Solid-supported synthesis of bicyclic peptides containing three parallel peptide chains

Article abstract of DOI:10.1002/ejoc.200210517

Four homodetic bicyclic peptides 9a-d, containing three parallel peptide chains, were synthesized on a hydroxymethyl-functionalized polystyrene support. α,α-Bis(aminomethyl)-β-alanine, bearing orthogonal protections (Alloc, Boc, Fmoc) on the three amino groups (1), was attached to the support through an H₂N-Leu-Leu-Gly-OH spacer, and the peptide chains were assembled on the amino groups of 1 by either a stepwise coupling or coupling of a segment, keeping the amino protection within each chain unchanged. N-(4-Allyloxy-4-oxobutanoyl)iminodiacetic acid (2) was then coupled to the Fmoc-protected chain. The Boc protecting group was removed, and the exposed amino group was coupled with the remaining free carboxylic acid function of 2. Finally, the Alloc and allyl ester protections were removed from the carboxylic and amino functions, and a second cyclization was performed. Release from the support gave 9a-d as free carboxylic acids. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003.

Full text of DOI:10.1002/ejoc.200210517

FULL PAPER
Solid-Supported Synthesis of Bicyclic Peptides Containing Three Parallel
Peptide Chains
Tuomas Karskela,*[a] Petri Heinonen,[a] Pasi Virta,[a] and Harri Lönnberg[a]
Keywords: Amino acids / Cyclic peptides / Cyclization / Solid-phase synthesis
Four homodetic bicyclic peptides 9a−d, containing three par-
allel peptide chains, were synthesized on a hydroxymethyl-
Fmoc-protected chain. The Boc protecting group was re-
moved, and the exposed amino group was coupled with the
functionalized polystyrene support. α,α-Bis(aminomethyl)-β- remaining free carboxylic acid function of 2. Finally, the Alloc
alanine, bearing orthogonal protections (Alloc, Boc, Fmoc) on
the three amino groups (1), was attached to the support
through an H2N-Leu-Leu-Gly-OH spacer, and the peptide
chains were assembled on the amino groups of 1 by either a
stepwise coupling or coupling of a segment, keeping the am-
and allyl ester protections were removed from the carboxylic
and amino functions, and a second cyclization was per-
formed. Release from the support gave 9a−d as free carb-
oxylic acids.
ino protection within each chain unchanged. N-(4-Allyloxy- ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
4-oxobutanoyl)iminodiacetic acid (2) was then coupled to the
Germany, 2003)
Introduction
the bicyclic peptides from the solid support as free car-
boxylic acids makes them suitable for possible further con-
jugation.
Conformational rigidity may give biologically active
homodetic cyclic peptides increased potency, receptor selec-
tivity, metabolic stability and bioavailability.[1Ϫ4] Additional
cyclization resulting in bicyclic homodetic peptides in-
creases the rigidity further making such peptides interesting
subjects of research.[5Ϫ9] In spite of this appeal, relatively
few syntheses of homodetic bicyclic peptides either in
solution[10Ϫ14] or on a solid support[15Ϫ17] have been de-
scribed. Recently, we have widened their scope by reporting
a synthesis of homodetic bicyclic spiropeptides on a solid
support.[18]
Figure 1. Branching units used in the synthesis of bicyclic peptides
To find out whether libraries of homodetic bicyclic pep-
tides containing three parallel amino acid chains could be
prepared conveniently on a solid support, we have synthe-
sized four such peptides on hydroxymethyl-functionalized
polystyrene. These peptides consist of two different branch-
ing units, viz. α,α-bis(aminomethyl)-β-alanine (cf. 1 in
Figure 1) and N-succinyliminodiacetic acid (cf. 2), used to
cap the carboxylic and amino termini of the peptide chains,
respectively. The synthesis is initiated by assembling the
peptide chains one after another on the three orthogonally
protected amino groups of solid-supported α,α-bis(amino-
methyl)-β-alanine; N-succinyliminodiacetic acid is then cou-
pled to one of the chains and cyclizations with the remain-
ing two branches are carried out on the support. Release of
Results and Discussion
Synthesis of Building Blocks
The preparation of the orthogonally protected α,α-bis(a-
minomethyl)-β-alanine branching unit 1, used as a solid-
supported scaffold for the peptide synthesis, has been de-
scribed previously.[19] The second branching unit 2, used to
cap the amino termini of the peptide chains, was synthe-
sized as depicted in Scheme 1. Succinic acid monoallyl es-
ter[20] and the di-tert-butyl ester of iminodiacetic acid[21]
were condensed by HOAt/DCC[22] activation. Subsequent
removal of the tert-butyl protecting groups gave 2 in a mod-
erate 40% overall yield. The amino-protected dipeptides
were obtained by established methods based on HOSu/
DCC chemistry.
