Application of cyano ylide methodology to the synthesis of cyclotheonamides E2 and E3

Article abstract of DOI:10.1016/S0040-4020(02)00627-0

A total synthesis of cyclotheonamides E₂ and E₃ is reported. A key step in the synthesis involves the formation of the α-keto amide linkage by application of the cyano ylide activation of a carboxyl group as developed in our earlier syntheses of cyclic peptide protease inhibitors.

Full text of DOI:10.1016/S0040-4020(02)00627-0

Tetrahedron 58 (2002) 6277–6283
Application of cyano ylide methodology to the synthesis of
cyclotheonamides E2 and E3
*
Harry H. Wasserman and Rui Zhang
Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520-8107, USA
Received 1 April 2002; accepted 29 April 2002
Abstract—A total synthesis of cyclotheonamides E2 and E3 is reported. A key step in the synthesis involves the formation of the a-keto
amide linkage by application of the cyano ylide activation of a carboxyl group as developed in our earlier syntheses of cyclic peptide protease
inhibitors. q 2002 Elsevier Science Ltd. All rights reserved.
The cyclotheonamides (A–E), recently isolated from
marine sources, are cyclic pentapeptides which show
notable activity as inhibitors of serine proteases.1,2 Because
of their biological properties and unusual structural features,
considerable attention has been given to the study of the
mode of enzyme inhibition and synthesis of these
macrocyclic a-keto lactams. Total syntheses of cyclo-
theonamides A and B have been reported2 by Schreiber,
Maryanoff, Wipf, Ottenheijm and Shioiri.
Adding to the interest in this area has been the recent
isolation of two new cyclotheonamides, E2 (1a) and E3
(1b),1b which contain most of the elements of cyclo-
theonamides A and B, except for the presence of a
D-alloisoleucine residue in the place of D-phenylalanine,
and side-chains containing benzoylalanine and isovaleryl-
alanine residues. A unique functional unit in all cyclo-
In a preliminary communication,3 we have outlined a total
theonamides is associated with the extra carbonyl a- to the
synthesis of cyclotheonamides E2 and E3. We now provide
details of this synthesis involving cyano ylide activation4 of
an arginine carboxyl in the formation of the key a-keto
lactam unit. A number of the steps in our synthesis make use
of protecting groups in common with the procedures
published in the early synthetic work. However, our strategy
has unique features which should be of considerable value
in future syntheses of compounds in this family. In
particular, the formation of the relatively robust a-keto
amide at an early stage of the synthesis avoids problems in
earlier syntheses associated with a-hydroxy precursors
generated en route to the a-keto amide residue. It precludes
the necessity for carrying a mixture of diasteromers through
subsequent steps in the synthesis, as well as the protection of
the hydroxyl group needed to avoid the possibility of
competing intramolecular lactone formation. Overall, the
simplicity of forming the a-keto amide residue using the
cyano ylide activation procedure avoids the extra steps
needed to generate, protect, deprotect and oxidize an
alcohol intermediate to the a-keto function.5,6
amide linkage in the arginine residue. It has been suggested
that this active carbonyl group is involved in the deactiva-
tion of a protease by imparting the characteristics of an
enzyme transition state analog.2b It is also noteworthy that
the cyclotheonamides have structural features similar to
those of the immunosuppressants bearing a-keto amide
functions such as FK-506 and rapamycin.2f
Keywords: cyano ylide methodology; cyclotheonamides; synthesis.
*
Corresponding author. Tel.: þ1-203-432-3973; fax: þ1-203-432-9990;
e-mail: harry.wasserman@yale.edu
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00627-0

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H. H. Wasserman, R. Zhang / Tetrahedron 58 (2002) 6277–6283
Scheme 1. Regents and conditions: (a) TIB, Py; then Boc2O, NaOH, 77%; (b) EDCI, HOBt, Pro-OBn, 88%; then Pd–C, H2, 100%; (c) EDCI, DMAP,
Ph3PvCHCN, 86%; (d) TFA; then EDCI, HOBt; 4, 78%.
Our synthesis began with the formation of the tripeptide
from arginine, proline and diaminopropionic acid (Scheme
1). The latter acid, formed by a Curtius-type degradation of
Teoc-aspargine (2)7 using bis(trifluoroacetyl) iodobenzene
(TIB), was then converted to the N-Boc derivative 3. The
acid 3 was then coupled (EDCI) with proline benzyl ester to
form the protected dipeptide which, on hydrogenation (Pd–
C), yielded 4. In a separate pathway, the ylide 6 was
prepared from arginine having a Boc-protected amino
group, and a doubly Cbz-protected guanidino residue (5).8
Reaction of 5 with (triphenylphosphoranylidine)acetonitrile
yielded the acyl cyano ylide 6 (86%).4a TFA removal of the
Boc group from 6 then yielded the free amine for coupling
with 4 (EDCI, HOBt) to form the tripeptide 7 (78%).
Tripeptide (7) was now ozonized to generate the strongly
electrophilic a,b-diketonitrile intermediate, not isolable,
which formed an amide bond with 11 to yield the
pentapeptide 12 (75%) (Scheme 3). At this point in the
synthesis, we found it expedient to change the allyl-
protecting group to a pentafluorophenoxy group for
enhanced activation of the ester. This was readily
accomplished, first by the use of Pd0 to regenerate the
carboxyl group (86%)10 and then DCC-promoted coupling
with pentafluorophenol (PFPOH) yielding 13 (88%).11 For
the ring closure of 13 to 14, we first selectively cleaved the
Boc group with HCl in Et2O–CH2Cl2 in the presence of the
acid-labile Teoc group12 and then carried out lactam
formation with DMAP, NaHCO3 (61%). The removal of
the Teoc protecting group to yield 17 and 18 took place with
TFA, permitting the installation of side-chains (EDCI,
HOBt) with N-benzoylalanine (15) to yield 17 and
isovaleryalanine (16), forming 18.
