3-Aminobicyclo[1.1.1]pentane-1-carboxylic Acid Derivatives: Synthesis and Incorporation into Peptides

Article abstract of DOI:10.1002/ejoc.200300554

We have developed an efficient synthesis of derivatives of 3-aminobicyclo[1.1.1]pentane-1-carboxylic acid (7, 8, 9, and 10) starting from [1.1.1]propellane (3). These rigid analogues of γ-aminobutyric acid have been incorporated into linear and cyclic peptides using solution chemistry and solid-phase techniques. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200300554

FULL PAPER
3-Aminobicyclo[1.1.1]pentane-1-carboxylic Acid Derivatives: Synthesis and
Incorporation into Peptides
Michael Pätzel,*[a] Maximilian Sanktjohanser,[a][‡] Alexander Doss,[a] Peter Henklein,[b] and
Günter Szeimies[a]
Keywords: Amino acids / Bicyclo[1.1.1]pentane / Peptides / Small ring systems
We have developed an efficient synthesis of derivatives of 3-
aminobicyclo[1.1.1]pentane-1-carboxylic acid (7, 8, 9, and
and cyclic peptides using solution chemistry and solid-
phase techniques.
10) starting from [1.1.1]propellane (3). These rigid analogues ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
of γ-aminobutyric acid have been incorporated into linear
Germany, 2004)
Introduction
through a multi-step procedure.[5] Treatment of the di-
bromocyclopropane 2 with 2.2 equiv. of methyllithium gen-
The chemistry of bicyclo[1.1.1]pentanes has gained con- erated the [1.1.1]propellane 3, which was reacted with biace-
siderable attention over the years and a broad range of dif- tyl under irradiation with an UV lamp to afford the dike-
ferently substituted derivatives have been synthesized.[2] tone 4. Compound 4 was then subjected to the haloform
Among them, the recently prepared 2-(3Ј-substituted bi- reaction followed by esterfication of both carboxylic acid
cyclo[1.1.1]pentyl)glycines represent the only examples of functions. Basic hydrolysis of the diester with one equiv. of
amino acids bearing the bicyclo[1.1.1]pentane moiety.[3]
That study has prompted us to report our investigations and led to 6.[5]
NaOH led to the selective saponification of one ester group
concerning the synthesis of derivatives of 3-aminobicyclo-
An alternative synthesis of the carboxylic acid 6 was car-
[1.1.1]pentane-1-carboxylic acid 1. This amino acid can be ried out by using the addition of a Grignard reagent to the
considered as a rigid analogue of 4-aminobutyric acid, [1.1.1]propellane 3 and subsequent trapping of the inter-
which is known to act as a neurotransmitter in the brain.
mediate with an electrophile.[6,2b] Specifically, the addition
of phenylmagnesium bromide to the central bond of the
propellane
3 afforded 3-phenylbicyclo[1.1.1]pentylmag-
nesium bromide, which could be trapped by methyl chloro-
formate to give compound 5. Oxidation of the precursor
5 with NaIO4/RuCl3 was performed according to standard
procedures and gave 6 in 49% yield.[7]
We investigated two different Curtius rearrangement pro-
cedures for the conversion of carboxylic acid 6 to the differ-
ently protected amino acid ester 7. According to the Yam-
ada-variation,[8] an isocyanate intermediate is produced
upon refluxing one equiv. of acid 6, diphenyl phosphorazid-
ate (DPPA) and triethylamine. This intermediate is trapped
by the alcoholic solvent (Method A) to afford the protected
amino acid 7.
We also applied a modification of this procedure in an
attempt to improve the yield.[9] Refluxing acid 6 with DPPA
in toluene for 2 h afforded the corresponding isocyanate,
which was detected by IR spectroscopy. tert-Butyl alcohol
was then added and the mixture was heated under reflux
for a further 12 h (Method B). After workup, however, the
amino acid ester 7 was isolated in lower yield than it was
when prepared according to Method A. Attempts to add
fluorenylmethanol to the isocyanate, to obtain the Fmoc-
Furthermore, unnatural amino acids having special
properties, such as rigidity, bulkiness or conformational
constraints, are of interest as building blocks for the syn-
thesis of modified peptides.[4]
Results and Discussion
We chose the 1,3-bicyclo[1.1.1]pentane-1-carboxylic acid
6 as the key intermediate for the synthesis of target com-
pound 1. This substance has been prepared by Michl et al.
[a]
Institut für Chemie, Humboldt-Universität Berlin,
Brook Taylor-Str.2 12489 Berlin
Fax: (internat) ϩ 49-30-20936940
E-mail: Michael.Paetzel@rz.hu-berlin.de
[b]
Institut für Biochemie, Humboldt-Universität Berlin,
Monbijoustr. 2, 10117 Berlin
[‡]
See also ref.[1]
Eur. J. Org. Chem. 2004, 493Ϫ498
DOI: 10.1002/ejoc.200300554
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
493

M. Pätzel, M. Sanktjohanser, A. Doss, P. Henklein, G. Szeimies
FULL PAPER
Scheme 1
Scheme 2
protected amino ester directly, failed and complex product
mixtures were obtained.[10] The structure of compound 7
was established by spectroscopic methods and by X-ray
analysis. The signals of the methylene groups of the bicyclo-
[1.1.1]pentane cage at δ ϭ 2.13 ppm are typical for this su-
bunit, as are the chemical shifts of the bridged carbon
atoms at δ ϭ 35 and 45 ppm. The X-ray analysis revealed
[11]
˚
that, at 1.852 A, the inter-bridgehead distance C1ϪC3
is in the range found for similar bicyclo[1.1.1]pentanes.[2,12]
The orthogonal protective groups of compound 7 were
removed according to standard procedures to give both the
N-Boc-protected amino acid 8 and the amino acid ester 9.
