Nitrodibenzofuran: A One-and Two-Photon Sensitive Protecting Group That Is Superior to Brominated Hydroxycoumarin for Thiol Caging in Peptides

Article abstract of DOI:10.1021/jacs.5b11759

Photoremovable protecting groups are important for a wide range of applications in peptide chemistry. Using Fmoc-Cys(Bhc-MOM)-OH, peptides containing a Bhc-protected cysteine residue can be easily prepared. However, such protected thiols can undergo isomerization to a dead-end product (a 4-methylcoumarin-3-yl thioether) upon photolysis. To circumvent that photoisomerization problem, we explored the use of nitrodibenzofuran (NDBF) for thiol protection by preparing cysteine-containing peptides where the thiol is masked with an NDBF group. This was accomplished by synthesizing Fmoc-Cys(NDBF)-OH and incorporating that residue into peptides by standard solid-phase peptide synthesis procedures. Irradiation with 365 nm light or two-photon excitation with 800 nm light resulted in efficient deprotection. To probe biological utility, thiol group uncaging was carried out using a peptide derived from the protein K-Ras4B to yield a sequence that is a known substrate for protein farnesyltransferase; irradiation of the NDBF-caged peptide in the presence of the enzyme resulted in the formation of the farnesylated product. Additionally, incubation of human ovarian carcinoma (SKOV3) cells with an NDBF-caged version of a farnesylated peptide followed by UV irradiation resulted in migration of the peptide from the cytosol/Golgi to the plasma membrane due to enzymatic palmitoylation. Overall, the high cleavage efficiency devoid of side reactions and significant two-photon cross-section of NDBF render it superior to Bhc for thiol group caging. This protecting group should be useful for a plethora of applications ranging from the development of light-Activatable cysteine-containing peptides to the development of light-sensitive biomaterials.

Full text of DOI:10.1021/jacs.5b11759

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Article
Nitrodibenzofuran: a One- and Two-Photon Sensitive Protecting Group that
is Superior to Brominated Hydroxycoumarin for Thiol Caging in Peptides
M. Mohsen Mahmoodi, Daniel Abate-Pella, Tom J Pundsack, Charuta
C. Palsuledesai, Philip C. Goff, David A Blank, and Mark D. Distefano
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b11759 • Publication Date (Web): 30 Mar 2016
Downloaded from http://pubs.acs.org on March 30, 2016
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Nitrodibenzofuran: a One- and Two-Photon Sensitive Protecting
Group that is Superior to Brominated Hydroxycoumarin for Thiol
Caging in Peptides
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M. Mohsen Mahmoodi, Daniel Abate-Pella, Tom J. Pundsack, Charuta C. Palsuledesai, Philip C. Goff, David A.
Blank and Mark D. Distefano*
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, 55455, USA
ABSTRACT: Photoremovable protecting groups are important for a wide range of applications in peptide chemistry. Using Fmoc-Cys(Bhc-
MOM)-OH, peptides containing a Bhc-protected cysteine residue can be easily prepared. However, such protected thiols can undergo
isomerization to a dead-end product (a 4-methylcoumarin-3-yl thioether) upon photolysis. To circumvent that photo-isomerization problem,
we explored the use of nitrodibenzofuran (NDBF) for thiol protection by preparing cysteine-containing peptides where the thiol is masked
with an NDBF group. This was accomplished by synthesizing Fmoc-Cys(NDBF)-OH and incorporating that residue into peptides by
standard solid phase peptide synthesis procedures. Irradiation with 365 nm light or two-photon excitation with 800 nm light resulted in
efficient deprotection. To probe biological utility, thiol group uncaging was carried out using a peptide derived from the protein K-Ras4B to
yield a sequence that is a known substrate for protein farnesyltransferase; irradiation of the NDBF-caged peptide in the presence of the
enzyme resulted in the formation of the farnesylated product. Additionally, incubation of human ovarian carcinoma (SKOV3) cells with an
NDBF-caged version of a farnesylated peptide followed by UV irradiation resulted in migration of the peptide from the cytosol/Golgi to the
plasma membrane due to enzymatic palmitoylation. Overall, the high cleavage efficiency devoid of side reactions and significant TP cross-
section of NDBF render it superior to Bhc for thiol group caging. This protecting group should be useful for a plethora of applications ranging
from development of light-activatable cysteine-containing peptides to the development of light-sensitive biomaterials.
enzymology19. Hence, significant efforts have gone into the
preparation of proteins and ligands/substrates containing caged
INTRODUCTION
thiols that can be triggered with light to reveal bioactive species;20–24
for that purpose, several protecting groups have been explored.25–29
The most widely used approach for thiol protection involves caging
with o-nitrobenzyl (ONB) groups. Among the advantages of ONB
groups are high one-photon quantum efficiency and high yield of
free compound produced upon photolysis.30 However, they are
poor chromophores with low two-photon (TP) sensitivities which
limit their biological applications. To address this issue, coumarin-
based protecting groups have been recently employed, which
absorb light at longer wavelengths and possess significantly higher
one- and two-photon absorptivity.31 In one important study, Hagen
and coworkers harnessed the chromatic orthogonality of ONB- and
coumarin-based protecting groups to introduce a wavelength
selective thiol caging system.32 Using a combination of those
protecting groups, they were able to selectively uncage different
thiols in a peptide sequence using different wavelengths for UV
irradiation; however, no TP photochemistry was explored. In
another novel study, Shoichet and coworkers incorporated
brominated hydroxycoumarin (Bhc) protected thiols into
hydrogels that allowed them to perform light induced protein
patterning within those materials with high spatial control.33,34 The
high TP sensitivity of Bhc allowed them to create 3D protein
patterns inside these polymeric scaffolds for tissue engineering
applications.
The ability of light to traverse various chemical and biological
barriers and be modulated by time and amplitude makes light-
regulated molecules unique tools for a plethora of applications in
the areas of chemistry and biology.1–4 Photoremovable protecting
groups, also known as caging groups, are one of the most important
light-regulated tools, which can be utilized to mask specific
functional groups in molecules such that they can be cleaved on
demand upon irradiation.5,6 In biological applications, this typically
involves masking a biomolecule with a caging group to produce a
compound whose biological activity is either increased or
decreased upon uncaging.7–9 The recent development of two-
photon sensitive protecting groups, which allow uncaging using
near IR irradiation, has resulted in significant improvements in the
spatiotemporal resolution of uncaging as well as increased
penetration with lower phototoxicity;10–14 the latter attribute is of
particular importance for the use of caged molecules in tissue
samples or intact organisms that are essentially opaque to UV light.
Due to inherent differences in the chemical reactivity of various
functional groups, there is no single photocleavable protecting
group that works efficiently for caging all functionalities. Hence,
protecting group selection must be performed on a case by case
basis.15,16
Thiol-containing compounds play vital roles in many aspects of
biology (e.g. controlling cellular redox state),17 protein chemistry
(e.g. protein and peptide folding, native chemical ligation18) and
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In recent work, we demonstrated that Bhc could also be used for
experiments were purchased from Sigma Aldrich (St. Louis, MO).
thiol protection of a peptidomimetic enzyme inhibitor.35 The high
one- and two-photon sensitivity of Bhc allowed efficient uncaging
of the inhibitor inside cells for biological applications. Inspired by
these results, we reasoned that Bhc could also be used for thiol
protection of cysteine-containing peptides. In the work reported
here, we first explored the use of Bhc-protected cysteine in
peptides. While straightforward to prepare using Fmoc-Cys(Bhc)-
OH as a building block in solid phase peptide synthesis, irradiation
of such caged peptides was complicated by the generation of an
unwanted photo-isomer instead of the free thiol. Using NMR
analysis, we were able to confirm the structure of the photo-isomer
to be a 4-methylcoumarin-3-yl thioether, in agreement with a
previous prediction by Hagen and coworkers.32 Further analysis of
several different peptide sequences revealed that the photo-
cleavage efficiency of Bhc-protected thiols is context dependent
and typically leads to photoisomer formation as the major product.
