Fluorogenic tagging of peptides via Cys residues using thiol-specific vinyl sulfone affinity tags

Article abstract of DOI:10.1016/j.tet.2014.05.103

Fluorescent tagging of Cys-containing peptides is presented herein. The procedure follows a two-step sequential reaction scheme using thiol specific bifunctional chemical reporters and fluorogenic labels. Vinyl-sulfone bearing chemical reporters have been synthesized and demonstrated to selectively modify cysteine under physiological conditions in the presence of other nucleophilic amino acids. Bifunctional chemical reporters decorated with a terminal alkyne moiety, suitable for modification with azide containing fluorogenic labels were also synthesized. Such fluorogenic (turn on) labels in combination with these new vinyl-sulfone tags can be used generally in fluorescent modulation schemes of thiol-bearing biomolecules.

Full text of DOI:10.1016/j.tet.2014.05.103

Tetrahedron xxx (2014) 1e5
Contents lists available at ScienceDirect
Tetrahedron
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Fluorogenic tagging of peptides via Cys residues using thiol-specific
vinyl sulfone affinity tags
a
b
a
b
b
ꢀ
ꢀ
ꢀ
ꢀ
}
Gergely B. Cserep , Zsuzsa Baranyai , David Komaromy , Kata Horvati , Szilvia Bosze ,
a
,
*
ꢀ
Peter Kele
a
€
“Lendulet” Chemical Biology Research Group, Hungarian Academy of Sciences, Research Centre for Natural Sciences, Institute of Organic Chemistry,
ꢀ
€ ꢀ
Magyar tudosok korutja 2, Budapest H-1117, Hungary
b
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
MTA-ELTE Research Group of Peptide Chemistry, Pazmany Peter setany 1a, Budapest H-1117, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorescent tagging of Cys-containing peptides is presented herein. The procedure follows a two-step
sequential reaction scheme using thiol specific bifunctional chemical reporters and fluorogenic labels.
Vinyl-sulfone bearing chemical reporters have been synthesized and demonstrated to selectively modify
cysteine under physiological conditions in the presence of other nucleophilic amino acids. Bifunctional
chemical reporters decorated with a terminal alkyne moiety, suitable for modification with azide con-
taining fluorogenic labels were also synthesized. Such fluorogenic (turn on) labels in combination with
these new vinyl-sulfone tags can be used generally in fluorescent modulation schemes of thiol-bearing
biomolecules.
Received 28 February 2014
Received in revised form 13 May 2014
Accepted 27 May 2014
Available online xxx
Keywords:
Fluorogenic dyes
Vinyl-sulfones
Thiol-ene chemistry
Bioorthogonal tagging
Fluorescence
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
e.g., by taking advantage of the metabolic machinery of living
systems using bioorthogonalized building blocks of biopolymers.9
Tagging of biomolecules with fluorescent probes by means of
bioorthogonal methods has drawn much interest lately.1 Static or
dynamic, in vitro or in vivo real-time fluorescence imaging of bi-
ological structures requires chemical transformations using bi-
ologically inert, non-toxic, and non-reactive functional groups.2 At
the same time these bioorthogonal groups need strong affinities
towards their counterpart function in a high yielding reaction at
rates comparable to that of biological processes.2 To date only
a limited set of reactions is available that satisfy the above men-
tioned criteria. Among these, the Staudinger ligation3 of azides and
phosphanes, the inverse electron demand DielseAlder reaction
between tetrazines and strained alkenes,4 the photoactivatable
dipolar cycloaddition of tetrazoles and alkenes,5 and the 1,3-dipolar
cycloaddition of azides and alkynes (CuAAC or SPAAC)6,7 are
probably the most widely utilized. Bioorthogonal, fluorescent
modulation schemes most often follow a two-step process. The first
step involves the implementation of a chemical reporter that can
subsequently be labeled with a specific tag carrying a bio-
orthogonal function.8 Installation of such handles can be achieved,
However, these modified building blocks are not always accepted
by the metabolic pathways of live systems. Another option for site
specific introduction of chemical reporters is offered by genetically
encoded unnatural amino acids bearing a bioorthogonal function.10
This method, however, is limited solely to proteins that are directly
encoded in the genome and cannot be applied to other bio-
molecules or to follow, e.g., post-translational modifications. In
such cases, introduction of bioorthogonalized handles through se-
lective chemical reactions is the method of choice. There are only
a few naturally occurring moieties with distinct reactivity that
enable selective manipulation of target structures. For example, N-
terminal modification of proteins becomes possible by making use
of the relatively lowered pKa of N-termini.11 Single-site modifica-
tion of proteins, however, still remains a challenging task. The thiol
function of cysteine residues as being the second least abundant
amino acid can offer a functionality that can be selectively modified
with chemical reporters.12 To target thiol groups under physiolog-
ical conditions chemical biologists can use native chemical ligation
techniques with synthetic surrogates, disulfide exchange13 or direct
alkylation schemes with iodoacetamides or maleimides.11 Other
techniques involve the treatment of Cys containing sequences with
O-mesitylenesulfonylhydroxylamine.14 Lately, thiol-ene/thiol-yne
* Corresponding author. Tel.: þ36 1 382 6986; e-mail address: kele.peter@ttk.
