4-Methylpseudoproline derived from α-methylserine - Synthesis and conformational studies

Article abstract of DOI:10.1039/c2ob25732g

This paper presents the synthesis and solution conformational studies of the tripeptides Fmoc-Ala-(R)-(αMe)Ser(Ψ^H,HPro)-Ala-OBu ^t (6a) and Fmoc-Ala-(S)-(αMe)Ser(Ψ^H,HPro)-Ala- OBu^t (6b). Additionally, the X-ray structure of 6a is given. NMR analysis corroborated by theoretical calculations (XPLOR) shows that in both peptides the amide bond between pseudoproline and the preceding amino acid is in the trans conformation. The same amide bond geometry was observed in the crystal state of 6a. The latter is additionally influenced by the presence of two symmetrically independent molecules in an asymmetric unit. Both molecules adopt a conformation which resembles β-turn type II, stabilized by hydrogen bonding. The conformational preferences and prolyl cis-trans isomerization of Ac-(αMe)Ser(Ψ^H,HPro)-NHMe (7) were explored at the IEFPCM/B3LYP/6-31+G(d) level of theory in vacuum, water and chloroform. It has been shown that the trans isomer predominates in water solutions and the cis isomer is preferred in chloroform. The conformation of 7 is down-puckered independently of the geometry of the amide bonds, with lower puckering in the transition state of the cis-trans isomerization.

Full text of DOI:10.1039/c2ob25732g

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PAPER
4-Methylpseudoproline derived from α-methylserine – synthesis and
conformational studies†
Joanna Katarzyńska,a Adam Mazur,a Wojciech M. Wolf,b Simon J. Teat,‡c Stefan Jankowski,*a
Mirosław T. Leplawya and Janusz Zabrocki*a
Received 15th April 2012, Accepted 20th June 2012
DOI: 10.1039/c2ob25732g
This paper presents the synthesis and solution conformational studies of the tripeptides Fmoc-Ala-(R)-
(αMe)Ser(ΨH,HPro)-Ala-OBut (6a) and Fmoc-Ala-(S)-(αMe)Ser(ΨH,HPro)-Ala-OBut (6b). Additionally,
the X-ray structure of 6a is given. NMR analysis corroborated by theoretical calculations (XPLOR) shows
that in both peptides the amide bond between pseudoproline and the preceding amino acid is in the trans
conformation. The same amide bond geometry was observed in the crystal state of 6a. The latter is
additionally influenced by the presence of two symmetrically independent molecules in an asymmetric
unit. Both molecules adopt a conformation which resembles β-turn type II, stabilized by hydrogen
bonding. The conformational preferences and prolyl cis–trans isomerization of Ac-(αMe)Ser(ΨH,HPro)-
NHMe (7) were explored at the IEFPCM/B3LYP/6-31+G(d) level of theory in vacuum, water and
chloroform. It has been shown that the trans isomer predominates in water solutions and the cis isomer is
preferred in chloroform. The conformation of 7 is down-puckered independently of the geometry of the
amide bonds, with lower puckering in the transition state of the cis–trans isomerization.
between the isomer geometry and biological activity of prolyl
Introduction
peptides. The Xaa–Pro amide bond was replaced by rigid cis or
trans amide bond surrogates: substituted proline rings as in I or
pseudoprolines (ΨPro) II (Scheme 1).1
Proline residues are widely recognized as the units controlling
the dynamic behavior of peptides and proteins due to the inher-
ently slow cis–trans isomerization of the Xaa–Pro bond. The
side chain of proline is covalently bonded to a nitrogen atom and
the N–Cα rotation (Φ angle) is restricted to about −60°. Proline
residues are usually encountered in loops or turn motifs at the
i + 1 position for β-turn type I or II when the Xaa–Pro amide
bond is in the trans conformation, or at the i + 2 position of turn
type VI in the cis form. What is characteristic of proline is a rela-
tively low activation barrier for isomerization (20–22 kcal
mol−1) combined with a small difference in free energy between
the isomers. As a consequence, the presence of proline residues
multiplies the number of conformers and can complicate the
characterization of a peptide structure by means of NMR. A
range of approaches have been applied to study the relationship
The effect of alkyl substitution on cis–trans isomerization was
examined by introducing a methyl group at all prolyl carbons in
Ac-(Me)Pro-NHMe. Only trans peptide bond isomers were
observed in the crystal state.2 The presence of methyl groups in
positions 3 or 4 does not alter significantly the fraction of the
cis isomer in comparison with native proline in water (about 25%)
or in chloroform (15–20%).3 The R or S configuration at carbon
atom 4, as in Ac-(4-Me)Pro-NHMe, has a strong effect on the
puckering of the proline ring. In water and in the crystal state,
(4S-Me)Pro and (4R-Me)Pro residues prefer up- and down-puck-
ering, respectively.4 The introduction of a methyl group5 in posi-
tion 5 increases the population of the cis-isomer by about 5%,
but 5,5-dimethyl5 or bulky tert-butyl6 substituents raise the cis-
isomer population up to 66%. The methyl group in position 2
exclusively favors the trans-isomer in different solvents.3 The
presence of Cα-methyl-L-proline ((αMe)Pro) in Nα-acyl dipeptide
N′-alkylamides of the RCO-(αMe)Pro-Xxx-NHR′ or RCO-Xxx-
(αMe)Pro-NHR′ type (Xxx = L (or D)-Ala, Aib (α-aminoisobuty-
ric acid) or L (or D)-(αMe)Pro) promotes the β-turn conformation
and the trans conformation of the preceding tertiary peptide
bond.7
aInstitute of Organic Chemistry, Lodz University of Technology, 90-924
Lodz, Poland. E-mail: stefan.jankowski@p.lodz.pl,
janusz.zabrocki@p.lodz.pl
bInstitute of General and Ecological Chemistry, Lodz University of
Technology, 90-924 Lodz, Poland
cCLRC Daresbury Laboratory, Daresbury, Warrington, Cheshire
WA4 4AD, UK
†Electronic supplementary information (ESI) available. CCDC 870748.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c2ob25732g
‡Now at: Advanced Light Source, Lawrence Berkeley National Labora-
tory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
Synthetic proline analogues II, known as pseudoprolines
(ΨPro), readily obtained by cyclocondensation of the amino
acids cysteine, threonine or serine with aldehydes or ketones are
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Scheme 1 Modified proline rings.
