Solid-Phase Azopeptide Diels-Alder Chemistry for Aza-pipecolyl Residue Synthesis to Study Peptide Conformation

Article abstract of DOI:10.1021/acs.joc.8b03283

Solid-phase chemistry for the synthesis and Diels-Alder reaction of Fmoc-protected azopeptides has been developed and used to construct aza-pipecolyl (azaPip) peptides. Considering their ability to induce electronic and structural constraints that favor c

Full text of DOI:10.1021/acs.joc.8b03283

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Article
Solid-Phase Azopeptide Diels-Alder Chemistry for Aza-
Pipecolyl Residue Synthesis to Study Peptide Conformation
Ramesh Chingle, Mukandila Mulumba, Nga N. Chung, Thi Mai-Dung
Nguyen, Huy Ong, Steven Ballet, Peter W. Schiller, and William D. Lubell
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b03283 • Publication Date (Web): 08 Apr 2019
Downloaded from http://pubs.acs.org on April 9, 2019
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Solid-Phase Azopeptide Diels-Alder Chemistry for Aza-Pipecolyl
Residue Synthesis to Study Peptide Conformation
†
‡
§
§
, ‡
,┴
,§,ǁ
Ramesh Chingle, Mukandila Mulumba, Nga N. Chung, Thi M.-D. Nguyen, Huy Ong,* Steven Ballet,* Peter W. Schiller,*
and William D. Lubell*,†
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‡
ǁ
Dꢀpartement de Chimie, Facultꢀ de Pharmacie and Département de Pharmacologie et Physiologie, Universitꢀ de Montrꢀal, C.P.
6128, Succursale Centre-Ville, Montrꢀal, Quꢀbec H3C 3J7, Canada
§
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montrꢀal,
Quꢀbec H2W 1R7, Canada
┴
Research Group of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
KEYWORDS: GHRP-6, Growth hormone releasing peptide 6, CD36 receptor, cluster of differentiation 36, N,N’-disuccin-
imidyl carbonate, agonist opioid peptides, structure-activity relationships.
ABSTRACT: Solid-phase chemistry for the synthesis and Diels-Alder reaction of Fmoc-protected azopeptides has been devel-
oped and used to construct aza-pipecolyl (azaPip) peptides. Considering their ability to induce electronic and structural con-
straints that favour cis-amide isomer geometry and type VI β-turn conformation in model peptides, azaPip residues have now
been introduced into biologically relevant targets by this enabling synthetic method. Turn conformers were shown to be
important for receptor affinity, selectivity and activity by employing azaPip residues to study the conformational require-
ments of opioid and cluster of differentiation 36 receptor peptide ligands.
INTRODUCTION
The therapeutic potential of peptides can be enhanced by synthetic modifications that preserve activity and improve bioa-
1
2
vailability, stability and selectivity. In light of the privileged role of β-turn secondary structures in molecular recognition,
3
many methods have been particularly developed to synthesize rigid peptide turn surrogates. Effective approaches are how-
ever still needed for the introduction of specific turn geometry systematically into peptide structures using solid-phase chem-
4
istry to enable examination of their relevance for activity by library synthesis in biomedical research. Effective methods for
introducing linear semicarbazide residues into azapeptides have for example expanded the use of such amino amide surro-
gates in mimics of type I and II’ β-turns.5
Although rarer than their trans counterparts, cis isomers of the amide N-terminal to proline (the so-called prolyl amide)
6
and the corresponding type VI β-turns play important roles in peptide folding and molecular recognition. Type VI β-turns
have been favored in model peptides using aza-proline (azaPro) and its homologue aza-pipecolic acid (azaPip), because re-
placement of the C
α
H by nitrogen in these cyclic residues causes electrostatic repulsion that disfavors the prolyl amide trans-
7
isomer to promote the cis-isomer (Figure 1). The biological relevance of prolyl amide cis-isomers has been studied with
8
azaPro in several peptides: hCGRP, TRH, collagen and mucin. In spite the presence of pipecolate (Pip) in numerous biologi-
cally active peptides,8b,9 and the potential of Pip as a Pro surrogate,7h,10 azaPip has never been employed to study biological
1
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activity, except in small molecule PPIase ligands. Although merited, study of azaPip has been handicapped by difficulties
to synthesize and couple cyclic aza-amino acids,7d,7g,7h which have required building block preparation in solution prior to
11
introduction into peptides. To enable the use of azaPip to study peptide chemical biology, a novel solid-phase Fmoc-based
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approach has been conceived, featuring the Diels-Alder chemistry of azo-glycine residues, and targeted on representative
opioid and cluster of differentiation-36 receptor (CD36) ligands.
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Figure 1. a) In Pro (n = 1) and Pip (n = 2), trans-amide isomer is favored. b) In azaPro and azaPip, electrostatic repulsion in the
trans-amide isomer drives the equilibrium towards the cis-isomer.
Potent and subtype selective opioid peptides were pursued in the context of programs to control pain and suppress detri-
mental effects of clinically applied analgesics. Morphiceptin (H-Tyr-Pro-Phe-Pro-NH
Phe-NH ) and endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH ) were selected due to their agonist activity and high selectivity for
the µ-opioid (MOP) receptors. Dynamic equilibria about the Tyr-Pro prolyl amide and a cis-conformer have been supported
2
),13 endomorphin-1 (H-Tyr-Pro-Trp-
2
2
1
4
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for the activity of these opioid peptides using a series of Pro surrogates, combined with ring substituted Tyr and Phe resi-
1
6
dues. Moreover, an active Dmt-Tic amide cis-isomer was concluded based on the crystal structure of the bifunctional MOP
agonist/δ-opioid (DOP) receptor antagonist Dmt-Tic-Phe-Phe-NH
2
(Dmt = 2′,6′-dimethyltyrosine, Tic = 1,2,3,4-tetrahydroi-
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soquinoline-3-carboxylate) in complex with the human DOP receptor.
Aza-analogs of growth hormone releasing peptide-6 (GHRP-6, H-His-ᴅ-Trp-Ala-Trp-ᴅ-Phe-Lys-NH ) bind CD36 selectively
2
and exhibit promise for treating indications implicating macrophage-driven inflammation, such as atherosclerosis, in part
due to the potential of modulating this co-receptor of the Toll-like receptor (TLR)-2/6 heterodimer involved in the recognition
19
and phagocytosis of multiple ligands (e.g., oxidized lipoproteins and thrombospondin). In contrast to GHRP-6, which exhibits
3
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a random coil conformation that lacks receptor selectivity, azaPro and azaPhe analogs have selective binding affinity for
CD36, likely due to preferred turn conformations as observed by circular dichroism (CD) and NMR spectroscopy.5e,5f
Peptide conformation–activity relationships have thus been examined in opioid and CD36 ligands by installing azaPip in
2
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place of respectively Pro in [ᴅ-Phe ]morphiceptin, and endomorphin-1 and -2, and Ala and Trp in GHRP-6. Diels-Alder re-
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actions between azoGly and butadiene or 2,3-dimethylbutadiene gave respectively (Δ )-azaPip and (4,5-dimethyl-Δ )-aza-
4
dehydropipecolate [(Δ )-azaDmp] residues, and the former were reduced after resin cleavage to azaPip peptides. Agonist
potency and selectivity were shown to be influenced by azaPip in bioassays based on inhibition of electrically induced con-
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tractions of the guinea pig ileum (GPI), and mouse vas deferens (MVD), in which opioid activity is respectively mediated
predominantly by the MOP and DOP receptor subtypes. Moreover, [azaPip]GHRP-6 analogs were shown to modulated CD36-
mediated overproduction of nitric oxide (NO), a marker of inflammatory response in macrophage cells activated with the
TLR-2/6 heterodimer agonist R-FSL-1, as quantified in a nitrite assay.22 An effective solid-phase strategy for synthesizing
azaPip peptide libraries has thus been conceived and used to identify pertinent conformers for biological activity.
2
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RESULT AND DISCUSSION
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A three-step process was devised to insert azaPip into peptides by modifying conventional Fmoc-based Solid-Phase Pep-
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tide Synthesis (SPPS): 1) aza-amino acylation, 2) azaGly to azoGly oxidation, and 3) [4+2]-cycloaddition (Scheme 1). Subse-
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quent Fmoc removal, semicarbazide acylation, elongation, deprotection and cleavage gave (Δ )-azaPip peptides, which were
hydrogenated in solution to aza-Pip analogs (Schemes 3 and 4).
