High Affinity Agonists of the Neuropeptide y (NPY) Y4 Receptor Derived from the C-Terminal Pentapeptide of Human Pancreatic Polypeptide (hPP): Synthesis, Stereochemical Discrimination, and Radiolabeling

Article abstract of DOI:10.1021/acs.jmedchem.6b00309

The diastereomeric mixture of d/l-2,7-diaminooctanedioyl-bis(YRLRY-NH₂) (BVD-74D, 2) was described in the literature as a high affinity Y₄ receptor agonist. Here we report on the synthesis and pharmacological characterization of the pure diastereomers (2R,7R)- and (2S,7S)-2 and a series of homo- and heterodimeric analogues in which octanedioic acid was used as an achiral linker. To investigate the role of the Arg residues, one or two arginines were replaced by Ala. Moreover, N^ω-(6-aminohexylaminocarbonyl)Arg was introduced as an arginine replacement (17). (2R,7R)-2 was superior to (2S,7S)-2 in binding and functional cellular assays and equipotent with 17. [³H]Propionylation of one amino group in the linker of (2R,7R)-2 or at the primary amino group in 17 resulted in high affinity Y₄R radioligands ([³H]-(2R,7R)-10, [³H]18) with subnanomolar K_d values.

Full text of DOI:10.1021/acs.jmedchem.6b00309

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Article
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High Affinity Agonists of the Neuropeptide Y (NPY) Y Receptor Derived
from the C-terminal Pentapeptide of Human Pancreatic Polypeptide
(hPP): Synthesis, Stereochemical Discrimination and Radiolabeling
Kilian Konrad Kuhn, Thomas Ertl, Stefanie Dukorn, Max Keller,
Guenther Bernhardt, Oliver Reiser, and Armin Buschauer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00309 • Publication Date (Web): 25 May 2016
Downloaded from http://pubs.acs.org on June 5, 2016
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High Affinity Agonists of the Neuropeptide Y
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(
NPY) Y Receptor Derived from the Cꢀterminal
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Pentapeptide of Human Pancreatic Polypeptide
(
hPP): Synthesis, Stereochemical Discrimination and
Radiolabeling
†
‡
†
†
†
Kilian K. Kuhn, Thomas Ertl, Stefanie Dukorn, Max Keller, Günther Bernhardt, Oliver
‡
*,†
Reiser, and Armin Buschauer
†
Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053 Regensburg,
Germany
‡
Institute of Organic Chemistry, University of Regensburg, Universitätsstraße 31, 93053
Regensburg, Germany
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ABSTRACT
The diastereomeric mixture of D/Lꢀ2,7ꢀdiaminooctanedioylꢀbis(YRLRYꢀNH ) (BVDꢀ74D, 2)
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was described in the literature as a high affinity Y receptor agonist. Here we report on the
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synthesis and pharmacological characterization of the pure diastereomers (2R,7R)ꢀ and (2S,7S)ꢀ2
and a series of homoꢀ and heterodimeric analogues in which octanedioic acid was used as an
achiral linker. To investigate the role of the Arg residues, one or two arginines were replaced by
ω
Ala. Moreover, N ꢀ(6ꢀaminohexylaminocarbonyl)Arg was introduced as an arginine replacement
(17). (2R,7R)ꢀ2 was superior to (2S,7S)ꢀ2 in binding and functional cellular assays and
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equipotent with 17. [ H]propionylation of one amino group in the linker of (2R,7R)ꢀ2 or at the
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primary amino group in 17 resulted in high affinity Y R radioligands ([ H]ꢀ(2R,7R)ꢀ10, [ H]18)
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with subnanomolar K values.
d
KEYWORDS
Bioisosteric replacement, carbamoylguanidine, crossꢀmetathesis, diastereomers, neuropeptide Y,
NPY Y receptor agonist, pancreatic polypeptide, radioligand
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1
. INTRODUCTION
Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) consist of 36
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ꢀ3
amino acids and share considerable sequence similarities (Figure 1). NPY, PYY and PP
activate NPY receptors, in humans comprising four functional subtypes of Gꢀprotein coupled
receptors (Y R, Y R, Y R, Y R), which are involved in the regulation of numerous central and
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peripheral biological processes. The Y R shares only low sequence identity with the other
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subtypes (e.g. 42% sequence identity of hY R with hY R ) and is an exception as it prefers PP
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over NPY and PYY. The Y R is considered to play a role, for example, in feeding behavior and
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the regulation of energy metabolism. Recently, Y R agonists were proposed as putative antiꢀ
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obesity agents. Therefore, there is a need for high affinity Y R ligands, agonists as well as
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antagonists, to explore the potential of the Y R as a biological target.
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Previous work from different groups suggests that connecting two pharmacophoric moieties
favors Y R binding, in particular, in case of peptidic agonists such as 1 and 2 (Figure 2), which
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are derived from the Cꢀterminal pentapeptide of hNPY or hPP, respectively. Compound 3 (URꢀ
MK188) is an example of a Y R antagonist, composed of two molecules of an argininamideꢀtype
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Y R
antagonist
((R)ꢀ4ꢀguanidinoꢀ1ꢀ({[(4ꢀhydroxyphenyl)methyl]amino}carbonyl)butylꢀαꢀ
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phenylbenzeneacetamide, BIBP3226), which are connected via the guanidine groups.
Generally, the arginine residues in positions 33 and 35 of the endogenous peptides are important
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for NPY receptor binding. Similarly, the arginine moieties are considered crucial structural
features of the Y R ligands 1-3. The “dimeric peptide” 1 (GW1229, also known as GR23118 or
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229U91) is both, a highꢀaffinity antagonist at the Y R and an agonist at the Y R. The
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pentapeptide sequence TyrꢀArgꢀLeuꢀArgꢀTyrꢀNH in 1 is very similar to the Cꢀterminal
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sequence of hPP (…Thr ꢀArg ꢀPro ꢀArg ꢀTyr ꢀNH ). D/Lꢀ2,7ꢀdiaminooctanedioylꢀ
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bis(YRLRYꢀNH ) (BVDꢀ74D, 2), comprising the same pentapeptide moieties as 1, but a
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different linker, is more selective for the Y R. Data reported for 2 refer to the diastereomeric
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mixture of (2R,7R)ꢀ2 and (2S,7S)ꢀ2, not to a single stereoisomer (note: in the following,
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positions 2 and 7 refer to the stereo centers in the 2,7ꢀdiaminosuberic acid moiety).
Figure 1. Amino acid sequences of human NPY, PYY and PP
Figure 2. Structures of the described NPY Y R antagonist/Y R agonist 1, the Y R agonist 2, and
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the Y R/Y R antagonist 3. Dpr: Lꢀ2,3ꢀdiaminopropionic acid; 2 was previously described as a
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mixture of two diastereomers due to (2R,7R)ꢀ and (2S,7S)ꢀconfiguration of the 2,7ꢀ
diaminosuberic acid linker.
