Synthesis of spirobicyclic peptides on a solid support

Article abstract of DOI:10.1002/1099-0690(200211)2002:21<3616::AID-EJOC3616>3.0.CO;2-J

The spirobicyclic peptides 2-6 were synthesized as stereoisomeric pairs using an orthogonally protected bis(aminomethyl)malonic acid building block 1 as the branching unit. Peptide 2 was synthesized by two methods. Either the chain assembly and first cyclization were carried out on a Wang resin, and the second cyclization in solution (Scheme 1), or the whole synthesis was performed on a solid-supported backbone amide linker derived from 4-alkoxybenzaldehyde (Scheme 3). The applicability of the latter method was further evaluated by synthesis of four additional spirobicyclic peptides 3-6.

Full text of DOI:10.1002/1099-0690(200211)2002:21<3616::AID-EJOC3616>3.0.CO;2-J

FULL PAPER
Synthesis of Spirobicyclic Peptides on a Solid Support
Pasi Virta,*[a] Jari Sinkkonen,[a] and Harri Lönnberg[a]
Keywords: Amino acids / Cyclic peptides / Cyclizations / Solid-phase synthesis
The spirobicyclic peptides 2−6 were synthesized as stereoiso-
meric pairs using an orthogonally protected bis(aminome-
thyl)malonic acid building block 1 as the branching unit.
Peptide 2 was synthesized by two methods. Either the chain
assembly and first cyclization were carried out on a Wang
resin, and the second cyclization in solution (Scheme 1), or
the whole synthesis was performed on a solid-supported
backbone amide linker derived from 4-alkoxybenzaldehyde
(Scheme 3). The applicability of the latter method was further
evaluated by synthesis of four additional spirobicyclic pep-
tides 3−6.
Introduction
Conformationally constrained cyclic analogs of biologic-
ally active peptides are useful tools for defining the struc-
tural requirements for binding to receptors.[1] Additional
cyclization still increases the rigidity, and hence the binding
properties of bicyclic peptides and their analogs are of spe-
cial interest.[2] In spite of this, relatively few synthesis of
bicyclic peptides have been described either in solution[3] or
on a solid support.[4]
Homodetic spiropeptides, i.e. spirobicyclic peptides ob-
tained by backbone cyclization, constitute an interesting
subclass of bicyclic peptides that may exhibit binding prop-
erties characteristic for spiroheteromacrocyclic com-
pounds.[5] We now report on the synthesis of such peptides
by two alternative manners. Previously, the orthogonally
protected bis(aminomethyl)malonic acid building block 1
has been prepared by us and exploited in the construction
of backbone cyclized/branched peptide conjugates.[6] The
same building block has now been used to obtain homode-
tic spirobicyclic peptides (Figure 1). In fact, it appears to be
the smallest building block that may be designed for such a
purpose. The peptide chain containing 1 may be assembled
and the first cyclization is carried out on a Wang resin,
while the second cyclization is done in solution after release
of the lariat-like peptide from the support. Alternatively,
the whole synthesis may be performed on a solid support
bearing a 4-alkoxybenzaldehyde-derived backbone amide
handle. Although the former method appears to give some-
what higher yields, as indicated by the preparation of hepta-
peptide 2 by both procedures, the latter approach, as a pure
solid support synthesis, undoubtedly is the method of cho-
ice for parallel synthesis of peptide libraries. Hence, the ap-
Figure 1. Orthogonally protected bis(aminomethyl)malonic acid 1
as a constituent of the spirobicyclic peptides 2؊6
plicability of this method has been further tested by pre-
paration of the four additional spirobicyclic peptides 3Ϫ6.
To the best of our knowledge, this is the first description
for the synthesis of spiropeptides.