The azaGly precursor, FmocNHNH
, which exhibited the right balance of stability and reactivity in couplings to peptides linked to Rink amide resin, to provide
azaGly peptides 2a-e without oxadiazalone formation (Scheme 1).24 Although N-(Boc)- and N-(Cbz)-azaGly had previously
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was treated with N,N’-disuccinimidyl carbonate (DSC) to provide activated carbazate
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oxidized to azoGly using N-bromosuccinimide (NBS) in a pyridine / dichloromethane solution, judicious employment of the
sterically hindered base 2,6-lutidine at −40 °C to rt was needed to circumvent Fmoc loss and enable oxidation of 2a-e on resin
to N-(Fmoc)-azoGly peptides 3a-e, respectively.
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Scheme 1. Installation of (Δ )-aza-pipecolyl residue
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Azopeptide resins 3a-e exhibited characteristic IR N=N stretch bands between 1700 to 1760 cm−1 [see supporting infor-
26
mation (SI)]. Diels–Alder reactions on 3a-e with butadiene and 2,3-dimethylbutadiene in CH
2
2
Cl at –78 °C with warming to
4
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rt gave respectively (Δ )-azaPip and (Δ )-azaDmp resins 4a-e and 5a-c,e. Removal of the Fmoc group using 20% piperidine
in DMF provided semicarbazides 6a-e and 7a-c,e.25
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Scheme 2. Synthesis of Fmoc-(∆ )azaPip-ᴅ-Phe-Lys-NH
2
(8):
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(8) was examined using H and 13C NMR spec-
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The structure of aza-pipecolyl tripeptide Fmoc-(∆ )azaPip-ᴅ-Phe-Lys-NH
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troscopy, after cleavage from resin 4d using a freshly made solution of TFA/H
2
O/TES (95:2.5:2.5, v/v/v, 20 mL/g resin) at
1
room temperature for 2 h and purification by preparative RP-HPLC (Scheme 2). In the H NMR spectrum of 8, the presence of
1
the vinyl protons at 5.87 – 5.81 and 5.81 – 5.74 ppm were indicative of the successful cycloaddition. The sets of sharp H and
13C NMR signals for 8 may be indicative of a single conformational isomer, rather than rapid equilibration of urea and carba-
mate isomers on the NMR timescale.27 ,
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Scheme 3. Conversion of (Δ )-azaPip (6a-c) and (Δ )-azaDmp (7a-c) peptides to [azaPip ]-morphiceptin and endo-
morphin analogs 14-19
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The poorly nucleophilic cyclic semicarbazide structure rendered acylation of (Δ )-azaPip and (Δ )-azaDmp peptides 6 and
challenging, such that common coupling conditions gave little success,28 e.g., N,N′-diisopropylcarbodiimide
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(DIC)/hydroxybenzotriazole (HOBt), DIC/ethyl cyano(hydroxyimino)acetate (oxyma), 1-hydroxy-7-azabenzotriazole
(HATU), benzotriazol-1-yloxytris(pyrrolidinophosphonium-hexafluorophosphate (PyBOP), n-propylphosphonic cyclic anhy-
dride (T3P), (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylaminomorpholino-carbenium hexafluorophosphate
COMU)],28 as ascertained by LC/MS analysis after resin cleavage. In contrast, the cyclic semicarbazides reacted effectively
(
with N-Fmoc-amino acid chlorides (300 mol%), which were generated with bis-(trichloromethyl)carbonate (BTC), and
coupled under microwave irradiation with the resin swollen in 2,4,6-collidine and THF (Schemes 3 and 4).8d,29 For example,
4
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Fmoc-Tyr(t-Bu)-Cl reacted with (Δ )-azaPip (6a-c) and (Δ )-azaDmp (7a-c) resins to provide respectively azapeptides 10a-c
and 11a-c in good conversion (see SI). Acid chlorides from Fmoc-Ala-OH and Fmoc-ᴅ-Trp(Boc)-OH reacted similarly with
4
4
(
Δ )-azaPip (6d-e) and (Δ )-azaDmp (7e) peptides to give the corresponding azapeptides 20-22. On the other hand, acid
4
chloride that was derived from sterically demanding Fmoc-Dmt-OH (9) reacted respectively with (Δ )-azaPip peptides 6a and
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a to provide azapeptides 12a and 13a in lower conversion, as indicated by the relatively lower final [Dmt ]morphiceptin
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yields (See SI). In spite the challenging coupling with Fmoc-Dmt-Cl, three [Dmt , azaPip , ᴅ-Phe ]morphiceptin derivatives
were prepared for the initial study and exhibited notably high affinity and selectivity as discussed below.30
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Scheme 4. Conversion of (Δ )-azaPip (6d-e) and (Δ )-azaDmp (7e) peptides to [azaPip]-GHRP-6 analogues 23-27
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After Fmoc removal, resin cleavage and removal of side chain protection were performed with a freshly made
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TFA/TES/H
2
O (95:2.5:2.5, v/v/v) cocktail to provide (Δ )-azaPip morphiceptins and endomorphins 14-17 (Scheme 3). Using
2
3
standard Fmoc-based SPPS, azapeptides 20-22 were elongated by couplings with DIC and HOBt in DMF. Resin cleavage as
4
described above, precipitation with Et
2
O, dissolution in 1:1 MeCN/H O, and freeze-drying gave [(Δ )-azaPip]GHRP-6 analogs
2
2
3-25 as white foams. After preparative RP-HPLC purification, pure fractions were combined and freeze-dried to white fluffy
4
powders. Final (Δ )-azaPip peptide purity was analyzed by analytical RP-HPLC in two different solvent systems (See Table 1).
4
After purification, (Δ )-azaPip opioid and GHRP-6 peptides 14a-c, 16a, 23 and 24, all were respectively hydrogenated using
Pd/C (10 wt%) in MeOH under 1 atm of hydrogen. Saturated azaPip peptides 18a-c, 19a, 26 and 27 were isolated after cata-
lyst filtration over CeliteTM, washing with MeOH, and evaporation of the combined filtrate and washings. The residue was
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freeze-dried to furnish off-white solids of >95% purity, which were examined for activity without further purification
Schemes 3 and 4, See Table 1).
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(
Table 1. Overall yields, final purity, retention times, and mass spectrometric analyses of aza-pipecolate
morphiceptin, endomorphin-1, endomorphin-2 and GHRP-6 analogues 14-19 and 23-27
HPLC
RT (min)a
CH OH CH CN
+
+
MS [M + 1] or [M + Na]
Isolated
Yield
No.
Compound
purity
0
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at 214b
m/z (calcd) m/z (obsd)
(
%)
nm
4
12
8.2
9
7.55
8.37
8.25
6.70
7.38
7.32
6.14
6.42
6.61
5.60
4.76
5.12
5.18
4.34
>95
535.2663 535.2679
624.2929 624.2944
585.2820 585.2835
563.2976 563.2964
652.3242 652.3234
613.3133 613.3112
563.2976 563.2962
591.3289 591.3284
537.2820 537.2826
626.3085 626.3099
587.2976 587.2991
565.3133 565.3138
797.4206 797.4189
912.4628 912.4628
962.4760 962.4764
799.4362 799.4381
914.4784 914.4780
1
4a H-Tyr-(∆ )azaPip-ᴅ-Phe-Pro-NH
4
>99
>99
>99
>98
>98
>99
>96
>95
>99
>99
>98
>99
>98
>95
>99
>96
14b H-Tyr-(∆ )azaPip-Trp-Phe-NH
2
4
1
1
1
4c H-Tyr-(∆ )azaPip-Phe-Phe-NH
2
4
8.6
6.5
7.2
4.6
2
5a H-Tyr-(4,5-dimethyl-∆ )azaPip-ᴅ-Phe-Pro-NH
2
4
5b H-Tyr-(4,5-dimethyl-∆ )azaPip-Trp-Phe-NH
2
4
15c H-Tyr-(4,5-dimethyl-∆ )azaPip-Phe-Phe-NH
2
4
1
1
1
6a H-Dmt-(∆ )azaPip-ᴅ-Phe-Pro-NH
2
4
7.01 4.96
7a H-Dmt-(4,5-dimethyl-∆ )azaPip-ᴅ-Phe-Pro-NH
2
1
2
5.73
6.61
6.47
6.09
4.87
5.98
4.91
5.15
6.07
4.07
4.68
4.58
4.29
3.92
5.82
5.26
3.97
4.70
8a H-Tyr-azaPip-ᴅ-Phe-Pro-NH
2
8
.2
18b H-Tyr-azaPip-Trp-Phe-NH
2
9
1
1
8c H-Tyr-azaPip-Phe-Phe-NH
2
4
.6
9a H-Dmt-azaPip-ᴅ-Phe-Pro-NH
2
4
4.2
3.9
4.6
23 His-ᴅ-Trp-Ala-(∆ )azaPip-ᴅ-Phe-Lys-NH
2
4
2
2
2
4 His-ᴅ-Trp-(∆ )azaPip-Trp-ᴅ-Phe-Lys-NH
2
4
5 His-ᴅ-Trp-(4,5-dimethyl-∆ )azaPip-Trp-ᴅ-Phe-Lys-NH
2
4
3
.2
.9
6 His-ᴅ-Trp-Ala-azaPip-ᴅ-Phe-Lys-NH
2
27 His-ᴅ-Trp-azaPip-Trp-ᴅ-Phe-Lys-NH
2
aAnalytical HPLC conditions: 1) 14a-c, CSH C18 column (4.6 mm × 100 mm, 5 μm), flow rate 0.5 mL/min, linear gradient of 20−95% water (0.1% FA)
in CH CN (0.1% FA) or in MeOH (0.1% FA); 2) 15-19, 23-24, and 26-27, Sunfire™ C18 column (2.1 mm x 50 mm, 3.5 μm), flow rate of 0.4 mL/min,
linear gradient 10−90% water (0.1% FA) in CH CN (0.1% FA) or in MeOH (0.1% FA); 3) 25, BEH C18 column (2.1 mm x 100mm, 1.7 μm), flow rate
of 0.5 mL/min, linear gradient 2−98% water (0.1% FA) in CH
3
3
b
c
3
CN (0.1% FA) or in MeOH (0.1% FA). Purified peptide HPLC purity at 214 nm. Ob-
+
+
served mass of H and Na adduct.