mta.hu (P. Kele).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.05.103
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G.B. Cserep et al. / Tetrahedron xxx (2014) 1e5
2
chemistry has also become an emerging tool of chemical biology
research.15 The reaction between thiols and alkenes/alkynes can be
triggered either by radical initiators (e.g., UV-light) or nucleophilic
catalysts (e.g., PPh3). Another group of Michael-acceptors is rep-
resented by reagents bearing vinyl-sulfone motif. Earlier works by
Masri et al.16a and Hemelaar et al.16b demonstrated that the excel-
lent reactivity of electron deficient vinyl sulfones towards thiols
allows fast and high yielding modulation of sulfhydryls without the
need for any further auxiliaries.16 A report by Santoyo-Gonzales
et al. provides a recent example for a vinyl-sulfone warhead with
multiple functionality.16c,d Contrary to its superior activity only few
examples were reported on the use of vinyl sulfones in bio-
orthogonal ligation schemes. Besides their reactivity, vinyl-sulfones
are mostly water soluble, highly stable compounds, and their
Michael-addition reaction with thiols proceeds almost quantita-
tively and basically without any by-products.16c,d The Michael
product of vinyl-sulfones is also much more stable compared to
other thiol specific reagents (e.g., maleimides).16 Herein we report
on the synthesis of hydrophilic vinyl-sulfone and alkyne containing
linkers that can site-selectively target thiols. These bifunctional
linkers can serve as chemical reporters enabling further function-
alization by means of alkyne-azide 1,3-dipolar cycloaddition, e.g.,
with fluorogenic labels. Recently, we have reported on the syn-
thesis of new fluorogenic systems that offered low-background
fluorescent tagging schemes with genetically encoded cyclo-
octyne bearing non-canonical amino acids.17 Combination of the
specificity of water soluble vinyl-sulfone based bifunctional
chemical reporters with the power of fluorogenic probes provides
us with a generally applicable technique for site specific and se-
lective fluorescent modulation of peptides and proteins. Further-
more the use of fluorogenic probes is also advantageous, for
example, in the emerging field of super-resolution microscopy
techniques.
A
B
C
Scheme 1. Syntheses of bifunctional chemical reporters and fluorescently labeled thiol
active reagents.
disulfides.18,19 Former HPLC and MS studies have shown that no
dimerization was observed following a 24 h incubation time.18
When an excess amount of 4a was incubated with DTHFPIC in
a buffered medium (50 mM HEPES, pH 8), the peptide could still be
detected after 15 min, however, it was completely consumed after
1 h, with simultaneous appearance of a new peak in the HPLC
chromatogram (ESI). Mass spectrometry confirmed that the new
peak corresponds to a DTHFPIC-4a adduct. Considering that the
heptapeptide contains further side chains with nucleophilic char-
acter, e.g., His, Thr, we compared the MS/MS spectra of the free
peptide and the labeled adduct. To our delight, fragmentation
patterns confirmed that the peptide forms a covalent bond
uniquely with Cys (ESI).
Next, we studied the pH range of reactions, especially in the
presence of other nucleophilic residues, e.g., Lys side chain. For this,
we have synthesized another peptide sequence that contained an
N-terminal Lys-Gly elongation (KGDTHFPIC). First, we explored
whether the altered sequence retained stability.19 Gratifyingly,
minimal disulfide-formation was observed after 24 h incubation
(ESI). At first, we examined the reaction between 1 and KGDTHFPIC
nonapeptide at pH 8, using 1.5 equiv vinyl sulfone reagent. The
reaction conducted this way was almost instantaneous as HPLC
chromatogram showed nearly complete consumption of the pep-
tide within 1 min (ESI). Then we lowered the pH to 7.4 and used
1.5 equiv of 1 to label the nonapeptide. As expected, at lower pH the
reaction required longer time and complete consumption of the
peptide could be observed only after 90 min (ESI). Using equimolar
amounts of the reagents at the same pH required 120 min for
complete conversion of the starting materials. MS, and MS/MS
analysis of the products have confirmed that the peptide was la-
beled with 1 exclusively through the Cys side chain (ESI).
