SSTR1- and SSTR3-Selective Somatostatin Analogues

Article abstract of DOI:10.1002/cbic.201000597

We prepared the two enantiomers of 3-(3'-quinolyl)-alanine (Qla, 1) in multigram scale by asymmetric hydrogenation. These amino acids, protected as Fmoc derivatives, were then used in the solid-phase synthesis of two new somatostatin 14 (SRIF-14) analogue

Full text of DOI:10.1002/cbic.201000597

DOI: 10.1002/cbic.201000597
SSTR1- and SSTR3-Selective Somatostatin Analogues
Rosario Ramꢀn,[a, b] Pablo Martꢁn-Gago,[a] Xavier Verdaguer,[a, b] Maria J. Macias,[a, c]
Pau Martin-Malpartida,[a] Jimena Fernꢂndez-Carneado,[d] Marc Gomez-Caminals,[d]
Berta Ponsati,[d] Pilar Lꢀpez-Ruiz,[e] Maria Alicia Cortꢃs,[e] BegoÇa Colꢂs,[e] and
Antoni Riera*[a, b]
We prepared the two enantiomers of 3-(3’-quinolyl)-alanine
(Qla, 1) in multigram scale by asymmetric hydrogenation.
These amino acids, protected as Fmoc derivatives, were then
used in the solid-phase synthesis of two new somatostatin 14
(SRIF-14) analogues 8a and 8b, tetradecapeptides in which
the tryptophan residue (Trp8) is replaced by one of the two
enantiomers of 3-(3’-quinolyl)-alanine (Qla8) and therefore lack
the NÀH bond in residue 8. The selectivity of these new ana-
logues for the somatostatin receptors, SSTR1–5, was measured.
Substitution with l-Qla8 yielded peptide 8a, which was highly
selective for SSTR1 and SSTR3, with an affinity similar to that of
SRIF-14. Substitution by d-Qla gave the relatively selective ana-
logue 8b, which showed high affinity for SSTR3 and significant
affinity for SSTR1, SSTR2 and SSTR5. The biological results dem-
onstrate that bulky and electronically poor aromatic amino
acids at position 8 are compatible with strong activity with
SSTR1 and SSTR3. Remarkably, these high affinity levels were
achieved with peptides in which the conformational mobility
was increased with respect to that of SRIF-14. This observation
suggests that conformational rigidity is not required, and
might be detrimental to the interaction with receptors SSTR1
and SSTR3. The absence of an indole N proton in Qla8 might
also contribute to the increased flexibility observed in these
analogues.
Introduction
Somatostatin, a 14-residue peptidic hormone produced in the
hypothalamus, was discovered in 1973 in an active form.[1]
Since then other forms of various lengths have been identi-
fied,[2] thus the name “somatostatin” refers to a family of heter-
ogeneous peptide hormones that differ in both length (from
14 to 37 residues) and amino acid composition, depending on
the species. In mammals, the different hormone forms are N-
least five characterized G protein-coupled receptors, SSTR1 to
SSTR5.[3] These receptors differ in their tissue distribution and
physiological functions. For instance, receptors SSTR2 and
SSTR5 participate in the inhibition of secretion of growth hor-
mone,[4] glucagon, and insulin.[5] SSTR3 and, to a lesser extent,
SSTR2 are involved in cellular apoptosis, while SSTR1 arrests
the cell cycle and regulates angiogenesis.[6]
SRIF-14 has been thoroughly studied and successfully syn-
thesized by the pharmaceutical industry as a therapeutic drug.
[a] Dr. R. Ramꢀn, P. Martꢁn-Gago, Prof. X. Verdaguer, Prof. M. J. Macias,
P. Martin-Malpartida, Prof. A. Riera
Institute for Research in Biomedicine (IRB Barcelona)
Baldiri Reixac, 10, 08028 Barcelona (Spain)
Fax: (+34)3094395
E-mail: antoni.riera@irbbarcelona.org
[b] Dr. R. Ramꢀn, Prof. X. Verdaguer, Prof. A. Riera
Departament de Quꢁmica Orgꢂnica, Universitat de Barcelona
Baldiri Reixac, 10, 08028 Barcelona (Spain)
[c] Prof. M. J. Macias
Catalan Institution for Research and Advanced Studies (ICREA)
Catalonia (Spain)
[d] Dr. J. Fernꢃndez-Carneado, M. Gomez-Caminals, Dr. B. Ponsati
BCN Peptides, S.A. (Lipotec Group)
Polꢁgon Industrial els Vinyets-Els Fogars 2
08777-Sant Quintꢁ de Mediona, Barcelona, (Spain)
[e] Prof. P. Lꢀpez-Ruiz, M. A. Cortꢄs, B. Colꢃs
terminal variations that result from differential processing of
the same precursor, preprosomatostatin I.[2] Somatostatin is
also known as somatotropin release-inhibiting factor (SRIF) and
growth-hormone-inhibiting hormone (GHIH).
Departamento de Bioquꢁmica y Biologꢁa Molecular
Universidad de Alcalꢃ de Henares, Facultad de Medicina
Campus Universitario, 28871 Madrid (Spain)
Supporting information for this article is available on the WWW under
1
http://dx.doi.org/10.1002/cbic.201000597: H and 13C NMR spectra of com-
1
SRIF-14 is involved in multiple biological functions that are
mediated by direct interactions between the hormone and at
pounds 4a–b, 5a–b, 6 and 7. H NMR TOCSY/NOESY spectra of SOM-14,
8a and 8b.
ChemBioChem 2011, 12, 625 – 632
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
625

A. Riera et al.
It has three main applications in clinical practice: 1) as a gastric
anti-secretory drug in the treatment of esophageal varices and
in acute variceal bleeding in cirrhotic patients; 2) in the treat-
ment of growth hormone secretion disorders; and 3) in the
treatment of primary thyroid-stimulating hormone (THS)-se-
creting pituitary tumors and neuroendocrine tumors of the
gastrointestinal tract.[7] As it is a natural hormone, SRIF-14 has
very low toxicity compared to synthetic drugs. However, be-
cause of its low serum stability (1–3 min half-life in human
plasma)[8] it has found limited clinical use since continuous in-
fusion is needed. This fact, coupled with its broad spectrum of
biological activity, has fostered research into SRIF-14 analogues
with distinct selectivity profiles and/or improved stability; but
only octreotide (trade name: Sandostatin) and lanreotide
(trade name: Somatuline) have reached the market.
Several structural studies[9] have provided information about
the conformational flexibility of SRIF-14 in solution. Although
SRIF-14 shows some intrinsic flexibility, which probably ac-
counts for its functional versatility,[10] residues Phe7-Trp8-Lys9-
Thr10 constitute a conserved b-turn that is stabilized by a net-
work of interactions between the Trp8 and Lys9 side chains
and with neighboring residues.[11] The pioneering studies of
Vale et al. demonstrated that a region including the turn is the
pharmacophore of the hormone.[12] Therefore, attempts to de-
velop molecules that retain these residues and the conforma-
tional structure of the hormone but of reduced size have yield-
ed a myriad of shorter SRIF-14 analogues, including octreotide
and lanreotide.[13]
the indole and aliphatic Lys9 side chain,[15] or possibly by in-
hibiting or delaying the recognition of these molecules by the
cellular degradation machinery.
