Solid-phase unnatural peptide synthesis (UPS)

Full text of DOI:10.1021/ja9601245

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070
J. Am. Chem. Soc. 1996, 118, 6070-6071
Solid-Phase Unnatural Peptide Synthesis (UPS)
,
†
†
Martin J. O’Donnell,* Changyou Zhou, and
William L. Scott*,
‡
Department of Chemistry, Indiana UniVersity-Purdue
UniVersity at Indianapolis, Indianapolis, Indiana 46202
Technology Core Research, Lilly Research Laboratories
Lilly Corporate Center, Eli Lilly and Company
Indianapolis, Indiana 46285
ReceiVed January 16, 1996
Solid-phase peptide synthesis (SPPS) has evolved into a
1,2
powerful synthetic method since it was first reported in 1963.
Figure 1. General scheme for solid-phase unnatural peptide synthesis
UPS). Abbreviations: shaded circle ) resin (Merrifield, Wang,...);
) O, NH, peptide, etc.; PG ) protecting group; UN ) unnatural
It is considered the method of choice for the automated
preparation of peptides in numerous laboratories around the
world. SPPS is now being used to rapidly synthesize peptides
in a combinatorial fashion.3 Peptide libraries constructed from
naturally occurring amino acids have been widely used to screen
for biological activity. However, increased activity, bioavail-
ability, and degradative resistance often require incorporation
(
X
n
side chain (an electrophile, such as an alkyl halide UN-X, is used in
the alkylation step); Y ) natural amino acid or peptide or N-protected
terminus; Y* ) resin-free N-terminal end; X * ) resin-free C-terminal
n
end. For experimental conditions, see ref 9.
4
of unnatural residues in the peptide framework. Currently, most
9
In this procedure, three new steps (Figure 1, reactions in
box), activation (C), alkylation (D), and imine hydrolysis of
the resin-bound Schiff base (E), have been added to the normal
SPPS sequence involving deprotection (B) and coupling (F or
G) steps. Following step E in the loop a number of options are
possible: repeat the steps in the UPS to add another unnatural
residue to the growing peptide chain (step F), couple a natural
amino acid or other residue via normal solid-phase methodology
(step G), or protect the free amino group subsequent to other
reactions on the resin-bound product (step H). Finally, the
product is cleaved from the resin (step I).
synthetic routes to modified peptides incorporate the individually
prepared unnatural amino acid residue5 by normal peptide
synthesis.
A more direct and flexible method for the preparation of
peptides containing modified amino acid residues would be to
build the new side chains onto the growing peptide chain during
the peptide synthesis.6 One major obstacle must be overcome
to practically realize this goal: a mild methodology of carbon-
carbon bond formation on solid phase is needed to selectiVely
introduce the side chain at the R-carbon of a particular residue
in the growing peptide chain. To accomplish this we have
adapted the solution phase chemistry employed in the selective
CR-Câ bond formation reaction of Schiff base activated amino
acids7 and peptides8 to a new methodology: “solid-phase
unnatural peptide synthesis” or solid-phase UPS (Figure 1).
To simplify analysis in the initial resin-bound alkylations, a
known dipeptide [(D,L)-Phe-Leu] was constructed by backbone
alkylation of a Gly-Leu derivative (Figure 2). The benzophe-
†
Indiana University-Purdue University at Indianapolis.
‡
Eli Lilly and Company.
(1) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. (b)
Figure 2. Alkylation step in solid-phase UPS.
Merrifield, R. B. Science 1986, 232, 341-347.
2) (a) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Tetrahedron 1993,
9, 11065-11133. (b) Benz, H. Synthesis 1994, 337-358.
3) (a) Ellman, J. A. CHEMTRACTS: Org. Chem. 1995, 8, 1-4. (b)
Pirrung, M. C. Ibid. 1995, 8, 5-12. (c) Czarnik, A. W. Ibid. 1995, 8, 13-
(
4
none imine of Gly-Leu-Merrifield resin (1) was conveniently
prepared from TFA‚H2N-Gly-Leu-resin by reaction with
(
10,11
benzophenone imine.
