Solid-phase total synthesis of trunkamide A

Article abstract of DOI:10.1021/jo015703t

Marine organisms are a rich source of novel, biologically active compounds. Herein, the solid-phase total synthesis of trunkamide A, currently in preclinical trials, is presented. Trunkamide A contains a thiazoline heterocycle and two residues of Ser and Thr with the hydroxy function modified as reverse prenyl (rPr). Cornerstones of the synthesis are as follows: (i) solid-phase peptide chain elongation using a quasi-orthogonal protecting scheme with tert-butyl and fluorenyl based groups, on a chlorotrityl resin; (ii) concourse of HOAt-based coupling reagents; and (iii) cyclizations in solution. Furthermore, the following synthetic steps are discussed: (i) preparation of the reverse prenyl derivatives of Ser and Thr; (ii) introduction of precursor of thiazoline as a protected amino thionoacid derivative; and (iii) formation of the thiazoline ring with DAST. All these features make this strategy particularly suitable for the large-scale synthesis of trunkamide A and other peptides containing the same motifs.

Full text of DOI:10.1021/jo015703t

7568
J . Org. Chem. 2001, 66, 7568-7574
Solid -P h a se Tota l Syn th esis of Tr u n k a m id e A1
J osep M. Caba,† Ignacio M. Rodriguez,‡ Ignacio Manzanares,‡ Ernest Giralt,*,† and
Fernando Albericio*,†
The Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain, and
Pharma Mar, s.a. c/ de la Calera 3, 28760-Tres Cantos, Madrid, Spain
albericio@qo.ub.es
Received April 22, 2001
Marine organisms are a rich source of novel, biologically active compounds. Herein, the solid-phase
total synthesis of trunkamide A, currently in preclinical trials, is presented. Trunkamide A contains
a thiazoline heterocycle and two residues of Ser and Thr with the hydroxy function modified as
reverse prenyl (rPr). Cornerstones of the synthesis are as follows: (i) solid-phase peptide chain
elongation using a quasi-orthogonal protecting scheme with tert-butyl and fluorenyl based groups,
on a chlorotrityl resin; (ii) concourse of HOAt-based coupling reagents; and (iii) cyclizations in
solution. Furthermore, the following synthetic steps are discussed: (i) preparation of the reverse
prenyl derivatives of Ser and Thr; (ii) introduction of precursor of thiazoline as a protected amino
thionoacid derivative; and (iii) formation of the thiazoline ring with DAST. All these features make
this strategy particularly suitable for the large-scale synthesis of trunkamide A and other peptides
containing the same motifs.
In tr od u ction
halalide F, isolated from the Sacoglossan mollusc Elysia
rufescens and the green alga Bryopsis sp.5 These com-
pounds are presently in phase III, II, and I clinical trials,
respectively.
Marine organisms are increasingly recognized as a
useful source of compounds of biomedical interest with
novel pharmaceutical properties.2 Thus, for instance, new
candidates for cancer therapy include the following
compounds: ecteinascidine-743, which was isolated from
the tunicate Ecteinascidia turbinata,3 aplidine, also
isolated from a tunicate (Aplidium albicans),4 and ka-
In 1996, Bowden and co-workers6 isolated trunkamide
A from the colonial ascidian Lissoclinum sp., which was
obtained from the Great Barrier Reef, Australia. Trunka-
mida A has been initially selected by the National Cancer
Institute (NCI) for further testing due to a good COM-
PARE correlation analysis and specificity against UO-
31 renal cell line, that is an multidrug resistant (MDR)
line. This could suggest a potential novel mechanism of
action and a possible non-MDR activity, as well. Trunka-
mide A is a cyclic heptapeptide that incorporates motifs
that are common to other members of the Patellin family,
which are also isolated from ascidians. Thus, trunkamide
A contains hydroxy side-chain amino acids with the
hydroxy function modified as dimethylallyl ethers [re-
verse prenyl, (rPr)] and a thiazoline heterocycle. Initially,
Bowden and co-workers assigned the L-configuration to
the seven stereogenic carbons present in the macrocyclic
ring (structure 1a ). More recently, and when the work
described here was close to completion, Wipf and Uto7
were the first to demonstrate that the initial assignment
was erroneous and, later, that the stereocenter exocyclic
to the thiazoline ring has a D-configuration (structure
1b).8 Furthermore, during the preparation of this manu-
script, McKeever and Pattenden have published a solu-
tion synthesis of trunkamide A.9
† University of Barcelona.
‡ Pharma Mar.
(1) Abbreviations used for amino acids and the designations of
peptides follow the rules of the IUPAC-IUB Commission of Biochemi-
cal Nomenclature in J . Biol. Chem. 1972, 247, 977-983. The following
additional abbreviations are used: ACH, R-cyano-4-hydroxycinnamic
acid; CI, chemical ionization; Cl-TrtCl-resin, 2-chlorotrityl chloride
resin; DAST, (diethylamino)sulfur trifluoride; DCC, N,N′-dicyclohexyl-
carbodiimide; DHB, 2,5-dihydroxybenzoic acid; DIEA, N,N-diisopro-
pylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; DMAP, 4-(di-
methylamino)pyridine; DMF, N,N-dimethylformamide; ES-MS, electro-
spray mass spectrometry; EtOAc, ethyl acetate; FAB, fast atom
bombardment; Fmoc, 9-fluorenylmethoxycarbonyl; Fmoc-Phe(S)-NBt,
1-(N-Fmoc-D-thionophenylalanyl)-6-nitrobenzotriazole; HFIP, 1,1,1,3,3,3-
hexafluoro-2-propanol; HPLC, high performance liquid chromatogra-
phy; HOBt, 1-hydroxybenzotriazole; HRMS, high-resolution mass
spectrometry; rPr, 1,1-dimethylallyl, reverse prenyl; MALDI-TOF,
matrix assisted laser desorption ionitzation-time-of-flight; MeOH,
methanol; NMM, N-methylmorpholine; NMR, nuclear magnetic reso-
nance; PyAOP, 7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phospho-
nium hexafluorophosphate; SPS, solid-phase synthesis; Tce, trichlo-
roethanol; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMS,
trimethylsilyl; Tzn, thiazoline; UV, ultraviolet. Amino acid symbols
denote the L-configuration unless stated otherwise. All solvent ratios
are volume/volume unless stated otherwise.
(2) See, for example: (a) Reviews on marine natural products:
Faulkner, J . Nat. Prod. Rep. covered yearly since 1977. (b) Wipf, P.
Chem. Rev. 1995, 95, 2115-2134. (c) Carte, B. K. Bioscience 1996, 46,
271-286. (d) Riguera, R. J . Mar. Biotechnol. 1997, 5, 187-193.
(3) (a) Rinehart, K. L.; Holt, T. G.; Fregeau, N. L.; Stroh, J . G.;
Keifer, P. A.; Sun, F.; Li, L. H.; Martin, D. G. J . Org. Chem. 1990, 55,
4512-4515. (b) Rinehart, K. L.; Holt, T. G.; Fregeau, N. L.; Stroh, J .
G.; Keifer, P. A.; Sun, F.; Li, L. H.; Martin, D. G. J . Org. Chem. 1991,
56, 1676.
(5) Hamann, M. T.; Scheuer, P. J . J . Am. Chem. Soc. 1993, 115,
5825-5826.
(6) Carroll, A. R.; Coll, J . C.; Bourne, D. J .; McLeod, J . K.; Zabriskie,
T. M.; Ireland, C. M.; Bowden, B. F. Aust. J . Chem. 1996, 49, 659-
667.
(7) Wipf, P.; Uto, Y. Tetrahedron Lett. 1999, 40, 5165-5169.
(8) Wipf, P.; Uto, Y. J . Org. Chem. 2000, 65, 1037-1049.
(9) McKeever, B.; Pattenden, G. Tetrahedron Lett. 2001, 42, 2573-
2577.
