̈
r Bildung und
1
3N12995; to S.W.H.) and thank J. Bienert (MPIBPC), Dr.
H. Frauendorf, Dr. M. John and co-workers (Institut fu
Organische und Biomolekulare Chemie, Georg-August-Uni-
versitat, Gottingen, Germany) for recording the MS and NMR
spectra.
̈
r
̈
̈
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DOI: 10.1021/acs.orglett.8b00270
Org. Lett. XXXX, XXX, XXX−XXX