Fluorescent labeling of peptides on solid phase

Article abstract of DOI:10.1039/b811693h

N ^α-Fmoc-N^ε-[(7-methoxycoumarin-4-yl) acetyl]-l-lysine (N^α-Fmoc-l-Lys(Mca)-OH) 3 is conveniently prepared by benzotriazole methodology (52% over two steps). N-Acylbenzotriazoles Mca-Bt 2, N^α-Fmoc-l-Lys(Mca)-Bt 4

Full text of DOI:10.1039/b811693h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Fluorescent labeling of peptides on solid phase†
Alan R. Katritzky,*a Megumi Yoshioka,a Tamari Narindoshvili,a Alfred Chungb and Jodie V. Johnsonc
Received 9th July 2008, Accepted 16th September 2008
First published as an Advance Article on the web 30th October 2008
DOI: 10.1039/b811693h
Na-Fmoc-Ne-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine (Na-Fmoc-L-Lys(Mca)-OH) 3 is conveniently
prepared by benzotriazole methodology (52% over two steps). N-Acylbenzotriazoles Mca-Bt 2,
Na-Fmoc-L-Lys(Mca)-Bt 4, coumarin-3-ylcarbonyl (Cc)-Bt 5, Na-Fmoc-L-Lys(Cc)-Bt 7 and
Na-(Cc)-L-Lys(Fmoc)-Bt 9 enable the efficient microwave enhanced solid-phase fluorescent labeling of
peptides.
transfer between an excited donor fluorophore to a proximal ac-
Introduction
ceptor fluorophore.21 Usually, the donor fluorescence is quenched
Proteins modified with fluorescent dyes, enzymes, and other
by the acceptor without subsequent fluorescence emission. Donor
and acceptor groups,13–17,22–24 attached to a synthetic peptide
undergo FRET, producing a unique fluorescence spectrum. En-
hanced donor fluorescence indicates proteolysis accompanied by
the loss of FRET as a result of separation of the donor and
acceptor groups.
Many donor and acceptor groups have been incorporated into
quenched fluorogenic substrates.13–24 Coumarins have extensive
and diverse applications as fluorescent probes or labels;25 they
exhibit an extended spectral range, are photostable and have high
emission quantum yields. 7-Methoxycoumarin-4-ylacetyl (Mca,
e325 = 14500 M-1cm-1 and Uf = 0.49) was proposed as a fluorophore
for thimet peptidase, pitrilysin and MMP substrates.14,16a,17 The
combination of Mca as a fluorophore and (2,4-dinitrophenyl)
(Dnp) as a quencher has several advantages over the more common
fluorogenic substrate pair of tryptophan (Trp)/Dnp: the Mca
residue is more fluorescent and more chemically stable than Trp,
and Mca is efficiently quenched by Dnp.
reporter groups are valuable tools with widespread uses in im-
munology and biochemical research. Protein-capture microarrays
have become a promising tool for protein analysis in drug dis-
covery, diagnostics and biological research in the last few years.1a
Fluorescent derivatives of biologically active peptides are useful
experimental tools for studying biological structure and function1b
and for visualization of intracellular processes or molecular
interactions.1c,d
Matrix metalloprotease (MMP) proteins are implicated in
many diseases, including arthritis, periodontal disease, tumor cell
invasion, and metastasis.2–7 The detection of a protein bound by a
specific capture agent is key to microarray-based methods, for tra-
ditional immunoassays and biosensor applications.8–12 Synthetic
peptide-based assays can differentiate enzyme types and monitor
their activities.13–17 Fluorogenic substrates can be monitored18
continuously and utilized at low concentrations, thus providing
a particularly convenient enzyme assay method.
