Design, synthesis, and pharmacological characterization of fluorescent peptides for imaging human V1b vasopressin or oxytocin receptors

Article abstract of DOI:10.1021/jm1016208

Among the four known vasopressin and oxytocin receptors, the specific localization of the V1b isoform is poorly described because of the lack of selective pharmacological tools. In an attempt to address this need, we decided to design, synthesize, and characterize fluorescent selective V1b analogues. Starting with the selective V1b agonist [deamino-Cys¹,Leu ⁴,Lys⁸]vasopressin (d[Leu⁴,Lys⁸]VP) synthesized earlier, we added blue, green, or red fluorophores to the lysine residue at position 8 either directly or by the use of linkers of different lengths. Among the nine analogues synthesized, two exhibited very promising properties. These are d[Leu⁴,Lys(Alexa-647)⁸]VP (3) and d[Leu⁴,Lys(11-aminoundecanoyl-Alexa-647)⁸]VP (9). They remained full V1b agonists with nanomolar affinity and specifically decorated the plasma membrane of CHO cells stably transfected with the human V1b receptor. These new selective fluorescent peptides will allow the cellular localization of V1b or OT receptor isoforms in native tissues.

Full text of DOI:10.1021/jm1016208

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Pharmacological Characterization of
Fluorescent Peptides for Imaging Human V1b Vasopressin or
Oxytocin Receptors
Maithꢀe Corbani,† Miguel Trueba,‡ Stoytcho Stoev,§ Brigitte Murat,† Julie Mion,† Vꢀera Boulay,†
||
||
Gilles Guillon,†, and Maurice Manning*,§,
†Institute of Functional Genomics, CNRS UMR5203- INSERM U661, University of Montpellier I and II, 141 Rue de la Cardonille,
34094 Montpellier Cedex 05, France
‡Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, Basque Country University, Leioa, Spain
§Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, Ohio, United States
ABSTRACT: Among the four known vasopressin and oxytocin receptors, the
specific localization of the V1b isoform is poorly described because of the lack of
selective pharmacological tools. In an attempt to address this need, we decided to
design, synthesize, and characterize fluorescent selective V1b analogues. Starting
with the selective V1b agonist [deamino-Cys1,Leu4,Lys8]vasopressin (d[Leu4,
Lys8]VP) synthesized earlier, we added blue, green, or red fluorophores to the
lysine residue at position 8 either directly or by the use of linkers of different
lengths. Among the nine analogues synthesized, two exhibited very promising
properties. These are d[Leu4,Lys(Alexa 647)8]VP (3) and d[Leu4,Lys(11-ami-
noundecanoyl-Alexa 647)8]VP (9). They remained full V1b agonists with
nanomolar affinity and specifically decorated the plasma membrane of CHO
cells stably transfected with the human V1b receptor. These new selective
fluorescent peptides will allow the cellular localization of V1b or OT receptor isoforms in native tissues.
’ INTRODUCTION
Fluorescent tools have been synthesized for other receptors of
the VP/OT family.14ꢀ16 However, although good fluorescent
V1a and OT ligands have been produced, to date, no good
fluorescent specific ligand is available to selectively detect central
and peripheral V1b receptors. Here we describe the synthesis of
nine new fluorescent analogues for the human V1b receptor
(hV1b-R). Two of them are promising new tools for the
detection of V1b or OT receptors in native tissue. In our previous
work,17,18,3,19 by replacing the glutamine4 of the natural arginine
vasopressin (AVP) with a cyclohexylalanine or a leucine, by
replacing the arginine8 with a lysine, and by removing the NH2 of
the cysteine1 to increase stability toward aminopeptidases, we
produced analogues showing an increased selectivity for the rat
V1b receptor (rV1b-R). Namely, the [deamino-Cys1,4-leucine,8-
lysine]vasopressin (d[Leu4,Lys8]VP) analogue, previously
synthesized in the du Vigneaud laboratory,20 was found to be
selective for the rV1b-R3,19 and, to a lesser extent, for the hV1b-R.
It conserved a nanomolar affinity for these receptor isoforms.18
By studying the structure/activity of the ligand/V1b receptor
complex, we have also been able to propose a model of the
ligandꢀreceptor interaction.21 This model predicts that the first
six amino acids would be located into a “binding pocket” inside
the membrane, whereas the residues at positions 7, 8, and 9
Vasopressin (VP) and the VP receptor family including V1a,
V1b, V2, and the related oxytocin (OT) receptor are involved in
many different physiological functions. Among them, the V1b
receptor is well-known for the activation of adrenocorticotropin
hormone (ACTH) secretion in the pituitary, thus strongly
participating, with corticotrophin releasing hormone (CRF), in
the activation of the pituitary/corticotrope axis in stress and
anxiety.1 Vasopressin also regulates adrenal function by mediat-
ing catecholamines secretion2 and induces pancreatic insulin
secretion via V1b receptors.3 Besides this neuroendocrine effect,
V1b receptors have been suspected to be localized centrally and
to participate in cognitive and behavioral functions involved in
rewarding, cognition, and sociality (see ref 4 for review).
Although several publications describe the vasopressin V1a,
V2, and OT receptor distribution by using autoradiography5ꢀ7
or immunodetection8 techniques, the lack of selective radiola-
beled VP analogues or of receptor antibodies has hindered
progress in the detection of V1b receptor distribution in native
tissues. Results obtained by molecular approaches such as
polymerase chain reaction after reverse transcription (RT-
PCR)9,10 or messenger ribonucleic acid (mRNA) detection by
in situ hybridization,11ꢀ13 although more accurate, did not
provide clear information regarding the brain regions detected
by immunostaining. This was partially due to the fact that the
probes shared common sequences with V1a and OT receptors.9
Received: December 21, 2010
Published: March 23, 2011
r
2011 American Chemical Society
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Figure 1. General structure of fluorescent peptides (1ꢀ3, 5, 7, 9ꢀ11, 13) and parent peptides (A, 4, 6, 8, B, 12). X represents the modification on Cys1
by either H atom or OH group, and Y represents the types of linker and fluorophore attached at position Lys8 of the parent peptides d[Leu4,Lys8]VP (A)
and HO1[Leu4,Lys8]VP (B).
Table 1. Physicochemical Properties of Fluorescent Peptides 1ꢀ3, 5, 7, 9ꢀ11, 13, Parent Peptides A and B, and Intermediates 4,
6, 8, 12
b
TLC, Rf
compd
peptide
d[Leu4,Lys8]VPd
d[Leu4,Lys(Atn)8]VPe
d[Leu4,Lys(Alexa 488)8]VP
d[Leu4,Lys(Alexa 647)8]VP
d[Leu4,Lys(β-Ala)8]VPe
d[Leu4,Lys(β-Ala-Alexa 647)8]VP
d[Leu4,Lys(Aha)8]VPe
d[Leu4,Lys(Aha-Alexa 647)8]VP
d[Leu4,Lys(Aud)8]VPe
d[Leu4,Lys(Aud-Alexa 647)8]VP
[HO1][Leu4,Lys8]VP
[HO1][Leu4,Lys(Alexa 488)8]VP
[HO1][Leu4,Lys(Alexa 647)8]VP
[HO1][Leu4,Lys(Aud)8]VPe
[HO1][Leu4,Lys(Aud-Alexa 647)8]VP
yield,a %
a
b
c
d
HPLCc tR, min
formula
MW (calcd) MW (MS) (found)
A
1
60.6
24.3
74.4
43.1
67.9
64.4
36.5
35.0
41.9
55.9
54.2
39.8
49.6
54.4
55.1
0.35 0.37 0.30 0.34
0.44 0.55 0.66 0.47
0.20 0.14 0.07 0.13
0.08 0.22 0.03 0.04
0.21 0.40 0.12 0.48
0.17 0.29 0.02 0.12
0.25 0.23 0.30 0.38
0.11 0.02 0.03 0.06
0.40 0.51 0.53 0.53
0.18 0.26 0.12 0.17
0.26 0.22 0.18 0.38
0.17 0.25 0.13 0.22
0.16 0.28 0.03 0.14
0.38 0.51 0.53 0.50
0.14 0.28 0.09 0.15
12.4
13.6
11.8
10.1
15.0
10.1
13.1
10.5
14.4
11.7
11.1
11.3
11.0
12.7
11.2
C47H67O11N11S2
C54H72O12N12S2
C68H77O21N13S4
1026.3
1145.4
1540.7
1026.4
1145.8
1540.5
2
3
4
C50H72O12N12S2
C54H80O12N12S2
C58H88O12N12S2
1097.3
1153.5
1209.5
1097.7
1153.4
1209.9
5
6
7
8
9
B
10
11
12
13
C47H67O12N11S2
C68H77O22N13S4
1042.3
1556.7
1042.6
1556.6
C58H88O13N12S2
1225.6
1226.0
a Yields are based on the amount of peptide used in the reaction and are uncorrected for acetic acid, TFA, and water content. Yields of peptides 3, 5, 7, 9,
11, and 13 are approximated and based on the MW (∼1250) of Alexa 647 given by Invitrogen/Molecular Probes (pending patent). b Solvent systems
and conditions are given in Experimental Section. c All peptides were at least 95% pure. For elution, a linear gradient 90:10 to 30:70 (0.05% aqueous
TFA/0.05% TFA in CH3CN) over 30 min with flow rate 1.0 mL/min was applied. d d[Leu4,Lys8]VP (A) was resynthesized as previously described.3,19
For original synthesis see ref 20. Abbreviations of position 8 substituents and linkers are as follows: Atn (anthraniloyl), β-Ala (β-alanine), Aha (7-
aminoheptanoic acid), and Aud (11-aminoundecanoyl).
