Silyl-based alkyne-modifying linker for the preparation of C-terminal acetylene-derivatized protected peptides

Article abstract of DOI:10.1021/jo302305d

A novel linker for the synthesis of C-terminal acetylene-functionalized protected peptides is described. This SAM1 linker is applied in the manual Fmoc-based solid-phase peptide synthesis of Leu-enkephalin and in microwave-assisted automated synthesis of Maculatin 2.1, an antibacterial peptide that contains 18 amino acid residues. For the cleavage, treatment with tetramethylammonium fluoride results in protected acetylene-derivatized peptides. Alternatively, a one-pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification.

Full text of DOI:10.1021/jo302305d

Brief Communication
pubs.acs.org/joc
Silyl-Based Alkyne-Modifying Linker for the Preparation of
C‑Terminal Acetylene-Derivatized Protected Peptides
Martin Strack,† Sina Langklotz,‡ Julia E. Bandow,‡ Nils Metzler-Nolte,† and H. Bauke Albada*,†
†Inorganic Chemistry 1, Bioinorganic Chemistry, Department of Chemistry and Biochemistry, and ‡Biology of Microorganisms, Ruhr
University Bochum, Universitatsstrasse 150, D-44780 Bochum, Germany
̈
S
* Supporting Information
ABSTRACT: A novel linker for the synthesis of C-terminal
acetylene-functionalized protected peptides is described. This
SAM1 linker is applied in the manual Fmoc-based solid-phase
peptide synthesis of Leu-enkephalin and in microwave-assisted
automated synthesis of Maculatin 2.1, an antibacterial peptide
that contains 18 amino acid residues. For the cleavage,
treatment with tetramethylammonium fluoride results in
protected acetylene-derivatized peptides. Alternatively, a one-
pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification.
ince the dawn of solid-phase peptide synthesis (SPPS), this
preferred method for preparing peptides significantly
peptide chemistry has not been described.17 Therefore, we
S
designed a silyl-protected acetylene-based linker that is
compatible with standard and microwave-assisted SPPS
procedures, which upon cleavage with fluoride anions affords
an acetylene moiety on the C-terminal amide of protected
peptides. In addition, it is also compatible with a one-pot
cleavage-click procedure, affording the 1,2,3-triazole conjugate
in high yields.
contributed to the identification of the role of peptides in
numerous diseases.1 Of these, a prominent class that is gaining
momentum is the antibacterial and anticancer peptides,2,3
which often need a free N-terminal amino group for their
activity.4 Together with the increasing number of bioactive
peptides known, sophisticated techniques for conjugated
peptides have become available.5 Just over a decade ago,
Meldal et al. reported a DiPEA-assisted Cu(I)-catalyzed version
of Huisgen’s alkyne−azide cycloaddition reaction that was
compatible with standard SPPS procedures and selectively
provided the 1,4-regioisomer of the 1,2,3-triazole.6 Together
with a solution-phase version discovered by Sharpless et al.,7
this became known as the copper-catalyzed alkyne−azide
cycloaddition (abbreviated as CuAAC) reaction8 and is now
applied across the chemical landscape.9 Importantly, click
reactions compatible with biological systems10 and a
ruthenium-based method that affords the 1,5-regioisomer of
the 1,2,3-triazole have become available.11
At this moment, many methods are available for the
introduction of specific reactive functionalities for bioconjuga-
tion strategies.12 Within the spectrum of available strategies, the
alkyne is among the most popular functionalities due to its
inertness under many conditions. Among the methods that are
currently available to introduce the alkyne and azide moieties
for which incorporation of artificial amino acid residues13 or N-
terminal acylation of peptides with azido- or acetylene-
containing moieties are the most straightforward methodsa
diazo transfer reaction on the N-terminal amino group of a
resin-bound peptide14 and modified BAL linkers are
applicable.15 Concerning the latter, formation of side products
during the on-resin reductive amination and acylation of the
resulting secondary amine have been reported.16 Whereas silyl
protection has proven to be very useful in acetylene chemistry,
a linker based on this principle and that is compatible with
For the synthesis of the linker, we used commercially
available (3-cyanopropyl)diisopropylchlorosilane 1 and 4-
iodobenzylamine hydrochloride 2. The bulky isopropyl groups
on the silicon atom were chosen in order to ensure stability of
the linker during SPPS procedures. For the preparation of the
silyl-protected acetylene moiety, chlorosilane 1 was reacted
with ethynylmagnesium bromide 3 to give 4-(ethynyldiiso-
propylsilyl)butanenitrile 4 in high yields (Scheme 1).18 The
other part of the linker was prepared using 4-iodobenzylamine
2, which was protected with the trityl (Tr) group, providing Tr-
protected 4-iodobenzylamine 5. After this, benzylamine 5 and
silyl-protected acetylene 4 were coupled using the Sonogashira
cross-coupling reaction,19 which gave Tr-protected cyano-
functionalized silyl alkyne 6. Reduction of the nitrile group
with DiBAL-H gave the target aldehyde 7 in good yields with
an overall yield from chlorosilane 1 to SAM linker 7 of 44%.