2. Results and discussion
2.1. Synthesis of bifunctional linkers
In the design of these hydrophilic bifunctional chemical re-
porters we have considered bioorthogonal functions that offer
concise synthesis, provide high yielding reactions within reason-
able timeframes, and most importantly that can be targeted by
fluorogenic dyes. More particularly, we aimed at constructing thiol-
specific chemical reporters that can be further modified with flu-
orogenic azides.17 Therefore we have designed chemical reporters
with ethylene glycol cores decorated with vinyl-sulfone and ter-
minal alkyne moieties as thiol and alkyne specific moieties, re-
spectively. The synthesis of 1 was achieved by reacting propargyl
alcohol with divinyl-sulfone (DVS, Scheme 1A). The reaction was
found to give the best yield (50%) when carried out in THF in the
presence of 4 equiv of DVS and catalytic amount of tBuOK. To
construct a more hydrophilic chemical reporter, ethylene glycol
was reacted with propargylbromide to provide 2 (Scheme 1B).
Subsequent reaction of 2 with DVS under similar conditions used
for the synthesis of 1 provided 3 in 60% yield. A portion of 3 and 1
was further subjected to cycloaddition reaction with 3-azido-7-
diethylaminocoumarin, 5 to furnish 4a and 4b (Scheme 1C).
2.3. Sequential labeling of peptide
Our next aim was to demonstrate the possibility of fluorescence
tagging of Cys-residues via sequential labeling schemes. For this, we
have used the KGDTHFPIC nonapeptide sequence. We anticipated
that a two-step tagging scheme of the peptide through its thiol
function by appropriately functionalized vinyl sulfone chemical re-
porter and fluorogenic labels would provide us with reaction mix-
tures that contain a sole fluorescent entity, i.e., the fluorescently
labeled peptide (Scheme 2). First, we have prepared and purified two
derivatives of the peptide: a) KGDTHFPIC-3 and b) KGDTHFPIC-4b.
Secondly, KGDTHFPIC-3 was mixed with 2 equiv of fluorogenic
dye 5 in the absence and in the presence of a copper catalyst. In
2.2. Reactivity and selectivity studies
In order to elaborate the reactivity and selectivity of bifunctional
vinyl-sulfones we have chosen a heptapeptide sequence (DTHFPIC)
containing a single Cys residue. Unlike other Cys-containing pep-
tides, this sequence has the unique feature of existing in mono-
meric form for longer time periods without the formation of
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G.B. Cserep et al. / Tetrahedron xxx (2014) 1e5
3
inefficiency (very low fluorescence) of label 7 is probably due to
poor compatibility of excitation and emission channels with the
fluorescent product. Differences in fluorescent quantum yields
could also be accounted for the lower intensities (for full and color
images see ESI, color of compound 6 is more intense).
3. Conclusions
We have presented a fluorescent tagging scheme using clickable
fluorogenic labels and hydrophilic, clickable, chemical reporters
with thiol-specific vinyl-sulfone warhead. Systematic studies have
demonstrated that these water soluble vinyl-sulfones have strong
and chemoselective affinity towards thiol groups under physio-
logical conditions. In peptide tagging experiments these bi-
functional linkers have been shown to be suitable for further
modification, e.g., with azide containing fluorogenic labels. The use
of these fluorogenic (turn on) labels may offer reduction of back-
ground fluorescence resulting in higher signal-to-noise ratios,
a feature highly demanded, e.g., in the field of super-resolution
microscopy. These results suggest a generalizable chemical modu-
lation scheme of cysteine containing peptides and proteins that
allow further manipulation, e.g., with fluorogenic labels in a se-
quential manner.
Scheme 2. Sequential (Route A) and direct (Route B) labeling strategies of Cys con-
taining peptides.
4. Experimental
4.1. General
a parallel experiment KGDTHFPIC was labeled with excessive
amount (2 equiv) of 4b. All reaction mixtures were loaded directly
onto SDS-gel. Following gel electrophoresis the gel was irradiated
with UV light to trigger fluorescence of the products. As expected,
the purified sample of KGDTHFPIC-4b contains a single fluorescent
product (Fig. 1A, line 3) indicating the position of the product of
KGDTHFPIC-3þ5 reaction in the presence of copper catalyst
(Fig. 1A, line 5).
Unless otherwise indicated, all starting materials were obtained
from commercial suppliers (SigmaeAldrich, Alfa Aesar, Merck) and
used without further purification. Analytical thin-layer chroma-
tography (TLC) was performed on Kieselgel 60 F254 precoated glass
TLC plates with 0.25 mm silica. Column chromatography was car-
ried out with flash silica gel (0.040e0.063 mm) from Merck. Sem-
ipreparative RP-HPLC purification was carried out using a Knauer
HPLC system under the following conditions: gradient elution:
5 min equilibration at 0% B, 0e90% B 90 min (A eluent: 0.1% TFA/
water v/v; B eluent: 0.1% TFA/acetonitrile:water/80:20 v/v), flow
rate: 4 ml/min, detection: 220 nm, column: Phenomenex Jupiter
ꢁ
C18, 10 mm IDꢀ250 mm, 10
m
m, 300 A. The NMR spectra were
recorded on a Bruker DRX-250 spectrometer. Chemical shifts (d) are
given in parts per million (ppm) using solvent signals as the ref-
erence. Coupling constants (J) are reported in Hertz (Hz). Splitting
patterns are designated as s (singlet), d (doublet), t (triplet), q
(quadruplet), quint (quintuplet), m (multiplet), dd (doublet of
a doublet), and td (triplet of doublets). Mass spectrometry of the
peptide derivatives was carried out on a Bruker Daltonics Esquire
3000þ ion trap mass spectrometer while high resolution mass
spectrometry (HRMS) measurements were carried out on an Agi-
lent 6230 MS-TOF system. All melting points were measured on
Fig. 1. Fluorescence images of gels. (A) Comparison between sequential (lane 5) and
direct labeling routes (lane 6). (B) Sequential labeling with further fluorogenic labels
(lanes 9e12) in comparison with direct labeling (lane 8).
