Expedient solid-phase synthesis of fluorogenic protease substrates using the 7-amino-4-carbamoylmethylcoumarin (ACC) fluorophore

Article abstract of DOI:10.1021/jo016140o

A highly efficient solid-phase synthesis method for the preparation of fluorogenic protease substrates based upon the bifunctional leaving group 7-amino-4-carbamoylmethylcoumarin (ACC) is reported. Methods for the large-scale preparation of the novel fluorogenic leaving-group ACC are provided (Scheme 1). Detailed procedures are also provided for loading a diverse set of amino acids to support-bound ACC in good yields and with minimal racemization. Finally, procedures are included for the preparative synthesis of optimized ACC substrates for HIV-1 protease and plasmin.

Full text of DOI:10.1021/jo016140o

910
J . Org. Chem. 2002, 67, 910-915
Exp ed ien t Solid -P h a se Syn th esis of F lu or ogen ic P r otea se
Su bstr a tes Usin g th e 7-Am in o-4-ca r ba m oylm eth ylcou m a r in (ACC)
F lu or op h or e
Dustin J . Maly,† Francesco Leonetti,† Bradley J . Backes,† Deborah S. Dauber,§
J ennifer L. Harris,§ Charles S. Craik,§ and J onathan A. Ellman*,†
Department of Chemistry, University of California, Berkeley, California 94720 and Department of
Pharmaceutical Chemistry, Program in Chemistry and Chemical Biology, University of California,
San Francisco, California 94143
jellman@ucink4.berkeley.edu
Received September 23, 2001
A highly efficient solid-phase synthesis method for the preparation of fluorogenic protease substrates
based upon the bifunctional leaving group 7-amino-4-carbamoylmethylcoumarin (ACC) is reported.
Methods for the large-scale preparation of the novel fluorogenic leaving-group ACC are provided
(Scheme 1). Detailed procedures are also provided for loading a diverse set of amino acids to support-
bound ACC in good yields and with minimal racemization. Finally, procedures are included for the
preparative synthesis of optimized ACC substrates for HIV-1 protease and plasmin.
In tr od u ction
Proteases, a family of enzymes that catalyze the
hydrolysis of amide bonds in proteins and peptides, are
ubiquitous in nature and constitute approximately 2%
of all known gene products.1 These enzymes play a
critical role in virtually all biological processes and
represent important drug targets in numerous therapeu-
tic areas,2 including cancer,3 AIDS,4 cardiovascular dis-
ease,5 neurodegenerative disease,6 and osteoperosis.7
Fluorogenic substrates that provide a spectrophoto-
metric readout upon proteolysis are one of the most
important chemical tools for the characterization of
proteases.8 Fluorogenic substrates allow for the continu-
ous kinetic analysis of proteases and are useful reagents
in the screening of potential protease inhibitors. In
addition, these substrates can serve as powerful tools for
determining protease substrate specificity,9 which can
provide valuable insight into biological function and also
aid in the design of potent and selective substrates and
inhibitors.
F igu r e 1. Fluorogenic substrates peptide-AMC, 1, and
peptide-ACC, 2.
these substrates because the coumarin lacks the neces-
sary functionality for linkage to a solid support. Attach-
ment to solid support through the side chain functionality
of the C-terminal amino acid has been employed,11 but
this method is difficult to apply for a majority of amino
acids.12 Therefore, we have recently developed a modified
aminocoumarin leaving group, 7-amino-4-carbamoylm-
ethylcoumarin (ACC), that can be directly attached to a
Fluorogenic 7-amino-4-methylcoumarin (AMC) peptide
substrates 1 (Figure 1) are extensively utilized in the
study of proteases.10 However, general solid-phase syn-
thesis methods are not available for the preparation of
(9) (a) Harris, J . L.; Backes, B. J .; Leonetti, F.; Mahrus, S.; Ellman,
J . A.; Craik, C. S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7754. (b)
Backes, B. J .; Harris, J . L.; Leonetti, F.; Craik, C. S.; Ellman, J . A.
Nat. Biotechnol. 2000, 18, 187. (c) Thornberry, N. A.; Rano, T. A.;
Peterson, E. P.; Rasper, D. M.; Timkey, T.; Garcia-Calvo, M.; Houtza-
ger, V. M.; Nordstrom, P. A.; Roy, S.; Vaillancourt, J . P. et al. J . Biol.
Chem. 1997, 272, 17907. (d) Meldal, M.; Svendsen, I.; Breddam, K.;
Auzanneau, F. I. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3314. (e) St.
Hilaire, P. M.; Willert, M.; J uliano, M. A.; J uliano, L.; Meldal, M. J .
Comb. Chem. 1999, 1, 509.
(10) Zimmerman, M.; Ashe, B.; Yurewicz, E.; Patel, G. Anal.
Biochem. 1977, 78, 47.
(11) (a) Rano, T. A.; Timkey, T.; Peterson, E. P.; Rotonda, J .;
Nicholson, D. W.; Becker, J . W.; Chapman, K. T.; Thornberry, N. A.
Chem. Biol. 1997, 4, 149. (b) Edwards, P. D.; Mauger, R. C.; Cottrell,
K. M.; Morris, F. X.; Pine, K. K.; Sylvester, M. A., Scott, C. W., Furlong,
S. T. Biorg. Med. Chem. Lett. 2000, 10, 2291.
† University of California, Berkeley.
§ University of California, San Francisco.
(1) Barrett, A. J .; Rawlings, N. D.; Woessner, J . F. Handbook of
Proteolytic Enzymes; Academic Press: London, 1998.