Results and Discussion
Synthesis of Spirobicyclic Peptides
Two alternative methods were applied to obtain the spi-
robicyclic heptapeptide 2. The first of them (Method 1) is
outlined in Scheme 1. Accordingly, a Wang resin was first
loaded with N-Fmoc-β-alanine using a standard symmet-
rical anhydride method (7, loading 90 µmol·gϪ1). The linear
peptide chain containing 1 was then assembled by the Fmoc
[a]
Department of Chemistry, University of Turku,
20014 Turku, Finland
Fax: (internat.) ϩ 358-2/333-6700
E-mail: pasi.virta@utu.fi
3616
 2002 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1434Ϫ193X/02/1121Ϫ3616 $ 20.00ϩ.50/0 Eur. J. Org. Chem. 2002, 3616Ϫ3621

Synthesis of Spirobicyclic Peptides on a Solid Support
FULL PAPER
Scheme 1. i) SPPS; ii) (Ph3P)4Pd0, 0.5  HCl, morpholine, DMSO,
THF; iii) piperidine, DMF; iv) HATU, DIPEA, DMF; v) TFA,
CH2Cl2
Figure 2. HPLC chromatograms of the synthesis products as dias-
tereomeric pairs; A: Lariat-like peptide (10, tr ϭ 13.64 and
14.00 min); B: spirobicyclic peptide (2a, tr ϭ 11.5 min and 2b, tr ϭ
12.4 min) from the cyclization in solution (Method 1); C: an ex-
ample of a spirobicyclic peptide (6, tr ϭ 14.9 and 15.2 min) synthe-
chemistry exploiting established activation methods
(HATU activation[7] for 1 and DCC/HOBt activation[8] for
the other amino acids). After completion of the chain as-
sized by Method 2; D: purified 6 in isocratic system (6a: tr
ϭ
9.0 min; 6b: tr ϭ 10.4 min); gradient from aqueous TFA to aceton-
sembly, the N-terminal Fmoc group and the allyl protec- itrile in 30 min (A and C), 5% acetonitrile in 0.1% aqueous TFA
tion[9] of the branching unit were removed, and the first
(B), 20% acetonitrile in 0.1% aqueous TFA (D), Hypersil Hypurity
C18 (150 ϫ 4.6 mm, 5 µm), flow 1.0 mL·minϪ1, detection at
215 nm
cyclization was carried out (5 equiv. HATU, 10 equiv. DI-
PEA in DMF, 4 h at room temperature). Insertion of 1 in
the peptide chain creates a stereogenic center at the branch-
ing carbon atom, and therefore the monocyclic lariat-like
peptide 10 when cleaved from the support was obtained as
a pair of diastereomers. The side chain (R1) of the Phe res-
idue and carbonyl carbon atom 16 (for numbering, see Fig-
ure 1) can either be on the same or opposite site of the ring.
The overall yield of the diastereomers purified by HPLC
was 50%. Before the second cyclization in solution, the so-
lution of 10 was neutralized and desalted. The cyclization
was performed in diluted solution (1 m 10 in DMF, 5
equiv. HATU, 10 equiv. DIPEA, 4 h at room temperature)
to avoid dimerization. The spirobicyclic diastereomers were
easily separated by isocratic HPLC (2a and 2b, see B in
Figure 2). Both isomers were obtained in a 13% overall isol-
ated yield.
duced originally by Bourne et al.[10] for the Boc chemistry.
The first building block, β-Ala-OtBu, was, however,
coupled to the linker precursor, viz. 4-[5-(4-methoxytri-
tyl)oxypentyloxy]benzaldehyde (13), by reductive amination
in solution (Scheme 2), and the secondary amine was then
protected with an Fmoc group. Detritylation and Jones ox-
idation then gave the linker 16, which was attached to an
Scheme 2. Synthesis of the linker 16: i) MMTrCl, Py; ii) 4-hydro-
xybenzaldehyde, DEAD, Ph3P, THF; iii) H-β-Ala-OtBu·HCl, DI-
PEA, Na2SO4, CH2Cl2; iv) NaBH3CN, AcOH, MeOH; v) AcOH;
The same spiroheptapeptide 2 was also prepared entirely
on a solid support (Method 2). The linker employed was
the 4-alkoxybenzaldehyde backbone amide linker, intro- vi) FmocCl, DIPEA, dioxane; vii) CrO3, H2O, H2SO4, acetone
Eur. J. Org. Chem. 2002, 3616Ϫ3621 3617

P. Virta, J. Sinkkonen, H. Lönnberg
FULL PAPER
HATU, 10 equiv. DIPEA in DMF, 4 h at room temper-
ature). Cleavage by HBr/AcOH/TFA (1:3:40, v/v/v, 3 h at 4
°C, repeated three times) released the bicyclic peptide as a
stereoisomer pair (2). The overall isolated yield after HPLC
purification was 6% for both isomers.
As indicated above, 2 was obtained in a higher yield by
performing only the first cyclization on a solid support. The
drawback of this method, however, is that a laborious
HPLC purification is needed before the second cyclization.
