Preparation of peptide p-nitroanilides using an aryl hydrazine resin

Article abstract of DOI:10.1021/ol048417n

(Chemical Equation Presented) Peptide p-nitroanilides are useful compounds for studying protease activity; however, the poor nucleophilicity of p-nitroaniline makes their preparation difficult. We describe a new efficient approach for the Fmoc-based synthesis of peptide p-nitroanilides using an aryl hydrazine resin. Mild oxidation of the peptide hydrazide resin yields a highly reactive acyl diazene that efficiently reacts with weak nucleophiles. We have prepared several peptide p-nitroanilides, including substrates for the Lethal Factor protease from B. anthracis.

Full text of DOI:10.1021/ol048417n

ORGANIC
LETTERS
2004
Vol. 6, No. 21
3801-3804
Preparation of Peptide p-Nitroanilides
Using an Aryl Hydrazine Resin
Youngeun Kwon, Kelly Welsh, Alexander R. Mitchell, and Julio A. Camarero*
Chemical Biology and Nuclear Sciences DiVision, Lawrence LiVermore National
Laboratory, Lawrence LiVermore National Laboratory, 7000 East AVenue,
LiVermore, California 94550
camarero1@llnl.goV
Received August 10, 2004
ABSTRACT
Peptide p-nitroanilides are useful compounds for studying protease activity; however, the poor nucleophilicity of p-nitroaniline makes their
preparation difficult. We describe a new efficient approach for the Fmoc-based synthesis of peptide p-nitroanilides using an aryl hydrazine
resin. Mild oxidation of the peptide hydrazide resin yields a highly reactive acyl diazene that efficiently reacts with weak nucleophiles. We
have prepared several peptide p-nitroanilides, including substrates for the Lethal Factor protease from B. anthracis.
Peptide p-nitroanilides are useful chromogenic substrates for
studying the activity and selectivity of proteolytic enzymes.1
The preparation of a peptide p-nitroanilide on the solid-phase
peptide synthesis (SPPS) platform is challenging due to the
poor nucleophilicity of the aromatic amino group of p-
nitroaniline (pNA), which is further deactivated by the
electron-withdrawing nitro group.2
Fmoc-based SPPS using Wang and Sasrin resins. However,
this method required modifications of commercial resins prior
to peptide synthesis. Abbenante et al.6 improved Mergler’s
method by using commercially available trityl chloride resin
(TCP resin) and the milder oxidizing reagent Oxone.
However, this oxidation method is not compatible with amino
acids sensitive to oxidative conditions.
So far, several strategies have been reported for the
preparation of peptide p-nitroanilides using SPPS. Burdick
et al.3 prepared a urethane-linked p-diaminobenzene resin
for the synthesis of peptide 4-aminoanilides using Boc/benzyl
chemistry. These peptide 4-aminoanilides were then oxidized
with sodium perborate4 to give the corresponding peptide
p-nitroanilides. Mergler et al.5 modified this protocol for
Other investigators have used Fmoc-protected amino acid
p-nitroanilides that were attached through side-chain func-
tional groups (-SH, -CO2H, -NH2) to a chlorotrityl resin.7,8
This approach, however, requires the preparation of Fmoc-
NR-protected amino acid p-nitroanilides, and it can be only
used when the last residue of the peptide contains a suitable
side-chain functional group (i.e., Cys, Asp, Glu, or Lys).
Alsina et al.9 modified this strategy by using a backbone
amide linker (BAL) to attach an R-carboxylic group protected
(1) Tonello, F.; Ascenzi, P.; Montecucco, C. J. Biol. Chem. 2003, 278,
40075.
(2) Voyer, N.; Lavoie, A.; Pinette, M.; Bernier, J. Tetrahedron Lett. 1994,
35, 355.
(3) Burdick, D. J.; Struble, M. E.; Burnier, J. P. Tetrahedron Lett. 1993,
34, 2589.
(4) McKillop, A.; Tarbin, J. A. Tetrahedron Lett. 1983, 24, 1505.
(5) Mergler, M.; Dick, F.; Gosteli, J.; Nyfeler, R. Lett. Pept. Sci. 2000,
7, 1.
(6) Abbenante, G.; Leung, D.; Bond, T.; Fairline, D. P. Lett. Pept. Sci.
2001, 7, 347-351.
(7) Kaspari, A.; Schierhorn, A.; Schutkowski, M. Int. J. Pept. Protein
Res. 1996, 48, 486.
(8) Bernhardt, A.; Drewello, M.; Schtkowsky, M. J. Peptide Res. 1997,
50, 143.
10.1021/ol048417n CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/18/2004