Treatment of 8 with TFA and Fmoc protection resulted in
the formation of compound 10, which is a suitable deriva-
tive for solid-phase peptide synthesis.
Scheme 4
We investigated the formation of dipeptides to get more
insight into the reactivity of the sterically demanding mono-
protected derivatives 8 and 9. A series of dipeptides 11Ϫ14
were synthesized, mostly in high yields, by coupling differ-
ent amino acids with both the N- and C-termini of the 3-
aminobicyclo[1.1.1]pentanecarboxylic acid using DPPA as
condensation reagent.
The lower yield of the bis-bicyclo[1.1.1]pentane dipeptide
14 is possibly a consequence of steric congestion of the bi-
cyclo[1.1.1]pentane cages. Compound 14 is practically in-
soluble in most organic solvents. Interestingly, we observed
two signals having a significant shift difference (almost
1
0.2 ppm) for the methylene groups in the H NMR spec-
trum.
In the further course of our investigations, we explored
the possibility of incorporating the protected 3-aminobicy-
clo[1.1.1]pentane-1-carboxylic acid 10 into longer peptides.
Liquid-phase chemistry was applied for the synthesis of
the tetrapeptide 15, whereas we used solid-phase chemistry
for the preparation of the peptides 16 and 17. The se-
Scheme 3
494
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Carboxylic Acid Derivatives and Peptides
FULL PAPER
Methyl 1-Phenylbicyclo[1.1.1]pentane-3-carboxylate (5): Phenyl-
magnesium bromide (100 mL, 0.107 mol, 1.07  in diethyl ether)
was added at 0 °C to a solution of [1.1.1]propellane (3) in diethyl
ether,[16] which was prepared from 2 (40.5 g, 0.15 mol) and MeLi
(232 mL, 0.33 mol, 1.42  in diethyl ether). The mixture was stirred
for 4 days at room temperature before being added dropwise within
5 h to a solution of methyl chloroformate (9.92 g, 0.105 mol) in
diethoxymethane (100 mL). The mixture was warmed to room tem-
perature and then stirred for a further 12 h. The reaction mixture
was quenched by the addition of HCl (2 , 100 mL) and the organic
phase was washed with brine (3 ϫ 80 mL). The combined aqueous
phases were extracted with diethyl ether. After drying the combined
organic phases with MgSO4, the solvent was evaporated under re-
duced pressure and the residue was distilled (b.p. 115 °C/10Ϫ3 Torr)
to afford 5 as a yellowish oil. [9.7 g, 32% (from 2)]. The NMR
spectra are in accordance with literature data.[7]
quences for 16 and 17 are derived from the neuropeptide
cholecystokinine (CCK).[13] The CCK-B subreceptor in the
brain acts as neurotransmitter and neuromodulator. The
shortest CCK fragment that exhibits a high affinity for the
CCK-B receptor is the C-terminal CCK-4 having the se-
quence TrpϪMetϪAspϪPhe.[14]
3-(Methoxycarbonyl)bicyclo[1.1.1]pentane-1-carboxylic Acid (6):
Ester
5 (1.01 g, 5 mmol), NaIO4 (19.25 g, 90 mmol) and
RuCl3·H2O (41% Ru, 37 mg, 0.15 mmol) were added to a mixture
of CCl4 (30 mL), MeCN (30 mL) and water (45 mL). After stirring
the mixture for 48 h, a further charge of RuCl3·H2O (20 mg) was
added and the reaction was stirred for a further 48 h. The reaction
mixture was diluted with water (90 mL) and was extracted with
dichloromethane (1 ϫ 150 mL, 1 ϫ 100 mL, 3 ϫ 50 mL). The sol-
vents of the combined organic phases were evaporated almost com-
pletely, the residue was dissolved in diethyl ether (200 mL) and
charcoal was added. After drying (MgSO4) and filtration through
a pad of silica gel, followed by evaporation of the solvent, the crude
product 6 was obtained. Crystallization from heptane/chloroform
gave the pure product 6 (338 mg, 49%). M.p. 138Ϫ139 °C (ref.[7]
139.5Ϫ140 °C). The NMR spectra are in accordance with litera-
ture data.[7]
Methyl 3-(tert-Butoxycarbonylamino)bicyclo[1.1.1]pentane-1-carb-
oxylate (7): Method A: DPPA (22.0 mL, 28.1 g, 102 mmol) was
added dropwise to a solution of carboxylic acid 6 (17.3 g,
102 mmol) and triethylamine (14.2 mL, 10.4 g, 102 mmol) in dry
tBuOH (320 mL). The solution was stirred at room temperature
for 4 h and then heated under reflux for 24 h. The solvent was
evaporated under reduced pressure and the residue was extracted
with tert-butyl methyl ether (3 ϫ 50 mL). The solution was washed
with aqueous sodium hydrogencarbonate solution and dried
(MgSO4). Evaporation of the solvent under reduced pressure af-
forded amino acid derivative 7 as colourless crystals (20.3 g, 83%).
