Solid-Supported Synthesis of Cryptand-like Macrobicyclic Peptides

Article abstract of DOI:10.1021/jo0344945

A straightforward method for the solid-supported synthesis of cryptand-like bicyclic peptides (1-5) on a backbone amide linker has been described. For the branching, two novel easily available building blocks, viz. N-(4-methoxytrityl)-N-(2-nitrobenzenesul

Full text of DOI:10.1021/jo0344945

Solid -Su p p or ted Syn th esis of Cr yp ta n d -lik e Ma cr obicyclic
P ep tid es
Pasi Virta* and Harri Lo¨nnberg
Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
pasi.virta@utu.fi
Received April 17, 2003
A straightforward method for the solid-supported synthesis of cryptand-like bicyclic peptides (1-
5) on a backbone amide linker has been described. For the branching, two novel easily available
building blocks, viz. N-(4-methoxytrityl)-N-(2-nitrobenzenesulfonyl)-protected N,N-bis(2-aminoethyl)-
â-alanine (6) and N-(9-fluorenylmethoxycarbonyl) protected iminodiacetic acid monoallyl ester (7),
have been employed. The key steps of the synthesis are as follows: (i) stepwise coupling of one
amino acid and 6 to the secondary amino group of the linker; (ii) removal of the 2-nitrobenzene-
sulfonyl group and SPPS by the Fmoc chemistry, using 7 as the penultimate and tert-butoxycarbonyl
(Boc) protected glycine as the last amino acid; (iii) removal of the 4-methoxytrityl protection and
subsequent SPPS by the Fmoc chemistry; (iv) removal of the allyl and Fmoc groups, followed by
cyclization; and (v) removal of the Boc and tert-butyl groups, followed by cyclization. Final cleavage
from the support and removal of benzyl-derived protecting groups gives the desired bicyclic products.
In tr od u ction
propriately protected lysine,3 Nꢀ-(1,3-diaminopropionyl)-
lysine,4 or 2-amino-3-[4-(aminomethyl)phenylacetyl]amino-
propionic acid5 building blocks in the peptide chain and
coupling the side chain amino groups to the â-carboxy
group of two aspartate residues. Another approach
exploits conjugate group mediated cyclization: a support-
bound peptide bearing one 3,5-dihydroxybenzoyl and two
2-fluoro-5-nitrobenzoyl residues is subjected to a SnAr
displacement of the fluoro substituents by the benzoyl
hydroxy groups.6 We have recently reported on a solid-
supported synthesis of spirobicyclic peptides7 and bicyclic
peptides containing three parallel peptide chains.8 These
syntheses have been based on bis(aminomethyl)malonic
acid9 and bis(aminomethyl)-â-alanine10 as branching
units. While the former compound allowed rather con-
venient solid-supported cyclization to spiro bicyclic pep-
tides, bicyclic peptides containing three parallel chains
were obtained in an overall yield less than 5% on using
the latter building block for branching. We now report
on a more efficient solid-supported synthesis that allows
preparation of cryptand-like bicyclic peptides having two
parallel and one opposite peptide chains. The method is
based on exploitation of a nitrogen atom as the branching
Conformationally constrained cyclic peptides have
gained interest as structural probes with which the
binding characteristics of receptors may be elucidated.1
Additional cyclization may increase their rigidity further,
and hence bicyclic peptides and their analogues consti-
tute an even more useful set of tools for such studies.2
The spatially defined locations of the side chain func-
tional groups may be systematically varied by changing
the amino acid content, which can be utilized as a basis
for the interpretation of the structure-affinity studies.
Bearing this in mind, it is quite surprising that only a
few studies on the solid-supported synthesis of bicyclic
peptides are available. Side-chain lactam-bridged bicyclic
peptides have been obtained by incorporating two ap-
(1) (a) Hruby, V. J .; Al-Obeidi, F.; Kazmierski, W. Biochem. J . 1990,
268, 249. (b) Rizo, J .; Gierasch, L. M. Annu. Rev. Biochem. 1992, 61,
387. (c) Blakburn, C.; Kates, S. A. Methods Enzymol. 1997, 289, 175.