For this reason, and since a pure solid support undoubtedly
is the method of choice for the parallel synthesis of peptide
libraries, the rest of the spirobicyclic peptides 3؊6 were syn-
thesized by Method 2. It is also worth noting that perform-
ance of two subsequent homodetic cyclizations on a single
support still is a challenge, and hence additional examples
of such procedures are desirable. The yields of the isolated
diastereomeric pairs ranged from 10 to 15%. In each case
diastereomeric mixtures were first purified and the isomers
were then separated by isocratic elution. The attempts to
obtain the enantiomerically pure bis(aminomethyl)malonic
acid building block 1 failed, and hence separation of the
final products 2؊6 by simple isocratic elution (4Ϫ20%
MeCN in 0.1% aqueous TFA) to pure diastereomers was
applied. The authenticity of all products was verified by
HPLC/ESI-MS (Table 1). In addition, 2a and 2b prepared
1
in a larger quantity were chacterized by HRMS, H NMR
and 13C NMR (including PDQF, HMQC and HMBC spec-
tra).
Table 1. Required (Mreq) and found ([M ϩ H]ϩfound, LC/ESI-MS/
MS) molecular masses for the spirobicyclic peptides 2؊6
Diastereomer pair
[M ϩ H]ϩfound
Mreq
Scheme 3. i) HATU, DIPEA, DMF; ii) SPPS; iii) TFA, CH2Cl2;
iv) HBr, AcOH, TFA
2
3
4
5
6
615.7
705.8
581.7
637.8
671.8
614.3
704.3
580.3
636.4
670.3
aminomethyl-PS support bearing a short peptide spacer
(Leu-Leu-Gly) (17, loading 90 µmol·gϪ1, Scheme 3). To the
subsequent chain assembly, the Fmoc chemistry was ap-
plied. The use of the tBu ester of β-Ala as the first residue
is essential, since it eliminates the diketopiperazine forma-
tion that has frequently been encountered with backbone
amide linkers during the Fmoc deprotection of the penul-
timate amino acid.[11] Diketopiperazine is a six-membered
ring, and therefore it is not formed when using a β-amino
acid. After the Fmoc deprotection, the exposed secondary
amino group of 17 was acylated with (FmocGly)2O [15
equiv. in DCM/DMF (9:1), 5 h at room temperature]. An
acid anhydride was used as the acylating agent, since sym-
metrical anhydrides have been reported to give higher yields
in this kind of difficult acylations than the corresponding
acids activated with normal coupling reagents.[11a,12] The
monocyclic lariat-like peptide 18 was then obtained as de-
scribed above for the preparation of 9 by Method 1. The
N-Boc protection of the branching unit and the OtBu pro-
tection of the linker-bound β-alanine were removed in a
single step (25% TFA in DCM, 1 h at room temperature),
All the spirobicyclic peptides prepared contain four β-
alanine residues, having only two positions to be random-
ized with natural α-amino acids. While this to some extent
reduces the diversity of the library obtained, it ensures effi-
cient backbone cyclization.[6] Further studies are un-
doubtedly needed to find out whether the diversity may be
still increased by using only natural amino acids.
Experimental Section
General Remarks: The NMR spectra were recorded with Bruker
200 NMR, JEOL JNM-GX 400 and JEOL JNM-A 500 spectro-
meters. The chemical shifts are given in ppm from internal TMS.
The mass spectra were recorded with 7070E VG or PE SCIEX API
365 LC/ESI-MS/MS mass spectrometers. Hypersil C18 columns
(150 ϫ 4.6 mm, 5 µm, or 250 ϫ 10 mm, 5 µm) were used in the
and the second on-resin cyclization was performed (5 equiv. RP HPLC analyses and purifications.
3618 Eur. J. Org. Chem. 2002, 3616Ϫ3621

Synthesis of Spirobicyclic Peptides on a Solid Support
FULL PAPER
Synthesis of the Spirobicyclic Peptides 2؊6
were added, and the mixture was shaken for 4 h at 25 °C. The resin
was filtered, washed with DMF, CH2Cl2, and MeOH, and dried.
Cleavage by HBr/AcOH/TFA (1:3:40, v/v/v, 4 °C, 3 h, three times)
released the stereoisomer pair 2. The overall isolated yield after
HPLC purification was 6% for each stereisomer.
