Configurational and conformational control on formation and oligomerization of 2-C mono-arylated pseudo-proline dipeptide building units by aromatic stacking interactions

Article abstract of DOI:10.1055/s-0031-1289595

Electrophilically induced cyclic acetal formation of the O-benzyl dipeptide esters Fmoc-NMeIle-Thr-OBn (1) and of Fmoc-Pro-Thr-OBn (6) has been observed to lead predominantly to the (R) diastereomers 2b and 8b at the 2-C position of the resulting substitu

Full text of DOI:10.1055/s-0031-1289595

LETTER
935
Configurational and Conformational Control on Formation and
Oligomerization of 2-C Mono-Arylated Pseudo-Proline Dipeptide Building
Units by Aromatic Stacking Interactions
Michael Keller, Manfred Mutter, Christian Lehmann*
Institut de Chimie Organique, Université de Lausanne, BCH-Dorigny, CH-1015 Lausanne, Switzerland
Fax +41(21)692 3955; E-mail: Christian.Lehmann@ico.unil.ch and Manfred.Mutter@ico.unil.ch
Received 8 February 1999
libria by synthesis of mimetics with defined stereochemi-
cal properties.
Abstract: Electrophilically induced cyclic acetal formation of the
O-benzyl dipeptide esters Fmoc-NMeIle-Thr-OBn (1) and of Fmoc-
Pro-Thr-OBn (6) has been observed to lead predominantly to the
(R) diastereomers 2b and 8b at the 2-C position of the resulting
substituted 1,3-oxazolidine (ΨPro) unit, while upon acetalization of
the corresponding O-methyl ester 4 the 2-C(S) epimer 5a is pre-
dominantly formed under the same proton catalyzed cyclization
conditions. With boron trifluoride etherate as Lewis acid the
reaction is particularly fast and leads selectively to the prolyl
threonine derived 2-C(R) dipeptide building block 8b, which could
conveniently be assembled into a nonamer with a virtually solvent
independent CD-spectrum of the polyproline type I (cis amide
bonds).
In the context of our studies to incorporate hetero-ali-
cyclic proline analogues and their chiral substituted de-
rivatives (Scheme 1) into peptide sequences, we recently
found a remarkable stereogenic transfer:4 when the Fmoc
protected dipeptide benzyl ester Fmoc-NMeIle-Thr-OBn
1 (Scheme 2) was treated with anisaldehyde dimethylace-
tal in refluxing tetrahydrofuran in the presence of catalytic
amounts of pyridinium p-toluenesulfonic acid (PPTS), an
initially formed 10:1 mixture of the diastereomeric cyclic
2-aryl-1,3-oxazolidines 2a (C-2(S)) : 2b (C-2(R)) was
nearly quantitatively (2:98) transformed into the C-2(R)
derivative 2b.
Key words: acetals, peptide analogues/mimetics, substituent
effects, stereocontrol, cis/trans-polyproline
Proline represents the only proteinogenic side chain N-cy-
clized amino acid and therefore has special intrinsic con-
formational properties extending to the peptide secondary
structure segment where it is incorporated.1 With respect
to its other proteinogenic congeners, the conformational
space of proline is on one hand restricted around the en-
docyclic N-C bond to a dihedral angle Φi = -60 15°, on
the other hand, its adjacent exocyclic tertiary amide Ωi-1
dihedral angle is subject to greater variation and can un-
dergo receptor or solvent induced conformational switch-
ing between the trans (Ωi-1 = 180°) and the cis (Ωi-1 = 0°)
conformation. The investigation of such transitions are of
foremost importance not only to relate observable macro-
scopic properties such as CD-spectra with molecular con-
formation,2 but also to understand biological signaling
pathways dependent on ligand-receptor interactions.3
Biostructural chemistry aims at understanding these equi-
Scheme 2
The latter diastereomer was isolated by a standard work-
up and both diastereomers were characterized after HPLC
separation and hydrogenolysis of the benzyl ester to the
corresponding acids 3a and 3b.4,8 1H 2D NMR analysis
showed that the b-proton of the cyclized threonine residue
gives rise to a very strong NOE cross peak to the ortho-
protons of the p-methoxyphenyl (pmp) residue, which is
therefore oriented to the same side of the five-membered
ring (Figure 1).