(d) Lambert, J . N.; Mitchell, J . P.; Roberts, K. D. J . Chem. Soc., Perkin
Trans. 1 2001, 471.
(2) (a) Ovchinnikov, Y. A.; Ivanov, V. T. Tetrahedron 1975, 31, 2177.
(b) Zanotti, G. C.; Pinnen, F.; Lucente, G.; Paolillo, L.; D’Auria, G.;
Trivellone, E.; D’Alagni, M. Biopolymers 1989, 28, 305. (c) Itokawa
H.; Morita, H.; Takeya, K.; Tomioka, N.; Itai, A.; Iitaka, Y. Tetrahedron
1991, 47, 7007. (d) Pavone, V.; Lombardi, A.; Nastri, F.; Saviano, M.;
Maglio, O.; D’Auria, G.; Quartara, L.; Maggi, C. A.; Pedone, C. J . Chem.
Soc., Perkin Trans. 2 1995, 987. (e) Quartara, L.; Pavone, V.; Pedone,
C.; Lombardi, A.; Renzetti, A. R.; Maggi, C. A. Regul. Pept. 1996, 65,
55. (f) Kolossvary, I.; Guida, W. C. J . Am. Chem. 1996, 118, 5011. (g)
Lombardi, A.; D’Auria, G.; Saviano, M.; Maglio, O.; Nastri, F.;
Quartara, L.; Pedone, C.; Pavone, V. Biopolymers 1997, 40, 505. (h)
Lombardi, A.; D’Auria, G.; Maglio, O.; Nastri, F.; Quartara, L.; Pedone,
C.; Pavone, V. J . Am. Chem. 1998, 120, 5879. (i) Oliva, R.; Falcigno,
L.; D’Auria, G.; Zanotti, G.; Paolillo, L. Biopolymers 2001, 56, 27. (j)
Oliva, R.; Falcigno, L.; D’Auria, G.; Saviano, M.; Paolillo, L.; Ansanelli,
G.; Zanotti, G. Biopolymers 2000, 53, 581. (k) Akaji, K.; Aimoto, S.
Tetrahedron 2001, 57, 1749.
(3) Bisello, A.; Nakamoto, C.; Rosenblatt, M.; Chorev, M. Biochem-
istry 1997, 36, 3293.
(4) Zhang, W.; Taylor, J . W. Tetrahedron Lett. 1996, 37, 2173.
(5) Yu, C.; Taylor, J . W. Tetrahedron Lett. 1996, 37, 1731.
(6) Kohn, W. D.; Zhang, L.; Weigel, J . A. Org. Lett. 2001, 3, 971.
(7) Virta, P.; Sinkkonen, J .; Lo¨nnberg H. Eur. J . Org. Chem. 2002,
3616.
(8) Karskela, T.; Heinonen, P.; Virta, P.; Lo¨nnberg, H. Eur. J . Org.
Chem. 2003, 9, 1687.
(9) Virta, P.; Rosenberg, J .; Karskela, T.; Heinonen, P.; Lo¨nnberg,
H. Eur. J . Org. Chem. 2001, 3467.
(10) Katajisto, J .; Karskela, T.; Heinonen, P.; Lo¨nnberg, H. J . Org.
Chem. 2002, 67, 7995.
