A novel dipeptidomimetic containing a cyclic threonine

Article abstract of DOI:10.1039/b915220b

An efficient and simple two-step procedure for the formation of hydroxy-Freidinger lactams is presented. The methodology allows assembly of the cyclic threonine motif (cThr) in solution and on solid support during conventional peptide synthesis.

Full text of DOI:10.1039/b915220b

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A novel dipeptidomimetic containing a cyclic threoninew
Frank Sicherl,ab Tommaso Cupidoab and Fernando Albericio*abc
Received (in Cambridge, UK) 27th July 2009, Accepted 10th December 2009
First published as an Advance Article on the web 13th January 2010
DOI: 10.1039/b915220b
An efficient and simple two-step procedure for the formation of
hydroxy-Freidinger lactams is presented. The methodology
allows assembly of the cyclic threonine motif (cThr) in solution
and on solid support during conventional peptide synthesis.
feature major structural differences in steric and electronic
properties when compared to the natural analogs. For
instance, these compounds contain an additional a-substituent
which result in a tertiary a-carbon like in 1-amino-2-hydroxy-
cyclohexane carboxylic acid derivatives.17–20 Apart from this,
Thr has been used for the assembly of several cyclic systems
into the peptidic backbone,21–23 however, in all of these
examples the hydroxyl either vanishes or remains part of the
heterocycle formed. Since our strategy for finding biologically
active peptidomimetics is based on nature-like structures,24 the
simple b-hydroxy-g-lactam 125 was considered as a cyclic Thr
(2) motif (cThr) (Fig. 1).26
Recent years have witnessed a considerable growth in the
number of peptide pharmaceuticals available, which currently
cover a broad range of therapeutic indications.1,2 Some of
these peptides contain conformational restrictions through
mainly cyclization3 or introduction of backbone constraints,4–6
which can overcome problems such as poor metabolic stability
and low oral availability.4–6 These constrained peptides have
the potential of enhanced interaction with the receptor and
better pharmacokinetic properties than their corresponding
peptides.7 Furthermore, these compounds very often adopt
preferred conformations such as turns, which may enhance
their binding to a biological target.8 Ideally, these con
formational constraints should maintain the appropriate
topological arrangement of functional groups that are essen-
tial for recognition, while removing some of the unfavorable
characteristics of peptides.9,10 In this regard, the Freidinger
lactam,4,5,8 which consists basically of an alkyl side-chain
bridge to the amide nitrogen of the n À 1 amino acid, has
been widely used for the preparation of a large number of
peptidomimetics with biological activity.
To the best of our knowledge, except for the synthesis of
b-substituted a-amino-b-hydroxy-g-lactam by Giese’s group,27,28
no efficient method has been reported for the introduction of
this structure into peptides. Despite displaying a conventional
Freidinger lactam containing a b-hydroxy functionality and its
obvious structural relation to Thr, 1 has never been considered
as a peptidomimetic element. Thus, its synthesis and assembly
into peptides respectively, represent a challenge.
Our synthetic strategy is based on the addition of an amino
group to epoxide 3,29,30 which can be obtained as a mixture of
the threo- and erythro-isomers in a ratio of 4/1 from Met in five
steps. Initial attempts to carry out the epoxide opening reac-
31
tion at reflux in the presence of LiClO4 always proceeded
incompletely and gave maximum yields of 33%. Finally,
performing the reaction in a microwave reactor showed
acceptable efficiency for this conversion. Moreover, from a
screening of commonly used activators32 for additions to
epoxides only LiClO4 and Ca(OTf)233,34 showed the formation
of the desired product as shown by TLC. In contrast, no
product formation was detected with Yb(OTf)3, Ti(OiPr)4,
BF3ÁOEt2 or Sc(OTf)3. On the basis of these findings, the
reaction conditions were optimized using Ca(OTf)2 to obtain
Thr and Ser moieties play a key role in several biologically
relevant processes. The hydroxyl is involved in biocatalysis as it is
located in the active pockets of a variety of enzymes, popular
examples are proteases,11,12 or it acts as an acceptor for covalent
modifications as for Ser/Thr directed kinases.13 The carbohydrate
part of O-type glycopeptides and glycoproteins is linked mostly to
a Thr, or a Ser residue respectively.14 Thr and Ser participate in
the regulation of biological events through phosphorylation
during signal cascades that control activation or deactivation of
proteins.15,16 Furthermore, Thr and Ser are components of a
variety of biologically active peptides.