would be outside. Thus, the Lys8 residue provides an epsilon
NH2 group which could be used for further additions. Accord-
ingly, we have taken advantage of the stability and good
selectivity of d[Leu4,Lys8]VP3,18 to introduce fluorophores on
the side chain of the Lys8 residue to create fluorescent tools that
would conserve the pharmacology of d[Leu4,Lys8]VP, resist
degradation, and selectively decorate with an excellent resolution
the plasma membrane of CHO cells expressing V1b and/or OT
receptors.
analogues, since we had previously shown that this peptide is a
V1b selective agonist for both rat and human VP/OT
receptors.3,18,19 First, we carried out the addition of a hydroxyl
(OH) group at the N terminal position of d[Leu4,Lys8]VP to
give [1-L-(ꢀ)-2-hydroxy-3-thiopropanoic acid,4-leucine,8-lysi-
ne]vasopressin ([HO1][Leu4,Lys8]VP) (peptide B in Tables 1
and 2). We had shown previously that this modification was
helpful in the design of fluorescent agonists for the hOT-R.16,22
We observed that the OH modification at position 1 did not
affect the ligand’s affinity but strongly increased its selectivity for
the hV1b-R (see Table 2). We thus also synthesized a short series
of OH-modified fluorescent compounds. We selected different
Rationale for Fluorescent Selective V1b Synthesis. We
used d[Leu4,Lys8]VP (peptide A in Figure 1 and Tables 1, 2,
and 3) as the parent molecule for designing fluorescent V1b
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Table 2. Binding Properties of Fluorescent Vasopressin Analogues for Human Vasopressin and Oxytocin Receptorsa
affinity Ki for [3H]AVP (nM)
hV1b-R selectivity index (SI)
analogue
peptide
hV1a-R
1.1 ( 0.1
hV1b-R
hV2-R
1.2 ( 0.2
hOT-R
V1a/V1b
V2/V1b
OT/V1b
AVP
A
AVPb
0.68 ( 0.01
0.52 ( 0.07
0.65 ( 0.22
1.2 ( 0.2
165 ( 16
1,3 ( 0,5
65 ( 11
1.7 ( 0.5
29 ( 6
1.6
1.8
2.5
56
d[Leu4,Lys8]VPb
d[Leu4,Lys(Atn)8]VP
d[Leu4,Lys(Alexa488)8]VP
d[Leu4,Lys(Alexa647)8]VP
d[Leu4,Lys(β-Ala)8]VP
d[Leu4,Lys(β-Ala-Alexa647)8]VP
d[Leu4,Lys(Aha) 8]VP
69 ( 16
6714 ( 806
4918 ( 1447
>100000
133
378
215
73
12912
7566
>83333
>606
4000
>1538
36082
955
1
246 ( 53
21 ( 5
32
2
258 ( 36
30 ( 9
25
3
12,033 ( 1903
215 ( 50
>100000
36 ( 7.5
12.6 ( 2.1
62 ( 9
0.2
9.7
1
4
5200 ( 190
>100000
165
123
436
29
5
8011 ( 2035
266 ( 28
6
0.61 ( 0.05
74 ( 6
22010 ( 3310
70705 ( 17325
31100 ( 7900
>100000
22 ( 7
36
7
d[Leu4, Lys(AhaꢀAlexa647)8]VP
2158 ( 369
384 ( 93
121 ( 16
210 ( 67
86 ( 27
179 ( 20
963 ( 197
324 ( 29
3742 ( 713
3415 ( 965
1.6
57
8
d[Leu4,Lys(Aud)8]VP
3.7 ( 1.5
13.0 ( 3.9
1.7 ( 0.5
149 ( 26
1799 ( 255
8.4 ( 2.7
668 ( 89
104
148
517
371
16
8405
>7692
934
9
d[Leu4,Lys(Aud-Alexa647)8]VP
HO[Leu4, Lys8]VP
HO[Leu4,Lys(Alexa488)8]VP
HO[Leu4,Lys(Alexa647)8]VP
HO[Leu4,Lys(Aud)8]VP
HO[Leu4,Lys(Aud-Alexa647)8]VP
1930 ( 326
879 ( 123
55300 ( 11250
29300 ( 5700
2790 ( 381
>100000
6.6
105
671
0.2
445
5.1
B
1558 ( 137
>100000
10
11
12
13
>336
>56
>100000
45000 ( 13400
>100000
332
5357
>150
>150
a Binding assays were performed on plasma membranes derived from CHO cells stably expressing the different VP/OT receptors as described in the
Experimental Section. Ki values are the mean ( SEM of at least three independent experiments, each performed in triplicate. For each analogue, the
hV1b-R SI was calculated as follows: SI = (Ki analogue for hVx-R)/(Ki analogue for hV1b-R), where hVx-R is hV1a-R, hV2-R, or hOT-R. Abbreviations of
the linkers are as follows: β-Ala (β-alanine, 4A), Aha (7-aminoheptanoic acid, 8A), and Aud (11-aminoundecanoyl, 12A). Atn is for anthraniloyl. b Data
from reference.3
Table 3. Functional Properties of Some Fluorescent Analogues on Phospholipase C Activity in CHO Cells Stably Expressing
Human V1b or OT Receptorsa
CHO hV1b-R
CHO hOT-R
analogue
peptide
EC50 (nM)
Emax (%)
100
EC50 (nM)
Emax (%)
AVPb
2.0 ( 0.6
24.5 ( 2.6
1.5 ( 0.4
2.48 ( 2.1
6.93 ( 4
8.3 ( 2.2
10.8 ( 1.3
nd
27.5 ( 1.8
OT
A
1
31 ( 1
100
d[Leu4,Lys8]-VPb
d[Leu4,Lys(Atn)8]-VP
d[Leu4,Lys(Alexa 488)8]-VP
d[Leu4,Lys(Alexa 647)8]-VP
d[Leu4,Lys(Aha-Alexa 647)8]-VP
d[Leu4,Lys(Aud-Alexa 647)8]-VP
99 ( 6
nd
108 ( 8.5
93.1 ( 4.6
112 ( 9
106.7 ( 4.3
102 ( 2
nd
nd
2
nd
nd
3
401 ( 14
267 ( 44
11.4 ( 2.6
70.6 ( 19
nd
73.4 ( 5.2
nd
7
9
172 ( 22
69.7 ( 3.05
a Phospholipase C stimulation was performed on CHO cells stably expressing hV1b-R or hOT-R. For each analogue, EC50 values and Emax were
calculated as described in Experimental Section. Results are the mean ( SEM of at least three independent experiments, each performed in triplicate.
b Data from ref 3.
fluorophores to attach to the d[Leu4,Lys8]VP (peptide A,
Figure 1). First, we chose the anthraniloyl (Atn) group, a small
fluorescent molecule with MW = 97 Da and a quantum yield (R)
of 0.358, used for the design of analogue 1 and highly sensitive to
microenvironmental changes.23 We have also selected the Alexa
fluorophores (Molecular Probes) for their brightness and their
resistance to photobleaching.24 We have used Alexa Fluor 488
(Alexa 488) (MW = 643; quantum yield R = 0.60; molar
extinction coefficient ε = 71 000) and Alexa Fluor 647
(Alexa 647) (MW ≈ 1250; R not determined, ε = 239 000),
the latter being one of the brightest fluorescent molecules
reported so far.25 Moreover, Alexa fluorophores are charged
molecules, soluble in water, and not prone to create molecular
stacking as the tetramethylrhodamine is (which they are derived
from). The aliphatic ω-aminocarboxylic acids, 3-aminopropionic
acid (β-Ala), 7-aminohexanoic acid (Aha), and 11-aminounde-
canoic acid (Aud) were selected as linkers of various sizes to
optimize the interaction of the fluorescent ligand to the V1b
receptor. Their spacer arms consist of 4, 8, and 12 atoms,
respectively, and will be referred to as 4A, 8A, and 12A further
in the text. The structures of the fluorescent peptides and of their
parent analogues, designed according to the above rationale, are
given in Figure 1 and Table 1.
’ RESULTS
Binding Pharmacological Properties of Fluorescent Ana-
logues. We first characterized the binding properties of each
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Figure 2. Binding pharmacological profiles of d[Leu4,Lys(Alexa 647)8]VP (analogue 3) and d[Leu4,Lys(Aud-Alexa 647)8]VP analogue 9 for human
vasopressin and oxytocin receptors stably expressed in CHO cells lines. The binding properties of analogues 3 and 9 were determined by competition
experiments on plasma membranes prepared from CHO cells stably expressing hV1a-R, hV2-R, hV1b-R, or hOT-R. Plasma membranes expressing the
3
different VP/OT receptor isoforms were incubated for 1 h at 30 °C with ∼1 nM [H]AVP in the presence or absence (control, C) of increasing
concentrations of unlabeled analogue 3 or 9. Nonspecific and total binding were determined respectively with and without 1 μM of either AVP or OT,
depending on the nature of the receptor studied. Specific binding calculated as the difference between total and nonspecific binding is expressed as
percent of the corresponding control specific binding and is the mean ( SEM of at least three independent experiments each performed in triplicate.
analogue and compared their affinities to those of AVP or of the
corresponding parent peptide A or peptide B taken as references
(Figure 2, Table 2). Attaching the Atn fluorophore directly to the
d[Leu4,Lys8]VP (A) to give d[Leu4,Lys(Atn)8]VP (analogue 1)
did not affect the affinity for the hV1b-R (0.65 vs 0.52 nM) and
poorly changed the selectivity versus the hV1a, hV2, and hOT
receptors. Thus, since there was no major decrease in affinity or
selectivity, we did not perform further modifications of analogue 1.
