Synthesis and NMR studies of cyclopeptides containing a sugar amino acid

Article abstract of DOI:10.1021/ol026126d

(Matrix presented) R² = H, Me, Bn or aminobutyl Cyclopeptides containing Glucuronic acid methylamine (Gum) alternating with Gly, L-Ala, D-Ala, L-Phe, D-Phe, L-Lys, or D-Lys were synthesized by a combination of solid-phase synthesis and solution

Full text of DOI:10.1021/ol026126d

ORGANIC
LETTERS
2002
Vol. 4, No. 15
2501-2504
Synthesis and NMR Studies of
Cyclopeptides Containing a Sugar
Amino Acid
Matthias Sto1ckle, Georg Voll, Robert Gu1nther, Elisabeth Lohof, Elsa Locardi,
Sibylle Gruner, and Horst Kessler*
Institut fu¨r Organische Chemie und Biochemie, Technische UniVersita¨t Mu¨nchen,
Lichtenbergstr. 4, D-85747 Garching, Germany
kessler@ch.tum.de
Received May 2, 2002
ABSTRACT
Cyclopeptides containing Glucuronic acid methylamine (Gum) alternating with Gly, L-Ala, D-Ala, L-Phe, D-Phe, L-Lys, or D-Lys were synthesized
by a combination of solid-phase synthesis and solution chemistry. A more effective pathway to synthesize the sugar amino acid Gum in
higher yields and in a shorter period of time was developed. Gum is employed in the benzylated and deprotected form. The cyclopeptides
were characterized by NMR and the structure of one deprotected cyclic peptide solved.
The aim of this work is the synthesis of new cyclic peptides
containing sugar amino acids (SAAs) and amino acids (AAs)
to provide new structural scaffolds. SAAs are pyranoid and
furanoid sugars, which contain at least one amino and one
carboxyl group.1 They are used as turn mimetica,1 peptido-
mimetic, and glycomimetic scaffolds1,2 and in combinatorial
chemistry.1,3 Modification of backbone or side chains of
cyclopeptide agents by SAAs could improve their pharma-
cological properties. The SAA Gum was designed in our
laboratory as a dipeptide isostere of the Gly-Ser sequence
held in a “flexible” â-turn conformation.1a,b
Cyclic compounds containing SAAs could be used to
modify cyclic peptides to provide a scaffold for template-
assembled synthetic proteins (TASP).4 The use of sugar rings
has distinct advantages over that of conventional AAs,
including hydrophilicity and introduction of lipophilicity by
use of protected SAAs.1d,e The easiest way to present
functional groups for the attachment of protein-mimicking
elements is to use conventional amino acids. To explore the
synthesis and conformation of such ring systems we present
here cyclic oligomers (1-16) of alternating Gum as SAA
and Xaa, where Xaa is an amino acid (Gly, Ala, Phe, or
Lys) in the D- or L-conformation (Table 1).
(1) (a) Graf von Roedern, E.; Kessler, H. Angew. Chem., Int. Ed. Engl.
1994, 33, 687-689. (b) Graf von Roedern, E.; Lohof, E.; Hessler, G.;
Hoffmann, M.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 10156-10167.
(c) Lohof, E.; Planker, E.; Mang, C.; Burkhart, F.; Dechantsreiter, M. A.;
Haubner, R.; Wester, H.-J.; Schwaiger, M.; Ho¨lzemann, G.; Goodman, S.;
Simon, L.; Kessler, H. Angew. Chem., Int. Ed. 2000, 39, 2761-2764. (d)
Gruner, S. A. W.; Locardi, E.; Lohof, E.; Kessler, H. Chem. ReV. 2002,
102, 491-514. (e) Lohof, E.; Burkhart, F.; Born, M. A.; Planker, E.; Kessler,
H. AdV. Amino Acid Mimetics Peptidomimetics 1999, 2, 263-292.
(2) (a) Locardi, E.; Sto¨ckle, M.; Gruner, S.; Kessler, H. J. Am. Chem.
Soc. 2001, 123, 8189-8196. (b) Suhara, Y.; Ichikawa, M.; Hildreth, J. E.
K.; Ichikawa, Y. Tetrahedron Lett. 1996, 37, 2549-2552. (c) Mu¨ler, C.;
Kitas, E.; Wessel, H. P. J. Chem. Soc., Chem. Commun. 1995, 23, 2425-
2426. (d) Smith, M. D.; Fleet, G. W. J. J. Peptide Sci. 1999, 5, 425-441.
