Facile synthesis of 7-amino-4-carbamoylmethylcoumarin (ACC)-containing solid supports and their corresponding fluorogenic protease substrates

Article abstract of DOI:10.1016/S0960-894X(03)00087-8

The bifunctional fluorophore, 7-amino-4-carbamoylmethylcoumarin (ACC) without any protection groups, was regioselectively attached to different solid supports functionalized with a primary amino group. The resulting resins were used to synthesize fluoroge

Full text of DOI:10.1016/S0960-894X(03)00087-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1033–1036
Facile Synthesis of 7-Amino-4-carbamoylmethylcoumarin
(ACC)-Containing Solid Supports and Their Corresponding
Fluorogenic Protease Substrates
Qing Zhu,a Dong B. Li,a Mahesh Uttamchandanib and Shao Q. Yaoa,b,
*
aDepartment of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
bDepartment of Biological Sciences, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
Received 14 December 2002; revised 20 January 2003; accepted 20 January 2003
Abstract—The bifunctional fluorophore, 7-amino-4-carbamoylmethylcoumarin (ACC) without any protection groups, was regio-
selectively attached to different solid supports functionalized with a primary amino group. The resulting resins were used to syn-
thesize fluorogenic protease substrates with high yield and purity.
# 2003 Elsevier Science Ltd. All rights reserved.
From the genomic data collected so far, it is estimated
that more than 2% of the gene products are proteases.1
Proteases are enzymes that hydrolyze the amide back-
bone of proteins. Many of them are vital to every aspect
of an organism’s life, being involved in a plethora of
cellular processes. Consequently, a number of proteases
are avidly investigated currently as major therapeutic
targets for diseases such as AIDS, Alzheimer’s disease
and cancer.2 With the completion of the human genome
project recently, many new putative proteases have been
identified. There is thus a pressing need to develop rapid
and general methods for high-throughput determination
of protease substrate specificity. To date, a number of
methods have been developed, some of which are bio-
chemical-based, such as those based on phage-display
peptide libraries on the surface of filamentous phage
particles,3 whilst others are chemical-based, in which
combinatorial chemical synthesis is used to generate
libraries of potential protease substrates on suitable
solid supports.4
group of a peptide, however, the fluorescence of the
molecule is essentially quenched, thereby rendering the
resulting peptide–AMC conjugate practically non-
fluorescent. Incubation of an AMC-containing peptide
substrate with a protease leads to specific cleavage of
the anilide bond between AMC and the conjugated
peptide, which liberates the fluorogenic AMC leaving
group, allowing for the simple determination of clea-
vage rates for individual substrates. Libraries of fluoro-
genic AMC peptide substrates have also been
synthesized to investigate the substrate specificity of
proteases.6,7 In particular, positional-scanning, syn-
thetic combinatorial libraries (PS-SCLs) have been used
to establish the substrate profiles of proteases in an
extremely rapid fashion, as only a few libraries, as well
as assays, are needed in order to provide information
for all side chains at every position of the peptide. In an
elegant demonstration of this approach, Thornberry et
al. synthesized Ac-X-X-X-D-AMC positional-scanning
libraries and used them to investigate caspases.7 Their
design of these libraries was made possible by the abso-
lute specificity of caspases for an aspartic acid residue at
the P1 position. By attaching the carboxylic acid side
chain of the C-terminal residue Asp to the solid support,
the authors were able to generate AMC-containing
peptide libraries and used them to screen against differ-
ent caspases. However, this method is not applicable for
the screening of most other proteases, as libraries hav-
ing other amino acids at the C terminus can not be
generated this way.8 Recently, a more efficient method
The use of 7-amino-4-methyl coumarin (AMC) peptide
substrates for the assay of protease activity is a well-
established method.5 AMC, a fluorogenic molecule, is
highly fluorescent in its free form. Upon conjugation of
its aromatic amino group to the C-terminal carboxyl
*Corresponding author: Tel.: +65-68741683; fax: +65-67791691;
e-mail: chmyaosq@nus.edu.sg
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00087-8

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Q. Zhu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1033–1036
has been developed which allows for direct incor-
poration of any amino acid at all possible positions in a
peptide substrate.9À11 This was accomplished by the use
introduction of an extra amino acid linker between the
resin and ACC is not expected to affect the protease
profile of ACC-conjugated peptides.
of
a
bifunctional fluorophore, 7-amino-4-carba-
moylmethylcoumarin (ACC), to replace the original
AMC in the library synthesis.10 With this method, the
aromatic amine of ACC was first protected with a Fmoc
group, followed by loading onto an amine-containing
resin such as Rink amide resin, via the carboxyl group
of ACC. This was then followed by piperidine depro-
tection of the Fmoc group and coupling of the first
amino acid to the resin using Fmoc-based peptide
chemistry. Compared with that of a regular aliphatic
amine, the much lower nucleophilicity of an aromatic
amine, such as that of ACC, has been well docu-
mented.12 We reasoned that, given the obvious differ-
ence in reactivity, it should be possible to directly couple
ACC, via its carboxyl group, to an aliphatic amine-
containing resin without the need of protecting the aro-
matic amine on ACC. In this way, tedious steps needed
to protect/deprotect the Fmoc group may be avoided.