Scheme 1
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936
M. Keller et al.
LETTER
The above bifurcation of the reaction coordinate seems to
hold only partially for the Lewis acid driven reaction con-
ditions, where under prolonged acetalization under the
conditions given in Scheme 3 even in the methyl ester
case predominant formation of the R-isomer is observed.
The more so, when the proline containing dipeptide
Fmoc-Pro-Thr-OBn 6 (Scheme 3) was subjected to acetal-
ization with p-fluoro-benzaldehyde dimethyl acetal in the
presence of boron trifluoro etherate, the reaction was im-
proved to occur to completion already at room tempera-
ture without the detection of an alternant stereoisomer by
HPLC analysis. The more electron deficient aromatic al-
dehyde was used to increase stability towards acid-cata-
lyzed ring opening in the further elongated oligomer. The
dipeptide isomer was further isolated by hydrogenolysis
and chromatography on silicagel.9 Although stereochem-
Figure 1
Given this stereochemistry for the main acetalization ically uniform according to HPLC and NMR, the product
product 2b, molecular modeling using the MAB-force 8b was observed to be conformationally inhomogeneous
field5 lead us to a plausible rationalization for this stereo- (main conformer: ω-trans; 80%), similarly as has been
selectivity: The two diastereomers C-2(R) and C-2(S) dif- documented previously by 1H 2D NMR for a series of re-
fer in energy primarily in their non-bonding distant (nbonds lated R-configured ΨPro derivatives.6
> 4) interactions term which was calculated to amount to
6.5 kcal in favor of the C-2(R) isomer (Figure 2). For re-
actions proceeding exergonically, it is generally accepted
to assume a product-like transition state which is bound to
be different for the two pathways leading to the (R)- and
the (S)-diastereomers respectively. Therefore the
transacetalization leading to the C-2(R) isomer is estimat-
ed to be favored significantly with respect to the epimeric
conversion, and this result emerges even without explicit
inclusion of solvent in the force-field approach. Since the
Scheme 3
reaction does not proceed at all in an aromatic lipophilic
solvent like toluene,4 a more direct proof by solvent inter-
ference is not possible. However, the mechanistic hypoth-
esis is confirmed by the fact that under the same
conditions the methyl ester substrate 4 is predominantly
transformed to the C-2(S) isomer 5a which is devoid of
such a transannular stacking relay, but can on the other
hand dispose all of its five-membered ring substituents in
an equatorial mode.
Scheme 4
Figure 2
Synlett 1999, S1, 935–939 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Stereocontrol in Pseudo-Proline Containing Peptides
937
Can we further take advantage of intramolecular stacking ΨPro can further be exploited to the general problem of
of aromatic substituents to increase stereochemical homo- secondary structure stabilization. In particular, the results
geneity? When assembled into peptide oligomers of alter- presented here are most promising for the understanding
nating Pro-ΨPro constitution (Scheme 4), a related Pro- and control of the polyproline I/II conformational transi-
aromatic stacking effect may be responsible for directing tion.
the cis/trans equilibrium along the substituted amide
bonds into a homogeneous cis (polyproline type I) confor-
mation. The CD-spectrum of the nonamer Ac-[Pro-
Thr(Ψ(R)-p-F-Ph,Hpro)]-Pro-OH 9 (Figure 4) is virtually sol-
vent independent and altered only marginally to a station-
ary curve with a slight decrease of absorption at 215 nm.
This result is significant not only because the CD-curve of
the all-trans-polyproline has been reported2 to virtually
take up the shape of a mirror image with respect to the
wavelength axis (at least above 200 nm where no aromatic
absorbance interferes), but also because in high-resolution
1H 2D NMR10 only one set of signal is observed for the C-
2 proton of each of the ΨPro residues. In addition, a co-
herent series of cis-characteristic CαH-CαH NOE signals is
observed. In contrast to the parent oligoproline constitu-
tion, no transition to the more extended, backbone ex-
posed trans (polyproline II) conformation could be
enforced by solvent exchange, an observation which we
correlate to the stabilization of the more compact polypro-
line I like helix by intramolecular interactions of the ad-
dressed aromatic stacking type.