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Opioid peptide biology
Table 2. GPI and MVD Assay of Opioid Peptide Analogues 14-19
GPI
MVD
MVD/GPI
IC50 ratio
No. Compound
IC50 (nM)a
IC50 (nM)a
Morphiceptin30a
318 ± 22
109 ± 16
4800 ± 440
594 ± 77
15.09
5.45
2.84
0.51
2.45
0.36
5.81
2.59
94.43
1.95
1.53
3.07
-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[
ᴅ-Phe3]30b
4
2
3
1
4a [(∆ )-azaPip , ᴅ-Phe ]
113 ± 29
321 ± 45
4
2
3
15a (∆ )-azaDmp , ᴅ-Phe ]
737 ± 108
0.723 ± 0.073
5.52 ± 0.19
17.4 ± 4.1
0.845 ± 0.058
6.46 ± 0.51
0.367 ± 0.014
17.2 ± 2.4
85.7 ± 13.7
Inactive
373 ± 44
1
4
2
3
1
6a [Dmt ,(∆ )-azaPip , ᴅ-Phe ]
1.77 ± 0.155
1.96 ± 0.33
101 ± 9
1
4
2
3
1
1
1
7a [Dmt , (∆ )-azaDmp , ᴅ-Phe ]
2
3
8a [azaPip , ᴅ-Phe ]
1
2
3
9a [Dmt , azaPip , ᴅ-Phe ]
2.19 ± 0.12
610 ± 16
1
2
[Dmt , Pip ]
1
2
3
[
Dmt , Pip , ᴅ-Phe ]
0.717 ± 0.077
26.3 ± 3.9
263 ± 38
Endomorphin-130c
4
2
1
1
4b [(∆ )-azaPip ]
4
2
= 1210± 79b
460 ± 122
15.3 ± 1.8
393 ± 16
5b [(4∆ )-azaDmp ]
K
e
18b [azaPip2]
93.8 ± 10.0
7.71 ± 1.47
116 ± 31
4.90
1.98
3.39
-
Endomorphin-230c
4
2
1
1
1
4c [(∆ )-azaPip ]
4
2
5c [(∆ )-azaDmp ]
3980 ± 30
961 ± 28
Inactive
8c [azaPip2]
2160 ± 340
2.25
a
b
4
Mean ± SEM; Antagonist; K
e
determined against DPDPE ([2-ᴅ-penicillamine,5- ᴅ-penicillamine]enkephalin); [(Δ )-azaDmp] = (4,5-
4
dimethyl-Δ )-aza-dehydropipecolate.
2
3
The influence of replacing Pro by azaPip residues in [ᴅ-Phe ]morphiceptin, as a stabilized and more potent analogue of
morphiceptin, and endomorphins-1 and -2 was ascertained by measuring potency and µ- and δ-receptor subtype selectivity
in the GPI and MVD assays.20-21 The activity of azaPip was compared relative to unsaturated variants (Δ )-azaPip and (Δ )-
4
4
1
2
3
azaDmp (Table 2). Replacement of Dmt for Tyr in [azaPip , ᴅ-Phe ]morphiceptin was performed to assess influences of 2’,6’-
dimethyl substituent hydrophobicity and constraint on -dihedral angle geometry on receptor subtype selectivity and affin-
ity.16a,31
3
2
2
In [ᴅ-Phe ]morphiceptin, the Pro to azaPip switch caused a nearly 6-fold improvement in potency in 18a in the GPI and
2
4
2
3
MVD assays with little change in MOP selectivity. Relative to azaPip 18a, unsaturated [(Δ )-azaPip , ᴅ-Phe ]morphiceptin
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4
2
4
analogs were less potent and less selective: (Δ )-azaPip 14a and (Δ )-Dmp 15a were respectively equipotent and about 7-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
fold less potent in the GPI assay, and both were about 2-fold more potent in the MVD assay, relative to [ᴅ-Phe ]morphiceptin.
2
2
On the other hand, replacement of Pro by azaPip in endomorphin-1 analog 18b resulted respectively in 5- and 17-fold
decreases in potency in the GPI and MVD assays, with a net increase in µ selectivity. In endomorphin-2 analog 18c, similar
substitution caused >100-fold reduction in potency without significant influence on µ/δ selectivity. Endomorphin analogs
4
4
2
1
5b and 15c containing (Δ )-Dmp were nearly inactive; however, (Δ )-azaPip counterparts 14b and 14c were nearly and
2
strikingly more potent than their saturated counterpart, yet not as active as the natural Pro endomorphins.
1
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Replacement of Tyr for Dmt in [azaPip , ᴅ-Phe ]morphiceptin analogs 16a, 17a and 19a augmented significantly potency
in both the GPI (20 to 150-fold increase) and MVD (>250-fold increase) assays with about a 2- to 15-fold reduction in
2
MOP/DOP selectivity. For comparison, [Pip ]morphiceptins (H-Dmt-Pip-Phe-Pro-NH
2
and H-Dmt-Pip-ᴅ-Phe-Pro-NH ) were
2
3
1
synthesized. Paralleling the activity of morphiceptin and [ᴅ-Phe ]-morphiceptin, insertion of ᴅ-Phe at position 3 of the [Dmt ,
2
1
2
Pip ]morphiceptin analogs ameliorated potency and lowered MOP/DOP selectivity. Notably, [Dmt , Pip ]morphiceptin exhib-
1
2
3
ited the most significant µ/δ selectivity of all analogs studied. The peptide analogue, [Dmt , Pip , ᴅ-Phe ]morphiceptin dis-
2
played slightly enhanced potency relative to azaPip 19a, but slightly reduced MOP/DOP selectivity (Table 2). Balanced
MOP/DOP agonists have shown beneficial pharmacological profiles, as compared to highly selective MOP agonists.32
Binding affinities for the µ-, δ- and κ-opioid receptors were determined for the most potent analogs in displacement stud-
3
3
2
2
1
3
ies using tritium-labeled standards (Table 3). The Pip and azaPip [Dmt , ᴅ-Phe ]morphiceptins had 4- and 34-fold greater
affinity for the µ-receptor subtype relative to the literature value for morphiceptin, which binds with about 2-fold better af-
3
2
finity than [ᴅ-Phe ]morphiceptin. They also bound the δ-receptor subtype with significant affinity. Replacement of Pip by
2
1
3
azaPip in the [Dmt , ᴅ-Phe ]morphiceptin analogs resulted respectively in 3- to 8-fold, 1- to 2-fold, and 3- to 4-fold decreases
in binding affinity at the µ, δ and κ-opioid receptor subtypes (Table 3).
Table 3: Opioid receptor μ, , and κ binding affinities of morphiceptin analogues 16a, 17a and 19a
[3
H]DAMGO
[3H]DSLET
[3H]U69,593
No.