10.1021/jo0344945 CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/04/2003
8534
J . Org. Chem. 2003, 68, 8534-8538

Solid-Phase Synthesis of Bicyclic Peptides
SCHEME 1a
triazolo-[4,5-b]pyridinium hexafluorophosphate 3-oxide
(HATU)-promoted coupling13 to aminomethyl polystyrene
derivatized with a Leu-Leu-Gly spacer. A loading of 180
µmol‚g-1 was obtained, according to the amount of
benzofulvene released from the Fmoc-protected support-
bound linker (12) upon deblocking. The exposed second-
ary amino group of the pre-linked â-alanine residue was
acylated with either N-Fmoc-Phe-OH (13) or N-Fmoc-
Asp(OBn)-OH (14), using a standard anhydride method.
It is worth noting that removal of the Fmoc group by a
piperidine treatment did not result in aspartimide forma-
tion in this position, which often is encountered with
peptides having a benzyl-protected aspartate residue.14
Branching unit 6 was then coupled by HATU activation
to the terminal R-amino group to obtain 15 and 16. The
o-NBS protection was removed with mild basic thiophenol
treatment15 (PhSH-DBU-DMF 1:1:18), and the first
peptide arm was assembled by the HATU/Fmoc chem-
istry, using branching unit 7 as the penultimate amino
acid (17, 18). The N-MMTr protection that well tolerated
the coupling and deprotection conditions employed was
then removed with dichloroacetic acid in dichloromethane
(3:97), and the second peptide arm was constructed (19-
23). The allyl group of branching unit 7 and the Fmoc
group of the second peptide arm were removed in this
order, and then cyclization efficiencies were studied by
using bicyclic peptide (1) as a model. Three different
coupling reagents, viz. HATU, benzotriazol-1-yl-N-oxy-tris-
(pyrrolidino)phosphonium hexafluorophosphate (PyBOP),16
and 7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phos-
phonium hexafluorophosphate (PyAOP),17 were employed
without additives. A mixture of DMSO and NMP (1:4,
v/v) was used as a solvent. This mixture has been
reported to minimize on-resin oligomerization,4 since a
combination of high solvent polarity and good peptide-
resin swelling impede hydrogen bond formation between
the support-bound chains.18 The first cyclization was
rather facile. Each of the coupling reagents at a 2-fold
excess in the presence of N,N-diisopropylethylamine
(DIEA, 4 equiv) gave almost complete cyclization in 5 h,
as shown by HPLC analysis of small aliquots released
from 24. No sign of imide formation at the iminodiacetyl
residue (7) was observed during the peptide chain as-
sembly or cyclization. After completion of the first cy-
clization, the Boc and tert-butyl protections were removed
in a single step with TFA in dichloromethane (DCM), and
then the efficiency of the second cyclization was studied.
The coupling procedures indicated above now resulted
in incomplete cyclization: 87%, 75%, and 69% with
PyBOP, PyAOP, and HATU, respectively [yields calcu-
lated from peak areas of 1 (b) and the corresponding
starting material (a), Figure 2]. These coupling efficien-
cies are inconsistent with the earlier results, according
a
Reagents and conditions: (i) MMTrCl, Py. (ii) 2-Nitrobenzene-
sulfonyl chloride, Et3N, dioxane. (iii) (1) Ethyl acrylate, DBU,
DCM, EtOH; (2) KOH, H2O, dioxane; (iv) (1) DIC, DMF, DCM;
(2) AlIOH.
point. Accordingly, peptides 1-5, containing leucine,
aspartate, phenylalanine, serine, histidine, and â-alanine
residues, were prepared on a backbone amide linker,11
using branching units 6 and 7 to construct the cryptand-
like structure. The otherwise difficult second cyclization
is in all likelihood facilitated by rapid pyramidal inver-
sion around the central nitrogen atom in 6 and 7, which
allows the peptide chains to adopt a mutual orientation
required for the lactamization without any retardation
by incompatible chirality of the branching units.