To continue the synthesis, L-Boc-tyrosine methyl ester was
converted to the TIPS derivative,9 and then reduced with
DIBAL-H to the aldehyde 8. A Wittig reaction of 8 with
allyl (triphenylphosphoranylidine)acetate (Scheme 2)
yielded the allyl protected a,b-unsaturated ester 9 which,
on treatment with TFA and coupling (EDCI, HOBt, Et3N)
with D-Boc-alloisoleucine gave the dipeptide 10 containing
a vinylogous tyrosine. Removal of the Boc group yielded 11
for reaction in the next stage with 7.
The Cbz and TIPS protecting groups in 17 and 18 were then
removed smoothly with HF·Py, yielding synthetic
cyclotheonamides E2 (1a) and E3 (1b) which were fully
characterized by HRMS, 1H NMR and 13C NMR. The NMR
spectra of 1a and 1b were identical in all significant respects
Scheme 2. Reagents and conditions: (a) Ph3PvCHCO2All, 90%; (b) TFA; then EDCI; HOBt, Et3N, D-Boc-alle, 85%; (c) TFA; then aq. NaHCO3, 100%.

H. H. Wasserman, R. Zhang / Tetrahedron 58 (2002) 6277–6283
6279
Scheme 3. Regents and conditions: (a) O3; then 11, 75%; (b) Pd(PPH3)4, PhSiH3, 86%; then DCC, PFP-OH, 88%; (c) HCl in Et2O–CH2Cl2; then DMAP,
NaHCO3, 61%; (d) TFA; then aq. NaHCO3; then EDCI, HOBt, 15 or 16, 83–85%; (e) HF·Py, 70–72%.
with the corresponding spectra of the natural materials,
kindly sent to us by Professors Fusetani and Nakao.
organic extracts were dried and concentrated. The residue
was purified by flash chromatography (hexanes–EtOAc,
5:4) to give 3 (3.65 g, yield 77%) as a white foam. 1H NMR
(500 MHz, CDCl3) d 11.14 (br s, 1H), 6.39 (br s, 0.5H), 6.04
(br s, 0.5H), 5.70 (br s, 0.5H), 5.28 (br s, 0.5H), 4.35 (m,
1H), 4.14 (m, 2H), 3.57 (m, 1H), 3.50 (m, 1H), 1.43 (s,
3.6H), 1.39 (s, 5.4H), 0.96 (t, J¼8.0 Hz, 2H), 0.0 (s, 9H);
13C NMR (125 MHz, CDCl3) d 173.5, 173.0, 158.2, 157.1,
157.0, 156.1, 81.9, 80.2, 63.8, 63.5, 54.9, 54.5, 54.1, 43.1,
42.1, 28.2, 28.1, 17.6, 21.6; HRMS (ES) calcd for
C14H28N2O6SiNa (MþNa)þ: 371.1614 (MþNa)þ, found
371.1609.
1. Experimental
1.1. General
1.1.1. Curtius degradation of Teoc-asparagine to N-Boc
derivative 3. Teoc-Asn
a
2 (3.77 g, 13.6 mmol) was
dissolved in mixture of DMF (3 mL), dioxane
(50 mL) and distilled water (53 mL). [Bis(trifluoro-
acetoxy)iodo]benzene (9.07 g, 21.1 mmol) was added in
one pot. After 15 min, pyridine (2.2 mL, 27.2 mmol) was
added, and the reaction mixture was stirred at room
temperature for 3 h. The solvents were evaporated under
reduced pressure and the residue was then dissolved in
water (50 mL) and extracted with ether (3£50 mL). The
aqueous layer was concentrated under reduced pressure
to afford a residue, which was used without purification.
1.1.2. Coupling of 3 with proline benzyl ester. Compound
3 (2.82 g, 8.09 mmol) was dissolved in CH2Cl2 (10 mL),
and EDCI (1.86 g, 9.71 mmol) and HOBt (1.31 g,
9.71 mmol) were added, followed by the addition of a
mixture of Pro-OBn hydrochloride (2.35 g, 9.71 mmol) and
triethylamine (1.36 mL, 9.71 mmol). The resulting mixture
was stirred overnight. 1N HCl (50 mL) was added, and after
extraction with CH2Cl2 (50 mL), the organic layer was
washed with sat. NaHCO3, brine, and then dried over
Na2SO4 and concentrated. Flash chromatography of the
residue afforded the benzyl ester of protected dipeptide 4
The residue was basified with 1N NaOH (35 mL) at 08C. Di-
tert-butyl dicarbonate (4.46 g, 20.4 mmol) in dioxane
(10 mL) was added dropwise. Then the reaction mixture
was warmed to room temperature, and stirred for 2 h. After
it was acidified with 1N HCl, the reaction mixture was
extracted with EtOAc (3£50 mL), and the combined
1
(3.81 g, yield 88%) as a glassy solid. H NMR (500 MHz,
CDCl3) d 7.31 (m, 5H), 5.90 (m, 1H), 5.34 (m, 1H), 5.18 (d,
J¼12.3 Hz, 1H), 5.06 (d, J¼12.3 Hz, 1H), 4.65 (m, 1H),

6280
H. H. Wasserman, R. Zhang / Tetrahedron 58 (2002) 6277–6283
4.58 (m, 1H), 4.11 (m, 2H), 3.75 (m, 2H), 3.42 (m, 1H), 3.23
(m, 1H), 2.18 (m, 1H), 1.96 (m, 3H), 1.41 (s, 9H), 0.95 (t,
J¼8.5 Hz, 2H), 0.04 (s, 9H); 13C NMR (125 MHz, CDCl3)
d 171.6, 169.9, 156.7, 156.1, 135.5, 128.6, 128.3, 128.1,
128.0, 79.3, 77.4, 66.9, 63.3, 59.0, 52.8, 52.2, 47.1, 46.5,
42.3, 31.1, 28.9, 28.4, 24.9, 22.0, 17.7, 21.5; HRMS (ES)
calcd for C26H41N3O7SiNa: 558.2612 (MþNa)þ, found
558.2606.