[a]
Department of Chemistry, University of Turku,
20014 Turku, Finland
Fax: (internat.) ϩ 358-2/333-7631
E-mail: tuokarsk@utu.fi
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2003, 1687Ϫ1691
DOI: 10.1002/ejoc.200200517
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T. Karskela, P. Heinonen, P. Virta, H. Lönnberg
FULL PAPER
Table 1. Dipeptide branches used for the synthesized peptides
(5؊9, cf. Scheme 2)
Entry
P1
P2
P3
5Ϫ9a
5Ϫ9b
5Ϫ9c
5Ϫ9d
β-Ala-Gly
Ser-β-Ala
Ala-Gly
Gly-β-Ala
Gly-β-Ala
Asn-Gly
Phe-β-Ala
Phe-β-Ala
Phe-β-Ala
Phe-β-Ala
Gly-Ala-Gly
Gly-Asn-Gly
Scheme 1. Synthesis of the branching unit 2: (i) HOAt, DCC, Py,
DMF; (ii) TFA, CH2Cl2
but segment coupling was considered safer and more repro-
ducible for manual SPPS since the number of steps on the
solid support was smaller.
After completing the chain assembly, the Fmoc protec-
Solid-Phase Synthesis of Bicyclic Peptides
tion of chain P1 was removed. The second branching unit
The hydroxymethyl-functionalized polystyrene was first 2 was converted into a cyclic anhydride with EDAC acti-
derivatized with an H2N-Leu-Leu-Gly-OH spacer.[23] Ac- vation, and allowed to react with the exposed amino group.
cordingly, glycine was attached to the resin by the sym- In this manner, protection of the carboxymethyl groups
metrical anhydride method, and the chain was elongated could be avoided. Cleavage of the Boc protecting group
by two leucine residues using Fmoc chemistry with HATU/ from chain P2 then enabled the first cyclization reaction,
DIEA[24,25] activation. The branching unit 1 was then cou- which was performed by HATU/DIEA activation. After re-
pled similarly to obtain tetrapeptide 4 (Scheme 2) bearing action at room temperature for 5 h, no starting material
three orthogonally protected amino groups. Two ap- could be detected. Because 2 is not chiral, there is no risk
proaches were used for the construction of the parallel pep- for epimerization even though the cyclization is slow.[26]
tide chains on these amino functions, which can be depro- While the cyclization of 6a and 6b, which contain β-alanine
tected one after another. With peptides 5a and 5b (Table 1), residues in both of the peptide chains participating in the
the chains were introduced as dipeptide segments. With reaction, proceeded well, the corresponding reaction with
peptides 5c and 5d, the chains on the Fmoc- and Boc-pro- 6c and 6d, having a glycine residue in place of β-alanine,
tected amino groups were assembled by stepwise coupling yielded a more complex product mixture. The fact that
using Fmoc- and Boc-protected amino acids, respectively. 1,1,3,3-tetramethylguanidino derivatives[27] could be de-
The Alloc-protected branch P3 was introduced as a seg- tected among the side products led us to use a phos-
ment. Both approaches gave satisfactory coupling yields, phonium coupling reagent for the second cyclization with
Scheme 2. Synthesis of bis(aminomethyl)-β-alanine conjugates: (i) SPPS; (ii) piperidine, DMF; (iii) N-(4-allyloxy-4-oxobutanoyl)imino-
diacetic anhydride, DMF; (iv) TFA, CH2Cl2; (v) HATU, DIEA, DMF; (vi) Pd(OAc)2, PPh3, Bu3SnH, AcOH, CH2Cl2; (vii) 30% HBr/
AcOH, thioanisole, pentamethylbenzene, TFA
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Eur. J. Org. Chem. 2003, 1687Ϫ1691

Bicyclic Peptides Containing Three Parallel Peptide Chains