the targets of interest as building blocks improving the efficiency
of solid phase synthesis by preventing peptide aggregation and
self-association.8 To adjust the cis–trans isomer ratio, the pseudo-
proline ring was modified in position 2 by the incorporation of
one or two methyl groups or an aryl substituent. Unsubstituted
pseudoprolines are similar to proline in terms of their trans
population,9 whereas the 2,2-dimethyl derivatives adopt almost
exclusively a cis amide conformation.10,11 For 2-monosubstituted
pseudoprolines containing a methyl or p-methoxyphenyl group,
the trans–cis ratio depends on the stereochemistry at carbon 2:
for the (2S)-diastereoisomer both forms are almost equally popu-
lated, whereas the (2R)-epimer preferentially adopts the trans
form.10 2-Trifluoromethyl pseudoproline strongly influences the
cis–trans rotational barriers with a moderate effect on the cis–
trans population ratio.12 For dipeptides containing threonine-
derived pseudoprolines Xaa-Thr(ΨMe,MePro) (Xaa = Gly, Phe,
Val) the cis conformer is the predominant form in solution.13
The oxazolidin-2-one ring (Oxd) can be regarded as a pseudo-
proline with an exclusively trans conformation of the preceding
peptide bond. Oligomers containing Oxd exhibit a tendency to
form intra- rather than intermolecular hydrogen bonds when the
chain length increases.14
Substantial effort was put into theoretical calculations at
different theory levels for unmodified proline15 and substituted
proline or pseudoproline16 Ac-Xxx-NHMe in order to determine
the substituent and solvent effects on the cis–trans isomerization
and the ring puckering The calculated rotational barriers and
populations of cis conformers in pseudoprolines II (O > S >
CH2) in water and chloroform are consistent with experimental
data. The calculated energy barriers for ring flips show that
down-to-up puckering transitions are dominant in the orders
CH2 < O < S in vacuo and CH2 ≈ O < S in water.17
based on the HIV-1MN V3 variant.20 Modification of cyclospor-
ine C21 and cyclosporine A22 with ΨPro led to more constrained
molecules retaining partial biological activity. The incorporation
of pseudoproline at position 7 of the peptide hormone oxytocine
led to a reduction in its agonistic activity, consistent with a
decrease of the trans conformation.23 The presence the
Cys-derived pseudoproline Cys(ΨMe,MePro) facilitated the
synthesis of the cyclic octapeptide, cyclogossine B, as a traceless
turn-inducer.24 The introduction of Cys(ΨMe,MePro) or
Ser(ΨMe,MePro) in position 3 of the insect kinin core pentapep-
tide led to a 100% prevalence of the cis isomer of Phe–Xxx-
(ΨMe,MePro) peptide bond.25 When Cys(ΨH,HPro) was present
isomers cis and trans were equally populated. The pseudoproline
Thr(ΨMe,MePro) was used in research into the structure and
activity studies of sansalvamide A analogues.26
Research into the structure and activity studies of pseudopro-
line-containing analogues of opioid peptides, morphiceptin and
endomorphin-2, showed the role of the cis conformation of the
Tyr–Pro peptide bond in their bioactive conformations.27 The
endomorphin-2 analogue H-Tyr-Ser(ΨH,HPro)-Phe-Phe-NH2,
which exists in an conformational equilibrium around the Tyr–
Ser(ΨH,HPro) bond with a cis–trans ratio of 45 : 55, displayed a
5-fold lower μ agonist potency in the GPI assay and a 10-fold
lower δ potency in the MVD assay than the native endomorphin-
2. Affinity towards both receptors decreased even more for the
dimethylated endomorphin-2 analogue H-Tyr-Ser(ΨMe,MePro)-
Phe-Phe-NH2, although the cis–trans isomerization was almost
completely shifted towards the cis form. Preliminary studies of
biological activity showed that the incorporation of (αMe)Ser-
(ΨH,HPro) at position 2 in endomorphin-2 led to a new insight
into the role of steric hindrance and configuration of carbon 4 in
the pseudoproline moiety.28 The (R)-Ser(ΨH,HPro) isomer inter-
acts with the μ-receptor half as readily as the (S)-isomer and both
isomers are much less active than the unmethylated analogue
H-Tyr-Ser(ΨH,HPro)-Phe-Phe-NH2. However, it should be noted
that H-Tyr-(S)-(αMe)Ser(ΨH,HPro)-Phe-Phe-NH2 possesses
similar μ-receptor potency as [Leu5]enkephalin. This observation
suggests that the presence of a pseudoproline (S)-(αMe)-
Ser(ΨH,HPro) moiety enables the adoption of the bioactive confor-
mation, but makes the interaction with the μ-receptor more difficult.
In order to explore factors that determine the conformation of
the pseudoproline ring derived from α-methylserine, two
Ab initio calculations were carried out to analyze the effect of
(4R)-substitution of the prolyl ring by electron-withdrawing
groups (OH, F). Prolyl trans–cis isomerization is an entirely
enthalpy driven process and the presence of electron-withdraw-
ing substituents increases the population of the up puckered con-
formations. This factor is responsible for stabilization of triple
helices of (Pro-Xaa-Gly)n sequences.18,19
Pseudoprolines have been introduced as proline analogues
into molecules of the eleven-residue cyclopeptide cyclo(-Arg-
His-Ile-Gly-Xaa-Gly-Arg-Ala-Phe-Cys-Tyr-) with a sequence
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Scheme 2 Transformation of α-alkyl-α-hydroxymethyl amino acids to 4-alkylpseudoproline.
tripeptides (6a,b) were synthesized and characterized by NMR
spectroscopy.
using TBTU as a coupling reagent32 resulted in a 70% and 74%
yield of the last step of synthesis of tripeptides 6a and 6b,
respectively.
In contrast to the first method, second approach was accom-
plished in good yield when the stepwise method longer than
Mutter’s technique, was applied to the synthesis of peptides
6a and 6b. (R)- and (S)-4-Methylpseudoproline were used as the
N-benzyloxycarbonyl derivatives 4a and 4b obtained in good
yield (70%) by cyclocondensation of the corresponding (R)- and
(S)-methylserine with formaldehyde in alkaline solution accord-
ing to a known procedure.33
Fmoc-Ala-(R)-(αMe)Ser(ΨH,HPro)-Ala-OBut 6a
Fmoc-Ala-(S)-(αMe)Ser(ΨH,HPro)-Ala-OBut 6b
Additionally X-ray analysis was performed for peptide 6a.
Density functional computations were carried for Ac-(αMe)-
Ser(ΨH,HPro)-NHMe 7 out to characterize its cis–trans isomeri-
zation process.
Results and discussion
The dipeptides Z-(R)-(αMe)Ser(ΨH,HPro)-Ala-OBut 5a and
Z-(S)-(αMe)Ser(ΨH,HPro)-Ala-OBut 5b with 4-methylpseudo-
proline as the N-terminus were synthesized conventionally in
good yield using the TBTU carboxyl activation method. The Z-
group was removed by catalytic hydrogenation.34
The NH-group in the 4-methylpseudoproline residue was acy-
lated (“difficult coupling”) by Fmoc-Ala C-activated by 1-propane-
phosphonic acid cyclic anhydride (PPAA),35 the most efficient
coupling reagent in our synthesis (with yields 38% and 34%,
respectively). It is worth emphasizing that the additional advan-
tage of this strategy is the fact that all by-products are easily
accessible by commonly used purification techniques.
Synthesis
Mutter and coworkers have been introduced pseudoprolines
(ΨPro, 1,3-oxazolidines, Oxa and 1,3-thiazolidines, Thz), as a
solubilizing protection technique for Ser, Thr and Cys in peptide
synthesis.8,29 We have used the pseudoproline concept for
α-alkyl-α-hydroxymethyl amino acids, members of the family of
nonproteinogenic, α,α-disubstituted amino acids with a hydro-
philic side-chain.30 α-Alkyl-α-hydroxymethyl amino acids, such
as serine and threonine, can be transformed into a corresponding
pseudoproline (oxazolidine) unit named 4-alkylpseudoproline31
(Scheme 2).
For the synthesis of tripeptides 6a and 6b in solution two
different coupling strategies were tested (Scheme 3). The first
method was a “2 + 1” coupling strategy. Initially, dipeptides
Fmoc-Ala-(R)-(αMe)Ser(ΨH,HPro)-OH (3a) and Fmoc-Ala-(S)-
(αMe)Ser(ΨH,HPro)-OH (3b) were synthesized according to
Mutter’s procedure,30 and oxazolidine, which was obtained in
situ, was acylated by means of Fmoc alanine fluoride. (R)- and
(S)-α-Methylserine (1a and 1b respectively) were easily accessi-
ble by the Kaminski’s method.31 The introduction of pseudopro-
line as a dipeptide unit was a very significant advantage, because
the problem of the secondary amine acylation was eliminated
and the peptide chain was elongated by two residues in one step.