Fluorescent biosensors can be composed of a binding molecule,
such as an antibody or enzyme, derivatized with a single fluores-
cent probe, which is sensitive to changes in the local environment.19
Fluorogenic groups can often be attached at the cleavage sites
of proteases and esterases. However, this simple approach is not
applicable if the enzyme requires binding interactions at both sides
of the cleavage site. For such cases, quenched fluorescent peptides
are designed as short sequences of amino acids, containing enzyme
cleavable specific sites, with fluorescent donor and acceptor probes
linked at the N- and C-termini.13–17
Fluorescently labeled peptides might be achieved by the reaction
of the peptide in solution with an activated form of fluorophore,
however a potentially more effective approach is to assemble the
peptide chain on a solid phase and incorporate the fluorophore
into the peptide attached to the solid support.26a,26b
For the solid phase peptide labeling by Mca, the N-termini
of peptide–resins have been acylated with 7-methoxycoumarin-
4-ylacetic acid using standard synthetic cycles.14,16a,17 However,
inefficient acylation of the peptide-resin led Malkar and Fields
to incorporate Mca into Na-Fmoc-lysine molecules by a 4-step
method providing Na-Fmoc-L-Lys(Mca)-OH (17% overall).27
Coumarin-labeled lysines are of considerable general interest
for the design and synthesis of fluorogenic triple-helical substrates
for the analysis of matrix metalloproteinase family members.27–29
Thus, Ne-coumarin-labeled-Na-Fmoc lysines allow the successful
labeling of peptide substrates by solid phase peptide synthesis for
an extracellular matrix metalloprotease and present a powerful
tool for proteolysis monitoring.27,28
In other biosensors20 the binding molecule is labeled with two
fluorophores, suitable for fluorescence resonance energy transfer
(FRET). The acceptor–quencher pair enables nonradiative energy
aUniversity of Florida, Gainesville, FL 32611-7200, USA. E-mail:
katritzky@chem.ufl.edu; Fax: +1 352-392-9199; Tel: +1 352-392-0554
bPROTEOMICS, Interdisciplinary Center for Biotechnology, Research
University of Florida, Gainesville, FL 32611-1376, USA. E-mail: achung@
biotech.ufl.edu
cDepartment of Chemistry, University of Florida, Gainesville, FL 32611-
7200, USA. E-mail: jvj@chem.ufl.edu
We have previously reported the extensive use of N-acy-
lbenzotriazoles for N-acylation,30 C-acylation31 and O-acylation32
reactions. (Na-Fmoc-aminoacyl)benzotriazoles and their Boc-
and Cbz- analogs enabled the preparation of chiral di-, tri- and
tetrapeptides in average yields of 88% from natural amino acids
† Electronic supplementary information (ESI) available: Synthetic proce-
dures for 2, 4, 7, 9; compound characterization data for 2, 4, 7–9; HPLC
profiles of 10–17; absorption spectra of 15 and fluorescence emission
spectra of 10, 11, 14–17. See DOI: 10.1039/b811693h
4582 | Org. Biomol. Chem., 2008, 6, 4582–4586
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in solution phase.33 Recently we have also prepared tri-, tetra-,
penta-, hexa-, and heptapeptides in 71% average crude yields by
microwave-assisted solid phase peptide synthesis utilizing (Na-
Fmoc-aminoacyl)benzotriazoles.34 These easily obtained, chirally
stable, and moisture insensitive reagents acylate NH2-peptide-
resins without using coupling agents or additives, thus avoiding
side reactions and epimerization.34
lysine inaqueous MeCN at 20 ◦C inthe presenceofEt3N to provide
lysine-scaffold based fluorescent building blocks 6 (see ref. 35)
and novel 8 (87 and 79%), respectively (Scheme 2), convertible
conveniently into the corresponding N-acylbenzotriazoles 7 (see
ref. 36) and 9 (87 and 71%).
We now report the efficient fluorescent labeling of peptides on
solid phase by acylation with benzotriazole activated derivatives
of (i) coumarin-3-ylcarboxylic acid and 7-methoxycoumarin-
4-ylacetic acid and (ii) coumarin-3-ylcarbonyl (Cc) and 7-
methoxycoumarin-4-ylacetyl (Mca) labeled lysines.
