Selective and reversible photochemical derivatization of cysteine residues in peptides and proteins

Article abstract of DOI:10.1039/c3sc51691a

The selective derivatization of solvent-exposed cysteine residues in peptides and proteins is achieved by brief irradiation of an aqueous solution containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs) with a 350 nm fluorescent lamp. NQMP can be conjugated with various moieties, such as PEG, dyes, carbohydrates, or possess a fragment for further selective derivatization, e.g., biotin, azide, alkyne, etc. Attractive features of this labeling approach include an exceptionally fast rate of the reaction and a requirement for a low equivalence of the reagent. The NQMP-thioether linkage is stable under ambient conditions, and survives protein digestion and MS analysis. Irradiation of an NQMP-labeled protein in a dilute solution (<40 μM) or in the presence of a vinyl ether results in a traceless release of the substrate. The reversible biotinylation of bovine serum albumin, as well as the capture and release of this protein using NeutrAvidin Agarose resin beads has been demonstrated.

Full text of DOI:10.1039/c3sc51691a

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Selective and reversible photochemical
derivatization of cysteine residues in peptides and
Cite this: Chem. Sci., 2014, 5, 1591
proteins†
Selvanathan Arumugam,a Jun Guo,b Ngalle Eric Mbua,b Frederic Friscourt,b
´ ´
a
a
ab
a
*
*
and Vladimir V. Popik
Nannan Lin, Emmanuel Nekongo, Geert-Jan Boons
The selective derivatization of solvent-exposed cysteine residues in peptides and proteins is achieved by
brief irradiation of an aqueous solution containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs)
with a 350 nm fluorescent lamp. NQMP can be conjugated with various moieties, such as PEG, dyes,
carbohydrates, or possess a fragment for further selective derivatization, e.g., biotin, azide, alkyne, etc.
Attractive features of this labeling approach include an exceptionally fast rate of the reaction and a
requirement for a low equivalence of the reagent. The NQMP-thioether linkage is stable under ambient
conditions, and survives protein digestion and MS analysis. Irradiation of an NQMP-labeled protein in a
dilute solution (<40 mM) or in the presence of a vinyl ether results in a traceless release of the substrate.
The reversible biotinylation of bovine serum albumin, as well as the capture and release of this protein
using NeutrAvidin Agarose resin beads has been demonstrated.
Received 18th June 2013
Accepted 17th December 2013
DOI: 10.1039/c3sc51691a
www.rsc.org/chemicalscience
derivatization include alkylation with bromo- and iodoacetyl
derivatives, mixed disulde formation, or Michael additions to
substituted maleimides.7,9 Some of these methods allow for the
Introduction
The site-specic modication of proteins provides unique
opportunities for modifying the properties and expanding the
function of these biomolecules.1,2 Among other applications,
site-specic modication has been employed for installing tags
to study protein trafficking, to alter the bioavailability and
pharmacokinetics by PEGylation, or for selective drug delivery
by the attachment of pharmaceuticals. In addition, many
proteins undergo co- or post-translational modication, thereby
altering their biochemical role and the introduction of such
moieties by synthetic means offers exciting opportunities to
access well-dened macromolecules for activity studies.3,4
The high nucleophilicity of the thiol moiety of cysteine has
long been used for installing a wide variety of functionalities.1,5
Cysteine has a relatively low natural abundance and most of the
thiol groups are involved in the formation of intra-protein
disulde bonds. As a result, solvent-exposed free cysteines are
rather uncommon in wild-type proteins.6 A unique cysteine
residue can, however, be readily introduced by site directed
mutagenesis, providing opportunities for installing novel
functionalities.7,8 Approaches commonly employed for cysteine
subsequent removal of the installed tag under different condi-
tions.10 Alternatively, cysteine can be converted into dehy-
droalanine, which then is used for thiol–ene conjugations.11
o-Quinone methides (oQMs) are very susceptible to nucleo-
philic attack, due to the signicant positive charge on the meth-
ylene carbon atom.12,13 Their reactivity towards DNA is well
documented,12,14 while their interaction with proteins and
peptides is virtually unknown. There are a few reports that
isomeric p-quinone methides, which are approximately two
orders of magnitude less reactive than oQMs,15 alkylate proteins.16
Labeling occurred mostly at the cysteines residues, however some
nucleophilic amino acid side chains were also tagged.
