8
762 J . Org. Chem., Vol. 64, No. 24, 1999
Alsina et al.
nium ion4 may form by attack of the oxygen from the
BAL-amide function onto the activated carboxyl;
despite this risk, we were able to develop effective
protocols which are found empirically to proceed with
minimal racemization.
reports of Fmoc SPS of peptide p-nitroanilides12 depend
on side-chain anchoring (which requires an appropriately
positioned trifunctional amino acid residue; moreover
highly variable yields are observed), and use modified
deprotection conditions [morpholine-NMP (1:1) instead
of the usual piperidine-DMF (1:4)] due to the apparent
partial instability of the p-nitroanilide to standard Fmoc
5
,6
To illustrate our general approach, we report here
7
Fmoc SPS of unprotected peptide p-nitroanilides and
peptide thioesters.8 In the first case, the C-terminal
13
removal chemistry. In the case of peptide thioesters,
standard Fmoc SPS protocols have been inappropriate
modification is particularly challenging due to the low
nucleophilicity of the aromatic amino nitrogen of p-nitro-
because of the lability of the sulfur-carbonyl bond to
aniline.9
-11
To the best of our knowledge, the only prior
14
prolonged exposure to secondary amines. These com-
pounds have been previously accessed in concert with
Fmoc chemistry either by modifying Fmoc deprotection
conditions [i.e., use of 1-methylpyrrolidine (25%),
(
1) Abbreviations used for amino acids and the designations of
peptides follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J . Biol. Chem. 1972, 247, 977-983. The following
additional abbreviations are used: BAL, Backbone Amide Linker; Bzl,
benzyl; Ddz, 2-(3,5-dimethyloxyphenyl)propyl(2)oxycarbonyl; DIEA,
N,N-diisopropylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide;
EDC, 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride;
ESMS, electrospray mass spectrometry; FABMS, fast atom bombard-
ment mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; HATU,
N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-
methylmethanaminium hexafluorophosphate N-oxide; HBTU, N-[(1H-
benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanamin-
ium hexafluorophosphate N-oxide; HOAt, 1-hydroxy-7-azabenzotriazole
hexamethyleneimine (2%), and 1-hydroxybenzotriazole
15
(
HOBt) (2%, w/v) in NMP-DMSO (1:1)], or by an
indirect route whereby an otherwise protected peptide
R
with a free C -carboxyl group, as made by stepwise Fmoc
16
chemistry on a 2-chlorotrityl (Cl-Trt) resin and then
released from the support by dilute acid, was extended
by a solution coupling to either an alkanethiol or an
amino acid S-alkyl thioester, followed by final deprotec-
tion.17
(
3-hydroxy-3H-1,2,3-triazolo-[4,5-b]pyridine); HOBt, 1-hydroxybenzo-
triazole; IRAA, “internal reference” amino acid (ref 19); NMM, N-
methylmorpholine; o,p-PALdehyde, mixture of 4-(4-formyl-3,5-dimethoxy-
phenoxy)butyric acid and 4-(2-formyl-3,5-dimethoxyphenoxy)butyric
acid; PEG-PS, poly(ethylene glycol)-polystyrene (graft resin support);
Ph, phenyl; pNA, p-nitroanilide; Py, pyrrolidino; PyAOP, 7-azabenzo-
triazol-1-yl-N-oxytris(pyrrolidino)phosphonium hexafluorophosphate;
PyBOP, benzotriazol-1-yl-N-oxytris(pyrrolidino)phosphonium hexafluo-
rophosphate; SPS, solid-phase synthesis; TFFH, 1,1,3,3-tetramethyl-
Resu lts a n d Discu ssion
Solid -P h a se Assem b ly of Tr is(a lk oxy)b en zyl-
a m id e BAL-An ch or ed C-Ter m in a l Allyl Ester P ep -
tid e-Resin s. The target for the first phase of this work
was Boc-Val-Tyr(tBu)-Phe-(BAL-Ile-PEG-PS)Ala-OAllyl,
although the method used is likely to be successful for
2
-fluoroformamidinium hexafluorophosphate; TMP, 2,4,6-trimethyl-
pyridine (collidine). Amino acid symbols denote the L-configuration
unless stated otherwise. All solvent ratios are volume/volume unless
stated otherwise.
(
(
F.; Barany, G. J . Am. Chem. Soc. 1998, 120, 5441-5452 and references
therein.
(
2) (a) University of Minnesota. (b) University of Barcelona.
3) J ensen, K. J .; Alsina, J .; Songster, M. F.; V a´ gner, J .; Albericio,
3
most sequences. As already described, 4-[(4 or 2)-formyl-
3
,5-dimethoxyphenoxy]butyric acid (o,p-PALdehyde, 4
3
,18
4) As described to account for racemization in the coupling of
equiv) was added to an amino-functionalized PEG-PS-
N-acyl-N-methylamino acids; see: (a) McDermott, J . R.; Benoiton, N.
L. Can. J . Chem. 1973, 51, 2562-2570. (b) Davies, J . S.; Mohammed,
A. K. J . Chem. Soc., Perkin Trans. 1 1981, 2982-2990.
(10) An indirect Boc SPS route to p-nitroanilides involves a urethane-
linked p-aminoanilide resin, and a late-stage solution oxidation step
to convert an aromatic amine to a nitro group. See: Burdick, D. J .;
Struble, M. E.; Burnier, J . P. Tetrahedron Lett. 1993, 34, 2589-2592.
