Synthesis and conformational analysis of linear and cyclic peptides containing sugar amino acids

Article abstract of DOI:10.1021/ja961068a

Sugar amino acids (SAAs) were designed and synthesized as new non-peptide peptidomimetics utilizing carbohydrates as peptide building blocks. They represent sugar-like ring structures that carry an amino and a carboxylic functional group and have a specific conformational influence on the backbone of peptides due to their distinct substitution patterns in rigid pyranose sugar rings. Five different SAAs (SAA1α, SAA1β, SAA2, SAA3, and SAA4) have been synthesized that show the ability to constrain linear backbone conformations or distinct turn structures. Linear and cyclic peptides involving SAAs have been prepared in solution as well as by solid phase synthesis. SAA1α and SAA2 were incorporated into two linear Leu-enkephalin analogs, replacing the natural Gly-Gly dipeptide. NMR studies provide evidence for the conformation-inducing effect of the carbohydrate moiety. SAA2 and SAA3 have been placed in cyclic hexapeptide analogs of somatostatin; SAA4 was incorporated in a model peptide. The conformation of the cyclic peptides cyclo(-SAA2-Phe-D-Trp-Lys-Thr-), cyclo(-SAA3-Phe-D-Trp-Lys(Boc)-Thr(tBu)-), and cyclo(-SAA4-Ala-D-Pro-Ala-Ala-) have been analyzed by various NMR techinques in combination with distance geometry calculations and subsequent molecular dynamic simulations. The determined solution conformations were compared to representative idealized peptide backbones. SAA2 and SAA3 induce a β-turn structure while SAA4 mimics a γ-turn. Both enkephalin analogs were not active in the guinea pig ileum assay. The somatostatin analog containing SAA2 has an inhibition constant (IC₅₀) of 0.15 μM for the inhibition of the release of growth hormone.

Full text of DOI:10.1021/ja961068a

10156
J. Am. Chem. Soc. 1996, 118, 10156-10167
Synthesis and Conformational Analysis of Linear and Cyclic
Peptides Containing Sugar Amino Acids
Erich Graf von Roedern,† Elisabeth Lohof, Gerhard Hessler,
Matthias Hoffmann, and Horst Kessler*
Contribution from the Institut fu¨r Organische Chemie und Biochemie, Technische UniVersita¨t
Mu¨nchen, Lichtenbergstrasse 4, D-85747 Garching, Germany
ReceiVed April 1, 1996X
Abstract: Sugar amino acids (SAAs) were designed and synthesized as new non-peptide peptidomimetics utilizing
carbohydrates as peptide building blocks. They represent sugar-like ring structures that carry an amino and a carboxylic
functional group and have a specific conformational influence on the backbone of peptides due to their distinct
substitution patterns in rigid pyranose sugar rings. Five different SAAs (SAA1R, SAA1â, SAA2, SAA3, and SAA4)
have been synthesized that show the ability to constrain linear backbone conformations or distinct turn structures.
Linear and cyclic peptides involving SAAs have been prepared in solution as well as by solid phase synthesis.
SAA1R and SAA2 were incorporated into two linear Leu-enkephalin analogs, replacing the natural Gly-Gly dipeptide.
NMR studies provide evidence for the conformation-inducing effect of the carbohydrate moiety. SAA2 and SAA3
have been placed in cyclic hexapeptide analogs of somatostatin; SAA4 was incorporated in a model peptide. The
conformation of the cyclic peptides cyclo(-SAA2-Phe-D-Trp-Lys-Thr-), cyclo(-SAA3-Phe-D-Trp-Lys(Boc)-
Thr(tBu)-), and cyclo(-SAA4-Ala-D-Pro-Ala-Ala-) have been analyzed by various NMR techniques in combination
with distance geometry calculations and subsequent molecular dynamic simulations. The determined solution
conformations were compared to representative idealized peptide backbones. SAA2 and SAA3 induce a â-turn
structure while SAA4 mimics a γ-turn. Both enkephalin analogs were not active in the guinea pig ileum assay. The
somatostatin analog containing SAA2 has an inhibition constant (IC50) of 0.15 µM for the inhibition of the release
of growth hormone.
In recent years the interest in a rational design of amino acid
and peptide mimetics has steadily grown due to the pharma-
cological limitations of bioactive peptides. A large variety of
modifications of peptide structures has been used for confor-
mationally directed drug design to investigate the active peptide-
receptor binding conformation.1 Constrained peptidomimetics
and cyclization of peptides remain of special interest to obtain
a distinct, bioactive conformation, especially in the field of
combinatorial synthesis for high throughput screening.2 Car-
bohydrates present as an attractive option for non-peptide
scaffolding as they contain well-defined and readily convertible
substituents3 with a rigid pyran ring.
Carbohydrates are frequently found in proteins as a result of
enzyme-mediated glycosylation in post-translational modifica-
tion processes.4 Sugar amino acids (SAAs) in particular occur
in nature as subunits of oligosaccharides (neuraminic acid), in
cell walls of bacteria (muraminic acid), and in some antibiotics.5
The syntheses of SAAs6 so far concentrated on the use of SAA
Figure 1. Example of a sugar amino acid (SAA2).
analogs as biopolymer building blocks7,8 to mimic oligo- and
polysaccharide structures via amide bond linkages.
Two years ago we reported one example of a sugar amino
acid as a new type of peptidomimetic (Figure 1).9,10 The novel
SAA was successfully incorporated into a cyclic peptide with
the â-turn motif of the somatostatin containing tetrapeptide Phe-
(4) For reviews, see: Varki, A. Glycobiology 1993, 3, 97-130. Suzuki,
T.; Kitajima, K.; Inoue, S.; Inuoe, Y. Glycoconjugate J. 1995, 12, 183-
193. Examples for synthetic applications: Perczel, A.; Kollat, E.; Hollosi,
M.; Fasman, G. D. Biopolymers 1993, 33, 665-685. Skuric, M.; Horvat,
J.; Horvat, S.; Chung, N. N.; Schiller, P. W. Int. J. Pept. Protein Res. 1994,
43, 402-409. Polt, R.; Porreca, F.; Szabo, L. Z.; Bilsky, E. J.; Davis, P.;
Abbruscato, T. J. Davis, T. P.; Horvath, R.; Yamamura, H. I.; Hruby, V. J.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7114-7118. Danishefsky, S. J.;
Roberge, J. Y. Pure Appl. Chem. 1995, 67, 1647-1662.
(5) Fox, J. J.; Kuwada, Y.; Watanabe, K. A. Tetrahedron Lett. 1968, 2,
6029-6032. Isono, K.; Asahi, K.; Suzuki, S. J. Am. Chem. Soc. 1969, 91,
7490-7505. Lichtenthaler, F. W.; Morino, T.; Menzel, H. M. Tetrahedron
Lett. 1975, 9, 665-668. Waltho, J. P.; Williams, D. H.; Selva, E.; Ferrari,
P. J. Chem. Soc., Perkin Trans. 1 1987, 9, 2103-2107.
(6) First synthesis of SAAs: Heyns, K.; Paulsen, H. Chem. Ber. 1955,
88, 188-195.
* Author to whom correspondence should be addressed. Tel.: ++49-
89-289 13301. Fax: ++49-89-289 13210. E-mail: kessler@
joshua.org.chemie.tu-muenchen.de.
† Present address: Max Planck Institut fu¨r Biochemie, Am Klopferspitz
18 a, D-82152 Martinsried, Germany.
X Abstract published in AdVance ACS Abstracts, October 1, 1996.
(1) Rich, R. A. Med. Res. ReV. 1993, 13, 327-384. Marshall, G. R.
Tetrahedron 1993, 49, 3547-3558. Gante, J. Angew. Chem., Int. Ed. Engl.
1994, 33, 1699-1720. Liskamp, R. M. J. Recl. TraV. Chim. Pays-Bas 1994,
113, 1-19.
(2) Gallop, M. A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gordon,
E. M. J. Med. Chem. 1994, 37, 1233-1251. Gordon, E. M.; Barrett, R.
W.; Dower, W. J.; Fodor, S. P. A.; Gallop, M. A. J. Med. Chem. 1994, 37,
1385-1401.
(3) Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Leahy, E. M.;
Salvino, J.; Arison, B.; Cichy, M. A.; Spoors, P. G.; Shakespeare, W. C.;
Sprengeler, P. A.; Hamley, P.; Smith, A. B.; Reisine, T.; Raynor, K.;
Maechler, L.; Donaldson, C.; Vale, W.; Freidinger, R. M.; Cascieri, M. A.;
Strader, C. D. J. Am. Chem. Soc. 1993, 115, 12550-12568.
(7) First oligomers: Fuchs, E. F.; Lehmann, J. Chem. Ber. 1975, 108,
2254-2260. Fuchs, E. F.; Lehmann, J. Carbohydr. Res. 1975, 45, 135-
141. Fuchs, E. F.; Lehmann, J. Carbohydr. Res. 1976, 49, 267-273.
(8) Mu¨ller, C.; Kitas, E.; Wessel, H. P. J. Chem. Soc., Chem. Commun.
1995, 2425-2426. Wessel, H. P.; Mitchell, C.; Lobota, C. M.; Schmid, G.
Angew. Chem., Int. Ed. Engl. 1995, 34, 2712-2713. Nicolaou, K. C.; Flo¨rke,
H.; Egan, M. G.; Barth, T.; Estevez, V. A. Tetrahedron Lett. 1995, 36,
1775-1778. Suhara, Y.; Hildreth, J. E. K.; Ichikawa, Y. Tetrahedron Lett.
1996, 37, 1575-1578.
S0002-7863(96)01068-2 CCC: $12.00 © 1996 American Chemical Society

