Regioselective synthesis of antiparallel loops on a macrocyclic scaffold constrained by oxazoles and thiazoles

Article abstract of DOI:10.1021/ol026463m

(matrix presented) The regioselective syntheses and structures are reported for two tris-macrocylic compounds, each possessing two antiparallel loops on a macrocyclic scaffold constrained by two oxazoles and two thiazoles. NMR solution structures show the

Full text of DOI:10.1021/ol026463m

ORGANIC
LETTERS
2002
Vol. 4, No. 20
3367-3370
Regioselective Synthesis of Antiparallel
Loops on a Macrocyclic Scaffold
Constrained by Oxazoles and Thiazoles
Yogendra Singh, Martin J. Stoermer, Andrew J. Lucke, Matthew P. Glenn, and
David P. Fairlie*
Centre for Drug Design and DeVelopment, Institute for Molecular Bioscience,
UniVersity of Queensland, Brisbane, Qld 4072, Australia
d.fairlie@imb.uq.edu.au
Received July 3, 2002
ABSTRACT
The regioselective syntheses and structures are reported for two tris-macrocylic compounds, each possessing two antiparallel loops on a
macrocyclic scaffold constrained by two oxazoles and two thiazoles. NMR solution structures show the loops projecting from the same face
of the macrocycle. Such molecules are shown to be prototypes for mimicking multiple loops of proteins.
Protein loops are common recognition motifs in biology, yet
creation of small molecules to structurally mimic bioactive,
multiloop surfaces remains a challenging objective.1,2 We
were inspired by Nature’s use in bacteria, fungi, plants, and
marine organisms of unusual amino acids containing ox-
azoles/thiazoles and their reduced analogues as constraints
to regulate the shapes3 of macrocycles4 like ascidiacyclamide
1,4b nostocyclamide,4c and thiopeptide antibiotics.4d Related
cyclic peptides show antitumor, antiinflammatory, immu-
noregulating, and enzyme-inhibiting activities.5 In ulithia-
cyclamide 2,4e the macrocycle is a scaffold supporting one
disulfide bridge. Here we report an efficient regioselective
synthesis and solution structure of tris-macrocycle 3, with
(4) (a) Wipf, P. Chem. ReV. 1995, 95, 2115-2134. (b) Ishida, T.; Inoue,
M.; Hamada, Y.; Kato, S.; Shiori, T. J. Chem. Soc., Chem. Commun. 1987,
370-371. (c) Todorova, A. K.; Juttner, F.; Linden, A.; Pluss, T.; von
Philipsborn. W. J. Org. Chem. 1995, 60, 7891-7895. (d) Toske, S. G.;
Fenical, W. Tetrahedron Lett. 1995, 36, 8355-8. (e) Ireland, C. M.; Scheuer,
P. J. J. Am. Chem. Soc. 1980, 102, 5688-5691. (f) Sowinski, J.; Toogood,
P. L. Tetrahedron Lett. 1995, 36, 67-70. (g) Fate, G. D.; Benner, C. P.;
Grode, S. H.; Gilbertson, T. J. J. Am. Chem. Soc. 1996, 118, 11363-11368.
(h) McGeary, R. P.; Fairlie, D. P. Curr. Opin. Drug Discuss. DeV. 1998, 1,
208-217.
(5) (a) Mocek, U.; Zeng, Z.; O’Hagan, D.; Zhou, P.; Fan, L.-D. G.; Beale,
J. M.; Floss, H. G. J. Am. Chem. Soc. 1993, 115, 7992-8001. (b) Rinehart,
J. B.; Gloer, J. B.; Cock, J. C.; Mizsak, S. A.; Scahill, T. A. J. Am. Chem.
Soc. 1981, 103, 1857. (c) Rinehart, K. L.; Kishure, V.; Bible, K. C.; Sakai,
R.; Sullins, D. W.; Li, K. M. J. Nat. Prod. 1988, 51, 1-21. (d) Ireland, C.
M.; Durso, A. R.; Newman, R. A.; Hacker, M. P. J. Org. Chem. 1982, 47,
1807-1811. (e) Shioiri, T.; Hamada, Y.; Kato, S.; Shibata, M.; Kondo, Y.;
Nakagawa, H.; Kohda, K. Biochem. Pharm. 1987, 36, 4181-4185.