Resu lts a n d Discu ssion
Syn th esis of th e Br a n ch ig Un its (6, 7). The branch-
ing units (6, 7) used to construct the bicyclic peptides (1-
5) were easily obtained from commercially available
starting materials (Scheme 1). N,N-Bis(2-aminoethyl)-
3-aminopropionic acid bearing the 4-methoxytrityl (MMTr)
and 2-nitrobenzenesulfonyl (o-NBS) protections at the
primary amino groups (6) was prepared from diethylene-
triamine. The 4-methoxytrityl group was first introduced
by treating the starting material with 4-methoxytrityl
chloride in pyridine (8),12 and the remaining primary
amino group was reacted with 2-nitrobenzenesulfonyl
chloride in dioxane, resulting in precipitation of 3-aza-
N1-(4-methotrityl)-N5-(2-nitrobenzenesulfonyl)pentane-
1,5-diamine (9) as a hydrochloride. Michael-type addition
of 9 to the â-carbon atom of ethyl acrylate, followed by
alkaline hydrolysis of the ester linkage, then gave N-(4-
methoxytrityl)-N-(2-nitrobenzenesulfonyl)-3-aminopropi-
onic acid (6) in an overall 55% yield (calculated from 8).
The monoallyl ester of N-(9-fluorenylmethoxycarbonyl)
(Fmoc) protected iminodiacetic acid (7) was, in turn,
obtained in a 90% yield by diisopropylcarbodiimide (DIC)
promoted conversion of the commercially available N-
protected iminodiacetic acid (10) to its anhydride that
was reacted with allyl alcohol.
Syn th esis of th e Bicyclic P ep tid es (1-5). The
synthesis of bicyclic peptides (1-5) is outlined in Scheme
2. A 4-alkoxybenzaldehyde derived backbone amide
linker (11),11b prepared as described previously,7 was first
attached by 1-[bis(dimethylamino)methylene]-1H-1,2,3-
(13) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J .
Chem. Soc., Chem. Commun. 1994, 201.
(14) (a) Yang, Y.; Sweeney, W. V.; Schneider, K.; Tho¨rnqvist, S.;
Chait, B. T.; Tam, J . P. Tetrahedron Lett. 1994, 35, 9689. (b) Quibell,
M.; Owen, D.; Peckman, L.; J ohnson, T. J . Chem. Soc., Chem. Commun.
1994, 2343. (c) Packman, L. C. Tetrahedron Lett. 1995, 36, 7523.
(15) Seitz, O. Angew. Chem., Int. Ed. 1998, 37, 3109.
(16) Coste, J .; Le-Nguen, D.; Castro, B. Tetrahedron Lett. 1990, 31,
205.
(17) Albericio, F.; Cases, M.; Alsina, J .; Triolo, S. A.; Carpino, L. A.;
Kates, S. A. Tetrahedron Lett. 1997, 38, 4853.
(18) (a) Fields, G. B.; Fields, C. G. J . Am. Chem. Soc. 1991, 113,
4202. (b) Santini, R.; Griffith, M. C.; Qi, M. Tetrahedron Lett. 1998,
39, 8951. (c) Vaino, A. R.; J anda, K. D. J . Comb. Chem. 2000, 2, 579.
(11) (a) J ensen, K. J .; Alsina, J .; Songster, M. F.; Vagner, J .;
Albericio, F.; Barany, G. J . Am. Chem. Soc. 1998, 120, 5441. (b) Bourne,
G. T.; Meutermans, W. D. F.; Alewood, P. F.; McGeary, R. P.; Scanlon,
M.; Watson, A. A.; Smythe, M. L. J . Org. Chem. 1999, 64, 3095.
(12) Verheijen, J . C.; Deiman, B. A. L. M.; Yeheskiely, E.; van der
Marel, G. A.; van Boom, J . H. Angew. Chem., Int. Ed. 2000, 39, 369.
