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

Article abstract of DOI:10.1021/ol047880x

(Chemical Equation Presented) The protected hydroxyethylene dipeptide isostere of Gln-Arg and the tripeptide derivative 1 were synthesized as components of potential peptidase inhibitors.

Full text of DOI:10.1021/ol047880x

ORGANIC
LETTERS
2004
Vol. 6, No. 25
4779-4782
Synthesis of a Tripeptide Derivative
Containing the Gln-Arg Hydroxyethylene
Dipeptide Isostere
|
Matthias Brewer,†,‡ Clint A. James,†,§ and Daniel H. Rich*,†,
Department of Chemistry and School of Pharmacy, UniVersity of Wisconsin-Madison,
Madison, Wisconsin 53706
dhrich@wisc.edu
Received October 13, 2004
ABSTRACT
The protected hydroxyethylene dipeptide isostere of Gln-Arg and the tripeptide derivative 1 were synthesized as components of potential
peptidase inhibitors.
The botulinum family of neurotoxins (BoNT-A through G)
are among the most lethal toxins known with a mouse LD50
equal to 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 metal-
lopeptidases 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
peptide2 and for BoNT-A a 17-mer peptide.3 The native
substrate for BoNT-A is SNAP-25, and the scissile bond lies
between a Gln and Arg residue. During the course of an
investigation aimed at developing BoNT-A inhibitors, we
became interested in synthesizing the Gln-Arg hydroxyeth-
ylene isostere as a possible transition-state mimetic inhibitor.
The hydroxyethylene isostere is defined as a dipeptide unit
in which the central amide bond has been replaced by a CH2-
CH(OH) group. First synthesized as a potential transition-
state analogue inhibitor of renin,4-6 the hydroxyethylene
moiety (HE) has also been applied with success to develop
HIV protease7 and â-secretase8 inhibitors. Although the
synthesis of hydroxyethylene isosteres has received consider-
able attention in the literature, most HE analogues synthe-
sized to date contain unfunctionalized side chains. Herein
(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.
(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.
(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.
* To whom correspondence should be addressed.
† Department of Chemistry.
‡ Present Address: University of California-Irvine, Department of
Chemistry, 516 Rowland Hall, Irvineb CA, 92697.
§ Present Address: Bristol-Myers Squibb Pharmaceutical Research
Institute, 100 Boulevard de l’Industrie, Candiac, Quebec, Canada J5R 1J1.
| School of Pharmacy.
(1) Montecucco, C.; Schiavo, G. Quart. 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.
10.1021/ol047880x CCC: $27.50
© 2004 American Chemical Society
Published on Web 11/12/2004

