Synthesis of trifunctional cyclo-β-tripeptide templates

Article abstract of DOI:10.3762/bjoc.8.180

The concept of template-assembled synthetic proteins (TASP) describes a central scaffold that predefines the three dimensional structure for diverse molecules linked to this platform. Cyclic β-tripeptides are interesting candidates for use as templates due to their conformationally defined structure, stability to enzymatic degradation, and ability to form intermolecular stacked tubular structures. To validate the applicability of cyclic β-tripeptides within the TASP concept, an efficient synthesis of the cyclopeptide with orthogonal functionalization of the side chains is desired. A solid-phase-supported route with on-resin cyclization is described, employing the aryl hydrazide linker cleavable by oxidation. An orthogonal protection-group strategy allows functionalization of the central cyclic β-tripeptide with up to three different peptide fragments or fluorescent labels.

Full text of DOI:10.3762/bjoc.8.180

Synthesis of trifunctional cyclo-β-tripeptide
templates
Frank Stein1, Tahir Mehmood1,2, Tilman Plass1, Javid H. Zaidi2
and Ulf Diederichsen*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 1576–1583.
1Institute for Organic and Biomolecular Chemistry, University of
Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germany and
2Department of Chemistry, Quaid-i-Azam University, Islamabad,
45320, Pakistan
doi:10.3762/bjoc.8.180
Received: 15 June 2012
Accepted: 20 August 2012
Published: 19 September 2012
Email:
Ulf Diederichsen* - udieder@gwdg.de
Associate Editor: S. Flitsch
* Corresponding author
© 2012 Stein et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
β-amino acids; cyclic β-tripeptide scaffold; orthogonal protection
groups; peptide synthesis; template-assembled synthetic proteins
(TASP)
Abstract
The concept of template-assembled synthetic proteins (TASP) describes a central scaffold that predefines the three dimensional
structure for diverse molecules linked to this platform. Cyclic β-tripeptides are interesting candidates for use as templates due to
their conformationally defined structure, stability to enzymatic degradation, and ability to form intermolecular stacked tubular
structures. To validate the applicability of cyclic β-tripeptides within the TASP concept, an efficient synthesis of the cyclopeptide
with orthogonal functionalization of the side chains is desired. A solid-phase-supported route with on-resin cyclization is described,
employing the aryl hydrazide linker cleavable by oxidation. An orthogonal protection-group strategy allows functionalization of the
central cyclic β-tripeptide with up to three different peptide fragments or fluorescent labels.
Introduction
Cyclic β-tripeptides form structurally well-defined secondary ation [3,4]. These properties make cyclic β-tripeptides interest-
structures with the potential for alignment of the rings to form ing candidates for the concept of template-assembled synthetic
intermolecular aggregates [1]. Due to the unidirectional align- proteins (TASP) [5,6]. The TASP concept describes a central
ment of the carbonyl groups and the flattened-ring con- scaffolding molecule directing all further attached molecules
formation, cyclic β-tripeptides form assemblies of stacked rings into a spatially predefined structure. Cyclic β-tripeptides are
through hydrogen bonding [2]. Furthermore, they are exceed- suitable for use as a central scaffold that carries different kinds
ingly stable against proteolytic cleavage and enzymatic degrad- of molecules and functionalities to direct them into a trigonal
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Results and Discussion
planar assembly. This idea was already used for the synthesis of
a C3-symmetric ligand for the immune response receptor CD40 General strategy
[7]. However, the synthetic route to these kinds of molecules is To equip the cyclic β-tripeptide with three different functional
demanding due to solution-phase synthesis of the β-tripeptides units, each amino acid residue needs to be specifically
and their final cyclization with low efficiency [8,9]. Moreover, addressed. Furthermore, all three amino acids should have side
only homofunctionalized cyclic β-tripeptides have been chains long enough to avoid steric hindrance between the
described so far [9,10]. To further investigate and exploit the moieties and the core. Thus, homo-β-lysine was chosen as the
potential of this class of circular peptides, a synthetic route underlying amino acid to build up the scaffold allowing side-
should fulfil the requirements of (i) fast synthetic access and (ii) chain functionalization by amide bond formation. To protect the
the possibility to attach different molecules on each side chain lysine side chain for selective and orthogonal amide-bond for-
of the central scaffold. Here we report a new effective synthesis mation following the solid-phase peptide synthesis (SPPS), the
for cyclic β-tripeptides on a solid support, employing the oxi- protection groups fluorenylmethoxycarbonyl (Fmoc) and
dation-labile aryl hydrazide linker [11]. We also describe an carbobenzyloxy (Cbz) were applied. Alteration of the amine in
orthogonal protection-group strategy to synthesize a trifunc- the third β-homolysine side chain to an azide [12-14] employing
tional cyclic β-tripeptide that has the potential of forming inter- the Wong azidation [15] enables Huisgen [3 + 2]-cycloaddition
molecular hydrogen-bonded stacks (Figure 1).