Here, we report the photochemical derivatization of peptides
and proteins based on the selective reaction of photochemically
generated 2-napthoquinone-3-methides (oNQMs, 2) with
solvent-exposed cysteine residues (Scheme 1). oNQMs (2) are
generated by the efficient photochemical dehydration (F ¼
0.20) of 3-(hydroxymethyl)-2-naphthols (naphthoquinone
methide precursors, NQMP 1, Scheme 1). The attractive features
of the labeling approach include an exceptionally fast rate of the
reaction and a requirement of a low equivalence of the reagent
for the quantitative functionalization of the available Cys resi-
dues. Furthermore, it is shown that photolysis of NQMP-caged
substrates in the absence of a labeling reagent restores the free
cysteines. A potential utility of this reversible derivatization
technique is demonstrated by the capture and subsequent
release of a protein (BSA).
aDepartment of Chemistry, University of Georgia, Athens, GA 30602, USA. E-mail:
vpopik@uga.edu
bComplex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road,
Athens, GA 30602, USA. E-mail: gjboons@ccrc.uga.edu
† Electronic supplementary information (ESI) available: Experimental procedures,
preparation and NMR spectra of newly synthesized compounds. See DOI:
10.1039/c3sc51691a
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While the parent NQMP 1a and 6-(hydroxymethyl)naphthalene-
1,7-diol 1b have rather low aqueous solubility, triethylene glycol
(TEG-NQMP, 1c), biotin (Biotin-NQMP, 1d),20 and mannoside
(Mann-NQMP, 1e)21 derivatives are freely soluble in wholly
aqueous solutions, up to at least 5 mM concentrations. The 300
or 350 nm irradiation of NQMP 1a–f results in an efficient
dehydration and the formation of o-naphthoquinone methides
(oNQM 2a–e).
The selectivity of oNQM towards nucleophilic amino acid
residues commonly present in proteins was tested using four
nonapeptide Ac-Cys-Lys-Tyr-Trp-Gly-Arg-Gly-Asp-Ser (4), Met-
Lys-Tyr-Trp-Gly-Arg-Gly-His-Ser (4-His), Ac-Met-Lys-Tyr-Trp-Gly-
Arg-Gly-Asp-Ser (6), and Met-Lys-Tyr-Trp-Gly-Arg-Gly-His-Ser
(6-His, Scheme 2). These nonapeptide contain amino acid
residues with nucleophilic side chains: thiol (cysteine), primary
amine (lysine and a free N-terminus), phenol (tyrosine), indole
(tryptophan), guanidine (arginine), carboxylate ion (aspartate),
Scheme 1 Photochemical alkylation and release of thiol-containing
substrates using 300 or 350 nm light.
Results and discussion
The lifetime of oNQM (2a) in neutral aqueous solutions is only 7 imidazole (histidine), and primary alcohol (serine).
ms, as it rapidly adds water (kH O ꢀ 2.6 Mꢁ1 sꢁ1) to regenerate
The irradiation of a PBS solution (pH ¼ 7.4) of peptides 4 and
2
the starting NQMP (1a).17 Among endogenous nucleophiles, 4-His (0.1 mM) containing 4 equivalents of NQMP 1c with a
only thiols are reactive enough (kRSH ꢀ 2.2 ꢂ 105 Mꢁ1 sꢁ1
)
to uorescent 300 nm lamp21 results in the quantitative conver-
17
outcompete water in a Michael addition to oNQM. The thioether sion of the starting peptide into the mono TEG-NQMP-labeled
(3), produced in the reaction of thiols with oNQM, is hydro- derivatives 5 and 5-His (Scheme 2, Table 1). Peptide 5 was iso-
lytically stable, but can be quantitatively cleaved under 300 or lated by preparative HPLC in a 95% yield and its structure
350 nm irradiation back to 2, with a 10% quantum yield conrmed by ESI-HRMS and MS/MS.21
(Scheme 1).18,19
When the cysteine in the oligopeptide was replaced with
The 3-hydroxy-2-naphthalenemethanol chromophore (1a) methionine (peptides 6 and 6-His), or free thiol was converted
has two major absorption bands in the UV region above 210 nm: into a disulde bond by oxidative homo-dimerization (peptide
at lmax ¼ 275 nm (log 3 ¼ 4.06) and at lmax ¼ 324 nm (log 3 ¼ 7), no signicant labeling was observed upon irradiation in the
3.70). The introduction of an alkoxy substituent at the 5-posi- presence of 4 to 12 equivalents of TEG-NQMP (1c).20 These
tion of the chromophore (e.g., 1c,19 Scheme 1) results in a ca. 10 experiments show the high cysteine specicity of the oNQM-
