Conformations and receptor activity of desmopressin analogues, which contain γ-turn mimetics or a ψ[CH2O] isostere

Article abstract of DOI:10.1021/jm011073b

Three analogues of the antidiuretic drug desmopressin ([1-desamino,8-D-arginine]vasopressin) have been prepared. In two of these, γ-turn mimetics based on a morpholin-3-one framework have been inserted instead of residues Phe3-Asn5, whereas the third anal

Full text of DOI:10.1021/jm011073b

J . Med. Chem. 2002, 45, 2501-2511
2501
Con for m a tion s a n d Recep tor Activity of Desm op r essin An a logu es, Wh ich
Con ta in γ-Tu r n Mim etics or a Ψ[CH
2
O] Isoster e
†
†
†,‡
§
§
Mattias Hedenstr o¨ m, ZhongQing Yuan, Kay Brickmann, J olanta Carlsson, Kjell Ekholm,
§
§
§
†
,†
Birgitta J ohansson, Eva Kreutz, Anders Nilsson, Ingmar Sethson, and J an Kihlberg*
Organic Chemistry, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden,
and Ferring AB, P.O. Box 30047, SE-20061 Limhamn, Sweden
Received October 26, 2001
Three analogues of the antidiuretic drug desmopressin ([1-desamino,8-D-arginine]vasopressin)
have been prepared. In two of these, γ-turn mimetics based on a morpholin-3-one framework
have been inserted instead of residues Phe3-Asn5, whereas the third analogue has a methylene
ether isostere in place of the amide bond between residues 3 and 4. The three analogues were
used to probe if the structure determined for desmopressin in aqueous solution, which contains
an inverse γ-turn centered around Gln4, is important in interactions with the vasopressin V
2
receptor. Conformational studies revealed that the analogues that contain either an inverse
γ-turn mimetic or a methylene ether isostere mimicked the conformation of desmopressin fairly
well and very well, respectively. Despite this, the analogues displayed only very low agonistic
activities at the vasopressin V
Phe3-Asn5 does not appear to be important when desmopressin is bound to the V
In addition, it was concluded that the amide bond between Phe3 and Gln4 in desmopressin is
crucial for interactions with the antidiuretic V receptor.
2
receptor. Consequently, an inverse γ-turn involving residues
2
receptor.
2
In tr od u ction
The neurohypophyseal peptide hormone vasopressin1
(
1, Figure 1) interacts with several members of the
2
G-protein-coupled receptor family. Activity at the V2
receptor is responsible for the antidiuretic properties of
vasopressin, whereas interactions with the V1a and V1b
receptors control blood pressure, as well as some other
properties. Deamination of Cys1 and simultaneous
replacment of L-Arg8 with D-Arg in 1 yields the potent
antidiuretic drug desmopressin 2.3 Desmopressin does
not cause vasoconstriction and contraction of smooth
muscles in the uterus or in the intestines as the original
peptide hormone does and is used in treatment of
diabetes insipidus, haemophilia A, von Willebrand’s
disease, and thrombocyte dysfunction prior to sur-
gery.3
,4
,5,6
F igu r e 1. Structures of the peptide hormone vasopressin and
The use of peptides as drugs is often limited by their
poor pharmacokinetic properties, namely, low uptake
on oral administration, rapid enzymatic degradation,
and facile excretion.7 In addition, flexibility of the
peptide may reduce the biological activity and the
receptor selectivity. To circumvent these problems, large
efforts have been made to design and synthesize pep-
tidomemetics, which have different structures than the
parent peptides but nevertheless act as ligands to the
the drug desmopressin. Differences are indicated in bold.
arrangement. It is also an advantage if the synthetic
route is short and efficient.
The conformation of desmopressin when bound to a
G-protein-coupled receptor is not known, but the struc-
ture of desmopressin alone has been studied by two-
dimensional nuclear magnetic resonance (NMR) spec-
troscopy in aqueous solution with or without addition
8
-12
same receptor.
Several requirements should be
1
3,14
of trifluoroethanol.
In addition, the structure of
fulfilled by peptidomimetics. The topographical relation-
ship between the functional groups of the mimetic
should resemble those of the peptide, and it is essential
that functionalities corresponding to side chains can be
introduced at desired positions with correct spatial
desmopressin when bound to the carrier protein neu-
rophysin has been determined by NMR spectroscopy.15
A large number of similar conformational studies have
also been performed for vasopressin and several of its
1
6-21
other analogues.
In general, all of these studies
reveal that the macrocyclic ring of desmopressin and
vasopressin adopts â- or γ-turn structures, with a highly
flexible acyclic tail.
In aqueous solution, and at a pH close to physiological
conditions, desmopressin (2) was found to contain a
*
To whom correspondence should be addressed. Tel.: +46-90-786
6
8 90. Fax: +46-90-13 88 85. E-mail: jan.kihlberg@chem.umu.se.
†
Umeå University.
Present address: Medicinal Chemistry, AstraZeneca R&D M o¨ ln-
‡
dal, SE-431 83 M o¨ lndal, Sweden.
§
Ferring AB.
1
0.1021/jm011073b CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/04/2002

2
502 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Hedenstr o¨ m et al.
Sch em e 1. Three Analogues of Desmopressin (4, 6, and 8) Prepared by Solid Phase Synthesis
Ta ble 1. In Vivo Antidiuretic Activity of Vasopressin and
Analogues
stable inverse γ-turn centered around Gln4.14 Interest-
ingly, such a γ-turn was also predicted when vaso-
b
peptide
na
agonist activity (IU/mg)
pressin was docked into a three-dimensional computer
model of the V1a receptor.22 Formation of a hydrogen
vasopressin (1)
561
desmopressin (2)
1000
645
bond between the CO of Phe3 and the NH of Asn5,
which generates a γ-turn (Figure 1), is thus conforma-
tionally favorable in desmopressin and may be compat-
ible with receptor binding. To probe if a Phe3-Asn5
γ-turn in desmopressin is important in interactions with
the V2 receptor, we have prepared three analogues of
desmopressin, two of which contain mimetics of γ-turns
in place of residues Phe3-Asn5. The antidiuretic activi-
ties of the analogues were determined, and their solu-
4
[Ala ]desmopressin
-3
4
6
8
5
6
7
<2 × 10
2.6 × 10-3 ((3.7 × 10-4)
-
2
0.56 ((7.2 × 10
)
a
Number of experiments. b The antidiuretic activity was cal-
culated using a vasopressin house standard (561 IU/mg) calibrated
against an international vasopressin standard. Values are mean
((SEM).
2
with a methylene bridge. Consequently, the seven-
1
tion structures were investigated by H NMR spectros-
membered, hydrogen-bonded γ-turn in 2 has been
replaced by a six-membered morpholin-3-one ring.
copy.