excellent yields (76–81%) of the secondary amines
4
with distinct amino acid esters (Scheme 1). Ring closure
was subsequently achieved by heating at reflux in toluene
to give the desired lactam. Dipeptidomimetics 5a–d were
isolated in yields of 76–98% from the corresponding amino
acid esters, whereas the threo-isomers were separated by
performing conventional column chromatography in mixtures
of hexane–EtOAc.
So far, several non-proteogenic amino acids have been
introduced into peptides as Thr-mimetics. However, most
a Institute for Research in Biomedicine, Barcelona Science Park,
Baldiri Reixac 10, 08028-Barcelona, Spain.
E-mail: frank.sicherl@contractors.roche.com,
albericio@irbbarcelona.org; Fax: +34 93 4037126;
Tel: +34 93 4037088
b CIBER-BBN, Networking Centre on Bioengineering,
Biomaterials and Nanomedicine, Barcelona Science Park,
Baldiri Reixac 10, 08028-Barcelona, Spain
c Department of Organic Chemistry, University of Barcelona,
´
´
Martı i Franques 1, 08028-Barcelona, Spain
w Electronic supplementary information (ESI) available: Detailed
experimental procedures and NMR data, including copies of NMR
spectra. See DOI: 10.1039/b915220b
Fig. 1 Cyclic hydroxylactam 1 as a natural-like Thr-mimetic.
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Scheme 1 Two-step sequence for the formation of cThr 5.
The successful assembly of the hydroxylactam into longer
peptides was further demonstrated, by coupling of an
additional amino acid to threo-5a, and threo-5b respectively
(Scheme 2). First of all, the Z group was cleaved by hydro-
genolysis on Pd/C, then the peptide bond formation was
achieved using HATU/HOAt35 as coupling reagents to give
tripeptidomimetics 7a–c.
Scheme 3 Synthesis of Fmoc protected epoxide 9.
obtain Fmoc protected vinyl glycine 11 was carried out by
heating at reflux in xylene. Epoxidation with mCPBA finally
led to 9 in a diastereomeric ratio of 6/1 (threo/erythro).
The optimized synthetic pathway gave access to the Fmoc
protected epoxide building block 9 in an excellent overall yield
of 80% on a multigram scale.
After finding efficient conditions for the assembly of cThr in
solution, we sought to develop a methodology to carry out the
epoxide opening reaction on solid support as a type of
‘‘coupling sequence’’ during peptide synthesis. Since the Z
group is not appropriate as temporary protection on solid
In the attempt to develop a methodology for the construc-
tion of the cyclic threonine building block on solid-phase, the
mixture of epoxides 9 was used for the addition reaction of the
amino group of Phe bound to Rink amide-aminomethyl-
polystyrene (AM-PS) resin. However, it was not possible to
achieve a complete conversion, even when using 10 equiv. of
epoxide. The maximum loading determined after the reaction
was 0.25 mmol gÀ1 which corresponds to a 40% yield. Given
the low solubility of Ca(OTf)2 in dioxane, we hypothesized
that employing a cosolvent would improve the outcome of the
reaction. However, addition of 5–10% of either DMF or
DMSO to obtain a clear solution did not enhance the reaction.
We consider that these findings indicate that the mechanism
for the reaction of an epoxide with an amine in the presence of
Ca(OTf)2 is based on a heterogeneous activation. Neither did
the attempts to carry out LiClO4-mediated epoxide-opening
on solid support lead to an improvement.
phase, we synthesized the Fmoc protected epoxide
9
(Scheme 3A). However, following a procedure employing
Pd(OH)2 as catalyst with 3,36 which was used for the removal
of the Z group of a-substituted derivatives of 3 without
affecting the epoxide, only amino butyric acid 8 was detected
after treatment with FmocOSu.