FULL PAPER
0.1% aq. TFA to acetonitrile over 30 min at a flow rate of 1.0 mL/
min. The detection wavelength was 215 nm. For abbreviations used
see ref.[22]
these peptides. The Alloc and allyl ester protecting groups
were removed simultaneously from the monocyclic peptides
using palladium/tributyltin hydride chemistry.[28] The se-
cond cyclization to 8a and 8b was performed over 16 h
using similar HATU/DIEA chemistry as before. To obtain
Di-tert-butyl
N-(4-Allyloxy-4-oxobutanoyl)iminodiacetate
(3):
Monoallyl succinate (2.9 g, 18 mmol), HOAt (2.6 g, 19 mmol),
8c and 8d, HATU was replaced with PyBOP[20] and the DCC (3.9 g, 19 mmol), di-tert-butyl iminodiacetate (4.2 g,
17 mmol) and pyridine (3.1 mL, 38 mmol) were stirred in DMF
(50 mL) overnight. The reaction mixture was filtered and concen-
trated by rotary evaporation. The residue was dissolved in CH2Cl2
and washed with 1  aq. AcOH and saturated aq. NaHCO3. The
organic phase was dried with Na2SO4, and the solvent was evapo-
rated. Silica gel chromatography (EtOAc/petroleum ether, 3:7, v/v)
yielded 3 (3.4 g, 48%) as a clear oil. Rf ϭ 0.49 (EtOAc/petroleum
ether, 3:7). H NMR (400 MHz, CDCl3): δ ϭ 1.45 and 1.49 [2 s, 2
ϫ 9 H, C(CH3)3], 2.67 (m, 4 H, CH2CH2CO2), 4.01 and 4.05 (2 s,
2 ϫ 2 H, CH2CO2tBu), 4.59 (td, J ϭ 1.4, 5.7 Hz, 2 H, 4
solvent was changed from DMF to a 1:4 mixture of DMSO
and NMP. Furthermore, the reaction time was extended to
22 h. The cyclization reaction was repeated with 7d since
not all the starting material had disappeared during the re-
action. In spite of these changes, the second cyclization re-
action of 7c and 7d led to a complex reaction mixture and
markedly decreased yield, probably because of polymeriz-
ation on the solid support. Acid-catalysed cleavage from the
support gave 9aϪd as free carboxylic acids. Accordingly,
1
the bicyclic peptides bear a functionalized side arm that OCH2CHϭCH2), 5.22 (tdd, J ϭ 1.4, 2.7, 10.4 Hz, 1 H, CHϭ
CHH), 5.31 (tdd, J ϭ 1.5, 3.1, 17.2 Hz, 1 H, CHϭCHH), 5.91 (tdd,
may be exploited for further conjugation in solution.
All four peptides were obtained in pure states by HPLC,
but in low yields. Only peptide 9a, which had the simplest
amino acid composition, was obtained as a reasonably pure
crude product. Owing to the chiral centre of branching unit
1, the compound appeared as a pair of diastereoisomers
that could be separated by HPLC (Figure 2). In addition to
these four bicyclic peptides, two attempts were made, unsuc-
cessfully, to synthesize a bicyclic peptide having a smaller
ring size and no β-alanine residues. Accordingly, the meth-
odology described seems to allow preparation of homodetic
bicyclic peptides on a solid support, but its applicability to
library synthesis is severely limited. Only in favourable cases
is the crude product reasonably pure, and usually HPLC
purification is needed.
J ϭ 5.7, 10.5, 17.2 Hz, 1 H, CHϭCH2) ppm. 13C NMR (100 MHz,
CDCl3):
δ
ϭ
27.6 (CH2CH2CON), 27.9 (CCH3), 29.1
(CH2CH2CO2), 48.8 and 50.7 (NCH2CO2), 65.1 (CH2ϭCHCH2O),
81.7 and 82.6 (CCH3), 118.0 (CHϭCH2), 132.1 (CHϭCH2), 167.8
and 168.3 (CO2tBu), 171.7 (CO2All), 172.3 (CON) ppm. MS (EIϩ):
m/z ϭ 385 [Mϩ] (0.1), 329 (6), 328 (5), 312 (12), 273 (46), 256 (22),
228 (47), 141 (100), 57 (72). HRMS (EIϩ): C19H31NO7 requires
385.2101; found 385.2101 [Mϩ].
