Folded conformation in peptides containing furanoid sugar amino acids [4]

Full text of DOI:10.1021/ja9816685

12962
J. Am. Chem. Soc. 1998, 120, 12962-12963
Scheme 1. Synthesis of Protected Furanoid Sugar Amino
Acids
Folded Conformation in Peptides Containing
Furanoid Sugar Amino Acids
T. K. Chakraborty,*† S. Jayaprakash,† P. V. Diwan,†
R. Nagaraj,‡ S. R. B. Jampani,† and A. C. Kunwar*†
Indian Institute of Chemical Technology and
Centre for Cellular and Molecular Biology
Hyderabad 500 007, India
ReceiVed May 14, 1998
Introduction of conformationally restricted nonpeptide isosteres
into the peptide backbone in order to achieve desirable secondary
structures is of great interest in structure-activity relationship
studies of peptides.1 Furanoid sugar amino acids, 6-amino-2,5-
anhydro-3,4-di-O-benzyl-6-deoxy-D-gluconic acid (1, Gaa) and
its mannonic congener 2 (Maa), can serve as rigid scaffolds in
the design and synthesis of carbopeptoids. The protected/
unprotected hydroxyl groups of sugar rings can also influence
the hydrophobic/hydrophilic nature of peptides.2 Herein, we report
an efficient synthesis of these furanoid sugar amino acids from
their acyclic precursors 3 and 4, followed by their incorporation
into Leu-enkephalin in its Gly-Gly position as dipeptide isosteres
leading to the formation of peptidomimetic analogues 5 and 6.
One of these analogues, Boc-Tyr-Gaa-Phe-Leu-OMe (5a), shows
an unusual and interesting turn structure determined by CD and
extensive NMR studies.
D-glucopyranoside (7),3 was transformed, in two steps, into
intermediate diol 8 in 72% yield. Treatment of 8 with Ph3P in
refluxing toluene led to the formation of an aziridine ring, which
was protected in situ to give the target intermediate 3 in 85%
yield. The crucial cycloetherification proceeded smoothly with
simultaneous debenzylation and oxidation of the primary hydroxyl
to the carboxylic acid moiety using an excess of PDC (5 molar
equiv) in DMF as solvent. After workup, the crude acid 9 was
treated with diazomethane. Chromatographic purification gave
methyl 6-(tert-butyloxycarbonyl)amino-2,5-anhydro-3,4-di-O-ben-
zyl-6-deoxy-D-gluconate (10) along with another compound,
which was identified as bicyclic compound 11, in about 75% yield
(10:11 ≈ 2:1).4 Treatment of 11 with K2CO3/MeOH converted it
to 10, leading to an overall yield of 72% from 3.
Similar oxidative rearrangement of 4, prepared from D-mannose
following the same route as outlined for 3, furnished the expected
mannonate 13 as the only product in 78% yield. In this case, the
“2,5-trans” geometry in the furanoid framework prevented the
formation of “11-type” bicyclic product.
Next, these furanoid gluconic and mannonic amino acids, 1
and 2, respectively, were incorporated into Leu-enkephalin,
replacing its Gly-Gly portion which is known to be flexible and
amenable to different conformations depending on the binding
environment.5,6 The analogues 5 and 6 were synthesized by
solution methods using EDCI/HOBt as coupling agents and DMF
as solvent. Reaction of 9 with H2N-Phe-Leu-OMe gave the
protected tripeptide Boc-Gaa(Bzl2)-Phe- Leu-OMe, which was
deprotected at the N-terminus with TFA/CH2Cl2 and subsequently
coupled with Boc-Tyr(Br-Z) to give Boc-Tyr(Br-Z)-Gaa(Bzl2)-
Phe-Leu-OMe. Finally, hydrogenation using H2/Pd(OH)2/C in
methanol yielded the side-chain-deprotected compound Boc-Tyr-
Gaa-Phe-Leu- OMe (5a). A similar reaction sequence starting with
Boc-protected mannonic amino acid 12 led to the formation of
Boc-Tyr-Maa-Phe-Leu-OMe (6a).7
The synthesis followed a novel reaction path in which an
intramolecular 5-exo opening of the hexose-derived aziridine ring
by the γ-benzyloxy oxygen with concomitant debenzylation
occurred during pyridinium dichromate (PDC) oxidation of the
primary δ-hydroxyl groups of 3 and 4. Complete stereocontrol
achieved in this electrophile-activated SN2 opening of the aziridine
ring under remarkably mild conditions outlines the essence of
our synthesis. Scheme 1 unfolds the details of the route. The
starting material, methyl 6-deoxy-6-azido-2,3,4-tri-O-benzyl-R-
Conformational analysis of these peptide analogues were carried
out by studying their circular dichroism (CD) spectra in trifluo-
roethanol (TFE).8 The CD spectrum of 5a exhibits a strong
† Indian Institute of Chemical Technology.