(2) Leung, D.; Abbenante, G.; Fairlie, D. P. J . Med. Chem. 2000,
43, 305.
(3) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing A. J . H. Chem.
Rev. 1999, 99, 2735.
(4) (a). Molla, A.; Granneman, G. R.; Sun, E.; Kempf, D. J . Antiviral
Res. 1998, 39, 1. (b) Kempf, D. J .; Sham, H. L. Curr. Pharm. Des. 1996,
2, 225.
(5) Ripka, W. C. Curr. Opin. Chem. Biol. 1997, 1, 242.
(6) Vassar, R.; Bennett, B. D.; Babu-Kahn, S.; Kahn, S.; Mendiaz,
E. A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.; et
al. Science 1999, 286, 735.
(12) Safety-catch linkers can be used to access virtually any ami-
nomethyl coumarin substrate by nucleophilic attack of an aminocou-
marin amide derivative of the relevant amino acid upon a support-
bound peptide substrate. However, this method requires additional
effort to scavenge the aminocoumarin amide nucleophile. See ref 9b.
(7) Drake, F. H.; Dodds, R. A.; J ames, I. E.; Connor, J . R.; Debouck,
C.; Richardson, S.; LeeRykaczewski, E.; Coleman, L.; Rieman, D.;
Barthlow, R.; Hastings, G.; Gowen, M. J . Biol. Chem. 1996, 271, 12511.
(8) Knight, C. G. Methods Enzymol. 1995, 248, 18.
10.1021/jo016140o CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/12/2002

Synthesis of Fluorogenic Protease Substrates
J . Org. Chem., Vol. 67, No. 3, 2002 911
Sch em e 1a
optimize conditions for this reaction. The two most
significant factors were the choice of â-keto electrophile
and the concentration of the reaction mixture. Using a
â-ketodiester (e.g., diethyl 1,3-acetonedicarboxylate) or
running the reaction at a concentration greater than 0.2
M caused significantly increased formation of byproducts.
However, by using 1,3-acetonedicarboxylic acid13 at a
concentration of 0.2 M the reaction can be run on 100 g
scale to afford pure 4 in 63% yield after precipitation and
crystallization from CH3CN. The carbamate of 4 was
hydrolyzed using 10 M NaOH with heating to reflux, and
the resulting free amine 5 was protected with FmocCl
utilizing the Meienhofer procedure to provide acid 6.14
ACC-resin 7 was then prepared by coupling acid 6 to
Rink amide AM resin using standard DICI and HOBt
coupling conditions. The substitution level of the resin
was 0.58 mmol/g (>95%) as determined by Fmoc analy-
sis.15
a
(a) Ethyl chloroformate (0.5 equiv), EtOAc, reflux; (b) 1,3-
Loa d in g of th e F ir st Am in o Acid . Due to the poor
nucleophilicity of the support-bound aminocoumarin,
specialized coupling conditions were necessary to ef-
ficiently load the first amino acid. Specifically, the use
of the in situ activating agent HATU (N-[(dimethy-
lamino)-1-H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-
N-methylmethanaminium hexafluorophosphate N-oxide)
in the presence of an equimolar amount of the hindered
organic base 2,4,6-collidine gave the best substitution
levels with minimal racemization.16 The level of racem-
ization during the loading of the first amino acid was
evaluated for the representative amino acids Fmoc-Asn-
(Trt)-OH and Fmoc-Leu-OH (Scheme 2). ACC-resin
loaded with Fmoc-L-Asn(Trt)-OH 8a and Fmoc-L-Leu-OH
8b was coupled with Fmoc-L-Phe-OH under standard
solid-phase conditions. No DL epimer (<1.0%) was ob-
served by reverse-phase HPLC anaysis of the resulting
dipeptide products 10a and 10b. Similarly, Fmoc-L-Phe-
OH was coupled to ACC-resin loaded with Fmoc-D-Asn-
(Trt)-OH 9a and Fmoc-D-Leu-OH 9b. No LL-epimer
(<1.0%) was observed for the dipeptide products 11a and
11b.
acetonedicarboxylic acid (1.1 equiv), 70% H2SO4; (c) 10 M NaOH,
reflux; (d) i. TMSCl (2.1 equiv), i-Pr2EtN (2.1 equiv); ii. FmocCl
(1.1 equiv); (e) Rink amide AM resin (0.5 equiv), HOBt (1 equiv),
DICI (1 equiv), DMF.
solid support, enabling solid-phase synthesis of peptide-
ACC substrates 2. Notably, for a number of proteases,
including thrombin and plasmin, fluorogenic peptide
substrates based upon ACC 2 were shown to have similar
kinetic parameters to the corresponding AMC peptide
substrates 1.9a Furthermore, the substrate specificities
of numerous proteases have been determined using
positional-scanning libraries of ACC fluorogenic peptide
substrates 2. These results were validated by the high
level of correlation between substrate specificity profiles
obtained with ACC 2 and AMC 1 substrate libraries for
thrombin and plasmin and by the consistency with
previously determined specificity information.
Herein, we report a method for the efficient large-scale
preparation of the novel fluorogenic leaving group ACC.
Detailed procedures are also provided for loading a
diverse set of Fmoc-amino acids to support-bound ACC
in good yields and with minimal racemization. Finally,
in previous work we identified optimized fluorogenic ACC
peptide substrates for numerous therapeutically impor-
tant proteases.9a Procedures are provided for the pre-
parative synthesis of optimized ACC substrates for HIV-1
protease and plasmin.