Synthesis of the Diastereomer Pair 2a,b. Method 1: N-Fmoc-pro-
tected β-alanine (0.24 mmol) and diisopropylcarbodiimide (DIC,
0.12 mmol) were dissolved in CH2Cl2 (1.0 mL) and allowed to react
for 20 min at 25 °C. Volatile materials were removed, and the res-
idue was suspended in DMF (1.0 mL). The dissolved material was
added onto the Wang resin (265 mg, initial loading of the hydroxy
groups: 1.03 mmol·gϪ1) pre-swelled in DMF. The suspension was
allowed to react for 1 h at 25 °C, giving a loading of 90 µmol·gϪ1
(determined on the basis of Fmoc). The unchanged hydroxy groups
on the support were capped by acetic anhydride before the syn-
thesis. The linear peptide chain 8 was first assembled by Fmoc
chemistry using well-established methods of activation, viz. HATU
activation for building block 1 and DCC/HOBt activation for the
other amino acids. After chain assembly, the allyl protecting group
of the carboxy function of the branching residue derived from 1
was removed by treatment with (Ph3P)4Pd0 (55 mg, 2 equiv.) in a
mixture of DMSO, THF, 0.5 mol·LϪ1 aq. HCl, and morpholine
(2:2:1:0.1, v/v/v/v; 5.0 mL) for 45 min at 25 °C under argon. The
resin was then washed with DMSO, THF, DMF, Et3N/DMF (1:1,
v/v), CH2Cl2, and MeOH in this order, and the Fmoc group was
removed with a mixture of piperidine and DMF (1:5, v/v, 15 min),
followed by washings with DMF, Et3N/DMF (1:1, v/v), CH2Cl2,
and MeOH in this order. After allyl and Fmoc deprotections, the
resin was suspended in DMF (4.0 mL), and HATU (46 mg, 5
equiv.) and N,N-diisopropylethylamine (DIPEA; 42 µL, 10 equiv.)
in DMF (1.0 mL) were added to the mixture. After 4 h of shaking
at 25 °C, the resin was filtered, washed with DMF, CH2Cl2, and
MeOH, and dried. The product was cleaved from the resin with a
mixture of TFA and CH2Cl2 (1:1, v/v, 25 °C, 30 min), and the crude
monocyclic peptide was purified by HPLC (Figure 2, A), yielding
9.2 mg (50%) of the TFA salt of the two stereoisomers of 10 (ESI-
MS: m/z ϭ 633.7 [M ϩ H]ϩ). The monocyclic peptide 10 was re-
leased from the TFA salt by adding sodium hydroxide (5 m, 2
equiv.) and desalting the mixture by HPLC (elution with water for
10 min, then a linear gradient from water to MeCN in 10 min, flow
rate 1 mL·minϪ1). The eluent was removed under reduced pressure,
and the residue was dissolved in DMF (12 mL). To achieve cycliza-
tion through the terminal amino and carboxy functions, HATU
(23 mg, 5 equiv.) and DIPEA (21 µL, 10 equiv.) were added, and
the mixture was stirred for 4 h at 25 °C. The solvents were evapor-
ated to dryness, the residue was dissolved in 0.1% aq. TFA, and
1
Diastereomer 2a: H NMR (500 MHz, [D6]acetone ϩ D2O, ppm):
δ ϭ 7.30Ϫ7.26 (m, 4 H, Ph), 7.22Ϫ7.18 (m, 1 H, Ph), 4.51 (dd,
J ϭ 9.7 Hz, 1 H and 5.4 Hz, HC-7), 3.88 (d, J ϭ 16.4 Hz, 1 H,
HHC-22), 3.79 and 3.70 (d, 1 H, J ϭ 14.4 Hz and d, 1 H, J ϭ
14.5 Hz, HHC-14 and HHC-29), 3.71 (d, J ϭ 16.4 Hz, 1 H, HHC-
22), 3.67Ϫ3.63 (m, 1 H, HHC-3), 3.57 and 3.56 (d, 1 H, J ϭ 14.5
Hz and d, 1 H, J ϭ 14.4 Hz, HHC-14 and HHC-29), 3.60Ϫ3.35
(m, 6 H, H2C-16, H2C-18 and H2C-25), 3.34Ϫ3.27 (m, 1 H, HHC-
3), 3.19 (dd, J ϭ 13.9 Hz, 1 H and 5.4 Hz, CHHPh), 2.89 (dd, J ϭ
13.9 Hz, 1 H and 9.7 Hz, CHHPh), 2.67Ϫ2.55 and 2.48Ϫ2.36 (m,
3 H and m, 2 H, HHC-4, H2C-11 and H2C-19), 2.43Ϫ2.39 (m, 2
H, H2C-26), 2.30Ϫ2.24 (m, 1 H, HHC-4) (Figure 3). 13C NMR
(125 MHz, [D6]acetone ϩ D2O, ppm): δ ϭ 177.03 and 176.99 (C-
12 and C-27), 176.4 (C-20), 175.5 (C-5), 174.9 (C-8), 173.4 (C-23),
173.1 and 172.9 (C-1 and C-16), 140.5, 131.9, 131.1, and 129.3
(Ph), 62.1 (C-15), 58.4 (C-7), 46.4 (C-22), 44.5 and 44.1 (C-14 and
C-29), 39.4 (CH2Ph), 39.1 and 37.9 (C-10 and C-18), 38.9 (C-3),
38.4 (C-25), 38.3 and 36.8 (C-11 and C-19), 38.2 (C-26), 37.3 (C-
4). PDQF, HMQC and HMBC spectra were also utilized. HRMS
(EI): calcd. for C28H38N8O8 [Mϩ] 614.281; found 614.283.