nm bathochromic shi of both bands. The longer wavelength tagging procedure. It is important to note that peptides 4 and
absorbance band extends past 360 nm, making this chromo- TEG-NQMP (1c) are photochemically stable under these condi-
phore suitable for activation using a 350 nm uorescent lamp, tions. No decomposition or losses of materials were detected by
or a 355 nm emission from a frequency tripled Nd:YAG laser. HPLC aer separate irradiation of 0.1 mM PBS solutions of 1c
o-Naphthoquinone methide precursors 1a–d also show signi- and peptide 4 for 12 min using both 300 and 350 nm uorescent
cant uorescence with a short lifetime (FFl ¼ 0.230 ꢃ 0.002 with lamps. Obviously, NQMP 1c still forms oNQM 2c, but in the
sFL ꢀ 7 ns for 1a).17 The emission spectrum of 1a in aqueous absence of thiols, the latter rapidly undergoes hydration back to
solutions contains two major bands at 360 nm and 423 nm. the starting materials.
Scheme 2 Selective alkylation of cysteine residue in peptides.
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Achieving a complete conversion of a 0.1 mM solution of 4 to achieved at a starting NQMP concentration equal [NQMP] ¼ 3 ꢂ
5 requires 2 min of irradiation at 300 nm (Table 1). Photolysis 10ꢁ4 Mꢁ1 + [Substrate]. In biochemical labeling experiments,
using a 350 nm lamp takes somewhat longer, apparently due where the concentration of a substrate is usually in the mM
to the lower extinction coefficients of NQMP 2c at this range or lower, 400 mM of the NQMP derivative should be
wavelength.18
The TEG-NQMP-peptide conjugate (5) is photoreactive itself available cysteine residues.
sufficient to achieve the complete functionalization of all the
and produces oNQM 2 upon irradiation. The formation of 5,
Thus, at a 0.1 mM concentration of peptide 4, almost
therefore, represents a photochemically driven equilibrium quantitative conversion to 5 is achieved with only four
between the peptide 4 and NQMP 1c on one side, and 5 on the equivalents of 1c (Table 1). At lower NQMP to peptide ratios,
other (Scheme 3).
the conversion is reduced, and at equimolar concentrations
of both components, the yield of the labeled peptide 5 drops
to 20%. These results agree well with the conversion pre-
dicted by eqn (2). A prolonged irradiation has virtually no
effect on the peptide 4 to 5 ratio, suggesting that system
reaches a photostationary state aer 2 min of irradiation with
a 300 nm light (Table 1).
Eqn 2 also suggests that at a low concentration of free
NQMP, the process can be reversed. The photolysis of the TEG-
NQMP-peptide conjugate (5) at a low concentration of free
NQMP (1) should result in the traceless release of the peptide 4
(Scheme 4). Indeed, irradiation of 0.1 mM solution of 5 for 2
min with 300 nm lamp produced the mixture containing 75%
the free peptide 4 and 21% of caged 5. Longer irradiation does
not signicantly affect the 4 to 5 ratio (Table 2). A very similar
product ratio was obtained in the photolysis of a 0.1 mM
peptide 4 solution, in the presence of an equimolar amount of
TEG-NQMP (1c) (Table 1).
The photo-release of the free cysteine residues can be ach-
ieved at any concentration of the NQMP-labeled protein, when
vinyl ethers are employed as oNQM (2) trapping agents. A very
facile Diels–Alder addition of the ethyl vinyl ether (EVE) to
intermediate oNQM converts the latter into a photo-stable
benzochroman 8 (Scheme 4).17,18 This reaction efficiently
removes the oNQM precursors from the reaction mixture and
shis the photo-equilibrium towards the substrates, with free
thiol. Due to the irreversibility of Diels–Alder addition, even one
equivalent of the ethyl vinyl ether is sufficient to achieve a
complete conversion of the NQMP-caged peptide 5. The
cleavage of NQMP-SR thioethers with, or without vinyl ether
derivatives can be accomplished using a 350 nm light. The
complete conversion of the starting material under these
conditions requires 2–3 times longer irradiation than with a
300 nm lamp (Table 2).