Scheme 1. Preparation of Peptide Anilides Using an Aryl Hydrazine Resin
amino acid to polystyrene resin through the R-amino group.
hydrazinobenzoyl resin and then activated by oxidation with
2 equiv of NBS in the presence of anhydrous pyridine for
10 min at room temperature. When the oxidation was
completed, the activated resin was washed with anhydrous
dichloromethane (DCM) and treated with 40 equiv of pNA
dissolved in DCM containing 5% dimethylformamide
(DMF).20 The cleavage reaction was monitored by analytical
HPLC.21 As anticipated, the cleavage reaction was slower
than observed for aliphatic N-nucleophiles,13,17-19 requiring
24 h for completion. The solvent was then removed and the
residue was treated with trifluoroacetic acid (TFA) containing
5% H2O. As shown in Figure 1A, the oxidation and cleavage
reaction with pNA was very clean and the main product was
identified by ES-MS as the corresponding peptide 1a with a
cleavage yield around 66% and purity around 95% (Table
1). We also reacted the same peptide diazene resin with
7-amino-4-methylcoumarin (AMC). Peptide AMC deriva-
tives are also desirable chromogenic and fluorogenic sub-
strates for exploring proteolytic activity.1,22
After conventional Fmoc-SPPS, the carboxyl group was
selectively deprotected, activated, and coupled with the
appropriate amino acid p-nitroanilide. However, some epimer-
ization on the penultimate residue can occur.
The methods presently available for the preparation of
peptide p-nitroanilides either require synthetic manipulation
steps or have limitations on the nature of amino acids that
can be incorporated or have racemization problems. In the
present work, we describe a novel and straightforward
strategy for the synthesis of peptide p-nitroanilides using an
aryl hydrazine linker that is compatible with Fmoc/t-Bu and
Boc/Bz SPPS.10-19
Our new approach (see Scheme 1) involves the direct
assembly of the peptide on an aryl hydrazine resin12-19 using
standard Fmoc-protocols. When the synthesis is completed,
the fully protected peptide hydrazide resin is activated by
mild oxidation with NBS in the presence of pyridine. The
resulting acyl diazene resin is then cleaved with pNA. The
fully deprotected peptide p-nitroanilide is finally obtained
by treatment with trifluoroacetic acid (TFA).
Acyl diazenes are reactive intermediates that readily react
with O- and N-nucleophiles.13,17-19 However, Millington et
al.13 showed that highly inactivated or sterically hindered
O-nucleophiles (such as t-BuOH or pentafluorophenol) are