This first generation of the silyl-based alkyne-modifying linkers
is called the SAM1 linker.
Compatibility of SAM1 linker 7 with SPPS was initially
assessed by preparing Leu-enkephalin 10 using standard SPPS
procedures under ambient conditions. For this, TentaGel HL
NH2 resin (loading: 0.55 mmol/g) was loaded using 1 equiv of
the SAM1 linker in the reductive amination reaction. After Tr
removal of SAM1-loaded resin 8 and coupling of the first amino
Received: October 17, 2012
Published: November 1, 2012
© 2012 American Chemical Society
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Brief Communication
Scheme 1. Synthesis of SAM1 Linker 7
Scheme 2. Solid-Phase Peptide Synthesis of Leu-Enkephalin
9 Using SAM1 Linker 7 and Its CuAAC Conjugation to
Azidomethylferrocene 11 (Structure of the Maculatin
Derivative 13 Is Also Shown)
acid (Fmoc-Leu-OH) to the H2N-SAM1-TentaGel resin, Fmoc
determination revealed that a loading of 0.36 mmol/g was
achieved. Although five steps preceded this Fmoc loading
determinationthe imine forming immobilization of the
linker, imine reduction using NaB(OAc)3H, acetylation of
reactive amino functionalities, trityl removal, and coupling of
Fmoc-Leu-OHa yield of 67% for loading of the resin with
the SAM1 linker can be assumed since the last three steps
usually proceed quantitatively. Fmoc loading determination
during the synthesis indicated that the SAM1 linker was stable
during the Fmoc-based SPPS procedures (see Supporting
Information). After completion of the synthesis, Leu-
enkephalin 10 was cleaved from 100 mg of resin using
tetramethylammonium fluoride (Me4NF), resulting in 18.1 mg
(87%) of crude material. Cleavage with TFA results in
hydration of the arylacetylene moiety.20 According to HPLC
analysis, the crude peptide was 72% pure, and subsequent
purification by HPLC gave pure peptide 10 in 25% yield.
Importantly, no Me4N ions were shown by 1H NMR analysis of
this sample (see Supporting Information).
Applicability of the generated arylacetylene-derivatized
peptide in the CuAAC reaction was assessed by conjugation
of peptide 10 to azidomethylferrocene 11 (Scheme 2).21 Using
2.3 equiv of azide 11 and conditions that were described by
Meldal et al.,6 complete consumption of the peptide was
obtained under formation of a species for which the HRMS m/
z value and isotope pattern correspond to the expected 1,2,3-
triazole-linked conjugate 12 (see Supporting Information).
Purification afforded a yield of 63%, which results in a yield of
only 16% over two reaction steps. However, a one-pot cleavage-
click procedure using Me4NF, azide 11, CuI, and DiPEA in
MeOH under 20 min of microwave-assisted heating to 60 °C
provided a yield of 59% of the desired click product (12) after
purification.22
NH2. This peptide allowed us to assess if the integrity of the
protecting groups in this peptide is maintained during the
course of the synthesis. Using Fmoc-Thr-SAM1-TentaGel,
automated synthesis of the protected peptide was performed.
