The "azirine/oxazolone method" under solid-phase conditions

Article abstract of DOI:10.1002/ejoc.200400330

Aib-containing peptides have been synthesized from the N- to the C-terminus by the "azirine/oxazolone method" under solid-phase conditions. In this new and convenient method for the synthesis of sterically demanding peptides on solid phase, 2H-azirine-3-amines are used to introduce aminoisobutyric acid, an α,α-disubstituted α-amino acid, into the peptide without the need for further reagents. Segments of naturally occurring peptaibols have been prepared by this method. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200400330

FULL PAPER
The ‘‘Azirine/Oxazolone Method’’ under Solid-Phase Conditions
Simon Stamm[a] and Heinz Heimgartner*[a]
Keywords: Aminoisobutyric acid (Aib) / Azirine / Oxazolone method / Inverse peptide synthesis / Peptides / Solid-phase
synthesis
Aib-containing peptides have been synthesized from the N-
to the C-terminus by the ‘‘azirine/oxazolone method’’ under
solid-phase conditions. In this new and convenient method
for the synthesis of sterically demanding peptides on solid
phase, 2H-azirine-3-amines are used to introduce aminoiso-
butyric acid, an α,α-disubstituted α-amino acid, into the pep-
tide without the need for further reagents. Segments of nat-
urally occurring peptaibols have been prepared by this
method.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2004)
Introduction
selectively to give extended peptide acids 5. In solution-
phase chemistry, the ‘‘azirine/oxazolone method’’ has pro-
ven successful for the introduction of a multitude of steri-
cally demanding α,α-disubstituted α-amino acids into pep-
tides and has found successful application in the synthesis
of some antibiotic active peptaibols or segments of
them.[24Ϫ28] A drawback of this method was that it was not
yet applicable to solid-phase synthesis.
Solid-phase peptide synthesis (SPPS) allows rapid access
to peptides and peptide conjugates.[1Ϫ3] Its importance is
particularly evident in combinatorial chemistry, where it
made the synthesis of compound libraries possible. SPPS is
exclusively carried out from the C- to the N-terminus, at-
tempts to reverse the strategy having largely failed because
of incomplete couplings and significant epimerization dur-
ing the coupling steps. The synthesis of C-terminal-modi-
fied peptides and peptide libraries is therefore not routine,
but methods for the synthesis of peptides from the N- to
the C-terminus have nevertheless been investigated.[4Ϫ9]
Moreover, the introduction of sterically demanding α,α-di-
substituted α-amino acids into peptides under solid-phase
conditions still remains a challenge, although progress has
been made in the case of aminoisobutyric acid (Aib) and
isovaline (Iva).[10Ϫ12]
Peptides containing α,α-disubstituted α-amino acids are
restricted in their conformational freedom.[13Ϫ16] As a
consequence of the rigidity of the peptide backbone, sec-
ondary structures such as β-turns and helices are stabilized
or even promoted.[17Ϫ20] The synthesis of such confor-
mationally restricted peptides is a basic approach when
searching for the biologically active conformation of a pep-
tide. One useful method for the introduction of α,α-disub-
stituted α-amino acids into peptides is the ‘‘azirine/oxazo-
lone method’’, in which 2H-azirin-3-amines 1 are used as
amino acid synthons[21Ϫ23] (Scheme 1). Thus, the reaction
between 2H-azirin-3-amines, such as the Aib synthon 2,
and amino or peptide acids 3 gives rise to peptide amides
4, the terminal amide bonds of which can be hydrolyzed
Scheme 1. a) 1, CH2Cl2; b) HCl (3 ), THF/H2O
Here we report a successful attempt to synthesize pep-
tides by the ‘‘azirine/oxazolone method’’ on solid phase Ϫ
a synthesis from the N- to the C-terminus, in which 2H-
azirin-3-amines are used to introduce α,α-disubstituted α-
amino acids into peptides without the need for further re-
agents.
Results and Discussion
[a]
Institute of Organic Chemistry, University of Zürich,
Winterthurerstrasse 190, 8057 Zürich, Switzerland
Fax: (internat.) ϩ 41-1-635-6836
The first questions we asked concerned chemical reactiv-
ity: do 2H-azirin-3-amines react in a similar way on a solid
E-mail: heimgart@oci.unizh.ch
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200400330
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The “Azirine/Oxazolone Method” under Solid-Phase Conditions
FULL PAPER
Scheme 2. a) 2, CH2Cl2; b) HCl (3 ), THF/H2O
support as they do in solution, that is to say, is a terminal
A wide variety of amines have been immobilized on solid
amide formed by the reaction between a 2H-azirin-3-amine supports through carbamate linkers, synthesized variously
and a solid-phase-bound carboxylic acid? Is selective hy- with phosgene,[31Ϫ34] 1,1Ј-carbonyldiimidazole (CDI),[35] or
drolysis of the terminal amide possible? Attenuated total 4-nitrophenyl chloroformate.[36Ϫ39] The use of a carbamate
reflectance (ATR) FT-IR measurements of the correspond- linker to attach amino acids through their N-termini was
ing intermediates 7, 8, 9, and 10 (Scheme 2), as well as the first reported by Letsinger.[5,31] We therefore also planned
synthesis of a resin-bound tripeptide and its hydrolysis,[29] the use of a carbamate linker, but the stability of the carba-
confirmed our hopes.
mate had to be tuned carefully. The carbamate has to be
The linker is of crucial importance in all variations of stable during hydrolysis of the terminal amide with 3  HCl
solid-phase synthesis, and so this was the second problem and deprotection of the tBu ester with TFA, but should
we dealt with. The 1-(4,4-dimethyl-2,6-dioxocyclohexylid- finally be cleavable under conditions not affecting the pep-
ene)ethyl (Dde) linker, developed by Bycroft et al.,[30] is tide. It was shown that the carbamate linker formed from
stable in trifluoroacetic acid (TFA), but can be cleaved with [4-(hydroxymethyl)phenyl]acetamidomethyl (PAM) resin 16
2% N2H4 in DMF. Its use resulted in the successful syn- fitted these requirements the best.[40] (Scheme 4).
thesis of HϪAlaϪAibϪN(Me)Ph (15, Scheme 3). However,
Phosgene in toluene (1.9 ) was added to PAM resin 16
the selective hydrolysis of the terminal amide group of 14
to generate a chloroformate intermediate, and treatment
with HϪAlaϪOtBu afforded resin 17. Amino acid tBu es-
was not possible, since the Dde-linker was not stable in
aqueous, acidic medium.
ters have proven to be useful building blocks for inverse
peptide synthesis,[4] because their chemistry is well known
and they are readily available from commercial suppliers.
Deprotection with TFA afforded resin 18, which was treated
with a solution of N,2,2-trimethyl-N-phenyl-2H-azirin-3-
amine (2, 4 equiv.) in DCM (c2 ϭ 0.2 ). Unconsumed 2
can easily be recovered and used in further coupling steps.