Figure 4
Acknowledgement
We thank Dipl. Chem. Cédric Sager and Drs. Yoshiro Tatsu and
Luc Patiny for assistance in confirming stereochemical assignments
1
by 2D H NMR, and the Swiss National Science Foundation for
financial support.
References and Notes
(1) Rose, G.D.; Gierasch, L.M.; Smith, J.A. Adv. Prot. Chem.
1985, 37, 1-109. Richardson, J.S., ibid. 1981, 34, 116-339.
Smith, J.A.; Pease, L.G. CRC Crit. Rev. Biochem. 1980, 8,
315-399.
(2) Sreerama, N.; Woody, R.W. Biochemistry 1994, 33, 10022-
10025. Thomasson, K.A.; Applequist, J. Biopolymers 1991,
31, 529-535. Rothe, M.; Rott, H.; Mazanek, J. Peptides, Proc.
Eur. Pept. Symp. 14th 1976 (A. Loffet, Ed., Editions de
l’Université de Bruxelles), pp. 309-318.
(3) Fischer, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 1415-
1436. Chen, J.K.; Schreiber, S.L. ibid. 1995, 34, 953-969.
(4) Keller, M.; Lehmann, C.; Mutter, M. Tetrahedron 1999, 55,
413-422.
lizing, structure disrupting protection technique to render
(5) Gerber, P.R.; Müller, K. J. Comput.-Aided Mol. Design 1995,
Historically, the ΨPro-concept was developed as a solubi-
difficult sequences accessible to solid phase peptide syn-
9, 251-268. Gerber, P.R. ibid. 1998, 12, 37-51.
(6) Dumy, P.; Keller, M.; Ryan, D.E.; Rohweder, B.; Wöhr, T.;
thesis and to set free the protected hydroxyl groups by
concentrated acidolysis after the oligomer assembly.7 In
the present work we showed that the synthetic concept of
Mutter M. J. Am. Chem. Soc. 1997, 119, 918-925.
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938
M. Keller et al.
LETTER
After 7h at 80 °C, only one isomer could be observed by
(7) Wöhr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun,
X.; Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218-9227, and
ref. cited therein.
HPLC. Heating at 80 °C was continued for another 10 h; very
weakly, a second peak close to the peak assigned to the (R)-
epimer 5b was observed. To the yellowish liquid was added
EtOAc (20 ml), and the solution washed with Na2CO3 (10%,
20 ml) and water (20 ml) before drying the organic layer over
MgSO4. Deprotection of the methylester was achieved using
LiOH (3.5 equiv in THF/H2O = 4:1, 5 ml, 2 h). The epimers
now were separated on the same gradient as above. The
relation between the 2-C (R) and (S)-iomer only changed
slightly to 8:92. (S)-epimer 3a: tR = 15.13 min, (R)-epimer
3b: tR = 15.84 min (50-100% B, 20 min, C18). The product was
purified by flash chromatography over silica using CHCl3/
CH3OH (100:15) as eluent to obtain 2-C(S) Fmoc-NMeIle-
Thr(ΨΗ,pmppro)-OH. C35H38N2O7 = 586.7. Yield:56 mg, 96%.
MS-ESI (m/z) 587.6 [M+H]+. Co-injection of the purified 2-
C(S) compound with the isolated kinetic product of the O-
benzyl reaction gave one single peak in HPLC.