[16] as an orthogonal coupling method. In order to build up the
peptide sequence in the presence of Fmoc, Cbz and the azide,
an acid-labile protecting group was required for temporary
protection of the primary α-amino group. Therefore, tert-buty-
loxycarbonyl (Boc) protection was used to mask all α-amino
groups during solid-phase synthesis of the tripeptide.
Synthesis of the cyclo-β-peptide scaffold
The Boc protected β-amino acid building blocks for SPPS of the
cyclic β-tripeptide were prepared from the respective β-amino
acids by Arndt–Eistert homologation [17-19]. The β-amino
acids were transformed into the respective diazoketones with
isobutyl chloroformate, triethylamine and diazomethane. The
ketones were further converted into the β-amino acids by Wolff
rearrangement using silver benzoate and water as a nucleophile
[19-23] yielding the homo-β-lysine derivatives 1 and 2 along-
side the azide β-amino acid 3 (Figure 2).
Figure 1: Trifunctional cyclic β-tripeptide forming an intermolecular
stack of rings by backbone hydrogen bonding.
In previous studies, synthesis of the cyclic β-tripeptide scaffold
was provided by cyclization of the linear β-tripeptide obtained
Figure 2: β-Amino acids 1–3 with orthogonal side-chain protection obtained by Arndt–Eistert homologation followed by Wolff rearrangement; cyclo-β-
tripeptide template 4 as obtained by coupling of amino acids 1–3 followed by cyclization.
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Scheme 1: Synthesis of cyclic peptides employing the oxidation-labile aryl hydrazide linker [11,24].
by solution-phase chemistry [8,9]. Purification by chromatogra- Functionalization of the β-tripeptide template
phy is required after each coupling step, and the final cycliza- The cyclic β-tripeptide template 4 has the potential for orthog-
tion reaction often results in poor yields. Herein, an alternative onal functionalization at the side chains with up to three
approach is described based on SPPS followed by on-resin different moieties, by successive amide-bond formation and by
cyclization. Recently, Waldmann et al. discovered an on-resin employing the Huisgen [3 + 2]-cycloaddition. Aggregation by
head-to-tail cyclization based on a Boc protocol using the oxi- stacking of the functionalized peptide rings will further provide
dation-labile aryl hydrazide linker [24] and cleavage from the a higher density of organized recognition motifs and labels. As
resin under simultaneous cyclization [11]. This method was a proof-of-concept, the successive coupling of a fluorophore
adapted for the synthesis of the cyclic β-tripeptide 4 (Figure 2). 5(6)-tetramethylcarboxyrhodamine 5 (TAMRA-COOH) and a
cell-penetrating peptide to the template 4 is reported, as well as
The synthesis of the cyclic β-tripeptide was performed the functionalization with the nucleobase recognition units
according to Scheme 1 by using the commercially available thymine-1-yl acetic acid (6) and (N4-benzyloxycarbonyl)cyto-
4-Fmoc-hydrazinobenzoyl AM NovaGel resin from Merck sine-1-yl acetic acid (7) (Figure 3). In all cases, the template
Biosciences. After coupling of the three β-amino acids by using contains a third option for functionalization by covalent attach-
the standard Boc-protocol, the hydrazide linker was oxidized to ment of molecules through [3+2]-cycloaddition under mild
generate nitrogen as a good leaving group. Nucleophilic attack conditions.
