Synthesis and Conformational Studies of a β-Turn Mimetic Incorporated in Leu-enkephalin

Article abstract of DOI:10.1021/jo0356863

A β-turn mimetic in which the four amino acids of a β-turn have been replaced by a 10-membered ring has been designed, synthesized, and subjected to conformational studies. In the mimetic, the intramolecular CO _i - HN_i+3 hydrogen bon

Full text of DOI:10.1021/jo0356863

Syn th esis a n d Con for m a tion a l Stu d ies of a â-Tu r n Mim etic
In cor p or a ted in Leu -en k ep h a lin
David Blomberg,† Mattias Hedenstro¨m,† Paul Kreye,† Ingmar Sethson,† Kay Brickmann,‡ and
J an Kihlberg*,†,‡
Organic Chemistry, Department of Chemistry, Umea˚ University, SE-901 87 Umea˚, Sweden, and
AstraZeneca R&D Mo¨lndal, SE-431 83 Mo¨lndal, Sweden
jan.kihlberg@chem.umu.se; jan.kihlberg@astrazeneca.com
Received November 16, 2003
A â-turn mimetic in which the four amino acids of a â-turn have been replaced by a 10-membered
ring has been designed, synthesized, and subjected to conformational studies. In the mimetic, the
intramolecular COi - HNi+3 hydrogen bond that is often found in â-turns has been replaced by an
ethylene bridge. In addition, the amide bond between residues i and i + 1 was exchanged for a
methylene ether isoster. Such a â-turn mimetic, based on the first four residues of Leu-enkephalin
(Tyr-Gly-Gly-Phe-Leu), was prepared in 15 steps. The synthesis relied on a â-azido alcohol prepared
in five steps from Cbz-Tyr(tBu)-OH as a key, i-position building block. tert-Butyl bromoacetate,
glycine, and a Phe-Leu dipetide were then used as building blocks for positions i + 1, i + 2, and i
1
+ 3, respectively. Conformational studies based on H NMR data showed that the â-turn mimetic
was flexible, but that it resembled a type-II â-turn at low temperature. This low energy conformer
closely resembled the structure determined for crystalline Leu-enkephalin.
In tr od u ction
pressin,11,12 which regulates water readsorption in the
kidneys, and LHRH,13,14 which releases sex-specific hor-
mones from the testes and ovaries. â-Turns have also
been found for the endogenous morphine-like substance
Leu-enkephalin.15-17 In view of these, and other ex-
amples, it is not surprising that large efforts have been
devoted to the design and synthesis of mimetics of
â-turns.
Several requirements should be fulfilled by a successful
â-turn mimetic. The conformation of the mimetic should
of course resemble that of the peptide backbone in the
â-turn. Because the side chains of biologically active
peptides have crucial roles in receptor recognition it is
Turns constitute an important class of polypeptide
secondary structure, defined as regions where a peptide
chain reverses its overall direction.1,2 When chain rever-
sal occurs over four residues in such a way that the
carbonyl oxygen atom of the first residue (i) and the
amide NH proton of the fourth residue (i + 3) come close
in space a â-turn is formed. In most cases, this involves
formation of an intramolecular hydrogen bond between
residues i and i + 3 to give a pseudo-ten-membered ring.
Different â-turns are distinguished by the values of the
Φ and Ψ torsional angles for the i + 1 and i + 2
residues1,3 or by the topology of the side chains in the
turn.4
â-Turns are often located at the surface of proteins
where they can undergo posttranslational modification
and serve as sites of recognition in interactions with
receptors and antibodies.1,2,5,6 Structural studies have also
revealed that small peptides functioning as hormones,
neurotransmitters, or having other regulatory roles in
the organism may contain â-turn structural motifs.
Examples include the hormones oxytocin,7-10 which
induces labor and milk production in mammals, vaso-
(7) Wood, S. P.; Tickle, I. J .; Treharne, A. M.; Pitts, J . E.; Mascaren-
has, Y.; Li, J . Y.; Husain, J .; Cooper, S.; Blundell, T. L.; Hruby, V. J .;
Buku, A.; Fischman, A. J .; Wyssbrod, H. R. Science 1986, 232, 633-
636.
(8) Husain, J .; Blundell, T. L.; Cooper, S.; Pitts, J . E.; Tickle, I. J .;
Wood, S. P.; Hruby, V. J .; Buku, A.; Fischman, A. J .; Wyssbrod, H. R.;
Mascarenhas, Y. Philos. Trans. R. Soc. London B 1990, 327, 625-
654.
(9) Lippens, G.; Hallenga, K.; Van Belle, D.; Wodak, S. J .; Nirmala,
N. R.; Hill, P.; Russell, K. C.; Smith, D. D.; Hruby, V. J . Biochemistry
1993, 32, 9423-9434.
(10) Rose, J . P.; Wu, C.-K.; Hsiao, C.-D.; Breslow, E.; Wang, B.-C.
Nature Struct. Biol. 1996, 3, 163-169.
(11) Langs, D. A.; Smith, G. D.; Stezowski, J . J .; Hughes, R. E.
Science 1986, 232, 1240-1242.
† Umea˚ University.
‡ AstraZeneca R&D Mo¨lndal.
(12) Schmidt, J . M.; Ohlenschla¨ger, O.; Ru¨terjans, H.; Grzonka, Z.;
Kojro, E.; Pavo, I.; Fahrenholz, F. Eur. J . Biochem. 1991, 201, 355-
371.
(1) Rose, G. D.; Gierasch, L. M.; Smith, J . A. In Advances in Protein
Chemistry; Anfinsen, C. B., Edsall, J . T., Richards, F. M., Eds.;
Academic Press: Orlando, FL, 1985; Vol. 37, pp 1-109.
(2) Smith, J . A.; Pease, L. G. CRC Crit. Rev. Biochem. 1980, 8, 315-
399.
(13) Nikiforovich, G. V.; Marshall, G. R. Int. J . Peptide Protein Res.
1993, 42, 181-193.
(14) Guarnieri, F.; Weinstein, H. J . Am. Chem. Soc. 1996, 118,
5580-5589.
(15) Smith, G. D.; Griffin, J . F. Science 1978, 199, 1214-1216.
(16) Aubry, A.; Birlirakis, N.; Sakarellos-Daitsiotis, M.; Sakarellos,
C.; Marraud, M. Biopolymers 1989, 28, 27-40.
(17) Amodeo, P.; Naider, F.; Picone, D.; Tancredi, T.; Temussi, P.
A. J . Peptide Sci. 1998, 4, 253-265.
(3) Hanessian, S.; McNaughton-Smith, G.; Lombart, H.-G.; Lubell,
W. D. Tetrahedron 1997, 53, 12789-12854.
(4) Ball, J . B. Tetrahedron 1993, 49, 3467-3478.
(5) Stanfield, R. L.; Fieser, T. M.; Lerner, R. A.; Wilson, I. A. Science
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10.1021/jo0356863 CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/20/2004
3500
J . Org. Chem. 2004, 69, 3500-3508

Synthesis and Conformational Studies of a â-Turn Mimetic
essential that they can be introduced at desired positions
in the mimetic with correct orientation. Turn mimetics,
in which amide bonds have been modified, can also be
expected to suffer less from the pharmacokinetic prob-
lems that affect peptide drug candidates, i.e., enzymatic
degradation and low uptake.18,19 Due to their rigid nature,
mimetics are useful tools in investigating the bioactive
conformation of peptides. Furthermore, since rigid ana-
logues pay a lower entropy cost upon binding to a receptor
they should be more potent and also more selective for a
specific type of receptor.20-22 It is, however, important to
avoid over-rigidification resulting in compromised inter-
actions between the ligand and the receptor.23
Many â-turn mimetics constitute replacements of
residues i + 1 and i + 2 in â-turns by aromatic,24,25
bicyclic,26-32 or spirocyclic33,34 systems (references are to
selected recent publications). Such mimetics attempt to
position their amino- and carboxyl-termini correctly so
that a reversal of the peptide backbone is enforced when
they are incorporated in a polypeptide. In general, this
kind of mimetic does not allow introduction of side chains
at the i + 1 and i + 2 positions. Reports of â-turn
mimetics that provide close resemblance of the backbone
of the turn, allow incorporation of side chains with correct
topology, and also incorporation into a peptide chain,
have appeared but are less frequent.23 Herein we describe
a novel type of â-turn mimetic that resembles the peptide
backbone in a turn and allows ready incorporation into
a peptide.35 Introduction of different side chains is
possible at the i, i + 2, and i + 3 positions, while side
chains other than hydrogen and methyl at the i + 1
position will require larger synthetic efforts. The ap-
proach is exemplified by incorporation of a â-turn mimetic
in place of the first four residues of Leu-enkephalin (Tyr-
Gly-Gly-Phe-Leu). Several mimetics of Leu-enkephalin
have been synthesized earlier by others including cyclized
analogues,36 a mimetic containing a conformationally
F IGURE 1. Transformation of a â-turn into conformationally
restricted mimetic 1.