For analogue 2, d[Leu4,Lys(Alexa 488)8]VP, with Alexa 488
replacing Atn, the affinity for the hV1b receptor was still very
good, with a Ki of 1.2 nM compared to 0.52 nM for the parent
analogue A. Its selectivities toward the hV1a-R and hV2-R were
excellent (Table 2). For analogue 3, d[Leu4,Lys(Alexa 647)8]VP,
the affinity for the hV1b-R dropped dramatically to 165 nM
(Table 2 and Figure 2), probably because of the relative size of
the fluorescent moiety. The Alexa 647 fluorophore is indeed
about twice as big as Alexa 488, thus conferring a MW of ∼2200
to analogue 3 vs 1500 for analogue 2. In spite of its loss of affinity,
analogue 3 retained good selectivity for the hV1b versus the hV1a
and hV2 receptors. Moreover, both fluorescent V1b analogues 2
and 3 exhibited a good affinity for the hOT-R: 30 nM for
analogue 2 (Alexa 488) and 36 nM for analogue 3 (Alexa 647).
Introducing an OH group at position 1 of analogue A to give B
([HO][Leu4,Lys8]VP) reduced the affinity of this analogue for
the hOT-R (Ki of 179 nM for analogue B vs 29 nM for analogue
A). Thus, we synthesized analogues of B coupled to Alexa 488
(analogue 10) and to Alexa 647 (analogue 11). Introducing an
Alexa 488 or Alexa 647 to B led to decreases of the affinities for
the hOT-R of the resultant fluorescent peptides 10 and 11. For
the Alexa 488 analogue 10, the Ki for hOT-R was 963 nM
compared with 30 nM for analogue 2. The Alexa 647 derivative
(analogue 11) exhibited a Ki for hOT-R of 324 nM compared
with 36 nM for analogue 3. However, the affinities of peptides 10
and 11 for hV1b-R also dropped dramatically (from 1.2 to 149
nM for the Alexa 488 analogues (2 and 10) and from 165 to 1799
nM for the Alexa 647 analogues (3 and 11).
βAla to d[Leu4,Lys(Alexa647)8]VP analogue 3 to give d[Leu4,
Lys(β-Ala-Alexa 647)8]VP (analogue 5) improved its affinity (65
nM for analogue 5 compared with 165 nM for analogue 3) but
did not improve its selectivity for hV1b versus hOT receptors
(selectivity index of 1 for analogue 5 vs 0.2 for analogue 3).
Increasing the length of the spacer to eight atoms with a
7-aminohexanoyl (Aha) linker to give d[Leu4,Lys(Aha-Alexa
647)8]VP (analogue 7) modestly improved the affinity and
selectivity of analogue 7. Further increasing the length of the
spacer to 12 atoms with an 11-aminoundecanoyl (Aud) linker to
give d[Leu4,Lys(Aud-Alexa 647)8]VP (analogue 9) significantly
improved the affinity and selectivity of analogue 9. The Ki was 13
nM for analogue 9 compared to 165 nM for analogue 3. This
improvement might result from the fact that the high MW
fluorescent moiety is now more distant from the core peptide
(A) and does not hamper the ligandꢀreceptor interaction as
much (Figure 3). The selectivity was also much better (SI of 6.6
compared to 0.2, Table 2 and Figure 2). In order to try to gain
more V1b-R selectivity vs OT-R, an OH-version of analogue 9
(HO[Leu4,Lys(Aud-Alexa 647)8]VP, analogue 13) was also
synthesized. The affinity severely dropped (from 13 to 668
nM) without any improvement in its selectivity. We thus decided
to forego using HO[Leu4,Lys8]VP (peptide B) for any further
designs of fluorescent V1b ligands.
Functional Properties of Fluorescent Peptides: Capacity
To Stimulate Phospholipase C. As both the hV1b and hOT
receptors are directly coupled to phospholipase C (PLC),26 we
measured the ability of each fluorescent analogue to activate PLC
in stably transfected CHO cells. First we looked at the capacity of
the most promising analogues to stimulate PLC in a CHO hV1b-
R cell line. As illustrated in Figure 4 and summarized in Table 3,
with the exception of OT, which behaved as a partial agonist of
the hV1b-R (31% of maximal VP-stimulated PLC activation), all
analogues tested were full agonists with analogue 3 having a
higher EC50. The Atn analogue (1) and the Alexa 488 analogue
(2) showed EC50 in the same range as analogue A and AVP
(Table 3). The Alexa 647 analogues 3, 7, and 9, although full
agonists, showed EC50 values of 401 nM for analogue 3
(Alexa 647 directly attached to analogue A) and 267 nM for
analogue 7 (having the 8A, Aha linker). Only the 12A Aud-
peptide (analogue 9) displayed a good EC50 of 11.4 nM for
hV1b-R coupling. Second, as the Alexa 647 compounds
In order to improve the affinity of the Alexa 647 analogues for
the hV1b-R, we introduced linkers of different lengths. We first
verified that attaching linkers to the reference analogue d[Leu4,
Lys8]VP (A) (Table 1) maintained the affinity of the nonfluor-
escent analogues for the hV1b-R within the nanomolar range
(Figure 3 and Table 2). Adding a 4A-spacer (3-aminopropionyl)
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spectrofluorimetry of cell suspensions in cuvettes. Living CHO
cells, stably expressing hV1b receptors, were exposed to analogue
1 for 1 h at 12 °C, washed, and evaluated for the fluorescence
emitted at the wavelength (λ) of 412 nm by spectrofluorimetry.
Complete excitation and emission spectra were established
previously, and the value of 412 nm was found to be the λmax
of experimental emission for analogue 1 whereas the λmax of
excitation was confirmed to be 343 nm (data not shown). As
shown in Figure 5 (A), analogue 1 can display a saturable
fluorescent binding in Dulbecco’s modified Eagles medium/
bovine serum albumin/4-2-hydroxylethyl-l-piperazineethanesul-
fonic acid (DMEM/BSA/HEPES) buffer with a fluorescence
(EC50) of 1.7 nM, a value very close to the one obtained by
binding experiments performed on membrane preparations
derived from CHO cells stably expressing hV1b-R (0.65 nM).
This binding (analogue 1 used at the saturating concentration of
10 nM) can be completely suppressed with an excess of the
nonfluorescent analogue A (1 μM) demonstrating its specificity
(Figure 5A).
Fluorescent derivatives of the V1b agonist d[Leu4,Lys8]VP
(A) were designed to provide tools for imaging the V1b-R on
cells in culture. To verify this point, confocal imaging was
conducted on living CHO stable cell lines, expressing either
one of the four human receptors, i.e., V1a-R, V1b-R, V2-R, or
OT-R. Only CHO cells expressing the hV1b-R and the hOT-R
could be labeled. The CHO hV1a-R and the CHO hV2-R
remained unlabeled, even at the concentration of 500 nM of
either Alexa 488 or Alexa 647 analogues (data not shown). In
CHO hV1b-R, the binding appeared to be patchy and mainly
localized at the plasma membrane (Figure 5B and Figure 6 right
panels). This “patchy” effect was not due to internalization,
since the experiments were conducted at 12 °C, a temperature
below the chain-melting transition temperature of the mem-
brane lipids (16 °C). This aspect was also observed after one
night of incubation at 4 °C, a condition where internalization
cannot take place either (data not shown). Moreover, the
distribution of the fluorescent labeling with either Alexa 488
or Alexa 647 compounds was found to be similar and was
completely suppressed in the presence of 1 μM analogue A
(Figure 5B).
Figure 6 further shows the ability of the fluorescent analogues
3 and 9 to selectively label hV1b or hOT receptors at the plasma
membrane of stably transfected CHO cells. Plasma membrane
labeling observed on CHO hV1b-R cells incubated with 250 nM
of analogue 3 (a mixed OT/V1b agonist; see Table 2) is totally
suppressed by co-incubation with 100 nM analogue A but only
very partially when similar experiments were performed on CHO
hOT-R cells (Figure 6, top rows). The complete displacement of
analogue 3 from CHO hOT-R was obtained only with 500 nM or
more of A, in agreement with its respective affinities for hV1b-R
(0.52 nM) compared to hOT-R (29 nM).3 As expected, [1-β-
mercapto-β,β-cyclopentamethylenepropionyl,2-0-methyl tyro-
sine,8-arginine]vasopressin (Manning compound) (500 nM), a
mixed OT/V1a antagonist,26,27a,27b totally displaced analogue 3
from CHO hOT-R cells but not from CHO hV1b-R cells.
Similarly, total displacement of analogue 3 from CHO hOT-R
cells was also obtained with a specific non-peptide OT antago-
nist, 4-chloro-3-[(3R)-(þ)-5-chloro-1-(2,4-dimethoxybenzyl)-
3-methyl-2-oxo-2,3-dihydro-1H-indol-3-yl]-N-ethyl-N-(3-pyri-
dylmethyl)benzamide, hydrochloride (14) (SR126768A)28
(100 nM), and this displacement was not seen on CHO hV1b-
R cells (not shown). Analogue 3 labeling was not displaced by
Figure 3. Influence of the size of the linker between Alexa 647 and
peptide A on its V1b affinity and selectivity for human vasopressin and
oxytocin receptors. (A) The V1b affinity of parent analogues A, 4, 6, and
8 and that of their corresponding peptides with Alexa 647 attached to
linkers of different lengths (analogues 3, 5, 7, 9) are plotted as the
numbers of atoms in the spacer bound to Lys8. Values are from Table 2.