The Fmoc loading of the resin before and after the automated
synthesis dropped marginally, that is, from 0.14 to 0.13 mmol/
g, indicating that the SAM1 linker is applicable in microwave-
assisted SPPS of long peptides. Cleavage of the peptide was
affected using an excess of Me4NF dissolved in 9:1 DMF/
MeOH (v/v) under heating of the sample to 60 °C in a
microwave (20 min). HPLC analysis revealed that the crude
peptide was ∼80% pure, and HRMS analysis confirmed
formation of the 18-meric peptide (see Supporting Informa-
tion). Interestingly, the m/z value of the obtained product
corresponds to the mass of the desired peptide lacking one tBu
group. We assume that ester hydrolysis of the aspartic acid side-
chain tert-butyl ester takes place under the alkaline conditions
of the cleavage reaction.
In conclusion, we have developed a new linker that is
compatible with standard and microwave-assisted Fmoc/t-Bu-
based solid-phase peptide synthesis. When treated with Me4NF,
peptides are released, having a C-terminal amide that is
alkylated with an arylacetylene moiety. Alternatively, a one-pot
cleavage-click procedure can be applied in which the 1,2,3-
triazole-linked conjugate of the protected peptide is obtained in
Lastly, we assessed the applicability of the SAM1 linker in
automated microwave-assisted SPPS by preparing a peptide of
intermediate length (i.e., 18 amino acid residues). For this, we
prepared derivative 13 (Scheme 2 for the structure) of the
antibacterial Maculatin 2.1 peptide,23 a C-terminally amidated
peptide with the sequence H-GFVDFLKKVAGTIANVVT-
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The Journal of Organic Chemistry
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This solution was washed with water (25 mL) and brine (15 mL),
dried over MgSO4, and after filtration, the solvent was removed by
rotary evaporation. Column chromatography over silica using an
eluent of 95:5 hexanes/ethyl acetate (v/v) yielded product 6 as
colorless oil which solidified slowly (896.3 mg, 77%): Rf = 0.43 (silica;
eluent, 4:1 hexanes/ethyl acetate (v/v)); mp 92.3−93.2 °C; IR (ATR)
3316 (m), 3058 (m), 2942 (s), 2863 (s), 2243 (m), 2151 (s), 1596 (s)
high yields. The linker can be used for the synthesis of peptides
of intermediate length, as was shown by the synthesis of 18-
meric Maculatin 2.1 derivative 13. We anticipate that silyl-based
alkyne-modifying (SAM) linkers will find widespread applica-
tions in the preparation of C-terminally labeled peptides and
peptide conjugates.
1
cm−1; H NMR (200 MHz, CD2Cl2) δ 7.73−7.03 (m, 19H), 3.36 (s,
EXPERIMENTAL SECTION
br, 2H), 2.44 (t, J = 3.5 Hz, 2H), 2.03−1.77 (m, 3H), 1.24−1.01 (m,
14H), 0.94−0.79 (m, 2H); 13C NMR (50 MHz, CD2Cl2) δ 146.6,
142.6, 132.5, 129.2, 128.5, 128.4, 127.0, 122.0, 120.4, 108.5, 89.7, 71.7,
48.3, 22.0, 21.2, 18.6, 18.4, 12.4, 10.2; MS (ESI+) m/z = 577.4 (calcd
577.3 for [M + Na]+). Anal. Calcd for C38H42N2Si: C, 82.26; H, 7.63;
N, 5.05. Found: C, 82.54; H, 7.65; N, 5.10.
