Synthesis of alkyne-bridged cyclic tripeptides toward constrained mimics of vancomycin

Article abstract of DOI:10.1021/jo051933m

The synthesis of a range of highly constrained cyclic tripeptides has been performed using either an intramolecular Sonogashira coupling or a macrolactamization as the final ring-closing reaction. Our approach gives access to rigidified 15-membered peptid

Full text of DOI:10.1021/jo051933m

Synthesis of Alkyne-Bridged Cyclic Tripeptides toward Constrained
Mimics of Vancomycin
Hefziba T. ten Brink, Dirk T. S. Rijkers, and Rob M. J. Liskamp*
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht UniVersity,
P.O. Box 80082, 3508 TB Utrecht, The Netherlands
r.m.j.liskamp@pharm.uu.nl
ReceiVed September 15, 2005
The synthesis of a range of highly constrained cyclic tripeptides has been performed using either an
intramolecular Sonogashira coupling or a macrolactamization as the final ring-closing reaction. Our
approach gives access to rigidified 15-membered peptidic macrocycles based on the central ring system
of vancomycin. Tripeptides 3a-c and dipeptide 11 were cyclized via an intramolecular Sonogashira
reaction, and the cyclic peptides 4a-c and 15a were obtained in 6-23% yield. In contrast,
macrolactamization of 12 and 17 resulted in the desired peptidic macrocycles 15b and 18 with 54-61%
yield. Modeling studies hint at a distorted triple bond, which explains the low yield of the Sonogashira-
based cyclization. Moreover, modeling data also showed that this class of peptidic macrocycles formed
a cavity-like structure in which guest molecules may bind.
Introduction
There is a growing interest in the development of novel
synthetic methodology for conformational restriction of peptides
to mimic the bioactive conformation as closely as possible. Ring-
closing metathesis (RCM) has often been used for this purpose
since it displays an extraordinary functional group tolerance and
high yield of cyclization.6 RCM has been used in our group
previously7,8 and more recently to synthesize pentapeptides to
mimic the central ring system of vancomycin.9 Some other C-C
coupling strategies for constraining peptides from the literature
include, e.g., a Suzuki coupling between two peptide fragments
followed by macrolactamization10,11 and peptide cyclizations
featuring a Heck,12,13 Suzuki,14 Stille,15 or Sonogashira12b,16
reaction.
Incorporation of covalent constraints into bioactive peptides
is an important design consideration to reduce the unfavorable
entropy loss upon receptor-binding.1 The resulting reduction of
conformational flexibility is important to increase the affinity
of the peptide for its natural receptor.2,3 In addition, covalent
constraints also play a decisive role in controlling the three-
dimensional structure of a molecule, e.g., forming cavity or
shell-like structures which are capable of binding other (small)
molecules. In Nature many examples of covalent constraints
are known, e.g., disulfide bridges, thioether bridges, lactone and
lactam bridges, or biaryl ether bridges. An outstanding example
of a compound having the latter constraint is the peptide
antibiotic vancomycin (Figure 1).4,5
(4) (a) Nicolaou, K. C.; Mitchell, H. J.; Jain, N. F.; Winssinger, N.;
Hughes, R.; Bando, T. Angew. Chem., Int. Ed. 1999, 38, 240. (b)
Blankenstein, J.; Zhu, J. Eur. J. Org. Chem. 2005, 1949.
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Chang, S. Tetrahedron 1998, 54, 4413. (c) Armstrong, S. K. J. Chem. Soc.,
Perkin Trans 1 1998, 371. (d) Fu¨rstner, A. Angew. Chem., Int. Ed. 2000,
39, 3012. (e) Trinka, T. M.; Grubbs, R. H. Acc. Chem. Res. 2001, 34, 18.
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10.1021/jo051933m CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/13/2006
J. Org. Chem. 2006, 71, 1817-1824
1817

ten Brink et al.
FIGURE 1. Target macrocyclic peptides inspired by the vancomycin DE ring system.
Here we describe the synthesis of highly constrained tripep-
tides in which an alkyne moiety is used as a cyclic constraint
(Figure 1). To accomplish this goal, two approaches of tripeptide
cyclization have been developed. Our first approach comprises
an intramolecular Sonogashira reaction as the cyclization step.
As an alternative, an intermolecular Sonogashira coupling which
is followed by an intramolecular amide bond formation has been
developed.
Similar to the ring-closing metathesis reaction, the Sono-
gashira coupling reaction displays also a broad functional group
tolerance.17-22 However, in contrast to an RCM-based cycliza-
tion, the Sonogashira coupling enables the stereocontrol of the
newly formed cyclic system, since a triple bond can be
selectively reduced to either the E- or Z-isomer.23-25 Addition-
ally, partial or complete reduction of the triple bond gradually
releases the ring strain of the cyclic peptide. Such conforma-
tionally relaxed structures can be used to probe conformation-
bioactivity relationships.
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Liskamp, R. M. J. Angew. Chem., Int. Ed. 1999, 38, 3684. (b) Reichwein,
J. F.; Liskamp, R. M. J. Eur. J. Org. Chem. 2000, 2335. (c) Reichwein, J.
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Dekker, F. J.; de Mol, N. J.; Fischer, M. J. E.; Kemmink, J.; Liskamp, R.
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J. Bioorg. Med. Chem. 2005, 13, 4221. (g) Ghalit, N.; Rijkers, D. T. S.;
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Results and Discussion
The retrosynthetic analysis of the cyclic tripeptides is depicted
in Scheme 1. Three different ways of cyclization are possible:
a linear tripeptide is cyclized via a Sonogashira coupling in
which a new C-C bond will be formed (route I), or two amide
bond formation reactions in which either a benzylic amine (route
IIa) or an aliphatic amine (route IIb) forms the point of
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4467. (b) Sonogashira, K. J. Organomet. Chem. 2002, 653, 46.
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Chem. 2003, 68, 1843.
1818 J. Org. Chem., Vol. 71, No. 5, 2006

Synthesis of Alkyne-Bridged Cyclic Tripeptides
SCHEME 1. Retrosynthetic Analysis of the Peptide Macrocycles
cyclization. First, route I was explored since it did not have
special requirements with respect to the choice of protecting
groups (vide infra).
Optimization of the ring-closing conditions leading to 4a-c
is shown in Table 1. The first Sonogashira reactions (entries
1-3) were carried out in 1,1,2-trichloroethane (TCE) as solvent
with 10 mol % Pd(Ph3P)4/CuI as the catalytic system in the
presence of triethylamine (5 equiv) as a base at room temper-
ature. According to TLC, the linear precursors were absent after
16 h. However, the cyclic products 4a-c were obtained in low
yields ranging from 6% (4a) to 23% for 4b. Moreover, a linear
diyne, as a byproduct, with general structure 5 (Figure 2) could
also be isolated (5b, 24%; 5c, 20%).29 When the Sonogashira
ring closure was carried out using a more diluted reaction
mixture, a higher product yield was not obtained, but indeed a
decreased amount of diyne 5c was formed (entry 4).
The syntheses started with the conversion of 4-hydroxyphen-
ylglycine into the corresponding Boc-protected amino acid
methyl ester using standard protection protocols26 (88% over
two steps). Then, iodonation was performed analogously to the
method of Nishiyama et al.27 with N-iodosuccinimide in acetone
in 80% yield. It was found necessary to methylate the hydroxy
group using MeI in the presence of K2CO3 to yield diiodo
compound 1. Not only did this decrease the light sensitivity of
1 but, more importantly, the electron-donating character of the
methoxy functionality increased the coupling yield of the
Sonogashira reaction (data not shown). The basic conditions of
this reaction did not influence the chiral integrity of the
phenylglycine residue, since the optical rotation of Boc-3,5-
diiodo-4-hydroxyphenylglycine methyl ester was not affected
by treatment with K2CO3 in acetone.
Our experiences with Sonogashira-based dendrimer synthe-
ses30 made us decide to change the solvent from TCE to CH3-
CN and to run the cyclization reaction at a concentration of 10
mM (entries 5 and 6). Unfortunately, the yields of 4b,c did not
improve, but the precursors could be recovered by column
chromatography.
After removal of the Boc group by treatment with hydro-
chloric acid in diethyl ether, the corresponding hydrochloride
of 1 was coupled with BOP/HOBt/DIPEA to Boc-Gly-OH, Boc-
D-Ala-OH, and Boc-D-Leu-OH, respectively, and dipeptides
2a-c were obtained in good overall yields (Scheme 2). Next,
the precursors for the Sonogashira cyclization, 3a-c, were
obtained by coupling of the N-terminally deprotected dipeptides
2a-c with BOP to alkyne 7a. The latter compound was
synthesized from Boc-Ser-OH/NaH and propargyl bromide
analogously to the method of Sugano and Miyoshi28a (Scheme
3).
Longer reaction times (16 f 36 h), increasing the amount of
catalyst (10 f 50 mol %), running the cyclization at a higher
dilution (10 f 0.5 mM), and the application of a different Pd
(28) (a) Sugano, H.; Miyoshi, M. J. Org. Chem. 1976, 41, 2352. (b)
Glenn, M. P.; Pattenden, L. K.; Reid, R. C.; Tyssen, D. P.; Tyndall, J. D.
A.; Birch, C. J.; Fairlie, D. P. J. Med. Chem. 2002, 45, 371.
(29) This product is formed via a Glaser-type oxidative dimerization of
the alkyne; see: (a) Siemsen, P.; Livingston, R. C.; Diederich, F. Angew.
Chem., Int. Ed. 2000, 39, 2632. (b) Batsanov, A. S.; Collings, J. C.; Fairlamb,
I. J. S.; Holland, J. P.; Howard, J. A. K.; Lin, Z.; Marder, T. B.; Parsons,
A. C.; Ward, R. M.; Zhu, J. J. Org. Chem. 2005, 70, 703. (c) Li, J.-H.;
Liang, Y.; Xie, Y.-X. J. Org. Chem. 2005, 70, 4393.
(26) Greene, T. W.; Wuts, P. G. M. ProtectiVe Groups in Organic
Synthesis; Wiley-Interscience: New York, 1999.
(27) Nishiyama, S. K.; Moon, H.; Yamamura, S. Tetrahedron Lett. 1994,
35, 8397.
(30) (a) Vrasidas, I.; Kemmink, J.; Liskamp, R. M. J.; Pieters, R. J. Org.
Lett. 2002, 4, 1807. (b) Vrasidas, I.; Andre´, S.; Valentini, P.; Bo¨ck, C.;
Lensch, M.; Kaltner, H.; Liskamp, R. M. J.; Gabius, H.-J.; Pieters, R. J.
Org. Biomol. Chem. 2003, 1, 803.
J. Org. Chem, Vol. 71, No. 5, 2006 1819

ten Brink et al.
SCHEME 2. Synthesis of the Peptide Macrocycles via the Sonogashira Ring Closure
FIGURE 2. Byproducts obtained in the Sonogashira ring-closure reaction.
SCHEME 3. Synthesis of the Serine(O-propynyl) Derivatives
source (Pd(Ph3P)2Cl2) were also not successful for increasing
the yields of the Sonogashira ring-closing reaction.
also reported that THF prevented diyne formation.32 Therefore,
it was decided to use THF and the first attempts also showed
that triethylamine as a base had to be replaced by diethylamine
to obtain any cyclic product (entries 7 and 8). Under these
conditions 4b was obtained in 27% yield. However, formation
of byproducts could not be prevented, as was apparent from
isolation of cyclic dimer 6 in 26% yield.29a
In the literature several high-yielding Sonogashira reactions
have been described with THF as solvent.31,32 Moreover, it was
(31) Miller, M. W.; Johnson, C. R. J. Org. Chem. 1997, 62, 1583.
(32) Thorand, S.; Krause, N. J. Org. Chem. 1998, 63, 8551.
1820 J. Org. Chem., Vol. 71, No. 5, 2006

Synthesis of Alkyne-Bridged Cyclic Tripeptides
SCHEME 4. Synthesis of Macrocycle 15
TABLE 1. Conditions for the Sonogashira Cyclization Reactiona
a cyclic dimer similar to 16 (Figure 4)shaving Boc instead of
Cbz groupsswas also present as was evidenced by ESI-MS.
Thus, not unexpectedly, the presence of the second iodine
moiety was not responsible for the low yields of the Sono-
gashira-based macrocyclizations. More likely, the linear geom-
etry of the triple bond hampers ring-closing. Indeed, preliminary
modeling studies using MacroModel33 of 4b showed a distorted
triple bond (Figure 3) in the resulting peptide macrocycle. The
bond angle of the triple bond was ca. 170°, thus deviating
considerably from linearity. As it turned out, such a strained
15-membered peptidic macrocycle was therefore difficult to
synthesize, and led to formation of nonstrained byproducts 5
and 6 (Figure 2).
concn,
product
(yield, %)
byproductb
(yield, %)
entry precursor mM solvent base
1
2
3
4
5
6
7
8
3a
3b
3c
3c
3b
3c
3a
3b
10
15
15
10
10
10
10
10
TCE
TCE
TCE
TCE
MeCN Et3N
MeCN Et3N
Et3N
Et3N
Et3N
Et3N
4a (6) [3a (2)]c
4b (23)
4c (14)
5b (24)
5c (20)
5c (8)
4c (14)
4b (20) [3b (73)]c
4c (12) [3c (32)]c
5c (17)
THF
THF
Et2NH 4a (13) [3a (30)]c
Et2NH 4b (27)
6 (26)
a Reaction time 16 h, 10 mol % Pd(Ph3P)4/10 mol % CuI employed as
the catalyst, 5 equiv of base used. b Byproducts were isolated by column
chromatography and characterized by mass spectrometry and 1H NMR.
c Percent of recovered starting material.
The conformational search for obtaining the global minimum
of 4b was carried out in chloroform, which was the solvent for
recording its NMR spectrum. The observed ring-coupling
constants and those calculated from the global minimum were
in excellent agreement (Figure 3). This indicates that the
conformation of this global minimum reflects the structure in
solution.
As was shown in Scheme 1, in addition to approach I, two
alternative approaches (IIa and IIb) are possible for the synthesis
of the desired 15-membered peptidic macrocycles. Both routes
feature an intermolecular Sonogashira reaction followed by a
macrolactamization, with either a benzylic amine (approach IIa)
or the peptide amine (approach IIb). Despite the outcome of
modeling studies (vide supra) showing that a strained peptide
macrocycle is obtained, the advantage of this approach might
be that the Sonogashira reaction is carried out first, followed
by a macrocyclization using a peptide coupling. Peptide
couplings are among the most widely studied reactions for which
many coupling reagents and conditions are available. The
precursor molecules were designed in such a way that both
protecting groups on the amine moiety (Boc) and on the
carboxylic acid moiety (tBu) were removed simultaneously by
a single acid treatment. The amino functionality that must be
excluded from the reaction was protected by a Cbz group
The configuration of the chiral centers in the linear precursors
was chosen as L, D, L. It is known that a D-amino acid is capable
of inducing a bend in the peptide sequence. Our results showed
(Table 1, entries 2, 5, and 8) that the best cyclization yield was
achieved in the presence of the smallest D-amino acid, alanine
(3b). Increasing steric bulk (leucine, 3c) or the absence of a
turn-inducing motif (glycine, 3a) resulted in a lower cyclization
yield.
A possible explanation of the low yield could also be the
presence of the second iodine moiety, either electronically or
sterically. Therefore, it was decided to synthesize dipeptide 11
(Scheme 4) to test this hypothesis. For this purpose, 3-iodo-
benzylamine was coupled to Boc-D-Ala-OH with BOP to give
3-iodobenzylamide 10, and after removal of the Boc group by
treatment with hydrochloric acid, 7a was coupled to the
corresponding hydrochloride with BOP/DIPEA to afford linear
precursor 11 in 66% overall yield. The Sonogashira-based
cyclization reaction was performed with the best reaction
conditions as was found in previous experiments (entry 8, Table
1). After a reaction time of 16 h, the linear precursor was absent
on TLC and two new spots were found and isolated. It turned
out that cyclic product 15a was formed in only 14% yield while
(33) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput.
Chem. 1990, 11, 440.
(34) Armstrong, A.; Brackenridge, I.; Jackson, R. F. W.; Kirk, J. M.
Tetrahedron Lett. 1988, 29, 2483.
J. Org. Chem, Vol. 71, No. 5, 2006 1821

ten Brink et al.
FIGURE 3. Representation of the lowest energy conformation of macrocycle 4b and the observed and calculated (in parentheses) coupling constants.
improvement with respect to the Sonogashira-induced macro-
cyclization (14%), and 16 was not isolated.
Indeed, an intermolecular Sonogashira coupling followed by
a macrolactamization via the peptide amine was the best
approach for the synthesis of alkyne-bridged cyclic tripeptides.
These cyclic tripeptides represent mimics of the right-half
macrocycle of vancomycin (Figure 1). To ultimately realize the
synthesis of a bicyclic system, a double Sonogashira reaction
is needed. For initial approaches toward the bicyclic multiple
side chain knotted framework, macrocycle 18 was synthesized
(Scheme 5), which is a versatile starting compound to construct
the left macrocycle mimic of vancomycin (Figure 5).
To this end, diiodo compound 2b was coupled to alkyne 8
and linear precursor 17 was obtained in an isolated yield of
34%. It should be mentioned that the stoichiometry of the
catalyst, the alkyne derivative, and the diiodo compound is an
important factor to avoid diaddition of the alkyne and was found
to be catalyst:8:2b ) 0.1:1.5:1. After removal of both protecting
groups, cyclization was performed with the same reaction
conditions as described for 15, and cyclic tripeptide 18 was
obtained in 61% yield. Unexpectedly, purification of tripeptide
18 was rather difficult since it was poorly soluble in most
organic solvents.
In conclusion, we designed two synthetic approaches for the
preparation of alkyne-bridged cyclic tripeptides. It was found
that an intramolecular Sonogashira reaction followed by a
macrolactamization gave the best results to obtain these highly
constrained 15-membered peptidic macrocycles. In this way a
mimic of the right half of the vancomycin cavity was obtained.
Incorporation of a triple bond opens an avenue to a diversity of
subsequent compounds accessible by, for example, click chem-
istry, selective reduction, oxidation, etc. In addition, the use of
diiodotyrosine (cf. the preparation of 18) may provide an entry
into a constrained mimic of vancomycin’s carboxylate-binding
pocket, which is under investigation.
FIGURE 4. Cyclic dimer 16 as a possible byproduct of macrolac-
tamization of 12.
TABLE 2. Conditions for the Macrolactamizationa
concn,
mM
15b
yield, %
cyclic dimer
Entry
16b yield, %
1
2
3
4
5
10
5
1
0.5
0.3
trace
8
44
54
10
95
79
50
not detected
not detected
a Reaction time 16 h, 1 equiv of HATU/HOAt, 2 equiv of DIPEA in
DMF at rt. b Cyclic dimer 16 was characterized by mass spectrometry only.
(Scheme 4). Cbz-Ser-OH was converted into Cbz-Ser(O-
propynyl)-OH (7b) analogously to 7a (Scheme 3). The tert-
butyl ester was introduced using tert-butyl 2,2,2-trichloro-
acetamidate/BF3‚etherate34 in tert-butyl alcohol, since POCl3/
tert-butyl alcohol35 gave unsatisfactory yields, and alkyne 8 was
obtained in 90% yield.
First, approach IIa was attempted for the synthesis of
macrocycle 15b depicted in Scheme 4. For this purpose Boc-
protected 3-iodobenzylamine (13) was coupled to dipeptide 9
(Scheme 3) via an intermolecular Sonogashira reaction. THF
was replaced by DCM since iodo compound 13 did not dissolve
in THF. Unfortunately, the coupling was very sluggish, and a
yield of only 6% was obtained. Formation of coupling product
14 could only be confirmed by mass spectrometry.
This route was abandoned in favor of approach IIb, and iodo
compound 10 was coupled with alkyne 8 to give linear dipeptide
12 (80%). Subsequently, dipeptide 12 was treated with acid to
remove the Boc/tBu protecting groups and subjected to an
HATU/HOAt-based macrolactamization at different concentra-
tions (Table 2). The cyclic dimer 16 (Figure 4) was observed
using a concentration of the deprotected starting material 12 of
as low as 1 mM. Gratifyingly, at a concentration of 0.5 mM in
DMF, 15b was obtained in 54% yield, which was a significant
Experimental Section
(S)-N-(tert-Butyloxycarbonyl)-3,5-diiodo-4-methoxyphenyl-
glycine Methyl Ester (1). This compound was synthesized on the
basis of a procedure described in the literature.27 (S)-N-(tert-
Butyloxycarbonyl)-4-methoxyphenylglycine methyl ester (10.6 g,
35.8 mmol) was dissolved in acetone (400 mL). The solution was
cooled to -79 °C, and the reaction mixture was protected from
light by aluminum foil. To this mixture was added a solution of
N-iodosuccinimide (17.7 g, 78.8 mmol) in acetone (100 mL)
dropwise over 5 h at -79 °C. After the mixture was stirred
overnight at rt, the reaction was complete according to TLC and
(35) Taschner, E.; Biernat, J. F.; Rzeszotarska, B.; Wasielewski, C.
Liebigs Ann. Chem. 1961, 646, 123.
1822 J. Org. Chem., Vol. 71, No. 5, 2006

Synthesis of Alkyne-Bridged Cyclic Tripeptides
SCHEME 5. Synthesis of Macrocycle 18
FIGURE 5. Future extension of macrocycle 18 to a bicyclic side chain knotted pentapeptide representing a constrained mimic of vancomycin’s
carboxylate-binding pocket.
the solvent was evaporated in vacuo. The residue was dissolved in
EtOAc, washed with a saturated solution of Na2S2O3, H2O, and
brine, and dried (Na2SO4). The solvent was removed under reduced
pressure, and diiodo compound 1 was obtained as a white solid
after purification by column chromatography (EtOAc/hexane, 1:4,
v/v) in 80% yield (15.7 g): Rf(EtOAc/hexane, 1:4, v/v) 0.50; mp
167 °C; [R]20D +88.5 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3)
δ 7.68 (s, 2H), 5.77 (d, 1H, J ) 6.3 Hz), 5.16 (d, 1H, J ) 6.6 Hz),
3.75 (s, 3H), 3.67 (s, 3H), 1.35 (s, 9H); 13C NMR (75.5 MHz,
CDCl3) δ 28.1, 53.0, 55.4, 60.5, 80.3, 90.7, 136.5, 138.2, 154.4,
158.6, 170.4; HRMS (TOF ES+) m/z 569.9236 [M + Na]+, calcd
for C15H19I2NO5Na+ 569.9250. Anal. Calcd for C15H19I2NO5: C,
32.93; H, 3.50; N, 2.56. Found: C, 33.07; H, 3.46, N, 2.47.
N-(tert-Butyloxycarbonyl)-glycyl-(S)-3,5-diiodo-4-methoxy-
phenylglycine Methyl Ester (2a). To remove the Boc group,
methyl ester 1 (1.20 g, 2.2 mmol) was dissolved in DCM (10 mL),
then 6 N HCl/diethyl ether (30 mL) was added, and the mixture
was stirred overnight at rt. The formed precipitate was isolated by
filtration, washed with diethyl ether, and subsequently dissolved
in DMF (1 mmol in 25 mL). To this solution were added Boc-
Gly-OH (424 mg, 1.1 equiv) and BOP (1.07 g, 1.1 equiv) followed
by DIPEA (1.17 mL, 3 equiv). After 2 h the reaction was complete,
and the solvent was evaporated in vacuo. The residue was dissolved
in EtOAc, and the organic layer was successively washed with 1
N KHSO4, saturated NaHCO3 and brine, dried (Na2SO4), and
evaporated to dryness under reduced pressure. Dipeptide 2a was
obtained as a white solid in 92% yield (1.22 g) after purification
by column chromatography (EtOAc/hexane, 1:2, v/v f EtOAc/
hexane, 2:1, v/v): Rf(EtOAc/hexane, 2:1, v/v) 0.54; HPLC showed
63 °C; [R]D +58.3 (c 0.5, CHCl3); 1H NMR (300 MHz, CDCl3,) δ
7.75 (s, 2H), 7.59 (d, 1H, J ) 6.3 Hz), 7.16 (br s, 1H), 5.53 (m,
2H), 4.38 (m, 1H), 4.19-4.00 (m, 5H), 3.83 (s, 3H), 3.72 (m, 4H),
2.47 (m, 1H), 1.45 (s, 9H); 13C NMR (75.5 MHz, CDCl3) δ 28.2,
43.1, 53.2, 54.3, 58.3, 60.6, 69.5, 75.5, 78.9, 80.6, 90.8, 135.7,
138.5, 155.5, 158.9, 168.1, 170.0, 170.7; ESI-MS m/z calcd for
C23H29N3O8 729, found [M + H]+ 730, [M + Na]+ 752, [(M -
C4H8) + H]+ 674, [(M - C5H8O2) + H]+ 630, [2M + H]+ 1459,
[2M + Na]+ 1480; HRMS (TOF ES+) m/z 751.9902 [M + Na]+,
calcd for C23H29I2N3O8Na+ 751.9942. Anal. Calcd for C23H29-
I2N3O8: C, 37.88; H, 4.01; N, 5.76. Found: C, 37.76; H, 4.11, N,
5.70.
Cyclic Tripeptide 4a. General Procedure for the Sonogashira
Cyclization Reaction. In a flame-dried nitrogen-filled flask tri-
peptide 3a (93 mg, 0.13 mmol, 1 equiv) was dissolved in a dry
solvent (purged with dry nitrogen gas for 25 min prior to use) to
obtain a final concentration of 0.5-15 mM. The exact reaction
conditions are given in Table 1. The palladium catalyst (15 mg,
0.1 equiv) was added followed by CuI (2.5 mg, 0.1 equiv) and
finally the base (5 equiv). The resulting reaction mixture was stirred
for 16 h. After this period of stirring, the solvent was removed
under reduced pressure, and the residue was subsequently purified
by column chromatography or preparative TLC. Cyclic tripeptide
4a (10 mg, 13%) was obtained as a white solid: Rf(EtOAc/hexane,
2:1, v/v) 0.46; mp 223 °C; [R]20 -24.0 (c 0.1 CHCl3); H NMR
1
D
(500 MHz, CDCl3) δ 7.81 (m, 1H), 7.49 (br s, 1H), 7.31 (s, 1H),
7.23 (s, 1H), 5.55 (d, 1H, J ) 6.3 Hz), 5.43 (br s, 1H), 4.54 and
4.48 (m, 1H), 4.18 and 4.12 (m, 1H), 4.41 (br s, 1H), 4.28-4.22
(m, 1H), 3.97-3.74 (m, 2H), 3.96 (s, 3H), 3.84 (s, 3H), 3.61-
3.33 (m, 1H), 1.46 (s, 9H); 13C NMR (75.5 MHz, CDCl3) δ 28.1,
45.5, 53.3, 54.2, 54.8, 59.2, 61.0, 66.8, 80.1, 85.3, 93.5, 131.3,
134.4, 138.2, 157.3, 168.3, 171.0, 173.8 (not all carbons were
detectable with HMBC/HSQC); ESI-MS m/z calcd for C23H28IN3O8
601, found [M + Na]+ 624, [M + MeCN + Na]+ 665, [(M -
C5H8O2) + H]+ 502; HRMS (TOF ES+) m/z 624.0839 [M + Na]+,
calcd for C23H28IN3O8Na+ 624.0819.
Linear Tripeptide 12. In a flame-dried nitrogen-filled flask
compound 10 (188 mg, 0.46 mmol) was dissolved in THF (15 mL),
which was purged with dry nitrogen gas. First Pd(PPh3)4 (53 mg,
0.046 mmol) was added followed by CuI (9 mg, 0.046 mmol), Et2-
NH (320 µL, 2.3 mmol), and finally alkyne 8 (202 mg, 0.70 mmol).
The reaction mixture was stirred for 16 h. Subsequently, the solvent
was removed under reduced pressure, and the residue was purified
that the product was more than 99% pure detected by UV and
1
ELSD; mp 72 °C; [R]20 +44.9 (c 0.25, CHCl3); H NMR (300
D
MHz, CDCl3) δ 7.76 (s, 2H), 7.58 (br s, 1H) 5.49 (d, 2H, J ) 6.9
Hz), 3.86 (m, 5H), 3.76 (s, 3H), 1.45 (s, 9H); 13C NMR (75.5 MHz,
CDCl3) δ 28.2, 44.1, 53.2, 54.2, 60.5, 80.3, 90.8, 135.8, 138.4,
156.9, 158.9, 169.2, 170.1; ESI-MS m/z calcd for C17H22I2N2O6
604, found [M + H]+ 605, [M + Na]+ 627, [M + Na + MeCN]+
668, [(M - C4H8) + H]+ 549, [(M - C5H8O2) + H]+ 505, [2M +
Na]+ 1230; HRMS (TOF ES+) m/z 626.9434 [M + Na]+, calcd
for C17H22I2N2O6Na+ 626.9465.
N-(tert-Butyloxycarbonyl)seryl-(O-propynyl)-glycyl-(S)-3,5-di-
iodo-4-methoxyphenylglycine Methyl Ester (3a). Tripeptide 3a
(1.94 mmol) was synthesized analogously to 2a and obtained as a
white solid (1.30 g, 92%): Rf(EtOAc/hexane, 2:1, v/v) 0.53; mp
J. Org. Chem, Vol. 71, No. 5, 2006 1823

ten Brink et al.
by column chromatography. Tripeptide 12 was obtained as a
colorless oil in 80% yield (225 mg) after purification by column
chromatography (EtOAc/hexane, 1:2, v/v f EtOAc/hexane, 1:1,
v/v): Rf(EtOAc/hexane, 1:1, v/v) 0.38; [R]20D +11.8 (c 1.9 CHCl3);
1H NMR (300 MHz, CDCl3) δ 7.34-7.20 (m, 9H), 6.83 (br s, 1H),
5.68 (d, 1H, J ) 6.3 Hz), 5.20 (m, 3H), 4.45-4.34 (m, 5H), 4.21
(br s, 1H), 4.04 (m, 1H), 3.78 (m, 1H), 1.46-1.36 (m, 21H); 13C
NMR (75.5 MHz, CDCl3) δ 18.2, 27.8, 28.1, 42.6, 50.0, 54.6, 59.0,
66.7, 69.7, 79.9, 82.2, 86.35, 122.5, 127.6, 127.8, 127.9, 128.3,
128.4, 128.5, 130.5, 136.1, 138.5, 155.5, 155.6, 169.0, 172.8; ESI-
MS m/z calcd for C33H43N3O8 609, found [M + Na]+ 632, [(M -
C4H8) + Na]+ 576; HRMS (TOF ES+) m/z 632.3097 [M + Na]+,
calcd for C33H43N3O8Na+ 632.2948.
Cyclic Tripeptide 15b. Linear tripeptide 12 (70 mg, 0.11 mmol)
was dissolved in DCM (3 mL). TFA (3 mL) was added, and the
mixture was stirred overnight at rt. The reaction mixture was
evaporated in vacuo, and the residue was coevaporated with DCM
(twice) to remove any residual TFA. Subsequently, the residue was
dissolved in DMF (220 mL); HOAt (17 mg, 0.12 mmol) and HATU
(48 mg, 0.13 mmol) followed by DIPEA (50 µL, 0.32 mmol) were
added, and the reaction mixture was stirred overnight. Then the
solvent was removed by evaporation in vacuo, and the residue was
triturated with EtOAc. The cyclic dimer was removed by filtration,
and the EtOAc layer was washed with 1 N KHSO4, saturated
NaHCO3, and brine, dried (Na2SO4), and evaporated under reduced
pressure. Compound 15b was obtained as a white solid in 54%
yield (23 mg) after purification by column chromatography (MeOH/
residue was purified by column chromatography. Compound 17
was obtained as a colorless oil in 34% yield (90 mg) after
purification by column chromatography (EtOAc/hexane, 1:4, v/v
f EtOAc/hexane, 1:1, v/v): Rf(EtOAc/hexane, 1:1, v/v) 0.3; HPLC
showed that the product was 97% pure detected by ELSD; 1H NMR
(300 MHz, CDCl3) δ 7.73 (s, 1H), 7.47 (br s, 1H), 7.27 (m, 6H),
5.64 (m, 1H), 5.36 (m, 1H), 5.44 (m, 3H), 54.40 (m, 3H), 4.25 (br
s, 1H), 3.94 (m, 1H), 3.88 (s, 3H), 3.84-3.80 (m, 1H), 3.73 (s,
3H), 1.45-1.33 (m, 21H); 13C NMR (75.5 MHz, CDCl3) δ 17.6,
27.9, 28.2, 53.0, 54.6, 59.2, 60.9, 66.8, 70.1, 81.8, 80.3, 82.0, 82.4,
90.0, 116.8, 128.0, 128.4, 132.6, 132.8, 133.8, 136.2, 138.2, 155.9,
160.5, 168.9, 170.2, 172.1; ESI-MS m/z calcd for C36H46N3O11 823,
found [M + Na]+ 846, [(M - C4H8) + Na]+ 724, [(M - C4H8) +
H]+ 668.
Cyclic Tripeptide 18. Cyclization of linear tripeptide 17 into
compound 18 was carried out as described for compound 15b.
Cyclic tripeptide 18 was obtained as a white solid in 36% yield
(31 mg): 1H NMR (300 MHz, CDCl3) δ 7.80 (s, 1H), 7.47 (d, 1H,
J ) 6 Hz), 7.36-7.31 (m, 6H), 6.72 (d, 1H, J ) 6 Hz), 5.74 (d,
1H, J ) 9 Hz), 5.51 (d, 1H, J ) 9 Hz), 5.15 (s, 2H), 4.53-4.40
(m, 2H), 4.21-4.17 (m, 1H), 4.46 (m, 1H), 4.12 (m, 1H), 3.93 (s,
3H), 3.88 (m, 1H), 3.83 (s, 3H), 1.34 (m, 3H); 13C NMR (75.5
MHz, CDCl3) δ 16.6, 49.8, 52.3, 54.3, 59.2, 61.0, 67.5, 85.2, 91.5,
92.3, 115.0, 128.2, 128.4, 128.6, 131.8, 133.6, 137.9, 157.7, 169.1,
170.8, 171.3; ESI-MS m/z calcd for C27H28IN3O8 649, found [M
+ H]+ 643, [M + Na]+ 672. Anal. Calcd for C27H28IN3O8: C,
49.93; H, 4.35; N, 6.47. Found: C, 49.86; H, 4.43; N, 6.38.
DCM, 95:5, v/v) and preparative TLC: Rf(MeOH/DCM, 95:5, v/v)
1
0.64; mp 122 °C; [R]20 -223.5 (c 0.3, CHCl3); H NMR (300
D
Acknowledgment. We thank Dr. Johan Kemmink for his
assistance in measuring the 500 MHz NMR spectra, and Cees
Versluis is acknowledged for performing the HRMS experi-
ments. We thank Hans Hilbers for his support with the modeling
experiments. These investigations were supported by the Council
for Chemical Sciences of The Netherlands Organization for
Scientific Research (CW-NWO).
MHz, CDCl3) δ 7.90 (br s, 1H), 7.46-6.82 (m, 10H), 6.36 (br s,
1H), 5.18-5.08 (m, 2H), 4.76 (m, 1H), 4.64 (m, 1H), 4.36 (m,
1H), 4.05-3.87 (m, 2H), 3.71-3.66 (m, 1H), 3.52-3.42 (m, 2H),
1.32 (s, 3H); 13C NMR (75.5 MHz, CDCl3) δ 17.3, 41.7, 49.5,
54.9, 58.6, 67.1, 68.0, 79.9, 87.2, 89.0, 122.6, 125.8, 128.0, 128.2,
128.3, 128.5, 131.0, 132.0, 132.1 136.3, 137.4, 156.2, 171.8, 173.2;
ESI-MS m/z calcd for C24N25N3O5 435, found [M + H]+ 436, [M
+ Na]+ 458; HRMS (TOF ES+) m/z 458.1690 [M + Na]+, calcd
for C24H25N3O5Na+ 458.1692. Anal. Calcd for C24H25N3O5: C,
66.19; H, 5.79; N, 9.65. Found: C, 66.10; H, 5.71, N, 9.61.
Linear Tripeptide 17. In a flame-dried nitrogen-filled flask,
dipeptide 2b (200 mg, 0.32 mmol) was dissolved in DCM (3 mL),
which was purged with dry nitrogen gas. First Pd(PPh3)4 (37 mg,
0.032 mmol) was added followed by CuI (6 mg, 0.032 mmol), Et2-
NH (111 µL, 0.8 mmol), and finally alkyne 8 (140 mg, 0.48 mmol),
and the resulting reaction mixture was stirred for 3 days. Subse-
quently, the solvent was removed under reduced pressure, and the
Supporting Information Available: Experimental details of
compounds 2b,c, 3b,c, 4b,c, 7a,b, 8-11, and 15a, complete
characterization of compounds 1-4, 7a, 8-12, 15, 17, and 18, 1H
NMR, 13C NMR, 1H COSY, HMBC, HSQC, and HPLC data, and
modeling data of 4b using MacroModel together with an atom
coordinates file. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO051933M
1824 J. Org. Chem., Vol. 71, No. 5, 2006