Synthesis of N-hydroxy peptides: chemical ligation of O-acyl hydroxamic acids.

Article abstract of DOI:10.1021/ol005722+

[process--see text] A novel chemical ligation process is described that results in the construction of N-hydroxy peptides.

Full text of DOI:10.1021/ol005722+

ORGANIC
LETTERS
2000
Vol. 2, No. 10
1399-1401
Synthesis of N-Hydroxy Peptides:
Chemical Ligation of O-Acyl Hydroxamic
Acids
Rebecca Braslau,* Jonathan R. Axon, and Bonnie Lee
Department of Chemistry, UniVersity of California, Santa Cruz, California 95064
braslau@chemistry.ucsc.edu
Received February 25, 2000
ABSTRACT
A novel chemical ligation process is described that results in the construction of N-hydroxy peptides.
Hydroxamic acids have received much attention as biologi-
cally active compounds such as ACE,1 lipoxygenase,2
peptidase,3 and PDE inhibitors,4 chelating agents,5 and
anticancer,6 antiarthritic,7 and antimalarial8 agents. While
working on O-acyl hydroxamic amino acids, we discovered
a new variation on chemical ligation that results in the
formation of a new peptide bond and leaves a residual
hydroxamic acid in the peptide chain. Previous chemical
ligation methods include the elegant work by Kent9 based
on a cysteine-mediated rearrangement (Scheme 1), the
Scheme 1. Cysteine-Based Native Chemical Ligation
(1) Parvathy, S.; Hussain, I.; Karran, E. H.; Turner, A. J.; Hooper, N.
M. Biochem. Soc. Trans. 1998, 26, S 242; Biochemistry 1998, 37, 1680.
(2) Connolly, P. J.; Wetter, S. K.; Beers, K. N.; Hamel, S. C.; Chen, R.
H. K.; Wachter, M. P.; Ansell, J.; Singer, M. M.; Steber, M.; Ritchie, D.
M.; Argentieri, D. C. Bioorg. Med. Chem. Lett. 1999, 9, 979. Yatabe, T.;
Kawai, Y.; Oku, T.; Tanaka, H. Chem. Pharm. Bull. 1998, 46, 966.
(3) Kim, D. H.; Jin, Y. H. Bioorg. Med. Chem. Lett. 1999, 9, 691.
(4) Groneberg, R. D.; Burns, C. J.; Morrissette, M. M.; Ullrich, J. W.;
Morris, R. L.; Darnborough, S.; Djuric, S. W.; Condon, S. M.; McGeehan,
G. M.; Labaudiniere, R.; Neuenschwander, K.; Scotese, A. C.; Kline, J. A.
J. Med. Chem. 1999, 42, 541. Kleinman, E. F.; Campbell, E.; Giordano, L.
A.; Cohan, V. L.; Jenkinson, T. H.; Cheng, J. B.; Shirley, J. T.; Pettipher,
E. R.; Salter, E. D.; Hibbs, T. A.; Dicapua, F. M.; Bordner, J. J. Med.
Chem. 1998, 41, 266.
(5) Moy, F. J.; Chanda, P. K.; Chen, J. M.; Cosmi, S.; Edris, W.;
Skotnicki, J. S.; Wilhelm, J.; Powers, R. Biochemistry 1999, 38, 7085.
(6) Joshi, B.; Li, L.; Zhu, Z. Y.; Wahl, S.; Tian, H.; Ben-Josef, E.; Taylor,
J. D.; Porter, A. T.; Tang, D. G. Cancer Res. 1999, 59, 4343. Barlaam, B.;
Koza, P.; Berriot, J. Tetrahedron 1999, 55, 7221. Li, L.; Zhu, Z. Y.; Joshi,
B.; Zhang, C.; Johnson, C. R.; Marnett, L. J.; Honn, K. V.; Crissman, J.
D.; Porter, A. T.; Tang, D. G. Anticancer Res. 1999, 19, 51, 60. Ghosh, P.;
Bag, S. P.; Sur, B.; Sur, P. Neoplasma 1998, 45, 68. Roberts, K. P.; Iyer,
R. A.; Prasad, G.; Liu, L. T.; Lind, R. E.; Hanna, P. E. Prostate 1998, 34,
92.
cysteine disulfide work by Kemp,10 the pseudoproline
methods of Tam,11 the oxime method as exemplified by
Rose,12 and the 6-nitro-2-hydroxybenzyl approach by Meu-
termans and Smythe.13 Chemical ligation has been utilized
as a key tool for syntheses of many biologically crucial
compounds such as lipopeptides,14 proteins,15 DNA strands
(9) Schno¨lzer, M.; Kent, S. B. H. Science 1992, 256, 221-225. Dawson,
P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776.