N-(4-Allyloxy-4-oxobutanoyl)iminodiacetic Acid (2): Compound 3
(3.4 g, 8.8 mmol) was stirred in a mixture of TFA and CH2Cl2 (2:1,
21 mL) for 30 min. The reaction mixture was concentrated by ro-
tary evaporation. The crude product was purified by silica gel chro-
matography (MeOH/CH2Cl2, 1:9) to give 2 (1.9 g, 79%) as a clear
1
viscous oil. Rf ϭ 0.28 (MeOH/CH2Cl2, 1:9). H NMR (400 MHz,
[D6]DMSO): δ ϭ 2.52 (m, 4 H, CH2CH2CO2), 3.96 and 4.18 (2 s,
2 ϫ 2 H, NCH2CO2), 4.51 (td, J ϭ 1.5, 5.3 Hz, 2 H, OCH2CHϭ
CH2), 5.18 (tdd, J ϭ 1.4, 2.8, 10.5 Hz, 1 H, CHϭCHH), 5.29 (tdd,
J ϭ 1.7, 3.4, 17.2 Hz, 1 H, CHϭCHH), 5.88 (tdd, J ϭ 5.3, 10.5,
17.3 Hz, 1 H, CHϭCH2), 12.70 (br. s, 1 H, CO2H) ppm. 13C NMR
(100 MHz, [D6]DMSO):
δ
ϭ
27.1 (CH2CH2CON), 28.7
(CH2CH2CO2), 48.2 and 49.7 (NCH2CO2), 64.3 (CH2ϭCHCH2O),
117.5 (CHϭCH2), 132.7 (CHϭCH2), 170.6 and 170.8 (CO2H),
171.7 (CO2All), 172.3 (CON) ppm. MS (EIϩ): m/z ϭ 273 [Mϩ]
(11), 255 (69), 229 (56), 216 (12), 141 (34), 101 (7), 41 (100). HRMS
(EIϩ): C11H15NO7 requires 273.0849; found 273.0846 [Mϩ].
N-[N-(Allyloxycarbonyl)phenylalanyl]-β-alanine (10): N-Fmoc-phe-
nylalanine (1.1 g, 2.8 mmol), DCC (0.68 g, 3.3 mmol) and HOSu
(0.38 g, 3.3 mmol) were dissolved in cooled dioxane and stirred for
2 h and the filtered. β-Alanine tert-butyl ester hydrochloride
(0.50 g, 2.8 mmol) and K2CO3 (0.38 g, 2.8 mmol) were dissolved in
aq. dioxane, and then added to the filtered solution. After stirring
overnight, the solvent was evaporated. The residue was dissolved
in CH2Cl2 (100 mL) and washed with half-saturated brine. The or-
ganic layer was dried with Na2SO4 and the solvent was evaporated.
The crude N-(N-Fmoc-phenylalanyl)-β-alanine tert-butyl ester was
purified by silica gel chromatography (EtOAc/petroleum ether, 6:4).
The Fmoc protecting group was removed by stirring for 30 min in
piperidine/CH2Cl2 (1:4). The volatile components were removed by
rotary evaporation. The product was purified by silica gel chroma-
tography (EtOAc/petroleum ether, 3:7, then MeOH/CH2Cl2, 1:19),
which gave N-phenylalanyl-β-alanine tert-butyl ester in an overall
yield of 84% (0.69 g). The latter compound (0.68 g, 2.3 mmol) was
dissolved in dioxane and NaOH (2.8 mmol) in H2O (10 mL) was
added. Allyl chloroformate (0.42 g, 2.8 mmol), diluted with diox-
Figure 2. Chromatogram of the crude diastereoisomeric pair of 9a
recorded by using a Hypersil HyPurity Elite C18 column (150 ϫ
4.6 mm, 5 µm), applying a linear gradient from 0.1% aq. TFA to
acetonitrile over 30 min at a flow rate of 1.0 mL/min; the detection
wavelength was 215 nm
Experimental Section
General Remarks: The NMR spectra were recorded with a JEOL
JNM-GX 400 or JNM-A 500 spectrometer. The chemical shifts are
given in ppm downfield from that of internal (CH3)4Si. The mass
spectra of low-molecular-weight compounds were recorded with a
VG 7070E mass spectrometer. Finnigan Mat Lasermat was used
for mass analyses of reactions on solid support. For high-resolution
mass spectrometry of bicyclic peptides, an Applied Biosystems
Mariner 5272 mass spectrometer was used. RP HPLC analyses and