Scheme 5
In summary, we have described the synthesis of some C-
and N-terminal protected derivatives of 3-aminobicyclo-
[1.1.1]pentanecarboxylic acid (1). These compounds re-
present rigid derivatives of 4-aminobutyric acid and can
easily be incorporated into peptides by classical coupling
methods as well as by solid-phase techniques.
Method B: A solution of carboxylic acid 6 (6.87 g, 40 mmol), tri-
ethylamine (6.1 mL, 4.4 g, 44 mmol) and DPPA (8.65 mL, 11.0 g,
40 mmol) in toluene (200 mL) was stirred for 30 min at ambient
temperature and then under reflux for 2 h. After formation of the
intermediate isocyanate (monitored by IR spectroscopy), dry tert-
butyl alcohol (8.88 g, 120 mmol) was added. The solution was
heated under reflux for 24 h and then the solvent was evaporated
under reduced pressure. The residue was extracted with tert-butyl
methyl ether (3 ϫ 20 mL). The solution was washed with aqueous
sodium hydrogencarbonate solution and dried (MgSO4). Evapor-
ation of the solvent under reduced pressure afforded the amino
acid derivative 7 (4.03 g, 48%) as colourless crystals. M.p. 123 °C.
IR (KBr): ν˜ ϭ 3442, 1730, 1685, 1653 cmϪ1. 1H NMR (400 MHz):
δ ϭ 1.37 (s, 9 H), 2.13 (s, 6 H), 3.60 (s, 3 H) ppm. 13C NMR
(400 MHz): δ ϭ 28.1 (CH3), 34.9 (C), 45.2 (C), 51.4 (CH3), 53.5
(CH2), 77.9 (C), 154.4 (C), 169.4 (C) ppm. MS (79 eV): m/z ϭ 168
(4), 154 (14), 57 (100), 41 (22). C12H19NO4 (241.28): calcd. C 59.73,
H 7.94, N 5.80; found C 59.67, H 7.91, N 5.60.
Experimental Section
Melting points are uncorrected. NMR: Bruker AC-300, DPX-300;
spectra were recorded in [D6]DMSO if not stated otherwise. MS:
Finnegan MAT 90. HRMS: Finnegan MAT 95. Elemental analy-
sis: CHNS-932 Analyser (Leco). TentaGel-S-Trt-(L)-Phe-Fmoc re-
sin used in the synthesis of peptides 16 and 17 was purchased at
Fa. PepChem, Tübingen, Germany. Protected amino acids and
coupling reagents are from Nova Biochem.
Abbreviations: DIEA ϭ N,N-diisopropylethylamine; DPPA ϭ di-
phenylphosphoryl azide; HBTU
N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate; Fmoc ϭ 9-
fluorenylmethoxycarbonyl; DIC Diisopropylcarbodiimide;
HOAt ϭ 1-Hydroxy-7-azabenzotriazole.
ϭ O-(1H-benzotriazol-1-yl)-
ϭ
Compounds 2,[15] 3[16] and 6[5] were prepared according to litera-
ture procedures.
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M. Pätzel, M. Sanktjohanser, A. Doss, P. Henklein, G. Szeimies
FULL PAPER
3-(tert-Butoxycarbonylamino)bicyclo[1.1.1]pentane-1-carboxylic
Acid (8): NaOH (2.40 g, 60 mmol) in MeOH (40 mL) was added
pound (4.6 mmol) in DMF (20 mL). Subsequently, triethylamine
(1.40 mL, 1.01 g, 10 mmol) dissolved in DMF (15 mL) was added
dropwise within 10 min to a solution of 7 (11.2 g, 46.2 mmol) in dropwise within 10 min to the reaction mixture. The mixture was
MeOH (200 mL). The mixture was heated under reflux for 3 h.
After evaporation of the solvent under reduced pressure, the resi-
warmed to room temp. and stirred for 12 h before a mixture of
ethyl acetate and benzene (2:1, 200 mL) was added. The solution
due was dissolved in water. The aqueous phase was extracted three was washed with 2  HCl (2 ϫ 50 mL), water (2 ϫ 50 mL) and
times with dichloromethane and then acidified with dilute HCl (pH
6). The combined organic phases were dried (MgSO4), and the sol-
vents were evaporated under reduced pressure. The residue was
recrystallised from diethyl ether to yield carboxylic acid 8 (9.30 g,
brine (50 mL). (In the case of the Boc-protected dipeptide 14, 5%
aqueous citric acid was used instead of 2  HCl.) The organic phase
was dried (MgSO4). After evaporation of the organic solvents un-
der reduced pressure, the dipeptides 11Ϫ14 were obtained as color-
88%). M.p. 172 °C (decomp.). IR (KBr): ν˜ ϭ 3600Ϫ3200, 2987, less crystals.