unable to react with acyl diazenes in an efficient way.
Intrigued by this finding, we explored the reactivity of
peptidyl diazenes to poorly reactive arylamines such a pNA
(the pKa of the conjugated acid of pNA’s amino group is
∼1.0). Model tripeptide 1a (Table 1) was synthesized on a
The cleavage reaction with AMC only took 6 h for
completion. This result can be attributed to the higher
nucleophilicity of the aromatic amino group in AMC (pKa
of the conjugated acid of the AMC’s amino group is 3.8).
Similar to the cleavage with pNA, the major product in the
crude reaction mixture corresponded to the peptide-AMC 1b
(Figure 1B) with a cleavage yield of 77% and a purity of
96% (Table 1).
(9) Alsina, J.; Yokum, T. S.; Albericio, F.; Barany, G. J. Org. Chem.
1999, 64, 8671.
(10) Wieland, T.; Leawalter, J.; Birr, C. Liebigs Ann. Chem. 1970, 740,
31.
(11) Milne, H. B.; Most, C. F. J. Org. Chem. 1968, 33, 169-175.
(12) Semenov, A. N.; Gordeev, K. Y. Int. J. Peptide Protein Res. 1995,
45, 303.
(13) Millington, C. R.; Quarrell, R.; Lowe, G. Tetrahedron Lett. 1998,
39, 7201.
(14) Stieber, F.; Grether, U.; Waldmann, H. Angew. Chem., Int. Ed. 1999,
38, 1073.
(15) Berst, F.; Holmes, A. B.; Ladlow, M.; Murray, P. J. Tetrahedron
Lett. 2000, 41, 6649.
(16) Rosenbaum, C.; Waldmann, H. Tetrahedron Lett. 2001, 42, 5677.
(17) Peters, C.; Wladmann, H. J. Org. Chem. 2003, 68, 6053.
(18) Ludolph, B.; Waldmann, H. Chem. Eur. J. 2003, 9, 3683.
(19) Camarero, J. A.; Hackel, B. J.; de Yoreo, J. J.; Mitchell, A. R. J.
Org. Chem. 2004, 69, 4145.
(20) The cleavage reaction was performed under dry N2 atmosphere to
minimize the hydrolysis of the acyl diazene resin.
(21) Small aliquots from the crude reaction were taken at various times.
The solvent was removed under vacuum, the peptide p-nitroanilide
deprotected with TFA (TFA/H2O, 95:5 v/v) and then analyzed by analytical
RP-HPLC.
Table 1. Sequences, Molecular Weights, and Yields of
Peptides Used in This Study.
purityc yieldd
peptide
sequence
H-LYA-pNA
H-LYA-AMC
Ac-FA-pNA
Ac-F-D-A-pNA
H-LWA-pNA
H-LMYKA-pNA
Ac-RRRRVLR-pNA
Mw/Da
(%)
(%)
1a
1b
2
3
4
5
6
7
485.5a 486.0b
522.6a 522.0b
398.4a 398.0b
398.4a 398.0b
508.6a 509.0b
744.9a 745.0b
1173.4a 1173.5b
95
96
98
97
95
85
90
85
66
77
46
46
81
60
43
45
Ac-LARRRPVLP-pNA 1239.5a 1239.3b
a Expected. b Found. c Based on HPLC purity. d Based on resin substitu-
tion.
(22) Nonneau, P. R.; Plouffe, C.; Pelletier, A.; Wernic, D.; Poupart, M.
A. Anal. Biochem. 1998, 255, 59.
3802
Org. Lett., Vol. 6, No. 21, 2004