Attempts to clarify the relevance of the Trp residue to the
biological activity of SRIF-14 and its analogues have been de-
scribed.[16] For instance, by measuring gastric acid and pepsin
inhibitory activities of several analogues of SRIF-14 that incor-
porated Trp8 modifications, Hirst et al. concluded that the NÀH
bond in the indole ring plays a crucial role.[17] However, ques-
tions remain concerning the effect of an electron-deficient het-
erocycle,[18] and whether the NÀH bond is strictly necessary for
activity with all receptors.
To further characterize these components, we set out to
change the electronic properties of the heterocycle by replac-
ing the native SRIF-14 Trp8 with 3-(3’-quinolyl)-alanine (Qla, 1).
Although Trp and Qla have similar shapes, the electronic prop-
erties of the two rings are reversed. Furthermore, the quinolyl
fragment lacks the NÀH bond present in the indole ring, which
seems to be essential for activity because of its involvement in
either binding to its receptors, or in adopting the biologically
active conformation of SRIF-14).[17] To elucidate the role of the
amino acid at position 8 in receptor recognition and conforma-
tional stability, we replaced Trp8 with the two 3-(3’-quinolyl)a-
lanine enantiomers (l-1 and d-1). To this end, we developed a
Recent advances in solid-phase peptide synthesis (SPPS)
have prompted us to revisit the full-length SRIF-14 structure in
order to design new analogues with the aim of overcoming
some of the limitations of the short-ring molecules. For in-
stance, octreotide and its derivatives do not retain SRIF-14’s
recognition for all receptors. Thus a gain of stability and rigidi-
ty has come at a cost, namely a loss of function with certain re-
ceptors. A second focus in the design of full length SRIF-14 an-
alogues concerns the use of these derivatives as tumor mark-
ers: around 80% of gastroenteropancreatic neuroendocrine
tumors (GEP-NET) express SSTR receptors. SRIF-14 is known to
interact with all five receptors while octreotride derivatives rec-
ognize just SSTR2 with high binding affinity, and to a lesser
extent SSTR5 and SSTR3.[7d] Furthermore, tumors that express
new synthetic strategy to obtain these unnatural amino acids
in multigram scale and high enantiomeric purity. Using NMR
spectroscopy, we analyzed the conformational changes in-
duced by the l-Qla and d-Qla substitution, and we performed
receptor binding assays with membranes isolated from Chi-
nese hamster ovary (CHO) cells that expressed each of the
SSTR receptors (SSTR1–5).
SSTRs often lose particular receptor subtypes, thus making full Results and Discussion
length SRIF-14 analogues an attractive choice as potential
Synthesis
markers for tumor identification and monitoring their progres-
sion.[14]
The racemic synthesis of the unnatural amino acid 3-(3’-quino-
We therefore centered our attention on the production of
lyl)alanine[19] 1 and its enzymatic resolution[20,21] have been de-
scribed previously. Nonetheless, we wished to develop an
asymmetric multigram-scale synthesis method that would pro-
vide each of the two enantiomers separately. To this end, 3-
quinoline carbaldehyde 2 was subjected to Horner–Emmons
olefination by using 3a and 3b, phosphonates that differ only
by the protecting groups at the nitrogen atom (Scheme 1).
The resulting dehydroamino acids, 4a and 4b, were hydrogen-
ated by using commercially available [Rh(COD)Et-DuPHOS]OTf
(cat-I) as a pre-catalyst. The Cbz-protected dehydroamino acid
4a gave low conversion both in methanol and in ethyl acetate
full-length SRIF-14 analogues with specificity towards some of
the receptors and improved stability. Since the presence of the
turn and the contacts between residues Trp8 and Lys9 are criti-
cal for this function, we focused on modifying these residues.
However, the critical role of Trp8 in the formation and stabiliza-
tion of the b-turn is still not fully understood, since a change
in the configuration of this amino acid does not have a signifi-
cant effect on the activity. Indeed, the substitution with d-Trp8
in SRIF-14 and octreotride appears simply to increase the
stability of these molecules, either by favoring the proximity of
626
www.chembiochem.org
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 625 – 632

Somatostatin Analogues
(Scheme 2). The Fmoc derivatives, l-7 and d-7, were obtained
in excellent yield under standard conditions.
The syntheses of [l-Qla8]-SRIF 8a and [d-Qla8]-SRIF 8b were
performed by SPPS on a 2-chlorotrityl chloride resin by using
Scheme 2. Protection of (3-3’quinolyl)-alanines 5 as Fmoc derivatives 7.
a) HCl, 1108C (100%); b) FmocOsu, Na2CO3, dioxane (93%).
the Fmoc/tBu, strategy (Scheme 3). The coupling of the first
amino acid, Fmoc-Cys(Trt)-OH, was performed in CH2Cl2 in the
presence of N-ethyldiisopropylamine (DIPEA). After coupling,
the remaining free chlorides were capped with methanol. The
next five Fmoc-protected amino acids (those present in the
natural hormone, up to Lys9) were added by using N,N’-di-
isopropylcarbodiimide (DIPCDI) and 1-hydroxybenzotriazole
(HOBt) in DMF, with 20% piperidine in DMF used for Fmoc re-
moval. The resin was split in two, and l-7 or d-7 amino acids
were incorporated into each half. The remaining protected
amino acids were coupled following the natural sequence. The
Scheme 1. Enantioselective synthesis of 3-(3’-quinolyl)alanine derivatives 5.
a) HCl, 1108C, 100%; b) FmocOSu, Na2CO3, dioxane, 93%.
because of its low solubility (Table 1). Acetamido derivative 4b
also gave low conversions in ethyl acetate but its enantiomeric
purity was high (96% ee at 258C). A lower enantiomeric excess
was achieved when raising the temperature to increase the
solubility (88% ee at 508C). However, working in methanol at
room temperature the yield improved to 99%, and the enan-
tiomeric excess was 95%. Lowering or increasing the tempera-
ture and/or pressure with cat-I as catalyst did not improve the
enantiomeric excess. Finally, the hydrogenation was assayed
with our recently developed rhodium complex (cat-II), which
was derived from a chiral aminodiphosphine (MaxPHOS).[22]
Gratifyingly, at 15 bar hydrogen with [Rh(COD)(MaxPHOS)]BF4
(cat-II) as a precatalyst, the enantioselectivity improved to a
noteworthy 99% ee (Table 1, entries 6 and 7).
Enantiomerically enriched acetamido esters 5b were hydro-
lyzed with hydrochloric acid, and the amino group was
conveniently protected for use in solid-phase synthesis
Table 1. Asymmetric hydrogenation of dehydroquinolylalanine deriva-
tives (4). Reaction conditions, yields and enantiomeric excesses of com-
pound 5.