Compound
/

a

a

a
Ki (nM)
Ki (nM)
Ki (nM)
Morphiceptin30a
19.8 ± 0.9
35.2 ± 6.3
>10000
>10000
-
-
-
-
[
[
ᴅ-Phe3]30b
1
2
3
Dmt , Pip , ᴅ-Phe ]
0.576 ± 0.021
1.72 ± 0.24
4.80 ± 0.26
1.61 ± 0.08
1.40 ± 0.12
2.89 ± 0.38
1.47 ± 0.03
2.61 ± 0.29
45.1 ± 0.8
166 ± 6
1/2.4/78
1/1.7/97
1/0.3/32
1
4
2
3
1
1
6a [Dmt ,(∆ )-azaPip , ᴅ-Phe ]
1
4
2
3
7a [Dmt , (∆ )-azaDmp , ᴅ-Phe ]
153 ± 34
193 ± 10
1
2
3
1/1.6/12
0
6
19a [Dmt , azaPip , ᴅ-Phe ]
a
2
4
5
2
5
Mean of 3-4 determinations ± SEM; DAMGO = [ᴅ-Ala ,N-MePhe ,Gly-ol ]enkephalin; DSLET = [ᴅ-Ser ,Leu ]enkephalin-Thr ;
U69593 = (5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8 yl]benzeneacetamide.
2
2
3
Improved potency and affinity on switching Pro for azaPip in [ᴅ-Phe ]morphiceptin may be explained by stabilization of
amide cis-isomer geometry, confirming the requisite for a natural Tyr-Pro cis-peptide bond for bioactive receptor binding.30a
1
2
3
Notable activity and affinity of [Dmt , Pip , ᴅ-Phe ]morphiceptin may reflect the lower barrier for amide isomerization N-
terminal to Pip relative to Pro,27b because rapid isomer equilibration may facilitate cis isomer binding. The enhanced µ/κ
1
3
selectivity ratios (32 – 120) of the azaPip relative to the Pip [Dmt , ᴅ-Phe ]morphiceptins are therapeutically relevant, because
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centrally active κ-opioid receptor agonists have been shown to cause dysphoria and sedation, side effects that preclude ther-
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5
6
34
apeutic development. Differences between the L-Pip and azaPip analogs may be due to the latter having greater polarity and
inability to completely adopt (S)-configuration.35
CD36 modulator activity
3
4
Five analogs of GHRP-6 were made by azaPip insertion respectively at the Ala and Trp positions, and examined for ability
to modulate NO overproduction in RAW macrophage cell lines after stimulation with the TLR-2/6 heterodimer agonist R-FSL-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
4
3
1
(Figure 2).5f,5i Notably, [(Δ )-azaPip ] and [(Δ )-azaPip ]GHRP-6 (23 and 24) caused similarly significant reductions (30%)
4
–6
of NO production as the positive control [azaY ]GHRP-6 at [10 M]. Saturated azaPip counterparts 26 and 27 also inhibited
4
3
R-FSL-1-induced NO production relative to control, but [(Δ )-azaDmp ]GHRP-6 26 was inactive. The effects of azaPip analogs
on NO production demonstrate further the importance of turn geometry for activity.5f
1
1
2 0
0 0
* *
*
***
8
6
4
2
0
0
0
0
0
*
**
R -F S L -1 (3 0 0 n g /m L )
Figure 2. Effects of [azapipecolyl]-GHRP-6 analogues 23-27[10–6 M] on the overproduction of NO induced by the TLR-2 agonist R-
FSL-1 in RAW macrophage cell line.
CONCLUSION
The high interconversion barrier and low energy differences of prolyl amide isomers accounts for observance of 25-40%
cis conformer in peptides.30b,36 In solution, the trans-isomer is favored due to steric and electronic reasons,36b,37 but prolyl cis-
6
6a,30b,36-38
isomers may favor receptor binding and activity. Steric interactions can favor prolyl amide cis-isomer,
but may in-
terfere with receptor binding giving unclear biological outcomes.38e,38f Employing electronic effects to stabilize cis-isomer ge-
ometry, azaPip offers effective means to study active conformers without complications due to steric effects on peptide–re-
ceptor interactions.
A new SPPS method was developed to make azaPip peptides by a route featuring DSC activation for aza-amino acylation,
4
NBS and 2,6-lutidine for aza- to azo-glycine oxidation, Diels−Alder chemistry with different dienes, (Δ )-azaPip acylation with
N-(Fmoc)amino acid chlorides and hydrogenation to azaPip peptides. Among seventeen azaPip derivatives synthesized from
1
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opioid and CD36 ligands, many exhibited improved biological activity lending insight about conformational requirements for
activity and receptor selectivity. Considering the plethora of biologically active Pro- and Pip-peptides, effective azaPip access
offers valuable means to study the cis-isomer backbone geometry and type VI β-turns for conception of potent and selective
prototypes for drug discovery.
1
2
3
4
5
6
7
8
9
1
1
1
1
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5
6
EXPERIMENTAL SECTION
In Vitro Bioassays and Receptor Binding Assays The GPI and MVD bioassays were performed by measuring inhibitory
concentrations on electrically induced contractions as reported in detail elsewhere.33,39 The MVD assay was performed es-
sentially as described in the literature.21,39a Briefly, adult, male albino mice (Swiss Webster, 30-50 g: Canadian Breeding La-
boratories, Montreal) were killed by cervical dislocation, and the vasa deferentia were dissected out. After removal of extra-
neous fat and connective tissue, the vas was stripped of its associated blood vessel, and the semen was gently expressed from
the lumen. The vas was then mounted under 0.5-g tension in a 5 mL organ bath containing warmed (37 C), oxygenated (95%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
+
0
2, 5% CO
2
), Mg -free Krebs solution of the following composition [mM]: NaC1, 118; CaCl
2
, 2.54; KCl, 4.75; KH
2
P0 , 1.19;
4
NaHCO , 25; glucose, 11; L-tyrosine, 0.2. A modified Harvard apparatus stimulator delivered repetitive field stimulation
3
through platinum wire ring electrodes at the top and bottom of the bath, consisting of twin, rectangular pulses (80 V, 0.15 Hz,
10-ms delay, 1.0-ms duration). Contractions of the muscle were recorded via a Hewlett-Packard Model FTA-1-1 force trans-
ducer connected to a Hewlett-Packard 7702B recorder. Determination of the reduction in the twitch height at various doses
5
permitted the construction of log dose-response curves. A log dose-response curve was determined with [Leu ]enkephalin as
standard for each ileum or vas preparation, and IC50 values of the enkephalin analogues being tested were normalized accord-
ing to a published procedure.40
Opioid Receptor Binding Assays Opioid receptor binding studies were performed using established protocols described in
33
3
detail elsewhere. Binding affinities for μ and δ receptors were determined by displacing, respectively, [ H]DAMGO (Multiple
3
Peptide Systems, San Diego, CA) and [ H]DSLET (Multiple Peptide Systems) from rat brain membrane binding sites, and κ
opioid receptor binding affinities were measured by displacement of tritium-labeled (5α,7α,8β-(‒)-N-methyl-N-[7-(1-pyrrol-
3
41
idinyl-1-oxaspiro[4.5]-dec-8-yl]benzeneacetamide ([ H]U69,593) (Amersham) from guinea pig brain membrane binding
3
3
3
sites. Incubations were performed for 2 h at 0° C with [ H]DAMGO, [ H]DSLET and [ H]U69,593 at respective concentrations
of 0.72, 0.78 and 0.80 nM. IC50 values were determined from log dose-displacement curves and K values were calculated from
the obtained IC50 values by means of the equation of Cheng and Prusoff, using values of 1.3, 2.6 and 2.9 nM for the dissocia-
i
4
2
3
3
3
tion constants of [ H]DAMGO, [ H]DSLET and [ H]U69,593, respectively.
Effects of aza-pipecolyl GHRP-6 analogs on downstream signaling; influence of aza-pipecolyl peptide on the overpro-
duction of NO induced by the TLR-2 agonist R-FSL-1 in macrophage cell line Murine RAW264.7 macrophage cell lines
(
American Type Cell Collection catalogue no. TIB-71) were seeded at 1.5 × 105 cells/well in DMEM medium supplemented
with penicillin and streptomycin in a 48-well plate format and incubated at 37 °C with 5% CO . After 2 h, the medium of
2
adhered cells was changed to DMEM-Pen/ Strep with 0.2% of bovine serum albumin (BSA), and the cells were treated with
4
aza-pipecolyl-GHRP-6 analogs (21-25), or with [azaY ]GHRP-6 (MPE-001) as positive control at a final concentration of 10−6
M. After a 1 h pre-incubation period, the cells were stimulated overnight with the TLR-2 ligand fibroblast-stimulating lipopep-
tide (R-FSL-1, 300 ng/mL, Invivogen catalogue no. L7022). Supernatants were collected for nitrite determination by reaction
with 2,3-diaminonaphthalene (DAN) and measurement of fluorescence. Briefly, 25 μL of sample were incubated with 0.5 μg
of DAN in a 100 μL final volume of phosphate buffer (50 mM, pH 7.5) at room temperature in the dark. After 15 min, the
reaction was stopped with 20 μL of NaOH (2.8N) and fluorescence was read using a fluorescence plate reader (TECAN Safire,
λ
exc 365 nm and λem 430 nm).