SCHEME 1. Retr osyn th etic An a lysis Revea ls Th a t
â-Tu r n Mim etic 1 Ca n Be P r ep a r ed fr om â-Azid o
Alcoh ols 2, r-Br om o Acid s 3, Am in o Acid s 4, a n d
Am in o Acid Der iva tives 5
restricted tyrosine analogue,37 and those containing
â-turn mimetics.38 Conformational studies and biological
evaluation of these mimetics have not produced a definite
picture of the bioactive conformation of Leu-enkephalin,
but the biological relevance of the â-turn observed in the
crystal structure of Leu-enkephalin15 has been ques-
tioned.36,38
Resu lts a n d Discu ssion
Design of th e â-Tu r n Mim etic. As discussed above,
â-turns are often stabilized by an intramolecular hydro-
gen bond between the carbonyl group of residue i and
the amino group of residue i + 3. The distance between
the carbonyl carbon atom of residue i and the nitrogen
atom of the amino group of residue i + 3 falls between
3.5 and 3.8 Å in type-I, -II, and -III â-turns. This indicates
that the intramolecular hydrogen bond found in these
turns could be replaced by an ethylene bridge. Simulta-
neous replacement of the amide bond between residues
i and i + 1 with a methylene ether isostere suggested
that the 10-membered ring in 1 could serve as a novel
â-turn mimetic (Figure 1). Replacement of the amide
bond with a methylene ether isostere generates a new
stereogenic center, the configuration of which should
influence the conformation of the turn mimetic.
(18) Spatola, A. F. In Chemistry and Biochemistry of Amino Acids,
Peptides, and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York,
Basel, 1983; Vol. 7, pp 267-357.
(19) Patani, G. A.; LaVoie, E. J . Chem. Rev. 1996, 96, 3147-3176.
(20) Ball, J . B.; Alewood, P. F. J . Mol. Recog. 1990, 3, 55-64.
(21) Rizo, J .; Gierasch, L. M. Annu. Rev. Biochem. 1992, 61, 387-
418.
(22) Hruby, V. J . Life Sci. 1982, 31, 189-199.
(23) Han, Y.; Giragossian, C.; Mierke, D. F.; Chorev, M. J . Org.
Chem. 2002, 67, 5085-5097.
(24) Kaul, R.; Deechongkit, S.; Kelly, J . W. J . Am. Chem. Soc. 2002,
124, 11900-11907.
(25) J ean, F.; Buisine, E.; Melnyk, O.; Drobecq, H.; Odaert, B.;
Hugues, M.; Lippens, G.; Tartar, A. J . Am. Chem. Soc. 1998, 120,
6076-6083.
(26) Wang, W.; Yang, J .; Ying, J .; Xiong, C.; Zhang, J .; Cai, C.;
Hruby, V. J . J . Org. Chem. 2002, 67, 6353-6360.
(27) Martin-Martinez, M.; Latorre, M.; Garcia-Lopez, M. T.; Cen-
arruzabeitia, E.; Del Rio, J .; Gonzalez-Muniz, R. Bioorg. Med. Chem.
Lett. 2002, 12, 109-112.
(28) Eguchi, M.; Shen, R. Y. W.; Shea, J . P.; Lee, M. S.; Kahn, M. J .
Med. Chem. 2002, 45, 1395-1398.
A retrosynthetic analysis revealed that azido alcohols
2, R-halo acids 3, protected amino acids 4, and peptides
(or amino acids) 5 constitute suitable building blocks for
synthesis of â-turn mimetic 1 (Scheme 1). The azido
group in 2 serves as a precursor for the amino group of
(29) Hoffmann, T.; Lanig, H.; Waibel, R.; Gmeiner, P. Angew. Chem.,
Int. Ed. 2001, 40, 3361-3364.
(30) Gosselin, F.; Lubell, W. D. J . Org. Chem. 2000, 65, 2163-2171.
(31) Estiarte, M. A.; Rubiralta, M.; Diez, A.; Thormann, M.; Giralt,
E. J . Org. Chem. 2000, 65, 6992-6999.
(32) J ohannesson, P.; Lindeberg, G.; Tong, W.; Gogoll, A.; Karlen,
A.; Hallberg, A. J . Med. Chem. 1999, 42, 601-608.
(33) Fernandez, M. M.; Diez, A.; Rubiralta, M.; Montenegro, E.;
Casamitjana, N.; Kogan, M. J .; Giralt, E. J . Org. Chem. 2002, 67,
7587-7599.
(36) Pawlak, D.; Oleszczuk, M.; Wojcik, J .; Pachulska, M.; Chung,
N. N.; Schiller, P. W.; Izdebski, J . J . Peptide Sci. 2001, 7, 128-140.
(37) Nicholls, I. A.; Alewood, P. F. Bioorg. Chem. 1994, 22, 300-
317.
(38) Gardner, B.; Nakanishi, H.; Kahn, M. Tetrahedron 1993, 49,
3433-3448.
(34) Alonso, E.; Lopez-Ortiz, F.; del Pozo, C.; Peralta, E.; Macias,
A.; Gonzalez, J . J . Org. Chem. 2001, 66, 6333-6338.
(35) Kreye, P.; Kihlberg, J . Tetrahedron Lett. 1999, 40, 6113-6116.
J . Org. Chem, Vol. 69, No. 10, 2004 3501

Blomberg et al.
Ketone 8 was then reduced with K-Selectride42,43 at -78
°C, which furnished allylic syn and anti amino alcohols
9 and 10 together with oxazolidinone 11. The latter was
assumed to be formed from syn alcohol 9 by cyclization.
Formation of the corresponding, diastereomeric oxazoli-
dinone from 10 was not observed under these reaction
conditions. The diastereomeric amino alcohols 9 and 10,
and oxazolidinone 11, were separated by careful flash
chromatography providing the three compounds in 36,
8, and 14% yields, respectively. Thus, the overall syn/
anti ratio ((9 + 11)/10) obtained in the reduction of ketone
8 was >6:1. The stereochemistry at the newly formed
stereogenic center of 9 and 10 was determined by
converting them to the corresponding oxazolidinones by
using aqueous potassium hydroxide in a mixture of
methanol and THF (1:2:4) at room temperature. Under
these conditions, syn amino alcohol 9 gave 11, whereas
anti amino alcohol 10 gave the diastereomeric oxazoli-
dinone. Irradiation of H-4 in the oxazolidinone ring of
11 gave a 5% NOE for H-5, whereas a 14% enhancement
of H-5 was obtained for the diastereomeric oxazolidinone,
thereby establishing that H-4 and H-5 in 11 had an anti
orientation. When reduction of 8 was performed with
DIBAL or Zn(BH4)2, i.e., with reducing agents which
contain chelating metals, anti amino alcohol 10 was
instead obtained as the main product. Best results were
obtained with Zn(BH4)2 which gave 10 in 64% yield
together with 25% of 9. No oxazolidinones were formed
when using these two reducing agents. A route to 9 and
10 based on addition of allyl zinc bromide to an aldehyde
corresponding to Weinreb amide 7 was investigated
previously but was then found to be inferior to the above
results.35
Removal of the carbamate protective group from the
amino functionality of allylic alcohol 9 was accomplished
by refluxing in a 1:1 mixture of aqueous potassium
hydroxide and ethanol to give amino alcohol 12 in 80%
yield (Scheme 3). Amino alcohol 12 was also prepared in
75% yield starting from oxazolidinone 11 using the same
conditions. Because 11 is formed from 9 under basic
conditions, oxazolidinone 11 is most likely a common
reaction intermediate in the deprotection of 9. The amino
group of 12 was converted to an azido group by treatment
with triflyl azide under copper sulfate catalysis in a
biphasic system.44,45 This reaction is known to proceed
with retention of configuration,44 and azide 13 was
obtained in 85% yield. The hydroxyl group of 13 was
alkylated under phase-transfer conditions46 using tert-
butyl bromoacetate to give compound 14, which contained
tyrosine and glycine equivalents at the i and i + 1 turn
positions. Introduction of the azido functionality proved
to be essential for a successful alkylation of syn-configu-
rated 13. When alkylation of Cbz-protected amino alcohol
9 was attempted under the same conditions as for 13 an
intramolecular reaction involving the Cbz group occurred
to give oxazolidinone 11 as the major product. Alkylated
azido alcohol 14 was found to be thermally sensitive.
SCHEME 2a
a
Key: (i) N-methylmorpholine, isobutyl chloroformate, N,O-
dimethylhydroxylamine hydrochloride, CH2Cl2, -15 °C to rt, 81%;
(ii) allylmagnesium bromide, THF -78 °C, 74%; (iii) K-Selectride,
THF, -78 °C, 9 36%, 10 8%, 11 14%.
the first residue of the turn, while the alkene serves as
a masked aldehyde to be used in a reductive amination
with 5. Alkylation of the hydroxyl group in 2 with R-halo
acids 3 links the residues found at position i and i + 1 of
the turn to each other. This step should be facile for
derivatives of R-halo acetic and propionic acid,39 i.e., when
glycine and alanine are found at the i + 1 position of the
â-turn, but can be expected to be more complex when
other amino acids are found in the i + 1 position.
