Synthesis of a tripeptide derivative containing the Phe-Arg hydroxyethylene dipeptide isostere.

Article abstract of DOI:10.1021/ol015612i

The protected hydroxyethylene dipeptide isostere of Phe-Arg and the tripeptide derivative 1 were synthesized as components of potential peptidase inhibitors. Key to the success of these syntheses is selective rhodium-catalyzed hydroboration in the presenc

Full text of DOI:10.1021/ol015612i

ORGANIC
LETTERS
2001
Vol. 3, No. 6
945-948
Synthesis of a Tripeptide Derivative
Containing the Phe-Arg Hydroxyethylene
Dipeptide Isostere
,†,§
Matthias Brewer† and Daniel H. Rich*
Department of Chemistry and School of Pharmacy, UniVersity of WisconsinsMadison,
Madison, Wisconsin 53706
dhrich@facstaff.wisc.edu
Received January 24, 2001
ABSTRACT
The protected hydroxyethylene dipeptide isostere of Phe-Arg and the tripeptide derivative 1 were synthesized as components of potential
peptidase inhibitors. Key to the success of these syntheses is selective rhodium-catalyzed hydroboration in the presence of a readily reduced
lactone. A convenient one-pot conversion of azides to diprotected guanidines was developed on the basis of the Staudinger reaction.
The botulinum family of neurotoxins (BoNT-A through G)
are among the most lethal toxins known with a mouse LD50
) 0.1-0.5 ng/kg. Upon metabolic activation, the toxins
produce zinc metalloproteases that cleave proteins involved
in the release of acetylcholine at the neuromuscular junction,
resulting in muscular paralysis.1 The BoNT metallopeptidases
are among the most selective peptidases yet identified as
judged by their unusually large substrate size. The minimum
cleavable substrate for BoNT-B is a 35-mer peptide,2 and
for BoNT-A a 17-mer peptide.3 During the course of an
investigation aimed at developing BoNT-A inhibitors, we
became interested in synthesizing the Phe-Arg hydroxyeth-
ylene isostere as a possible transition-state mimetic inhibitor.
The hydroxyethylene isostere, first synthesized for inhibition
of renin,4-6 has also been applied with success to the
development of HIV protease7 and â-secretase8 inhibitors.
Although the synthesis of hydroxyethylene isosteres (HE)
has received considerable attention in the literature, most HE
synthesized to date contain relatively unfunctionalized side
chains. Herein we report the first synthesis of the fully
functionalized Phe-Arg hydroxyethylene isostere as a tri-
peptide derivative.
The synthesis began from known lactone 29 (Scheme 1)
which was synthesized as described previously.10 Alkylation
of 2 with allyl bromide gave a mixture of diastereomers
(5) Holladay, M. W.; Rich, D. H. Tetrahedron Lett. 1983, 24, 4401.
(6) Greenlee, W. J. Med. Res. ReV. 1990, 10, 173.
(7) (a) Huff, J. R. J. Med. Chem. 1991, 34, 2305. (b) Vacca, J. P.; Dorsey,
B. D.; Schleif, W. A.; Levin, R. B.; McDaniel, S. L.; Darke, P. L.; Zugay,
J.; Quintero, J. C.; Blahy, O. M.; Roth, E.; Sardana, V. V.; Schlabach, A.
J.; Graham, P. I.; Condra, J. H.; Gotlib, L.; Holloway, M. K.; Lin, J.; Chen,
I.-W.; Vastag, K.; Ostovic, D.; Anderson, P. S.; Emini, E. A.; Huff, J. R.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4096.
† Department of Chemistry.
§ School of Pharmacy.
(8) (a) Ghosh, A. K.; Shin, D.; Downs, D.; Koelsch, G.; Lin, X.;
Ermolieff, J.; Tang, J. J. Am. Chem. Soc. 2000, 122, 3522. (b) Shearman,
M. S.; Beher, D.; Clarke, E. E.; Lewis, H. D.; Harrison, T.; Hunt, P.; Nadin,
A.; Smith, A. L.; Stevenson, G.; Castro, J. L. Biochemistry 2000, 39, 8698.
(c) Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.; Ghosh, A. K.;
Zhang, X. C.; Tang, J. Science 2000, 290, 150.
(1) Montecucco, C.; Schiavo, G. Q. ReV. Biophys. 1995, 28, 423.
(2) Shone, C. C.; Roberts, A. K. Eur. J. Biochem. 1994, 225, 263.
(3) Schmidt, J. J.; Bostian, K. A. J. Protein Chem. 1995, 14, 703.
(4) (a) Szelke, M.; Jones, D. M.; Hallett, A. European Patent Application
EP 45665, 1982; Chem. Abstr. 1982, 97, 39405p. (b) Szelke, M.; Jones, D.
M.; Atrash, B.; Hallett, A.; Leckie, B. J. Proc. Am. Pept. Symp. 8th 1983,
579.
(9) Fray, A. H.; Kaye, R. L.; Kleinman, E. F. J. Org. Chem. 1986, 51,
4828.
10.1021/ol015612i CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/23/2001