To demonstrate the generality of our coupling condi-
tions, a diverse set of Fmoc-amino acids were coupled to
7 and the loading efficiencies were determined (Scheme
3, Table 1). For the majority of the amino acids tested,
good yields were achieved with a single coupling step.
For those amino acids that provided lower coupling yields
(Pro, Thr, Ile, Val, and Arg), a second coupling was
carried out to increase the loading level.
Resu lts a n d Discu ssion
Although our chosen coupling conditions gave high
coupling yields for most amino acids, the coupling of
Fmoc-His(Trt)-OH (Table 1, entry 8i) was accompanied
by an unexpected competitive transfer of the side chain
trityl group to the support-bound ACC.17 To efficiently
load His onto the support-bound coumarin without trityl
Syn th esis of ACC-Resin . Three criteria were deemed
necessary for the preparation of the support-bound ACC.
First, the synthesis sequence must be efficient and allow
for the preparation of multigram quantities of compound
without the need for column chromatography. Second,
the ACC must be loaded to support in an efficient manner
without the formation of byproducts. Finally, the resin-
bound ACC should be amenable to standard solid-phase
Fmoc-peptide synthesis. To meet these criteria, N-Fmoc-
7-amino-4-carboxymethylcoumarin 6 was prepared as a
bifunctional molecule to be loaded onto solid support via
the acid-labile Rink linker (Scheme 1). Starting from
commercially available 3-aminophenol, treatment with
ethyl chloroformate provides carbamate 3. Coumarin 4
was formed via a Pechmann reaction of carbamate 3 with
1,3-acetonedicarboxylic acid in 70% H2SO4. An extensive
study of experimental parameters was undertaken to
(13) It is important that the 1,3-acetonedicarboxylic acid used in
this step is pure. Contamination with the decarboxylation product,
acetoacetic acid, leads to the formation of 7-carbethoxyamido-4-
methylcoumarin, which is difficult to remove at this step.
(14) Bolin, D. R.; Sytwu, I.-I.; Humiec, F.; Meienhofer, J . Int. J . Pept.
Protein Res. 1989, 33, 353.
(15) Bunin, B. A. The Combinatorial Index; Academic Press: San
Diego, 1998; pp 213-236.
(16) Carpino, L. A.; Ionescu, D.; El-Faham, A. J . Org. Chem. 1996,
61, 2460.
(17) Trityl transfer was established by attempted acetylation to cap
the unreacted coumarin. However, after acidic cleavage from support
only His-ACC and free ACC were detected without any presence of
acetyl-ACC.

912 J . Org. Chem., Vol. 67, No. 3, 2002
Maly et al.
Sch em e 2a
a
(a) 20% Piperidine/DMF; (b) Fmoc-L-Asn(Trt)-OH (5 equiv) or Fmoc-L-Leu-OH (5 equiv), HATU (5 equiv), 2,4,6-collidine (5 equiv),
DMF, 24 h; (c) Fmoc-D-Asn(Trt)-OH (5 equiv) or Fmoc-D-Leu-OH (5 equiv), HATU (5 equiv), 2,4,6-collidine (5 equiv), DMF, 24 h; (d) i.
20% piperidine/DMF; ii. Fmoc-L-Phe-OH, DICI, HOBt, DMF; iii. TFA/i-Pr3SiH/H2O (95:2.5:2.5).
Sch em e 3a
Ta ble 2. Op tim iza tion of Rea ction Con d ition s to Loa d
Histid in e to Su p p or t-Bou n d ACC
collidine/
HATUa
DL-epimer,
trityl transfer,
b
c
%
%
Fmoc-His(Trt)-OH
Fmoc-His(Trt)-OH
Fmoc-His(Trt)-OH
Fmoc-His(Boc)-OH
Fmoc-His(Boc)-OH
1:1
5:1
10:1
2:1
17
27
40
4.7
4.0
15
4
<1
-
1:1
-
Equivalent of 2,4,6-collidine/equivalent of HATU. b Percentage
of DL-epimer was determined by reverse-phase HPLC analysis of
Fmoc-L-Phe-L-His-ACC dipeptide. c Trityl transfer determined by
HPLC quantitation of unacylated ACC.17
a
a
(a) 20% Piperidine/DMF; (b) HATU (5 equiv), 2,4,6-collidine
(5 equiv), Fmoc-amino acid (5 equiv), DMF, 24 h.
Ta ble 1. Loa d in g Efficien cies for a Diver se Set of Am in o
Acid s to Su p p or t-Bou n d ACC
ization-prone amino acid (Table 2).18 We then explored
the use of alternative side-chain protecting groups. When
Fmoc-His(Boc)-OH was used with our standard coupling
conditions, we obtained a high loading level of 8j, with a
significant decrease in racemization (4.0% DL-epimer).
Thus, by using Fmoc-His(Boc)-OH rather than Fmoc-
His(Trt)-OH for the coupling of the first amino acid,
uniform coupling conditions can be employed to load all
tested Fmoc-amino acids.