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General experimental procedures.
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2
2
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5
5
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5
5
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5
6
Chemicals were used as received from commercial sources without further purification unless stated otherwise. Polysty-
rene Rink amide resin (0.5 mmol/g 75−100 mesh) was purchased from Advanced Chemtech™ (Louisville, Kentucky, USA),
and the manufacturer’s reported loading was used in the calculation of final product yields. Solvents were obtained from
Fisher Scientific. Anhydrous DCM, THF and DMF were obtained by passage through a solvent filtration system (Glass-Contour,
4
3
Irvine, CA). 9-H-Fluoren-9-ylmethyl carbazate was synthesized according to the literature method. Triphosgene, 2,4,6-col-
lidine, NBS, 2,6-lutidine, N,N-diisopropylethylamine (DIEA), TFA, 1,3-butadiene, and 2,3-dimethylbutadiene, all were pur-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
®
chased from Aldrich and used as received. Amino acids [e.g., Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-ᴅ-Phe-
OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-D-Trp(Boc)-OH, Boc-His(Trt)-OH], and coupling reagents including DIC, DSC,
HOBt, COMU, HATU, Oxyma, PyBOP, T P and 9-fluorenylmethyl N-succinimidyl carbonate (Fmoc-OSu), all were purchased
3
™
from Chem Impex and CS Bio and used as received. N-Fmoc-2′,6′-dimethyl-L-tyrosine was synthesized in house as described
below from N-Boc-2′,6′-dimethyl-L-tyrosine, which was received from Santa Cruise Biotechnology. Nuclear magnetic reso-
1
13
1
nance spectra ( H, C and Dept NMR) were recorded on a Bruker AV 500 spectrometer. H NMR spectra were referenced to
DMSO-d (39.52 ppm), as specified below. Coupling constants, J
(2.50 ppm) and 13C NMR spectra were measured in DMSO-d
6
6
values were measured in Hertz (Hz) and chemical shift values in parts per million (ppm). Accurate mass measurements were
performed on a liquid chromatography-mass spectrometry (LC-MS) instrument from Agilent technologies 1200 series in pos-
itive electrospray ionisation (ESI-TOF) mode at the Université de Montréal Mass Spectrometry facility. Sodium and proton
+
+
adducts ([M+Na] and [M+H] ) were used for empirical formula confirmation.
Analyses and characterization of crude and pure azapeptides were performed on either a Agilent™ 1100 Series LC-MS
instrument with ESI ion-source, single quadrupole mass detection and positive mode ionization or a AgilentTM technologies
1
200 series LC-MS instrument with ESI-TOF ion source and positive mode ionization equipped or a WatersTM ACQUITY UPLC
instrument series equipped with a SYNAPT G2-S Mass spectrometry with ESI-TOF ion source and positive mode ionization.
Azapeptide samples were dissolved in 10% H O in methanol prior to injections. The LC-MS analyses for all Diels-Alder reac-
tions and coupling reactions were performed on a Sunfire™ C18 column (2.1 mm x 50 mm, 3.5 μm), using binary solvent
2
system consisting of 0.1% formic acid (FA) in H O, and 0.1% FA in methanol at a flow rate of 0.4 mL/min and UV detection
2
at λ = 214 nm.
Purification of azapeptides was conducted on a WatersTM PrepLC instrument equipped with a reverse-phase GeminiTM
C18 column (Phenomenex® Inc., pore size: 110 Å, particle size: 5 μm, 250 mm ×21.2 mm) or a reverse-phase SunfireTM C18
column (pore size: 110 Å, particle size: 5 μm; 150 × 19 mm) employing a specified linear gradient of 5-60% of MeOH contain-
ing 0.1% FA in water containing 0.1% FA, at a flow rate of 10.0 mL/min and monitored with a UV detector at 214 nm and 254
nm. Fractions containing pure peptide were combined, freeze-dried and lyophilized to a white powder. Purified azapeptide
samples were analyzed for purity by LC-MS using a CSH C18 column (4.6 mm × 100 mm, 5 μm), with a flow rate of 0.5 mL/min
using a 20−95% linear gradient of water (0.1% FA) in CH
fire™ C18 column (2.1 mm x 50 mm, 3.5 μm) with a flow rate of 0.4 mL/min using a 10−90% linear gradient of water (0.1%
FA) in CH CN (0.1% FA) or in MeOH (0.1% FA) for compounds 15-19, 23-24, and 26-27; or a BEH C18 column (2.1 mm x
00mm, 1.7 μm) with a flow rate of 0.5 mL/min using a 2−98% linear gradient of water (0.1% FA) in CH CN (0.1% FA) or in
3
CN (0.1% FA) or in MeOH (0.1% FA) for compounds 14; or a Sun-
3
1
3
MeOH (0.1% FA) for compound 25; and monitored with a UV detection at λ = 214 nm. Infrared spectra were recorded in the
neat on a Perkin Elmer Spectrum One FTIR instrument and reported in reciprocal centimetres (cm–1).
Fmoc-based peptide synthesis. On an automated shaker, polystyrene Rink amide resin (0.50 mmol/g, 75-100 mesh) and Fmoc-
2
3
based peptide synthesis was employed under standard conditions. Couplings of amino acids (3 equiv) were performed in DMF
using DIC (3 equiv) and HOBt (3 equiv). The Fmoc groups were removed by treating the resin twice with 20% piperidine in DMF
1
2
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The Journal of Organic Chemistry
for 30 min. After each coupling and deprotection step, the resin was washed using 15 sec. agitations with DMF (3 times), MeOH (3
times) and DCM (3 times), sequentially.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
General protocol for cleavage of aliquots of resin-bound peptide for analysis by LC-MS. An aliquot of peptide bound
resin (3-5 mg) was treated with a freshly made solution of TFA/H
2
O/TES (95:2.5:2.5, v/v/v, 0.5 mL) for 30 min at room
®
temperature. The resin was removed by filtration. The filtrate was collected in a 1.5 mL Eppendorf tube. The filtrate volume
was concentrated, and the crude peptide was precipitated with cold ether (1.5 mL). After agitation on a vortex shaker, the
mixture was spun in a centrifuge. The supernatant was decanted leaving a pellet, which was dissolved in methanol (or H
mg/mL) and subjected to LC/MS analysis.
2
O, 1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of [azapipecolyl]-GHRP-6 analog 26 as a representative for the synthesis of aza- GHRP-6 and opioids, em-
ploying solid-phase Diels-Alder chemistry of Fmoc-protected azopeptides
Representative synthesis of fluorenylmethyl carbazate protected aza-Gly on solid phase, synthesis of Fmoc-
azaGly-ᴅ-Phe-Lys(Boc)-NH-rink resin 2d:
A solution of DSC (3 eq., 0.769 g, 3 mmol) in DMF (15 mL) was treated dropwise with a solution of 9-H-fluoren-9-ylmethyl
carbazate (3 eq., 0.763 g, 3 mmol) in 15 mL of DMF over 15 min at 0 °C, stirred for 1 h at rt, and the activated carbazate 1 was
transferred to a syringe tube, equipped with a Teflon™ filter, stopper and stopcock, containing ᴅ-Phe-Lys(Boc)-NH-Rink resin
(
1 eq., 2.15 g, 1 mmol) swollen in 12 mL of DMF. The resin mixture was treated with DIEA (6 eq., 0.775 g, 0.992 mL, 6 mmol),
shaken on an automated shaker for 12 h and filtered. After filtration, the resin was washed sequentially with DMF (3 times),
-
1
MeOH (3 times), and DCM (3 times), to afford aza-glycine resin 2d; IR (neat) vmax/cm 2921, 1671, 1603, 1506, 1492, 1451,
1243, 1207, 1171. LC-MS analysis of a cleaved resin aliquot as described above indicated complete conversion; LC-MS [10–
9
=
0% MeOH (0.1% FA) in water (0.1% FA) over 10 min, followed by 10% MeOH (0.1% FA) in water (0.1% FA) for 4 min] R. T.
+
6.44 min. ESI-MS m/z: calculated for C31
H
37
N
6
O
5
[M+H] 573.27; found 573.5.
Representative protocol for the oxidation of aza residue and [4+2]-cycloaddition reaction on solid phase, synthe-
4
sis of Fmoc-(∆ )azaPip-ᴅ-Phe-Lys(Boc)-NH-rink resin 4d.
