Cis-4-amino-l-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues: Part 2

Article abstract of DOI:10.1021/jm300947s

Recently, we reported synthesis and activity of a constrained cyclic analogue of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH₂) and related linear models containing the cis-4-amino-l-proline (cAmp) in place of native Pro². In the present

Full text of DOI:10.1021/jm300947s

Article
pubs.acs.org/jmc
cis-4-Amino‑L‑proline Residue As a Scaffold for the Synthesis of
Cyclic and Linear Endomorphin‑2 Analogues: Part 2
Adriano Mollica,*,† Francesco Pinnen,† Azzurra Stefanucci,† Luisa Mannina,‡,§ Anatoly P. Sobolev,§
Gino Lucente,∥ Peg Davis,⊥ Josephine Lai,⊥ Shou-Wu Ma,⊥ Frank Porreca,⊥ and Victor J. Hruby#
†Dipartimento di Scienze del Farmaco, Universita
̀
di Chieti-Pescara “G. d′Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy
‡Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita
̀
di Roma, P.le A. Moro 5, 00185 Rome, Italy
§Laboratorio di Risonanza Magnetica “Annalaura Segre”, Istituto di Metodologie Chimiche, CNR, Via Salaria Km 29.300, 00015
Monterotondo, Rome, Italy
∥Dipartimento di Chimica e Tecnologie del Farmaco e Istituto di Chimica Biomolecolare, CNR Sezione di Roma, “Sapienza”,
̀
Universita di Roma, P.le A. Moro 5, 00185 Roma, Italy
⊥
#
Departments of Pharmacology and Chemistry and Biochemisty, University of Arizona, Tucson, Arizona 85721, United States
S
* Supporting Information
ABSTRACT: Recently, we reported synthesis and activity of a
constrained cyclic analogue of endomorphin-2 (EM-2: Tyr-
Pro-Phe-Phe-NH2) and related linear models containing the
cis-4-amino-L-proline (cAmp) in place of native Pro2. In the
present article, the adopted rationale is the possible
modulation of the receptor affinity of the cAmp containing
EM-2 analogues by assigning a different stereochemistry to the
Phe3 and Phe4 residues present in the ring. Thus, eight more
analogues with different absolute configuration at the chiral
center of the aromatic residues in positions 3 and 4 have been
synthesized and their opioid activity examined. The stereo-
chemical change at the α-carbon atoms leads to a meaningful enhancement of the affinity and activity toward μ opioid receptors
with respect to the prototype compound 9: e.g., 9a, Kμi = 63 nM, GPI (IC50) = 480 nM; 9b, Kμi = 38 nM, GPI (IC50) = 330 nM.
In the specific field of EM-2, the study of linear analogues
containing cAmp at position 2 can give information on the
biological consequences of the presence of an additional free
primary amino group adjacent to that of the native N-terminal
tyrosine. Furthermore, an intramolecular side chain-to-tail
coupling reaction between the cAmp-γ-NH2 and the Phe4 C-
terminal carboxyl group gives rise to an 11-membered cyclic
system further constrained by the presence of the cAmp inside
the backbone8 (see Figure 1).
A series of selected cyclic pentapeptide EM-1 analogues has
been previously reported.14−16 These cyclopeptides maintain
the amino acid sequence of the parent and are characterized by
an amino acidic bridge of different length and chirality inserted
between the N-terminal Tyr1 and the C-terminal Phe4. Notable
structural feature of these analogues is the D absolute
configuration at position 2 and 3 of the backbone as well as
the absence of the N-terminal free amino group, which is
intramolecularly bound to the fifth amino acid used as a bridge.
As compared with these models, the here reported cyclic
system, containing the cAmp residue, presents two main
differences: (i) a more constrained peptide skeleton with lower
INTRODUCTION
■
In the light of the intrinsic limitations of native opioid peptides
as therapeutic agents,1−3 the design of cyclic analogues appears
particularly appealing.4−7 In a recent article,8 our research team
reported the design and synthesis of a cyclic model and related
linear structures, based on the sequence of endomorphin-2 (H-
Tyr-Pro-Phe-Phe-NH2 ; EM-2)9,10 containing a modification at
the Pro2 (see Figure 1). The relevant structural feature of those
models was based on the utilization of a cis-4-amino-L-proline
(cAmp) residue to replace the native proline. This synthetic
amino acid11 combines the conformational rigidity of the
pyrrolidine Pro ring with the presence at position 4 of the
heterocyclic ring of a cis-oriented primary amino group, thus
realizing an arrangement corresponding to a proline/γ-amino-n-
butyric acid chimera,12,13 namely, proline/GABA cis-chimera.11
The presence of the cAmp at position 2 of the tetrapeptide
backbone should not alter significantly the EM-2 amino acid
sequence and offers, at the same time, as compared with the
native Pro residue, an additional amino group available for a
cyclization reaction.