(8) Experimentals to Scheme 2: Fmoc-NMeIle-Thr(ΨΗ,pmppro)-
OBn (2) C41H44N2O7 = 676.8 Fmoc-NMeIle-Thr-Bn (1; 50
mg, 0.09 mmol) was dissolved in THF (2 ml). PPTS (6.8 mg,
0.3 equiv) and anisaldehyde dimethylacetal (0.096 ml, 5
equiv) added and heated under reflux for 7h. Samples were
taken after 1h, 3h and 7h in order to follow the reaction by
HPLC: isomer 2-C(S) 2a: rt = 22.48 min, isomer 2-C(R) 2b:
rt = 23.47 min, 50-100% B, C18 (A = water containing 0.09%
TFA; B = acetonitrile for HPLC-R containing 10% water and
0.09% TFA; cf. Scheme 2 in lit.4). Complete conversion to 2-
C(R) 2b was stated after 7 hours. The solvent was evaporated
and replaced by AcOEt (20 ml) and washed subsequently with
aqueous Na2CO3 (0.5 M, 20 ml, 3x) and water (20 ml, 3x). The
organic layer was dried over MgSO4, filtered and the solvent
removed in vacuo. To the slightly yellow oily residual was
added methanol (0.10 ml), before addition of ether (5 ml) to
yield oxazoline 2b as a white precipitate (98%), which was
collected on a glass filter and recrystallized from methanol
(0.1 ml) / ether (5 ml). HPLC: 23.52 min (95% purity; no other
isomer detected). MS-ESI (m/z): 676.8 [M+H]+. Separation of
the diastereomers: In a separate reaction, the same conditions
as above described were used, but the reaction was stopped
after 1.5 h and the isomers separated by means of reversed
phase HPLC, using an isocratic gradient 40% A and 60% B to
100% B, to obtain 20 mg of each epimer (2a and 2b)
(9) Experimentals to Scheme 3: Fmoc-Pro-Thr(ΨH,p-F-Phpro)-
OBn (7) C38H35N2O6F = 634.7. Fmoc-Pro-Thr-OBn (6; 1.3g,
2.05 mmol) was dissolved in CH2Cl2 (130 ml) before adding
para-fluorobenzaldehyde dimethylacetal 2.1 ml, 10 equiv)
under nitrogen atmosphere. To the clear solution was added
BF3.OEt2 (965 ml, 7.8 mmol) and stirred. The solution first
turned to yellow and after two minutes to bordeaux red. After
20 min, the reaction was stopped by adding a solution of
Na2CO3 (10%, 50 ml). The organic layer was washed with
water (50 ml, 2) and dried over MgSO4. All solvent was
evaporated and replaced with CH3OH. To this solution of
crude acetal 7, Pd-C (130 mg) was added and hydrogen
bubbled through the solution for 1.5 h. After filtration over
Celite and evaporation of the filtrate, a yellowish oil was
reconstituted, which was purified on silica using CHCl3/
CH3OH/HOAc (100/10/1 ml) as eluent. 660 mg (1.21 mmol,
49%) of a white solide was isolated and identified as Fmoc-
Pro-Thr(ΨH,p-F-Phpro)-OH (8b; assigned to 2-C(R) based on
a preliminary 1H 2D NMR NOESY experiment)
separately. Fmoc-NMeIle-Thr(ΨΗ,pmppro)-OH (3)
C35H38N2O7 = 586.7 Deblocking of the benzyl protecing
