Solid-phase synthesis of C-terminal azapeptides

Article abstract of DOI:10.1002/psc.2711

The solid-phase synthesis of azapeptides possessing a C-terminal aza-residue has been accomplished by a protocol featuring regioselective alkylation of benzhydrylidene-aza-glycinamide and illustrated by the syntheses of [aza-Lys⁶] growth-hormone-releasing peptide-6 analogs.

Full text of DOI:10.1002/psc.2711

Protocol
Received: 25 September 2014
Revised: 4 October 2014
Accepted: 13 October 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2711
Solid-phase synthesis of C-terminal azapeptides‡
Ngoc-Duc Doan, Jinqiang Zhang, Mariam Traoré, Winnie Kamdem
and William D. Lubell*
The solid-phase synthesis of azapeptides possessing a C-terminal aza-residue has been accomplished by a protocol featuring
regioselective alkylation of benzhydrylidene-aza-glycinamide and illustrated by the syntheses of [aza-Lys6] growth-hormone-
releasing peptide-6 analogs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: C-terminal azapeptides; regioselective alkylation; benzhydrylidene-protecting group; acylation of semicarbazide
exhibited longer duration of action relative to its parent peptide, albeit
Scope and Comments
with lower potency [6]. Furthermore, C-terminal aza-analogs of
luteinizing hormone releasing hormone (LHRH), such as [aza-Gly10]
Azapeptides are peptide derivatives in which the Cα atom of one or
more amino acids in the sequence has been replaced by a nitrogen
atom [1]. In azapeptides, the use of a semicarbazide as an amino
acid surrogate causes profound effects on the conformation and
physical properties of the peptide because of the nature of the
aza-residue. For example, spectroscopic, X-ray crystallographic and
computational studies all have indicated that the semicarbazide con-
straint can favor a turn conformation within the azapeptide [2–4].
Moreover, the aza-residue may enhance the stability of the peptide
against protease degradation [1,5]. In this regard, azapeptides offer
potential for improving peptide-based drug candidates. For example,
[aza-Val3] angiotensin-II (bovine) [Asp-Arg-aza-Val-Tyr-Val-His-Pro-Phe]
LHRH [7,8], retained significant biological activity and exhibited
*
Correspondence to: William D. Lubell, Département de Chimie, Université de
Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7.
E-mail: william.lubell@umontreal.ca
‡
Special issue of contributions presented at the 14th Naples Workshop on Bioactive
Peptides “The renaissance era of peptides in drug discovery”, June 12–14, 2014, Naples.
Département de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville,
Montréal, Québec, H3C 3J7, Canada
J. Pept. Sci. 2014
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

DOAN ET AL.
Figure 1. Chemical structure of Zoladex.
improved metabolic stability likely by avoiding degradation by
C-terminal peptidases. Studies of C-terminal aza-analogs of LHRH
led to the potent long-acting agonist pyroGlu-His-Trp-D-Ser(t-Bu)-
Tyr-Gly-Leu-Arg-Pro-aza-Gly-NH2 (goserelin acetate or Zoladex,
AstraZeneca, Figure 1), which is currently used in the clinic for the
treatment of prostate and breast cancer [9].
Benzhydrylidene-aza-glycinamide Rink resin 1 was synthesized by
activation of benzophenone hydrazone with DSC (Figure 2) [17] and
coupling of the activated benzhydrylidene-carbazate to the amine of
the Rink resin. Qualitative completion of the reaction was ascertained
by a negative Kaiser test; moreover, formation of benzhydrylidene-aza-
glycinamide was validated by cleavage of a resin aliquot with a freshly
made solution of TFA/H2O/triethylsilane (TES; 95/2.5/2.5), removal of
the resin by filtration, and analysis of the residue by LC-MS, which
indicated one single peak (retention time (R.T.) = 4.97 min, 50–90%
MeOH in water containing 0.1% formic acid (FA) over 10 min) having
the desired molecular ion (ESI-MS m/z calcd for C14H14N3O [M+H]+
240.1, found 240.1).
Three different side chains were initially introduced onto the
aza-residue by treating aza-glycinamide resin 1 with TEAH
(120 mol %, as a 40% aqueous solution) in THF followed by alkyl-
ation with the respective alkyl halide: 1-bromo-4-chlorobutane,
propargyl bromide, and 4-methylbenzyl bromide. Conversion
to the desired alkyl aza-glycinamide resins 2 was ascertained af-
ter cleavage of a resin aliquot with acid followed by LC-MS anal-
ysis (Figure 2). Moreover, resin 2c could be cleaved using a
freshly made solution of TFA/H2O/TES (95/2.5/2.5), and purifica-
tion of the residue by preparative HPLC gave aza-(4-methyl)
phenylalanine derivative 3c in 24% overall yield.