Here we report on the synthesis and pharmacological characterization of the pure
stereoisomers (2R,7R)ꢀ2 and (2S,7S)ꢀ2. Very recently, a pilot study from our laboratory
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suggested that the amino groups in the linker of 2 are dispensable. Therefore, we synthesized a
set of analogues using octanedioic acid instead of 2,7ꢀdiaminosuberic acid as linker. To
investigate the contribution of the individual arginines to the Y R affinity of the dimers, we
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prepared compounds in which one or two Arg moieties in the pentapeptide building block were
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replaced by Ala. Furthermore, the suitability of the ‘dimeric’ pentapeptides as precursors for the
preparation of radiolabeled compounds was explored, the target compounds were characterized
in functional assays, and two tritiated derivatives were synthesized and used to perform Y R
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binding studies.
2. RESULTS AND DISCUSSION
Chemistry. The enantiopure 2,7ꢀdiaminooctanedioic acids were synthesized from (S)ꢀ and (R)ꢀ
2ꢀallylglycine via the N-Boc protected esters (S)ꢀ5 and (R)ꢀ5 (Scheme 1) by analogy with
17,18
procedures from the literature.
The enantiomers of 5 were submitted to a crossꢀmetathesis
reaction, employing Grubbs’ II catalyst to build up the core structure 6. After exchanging the
solvent (DCM) with ethanol, compound 6 was not isolated, but hydrogenated at 20 bar in the
19,20
presence of the same catalyst,
in contrast to reported procedures requiring workup of 6 and
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Pd/C catalyzed hydrogenation. Besides the convenience of this protocol, overall yields were
improved, affording (2R,7R)ꢀ7 and (2S,7S)ꢀ7 in 95% and 74% yield, respectively, over two steps.
Hydrolysis of the ethyl ester quantitatively provided the diacids (2R,7R)ꢀ8 and (2S,7S)ꢀ8.
a
Scheme 1. Enantioselective synthesis of the diacids (2R,7R)ꢀ8 and (2S,7S)ꢀ8
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2S,7S)ꢀ8 was prepared according to the same protocol using (S)ꢀ2ꢀallylglycine as starting
material. Reagents and conditions: (a) Boc O, 1 M NaOH/dioxane (2:1), 0 °C to rt, 18 h; (b)
DCC, 4ꢀDMAP, EtOH, DCM, 0 °C to rt, 12 h; (c) 0.1 equiv Grubbs’ II catalyst, anhydrous
DCM, N atmosphere, reflux, 12 h; compd. 6 was not isolated, but subjected to hydrogenation in
the next step; (d) 0.1 equiv Grubbs’ II catalyst (from reaction step (c)), ethanol, H (20 bar), rt,
5 h (95% for (R,R)ꢀ7, 74% for (S,S)ꢀ7 over two steps, (c) and (d)); (e) 1 M NaOH, EtOH, rt, 21 h
quant.).
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Peptide synthesis was carried out manually on a Sieberꢀamide resin applying an Fmoc protocol
and ‘double’ coupling procedures with HBTU/HOBt/DIPEA (Scheme 2). After cleavage from
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the resin, two equivalents of the protected pentapeptide 9 were crossꢀlinked with the in situ
activated (R,R)ꢀ and (S,S)ꢀconfigured enantiomers of 2,7ꢀdiaminosuberic acid (8) to obtain the
corresponding peptides (2R,7R)ꢀ2 and (2S,7S)ꢀ2 after deprotection. Subsequent acylation
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propionamides (2R,7R)ꢀ10 (for preparation of [ H]ꢀlabeled form see below) and (2S,7S)ꢀ10.
With respect to labeling of compounds devoid of amino groups in the linker (Scheme 3), we
introduced the recently reported arginine derivative 11 (Figure 3), in which the guanidine group
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is replaced by a nonclassical bioisostere, a functionalized carbamoylguanidine moiety.
Scheme 2. Synthesis of (2R,7R)ꢀ2 and (2S,7S)ꢀ2 and the propionylated analogues (2R,7R)ꢀ10 and
a
(
2S,7S)ꢀ10
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a
Reagents and conditions: (a) SPPS (Fmoc strategy), Fmocꢀaa/HBTU/HOBt/DIPEA (5/5/5/10
equiv), solvent: DMF/NMP (8:2), ‘double’ coupling at rt, 60 min; Fmoc deprotection was carried
out with 20% piperidine in DMF/NMP (8:2), rt, 2 × 10 min; (b) DCM/TFA (97:3), 10 × 6 min;
(c) HBTU, HOBt, DIPEA, anhydrous DMF, 35 °C, 16 h; (d) TFA/H O (95:5), rt, 2.5 h; (e)
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succinimidyl propionate, 3% DIPEA in anhydrous DMF, rt, 2 h.
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ω
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Figure 3. Structure of the N ꢀcarbamoylated arginine building block 11.
For the preparation of the homodimeric ligands (16, 19, 21) shown in Scheme 3, octanedioic
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acid, activated as diꢀsuccinimidyl ester (12 ), was used for crossꢀlinking (Scheme 3). By
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contrast, as a building block for the synthesis of heterodimeric ligands (17, 18, 20, 22,),
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compound 13 was prepared by coupling suberic acid monomethyl ester to the resinꢀbound
peptide sequence Tyr(tBu)ꢀArg(Pbf)ꢀLeuꢀArg(Pbf)ꢀTyr(tBu). The sideꢀchain protected peptides
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4 and 15 were treated with 12 to give 16, 19 and 21), whereas in situ activated 13 was coupled
to the sideꢀchain protected peptides 14 and 15 for the preparation of the heterodimeric ligands 20
and 22, respectively. By analogy with the synthesis of the potential radioligands (2S,7S)ꢀ10 and
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(
2R,7R)ꢀ10, the precursor 17 was treated with succinimidyl propionate to give 18 as described
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very recently.
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Scheme 3. Synthesis of homoꢀ and heterodimeric Y R ligands
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Reagents and conditions: (a) cf. legend to Scheme 2; (b) DCM/TFA (97:3), 10 × 6 min; (c) 1
equiv of 12 in 1% DIPEA in anhydrous DMF, followed by 2.5 equiv of 9, 14 or 15, 35 °C, 16 h;
d) TFA/H O (95:5), rt, 2.5 h; (e) suberic acid monomethyl ester/HBTU/HOBt/DIPEA (4/4/4/8
equiv), solvent: DMF/NMP (8:2), single coupling at 30 °C, 14 h; (f) 0.5 M NaOH/MeOH (1:5),
reflux, 3 h; (g) 11/HBTU/HOBt/DIPEA (3/3/3/6 equiv), solvent: DMF/NMP (8:2), single
coupling at 35 °C, 16 h; (h) 13/HBTU//HOBt/DIPEA (1/1/1/2 equiv), solvent: DMF/NMP (8:2),
single coupling at 35 °C, 16 h; (i) succinimidyl propionate, DMF, DIPEA, rt, 2 h; (j)
(
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3/HBTU/HOBt/DIPEA (1/1/1/3.5 equiv), solvent: DMF, rt, 5 min, subsequent addition of 1.1
equiv of 14 or 15, rt, 17 h.
