Synthesis of reversible cyclic peptides

Article abstract of DOI:10.1016/j.tetlet.2004.07.041

Reversible cyclic peptides were synthesized by introducing a disulfide bond into the backbone of a peptide containing alternating d/l amino acids. The peptides exist in two distinct conformations (linear/cyclic) that are controlled by reduction/oxidation of the disulfide bond. A novel approach for the synthesis of cyclic peptides that can exist in either linear or cyclized conformations is described. Synthesis of the peptides was achieved via a modified solid phase methodology. The reversible linear/cyclized (i.e., open/closed) states are controlled via the reduction/oxidation of a disulfide bond incorporated into the backbone of the peptide chain.

Full text of DOI:10.1016/j.tetlet.2004.07.041

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 6795–6798
Synthesis of reversible cyclic peptides
Alfonso Ortiz-Acevedo and Gregg R. Dieckmann*
Department of Chemistry, The University of Texas at Dallas, 2601 North Floyd Road, Richardson, TX 75080, USA
Received 21 May 2004;revised 30 June 2004;accepted 2 July 2004
Available online 28 July 2004
Abstract—A novel approach for the synthesis of cyclic peptides that can exist in either linear or cyclized conformations is described.
Synthesis of the peptides was achieved via a modified solid phase methodology. The reversible linear/cyclized (i.e., open/closed)
states are controlled via the reduction/oxidation of a disulfide bond incorporated into the backbone of the peptide chain.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The use of biological macromolecules, such as DNA,
polysaccharides, and polypeptides, to modify the prop-
erties of nanoscale particles has provided notable suc-
cess and generated increased interest in the interface
between biology and nanotechnology.1–3 Recently we
have designed/synthesized amphiphilic helical peptides
that noncovalently bind single-walled carbon nanotubes
(SWNTs) and effectively solubilize SWNTs in water,
yielding unbundled, individual SWNTs several microns
in length.4,5 One property that we would like to improve
is the affinity of peptides for the SWNTs. The utility of
peptide-wrapped nanotubes should increase significantly
if the peptides could be stabilized in a nanotube-bound
state. We hypothesize this could be achieved by peptides
designed to wrap around the circumference of a SWNT
and then form head-to-tail covalent attachments, yield-
ing closed rings on the nanotube. Toward that end, we
have designed and synthesized a new class of peptides,
called reversible cyclic peptides (RCPs), which have
the ability to switch between a linear (open) state and
a cyclic (closed) state.
The design of the RCPs is based on a specific class of
cyclic peptides, which contain equal numbers of L- and
D-amino acids arranged in an alternating L/D se-
quence.1–3,6,7 L/D-peptides place all amino acid side
chains on one face of the backbone, verses all L- or D-
peptides where side chains radiate from alternating
faces. In L/D-peptides, side chain sterics encourage a
ring-like conformation with all side chains on the out-
side surface of the backbone. Although Cys side chains
have been used previously to induce peptide cycliza-
tion,1,3 the unique approach presented here involves
the introduction of two thiols via modifications to the
N- and C-termini of a linear L/D-peptide. The thiols
are incorporated into the peptide backbone, thus facili-
tating reversible peptide cyclization via formation of a
disulfide bond, while allowing the adoption of backbone
dihedral angles appropriate for an unstrained cyclic con-
formation (not possible if a Cys-Cys side chain disulfide
were used).
Abbreviations: Reversible cyclic peptide (RCP);9-Fluorenylmethoxy-
carbonyl (Fmoc);Dimethyl sulfoxide (DMSO);Dimethylformamide
(DMF);Diisopropylethylamine (DIEA);1H-Benzotriazolium, 1-[bis-
(dimethylamino) methylene]-6-chloro-, 3-oxide, hexafluorophos-
phate(1-) (9CI) (HCTU);Dichloromethane (DCM);Ethyl acetate
(EtOAc);Thin layer chromatography (TLC);Trifluoroacetic acid
(TFA);Solid phase peptide synthesis (SPPS);2-(1H-Benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU);N-Hy-
droxybenzotriazole H2O (HOBt); N-Methylpyrrolidone (NMP); b-
Mercaptoethanol (BME);Electrospray ionization-mass spectrometry
(ESI-MS);Tris(2-carboxyethyl)-phosphine hydrochloride (TCEP);
5,50-Dithiobis(2-nitrobenzoic acid) (DTNB).
RCPs were synthesized using a modified solid phase
methodology. The modified C-terminus of each RCP
was generated by coupling an orthogonally protected
N-a-Fmoc-L-glutamic acid c-tert-butyl ester (Fmoc-
Glu(t-Bu)-OH) to the amino end of a cysteamine. This
orientation provides a thiol directed along the peptide
backbone that can be utilized for disulfide bond forma-
tion. The N-terminal thiol was generated by coupling
2-mercaptoacetic acid to the free amine of the extended
peptide backbone. Upon cleavage of the peptide from
the resin and reduction of all disulfides, RCPs were
produced containing two free thiols oriented in an
Keywords: Cyclic peptides;Disulfide bond;Solid phase synthesis.
*
Corresponding author. Tel.: +1-972-883-2903;fax: +1-972-883-
2925;e-mail: dieckgr@utdallas.edu
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.07.041

