Conformational properties of 4-mercaptoproline and related derivatives

Article abstract of DOI:10.1002/anie.200704310

(Chemical Equation Presented) Spot the difference: Conformational analysis of the 2S,4R and 2S,4S epimers of N-acetyl-4-mercaptopyrrolidine-2-carboxylic acid methyl esters reveals ring-pucker preferences that are opposite of those of the hydroxyproline de

Full text of DOI:10.1002/anie.200704310

Angewandte
Chemie
DOI: 10.1002/anie.200704310
Proline Conformations
Conformational Properties of 4-Mercaptoproline and Related
Derivatives**
Sergio A. Cadamuro, Rudolf Reichold, Ulrike Kusebauch, Hans-Jürgen Musiol,
Christian Renner, Paul Tavan, and Luis Moroder*
Proline residues have long been recognized to play a unique
and important role in the structural properties of peptides and
proteins. The cis/trans isomerization of the aminoacyl–proline
bonds is critically involved in folding and stabilizing protein
structures.[1] This general notion has inspired an intensive
search for proline analogues that influence the equilibrium
conformational populations of the cis/trans prolyl bonds and
of the pyrrolidine ring-pucker isomers (endo/exo of C4) in
order to possibly restrict at will the degree of conformational
freedom of polypeptide chains and thus to modulate the
thermodynamic stability of peptide and protein structures.[2]
Of the various modified prolines found in nature, the most
common are (2S,4R)-hydroxyproline (Hyp) and (2S,3S)-Hyp,
which are generated in post-translational processes exclu-
sively in Y and X positions of the collagen (Xaa-Yaa-Gly)
repeats, respectively, with the enzymatic 4R hydroxylation
being by far the dominant modification.[3] The stereoelec-
tronic effects of this electronegative substituent at C4 or C3 of
the pyrrolidine ring have been the subject of comparative
analysis, particularly in synthetic model compounds with the
fluorine substituent, in terms of (de)stabilization of the
collagen triple helix.[2a,h,i,k,4] These studies on collagen model
peptides have been extended to other proteins by exploiting
the strong effects of 4-fluoroprolines (Flp).[2g,5]
the less electronegative thiol group results in altered con-
formational preferences: The 4R epimer of Mpc induces a Cg-
endo pucker while (4R)-Hyp and similar 4R substitutions
generate the exo pucker (Scheme 1). This may have interest-
Scheme 1. Conformational equilibria of Ac-(4R/S)-Mpc-OMe (1) and
Ac-(4R/S)-Hyp-OMe (5).
ing structural implications for the design of peptides and
proteins, particularly when the thiol group is exploited for
thioether or disulfide intra- and interchain cross-bridging of
polypeptide chains.
To evaluate the effect of 4-mercap-
to substitution on the pyrrolidine ring
conformation the epimeric Ac-(2S,4R/
S)-Mpc-OMe derivatives 1 were syn-
thesized (Figure 1). In addition, to
Rather surprisingly, the non-natural synthetic 3- and 4-
mercaptopyrrolidine-2-carboxylic acids (Mpc),[6] chalcogen
analogues of hydroxyprolines, have been used only sporadi-
cally for side chain/side chain cyclization of peptides through
thioether or disulfide bridges in attempts to restrict the
conformational space of peptidic macrocycles.[7] Herein we
report a structural comparison of the (2S,4R)- and (2S,4S)-
Mpc epimers, in which replacement of the hydroxy group with
mimic the effect of side-chain bridging
of such Mpc residues by thioethers or
disulfides in peptides, the related alkyl
and alkylthio epimer pairs Ac-(2S,4R/
Figure 1. Chemical
structure of N-acetyl-
proline methyl ester
(4, X=Y=H) and var-
ious derivatives, in
which either X or Y is
replaced by SH (1),
SMe (2), SSMe (3),
OH (5), or F (6).
S)-Mpc(Me)-OMe (2) and Ac-(2S,4R/
S)-Mpc(SMe)-OMe (3) were pre-
pared, and their conformational pref-
erences were compared with those of
the known Ac-Pro-OMe (4), Ac-
(2S,4R/S)-Hyp-OMe (5), and Ac-
(2S,4R/S)-Flp-OMe (6) by NMR structural analysis in aque-
ous solution. In the model compounds the known effect of pH
on the isomerization of Xaa–Pro bonds was prevented by the
N and C derivatization.[8] Similarly, complications arising
from hydrogen bonding in amide derivatives,[9] although weak
in aqueous environments, are suppressed with C-terminal
esters.