Incorporation of the i + 2 residue requires formation of
two amide bonds. The overall success of this approach
to â-turn mimetics relies on a route that provides ready
access to building block 2 in enantiomerically pure form,
as well as on procedures for linking the four building
blocks to each other. The first four residues of Leu-
enkephalin (Tyr-Gly-Gly-Phe-Leu) have been shown to
adopt a â-turn both in the crystal and in solution,15-17
and incorporation of a â-turn mimetic in Leu-enkephalin
was therefore chosen as our first synthetic target. This
requires preparation of an azido alcohol in which Ri
contains a protected phenol (cf. 13, Scheme 3), for
instance from a derivative of tyrosine. The remaining
building blocks would be derivatives of R-bromo acetic
acid, glycine, and the dipeptide Phe-Leu, respectively.
Syn th esis of a â-Tu r n Mim etic a n d Its In cor p or a -
tion in Leu -en k ep h a lin . The synthesis started by
conversion of protected tyrosine 6 to the corresponding
Weinreb amide 7 by activation using isobutyl chlorofor-
mate and N-methylmorpholine followed by treatment
with N,O-dimethylhydroxylamine hydrochloride (Scheme
2).40 Treatment of 7 with allylmagnesium bromide at -78
°C afforded allyl ketone 8 in 68% yield over two steps.41
(42) Mohapatra, D. K.; Datta, A. J . Org. Chem. 1998, 63, 642-646.
(43) Achmatowicz, B.; Wicha, J . Tetrahedron Lett. 1987, 28, 2999-
3002.
(44) Cavender, C. J .; Shiner, V. J . J . Org. Chem. 1972, 37, 3567.
(45) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996,
37, 6029-6032.
(46) Pietraszkiewicz, M.; J urczak, J . Tetrahedron 1984, 40, 2967-
2970.
(39) Hedenstro¨m, M.; Holm, L.; Yuan, Z.; Emtena¨s, H.; Kihlberg, J .
Bioorg. Med. Chem. Lett. 2002, 12, 841-844.
(40) Melnyk, P.; Ducrot, P.; Thal, C. Tetrahedron 1993, 49, 8589-
8596.
(41) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815-
3818.
3502 J . Org. Chem., Vol. 69, No. 10, 2004

Synthesis and Conformational Studies of a â-Turn Mimetic
SCHEME 3a
SCHEME 4a
a
Key: (i) EtOH/KOH (1 M), reflux, 81%; (ii) NaN3, Tf2O, H2O,
CH2Cl2, CuSO4, DMAP, 0 °C to rt, 85%; (iii) tert-butyl R-bromoac-
etate, QHSO4, 50% NaOH (aq), benzene; (iv) N-methylmorpholine
oxide, OsO4, H2O, acetone, THF, 70% from 13; (v) Na2CO3,
Pb(OAc)4, benzene, 85%.
Therefore, the solvents were removed at 0 °C after
workup and the product was used without any further
purification in the following oxidation step. Attempted
alkylation of the anti-configurated azido alcohol gener-
ated from 10 resulted in decomposition and the diaste-
reomer of 14 could therefore not be prepared. The alkene
moiety in 14 was transformed to a diol giving 15 (70%
from 13) using catalytic amounts of osmium tetraoxide
with N-methylmorpholine N-oxide as co-oxidant. Diol 15
was then cleaved by lead tetraacetate to give aldehyde
16 (85%). Somewhat surprisingly, attempts to generate
aldehyde 16 directly from alkene 14 by ozonolytic cleav-
age failed.
a
Key: (i) H-Phe-Leu-OMe, NaBH(OAc)3, NEt3, ClCH2CH2Cl,
82%; (ii) Fmoc-Gly-OH, HATU, DIEA, DMF, 85%; (iii) formic acid,
99%; (iv) PfpOH, DCC, EtOAc, 0 °C; (v) DBU, dioxane, reflux, 56%
from 19; (vi) SnCl2, PhSH, TEA, THF; (vii) LiOH (0.1 M), THF,
57% from 21.
Reductive alkylation47 of the amino group of the
dipeptide H-Phe-Leu-OMe48 with aldehyde 16 using
sodium triacetoxyborohydride as reducing agent gave
secondary amine 17 in 82% yield (Scheme 4). In this way
the C-terminal leucine, and also the phenylalanine at
position i + 3 of the â-turn mimetic, were connected to
building block 16 containing the i position tyrosine and
the i + 1 glycine of the desired Leu-enkephalin mimetic.
Introduction of glycine at the i + 2 position was achieved
by amide bond formation between Fmoc-protected glycine
and the secondary amine in 17. Coupling using diisopro-
pyl carbodiimide as activating agent gave the desired
amide 18 in moderate yields. However, replacing diiso-
propyl carbodiimide with the more potent HATU49,50
furnished tertiary amide 18 in 85% yield. The tert-butyl
ester and the phenolic tert-butyl ether of 18 were cleaved
simultaneously, without affecting the methyl ester, by
treatment with formic acid to afford 19 in almost
quantitative yield. Activation of the carboxylic acid in 19,
to give pentafluorophenyl ester51 20, followed by treat-
ment with DBU in refluxing dioxane under high dilution
conditions gave the desired cyclized product 21 in 56%
yield. Attempts to achieve this critical macrolactamisa-
tion starting with Fmoc-cleavage from 19, followed by
intramolecular formation of the amide bond promoted by
coupling reagents as HATU or DIC, proved to be less
successful. Finally, the N- and C-termini of 21 were
deprotected in two steps. First the azide was reduced52
using tin chloride, thiophenol and triethylamine to give
amine 22. Then the methyl ester in 22 was hydrolyzed
with lithium hydroxide which gave the target â-turn
mimetic 23 (57% yield from 21). Mimetic 23 was thus
prepared with an overall yield of 3.2% over the 15-step
reaction sequence.
Con for m a tion a l Stu d ies. A previous structure de-
termination of a γ-turn mimetic incorporated in the
peptide drug desmopressin, performed in our laboratory,
was complicated by the flexibility and size of the pepti-
domimetic.53 Attempted structural determination of Leu-
enkephalin 23 turned out to be complex for the same
(47) AbdelMagid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C.
A.; Shah, R. D. J . Org. Chem. 1996, 61, 3849-3862.
(48) Katritzky, A. R.; Xu, Y. J .; He, H. Y.; Steel, P. J . J . Chem. Soc.,
Perkin Trans. 1 2001, 1767-1770.
(51) Kisfaludy, L.; Scho¨n, I. Synthesis 1983, 325-327.
(52) Bartra, M.; Romea, P.; Urpi, F.; Vilarrasa, J . Tetrahedron 1990,
46, 587-594.
(53) Hedenstro¨m, M.; Yuan, Z.; Brickmann, K.; Carlsson, J .; Ekholm,
K.; J ohansson, B.; Kreutz, E.; Nilsson, A.; Sethson, I.; Kihlberg, J . J .
Med. Chem. 2002, 45, 2501-2511.
(49) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J .
Chem. Soc., Chem. Commun. 1994, 201-203.
(50) Carpino, L. A.; El-Faham, A. Tetrahedron 1999, 55, 6813-6830.
J . Org. Chem, Vol. 69, No. 10, 2004 3503

Blomberg et al.
TABLE 1. 1H NMR Ch em ica l Sh ifts (δ, p p m ) of
Com p ou n d 21 in MeOH-d 3 a t -70 °C
TABLE 2. Dista n ce Restr a in ts Used in th e Str u ctu r e
Ca lcu la tion of Com p ou n d 21
distance
proton paira,b
restraintc,d (Å)
Gly3 HN-Gly3 HR1
Gly3 HN-Gly3 HR2
Leu5 HN-H2
Leu5 HN-Leu5 Hâ1
Leu5 HN-Leu5 Hâ2
Phe4 Hδ-Phe4 Hâ
Tyr1 Hδ-Tyr1 HR
Tyr1 Hδ-Tyr1 Hâ1
Tyr1 Hδ-Tyr1 Hâ2
H1-Gly2 HR
2.36
2.98
2.70
2.91
2.82
2.31
3.16
2.84
2.58
2.19
2.22
2.03
2.38
2.72
2.53
2.66
2.69
2.71
2.81
3.41
2.80
3.19
2.63
3.16
2.93
2.92
2.75
2.48
2.26
H1-Gly3 HR2
H5-Gly3 HR2
Phe4 Hâ-Phe4 HR
H4-Gly3 HR2
H4-Phe4 HR
residue NH
CRH
4.01
4.51b
CâH
CγH
other
Tyr1
2.49, 2.96
7.18 (Hδ), 6.78 (Hꢀ)
H2-Phe4 HR
Gly2a
Leu5 Hγ-Leu5 HR
Leu 5 Hδ-Leu5 HR
H2-Tyr1 Hâ1
H3-Tyr1 Hâ1
H1-Tyr1 Hâ2
H1-H2
Gly3
Phe4
Leu5
8.86 3.52, 4.75
4.30
7.32 4.69
3.46b
7.28 (Hδ), 7.38 (Hꢀ)
1.65, 1.74 1.63 0.98c (Hδ)
bridge H1
H2
H3
H4
H5
3.95 2.15
1.70
2.41 3.77
H1-H3
Tyr1 HR-H3
Tyr1 HR-H2
Tyr1 HR-Tyr1 Hâ2
H2-Tyr1 Hâ2
H2-H4
a
Refers to the methylene group corresponding to Gly2 HR in
b
Leu-enkephalin. Degeneracy has been assumed.