However, this method gave dipeptides 3a and 3b in very low
yield (15% and 14%, respectively). This was probably a conse-
quence of decreased nucleophilicity of the nitrogen atom caused
by the presence of an oxygen atom in the oxazolidine ring and
the steric hindrance of the α-methyl group. Efforts to optimize
the reaction conditions or use other acylating reagents were
unsuccessful. Moreover, the isolation of the desired product from
a heterogeneous mixture of components with very similar HPLC
retention times became a very labor-intensive process. However,
In summary based on our observations we can conclude that
4-methylpseudoproline derived from α-methylserine after protec-
tion of the NH-group is stable and can be isolated and stored for
a long time. N-protected C-activated 4-methylpseudoproline is
an efficient acylating component in the formation of peptide
bonds. This result proves its usefulness in the synthesis of pep-
tides with N-terminal 4-methylpseudoproline. On the other hand,
acylation of the NH-group in 4-methylpseudoproline with C-acti-
vated amino acids gives the desired dipeptides with C-terminal
pseudoproline in very low yield and this can be termed as
“difficult coupling”.
NMR analysis
1H and 13C NMR spectra recorded at 27 °C indicate the presence
of one set of signals for dipeptides 3a and 3b as well as for tri-
peptides 6a and 6b, demonstrating that the peptides are rigid or
undergo fast exchange, independently of the configuration at the
C-4 carbon atom. Amide bond configuration was determined by
analysis of cross-peak patterns in the corresponding ROESY
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Scheme 3 Two strategies employed for the synthesis of tripeptides 6a and 6b.
spectra, namely the presence of strong correlation signals
between Ala1 Hα (6a and 6b) or Ala1 Hβ (3a and 3b) protons
and methylene protons at the position 2 in the pseudoproline
ring clearly identified the trans geometry of the Ala-(αMe)-
Ser(ΨH,HPro) amide bond. Further inspection of the ROESY
spectrum of tripeptide 6b did not reveal any other signals.
However, for 6a additional ROE connectivities were detected
and the interproton interactions were divided into three groups of
distance restraints: strong (Ala1Hβ–(αMe)Ser2Hδ), medium
(Ala1Hβ–(αMe)Ser2Hβ, Ala1Hβ–(αMe)Ser2Hδ, (αMe)Ser2Hβ–
Ala3Hα, (αMe)Ser2Hβ–Ala3HN) and weak ((αMe)Ser2Hβ–
Ala1Hα, (αMe)Ser2Hβ–Ala3Hβ, (αMe)Ser2Hβ–Fmoc “ortho”),
based on the peak integrals.
addition in IR spectra of 6a and 6b recorded in CDCl3 the
additional broad signal below 3400 cm−1 (hydrogen-bonded
NHs) was observed for 6a only (Fig. S8, ESI).†
Crystal-state studies
It is remarkable that only a limited number of crystal structures
of peptides containing a pseudoproline residue have been estab-
lished so far. In particular, the dipeptides Cbz-Val-Thr(ΨMe,MePro)-
OMe and Cbz-Val-Thr(ΨMe,MePro)-OH adopt a trans-confor-
mation,13 while a cis-amide bond conformation has been estab-
lished for Fmoc-Ala-Cys(ΨMe,MePro)-OH.30b
Fmoc-Ala-(R)-(αMe)Ser(ΨH,HPro)-Ala-OBut (tripeptide 6a)
crystals suitable for X-ray analysis were grown and used to deter-
mine its crystal-state structure. The peptide bond preceding ΨH,
HPro adopted the trans conformation as in solution. A view of
the two symmetrically independent molecules A and B located
in the asymmetric unit is given in Fig. 1 with atom labeling.
Both symmetrically independent molecules are the same dia-
stereoisomers with relative configurations (S*,R*,S*) at atoms
C16, C21, C24 and C46, C51, C54 in A and B, respectively.
The temperature dependence of NH chemical shifts was ana-
lyzed in DMSO-d6 within the range of 300–320 K. The tempera-
ture coefficients of NH1 and NH3 proton chemical shifts were
found to be different and equal to −8.3/−0.50 and −4.8/−7.5
ppb K−1 for 6a and 6b, respectively. The observed temperature
coefficients imply the presence of a strong hydrogen bonding
interaction for 6a and a weaker, if any, hydrogen bonding for
1
6b.36 The temperature effect on the H NMR spectra of 6a and
6b in DMSO-d6 is presented in Fig. S6 and S7 (ESI).† In
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resembles the β-turn type II38 stabilized by a hydrogen bonding
between the carbonyl group of the Fmoc protecting group first
residue and the main chain nitrogen atom of the third residue
(Fig. 1, ESI, Table S2†). The 1,3-oxazolidine rings adopt dis-
torted envelope conformations with the 4-Ala substituent occu-
pying axial positions. The main pseudo-symmetry elements are a
mirror image passing through the C20 atom and a two fold axis
passing through the N5 atom in the rings of molecules A and B,
respectively. The endocyclic torsion angles and the three lowest
asymmetry parameters39 are summarized in Table S3 (ESI†). In
the crystal, both symmetrically independent Fmoc residues are
located almost perpendicular to one another. Their arrangement
is stabilized by an unusual short intermolecular contact between
the C5–H51 bond and the C31–C36 ring. The distance between
the ring centroid Cg and the H51 atom is 2.76 Å, and the C5–
H51⋯Cg angle is 169°. The environment of the molecules in
the crystal with the hydrogen bond network is shown in Fig. S10
(ESI†). The hydrogen atoms bonded to atoms N1 and N4 are
involved in intermolecular hydrogen bonds with carbonyl
oxygen atoms. The hydrogen bonds form infinite chains, which
extend along the b crystal axis.
Theoretical calculations
ROE cross-peaks and vicinal coupling constants were used as
the geometrical restraints in theoretical calculations of 6a and 6b
structures by means of the default simulated annealing protocol
(sa.inp) implemented within the XPLOR-NIH program.40 Struc-
tures of the main calculated clusters of peptides 6a and 6b are
presented in Fig. 2 and 3.
Fig. 4 illustrates the good agreement between the structures
determined in the crystal state and in solution for 6a. The intro-
duction of a methyl group at position 4 in the pseudoproline ring
in tripeptides 6a and 6b caused a shift of cis–trans isomerization
exclusively towards the trans isomer, as observed both in sol-
ution and in the crystal state.
We decided to investigate theoretically the influence of both
modifications of the proline residue, the replacement of a carbon
atom by an oxygen atom and the introduction of methyl group at
the α-carbon atom on the puckering of the pseudoproline ring,
cis–trans equilibrium, and rotational barriers of the amide bond
in vacuo, in chloroform and in water. Calculations were carried
out in vacuo for Ac-(R)-(αMe)Ser(ΨH,HPro)-NHMe 7 (Fig. 5) at
the B3LYP/6-31+G(d) level of theory and compared with litera-
ture data for proline (Pro), (S)-oxazolidine-4-carboxylic acid
(Oxa) and Cys-derived (R)-thiazolidine-4-carboxylic acid (Thz),
calculated at the HF/6-31+G(d) level.14f
The trans and cis isomer geometries were optimized for the
R enantiomer. Transition states were located by calculating
energy profiles along the ω′ angle. The trans isomer was chosen
as a starting point and the potential energy was scanned in two
possible directions using 15° and −15° ω′ steps. The resulting
structures were further optimized as saddle points. The free ener-
gies of transition states differed by 2.5 kcal mol−1 in CDCl3 and
4 kcal mol−1 in H2O, depending on the direction of rotation. The
lower reported values correspond to the transition state TS1 with
the methyl and acetyl groups placed on the same side. The opti-
mized trans, cis and TS structures of 7 are shown in Fig. 5. The
Fig. 1 Two symmetrically independent molecules A and B of Fmoc-
Ala-(R)-(αMe)Ser(ΨH,HPro)-Ala-OBut (6a) with atom numbering. The
intramolecular hydrogen bonding is indicated with a dashed line. Displa-
cement ellipsoids are drawn at a 50% probability level.