separations were performed with a Hypersil HyPurity Elite C18
column (150 ϫ 4.6 mm, 5 µm), applying a linear gradient from
Eur. J. Org. Chem. 2003, 1687Ϫ1691
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1689

T. Karskela, P. Heinonen, P. Virta, H. Lönnberg
FULL PAPER
ane (1.0 mL) was slowly added to the stirred reaction mixture. After
the reaction had reached completion, the mixture was concen-
trated. The residue was dissolved in CH2Cl2 and washed with
water. The organic layer was then dried with Na2SO4, the solvent
was evaporated, and the residue was purified by silica gel chroma-
tography (EtOAc/petroleum ether, 3:7), yielding N-(N-alloc-phe-
nylalanyl)-β-alanine tert-butyl ester (0.78 g, 90%). The product was
allyl ester deprotection was performed by palladium/tributyltin hy-
dride chemistry.[28] Palladium() acetate, reduced with tri-
phenylphosphane (6 equiv.), was used as the catalyst. HATU/DIEA
activation[24,25] was utilized for coupling reactions using amino acid
(2.5 equiv.), HATU (2.5 equiv.) and DIEA (5 equiv.). The resin
beads were swelled with the reaction solvent before addition of the
coupling reagents. The total volume of the solvent was 10 µL/mg
stirred for 45 min in TFA/CH2Cl2 (1:1; 8 mL), after which time the of resin. Reaction time was 2 h. The solid support was then washed
CH2Cl2 and some TFA was evaporated, and some diethyl ether was
added. The product was left to crystallize in a refrigerator over-
night. The crystals that formed were suction-filtered and washed
with DMF, CH2Cl2 and MeOH. The resin was always dried under
reduced pressure after washes. Coupling reactions were monitored
either by determining the Fmoc loading or by removing small resin
with cold diethyl ether. Silica gel chromatography (MeOH/CH2Cl2, aliquots that were cleaved and analysed by HPLC to evaluate the
1:9) gave N-[N-(allyloxycarbonyl)phenylalanyl]-β-alanine (0.45 g, quality of the on-resin product. The theoretical loading was calcu-
68%). Rf ϭ 0.47 (MeOH/CH2Cl2, 1:9). 1H NMR (400 MHz, lated by Equation (1), where L stands for the loading [mol gϪ1] and
[D6]DMSO): δ ϭ 2.33 (t, J ϭ 6.8 Hz, 2 H, CH2CO2), 2.71 (dd, J ϭ ∆M for the change in the molecular mass of the product. The yield
10.3, 13.7 Hz, 1 H, CHHPh), 2.91 (dd, J ϭ 4.5, 13.7 Hz, 1 H, was then calculated by Equation (2).
CHHPh), 3.24 (m, 2 H, CH2CON), 4.14 (m, 1 H, NCHCO2), 4.36
L
theoretical ϭ Linitial/(∆M ϫ Linitial ϩ 1)
(1)
(d, J ϭ 5.1 Hz, 2 H, OCH2CHϭCH2), 5.11 (dd, J ϭ 1.3, 10.5 Hz,
1 H, CHϭCHH), 5.18 (dd, J ϭ 1.7, 17.3 Hz, 1 H, CHϭCHH),
5.81 (tdd, J ϭ 5.1, 10.5, 17.3 Hz, 1 H, CHϭCH2), 7.15Ϫ7.25 (m,
5 H, Ar), 7.39 (d, J ϭ 8.5 Hz, 1 H, Phe NH), 8.04 (t, J ϭ 5.4 Hz,
1 H, β-Ala NH), 12.17 (br. s, CO2H) ppm. 13C NMR (100 MHz,
[D6]DMSO): δ ϭ 33.8 (CH2CH2N), 34.8 (CH2CH2CO2), 37.6
(CH2Ph), 56.1 (NCHCO), 64.3 (CH2ϭCHCH2O), 116.8 (CHϭ
CH2), 126.2 (C4 Ph), 128.0 (C2,6 Ph), 129.1 (C3,5 Ph), 133.5 (CHϭ
CH2), 138.1 (C1 Ph), 155.7 (NCO2CH2), 171.4 (CON), 172.9
(CO2H) ppm. MS (EIϩ): m/z ϭ 320 [Mϩ] (1), 219 (8), 204 (9), 160
(9), 91 (65), 41 (100). HRMS (EIϩ): C16H20N2O5 requires 320.1372;
found 320.1368 [Mϩ].