Figure 1. HPLC analysis of the crude product obtained by NBS
oxidation and pNA/AMC cleavage. Peptide 1 was cleaved using
pNA (A) and AMC (B). The asterisk denotes the corresponding
peptide arylamide. Linear gradient of 0-70% buffer B (90% CH3-
CN, 10% H2O, 0.1% TFA) in buffer A (H2O, 0.1% TFA) over 30
min was used for HPLC.
Although peptidyl diazenes have been shown to be free
of racemization during cleavage with aliphatic amines,11,16,19,23
the slow cleavage reaction with pNA raised the possibility
of racemization through oxazolone formation24 during such
a long reaction time (24 h). In order to exclude this
possibility, two dipeptide diastereomers (peptides 2 and 3)
were assembled on the hydrazine resin, oxidized with NBS,
and then cleaved with pNA for 24 h. As shown in Figure 2,
Figure 3. HPLC analysis of the crude product obtained by NBS
oxidation and pNA cleavage: (A) peptide 4, (B) peptide 5, (C)
peptide 6, and (D) peptide 7. The asterisk denotes the corresponding
peptide arylamide. Linear gradient of 0-70% buffer B over 30 min
was used for HPLC.
1). In the same way, Met-containing peptide 5 was also
obtained in good yield (Figure 3B, Table 1). It is interesting
to note that although the Met residue is initially oxidized by
NBS to the corresponding sulfoxide, during the TFA
deprotection step, the sulfoxide is reduced when the reaction
is carried out for 3 h at room temperature in the presence of
2% EtSH. This result is notable because Met-containing
peptide p-nitroanilides cannot be prepared by oxidation
reactions performed with sodium perborate or Oxone.3,4,6
We also prepared peptides 6 and 7 (Figure 3C,D, Table
1). The sequence of these peptides contains the recognition
sequence for the Lethal Factor (LF) protease,25 and they have
been fully characterized as substrates for this endometallo-
protease.1 The C-terminal modified peptides were obtained
in good yield using the same protocol as for model peptide
1 (Table 1). In both cases, the crude material was purified
by semipreparative RP-HPLC and used to test the activity
of recombinantly generated LF protein (Figure 4).26
Figure 2. Epimerization studies of the C-terminal residue during
activation and cleavage. HPLC traces of the crude products for the
oxidation and cleavage with NBS and pNA of Ac-Phe(L)-Ala(L)-
pNA (A) and Ac-Phe(L)-Ala(D)-pNA (B). Linear gradient of 30-
70% buffer B (90% CH3CN, 10% H2O, 0.1% TFA) over 30 min
was used for HPLC.
HPLC analysis of the crude cleavage reactions indicated no
racemization of the C-terminal residue (<0.5% by HPLC
analysis).
We have also recently shown that mild oxidation of peptide
hydrazides with NBS is totally compatible with oxidative-
sensitive residues (i.e., Tyr, Trp, Met, and Cys) when the
appropriate protecting groups and oxidative conditions are
employed.19 Thus, peptide 4, which contains an Nim-Boc
protected Trp residue, was totally unaffected by the NBS
oxidation and pNA cleavage, and it was obtained as the major
component in the crude reaction mixture (Figure 3A, Table
Figure 4. LF-catalyzed hydrolysis of peptide 6. (A) Hydrolysis
reaction was spectrophotometrically monitored at 405 nm. (B)
HPLC analysis of the hydrolysis crude reaction after 20 min. HPLC
conditions were similar to those employed in Figure 3.
(23) Wolman, Y.; Gallop, P. M.; Patchornik, A. J. Am. Chem. Soc. 1961,
83, 1263.
(24) Benoiton, N. L. Biopolymers 1996, 40, 245.
Org. Lett., Vol. 6, No. 21, 2004
3803

In conclusion, we have developed a straightforward
method to prepare both peptide derivatives of pNA and AMC
without limitation of size and amino acid composition. The
mild oxidation procedure involved is totally compatible with
sensitive amino acid residues under the conditions employed.
Moreover, no detectable racemization is observed at the
C-terminal residue during the activation and cleavage of the
hydrazide linker by pNA. Finally, this new method requires
neither premodification of commercial resins nor additional
manipulation after the cleavage and deprotection steps.
Acknowledgment. This work was performed under the
auspices of the U.S. Department of Energy by the University
of California, Lawrence Livermore National Laboratory,
under Contract No. W-7405-Eng-48.
(25) Lethal factor is a Zn-metalloprotease secreted by virulent strains of
B. anthacis that targets the N-terminal region of mitogen-activated protein
kinase kinases (MAPKKs).
(26) Recombinant LF was prepared by cloning the LF gene into the
multiple cloning site of pTXB1 plasmid (New England Biolabs). LF was
expressed as an intein fusion prtotein and purified using chitin beads.
Supporting Information Available: Experimental de-
tails. This material is available free of charge via the Internet
at http://pubs.acs.org.
OL048417N
3804
Org. Lett., Vol. 6, No. 21, 2004