To circumvent this problem, we next explored using
nitrodibenzofuran (NDBF)36 as a thiol caging group since it
manifests a two-photon cross-section comparable to that of Bhc.
Thus, cysteine-containing peptides were prepared where the thiol
was protected with an NDBF group. This was accomplished by
preparing Fmoc-Cys(NDBF)-OH and incorporating it into
peptides via standard solid phase peptide synthesis. In contrast to
Bhc-caged thiols, irradiation of NDBF-protected thiols at 365 nm
resulted in clean conversion to the free mercaptan. Deprotection
was also obtained via irradiation at 800 nm where the TP action
cross-section was measured to be comparable to that of Bhc
protected acetate (Bhc-OAc). To probe the utility of this
protecting group for biological experiments, thiol group uncaging
was carried out using a K-Ras-derived peptide containing an
NDBF-protected cysteine. Irradiation of that molecule in the
presence of protein farnesyltransferase (PFTase) and farnesyl
diphosphate (FPP) resulted in the formation of the free thiol form
and subsequent enzymatic conversion to a prenylated species.
Those experiments indicate that one- and two-photon
deprotection can be performed under mild conditions that allow
enzymatic activity to be retained. In order to illustrate the utility of
this strategy for the development of caged peptides that can be
activated via irradiation inside live cells, the thiol of a cell-
penetrating peptide known to be a substrate for palmitoyl
acyltransferase (PAT) was protected as a NDBF thioether.
Irradiation of human ovarian carcinoma (SKOV3) cells, pre-
incubated with the probe, resulted in migration of the peptide from
the cytosol/Golgi to the plasma membrane (visualized via confocal
microscopy) due to enzymatic palmitoylation. These data suggest
that the NDBF group should be useful for caging thiols in peptides
and potentially larger proteins assembled via native chemical
ligation18 for biological applications. The high uncaging yield of
NDBF caged thiols upon one- and two-photon irradiation, together
with the facile incorporation of caged cysteine via standard solid
phase peptide synthesis into peptides containing multiple cysteines,
make this a highly versatile strategy for studying cysteine-
containing peptides and proteins.
HPLC analysis (analytical and preparative) was performed using a
Beckman model 125/166 instrument, equipped with a UV detector
and C18 columns (Varian Microsorb-MV, 5 µm, 4.6 x 250 mm and
1
Phenomenex Luna, 10 µm, 10 x 250 mm respectively). H NMR
data of synthetic compounds were recorded at 500 MHz on a
1
Varian Instrument at 25 °C. H NMR data for the products of
photolysis reactions were recorded using a Bruker Advance III 700
MHz spectrometer with 1.7 mm TCI cryoprobe.
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General procedure for solid phase peptide synthesis. Peptides
were synthesized using an automated solid-phase peptide
synthesizer (PS3, Protein Technologies Inc., Memphis, TN)
employing Fmoc/HCTU based chemistry. Fmoc-Met-Wang resin
(0.25 mmol) was transferred into a reaction vessel and peptide
chain elongation was performed using HCTU and N-
methylmorpholine. Standard amino acid coupling was performed
by incubation of 4 equiv of both HCTU and Fmoc protected
amino acid with the resin for 30 min. Coupling of Fmoc-Cys(Bhc)-
OH or Fmoc-Cys(NDBF)-OH was performed by incubation of 1.5
equiv of both the amino acid and HCTU with the resin for 6 h.
Peptide chain elongation was completed by N-terminus
deprotection using 10% piperidine in DMF (v/v). 5-Fam coupling
was conducted by incubation of 1.2 equiv of 5-Fam, 1.2 equiv of
HCTU and 2 equiv of N,N-diisopropylethylamine (DIEA) with
the resin for 8 h. Resin was then transferred into a syringe filter and
washed three times with DCM and dried in vacuo. Global
deprotection and resin cleavage was accomplished via treatment
with Reagent K. Peptides were then precipitated with Et2O,
pelleted by centrifugation and the residue rinsed twice with Et2O.
The resulting crude peptide was dissolved in MeOH and purified
by preparative RP-HPLC.
General procedure for synthesis of peptides containing C-
terminal methyl esters. Trityl chloride resin (1eq) was transferred
into a fitted syringe and washed 3 times with DMF. In a separate
flask, Fmoc-Cys-OMe (3 eq) was treated with DIEA (6 eq) in
DCM, then transferred into the resin containing fitted syringe, then
mixed overnight using a rotisserie. Resins were treated with MeOH
to cap any unreacted trityl moiety, followed by washing with DMF
(3 ×). The prepared Fmoc-Cys-OMe loaded resins were used to
synthesize peptides containing C-terminal methyl esters via
traditional Fmoc/HCTU based chemistry as described in “general
procedure for peptide synthesis”.
5-Fam-KKKSKTKC(Bhc)VIM (5). ESI-MS: calcd for
[C87H126BrN16O23S2 + 3H]3+ 635.2597, found 635.2568.
5-Fam-KKKSKTKC(NDBF)VIM (17b). ESI-MS: calcd for
[C91H130N17O23S2 + 3H]3+ 630.9650, found 630.9658.
C(Bhc)VLS. ESI-MS: calcd for [C27H37BrN4O9S + H]+ 673.1537,
found 673.1575.
Dansyl-GC(Bhc)VLS. ESI-MS: calcd for [C41H51BrN6O12S2 + H]+
963.2363, found 963.2302.
Dansyl-GC(NDBF)VLS. ESI-MS: calcd for[C45H55N7O12S2 + Na]+
972.3248, found 972.3280.
KKKSKC(Bhc)CVIM (5). ESI-MS: calcd for [C63H109BrN15O17S3
EXPERIMENTAL SECTION
+ 3H]3+ 507.5485, found 507.5497.
General. All reagents needed for solid phase peptide synthesis
were purchased from Peptide International (Louisville, KY). All
other solvents and reagents used for synthesis and other
KKKSKTCC(Bhc)IM. ESI-MS: calcd for [C64H112BrN16O17S3 +
3H]3+ 517.2240, found 517.2240.
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KKKSKTKC(NDBF)VIM
(17a)
ESI-MS:
calcd
for
Fmoc-Cys(NDBF)-OH (16). Ester 15 (900 mg, 1.50 mmol) was
dissolved in 25 mL of CH2Cl2 and Me3SnOH (678 mg, 3.75 mmol)
was added. The reaction was refluxed for 7 h and monitored by
TLC (1:1 Hex/EtOAc) at which point the solvent was removed in
vacuo and the resulting oil dissolved in EtOAc (30 mL). The
organic layer was washed with 5% HCl (3 x 10 mL) and brine (3 x
10 mL), dried with Na2SO4 and evaporated to give 786 mg of 16 as
[C70H117N17O17S2 + 3H]3+ 511.6158, found 511.6238.
NBD-Hex-C(NDBF)LC-OMe (20) ESI-MS: calcd for
[C54H70N8O11S2 + H]+ 1071.4684, found 1071.4284.