(B) The hV1b-R selectivity index of analogues described above is plotted
as the number of atoms of the spacer bound to Lys8. Values are from
Table 2 with V1b selectivity index calculated as log[Ki(hVx-R)/Ki-
(hV1b-R)], where Vx is hV2-R, hV1a-R, or hOT-R.
displayed some affinity for the hOT-R too, we also compared
their capacities to activate PLC in a CHO hOT-R cell line. We
observed that, compared to OT, the natural ligand AVP was a
partial agonist (27.5% of maximal OT-stimulated PLC
activation). Analogues 3 and 9 also displayed partial agonism,
with ∼70% of PLC activation compared to the one induced by
the natural agonist OT. In general, the respective EC50 of all
analogues for either the hV1b-R or the hOT-R remained well
correlated with their binding properties (compare Tables 2 and 3).
Labeling Properties of Fluorescent Peptides Evaluated by
Spectrofluorimetry and Cell Imaging. The Atn analogue 1,
d[Leu4,Lys(Atn)8]VP, was designed to provide a fluorescent
V1b selective ligand with blue emission that could be a possible
partner of an Alexa 488 analogue to record experimental fluor-
escence resonance energy transfer (FRET) between V1b homo-
dimers. Since UV confocal microscopes are not easily available,
the Atn analogue 1 cannot be tested by imaging but by
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Figure 4. Functional properties of d[Leu4,Lys(Alexa 647)8]VP (analogue 3) and d[Leu4,Lys(Aud-Alexa 647)8]VP (analogue 9) on phospholipase C
activity in CHO cells stably expressing the human V1b and OT receptors. Left: CHO cells stably expressing the hV1b-R were incubated with or without
(control) increasing amounts of fluorescent or parent peptides. Total inositol phosphate (InsP) that accumulated was measured as described in
Experimental Section and expressed as % of maximal AVP stimulation (1 μM). Data are the mean ( SEM of three independent experiments, each
performed in triplicate. Right: Similar experiments were performed with CHO cells stably expressing the hOT-R. Total inositol phosphate that
accumulated was measured as described in Experimental Section and expressed as the % of maximal OT stimulation (1 μM). Data are the mean ( SEM
of three independent experiments, each performed in triplicate.
Figure 5. Fluorescent properties of d[Leu4,Lys(Atn)8]VP (analogue 1), d[Leu4,Lys(Alexa 488)8]VP (analogue 2), and d[Leu4,Lys(Alexa 647)8]VP
(analogue 3) on CHO cells stably expressing the human V1b receptor. (A) Histogram (top): Cells in suspension were incubated for 1 h at 12 °C with 10
nM analogue 1 in the absence (dark bar) or in the presence (hatched bar) of 1 μM d[Leu4,Lys8]VP (analogue A) and compared to control cells
incubated without fluorescent ligand (white bar). Cells were washed three times with cold PBS and measured for fluorescent emission at 412 nm in
cuvettes of spectrofluorimetry. Saturation curve (bottom): Aliquots of cells were incubated with increasing concentrations of analogue 1 for 1 h at 12 °C,
washed three times, and counted for fluorescence emission at 412 nm. The endogenous fluorescence at 412 nm of control cells, not exposed to
fluorescent ligand, was deduced for each concentration. (B) CHO hV1b-R cells grown on coverslips were incubated in the presence of analogue 2 (50
nM) or of analogue 3 (250 nM) for 1 h at 12 °C in the absence or in the presence of 1 μM d[Leu4,Lys8]VP (analogue A). Confocal imaging was
performed with Zeiss LSM510 Meta microscope using a 63ꢁ (NA 1.4) objective with an argon laser excitation at 488 nm and a BP 505ꢀ530 nm
emission filter for analogue 2 (green) and with a helium/neon laser excitation at 633 nm with a LP650 for emission for analogue 3 (purple).
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Figure 6. Displacement of d[Leu4,Lys(Alexa 647)8]VP (analogue 3) and of d[Leu4,Lys(Aud-Alexa 647)8]VP (analogue 9) binding with nonfluor-
escent ligands on CHO hV1b-R and hOT-R stable cell lines. CHO cells on coverslips were incubated in the presence of analogue 3 (250 nM) or of
analogue 9 (150 nM) for 1 h at 12 °C in the absence or in the presence of 100 nM selective V1b agonist d[Leu4,Lys8]VP (analogue A) or of 1 μM non-
peptide AVP V2 antagonist 1529 or of 500 nM V1a/OT peptide antagonist Manning compound.27a Confocal imaging was performed with Zeiss LSM510
Meta microscope using a 63ꢁ (NA 1.4) with laser excitation at 633 nm and LP650 for emission. The bar represents 10 μm (zoom 4ꢁ). Images are
representative of three independent experiments.
the selective non-peptide AVP V2 antagonist (1-[4-N-tert-
butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-
[4-(2-morpholinoethoxy)cyclohexane]indol-2-one, phosphate
monohydrate (15) (SR121463)29 (1 μM), neither from CHO
hV1b-R nor from CHO hOT-R cells.
The labeling of hV1b-R CHO cells with 150 nM analogue 9 (a
selective V1b agonist; see Table 2) was intense and completely
displaced by 100 nM A but neither by 500 nM Manning
compound nor by 1 μM 15 (Figure 6, bottom row left). When
similar experiments were performed on CHO hOT-R cells, the
labeling was very low and both A (V1b selective) and Manning
compound (OT/V1a peptide antagonist) totally displaced ana-
logue 9 labeling (Figure 6, bottom row right). However, this
labeling was not displaced by the non-peptide AVP V2 antagonist
15 (1 μM) neither from CHO hV1b-R nor from CHO hOT-R
cells as previously shown for analogue 3.
fluorescence was quantified with a laser scanning microscope
(LSM) browser after confocal imaging using helium/neon laser
633 nm for detecting Alexa 647 analogues (Figure 7). Analogue
9, which displays a better affinity for the hV1b-R as compared to
analogue 3 (as measured by competition studies on membrane
preparations with 3[H]-AVP, see Table 2), also showed a better
capacity to bind membrane V1b-R sites on living cells at low
concentration (affinity of the fluorescent ligand of 14.23 ( 2.19
nM for analogue 9 compared to 172.3 ( 19 nM for analogue 3,
Figure 7 left panel). On CHO hOT-R cells, the affinities of the
two analogues were quite similar, with a slight preference for
analogue 3 (109 ( 9.7 nM) compared to analogue 9 (162 ( 17
nM) (Figure 7, right panel), reflecting the results obtained in the
displacement studies performed with 3[H]AVP (Table 2).
’ DISCUSSION AND CONCLUSION
In order to fully characterize the imaging properties of the two
analogues 3 and 9, we have established saturation curves of each
analogue in the conditions of living cells (DMEM/BSA/HEPES)
on CHO cells expressing either V1b-R or OT-R. The membrane
Receptors of the vasopressin and OT family are important in
the regulation of the stress processes (review in ref 30). Centrally,
the V1b receptors have been involved in stress and especially in
learning and memory processes. Important data have been
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Figure 7. Fluorescent binding properties of d[Leu4,Lys(Alexa 647)8]VP (analogue 3) and d[Leu4,Lys(Aud-Alexa 647)8]VP (analogue 9) on CHO
cells stably expressing the human V1b and OT receptors. CHO hV1b-R cells on coverslips were incubated in the presence of increasing concentrations of
analogue 3 or of analogue 9 for 1 h at 12 °C, washed 3 times with PBS, and fixed with 4% PFA. Confocal imaging was performed with a Zeiss LSM510
Meta microscope using a 63ꢁ objective with a helium/neon laser excitation at 633 nm with a LP650 for emission. Membrane fluorescence was evaluated
on 30ꢀ40 cells for each concentration of analogue by LSM Browser and ImageJ using the 1ꢀ250 fluorescent unit dynamics. After deduction of the values
of control unlabeled lines 5ꢀ7 cells, saturation curves were constructed with the mean of three distinct experiments and expressed as % of the maximal
labeling.
obtained by the use of knock-out (KO) animals, but after a period
of cloning and pharmacological characterization in the past
decade, it became necessary to elucidate the distribution of these
receptors to better understand their central functions in vivo.
To do so, tracers are necessary and the radiolabeled natural
hormones were the first candidates to detect receptors in situ.
However, because of the close affinity between AVP and OT for
the OT receptors,26,31 it is very difficult to discriminate between
their receptor sites. Other studies were conducted using receptor
antibodies, but since no good antibody against the V1b-R is
available, the information about V1b receptor localization re-
mains uncertain. Moreover, data obtained by in situ hybridization
experiments were not always reliable, because of the partial
similarities between the VP and OT receptor probes,9,10 and
reflect mRNA levels encoding the receptor and not the expres-
sion of the receptor itself.