Peptides Containing Sugar Amino Acids
J. Am. Chem. Soc., Vol. 118, No. 42, 1996 10157
Figure 2. Structures of sugar amino acids (SAAs) discussed in this work.
D-Trp-Lys-Thr. The conformational analysis clearly typified
two â-turns. We herein report a systematic approach using
several sugar amino acids (SAAs) as a new class of building
blocks for peptide scaffolds and conformational restrained
peptidomimetics.9,10 SAAs unit the functional groups of amino
acids with the rigidity of pyranoid ring structures.11 Figure 2
shows a construction kit for predetermined constrained local
conformations in synthetic peptides containing a series of six
SAAs;10 these units offer possibilities as mimetic structures for
both amino acids and dipeptide isosteres. The syntheses of all
SAAs were performed using readily available starting materials.
SAA compounds of type 1R,6 3,12 and 513 have already been
synthesized by other groups, although they have not been used
as structural units in peptides.
The SAAs shown in Figure 2 contain a six-membered ring
with all substituents in equatorial positions, except for the
methoxy group in SAA1R. Therefore, the chair conformation
is very stable and rigid, consequently allowing a prediction on
the conformational restriction introduced to peptides. As will
be demonstrated, SAA1R and SAA1â constrain a linear peptide
conformation, whereas the others are turn mimetics. Thereby
the turn diameter is decreasing from SAA2 to SAA5 (Figure
2). SAA2 and SAA3 serve as â-turn mimetics and SAA4 as a
γ-turn mimetic, and SAA5 can be regarded as a homoproline
derivative (or hydroxylated pipecolic acid).
Figure 3. SAA1R and SAA2 incorporated into enkephalin anlogs 19
and 20.
In order to explore the effect of the dipeptide isosteres SAA1R
and SAA2 on the conformation of linear peptides by NMR
spectroscopy, we synthesized the Leu-enkephalin analogs H-Tyr-
SAA1R-Phe-Leu-OMe (19) and H-Tyr-SAA2-Phe-Leu-OMe
(20) in which the SAAs replace the Gly-Gly dipeptide of the
natural sequence H-Tyr-Gly-Gly-Phe-Leu-OH (Figure 3).
The dipeptide Gly-Gly serves as a spacer in enkephalin
between the messenger amino acid Tyr which is essential for
Figure 4. SAA2, SAA3 and SAA4 incorporated in cyclic peptides
26, 33, and 36.
the activity and the address sequence Phe-Leu responsible for
the selectivity.14 The conformational influence of SAA2, SAA3,
and SAA4 on the peptide backbone of three cyclic peptides
(Figure 4) was investigated in more detail by NMR spectros-
copy, distance geometry, and subsequent molecular dynamic
calculations.
(9) Graf von Roedern, E.; Kessler, H. Angew. Chem., Int. Ed. Engl. 1994,
33, 670-671. (In this article SAA2 was referred to as “GUM” for
glucosyluronic acid methylamine.) Kessler, H.; Diefenbach, B.; Finsinger,
D.; Geyer, A.; Gurrath, M.; Goodman, S. L.; Ho¨lzemann, G.; Haubner, R.;
Jonczyk, A.; Mu¨ller, G.; Graf von Roedern, E.; Wermuth, J. Lett. Pept.
Sci. 1995, 2, 155-160.
(10) Presented at the XVIIth International Carbohydrate Symposium,
1994, Book of Abstracts A1.12. 14th American Peptide Symposium, 1995.
Graf von Roedern, E.; Lohof, E.; Hoffmann, M.; Hessler, G.; Kessler, H.
In Peptides: Chemistry, Structure and Biology; Kaumaya, P. T. P.; Hodges,
R. S., Eds.; Mayflower Scientific Ltd., 1996; pp 682-684.
(11) Furanoid building block: L. Poitout, Y. Le Merrer, J.-C. Depezay,
Tetrahedron Lett. 1995, 36, 6887-6890. During the review and revision
of this manuscript, a similar approach was published: McDevitt, J. P.;
Lansbury, P. T., Jr. J. Am. Chem. Soc. 1996, 118, 3818-3828.
(12) Nitta, Y.; Kuranari, M.; Kondo, T. Yakugaku Zasshi 1961, 81, 1189-
1194.
The highly active somatostatin cyclic hexapeptide cyclo(-Phe-
Pro-Phe-D-Trp-Lys-Thr-)15 was used as a classical peptide for
design of peptidomimetics,16 since the solution structure revealed
two â-turns.17 The sequence Phe-D-Trp-Lys-Thr remained in
(14) Portoghese, P. S.; Sultana, M. S.; Takemori, A. E. J. Med. Chem.
1990, 33, 1714-1720. Portoghese, P. S. J. Med. Chem. 1992, 35, 1927-
1937.
(15) Veber, D. F.; Freidinger, R. M.; Perlow, D. S.; Paleveda, W. J.;
Holly, F. W.; Strachan, R. G.; Nutt, R. F.; Arison, B. H.; Homnick, C.;
Randall, W. C.; Glitzer, M. S.; Saperstein, R.; Hirschmann, R. Nature 1981,
292, 55-58.
(13) Bashyal, B. P.; Chow, H. F.; Fleet, G. W. J. Tetrahedron Lett. 1986,
27, 3205-3208.

10158 J. Am. Chem. Soc., Vol. 118, No. 42, 1996
Graf Von Roedern et al.
Scheme 1. Synthesis of the Protected Building Block
SAA1â
R-anomer. Other acetylation methods, i.e., acetic anhydride/
pyridine or acetic anhydride/perchloric acid also provide the
acetylated glucuronolactone along with R-acetate. The â-azide
7 was obtained from 6 with tin tetrachloride and trimethylsilyl
azide22 in an overall yield of 43%. Catalytic reduction at low
temperature provided H-SAA3-OMe (8), which was used
without further purification.
The synthesis of Fmoc-SAA4-(tri-O-benzyl)-OH, 15 (Scheme
3), followed a procedure published for the stereoselective
C-glycosidation of 2-acetamido-2-deoxy-D-glucose.23 With
D-glucosamine as the starting material, the partially benzylated
sugar 9 was obtained in two steps according to a procedure of
Fletcher and Inch.24 The amino function was subsequently
protected by Cbz-Cl to obtain 10 in 90% yield. Chlorination
of the anomeric hydroxyl group provided the R-chloro com-
pound which was treated with tributyltin lithium to afford 11
in 79% yield.23 The generation of the glycosyl dianion 12 was
accomplished in two separate temperature steps: first, deproto-
nation of the urethane nitrogen at -78 °C using 1 equiv of BuLi;
second, transmetalation at -55 °C using 1.2 equiv of BuLi.
The dianion 12 was visualized by a deep red color of the solution
and was subsequently trapped by carbon dioxide to afford 13
in 83% yield. For the application of SAA4 in solid phase
peptide synthesis, 13 was transformed into the Fmoc derivative
15. TFA/thioanisole25 or catalytic hydrogenolysis on Pd/C26
were not selective for removal of the Cbz group in 13. The
best result for cleaving the Cbz group was obtained by using
trimethylsilyl iodide in CH3CN.27 However, the C7-O-benzyl
ether of 13 was cleaved to some extend. While the amount of
side product was temperature independent, the yield was opti-
mized by varying the reaction time. The crude reaction mixture
was treated with Fmoc-ONSu28 to afford 15 in 48% yield.
The synthesis of SAA5 has been published by Fleet et al.13
As part of our ongoing study this azasugar will be incorporated
into peptides to compare its conformational and pharmaceutical
influences as a proline and pipecolic acid surrogate.
Synthesis of the SAA Peptides. The enkephalin analogs
19 and 20 (Figure 3) were assembled starting from dipeptide
H-Phe-Leu-OMe, which was coupled with the monoprotected
SAAs using standard peptide solution protocol with EDCl‚HCl
(1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochlo-
ride) and HOBt (1-hydroxybenzotriazole) as coupling reagents
in an overall yield of 34% 19 and 50% 20. Protection of the
hydroxyl groups of SAA1R and SAA2 was not necessary. In
these syntheses the chemical behavior of the SAAs was similar
to that of unprotected threonine.
Cyclo(-SAA2-Phe-D-Trp-Lys-Thr-) (27) was synthesized us-
ing THF and DMF as a solvent mixture in the coupling
reactions. EDCl‚HCl and HOBt were used as coupling agents,
and the cyclization step was activated with TBTU (2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate).
By using such strong activation, some â-elimination in SAA2
a âII′-turn being part of many other active somatostatin ana-
logs.18 In cyclo(-SAA2-Phe-D-Trp-Lys-Thr-) and cyclo(-SAA3-
Phe-D-Trp-Lys-Thr-) the SAAs replaced the two neighboring
amino acids Phe-Pro to investigate the resulting turn pattern.
SAA4 was incorporated in a model peptide of the sequence
cyclo(-SAA4-Ala-D-Pro-Ala-Ala-).
Synthesis of the SAA Building Blocks. Cbz-SAA1R-OH
was synthesized as described by Heyns and Paulsen starting
from the R-methyl glycoside in an overall yield of 37%.6 The
â-anomer Cbz-SAA1â-OH (4) was prepared from glucosamine
which was transformed to glycosyl bromide 1 with acetyl
bromide (Scheme 1).19
The â-methyl glycoside20 was obtained by treatment of
bromide 1 with methanol and pyridine and further protected by
the benzyloxycarbonyl (Cbz) group. Deacetylation of 2 was
achieved by methanolysis, and the resulting compound 3 was
selectively oxidized at the free primary hydroxyl group with
oxygen on a platinum catalyst in aqueous solution by the method
of Heyns and Paulsen6 in an overall yield of 49%.
The synthesis of SAA2 has already been published by our
group.9 SAA2 was obtained as Cbz-SAA2-OMe in an overall
yield of 12% and adequately deprotected for further synthesis.
The enantiomer of SAA2 was prepared by Fuchs and Lehmann7
in 11 steps starting from glucose.
H-SAA3-OMe (8) has already been described by Nitta et al.,12
who prepared the azide from the unstable bromide (obtained
from R/â acetate mixture) using NaN3 in 53% yield for this
step. Because of the unsatisfactory yields, we have improved
the synthesis following the route outlined in Scheme 2. The
glucuronolactone was converted to the methyl ester with
methanol via base catalysis and was then acetylated by a mixture
of acetic anhydride and sodium acetate.21 Crystallization
allowed an excellent separation of the â-acetate 6 from the
(22) Paulsen, H.; Gyo¨rgydeak, Z.; Friedmann, M. Chem. Ber. 1974, 107,
1568-1578.
(23) Hoffmann, M. Kessler, H., Tetrahedron Lett. 1994, 35, 6067-6070.
For a similar approach to C-glycosides of glucose, see also: Wittmann,
V.; Kessler, H. Angew. Chem. Int. Ed. Engl. 1993, 32, 1091-1093. Frey,
O.; Hoffmann, M.; Wittmann, V.; Kessler, H.; Uhlmann, P.; Vasella, A.
HelV. Chim. Acta 1994, 77, 2060-2069. Frey, O.; Hoffmann, M.; Kessler,
H. Angew. Chem., Int. Ed. Engl. 1995, 34, 2026-2028.
(24) Inch, T. D.; Fletcher, H. G. J. Org. Chem. 1966, 31, 1810-1815.
(25) Kiso, Y., Ukawa, K.; Akita, T. J. Chem. Soc., Chem. Commun. 1980,
101-102.
(16) For a recent example, see: Beusen, D. D.; Zabrocki, J.; Slomczynka,
U.; Head, R. D.; Kao, J. L.-F.; Marshall, G. R. Biopolymers 1995, 36, 181-
200.
(17) Kessler, H.; Bernd, M.; Kogler, H.; Zarbock, J.; Sorensen, O. W.;
Bodenhausen, G.; Ernst, R. R. J. Am. Chem. Soc. 1983, 105, 6944-6952.
(18) Veber, D. F. In Peptides, Chemistry and Biology, Proceedings of
the Twelfth American Peptide Symposium; Smith, J. A.; Rivier, J. E., Eds.;
Escom: Leiden, 1992; pp 3-14.
(19) Irvine, J. C.; McNicoll, D.; Hynd, A. J. Chem. Soc., London 1911,
11, 250-261.
(20) Fodor, G.; O¨ tvo¨s, L. Chem. Ber. 1956, 89, 701-708.
(21) Wolfom, M. L.; Thompson A. In Methods in Carbohydrate
Chemistry Vol. II; Whistler, R. L.; Wolfrom, M. L.; BeMiller, J. N., Eds.;
Academic Press: New York, 1963; pp 211-215.
(26) Bergmann, M.; Zervas, L. Ber. 1932, 65, 1192-1201.
(27) Lott, R. S.; Chauhan, V. S.; Stammer, C. H. J. Chem. Soc., Chem.
Commun. 1979, 495-496.
(28) Ten Kortenaar, P. B. W.; Van Dijk, B. G.; Peeters, J. M.; Raaben,
B. J.; Adams, P. J. H. M.; Tesser, G. I. Int. J. Pept. Protein Res. 1986, 27,
398-400.

