Diversity-oriented synthesis of azapeptides with basic amino acid residues: Aza-lysine, aza-ornithine, and aza-arginine

Article abstract of DOI:10.1021/ol501586y

Aza-peptides with basic amino acid residues (lysine, ornithine, arginine) and derivatives were synthesized by an effective approach featuring alkylation of a hydrazone-protected aza-glycine residue with α-bromo ω-chloro propane and butane to provide the corresponding alkyl chloride side chains. Displacement of the chloride with azide and various amines gave entry to azaOrn, azaLys, and azaArg containing peptides as demonstrated by the solution and solid-phase syntheses of 29 examples, including an aza-library of Growth Hormone Releasing Peptide-6 analogs.

Full text of DOI:10.1021/ol501586y

Letter
pubs.acs.org/OrgLett
Diversity-Oriented Synthesis of Azapeptides with Basic Amino Acid
Residues: Aza-Lysine, Aza-Ornithine, and Aza-Arginine
Mariam Traore, Ngoc-Duc Doan, and William D. Lubell*
́
́ ́ ́ ́ ́
Departement de Chimie, Universite de Montreal, C.P. 6128, Succursale Centre-Ville, Montreal, Quebec, Canada H3C 3J7
S
* Supporting Information
ABSTRACT: Aza-peptides with basic amino acid residues (lysine, ornithine, arginine) and derivatives were synthesized by an
effective approach featuring alkylation of a hydrazone-protected aza-glycine residue with α-bromo ω-chloro propane and butane
to provide the corresponding alkyl chloride side chains. Displacement of the chloride with azide and various amines gave entry to
azaOrn, azaLys, and azaArg containing peptides as demonstrated by the solution and solid-phase syntheses of 29 examples,
including an aza-library of Growth Hormone Releasing Peptide-6 analogs.
asic amino acid residues, such as lysine and arginine,
Bfunction in numerous biological processes including post-
translational modifications,1 transport across membranes,2 and
as enzymatic cleavage sites.3 For example, acylation and
methylation of lysine residues of histone proteins alter chromatin
structure and gene regulation.1,4 Cell-penetrating peptides,5,6
such as Tat and penetratin, are composed of multiple basic
residues. The protease trypsin cleaves peptides specifically at the
C-terminal of lysine and arginine residues.7 Peptide mimics with
constrained conformations and chemical modifications at lysine
and arginine residues are thus desirable tools for studying and
regulating such phenomena.8−14
Azapeptides employ the electronic constraints of a semi-
carbazide residue to rigidify peptide geometry in favor of turn
conformations, with the added benefit of enhanced stability
against protease degradation.15 Efforts to prepare basic aza-
amino acid residues have typically involved building block
synthesis in solution,16−18 which restricts analog generation.
Figure 1. (A) Ornithine and lysine in native peptide, and aza-lysine
analogs possessing (B) saturated (C) alkyne and (D) Z-olefin side
Recently, our laboratory reported the synthesis of aza-Lys
chains.
peptides by a route featuring the copper-catalyzed addition of
Mannich reagents to an aza-propargylglycine residue.19 This so-
called A3 reaction20 was amenable to solid phase to provide rigid
semicarbazone to prepare the heterocycle. Considering the rate
of nitrogen alkylation, we perceived that the more acidic
semicarbazone nitrogen could be alkylated with an α-bromo-
ω-chloro alkane to furnish an alkyl chloride side chain.
Subsequent displacement of the chloride using a variety of
amines may then provide access to a diverse array of aza-residues
with basic side chains. This strategy has now been realized using
lysine analogs of type C (Figure 1) possessing a variety of tertiary
amines on the side chain. Moreover, hydrogenation of the triple
bond gave access to the Z-olefin analogs D. In principle, further
reduction of the triple bond may give access to aza-lysine analogs
of type B. The A3-route does however have limitations in
requiring typically secondary amines, a four-carbon side chain
length, and extra steps to prepare saturated analogs.
In our efforts to make constrained N-aminoimidazolidin-2-one
peptides,21 we have recently explored the application of 1,2-
dibromoethane in the alkylation of both urea nitrogen of a
Received: June 2, 2014
© XXXX American Chemical Society
A
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Letter
both solution- and solid-phase methods, which have provided a
wide diversity of peptide analogs possessing basic aza-residues.