SM Catalyst[a] Solvent T [8C] P [bar] Yield [%] ee [%] (conf)[b]
[c]
1
2
3
4
5
6
7
4a
4a
4b
4b
4a
4b
4b
(S,S)-I
(S,S)-I
(R,R)-I
(R,R)-I
(S,S)-I
(R)-II
MeOH
AcOEt
AcOEt
AcOEt
MeOH
MeOH
MeOH
25
50
25
50
25
25
25
10
5
40
40
5
60
60
n.d.[c]
n.d.[c]
99
n.d.
n.d.
[c]
96.0 (R)
88.0 (R)
95.0 (S)
98.6 (S)
99.0 (S)
25
15
99
99
Scheme 3. SPPS of somatostatin analogues. a) i: Fmoc-l-Cys(Trt)-OH
(3 equiv), DIPEA (3 equiv); ii: MeOH; b) i: piperidine 20% DMF; ii: Fmoc-AA-
OH (3 equiv), DIPCDI (3 equiv), HOBt (3 equiv), DMF; c) i: piperidine 20%
DMF; ii: Fmoc-Qla-OH (3 equiv), DIPCDI (3 equiv), HOBt (3 equiv), DMF;
d) CH2Cl2/TFE/AcOH; e) I2; f) TFA/CH2Cl2/anisole/H2O.
(R)-II
n.d.: not determined. [a] 3 mol% catalyst. [b] Measured by HPLC (CHIRAL-
PAK-IA) [c] Incomplete reaction.
ChemBioChem 2011, 12, 625 – 632
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
627

A. Riera et al.
disulfide bridge in the two analogues was formed in solution
at room temperature after cleavage (CH2Cl2/TFE/AcOH) of the
fully protected linear peptide from the resin with iodine. Final-
ly, side-chain deprotection by using TFA/CH2Cl2/anisole/H2O for
4 h afforded SRIF-14 analogues 8a and 8b respectively, in
good yield (74 and 67%) and purity (95 and 84%), as assessed
by RP-HPLC.
a highly populated beta hairpin for residues 6 to 11 (Figure 1;
a larger image is available in the Supporting Information).
Along the backbone we observed HNÀHN and HAÀHN con-
tacts across the turn. In addition, the turn was stabilized by a
network of contacts involving several side chains, for example
NOEs between Trp8 and Lys9 were abundant, and further sta-
bilized by interactions between the Phe6 and Phe11 aromatic
side chains. These phenylalanines also showed contacts with
the tryptophan residue. Some of the contacts observed be-
tween side chains are compatible with the presence of several
rotamers, as previously described for SRIF-14 under other
experimental conditions.[9a]
Structure
In order to examine the functional effects of the Trp8 muta-
tions at the structural level, we analyzed the NOE pattern of
SRIF-14 (trifluoroacetate counterion) and 8a and b (also as tri-
fluoroacetates) in aqueous solution (buffered at pH 6.5, 128C).
Spin systems and sequential assignments for the two mole-
cules were obtained from 2D TOCSY and NOESY homonuclear
experiments.
The substitution of Trp8 with l-Qla8 (8a) dramatically re-
duced the rigidity of this molecule compared to that of SRIF-
14, as reflected in differences in the intensities of several
medium- and long-range contacts. Although both Phe6 and
Phe11 still displayed weak NOEs with the Lys9 side chain,
those corresponding to Phe11 were weaker than their Phe6–
Lys9 counterparts. No NOEs were detected from l-Qla8 to the
Under our experimental conditions, the NMR data for SRIF-
14 showed a pattern of NOEs consistent with the presence of
Figure 1. NOESY (600 MHz, D2O) of SRIF-14 and [l-Qla8]-SRIF (8a). Characteristic NOEs in several regions of the spectrum of 8a are assigned in order to com-
pare them in number and intensity with those for SRIF-14. Upper left and lower left quadrants of the spectra are shown.
628
www.chembiochem.org
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 625 – 632

Somatostatin Analogues
Lys9 side chain. The absence of this interaction was corroborat-
ed by the enhanced downfield shift of the Lys9 g-methylene of
the analogue (1.186 ppm) in comparison with SRIF-14
(0.887 ppm).[15] Also, the NOEs between Phe6 and Phe11 and
from these to the l-Qla8 side chain were absent in 8a. The
lack of contacts from the l-Qla8 side chain to neighboring resi-
dues indicates that its bulky side chain is somehow forced to
adopt a conformation with the side chain pointing away from
the residues in the turn.
are required for efficient interactions between residue 8 and
the binding pocket of SSTR4, by means of a predicted aromat-
ic-aromatic interaction to a residue within the receptor. A lack
of affinity for SSTR4 would thus be expected for 8a and 8b,
which contain electron-poor quinoline heterocycles at posi-
tion 8, and this was indeed fully confirmed here. Analogue 8a
also had no affinity for SSTR2 and SSTR5 although, interesting-
ly, it retained the SRIF-14 binding affinity for SSTR1 and SSTR3,
with Ki values in the nanomolar range. Thus, 8a presents a
binding profile that is complementary to that of octreotide.
(Octreotide has high affinity for SSTR2 and is moderately active
with SSTR3 and 5, while peptide 8a was highly active with
SSTR1 and 3 and showed no affinity for SSTR2, 4 or 5.) In
contrast, 8b showed a broader binding profile. It retained the
binding affinity of SRIF-14 for SSTR3, and showed a remarkable
activity with SSTR1. Moreover, in contrast to 8a, 8b also had
binding affinity for SSTR2 and 5 with Ki values in the nanomo-
lar range (although much lower than SRIF-14). The binding
profile of 8b is quite similar to Pasireotide (SOM-230), a cyclo-
hexapeptide developed by Novartis that has completed phase
II trials.[23]
A similar situation was observed for 8b, in which Trp8 was
substituted by d-Qla8: no NOEs were detected for any of the
quinoline-ring protons to those in the nearby side chains. The
interactions with the Lys9 side chain, which are very strong in
SRIF-14, were absent in the spectra of 8b. This observation
indicates that, independently of the configuration of Qla, the
bulky and electron-poor side chain of 8a cannot adopt a con-
formation that favors contacts with the neighboring residues.
It is also noteworthy that interactions between the phenylala-
nine residues were minimal, as shown by the extremely weak
NOEs between them. These two effects might contribute to
the high flexibility of 8b,[15] which is consistent with, for exam-
ple, the absence of NOEs between facing residues and the dis-
appearance of the NHÀNH contacts.
Finally, the stability in serum was measured. Half-lives in
human serum of 3.4 h and 10.0 h were found for peptides 8a
and 8b, respectively. Somewhat surprisingly, although the sta-
bilities of these new peptides were superior to that of native
SRIF-14 (2.7 h under the same conditions), they were lower
than that of d-[Trp8]-SRIF-14 (19.7 h).
Together, these features suggest that the flexibilities of 8a
and 8b are much greater than that of SRIF-14, and that the
Qla8 substitution introduces a rearrangement at the side-chain
level that has a strong impact on the conformation variability
of the whole molecule.
