Development of a Visible Light Triggerable Traceless Staudinger Ligation Reagent

Article abstract of DOI:10.1021/acs.joc.8b01370

A series of substituted 9-methylenylanthracene photocages for diphenylphosphinothioesters have been synthesized to explore their photo-uncaging properties by visible light. Substituents such as phenyl, p-trifluoromethylphenyl, p-methoxyphenyl, ethyn-1-ylbenzene, and 3,3-dimethylbut-1-yn-1-yl have been introduced in order to extend the π-conjugation of the photocage and to shift the wavelength response of the uncaging process to the visible spectral range. Among these new photocages, the (10-(3,3-dimethylbut-1-yn-1-yl)anthracen-9-yl)methyl has been shown to have the best performance in terms of fast photo-uncaging and minimal byproduct formation. It is responsive to both UV and visible photoexcitation. Quantum yields of the photoinduced heterolytic anthracenylmethyl-phosphorus bond cleavage at 366 and 416 nm were found to be 0.08 and 0.025, respectively. This photocage enables traceless Staudinger ligation to be triggered by photoirradiation in the visible spectral range for bioconjugation applications. We demonstrate this with a series of visible-light-induced oligopeptide syntheses via the conjugation of amino acid/oligopeptide building blocks by the characteristic peptide linkage attained by traceless Staudinger ligation. Yields of the resultant conjugated oligopeptides ranged from 31 to 43%. This new photocage opens up the possibility of in situ synthesis of functional proteins/peptides mediated by visible-light-induced photoclick processes for the regulation of cellular/metabolic functions of life systems.

Full text of DOI:10.1021/acs.joc.8b01370

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Article
Development of a Visible Light Triggerable
Traceless Staudinger Ligation Reagent
PENG HU, Karsten Berning, Yun-Wah Lam, Isabel Hei-
Ma NG, Chi-Chung YEUNG, and Michael Hon-Wah Lam
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01370 • Publication Date (Web): 15 Oct 2018
Downloaded from http://pubs.acs.org on October 15, 2018
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Development of a Visible Light Triggerable Traceless Staudinger
Ligation Reagent
Peng HU, Karsten BERNING, Yun-Wah LAM, Isabel Hei-Ma NG, Chi-Chung YEUNG,
Michael Hon-Wah LAM*
Department of Chemistry, City University of Hong Kong, Hong Kong SAR, China
* Corresponding author:
Prof. Michael Hon-Wah LAM
Department of Chemistry,
City University of Hong Kong
Tat Cee Ave., Kowloon
Hong Kong SAR
China
Tel: (852)-3442-7329
Fax: (852)-3442-0522
Email: bhmhwlam@cityu.edu.hk
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Abstract
A series of substituted 9-methylenylanthracene photo-cage for diphenylphosphinothioesters have
been synthesized to explore their photo-uncaging properties by visible light. Substituents such as
phenyl, p-trifluoromethylphenyl, p-methoxyphenyl, ethyn-1-ylbenzene and 3,3-dimethylbut-1-yn-
1-yl have been introduced in order to extend the -conjugation of the photo-cage and to shift the
wavelength response of the uncaging process to the visible spectral range. Amongst these new
photo-cages, the (10-(3,3-dimethylbut-1-yn-1-yl)-anthracen-9-yl)methyl has shown the best
performance in terms of fast photo-uncaging and minimal by-product formation. It is responsive
to both UV & visible photo-excitation. Quantum yields of the photo-induced heterolytic
anthracenylmethyl – phosphorus bond cleavage at 366 & 416 nm were found to be 0.08 and 0.025
respectively. This photo-cage enables traceless Staudinger ligation to be triggered by photo-
irradiation in the visible spectral range for bioconjugation applications. We demonstrate this with
a series of visible light-induced oligopeptide synthesis via the conjugation of amino
acid/oligopeptide building blocks by the characteristic peptide linkage attained by traceless
Staudinger ligation. Yields of the resultant conjugated oligopeptides were ranged from 31 to 43%.
This new photo-cage opens up the possibility of in situ synthesis of functional proteins/peptides
mediated by visible light-induced photo-click processes for the regulation of cellular/metabolic
functions of life systems.
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Introduction
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The versatile, stereospecific and high yield chemical linking of molecular units, with minimal by-
product formation, under mild reaction conditions in benign solvents is the scope of “Click
Chemistry”. Since the promotion of this concept in 2001, click chemistry has been extensively
utilized in organic synthesis, bioconjugation, polymer fabrication and functional surface
modification, and nanotechnology.1,2 The photo-inducibility of some of the “spring-loaded” click
reactions has further enabled precise spatial and temporal control of the molecular conjugation
events.3 Numerous elegant processes, such as copper-free acetylene-azide cycloaddition4-6, photo-
induced nitrile imine tetrazole-ene cycloaddition (NITEC)7-9, azirine-alkene cycloaddition10,
photo-induced thiol-ene/thiol-yne coupling11-13, photo-enol reaction14, and photo-induced Diels-
Alder cycloaddition15, have been developed in recent years for “photo-click” conjugation. Yet,
most of these processes require UV excitation (excitation < 400 nm), which seriously hinders their
use in in vitro / in vivo bioimaging and other bioconjugation applications. So far, few visible light-
activated “photo-click” reactions have been reported. For example, Lin and co-workers
demonstrated the photo-induced fluoro-tagging of microtubules in living CHO cells by a
terthiophene-tetrazole “photo-click” reagent at 405 nm16 and a naphthalene-tetrazole “photo-click”
reagent at 700 nm via two-photon excitation17. Fraser et al., used eosin-Y as the photo-initiator for
thiol-ene photo-click reaction at 400 – 700 nm.18 Arslan et al., reported the use of
dibenzoyldiethylgermane as the visible-light responsive photo-reducer/photo-initiator for copper
catalyzed azide-alkyne cycloaddition (CuAAC) click reactions.19 Zhang et al., reported a
coumarin-caged tetramethyl guanidine photobase generator at 400 – 500 nm for thiol-Michael
addition reactions.20
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Photo-caging of phosphines to bring about photo-activation of non-traceless and traceless
Staudinger ligation has recently been explored by Carrico and co-workers21 and our research
team22 respectively. Both the 4,5-dimethoxy-2-nitrobenzyl and anthracenylmethyl photo-cages
involved only respond to UV excitation. In the latter case, the most efficient excitation at 376 nm is
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attributable to the (anthryl)  *(anthryl) and (anthryl)  *(diarylphosphine) transitions.22
Therefore, it may be possible to extend the wavelength responses of the photo-uncaging process
to the visible spectral range via the fine-tuning of the energy of these electronic transitions. In their
study of the laser flash photolysis of benzhydryl phosphonium salts at 266 nm, Ammer et al. also
reported that electronic effects due to substituents on the benzhydryl rings perturbed their quantum
efficiency.23 In this context, we explored the photolytic reactivity of a series of anthracenylmethyl-
based photo-cage with extended -conjugation for photo-triggered traceless Staudinger ligation
(Scheme 1) via single-photon photo-excitation in the visible spectral range.
