Microwaves enhance cyclisation of tetrapeptides

Article abstract of DOI:10.1016/j.tetlet.2009.10.026

Head-to-tail cyclisation tetrapeptides can be improved using microwave dielectric heating. Cyclisation occurs rapidly at millimolar dilution giving the product in higher purity and yields if compared with standard conditions. The reaction can be applied t

Full text of DOI:10.1016/j.tetlet.2009.10.026

Tetrahedron Letters 50 (2009) 7159–7161
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Microwaves enhance cyclisation of tetrapeptides
Elena Cini a, Cinzia B. Botta b, Manuela Rodriquez b, Maurizio Taddei a,
*
a Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy
b Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte don Melillo, 84084 Fisciano, Salerno, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Head-to-tail cyclisation tetrapeptides can be improved using microwave dielectric heating. Cyclisation
occurs rapidly at millimolar dilution giving the product in higher purity and yields if compared with stan-
dard conditions. The reaction can be applied to sequences containing at least one D-amino acid.
Received 1 September 2009
Revised 29 September 2009
Accepted 5 October 2009
Available online 9 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Microwaves
Cyclopeptides
Solid-phase synthesis
The application of microwave dielectric heating to solid phase
peptide synthesis is a well established procedure that gives better
yields and purity if compared with standard room temperature
methods.1 The most remarkable achievement is the chance to pre-
pare very long peptides in short time minimizing elongation issues
such as incomplete peptide coupling and formation of stable b-
sheets which tend to aggregate and prevent further acylation.2 Sev-
eral cyclopeptides, important molecules with interesting biological
activities, have also been prepared under microwave dielectric
heating through side chain-to-tail (or head-to-tail) or disulfide
bridge cyclisations on solid support.3 Moreover, microwave-as-
sisted nucleophilic aromatic substitution or Heck reactions have
also been used to close the peptide cycle on the resin.4
However, the backbone (head-to-tail) cyclisation of linear pep-
tides in solution is the more extensively employed approach for
the synthesis of cyclopeptides. All coupling agents are suitable
for this cyclisation, although the reaction proceeds more slowly
when compared to normal peptide couplings. Undesired by prod-
ucts are oligomers derived by intermolecular cyclodimerization.
This problem can be reduced carrying out the cyclisation in solu-
tion under high dilution.5 While head-to-tail cyclisations of hexa-
peptides occur without particular sequence-specific problems,
tetra- and pentapeptides can be assembled only when preorgani-
zation of the linear precursor is possible and tripeptides do not cy-
clize to the nine-membered ring with few exceptions.6 Peptide
hesitancy to cyclise is attributed to the predominantly trans-con-
figured peptide bonds favouring an extended conformation of the
precursor. Since heating a solution containing a linear peptide
would accelerate the trans–cis-equilibration,7 we decided to inves-
tigate the influence of microwave dielectric heating on the head-
to-tail cyclisation of different tetrapeptides (see Scheme 1).
Being interested in the preparation of cyclopeptide analogues of
the potent histone deacetylase (HDAC) inhibitor FR235222,8 we
synthesized the simplified linear tetrapeptide H-Ahoda-Iva-Phe-
(D)-Pro-OH (1 in Table 1) following a microwave assisted solid
phase synthesis Fmoc protocol.9 The reference peptide 12 was pre-
pared under standard conditions using HATU (3.5 equiv) and DIEA
(4 equiv) in DCM/DMF at 8 Â 10À5 M concentration.10 Stirring the
reaction mixture at 4 °C for 1 h and at rt for 4 h gave 55% conver-
sion of 1 into 12. HPLC purification from higher molecular weight
oligomers (dimers and trimers) gave cyclopeptide 12 in 25% yield
(entry 1 in Table 1).
The reduction of the solvent amount required for an efficient
cyclisation was an additional achievement of this study. Submit-
ting 5 mg of peptide 1 to HATU (3.5 equiv) and DIEA (4 equiv) in
5 mL of DMF (1.8 Â 10À3 M) to microwave dielectric heating
(25 W) at 75 °C for 10 min, the cyclopeptide 12 was obtained as
the main product of the reaction (much less dimers or trimers
OTBDMS
O
O
See Entry 3 in
Table 1
N
O
4
O
H
N
NH
N
HN
OTBDMS
O
H2N
N
H
H
N
O
O
COOH
4
O
O
1
12
* Corresponding author. Tel.: +39 0577234280; fax: +39 0577234275.
E-mail address: taddei.m@unisi.it (M. Taddei).