Yield ϭ (Lmeasured/Ltheoretical) ϫ 100%
(2)
Synthesis of Bicyclic Peptide 9a: Hydroxymethyl-functionalized
polystyrene resin was first loaded with Fmoc-glycine (loading 340
µmol gϪ1) by the symmetrical anhydride method. The unchanged
hydroxy groups were acetylated (Ac2O/2,6-lutidine/1-methylimidaz-
ole/THF, 5:5:8:100; 30 min), and the support was washed with
CH2Cl2 and MeOH. Two Fmoc-leucine units and branching unit
1 were subsequently attached in a stepwise manner to the glycine-
loaded resin (120 mg, 42 µmol). Boc-Gly-β-AlaOH[30] and Fmoc-
β-Ala-GlyOH[31] were coupled to the branching unit as dipeptide
segments after removal of corresponding protecting groups. At this
point, the Fmoc loading was 95% of the theoretical loading. The
Alloc protecting group was then removed, and Alloc-Phe-β-AlaOH
(10) was coupled. The Fmoc protecting group was removed from
the branched peptide 5a. Branching unit 2 (45 mg, 160 µmol) was
dissolved in DMF and converted into its anhydride by treatment
with EDAC·HCl (30 mg, 160 µmol) for 40 min. The solution was
added to the peptide-derivatized solid support (130 mg, 32 µmol)
that had been swollen in DMF. After 40 min of shaking, the resin
was washed with DMF, CH2Cl2 and MeOH. This anhydride coup-
ling was repeated. After removal of the last Boc protecting group,
the first cyclization was performed by shaking the resin overnight
with a mixture of HATU and DIEA (5 and 10 equiv., respectively)
in DMF. Alloc and allyl ester protections were removed from the
solid-supported peptide (7a; 110 mg, 29 µmol) and the second
cyclization was carried out in a similar manner. The product was
cleaved from the support with 0.1  TBAF in THF containing 0.1
 water (6 h, 55 °C). After cleavage, the resin was rinsed with THF
(5 mL), neutralized with TFA and the solvent was evaporated un-
der reduced pressure. The cleavage reaction was repeated four
times. HPLC purification yielded the two diastereoisomeric prod-
ucts (1.5 mg and 0.5 mg, combined yield of 4.7% calculated from
the original Fmoc loading). Diastereoisomer A: 1H NMR
N-(N-Fmoc-serinyl)-β-alanine (11): N-Fmoc-O-tBu-serine (0.84 g,
2.2 mmol), DCC (0.54 g, 2.6 mmol) and HOSu (0.30 g, 2.6 mmol)
were dissolved in cooled dioxane (25 mL) and stirred for 2 h and
then filtered. β-Alanine tert-butyl ester hydrochloride (0.40 g,
2.2 mmol) and K2CO3 (0.30 g, 2.2 mmol) were dissolved in aq. di-
oxane (4 mL), and then added to the filtered solution. After stirring
overnight, the solvent was evaporated. The residue was dissolved
in CH2Cl2 (100 mL) and washed with half-saturated brine. The or-
ganic layer was dried with Na2SO4 and the solvent was evaporated.
Crude N-(N-Fmoc-O-tert-butylserinyl)-β-alanine tert-butyl ester
was purified by silica gel chromatography (EtOAc/petroleum ether,
3:7). The tert-butyl protecting groups were removed by stirring for
2 h in TFA, after which time the TFA was evaporated. Silica gel
chromatography (MeOH/CH2Cl2, 1:9) gave the title compound
(0.72 g, 82%). Rf
ϭ
0.28 (MeOH/CH2Cl2, 1:9). 1H NMR
(400 MHz, [D6]DMSO): δ ϭ 2.36 (m, 2 H, CH2CO2), 3.25 (m, 2
H, CH2CON), 3.54 (m, 2 H, CH2OH), 3.99 (m, 1 H, NCHCO2),
4.21 (m, 1 H, Fmoc CH), 4.25 (m, 2 H, Fmoc CH2), 7.32 (m, 3 H,
Fmoc Ar, Ser NH), 7.41 (m, 2 H, Fmoc Ar), 7.73 (m, 2 H, Fmoc
Ar), 7.88 (m, 2 H, Fmoc Ar), 7.94 (m, 1 H, β-Ala NH) ppm. 13C
NMR (100 MHz, [D6]DMSO):
δ ϭ 33.9 (CH2CH2N), 34.9
(CH2CH2CO2), 46.6 (CH Fmoc), 57.2 (NCHCO), 61.8 (CH2OH),
65.8 (CH2 Fmoc), 120.1, 125.4, 127.1, 127.7, 140.7, 143.8, and
143.9 (Ar Fmoc), 155.9 (NCO2CH2), 170.1 (CON), 173.0 (CO2H)
ppm. MS (EIϩ): m/z ϭ 398 [Mϩ] (1), 354 (1), 178 (100), 165 (25).
HRMS (EIϩ): C21H22N2O6 requires 398.1478; found 398.1480
[Mϩ].
