Aza-amino acid scanning of chromobox homolog 7 (CBX7) ligands

Article abstract of DOI:10.1002/psc.2982

An aza-amino acid scan of peptide inhibitors of the chromobox homolog 7 (CBX7) was performed to study the conformational requirements for affinity to the methyllysine reader protein. Twelve azapeptide analogues were prepared using three different approaches employing respectively N-(Fmoc)aza-amino acid chlorides and submonomer azapeptide synthesis to install systematically aza-residues at the first four residues of the peptide, as well as to provide aza-lysine residues possessing saturated and unsaturated side chains. The aza-peptide ligands were evaluated in a chromobox homolog 7 binding assay, providing useful insight into structural requirements for affinity. Copyright

Full text of DOI:10.1002/psc.2982

Special Issue Article
Received: 30 November 2016
Revised: 23 January 2017
Accepted: 25 January 2017
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2982
Aza-amino acid scanning of chromobox
homolog 7 (CBX7) ligands
Mariam Traoré,a,c Michael Gignac,b Ngoc-Duc Doan,a,d Fraser Hofb
and William D. Lubella*
An aza-amino acid scan of peptide inhibitors of the chromobox homolog 7 (CBX7) was performed to study the conformational
requirements for affinity to the methyllysine reader protein. Twelve azapeptide analogues were prepared using three different
approaches employing respectively N-(Fmoc)aza-amino acid chlorides and submonomer azapeptide synthesis to install systemat-
ically aza-residues at the first four residues of the peptide, as well as to provide aza-lysine residues possessing saturated and
unsaturated side chains. The aza-peptide ligands were evaluated in a chromobox homolog 7 binding assay, providing useful
insight into structural requirements for affinity. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: aza-peptides; chromobox homolog 7 ligands; submonomer solid-phase synthesis; trimethyllysine peptides mimics
Mannich reaction on aza-propargylglycine provided the
corresponding lysine analogues possessing a triple bond in the side
Introduction
chain [16]. Finally, aza-phenylalanine and aza-leucine residues were
introduced through the application of N-(Fmoc)aza-amino acid
chlorides [17]. This combination of different azapeptide synthetic
Enzymes involved in protein post-translational modification are
promising targets for therapeutic intervention. The chromobox
homolog 7 (CBX7) is a member of the family of histone reader
approaches has provided 12 analogues (2–7, Figure 1) that were
proteins and binds N-trimethyllysine-27 of histone-3 (H3K27me3)
subsequently evaluated in a competitive binding assay, which has
in a recognition event that silences tumor suppressors [1–3].
Several lines of evidence suggest targeting CBX7 for cancer therapy
[3–5]. For example, overexpression of CBX7 in hematopoietic stem
demonstrated the importance of backbone conformation for
receptor affinity.
cells drives proliferation and genesis of T-cell leukemia [5]. On the
other hand, diminished expression of CBX7 induces a senescent
phenotype with reduced cell proliferation in prostate cancer cell
lines [1].
Chemistry
A series of short N-trimethyllysine containing peptides were
recently reported that inhibited formation of the CBX7-H3K27me3
complex [6]. For example, pentapeptide 1 (Figure 1) inhibited the
association of CBX7 and FITC-labeled H3K27me3 with IC50
11 0.4 μM and exhibited almost twofold selectivity for CBX7 over
CBX4. Peptide 1 and analogues represent the first examples of
inhibitors of any chromodomain [6] and form an initial set of
research tools for studying therapeutic hypotheses related to
chromodomain-containing proteins [7–12].
Employing 1 as lead peptide, we have performed an aza-amino
acid scan in which each amino acid residue was replaced by its cor-
responding semicarbazide counterpart in order to gain insight into
the conformational preferences of the parent peptide. A set of
constrained aza-lysine analogues was specifically made to study
the influence of side chain geometry and ε-amine methylation on
affinity. Two strategies were employed to make aza-lysine
analogues possessing saturated and unsaturated side chains.