(m, 12H), 7.29 (m, 1H), 6.77 (d, J¼7.5 Hz, 1H), 6.13 (m,
1H), 5.33 (d, J¼7.9 Hz, 1H), 5.23 (s, 2H), 5.15 (m, 1H),
5.06 (d, J¼3.0 Hz, 2H), 4.63 (m, 1H), 4.38 (m, 1H), 4.10
(m, 2H), 4.00 (m, 2H), 3.57 (m, 2H), 3.20 (m, 1H), 2.79 (m,
1H), 1.98 (m, 2H), 1.93 (m, 1H), 1.83 (m, 1H), 1.75 (m, 2H),
1.64 (m, 1H), 1.33 (s, 9H), 0.93 (t, J¼8.3 Hz, 2H), 0.0 (s,
9H); 13C NMR (125 MHz, CDCl3) d 193.4, 170.7, 170.4,
163.9, 160.5, 156.3, 156.0, 155.8, 136.7, 134.7, 133.4, 133.3,
133.2, 129.2, 129.1, 128.6, 128.4, 128.2, 128.0, 127.8, 127.6,
122.6, 121.9, 120.1, 120.0, 78.8, 77.4, 68.7, 66.9, 63.1, 60.7,
54.3, 54.2, 51.3, 48.8, 47.8, 47.3, 44.5, 43.0, 30.3, 29.1, 28.2,
24.6, 24.4, 17.6, 21.6; HRMS (ES) calcd for C61H73N8O11-
PSiNa: 1175.4803 (MþNa)þ, found 1175.4802.
1.1.3. Debenzylation of the benzyl ester of 4. The benzyl
ester of 4 (3.24 g, 6.05 mmol) in EtOAc (50 mL) was treated
with 10% Pd–C (322 mg, 5 mol%), and the reaction
mixture was stirred under a balloon of H2 overnight, and
then filtered through Celite followed by washing of the
Celite with EtOAc–MeOH. The filtrate was concentrated to
1.1.6. Wittig reaction of aldehyde 8 to form unsaturated
ester 9. The TIPS derivative of L-Boc-tyrosine methyl
ester9 was reduced with DIBAL-H to the aldehyde 8 (90%).
A solution of aldehyde 8 (2.60 g, 8.54 mmol) in DMF
(20 mL) was treated with [(allyloxycarbonyl)methylene]-
triphenylphosphorane (3.38 g, 9.39 mmol) and stirred at
room temperature for 5 h. The reaction mixture was diluted
with water (80 mL), and extracted with ether (3£100 mL).
The combined organic extracts were dried over Na2SO4,
concentrated, and purified by flash chromatography (5:1
hexanes–EtOAc) to give the allyl ester 9 (4.0 g, 95%) as a
colorless oil. 1H NMR (500 MHz, CDCl3) d 7.01 (d,
J¼8.2 Hz, 2H), 6.92 (dd, J¼5.1, 15.6 Hz, 1H), 6.81 (d,
J¼8.2 Hz, 2H), 5.92 (m, 1H), 5.86 (dd, J¼1.5, 15.6 Hz,
1H), 5.31 (dd, J¼1.5, 17.2 Hz, 1H), 5.22 (dd, J¼1.1,
10.4 Hz, 1H), 4.67 (d, J¼8.6 Hz, 1H), 4.62 (d, J¼5.4 Hz,
2H), 4.56 (br s, 1H), 2.81 (d, J¼6.3 Hz, 2H), 1.41 (s, 9H),
1.23 (m, 3H), 1.10 (s, 9H), 1.09 (s, 9H); 13C NMR
(125 MHz, CDCl3) d 165.9, 155.1, 155.0, 148.5, 132.3,
130.4, 128.9, 120.8, 120.1, 118.4, 118.2, 79.8, 77.4, 65.2,
52.7, 40.1, 28.4, 18.0, 12.7; HRMS (ES) calcd for C28H45-
NO5SiNa: 526.2965 (MþNa)þ, found 526.2966.
1
give the free acid 4 (2.70 g, 100%) as a white foam. H
NMR (500 MHz, CDCl3) d 5.65 (d, J¼7.0 Hz, 1H), 5.37 (br
s, 1H), 4.66 (m, 1H), 4.56 (t, J¼6.2 Hz, 1H), 4.11 (m, 2H),
3.76 (m, 2H), 3.37 (m, 2H), 2.16 (m, 2H), 2.05 (m, 2H), 1.46
(s, 1.8H), 1.40 (s, 7.2H), 0.95 (t, J¼8.5 Hz, 2H), 0.0 (s, 9H);
13C NMR (125 MHz, CDCl3) d 174.3, 171.1, 156.8, 156.5,
79.6, 63.6, 59.3, 52.4, 47.6, 42.2, 28.9, 28.5, 24.9, 17.8,
21.4; HRMS (ES) calcd for C19H35N3O7 SiNa: 468.2142
(MþNa)þ, found 468.2141.
1.1.4. Conversion of protected arginine to the cyano ylide
6. A solution of Boc-Arg(Cbz)2-OH 5 (56.46, 11.9 mmol) in
CH2Cl2 (20 mL) was treated with DMAP (1.45.4 mg,
10 mol%) and EDCI (2.51 g, 13.1 mmol), followed by
(cyanomethylene)triphenylphosphorane (7.17 g, 23.8 mmol).