■
General Experimental Details. All reagents were obtained from
commercial sources and used without purification. Solvents were dried
according to standard protocols, distilled, and stored over molecular
sieves (4 Å). For the Sonogashira reaction, trans-dichlorobis(triphenyl-
phosphine)palladium(II), Premion (Alfa Aesar) was used. TLC
analysis was performed on silica gel 60 F254 aluminum sheets, and
spots were visualized by UV light. Kaiser tests24 on the resin-bound
material were carried out with a mixture of ninhydrin (1 g) in t-
BuOH/H2O/AcOH (95:4.5:0.5, % v/v/v, Vtot = 500 mL). Elemental
analysis was performed in CHN mode. FT-IR spectroscopy was
carried out on a spectrophotometer equipped with an attenuated total
reflection (ATR) unit at 4 cm−1 resolution; abbreviations are s
N-(Trityl)-4-(2-(3-formylpropyl)diisopropylsilylethynyl)-
benzylamine 7. Nitrile 6 (720.6 mg, 1.3 mmol) was dissolved in dry
DCM (20 mL) and cooled to −78 °C, after which 2.1 equiv of DiBAL-
H (2.73 mL, 2.73 mmol, 1 M in n-hexane) was added dropwise. The
solution was maintained at −78 °C for 2 h followed by cautious
addition of 2-propanol (1 mL). Upon warming, an aqueous solution
saturated with citric acid (8 mL) was added, and the mixture was
allowed to reach rt within 1 h. The crude organic material was
extracted with DCM (2 × 20 mL), after which the combined organic
phases were washed with brine (15 mL) and dried over MgSO4. Then,
the drying agent was removed by filtration, and the filtrate was
concentrated by rotary evaporation under reduced pressure. Column
chromatography (silica) using an eluent of 15:1 petroleum ether (40−
60)/ethyl acetate (v/v) yielded product 7 as colorless oil (476.0 mg,
66%): Rf = 0.26 (silica; eluent, 7:1 hexanes/ethyl acetate (v/v)); IR
(ATR) 3329 (w), 3057, 3029 (m), 2940, 2863 (s), 2152 (s), 1725 (s),
1595, 1505 (m) cm−1; 1H NMR (200 MHz, CD2Cl2) δ 9.75 (t, J = 1.9
Hz, 1H), 7.62−7.12 (m, 19H), 3.33 (s, br, 2H), 2.51 (dt, J = 7.1 Hz,
1.8 Hz, 2H), 1.93−1.73 (m, 3H), 1.22−0.95 (m, 14H), 0.80−0.65 (m,
2H); 13C NMR (50 MHz, CD2Cl2) δ 203.2, 146.6, 142.4, 132.5, 129.2,
128.5, 128.4, 127.0, 122.2, 108.0, 90.3, 71.7, 48.3, 48.0, 18.6, 18.4, 12.4,
10.5; HRMS (ESI+) m/z = 558.3184 (calcd 558.3187 for [M + H]+, M
= C38H43NOSi).
Loading of the Resin with SAM1 Linker 7 To Provide SAM1-
Loaded Resin 8. For the attachment of the SAM1 linker to the resin,
1 equiv of aldehyde 7 (307 mg, 0.55 mmol) was dissolved in dry THF
(5 mL) and the solution was added to the TentaGel HL NH2 resin (1
g, 0.55 mmol, loading = 0.55 mmol/g). The mixture was agitated at rt
for 2 h, after which 1.2 equiv of NaB(OAc)3H (140 mg, 0.66 mmol)
was added (overnight, rt). After this, the reagents were removed and
the resin was washed with THF (5 × 6 mL × 2 min), DMF (5 × 6 mL
× 2 min), and DCM (5 × 6 mL × 2 min). Immediately after this, all
reactive amino groups were capped using a solution of Ac2O (0.5 M),
DiPEA (0.125 M), and HOBt (0.015 M) in NMP (rt, 2 × 6 mL × 10
min). Lastly, the resin was washed with NMP (5 × 6 mL × 2 min) and
DCM (5 × 6 mL × 2 min). Infrared analysis showed the presence of a
weak band at 2154 cm−1, which can be assigned to the alkyne
functionality (see Supporting Information).
SPPS of Leu-Enkephalin 10 Using the SAM1 Linker 7.