(3) Kobertz, W. R.; Bertozzi, C. R.; Bednarski, M. D. J. Org. Chem. 1996,
61, 1894-1897.
‡ Centre for Cellular and Molecular Biology.
(1) (a) Hruby, V. J.; Li, G.; Haskell-Luevano, C.; Shenderovich, M.
Biopolymers 1997, 43, 219-248. (b) Hanessian, S.; McNaughton-Smith, G.;
Lombart, H.-G.; Lubell, W. D. Tetrahedron 1997, 53, 12789-12854. (c)
Giannis, A.; Kolter, T. Angew. Chem., Int. Ed. Engl. 1993, 32, 1244-1267.
(2) (a) Graf von Roedern, E.; Lohof, E.; Hessler, G.; Hoffmann, M.; Kessler,
H. J. Am. Chem. Soc. 1996, 118, 10156-10167. (b) McDevitt, J. P.; Lansbury,
P. T., Jr. J. Am. Chem. Soc. 1996, 118, 3818-3828. (c) Poitout, L.; Le Merrer,
Y.; Depezay, J.-C. Tetrahedron Lett. 1995, 36, 6887-6890. (d) Fuchs, E.-F.;
Lehmann, J. Chem. Ber. 1975, 108, 2254-2260.
(4) For earlier works on acid-catalyzed opening of aziridine rings, see: (a)
Tanner, D. Angew. Chem., Int. Ed. Engl. 1994, 33, 599-619. (b) Dehmlow,
H.; Mulzer, J.; Seilz, C.; Strecker, A. R.; Kohlmann, A. Tetrahedron Lett.
1992, 33, 3607-3610 and references therein.
(5) Aldrich, J. V. In Burger’s Medicinal Chemistry and Drug DiscoVery,
Vol. 3; Wolff, M. E., Ed.; John Wiley & Sons: New York, 1996; pp 321-
441 and references therein.
(6) Smith, G. D.; Griffin, J. F. Science 1978, 199, 1214-1216.
(7) New compounds exhibited satisfactory spectral and exact mass data.
10.1021/ja9816685 CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/25/1998

Communications to the Editor
J. Am. Chem. Soc., Vol. 120, No. 49, 1998 12963
Figure 1. Stereoview of the five superimposed minimum energy conformations of 5a selected from the 20 energy-minimized structures sampled during
20-ps MD simulations.
positive band at 216 nm characteristic of a type II â-turn.9 The
corresponding deprotected peptide H2N-Tyr-Gaa-Phe-Leu-OMe
(5b) also shows a positive band, although of reduced ellipticity
at ∼217 nm. The spectra of the Maa-containing analogues, both
protected (6a) and deprotected H2N-Tyr-Maa-Phe-Leu-OMe (6b),
indicate a lower tendency to form turn structures.
ROESY cross-peak between Tyrâ protons and Phe aromatic
protons suggests their close proximity.
Using ROESY cross-peak intensities as constraints, molecular
dynamics (MD) simulations were carried out for a 20-ps period,
sampling 20 frames at equal intervals which were energy-
minimized. Five minimum energy conformations out of these 20
structures were selected and superimposed as shown in Figure
1.11 A close look shows that the peptide takes a turn-like structure
stabilized by aromatic-aromatic as well as hydrophobic interac-
tion between the Leu side chain and the sugar ring. This structure
very strongly resembles the conformation of Met-enkephalin in
the presence of SDS micelles,12 which is also stabilized by
hydrophobic aromatic-aromatic interaction and corresponds to
one of the two theoretical binding conformations13 that enkephalin
may adopt while interacting with cell membranes in which opioid
receptors are located. We find that such a structure is preintro-
duced in 5a, making it a potential ligand for the δ-receptor which
requires folded conformations with close proximity (<10 Å) of
the two aromatic rings having nearly parallel orientation.14
The analgesic activities of these analogues were determined
by mouse hot-plate test15 following i.p. administration. Com-
pounds 5a,b showed activities (ED50 ) 1.14 and 1.48 µmol/
animal, respectively) similar to that of Leu-enkephalin methyl
ester (ED50 ) 1.35 µmol/animal). The same trend was observed
when analgesic activities were assayed by the tail clip method.16
Compounds 6a,b showed no significant activity in either of these
tests. These positive results are probably due to the above-
mentioned preintroduced folded conformations present in these
peptides which were absent in the biologically inactive pyranoid
sugar amino acid-containing Leu-enkephalin analogues reported
earlier.2a
Finally, solution conformation of 5a, which shows the best turn
1
structure in CD, was determined by studying, in detail, its H
NMR spectra in DMSO-d6 (10 mM). The resonance assignments
were carried out with the help of TOCSY and ROESY experi-
ments. The temperature coefficients of amide proton chemical
shifts (∆δ/∆T) were measured between 21 and 70 °C. The small
∆δ/∆T for Leu amide (-1.3 ppb/K) shows its involvement in
strong H-bonding, while other amide protons show medium to
large values of temperature coefficients.
