Synthesis and alkylation of aza-glycinyl dipeptide building blocks

Article abstract of DOI:10.1002/psc.2572

Aza-glycinyl dipeptides are useful building blocks for the synthesis of a diverse array of azapeptides. The construction of the aza-glycine residue is however challenging, because of the potential for side reactions, such as those leading to formation of oxadiazalone, hydantoin and symmetric urea by-products. Employing N,N′-disuccinimidyl carbonate to activate benzophenone hydrazone, we have developed a more efficient approach for the synthesis of aza-glycinyl dipeptides. Alkylation of the semicarbazone of the resulting protected aza-glycinyl dipeptides using tetraethylammonium hydroxide and propargyl bromide provided an efficient entry into the aza-propargylglycinyl peptide building blocks, which have served previously in various reactions including Sonogashira cross-couplings, dipolar cycloadditions and intramolecular exo-dig cycloadditions to furnish a variety of azapeptide building blocks. Copyright

Full text of DOI:10.1002/psc.2572

Protocol
Received: 12 June 2013
Revised: 12 September 2013
Accepted: 14 September 2013
Published online in Wiley Online Library: 6 November 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2572
Synthesis and alkylation of aza-glycinyl
dipeptide building blocks
Yesica Garcia-Ramos and William D. Lubell*
Aza-glycinyl dipeptides are useful building blocks for the synthesis of a diverse array of azapeptides. The construction of the
aza-glycine residue is however challenging, because of the potential for side reactions, such as those leading to formation of
oxadiazalone, hydantoin and symmetric urea by-products. Employing N,N′-disuccinimidyl carbonate to activate benzophe-
none hydrazone, we have developed a more efficient approach for the synthesis of aza-glycinyl dipeptides. Alkylation of
the semicarbazone of the resulting protected aza-glycinyl dipeptides using tetraethylammonium hydroxide and propargyl
bromide provided an efficient entry into the aza-propargylglycinyl peptide building blocks, which have served previously
in various reactions including Sonogashira cross-couplings, dipolar cycloadditions and intramolecular exo-dig cycloadditions
to furnish a variety of azapeptide building blocks. Copyright © 2013 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-glycinyl dipeptide; benzophenone hydrazone; alkylation; N,N′-disuccinimidyl carbonate (DSC); azapeptide
properties, such as longer duration of action and resistance to
proteases relative to natural peptides. [1,8–11]
Scope and Comments
Methods for the synthesis of azadipeptides have been
reviewed with emphasis on the issues of side product formation,
such as hydantoin from intramolecular cyclization of activated
carbamato and isocyanato amides, and oxadiazalone on
Azapeptides possess one or more semicarbazide residues from
replacement of a backbone α-carbon by nitrogen in the peptide
sequence (Figure 1) [1–11]. The conformation of the azapeptide
is thus rigidified, because of the planarity of the urea and the
lone-pair repulsion between adjacent nitrogen in the
*
Correspondence to: William D. Lubell, Département de Chimie, Université de
Montréal, C.P. 6128, Succursale Centre-Ville, Montreal, Quebec H3C 3J7,
Canada. E-mail: william.lubell@umontreal.ca
semicarbazide moiety, such that the φ and ψ dihedral angles of
the aza-residue backbone have a tendency to adopt turn
geometry, as shown by computational, spectroscopic and X-ray
crystallography [1–7,11]. Moreover, the semicarbazide residue
can bestow the azapeptide with improved pharmacokinetic
Département de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-
Ville, Montreal, Quebec, H3C 3J7, Canada
J. Pept. Sci. 2013; 19: 725–729
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

GARCIA-RAMOS AND LUBELL
Figure 1. Azapeptide examples and structural comparison with peptides.
Figure 2. Strategies for the synthesis of azadipeptides.
activation of carbazates with phosgene equivalents (Figure 2) [1,11].
The issue of oxadiazalone formation has been surmounted in the
synthesis of the aza-glycinyl dipeptide in solution by employing
benzophenone hydrazone as the aza-Gly precursor [12]. The for-
mation of symmetric urea 5 has however plagued this method,
particularly, from using reactive activating agents, such as phos-
gene/triphosgene and carbonyldiimidazole. Symmetric urea 5
was minimized by using p-nitrophenyl chloroformate, as a safer
agent than phosgene, to activate benzophenone hydrazone at
0 °C, because the p-nitrophenyl alkylidene carbazate intermediate
was relatively stable and reacted effectively with amino esters
and amino amides to provide aza-glycinyl dipeptides [12,13].