Peptides Containing Sugar Amino Acids
J. Am. Chem. Soc., Vol. 118, No. 42, 1996 10159
Scheme 2. Synthesis of the Protected Building Block SAA3
Scheme 3. Synthesis of the Protected Building Block SAA4
wasobserved as TBTU reacted with the unprotected sugar
hydroxyl groups.
The dihedral angles æi and ψi+1 display no conformational
restriction. Therefore the peptidomimetic SAA1R can be used
to replace two adjacent amino acids introducing conformational
constraints at the ψi, ωi, and æi+1 angle. These restrictions result
in an extended peptide conformation. In agreement with a linear
conformation, ROE cross signals were only found between
adjacent amino acids.
Cyclo(-SAA3-Phe-D-Trp-Lys(Boc)-Thr(tBu)-) (33) was also
synthesized in solution. Contrary to the C-glycosidic SAA2,
the free amine of SAA3 was unstable due to epimerization.
Therefore, hydrogenation of the azide 7 on Pd/C was performed
in THF since anomerization is known to occur preferably in
protic solvents. After isolation of compound 8, the amine was
immediately coupled with IIDQ (1-(isobutoxycarbonyl)-2-
isobutoxy-1,2-dihydroquinoline).29 Coupling with EDCl‚HCl
in THF gave more side products than IIDQ, because of the low
nucleophilicity of the anomeric amine. Further couplings were
performed with standard peptide coupling procedures (see
Experimental Section) and by adding H2O as a scavenger for
the hydroxyl groups during cyclization with TBTU.
3
Analysis of the J(H,H) coupling constants in peptide 20
indicates that the pyranoid ring of SAA2 is in the all-equatorial
chair conformation, fixing the backbone angles ωi and æi+1
around 180°. The æi and both ψ angles are not restricted, which
allows SAA2 to form loop structures. The homonuclear 3J(H,H)
coupling constants between the methylene protons of C7 and
the H6 in 20 were 1.0 and 8.5 Hz, respectively. This indicates
a preferred conformation about the C6-C7 bond in 20 with one
of the methylene protons oriented antiperiplanar to H6 (ø1 )
-60° or ø1 ) -180°). The assignment of the two diastereotopic
protons was performed using the distance information of a
ROESY spectrum (see Experimental Section). The ROE cross
signals between aromatic protons of the tyrosine side chain and
H2 of SAA2 show a distance of 3.8 and 3.9 Å. Obviously SAA2
induces a bent structure, without acting as a rigid â-turn mimetic.
Presuming a trans amide bond between Tyr and SAA2, such
short distances are only possible if H7(pro-S) is antiperiplanar
to H6 (ψi ) -60°). In the case of the other rotamer (ψi )
180°), the distance would be larger than 6.5 Å. This turn
structure is also in agreement with Newman’s 1,5-repulsion
theory.30 The other possible rotamer would result in a strong
sterical repulsion between the NH- and HO- substituent of SAA2
(Figure 5).
The peptide cyclo(-SAA4-Ala-D-Pro-Ala-Ala-) (36) was
synthesized by solid phase synthesis. The pentapeptide analog
H-Ala-SAA4(tri-O-benzyl)-Ala-D-Pro-Ala-OH (34) was as-
sembled on the 2-chlorotritylchloride resin using Fmoc-
chemistry and cleaved from the resin by HOAc/trifluoroethanol
in CH2Cl2. Compound 15 was used in 1.1 equiv and all other
amino acids in 1.7 equiv relative to the determined loading of
the resin. The subsequent cyclization was performed at high
dilution using TBTU as a coupling agent. The protecting groups
were removed by hydrogenolysis in the presence of Pd/C.
Conformational Analysis. The 1H NMR spectrum in
DMSO-d6 of the linear peptide 19 shows large 3J(H,H) coupling
constants around 10 Hz between the ring protons of SAA1R,
and this implies that the pyranoid ring of SAA1R is in the
4
predicted C1 chair conformation. In Figure 5 the dihedral
The conformational analyses of the cyclic peptides were
carried out using the side chain protected cyclo(-SAA2-Phe-D-
Trp-Lys(Cbz)-Thr-) (26), cyclo(-SAA3-Phe-D-Trp-Lys(Cbz)-
Thr(tBu)-) (33), and cyclo(-SAA4-Ala-D-Pro-Ala-Ala-) (36). The
angles of SAA1R are compared to those of the backbone of a
dipeptide with a cis amide bond. The ωi angle of SAA1R is
fixed to -60°, whereas the ψi and æi+1 angles are about 180°.
(29) Bodanszky, M.; Bodanszky, A. Practice in Peptide Synthesis, 2nd
ed.; Springer Verlag: Heidelberg, 1994.
(30) Newman, M. S. J. Am. Chem. Soc. 1950, 72, 4783-4786.

10160 J. Am. Chem. Soc., Vol. 118, No. 42, 1996
Graf Von Roedern et al.
from averaging the J values of each individual conformation
over the trajectory. Moreover radial distribution functions
(radf)37 were calculated to judge solvent accessibility of the
amide protons.
Cyclo(-SAA21-Phe2-D-Trp3-Lys4(Cbz)-Thr5-) (26) shows only
one conformer in the 1H NMR spectrum. Large 3J(H,H)
coupling constants (approximately 9 Hz) between the carbo-
hydrate protons indicated a 4C1 analog chair conformation. The
backbone amide bonds of 26 are all trans configurated as no
strong HR-HR NOE were detectable.
Figure 6 shows the averaged and minimized structure of 26
consistent with a pseudo-â/âII′-turn arrangement with D-Trp in
the i + 1 position of the distorted âII′-turn and the dipeptide
isostere SAA2 in the i and i + 1 positions of a pseudo-â-turn
as indicated by the backbone dihedral angles in Table 1. As
expected, radial distribution functions indicated solvent acces-
sibility only for amide protons with large negative temperature
coefficients. The analysis of the trajectory revealed large
fluctuations for the æ-angles of D-Trp and Phe. This flexibility
was most likely to cause large deviations between the experi-
mental and calculated 3J(HN,HR) coupling constants (Phe
3J(HN,HR) ) 7.2 Hz (exp) and 10.1 Hz (calcd), D-Trp 3J(HN,HR)
) 5.9 Hz (exp) and 9.2 Hz (calcd)), especially since the Karplus
curve has a steep slope for the relevant coupling constants. For
the side chain of Lys (diastereotopic assignment according to
Wu¨thrich et al.38 ), the Pachler equations39 predicted a population
of 51% for the preferential ø1 angle of -60°, whereas according
to the Pachler equations no predominant side chain orientation
exists for D-Trp and Phe in solution. The synclinal orientation
of the Thr5HR-Hâ protons was confirmed by the HR-Hâ
coupling constant of 4.6 Hz and the ROEs between Thr5HR-
Hâ and Thr5Hâ-SAA2HN.
Figure 5. Comparison of SAA1R with the natural dipeptide D-Ser-
Ser and SAA2 with Gly-Xaa and characteristic long-range NOE effects
in the linear peptide 20.
hydroxyl groups of the sugar moiety were unprotected in all
cases. The conformational analyses were based on NMR
spectroscopy in DMSO-d6 at 300 K: Interproton distances were
calculated from NOESY and ROESY cross peak volumes,
homonuclear proton coupling constants were obtained from 1D
spectra or P.E. COSY spectra, and temperature dependence of
the amide protons has been measured by 1D spectra in the range
300-340 K.
1
The H NMR spectrum of cyclo(-SAA31-Phe2-D-Trp3-Lys4-
(Boc)-Thr5(tBu)-) (33) in DMSO-d6 at 300 K showed three
conformations with ratios of 80:15:5. The ROESY spectrum
proved them to be different conformers of the same molecule
by exchange peaks. Only the major conformation could be
3
completely assigned. The large J(H,H) coupling constants
(approximately 9 Hz) between the carbohydrate protons were
4
The starting geometries for the MD simulations were obtained
by distance geometry (DG) with a modified version31 of the
DISGEO32 program using proton-proton distances and
characteristic for a C1 analog chair conformation.
Figure 7 shows the averaged backbone conformation of 33
with a âII′/pseudo-â-turn arrangement. D-Trp occupies the i
+ 1 position of a distorted âII′-turn. SAA3 is acting as a â-turn
mimetic (Table 2). The corresponding hydrogen bond between
Thr carbonyl oxygen and Phe amide proton is present to a degree
of 13% during the rMD simulation. Only the ROE between
D-Trp3HR and D-Trp3H2 is significantly violated due to flexibility
which was indicated by the homonuclear HR-Hâ coupling
constant and the fact that the D-Trp3HR-D-Trp3H2 and D-Trp3HR-
D-Trp3H4 ROEs cannot be met by a single side chain conformer.
Both Thr and Phe had a flexible side chain as indicated by the
3
3J(HN,HR) and J(HR,Hâ) proton-proton coupling constants.33
The following distance restraint MD simulations (rMD) were
performed in explicit DMSO solvent34 with the CVFF35 force
field of the Discover36 program. The last 150 ps of the rMD
simulation were collected and analyzed. The depicted structures
were obtained by averaging over the last 50 ps and energy-
minimized by 300 steps steepest descent (for details, see
Experimental Section). In order to compare the rMD calcula-
tions with experimental data, distances were calculated by r-3
averaging over the trajectory; coupling constants were obtained
3
3J(HR,Hâ) coupling constants (Thr J(HR,Hâ) ) 6.5 Hz, Phe
3J(HR,Hâ) ) 7.8t and 5.6h Hz). As in the case of 26 only the
Lys side chain occupied a preferred rotamer. According to the
Pachler equations, ø1 ) -60° was populated to 81%. Similar
to compound 26, large fluctuations were observed for the
æ-angles of D-Trp and Phe during the simulation. For all amide
protons radial distribution functions were calculated from the
150 ps rMD trajectory. They agree well with the temperature
coefficients.
(31) Mierke, D. F.; Kessler, H. Biopolymers 1993, 33, 1003-1017.
(32) Havel, T. F. Prog. Biophys. Mol. Biol. 1991, 56, 43-78. Crippen,
G. M.; Havel, T. F. Distance Geometry and Molecular Conformation;
Research Studies Press LTD.: Somerset, England; John Wiley & Sons:
New York, 1988. Havel, T. F. DISGEO, Quantum Chemistry Exchange
Program, Exchange No. 507, Indiana University, 1986. Havel, T. F.;
Wu¨thrich, K. Bull. Math. Biol. 1984, 46, 673-698.
(33) Bystrov, V. F. Prog. Nucl. Magn. Reson. Spectrosc. 1976, 10, 41-
81.
(34) Mierke, D. F.; Kessler, H. J. Am. Chem. Soc. 1991, 113, 9466-
9470.
(35) Maple, J. R.; Dinur, U.; Hagler, A. T. Proc. Natl. Acad. Sci. U.S.A.
1988, 85, 5350-5354. Dauber Osguthorpe, P.; Roberts, V. A.; Osguthorpe,
D. J.; Wolff, J.; Genest, M.; Hagler, A. T. Proteins: Struct. Funct. Genetics
1988, 4, 31-47.
(36) DISCOVER Version 2.9.0, BIOSYM Technologies, 10065 Barnes
Canyon Road, San Diego, CA 92121.
(37) Haile, J. M. Molecular Dynamics Simulation; John Wiley & Sons,
Inc.: New York, 1992; pp 260-263.
(38) Wagner, G.; Braun, W.; Havel, T. F.; Schaumann, T.; Wu¨thrich,
K. J. Mol. Biol. 1987, 196, 611-639.
(39) Pachler, K. G. R. Spektrochim. Acta 1963, 19, 2085-2092. Pachler,
K. G. R. Spektrochim. Acta 1964, 20, 581-587.