h
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Scheme 1. Visible-light triggered traceless Staudinger ligation.
Results and Discussion
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A series of new substituted anthracenylmethyl photo-caged diphenylphosphinothioesters, 2 – 6,
have been synthesized via the nucleophilic substitution of the corresponding 9-
(chloromethyl)anthracene by the phosphinothioester (with R2 = methyl) (Scheme 2). All the
substituted anthracenylmethylphosphionthioester (chloride salt) are soluble in common organic
solvents such as THF, DMF, DMSO, methanol, and their aqueous organic mixtures. They are also
soluble in water. The purpose of introducing aryl substituents at the 10-position of the anthracenyl
chromophore is to extend the -conjugation of the photo-cage so as to lower the (anthryl) –
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*(anthryl) and (anthryl) – *(diarylphosphine) energy gaps. Fig. 1 shows the UV-vis absorption
spectra of the phosphonium compounds 1 – 6. All the substituted compounds show red-shifted
absorption bands compared to 1 with unsubstituted anthracenylmethyl chromophore. The extent
of such a red-shift in phosphonium compound 2 – 4 was very much similar despite the presence
of electron-withdrawing or electron-donating groups at the para-position of the phenyl substituent.
This is most probably caused by to the orthogonal orientation of the phenyl substituent with respect
to the anthracenyl ring due to their steric interaction which limits -conjugation. For 5 and 6 where
such steric hindrance is removed because of the acetylene bridge, absorption bands of the
anthracenylmethyl chromophores display ca. 30 – 40 nm of red-shift.
(b)
(a)
(c)
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Scheme 2. Synthesis of substituted anthracenylmethyl photocaged diarylphosphinothioesters, 2 – 6: (a) substituted
phenylboronic acid, Pd(OAc)2, BINAP, K2CO3, THF, reflux, 16 h; (b) pyridine, SOCl2, CH2Cl2, -10 C for 7 h; (c)
diphenylphosphinothioester, CHCl3, RT, 16-48 h.
Photolysis of the new phosphonium compounds at > 400 nm broad band irradiation at room
31
temperature was monitored by P NMR (Fig. 2). Complete uncaging was achieved in 20 to 40
min. giving the diphenylphosphinothioester and its oxide, as well as other uncharacterized by-
products.
Figure 1. Spectroscopic properties of the anthracenylmethyl photo-caged diphenylphosphinothioesters 1 – 6: UV-
vis absorption spectra (10-5 M) in 3:1 THF/H2O.
Amongst the five new phosphonium compounds,
4
gave the best yield of
diphenylphosphinothioester, while 3 produced the least. This may be due to the presence of the
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electron-donating methoxy group in 4 that favours heterolytic cleavage of the anthracenylmethyl –
phosphine bond via the stabilization of the carbocation intermediate. The electron-withdrawing
trifluoromethyl group in 3 renders it prone to the homolytic pathway.23 Both 5 and 6 underwent
heterolytic photolysis with minimal uncharacterized by-product formation. Compound 5 required
the longest photo-irradiation duration for photolysis, while 6 required the least.
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Figure 2. 31P NMR monitoring of photolysis of the phosphonium compound 2 – 6 in degassed 3:1 d8-THF/D2O.
Broadband photo-excitation (ex > 400 nm) was administered by a 500 W Hg lamp equipped with proper optical
filters. Spectra shown were obtained upon complete disappearance of the corresponding phosphonium compounds.
As compound 4 – 6 showed cleaner photolysis with less by-product formation, they were further
examined with visible photo-excitation (excitation > 420 nm). Photolysis of 1 (R1 = –H) under
similar photo-irradiation condition was also evaluated for comparison (Fig. 3). Only small amount
of the diphenylphosphinothioester was produced by 1 even after 90 min. of broad band irradiation
by visible light.
On the other hand, compound 4 – 6 were able to produce the
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diphenylphosphinothioester in 40, 50 and 25 min., respectively. The (10-(3,3-dimethylbut-1-yn-1-
yl)anthracen-9-yl)methyl chromophore (in compound 6) gives the best performance in terms of
fast photo-uncaging and yield of the traceless Staudinger reagent.
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Figure 3. 31P NMR monitoring of the photolysis of phosphonium compound 1 and 4 – 6 in degassed 3:1 d8-
THF/D2O. Broadband photo-excitation (ex > 420 nm) was administered by a 500 W Hg lamp equipped with proper
optical filters. Spectra shown were obtained upon complete disappearance of the corresponding phosphonium
compounds.