Ca p p in g of Un r ea cted Am in ocou m a r in s. When
purification of fluorogenic substrates is not straightfor-
ward, (e.g., when combinatorial libraries of peptide
substrates are generated), a method for completely
acetylating all unreacted support-bound aminocoumarin
is essential. Quantitative capping of unacylated coumarin
is necessary because the presence of unreacted ACC
creates a high background fluorescence that reduces the
sensitivity of the assay. A number of conditions were
explored for the complete acylation of the support-bound
aminocoumarin. The optimal results were obtained using
the nitrotriazole ester of acetic acid.19 Treating the resin
resin product
Fmoc-amino acid
yield,a
%
8c
8d
8a
8e
8f
8g
8h
8i
Fmoc-Ala-OH
80
50b
85
92
82
81
81
78
90
57b
83
84
94
83
98
75b
92
70b
94
98
57b
Fmoc-Arg(Pbf)-OH
Fmoc-Asn(Trt)-OH
Fmoc-Asp(O-t-Bu)-OH
Fmoc-Glu(O-t-Bu)-OH
Fmoc-Gln(Trt)-OH
Fmoc-Gly-OH
Fmoc-His(Trt)-OH
Fmoc-His(Boc)-OH
Fmoc-lle-OH
Fmoc-Leu-OH
Fmoc-Lys(Boc)-OH
Fmoc-Met-OH
Fmoc-Nle-OH
Fmoc-Phe-OH
8j
8k
8b
8l
8m
8n
8o
8p
8q
8r
Fmoc-Pro-OH
Fmoc-Ser(O-t-Bu)-OH
Fmoc-Thr(O-t-Bu)-OH
Fmoc-Trp(Boc)-OH
Fmoc-Tyr(O-t-Bu)-OH
Fmoc-Val-OH
8s
8t
8u
a
b
Loading efficiency determined by Fmoc analysis. Yield for
double coupling.
transfer, several different coupling conditions were ex-
plored (Table 2). Trityl transfer could be completely
suppressed by using 10 equiv of base to HATU. However,
under all of these conditions the use of a large excess of
base resulted in extensive epimerization of this racem-
(18) Benoitin, N. L. Quantitation and Sequence Dependence of
Racemization in Peptide Synthesis. In The Peptides: Analysis, Syn-
thesis, Biology; Gross, E., Meienhofer, J ., Eds.; Academic Press: New
York, 1983; vol. 5, p 260.
(19) Blankemeyer-Menge, B.; Nimtz, M.; Frank, R. Tetrahedron Lett.
1990, 31, 1701.

Synthesis of Fluorogenic Protease Substrates
J . Org. Chem., Vol. 67, No. 3, 2002 913
Sch em e 4a
amino acids were purchased from Calbiochem-Novabiochem
Corp. (San Diego, CA). The amine substitution level of the
Fmoc-substituted Rink resin was determined (0.63 mmol/
gram) by a spectrophotometric Fmoc-quantitation assay.15
Anhydrous, low-amine content DMF was purchased from EM
Science. HATU was purchased from Perseptive Biosystems
(Framingham, MA). DICI, HOBt, AcOH, FmocCl, TFA, 2,4,6-
collidine, and i-Pr3SiH were purchased from Aldrich and used
without further purification. TMSCl was freshly distilled under
N2 from CaH2. Prior to use, 1,3-acetonedicarboxylic acid, which
a
(a) AcOH (10 equiv), 3-nitro-1,2,4-triazole (10 equiv), DICI (10
equiv), DMF, 24 h; (b)i-Pr2EtN, 3 h.
1
was purchased from Aldrich, was assessed by H NMR to be
free of acetoacetic acid.13 Thin-layer chromatography was
carried out on Merck 60 F254 250-µm silica gel plates. IR
spectra were recorded as KBr pellets. NMR chemical shifts
are reported in ppm downfield from an internal solvent peak
and J values are in hertz. All HPLC analyses were performed
utilizing a Rainin Dynamax Microsorb C18 reverse-phase
analytical column (4.6 mm × 25 cm). All HPLC purifications
were carried out using a Varian Microsorb C18 reverse-phase
preparatory column (21.4 mm × 25 cm). The human enzyme
plasmin was purchased from Haematologic Technologies Inc.
(Essex J ct., VT) and used as received.
with AcOH, 3-nitro-1,2,4-triazole, and DICI in DMF,
followed by addition of i-Pr2EtN after 24 h, quantitatively
acetylated any unreacted ACC (Scheme 4).
Syn th esis a n d Eva lu a tion of F lu or ogen ic P ep tid e
Su bstr a tes of HIV-1 P r otea se a n d P la sm in . To il-
lustrate the facility of the solid-phase synthesis method
for the production of useful quantities of fluorogenic
substrates, ACC-peptide substrates Ac-Lys-Gln-Trp-Lys-
ACC 12 for plasmin and Ac-Arg-Lys-Ser-Leu-Val-Nle-
ACC 13 for HIV-1 protease were prepared. These ACC-
peptides were determined to be optimal substrates, as
reported previously, using positional scanning combina-
torial substrate libraries.9a,20 Substrates 12 and 13 were
prepared using the appropriately substituted Fmoc-
amino acid ACC-resins (8l and 8n ) and standard HOBt
and DICI coupling conditions. After purification by
preparative HPLC, the substrates were obtained in 53%
and 55% overall yield, respectively. The kinetic param-
eters for each substrate were determined as shown in
Table 3. The cleavage of ACC substrate 13 by HIV-1
protease is particularly noteworthy since AMC substrates
have not previously been reported for HIV-1 protease.
Because the ACC fluorogenic substrate is more straight-
forward to prepare and is significantly more stable than
the standard fluorescence-quenched peptide substrates
for HIV-1 protease,21 fluorogenic substrate 13 may be of
utility for inhibitor characterization and screening efforts.