Peptides Containing Sugar Amino Acids
J. Am. Chem. Soc., Vol. 118, No. 42, 1996 10161
Figure 6. Stereoplot of cyclo(-SAA2-Phe-D-Trp-Lys(Cbz)-Thr-) (26) obtained by averaging the last 150 ps of the rMD tarjectory and subsequent
energy minimization by 300 steps steepest descent. The depicted orientation of the side chains of D-Trp and Phe is arbitrary due to their flexibilty.
Table 1. Backbone Dihedral Angles for 26
æ
ψ
ω
SAA2
111 (ThrCO-HN-C7-C6)
-63 (HN-C7-C6-O)
-177 (C7-C6-O-C2)
177 (C6-O-C2-CO)
-26 (O-C2-CO-PheHN)
172 (C2-CO-HN-PheCR)
Phe
D-Trp
Lys
-122
105
-112
-37
-173
-177
-173
175
95
-108
-123
Thr
126
Figure 7. Stereoplot of cyclo(-SAA3-Phe-D-Trp-Lys(Boc)-Thr(tBu)-) (33) obtained by averaging the last 150 ps of the rMD tarjectory and subsequent
energy minimization by 300 steps steepest descent. The depicted orientation of the side chains of D-Trp and Phe is arbitrary due to their flexibility.
The 1D-proton spectrum of cyclo(-SAA41-Ala2-D-Pro3-Ala4-
Ala5-) (36) shows two conformations in a ratio of 80:20, which
were both assigned. Surprisingly the second conformation does
not result from a cis Pro bond as would be expected. Exchange
peaks in the ROESY spectrum proved that the two sets of signals
result from two conformers of the same compound. The major
conformation formed an all-trans configuration around the
amide bonds, since no strong HR-HR ROEs were observed.

10162 J. Am. Chem. Soc., Vol. 118, No. 42, 1996
Graf Von Roedern et al.
Table 2. Backbone Dihedral Angles for the Major Conformation of 33
æ
ψ
ω
SAA3
53 (ThrCO-HN-C1-O)
178 (HN-C1-O-C5)
-174 (C1-O-C5-CO)
-58 (O-C5-CO-PheHN)
173 (C5-CO-HN-PheCR)
Phe
D-Trp
Lys
-139
109
-94
-114
118
-178
179
179
-117
-54
Thr
139
-168
Figure 8. Stereoplot of cyclo(-SAA4-Ala-D-Pro-Ala-Ala-) (36) obtained by averaging the last 150 ps of the rMD tarjectory and subsequent energy
minimization by 300 steps steepest descent.
Table 3. Backbone Dihedral Angles of the Major Conformation of 36
æ
ψ
ω
SAA4
114 (Ala5CO-HN-C2-C1)
-53 (HN-C2-C1-CO)
-63 (C2-C1-CO- Ala2HN)
-177 (C1-CO-HN- Ala2CR)
Ala2
-85
76
-86
-142
136
-111
-39
178
173
D-Pro3
Ala4
-178
-178
Ala5
124
The chemical shifts of the proline â- and γ-carbons indicated
that the preceding amide bond is trans-configurated.40 The
distance of 273 pm for SAA4H2 and SAA4H4 (see Supporting
Information) was also indicative for a C1 chair conformation
close to the corresponding value of â-D-glucose.
Figure 8 shows the averaged and minimized structure of the
major conformation of 36 with a âII′/pseudo-γ-turn arrangement.
D-Pro occupies the i + 1 position of a âII′-turn SAA4 acting as
a γ-turn mimetic (Table 3).
During the simulation a frequent switch between a âII′- and
a γ-turn was observed for residues located in the â-turn region.
Hence, hydrogen bonding occurred for both types of turns. This
flexibility of the âII′-turn might cause the violation of the
3J(Ala5HN,HR) coupling constant.41
Furthermore, the carbon chemical shifts of D-Pro3 indicate a
trans configuration at the preceding amide bond. No strong
HR-HR NOEs were detectable for the second conformation. Due
to the low population, no structural calculations were performed.
Carbon chemical shifts of D-Pro3 were very similar for both
conformers, so no change of the local structure is expected.
Available ROE data (Ala5HN-Ala4HN, 306 pm; Ala4HN-
D-Pro3, 231 pm; Ala5HN-D-Pro3, 389 pm; Ala4HN-Ala4HR, 315
pm) and the small negative temperature coefficient Ala5HN are
in agreement with a âII′-turn with D-Pro3 in the i + 1 position.
The experimental data indicate a different conformation for both
conformers at SAA4HN. The chemical shift changed drastically
(from 7.99 ppm to 6.23 ppm). The change of the temperature
coefficient for SAA4HN from -6.5 ppb/K to -1.0 ppb/K
indicated an internal orientation of this amide proton, which
was confirmed by ROE data (shortening of the Ala5HN-
SAA4HN distance from 370 pm to 251 pm). The existence of
such conformers suggests that the rotation about the bond
between SAA4HN and the SAA4C1 is hindered.
4
This clearly shows that SAA4 occupies the i + 1 position of
the pseudo-γ-turn forming a hydrogen bond between the Ala2
amide proton and the SAA4 carbonyl populated to 38.8%.
In the minor conformation, the carbohydrate moiety exists
4
in a C1 chair conformation as confirmed by the large homo-
nuclear coupling constants between the carbohydrate protons.
(41) Kopple, K. D.; Ohnishi. M.; Go, A. J. Am. Chem. Soc. 1969, 91,
4264-4272. Ovchinninkov, Y. A.; Ivanov, V. T. Tetrahedron 1975, 31,
2177-2209. Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512-
523.
(40) Deber, C. M.; Madison, V.; Blout, E. R. Acc. Chem. Res. 1976, 9,
106-113. Dorman, D. E.; Borvey, F. A. J. Org. Chem. 1973, 38, 1719-
1722. Dorman, D. E.; Borvey, F. A. J. Org. Chem. 1973, 38, 2379-2383.

Peptides Containing Sugar Amino Acids
J. Am. Chem. Soc., Vol. 118, No. 42, 1996 10163
Figure 9. Superposition of 26 (black) and an idealized âII′/âII′-turn arrangement (gray); superposition of 33 (black) and an idealized âII′/âII′-turn
arrangement (gray); and superposition of 36 (black) and an idealized âII′/γ-turn-arrangement (gray).
Biological Tests. The two enkephalin analogs 19 and 20
and the somatostatin analog 27 were tested for their biological
activity. The enkephalin analogs 19 and 20 show no activity
in the guinea pig ileum assay.42 The somatostatin analog 27
has an inhibition constant (IC50) of 0.15 µM in displacing the
receptor-bound radioligand [125I]Try11 somatostatin-14 in AtT20
cell membranes obtained from mice hypophyses. In fact, com-
pound 27 is only 75 times less active than the highly potent
somatostatin analog cyclo(-Phe-Pro-Phe-D-Trp-Lys-Thr-). This
is particularly remarkable, since 27 does not contain the lipo-
philic residues on both sides of the active tetrapeptide sequence
that are considered to be important for high somatostatin
activity.15,18
acid, the superposition showed that SAA4 meets the geometric
requirements to form a γ-turn.
Obviously the dipeptide isostere SAA2 mimics a â-turn in
peptide 26. SAA3 whose backbone is one atom shorter than
that of a dipeptide isostere is suited as a â-turn mimetic in 33
just as well. In comparison to SAA3 in 33 the carbohydrate
moiety of SAA2 in 26 is slightly out of plane of the â/âII′-turn
arrangement. Apparently 26 and 33 form very similar backbone
structures.
The superposition of the cyclic SAA containing peptides with
the idealized turns show that the SAA building blocks form
the proposed turn structures. The SAAs of the peptide construc-
tion kit may thus become a tool for a rational design of peptide
conformations. A main advantage of the SAAs is that the con-
formational restriction changes significantly while the structure
of the sugar moiety is more or less preserved. The protocol
developed also allows the use of SAAs in solid phase peptide
syntheses as well as in combinatorial synthesis. Libraries may
be composed of different SAAs alone or including other natural
or unnatural amino acids. The hydroxyl groups of the SAA
can be modified, e.g., by benzylation or other derivatives. Such
modifications will change the physical and chemical properties
without changing the backbone structure in the cyclic peptide.
The protecting groups could even serve as mimics for additional
peptidic structures. Moreover, the presented SAAs can also be
used as building blocks of oligo- and polysaccharide analogs.8
Conclusion
The conformational analyses of the cyclic peptides presented
here show that the replacement of the amino acids by SAAs
introduced the proposed turn motifs combining the structural
features of peptides and carbohydrates. As confirmed by the
large 3J(H,H) coupling constants, the carbohydrate ring remains
4
in the C1 conformation rendering a significant influence on
the peptide backbone. Figure 9 shows the superpositions of
the averaged and minimized structures of 26, 33, and 36 with
cyclic peptide backbones consistent with idealized turn struc-
tures.43 26 and 33 were compared with the appropriate âII′/
âII′-turn replacing the Phe-Pro residues of the cyclic hexapeptide
cyclo(-Phe-Pro-Phe-D-Trp-Lys-Thr-).18 The backbone dihedral
angles and the temperature coefficients were in agreement with
the corresponding data in the literature.44 Peptide 36 containing
SAA4 was superimposed with the backbone of a cyclic penta-
peptide with an idealized âII′/γ-turn arrangement. Although
SAA4 has one more backbone atom than a natural R-amino
Experimental Section45
General Methods. Solvents for moisture sensitive reactions were
distilled and dried according to standard procedures. All other solvents
were distilled before use. Pt/C and Pd/C were donated by Degussa,
Frankfurt/M., Germany. Flash column chromatography (FC) was per-
formed with indicated solvents on silica gel 60, 230-400 mesh (Merck
KGaA, Darmstadt). All reactions were monitored by thin-layer chro-
matography with 0.25 mm precoated silica gel 60 plates with F254 indi-
cator (Merck KGaA, Darmstadt). Melting points were obtained on a
Bu¨chi-Tottoli apparatus and are uncorrected. Optical rotation were
determined with a Perkin-Elmer 241 polarimeter. IR spectra were
recorded on a Perkin-Elmer 257 spectrophotometer. FAB mass spectra
were recorded on Varian MAT 311 A using NOBA or glycol matrices.
Elemental analysis were performed on a Heraeus EA415-0 analyzer.
RP-HPLC analysis were carried out on Beckman System Gold using a
Nucleosil-7 C18 column; solvent A, H2O + 0.1% CF3COOH, and
(42) Biological test performed at the laboratories of Prof. P. Schiller,
Clinical Research Institute of Montreal, Canada.
(43) The structures of the cyclic model peptides were assembled from
five, respectively six alanines. The torsion angles of the idealizied turns
were adjusted on the linear peptides which were subsequently cyclized and
minimized by a few steps. The torsion angles were taken from the
following: Richardson, J. S. AdV. Protein Chem. 1981, 34, 167-339.
(44) Freidinger, R. M.; Veber, D. F. Conformationally Directed Drug
Design: Peptides and Nucleic Acids as Templates or Targets; Vida, J. A.;
Gordon, M., Eds.; American Chemical Society: Washington, DC, 1984;
pp 169-187.
(45) Complete spectral details are available in the Supporting Information.