trans/cis Equilibrium constants (Kt,c) and predominant
ring puckerings were extracted from NMR spectral data to
estimate the stereoelectronic effect of the thiol group on the
conformational preference of the prolyl bond and on the ring
puckering. These values are reported in Table 1 and com-
pared to those known for the 4-hydroxy- and 4-fluoroproline
epimer pairs. The equilibrium constants show that the 4R/S
thiol group has a much weaker effect on the trans/cis
[*] Dr. S. A. Cadamuro, Dr. U. Kusebauch, H.-J. Musiol,
Priv.-Doz. Dr. C. Renner,[+] Prof. Dr. L. Moroder
Max-Planck-Institut für Biochemie
Am Klopferspitz 18, 82152 Martinsried (Germany)
Fax: (+49)89-8578-2847
E-mail: moroder@biochem.mpg.de
Homepage: http://www.biochem.mpg.de/en/rg/moroder/
R. Reichold, Prof. Dr. P. Tavan
Lehrstuhl für Biomolekulare Optik
Ludwig-Maximilians-Universität
Oettingenstrasse 67, 80538 München (Germany)
[+] Current address: Deutsche Forschungsgemeinschaft
Kennedyallee 40, 53170 Bonn/Bad-Godesberg (Germany)
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(SFB533, A8, and C3).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2008, 47, 2143 –2146
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Communications
Table 1: Thermodynamic parameters and conformational preferences of
compounds 1–6.
ÀDH0[b]
DS0[b]
[a]
Cmpd
Kt/c
Dominant
conformation
[kJmolÀ1
]
[JmolÀ1 KÀ1
]
1(4R)
1(4S)
2(4R)
2(4S)
3(4R)
3(4S)
4[c]
5.4
4.7
4.1
3.1
4.3
3.6
4.8
6.1
2.4
7.3
2.6
trans, Cg-endo
trans, Cg-exo
trans, Cg-endo
trans, Cg-exo
trans, Cg-endo
trans, Cg-exo
trans, Cg-endo
trans, Cg-exo
trans, Cg-endo
trans, Cg-exo
trans, Cg-endo
4.84Æ0.8%
3.30Æ1.0%
1.93Æ4.8%
4.99Æ2.5%
4.88Æ2.6%
1.62Æ4.6%
5.04Æ1.0%
7.84Æ1.0%
n.a.[e]
À2.19Æ5.9%
1.89Æ5.4%
2.86Æ2.8%
À5.08Æ5.0%
À4.32Æ4.3%
5.25Æ5.2%
À3.82Æ4.3%
À10.7Æ1.0%
n.a.[e]
5(4R)[d]
5(4S)[d]
6(4R)[c]
6(4S)[c]
7.73Æ3.3%
À9.81Æ8.3%
3.04Æ1.1%
À2.47Æ4.3%
[a] Determined by integration of well-resolved signals in the 1H NMR
spectra in D2O at 298 K. [b] The enthalpy (DH0) and entropy (DS0)
contributions to the free energy difference between the trans and cis
conformers were derived from van’t Hoff plots (see Figure 1 in the
Supporting Information); error limits were obtained from the residuals
of the linear least-squares fitting. [c] Values from Ref. [2g]. [d] Values
from Ref. [2k]. [e] Not available.
Figure 2. Optimized geometries of the l-proline derivatives as trans
g
À
amide conformers: The strength and direction of the X C bond
dipoles m are indicated by arrows. The atomic partial charges are
indicated by a color code ranging from red (negative) through white
(neutral) to blue (positive); adjacent red and blue atoms represent
dipoles. The spheres represent “compound atoms”; the charges of
hydrogen atoms are added to those of the neighboring heavy atoms.
The experimentally observed conformers are marked by boxes.
conformational preference than the significantly more elec-
tronegative hydroxy and fluorine substituents. Indeed the Kt,c
values of 1(4R) and 1(4S) differ only slightly from that of
unsubstituted proline. However, the ring-pucker preferences
were reversed between the ring-substituted Ac-Pro-OMe
species with similar anti (1(4R), 5(4R), and 6(4R)) and syn
(1(4S), 5(4S), and 6(4S)) orientation of the electronegative
substituent relative to the fixed l configuration of the Ca
atom. Thus, an anti orientation of the 4-substituent resulted in
a predominant Cg-endo pucker for Mpc and in the known
predominant Cg-exo pucker for Hyp and Flp, whereas the
opposite was true for the respective syn-oriented species.
The transformation of the thiol group into a methyl-
thioether or methyldisulfide shifted the trans/cis equilibrium
of the prolyl bond toward the cis conformation with the effect
of the thioether being milder than that of the methyl disulfide.
The ring-pucker preference was not affected by this type of
derivatizations of the thiol group.