J . Org. Chem, Vol. 68, No. 22, 2003 8535

Virta and Lo¨nnberg
SCHEME 2a
a
Reagents and conditions: (a) (i) HATU, DIEA, DMF. (ii) Piperidine, DMF. (iii) (FmocPhe)2O or (FmocAsp)2O, DMF, DCM. (iv) 6,
HATU, DIEA, DMF. (v) PhSH, DBU, DMF. (vi) Fmoc SPPS. (vii) DCA, DCM. (viii) Pd(AcO)2, Ph3P, Bu3SnH, AcOH, DCM. (ix) PyAOP,
DIEA, DMSO, NMP. (x) TFA, DCM. (xi) HBr, AcOH, anisole, TFA.
to which the cyclization with HOBt-based reagents is
usually slower than that with HOAt-based reagents, and
with uronium salts slower than with phosphonium
salts.1c,19 The excess of the coupling reagent and DIEA
was then increased to 5- and 10-fold, respectively. Under
such conditions, the yield of cyclization exceeded 90%
with all the coupling agents employed. No marked
differences in the overall isolated yields of 1 (15-17%)
were observed. Even though PyBOP seemed to be the
most efficient cyclization reagent, the isolated yield was
highest with PyAOP.20 Accordingly, PyAOP/DIEA (5/10
equiv, 5 h) was then used for the cyclization of monocyclic
peptides 24-28 to their bicyclic counterparts. The same
treatment was used also for the first cyclization of
deprotected 19-23, even though each of the coupling
reagents gave already almost complete cyclization at a
2-fold excess indicated above for 24. Acidolytic cleavage
and deprotection, followed by RP HPLC purification
(Figures 3 and 4), gave the bicyclic peptides (1-5) in a
(19) Arttamangkul S.; Arbogast, B.; Barofsky, D.; Aldrich, J . V. Lett.
Pept. Sci. 1997, 3, 357.
(20) Plaue, S. Int. J . Pept. Protein Res. 1990, 35, 510
8536 J . Org. Chem., Vol. 68, No. 22, 2003

Solid-Phase Synthesis of Bicyclic Peptides
F IGURE 3. An example of analytical RP HPLC chromato-
grams of a crude product mixture of bicyclic peptide 3.
Notation: (a) bicyclic product (3), tr ) 13.98 min, (b) mainly
polymerized material. For the chromatographic conditions, see
protocol B in the Experimental Section.
F IGURE 1. Strucures 1-7.
F IGURE 2. Analytical HPLC chromatograms of the crude
product mixtures of 1 obtained by using 2 equiv of different
coupling reagents in the second cyclization: (i) HATU, (ii)
PyAOP, and (iii) PyBOP. The reaction time was 5 h (at room
temperature), 4 equiv of DIEA was used as a base, and
DMSO-NMP (1:4) was used as a solvent. Notation: (a)
starting material, tr ) 13.56 min; (b) bicyclic product (1), tr )
13.94 min. For the chromatographic conditions, see protocol
A in the Experimental Section.
F IGURE 4. Analytical RP HPLC chromatograms of purified
bicyclic peptides 1-5. Notation: (a) 2, tr ) 12.46 min, (b) 5, tr
) 14.18 min, (c) 3, tr ) 14.22 min, (d) 1, tr ) 15.90 min, (e) 4,
tr ) 18.19 min. For the chromatographic conditions, see
protocol B in the Experimental Section.
TABLE 1. Requ ir ed {[(M + 2H+)/2]r eq} a n d F ou n d {[(M +
2H+)/2]fou n d } LC/ES-HRMS Molecu la r Ma sses a n d Isola ted
Yield s of th e Bicyclic P ep tid es (1-5) P r ep a r ed
5-17% yield (Table 1). The cyclization efficiency itself
was not the reason for the lower yields of 2-5 compared
to 1, but the presence of trifunctional amino acid residues
(Asp, His) and sequence-dependent competition between
on-support polymerization and the desired cyclization
increased the amount of impurities, as indicated by the
presence of a broad signal in the HPLC chromatogram
of the crude product (Figure 3). The final products (1-5)
were characterized, in addition to RP HPLC (Figure 4),
bicyclic
peptide
yield/%
[(M + 2H+)/2]found
[(M + 2H+)/2]req
1
2
3
4
5
17
8
5
8
7
436.7350
448.7240
437.7075
467.7262
462.7199
436.7349
448.7223
437.7063
467.7245
462.7198
1
by HRMS (Table 1) and H NMR spectroscopy (Support-
with structurally related aspartate and glutamate.21 The
amino acids used to construct the peptide bridges may
well contain side chain functionalities, allowing random-
ization of the amino acid composition and hence a library
synthesis.
ing Information).