A closer examination of the spectroscopic properties of 6 reveals its absorption bands in 350 – 450
nm, with the lowest-energy absorption maximum at 423 nm. Quantum yields of the photo-
uncaging process of 6 at 366 and 416 nm were measured to be 0.08 and 0.025, respectively, via
reference to the absolute FeII at the corresponding excitation of ferrioxalate actinometry32-33. Thus,
the UV photo-uncaging responses of 6 is quite comparable to that of our previous UV-only photo-
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triggered traceless Staudinger reagent 1 (  0.07), while it also possesses reasonable photo-
uncaging efficiency in the visible spectral range. For comparison, the quantum efficiency of the
photo-uncaging of the dimethoxy-substituted p-hydroxyphenacyl photocaged -amino acids, L-
glutamic acid and GABA at 400 nm was 0.03 – 0.04.24 The photo-uncaging of cyclic nucleotides
from dialkylamino-substituted coumarinylmethyl photocages at 350 – 400 nm were found to
proceed with quantum yields of 0.20 – 0.28.25 Olson et al. reported a 7-diethylaminocoumarin-4-
ylmethyl photocage for the photo-release of cAMP at 450 nm with a quantum efficiency of 0.78.26
Recently, Slanina et al. reported a series of BODIPY-based photocages that showed remarkable
photo-uncaging quantum efficiency, ranging from 0.01 to 0.95, for a variety of simple leaving
groups at > 500 nm.27
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Despite its relatively lower photo-uncaging quantum efficiency by visible light (  0.025)
compared to that by UV excitation, 6 offers the possibility visible-light-triggered traceless
Staudinger ligation. This is demonstrated by its photo-induced reactivity towards
azidomethylbenzene and 2-azido-N-benzylacetamide (azido-Gly-NHBn) under broadband
irradiation at  > 420 nm (Table 1). Product yields of 52 and 49% respectively, which is
comparable to those obtained by our previous UV-only photo-triggered traceless Staudinger
reagent.22 Reactions of the un-caged diphenylphosphinothioester with the azide substrates have
also been carried out. The yields of the corresponding conjugation products were  75% for both
reactions. The reduction in product yield from the photocaged traceless Staudinger ligation reagent
suggests that quantitative photo-uncaging of all the caged reagent was not achieved. In our
previous study of the UV-only photocage, the overall photolytic yield of the corresponding
uncaged product was found to be 58%.22 Interference of the subsequent ligation process by side-
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products generated during photolysis may also be the cause of the lower photo-triggered ligation
yield.
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Table 1 Visible light-triggered traceless Staudinger ligation of 6 with selected aliphatic azides.
h (> 420 nm)
THF/buffer (pH 7.4)
16 h, RT
Traceless Staudinger
ligation product
6
Reagent†
Azide‡
Traceless Staudinger ligation
product yield (%)
excitation (duration)
--
--
6
> 420 nm (120 min)
> 420 nm (120 min)
--
N.D.
N.D.
N.D.
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--
N.D.
> 420 nm (120 min)
> 420 nm (120 min)
52%
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49%
N.D. = not detected
† concentration of phosphinothioester 6 = 0.2 mmol
‡ concentration of azide = 0.2 mmol
One of the advantageous features of traceless Staudinger ligation reaction is its ability to conjugate
molecule units by a simple peptide bond without any other residue atoms at the conjugation.
Because of this, it has long been recognized as a tool for convergent synthesis of peptides and
proteins.28-30 By caging the phosphinothioester traceless Staudinger ligation reagents with the new
3,3-dimethylbutynyl substituted anthracenylmethyl photo-cage, it becomes possible to regulate
peptide and protein synthesis by visible light. This cannot be achieved by any other photo-click
processes currently available. This concept is demonstrated by the phosphonium salts 7 and 8.
Instead of a simple (acetylthio)methyl moiety in 6, compound 7 and 8 contain R2 = Ac-Gly and
Ac-Ala-Gly, respectively, as the “click-able” amino acid and dipeptide units, which tether to the
phosphonium via a thiol group to their C-termini (Table 2 & Table 3). Their solubility properties
in organic and aqueous organic solvents are comparable to that of 6, despite of the larger non-polar
R2 that they possess. Both reagents underwent photo-induced traceless Staudinger ligation with
six azidylated amino acids and dipeptides (9a – 9f) at excitation > 420 nm to give the corresponding
oligopeptides, which were isolated from the reaction mixtures and characterized by mass
spectrometry and 1H- and 13C NMR spectrometry. Yields of those oligopeptides are ranged from
31 to 43%, which are consistent with those obtained with traditional traceless Staudinger ligation
processes.28-30
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In summary, we demonstrated that by extending the -conjugation of the anthracenyl chromophore
of the photo-cage of diarylphosphinothioester traceless Staudinger ligation reagents, the
wavelength responses of the photo-uncaging process can be shifted into the visible spectral range.
This should render the photo-triggered traceless Staudinger ligation process more bio-compatible.
The new (10-(3,3-dimethylbut-1-yn-1-yl)anthracen-9-yl)methyl photo-cage on phosphonium salts
can be uncaged with excitation > 420 nm. Visible light-induced conjugation of amino acids and
peptides by peptide linkages have been achieved with moderate yields. This opens up the
possibility of in situ synthesis of functional proteins/peptides mediated by photo-click processes
for the regulation of cellular/metabolic functions of life systems.
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Table 2 Visible light-triggered traceless Staudinger ligation of 7† with azidylated amino acids and peptides.
5
6
7
h (> 420 nm)*
8
9
THF/buffer (pH 7.4)
16 h, RT
Traceless Staudinger
ligation product
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7
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Entry
Azide compound‡
Oligopeptide product
Yield (%)
Ac-Gly-Gly-NHBn
1
41%
10a
9a
Ac-Gly-Phe-NHBn
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3
4
38%
32%
43%
10b
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9c
Ac-Gly-Leu-NHBn
10c
Ac-Gly-Gly-Phe-OMe
10d
9d
9e
Ac-Gly-Phe-Val-NH2
5
6
39%
31%
10e
Ac-Gly-Leu-Phe-OMe
10f
9f
* duration of photolysis = 120 min
† concentration of phosphinothioester 7 = 0.2 mmol
‡ concentration of azide = 0.2 mmol
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4
Table 3 Visible light-triggered traceless Staudinger ligation of 8† with azidylated amino acids and peptides.