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A. Ortiz-Acevedo, G. R. Dieckmann / Tetrahedron Letters 45 (2004) 6795–6798
the ESI-MS of IV, verified the identities of III and
IV.13,14
The molecule IV was then used to synthesize RCPs of
desired lengths utilizing established Fmoc-based SPPS
protocols (Scheme 2). Two peptides of 11 and 15 amino
acids were synthesized in the following way to yield
RCPs RC5 and RC7 (Fig. 1). Compound IV was loaded
on amide resin using a standard solid phase coupling
procedure involving HBTU, HOBt, and DIEA in
NMP, allowing the coupling reaction to proceed over-
night. Peptides of the desired lengths were then synthe-
sized by automated-SPPS utilizing amino acids of
alternating chirality. Removal of the Fmoc group from
the N-terminal amino acid with a solution of 20% pip-
eridine in DMF afforded the free amine V that was sub-
sequently coupled with a dithio diglycolic acid to yield
VI. Compound VI was washed with BME and several
DCM washes prior to being dried under high vacuum.
The BME washes were incorporated to remove the
N-terminal thiol protection via disulfide exchange with
BME and to cleave any peptide that was not directly
coupled to the resin. Allowing these peptides to continue
into the next step would generate an undesired glutamic
acid derivative of the target peptide. Cleavage of the
peptides from the solid support was accomplished using
a standard 10mL cleavage cocktail of TFA, thioanisole,
ethanedithiol, and anisole (90:5:3:2) yielding the crude
product VII. Purification of VII by HPLC yielded the
pure product (overall yield = 6.3% for RC5, 5.4% for
RC7) displaying the appropriate molecular weight of
the linear (reduced) peptide, m/z = 1310 [reduced
RC5 + H]+ and m/z = 1710 [reduced RC7 + H]+, as indi-
cated by ESI-MS analysis.
Scheme 1. Reaction conditions: (a) DMSO/H2O;(b) HCTU, DIEA in
DMF;(c) TFA/DCM;overall yield for steps a–c: 91.3%.
appropriate way to form a closed ring upon intramolec-
ular disulfide formation.
The C-terminal thiol of each RCP was introduced via a
glutamic acid residue that has been coupled to a cyste-
amine molecule (Scheme 1). Cystamine dihydrochloride
(II), the disulfide of cysteamine hydrochloride (I), was
generated by oxidizing I with DMSO in a minimal
amount of water with stirring overnight.8–10 The solu-
tion was monitored for the presence of free thiol with
the Ellman test.11,12 Once a negative test was achieved,
cold absolute ethanol was slowly added to precipitate
the product (II), which was collected on a glass fritted
funnel and dried under high vacuum. A solution of II
in DMF was then added to a round bottom flask con-
taining Fmoc-Glu(t-Bu)-OH (2.2equiv), DIEA, and