1698, 1645 cmϪ1. H NMR (400 MHz, CDCl3): δ ϭ 1.46 (s, 9 H),
1
Methyl
{[3-(tert-Butoxycarbonylamino)bicyclo[1.1.1]pent-1-yl]-
2.30 (s, 6 H) ppm. 13C NMR (400 MHz): δ ϭ 28.1 (CH3), 35.2 (C),
carbonyl}glycinate (11): Yield: 89%. M.p. 160 °C (MeOH). IR
(KBr): ν˜ ϭ 3323, 2981, 1691 cmϪ1. 1H NMR (400 MHz): δ ϭ 1.38
(s, 9 H), 2.06 (s, 6 H), 3.62 (s, 3 H), 3.77 (d, J ϭ 6 Hz, 2 H) ppm.
13C NMR (100 MHz): δ ϭ 28.6 (CH3), 37.1 (C), 40.9 (CH2), 45.2
(C), 52.1 (CH3), 53.6 (CH2), 78.3 (C), 155.0 (C), 169.6 (C), 170.7
(C) ppm. MS (70 eV): m/z ϭ 299 ([Mϩ ϩ 1], 1), 109 (100).
C14H22N2O5 (298.34): calcd. C 56.36, H 7.42, N 9.39; found C
56.23, H 7.39, N 9.12.
45.0 (C), 53.3 (CH2), 77.7 (C), 154.5 (C), 170.8 (C) ppm. MS
(79 eV): m/z ϭ 127 (4), 126 (5), 57 (100), 41 (23). C11H17NO4
(227.26): calcd. C 58.14, H 7.54, N 6.16; found C 58.44, H 7.48,
N 6.22.
Methyl 3-Aminobicyclo[1.1.1]pentane-1-carboxylate Hydrochloride
(9): Gaseous HCl was bubbled for 1 h through a solution of the
Boc-protected amino acid 7 (21.4 g, 88.4 mmol) in dry methanol
(370 mL) at Ϫ78 °C. The temperature was maintained below Ϫ50
°C. The solution was warmed to room temperature and evapor-
ation of the solvents under reduced pressure afforded the spectro-
scopically pure amino acid 9 (15.4 g, 98%). For further purification,
compound 9 was recrystallised from methanol. M.p. 209 °C (de-
Compound 12: Yield: 83%. M.p. 117 °C (diethyl ether/pentane). IR
(KBr): ν˜ ϭ 3305, 2922, 1666 cmϪ1. 1H NMR (400 MHz): δ ϭ 2.23
(s, 6 H), 2.70Ϫ2.97 (m, 2 H), 3.61 (s, 3 H), 4.12 (m, 1 H), 4.95 (m,
2 H), 7.18Ϫ7.68 (m, 10 H) ppm. 13C NMR (100 MHz): δ ϭ 35.5
(C), 37.5 (CH2), 45.2 (C), 51.4 (CH3), 53.7 (CH2), 56.1 (CH), 65.1
(CH2), 126.1 (CH), 127.4 (CH), 127.6 (CH), 127.9 (CH), 128.2
(CH), 129.1 (CH), 136.9 (C), 137.8 (C), 155.7 (C), 169.2 (C), 171.8
(C) ppm. HRMS: calcd. 422.1842; found 422.1803. C24H26N2O5
(422.48): calcd. C 68.23, H 6.20, N 6.63; found C 67.91, H 6.18,
N 6.85.
comp.) IR (KBr): ν˜ ϭ 3016, 2990, 1739, 1630 cmϪ1 1H NMR
.
(400 MHz): δ ϭ 2.25 (s, 6 H), 3.63 (s, 3 H, COOMe) ppm. 13C
NMR (100 MHz): δ ϭ 34.7 (C), 43.1 (C), 51.7 (CH3), 52.6 (CH2),
168.0 (C) ppm. MS (70 eV): m/z ϭ 163 (12), 82 (100). C7H12ClNO2
(177.63): calcd. C 47.33, H 6.81, N 7.89, Cl 19.96; found C 47.55,
H 6.77, N 7.95, Cl 19.93.
Compound 13: Yield: 94%. M.p. 137 °C (MeOH). IR (KBr): ν˜ ϭ
3-[(9-Fluorenylmethoxycarbonyl)amino]bicyclo[1.1.1]pentane-1-
carboxylic Acid (10): 3-(N-Boc-amino)bicyclo[1.1.1]pentane-1-
carboxylic acid (8, 2.01 g, 8.8 mmol) was treated with TFA (95%
in water; 5 mL) for 1 h followed by coevaporation of the excess
of TFA with heptane. The crude 3-aminobicyclo[1.1.1]pentane-1-
carboxylic acid trifluoroacetate was dissolved in a mixture of ace-
tone/water (20 mL, 1:1) and Na2CO3·10H2O (2.52 g) was added.
The pH was adjusted to 9 by addition of a saturated Na2CO3 solu-
tion. Fmoc-O-succinimide (2.97 g, 8.8 mmol) was added over a per-
iod of 30 min. The mixture was stirred for 12 h and the value of
the pH was maintained all this time at 9 by further addition of a
few drops of saturated Na2CO3 solution. The mixture was diluted
with ethyl acetate (50 mL) and acidified carefully with 6  HCl.