of the N-terminal amino group at the activated carbonyl group
provided cleavage of the cyclized β-tripeptide from the resin. The template approach allows for the preparation of constructs
Purification did not require any chromatography since organiza- that, e.g., combine biological activity, fluorescence labelling
tion by intermolecular hydrogen bonding resulted in decreased and cell-penetrating or cell-directing units in one molecule to be
solubility and precipitation of the β-tripeptide template 4 from used for in vivo studies. The defined spatial organization of
methanol.
recognition units such as nucleobases on the template provides
Figure 3: Functional units provided as carboxylic acids for the attachment to the cyclo-β-peptide: 5(6)-tetramethylcarboxyrhodamine 5, thymine 6,
Cbz protected cytosine with a carboxylic acid linker 7 [25], and fully protected peptide sequence of penetratin 8 [26].
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Scheme 2: Functionalization of the cyclic β-tripeptide 4.
a specific hydrogen-bonding network orthogonal to the peptide trating peptide penetratin 8 [26] with the homolysine side chain
ring aggregation, which will be of value for aggregate forma- of the cyclic β-tripeptide 15 was accomplished by HOBt and
tion and molecular architectures defined by a three-dimensional HBTU activation in solution using a 10-fold access of pene-
network of hydrogen bonds.
tratin. The β-peptide template 16 functionalized at two side
chains was obtained, purified by HPLC, and characterized by
Selective release of the first amino group was achieved by mass spectrometry.
removal of the Cbz protecting group using TFA and m-cresol
(95:5 v/v). The first functional unit of a recognition moiety was Conclusion
attached to the template as a carboxylic acid by amide-bond for- Cyclic β-tripeptides provide an interesting platform for the
mation using PyBOP and DIEA, or PyBrOP and DIEA for concurrent arrangement of side-chain bound functionalities in
coupling (Scheme 2). Exemplarily, TAMRA-COOH (5) and the combination with the ability of intermolecular tubular aggrega-
nucleobase moieties thymine-1-yl acetic acid (6) and (N4- tion of the rings. An easy and fast approach towards cyclic
benzyloxycarbonyl)cytosine-1-yl acetic acid (7) were attached β-tripeptides was presented. The tripeptide scaffold was assem-
to the cyclo-β-peptide yielding monofunctionalized templates bled completely on a solid support, thereby saving purification
14, 10 and 12 (Figure 4).
steps following amino acid coupling. Moreover, cleaving of the
peptide from the resin and cyclization was performed in a single
The second amino group was Fmoc-deprotected with 20% step employing the oxidation-labile aryl hydrazide linker [11].
piperidine in DMF. Coupling of the fully protected cell-pene- Together with a convenient and efficient purification procedure,
Figure 4: Cyclic β-tripeptides varying in two side-chain functionalities and containing an additional azide moiety, e.g., for ligation with click chemistry.
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Beilstein J. Org. Chem. 2012, 8, 1576–1583.
this method is beneficial with respect to yield and speed of the ical columns and 10 mL/min for preparative columns. All crude
synthesis. Further, an orthogonal protection strategy was intro- samples were dissolved in methanol or acetonitrile and filtered
duced to equip the β-peptide template with three different func- prior to use. Electrospray ionization (ESI) mass spectra were
tional molecules allowing testing of the cyclic β-tripeptides as a obtained on a Finnigan LCQ instrument. High-resolution mass
scaffold for the defined orientation of functional units and the spectra (HRMS) were obtained on a Bruker Apex IV FT-ICR-
combination of up to three modules in one molecule. The MS instrument. 1H NMR and 13C NMR spectra of samples
advantages of using the β-peptide template within the TASP dissolved in DMSO-d6, CDCl3 or acetone-d6 were recorded on
concept are the spatially defined and rigid structure, with a high a Varian Unity 300 (300 MHz) spectrometer. Residual solvent
stability against enzymatic degradation, and their ability to form proton signals were used as an internal standard.
intermolecular staples by backbone hydrogen bonding. This
might be of special advantage to target or imitate multivalent Synthesis of compounds
and/or cooperative processes.
(S)-tert-Butyl(7-azido-1-diazo-2-oxoheptan-3-yl)carbamate.