(10) Kemp, D. S.; Carey, R. I. J. Org. Chem. 1993, 58, 2216.
(11) Tam, J. P.; Miao, Z. J. Am. Chem. Soc. 1999, 121, 9013.
(12) Rose, K. J. Am. Chem. Soc. 1994, 116, 30.
(13) Meutermans, W. D. F.; Golding, S. W.; Bourne, G. T.; Miranda, L.
P.; Dooley, M. J.; Alewood, P. F.; Smythe, M. L. J. Am. Chem. Soc. 1999,
121, 9790.
(14) Melnyk, O.; Bossus, M.; David, D.; Rommens, C.; Grasmasse, H.
J. Pept. Res. 1998, 52, 180. Zhang, L. S.; Torgerson, T. R.; Liu, X. Y.;
Timmons, S.; Colosia, A. D.; Hawiger, J.; Tam, J. P. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 9184.
(7) Dimartino, M.; Wolff, C.; High, W.; Stroup, G.; Hoffman, S.; Laydon,
J.; Lee, J. C.; Bertolini, D.; Galloway, W. A.; Crimmin, M. J.; Davis, M.;
Davies, S. Inflammation Res. 1997. 46, 211.
(8) Holland, K. P.; Elford, H. L.; Bracchi, V.; Annis, C. G.; Schuster, S.
M.; Chakrabarti, D. Antimicrob. Agents Chemother. 1998, 42, 2456.
10.1021/ol005722+ CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/26/2000

and templates,16 oligonucleotides,17 enzyme inhibitors,18 and
HIV targets19 and for solid-phase syntheses of unprotected
peptides20 and glycopeptides.21
in 73% yield as a deep blue solid. Treatment of 3 with warm
toluene resulted in the formation of a mixture of the parent
hydroxamic acid 2b and the corresponding O-benzyl hy-
droxamic acid 4 (33% yield) (Scheme 3). Mechanistically,
The O-acyl hydroxamic acid substrates 1 were prepared
by converting Fmoc-glycine or Fmoc-alanine into the stable
acid chlorides with oxalyl chloride.22 Coupling to O-benzoyl
N-tert-butyl hydroxylamine with pyridine in refluxing ben-
zene gave the O-protected hydroxamic acid derivatives 1
(Gly 82%, Ala 96% yields). Removal of the Fmoc group
with dimethylamine resulted in rearrangement through a six-
membered transition state (Scheme 2).23 The identity of the
Scheme 3. Formation and Trapping of Acyl Nitroxide 3
Scheme 2. Hydroxamic Acid Based Chemical Ligation
acyl nitroxide 3 abstracts a benzylic hydrogen25 from toluene,
forming 1 equiv of hydroxamic acid 2b and 1 equiv of benzyl
radical. The benzyl radical is scavenged by unreacted
persistent acyl nitroxide 3 in a coupling reaction. In conjunc-
tion with stereoselectivity studies in our laboratories on the
coupling of prochiral radicals with chiral nitroxides,26 acyl
nitroxide 3 was trapped with 1-phenethyl radical generated
from ethylbenzene both at room temperature and at -78 °C
(Scheme 4). No stereoselectivity was observed; O-alkyl
resultant N-acyl amino hydroxamic acids 2 (Gly 88%, Ala
83% yields) was confirmed both by IR (peaks at 3200 and
1634 cm-1) and by visualization of the TLC by acidic ferric
chloride.24 Mild oxidation of 2b with saturated aqueous
potassium ferricyanide solution gave acyl nitroxide 3, isolated
(15) Dawson, P. E.; Churchill, M. J.; Ghadiri, R.; Kent, S. B. H. J. Am.
Chem. Soc. 1997, 119, 4325. Canne, L. E.; Bark, S. J.; Kent, S. B. H. J.
Am. Chem. Soc. 1996, 118, 5891. Hackeng, T. M.; Griffin, J. H.; Dawson,
P. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10068. Futaki, S.; Aoki, M.;
Ishikawa, T.; Kondo, F.; Asahara, T.; Niwa, M.; Nakaya, Y.; Yagami, T.;
Kitagawa, K. Bioorg. Med. Chem. 1999, 7, 187. Xu, R.; Cowburn, D.; Muir,
T. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 388.