Alkylation of aza-glycine semicarbazone 12 (Scheme 1) with
α,ω-dihaloalkanes gave the corresponding aza-ω-haloalkylglycine
peptides that were reacted with various amines or sodium azide
to give lysine residues with both saturated and double bond
containing side chains [13–15]. Alternatively, copper-catalyzed
Aza-amino amides are semicarbazides. On introduction into
so-called azapeptides, the semicarbazide induces conformational
constraint about the peptide backbone due to its planar urea and
the repelling nitrogen lone pairs of the N,N0-diacylhydrazine
component [18]. Substitution of aza-residues into peptides has
been shown to induce turn conformation, enhance molecular
recognition and prevent protease degradation [18–20].
*
Correspondence to: William D. Lubell, Département de Chimie, Université de
Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada.
E-mail: william.lubell@umontreal.ca
The authors declare no competing financial interest.
a Département de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville,
Montréal, Québec, H3C 3J7, Canada
b Department of Chemistry, University of Victoria, Victoria, British Columbia, V8W
3V6, Canada
c
NuChem Therapeutics Inc., 6100 Royalmount Ave, Montreal H4P 2R2, Canada
d Department of Chemistry, Massachusetts Institute of Technology, 77
Massachusetts Ave, Cambridge 02139, USA
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M. TRAORÉ ET AL.
Figure 1. CBX7 ligand parent pentapeptide 1 and aza-anologues 2–7.
To study peptide 1 with semicarbazides, a variety of methods
were employed for the synthesis of azapeptides on solid-phase.
Considering the importance of the geometry and amine
substituents of the lysine residue side chain, a submomomer strat-
egy for the solid-phase incorporation of aza-lysine residues was
employed featuring alkylation of azaglycine semicarbazone 12 with
1-bromo-4-chlorobutane, as well as E- and Z-1,4-dichlorobutene to
install, respectively, saturated and unsaturated ω-haloalkyl side
chains that were subsequently displaced by various amines
(Scheme 1) [13–15].
hydroxylamine hydrochloride in pyridine, and the resulting
semicarbazides were coupled to Fmoc-Leu-OH by way of its sym-
metric anhydride, which was prepared using diisopropylcarbodii-
mide (DIC). The resulting azatripeptides were elongated using
standard Fmoc-based solid-phase peptide synthesis protocols
[21]. Acetylation of the N-terminal phenylalanine residue was per-
formed with acetic anhydride and DIEA in DCM. Azide 19 was re-
duced chemoselectively using tris(2-carboxy)ethylphosphine
(TCEP) in THF/H2O (9:1) to provide lysine analogue 20 [13]. Resin
cleavage was performed using
a cocktail of TFA/H2O/TES
On Rink amide resin, supported benzhydrylidenyl-azaGly-
Ser(OtBu)-amide 12 was synthesized by acylation of serine resin
11 with activated carbazate 10 generated from mixing benzophe-
nonehydrazone (8) withp-nitrophenyl chloroformate (9, Scheme 1).
Alkylation of resin 12 was, respectively, performed with 1-
bromo-4-chlorobutane and 1,4-dichlorobut-2-ene at room temper-
ature using tetraethylammonium hydroxide (300 mol % of a 35%
aqueous solution) in tetrahydrofuran (THF) to provide the
corresponding aza-chloroalkylglycine resins 13, and E- and Z-14.
Conversion was assessed to be complete by liquid
chromatography–mass spectrometry examination of the residue
obtained on treatment of resin aliquots with trifluoroacetic
acid/water/triethylsilane [TFA/H2O/TES (95:2.5:2.5)] and resin
filtration. Subsequently, the resin 13 was treated with sodium azide
in dimethylformamide (DMF) to displace the chloride and provide
the corresponding azide 15. Access to other degrees of ω-amine
methylation was gained by displacement of chlorides 13, and E-
and Z-14 with methylamine, dimethylamine and trimethylamine.