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Synthesis of the Radiolabeled Ligands [ H](2R,7R)-10 and [ H]18. Based on the results
obtained in the functional assays (see below), (2R,7R)ꢀ10 and 18 were selected for the synthesis
of the corresponding tritiated versions. An 8ꢀfold excess of the precursor peptides (2R,7R)ꢀ2 and
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was treated with commercially available succinimidyl [ H]propionate (Figure 4A). The
radioligands were obtained in high radiochemical purities (Figure 4B and Figure 4D). After
storage of the radioligands in a mixture of EtOH/H O (1:1) at ꢀ20 °C over 9 months,
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approximately 10% were decomposed (Figure 4C and Figure 4E). No decomposition was
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observed after an incubation period of 48 h, when the ‘cold’ analogues, (2R,7R)ꢀ10 and 18, were
investigated for stability (Figure 5A and Figure 5B) in a HEPES buffer (buffer I, pH 7.4).
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Figure 4. Preparation, purity, identity control and longꢀterm stability of the radiolabeled peptides
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[
H](2R,7R)ꢀ10 and [ H]18. (A) Synthesis of the radioligands [ H](2R,7R)ꢀ10 and [ H]18 by
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propionylation of the precursors (2R,7R)ꢀ2 and 17. Radiochemical yield: 34% ([ H](2R,7R)ꢀ10),
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0% ([ H]18). (B) HPLC analysis of [ H](2R,7R)ꢀ10 (ca. 0.35 µM) spiked with ‘cold’ (2R,7R)ꢀ
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[
H](2R,7R)ꢀ10 (ca. 0.25 µM) spiked with ‘cold’ (R,R)ꢀ10 (13 µM), analyzed after 9 months of
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storage at ꢀ20 °C in EtOH/H O (1:1). Radiochemical purity: 87%. (D) HPLC analysis of [ H]18
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ca. 0.35 µM) spiked with ‘cold’ 18 (5 µM), analyzed 10 days after synthesis. Radiochemical
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purity > 99%. (E) HPLC analysis of [ H]18 (ca. 0.25 µM) spiked with ‘cold’ 18 (13 µM),
analyzed after 9 months of storage at ꢀ20 °C in EtOH/H O (1:1). Radiochemical purity: 90%.
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The minor differences in t result from serial detection of the UV and radiometric signals.
R
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Figure 5. HPLC analysis of ‘cold’ (2R,7R)ꢀ10 (A) and 18 (B) after incubation in buffer I
(HEPES buffer, sodiumꢀfree) (pH 7.4) for up to 48 h. Both compounds showed no
decomposition. Peaks between 0 and 5 min correspond to buffer components.
Functional Studies at the hY R. The target compounds were investigated for Y R agonism in
4
4
2
+
23
an aequorin Ca assay and a luciferase gene reporter assay on genetically engineered CHO and
HEK293 cells, respectively (data cf. Table 1 and Figure 6).
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6
Table 1. NPY Y R agonist potencies (EC ) and intrinsic activities (α) of ’dimeric‘
4
50
pentapeptides and reference compounds PP and 1.
a
b
Aequorin assay
Luciferase assay
EC50 [nM]
0.73 ± 0.2
5.5 ± 0.8
1.6 ± 0.6
19 ± 4.8
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
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7
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9
0
1
2
3
4
5
6
7
8
9
0
Compd.
EC50 [nM]
9.7 ± 0.4
α
α
PP
1
1
c
1
8.5
0.62
0.75
0.78
0.76
0.70
0.73
0.80
0.67
ꢀ
1.01
1.03
1.07
1.00
0.98
0.93
1.06
1.05
ꢀ
(2R,7R)ꢀ2
(2S,7S)ꢀ2
(2R,7R)ꢀ10
6.9 ± 0.6
18 ± 1.6
14 ± 2.0
59 ± 5.6
49 ± 13
6.4 ± 1.3
13 ± 1.1
> 3000
4.0 ± 0.9
27 ± 4.6
(2S,7S)ꢀ10
1
6
7
86 ± 16
1
1.7 ± 0.2
7.1 ± 1.9
> 3000
18
19
20
21
22
290 ± 33
> 3000
0.85
ꢀ
160 ± 29
> 3000
0.98
ꢀ
310 ± 38
0.85
150 ± 18
0.99
a
23
b
Aequorin calcium mobilization assay on CHOꢀhY ꢀG ꢀmtAEQ cells.
CREꢀluciferase
4
qi5
reporter gene assay on HEK293 cells stably coꢀexpressing the hY R and the CREꢀcontrolled
4
luciferase gene reporter. Y R agonist potency was determined from the inhibition of forskolin (2
4
µM) stimulated luciferase activity. The maximal response (intrinsic activity, α) is referred to the
c
23
effect of PP set to α = 1.0. EC value reported by Ziemek et al.
50
The configuration of the stereo centers in the 2,7ꢀdiaminosuberic acid linker in 2 had an impact
on Y R potency. In the luciferase assay (2R,7R)ꢀ2 (EC = 1.6 nM) was 12 times more potent
4
50
than (2S,7S)ꢀ2 (EC50 = 19 nM). In the aequorin assay the difference in potency was less
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pronounced (EC50 values: 6.9 nM ((2R,7R)ꢀ2) vs. 18 nM ((2S,7S)ꢀ2)). The same tendency
became obvious for the propionylated analogues (2R,7R)ꢀ10 and (2S,7S)ꢀ10 in both assays.
Generally, propionylation of an amino group of the linker did only slightly (by a factor of two)
10
11
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15
16
17
18
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affect Y R potency in both assays.
4
Except for 17 and 18, compounds devoid of an amino group in the linker were significantly
less potent Y R agonists. Interestingly, replacement of one of the ’inner arginines‘ in 16 by the
4
G
N ꢀcarbamoylated arginine 11 considerably increased Y R agonist potency (17, EC : 6.4 nM
4
50
and 1.7 nM in the aequorin assay and the luciferase assay, respectively), that is, 17 was
equipotent with (2R,7R)ꢀ2. As observed for the stereoisomers of 2 and 10, propionylation of 17,
resulting in the derivative 18, led only to a minor decrease in Y R potency.
4
The homodimeric compounds 19 and 21, in which two arginines were replaced by alanine,
proved to be inactive at the Y R, suggesting that the presence of at least one unaltered
4
pentapeptide sequence TyrꢀArgꢀLeuꢀArgꢀTyrꢀNH is indispensable. Compounds 20 and 22, in
2
which only one Arg was replaced by Ala, were less potent than 16 by a factor of 5 to 6 in the
aequorin assay and by a factor of 2 in the luciferase assay.