Scheme 1
Scheme 3
we report the synthesis of the fully functionalized Gln-Arg
hydroxyethylene isostere as a tripeptide derivative.
Our synthetic approach to Asn-Gln-HE-Arg 1 was based
on a Horner-Wadsworth-Emmons strategy (Scheme 1).9
In a retrosynthetic manner, the Gln-HE-Arg portion of
derivative 1 would be derived from lactone 2 (Scheme 1).
Clearing two stereocenters from lactone 2 reveals the
unsaturated 1,4-dicarbonyl derivative 3. This in turn could
be derived from a Horner-Wadsworth-Emmons reaction
that, in a single step, provides the entire carbon framework
of the desired inhibitor. The compounds required for this
reaction would be Gln-derived ketophosphonate 4 and an
R-ketoester of type 5.
reaction. Of these, only the siloxy derivative 11 provided
good yields of the desired product in a reproducible manner.
The synthesis of siloxy 11 is shown in Scheme 3. Acid-
catalyzed methanolysis of δ-valerolactone12 afforded the
corresponding hydroxyester, which was directly silyated to
afford silyl ether 9 in 76% yield over two steps. Treating
the potassium enolate of ester 9 with Davis oxaziridine13
afforded R-hydroxyester 10 in a satisfactory yield of 56%.
Oxidation of the hydroxyl group with Dess-Martin perio-
dinane afforded R-ketoester 11 in high yield (96%) and
excellent purity.14,15
Treatment of the sodium salt of phosphonate 4 with
ketoester 11 provided the (Z)-isomer of desired olefin 12 in
80% yield with none of the (E)-isomer observed. This
condensation was strongly temperature dependent; decom-
position occurred when the temperature was raised above
-30 °C. The next step in the synthesis was reduction of the
ketone functionality. Treating ketone 12 with NaBH4 at -30
°C and allowing the reaction to warm to room temperature
produced an inseparable mixture of diastereomeric lactones
13a and 13b in a 2:3 ratio.9b Interestingly, the ratio of
diastereomers obtained changes to 2:1 favoring diastereomer
13a when the reaction was instead maintained at -30 °C
and quenched at that temperature with a saturated aqueous
solution of NH4Cl. We hypothesize that on warming to room-
temperature, adventitious methoxide causes epimerization of
the allylic center, while at low-temperature, epimerization
does not occur. If so, then lactone 13b appears to be the
thermodynamically favored product.
The synthesis of ketophosphonate 4 is shown in Scheme
2. BocGln(Trt)OH (6)10 was reacted with TMS-diazo-
Scheme 2
methane to form methyl ester 7 in quantitative yield. Claisen
condensation of this ester with lithio dimethyl methylphos-
phonate afforded the desired ketophosphonate 4 in 87%
yield.11
With the phosphonate in hand, several keto-esters were
evaluated as partners for the Horner-Wadsworth-Emmons
Attempts were made to control the product ratio using
bulkier hydride sources.16 Interestingly, DIBAL favored
formation of 13b over 13a (6:1 ratio), while LS-Selectride
and LiAlH(OtBu)3 both favored 13a (11.5:1 and 10:1, 13a
to 13b, respectively). Unfortunately, reductions with DIBAL
and LS-Selectride were both low-yielding reactions, provid-
ing only 37 and 30% yields of product, respectively.
(9) (a) Litera, J.; Budesinsky, M.; Urban, J.; Soucek, M. Collect. Czech.
Chem. Commun. 1998, 63, 231. (b) Chakravarty, P. K.; Delaszlo, S. E.;
Sarnella, C. S.; Springer, J. P.; Schuda, P. F. Tetrahedron Lett 1989, 30,
415. (c) Plata, D. J.; Leanna, M. R.; Morton, H. E. Tetrahedron Lett 1991,
32, 3623. (d) Captain, L. F.; Xia, X. Y.; Liotta, D. C. Tetrahedron Lett
1996, 37, 4293.
(12) Huckstep, M.; Taylor, R. J. K.; Caton, M. P. L. Synthesis 1982,
881.
(13) Vishwakarma, L. C.; Stringer, O. D.; Davis, F. A. Org. Synth. 1988,
66, 203.
(10) Sieber, P.; Riniker, B. Tetrahedron Lett 1991, 32, 739.
(11) Chakravarty, P. K.; Combs, P.; Roth, A.; Greenlee, W. J. Tetrahe-
dron Lett. 1987, 28, 611.
(14) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
(15) Ireland, R. E.; Liu, L. B. J. Org. Chem. 1993, 58, 2899.
(16) Nishi, T.; Kataoka, M.; Morisawa, Y. Chem. Lett. 1989, 1993.
4780
Org. Lett., Vol. 6, No. 25, 2004