The reaction mixture was stirred for 4 h, and then
concentrated. Flash chromatography of the residue with
1:1 hexane–EtOAc gave 6 (8.45 g, yield 86%) as a white
foam: IR (neat); 3388 (br), 3275 (shoulder), 2177, 1715,
1607, 1507, 1497, 1439, 1378, 1367, 1253, 1176,
1108 cm21 1H NMR (500 MHz, CDCl3) d 9.53 (br s,
.
1H), 9.29 (br s, 1H), 7.60, 7.56 (m, 9H), 7.50–7.46 (m, 6H),
7.42–7.30 (m, 6H), 7.32–7.20 (m, 4H), 5.33 (d, J¼7.7 Hz,
1H), 5.23 (s, 2H), 5.13 (s, 2H), 4.92 (m, 1H), 4.13 (m, 1H),
3.97 (m, 1H), 2.02 (m, 1H), 1.78 (m, 3H), 1.44 (s, 9H); 13C
NMR (125 MHz, CDCl3) d 194.3, 163.8, 160.5, 160.4,
155.8, 155.5, 136.9, 134.7, 133.5, 133.4, 133.2, 129.0,
128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 127.6, 127.5,
123.0, 122.2, 120.8, 120.6, 78.8, 77.4, 68.7, 67.0, 55.9, 55.8,
47.8, 46.8, 44.5, 30.3, 28.4, 28.3, 24.8; HRMS (ES) calcd
for C47H48N5O7PNa: 848.3189 (MþNa)þ, found 848.3184.
1.1.7. Coupling of 9 with D-Boc-alloisoleucine. The
unsaturated ester 9 (4.18 g, 8.25 mmol) was treated with
CH2Cl2–TFA (1:1, 50 mL) for 1 h, then concentrated under
vacuum. The residue was dissolved in CH2Cl2 (10 mL), and
triethylamine (1.26 mL, 9.08 mmol) was added. The reac-
tion mixture was added to a solution of Boc-D-AIle (1.73 g,
7.50 mmol), EDCI (1.73 g, 9.00 mmol), HOBt (1.22 g,
9.00 mmol) in CH2Cl2 (50 mL). The resulting mixture was
stirred at room temperature overnight and then diluted with
CH2Cl2 (50 mL), and washed with 1N HCl (100 mL), sat.
NaHCO3 (100 mL) and brine (100 mL). The organic layer
was dried over Na2SO4 and concentrated. Flash chroma-
tography of the residue with 7:1 hexanes–EtOAc afforded
coupling product 10 (3.93 g, 85% yield) as a white foam. 1H
NMR (500 MHz, CDCl3) d 6.99 (d, J¼8.4 Hz, 2H), 6.94
(dd, J¼4.9, 15.7 Hz, 1H), 6.79 (d, J¼8.4 Hz, 2H), 6.30 (m,
1H), 5.89 (m, 2H), 5.29 (dd, J¼1.5, 17.2 Hz, 1H), 5.22 (dd,
J¼1.1, 10.5 Hz, 1H), 5.08 (m, 1H), 4.91 (m, 1H), 4.60 (d,
J¼5.7 Hz, 2H), 3.96 (m, 1H), 2.88 (m, 1H), 2.79 (m, 1H),
1.78 (m, 1H), 1.44 (s, 9H), 1.23 (m, 4H), 1.09 (s, 9H), 1.08
(s, 9H), 1.05 (m, 1H), 0.84 (t, J¼6.5 Hz, 3H), 0.77 (d,
J¼6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) d 171.6,
165.8, 156.1, 155.2, 147.6, 132.3, 130.3, 128.7, 121.1,
120.1, 118.3, 80.1, 65.2, 58.8, 51.2, 39.7, 36.9, 28.5, 26.2,
18.0, 14.5, 12.8, 11.6; HRMS (ES) calcd for C34H56N2O6-
SiNa: 639.3805 (MþNa)þ, found 639.3804.
1.1.5. Coupling of cyano ylide 6 with acid 4 to form 7.
Cyano ylide 6 (2.58 g, 3.12 mmol) was treated with
CH2Cl2–TFA (1:1, 50 mL). After 1 h, the reaction mixture
was concentrated under vacuum, and the residue was treated
with 10% Na2CO3 (50 mL) and extracted with CH2Cl2
(3£60 mL). The combined organic extracts were dried over
Na2SO4, and concentrated to yield a foam. Without
purification, it was added to a reaction mixture of 4
(1.32 g, 2.97 mmol), EDCI (626 mg, 3.27 mmol), and HOBt
(441.8 mg, 3.27 mmol) in CH2Cl2 (25 mL). The reaction
mixture was stirred overnight, and then concentrated. Flash
chromatography of the residue with 2.5:1 EtOAc–hexane
afforded 7 (2.67 g, 78% yield) as a white foam: IR (neat)
3391 (br), 3307 (shoulder), 2181, 1717, 1652, 1646, 1608,
1507, 1437, 1250 cm21. 1H NMR (500 MHz, CDCl3) d 9.46
(br s, 1H), 9.27 (br s, 1H), 7.62–7.60 (m, 3H), 7.55–7.45

H. H. Wasserman, R. Zhang / Tetrahedron 58 (2002) 6277–6283
6281
1.1.8. Formation of the free amine 11 from Boc
derivative 10. Product 10 containing a vinylogous tyrosine
residue (980.0 mg, 1.59 mmol) was treated with CH2Cl2–
TFA (1:1, 20 mL) at room temperature for 1 h, then the
solvents were evaporated under vacuum. The residue was
basified with 10% Na2CO3 (15 mL), and extracted with
CH2Cl2 (3£15 mL). The combined extracts were dried over
Na2SO4, and concentrated to give the free amine 11
(822 mg, 100% yield) as a pale yellow oil. 1H NMR
(500 MHz, CDCl3) d 7.57 (d, J¼8.6 Hz), 6.99 (d,
J¼8.2 Hz, 2H), 6.93 (dd, J¼5.2, 15.7 Hz, 1H), 6.78 (d,
J¼8.2 Hz, 2H), 5.89 (m, 1H), 5.79 (d, J¼15.7 Hz, 1H),
5.28 (d, J¼18.2, 1H), 5.20 (d, J¼10.4 Hz, 1H), 4.87 (m,
1H), 4.60 (d, J¼5.6 Hz, 2H), 3.25 (m, 1H), 2.88 (dd,
J¼6.7, 13.8 Hz, 1H), 2.80 (dd, J¼7.2, 13.8 Hz, 1H), 2.03
(m, 1H), 1.30–1.19 (m, 7H), 1.07 (s, 9H), 1.06 (s, 9H), 0.89
(t, J¼7.4 Hz, 3H), 0.72 (d, J¼6.9 Hz, 3H); 13C NMR
(125 MHz, CDCl3) d 174.3, 165.8, 155.1, 148.2, 132.3,
130.4, 130.3, 129.0, 120.8, 120.6, 120.1, 118.2, 77.4, 65.2,
57.9, 50.9, 39.9, 37.1, 26.9, 18.0, 17.6, 13.3, 13.0, 12.0;
HRMS calcd for C29H49N2O4Si: 517.3461 (MþH)þ, found
517.3467.