Except for 19 and 21, all investigated compounds were partial agonists in the aequorin assay
(intrinsic activities between 62% and 85% referred to hPP), but full agonists in the luciferase
assay. Moreover, the potencies obtained in the luciferase assay were on average slightly higher
than those obtained in the aequorin assay, presumably due to more pronounced signal
24
amplification in the gene reporter assay.
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0
Figure 6. Y R agonism of PP, 1, 2, 10 and 16ꢀ22 determined in a calcium (aequorin) assay and a
4
luciferase reporter gene assay. (A) and (B) Concentrationꢀresponse curves (CRCs) from an
aequorin assay on CHOꢀhY RꢀmtAEQꢀGqi5 cells. Mean values ± SEM were from at least 3
4
independent experiments (performed in triplicate). (C) and (D) Inhibition of forskolinꢀstimulated
(
2 µM) luciferase activity (corresponding to 100%) in hY R expressing HEK293 cells by PP, 1,
4
2, 10 and 16ꢀ22, with the maximum inhibitory effect of the endogenous ligand PP which is set to
% luciferase activity and corresponds to full agonism (α = 1.0). Data points shown are the mean
SEM of at least three independent experiments performed in triplicate.
0
±
3
3
Characterization of [ H](2R,7R)-10 and [ H]18. Saturation binding experiments with the
tritiated radioligands (Figure 7A and Figure 7D), performed on intact CHOꢀhY ꢀG ꢀmtAEQ
4
qi5
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cells in buffer I (HEPES, sodiumꢀfree) as used by Balasubramaniam et al. to investigate the
diastereomeric mixture 2, revealed a K value of 0.67 ± 0.1 nM for both compounds,
d
3
3
3
[
[
H](2R,7R)ꢀ10 and [ H]18. The number of specific binding sites per cell, determined with
10
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3
H](2R,7R)ꢀ10 or [ H]18, was 320,000 ± 20,000 and 340,000 ± 15,000, respectively. At
3
concentrations around the K value, unspecific binding amounted to 10% ([ H](2R,7R )ꢀ10) and
d
3
2
0% ([ H]18), respectively, of the total binding. Association to the receptor was complete after
1
00 min (Figure 7B and Figure 7E). Dissociation from the receptor was biphasic (Figure 7C and
ꢀ1
3
ꢀ1
3
Figure 7F) with a fast k (0.0329 min for [ H](2R,7R)ꢀ10 and 0.0309 min for [ H]18) and a
off
ꢀ1
3
ꢀ1
3
slow k (0.0024 min for [ H](2R,7R)ꢀ10 and 0.0026 min for [ H]18).
off
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3
Figure 7. Y R binding characteristics of the radioligands [ H](2R,7R)ꢀ10 and [ H]18 determined
4
at live CHOꢀhY ꢀG ꢀmtAEQ cells at 22 °C in buffer I (HEPES, sodiumꢀfree). (A)
4
qi5
3
Representative saturation binding experiment with [ H](2R,7R)ꢀ10. (B) Y R association kinetics
4
3
of [ H](2R,7R)ꢀ10 (c = 1.5 nM). Inset: Linearization ln[B /(B ꢀB )] versus time. (C) Y R
eq
eq
t
4
3
dissociation kinetics of [ H](2R,7R)ꢀ10 (c = 1.5 nM, preꢀincubation time: 2 h) determined in the
presence of a 200ꢀfold excess of (2R,7R)ꢀ2. Fitting of the data according to a biphasic
ꢀk (1)t
ꢀk (2)t
off
2
exponential decay (black curve, B = B (1) ·e off + B (2) · e
, R = 0.9532) resulted in a
0
0
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much better correlation than an analysis according to a monophasic exponential decay (blue
ꢀ
k
t
2
off
dotted curve, B = B · e
, R = 0.8321). Inset: Linearization ln[B/B ] versus time. (D)
0
0
3
Representative saturation binding experiment with [ H]18. (E) Y R association kinetics of
4
10
11
12
13
14
15
16
17
18
19
20
21
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3
[
H]18 (c = 1.5 nM). Inset: Linearization ln[B /(B ꢀB )] versus time. (F) Y R dissociation
eq eq t 4
3
kinetics [ H]18 (c = 1.5 nM, preꢀincubation time: 2 h) determined in presence of 200ꢀfold excess
ꢀ
of 17. Fitting of the data according to a biphasic exponential decay (black curve, B = B (1) · e
0
k
(1)t
off
ꢀk (2)t
off
2
+
B (2) · e
0
, R = 0.9819) resulted in a much better correlation than an analysis
ꢀk
t
2
off
according to a monophasic exponential decay (blue dotted curve, B = B · e
, R = 0.9166).
0
Inset: Linearization ln[B/B ] versus time.
0
3
3
Table 2. Binding data of [ H](2R,7R)ꢀ10 and [ H]18.
Saturation binding
Binding kinetics
ꢀ1 c
ꢀ1 d
ꢀ1 e
radioligand
K [nM]
kobs [s ]
0.0467
0.0262
koff1 [s ]
koff2 [s ]
d
3
a
b
[
[
H](2R,7R)ꢀ10 0.67 ± 0.1
9.8 ± 2.5
0.0329
0.0391
0.0024
0.0032
3
a
H]18
0.67 ± 0.1
a
Equilibrium dissociation constant determined on CHOꢀhY ꢀG ꢀmtAEQ cells in buffer I;
4
qi5
b
mean ± SEM from at least 4 independent experiments (performed in triplicate). Equilibrium
dissociation constant determined on CHOꢀhY ꢀG ꢀmtAEQ cells in Leibovitz’ Lꢀ15 medium;
mean ± SEM from at least 4 independent experiments (performed in triplicate). Apparent
association constant, mean from 3 independent experiments (performed in triplicate). Fast
dissociation constant, mean from 3 independent experiments (performed in triplicate), calculated
4
qi5
c
d
e
according to a fourꢀparameter equation describing a biphasic exponential decay. Slow
dissociation constant, mean from 3 independent experiments (performed in triplicate), calculated
according to a fourꢀparameter equation describing a biphasic exponential decay. Note: A
kinetically derived dissociation constant Kd(kin) and the association rate constant kon was not
calculated, as k was ambiguous due to a fast (koff1) and a slow (koff2) component.
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Competition Binding Studies at NPY Receptor Subtypes. K values of the ‘dimeric’
i
pentapeptides were determined in competition binding experiments on live cells expressing
human Y , Y , Y or Y receptors to determine the NPY receptor subtype selectivity. The
1
2
4
5
0
1
2
3
4
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unspecific binding at the respective NPY receptor expressing cells was low, and there was no
hint to an internalization of the used radioligands, which might lead to a misinterpretation of the
binding data. Therefore, investigations on a potential internalization, e. g. by acid wash
16
experiments, were not considered necessary.