reasons. Several different solvents and temperatures
were explored to find suitable conditions for NMR-based
structure determination of 23, i.e., aqueous solution,
mixtures of water with DMSO-d6 (1:1) or TFE-d3 (1:1),
as well as MeOH-d4. Although the spectrum of 23 could
be assigned in aqueous solution to confirm the structure,
spectral data of sufficient quality for structure determi-
nation was not obtained in any of the solvents. Broad
peaks and cross-peaks with both positive and negative
signs in the NOESY spectrum indicated slow to inter-
mediate exchange rates between several conformers. This
conformational mobility may be advantageous from a
molecular recognition perspective since it allows some
flexibility for the mimetic in receptor interactions.
Our attention then shifted to the protected mimetic 21,
since it should be able to serve as a model for the
conformational preferences of the turn mimetic part
which is common to 21 and 23. We reasoned that the
fact that the N- and C-termini of 21 were protected,
should have no or only minor influence on the low-energy
conformations of the 10-membered ring that constitutes
the â-turn mimetic. Fortunately, the dynamic properties
of 21 were favorable in MeOH-d3 at -70 °C, and spectra
with sufficient quality could be acquired to assign the
resonances of 21 (Table 1) and extract distance restraints.
By inspection of the NOE-buildup curves when mixing
times ranging from 30 to 250 ms were used, it was
apparent that many of the observed cross-peaks origi-
nated from spin-diffusion54 due to the slow tumbling rate
of 21. To avoid problems with spin-diffusion, all distance
restraints were extracted from the NOESY spectrum
obtained with a mixing time of 30 ms. In total, 29
distance restraints for the structure calculation were
derived and classified as short or long distances with 4
or 5 Å as the upper distance limit, respectively (Table
H3-H4
a
b
Hydrogen atoms H1-H5 are defined in Table 1. The hydro-
gen atoms in the ethylene bridge (H2-H5) were also treated as
pseudoatoms in the structure calculations. This resulted in a
similar overall conformation although a slightly larger flexibility
was observed in the part of the mimetic containing the bridge.
c Determined with the Gly3 HR1-HR2 cross-peak volume as
d
reference, and a Gly3 HR1-HR2 reference distance of 1.772 Å. In
the structure calculations the upper limit was set to 4 Å for
distances determined to be below 3 Å. For distances determined
to be above 3 Å, the upper limit was set to 5 Å.
2). Simulated annealing with the X-PLOR55 force field
resulted in an ensemble of low-energy structures with a
well defined backbone conformation, especially in the
part of the molecule which contains the â-turn mimetic
(Figure 2). The average rmsd for all heavy atoms was
2.64 Å but for the backbone of the turn mimetic it was
as low as 0.23 Å. Since relatively few distance restraints
were used in the structure calculation, unrestrained
simulated annealing was performed. This confirmed that
the extracted distance restraints provided the necessary
information to find the observed low-energy conforma-
tions of 21.
To find out how well the 10-membered ring of 21
mimics a â-turn, we superimposed the â-turn mimetic
part in the low energy conformers with different idealized
â-turns. This revealed that the backbone of the â-turn
in 21, and the position of the i + 2 side chain, super-
imposes well with an idealized type-II â-turn (Figure 3).
The backbone rmsd from CR of residue i to CR of residue
i + 3 is only 0.43 Å for the ensemble of low energy
conformers. The only larger discrepancy is the orientation
of the i + 1 side chain that differs approximately 60°
(54) Spin-diffusion refers to transfer of magnetization through
alternative pathways in a network of nuclei in close proximity to each
other and reveals itself as delayed NOEs.
(55) Bru¨nger, A. T. X-PLOR, Version 3.1. A system for X-ray
crystallography and NMR; Yale Universtity Press: New Haven, CT,
1992.
3504 J . Org. Chem., Vol. 69, No. 10, 2004

Synthesis and Conformational Studies of a â-Turn Mimetic
F IGURE 4. Superimposition of the structure of crystalline
Leu-enkephalin (yellow) and one of the 10 lowest energy
conformers of 21 (green). The conformer of 21 having the
lowest rmsd value (0.37 Å) when compared to crystalline Leu-
enkephalin was chosen for the superimposition.
+ 1 side chain of a type-II â-turn. The conformation of
the â-turn mimetic in 21 also coincides well with the
â-turn found in the crystal structure of Leu-enkephalin
(Figure 4).15 The average rmsd for the heavy atoms in
the turn is 0.55 Å for the ensemble of conformers for 21
compared to the structure of crystalline Leu-enkephalin.
The rmsd for the whole backbone when comparing 21 and
Leu-enkephalin ranges from 0.61 to 1.68 Å, a value that
is strongly correlated to the orientation about the flexible
Phe φ-angle.
F IGURE 2. Superimposition of the 10 lowest energy conform-
ers of Leu-enkephalin mimetic 21. The backbone atoms for
residues i f i + 2 were used for the superimposition.
Con clu sion s
A novel mimetic of a â-turn has been designed. In the
mimetic, the intramolecular hydrogen bond that is often
found between residues i and i + 3 of a â-turn has been
replaced by an ethylene bridge to give a 10-membered
ring. In addition, the amide bond between residues i and
i + 1 was exchanged for a methylene ether isoster. A
synthetic route to such a â-turn mimetic corresponding
to the first four residues of Leu-enkephalin was developed
based on assembly of building blocks. The key, i-position
building block was prepared from Cbz-Tyr(tBu)-OH in
five steps and contained a â-azido alcohol moiety as well
as an ethylene bridge equivalent. Assembly of the â-turn
mimetic then begun by alkylation of the hydroxyl group
of the i position tyrosine building block with tert-butyl
bromoacetate thus incorporating a glycine equivalent at
the i + 1 position. The i + 3 phenylalanine in the turn,
together with the C-terminal leucine of Leu-enkephalin,
were incorporated by reductive amination. Finally, gly-
cine at position i + 2 of the â-turn mimetic was intro-
duced by formation of two amide bonds, one of which
closed the 10-membered ring. It is envisioned that this
approach to â-turn mimetics should allow facile incor-
poration of different side chains at positions i, i + 2, and
i + 3 of a â-turn. However, it is likely that, in its present
form, the approach will be restricted to incorporation of
glycine and alanine equivalents at the i + 1 position.39
Conformational studies showed that the â-turn mimetic
F IGURE 3. Superimposition of the â-turn mimetic part from
the lowest energy conformer of 21 with a tetrapeptide oriented
as an idealized type-II â-turn. Backbone atoms from Tyr1 CR
to Phe4 CR in 2 were used for the superimposition. The dashed
line indicates the hydrogen-bond found in the ideal â-turn. The
location of the side-chains of residues i + 1 and i + 2 in the
tetrapeptide are marked in gray. Positions for side chains at
these positions in mimetic 21 are marked in light yellow.
between 21 and a type-II â-turn. Since 21 has a glycine
in this position, this deviation should not be critical for
the receptor interactions of the present Leu-enkephalin
mimetic. If a properly oriented side-chain is required in
the i + 1 position, incorporation of a D-amino acid
equivalent should provide a better agreement with the i
J . Org. Chem, Vol. 69, No. 10, 2004 3505

Blomberg et al.
dissolved in THF (80 mL) and cooled to -78 °C. K-Selec-
tride42,43 (20 mL, 20 mmol, 1 M solution in THF) was added
slowly, and the reaction was stirred for 4 h. Water and 10%
aqueous citric acid were added to quench the reaction and
adjust pH > 4. The aqueous phase was extracted with CH2-
Cl2. The combined organic layers were washed with saturated
aqueous NaHCO3 and brine and dried over Na2SO4. The
solvent was removed under reduced pressure, and the residue
was purified by flash chromatography (heptane/ethyl acetate
10:1 f 2:1) to yield 9 (1.55 g, 36%) as a colorless oil, 10 (0.37
g, 8.5%) as a white solid, and 11 (0.45 g, 14%) as a colorless
oil.
was flexible when incorporated in Leu-enkaphlin but that
a type-II â-turn was adopted in methanol-d3 at low
temperature. This low energy conformer closely re-
sembled the structure found for crystalline Leu-enkepha-
lin. It is suggested that the inherent flexibilty of this type
of â-turn mimetics could be an advantage in ligand-
receptor interactions.