(S)-6-Azido-2-(tert-butoxycarbonylamino)hexanoic acid
As an initial effort, we equipped the cyclic β-tripeptide with a (7.38 g, 27.1 mmol, 1.00 equiv) was dissolved in dry THF
fluorophore and a protected cell-penetrating peptide, which can (108 mL) under an argon atmosphere. The solution was cooled
be further functionalized with any biologically active molecule at −15 °C and dry triethylamine (4.19 mL, 29.8 mmol,
bearing an alkyne. Potentially, this generates a fluorescent and 1.10 equiv) and isobutyl chloroformate (3.91 mL, 29.8 mmol,
cell-permeable drug applicable for in vivo experiments and 1.10 equiv) were slowly added and the whole mixture was
other biochemical assays. In addition, different nucleobases stirred for 45 min. The reaction mixture was warmed to 0 °C,
were linked to the central core in order to generate a and under exclusion of light an approximately 0.35 M solution
template that can form a three dimensional network of hydrogen of diazomethane (181 mL, 54.2 mmol, 2.00 equiv) in Et2O was
bonds.
added. After 15 min, the reaction mixture was stirred for an
additional 6 h at room temperature. Excess diazomethane was
decomposed by dropwise addition of AcOH. The solvent was
removed under reduced pressure, and the residue was dissolved
Experimental
General remarks
All technical solvents were distilled prior to use. The solvents in Et2O and washed with saturated solutions of NaHCO3 (3 ×
of analytical and HPLC grade were used as supplied. The 50 mL), NH4Cl (2 × 50 mL) and NaCl (1 × 50 mL). The
solvents used for the synthesis were obtained in quality puriss. combined organic layers were dried over MgSO4, filtered and
abs. from Acros Organics, Sigma Aldrich, Merck, or VWR. All concentrated under reduced pressure to furnish a yellow oil. The
chemicals were of the highest grade available and used as title compound was used for the next reaction without further
supplied. All moisture- and oxygen-sensitive reactions were purification. DC (EtOAc/n-pentane 2:3 + 1% AcOH): Rf = 0.73.
carried out under an inert gas atmosphere (nitrogen or argon).
Analytical TLC was performed on Merck TLC aluminium (S)-7-Azido-3-(tert-butoxycarbonylamino)heptanoic acid (3).
sheets silica gel 60 F254. Detection was performed under A solution of silver trifluoroacetate (219 mg, 990 µmol,
UV light (254 nm) or by dipping into a solution of ninhydrin 0.110 equiv) in triethylamine (3.79 mL, 27.0 mmol, 3.00 equiv)
(3% in ethanol) followed by heating with a heat gun. Eluents was slowly added to a solution of (S)-tert-butyl 7-azido-1-
and the appropriate Rf values are indicated. The columns for diazo-2-oxoheptan-3-yl carbamate (2.67 g, 9.00 mmol,
flash chromatography were packed with silica gel 60 from 1.00 equiv) in THF/H2O 9:1 (45.0 mL) under exclusion of light
Macherey–Nagel with a grit size of 0.063 to 0.2 mm and were at −15 °C. The reaction solution was warmed to room tempera-
run under a pressure of 1 to 1.5 bar. The substance was applied ture after 30 min and stirred for a further 12 h until completion.
as a concentrated solution or adsorbed on silica gel. Eluents are The solvent was removed under reduced pressure. The residue
indicated. Reverse-phase HPLC was performed on an Äkta was dissolved in Et2O and washed with a saturated solution of
Basic 900 from Pharmacia Biotech. UV detection was NaHCO3 (3 × 50 mL). The combined aqueous layers were acid-
performed at 215, 254 and 280 nm wavelength. The solvents ified with 2 M hydrochloric acid to pH 2–3 and were extracted
used for HPLC were of HPLC grade and degassed while being with EtOAc (5 × 50 mL). The combined organic layers were
stirred in vacuo. Demineralized Water for HPLC use was dried over MgSO4, filtered and concentrated under reduced
preprocessed by the water treatment plant “Simplicity” from pressure. The title compound was obtained after purification by
Millipore. All HPLC runs were performed by using linear gradi- flash chromatography (silica gel, 400 g, DCM/MeOH 9:1 + 3%
ents between A (0.1% aq TFA), B (0.1% TFA in methanol) or AcOH) to furnish a yellow oil (0.714 g, 2.49 mmol, 28% yield).