Y R Binding. The binding data of the ‘dimeric’ pentapeptides and the reference compounds
4
3
3
PP, 1 and 3, were determined at the Y R, using the new radioligands [ H](2R,7R)ꢀ10 and [ H]18
4
(
Figure 8). The K value of PP (0.65 nM) was in good agreement with published data (K : 0.50
i
i
1
6
25
nM, 0.53 nM ), whereas the affinity of 3 (K = 660 nM) was slightly lower than reported (K =
i
i
10
1
30 nM ). In case of the ‘cold’ analogues of the radioligands, (2R,7R)ꢀ10 (0.87 nM) and 18 (1.2
nM), the K values were in good agreement with the K values (cf. Figure 8, Table 2). Generally,
i
d
the K values of the ‘dimeric’ pentapeptides at the Y R were by a factor of approximately 10
i
4
lower than the EC values determined in the functional assays (aequorin or luciferase assay,
5
0
Table 1). Nevertheless, the Y R affinities correlated with the order of potency.
4
1
5
The diastereomeric mixture of 2 was previously reported to show Y R agonism in the twoꢀ
4
digit nM range (EC50 = 14.8 nM, cAMP assay), but much higher affinity (K = 0.05 nM,
i
1
25
radioligand [ I]PP) determined in sodiumꢀfree HEPES buffer (buffer I), which has a broad
application in binding experiments in the NPY field. Experiments performed with different
buffers in our laboratory revealed that the discrepancies between functional and binding data are,
at least in part, caused by the absence (buffer I) or presence (Leibovitz’ Lꢀ15 medium) of sodium
+
ions. An exchange of the sodiumꢀfree buffer I with the Na containing Lꢀ15 medium led to an
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increase in the K value of [ H](2R,7R)ꢀ10 from 0.67 nM to 9.8 nM (cf. Table 2, footnote b).
d
3
Similarly, K values determined in competition binding experiments using [ H](2R,7R)ꢀ10 in Lꢀ
i
1
5 medium were by a factor of almost 10 higher (K values, (2R,7R)ꢀ2: 2.0 nM; (2S,7S)ꢀ2: 17
i
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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57
58
59
60
nM; (2R,7R)ꢀ10: 6.3 nM). It should be stressed that the binding constants determined in Lꢀ15
medium were in good agreement with the agonist potencies determined in the functional assays
+
using Na containing buffer III or DMEM.
Interestingly, the affinity of the Y R antagonist 3 was not affected in the same way by an
4
exchange of the binding buffer. The K value of 3 in competition binding experiments using the
i
Lꢀ15 medium was even lower (K = 150 nM) than in experiments using buffer I. These results
i
are compatible with recent insights obtained from GPCR crystal structures. Several highꢀ
+
resolution crystal structures described Na ions as allosteric stabilizers of inactiveꢀstate receptor
26,27
+
conformations.
active state.
Therefore, Na is expected to reduce the affinity of agonists binding to the
3
3
As an alternative to [ H](2R,7R)ꢀ10, the radioligand [ H]18 is also suited for competition
3
binding. The K values determined by displacement of [ H]18 were on average slightly lower
i
(Table 3).
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0
Figure 8. Radioligand displacement curves from competition binding experiments performed
3
3
with [ H](2R,7R)ꢀ10 or [ H]18 at CHOꢀhY RꢀG ꢀmtAEQ cells in buffer I. (A) and (B)
4
qi5
3
3
Displacement of [ H](2R,7R)ꢀ10 (K = 0.67 nM, c = 0.6 nM). (C) Displacement of [ H]18 (K =
d
d
0.67 nM, c = 1 nM). Data represent mean values ± SEM of at least 3 independent experiments,
each performed in triplicate.
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NPY receptor subtype selectivity. The K values of (2R,7R)ꢀ10 and 18, the ’cold’ analogues
i
of the new Y R radioligands at the Y R, Y R and Y R were in the threeꢀdigit nanomolar range
4
1
2
5
(
Table 3). The ‘heterodimeric’ ligands 20 and 22 were comparable with (2R,7R)ꢀ10 and 18 at the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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Y R and Y R, but only very weak binders at the Y R. K values were not determined for the
1
5
2
i
‘homodimeric’ ligand 21 at all NPY receptors due to low affinity. The same holds for compound
1
9 with exception of the Y R. Peptide dimers derived from the Cꢀterminus of NPY such as 1
5
1
3,28
were among the first high affinity Y R antagonists described in literature.
Therefore, we
1
selected (2R,7R)ꢀ2 and 17 as examples for functional investigations at the Y R. In a standard
1
Y R model, the Furaꢀ2 calcium assay on HEL cells, both compounds revealed antagonism with
1
K values of 9.5 nM and 25.3 nM, respectively (cf. Table 3, footnote, and Supporting
b
Information, Figure S3). In case of (2R,7R)ꢀ2 the result disagrees with published data for the
diastereomeric mixture 2, which was reported to be an agonist in a cAMP assay on Y R
1
15
expressing HEK 293 cells. It may be speculated, whether the opposing qualities of action are
2
9
related to the specific repertoire of G and G coupling in HEL cells, endogenously expressing
q
i
the Y R, compared to genetically engineered HEK293 cells.
1
Table 3. NPY receptor binding data.
Y R
1
Y R
2
Y R, K [nM]
Y R,
5
4
i
a
b
3
c
3
d
3
e
f
Compd.