Exp er im en ta l Section
[(S)-2-(4-ter t-Bu toxyph en yl)-1-(m eth oxym eth ylcar bam -
oyl)eth yl]ca r ba m ic Acid Ben zyl Ester (7). Cbz-Tyr(tBu)-
OH*DCHA (6, 5.00 g, 9.05 mmol) was dissolved in EtOAc (250
mL), and 10% aqueous citric acid (150 mL) was added. The
mixture was stirred for 30 min at room temperature. The two
phases were separated, and the aqueous phase was extracted
with EtOAc. The combined organic phases were washed with
10% aqueous citric acid and dried over Na2SO4. The solvent
was removed under reduced pressure, and the residue was
dissolved in CH2Cl2 (72 mL) and cooled to -20 °C. N-
Methylmorpholine (2.50 mL, 22.6 mmol) was added followed
by slow addition of isobutyl chloroformate (1.67 mL, 11.3
mmol), and the reaction was stirred for 15 min. N,O-Dimeth-
ylhydroxylamine HCl (1.10 g, 11.3 mmol) was added in one
portion,40 and after 15 min the cooling bath was removed and
the reaction was stirred for another 3 h at room temperature.
The reaction was quenched with water and the phases were
separated. The aqueous phase was extracted with CH2Cl2 and
the combined organic layers were washed with satd. aqueous
NaHCO3, brine and dried over Na2SO4. The solvent was
removed under reduced pressure and the residue was purified
by flash chromatography (heptane/ethyl acetate 3:1) to give 7
Compound 9: [R]20 ) -17.6 (c ) 0.42 in CHCl3); IR (neat)
D
1
3419, 3343, 1691 cm-1; H NMR (400 MHz, CDCl3, 25 °C) δ
7.38-7.28 (m, 5H), 7.11 (d, J ) 8.3 Hz, 2H), 6.90 (d, J ) 8.3
Hz, 2H), 5.79-5.64 (m, 1H), 5.25 (d, J ) 7.5 Hz 1H), 5.10-
5.00 (m, 4H), 3.87-3.78 (m, 1H), 3.63-3.56 (m, 1H), 2.89 (dd,
J ) 13.7 and 7.4 Hz, 1H), 2.84 (dd, J ) 13.7 and 8.1 Hz, 1H),
2.52 (sb, 1H), 2.24 (dd, J ) 4.9 and 13.6 Hz, 1H), 2.19 (dd, J
) 8.1 and 13.6 Hz, 1H), 1.32 (s, 9H); 13C NMR (100 MHz,
CDCl3, 25 °C) δ 156.4, 153.6, 136.4, 134.2, 133.0, 129.6, 128.4,
127.9, 127.8, 124.1, 118.2, 78.2, 69.6, 66.6, 55.6, 39.1, 38.0, 28.7;
HRMS (FAB) calcd for C24H30NO4 (M+H) 398.2331, found
398.2336. Anal. Calcd for C24H29NO4: C, 72.52; H, 7.86; N,
3.52. Found: C, 72.83; H, 7.99; N, 3.46.
Compound 10: [R]20D ) -14.7 (c ) 0.075 in CHCl3); IR (neat)
1
3318, 1687, 1535, 1504 cm-1; H NMR (400 MHz, CDCl3, 25
°C) δ 7.35-7.20 (m, 5H), 7.07 (d, J ) 8.3 Hz, 2H), 6.90 (d, J )
8.3 Hz, 2H), 5.88-5.77 (m, 1H;), 5.24 (d, J ) 7.5 Hz, 1H), 5.20-
4.96 (m, 4H), 3.93-3.83 (m, 1H), 3.76-3.67 (m, 1H), 3.05 (sb,
1H), 2.92 (dd, J ) 14.2 and 4.6 Hz, 1H), 2.77-2.66 (m, 1H)
2.36-2.18 (m, 2H), 1.32 (s, 9H); 13C NMR (100 MHz, CDCl3,
25 °C) δ 156.2, 153.5, 136.3, 134.4, 132.8, 129.5, 128.3, 127.8,
127.7, 124.0, 117.9, 78.1, 72.4, 66.4, 56.1, 38.1, 34.5, 28.6;
HRMS (FAB) calcd for C24H30NO4 (M + H) 398.2331, found
398.2331. Anal. Calcd for C24H29NO4: C, 72.52; H, 7.86; N,
3.52. Found: C, 72.38; H, 8.07; N, 3.60.
(3.18 g, 85%) as a colorless oil: [R]20 ) +2.4 (c ) 0.25 in
D
CHCl3); IR (neat) 1716, 1658, 1506 cm-1; 1H NMR (400 MHz,
CDCl3, 25 °C) δ 7.34-7.31 (m, 5H), 7.04 (d, J ) 8 Hz, 2H),
6.89 (d, J ) 8 Hz, 2H), 5.39 (d, J ) 9 Hz, 1H), 5.08 (d, J ) 12
Hz, 1H), 5.02 (d, J ) 12 Hz, 1H), 5.02-4.93 (m, 1H), 3.62 (s,
3H), 3.13 (s, 3H), 3.01 (dd, J ) 6.4 and 6.5 Hz, 1H), 2.87 (dd,
J ) 6.4 and 6.5 Hz, 1H), 1.31 (s, 9H); 13C NMR (100 MHz,
CDCl3, 25 °C) δ 171.9, 155.6, 154.1, 136.3, 131.1, 129.7, 128.3,
127.9, 127.8, 124.0, 78.1, 66.6, 61.3, 52.0, 38.0, 31.8, 28.7;
HRMS (FAB) calcd for C23H30N2O (M + H) 415.2233, found
415.2244.
Compound 11: [R]20D ) -64.1 (c ) 0.105 in CHCl3); IR (neat)
1
1753, 1508 cm-1, H NMR (400 MHz, CDCl3, 25 °C) δ 6.99 (d,
J ) 8.5 Hz, 2H), 6.90 (d, J ) 8.5 Hz, 2H), 6.62 (s, 1H), 5.57-
5.44 (m, 1H), 5.00-4.91 (m, 2H), 4.25-4.19 (m, 1H), 3.64-
3.58 (m, 1H; CHNH), 2.80 (dd, J ) 13.5 and 6.1 Hz, 1H), 2.63
(dd, J ) 13.5 and 7.5 Hz, 1H), 2.23-2.07 (m, 2H), 1.25 (s, 9H);
13C NMR (100 MHz, CDCl3, 25 °C) δ 158.8, 154.0, 131.1, 130.4,
129.4, 124.2, 118.7, 80.1, 78.1, 57.7, 40.3, 38.2, 28.4; HRMS
(FAB) calcd for C17H24O3N (M + H) 290.1756, found 290.1727.
[(S)-1-(4-ter t-Bu toxyben zyl)-2-oxop en t-4-en yl]ca r ba m -
ic Acid Ben zyl Ester (8). Weinreb amide 7 (5.69 g, 13.7
mmol) was dissolved in THF (100 mL) and cooled to -78 °C.
Allylmagnesium bromide (41 mL, 41.0 mmol, 1 M solution in
THF) was added slowly,41 and after complete addition stirring
was continued for 4 h at -78 °C. The reaction was quenched
with saturated aqueous NH4Cl followed by extraction with
CH2Cl2. The combined organic layers were washed with
saturated aqueous NaHCO3 and brine and dried over Na2SO4.
The solvent was removed under reduced pressure, and the
residue was purified by flash chromatography (heptane/ethyl
acetate 15:1 f 6:1) to give 8 (4.34 g, 80%) as a white solid:
(2S,3S)-2-Am in o-1-(4-ter t-bu toxyp h en yl)h ex-5-en -3-ol
(12). Amino alcohol 9 (1.47 g, 3.70 mmol) was dissolved in a
mixture of 1 M aqueous KOH (30 mL) and ethanol (30 mL).