As a consequence of the structural properties of the poly
functional cAmp residue, its insertion into a peptide backbone
gives rise, in addition to usual linear analogues, to structurally
interesting cyclic models.
Received: July 4, 2012
© XXXX American Chemical Society
A
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Figure 1. Here reported EM-2 linear (13a−c and 15 a-c) and cyclic (9a−b) analogues obtained by adopting the side chain-to-tail strategy. For the
sake of clarity, the structures of the previously reported analogue (9) and its precursors (13−15) are also indicated. Product 9c was not synthesized
as reported in the text.
Table 1. Binding Affinity and in Vitro Activity for Compounds 9a-b, 13a-c and 15a-c.
ab
,
receptor affinity (nM)
dfi
functional bioactivity (nM)
cfg
, ,
efi
, ,
g
g
, ,
compound
Kiμ
Kiδ
>500
Kiκ
GPI (IC50)
MVD (IC50)
hi
,
EM-2
9.6 90.98
660 79
63 13
>500
nb
15
2
510 35
h
j
9
nb
1.4% at 1 μM
480 95
0% at 1 μM
1400 170
950 120
9a
9b
1010 150
>10000
>10000
nb
38
9
860 110
330 65
h
13
1960 2254
>10000
nb
0% at 1 μM
2% at 1 μM
11% at 1 μM
3.2% at 1 μM
890 130
3.6% at 1 μM
3.6% at 1 μM
3.6% at 1 μM
1.3% at 1 μM
20% at 1 μM
0% at 1 μM
14% at 1 μM
5.4% at 1 μM
13a
13b
13c
>10000
nb
nb
1010 200
760 120
315 81
3700 340
760 90
>10000
nb
nb
nb
h
15
nb
nb
15a
15b
15c
nb
nb
0.9% at 1 μM
12% at 1 μM
2300 370
nb
nb
nb
nb
a
Competition analyses was carried out using membrane preparations from transfected HN9.10 cells that constitutively expressed the DOR and
b
c
d
MOR, respectively. Competition analyses for κ receptors were carried out using rat recombinant CHO cells. Kd = 0.85 0.2 nM. Kd = 0.50 0.1
nM. Kd = 2.0 0.05 nM. The Ki values are calculated using the Cheng and Prusoff equation to correct for the concentration of the radioligand used
in the assay.
e
f
g
h
i
j
SEM. Data from ref 8. Data from ref 17. nb = no binding detected.
conformational flexibility and more defined spatial orientation
of the aromatic side chains; (ii) the presence of the positively
charged Tyr N-terminal amino group, an element which,
despite the structural diversity, is characteristic of practically all
the endogenous opioid ligands including EMs and enkephalins.
The first example of cyclic EM-2 analogue incorporating the
cAmp residue in place of the native Pro2 has been described by
us in a previous paper and its structure, together with those of
its related linear derivatives, is reported in Figure 1
(compounds 9, 13, and 15, respectively). Both 9 and its linear
derivatives showed, as compared with the parent EM-2, a
sensible decrease of binding affinity toward the three (μ, δ, and
κ) examined opioid receptors and only modest in vitro
functional bioactivity (Table 1). As indicated in Figure 1, the
three previously reported peptides (9, 13, and 15) maintain the
homochiral all-L sequence found in the parent EM-2.
consequences of the cyclization on the conformation of short
linear peptides. As compared with more extended systems, the
resulting cyclic structures are characterized with reduced
conformational heterogeneity as well as more defined top-
ography of the attached side chains. This feature sensibly
reduces the population of conformers available for a proper
receptor−ligand interaction. In the case under study, the
cyclization was designed so as to involve the Pro2-Phe3-Phe4
sequence strongly influencing the spatial orientation of the
attached benzylic side chains and to leave external to the
cyclopeptide moiety the N-terminal Tyr1 residue. This
arrangement changes profoundly the shape of the electron-
rich zone bound to interact with the counterpart located in the
binding pocket of μ opioid receptors. However, results reported
in Table 1 clearly show that the parent linear ligand EM-2
adopts sensibly different and more favorable side chain
topography relative to the cyclic prototype 9.