C (0.1% TFA in MeCN) and water (0.1% TFA) within DC (DCM/MeOH 9:1, 3% AcOH): Rf = 0.51; 1H NMR
30 minutes. Flow rates were taken as 1 mL/min for the analyt- (300 MHz, acetone-d6) δ 1.38 (s, 9H, Boc-CH3), 1.42–1.70 (m,
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Beilstein J. Org. Chem. 2012, 8, 1576–1583.
6H, γ-H2, δ-H2, ε-H2), 2.41–2.56 (m, 2H, -H2), 3.30–3.38 (t, pound was obtained after recrystallization (MeOH, 3.00 mL) to
3JH,H = 6.6 Hz, 2H, ζ-H2), 3.93 (m, 1H, β-H), 5.89 (d, 3JH,H = furnish a yellow powder (114 mg, 141 μmol, 29% yield). DC
7.2 Hz, 1H, NHBoc); ESIMS m/z: 309.2 [M + Na]+, 285.2 [M − (DCM/MeOH 9:1): Rf = 0.42; ESIMS m/z: 831.4 [M + Na]+;
H]−; HRMS: C12H22N4O4: [M + Na]+ calcd: 309.1533; found: HRMS: C44H56N8O7 [M + Na]+ calcd: 831.4164; found:
309.1534, [M − H]− calcd: 285.1568; found: 285.1565.
831.4171.
Fmoc-R(Pbf)Q(Trt)IK(Boc)IW(Boc)FQ(Trt)N(Trt)R(Pbf)R cyclo(β3-HLys(Fmoc)-β3-HLys(N3)-β3-HLys) (10). The
(Pbf)MK(Boc)W(Boc) K(Boc)K(Boc)-OH (8). The fully completely protected cyclic β-tripeptide 4 (79.4 mg, 98.0 μmol)
protected penetratin 8 was synthesized on H-Lys(Boc)-2-ClTrt was dissolved in a mixture TFA and m-cresol (95:5 v/v 5 mL)
resin (0.61 mmol/g, 163.9 mg, 0.100 mmol, 1.00 equiv) using a and stirred for 6 h. The crude peptide was precipitated with ice-
Liberty peptide synthesizer (CEM, Kamp-Lintfort, Germany) cold MTBE (3 × 13 mL) and the title compound was obtained
equipped with a Discover microwave reaction cavity (CEM). by RP-HPLC purification to give a yellow solid (43.2 mg,
Standard reagents, protocols and procedures were used for 64.0 μmol, 65% yield). RP-HPLC: tR = 23.20 min (40→100%
deprotection (20% piperidine in NMP, 210 s, 50 °C, 20 W) and B in 30 min); ESIMS m/z: 675.4 [M]+; HRMS: C26H50N8O5
coupling (HBTU/HOBt/DIEA/NMP, 300 s, 50 °C, 20 W). [M]+ calcd: 675.3977; found: 675.3974.
Double couplings were performed for arginine. No final Fmoc
deprotection step was used. After the automated peptide syn- cyclo(β3-HLys(Fmoc)-β3-HLys(N3)-β3-HLys(TAMRA)) (14).
thesis, the peptide was cleaved from the resin using a 30% solu- The cyclic β-peptide 9 (43.2 mg, 64.0 μmol, 1.00 equiv),
tion of HFIP in DCM (5.00 mL) for 45 min. Afterwards, the PyBOP (48.3 mg, 93.0 μmol, 1.45 equiv) and 5(6)-TAMRA
cleavage cocktail was filtered, the resin was washed (3 × 3 mL (40.0 mg, 93.0 μmol, 1.45 equiv) were dissolved in DMF
30% HFIP in DCM) and all phases combined. The excess (500 μL) and DIEA (32.0 μL, 186 μmol, 2.90 equiv) was added.
solvent was removed under a nitrogen stream and the crude pro- The reaction mixture was for 6 h at room temperature. The
duct was precipitated by adding MTBE (10 mL). The title com- crude peptide was precipitated with ice-cold MTBE (3 ×
pound was obtained after RP-HPLC purification to yield a 14 mL) and the title compound was obtained by RP-HPLC
white solid (117 mg, 25.6 µmol, 26% yield); RP-HPLC: tR = purification to give a pink solid (39.4 mg, 36.0 μmol, 57%
34.7 min (98→100% C in 40 min); ESIMS m/z: 4552.28 [M]−; yield). RP-HPLC: tR = 22.95→23.43 min (40→90% B in
HRMS: C245H316N34O43S4 [M]+ calcd: 4550.25; found: 30 min); ESIMS m/z: 1087.4 [M]+, 1109.4 [M + Na]+; HRMS:
4550.29.