26
This reduces the flexibility of the peptide backbone and
should prevent enzymatic degradation after Phe3, a
position which is otherwise susceptible to cleavage by
Resu lts a n d Discu ssion
Design a n d Syn th esis. Analogues 4, 6, and 8
2
7
(Scheme 1) were designed to investigate if the inverse
chymotrypsin. Ab initio calculations have shown that
morpholin-3-ones constitute excellent mimetics of either
inverse or classical γ-turns, depending on the choice of
stereochemistry at C-6 of the morpholinone ring.26 In
4, the i + 1 side chain is located in a pseudoequatorial
position, as in the inverse γ-turn found in desmopressin
in aqueous solution, whereas the i + 1 side chain in 6
is in a pseudoaxial orientation, just as in a classical
γ-turn. In desmopressin analogue 8, the amide bond
between residues 3 and 4 is instead replaced by a
methylene ether isostere. J ust as for 4 and 6, this
prevents enzymatic cleavage after Phe3, but the lack
of conformational restraints should allow 8 to adopt
other conformations than mimetics 4 and 6.
γ-turn found for residues Phe3-Asn5 in aqueous solu-
tions of desmopressin is essential also in interactions
with G-protein-coupled receptors. To simplify the syn-
thesis of these analogues, Gln at position 4 of desmo-
pressin was replaced by Ala. This replacement still
allows structure-activity comparisons of desmopressin
with the three analogues since a Gln4fAla substitution
in desmopressin has only a minor effect on the activity
at the kidney V2 receptor (cf. Table 1).2
3,24
In addition,
it is most unlikely that the secondary structure of
desmopressin is influenced by a Gln4fAla substitution
since Gln4 can be replaced by such a structurally
different residue as a glycosylated serine without af-
fecting the conformation.25
In analogues 4 and 6, the amide bond between
residues i and i + 1 of the turn in 2 has been exchanged
for a Ψ[CH2O] isostere, simultaneously with replace-
ment of the hydrogen bond between residues i and i +
γ-Turn mimetics 3 and 5 were prepared as described
2
6
earlier. Synthesis of the methylene ether isostere 7
started from commercially available (2S)-phenylalaninol
(9, Scheme 2). Initial attempts to prepare analogues of
7, in which the amino group was protected with either

Conformations and Activity of Desmopressin Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2503
Sch em e 2a
phase synthesis (Scheme 1). This was performed on a
polystyrene resin grafted with poly(ethylene glycol)
chains (TentaGel S NH2) functionalized with the Rink
3
3,34
R
amide linker.
N -Fmoc amino acids carrying stan-
dard side chain protective groups were coupled to the
3
5
resin as benzotriazolyl (HOBt) esters, whereas mi-
a
metics 3, 5, and 7 were activated as the more reactive
Reagents: (a) TfN3, DMAP, CuSO4, CH2Cl2, room tempera-
3
6
ture, 2 h; 68%. (b) R-(-)-2-Chloropropionic acid, NaH (50% oil
suspension), 1,4-dioxane, room temperature, 20 h; 48%.
7-azabenzotriazolyl (HOAt) esters. When the mimetics
had been attached to the solid phase, the azido group
was reduced by treatment with tin(II) chloride in the
a tert-butoxycarbonyl (Boc) or a 9-fluorenylmethoxycar-
bonyl (Fmoc) group, by different routes were hampered
by low overall yields and formation of impure products.
Because azides can be conveniently reduced to amines
2
9
presence of thiophenol and triethylamine. The progress
of the reduction was conveniently monitored by the
disappearance of the N3 stretch in the IR spectrum
2
8
3
7
obtained from a few resin beads. After cleavage from
the solid phase and side chain deprotection by treatment
with trifluoroacetic acid, disulfide bond formation was
affected by oxidation with iodine in methanol.25 Finally,
purification by reversed-phase high-performance liquid
chromatography (HPLC) gave desmopressin analogues
on solid phase, the azido group was instead chosen as a
protective group for 7.26,29 Conversion of 9 to azido
alcohol 10 was achieved in a copper-catalyzed diazo
transfer reaction using freshly prepared triflic azide.
Base-promoted O-alkylation of 10 to give 7 was carried
out with both (2R)-2-chloro- and (2R)-2-bromopropionic
acid to establish if the choice of leaving group influenced
3
0,31
2
6
4
,
6, and 8, each in ∼20% overall yield. The structures
of the three desmopressin analogues were confirmed by
mass and NMR spectroscopy (cf. Tables 2, 3, and 5).
the amount of epimerization of the propionic acid
moiety.32 In this case, use of 2-chloropropionic acid
together with sodium hydride as base gave 7 in 48%
P h a r m a cologica l Stu d ies. The antidiuretic effect
of desmopressin analogues 4, 6, and 8 at the V2 receptor
was determined after intravenous administration to
anaesthetized rats (Table 1). Analogues 4 and 6, which
contain γ-turn mimetics, were both more than 5 orders
of magnitude less active than vasopressin and desmo-
pressin. The methylene ether isostere 8, which lacks the
conformational restriction imposed by the γ-turn mi-
yield and excellent diastereoselectivity (97:3) according
1
to H NMR spectroscopy. When 2-bromopropionic acid
was used under identical conditions, 7 was obtained in
6
8% yield but as a 4:1 diastereomeric mixture, which
was most difficult to separate.
Compounds 3, 5, and 7 were then used as building
blocks to prepare 4, 6, and 8, respectively, using solid
Ta ble 2. Temperature Coefficients (∆δ/∆T, ppb/K) and 1H and 13C NMR Chemical Shifts (δ, ppm) for Peptide 4 in Aqueous Solutiona
residue
Mpr1
∆δ/∆Tb
NH
CRH
2.61c
CâH
CγH
other
2.82, 3.30
3
5
5
5.6
4.13
9.1
4.26
2.7
36.2
2.41c
37.9
2.88, 2.95
38.2
Tyr2
Phe3
-6.7
-6.7
8.32
7.73
6.72(Hδ), 6.66(Hꢀ)
131.0(Cδ), 117.1(Cꢀ)
7.26(Hδ), 7.31(Hꢀ), 7.20(Hú)
131.3(Cδ), 130.5(Cꢀ), 128.7(Cú)
methylene bridge
Ala4
3.91(H1), 3.69(H2), 3.26(H2′), 76.0(C1), 52.4(C2)
4.28
6.3
4.27
1.1
4.64
7.1
4.37
2.5
4.19
5.7
3.81, 3.86
3.4
1.43c
18.2
2.65, 2.90
39.8
2.86, 3.13
37.9
1.83, 2.24
31.0
1.70, 1.85
29.1
7
6
5
6
5
4
Asn5
Cys6
Pro7
6.91 and 7.72(NδH2)
-7.8
8.12
1.99c
26.4
3.56 and 3.83(Hδ)
49.7(Cδ)
D-Arg8
Gly9
-11.0
-4.7
8.89
8.33
1.58c
26.2
3.12 (Hδ), 7.23(NꢀH)
c
42.1(Cδ)
7.18 and 7.32(CONH2)
a
b
Obtained at 600 MHz in water containing 10% D2O at 278 K and pH 6.7 with H2O (δH 4.98 ppm) as internal standard. The amide
2
c
proton coefficients were all linear with R > 0.99. Degeneracy has been assumed.