Fmoc-Cys(MOM-Bhc)-OCH3 (3). Chloride 2 (93 mg, 0.28
mmol) and Fmoc-Cys-OCH3 (200 mg, 0.56 mmol) were dissolved
in 3 mL of a solution of 2:1:1 DMF/CH3CN/H2O/0.1% TFA
(v/v). Zn(OAc)2 was then added (154 mg, 0.70 mmol) and the
reaction monitored by TLC (1:1 Hex/EtOAc). After two days, the
solvent was removed and the reaction purified via column
chromatography (1:1 Hex/EtOAc) to give 149 mg of 3 as yellow
1
a yellow powder (90% yield) as a diastereomic mixture. H NMR
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(CDCl3) δ 8.36 – 8.39 (1H), 7.97 - 8.03 (2H, m), 7.75 – 7.77 (2H,
m), 7.53 – 7.62 (4H, m), 7.30 – 7.42 (5H, m), 5.58 – 5.62 (1H, m),
4.88 – 4.91 (1H, m), 4.54 - 4.66 (1H, m), 4.16 – 4.40 (3H, m), 2.88
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-3.04 (2H, m), 1.71 – 1.74 (3H, m). C NMR (CDCl3) δ 174.42,
1
powder (81% yield). H NMR (CDCl3) δ 7.83 (1H, s) 7.76 (2H, d,
158.28, 155.88, 153.66, 147.66, 143.75, 141.31, 133.37, 129.45,
128.95 127.73, 127.11, 125.21, 123.77, 122.41, 121.87, 120.99,
119.97, 112.18, 108.25, 67.49, 53.48, 47.02, 39.60, 33.70, 23.66.
HR-MS (ESI) m/z calcd for [C32H26N2O7S + Na]+ 605.1358, found
619.1335.
J = 7.5), 7.6 (2H, d, J=7.5 Hz), 7.38 (2H, m), 7.29 (2H, m), 7.13
(1H, s), 6.36 (1H, s), 5.74 (2H, s), 4.68 (1H, m), 4.38-4.48 (2H,
m), 4.20 (1H, t), 3.74 (3H, s), 3.50 (3H, s). HR-MS (ESI) m/z
calcd for (C31H28BrNO8S + Na)+ 676.0611 (79Br) and 678.0596
(81Br), found 676.0639 (79Br) and 678.0636 (81Br).
General procedure for UV photolysis of caged molecules. The
caged compound was dissolved in photolysis buffer (50 mM
phosphate buffer, pH 7.4 containing 1 mM DTT) at a final
concentration of 25-250 µM. The solutions were transferred into a
quartz cuvette (10 x 50 mm) and irradiated with 365 nm UV light
using a Rayonet reactor (2 x 14 watt RPR-3500 bulbs). After each
reaction the samples were analyzed by RP-HPLC or LC-MS.
Fmoc-Cys(MOM-Bhc)-OH (4). Ester 3 (100 mg, 0.15 mmol)
and Me3SnOH (69 mg, 0.38 mmol) were dissolved in CH2Cl2 (5
mL) and brought to reflux. After 7 h the reaction was judged
complete by TLC (1:1 Hex/EtOAc), the solvent removed in vacuo
and the resulting oil redissolved in EtOAc (20 mL). The organic
layer was washed with 5% HCl (3 x 10 mL) and brine (3 x 10 mL),
dried with Na2SO4 and evaporated to give 92 mg of 4 as a yellow
powder (90% yield). 1H NMR (d6-acetone) δ 8.12 (1H, s), 7.86
(2H, d, J=7.5), 7.73 (2H, t, J=7), 7.41 (2H, t, J=7.5), 7.33 (2H, m),
7.16 (1H, s), 6.42 (1H, s), 5.64 (1H, s), 5.42 (2H, s), 4.51 (1H, b),
4.37-4.41 (2H, m), 4.32 (1H, t), 4.25 (1H, t), 4.07 (2H, d), 3.49
(3H, s). HR-MS (ESI) m/z calcd for [C30H26BrNO8S + Na]+
662.0455 (79Br) and 664.0439 (81Br), found 662.0472 (79Br) and
664.0428 (81Br).
General procedure for LC-MS analysis. Aliquots (100 µL)
containing 5-20 µM caged compound in photolysis buffer were
irradiated in a Rayonet UV photo-reactor or using an 800 nm laser
(see below for description). Each irradiated sample was then
analyzed by LC-MS. The general gradient for LC-MS analysis was
0–100% H2O (0.1% HCO2H) to CH3CN (0.1% HCO2H) in 25
min.
Photolysis study of Bhc protected Boc-cysteamine (11) and
NMR analysis of the photo-isomerization reaction. Aliquots
(200 µL) containing compound 11 (200 µM in photolysis buffer)
were irradiated at 365 nm for 80 and 400 s. After each illumination,
samples were analyzed via RP-HPLC by monitoring the
absorbance at 220 nm. To obtain sufficient photo-isomer for NMR
analysis, 10 mL of a 300 µM solution of 11 was irradiated for 6 min
and purified via preparative RP-HPLC. The collected eluate was
lyophilized to yield ~ 1 mg of the desired compound that was then
Fmoc-Cys(NDBF)-OCH3 (15). NDBF-Br (1.00 g, 3.12 mmol)
and Fmoc-Cys-OCH3 (2.2 g, 6.25 mmol) were dissolved in 60 mL
of a solution of 2:1:1 DMF/ACN/0.1% TFA in H2O (v/v/v). A 0.5
M aqueous solution of Zn(OAc)2 was prepared in 0.1% TFA (v/v)
and 25 µL of that solution was added to the reaction mixture. The
reaction was monitored by TLC (1:1 Hex/Et2O) and stopped after
36 h of stirring at rt. Solvent was evaporated in vacuo and the final
product was purified via column chromatography (1:1 Hex/ Et2O)
to give 0.90 g of a diastereomeric mixture of 15 as a yellow oil (48
1
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dissolved in 500 µL of d6-acetone and subjected to H NMR
% yield). H NMR (CDCl3) δ 8.38 – 8.40 (1H), 8.01 - 8.06 (2H,
analysis.
m), 7.77 (2H, m), 7.54 – 7.63 (4H, m), 7.30 – 7.45 (5H, m), 5.58 –
5.59 (1H, m), 4.84 – 4.88 (1H, m), 4.53 – 4.59 (1H, m), 4.14 –
4.40 (3H, m), 3.72 – 3.78 (3H), 2.84 -3.03 (2H, m), 1.72 – 1.74
(3H, m). HR-MS (ESI) m/z calcd for [C33H28N2O7S + Na]+
619.1515, found 619.1537.
Photolysis rate and quantum efficiency of 17b using UV
excitation. Aliquots (100 µL) containing 17b (200 µM in
photolysis buffer) were irradiated at 365 nm in a rayonet UV
reactor. The duration of irradiation ranged from 5 to 90 sec. After
each irradiation interval, 80 µL of the sample was analyzed by RP-
HPLC. The reaction samples were eluted with gradient of 0.1%
TFA in H2O (Solvent A) and 0.1% TFA in CH3CN (Solvent B)
(gradient of a 3% increase in Solvent B/min, flow rate 1 mL/min)
and monitored by fluorescence (λex = 492 nm, λem = 518 nm).
Reaction progress data was plotted in KaleidaGraph 3.0 and fitted
via non-linear regressional analysis to a first-order process. The
quantum efficiency (Qu) was calculated using the formula
Qu=(Iσt90%)-1, where I is the irradiation intensity in ein cm-2s-1, σ is
the decadic extinction coefficient (103 times ɛ, molar extinction
Fmoc-Cys(NV)-OMe. This compound was synthesized following
the same procedure described above for synthesis of 15, except
NDBF-Br was replaced with 1-(bromomethyl)-4,5-dimethoxy-2-
1
nitrobenzene (NV-Br). Yield was 80 %. H NMR (CDCl3) δ 7.78
(2H, d, J = 7.5), 7.68 (1H, s), 7.62 (2H, t, J = 8.0 Hz), 7.42 (2H, t, J
= 7.5 Hz), 7.33 (2H, t, J = 8 Hz), 6.89 (1H, s), 5.66 (H, d, J = 8.0
Hz), 4.64 (1H, q, J = 7.5 Hz), 4.42 (2H, d, J = 7.0 Hz), 4.25 (1H, t,
J = 7.5 Hz), 4.00 (3H, s), 3.94 (3H, s), 3.81 (3H, s), 3.00 (2H, m).