Scheme 1a
Scheme 2a
a (a) LDA, THF, -78 °C, 30 min; allyl bromide, DMPU, -78
°C, 15 min, then -50 °C, 1.5 h (62%); (b) Rh(Ph3P)3Cl, cate-
cholborane, THF, 0 °C, then rt, 30 min; 30% H2O2, THF, EtOH,
pH 7.2 buffer, rt, overnight (70%).
a (a) MsCl, TEA, toluene, 0 °C to rt, 15 min; NaN3, Bu4NBr,
H2O, reflux 2 h (90%); (b) 4 N HCl, dioxane; (c) Cbz-Asn(Trt)-
OH, HOBT, EDCI, DIEA, DMF, 0 °C to rt, overnight (89%, 2
steps); (d) 1 M LiOH, dioxane; (e) TBSCl, imidazole, DMF, rt, 36
h; MeOH, 4 h (74%, 2 steps).
(5.3:1 trans to cis) that were separated by flash column
chromatography to give the desired trans lactone 3 in 62%
yield.
Cleavage of the Boc group from 6a with 4 N HCl in dioxane,
followed by reprotection as the Cbz derivative, produced 6b
in 88% yield over two steps. Solvent diffusion crystallization
of 6b from hexane/ethyl acetate produced X-ray diffraction
quality crystals, which allowed for the unambiguous assign-
ment of this advanced intermediate in which all of the chiral
centers have been set (Figure 1).
Conversion of lactone 3 to alcohol 4 by hydroboration
followed by oxidation was initially problematic. Use of
disiamylborane, 9-BBN, dicyclohexylborane, and (S)-alpine-
borane gave only low, variable yields of alcohol 4 due to
competitive reduction of the γ-lactone to its corresponding
hemiacetal 5.11 Attempts to suppress the formation of this
side product by controlling the reaction temperature and the
amount of borane used were unfruitful.
This problem was circumvented by using rhodium-
catalyzed hydroboration conditions12 followed by neutral
oxidation. Under these conditions, olefin 3 was converted
to alcohol 4 in 70% yield with no observable formation of
5.
Alcohol 4 was readily converted to azide 6a in 90% yield
in a one-pot mesylation-displacement reaction (Scheme 2).13
Figure 1. ORTEP plot of compound 6b.
(10) DeCamp, A. E.; Kawaguchi, A. T.; Volante, R. P.; Shinkai, I.
Tetrahedron Lett. 1991, 32, 1867.
(11) (a) Brown, H. C.; Bigley, D. B. J. Am. Chem. Soc. 1961, 83, 486.
(b) Kohn, P.; Samaritano, R. H.; Lerner, L. M. J. Am. Chem. Soc. 1965,
87, 5475. (c) Brown, H. C.; Bigley, D. B.; Arora, S. K.; Yoon, N. M. J.
Am. Chem. Soc. 1970, 92, 7161.
(12) (a) Ma¨nnig, D.; No¨th, H. Angew. Chem., Int. Ed. Engl. 1985, 24,
878. (b) Burgess, K.; Ohlmeyer, M. J. Chem. ReV. 1991, 91, 1179. (c) Evans,
D. A.; Fu, G. C.; Hoveyda, A. H. J. Am. Chem. Soc. 1992, 114, 6671. (d)
Evans, D. A.; Fu, G. C.; Anderson, B. A. J. Am. Chem. Soc. 1992, 114,
6679.
Compound 6b, however, was not a viable synthetic
intermediate as all attempts to convert it to the free acid via
treatment with LiOH resulted in significant formation of the
side product oxazolidinone 7. Attempts to suppress oxazo-
lidinone formation by controlling the reaction temperature
and amount of hydroxide used failed.
(13) Mattingly, P. G. Synthesis 1990, 366.
Org. Lett., Vol. 3, No. 6, 2001
946