HCTU (2.2equiv) in DMF and the reaction was allowed
to stir overnight at room temperature, generating the
side chain protected derivative (III).
Characterization of the solution behavior of the RCPs
provided verification of the intended design. All RCP
solutions were prepared using degassed deionized
water to prevent oxidation of the free thiols. The peptide
concentration of RCP stock solutions was determined
by utilizing the tyrosine absorbance (e = 1420Mꢀ1 cmꢀ1
at 275nm). The Ellman test was used to determine
the free thiol concentration of stock solutions, as
well as to monitor the oxidation of experimental
solutions.11
Isolation of III was achieved by a series of liquid–liquid
extractions followed by column chromatography. The
reaction mixture was dissolved in a 3 · volume of
DCM, and excess reagents were extracted by washing
with a 5% sodium bicarbonate solution followed by sev-
eral water washes. Excess DCM was removed by a slow
air stream over the solution, and the impure product
concentrate was loaded onto a silica column and eluted
using 1:1 DCM–EtOAc. TLC and HPLC monitoring of
column fractions confirmed the presence of III in early
fractions. Product fractions were consolidated and the
elution solvent removed under reduced pressure, gener-
ating a white solid. The solid (III) was redissolved in
DCM and crystallized from DCM/hexanes. The desired
bis-(N-a-Fmoc-L-glutamic acid a-amidoethyl) disulfide
(IV) was generated by stirring a solution of III in 50%
TFA in DCM for 2h. Monitoring the side chain depro-
tection by HPLC indicated the reaction was complete
after 1h. The final product (IV) was isolated in 91.3%
overall yield as a solid by removing the TFA and
DCM under reduced pressure. Comparisons of the
NMR spectra (1H and 13C) of III and IV, as well as
Direct transitions from the linear to the cyclized states of
RC5 and RC7 were observed by shifts in the HPLC
retention times of 100lM peptide solutions that were
oxidized by atmospheric oxygen (Fig. 2). Aliquots of
the HPLC peaks corresponding to the oxidized RCPs
were submitted for mass spectral analysis. The molecu-
lar ion for each cyclized peptide appeared at 2mass units
less than that of the corresponding linear RCP mono-
mer, m/z = 1308 [oxidized RC5 + H]+ and m/z = 1708
[oxidized RC7 + H]+ (Fig. 2). The reversibility of the
proposed linear/cyclized states of the peptides was ob-
served when solutions of cyclized RCP monomers were
treated with the reducing agent TCEP to regenerate the
linear state. HPLC traces of the solutions after TCEP
addition (data not shown) clearly showed the peptides
eluting at the retention times pertaining to the linear
states.