Therefore, we followed a synthetic route starting from
H-Met-OMe in analogy to the procedure described for 3
(Scheme 3B). After introducing the Fmoc group, the sulfur
was oxidized by NaIO4 to give 10. Subsequently, pyrolysis to
Therefore, we turned to the dipeptide mimetic building
block, which was previously synthesized in solution, for the
assembly of the hydroxylactam. Using H-Phe-OtBu and 9
the Fmoc protected tBu ester 12 was obtained following the
procedure described above (Scheme 4).
In contrast to the Z-protected derivatives the two diastereo-
isomers of 12 were not separable by column chromatography
as a result of no observable difference in Rf-values. Therefore,
12 was used as mixture for peptide coupling. Acidic hydrolysis
Scheme 2 Tripeptidomimetics 7a–c obtained from threo-5a and
threo-5b.
Scheme 4 Synthesis of dipeptide mimetic building block 13.
Chem. Commun., 2010, 46, 1266–1268 | 1267
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Notes and references
1 T. Cupido, J. Tulla-Puche, J. Spengler and F. Albericio, Curr.
Opin. Drug Discovery Dev., 2007, 10, 768.
2 T. Bruckdorfer, O. Marder and F. Albericio, Curr. Pharm.
Biotechnol., 2004, 5, 29.
3 R. A. Hughes and C. J. Moody, Angew. Chem., Int. Ed., 2007, 46,
7930.
4 R. M. Freidinger, J. Med. Chem., 2003, 46, 5553.
5 R. M. Freidinger, D. S. Perlow and D. F. Veber, J. Org. Chem.,
1982, 47, 104.
6 W. L. Scott, J. G. Martynow, J. C. Huffman and M. J. O’Donnell,
J. Am. Chem. Soc., 2007, 129, 7077.
7 E. Benedetti, C. Pedone and M. Saviano, Front. Drug
Des. Discovery, 2007, 3, 539.
8 R. M. Freidinger, D. F. Veber, D. S. Perlow, J. R. Brooks and
R. Saperstein, Science, 1980, 210, 656.
9 A. Piserchio, Y. Han, M. Chorev and D. F. Mierke, Biopolymers,
2002, 64, 16.
10 M. Goodman, C. Zapf and Y. Rew, Biopolymers, 2001, 60, 229.
11 A. J. Barrett, N. D. Rawlings and J. F. Woessner, Cysteine, Serine
and Threonine Peptidases, Elsevier, London, UK, 2nd edn, vol. 2:
Handbook of Proteolytic Enzymes, 2004.
12 J. C. Powers, J. L. Asgian, O. D. Ekici and K. E. James, Chem.
Rev., 2002, 102, 4639.
Scheme
5 Solid-phase synthesis of pentapeptide mimetic 15
employing 13.
13 E. J. Goldsmith, R. Akella, X. Min, T. Zhou and
J. M. Humphreys, Chem. Rev., 2007, 107, 5065.
14 H. C. Hang and C. R. Bertozzi, Bioorg. Med. Chem., 2005, 13, 5021.
15 H. D. Kulasekara and S. I. Miller, Nat. Cell Biol., 2007, 9, 734.
16 J. Song, C. Chao and Y. Xu, Cell Cycle, 2007, 6, 1412.
17 P. R. Guzzo, M. P. Trova, T. Inghardt and M. Linschoten,
Tetrahedron Lett., 2002, 43, 41.
18 A. B. Avenoza, J. I. Barriobero, C. Cativiela, M. A. Fernandez-
Recio, J. M. Peregrina and F. Rodriguez, Tetrahedron, 2001, 57,
2745.
of the tBu ester with TFA in DCM led to acid 13 which was
directly activated without purification and coupled to resin
bound Phe (Scheme 5). Subsequently, for the capping of free
amino groups, the resin was incubated with Ac2O in pyridine
which acetylated the b-hydroxyl as well (14). Following a
standard Fmoc procedure the peptide sequence was elongated
by Gly and Ala. The Fmoc-protected pentapeptide mimetic 15
was obtained by cleavage with TFA containing 2.5% of each
TIS and water and finally isolated by RP-HPLC in a 37%
overall yield.