A preliminary consideration to explain the lower activity of 9,
as compared with the parent, may be attributed to the
B
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a
Scheme 1. Chemical Synthesis of Intermediates 1−5a−c, 6−8a−b, and Final Products 9a−b
a
Reagents and conditions: (a) 1a−c, HCl·D-Phe-OMe or HCl·L-Phe-OMe, EDC, HOBt·H2O, NMM, DMF, 0 °C, 20 min, then room temperature,
24 h, 90−95%. (b) 2a−c, TFA/DCM 1:1, room temperature, 3 h, quantitative. (c) 3a−c, Boc-cAmp (Cbz)−OH, EDC, HOBt·H2O, NMM, DMF, 0
°C, 20 min, then room temperature, 24 h, 77−88%. (d) 4a−c, 1 N NaOH, MeOH, room temperature, 3 h, quantitative. (e) 5a−c, 10% Pd/C,
MeOH, H2, room temperature, 2−6 h, 73−81%. (f) 6a−b, pyBop, DIPEA, DMF, room temperature, 12 h, 58−60%. (g) 7a−b, TFA/DCM 1:1,
room temperature, 3 h, quantitative. (h) 8a−b, Boc-Tyr-OH, EDC, HOBt.H2O, NMM, DMF, 0 °C, 20 min, then room temperature, 24 h, 80−83%.
(i) 9a−b, TFA/DCM 1:1, room temperature, 3 h, 76−93%.
On the basis of the above considerations and being confident
on the efficient role that the constrained cyclic system of 9 may
exert in controlling the spatial orientation of the side chains at
positions 3 and 4 of the backbone, we considered interesting to
examine a new series of EM-2 cyclic analogues, which maintains
the cAmp at position 2 but is characterized by a different
stereochemistry of the phenylalanine residues. As shown in
Figure 1, the new products (9a−b, 13a−c, and 15a−c), as
compared with the previously reported,8 contain, at positions 3
and 4, a combination of phenylalanine residues with different L
and D absolute configuration. Synthesis of the new linear and
cyclic compounds is reported in Schemes 1 and 2. Table 1
reports the biological properties of the three cyclopeptides 9
and 9a−b. In order to evaluate the effects of the introduced
stereochemical modidifications on the linear models, the
activity of the linear tetrapeptides 13a−c and 15a−c, possessing
a C-terminal free carboxyl or an amide group, respectively, is
also reported in Table 1.
15a−c are reported in Table 1. All compounds showed greater
activity in the GPI than the MVD, defining their predominantly
μ agonist character.
The data show that all the linear tetrapeptides 13a−c
containing a C-terminal free carboxyl are inactive at the μ, δ,
and κ opioid receptors. Compounds 13a−c and 15a−c possess
similar activity toward μ receptor; as already noted,8,17 the
cationic center in position 4 of the cAmp ring, which adds to
that of the N-terminal Tyr, seems not to favor the binding. This
could be related to repulsive interaction with an electropositive
area present in the involved binding pocket. On the contrary,
the cyclic compounds 9a−b showed very good affinity for μ
receptors and weak to scarce affinity for both δ and κ receptors.
The highest activity is shown by the cyclic model 9b with good
binding values for μ receptors (Ki) and only modest activity at
δ receptors with 20-fold selectivity. Product 9b, with D-Phe3
and L-Phe4, show a Ki larger that 9a but still in the nanomolar
range.
As the data in Table 1 show, the affinity toward the μ opioid
receptors of the analogues 13a−b, linear precursors of 9a−b, is
10 to 20 times lower as compared with the corresponding cyclic
models. Thus, the cyclization, locking the peptide backbone
RESULTS AND DISCUSSION
■
Binding affinity and in vitro fuctional activity of both cyclic
tetrapeptides 9a−b and related linear analogues 13a−c and
C
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a
Scheme 2. Chemical Synthesis of Intermediates 10−12a−c, 14a−c, and Final Products 13a−c and 15a−c
a
Reagents and conditions: (a) 10a−c, TFA/DCM 1:1, room temperature, 3 h, quantitative. (b) 11a−c, Cbz-Tyr-OH, EDC, HOBt·H2O, NMM,
DMF, 0 °C, 20 min, then room temperature, 24 h, 80−89%. (c) 12a−c, 1N NaOH, MeOH, room temperature, 3 h, quantitative. (d) 13a−c, HBr/
AcOH 33%, room temperature, 3 h, 70−75%. (e) 14a−c, IBCF, NMM, THF, NH4OH, 30 min, −10 °C, then room temperature, 3 h, 85−87%. (f)
15a−c, HBr/AcOH 33%, room temperature, 3 h, 65−73%.