(1) (a) Fairlie, D. P.; West, M. L.; Wong, A. K Curr. Med. Chem. 1998,
5, 29-62. (b) Reineke, U.; Schneider-Mergener, J. Angew. Chem., Int. Ed.
1998, 37, 769-771.
(2) (a) Bisang, C.; Weber, C.; Robinson, H. A. HelV. Chim. Acta 1996,
79, 1825-1841. (b) Mutter, M.; Dumy, P.; Garrouste, P.; Lehmann, C.;
Mathieu, M.; Peggion, C.; Peluso, S.; Razaname, A.; Tuchscherer, G. Agnew.
Chem., Int. Ed. Engl. 1996, 35, 1482-1485. (c) Hamuro, Y.; Calama, M.
C.; Park, H. S.; Hamilton, A. D. Agnew. Chem., Int. Ed. Engl. 1997, 36,
2680-2683. (d) Favre, M.; Moehle, K.; Jiang, L.; Pfeiffer, B.; Robinson,
J. A. J. Am. Chem. Soc. 1999, 121, 2679-2685.
(3) (a) Fairlie, D. P.; Abbenante, G.; March, D. R. Curr. Med. Chem.
1995, 2, 654-86. (b) Abbenante, G.; Fairlie, D. P.; Gahan, L. R.; Hanson,
G. R.; Pierens, G.; van den Brenk, A. L. J. Am. Chem. Soc. 1996, 118,
10384-10388.
10.1021/ol026463m CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/11/2002

two antiparallel loops containing oxazole projecting perpen-
dicularly from the same face of a macrocyclic scaffold 6
constrained by oxazoles and thiazoles. The alternative tris-
macrocycle isomer 7, in which the loops contain thiazole
instead of oxazole, was independently synthesized and
structurally characterized by NMR spectroscopy. Molecular
models suggest that scaffold 6 could be suitable for mimick-
ing multiloop surfaces of proteins such as interhelical loops
from helix bundles and complementarity-determining loops
of antibodies.
Functionalized dipeptide surrogates Boc-L-Lys(Cbz)(Ox)-
OH (4a X ) O, R ) -(CH2)4NHCO2Bn)6 and L-Glu(OcHx)-
(Thz)-OH (4b, X ) S, R ) -(CH2)2CO2cHx)7a were
elaborated to tetrapeptide analogue 5 (R1 ) -(CH2)4NHCO2-
Bn, R2 ) -(CH2)2CO2cHx). Cyclodimerization of 5 (Scheme
1) followed by deprotection gave a high isolated yield (84%)
Figure 1. Natural products 1 and 2 and synthetic target 3.
chains are directed from the same face of the macrocycle,
as in other oxazole- or thiazole-containing macrocycles,10
suggesting 6 as a likely scaffold for supporting discontinuous
loops.
Scheme 1a
As a prelude to grafting loops from protein surfaces onto
scaffold 6, we prepared prototypes 3 and 7 (isolated yields:
71 and 1%, respectively)11 by coupling L-Lys and L-Glu side
chains of 6 (Scheme 2).
Compound 3 was highly symmetrical, displaying one set
1
of H and 13C resonances, indicative of C2 fold symmetry.
3
The chemical shifts for Lys RNH (δ 8.33, JNH-RH ) 9.06
Hz) and Lys ꢀNH (δ 7.73) resonances of 3 in DMSO-d6 are
temperature dependent (∆δ/T 4.9, 3.6 ppb/K, respectively)
and exchange rapidly upon addition of 5% D2O. By contrast,
a Reaction conditions: (a) BOP, DIPEA, DMF, 4b, rt (97%).
(b) TFA, CH2 Cl2, ∼0 °C (100%). (c) BOP, DIPEA, DMF (7 ×
10-3 M), rt. (d) NaHCO3. (e) HF, p-cresol (84% from 5).