group was achieved under hydrogen atmosphere in methanol
(5 ml) using Pd/C as catalyst. After completion of the
deprotection, the suspension was filtered over Celite and all
solvent evaporated. HPLC: 2-C(R) epimer 3b 15.9 min (95%
purity; no other isomer), 2-C(S) epimer 3a 15.1 min (single
peak), 50-100% B, C18; MS-ESI m/z) 559.8 [M+H]+. 1H
NMR(400MHz, 10mg/ml, CDCl3, 300 K):2-C(R)-stereo-
isomer (3b; all-trans in CDCl3): δ (ppm) 7.8 (d, 2H, J = 7.6
Hz, Fmoc), 7.62 (d, 2H, J = 7.6 Hz, Fmoc), 7.55 (d, 2H,
J = 8.8 Hz, o-pmp), 7.42 (d×d, 2H, Fmoc), 7.35 (d×d, 2H,
Fmoc), 7.28 (s, CHCl3), 6.94 (d, 2H, J = 8.8 Hz, m-pmp), 6.73
(s, 1H, 2-H), 4.6 (d, 1H, β-Thr), 4.59 (d, 1H, Fmoc-CH), 4.46
(m, 1H, Fmoc-CH2), 4.4 (m, 1H, β-Ile), 4.3 (m, 1H, Fmoc-
CH2’), 4.29 (d, 1H, J = 8 Hz, α-Thr), 3.835 (s, 3H, OMe), 2.88
(s, 3H, NMe), 2.03 (m, 1H, β-Ile), 1.46 (d, 3H, J = 6 Hz, β-
Thr), 0.721 (m, 3H, δ-Ile), 0.472 (d, 3H, J = 6.8Hz, γ-Ile). 2-
C(S)-stereoisomer (3a; 40% cis and 60% trans in DMSO-d6):
7.8 (d, 1H, J = 6.8 Hz, m-pmp trans), 7.74 (d, 1-2H, J = 6.0
Hz, m-pmp cis), 7.6 (d, 1H, J = 6.2 Hz, o-pmp trans), 7.52 (d,
1-2H, J = 6.4 Hz, o-pmp cis), 7.29-7.33 (m, 4-6H, Fmoc cis
and trans), 7.04 (d, 1-1.5H, J = 8.8 Hz, Fmoc), 6.86 (d, 1-
1.6H, J = 8.8 Hz, Fmoc), 6.15 (s, 0.2-0.5H, α-Thr trans), 6.09
(s, 1-2H, α-Thr cis), 5.89 (s, 1-1.5H, 2-H trans), 5.49 (s, 0.8-
1.2H, 2-H cis), 4.28 (d, 1H, J = 7.8 Hz, β-Thr), 4.11 (m, 0.5-
1H, α-Ile cis), 4.05 (m, 0.3-0.7H, α-Ile trans), 3.87 (m, 1-2 H,
Fmoc-CH cis and trans), 3.79 (s, 3H, NMe), 3.74 (s, 3H,
OMe), 3.66 (m, 2-3H, Fmoc-CH2), 3.3-3.5 (s, H2O), 2.65 (m,
1H, β-Ile cis), 2.65 (m, 1H, β-Ile trans), 2.49 (s, DMSO-d6),
1.79 (m, 2-3H, β-Ile-CH3, cis), 1.72 (m, 3-4H, β-Ile-CH3
trans), 1.43 (d, 3-4H, J = 5.6 Hz, β-Thr-CH3 trans), 0.91 (d, 2-
3H, β-Thr-CH3 cis), 0.8 (m, 3-4H, γ-Ile-CH3 trans), 0.72 (d, 2-
3H, J = 6.8 Hz, γ-Ile-CH3 cis). Fmoc-NMeIle-
C31H29N2O6F = 544.7. MS-ESI (m/z) 545.2 M+H+. HPLC
tR = 12.38 min (50-100% CH3CN), 93% purity (only one
isomer). 1H NMR (400 MHz, CDCl3, 10 mg/ml, 295 K; two
conformers; 80% ω-trans): δ (ppm) 7.8 (d, 2H, aromatic
Fmoc), 7.65 (dd, 2H, ortho H-p-F-Ph), 7.6 (t, 2H, aromatic
Fmoc), 7.4 (t, 2H, aromatics Fmoc), 7.3 (t, 2H, aromatic
Fmoc), 7.27 (s, CDCl3), 7.1 (t, 2H, meta H-p-F-Ph), 6.7 (s, 1H,
2-H oxazolidine; ω-trans), 6.45 (s, 0.08H, 2-H oxazolidine;
further conformational isomer), 6.0 (s, 0.17H, 2-H
oxazolidine, ω-cis), 4.5 (t, 1H, β-Thr), 4.4 (dd, 1H, α-Thr and
Fmoc-CH), 3.6 (m, 1H, γ-Pro), 3.5 (m, 1H, γ-Pro), 2.1 (m, 1H,
δ-Pro), 1.95 (m, 1H, δ’-Pro), 1.75 (m, 1H, β’-Pro), 1.5 (m, 1H,
β’-Pro), 1.45 (d, 3H, β-CH3-Thr; ω-trans), 1.25 (d, 0.8H, β-
CH3-Thr; ω-cis).