The 4-chlorobutyl and propargyl groups were selected to dem-
onstrate further methods for adding side chain diversity at the
C-terminal reside, respectively, by displacement of the chloride with
different amines and copper-catalyzed addition of Mannich reagents
to the alkyne (so-called A3-reaction) [19,20]. In principle, these side
chains may be respectively diversified using other nucleophiles to
displace the chloride [18], as well as by copper-catalyzed azide-
alkyne cycloaddition reactions [18,21].
In the case of 4-chlorobutyl glycinamide 2a, displacement of the
chloride with sodium azide gave the corresponding azalysine 4 in
90% conversion as determined by LC-MS of the crude residue after
TFA cleavage (Figure 3). Semicarbazone resin 4 was then converted
to semicarbazide 5 by transimination employing NH2OH · HCl in
pyridine at 60 °C for 12 h [11]. Semicarbazide 5 was coupled to
Fmoc-D-Phe-OH by way of its symmetric anhydride, which was pre-
pared using N,N′-diisopropylcarbodiimide (DIC) [18,22]. Azapeptide
6 was elongated by standard Fmoc-based solid-phase peptide syn-
thesis [18,20,22]. Azide 7 was reduced using tris(2-carboxy)
The application of azapeptides in drug discovery is expected to
grow with the development of effective solid-phase methods [10].
In particular, the discovery of a submonomer strategy featuring
regioselective alkylation of an aza-glycine moiety on a solid support
has unleashed azapeptide synthesis from the difficulties of solution-
phase hydrazine chemistry and enabled broader side chain diversity
to be introduced at aza-residues [11,12]. Nevertheless, the solid-
phase synthesis of C-terminal azapeptides has remained a challenge.
Notably, a limited number of C-terminal azapeptides have been
previously synthesized using N-(Fmoc)-aza-amino acid and (Ddz)-aza-
amino acid chlorides that were respectively prepared from N′-alkyl
9-fluorenylmethyl and 2-(3,5-dimethoxyphenyl)propan-2-yl carbazates
on activation with phosgene prior to coupling to the amine of the Rink
amide resin [13–15]. Side chain diversity is, however, limited in scope
using this approach, which necessitates synthesis of hydrazine build-
ing blocks in solution [16]; moreover, the toxicity of phosgene and side
products such as oxadiazole analogs from phosgene activation inhibits
the versatility of this method [1,17].
In efforts to further develop the submonomer strategy, we be-
came interested in C-terminal azapeptides and have recently
communicated an approach for synthesizing growth-hormone-
releasing peptide-6 (GHRP-6) analogs possessing C-terminal aza-
residues [18]. Key to this approach has been the application of
benzhydrylidene-aza-glycinamide Rink resin because of the ability
to measure conversion to the alkylated residue by comparison of
the starting and modified benzhydrylidene-aza-amino amides after
cleavage of the resin with acid and analysis by LC-MS. In this proto-
col, details are provided for the solid-phase method to prepare
C-terminal azapeptides having a broader side chain diversity, by a
route featuring (i) synthesis of the benzhydrylidene-aza-glycinamide
Rink resin, (ii) regioselective alkylation of the aza-glycinamide moiety,
(iii) removal of the semicarbazone, and (iv) coupling of amino acids to
the resulting semicarbazide employing the symmetric anhydride strat-
egy. In addition, the utility of this method has been demonstrated by
the synthesis of [aza-Lys6]GHRP-6 derivatives.
Figure 2. Synthesis and alkylation of a benzhydrylidene-protected aza-Gly resin.
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Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2014

C-TERMINAL AZAPEPTIDES
Figure 3. Synthesis of [aza-Lys6]GHRP-6.
ethylphosphine (TCEP) in THF/H2O (9/1) for 4 h to provide peptidyl-
resin 8 [18]. After deprotection and cleaving from the resin,
followed by purification by preparative HPLC, azapeptide 9 was iso-
lated in 7% overall yield and >95% purity (Figure 3).