1
Figure 3. H NMR (500 MHz, [D6] ϩ D2O) spectrum of 2a
1
purified by HPLC (Figure 2, B). The spirobicyclic products 2a and Diastereomer 2b: H NMR (500 MHz, [D6]acetone ϩ D2O, ppm):
2b were separated. The isolated yield for both stereoisomers was
2.0 mg (26% from 10, overall 13%). Method 2: A short peptide
spacer (Leu-Leu-Gly) was first attached to an aminomethylpolys-
δ ϭ 7.32Ϫ7.26 (m, 4 H, Ph), 7.21Ϫ7.18 (m, 1 H, Ph), 4.51 (dd,
J ϭ 9.5 Hz, 1 H and 5.5 Hz, HC-7), 3.87 (d, J ϭ 14.4 Hz, 1 H,
HHC-29), 3.81 (s, 2 H, H2C-22), 3.74 (d, J ϭ 14.7 Hz, 1 H, HHC-
tyrene resin. Initial loading (0.6 mmol·gϪ1) was reduced (90 14), 3.75Ϫ3.70 (m, 1 H, HHC-18), 3.69Ϫ3.64 (m, 1 H, HHC-25),
µmol·gϪ1) and the unchanged amino groups were carefully capped
with acetic anhydride. The solid support 17 (130 mg, loading 90
µmol·gϪ1) employed in the peptide synthesis was then obtained by
acylating the tripeptide-derivatized aminomethylpolystyrene resin
with 16 (HATU/DIPEA activation in DMF). Treatment with piper-
idine/DMF (1:5, 15 min) exposed the secondary amino group,
which was subsequently acylated with (Fmoc-Gly)2O (15 equiv.) at
3.57 (d, J ϭ 14.7 Hz, 1 H, HHC-14), 3.58Ϫ3.52 (m, 1 H, HHC-
10), 3.51Ϫ3.46 (m, 1 H, HHC-3), 3.48 (d, J ϭ 14.4 Hz, 1 H, HHC-
29), 3.43Ϫ3.38 (m, 1 H, HHC-3), 3.37Ϫ3.24 (m, 3 H, HHC-18,
HHC-25 and HHC-10), 3.17 (dd, J ϭ 14.0 Hz, 1 H and 5.4 Hz,
CHHPh), 2.87 (dd, J ϭ 14.0 Hz and 9.4 Hz, CHHPh), 2.60Ϫ2.48
(m, 4 H, HHC-4, H2C-19 and HHC-11), 2.45Ϫ2.37 (m, 3 H, HHC-
4 and H2C-26), 2.26 (ddd, 1 H, J ϭ 15.2, 5.2 Hz and 2.7 Hz, HHC-
25 °C for 6 h. The resin-bound lariat-like monocyclic peptide 18, 4) (Figure 4). 13C NMR (125 MHz, [D6]acetone ϩ D2O, ppm): δ ϭ
bearing an N-terminal Boc and O-terminal tBu protection, was
then assembled using the same activation and deprotection
methods as described above for the preparation of 9 by Method 1.
The Boc and tBu protections were removed in a single step by TFA/
CH2Cl2 (1:4, v/v, 25 °C, 1 h), followed by washings with CH2Cl2,
Py/CH2Cl2 (1:19, v/v), and MeOH in this order. The resin was dried
under reduced pressure, and suspended in DMF (1.5 mL). HATU
177.1 (C-12), 176.7 (C-27), 176.3 (C-20), 175.5 (C-5), 174.7 (C-8),
173.3 (C-23), 173.03 and 172.98 (C-1 and C-16), 140.5, 131.9,
131.1, and 129.2 (Ph), 62.2 (C-15), 58.2 (C-7), 46.3 (C-22), 44.2 (C-
14), 44.0 (C-29), 39.04 and 39.03 (C-3 and CH2Ph), 38.84 and 38.77
(C-18 and C-26), 38.11 and 38.07 (C-11 and C-25), 38.0 (C-10),
37.5 (C-19), 36.9 (C-4). PDQF, HMQC and HMBC spectra were
also utilized. HRMS (EI): calcd. for C28H38N8O8 [Mϩ] 614.281;
(22 mg, 5 equiv.) in DMF (0.4 mL) and DIPEA (20 µL, 10 equiv.) found 614.284.