10164 J. Am. Chem. Soc., Vol. 118, No. 42, 1996
Graf Von Roedern et al.
solvent B, CH3CN + 0.1% CF3COOH, with UV detection at 220 and
254 nm.
with H2O (100 mL), and dried (MgSO4). The solvent was evaporated
to yield 115 g (94%) as a colorless solid. 1H NMR (250 MHz, DMSO-
d6, 300 K): δ 7.45 (d, 1H, HN), 7.23-7.38 (m, 5H, C6H5), 4.77-5.15
(m, 4H, PhCH2,H3,H4), 4.47 (d, 1H, H1), 4.20 (dd, 1H, H6t), 4.02 (dd,
1H, H6h), 3,79 (ddd, 1H, H5), 3.48 (ddd, 1H, H2), 3.32 (s, 3H, CH3),
Proton signals were assigned by the combined use of TOCSY,46
z-TOCSY,47 DQF-COSY,48 and/or P.E. COSY49 spectra. Carbon shifts
were obtained with HMQC50 and HMQC-COSY51 experiments. The
HMQC experiment was performed with a BIRD-puls.52 In addition,
for peptide 36 a HMBC53 spectrum with a low-pass J filter54 was re-
corded and used for the assignment of the 13C resonances. In this case,
sequential assignment was performed with the HMBC spectrum whereas
in the other cases the correct sequence was confirmed by a ROESY55
with a pulsed spinlock56 (33) and a NOESY57 experiment (26).
Quantitative information on interproton distances was obtained from
NOESY and ROESY spectra with mixing times of 120 and 150 ms,
respectively. Integrals from the ROESY experiment were offset
corrected.58 For all distance calculations the isolated two-spin ap-
proximation was used. Interproton distances and homonuclear coupling
constants were employed (for details, see Supporting Information) for
structure calculation.
3
1.84 + 1.97 + 2.02 (3 s, 9H, CH3CO); J(SAA1âH1,H2) ) 8.3 Hz,
3J(SAA1âHN,H2) ) 9.0 Hz, 3J(SAA1âH2,H3) ) 9.0 Hz, 3J(SAA1âH3,H4)
3
3
) 9.0 Hz, J(SAA1âH4,H5) ) 9.0 Hz, J(SAA1âH5,H6a) ) 4.7 Hz,
3J(SAA1âH5,H6b)
) ) 12.4 Hz,
1.9 Hz, 3J(SAA1âH6a,H6b)
3J(SAA1âHN,H2) ) 9.0 Hz. Anal. Calcd for C21H27NO10: C, 55.63;
H, 6.00; N, 3.09. Found: C, 55.75; H, 6.00; N, 3.07.
Methyl 2-[N-(Benzyloxycarbonyl)amino]-2-deoxy-â-D-glucopy-
ranoside (3). Acetate 2 (62 g, 0.14 mol) was dissolved in dry MeOH
(400 mL), and dimethylethylamine (20 mL) was added. After 24 h
the solvent was evaporated, and the residue was recrystallized from
H2O to yield 44 g (99%) as colorless needles. 1H NMR (250 MHz,
DMSO-d6 + 10% D2O, 300 K): δ 7.27-7.36 (m, 5H, C6H5), 4.98 (s,
2H, PhCH2), 4.13 (d, 1H, H1), 3.66 (dd, 1H, H6t), 3.43 (dd, 1H, H6h),
3.31 (s, 3H, CH3), 3.31-3.02 (m, 4H, H2,H3,H4;H5); 3J(SAA1âH1,H2)
) 7.6 Hz, 3J(SAA1âH5,H6a) ) 1.0 Hz, 3J(SAA1âH6a,H6b) ) 11.7 Hz.
Anal. Calcd for C15H21N1O7: C, 55.04; H, 6.47; N, 4.28. Found: C,
55.00; H, 6.56; N, 4.31.
2-[N-(Benzyloxycarbonyl)amino]-1-O-methyl-2-deoxy-â-D-glu-
copyranuronic Acid (4). Compound 3 (10.0 g, 31 mmol) was
dissolved in H2O (100 mL) and stirred with 10% Pt/C (5.0 g, ca. 50%
H2O) at 90 °C under a stream of O2. The gas was pumped through the
closed apparatus and purified by passing through 4 N NaOH. The pH
of the mixture was maintained between 7 and 8 by addition of 10%
NaHCO3. Catalyst (2 g) was added after 15 and 30 h. After 50 h the
catalyst was removed by filtration. The filtrate was neutralized with
ion exchange resin (20 g, Aldrich, Amberlyst 15, strongly acidic, H+
form), stirred for 10 min, filtered, and concentrated in vacuo to 30
mL. The product crystallized at 4 °C, yielding 5.2 g (54%) as colorless
needles. Mp: 142 °C dec. [R]20D: -35.7° (c ) 1.0, MeOH). 1H
NMR (250 MHz, DMSO-d6 + 10% D2O, 300 K): δ 7.37-7.31 (m,
5H, C6H5), 4.98 (s, 2H, PhCH2), 4.61 (s, 1H, H1), 3.76 (d, 1H, H5),
3.45-3.35 (m, 3H, H2,H3,H4), 3.23 (d, 1H, CH3); 3J(SAA1âH1,H2) )
2.1 Hz, 3J(SAA1âH4,H5) ) 9.2 Hz. FAB-MS: 342 [M + H]+. Anal.
Calcd for C15H19NO8: C, 52.79; H, 5.61; N, 4.10. Found: C, 52.47;
H, 5.68; N, 4.21.
Structure Calculations. Structure calculations were performed
using distance geometry to generate a starting structure for subsequent
restrained MD refinements. For distance geometry (DG), a modified
version of the DISGEO program using proton-proton distances and
3J(HN,HR) and 3J(HR,Hâ) homonuclear coupling constants as restraints
using the Karplus equation. For the following MD simulations in
explicit DMSO59 the Discover program with the CVFF force field was
used. It is especially parametrized for peptides and small organic
molecules, but proved to be useful in simulations for saccharides.60
Hence, it is an adequate choice for the mainly peptidic compounds
investigated here.
Synthesis of SAA1â (3,4,6-Tri-O-acetyl-2-[N-(benzyloxycarbon-
yl)amino]-2-deoxy-1-O-methyl-â-D-glucopyranoside, 2). 3,4,6-Tri-
O-acetyl-2-amino-2-deoxy-R-D-glucopyranosyl bromide hydrobromide
119 (123 g, 0.27 mol) was dissolved in dry MeOH (1 L) and pyridine
(22 mL, 0.27 mol) added. After 6 h the solvent was evaporated and
the residue dried in vacuo. NaHCO3 (112 g, 1.12 mol) was dissolved
in H2O (1 L) and added to EtOAc (1.4 L). Benzyl chloroformate (100
mL, 300 mmol, 50% solution in toluene) was added under vigorous
stirring. After the evolution of CO2 stopped, the organic phase was
separated, washed three times with 0.5 N HCl (100 mL) and three times
(46) Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521-
528. Bax, A.; Byrd, R. A.; Aszalos, A. J. Am. Chem. Soc. 1984, 106, 7632-
7633. Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355-360.
(47) Titman, J. J.; Neuhaus, D.; Keeler, J. J. Magn. Reson. 1989, 85,
111-131. Titman, J. J.; Keeler, J. J. Magn. Reson. 1990, 89, 640-646.
(48) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1975, 64,
2229-2246. Piantini, U.; Sørensen, O. W.; Ernst, R. R. J. Am. Chem. Soc.
1982, 104, 6800-6801. Rance, M.; Sørensen, O. W.; Bodenhausen, G.;
Wagner, G.; Ernst, R. R.; Wu¨thrich, K. Biochem. Biophys. Res. Commun.
1983, 117, 458-479.
(49) Mu¨ller, L. J. Magn. Reson. 1987, 72, 191-196.
(50) Bendall, M. R.; Pegg, D. T.; Dodrell, D. M. J. Magn. Reson. 1983,
52, 81-117. Bax, A.; Griffey, R. H.; Hawkins, B. L.; J. Am. Chem. Soc.
1983, 105, 7188-7190. Bax, A.; Griffey, R. H.; Hawkins, B. L. J. Magn.
Reson. 1983, 55, 301-315.
(51) Clore, G. M.; Bax, A.; Wingfield, P.; Gronenborn, A. M. FEBS
Lett. 1988, 238, 17-21.
(52) Garbow, J. R.; Weitkamp, D. P.; Pines, A. Chem. Phys. Lett. 1982,
93, 504-509. Bax, A.; Subramanian, S. J. Magn. Reson. 1986, 67, 565-
569.
(53) Bax, A.; Summers, M. T. J. Am. Chem. Soc. 1986, 108, 2093-
2094.
Synthesis of SAA2 (7-Amino-2,6-anhydro-L-glycero-L-gulo-7-de-
oxyheptonic Acid, 5). Cbz-SAA2-OMe9 (0.36 g, 1.0 mmol) was
dissolved in MeOH (4 mL) and treated with 1 N NaOH (1.2 mL, 1.2
mmol). After 1 h, ion exchange resin (0.5 g, Aldrich, Amberlyst 15,
strongly acidic, H+ form) was added and stirred for 10 min. The resin
was filtered off and washed with MeOH. 10% Pd/C catalyst (50 mg)
was added to the filtrate, and the reaction mixture was stirred for 1 h
under an hydrogen atmosphere. The catalyst was filtered off, and the
solvent was removed in vacuo to yield 201 mg (97%) as colorless
crystals. Mp: >250 °C dec. [R]20D: -41.2° (c ) 1, H2O). FAB-
MS: 230 ([M + Na]+).
Synthesis of SAA3 (1,2,3,4-Tetra-O-acetyl-â-D-glucuronic Acid
Methyl Ester, 6). Glucuronolactone (43 g, 240 mmol) was suspended
in dry MeOH (1.5 L), and dimethylethylamine (0.5 mL) was added.
The reaction mixture was stirred for 3 h until the glucuronolactone
was dissolved. The solvent was evaporated and the foam used without
purification. Acetic anhydride (210 mL, 2.2 mol) and sodium acetate
(21 g, 260 mmol) were added, and the suspension was stirred for 8
days. The reaction mixture was poured onto 1 L ice water and stirred
overnight. The â-acetate was separated by filtration, washed with water,
and recrystallized from EtOAc/hexanes. The mother liquor was
extracted three times with ether (200 mL), and the combined ether
extracts were washed with saturated sodium bicarbonate solution and
brine, dried (MgSO4), and evaporated. The resulting solid was
recrystallized from EtOAc/hexanes to yield 42.8 g (47%). Rf (EtOAc/
(54) Kogler, H.; Sørensen, O. W.; Bodenhausen, G.; Ernst, R. R. J. Magn
Reson. 1983, 55, 157-163.
(55) Griesinger, C.; Ernst, R. R. J. Magn. Reson. 1987, 75, 261-271.
(56) Kessler, H.; Griesinger, C.; Kersebaum, R.; Wagner, K.; Ernst, R.
R. J. Am. Chem. Soc. 1987, 109, 607-609.
(57) Jeener, J.; Maier, B. H.; Bachman, P.; Ernst, R. R. J. Chem. Phys.
1979, 71, 4546-4553.
(58) Bax, A. J. Magn. Reson. 1988, 77, 134-147.
1
hexanes, 1:1): 0.53; H NMR (250 MHz, DMSO-d6): δ 6.02 (d, J )
(59) Mierke, D. F.; Kessler, H. J. Am. Chem. Soc. 1991, 113, 9466-
9470.
8.1 Hz, 1H, H1), 5.51(dd, J ) 9.4 Hz, 1H, H3), 5.01 (dd, J ) 9.6 Hz,
1H, H4), 4.97 (dd, J ) 8.2 Hz, 1H, H2), 4.67 (d, J ) 9.8 Hz, 1H, H5),
3.63 (s, 3H, OCH3), 2.08-1.97 (4s, 12H, CH3CO).
(60) Siebert, H. C.; Reuter, G.; Schauer, R.; von der Lieth, C. W.;
Dabrowski, J. Biochemistry 1992, 31, 6962-6971. Aulabaugh, A. E.;
Crouch, R. C.; Martin, G. E.; Ragouzeos, A.; Shockcor, J. P.; Spitzer, T.
D.; Farrant, R. D.; Hudson, B. D.; Lindon, J. C. Carbohydr. Res. 1992,
230, 201-212. Ferna´ndez, P.; Jime´nez-Barbero, J. Carbohydr. Res. 1993,
248, 15-16.
2,3,4-Tri-O-acetyl-1-azido-1-deoxy-D-glucuronic Acid Methyl Es-
ter (7). Trimethylsilyl azide (15.5 mL, 190 mmol) was added to a
stirred solution of acetate 6 (31 g, 82 mmol) in dry CH2Cl2 (450 mL)

