Restriction of a Peptide Turn Conformation and Conformational Analysis of Guanidino Group Using Arginine-Proline Fused Amino Acids: Application to Mini Atrial Natriuretic Peptide on Binding to the Receptor

Article abstract of DOI:10.1021/jm030232j

Compounds (2S,4S)- and (2S,4R)-4-(2′-guanidinoethyl)proline have been synthesized as a conformationally restricted arginine, Their backbones fit the i + 1 position in a turn, and the side chains are restricted compared to that of arginine. These analogues were incorporated into mini atrial natriuretic polypeptide, which has an important turnlike conformation at Gly⁶-Arg⁷-Met⁸-Asp⁹. Structural analysis revealed that the size of the conformational space of Arg⁷ on binding to the receptor was approximately one-third of the entire conformational space.

Full text of DOI:10.1021/jm030232j

J . Med. Chem. 2004, 47, 489-492
489
Brief Articles
Restr iction of a P ep tid e Tu r n Con for m a tion a n d Con for m a tion a l An a lysis of
Gu a n id in o Gr ou p Usin g Ar gin in e-P r olin e F u sed Am in o Acid s: Ap p lica tion to
Min i Atr ia l Na tr iu r etic P ep tid e on Bin d in g to th e Recep tor
Kenji Sugase,† Manabu Horikawa,† Masako Sugiyama,‡ and Masaji Ishiguro*,†
Suntory Institute for Bioorganic Research and Daiichi Suntory Biomedical Research Co., Ltd,
Shimamoto, Mishima 618-8503, J apan
Received May 14, 2003
Compounds (2S,4S)- and (2S,4R)-4-(2′-guanidinoethyl)proline have been synthesized as a
conformationally restricted arginine. Their backbones fit the i + 1 position in a turn, and the
side chains are restricted compared to that of arginine. These analogues were incorporated
into mini atrial natriuretic polypeptide, which has an important turnlike conformation at Gly6-
Arg7-Met8-Asp9. Structural analysis revealed that the size of the conformational space of Arg7
on binding to the receptor was approximately one-third of the entire conformational space.
In tr od u ction
Influence of the absence of the side chain was estimated
by comparing the activity of [Pro7]miniANP to that of
[Ala7]miniANP. However, the activity of [Pro7]miniANP
was lower than that of miniANP (18). Since the side
chain of Arg7 is also important for the activity, we have
focused on the role of the side chain of Arg7 and have
investigated the conformational space of the guanidino
group of Arg7 on binding. [4S-GEPro7]miniANP (19) and
[4R-GEPro7]miniANP (20) were synthesized, and their
conformations were analyzed by NMR and systematic
conformational search. The structural analysis together
with the biological activity indicated that the position
of the side chain of Arg7 is limited to one-third of the
entire conformationally accessible space on binding to
the receptor.
Turn conformations play an important role in peptides
and proteins; they have been implicated in molecular
recognition and in protein folding.1 Proline substitution
has been widely used to search for turns2 because
proline is frequently found at the i +1 position of a
â-turn.3 Proline can be easily incorporated into peptides
via solid-phase synthesis, but functional side chains may
be lost by proline substitution. To alleviate the problem,
several proline analogues have been synthesized that
retain the side chain moieties of R-amino acids.4 How-
ever, detailed structural analysis of the side chain
moieties has not been reported yet, despite of the
importance of their direction on biological activity.
We have synthesized (2S,4S)- and (2S,4R)-4-(2′-
guanidinoethyl)proline (4S-GEPro (1) and 4R-GEPro
(2)) as arginine analogues, aiming at restricting the side
chains as well as the backbone (Scheme 1). These
analogues were used to determine the conformational
space of the guanidino group of mini atrial natriuretic
polypeptide (miniANP) on binding to the receptor.
Resu lts a n d Discu ssion
Syn th esis of Nω-P bf-F m oc-GEP r o-OH (3,4) for
th e Solid -P h a se P ep tid e Syn th esis. The synthesis
of cis-4-substituted proline analogue 3 started with
trans-4-hydroxy-L-proline (5) as illustrated in Scheme
1. Compound 5 was protected by the general procedures
to afford compound 6. Improved Mitsunobu type reac-
tion8 of 6 with phenylsulfonylacetonitrile and cyanom-
ethylenetributylphoshorane in toluene at 100 °C ste-
reospecifically afforded cis-4-substituted proline derivative
7. Removal of the phenylsulfonyl group9 of 7 gave nitrile
8. Reduction of 8 followed by guanylation10 gave guani-
dine 9. Conversion of 9 to Fmoc-amino acid 3 was
accomplished by general procedures,11 and free amino
acid 1 was also obtained by deprotection of 10. Com-
pounds 2 and 4 were synthesized from cis alcohol 12,
which was easily prepared from trans alcohol 6, by the
same procedure as described for the synthesis of 1 and
3 from 6 as illustrated in Scheme 1.
MiniANP (18) (MCHFGGRMDRISCYR-NH2), opti-
mized by monovalent phage display, is the smallest
analogue of ANP-related peptides.5 MiniANP binds
selectively to natriuretic peptide receptor A (NPR-A),
which is a transmembrane protein composed of ap-
proximately 1060 residues,6 causing cGMP synthesis.
Our previous study by proline-scanning mutagenesis
revealed that the receptor-bound conformation of min-
iANP had a turnlike conformation at residues 6-9.7 In
the study, proline substitution for Arg7, corresponding
to the i + 1 position of the turn, resulted in the most
potent activity of all proline-substituted analogues.
P ep tid e Syn tesis. MiniANP (18), [4S-GEPro7]-
miniANP (19), [4R-GEPro7]miniANP (20), [Pro7]miniANP
(21), and [Ala7]miniANP (22) were synthesized using
* To whom correspondence should be addressed. Phone: +81-75-
962-6028. Fax: +81-75-962-2115. E-mail: ishiguro@sunbor.or.jp.
† Suntory Institute for Bioorganic Research.
‡ Daiichi Suntory Biomedical Research Co., Ltd.
10.1021/jm030232j CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/11/2003