Scheme 1. Microwave-assisted cyclisation of tetrapeptides.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.10.026

7160
E. Cini et al. / Tetrahedron Letters 50 (2009) 7159–7161
Table 1
Head-to tail microwave-assisted cyclisation of linear tetrapeptides
En.
1
Linear peptide
Reaction conditions
Cyclopeptide
Yielda
25%
H-Ahoda(OTBDMS)-Iva-Phe-
DCM/DMF 1/1 (8 Â 10À5 M) HATU (3.5 equiv), DIEA (4 equiv) 4 °C for 1 h,
then rt for 4 h
cyclo(Ahoda(OTBDMS)-Iva-Phe-
(
D
)-Pro-OH 1
H-Ahoda(OTBDMS)-Iva-Phe-
)-Pro-OH 1
H-Ahoda(OTBDMS)-Iva-Phe-
)-Pro-OH 1
H-Ahoda(OTBDMS)-Iva-Phe-
)-Pro-OH 1
H-Ahoda(OTBDMS)-Iva-Phe-
)-Pip-OH 2
H-Ahoda(OTBDMS)-Trp-Trp-
)-Pro-OH 3
H-Ahoda(OTBDMS)-Pro-Phe-
)-Pro-OH 4
H-Ahoda(OTBDMS)-Phe-Phe-
)-Pro-OH 5
H-Ahoda(OTBDMS)-Iva-Phe-
)-Trp-OH 6
H-Ahoda(OTBDMS)-Trp-Phe-
)-Pro-OH 7
H-Ala-Val-Phe-(
(D
)-Pro-) (12)
cyclo(Ahoda(OTBDMS)-Iva-Phe-
)-Pro-) 12
cyclo(Ahoda(OTBDMS)-Iva-Phe-
)-Pro-) 12
cyclo(Ahoda(OTBDMS)-Iva-Phe-
)-Pro-) 12
cyclo(Ahoda(OTBDMS)-Iva-Phe-
)-Pip-) 13
cyclo(Ahoda(OTBDMS)-Trp-Trp-
)-Pro-) 14
cyclo(Ahoda(OTBDMS)-Pro-Phe-
)-Pro-) 15
cyclo(Ahoda(OTBDMS)-Phe-Phe-
)-Pro-) 16
cyclo(Ahoda(OTBDMS)-Iva-Phe-
)-Trp-) 17
cyclo(Ahoda(OTBDMS)-Trp-Phe-
)-Pro-) 18
cyclo(Ala-Val-Phe-(
2
DMF, (1.8 Â 10À3 M), HATU (3.5 equiv), DIEA (4 equiv), microwave,
10 min, 75 °C, 25 W
45%
43%
22%b
45%
43%
38%
35%
46%
48%
39%
40%
36%
0
(
D
(D
3
CH2Cl2, (4 Â 10À3 M), HATU (3.5 equiv), DIEA (4 equiv), microwave,
10 min, 75 °C, 25 W
(
D
(D
4
CH2Cl2, (4 Â 10À3 M), HATU (3.5 equiv), DIEA (4 equiv), 4 °C for 1 h, then
rt for 6 h
(
D
(D
5
CH2Cl2, (4 Â 10À3 M), HATU (1.5 equiv), DIEA (2 equiv), microwave,
2 Â 10 min, 75 °C, 25 W
(
D
(D
6
CH2Cl2, (4 Â 10À3 M), HATU (1.5 equiv), DIEA (2 equiv), microwave,
2 Â 10 min, 75 °C, 25 W
(
D
(D
7
CH2Cl2, (4 Â 10À3 M), HATU (1.5 equiv), DIEA (2 equiv), microwave,
2 Â 10 min, 75 °C, 25 W
(
D
(D
8
CH2Cl2, (4 Â 10À3 M), HATU (1.5 equiv), DIEA (2 equiv), microwave,
2 Â 10 min, 75 °C, 25 W
(
D
(D
9
CH2Cl2, (4 Â 10À3 M), HATU (1.5 equiv), DIEA (2 equiv), microwave,
2 Â 10 min, 75 °C, 25 W
(
D
(D
10
11
12
13
14
CH2Cl2, (4 Â 10À3 M), HATU (1.5 equiv), DIEA (2 equiv), microwave,
2 Â 10 min, 75 °C, 25 W
(
D
(D
D
)-Pro-OH 8
CH2Cl2/DMF 1/1 (4 Â 10À3 M), HATU (1.5 equiv), DIEA (2 equiv),
microwave, 2 Â 10 min, 75 °C, 25 W
D
)-Pro-) 19
H-Ala-Pro-Phe-(
H-Ala-Val-Phe-(
D
)-Val-OH 9
CH2Cl2/DMF 1/1 (4 Â 10À3 M), HATU (1.5 equiv), DIEA (2 equiv),
microwave, 2 Â 10 min, 75 °C, 25 W
cyclo(Ala-Pro-Phe-(D)-Val-) 20
D
)-Trp-OH 10
DMF (10À5 M), HATU (3.5 equiv), DIEA (4 equiv), microwave, 2 Â 10 min,
75 °C, 25 W
cyclo(Ala-Val-Phe-Pro-) 21
cyclo(Ala-Val-Phe-Trp-) 22
H-Ala-Val-Phe-Pro-OH 11
CH2Cl2/DMF 1/1 (4 Â 10À3 M), HATU (3.5 equiv), DIEA (4 equiv),
microwave, 2 Â 10 min, 75 °C, 25 W
a
Yields of isolated product.