The phases were separated and the organic phase was washed with
brine (3 ϫ 30 mL) and dried with MgSO4. After evaporation of
the solvent under reduced pressure, the crude product 10 (2.83 g,
92%) was obtained. Compound 10 was further purified by recrys-
tallisation from acetonitrile. M.p. 233Ϫ235 °C. 1H NMR
(300 MHz): δ ϭ 2.09 (s, 6 H), 2.50 (s, 1 H), 3.34 (m, 2 H), 4.27 (m,
1 H), 7.33Ϫ7.90 (m, 8 H), 12.37 (s, 1 H) ppm. 13C NMR (75 MHz):
δ ϭ 30.7 (C), 39.8 (C), 46.7 (CH), 53.4 (CH2), 65.2 (CH2), 120.2
(CH), 125.2 (CH), 127.1 (CH), 127.7 (CH), 140.9 (C), 144.0 (C),
155.2 (C), 170.8 (C) ppm. MS (70 eV): m/z ϭ 349 (4) [Mϩ], 178
(100), 165 (14). HRMS (70 eV): calcd. 349.13124, found 349.13114
C21H19NO4 (349.37): calcd. C 72.24, H 5.49, N 4.01; found C
72.05, H 5.61, N 4.01.
1
3600Ϫ3300, 2926, 1670 cmϪ1. H NMR (CDCl3, 400 MHz): δ ϭ
2.35 (s, 6 H), 3.68 (s, 3 H), 3.75 (m, 2 H), 5.13 (s, 2 H), 7.26Ϫ7.36
(m, 5 H) ppm. 13C NMR (100 MHz): δ ϭ 36.0 (CH3), 43.8 (CH2),
45.7 (C), 51.9 (CH3), 54.3 (CH2), 65.9 (CH2), 128.2 (CH), 128.2
(CH), 128.8 (CH), 137.5 (C), 156.9 (C), 169.7(C), 170.0 (C) ppm.
MS (70 eV): m/z ϭ 332 (2) [Mϩ], 301 (5), 91 (100). C17H20N2O5
(332.36): calcd. C 61.44, H 6.06, N 8.43; found C 61.31, H 5.88,
N 8.46.
Methyl 3-{-N-[3Ј-N-(tert-Butoxycarbonyl)aminobicyclo[1.1.1]pent-
1Ј-yl-carbonyl]amino}bicyclo[1.1.1]pentane-1-carboxylate
(14):
Yield: 37%. M.p. 285 °C (dec., CHCl3). IR (KBr): ν˜ ϭ 3321, 2921,
1
1690 cmϪ1. H NMR (400 MHz): δ ϭ 1.37 (s, 9 H), 2.02 (s, 6 H),
2.21 (s, 6 H), 3.60 (s, 3 H) ppm. 13C NMR (100 MHz): δ ϭ 28.1
(CH3), 35.6 (C), 44.2 (C), 44.6 (C), 45.2 (C), 51.0 (CH3), 53.0
(CH2), 53.8 (CH2), 154.4 (C), 163.8 (C), 169.2 (C) ppm.
C18H26N2O5 (350.41): calcd. C 61.70, H 7.48, N 7.99; found C
61.48, H 7.57, N 8.27.
Synthesis of Peptide Fmoc-L-Ala-Bcp-L-Tyr(OtBu)-L-Leu-OMe 15
by Liquid-Phase Chemistry. General Procedure: Deprotection Ϫ Re-
moval of the Fmoc Group: The protected peptide was dissolved in
20% piperidine in DMF (c ഠ 0.2 ) and the reaction mixture was
stirred for 15 min. The course of the reaction was monitored by
TLC. The volatiles were evaporated under reduced pressure and
the residue was used in the next coupling steps as the amine compo-
nent.
Synthesis of Dipeptides 11؊14
Coupling Procedure: The amino and the carboxyl components, as
well as the coupling reagent, were dissolved in DMF in 3-necked
flask under an Ar atmosphere at 0 °C. A solution of Hünig’s base
dissolved in DMF was then added dropwise within 10 min. The
General Procedure: DPPA (1.08 mL, 1.38 g, 5.00 mmol) dissolved
in DMF (15 mL) was added within 10 min to an ice-cooled solu-
tion of the carboxyl component (4.5 mmol) and the amino com-
496
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Carboxylic Acid Derivatives and Peptides
FULL PAPER
mixture was stirred at 0 °C for 2 h and then warmed to room tem-
perature and stirred for a further 12 h. Purification: The reaction [α]D ϭ Ϫ33.8 (c ϭ 1, CH2Cl2). H NMR (300 MHz, CDCl3): δ ϭ
colourless foam. Rf ϭ 0.33 (petroleum ether/ethyl acetate, 1:4).
1
mixture was dissolved in ethyl acetate/benzene (2:1, 30 mL) and
washed with 5% aqueous citric acid (2 ϫ 10 mL), water (2 ϫ
10 mL), saturated NaHCO3 solution (2 ϫ 10 mL), and brine (2 ϫ
10 mL). After drying the organic phase (MgSO4) and evaporating
the solvent under reduced pressure, the product was purified by
chromatography.