490 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Sch em e 1. Synthesis of Protected Amino Acids 3 and 4a
Brief Articles
a
Conditions: (a) CbzCl, 2 N NaOH (aq), room temp, 20 h; (b) Me2NCH(OtBu)2, 80 °C, 6 h, 66% from 5; (c) Mg, HgCl2 (1 mol %), MeOH,
THF, room temp, 2 h, 75% 8 (from 6), 62% 14 (from 12); (d) NaBH4, CoCl2, MeOH, -20 °C, 2 h; (e) N,N′-bis(tert-butoxycarbonyl)-1H-
pyrazole-1-carboxamidine, THF, room temp, 12 h, 61% 9 (from 8), 71% 15 (from 14); (f) TFA, CH2Cl2, 0 °C, 10 h; (g) PbfCl, NaOH (aq),
THF, 0 °C, 10 h, 56% 11 (from 9), 61% 17 (from 15); (h) H2, 10% Pd/C, MeOH, room temp, 2 h; (i) FmocOSu, 10% Na2CO3 solution, DME,
room temp, 12 h, 56% 3 (from 11), 58% 4 (from 17); (j) PDC, MS4A, CH2Cl2, room temp, 2 h, 84%; (k) LiAlH(OtBu), THF, -78 °C, 2 h,
98%, 99% ee; (l) TMSOTf, PhSMe, TFA, 0 °C, 2 h, 49% 1 (from 9), 49% 2 (from 15).
Ta ble 1. Biological Activity of MiniANP and Analogues
relative biological
biological
activitya
EC50 (nmol)
activity
EC50(analogue)/
EC50(miniANP)
peptide
miniANP (18)
3.1 ( 0.5
4.6 ( 0.4
1.0
1.5
0.4
18.7
44.3
[4S-GEPro7]miniANP (19)
[4R-GEPro7]miniANP (20)
[Pro7]miniANP(21)
1.3 ( 0.6
58.1 ( 17.2
137.3 ( 11.3
[Ala7]miniANP(22)
a
The mean values of three independent experiments.
FastMoc chemistry by a solid-phase peptide synthesizer
(model 433A PE Biosystems, Tokyo, J apan). After the
formation of a disulfide bond by DMSO/HCl, they were
purified to homogeneity by reverse-phase HPLC.
Biologica l Activity. The production of cGMP in
Chinese hamster ovary cells expressing NPR-A in
response to peptides 18-22 was measured. Table 1
shows that the biological activity of [Pro7]miniANP (21)
is approximately 19 times lower than that of miniANP
(18), but [4S-GEPro7]miniANP (19) and [4R-GEPro7]-
miniANP (20) are as potent as miniANP (18) despite
the chiral difference of the C4 atom. Therefore, it
appears important that residue 7 supports a â-turn
structure and presents a guanidino group to achieve full
activity.
Solu tion Str u ctu r e. Although the overall conforma-
tion of the analogues 19 and 20 could not be determined
by NMR, in each case the distance constraints were
consistent with and converged well onto a type I â-turn;
this structure was not so clear in the case of miniANP
itself (Figure 1). Heavy atom root-mean-square devia-
tions (rmsd) of the turn region was 0.69 Å for [4S-
GEPro7]miniANP (19) and 0.44 Å for [4R-GEPro7]-
miniANP (20). The stability difference would be due to
van der Waals interaction between the guanidinoethyl
group and the carbonyl group (van der Waals interaction
reaches ∼15 Å, while NOE reaches ∼6 Å); namely, the
guanidinoethyl group and the carbonyl group were
F igu r e 1. Turn conformation of [4S-GEPro7]miniANP (A) and
[4R-GEPro7]miniANP (B) on their mean coordinates. Backbone
heavy atoms in residues 6-9 were used for alignment.
Hydrogens are omitted for clarity.
located on the same side of the proline ring for 4S-
GEPro (1), while they were on the opposite side for 4R-
GEPro (2).
The averaged turn of the analogues 19 and 20
overlapped well (rmsd ) 0.23 Å). Moreover, their HR and
HN chemical shifts including miniANP (18) were in good
agreement with each other within 0.04 ppm except for
residues 5-9. Therefore, the analogues 19 and 20

