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
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