€
Buchi 501 apparatus and are uncorrected. IR spectra were obtained
The fact that line 5 of Fig. 1A shows only a single fluorescing
band, implies that the excess amount of fluorogenic label 5 does not
contribute to the fluorescent signal. The image also shows that the
same reaction in the absence of copper does not result in fluores-
cent species (Fig. 1A, line 4). On the other hand, when the peptide
was reacted directly with 2 equiv of fluorescent chemical reporter
4b a second fluorescent band appears just above the product line
indicating the presence of unreacted 4b (Fig. 1A, line 6). These
findings support the superiority of sequential modification routines
(Route A) over direct fluorescent labeling schemes.
on Bruker IFS55 spectrometer on a single-reflection diamond ATR
unit.
For peptide synthesis, N,N0-diisopropylcarbodiimide (DIC), trii-
sopropylsilane (TIS) were purchased from Fluka (Buchs, Switzer-
land). The amino acid derivatives were obtained from Reanal
(Budapest, Hungary) and IRIS Biotech (Marktredwitz, Germany). 1-
Hydroxybenzotriazole (HOBt) and 1,8-diazabicyclo-[5.4.0]undec-7-
ene (DBU) were purchased from IRIS Biotech. Fmoc-Rink Amide
€
MBHA resin was purchased from NovaBiochem (Laufelfingen,
Switzerland). Acetonitrile, trifluoroacetic acid (TFA), N-methyl-
pyrrolidone (NMP), and dimethyl sulfoxide (DMSO) were obtained
from Merck (Darmstadt, Germany). Ninhydrin was bought from
SigmaeAldrich (St. Louis, MO, USA). N,N-Dimethylformamide
(DMF) was purchased from Reanal (Budapest, Hungary).
In a separate experiment we have tested the performance of
other fluorogenic labels 6e8 that have recently been developed in
our laboratory.17 Fig. 1B shows that all fluorogenic labels gave
similar results, with label 8 giving the best performance. The
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4
4.2. Synthesis
J1¼8.9 Hz, J2¼2.4 Hz), 6.76 (1H, dd, J1¼16.6 Hz, J2¼9.9 Hz), 7.41 (1H,
d, J¼8.9 Hz), 8.39 (1H, s), 8.5 (1H, s). 13C NMR (CDCl3, 62.5 MHz):
4.2.1. 3-(2-(Vinylsulfonyl)ethoxy)prop-1-yne (1). To
propargyl alcohol (120
in anhydrous THF (15 mL) under argon atmosphere was added
dropwise a suspension of BuOK (46 mg, 0.41 mmol) in anhydrous
a
solution of
d
¼12.4, 45.0, 55.0, 63.9, 64.4, 97.0, 107.0, 110.1, 116.6, 123.7, 129.1,
m
L, 2.05 mmol) and DVS (820
mL, 8.20 mmol)
130.0, 134.8, 137.7, 143.7, 151.7, 155.8, 156.9. HRMS (ESI): [MþH]þ
calcd for C20H25N4O5Sþ: 433.1546, found: 433.1544.
t
THF (20 mL). The reaction mixture was stirred at room temperature
for 45 min when TLC (DCM:MeOH/200:1) showed total conversion
of the alcohol. After evaporation of the solvent the product was
extracted with EtOAc (5ꢀ10 mL) and purified on silica (DCM:MeOH/
500:1) to give 174 mg (49%) of the desired product as a colorless oil.