Removal of the p-nitrophenol by-product has however compli-
cated this process.
aza-glycinyl-proline tert-butyl ester has served as a precursor in
the synthesis of azapeptides 3 (Figure 1), which block myometrial
contractions by modulating the activity of the prostaglandin F2α
receptor through a mechanism featuring biased signalling and
which exhibit potential as prototypes to develop inhibitors of
preterm labour [18]. In the alkylation of benzhydrylidene aza-
glycinyl phenylalanine tert-butyl ester 6a with propargyl bromide
(Scheme 1), racemisation has been avoided by employing
tetraethylammonium hydroxide as a milder base, instead of potas-
sium tert-butoxide or tert-butylaminotri(pyrrolidino)phosphorane
[13,19]. The resulting aza-propargylglycinyl dipeptide 7a and
related analogues have served as versatile building blocks
for broadening further the diversity of azapeptides, because
the propargyl moiety has reacted effectively in Sonogashira
cross-coupling chemistry with various aryl and heteroaryl
halides [14,21], in copper-catalyzed azide-alkyne cycloadditions
[20] and in 5-exo-dig cyclizations (Scheme 2) [14,21]. The latter
reaction has provided a series of N-amino imidazolin-2-one
dipeptide building blocks, which were shown by X-ray
Aza-glycinyl dipeptides have been employed in sub-monomer
syntheses of a variety of structurally diverse and biologically
active azapeptides by approaches featuring chemoselective alkyl-
ation and arylation of the semicarbazone to install various side
chains at the aza-residue [13–17]. For example, benzhydrylidene
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SYNTHESIS AND ALKYLATION OF AZA-GLYCINYL DIPEPTIDE BUILDING BLOCKS
Scheme 1. Optimized azadipeptide synthesis.
Scheme 2. Applications of aza-propargylglycinyl dipeptides in Sonogashira coupling, 5-exo-dig cyclization and 1,3-dipolar cycloaddition reactions [14, 20, 21].
crystallography and NMR spectroscopy to mimic turn geometry
in model peptide 4 [14,21].
aza-glycine to tert-butyl glycinate and α-amino iso-butyrate to pro-
vide the respective aza-glycinyl dipeptides 6b and 6c, which were
similarly alkylated to provide aza-propargylglycinyl dipeptides 7b
and 7c. In light of the practical advantages of these innovations as
well as the utility of aza-propargylglycinyl dipeptides 7, which have
previously proven effective in Sonogashira coupling, 5-exo-dig cycli-
zation and 1,3-dipolar cycloadditions (Scheme 2), these protocols
should be of general use for the effective synthesis of azapeptides.
In this protocol, two innovations are highlighted, which have
improved the synthesis and alkylation of aza-glycinyl dipeptides.
By employing N,N’-disuccinimidyl carbonate (DSC) instead of
p-nitrophenyl chloroformate in the activation of benzophenone
hydrazone, symmetric urea 5 and p-nitrophenol, both can be
avoided such that higher yields of aza-glycinyl dipeptide ester
are obtained after a simpler chromatography (Scheme 1).
O-Succinimidyl carbamates have previously been used in the syn-
thesis of oligoureas from N-protected β-amino alcohols, because
of their stability and tendency to form crystalline solids [22,23]. Acti-
vation of the ε-amine of lysine residues in peptides with DSC has also
provided stable O-succinimidyl carbamates, which reacted effectively
to make peptide–protein conjugates [24]. The application of DSC in
the activation of diphenyl hydrazone has now been shown to pro-
vide a useful activated aza-glycine intermediate, which has been
used to acylate primary amino esters, including α-aminoisobutyric
tert-butyl ester, to provide aza-glycinyl dipeptides in high yields.
In light of the value of aza-propargylglycinyl dipeptide 7a, a pro-
cedure is also described that employs tetraethylammonium hydrox-
ide as base and microwave heating for the racemisation-free
alkylation of aza-glycinyl dipeptide ester 6a with propargyl bromide.