Conclusions
Biological activity
In summary, we prepared two new tetradecapeptide SRIF-14
analogues, 8a and 8b, in which the tryptophan residue (Trp8)
was replaced with each of the two enantiomers of 3-(3’-quino-
lyl)alanine (Qla8) and which therefore lack the NÀH bond in
residue 8. The selectivity for the receptors SSTR1–5 of these
new somatostatin analogues was measured. Substitution of
Trp8 by l-Qla8 in SRIF-14 provided peptide 8a, which was
highly selective for receptors SSTR1 and SSTR3, with an affinity
similar to that of SRIF-14. Substitution by d-Qla gave a relative-
ly selective analogue 8b, which showed high affinity for SSTR3
and considerable affinity for receptors SSTR1, SSTR2 and
SSTR5. The biological results demonstrated that bulky and
electronically poor aromatic amino acids at position 8 do not
compromise strong activity with receptors SSTR1 and SSTR3.
Remarkably, this was achieved in
The receptor selectivities of 8a and 8b were assessed by bind-
ing assays. For comparative purposes, the same test was ap-
plied to SRIF-14, [d-Trp8]-SRIF and octreotide. Stable CHO cell
lines, each of which expressed one of the five SSTR receptors,
were cultured. Membrane preparations from these were used
to evaluate the efficacy of the interaction with each receptor
by competitive binding assays with 125I-labeled and unlabeled
SRIF-14 (Table 2).
The two analogues showed negligible affinity for SSTR4. Pre-
viously, Hirschmann and co-workers reported a correlation be-
tween the electrostatic potentials of various simulated substi-
tutions at position 8 of SRIF-14, and the resulting binding to
the SSTR pocket.[18] This suggested that electron-rich p systems
spite of the increased conforma-
tional mobility compared to that
Table 2. Affinity of 8a, 8b, somatostatin, [d-Trp8]-SRIF and octreotide to receptors SSTR1–5. Values are meanÆ
of SRIF-14. This observation sug-
gests that conformational rigidi-
ty is not required and might
even be detrimental to interac-
tion with SSTR1 and SSTR3. The
absence of the indole NH proton
of Qla8 may also contribute to
the flexibility increase observed
in these analogues.[18]
S.E.M.
Ki [nm]
SSTR1
SSTR2
SSTR3
SSTR4
SSTR5
SRIF-14
[d-Trp8]-SRIF
octreotide
[l-Qla8]-SRIF 8a
[d-Qla8]-SRIF 8b
0.43Æ0.08
0.32Æ0.11
0.0016Æ0.0005
0.001Æ0.0007
0.053Æ0.011
0.53Æ0.21
0.61Æ0.02
15.2Æ5.9
0.74Æ0.07
5.83Æ0.44
0.23Æ0.04
0.46Æ0.24
11.53Æ1.91
300Æ85
>1000
1.33Æ0.60 >1000
1.95Æ0.58 >1000
0.65Æ0.19 >1000
>1000
13.66Æ4.32
0.68Æ0.10
1.16Æ0.39
0.09Æ0.01
14.52Æ2.97
Kd [nm]
0.41Æ0.21
0.96Æ0.15
0.27Æ0.005
ChemBioChem 2011, 12, 625 – 632
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
629

A. Riera et al.
purged and pressurized to 7 bar with hydrogen. After four days at
room temperature the reaction mixture was purged with nitrogen,
the solvent was then evaporated, and the resulting crude product
was purified by chromatography (SiO2/NEt3 2.5% v/v) with hex-
anes/AcOEt to afford 0.20 g of (2S)-5a as a white solid and 0.17 g
of starting material 4a (34% conversion, 40% yield). [a]D =+10.1
(c=0.50, CHCl3); 1H NMR (400 MHz, CDCl3): d=8.68 (s, 1H), 8.08
(d, J=8.4 Hz, 1H), 7.89 (s, 1H), 7.64–7.77 (m, 2H), 7.53 (t, J=7.2,
7.4 Hz, 1H), 7.32 (s, 5H), 5.47 (d, J=6.8 Hz, 1H), 5.10 (s, 2H), 4.76
(q, J=7.2, 6.6 Hz, 1H), 3.73 (s, 3H), 3.21–3.41 ppm (m, 2H);
13C NMR (100 MHz, CDCl3): 171.7, 155.9, 151.8, 147.4, 136.3, 129.6,
129.4, 129.1, 128.8, 128.5, 128.3, 128.1, 127.8, 127.1, 67.3, 54.9, 52.8,
35.9 ppm; IR (film): nmax =3328(b), 2925, 1717, 1215, 1056 cmÀ1; MS
(CI-NH3) m/z (%): 365.1 (100) [M+H]+; HRMS: calcd for C21H21N2O4:
365.1496 [M+H]+, found: 365.1489.
Experimental Section
General procedures: Dry solvents were distilled before use. All
other reagents were used as received. All reactions were per-
formed with flame-dried glassware under argon. Optical rotations
were measured at room temperature (238C) in a Perkin–Elmer 241
MC polarimeter. Infrared spectra were recorded by using an NaCl
film in a Nicolet Nexus FTIR spectrometer. H and 13C NMR were re-
1
corded on a Varian Mercury 400 spectrometer. 1H NMR spectra
were obtained at 400 MHz with tetramethylsilane as internal stan-
dard. 13C NMR spectra were obtained at 100.6 MHz in CDCl3, and
referenced to the solvent signal. 2D TOCSY and NOESY homonu-
clear experiments were performed in a Bruker Avance III spectrom-
eter. Chemical shifts were recorded in ppm.
Methyl
(Z)-2-benzyloxycarbonylamino-3-(3-quinolyl)-2-prope-
Methyl (2S)-2- acetamide-3-(3-quinolyl)-propanoate, (2S)-5b
noate, 4a: 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU; 0.57 mL,
3.82 mmol) was added dropwise to a solution of methyl (Æ)-Z-a-
phosphonoglycine trimethyl (1.27 g, 3.82 mmol) in CH2Cl2 (10 mL).
After 10 min of stirring at room temperature, a solution of quinol-
yl-3-carboxaldehyde (500.0 mg, 3.18 mmol) in CH2Cl2 (8 mL) was
added. After 2 h of stirring at room temperature, the reaction was
complete. The solvent was removed at reduced pressure, and the
residue was diluted with EtOAc (23 mL). The organic layer was
washed with 1m HCl and brine, and then dried and evaporated.
The crude product was purified by chromatography (SiO2/NEt3
2.5% v/v) with hexanes/AcOEt mixtures to afford 580.0 mg of 4a
(50% yield) as a white solid. M.p. 118–1208C; 1H NMR (400 MHz,
CDCl3): d=9.02 (s, 1H), 8.20 (s, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.65–
7.76 (m, 2H), 7.53 (t, J=7.2, 7.4 Hz, 2H), 7.30 (s, 5H), 6.81 (s, 1H),
5.09 (s, 2H), 3.87 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
165.6, 164.3, 153.6, 151.2, 141.2, 136.8, 135.9, 135.8, 135.3, 130.7,
129.4, 128.8, 128.7, 128.6, 128.5, 127.7, 127.3, 125.7, 120.2, 68.0,
53.2 ppm; IR (film): nmax =3295(b), 2904, 1721, 1260 cmÀ1; MS (ESI)
m/z (%): 363.0 (100) [M+H]+; elemental analysis calcd (%) for
C21H18N2O4: C 69.60, H 5.01, N 7.73; found: C 69.30, H 5.09, N 7.77.