Peptides Containing Sugar Amino Acids
J. Am. Chem. Soc., Vol. 118, No. 42, 1996 10165
with SnCl4 (4.0 mL, 29 mmol). The solution was stirred at room
temperature for 3 h, then diluted with CH2Cl2 (300 mL), and washed
three times with 10% K2CO3 solution (100 mL) and twice with brine
(50 mL). After drying (MgSO4), the solution was concentrated in vacuo
and recrystallized from EtOAc/hexanes to yield 26.8 g (91%) as white
138.5, 138.2, 138.1, 137.1, 128.3-127.2, 83.7, 78.0, 77.7, 74.2, 73.9,
72.3, 68.7, 65.2, 54.5. FAB-MS: 634 (28, [M + Na]+), 517 (18), 182
(34), 147 (100). Anal. Calcd for C36H37NO8: C, 70.69; H, 6.09; N,
2.29. Found: C, 70.71; H, 6.03; N, 2.33.
Treatment of 13 with Trimethylsilyl Iodide, followed by Fmoc-
ONSu. Acid 13 (1.0 g, 1.6 mmol) was dissolved in dry CH3CN (10
mL) at room temperature in a Falcon tube. Trimethylsilyl iodide (0.49
mL, 4.0 mmol) was added, and the reaction mixture was stirred for 12
min, treated with MeOH (100 µL), diluted with THF (10 mL), and
treated with Fmoc-ONSu (720 mg, 2.0 mmol). The pH of the solution
was maintained at 7-8 by addition of DIEA, and the completion of
the reaction was followed by HPLC (30 f 90, B in A, 30 min: tR-
(amine) ) 20.4 vs tR(urethane) ) 26.4 min). After 14 and 24 h
additional Fmoc-ONSu was added. Purification was achieved by FC
(acetone/hexanes, 1:3, and acetone/hexanes (+0.1% TFA), gradient 1:2
to 2:1) to yield 15 in 546 mg (48%), 16 in 62 mg (6.3%), and 17 in
223 mg (17%).
1
solid. Rf (EtOAc/hexanes, 1:1): 0.60; H NMR (250 MHz, DMSO-
d6): δ 5.40 (dd, J ) 9.6 Hz, 1H, H3), 5.19 (d, J ) 8.8 Hz, 1H, H1),
5.05 (dd, J ) 9.8 Hz, 1H, H4), 4.87 (dd, J ) 9.2 Hz, 1H, H2), 4.57 (d,
J ) 9.9 Hz, 1H, H5), 3.66 (s, 3H, OCH3), 2.04-1.98 (3s, 9H, CH3-
CO); FAB-MS: 360 (3, [M + H]+), 317 (66, [M - N3]+), 257 (23),
154 (100). Anal. Calcd for C13H17N3O9: C, 43.46; H, 4.77; N, 11.70.
Found: C, 43.27; H, 4.77; N, 11.61.
Synthesis of SAA4 (2-[(Benzyloxycarbonyl)amino]-3,4,6-tri-O-
benzyl-2-deoxy-D-glucosamine, 10). Benzyl chloroformate (4.9 mL,
14 mmol, 50% solution in toluene) diluted in CHCl3 (100 mL) was
slowly added to a solution of 924 (7.0 g, 14 mmol) and NaHCO3 (6.0
g, 71 mmol) in MeOH (200 mL) at 0 °C. After 3 h additional NaHCO3
(6.0 g, 71 mmol) and benzyl chloroformate (2.5 mL, 7.2 mmol) were
added, and the suspension was stirred overnight at room temperature.
The reaction mixture was concentrated, suspended in EtOAc (1 L),
filtered, and concentrated again. The crude product was crystallized
from EtOAc/hexanes to yield 8.03 g (90%) as colorless crystals. Rf
(hexanes/acetone, 1:2): 0.39. Mp: 192-193 °C (hexanes/EtOAc).
[R]20D: +57.7 (c ) 1.06, CHCl3). 1H NMR (250 MHz, DMSO-d6):
7.42 (d, J ) 9.2 Hz, 1H, HN), 7.34-7.18 (m, 20H, Harom), 6.75 (d, J )
4.0 Hz, 1H, HO), 5.12-4.95 (m, 3H, PhCH2,H1), 4.77-4.43 (m, 6 H,
PhCH2), 3.44-3.35 (m, 6H); 13C NMR (76.7 MHz, DMSO-d6): 156.2,
138.9, 138.4, 138.3, 137.2, 128.3-127.3, 91.3, 79.8, 78.7, 74.1, 74.0,
72.4, 69.9, 69.3, 65.3, 55.8. FAB-MS: 566 (4, [M - H2O + H]+).
Anal. Calcd for C35H37NO7: C, 72.02; H, 6.39; N, 2.40. Found: C,
72.14; H, 6.44; N, 2.41.
3-[(9-Fluorenylmethoxycarbonyl)amino]-2,6-anhydro-4,5,7-tri-O-
benzyl-3-deoxy-D-glycero-D-gulo-heptonic Acid (15). Rf (CHCl3/
MeOH, 1:3): 0.79. tR 20.9 min (58 f 72, B in A, 30 min). [R]20
:
D
+13.8 (c ) 0.92, THF). 1H NMR (500.13 MHz, DMSO-d6): δ 12.84
(s, 1 H, HOOC), 7.87 (d, J ) 7.4 Hz, 2H), 7.71 (d, J ) 7.4 Hz, 1H),
7.66-7.62 (m, 2H), 7.41-7.16 (m, 19H, Harom), 4.71-4.54 (m, 6H,
PhCH2), 4.31 (dd, J ) 6.7 Hz, J ) 10.2 Hz, 1H), 4.25-4.16 (m, 2H,
PhCH2), 3.86 (d, J ) 9.3 Hz, 1H, H2), 3.77-3.60 (m, 4H, H3,H4,H5,H6),
3.49 (s, 2H, H7). 13C NMR (125 MHz, DMSO-d6): 169.7, 155.7, 143.9,
143.7, 140.7, 138.4, 138.1, 138.0, 128.5-126.9, 125.2, 125.0, 120.1,
83.2 (C4), 78.2 (C2), 77.6 (C5, C6), 74.2, 74.0, 72.3, 68.8 (C7), 65.7,
54.4 (C3), 46.6 (CH). FAB-MS: 722 (6, [M + Na]+), 700 (24, [M +
1]+), 478 (10), 179 (100). Anal. Calcd for C36H37NO8: C, 73.80; H,
5.90; N, 2.00. Found: C, 73.70; H, 5.92; N, 2.01.
Cbz-SAA1r-Phe-Leu-OMe. General Procedure for Coupling of
Peptide and SAA. In a representative experiment, H-Phe-Leu-OMe
was dissolved in THF (5 mL) and cooled to 0 °C. Cbz-SAA1R-OH
(0.68 g, 2.0 mmol), HOBt (0.3 g, 2.0 mmol), and EDCl‚HCl (0.40 g,
2.1 mmol) were added. The pH was adjusted to 7 by dropwise addition
of NMM (N-methylmorpholine). After 10 h the solvent was evaporated
and the residue dissolved in EtOAc (100 mL). The solution was washed
three times with 0.5 N HCl (20 mL) and three times with aqueous 5%
NaHCO3 (20 mL) and with H2O (20 mL). The organic layer was dried
(MgSO4) and concentrated to yield 0.90 g (73%) as a colorless solid.
Cbz-Tyr-SAA1r-Phe-Leu-OMe. General Procedure for Re-
moval of Cbz Protecting Group. In a representative experiment Cbz-
SAA1R-Phe-Leu-OMe (0.62 g, 1.0 mmol) was dissolved in MeOH (5
mL), 10% Pd/C catalyst (50 mg) was added, and the reaction mixture
was stirred for 1 h under an H2 atmosphere. The catalyst was filtered
off, and the solvent was removed in vacuo. The residue was coupled
with Cbz-Tyr-OH (0.32 g, 1.0 mmol) as mentioned above. After 10 h
the solvent was evaporated, and the residue was chromatographed on
silica gel (CHCl3/MeOH, 12:1) to afford 0.41 g (53%) as a colorless
solid. Rf (CHCl3/MeOH, 12:1): 0.20.
H-Tyr-SAA1r-Phe-Leu-OMe‚HCl (19). Cbz-Tyr-SAA1R-Phe-
Leu-OMe (155 mg, 0.2 mmol) was hydrogenated as above. The catalyst
was filtered off, a saturated solution of HCl in Et2O (0.5 mL) was added,
and the solvent was removed in vacuo to give 112 mg (94%) as a
colorless solid. 1H NMR (600 MHz, DMSO-d6, 300 K): 9.25 (s, 1H,
HO-Tyr), 8.49 (d, 1H, SAA1RΗΝ) 8.27 (d, 1H, LeuHN), 8.17 (d, 1H,
PheHN), 7.97 (s, 3H, TyrHN), 7.28-7.15 (m, 5H, C6H5), 7.13 (d, 2H,
TyrH3′), 6.68 (d, 2H, Tyr TyrH2′), 5.23-5.10 (m, 2H, HO-SAA1R),
4.58 (m, 2H, PheHR,SAA1RH1), 4.27 (ddd, 2H, LeuHR), 3.96 (ddd,
1H, TyrHR), 3.89 (d, 1H, SAA1RH5), 3.72 (ddd, 1H, SAA1RH2), 3.63
(s, 3H, LeuOMe), 3.57 (dd, 1H, SAA1RH3), 3.38 (d, 1H, SAA1RH4),
3.26 (s, 3H, SAA1ROMe), 3.08, 2.82 (m, 4H, PheHâ,TyrHâ), 1.70-
1.45 (m, 3H, 2 LeuHâ,LeuHγ), 0.85 + 0.90 (2d, 6H, 2 LeuHδ);
Tributyl[2-[(benzyloxycarbonyl)amino]-3,4,6-tri-O-benzyl-2-deoxy-
â-D-glucopyranosyl]stannane (11). Compound 10 (7.0 g, 12 mmol)
was treated with SOCl2 (100 mL) at room temperature for 30 min and
concentrated to dryness, and the product was coevaporated with dry
CHCl3 (20 mL). The yellow solid was dissolved in dry THF (100
mL) and added within 20 min to a solution of Bu3SnLi23b,61 in THF
(ca. 3 equiv) at -78 °C. The mixture was stirred for 1 h at -78 °C,
quenched with saturated NH4Cl solution (10 mL), warmed to room
temperature, diluted with H2O, and extracted twice with EtOAc (700
mL). The combined organic layers were dried (MgSO4) and concen-
trated to afford a yellow oil. Purification was achieved by FC (hexanes/
EtOAc, gradient 1:0 to 4:1) to yield 8.10 g (79%) as colorless oil. Rf
(hexanes/acetone, 3:1): 0.91. [R]20D: -2.7 (c ) 1.0, CHCl3). 1H NMR
(500 MHz, CDCl3): δ 7.64 (d, J ) 8.0 Hz, 1H, HN), 7.34-7.12 (m,
20H, Harom), 5.06-4.44 (m, 8H, PhCH2), 3.79 (m, 2H), 3.67-3.57 (m,
3H), 3.49-3.41 (m, 2H). 13C NMR (125 MHz, CDCl3): 170.6, 155.8,
138.5, 138.2, 138.1, 137.1, 128.3-127.2, 83.7, 78.0, 77.7, 74.2, 73.9,
72.3, 65.2, 54.5. FAB-MS (calcd for C47H63NO6120Sn): 801 (40, [M
- Bu + H]+), 711 (6), 291 (20, [SnBu3]+), 236 (40), 180 (100). Anal.
Calcd for C47H63NO6Sn: C, 65.89; H, 7.41; N, 1.63; Found: C, 66.12;
H, 7.33; N, 1.65.
Conversion of 11 with BuLi and Addition of CO2. Stannane 11
(8.1 g, 9.5 mmol) was dissolved in dry THF (60 mL). At -78 °C,
BuLi (5.9 mL 1.6 M solution in hexanes, 9.5 mmol) was added within
10 min. The reaction mixture was then warmed to -55 °C, and BuLi
(7.1 mL, 11 mmol) was added within 5 min while the color of the
solution changed to deep red. CO2 was pumped for 15 min through
the reaction mixture which was quenched after 1 h with 10% KHSO4
solution (50 mL) at -55 °C, warmed to room temperature, and extracted
twice with EtOAc (500 mL). Purification was achieved by FC
(hexanes/acetone, 1:0, 1:1, CHCl3/MeOH/HOAc, 50:7:3) to afford 13
in 4.77 g (83%) as colorless solid, 11 in 0.46 g (5.7%) and 14 in 0.59
g (11%).
3J(SAA1RΗΝ,H1)
)
8.3 Hz, 3J(SAA1RH1,H2)
)
10.1 Hz,
3-[(Benzyloxycarbonyl)amino]-2,6-anhydro-4,5,7-tri-O-benzyl-D-
glycero-D-gulo-heptonic Acid (13). Rf (CHCl3/MeOH, 1:3): 0.59.
Mp: 155 °C. [R]20D: +15.1 (c ) 1.0, CHCl3). 1H NMR (500 MHz,
DMSO-d6): δ 7.64 (d, J ) 8.0 Hz, 1H, HN), 7.34-7.12 (m, 20H, Harom),
5.06-4.44 (m, 8H, PhCH2), 3.80-3.78 (m, 2H), 3.67-3.57 (m, 3H),
3.49-3.41 (m, 2H). 13C NMR (125 MHz, DMSO-d6): 170.6, 155.8,
3J(SAA1RH4,H5) ) 9.7 Hz, J(PheHN,HR) ) 7.5 Hz, J(LeuHγ,Hδ) )
6.1 Hz. 13C-NMR (150 MHz, DMSO-d6, 300 K): δ 72.1 (C4), 71.2
(C5), 70.2 (C3), 54.7 (SAA1ROMe), 53.5 (C2), 53.4 (PheCR), 53.0
(TyrCR), 51.7 (LeuOMe), 50.3 (LeuCR), 39.4 (LeuCâ), 36.9 (TyrCâ),
36.0 (PheCâ), 23.9 (LeuCγ), 22.5 (LeuCδ), 21.1 (LeuCδ). Anal. Calcd
for C32H45ClN4O10: C, 56.42; H, 6.66; N, 8.22. Found: C, 56.44; H,
6.68; N, 8.21.
3
3
(61) Prahash, H.; Silser, H. H. Inorg. Chem. 1972, 11, 2258-2259.