Hydrolysis of resin 19 with 3  HCl in H2O/THF afforded
resin 20, which was conventionally coupled to
HϪPheϪOtBu with PyBOP as coupling reagent. Cleavage
from the solid support was achieved with HBr (33%) in
acetic acid. After HPLC purification and lyophilization,
model peptide 21 was isolated in 50% yield. A second model
peptide [HϪAlaϪAibϪValϪAibϪPheϪOH (22)] contain-
ing two Aib residues was synthesized analogously (Table 1)
in 33% yield after HPLC and lyophilization.
A crucial point in the context of this strategy was the
question of epimerization of the Cα center(s). In order to
determine the extent of racemization/epimerization, the tri-
Scheme 3. a) HϪAlaϪOtBu·HCl, CH2Cl2, DIPEA; b) TFA/
peptide 21 was hydrolyzed and the amino acids were ana-
CH2Cl2 (1:1), TIPS; c) 2, CH2Cl2; d) N2H4 (2%), DMF. DIPEA ϭ
lyzed by capillary gas chromatography with enantiomer la-
N,N-diisopropylethylamine, TFA ϭ trifluoroacetic acid, TIPS ϭ
triisopropylsilane, DMF ϭ N,N-dimethylformamide
beling.[41] The results showed that alanine and phenylala-
Eur. J. Org. Chem. 2004, 3820Ϫ3827
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S. Stamm, H. Heimgartner
FULL PAPER
Scheme 4. a) COCl2 (1.9  in PhMe), THF; b) HϪAlaϪOtBu·HCl, DIPEA, CH2Cl2; c) TFA, CH2Cl2, TIPS; d) 2, CH2Cl2; e) HCl
(3 ), THF/H2O; f) HϪPheϪOtBu·HCl, PyBOP, DIPEA, CH2Cl2; g) HBr (33%) in acetic acid. PyBOP ϭ (1H-benzotriazol-1-yloxy)tri-
pyrrolidinophosphonium hexafluorophosphate
Table 1. General procedures
Description
Reagents[a]
Duration and
repetition
General procedure for the attachment of the first amino acid
Chloroformate formation
Washes
10 equiv. COCl2 (1.9 ) in toluene, THF
1 ϫ 2 h
2 ϫ, each
1 ϫ
THF, CH2Cl2
4 equiv. HϪAAϪOtBu·HCl, 8 equiv. DIPEA, CH2Cl2
Coupling
Washes
CH2Cl2, DMF, CH2Cl2
3 ϫ, each
General procedure for removal of the tBu protecting group
Hydrolysis
Washes
TFA/CH2Cl2, TIPS (5%)
1 ϫ 5 s (25%),
1 ϫ 30 min (50%)
3 ϫ, each
CH2Cl2, DMF, CH2Cl2
General procedure for peptide synthesis with 2H-azirin-3-amine 2
Coupling
Washes
4 equiv. 2, CH2Cl2
CH2Cl2
1 ϫ
3 ϫ
Hydrolysis
Washes
HCl (3 ) in THF/H2O
THF, DMF, CH2Cl2
1 ϫ
3 ϫ, each
4 equiv. PyBOP, 4 equiv. HϪAAϪOtBu·HCl, 12 equiv. DIPEA, CH2Cl2
1 ϫ
2 ϫ, each
1 ϫ
2 ϫ, each
General procedure for cleavage from the support
Cleavage
Washes
HBr (33%) in acetic acid, 2 drops of H2O
Acetic acid/CH2Cl2 (1:1), CH3CN/CH2Cl2 (1:1)
1 ϫ 6 h
3 ϫ, each
[a]
DIPEA ϭ N,N-diisopropylethylamine, HOAt ϭ 1-hydroxy-7-azabenzotriazole, HOBt ϭ 1-hydroxybenzotriazole, NMM ϭ N-methyl-
morpholine, PyBOP ϭ (1H-benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, TIPS ϭ triisopropylsilane.
nine had racemized by 0.6% and 2.8%, respectively. It was
To examine the use of the ‘‘azirine/oxazolone method’’
not possible to achieve resolution of the epimers by under solid-phase conditions we attempted to synthesize a
HPLC[42] on the crude product, the derivatized crude prod- Peptaibolin derivative and two segments of other pept-
uct,[43] or the purified product. We therefore assume that aibols.[44] The hexapeptide HϪAlaϪAibϪAlaϪGlnϪAibϪ
the extent of racemization/epimerization in the crude prod- ValϪOH (A4Ϫ9) (23) of the peptaibol antibiotic Alame-
uct is similar to that in the purified product, and hence that thicin[45,46] was synthesized in 25% yield by the procedures
the synthesis had been carried out with an acceptable de- listed in Table 1 (Table 2). Both Aib residues were intro-
gree of racemization, similar to that seen in classical SPPS. duced by the ‘‘azirine/oxazolone method’’, Ala (A3) by
3822
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2004, 3820Ϫ3827

The “Azirine/Oxazolone Method” under Solid-Phase Conditions
FULL PAPER
Table 2. Synthesized peptides
Description
Sequence
Yield [%][a]
Model tripeptide
HϪAlaϪAibϪPheϪOH (21)
50
33
25
20
30
Model pentapeptide
A4Ϫ9 of Alamethicin
Peptaibolin derivative
A9Ϫ13 of Stilboflavin A3
HϪAlaϪAibϪValϪAibϪPheϪOH (22)
HϪAlaϪAibϪAlaϪGlnϪAibϪValϪOH (23)
HϪLeuϪAibϪLeuϪAibϪPheϪOH (24)
HϪValϪAibϪGlyϪAibϪAlaϪOH (25)
[a]
Yield of product isolated after HPLC purification.
method A (Table 1) and both Gln and Val by method B.[47]
Method B was applied to prevent nitrile formation, as the
side chain of Gln was not protected.[48] The pentapeptide
HϪLeuϪAibϪLeuϪAibϪPheϪOH (24) is a derivative of
Peptaibolin[49] (AcϪLeuϪAibϪLeuϪAibϪPheol), and was
also synthesized by the procedures listed in Table 1. Both
Aib residues were introduced by the ‘‘azirine/oxazolone
method’’, while method A was used for the coupling of the
other amino acids. The lower yield (20%) is the result of
Reaction vessels for solid-phase synthesis: single fritted (20 µm)
PE reservoirs (15 mL) (Separtis, Grenzach-Wyhlen, Germany) were
used on an Advanced ChemTech PLS 4 ϫ 6 Shaker (Advanced
ChemTech, Inc., Louisville, KY, USA) with a custom-made ad-
apter. The original Advanced ChemTech reaction vessels were used
for reactions under N2. High-performance liquid chromatography
(HPLC): instrument: Waters 600E multisolvent delivery system
equipped with a Waters 996 PDA (Waters, Milford, CA, USA);
˚
column: Interchim Uptisphere ODB C18, 300 A, 10 µm, 250 ϫ
4.6 mm (Interchim, Montluc¸on, France), Interchim Uptisphere
˚
incomplete coupling of phenylalanine, but a repeated coup- WOD C18, 300 A, 10 µm, 250 ϫ 21.2 mm (prep. HPLC), or Vydac
˚
218TP C18, 300 A, 10 µm, 250 ϫ 22 mm (prep. HPLC) (Vydac,
Hesperia, CA, USA). Column chromatography (CC): silica gel C-
560 (0.04Ϫ0.063 mm, 230Ϫ400 mesh) from Chemie Uetikon (CU
Chemie Uetikon GmbH, Uetikon, Switzerland). Prep. TLC: Merck
TLC plates (glass), silica gel 60 F254, 0.25 mm (Merck KGaA,
Darmstadt, Germany). IR Spectra: PerkinϪElmer, Spectrum one
FT-IR spectrophotometer (PerkinϪElmer, Wellesley, MA, USA);
ATR-FT-IR with a Bio-Rad FTS-45 (Bio-Rad, Hercules, CA,
USA) instrument equipped with a MKII Golden Gate single reflec-
tion ATR system from Specac (Specac Inc., Smyrna, GA, USA).