Scheme 4. Nondiastereoselective Coupling of Optically
Active Acyl Nitroxide 3 with a Prochiral Radical
(16) Selvasekaran, J.; Turnbull, K. D. Nucl. Acids Res. 1999, 27, 624.
Kuznetsova, S. A.; Merenkova, I. N.; Kanevsky, I. E.; Shabarova, Z. A.;
Blumenfeld, M. Anti. Nucl. Acid Drug DeV. 1999, 9, 95. McPherson, M.;
Wright, M. C.; Lohse, P. A. Synlett 1999, 978. Dolinnaya, N. G.; Kuchumov,
A. R.; Shabarova, Z. A. Mol. Biol. 1998. 32, 273, 626. Dolinnaya, N. G.;
Shabarova, Z. A. Russ. Chem. Bull. 1996, 45, 1787. Futaki, S.; Ishikawa,
T.; Niwa, M.; Kitagawa, K.; Yagami, T. Bioorg. Med. Chem. 1997, 5, 1883.
Bordusa, F.; Ullmann, D.; Elsner, C.; Jakubke, H. D. Angew. Chem., Int.
Ed. Engl. 1997, 36, 2473. Severin, K.; Lee, D. H.; Martinez, J. A.; Ghadiri,
M. R. Chem. Eur. J. 1997, 3, 1017. Dolinnaya, N. G.; Merenkova, I. N.;
Shabarova, Z. A. Nucleosides Nucleotides 1994, 13, 2169.
(17) Ueno, Y.; Nakagawa, A.; Matsuda, A. Nucleosides Nucleotides 1998,
17, 283. James, K. D.; Ellington, A. D. Chem. Biol. 1997, 4, 595. Letsinger,
R. L.; Wu, T. F.; Elghanian, R. Nucleosides Nucleotides 1997, 16, 643. Li,
T. H.; Weinstein, D. S.; Nicolaou, K. C. Chem. Biol. 1997, 4, 209.
(18) Lu, W. Y.; Starovasnik, M. A.; Kent, S. B. H. FEBSLett. 1998,
429, 31. Baca, M.; Muir, T. W.; Schno¨lzer, M.; Kent, S. B. H. J. Am. Chem.
Soc. 1995, 117, 1881.
hydroxamic acid 5 was produced as a 1:1 mixture of
diastereoisomers, reflecting the conformational mobility of
acyclic acyl nitroxide 3.
The O to N acyl rearrangement process was then extended
to the formation of new peptide bonds. O-Acylation of tert-
butylhydroxylamine with N-acetyl glycine or alanine 6 under
coupling conditions (EDCI, CH2Cl2) yielded hydroxylamine
esters 7 (Table 1). A small amount of DMF was added when
necessary to achieve solubility of all reactants. No products
resulting from N-acylation of the tert-butylhydroxylamine
were observed. Treatment of hydroxylamine esters 7 with
(19) Liu, C. F.; Rao, C.; Tam, J. P. J. Am. Chem. Soc. 1996, 118, 307.
Weinberg, J.; Liao, H. X.; Torres, J. V.; Matthews, T. J.; Robinson, J.;
Haynes, B. F. AIDS Res. Hum. RetroViruses 1997, 13, 657.
(20) Canne, L. E.; Botti, P.; Simon, R. J.; Chen, Y.; Dennis, E. A.; Kent,
S B. H. J. Am. Chem. Soc. 1999, 121, 8720. Camarero, J. A.; Cotton, G. J.;
Adeva, A.; Muir, T. W. Int. J. Pept. Protein Res. 1998, 51, 303. Lelievre,
D.; Chabane, H.; Delmas, A. Tetrahedron Lett. 1998, 39, 9675. Walker,
M. A. Angew. Chem., Int. Ed. Engl. 1997, 36, 1069. Klinguer, C.; Melnyk,
O.; Loing, E.; Grasmasse, H. Tetrahedron Lett. 1996, 37, 7259. Lu, W. Y.;
Qasim, M. A.; Kent, S. B. H. J. Am. Chem. Soc. 1996, 118, 8518. Liu, C.
F.; Rao, C.; Tam, J. P. J. Am. Chem. Soc. 1996, 118, 307. Liu, C. F.; Tam,
J. P. J. Am. Chem. Soc. 1994, 116, 4149.
(24) For recent application of ferrous complexes of hydroxamic acids
in DNA cleavage, see: Hashimoto, S.; Nakamura, Y. Chem. Pharm. Bull.