Secondary ε-methylamine 17b was protected using Boc2O and
diisopropylethylamine (DIEA) in DCM to give carbamate 18b. The
(95:2.5:2.5). Peptides 2a–d, E- and Z-3c, and E-3d were, respectively,
shown to be of 25–80% crude purity, purified on a preparative col-
umn (250 × 21.2 mm, 5 μm, Gemini™ C18) using gradients of 10–
80% distilled water containing 0.1% formic acid in methanol
(0.1% formic acid) at a flow rate of 10.0 ml/min, and isolated in 1–
9% yields (see Supporting Information).
To introduce a triple bond into the aza-lysine side chain, a
copper-catalyzed Mannich addition was performed on
aza-propargylglycine 21 (Scheme 2) [16]. Propargylation was
performed on semicarbazone 12 using tetrabutylammonium
hydroxide and propargyl bromide to give aza-dipeptide 21 [22],
which was treated with CuI (20 mol %), dimethylamine (600 mol
%) and 37% aqueous formaldehyde (600 mol %) in
dimethylsulfoxide at rt for 3 h, to furnish the azalysine dipeptide
22 [16]. Peptide elongation using solid-phase synthesis protocols
provided azapeptide resin 23 [21], which was treated with methyl
iodide (1 equiv) in DMF for 1 h to give the tetra-alkylamonium salt
24. Azapeptide 4 was cleaved from the resin using a solution of
TFA/H2O/TES (95/2.5/2.5), characterized to be of 75% crude purity
by analytical high performance liquid chromatography, and iso-
lated as described above in 9% yield.
semicarbazone was removed using
a 1.5 M solution of
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J. Pept. Sci. 2017

CHROMOBOX HOMOLOG 7 (CBX7) AZAPEPTIDE LIGANDS
Scheme 1. Solid-phase synthesis of aza-Lys derivatives 2 and 3.
The N-terminal portion of the peptide was scanned with
aza-residues by employing N-(Fmoc)aza-amino acid chlorides to
install the aza-phenylalanine [17], and aza-leucine residues, and
by using a submonomer approach to add the aza-alanine residue
(Schemes 3 and 4) [22].
The aza-amino acid chlorides were, respectively, prepared from
fluorenylmethylcarbazate 28 [17]. Reduction of the corresponding
semicarbazones from condensation of 28 with iso-butanal and
benzaldehyde using sodium cyanoborohydride gave N0-alkyl
carbazates 29, which were treated with phosgene in toluene to
give the corresponding N-(Fmoc)aza-amino acid chlorides 30 after
evaporation of the volatiles [17]. Aza-amino acid chlorides 30
were used without further purification, dissolved in dichlorometh-
ane (DCM) and added to resin-bound peptides 26 and 27. The
latter were made from resin 25, which was obtained by coupling
of Fmoc-Lys[N,N-(CH3)2] [23] to H-Ser(tBu)-resin, treatment with
methyl iodide and removal of the Fmoc group to give
corresponding trimethyllysine resin 26 that was elongated to give
peptide 27. Acylation of peptide 26 with Fmoc-aza-leucine amino
acid chloride 30a gave aza-tripeptide resin 31. Similarly, treat-
ment of the resin 27 with Fmoc-aza-phenylalanine amino acid
chloride 30b furnished aza-pentapeptide 32. Azapeptide 5 was
prepared from 31 by Fmoc group removals, acylation with the
symmetric anhydride from treatment of Fmoc-Ala-OH with DIC,
coupling with Fmoc-Phe-OH using HBTU, acetylation with acetic
anhydide and DIEA, resin cleavage and purification as described
for the synthesis of peptide 2 above. Similarly, azapeptide 6 was
prepared, respectively, from 32 by Fmoc group removal, acetyla-
tion with acetic anhydide and DIEA, resin cleavage and
purification.
The aza-alanine residue was introduced at the peptide 2-
position by alkylation of aza-glycine semicarbazone 33 [22], which
was prepared from Leu-Lys(N+Me3)-H-Ser(tBu)-resin using similar
protocols as described for the synthesis of semicarbazone 12
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M. TRAORÉ ET AL.