Eur. J. Org. Chem. 2002, 3616Ϫ3621
3619

P. Virta, J. Sinkkonen, H. Lönnberg
FULL PAPER
ine (DIPEA, 0.33 mL, 1.9 mmol), and excess of Na2SO4 were
stirred in CH2Cl2 (20 mL) for 2 d at room temperature. Solids were
filtered off, and the filtrate was concentrated to yield a yellow oil.
The oil was dissolved in a cold (ϩ4 °C) mixture of AcOH and
MeOH (AcOH/MeOH, 3:200, v/v, 40 mL), and NaBH3CN (0.24 g,
3.7 mmol) was added to the mixture, which was allowed to warm
to room temperature, stirred for additional 5 h, and the solvents
were evaporated to dryness. The resulting oil was diluted with
AcOH (30 mL), stirred overnight at room temperature, and then
the volatiles were removed. The residue was diluted with CH2Cl2,
washed with water and brine, dried with Na2SO4, and the solvents
were evaporated to dryness. The crude product was purified by sil-
ica gel chromatography (10% MeOH in CH2Cl2) to yield 0.52 g
(83%) of 14 as a colourless oil. 1H NMR (400 MHz, CDCl3, ppm):
δ ϭ 7.37 (d, J ϭ 8.5 Hz, 2 H, Ph), 6.90 (d, J ϭ 8.5 Hz, 2 H, Ph),
4.11 (s, 2 H, PhCH2NH), 3.94 (t, J ϭ 6.4 Hz, 2 H, CH2OPh), 3.66
(t, J ϭ 6.4 Hz, 2 H, HOCH2), 3.12 (t, J ϭ 6.7 Hz, 2 H,
NHCH2CH2), 2.75 (t, J ϭ 6.7 Hz, 2 H, CH2COO), 1.80 (m, 2 H,
CH2CH2OPh), 1.62 (m, 2 H, HOCH2CH2), 1.53 [m, 2 H,
CH2(CH2)2OPh], 1.43 [s, 9 H, C(CH3)3]. 13C NMR (100 MHz,
CDCl3, ppm): δ ϭ 170.7 (COOtBu), 159.9, 131.3, 122.8, 115.1 (Ph),
82.5 (Cq in tBu), 67.9 (CH2OPh), 62.6 (HOCH2), 51.5
(PhCH2NH), 42.6 (NHCH2CH2), 32.3 (CH2COO), 31.6
(CH2CH2OPh), 28.9 (HOCH2CH2), 28.0 [C(CH3)3], 22.3
1
Figure 4. H NMR (500 MHz, [D6] ϩ D2O) spectrum of 2b
Syntheses of the Diastereomer Pairs 3؊6: The spirobicyclic peptides
3Ϫ6 were synthesized analogously to 2 by Method 2. The overall
isolated yields as stereoisomeric pairs were 10Ϫ15%. All stereoiso-
mers were first purified (Figure 2, C), and the stereoisomers were
then separated by an isocratic elution (4Ϫ20% MeCN in 0.1%
aqueus TFA, Figure 2, D). The authenticity of the products was
verified by HPLC/ESI-MS. Calulated and found molecular masses
Mreq and [M ϩ H]ϩ for all the spirobicyclic peptides 2؊6 are
shown in Table 1.
[CH2(CH2)2OPh]. MS (EI): m/z (%)
ϭ 208 (100) [M Ϫ
(CH2)2COOtBu]ϩ, 280 (28) [M Ϫ tBu]ϩ, 337 (3) [Mϩ]. HRMS (EI):
Synthesis of the Linker 16
calcd. for C19H31NO4 [Mϩ] 337.2253; found 337.2251.