HR-MS (ESI) m/z calcd for [C28H28N2O8S + Na]+ 575.1464,
found 575.1493.
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coefficient) in cm2 mol-1 and t90% is the irradiation time in sec for
of the peptide with the plasma membrane, in the presence and
absence of UV exposure, was statistically quantified using Pearson
correlation coefficient analysis calculated using FIJI software.
90% conversion to the product.31 The UV intensity of the lamps (I)
in the photo-reactor was measured using potassium ferrioxalate
actinometry.37
Cell viability assay. SKOV3 cells were grown and irradiated for 0,
5 and 50 min following same procedure described above. In each
sample, media was replaced with 1 mL Mcoy’s 5a medium (10 %
FBS) containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) and incubated for one hour
at 37 °C under CO2 (5.0%). Media was removed from each plate
and cells were washed with 1X warm PBS buffer. 1.5 mL DMSO
was added to each plate to lyse the cells. The cells were placed on
an orbital shaker for 15 min until cells get completely dissolved.
Absorbance were obtained at 570 nm using UV spectrometer. Data
were normalized such that cells that were not exposed to irradiation
had a cell viability of 100 %.
Laser apparatus for two-photon irradiations. The light source
that was utilized for two-photon irradiation is a homebuilt,
regeneratively amplified Ti:sapphire laser system. This laser
operates at 1 kHz with 210 mw pulses centered at a wavelength of
800 nm. The laser pulses have a Gaussian full width at half
maximum of 80 fs. Samples were irradiated in a 15 µL quartz
cuvettes (Starna Cells corp.).
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Two-photon uncaging cross-section (δu) of 17a at 800 nm. The
two-photon action cross-section for 17a was measured by
comparing the photolysis rate of 17a with Bhc-OAc as a reference
(δu = 0.45 at 800 nm). Aliquots (100 µL) containing 17a (300 µM
in photolysis buffer) were irradiated with the 800 nm laser system
for varying amounts of time ranging from 2.5 to 30 min. Each
sample was analyzed by HPLC using the method described above.
Similar photolysis experiments were conducted using 100 µL
aliquots of Bhc-OAc (100 µM in 50 mM phosphate buffer, pH 7.2).
Photolyzed Bhc-OAc solutions were also analyzed by RP-HPLC.
The compounds were eluted with a gradient of Solvent A and
Solvent B (gradient of a 1% increase in Solvent B/min, flow rate 1
mL/min) monitored by absorbance at 220 nm. Reaction progress
data was analyzed as described above and the first order decay
constants for the two compounds were used in the formula δuΦu
(17a) = δuΦu (reference) × Kobs (17a)/ Kobs(reference) to calculate
the value of δu for 17a where δuΦu (reference) = 0.45 GM.
RESULTS AND DISCUSSION
1. Synthesis and Studies of the Photolysis of Bhc-
Protected Cysteine-Containing Peptides.
1.1. Synthesis of Bhc-Protected Fmoc-Cysteine and
Incorporation into Peptides. Previous studies in our
laboratory have shown that Bhc can be used for photolabile thiol
protection of a peptidomimetic enzyme inhibitor. The caged
molecule manifested efficient cleavage to yield a free thiol upon
one- and two-photon irradiation allowing it to be used for
biological applications in cell culture. Therefore, in order to
develop photolabile S-protected cysteine-containing peptides, we
initially used Bhc as a caging group. Our strategy was to prepare
Bhc-protected Fmoc-cysteine and incorporate that into a peptide
of interest through solid phase peptide synthesis (SPPS); the
synthesis of a form of cysteine suitable for SPPS is depicted in
Scheme 1. The phenolic hydroxyl group of Bhc-chloride (1) was
protected using chloromethyl methyl ether and triethylamine to
give MOM-protected Bhc-Cl (2) in 89% yield. This chloride was
subsequently used to alkylate Fmoc-cysteine methyl ester under
mild acidic conditions using Zn(OAc)2 as a catalyst38 to produce 3
in 80 % yield. Saponification of the methyl ester using trimethyl tin
hydroxide39 under reflux generated the desired caged Fmoc-
Cysteine derivative (4) in 81% yield.
UV and two-photon triggered farnesylation of 17. A 7.5 µM
solution of compound 17 was prepared in prenylation buffer (15
mM DTT, 10 mM MgCl2, 50 µM ZnCl2, 20 mM KCl and 22 µM
FPP) and divided into three 100 µL aliquots. Yeast PFTase was
added to the first aliquot to give a final concentration of 30 nM but
the resulting sample was not subjected to photolysis, The second
aliquot was irradiated in absence of yeast PFTase while the third
sample was supplemented with yeast PFTase (50 nM) and then
photolyzed with either UV or laser light. UV photolysis was
conducted for 1 min at 365 nm while two-photon irradiation was
performed for 2.5 min at 800 nm. Each sample was incubated for
30 min at rt and then analyzed by LC-MS as described above.
Cell culture and microscopy. SKOV3 cells were grown in Mcoy’s
5a medium containing 10 % FBS at 37 ºC under CO2 (5.0%). For
microscopy experiments cells were seeded into 35 mm glass
bottomed dishes at a density of 8 × 103 cells/ cm2. To monitor the
trafficking of 20 before and after UV irradiation inside cells,
SKOV3 cells were incubated with 5 µM of 20 for 3 h. The medium
was then replaced with RPMI (10 % FBS) medium without phenol
red. Half of the plates were irradiated at 330 nm for 5 min using a
transilluminator (Fotodyne Inc.), then all of the plates were
incubated for 120 min at 37 ºC under CO2 (5.0%). Cells were then
incubated with Hoecht 33342 (2 µg/mL) and AF488-WGA (15
µg/mL) in Mcoy’s 5a (10 % FBS) medium for 10 min. The
medium was removed and cells were washed 3 times with warm
PBS buffer followed by RPMI medium (10 % FBS, no phenol red).
Cells were directly imaged using an Olympus FluowView IX2
inverted confocal microscope with a 60X objective. Co-localization
Scheme 1.
Synthesis of Bhc-Protected Fmoc-Cys and
Incorporation into a Peptide via SPPS.
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Extracted ion current (EIC) chromatograms obtained by LC-
MS analysis (Figure 1B, C), clearly revealed the disappearance of
the starting peptide (tR = 8.45 min, m/z = 635.26) upon photolysis
and concomitant formation of a new peak with identical mass (tR =
8.70 min, m/z = 635.26) that corresponds to the photo-
isomererized product. To study whether any free (uncaged)
peptide (6) was produced upon irradiation, 6 was synthesized by
an independent route and subjected to LC-MS. Analysis of the LC-
MS traces of the irradiated peptide, showed no evidence for the
presence of free peptide ions that match with the authentic
standard (Figure 1C, D).
We initially hypothesized that the observed isomerization might be
due to photo-induced migration of the Bhc group to a side chain
amine group of a neighboring lysine residue. This hypothesis was
tested by MS/MS fragmentation analysis, since migration of Bhc to
other residues would change the fragmentation of the photo-
isomer relative to the starting peptide. Interestingly, MS/MS
analysis revealed that the two isomers have the same backbone
fragmentation pattern (Table S1). Of particular importance, two of
the main fragments present in both the photo-isomer and the caged
peptide are the doubly charged X4 and B7 ions (Figure S2), which
clearly indicate that the Bhc group remains connected to cysteine
even after irradiation. This observation is not consistent with the
hypothesis of photo-triggered Bhc migration to lysine residues and
indicates that a rearrangement occurs directly on the caged cysteine
residue.
The high extent of photo-isomerization observed upon photolysis
of compound 5, was somewhat contrary to our previous results
with Bhc protected inhibitors, where photolysis led to efficient
(>85%) uncaging. Thus, we considered the possibility that the
photo-cleavage of Bhc caged thiols may be context dependent. To
test this hypothesis, two additional peptides were synthesized in
which the location of caged cysteine was moved by one residue
along the peptide sequence. Each peptide was photolyzed
separately and the resulting products were analyzed by LC-MS.