Removal of the Tr group was affected using a solution of DCM/TFA/
TIS (94:4:2, v/v/v) that was added to the resin followed by agitation
at rt (2 × 6 mL × 10 min). The reagents were removed by filtration,
and the resin was washed with DCM (5 × 6 mL × 2 min), 10% DiPEA
in DCM (v/v, 1 × 6 mL × 2 min), and DCM (5 × 6 mL × 2 min).
For coupling of the amino acid residues, a solution of the Fmoc-
protected amino acid (4 equiv), HOBt (4 equiv), and HBTU (3.8
equiv) in DMF (6 mL) was prepared and preactivated with DiPEA (10
equiv) shortly before the solution was added to the resin. Coupling
was performed under agitation (rt, 2 h). The chemicals were removed
by filtration, and the resin was washed with DMF (3 × 6 mL × 2 min)
and DCM (2 × 6 mL × 2 min). Removal of the Fmoc group was
affected using a solution of 20% piperidine in NMP (2 × 6 mL × 10
min). The resin was gently agitated (rt, 10 min), after which the
chemicals were removed by filtration and the resin was washed with
DMF (3 × 6 mL × 2 min) and DCM (2 × 6 mL × 2 min). Lastly,
peptide-bound resin (100 mg, 28.7 μmol) was treated once with a
1
(strong), m (medium), and w (weak). H NMR spectroscopy was
performed in deuterated solvents at 30 °C. Chemical shifts (δ) are
given in parts per million (ppm) relative to TMS, and the solvent
residual signal is used as a reference. Abbreviations for peak
multiplicities are s (singlet), d (doublet), t (triplet), dt (doublet of
triplets), m (multiplet), and br (broad). Mass spectra were measured
using an electrospray ionization (ESI) or electron impact (EI) mass
spectrometer. Details for the spectrometers and methods that were
used for HRMS analysis are given in the Supporting Information.
Analytical HPLC was performed on an automated HPLC system using
a C18-AQ RP column (250 × 4.6 mm) at a flow-rate of 1 mL/min. A
linear gradient of 5% buffer B per min starting at 5 min of buffer A (A:
H2O/MeCN/TFA, 95:5:0.1, v/v/v; B: MeCN/H2O/TFA, 95:5:0.1, v/
v/v) was used. Purification of the peptides was performed on an
HPLC machine equipped with PDA detector that was coupled to an
RP-18e reversed phase column (250 × 25 mm), using a similar
gradient as for the analytical HPLC but with a flow rate of 20 mL/min.
4-(Ethynyldiisopropylsilyl)butanenitrile 4. This compound was
prepared according to a literature procedure.19 The obtained material
1
was analyzed using TLC, H NMR, 13C NMR, IR, and EI-MS.
N-(Triphenylmethyl)-4-iodobenzylamine 5. A mixture of 4-
iodobenzylamine hydrochloride 2 (1 g, 3.7 mmol) and Et3N (1.28 mL,
9.25 mmol) in DCM (15 mL) was prepared and to this
triphenylmethylchloride (1.14 g, 4.08 mmol) was added in portions.