A sealed flask containing swollen Fmoc-azaGly-ᴅ-Phe-Lys-NH-Rink resin 2d (1 eq., 2.43 g, 1 mmol) in dry DCM (24 ml)
under argon was cooled to –78 °C, treated with 2,6-lutidine (0.321 g, 0.349 mL, 3 mmol), stirred for 1 min, and treated with a
single portion of NBS (3 eq., 0.534 g, 3 mmol). The bath was removed. The reaction mixture warmed to room temperature
with shaking on an automated shaker for 1 h and filtered. The filtered resin was washed successively using 15 sec agitations
-
1
with DCM (3 times) and dried under vacuum to provide azopeptide resin 3d: IR (neat) vmax/cm 2922, 1710, 1678, 1601,
1492, 1451, 1243, 1207, 1172.
Vacuum dried azopeptide resin 3d (~2.5 g, 1 mmol) was suspended in anhydrous DCM (5 mL), placed in a sealed flask
under argon, cooled to –78 °C, and treated dropwise via a cannula with a solution of 1,3-butadiene (1 M, 10 mL, 10 mmol) in
dry DCM (5 mL). The reaction mixture was warmed to room temperature, shaken on an automated shaker overnight, and
filtered. The resin was washed and filtered successively using 15 sec agitations with DCM (3 times) to afford resin 4d. A
cleaved resin aliquot was analyzed as described above by LC-MS, which indicated complete conversion. A peak was observed
4
with molecular ion consistent with Fmoc-(∆ )azaPip-ᴅ-Phe-Lys-NH
2
(8): LC-MS [10–90% MeOH (0.1% FA) in water (0.1%
FA) over 10 min, followed by 10% MeOH (0.1% FA) in water (0.1% FA) for 4 min] R. T. = 7.99 min. ESI-MS m/z: calculated for
+
C
35
H
41
N
6
O
5
[M+H] 625.31; found 625.3.
4
Fmoc-(∆ )azaPip-ᴅ-Phe-Lys-NH
2
(8)
1
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Page 14 of 20
Resin 4d, (~0.5 g) was treated with a freshly made solution of TFA/H O/TES (95:2.5:2.5, v/v/v, 20 mL/g of peptide resin)
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
at room temperature for 2 h, filtered and washed with TFA. The filtrate and washes were concentrated to an oil, from which
a precipitate was obtained upon addition of cold ether (10-15 mL). After centrifugation, the supernatant was decanted, and
the precipitated peptide was taken up in aqueous acetonitrile (10% v/v) and freeze-dried prior to analysis and purification.
®
®
The crude sample was purified by preparative RP-HPLC on a reverse-phase Gemini C18 column (Phenomenex Inc., pore
size: 110 Å, particle size: 5 μm, 250 mm x 21.2 mm) using a binary solvent system consisting of a gradient of 5%-60% MeOH
(
0.1% FA) in water (0.1% FA), with a flow rate of 10.0 mL/min, and UV detection at 214 nm. The desired fractions were
4
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
combined and freeze-dried to white fluffy powder: Fmoc-(∆ )azaPip-ᴅ-Phe-Lys-NH
DMSO-d ) δ 8.35 (s, 1H), 7.98 – 7.92 (m, 1H), 7.90 – 7.85 (m, 2H), 7.85 – 7.81 (m, 2H), 7.44 – 7.37 (m, 2H), 7.37 – 7.32 (m, 2H),
.29 – 7.23 (m, 2H), 7.23 – 7.15 (m, 3H), 6.92 – 6.80 (m, 2H), 5.87 – 5.81 (m, 1H), 5.81 – 5.74 (m, 1H), 4.82 (s, 1H), 4.46 – 4.36
2
(8, 15 mg, 12%). H NMR (500 MHz,
6
7
(m, 1H), 4.15 – 4.05 (m, 2H), 3.96 – 3.66 (m, 4H), 3.29 – 3.17 (m, 1H), 3.17 – 3.08 (m, 1H), 3.03 – 2.92 (m, 1H), 2.92 – 2.84 (m,
1
1
H), 2.74 – 2.59 (m, 2H), 1.70 – 1.55 (m, 1H), 1.51 – 1.36 (m, 3H), 1.36 – 1.22 (m, 1H), 1.22 – 1.07 (m, 2H); 13C{ H}-NMR (125
MHz, DMSO-d
6
) δ 172.9, 171.1, 161.3, 156.7, 139.1, 137.3, 137.1, 128.7, 128.3, 127.5, 126.7, 125.7, 125.2, 123.5, 120.7, 119.3,
+
6
1.9, 54.4, 52.0, 44.3, 40.1, 37.8, 37.0, 31.0, 30.3, 21.9, 21.3. HRMS m/z calcd for C35
H
41
N
6
O
5
[M+H] 625.3133; found 625.3126.
N-Fmoc-2′,6′-dimethyl-L-tyrosine (Fmoc-Dmt-OH, 9):
N-Boc-2′,6′-Dimethyl-L-tyrosine (0.22 g, 0.711 mmol) was dissolved in DCM (10 mL), stirred and treated with bubbles of
dry HCl gas at rt for 3h. The volatiles were removed under vacuum to give hydrochloride salt (~0.15 g, 99%) as white solid.
The hydrochloride salt (1 eq., ~0.15 g, 0.711 mmol) was dissolved in 10 mL of acetone/water (1:1 v/v), treated with sodium
carbonate (1.1 eq., 0.0829 g, 0.783 mmol) and Fmoc-OSu (1 eq., 0.24 g, 0.711 mmol), and stirred for 12h. The volatiles were
evaporated. Ethyl acetate was added to the mixture, which was acidified using 6 M aqueous HCl. The organic phase was sep-
arated and washed with 1N HCl and brine, dried over Na
2
SO
4
, filtered and evaporated to a residue, that was purified by chro-
matography on silica gel using a mixture of CH
2
Cl
2
/MeOH/AcOH 95.5:2.5:2 v/v to afford N-Fmoc-2′,6′-dimethyl-L-tyrosine 9
1
(
0.24 g, 78 %). H NMR (500 MHz, DMSO) δ 12.67 (br s 12.67), 8.95 (s, 1H), 7.92 – 7.86 (m, 2H), 7.74 (d, J = 8.7 Hz, 1H), 7.71 –
7.65 (m, 2H), 7.47 – 7.38 (m, 2H), 7.36 – 7.27 (m, 2H), 6. 42 – 6.37 (m, 2H), 4.23 – 4.17 (m, 3H), 4.13 – 4.01 (m, 1H), 3.00 (dd,
J = 14.3, 6.0 Hz, 1H), 2.82 (dd, J = 14.3, 8.8 Hz, 1H), 2.20 (s, 6H); 13C{ H}-NMR (125 MHz, DMSO-d
1
) δ 173.7, 155.9, 155.1, 143.9,
6
-
1
1
1
40.7, 137.8, 127.6, 127.1, 125.3, 120.1, 114.8, 65.7, 59.9, 46.6, 29.7, 20.1; IR (neat) vmax/cm 3321, 1702, 1596, 1536, 1449,
+
261, 1140, 1025; HRMS m/z calculated for C26
H
26NO [M+H] 432.1805; found 432.1815.
5
Representative protocol for acylation of semicarbazides with N-(Fmoc)amino acid chlorides on solid phase, syn-
4
thesis of Fmoc-Ala-(∆ )azaPip-ᴅ-Phe-Lys(Boc)-NH-rink resin 20d:
Resin 4d was treated with 20% piperidine in DMF to remove the Fmoc group using the protocol above. The resulting H-
(
∆4)azaPip-ᴅ-Phe-Lys(Boc)-NH-Rink resin (6d, ~387 mg, 0.171 mmol) was transferred into a microwave vial, and swollen in
THF. A premixed 0 °C solution of Fmoc-Ala-OH (5 eq., 266 mg, 0.857 mmol) and triphosgene (1.66 eq., 84.4 mg, 0.284 mmol)
in THF (8 mL) was treated dropwise with 2,4,6-collidine (23 eq., 477 mg, 0.525 mL, 3.94 mmol) over 1 min, and transferred
into the microwave vial containing the swollen resin. The vial was sealed and irradiated (25W) at 75 °C for 2 h. After cooling,
the resin was filtered and washed sequentially with DMF (3 times), MeOH (3 times), and DCM (3 times) to afford resin 20d.