(30.0 mg, 0.03 mmol). The resulting mixture was stirred at
room temperature for 15 min, then 1N HCl (10 mL) was
added, and stirred continuously for 1 h at room temperature.
The aqueous layer was extracted with CH2Cl2 (3£10 mL),
and the combined extracts were dried over Na2SO4, filtered
and concentrated to give the acid which was dissolved in
CH2Cl2 (2 mL). DCC (96.0 mg, 0.47 mmol) and penta-
fluorophenol (114.1 mg, 0.62 mmol) were then added, and
the resulting mixture was stirred overnight. The solvent was
evaporated under reduced pressure and the residue was
separated on silica gel with 1.5:1 hexane–EtOAc to give the
pentafluorophenoxy derivative 13 (358.8 mg, 76% yield for
the two steps) as a white foam. 1H NMR (500 MHz,
CD3OD) d 7.40–7.27 (m, 11H), 7.12 (d, J¼8.4 Hz, 2H),
6.81 (d, J¼8.4 Hz, 2H), 6.20 (m, 1H), 5.27 (br s, 2H), 5.11
(br s, 2H), 4.56 (m, 1.4H), 4.40–4.27 (m, 2.6H), 4.12 (m,
2H), 3.97 (m, 2H), 3.73 (m, 2H), 3.40 (m, 1H), 3.14 (m, 1H),
3.01 (m, 1H), 2.98 (m, 1H), 2.30–1.60 (m, 9H), 1.41 (s, 9H),
1.25 (m, 4H), 1.14 (m, 1H), 1.11 (s, 9H), 1.09 (s, 9H), 0.96
(m, 3H), 0.85–0.62 (m, 6H), 0.03 (s, 8H), 20.04 (s, 1H);
13C NMR (125 MHz, CD3OD) d 196.5, 174.4, 173.2, 173.1,
172.8, 172.1, 171.6, 165.2, 163.3, 163.2, 162.1, 162.0,
158.7, 158.5, 157.3, 157.2, 156.4, 154.9, 154.7, 143.7,
141.8, 140.3, 138.6, 136.9, 136.7, 131.5, 131.3, 131.2,
129.8, 129.7, 129.6, 129.5, 129.2, 129.1, 129.0, 121.0,
118.6, 100.2, 99.5, 80.5, 70.1, 68.4, 64.4, 62.1, 62.0, 61.5,
58.9, 58.7, 58.5, 57.2, 55.9, 54.4, 53.9, 53.7, 46.1, 45.7,
42.8, 39.8, 38.8, 38.6, 38.3, 34.9, 30.9, 30.7, 28.9, 27.5,
27.3, 26.8, 26.7, 26.2, 26.0, 18.8, 18.6, 15.4, 15.2, 15.0,
14.0, 12.2, 12.1, 12.0, 21.2; MS (ES) calcd for C75H104-
N9O17F5Si2Na: 1576.6907 (MþMeOHþNa)þ, found
1576.76.
1.1.9. Formation of the pentapeptide 12. A solution of the
cyano ylide 7 (540 mg, 0.47 mmol) in CH2Cl2 (15 mL) was
cooled to 2788C, then ozone was bubbled through until the
solution became bluish. Nitrogen was then bubbled through
for 10 min to remove extra ozone. A solution of amine 11
(255 mg, 0.49 mmol) in CH2Cl2 (2 mL) was added, and the
resulting reaction was kept at 2788C for 15 min, then
warmed to room temperature, and the solvent was
evaporated. AgNO3 (5 mL, 1 M in 4:1 of THF–H2O) was
added, and the reaction stirred for 4 h. The reaction mixture
was extracted with EtOAc (3£10 mL). The combined
organic layers were dried over Na2SO4 and concentrated.