(3) (a) McDevitt, J. P.; Lansbury, P. T., Jr. J. Am. Chem. Soc. 1996,
118, 3818-3828. (b) Schweizer, F.; Hindsgaul, O. Curr. Opin. Chem. Biol.
1999, 3, 291-298.
The tetrameric ring size was chosen to mimic cyclic
hexapeptides. This comparison is based on the chain length
between the Gum amino group and the carboxylate carbon
(4) (a) Tuchscherer, G. Tedrahedron Lett. 1993, 34, 8419-8422. (b)
Tuchscherer, G.; Grell, D.; Mathieu, M.; Mutter. M. J. Peptide Res. 1999,
54, 185-194.
10.1021/ol026126d CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/03/2002

Fmoc protection followed by TEMPO oxidation (TEMPO
) 2,2,6,6-tetramethyl-piperidine-1-oxyl) leads to unprotected
Fmoc-Gum-OH.1a To date, Fmoc-Gum(Bn)3-OH 19 was
synthesized in a 12-step synthesis starting from D-gluco-
pyranose (overall yield 3%).1d,e Herein we present an
alternative pathway starting from compound 17 (Scheme 2).
Table 1. Synthesized Cyclic Oligomers
Cyclo[Gum(R)3-Xaa1-Gum(R)3-Xaa2] (R ) H or Bn; XaaX )
Gly, L-Ala, D-Ala, L-Phe, D-Phe, L-Lys, or D-Lys)
cyclo[Gum(R)3-Xaa1-Gum(R)3-Xaa2]
R ) H
R ) Bn
Xaa1
Xaa2
1
2
3
4
9
10
11
12
5
6
7
8
Gly
L-Ala
L-Ala
D-Ala
L-Lys
L-Lys
D-Lys
D-Lys
L-Lys(Boc)
L-Lys(Boc)
D-Lys(Boc)
D-Lys(Boc)
Gly
L-Ala
D-Ala
D-Ala
L-Phe
D-Phe
L-Phe
D-Phe
L-Phe
D-Phe
L-Phe
D-Phe
Scheme 2. Synthesis of Fmoc-Gum(Bn)3-OH 19a
13
14
15
16
a Reaction conditions: (a) Boc2O, THF, H2O, 81% starting from
compound 18. (b) MMTr-Cl, TEA, DMAP, DMF, 78%. (c) BnBr,
18-crown-6, KOH, THF, 63%. (d) (1) 20 vol % TFA in DCM,
H2O; (2) Fmoc-Cl, NaHCO3, THF, H2O, 71%. (e) TEMPO, KBr,
TBABr, NaOCl, DCM, NaCl, NaHCO3, H2O, 96%.
(six atoms, similar to a dipeptide unit; see above). Lys was
chosen to allow further side chain functionalization, whereas
Phe was used to simplify the procedure of HPLC separation,
since Phe can easily be detected by UV spectroscopy.
Furthermore, D-Phe shows structurally induced tendencies
and exhibits better separation of NMR signals. Similar
compounds with furanoid SAAs were already described
previously.5
Boc protection of 17 was realized in situ under standard
conditions8 with Boc2O. Reaction of monomethoxytrityl
chloride (1.1 equiv) with Boc-N-(â-D-glucopyranosyl)-
methylamine in DMF solution overnight at room temperature
in the presence of catalytic amounts of 4-(dimethylamino)-
pyridine (DMAP) and triethylamine (TEA, 1.5 equiv)
produced Boc-N-(6-O-MMTr-â-D-glucopyranosyl)methyl-
amine in good yield (78%). Compound 20 was obtained in
63% yield by treating the last compound in THF solution
overnight at room temperature with benzylbromide (3.3
equiv), KOH powder (5.4 equiv), and catalytic amounts of
18-crown-6 under argon. After simultaneous cleavage of the
monomethoxy triphenylmethyl ether and Boc deprotection,
the intermediate was Fmoc protected with Fmoc chloride to
yield Fmoc-N-(2,3,4-tri-O-benzyl-â-D-glucopyranosyl)meth-
ylamine in 71% over two steps. Finally, TEMPO oxidation
in a two-phase system (water/dichloromethane) with tetra-
butylammonium bromide (TBABr) as a phase transfer
catalyst led to the final product Fmoc-Gum(Bn)3-OH 19 in
excellent yields (96%). The overall yield starting from
2,3,4,6-tetra-O-acetyl-R-D-glucopyranosyl bromide was 22%.