The reaction was heated to reflux for 5 h. The solvent volume
was reduced to ∼35 mL under reduced pressure. Water and
CH2Cl2 were added, and the two phases were separated. The
aqueous layer was extracted with CH2Cl2. The combined
organic phases were dried over Na2SO4, concentrated under
reduced pressure, and purified by flash chromatography
(toluene/ethanol 4:1 f 2:1) to give 12 (0.78 g, 80%) as colorless
oil. Compound 12 (0.26 g, 75%) was also prepared starting from
oxazolidinone 11 (0.35 g, 1.2 mmol) following the procedure
[R]20 ) +64.7 (c ) 0.6 in CHCl3); IR (neat) 1707, 1504 cm-1
;
D
1H NMR (400 MHz, CDCl3, 25 °C) δ 7.38-7.27 (m, 5H), 7.02
(d, J ) 8 Hz, 2H), 6.80 (d, J ) 8 Hz, 2H), 5.82 (m, 1H), 5.38 (d,
J ) 7.5 Hz), 5.16 (d, J ) 10.2 Hz, 1H), 5.08 (s, 2H), 5.04 (d, J
) 16.2 Hz, 1H), 4.62 (dd, J ) 7.3 and 6.8 Hz, 1H), 3.15 (dd, J
) 6.8 and 17.5 Hz, 1H), 3.05 (dd, J ) 6.8 and 17.5 Hz, 1H),
3.00 (d, J ) 6.6 Hz, 2H), 1.31 (s, 9H); 13C NMR (100 MHz,
CDCl3, 25 °C) δ 206.7, 155.6, 154.3, 136.1, 130.5, 129.6, 129.4,
128.4, 128.0, 127.9, 124.2, 119.2, 78.3, 66.8, 60.0, 45.5, 37.0,
28.7; HRMS (FAB) calcd for C24H29NO4 (M + H) 396.2175,
found 396.2188.
above: [R]20 ) -29.9 (c ) 0.107 in CHCl3); IR (neat) 3357,
D
1
1504 cm-1; H NMR (400 MHz, CDCl3, 25 °C) δ 6.96 (d, J )
8.4 Hz, 2H), 6.80 (d, J ) 8.4 Hz, 2H), 5.82-5.71 (m, 1H), 5.03-
4.91 (m, 2H), 3.39-3.32 (m, 1H), 2.81-2.68 (m, 2H), 2.41-
2.11 (m, 6H), 1.21 (s, 9H); 13C NMR (100 MHz, CDCl3, 25 °C)
δ 153.3, 134.8, 133.5, 129.2, 123.8, 116.6, 77.7, 72.0, 55.7, 39.6,
38.6, 28.4; HRMS (FAB) calcd for C16H26NO2 (M+H) 264.1964,
found 264.1978. Anal. Calcd for C16H25NO2: C, 72.97; H, 9.57;
N, 5.32. Found: C, 73.17; H, 9.38; N, 5.28.
[(1S,2S)-1-(4-ter t-Bu toxyben zyl)-2-h yd r oxyp en t-4-en yl]-
ca r ba m ic Acid Ben zyl Ester (9), [(1S,2R)-1-(4-ter t-Bu -
toxyben zyl)-2-h yd r oxyp en t-4-en yl]ca r ba m ic Acid Ben zyl
Ester (10), a n d (4S,5S)-5-Allyl-4-(4-ter t-bu toxyben zyl)-
oxa zolid in -2-on e (11). Allyl ketone 8 (4.34 g, 11 mmol) was
(2S,3S)-2-Azid o-1-(4-ter t-Bu t oxyp h en yl)h ex-5-en -3-ol
(13). A solution of NaN3 (5.00 g, 76.9 mmol) in H2O (11.5 mL)
was treated with CH2Cl2 (19 mL) at 0 °C. To this vigorously
stirred solution was added triflic anhydride (2.60 mL, 15.4
mmol, freshly distilled from P2O5), and stirring was continued
3506 J . Org. Chem., Vol. 69, No. 10, 2004

Synthesis and Conformational Studies of a â-Turn Mimetic
for 2 h. The two phases were separated, and the aqueous layer
was extracted with CH2Cl2 to give a triflyl azide solution of a
total volume of ∼30 mL (do not evaporate to dryness; explo-
sions have been reported). The combined organic layers were
washed with saturated aqueous NaHCO3 and dried over Na2-
SO4 to give a triflyl azide solution. The triflyl azide solution
was carefully added to amino alcohol 12 (0.91 g, 3.46 mmol),
DMAP (0.24 g, 1.96 mmol), and CuSO4 (0.03 g, 0.19 mmol) in
CH2Cl2 (10 mL).44,45 The reaction was stirred for 2 h and
quenched with 10% aqueous citric acid. The aqueous phase
was extracted with CH2Cl2. The combined organic phases were
washed with saturated aqueous NaHCO3, dried over Na2SO4,
and concentrated under reduced pressure. The residue was
purified by flash chromatography (heptane/ethyl acetate 9:1
tography (heptane/ethyl acetate 3:1) to give 16 (0.69 g, 85%)
as a colorless oil: [R]20D ) -17.5 (c 0.21, CHCl3); IR (neat) 2728,
1
2112, 1747, 1726 cm-1; H NMR (400 MHz, CDCl3, 25 °C) δ
9.75 (s, 1H), 7.09 (d, J ) 8.4 Hz, 2H), 6.88 (d, J ) 8.4 Hz, 2H),
4.03 (s, 2H), 3.98-3.91 (m, 1H), 3.61-3.54 (m, 1H), 3.01 (dd,
J ) 4.8 and 14.3 Hz, 1H), 2.84-2.68 (m, 3H), 1.42 (s, 9H),
1.27 (s, 9H); 13C NMR (100 MHz, CDCl3, 25 °C) δ 199.7, 168.9,
154.0, 132.1, 129.4, 124.1, 81.5, 78.0, 76.5, 68.6, 65.6, 45.3, 35.4,
28.6, 27.8. Anal. Calcd for C37H56O7N5: C, 62.20; H, 7.71; N,
10.36. Found: C, 62.47; H, 7.88; N, 10.17.
(S )-2-[(S )-2-[(3S ,4S )-4-Azid o-3-t er t -b u t oxyca r b on yl-
m eth oxy-5-(4-ter t-bu toxyp h en yl)p en tyla m in o]-3-p h en yl-
p r op ion yla m in o]-4-m eth ylp en ta n oic Acid Meth yl Ester
(17). Triethylamine (0.32 mL, 2.22 mmol) and H-Phe-Leu-
OMe48 (0.72 g, 2.16 mmol) were added to a solution of aldehyde
16 (0.67 g, 1.66 mmol) in 1,2-dichloroethane (25 mL). The
solution was stirred for 5 min, after which time NaBH(OAc)3
(0.57 g, 2.67 mmol) was added in one portion.56 Stirring was
continued for 45 min followed by addition of saturated aqueous
NaHCO3 and separation of the two phases. The aqueous phase
was extracted with CH2Cl2. The combined organic phases were
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash chroma-
tography (heptane/ethyl acetate 3:1 f 1:1) to give 17 (0.93 g,
f 4:1) to furnish 13 (0.85 g, 85%) as a colorless oil: [R]20
)
D
+18.8 (c ) 0.18 in CHCl3); IR (neat) 3425, 2102, 1504 cm-1
;
1H NMR (400 MHz, CDCl3, 25 °C) δ 7.11 (d, J ) 8.4 Hz, 2H),
6.92 (d, J ) 8.4 Hz, 2H), 5.80-5.68 (m, 1H), 5.15-5.05 (m,
2H), 3.59-3.53 (m, 1H), 3.43-3.48 (m, 1H), 2.94 (dd, J ) 13.8
and 6.0 Hz, 1H), 2.85 (dd, J ) 13.8 and 8.7 Hz, 1H), 2.34-
2.28 (m, 2H), 1.32 (s, 9H); 13C NMR (100 MHz, CDCl3, 25 °C)
δ 153.74, 133.7, 132.2, 129.5, 124.2, 118.0, 78.2, 71.4, 66.9, 38.7,
36.3, 28.5; HRMS (FAB) calcd for
290.1869, found 290.1889.
C16H24N3O2 (M + H)
82%) as a slightly yellow oil: [R]20 ) -47.6 (c ) 0.17 in
D
(4S,5S)-5-Azid o-6-(4-ter t-b u t oxyp h en yl)-4-(oxya cet ic
a cid ter t-bu tyl ester )h exa n e-1,2-d iol (15). Azido alcohol 13
(0.800 g, 2.76 mmol) was dissolved in benzene (30 mL) and
added to 50% aqueous NaOH (30 mL). NBu4HSO4 (165 mg,
0.49 mmol) was added followed by addition of tert-butyl
bromoacetate (0.73 mL, 1.67 mmol), and the reaction was
vigorously stirred.46 After 1 h, H2O (30 mL) and CH2Cl2 (30
mL) were added, and the two phases were separated. The
aqueous phase was extracted with CH2Cl2. The combined
organic phases were washed with brine and dried over Na2-
SO4. The solvent was removed under reduced pressure at 0
°C to give the sensitive alkylated azide 14. The freshly
generated 14 (0.85 g, 1.98 mmol), OsO4 (cat. amount), and
N-methylmorpholine N-oxide (0.50 g, 4.27 mmol) were im-
mediately dissolved in a mixture of acetone (16 mL), THF (16
mL), and H2O (16 mL). After the mixture was stirred for 10
h, 0.2 M aqueous HCl was added to adjust the pH to ∼2. The
aqueous layer was extracted with EtOAc. The combined
organic phases were washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was
purified by flash chromatography (heptane/ethyl acetate 1:1
f 1:2) to give 15 (0.88 g, 73%, from 13) as a 1:1 diastereomeric
mixture: IR (neat) 3428, 2110, 1730 cm-1; 1H NMR (400 MHz,
CDCl3, 25 °C) δ 7.14-7.09 (m, 2H), 6.94 (d, J ) 8.6 Hz, 2H),
4.40 (s, 0.5H), 4.24 (s, 0.5H), 4.24 (d, J ) 16.5 Hz, 0.5H), 4.22
(d, J ) 16.9 Hz, 0.5H), 4.17 (d, J ) 16.9 Hz, 0.5H), 4.17-4.09
(m, 0.5H), 4.04 (d, J ) 16.5 Hz, 0.5H), 3.98-3.90 (m, 0.5H),
3.78-3.72 (m, 0.5H), 3.69-3.59 (m, 2.5H), 3.56-3.47 (m, 1H),
3.00 (dd, J ) 2.6 and 14.2 Hz, 0.5H), 2.96 (dd, J ) 3.2 and
14.2 Hz, 0.5H), 2.67-2.56 (m, 1H), 2.44 (s, 0.5H), 2.39 (s, 0.5H),
1.82-1.56 (m, 2H), 1.49 (s, 4.5H), 1.48 (s, 4.5H), 1.32 (s, 9H);
13C NMR (100 MHz, CDCl3, 25 °C) δ 170.6, 170.0, 153.9, 132.6,
132.5, 129.5, 124.3, 82.4, 82.3, 82.0, 78.5, 78.2, 70.5, 67.9, 67.8,
66.5, 66.5, 65.7, 60.2, 35.6, 35.3, 34.1, 33.5, 28.6, 27.9; HRMS
(FAB) calcd for C22H35N3O6 (M + H) 438.2604, found 438.2598.