PP
K [nM]
K [nM]
[ H](2R,7R)ꢀ10
[ H](2R,7R)ꢀ10
[ H]18
K [nM]
i
i
i
g
g
g
440
>5000
0.65 ± 0.13
ꢀ
0.37 ±
17
0.02
1
2
ꢀ
ꢀ
1.6 ± 0.34
ꢀ
ꢀ
1.3 ± 0.78
ꢀ
ꢀ
h
h
h
7.5
1100
1.06 ± 0.47
>1000
h
(0.05 )
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9
1
1
1
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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
i
(2R,7R)ꢀ2
2S,7S)ꢀ2
440 ± 81
830 ± 250
950 ± 520
0.45 ± 0.14
2.0 ± 0.04
0.18 ±
0.02
1500 ± 300
2100 ± 980
(
930 ± 270
2.3 ± 0.16
660 ± 140
0.87 ± 0.16
17 ± 4.3
150 ± 36
6.3 ± 0.8
ꢀ
j
j
j
3
24
920
630 ± 80
>5000
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
(
(
2R,7R)ꢀ10 400 ± 46
2S,7S)ꢀ10 290 ± 110
420 ± 50
0.32 ±
0.02
710 ± 96
750 ± 380
1700 ± 70
730 ± 150
2.8 ± 0.44
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
400 ± 71
280 ± 40
140 ± 1.4
220 ± 40
k
1
6
7
720 ± 100
410 ± 150
360 ± 46
3.5 ± 0.59
l
k
1
1.2 ± 0.37
k
18
1300 ± 760 1.2 ± 0.49
0.66 ±
0.13
1
9
0
>3000
>10000
>3000
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
550 ± 5.5
220 ± 40
>10000
2
1400 ± 270 >3000
12 ± 1.6
>3000
21
22
>10000
>10000
>3000
1600 ± 30
11 ± 1.4
ꢀ
460 ± 50
a
3
30
Radioligand competition binding assay with [ H]URꢀMK136 (K = 6.2 nM, c = 4 nM) using
MCFꢀ7ꢀhY cells in buffer II. Radioligand competition binding assay with [ H]propionylꢀ
pNPY (K = 1.4 nM, c = 1 nM) using CHOꢀhY ꢀG ꢀmtAEQ cells in buffer I. Radioligand
competition binding assay with [ H](2R,7R)ꢀ10 (K = 0.67 nM, c = 0.6 nM) using CHOꢀhY Rꢀ
G ꢀmtAEQ cells in buffer I. Radioligand competition binding assay with [ H](2R,7R)ꢀ10 (K =
.8 nM, c = 10 nM) using CHOꢀhY RꢀG ꢀmtAEQ cells in Lꢀ15 medium. Radioligand
competition binding assay with [ H]18 (K = 0.67 nM, c = 1 nM) using CHOꢀhY RꢀG ꢀmtAEQ
cells in buffer I. Radioligand competition binding assay with [ H]propionylꢀpNPY (K = 4.83
nM, c = 4 nM) using HECꢀ1b hY R cells in buffer II. K value reported by Berlicki et al. Ki
value reported by Balasubramaniam et al.; these authors used [ I]PYY (Y R expressed in
HEK 293 cells, Y R or Y R expressed in CHO cells) and [ I]PP (Y R expressing CHO cells)
for the determination of the binding data. Y R antagonism at HEL cells (Furaꢀ2 calcium assay):
K = 9.5 ± 0.12 nM (cf. Supporting Information, Figure S3); in contrast to data reported in ref.
for the diastereomeric mixture 2 (cAMP assay, Y R expressing HEK 293 cells) no Y R agonist
activity. K value reported by Keller et al. Ref. : K values determined by flow cytometry
using fluorescenceꢀlabeled K4ꢀhPP in buffer I: 16: 7.4 nM, 17: 1.5 nM, 18: 3.6 nM. Y R
d
3
1 b
3
1
3
2
33 c
d
2
qi5
3
d
4
d
3
qi5
d
e
9
4 qi5
3
d
4
qi5
f
3
d
3
4 g
25 h
5
i
15
125
1
1
25
2
5
4
i
1
1
5
b
1
1
j
10
k
16
i
i
l
1
antagonism at HEL cells (Furaꢀ2 calcium assay): K = 25.3 ± 3.8 nM (cf. Supporting
Information, Figure S3).
b
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3
. CONCLUSIONS
The synthesis of the enantiopure diaminosuberic acids (R,R)ꢀ8 and (S,S)ꢀ8, accomplished in
10
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17
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excellent yield via Grubbs’ II catalyzed coupling, gave access to the individual diastereomers of
compound 2 with (2R,7R)ꢀ or (2S,7S)ꢀconfiguration in the linker. In the same way, octanedioic
acid was used as an achiral linker to combine two identical or two different pentapeptide
moieties, in which the Arg residues were either retained or replaced at one or two positions by
Ala or by an aminoꢀfunctionalized carbamoylated Arg. Interestingly, the latter turned out to
compensate for the absence of the two amino groups in the connecting diacid with respect to
affinity and potency (cf. compound 17). Moreover, propionylation of one amino group in the
linker of 2 (cf. compound 10) or at the primary amino group in 17 gave access to the
3
3
3
corresponding tritiated high affinity radioligands. [ H](2R,7R)ꢀ10 ([ H]URꢀKK193) and [ H]18
3
(
[ H]URꢀKK200) are useful new pharmacological tools for the Y R. (2R,7R)ꢀ2 was superior to
4
(2S,7S)ꢀ2 and, in contrast to published data for the mixture 2, affinities and agonist potencies
were essentially in the same range. As demonstrated by saturation and competition binding
experiments, the composition of the buffer may account for discrepancies between functional
and binding data. It should be kept in mind that the affinity of peptidic Y R agonists may be
4
considerably higher in the absence of sodium ions, conditions widely used for the investigation
of NPY receptor ligands.
4. EXPERIMENTAL SECTION
Chemistry: General Conditions. Chemicals and solvents were purchased from commercial
suppliers and used without further purification unless otherwise indicated. MeCN for HPLC
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2
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6
(
gradient grade) was obtained from Merck (Darmstadt, Germany). DMF for peptide synthesis,
NMP for peptide synthesis and HOBT hydrate were obtained from Acros Organics/Fisher
Scientific (Nidderau, Germany). FmocꢀSieberꢀPS resin (0.61 mmol/g), (S)ꢀallylglycine, (R)ꢀ
allylglycine, FmocꢀArg(Pbf)ꢀOH, FmocꢀTyr(tBu)ꢀOH and HBTU were purchased from Iris
Biotech (Marktredwitz, Germany). FmocꢀAlaꢀOH, FmocꢀLeuꢀOH, dicyclohexylcarbodiimide,
dioxane, sodium hydroxide and methanol were obtained from Merck (Darmstadt, Germany).
Trifluoroacetic acid, dichloromethane, diethyl ether, ethanol and Triton Xꢀ100 were obtained
from Sigma Aldrich (Deisenhofen, Germany), N,Nꢀdiisopropylethylamine (DIPEA) (99%) was
from ABCR (Karlsruhe, Germany), diꢀtertꢀbutyl dicarbonate (>97%) and 8ꢀmethoxyꢀ8ꢀ
oxooctanoic acid (98%) were from Alfa Aesar (Karlsruhe, Germany). Bovine serum albumin and
bacitracin were purchased from Serva (Heidelberg, Germany), coelenterazin h was obtained
from Biotrend (Cologne, Germany), human pancreatic polypeptide and porcine neuropeptide Y
were synthesized by Prof. Dr. C. Cabrele (University Salzburg, Salzburg, Austria). Compound 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
10
was a gift from Dr. A. J. Daniels (Glaxo Wellcome, NC, USA). The synthesis of compounds 3,
16
22
16
16
3
32
1
1,
12,
13,
16-18
and [ H]propionylꢀpNPY
was previously described. (4,5ꢀ
DihydroIMES)(PCy )Cl Ru=CHPh (Grubbs’ II catalyst) was synthesized according to literature
3
2
3
5
procedure. Millipore water was used throughout for the preparation of buffers and HPLC
eluents. Polypropylene reaction vessels (1.5 or 2 mL) with screw cap (SüdꢀLaborbedarf, Gauting,
3
3
Germany) were used for the synthesis of the radioligands [ H](2R,7R)ꢀ10 and [ H]18, for small
scale reactions (e.g. the preparation of 16), the investigation of chemical stabilities and for the
storage of stock solutions. TL chromatography was performed on Merck silica gel 60 F254 TLC
aluminum plates, silica gel 60 (40ꢀ63 µm, Merck) was used for column chromatography.