(500 MHz, [D6]acetone/D2O):
δ ϭ 0.86Ϫ0.94 (CH3 Leu),
1.61Ϫ1.75 (CH2, CH Leu), 2.30Ϫ2.90 (CH2CO β-Ala, CH2CO
succinyl), 2.86 (CHH Phe), 3.20Ϫ4.10 (CH2N β-Ala, Gly, branch-
ing unit), 3.25 (CHH Phe), 4.05Ϫ4.45 [CON(CH2CO)2], 4.36
(CHN Leu), 4.52 (CHN Leu), 4.55 (CHN Phe), 7.15Ϫ7.30 (H-Ar
Phe), 7.30Ϫ7.37 (NH) and 7.80Ϫ8.15 (NH) ppm. Diastereoisomer
B: 1H NMR (500 MHz, [D6]acetone/D2O): δ ϭ 0.88Ϫ0.95 (CH3
Leu), 1.68Ϫ1.75 (CH2, CH Leu), 2.18Ϫ2.80 (CH2CO β-Ala,
Solid-Phase Syntheses: Fmoc protecting groups were removed by
treatment with piperidine/DMF (1:4) for 20 min. After deprotec-
tion, the solid support was washed with DMF, CH2Cl2 and MeOH.
Boc protecting groups were removed by treatment with TFA/ CH2CO succinyl), 2.83 (CHH Phe), 3.20Ϫ4.25 (CH2N β-Ala, Gly,
CH2Cl2 (1:3) for 1 h. The support was then neutralized with pyri-
dine in CH2Cl2 and washed with CH2Cl2 and MeOH. Alloc and
branch), 3.37 (CHH Phe), 4.07Ϫ4.47 [CON(CH2CO)2], 4.29 (CHN
Leu), 4.54 (CHN Leu), 4.57 (CHN Phe), 7.19Ϫ7.31 (H-Ar Phe),
1690
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 1687Ϫ1691

Bicyclic Peptides Containing Three Parallel Peptide Chains
FULL PAPER
7.34 (NH) and 7.95Ϫ8.35 (NH) ppm. HRMS (ESIϩ): [M ϩ H]ϩ
found 1084.5442; C49H73N13O15ϩH requires 1084.5422.
C. Pedone, V. Pavone, J. Am. Chem. Soc. 1998, 120,
5879Ϫ5886.
R. Oliva, L. Falcigno, G. D’Auria, G. Zanotti, L. Paolillo, Bi-
opolymers 2001, 56, 27Ϫ36.
V. Pavone, A. Lombardi, F. Nastri, M. Saviano, O. Maglio, G.
D’Auria, L. Quartara, C. A. Maggi, C. Pedone, J. Chem. Soc.,
Perkin Trans. 2 1995, 987Ϫ993.
L. Quartara, V. Pavone, C. Pedone, A. Lombardi, A. R.
Renzetti, C. A. Maggi, Regul. Pept. 1996, 65, 55Ϫ59.
A. Lombardi, G. D’Auria, M. Saviano, O. Maglio, F. Nastri,
L. Quartara, C. Pedone, V. Pavone, Biopolymers 1997, 40,
505Ϫ518.
R. Oliva, L. Falcigno, G. D’Auria, M. Saviano, L. Paolillo, G.
Ansanelli, G. Zanotti, Biopolymers 2000, 53, 581Ϫ595.
K. Akaji, S. Aimoto, Tetrahedron 2001, 57, 1749Ϫ1755.
C. Yu, J. W. Taylor, Tetrahedron Lett. 1996, 37, 1731Ϫ1734.
W. Zhang, J. W. Taylor, Tetrahedron Lett. 1996, 37, 2173Ϫ2176.
A. Bisello, C. Nakamoto, M. Rosenblatt, M. Chorev, Biochem-
istry 1997, 36, 3293Ϫ3299.
[9]
Synthesis of Bicyclic Peptide 9b: The synthesis was conducted as
described for 9a, with the exception that the initial loading was 210
µmol gϪ1. Fmoc-Ser-β-AlaOH (11) was used instead of Fmoc-β-
Ala-GlyOH, and the reaction time for the first cyclization was 5 h.
The peptide was cleaved from the resin with acid (30% HBr in
AcOH/pentamethylbenzene/thioanisole/TFA, 4:5:6:100; room tem-
perature, 3 h). The resin was rinsed with CH2Cl2 and MeOH. The
solvents were evaporated under reduced pressure, and the residue
was extracted with EtOH/0.1% aq. TFA (1:4). The extract was
washed with diethyl ether before HPLC purification. The dia-
stereoisomers could not be separated by the HPLC system used.
HRMS (ESIϩ): (C50H75N13O16 ϩ 2 H)/2 requires 557.7800; found
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
557.7813 [M ϩ 2 H]2ϩ
.