The mixture was allowed to stir at rt overnight. Subsequently, water
(20 mL) was added to the reaction mixture, the organic layer was
separated, and remaining organic material present in the water phase
was extracted using DCM (2 × 25 mL). The organic phases were
combined and washed with brine (15 mL), dried over MgSO4, and
after filtration, the solvent was removed by rotary evaporation under
reduced pressure. Column chromatography over silica with 9:1
hexanes/ethyl acetate (v/v) as eluent yielded product 5 as a white
solid (1.695 g, 96%): Rf = 0.47 (silica; eluent, 9:1 hexanes/ethyl
acetate (v/v)); mp 159.6−160.7 °C; IR (ATR) 3299 (s), 3057 (s),
1
2849 (s), 1596 (s) cm−1; H NMR (200 MHz, CD2Cl2) δ 7.74−7.43
(m, 8H), 7.41−7.08 (m, 11H), 3.28 (d, J = 7.3 Hz, 2H), 1.91 (t, J = 7.6
Hz, 1H); 13C NMR (50 MHz, CD2Cl2) δ 146.6, 141.5, 137.9, 130.5,
129.2, 128.5, 127.0, 92.3, 71.6, 47.9; MS (EI+) m/z = 476.2 (calcd
476.1 for [M + H]+). Anal. Calcd for C26H22IN: C, 65.69; H, 4.66; N,
2.95. Found: C, 65.90; H, 4.63; N, 2.78.
N-(Trityl)-4-(2-(3-cyanopropyl)diisopropylsilylethynyl)-
benzylamine 6. A solution of 4-(ethynyldiisopropylsilyl)butanenitrile
4 (655.4 mg, 3.16 mmol) and N-(triphenylmethyl)-4-iodobenzylamine
5 (1 g, 2.1 mmol) in THF/NEt3 (3:1, v/v, Vtot = 32 mL) was prepared
and degassed thoroughly. Then, copper(I) iodide (40 mg, 0.21 mmol)
and bis(triphenylphosphine)palladium(II) chloride (36.8 mg, 0.05
mmol) were added, and the yellowish solution was again thoroughly
degassed. The reaction mixture was allowed to stir at room
temperature for 2 h, after which it was diluted with Et2O (30 mL).
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The Journal of Organic Chemistry
Brief Communication
solution containing excess Me4NF (9.1 mg, 55 μmol) in DMF/MeOH
(9:1, 1 mL, v/v) for 30 min. The solution was separated from the resin
by filtration, and the latter was washed with DMF (3 × 0.5 mL × 2
min). After combining the organic phases, the solvents were removed
in vacuo (T < 40 °C) and the solid material was lyophilized from
H2O/MeCN (1:1, v/v) to give acetylene-functionalized peptide 10
with a crude yield of 18.1 mg with 72% purity according to HPLC.
Preparative HPLC yielded 6.1 mg (7.3 μmol, 25%) of pure peptide 10:
HPLC (C18) tR = 21.4 min; MS (ESI+) m/z = 725.4 (calcd 725.4 for
[M + H]+); HRMS (ESI+) m/z 725.4031 (calcd 725.4021 for [M +
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed procedures, NMR spectra of all new compounds, IR
spectrum of the resin-bound SAM1 linker 8, 2D NMR spectra
of peptides 10 and 12, UPLC-HR-MS analysis of ferrocene
conjugate 12, and the crude HPLC trace and HRMS analysis of
peptide 13. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
H]+, M = C41H52N6O6). H NMR, COSY, and TOCSY spectra of
AUTHOR INFORMATION
Corresponding Author
*E-mail: h.b.albada@gmail.com.
Notes
■
peptide 10 are available in the Supporting Information.
Azidomethylferrocene 11. This compound was prepared
according to a literature procedure.25 The obtained material was
1
analyzed using TLC, HPLC, H NMR, IR, and ESI-MS.
Solution-Phase Labeling of Leu-Enkephalin 12. A solution of
Leu-enkephalin derivative 10 (0.99 mg, 1.18 μmol), DiPEA (0.48 μL,
2.76 μmol), and azidomethylferrocene 11 (0.67 mg, 2.76 μmol) in
MeCN (1 mL) was prepared and degassed. Then, copper(I) iodide
(0.13 mg, 0.69 μmol) was added, and the yellow solution was stirred at
rt overnight. The crude reaction mixture was directly subjected to
semipreparative HPLC, affording the desired product with a yield of
0.8 mg (0.74 μmol, 63%): UPLC-HRMS (ESI+) tR = 18.8−18.9 min;
m/z = 966.4312 (calcd 966.4323 for [M + H]+, M = C52H63FeN9O6).
Cleavage-Click Labeling of Leu-Enkephalin 9. In a reaction
vessel, peptide-loaded resin 9 (75.2 mg, 21.5 μmol), TMAF·4H2O
(11.9 mg, 72 μmol), azidomethylferrocene 11 (8.7 mg, 36 μmol),
DiPEA (1.5 μL, 9.6 μmol), and MeOH (0.7 mL) were mixed, and the
suspension was degassed. Then, copper(I) iodide (0.91 mg, 4.8 μmol)
was added, the vessel sealed, and the mixture heated to Tmax = 60 °C
(external surface sensor) under microwave irradiation for 20 min (in a
CEM Discover microwave reactor). The resin was filtered, washed
with MeCN (3 × 1 mL), and the solvents were removed in vacuo.
