Solid phase synthesis of hydroxamate peptides for histone deacetylase inhibition

Article abstract of DOI:10.1016/j.tetlet.2012.10.113

An orthogonal protecting group strategy was devised to synthesize hydroxamic acid containing peptides for biomimetic histone deacetylase (HDAC) inhibition. The basic building block was a protected aminosuberic acid (Asu) derivative bearing a protected hydroxamate in the side-chain, related closely to HDAC inhibitors that are transition-state analogs of acetyllysine. These inhibitors include suberoylanilide hydroxamic acid (SAHA), currently being used to treat a variety of human cancers. This strategy was employed to synthesize a series of nonameric peptides related to actual HDAC substrates, derived from known sites of acetylation/deacetylation on the N-terminal tails of the histone core proteins H2A, H2B, H3, and H4. In each case the lysine residue was replaced by a hydroxamate-bearing side chain, to mimic the endogenous site of deacetylation. Mass spectrometry and high performance liquid chromatography (HPLC) confirmed the success of automated solid-phase synthesis. These results suggest facile synthesis of a new class of HDAC inhibitors that may have enhanced selectivity for specific HDAC isoforms.

Full text of DOI:10.1016/j.tetlet.2012.10.113

Tetrahedron Letters 54 (2013) 151–153
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Solid phase synthesis of hydroxamate peptides for histone deacetylase
inhibition
David M. Wilson a, , Lisa N. Silverman b, Markus Bergauer b, Kayvan R. Keshari a
⇑
a Department of Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, CA 94143, USA
b Department of Chemistry, Columbia University, New York, NY 10027, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An orthogonal protecting group strategy was devised to synthesize hydroxamic acid containing peptides
for biomimetic histone deacetylase (HDAC) inhibition. The basic building block was a protected amino-
suberic acid (Asu) derivative bearing a protected hydroxamate in the side-chain, related closely to HDAC
inhibitors that are transition-state analogs of acetyllysine. These inhibitors include suberoylanilide
hydroxamic acid (SAHA), currently being used to treat a variety of human cancers. This strategy was
employed to synthesize a series of nonameric peptides related to actual HDAC substrates, derived from
known sites of acetylation/deacetylation on the N-terminal tails of the histone core proteins H2A, H2B,
H3, and H4. In each case the lysine residue was replaced by a hydroxamate-bearing side chain, to mimic
the endogenous site of deacetylation. Mass spectrometry and high performance liquid chromatography
(HPLC) confirmed the success of automated solid-phase synthesis. These results suggest facile synthesis
of a new class of HDAC inhibitors that may have enhanced selectivity for specific HDAC isoforms.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 26 September 2012
Revised 23 October 2012
Accepted 25 October 2012
Available online 2 November 2012
Keywords:
Histone deacetylase
Solid phase
Peptide
Hydroxamic acid
Cancer
The reversible acetylation of histones has been shown to play a
critical role in transcriptional regulation.1,2 This process is regu-
lated by a number of nucleosome remodeling complexes and by
post translational modifications to the core histones them-
selves,3–5 a process regulated by the competing enzymatic activi-
ties of histone acetyltransferases (HATs) and histone deacetylases
(HDACs).6 The targets of these enzymes are the N-terminal do-
mains of all four core histone types, with an additional two
C-terminal domains found on the C-termini of the histone 2As. It
has been speculated that the specific patterns of post-translational
events occurring at these sites comprise a code utilized in the
recruitment of particular transcriptional complexes.7
Inhibitors of HDACs represent a promising modality for the
treatment of human cancers, with several compounds in the clinic
most notably suberoylanilide hydroxamic acid (SAHA), a zinc che-
lator.8–10 HDAC inhibitors have shown to lead to cell cycle arrest,
differentiation and/or apoptosis in tumor cells. Because of the zinc
chelating properties of hydroxamic acids, considerable effort has
been made to synthesize hydroxamic acid containing peptides as
inhibitors of HDAC and other zinc metalloenzymes.11–13 Efforts to
improve the potency of SAHA-related inhibitors were facilitated
by crystallographic studies of histone deacetylase-like protein
(HDLP), a bacterial homolog of human HDAC1, with bound trichos-
tatin A (TSA) or SAHA.14 These studies revealed that chelation of
zinc in the catalytic pocket is the primary mechanism of inhibition,
and suggested a ‘biomimetic’ approach to inhibitor design,
whereby the actual HDAC substrate (acetylated lysine) is used to
construct related inhibitors.15 This was accomplished via introduc-
tion of a chiral center using (L)-aminosuberic acid (Asu), leading to
inhibitors that mimic the transition state for the hydrolysis of
acetyllysine, as depicted in Figure 1.16
In this Letter we have extended this biomimetic approach to
peptide-based inhibitors whose structures are derived from actual
histone tail sequences, and report a novel methodology for incor-
porating hydroxamic acids in the amino acid side chains of pep-
tides. This was accomplished using an orthogonal protecting
group strategy, by synthesizing an Fmoc-protected building block.