19 F. Clerici, M. L. Gelmi and A. Gambini, J. Org. Chem., 1999, 64,
5764.
20 F. Clerici, M. L. Gelmi and A. Gambini, J. Org. Chem., 2000, 65,
6138.
Pentapeptide mimetic 15 was fully characterized by NMR
spectroscopy (see ESIw). Further ROESY experiments showed
several NOE contacts from Phe5 to the hydroxylactam. These
observations indicate that the C-terminal part of 15 exhibits a
turn-type conformation that is stabilized by the lactam ring.
In conclusion, with the objective to develop a synthetic
access to a nature-like constrained Thr motif, we developed
an efficient synthesis for b-hydroxy-g-lactam from easily
obtainable amino acid epoxides 3 and 9. The epoxide opening
reaction with an amino acid amine as the key-step was
optimized to good yields employing a microwave reactor
and Ca(OTf)2 as activator. The methodology presented here
allowed the assembly of this motif in solution and its sub-
sequent incorporation as a dipeptide building block on solid
support as demonstrated by the syntheses of several examples.
Structural analysis of pentapeptide mimetic 15 by NMR
experiments gives strong evidence of the occurrence of a
preferred conformation. Ongoing research includes the bio-
logical evaluation as well as detailed structural analysis of
peptides containing the cThr motif.
21 G. Haberhauer and F. Rominger, Synlett, 2003, 780.
22 T. Doi, Y. Miura, S. Kawauchi and T. Takahashi, Chem.
Commun., 2005, 4908.
23 E. Biron, J. Chatterjee and H. Kessler, J. Pept. Sci., 2006, 12, 213.
24 R. Breinbauer, I. R. Vetter and H. Waldmann, Angew. Chem., Int.
Ed., 2002, 41, 2878.
25 Compound
1 was reported as an undesired side product:
T. Tashiro, S. Fushiya and S. Nozoe, Chem. Pharm. Bull., 1988,
36, 893.
26 The term ‘cyclothreonine’ has been used for a Thr were the
hydroxyl-O is linked to the amide-N: U. Anthoni, T. M. Fatum
and C. Flensburg, Acta. Crystallogr., Sect. C: Cryst. Struct.
Commun., 1998, C54, 240.
27 C. Wyss, R. Batra, C. Lehmann, S. Sauer and B. Giese, Angew.
Chem., Int. Ed. Engl., 1996, 35, 2529.
28 S. Sauer, A. Schuhmacher, F. Barbosa and B. Giese, Tetrahedron
Lett., 1998, 39, 3685.
29 K. J. Shaw, J. R. Luly and H. Rapoport, J. Org. Chem., 1985, 50,
4515.
30 T. Van Truong and H. Rapoport, J. Org. Chem., 1993, 58, 6090.
31 S. Rodriguez-Escrich, D. Popa, C. Jimeno, A. Vidal-Ferran and
M. A. Pericas, Org. Lett., 2005, 7, 3829.
32 C. Schneider, Synthesis, 2006, 3919.
33 M. Sova, A. Babic, S. Pecar and S. Gobec, Tetrahedron, 2007, 63,
141.
F.S. thanks the German Academic Exchange Service
(DAAD) for a fellowship. This work was partially supported
by CICYT (CTQ2006-03794/BQU), the Instituto de Salud
Carlos III (CB06_01_0074), the Generalitat de Catalunya
(2009SGR 1024), the Institute for Research in Biomedicine,
and the Barcelona Science Park.
34 A. Babic, M. Sova, S. Gobec and S. Pecar, Tetrahedron Lett., 2006,
47, 1733.
35 L. Carpino, A. El-Faham, C. A. Minor and F. Albericio, J. Chem.
Soc., Chem. Commun., 1994, 201.
36 R. G. Shea, J. N. Fitzner, J. E. Fankhauser, A. Spaltenstein,
P. A. Carpino, R. M. Peevey, D. V. Pratt, B. J. Tenge and
P. B. Hopkins, J. Org. Chem., 1986, 51, 5243.
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1268 | Chem. Commun., 2010, 46, 1266–1268