In addition, since no Fmoc group is used to protect
ACC before its conjugation to a solid support, other
types of peptide chemistry (i.e., Boc-based chemistry)
may be readily adopted for the synthesis of ACC-con-
taining peptides.
The feasibility of this strategy was first examined and
optimized in solution,13 then applied to solid-phase
reactions (Scheme 1a). Unprotected ACC, 1, was cou-
pled with glycine benzyl ester at room temperature to
give the desired product 2 in moderate yield. Critically,
no self-condensation of ACC was observed, indicating
the feasibility of its direct conjugation to the solid sup-
port without protecting the aromatic amine. Following
published protocols,11 2 was added to a mixture con-
taining Fmoc-Asp(tBu)-OH, HATU and collidine in
DMF and the reaction was run at room temperature for
three days, generating the desired product 3 with an
yield comparable to reported data.11 To demonstrate
the generality of this strategy for solid-phase peptide
synthesis, we next investigated the ACC coupling with
different types of solid supports.14 Wang, Rink amide
and 2-chlorotrityl chloride resins were chosen because
each contains a different functional group (Scheme 1b).
For Rink amide resin, which contains an aliphatic
amine, ACC was directly coupled to the resin. For
Wang and 2-chlorotrityl chloride resins, which do not
contain amine groups, an amino acid was first con-
veniently attached to the solid support to provide an
amine handle for the subsequent ACC coupling. Five
different Fmoc-protected amino acids were attached to
each resin. Loadings of the resins were determined to be
between 0.6–0.8 mmol/g. After deprotection of the
Fmoc group, 1 was coupled directly to resins without
any protection, followed by direct coupling of the first
amino acid, Fmoc-Asp(tBu)-OH using HATU coupling
as previously reported.11 The loading efficiency and
purity of the steps were determined by both Fmoc UV
analysis, as well as LC-MS analysis of the cleavage pro-
ducts.15 Results are summarized in Table 1. Loadings of
In this paper, we developed a facile method for the
synthesis of ACC-containing solid supports and their
corresponding fluorogenic peptide substrates. By con-
jugation of unprotected ACC directly to aliphatic
amine-containing solid supports, we were able to suc-
cessfully synthesize ACC-containing peptides with high
yield and purity. Furthermore, we demonstrated that
this strategy was also adaptable to non-amine-contain-
ing solid supports such as Wang and 2-chlorotrityl
resins, which were conveniently functionalized with an
amino acid linker, followed by ACC conjugation. The
Scheme 1.

Q. Zhu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1033–1036
Table 1. The efficient loading to generate Asp-ACC-AA-resins
1035
Resin
Rinka
Wang
Gly
2-Chlorotrityl chloride
Amino acid
Ala
Asp
Leu
0.62
Lys
Ala
Asp
Gly
Ile
Leu
0.15
Loading
(mmol/g)
Step 1
Step 2–3
0.67
0.62
0.75
0.68
0.720.67
0.68
0.76
0.63
0.9
0.80
0.75
0.69
0.62
0.68
0.20
0.18
0.21
0.16
aThe loading efficiency of double coupling. See Scheme 1b and main text for detailed steps.
Figure 1. LC-MS analysis of products cleaved from: (a) Wang resin; (b) 2-chlorotrityl resin; (c) Rink resin.
1 with different resins, followed by first amino acid
loading, were generally good, except in the case of
2-chlorotrityl resins, where loadings were ꢀ0.2mmol/g
for Steps 2–3 combined. LC-MS analysis (vide infra) of
the cleavage product, however, confirmed the formation
of >90% of the desired product (Fig. 1b), indicating
the low loading efficiency of ACC+first amino acid was
not due to side product formation. The exact cause is
still under investigation.
Acknowledgements
Funding support from the National University of Sin-
gapore and the Agency for Science, Technology and
Research (A*STAR) of Singapore is acknowledged.