5
6
7
h (> 420 nm)*
8
THF/buffer (pH 7.4)
9
16 h, RT
Traceless Staudinger
ligation product
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Entry
Azide compound‡
Oligopeptide product
Yield (%)
Ac-Ala-Gly-Phe-NHBn
1
35%
11a
9b
Ac-Ala-Gly-Leu-NHBn
2
3
30%
33%
11b
9c
9e
Ac-Ala-Gly-Phe-Val-NH2
11c
Ac-Ala-Gly-Leu-Phe-OMe
4
31%
11d
9f
* duration of photolysis = 120 min
† concentration of phosphinothioester 8 = 0.2 mmol
‡ concentration of azide = 0.2 mmol
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Experimental Section
Methods
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All reagents were purchased and used without further purification unless specified otherwise.
Solvents for photolysis studies were degassed with nitrogen. S-((Diphenylphosphaneyl)-
methyl)ethanethioate was prepared according to literature methods.22 (10-Bromoanthracen-9-
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31
yl)methanol was prepared according to literature method.31 1H NMR, C NMR and P NMR
spectra were recorded on a 300 MHz and a 400 MHz Bruker nuclear resonance spectrometers. 31P
NMR spectra were proton-decoupled. Mass spectra were measured by an PE SCIEX API 150EX
mass spectrometer and an Applied Biosystems QSTAR Elite hybrid quadrupole time-of-flight
mass spectrometer. UV-vis spectra were measured by an Agilent 8453 ultraviolet-visible
spectrophotometer. Light source used for photolysis studies was a Newport 67005 500W Hg lamp
equipped with a Newport 61945 IR reducer. Broadband irradiation was achieved using optical
filters of appropriate cut-off wavelengths (Newport & Edmund). Quantum yield measurements at
specific excitation wavelength were carried out using a Newport 77200 monochromator.
1
Qualitative and quantitative determination of reaction products were characterized by H NMR
and mass spectrometry.
Synthesis of photocaged phosphinothioester (2 – 6)
Synthesis of (10-phenylanthracen-9-yl)methanol
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(10-Bromoanthracen-9-yl)methanol (2.01 g, 7 mmol) and phenylboronic acid (2.56 g, 21 mmol)
were dissolved in 35 mL of degassed THF. Pd(OAc)2 (157 mg, 0.7 mmol), BINAP (436 mg, 0.7
mmol) and potassium carbonate (2.90 g, 21 mmol) were then added. The suspension was refluxed
under N2 for 16 h. The reaction mixture was concentrated under vacuum and purified by
chromatography on silica gel eluting with hexane/ethyl acetate (v/v 3/1) to give (10-
phenylanthracen-9-yl) methanol as a pale yellow amorphous solid (1.82 g, yield 92%). 1H NMR
(300 MHz, CDCl3): δ 8.50 (d, J = 9.0 Hz, 2H), 7.68 (d, J = 9.0 Hz, 2H), 7.53-7.62 (m, 5H), 7.33-
7.42 (m, 4H), 5.76 (d, J = 6.0 Hz, 2H), 1.82 (t, J = 6.0 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3):
δ 138.9, 131.1, 130.9, 130.2, 129.9, 128.4, 127.9, 127.6, 126.2, 125.0, 123.9, 57.6 ppm.
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Synthesis of (10-(4-(trifluoromethyl)phenyl)anthracen-9-yl)-methanol
(10-Bromoanthracen-9-yl)methanol (574 mg, 2 mmol) and (4-(trifluoromethyl)phenyl)boronic
acid (1.14 g, 6 mmol) were dissolved in 10 mL of degassed THF. Pd(OAc)2 (45 mg, 0.2 mmol),
BINAP (124 mg, 0.2 mmol) and potassium carbonate (829 mg, 6 mmol) were then added. The
suspension was reflux under N2 for 48 h. The reaction mixture was concentrated under vacuum
and purified by chromatography on silica gel eluting with hexane/ethyl acetate (v/v 3/1) to give
(10-(4-(trifluoromethyl)phenyl)anthracen-9-yl)methanol as a pale yellow amorphous solid (410
mg, yield 58%). 1H NMR (400 MHz, CDCl3): δ 8.51 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 8.0 Hz, 2H),
7.51-7.62 (m, 6H), 7.39 (t, J = 8.0 Hz, 2 Hz), 5.76 (d, J = 4.0 Hz, 2H), 1.87 (t, J = 4.0 Hz, 1H)
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ppm. 13C NMR (100 MHz, CDCl3): δ 143.0, 136.9, 131.7, 131.6, 130.1, 129.9, 129.9, 127.2, 126.3,
5
6
125.5, 125.5, 124.1, 123.0, 57.5 ppm.
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Synthesis of (10-(4-methoxyphenyl)anthracen-9-yl)methanol
(10-Bromoanthracen-9-yl)methanol (2.01 g, 7 mmol) and 4-(methoxyphenyl)boronic acid (3.19 g,
21 mmol) were dissolved in 35 mL of degassed THF. Pd(OAc)2 (157 mg, 0.7 mmol), BINAP (436
mg, 0.7 mmol) and potassium carbonate (2.90 g, 21 mmol) were then added. The suspension was
reflux under N2 for 16 h. The reaction mixture was concentrated under vacuum and purified by
chromatography on silica gel eluting with hexane/ethyl acetate (v/v 3/1) to give (10-(4-
methoxyphenyl)anthracen-9-yl)methanol as a pale yellow amorphous solid (1.64 g, yield 85%).
1H NMR (300 MHz, CDCl3): δ 8.45 (d, J = 9.0 Hz, 2H), 7.73 (d, J = 9.0 Hz, 2H), 7.48-7.56 (m,
2H), 7.31-7.39 (m, 2H), 7.23-7.31 (m, 2H), 7.07-7.13 (m, 2H), 5.68 (d, J = 3.0 Hz, 2H), 3.93 (s, 3
H), 1.99 (t, J = 3.0 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3): δ 159.1, 138.7, 132.2, 130.9, 130.8,
130.5, 130.0, 127.9, 126.2, 125.0, 123.9, 113.9, 57.5, 55.4 ppm.