Quantum chemical calculations using density functional
theory (DFT) were used to explain the experimental data
obtained for compound 1 and 4–6. In the case of Mpc (1) and
Hyp (5) derivatives, potential curves were calculated for the
dihedral angles w, y, and a (Figure 1). The gas-phase energies
and molecular geometries obtained by our DFT descriptions
of 4, 6(4R), and 6(4S) (see Tables II and III in the Supporting
Information) agree very well with the results of previous DFT
calculations,[2h] confirming our theoretical approach. The
populations of the different conformers derived from NMR
experimental data and those computed from DFT results are
congruent with few exceptions (see Table IV in the
Supporting Information). Most importantly, the calculations
predict the initially unexpected but experimentally observed
preference of 1(4R) for the Cg-endo pucker and of 1(4S) for
the Cg-exo pucker.
site and the dipole of the preceding amide bond. Figure 2
shows how the combination of (4R)-Hyp or (4R)-Flp with a
Cg-endo pucker leads to an unfavorable antiparallel orienta-
tion of these dipoles, while the Cg-exo pucker results in close
to perpendicular dipole moments with an almost neutral
contribution to the total energy. This results in the Cg-exo
pucker being more favorable for the (4R)-Hyp and the (4R)-
Flp derivatives. In contrast, the dipole moment at the
substitution site of Mpc is very weakand causes almost no
energetic penalty for the 4R-endo combination. The exper-
imentally observed preference of Mpc for the 4R-endo
geometric variant implies that the net contributions of all
interactions (electrostatic, van der Waals, bond geometries,
etc.) except the decisive dipole–dipole interaction discussed
before are slightly in favor of the Cg-endo pucker for the (4R)-
Mpc derivative 1. Therefore, the preference of the (4R)-Hyp
(5) and (4R)-Flp (6) derivatives for the Cg-exo pucker is
explained by a reduced unfavorable dipole–dipole orientation
rather than a specific favorable interaction. One would expect
that other 4R or 4S substitutions that introduce small dipole
moments will also prefer the 4R-endo and 4S-exo combina-
tions that had been hitherto considered unfavorable and
unusual, unless steric effects prevail over electrostatic inter-
action as it is the case in the pair of 4-methylproline
epimers.[2n,9]
In our preceding synthetic efforts to control the folding/
unfolding of a collagen triple helix by applying light we
introduced two (4S)-Mpc residues, as the synthetically more
readily accessible epimer, into the Ac-(Gly-Pro-Hyp)7-Gly-
Gly-NH2 model collagen peptide for side chain/side chain
cross-bridging of the two thiol groups with a suitable thiol-
reactive azobenzene derivative (Figure 3).[11] Optimal loca-
tions for the Mpc residues according to modeling experiments
are the i and i + 7 positions corresponding to Xaa and Yaa
residues of the classical (Xaa-Yaa-Gly) collagen triplets.
High-resolution X-ray analysis of collagen model peptides[12]
The DFT calculations reveal that the pucker preference of
a given substitution at the 4-position is mainly determined by
the interaction between the bond dipole at the substitution
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peptide containing one Gly-Pro-Pro repeat,[4k] should be fully
compensated by the favored Cg-exo pucker of the (4S)-Mpc.
Similarly, like a Pro residue in X position combined with Hyp
in Y, (4S)-Mpc should marginally affect the triple-helical fold
despite its favored Cg-exo pucker. The rather strong exper-
imentally observed destabilization must therefore be assigned
mainly to steric effects, fully supporting a strong interplay
between stereoelectronic and steric effects in the assembly of
collagen triple helices.
Despite the limitations of the predictive power of the
simple Ac-Pro-OMe system for values in protein environ-
ments, the results of this study offer a more general view of
the relation between 4-substitutions of proline and resulting
conformational properties of the amide bond and, especially,
of the proline ring pucker. The increased understanding of the
determinants of proline geometry together with the decisive
role of proline residues and related analogues in peptide and
protein structures can provide a powerful tool in the design
and folding studies of polypeptides. In contrast, their appli-
cation in proteins must await improved methodologies for an
efficient incorporation of such non-natural amino acids into
expressed proteins unless synthetic and semisynthetic ligation
strategies suffice for the purpose.
Figure 3. Thermal unfolding of the triple-helical Ac-(Gly-Pro-Hyp)7-Gly-
*
Gly-NH2 ( ) (Tm =438C) and of its analogue Ac-(GPO)2-(G-(4S)-Mpc-
O)-(GPO)-(GP-(4S)-MPC)-(GPO)2-GG-NH2 with (4S)-Mpc residues
located in the X and Y positions (&) (Tm =34.58C). The unfolding was
monitored by CD at 225 nm in aqueous solution at a peptide
concentration of 1 mm; O=(4R)-hydroxyproline.