Ta ble 3. 1H NMR Chemical Shifts (δ, ppm) for Peptide 6 in Aqueous Solutiona
residue
Mpr1
Tyr2
Phe3
NH
CRH
CâH
CγH
other
2.92b
4.17
4.47
4.40
2.47, 2.55
2.47, 2.51
2.68, 2.98
1.34b
8.22
7.67
6.74(Hδ), 6.65(Hꢀ)
7.28(Hδ), 7.35(Hꢀ), 7.25(Hú)
Ala4
methylene bridge
Asn5
Cys6
Pro7
D-Arg8
Gly9
4.19(H1), 3.40(H2), 3.46(H2′)
6.99 and 7.74(NδH2)
5.36
5.03
4.41
4.26
2.75, 2.92
2.76, 3.27
1.88, 2.27
1.71, 1.87
8.69
2.00b
1.60b
3.64, 3.75(Hδ)
b
8.90
8.46
3.16 (Hδ), 7.28(NꢀH)
b
3.85
7.20 and 7.42(CONH2)
a
Obtained at 600 MHz in water containing 10% D2O at 278 K and pH 6.7 with H2O (δH 4.98 ppm) as internal standard. b Degeneracy
has been assumed.

2
504 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Hedenstr o¨ m et al.
Ta ble 4. Hydrogen Bonds and Their Relative Frequency in the
Accepted Structures of Peptide 4
the other hand, the chemical shifts of the three residue
acyclic tail had only small deviations from random coil
values, which is an indication of considerable flexibility.
A total of 74 distance restraints were derived from the
NOESY spectrum. These restraints consisted of 33
intraresidual, 29 sequential, 11 medium, and 1 long-
range connectivity. When determining distance re-
straints, C6 and C5 in the morpholinone ring of the
γ-turn mimetic were considered as part of Phe3, and
the NOE between Mpr1 HR and Cys6 NH was regarded
a
b
c
c
donor
D-Arg8 HN Cys6 O
Gly9 HN Cys6 O
acceptor angle (deg) dist. (Å) A (%) B (%)
135
154
1.8
2.5
100
13
100
29
a
The donor-hydrogen-acceptor angle criterion was 120-180°.
b
The hydrogen-acceptor distance criterion was e3.0 Å. c Struc-
ture families A and B, respectively.
metics, had a substantially higher antidiuretic effect
than 4 and 6 but was still 3 orders of magnitude less
potent than 1 and 2. However, a dose-response curve
was obtained for both analogues 6 and 8, for doses below
3
as a long-range connectivity. All J NHR coupling con-
stants, except that of Phe3, are consistent with confor-
mational averaging and were therefore not utilized in
0
.2 mg/rat, revealing that they were able to bind to the
40
3
the structure calculations. For Phe3, J NHR was found
to be 9.6 Hz, which was converted to a φ dihedral angle
of -109 ( 30°. Because of spectral overlap and degener-
receptor and elicit a message. It was also found that
the three analogues did not show any activity as
vasopressin receptor antagonists when evaluated in vivo
in rats. The low activity at the V2 receptor displayed by
3
ated chemical shifts of the â-protons, J Râ could only be
determined for Phe3, Cys6, and D-Arg8. Both Phe3 and
4
, 6, and 8 can be due to the structural modifications of
3
Cys6, but not D-Arg8, had a J Râ that indicated popula-
the analogues as compared to desmopressin or to an
influence from these modifications on the conforma-
tional features of the analogues. To differentiate be-
tween these possibilities, it was decided to investigate
tion of one preferred rotamer about the CR-Câ bond,
1
and the ø torsion angles for these two residues were
determined to be -50 ( 30 and -70 ( 30°, respectively.
Because all amide proton temperature coefficients were
more negative than -4.7 ppb/K, no indications of
intramolecular hydrogen bonds or steric shielding of
protons from solvent that could be used as restraints
were found for 4.
1
the conformation(s) of analogues 4 and 8 by using H
NMR spectroscopy. Because an inverse γ-turn was
found for desmopressin in aqueous solution, analogue
4
, which contains a mimetic of an inverse γ-turn, was
selected for the NMR study instead of 6, which contains
a mimetic of a classical γ-turn.
The 74 NOE-derived distance restraints and the three
dihedral angle restraints mentioned above were used
to determine starting structures for 4 with the program
1
Str u ctu r e Deter m in a tion of An a logu e 4. The H
resonances of desmopressin analogue 4 were assigned
4
1
by using the conventional strategy38 from a set of COSY,
X-PLOR. These structures were then subjected to
simulated annealing using an ab initio protocol followed
TOCSY, NOESY, and ROESY spectra obtained in
4
2,43
3
9
by simulated annealing refinement.
Somewhat sur-
aqueous solutions at 5 °C and pH 6.7 (Table 2). The
proton resonances were then used in combination with
prisingly, none of the resulting refined structures
fulfilled the acceptance criterion of no NOE distance
violations >0.3 Å. Such a result could be explained if 4
is flexible so that it can assume two or more conforma-
tions that are in fast exchange on the NMR time scale.
This would result in time-averaged NOEs that, due to
gradient-enhanced 13C-HSQC spectra to identify the
13
C
resonances. Large deviations from random coil chemical
shifts of backbone NH and HR in the macrocyclic ring
(residues 1-6), even for those residues not involved in
the γ-turn mimetic inserted at positions 3-5, indicated
a well-ordered structure in this part of 4. This observa-
tion was further supported by the presence of several
medium range nuclear Overhauser effects (NOEs). On
-
6
the r dependence of the NOE intensities, will over-
emphasize the restraints that represent short dis-
4
4
tances. As a consequence, incompatibilities in the
Ta ble 5. Temperature Coefficients (∆δ/∆T, ppb/K) and 1H and 13C NMR Chemical Shifts (δ, ppm) for Peptide 8 in Aqueous Solutiona
residue
Mpr1
∆δ/∆Tb
NH
CRH
CâH
CγH
other
2.57
2.86, 3.15
3
6.1
4.23
8.4
35.4
2.59, 2.64
37.4
2.60, 3.29
Tyr2
-7.3
-1.5
8.35
7.19
6.74(Hδ), 6.68(Hꢀ)
132.1(Cδ), 117.2(Cꢀ)
7.27(Hδ), 7.37(Hꢀ), 7.29(Hú)
5
5
Phey[CH2O]
4.22
3
.29 and 3.42(CH2O)
131.3(Cδ), 130.7(Cꢀ), 128.8(Cú)
2.9(CH2O)
2.0
38.6
1.33c
7
Ala4
3.95
9.2
4.82
1.4
5.02
2.3
4.44
2.6
7
5
5
6
5
4
20.3
2.70, 2.75
37.7
2.68, 3.18
40.1
1.9, 2.28
31.3
1.73, 1.89
29.5
Asn5
Cys6
Pro7
-5.6
-6.2
8.38
8.65
6.96 and 7.74(NδH2)
2.01c
27.6
3.67 and 3.79(Hδ)
49.8(Cδ)
D-Arg8
Gly9
-9.1
-5.3
8.91
8.48
4.26
5.7
1.60c
26.2
3.16 (Hδ)
c
42.3(Cδ)
3.86c
4.1
7.20 and 7.43(CONH2)
a
b
Obtained at 600 MHz in water containing 10% D2O at 278 K and pH 6.7 with H2O (δH 4.98 ppm) as internal standard. The amide
2
c
proton coefficients were all linear with R > 0.99. Degeneracy has been assumed.