3-N-(Ca r beth oxy)a m in op h en ol (3). To a 2 L round-
bottom flask fitted with a stir bar were added 3-aminophenol
(150 g, 1.37 mol) and EtOAc (500 mL). The flask was fitted
with a condenser, and the mixture was heated to reflux for 30
min. Ethyl chloroformate (74.6 g, 0.687 mol) was added
dropwise over a 1 h period. The reaction mixture was allowed
to cool to room temperature at which time a white precipitate
formed. The precipitate was removed by filtration and washed
with EtOAc (3 × 300 mL) and petroleum ether (3 × 300 mL).
The combined filtrate was concentrated to afford 123 g (99%)
of 3 as a white solid. The product was sufficiently pure by NMR
analysis to be taken on to the next step without further
purification. The analytical data are in agreement with
literature values.22 1H NMR (300 MHz, DMSO-d6): δ 1.19 (t,
3, J ) 7.1), 4.06 (quartet, 2, J ) 7.1), 6.32-6.36 (m, 1), 6.75-
6.82 (m, 1), 6.95-6.99 (m, 2), 9.29 (br s, 1), 9.44 (s, 1).
7-N-(Ca r beth oxy)a m in ocou m a r in -4-a cetic Acid (4). To
a 5 L round-bottom flask fitted with a stir bar were added 3
(100 g, 0.552 mol) and 70% H2SO4 (2.75 L). The rapidly stirred
reaction mixture was cooled in an ice bath, and 1,3-acetonedi-
carboxylic acid (88.7 g, 0.607 mol) was added in portions. The
reaction mixture was allowed to warm to room temperature
and stirred for 8 h, after which the reaction mixture was
poured onto ice (4 kg) and stirred for 30 min. The resultant
white precipitate was collected by filtration and washed with
Et2O (3 × 2 L). The crude material was then partially dissolved
in hot CH3CN (700 mL). Upon cooling to room temperature,
the precipitate was collected to afford 101 g (63%) of 4 as a
Con clu sion
An efficient strategy has been developed for the
straightforward solid-phase synthesis of single fluoro-
genic substrates. This methodology allows for the straight-
forward extension to fluorogenic-substrate libraries. A
large number of diverse amino acids can be efficiently
coupled to the ACC-resin with acceptable loading levels
and minimal racemization. A capping protocol has also
been developed to quantitatively acetylate any unreacted
ACC. As previously reported, the resulting ACC-pep-
tides can be utilized to rapidly identify preferred peptide
substrates using positional scanning libraries. Further-
more, fluorogenic substrates prepared with ACC should
be of considerable use for biochemical studies, as dem-
onstrated by the straightforward preparation of the
fluorogenic substrates 12 and 13 for plasmin and HIV-1
protease, respectively.
1
white solid. mp 194-195 °C. H NMR (300 MHz, DMSO-d6):
δ 1.21 (t, 3, J ) 7.1), 3.81 (s, 2), 4.11 (quartet, 2, J ) 7.1), 6.28
(s, 1), 7.31-7.35 (m, 1), 7.52-7.58 (m, 2), 10.12 (s, 1). 13C NMR
(125 MHz, DMSO-d6): δ 14.4, 37.1, 60.8, 104.5, 113.7, 113.7,
114.3, 126.1, 143.0, 149.8, 153.4, 154.1, 160.1, 170.7. Anal.
Calcd for C14H13NO6: C, 57.73; H, 4.50; N, 4.81. Found: C,
57.57; H, 4.57; N, 4.70.
If the 1,3-acetonedicarboxylic acid used in this reaction is
contaminated with acetoacetic acid, the formation of 7-carbe-
thoxyamido-4-methylcoumarin can result. This byproduct can
be removed in the next step.
7-Am in ocou m a r in -4-a cetic Acid (5). To a 2 L round-
bottom flask fitted with a condenser were added 4 (101 g, 0.345
mol), NaOH (138 g, 3.45 mol), and H2O (900 mL). The reaction
mixture was stirred at reflux for 16 h. After cooling to room
temperature, the pH of the reaction mixture was adjusted to
2, by the dropwise addition of H2SO4. The resultant yellow
precipitate that formed was collected by filtration to afford 64.3
g (85%) of 5 as a fluffy yellow powder. The product was
sufficiently pure by NMR analysis to be taken on to the next
step without further purification. The analytical data are in
Exp er im en ta l Section
Rea gen ts a n d Gen er a l Meth od s. Unless otherwise noted,
materials were obtained from commercial suppliers and used
without further purification. Rink Amide AM resin and Fmoc-
(20) Dauber, D. S.; Ziermann, R.; Parkin, N.; Maly, D. J .; Mahrus,
S.; Harris, J . L.; Ellman, J . A.; Petropoulos, C.; Craik, C. S. J . Virol.
2002, 76, 1359.
(21) Krafft, G. A.; Wang, G. T. Methods Enzymol. 1994, 241, 70.
(22) Atkins, R. L.; Bliss, D. E. J . Org. Chem. 1978, 43, 1975.

914 J . Org. Chem., Vol. 67, No. 3, 2002
Ta ble 3. Kin etic P a r a m eter s for F lu or ogen ic Su bstr a tes
Maly et al.
substrate
yield, %
kcat, s-1
Km, mM
kcat/Km, M-1 s-1
Ac-Lys-Gln-Trp-Lys-ACC (12)
Ac-Arg-Lys-Ser-Leu-Val-Nle-ACC (13)
53
55
11.0 ( 0.4
0.040 ( 0.005
280000 ( 10000
296 ( 83.4
nda
nda
a
Not determined.
agreement with literature values.23 1H NMR (300 MHz,
DMSO-d6): δ 3.73 (s, 2H), 5.97 (s, 1), 6.16 (br s, 2), 6.41 (d, 1,
J ) 0.7), 6.54 (dd, 1, J ) 8.6, 2.1), 7.33 (d, 1, J ) 8.6), 12.65 (s,
1).