References and Notes
After three steps of ACC loading and amino acid cou-
plings as indicated in Scheme 1b, the products were
cleaved off the resins and analyzed by LC-MS in order
to further assess the loading efficiency and purity.15 All
three resins produced >90% of the desired products
(Fig. 1). One of them, the Asp-ACC-Rink resin was
used further to synthesize a known ACC-conjugated
tetrapeptide substrate of Caspase 1,7 Ac-Asp-Glu-Val-
Asp-ACC, by standard solid-phase peptide synthesis on
an automatic peptide synthesizer.16 Upon cleavage, the
crude peptide product was analyzed by LC-MS (Fig.
1c), which indicated the desired product with >90%
purity, further demonstrating the highly efficient nature
of the ACC loading and our strategy.
1. Barrett, A. J.; Rawlings, N. D.; Woessner, J. F. Handbook
of Proteolytic Enzymes; Academic Press: London, 1998.
2. (a) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing,
A. J. H. Chem. Rev. 1999, 99, 2735. (b) Molla, A.; Granne-
man, G. R.; Sun, E.; Kempf, D. J. Antiviral Res. 1998, 39, 1.
(c) Kempf, D. J.; Sham, H. L. Curr. Pharm. Des. 1996, 2, 225.
3. (a) Matthews, D. J.; Wells, J. A. Science 1993, 260, 1113.
(b) Ding, L.; Coombs, G. S.; Strandberg, L.; Navre, M.;
Corey, D. R.; Madison, E. L. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 7627.
4. Lam, K. S.; Lebl, M. Methods Mol. Biol. 1998, 87, 1.
5. Zimmerman, M.; Ashe, B.; Yurewicz, E.; Patel, G. Anal.
Biochem. 1977, 78, 47.
6. Rano, T. A.; Timkey, T.; Peterson, E. P.; Rotonada, J.;
Nicholson, D. W.; Becker, J. W.; Chapman, K. T.; Thorn-
berry, N. A. Chem. Biol. 1997, 4, 149.
In conclusion, we have developed a facile strategy that
allows efficient conjugation of unprotected ACC
directly on a number of resins with different types of
functionalities. The strategy was simple and highly effi-
cient, generating little or no side products. One of the
ACC-conjugated resins was used to successfully synthe-
size a known fluorogenic tetrapeptide substrate of Cas-
pase 1 with high efficiency and purity. This method
should be amendable to the synthesis of other fluoro-
genic protease substrates with different types of solid-
phase peptide chemistry. It should also be adaptable to
the combinatorial synthesis of fluorogenic peptide
libraries for potential high-throughput profiling of
protease specificities.
7. Thornberry, N. A.; Rano, T. A.; Peterson, E. P.; Rasper,
D. M.; Timkey, T.; Garcia-Calvo, M.; Houtzager, V. M.;
Nordstrom, P. A.; Roy, S.; Vaillancourt, K. T.; Chapman,
K. T.; Nicholson, D. W. J. Biol. Chem. 1997, 272, 17907.
8. Edwards, P. D.; Mauger, R. C.; Cottrell, K. M.; Morris,
F. X.; Pine, K. K.; Sylvester, M. A.; Scott, C. W.; Furlong,
S. T. Bioorg. Med. Chem. Lett. 2000, 10, 2991.
9. Harris, J. L.; Backes, B. J.; Leonetti, F.; Mahrus, S.; Ell-
man, J. A.; Craik, C. S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
7754.
10. Beckes, B. J.; Harris, J. L.; Leonetti, F.; Craik, C. S.; Ell-
man, J. A. Nat. Biotechnol. 2000, 18, 187.
11. Maly, D. J.; Leonetti, F.; Backes, B. J.; Dauber, D. S.;
Harris, J. L.; Craik, C. S.; Ellman, J. A. J. Org. Chem. 2002,
67, 910.

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Q. Zhu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1033–1036
12. For recent examples see: (a)Winum, J. V.; Toupet, L.; Bar-
ragan, V.; Dewynter, G.; Montero, J. Org. Lett. 2001, 3, 2 2 41.
(b) Lee, H. B.; Balasubramanian, S. Org. Lett. 2000, 2, 323.
13. Glycine benzyl ester (305 mg, 1.1 mmol) was dissolved in
DMF in an ice–salt bath under nitrogen. DIEA (500 mL, 2.88
mmol) was slowly added and the resulting mixture was stirred
for 10 min, followed by addition of 1 (200 mg, 0.91 mmol),
DMAP (56 mg, 0.46 mmol) and EDC (210 mg, 1.1 mmol).