Procedure A: (S,S)-[[(COD)Et-DuPHOS]RhI]OTf (7.33 mg, 0.01 mmol)
was added to a solution of dehydro amino acid 4b (100 mg,
0.37 mmol) in MeOH (6 mL). The reaction mixture was purged and
pressurized with hydrogen to 5 bar. After 24 h of stirring at room
temperature, the reaction mixture was purged with nitrogen, the
solvent was evaporated, and the resulting crude product was puri-
fied by chromatography (SiO2/NEt3 2.5%, v/v) with hexanes/AcOEt
to afford 99 mg of (2S)-5b as a white solid (99% conversion, 99%
yield, ee>95%).
Procedure B: (R)-[Rh(COD)(MaxPHOS)]BF4 (6.25 mg, 0.011 mmol) was
added to a solution of the dehydro amino acid 4b (100 mg,
0.37 mmol) in MeOH (12 mL). The reaction was carried out as
described above but pressurized to 25 or 15 bar. (2S)-5b was ob-
tained in 99% yield with an enantiomeric purity of 98.6%.
[a]D =+104.5 (c=1.00, CHCl3); m.p. 1208C; 1H NMR (400 MHz,
CD3OD): d=8.72 (s, 1H; CH Ar), 8.19 (s, 1H; CH Ar), 7.99 (d, J=
8.4 Hz, 1H; CH Ar), 7.90 (d, J=8.0 Hz, 1H; CH Ar), 7.74 (t, J=7.2,
8.4 Hz, 1H; CH Ar), 7.60 (t, J=7.2, 8.4 Hz, 1H; CH Ar), 5.48 (s, 1H;
NH), 4.80–4.82 (m, 1H; CHa), 3.71 (s, 3H; CH3), 3.37–3.44 (m, 1H;
CH2 (CHb)), 3.12–3.23 (m, 1H; CH2 (CH2b)), 1.89 ppm (s, 3H; CH3);
13C NMR (100 MHz, CD3OD): d=171.9 (CO), 171.8 (CO), 151.4 (CH),
146.5 (C), 136.8 (CH), 130.6 (C), 129.7 (CH), 128.4 (C), 127.8 (CH),
127.7 (CH), 127.1 (CH), 53.5 (CH3) , 51.7 (CH), 34.5 (CH2), 21.0 ppm
Methyl (Z)-2-acetamide-3-(3-quinolyl)-2-propenoate, 4b: DBU
(3.20 mL, 21.00 mmol) was addded drop-wise to a solution of
methyl-2-N-(acetylamino)dimethylphosphonoacetate
18.21 mmol) in CH2Cl2 (10 mL). After 30 min of stirring at room
temperature, solution of quinolyl-3-carboxaldehyde (2.20 g,
(4.35 g,
a
14.00 mmol) in CH2Cl2 (8 mL) was added. After 3 h of stirring at
room temperature, the reaction was complete. The solvent was
removed at reduced pressure, and the residue was diluted with
EtOAc (23 mL). The organic layer was washed with 1m HCl and
brine, then dried and evaporated. The crude product was purified
by chromatography (SiO2/NEt3 2.5% v/v) with hexanes/AcOEt to
afford 2.90 g of 4b (80% yield) as a white solid. M.p. 165–1688C.
1H NMR (400 MHz, CD3OD): d=8.99 (s, 1H; CH Ar), 8.49 (s, 1H; CH
Ar), 7.96–8.02 (m, 2H; CH Ar), 7.80–7.81 (m, 1H; CH Ar), 7.61–7.67
(m, 1H; CH Ar), 7.57 (s, 1H; CHb), 3.86 (s, 3H; CH3), 2.14 ppm (s,
3H; CH3); 13C NMR (100 MHz, CD3OD): d=171.8 (CO), 165.5 (CO),
150.4 (CH), 147.0 (C), 137.6 (CH), 131.0 (CH), 129.3 (CH), 128.7 (CH),
128.0 (C), 127.8 (CH), 127.7 (C), 127.6 (CH), 127.6 (C), 48.5 (CH3),
21.4 ppm (CH3); IR (film): nmax =3244(b), 1718, 1652, 1283 cmÀ1; MS
(ESI) m/z (%): 271.1 (100) [M+H]+; HRMS: calcd for C15H15N2O3:
271.1077 [M+H]+, found: 271.1082; elemental analysis calcd (%)
for C15H14N2O3: C 66.66, H 5.22, N 10.36; found: C 66.14, H 5.08, N
10.15.
(CH3); IR (film):nmax =3274(b), 3033, 2948, 1744, 1658, 1541 cmÀ1
;
MS (CI-NH3) m/z (%): 273.1 (100) [M+H]+; HRMS: calcd for
C15H17N2O3: 273.1233 [M+H]+, found: 273.1239; elemental analysis
calcd (%) for C15H16N2O3: C 66.16, H 5.92, N 10.29; found: C 65.92,
H
5.94, N 9.91; HPLC (CHIRALPAK-IA, heptane/EtOH 70:30,
0.5 mLminÀ1, l=254 nm, tR(S)=24 min, tR(R)=29 min).
(2S)-2-amino-3-(3-quinolyl)-propanoic acid, (2S)-6: A solution of
(2S)-5b (1.30 g, 4.78 mmol) in concentrated HCl (32 mL) was
heated under reflux for 6 h. After cooling, the solution was concen-
trated under reduced pressure to furnish 1.20 g of (2S)-6 (quantita-
tive yield) as a white solid. [a]D =+13.8 (c=0.50, D2O); m.p. 1608C;
1H NMR (400 MHz, D2O): d=8.97 (s, 2H; CH Ar), 8.00–8.21 (m, 3H;
CH Ar), 7.86 (t, J=7.6, 8.0 Hz, 1H; CH Ar), 4.16–4.26 (m, 1H; CHa),
3.51 ppm (t, J=4.4, 5.4 Hz, 2H; CH2 (CH2b)); 13C NMR (100 MHz,
D2O): d=171.1 (CO), 148.1 (CH), 144.9 (C), 136.9 (C), 135.6 (CH),
130.7 (CH), 129.5 (CH), 129.2 (CH), 129.1 (CH), 129.0 (C), 120.2 (CH),
53.7(CH3), 32.9 ppm (CH2); IR (film): nmax =3264(b), 1720, 1530 cmÀ1
;
MS (CI-NH3) m/z (%): 217.1 (100) [M+H]+; HRMS: calcd for
C12H13N2O2: 217.0899 [M+H]+, found: 217.0971; elemental analysis
calcd (%) for C12H21ClN2O6: C 44.38, H 6.02, N 8.63; found: C 43.95,
H 5.54, N 8.28.