4.2.5. 7-(Diethylamino)-3-(4-((2-(2-(vinylsulfonyl)ethoxy)ethoxy)-
methyl)-1H-1,2,3-triazol-1-yl)-coumarin
(4b). 7-Diethylamino-3-
azido-coumarin (70 mg, 0.28 mmol) and 3-(2-(vinylsulfonyl)-eth-
oxy)-prop-1-yne (1) (63 mg, 0.28 mmol) were dissolved in MeCN
(10 mL) and triethylamine (50 mL) and CuI (53 mg, 0.28 mmol) were
Rf¼0.28 (DCM:MeOH/500:1). IR:
1386; 1445; 1612; 2116; 2877; 3060; 3269 cmꢁ1
250 MHz):
n
(neat)¼1095; 1126; 1311; 1358;
added. The mixture was stirred at room temperature for 1 h when
TLC (Hexane:EtOAc/1:1) showed completion of the reaction. After
evaporation of the solvent the product was extracted by EtOAc
(3ꢀ5 mL) from the brown residue and was purified on silica (Hex-
ane:EtOAc/1:1/1:3 gradient) to yield 67 mg (50%) product as
a yellowish powder. Rf¼0.12 (Hexane:EtOAc/1:2). Mp¼101e103 ꢂC.
.
1H NMR (CDCl3,
d
¼2.47 (1H, d, J¼2.3 Hz), 3.26 (2H, t, J¼5.6 Hz), 3.91 (2H, t,
J¼5.6 Hz), 4.14 (2H, d, J¼2.3 Hz), 6.09 (1H, d, J¼9.9 Hz), 6.38 (1H, d,
J¼16.6 Hz), 6.70 (1H, dd, J1¼16.6 Hz, J2¼9.9 Hz). 13C NMR (CDCl3,
62.5 MHz):
d
¼54.7, 58.4, 63.2, 75.4, 78.4, 129.2, 137.4. HRMS (ESI):
[MþH]þ calcd for C7H11O3Sþ: 175.0429, found: 175.0431.
IR:
n
(neat)¼1188,1243,1265,1291,1346,1381,1472,1596,1624,1695,
1714, 2870, 2904, 2976, 3060, 3160 cmꢁ1. 1H NMR (CDCl3, 250 MHz):
4.2.2. 2-(Prop-2-ynyloxy)ethanol (2). A solution of propargylbromide
(2.5 mL, 80% in toluene, 22.5 mmol) and ethylene glycol (2.5 mL) was
flushed with argon and cooled to 0 ꢂC in an ice bath. Powdered NaOH
(1.08 g, 45.0 mmol) was added and the reaction mixture was stirred
at 45 ꢂC for 3 h. The precipitate was filtered, washed with DCM
(2ꢀ3 mL), and the filtrate was extracted with DCM (3ꢀ10 mL). After
evaporation of the solvent the crude product was purified using silica
gel column chromatography (Hexane:EtOAc/2:1) to yield 1.34 g (60%)
d
¼1.22 (6H, t, J¼7.0 Hz), 3.25 (2H, t, J¼5.6 Hz), 3.44 (4H, q, J¼7.0 Hz),
3.67 (4H, m), 3.88 (2H, t, J¼5.6 Hz), 4.72 (2H, s), 6.07 (1H, dd,
J¼9.9 Hz), 6.36 (1H, d, J¼16.6 Hz) 6.53 (1H, s), 6.66 (1H, d, J¼8.9 Hz),
6.79 (1H, dd, J1¼16.6 Hz, J2¼9.9 Hz), 7.39 (1H, d, J¼8.9 Hz), 8.35 (1H,
s), 8.53 (1H, s). 13C NMR (CDCl3, 62.5 MHz):
d¼12.3, 44.9, 54.9, 64.3,
64.6, 69.2, 70.4, 96.9, 106.9, 110.0, 116.7, 123.6, 128.8, 130.0, 134.8,
137.8, 144.4, 151.5, 155.7, 156.9. HRMS (ESI): [MþH]þ calcd for
C
22H29N4O6Sþ: 477.1808, found: 477.1810.
of 2 as a pale yellow oil. Rf¼0.25 (Hexane:EtOAc/2:1). IR:
n
(neat)¼
1106; 1246; 1729; 2115; 2868; 2935; 3287; 3400 cmꢁ1
.
1H NMR
4.3. Peptide synthesis, tagging reactions
(CDCl3, 250 MHz):
d
¼2.42 (1H, t, J¼2.2 Hz), 2.99 (1H, s), 3.55 (2H, t,
J¼4.4 Hz), 3.66 (2H, t, J¼4.4 Hz), 4.12 (2H, d, J¼2.2 Hz). 13C NMR
4.3.1. Peptide synthesis. The peptides (DTHFPIC, KGDTHFPIC) were
synthesized manually by Fmoc/tBu solid phase strategy on Fmoc-
Rink Amide MBHA resin. The protocol of the synthesis was the
following: (i) Fmoc deprotection with 2% piperidine/2% DBU/DMF
(v/v), 2þ2þ5þ10 min; (ii) washing with DMF (5ꢀ1 min); (iii)
coupling with 3 equiv of Fmoc-amino acid derivativeeDICeHOBt
dissolved in NMP, 60 min; and (iv) washing with DMF (5ꢀ1 min).