Flash chromatography of the residue with 1.5:1 EtOAc–
hexanes gave pentapeptide 12 (492.3 mg, 75% yield) as a
white foam. 1H NMR (500 MHz, CD3OD) d 7.41–7.27 (m,
10H), 7.09 (m, 2H), 6.94 (m, 1H), 6.79 (m, 2H), 5.91 (m,
2H), 5.27 (m, 3H), 5.18 (m, 1H), 5.12 (s, 2H), 4.79 (m, 1H),
4.57 (m, 3H), 4.33 (m, 2H), 4.14 (m, 2H), 3.90 (m, 2H), 3.70
(m, 2H), 3.41 (m, 1H), 3.21 (m, 1H), 2.94 (m, 1H), 2.69 (m,
1H), 2.18–1.69 (m, 5H), 1.62 (m, 2H), 1.52 (m, 2H), 1.42 (s,
9H), 1.24 (m, 4H), 1.14 (m, 1H), 1.11 (s, 9H), 1.09 (s, 9H),
0.95 (m, 3H), 0.80 (m, 3H), 0.65 (m, 3H), 0.02 (s, 9H); 13C
NMR (125 MHz, CD3OD) d 196.5, 175.4, 174.3, 172.9,
172.5, 171.9, 171.6, 167.5, 167.3, 165.2, 165.1, 162.1,
158.7, 158.5, 157.2, 157.1, 157.0, 156.2, 149.5, 149.3,
149.2, 138.6, 136.9, 136.7, 136.6, 133.8, 133.7, 131.4,
131.3, 129.9, 129.7, 129.6, 129.5, 129.4, 129.2, 129.1,
129.0, 121.9, 121.8, 120.9, 118.6, 118.5, 100.2, 99.4, 80.5,
80.4, 70.2, 70.1, 70.0, 68.4, 66.3, 66.2, 64.4, 62.1, 62.0,
61.4, 58.7, 58.4, 57.2, 56.2, 55.8, 54.4, 54.3, 54.2, 53.5,
53.4, 46.2, 45.7, 42.8, 40.2, 40.1, 38.3, 38.4, 30.9, 30.4,
29.0, 27.5, 26.0, 18.8, 18.6, 15.3, 15.2, 14.0, 12.2, 12.1,
21.3; HRMS (ES) calcd for C72H109N9O17SiNa:
1450.7378 (MþMeOHþNa)þ, found 1450.7375.
1.1.11. Ring-closure of 13 to the protected macrocyclic
pentapeptide 14. The open-chain pentapeptide 13 (175 mg,
0.115 mmol) was dissolved in CH2Cl2 (10 mL), sat. HCl in
Et2O (6 mL) was added, and the mixture was stirred for
15 min, then concentrated under vacuum. The residue in
DMF (50 mL) was added slowly through a syringe pump
over 1 h to a mixture of NaHCO3 (145 mg, 1.72 mmol) and
DMAP (2.81 mg, 0.02 mmol) in DMF (50 mL) which was
cooled to 258C, and the reaction mixture was stirred at
258C for 20 h, then quenched with 1N HCl and extracted
with Et2O (3£150 mL). The organic extracts were com-
bined, dried over Na2SO4 and concentrated. The residue was
purified on silica gel with EtOAc–hexane (7:3) to give the
macrocyclic product 14 (86.8 mg, yield 61%) as a white
foam. 1H NMR (500 MHz, CD3OD) d 7.37–7.22 (m, 10H),
7.09 (d, J¼8.4 Hz, 2H), 6.77 (m, 3H), 6.16 (d, J¼15.3 Hz,
1H), 5.21 (s, 2H), 5.09 (m, 2H), 4.75 (m, 1H), 4.42 (m, 2H),
4.28 (m, 1H), 4.16–3.90 (m, 5H), 3.88 (m, 1H), 3.72 (m,
1H), 3.43 (m, 1H), 3.06 (m, 1H), 2.75 (m, 1H), 2.58 (m, 1H),
2.13 (m, 1H), 2.05 (m, 1H), 1.95–1.72 (m, 4H), 1.67 (m,
1H), 1.55 (m, 1H), 1.49–1.31 (m, 2H), 1.21 (m, 4H), 1.08 (s,
9H), 1.06 (s, 9H), 0.90 (m, 2H), 0.77 (m, 3H), 0.60 (d,
J¼6.6 Hz, 0.9H), 0.54 (d, J¼6.6 Hz, 2.1H), 20.03 (s, 9H);
13C NMR (125 MHz, CD3OD) d 174.1, 173.9, 172.9, 172.2,
171.9, 171.8, 170.5, 168.0, 165.1, 162.3, 158.5, 157.2,
156.2, 144.4, 144.0, 138.6, 138.5, 136.7, 131.9, 131.3,
131.2, 129.9, 129.8, 129.7, 129.6, 129.5, 129.2, 129.1,
129.0, 125.3, 124.7, 120.8, 99.8, 99.6, 70.1, 68.4, 68.3, 64.5,
61.8, 58.4, 57.8, 56.8, 56.1, 53.1, 52.9, 52.6, 52.5, 46.3,
46.2, 41.5, 41.2, 40.8, 40.0, 39.6, 31.5, 30.9, 27.3, 26.7,
1.1.10. Formation of the pentafluorophenoxy derivative
13. Pentapeptide 12 (435 mg, 0.31 mmol) was dissolved in
CH2Cl2 (8 mL), the flask was wrapped with aluminum foil,
and phenylsilane (39 mL, 0.62 mmol) was added, followed
by the addition of tetrakis (triphenylphosphine)palladium

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H. H. Wasserman, R. Zhang / Tetrahedron 58 (2002) 6277–6283
26.4, 26.2, 24.9, 24.7, 18.8, 18.7, 18.6, 15.1, 14.8, 14.0,
12.7, 12.4, 21.3; HRMS (ES) calcd for C63H95N9O14Si2Na:
1292.6435 (MþMeOHþNa)þ, found 1292.6444.
26.4, 26.1, 24.9, 24.7, 23.8, 22.9, 22.8, 18.5, 18.1, 15.1,
14.8, 14.0, 12.6, 12.4; HRMS (ES) calcd for C66H96N10-
O14SiNa: 1303.6774 (MþMeOHþNa)þ, found 1303.6775.
1.1.12. Removal of Teoc protecting group to form the
protected cyclotheonamides 17 and 18. The macrocyclic
pentapeptide with TIPS, Teoc, and Cbz protection (40 mg,
0.032 mmol) was treated with TFA–CH2Cl2 (1:1, 6 mL),
and the reaction mixture was stirred for 90 min, then
concentrated. The residue was dissolved in CH2Cl2
(15 mL), and washed with aqueous NaHCO3 (2£10 mL).