indicated that reagent 11 was stable in these reduction
conditions, and these conditions were chosen for the desired
overall transformation.
In the event, triphenylphosphine was added to a room
temperature mixture of 6a, 11, and H2O in THF (Scheme
3). Evolution of nitrogen commenced immediately, and the
reaction was allowed to stir overnight at room temperature.
The mixture was concentrated in vacuo and the residue
purified by flash column chromatography to yield 70% of
the desired product 10a.
Replacement of the N-terminal protecting group with Cbz
had little effect on the reaction yield. Compound 6b was
successfully converted to its corresponding guanidine 10b
under the same reaction conditions in 71% yield.
The N-Fmoc protecting group, however, is not stable to
these reaction conditions. When 6c was used as the starting
azide, the desired product 10c was only recovered in 20%
yield and significant Fmoc cleavage was apparent by TLC.
This is presumed to be due to the basic nature of the
intermediate phosphazene.
As our first use of the hydroxyethylene isostere will be to
incorporate it into a peptide, the synthetic approach was
altered to include the next N-terminal amino acid in the
native sequence (Scheme 2).
Deprotection of 6a followed by an EDCI/HOBT-mediated
peptide coupling with the Z(Trt)-diprotected derivative of
asparagine provided 8 in 89% yield over two steps. In this
case, lactone opening with LiOH proceeded cleanly and the
newly formed secondary alcohol was protected as the TBS
ether 9 in 74% yield over two steps.
Attempts were first made to convert azide 6a into the
diprotected guanidine 10a (Scheme 3). However, reduction
Surprisingly, when azide 9 was treated according to the
conditions in Scheme 3, none of the desired guanidine 12
was obtained. Instead, the major product was identified as
the stable betaine 13 in which the phosphazene has depro-
tonated the carboxylic acid. This highly polar compound was
isolated by flash column chromatography (85:15 CH2Cl2/
MeOH) in 85% yield.
Scheme 3a
a (a) 11, Ph3P, H2O, THF, rt, overnight.
of the azide to the corresponding primary amine via catalytic
hydrogenation (10% or 5% Pd/C with 1 atm of H2) or
tin(II) chloride consistently gave only complex reaction
mixtures and low yields of the desired amine, presumably
due to an intramolecular (or intermolecular) attack of the
newly formed amine on the lactone carbonyl. To minimize
these possible side reactions, a one-step reduction/guanidation
procedure was envisioned which would eliminate the need
to isolate the free amine. The bis-Boc carboxamidine 1114
was chosen as the electrophilic guanidation reagent for this
transformation. However, this reagent was found to be
incompatible with the reduction methods thus far described.
The betaine structure is further supported by 31P NMR in
which the phosphorus of 13 displays a chemical shift
consistent with know betaines17 (Figure 2, Spectrum A). The
The Staudinger reaction is a mild conversion of azides to
phosphazenes via treatment with phosphines.15 While phos-
phazenes display a wide range of reactivities, they are readily
hydrolyzed to the corresponding primary amine and phos-
phine oxide in the presence of water.16 Preliminary studies
Figure 2. 31P NMR spectra of betaine 13 in THF-d8. Spectrum A
peak assignments: (a) 13, (b) Ph3PO, (c) Ph3P. Spectrum B peak
assignments: (a) Ph3PO, (b) 14, (c) Ph3P.
Org. Lett., Vol. 3, No. 6, 2001
947

by flash column chromatography and crystallization. Re-
moval of the Cbz group from 12 via hydrogenolysis,
followed by protection as the Fmoc derivative, produced the
desired final product 1 in 71% yield over two steps (Scheme
4).
Scheme 4a
The Phe-Arg HE (1) reported here is the first HE to contain
a highly functionalized natural amino acid side chain and is
the most complex HE synthesized to date. Several of the
intermediates in this synthesis are versatile building blocks
that may prove useful for the synthesis of other side chain
modified HE analogues. The simple one-pot conversion of
azides to diprotected guanidines takes advantage of the mild
conditions associated with the Staudinger reduction, which
allows azides to be reduced to free amines in the presence
of guanidation reagent 11. The protecting group scheme
employed in this synthesis should render tripeptide derivative
1 amendable to solid-phase peptide synthesis.
Acknowledgment. We thank Dr. Douglas Powell for
obtaining the X-ray crystal structure of compound 6b, Dr.
Martha Vestling for obtaining the mass spectra, and Dr.
Charlie Fry for his assistance in obtaining NMR spectra. This
work was supported by a research grant from NIHGMS (GM
59 956) and instrumentation grants NSF CHE-9208463 and
NIH 1 S1O RR0 8389-01.
a (a) 11, Ph3P, 0.5 M LiOH, THF, rt, 48 h (78%); (b) H2 (1 atm),
10% Pd/C, MeOH, acetic acid. (c) Fmoc-O-succinimide, 10%
Na2CO3, dioxane, rt (69%, 2 steps).
Supporting Information Available: Detailed experi-
mental procedures for the synthesis and characterization of
compounds 1, 3, 4, 6a, 8, 9, and 12. This material is available
free of charge via the Internet at http://pubs.acs.org.
31P NMR spectrum of 14 is provided for comparison (Figure
2, Spectrum B) and shows the significant difference in
chemical shifts between betaines and phosphazenes.
Treatment of 13 with LiOH in dioxane produced tri-
phenylphosphine oxide and the corresponding free amine.
On the basis of these findings, the reaction conditions were
altered to include 1 M LiOH, and the reaction time was
increased to 2 days. Under these conditions, azide 9 is
converted to guanidine 12 in 62% yield after purification
OL015612I
(15) (a) Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F. Tetrahedron
1981, 37, 437. (b) Barluenga, J.; Palacios, F. Org. Prep. Proced. Int. 1991,
23, 1.
(16) (a) Vaultier, M.; Knouzi, N.; Carrie, R. Tetrahedron Lett. 1983,
24, 763. (b) Knouzi, N.; Vaultier, M.; Carrie, R. Bull. Soc. Chim. Fr. 1985,
815.
(14) (a) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Tetrahedron Lett.
1993, 34, 3389. (b) Drake, B.; Patek, M.; Lebl, M. Synthesis 1994, 579.
(17) Garcia, J.: Urpi, F.; Vilarrasa, J. Tetrahedron Lett. 1984, 25, 4841.
948
Org. Lett., Vol. 3, No. 6, 2001