NMR Spectra: Bruker ARX-300, Bruker DRX-500, or Bruker
DRX-600 machines (Bruker Biospin, Karlsruhe, Germany).
ling with additional pentafluorophenol did not increase the
yield. The segment HϪValϪAibϪGlyϪAibϪAlaϪOH
(A9Ϫ13) (25) of Stilboflavin A3[50] was prepared in 30%
yield by the ‘‘azirine/oxazolone method’’ and method A.
Conclusion
In conclusion, we have been able to adapt the ‘‘azirine/
oxazolone method’’ under solid-phase conditions, and have
consequently developed a new and convenient method for
the synthesis of sterically demanding peptides on solid Chemical shifts are given in ppm relative to tetramethylsilane
(TMS) as internal standard. 2D NMR experiments were performed
for assignment of the signals. Some spin systems were simulated
with NMR-Sim from Bruker. HPLC-MS: instrument:
HewlettϪPackard HP 1100 HPLC system (HewlettϪPackard, Palo
Alto, CA, USA) equipped with a HTS PAL autosampler (CTC
Analytics, Zwingen, Switzerland), connected to a Bruker ES-
QUIRE-LC quadrupole ion trap instrument (Bruker Daltonik
GmbH, Bremen, Germany) equipped with a HewlettϪPackard
Electrospray Ionization (ESI) source (HewlettϪPackard Co., Palo
phase. The simple procedure may possibly be automatable.
It has been shown that this approach for the synthesis of
Aib-containing peptides from the N- to the C-terminus can
be combined with conventional coupling methods. The
method found a successful application in the synthesis of
different peptaibol segments.
˚
Experimental Section
Alto, CA, USA); column: Interchim Uptisphere HDO C18, 120 A,
3 µm, 200 ϫ 2.0 mm column (Interchim, Montluc¸on, France);
eluents: A ϭ H2O/HCOOH (99.95:0.05), B ϭ CH3CN/HCOOH
(99.95:0.05); flow rate: 0.18 mL/min, gradient (A:B): 0Ϫ10 min:
95:5Ϫ50:50, 10Ϫ20 min: 50:50Ϫ0:100, 20Ϫ30 min: 0:100. MS:
Bruker ESQUIRE-LC quadrupole ion trap instrument (Bruker
Daltonik GmbH, Bremen, Germany) or Finnigan TSQ-700 triple
quadrupole instrument (Finnigan MAT, San Jose, CA, USA). Di-
rect infusion ESI-MS were performed with a syringe infusion pump
at a flow rate of 5 µL/min.
General Remarks: Unless otherwise noted, materials were obtained
from commercial suppliers and were used without further purifi-
cation. Hydroxymethylpolystyrene, 1% divinylbenzene, 100Ϫ200
mesh, loading 0.98 mmol/g was from Novabiochem (CalbiochemϪ
Novabiochem, Läufelfingen, Switzerland), [4-(hydroxymethyl)phe-
nyl]acetamidomethylpolystyrene [4-(oxomethyl)phenylacetamido-
methylpolystyrene], 1% divinylbenzene, 100Ϫ200 mesh, loading
0.62 mmol/g from Acros Organics (Acros Organics, Geel, Belgium),
and carboxylpolystyrene, 1% divinylbenzene, 200Ϫ400 mesh, load-
ing 4.0 mmol/g from Lipal Biochemicals (Lipal Biochemicals, Gun-
detswil, Switzerland). Aminomethylpolystyrene, 1% divinylben-
zene, 100Ϫ200 mesh, loading 1.14 mmol/g, hydroxyethylpoly-
styrene, 1% divinylpolystyrene, 100Ϫ200 mesh, loading 1.30 mmol/
Abbreviations: Aib: α-aminoisobutyric acid; ATR-FT-IR: attenu-
ated total reflectance Fourier transform infrared spectroscopy; CC:
column chromatography; DCM: dichloromethane; Dde: N-1-(4,4-
dimethyl-2,6-dioxocyclohexylidene)ethyl; DIPCDI: diisopropylcar-
g, and carboxylpolystyrene, 1% divinylbenzene, 100Ϫ200 mesh, bodiimide; DIPEA: N,N-diisopropylethylamine; HM: hydroxy-
loading 1.96 mmol/g were from Rapp Polymere (Rapp Polymere,
methyl; HOAt: 1-hydroxy-7-azabenzotriazole; HOBt: 1-hydroxy-
Tübingen, Germany). N,2,2-Trimethyl-N-phenyl-2H-azirin-3-am-
benzotriazole; NMM: N-methylmorpholine; PAM: [4-(hydroxy-
ine was synthesized by Villalgordo and Heimgartner’s method.[51,52] methyl)phenyl]acetamidomethyl;
PfpOH:
pentafluorophenol;
Eur. J. Org. Chem. 2004, 3820Ϫ3827
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S. Stamm, H. Heimgartner
FULL PAPER
PyBOP:
(1H-benzotriazol-1-yloxy)tripyrrolidinophosphonium
Synthesis of H؊Ala؊Aib؊N(Me)Ph (15) with Use of the Dde-
Linker: The immobilized linker system 11 was synthesized by the
method of Chhabra et al.[55] with aminomethylpolystyrene (500 mg,
0.57 mmol), 5-(4,4-dimethyl-2,6-dioxocyclohexylidene)-5-hydroxy-
pentanoic acid (291 mg, 1.14 mmol), DIPCDI (177 µL,
1.14 mmol), and HOAt (2.28 mL, 1.14 mmol of a 0.5  solution in
DMF). Resin 12: Resin 11 was swollen in DMF,
HϪAlaϪOtBu·HCl (416 mg, 2.29 mmol) and DIPEA (390 µL,
2.28 mmol) in DMF (5 mL) were then added, and the resin was
agitated at room temp. for 1 d. The resin was separated by filtration
and washed with DMF (3 ϫ) and DCM (3 ϫ). Resin 13: Resin 12
was swollen in DCM. TFA/DCM (5 mL, 1:1) and TIPS (230 µL)
were added, and the resin was agitated for 2 h. The resin was sepa-
rated by filtration and washed with DCM (3 ϫ), DMF (3 ϫ), and
DCM (2 ϫ). Resin 14: Resin 13 was swollen in DCM. A solution
of N,2,2-trimethyl-N-phenyl-2H-azirin-3-amine (2, 204 mg,
0.17 mmol) in DCM (4 mL) was added, and the resin was agitated
at room temp. overnight. The resin was separated by filtration and
washed with DCM (3 ϫ). H؊Ala؊Aib؊N(Me)Ph (15): Resin 14
was swollen in DMF. A solution of N2H4·H2O (100 µL) in DMF
(5 mL) was added, and the resin was agitated for 20 min. The resin
was separated by filtration and the cleavage was repeated. Finally,
the resin was washed with DMF (3 ϫ), and the filtrate was concen-
trated. CC (DCM/MeOH, 40:1, 1% Et3N) yielded 15 (91 mg, 61%).