In comparison with the conventional procedures for the
preparation of fluorescent peptides on solid phase,24–26,28 our
methodology optimized under mild microwave irradiation, utilizes
benzotriazole activated fluorogenic substrates which are stable,
moisture insensitive acylation reagents enabling solid phase flu-
orescent labeling of peptides without using coupling agents or
additives and avoiding side reactions and epimerization.
Scheme 2
Results and discussion
Benzotriazole activated fluorogenic substrates 2, 4, 5, 7, 9 were
prepared and demonstrated to be useful acylation reagents, for
the efficient fluorescent labeling of peptides on solid phase.
Solid Phase Peptide Synthesis
Solid phase peptide synthesis, microwave-assisted as optimized
previously in our laboratory,34 enables efficient acylation of NH2-
groups on solid phase by the benzotriazole activated fluorogenic
substrates 2, 4, 5, 7, 9. We used 2 or 5 to couple the fluorophore
directly to diverse peptides. Alternatively, we used 4, 7, or 9
to couple the fluorophore already attached to a lysine moiety
to the peptides. Coumarin-labeled peptides were synthesized as
C-terminal amides using Fmoc solid-phase methodology under
microwave irradiation.
Na-Fmoc-Ne-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine
(Na-Fmoc-L-Lys(Mca)-OH) 3
7-Methoxycoumarin-4-ylacetic acid 1 was converted by 1H-
benzotriazole and thionyl chloride in THF at 20 ◦C into crys-
talline, stable 4-(benzotriazole-1-ylacetyl)-7-methoxycoumarin 2
(78%) (Scheme 1); 2 was then coupled with Na-Fmoc-L-lysine
in aqueous MeCN in the presence of Et3N for 20 min, to
afford Na-Fmoc-L-Lys(Mca)-OH 3 (overall 51%). In comparison
with the recent literature procedure27 for the preparation of Na-
Fmoc-L-Lys(Mca)-OH 3, our two-step methodology uses Na-
Fmoc-L-lysine, offers simple preparative and workup procedures,
short times to completion, the use of inexpensive reagents and
high yields. Conventional benzotriazole activation of 3 gave Na-
Fmoc-L-Lys(Mca)-Bt 4 (70%) (Scheme 1).
Solid phase fluorescent labeling with 4 and preparation of labeled
dipeptide H-L-Ala-L-Lys(Ne-Mca)-NH2 10
The convenience of 4 for fluorescent labeling on solid phase
was demonstrated for a model dipeptide H-L-Ala-L-Lys(Ne-Mca)-
NH2 10. Compound 10 was synthesized using microwave-assisted
SPPS conditions.34 After initial removal of the Fmoc protecting
group, free Rink resin-NH2 was coupled with 4 in DMF under mi-
crowave irradiation for 10 min at 70 ◦C. The second coupling was
performed with the (Na-Fmoc-aminoacyl)benzotriazole reagent
derived from Fmoc-L-Ala, finally the desired peptide was cleaved
from the resin to produce peptide amide 10 (26%) (Scheme 3,
Table 1, Fig. 1). Conditions were optimized to maximize the rate
while avoiding epimerization.
Solid phase fluorescent labeling with 7 to synthesize labeled
peptides 11–14
Scheme 1
In a similar manner, microwave-assisted SPPS was achieved
(3 min coupling time for each step) with 7 to obtain the
fluorescently labeled di, tri-, tetra- and hexa-peptides: H-L-Ala-
L-Lys(Ne-Cc)-NH2 11, H-L-Pro-L-Phe-L-Lys(Ne-Cc)-NH2 12, H-
L-Trp-L-Lys(Ne-Cc)-L-Met-L-Phe-NH2 13 and H-L-Lys(Ne-Cc)-L-
Pro-Gly-L-Leu-L-Met-L-Trp-NH2 14 in yields of 18–45% (after
HPLC purification) (Table 1). The successive coupling steps
Na-Fmoc-Ne-(coumarin-3-ylcarbonyl)-L-lysine benzotriazolide
(Na-Fmoc-L-Lys(Cc)-Bt) 7 and Na-(coumarin-3-ylcarbonyl)-
Ne-Fmoc-L-lysine benzotriazolide (Na-Cc-L-Lys(Fmoc)-Bt) 9
3-(Benzotriazole-1-ylcarbonyl)chromen-2-one35 5 was coupled
with commercially available Na-Fmoc-L-lysine and Ne-Fmoc-L-
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coupling with Na-coumarin-attached lysine 9 through its free Ne-
position to prepare labeled tripeptide H-L-Phe-L-Leu-L-Lys(Na-
Cc)-NH2 15 (35%) (Table 1).