10166 J. Am. Chem. Soc., Vol. 118, No. 42, 1996
Graf Von Roedern et al.
Table 4. Proton and Carbon Chemical Shifts of 26 and Temperature Coefficients
SAA2
Phe
D-Trp
Lys
Thr
1H/13C
HN
7.62
3.49
(H2)
3.09
(H3)
3.16
(H4)
2.98
(H5)
3.14
(H6)
3.28
7.38
4.53
8.51
4.32
8.54
54.6 3.91
7.26
4.20 57.6
HR/CR
77.2
52.3
37.8
53.3
(C2)
72.3
(C3)
76.7
(C4)
70.6
(C5)
78.3
(C6)
39.6
Hâ/Câ
Hγ/Cγ
Hδ/Cδ
Hꢀ/Cꢀ
H7/C7
2.96/2.81
2.99/2.83 26.5 1.60proS/1.39proR 30.8
3.90 66.5
0.96 18.5
10.78
(HN)
0.94
22.8
28.8
39.9
6.85-7.10 125.7-128.7 7.09
123.3 1.24
(C2)
4.95
(OH)
(H2)
7.55
(H4)
7.02
(H5)
7.09
(H6)
7.35
(H7)
117.9 2.88
(C4)
117.7 4.98
(H7proR) (C7)
3.41
(C5)
(HN)
120.4 4.98
65.0
(CH2(Cbz))
127 - 128 + 110.0
(C6H5(Cbz))
(H7proS
)
(C6)
(CH2(Cbz))
110.9
(C7)
∆δ/∆T [ppb/K] for the amide H -6.8
-1.8
-9.8
-6.8
-0.5
Cbz-SAA2-Phe-Leu-OMe. Cbz-SAA2-OMe (180 mg, 0.5 mmol)
was dissolved in MeOH (5 mL) and 1 N NaOH (0.75 mL). After 2 h
HOBt (75 mg, 0.5 mmol) was added, and the solvent was removed in
vacuo. The Cbz group of Cbz-Phe-Leu-OMe (320 mg, 0.75 mmol)
was removed as described above, and the deprotected compound was
dissolved in THF (5 mL). This solution was given to the residue of
the saponification and coupled with EDCI (see above). After 10 h the
solvent was evaporated, and the residue was purified by FC (CHCl3/
MeOH, 9:1) to yield pure 30 in 220 mg (71%) as a colorless solid.
FAB-MS: 638 [M + Na]+.
solution was washed three times with 0.5 N HCl (20 mL) and three
times with aqueous 5% NaHCO3 (20 mL) and with H2O (20 mL). The
organic layer was dried (MgSO4) and concentrated. The product was
precipitated in Et2O to yield 0.81 g (59%) as a colorless solid.
Boc-Lys(Cbz)-Thr-SAA2-Phe-D-Trp-OMe (24). Compound 23
(0.50 g, 1.0 mmol) was dissolved in MeOH (5 mL), THF (5 mL), and
1 N NaOH (1.5 mL). After 2 h HOBt (150 mg, 1.0 mmol) was added,
and the solvent was removed in vacuo. The Cbz group of 22 (0.69 g,
1.0 mmol) was removed as described above, and the deprotected
compound was dissolved in THF (8 mL) and DMF (2 mL). This
solution was added to the residue of the saponification and coupled as
above. After 10 h the solvent was evaporated, and the residue was
chromatographed (CHCl3/MeOH, 4:1) to give 0.74 g (73%) of 24 as a
colorless solid.
Cyclo(-SAA2-Phe-D-Trp-Lys(Cbz)-Thr-) (26). 24 (0.51 g, 0.5
mmol) was dissolved in MeOH (5 mL), THF (5 mL), and 1N NaOH
(0.75 mL). After 4 h ion exchange resin (1 g, Aldrich, Amberlyst 15,
strongly acidic, H+ form) was added and stirred for 10 min. The resin
was filtered off, and the filtrate was concentrated in vacuo. The residue
was treated with saturated HCl/Et2O (5 mL) and MeSH (0.5 mL) for
1 h. The solvent was removed in vacuo, and the residue was dissolved
in DMF (0.5 L). HOBt (90 mg, 0.6 mmol), DIEA (0.43 mL), H2O
(0.5 mL), and TBTU (190 mg, 0.6 mmol) were added under stirring.
After 1 h the solvent was removed in vacuo. The residue was chromat-
ographed (CHCl3/MeOH, 4:1) to give 246 mg (56%) of 26 as a color-
less solid. FAB-MS: 887 [M + H]+. Anal. Calcd for C45H55N7O12:
C, 61.01; H, 6.26; N, 11.07. Found: C, 59.39; H, 6.14; N, 10.40.
Cyclo(-SAA2-Phe-D-Trp-Lys-Thr-) (27). The Cbz group of 26 (30
mg, 0.033 mmol) was removed as described above. The product was
lyophilized from H2O/tBuOH (1:1) to give 24 mg (97%) of 27 as a
colorless solid. FAB-MS: 753 [M + H]+.
Cbz-Thr(tBu)-SAA3(tri-O-acetyl)-OMe (28). Azide 7 (3.7 g, 10.4
mmol) was dissolved in THF (140 mL), and 10% Pd/C (1 g) was added.
After 10 min of ultrasonification the reaction mixture was hydrogenated
on an ice bath for 2 h. The solution was reduced in vacuo. Cbz-Thr-
(tBu)-OH was prepared from DCHA salt by dissolving Cbz-Thr(tBu)
OH‚DCHA (5.1 g, 10.4 mmol) in EtOAc (600 mL) and washing the
organic phase four times with 1 N HCl and once with brine. After
being dried with MgSO4, the solvent was evaporated. The foam was
dissolved in CH2Cl2 (30 mL), IIDQ (3.04 g) was added on an ice bath,
and the reaction mixture was stirred overnight. The solvent was
evaporated. FC (EtOAc/hexanes, 1:1) yielded 3.5 g (54%) as a white
solid. Rf (EtOAc/hexanes, 2:1): 0.60.
Cbz-Lys(Boc)-Thr(tBu)-SAA3(tri-O-acetyl)-OMe (30). Com-
pound 28 (1.9 g, 3.05 mmol) was dissolved in EtOAc (60 mL) and
after addition of 10% Pd/C (900 mg) hydrogenated for 1 h. The solution
was filtered through Celite. Cbz-Lys(Boc)-ONSu (1.45 g, 3.04 mmol)
was dissolved in EtOAc (30 mL), and NMM was added to adjust the
pH to 8. After being stirred overnight the solvent was evaporated and
subjected to FC (EtOAc/hexanes, 1:1) to yield 2.2 g (85%). Rf (EtOAc/
hexanes, 2:1): 0.33.
H-Tyr-SAA2-Phe-Leu-OMe‚HCl (20). The Cbz group of Cbz-
SAA2-Phe-Leu-OMe (220 mg, 0.36 mmol) was removed and coupled
with EDCI as above. The solvent was evaporated, and the residue
was chromatographed on silica gel (CHCl3/MeOH, 9:1). For the
removal of the Boc group the purified peptide was treated with saturated
HCl/Et2O (5 mL) and MeSH (0.5 mL) for 1 h. The solvent was
removed in vacuo to give 185 mg (75%) 20 as a colorless solid. FAB-
MS: 645 [M + H]+; 1H NMR (500 MHz, DMSO-d6, 300 K): δ 9.37
(s, 1H, HO-Tyr), 8.43 (d, 1H, LeuHN), 8.41 (dd, 1H, SAA2HN), 8.06
(d, 1H, PheHN), 8.04 (s, 3H, TyrHN), 7.28-7.15 (m, 5H, C6H5), 7.02
(d, 2H, TyrH3′), 6.72 (d, 2H, TyrH3′), 5.14 (m, 3H, 3 HO-SAA2), 4.55
(ddd, 1H, PheHR), 4.30 (ddd, 2H, LeuHR), 3.93 (dd, 1H, TyrHR), 3.65
(dd, 1H, H7t(pro-R)), 3.61 (s, 3H, LeuOMe), 3.57 (d, 1H, H2), 3.19
(ddd, 1H, H4), 3.16 (ddd, 1H, H3), 3.11 (dd, 1H, PheHât), 3.03 (ddd,
1H, H6), 2.98 (dd, 1H, H7h(pro-S)), 2.95 (ddd, 1H, H5), 2.91 (dd, 1H,
TyrHât), 2.83 (dd, H, TyrHâh), 2.82 (dd, 1H, PheHâh), 1.65-1.50 (m,
3
3H, 2 LeuHâ,LeuHγ), 0.83 + 0.88 (2d, 6H, 2 LeuHδ); J(TyrHR,Ηât)
) 7.0 Hz, 3J(TyrHR,Ηâh) ) 7.0 Hz, 3J(TyrHât,Hâh) ) 14.0 Hz,
3
3J(SAA2HN,H7t(pro-R)) ) 6.2 Hz, J(SAA2HN,H7(pro-S)) ) 4.7 Hz,
3J(SAA2H7t,H7h) ) 12.4 Hz, 3J(SAA2H7t(pro-R),H6) ) 1.0 Hz,
3J(SAA2H7h(pro-S),H6) ) 8.5 Hz, 3J(SAA2H4,H3) ) 8.8 Hz,
3
3
3J(SAA2H3,H2) ) 9.0 Hz, J(PheHN,HR) ) 8.4 Hz, J(PheHR,Ηât) )
9.0 Hz, J(PheHR,Ηâh) ) 5.5 Hz, J(PheHât,Hâh) ) 13.3 Hz, J(Leu-
HN,HR) ) 7.8 Hz. 13C-NMR (125 MHz, DMSO-d6, 300 K): δ ) 130.0
(TyrC2′), 125.3 + 127.7 + 128.7 (Phearom), 115.0 (TyrC2′), 78.1 (C2),
78.1 (C6), 77.1 (C4), 72.0 (C3), 71.2 (C5), 53.8 (PheCR), 53.5 (TyrCR),
51.7 (LeuOCH3), 50.2 (LeuCR), 40.8 (C7), 39.3 (LeuCâ), 37.1 (PheCâ),
36.2 (TyrCâ), 24.3 (LeuCγ), 22.5 (LeuCδa), 21.2 (LeuCδb). Anal. Calcd
for C32H45ClN4O10: C, 56.42; H, 6.66; N, 8.22. Found: C, 52.78; H,
5.96; N, 7.32.
3
3
3
Cbz-SAA2-Phe-D-Trp-OMe (22). Cbz-SAA2-OMe (0.72 g, 2.0
mmol) was dissolved in MeOH (10 mL) and 1 N NaOH (3.0 mL).
After 2 h HOBt (300 mg, 2.0 mmol) was added, and the solvent was
removed in vacuo. The Boc group of 21 (0.92 g, 1.84 mmol) was
removed by adding HCl in ether (4 mL, saturated at 0 °C) to a stirred
solution of the protected peptide with addition of MeSH (0.5 mL) as
a scavenger. The solution was stirred at room temperature for 30 min
and evaporated in vacuo. The deprotected compound was dissolved
in THF (10 mL). This solution was added to the residue of the
saponification and coupled as above. After 10 h the solvent was
evaporated, and the residue was dissolved in EtOAc (100 mL). The