Employing benzhyldrylidene aza-propargylglycinyl resin 2b, the
A3-reaction was performed in DMSO using diethylamine, aqueous
formaldehyde, and CuI to give resin 10 in complete conversion,
as assessed by LC-MS of the residue after resin cleavage (Figure 4)
[20]. Semicarbazone 10 was converted to its semicarbazide coun-
terpart, acylated with Fmoc-D-Phe and DIC, elongated, and cleaved
as described previously to provide [N,N′-diethylamino-aza-Lys6]
GHRP-6 analog 12 in 13% overall yield (>99% purity) after purifica-
tion by preparative HPLC.
to ascertain resin capacity. Reagents, including DSC, benzophenone
hydrazone, TEAH, hydroxylamine hydrochloride, pyridine, FA, N,
N-diisopropylethylamine (DIEA), diethylamine, 1-bromo-4-chlorobutane,
and 4-methylbenzyl bromide, all were purchased from Aldrich and used
without further purification. Protected amino acids Fmoc-L-Trp(Boc)-OH
and Fmoc-D-Trp(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-Ala-OH, and
Boc-His(Trt)-OH were purchased from Novabiochem (EMD Bioscience
Inc., San Diego, California) or GL Biochem, Ltd. (Shanghai, China). All
solvents were obtained from VWR International. Analytical LC-MS
analyses were performed on a 100-Å, 3-μm, 100 × 4.6-mm C18 SunFire
column™ with a flow rate of 0.35 ml/min using a gradient of distilled
water with 0.1% FA in acetonitrile with 0.1% FA. Peptide analogs were
purified on a preparative column (250 × 21.2 mm, 5 μm, Gemini™ C18)
using various gradients of distilled water with 0.1% FA in acetonitrile
with 0.1% FA at a flow rate of 10.0 ml/min.
Experimental Procedure
General
Synthesis of Benzhydrylidene-Protected aza-Gly Resin 1
Polystyrene Rink amide resin (0.6 mmol/g, 75–100 mesh) was purchased
The synthesis of benzhydrylidene-protected aza-Gly resin 1 was
performed using a free-amine Rink resin and benzophenone
from Advanced Chemtech™, and a standard Fmoc loading test was used
Figure 4. Synthesis of [N,N′-diethylamino-aza-Lys6]GHRP-6 analog 12.
J. Pept. Sci. 2014
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

DOAN ET AL.
hydrazone that was preactivated with DSC. In a round-bottom flask,
a solution of DSC (510mg, 2 mmol, 3.3 equiv) in DMF (8ml) was
treated dropwise with a solution of benzophenone hydrazone
(353 mg, 1.8 mmol, 3 equiv), stirred for 2 h at room temperature,
treated with DIEA (316 μl, 1.8 mmol, 3 equiv), and the resulting
solution was quickly transferred to a plastic syringe tube equipped
with a Teflon™ filter, stopper, and stopcock containing the free
amine of the Rink resin (1 g, 0.6 mmol/g, 1 equiv). After agitation
on an automated shaker for 16 h at room temperature, the resin
was filtered, washed with DMF (3 × 10 ml), MeOH (3 × 10 ml), and
DCM (3 × 10 ml), dried under vacuum, and stored in the fridge. The
reaction was monitored by the Kaiser test. The residue obtained
from cleavage of a resin aliquot (3 mg), after treatment with 1 ml
of TFA/TES/H2O (95 : 2.5 : 2.5, v/v/v), resin filtration, and evaporation,
was analyzed by LC-MS [50–90% MeOH (0.1% FA) in water (0.1%
FA) over 10 min], which indicated a single peak (R.T. = 4.97 min)
with a molecular ion corresponding to the desired product.
column (100 Å, 3.5 μm, 4.6 × 100 mm) demonstrated 3c to be of
>99% purity.
A3-Coupling of Benzhydrylidene-Protected aza-Propargylglycinamide
Resin (2a) Using Diethylamine and Formic Aldehyde
A syringe containing resin 2b (400 mg, 0.168 mmol) swollen in 4 ml of
DMSO was treated sequentially with diethylamine (104 μl, 1.01 mmol),
aqueous formaldehyde (82 μl, 1.01 mmol, 37% in H2O), and CuI
(6.5 mg, 0.034 mmol) and shaken on an automated shaker for 3 h. After
filtration, the resin was washed sequentially with AcOH/H2O/DMF
(5 : 15 : 80, v/v/v, 3 × 5 ml), THF (3 × 5 ml), MeOH (3 × 5 ml), and DCM
(3 × 5 ml). Examination by LC-MS of an aliquot of the residue from resin
cleavage using DCM/TFA (v/v, 1 : 1) for 20 min showed the desired am-
ide from azalysine analog 10 [R.T. = 5.24 min, 30–95% MeOH (0.1% FA)
in water (0.1% FA) over 10 min, ESI-MS m/z calcd for C22H27N4O [M
+H]+ 363.2, found 363.2], together with benzophenone as a side prod-
uct from semicarbazone hydrolysis. After cleavage of the resin
(200 mg, 0.084 mmol) using DCM/TFA (v/v, 1 : 1) for 30 min, filtration
of the resin, and evaporation of the filtrate and washings, the residue
was purified by preparative TLC. The amide from cleavage of aza-lysine
analog 10 was isolated as yellow oil (8 mg, 26%): 1H NMR (400 MHz,
CDCl3) δ 7.58–7.33 (m, 10H), 4.09 (t, J= 1.9 Hz, 2H), 3.36 (t, J=1.9Hz,
2H), 2.46 (q, J= 7.2 Hz, 4H), and 1.01 (t, J=7.2Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ 159.58, 158.85, 138.60, 135.45, 130.23, 129.84,
129.10, 128.70, 128.64, 128.15, 79.21, 78.28, 47.13, 40.67, 35.02, and
12.58. HRMS m/z calcd for C22H27N4O [M+H]+ 363.2185, found
363.2179.