Agonists and antagonists for different classes of receptors have
been produced, but still, all were not completely selective,
especially toward the V1b-R.27b A selective V1b agonist was
produced by replacement in AVP of the glutamine residue in
position 4 by a leucine18 and made highly resistant to degradation
by deamination at the Cys1 position. The subsequent re-
placement of Arg8 by a Lys8, leading to the V1b agonist
A,3,19,20 did not modify its V1b selectivity. This allowed the
covalent addition of a fluorophore to the free ε NH2 group of the
Lys residue at position 8 in A, thus making possible the synthesis
of new fluorescent V1b agonists.
from binding to the V1b-R with a similar affinity as the parent
peptide A (0.65 vs 0.52 nM). It may be a good compound to
probe ligand binding interactions with V1b receptors and to
confirm the data about the vasopressin V1b binding pocket we
have obtained by modeling.21 It may also be a good donor to a
green acceptor in FRET experiments for identification of V1b-R
homodimers in vivo. Thus, although other receptors of the
vasopressin/oxytocin family are known to be expressed as
homodimers (OT-R/OT-R)34,35 or heterodimers (V1a-R/
V2-R),36,37 nothing is known about V1b-R monomers or dimers.
Only heterodimers of V1b-R with corticotrophin-releasing hor-
mone receptor type 1 (CRHR1) have been described.38 We have
also chosen the “Alexa family” of fluorophores (Molecular
Probes) that develop very good brightness and good resistance
to degradation and to photobleaching, compared to Cyanines 3
and 5, for example,24,39 and that are relatively easy to attach to
peptides. The Alexa 488 was selected because it is excited in the
488 nm range, a wavelength very common on any fluorescent
microscope, including the electrophysiological settings, where it
can be an excellent tool to reveal cells to be recorded (data not
shown). It was also a good acceptor for energy transfer with Atn
(good overlapping of the emission of Atn/excitation of Alexa 488
spectra) so that we could conduct studies on V1b-R homodimers
in natural tissues later. Alexa 647 was also selected because it
provides an extremely bright signal (ε = 239 000) compared to
other Alexa fluorophores including Alexa 488 (ε = 165 000), a
quality that will be essential for detecting low levels of receptors
in tissue slices. As Alexa 647 excitation is in the far red, cell
autofluorescence is also very low.
Taking advantage of the progress in confocal microscopy and
in the production of new fluorophores, we elected to design and
synthesize the fluorescent V1b analogues reported here. We
selected three different colors for complementary purposes. The
Atn, successfully attached to the μ opioid receptor agonist
DALDA (H-Dmt-D-Arg-Phe-Lys-NH2 with Dmt = 20,60-
dimethyltyrosine),32,33 is a good reporter of environment23
displaying a relatively small size. Thus, we confirm here that
the Atn fluorophore does not prevent d[Leu4,Lys(Atn)8]VP (1)
The pharmacological properties (binding, coupling to PLC)
were determined for the 13 analogues and compared with the
parent peptide d[Leu4,Lys8]VP (analogue A) and to the two
natural peptides AVP and OT (Table 2). First it appears that all
analogues conserved a very good selectivity for V1b-R vs V1a-R
and V2-R. Directly attaching the fluorophore reduced slightly
the selectivity, from 56-fold in d[Leu4,Lys8]VP (analogue A) to
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32-fold (analogue 1, Atn) and to 25-fold in analogue 2 (Alexa 488).
The selectivity was totally inverted for analogue 3 (Alexa 647),
becoming 5 times better for the OT-R compared to the V1b-R.
As a possible explanation, it has been previously proposed that in
the OT molecule, compared to AVP, the neutral Leu8 is an
important amino acid to keep OT-R selectivity, while this
position has to be positively charged to interact with vasopressin
receptors.40,41 As the Lys8 of d[Leu4,Lys8]VP (A) (that has
replaced the Arg8 naturally present in AVP) is now neutralized by
attaching a bulky residue on the free ε NH2, it may explain the
modification of V1b-R toward OT-R selectivity. It is also possible
that the bulky fluorescent molecule (especially big in the case of
Alexa 647) prevents the 1ꢀ6 sequence of the peptide A to fit into
the binding pocket situated at the top of TM5 and TM7 as
described in our V1b-R model.21,42 To overcome this problem,
we added linkers of different sizes, 4A (βAla), 8A (Aha), and 12A
(Aud). These linkers, comprising carbon chains of different
lengths, all have a C-terminal NH2 group to which a fluorophore
could be covalently attached. We observed that, as shown in
Figure 3A, the longer the linker, the more the affinity of the
Alexa 647 analogue for the V1b-R becomes closer to that of the
parent peptide A. This affinity reaches 13 nM for analogue 9,
compared to 0.52 nM for A and compared to 3.7 nM observed for
analogue 8, which corresponds to an analogue of A simply
connected to the Aud linker with no fluorophore. The selectivity
toward the hV1b-R also increased. Thus ,analogue 3 (Alexa 647
without a linker) is more selective for the hOT-R compared to
the hV1b-R (36 nM vs 165 nM). With the Aud linker (analogue 9),
it became more selective for the hV1b-R (13 nM) compared to
the hOT-R (86 nM). Thus, we totally inverted the compound
selectivity (Figure 3B). The selectivity for the hV1a-R and the
hV2-R was not strongly modified, and both analogues 3 and 9
remained low-affinity agonists for these two receptor isoforms
(over 2000-fold).
We also noticed that replacing one of the hydrogens on the R
carbon at position 1 by a hydroxyl group in peptide A to give
peptide B enhanced the hV1bR/hOTR selectivity. Thus, for
analogue B, the affinity for the hV1b-R was conserved (1.7 nM vs
0.52 nM), whereas the hV1bR/hOTR selectivity was doubled
(V1b-R SI of 105 instead of 56). We thus synthesized OH-
substituted versions of the analogues coupled to Alexa 488 or
Alexa 647 (peptides 10ꢀ13). Unfortunately, although the affi-
nity for the hOT-R remained weak, the affinity for the hV1b-R
also dropped (to 149 nM for the Alexa 488 analogue (10) and to
1799 nM for the Alexa 647 analogue (11). Adding a 12A linker
(analogue 13) did not improve the affinities, neither for the hOT-
R (3415 nM) nor for the hV1b-R (668 nM). So we did not
pursue this track any further.
agonism, with ∼70% of activity compared to 100% observed with
OT. AVP was also a partial agonist at hOT-R with an Emax of only
27.5%. It is well-known that the OT-R can couple to Gq (PLC)
and also to Gi,40,41 which is not the case for the V1b-R. These
data confirm that OT can be a partial agonist for AVP receptors
and, on the contrary, AVP can be a partial agonist for the OT-R.
Since it is known that there is a functional antagonism between
AVP and OT, the first (AVP) being released during the stress and
the second (OT) being present in nursing and calming behaviors,
each acting at its own receptors, the partial agonism shown here
could play a role in this functional antagonism by competing for
the opposite site and preventing the “opposite effect” hormone
from interacting with its own receptor, thus reinforcing the
physiological effects.
The membrane hV1b receptors are labeled as patches. This
patchy effect could reveal domains of the membrane where the
receptors are concentrated. However, it does not seem to be due
to the fact that the ligand is an agonist, since it also happened with
other peptides from the vasopressin family including the V1a
antagonist Rhm8-PVA (4OH-Ph(CH2)2CO-DTyr(Me)-Phe-
Gln-Asn-Arg-Pro-Lys-(5-carboxytetramethylrhodamyl)-NH2)
on rat hepatocytes cells,15 V1a/OT derivatives attached to
Rhm (tetramethylrhodamyl)22 and with other ligands such as
Alexa 488-opioid dermorphin and deltorphin43 on CHO cells.
However, this aspect was similar to the one observed on living
cells before paraformaldehyde (PFA) fixation (not shown) and
we preferred this technique to the one using antibodies, where
fixation and Triton permeation are necessary for receptor detection
and may give distortions. Thus, we depict here functional receptors
capable of PLC activation and of internalization (not shown).
In this study, in contrast to studies performed only on
membranes, we have validated the capacity of the fluorescent
ligands 3 and 9 to label directly specific living cells and we have
verified that the pharmacological and functional data fit well with
the imaging data. Often, many new compounds lack full char-
acterization on all VP and OT receptor isoforms, and because
they display only a partial specificity (V1b SI < 50), they could
induce nonspecific labeling of several OT/AVP receptors.27b
From our data, analogues 3 and 9 seem really promising for
detecting human V1b or OT receptors. d[Leu4,Lys(Aud-
Alexa 647)8]VP analogue 9 seems more selective for the hV1b-
R but also presents some affinity for hOT-R. Utilized at 150 nM
and in the presence of 100 nM OT-R selective OT antagonist 14
(or of the Manning compound), it provides a very selective
labeling of the hV1b-R. d[Leu4,Lys(Alexa 647)8]VP (analogue
3) presents a mixed affinity for hV1b and hOT receptors and can
also be used in the presence of 100 nM of the non-peptide OT
antagonist 14 or of the Manning compound to selectively label
V1b sites. It can also be used to label OT sites in the presence
of the selective non-peptide AVP V1b antagonist (2S,4R)-
1-[5-chloro-1-[2,4-dimethoxyphenyl)sulfonyl]-3-(2-meth-
oxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3yl]-4-hydroxy-N,N-
dimethyl-2-pyrrolidine carboxamine (16) (SR149415).44 Thus,
the labeling of analogue 3 on CHO hOT-R cells was only
completely suppressed with high concentrations of analogue A,
more than 500 nM or with the non-peptide V1b antagonist 16 at
a concentration higher than 500 nM (not shown) at which it can
also compete with hOT-R as described.44
To fully characterize the analogues, functional coupling was
evaluated for the most promising compounds (Table 3). The
activation of PLC was measured for the parent peptide A and the
fluorophore-coupled analogues Atn (analogue 1), Alexa 488
(analogue 2), and Alexa 647 (analogue 3) and with linkers
(analogues 7 and 9). The activity was compared to that of the
corresponding native peptides AVP and OT in two different
stable cell lines: one expressing hV1b-R, the other hOT-R. First,
it appears that the EC50 values were in the same range compared
to the Ki values. All analogues (original A, Atn-, Alexa 488-, or
Alexa 647-coupled analogues) were full agonists at the hV1b-R
compared to the natural peptide AVP (Table 3). Only OT
displayed partial agonism. The two Alexa 647 analogues 3 and
9 were also tested on CHO hOT-R, and both displayed partial
To conclude, by inserting different fluorophores at position 8
in d[Leu4,Lys8]VP (A), we generated two new promising
fluorescent analogues d[Leu4,Lys(Alexa 647)8]VP (3) and
d[Leu4,Lys(Aud-Alexa 647)8]VP (9) capable of selectively
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labeling hV1b-R or hOT-R expressed on CHO cells. We
completely characterized their conditions of utilization. In nat-
ural conditions on nonfixed tissue, these fluorescent peptides 3
and 9, having the Alexa 647 fluorophore attached to the lysine
residue at position 8, will allow the detection of very low levels of
V1b or OT binding sites on native tissues in the CNS, with
almost no background, and will permit, for the first time, the
cartography of the V1b receptor within the brain.