Scheme 2. Synthesis of azalysine 4 possessing a triple bond.
above. Exposure of 33 to tetraethylammonium hydroxide
followed by iodomethane in THF provided aza-alanine resin 34
(Scheme 4). The semicarbazone was removed using NH2OH•HCl
in pyridine, and the resulting semicarbazide was coupled to
Fmoc-phenylalanine using DIC as described above. After Fmoc
removal using 20% piperidine in DMF, resin 35 was, respectively,
acylated with acetic anhydride and DIEA in DMF to give
acetamide 36, and p-bromobenzoic acid using O-(Benzotriazol-
1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HBTU)
and DIEA in DMF to provide the benzamide 37. Resins 36 and
37 were, respectively, cleaved and the peptides purified by high
performance liquid chromatography as described above to
provide aza-alanine peptides 7a and 7b.
Scheme 4. Submonomer synthesis of aza-alanine peptide 7.
Different methods were employed to synthesize azapeptides
2–7 to best introduce specific functional groups effectively. For
example, submonomer solid-phase synthesis enabled access to
aza-lysine peptides 2 and 3 possessing saturated and olefin
aza-lysine side chains having diverse ω-amino substituents,
because alkylation of a common aza-glycine intermediate with
Scheme 3. Solid phase synthesis of aza-Phe, and aza-Leu analogues 5 and 6.
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J. Pept. Sci. 2017

CHROMOBOX HOMOLOG 7 (CBX7) AZAPEPTIDE LIGANDS
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7 Simhadri C, Gignac MC, Anderson CJ, Milosevich N, Dheri A, Prashar N,
Flemmer RT, Dev A, Henderson TG, Douglas SF, Wulff JE, Hof F.
Structure–activity relationships of Cbx7 inhibitors, including selectivity
studies against other Cbx proteins. ACS Omega 2016; 1: 541–551. DOI:
10.1021/acsomega.6b00120.
8 Stuckey JI, Simpson C, Norris-Drouin JL, Cholensky SH, Lee J, Pasca R,
Cheng N, Dickson BM, Pearce KH, Frye SV, James LI. Structure–activity
relationships and kinetic studies of peptidic antagonists of CBX
chromodomains. J. Med. Chem. 2016; 59: 8913–8923. DOI: 10.1021/acs.
jmedchem.6b00801.
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Aguilo F, Walsh MJ, Zhou MM. Structure-guided discovery of selective
antagonists for the chromodomain of Polycomb repressive protein
CBX7. ACS Med. Chem. Lett. 2016; 7: 601–605. DOI: 10.1021/
acsmedchemlett.6b00042.
10 Milosevich N, Gignac MC, McFarlane J, Simhadri C, Horvath S, Daze KD,
Croft CS, Dheri A, Quon TTH, Douglas SF, Wulff JE, Paci I, Hof F.
Selective inhibition of CBX6: a methyllysine reader protein in the
Polycomb family. ACS Med. Chem. Lett. 2016; 7: 139–144. DOI: 10.1021/
acsmedchemlett.5b00378.
11 Stuckey JI, Dickson BM, Cheng N, Liu Y, Norris JL, Cholensky SH,
Tempel W, Qin S, Huber KG, Sagum C, Black K, Li F, Huang XP, Roth BL,
Baughman BM, Senisterra G, Pattenden SG, Vedadi M, Brown PJ,
Bedford MT, Min J, Arrowsmith CH, James LI, Frye SV. A cellular
chemical probe targeting the chromodomains of Polycomb repressive
complex 1. Nat. Chem. Biol. 2016; 12: 180–187. DOI: 10.1038/
nchembio.2007.
12 Barnash KD, Lamb KN, Stuckey JI, Norris JL, Cholensky SH, Kireev DB,
Frye SV, James LI. Chromodomain ligand optimization via target-class
directed combinatorial repurposing. ACS Chem. Biol. 2016; 11:
2475–2483. DOI: 10.1021/acschembio.6b00415.
13 Traore M, Doan N-D, Lubell WD. Diversity-oriented synthesis of
azapeptides with basic amino acid residues: aza-lysine, aza-ornithine,
and aza-arginine. Org. Lett. 2014; 16: 3588–3591. DOI: 10.1021/
ol501586y.