After unsuccessful attempts to effect
1
8. (d) Mitscher, L. A. Ibid. 1995, 8, 19-25. (e) Pinilla, C; Appel, J.;
Blondelle, S.; Dooley, C.; D o¨ rner, B.; Eichler, J.; Ostresh, J.; Houghten,
R. A. Biopolymers 1995, 37, 221-240. (f) Schultz, P. G.; Lerner, R. A.
Science 1995, 269, 1835-1842. (g) Terrett, N. K.; Gardner, M.; Gordon,
D. W.; Kobylecki, R. J.; Steele, J. Tetrahedron 1995, 51, 8135-8173.
benzylation of 1 with melted KOH/K2CO3 (incomplete conver-
8
12
sion) or strong ionic bases (racemization of peptides),
simultaneous deprotonation and alkylation of 1 with benzyl
bromide was accomplished using the organic-soluble, nonionic
(4) (a) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J.
Am. Chem. Soc. 1992, 114, 10646-10647. (b) Kerr, J. M.; Banville, S. C.;
Zuckermann, R. N. J. Am. Chem. Soc. 1993, 115, 2529-2531. (c) Karle,
I. L.; Rao, R. B.; Prasad, S.; Kaul, R.; Balaram, P. J. Am. Chem. Soc. 1994,
(9) Reaction conditions: C, (a) TFA‚H2N-Gly-Merrifield resin,
Ph2CdNH (1.5 equiv)/1-N-methyl-2-pyrrolidinone (NMP), overnight; (b)
H2N-Gly-Wang resin, Ph2CdNH (1.5 equiv)/AcOH (1.3 equiv)/NMP,
overnight. D, UN-X (2.0 equiv)/2-tert-butylimino-2-diethylamino-1,3-
dimethylperhydro-1,3,2-diazaphosphorine (BEMP) (2.0 equiv)/NMP, over-
night. E, (a) Merrifield resin, 1 N aqueous HCl/THF (3/7), overnight, then
DIEA (10%)/NMP; (b) Wang resin, 1 N aqueous NH2OH‚HCl/THF (3/7),
5 h, then DIEA (10%)/NMP. H, (a) Merrifield resin, Boc2O (3.0 equiv)/
NMP or CbzOSu (3.0 equiv)/NMP; (b) Merrifield or Wang resin, FmocCl
(3.0 equiv)/DIEA (6.0 equiv)/NMP. I, (a) Merrifield or Wang resin, Ti-
(OEt)4 (5.0 equiv)/allyl alcohol, 120 °C, pressure tube, 2 h; (b) only for
Wang resin, TFA (95%)/H2O, 5 h.
1
16, 10355-10361. (d) Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.
Chem., Int. Ed. Engl. 1995, 34, 621-633. (e) Voyer, N.; Lamothe, J.
Tetrahedron 1995, 51, 9241-9284.
(5) (a) Duthaler, R. O. Tetrahedron 1994, 50, 1539-1650. (b) Williams,
R. M. in AdVances in Asymmetric Synthesis; Hassner, A., Ed.; Jai Press
Inc.: Greenwich, CT, 1995; Vol. 1, pp 45-94. (c) Ojima, I. in AdVances
in Asymmetric Synthesis; Hassner, A., Ed.; Jai Press Inc.: Greenwich, CT,
1
995; Vol. 1, pp 95-146.
6) CR-C bond formation in protected peptides: reference 8, footnote
. (a) Ager, D. J.; Froen, D. E.; Klix, R. C.; Zhi, B.; McIntosh, J. M.;
(
1
Thangarasa, R. Tetrahedron 1994, 50, 1975-1982. (b) Steglich, W.; J a¨ ger,
M.; Jaroch, S.; Zistler, P. Pure Appl. Chem. 1994, 66, 2167-2170. (c)
Kazmaier, U. J. Org. Chem. 1994, 59, 6667-6670. (d) Seebach, D.; Beck,
A. K.; Studer, A. In Modern Synthetic Methods 1995; Ernst, B., Leumann,
C., Eds.; VCH: Weinheim, 1995; Vol. 7, pp 1-178.
(10) O’Donnell, M. J.; Polt, R. L. J. Org. Chem. 1982, 47, 2663-2666.
(11) Other recent applications involving resin-bound imines: Murphy,
M. M.; Schullek, J. R.; Gordon, E. M.; Gallop, M. A. J. Am. Chem. Soc.
1995, 117, 7029-7030.