A resin aliquot was cleaved as described above and analyzed by LC-MS which indicated complete conversion; LC-MS [10–90%
MeOH (0.1% FA) in water (0.1% FA) over 10 min, followed by 10% MeOH (0.1% FA) in water (0.1% FA) for 4 min] R. T. =
+
8
.04 min. ESI-MS m/z: calculated for C38
H
46
N
7
O
6
[M+H] 696.34; found 696.3.
1
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1
The Journal of Organic Chemistry
Representative protocol for azapeptide elongation, deprotection and cleavage from resin, synthesis of His-D-Trp-
4
Ala-(∆ )azaPip-ᴅ-Phe-Lys-NH
2
(23):
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Aza-4,5-dehydropipecolate peptide 23 was synthesized from resin 20d (~400 mg) by sequential Fmoc group removals,
DIC/HOBt couplings of Fmoc-D-Trp(Boc), followed by Boc-His(Trt), and resin cleavage using the respective general protocols
described above. A resin aliquot was cleaved as described above and analyzed by LC-MS which indicated complete conversion,
4
and a peak with molecular ion consistent with His-D-Trp-Ala-(∆ )azaPip-ᴅ-Phe-Lys-NH
2
(23) of 44% purity: R. T. 4.58 min on
a Sunfire™ column with a gradient of 5-50% MeOH (0.1% FA) in water (0.1% FA) over 9 min, followed by 5% MeOH (0.1%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
FA) in water (0.1% FA) for 5 min. ESI-MS m/z: calcd for C40
H
53
N
12
O
6
[M+H] 797.41, found 797.4.
4
His-D-Trp-Ala-(∆ )azaPip-ᴅ-Phe-Lys-NH
2
(23) was cleaved from the resin using a freshly made solution of TFA/H O/TES
2
(
95:2.5:2.5, v/v/v, 20 mL/g of peptide resin) at room temperature for 2 h. The resin was filtered and rinsed with TFA. The
filtrate and rinses were concentrated to an oil, from which a precipitate was obtained by addition of cold ether (10-15 mL).
After centrifugation, the supernatant was decanted, and the precipitated peptide was taken up in aqueous acetonitrile (10%
v/v) and freeze-dried prior to analysis and purification. The crude sample was purified by RP-HPLC on a reverse-phase Gem-
®
®
ini C18 column (Phenomenex Inc., pore size: 110 Å, particle size: 5 μm, 250 x 21.2 mm) using a binary solvent system
consisting of a gradient of 5%-60% MeOH (0.1% FA) in water (0.1% FA), with a flow rate of 10.0 mL/min, and UV detection
at 214 nm. The desired fractions were combined and freeze-dried to white fluffy powder: azapeptide 23 (5.8 mg, 4.2%). The
purified product was analyzed to be of >99% purity by analytical RP-HPLC on a Sunfire™ column using gradient 1: 5-50% of
MeOH (0.1% FA) in H
0.1% FA) in H O (0.1% FA) over 9 min, followed by 5% MeCN for 5 min, R.T. = 3.92 min; HRMS m/z calcd for C40
M+H] 797.4206, found 797.4189.
2
O (0.1% FA) over 9 min, followed by 5% MeOH for 5 min, R.T. = 4.87 min; gradient 2: 5-50% of MeCN
(
2
H
53
N
12
O
6
+
[
4
Representative protocol for hydrogenation of (Δ )-azaPip to azaPip peptides, synthesis of His-D-Trp-Ala-azaPip-
ᴅ-Phe-Lys-NH (26):
2
4
A solution of [aza-4,5-dehydropipecolyl ]GHRP-6 (23, 1 eq., 2 mg, 0.00251 mmol) in methanol (0.2 mL) was treated care-
fully with 10 wt% palladium-on-carbon (1 eq., ~ 1 mg) under a stream of argon. The resulting suspension was placed under
hydrogen atmosphere (1 atm), stirred under a balloon of hydrogen at room temperature for 1–2 h, and filtered on CeliteTM
,
which was washed with methanol. The filtrate and washings were combined and concentrated under vacuum. The residue
4
was freeze-dried to furnish [azapipecolyl ]-GHRP-6 (26, 2 mg, 99 %) as off white solid: RP-HPLC on a Sunfire™ column re-
vealed >99% purity using gradient 1: 5-50% of MeOH (0.1% FA) in H O (0.1% FA) over 9 min, followed by 5% MeOH over 5
O (0.1% FA) over 9 min, followed by 5% MeCN over 5 min, R.T.
2
min, R.T. = 5.15 min; gradient 2: 5-50% MeCN (0.1% FA) in H
2
+
=
3.97 min; HRMS m/z calcd for C40
H
55
N
12
O
6
[M+H] 799.4362, found 799.4381.
Analytical LC-MS characterization of purified aza-pipecolyl morphiceptin and endomorphin analogues (14-19).
4
Tyr-(∆ )azaPip-ᴅ-Phe-Pro-NH (14a): Isolated yield (7.6 mg, 12%), LC-MS analysis (a) 20-95% of MeOH (0.1% FA) in
2
H
2
O (0.1% FA) over 11 min, then 20% MeOH for 4 min, R.T. = 7.55 min; (b) 10-95% MeCN (0.1% FA) in H O (0.1% FA) over
2
+
1
1 min, then 10% MeCN for 4 min, R.T. = 6.42 min; HRMS m/z calcd for C28
H
35
N
6
O
5
[M+H] 535.2663, found 535.2679.
4
Tyr-(∆ )azaPip-Trp-Phe-NH
2
(14b): Isolated yield (5.2 mg, 8.2%), LC-MS analysis (a) 20-95% of MeOH (0.1% FA) in H O
2
(
0.1% FA) over 11 min, then 20% MeOH for 4 min, R.T. = 8.37 min; (b) 20-95% MeCN (0.1% FA) in H O (0.1% FA) over 15
2
+
min, then 20% MeCN for 3 min, R.T. = 6.61 min; HRMS m/z calcd for C34
H
38
N
7
O
5
[M+H] 624.2929, found 624.2944.
1
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Page 16 of 20
4
Tyr-(∆ )azaPip-Phe-Phe-NH
2
(14c): Isolated yield (5.3 mg, 9%), LC-MS analysis (a) 20-95% of MeOH (0.1% FA) in H O
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
0.1% FA) over 11 min, then 20% MeOH for 4 min, R.T. = 8.25 min; (b) 20-95% MeCN (0.1% FA) in H
2
O (0.1% FA) over 11
+
min, then 20% MeCN for 4 min, R.T. = 5.60 min; HRMS m/z calcd for C32
H
37
N
6
O
5
[M+H] 585.2820, found 585.2835.
4
Tyr-(4,5-dimethyl-∆ )azaPip-ᴅ-Phe-Pro-NH
2
(15a): Isolated yield (4.9 mg, 8.6%), LC-MS analysis (a) 10-90% of MeOH
(0.1% FA) in H
2
2
O (0.1% FA) over 9 min, then 10% MeOH for 5 min, R.T. = 6.70 min; (b) 10-90% MeCN (0.1% FA) in H O (0.1%
+
FA) over 9 min, then 5% MeCN for 5 min, R.T. = 4.76 min; HRMS m/z calcd for C30
H
39
N
6
O
5
[M+H] 563.2976, found 563.2964.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
Tyr-(4,5-dimethyl-∆ )azaPip-Trp-Phe-NH (15b): Isolated yield (3.3 mg, 6.5%), LC-MS analysis (a) 10-90% of MeOH
2
(0.1% FA) in H
2
O (0.1% FA) over 9 min, then 10% MeOH for 5 min, R.T. = 7.38 min; (b)10-90% MeCN (0.1% FA) in H O (0.1%
2
+
FA) over 9 min, then 10% MeCN for 5 min, R.T. = 5.12 min; HRMS m/z calcd for C36
H
42
N
7
O
5
[M+H] 652.3242, found 652.3234.
4
Tyr-(4,5-dimethyl-∆ )azaPip-Phe-Phe-NH (15c): Isolated yield (3.6 mg, 7.2%), LC-MS analysis (a) 10-90% of MeOH
2
(
0.1% FA) in H
2
O (0.1% FA) over 9 min, then 10% MeOH for 5 min, R.T. = 7.32 min; (b) 10-90% MeCN (0.1% FA) in H O (0.1%
2
+
FA) over 9 min, then 10% MeCN for 5 min, R.T. = 5.18 min; HRMS m/z calcd for C34
H
41
N
6
O
5
[M+H] 613.3133, found 613.3112.
4
Dmt-(∆ )azaPip-ᴅ-Phe-Pro-NH (16a): Isolated yield (2.4 mg, 4.6%), LC-MS analysis (a) 10-90% of MeOH (0.1% FA) in
2
H
2
O (0.1% FA) over 9 min, then 10% MeOH for 5 min, R.T. = 6.14 min; (b) 10-90% MeCN (0.1% FA) in H O (0.1% FA) over 9
2
+
min, then 10% MeCN for 5 min, R.T. = 4.34 min; HRMS m/z calcd for C30
H
39
N
6
O
5
[M+H] 563.2976, found 563.2962.