Solid-Phase Synthesis Procedures. The schematic structures of
d[Leu4,Lys8]VP (A) and parent molecules including all fluorescent
analogues are illustrated in Figure 1. Their syntheses will be
described below.
HO-Mpr(Mob)-Tyr(Bzl)-Phe-Leu-Asn-Cys(Mob)-Pro-Lys(2CIZ)-Gly-
NH2 (Protected Precursor for Peptide B (PrB)). Boc-Gly-resin (0.7 g, 0.5
mmol) was subjected to eight cycles of deprotection, neutralization, and
coupling with Boc-Lys(2CIZ), Boc-Pro, Boc-Cys(Mob), Boc-Asn-ONp,
Boc-Leu, Boc-Phe, Boc-Tyr(Bzl), and HO-Mpr(Mob), respectively. A 1
M HCl/AcOH mixture was used in all the deprotection steps.46
Neutralizations were carried out with 10% Et3N/CH2Cl2. All coupling
reactions (except when Boc-Asn was involved) were performed by the
DCC/HOBt procedure50 in CH2Cl2/DMF (9:1, v/v). Boc-asparagine
was coupled as its nitrophenyl ester51 in DMF. The resulting protected
peptidyl resin was cleaved by ammonolysis in methanol.52 The protected
peptide was extracted with hot DMF (30 mL), and the product was
precipitated by the addition of hot water (∼300 mL). After cooling, the
product was collected, dried in vacuo over P2O5, and reprecipitated from
DMF (30 mL) and ether (∼200 mL). Collection and drying in vacuo
over P2O5 gave the required protected nonapeptide amide (PrB) 0.53 g
68.5% yield.** Mp 112ꢀ114 °C. TLC, Rf (solvent system): 0.73 (a);
0.78 (b); 0.70 (c); 0.77 (d).
’ EXPERIMENTAL SECTION
Materials. All reagents used were analytical grade. Most standard
chemicals were purchased from Sigma (St. Louis, MO) or Merck
(Darmstadt, Germany) unless otherwise indicated. AVP and OT were
from Bachem (Bubendorf, Switzerland) and [3H]AVP (44 Ci/mmol)
and myo-[2-3H]inositol (20 Ci/mmol) from PerkinElmer (Courtab!uf,
France). Fetal calf serum and polyornithine came from Sigma
(Saint-Quentin Fallavier, France). Dulbecco’s modified Eagle medium
(DMEM) and penicillinꢀstreptomycin were purchased from Invitrogen
(Cergy Pontoise, France). Inositol-free DMEM came from ICN Bio-
chemicals (Orsay, France). Dowex AG1-X8 formate form 200ꢀ400
mesh was purchased from Bio-Rad (Munich, Germany). All peptides
and fluorescent derivatives listed in Table 1 and Manning compound27a
were synthesized in the laboratory of Dr. M. Manning (College of
Medicine, University of Toledo, OH, U.S.) as described below. The non-
peptide OT antagonist 14,28 the non-peptide AVP V2 antagonist 15,29
and the non-peptide AVP V1b antagonist 1644 were from Sanofi-
Synthelabo (Toulouse, France).
L-(ꢀ)-2-Hydroxy-3-methoxybenzylthiopropanoic acid (HO-Mpr-
(Mob) was synthesized as previously described for HO-Mpr(Bzl).45
The Mpr(Meb) and Boc-11-aminoundecanoic acid were from Chem-
Impex International (Wood Dale, IL). The NR-Boc protected amino
acids were purchased from Bachem (Torrance, CA) and Chem-Impex
International (Wood Dale, IL). The Boc-Gly-resin was from Calbio-
chem-Novabiochem Corp. (San Diego, CA). The Alexa Fluor 488
carboxylic acid, 2,3,5,6-tetrafluorophenyl ester (Alexa Fluor 488 5-TFP)
(5-isomer), and Alexa Fluor 647 carboxylic acid succinimidyl ester were
from Invitrogen/Molecular Probes, Inc. (Eugene, OR). The N-carboxy-
anthranilic anhydride (isatoic anhydride) and Boc-β-alanine (Boc-3-
aminopropionic acid) were from Sigma-Aldrich, Inc. (St. Louis, MO).
The Boc-7-aminoheptanoic acid (BOC-Aha) was from Bachem
(Torrance, CA). Peptide d[Leu4,Lys8]VP20 (A) was resynthesized as
previously described.3,19 Peptide [HO1]Leu4,Lys8]VP (B) was synthe-
sized specifically for this study by the Merrifield solid-phase method46,47
with modifications previously described48,49,3,19 as shown in Solid-Phase
Synthesis Procedures below. TLC was run on precoated silica gel plates
(60F-254, 3 Merck) with the following solvent systems: (a) 1-butanol/
AcOH/H2O (upper phase) (4:1:5); (b) 1-butanol/AcOH/H2O
(4:1:2); (c) 1-butanol/AcOH/H2O/pyridine (15:3:3:10); (d) 1-buta-
nol/AcOH/H2O (2:1:1). Loads of 10ꢀ15 μg each were applied, and
chromatograms were developed at a minimal length of 10 cm. For
detection, a combination of monitoring on a UV lamp (model UVGL-
58, UVP Inc., San Gabriel, CA) and the chlorine gas procedure for the
KI-starch reagent47 was used. Analytical HPLC was performed on a
Hitachi D-7000 HPLC system under the following conditions: 90:10 to
30:70 0.05% aqueous TFA/0.05% TFA in MeCN, linear gradient over
30 min at 1.0 mL/min (λ = 254 or 214 nm) on a Vydac 218TP54 C18
column (Grace Vydac, Hesperia, CA). For semipreparative HPLC the
same apparatus and gradient were used over 30 min at 5.0 mL/min (λ =
214 nm) on a Vydac 218TP510 C18 column. All peptides were at least
95% pure. Mass spectra (MS) were done by the Tuft’s Core Facility,
Department of Physiology (Boston, MA) on a MALDI-TOF Voyager
mass spectrometer (Perspective Biosystems/Applied Biosystems) using
dihydrobenzoic acid as the matrix.
[HO1][Leu4,Lys8]VP (Free Peptide B). The Na/liquid NH3
procedure53 with the modifications previously described5,48,49,54 was
used for the deprotection of the protected precursor PrB. A solution of
PrB in sodium-dried ammonia (∼400 mL) was treated at the boiling
point and with stirring with sodium from a stick of metal contained in a
small-bore glass tube until a light-blue color persisted in the solution for
about 30 s. NH4Cl was added to discharge the color. Reoxidation of the
resulting deblocked disulfydryl peptide (B) was performed with potas-
sium ferricyanide (K3[Fe(CN)6]) using the modified reverse
procedure55 as follows. The peptide residue was dissolved in 25 mL of
50% AcOH, and the solution was diluted with 50 mL of H2O. The
peptide solution was added dropwise with stirring over a period of
15ꢀ30 min to a 600 mL aqueous solution that contained 20 mL of a 0.01
M solution of K3[Fe(CN)6]. Meanwhile, the pH was adjusted to
approximately 7.0 with concentrated ammonium hydroxide. Following
oxidation, the free peptide (B) was isolated and purified as follows. After
acidification with AcOH to pH 4.5 and stirring for 20 min with an anion
exchange resin (Bio-Rad, AG 3 ꢁ 4, Clꢀ form, 5 g damp weight), the
suspension was slowly filtered and washed with 0.2 M AcOH (3 ꢁ
30 mL). The combined filtrate and washings were lyophilized. The
resulting powder was desalted on a Sephadex G-15 column (110 cm ꢁ
2.7 cm), eluting with aqueous AcOH (50%), with a flow rate of 5 mL/h.56
The eluate was fractionated and monitored for absorbance at 254 nm.