Conformations and Activity of Desmopressin Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2505
temperature (Figure 2) and becomes substantially nar-
rower when the temperature is raised. The fact that only
Asn5 HR displays significant line broadening, although
many other atoms also experience different environ-
ments in the two structure families, is due to the large
chemical shift difference for Asn5 HR, as revealed by
4
5
Gaussian98 calculations for structure families A and
B. Accordingly, the exchange process is close to coales-
cence for Asn5 HR but in the fast exchange region for
the other protons, which experience smaller shift dif-
ferences upon exchange. However, the observed broad-
ening of the Asn5 HR resonance implies that the mean
lifetime of each conformer is in the millisecond time
range. Unfortunately, this relatively slow exchange rate
prevented simulations of the exchange process by time-
averaged NOE simulations or unrestrained molecular
dynamics (MD) calculations, since it would have re-
quired prohibitively long simulations to sample the
transitions between the conformations.
The structures belonging to each of families A and B
differ substantially from each other, especially for the
residues found in the macrocyclic ring of 4 (Figure 3).
In structure family A, the side chain of Asn5 points
toward the center of the macrocyclic ring and the
morpholinone ring in the turn mimetic is located in the
same plane as the macroyclic ring. In family B, Asn5
points away from the macrocyclic ring whereas the
morpholinone ring is perpendicular to the plane of the
macrocycle. As a consequence, the backbone of residues
F igu r e 2. Amide region of the NOESY spectrum of peptide 4
in H O/D O (9:1) at 278 K and pH 6.7. The boxed cross-peak
2 2
is between Cys6 NH and Asn5 HR. The line broadening of Asn5
HR is easily distinguished.
NOE-derived distance restraints may occur if they
represent short distances from different conformations.
A closer inspection of the structures calculated for 4
revealed that they could be divided into two families
with complementary NOE violations. That is, the dis-
tance restraint violations found in one structure family
were not found in the other and vice versa. Subse-
quently, it was possible to derive two sets of distance
restraints, each of which was used to calculate two
structure families, A and B, that did not show any NOE
violations. It appears likely that this conformational
exchange process accounts for the large difference in the
temperature coefficient of Phe3 NH in 4 (-6.7 ppb/K)
as compared to in desmopressin (-2.7 ppb/K). For
desmopressin, shielding from the surrounding aqueous
solution was proposed to explain the small value of the
temperature coefficient of Phe3 NH.14 In mimetic 4, this
shielding appears to be decreased due to the conforma-
tional exchange process between families A and B.
Further experimental support for the presence of such
a conformational exchange is provided by the observa-
tion that the resonance of Asn5 HR is very broad at low
1
-6 for the structures in family B assumes a saddlelike
fold, which provides a completely different location for
the side chain of Ala4 than in family A. In both structure
families, the side chains of residues Tyr2 and Phe3
adopt similar orientations with respect to the macro-
cyclic ring and the morpholinone ring retains its half-
2
6
chair conformation, with the C-2 methyl group that
constitutes the side chain of Ala4 in a pseudoequatorial
orientation. The average backbone rmsd from the mean
structure of residues 1-6 in the accepted structures of
4 is 0.34 and 0.21 Å for families A and B, respectively,
indicating a well-ordered structure for each family,
although a certain flexibility can be seen for the disul-
fide bridge. The rmsd of all heavy atoms in the macro-
cyclic ring is 0.71 and 0.63 Å for families A and B,
respectively. A hydrogen bond between D-Arg8 NH and
Cys6 O appears in both structure families (Table 4) and
F igu r e 3. Superimposition of 20 structures, randomly chosen from those calculated for families A (left) and B (right) of peptide
4
.

2
506 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Hedenstr o¨ m et al.
Ta ble 6. Hydrogen Bonds and Their Relative Frequency in the
Accepted Structures of Peptide 8
anglea
(deg)
dist.b
(Å)
relative
frequency (%)
donor
acceptor
Cys6 HN
D-Arg8 HN
Gly9 HN
Gly9 HN
Ala4 O
Cys6 O
Cys6 O
Pro7 O
135
134
148
138
2.0
2.0
2.6
2.3
91
36
50
9
a
The donor-hydrogen-acceptor angle criterion was 120-180°.
The hydrogen-acceptor distance criterion was e3.0 Å.
b
macrocyclic ring, whereas Asn5 points toward the center
of the macrocycle. The average backbone rmsd from the
mean structure of residues 1-6 in 8 was 0.29 Å,
whereas the rmsd of the heavy atoms of these residues
was 0.56 Å; both values indicated a well-ordered struc-
ture. As expected, the three residue tail is quite flexible
but some conclusions can be made with regard to its
structural preferences. The same hydrogen bonds were
found in 8 as in 4, i.e., between D-Arg8 NH and Cys6 O
and between Gly9 NH and Cys6 O (Table 6). The latter
hydrogen bond is, however, more frequent, suggesting
that the tail has some preference for adopting a â-turn.
In addition, a hydrogen bond was found between Cys6
NH and Ala4 O in almost all accepted structures, a fact
that can explain the large difference in chemical shift
of Cys6 NH as compared to 4.
F igu r e 4. Superimposition of 20 structures, randomly chosen
from those calculated for peptide 8.
a γ-turn is thus formed over residues 6-8.46 In some of
the accepted structures, a hydrogen bond is also found
between Gly9 NH and Cys6 O. This hydrogen bond
pattern describes a three residue tail that is quite well-
defined for the first residue, Pro7, but which then
becomes increasingly more flexible toward the C-ter-
minal Gly9. The similar structure and flexibility of the
tail in both structure families indicate that the tail does
not participate in the conformational exchange involving
the macrocyclic ring. One might speculate that the
energy barrier between the two structure families is low
since the hydrogen bonds in the tail do not have to be
broken to allow the observed conformational exchange.
Com p a r ison of Str u ctu r e a n d Activity for An a -
logu es 4 a n d 8 w ith Desm op r essin . Previously, the
structure of 2 has been determined by 2D NMR spec-
1
14
Str u ctu r e Deter m in a tion of An a logu e 8. The H
troscopy both in aqueous solution and in aqueous
1
3
13,47
and C resonances of 8 were assigned in the same
manner as for desmopressin analogue 4 (Table 5). As
in the case of 4, large deviations from random coil
chemical shifts were observed for the residues in the
macrocyclic ring, whereas only minor deviations were
seen for the three residue tail. Because of some unfor-
tunate spectral overlap, only 55 distance restraints (32
intraresidue, 20 sequential, 2 medium, and 1 long-
range) derived from the ROESY spectrum could be used
in the structure calculations using a simulated anneal-
trifluoroethanol.