Visualization was accomplished by UV light (λ = 254 nm or 366 nm) and ninhydrin/acetic acid
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solution. NMRꢀspectra were recorded on a Bruker Avance 300 (7.05 T, H: 300.1 MHz, C:
1
7
5.5 MHz) or a Bruker Avance 600 instrument with cryogenic probe (14.1 T, H: 600.3 MHz,
1
3
C: 150.9 MHz) (Bruker, Karlsruhe, Germany). ATRꢀIR spectroscopy was performed on a
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17
18
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56
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60
Biorad Excalibur FTS 3000 spectrometer (Biorad, Munich, Germany), equipped with a Specac
Golden Gate Diamond Single Reflection ATRꢀSystem. Mass spectra were recorded on a
Finnigan MAT95 (EIꢀMS 70eV) or a Finnigan MAT SSQ 710 A (CIꢀMS (NH )). For HRMS an
3
Agilent QꢀTOF 6540 UHD (Agilent, Waldbronn, Germany) equipped with an ESI source was
20
3
ꢀ1 ꢀ1
used. Optical rotations (given as specific rotation [α] , [° cm dm g ], concentrations (c) given
as g/100 mL) were determined in an MCP 500 (Anton Paar, OstfildernꢀScharnhausen, Germany)
or a 241 polarimeter (Perkin Elmer, Rodgau, Germany) at 589 nm in a 1.0ꢀdm cuvette.
Preparative HPLC was performed on a system from Knauer (Berlin, Germany) consisting of two
Kꢀ1800 pumps, a Kꢀ2001 detector and a RPꢀcolumn (KinetexꢀXB C , 5 µm, 250 mm × 21 mm,
1
8
Phenomenex, Aschaffenburg, Germany) at a flowꢀrate of 15 mL/min using mixtures of
acetonitrile and 0.1% aqueous TFA solution as mobile phase. A detection wavelength of 220 nm
was used throughout. The collected fractions were lyophilised using an alpha 2ꢀ4 LD apparatus
(Martin Christ, Osterode am Harz, Germany) equipped with a RZ 6 rotary vane vacuum pump
(vacuubrand, Wertheim, Germany). Analytical HPLC analysis was performed on a system from
MerckꢀHitachi (Hitachi, Düsseldorf, Germany) composed of a Lꢀ6200ꢀA pump, a ASꢀ2000A
autosampler, a Lꢀ4000A UV detector, a Dꢀ6000 interface and a RPꢀcolumn KinetexꢀXB C , 5
1
8
µm, 250 mm × 4.6 mm (Phenomenex, Aschaffenburg, Germany) at a flow rate of 0.8 mL/min.
Mixtures of acetonitrile (A) and 0.1% aqueous TFA solution (B) were used as mobile phase. The
following gradient was applied: A/B: 0ꢀ25 min: 10:90 ꢀ 60:40, 25ꢀ35 min: 60:40 ꢀ 95:5, 35ꢀ45
min: 95:5. Detection was performed at 220 nm, the oven temperature was 30 °C.
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1
Compound Characterization. New nonꢀpeptide intermediates were characterized by Hꢀ and
1
3
CꢀNMR Spectroscopy, HRMS and optical rotation (if applicable). New peptidic compounds
(
fully deprotected) were characterized by HRMS and RPꢀHPLC analysis. In addition,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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9
0
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0
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9
0
1
13
1
compounds 20 and 22 were characterized by Hꢀ, Cꢀ and 2DꢀNMR ( HꢀCOSY, HSQC,
HMBC). Purity of tested compounds was >95% as determined by HPLC.
Chemistry: Experimental Protocols and Analytical data. General procedure for solid
phase peptide synthesis. Peptides were synthesized by manual SPPS using the Fmoc strategy on
an FmocꢀSieberꢀPS resin. 5ꢀml or 20ꢀml Discardit II syringes (Becton Dickinson, Heidelberg,
Germany) were equipped with 35 µm polyethylene frits (Roland Vetter Laborbedarf,
Ammerbuch, Germany) and used as reaction vessels. DMF/NMP (8:2) was used as solvent.
Protected standard Lꢀamino acids (5 equiv) were preꢀactivated with HBTU (5 equiv)/HOBt (5
equiv)/DIPEA (10 equiv) for 5 min and added to the resin. ‘Double’ coupling at rt was
performed for all standard amino acids for 45 min. After completed coupling of an Fmocꢀaa, the
resin was washed with DMF/NMP and treated with 20% piperidine in DMF/NMP (8:2) at rt (2×)
for 10 min to remove the Fmoc group, followed by thorough washing of the resin.
General procedure for cleavage of protected peptides from the resin. After the last
coupling step and Fmoc deprotection, the resin was washed with DCM. Sideꢀchain protected
peptides were cleaved from the resin with CH Cl /TFA (97:3) (rt, 10 × 6 min). The peptides
2
2
were separated from the resin by filtration. The combined filtrates were collected in a round
bottom flask containing water (10 times the volume of the combined filtrates). The organic
solvent was removed on a rotary evaporator; the aqueous phase was lyophilised followed by
purification by preparative HPLC.
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(
2R,7R)-Diaminooctanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide)
hexa(hydrotrifluoroacetate) ((2R,7R)-2). (R,R)ꢀ8 (4.3 mg, 10.7 µmol), HBTU (8.5 mg, 2.1
equiv) and HOBt (3.5 mg, 2.1 equiv) were dissolved in anhydrous DMF (400 µL). DIPEA (10
µL, 5 equiv) was added and the mixture was stirred at room temperature for 5 min followed by
the addition of a solution of 9 (40 mg, 2.5 equiv). The resulting mixture was stirred at 35 °C for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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1
6 h. Water (10 mL) was added and the protected intermediate was extracted with DCM (2 × 10
mL). The combined organic layers were evaporated, and the residue was dried in vacuo.
TFA/water (95:5, 2 mL) was added and the mixture was stirred at room temperature for 2.5 h.
Water (100 mL) was added followed by lyophilisation. The product was purified by preparative
HPLC (column: KinetexꢀXB C18 250 × 21 mm; gradient: 0–20 min: MeCN/0.1% aq TFA
1
0:90–35:65, t = 15.8 min). Lyophilisation of the eluate afforded (2R,7R)ꢀ2 as a white fluffy
R
4
+
solid (7.5 mg, 29.3%). HRMS (m/z): [M+4H] calcd. for C H N O , 427.2495; found,
80 128 26 16
4
27.2510. RPꢀHPLC (220 nm): 96% (t = 18.93 min, k = 5.6). C H N O · C H F O
R
80 124 26 16
12
6
18 12
(
1706.04 + 684.14).