1H NMR (300 MHz, CDCl3, 25 °C, TMS): δ ϭ 1.19 [d, J ϭ 7.0 Hz,
3 H, CH3 (Ala)], 1.51, 1.54 [2s, 6 H, 2 CH3 (Aib)], 3.13 [q, J ϭ
7.0 Hz, 1 H, CHα (Ala)], 3.26 [s, 3 H, CH3 of N(CH3)Ph],
7.41Ϫ7.23 (m, 5 arom. H) ppm. NH and NH2 could not be de-
tected. MS (ESI): m/z (%) ϭ 157 (46) oxazolone of [M Ϫ
HN(CH3)Ph ϩ H]ϩ, 264 (100) [M ϩ H]ϩ, 286 (6) [M ϩ Na]ϩ, 527
(5) [2M ϩ H]ϩ.
hexafluorophosphate; TIPS: triisopropylsilane.
Synthesis of Resin 10, IR Analysis. Resin 7: Carboxylpolystyrene
(50 mg, 0.20 mmol) was swollen in DCM. A solution of N,2,2-tri-
methyl-N-phenyl-2H-azirin-3-amine (2, 178 mg, 1.02 mmol) in
DCM (1.5 mL) was added, and the resin was agitated at room
temp. for 15 h. The resin was separated by filtration, washed with
DCM (3 ϫ) and Et2O (3 ϫ), and dried in vacuo. ATR-FT-IR (be-
ads): ν˜ ϭ 1632 cmϪ1 (carboxylpolystyrene, beads: ν˜ ϭ 1692 cmϪ1).
Resin 8: Resin 7 was swollen in THF. A solution of HCl (2 mL, 3
 in THF/H2O, prepared from concd. HCl and THF) was added,
and the resin was agitated at room temp. for 16 h. The resin was
separated by filtration, washed with THF (3 ϫ), DCM (3 ϫ), and
Et2O (3 ϫ), and dried in vacuo. ATR-FT-IR (beads): ν˜ ϭ 1716,
1645 cmϪ1. Resin 9: Remaining resin 8 (ca. 5 mg) was swollen in
DCM. A solution of 2 (13 mg, 0.08 mmol) in DCM (1.5 mL) was
added, and the resin was agitated at room temp. for 16 h. The resin
was separated by filtration, washed with DCM (3 ϫ) and Et2O (3
ϫ), and dried in vacuo. ATR-FT-IR (beads): ν˜ ϭ 1636 cmϪ1. Resin
10: Resin 9 was swollen in THF. A solution of HCl (2 mL, 3  in
THF/H2O, prepared from concd. HCl and THF) was added, and
the resin was agitated at room temp. for 7 h. The resin was sepa-
rated by filtration, washed with THF (3 ϫ), DCM (3 ϫ), and Et2O
(3 ϫ), and dried in vacuo. ATR-FT-IR (beads): ν˜ ϭ 1728, 1645
cmϪ1
.
Synthesis of Resin-bound Ala؊Aib؊Phe؊OtBu and its Hydrolysis:
Carboxylpolystyrene (103 mg, 0.20 mmol) was swollen in DCM. A
solution of HϪAlaϪOtBu·HCl (76 mg, 0.42 mmol) in DCM
(1 mL), a solution of PyBOP (208 mg, 0.40 mmol) in DCM (1 mL),
and DIPEA (0.2 mL, 1.17 mmol) were added, and the resin was
agitated at room temp. overnight. The resin was separated by fil-
tration, washed with DMF (4 ϫ), DCM (3 ϫ), and Et2O (2 ϫ),
and dried in vacuo. ATR-FT-IR (beads): ν˜ ϭ 1729, 1663, 1652
cmϪ1 (carboxylpolystyrene, beads: ν˜ ϭ 1685 cmϪ1). The new resin
was swollen in DCM. TFA/DCM (3 mL, 1:1) and TIPS (0.2 mL)
were added, and the resin was agitated for 2 h. The resin was sepa-
rated by filtration, washed with DMF (3 ϫ), DCM (3 ϫ), and Et2O
(2 ϫ), and dried in vacuo. ATR-FT-IR (beads): ν˜ ϭ 1733, 1641
cmϪ1. Half of the obtained resin was swollen in DCM. A solution
General Procedures
a) Attachment of the First Amino Acid: All manipulations were car-
ried out under N2. PAM or HM resin was swollen in THF. After
filtration, a solution of COCl2 in toluene (1.9 , 10 equiv.) and
THF (ca. 2.5 mL/1 g resin) were added to the resin, which was agi-
tated at room temp. for 2 h, and then washed with THF (2 ϫ) and
DCM (2 ϫ). In a separate vial, HϪAAϪOtBu·HCl (4 equiv.) was
dissolved in DIPEA (8 equiv.) and DCM [cAA ϭ 0.2 ]. This mix-
ture was added to the resin, any possibly occurring ammonium salt
being removed by filtration. The resin was agitated at room temp.
overnight and was then washed with DMF (3 ϫ) and DCM (3 ϫ).
of
N,2,2-trimethyl-N-phenyl-2H-azirin-3-amine
(2,
40 mg,
0.23 mmol) in DCM (2 mL) was added, and the resin was agitated
at room temp. overnight. The resin was separated by filtration,
washed with DCM (3 ϫ) and Et2O (2 ϫ), and dried in vacuo. ATR-
FT-IR (beads): ν˜ ϭ 1637 cmϪ1. The resulted resin was swollen in
THF. A solution of HCl (3 mL, 3  in THF/H2O, prepared from
concd. HCl and THF) was added, and the resin was agitated at
room temp. overnight. The resin was separated by filtration,
washed with THF (3 ϫ), DCM (3 ϫ), and Et2O (2 ϫ), and dried
in vacuo. ATR-FT-IR (beads): ν˜ ϭ 1719, 1636 cmϪ1. The resin was
swollen in DCM. A solution of PyBOP (103 mg, 0.20 mmol) in
DCM (1 mL), HϪPheϪOtBu·HCl (51 mg, 0.20 mmol) in DCM
(1 mL), and DIPEA (0.1 mL, 0.58 mmol) were added, and the resin
was agitated at room temp. overnight. The resin was separated by
filtration, washed with DMF (3 ϫ), DCM (3 ϫ), and Et2O (2 ϫ),
and dried in vacuo. ATR-FT-IR (beads): ν˜ ϭ 1730, 1652, 1646
cmϪ1. The resin-bound peptide was hydrolyzed by the method of
Westall et al.[53] (propionic acid/HCl, 1:1, at 130 °C, sealed tube).