C61H70N10O9 [M]+ calcd: 1087.5400; found: 1087.5419.
cyclo(β3-HLys(Fmoc)-β3-HLys(N3)-β3-HLys(Cbz)) (4). The cyclo(β3-HLys-β3-HLys(N3)-β3-HLys(TAMRA)) (15). The
peptide sequence H-β3-HLys(Fmoc)-β3-HLys(N3)-β3- cyclo-β-tripeptide 14 (39.4 mg, 36.0 μmol, 1.00 equiv) was
HLys(Cbz)-OH was synthesized on 4-Fmoc-hydrazinobenzoyl dissolved in a solution of 20% piperidine in DMF (500 μL) and
AM NovaGel resin (0.700 mmol/g, 1.00 g, 7.00 mmol, stirred for 3 min at 50 °C (25 W) by using a manual discover
1.00 equiv). After N-terminal Fmoc deprotection (20% piperi- SPS ultrasound peptide synthesizer. Afterwards, the crude
dine in DMF, 3.50 mL, 20 min), standard Boc protocols were peptide was precipitated with ice-cold MTBE (1 × 15 mL)
used for coupling (β-amino acid 5.00 equiv, HBTU 4.50 equiv, and the title compound was obtained after RP-HPLC purifica-
HOBt 5.00 equiv, DIEA 10.0 equiv, 2.80 mL DMF, 18 h) and tion (23.2 mg, 27.0 μmol, 74% yield). RP-HPLC: tR =
Boc cleavage (3.00 mL TFA/m-cresole–95/5 v/v, 2 × 2 min) of 14.50→15.34 min (40→95% B in 30 min); ESIMS m/z: 433.2
β-amino acids Boc-β3-HLys(Fmoc)-OH, Boc-β3-HLys(Cbz)- [M + 2H]2+, 865.4 [M]2+; HRMS: C46H60N10O7 [M]+ calcd:
OH and Boc-β3-HLys(N3)-OH. After N-terminal deprotection 865.4719; found: 865.4718.
of the last amino acid, the resin was washed with DCM (3 ×
3 mL) and the hydrazinobenzoyl linker was oxidized to the cyclo(β3 -HLys(penetratin 8)-β3 -HLys(N3 )-β3 -
acyldiazene by stirring the resin in a solution of N-bromosuc- HLys(TAMRA)) (16). The fully protected peptide penetratin 8
cinimide (0.249 g, 1.40 mmol, 2.00 equiv) and pyridine (9.00 mg, 1.98 μmol, 1.43 equiv) was dissolved in DMF
(113 μL, 1.40 mmol, 2.00 equiv) in DCM (3.50 mL) for 7 min. (30.0 μL) and DIEA (2.42 μL, 14.0 μmol, 10.0 equiv) and stock
The resin was washed with DCM (3 × 3 mL). Then, a solution solutions of HOBt and HBTU in DMF (4.16 μL, 2.08 μmol,
of DIEA (244 μL, 1.40 mmol, 2.00 equiv) in DCM (3.50 mL) 1.50 equiv) were added. After 5 min of preactivation, the solu-
was added to the resin and the reaction mixture was stirred for tion was added to the cyclic β-tripeptide 15 (1.20 mg,
48 h. The resin was filtered and washed with DCM (5 × 1.39 μmol, 1.00 equiv) and stirred under exclusion of light for
3.00 mL).The washing fractions were combined and the organic 48 h. The crude product was precipitated with ice-cold MTBE
solvent was removed under reduced pressure. The title com- (1 × 14 mL) and the title compound was obtained by RP-HPLC
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Beilstein J. Org. Chem. 2012, 8, 1576–1583.