Scheme 4
Scheme 5
Reduction with LiAlH(OtBu)3 provided good product yield
(84%), but the high diastereomeric ratio was not reproducible
on scale-up.
The stereochemical identification of these compounds was
initially established by conversion to the corresponding
1
oxazolidinones 14a and 14b, which have characteristic H
NMR spectra.17 Comparison to analogous compounds in the
literature allowed for identification of diastereomer 13a as
the (4R,5S)-lactone, and 13b as the (4S,5S)-lactone.18 This
initial stereochemical assignment was confirmed by X-ray
crystallography of the enantiomer of 15a.19 This also proved
correct our initial assumption that hydrogenation of the
unsaturated lactones occurs on the face opposite the bulky
substituent at the C-4 position to provide the cis-lactone
products.
successful use of an azide as a protected amine equivalent
in our previous synthesis of the Phe-Arg HE20 made this an
attractive strategy for the current synthesis as well. Alcohol
16 was converted to azide 17 in 72% yield by a one-pot
mesylation-displacement reaction (Scheme 5).21
The N-Boc protecting group of 17 was converted to the
Cbz-protecting group in a one-pot procedure developed by
Sakaitani and Ohfune22 to provide 18 in 72% yield (Scheme
6). Unfortunately, lactone opening of this compound resulted
in a significant amount of the undesired oxazolidinone 19.
Scheme 6
Hydrogenation of the inseparable mixture of 13a and 13b
occurs in a completely stereospecific manner, providing a
separable mixture of only two diastereomeric lactones 15a
and 15b. The preferred catalyst for this transformation was
found to be Pt(IV) oxide, as Pd/C resulted in varying degrees
of silyl ether cleavage and no conditions could be found that
allowed separation of the diasteromeric free alcohols.
The lability of the silyl ether was also noted during HPLC
studies of lactone 15a. Incubation of 15a in a 1:1 mixture
of methanol and an HPLC eluent composed of acetonitrile
(70%), water (30%), and TFA (0.1%) provided clean
cleavage of the silyl ether. This proved to be an exceptionally
mild and convenient method for alcohol deprotection and
was thereafter used as the method of choice for the
conversion of lactone 15a to alcohol 16 (Scheme 5). The
As the final goal of this synthesis was to form a 17-mer
peptide containing the Gln-Arg dipeptide analogue, the next
amino acid in the 17-mer sequence was added at this point.
TBSOTf-mediated Boc deprotection of 17 followed by
EDCI/HOBT-mediated peptide coupling provided Asn-
derived tripeptide 20 in 94% yield over two steps (Scheme
(17) Rich, D. H.; Sun, E. T. O.; Ulm, E. J. Med. Chem. 1980, 23, 27.
(18) Futagawa, S.; Inui, T.; Shiba, T. Bull. Chem. Soc. Jpn. 1973, 46,
3308.
(19) X-ray crystallographic data for compound ent-15a is included in
Supporting Information.
(20) Brewer, M.; Rich, D. H. Org Lett 2001, 3, 945.
(21) Mattingly, P. Synthesis 1990, 366.
(22) (a) Sakaitani, M.; Ohfune, Y. Tetrahedron Lett 1985, 26, 5543. (b)
Sakaitani, M.; Ohfune, Y. J. Org. Chem. 1990, 55, 870. (c) Sakaitani, M.;
Ohfune, Y. J. Am. Chem. Soc. 1990, 112, 1150.
Org. Lett., Vol. 6, No. 25, 2004
4781

under basic conditions to provide the desired product in near
quantitative yield. The secondary alcohol was subsequently
protected as the TBS-silyl ether to provide acid 21 in 80%
yield over two steps.
Scheme 7
Converting azide 21 to guanidine 22 was successfully
achieved in 56% isolated yield via the Staudinger reduction-
based protocol previously described by us.20 The synthesis
was finished by removing the Cbz-protecting group via
hydrogenolysis over Pd(OH)2 on carbon and reprotecting the
resulting primary amine as the Fmoc derivative. In this
manner, the desired Asn-Gln-Arg HE derivative 1 was
obtained in 31% yield over two steps in a form suited for
peptide synthesis.
The protected Gln-Arg HE analogues 1 and 22 reported
here contain two highly functionalized natural amino acid
side chains and are the most complex HE derivatives
synthesized to date. Several of the intermediates in this
synthesis are versatile building blocks that may prove to be
useful for the synthesis of other side chain-modified HE
analogues. Incorporation of protected HE analogue 1 into
the 17-mer peptide substrate of BoNT-A and the bioactivity
of the resulting substrate based inhibitor will be reported
elsewhere.
Acknowledgment. We thank Dr. Ilia A. Guzei for
obtaining the X-ray crystal structure of compound ent-15a,
Dr. Martha Vestling for obtaining the mass spectra, and Drs.
Charlie Fry and Thomas Stringfellow for their assistance in
obtaining NMR spectra. This work was supported by a
research grant from NIHGMS (GM 59 956) and instrumen-
tation grants NSF CHE-9208463 and NIH 1 S1O RR0 8389-
01.
7). Lactone opening to provide the free carboxylic acid
initially proved to be problematic, as a significant amount
of the reverse reaction was found to occur during workup.
Carefully monitoring the pH of the reaction during the
quench step was unsuccessful and formation of lactone 20
was still observed. Ultimately, an optimized workup proce-
dure was developed that allowed isolation of the free acid
Supporting Information Available: Detailed experi-
mental procedures for the synthesis and characterization data
of compounds 1, 4, 7, 9-13, 15-18, and 20-22 and
crystallographic data for compound ent-15a. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL047880X
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Org. Lett., Vol. 6, No. 25, 2004