3
the Glu RNH resonance (δ 8.22, JNH-RH ) 7.96 Hz) for 3
is almost temperature independent (∆δ/T 0.46 ppb/K) and
only exchanges slowly with D2O, features consistent with a
hydrogen bond, the H-bond acceptor being identified as the
Glu-γ-CO by ROEs (see below). The different 3JNH-RH values
for Glu and Lys amide-NHs of the scaffold translate, via
the Karplus equation, to NHRCH dihedral angles of 148 and
158°, respectively, which in turn correspond to Φ angles of
-150 (Glu) and -140° (Lys). The conformational strain in
3 is evidenced by significant chemical shift differences for
several pairs of geminal protons within the loop portion of
the molecule (Glu âHs 0.54, Glu γHs 0.23, Lys âHs 0.83,
Lys ꢀHs 1.13 ppm), consistent with their differing orienta-
tions above the macrocyclic template.
of cyclic octapeptide analogue 6 (R1 ) -(CH2)4NH2, R2 )
-(CH2)2CO2H).8 Dimerization is favored over cyclooligo-
merization7b (∼16%) by the presence of two turn-inducing
heterocyclic oxazole/thiazole constraints. NMR spectra (1H,
13C) for 6 indicate C2 fold symmetry.
Molecular modeling9 (Figure 2) identified 6 as a rigid,
rhombus-shaped macrocycle (∼6.5 × ∼6.6 Å) similar to the
marine natural product ascidiacyclamide (1).4b It has a C2
fold axis of symmetry with the two thiazoles tilted ∼90° out
of the macrocycle plane. The flexible L-Lys and L-Glu side
ROE data support the presence of a H-bond between Glu
NH‚‚‚OCγ Glu, with correlations between Glu γH (δ 2.16)
(6) The oxazole was prepared from Boc-Lys(Z)Ser-OMe by cyclodehy-
dration (DAST) followed by oxidation (BrCCl3/DBU): Phillips, A. J.; Uto,
Y.; Wipf, P.; Reno, M. J.; Williams, D. R. Org. Lett. 2000, 2, 1165-1168.
(7) (a) Singh, Y.; Sokolenko, N.; Kelso, M. J.; Gahan, L. R.; Abbenante,
G.; Fairlie, D. P. J. Am. Chem. Soc. 2001, 123, 333-334. (b) Sokolenko,
N.; Abbenante, G.; Scanlon, M. J.; Jones, A.; Gahan, L. R.; Hanson, G. R.;
Fairlie, D. P. J. Am. Chem. Soc. 1999, 121, 2603-2604.
(8) TFA‚H-Lys(Z)(Ox)Glu(OcHx)(Thz)-OH (5, 1.4 g, 1.9 mmol) and
BOP (1.1 g, 2.5 mmol) were dissolved in DMF (260 mL) and stirred with
DIPEA (1.1 mL, 6.3 mmol) at 20 °C for 24 h. Workup, HF cleavage, and
HPLC purification yielded 6 (812 mg, 84%; HRMS: (M + H) found
815.2599, calcd 815.2605).
(9) (a) MacroModel: Mohamadi, F.; Richards, N. G. J.; Guida, W. C.;
Kiskamp, R.; Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still,
W. C. J. Comput. Chem. 1990, 112, 440-447. (b) Monte Carlo (MCMM):
Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc. 1989, 111, 4379-
4386. (c) Search, AMBER*: McDonald, D. Q.; Still, W. C. Tetrahedron
Lett. 1992, 33, 7743-7746. (d) Force field, water GB/SA solvent
continuum: Still, W. C.; Tempczyk, A.; Hawely, R. C.; Hendrickson, T. J.
Am. Chem. Soc. 1990, 112, 6127-6129. (e) Minimization: Polak, E.;
Ribiere, G. ReV. Fr. Inf. Rech. Oper. 1969, 16-R1, 35.
Figure 2. Superposition of 59 low energy conformations of 6 (∆E
< 11.5 kJ/mol, RMSD < 0.7 Å for 24 atoms) viewed top down
(left) and side on (right); side chains are omitted for clarity.
3368
Org. Lett., Vol. 4, No. 20, 2002

Scheme 2. Cyclization of 6‚2TFA (1 × 10-3 M) from 0 to 20
°C gives a 10:1 ratio of 3 and 7.11
Scheme 3a
a Reaction conditions: (a) BOP, DIPEA, DMF, rt (98%). (b)
TFA, CH2Cl2, ∼0 °C (100%). (c) BOP, DIPEA, DMF (6 × 10-3
M), rt (91%). (d) LiOH, EtOH/H2O (3:1), ∼0 °C. (e) HF, p-cresol
(90% from 10). (f) BOP, DIPEA, DMF (6 × 10-3 M), rt (78%).