Linear peptide 1 was present in the crude in about 50% (HPLC analysis).
b
present in the crude comparing with rt reaction). After medium
pressure chromatography on a reverse phase cartridge, cyclopep-
tide 12 was isolated in 45% yield (entry 2 in Table 1).
under the same conditions giving a mixture of starting material,
cyclopeptide 12 and several oligomers (entry 4 in Table 1). When
the cyclisation was carried out at 75 °C under conventional heating
(sealed tube, 4 Â 10À3 M concentration) a strong influence of the
solvent was observed. In CH2Cl2 (or CHCl3), compound 12 was
formed in very small amount, while in DMF a result comparable
to microwave irradiation was obtained after 3 h of heating (36%,
isolated yields of 12).12
Using the millimolar as the limit concentration, we repeated the
microwave cyclisation on tetrapeptides 2–713 obtaining always
cyclic compounds 13–18 in high purity and yields. Moreover, other
peptides without the Ahoda amino acid (8–10), were submitted to
the microwave assisted head-to-tail cyclisation conditions, giving
the desired cycles 19–21 in acceptable yields (entries 11–13 in
Moreover, the cyclisation was repeated in dichloromethane,
still at 10À3 M concentration obtaining a promising HPLC/ESMS
layout (Fig 1). Cyclopeptide 12 was isolated in 43% yield after chro-
matography (entry 3 in Table 1) allowing us to reduce the potential
inconvenience associated with the use of large amounts of DMF
and to cut down on HATU and DIEA up to 1.5 and 2 equiv, respec-
tively.11 However, these seem to be the best conditions to run this
kind of cyclisation since further attempts to reduce the amount of
solvent employed gave worse results in terms of crude composi-
tion and cyclopeptide yields. In order to give credit to microwaves
for this result, compound 1 was stirred at room temperature for 6 h
Figure 1. Comparison between HPLC/ESMS profiles of the crude reaction mixture: (A) standard high dilution conditions (entry 1 in Table 1); (B) microwave dielectric heating.

E. Cini et al. / Tetrahedron Letters 50 (2009) 7159–7161
7161
4. Byk, G.; Cohen-Ohana, M.; Raichman, D. Biopolymers 2006, 84, 274–282;
Rajamohan Reddy, P.; Balraju, V.; Madhavan, G. R.; Banerji, B.; Iqbal, J.
Tetrahedron Lett. 2003, 44, 353–356.
Table 1). In some cases the solvent was changed from CH2Cl2 to
DMF (or a mixture CH2Cl2/DMF), as the starting peptide was not
soluble in CH2Cl2 alone at millimolar concentration. The only lim-
5. Davies, J. S. J. Pept. Sci. 2003, 9, 471–501.
6. Sewald, N.; Jakubke, H.-D. Peptides: Chemistry and Biology; Wiley-VCH:
Weinheim, 2002.
7. Lampariello, L. R.; Piras, D.; Rodriquez, M.; Taddei, M. J. Org. Chem. 2003, 68,
7893–7895; Falorni, M.; Giacomelli, G.; Nieddu, F.; Taddei, M. Tetrahedron Lett.
1997, 38, 4663–4666.
itation was (as expected) the presence of one
D-amino acid in the
sequence. In fact, all -series amino acid sequence, such as tetra-
L
peptide 11, did not cyclise under described conditions (entry 14
in Table 1). Analogously no results were obtained even at higher
dilution.
In summary we found that it is possible to enhance the head-to-
tail cyclisation of tetra- (and higher oligo-) peptides under con-
trolled microwave dielectric heating shortening the time required
for the reaction, decreasing the amount of solvent and streamlining
the procedure for work-up and isolation.