Scheme 3 Light-driven equilibrium between NQMP and NQMP-SR.
The position of the equilibrium is dened by the rate of
reaction of the common intermediate 2 with water (khydr
¼
kH O[H2O]) and cysteine (kRSH), as well as by the efficiency of the
2
photoelimination reaction of NQMP (1, khv) and NQMP-SR (5,
kh0 v). Since under the established equilibrium conditions, the
concentrations of 1 and 5 remain constant, we can write the
following equation (eqn (1)), where [NQMP], [RSH], and [NQMP-
SR] are the equilibrium concentrations of corresponding
substrates:
khv½NQMPꢄ
kh0 v½NQMP-SRꢄ
kRSH½RSHꢄ
¼
¼
(1)
khydr
½NQMP-SRꢄ
½RSHꢄ
khvkRSH½NQMPꢄ
(2)
kh0 vkhydr
The ratio of the caged to free thiol can be further expressed
as a function of the NQMP concentration (eqn (2)). Since kRSH is
about ve orders of magnitude larger than kH O, and khv/kh0 v
ꢀ
2
2,17 the equilibrium is shied in the favor of NQMP-SR (5)
formation at relatively low concentrations of thiol and NQMP in
aqueous solutions. Using the experimental rates of oNQM
hydration and reactions with simple thiols,16 as well as the
photochemical efficiencies of the NQMP and NQMP-SR reac-
tions, we can evaluate that a 90+% conversion of a substrate is
Table 1 Efficiency of photo-labeling of peptide 4 with TEG-NQMP (1c) under different conditionsa
Irradiation
wavelength (nm)
Irradiation
time (min)
4 to 1c
ratio
Yield of
conjugate 5 (%)
Unreacted
4 (%)
300
350
300
300
300
300
2
5
2
2
2
1 : 4
1 : 4
1 : 3
1 : 2
1 : 1
1 : 1
94 ꢃ 2
95 ꢃ 2
79 ꢃ 3
47 ꢃ 2
20 ꢃ 2
18 ꢃ 2
ndb
ndb
17 ꢃ 2
50 ꢃ 2
76 ꢃ 2
79 ꢃ 2
12
a
b
Starting concentration of peptide 4 ¼ 0.1 mM in PBS (pH ¼ 7.4) at r.t. Not detected.
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Scheme 4 Photochemical uncaging of peptide cysteine moiety.
Scheme 5 Selective photochemical derivatization of bovine serum
albumin.
These observations indicate that the system photo-equili-
brates to the same photo-stationary state starting either from
the NQMP + peptide 4 or from the caged peptide 5 side. As
expected, further dilution of the solution of 5 results in higher
yields of release of the thiol moiety. At a 20 mM concentration or
below, no caged substrate 5 can be detected aer irradiation
(Table 2). For practical applications, we can evaluate that a
90+% release of the NQMP-caged thiol is achieved at a 40 mM or
lower substrate concentration.
derivatization of the protein was conrmed by MALDI-TOF and
ESI mass spectra, as well as by digestion analysis.21
It is interesting to note that the BSA dimer, which is clearly
seen on both gels, apparently possesses at least one free
cysteine residue that is photo-labeled with 1d and 1f (Fig. 1,
lanes 3A and 6B).
The efficiency of the BSA labelling with NQMP-biotin (1d)
was highest at pH ꢀ 7 but was progressively reduced at higher
acidities. We believe that this phenomenon is linked to the
acidity of the Cys-34 in the BSA (pKa ꢀ 5).24 Under neutral
conditions, this residue apparently exists in a very reactive thi-
olate form. In a more acidic medium, the equilibrium shis to
the less nucleophilic thiol, which loses competition for the
oNQM intermediate (2, Scheme 3) to the aqueous solvent. The
pH-dependence of the NQMP-based cysteine labeling system is
currently under investigation.
To further test the specicity of NQMP labeling of proteins,
we have prepared a BSA analog 9b, in which the Cys-34 has been
converted into a TEG ether (Scheme 6).21 The incubation of 9b
with NQMP-biotin (1d) in the dark, or under irradiation,
produces no biotinylated BSA (Fig. 2A, lanes 2 and 5). This result
conrms the cysteine selectivity of the NQMP photo-derivati-
zation technique.