Based on the EIC data (Figures S3 and S4), photolysis of
KKKSKTCC(Bhc)IM produced only the photo-isomer and no
detectable uncaged peptide. In contrast, photolysis of
KKKSKTC(Bhc)CVIM generated a mixture of both the photo-
isomer and free peptide. This hypothesis was also tested with
shorter peptides where C(Bhc)VLS showed formation both
uncaged and isomerized product (Figure S5), whereas photolysis
of dansyl-GC(Bhc)VLS did not produce any uncaged peptide
(Figure S6). These data confirm that the efficiency of Bhc photo-
cleavage is highly dependent on its surrounding chemical
environment. It is worth noting that photoisomerization was also
observed upon two photon excitation of C(Bhc)VLS (Figure S7).
Previously reported mechanistic studies have demonstrated that
the photo-cleavage of coumarin-based protected carboxylic acids
results in scission of the C-O bond to produce a reactive
carbocation which is rapidly quenched by water when the latter is
used as the solvent.40 However, an alternative pathway could
involve reaction of the cationic intermediate with an internal
nucleophile via an intramolecular process; such a mechanism
would give rise to an isomeric product consistent with the
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The general route for synthesis of caged peptides employed
standard SPPS conditions, in which Fmoc protected residues were
added sequentially to a peptide anchored on Wang resin. The only
exception involved the incorporation of the caged Fmoc-Cys
residue where the coupling time was increased to 6 hours to ensure
quantitative incorporation of the non-standard residue. Final
treatment of the resin-bound peptide with acid (standard
conditions using Reagent K) removed all side-chain protecting
groups, including the MOM group present on the Bhc moiety, and
cleaved the polypeptide from the resin to generate the desired
caged molecule. This strategy was successfully employed to
synthesize a caged form of K-Ras peptide, 5 that includes an N-
terminal fluorescein group. While the uncaged form of that peptide
is a known substrate for the enzyme, PFTase, the Bhc-protected
form is not. The goal was to use light to uncage the peptide and
restore its ability to be recognized by the enzyme and undergo
farnesylation.
1.2. Photolysis of Bhc Protected Cysteine Containing
Peptides. Having successfully synthesized and purified the
fluorescein-labeled caged peptide, the next step was to verify its
uncaging efficiency upon photolysis. Hence, solutions of caged
peptide in photolysis buffer (1 mM DTT in 50 mM phosphate
buffer at pH 7.2) were irradiated using 365 nm light in a Rayonet
photoreactor for varying amount of time (Figure 1A). Each sample
was analyzed by reversed-phase HPLC and monitored by
fluorescence. Inspection of the HPLC traces (Figure S1) revealed
that the starting peptide peak disappeared over time with
concomitant formation of a new peak with a later retention time.
Surprisingly, further analysis of the reaction mixture via ESI-MS
revealed that the photolytic product and non-irradiated starting
peptide had identical masses indicating that irradiation causes
isomerization instead of uncaging.
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observations reported here. To examine this possibility, photolysis
photo-isomerization. These results suggest that the photo-
reactions were performed in the presence of high concentrations of
thiols in order to increase the rate of trapping. Thus, aqueous
solutions of 5 were irradiated in presence of excess DTT (up to 200
mM), and analyzed by RP-HPLC. Interestingly, none of those
experiments revealed any measurable change in the extent of
rearrangement may proceed through a concerted intramolecular
mechanism, however, additional experiments are needed to
thoroughly address this question.
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Figure 1. (A) Uncaging reaction of Bhc protected cysteine containing peptide (5) upon UV irradiation. LC-MS analysis of photolysis of peptide 5:
(B) EIC chromatogram (m/z = 635.20-635.30) of a sample of 5 in photolysis buffer, (C) EIC chromatogram (m/z = 635.20-635.30) of sample of 5
after 120 s photolysis showing the formation of a photo-isomer, (D) EIC chromatogram (m/z = 551.20-551.30) of a standard sample of free peptide
6b showing that no uncaged product was detected upon photolysis of 5.
suggested by Hagen and coworkers.32 In such a case, the resonance
1.3. NMR Analysis of Bhc Photo-Rearrangement.
The possibility of photo-rearrangement of related amino-
coumarin-protected cysteines has been previously suggested by
Hagen and coworkers, however, in their publication, no analysis
was performed to conclusively identify the structure of photo-
isomer generated.32 Therefore, after first observing the photo-
rearrangement of Bhc caged thiols by mass-spectroscopy, we
decided to determine the structure of the isomeric product using
NMR methods. In order to obtain sufficient material for NMR
analysis, a solution of caged peptide 5 was irradiated and the photo-
at 2.4 ppm could be attributed to the presence a methyl group in
the final photo-product (10).
1
Due to the complicated H NMR spectra of the peptides, we
decided to validate the proposed hypothesis using a simpler model
system. Hence, Bhc protected cysteamine (11, Figure 2), which has
a simple structure and a straightforward synthetic route, was
chosen as the model system. Additionally, this specific molecule
has been previously used by Shoichet and coworkers for photo-
triggered uncaging of thiol functionality inside hydrogels.28
Therefore, knowing all the complexities due to context dependence
of Bhc photo-cleavage, we were also interested to see how efficient
this compound could undergo uncaging.
1
isomer was isolated via preparative RP-HPLC purification. H
NMR spectra were obtained using a sensitive cryo-probe
instrument. As shown in Figure S8A (5) and S8B (photo-isomer),
both compounds have very similar spectra, with the exception of a
distinctive peak at 6.2 ppm present in the spectrum of 5 that is
missing in the spectrum of the photo-isomer; concomitantly, a new
signal appears at 2.4 ppm in the spectrum of the isomer that is not
present in the spectrum of 5. Comparison of those spectra with the
Compound 11 was synthesized following a previously reported
procedure.33 Solutions of 11 were irradiated using 365 nm light in a
Rayonet photoreactor for varying amounts of time followed by
analysis via RP-HPLC with UV detection. That allowed the
disappearance of 11 as well as the formation of the isomeric
rearrangement product (12) and Bhc-OH (7, formed from the
desired uncaging reaction) to be monitored. Inspection of the
HPLC data (Figure S9) indicates that the major product of
photolysis of compound 11 under these conditions is the photo-
isomer (12, the product with a higher retention time) with a
smaller amount of the desired uncaged product formed, as
indicated by the low intensity peak corresponding to Bhc-OH (7).
1
Bhc H NMR spectrum31 indicates that the signal at 6.2 ppm
corresponds to the aryl proton at the 3 position of Bhc (Scheme 2).
The disappearance of that peak and the appearance of the new
signal at 2.4 ppm are consistent with a photo-induced 1,3 shift of
the sulfur atom from the exocylic position to the 3 position to give
intermediate 9 followed by tautomerization to yield a 4-
methylbromohydroxycoumarin-3-yl thioether (10) as previously
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Scheme 2. Hypothesized mechanism of photo-isomerization of
Bhc caged cysteine.
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Reactions containing Bhc-cysteamine (11) and its corresponding
photo-isomer (12) were separated by reversed-phase HPLC and
the purified compounds analyzed by NMR spectroscopy.
Comparison of the 1H NMR spectrum of 11 with that of
compound 12 revealed characteristic changes in the proton signals
corresponding to those observed in the peptide experiment (Figure
2). Methylene (Hd) and aryl (He) protons present in the starting
material are absent in the spectrum of the photo-isomer. In
addition, a new signal at 2.72 ppm (Hh) corresponds to the new
methyl group that is generated. Also of note, the triplet signal
(H’b), corresponding to the methylene protons of cysteamine,
shifts downfield relative to that of the starting compound (Hb) as a
result of thiol conjugation with double bonds which renders the
thiol a stronger electron-withdrawing group; alternatively, this shift
may be due to a ring current effect. These observations
convincingly support the suggested mechanism for photo-
rearrangement and the proposed structure of the photo-isomer. A
similar photolysis experiment was performed in D2O. LC-MS
analysis clearly indicates formation of a mono-deuterated photo-
isomer (Figure S10). This data demonstrates that there is a solvent-
derived proton incorporated in the product, consistent with the
mechanism proposed for photo-rearrangement described in
Scheme 2.