The mixture was filtered, and the filtrate was concentrated and
dried in vacuo to yield a colorless powder. MS (ESI): m/z (%) ϭ
b) Removal of the tBu Protecting Group: The resin was swollen in
DCM. TFA in DCM (1 ϫ 5 s, 25%; 1 ϫ 30 min, 50%) and TIPS
(5%, in each case) were added, and the resin was agitated at room
temp. Afterwards, the resin was washed with DCM (3 ϫ), DMF
(2 ϫ), and DCM (3 ϫ).
c) Coupling with N,2,2-Trimethyl-N-phenyl-2H-azirin-3-amine (2):
The resin was swollen in DCM. A solution of 2 (4 equiv.) in DCM
(c2 ϭ 0.2 ) was added, and the resin was agitated at room temp.
overnight, and then washed with DCM (3 ϫ). Unconsumed 2 can
easily be recovered.
d) Hydrolysis of the Terminal Amide: The resin was swollen in THF.
HCl (ca. 3Ϫ4 mL/200 mg resin, 3  in THF/H2O, prepared from
concd. HCl and THF) was added, and the resin was agitated at
room temp. overnight, and then washed with THF (3 ϫ), DMF
(3 ϫ), and DCM (3 ϫ).
e) Coupling with H؊AA؊OtBu·HCl (Method A): The resin was
90 (2) [Ala ϩ H]ϩ, 104 (17) [Aib ϩ H]ϩ, 120 (60),[54] 166 (100) [Phe swollen in DCM. PyBOP (4 equiv.) in DCM was added, followed
ϩ H]ϩ. ATR-FT-IR (beads): ν˜ ϭ 1686 cmϪ1
.
by HϪAAϪOtBu·HCl (4 equiv.) in DCM and DIPEA (12 equiv.)
3824
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 3820Ϫ3827

The “Azirine/Oxazolone Method” under Solid-Phase Conditions
FULL PAPER
(cAA ϭ 0.2 ), and the resin was agitated at room temp. overnight,
and then washed with DCM (2 ϫ), DMF (2 ϫ), and DCM (3 ϫ).
[M Ϫ (AcϪAla) ϩ H]ϩ, 378 (52) [M ϩ H]ϩ, 400 (71) [M ϩ Na]ϩ.
Prep. TLC (DCM/MeOH, 20:1) yielded 26 (9 mg, 38%) as a color-
less powder. HPLC-MS: tR ϭ 14.8 min, m/z (%) ϭ 120 (37), 180
(100) [H Ϫ Phe Ϫ OMe ϩ H]ϩ, 199 (63) oxazolone of [M Ϫ
(PheϪOMe) ϩ H]ϩ, 265 (82) [M Ϫ (AcϪAla) ϩ H]ϩ, 378 (48)
[M ϩ H]ϩ, 400 (89) [M ϩ Na]ϩ.
f) Coupling with H؊AA؊OtBu·HCl) (Method B, according to Gau-
sepohl et al.):[47] The resin was swollen in DMF. HOBt (6 equiv.) in
DMF, PyBOP (4 equiv.) in DMF, NMM (2.3 equiv.), and then
HϪAAϪOtBu·HCl (4 equiv.) in DMF and NMM (4 equiv.) were
added (cAA ϭ 0.2 ). The resin was agitated at room temp. and,
after 10 and 20 min, additional NMM (each 2.3 equiv.) was added.
The resin was agitated at room temp. for 1 h (5 h for coupling to
Aib). The resin was washed with DMF (3 ϫ) and DCM (3 ϫ).
H؊Ala؊Aib؊Val؊Aib؊Phe؊OH (22): PAM resin (402 mg,
0.249 mmol) was treated as in General Procedure a) and dried in
vacuo. The synthesis was continued with half of the resin as in
General Procedures b), c), d), e), b), c), d), e), and h) to yield 22
(25 mg, 33%) as a colorless powder after prep. HPLC purification
and lyophilization.
HM resin (501 mg, 0.491 mmol) was treated as in General Pro-
cedure a) and dried in vacuo. The synthesis was continued with
25% of the resin as described in General Procedures b), c), d), e),
b), c), d), e), and h) to yield 22 (16 mg, 21%) as a colorless powder
after prep. HPLC purification and lyophilization. HPLC-MS: tR ϭ
10.4 min, m/z (%) ϭ 256 (11), 341 (48) oxazolone of [M Ϫ Phe ϩ
H]ϩ, 506 (100) [M ϩ H]ϩ. MS (ESI): m/z (%) ϭ 506 (100) [M ϩ
g) Coupling with H؊AA؊OtBu·HCl (Method C): The resin was
swollen in DCM. PfpOH (4 equiv.) in DCM, PyBOP (4 equiv.) in
DCM, and then HϪAAϪOtBu·HCl (4 equiv.) in DCM and DI-
PEA (12 equiv.) were added (cAA ϭ 0.2 ). The resin was agitated
at room temp. overnight, and then washed with DCM (2 ϫ), DMF
(2 ϫ), and DCM (3 ϫ).
h) Cleavage: The resin was swollen in DCM. HBr in AcOH (33%,
1 mL/100 mg resin) and two drops of water were added, and the
resin was agitated for 5 to 6 h. The resin was separated by filtration H]ϩ, 528 (31) [M ϩ Na]ϩ, 550 (9) [MNa ϩ Na]ϩ. IR (KBr): ν˜ ϭ
and washed with AcOH/DCM (1:1, 3 ϫ) and MeCN/DCM (1:1, 3
ϫ). The solvents were evaporated under reduced pressure and the
crude product was purified by HPLC. The purified product was ly-
ophilized.
3428 m, 3308 s, 3065 s, 2976 s, 2940 s, 2622 w, 1722 s, 1668 vs, 1529
vs, 1468 m, 1458 m, 1389 m, 1367 m, 1322 w, 1263 m, 1202 vs,
1139 s, 1004 w, 929 w, 837 w, 800 w, 722 m, 701 w, 598 w cmϪ1
.