BSA is notorious for binding lipophilic compounds.
However, BSA incubated with NQMP-DNS (1f) in the dark shows
virtually no uorescence, while an irradiated sample exhibits a
strong emission at 490 nm (Fig. 3). Both samples were puried
by a triple ltration through a Sephadex column and had the
same concentration (9.7 mM). The difference in the absorbance
at 335 nm (lmax of the dansyl chromophore) between 10f and a
control allows us to evaluate concentration of the dansyl groups
The successful peptide labeling prompted us to explore the
feasibility of the selective photo-caging of the free cysteine
residues in the proteins. Bovine serum albumin (BSA, 9a),
which contains a solvent-exposed cysteine residue (Cys-34), was
employed as a model protein.22 Solutions of the protein (11 mM)
and NQMP-biotin (1d, 100 mM)20 in PBS (pH ¼ 7.4) were incu-
bated in the dark for 15 min, or irradiated with a 350 nm
uorescent lamp for 2 min (Scheme 5). Low molecular weight
compounds were removed by spin ltration or by size-exclusion
chromatography (SEC) and the resulting protein sample was
analyzed by Western blotting, using an antibiotin antibody –
HRP conjugate (Fig. 1A).21 As expected, the BSA-NQMP-biotin
adduct 10d was formed only in the irradiated samples (compare
lanes 2 and 3, Fig. 1A; lanes 1 and 4, Fig. 2A). The direct uo-
rescent labeling of a protein was demonstrated using the
conjugate of NQMP with the uorescent dansyl group (NQMP-
DNS, 1f).23 Solutions of the protein (12 mM) and 1f (100 mM) were
incubated in the dark for 30 or 90 min, or irradiated with 350
nm uorescent lamps for 6 min (Scheme 5). Aer the removal of
unreacted 1f, the protein samples were resolved using a 4–20%
Tris–HCl gel. The uorescent image of the gel clearly demon-
strates that the dansyl labelling of the protein occurred only
under irradiation (Fig. 1B, lane 6).21 The NQMP-DNS
Table 2 Uncaging of o-NQMP labeled peptide 5a
Irradiation
time (min)
Concentration
of 5 (mM)
Concentration
of EVE (mM)
Unreacted
5 (%)
Yield of
peptide 4 (%)
2
12
2
2
5
100
100
50
20
20
0
0
0
0
0
21 ꢃ 2
19 ꢃ 2
4 ꢃ 1
ndb
75 ꢃ 3
77 ꢃ 3
92 ꢃ 2
96 ꢃ 2
94 ꢃ 2c
97 ꢃ 2
96 ꢃ 2c
ndb
2
5
100
100
100
100
ndb
ndb
Using 300 nm uorescent lamps in PBS solution at r.t. b Not detected. c 350 nm uorescent lamps were used.
a
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Fig. 3 Emission spectra (lexc ¼ 350 nm) of ꢀ10 mM PBS solutions of
BSA derivative 10f (solid line) and a control BSA (9a) isolated after
incubation with 1f in the dark (dashed line).
in the sample of 10f at 9.0 mM. This corresponds to an almost
quantitative labeling of the BSA (see discussion below).
The exhaustive NQMP derivatization of BSA (as tested by
Ellman's reagent) requires lower concentrations (100 mM) of
NQMP than needed for the peptide 4 (400 mM, vide supra). There
are two potential explanations for this phenomenon. Firstly, the
hydrophobic interaction between the aromatic chromophore
and the protein might increase the efficient concentration of
NQMP 1d–f at the protein surface, thus shiing the position of
the photo-equilibrium in favor of 10d–f. On the other hand, the
Cys-34 residue in BSA is apparently more acidic than the
cysteine in peptide 4.24 Therefore, at pH ¼ 7.4, BSA has a
signicantly higher fraction of cysteine in the reactive thiolate
form than 4. We are currently investigating the effect of cysteine
S–H acidity in peptides and proteins on the efficiency of NQMP
labeling.