Solid phase fluorescent labeling with 5 to synthesize labeled
dipeptide (Cc)-L-Leu-L-Leu-NH2 16 and labeling with 2 to
synthesize labeled dipeptide (Mca)-L-Leu-L-Leu-NH2 17
Fluorescent labeling with benzotriazole-activated coumarin-3-
ylcarboxyl acid 5 was demonstrated by the preparation of
coumarin-3-ylcarbonyl-labeled dipeptide (Cc)-L-Leu-L-Leu-NH2
16. After initial removal of the Fmoc protecting group from
Rink resin, Fmoc-L-Leu-Bt was utilized for each of two successive
coupling steps (3 min coupling time for each step). After the final
coupling with 5 (10 min coupling time), the desired fluorescent-
labeled peptide 16 (29%) (Table 2) was cleaved from the resin.
Fluorescent labeling with benzotriazole activated 7-methoxy-
coumarin-4-ylacetic acid 2 was demonstrated to allow the prepa-
ration of 7-methoxycoumarin-4-ylacetyl-labeled dipeptide (Mca)-
L-Leu-L-Leu-NH2 17 (26%) (Scheme 4, Table 2), under similar
conditions to those utilized for the preparation of 16.
Scheme 3
Fig.
1
Bottom: the HPLC profile of crude peptide 10 (H-L-
Ala-L-Lys(Ne-Mca)-NH2). Top: the HPLC profile of peptide 10 after
purification.
utilized the appropriate N-acylbenzotriazoles derived from Fmoc-
protected L-Met, L-Trp, L-Phe, L-Leu, L-Pro and Gly prepared
by our previously published procedures.34 The mild synthetic
conditions allowed utilization of the unprotected indole-NH of
L-Trp, and no complications were observed with L-Met or any
other of the amino-N-protected amino acids utilized.
Scheme 4
Absorption (lAbs.) and fluorescence (lEm.) wavelength maxima
were measured of all synthesized fluorescent peptides 10–17.
(Table 3) (Figures showing fluorescence are included in the ESI.†)
Solid phase fluorescent labeling with 9 to synthesize labeled
tripeptide 15
Conclusions
Microwave-assisted SPPS (3 min coupling time for each step) and
Fmoc strategy also successfully allowed fluorescent labeling by
In conclusion we have described the convenient and efficient
preparation of a variety of coumarin fluorescent probes or
Table 1 Preparation of fluorescent peptides 10–15
Purified peptide
Labeled peptide
Structure (N–C terminus)
Yield (%)a
Purity (%)b
tRc/min
HRMSd [M + H]+
10
11
12
13
14
15
H-L-Ala-L-Lys(Ne-Mca)-NH2
26
45
23
18
20
35
99
99
99
94
99
99
9.10
9.82
13.38
18.02
17.00
14.28
433.2103
389.1825
562.2680
782.3328
902.4212
578.2987
H-L-Ala-L-Lys(Ne-Cc)-NH2
H-L-Pro-L-Phe-L-Lys(Ne-Cc)-NH2
H-L-Trp-L-Lys(Ne-Cc)-L-Met-L-Phe-NH2
H-L-Lys(Ne-Cc)-L-Pro-Gly-L-Leu-L-Met-L-Trp-NH2
H-L-Phe-L-Leu-L-Lys(Na-Cc)-NH2
a Isolated yields after HPLC purification; b Purity after HPLC purification; c tR = retention time. For conditions see experimental. d For the calculated
values, see the ESI.†