H-bonds described above. The 20 lowest energy (E < 10
kJ/mol) calculated structures, shown superimposed in Figure
3, had no residual distance (>0.16 Å), H-bond, or dihedral
and Glu NH (medium) and between Glu NH and Lys ꢀNH
(weak). The latter indicates that the loops incorporate oxazole
rather than thiazole. Other intraloop correlations between Lys
RNH and Lys δH (δ1.44, weak; 1.14, strong) and Lys âH
(δ 2.54) and Lys ꢀH (δ 3.86, strong) as well as interloop
correlations between Glu RNH and Lys âH (δ 2.54, vs weak)
and Glu RNH and Lys γH (δ 1.14, vs weak) define the
locations of the two loops.
The identity of the loops was confirmed by an alternative
independent synthesis of 3 (Scheme 3) by coupling Cbz-L-
Lys(Boc)(Ox)-OH 8 to L-Glu(OtBu)(Thz)-OEt 9 to give Cbz-
Lys(Boc)(Ox)Glu(OtBu)(Thz)OEt 10. Subsequent loop for-
mation to 11 preceded dimerization to 3.
Figure 3. Superimposition of the 20 lowest energy NMR structures
of 3 shown top down (left) and side on (right).
angle violations. There was tight convergence of the mac-
rocyclic scaffold (RMSD 0.2 Å), while the loops retained
some flexibility (RMSD 1.8 Å).
After modification of standard topology and parameter
files (Supporting Information) to accommodate the unnatural
dipeptide surrogates, the structure of 3 was calculated in
XPLOR12 using only the two Lys dihedral angle restraints
(F ) -120 ( 30°). The resulting convergent structures were
refined using 60 ROE distance constraints and the two
The thiazole-containing macrocyclic component of 3
differs from the modeled template 6 in that the oxazoles are
now (90° out of the plane of the thiazole-containing
macrocycle and in the plane of the oxazole-containing loops.
The thiazoles are tilted 20-30° (instead of 90°) down from
the plane of the macrocycle corresponding to 6. The CR-
Câ vectors are 6 Å apart and project from the same face of
the scaffold, directing both oxazole-containing loops per-
pendicularly and staggered relative to one another.
(10) (a) Mink, D.; Mecozzi, S.; Rebek, J., Jr. Tetrahedron Lett. 1998,
39, 5709-5712. (b) Haberhauer, G.; Somogyi, L.; Rebek, J., Jr. Tetrahedron
Lett. 2000, 41, 5013-5016. (c) Somogyi, L.; Haberhauer, G.; Rebek, J.,
Jr. Tetrahedron Lett. 2001, 57, 1699-1708. (d) Boss, C.; Rasmussen, P.
H.; Wartini, A. R.; Waldvogel, S. R. Tetrahedron Lett. 2000, 41, 6327-
6331. (e) Bertram, A.; Hannam, J. S.; Jolliffe, K. A.; Gonzalez-Lopez de
Turiso, F.; Pattenden, G. Synlett 1999, 11, 1723-1726.
(11) DIPEA (0.2 mL, 1.15 mmol) was added to 6‚2TFA (90 mg, 0.085
mmol) and DPPA (60 µL, 2.78 mmol) in dry DMF (70 mL) at 0 °C and
stirred (2 h, 0 °C.; 48 h, 20 °C), concentrated, and purified by rpHPLC to
give 3 (48 mg, 71%; HRMS: (M + H) found 779.2388, calcd 779.2392)
and side product 7 (1% yield). Both 3 and 7 were subsequently synthesized
independently (1f-1t, Supporting Information). When BOP was used for
coupling, hydroxybenzotriazole was trapped by 3, coeluting from rpHPLC
(12) XPLOR 3.851 using a dynamic simulated annealing protocol in a
geometric force field and minimized using CHARm: (a) Bru¨nger, A. T.
X-PLOR Manual Version 3.1; Yale University: New Haven, CT, 1992.
(b) Nilges, M.; Gronenborn, A. M.; Bru¨nger, A. T.; Clore, G. M. Protein
Eng. 1988, 2, 27-38. (c) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.;
States, D. J.; Swaminathan, S.; Karplus, M. J. Comput. Chem. 1983, 4,
187-217.