(4) (a) Rinehart, K. L.; Bertelloni, L. Chem. Abstr. 1991, 115,
248086q. (b) Schmitz, F. J .; Yasumumoto, T. J . Nat. Prod. 1991, 54,
1469-1490.
10.1021/jo015703t CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/25/2001

Solid-Phase Total Synthesis of Trunkamide A
J . Org. Chem., Vol. 66, No. 23, 2001 7569
Sch em e 1
Resu lts a n d Discu ssion
With the aim of preparing a sufficient quantity of
trunkamide A (1b) to carry out a complete study of its
biological activity, as well as a conformational study, an
efficient synthesis was developed. The retrosynthetic
analysis (Scheme 1) shows the cornerstones of this
approach: (i) the use of the solid-phase strategy for the
elongation of the peptide chain,10 where the molecule
under construction is bound to an insoluble support
during all synthetic operations. The use of this technique
means that excess reagents and soluble byproducts can
be removed simply by washing the molecule-resin with
suitable solvents. Large excesses of the soluble reagents
can, therefore, be used in order to drive the reactions to
completion in relatively short periods of time, thus
avoiding racemization (where applicable) and other sec-
ondary reactions; (ii) the use of a fluorenylmethyloxy-
carbonyl (Fmoc) base strategy,10 because the high acid
lability of the reverse prenyl ether prevents the use of
acid-labile protecting groups; (iii) the use of the super-
acid-labile chlorotrityl chloride resin (ClTrt-Cl-resin),11
which allows the cleavage of the peptide under extremely
mild acidic conditions, such as mixtures of hexafluoro2-
propanol (HFIP) and CH2Cl2;12 (iv) macrocyclization
through the formation of the peptide bond between the
amino function of the thiophenylalanyl residue and the
carboxylic acid function of the Pro in the penultimate
step. The choice of this cyclization point was based on a
number of desirable features. For example, the benzyl
group in the R-postion with respect to the amino function
is rather unhindered in comparison with â-branched
residues and/or reverse prenyl-protected hydroxy func-
tions. Likewise, the carboxylic acid function forms part
of the Pro and therefore cannot give an oxazolone during
the activation step, which is one of the mechanisms
through which the epimerization occurs.13 Furthermore,
the protected amino thionoacid is incorporated in the last
step through a very mild activation process, which allows
the presence of the free hydroxyl function of the Ser; and
(v) formation of the thiazoline ring in the last step
through the exposure of a â-hydroxy thiopeptide to an
activating reagent of the hydroxy function. If the thi-
azoline ring is formed in an earlier step of the synthetic
process, the exposure of the ring to further treatments
could cause the epimerization of the two stereocenters
of the Phe-Tzn moiety.14
To fulfill the requirements of Scheme 1, Fmoc-Ser/Thr-
(rPr)-OH and an activated species of the Fmoc-D-thio-
phenylalanine acid [Fmoc-D-Phe(S)-OH] were synthe-
sized. The reverse-prenylated derivatives were prepared
according to Scheme 2 from the commercially available
Fmoc-Ser/Thr(tBu)-OH (8). Thus, the following steps
were undertaken: (i) orthogonal protection of the car-
boxyl group under the form of its trichloroethyl ester by
reaction with the appropriate alcohol, DCC, and DMAP;
(ii) removal of the tert-butyl groups with TFA-H2O (19:
1); (iii) formation of the 1,1-dimethylpropionyl ether by
reaction with the corresponding trichloroacetimidate,
which was prepared by a slightly modified version of the
protocol described by Armstrong and co-workers;15 (iv)
partial reduction of the triple bond to the double bond
by catalytic hydrogenation in the presence of Pd/C and
quinoline;16 and (v) removal of the trichloroethyl ester
with Zn and NH4OAc. The preparation of this propionyl
t
(14) Alternatively, the synthesis of the linear sequence of the Bu
analogue of Trunkamide A was attempted in the solid phase by
incorporation of the Fmoc-Phe-Tzn-OH (the configuration of the
stereocenter of Phe corresponded to that originally described by
Bowden, ref 6), which was synthesized according to a published method
(Lee, J .; Griffin, J . H.; Nicas, T. I. J . Org. Chem. 1996, 61, 3983-3986),
onto the H-Thr(tBu)-Ser(tBu)-Ile-Ala-Pro-ClTrt-resin. The analysis by
HPLC of the cleaved material showed four major peaks with a mass
of (M + 18). This could indicate the opening of the thiazoline ring due
to the acid treatment and the formation of the four diastereomers due
to the epimerization of the two stereocenters during the coupling
reaction and/or cleavage from the solid support.
(15) Armstrong, A.; Brackenridge, I.; J ackson, R. F. W.; Kirk, J . M.
Tetrahedron Lett. 1988, 29, 2483-2486.
(16) The use of the propionyl derivative was also reported by Wipf
and Venkatraman for the preparation of reverse prenylamines. Wipf,
P.; Venkatraman, S. J . Org. Chem. 1996, 61, 6517-6522.
(10) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches
to the Synthesis of Peptides and Proteins; CRC Press: Boca Raton, FL,
1997.
(11) Barlos, K.; Gatos, D.; Scha¨fer, W. Angew. Chem., Int. Ed. Engl.
1991, 30, 590-593.
(12) Bollhagen, R.; Schmiedberger, M.; Barlos, K.; Grell, E. J . Chem.
Soc., Chem. Commun. 1994, 2559-2560.
(13) Albericio, F.; Carpino. L. A. In Methods in Enzymology; Fields,
G. B., Ed.; Academic Press: New York, 1997; pp 104-126.

7570 J . Org. Chem., Vol. 66, No. 23, 2001
Caba et al.
Sch em e 2
ether and its subsequent reduction to the allyl derivative
avoids the lack of regioselectivity encountered with the
use of 1,1-dimethylallyl derivatives.17 This five-step
scheme allowed the preparation of these derivatives with
an overall, nonoptimized yield of 28% for Ser and 25%
for Thr.
For the activation of the thio acid, the 1-(N-Fmoc-D-
thionophenylalanyl)-6-nitrobenzotriazole [Fmoc-D-Phe(S)-
NBt] (9) derivative was used.18 This kind of derivative,
which had been previously used only in the Boc form in
solution, is perfectly stable, can be stored, and is compat-
ible with the solid-phase approach, as demonstrated for
first time in the present work.
Cleavage of the reverse-prenylated peptide was carried
out very smoothly with HFIP-CH2Cl2 (1:4) and gave a
yield of 51%. HPLC analysis of the crude product showed
a purity of 63%.
The macrocyclization was carried out with PyAOP/
DIEA.22 PyAOP is a phosphonium salt based on HOAt,
which reduces the risk of racemization23 and does not
lead to the termination of the peptide often associated
with the corresponding uronium/aminium salts.24
The formation of the thiazoline ring was performed in
a single step by exposure of the â-hydroxy thiopeptide to
DAST.25,26 This cyclization was also attempted by the use
of other methods such as Burgess27,28 or Mitsunobu29
reactions. In both cases, although traces of product were
obtained, the quality of the product was clearly inferior
to that obtained with DAST. The reaction mixture was
not worked-up and the crude product was purified by
semipreparative HPLC to give Trunkamide A (1b) (over-
all yield of 5%),30 which showed an excellent purity by
1
HPLC (Figure 1), was characterized by MS and H NMR
spectroscopy (600 MHz) (Table 2). The resulting data
confirmed the configuration proposed by Wipf.8
The linear sequence was synthesized on the ClTrt-Cl-
resin.11 The partial incorporation of Fmoc-Pro-OH (0.5
equiv) was performed in the presence of DIEA (2
equiv).19,20 The incorporation of the first six protected
amino acids was carried out using DIPCDI as the
coupling reagent. The reverse-prenylated derivatives
were incorporated with an excess of 1.7 equiv. The last
Ser unit was incorporated with an unprotected hydroxy
side chain. Finally, the Fmoc-D-Phe(S)-NBt coupled very
smoothly to give a negative ninhydrin test after 90 min.21
In conclusion, a total solid-phase synthesis of trunk-
amide A (1b) is reported. The incorporation of the
modified residues Ser, Thr, and Phe into the peptidic
chain avoided further manipulation of these residues in
subsequent steps of the synthesis. This process takes
advantage of the benefits of the solid-phase approach,
(22) Albericio, F.; Cases, M.; Alsina, J .; Triolo, S. A.; Carpino, L. A.;
Kates, S. A. Tetrahedron Lett. 1997, 38, 4853-4856.