4
Dmt-(4,5-dimethyl-∆ )azaPip-ᴅ-Phe-Pro-NH (17a): Isolated yield (1.1 mg, 2%), LC-MS analysis (a) 10-90% of MeOH
2
(
0.1% FA) in H
2
O (0.1% FA) over 9 min, then 10% MeOH for 5 min, R.T. = 7.01 min; (b) 10-90% MeCN (0.1% FA) in H O (0.1%
2
+
FA) over 9 min, then 10% MeCN for 5 min, R.T. = 4.96 min; HRMS m/z calcd for C32
H
43
N
6
O
5
[M+H] 591.3289, found 591.3284.
Tyr-azaPip-ᴅ-Phe-Pro-NH
2
(18a): Isolated yield (2.5 mg, 12%), LC-MS analysis (a) 10-90% of MeOH (0.1% FA) in H O
2
(
0.1% FA) over 9 min, then 10% MeOH for 5 min, R.T. = 5.73 min; (b) 10-90% MeCN (0.1% FA) in H O (0.1% FA) over 9 min,
2
+
then 10% MeCN for 5 min, R.T. = 4.07 min; HRMS m/z calcd for C28
H
37
N
6
O
5
[M+H] 537.2820, found 537.2826.
Trp-azaPip-Trp-Phe-NH
2
(18b): Isolated yield (2 mg, 8.2%), LC-MS analysis (a) 10-90% of MeOH (0.1% FA) in H
2
O (0.1%
FA) over 9 min, then 10% MeOH for 5 min, R.T. = 6.61 min; (b) 10-90% MeCN (0.1% FA) in H
2
O (0.1% FA) over 9 min, then 10%
+
MeCN for 5 min, R.T. = 4.68 min; HRMS m/z calcd for C34
H N
40 7
O
5
[M+H] 626.3085, found 626.3099.
Tyr-azaPip-Phe-Phe-NH
2
(18c): Isolated yield (1.5 mg, 9%), LC-MS analysis (a) 10-90% of MeOH (0.1% FA) in H
2
O (0.1%
FA) over 9 min, then 10% MeOH for 5 min, R.T. = 6.47 min; (b) 10-90% MeCN (0.1% FA) in H
2
O (0.1% FA) over 9 min, then
+
10% MeCN for 5 min, R.T. = 4.58 min; HRMS m/z calcd for C32
H
39
N
6
O
5
[M+H] 587.2976, found 587.2991.
Dmt-azaPip-ᴅ-Phe-Pro-NH
2
(19a): Isolated yield (1.2 mg, 4.6%), LC-MS analysis (a) 10-90% of MeOH (0.1% FA) in H O
2
(
0.1% FA) over 9 min, then 10% MeOH for 5 min, R.T. = 6.09 min; (b) 10-90% MeCN (0.1% FA) in H O (0.1% FA) over 9 min,
2
+
then 10% MeCN for 5 min, R.T. = 4.29 min; HRMS m/z calcd for C30
H
41
N
6
O
5
[M+H] 565.3133, found 565.3138.
Analytical LC-MS characterization of purified aza-pipecolyl GHRP-6 analogues (23-27).
4
His-ᴅ-Trp-Ala-(∆ )azaPip-ᴅ-Phe-Lys-NH
2
(23): Isolated yield (5.8 mg, 4.2%), LC-MS analysis (a) 5-50% of MeOH (0.1%
O (0.1% FA)
[M+H] 797.4206, found 797.4189. LC-
FA) in H
2
O (0.1% FA) over 9 min, then 5% MeOH over 5 min, R.T. = 4.87 min; (b) 5-50% MeCN (0.1% FA) in H
2
+
over 9 min, then 5% MeCN for 5 min, R.T. = 3.92 min; HRMS m/z calcd for C40
H
53
N
12
O
6
+
MS m/z calcd for C40
H
53
N
12
O
6
[M+H] 797.41, found 797.4.
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4
His-ᴅ-Trp-(∆ )azaPip-Trp-ᴅ-Phe-Lys-NH
2
(24): Isolated yield (5.1 mg, 3.9%), LC-MS analysis (a) 5-50% of MeOH (0.1%
O (0.1% FA) over 9 min, then 5% MeOH for 5 min, R.T. = 5.98 min; (b) 5-50% MeCN (0.1% FA) in H O (0.1% FA) over
[M+H] 912.46275, found 912.4628. LC-MS
FA) in H
2
2
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
9 min, then 5% MeCN for 5 min, R.T. = 5.82 min; HRMS m/z calcd for C48
H
58
N
13
O
6
+
m/z calcd for C48
H
58
N
13
O
6
[M+H] 912.46, found 912.4.
4
His-ᴅ-Trp-(4,5-dimethyl-∆ )azaPip-Trp-ᴅ-Phe-Lys-NH
2
(25): Isolated yield (6.2 mg, 4.6%), LC-MS analysis (a) 2-98%
of MeOH (0.1% FA) in H
2
O (0.1% FA) over 11 min, then 2% MeOH for 4 min, R.T. = 4.91 min; (b) 2-50% MeCN (0.1% FA) in
+
H
2
O (0.1% FA) over 11 min, then 2% MeCN for 4 min, R.T. = 5.26 min; HRMS m/z calcd for C50
H
61
N
13
O
6
[M+Na] 962.4760,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
found 962.4764. LC-MS m/z calcd for C50
H
61
N
13
6
O [M+Na] 962.49, found 962.52.
His-ᴅ-Trp-Ala-azaPip-ᴅ-Phe-Lys-NH
in H O (0.1% FA) over 9 min, then 5% MeOH for 5 min, R.T. = 5.15 min; (b) 5-50% MeCN (0.1% FA) in H
min, then 5% MeCN for 5 min, R.T. = 3.97 min; HRMS m/z calcd for C40
2
(26): Isolated yield (2 mg, 4.2%), LC-MS analysis (a) 5-50% of MeOH (0.1% FA)
O (0.1% FA) over 9
[M+H] 799.4362, found 799.4381. LC-MS
2
2
+
H
55
N
12
O
6
+
m/z calcd for C40
H
55
N
12
O [M+H] 799.43, found 799.4.
6
His-ᴅ-Trp-azaPip-Trp-ᴅ-Lys-NH
2
(27): Isolated yield (2 mg, 3.9%), LC-MS analysis (a) 5-50% of MeOH (0.1% FA) in H
O (0.1% FA) over 9 min,
[M+H] 914.4784, found 914.4780. LC-MS m/z
2
O
(
0.1% FA) over 9 min, then 5% MeOH for 5 min, R.T. = 6.07 min; (b) 5-50% MeCN (0.1% FA) in H
2
+
then 5% MeCN for 5 min, R.T. = 4.70 min; HRMS m/z calcd for C48
H
60
N
13
O
6
+
calcd for C48
H
60
N
13
O
6
[M+H] 914.47, found 914.5.
ASSOCIATED CONTENT
Supporting Information
1H and 13C NMR spectra for compounds 8, and Fmoc-Dmt-OH 9. LC-MS data for compounds 14-19 and 23-27. IR Spectra for com-
pounds 2d and 3d. The Supporting Information is available free of charge via the Internet at http://pubs. acs.org.
AUTHOR INFORMATION
Corresponding Author
*
E-mail: william.lubell@umontreal.ca
ORCID ID
Ramesh Chingle: 0000-0003-1175-2454
Mukandila Mulumba: 0000-0001-7708-7480
Nga N. Chung: 0000-0001-7293-4855
Thi M.-D. Nguyen: 0000-0002-7140-3515
Huy Ong: 0000-0001-8459-2645
Steven Ballet: 0000-0003-4123-1641
Peter W. Schiller: 0000-0001-7311-869XWilliam D. Lubell: 0000-0002-3080-2712
Author Contributions
The manuscript was written through contributions of all authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENT
Support is acknowledged from NSERC Canada, CIHR (MOP89716), MDEIE Quebec (No. 878-2012), Amorchem, Mperia
Therapeutics Inc. and NIH USA (DA-004443). SB thanks the Strategic Research Program – Growth Funding, provided by VUB.
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Page 18 of 20
We thank M.-C. Tang, K. Venne, S. Comtois-Marotte, P. Aguiar, and S. Bilodeau for the HRMS and LC-MS spectrometry, and
NMR spectroscopy at Universite de Montreal regional centers.
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