CHCl3); IR (neat) 3329, 2107, 1743, 1673 cm-1; 1H NMR (400
MHz, CDCl3, 25 °C) δ 7.60 (d, J ) 9.1 Hz, 1H), 7.29-7.15 (m,
5H), 7.08 (d, J ) 8.2 Hz, 2H), 6.88 (d, J ) 8.2 Hz, 2H), 4.64-
4.56 (m, 1H), 3.90 (d, J ) 16.3 Hz, 1H), 3.84 (d, J ) 16.3 Hz,
1H), 3.64 (s, 3H), 3.49 (dt, J ) 10.2 and 4.1 Hz, 1H), 3.30 (dd,
J ) 9.3 and 3.8 Hz, 1H), 3.23 (dt, J ) 8.0 and 4.3 Hz, 1H),
3.14 (dd, J ) 13.6 and 3.8 Hz, 1H), 2.92 (dd, J ) 14.0 and 3.6
Hz, 1H), 2.78-2.55 (m, 4H), 1.76-1.46 (m, 6H), 1.43 (s, 9H),
1.28 (s, 9H), 0.90 (d, J ) 5.8 Hz, 3H), 0.88 (d, J ) 5.8 Hz, 3H);
13C NMR (100 MHz, CDCl3, 25 °C) δ 173.4, 173.1, 169.1, 153.9,
137.4, 132.6, 129.4, 129.0, 128.5, 126.7, 124.1, 81.4, 80.5, 78.0,
68.0, 65.7, 63.7, 51.9, 49.8, 44.7, 41.2, 39.2, 35.3, 30.2, 28.6,
27.9, 24.6, 22.7, 21.5; HRMS (FAB) calcd for C37H56N5O7 (M
+ H) 682.4180, found 682.4175. Anal. Calcd for C37H55N5O7:
C, 65.18; H, 8.13; N, 10.27. Found: C, 65.42; H, 8.35; N, 10.44.
(S)-2-((S)-2-[[(3S,4S)-4-Azid o-3-ter t-b u t oxyca r b on yl-
m eth oxy-5-(4-ter t-bu toxyp h en yl)p en tyl]-[2-(9H-flu or en -
9-ylm e t h oxyca r b on yla m in o)a ce t yl]a m in o]-3-p h e n yl-
p r op ion yla m in o)-4-m eth ylp en ta n oic Acid Meth yl Ester
(18). Amine 17 (0.85 g, 1.25 mmol) was dissolved in DMF (5
mL) and added to
a mixture of diisopropylethylamine
(0.56 mL, 3.23 mmol), HATU49,50 (O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate, 0.62 g,
1.64 mmol), and Fmoc-Gly-OH (0.53 g, 1.78 mmol) in DMF (3
mL) at 0 °C. The reaction was allowed to reach room temper-
ature, and stirring was continued for another 15 h. To the
reaction were added saturated aqueous NaHCO3 and EtOAc.
The two phases were separated, and the aqueous layer was
extracted with EtOAc. The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash chroma-
tography (heptane/ethyl acetate 2:1 f 1:1) to give 18 (1.06 g,
85%) as white solid of a ∼4:1 mixture of rotamers: [R]20
)
D
-57.0 (c ) 0.32 in CHCl3); IR (neat) 2108, 1724, 1678, 1651
cm-1; 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.79-7.73 (m, 2H),
7.65-7.54 (m, 2H), 7.43-7.36 (m, 2H), 7.35-7.18 (m, 7H),
7.18-7.09 (m, 2H), 6.99 (d, J ) 8.4 Hz, 1H), 6.93 (d, J ) 7.0
Hz, 2H), 5.74 (t, J ) 4.3 Hz, 0.8H), 5.62 (t, J ) 4.3 Hz, 0.2H),
4.76-4.62 (m, 0.8H), 4.60-4.47 (m, 1.2H), 4.42-4.30 (m, 2H),
4.28-4.17 (m, 1H), 4.15-3.94 (m, 4H), 3.71 (s, 0.5H), 3.67 (s,
2.5H), 3.64-3.45 (m, 2H), 3.43-3.30 (m, 3H), 3.21-3.08 (m,
1H), 3.05 (dd, J ) 3.3 and 14.0 Hz, 0.2H), 2.96 (dd, J ) 3.3
and 14.0 Hz, 0.8H), 2.70 (dd, J ) 10.4 and 13.9 Hz, 0.2H),
2.55 (dd, J ) 10.4 and 13.9 Hz, 0.8H), 1.80-1.52 (m, 5H), 1.49
[(1S,2S)-2-Azid o-3-(4-ter t-b u t oxyp h en yl)-1-(2-oxoet h -
yl)p r op oxy]a cet ic Acid ter t-Bu t yl E st er (16). The di-
astereomeric mixture of diols 15 (0.87 g, 2.0 mmol) was
dissolved in benzene (60 mL) followed by addition of Na2CO3
(0.53 g, 5.0 mmol). The reaction was cooled to 0 °C, and Pb-
(OAc)4 (1.4 g, 3.16 mmol) was added. The reaction was kept
at 0 °C for 10 min and then allowed to reach room temperature
and stirred for another 3 h. The reaction was quenched with
ethylene glycole (20-25 drops), and after 5 min Et2O and
saturated aqueous NaHCO3 were added. The aqueous phase
was extracted with Et2O. The combined organic phases were
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash chroma-
(56) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C.
A.; Shah, R. D. J . Org. Chem. 1996, 61, 3849-3862.
J . Org. Chem, Vol. 69, No. 10, 2004 3507

Blomberg et al.
(s, 1.5H), 1.46 (s, 7.5H), 1.33 (s, 9H), 0.93 (d, J ) 2.5 Hz, 2.5H),
0.92 (d, J ) 2.5 Hz, 2.5H), 0.89-0.85 (m, 1H); 13C NMR (100
MHz, CDCl3, 25 °C, CHCl3) δ 173.1, 170.3, 170.1, 169.3, 156.3,
154.3, 144.0, 141.4, 137.3, 132.6, 129.8, 129.1, 128.8, 127.8,
127.2, 126.9, 125.2, 124.5, 124.4, 120.1, 82.0, 79.8, 78.5, 68.0,
67.3, 65.9, 52.3, 51.0, 47.2, 43.3, 41.1, 35.4, 34.2, 30.3, 28.9,
25.0, 22.9, 21.9; HRMS (FAB) calcd for C54H69N6O10 (M + H)
961.5075, found 961.5048. Anal. Calcd for C54H68N6O10: C,
67.48; H, 7.13; N, 8.74. Found: C, 67.81; H, 7.28; N, 8.55.
(S)-2-((S)-2-[[(3S,4S)-4-Azid o-3-ca r b oxym et h oxy-5-(4-
h yd r oxyp h en yl)p en t yl][2-(9H -flu or en -9-ylm et h oxyca r -
bon yla m in o)a cetyl]a m in o]-3-p h en ylp r op ion yla m in o)-4-
m eth ylp en ta n oic Acid Meth yl Ester (19). Compound 18
(1.10 g, 1.14 mmol) was dissolved in formic acid (19 mL) and
stirred at room temperature for 20 h. Toluene (3 × 5 mL) was
added, and the solvents were removed under reduced pressure.