1H NMR (500 MHz, [D6]DMSO, 25 °C, TMS): δ ϭ 0.81, 0.85 [2d,
J ϭ 6.8 Hz, 6 H, 2 CH3 (Val)], 1.34 [d, J ϭ 6.9 Hz, 3 H, CH3
(Ala)], 1.29, 1.35, 1.38, 1.41 [4s, 12 H, 4 CH3 (Aib)], 2.01 [oct., J ϭ
6.8 Hz, 1 H, CHβ (Val)], 2.94 [dd, J (CHAHB,CHα) ϭ 8.1, 2J ϭ
13.8 Hz, 1 H, CHAHB (Phe)], 3.03 [dd, J (CHAHB,CHα) ϭ 5.7,
2J ϭ 13.8 Hz, 1 H, CHAHB (Phe)], 3.85 [q, J ϭ 6.9 Hz, 1 H, CHα
(Ala)], 4.02 [t, J ϭ 7.3 Hz, 1 H, CHα (Val)], 4.41 [ddd, J
(CHα,CHAHB) ϭ 8.1, J (CHα,NH) ϭ 7.9, J (CHα,CHAHB) ϭ
5.7 Hz, 1 H, CHα (Phe)], 7.18Ϫ7.27 (m, 5 arom. H), 7.30 [d, J ϭ
7.7 Hz, 1 H, NH (Val)], 7.48 [d, J (NH,CHα) ϭ 7.9 Hz, 1 H, NH
H؊Ala؊Aib؊Phe؊OH (21): PAM resin (202 mg, 0.125 mmol)
was treated as in General Procedures a), b), c), d), e), and h) to
yield 21 (32 mg, 50%) as a colorless powder after prep. HPLC puri-
fication and lyophilization.
HM resin (201 mg, 0.197 mmol) was treated as in General Pro-
cedures a), b), c), d), e), and h) to yield 21 (33 mg, 40%) as a color-
less powder after precipitation in ether or prep. HPLC purification
and lyophilization. HPLC-MS: tR ϭ 11.6 min, m/z (%) ϭ 129 (31),
157 (47) oxazolone of [M Ϫ Phe ϩ H]ϩ, 251 (28) [M Ϫ Ala ϩ H]ϩ,
322 (100) [M ϩ H]ϩ, 344 (8) [M ϩ Na]ϩ. MS (ESI): m/z (%) ϭ
322 (100) [M ϩ H]ϩ, 344 (60) [M ϩ Na]ϩ. IR (KBr): ν˜ ϭ 3422 s,
3575 s, 3237 s, 3068 s, 3034 s, 2992 s, 2945 s, 2617 w, 1724 vs, 1671
vs, 1534 vs, 1500 s, 1467 m, 1457 m, 1443 m, 1392 m, 1369 m, 1332
w, 1266 s, 1201 vs, 1142 vs, 1031 m, 1003 w, 839 w, 800 m, 723 m,
ϩ
(Phe)], 7.5Ϫ8.5 [br. s, 3 H, NH3 (Ala)], 7.91, 8.48 [2s, 2 H, 2 NH
(Aib)], ca. 11.0Ϫ13.5 (br. s, 1 H, COOH) ppm. 13C NMR
(125 MHz, [D6]DMSO, 25 °C, TMS): δ ϭ 17.0 [q, CH3 (Ala)], 18.4,
19.3 [2q, 2 CH3 (Val)], 24.3, 24.9, 25.0, 25.1 [4q, 4 CH3 (Aib)], 30.1
[d, CHβ (Val)], 36.8 [t, CH2 (Phe)], 48.4 [d, CHα (Ala)], 53.6 [d,
CHα (Phe)], 56.1, 56.5 [2s, 2 Cα (Aib)], 58.4 [d, CHα (Val)], 126.4
(d, arom. CHp), 128.1 (d, arom. CHm), 129.2 (d, arom. CHo), 137.5
(s, arom. C), 169.1 [s, CO (Ala)], 170.4 [s, CO (Val)], 172.6 [s, CO
(Phe)], 173.3, 173.5 [2s, 2 CO (Aib)] ppm.
1
702 m, 641 m, 518 w cmϪ1. H NMR (600 MHz, [D6]DMSO, 25
°C, TMS): δ ϭ 1.32 [d, J ϭ 7.0 Hz, 3 H, CH3 (Ala)], 1.34, 1.35 [2s,
2
6 H, CH3 (Aib)], 2.95 [dd, J (CHAHB,CHα) ϭ 8.3, J ϭ 13.7 Hz,
1 H, CHAHB (Phe)], 3.08 [dd, J (CHAHB,CHα) ϭ 5.3, 2J ϭ 13.7 Hz,
1 H, CHAHB (Phe)], 3.82Ϫ3.83 [m, 1 H, CHα (Ala)], 4.44 [ddd, J
(CHα,CHAHB) ϭ 8.3, J (CHα,NH) ϭ 8.0, J (CHα,CHAHB) ϭ
5.3 Hz, 1 H, CHα (Phe)], 7.18Ϫ7.28 (m, 5 arom. H), 7.53 [d, J (NH,
CHα) ϭ 8.0 Hz, 1 H, NH (Phe)], 8.04 [br. s, 3 H, NH3ϩ (Ala)], 8.35
[s, 1 H, NH (Aib)], ca. 12.0Ϫ13.5 (br. s, 1 H, COOH) ppm. 13C
NMR (150 MHz, [D6]DMSO, 25 °C, TMS): δ ϭ 17.1 [q, CH3
(Ala)], 24.4, 25.0 [2q, 2 CH3 (Aib)], 36.7 (t, CH2), 48.3 [d, CHα
(Ala)], 53.5 [d, CHα (Phe)], 56.3 [s, Cα (Aib)], 126.4 (d, arom. CHp),
128.1 (d, arom. CHm), 129.3 (d, arom. CHo), 137.5 (s, arom. C),
168.9 [s, CO (Ala)], 172.7 [s, CO (Phe)], 172.9 [s, CO (Aib)] ppm.
H؊Ala؊Aib؊Ala؊Gln؊Aib؊Val؊OH (23): PAM resin (202 mg,
0.125 mmol) was treated as described in General Procedures a), b),
c), d), e), b), f), b), c), d), f) and h) to yield 23 (21 mg, 25%) as a
colorless powder after prep. HPLC purification and lyophilization.