A. Ortiz-Acevedo, G. R. Dieckmann / Tetrahedron Letters 45 (2004) 6795–6798
6797
Scheme 2. Reaction conditions: (a) HBTU, HOBt and DIEA in NMP;(b) standard SPPS protocols using alternating L- and D-amino acids;(c) 20%
piperidine in DMF;(d) HBTU, HOBt, DIEA, and dithio diglycolic acid in NMP;(e) BME;(f) thioanisole, ethanedithiol, and anisole in TFA;overall
yield for steps a–f: 6.3% (RC5), 5.4% (RC7).
Figure 1. (a) Sequences of RC5 (11 amino acids) and RC7 (15 amino acids);(b) molecular model 15 of RC5 demonstrating the cyclized conformation
of the peptide. The final model highlights the placement of amino acid side chains on the outside of the ring, as well as the orientation of the
backbone carbonyl and NH groups perpendicular to the ring plane. Color scheme: green, carbon;red, oxygen;blue, nitrogen;white, hydrogen;
yellow, sulfur.

6798
A. Ortiz-Acevedo, G. R. Dieckmann / Tetrahedron Letters 45 (2004) 6795–6798
Figure 2. Chromatograms of 100lM RCP solutions displaying the presence of both linear and cyclized conformations of the peptides in solution.
9. Mills, E. J.;Bogert, M. T. J. Am. Chem. Soc. 1940, 62,
1173–1180.
10. Yiannios, C. N.;Karabinos, J. V. J. Org. Chem. 1963,
28(11), 3246–3248.
11. Ellman, G. L.;Courtney, K. D.;Andres, V.;Featherstone,
R. F. Biochem. Pharm. 1961, 7, 88–90.
In summary, we have described the design and synthesis
of a new class of cyclic peptide incorporating a back-
bone disulfide bond to introduce reversibility of linear/
cyclized states. Synthesis was achieved by modifying
the N- and C-termini of the peptide backbones, intro-
ducing thiols at both ends, which then form a disulfide
bond through oxidation. Characterization of the pep-
tides by HPLC and MS analysis verified the design of
the RCPs, which can be used in their linear or cyclized
conformations.
12. Free thiol concentration was determined using the Ellman
test. A stock solution of DTNB was prepared by dissolv-
ing 20mg of DTNB in 5mL of 100mM potassium
phosphate buffer at pH8. Analytical solutions were
prepared by mixing 100lL of sample with 100lL of
DTNB stock solution and 2.8mL of 100mM potassium
phosphate buffer at pH8. A reference solution was also
prepared incorporating 100lL of water in place of the
sample solution. After 15min the absorbance (k = 410nm)
of the analytical solutions was measured using a 1cm
cuvette. Free thiol concentration (mol/L) was determined
by the equation;[SH] = ( Asample ꢀ Areference)/136,50.
13. A sample of compound III was analyzed by NMR and
Acknowledgements
We would like to thank Dr. Zoltan Kovacs and Dr.
Mark Woods for helpful discussions. Support for this
research by the MIRROR Federal Initiative (A.O.-A.)
and the American Chemical Society Petroleum Research
Fund (PRF# 40835-AC3;G.R.D.) is gratefully
acknowledged.
1
ESI prior to being converted to IV. H NMR (270MHz,
DMSO-d6): d 8.09 (2H, t, J = 5Hz), 7.88 (4H, d, J = 8Hz),
7.72 (4H, m), 7.50 (2H, J = 7Hz), 7.41 (4H, t, J = 7Hz),
7.32 (4H, t, J = 7 Hz), 4.25 (6H, m), 3.96 (2H, m), 2.77
(4H, t, J = 6Hz), 2.22 (4H, t, J = 7Hz), 1.80 (4H, m), 1.37
(18H, s); 13C NMR (270MHz, DMSO-d6): d 172.2, 172.0,
156.5, 144.4, 144.3, 141.3, 128.2, 127.6, 125.9, 120.7, 80.3,
66.2, 54.5, 47.2, 38.4, 37.5, 31.9, 28.3, 27.9;ESI-MS ( m/z)
[M + H]+ = 967 (MWcalcd = 966gmolꢀ1).
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1
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NMR (270MHz, DMSO-d6): d 8.11 (2H, t, J = 5Hz), 7.85
(4H, d, J = 8Hz), 7.70 (4H, m), 7.50 (2H, J = 8Hz), 7.40
(4H, t, J = 7Hz), 7.30 (4H, t, J = 7Hz), 3.95 (4H, m), 3.37
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(4H, m); 13C NMR (270MHz, DMSO-d6): d 174.5, 172.3,
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54.6, 47.2, 38.4, 37.5, 30.8, 27.7;ESI-MS
[M + H]+ = 855 (MWcalcd = 854gmolꢀ1).
(
m/z)
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w = ꢀ156ꢁ.
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Proteins 1988, 4,