Methyl
noate, (2S)-5a: (S,S)-[[(COD)Et-DuPHOS]RhI]OTf (9.7 mg, 0.01 mmol)
was added to solution of dehydro amino acid 4a
(500.0 mg,1.37 mmol) in MeOH (200 mL). The reaction mixture was
(2S)-2-benzyloxycarbonylamino-3-(3-quinolyl)-propa-
a
630
www.chembiochem.org
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 625 – 632

Somatostatin Analogues
N-Fmoc-l-3-(3-quinolyl)-alanine, l-7:
A solution of FmocOsu
Table 3. NMR data for 8a from 1H NMR, TOCSY, NOESY (D2O, 600 MHz).
(2.72 g 8.07 mmol) in dioxane (25 mL) was added to a suspension
of (2S)-6 (1.36 g, 5.38 mmol) in 10% Na2CO3 (25 mL) at 08C. The
mixture was stirred for 48 h at room temperature. Water (40 mL)
was then added and the mixture was extracted with hexanes (3ꢅ
20 mL). The remaining aqueous phase was cooled and adjusted to
pH 2 with 1m hydrochloric acid and then extracted with ethyl ace-
tate (3ꢅ20 mL). The combined organic layers were dried over
MgSO4 and evaporated to yield 2.20 g of l-7 (93% yield) as a white
solid. [a]D =+56.0 (c=1.00, CHCl3); m.p. 146–1488C. 1H NMR
(400 MHz, DMSO): d=8.96 (s, 1H; CH), 8.34 (s, 1H; CH), 8.08 (d,
J=8.3 Hz, 1H; CH), 7.94 (d, J=8.6 Hz, 1H; CH), 7.87 (d, J=7.5 Hz,
2H; CH), 7.78 (t, J=7.4 Hz, 1H; CH), 7.62 (m, 3H; CH), 7.39 (dd, J=
13.6, 6.9 Hz, 2H), 7.28 (t, J=7.3 Hz, 1H), 7.20 (t, J=7.4 Hz, 1H), 4.40
(m, 1H; CHa), 4.19 (m, 3H; CH, CH2), 3.41 (dd, J=4.2, 13.9 Hz, 1H;
CH2b), 3.15 ppm (m, 1H; CH2b); 13C NMR (100 MHz, DMSO): d=
173.6 (CO), 173.4 (CO),156.7 (C), 152.2 (CH), 146.1 (C), 144.3 (C),
144.2 (C), 141.3(2C), 137.1 (CH), 131.9 (C), 130.1 (CH), 128.5 (2CH),
128.3 (2CH), 127.7 (2CH), 127.5 (CH), 125.9 (CH), 125.8 (CH), 120.7
(2CH), 66.6 (CH2), 55.8 (CHa), 47.2 ppm (CH), 34.5(CH2b); IR (film):
nmax 3319 (b), 3010, 1715, 1441 cmÀ1; MS (CI-NH3) m/z (%): 439.2
(100) [M+H]+; HRMS: calcd for C27H23N2O4: 439.1658 [M+H]+,
found: 439.1647.
Chemical shift (ppm)
HN
Ha
Hb
other
1 Ala
2 Gly
3 Cys
4 Lys
5 Asn
6 Phe
7 Phe
8 Qla
7.894
8.466
8.284
8.484
8.051
7.871
7.973
7.988
3.898
3.760
4.430
3.989
4.292
4.060
4.184
4500
1.291
2.841
1.393
2.321
2.385
2.634
3.081
Hg 1.087, Hd 1.038
Hg 7.021
Hg 6.656, Hd 6.995
HD 6.861, HE 6.995
H
D1 8.560, HD2 8.525, HH3 6.972,
HH2 6.892, HE3 7.895
Hg 1.186, Hd 1.387
9 Lys
10 Thr
11 Phe
12 Thr
13 Ser
14 Cys
8.226
7.785
7.919
7.966
8.057
8.1754
3.903
4.107
4.473
3.989
4.299
4.323
1.591
4.013
2.809
3.836
3.710
2.886
Hg1 0.851, Hg2 0.870
HD 6.952, HE 7.000, HZ 0838
Table 4. NMR data for 8b from 1H NMR, TOCSY, NOESY (D2O, 600 MHz).
Chemical shift (ppm)
HN
Ha
Hb
other
General procedure for the synthesis of peptides l-8 and d-8:
The syntheses were performed by SPPS on a 2-Cl-Trt resin
(1.60 mmolgÀ1) by using the Fmoc/tBu, strategy. The first amino
acid, Fmoc-l-Cys-OH, (3 equiv) was coupled for 2 h in the presence
of DIPEA (6 equiv) in CH2Cl2 as solvent, then end-capped with
methanol (0.8 mLgÀ1). Fmoc removal was then performed by treat-
ing the peptidyl resin with 20% piperidine in DMF (2ꢅ15 min). The
second amino acid Fmoc-Ser-OH (3 equiv) was coupled using
DIPCDI (3 equiv) and HOBt (3 equiv), as activating reagents, in DMF
for 1–2 h. The Kaiser test was used to check for coupling comple-
tion.[24] This procedure was repeated for the following 11 Fmoc-
protected amino acids and for the final Boc-Ala-OH. The fully pro-
tected linear peptide was then cleaved from the resin by a cleav-
age cocktail (CH2Cl2/TFE/AcOH, 16:5:2 (v/v)) for 2 h. The formation
of the disulfide bridge in each of the two analogues was achieved
by using iodine (4 equiv) in solution at room temperature for
30 min, then the reaction was quenched with an aqueous solution
of sodium thiosulfate (1n). The aqueous layer was extracted with
CH2Cl2 (3ꢅ150 mL) and the combined organic layer was washed
with a mixture of 5% aqueous citric acid and 5% sodium chloride
(1:1) and evaporated under reduced pressure. Finally, total depro-
tection of the side chains was performed by using an acidic mix-
ture (TFA/CH2Cl2/anisole/H2O 12:6:2:1, v/v) for 4 h, and the remain-
ing solution was then washed with heptane (5 mL) and the aque-
ous layer was precipitated in Et2O (À108C) to afford SRIF-14 ana-
logues 8a and 8b.
1 Ala
2 Gly
3 Cys
4 Lys
5 Asn
6 Phe
7 Phe
8 Qla
7.906
8.479
8.295
8.400
8.287
8.155
8.050
8.448
3.898
3.764
4.433
4.279
4.528
4.341
4.191
4.433
1.305
2.849
1.418
2.450
2.644
2.648
3.022
Hd 1.274, HE 2.596
Hg 7.060
Hd 6.722, HE 6.998
HD 6.798, HE 6.610
HD1 8.609, HD2 8.505, HZ3 7.714,
HH2 7.898, HE3 7.961
Hg 0.752, Hd 1.485, HE 2.462
Hg1 0.889
9 Lys
10 Thr
11 Phe
12 Thr
13 Ser
14 Cys
8.344
7.815
8.150
8.133
8.044
8.077
4.100
4.161
4.637
4.180
4.301
4.293
1.213
3.911
2.734
3.972
3.669
2.929
HD 6.930, HE 7.068
Hg 0.928
HCOOH (99.9: 0.1), B: ACN-HCOOH (99.3:0.07)), flow: 2 mL.minÀ1
l=220 nm]; HRMS: calcd for C77H106N18O19S2: 825.3583; found:
,
825.6195.