Figure 2. (A) Photolysis of Bhc-protected Boc-cysteamine and the
resulting photolytic products. (B) 1H NMR spectrum of Bhc protected
Boc-cysteamine (top) and the corresponding photo-isomer (bottom).
2. Use of Nitrodibenzofuran for Development of Caged
Cysteine-Containing Peptides.
2.1. Alternative Strategy using NDBF. The initial goal of our
work was to identify a protecting group that could be used to cage
the thiol group of cysteine when present within a peptide that
would be efficiently deprotected through UV irradiation as well as
near infrared light via a two-photon process. Although Bhc has
shown reasonable one- and two-photon uncaging efficiencies for
protection of various functionalities including carboxylates,
phosphates and carbamates, the experiments described above
revealed its photo-cleavage efficiency is unpredictable when used
with thiols; moreover, the main product formed upon irradiation is
often an unwanted rearrangement byproduct in lieu of the desired
free thiol. To address these limitations, we elected to examine
another type of caging group that undergoes uncaging via a process
significantly different from coumarin-based compounds.
o-Nitrobenzyl (ONB) based caging groups have been extensively
used for thiol photo-caging. Despite, their relatively slower
uncaging rate (compared with coumarins), they undergo
photolysis with minimal byproduct formation. However, ONB-
based compounds suffer from low one-photon and especially low
two-photon absorptivity which limits their applications in cellular
To obtain an accurate ratio of the extent of uncaging versus photo-
rearrangement, a sample of compound 11 that had been subjected
to irradiation, and thus contained both uncaged and photo-
isomerized product, was analyzed by 1H-NMR spectroscopy
(Figure S11). Using integration values obtained from characteristic
protons from each product, a ratio for uncaging versus photo-
isomerization of 1:10 was calculated. Thus, while Shoichet and
coworkers have used 11 (under different conditions) to
successfully uncage a thiol upon photolysis, the experiments
reported here revealed that the major product of this reaction is an
unwanted photo-isomer. Overall, the variability obtained using Bhc
suggests that it is not generally applicable as a caging group for
thiols and that there is a real need for an alternative caging group
for general usage.
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media and live tissue. In 2006, Momotake et. al. introduced
nitrodibenzofuran (NDBF), a more extensively conjugated form of
ONB, as a new caging group with high one- and two-photon
sensitivity.36 This compound has previously been used for
protection of hydroxyl functionalities and showed rapid and
efficient uncaging upon one- and two-photon irradiation.41 Due to
its advantages over traditional ONB based molecules, we decided
to explore its applicability for photo-caging of thiols in order to
prepare peptides containing caged cysteine residues.
2.3. One- and Two-Photon Photolysis of NDBF-Caged
Cysteine Peptides.
Scheme 4. Light triggered uncaging of NDBF protected K-Ras
peptide (17b).
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2.2. Synthesis of NDBF-Protected Fmoc-Cys-OH and
Incorporation via SPPS. Similar to the synthesis of Bhc-caged
peptides, the strategy pursued here was to first synthesize Fmoc-
cysteine containing an NDBF-protected thiol [Fmoc-Cys(NDBF)-
OH] and then incorporate that into a peptide through standard
SPPS. Starting from dibenzofuran, NDBF-Br (13) was synthesized
in four steps (Scheme S1). Next, as described in Scheme 3, Fmoc-
cysteine methyl ester was first reacted with NDBF-Br under acidic
conditions, to produce compound 15 in 70 % yield. The resulting
methyl ester was then hydrolyzed using (CH3)3SnOH to yield
Fmoc-Cys(NDBF)-OH (16) in 75 % yield.
Scheme 3. Synthesis of NDBF Protected Fmoc-Cys-OH and
Incorporation into Peptide Sequence via SPPS.
After completing the synthesis of the fluorescently labeled caged
peptide 17b, photolysis experiments were conducted to probe for
the formation of the uncaged peptide containing a free thiol upon
photolysis (Scheme 4). In this experiment, a solution of 17b was
irradiated at 365 nm for 45 seconds and subjected to RP-HPLC. As
can be seen from the chromatograms shown in Figure S12,
photolysis resulted in the disappearance of the peak corresponding
to the starting peptide 17b and concomitant appearance of a new
peak tentatively assigned as 6b. ESI-MS/MS analysis confirmed
that the newly formed peak corresponds to the expected free
peptide (Figure S12, Table S3). The absence of any unwanted
photo-products based on an HPLC trace devoid of any other
significant products, suggests that that photolysis of NDBF-caged
peptides undergo conversion to free peptide upon UV irradiation
with high efficiency. In order to further evaluate the general
applicability of this strategy,
a second peptide, dansyl-
GC(NDBF)VLS was also synthesized and studied. Analysis of a
photolysis reaction containing that peptide showed complete
conversion to the free peptide upon irradiation (Figure S13),
unlike its Bhc-protected counterpart (compare with Figure S6).
This data suggests that NDBF lacks the limitations and undesired
reactivity manifested by Bhc for thiol caging.
One-photon uncaging kinetics of compound 17b were evaluated
by irradiating solutions of 17b for varying periods of time followed
by analysis via RP-HPLC (Figure 3A). Based on that data,
summarized in Figure 5A, the uncaging quantum yield (ɛΦ) of
peptide 17b was measured to be 0.2. The quantum yield measured
in this experiment is somewhat lower than the value reported for
NDBF used for caging hydroxyl functionality (0.7)36, which may be
due to the light absorption of fluorescein attached to the peptide.
However, due to the high molar absorptivity of NDBF, which
results in a high ɛΦ value, the uncaging t1/2 was quite short (25
The resulting protected cysteine residue was successfully
incorporated into several K-Ras-derived peptides (17a,b) via
standard SPPS as described for the related Bhc-protected peptides
noted above; the final products were characterized by ESI-MS-MS
to confirm the presence of the NDBF group after the global
deprotection step (Table S2). Since, NBDF protection of cysteine
involves a thioether bond, there was no evidence of any S- to N-
shift or deprotection occurring during synthesis, a problem that
does occur when thiocarbamate-based protection strategies are
used.32
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seconds) under the photolysis conditions (standard Rayonet
reactor).
2.4. One- and Two-Photon Triggered In Vitro
Farnesylation of a Caged K-Ras Peptide. Since the NDBF-
caged peptide demonstrated good uncaging efficiency, we next
sought to examine its utility in a more biologically relevant context.
Protein prenylation is a critical process that affects key signaling
mechanisms within cells involved in a plethora of functions from
growth to differentiation.42 Prenyl groups are appended to proteins
via thioether linkages formed by alkylation of specific cysteine
residues catalyzed by prenyltransferases including protein
farnesyltransferase (PFTase), which transfers a farnesyl (C15)
group.43 Thus, a K-Ras-derived peptide incorporating a caged
cysteine residue at the site of prenylation should not be a substrate
for PFTase; however, upon irradiation, the resulting peptide
manifesting a free thiol produced by photocleavage of the
protecting group should be an efficient substrate and become
farnesylated (Figure 4A). In order to test this, a series of
experiments was conducted in which a caged peptide was treated
with farnesyl diphosphate (FPP) in prenylation buffer (15 mM
DTT, 10 mM MgCl2, 50 µM ZnCl2, 20 mM KCl), with or without
enzyme addition and UV light exposure.