1
columns and detectable in H NMR spectra.
Org. Lett., Vol. 4, No. 20, 2002
3369

Figure 4. Superimposition of the 20 lowest energy NMR structures
of 7 shown end on (left) and side on (right).
Figure 5. Scaffold from 3 (NMR structure) with two grafted loops
(green) excised from crystal structures: (a, left) interhelical regions
(A167-171, A230-234) of R-catenin (pdb: 1DOW),14a and (b,
right) CDR loops L3 (L92-96) and L1 (L24-27) of antibody Igg1
Fab’ fragment B13I2 (pdb: 2IGF);14b both were modeled using
template forcing (Supporting Information) on corresponding 4-5
residue loop fragments (blue ribbons) from crystal structures.14
Regioselective formation of oxazole- rather than thiazole-
containing loops in 3 may be due to formation of the Glu
NH‚‚‚OC Glu H-bond during loop formation, though we
were unable to detect it at low temperatures. Alternatively,
steric hindrance from the thiazoles may reduce access of the
amine to the bulky activated ester. It is possible that
π-complex formation13 between oxazole and activated ester
stabilizes an intermediate for condensation, as suggested by
trapping of hydroxybenzotriazole during synthesis.11
Isomer 7 was independently synthesized (Scheme 3) from
L-Lys(Boc)(Ox)-OMe and Cbz-L-Glu(OtBu)(Thz)-OH in-
stead of 8 and 9. Like 3, isomer 7 displayed C2 fold
symmetry in 1H and 13C NMR spectra. NMR parameters for
7 were significantly different from those for 3, with the Lys
RNH resonance (δ 7.95, 3JNH-RH ) 9.32 Hz) being relatively
independent of temperature (∆δ/T 1.0 ppb/K) and likely
H-bonded. The Glu RNH and Lys ꢀNH resonances are more
temperature dependent (∆δ/T 4.0, 3.9 ppb/K, respectively)
and also display faster D2O exchange than Lys RNH.
Interestingly, Lys RNH retained its high coupling constant
despite the change in loop conformation from over the
oxazole to over the thiazole. Like 3, the loop regions within
7 display significant chemical shift differences for several
pairs of geminal protons (Glu-âHs 0.49, Glu-γHs 0.65, Lys-
âHs 0.23, and Lys-ꢀHs 0.76 ppm), although these differences
themselves have changed with respect to 3.
restraints (Supporting Information). The NMR structures of
7 (Figure 4) indicated that the loops project from one face
of the macrocycle but were not as perpendicular as in 3.
The calculated structures for the loops in 7 converged well
(loop heavy atom RMSD ) 0.9 Å); however, some ROE
signals for Lys γCH2/δCH2 could not be assigned. Compared
with 3, the macrocyclic scaffold structures of 7 did not
converge as well (backbone RMSD, 0.3 Å), particularly the
oxazole rings, which tilt (20° from the plane of the cyclic
scaffold.
Molecular models (Figure 5) suggest that the dimensions
and directionality of side chains of the scaffold in 3 match
the positions of the interhelical protein loops of helix bundles
(Figure 5a) and complementarity-determining loops of
antibodies (Figure 5b). The cyclic scaffold 6 in structures 3
and 7 therefore seems appropriate for devising structural
mimics of even larger multiloop protein surfaces.
Acknowledgment. We thank Michael Kelso for modify-
ing XPLOR and ARC for some financial support.
Supporting Information Available: Compound synthe-
ses, characterization (NMR, MS, HPLC), computer modeling
data, NMR spectra, and structure calculation data for 3 and
7. This material is available free of charge via the Internet
at http://pubs.acs.org.
Due to significant spectral overlap, principally in the Lys
γCH2/δCH2 region, NMR structure calculations in XPLOR
were based on a smaller subset of 30 distance restraints,
together with the Lys dihedral angle and Lys RNH H-bond
OL026463M
(14) (a) Pokutta, S.; Weis, W. I. Mol. Cell 2000, 5, 533-43. (b) Stanfield,
R. L.; Fieser, T. M.; Lerner, R. A.; Wilson, I. A. Science 1990, 248, 712-
719.
(13) Hunter, C. A.; Sanders, J. K. M. J. Am. Chem. Soc. 1990, 112,
5525-5534.
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