Peptides were cleaved from the resin with the TFA/H2O/TIS
(9.5:2.5:2.5 v/v) mixture (2 h, RT). After filtration, compounds were
precipitated in cold diethyl ether, centrifuged (4000 rpm, 5 min),
and freeze-dried in water. Crude products were purified by semi-
preparative RP-HPLC. Purified peptides were analyzed by analyti-
cal RP-HPLC, ESI MS, and amino acid analysis (ESI).
(CDCl3, 62.5 MHz):
d
¼58.1, 61.2, 71.0, 74.6, 79.3. HRMS (ESI): [MþH]þ
calcd for C5H9Oþ2 : 101.0603, found: 101.0602.
4.2.3. 3-(2-(2-(Vinylsulfonyl)ethoxy)ethoxy)prop-1-yne (3). To a so-
lution of 2 (320 mg, 3.2 mmol) and DVS (480
mL, 4.8 mmol) in
anhydrous THF (15 mL) under argon atmosphere was added
t
dropwise a suspension of BuOK (72 mg, 0.71 mmol) in anhydrous
THF (30 mL). The reaction mixture was stirred at room temperature
for 45 min when TLC (Hexane:EtOAc/5:3) showed total conversion
of the alcohol. After evaporation of the solvent the product was
extracted by EtOAc (5ꢀ10 mL) and purified on silica (Hex-
ane:EtOAc/2:1) to give 402 mg (58%) pale yellow oil. Rf¼0.27
(Hexane:EtOAc 5:3). IR:
n
(neat)¼1122; 1353; 1385; 1461; 2115;
2872; 3266 cmꢁ1 1H NMR (CDCl3, 250 MHz):
.
d
¼2.43 (1H, d,
4.3.2. Synthesis of KGDTHFPIC-3. 7.6 mg (7.5
peptide was dissolved in 1000 L 50 mM HEPES buffer (pH 8.0) and
2.4 mg (11.2 mol, 1.5 equiv) 3 in 100 L DMF was added to the
mmol) KGDHTFPIC
J¼2.4 Hz), 3.24 (2H, t, J¼5.7 Hz), 3.64 (4H, m), 3.87 (2H, t, J¼5.7 Hz),
4.16 (d, 2H, J¼2.4 Hz), 6.06 (1H, d, J¼9.9 Hz), 6.36 (1H, d, J¼16.6 Hz),
6.78 (1H, dd, J1¼16.6 Hz, J2¼9.9 Hz). 13C NMR (CDCl3, 62.5 MHz):
m
m
m
peptide. The reaction was stirred at room temperature and moni-
tored by analytical RP-HPLC. The reaction was totally completed
after 1 h. The product was purified by semi-preparative RP-HPLC
and analyzed by analytical RP-HPLC, ESI MS, MS/MS (see ESI).
d
¼54.9, 58.2, 64.6, 68.6, 70.2, 74.7, 79.3, 128.7, 137.8. HRMS (ESI):
[MþH]þ calcd for C9H15O4Sþ [MþH]þ: 219.0691, found: 219.0689.
4.2.4. 7-(Diethylamino)-3-(4-((2-(vinylsulfonyl)ethoxy)methyl)-1H-
1,2,3-triazol-1-yl)-coumarin (4a). 7-Diethylamino-3-azido-couma-
rin (100 mg, 0.39 mmol) and 3-(2-(vinylsulfonyl)ethoxy)-prop-1-
yne (1) (67 mg, 0.39 mmol) were dissolved in MeCN (10 mL) and
4.3.3. Synthesis of KGDTHFPIC-4b. Route A: 1.0 mg (0.8
KGDHTFPIC-3 was dissolved in 287 L 50 mM HEPES buffer (pH
8.0). 203 L 10 mM sodium -ascorbate (2.5 equiv) solution in
50 mM HEPES buffer (pH 8.0) and 203 L 10 mM CuSO4eTBTA
(2.5 equiv) solution (55% DMSO, 45% distilled water) was prepared
and combined. To this mixture 418 g (1.6 mol, 2 equiv) 5 in 287
DMSO and the 287 L solution of KGDTHFPIC-3 was added finally.
The reaction was stirred at room temperature and monitored by
analytical RP-HPLC. The reaction was completed after 24 h. The
product was purified by semipreparative RP-HPLC and analyzed by
analytical RP-HPLC, ESI MS, MS/MS (ESI).
mmol)
m
m
L
triethylamine (50
mL) and CuI (7.4 mg, 0.039 mmol) were added.
m
The mixture was stirred at room temperature for 1 h when TLC
(Hexane:EtOAc/1:1) showed completion of the reaction. After
evaporation of the solvent the product was extracted by EtOAc
(3ꢀ5 mL) purified on silica (Hexane:EtOAc/1:1/1:3 gradient) to
yield 62 mg (37%) yellowish powder. Rf¼0.14 (Hexane:EtOAc 1:1).