Synthesis of (10-(phenylethynyl)anthracen-9-yl)methanol
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(10-Bromoanthracen-9-yl)methanol (1.15 g, 4 mmol) and ethynylbenzene (879 μL, 8 mmol) were
dissolved in 15 mL of degassed THF. PdCl2(PPh3)2 (281 mg, 0.4 mmol), Pd(PPh3)4 (462 mg, 0.4
mmol), CuI (76 mg, 0.4 mmol) and 5 mL trimethylamine were then added. The suspension was
reflux under N2 for 16 h. The reaction mixture was concentrated under vacuum and purified by
chromatography on silica gel eluting with hexane/ethyl acetate (v/v 3/1) to give (10-
(phenylethynyl)anthracen-9-yl)methanol as a yellow amorphous solid (699 mg, yield 79%). 1H
NMR (400 MHz, CDCl3): δ 8.75 (dd, J1 = 4.0 Hz, J2 = 4.0 Hz, 2H), 8.46 (dd, J1 = 4.0 Hz, J2 = 4.0
Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.63 (dd, J1 = 4.0 Hz, J2 = 4.0 Hz, 4H), 7.39-7.49 (m, 3H), 5.70
13
(d, J = 4.0 Hz, 2H), 1.80 (t, J =4.0 Hz, 1H) ppm. C NMR (100 MHz, CDCl3): δ 132.4, 132.1,
131.7, 129.9, 128.7, 128.6, 127.7, 126.7, 126.3, 124.3, 123.5, 119.2, 86.3, 57.5 ppm.
Synthesis of (10-(3,3-dimethylbut-1-yn-1-yl)anthracen-9-yl)methanol
(10-Bromoanthracen-9-yl)methanol (574 mg, 2 mmol) and 3,3-dimethylbut-1-yne (709 μL, 6
mmol) were dissolved in 9 mL of degassed THF. PdCl2(PPh3)2 (140 mg, 0.2 mmol), Pd(PPh3)4
(231 mg, 0.2 mmol), CuI (38 mg, 0.2 mmol) and 3 mL trimethylamine were then added. The
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suspension was reflux under N2 for 16 h. The reaction mixture was concentrated under vacuum
and purified by chromatography on silica gel eluting with hexane/ethyl acetate (v/v 3/1) to give
(10-(3,3-dimethylbut-1-yn-1-yl)anthracen-9-yl)methanol as an orange amorphous solid (495
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mg, yield 86%). 1H NMR (400 MHz, CDCl3): δ 8.57-8.63 (m, 2H), 8.36-8.45 (m, 2H), 7.54-7.62
(m, 4H), 5.66 (d, J = 4.0 Hz, 2H), 1.76 (t, J =4.0 Hz, 1H), 1.55 (s, 9H) ppm. 13C NMR (100 MHz,
CDCl3): δ 132.2, 131.0, 129.8, 127.8, 126.5, 125.9, 124.2, 120.2, 111.3, 75.8, 57.4, 31.4, 29.0 ppm.
9-(Chloromethyl)-10-phenylanthracene
(10-phenylanthracen-9-yl)methanol (1.14 g, 4 mmol) was dissolved in 200 mL of dry
dichloromethane and cooled to -10 ℃. Pyridine (338 μl, 4.2 mmol) and SOCl2 (305 μl, 4.2 mmol)
were then added. The resulting solution was stirred in an ice bath for 7 h. The mixture was then
concentrated and purified by flash chromatography on silica gel eluting with
dichloromethane/ethyl acetate (v/v 1/1) to give 9-(chloromethyl)-10-phenylanthracene as a yellow
amorphous solid (1.15 g, yield 95%). 1H NMR (400 MHz, CDCl3): δ 8.39 (d, J =8.0 Hz, 2H), 7.69
13
(d, J = 12.0 Hz, 2H), 7.52-7.65 (m, 5H), 7.34-7.42 (m, 4H), 1.98 (d, 2H) ppm. C NMR (100
MHz, CDCl3): δ 139.8, 138.7, 131.1, 130.2, 129.7, 128.4, 128.1, 127.7, 127.6, 126.7, 125.1, 123.4,
39.3 ppm.
9-(Chloromethyl)-10-(4-(trifluoromethyl)phenyl)anthracene
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(10-(4-(trifluoromethyl)phenyl)anthracen-9-yl)-methanol (352 mg, 1 mmol) was dissolved in 50
mL of dry dichloromethane and cooled to -10 ℃. Pyridine (85 μl, 1.05 mmol) and SOCl2 (76 μl,
1.05 mmol) were then added. The resulting solution was stirred in an ice bath for 7 h. The mixture
was then concentrated and purified by flash chromatography on silica gel eluting with
dichloromethane/ethyl
acetate
(v/v
1/1)
to
give
9-(chloromethyl)-10-(4-
1
(trifluoromethyl)phenyl)anthracene as a yellow amorphous solid (330 mg, yield 89%). H NMR
(400 MHz, CDCl3): δ 8.41 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 8.0 Hz, 2H), 7.63 (t, J = 8.0 Hz, 2 Hz),
7.58 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.41 (t, J = 8.0 Hz, 2H), 5.70 (s, 2H) ppm. 13C
NMR (100 MHz, CDCl3): δ 142.7, 137.8, 131.6, 130.2, 129.9, 129.6, 128.4, 127.4, 126.8, 125.6,
125.5, 125.5, 123.6, 123.0, 39.1 ppm.
9-(Chloromethyl)-10-(4-methoxyphenyl)anthracene
(10-(4-methoxyphenyl)anthracen-9-yl)methanol (943 mg, 3 mmol) was dissolved in 150 mL of
dry dichloromethane and cooled to -10 ℃. Pyridine (254 μl, 3.15 mmol) and SOCl2 (229 μl, 3.15
mmol) were then added. The resulting solution was stirred in an ice bath for 7 h. The mixture was
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then concentrated and purified by flash chromatography on silica gel eluting with
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6
dichloromethane/ethyl
acetate
(v/v
1/1)
to
give
9-(chloromethyl)-10-(4-
7
8
9
methoxyphenyl)anthracene as a yellow amorphous solid (998 mg, yield 80%). 1H NMR (400 MHz,
CDCl3): δ 8.38 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H), 7.61 (t, J = 8.0 Hz, 2H), 7.38 (t, J =
8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 5.69 (s, 2H), 3.95 (s, 3 H) ppm. 13C
NMR (100 MHz, CDCl3): δ 159.2, 139.7, 132.2, 130.7, 130.5, 129.7, 128.1, 127.4, 126.6, 125.1,
123.4, 113.9, 55.4, 39.4 ppm.