Oligomerization of Gum(Bn)3-OH alternating with L-
and D-forms of Ala, Lys, Phe, or Gly, respectively, was
realized by solid-phase synthesis with the Fmoc strategy
using TentaGel S Trt resin (Rapp Polymere), O-(7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluoro-
phosphate (HATU),9 and 1-hydroxy-7-azabenzotriazole
(HOAt)10 as coupling reagents and 2,4,6-collidine as a base
in DMF (Scheme 3).
For the synthesis of Gum, â-D-glucopyranosylmethylamine
17 is an important intermediate product for which we present
here a more efficient synthesis (Scheme 1). 2,3,4,6-Tetra-
Scheme 1. Improved Synthesis of
â-D-Glucopyranosylmethylamine 17a
a Reaction conditions: (a) Hg(CN)2, melt, 85 °C, argon atmo-
sphere, 1 h, 81%; (b) LiAlH4, THF, 0 °C.
O-acetyl-R-D-glucopyranosyl bromide reacts with Hg(CN)2
in melt6 in only 1 h and excellent yields (81%) to form
2,3,4,6-tetra-O-acetyl-â-D-glucopyranosylcyanide 18, fol-
lowed by reduction to compound 17. This pathway is much
more efficient than all alternative routes such as the synthesis
of 18 by reaction of pentaacetylglucose with trimethylsilyl-
cyanide (TMSCN)7 or the synthesis of compound 17 by
reduction of â-D-glucopyranosylnitromethane, which is pre-
pared by nitroaldol reaction of glucose in low yield (27%)
with high experimental effort.1a
The azabenzotriazol-based coupling reagents were the most
suitable, since they permit high coupling yields and low
(7) Ko¨ll, P.; Fo¨rtsch, A. Carbohydr. Res. 1987, 171, 3001-315.
(8) Moroder, L.; Hallett, A.; Wu¨nsch, E.; Keller, O.; Wersin, G. Hoppe-
Seyler’s Z. Physiol. Chem. 1976, 357, 1651-1653.
(5) (a) Gruner, S.; Kessler H. Peptides 2000, 313-314. (b) Gruner, S.
A. W.; Truffault, V.; Voll, G.; Locardi, E.; Sto¨ckle, M.; Kessler, H. Eur. J.
Chem., submitted for publication. (c) van Well, R. M.; Overkleeft, H. S.;
Overhand, M.; Vang Carstenen, E.; van der Marel, G. A.; van Boom, J. H.
Tetrahedron Lett. 2000, 41, 9331-9335.
(9) (a) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J. Chem.
Soc., Chem. Commun. 1994, 201. (b) Albericio, F.; Bofill, J. M.; El-Faham,
A.; Kates, S. A. J. Org. Chem. 1998, 63, 9678-9683.
(6) Fuchs, E.-F.; Lehmann, J. Chem. Ber. 1975, 108, 2254-2260.
(10) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.
2502
Org. Lett., Vol. 4, No. 15, 2002

symmetrical conformation on the NMR time scale at 300
K. Even the unprotected, mixed cycle of L-Ala and D-Ala 3
shows only a very small asymmetric tendency. As expected,
the incorporation of Phe induced a better separation of NMR
signals. The tetrameric ring size of the cyclic oligomers
described here was chosen to mimic cyclic hexapeptides, with
Gum as a â-turn-inducing agent (see above). To test, whether
the Gum residue displays its â-turn-inducing properties also
in these compounds, we further investigated the conformation
of the unprotected oligomer cyclo(Gum-L-Lys-Gum-D-Phe)
10 using distance geometry and molecular modeling.
A total of 18 distance restraints derived from the ROESY
spectrum were used in combination with selected coupling
constants for metric matrix distance geometry calculations
of 1000 starting structures using a modified12 version of
DISGEO.13 After a cluster algorithm14 was applied to the
backbone atoms (including sugar atoms within the backbone
ring), this first pool of conformations was used as a starting
point for further structural refinement. A 150 ps restrained
molecular dynamics simulation of cyclo(Gum-L-Lys-Gum-
D-Phe) 10 was then carried out in the CVFF force field as
incorporated in the Discover program package.15 The simula-
tion was performed in an explicit water box (3199 H2O
molecules, box size 46 × 46 × 46 Å3, cubical) with an
explicit image of periodic boundary conditions. All ROE
violations, calculated over the trajectory with r-3 1/3 averag-
ing, remained below 0.13 Å. The averaged and energy-
minimized structure from the restrained MD trajectory (see
Figure 1) is in agreement with the temperature gradients of
the amide protons, which indicate good solvent accessibility
for all HN.