Experimental Section
and computational analysis[13] revealed alternate Cg-endo and
Cg-exo puckers of the Pro and Hyp residues in the X and Y
positions of the triplets as a repetitive motif possibly involved
in stabilizing the triple helix. Extensive comparative studies of
synthetic collagen peptides containing (4R)-Hyp or (4S)-Hyp,
and (4R)- or (4S)-Flp have shown that occupancy of the X and
Y positions with proline analogues characterized by prefer-
ences for Cg-endo and Cg-exo puckers, respectively, leads to
markedly increased thermal stabilities of the triple helix,
while the opposite effect is induced with reversed ring
puckerings.[2h,i,k,4a,b,12] However, more recently, in X-ray
structures of [Gly-(4R)-Hyp-(4R)-Hyp]n peptides both Hyp
residues assume exo conformations.[4g,j] In view of the still-
evolving understanding of the mechanism of triple-helix
stabilization and the weakelectron-withdrawing property of
the thiol group we assumed that replacement of a Pro and
Hyp residue in Ac-(Gly-Pro-Hyp)7-Gly-Gly-NH2 with (4S)-
Mpc should affect only marginally the triple-helix stability. In
contrast, a rather substantial decrease in the thermal stability
was observed as shown in Figure 3.
Taking into account that Pro and/or Hyp replacements in
single triplets of (Gly-Pro-Hyp)n peptides, that is, in host–
guest peptides, lead to results that are significantly different
than those from repetitive replacements,[4k] a rational inter-
pretation of the drop of the Tm value by about 88C can be put
forward. The stereoelectronic effects of substituted prolines
in the (Gly-Pro-Hyp) collagen repeats are not additive in
terms of triple-helix (de)stabilization,[4i] but the steric effects
seem to be so.[2n] By the single substitution of a Hyp residue in
Y with (4S)-Mpc the trans peptide bond is less favored and
similar to that of a Pro residue (Table 1). This negative effect,
which leads to a 28C lower Tm in a (Gly-Pro-Hyp)8 host
Details of the synthesis of Ac-(4R/S)-Mpc-OMe (1), Ac-(4R/S)-
Mpc(Me)-OMe (2), and Ac-(4R/S)-Mpc(SMe)-OMe (3) are reported
in Supporting Information. Solutions in D2O were used for NMR
measurements, and in the case of Ac-Mpc-OMe tris-(2-carboxyethyl)-
phosphine hydrochloride (1 equiv) was added to prevent oxidation.
NMR experiments were performed with a Bruker DRX500 spec-
trometer using a triple-resonance (15N/13C/1H) inverse probe. Assign-
ment of 1H and 13C NMR resonances was based on homonuclear 2D
1H-1H NOESY and TOCSY experiments and heteronuclear 2D 13C-
1H COSY experiments.[15]
Thermodynamics and kinetics of amide-bond isomerization:
Equilibrium constants (Kt/c) for the trans/cis conformer ratios at
various temperatures were determined by integration of the signals of
the a and g protons in 1D 1H NMR spectra. The enthalpic and
entropic contributions to the free energy difference between the cis
and trans conformers were obtained from vanꢀt Hoff plots according
to ln(KZE) = (ÀDH0/R)(1/T) + DS0/R.
NMR conformation analysis: The pucker of the proline ring was
identified by the method of Gerig and McLeod[16] by means of the
distinct pattern of the 1H–1H coupling constants observed in 1D
1H NMR spectra; for example, the Cg-exo pucker results in large and
similar coupling constants J(Ha,Hb1) and J(Ha,Hb2), whereas for the
Cg-endo pucker one of the coupling constants is large and the other
small. The cis-to-trans isomerization of the amide bond is a slow
process on the NMR time scale, and therefore two distinct signals are
observed for the two conformers. On the other hand, the pucker
inversion is a fast process, and therefore the signal obtained for the
trans conformer is an averaging of the trans, Cg-endo and trans, Cg-exo
pucker; the same is true for the cis conformer. An estimation of the
ratio of the two puckerings for the trans and cis conformers was
obtained by the equation DJexp = xDJendo + yDJexo where DJ refers to
the difference between J(Ha,Hb1) and J(Ha,Hb2), while DJendo and
DJexo were calculated with the program MestRe-J[17] using the
dihedral angles obtained from the lowest energy conformers pro-
duced by quantum chemical calculations (see Table I in the
Supporting Information).
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Communications
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Received: September 18, 2007
Published online: January 28, 2008
Keywords: conformation analysis · peptides · proline ·
.
pyrrolidine · stereoelectronic effects
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