Furthermore, variations of the protocols are given for coupling of
Experimental Procedure
Benzhydrylidene aza-glycinyl-L-phenylalanine tert-butyl ester (6a)
In a flame-dried round-bottom flask, at 0 °C, a solution of DSC
(7.18 g, 28.03 mmol, 1.1 eq) in dry CH2Cl2 (80 ml) and DMF (15 ml)
was treated dropwise by cannula with a 0 °C solution of benzophe-
none hydrazone (5g, 25.5mmol, 1 eq) in dry CH2Cl2 (112 ml). The
ice bath was removed, and the reaction mixture was allowed to
warm to room temperature. After stirring for 1 h, the mixture was
cooled to 0 °C and treated dropwise by cannula with a premixed
0 °C solution of L-Phe-Ot-Bu·HCl (6.57 g, 25.48mmol, 1 eq) and DIEA
(8.4ml, 50.96 mmol, 2 eq) in CH2Cl2 (32 ml). The ice bath was
removed. The reaction mixture was allowed to warm to room tem-
perature and stirred for 16h. The volatiles were evaporated, and the
J. Pept. Sci. 2013; 19: 725–729 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

GARCIA-RAMOS AND LUBELL
residue was purified on a column of silica gel using flash chroma-
tography with 10–50% EtOAc in hexane as solvent system. Ester
6a was obtained as oil (8.60 g, 76% yield): the physical and spectro-
scopic properties of 6a were identical to those reported in [19].
1.13 mmol, 1 eq) in THF (8ml), 40% tetraethylammonium hydroxide
in H2O (416 μl, 1.31mmol, 1 eq) and 80% propargyl bromide in tol-
uene (168 μl, 1.31 mmol, 1 eq). Purification by flash chromatogra-
phy, using 50% EtOAc in hexane as solvent system, gave 7b
(369mg, 83% yield). Rf = 0.51 (7: 3 hexane/EtOAc); 1H NMR
(400MHz, CDCl3) δ 7.53 (m, 2H), 7.47–7.41 (m, 4H), 7.39–7.34
(m, 4H), 7.0 (t, J = 5.1 Hz, 1H), 4.05 (m, 4H), 2.06 (t, J = 2.2Hz, 1H),
1.49 (s, 9H); 13C NMR (300MHz, CDCl3) δ 169.8, 159.4, 158.5, 138.9,
135.8, 130.5, 130.1, 129.5, 129.1, 129.0, 128.5, 82.2, 78.9, 72.3, 43.6,
35.6, 28.4. IR (thin film) ν 2984, 1736, 1682, 1500, 1448, 1370,
Benzhydrylidene aza-glycinylglycine tert-butyl ester (6b)
This was prepared using the protocol described earlier for
phenylalanine dipeptide 6a using benzophenone hydrazone
(697 mg, 3.55 mmol, 1 eq), DSC (1g, 3.90 mmol, 1.1 eq), DIEA
(1.2 ml, 7.1mmol, 2 eq) and glycine tert-butyl ester (595 mg,
3.55 mmol, 1 eq) in dry CH2Cl2 (28 ml) and DMF (5ml). Flash
chromatography, using 20–50% EtOAc in hexane as solvent system,
and evaporation of the collected fractions gave aza-dipeptide 6b
(873 mg, 70% yield); mp 141–142, Rf = 0.14 (8: 2 hexane/EtOAc);
δ 7.66 (s, 1H), 7.56 (m, 5H), 7.37 (m, 3H), 7.28 (m, 2H), 6.79 (s, 1H),
4.07 (d, J = 4.9Hz, 2H), 1.54 (s, 9H); 13C NMR (300 MHz, CDCl3)
δ 169.1, 155.0, 148.2, 136.5, 131.5, 129.5, 129.4, 129.0, 128.1, 127.9,
126.8, 81.7, 42.0, 27.7. IR (thin film) ν 2986, 1739, 1688, 1524, 1370,
1226, 1164, 1153, 1116, 692 cmÀ1; HRMS (LC-ESI) m/z calcd for
C20H24N3O3 [MH]+ 354.1812, found 354.1814.
1222, 1152, 737, 698 cmÀ1
; HRMS (LC-ESI) m/z calcd for
C23H26N3O3 [MH]+ 392.1969, found 392.1969.
tert-Butyl benzhydrylidene aza-propargylglycinylamino
iso-butyrate
This was prepared using the protocol described earlier for phenylal-
anine dipeptide 7a employing the aza-dipeptide ester 6c (552 mg,
1.45 mmol, 1 eq) in THF (10 ml), 40% tetraethylammonium hydrox-
ide in H2O (1.60 ml, 4.35 mmol, 3 eq) and 80% propargyl bromide in
toluene (647 μl, 4.35 mmol, 3 eq). Flash chromatography, using
15–50% EtOAc in hexane as solvent system, and evaporation of
the collected fractions gave 7c (609mg, 91% yield); mp 125–127,
Rf = 0.62 (7: 3 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) δ 7.57
(m, 2H), 7.48–7.43 (m, 4H), 7.40–7.37 (m, 4H), 7.3 (s, 1H), 4.06 (d,
J = 1.8Hz 2H), 2.06 (t, J = 2.2Hz, 1H), 1.62 (s, 6H), 1.50 (s, 9H); 13C
NMR (300 MHz, CDCl3) δ 174.4, 157.8, 157.6, 139.1, 135.9, 130.3,
130.0, 129.5, 128.9, 128.9, 128.5, 81.4, 79.1, 72.1, 57.4, 34.9, 28.2,
25.6. IR (thin film) ν 2985, 1726, 1687, 1498, 1461, 1301, 1155,
1094, 949, 697 cmÀ1; HRMS (LC-ESI) m/z calcd for C25H29N3O3Na
[MNa]+ 442.2101, found 442.2107.