Biological assays
Preparation of cells stably expressing each SRIF-14 receptor: CHO-K1
cells (American Type Culture Collection, Rockville, MD) were main-
tained in Kaighn’s modification of Ham’s F12 medium (F12K) sup-
plemented with 10% fetal bovine serum. pcDNA3 vectors encod-
ing each of the SSTR receptors were obtained from UMR cDNA Re-
source Center (University of Missouri–Rolla, MO, USA). CHO-K1 cells
were stably transfected with these vectors by using Lipofectamine
(Invitrogen). Colonies that were viable in F12K containing the anti-
biotic G418 (700 mgmLÀ1) were screened for SRIF-14 receptor ex-
pression, and then maintained in a G418 (400 mgmLÀ1)-containing
medium. Expression was detected by RT-PCR and Western blot,
and confirmed by radio ligand binding assay.
[l-Qla8]-Somatostatin, 8a: SRIF-14 analogue [l-Qla8]-SRIF was
synthesized from 0.33 g of 2-Cl-Trt resin (1.60 mmolgÀ1) following
the general procedure and with Fmoc-l-Qla-OH, l-7, affording
0.43 g of 8a in 74% yield and 95% purity. NMR: see Table 3; HPLC:
tR =8.32 min [gradient from 25% to 60% of B in 20 min (A: H2O-
HCOOH (99.9: 0.1), B: ACN-HCOOH (99.3:0.07)), flow: 2 mLminÀ1
,
l=220 nm]; HRMS: calcd for C77H106N18O19S2: 825.3583; found:
825.6195.
Receptor ligand-binding assay: All receptor-binding assays were per-
formed with membranes isolated from CHO-K1 cells expressing the
cloned human SRIF-14 receptors, as previously described.[25] The
assay buffer consisted of Tris (50 mm, pH 7.5) with EGTA (1 nm),
MgCl2 (5 mm), leupeptin (10 mgmLÀ1), pepstatin (10 mgmLÀ1), baci-
tracin (200 mgmLÀ1), aprotinin (0.5 mgmLÀ1), and BSA (0.2%). CHO-
[d-Qla8]-Somatostatin, 8b: SRIF-14 analogue [d-Qla8]-SRIF was
synthesized from 2-Cl-Trt resin (0.33 g, 1.60 mmolgÀ1) following
the general procedure and with Fmoc-d-Qla-OH, d-7, affording
0.42 g of 8a in 67% yield and 84% purity. NMR: see Table 4; HPLC:
tR =8.94 min [gradient from 25% to 60% of B in 20 min (A: H2O-
ChemBioChem 2011, 12, 625 – 632
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
631

A. Riera et al.
608–613; d) W. W. de Herder, L. J. Hofland, A. J. van der Lely, S. W. J.
Lamberts, Endocr.-Relat. Cancer 2003, 10, 451–458; e) M. K. Kole, J.
Goldman, J. P. Rock, Skull Base Surg. 1997, 7, 89–93.
K1 cell membranes, radiolabeled SRIF-14, and unlabeled test com-
pounds were suspended/diluted in the buffer. Assays (200 mL) were
performed in 96-well polypropylene plates. Ten micrograms of
membrane protein were incubated (1 h, 308C) with 125I-Tyr11-SRIF
(0.1 nm, 2000 CimmolÀ1) in the presence or absence of various
concentrations of unlabeled peptides (1 pm–1000 nm). The binding
reaction was terminated by vacuum filtration over Whatman GF/F
glassfiber filters, pre-soaked in 0.5% (w/v) polyethyleneimine and
0.2% bovine serum albumin, by using a 98-well harvester (Inotech).
The filters were washed with ice-cold Tris-HCl (50 mm, pH 7.5) and
dried, after which scintillator sheets were adhered to the filter and
the bound radioactivity was analyzed in a liquid scintillation coun-
ter (microb plus, Wallac). Specific binding was defined as total
bound 125I-Tyr11-SRIF minus the amount bound in the presence of
1000 nm SRIF (nonspecific binding). Inhibition curves were ana-
lyzed, and IC50 values were calculated by using a curve-fitting pro-
gram (“Prism”, GraphPad, La Jolla, CA). Ki values were determined
as described by Cheng and Prusoff.[26] Data are the meanÆS.E.M.
of at least three separate experiments, each performed in tripli-
cate.
[8] I. M. Chapman, A. Helfgott, J. O. Willoughby, J. Endocrinol. 1991, 128,
369–374.
[9] a) M. Knappenberg, A. Michel, A. Scarso, J. Brison, J. Zanen, K. Hallenga,
P. Deschrijver, G. Van Binst, Biochim. Biophys. Acta Protein Struct. Mol. En-
zymol. 1982, 700, 229–246; b) K. Hallenga, G. Van Binst, A. Scarso, A.
Michel, M. Knappenberg, C. Dremier, J. Brison, J. Dirkx, FEBS Lett. 1980,
119, 47–52; c) L. A. Buffington, V. Garsky, J. Rivier, W. A. Gibbons, Bio-
phys. J. 1983, 41, 299–304; d) L. A. Buffington, V. Garsky, J. Rivier, W. A.
Gibbons, Int. J. Pept. Protein Res. 1983, 21, 231–241.
[10] G. V. Nikiforovich, G. R. Marshall, S. Achilefu, Chem. Biol. Drug Des. 2007,
69, 163–169.
[11] R. M. Freidinger, D. S. Perlow, W. C. Randall, R. Saperstein, B. H. Arison,
D. F. Veber, Int. J. Pept. Protein Res. 1984, 23, 142–150.
[12] W. Vale, J. Rivier, N. Ling, M. Brown, Metab. Clin. Exp. 1978, 27, 1391–
1401.
[13] Selected references: a) W. Bauer, U. Briner, W. Doepfner, R. Haller, R. Hu-
guenin, P. Marbach, T. J. Petcher, J. Pless, Life Sci. 1982, 31, 1133–1140;
b) R. F. Nutt, D. F. Veber, P. E. Curley, R. Saperstein, R. Hirschmann, Int. J.
Pept. Protein Res. 1983, 21, 66–73; c) G. Melacini, Q. Zhu, G. Osapay, M.
Goodman, J. Med. Chem. 1997, 40, 2252–2258; d) D. D’Addona, A. Caro-
tenuto, E. Novellino, V. Piccand, J. C. Reubi, A. Di Cianni, F. Gori, A. M.
Papini, M. Ginanneschi, J. Med. Chem. 2008, 51, 512–520; e) M. Gairi, P.
Saiz, S. Madurga, X. Roig, J. Erchegyi, S. C. Koerber, J. C. Reubi, J. E.