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Table 2 Preparation of fluorescent peptides 16 and 17
Purified peptide
Yield (%)a
Labeled peptide
Structure (N–C terminus)
Purity (%)b
tRc/min
HRMSd [M + H]+
16
17
(Cc)-L-Leu-L-Leu-NH2
(Mca)-L-Leu-L-Leu-NH2
29
26
> 99
> 99
20.67
17.47
416.2223
460.2455
a Isolated yields after HPLC purification; b Purity after HPLC purification; c tR = retention time. For conditions see experimental. d For the calculated
values, see the ESI.†
Table 3 Absorption and fluorescence data of fluorescent labeled peptides
a variety of coupling steps were optimized to increase rate and
eliminate epimerization. Single mode irradiation with monitoring
of temperature, pressure, and irradiation power versus time was
used throughout, making the procedure highly reproducible. To
customize, we varied Tmax from 50 to 70 ◦C, power from◦80–100 W,
10–17
Entry
Compound
l
Abs.[nm]a
l
Em. [nm]a
1
2
3
4
5
6
7
8
10
11
12
13
14
15
16
17
323
294
295
289
290
299
295
322
383
409
407
409
405
407
413
383
and time from 3–10 min. The conditions (Tmax = 70 C, Pmax
100 W, and time = 3–10 min) were found to be optimal.
=
MS/MS peptide fragmentation was obtained on the crude pep-
tides by way of low resolution MS and tandem mass spectrometry
(MSn) data obtained via HPLC/UV/(+)ESI-MS and –MSn on a
ThermoFinnigan (San Jose, CA) LCQ Classic quadruple ion trap
mass spectrometer in electrospray ionization (ESI) mode. High
resolution mass spectrometry (HRMS) via flow-injection positive
[(+)ESI]-time of flight (TOF) was obtained on an Agilent 1200
series spectrometer.
a Determined in 95% methanol.
labels, including coumarin-3-carbonyl and 7-methoxycoumarin-
4-yl-acetyl labeled lysines as fluorogenic substrates in solution
phase and demonstrated that their benzotriazole derivatives are
appropriate materials for peptide labeling thus enabling efficient
peptide a-amino group acylation under microwave irradiation on
solid phase without using coupling agents or additives and without
side reactions or epimerization.
(S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(2-(7-me-
thoxy-2-oxo-2H-chromen-4-yl)acetamido)pentanoic acid (Na-
Fmoc-L-Lys(Mca)-OH) (3). Compound 2 (0.36 g, 1.1 mmol)
was added in one portion to a solution of Na-Fmoc-L-lysine
(0.57 g, 1.8 mmol) in MeCN–H2O (24 mL:5 mL), in the presence
of Et3N (0.75 mL, 5.4 mmol). The reaction mixture was stirred at
20 ◦C for 20 min. A solution of 6M HCl (2 mL) was then added
and the MeCN was removed under reduced pressure. The residue
was extracted with EtOAc (100 mL) and the organic extract was
washed with 6M HCl (2 ¥ 50 mL), brine (50 mL) and dried over
MgSO4. Evaporation of the solvent gave white microcrystals of
3 (0.41 g, 65%), ◦which were recrystallized from EtOAc–hexanes.