and the aromatic side chains in a semirigid conformation,
strongly improves the binding. This puts in evidence how in the
EM-2 molecule, which, differently from enkephalins, possesses
an additional Phe residue in position 3, favorable and probable
critical π−π interactions between adjacent aromatic residues
may take place.
CONCLUSIONS
■
As previously mentioned, we have examined here a series of
cyclic EM-2 analogues characterized by relevant new structural
features specifically bound to the presence inside the backbone
of the cAmp residue. The previously reported compound 9 as
well as its here examined diastereisomers 9a−b are the first
cyclic EM analogues obtained without addition, in order to help
the cyclization reaction, of extra residues to the native
tetrapeptidic sequence. Here, the use of a proline/GABA
chimera replacing the native Pro2 as well as an effective
cyclization step, leads to a highly constrained cyclic backbone
surrounded by three aromatic rings pertaining to Tyr1, Phe3,
and Phe4 residues. Results confirm the high influence of the
relative stereochemistry of the residues on both the output of
the cyclization reaction and the proper fitting of the resulting
cyclopeptide with the involved receptor area. Failure to obtain
the cyclotripeptide 9c and the different values of bioactivity
shown by 9 and 9a−b are clearly related to this effect.
However, with the identification of the two active
compounds in the nanomolar range (9a and 9b), a step
further has been done after the design and synthesis of the
prototype cyclotripeptide 9.8 Still other studies need to be done
in order to better understand the stereospecific and topo-
graphic requirements of the opioid receptors and the rules
governing the conformational preferences in here reported
cyclic tripeptides.
Chemistry. Linear peptide analogues were prepared as
previously described1 by following usual peptide chemistry (see
Schemes 1 and 2). The two cyclic products 9a−b (Figure 1)
were obtained as follows: deprotecting the tripeptide 3a−c at
the COOMe terminal by hydrolysis with 1 N NaOH in MeOH,
followed by hydrogenolysis with Pd/C gave 5a−c. The
cyclization reaction was performed by using the pyBop
coupling reagent in a highly diluted DMF (10−3 mol) solution.
Both the precursor tripeptide acids containing D-Phe3/D-Phe4
(5a) and D-Phe3/Phe4 (5b) gave the corresponding N-Boc
protected cyclotripeptide intermediates 6a,b in good yields (60
and 58%, respectively). A different behavior was shown by the
Phe3/D-Phe4 containing tripeptide (5c) which, differently from
the other three studied diastereomeric tripeptides of this series,
namely, the here reported D-Phe3/D-Phe4 (5a) and D-Phe3/
Phe4 (5b) intermediates, as well as the previously examined
precursor of (9) (i.e., N-Boc-cAmp-Phe-Phe-OH), failed to
give, under the adopted experimental conditions, the
corresponding cyclic derivatives. This result is not unexpected,
e.g., refs 18−23, and puts in evidence that the cyclization
reaction, which leads to a very small ring and low flexible
cyclopeptide, is highly influenced by the spatial orientation of
the three adjacent aromatic chains.
EXPERIMENTAL SECTION
■
General Procedure for the Synthesis of 9a−b, 13a−c, and
15a−c. Nα-Boc-cAmp(Z)−OH was synthesized as previously
reported.11 All couplings of the linear intermediates and linear
D
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products were performed using the standard coupling method of
carbodiimmide (EDC/HOBt/NMM) in DMF as described below.