0.78 [d, J ϭ 5.2 Hz, 6 H, Leu-CH(CH3)2], 1.24 [s, 9 H, Tyr-
C(CH3)3], 1.27 (d, J ϭ 6.9 Hz, 3 H, Ala-CH3), 1.35Ϫ1.70 [m, 3 H,
Leu-CH2, Leu-CH(CH3)2], 2.18 (s, 6 H, Bcp-CH2), 2.95 (d, J ϭ
6.8 Hz, 2 H, Tyr-CH2), 3.62 (s, 3 H, Leu-OCH3), 4.15 (t, 1 H,
Tyr-CH), 4.20Ϫ4.60 (m, 5 H, Fmoc-CH, Fmoc-CH2, Leu-CH,
Ala-CH), 6.80Ϫ7.80 (m, 12 H, Fmoc CHarom., Tyr-CHarom.) ppm.
13C NMR (75.5 MHz, CDCl3): δ ϭ 18.7 (Ala-CH3), 21.9, 22.6
[Leu-CH(CH3)2], 24.8 (Leu-CH(CH3)2], 28.8 [Tyr-C(CH3)3], 37.5
(Tyr-CH2), 41.1 (Leu-CH2), 45.0 (Bcp-C-3), 47.0 (Tyr-CH), 50.0
(Ala-CH), 50.9 (Leu-CH), 52.3 (Leu-OCH3), 53.7 (Bcp-CH2), 54.0
(Fmoc-CH), 67.1 (Fmoc-CH2), 78.4 [Tyr-C(CH3)3], 120.0, 124.3,
125.0, 127.1, 127.8, 129.9 (Fmoc-CHarom., Tyr-CHarom.), 131.2,
141.3, 143.6, 143.7 (Fmoc-Carom., Tyr-Carom.), 154.4 (Fmoc-OCO),
169.0 (Bcp-CON), 170.0 (Tyr-CON), 170.2 (Ala-CON), 172.7 (Leu-
COOMe) ppm. HRMS (C45H55N4O8 ϭ M ϩ 1): calcd. 767.4019,
found 767.4029.
Synthesis of Dipeptide Fmoc-NH-L-Tyr(OtBu)-L-Leu-OMe: Ac-
cording to the general procedure, N-Fmoc-O-tBu--tyrosine
(0.504 g, 1.10 mmol), methyl -leucinate (0.222 g, 1.23 mmol),
DPPA (0.240 mL, 0.306 g, 1.11 mmol), and DIEA (0.420 mL,
0.31 g, 2.40 mmol) were reacted in DMF (20 mL). After the evap-
oration of the solvent under reduced pressure, the dipeptide were
obtained as a colourless foam (0.609 g, 94.6%). The substance was
used in the next step without further purification. Rf (petroleum
1
ether/EtOAc, 1:4) ϭ 0.83. H NMR (300 MHz, CDCl3): δ ϭ 0.80
[d, J ϭ 5.6 Hz, 6 H, Leu-CH(CH3)2], 1.25 [s, 9 H, Tyr-C(CH3)3],
1.30Ϫ1.70 [m, 3 H, Leu-CH2, Leu-CH(CH3)2], 2.95 (d, 2 H, Tyr-
CH2), 3.58 (s, 3 H, Leu-OCH3), 4.10Ϫ4.55 (m, 5 H, Fmoc-CH,
Fmoc-CH2, Leu-CH, Tyr-CH), 5.30 (s, 1 H, Leu-NH), 6.05 (s, 1
Synthesis of H2N-Gly-Bcp-L-Met-L-Asp(OtBu)-L-Phe-COOH (16)
by Solid-Phase Synthesis: CP--Phe-Fmoc resin (1.50 g,
0.900 mmol) was placed in a column fitted at the bottom with a
special tap. The tap had the function of controlling an inlet stream
of nitrogen and the outlet of solvents and reaction washings. DMF
was added to the resin, which was left to swell under a continuous
stream of nitrogen. The following deprotection and coupling pro-
cedures were performed for each of the four coupling steps.
H, Tyr NH), 6.82Ϫ7.70 (m, 12 H, Fmoc CHarom., Tyr-CHarom.
)
ppm. 13C NMR (75.5 MHz, CDCl3): δ ϭ 21.9, 22.7 [Leu-
CH(CH3)2], 24.7 [Leu-CH(CH3)2], 28.8 [Tyr-C(CH3)3], 37.8 (Tyr-
CH2), 41.5 (Leu-CH2), 47.1 (Tyr-CH), 50.9 (Leu-CH), 52.3 (Leu-
OCH3), 55.0 (Fmoc-CH), 67.1 (Fmoc-CH2), 78.4 [Tyr-C(CH3)3],
120.0, 124.3, 125.0, 127.1, 127.7, 129.9 (Fmoc-CHarom., Tyr-
CHarom.), 130.5, 141.3, 143.7, 143.8 (Fmoc-Carom., Tyr-Carom.),
154.5 (Fmoc-OCO), 170.5 (Tyr-CON), 172.7 (Leu-COOMe) ppm.