The bond lengths and angles are quite close to those commonly
encountered in related compounds.37 The important feature
influencing the peptide conformation, and especially crystal
packing, is the existence of two symmetrically independent
molecules in the asymmetric unit. For comparison, a superposi-
tion of the two independent molecules as determined by the
least-squares fitting of the common fragments is presented in
Fig. S9 (ESI).†
The observed backbone torsion angles, ψ, ψ, ω (°) for mole-
cules A and B are: Ala1, −57.4, 139.5, 179.0 (A), −58.8, 129.1,
−179.6 (B); (αMe)Ser(ΨH,HPro), 78.8, 1.4, 176.0 (A), 69.6,
10.4, 178.2 (B); Ala3, −124.5, −16.8, 177.6 (A), −131.9, 59.5,
171.9 (B). Both molecules adopt a conformation which
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Fig. 2 Structures of the calculated main clusters (left) and the main central cluster structure (right) of Fmoc-Ala-(R)-(αMe)Ser(ΨH,HPro)-Ala-OBut
(6a).
Fig. 3 Structures of the calculated main clusters (left) and the main central cluster structure (right) of Fmoc-Ala-(S)-(αMe)Ser(ΨH,HPro)-Ala-OBut
(6b).
angle N–H⋯N are 2.410 Å and 102.1° for the trans isomer, and
2.401 Å and 103.1° for the cis isomer, respectively. The stabiliz-
ing effect of the hydrogen bond was found for TS1 (2.257 Å and
105.6°), but not for TS2 (Fig. 5).
The calculated free energies of activation for the trans-to-cis
rotation of Ac-Xxx-NHMe peptides are listed in Table 1. In
chloroform and water the barriers of rotation for pseudoprolines
are lower than for proline, except (2R)-CF3-Oxa. The preferences
of cis isomers in chloroform are in the order 7 > Oxa, CF3-Oxa
> Thz, Pro. In water with predominant trans isomers the highest
populations of cis isomers are found for Oxa, CF3-Oxa and Thz.
The fractions of the cis isomer in pseudoproline 7 and proline
are lower and comparable. This means that the effect of the pres-
ence of the oxygen atom was compensated by the steric effect of
the methyl group.
The following parameters were used to characterize the pseu-
doproline ring: backbone torsion angles (ω, ϕ and ψ), endocyclic
torsion angles χi (i = 0–4) (Fig. 6). and puckering amplitudes qi
(i = a, z or m). The parameter qa of Han and Kang is the
maximum angle between the mean plane and the line joining the
geometrical center and each atom of the ring.41 The parameter qz
of Cremer and Pople corresponds to the maximum z-displace-
ment perpendicular to the mean plane of the ring.42 The par-
ameter χm of Altona and Sundaralingam is the maximum value
attainable by the endocyclic torsion angles of the ring.43 The
trans (“t”) and cis (“c”) conformations of Ac–Xxx peptide
Fig. 4 Comparison of structures of 6a determined in the crystal state
by X-ray (red) and calculated from NMR data (blue).
conformation of 7 is down-puckered (Cγ-endo) independently of
the geometry of amide bonds. The structures are stabilized by a
weak hydrogen bond between NH (NHMe) and the nitrogen
atom in the pseudoproline ring. The distance d(N–H⋯N)
between the hydrogen atom and the acceptor nitrogen atom and
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Table 1 Free energies of activation (ΔG≠, kcal mol−1) of cis–trans
isomerization of Ac-Xxx-NHMe peptides and the fraction of the cis
conformer in chloroform and water at IEFPCM/B3LYP/6-31+G(d) or
determined experimentally
cis, %
Chloroform
cis, %
Water
Peptide
ΔG≠
ΔG≠
7
62
17.49
19.32
16.85
16.52
18.47
14.22
29
18.06
21.61
18.45
16.12
21.66
16.70
Proa
9
23
Oxaa
18 (27c)
11
39 (34c)
45
Thza
(2S)-CF3-Oxab
(2R)-CF3-Oxab
40c
24c
43c
45c
a From ref. 15f. b From ref. 12, calculations at B3LYP/6-311++G(d,p)//
CPMC HF/6-31+G(d). c Cis content determined from NMR, ref. 12, in
other cases calculated from ΔG = −RT lnK where K = [cis]/[trans].
Fig. 5 Optimized structures of trans and cis isomers and transition
states for trans–cis isomerization of Ac-(R)-(αMe)Ser(ΨH,HPro)-NHMe
7, optimized at the IEFPCM/B3LYP/6-31G(d) level.
Fig. 6 Definition of torsion angles in peptide 7.
bonds are defined by the orientation of the methyl carbon of the
acetyl group and the Cα of the proline residue. If the γ-oxygen
and the CvO group of the prolyl residue are on the same side of
the plane defined by the three atoms Cδ, N and Cα, the confor-
mation is defined as down-puckered. The opposite orientation of
the oxygen atom corresponds to the up-puckered conformation.
The backbone conformations are described by capital letters
in terms of the position on the ϕ − ψ map. Regions A and C are
defined to lie within the following angles −110° ≤ ϕ < −40° and
−90° ≤ ψ < −10° (region A) or 50° ≤ ψ < 130° (region C).
Region B is defined as a bridge region between A and C.44 The
replacement of CH2 in Pro by an oxygen atom, as in Oxa, or by
a sulfur atom as in Thz, did not change significantly the back-
bone torsion angle values. Conformations of the trans and cis
isomers of peptide 6 belong to regions B and A on the ϕ − ψ
map, respectively. The ψ value of the trans isomer is very close
to the boundary ψ = 50° for the conformation C. For this reason
conformation of the trans isomer of peptide 7 can be considered
as C, equivalent to the γ-turn.
Conclusions
4-Methylpseudoproline derived from α-methylserine after the
protection of the NH-group is stable and can be isolated and
stored for a long time. N-protected C-activated 4-methylpseudo-
proline is an efficient acylating component in the formation
of peptide bonds. In the tripeptides Fmoc-Ala-(R)-(αMe)-
Ser(ΨH,HPro)-Ala-OBut (6a) and Fmoc-Ala-(S)-(αMe)Ser(ΨH,HPro)-
Ala-OBut (6b) in solution, the amide bonds between Ala and
(αMe)Ser(ΨH,HPro) adopt a trans geometry. The same geometry
of this amide bond has been found for 6a in the crystal state.
Theoretical calculations for the peptide Ac-(αMe)Ser(ΨH,
HPro)-NHMe (7) have revealed that all the puckering parameters
qi of the pseudoproline ring are lower in the transition state than
in the trans and cis isomers. The lower puckering in the tran-
sition state should facilitate cis–trans isomerization in a more
crowded environment, in contrast to proline and unsubstituted
pseudoprolines.
All puckering parameters qi of the pseudoproline ring in 7 are
lower in the transition state than in the trans and cis isomers
(Table 2). The lower puckering in the transition state
should facilitate cis–trans isomerization in a more crowded
environment, in contrast to proline and unsubstituted
pseudoprolines.12,15f
In chloroform, the cis isomer predominates for pseudoproline
7, as opposed to proline existing almost exclusively as the trans
isomer. In water, the fractions of the cis isomer are comparable
for pseudoproline 7 and proline, suggesting that the effect of the
presence of the oxygen atom in the proline ring is compensated
by the steric effect of the methyl group.