HPLC-MS: tR ϭ 8.3 min, m/z (%) ϭ 441 (10) oxazolone of [M Ϫ
Val ϩ H]ϩ, 558 (100) [M ϩ H]ϩ. MS (ESI): m/z (%) ϭ 558 (29)
[M ϩ H]ϩ, 580 (100) [M ϩ Na]ϩ, 602 (74) [MNa ϩ Na]ϩ. IR
(KBr): ν˜ ϭ 3315 s, 3065 m, 2984 m, 2942 m, 2614 w, 1665 vs, 1533
s, 1467 m, 1455 m, 1424 w, 1390 m, 1368 w, 1332 w, 1267 m, 1201
s, 1140 s, 1042 w, 1004 w, 930 w, 836 w, 800 w, 722 w, 598 w, 518
w cmϪ1. 1H NMR (500 MHz, [D6]DMSO, 25 °C, TMS): δ ϭ 0.82,
0.85 [2d, J ϭ 6.8 Hz, 6 H, 2 CH3 (Val)], 1.23 [d, J ϭ 7.0 Hz, 3 H,
Ac؊Ala؊Aib؊Phe؊OMe (26) (Derivatization of 21): The crude
product 21 (from a 0.062 mmol batch) was dissolved in (NH4)2CO3 CH3 (Ala)], 1.36, 1.38 [2s, 6 H, 2 CH3 (Aib)], 1.38 [d, J ϭ 6.9 Hz,
(2.4 mL, 0.1 ), Ac2O/MeOH (6 mL, 1:3) was then added, and the
solution was stirred at room temp. for 1 h. After lyophilization, the
residue was dissolved in MeOH/H2O (2 mL, 10:1), and a solution
of CH2N2 in Et2O (ca. 2 mL, ca. 1 ) was added. The solution was
stirred at room temp. and then concentrated in vacuo. HPLC-MS:
tR ϭ 14.8 min, m/z (%) ϭ 120 (45), 180 (92) [H Ϫ Phe Ϫ OMe ϩ
H]ϩ, 199 (84) oxazolone of [M Ϫ (PheϪOMe) ϩ H]ϩ, 265 (100)
3 H, CH3 (Ala)], 1.40 [s, 6 H, 2 CH3 (Aib)], 1.84Ϫ1.93 [m, 2 H,
CH2 (Gln)], 2.01Ϫ2.08 [m, 1 H, CHβ (Val)], 2.10Ϫ2.14 [m, 2 H,
CH2 (Gln)], 3.84 [q, J ϭ 7.0 Hz, 1 H, CHα (Ala)], 4.06Ϫ4.10 [m, 2
H, CHα (Val), CHα (Gln)], 4.15 [quint., J ϭ 6.9 Hz,1 H, CHα (Ala)],
6.89 [s, 1 H, NH2 (Gln)], 7.12 [d, J ϭ 8.5 Hz, 1 H, NH (Val)], 7.40
(s, 1 H, NH2 (Gln)], 7.66 [d, J ϭ 6.3 Hz, 1 H, NH (Ala)], ca.
ϩ
7.7Ϫ8.4 [br. s, 3 H, NH3 (Ala)], 7.84 [d, J ϭ 7.1 Hz, 1 H, NH
Eur. J. Org. Chem. 2004, 3820Ϫ3827
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3825

S. Stamm, H. Heimgartner
FULL PAPER
(Gln)], 7.86 [s, 1 H, NH (Aib)], 8.63 [s, 1 H, NH (Aib)], ca.
11.8Ϫ13.1 (br. s, 1 H, COOH) ppm. 13C NMR (125 MHz,
[D6]DMSO, 25 °C, TMS): δ ϭ 17.0, 17.5 [2q, 2 CH3 (Ala)], 18.0,
19.1, [2q, 2 CH3 (Val)], 24.2, 24.6, 24.9, 25.7 [4q, 4 CH3 (Aib)],
27.4 [t, CH2 (Gln)], 29.9 [d, CHβ (Val)], 31.8 [t, CH2 (Gln)], 48.3,
48.9 [2d, 2 CHα (Ala)], 53.1 [d, CHα (Gln)], 56.2, 56.2 [2s, 2 Cα
(Aib)], 57.3 [d, CHα (Val)], 169.3 [s, CO (Ala)], 171.0 [s, CO (Gln)],
172.3 [s, CO (Ala)], 172.8 [s, CO (Val)], 173.4, 173.7 [2s, 2 CO
(Aib)], 174.2 (s, CONH2) ppm.
25.8 [4q, 4 CH3 (Aib)], 29.6 [d, CHβ (Val)], 43.4 [t, CH2α (Gly)],
47.7 [d, CHα (Ala)], 55.9, 56.3 [2s, 2 Cα (Aib)], 57.6 [d, CHα (Val)],
167.8 [s, CO (Val)], 168.3 [s, CO (Gly)], 173.7 [s, CO (Aib)], 174.0
[s, CO (Ala)], 174.1 [s, CO (Aib)] ppm.
Acknowledgments
We thank Dr. Daniel Obrecht, Polyphor Ltd., Allschwil (Switzer-
land) for ongoing advice and valuable discussion. Financial sup-
port by the Swiss National Science Foundation and F. Hoffmann-
La Roche AG, Basel (Switzerland) is gratefully acknowledged.
H؊Leu؊Aib؊Leu؊Aib؊Phe؊OH (24): PAM resin (201 mg,
0.125 mmol) was treated as described in General Procedures a), b),
c), d), e), b), c), d), and e) and dried in vacuo. One part (14%) was
cleaved as described in General Procedure h), and the crude prod-
uct was analyzed by HPLC-MS. With the other part (86%) a se-
cond coupling was performed as described in General Procedure
g). Cleavage as described in General Procedure h) yielded 24
(15 mg, 20%) as a colorless powder after prep. HPLC purification
and lyophilization. HPLC-MS: tR ϭ 12.0 min, m/z (%) ϭ 397 (15)
oxazolone of [M Ϫ Phe ϩ H]ϩ, 562 (100) [M ϩ H]ϩ. IR (KBr):
ν˜ ϭ 3423 m, 3320 s, 3064 m, 3034 m, 2962 s, 2939 s, 2874 m, 1723
sh, 1668 vs, 1529 vs, 1468 m, 1441 m, 1388 m, 1367 m, 1269 m,
1202 vs, 1140 s, 1081 w, 1031 w, 941 w, 878 w, 837 w, 800 w, 722
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[8]
˚
A. Johansson, E. Akerblom, K. Ersmark, G. Lindeberg, A.
[D6]DMSO, 25 °C, TMS): δ ϭ 0.83, 0.86, 0.90, 0.92 [4d, J ϭ
6.3 Hz, 12 H, 4 CH3 (Leu)], 1.30, 1.36, 1.38, 1.39 [4s, 12 H, 4 CH3
(Aib)], 1.40Ϫ1.65 [m, 6 H, 2 CH2 (Leu), 2 CHγ (Leu)], 2.95 [dd, J
(CHAHB,CHα) ϭ 8.3, 2J ϭ 13.8 Hz, 1 H, CHAHB (Phe)], 3.04 [dd,
J (CHAHB,CHα) ϭ 5.4, 2J ϭ 13.8 Hz, 1 H, CHAHB (Phe)], 3.77
[br. s, 1 H, CHα (Leu)], 4.15 [m, 1 H, CHα (Leu)], 5.41 [ddd, J
(CHα,CHAHB) ϭ 8.3, J (CHα,NH) ϭ 7.8, J CHα,CHAHB) ϭ
5.4 Hz, 1 H, CHα (Phe)], 7.18Ϫ7.26 (m, 5 arom. H), 7.51 [d, J
(NH,CHα) ϭ 7.8 Hz, 1 H, NH (Phe)], 7.59 [d, J ϭ 7.4 Hz, 1 H,
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[11]
[12]
ϩ
NH (Leu)], 7.80 [s, 1 H, NH (Aib)], 8.08 [br. s, 3 H, NH3 (Leu)],
1997, 38, 2531Ϫ2534.