mp 184.9–185.7 C. 1H NMR (300 MHz, DMSO-d6) d 1.20–1.46
(m, 4H), 1.46–1.80 (m, 2H), 3.02–3.15 (s, 2H), 3.70 (s, 2H),
3.88 (s, 3H), 3.90–3.98 (m, 1H), 4.20–4.36 (m, 3H), 6.28 (s, 1H),
6.97–7.06 (m, 2H), 7.36 (t, J = 7.2 Hz, 2H), 7.45 (t, J = 7.2 Hz,
2H), 7.64–7.74 (m, 2H), 7.76 (d, J = 7.4 Hz, 2H), 7.93 (d, J =
7.4 Hz, 2H), 8.24–8.31 (m, 1H); 13C NMR (75 MHz, DMSO-d6)
d 23.1, 28.5, 30.4, 46.6, 53.8, 55.9, 65.6, 100.9, 112.2, 112.6, 112.7,
120.1, 125.3, 126.5, 127.1, 127.7, 140.7, 143.8, 143.9, 151.2, 154.9,
156.2, 160.1, 162.4, 167.4, 174.0. Found: C, 67.55; H, 5.60; N,
4.40. Anal. calcd. for C33H32N2O8: C, 67.80; H, 5.52; N, 4.79%.
Experimental
General Methods
Reagents were purchased from Peptides International or Aldrich
and used without further purification. Rink-amide-MBHA resin
(200–400 mesh, 0.35 meq/g) was purchased from Peptide Inter-
national (Louisville, KY, USA). Melting points were determined
on MEL-TEMP II apparatus. NMR spectra were recorded on
OXFORD 300 in CDCl3 with TMS as the internal standard for
1H (300 MHz) or a solvent as the internal standard for 13C NMR
(75 MHz). UV and fluorescence measurements were recorded
on Cary 100 UV-Vis and FluoroMax spectrophotometers respec-
tively. Elemental analyses were performed on a Carlo Erba-1106
instrument. MALDI analyses were performed on Bruker Reflex II
TOF mass spectrometer retrofilled with delayed extraction.
Analytical reversed-phase HPLC was performed on a Rainin
HPXL system with a Vydac C-18 (5 mm, 2.1 ¥ 250 mm) silica
column at a 1ml/min flow rate. Peptides were eluted using a 10–
80% gradient of solvent B (0.1% TFA in acetonitrile) vs solvent
A (0.1% TFA in water) and peaks were detected at a wavelength
of 214 nm. The identification of the products was achieved by
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF, ABI 4700 Proteomics Analyzer)
with a-cyano-4-hydroxy cinnamic acid as the matrix. Synthesis
of the peptides was performed in a Discover BenchMate peptide
synthesizer from CEM (Matthews, NC, USA). The conditions for
General procedure for the synthesis of fluorescent peptides on
solid phase
Labeled peptides were synthesized using Fmoc solid-phase
methodology as C-terminal amides utilizing Rink-amide-HMBA
resin. The commercial Rink-amide-HMBA resin was deprotected
◦
by 20% piperidine/DMF (15 min, 20 C) (0.05 mmol) and then
coupled with 5 eq. of benzotriazole activated fluorogenic substrate
(2, 4, 5, 7, 9) or with Na-Fmoc-protected (aminoacyl)benzotriazole
reagent derived from Fmoc-protected amino acids, prepared
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following previously published procedures,34 (for the sequences
see Tables 1 and 2) in DMF, under microwave irradiation for
10 min for fluorescent labeling and 3 min for other couplings. When
complete coupling was verified by a negative Kaiser (ninhydrin)
test (10 min), the solid resin was washed with DMF (3 ¥ 5 mL)
and DCM (3 ¥ 5 mL) followed by another coupling. After the
coupling step, the desired peptide was cleaved from the resin
using established cleavage cocktails: (i) TFA–anisole–thioanisole-
2,3-dimercaptopropanol (from Alfa Aesar, Ward Hill, MA USA)
(90:2:3:5) (for peptide sequences including Trp or Met) or (ii)
TFA:water:TIPS (95:2.5:2.5) (for the other peptide sequences)
at 20 ◦C for 2 h. The resin was filtered, then the cocktail was
concentrated under nitrogen, and cold diethyl ether was added to
achieve precipitated peptide (10–17), under conditions optimized
to increase rate and eliminate epimerization.
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Acknowledgements
We thank Dr. C. D. Hall for helpful suggestions and Dr.
N. M. Khashab for some preliminary experiments.
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