HRMS (C35H42N2O6) calcd. 586.3043; found 586.3059.
Deprotection ؊ Removal of the Fmoc Group: A solution of piperi-
dine in DMF (20%, 5 mL) was added to the resin free of solvents.
After passage of a stream of nitrogen in the column for 5 min, the
resulting solution was drained off. The residue was washed with
DMF (2 ϫ 20 mL); each time a stream of nitrogen was passed
through the column for 2 min.
Synthesis of Tripeptide Fmoc-Bcp-L-Tyr(OtBu)-L-Leu-OMe: The
dipeptide Fmoc--Tyr(OtBu)--Leu-OMe (0.507 g, 0.863 mmol)
was deprotected according to the general procedure. The depro-
tected amino acid, 3-(Fmoc-amino)bicyclo[1.1.1]pentane-1-car-
boxylic acid (10, 0.295 g, 0.845 mmol), HBTU (0.330 g,
0.871 mmol) and DIEA (0.15 mL, 0.11 g, 0.86 mmol) were reacted
as described above. After workup and chromatography (petroleum
ether/ethyl acetate, 1:4), the tripeptide was obtained as a colourless
foam (0.405 g, 69%). Rf ϭ 0.51 (petroleum ether/ethyl acetate, 1:4).
Coupling Procedure: A solution of HBTU (3.0 equiv.), Fmoc-pro-
tected amino acid (3.0 equiv.) and DIEA (6.0 equiv.) dissolved in
DMF (6 mL; see Table 1) was added to the deprotected N-terminus
resin. A stream of nitrogen was passed through the resulting mix-
ture for 1 h. After filtration, the resin was washed three times with
DMF (3 mL) and once with dichloromethane (3 mL). Each coup-
ling reaction was carried out twice.
1
[α]D ϭ Ϫ20.5 (c ϭ 1, CH2Cl2). H NMR (300 MHz, CDCl3): δ ϭ
0.81 [d, J ϭ 5.7 Hz, 6 H, Leu-CH(CH3)2], 1.24 [s, 9 H, Tyr-
C(CH3)3], 1.35Ϫ1.70 [m, 3 H, Leu-CH2, Leu-CH(CH3)2], 2.15 (s,
6 H, Bcp-CH2), 2.94 (d, J ϭ 6.9 Hz, 2 H, Tyr-CH2), 3.64 (s, 3 H,
Leu-OCH3), 4.15 (m, 1 H, Tyr-CH), 4.20Ϫ4.65 (m, 4 H, Fmoc-
Removal of the peptide from the resin was effected by treatment
with a mixture of acetic acid, methanol and dichloromethane
(5:1:4) within 2 h (about 10 mL/g resin). The resin was filtered off
and washed with the same mixture and dichloromethane (10 mL).
The combined organic solvents were diluted with heptane (300 mL,
to remove the acetic acid azeotropically). The solvents were then
evaporated almost completely under reduced pressure. A further
charge of heptane (25 mL) was added and the solvent was evapo-
rated again. The residue was dissolved in water and subsequently
lyophilized. A grey solid was formed, which was further purified
by preparative HPLC chromatography to give the pentapeptide 16.
HRMS (C30H44N5SO8 ϭ [Mϩ ϩ 1]): calcd. 634.2910, found
634.2909.
CH, Fmoc CH2, Leu-CH), 6.80Ϫ7.80 (m, 12 H, Fmoc CHarom.
,
Tyr-CHarom.) ppm. 13C NMR (75.5 MHz, CDCl3): δ ϭ 21.9, 22.7
[Leu-CH(CH3)2], 24.8 [Leu-CH(CH3)2], 28.8 [Tyr-C(CH3)3], 37.5
(Tyr-CH2), 41.2 (Leu-CH2), 45.0 (Bcp-C-3), 47.1 (Leu-OCH3), 50.9
(Leu-CH), 52.3 (Tyr-CH), 53.4 (Bcp-CH2), 54.0 (Fmoc-CH), 66.5
(Fmoc-CH2), 78.4 [Tyr-C(CH3)3], 120.0, 124.3, 125.0, 127.1, 127.8,
129.9 (Fmoc-CHarom., Tyr-CHarom.), 133.1, 141.3, 143.8, 143.8
(Fmoc-Carom., Tyr-Carom.), 154.4 (Fmoc-OCO), 169.0 (Bcp-CON),
170.0 (Tyr-CON), 172.7 (Leu-COOMe) ppm. HRMS
(C41H49N3O7) calcd. 695.3568; found 695.3569.
Synthesis of H2N-Bcp-L-Met-Gly-L-Trp(Boc)-L-Met-L-Asp(OtBu)-L-
Synthesis of Tetrapeptide Fmoc-
OMe (15): The tripeptide Fmoc-Bcp--Tyr(OtBu)--Leu-OMe
(0.507 g, 0.720 mmol) was deprotected according to the general
L
-Ala-Bcp-
L
-Tyr(OtBu)-
L
-Leu-
Phe-COOH (17) by Automated Solid-Phase Synthesis: The peptide
synthesis was performed on a Syro II synthesizer (MultiSynTec,
Witten, Germany) by using Fmoc strategy and HBTU/DIEA coup-
procedure. The free amino component, Fmoc--alanine hydrate ling protocols. We used TentaGel-S-Trt-(L)-Phe-Fmoc resin
(0.254 g, 0.772 mmol), HBTU (0.320 g, 0.844 mmol), and DIEA (100 mg; Rapp Polymere, Tübingen, Germany) with a capacity of
(0.13 mL, 0.096 g, 0.77 mmol) were reacted as described above.