4-[5-(4-Methoxytrityloxy)pentyloxy]benzaldehyde (13): 4-Methoxy-
trityl chloride (5.0 g, 16 mmol) was added to a stirred mixture of
1,5-pentanediol (11, 7.4 mL, 70 mmol) and pyridine (50 mL). The
mixture was agitated overnight at room temperature, and then all
volatile substances were removed. The residue was dissolved in
CH2Cl2, washed with water and brine, dried with Na2SO4, and the
solvents were evaporated to dryness. The residue was purified by
silica gel chromatography (30% EtOAc in petroleum ether) to yield
tert-Butyl
3-{(9-Fluorenylmethoxycarbonyl)[4-(5-hydroxypentyl-
oxy)benzyl]amino}propanoate (15): 9-Fluorenylmethoxycarbonyl
chloride (FmocCl; 1.8 g, 7.1 mmol) was added portionwise to a
stirred solution of 14 (2.2 g, 6.4 mmol) and DIPEA (1.1 mL,
6.4 mmol) in dioxane (30 mL). The reaction mixture was stirred
overnight at room temperature, and the solvents were evaporated
to dryness. The residue was purified by silica gel chromatography
(0 to 4% MeOH in CH2Cl2) to yield 2.2 g (61%) of 15 as a colour-
less oil. 1H NMR (400 MHz, CDCl3, ppm): δ ϭ 7.74 (m, 2 H,
Fmoc), 7.59 (d, J ϭ 7.1 Hz, 1 H, Ph), 7.48 (d, J ϭ 7.1 Hz, 1 H,
Ph), 7.38 (m, 2 H, Fmoc), 7.33Ϫ7.24 (m, 2 H, Fmoc), 7.11 (d, J ϭ
8.0 Hz, 1 H, Ph), 6.94 (d, J ϭ 7.9 Hz, 1 H, Ph), 6.79 (m, 2 H,
Fmoc), 4.53 (m, 2 H, CH2O in Fmoc), 4.39, 4.30 (s and s, 2 H,
PhCH2N), 4.24 (m, 1 H, H9 Fmoc), 3.94 (t, J ϭ 6.4 Hz, 2 H,
CH2OPh), 3.67 (m, 2 H, HOCH2), 3.44, 3.27 (m and m, 2 H,
NCH2CH2), 2.46, 2.17 (m and m, 2 H, CH2CH2COO), 1.80 (m, 2
H, CH2CH2OPh), 1.70Ϫ1.50 (m, 4 H, HOCH2CH2CH2), 1.43 [s,
9 H, C(CH3)3]. 13C NMR (100 MHz, CDCl3, ppm): δ ϭ 171.1,
170.8 (COOtBu), 158.3 (Ph), 156.4, 155.9 (CϭO in Fmoc), 143.9,
141.3, 129.1, 128.5, 127.6, 127.0, 124.8, 119.9, 114.5 (Fmoc and
Ph), 80.7 (Cq in tBu), 67.7 (CH2OPh), 67.1, 67.0 (CH2O in Fmoc),
62.7 (CH2OH, 50.3, 50.1 (PhCH2N), 47.4, 47.3 (C9, Fmoc), 43.2,
42.1 (NCH2CH2), 34.4, 34.1 (CH2COOtBu), 32.4 (CH2CH2OPh),
29.0 (HOCH2CH2), 28.0 [C(CH3)3], 22.3 [CH2(CH2)2OPh]. MS
(EI): m/z (%) ϭ 280 (100) [M Ϫ Fmoc Ϫ tBu]ϩ, 502 (5) [M Ϫ
tBu]ϩ, 559 (1.5) [Mϩ]. HRMS (EI): calcd. for C34H41NO6 [Mϩ]
559.2934; found 559.2933.
1
5.4 g (89%) of 12 as a colourless oil. H NMR (200 MHz, CDCl3,
ppm): δ ϭ 7.37Ϫ7.06 (m, 12 H, MMTr), 6.72 (m, 2 H, MMTr),
3.68 (s, 3 H, CH3O), 3.51 (t, J ϭ 6.2 Hz, 2 H, CH2OH), 2.96 (t,
J
ϭ 6.4 Hz, 2 H, MMTrOCH2), 1.62Ϫ1.28 (m, 6 H,
CH2(CH2)3CH2). The tritylated pentanol 12 (1.0 g, 2.7 mmol), 4-
hydroxybenzaldehyde (0.36 g, 2.9 mmol) and Ph3P (0.77 g,
2.9 mmol) were dissolved in THF (20 mL). Diethyl azodicarboxyl-
ate (DEAD, 0.60 g, 3.4 mmol) was added dropwise to the mixture,
which was stirred overnight at room temperature. The volatile sub-
stances were removed under reduced pressure, and the crude reac-
tion product was purified by silica gel chromatography (CH2Cl2)
to yield 0.99 g (78%) of 13 as a colourless oil. 1H NMR (400 MHz,
CDCl3, ppm): δ ϭ 9.87 (s, 1 H, CHO), 7.80 (d, J ϭ 8.6 Hz, 2 H,
Ph), 7.45Ϫ7.19 (m, 12 H, MMTr), 6.96 (d, J ϭ 8.6 Hz, 2 H, Ph),
6.81 (d, J ϭ 8.7 Hz, 2 H, MMTr), 4.01 (t, J ϭ 6.4 Hz, 2 H,
CH2OPh), 3.78 (s, 3 H, CH3O), 3.09 (t, J ϭ 6.3 Hz, 2 H,
MMTrOCH2), 1.79 (m, 2H,CH2CH2OPh), 1.69 (m,
2 H,
MMTrOCH2CH2), 1.57 (m, 2 H, CH2(CH2)2OPh). 13C NMR
(100 MHz, CDCl3, ppm): δ ϭ 190.8 (CHO), 164.2, 158.4, 144.9,
136.1, 131.9, 130.3, 129.7, 128.4, 127.7, 126.7, 114.7, 112.9 (MMTr
and Ph), 86.0 (Cq, MMTr), 68.2 (CH2OPh), 63.1 (MMTrOCH2),
55.2 (CH3O), 29.7 (CH2CH2OPh), 28.8 (MMTrOCH2CH2), 22.7
[CH2(CH2)2OPh]. MS (EI): m/z (%) ϭ 273 (100) [MMTrϩ], 480
(14) [Mϩ]. HRMS (EI): calcd. for C32H32O4 [Mϩ] 480.2301;
found 480.2307.