(2S,7S)-Diaminooctanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide)
hexa(hydrotrifluoroacetate) ((2S,7S)-2). (2S,7S)ꢀ2 was prepared in analogy to (2R,7R)ꢀ2 by
using (S,S)ꢀ8 (4.3 mg, 10.7 µmol). The product was purified by preparative HPLC (column:
KinetexꢀXB C18 250 × 21 mm; gradient: 0–20 min: MeCN/0.1% aq TFA 10:90–35:65, t = 15.5
R
min). Lyophilisation of the eluate afforded (2S,7S)ꢀ2 as a white fluffy solid (8.6 mg, 33.6%).
4+
HRMS (m/z): [M+4H] calcd. for C H N O , 427.2495; found, 427.2508. RPꢀHPLC (220
8
0
128 26 16
nm): 98% (t = 18.37 min, k = 5.4). C H N O · C H F O (1706.04 + 684.14).
R
80 124 26 16
12
6 18 12
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(
2R,7R)-Diethyl 2,7-bis[(tert-butoxycarbonyl)amino]oct-4-enedioate ((R,R)-6). According
3
6
to literature procedure in a flameꢀdried Schlenk flask (R)ꢀ5 (106 mg, 0.43 mmol, 2 equiv) was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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6
7
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9
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1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
dissolved in anhydrous DCM (8 mL) under N ꢀatmosphere. Grubbs’ II catalyst second
2
generation (18.5 mg, 0.022 mmol, 0.1 equiv) was added and the reaction mixture was stirred
under reflux for 11 h until all starting material had been consumed. The solvent was removed
under reduced pressure and the crude product was purified by column chromatography
(PE/EtOAc 5:1) to obtain (R,R)ꢀ6 (89 mg, 0.19 mmol, 89%) as colorless oil. R = 0.23
f
1
(
PE/EtOAc 5:1). HꢀNMR (300 MHz, CDCl ): δ (ppm) 1.21 (t, 6H, J 7.1 Hz), 1.38 (s, 18H), 2.31
3
ꢀ
2.49 (m, 4H), 4.13 (qd, 4H, J 1.8 Hz, 7.1 Hz), 4.2 – 4.31 (m, 2H), 5.01 – 5.11 (m, 2H), 5.31 –
1
3
5.44 (m, 2H). CꢀNMR (75 MHz, CDCl ): δ (ppm) 14.2, 28.3, 35.5, 53.1, 61.3, 79.8, 128.5,
3
1
55.2, 171.9. IR (neat) 3369, 2978, 2937, 1703, 1498, 1367, 1330, 1248, 1159, 1095, 1051,
ꢀ1
ͦͤ
+
1021, 969, 857, 779 cm . [α] ꢀ27.0 (c 1, chloroform). LRMS (ESI): m/z [M+H] 459.3,
ꢀ
+
+
+
+
[
M+Na] 481.3, [2M+Na] 939.5. HRMS (ESI): m/z [M+H] calcd. for [C H N O ] 459.2701,
22 39 2 8
found: 459.2711. C H N O (458.55).
2
2
38
2
8
(2S,7S)-Diethyl 2,7-bis[(tert-butoxycarbonyl)amino]oct-4-enedioate ((S,S)-6). Compound
(S,S)ꢀ(+)ꢀ6 was prepared according to (R,R)ꢀ6 from (S)ꢀ5 (105 mg, 0.43 mmol, 2 equiv) as
starting material to obtain the product as a colorless oil (47.3 mg, 0.1 mmol, 49%). The
spectroscopic data were identical to those for the enantiomer (R,R)ꢀ(ꢀ)ꢀ6. [α]ͦ +27.9 (c 1,
ͤ
ꢀ
chloroform).
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(
2R,7R)-Diethyl 2,7-bis[(tert-butoxycarbonyl)amino]octanedioate ((R,R)-7). In a flame
dried Schlenk flask under nitrogen atmosphere (R)ꢀ5 (300 mg, 1.23 mmol, 2 equiv) was
dissolved in anhydrous DCM (18 mL). Grubbs’ II catalyst (52 mg, 0.06 mmol, 0.1 equiv) was
added, and the reaction mixture was stirred under reflux for 12 h. After all starting material was
consumed, the solvent was removed under reduced pressure, and EtOH (10 mL) was added. The
mixture was transferred into an autoclave and stirred under hydrogen at 20 bar at room
temperature for 15 h. The reaction mixture was filtrated, and the solvent was removed under
reduced pressure. The crude product was purified by column chromatography (PE/EtOAc 3:1) to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
38
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41
42
43
44
45
46
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48
49
50
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55
56
57
58
59
60
yield (R,R)ꢀ7 (268 mg, 0.58 mmol, 95% over two steps) as colorless oil. R = 0.3 (PE/EtOAc
f
1
3
:1). HꢀNMR (300 MHz, CDCl ): δ (ppm) 1.21 (t, 6H, J 7.1 Hz), 1.24 – 1.33 (m, 4 H), 1.37 (s,
3
1
3
18H), 1.46 – 1.79 (m, 4 H), 4.05ꢀ4.24 (m, 4H + 2H), 4.94 – 5.04 (m, 2H). CꢀNMR (75 MHz,
CDCl ): δ (ppm) 14.2, 24.9, 28.3, 32.6, 53.3, 61.3, 79.8, 155.4, 172.8. IR (neat) 3354, 2978,
3
ꢀ1
ͦͤ
2937, 2870, 1699, 1505, 1457, 1367, 1248, 1159, 1021, 861, 779 cm . [α] ꢀ14.8 (c 1,
ꢀ
+
+
+
chloroform). LRMS (ESI): m/z [M+H] 461.3, [M+Na] 483.3, [2M+Na] 943.5. HRMS (ESI):
+
+
m/z [M+H] calcd. for [C H N O ] 461.2857, found: 461.2865. C H N O (460.56).
22
41
2
8
22 40
2
8
(2S,7S)-Diethyl 2,7-bis[(tert-butoxycarbonyl)amino]octanedioate ((S,S)-7). Compound
(S,S)ꢀ(+)ꢀ7 was prepared according to (R,R)ꢀ7 by using (S)ꢀ5 (303 mg, 1.2 mmol, 2 equiv) as
starting material to obtain the product as a colorless oil (212 mg, 0.46 mmol, 74% over two
ͦ
ͤ
steps). The spectroscopic data were identical to those for its enantiomer (R,R)ꢀ(ꢀ)ꢀ7. [α] +16.9
ꢀ
(
c 1, chloroform).
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