1998, 46, 1941.
(25) Perkins, M. J.; Berti, C.; Brooks, D. J.; Grierson, L.; Grimes, J.
A.-M.; Jenkins, T. C.; Smith, S. L. Pure Appl. Chem. 1990, 62, 195-200.
Berti, C.; Grierson, L.; Grimes, J. A.-M.; Perkins, M. J.; Terem, B. Angew.
Chem., Int. Ed. Engl. 1990, 29, 653.
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A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684.
(22) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161.
(23) While this manuscript was being completed, a paper by Phanstiel
appeared describing the identical rearrangement in an N-benzoyloxy-leucine
derivative: Wang, L.; Phanstiel IV, O. J. Org. Chem. 2000, 65, 1442.
(26) Braslau, R.; Burrill, L. C.; Mahal, L. K.; Wedeking, T. Angew.
Chem., Int. Ed. Engl. 1997, 36, 237. Braslau, R.; Burrill, L. C.; Chaplinski,
V.; Howden, R. H.; Papa, P. W. Tetrahedron: Asymmetry 1997, 8, 3209.
Braslau, R.; Naik, N.; Zipse, H., submitted for publication.
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Org. Lett., Vol. 2, No. 10, 2000

formation of rearranged product 10. In the preparation of
10a, we found that the N-hydroxy peptide was easily purified
by removal of the dibenzofulvene byproduct using trituration
of the residue with benzene followed by evaporation. Thus,
the ligation of Ac-glycine from O-acyl hydroxamic acid 9a
gave Ac-Gly-Ala-hydroxamic acid 10a in 75% isolated yield
following column chromatography, and ligation of the
dipeptide Z-Leu-Gly fragment gave Z-Leu-Gly-Ala-hydrox-
amic acid 10e in 24% isolated yield (purification by flash
column followed by HPLC). This hydroxamic acid based
rearrangement, like the cysteine rearrangement, is effective
due to the good leaving group properties of the hydroxamic
acid conjugate base, similar to that of thiolate. The pKa values
of alkyl hydroxamic acids27 are found at approximately 9.4,
whereas the thiol of cysteine has a pKa of 8.3.
Table 1. Preparation of O-Acyl Hydroxamic Acid Peptide
Substrates
compd
R1
Ac
BzGly
BocGly
ZLeu
Bz
Bz
ZGly
Ac
R2
% yield
7a
7b
7c
7d
7e
7f
7g
9a
9d
9e
H
H
H
H
42
71
75
62
84
63
95
49
24
27
H
Me
Me
H
H
H
We have been working on extending this methodology to
the formation of longer peptides in which the tert-butyl amino
group of precursor 7 is an N-hydroxy amino acid, derived
by the oxaziridine route.28
Bz
ZLeu
The resulting N-hydroxy peptides available by this new
ligation method are expected to be valuable as peptidomi-
metics and as metalloenzyme inhibitors by virtue of their
strong metal chelation properties. This new ligation meth-
odology adds to the existing arsenal of mild experimental
conditions for peptide bond formation.
Fmoc-amino acid chlorides 8 gave N-acylated products 9.
This reaction tended to be sluggish due to the sterically
hindered neopentyl hydroxylamine nitrogen in 7 and thus
required use of the preformed acid chlorides, and for
particularly hindered cases (9d and 9e), the use of toluene
at reflux in place of THF.
The key ligation step was effected by deprotection of the
Fmoc group under mild conditions (Table 2). Thus, brief
exposure of ester 9 to dimethylamine in THF gave immediate
Acknowledgment. We thank the National Science Foun-
dation (CHE-9527647) and the National Science Foundation
REU program (CHE-9300572) for providing financial sup-
port.
Table 2. Ligation To Form Di- and Tripeptides
Supporting Information Available: Experimental pro-
cedures with spectral data for compounds 1a 1b, 2a, 2b, 3,
4, 5, 7a-f, 9a, and 10a. This material is available free of
charge via the Internet at http://pubs.acs.org.
compd
R
% yield
OL005722+
10a
10d
10e
Ac
Bz
ZLeu
75
70
24
(27) Wise, W. M.; Brandt, W. W. J. Am. Chem. Soc. 1955, 77, 1058.
(28) Polonski, T.; Chimiak, A. Tetrahedron Lett. 1974, 28, 2453.
Hajipour, A. R.; Pyne, S. G. J. Chem. Res., Synop. 1992, 388.
Org. Lett., Vol. 2, No. 10, 2000
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