The reaction was allowed to warm up to room temperature
and stirred overnight. The solvent was removed under reduced
pressure and the residue was separated with ethyl acetate and
water. The organic layer was washed with 10% hydrochloric
acid and brine, dried (Na2SO4), and concentrated to afford a
yellow solid, which was further purified by flash chromato-
graphy (silica gel, ethyl acetate/hexane=3:1) to afford 2 as a
yellow solid (120 mg, 36%). Mp 239–241; 1H NMR
(300 MHz, DMSO) d 8.67 (t, J=5.6 Hz, 1H), 7.43 (d, J=8.7
Hz, 1H), 7.35 (s, 5H), 6.52(d, J=8.7 Hz, 1H), 6.41 (s, 1H),
6.13 (s, 2H), 6.00 (s, 1H), 5.12 (s, 1H), 3.93 (t, J=5.9 Hz, 1H),
3.64 (s, 1H); HRMS (EI): calcd for C20H18N2O5 366.1216,
found 366.1216. Fmoc-Asp(tBu)-OH (116 mg, 0.28 mmol) and
HATU (212 mg, 0.56 mmol) dissolved in DMF, were added
collidine (80 uL, 0.60 mmol) and the resulting solution was
agitated for 10 min. Compound 2 (50 mg, 0.14 mmol) was
then added and the mixture was agitated for three days. DMF
was removed under reduced pressure and the residue was
extracted with ethyl acetate and 10% HCl. The organic layer
was washed with brine, dried (Na2SO4), and concentrated to
afford a yellow oil, which was further purified by flash chro-
matography (silicon gel, ethyl acetate/hexane=3:1) to afford 3
as a white plate (52mg, 49%). Mp: 155–157. 1H NMR
(300 MHz, DMSO) d 10.57 (s, 1H), 8.72(t, J=5.6 Hz, 1H),
7.90–7.88 (m, 3H), 7.78–7.72(m, 4H), 7.50 (d, J=8.7 Hz, 1H),
6.37 (s, 1H), 5.12(s, 1H), 4.55–4.53 (M, 1 h), 4.33–4.20 (M, 3
h), 3.95 (t, J=5.9 Hz, 1H), 3.77 (s, 1H), 2.79–2.71 (m, 1H),
2.61–2.50 (m, 1H), 1.37 (s, 9H); HRMS (FAB): calcd for
C43H41N3O10Na 782.2690, found 782.2685.
14. Representative synthesis of Asp-ACC-AA-resin. Wang
resin (0.2g, 1.0 mmol/g) was suspended in a 2mL DCM/
DMF (9:1) solution. Fmoc-Gly-OH (0.3 g, 1.0 mmol), HOBt
(0.15 g, 1.0 mmol), DIC (0.156 mL, 0.2mmol), DMAP (2.4
mg, 0.1 mmol) were added. After stirring for 4 h at room
temperature, acetic anhydride (41 mg) and pyrindine (32mg)
were added and the mixture was reacted further for 30 min.
Filtration and wash of the resin with 3Â20 mL DMF (AR
grade), 3Â20 mL DCM, 3Â20 mL MeOH give Fmoc-Gly-
Wang resin. The substitution level of the resin was 0.72mmol/
g (72%) as determined by Fmoc UV analysis. To a 20 mL
conical tube was added the resin (0.1 g) and 10 mL 20%
piperidine in DMF. After shaking for 30 min, the resin was
filtrated and washed with DMF (3Â20 mL). Compound 1 (66
mg, 0.3 mmol), HOBt (46 mg, 0.3 mmol), DIC (38 mg, 0.3
mmol) in 2mL DMF were subsequently added. The mixture
was shaken for two days and then filtered, washed with DMF
(3Â20 mL), DCM (3Â20 mL), MeOH (3Â20 mL) and dried
over P2O5 to give ACC-Gly-Wang resin. To a 20 mL conical
test tube was added the above resin (0.1 g) and DMF (2mL).
Fmoc-Asp(tBu)-OH (0.288 g, 0.7 mmol), HATU (0.266 g, 0.7
mmol), and 2,4,6-collidine (85 mg, 0.7 mmol) were added,
followed by shaking for three days to give Fmoc-Asp(tBu)-
ACC-Gly-Wang resin. The resin was filtered and washed with
DMF (3Â20 mL). The substitution level of the resin was
determined by Fmoc UV analysis to be 0.68 mmol/g (>95%).
15. LC was performed on a WatersTM 600 HPLC equipped
with a Phenomenex RP-18 (5 mm, 4.6Â250 mm) column using
an acetontrile–water gradient (with 0.1% TFA). MS was run
on an Electrospray mass spectrometer (Finnigan, USA).
16. Peptides were synthesized on PioneerTM Peptide Synthesi-
zer (PerSeptive Biosystems, USA) using standard Fmoc pep-
tide chemistry with the HBTU/HOBt/DIEA coupling method.