The organic layer was dried over Na2SO4 and concentrated.
The solution of the residue in CH2Cl2 (1 mL) was added to a
mixture of EDCI (12.3 mg, 0.064 mmol), HOBt (8.6 mg,
0.064 mmol) and benzoylalanine 15 (6.8 mg, 0.0352 mmol)
or isovalerylalanine 16 (8.3 mg, 0.0352 mmol) in CH2Cl2
(1 mL), and the resulting mixture was stirred overnight. It
was then diluted with CH2Cl2 (15 mL) and washed with sat.
NaHCO3, and then brine. The organic layer was dried over
Na2SO4 and concentrated. Flash chromatography of the
residue in each case with 5:1 EtOAc–hexanes gave 17
(34.5 mg, yield 85%) or 18 (33.2 mg, yield 83%) as white
foams.
1.1.13. Cyclotheonamides E2 and E3. Product 17 (20 mg,
0.0158) or product 18 (20 mg, 0.016 mmol) was treated
with HF·Py (1 mL) and anisole (0.15 mL) and the
resulting mixture was stirred at room temperature for
3 h. Nitrogen was then bubbled through the solution for
1 h to remove excess HF, H2O (8 mL) was added, and
the resulting solution was lyophilized to give crude
products. HPLC purification (C18, gradient eluting with
CH3CN–H2O 1:99 to 40:60 over 25 min, 1% TFA)
furnished cyclotheonamide E2 (9.4 mg, 70%) or
cyclotheonamide E3 (9.5 mg, 72%) as white foams.
For E2 (1a). 1H NMR (500 MHz, CD3OD) d 7.87 (d,
J¼7.2 Hz, 2H), 7.56 (t, J¼7.3 Hz, 1H), 7.47 (m, 2H), 7.08
(d, J¼8.4 Hz, 2H), 6.82 (dd, J¼2.5, 15.5 Hz, 1H), 6.72 (d,
J¼8.5 Hz, 2H), 6.17 (dd, J¼2.2, 15.5 Hz, 1H), 4.75 (m,
1H), 4.67 (m, 1H), 4.58 (m, 1H), 4.52 (m, 1H), 4.28 (m, 2H),
4.14 (m, 0.7H), 4.06 (m, 0.3H), 3.86 (m, 1H), 3.55 (m, 1H),
3.15 (m, 2H), 3.08 (m, 1H), 2.87 (m, 1H), 2.59 (m, 1H), 2.25
(m, 1H), 1.97 (m, 4H), 1.70 (m, 1H), 1.58 (m, 2H), 1.45 (d,
J¼7.2 Hz, 3H), 1.41 (m, 1H), 1.31 (m, 1H), 0.83 (m, 4H),
0.63 (d, J¼6.7 Hz, 1H), 0.57 (d, J¼6.7 Hz, 2H); 13C NMR
(125 MHz, CD3OD) d 175.3, 175.2, 174.4, 174.3, 174.1,
173.2, 173.1, 172.4, 172.3, 171.8, 171.6, 170.5, 170.3,
168.1, 158.7, 157.5, 144.4, 144.0, 135.2, 133.0, 131.2,
131.1, 130.0, 129.7, 128.7, 125.2, 124.6, 116.4, 99.8, 99.7,
71.5, 62.0, 58.6, 58.1, 55.9, 55.3, 55.2, 53.6, 53.5, 53.4,
53.3, 51.8, 51.7, 50.9, 50.8, 42.2, 42.1, 41.6, 41.2, 41.0,
40.9, 40.0, 39.6, 31.6, 31.0, 27.4, 27.3, 26.6, 26.3, 26.2,
25.3, 24.8, 17.9, 14.8, 14.6, 12.5, 12.3; HRMS (ES) calcd
for C43H61N10O10: 877.4572 (MþMeOHþH)þ, found
877.4575.
For 17. 1H NMR (500 MHz, CD3OD) d 7.84 (m, 2H), 7.51
(m, 1H), 7.44–7.26 (m, 12H), 7.18 (d, J¼8.5 Hz, 2H), 6.8
(m, 1H), 6.79 (d, J¼8.5 Hz, 2H), 6.19 (dd, J¼1.5, 15.4 Hz,
1H), 5.24 (s, 2H), 5.11 (m, 2H), 4.81 (m, 1H), 4.66 (m, 1H),
4.57 (m, 1H), 4.47 (m, 1H), 4.31 (m, 1H), 4.25 (m, 1H),
4.09–3.92 (m, 2H), 3.91 (m, 1H), 3.78 (m, 1H), 3.50 (m,
1H), 3.09 (m, 1H), 2.86 (m, 1H), 2.62 (m, 1H), 2.20–1.73
(m, 6H), 1.67 (m, 1H), 1.58 (m, 1H), 1.40 (m, 5H), 1.25 (m,
3H), 1.11 (s, 9H), 1.10 (s, 9H), 0.81 (m, 4H), 0.64 (d,
J¼6.8 Hz, 1H), 0.58 (d, J¼6.6 Hz, 2H); 13C NMR
(125 MHz, CD3OD) d 175.5, 174.4, 174.2, 173.2, 172.3,
172.1, 171.8, 170.8, 170.4, 168.3, 165.4, 162.6, 157.5,
156.5, 144.5, 144.1, 138.7, 138.6, 136.7, 135.2, 133.0,
131.9, 131.3, 131.2, 129.9, 129.8, 129.7, 129.7, 129.6,
129.2, 129.1, 129.0, 128.7, 125.3, 124.6, 120.9, 99.8, 99.6,
70.1, 68.5, 68.4, 61.8, 58.5, 57.8, 56.8, 56.1, 53.2, 53.0,
51.5, 51.4, 50.9, 50.8, 46.3, 46.2, 41.6, 41.2, 41.1, 40.8,
40.0, 39.6, 31.5, 30.9, 27.3, 26.7, 26.4, 26.2, 24.9, 24.7,
18.6, 18.2, 15.1, 14.8, 14.0, 12.6, 12.6, 12.4; HRMS (ES)
calcd for C68H92N10O14SiNa: 1323.6461 (MþMeOHþ
Na)þ, found 1323.6459.