(12) Solution-phase model alkylation (solution UPS) of the dipeptide
Schiff base ester Ph2CdNCH2CO-Phe-OMe with 4-bromobenzyl bromide
showed that 15-68% racemization of the pre-existing chiral center was
observed with potassium tert-butoxide, 9-lithium fluorene, or lithium 2,6-
di-tert-butyl-4-methylphenoxide.
(
7) O’Donnell, M. J.; Wu, S.; Huffman, J. C. Tetrahedron 1994, 50,
507-4518.
8) O’Donnell, M. J.; Burkholder, T. P.; Khau, V. V.; Roeske, R. W.;
Tian, Z. Pol. J. Chem. 1994, 68, 2477-2488.
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S0002-7863(96)00124-2 CCC: $12.00 © 1996 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 118, No. 25, 1996 6071
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). The overall isolated yields for these products ranged from
7-55% based on the starting Boc-Gly-resin or Boc-Leu-
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resin.
Solid-phase UPS can also be used with Wang resins and
Fmoc-based SPPS methodology. Because of the acid sensitivity
of the Wang resin linkage, it was necessary to develop methods
for introduction and removal of the Schiff base group under
essentially neutral conditions. The activation step is ac-
complished by standard piperidine cleavage of the N-terminal
Fmoc group followed by reaction of the free amine with
benzophenone imine (1.5 equiv) in the presence of acetic acid
Figure 3. Organic-soluble, nonionic iminophosphorane bases.
bases P1-t-Bu, P1-t-Oct, (“Schwesinger bases”),13 or BEMP
(
Figure 3) to yield the benzophenone imine of (D,L)-Phe-Leu-
Merrifield resin (2).
(
1.3 equiv). Following the normal alkylation reaction, the imine
Compound 2 was hydrolyzed (aqueous HCl, THF) and then
neutralized to afford the free amino group, which was N-acylated
is hydrolyzed (aqueous NH2OH‚HCl, THF, pH = 6) and then
neutralized (iPr2NEt) to the free amino group, which can be
9
14
with Fmoc-Cl. Titanate-mediated transesterification conve-
niently cleaved the product from the resin to yield the O-allyl
subjected to the various available options. For example, Fmoc-
(D,L)-â-naphthylalanine allyl ester (4d) was prepared in 80%
overall purified yield (83% crude yield) starting from Fmoc-
Gly-Wang resin. Additionally, the free model dipeptide (D,L)-
Phe-Leu (7) was obtained by UPS from Gly-Leu-Wang resin
15
ester, Fmoc-(D,L)-Phe-Leu-Oallyl (3), in 55% overall crude
yield (54% purified yield).
The preparation of unnatural amino acids was demonstrated
by the synthesis of 4a-c (Figure 4). Thus, TFA‚H2N-Gly-
Merrifield resin was activated as its benzophenone imine and
alkylated to give the R-alkylated product. Hydrolysis and
N-acylation (Boc2O, Cbz-OSu or Fmoc-Cl), followed by titan-
ate-mediated transesterification gave the O-allyl esters of the
N-Boc, N-Cbz-, or N-Fmoc-protected unnatural amino acids
1
7
in 60% overall yield using TFA to cleave the product from
the resin.
The use of mild reagents and room temperature reactions in
solid-phase UPS chemistry allows for the easy automation of
this technique. In this regard, preliminary results are very
encouraging. Thus, the dipeptide (D,L)-Phe-Leu (7) and a series
of Phe-substituted Phe-Leu derivatives have been prepared
from Fmoc-Leu-Wang resin (Via the Gly-Leu-Wang resin
intermediate). The reactions were performed on an automatic
16
(4a-c) in 42-54% overall yield.
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peptide synthesizer using a variety of benzylic halides.
These preliminary studies demonstrate that UPS can be
utilized to convert resin-bound amino acids or peptides to
unnatural derivatives. The mild reagents and conditions used
are compatible with both Boc- and Fmoc-based SPPS strategies,
and the technique has been automated. A variety of structurally
1
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diverse combinatorial libraries derived from unnatural amino
acids or unnatural peptides are now accessible using this
technique. We are currently exploring side chain compatibility,
incorporation of R,R-disubstituted residues, and stereoselective
UPS.