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9-(Chloromethyl)-10-(phenylethynyl)anthracene
(10-(Phenylethynyl)anthracen-9-yl)methanol (617 mg, 2 mmol) was dissolved in 100 mL of
dry dichloromethane and cooled to -10 ℃. Pyridine (169 μl, 2.1 mmol) and SOCl2 (153 μl, 2.1
mmol) were then added. The resulting solution was stirred in an ice bath for 7 h. The mixture was
then concentrated and purified by flash chromatography on silica gel eluting with
dichloromethane/ethyl acetate (v/v 1/1) to give 9-(chloromethyl)-10-(phenylethynyl)anthracene as
1
a yellow amorphous solid (595 mg, yield 91%). H NMR (400 MHz, CDCl3): δ 8.75 (d, J = 8.0
Hz, 2H), 8.36 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.61-7.70 (m,
13
4H), 7.40-7.50 (m, 3H), 5.63 (s, 2H) ppm. C NMR (100 MHz, CDCl3): δ 132.3, 131.7, 129.6,
128.8, 128.6, 127.9, 127.2, 126.4, 123.8, 123.3, 120.2, 102.1, 86.2, 38.9 ppm.
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(10-(3,3-Dimethylbut-1-yn-1-yl)anthracen-9-yl)methanol (432 mg, 1.5 mmol) was dissolved
in 75 mL of dry dichloromethane and cooled to -10 ℃. Pyridine (127 μl, 1.58 mmol) and SOCl2
(115 μl, 1.58 mmol) were then added. The resulting solution was stirred in an ice bath for 7 h. The
mixture was then concentrated and purified by flash chromatography on silica gel eluting with
dichloromethane/ethyl acetate (v/v 1/1) to give 9-(chloromethyl)-10-(3,3-dimethylbut-1-yn-1-
yl)anthracene as an orange amorphous solid (311 mg, yield 85%). 1H NMR (400 MHz, CDCl3):
δ 8.61 (d, J = 8.0 Hz, 2H), 8.31 (d, J = 4.0 Hz, 2H), 7.56-7.66 (m, 4H), 5.60 (s, 2H), 1.55 (s, 9H)
ppm. 13C NMR (100 MHz, CDCl3): δ 132.2, 129.5, 128.0, 127.6, 127.0, 126.0, 123.7, 121.2, 111.9,
75.8, 39.1, 31.3, 29.0 ppm.
Photocaged phosphinothioester 2
Diphenylphosphinothioester (549 mg, 2 mmol) and 9-(chloromethyl)-10-phenylanthracene (666
mg, 2.2 mmol) were dissolved in 2 mL of chloroform. The resulting solution was stirred at ambient
temperature under N2 for 16 h. The reaction mixture was added to 60 mL of diethyl ether, stirred
for 2 h and filtered to give the photocaged phosphinothioester 2 as a yellow amorphous solid (1.14
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g, yield 99%). 1H NMR (400 MHz, CDCl3): δ 8.30 (d, J = 12.0 Hz, 2H), 7.49-7.60 (m, 10H), 7.27-
7.38 (m, 8H), 7.21 (t, J = 8.0 Hz, 2H), 6.19 (d, J = 16.0 Hz, 2H), 5.24 (d, J = 8.0 Hz, 2H), 2.15 (s,
3H) ppm. 13C NMR (100 MHz, CDCl3): δ 191.0 (d, J = 2.0 Hz), 139.3 (d, J = 7.0 Hz), 138.3 (d, J
= 2.0 Hz), 134.8 (d, J = 3.0 Hz), 133.9 (d, J = 9.0 Hz), 130.0 (d, J = 2.0 Hz), 130.5 (d, J = 6.0 Hz),
129.7 (d, J = 5.0 Hz), 129.3 (d, J = 12.0 Hz), 128.4, 127.8, 127.7, 126.7 (d, J = 2.0 Hz), 125.0,
124.5 (d, J = 3.0 Hz), 119.8 (d, J = 11.0 Hz), 115.8 (d, J = 83.0 Hz), 29.8, 25.9 (d, J = 43.0 Hz),
21.3 (d, J = 49.0 Hz) ppm. 31P NMR (400 MHz, CDCl3): δ 28.01 ppm. HRMS (ESI): m/z calcd
for C36H30OPS [M-Cl-]+ 541.1755, found 541.1750.
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Photocaged phosphinothioester 3
Diphenylphosphinothioester
(137
mg,
0.5
mmol)
and
9-(chloromethyl)-10-(4-
(trifluoromethyl)phenyl)anthracene (204 mg, 0.55 mmol) were dissolved in 1 mL of chloroform.
The resulting solution was stirred at ambient temperature under N2 for 16 h. The reaction mixture
was added to 30 mL of diethyl ether, stirred for 2 h and filtered to give the photocaged
phosphinothioester 3 as a yellow amorphous solid (300 mg, yield 93%). 1H NMR (400 MHz,
CDCl3): δ 8.35 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 8.0 Hz, 2H), 7.53-7.61 (m, 6H), 7.30-7.47 (m,
10H), 7.23-7.28 (m, 2H), 6.24 (d, J = 16.0 Hz, 2H), 5.25 (d, J = 8.0 Hz, 2H), 2.16 (s, 3H) ppm.