Scheme 3. Oligomerization and Cyclization of Gum(Bn3)-OH
Alternating with L-Ala, D-Ala, L-Phe, D-Phe, L-Lys, D-Lys, or
Gly on TentaGel S Trt resina
a Reaction conditions: (a) Fmoc-Gum(Bn3)-OH (2 equiv), HATU
(2 equiv), HOAt (2 equiv), 2,4,6-collidine (20 equiv), DMF, 15 h,
rt; (b) 1:4 piperidine/DMF, 2 × 30 min, rt; (c) Fmoc-AA-OH (3
equiv), HATU (3 equiv), HOAt (3 equiv), 2,4,6-collidine (30 equiv),
DMF, 15 h, rt; (d) 1:1:3 TFE/AcOH/DCM or 20 vol % HFIP in
DCM. (e) DIC (10 equiv), HOAt (1 equiv), NMM (3 equiv), 9:1
DCM/DMF, 4 days, 0-4 °C; (f) HATU (1 equiv), HOAt (1 equiv),
2,4,6-collidine (10 equiv), DMF, overnight, 0-4 °C.
racemization. As solution studies have shown,11 racemization
is lower when using 2,4,6-collidine rather than diisopropyl
ethylamine (DIPEA). Our studies on solid-phase synthesis
have confirmed this.1e
Oligomers were cleaved from resin with 1:1:3 2,2,2-
trifluoroethanol (TFE)/AcOH/DCM or with 20 vol %
hexafluoro 2-propanol in DCM. Cyclic tetramers were
obtained by cyclization of linear oligomers with diisopropyl
carbodiimide (DIC), HOAt, and N-methylmorpholine (NMM)
in a very diluted (0.1-0.2 mM) solution of 9:1 DCM/DMF
at 4 °C in 4 days or overnight by treatment of the linear
oligomers with HATU (1 equiv), HOAt (1 equiv), and 2,4,6-
collidine in a highly diluted (0.2 mM) solution in DMF.
Yields between 60 and 75% were obtained. Deprotected
cycles were received by hydrogenolytic cleavage of benzylic
ethers with Pd/C (5%, dry) and H2 (50 bar) overnight in 7:2:1
N,N-dimethylacetamid/MeOH/AcOH in excellent yields (95%).
We obtained unprotected cyclic oligomers also by the
described coupling protocol in Scheme 3 with the unprotected
Fmoc-Gum-OH. But the yields were only between 30 and
50% for oligomerization and 30-60% for cyclization. Boc
deprotection of the lysine side chain was obtained by
treatment with 50 vol % TFA in DCM almost quantitatively.
Figure 1. Stereopicture of the three-dimensional structure of 10.
To obtain more insight into the conformational flexibility
of 10, a subsequent 150 ps MD simulation in water without
applying any experimental restraints (free MD) was then
carried out. The structure remained stable and very similar
The cyclic oligomers were characterized by NMR spec-
troscopy in DMSO (protected cycles 5-8 and 13-16) and
water or D2O, respectively (unprotected cycles 1-4 and
9-12). NMR spectra of the cyclopeptides containing only
Gly, L-Ala, or D-Ala alternated with Gum indicate a
(12) Mierke, D. F.; Kessler, H. Bioploymers 1993, 33, 1003-1017.
(13) (a) Havel, T. F. DISGEO, Quantum Chemistry Exchange Program
1988, Exchange No. 507; Indiana University: Bloomington, IN, 1988. (b)
Havel, T. F. Prog. Biophys. Mol. Biol. 1991, 56, 43-78
(14) Kelley, L. A.; Gardner, S. P.; Sutcliffe, M. J. Protein Eng. 1996, 9,
1063-1065.
(11) Carpino, L. A.; El-Faham, A.; Albericio, F. Tetrahedron Lett. 1994,
35, 2279-2282.
(15) DiscoVer, version 2.98; BIOSYM/Molecular Simulations: San
Diego, CA, 1995.