For E3 (1b). 1H NMR (500 MHz, CD3OD) d 7.08 (d,
J¼8.5 Hz, 2H), 6.82 (m, 1H), 6.71 (d, J¼8.4 Hz, 2H), 6.16
(d, J¼15.4 Hz, 1H), 4.75 (m, 1H), 4.62 (m, 1H), 4.51 (m,
1H), 4.34 (m, 1H), 4.26 (m, 2H), 4.12 (m, 0.7H), 4.04 (m,
0.3H), 3.84 (m, 1H), 3.53 (m, 1H), 3.14 (m, 2H), 3.07 (m,
1H), 2.82 (m, 1H), 2.58 (m, 1H), 2.24 (m, 1H), 2.12–1.92
(m, 6H), 1.70 (m, 1H), 1.59 (m, 2H), 1.38 (m, 1H), 1.35 (m,
2H), 1.30 (d, J¼7.1 Hz, 3H), 0.95 (d, J¼5.6 Hz, 3H), 0.94
(d, J¼5.9 Hz, 3H), 0.82 (m, 4H), 0.62 (d, J¼6.7 Hz, 1H),
0.56 (d, J¼6.8 Hz, 2H); 13C NMR (125 MHz, CD3OD) d
175.7, 175.1, 175.0, 174.3, 174.1, 173.2, 173.1, 172.4,
172.3, 171.8, 170.5, 168.1, 158.7, 157.5, 144.4, 144.0,
131.2, 131.1, 130.0, 125.2, 124.6, 116.4, 99.8, 99.7, 71.4,
62.0, 58.5, 58.1, 55.9, 55.3, 55.2, 53.7, 53.6, 51.6, 50.9,
46.0, 42.2, 42.1, 41.6, 41.1, 40.9, 39.7, 39.6, 31.5, 31.0,
27.6, 27.4, 27.3, 26.6, 26.4, 26.2, 25.3, 22.9, 22.8, 17.8,
14.8, 14.6, 12.5, 12.3; HRMS (ES) calcd for C41H65N10O10:
857.4885 (MþMeOHþH)þ, found 857.4888.
1
For 18. H NMR (500 MHz, CD3OD) d 7.42–7.27 (m,
10H), 7.13 (d, J¼8.4 Hz, 2H), 6.83 (m, 1H), 6.80 (d,
J¼8.4 Hz, 2H), 6.18 (d, J¼15.1 Hz, 1H), 5.26 (s, 2H), 5.14
(m, 2H), 4.80 (m, 1H), 4.61 (m, 1H), 4.47 (m, 1H), 4.38–
4.25 (m, 2H), 4.22 (m, 1H), 4.10–3.85 (m, 3H), 3.77 (m,
1H), 3.51 (m, 1H), 3.09 (m, 1H), 2.83 (m, 1H), 2.62 (m, 1H),
2.14–2.00 (m, 5H), 1.95 (m, 1H), 1.86 (m, 2H), 1.68 (m,
1H), 1.60 (m, 1H), 1.48–133 (m, 2H), 1.32–1.21 (m, 7H),
1.11 (s, 9H), 1.10 (s, 9H), 0.99–0.90 (m, 6H), 0.81 (m, 4H),
0.64 (d, J¼6.8 Hz, 1H), 0.58 (d, J¼6.7 Hz, 2H); 13C NMR
(125 MHz, CD3OD) d 175.5, 175.1, 174.1, 173.9, 172.0,
171.8, 171.4, 170.5, 165.1, 162.3, 157.2, 156.2, 144.4,
144.1, 138.7, 138.6, 136.8, 131.9, 131.3, 131.2, 129.9,
129.8, 129.7, 129.6, 129.2, 129.1, 129.0, 125.3, 124.7,
120.9, 99.8, 99.6, 70.1, 68.5, 68.4, 61.8, 58.4, 57.8, 56.8,
56.1, 53.2, 53.0, 51.4, 51.3, 46.3, 46.0, 41.6, 41.2, 41.1,
40.8, 40.0, 39.6, 33.2, 31.5, 30.9, 30.8, 27.5, 27.3, 26.7,
Acknowledgments
This work was supported by grants from the NIH and NSF.
We thank Dr Walter McMurray of the Yale Cancer Center
Mass Spectrometry Resource for help in determining
HRMS spectra of new compounds prepared in this

H. H. Wasserman, R. Zhang / Tetrahedron 58 (2002) 6277–6283
6283
4. (a) Wasserman, H. H.; Ho, W.-B. J. Org. Chem. 1994, 59,
4364–4366. (b) Wasserman, H. H.; Petersen, A. K.
Tetrahedron Lett. 1997, 38, 953–956. (c) Wasserman, H. H.;
Petersen, A. K. J. Org. Chem. 1997, 62, 8972–8973.
5. An additional difference between our synthesis and earlier
work lies in the final macrolactamization site. In contrast to the
other synthetic routes, the ring closure in our sequence took
place between the NH terminus of the diaminopropionic acid
residue and the carboxyl group of the a,b-vinylogous tyrosine.
Di-Cbz protection of the guanidine residue provided excellent
protection throughout the sequence and permitted easy
removal at the last stage with HF·Py and anisole (70–72%).6
As outlined, all steps were accomplished in good to excellent
yields.
synthesis. We are grateful to Professors Fustetani and Nakao
1
for sending us H NMR and 13C NMR spectra of natural
cyclotheonamides E2 and E3 for comparison with our
synthetic products.
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