Synthesis of Bicyclic Peptide 9c: The synthesis was conducted as
[18]
[19]
[20]
described for 9a, except that the initial loading was 440 µmol gϪ1
.
P. Virta, J. Sinkkonen, H. Lönnberg, Eur. J. Org. Chem. 2002,
3616Ϫ3621.
Instead of segment coupling, the Fmoc- and Boc-protected
branches were assembled by stepwise couplings. The first cycliza-
tion was performed by shaking the resin with a mixture of HATU
and DIEA (5 and 10 equiv., respectively) in DMF for 5 h. The
second cyclization was carried out by shaking the resin with
PyBOP and DIEA (2 and 4 equiv., respectively) in DMSO/NMP
(1:4) for 22 h. Acid was used to release the product as described
for 9b. The diastereoisomers could not be separated by the HPLC
system. HRMS (ESIϩ): (C50H74N14O16 ϩ 2 H)/2 requires 564.2776;
J. Katajisto, T. Karskela, P. Heinonen, H. Lönnberg, J. Org.
Chem. 2002, 67, 7995Ϫ8001.
Succinic acid monoallyl ester was prepared, although it is com-
mercially available, by the reaction of succinic anhydride, allyl
alcohol (2 equiv.) and pyridine (1.1 equiv.) at room temp. for
2 h.
H. Takalo, P. Pasanen, J. Kankare, Acta Chem. Scand., Ser. B
1988, 42, 373Ϫ377.
[21]
[22]
Abbreviations: Alloc, allyloxycarbonyl; DCC, N,NЈ-dicyclo-
hexylcarbodiimide; DIEA, N,N-diisopropylethylamine; DMF,
found 564.2764 [M ϩ 2 H]2ϩ
.
N,N-dimethylformamide;
DMSO,
dimethyl
sulfoxide;
EDAC·HCl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride; HATU, 1-[bis(dimethylamino)methylene]-1H-
[1,2,3]triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide;
HOAt, 1-hydroxy-7-azabenzotriazole; HOSu, N-hydroxysuc-
cinimide; NMP, N-methylpyrrolidone; PyBOP, (1-benzotriazo-
lyl)-N-oxytris(pyrrolidino)phosphonium hexafluorophosphate;
SPPS, solid-phase peptide synthesis; TBAF, tetrabutylam-
monium fluoride; TFA, trifluoroacetic acid; THF, tetrahydro-
furan.
G. T. Bourne, W. D. F. Meutermans, P. F. Alewood, R. P.
McGeary, M. Scanlon, A. A. Watson, M. L. Smythe, J. Org.
Chem. 1999, 64, 3095Ϫ3101.
L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397Ϫ4398.
L. A. Carpino, A. El-Faham, C. A. Minor, F. Albericio, J.
Chem. Soc., Chem. Commun. 1994, 201Ϫ203.
A. Ehrlich, H.-U. Heyne, R. Winter, M. Beyermann, H. Haber,
L. A. Carpino, M. Bienert, J. Org. Chem. 1996, 61, 8831Ϫ8838.
F. Albericio, J. M. Bofill, A. El-Faham, S. A. Kates, J. Org.
Chem. 1998, 63, 9678Ϫ9683.
Synthesis of Bicyclic Peptide 9d: The synthesis was conducted as
described for 9c, except that the second cyclization was repeated.
The diastereoisomers could not be separated by the HPLC system.
HRMS (ESIϩ): (C54H80N16O18 ϩ H) requires 1241.5909; found
1241.5861 [M ϩ H]ϩ.
Acknowledgments
We thank H. Hakala and K. Wiinamäki for mass analyses and
[23]
J. Sinkkonen for NMR spectroscopic data.
[24]
[25]
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V. J. Hruby, F. Al-Obeidi, W. Kazmierski, Biochem. J. 1990,
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J. N. Lambert, J. P. Mitchell, K. D. Roberts, J. Chem. Soc.,
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[5]
Y. A. Ovchinnikov, V. T. Ivanov, Tetrahedron 1975, 31,
2177Ϫ2209.
[6]
H. Itokawa, H. Morita, K. Takeya, N. Tomioka, A. Itai, Y.
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[7]
´
I. Kolossvary, W. C. Guida, J. Am. Chem. Soc. 1996, 118,
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5011Ϫ5019.
[8]
A. Lombardi, G. D’Auria, O. Maglio, F. Nastri, L. Quartara,
Received September 17, 2002
Eur. J. Org. Chem. 2003, 1687Ϫ1691
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1691