Alkylation of Benzhydrylidene-Protected aza-Gly Resin 1
To a plastic syringe tube equipped with a Teflon™ filter, stopper, and
stopcock containing a suspension of benzhydrylidene-protected
aza-Gly resin 1 (200 mg, 0.6 mmol/g, 1 equiv) swollen in THF (3 ml),
a solution of 40% TEAH in water (233μl, 0.36 mmol, 3 equiv) was
added, and the mixture was agitated on an automated shaker for
30 min at room temperature. Propargyl bromide (80% in toluene,
40.5 μl, 0.36 mmol, 3 equiv), 4-methylbenzyl bromide (Figure 2,
bromide c; 100 μl, 0.72mmol, 6 equiv), or 4-bromo-chlorobutane
(44.3 μl, 0.38 mmol, 3.2 equiv) was respectively added to the resin
mixture, which was agitated for an additional 2 h for 4-bromo-
chlorobutane, or for an additional 12 h for propargyl and 4-
methylbenzyl bromides. The resin was filtered; washed with DMF
(3 × 10 ml), MeOH (3 × 10 ml), THF (3 × 10 ml), and DCM (3 × 10 ml);
and dried under vacuum. Aliquots of resins 2a and 2b were
respectively cleaved using a freshly made solution of TFA/H2O/TES
(95/2.5/2.5) for 1 h, and the residue was analyzed by LC-MS, which
indicated respectively single peaks for 3a [R.T. = 7.12 min, 20–80%
MeOH (0.1% FA) in water (0.1% FA) over 14 min, ESI-MS m/z calcd
for C18H20ClN3O [M+ H]+ 330.1, found 330.1] and for 3b [R.T.
= 5.79 min, 50–95% MeOH (0.1% FA) in water (0.1% FA) over
10 min, ESI-MS m/z calcd for C17H16N3O [M + H]+ 278.1, found 278.1].
Resin 2c (250 mg) was cleaved from the support using 5 ml of a
freshly made solution of TFA/H2O/TES (95/2.5/2.5) at room tempera-
ture for 2 h. The resin was filtered and rinsed twice with 2 ml of TFA.
The filtrate and rinses were evaporated to obtain yellow oil, which
was diluted in MeOH, and analyzed by LC-MS analysis [70–90% MeOH
(0.1% FA) in water (0.1% FA) over 14 min], which demonstrated 78%
conversion (R.T. = 9.47 min). The residue was purified on a Waters™
Prep LC instrument equipped with an RP Gemini™ C18 column
(250 × 21.2 mm, 5 μm) using a binary solvent system consisting of
70–90% MeOH (0.1% FA) in H2O (0.1% FA) at a flow rate of 10.0 ml/
min with UV detection at 254 nm over 50 min. Fractions containing
>99% pure product were combined, evaporated, and dried under
vacuum to give azapeptide 3c (14 mg, 0.04 mmol, 24% yield) as yellow
oil. 1H NMR (300 MHz, MeOD) δ ppm 2.27 (s, 3H), 3.32 (dd, J=3.3 and
1.6, 2H), 4.43 (s, 2H), 6.82 (d, J=7.0Hz, 2H), 7.04 (d, J= 7.0 Hz, 2H), 7.18
Conversion of Semicarbazone 4 to Semicarbazide 5
Benzhydrylidene-4-azidobutyl-aza-glycinamide Rink resin 4 (200 mg,
0.12 mmol) in a plastic syringe tube equipped with a Teflon™ filter,
stopper, and stopcock was treated with a freshly made solution of
1.5 M NH2OH · HCl in pyridine (5 ml), heated in a water bath at 60 °C
with sonication for 12 h, filtered, and washed with DMF (3 × 5 ml),
MeOH (3 × 5 ml), and DCM (3 × 5 ml). Full conversion to the corre-
sponding semicarbazide was determined by LC-MS analysis [20–
80% MeOH (0.1% FA) in water (0.1% FA) over 14 min, R.T. = 10.5 min]
of the residue obtained from cleavage of an aliquot of resin 5.