The residue was purified by flash chromatography (EtOAc/
heptane/formic acid 2:1:0 f 2:1:0.05) to give 19 (0.97 g, 99%)
7.43-7.08 (m, 8H), 6.93-6.84 (m, 2H), 4.27-4.17 (m, 3H),
4.00-3.91 (m, 1H), 3.89-3.81 (m, 1H), 3.52-3.45 (m, 1H),
3.42-3.30 (m, 3H), 3.26-3.17 (m, 1H), 2.90-2.81 (m, 1H),
2.79-2.71 (m, 1H), 2.47-2.36 (m, 1H), 1.59-1.43 (m, 2H),
1.41-1.20 (m, 2H), 0.90-0.79 (m, 6H), 0.79-0.69 (m, 1H);
HRMS (FAB) calcd for C30H41N4O7 (M + H) 569.2975, found
569.2964.
NMR Exp er im en ts. All spectra of 23 were recorded on a
Bruker DRX 600 MHz spectrometer equipped with a TXI
cryoprobe. The spectra obtained at -70 °C for 21 were recorded
on a Bruker AMX 500 MHz spectrometer equipped with a TXI
probe. The NMR sample of 23 was prepared by dissolving 2.3
mg in phosphate-buffered aqueous solution at pH 5.5 to give
9 mM final concentration. The NMR sample of 21 was
prepared by dissolving 2.8 mg of 21 in MeOH-d3 giving a 10
mM solution. TOCSY,57 NOESY,58 and DQF-COSY59 2D
experiments were all recorded with 2048 complex data points
in t2 and 512 increments in t1. Zero-filling resulted in datasets
with 2048 × 1024 points. A DIPSI spin-lock was used in the
TOCSY spectra with a mixing time of 85 ms. Apodization with
a shifted squared sine-bell function was applied in both
dimensions prior to Fourier transformation except in the
DQF-COSY experiment where an unshifted sine-bell function
was used. A 250 ms mixing time was used in the NOESY
spectrum for 23 and 30 ms for 21.
as a white solid:. [R]20 ) -50.4 (c ) 0.14 in CHCl3); IR (neat)
D
3322, 2106, 1723, 1649 cm-1; 1H NMR (400 MHz, (CD3)2SdO,
70 °C) δ 7.85 (d, J ) 7.3 Hz, 2H), 7.68 (d, J ) 7.1 Hz, 2H),
7.40 (t, J ) 7.4 Hz, 2H), 7.34-7.28 (m, 2H), 7.27-7.10 (m, 5H),
7.06 (d, J ) 8.4 Hz, 2H), 6.72-6.67 (m, 2H), 4.33-4.17 (m,
4H), 4.15-4.10 (m, 2H), 3.63-3.56 (m, 1H), 3.58 (s, 3H), 3.52-
3.46 (m, 2H), 3.31-3.24 (m, 2H), 3.03-2.86 (m, 2H), 2.64-
2.54 (m, 1H), 1.81-1.64 (m, 2H), 1.62-1.48 (m, 3H), 0.84 (d,
J ) 6.2 Hz, 3H), 0.81 (d, J ) 6.2 Hz, 3H); HRMS (FAB) calcd
for C46H53N6O10 (M + H) 849.3823, found 849.3835.
Str u ctu r e Ca lcu la tion s. Assignment and peak picking
were performed in Felix (Accelrys Inc.). Distance restraints
were extracted from the NOESY spectrum of 21 obtained at
-70 °C. NOESY cross-peak volumes were converted to inter-
proton distance restraints, dij, by using the isolated spin-pair
approximation
(S)-2-((S)-2-[(S)-10-[(S)-1-Azid o-2-(4-h yd r oxyp h en yl)-
eth yl]-3,6-d ioxo[1,4,7]oxa d ia zeca n -7-yl]-3-p h en ylp r op io-
n yla m in o)-4-m eth ylp en ta n oic Acid Meth yl Ester (21).
Acid 19 (0.3 g, 0.35 mmol) was dissolved in EtOAc (3 mL).
Dicyclohexylcarbodiimide (0.11 g, 0.52 mmol) and pentafluo-
rophenol (0.1 g, 0.52 mmol) were then added at 0 °C.51 The
reaction was stirred for 2 h and then filtered through Celite.
The solvent was then removed under reduced pressure, and
the residue was filtered through silica (heptane/ethyl acetate
2:1 f 1:1) to yield activated ester 20 (0.35 g, ∼0.35 mmol) still
containing a small amount of dicyclohexyl urea. A solution of
20 (0.35 g, ∼0.35 mmol) in dioxane (10 mL) was added, via a
syringe pump, to a refluxing solution of 1,8-Diazabicyclo[5.4.0]-
undec-7-ene (0.15 mL, 1.0 mmol) in dioxane (52 mL) over a
period of 12 h. After complete addition, the reaction was
allowed to reflux for another 15 min before the solvent was
removed under reduced pressure and the residue was purified
by flash chromatography (toluene/ethanol 20:1 f 10:1) to give
21 (0.12 g, 56%, from 19) as a white solid: [R]20D ) -76.9 (c )
dij ) dref(Vref/Vij)1/6
where dref corresponds to a reference distance in the molecule
with the known cross-peak volume Vref. The cross-peak be-
tween the R-protons of Gly2 was used as the reference volume
with a reference distance of 1.772 Å. The upper limit for the
distance restraints were set to 4 Å for all dij shorter than 3
and 5 Å for all dij exceeding 3 Å.
Starting structures for 21 were prepared with an un-
restrained MD simulation. The two structures with the largest
conformational difference were subjected to restrained simu-
lated annealing with the 29 experimentally derived distance
restraints. The initial temperature in the simulated annealing
protocol was set to 1000 K and the final temperature was 100
K. A soft square-welled potential with a force constant of 50
kcal mol-1 Å-2 was used for the distance restraints. All
structures were minimized with a 200 step Powell minimiza-
tion after the refinement. Structures were accepted if they did
not contain any distance restraint violations larger than 0.3
Å and no dihedral angle violations larger than 5°. X-PLOR55
was used for all structure calculations.
0.1 in CHCl3); IR (neat) 3509-3087, 2106, 1738, 1649 cm-1
;
1H NMR (400 MHz, CD3OH, -70 °C) δ 7.37-7.10 (m, 8H),
6.79-6.70 (m, 2H), 4.73-4.60 (m, 2H), 4.54-4.43 (m, 2H),
4.35-4.22 (m, 1H), 4.01-3.95 (m, 1H), 3.93-3.87 (m, 1H),
3.77-3.66 (m, 1H), 3.68 (s, 3H), 3.55-3.40 (m, 3H), 2.95-2.86
(m, 1H), 2.49-2.31 (m, 2H), 2.15-2.04 (m, 1H), 1.81-1.45 (m,
4H), 1.07-0.88 6H); HRMS (FAB) calcd for C31H41N6O7 (M +
H) 609.3037, found 609.3026.
(S)-2-((S)-2-[(S)-10-[(S)-1-Am in o-2-(4-h yd r oxyp h en yl)-
eth yl]-3,6-d ioxo[1,4,7]oxa d ia zeca n -7-yl]-3-p h en ylp r op io-
n yla m in o)-4-m eth ylp en ta n oic Acid (23). Compound 21
(0.06 g, 0.1 mmol) was treated with a mixture of SnCl2 (0.07
g, 0.37 mmol), triethylamine (0.15 mL, 1.1 mmol), and
thiophenol (0.15 mL, 1.5 mmol) in THF (6 mL) at room
temperature.52 After 2 h, the solvent was removed under
reduced pressure, and the residue was purified by HPLC and
lyophilized to give amine 22 (0.04 g, 0.07 mmol) which was
directly dissolved in THF (10 mL) and treated with 0.1 M
aqueous LiOH (1.9 mL, 0.19 mmol). After 3 h the reaction was
quenched with an excess of acetic acid (∼ 0.1 mL). The solvent
was removed under reduced pressure, and the residue was
purified by HPLC and lyophilized to give 23 (0.031 g, 57%,
Ackn owledgm en t. This work was founded by grants
from the Swedish Research Council and the Go¨ran
Gustafsson Foundation for Research in Natural Sciences
and Medicine.
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
and 13C NMR spectra for compounds 7-13, 15-19, 21 and
23. This material is available free of charge via the Internet
at http://pubs.acs.org.
J O0356863
(57) Braunschweiler, L.; Ernst, R. R. J . Magn. Reson. 1983, 53, 521-
528.
(58) Macura, S.; Ernst, R. R. Mol. Phys. 1980, 41, 95-117.
(59) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J . Chem. Phys. 1976,
64, 2229-2246.
from 21) as a white solid: [R]20 ) -47.6 (c ) 0.59 in MeOH);
D
IR (neat) 3261, 2958, 1658, 1616 cm-1
;
1H NMR (600 MHz,
H2O, 33 °C, H2O) δ 8.60-8.49 (m, 0.6H), 8.12-8.05 (m, 0.2H),
3508 J . Org. Chem., Vol. 69, No. 10, 2004