Aza-lysine analogs were pursued initially by a solution-phase
method (Scheme 1) featuring alkylation of benzhydrylidene-
particular, [azaLys]-GHRP-6 analogues were targeted, because
of the presence of two lysine residues in the related peptide
EP80317 (Haic-2Me-Trp-D-Lys-Trp-D-Phe-Lys-NH2), which
has exhibited CD36 selectivity.24,25 Azapeptide analogs of
GHRP-6 were thus targeted possessing respectively basic aza-
residues at the 3-, 4-, and 6-positions.
Scheme 1. Solution-Phase Synthesis of Aza-Lys and Aza-Orn
Dipeptides
Initially, the set of [azaOrn4]-, [azaLys4]-, and [azaArg4]-
GHRP-6 analogs was pursued in order to demonstrate a solid-
phase protocol for preparing aza-derivatives of the common
naturally occurring basic amino acids. On rink amide resin,
benzylidene-azaGly-D-Phe-Lys(Boc) 9 (Scheme 2) was synthe-
Scheme 2. Synthesis of [AzaLys4]-, [AzaOrn4]-, and
[AzaArg4]-GHRP-6 Analogs
a
Isolated yield calculated from resin loading and crude purity
ascertained by LC-MS.
azaGly-Phe-OtBu (1) and benzhydrylidene-azaGly-Phe-NiPr
(2).22 Employing tetraethylammonium hydroxide (120 mol %,
as a 40% aqueous solution) in THF, ester 1 and amide 2 were
alkylated with 1-bromo-3-chloropropane and 1-bromo-4-chlor-
obutane to provide the chloroalkyl semicarbazones 3−5, which
were converted into aza-Orn and aza-Lys dipeptides by chloride
displacement with different amines. Racemization of esters 3 and
4 (up to 15%) was however detected from the alkylation of tert-
butyl ester 1 at room temperature, but was avoided by decreasing
the temperature to 0 °C. On the other hand, no loss of
configuration was observed using amide 2 even at rt. Chloroalkyl
semicarbazones 3−5 were then converted to analogs of aza-Orn
and aza-Lys 6−8 from reactions with sodium azide, primary and
secondary amines, and trimethylamine in DMF at 60 °C
(Scheme 1). The relatively polar final dipeptides were purified by
preparative HPLC on a C18 column, except for compounds 6a
and 6d, which were purified by flash chromatography on silica
gel.
sized as previously described.26 Alkylation of resin 9 was
performed with 1-bromo-3-chloropropane and 1-bromo-4-
chlorobutane, respectively, at room temperature using tetra-
ethylammonium hydroxide (200 mol % of a 40% aqueous
solution) in THF to provide the chloroalkyl resins 10a and 10b.
Conversion was assessed to be complete by treating aliquots of
resin 10 with TFA/H2O/TES (9.5:0.25:0.25) and examination
of the residue after resin filtration by LC-MS. Resins 10 were
respectively treated with sodium azide in DMF to provide the
corresponding azides 11a and 11b. The semicarbazone was
removed using a 1.5 M solution of hydroxylamine hydrochloride
in pyridine, and the resulting semicarbazides 12 were coupled to
Fmoc-Ala by way of its symmetric anhydride using diisopropyl-
carbodiimide (DIC) to provide azatetrapeptides 13.26 Elonga-
tion of the sequence was then performed using conventional
Fmoc-based solid-phase peptide synthesis (SPPS).27
A method for the synthesis of azapeptides possessing basic aza-
amino acid residues was next developed on solid phase. Growth
Hormone Releasing Peptide-6 (GHRP-6, His-D-Trp-Ala-Trp-D-
Phe-Lys-NH2), a cluster of differentiation 36 receptor (CD36)
ligand,23 was chosen for this study, because of its interesting
biological activity and challenging chemical structure. In
Azides 14 were chemoselectively reduced with tris(2-
carboxy)ethylphosphine (TCEP) to provide the corresponding
amines 15.28,29 Resin cleavage was then performed using a
cocktail of TFA/H2O/TES (9.5:0.25:0.25) to provide respec-
tively [azaOrn4]- and [azaLys4]-GHRP-6 (17a and 17b) in 58%
and 54% crude purities. Purification of the crude azapeptides by
B
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HPLC on a C18-column gave 1% and 2% overall yields of 17a
and 17b. Alternatively, amine 15a was treated with N,N-
bis(Boc)-S-methylisothiourea in DMF at rt for 12 h to provide
[azaArg4]-GHRP-6 resin 16a,30 which was cleaved from the resin
using the same cocktail to provide [azaArg4]-GHRP-6 (18a) in
21% crude purity, and 3% overall yield after purification by
HPLC.
details). Employing a similar synthetic strategy [azaLys3]-
GHRP-6 analogues 23 were prepared from benzylidene-
azaGly-Trp(Boc)-D-Phe-Lys(Boc) rink amide resin (Table 1).31
Finally, [azaLys6]-GHRP-6 analogs 25 were prepared using
methyl-, dimethyl-, and trimethylamines (Scheme 4). Benzyli-
Scheme 4. Synthesis of [azaLys6]-GHRP-6 Analogues
Chloroalkyl resins 10 were subsequently employed to prepare
a library of diverse [azaLys]-GHRP-6 derivatives (Scheme 3).