This compound may be used in standard solid phase peptide syn-
thesis protocols, with liberation of the CO(NHOH) functionality un-
der typical cleavage conditions.
The syntheses of Fmoc protected 3 were achieved as shown in
Figure 2. In Method A we used the commercially available
Fmoc-(L)-Asu-OMe (1). The x-carboxylic acid was activated with
oxalylchloride and reacted with para-methoxybenzyl protected
hydroxylamine.17 The ester 2 was cleaved using LiOH to give
Fmoc-(
1 and the need for building block 3 in a gram scale range, an alter-
native route starting from enantiopure ( )-amino suberic acid (4)
L
)-Asu(NHOPMB)-OH in 71% yield.18 Given the high cost of
L
was considered. After Fmoc-protection under standard conditions,
simultaneous protection of the amino group and the carboxylic
acid was achieved by heating acid 5 with paraformaldehyde/TsOH
under water removal to give the oxazolidin-5-one. A similar strat-
egy with Cbz-protected derivatives was used before to enable the
⇑
Corresponding author. Tel.: +1 415 353 1668; fax: +1 415 353 8593.
E-mail address: david.m.wilson@ucsf.edu (D.M. Wilson).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.10.113

152
D. M. Wilson et al. / Tetrahedron Letters 54 (2013) 151–153
Figure 1. Molecular design of an aminosuberic acid-based inhibitor. (a) Action of HDACs on N-terminal acetyllysine residues, with zinc-mediated hydrolysis of side-chain
acetyl group; (b) chemical structure of suberoylanilide hydroxamic acid (SAHA), a transition state analog of acetyllysine. Chiral ( )-Asu based hydroxamates mimic the
L
endogenous substrate more closely and have been shown to be even more potent inhibitors of HDACs; (c) the X-ray crystallographic structure of histone deacetylase-like
protein (HDLP) with bound SAHA was used to describe the Zn-mediated hydrolysis of acetyllysine residues at the N-terminus of histone core proteins, and has assisted in the
design of HDAC inhibitors (adapted from Ref. 14).
Figure 2. Method A: (a) (1) (COCl)2 (1.1 equiv), CH2Cl2, cat. DMF, 0 °C to rt, 1 h; (2). H2NOPMB (1.2 equiv), DIEA (2.0 equiv), CH2Cl2, 0 °C to rt., 2 h (88%); (b) LiOH⁄H2O (0.2 M
in H2O, 2.0 equiv), THF, 0 °C, 8 h (71%). Method B: (a) FmocOSu (0.8 equiv), NaHCO3 (3.0 equiv), 1,4-dioxane:H2O (1:1), rt., 20 h (quant.); (b) paraformaldehyde (2.0 equiv),
TsOH⁄H2O (0.06 equiv), toluene, dean-stark, reflux, 3 h (93%); (c) (1) (COCl)2 (1.2 equiv), CH2Cl2, cat. DMF, 0 °C to rt, 1 h; (2) H2NOPMB (1.1 equiv), NEt3 (1.3 equiv), CH2Cl2,
0 °C to rt., 2 h (77%); (d) THF, 1 N NaOH (1:1), rt., 3 h; (e) FmocOSu (1.5 equiv), NaHCO3 (10.0 equiv), acetone:H2O (1:1), rt., overnight (60%).