In aqueous solution, an inverse
γ-turn centered around Gln4 and stabilized by a hydro-
gen bond between Phe3 CO and Asn5 NH was found.
In aqueous trifluoroethanol, a â-turn was observed with
Gln4 and Asn5 in the i + 1 and i + 2 positions. NMR
studies of a complex between desmopressin and the
carrier protein neurophysin II, which may serve as a
model for interactions between 2 and protein receptors,
revealed the same â-turn at Gln4 and Asn5 as found in
1
5
aqueous trifluoroethanol. In this complex, the three
residue tail of 2 provides important contacts with
neurophysin II and an additional â-turn is found for
residues Cys6-Gly9. Modeling of the complex between
vasopressin (1) and the rat V1a recpetor, based on data
from site-directed mutagenesis, suggested that 1 was
bound in a cleft between the transmembrane helices so
3
ing protocol. All of the measured J NHR coupling con-
stants were within 5.5-6.7 Hz and thus in the region
corresponding to conformational averaging, whereas
3
spectral overlap prevented determination of any J Râ
coupling constants. Hence, no φ dihedral angle re-
straints were used in the structure calculations. The
small value of the temperature coefficient for Phe3 NH
in 8 (∆δ/∆T -1.5 ppb/K, Table 5) coincides well with
that found for desmopressin (-2.7 ppb/K).1 Because
this small ∆δ/∆T value for desmopressin is most likely
due to shielding from the solvent rather than hydrogen
bonding, the Phe3 NH temperature coefficient was not
used as a restraint in the structure calculations for 8.
The similarity in the environment for Phe3 NH in 8 and
desmopressin was confirmed by the structure calcula-
tions described below. After refinement, the structural
calculations for 8 resulted in 88 accepted structures out
of the 100 that were calculated initially.
2
2
that residues Phe3-Asn5 formed a γ-turn, in a similar
way as found for 2 in aqueous solution. It should be
stressed that structural studies of desmopressin in
solution, when bound by neurophysin II or when mod-
eled in complex with a G-protein-coupled receptor,
might not reflect binding of desmopressin to the antidi-
4
uretic V receptor. Despite this, these structural studies
2
reveal that 2 has a preference to adopt either γ- or
â-turn structures for residues in the macrocylic ring and
that the conformation is influenced by the surrounding
environment.
Superimposition of the lowest energy structure de-
termined in aqueous solution for 2, with the minimum
energy structures in each of families A and B of
analogue 4, revealed that the inverse γ-turn mimetic
had some influence on the conformation of 4 (Figure 5).
The rmsd for the backbone atoms of residues 1-6 in
the superimposition was 1.31 and 1.80 Å for families A
In contrast to 4, no indications of a conformational
exchange process were found for 8; instead, the back-
bone of residues 1-6 adopts one well-defined conforma-
tion (Figure 4). This closely resembles the structures
belonging to family A of 4 and has the side chains of
residues Tyr2, Phe3, and Ala4 directed away from the

Conformations and Activity of Desmopressin Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2507
F igu r e 5. Comparison of the lowest energy structure found in aqueous solution for desmopressin14 with the lowest energy
structures from families A (left) and B (right) of 4. Desmopressin is represented in green, and the lowest energy structures from
families A and B are shown in blue and purple, respectively. The backbone atoms (N, CR, and CO) of residues 1-6 were used for
the superimposition and the rmsd calculations.
and B, respectively. Thus, the backbone of family A has
a better resemblance with desmopressin than family B,
especially for the part of the backbone belonging to the
γ-turn mimetic. In both structure families, the side
chains of residues Tyr2 and Phe3 are positioned only
slightly differently than in desmopressin. In contrast,
the side chain of Asn5, which is critical for the antidi-
uretic activity,48 points away from the macrocyclic ring
in family B whereas it lies over the macrocycle both in
desmopressin and in family A of 4. The three residue
tail is flexible both in 4 and in desmopressin, but
because the same hydrogen bonds are observed in this
region for both compounds, the conformation of the tail
appears to be unaffected by the inverse γ-turn mimetic.
Even though the covalently linked backbone of the
inverse γ-turn mimetic makes 4 more rigid than des-
mopressin, structural family A mimics the structure of
desmopressin fairly well. It therefore appears that
conformational influences from the inverse γ-turn mi-
metic in 4 are, on their own, insufficient to explain the
very low antidiuretic effect found for 4 (Table 1).
Instead, it seems that the covalent modifications in the
mimetic part of 4, i.e., the methylene ether isostere or
the methylene bridge, may be responsible for the loss
of antidiuretic activity; this was a hypothesis that was
probed further with desmopressin analogue 8.
Analogue 8 has a very good conformational resem-
blance to desmopressin, as revealed by comparison of
the lowest energy structures of 8 and desmopressin
Figure 6). In this case, the rmsd for the backbone atoms
F igu r e 6. Comparison of the lowest energy strutures found
in aqueous solution for desmopressin14 and peptide 8. Desmo-
pressin is represented in green, and 8 is represented in red.
The backbone atoms (N, CR, and CO) of residues 1-6 were
used for the superimposition and the rmsd calculations.
(
of residues 1-6 in the superimposition is only 0.66 Å.
The methylene ether isostere in 8 does not allow
formation of the hydrogen bond between Phe3 CO and
Asn5 NH, which is found in the inverse γ-turn of
desmopressin.1 Despite this, the backbone conformation
of an inverse γ-turn is well-conserved in 8. In addition,
the positions of the side chains of Tyr2, Phe3, and Asn5
deviate only slightly from those of desmopressin. The
close similarity between the conformations adopted by
role in interactions with the antidiuretic V2 receptor.
We interpret our data as suggesting that this amide
bond is involved in hydrogen bonding with the G-
protein-coupled receptor. Lack of an amide bond be-
tween residues 3 and 4 also explains the loss of
bioactivity displayed by analogues 4 and 6 (Table 1),
which contain γ-turn mimetics instead of residues
Phe3-Asn5. The fact that 4 and 6 are approximately 2
orders of magnitude less potent than 8 may be because
4
8
and desmopressin reveals that the low biological
activity of 8 is most certainly due to the amide bond
between Phe3 and Gln4 in desmopressin has a crucial

2
508 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Hedenstr o¨ m et al.
the γ-turn mimetic makes the rest of the macrocyclic
ring in 4 and 6 more rigid and/or the methylene bridge
provides additional hindrance for receptor binding. It
is also possible that analogue 8, just as desmopressin,
is able to adopt γ- or â-turn conformations in the
macrocyclic ring depending on the environment, thereby
explaining the increased potency of 8 as compared to 4
and 6. Finally, the data presented in this paper
suggest that desmopressin does not bind to the V2
receptor with residues Phe3-Asn5 oriented as a γ-turn.
Con clu sion s. Two analogues of desmopressin (4 and
(2S)-2-Azid o-3-p h en ylp r op a n ol (10). First, triflic azide
(
TfN
triflic anhydride (1.35 mL, 8.01 mmol) to a mixture of NaN
2.60 g, 40 mmol) in H O (6 mL) and CH Cl (10 mL) at 0 °C.