(23) Carpino, L. A.; El-Faham, A.; Albericio, F. Tetrahedron Lett.
1994, 35, 2279-2283.
(17) Initially, the introduction of the allyl moiety was attempted
directly from 1,1-dimethylallyl derivatives. Thus, reaction of Fmoc-
Ser/Thr-OTce with 1,1-dimethylallyl alcohol in the presence of acids
such as citric, phosphoric, sulfuric, or trifluoroacetic led in all cases to
a mixture of the target compound and its 3,3-dimethylallyl isomer.
Furthermore, the reaction with 1,1-dimethylallyl trichloroacetimidate
led regioselectively to the formation of the undesired isomer and the
attempt at displacement of the mesyl group from the alkoxide led to
the dehydroalanine derivative. Finally, the best yields were obtained
by using 3,3-dimethylallyl alcohol in the presence of 85% aqueous H3-
PO4 (3.3 equiv) for 4 h at 35-40 °C in a sealed tube. The two isomers
were purified by silica gel chromatography, followed by preparative
reverse phase HPLC. Thus, although the two target compounds were
obtained, the low yields obtained make this method unsuitable for their
large scale preparation.
(24) Albericio, F.; Bofill, J . M.; El-Faham, A.; Kates, S. A. J . Org.
Chem. 1998, 63, 9678-9683.
(25) (a) Halasz, S. P.; Glemser, O. Chem. Ber. 1970, 103, 594-602.
(b) Lafargue, P.; Guenot, P.; Lellouche, J . P. Heterocycles 1995, 41,
947-958.
(26) DAST has been used by Wipf and co-workers for the preparation
of the thiazoline from a â-hydroxy thiopeptide in an oxazoline f
thiazoline sequence strategy; refs 7 and 8.
(27) (a) Atkins, G. M.; Burgess, E. M. J . Am. Chem. Soc. 1968, 90,
4744-4745. (b) Burgess, E. M.; Penton, H. R.; Taylor, E. A. J . Org.
Chem. 1973, 38, 26-31.
(28) Burgess’ reagent has been used by Wipf and co-workers. (Wipf,
P.; Venkatraman, S. Synlett 1997, 1-10) and Pattenden and co-
workers (Boden, C. D. J .; Pattenden, G. Tetrahedron Lett. 1995, 36,
6153-6156) for the preparation of thiazolines.
(18) Shalaby, M. A.; Grote, C. W.; Rapoport, H. J . Org. Chem. 1996,
61, 9045-9048.
(29) Gale´otti, N.; Montagne, C.; Poncet, J .; J ouin, P. Tetrahedron
Lett. 1992, 33, 2807-2810.
(19) The use of ClTrt-Cl-resins of high loading (>0.5 mmol/g) for
the preparation of hydrophobic peptides such as Kahalalide F usually
leads to impure crude peptide. Chiva, C.; Vilaseca, M.; Giralt, E.;
Albericio, F. J . Pept. Sci. 1999, 5, 131-140.
(30) If the crude reaction mixture is washed with dilute aqueous
solutions of NaHCO3, conversion (40-70%) to a compound with a mass
ion (M + 16) took place. An NMR study of this compound, which shows
chromatographic behavior very similar to trunkamide A, only shows
different chemical shifts for the proton belonging to the D-Phe-Tzn,
indicating that an oxidation at the sulfur atom could have taken place.
(20) Unreacted reactive sites were capped with MeOH-DIEA.
(21) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595-598.

Solid-Phase Total Synthesis of Trunkamide A
J . Org. Chem., Vol. 66, No. 23, 2001 7571
Spain). Analytical HPLC was carried out on a Shimadzu
instrument comprising two solvent delivery pumps (model LC-
6A), automatic injector (model SIL-6B), variable wavelength
detector (model SPD-6A), system controller (model SCL-6B)
and plotter (model C-R6A). UV detection was performed at 220
nm, and linear gradients of CH3CN (+0.036% TFA) into H2O
(+0.045% TFA) were run at 1.0 mL/min flow rate from the
following: (condition A) 0:1 to 1:0 over 30 min; (condition B)
1:1 to 10:0 over 30 min. Flash chromatography was carried
out using silica gel 60 A C C 50-70 mm SDS (Peypin, France).
Optical rotations were measured on a Perkin-Elmer 241 MC
polarimeter. IR spectra were obtained using a Nicolet 510 FT-
IR spectrometer. MALDI-TOF- and ES(+)-MS analyses of
peptide samples were performed in a PerSeptive Biosystems
Voyager DE RP, using ACH or DHB matrixes, and in a
Micromass VG-quattro spectrometer. CI-MS analyses of amino
acid derivatives were performed using a Hewlett-Packard HP-
5988A spectrometer. 1H NMR (500 MHz, 200 MHz) and 13C
NMR (50 MHz) spectroscopy was performed on a Bruker DMX-
500 (11.7 T) and Varian Gemini 200 (4.7 T). Chemical shifts
(d) are expressed in parts per million downfield from TMS.
Coupling constants are expressed in Hertz.
N-Boc-D-p h en yla la n in e 2-a m in o-5-n itr oa n ilid e (10).
Boc-D-Phe-OH (6 g, 22.6 mmol) was dissolved in THF (180 mL),
and NMM (4.98 mL, 45.2 mmol, 2 equiv) was added at -20
°C under N2, followed by the dropwise addition of isobutyl
chloroformate (2.95 mL, 22.6 mmol, 1 equiv). The reaction
mixture was stirred for 10 min, a solution of 4-nitro-1,2-
phenylenediamine (3.46 g, 22.6 mmol, 1 equiv) in THF (60 mL)
was added, and the mixture stirred for a further 2 h at -20
°C and 16 h at 23 °C. The precipitate was filtered off, and the
filtrate was evaporated to dryness. The residue was dissolved
in EtOAc (200 mL) and extracted with aqueous solutions of 1
M NaH2PO4 (2 × 100 mL), saturated brine (2 × 100 mL),
saturated NaHCO3 (2 × 100 mL), and saturated NaCl (2 ×
100 mL), dried over Na2SO4, and evaporated to dryness to give
the title compound (8.77 g, 97%) as a yellow solid: analytical
HPLC (tR 22.1 min, condition A; tR 7.4 min, condition B); [R]D
-43 (c 0.01, DMSO); IR (KCl) νmax 3442, 1733, 1507, 1387, 1152
cm-1; CI-MS calcd for C20H24N4O5 400, found 401 [(M + H)+,
46], 418 [(M + NH4)+, 100]; HRMS (FAB) ES-MS calcd
401.1825, found 401.1812; 1H NMR (300 MHz, DMSO-d6) δ
9.37 (1H, s, NH-ar), 7.94 (1H, s, ar), 7.85 (1H, dd, J ) 9.0 Hz,
1.2 Hz, ar), 7.30-7.20 (6H, m, 5H ar, NH Phe), 6.72 (1H, d, J
) 9.0 Hz, ar), 6.35 (2H, s, NH2), 4.35-4.25 (1H, m, R-CH Phe),
3.10-3.00 (1H, m, â-CH2 Phe), 2.90-2.82 (1H, m, â-CH2 Phe),
1.33 (9H, s, 3CH3 Boc); 13C NMR (75 MHz, DMSO-d6) δ 171.8
(CO Phe), 156.1 (CO Boc), 150.3 (C ar), 138.2 (C ar), 135.8 (C
ar), 129.7 (CH ar), 128.5 (CH ar), 126.8 (CH ar), 123.8 (CH
ar), 122.8 (CH ar), 121.4 (C ar), 113.9 (CH ar), 78.8 (C Boc),
56.8 (R-C Phe), 37.4 (â-C Phe), 28.5 (3CH3 Boc).