purification to give a pink solid (1.38 mg, 0.256 μmol, 18% ESIMS m/z: 982.4 [M + Na]+; HRMS C50H61N11O9: [M + H]+
yield). For spectrometry, a small portion was deprotected by calcd: 960.4726; found: 960.4708, [M + Na]+ calcd: 982.4546;
TFA for 2 h, precipitated with ice-cold MTBE and analyzed by found: 982.4527.
mass spectrometry. RP-HPLC: tR = 31.8 min (95→100% B in
40 min). Deprotected compound: ESIMS m/z: 3314.83 Synthesis of cyclo(β3-HLysNH2-β3-HLys(N3)-β3-HLys((N4-
[M + H]+; HRMS: C165H236N44O28S [M]+ calcd: 3313.81; benzyloxycarbonyl) cytosine-1-yl acetate) (13). Cyclo(β3-
found: 3313.83.
HLys(Fmoc)-β3-HLys(N3)-β3-HLys-((N4-benzyloxycarbonyl)
cytosine-1-yl acetic acid) (12, 15.0 mg, 15.6 µmol, 1.00 equiv)
cyclo(β3-HLys(Fmoc)-β3-HLys(N3)-β3-HLys-(thymin-1-yl was dissolved in a solution of 20% piperidine in DMF
acetate)) (10). Cyclo(β3-HLys(Fmoc)-β3-HLys(N3)-β3-HLys) (1000 µL). The reaction mixture was sonicated for 35 min in an
(9, 6.24 mg, 9.25 µmol, 1.00 equiv) was dissolved in dry NMP ultrasonic cleaning bath. Then, ice-cold tert-butylmethylether
(700 µL). Thymin-1-ylacetic acid 6 (8.51 mg, 46.3 µmol, (MTBE) was added. The observed precipitate was separated by
5.01 equiv), PyBrOP (21.6 mg, 46.3 µmol, 5.01 equiv) and centrifugation (15 min, 4 °C, 4500 rpm) and the residue was
DIEA (16.1 µL, 92.5 µmol, 10.0 equiv) were added. After stir- dried in high vacuum. The title compound was obtained by
ring of the reaction mixture for 72 h at room temperature, the RP-HPLC purification (10.3 mg, 14.0 µmol, 90%) as a white
solvent was evaporated under reduced pressure and the crude solid. RP-HPLC: tR = 8.8 min (10→40% B in 30 min); ESIMS
title compound (6.61 mg, 7.86 µmol, 85%) was recrystallized m/z: 760.4 [M + Na]+; HRMS C35H51N11O7: [M − H]− calcd:
from a mixture of MeOH/TFA (4:1, 1.00 mL) to yield a 736.3900; found: 736.3888, [M + Na]+ calcd: 760.3865; found:
brownish solid. The crude product was directly used in the next 760.3866.
step without further purification. ESIMS m/z: 863.4 [M + Na]+;
HRMS: C43H56N10O8 [M + Na]+ calcd: 863.4175; found: Acknowledgements
863.4174, [M − H]− calcd: 839.4210; found: 839.4214.
Financial support from the Deutsche Forschungsgemeinschaft
(Research Center Molecular Physiology of the Brain) is grate-
cyclo(β3-HLys(NH2)-β3-HLys(N3)-β3-HLys(thymin-1-yl fully acknowledged.
acetate)) (11). Cyclo(β3-HLys(Fmoc)-β3-HLys(N3)-β3-HLys-
References
(thymin-1-yl acetate)) (10, 7.61 mg, 9.05 µmol, 1.00 equiv) was
1. Seebach, D.; Gardiner, J. Acc. Chem. Res. 2008, 41, 1366–1375.
dissolved in a solution of 20% piperidine in DMF (1000 µL).
The reaction mixture was sonicated for 35 min in an ultrasonic
cleaning bath. Then, ice-cold tert-butylmethylether (MTBE)
(14 mL) was added. The observed precipitate was separated by
centrifugation (20 min, −5 °C, 9000 rpm) and the residue was
dried in high vacuum. The title compound was obtained by
RP-HPLC purification (5.04 mg, 8.15 µmol, 90%) to obtain a
brownish solid. RP-HPLC: tR = 10.5 min (30→100% B in
30 min); ESIMS m/z: 641.4 [M + Na]+; HRMS C28H46N10O6:
[M + H]+ calcd: 619.3635; found: 619.3637, [M + Na]+ calcd:
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