D. M. Wilson et al. / Tetrahedron Letters 54 (2013) 151–153
153
selective functionalization of amino acid side chains.19,20 However,
to the best of our knowledge, this is the first time that this strategy
was applied to an Fmoc-protected amino acid. Activation of the
of lead compounds that may have enhanced selectivity for specific
HDAC isoforms.
free
x
-carboxylic acid and reaction with H2NOPMB, as in Method
Acknowledgements
A, afforded compound 3. Unfortunately treatment of the oxazoli-
din-5-one 6 with LiOH or NaOH did not give the free Fmoc-
protected acid 3, as reported previously for Cbz-protected
analogous compounds.19 Therefore the amino suberic acid deriva-
tive 6 was fully deprotected and subsequently reacted with
FmocOSu under basic conditions to give the final product 3.
The authors would like to acknowledge the support of Professor
Ronald Breslow.
References and notes
1. Grunstein, M. Nature 1997, 389, 349–352.
2. Kouzarides, T. Cell 2007, 131, 822.
HPLC-analysis of Fmoc-(L)-Asu(NHOPMB)-OH using a chiral sta-
tionary phase21 showed no racemization in the material obtained
via Method A (99.9% ee). With Method B slight racemization was
observed (93.8% ee).
3. Zhang, Y.; Ng, H. H.; Erdjument-Bromage, H.; Tempst, P.; Bird, A.; Reinberg, D.
Genes Dev. 1999, 13, 1924–1935.
4. Alland, L.; David, G.; Shen-Li, H.; Potes, J.; Muhle, R.; Lee, H. C.; Hou, H., Jr.;
Chen, K.; DePinho, R. A. Mol. Cell. Biol. 2002, 22, 2743–2750.
5. You, A.; Tong, J. K.; Grozinger, C. M.; Schreiber, S. L. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 1454–1458.
The amino suberic acid derivative (
thesize a series of peptides corresponding to the N-termini of the
histone core proteins, including all-( )-Ac-PAPK-Asu(NHOH)-
L)-3 was then used to syn-
6. Struhl, K. Genes Dev. 1998, 12, 599–606.
7. Strahl, B. D.; Allis, C. D. Nature 2000, 403, 41–45.
L
GSKK-CONH2 in a continuous flow synthesizer on PAL-PEG-PS re-
sin.22 Figure 3 shows a crude HPLC chromatogram of the resulting
hydroxamic acid-bearing peptide. The identities of all peptides
were confirmed by mass spectroscopy. Preliminary studies indi-
cate that these peptides are indeed potent inhibitors of human
HDACs in vitro, results will be reported separately.
In conclusion, we have developed a novel orthogonally pro-
tected unnatural aminosuberic acid derivative, compatible with
routine solid phase methods. This building block was used to syn-
thesize a series of hydroxamic acid bearing peptides that mimic ac-
tual HDAC substrates, namely the acetylated N-termini of histone
core proteins. This strategy may be employed to generate libraries
8. Duvic, M.; Talpur, R.; Ni, X.; Zhang, C.; Hazarika, P.; Kelly, C.; Chiao, J. H.; Reilly,
J. F.; Ricker, J. L.; Richon, V. M.; Frankel, S. R. Blood 2007, 109, 31–39.
9. Kirschbaum, M.; Frankel, P.; Popplewell, L.; Zain, J.; Delioukina, M.; Pullarkat,
V.; Matsuoka, D.; Pulone, B.; Rotter, A. J.; Espinoza-Delgado, I.; Nademanee, A.;
Forman, S. J.; Gandara, D.; Newman, E. J. Clin. Oncol. 2011, 29, 1198–1203.
10. Marks, P. A. Expert Opin. Investig. Drugs 2010, 19, 1049–1066.
11. Dankwardt, S. M.; Billedeau, R. J.; Lawley, L. K.; Abbot, S. C.; Martin, R. L.; Chan,
C. S.; Van Wart, H. E.; Walker, K. A. Bioorg. Med. Chem. Lett. 2000, 10, 2513–
2516.
12. Komatsu, Y.; Tomizaki, K. Y.; Tsukamoto, M.; Kato, T.; Nishino, N.; Sato, S.;
Yamori, T.; Tsuruo, T.; Furumai, R.; Yoshida, M.; Horinouchi, S.; Hayashi, H.
Cancer Res. 2001, 61, 4459–4466.
13. Furumai, R.; Komatsu, Y.; Nishino, N.; Khochbin, S.; Yoshida, M.; Horinouchi, S.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 87–92.
14. Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.; Rifkind, R. A.; Marks, P.
A.; Breslow, R.; Pavletich, N. P. Nature 1999, 401, 188–193.