(11) Another Boc SPS chemistry route involves oxime ester anchor-
ing, but suffers from low yields during the cleavage step using
p-nitroaniline as the attacking nucleophile. See: Voyer, N.; Lavoie,
A.; Pinette, M.; Bernier, J . Tetrahedron Lett. 1994, 35, 355-358.
(12) (a) Kaspari, A.; Schierhorn, A.; Schutkowski, M. Int. J . Pept.
Protein Res. 1996, 48, 486-494. (b) Bernhardt, A.; Drewello, M.;
Schutkowski, M. J . Pept. Res. 1997, 50, 143-152.
(13) Such instability is reported in ref 12a, which states that about
half of the p-nitroanilide moieties are lost after 1 h of treatment of
Fmoc-Glu-pNA with piperidine-NMP (1:4). However, this result is in
contrast to a claim that Boc-Ala-pNA is completely stable toward
piperidine-DMF (1:1) and pyrrolidine-DMF (1:3); see: Rijkers, D. T.
S.; Adams, H. P. H. M.; Hemker, H. C.; Tesser, G. I. Tetrahedron 1995,
51, 11235-11250.
(
5) Compare to discussions of the mechanism of racemization via
5
(4H)-oxazolone mechanisms upon activation of N-acylamino acids.
Reviews: (a) Kemp, D. S. In The Peptides: Analysis, Synthesis, Biology;
Gross, E., Meienhofer, J ., Eds.; Academic Press: New York, 1979; Vol.
, pp 315-383. (b) Barany, G.; Merrifield, R. B. In The Peptides:
1
Analysis, Synthesis, Biology; Gross, E., Meienhofer, J ., Eds.; Academic
Press: New York, 1980; Vol, 2, pp 1-284, especially pp 122-123. (c)
Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to
the Synthesis of Peptides and Proteins; CRC: Boca Raton, FL, 1997;
pp 116-119.
(6) It is precisely for this reason that stepwise SPPS is rarely carried
out in the N f C direction, as has been reviewed in ref 5b. For a few
examples of N f C SPPS, see: (a) Letsinger, R. L.; Kornet, M. J . J .
Am. Chem. Soc. 1963, 85, 3045-3046. (b) Felix, A. M.; Merrifield, R.
B. J . Am. Chem. Soc. 1970, 92, 1385-1391. (c) Henkel, B.; Zhang, L.;
Bayer, E. Liebigs Ann. Recl. 1997, 2161-2168. (d) L e´ ger, R.; Yen, R.;
She, M. W.; Lee, V. J .; Hecker, S. J . Tetrahedron Lett. 1998, 39, 4171-
4
174.
7) Amino acid and peptide p-nitroanilides are needed as chromoge-
(14) On the other hand, thioester intermediates required for chemi-
cal ligation are readily prepared by Boc chemistry, as reviewed in ref
8d. See also: (a) Blake, J .; Li, C. H. Proc. Natl. Acad. Sci. U.S.A. 1981,
78, 4055-4058. (b) Hojo, H.; Kwon, Y.; Kakuta, Y.; Tsuda, S.; Tanaka,
I.; Hikichi, K.; Aimoto, S. Bull. Chem. Soc. J pn. 1993, 66, 2700-2706.
(c) Canne, L. E.; Walker, S. M.; Kent, S. B. H. Tetrahedron Lett. 1995,
36, 1217-1220.
(
nic substrates to monitor the activity of numerous proteolytic enzymes.
See: Handbook of Synthetic Substrates for the Coagulation and
Fibrinolytic System; Hemker, H. C., Ed.; Martinus Nijhof Publishers:
Boston, 1983.
(
8) Chemical ligation of unprotected peptides in aqueous solution
has emerged as an extremely powerful method of peptide/protein
assembly. Thioesters are often the reactive C-terminal functionality
used in these chemical ligations. See: (a) Dawson, P. E.; Muir, T. W.;
Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776-779. (b) Tam,
J . P.; Lu, Y.-A.; Liu, C. F.; Shao, J . Proc. Natl. Acad. Sci. U.S.A. 1995,
(15) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39,
8669-8672.
(16) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J . Pept.
Protein Res. 1991, 37, 513-520.
(17) Futaki, S.; Sogawa, K.; Maruyama, J .; Asahara, T.; Niwa, M.;
Hojo, H. Tetrahedron Lett. 1997, 38, 6237-6240.
9
1
2, 12485-12489. (c) Zhang, L.; Tam, J . P. J . Am. Chem. Soc. 1997,
19, 2363-2370. Review: (d) Muir, T. W.; Dawson, P. E.; Kent, S. B.
(18) For all structural formulas in this paper, BAL handles are
depicted as the isomer in which the aminomethyl group is para to the
valeryl linking group. However, many of the experiments described
herein start with a mixture (1:2) of the ortho and para isomers of
PALdehyde, i.e., o,p-PALdehyde; furthermore, the side-chain moiety
had one fewer carbon (substitution of butyryl for valeryl). The latter
compound is commercially available from PE Biosystems (Framing-
ham, MA).
H. Methods Enzymol. 1997, 289, 266-298.
(
9) For example, the R(PAL) anchoring strategy for SPPS of N-
alkylamides was successful with R ) Me, Et, and C CH CH , but
when R ) C NO , although the reductive amination step gave the
6
H
5
2
2
6
H
4
2
expected N-PAL-p-nitroaniline intermediate, this compound could not
be acylated further. See: Songster, M. F.; V a´ gner, J .; Barany, G. Lett.
Pept. Sci. 1996, 2, 265-270.