, caution31) was prepared by addition of freshly distilled
3
3
(
2
2
2
The mixture was stirred vigorously for 2 h at 0 °C, and the
phases were separated. The water phase was extracted two
times with a small amount of CH
organic layers were washed with saturated aqueous NaHCO
Then, 9 (0.287 g, 1.90 mmol), 4-(dimethylamino)pyridine (0.126
g, 1.03 mmol), and CuSO (18 mg) were dissolved in CH Cl
2 mL). The TfN solution (10 mL) was added dropwise, and
2 2
Cl , and the combined
3
.
4
9
4
2
2
(
3
while it was stirred for 2 h at room temperature, the color
changed from blue to green. The solution was washed twice
with 10% aqueous citric acid, twice with saturated aqueous
3
6
), in which γ-turn mimetics were inserted instead of
residues Phe3-Asn5, have been prepared to probe the
interactions between desmopressin and the V2 G-protein-
coupled receptor. An analogue (8) having a methylene
ether isostere in place of the amide bond between
residues 3 and 4 was also synthesized to further aid the
structure-activity studies. Conformational studies based
on 2D NMR spectroscopy revealed that one of the two
structural families found for analogue 4 (which contains
a mimetic of an inverse γ-turn) mimicked the lowest
energy conformation determined for desmopressin in
aqueous solution fairly well. Moreover, the minimum
energy conformation found for analogue 8 resembled
that of desmopressin very well. Analogues 4 and 6 were
both more than 5 orders of magnitude less active as
antidiuretics than desmopressin when administered
intravenously to rats, whereas analogue 8 was more
active than 4 and 6 but still 3 orders of magnitude less
active than desmopressin. The good conformational
resemblance between the analogues and desmopressin,
together with the low antidiuretic activity displayed by
the analogues, revealed that the amide bond between
Phe3 and Gln4 in desmopressin has a crucial role in
interactions with the antidiuretic V2 receptor. Most
likely, this amide bond is involved in hydrogen bonding
with the G-protein-coupled receptor. In addition, this
investigation does not support the presence of an inverse
γ-turn involving residues Phe3-Asn5 when desmo-
pressin is bound to the V2 receptor.
NaHCO , and twice with brine. The organic layer was dried
over MgSO
4
, filtered, and concentrated. Flash column cro-
matography (heptane-EtOAc, 2:1) of the residue gave 10
2
0
(
0.229 g, 68%) as a colorless oil; [R]
D
2 2
) -1.9° (c 1.0, CH Cl ).
1
H NMR: δ 7.38-7.25 (m, 5H, Ph), 3.72 (m, 2 H, CH
CHN ), 3.57 (m, 1 H, CH
H, PhCH ), 2.84 (dd, J ) 13.6 and 6.1 Hz, 1 H, PhCH
2
O and
3
2
O), 2.89 (dd, J ) 13.6 and 6.1 Hz, 1
2
2
), 2.62
(br. s, 1 H, OH). 13C NMR: δ 137.2, 129.4 (2 C), 128.8 (2 C),
127.1, 65.4, 64.5, 37.1. IR (neat): n ) 3345 (OH), 2108 (N
HR FABMS calcd for C O (M + H), 178.0980; found,
78.0981.
2S)-2-[(2S)-2-Azido-3-p h en ylpr opyloxy]p r op ion ic Acid
7). (2R)-2-Chloropropionic acid (125 mg, 1.15 mmol) followed
3
).
9
11 3
H N
1
(
(
by NaH (50% in mineral oil, 610 mg, 12.7 mmol) was added
to 10 (89 mg, 0.50 mmol) in dry 1,4-dioxane (25 mL) at room
temperature. After it was stirred for 20 h, H O (8 mL) was
2
added to destroy the excess of NaH. The resulting aqueous
phase was washed with heptane and then acidified to pH 2-3
with aqueous HCl (2 M). The aqueous phase was extracted
with CH
dried over Na
chromatography (heptane-EtOAc-AcOH, 80:16:4) of the resi-
2
Cl
2
, and the organic phase was washed with water,
2
SO , filtered, and concentrated. Flash column
4
20
due gave 7 (60 mg, 48%) as a colorless oil; [R]
D
) -13.3° (c
). H NMR: δ ) 10.11 (br. s, 1 H, COOH), 7.40-
7.20 (m, 5 H, Ph), 4.05 (q, J ) 6.9 Hz, 1 H, CHCH ), 3.83 (m,
1
1
2 2
.0, CH Cl
3
2 H, CH
CH O), 2.77 (dd, J ) 13.5 and 8.0 Hz, 1 H, PhCH
J ) 13.5 and 5.8 Hz, 1 H, PhCH ), 1.52 (d, J ) 7.0 Hz, 3 H,
). C NMR: δ 178.7, 137.1, 129.3 (2 C), 128.7 (2 C), 127.0,
), 1722 (Cd
(M + H), 250.1192; found,
2
O and CHN
3
), 3.41 (dd, J ) 10.3 and 8.0 Hz, 1 H,
2
2
), 2.68 (dd,
2
1
3
CH
3
7
5.4, 72.5, 62.8, 37.2, 18.5. IR (neat): ν ) 2121 (N
3
O). HR FABMS calcd for C12
250.1192.
15 3 3
H N O
Solid P h a se Syn th esis of P ep tid es 6 a n d 8. Peptides 6
and 8 were synthesized on solid phase using the Fmoc strategy
with DMF as solvent in a mechanically agitated reactor.
ArgoGel-RINK (0.67 mmol/g, 388 mg, 260 mmol) was used
Exp er im en ta l Section
Gen er a l. The reactions in the synthesis of 7 were carried
out under an inert atmosphere with dry solvents under
anhydrous conditions. CH Cl and 1,4-dioxane were dried by
2 2
distillation from calcium hydride and sodium benzophenone,
respectively. Dimethylformamide (DMF) was distilled before
use in solid phase peptide synthesis. Thin-layer chromatog-
raphy (TLC) was performed on Silica Gel 60 F254 (Merck) with
detection by UV light and charring with phosphomolybdic acid
in EtOH (26 mM). Flash column chromatography (eluents
given in brackets) was performed on silica gel (Matrex, 60 Å,
as solid support for 6, whereas TentaGel S NH
2
(0.27 mmol/g,
3
3,34
2
96 mg, 80 mmol) functionalized with the Rink linker
was
R
used for 8. N -Fmoc amino acids with side chain protecting
groups were used as follows: 2,2,5,7,8-pentamethylchroman-
6
-sulfonyl (Pmc) for D-Arg; triphenylmethyl (Trt) for Asn, Cys,
and 3-mercaptopropionic acid; and tert-butyl (tBu) for Tyr.
R
The Rink linker, N -Fmoc amino acids, and mercaptopro-
pionic acid were coupled to the resin after activation as HOBt
esters.35 These were prepared in situ by treatment of the
appropiate acid (4 equiv) with 1-hydroxybenzotriazole (HOBt;
6 equiv) and 1,3-diisopropylcarbodiimide (DIC; 3.9 equiv) in
freshly distilled DMF (1-1.5 mL) during 30-60 min. Building
blocks 5 (40 mg, 65 mmol, 0.25 equiv) and 7 (32 mg, 130 mmol,
3
5-70 mm, Grace Amicon).