The fractions making up the major peak were checked by TLC, pooled,
and lyophilized. The residue was further subjected to gel filtration on
Sephadex LH-20 using a 100 cm ꢁ 1.5 cm column, eluting with aqueous
AcOH (2 M) with a flow rate of 4 mL/min. The peptide was eluted in a
single peak (absorbance at 254 nm). After lyophilization of the pertinent
fractions, the collected peptide was subjected to a final semipreparative
HPLC purification to give 54.9 mg (54.2%) of desired [HO1]Leu4,
Lys8]VP (B). TLC, Rf (solvent system): 0.26 (a); 0.22 (b); 0.18 (c);
0.38 (d). HPLC, tR: 11.1 min. MW (calculated): 1042.3. MW (found by
MS): 1042.6.
Fluorescent Analogue Synthesis
Peptide Labeling with Anthraniloyl (Atn) Fluorophore (Peptide 1,
Table 1). We adapted the synthesis of Boc-Dap(Atn)23 and Boc-
Lys(Atn)32 for the preparation of d[Leu4,Lys(Atn)8]VP (peptide 1)
as follows: To a stirred solution of 12.5 mg (12.2 μM) of d[Leu4,
Lys8]VP (A) and 1.3 mg (12.2 μM) of dry Na2CO3 in water/acetonitrile
(1:1 v/v, 150 μL), isatoic anhydride, 2.38 mg (14.6 μM) in 100 μL of
acetonitrile, was added. The mixture was stirred overnight at room
temperature at pH ∼8 and with TLC monitoring. The mixture was
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acidified to pH 2 with 5% aqueous KHSO4 and was subjected to a two-
step purification procedure, first on a Sephadex G-15 column with 25%
acetic acid as eluent and finally by semipreparative HPLC to give the
desired peptide 1.
General Procedure for Peptide Labeling with Alexa 488 and
Alexa 647 Fluorophores
fluorescent peptide d[Leu4,Lys(Aha-Alexa 647)8]VP (7) was isolated
by semipreparative HPLC and lyophilized. Yield 1 mg (∼35.0%)
(Table 1).
Peptides d[Leu4,Lys(β-Ala)8]VP (4), d[Leu4,Lys(Aud)8]VP (8),
and [HO1][Leu4,Lys(Aud)8]VP (12) were labeled utilizing Alexa 647
succinimidyl ester as described above for peptide 7 to give peptides
d[Leu4,Lys(β-Ala-Alexa 647)8]VP (5), d[Leu4,Lys(Aud-Alexa 647)8]VP
(9), and [HO1][Leu4,Lys(Aud-Alexa 647)8]VP (13). Yields of 5, 9, and
13 are given in Table 1.
Fluorescent Peptides with Alexa Fluorophore Directly Attached at
Position 8 of Parent Peptide d[Leu4,Lys8]VP (A) and [HO1][Leu4,
Lys8]VP (B) (Peptides 2, 3, 10, and 11 (Table 1)). The preparation of
the fluorescent peptides 2, 3, 10, and 11 was performed as described by
Albizu and co-workers57 with some modifications as follows: 1.2 μM (in
excess) parent peptide d[Leu4,Lys8]VP (A) or [HO1][Leu4,Lys8]VP
(B) was dissolved in 100 μL of anhydrous DMF and mixed with 5 μL of
DIPEA. The solution was added to 1 mg of the appropriate fluorophore.
For fluorescent peptides 2 and 3 the parent peptide was d[Leu4,Lys8]VP
(A). For fluorescent peptides 10 and 11 the parent peptide was
[HO1][Leu4,Lys8]VP (B). The fluorophores were as follows: for
peptides 2 and 10 Alexa 488 tetrafluorophenyl ester (5-carboxy pure
isomer) was used. For peptides 3 and 11, Alexa 647 succinimidyl ester
was utilized. The reaction mixture was whirled using a vortex for 1 h at
room temperature in the dark with pH, TLC, and HPLC monitoring.
After the reaction was over, the mixture was acidified (6 μL of TFA) and
evaporated in vacuo. The fluorescent peptide was isolated by semipre-
parative HPLC and lyophilized. Yields of 2, 3, 10, and 11 are given in
Table 1.
Fluorescent Peptides with the Alexa 647 Fluorophore Attached at
Position 8 of Parent Peptide d[Leu4, Lys8]VP (A) by Means of Spacer
(Peptides 5, 7, 9, and 13 (Table 1)). All peptides 5, 7, 9, and 13 were
prepared by a general two-step procedure as follows. First, the spacer-
containing peptides d[Leu4,Lys(β-Ala)8]VP (4), d[Leu4,Lys(Aha)8]VP
(6), d[Leu4,Lys(Aud)8]VP (8), and [HO1][Leu4,Lys(Aud)8]VP (12)
(Table 1) were synthesized as described below. Next, the Alexa 647
fluorophore was attached to peptides 4, 6, 8, and 12 as described above
to give the desired peptides 5, 7, 9, and 13.
The structures of peptides 1, 2, 4, 6, 8, 10, and 12 were confirmed by
MALDI-TOF mass spectrometry (MS). The structures of the Alexa 647
containing peptides 3, 5, 7, 9, 11, and 13 were not confirmed by mass
spectrometry, since the structure of the Alexa 647 fluorophore has not
been revealed by Invitrogen/Molecular Probes because of a pending
patent application. The analytical and some other physicochemical data
of the fluorescent peptides 1ꢀ3, 5, 7, 9ꢀ11, 13 and of the intermediates
4, 6, 8, 12 are presented in Table 1.
Membrane Preparation. Four different Chinese hamster ovary
(CHO) cell lines stably expressing the human V1a, V1b, V2, or OT
receptors were washed twice in phosphate buffered saline (PBS) without
CaCl2 and MgCl2, harvested in lysis buffer (15 mM Tris-HCl, pH 7.4;
2 mM MgCl2; 0.3 mM ethylene diaminetetraacetic acid (EDTA),
Polytron homogenized, and centrifuged at 44000g for 20 min at 4 °C
as previously described.3 Pellets were washed in 50 mM Tris-HCl (pH
7.4), 3 mM MgCl2 and centrifuged at 44000g for 20 min at 4 °C.
Membranes were resuspended in a small volume of the same buffer.
Protein concentration was determined by the method of Bradford (Bio-
Rad protein assay kit) using bovine serum albumin (BSA) as a standard,
and membranes were stored in liquid nitrogen.
Binding Assays. Membrane incubations with [3H]AVP were
performed as previously described.3 Briefly, 10ꢀ20 μg of membrane
proteins were incubated for 60 min at 30 °C in a medium containing
50 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 1 mg/mL BSA, 0.01 mg/mL
leupeptine, and 1 nM 3[H]AVP in the presence (nonspecific binding) or
in the absence (total binding) of 1 μM unlabeled AVP in a final volume
of 200 μL. For Ki determinations, increasing amounts of the unlabeled
analogue to be tested were added to the incubation medium to a fixed
concentration of tritiated ligand. The membrane-associated radioactivity
was collected by filtration through GF/C filters and counted. Specific
binding was calculated as the difference between total and nonspecific
binding values and expressed as percent of the specific binding deter-
mined without unlabeled analogue.
Phospholipase C Assay. Inositol phosphate (InsP) accumulation
was determined as described previously.3 CHO cells were seeded at
100 000 cells per well in 24-well plates and grown for 24 h in their regular
culture medium, then incubated for another 24 h period in serum- and
inositol-free medium supplemented with 2 μCi/ml myo-[2-3H]inositol.
Cells were then washed twice with HBS, incubated for 15 min in HBS
supplemented with 20 mM LiCl, and further stimulated for 15 min at
37 °C in the same buffer with increasing concentrations of vasopressin
analogues. The reaction was stopped by perchloric acid (5% vol/vol).
Total InsPs was extracted, purified on Dowex AG1-X8 anion exchange
chromatography column, and counted.
Cell Labeling and Imaging. CHO cells, seeded on 12 mm glass
coverslips precoated with polyornithine, were incubated in DMEM, BSA
0.2 mg/mL, HEPES 25 mM, pH 7.4, for 1 h at 12 °C with fluorescent
analogues in the absence or in the presence of nonfluorescent VP
analogues. In the latter case, a 30 min preincubation of the cells in the
same medium containing only these ligands was performed. After three
washes with cold PBS, the cells were finally fixed in 4% PFA at 4 °C
overnight and mounted with mowiol. The fluorescent cells were imaged
using a confocal microscope Zeiss LSM510 Meta equipped with an
Axiovert200 M microscope. The objective 63ꢁ (NA 1.4) for oil
immersion was used. For detection of Alexa 488-labeled cells, the
Preparation of d[Leu4,Lys(Aha)8VP (6). 7.35 mg (30 μM) of Boc-7
aminoheptanoic acid (Boc-Aha), 7.35 mg (30 μM) of DCC, and 4.1 mg
(30 μM) of HOBt were dissolved in ∼200 μL of dry DMF, and the
mixture was stirred for 30 min. Next, 23.6 mg (23 μM) of d-
[Leu4Lys8]VP (A) as a solution in ∼150 μL of dry DMF and neutralized
in advance with DIPEA was added, and the reaction mixture was
incubated overnight at room temperature with continuous stirring and
pH ∼8.5 monitoring. The desired pH was maintained using DIPEA.
After the reaction was over (TLC and HPLC monitoring), the reaction
mixture was separated on a Sephadex G-15 column using 25% AcOH as
an eluent. The fraction containing the peptide was collected and
lyophilized. The Boc-protecting group of the peptide was removed by
treatment with 50% TFA in CH2Cl2 for 30 min at room temperature.