F igu r e 1. HPLC chromatograms of pure trunkamide A (1b).
A reversed-phase C-18 column was used for the analysis with
elution by a linear gradient over 30 min of 0.036% TFA in ACN
and 0.045% TFA in H2O from 45:55 to 70:30, flow rate 1.0 mL
min-1
.
which allows rapid incorporation of the residues. Finally,
an efficient method for the preparation of reverse-
prenlylated derivatives of Ser and Thr has been devel-
oped and consists of five robust steps from commercially
available compounds. All these features make this strat-
egy particularly suitable for the large scale synthesis of
trunkamide A (1b) and other peptides containing the
same motifs.
Exp er im en ta l Section
Gen er a l P r oced u r es. Cl-TrtCl-resin, protected Fmoc-
amino acid derivatives, HOBt, and PyAOP were obtained from
PerSeptive Biosystems (Framingham, MA), Bachem (Buben-
dorf, Switzerland), Albatross (Montreal, Canada), and Nova-
Biochem (La¨ufelfingen, Switzerland). DIEA, DIPCDI, piperi-
dine, Fmoc-Cl, NMM, isobutyl chloroformate, 4-nitro-1,2-
phenylenediamine, P4S10, 2-methyl-3-butyn-2-ol, trichloroaceto-
nitrile, TFA, DMAP, DCC, HFIP, DAST, Pd-C 10%, CF3SO3H,
2,2,2-trichloroethanol, and quinoline were obtained from Al-
drich (Milwaukee, WI). DMF, CH2Cl2, CHCl3, and EtOAc were
obtained from SDS (Peypin, France). Acetonitrile (HPLC
grade) and THF were obtained from Scharlau (Barcelona,
Spain). Hexane, Et2O, and methanol were obtained from
Panreac (Moncada i Reixac, Barcelona). All commercial re-
agents and solvents were used as received with the exception
of DMF and CH2Cl2, which were bubbled with nitrogen to
remove volatile contaminants (DMF) and stored over activated
4A molecular sieves (Merck, Darmstadt, Germany) (DMF) or
CaCl2 (CH2Cl2). Et2O was stored over Na.
N-Boc-D-p h en yla la n in e 2-a m in o-5-n itr oth ioa n ilid e (11).
P4S10 (4.72 g, 10.6 mmol, 0.5 equiv) was added to a suspenssion
of Na2CO3 (1.13 g, 10.6 mmol, 0.5 equiv) in THF (350 mL) at
23 °C under a flow of N2. After 1 h the mixture was cooled to
0 °C and N-Boc-D-phenylalanine 2-amino-5-nitroanilide (10)
(8.50 g, 21.2 mmol) was added and the mixture stirred for 30
min at 0 °C and 2.5 h at 23 °C. The mixture was filtered
through Celite and the filtrate evaporated to dryness. The
residue was dissolved in EtOAc-hexane (2:1, 200 mL), ex-
tracted with 5% aqueous NaHCO3 (3 × 10 mL) and the
aqueous phase extracted with EtOAc-hexane (2:1, 100 mL).
The combined organic phases were dried over MgSO4, filtered
and evaporated to dryness to give N-Boc-D-phenylalanine
2-amino-5-nitrothioanilide (8.40 g, 95%) as a yellow-brown
solid: analytical HPLC (tR 25.4 min, condition A; tR 10.1 min,
condition B); [R]D - 80 (c 0.01, CHCl3DMF, 97:3); IR (KCl) νmax
3357, 1677, 1509, 1304, 1023 cm-1; CI-MS calcd for C20H24N4O4S
416, found 417 [(M + H)+, 46], 434 [(M + NH4)+, 20]; HRMS
Solution reactions were performed in round-bottomed flasks.
Organic solvent extracts were dried over anhydrous MgSO4,
followed by solvent removal under reduced pressure and at
<40 °C.
Solid-phase syntheses were carried out in polypropylene
syringes (10/20 mL) fitted with a polyethylene porous disk.
Solvents and soluble reagents were removed by suction.
Removal of the Fmoc group was carried out with piperidine-
DMF (2:8, v/v) (2 × 1 min, 2 × 20 min). Washings between
deprotection, coupling, and final deprotection steps were
carried out with DMF (5 × 1 min) and CH2Cl2 (5 × 1 min)
using 10 mL solvent/g resin for each wash. Peptide synthesis
transformations and washes were performed at 25 °C.
HPLC columns (Nucleosil C18 reversed-phase column, 4.6
× 250 mm, 10 mm) were obtained from Scharlau (Barcelona,
1
(FAB) ES-MS calcd 417.1596, found 417.1587; H NMR (300
MHz, CDCl3) δ 9.53 (1H, bs, NH-ar), 7.93 (1H, dd, J ) 9.2, 2.8
Hz, ar), 7.62 (1H, s, ar), 7.40-7.20 (5H, m, ar), 6.62 (1H, d, J
) 9.2 Hz, ar), 5.61 (1H, d, J ) 7.8 Hz, NH Phe), 4.85-4.75
(1H, m, R-CH Phe), 4.62 (2H, bs, NH2), 3.25-3.10 (2H, m,
â-CH2 Phe), 1.33 (9H, s, 3CH3 Boc); 13C NMR (75 MHz, CDCl3)

7572 J . Org. Chem., Vol. 66, No. 23, 2001
Caba et al.
δ 205.6 (CS Phe), 156.2 (CO Boc), 148.8 (C ar), 137.7 (C ar),
135.9 (C ar), 129.3 (CH ar), 128.8 (CH ar), 127.4 (CH ar), 125.4
(CH ar), 124.8 (CH ar), 121.9 (C ar), 114.6 (CH ar), 81.1 (C
Boc), 63.1 (R-C Phe), 41.3 (â-C Phe), 28.1 (3CH3 Boc).
sphere, the above solution was added dropwise during 20 min
to a solution of trichloroacetonitrile (10.3 mL, 102.2 mmol, 1
equiv) in anhydrous Et2O (20 mL), and the reaction was stirred
for 2 h, during which time the tempearture increased to 23
°C. The solvent was removed, MeOH-hexane (1:19, 10 mL)
was added, and the mixture was vigorously stirred for 1 min.
During this time, a precipitate appeared that was filtered off
and washed with cold hexane. The filtrates were combined and
evaporated to dryness to give 1,1-dimethylpropionyl 2,2,2-
trichloroacetimidate (15.9 g, 69%): IR (film) νmax 3307, 1669,
1366, 1314, 1140, 1077 cm-1; CI-MS calcd for C7H8NOCl3 227,
found 228 [(M + H)+, 100], 245 [(M + NH4)+, 19]; 1H NMR
(200 MHz, CDCl3) δ 8.56 (1H, bs, NH), 2.62 (1H, s, CH), 1.82
(6H, s, 2CH3); 13C NMR (50 MHz, CDCl3) δ 159.6 (CN), 92.0
(CCl3), 83.7 (C), 75.2 (C), 73.2 (CH), 28.5 (2CH3).