To remove any 7-amino-4-methylcoumarin, which is formed
by the hydrolysis of the 7-carbethoxyamido-4-methylcoumarin
byproduct formed in the last step, the following procedure was
followed. The yellow precipitate is dissolved in a minimal
volume of 2 M NaOH. The brown solution is then filtered to
remove any insoluble 7-amino-4-methylcoumarin. The pH of
the filtrate is then adjusted to 2, and the yellow precipitate
that forms is collected by filtration to afford pure 5. This
procedure generates product 5 free of any contaminants with
a minimal loss in yield (∼10%).
Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Thr(O-t-Bu)-OH, Fmoc-
Val-OH, and Fmoc-Pro-OH, a second coupling was carried out.
Gen er a l P r oced u r e for Ca pp in g Un r ea cted Am in ocou -
m a r in w ith a n Acetyl Gr ou p . In a separate 50 mL round-
bottom flask were added 3-nitro-1,2,4-triazole (3.0 g, 26 mmol),
AcOH (1.6 mL, 26 mmol), DMF (18 mL), and DICI (4.2 mL,
26 mmol). After stirring for 5 min, the mixture was added to
the resin and allowed to react for 24 h, whereupon i-Pr2EtN
(0.90 mL, 5.3 mmol) was added followed by stirring for an
additional 3 h. The resin was filtered and washed with DMF
(3 × 50 mL).
Rep r esen ta tive Syn th esis of Sin gle P ep tid e-ACC. As
a representative example, the synthesis of Fmoc-L-Phe-L-His-
ACC is reported. To a 6 mL syringe cartridge were added
Fmoc-L-His-ACC (0.06 mmol) and DMF (2 mL). The mixture
was agitated with N2 for 30 min and filtered. A 20% solution
of piperidine in DMF (2 mL) was added, followed by agitation
for 30 min. The resin was filtered and washed with DMF (3 ×
2 mL). In a separate scintillation vial were added Fmoc-L-Phe-
OH (112 mg, 0.290 mmol), HOBt (44 mg, 0.29 mmol), DMF
(1.2 mL), and DICI (45 µL, 0.29 mmol). After a preactivation
time of 5 min, the mixture was added to the resin, followed by
agitation for 3 h. The resin was filtered, washed with DMF (3
× 2 mL), THF (3 × 2 mL), and MeOH (3 × 2 mL), and then
dried in vacuo over P2O5. The crude products were subjected
to reverse-phase HPLC preparatory chromatography followed
by lyophilization. For the tetrapeptide 12, as well as the
hexapeptide 13, the amino terminus was capped as the acetyl
derivative. This was accomplished by premixing AcOH (5
equiv), DICI (5 equiv), and HOBt (5 equiv) in DMF and adding
the resulting mixture to the resin. After agitation for 4 h the
resin was filtered, washed, and dried. The purification of all
ACC-peptides was performed using reverse-phase HPLC
preparatory chromatography (CH3CN/H2O-0.1% TFA, 10-
60% for 20 min, 5 mL/min, 254 nm detection for 60 min).
Finally, all ACC-peptides have been analyzed by analytical
reverse-phase HPLC analysis (CH3CN/H2O-0.1% TFA, 10-
60% for 15 min, 0.8 mL/min, 254 nm detection for 60 min).
F m oc-L-P h e-L-Asn -ACC (10a ). Analytical reverse-phase
HPLC analysis of crude material according to General Condi-
tions (tR ) 23.3 min, 83% purity). 1H NMR (300 MHz, DMSO-
d6): δ 2.51 (m, 1) 2.62 (m, 1), 2.76 (m, 1), 3.0 (m, 1), 3.62 (s, 2),
4.11 (m, 3), 4.28 (m, 1), 4.68 (m, 1), 6.28 (s, 1), 6.95 (s, 1), 7.05-
7.30 (m, 8), 7.34-7.41 (m, 3), 7.47 (dd, 1, J ) 9.0, 1.8), 7.50-
7.70 (m, 5), 7.77 (d, 1, J ) 1.8), 7.84 (d, 2, J ) 7.5), 8.45 (d, 1,
J ) 7.7), 10.39 (s, 1). MS (ESI), m/z calcd for C39H35N5O8:
701.3. Found: m/z 702.2 (M + H)+.
7-N-(F lu or en ylm et h oxyca r b on yl)a m in ocou m a r in -4-
a cetic Acid (6). To a 1 L round-bottom flask fitted with a
condenser and heating mantle were added 5 (20.0 g, 91.2
mmol) and CH2Cl2 (150 mL). To this stirring suspension was
added freshly distilled TMSCl (21.8 g, 201 mmol) and i-Pr2-
EtN (25.9 g, 201 mmol). It is important that the TMSCl is
freshly distilled. The reaction mixture was stirred and heated
to reflux for 3 h, followed by cooling in an ice bath. FmocCl
(26.0 g, 100 mmol) was then added in portions, and the
reaction mixture was allowed to warm to room temperature,
followed by stirring for an additional 11 h. The reaction
mixture was then stirred rapidly, and MeOH (500 mL) was
added. The resultant white precipitate that formed was
collected by filtration and washed with MeOH (2 × 250 mL)
and Et2O (2 × 500 mL) to afford 35.4 g (88%) of 6. mp 273-
1
276 °C. IR 3305, 3038, 1735, 1701 cm-1. H NMR (400 MHz,
DMSO-d6): δ 3.86 (s, 2), 4.33 (t, 1, J ) 6.2), 4.55 (d, 2, J )
6.2), 6.34 (s, 1), 7.33-7.44 (m, 5), 7.56 (s, 1), 7.61 (d, 1, J )
8.6), 7.76 (d, 2, J ) 7.3), 7.91 (d, 2, J ) 7.4) 10.23 (s, 1), 12.84
(s, 1). 13C NMR (125 MHz, DMSO-d6): δ 37.1, 46.6, 65.9, 104.7,
113.8, 113.8, 114.4, 120.2, 125.1, 126.1, 127.1, 127.7, 140.8,
142.7, 143.6, 149.8, 153.2, 154.0, 160.0, 170.6. Anal. Calcd for
C
26H19NO6: C, 70.74; H, 4.34; N, 3.17. Found: C, 70.56; H,
4.18; N, 3.17.
ACC-Resin Syn th esis (7). To a 5 L round-bottom flask
were added Rink Amide AM resin (95 g, 60 mmol) and DMF
(1 L). The mixture was gently stirred with an overhead
mechanical stirring apparatus for 30 min and then filtered
using a filter cannula (Pharmacia, Uppsala, Sweden). A 20%
solution of piperidine in DMF (1 L) was then added, and the
mixture was agitated for 30 min. The resin was filtered and
washed with DMF (3 × 1 L). ACC (53 g, 120 mmol), HOBt
(18.5 g, 120 mmol), and DMF (1 L) were added to the flask
followed by DICI (19 mL, 120 mmol). The mixture was agitated
overnight and then filtered, washed with DMF (3 × 1 L), THF
(3 × 1 L), and MeOH (3 × 1 L), and dried over P2O5. The
substitution level of the resin was 0.53 mmol/g (>95%) as
determined by Fmoc-analysis.15
P r oced u r e for Cou p lin g F m oc-Am in o Acid s to th e
ACC-Resin (8a -u ). To a 100 mL round-bottom flask were
added ACC-resin (5.0 g, 2.6 mmol) and DMF (50 mL). The
mixture was stirred using a magnetic stir bar for 30 min and
filtered with a filter cannula. A 20% solution of piperidine in
DMF (50 mL) was added followed by agitation for 30 min. The
resin was filtered and washed with DMF (3 × 50 mL). An
Fmoc-amino acid (13 mmol), DMF (30 mL), HATU (5.0 g, 13
mmol), and 2,4,6-collidine (1.8 mL, 13 mmol) were added to
the flask followed by agitation for 24 h. The resin was then
filtered and washed with DMF (3 × 50 mL). To efficiently load
F m oc-L-P h e-D-Asn -ACC (11a ). Analytical reverse-phase
HPLC analysis of crude material according to General Condi-
tions (tR ) 22.7 min, 85% purity). 1H NMR (300 MHz, DMSO-
d6): δ 2.40-2.60 (m, 2), 2.70-2.85 (m, 1), 2.90-3.00 (m, 1),
3.61 (s, 2), 4.00-4.20 (m, 3), 4.25-4.35 (m, 1), 4.70-4.80 (m,
1), 6.28 (s, 1), 6.92 (s, 1), 7.10-7.40 (m, 11), 7.49 (d, 1, J )
8.9), 7.55-7.65 (m, 4), 7.72 (d, 1, J ) 8.2), 7.76 (s, 1), 7.82 (t,
2, J ) 7.1), 8.54 (d, 1, J ) 8.3), 10.23 (s, 1). MS (ESI), m/z
calcd for C39H35N5O8: 701.3. Found: m/z 702.2 (M + H)+.
F m oc-L-P h e-L-Leu -ACC (10b). Analytical reverse-phase
HPLC analysis of crude material according to General Condi-
tions (tR ) 26.1 min, 86% purity). 1H NMR (300 MHz, DMSO-
d6): δ 0.70-0.95 (m, 6), 1.40-1.75 (m, 3), 2.75 (m, 1), 3.00 (m,
1), 3.62 (s, 2), 4.12 (m, 3), 4.30 (m, 1), 4.47 (m, 1), 6.29 (s, 1),
7.10-7.33 (m, 8), 7.37 (t, 2, J ) 7.4), 7.45 (d, 1, J ) 8.9), 7.55-
7.65 (m, 4), 7.69 (d, 1, J ) 8.5), 7.78 (s, 1), 7.85 (d, 2, J ) 7.5),
8.29 (d, 1, J ) 7.9), 10.50 (s, 1). MS (ESI), m/z calcd for
(23) (a) Kanaoka, Y.; Kobayashi, A.; Sato, E.; Nakayama, H.; Ueno,
T.; Muno, D.; Sekine, T. Chem. Pharm Bull. 1984, 32, 3926. (b) Besson,
T.; J oseph, B.; Moreau, P.; Viaud, M. C.; Coudert, G.; Guillaumet.
Heterocycles 1992, 34, 273.
C
41H40N4O7: 700.3. Found: m/z 723.3 (M + Na)+.