Brief Articles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 491
In the case of shorter or longer side chains, such as
4-guanidinomethylproline or 4-(3′-guanidino-n-propyl)-
proline, their molecular volumes did not overlap well
with that of arginine (data not shown).
The size of the intersection region of all molecular
volumes (295 Å3) was approximately one-third of that
of arginine (969 Å3). For arginine, 36 conformations
were included inside the intersection region while 97
conformations were searched. At least one of those
conformations would bind to the receptor. The intersec-
tion region contains mainly the equatorial configuration
of the guanidinoethyl groups in 4S- and 4R-GEPro (1,
2) and arginine with an extended side chain. Since those
conformations were calculated to be relatively stable,
the guanidino group in each peptide (18-20) takes the
same arrangement easily.
Con clu sion
This report demonstrates the use of the arginine-
proline fused amino acids to determine the conforma-
tional space where the guanidino group interacts with
the receptor. The backbones of the arginine analogues
fit the turn because of the bent backbone of proline. The
systematic conformational search showed that the side
chains of 4R- and 4S-GEPro are restricted compared to
that of arginine, and the conformational spaces acces-
sible to the guanidino group differed depending on the
chirality of the C4 atom as shown in Figure 2. Since the
biological activity of the analogues (19, 20) was as potent
as that of miniANP (18), it can be concluded that the
receptor-bound conformation of the guanidinoethyl group
would be one of the conformations included in the
intersection region of each conformationally accessible
space.
F igu r e 2. Conformations accepted by the systematic confor-
mational search and molecular volumes of the guanidino
groups of Arg (A and E), 4S-GEPro (B and F), and 4R-GEPro
(C and G) and the intersection region of each volume (D and
H). Parts D and H contain all conformations. (A-D) Front view
of the turn. (E-H) Top view. The red, blue, green, and magenta
areas represent Arg, 4S-GEPro, 4R-GEPro, and the intersec-
tion region, respectively. The conformations are aligned using
backbone atoms. The side chains of Met and Asp are omitted
for clarity.
Analogous proline analogues can also be designed and
synthesized readily. Therefore, the method we proposed
here can be used widely and may shed light on the
structural analysis of ligands containing a turn.
Exp er im en ta l Section
should possess almost the same conformational proper-
ties as for the backbone.
NMR Mea su r em en ts a n d Str u ctu r e Ca lcu la tion s. 2D
DQF-COSY, TOCSY, and NOESY experiments were carried
out on 2.0 mM samples dissolved in 90% H2O/10% D2O using
a Bruker DMX 750 spectrometer. Proton signals were assigned
by the conventional assignment strategy. NOE volumes were
converted into upper-bound distance restraints classified as
strong, medium, weak, or very weak, corresponding to 2.7, 3.7,
5.0, or 6.0 Å, respectively. The lower bound distance restraints
System a tic Con for m a tion a l Sea r ch . The NMR
results above suggest that peptides 18-20 share a
similar backbone structure. Since the analogues 19 and
20 have similar biological activity as well, it seems likely
that their guanidino groups interact with the ANP
receptor in a similar conformational space. The confor-
mational space accessible to the guanidino groups of 4S-
and 4R-GEPro (1, 2) and arginine were mapped out
systematically and compared. It should be noted that
the receptor-bound conformation of residues 6-9 could
be different from the typical type I â-turn used in the
conformational search. However, our previous study
indicated that miniANP (18) has a turnlike conforma-
tion on binding12 and proline has a strong propensity
to form a â-turn as shown by the solution structures.
Therefore, the difference, if any, would be only to a
slight degree.
3
were set to 1.8 Å. J HN,Ha coupling constants were measured
from a 1D spectrum and the DQF-COSY spectrum and were
converted into dihedral angle restraints. An amide proton
temperature coefficient (dsHN/dT), whose value greater than
-4.6 ppb/K is a good indicator of a hydrogen bond, was
analyzed by measuring 1D NMR spectra at 15, 25, 35, 45, and
55 °C. Structure calculations were carried out using torsion-
angle molecular dynamics with the program XPLOR-NIH. A
total of 100 structures were generated, and the 10 lowest
energy structures were submitted to subsequent analysis.
System a tic Con for m a tion a l Sea r ch . Conformational
spaces accessible to the guanidinoethyl group at a turn were
searched for systematically with the program Search Compare
(Accelrys, San Diego, CA). To simplify the search, only a type
I â-turn (Gly-Pro-Met-Asp) was used. First, (4S)- and (4R)-
methylproline were constructed and the proline ring conforma-
tions were searched, during which the N1-C2 bond was
cleaved, allowing other bonds to rotate freely. While C3-C4
and C4-C5 bonds were rotated 360° every 10°, C2-C3 and C5-
N1 bonds were automatically rotated by the algorithm. The
Figure 2 shows the conformations accepted by the
search together with molecular volumes. Since the
potential energy of the conformations in the binding site
could not be estimated, all conformations were used in
the analysis. The volume of arginine can encompass
almost the total volumes of 4S- and 4R-GEPro (1, 2).

492 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Brief Articles
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J M030232J