Fig. 1 Western blot analysis of photochemical labeling of BSA (9a)
with NQMP-biotin (1d) and NQMP-DNS (1f). Membranes were stained
by Coomassie Brilliant Blue dye (lower panels) and a-antibiotin anti-
body-HPR – ECL Plus chemiluminescent substrate (A). Lanes 1A and
1B: molecular weight marker; 2A and 3B: unmodified BSA; 3A: BSA+1d
after 2 min of irradiation; 4B & 5B: BSA incubated with 1f in the dark for
30 and 90 min; 6B: BSA + 1f after 6 min of irradiation.
To quantify the extent of the photo-biotinylation of BSA, the
total protein concentration in the irradiated samples was
determined using Bradford and bicinchoninic acid assays. The
biotin concentration was measured using a biotin quantica-
tion kit. Alternatively, the BSA to NQMP ratio was determined
spectroscopically (Table S1†).21 Both methods report near
quantitative BSA biotinylation. This was a surprising result,
because previous reports suggest that only 60–80% BSA
contains a free thiol group. The rest of the Cys-34 moieties are
believed to form disuldes with cysteine or glutathione.21,25 It is
possible that the free thiol content of BSA is actually higher,
since Ellman's test, which is uniformly used in these measure-
ments, oen underestimates the SH content in proteins.26,27
This is even more plausible for BSA, since the Cys34 residue is
Fig.
2 Western blot analysis of photochemical labeling (A) and
uncaging (B) of BSA. Membranes were stained by antibiotin antibody-
HPR – ECL Plus chemiluminescent substrate (for a-biotin) and Coo-
massie Brilliant Blue dye (for protein).
Scheme 6 BSA with blocked Cys-34 does not react with NQMP.
Scheme 7 Photo-release of BSA from NQMP caged form 10d.
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removal of a biotin tag (Fig. 2B, lane 7). In fact, the electro-
phoretic mobility of the BSA derivative 11d is not affected by
irradiation, illustrating photochemical stability of this conju-
gate (Fig. 2B, lane 6 and 7).
The reversible derivatization of the cysteine residues
described in this report can be employed for pulling down and
purifying proteins containing or genetically engineered to
contain solvent-exposed cysteine residues. To explore the
capabilities of the method, we have captured BSA on Neu-
trAvidin Agarose beads and then photochemically cleaved the
protein from the solid support.
Scheme 8 Irreversible photo-biotinylation of BSA.
Following a standard protocol (vide supra), a PBS solution
containing BSA 9a (10 mM) and the Biotin-NQMP conjugate 1d
(100 mM) was irradiated for 5 min at 350 nm. NeutrAvidin
Agarose resin (ca. 10% excess) was added to the photolysate and
incubated in the dark for 30 min. The resulting resin was
separated by centrifugation and washed. The combined super-
natant contained no detectable amounts of BSA or 1d, according
to spectrophotometric analysis and Bradford assay (Scheme 9).
The resulting NeutrAvidin Agarose resin was re-suspended in
PBS buffer to achieve a ca. 10 mM concentration of bound BSA
(and ca. 100 mM of bound 1d). The suspension was exposed to
350 nm light for 5 min under ambient conditions. The analysis
of the supernatant showed the release of 70–80% of immobi-
Scheme 9 Photo-triggered catch and release of BSA using Neu-
trAvidin Agarose gel.
similar or even more acidic than the thiol group in 2-nitro-5- lized BSA (Scheme 9). When a BSA-derivatized NeutrAvidin
thiobenzoate (NTB).24,27 On the other hand, we cannot exclude Agarose resin suspension was diluted to ca. 5 mM concentration
the photochemically induced cleavage of the disulde bonds, of bound protein (ꢀ50 mM of 1d), 5 min of 350 nm irradiation
either directly or through NQMP sensitization.
resulted in a 95% release of BSA from the beads. A similar, high
Cysteine residues are oen used for the preparation of gly- yield of the protein photo-cleavage from the resin support was
coproteins.7,10b,28 To test the utility of thiol – oNQM photo-click obtained at 10 mM of the resin-bound BSA when diethylene
chemistry for protein glycosylation, a PBS solution of BSA (9a, glycol monovinyl ether (equimolar amount in respect to 1d) was
11 mM) and mannose-NQMP conjugate (1e, 100 mM)21 was added to a suspension before irradiation. This reagent appar-
exposed to a 350 nm light for 2 min (Scheme 5). The excess of 1e ently converts the resin-bound NQMP groups into photochem-
was removed by spin ltration and the resulting BSA-mannose ically stable benzochroman moieties (vide supra).