13C NMR (100 MHz, CDCl3): δ 191.0 (d, J = 2.0 Hz), 142.4, 137.3 (d, J = 6.0 Hz), 134.8 (d, J =
3.0 Hz), 134.0 (d, J = 9.0 Hz), 131.5 (d, J = 2.0 Hz), 130.5 (d, J = 6.0 Hz), 130.2 (d, J = 32.0 Hz),
129.5 (d, J = 4.0 Hz), 129.3 (d, J = 12.0 Hz), 127.0, 126.8, 125.6, 125.5 (d, J = 3.0 Hz), 124.8 (d,
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J = 3.0 Hz), 122.9, 110.3 (d, J = 11.0 Hz), 115.9 (d, J = 84.0 Hz), 29.8, 26.0 (d, J = 43.0 Hz), 21.3
5
6
31
(d, J = 49.0 Hz) ppm. P NMR (400 MHz, CDCl3): δ 28.16 ppm. HRMS (ESI): m/z calcd for
7
8
9
C37H29F3OPS [M-Cl-]+ 609.1629, found 609.1630.
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Photocaged phosphinothioester 4
Diphenylphosphinothioester
(549
mg,
2
mmol)
and
9-(chloromethyl)-10-(4-
methoxyphenyl)anthracene (204 mg, 2.2 mmol) were dissolved in 2 mL of chloroform. The
resulting solution was stirred at ambient temperature under N2 for 48 h. The reaction mixture was
added to 60 mL of diethyl ether, stirred for 2 h and filtered to give the photocaged
1
phosphinothioester 4 as a yellow amorphous solid (1.03 g, yield 85%). H NMR (400 MHz,
CDCl3): δ 8.28 (d, J = 8.0 Hz, 2H), 7.51-7.60 (m, 8H), 7.29-7.37 (m, 6H), 7.19-7.25 (m, 4H), 7.11
(d, J = 8.0 Hz, 2H), 6.18 (d, J = 16.0 Hz, 2H), 5.24 (d, J = 8.0 Hz, 2H), 3.95 (s, 3H), 2.15 (s, 3H)
ppm. 13C NMR (100 MHz, CDCl3): δ 191.1 (d, J = 2.0 Hz), 159.2, 139.2 (d, J = 6.0 Hz), 134.7 (d,
J = 3.0 Hz), 134.0 (d, J = 9.0 Hz), 132.2 (d, J = 2.0 Hz), 130.6 (d, J = 6.0 Hz), 130.3 (d, J = 2.0
Hz), 120.1 (d, J = 5.0 Hz), 129.3 (d, J = 12.0 Hz), 127.8, 126.7 (d, J = 2.0 Hz), 125.0, 124.5 (d, J
=13.0 Hz), 119.0 (d, J = 11.0 Hz), 116.0 (d, J = 83.0 Hz), 113.9, 55.4, 29.8, 25.9 (d, J = 43.0 Hz),
21.3 (d, J = 49.0 Hz) ppm. 31P NMR (400 MHz, CDCl3): δ 27.93 ppm. HRMS (ESI): m/z calcd
for C37H32O2PS [M-Cl-]+ 571.1861, found 571.1860.
Photocaged phosphinothioester 5
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Diphenylphosphinothioester
(274
mg,
1
mmol)
and
9-(chloromethyl)-10-
(phenylethynyl)anthracene (360 mg, 1.1 mmol) were dissolved in 1 mL of chloroform. The
resulting solution was stirred at ambient temperature under N2 for 16 h. The reaction mixture was
added to 30 mL of diethyl ether, stirred for 2 h and filtered to give the photocaged
phosphinothioester 5 as a yellow amorphous solid (535 mg, yield 89%). 1H NMR (400 MHz,
CDCl3): δ 8.57 (d, J = 8.0 Hz, 2H), 8.27 (d, J = 8.0 Hz, 2H), 7.75 (d, J = 8.0 Hz, 2H), 7.49-7.58
(m, 6H), 7.42-7.49 (m, 5H), 7.30-7.40 (m, 6H), 6.15 (d, J = 16.0 Hz, 2H), 5.12 (d, J = 8.0 Hz, 2H),
13
2.13 (s, 3H) ppm. C NMR (100 MHz, CDCl3): δ 190.8 (d, J = 2.0 Hz), 134.8 (d, J = 2.0 Hz),
134. (d, J = 9.0 Hz), 131.7, 131.65, 130.5 (d, J = 6.0 Hz), 129.4 (d, J = 13.0 Hz), 128.8, 128.6,
127.4, 127.0, 126.3, 125.0 (d, J = 3.0 Hz), 123.2, 120.3 (d, J = 11.0 Hz), 119.5 (d, J = 7.0 Hz),
115.7 (d, J = 83.0 Hz), 102.2 (d, J = 2.0 Hz), 86.0 (d, J = 4.0 Hz), 29.8, 25.9 (d, J = 43.0 Hz), 21.1
31
(d, J = 49.0 Hz) ppm. P NMR (400 MHz, CDCl3): δ 27.33 ppm. HRMS (ESI): m/z calcd for
C38H30OPS [M-Cl-]+ 565.1755, found 565.1755.
Photocaged phosphinothioester 6
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Diphenylphosphinothioester (137 mg, 0.5 mmol) and 9-(chloromethyl)-10-(3,3-dimethylbut-
1-yn-1-yl)anthracene (169 mg, 0.55 mmol) were dissolved in 1 mL of chloroform. The resulting
7
8
9
solution was stirred at ambient temperature under N2 for 16 h. The reaction mixture was added to
30 mL of diethyl ether, stirred for 2 h and filtered to give the photocaged phosphinothioester 6 as
an orange amorphous solid (273 mg, yield 94%). 1H NMR (400 MHz, CDCl3): δ 8.50 (d, J = 8.0
Hz, 2H), 8.26 (d, J = 8.0 Hz, 2H), 7.51-7.60 (m, 6H), 7.45 (t, J = 8.0 Hz, 2H), 7.31-7.40 (m, 6H),
6.14 (d, J = 16.0 Hz, 2H), 5.16 (d, J = 8.0 Hz, 2H), 2.15 (s, 3H), 1.56 (s, 9H) ppm. 13C NMR (100
MHz, CDCl3): δ 190.9 (d, J = 2.0 Hz), 134.8 (d, J = 3.0 Hz), 134.1 (d, J = 9.0 Hz), 131.7 (d, J =
5.0 Hz), 130.6 (d, J = 6.0 Hz), 129.3 (d, J = 12.0 Hz), 127.6, 126.9 (d, J = 2.0 Hz), 126.0, 124.8
(d, J = 3.0 Hz), 120.7 (d, J = 7.0 Hz), 119.0 (d, J = 11.0 Hz), 115.7 (d, J = 83.0 Hz), 112.1 (d, J =
3.0 Hz), 75.6 (d, J = 4.0 Hz), 31.2, 29.8, 29.0, 25.9 (d, J = 43.0 Hz), 20.9 (d, J = 49.0 Hz) ppm.
31P NMR (400 MHz, CDCl3): δ 27.02 ppm. HRMS (ESI): m/z calcd for C36H34OPS [M-Cl-]+
545.2068, found 545.2070.