In summary, easily available branching units 6 and 7
allow solid-supported synthesis of cryptand-like bicyclic
peptides in a reasonable yield. The cyclizations occur
more readily than with building blocks containing a spiro
carbon, such as tris(aminomethyl)acetate or bis(amino-
methyl)malonate. It is also worth noting that branching
unit 7 is not susceptible to imide formation encountered
(21) (a) Tam, J . P.; Riemen, M. W.; Merrifield, R. B. Pept. Res. 1988,
1, 6. (b) Ernesto, N.; Pedroso, E.; Girald, E. Tetrahedron Lett. 1989,
30, 497.
J . Org. Chem, Vol. 68, No. 22, 2003 8537

Virta and Lo¨nnberg
secondary amino group was acylated by an anhydride method,
i.e. by treating the support with 15 equiv of NR-Fmoc protected
anhydride of phenylalanine or monobenzylated aspartate in
DCM (25 °C, 6 h) to obtain 13 or 14, respectively. The Fmoc
group was removed, and branching unit 6 was coupled (5 equiv
of 6, 5 equiv of HATU, and 10 equiv of DIEA in DMF, 25 °C,
1 h), giving 15 and 16. The o-NBS group was removed by
treatment with a mixture of PhSH, DBU, and DMF (5:5:90,
25 °C, 2 h), and the first peptide arm was assembled by using
the HATU/Fmoc chemistry described above. Elongation of 15
in a stepwise manner with Fmoc-Ser(tBu)-OH, Fmoc-â-Ala-
OH, branching unit 7, and Boc-Gly-OH gave 17, while 18 was
obtained by using Fmoc-Phe-OH instead of Fmoc-Ser(tBu)-OH
for the otherwise similar derivatization of 16. Prolonged
reaction time and higher excess of reagents had to be used for
the coupling of Boc-Gly-OH to the secondary amino function
of the branching unit 7 (10 equiv of Boc-Gly-OH, 10 equiv of
HATU, 20 equiv of DIEA in DMF-DCM, 1:9, 25 °C, 4 h). The
4-methoxytrityl group was then removed from 17 and 18 with
DCA in DCM (3:97, v/v), and the second peptide arm was
constructed by again using the HATU/Fmoc chemistry. Cou-
pling of either Fmoc-Leu-OH or Fmoc-His(Bom)-OH, followed
by Fmoc-â-Ala, to 17 gave 19 and 20, and coupling of Fmoc-
Ser(tBu)-OH, Fmoc-Phe-OH, or Fmoc-His(Bom)-OH, again
followed by Fmoc-â-Ala, to 18 gave peptides 21-23. The allyl
ester protection was removed by adding Pd(OAc)2 (2 equiv),
P(Ph)3 (12 equiv), AcOH (12 equiv), and Bu3SnH (24 equiv) to
the resins (19-23) swelled in DCM.22 The suspensions were
allowed to shake for 1 h under argon, and the resins were then
filtered, washed with DCM, MeOH, and Et3N-DMF (1:3, v/v),
and dried. The Fmoc group was then removed from the second
peptide arm with the conventional piperidine treatment, and
the resins were washed with DMF, DCM, Et3N-DCM (1:3,
v/v), and MeOH and dried. For the first cyclization, PyAOP (5
equiv) and DIEA (10 equiv) were added onto the resins swelled
in DMSO-NMP (1:4, v/v), and the reaction was allowed to
proceed for 5 h at 25 °C. The resins (24-28) were filtered,
washed several times with DMF, DCM, and MeOH, and dried.