Org. Lett., Vol. 4, No. 15, 2002
2503

to the structure calculated from the restrained simulation.
The distance RMSD between the averaged and minimized
structures from both the free and the restrained simulations
is 0.46 Å for the backbone atoms. This good agreement
proves that the solution structure calculated from NMR data
represents a stable low-energy conformation in the CVFF
force field.
see Supporting Information). In combination with the
standard reduction according to Dunitz and Waser, this
procedure results in a hexacycle, consisting of the Xaa-CR
and the Gum C and X atoms. For this hexacycle, we have
found a nearly ideal twisted boat conformation (dihedral
angles (calcd/ideal), starting from Lys4CR-Gum1C-Gum1X-
D-Phe2CR: -53.1/-54.8; 17.7/27.0; 43.9/27.0; -64.6/-54.8;
41.9/27.0; 13.1/27.0).
Against our expectations, Gum does not act as a â-turn
mimetic in 10. This can be explained by the comparison
between an ideal âI′-turn in a regular tetrapeptide and the
analogous turn in Xaa-Gum-Xaa. In the tetrapeptide, the
amide bond between the amino acids in positions i + 1 and
i + 2 is planar, whereas the analogous bond in Xaa-Gum-
Xaa resembles an aliphatic chain. Therefore, the distance of
the CO(i)-HN(i + 2) hydrogen bond in the tetrapeptide
measures 1.85 Å, as opposed to the corresponding distance
of 2.43 Å in the Xaa-Gum-Xaa turn (see Supporting
Information). It therefore seems that the Gum replacement
of a single Xaa-Xaa sequence in a regular cyclic hexapeptide
results in an additional cyclic constraint, which can be
compensated for by the rest of the molecule, while still
maintaining â-turn geometry. On the contrary, the replace-
ment of two adjacent Xaa-Xaa sequences leads to a very
high ring tension in the usual conformation of two adjacent
â-turns, so this conformation becomes energetically un-
favored.
For a better rationalization of the conformation, we have
reduced the structure of 10 according to the principle of
Dunitz and Waser.16 Cyclic hexapeptides in such a model
resemble cyclohexane-like conformations.17 The Gum sugar
ring possesses five large substituents that can be in either
an all-axial or an all-equatorial position, with the latter being
highly energetically favored. Therefore, the Gum methylene
carbon C, the three ring atoms C1, O, and C5, and the Gum
carbonyl carbon C6 can be regarded as sterically fixed in
space relative to each other. Thus, it is possible to reduce
C1, O, and C5 to a single pseudoatom X (for further details
A close inspection of the ROESY spectrum reveals a
second conformation, which interchanges slowly on the
NMR time scale with the main conformation. The structure
of the second conformation could not be elucidated, since
the signals of the two conformations are not separated well
enough. The population of the second conformer increases
with the temperature (approximately 3% at 293 K until 5%
at 320 K). It should be noted that a conformation with D-Phe
in the standard position i + 1 of a âII′-turn cannot be formed
in 10. In such a conformation, there would exist two very
unfavorable, close contacts between the CO(i) and O(i + 4)
oxygen atoms, as opposed to the stabilizing hydrogen bonds
in a standard cyclic hexapeptide. However, a conformation
that corresponds to a chair conformation according to Dunitz
and Waser would explain the slow interchange of the two
conformers, since there is a high energy barrier between the
boat and the chair conformations in such a hexacycle.
Acknowledgment. The authors thank B. Cordes, M.
Kranawetter, and M. Wolff for technical assistance. Financial
support by the DFG and the Fonds der Chemischen Industrie
is acknowledged.
Supporting Information Available: Characterization and
detailed descriptions of the synthesis of key compounds;
tables of coupling constants, ROE data, and amide proton
temperature gradients of cyclo(Gum-D-Phe-Gum-L-Lys) 10
in the conformation described; all molecular dynamics
protocols; and a detailed description of the Dunitz-Waser
reduction of our calculated structure. This material is
available free of charge via the Internet at http://pubs.acs.org.
(16) Dunitz, J. D.; Waser, J. J. Am. Chem. Soc. 1972, 94, 5645-5650.
(17) Kessler, H.; Gratias, R.; Hessler, G.; Gurrath, M.; Mu¨ller, G. Pure
Appl. Chem. 1996, 68, 1201-1205.
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