Peptides Containing Sugar Amino Acids
J. Am. Chem. Soc., Vol. 118, No. 42, 1996 10167
Table 5. Proton and Carbon Chemical Shifts of the Major Conformation of 33 and Temperature Coefficients
SAA3
8.47
Phe
D-Trp
Lys
Thr
1H/13C
HN
7.55
8.27
4.42
8.36
3.86
7.17
4.00
HR/CR
4.37 81.0 4.35
53.4
35.8
54.2
53.9
54.0
66.3
19.0
(H1) (C1)
Hâ/Câ
Hγ/Cγ
Hδ/Cδ
Hꢀ/Cꢀ
3.60 70.6 2.98/2.87
(H2) (C2)
2.96/2.82 27.0
1.55proS/1.37proR 31.0
3.87
1.04
3.18 76.4
10.75
(HN)
0.9
22.8
29.0
39.5
(H3) (C3)
3.30 72.4 6.93/7.07/7.12 129.0/127.8/125.8 7.00
121.5 1.22
(C2)
(H4) (C4)
3.62 77.7
(H5) (C5)
(H2)
7.50
(H4)
6.98
(H5)
7.07
(H6)
7.35
(H7)
118.0 2.80
(C4)
118.0 HN: 6.74
(C5)
120.7 Boc: 1.35
Boc: 22 t-Bu 1.12
(C6)
111.2
(C7)
∆δ/∆T [ppb/K] for the amide H -7.0
0.0
-5.9
-7.3
0.0
Table 6. Proton and Carbon Chemical Shifts for the Major Conformation of 36 and Temperature Coefficients
SAA4
Ala2
Pro3
Ala4
8.58 172.2
Ala5
7.54 170.1
1H/13C
HN/CO
HR/CR
Hâ/Câ
Hγ/Cγ
Hδ/Cδ
7.99
167.3
7.73 170.4
4.31
1.22
171.9
59.0
28.2
24.6
46.2
3.53(H1)
3.97(H2)
3.17(H3)
3.25(H4)
3.12(H5)
79.7(C1)
51.5(C2)
75.4(C3)
69.6(C4)
80.9(C5)
60.8(C6)
46.2 4.13
3.92
1.25
49.1
17.0
4.06
1.15
49.4
17.3
16.0 1.98proR/1.79proS
2.12/1.88
3.65/3.51
3.65/3.49(H6)
∆δ/∆T [ppb/K] for the amide H -6.5
-5.2
-6.9
-0.1
Pro-OH (0.236 g, 0.7 mmol), Fmoc-Ala-OH (0.218 g, 0.7 mmol), and
15 (0.315 g, 0.46 mmol) for chain elongation; TBTU/HOBt was used
for activation. The peptide was cleaved with CH2Cl2/trifluoroethanol/
HOAc (8:1:1) to yield 265 mg (82%) of crude H-Ala-SAA4-(tri-O-
benzyl)-Ala-D-Pro-Ala-OH (34), which was purified by preparative
HPLC (40 f 70, B in A, 30 min, tR 13.6 min). FAB-MS: 810 (14,
[M + Na]+); 788 (100, [M + H]+).
34 (52.2 mg) was dissolved in NMP (100 mL) and added slowly to
a solution of TBTU (70.2 mg), HOBt (58.2 mg), and DIEA (pH of
solution was approximately 7.5) in NMP (300 mL). After 3 h at room
temperature the solution was treated with H2O (30 mL) and concentrated
and the product purified by preparative HPLC (40 f 70, B in A, 30
min, tR 20.7 min). FAB-MS for cyclo(SAA4-(tri-O-benzyl)-Ala-D-Pro-
Ala-Ala) (35): 792 (3, [M + Na]+).
Cbz-Lys(Boc)-Thr(tBu)-SAA3-Phe-D-Trp-OMe (31). Cbz-Lys-
(Boc)-Thr(tBu)-SAA3(tri-O-acetyl)-OMe (1.1 g, 1.3 mmol) was dis-
solved in THF (20 mL) and 1 N NaOH (5.5 mL) added. After 3 h
Amberlyst 15 (1.7 g) was added and the suspension stirred for 15 min.
The solid was removed by filtration. The solution was used without
further purification. Cbz-Phe-D-Trp-OMe (29) (640 mg, 1.3 mmol)
was dissolved in MeOH (20 mL), and after addition of 10% Pd/C (320
mg) the solution was hydrogenated for 1 h. The solvent was evaporated
and the foam redissolved in THF (10 mL) and added to the Cbz-Lys-
(Boc)-Thr(tBu)-SAA3-OH. The solution was cooled on an ice bath,
and EDCl‚HCl (290 mg, 1.5 mmol), HOBt (235 mg, 1.5 mmol), and
NMM (380 µL) were added. The reaction mixture was stirred
overnight. The solvent was evaporated and after redissolving EtOAc
(200 mL) washed three times (50 mL each) with 1 N HCl, three times
with saturated NaHCO3, and once with brine. The organic phase was
dried with MgSO4 and the solvent removed by evaporation. FC (CHCl3/
MeOH, 15:1) gave 1.05 g of a white solid in 77% yield. Rf (CH3CN/
H2O, 4:1): 0.71.
The cyclic peptide was dissolved in H2O/HOAc (30 mL, 1:1) and
hydrogenated for 24 h in the presence of Pd/C (30 mg of 5% Pd/C).
The product was purified by preparative HPLC (5 f 40, B in A, 30
min, tR 12.5 min) to yield 36 in 22.9 mg (68%).
H-Lys(Boc)-Thr(tBu)-SAA3-Phe-D-Trp-OH (32). Methyl ester 31
(360 mg, 0.34 mmol) was dissolved in MeOH (5 mL), and THF (20
mL) and 1 N NaOH (0.5 mL) were added. After 3 h Amberlyst 15
(0.6 g) was added, and the mixture was stirred for 15 min. The resin
was removed by filtration and the solvent evaporated. The foam was
redisolved in MeOH (20 mL), and after addition of 10% Pd/C (200
mg) hydrogenated for 1 h. After evaporation of the solvent the foam
was purified by preparative HPLC (35 f 55, B in A, 30 min, tR 11.2
min), yielding 150 mg (48%). Rf (CH3CN/H2O, 4:1): 0.59.
Cyclo(-Lys(Boc)-Thr(tBu)-SAA3-Phe-D-Trp-) (33). The linear
peptide 32 (45 mg, 0.05 mmol) was dissolved in NMP (250 mL) and
water (0.45 mL) added. The solution was stirred for 15 min and added
dropwise to the coupling mixture of HOBt (38.3 g, 0.25 mmol), TBTU
(48.2 g, 0.15 mmol), and DIEA (45 µL) in NMP (200 mL) over 1 h.
The solvent was evaporated, and the peptide was purified by preparative
HPLC (35 f 55, B in A, 30 min, tR 20.2 min) to yield 10 mg (22%).
FAB-MS: 894 [M + H]+.
Acknowledgment. We thank Dr. B. Kutscher and Dr. M.
Bernd, ASTA Medica AG, Frankfurt/M., Germany, for their
support. Financial support by the Deutsche Forschungsgemein-
schaft and the Fonds der Chemischen Industrie is gratefully
acknowledged.
Supporting Information Available: Comparisons between
experimentally determined and simulated NOE-derived distances
3
and homonuclear J coupling constants for 26, 33, and 36;
details of structure calculation; synthesis and characterization
of all precursor dipeptides; characterization of compounds 14,
16, and 17; table with proton and carbon chemical shifts for
the minor conformation of compound 36 and temperature
coefficients (13 pages). See any current masthead page for
ordering information and Internet access instructions.
Cyclo(-SAA4-Ala-D-Pro-Ala-Ala-) (36). Fmoc-Ala-OH (0.2 g, 0.7
mmol) was coupled to 2-chlorotrityl chloride resin (0.4 g, 1.25 mmol
Cl-/g resin) which was used to initiate the synthesis with Fmoc-D-
JA961068A