The peptide exhibits some unusual side-chain conformations,
especially for Leu, whose side chain appears very rigid, as its
JR,â(pro-R) ) 10.4 Hz, JR,â(pro-S) ) 4.6 Hz, Jâ(pro-R),γ ) 4.6 Hz,
and Jâ(pro-S),γ ) 9.6 Hz indicate the presence of predominantly
one single conformation about ø1 (g-) and ø2 with an anti
relationship between âH(pro-S) and γH. The Phe and Tyr side
chains also show the presence of g- conformation about ø1.
The small J2-3 of 3.8 Hz and a ROESY cross-peak between
sugar C2-H and C5-H indicates an envelope (C3-endo) confor-
mation of the sugar ring. Other cross-peaks between LeuRH-
LeuδCH3(pro-S), LeuâΗ(pro-R)-sugarC3-OH, LeuγH-sugarC6-
H(pro-R), LeuδCH3(pro-R)-sugarC3-OH, and LeuδCH3(pro-R)-
sugarC6-H(pro-R) show that these protons come close to each
other and result in the Leu side chain getting locked in a single
conformation. This conformation also shows H-bonding between
LeuNH f sugarC3-OH, leading to a nine-membered â-turn-like
ring structure. The sugar-hydroxyl can also possibly act as a
H-bond donor to Leu carbonyl or solvent molecules. There is a
downfield shift of the sugar C3-hydroxyl proton by 0.4 ppm
compared to its chemical shift in the 1H NMR spectrum of Boc-
Gaa-OMe in DMSO-d6 (10 mM), whereas the C4-OH signal did
not change position. Amino acids with hydroxyl groups in their
side chains (serine, threonine) serve as acceptors only ∼30% of
the time.10 Moreover, a main-chain NH f side-chain OH H-bond
leading to this type of turn structure is also very rare, mainly
because of the free rotation about ø1 in these amino acids. In
sugar amino acids, unlike in serine and threonine, the hydroxyls
are conformationally restricted, forcing one of them to participate
in the formation of an unusual secondary structure. This pseudo-
â-turn is probably responsible for the strong positive band at 216
nm in the CD spectrum of the molecule. Another long-range
In conclusion, furanoid sugar amino acids, now easily obtain-
able from hexose-derived aziridinyl precursors, can serve as useful
templates to induce interesting secondary structures in peptides.
Acknowledgment. We thank Dr. J. A. R. P. Sharma for calculations
and CSIR, New Delhi, for research fellowships (S.J. and S.R.B.J.) and a
Young Scientist Award Research Grant (T.K.C.).
Supporting Information Available: Listing of selected physical data
1
for 5a, 6a, 10, 11, and 13; CD spectra of 5a, 5b, 6a, and 6b; H NMR
spectra of 5a and 6a; TOCSY and ROESY spectra of 5a; details of
analgesic activity assay (11 pages, print/PDF). See any current masthead
page for ordering information and Web access instructions.
JA9816685
(11) MD simulations were carried out using the Insight II (97.0)/Discover
program on a Silicon Graphics Octane workstation.
(12) Graham, W. H.; Carter, E. S., II; Hicks, R. P. Biopolymers 1992, 32,
1755-1764.
(13) (a) Behnam, B. A.; Deber, C. M. J. Biol. Chem. 1984, 259, 14935-
14940. (b) Deber, C. M.; Behnam, B. A. Biopolymers 1985, 24, 105-116.
(14) Yamazaki, T.; Mierke, D. F.; Said-Nejad, O. E.; Felder, E. R.;
Goodman, M. Int. J. Pept. Protein Res. 1992, 39, 161-181.
(15) Dutta, A. S.; Gormley, J. J.; Hayward, C. F.; Morley, J. S.; Shaw, J.
S.; Stacey, G. J.; Turnbull, M. T. Life Sci. 1977, 21, 559-562.
(16) Bobrova, I.; Abissova, N.; Mishlakova, N.; Rozentals, G.; Chipens,
G. Eur. J. Med. Chem. 1998, 33, 255-266 and references therein.
(8) The CD spectra were recorded in a JASCO J 715 spectropolarimeter
in quartz cells of 1-mm path length at 25 °C using peptide concentration of
0.2 mM in TFE.
(9) Woody, R. W. In The Peptides: Analysis, Synthesis, Biology, Vol. 7;
Udenfriend, S.; Meienhofer, J.; Hruby, V. J., Eds.; Academic Press: New
York, 1985; pp 15-114.
(10) (a) McDonald, I. K.; Thornton, J. M. J. Mol. Biol. 1994, 238, 777-
793. (b) Burley, S. K.; Petsko, G. A. AdV. Protein Chem. 1988, 39, 125-
189.