After workup and chromatography (petroleum ether/ethyl acetate,
1:4) tetrapeptide 15 was obtained (0.120 g, 0.582 mmol, 69%) as a
0.47 mmol/g. The Fmoc group was deprotected using 20% piperi-
dine in DMF. The final cleavage from the resin was carried out
using a mixture of acetic acid, methanol and dichloromethane
Eur. J. Org. Chem. 2004, 493Ϫ498
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
497

M. Pätzel, M. Sanktjohanser, A. Doss, P. Henklein, G. Szeimies
FULL PAPER
Table 1. Reactions
Fmoc-protected amino acid
Mol. mass [g/mol]
Amount amino acid [g/mmol]
Amount HBTU [g]
Amount DIEA [ml]
-Asp(O-tBu) OH
-Methionine-OH
Bcp-OH
411
371
349
247.32
1.14/2.77
1.00 /2.70
0.942/2.70
0.667/2.70
1.11
1.12
1.11
1.14
0.939
0.942
0.940
0.940
Glycin-OH
[5]
P. Kaszynski, J. Michl, J. Org. Chem. 1988, 53, 4593Ϫ4594.
S. Guffler, Thesis, Humboldt-University, Berlin, 1999.
A. Godt, Diploma thesis, München, 1988.
Compounds 5 and 6 were prepared earlier by Applequist and
co-workers using the addition of dichlorocarbene to the corre-
sponding bicyclo[1.1.0]butanes, followed by reductive elimin-
ation of the chlorine atoms and saponification/esterfication, to
afford 5. Compound 5 was oxidized to the acid 6 using NaOCl/
RuO2. See: D. E. Applequist, T. L. Renken, J. W. Wheeler, J.
Org. Chem. 1982, 47, 4985Ϫ4995.
D. Banthorpe, The Chemistry of the Azide Group (Ed.: S. Patai),
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P. Luger, M. Weber, G. Szeimies, M. Pätzel, Acta Crystallogr.,
Sect. C 2000, 56, 1170Ϫ1172. For additional results of charge
density measurements, see: P. Luger, M. Weber, G. Szeimies,
M. Pätzel, J. Chem. Soc., Perkin Trans. 2 2001, 1956Ϫ1960.
J. D. D. Rehm, B. Ziemer, G. Szeimies, Eur. J. Org. Chem.
1999, 2079.
(5:1:4) within 2 h (about 10 mL/g resin).The further work up was
the same as described for compound 16. The crude peptide was
purified by reverse-phase chromatography on a Shimadzu LC 8
preparative HPLC (300 ϫ 40 mm column; VYDAC 218TPB1520;
The Separations Group, Hesperia, CA, USA) using a gradient from
20 to 80% B [A: TFA (1 mL) in water (1000 mL); B: TFA (2 mL)
in acetonitrile (800 mL) and water (200 mL)].The flow rate was
70 mL/min. HRMS (C51H70N8S2O12 ϩ Na): calcd. 1073.4452,
found 1073.4435.
[6] [6a]
[6b]
[7]
[8]
[9]
Acknowledgments
We are grateful to S. Hinze, T. Krahl, O. Krätke, and Dr. S. Szoladz
[10]
[11]
for experimental contributions.
[1]
The experiments performed by M. S. were carried out at the
Institut für Organische Chemie der Universität München. See
also: M. Sanktjohanser, PhD Thesis, Universität München,
1993.
[12]
[13]
[14]
[2] [2a]
M. D. Levin, P. Kaszynski, J. Michl, Chem. Rev. 2000, 100,
[2b]
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P. Henklein, M. Boomgaarden, B. Penke, R. Morgenstern, T.
Ott, Pharmazie 1989, 44, 61.
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[3]
[4]
[3a] R. Pellicciari, M. Raimondo, M. Marinozzi, B. Natalini, G.
Costantino, C. Thomsen, J. Med. Chem. 1996, 39, 2874Ϫ2876.
[3b] R. G. Costantino, K. Maltoni, M. Marinozzi, E. Camaioni,
L. Prezeau, J.-P. Pin, R. Pellicciari, Bioorg. Med. Chem. 2001,
9, 221Ϫ227.
[15]
[16]
K. M. Lynch, W. P. Daily, J. Org. Chem. 1995, 60, 4666Ϫ4668.
J. Belzner, U. Bunz, K. Semmler, G. Szeimies, K. Opitz, A.-D.
Schlüter, Chem. Ber. 1989, 11, 397Ϫ398.
K. H. Park, M. J. Kurth, Tetrahedron 2002, 58, 8629Ϫ8659.
Received September 2, 2003
498
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 493Ϫ498