8. Petrella, A.; D’Acunto, C. W.; Rodriquez, M.; Festa, M.; Tosco, A.; Bruno, I.;
Terracciano, S.; Taddei, M.; Gomez-Paloma, L. Eur. J. Cancer 2008, 44, 740–749;
Rodriquez, M.; Terracciano, S.; Cini, E.; Settembrini, G.; Bruno, I.; Bifulco, G.;
Taddei, M.; Gomez-Paloma, L. Angew. Chem., Int. Ed. 2006, 45, 423–427; Gomez-
Paloma, L.; Bruno, I.; Cini, E.; Khochbin, S.; Rodriquez, M.; Taddei, M.;
Terracciano, S.; Sadoul, K. ChemMedChem 2007, 2, 1511–1519.
9. The synthesis was performed using an automated microwave peptide
synthesizer (Liberty from CEM Corporation). The 2-chlorotrityl resin was
loaded with the first amino acid in DMF with a double coupling protocol at
23 W and 75 °C. Fmoc deprotection and HBTU/HOBT mediated couplings with
the other amino acids were both carried out in 5 min at 23 W and 75 °C. The
last amino acid introduced was N-FmocAhoda(OTBDMS) (Ahoda: 2-amino-9-
hydroxy-8-oxodecanoic acid). The resin was removed from the microwave
synthesizer and the tetrapeptide was cleaved from the support using AcOH/
TFE/DCM for 3 h at rt.
Acknowledgments
This work was financially supported by MIUR (Rome), within
PRIN project 2006.
10. Cyclic tetrapeptides are sufficiently small to be considered ‘druglike’ and have
been used in the search for novel bioactive molecules. Unfortunately their use
is limited by the short supply of the natural ones and by the difficulties in
synthesizing them: Norgren, A. S.; Búttner, F.; Prabpai, S.; Kongsaeree, P.;
Arvidsson, P. R. J. Org. Chem. 2006, 71, 6814; Cavelier-Frontin, F.; Pèpe, G.;
Verducci, J.; Siri, D.; Jacquier, R. J. Am. Chem. Soc. 1992, 114, 8885.
11. The crude product obtained form resin cleavage was dissolved in dry CH2Cl2
(4 Â 10À3 M) and to this solution, cooled to 0 °C, HATU (1.5 equiv) and DIEA
References and notes
1. Cemazar, M.; Craik, D. J. J. Pept. Sci. 2008, 14, 683–689. and references cited
therein; Sabatino, G.; Papini, A. M. Curr. Opin. Drug Disc. Dev. 2008, 11, 762–
770; Murray, J. K.; Gellman, S. H. Nat. Protocols 2007, 2, 624–631; See also:
Katritzky, A. R.; Haase, D. N.; Johnson, J. V.; Chung, A. J. Org. Chem. 2009, 74,
2028–2032.
(2 equiv) were added. The vial was inserted in the cavity of
a Discover
synthesizer (CEM Corporation) and heated at 75 °C (25 W power, max internal
pressure 100 psi) for two cycles of 10 min each (with a no irradiation interval
of 2 min). The solvent was evaporated and the product isolated by preparative
HPLC (Column Phenomenex C18, flow 1 mL/min, 40 °C, Solvent A: water with
0.1% TFA. Solvent B: MeCN. Gradien from 95/5 A/B to 0/100 A/B in 10 min).
2. Rizzolo, F.; Sabatino, G.; Chelli, M.; Rovero, P.; Papini, A. M. Int. J. Pept. Res. Ther.
2007, 13, 203–208.
3. Galanis, A. S.; Albericio, F.; Grøtli, M. Biopolymers 2008, 92, 23–34; Van Dijk, M.;
Mustafa, K.; Dechesne, A. C.; Van Nostrum, C. F.; Hennik, W. E.; Rijkers, D. T. S.;
Liskamp, R. M. J. Biomacromolecules 2007, 8, 327–330; Monroc, S.; Feliu, L.;
Planas, M.; Bardají, E. Synlett 2006, 1311–1314; Campiglia, P.; Gomez-
Monterrey, I.; Longobardo, L.; Lama, T.; Novellino, E.; Grieco, P. Tetrahedron
Lett. 2004, 45, 1453–1456; Grieco, P.; Campiglia, P.; Gomez-Monterrey, I.;
Lama, T.; Novellino, E. Synlett 2003, 2216–2218.
12. For
a critical comparison between microwaves and conventional heating
technologies see: Bacsa, B.; Horváti, K.; Bõsze, S.; Andreae, F.; Kappe, C. O. J. Org.
Chem. 2008, 73, 7532–7541.
13. All linear peptides were prepared using the automated microwave peptide
synthesizer.