The synthesis of the cyclic peptides 9a−b begins with the coupling
between D or L Boc-Phe-OH and D or L HCl·H-Phe-OMe (Scheme 1)
to obtain three diastereoisomeric dipeptides 1a−c. The dipeptides
obtained were N-terminal deprotected in TFA 1:1 DCM. The
resulting TFA salts 2a−c were coupled with Nα-Boc-cAmp(Z)−OH to
yield the three diastereoisomeric tripeptides 3a−c. Final peptides were
prepared from 3a−c by two different synthetic pathways; in the first
way, to give the cyclic products 9a−b (see Scheme 1) and in the
second way, to give the linear products 13a−c and 15a−c (see Scheme
2). The two cyclic products 9a−b (Scheme 1) were obtained by
deprotecting the tripeptide 3a−b at the COOMe terminal by
hydrolysis with NaOH 1 N in MeOH, followed by deprotection of
Z group in position 4 of the cAmp by hydrogenolysis with Pd/C 10%
in MeOH to give 5a−c. The deprotected tripeptides 5a−c were
cyclized using pyBop coupling reagent in a highly diluted DMF (10−3
mol) solution. As mentioned before, the reaction provided only the
two cyclic products 6a−b but not the 6c diasteromer. Then, 6a−b
were N-terminal deprotected in TFA/DCM mixture, and the resulting
TFA salts 7a−b coupled to Boc-Tyr-OH give the tetrapeptides 8a−b,
which were deprotected by TFA/DCM to give the two final products
9a−b. As shown in Scheme 2, the linear products were obtained by
deprotection of the N-terminal Boc group of 3a−c and coupling the
resulting TFA salts 10a−c with Cbz-Tyr-OH to obtain the three linear
fully protected products 11a−c. Then, the COOMe terminal ester
groups were hydrolyzed by NaOH 1 N in MeOH to give the free acids
12a−c. Free acids were transformed into an amide group by activation
of the free carboxylic function by mixed anhydride and subsequent
reaction with NH4OH to give the terminal amides 14a−c. Finally, the
Cbz groups on Tyr and cAmp side chain were removed by HBr in
glacial acetic acid to give the six final linear products 13a−c and 15a−c
as HBr salts.
[MH+], calcd 570.2712; found, 570.2715. LRMS (ESI) m/z = 570.3.
Anal. Calcd for C32H35N5O5: C, 67.47; H, 6.19; N, 12.29; O, 14.04.
Found: C, 67.45; H, 6.15; N, 12.32; O, 14.09. 9b: 1H NMR
((CD3)2SO) δ 2.04−2.19 (m, 2H, Pro C3H2), 2.79−3.14 (m, 2H,
βCH2 Tyr1), 2.89−3.07 (m, 2H, βCH2 Phe3), 3.06−3.18 (m, 2H,
βCH2 Phe4), 3.24−3.97 (m, 2H, Pro C5H2), 3.71 (m, 1H, αCH Phe4),
4.12 (m, 1H, αCH Tyr1), 4.38 (m, 1H, Pro C4H), 4.40 (m, 1H, αCH
Phe3), 4.69 (m, 1H, αCH Pro), 6.44 (d, 1H, NH Pro), 6.72 (d, 2H,
C3,5H Tyr1), 7.18 (d, 2H, C2,6H Tyr1), 7.87 (d, 1H, NH Phe4), 8.04
+
(br, 3H, NH3 Tyr1), 8.27 (d, 1H, NH Phe3). ESI-HRMS for
C32H36N5O5 [MH+], calcd 570.2712; found, 570.2710. LRMS (ESI)
m/z = 570.3. Anal. Calcd for C32H35N5O5: C, 67.47; H, 6.19; N, 12.29;
O, 14.04. Found: C, 67.51; H, 6.23; N, 12.25; O, 14.01.
Biological Activity and Binding Assays. Functional Guinea Pig
Ileum (GPI) and Mouse Vas Deferens (MVD) Assays. In vitro
biological assays were performed on 9a−b as TFA salts and 13a−c and
15a−c as hydrobromide salts. GPI and MVD in vitro bioassays were
performed as described previously.24,25 For a brief description, see
Supporting Information.
Radioligand Labeled Binding Assays. μ and δ Opioid
Receptors. Crude membranes were prepared as previously described26
from transfected cells that express the MOR or the DOR. For a brief
description, see Supporting Information.
κ Opioid Receptors. κ opioid receptor (KOR) binding affinities
were carried out by CEREP, Rue du Bois l′Eveque, BP 30001−86600
Celle l′Evescault (FRANCE), following a slightly modified procedure
previously reported by Meng et al.27 Experiments were performed on
Chinese hamster ovary (CHO) cell lines that stably express human
KOP, established as previously described.27 For a brief description, see
Supporting Information.