5-(4-{[(2-tert-Butoxycarbonylethyl)(9-fluorenylmethoxycarbonyl)-
amino]methyl}phenoxy)pentanoic Acid (16): A solution of CrO3
(0.42 g, 4.2 mmol), H2SO4 (0.42 mL) and water (1.3 mL) was added
dropwise to the stirred solution of 15 (2.1 g, 3.8 mmol) in acetone
(75 mL). The mixture was agitated for 2 h at room temperature.
tert-Butyl
3-{[4-(5-Hydroxypentyloxy)benzyl]amino}propanoate CHCl3 (75 mL) and pyridine (0.6 mL) were added to the mixture,
(14): The aldehyde 13 (0.90 g, 1.9 mmol), the tert-butyl ester of β-
alanine hydrochloride (0.34 g, 1.9 mmol), N,N-diisopropylethylam-
and then the resulting organic phase was filtered, washed with
brine, dried with Na2SO4, and the solvents were evaporated to dry-
3620
Eur. J. Org. Chem. 2002, 3616Ϫ3621

Synthesis of Spirobicyclic Peptides on a Solid Support
FULL PAPER
G. C. Zanotti, F. Pinnen, G. Lucente, L. Paolillo, G. D’Au-
[3] [3a]
ness. The residue was purified by silica gel chromatography (10%
MeOH in CH2Cl2) to yield 1.9 g (86%) of 16 as a colourless oil.
1H NMR (400 MHz, CDCl3, ppm): δ ϭ 7.74 (m, 2 H, Fmoc), 7.59
(d, J ϭ 7.1 Hz, 1 H, Ph), 7.48 (d, J ϭ 7.1 Hz, 1 H, Ph), 7.40Ϫ7.20
(m, 4 H, Fmoc), 7.11 (d, J ϭ 8.1 Hz, 1 H, Ph), 6.93 (d, J ϭ 7.9
Hz, 1 H, Ph), 6.79 (m, 2 H, Fmoc), 4.54 (m, 2 H, CH2O in Fmoc),
4.39, 4.30 (s and s, 2 H, PhCH2N), 4.24 (m, 1 H, 9-H Fmoc), 3.95
(m, 2 H, CH2OPh), 3.46, 3.27 (m and m, 2 H, NCH2CH2), 2.44
(m, 3 H, HOOCCH2 and NCH2CH2COOtBu), 2.18 (m, 1 H,
NCH2CH2COOtBu), 1.84 (m, 4 H, CH2CH2CH2OPh), 1.43 [s, 9 H,
C(CH3)3]. 13C NMR (100 MHz, CDCl3, ppm): δ ϭ 178.9 (COOH),
171.2, 170.9 (COOtBu), 158.3 (Ph), 156.4,156.0 (CϭO in Fmoc),
144.0, 141.4, 129.2, 128.6, 127.7, 127.1, 124.9, 120.0, 114.5 (Fmoc
and Ph), 80.7 (Cq in tBu), 67.3 (CH2OPh), 67.2 (CH2O in Fmoc),
50.4, 50.1 (PhCH2N), 47.5, 47.3 (C-9 Fmoc), 43.2, 42.2
(NCH2CH2), 34.4, 34.2 (CH2CH2COOtBu), 33.6 (CH2CH2OPh),
28.6 (HOOCCH2), 28.1 [C(CH3)3], 21.4 [CH2(CH2)2OPh]. MS (EI):
m/z (%) ϭ 178 (100) [benzofulveneϩ], 294 (17) [Mϩ Ϫ Fmoc Ϫ
tBu], 516 (1) [Mϩ Ϫ tBu], 573 (0.5) [Mϩ]. HRMS (EI): calcd. for
C34H39NO7 [Mϩ] 573.273; found 573.271.
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