m
m
mL
m
Mp¼152e154 ꢂC. IR:
(neat)¼1249; 1291; 1432; 1465; 1484; 1526;
n
1596; 1621; 1708; 2875; 2925; 3069; 3179 cmꢁ1. 1H NMR (CDCl3,
250 MHz):
d
¼1.24 (6H, t, J¼7.1 Hz), 3.28 (2H, t, J¼5.6 Hz), 3.46 (4H,
Route B: 2.5 mg (2.5
400 L 50 mM HEPES buffer (pH 8.0) and 2.3 mg (4.9
4b in 400 L DMF was added to the peptide. The reaction was
m
mol) KGDHTFPIC peptide was dissolved in
q, J¼7.1 Hz), 3.97 (2H, t, J¼5.6 Hz), 4.72 (2H, s), 6.10 (1H, d, J¼9.9 Hz),
m
mmol, 2 equiv)
6.40 (1H, d, J¼16.6 Hz), 6.55 (1H, d, J¼2.4 Hz), 6.68 (1H, dd,
m
ꢀ
Please cite this article in press as: Cserep, G. B.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.103

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G.B. Cserep et al. / Tetrahedron xxx (2014) 1e5
5
€
stirred at room temperature and monitored by analytical RP-HPLC.
The reaction was totally completed after 7 h. The product was pu-
rified by semi-preparative RP-HPLC and analyzed by analytical RP-
HPLC, ESI MS, MS/MS (ESI).
the ‘Lendulet’ Program of the Hungarian Academy of Sciences
(LP2013-55/2013) is greatly acknowledged.
Supplementary data
4.4. Gel chromatography
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2014.05.103.
SDS-PAGE gel electrophoresis was carried out using PhastGelÒ
high density gels and PhastGel SDS buffer strips on PhastSystem
electrophoresis system (Pharmacia, Uppsala, Sweden). The fol-
lowing samples were prepared:
References and notes
1. 1
2. 1
3. and 7. 1
4. 1 g/
m
m
g/
g/
m
m
L KGDTHFPIC in distilled water.
L KGDTHFPIC-3 in distilled water.
g/ L KGDTHFPIC-4b in distilled water.
L KGDTHFPIC-3 and 2 equiv 5 in distilled water/DMSO.
5. and 9. 1 g/ L KGDTHFPIC-3 in HEPES buffer, 2 equiv 5 in DMSO,
2.5 equiv 10 mM sodium -ascorbate solution, 2.5 equiv 10 mM
1. (a) Sahoo, H. RSC Adv. 2012, 2, 7017; (b) Sameiro, M.; Gonc¸ alves, T. Chem. Rev.
2009, 109, 190; (c) Kele, P.; Li, X.; Link, M.; Nagy, K.; Herner, A.; Lorincz, K.; Beni,
Sz; Wolfbeis, O. S. Org. Biomol. Chem. 2009, 7, 3486.
}
ꢀ
m m
2. (a) Debets, M. F.; van Hest, J. C. M.; Rutjes, F. P. J. T. Org. Biomol. Chem. 2013, 11,
6439; (b) Karver, M. R.; Weissleder, R.; Hilderbrand, S. A. Angew. Chem., Int. Ed.
2012, 51, 920; (c) Carroll, L.; Evans, H. L.; Aboagye, E. O.; Spivey, A. C. Org. Bi-
omol. Chem. 2013, 11, 5772.
m
m
m
m
L
€
3. Schilling, C. I.; Jung, N.; Biskup, M.; Schepers, U.; Brase, S. Chem. Soc. Rev. 2011,
CuSO4eTBTA solution, the sample was mixed for 20 h at room
40, 4840.
temperature before the gel electrophoresis.
6. and 8. 1 mg/mL KGDTHFPIC in HEPES buffer, 2 equiv 4b in DMSO,
the sample was stirred for 20 h at room temperature before the gel
electrophoresis.
4. Knall, A. C.; Slugovc, C. Chem. Soc. Rev. 2013, 42, 5131.
5. Lim, R. K.; Lin, Q. Acc. Chem. Res. 2011, 44, 828.
6. Hein, J. E.; Fokin, V. V. Chem. Soc. Rev. 2010, 39, 1302.
7. (a) Varga, B. R.; Kallay, M.; Hegyi, K.; Beni, Sz; Kele, P. Chem.dEur. J. 2012, 18,
822; (b) Dommerholt, J.; Schmidt, S.; Temming, R.; Hendriks, L. J. A.; Rutjes, F. P.
J. T.; van Hest, J. C. M.; Lefber, D. J.; Friedl, P.; van Delft, F. L. Angew. Chem., Int. Ed.
2010, 49, 9422; (c) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc.
2010, 132, 3688.
8. Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13.
9. Grammel, M.; Hang, H. C. Nat. Chem. Biol. 2013, 9, 475.
10. Xiao, H.; Chatterjee, A.; Choi, S.-H.; Bajjuri, K. M.; Sinha, S. C.; Schultz, P. G.
Angew. Chem., Int. Ed. 2013, 52, 14080.
11. Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974.
12. Chalker, J. M.; Bernardes, G. J. L.; Lin, Y. A.; Davis, B. G. Chem.dAsian J. 2009, 4,
630.
ꢀ
ꢀ
10. 1
mg/mL KGDTHFPIC-3 in HEPES buffer, 2 equiv 6 in DMSO,
2.5 equiv 10 mM sodium
L-ascorbate solution, 2.5 equiv 10 mM
CuSO4eTBTA solution, the sample was mixed for 20 h at room
temperature before the gel electrophoresis.
11. 1
mg/mL KGDTHFPIC-3 in HEPES buffer, 2 equiv 7 in DMSO,
2.5 equiv 10 mM sodium
L-ascorbate solution, 2.5 equiv 10 mM
CuSO4eTBTA solution, the sample was mixed for 20 h at room
temperature before the gel electrophoresis.
13. (a) Wong, C. T. T.; Tung, C. L.; Li, X. Mol. BioSyst. 2013, 9, 826; (b) Markey, L.;
Giordani, S.; Scanlan, E. M. J. Org. Chem. 2013, 78, 4270.
14. Bernardes, G. J. L.; Chalker, J. M.; Errey, J. C.; Davis, B. G. J. Am. Chem. Soc. 2008,
12. 1
m
g/
m
L KGDTHFPIC-3 in HEPES buffer, 2 equiv 8 in DMSO,
-ascorbate solution, 2.5 equiv 10 mM
2.5 equiv 10 mM sodium
L
130, 5052.
CuSO4eTBTA solution, the sample was mixed for 20 h at room
temperature before the gel electrophoresis.
15. Dondoni, A. Angew. Chem., Int. Ed. 2008, 47, 8995.
16. (a) Masri, M. S.; Friedman, M. J. Protein Chem. 1988, 7, 49; (b) Hemelaar, J.;
Borodovsky, A.; Kessler, B. M.; Reverter, D.; Cook, J.; Kolli, N.; Gan-Erdene, T.;
Wilkinson, K. D.; Gill, G.; Lima, C. D.; Ploegh, H. L.; Ovaa, H. Mol. Cell. Biol. 2004,
40e40
SDS, 0.01% Bromophenol Blue, pH 8.0) was added to 40
samples. The samples were heated at 100 ꢂC for 5 min. 4e4
m
L 2ꢀ running buffer (10 mM Tris/HCl, 1 mM EDTA, 2.5%
L of each
L of
~
24, 84; (c) Morales-Sanfrutos, J.; Lopez-Jaramillo, J.; Ortega-Munoz, M.; Megia-
m
Fernandez, A.; Perez-Balderas, F.; Hernandez-Mateo, F.; Santoyo-Gonzalez, F.
Org. Biomol. Chem. 2010, 8, 667; (d) Morales-Sanfrutos, J.; Lopez-Jaramillo, F. J.;
Hernandez-Mateo, F.; Santoyo-Gonzalez, F. J. Org. Chem. 2010, 18, 4039; (e)
Chatani, S.; Nair, D. P.; Bowman, C. N. Polym. Chem. 2013, 4, 1048; (f) Mather, B.
D.; Viswanathan, K.; Miller, K. M.; Long, T. E. Prog. Polym. Sci. 2006, 31, 487.
m
the samples were applied for the gel. The separation was carried
out according to the PhastSystem, Separation Technique File No.
112.20 The gel was irradiated with UV light and photographed. The
images were taken using Genesnap software (Syngene, Frederick,
USA).
ꢀ
ꢀ
17. (a) Herner, A.; Nikic, I.; Kallay, M.; Lemke, E. A.; Kele, P. Org. Biomol. Chem. 2013,
ꢀ
ꢀ
11, 3297; (b) Herner, A.; Estrada Girona, G.; Nikic, I.; Kallay, M.; Lemke, E. A.;
Kele P. under review.
ꢀ
}
}
18. Balogh, A.; Horvati, K.; Mezo, G.; Derzbach, L.; Szebeni, B.; Nagy, L.; Prechl, J.;
ꢀ ꢀ
Vasarhelyi, B.; Hudecz, F.; Bosze, S. J. Pept. Sci. 2009, 15, 285.
ꢀ
}
19. Horvati, K.; Bosze, Sz; Hudecz, F.; Medzihradszky-Schweiger, H. J. Pept. Sci.
Acknowledgements
2008, 14, 838.
20. PhastSystem Separation Technique File No. 112,https://www.gelifesciences.
com/gehcls_images/GELS/Related%20Content/Files/1314723116657/lit-
doc80131229_20110830190204.pdf.
Financial support of the Hungarian Scientific Research Fund
(OTKA, grant numbers K-100134, K-104275, and NN-110214) and
ꢀ
Please cite this article in press as: Cserep, G. B.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.103