[13]
8.58 [s, 1 H, NH (Aib)], ca. 12.4Ϫ13.0 (br. s, 1 H, COOH) ppm.
13C NMR (150 MHz, [D6]DMSO, 25 °C, TMS): δ ϭ 21.5, 21.9,
22.5, 23.1 [4q, 4 CH3 (Leu)], 23.6, 24.0 [2d, 2 CHγ (Leu)], 24.0,
24.2, 25.0, 25.5 [4q, 4 CH3 (Aib)], 36.7 [t, CH2 (Phe)], 39.5, 39.7
[2t, 2 CH2(Leu)], 51.1, 51.8 [2d, 2 CHα (Leu)], 53.6 [d, CHα (Phe)],
55.9, 56.4 [2s, 2 Cα (Aib)], 126.3 (d, arom. CHp), 128.0 (d, arom.
CHm), 129.1 (d, arom. CHo), 137.5 (s, arom. C), 168.4, 171.3 [2s, 2
CO (Leu)], 172.6 [s, CO (Phe)], 173.3, 173.8 [2s, 2 CO (Aib)] ppm.
W. F. DeGrado, Adv. Prot. Chem. 1988, 39, 51Ϫ124.
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R. T. N. Luykx, A. Linden, H. Heimgartner, Helv. Chim. Acta
H؊Val؊Aib؊Gly؊Aib؊Ala؊OH (25): PAM resin (200 mg,
0.124 mmol) was treated as described in General Procedures a), b),
c), d), e), b), c), d), e) and h) to yield 25 (20 mg, 31%) as a colorless
powder, after prep. HPLC purification and lyophilization. HPLC-
MS: tR ϭ 9.8 min, m/z (%) ϭ 327 (51) oxazolone of [M Ϫ Ala ϩ
H]ϩ, 416 (100) [M ϩ H]ϩ. IR (KBr): ν˜ ϭ 3315 s, 3066 s, 2985 s,
2943 s, 2642 w, 1667 vs, 1537 vs, 1468 m, 1387 m, 1367 m, 1335 w,
1296 m, 1248 m, 1202 vs, 1140 s, 1018 w, 980 w, 946 w, 837 w, 800
[19]
[20]
[21]
[22]
1
w, 722 m, 662 w, 598 w, 562 w, 518 w cmϪ1. H NMR (500 MHz,
[23]
[D6]DMSO, 25 °C, TMS): δ ϭ 0.94, 0.95 [2d, J ϭ 6.7 Hz, 6 H, 2
CH3 (Val)], 1.27 [d, J ϭ 7.3 Hz, 3 H, CH3 (Ala)], 1.38, 1.39, 1.41
[3s, 12 H, 4 CH3 (Aib)], 2.10 [oct., J ϭ 6.7 Hz, 1 H, CHβ (Val)],
3.52 [dd, J (NH,CHAHB) ϭ 5.6, 2J ϭ Ϫ16.3 Hz, 1 H, CHAHB
(Gly)], 3.60 [d, J ϭ 6.2 Hz, 1 H, CHα (Val)], 3.65 [dd, J
(CHAHB,NH) ϭ 5.7, 2J ϭ Ϫ16.3 Hz, 1 H, CHAHB (Gly)], 4.16
[quint., J ϭ 7.3 Hz, 1 H, CHα (Ala)], 7.47 [d, J ϭ 7.3 Hz, 1 H, NH
(Ala)], ca. 7.6Ϫ8.5 [br. s, 3 H, NH3ϩ (Val)], 7.70 [s, 1 H, NH (Aib)],
8.01 [dd, J (NH,CHAHB) ϭ 5.7, J (NH,CHAHB) ϭ 5.6 Hz, 1 H,
NH (Gly)], 8.70 [s, 1 H, NH (Aib)], ca. 11.8Ϫ13.0 (br. s, 1 H,
COOH) ppm. 13C NMR (125 MHz, [D6]DMSO, 25 °C, TMS): δ ϭ
17.0 [q, CH3 (Ala)], 17.6, 18.4, [2q, 2 CH3 (Val)], 23.7, 23.9, 25.8,
[24]
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1903Ϫ1915.
After PyBOP-mediated coupling of carboxylpolystyrene and
HϪAlaϪOtBu, the tBu ester was hydrolyzed in TFA/DCM.
The resin was treated with N,2,2-trimethyl-N-phenyl-2H-azirin-
[28]
[29]
3826
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 3820Ϫ3827

The “Azirine/Oxazolone Method” under Solid-Phase Conditions
FULL PAPER
3-amine (2), the terminal amide was then hydrolyzed with 3 
HCl, and PyBOP-mediated coupling with HϪPheϪOtBu fi-
nally yielded the resin-bound tripeptide AlaϪAibϪPheϪOtBu.
After every reaction, the resin was examined by ATR-FT-IR
spectroscopy. Acidic hydrolysis (propionic acid/HCl, 130 °C) of
the resin-bound tripeptide gave Ala, Aib and Phe, which were
detected by ESI-MS.
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Ac2O/MeOH (1:3) was then added, and the solution was stirred
at room temp. for 1 h. After lyophilization, the residue was
dissolved in MeOH/H2O (10:1) and a solution of CH2N2 in
Et2O was added. The solution was stirred at room temp. and
then concentrated in vacuo.
‘‘Special Issue: Peptaibols/Peptaibiotics’’: H. Brückner (Ed.), J.
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[31]
[46]
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[32]
[33]
Nevertheless a minor product (m/z ϭ 658, [M ϩ Val ϩ H]ϩ)
was detected. Probably the amide group in the side chain of
Gln had partially hydrolyzed in 3  HCl, so that two molecules
of Val could add in the subsequent coupling reaction.
M. Crisma, A. Barazza, F. Formaggio, B. Kaptein, Q. Broxter-
mann, J. Kamphuis, C. Toniolo, Tetrahedron 2001, 57,
2813Ϫ2825.
[34]
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´
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Some loss of peptide was observed during deprotection of tBu
esters with TFA on hydroxymethyl polystyrene resin. When
hydroxyethylpolystyrene resin was used no cleavage from the
support was achieved with either trifluoromethanesulfonic acid
or HBr in acetic acid.
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F. C. Westall, J. Scotchler, A. B. Robinson, J. Org. Chem. 1972,
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Fragment of Phe (shown by MS3).
[54]
[55]
[41]
[42]
[43]
Performed by C. A. T. GmbH & Co., Tübingen, Germany.
S. R. Chhabra, H. Parekh, A. N. Khan, B. W. Bycroft, B. Kel-
lam, Tetrahedron Lett. 2001, 42, 2189Ϫ2192.
˚
Interchim Uptisphere HDO C18, 120 A, 3 µm, 200 ϫ 2.0 mm.
The crude product was dissolved in aq. (NH4)2CO3 (0.1 ),
Received May 11, 2004
Eur. J. Org. Chem. 2004, 3820Ϫ3827
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3827