This journal is © The Royal Society of Chemistry 2012
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Table 2 Backbone torsion angles, endocyclic torsion angles and puckering amplitudes of pseudoprolines 7 in Ac-Xxx-NHMe optimized at the
B3LYP/6-31+G(d) level in vacuo and in solution
Torsion angles (°)
Puckering amplitudes
χm
Peptide Solvent Isomer or TS ω′
ϕ
ψ
ω
χ0
χ1
χ2
χ3
χ4
q
q
7a
vacuo
trans
cis
TS
trans
cis
TS
trans
cis
TS
−172.8 −75.2
47.3
175.9
−7.7
28.6 −40.9 35.1
−16.1 11.0 0.362 40.2
−21.9 11.1 0.363 40.5
−38.3 37.9 10.8 0.354 39.5
9.2
−75.3
−22.0 −174.1 −1.5
24.4 −39.8 37.9
−50.5
−137.4 18.4
177.1
−22.5 0.6
22.5
H2O
−171.2 −74.5
−24.0 −176.8 −0.6
−14.1 −177.0 −2.9
23.9 −40.0 38.7
25.7 −40.7 38.0
−23.0 11.2 0.368 41.0
−20.9 11.3 0.370 41.0
11.1
−79.2
−145.2 21.0
−50.3
176.7
−27.9 9.0
14.2
−33.0 38.1
10.4 0.346 38.0
−22.6 11.2 0.366 40.7
−21.6 11.2 0.367 40.8
−37.0 38.0 10.6 0.351 39.0
CHCl3
−171.1 −75.7
−16.4 −178.2 −0.9
−21.0 −175.2 −2.1
24.0 −39.9 38.4
25.0 −40.4 38.1
9.2
−75.6
−50.7
−139.7 18.1
177.2
−23.8 2.6
20.6
solution was cooled to 0–5 °C. In order to perform acylation
Fmoc-Ala-F (3.1 g, 10 mmol) solution in THF (10 mL) was
added dropwise within 60 min, while pH was adjusted between
8 and 9 using saturated NaHCO3 (∼5 mL). The reaction mixture
was stirred for another 30 min at 5 °C and then for 2 h at room
temperature. After THF evaporation the reaction mixture was
diluted with water (∼30 mL) and extracted with diethyl ether (3
× 15 mL), and the diethyl ether extracts were discarded. After
cooling to 0 °C, the solution was acidified with 6 N HCl
(∼15 mL) to pH 3–4. The suspension was extracted with ethyl
acetate (3 × 30 mL). The organic phases were combined and
washed with water (3 × 20 mL) and dried over MgSO4. After
evaporation the crude product was purified by flash chromato-
graphy (EtOAc–hexane–AcOH: 9 : 1 : 0.1) over silica gel to give
640 mg of 3a in 15% yield; [α]D = +4.59° (c 1, CHCl3); Rf (A)
= 0.53, Rf (B) = 0.22; HPLC (40–70% B, 25 min) tR = 13.42
(100%); IR ν 3292, 2975, 2871, 1716, 1654, 1529, 1450, 1241,
1072, 962, 759, 740 cm−1; 1H NMR (700 MHz, CDCl3, TMS) δ
1.37 (d, J = 6.5 Hz, 3H, Cβ–H Ala), 1.66 (s, 3H, Cβ–H (αMe)
Ser), 3.83, (bd, J = 6.9 Hz, 1H, O–CH2–C), 4.18 (bt, J = 6.7 Hz,
1H, CH Fmoc), 4.26–4.32 (m, 1H, Cα–H Ala), 4.32–4.42 (m,
3H, CH2 Fmoc overlapped with O–CH2–C), 5.08, (bs, 1H, N–
CH2–O), 5.22 (bs, 1H, N–CH2–O), 5.78 (bs, 1H, NH), 7.29 (t,
J = 7.4 Hz, 2H, C–H arom.), 7.37 (t, J = 7.4 Hz, 2H, C–H arom.),
7.57 (d, J = 7.4 Hz, 1H, C–H arom.), 7.59 (d, J = 7.4 Hz, 1H,
C–H arom.), 7.76 (d, J = 7.4 Hz, 2H, C–H arom.); 13C NMR
(175 MHz, CDCl3, TMS) δ 18.3 (Cβ–H Ala), 19.5 (Cβ–H (αMe)
Ser), 47.1 (CH Fmoc), 49.2 (Cα–H Ala), 65.1 (CIV (αMe)Ser),
67.2 (CH2 Fmoc), 77.8 (O–CH2–C), 80.2 (N–CH2–O), 120.0,
125.1, 127.1, 127.8 (C–H arom.), 141.3, 143.7 (C arom.), 155.8
(CvO Fmoc), 170.2 (CvO (αMe)Ser), 173.7 (CvO Ala).
Experimental section
General remarks
All solvents were purified by conventional methods. Evapor-
ations were carried out under reduced pressure. Optical rotations
were measured in a 1 dm cell (1 ml) on a Horiba high-speed
automatic polarimeter at 589 nm. For thin layer chromatography
250 nm silica gel GF precoated uniplates (Merck) were used
with the following solvent systems: (A) CHCl3–MeOH–AcOH
9 : 1 : 0.2, (B) EtOAc–hexane 9 : 1 : 0.1 (C) EtOAc–hexane 1 : 1,
(D) CHCl3–MeOH 19 : 1, (E) EtOAc–hexane 9 : 1. Chromato-
grams were visualized with chlorine followed by starch–KI and/
or ninhydrin. Flash chromatography was performed with Merck
silica gel 60 (230–400 mesh). HPLC was performed on a LDC/
Milton-Roy analytical instrument using a Vydac C18 column
(0.46 × 25 cm): flow 1.0 ml min−1, detection at 220 nm with the
following eluants (A) 0.05% trifluoroacetic acid in water and (B)
0.038% trifluoroacetic acid in acetonitrile–water 90 : 10 with a
gradient application. IR spectra of solids were measured by
using ATR method on FTIR spectrophotometer alpha (Bruker).
FTIR absorption spectra were recorded using Nicolet 6700
FT-IR spectrophotometer. Solutions in deuterochloroform
(99.8% d, Aldrich) (2 mM) were placed in a cell with KBr and
path length of 1 mm. FAB-MS spectra were recorded on a Finni-
gan MAT 95 spectrometer. NMR spectra were recorded on a
Bruker Avance II Plus spectrometer at 700 MHz (1H) and
175 MHz (13C), respectively. Measurements were carried out in
deuterochloroform (99.96% d, Aldrich) at 25 °C with tetra-
methylsilane as the internal standard. H and 13C NMR reson-
1
ances were assigned on the basis of COSY-DQF, ROESY,
HMQC and HMBC experiments. ROESY spectra (recorded with
400 ms mixing time) were assigned and integrated and cross-
peaks were classified cross as strong (1.8–2.5 Å), medium
(1.8–3.5 Å) or weak (1.8–5.0 Å).