Acknowledgment. We gratefully acknowledge the National Insti-
tutes of Health (GM 28193) for support of this research. We thank
John Richardson, David Smiley, and Larry Spangle for analytical
support and Stephen Kaldor, Charles Lugar, and David Mendel for
helpful discussions.
Figure 4. Preparation of amino acid derivatives (4) by solid-phase
amino acid synthesis and di- and tripeptides (5 and 6, respectively) by
solid-phase UPS.
Using this methodology (in conjunction with final Fmoc
derivatization), dipeptides 5a and 5b, in which the unnatural
residue was created as either the C- or N-terminal residue, were
prepared. Similarly, tripeptides containing unnatural residues
added during the second or third “loop” of the synthesis are
represented by compounds 6a and 6b. It is also possible to
prepare completely unnatural polypeptides (5c and 6c) (Figure
Supporting Information Available: Experimental procedures for
solid and solution phase UPS and H-NMR spectra and mass spectra
data for all new compounds (36 pages). Ordering information is given
on any current masthead page.
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JA9601245
(
17) LC-MS analysis of crude product (D,L)-Phe-Leu 7 showed a 1:1
mixture of (D)-Phe-Leu and (L)-Phe-Leu [>80%, molecular ion: 279.2
(
M+1)] containing (D,L)-Phe-Leu-Leu [molecular ion: 392.3 (M + 1),
(13) No racemization was observed with P1-t-Bu, P1-t-Oct, or BEMP.
also found in an independently prepared sample of Phe-Leu from Leu-
Wang resin by normal SPPS]. Elemental analysis of crude product 7. Calcd
for CF3CO2H‚(D,L)-Phe-Leu: C, 52.04; H, 5.91; N, 7.14. Found: C, 49.90;
H, 5.81; N, 7.04. Amino acid analysis: Phe/Gly ) 97/3.
In contrast, P2-Et and P4-t-Bu (Figure 3) resulted in overalkylation at the
amide bond and extensive racemization. For other uses of “Schwesinger
bases”, see: Schwesinger, R.; Willaredt, J.; Schlemper, H.; Keller, M.;
Schmidt, D.; Fritz, H. Chem. Ber. 1994, 127, 2435-2454.
(
18) The dipeptides 7a-f were prepared (from benzyl bromide or the
(14) (a) Seebach, D.; Hungerb u¨ hler, E.; Nafe, R.; Schnurrenberger, P.;
o-, m-, or p-methyl-, p-trifluoromethyl-, or p-nitrobenzyl bromides) on an
Advanced ChemTech 357 automatic peptide synthesizer using conditions
similar to those developed earlier. The products were identified by LC-MS
Weidmann, B.; Z u¨ ger, M. Synthesis 1982, 138-141. (b) Rehwinkel, H.;
Steglich, W. Synthesis 1982, 826-827. (c) Oppolzer, W.; Lienard, P. HelV.
Chim. Acta 1992, 75, 2572-2582.
1
and H-NMR.
(
15) Allyl esters in peptide synthesis: (a) Seitz, O.; Kunz, H. Angew.
(
19) Hydantoins 8 were prepared in 32-50% yields by solid-phase UPS
Chem., Int. Ed. Engl. 1995, 34, 803-805. (b) Beugelmans, R.; Neuville,
followed by the Parke-Davis cyclization methodology, see: Dewitt, S. H.;
Kiely, J. S.; Stankovic, C. J.; Schroeder, M. C.; Reynolds Cody, D. M.;
Pavia, M. R. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6909-6913.
L.; Bois-Choussy, M.; Chastanet, J.; Zhu, J. Tetrahedron Lett. 1995, 36,
129-3132.
3
(
16) Overall yields of amino acids from resin-bound reactions (crude
yield, isolated yield): 4a, 55%, 54%; 4b, 50%, 48%; 4c, 45%, 42%. Overall
yields of di- and tripeptides from resin-bound reactions (crude yield, isolated
yield): 5a, 47%, 42%; 5b, 55%, 53%; 5c, 40%, 37%; 6a, 67%, 56%; 6b,
1
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6%, 40%; 6c, 50%, 43%. All products gave satisfactory TLC, H-NMR,
and HRMS.