conjugate (10e) was analyzed for the total protein. The mannose
content in 10e was determined by HPLC, aer TFA hydrolysis,
conrming an almost quantitative derivatization.21
Conclusions
To explore the reversibility of the cysteine photo-derivatization We have described an efficient photochemical method for the
in proteins, a 2 mM PBS solution of biotinylated BSA 10d was irra- selective derivatization of free cysteine residues in peptides
diated with a 350 nm uorescent lamp for 2 min and the protein and proteins. The irradiation of a neutral aqueous solution
isolated and puried by spin ltration.21 Western blot analysis of containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs)
the product showed the complete removal of the biotin tag (Fig. 2B, and protein or peptide with a hand-held 300 or 350 nm uo-
lane 4), while Coomassie Brilliant Blue staining conrmed the rescent lamp results in the fast and efficient conversion of free
presence of protein in the sample. The product protein has the cysteines into NQMP-thioethers, while other amino acid resi-
same mobility as 9a (Fig. 2B, lane 2) and biotinylated 10d, thus dues remain unchanged. 0.4 mM of NQMP and 2 min of
conrming the clean regeneration of native BSA (Scheme 7).
exposure is sufficient for the quantitative derivatization of the
The photo-stable functionalization of cysteine residues can substrate. NQMP can be equipped with various moieties, such
be achieved via a two-step procedure involving derivatization as PEGs, dyes, carbohydrates, or possess a fragment for further
with a vinyl ether, followed by a photo-Diels Alder reaction with selective derivatization, e.g., biotin, azide, alkyne, etc. The
NQMP. Thus, BSA (9a) was converted into the BSA-vinyl ether utility of this method was demonstrated by derivatizing bovine
conjugate (9c), by the reaction with 2-(2-(vinyloxy)ethoxy)ethyl serum albumin with biotin, uorescent dye, and mannose in a
iodide.21 Two minutes of irradiation at 350 nm for 9c (11 mM) in high yield. Other substrates, which survive short exposure to
the presence of NQMP 1d (100 mM) in PBS resulted in the 350 nm light, can be conjugated with cysteine residues via this
complete conversion of the BSA-vinyl ether conjugate (9c) into technique. The orthogonality of the oNQM-based labeling
the Diels–Alder adduct 11d (Scheme 8, Fig. 2A, lane 6).21
technique to azide click chemistry20,29 allows for the two-step
In contrast to 10d, irradiation of the BSA-Biotin conjugate derivatization procedure. Firstly, a target protein is photo-
11d, even for extended periods of time, does not result in the chemically derivatized with NQMP containing an azide or
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alkyne functionality; the second substrate is then attached
using conventional copper-catalyzed or strain-promoted azide-
alkyne click chemistry. The NQMP label can be later removed
by irradiating the substrate in a dilute solution (40 mM or
lower) in the absence of free NQMP. The quantitative release of
NQMP-caged substrates at higher concentrations can be ach-
ieved in the presence of vinyl ethers. We have demonstrated
the reversible biotinylation of BSA, as well as the capture of
biotinylated BSA on NeutrAvidin Agarose resin and then
photochemical release of the protein from the beads. The
cleavage of the protein from the solid support does not require
any reagents, simplifying the purication. The reversible
modication of the cysteine residues in proteins can be
employed for the photo-regulation of protein activity, either by
the caging of catalytically active cysteine (e.g., in cysteine
proteases) or by attaching an activity-modifying fragment to it
(e.g., reversibly caging Cys in Acyl carrier proteins). The
reversible PEGylation of active peptides and proteins can be
employed for a light-directed drug delivery. The very fast rate
7 G. J. L. Bernardes, F. J. Grayson, S. Thompson, J. M. Chalker,
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carrying the uorescent NQMP derivative23 can be employed in
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Acknowledgements
This research project was supported by the National Science
Foundation (Grant no. CHE-0842590, V.V.P.), the National
Cancer Institute of the U. S. National Institutes of Health (Grant 12 Quinone Methides, Wiley Series of Reactive Intermediates in
no. RO1 CA88986, G.-J.B.), and the National Institute of General
Medical Sciences of the U. S. National Institutes of Health
(Grant no. R01 GM61761, G.-J.B.).
Chemistry and Biology, ed. S. E. Rokita, Wiley, Hoboken,
NJ, 2009, vol. 1.
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