The unreacted amino functions were then capped with Ac2O
in THF containing N-methylimidazole and lutidine. Before the
second cyclization, the Boc and t-Bu groups were removed in
a single step by treatment with TFA-DCM (1:3, v/v, 25 °C,
1.5h), and the resins were washed/neutralized with DCM,
pyridine-DCM, DIEA-DCM, and MeOH and dried. The
cyclization was then carried out as described above for the first
lactamization. The resins were washed several times with
DMF, DCM, TFA-DCM (1:9), and MeOH and dried. The
bicyclic peptides (1-5) were cleaved from supports 29-33 with
a mixture of 33% HBr in AcOH-anisole-TFA (5:5:90 v/v/v,
25 °C, 2 h), evaporated to dryness, and purified by RP HPLC.
Each compound was obtained as a white powder, the isolated
yields ranging from 5% to 17% (Table 1). The purity (>90%)
and retention times of the compounds synthesized are il-
lustrated in Figure 4. The authenticity of the products was
verified by HRMS (ESI) (Table 1) and 1H NMR spectroscopy
(Supporting Information).
Exp er im en ta l Section
3-Aza-N1-(4-m eth oxytr ityl)-N5-(2-n itr oben zen esu lfon yl)-
p en ta n e-1,5-d ia m in e (9). 2-Nitrobenzenesulfonyl chloride
(0.92 g, 4.1 mmol) in dioxane (30 mL) was slowly added to a
mixture of 3-aza-N1-(4-methoxytrityl)pentane-1,5-diamine12 (8,
1.6 g, 4.1 mmol) and dioxane (7 mL). Triethylamine (0.58 mL,
4.1 mmol) was added, and the reaction mixture was stirred at
room temperature overnight. The product precipitated as a
hydrochloride was filtered, washed several times with toluene,
and dried under reduced pressure. The salt was partitioned
between DCM (100 mL) and 0.1 mol L-1 aq sodium hydroxide
(100 mL), and the mixture was shaken vigorously. The organic
phase was washed with brine, dried over Na2SO4, and evapo-
rated to dryness, giving 9 (1.7 g, 71%) as a yellowish foam.
1
The product was used without further purification. H NMR
(CDCl3, 400 MHz) δ 8.11 (m, 1H), 7.78-7.66 (m, 3H), 7.44-
7.15 (m, 12H), 6.80 (m, 2H), 3.78 (s, 3H), 3.12 (m, 2H), 2.71
(m, 2H), 2.59 (m, 2H), 2.21 (m, 2H); 13C NMR (CDCl3, 100
MHz) δ 157.8, 148.1, 146.2, 138.1, 133.5, 133.4, 132.6, 131.0,
129.7, 128.5, 127.8, 126.2, 125.3, 113.1, 70.2, 55.2, 49.5, 47.9,
43.1; HRMS (ESI) [M + H]+ required 561.2166, found 561.2168.
N -(4-Me t h oxyt r it yl)-N -(2-n it r ob e n ze n e su lfon yl)-3-
a m in op r op ion ic Acid (6). Compound 9 (1.7 g, 3.0 mmol)
was dissolved in a mixture of DCM (2.0 mL) and EtOH (2.0
mL), and ethyl acrylate (0.48 mL, 4.4 mmol) and DBU (0.44
mL, 3.0 mmol) were added. The mixture was stirred at room
temperature for 3 days and then evaporated to dryness. The
residue was dissolved in dioxane (60 mL) containing water (4
mL) and KOH (0.8 g, 15 mmol), and the mixture was stirred
at room temperature overnight to hydrolyze the ester linkage.