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Synthesis of photocaged phosphinothioester (7) with an Ac-Gly moiety as “click-able” amino acid
Synthesis of (diphenylphosphino)methanethiol
Diphenylphosphine thioester (7.132 g, 26.0 mmol) was dissolved in anhydrous methanol (160 mL),
and N2 was bubbled through the solution for 1 h. Sodium hydroxide (2.08 g, 52.0 mmol) was then
added, and the mixture was stirred under N2 for 2 h. Solvent was removed under reduced pressure,
and the residue was dissolved in methylene chloride (200 mL). The resulting solution was washed
with 2 N HCl (2 × 200 mL) and brine (200 mL). The organic layer was dried over anhydrous
MgSO4 and filtered, and solvent was removed under reduced pressure. The residue was purified
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by flash chromatography (alumina, v/v 1/3) to give (diphenylphosphino)methanethiol as a clear
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oil (5.374 g, yield 89%). H NMR (400 MHz, CDCl3): δ 7.41-7.47 (m, 4H), 7.34-7.37 (m, 6H),
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3.05 (d, J = 8.0 Hz, 2H), 1.38 (q, J = 8.0 Hz, 1H) ppm. 13C NMR (100 MHz, CDCl3): δ 137.1 (d,
J = 19.0 Hz), 132.9 (d, J = 25.0 Hz), 129.2, 128.8 (d, J = 9.0 Hz), 21.0 (d, J = 32.0 Hz) ppm. 31P
NMR (400 MHz, CDCl3): δ -7.93 ppm.
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Synthesis of S-((diphenylphosphino)methyl)-2-acetamido-ethanethioate
(Diphenylphosphino)methanethiol (2.323 g, 10 mmol) and 2-acetamidoacetic acid (1.171 g, 10
mmol) were dissolved in 100 mL dichloromethane and 40 mL DMF. DCC (1.942 g, 10 mmol)
was then added. The mixture was stirred at room temperature under N2 for 16h. The resulting
mixture was filtered. The filtrate was concentrated and purified by chromatography on silica gel
eluting with hexane/ethyl acetate (v/v 1/2) to give the product as a white amorphous solid (2.61 g,
yield 79%). 1H NMR (300 MHz, CDCl3): δ 7.32-7.46 (m, 10H), 6.00 (br, 1H), 4.16 (d, J = 6.0 Hz,
2H), 3.52 (d, J = 3.0 Hz, 2H), 2.02 (s, 3H) ppm. 13C NMR (75 MHz, CDCl3): δ 196.2 (d, J = 3.75
Hz), 170.4, 136.5 (d, J = 13.5 Hz), 132.8 (d, J = 19.5 Hz), 129.3, 128.7 (d, J = 6.75 Hz), 49.1, 25.4
(d, J = 24.0 Hz), 23.0 ppm. 31P NMR (300 MHz, CDCl3): δ -15.24 ppm. MS (ESI): m/z calcd for
C17H19NO2PS [M+H]+ 332.0, found 332.1.
Synthesis of photocaged phosphinothioester 7
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S-((Diphenylphosphino)methyl)-2-acetamidoethanethioate (1.658 g,
5
mmol) and 9-
(chloromethyl)-10-(3,3-dimethylbut-1-yn-1-yl)anthracene (1.606 g, 5.25 mmol) were dissolved in
8 mL chloroform. The resulting solution was stirred at ambient temperature under N2 for 16 h. The
reaction mixture was added to 100 mL of diethyl ether/hexane(v/v 1/1), stirred for 2 h and filtered
to give the product as a yellow amorphous solid (3.045 g, yield 91%). 1H NMR (300 MHz, CDCl3):
δ 9.45 (t, J = 6.0 Hz, 1H), 8.40 (d, J = 9.0 Hz, 2H), 8.07 (d, J = 9.0 Hz, 2H), 7.41-7.53 (m, 6H),
7.21-7.40 (m, 8H), 5.76 (d, J = 15.0 Hz, 2H), 4.56 (d, J = 9.0 Hz, 2H), 3.95 (d, J = 6.0 Hz, 2H),
2.10 (s, 3H), 1.55 (s, 9H) ppm. 13C NMR (75 MHz, CDCl3): δ 195.9 (d, J = 1.5 Hz), 172.3, 135.0,
134.0 (d, J = 9.8 Hz), 131.4 (d, J = 4.5 Hz), 130.2 (d, J = 6.0 Hz), 129.4 (d, J = 12.0 Hz), 127.5,
126.8, 125.8, 124.3, 120.7 (d, J = 6.8 Hz), 118.2 (d, J = 11.3 Hz), 116.0, 114.9, 112.1 (d, J = 3.0
Hz), 75.4 (d, J = 3.8 Hz), 48.6, 31.2, 28.9, 24.3 (d, J = 44.3 Hz), 22.9, 20.6 (d, J = 50.3 Hz) ppm.
31P NMR (300 MHz, CDCl3): δ 24.22 ppm. HRMS (ESI): m/z calcd for C38H37NO2PS [M-Cl-]+
602.2283, found 602.2285.
Synthesis of photocaged phosphinothioester (8) with an Ac-Ala-Gly moiety as “click-able”
dipeptide
Synthesis
of
S-((diphenylphosphaneyl)methyl)-2-(2-((tert-butoxycarbonyl)amino)-
propanamido)ethanethioate
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