α,ω-dihaloalkane and alkene residues installed effectively an
ω-chloroalkyl side chain for diversification of the terminal amine
by nucleophilic displacements. Although a triple bond may in
principle be installed using a similar alkylation strategy, attempts
to alkylate the aza-glycine residue with 1,4-dichlorobutyne were
unsuccessful; instead, the copper-catalyzed Mannich reaction on
an aza-propargylglycine residue proved an effective alternative
for introducing the acetylene into the aza-lysine side chain in
azapeptide 4. Application of submonomer chemistry to install
the aza-phenylalanine and aza-leucine residues in the presence
of the trimethyl-lysine reside was however complicated likely by
the presence of the tetra-alkyl ammonium residue, such that
the application of N-(Fmoc)aza-amino acid chlorides was used to
provide azapeptides 5 and 6 (Scheme 3) with better crude purity
and isolated yield. On the other hand, alkylation of the
aza-glycine residue with iodomethane in the presence of the
trimethyl-lysine was successful and provided access to aza-alanine
peptide 7.
The 12 new azapeptide analogues of ligand 1 were evaluated
for their affinity for CBX7 in a competitive fluorescence polariza-
tion assay that measured the displacement of a dye-labeled
peptide ligand, as previously reported [6,7]. Most of the new
analogues were not measurably active in the assay up to the limits
of their most concentrated solutions (0.8–1.8 mM). At 1 mM,
azapeptide 2d showed 10% of the response of the positive
control pentapeptide. Azapeptides 6 and 7a showed 50 and
90% responses, respectively, at 1.5 mM, but their respective bind-
ing curves did not saturate and could not be fitted to provide IC50
values due to limits in their solubility. The potency of 7a suggests
that CBX7 is more tolerant of backbone conformational changes
in the area of the leucine residue, which may be explained
because unlike along the rest of the binding interface, the protein
does not make a hydrogen bond to the peptide’s backbone
carbonyl at this position [6, 24].
14 Doan N-D, Zhang J, Traoré M, Kamdem W-L, Lubell WD. Solid-phase
synthesis of C-terminal azapeptides. J. Pept. Sci. 2015; 21: 387–391.
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15 Doan N-D, Lubell WD. In “Solid-Phase Synthesis of Z-Alkene Aza-Lysine
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Symposium, NaydenovaE , PajpanovaT (eds.). Bulgarian Peptide
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16 Zhang J, Proulx C, Tomberg A, Lubell WD. Multicomponent diversity-
oriented synthesis of aza-lysine-peptide mimics. Org. Lett. 2014; 16:
298–301. DOI: 10.1021/ol403297v.
Although the azapeptides maintain all side-chain structural
components present in parent ligand 1 (IC50 11 μM), a loss of
affinity was generally seen across the series. Considering the back-
bone conformational preferences of azapeptides [18], as well as the
flatter nature of the semicarbazide residue [25], such constraints on
peptide 1 were not tolerated in the protein binding site, likely
because they disturb the preferred β-strand conformation [24].
17 Boeglin D, Lubell WD. Aza-amino acid scanning of secondary structure
suited for solid-phase peptide synthesis with Fmoc chemistry and aza-
amino acids with heteroatomic side chains. J. Comb. Chem. 2005; 7:
864–878. DOI: 10.1021/cc050043h.
18 Proulx C, Sabatino D, Hopewell R, Spiegel J, García-Ramos Y, Lubell WD.
Azapeptides and their therapeutic potential: current issues and new
possibilities. Future Med. Chem. 2011; 1139–1164. DOI: 10.4155/
fmc.11.74.
19 Chingle R, Lubell WD. Azopeptides: synthesis and pericyclic chemistry.
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20 Sabatino D, Proulx C, Pohankova P, Ong H, Lubell WD. Structure–activity
relationships of GHRP-6 azapeptide ligands of the CD36 scavenger
receptor by solid-phase submonomer azapeptide synthesis. J. Am.
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Supporting information
Additional supporting information may be found in the online ver-
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Table S1. Azapeptide yields and purities.
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