The reaction mixture was evaporated by a flow of N2, and the resulting
residue was subjected to a final purification by semipreparative HPLC to
give the spacer-containing peptide d[Leu4,Lys(Aha)8]VP (6) with a
yield of 9.7 mg (36.5%) (Table 1). Spacer-containing peptides d[Leu4,
Lys(β-Ala)8]VP (4) and d[Leu4,Lys(Aud)8]VP (8) were prepared in
the same manner as peptide (6), using the parent peptide d[Leu4,
Lys8]VP (A) and Boc-β-Ala and Boc-Aud, respectively. Peptide
[HO1]Leu4,Lys(Aud)8]VP (12) was prepared by the same approach,
utilizing Boc-Aud and the parent peptide [HO1][Leu4,Lys8]VP (B).
Yields of 4, 8, and 12 are given in Table 1.
Synthesis of d[Leu4,Lys(Aha-Alexa 647)8VP (7). 1.38 mg (1.2 μM)
(in excess) of peptide d[Leu4,Lys(Aha)8]VP (6) was dissolved in 100
μL of anhydrous DMF, mixed with 5 μL of DIPEA. The solution was
added to 1 mg of Alexa 647 succinimidyl ester, and the reaction mixture
was whirled using vortex for 1 h at room temperature in the dark at pH
∼8 with TLC and HPLC monitoring. After the reaction was over, the
mixture was acidified (6 μL TFA) and evaporated in vacuo. The
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excitation was performed with an argon laser at λ = 488 nm. The green
emission was collected using a BP 505ꢀ530 nm emission filter. For
detection of Alexa 647, helium/neon laser 633 nm was used. The
emission was collected with a LP650 nm filter. Images were captured
in multitracking mode to avoid channel cross-talk, and the settings were
established on unlabeled cells used as negative control. For fluorescence
quantification, the Zeiss LSM Browser was used as previously
described58 with 30ꢀ40 cells evaluated for each fluorescent binding
condition.
Alternatively, the binding of the Atn-labeled peptide (analogue 1) was
evaluated by cuvette spectrofluorimetry. Cells were detached with PBS,
10 mM EDTA and incubated with the fluorescent analogue in DMEM,
0.2 mg/mL BSA, 25 mM HEPES, pH 7.4, at 12 °C as previously
described. After three cold washes with PBS and gentle centrifugation
(900g, 4 min), the cells were resuspended in PBS and placed in a cuvette
at 4 °C. The fluorescence was measured using λ = 343 nm excitation and
λ = 412 nm emission in a Photon Technology International 810
spectrometer equipped with a UV lamp. These values were chosen after
full excitation and emission curves had been established with soluble
Atn-analogue 1. The specific detection was calculated by deducing the
fluorescence emission measured in CHO hV1b-R cells incubated with an
excess of nonfluorescent ligand.
Data Analysis. All data were were analyzed using Prism (GraphPad
Software, Inc., San Diego, CA). For the radioligand binding data, the
inhibitory dissociation constant (Ki) for unlabeled vasopressin analo-
gues was calculated from binding competition experiments according to
the Cheng and Prusoff equation: Ki = IC50(1 þ [L]/Kd), where IC50 is
the concentration of unlabeled analogue leading to half-maximal inhibi-
tion of specific binding, [L] the concentration of the radioligand present
in the assay, and Kd its affinity for the receptor studied. The respective Kd
values of 3[H]AVP for hV1a-R (1.1 nM), hV1b-R (0.68 nM), hV2-R (1.2
nM), and hOT-R (1.7 nM) had been previously determined by Pena
et al.3 and are listed in Table 2 (line 1). For the PLC assay, results are
expressed as the mean ( SEM of the number of distinct experiments
indicated (n). Statistical analysis of the data was performed using the
one-way analysis of variance test.
Chlebowski for her expert assistance in the preparation of this
manuscript.
’ ABBREVIATIONS USED
AcOH, acetic acid; ACTH, adrenocorticotropin hormone; β-Ala,
3-aminopropionyl; Alexa 488, Alexa Fluor 488 carboxylic acid,
2,3,5,6-tetrafluorophenyl ester (5-isomer), and the related
acyl group; Alexa 647, Alexa Fluor 647 carboxylic acid succini-
midyl ester and the related acyl group (structure unknown,
pending patent application); Aha, 7-aminohexanoyl; Atn, anthra-
niloyl; Aud, 11-aminoundecanoyl; AVP, arginine vasopressin;
Boc, tert-butyloxycarbonyl; Bzl, benzyl; cAMP, cyclic adenosine
monophosphate; BSA, bovine serum albumin; CHO, Chinese
hamster ovary; CHO hV1a-R, CHO hV1b-R, CHO hV2-R, and
CHO hOT-R, CHO cells stably expressing human vasopressin
V1a, V1b, V2, and oxytocin receptors, respectively; CRH (or
CRF), corticotrophin releasing hormone; CRHR1, corticotro-
phin-releasing hormone receptor type 1; 2CLZ, 2-chlorobenzy-
loxycarbonyl; DALDA, H-Dmt-D-Arg-Phe-Lys-NH2, Dmt, 20,60-
dimethyltyrosine; Dap, L-2,3-diaminopropionic acid; DCC, dicy-
clohexylcarbodiimide; DIPEA, N,N-diisopropylethylamine; d-
[Leu4,Lys8]VP, [deamino-Cys1,4-leucine,8-lysine]vasopressin;
DMF, dimethylformamide; DMEM, Dulbecco’s modified
Eagle’s medium; ε, molar extinction coefficient; EC50 or Kact,
concentration of agonist leading to half-maximal activity; Emax
,
maximal efficiency; EDTA, ethylenediaminetetraacetic acid;
EGFP, enhanced green fluorescent protein; ESMS, electron
spray mass spectrometry; Et3N, triethylamine; FRET, fluores-
cence resonance energy transfer; Gi, G-protein type i; Go, G-
protein type o; HOBt, 1-hydroxybenzotriazole; HBS, HEPES
buffer saline; HO1[Leu4,Lys8]VP, [1-L-(ꢀ)-2-hydroxy-3-thio-
propanoic acid,4-leucine,8-lysine]vasopressin; HO-Mpr, L-(ꢀ)-
2-hydroxy-3-thiopropanoyl; HPLC, high-performance liquid
chromatography; InsPs, total inositol phosphates; Kd, affinity at
concentration of ligand leading to half-maximal specific binding
deduced from saturation experiments; Ki, inhibitory dissociata-
tion constant at concentration of peptide leading to half-maximal
specific binding deduced from competition experiments; KO,
knock-out animals; λ, wavelength; MeCN, acetonitrile; LSM, la-
ser scanning microscope; mowiol, mounting medium for fluor-
escence microscopy; mRNA, messenger ribonucleic acid;
Manning compound, [1-β-mercapto-β,β-cyclopentamethylene-
propionyl,2-O-methyltyrosine,8-arginine]vasopressin (VP V1a/
OT antagonist); Mob, p-methoxybenzyl; Mpr, 3-mercaptopro-
pionyl; ONp, p-nitrophenyl ester; OT, oxytocin; OT-R, oxytocin
receptor; PBS, phosphate buffered saline; PFA, paraformalde-
hyde; PLC, phospholipase C; Rhm, tetramethylrhodamyl;
Rhm8-PVA, 4-HO-Ph(CH2)2-CO-D-Tyr(Me)-Phe-Gln-Asn-
Arg-Pro-Lys(5-carboxytetramethylrhodamyl]-NH2; D-Tyr(Me),
O-methyl-D-tyrosine; RT-PCR, polymerase chain reaction after
reverse transcription; SEM, standard error of the mean; SI,
selectivity index; TFA, trifluoroacetic acid; TLC, thin-layer chro-
matography; VP, vasopressin; V1a-R, V1b-R, and V2-R, vaso-
pressin receptor isoforms V1a (vascular), V1b (pituitary), and V2
(renal), respectively
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (419) 383-4131. Fax: (419) 383-6228. E-mail: maurice.
manning@utoledo.edu.
Author Contributions
Both authors collaborated equally on this work.
’ ACKNOWLEDGMENT
We thank the Agence Nationale pour la Recherche (ANR) for
Grant ANR09 MNPS031-01. We thank Rao Makineni, Robert
Tyner, and Frederick Paulsen for their generous research support
(to M.M.), and we acknowledge NIH Grant GM-25280 (to M.
M.). M.T. acknowledges financial support from Grant BFU2007-
63022/BFI from the Ministerio de Educacion y Ciencia (Madrid,
Spain), Grant IT-309-07, fellowship from the Departamento de
Educacion, Universidades e Investigacion de Gobierno Vasco
(Vitoria-Gasteiz, Spain), and the Invited Professor fellowship
from the University Montpellier II (France). We thank the
Montpellier RIO Imaging Facility, especially Julien Cau and
Nicole Lautredou. We acknowledge Nathalie Galeotti for the
quality control of the fluorescent peptides throughout this study.
We are grateful to Claudine Serradeil-leGal (Sanofi Synthꢀelabo,
Toulouse, France) for her generous gift of selective non-peptide
vasopressin and oxytocin antagonists. We also thank Anna
’ ADDITIONAL NOTE
Symbols and abbreviations are in accordance with the recom-
mendations of the IUPAC-IUB Commission on Biochemical
Nomenclature (Eur. J. Biochem. 1989, 180, A9ꢀA11) and
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IUPHAR (Trends Pharmacol. Sci. 2001). All amino acids are in
the L-configuration unless otherwise noted.
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