As predicted, the caged peptide (17b) was not farnesylated when
treated with yeast PFTase. LC-MS analysis of reaction mixture
indicates only the presence of the caged peptide (m/z = 630.87,
Figure 4A). Photolysis of 17b for 60 seconds at 365 nm, in the
absence of enzyme, generated the uncaged peptide as confirmed by
the formation of a new peak with the expected m/z value (m/z =
551.21, Figure S15). However, photolysis of 17b in the presence of
PFTase, led to the generation of a farnesylated peptide (19b). The
new peak with the retention time of 10.55 min has a mass to charge
ratio of 619.26 which is in good agreement with the calculated
value (C92H145N16O20S23+, 619.34) for the farnesylated peptide. This
observation clearly illustrates that the peptide undergoes UV-
dependent farnesylation which could make it a useful probe for
studying prenylation reactions in a spatio-temporally controllable
manner.
% Caged Peptide
% Uncaged Peptide
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Figure 3. (A) HPLC quantification of starting peptide (17b)
disappearance and formation of the uncaged peptide (6b) as a function
of irradiation time at 365 nm, and (B) HPLC quantification of
uncaging of 17a as a function of two-photon irradiation time (800 nm,
pulsed Ti:Saphire laser, 210 mw, 80 fs pulse width). Photolysis
reactions were performed in 200 µM, and 300 µM solutions of 17b,
17a respectively, containing 1 mM DTT in 50 mM PB, pH 7.5.
Since the NDBF-caged peptide showed useful uncaging properties
upon one-photon irradiation, further experiments were performed
to evaluate its two-photon uncaging efficiency. Thus, solutions of
17a were irradiated at 800 nm using a Ti:Saphire laser and the
photolysis products were again analyzed by RP-HPLC and
confirmed by LC-MS (Figure S14). The two-photon action cross-
section of 17a was measured using 6-bromo-7-hydroxycoumarin-4-
ylmethyl acetate (Bhc-OAc) as a reference.31 Even though the
extinction coefficient of the NDBF chromophore at 400 nm is less
than 10 % of that at λmax (325 nm), a value of 0.13 GM for 17a
uncaging at 800 nm was calculated (Figure 3B). It should be noted
that this value would be greater if the two-photon irradiation was
performed at lower wavelengths where the extinction coefficient is
closer to the maximum value although tissue penetration would be
less. Overall, these results demonstrate that NDBF is an efficient
thiol caging group that undergoes clean photo-cleavage to generate
a free thiol upon one- or two-photon irradiation.
Since, it would be useful to employ such caged peptides for studies
in tissue or whole organisms where UV light cannot efficiently
penetrate, the ability of 17a to undergo farnesylation by irradiation
at longer wavelengths via two-photon excitation was examined.
Accordingly, in vitro farnesylation reactions, similar to those
described above for UV irradiation, were conducted using an 800
nm laser light source.
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shown by the appearance of an ion of m/z = 499.99 (Figure 4D). In
summary, this data demonstrates that an NDBF-caged K-Ras
peptide (17a) is capable of undergoing farnesylation triggered by
800 nm light via two-photon excitation (also see Figure S16).
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One of the important goals for photo-caging of bioactive
molecules, including peptides, is to modulate their activity by
irradiation inside cells in order to study biological processes. Since
farnesylation of NDBF-caged K-Ras peptide was efficiently and
rapidly triggered by UV and IR irradiation in vitro, we decided to
explore the same strategy to develop peptides that can be efficiently
activated upon irradiation inside live cells.
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Protein palmitoylation is a post-translational modification that
plays critical roles in subcellular protein localization. In this
process, palmitoyl acyltransferases (PATs) covalently attach a C16
palmitate group via thioesterification to one or more specific
cysteine residues present in protein targets.44 This modification
causes proteins to be more hydrophobic and migrate to the plasma
membrane where they are active;45 prenylated proteins including
H-Ras and N-Ras are commonly palmitoylated. Draper et. al. have
developed several fluorescently labeled cell-penetrating peptides
including NBD-Hex-CLC(S-farnesyl)-OMe (21, Scheme 5),
which they have used to study palmitoylation inside cells.46 When
the free cysteine in the peptide is not modified, it localizes mainly in
the cytosol and the Golgi; however palmitoylation of the free
cysteine by PATs inside cells results in the migration of the peptide
to the plasma membrane. Therefore, a caged version of Hex-
CLC(S-farnesyl)-OMe (20), cannot be a substrate for PAT and
would thus localize in the cytosol/Golgi; however, irradiation
should uncage the peptide, revealing a free thiol that would become
palmitoylated and hence gradually migrate to plasma membrane.
While peptide 21 has previously been shown to traffic to the
plasma membrane, it was impossible to temporally control that
process since cellular uptake and trafficking could not be
uncoupled. However, the availability of a caged form makes this
possible.
Peptide 20 was prepared using a cysteine anchoring method
developed by our group for the synthesis of C-terminal methyl
esters (Figure S17).47 Trityl chloride resin was first treated with
Fmoc-Cys-OMe and DIEA in CH2Cl2 overnight to afford Fmoc-
cysteine loaded resin. The peptide was extended on the resin
through standard SPPS employing Fmoc-based chemistry. Reagent
K treatment cleaved the peptide from the resin which was then
farnesylated via treatment with farnesyl bromide and Zn(OAc)2
under acidic conditions. The final caged peptide was purified by
preparative RP-HPLC. Despite the presence of two cysteines in the
sequence, there was no evidence of NDBF scrambling between the
two thiols during the synthesis, consistent with the stability
afforded by the NDBF thioether linkage. Those results are in
contrast to those obtained when thiocarbonate strategies are used
for thiol protection in peptides containing multiple cysteines where
migration via acyl transfer readily occurs. Moreover, studies have
shown that carbonates and thiocarbonates are prone to hydrolysis
via esterases, thus limiting their applicability in living systems.48 In
general, the efficient assembly of caged peptide 20, highlights the
utility of NDBF-protected cysteine and how it can be employed for
Figure 4. (A) photo-uncaging of 17 and its subsequent farnesylation
by enzyme, (B) EIC chromatogram (m/z = 511.62)) of a 7.5 µM
solution of 17a, in prenylation buffer contining PFTase without
irradiation, (C) EIC chromatogram (m/z = 431.95) of a solution of
17a, after 2.5 min irradiation at 800 nm (Ti:Saphire laser, 170 mw, 90
fs) in prenylation buffer without PFTase, (D) EIC chromatogram
(m/z = 499.99) of 17a after 2.5 min irradiation at 800 nm (Ti:Saphire
laser, 210 mw, 90 fs) in presence of PFTase showing the formation of
farnesylated peptide (19a).
As was noted before, treatment of the caged peptide with enzyme
in the presence of FPP without irradiation did not alter the starting
peptide (Figure 4). Irradiation of 17a (m/z = 511.62) at 800 nm
for 2.5 min in the absence of enzyme, generated the free peptide
(6a) as confirmed from the EIC chromatogram (m/z = 431.95,
Figure 4C). Treatment of 17a with the enzyme along with 2.5 min
two-photon irradiation generated the farnesylated peptide (19a) as
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the synthesis of a variety of caged peptides including those
containing multiple cysteines, with no risk of caging group
migration.
palmitoylated and have been attributed to competing pathways
involving degradation versus membrane targeting.49
To study the localization process in more detail, samples of the
caged peptide were allowed to internalize for 3 hours and then
uncaged by UV exposure. Analysis of the cellular distribution of the
peptide in those samples at different times ranging from 30-120
min showed that the membrane colocalization reached a peak after
30 min followed by a slow decrease (Figure S18). Such behavior is
consistent with observations made with fluorescent proteins that
have been shown to undergo dynamic cycling involving
palmitoylation and concomitant membrane localization followed
by depalmitoylation and internalization.50,51 Overall, these live cell
experiments illustrate how NDBF-caging of cysteine allows an
enzyme substrate for palmitoylation to be temporally activated,
thus permitting the processes of cellular entry and subsequent
enzymatic modification to be deconvoluted.