Fmoc-Ala-(S)-(αMe)Ser(ΨH,HPro)-OH (3b)
For the synthesis of 3b the same procedure was applied as for 3a
using (S)-methylserine 1b (3.6 g, 30 mmol). The pure product
was obtained in 14% yield (600 mg); [α]D = −27.09° (c 1,
CHCl3); HPLC (40–70% B, 25 min) tR = 13.39 (100%); IR ν
3303, 2975, 2872, 1713, 1650, 1530, 1449, 1240, 1072, 946,
Fmoc-Ala-(R)-(αMe)Ser(ΨH,HPro)-OH (3a)
(R)-Methylserine 1a (3.6 g, 30 mmol) was dissolved in 2 N
NaOH (15 mL). Subsequently aqueous solution of formaldehyde
(37%, 9 mL) was added dropwise under vigorous stirring and
the resulting solution was stored for 48 h at room temperature,
after which time the pH was 8–9. Then, THF (20 mL) was
added to (R)-4-methylpseudoproline (2a) formed in situ and the
1
758, 739 cm−1; H NMR (700 MHz, CDCl3, TMS) δ 1.36 (d, J
= 6.8 Hz, 3H, Cβ–H Ala), 1.69 (s, 3H, Cβ–H (αMe)Ser), 3.91 (d,
J = 8.9 Hz, 1H, O–CH2–C), 4.26 (q, J = 6.8 Hz, 1H, Cα–H Ala),
4.28 (d, J = 8.9 Hz, 1H, O–CH2–C), 4.21 (t, J = 6.9 Hz, 1H, CH
6712 | Org. Biomol. Chem., 2012, 10, 6705–6716
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Fmoc), 4.36, 4.38 (dd, J = 1.8 Hz, J = 6.87 Hz, 2H, CH2 Fmoc),
5.08 (d, J = 3.4 Hz, 1H, N–CH2–O), 5.26 (d, J = 3.4 Hz, 1H,
N–CH2–O), 5.80 (bd, J = 7.2 Hz, 1H, NH), 7.29 (t, J = 7.4 Hz,
2H, C–H arom.), 7.37 (t, J = 7.4 Hz, 2H, C–H arom.), 7.58 (d,
J = 7.4 Hz, 1H, C–H arom.), 7.60 (d, J = 7.4 Hz, 1H, C–H
arom.), 7.76 (d, J = 7.4 Hz, 2H, C–H arom.); 13C NMR
(175 MHz, CDCl3, TMS) δ 17.6 (Cβ–H Ala), 19.7 (Cβ–H (αMe)
Ser), 47.1 (CH Fmoc), 49.2 (Cα–H Ala), 65.1 (CIV (αMe)Ser),
67.2 (CH2 Fmoc), 77.8 (O–CH2–C), 80.2 (N–CH2–O), 120.0,
125.1, 127.1, 127.8 (C–H arom.), 141.3, 143.7 (C arom.), 155.8
(CvO Fmoc), 170.1 (CvO (αMe)Ser), 173.8 (CvO Ala).
Ala1), 170.2 (CvO (αMe)Ser), 171.3 (CvO Ala3); HRFAB m/z
calcd for C30H38N3O7 [M + H]+ 552.2709, found 552.2704.
Fmoc-Ala-(S)-(αMe)Ser(ΨH,HPro)-Ala-OBut (6b from 3b) –
method I
As reported for the synthesis of 6a according to method I, the
same procedure was applied to 6b, starting from 3b. The pure
product was obtained in 73% yield (403 mg); [α]D = −52.69°
(c 1, CHCl3); Rf (C) = 0.46, Rf(D) = 0.71; HPLC (50–80% B,
25 min) tR = 14.18 (100%); IR ν 3303, 2977, 2887, 1719, 1657,
1
1527, 1450, 1248, 1152, 1071, 946, 759, 740 cm−1; H NMR
(700 MHz, CDCl3, TMS) δ 1.36 (d, J = 6.7 Hz, 3H, Cβ–H
Ala3), 1.39 (d, J = 7.0 Hz, 3H, Cβ–H Ala1), 1.46 (s, 9H, tBu),
1.72 (s, 3H, Cβ–H (αMe)Ser), 3.77 (d, J = 8.9 Hz, 1H, O–CH2–
C), 4.28 (q, J = 7.0 Hz, 1H, Cα–H Ala1), 4.24 (t, J = 6.8 Hz, 1H,
CH Fmoc), 4.37–4.45 (m, 3H, Cα–H Ala3 overlapped with CH2
Fmoc), 4.56 (d, J = 8.9 Hz, 1H, O–CH2–C), 5.14 (d, J = 2.8 Hz,
1H, N–CH2–O), 5.20 (d, J = 2.8 Hz, 1H, N–CH2–O), 5.62 (bd,
J = 7.0 Hz, 1H, NH1), 7.35 (t, J = 7.5 Hz, 2H, C–H arom.),
7.41–7.46 (m, 3H, NH3 overlapped with C–H arom.), 7.61 (d,
J = 7.5 Hz, 1H, C–H arom.), 7.62 (d, J = 7.5 Hz, 1H, C–H arom.),
7.79 (d, J = 7.5 Hz, 2H, C–H arom.); 13C NMR (175 MHz,
CDCl3, TMS) δ 17.8 (Cβ–H Ala3), 17.9 (Cβ–H Ala1), 19.0 (Cβ–
H (αMe)Ser), 27.5 (CH3–C But), 46.7 (CH Fmoc), 48.6 (Cα–H
Ala3), 49.0 (Cα–H Ala1), 65.6 (CH3–C (αMe)Ser), 66.6 (CH2
Fmoc), 80.0 (N–CH2–O), 77.2 (O–CH2–C), 81.6 (CH3–C But),
119.6, 124.7, 126.6, 127.3 (C–H arom.), 140.9, 143.4 (C arom.),
155.8 (CvO Fmoc), 169.5 (CvO Ala1), 170.2 (CvO (αMe)
Ser), 171.5 (CvO Ala3); HRFAB m/z calcd for C30H38N3O7
[M + H]+ 552.2709, found 552.2703.
Fmoc-Ala-(R)-(αMe)Ser(ΨH,HPro)-Ala-OBut (6a from 3a) –
method I
HCl·Ala-OBut (182 mg, 1 mmol) was added to the solution of
3a (424 mg, 1 mmol), DIEA (0.35 mL, 2 mmol) and TBTU
(321 mg, 1 mmol) in DCM (10 mL). After 12 h, the solvent was
evaporated under reduced pressure, and the residue was dis-
solved in EtOAc (50 mL). The organic solution was washed
with 5% NaHCO3 (3 × 15 mL), water (3 × 15 mL), 10%
KHSO4 (3 × 15 mL) and water (3 × 15 mL), dried over MgSO4
and concentrated under reduced pressure. The crude product was
purified on silica gel (EtOAc–hexane: 1 : 1) to yield 386 mg
(70%) of 6a as a foam. Crystallization from methanol–water
gave the crystal form, mp. = 160–161 °C; [α]D = +16.09° (c 1,
CHCl3); Rf (C) = 0.35, Rf(D) = 0.60; HPLC (50–80% B,
25 min) tR = 15.36 (100%); IR ν 3326, 2977, 2871, 1703, 1660,
1
1530, 1478, 1251, 1152, 1071, 948, 759, 740 cm−1; H NMR
(700 MHz, CDCl3, TMS) δ 1.27 (d, J = 7.0 Hz, 3H, Cβ–H
Ala3), 1.37 (d, J = 6.7 Hz, 3H, Cβ–H Ala1), 1.39 (s, 9H, But),
1.68 (s, 3H, Cβ–H (αMe)Ser), 3.77 (d, J = 9.1 Hz, 1H, O–CH2–
C), 4.11 (q, J = 6.7 Hz, 1H, Cα–H Ala1), 4.18 (t, J = 6.8 Hz, 1H,
CH Fmoc), 4.38 (q, J = 7.0 Hz, 1H, Cα–H Ala3), 4.28, 4.40 (dd,
ABX, J = 6.8 Hz, J = 7.3 Hz, 2H, CH2 Fmoc), 4.42 (d, J = 9.1
Hz, 1H, O–CH2–C), 5.03 (d, J = 3.5 Hz, 1H, N–CH2–O), 5.41
(d, J = 3.5 Hz, 1H, N–CH2–O), 5.44 (bd, J = 6.7 Hz, 1H, NH1),
7.17 (bd, J = 7.0 Hz, 1H, NH3), 7.31 (t, J = 7.5 Hz, 2H, C–H