ASSOCIATED CONTENT
* Supporting Information
■
S
All solvents, reagents, and starting materials were obtained from
commercial sources unless otherwise indicated. All reactions were
performed under N2 unless otherwise noted. Intermediate products
1a−c, 3a−c, 6a-b, and 8a−b were purified by silica gel
chromatography. Products 9a−b, 13a−c, and 15a−c used for the
biological assay were purified by RP-HPLC using a semipreparative
Vydac (C18-bonded, 300 Å) column and a gradient elution at a flow
rate of 10 mL/min. The gradient used was 10−90% acetonitrile in
0.1% aqueous TFA over 40 min. Approximately 10 mg of crude
peptide was injected each time, and the fractions containing the
purified peptide were collected and lyophilized to dryness. The purity
of the final products, determined by NMR analysis and by analytical
RP-HPLC (C18-bonded 4.6 × 150 mm) at a flow rate of 1 mL/min on
a Waters Binary pump 1525 using a isocratic elution of 20% CH3CN/
H2O 0.1% TFA, monitored with a Waters 2996 Photodiode Array
Detector, was found to be >95%.
Details of syntheses, general procedures, compound character-
ization, biochemistry, and experimental section. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +34-08713554476. Fax: +39-08713554477. E-mail: a.
mollica@unich.it.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
Boc, tert-butyloxycarbonyl; BSA, bovine serum albumin; Cbz,
carbobenzoxy; GPI, guinea pig ileum; DAMGO[3H], [3H]-[D-
Ala(2),N-Me-Phe-(4),Gly-ol(5)]enkephalin; [3H]-U69593,
[3H]-(+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-
oxaspiro[4.5]dec-8-yl]benzeneacetamide; DCM, dichlorome-
thane; DIPEA, diisopropylethylamine; [3H-]DPDPE, [3H]-[2-
D-penicillamine,5-D-penicillamine]enkephalin; DMF, N,N-di-
methylformamide; DMSO, dimethyl sulfoxide; DOR, δ opioid
receptor; EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide;
HOBt, 1-hydroxybenzotriazole; IBCF, isobutyl chloroformate;
MOR, μ opioid receptor; MVD, mouse vas deferens; NMM, N-
methylmorpholine; PMSF, phenylmethylsulfonyl fluoride; RP-
HPLC, reversed phase high performance liquid chromatog-
raphy; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMS,
tetramethylsilane; PyBop, benzotriazol-1-yloxytripyrrolidino-
phosphonium hexafluorophosphate
1H NMR spectra were performed in CDCl3 or DMSO-d6 solution
on a Varian Inova operating at the 1H frequency of 300 MHz and on a
1
Bruker AVANCE AQS600 operating at the H frequency of 600.13
MHz. Chemical shifts were referred to TMS as internal standard in the
case of CDCl3 solution and to the residual proton signal of DMSO at
2.5 ppm in the case of DMSO-d6 solution. Peptide structures were
determined by means of 2D NMR experiments, namely, 1H−1H
1
TOCSY and H−1H NOESY. Peptide structures were also confirmed
by high resolution-mass spectra (HR-MS)
2 ppm. For the final
products, 9a−b, 13a−c, and 15a−c, elementary analyses (within
0.4% of the theoretical values) were performed.
TFA·Tyr-c[4-NH-Pro-Phe-Phe] (9a−b). Compound 8a−b (1.0
equiv) was dissolved in 1:1 CH2Cl2/TFA mixture according to the
1
general procedure to give 9a (93%) and 9b (87%). 9a: H NMR
((CD3)2SO) δ 1.39−2.38 (m, 2H, Pro C3H2), 2.76−2.93 (m, 2H,
βCH2 Phe4), 2.84−3.09 (m, 2H, βCH2 Phe3), 2.89−2.97 (m, 2H,
βCH2 Tyr1), 3.42−3.50 (m, 2H, Pro C5H2), 3.50 (m, 1H, αCH Pro),
3.86 (m, 1H, αCH Tyr1), 3.89 (m, 1H, Pro C4H), 4.41 (m, 1H, αCH
Phe4), 4.62 (m, 1H, αCH Phe3), 6.15 (d, 1H, NH Pro), 6.74 (d, 2H,
C3,5H Tyr1), 6.98 (d, 2H, C2,6H Tyr1), 7.65 (d, 1H, NH Phe3), 7.94 (d,
1H, NH Phe4), 8.36 (br, 3H, NH3+ Tyr1). ESI-HRMS for C32H36N5O5
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■
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