(10) Experimentals to Scheme 4: Solid Phase Synthesis of Ac-Pro-
Thr(H,para-F-PhPro)4-OH (9). Standard protocols for Fmoc-
chemistry on Sasrin resin were used. Commercially available
Fmoc-Pro-Sasrin (0.64 mmol/g resin, BACHEM, Bubendorf,
Switzerland) was dried in vacuo over night before use. 50
mmol/g resin (ca. 0.03 mmol) was swelled in DMF
(dimethylformamide) for 30 min and washed with CH3OH (10
ml, 1x), CH2Cl2 (10 ml, 3x) and DMF (10 ml, 3x). Fmoc
deprotection was carried out using piperidine in DMF (20%,
3 × 5 min). Coupling reagent HBTU (3 equiv, 35 mg, ALEXIS,
Läufelfingen, Switzerland), deprotection base DIEA
(diisopropylethylamine, 6 equiv, 35 ml). For each coupling, 3
equivalents of Fmoc-Pro-Thr(H,para-F-PhPro)-OH (50 mg) were
taken. Solvent for the couplings was DMF (5 ml). Couplings
(1.5 h) were verified upon their completion by reversed phase
HPLC of a small aliquot cleaved from the resin be 2% TFA in
Thr(ΨΗ,pmppro)-OMe (5) C36H40N2O7 = 600.8. Fmoc-NMe-
Ile-Thr-OMe (4; 50 mg, 0.083 mmol), PPTS (7.8 mg, 0.3
equiv) and anisaldehyde dimethylacetal (0.11 ml, 5 equiv) in
THF (2.5 ml) were heated under reflux for 16 h. The reaction
was followed by HPLC (Gradient 50-100% B, 20 min, C18).
Synlett 1999, S1, 935–939 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
CH2Cl2 after each coupling step. After the last coupling and
Stereocontrol in Pseudo-Proline Containing Peptides
939
C71H79N9O15F4 = 1374.5 MS-ESI (m/z) 1375.5 M+H+. HPLC
tR = 10.46 min (50-100% acetonitrile, 20 min). 1H NMR (400
MHz, CDCl3, 5 mg/ml, 298 K): δ (ppm) 8.0 (m, 6-7H, ortho-
H p-F-Ph), 7.29 (s, CDCl3), 7.1 (m, 5-6H, meta-H p-F-Ph),
6.75 (s, 1H, 2-H oxazolidine), 6.67 (s, 2H, 2-H’/H’’
oxazolidine), 6.62 (s, 1H, 2-H’’’ oxazolidine),1.5-5 (33H,
α,β,γ,δ−Pro, α,β-Thr), 2.08 (s, 3H, Ac), 1.25-1.5 (4d, 12H, β-
CH3-Thr).
deprotection, the sequence was N-capped by acetylation using
acetic anhydride (3 equiv, 10 ml) in the presence of DIEA (5
equiv, 35 ml) in DMF (5 ml) during 16 h. Finally, the
assembly was cleaved from the support by 2% TFA in CH2Cl2
(2 min) and immediately neutralized with 3 equivalents of
DIEA (5%, CH2Cl2) to prevent ring opening of the
oxazolidine. The organic layer was washed with citric acid (20
ml, 3x) and water (millipur, 20 ml, 3x), dried over MgSO4 and
the solvent removed under vacuo. The peptide was further
purified by semipreparative HPLC using a gradient of 20-50%
(30 min) CH3CN containing 0.09% TFA, and the peptide
containing fractions lyophilized to obtain 6.2 mg of pure
peptide. Ac-Pro-Thr(H,para-F-PhPro)4-OH (9)
Article Identifier:
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Synlett 1999, S1, 935–939 ISSN 0936-5214 © Thieme Stuttgart · New York