The volatile substances were removed, and the residue was
dissolved in pyridine (50 mL). The mixture was shaken with
a Dowex-50 resin (pyridinium ion form) for 1 h. The resin was
filtered off, and the filtrate was evaporated to dryness. The
crude product was purified by Silica gel chromatography (0.2%
pyridine and 10% MeOH in DCM) to yield 6 (1.5 g, 78%) as a
white foam. 1H NMR (CDCl3, 400 MHz) δ 8.04 (m, 1H), 7.68-
7.57 (m, 3H), 7.45-7.12 (m, 12H), 6.77 (m 2H), 3.74 (s, 3H),
3.19 (m, 2H), 2.81-2.72 (m, 6H), 2.42-2.34 (m, 4H); 13C NMR
(CDCl3, 100 MHz,) δ 175.7, 157.9, 148.1, 145.8, 137.6, 133.4,
133.4, 132.6, 130.6, 129.8, 128.5, 127.9, 126.4, 124.8, 113.2,
70.6, 55.2, 53.9, 53.0, 50.2, 40.3, 39.6, 31.2; HRMS (FAB) [M
+ H]+ requires 633.238, found 633.237.
N-(9-F lu or en ylm eth oxyca r bon yl)im in od ia cetic Acid
Mon oa llyl Ester (7). DIC (0.44 mL, 2.8 mmol) was slowly
added to a solution of N-Fmoc-iminodiacetic acid (10, 1.0 g,
2.8 mmol) in a mixture of DMF (1.0 mL) and DCM (4.0 mL).
After 1 h of stirring, allyl alcohol (2.0 mL, 28 mmol) was added,
and the agitation of the reaction mixture was continued
overnight at room temperature. All volatile materials were
removed, and the residue was dissolved in a small amount of
ethyl acetate. Partially precipitated diisopropylurea was fil-
tered off, and the filtrate was evaporated to dryness. The crude
reaction product was purified by Silica gel chromatography
(5% MeOH in DCM) to yield 7 (1.0 g, 91%) as a colorless oil.
1H NMR (CDCl3, 400 MHz) δ 7.76-7.73 (m, 2H), 7.54-7.51
(m, 2H), 7.41-7.36 (m, 2H), 7.31-7.27 (m, 2H), 5.90 (m, 1H),
5.37-5.26 (m, 2H), 4.64 (m, 2H), 4.46 (m, 2H), 4.23 (m, 1H),
4.20 (s, 1H), 4.19 (s, 1H), 4.10 (s, 1H), 4.08 (s, 1H); 13C NMR
(CDCl3, 100 MHz) δ 173.8, 173.4, 170.1, 169.3, 155.9, 155.8,
143.5, 143.5, 141.3, 131.2, 131.2, 127.8, 127.1, 124.9, 120.0,
119.3, 68.5, 68.4, 66.4, 66.3, 49.8, 49.7, 49.6, 49.3, 47.0, 46.9;
HRMS (EI) M+ requires 395.1369, found 395.1367.
Ack n ow led gm en t. The authors wish to thank Dr.
Harri Hakala for performing the HRMS analyses.
Su p p or tin g In for m a tion Ava ila ble: General procedures
and spectral data for compounds 1-7. This material is
available free of charge via the Internet at http://pubs.acs.org.
Syn th esis of Bicyclic P ep tid es (1-5). A 300-mg sample
of resin 127 having a loading of 180 µmol g-1, determined by
the release of benzofulvene from the linker, was used for the
synthesis of each bicyclic peptide (1-5). The Fmoc group was
removed with piperidine in DMF (1:4, v/v), and the exposed
J O0344945
(22) (a) Guibe´, F.; Dangles, O.; Balavoine, G. Tetrahedron Lett. 1986,
27, 2365. (b) Guibe´, F.; Zhang, H. X.; Balavoine, G. Tetrahedron Lett.
1988, 29, 623.
8538 J . Org. Chem., Vol. 68, No. 22, 2003