15. Bertrand, P. Eur. J. Med. Chem. 2010, 45, 2095–2116.
16. Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem. 2003, 46, 5097–5116.
17. Ramsay, S. L.; Freeman, C.; Grace, P. B.; Redmond, J. W.; MacLeod, J. K.
Carbohydr. Res. 2001, 333, 59–71.
18.
(
L
)-2-N-Fmoc-amino-7-(4-methoxy-benzyloxycarbamoyl)-heptanoic acid ((
C31H34N2O7 (546.63): Mass (APCI+): 547 (M+1); HRMS (FAB+) calcd for
31H35N2O7: 547.2444, found: 547.2415; 1H NMR (DMSO-d6, 400 MHz): 1.1–
L)-3):
C
1.7 (m, 8H), 1.93 (t, J = 7.2 Hz, 2H), 3.74 (s, 3H), 3.8–4.0 (m, 1H), 4.1–4.4 (m,
3H), 4.69 (s, 2H), 6.92 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 8.5 Hz, 2H), 7.32 (dd, J = 7.5,
7.5 Hz, 2H), 7.41 (dd, J = 7.5, 7.5 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.72 (d,
J = 7.5 Hz, 2H), 7.88 (d, J = 7.5 Hz, 2H), 10.84 (s, 1H), 12.52 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz): 24.77, 25.24, 27.99, 30.60, 32.17, 46.63, 53.72, 55.02,
65.53, 76.34, 113.61, 120.07, 125.24, 127.02, 127.59, 128.00, 130.52, 140.68,
143.82, 156.12, 159.27, 169.18, 173.96.
19. Scholtz, J. M.; Bartlett, P. A. Synthesis-Stuttgart 1989, 542–544.
20. Itoh, M. Chem. Pharm. Bull. 1969, 17, 1679–1686.
21. (S,S)-Welk-O1 (25 cm  46 mm, Regis Technologies); 50% isopropanol, 50%
hexanes (+0.1% TFA), 1 ml/min, UV (254 nm): Rf 11.2 min ((L)-3) and 12.7 min
((
22. Peptide Synthesis: Fmoc-protected amino acids were acquired as free acids from
Bachem. The peptides were synthesized using the Fmoc strategy on
D)-3). A racemic sample was obtained using Method B with rac-4.
a
continuous flow Applied Biosystems Pioneer solid phase synthesizer using
PAL-PEG-PS resin (0.2 mmol/g scale, continuous flow, Applied Biosystems).
Single coupling cycles of 15 min using OH/HBTU activation chemistry (10-fold
excess amino acid:resin) were employed for all residues except Asu, whereas
OH/DIC/HOBt chemistry (four-fold excess amino acid:resin) and a 1 h coupling
time were employed for the aminosuberate building block 3. The side chain
protecting groups used were as follows: Lys (t-Boc); Ser (O-t-Bu). After peptide
assembly the N-terminus was manually acetylated using 1:1 acetic
anhydride:pyridine for 30 min followed by thorough washing with DMF,
methanol, and CH2Cl2. The peptide was cleaved from the resin and
simultaneously deprotected using 95:2.5:2.5 (v/v/v) trifluoroacetic
acid:triisopropyl silane:water for 3 h. The crude peptide was precipitated
and washed with cold ether, followed by dissolution in water (0.1% v/v TFA),
and lyophilization. Reversed phase C18 HPLC purification was used to purify the
peptide to homogeneity using aqueous-acetonitrile gradients (0–100%
acetonitrile, Rf: 14.4 min) containing 0.1% (v/v) TFA. After lyophilization, the
identity of the peptide was confirmed by mass spectrometry (TOF, ES+: 1039
(M+1)).
Figure 3. Synthesis of biomimetic peptide-based HDAC inhibitors. (a) Example of
hydroxamate-bearing peptide constructed using building block 3 and automated
solid-phase synthesis. The site of the artificial Asu-based amino acid corresponds to
a known site of acetylation/ deacetylation on the N-terminus of histone core protein
H2B; (b) Crude chromatogram, obtained by reversed phase
C18-HPLC using a
water(+0.1% TFA)-acetonitrile (+0.1% TFA) gradient. Preparative HPLC with collec-
tion of the dominant peak and subsequent mass spectrometry confirmed the
identity of all hydroxamate-bearing peptides.