arom.), 7.40 (t, J = 7.5 Hz, 2H, C–H arom.), 7.56 (d, J = 7.5 Hz,
1H, C–H arom.), 7.58 (d, J = 7.5 Hz, 1H, C–H arom.), 7.76 (d, J
Z-(R)-(αMe)Ser(ΨH,HPro)-OH (4a)
(R)-Methylserine 1a (1.2 g, 10 mmol) was dissolved in 2 N
NaOH (5 mL). Then, an aqueous solution of formaldehyde
(37%, 3 mL) was added dropwise under vigorous stirring and
the resulting solution was stored for 48 h at room temperature,
after which time the pH was 8–9. Then, NaHCO3 (2 g,
24 mmol) and acetone (10 mL) were added to (R)-4-methylpseudo-
proline (2a) formed in situ, and the solution was cooled to
0–5 °C. Subsequently Cbz–Cl (3 mL, 15 mmol) was added
dropwise under vigorous stirring, while the pH was adjusted to 9
with saturated NaHCO3 (∼1 mL). The reaction mixture was
stirred for the next 30 min at 5 °C and for 2 h at room tempera-
ture. Then, acetone was evaporated, the reaction mixture was
diluted with water (∼30 mL) and extracted with diethyl ether (3
× 10 mL), the diethyl ether extracts were discarded. After
cooling to 0 °C, the solution was acidified with 6 N HCl
(∼5 mL) to pH 3–4. The suspension was extracted with ethyl
acetate (3 × 15 mL). The organic phases were combined and
washed with water (3 × 10 mL) and dried over MgSO4. After
evaporation of the solvent the pure product was obtained in 72%
yield (3.8 g) as a foam, [α]D = +37.09° (c 1, CHCl3); Rf (A) =
0.65, Rf (B) = 0.43; HPLC (10–90% B, 25 min) tR = 15.15
(100%); IR ν 3317, 2971, 2871, 1704, 1408, 1352, 1240, 1064,
1
= 7.5 Hz, 2H, C–H arom.); H NMR (700 MHz, DMSO-d6) δ
1.12 (d, J = 7.3 Hz, 3H, Cβ–H Ala3), 1.18 (d, J = 6.9 Hz, 3H,
Cβ–H Ala1), 1.30 (s, 9H, But), 1.46 (s, 3H, Cβ–H (αMe)Ser),
3.79 (d, J = 8.8 Hz, 1H, O–CH2–C), 4.11 (q, J = 6.7 Hz, 1H,
Cα–H Ala1), 4.18 (t, J = 6.8 Hz, 1H, CH Fmoc), 4.38 (q, J = 7.0
Hz, 1H, Cα–H Ala3), 4.28, 4.40 (dd, ABX, J = 6.8 Hz, J = 7.3
Hz, 2H, CH2 Fmoc), 4.36 (dd, J = 9.5, 6.0 Hz, 1H, O–CH2–C),
5.03 (d, J = 3.5 Hz, 1H, N–CH2–O), 5.06 (d, J = 3.7 Hz, 1H,
N–CH2–O), 5.28 (d, J = 3.7 Hz, 1H, NH1), 7.18 (bd, J = 6.9 Hz,
1H, NH3), 7.32 (t, J = 7.4 Hz, 2H, C–H arom.), 7.41 (t, J = 7.4
Hz, 2H, C–H arom.), 7.67 (dd, J = 7.2, 4.2 Hz, 2H, C–H arom.),
7.86 (d, J = 5.0 Hz, 1H, NH), 7.88 (d, J = 7.6 Hz, 2H, C–H
arom.); 13C NMR (CDCl3, TMS) δ 16.8 (Cβ–H Ala3), 17.3 (Cβ–
H Ala1), 18.7 (Cβ–H (αMe)Ser), 27.5 (CH3–C But), 46.6 (CH
Fmoc), 48.6 (Cα–H Ala3), 49.2 (Cα–H Ala1), 65.2 (CH3–C
(αMe)Ser), 66.8 (CH2 Fmoc), 79.6 (N–CH2–O), 77.8 (O–CH2–
C), 81.0 (CH3–C But), 119.5, 124.5, 126.5, 127.5 (C–H arom.),
140.8, 143.2 (C arom.), 155.8 (CvO Fmoc), 169.0 (CvO
1
920,768, 716, 660 cm−1; H NMR (700 MHz, CDCl3, TMS) δ
1.72 (s, 3H, Cβ–H), 3.86 (d, J = 9.0 Hz, 1H, O–CH2–C), 4.34
Org. Biomol. Chem., 2012, 10, 6705–6716 | 6713
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(d, J = 9.0 Hz, 1H, O–CH2–C), 5.02 (d, J = 2.9 Hz, 1H, N–
CH2–O), 5.09 (d, J = 2.9 Hz, 1H, N–CH2–O), 5.18 (bs, 2H,
CH2–Ph), 7.37 (bs, 5H, C–H arom.); 13C NMR (175 MHz,
CDCl3, TMS) δ 19.5 (Cβ–H (αMe)Ser), 63.9 (CIV (αMe)Ser),
67.5 (CH2–Ph), 77.9 (N–CH2–O), 80.2 (O–CH2–C), 127.9,
128.2, 128.5 (C–H arom.), 135.8 (CIV Ph), 152.9 (CvO Z),
176.3 (CvO (αMe)Ser).
NMR (700 MHz, CDCl3, TMS) δ 1.33 (d, J = 6.7 Hz, 3H, Cβ–H
Ala), 1.46 (s, 9H, But), 1.65 (s, 3H, Cβ–H (αMe)Ser), 3.66–3.81
(bm, 2H, O–CH2–C), 4.36 (q, J = 6.7 Hz, 1H, Cα–H Ala), 4.97
(bd, J = 3.0 Hz, 1H, N–CH2–O), 5.13 (bd, J = 3.0 Hz, 1H, N–
CH2–O), 5.14 (d, J = 12.2 Hz, 1H, CH2–Ph), 5.22 (d, J = 12.2
Hz, 1H, CH2–Ph), 7.34–7.35 (bm, 1H, NH), 7.37–7.39 (m, 5H,
C–H arom.); 13C NMR (175 MHz, CDCl3, TMS) δ 18.1 (Cβ–H
Ala), 19.8 (Cβ–H (αMe)Ser), 28.0 (CH3–C But), 49.0 (Cα–H
Ala), 63.9 (CIV (αMe)Ser), 67.5 (CH2–Ph), 80.5 (N–CH2–O),
81.9 (O–CH2–C), 128.1, 128.3, 128.6 (C–H arom.), 135.9 (CIV
Ph), 153.0 (CvO Z), 171.4 (CvO Ala), 171.7 (CvO (αMe)
Ser).
Z-(S)-(αMe)Ser(ΨH,HPro)-OH (4b)
As reported for the synthesis of 4a, the same procedure was
applied to 4b, starting from (S)-methylserine 1b (1.2 g,
10 mmol). The pure product was obtained in 70% yield (3.7 g);
Fmoc-Ala-(R)-(αMe)Ser(ΨH,HPro)-Ala-OBut (6a from 5a) –
method II
[α]D = −36.69° (c 1, CHCl3); HPLC (10–90% B, 25 min) tR
15.13 (100%); IR ν 3339, 2978, 2871, 1705, 1411, 1358, 1241,
1066, 921,770, 713, 659 cm−1 1H NMR (700 MHz, CDCl3,
=
;
TMS) δ 1.72 (s, 3H, Cβ–H), 3.89 (d, J = 9.0 Hz, 1H, O–CH2–
C), 4.33 (d, J = 9.0 Hz, 1H, O–CH2–C), 5.02 (d, J = 2.9 Hz, 1H,
N–CH2–O), 5.09 (d, J = 2.9 Hz, 1H, N–CH2–O), 5.18 (bs, 2H,
CH2–Ph), 7.37 (bs, 5H, C–H arom.), C13 NMR (CDCl3, TMS)
19.5 (Cβ–H (αMe)Ser), 63.9 (CIV (αMe)Ser), 67.5 (CH2–Ph),
77.8 (N–CH2–O), 80.2 (O–CH2–C), 127.9, 128.2, 128.5 (C–H
arom.), 135.8 (CIV Ph), 152.9 (CvO Z), 176.4 (CvO (αMe)
Ser).
A suspension of 5a (432 mg, 1.1 mmol) and 10% cat. Pd/C
(50 mg) in EtOAc (20 mL) was stirred for 2 h under H2 (1 atm).