The H and 13C NMR spectra of compounds 7 and 10 were
1
recorded at 400 and 100 MHz, respectively, on a Bruker DRX-
4
7
00 spectrometer for solutions in CDCl
3
[residual CHCl
3
(δ
H
.26 ppm) or CDCl (δ 77.0 ppm) as internal standard] at 298
3
C
3
6
K. IR spectra were recorded on an ATI Mattson Genesis Series
FTIR spectrometer. Optical rotations were measured with a
Perkin-Elmer 343 polarimeter. Positive fast atom bombard-
ment mass spectra (FABMS) were recorded on a J EOL SX102
A mass spectrometer. Ions for FABMS were produced by a
beam of Xenon atoms (6 keV) from a matrix of glycerol and
thioglycerol. In the amino acid analysis, asparagine was
determined as aspartic acid.
1.6 equiv) were activated with 1-hydroxy-7-azabenzotriazole
(HOAt; 0.38 equiv for 5, 2.5 equiv for 7) and DIC (0.3 equiv
for 5, 1.5 equiv for 7) in DMF (1 mL). Couplings were
performed during 1-2 h and monitored by using bromophenol
blue50 (0.05% of the resin capacity) as indicator of unacylated
R
amino groups on the peptide resin. N -Fmoc deprotection was
achieved by a slow flow of 20% piperidine in DMF through
the reactor (4 min), followed by addition of the piperidine
solution to the reactor and rotation during 7 min. After each
deprotection, the resin was washed with DMF. After incorpo-
ration of building block 5, which was coupled to an excess of
2
6
26
Peptide 4 and compound 5 were prepared as described
in the cited reference. (2S)-2-Amino-3-phenylpropanol (9) was
purchased from Aldrich (U.S.A.).

Conformations and Activity of Desmopressin Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2509
resin, the remaining nonacylated amino groups on the peptide
resin were capped by treatment with acetic anhydride. The
two different occasions. When the peptide under investigation
had a low activity, only one dose of the peptide was given. One
injection of saline was also given to each animal.
The antagonistic activity was determined by giving each
animal three doses of 1 thereby creating a dose-response
curve. A dose of the peptide under investigation was given 10
min prior to a dose of 1, chosen in the linear part of the dose-
response curve.
resin was then washed with CH
2
Cl
2
(5 × 2 mL) and dried in
vacuo. Incorporation of 5 and 7 was confirmed by IR spectros-
3
7
copy on a few resin beads, which showed an N stretch at
3
-
1
29
2
119 cm . Reduction of the azido group was achieved by
sequential addition of triethylamine (280 mL, 2 mmol),
thiophenol (165 mL, 1.6 mmol), and anhydrous SnCl (75.8
mg, 0.4 mmol) to the resin suspended in tetrahydrofuran (THF;
2
P r ep a r a tion of NMR Sa m p les. Desmopressin analogue
4 (4.4 mg) was dissolved in phosphate buffer (20 mM, pH 6.7,
1
2
mL). After 20 h, the resin was washed with THF (8 × 2 mL),
0% piperidine in DMF (2 mL, 5 min), and CH
Cl
2 2
(8 × 2 mL)
550 mL), which contained 10% D
2
O to give an ∼8 mM solution.
and dried in vacuo. The disappearance of the N
3
stretch in
Another aqueous sample of 4, having a concentration of ∼20
mM, was preperad in the same manner and used for the
NOESY experiment. No aggregation of 4 was detected at these
concentrations. Analogue 6 was dissolved in the same buffer
as 4 to give an ∼10 mM sample. Analogue 8 (5 mg) was
dissolved in phosphate buffer (40 mM, pH 6.6, 550 mL), which
the IR spectrum indicated a successful reduction. The resin
was then suspended in DMF (1 mL), and the synthesis was
continued as described above.
After completion of the synthesis, the resin was washed with
CH
2
Cl
2
(8 × 2 mL) and dried in vacuo. Each of the peptides
were cleaved from a portion of the resins, and the amino acid
side chains were deprotected, by treatment with trifluoroacetic
acid/water/thioanisole/ethanedithiol (87.5:5:5:2.5) for 2 h fol-
lowed by filtration. Acetic acid was added to the filtrate, the
solution was concentrated, and acetic acid (3 × 5 mL) was
added again followed by concentration after each addition. The
residue was triturated with diethyl ether. The diethyl ether
solution was decanted, and the solid, crude peptide was
dissolved in acetic acid/water (1:5) and freeze-dried. At this
stage, the precursor of peptide 6 was purified with reversed-
phase HPLC prior to oxidation in order to remove the capped
contained 10% D
buffers contained 0.3 mM NaN
2
O to give an ∼9 mM solution. The phosphate
3
(0.3 mM) to prevent bacterial
growth. In all samples, the pH was adjusted to the original
value of the buffer by addition of small aliquots of solutions of
aqueous 0.1 M NaOH or HCl.
5
2
53
54
NMR Exp er im en ts. DQF-COSY, TOCSY, NOESY,
ROESY,55 and gradient-enhanced H- C-HSQC experi-
ments were recorded on a 600 MHz Bruker DRX instrument
at 5 °C for peptides, 4, 6, and 8. Temperature gradient
experiments were conducted with a 500 MHz Bruker AMX
spectrometer. The water resonance at 4.98 ppm was used as
an internal shift reference. NOESY spectra for 4 and 6 were
recorded with a mixing time of 200 ms. The ROESY spectrum
for 8 was recorded with a spin-lock field strength of ∼4.5 kHz
1
13
56
2
5
tetrapeptide. Cyclization was performed by alternating ad-
ditions of portions of the crude peptide in acetic acid and I in
methanol (0.1 M) to 10% acetic acid in methanol (2 mL/mg
cleaved resin). After the final addition of I , a light brown
2
5
7
and a mixing time of 150 ms. A DIPSI-2 mixing sequence
2
solution was obtained, which was neutralized and decolorized
was used in the TOCSY experiments with a spin-lock time of
1
13
by stirring with Dowex 2 × 8 anion exchange resin (Fluka,
86 ms. COSY, TOCSY, and H- C-HSQC experiments were
5
0-100 mesh, converted from Cl- to acetate form by washing
recorded with 2048 complex data points in t2 and 512 incre-
with 1 M aqueous NaOH, water, acetic acid, water, and
methanol), filtrated, and concentrated. The peptide was then
dissolved in water and freeze-dried.
ments in t
matrix of 2 K × 1 K complex data points. NOESY and ROESY
spectra were recorded with 2048 complex data points in t and
00 increments in t . Zero-filling and linear prediction up to 1
1 1
. Linear prediction in t resulted in a final data
2
8
1
Peptides 6 and 8 were purified on a Beckman System Gold
HPLC using a Kromasil C-8 column (100 Å, 5 µm, 20 mm ×
K resulted in a final data matrix of 4 K × 2 K. Apodization
with a shifted square sine bell function was performed in both
dimensions prior to Fourier transformation in all experiments
except COSY where a sine bell function was used. WATER-
GATE58 was used for water suppression in the ROESY,
NOESY, and TOCSY experiments. Amide proton temperature
2
50 mm) and a gradient of 0-80% of B in A over 60 min
(solvent systems: A, 0.1% aqueous trifluoroacetic acid, and
B, 0.1% trifluoroacetic acid in acetonitrile, flow rate: 11 mL/
min) with detection at 214 nm. This gave peptides 6 (11.3 mg,
1
7% based on the amount of 5 employed) and 8 (11 mg, 23%
based on the amount of resin employed).