F m oc-L-P h e-D-Leu -ACC (11b). Analytical reverse-phase
HPLC analysis of crude material according to General Condi-

Synthesis of Fluorogenic Protease Substrates
J . Org. Chem., Vol. 67, No. 3, 2002 915
1
tions (tR ) 24.8 min, 83% purity). 1H NMR (300 MHz, DMSO-
d6): δ 0.76 (d, 3, J ) 6.3), 0.79 (d, 3, J ) 6.5), 1.32 (m, 1),
1.35-1.60 (m, 2), 2.75-3.00 (m, 2), 3.62 (s, 2), 4.00-4.20 (m,
3), 4.25-4.60 (m, 2), 6.28 (s, 1), 7.10-7.40 (m, 10), 7.50 (d, 1,
J ) 8.7), 7.55-7.68 (m, 4), 7.72-7.85 (m, 4), 8.49 (d, 1, J )
8.2), 10.29 (s, 1). MS (ESI), m/z calcd for C41H40N4O7: 700.3.
Found: m/z 723.2 (M + Na)+, 739.2 (M + K)+.
F m oc-L-P h e-L-His-ACC. Analytical reverse-phase HPLC
analysis of crude material according to General Conditions (tR
) 20.3 min, 82% purity). 1H NMR (300 MHz, DMSO-d6): δ
2.65-2.82 (m, 1), 2.92-3.16 (m, 2), 3.17-3.50 (m, 1), 3.63 (s,
2), 4.10-4.40 (m, 4), 4.74 (m, 1), 6.31 (s, 1), 7.10-7.50 (m, 13),
7.55-7.80 (m, 6), 7.85 (d, 2, J ) 7.5), 8.62 (d, 1, J ) 7.5), 8.91
(s, 1), 10.48 (s, 1). MS (ESI), m/z calcd for C41H36N6O7: 724.3.
Found: m/z 725.3 (M + H)+.
F m oc-L-P h e-D-His-ACC. Analytical reverse-phase HPLC
analysis of crude material according to General Conditions (tR
) 19.7 min, 83% purity). 1H NMR (300 MHz, DMSO-d6): δ
2.65-2.8 (m, 1), 2.81-2.91 (m, 1), 2.92-3.05 (m, 1), 3.10-3.20
(m, 1), 3.63 (s, 2), 4.05-4.20 (m, 3), 4.21-4.35 (m, 1), 4.70-
4.85 (m, 1), 6.31 (s, 1), 7.10-7.40 (m, 12), 7.46 (dd, 1, J ) 8.8,
1.8), 7.58 (d, 2, J ) 7.4), 7.66 (d, 2, J ) 8.7), 7.75 (d, 1, J )
1.8), 7.76-7.85 (m, 3), 8.73 (d, 1, J ) 8.2), 8.90 (s, 1), 10.36 (s,
1). MS (ESI), m/z calcd for C41H36N6O7: 724.3. Found: m/z
725.3 (M + H)+.
9.2 min). H NMR (300 MHz, DMSO-d6): δ 1.15-1.35 (m, 5),
1.40-1.75 (m, 9), 1.84 (s, 3), 2.05-2.11 (m, 2), 2.60-2.80 (m,
4), 2.90-3.05 (m, 1), 3.06-3.20 (m, 1), 3.64 (s, 2), 4.10-4.25
(m, 2), 4.32-4.41 (m, 1), 4.46-4.60 (m, 1), 6.31 (s, 1), 6.78-
6.82 (m, 1), 6.88 (t, 1, J ) 7.30), 6.99 (t, 1, J ) 7.30), 7.10-
7.22 (m, 2), 7.20-7.30 (m, 2), 7.44 (dd, 1, J ) 8.9, 1.9), 7.54 (d,
1, J ) 7.7), 7.55-7.75 (m, 8), 7.80 (d, 1, J ) 1.8), 7.95 (d, 1, J
) 6.9), 8.00-8.10 (m, 2), 8.25-8.28 (m, 1), 10.46 (s, 1). MS
(ESI), m/z calcd for C41H54N10O9: 830.4. Found: m/z 831.4 (M
+ H)+.
Ac-Ar g-Lys-Ser -Leu -Va l-Nle-ACC (13). Resin 8n (32 mg,
0.012 mmol) was used to construct peptide 13 (8.0 mg, 0.0068
mmol, 55% yield) according to General Procedures. Analytical
reverse-phase HPLC analysis according to General Conditions
(tR ) 11.6 min). 1H NMR (300 MHz): δ 0.70-0.90 (m, 15),
1.13-1.33 (m, 6), 1.35-1.55 (m, 8), 1.55-1.75 (m, 5), 1.81 (s,
3), 1.91-1.97 (m, 1), 2.65-2.75 (m, 2), 3.02-3.10 (m, 2), 3.50-
3.54 (m, 2), 3.62 (s, 2), 4.12-4.33 (m, 6), 5.02 (br s, 1), 6.28 (s,
1), 7.16 (br s, 2), 7.41 (dd, 1, J ) 8.4, 1.8), 7.51 (t, 1, J ) 5.7),
7.60-7.80 (m, 8), 7.92-8.20 (m, 7), 10.45 (s, 1). MS (ESI), m/z
calcd for C45H72N12O11: 956.5. Found: m/z 957.4 (M + H)+.
Ack n ow led gm en t. We thank Cleo Salisbury for
helpful suggestions and review of the manuscript. This
work was supported by National Institutes of Health
Grants GM54051 (to J .A.E.) and CA72006 and AI35707
(to C.S.C.)
Ac-Lys-Gln -Tr p -Lys-ACC (12). Resin 8l (150 mg, 0.057
mmol) was used to construct peptide 12 (32 mg, 0.030 mmol,
53% yield) according to General Procedures. Analytical reverse-
phase HPLC analysis according to General Conditions (tR
)
J O016140O