Rivier, E. Giralt, J. Pept. Sci. 2006, 12, 82–91; f) J. Erchegyi, C. R. R. Grace,
M. Samant, R. Cescato, V. Piccand, R. Riek, J. C. Reubi, J. E. Rivier, J. Med.
Chem. 2008, 51, 2668–2675; g) C. R. R. Grace, J. Erchegyi, J. C. Reubi,
J. E. Rivier, R. Riek, Biopolymers 2008, 89, 1077–1087; h) C. R. R. Grace, J.
Erchegyi, S. C. Koerber, J. C. Reubi, J. Rivier, R. Riek, J. Med. Chem. 2006,
49, 4487–4496; i) C. R. R. Grace, L. Durrer, S. C. Koerber, J. Erchegyi, J. C.
Reubi, J. E. Rivier, R. Riek, J. Med. Chem. 2005, 48, 523–533; j) S. Gazal,
G. Gelerman, O. Ziv, O. Karpov, P. Litman, M. Bracha, M. Afargan, C.
Gilon, J. Med. Chem. 2002, 45, 1665–1671; k) L. Chen, C. Hoeger, J.
Rivier, V. D. Fitzpatrick, R. L. Vandlen, A. H. Tashjian Jr, Biochem. Biophys.
Res. Commun. 1999, 258, 689–694.
Serum stability assay: A peptide solution of 6 mgmLÀ1 in water
(1.8 mg in 300 mL) was sterilized by filtration (0.22 mm filter). 10 mL
Aliquots from this solution were added to 90 mL serum (human
male AB plasma, sterile filtered; SIGMA). These solutions were incu-
bated at 358C and samples were taken at 0, 1, 7, 17, 24 and 48 h.
Each sample was treated with acetonitrile (200 mL) and cooled to
08C for 30 min to precipitate the proteins. The suspensions were
centrifuged (10000 rpm, 10 min, 48C). This procedure was repeat-
ed twice. Solutions were filtered (0.45 mm PVDF), and analyzed by
RP-HPLC (eluent: 20–80% B (B=0.07%TFA in acetonitrile); 20 min
gradient; flow: 1 mLminÀ1). For each peptide the experiment was
repeated twice. The half-life of the peptide in serum was calculated
from the analysis of these degradation data.
[14] M. Appetecchia, R. Baldelli, J. Exp. Clin. Cancer Res. 2010, 29, 19.
[15] B. H. Arison, R. Hirschmann, D. F. Veber, Bioorg. Chem. 1978, 7, 447–451.
[16] C. A. Meyers, D. H. Coy, W. Y. Huang, A. V. Schally, T. W. Redding, Bio-
chemistry 1978, 17, 2326–2331.
Acknowledgements
[17] B. H. Hirst, B. Shaw, C. A. Meyers, D. H. Coy, Regul. Pept. 1980, 1, 97–
113.
[18] V. Prasad, E. T. Birzin, C. T. McVaugh, R. D. van Rijn, S. P. Rohrer, G. Chic-
chi, D. J. Underwood, E. R. Thornton, A. B. Smith III, R. Hirschmann, J.
Med. Chem. 2003, 46, 1858–1869.
[19] D. E. Levy, F. Lapierre, W. Liang, W. Ye, C. W. Lange, X. Li, D. Grobelny, M.
Casabonne, D. Tyrrell, K. Holme, A. Nadzan, R. E. Galardy, J. Med. Chem.
1998, 41, 199–223.
[20] S. A. Thomas, T. Li, K. W. Woods, X. Song, G. Packard, J. P. Fischer, R. B.
Diebold, X. Liu, Y. Shi, V. Klinghofer, E. F. Johnson, J. J. Bouska, A. Olson,
R. Guan, S. R. Magnone, K. Marsh, Y. Luo, S. H. Rosenberg, V. L. Giranda,
Q. Li, Bioorg. Med. Chem. Lett. 2006, 16, 3740–3744.
[21] a) E. Dyer, W. Yokayama, J. Org. Chem. 1961, 26, 2124–2125; b) U.
Jacoby, F. Zymalkowski, Arch. Pharm. Ber. Dtsch. Phrm. Ges. 1971, 304,
271–277.
[22] M. Revꢃs, C. Ferrer, T. Leꢀn, S. Doran, P. Etayo, A. Vidal-Ferran, A. Riera,
X. Verdaguer, Angew. Chem. 2010, 122, 9642–9645; Angew. Chem. Int.
Ed. 2010, 49, 9452–9455.
[23] C. Bruns, I. Lewis, U. Briner, G. Meno-Tetang, G. Weckbecker, Eur. J. Endo-
crinol. 2002, 146, 707–716.
[24] E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Anal. Biochem. 1970,
34, 595–598.
We thank MICINN (CTQ2008-00763/BQU), IRB Barcelona, the Gen-
eralitat de Catalunya (2009SGR 00901) and BCN Peptides S.A. for
financial support. R.R. and P.M.-G. thank MICINN for doctoral fel-
lowships.
Keywords: hormones · peptides · quinolylalanine asymmetric
hydrogenation · somatostatin · SRIF · unnatural amino acids
[1] a) P. Brazeau, W. Vale, R. Burgus, N. Ling, M. Butcher, J. Rivier, R. Guille-
min, Science 1973, 179, 77–79; b) R. Burgus, N. Ling, M. Butcher, R. Guil-
lemin, Proc. Natl. Acad. Sci. USA 1973, 70, 684–688.
[2] M. A. Sheridan, J. D. Kittilson, B. J. Slagter, Am. Zool. 2000, 40, 269–286.
[3] a) D. Hoyer, G. I. Bell, M. Berelowitz, J. Epelbaum, W. Feniuk, P. P. A. Hum-
phrey, A.-M. O’Carroll, Y. C. Patel, A. Schonbrunn, J. E. Taylor, T. Reisine,
Trends Pharmacol. Sci. 1995, 16, 86–88; b) Y. C. Patel, C. B. Srikant, Endo-
crinology 1994, 135, 2814–2817.
[4] I. Shimon, X. Yan, J. E. Taylor, M. H. Weiss, M. D. Culler, S. Melmed, J. Clin.
Invest. 1997, 100, 2386–2392.
[5] M. Z. Strowski, R. M. Parmar, A. D. Blake, J. M. Schaeffer, Endocrinology
2000, 141, 111 – 117.
[25] S. Rens-Domiano, S. F. Law, Y. Yamada, S. Seino, G. I. Bell, T. Reisine, Mol.
Pharmacol. 1992, 42, 28–34.
[26] Y.-C. Cheng, W. H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099–3108.
[6] G. Bocci, M. D. Culler, A. Fioravanti, P. Orlandi, A. Fasciani, R. Colucci, J. E.
Taylor, D. Sadat, R. Danesi, M. Del Tacca, Eur. J. Clin. Invest. 2007, 37,
700–708.
[7] a) G. Garcia-Tsao, A. J. Sanyal, N. D. Grace, W. Carey, Hepatology 2007,
46, 922–938; b) J. Ayuk, M. C. Sheppard, Postgrad. Med. J. 2006, 82, 24–
Received: October 4, 2010
´
30; c) M. Pawlikowski, G. Melen-Mucha, Curr. Opin. Pharmacol. 2004, 4,
Published online on January 21, 2011
632
www.chembiochem.org
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 625 – 632