Preparative HPLC yielded peptide 12 as yellowish solid (13.7 mg, 12.7
μmol, 59%): HPLC (C18) tR = 22.3 min; ESI-MS (ESI+) m/z = 966.3
(calcd 966.4 for [M + H]+). 1H NMR, COSY, and TOCSY spectra of
conjugate 12 are available in the Supporting Information.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Martin Gartmann for measuring the 400 MHz
spectra of Leu-enkephalin derivatives 10 and 12, and we thank
the State of North Rhine-Westphalia (NRW), Germany, and
the European Union, European Regional Development Fund,
“Investing in your future”.
REFERENCES
■
(1) Kastin, A. J., Ed. Handbook of Biologically Active Peptides, 1st ed.;
Elsevier Inc.: Burlington, MA, 2006.
(2) Hancock, R. E. W.; Sahl, H.-G. Nat. Biotechnol. 2006, 24, 1551.
(3) See for example: Wang, G., Ed. Antimicrobial Peptides: Discovery,
Design and Novel Therapeutic Strategies; CABI: Wallingford, England,
2010.
(4) Albada, H. B.; Chiriac, A.-I.; Wenzel, M.; Penkova, M.; Bandow, J.
E.; Sahl, H.-G.; Metzler-Nolte, N. Beilstein J. Org. Chem. 2012, 8, 1753.
(5) Aslan, M.; Dent, A. Bioconjugation: Protein Coupling Techniques for
the Biomedical Sciences, 1st ed.; Macmillan Reference: London, UK,
1998.
Microwave-Assisted SPPS of Maculatin 2.1 Derivative 13.
First, Fmoc-Thr-OH was coupled to the Tr-deprotected SAM1-
TentaGel resin using the manual procedure described above (for the
loading of Fmoc-Leu-OH). This resin was then subjected to
microwave-assisted SPPS in which Fmoc deprotection is affected by
two treatments of the resin with 20% piperidine in NMP and exposure
of the reaction mixture to microwave irradiation with a power of 50 W
for 180 s with a Tmax of 75 °C. Coupling of Fmoc-protected amino
acid residues is performed using a single treatment with 4 equiv of
Fmoc-amino acid, 4 equiv of HOBt, 3.8 equiv of HBTU, and 10 equiv
of DiPEA in DMF/NMP; the reaction mixture was exposed to
microwave irradiation with a power of 24 W for 300 s with a Tmax of 75
°C. All manipulations were performed using a sealed reaction vessel
with internal temperature sensor in a CEM Discover microwave
reactor. After completion of the automated synthesis program
(approximately 14 h), the peptide-containing resin (13.2 mg, 1.1
μmol, contains approximately 3 mg peptide) and Me4NF·4H2O (5.9
mg, 36 μmol) were mixed in DMF/MeOH (9:1, v/v, 1 mL) and the
mixture was heated in a sealed reaction vessel under microwave
irradiation (with Tmax = 60 °C, as measured with an external surface
sensor) for 20 min (in a CEM Discover microwave reactor). The resin
was filtered and washed with DMF (3 times 0.5 mL), after which the
solvents of the combined organic phases were removed in vacuo (T <
40 °C). The obtained material was lyophilized from MeCN:H2O (1:1,
v/v, 15 mL) and yielded the crude product as white solid (6.4 mg).
During the weighing process, the hygroscopic solid slowly increased in
weight, and the given yield corresponds to the first value obtained.
Since this material contains approximately 3.33 mg of ammonium salt
that originates from the excess of Me4NF used in the cleavage, it
contains approximately 3.1 mg of peptide. Therefore, quantitative
cleavage of the peptide from the resin can be assumed: HPLC (C18) tR
= 22.5 min; HRMS (ESI+) m/z = 1245.7078 (calcd 1245.7085 for [M
− tBu + 2H]2+, M = C130H188N22O27).
(6) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
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