N-F m oc-D-p h en yla la n in e 2-a m in o-5-n it r ot h ioa n ilid e
(12). N-Boc-D-phenylalanine 2-amino-5-nitrothioanilide (11)
(8.40 g, 20.2 mmol) was dissolved in TFA-CH2Cl2 (45:55, 50
mL), and the solution was stirred for 20 min. The solvent was
evaporated, Et2O was added, and the solvent was evaporated
again. This procedure was repeated two more times. The
residue was dissolved in 10% aqueous Na2CO3dioxane (2:1, 250
mL), Fmoc-Cl (5.23 g, 20.2 mmol, 1 equiv) in dioxane (65 mL)
was added, and the mixture was stirred for 1 h at 0 °C. The
dioxane was removed by evaporation under reduced pressure
and the resulting solid was dissolved in EtOAc (100 mL). The
solution was cooled to 0 °C, and the pH was adjusted to <3
with 5% aqueous KHSO4. The aqueous phase was extracted
with EtOAc (2 × 75 mL). The combined organic phases were
dried over Na2SO4, filtered, and evaporated to dryness to give
N-Fmoc-D-phenylalanine 2-amino-5-nitrothioanilide (8.80 g,
80%) as an orange-yellow solid: analytical HPLC (tR 27.3 min,
condition A; tR 15.1 min, condition B); [R]D -14.3 (c 0.01,
DMSO); IR (KCl) νmax 1702, 1509, 1322, 1031 cm-1; ES-MS
calcd for C30H26N4O4S 538.2, found 539.4 (M + H)+; HRMS
F m oc-Ser -OTce (5a ). Fmoc-Ser(tBu)-OH (1 g, 2.6 mmol)
was dissolved in CH2Cl2 (7 mL). DMAP (0.15 g, 1.3 mmol, 0.5
equiv) and 2,2,2-trichloroethanol (Tce) (0.3 mL, 3.1 mmol, 1.2
equiv) were added, followed by a solution of DCC (0.63 g, 3.1
mmol, 1.2 equiv) in CH2Cl2 (2.5 mL): (all additions were
performed under a N2 atmosphere at 0 °C). The reaction
mixture was stirred for 18 h at room temperature. The reaction
mixture was cooled to 0 °C, filtered and the filtrate was
concentrated in vacuo. The resulting crude material was
dissolved in EtOAc (7 mL) and washed with 10% aqueous citric
acid (2 × 10 mL), saturated aqueous NaHCO3 (2 × 10 mL)
and brine (2 × 10 mL), dried (MgSO4), and concentrated in
vacuo to give Fmoc-Ser(tBu)-OTce (4a ) (1.34 g), which was used
without further purification.
1
(FAB) ES-MS calcd 539.1753, found 539.1761; H NMR (200
MHz, CDCl3) δ 9.19 (1H, bs, NH-ar), 7.82 (1H, dd, J ) 9.2 Hz,
1.2 Hz, ar), 7.80-7.10 (14H, m, ar), 6.43 (1H, d, J ) 9.2 Hz,
ar), 5.93 (1H, bs, NH Phe), 4.95-4.85 (1H, m, R-CH Phe),
4.20-4.00 (3H, m, CH Fmoc, CH2 Fmoc), 3.30-3.10 (2H, m,
â-CH2 Phe); 13C NMR (50 MHz, CDCl3) δ 205.1 (CS Phe), 156.5
(CO Fmoc), 148.2 (C ar), 143.3 (C ar), 141.0 (C ar), 138.2 (C
ar), 135.7 (C ar), 129.3 (CH ar), 128.9 (CH ar), 127.8 (CH ar),
127.5 (CH ar), 127.0 (CH ar), 125.3 (CH ar), 124.9 (CH ar),
124.7 (CH ar), 121.0 (C ar), 120.0 (CH ar), 115.6 (CH ar), 67.0
(CH2 Fmoc), 62.9 (R-C Phe), 46.7 (CH Fmoc), 41.7 (â-C Phe).
1-(N -F m oc-D-t h ion op h e n yla la n yl)-6-n it r ob e n zot r i-
a zole (9). To a solution of N-Fmoc-D-phenylalanine 2-amino-
5-nitrothioanilide (12) (8.80 g, 16.4 mmol) in 95% aqueous
HOAc (185 mL) was added NaNO2 (1.70 g, 24.6 mmol, 1.5
equiv) portionwise during 5 min at 0 °C. The mixture was
stirred for 30 min at 0 °C, and during this time a precipitate
formed. Cold H2O (200 mL) was added to the mixture, and
the precipitate was filtered off, washed with cold H2O and
dissolved in CH2Cl2 (100 mL). The organic phase was extracted
with H2O (2 × 75 mL), and the combined aqueous phases were
extracted with CH2Cl2 (2 × 75 mL). The combined organic
phases were washed with H2O (2 × 75 mL) and dried over
Na2SO4, and the solvent was removed to give 1-(N-Fmoc-D-
thionophenylalanyl)-6-nitrobenzotriazole (7.70 g, 85%) as an
orange-brown solid: analytical HPLC (tR 29.7 min, condition
A; tR 19.2 min condition B); [R]D -97 (c 0.014, CHCl3); IR (KCl)
νmax 3309, 1696, 1537, 1348, 1254 cm-1; ES-MS calcd for
Fmoc-Ser(tBu)-OTce (4a ) was dissolved in TFA-H2O (19:1,
10 mL) and the solution was stirred for 5 h. The reaction
mixture was concentrated in vacuo and the residue purified
by flash chromatography (EtOAc-hexane, 3:7) to give Fmoc-
Ser-OTce (5a ) (0.83 g, 1.8 mmol, 69% overall yield for the two
steps): analytical HPLC (tR 12.2 min, condition B); [R]D -5.6
(c 0.01, CHCl3, 23 °C); IR (film) 3407, 1769, 1692, 1516, 1209,
1082 cm-1; CI-MS calcd for C20H18O5NCl3 457, found 475 [(M
+ NH4+, 100]; HRMS (FAB) ES-MS calcd 458.0329, found
458.0332; 1H NMR (200 MHz, CDCl3) δ 7.70-7.20 (8H, m, Ar),
6.05 (1H, d, J ) 8.0 Hz, NH), 4.85 (1H, d, J ) 12.0 Hz, CH2
Tce), 4.67 (1H, d, J ) 12.2 Hz, CH2 Tce), 4.60-4.54 (1H, m,
R-CH Ser), 4.45-4.30 (2H, m, CH2 Fmoc), 4.20-4.15 (1H, m,
CH Fmoc), 4.10-3.85 (2H, m, â-CH2 Ser), 3.50 (1H, bs, OH);
13C NMR (50 MHz, CDCl3) δ 169.0 (CO Ser), 156.2 (CO Fmoc),
143.5 (C Ar), 141.1 (C Ar), 127.6 (CH Ar), 126.9 (CH Ar), 124.9
(CH Ar), 119.8 (CH Ar), 94.2 (CCl3 Tce), 74.4 (CH2 Tce), 67.2
(CH2 Fmoc), 62.6 (â-CH2 Ser), 55.9 (R-CH Ser), 46.9 (CH Fmoc).
F m oc-Ser (r P r )-OH (8a ). Fmoc-Ser-OTce (5a ) (0.83 g, 1.8
mmol) was dissolved in CH2Cl2-hexane (2:1, 4 mL), and 1,1-
dimethylpropionyl trichloroacetimidate (13) (0.41 g, 1.8 mmol,
1 equiv) and CF3SO3H (40 µL) were added under a N2
atmosphere. The reaction mixture was stirred for 4 days, and
the same quantities of the trichloroacetimidate and CF3SO3H
were added every 24 h. The reaction mixture was filtered, and
the filtrate was concentrated in vacuo, dissolved in EtOAc (10
mL), and washed with saturated aqueous NaHCO3 (2 × 10
mL), H2O (2 × 10 mL), and brine (2 × 10 mL). The organic
solution was dried (MgSO4) and concentrated in vacuo to give
the 1,1-dimethylpropionyl ether of Fmoc-Ser-OTce (6a ) (0.95
g), which was used without further purification.
C
30H23N5O4S 549.1, found 550.2 (M + H)+; HRMS (FAB) ES-
MS calcd 550.1549, found 550.1569; 1H NMR (200 MHz,
CDCl3) δ 9.61 (1H, s, ar), 8.41 (1H, dd, J ) 8.9 Hz, 1.2 Hz, ar),
8.25 (1H, d, J ) 8.9 Hz, ar), 7.80-7.10 (13H, m, ar), 6.65-
6.50 (1H, m, R-CH Phe), 5.79 (1H, d, J ) 9.2 Hz, NH), 4.45-
4.30 (2H, m, CH2 Fmoc), 4.20-4.10 (1H, m, CH Fmoc), 3.41
(1H, dd, J ) 13.6, 5.0 Hz, â-CH2 Phe), 3.08 (1H, dd, J ) 13.6,
8.0 Hz, â-CH2 Phe); 13C NMR (50 MHz, CDCl3) δ 207.9 (CS
Phe), 155.3 (CO Fmoc), 149.4 (C ar), 148.8 (C ar), 143.5 (C
ar), 141.1 (C ar), 135.0 (C ar), 131.5 (C ar), 129.2 (CH ar), 128.5
(CH ar), 127.7 (CH ar), 127.3 (CH ar), 127.0 (CH ar), 124.0
(CH ar), 122.2 (CH ar), 121.4 (CH ar), 119.9 (CH ar), 112.5
(CH ar), 66.9 (CH2 Fmoc), 62.3 (R-C Phe), 47.1 (CH Fmoc),
42.6 (â-C Phe).
The 1,1-dimethylpropionyl ether of Fmoc-Ser-OTce (6a ) was
dissolved in MeOH (20 mL) and 10% Pd/C (38 mg, 4% of the
crude weight) and quinoline (0.44 mL, 0.45 mL per g of crude)
were added under a N2 atmosphere. The atmosphere was
changed from N2 to H2 and the mixture was stirred for 2 h.
The reaction mixture was concentrated in vacuo and the
residue purified by flash chromatography (CHCl3-hexane, 8:2)
to give Fmoc-Ser(rPr)-OTce (7a ) (0.38 g, 0.72 mmol): analytical
HPLC (tR 20.7 min, condition B); [R]D -26.4° (c 0.05, CHCl3,
23 °C); IR (film) 3442, 2979, 1782, 1506, 1451 cm-1; CI-MS
calcd for C25H26O5NCl3 525, found 526 [(M + H)+, 34], 543 [(M
+ NH4)+, 100]; HRMS (FAB) ES-MS calcd 526.0955, found
526.0940; 1H NMR (200 MHz, CDCl3) δ 7.80-7.22 (8H, m, Ar),
1,1-Dim eth ylp r op ion yl 2,2,2-tr ich lor oa cetim id a te (13).
A suspension of either KH in paraffin oil (35%, 1.17 g, 10.2
mmol, 0.1 equiv) or NaH (60%, 0.82 g, 10.2 mmol, 0.1 equiv)
was washed with hexane (3 × 4 mL) under a N2 atmosphere.
The resulting hydride was suspended in anhydrous Et2O (10
mL), and a solution of 2-methyl-3-butyne-2-ol (10 mL, 102.2
mmol, 1 equiv) in anhydrous Et2O (14 mL) was added
dropwise. The mixture was stirred for 10 min at 23 °C while
the precipitate redissolved. At -5 °C and under a N2 atmo-
i
5.82-5.65 (2H, m, CH Pr, NH), 5.20-5.10 (2H, m, CH2 iPr),
4.87 (1H, d, J ) 10.8 Hz, CH2 Tce), 4.74 (1H, d, J ) 10.8 Hz,

Solid-Phase Total Synthesis of Trunkamide A
Ta ble 1. 1H NMR (500 MHz, CDCl3) Da ta for 2
HR Hâ
J . Org. Chem., Vol. 66, No. 23, 2001 7573
residue
NH
5.90 (bs)
7.19 (d, J ) 7.0 Hz) 4.24-4.18 (m)
other
Ala
Ile
4.65 (qd, J ) 7.0, 7.0 Hz) 1.23 (3H, d, J ) 7.0 Hz)
2.22-2.16 (m)
1.42-1.34, 1.12-1.06 (2H, 2m, γCH2);
0.92 (3H, d, J ) 7.0 Hz, γCH3);
0.89 (3H, t, J ) 7.5 Hz, δCH3)
i
Ser(rPr) 7.98 (bs)
Thr(rPr) 6.78 (bs)
4.73 (ABX, J ) 8.0,
3.62-3.58 (1H, m);
3.42-3.37 (1H, m)
5.83-5.74 (1H, m, CH Pr);
8.0, 3.5 Hz)
5.17-5.09 (2H, m, CH2 rPr);
1.29 (6H, s, 2CH3 rPr)
4.36-4.44 (m)
4.35-4.32 (m)
5.83-5.74 (1H, m, CH rPr);
5.17-5.09 (2H, m, CH2 rPr);
1.29 (6H, s, 2CH3 rPr);
1.11 (3H, d, J ) 6.5 Hz, γCH3)
Ser
8.83 (bs)
5.66-5.60 (m)
5.08-5.00 (m)
4.18-4.14 (m)
3.99 (1H, dd, J ) 12.5, 4.0 Hz);
3.62-3.58 (1H, m)
D-Phe(S) 6.56 (bs)
Pro
3.54-3.46 (1H, m); 3.02 (1H, dd,
J ) 14.0, 9.0 Hz)
7.29-7.23 (5H, m, ar)
3.84-3.77 (1H, m, δCH2);
3.54-3.46 (1H, m, δCH2);
2.10-2.06, 2.02-1.96, 1.92-1.76
(4H, 3m, âCH2, γCH2)
CH2 Tce), 4.70-4.60 (1H, m, R-CH Ser), 4.50-4.35 (2H, m,
CH2 Fmoc), 4.32-4.25 (1H, m, CH Fmoc), 3.90 (1H, dd, J )
8.0, 2.8 Hz, â-CH2 Ser), 3.60 (1H, dd, J ) 8.0, 2.8 Hz, â-CH2
Ser), 1.26 (6H, s, 2 CH3 iPr); 13C NMR (50 MHz, CDCl3) δ 168.1
1261, 1092 cm-1; ES-MS calcd for C26H28O5NCl3 539.1, found
m/z 540.4 [M + H]+; HRMS (FAB) ES-MS calcd 540.1111,
found 540.1112; 1H NMR (250 MHz, CDCl3) δ 7.80-7.30 (8H,
i
m, Ar), 5.81-5.70 (1H, m, CH Pr), 5.66 (1H, d, J ) 9.5 Hz,
i
(CO Ser), 155.2 (CO Fmoc), 143.2 (C Ar), 142.6 (CH Pr), 141.1
NH), 5.2-5.12 (2H, m, CH2 iPr), 4.88 (1H, dd, J ) 12 Hz, CH2
Tce), 4.62 (1H, dd, J ) 12 Hz, CH2 Tce), 4.45-4.40 (3H, m,
R-CH Thr, CH2 Fmoc), 4.35-4.20 (2H, m, CH Fmoc, â-CH Thr),
1.30-1.27 (9H, m, γ-CH3 Thr, 2 CH3 iPr); 13C NMR (50 MHz,
CDCl3) δ 169.8 (CO Thr), 156.6 (CO Fmoc), 143.7 (C Ar), 143.5
(C Ar), 127.6 (CH Ar), 126.9 (CH Ar), 125.0 (CH Ar), 119.9
(CH Ar), 114.5 (CH2 iPr), 94.0 (CCl3 Tce), 75.6 (C Pr), 74.5
i
(CH2 Tce), 67.2 (CH2 Fmoc), 62.5 (â-CH2 Ser), 54.4 (R-CH Ser),
47.0 (CH Fmoc), 25.6 (CH3 iPr), 25.3 (CH3 iPr).
i
Fmoc-Ser(rPr)-OTce(7a ) (0.38 g, 0.72 mmol) was dissolved
in THF (6 mL), and Zn dust (1.56 g, 24 mmol, 33.1 equiv) and
1 M NH4OAc (1.35 mL, 1.3 mmol, 1.87 equiv) were added
under a N2 atmosphere. The reaction mixture was stirred for
14 h, filtered, and concentrated in vacuo. The crude material
was dissolved in EtOAc (10 mL), washed with 5% aqueous
KHSO4 (2 × 10 mL) and brine (2 × 10 mL), dried (MgSO4),
and concentrated in vacuo to give Fmoc-Ser(rPr)-OH (8a ) (0.28
g, 0.71 mmol, 40% overall yield for three steps): analytical
HPLC (tR 11 min, condition B); [R]D + 11 (c 0.009, CHCl3, 23
°C); IR (film) 2977, 1725, 1510, 1451, 1209 cm-1; CI-MS calcd
for C23H25O5N 395, found 396 [(M + H)+, 18], 413 [(M + NH4)+,
59]; HRMS (FAB) ES-MS calcd 396.1811, found 396.1814; 1H
NMR (200 MHz, CDCl3) δ 7.80-7.20 (8H, m, Ar), 5.80-5.60
(CH Pr), 141.3 (C Ar), 127.7 (CH Ar), 127.0 (CH Ar), 125.2
(CH Ar), 120.0 (CH Ar), 114.2 (CH2 iPr), 94.3 (CCl3 Tce), 76.0
(C iPr). 75.0 (CH2 Tce), 68.0 (â-CH Thr), 67.3 (CH2 Fmoc), 59.8
(R-CH Thr), 47.2 (CH Fmoc), 26.6 (CH3 iPr), 26.1 (CH3 iPr),
20.7 (γ-CH3 Thr).
According to the procedure used for the synthesis of Fmoc-
Ser(rPr)-OH (8a ), Fmoc-Thr(rPr)-OTce (7b) (0.34 g, 0.63 mmol)
provided Fmoc-Thr(rPr)-OH (8b) (0.24 g, 0.6 mmol, 35% overall
yield for three steps): analytical HPLC (tR 13.4 min, condition
B); ES-MS calcd for C24H27O5N 409.2, found m/z 410.7 [M +
H]+; HRMS (FAB) ES-MS calcd 410.1967, found 410.1974; [R]D
18.3° (c 0.009, CHCl3, 23 °C); IR (film) 2979, 1726, 1506, 1451,
1
1211 cm-1; H NMR (200 MHz, CDCl3) δ 7.80-7.20 (8H, m,
i
Ar), 5.90-5.70 (2H, m, CH Pr, NH), 5.20 (1H, d, J ) 10.2 Hz,
(2H, m, NH, CH Pr), 5.11 (1H, d, J ) 19.4 Hz, CH2 iPr), 5.00
CH2 iPr), 5.13 (1H, d, J ) 3.2 Hz, CH2 iPr), 4.40-4.10 (5H, m,
R-CH Thr, â-CH Thr, CH2 Fmoc, CH Fmoc), 1.31 (3H, d, J )
3.0 Hz, γ-CH3 Thr), 1.25 (6H, s, 2 CH3 iPr); 13C NMR (50 MHz,
CDCl3) δ 173.6 (CO Thr), 156.3 (CO Fmoc), 143.5 (C Ar), 142.4
i
(1H, d, J ) 11.6 Hz, CH2 iPr), 4.45-4.05 (4H, m, R-CH Ser,
CH2 Fmoc, CH Fmoc), 3.72 (1H, dd, J ) 9.4, 2.6 Hz, â-CH2
Ser), 3.48 (1H, dd, J ) 9.4, 2.6 Hz, â-CH2 Ser), 1.24 (6H, s, 2
CH3 iPr); 13C NMR (50 MHz, CDCl3) δ 174.0 (CO Ser), 156.0
i
(CH Pr), 141.1 (C Ar), 127.6 (CH Ar), 126.1 (CH Ar), 125.0
i
i
(CO Fmoc), 143.6 (C Ar), 142.7 (CH Pr), 141.1 (C Ar), 127.5
(CH Ar), 119.0 (CH Ar), 114.9 (CH2 iPr), 77.1 (C Pr), 67.7 (â-
(CH Ar), 126.9 (CH Ar), 125.0 (CH Ar), 119.8 (CH Ar), 114.4
CH Thr), 67.2 (CH2 Fmoc), 58.9 (R-CH Thr), 47.0 (CH Fmoc),
i
(CH2 iPr), 75.7 (C Pr), 67.2 (CH2 Fmoc), 62.6 (â-CH2 Ser), 54.3
26.6 (CH3 iPr), 25.6 (CH3 iPr), 19.1 (γ-CH3 Thr).
(R-CH Ser), 47.0 (CH Fmoc), 25.5 (2 CH3 iPr).
H-D-P h e(S)-Ser -Th r (r P r )-Ser (r P r )-Ile-Ala -P r o-OH (3).
Cl-TrtCl-resin (1 g, 1.6 mmol/g) was placed in a 20 mL
polypropylene syringe fitted with a polyethylene filter disk.
The resin was then washed with CH2Cl2 (5 × 1 min) and a
solution of Fmoc-Pro-OH (0.27 g, 0.8 mmol, 0.5 equiv) and
DIEA (0.28 mL, 1.6 mmol, 2 equiv) in CH2Cl2 (2.5 mL) was
added. The reaction mixture was stirred for 1 h. The reaction
was terminated by addition of MeOH (0.8 mL) and stirred for
a further 10 min. The Fmoc-Pro-O-TrtCl-resin (0.8 mmol) was
subjected to the following washings with CH2Cl2 (3 × 0.5 min),
DMF (3 × 0.5 min), piperidine-DMF (1:4, 2 × 1, 2 × 20 min),
DMF (5 × 1 min), 2-propanol (2 × 1 min), DMF (5 × 1 min),
MeOH (2 × 1 min), and CH2Cl2 (3 × 1 min) and then dried
under vacuum.
F m oc-Th r -OTce (5b). According to the procedure used for
the synthesis of Fmoc-Ser-OTce (5a ), Fmoc-Thr(tBu)-OH (1 g,
2.5 mmol) provided Fmoc-Thr-OTce (5b) (0.81 g, 1.7 mmol, 68%
overall yield for two steps): analytical HPLC (tR 14 min,
condition B); CI-MS calcd for C21H20O5NCl3 472, found 473 [(M
+ H)+, 76]; HRMS (FAB) ES-MS calcd 472.0485, found
472.0486; 1H NMR (200 MHz, CDCl3) δ 7.80-7.20 (8H, m, Ar),
5.80 (1H, d, J ) 8.8 Hz, NH), 4.90 (1H, dd, J ) 12 Hz, CH2
Tce), 4.70 (1H, dd, J ) 12 Hz, CH2 Tce), 4.57-4.40 (4H, m,
R-CH Thr, CH2 Fmoc, â-CH Thr), 4.25-4.18 (1H, m, CH Fmoc),
2.41 (1H, bs, OH), 1.27 (3H, d, J ) 7 Hz, γ-CH3 Thr); 13C NMR
(50 MHz, CDCl3) δ 169.4 (CO Thr), 156.7 (CO Fmoc), 143.4 (C
Ar), 141.0 (C Ar), 127.6 (CH Ar), 126.9 (CH Ar), 124.9 (CH
Ar), 119.8 (CH Ar), 94.3 (Cl3 Tce), 74.3 (CH2 Tce), 67.5 (â-CH
Thr), 67.2 (CH2 Fmoc), 59.2 (R-CH Thr), 46.9 (CH Fmoc), 20.0
(γ-CH3 Thr).
Fmoc-Ala-OH (1.24 g, 4 mmol, 5 equiv), Fmoc-Ile-OH (1.4
g, 4 mmol, 5 equiv), Fmoc-Ser(rPr)-OH (0.54 g, 1.4 mmol, 1.7
equiv), Fmoc-Thr(rPr)-OH (0.56 g, 1.4 mmol, 1.7 equiv), and
Fmoc-Ser-OH (1.05 g, 3.2 mmol, 4 equiv) were added sequen-
tially to the H-Pro-O-TrtCl-resin obtained above using DIPCDI
(0.62 mL, 4 mmol, 5 equiv, for Ala and Ile; 0.21 mL, 1.4 mmol,
1.7 equiv, for Ser(rPr) and Thr(rPr); 0.49 mL, 3.2 mmol, 4
equiv, for Ser) and HOBt (0.54 g, 4 mmol, 5 equiv, for Ala and
F m oc-Th r (r P r )-OH (8b). According to the procedure used
for the synthesis of Fmoc-Ser(rPr)-OTce (7a ), Fmoc-Thr-OTce
(5b) (0.81 g, 1.7 mmol) provided Fmoc-Thr(rPr)-OTce (7b) (0.34
g, 0.63 mmol): analytical HPLC (tR 21.5 min, condition B);
[R]D -6.9 (c 0.024, CHCl3, 23 °C); IR (film) 2929, 1732, 1507,

7574 J . Org. Chem., Vol. 66, No. 23, 2001
Ta ble 2. 1H NMR (600 MHz, CDCl3-DMSO-d 6 7:3) Da ta for 1b
HR Hâ
Caba et al.
residue
NH
other
Ala
Ile
7.29 (d, J ) 5.5 Hz) 4.32 (qd, J ) 7.4, 7.4 Hz) 1.09 (3H, d, J ) 8.4 Hz)
6.31 (d, J ) 9.9 Hz) 4.40 (dd, J ) 9.9, 3.3 Hz) 2.26-2.20 (m)
-
1.16, 1.04 (2H, 2m, γ-CH2);
0.88-0.82 (6H, m, γ-CH3, δ-CH3).
5.65 (1H, dd, J ) 17.6, 11.0 Hz,
Ser(rPr) 7.53 (d, J ) 8.1 Hz) 4.43 (ABX, J ) 8.1,
3.78 (1H, dd, J ) 10.5, 2.6 Hz);
i
8.1, 2.6 Hz)
3.38 (1H, dd, 10.5, 2.6 Hz)
CH Pr); 5.08 (1H, d, J ) 11.0 Hz,
CH2 iPr); 5.04 (1H, d, J ) 17.6 Hz,
CH2 iPr); 1.20 (6H, s, 2CH3 iPr)
5.83 (1H, dd, J ) 17.6, 11.0 Hz,
Thr(rPr) 7.82 (d,J ) 7.3 Hz) 4.55 (dd, J ) 7.3, 4.8 Hz) 3.85-3.80 (m)
i
CH Pr); 5.17 (1H, d, J ) 17.6 Hz,
CH2 iPr); 5.13 (1H, d, J ) 11.0 Hz,
CH2 iPr); 1.40 (3H, s, CH3 iPr); 1.30
(3H, s, CH3 iPr); 0.95 (3H, d,
J ) 8.1 Hz, γ-CH3)
Tzn
-
5.00-4.91 (m)
8.42 (d, J ) 8.8 Hz) 5.00-4.91 (m)
4.26 (t, J ) 9.4 Hz)
3.67 (1H, dd, J ) 12.3, 12.3 Hz);
3.54 (1H, dd, J ) 12.3, 12.3 Hz)
3.10 (1H, dd, J ) 16.6, 4.6 Hz);
2.72 (1H, dd, J ) 16.6, 12.5 Hz)
3.45-3.40 (1H, m, δ-CH2);
3.30-3.25 (1H, m, δ-CH2);
1.80-1.61 (3H, m, â-CH2, γ-CH2);
1.07 (1H, m, â-CH2)
D-Phe
Pro
7.20-7.10 (5H, m, Ar)
-
Ile; 0.18 g, 1.4 mmol, 1.7 equiv, for Ser(rPr) and Thr(rPr); 0.43
g, 3.2 mmol, 4 equiv, for Ser) in DMF (7 mL, for Ala, Ile and
Ser) or CH2Cl2 [4 mL, for Ser(rPr) and Thr(rPr)]. Finally, a
solution of Fmoc-D-Phe(S)-NBt (1.32 g, 2.4 mmol, 3 equiv) in
CH2Cl2 (7 mL) was added to the peptide resin. In all cases,
the ninhydrin test was negative after 90 min of coupling.
Removal of the Fmoc group and washings were carried out as
described in General Procedures.
The peptide was cleaved from the resin using HFIP-CH2-
Cl2 (1:4, 4 × 3 min). The combined filtrates were evaporated
to dryness under reduced pressure to give 0.34 g of the title
compound with a purity of >63% as determined by HPLC (tR
21.2 min, condition A): ES-MS calcd for C43H67N7O10S 873.5,
found m/z 874.6 [M + H]+.
(1 mL), and a solution of DAST (17 µL, 0.13 mmol, 1.1 equiv)
in CH2Cl2 (0.1 mL) was added dropwise at -20 °C under a N2
atmosphere. After 30 min, a further 1.1 equiv of DAST was
added under the same conditions and the reaction was stirred
for an additional 30 min. The solvent was removed by
evaporation under reduced pressure. The crude product was
purified by HPLC (Vydac C18 reversed-phase column, 15-20
µm, 250 × 10 mm), linear gradient from 55% to 70% of
acetonitrile in water in 30 min, 3 mL/min, detection at 220
nm, to give the title product (35 mg, 0.04 mmol, 33% yield):
analytical HPLC (tR 16.2 min, condition B); ES-MS calcd for
C
43H63N7O8S 837, found m/z 838 [M + H]+, 860 [M + Na]+;
HRMS (FAB) ES-MS calcd 838.4537, found 838.4556; 1H NMR
(600 MHz, CDCl3-DMSO-d6, 7:3) shown in Table 2.
Cyclo[D-P h e(S)-Ser -Th r (r P r )-Ser (r P r )-Ile-Ala -P r o] (2).
The crude linear peptide (0.34 g, 0.39 mmol) was dissolved in
CH2Cl2-DMF (9:1, 150 mL), and PyAOP (0.41 g, 0.78 mmol,
2 equiv) and DIEA (0.27 mL, 1.55 mmol, 4 equiv) were added.
The mixture was stirred for 1 h, and then the solvent was
removed by evaporation under reduced pressure. The crude
product was purified by flash chromatography (CHCl3-MeOH,
9.8:0.2) to give the title product (100 mg, 0.12 mmol, 15%
yield): analytical HPLC (tR 14.7 min, condition B); ES-MS
calcd for C43H65N7O9S 855.5, found 856.4 [M + H]+; HRMS
Ack n ow led gm en t. The excellent technical assis-
tance of Pablo Floriano (Pharma Mar, s.a.), Teresa
Ochoa (UB), and J avier Garc´ıa (UB) is especially
acknowledged. In addition to the support of Pharma
Mar, s.a., this work was partially supported by CICYT
(BIO99-484 and BQU2000-0235) and Generalitat de
Catalunya [Predoctoral fellowship to J .M.C., Grup Con-
solidat,and Centre de Refere`ncia en Biotecnologia].
1
(FAB) ES-MS calcd 856.4642, found 856.4637; H NMR (500
MHz, CDCl3) shown in Table 1.
Tr u n k a m id e A. Cyclo[D-P h e-Tzn -Th r (r P r )-Ser (r P r )-
Ile-Ala -P r o] (1b). The cyclic peptide was dissolved in CH2Cl2
J O015703T