Coupling of N-(Fmoc)Amino Acid to Semicarbazide Resin 5
and Azapeptide Elongation
In dry DCM (10 ml), Fmoc-D-Phe-OH (1.33g, 3.45 mmol, 10 equiv)
and DIC (270 μl, 1.73 mmol, 5 equiv) were stirred at room temperature
for 30 min. The resulting suspension was concentrated in vacuo to a
residue, which was dissolved in DMF (10 ml) and added to a plastic
syringe tube equipped with a Teflon™ filter, stopper, and stopcock
containing semicarbazide resin 5 (500 mg, 0.345 mmol). After agitation
for 16 h at room temperature on an automatic shaker, the reaction was
shown to have >90% conversion to azadipeptide 6 by LC-MS analysis
[10–80% MeOH (0.1% FA) in water (0.1% FA) over 12 min, R.T.
= 8.3 min] of the residue obtained from cleavage of a resin aliquot
(3 mg). Subsequent Fmoc removal and couplings to complete the
target sequence were performed according to conventional Fmoc-
based SPPS protocols [23]. Azide 7 was reduced to peptidyl-resin 8
using TCEP as previously reported [18]. Deprotection and resin cleav-
age with TFA/TES/H2O (95:2.5:2.5, v/v/v) gave a crude residue, which
was purified by preparative RP-HPLC using binary solvent systems
consisting of 5–30% MeOH (0.1% FA) in H2O (0.1% FA) over 60 min.
Pure fractions were combined, freeze-dried, and lyophilized to a white
(dd, J=8.0 and 1.6 Hz, 2H), 7.25–7.36 (m, 4H), and 7.36–7.57 (m, 4H). 13
C
NMR (100 MHz, MeOD) δ ppm 22.0, 128.2, 129.3, 130.0, 130.6, 130.7,
131.0, 132.0, 132.3, 135.8, 138.0, 138.7, 140.7, 163.5, and 164.0. High
resolution MS (HRMS) calcd m/z for C22H22N3O [M+H]+ 344.1757,
found 344.1772. Analysis by LC-MS [70–90% MeOH (0.1% FA) in H2O
(0.1% FA) over 14 min, R.T. = 6.0 min] on a SunFire C18 analytical
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Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2014

C-TERMINAL AZAPEPTIDES
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[0–50% MeCN (1% FA) in water (1% FA) over 14 min] R.T. =
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a
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over 10 min] R.T. = 7.63 min, purity >99%; [5–60% MeCN (1% FA) in
water (1% FA) over 10 min] R.T. = 8.65 min, purity >99%; HRMS m/z
calcd for C49H59N13NaO6 [M + Na]+ 948.4609, found 948.4615.
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Although the benzhydrylidene protection offers a useful means for
analyzing conversion of the alkylation of the aza-glycinamide resi-
due, attempts to isolate the corresponding benzhydrylidene-
protected aza-amino amides have met with varying success be-
cause of hydrolysis and loss of benzophenone. In the alkylation
step, steric hindrance from the resin and the reactivity of the alkyl
halide influence conversion and reaction times, limiting use of
bulky alkyl halides. The quality of the resin has influenced the per-
formance of the described chemistry, and certain resins that have
proven effective for normal SPPS have given lower yields of
azapeptide for reasons that are poorly understood at this time.
14 Boeglin D Lubell WD Aza-amino acid scanning of secondary structure
suited for solid-phase peptide synthesis with fmoc chemistry and aza-
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Acknowledgement
This research was supported by the Natural Sciences and Engineering
Research Council of Canada, the Canadian Institutes of Health Research
(grant no. 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.
17 Garcia-Ramos Y Lubell WD Synthesis and alkylation of aza-glycinyl
dipeptide building blocks. J. Pept. Sci. 2013; 19: 725–9.
18 Traore M, Doan ND Lubell WD Diversity-oriented synthesis of
azapeptides with basic amino Acid residues: aza-lysine, aza-ornithine,
and aza-arginine. Org. Lett. 2014; 16: 3588–91.
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