Scheme 3. Synthesis of [AzaLys4]-GHRP-6 Derivatives
Employing various primary and secondary amines to displace
chlorides 10a and 10b in DMF, substituted azaOrn and azaLys
resins 19 and 20 were respectively synthesized. In the case of
secondary amine analogs, the amine was subsequently protected
as the corresponding N-Boc derivative by treating the resin with
di-tert-butyldicarbonate in dichloromethane. The semicarbazone
protecting group was removed, with the peptides elongated and
cleaved from resin as previously described above. Final [azaOrn4]
and [azaLys4]-GHRP-6 analogs 21 and 22 were obtained in 23−
63% crude purities and isolated in 2−9% overall yields after
purification by HPLC (Table 1; see Supporting Information for
dene-azaGly rink amide resin 24 was obtained by removal of the
Fmoc protection from rink amide resin, and treatment of the
amine resin with 4-nitrophenyl 2-(diphenylmethylidene)-
carbazate, which was prepared from the reaction of benzophe-
none hydrazone with p-nitrophenylchloroformate (Scheme 4).
LCMS analysis of a cleaved resin aliquot of 24 showed 98%
conversion to the N-(diphenylmethylidene)glycinamide product
possessing the desired molecular ion. To introduce the side
chains, resin 24 was alkylated with 1-bromo-4-chlorobutane and
tetraethylammonium hydroxide (120 mol %, as a 40% aqueous
solution) in THF, followed by chloride conversion into an amine.
In the case of the methylamine analog, the amine was
subsequently protected as the corresponding N-Boc derivative
before peptide elongation. After removal of the semicarbazone
group, azapeptides were elongated and cleaved from the resin as
previously described.
Table 1. [AzaLys]-, [AzaOrn]-GHRP-6 Derivatives
In summary, 16 new GHRP-6 analogs were synthesized
possessing aza-residues with different basic amine side chains
(Scheme 2 and Table 1). This method has provided access to
azaLys, azaOrn, and azaArg peptide sequences from a common
diversity-oriented strategy featuring alkylation of an azaGly
residue with an α-bromo-ω-chloroalkane followed by displace-
ment of the resulting chloride with various amines. With this set
of GHRP-6 analogs possessing basic aza-residues in hand, their
impact on CD36 affinity and activity is currently being examined
and will be reported in due time. Considering the power of this
method for preparing azapeptides possessing diverse basic aza-
residues, this approach should find significant applications in the
study of various events featuring the post-translational
modification and activity of lysine and arginine containing
peptide structures.
ASSOCIATED CONTENT
■
a
Crude purity ascertained by LCMS analysis at 214 nm using H2O
S
* Supporting Information
Experimental procedures and characterization data. This material
is available free of charge via the Internet at http://pubs.acs.org.
(0.1% FA)/MeOH (0.1% FA) and H2O (0.1% FA)/MeCN (0.1% FA)
b
as eluents. Isolated purity ascertained in same manner as crude purity.
c
Isolated yields calculated from resin loading.
C
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Letter
(28) Saneyoshi, H.; Ochikubo, T.; Mashimo, T.; Hatano, K.; Ito, Y.;
Abe, H. Org. Lett. 2014, 16, 30−33.
AUTHOR INFORMATION
Corresponding Author
■
(29) Faucher, A.-M.; Grand-Maitre, C. Synth. Commun. 2003, 33,
3503−3511.
*E-mail: william.lubell@umontreal.ca.
Notes
(30) Gers, T.; Kunce, D.; Markowski, P.; Izdebski, J. Synthesis 2004,
37−42.
(31) Sabatino, D.; Proulx, C.; Pohankova, P.; Ong, H.; Lubell, W. D. J.
Am. Chem. Soc. 2011, 133, 12493−12506.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Natural Sciences and
Engineering Research Council of Canada (NSERC), the
Canadian Institutes of Health Research (Canada-UK partnership
on Antibiotic Resistance), the Minister
economique de l’innovation et de l’exportation du Quebec
(#878-2012, Traitement de la degenerescence maculaire), and
Amorchem.
̀ ́
e du developpement
́
́
́
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