Scheme 5. Schematic representation of NBD-Hex-
C(NDBF)LC-OMe uncaging and subsequent palmitoylation.
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Finally, in order to highlight the improved uncaging efficiency of
NDBF and its utility in live cell experiments, a comparison was
made between the uncaging efficiency of NDBF versus the 6-
nitroveratryl (NV) group which is one of the most frequently used
caging groups.15 We elected to study this experimentally since a
range of values for the quantum yield of NV have been reported.15
Thiol-protected forms of Fmoc-Cys-OMe were prepared using the
two caging groups which were then subjected to UV irradiation
(365 nm) and the extent of deprotection was monitored. Uncaging
data obtained by HPLC analysis shown in Figure S19, shows that
the NDBF uncaging efficiency is greater than 10-fold higher than
that of NV. This difference reflects the high molar absorptivity (ɛ =
15,300 M-1cm-1) and high quantum yield (Φ = 0.2) of NDBF
leading to a high ɛΦ (~ 3060) value versus that of NV (ɛΦ ~ 6000
× 0.01 = 60).52 Thus, the lower uncaging efficiency manifested by
NV requires longer irradiation times to obtain comparable levels of
uncaging. When SKOV3 cells were subjected to 50 min of
irradiation (10-fold longer than was used to uncage 20), a
significant decrease in cell viability was observed. Figure S20 shows
a 5% loss of viability after 5 min of irradiation and a 57% decrease
after 50 min. Clearly this excessive loss of viability precludes the use
of NV protection in this experiments and serves to underscore the
increased efficiency and utility of NDBF. However, it should be
noted that the two-photon action cross-section of NDBF thiol
uncaging is comparable to those manifested by Bhc-carboxylates31
and NDBF-alcohols36. To date, two-photon activation of such
caged molecules has been restricted to experiments where only a
small fraction of uncaging is required to obtain a biological
response. At present, it is unclear whether a large fraction of an
NDBF-caged bioactive thiol can be released via two-photon
excitation in cells since longer irradiation times may result in IR
heating or phototoxicity; cell-based experiments to answer these
questions are currently in progress. Nevertheless, the results
reported here highlight the utility of NDBF caging for a variety of
different biological applications. Coupled with its utility for the
synthesis of peptides containing multiple cysteines, the data
described here make it clear why NDBF is a superior choice for
thiol caging.
Next, light triggered intracellular palmitoylation of caged peptide
20 was studied using ovarian human carcinoma SKOV3 cells. Cells
were incubated with 20 in for 3 hours at 37 °C to allow cellular
uptake. The cells were then divided into two groups and one was
subjected to 5 min of UV irradiation at 330 nm. Both irradiated and
non-irradiated cells were incubated for an additional 120 min at 37
°C, stained with nuclear and membrane markers and imaged by
confocal microscopy. As observed in the fluorescence microscopy
images (Figure 5A), before irradiation, the peptides reside
primarily in the cytosol and Golgi. However, after UV exposure, the
peptides traffic to the plasma membrane where they colocalize with
the membrane dye; this change in colocalization occurs due to
enzymatic palmitoylation. The degree of colocalization of the
peptide and plasma membrane dye was quantified by calculating
Pearson’s coefficients for both the non-irradiated (0.26) and
irradiated cells (0.76) which clearly indicates a significant increase
in membrane localization of the peptide after irradiation (Figure
5B). It is important to note that while all of the peptide did not
localize to the membrane upon photolysis, this is unlikely to be due
to incomplete uncaging since UV-mediated uncaging is quite fast
and efficient (see Figure 3). In their original work, Draper et al46.
reported incomplete localization even when starting with the fully
deprotected form of the peptide used here.46 Similar results (only
partial localization in pulse-chase labeling experiments) have been
reported in work with fluorescent proteins that are prenylated and
CONCLUSION
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In this work, we analyzed the photolysis of several Bhc protected
completely converted to free peptide with a photolysis quantum
thiol-containing peptides and small molecules. Those experiments
revealed that Bhc-caged thiols exhibit variable uncaging yields and
that their photolysis frequently leads to the formation of an
unwanted rearrangement product. Using NMR analysis, the
structure of the photochemically produced isomer was established
to be a 4-methylcoumarin-3-yl thioether.
yield of 0.2. The two-photon action cross-section of the caged
peptide was measured to be 0.13 GM at 800 nm comparable to that
of Bhc-OAc. The one- and two-photon photolysis of a caged K-Ras
peptide in the presence of PFTase revealed that the free peptide
formed upon irradiation is efficiently converted by the enzyme to
its biologically relevant prenylated form. The NDBF protected
cysteine was also used to develop a light activatable cell-penetrating
peptide containing a caged and a farnesylated cysteine. Confocal
microscopy analysis showed that the caged peptide could be
activated inside cells upon light exposure which resulted in
intracellular migration due to enzymatic palmitoylation. Taken
together, this work for the first time reports an efficient, robust and
9
The poor uncaging yield of Bhc-caged thiols led us to search for a
more efficient thiol caging group that would be useful for biological
applications; accordingly, NDBF caging, which has previously been
shown to be effective for hydroxyl group protection, was explored.
NDBF-protected Fmoc-cysteine was synthesized and successfully
incorporated into a K-Ras-derived peptide via standard solid phase
synthesis. The resulting caged peptide was photolyzed and
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Figure 5. Live cell experiments showing temporal control of enzymatic palmitoylation via NDBF-thiol caging. A) Images obtained by
fluorescence confocal microscopy illustrating intracellular localization of fluorescently labeled peptide 20 in SKOV3 cells, before (top) and
after (bottom) UV exposure, (B) Quantification of co-localization of peptide and membrane dye via Pearson’s coefficient analysis, indicating
a significant increase in plasma membrane localization of peptide upon irradiation.
broadly applicable strategy for the synthesis of a variety of peptides
and related small molecules containing caged thiols that can be
activated by both one- and two- photon processes in live cells.
These results set the stage for a variety of studies where
spatiotemporal control of thiol reactivity is required including a
diverse span of applications ranging from chemical biology to
material science.
ACKNOWLEDGMENT
We thank Dr. Joseph Dalluge for helpful discussion regarding mass
spectrometry. We thank Dr. Guillermo Marquez and John Oja at
University of Minnesota imaging center for helpful discussion
regarding confocal microscopy experiments. We also thank Prof. Emil
Lou, Dr. Venugopal Thayanithy and Snider Desir for providing us with
SKOV-3 cells and helpful discussion regarding the cell culture.
Financial support for these studies through National Institutes of
Health Grant GM084152, the University of Minnesota Supercomputer
Institute, the University of Minnesota Imaging Center, a Nikon Center
of Excellence for confocal microscopy and the University of Minnesota
Graduate School is gratefully acknowledged. This work is dedicated to
Professor Peter B. Dervan on the occasion of his 70th birthday for his
insightful use of physical organic chemistry to develop new tools to
facilitate the study of challenging problems in biology and medicine.
ASSOCIATED CONTENT
Supporting Information. Synthesis procedures, HPLC and LC-MS
data, and MS/MS fragmentation table. This material is available free of
charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
* Tel (612) 624-0544; Fax (612) 626-7541; E-mail:
diste001@umn.edu
ABBREVIATIONS
NDBF, nitrodibenzofuran; Bhc, brominated hydroxycoumarin; PB,
phosphate buffer; PFTase, protein farnesyltransferase; TP, two-
photon; LC-MS, liquid chromatography–mass spectrometry; TLC,
thin layer chromatography; HPLC, high-performance liquid
chromatography.
Notes
The authors declare no competing financial interests.
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(28) Wosnick, J. H.; Shoichet, M. S. Chem. Mater. 2008, 20, 55.
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