Peptide 6 had FABMS calcd for C45
H); found, 1011. Amino acid analysis: Arg 0.99 (1), Cys 1.00
1
coefficients were determined from a series of H NMR spectra
acquired at temperatures ranging from 5 to 40 °C with
presaturation during the relaxation delay. The amide proton
shifts were adjusted with regard to the temperature depen-
dence of the water signal (-11.9 ppb/K).59 All processing was
63 12 11 2
H N O S 1011 (M +
1
(1), Gly 1.02 (1), Pro 1.00 (1), Tyr 0.98 (1). H NMR data are
given in Table 3.
2
performed in XWINNMR (Bruker) on a SGI O workstation.
Peptide 8 had FABMS calcd for C44
H); found, 999. Amino acid analysis: Arg 0.99 (1), Asp 1.02
1), Cys 0.96 (1), Gly 1.02 (1), Pro 1.02 (1), Tyr 0.98 (1). H
63 12 11 2
H N O S 999 (M +
Assign m en t a n d Str u ctu r e Deter m in a tion . Proton reso-
nance assignment for peptides 4, 6, and 8 was obtained using
1
(
38
the conventional strategy. Initial spin system recognition was
NMR data are given in Table 5.
Agon istic a n d An ta gon istic P r op er ties of P ep tid es 4,
obtained from the COSY and TOCSY spectra. Sequential
assignment could subsequently be made with use of the
NOESY or ROESY spectra. The complete proton resonance
assignment was used together with the gradient-enhanced
6
, a n d 8 a t th e Va sop r essin Recep tor . Vasopressin (1) was
used as control and agonist (561.3 IU/mg) and stock solutions
of 1, 4, 6, and 8 were made in 0.9% NaCl. Female Sprague-
Dawley rats (184-221 g) were anaesthetized i.p. with Inactin
in the afternoon before each study. A tracheostomy was
performed, and catheters were introduced in the jugular vein
and in the urethra. Hydration with a hypoosmotic solution was
started immediately after surgery had been completed. In the
morning of the study, the urethral catheters were connected
to a conductometer connected to an amplifier and a computer
for registration of urine conductivity, which was used as read-
out for the antidiuretic response. The compound investigated
was administered in 0.9% NaCl (0.2 mL) through the catheter
in the jugular vein. Each experiment was started by giving
the animal a “reactivity dose” of 1.
1
13
13
H- C-HSQC spectra to assign the C resonances.
Peak volumes from assigned cross-peaks in the NOESY (for
4
) and ROESY (for 8) spectra were converted to interproton
distances, dij, by using the isolated spin pair approximization
1
/6
ref
dij ) dref(V /Vij)
in which dref corresponds to a known interproton distance. Vref
and V are the volumes of the reference cross-peak and peak
ij
ij, respectively. The mean value of the cross-peak volumes from
the geminal â-methylene protons in Phe3 and Cys6 was used
as Vref for 4. Because of overlap, only the cross-peak from the
â-methylene protons in Phe3 could be used to determine Vref
for 8. The reference distance dref was set to 1.77 Å for both 4
and 8. In the structure calculations, the lower distance limit
for dij was set to 1.80 Å and the upper distance limit was set
The agonistic, i.e., the antidiuretic, activity was then
5
1
determined as described previously. Each animal received
two doses of 1 and two doses of the peptide under investigation
to give a 2 × 2 assay, and each peptide was investigated at

2
510 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Hedenstr o¨ m et al.
to 10% above the determined distance. Methyl, methylene, and
aromatic ring protons with degenerate shifts were treated as
pseudoatoms. Upper distance limits for distance restraints
involving these pseudoatoms were increased by 1.0, 0.9, and
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(
6
0
1
.2 Å, respectively.
NOESY peak picking and cross-peak volume integration for
4
were performed in XWINNMR. J NHR was measured from the
line widths in the NOESY spectrum according to the method
6
1
of Wang et al.
J Râ was determined by simulating the observed
6
2
COSY cross-peaks with the programs SPHINX and LINSHA.
Coupling constants were converted into dihedral angles using
3
the Karplus equation. Those values for J Râ that were compat-
ible with the NOEs were chosen as dihedral angle restraints
for the structure calculations. Peak picking and ROESY cross-
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6
3
peak integrations for 8 were performed in FELIX, which was
3
3
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derived from two-dimensional NMR spectroscopy and molecular
dynamics simulation. Eur. J . Biochem. 1991, 201, 355-371.
also used to extract J HNR. Because of spectral overlap, no J Râ
was estimated.
Three-dimensional starting structures were determined
4
1
from NMR data with the program X-PLOR. Structures
generated within X-PLOR were subjected to simulated an-
nealing using an ab initio simulated annealing protocol
4
2,43
followed by simulated annealing refinement.
Two starting
structures with different conformations were used to sample
more of the conformational space. The initial temperature in
the annealing protocol was set to 1000 K, and the final
temperature was 100 K. All structures were minimized with
a 200 step Powell minimization after refinment. A soft square-
(
18) Shenderovich, M. D.; Kasprzykowski, F.; Liwo, A.; Sekacis, I.;
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Sar7, Arg8]vasopressin by H NMR spectroscopy and molecular
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welled potential with a force constant of 50 kcal mol-
1
Å
-2
was
538.
(
19) Zieger, G.; Andreae, F.; Sterk, H. Assignment of proton NMR
resonances and conformational analysis of [Lys8]-vasopressin
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20) Yu, C.; Yang, T.-H.; Yeh, C.-J .; Chuang, L.-C. Combined use of
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used for the NOE distance restraints, and a square well
-
1
-2
potential with a force constant of 1 kcal mol rad was used
for the dihedral angle restraints. In all calculations, only
structures with no distance restraint violations larger than
(
0
5
.3 Å and no dihedral angle restraint violations larger than
° were accepted. All structure calculations were performed
on SGI O
2
workstations. Root mean square deviation values
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1
triglycyllysylvasopressin: H NMR spectroscopic and molecular
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dynamics study. Magn. Reson. Chem. 1993, 31, 481-488.
(
22) Mouillac, B.; Chini, B.; Balestre, M.-N.; Elands, J .; Trumpp-
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Ack n ow led gm en t. This work was funded by grants
from the Swedish Research Council, the Biotechnology
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Foundation for Research in Natural Sciences and Medi-
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