The palladium carbon catalyst was filtered off over Celite, and
the solution was concentrated in vacuo. The crude residue was
dissolved in DCM (10 mL) and HCl × Ala-OBut (182 mg,
1 mmol), DIEA (0.52 mL, 3 mmol) and 50% PPAA in DMF
(1.5 mL, 2.5 mmol) were added. After 12 h, the solvent was
evaporated under reduced pressure and the residue was dissolved
in EtOAc (50 mL). The organic solution was washed with 5%
NaHCO3 (3 × 15 mL), water (3 × 15 mL), 10% KHSO4 (3 ×
15 mL), water (3 × 15 mL), dried over MgSO4 and concentrated
under reduced pressure. The crude product was purified on silica
gel (EtOAc–hexane: 1 : 1) to yield 210 mg (38%) of 6a as a
foam.
Z-(R)-(αMe)Ser(ΨH,HPro)-Ala-OBut (5a)
HCl × Ala-OBut (0.91 g, 5 mmol) was added to a solution of 4a
(1.3 g, 5 mmol), DIEA (1.7 mL, 10 mmol) and TBTU (1.6 g,
5 mmol) in DCM (30 mL). After 12 h, the solvent was evapor-
ated under reduced pressure, and the residue was dissolved in
EtOAc (100 mL). The organic solution was washed with 5%
NaHCO3 (3 × 30 mL), water (3 × 30 mL), 10% KHSO4 (3 ×
30 mL) and water (3 × 30 mL), dried over MgSO4 and concen-
trated under reduced pressure. The crude product was purified on
silica gel (EtOAc–hexane: 1 : 1) to yield 1.5 g (76%) of 5a as a
foam; [α]D = +70.49° (c 1, CHCl3); Rf (C) = 0.35, Rf(D) = 0.60;
HPLC (40–80% B, 25 min) tR = 14.47 (100%); IR ν 3317,
2971, 2871, 1707, 1647, 1534, 1410, 1349, 1152, 1050, 946,
Fmoc-Ala-(S)-(αMe)Ser(ΨH,HPro)-Ala-OBut (6b from 5b) –
method II
As reported for the synthesis of 6a by using method II, the same
procedure was applied to the 6b, starting from 5b (1.1 mmol,
432 mg). The pure product was obtained in 34% yield (190 mg).
Molecular dynamics
1
809, 733, 695 cm−1; H NMR (700 MHz, CDCl3, TMS) δ 1.32
(d, J = 6.7 Hz, 3H, Cβ–H Ala), 1.48 (s, 9H, But), 1.67 (s, 3H,
Cβ–H (αMe)Ser), 3.77–3.80 (m, 2H, O–CH2–C), 4.39–4.40 (m,
1H, Cα–H Ala), 5.01–5.17 (m, 2H, N–CH2–O), 5.22–5.26 (m,
2H, CH2–Ph), 7.34–7.35 (bm, 1H, NH), 7.36–7.39 (m, 5H, C–H
arom.); 13C NMR (175 MHz, CDCl3, TMS) δ 18.1 (Cβ–H Ala),
19.7 (Cβ–H (αMe)Ser), 28.0 (CH3–C But), 49.0 (Cα–H Ala),
63.9 (CIV (αMe)Ser), 67.4 (CH2–Ph), 80.5 (N–CH2–O), 81.9
(O–CH2–C), 128.1, 128.3, 128.6 (C–H arom.), 135.9 (CIV Ph),
152.8 (CvO Z), 171.4 (CvO Ala), 171.8 (CvO (αMe)Ser).
All density functional calculations were carried out using the
Gaussian 03 package.45 Geometry optimizations employed the
B3LYP/6-31+G(d) level of theory.46 The solvation effects on
conformational preferences were estimated with a polarizable
continuum model (IEFPCM) in chloroform and water.47
Vibrational frequencies were calculated for optimized structures
at the same theory level in order to confirm that all of the opti-
mized structures are true stationary points at the potential energy
surface. Gibbs free energies, enthalpies and entropies were calcu-
lated at temperature of 298.15 K, 1 atm, with pressure and
vibrational frequencies were not scaled.
Z-(S)-(αMe)Ser(ΨH,HPro)-Ala-OBut (5b)
As reported for the synthesis of 5a, the same procedure was
applied to 5b, starting from 4b (1.3 g, 5 mmol). The pure
product was obtained in 82% yield (1.6 g), [α]D = −36.39° (c 1,
CHCl3); Rf (C) = 0.35, Rf(D) = 0.60; HPLC (40–80% B,
25 min) tR = 13.39 (100%); IR ν 3339, 2978, 2871, 1697, 1673,
X-ray single crystal structure analysis for Fmoc-Ala-(R)-(αMe)-
Ser(ΨH,HPro)-Ala-OBut (6a)
Formula: C30H37N3O7, Mw = 551.63, colorless crystal 0.10 ×
0.01 × 0.02 mm, a = 38.885(2), b = 7.501(1), c = 24.170(1) Å,
V = 5906.7(5) Å3, ρcalc = 1.241 g cm−3, μ = 1.03 cm−1, semi-
1
1517, 1407, 1349, 1152, 1051, 953, 764, 745, 697 cm−1; H
6714 | Org. Biomol. Chem., 2012, 10, 6705–6716
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14 (a) F. Bernardi, M. Garavelli, M. Scatizzi, C. Tomasini, V. Trigar,
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empirical absorption correction based on multiple scanned
equivalent reflections (0.927 < T < 0.973), Z = 8, crystal system:
monoclinic, space group: C2, λ = 0.68980 Å, T = 100 K,
ω scans, 28 384 reflections collected ( h, k, l), 2θmax = 55°,
7930 unique reflections (Rint = 0.076) and 7318 observed reflec-
tions [I ≥ 2σ(I)], 760 refined parameters, refinement in F2, Rall
=
0.061, wR(F2) = 0.1763, max (min) residual electron density
Δρmax = 0.88, Δρmin = −0.54) e Å−3, hydrogen atoms were
refined as riding on their parent carbon or nitrogen atoms. In the
absence of significant anomalous scattering effects, Friedel pairs
were merged for the final refinement cycles. Several crystalliza-
tion attempts from water–methanol and water–ethanol mixtures
yielded aggregates of small crystals, too weakly diffracting for
analysis with standard laboratory X-ray sources. The final X-ray
data were collected with a Bruker SMART APEX CCD area
detector diffractometer mounted at station 9.8 of the Synchrotron
Radiation Source at Daresbury Laboratory, Cheshire, England.
Computer programs used: data collection SMART APEX,48 data
reduction SAINT-Plus,49 absorption correction SADABS,50
structure solution, refinement and molecular graphics
SHELXTL.51 CCDC 870748.
19 K. Okuyama, C. Hongo, R. Fukushima, G. Wu, H. Narita, K. Noguchi,
Y. Tanaka and N. Nishino, Biopolymers, 2004, 76, 367–377.
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M. Mutter, Angew. Chem., Int. Ed., 2000, 39, 1111–1115.
21 M. Keller, T. Wöhr, P. Dumy, L. Patiny and M. Mutter, Chem.–Eur. J.,
2000, 6, 4358–4363.
Acknowledgements
This work was partially supported by grant 4 T09A 065 22 from
the State Committee for Scientific Research (Poland). Computer
time was provided by the Interdisciplinary Center for Mathemat-
ical and Computational Modeling, Warsaw University (Poland).
Authors are indebted to Dr Krzysztof Huben for final form of
graphics preparation.
22 L. Patiny, J.-F. Guichou, M. Keller, O. Turpin, T. Rückle, P. Lhote,
T. M. Buetler, U. T. Ruegg, R. M. Wenger and M. Mutter, Tetrahedron,
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23 A. Wittelsberger, L. Patiny, J. Slaninova, C. Barberis and M. Mutter,
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