tert-Butyl benzhydrylidene aza-glycinylamino iso-butyrate (6c)
This was prepared using the protocol described earlier for
phenylalanine dipeptide 6a using benzophenone hydrazone
(1.35 g, 6.90 mmol, 1 eq), DSC (1.94 g, 7.59 mmol, 1.1 eq), DIEA
(2.3 ml, 13.8 mmol, 2 eq) and α-aminoisobutyric tert-butyl ester
(1.35 g, 6.90 mmol, 1 eq) in dry CH2Cl2 (54 ml) and DMF (10 ml).
Aza-dipeptide 6c (2.42 g, 92% yield) was obtained after purifica-
tion by flash chromatography with 15–40% EtOAc in hexane as
solvent system: mp 141–142, Rf = 0.28 (8 : 2 hexane/EtOAc); 1H
NMR (400 MHz, CDCl3) δ 7.55–7.50 (m, 6H), 7.36 (m, 3H), 7.26 (m,
2H), 7.09 (s, 1H), 1.66 (s, 6H), 1.53 (s, 9H); 13C NMR (300 MHz, CDCl3)
δ 174.3, 154.4, 147.8, 137.3, 132.1, 130.0, 129.5, 128.7, 128.6, 127.3,
81.7, 57.0, 28.1, 25.5. IR (thin film) ν 2984, 1723, 1677, 1512, 1461,
1449, 1302, 1154, 1105, 1068, 701, 693 cmÀ1; HRMS (LC-ESI) m/z
calcd for C22H27N3O3Na [MNa]+ 404.1945, found 404.1948.
The 1H and 13C NMR spectra for compounds 6b, 6c, 7b and 7c
are presented in the Supporting information.
Limitations
Although activation with DSC has given high yields with primary
amino esters, including the sterically bulky tert-butyl α-amino
iso-butyrate, symmetric urea 5 was not completely avoided in reac-
tions with proline tert-butyl ester. Aza-glycinyl-proline analogues
have been made using p-nitrophenylchloroformate as activating
agent, which remains the reagent of choice for their synthesis [12,13].
Benzhydrylidene aza-propargylglycinyl-L-phenylalanine
tert-butyl ester (7a)
A solution of benzhydrylidene aza-glycinyl-L-phenylalanine t-butyl
ester (6a, 500 mg, 1.13 mmol, 1 eq) in THF (15 ml) at 0 °C was
treated with 40% tetraethylammonium hydroxide in H2O
(502 μl, 3.37 mmol, 3 eq), stirred for 30 min, treated with 80%
propargyl bromide in toluene (502 μl, 3.37 mmol, 3 eq), heated
to 60 °C using microwave irradiation in a 300 MW Biotage
(Uppsala, Sweden) apparatus on the high absorption level with
automated temperature monitoring for 3 h and cooled to room
temperature, and the solvent was reduced. The volume was
diluted with CH2Cl2 (10 ml), and the organic phase was washed
three times with H2O, dried and evaporated. The residue was
purified by flash chromatography eluting with 1 : 9 EtOAc/hexane.
Evaporation of the collected fractions gave ester 7a as colorless
oil (394mg, 73% yield): the physical and spectroscopic properties
of 7a were identical to those reported in [19].
Acknowledgements
This research was supported by the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC), the Canadian Institutes of
Health Research (CIHR, grant #TGC-114046), the Ministère du
développement économique de l′innovation et de l′exportation
du Quebec (#878-2012, Traitement de la dégénerescence
maculaire) and Amorchem. The authors thank Dr A. Fürtös, K.
Venne, and M-C. Tang for their assistance with mass spectrometry.
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Supporting Information
Additional supporting information may be found in the online
version of this article at the publisher’s web site.
J. Pept. Sci. 2013; 19: 725–729 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci