Cyclic peptides containing a δ-sugar amino acid - Synthesis and evaluation as artificial receptors

Article abstract of DOI:10.1016/j.tet.2004.11.024

An Fmoc-protected δ-sugar amino acid, prepared by oxidation of a glucosamine derivative, was coupled to three different tripeptide tert-butyl esters (H-Tyr-Tyr-Tyr-O^tBu, H-Tyr-Glu(OBzl)-Tyr-O^tBu and H-Tyr-Arg(Mtr)-Tyr-O^tBu) and the resulting sugar amino acid/amino acid hybrids were transformed into dimers that were subsequently cyclized to give three C₂-symmetric macrocycles. The macrocycles were deprotected and their binding properties towards p-nitrophenyl glycosides, nucleotides, and purines were examined. Of the ligands screened, only some of the purines showed weak, but significant, binding.

Full text of DOI:10.1016/j.tet.2004.11.024

Tetrahedron 61 (2005) 863–874
Cyclic peptides containing a d-sugar amino acid—synthesis and
evaluation as artificial receptors
*
Johan F. Billing and Ulf J. Nilsson
Organic and Bioorganic Chemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden
Received 25 August 2004; revised 21 October 2004; accepted 12 November 2004
Available online 10 December 2004
Abstract—An Fmoc-protected d-sugar amino acid, prepared by oxidation of a glucosamine derivative, was coupled to three different
tripeptide tert-butyl esters (H-Tyr-Tyr-Tyr-OtBu, H-Tyr-Glu(OBzl)-Tyr-OtBu and H-Tyr-Arg(Mtr)-Tyr-OtBu) and the resulting sugar amino
acid/amino acid hybrids were transformed into dimers that were subsequently cyclized to give three C2-symmetric macrocycles. The
macrocycles were deprotected and their binding properties towards p-nitrophenyl glycosides, nucleotides, and purines were examined. Of the
ligands screened, only some of the purines showed weak, but significant, binding.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Herein, we report the synthesis of polyamphiphilic water-
soluble macrocyclic sugar amino acid/amino acid hybrid
molecules 1a–c (see Fig. 1) and an investigation of their
binding properties against biomolecules. We chose to use
the d-sugar amino acid obtained by oxidation of a partially
protected methyl b-glycoside of glucosamine together with
the aromatic amino acid tyrosine as building blocks for our
macrocycles. The d-sugar amino acid was chosen because
of its extended geometry, which prevents turn formation and
presumably thus gives rise to more accessible cores of the
macrocycles. In addition, we introduced amino acids with
charged side chains to enhance solubility and potentially
Interactions of small ligands, such as carbohydrates,
metabolites or hormones, with binding sites in proteins are
vital to life processes and the synthesis of artificial receptors
that mimic such interactions has been an ongoing goal in
many research groups for a long time.1 A basic design for
biomimetic artificial receptors involves amphiphilic
molecules, often macrocycles, with both polar and non-
polar regions, thus enabling interactions with both polar and
non-polar regions of a ligand.
Sugar amino acids2–5 are carbohydrates that contain at least
one amino and one carboxylic acid functionality, which
allows for the use of peptide coupling chemistry in order to
combine them with amino acids or other building blocks.
Sugar amino acids have been used to prepare cyclic homo-
oligomers6–8 and cyclic sugar amino acid/amino acid
hybrids,9–15 that have been used in various studies. It has
been proposed that such molecules could be interesting
artificial receptors, and in one case it has been shown that a
cyclodextrin-like cyclic hexamer could bind to benzoic acid
and p-nitrophenol in water, although no binding constants
were given.6 We decided to explore the use of sugar amino
acids as polar structural elements combined with non-polar
aromatic amino acids for the construction of amphiphilic
molecules as biomimetic receptors.
Keywords: Sugar amino acids; Macrocycles; Cyclic peptides; Artificial
receptors; Molecular recognition.
* Corresponding author. Tel.: C46 46 2228218; fax: C46 46 2228209;
e-mail: ulf.nilsson@bioorganic.lth.se
Figure 1. Synthesized macrocycles.
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.11.024

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J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
also binding. Two monosaccharides and six amino acids
were used in each macrocyclic ring in order to obtain
macrocycles large enough to form a central pocket where
ligands might bind.
diimide hydrochloride (EDC$HCl), 1-hydroxybenzotriazole
(HOBt) and N-methylmorpholine in THF, tert-butyl esters
were cleaved with 33% TFA in CH2Cl2 using Et3SiH as a
scavenger,20 and piperidine was used to cleave the Fmoc
group. The 4-methoxy-2,3,6-trimethylbenzenesulfonyl
(Mtr) group has previously been reported to be stable at
25% TFA in CH2Cl2,15 and this was also the case at 33%
TFA in CH2Cl2.
2. Results and discussion
2.1. Synthesis
Sugar amino acid precursor 7 was oxidized with Jones’s
reagent and the crude sugar amino acid was directly coupled
to a C-protected tripeptide 10a–c (Scheme 3) to give sugar
amino acid/amino acid hybrids 14a–c. The hybrids 14a–c
were either deprotected at the N-terminal using DBU in
the presence of a solid-phase thiol as a scavenger for
the liberated dibenzofulvene21 to give 15a–c or at the
C-terminal using TFA/Et3SiH/CH2Cl220 to give 16a–c. The
N-deprotected hybrids 15a–c and the C-deprotected hybrids
16a–c were coupled together using N,N0-diisopropylcarbo-
diimide (DIC) and HOBt to give linear dimers 17a–c. The
EDC$HCl/HOBt/N-methylmorpholine coupling protocol
that had been used earlier in the synthetic scheme gave
excessive epimerization in this coupling (ca. 20% epimer of
17a formed according to NMR) and could not be used here.
The DIC/HOBt protocol without base has been reported to
give good results in difficult couplings22 and gave good
results with 17a–c (no epimerization according to 1H NMR
spectrum). The linear dimers were N-deprotected as above
to give 18a–c.
The synthetic strategy towards the macrocycles involved
two building blocks for each macrocycle, a C-protected
tripeptide and an amino sugar precursor, which upon
oxidation gives the N-protected sugar amino acid (SAA).
The tripeptide and the sugar amino acid were coupled
together to give a linear sugar amino acid/amino acid
hybrid, which was then transformed into a dimer that was
subsequently cyclized to give the desired macrocycle. To
achieve this, it was necessary to use orthogonal protecting
groups for N- and C-protection. The use of the base-labile
Fmoc group and acid-labile tert-butyl esters met this
requirement.
The starting material glucosamine hydrochloride was
transformed into the known tri-O-acetylated methyl pyrano-
side 3 in a two-step procedure using a combination of
previously described methods16,17 (Scheme 1). Attempts to
deacetylate 3 using base-catalyzed transesterfication with
Me2NEt in MeOH gave ca. 12% of an N-acetyl side-
product, while acidic transesterfication cleanly produced
known hydrochloride 4.18 The amine was selectively
protected using N-(9-fluorenylmethoxycarbonyloxy)-
succinimide (Fmoc-OSu) to give 5. The primary hydroxyl
group was protected as the triphenylmethyl ether and the
secondary hydroxyl groups as benzoates to give 6. Cleavage
of the triphenylmethyl ether using hydrogen bromide in
acetic acid completed the synthesis of sugar amino acid
precursor 7. A similar sequence leading to the a anomer of
the same sugar amino acid has been disclosed.19
In order to evaluate cyclization conditions, a portion of 18a
was C-deprotected and initial cyclization attempts were
made using the following conditions:
(a) EDC$HCl/HOBt and N-methylmorpholine in THF.
(b) DIC/HOBt both with and without N,N-diisopropylethyl-
amine (DIPEA) in both THF and DMF.
(c) Diphenylphosphoryl azide (DPPA) both with NaHCO3
in DMF and with DIPEA in THF.
(d) 1-[bis-(Dimethylamino)methylene]-1H-benzotriazo-
lium tetrafluoroborate 3-oxide (TBTU)†, HOBt, and
DIPEA in THF.
Three different tripeptide tert-butyl esters 10a–c were
prepared in solution in good yields (Scheme 2) to serve as
the required C-protected tripeptides. Peptide couplings were
made using N-(3-dimethylaminopropyl)-N0-ethyl carbo-
(e) 1-[bis-(Dimethylamino)methylene]-1H-1,2,3-tria-
zolo[4,5-b]pyridinium hexafluorophosphate 3-oxide
(HATU) with both DIPEA and 2,4,6-collidine in THF.
(f) 1-(1-Pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridinyl-
methylene)pyrrolidinium hexafluorophosphate 3-oxide
(HAPyU)‡ with both DIPEA and 2,4,6-collidine in
THF.
All attempts were carried out at 1 mM concentration of the
linear starting material. Only TBTU, HATU, and HAPyU
† HATU and HBTU were long believed to be uronium salts, but have been
shown to be guanidinium salts when prepared using the conventional
methods.23 This is likely to be true for TBTU and HAPyU as well. We
have chosen to name all these reagents as guanidinium salts.
‡ HAPyU was prepared from 1-hydroxy-7-azabenzotriazole potassium salt
(KOAt)23 and commercially available chloro-N,N,N0,N0-bis(tetramethyl-
ene)-formamidinium hexafluorophosphate using Knorr’s method for the
preparation of similar coupling reagents.24,25
Scheme 1. Synthesis of the d-sugar amino acid precursor 7: (a) AcBr (neat),
3 days, 82%; (b) MeOH, pyridine, 1 h, 76%; (c) HCl/MeOH, 24 h, 95%; (d)
Fmoc-OSu, NaHCO3, 15 h, 76%; (e) chlorotriphenylmethane, pyridine,
85 8C, 2 h, then BzCl, pyridine, r.t., 4 h, 63%; (f) HBr/AcOH, 3 min, 81%.

J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
865
Scheme 2. Synthesis of tripeptide tert-butyl esters 10a–c: (a) Fmoc-Tyr-OH, EDC$HCl, HOBt, N-methylmorpholine, THF, 16 h, 83–88%; (b) TFA, Et3SiH,
CH2Cl2, 4 h; (c) H-Tyr-OtBu, EDC$HCl, HOBt, N-methylmorpholine, THF, 16 h, 83–94%; (d) piperidine, CH2Cl2, 30 min, 83–90%.
Scheme 3. Synthesis of macrocycles 1a–c: (a) CrO3, H2SO4(aq), acetone, 1.5 h; (b) EDC$HCl, HOBt, N-methylmorpholine, THF, 16 h, 45–64% (two steps);
(c) DBU, N-(2-mercaptoethyl) aminomethyl polystyrene, 6 h, 82–99%; (d) TFA, Et3SiH, CH2Cl2, 3–4 h; (e) DIC, HOBt, THF, 16 h, 48–72%; (f) DBU, N-(2-
mercaptoethyl)aminomethyl polystyrene, 6 h, 88–99%; (g) (i) TFA, Et3SiH, CH2Cl2, 3–4 h; (ii) HAPyU, DIPEA, THF, 2 h, 39–53%; (h) NaOMe/MeOH, 24 h,
61%; (i) (i) HCOOH, Pd black, MeOH, 15 min; (ii) NaOMe/MeOH, 24 h, 81%; (j) (i) TFA, PhSMe, 24 h; (ii) NaOMe/MeOH, 24 h, 59%.
gave the macrocycle 19a as a major product according to
MALDI-TOF analysis of the reaction mixtures.
Compounds 18b–c could also be cyclized to 19b–c using
this method (see Table 1). In the cyclization of 18b to 19b,
HAPyU gave a better yield than HATU.
The HATU and HAPyU reagents have been shown to give
less epimerization than TBTU in the cyclization of
pentapeptides26 and these reagents were thus investigated
further. Furthermore, it has been shown that DIPEA gives
less epimerization than 2,4,6-collidine for the cyclization of
pentapeptides by HAPyU,26 while for segment conden-
sations the opposite is true.27 Hence, both DIPEA and 2,4,6-
collidine were evaluated as bases. In the cyclization of 18a
to 19a, DIPEA gave a higher yield and less epimerization.
The 1H NMR spectra of the protected macrocycles 19a–c only
gave poorly resolved spectra with broad peaks at room
temperature, presumably due to slow conformational
exchange. Resolved spectra of 19a and 19b could be obtained
at 150 and 120 8C, respectively, but compound 19c only gave
poorly resolved spectra even at these temperatures.
Macrocycle 19a was deprotected using 10 mM NaOMe in

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J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
Table 1. Cyclization conditions, yields and epimerizations
Starting material
Reagentsa
Reaction time (h)b
Isolated yield (%)c
Epimerization
18a
18a
18b
18b
18c
HAPyU/2,4,6-collidine
HAPyU/DIPEA
HAPyU/DIPEA
HATU/DIPEA
3
1
3
2.5
4
35
37
53
32
34
5% isolated yield
Not observed
Traces on TLC
Traces on TLC
Traces on TLC
HAPyU/DIPEA
a At room temperature in THF with 1 mM concentration of linear starting material.
b Reactions were monitored with MALDI-TOF until the deprotected starting material was consumed.
c For both C-deprotection and cyclization.
MeOH to afford 1a in 61% yield. The deprotection of
macrocycle 19b started with the cleavage of the benzyl
esters using catalytic transfer hydrogenation with palladium
black and formic acid as the hydrogen source,28 followed by
treatment of the crude product with NaOMe/MeOH to give
1b in 81% yield. In the case of the macrocycle 19c, the Mtr
groups were first cleaved using neat TFA with thioanisole as
a scavenger29 and the crude product was then treated with
NaOMe/MeOH to afford 1c in 59% yield after HPLC
purification.
acids and turns formed by the tripeptides (Fig. 3). In the
conformers with lowest energy, the axial hydrogen atoms in
the two sugar amino acids were facing each other, but
conformers where one of the sugar amino acids had rotated
to place the hydroxyl groups in position to form hydrogen
bonds to the tripeptide, or to the other sugar amino acid,
were also found. Hydrogen bonds were occasionally found
within the tripeptides, but no pattern could be discerned.
3
The JHH coupling constants for all three macrocycles
indicate that the sugar amino acids are in the 4C1
conformation. This was the case for the calculated
conformers of 1a–b, but many conformers of macrocycle
1c deviated from the expected 4C1 conformation of the
pyranose rings. As there is no support for this in the
coupling constants, we conclude that it is an artefact in
the calculations possibly induced by the strong hydrogen
bond formed between the arginine and tyrosine side chains.
2.2. Conformational analysis
The deprotected macrocycles 1a–c all gave well resolved
NMR spectra at room temperature. Macrocycle 1a in
MeOH-d4 and macrocycles 1b–c in D2O all gave the
1
expected H NMR spectra for symmetrical compounds. In
addition to the major peaks, macrocycle 1c in also gave
smaller peaks at 2.81 and 2.56 ppm, as well as some
overlapping smaller peaks at 3.74 ppm and in the aromatic
1
region. Heating of 1c in DMSO-d6 brought the H NMR
signals to coalescence, which shows that the multiple peaks
of 1c at ambient temperature were due to slow confor-
mational exchange (Fig. 2).
Figure 2. The aromatic region of the 1H NMR spectrum of 1c at different
temperatures (400 MHz, DMSO-d6).
Figure 3. Calculated global energy minimum of macrocycle 1a (side chains
omitted for clarity).
Monte–Carlo conformational searches were performed on
1a–c using MacroModel 8.5 (MMFFs force field with water
as solvent, 20,000 steps, all backbone torsions were selected
for random variation) to give for each macrocycle 110–240
conformers within 5 kcal/mol of the global minimum. When
these conformers were studied, a coherent picture emerged.
The dominant low-energy conformers for macrocycles 1a–c
were twisted, oblong structures with extended sugar amino
2.3. Molecular recognition properties
Compound 1a was not soluble in water and its binding
properties were not examined. Macrocycles 1b–c were
screened using NMR titrations against a number of putative
ligands: p-nitrophenyl glycosides, nucleotides, aromatic

J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
867
amino acids, aromatic amines and purines. Of all the ligands
screened, only 1b and caffeine and 1c and the purine
nucleotides 20-deoxyadenosine 50-monophosphate (dAMP)
and 20-deoxyguanosine 50-monophosphate (dGMP) showed
weak, but significant, interactions (Kaz10 MK1). For
comparison, reference peptide Ac-Tyr-Arg-Tyr-OMe§ was
also titrated with dAMP and dGMP and was found to bind
more weakly (Kaz5 MK1). The binding is most likely due
to hydrophobic interaction between the purine and tyrosine
rings, and the small increase in affinity for 1c is thus due to
its dimeric cyclic structure and/or the presence of the sugar
amino acid moieties. However, the weak affinities preclude
conclusions regarding detailed structure–affinity
relationships.
precipitated from the mixture to afford 2 (37.6 g, 82%)
as small white needles. Mp 143–148 8C (dec.), lit.16
149–150 8C (dec.); [a]D22ZC130 (c 1.0, acetone), lit.5
[a]DZC148.4 (c 5.01, acetone); 1H NMR (300 MHz,
CDCl3) d 8.66 (br s, 3H, NH3), 7.10 (d, JZ3.6 Hz, 1H, H1),
5.50 (t, JZ9.8 Hz, 1H, H3), 5.25 (t, JZ9.7 Hz, 1H, H4),
4.33 (m, 2H, H5CH6), 4.17 (d, JZ10.9 Hz, H6), 3.93 (dd,
JZ10.3, 3.7 Hz, 1H, H5), 2.26, 2.13, 2.09 (3s, 3H
each, OAc)¶; HRMS (FAB) calcd for C12H18BrNO7Na
(MKHBrCNa): 390.0164; found 390.0171.
4.2.2. Methyl tri-O-acetyl-2-amino-2-deoxy-b-D-gluco-
pyranoside (3). Hydrobromide 2 (37.6 g, 84 mmol) was
dissolved in MeOH (800 mL) and pyridine (8 mL, distilled
from CaH2) was added. After 1 h, toluene (150 mL) was
added and the mixture was concentrated. The residue was
dissolved in chloroform (750 mL), washed with Na2CO3-
(aq) (5%, 2!100 mL) and water (100 mL), dried over
Na2SO4, and concentrated. The crude product was
recrystallized in chloroform/heptane to give 3 (20.3 g in
three crops, 76%) as small white needles. Mp 146–148 8C,
lit.17 151–152 8C; [a]D22ZC14 (c 1.0, MeOH), lit.17
[a]2D7ZC15 (c 1, MeOH); 1H NMR spectrum is in
agreement with published data17; HRMS (FAB) calcd for
C13H21NO8Na (MCNa): 342.1165, found 342.1158.
3. Conclusions
We have described the synthesis of three d-sugar amino
acid/tripeptide dimeric macrocycles and evaluated their
binding properties. Macrocycles 1b–c were found to bind
some purine derivatives with weak, but significant, binding
constants. Apparently, the structures have to be modified in
order to present binding sites pre-organized for higher
affinity binding of biomolecules. However, although the
binding is weak, this shows that sugar amino acid containing
peptides can act as artificial receptors and serves as a
starting point for further research.
4.2.3. Methyl 2-amino-2-deoxy-b-D-glucopyranoside
hydrochloride (4). Acetyl chloride (67 mL, 0.95 mol) was
slowly added to MeOH (320 mL) at 0 8C. Compound 3
(11.2 g, 35.1 mmol) was added and the solution was stirred
for 24 h at room temperature. The solution was concentrated
and the crude product was recrystallized in MeOH/EtOAc to
give 4 (7.64 g in two crops, 95%) as small white needles.
Mp 192–193 8C, lit.18 190 8C; [a]2D2ZK25 (c 1.0, water),
lit.18 [a]D22ZK23.4 (c 1, water); 1H NMR (300 MHz, D2O)
d 4.51 (d, JZ8.6 Hz, 1H, H1), 3.79 (dd, JZ12.4, 2.0 Hz,
1H, H6), 3.61 (dd, JZ12.4, 5.2 Hz, 1H, H6), 3.54 (dd, JZ
10.5, 8.3 Hz, 1H, H3), 3.45 (s, 3H, OMe), 3.34 (m, 2H, H4C
H5), 2.88 (dd, JZ10.6, 8.5 Hz, 1H, H2); HRMS (FAB) calcd
for C7H15NO5Na (MKHClCNa): 216.0848; found
216.0855.
4. Experimental
4.1. General methods
˚
THF and CH2Cl2 were dried over 4 A molecular sieves
˚
before use and MeOH was dried over 3 A molecular sieves
before use. Other solvents were not dried unless specified.
˚
Matrex 35–70 mm 60 A silica (Millipore) was used for flash
chromatography. Sephadex LH-20 in CH2Cl2/MeOH 1:1
was used for size-exclusion chromatography. Sep-Pak Plus
C18 cartridges (Waters) were used for solid-phase extrac-
tion. Chemical shifts are reported relative to Me4Si and were
calculated using the residual solvent peak as a reference.
NMR spectra were assigned with the help of correlation
spectroscopy (COSY). All compounds were estimated to be
4.2.4. Methyl 2-(9-fluorenylmethoxycarbonyl)amino-2-
deoxy-b-D-glucopyranoside (5). Compound 4 (2.78 g,
12.1 mmol) was dissolved in water (22 mL) and NaHCO3
(1.02 g, 12.1 mmol) was added. After the evolution of gas
had ceased, additional NaHCO3 (1.02 g, 12.1 mmol),
acetone (22 mL), and N-(9-fluorenylmethoxycarbonyloxy)-
succinimide (4.08 g, 12.1 mmol) were added. The reaction
mixture was stirred overnight and solidified during the
reaction. The product was suspended in water (100 mL) and
chloroform (100 mL), filtered off, and washed with water
and chloroform. The crude product was recrystallized in
methanol to give 5 (3.81 g in three crops, 76%) as tiny white
1
O95% pure by H NMR spectroscopy.
4.2. Preparation of sugar amino acid precursor 7
4.2.1. Tri-O-acetyl-2-amino-2-deoxy-a-D-glucopyranosyl
bromide hydrobromide (2).16,17 Acetyl bromide (70 mL,
0.94 mol) was added to D-glucosamine hydrochloride
(21.9 g, 101.6 mmol, dried 24 h in vacuo at 70 8C over
P2O5) and the mixture was stirred for 3 days at room
temperature. Residual acetyl bromide was removed in vacuo
(water aspirator) and the crude product was dissolved in hot
chloroform (distilled from P2O5) and filtered while still hot.
Crystals began to form as the solution cooled and diethyl
ether was added to the stirred solution until the product
needles. Mp 163–164 8C; [a]2D2ZK20 (c 0.5, MeOH); H
1
NMR (400 MHz, MeOH-d4) d 7.79 (d, JZ7.5 Hz, 2H,
Fmoc), 7.68 (d, JZ6.9 Hz, 2H, Fmoc), 7.38 (t, JZ7.2 Hz,
2H, Fmoc), 7.30 (t, JZ7.5 Hz, 2H, Fmoc), 4.28 (m, 4H, 3!
Fmoc-HCH1), 3.88 (dd, JZ11.9, 2.1 Hz, 1H, H6), 3.68
(dd, JZ11.8, 5.8 Hz, 1H, H6), 3.46 (s, 3 H, OMe), w3.34
§ Ac-Tyr-Arg(Mtr)-Tyr-OMe was prepared from Ac-Tyr-OH, H-Arg(Mtr)-
OtBu and H-Tyr-OMe analogously to the synthesis of 9a–b. The Mtr
group was cleaved as in the synthesis of 1c.
¶ In agreement with previously published data,30 but a COSY experiment
shows that the signals had been incorrectly assigned.

868
J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
(H2CH3CH4CH5, partially obscured by solvent signal);
HRMS (FAB) calcd for C22H25NO7Na (MCNa): 438.1529;
found 438.1530.
300 MHz) d 7.77 (d, JZ7.5 Hz, 2H, Fmoc), 7.56 (d, JZ
7.7 Hz, 2H, Fmoc), 7.37 (t, JZ7.5 Hz, 2H, Fmoc), 7.28 (m,
2H, Fmoc), 7.03 (d, JZ8.5 Hz, 2H, Tyr-Hd), 7.00 (d, JZ
8.4 Hz, 2H, Tyr-Hd), 6.68 (d, JZ8.5 Hz, 2H, Tyr-H3), 6.67
(d, JZ8.5 Hz, 2H, Tyr-H3), 4.45 (t, JZ7.0 Hz, 1H, Tyr-Ha),
4.30 (m, 2H, 1!Tyr-HaC1!Fmoc-H), 4.16 (m, 2H,
Fmoc), 3.05–2.85 (m, 3H, 3!Tyr-Hb), 2.71 (dd, JZ13.9,
9.3 Hz, 1H, Tyr-Hb), 1.37 (s, 9H, OtBu). HRMS (FAB)
calcd for C37H39N2O7 (MCH): 623.2757; found 623.2748.
4.2.5. Methyl 3,4-di-O-benzoyl-2-(9-fluorenylmethoxy-
carbonyl)amino-2-deoxy-6-O-triphenylmethyl-b-D-
glucopyranoside (6). Compound 5 (4.00 g, 9.63 mmol) was
dissolved in pyridine (200 mL, distilled from CaH2).
Chlorotriphenylmethane (8.05 g, 28.9 mmol) was added
and the mixture was stirred at 85 8C for 2 h. Benzoyl
chloride (5.6 mL, 48.2 mmol) was added at 0 8C and the
mixture was stirred for 3.5 h in room temperature. The
mixture was poured over ice and the product was extracted
with EtOAc (3!300 mL). The extract was washed with
H2SO4(aq) (0.5 M, 2!200 mL), NaHCO3(aq) (sat., 2!
200 mL) and water (100 mL), dried over Na2SO4 and
evaporated. The product was purified with flash chroma-
tography (toluene/EtOAc 10:1, RfZ0.20) and lyophilized
from benzene to give 6 (5.27 g, 63%) as a fluffy white
4.3.2. Fmoc-Tyr-Tyr-Tyr-OtBu (9a). Compound 8a
(951 mg, 1.53 mmol) was dissolved in CH2Cl2 (12 mL)
and Et3SiH (0.61 mL, 3.8 mmol) and TFA (6 mL) were
added. The mixture was stirred for 4 h and coevaporated
with toluene. The crude free acid was dissolved in THF
(14 mL) and H-Tyr-OtBu (363 mg, 1.53 mmol), HOBt
(206 mg, 1.53 mmol), EDC$HCl (308 mg, 1.60 mmol) and
N-methylmorpholine (0.340 mL, 3.06 mmol) were added.
The mixture was stirred overnight and then evaporated. The
residue was dissolved in MeOH and impregnated on silica.
The product was purified with flash chromatography
(toluene/MeOH 5:1, RfZ0.36) to give 9a (1.13 g, 94%) as
1
powder. [a]2D2ZK20 (c 0.4, MeOH); H NMR (300 MHz,
DMSO-d6) d 7.82 (d, JZ7.5 Hz, 2H, Ar), 7.77 (d, JZ
7.5 Hz, 2H, Ar), 7.63 (m, 4H, NHC3!Ar), 7.53 (m, 2H,
Ar), 7.37 (m, 14H, Ar), 7.17 (m, 10H, Ar), 5.49 (m, 2H,
H3CH4), 4.68 (d, JZ8.4 Hz, 1H, H1), 4.22 (m, 2H, Fmoc),
4.04 (t, JZ6.5 Hz, 1H, Fmoc), 3.93 (br d, JZ7.8 Hz, 1H,
H5), 3.82 (q, JZ9.2 Hz, 1H, H2), 3.50 (s, 3H, OMe), w3.28
(H6, obscured by HDO signal), 2.95 (dd, JZ10.0, 3.4 Hz,
1H, H6); HRMS (FAB) calcd for C55H47NO9Na (MCNa):
888.3149; found 888.3161.
1
a white amorphous solid. [a]2D2ZK22 (c 0.5, MeOH); H
NMR (MeOH-d4, 300 MHz) d 7.77 (d, JZ7.5 Hz, 2H,
Fmoc), 7.55 (d, JZ7.5 Hz, 2H, Fmoc), 7.37 (t, JZ7.5 Hz,
2H, Fmoc), 7.26 (m, 2H, Fmoc), 7.00 (d, JZ8.5 Hz, 6H,
Tyr-Hd), 6.66 (m, 6H, Tyr-H3), 4.55 (t, JZ6.4 Hz, 1H, Tyr-
Ha), 4.42 (t, JZ6.9 Hz, 1H, Tyr-Ha), 4.20 (m, 4H, 1!Tyr-
HaCFmoc), 3.05–2.75 (m, 5H, 5!Tyr-Hb), 2.66 (dd, JZ
14.5, 9.5 Hz, 1H, Tyr-Hb), 1.35 (s, 9H, OtBu); HRMS
(FAB) calcd for C46H48N3O9 (MCH): 786.3391; found
786.3398.
4.2.6. Methyl 3,4-di-O-benzoyl-2-(9-fluorenylmethoxy-
carbonyl)amino-2-deoxy-b-D-glucopyranoside (7). Com-
pound 6 (3.66 g, 4.23 mmol) was dissolved in glacial acetic
acid (150 mL) and HBr in AcOH (4.1 M, 2.1 mL, 8.5 mmol)
was added. The mixture was stirred for 3 min and then
poured over ice. The product was extracted with chloroform
(4!100 mL) and the extract was dried over MgSO4 and
evaporated. The product was purified with flash chroma-
tography (toluene/EtOAc 2:1, RfZ0.14) and lyophilized
from benzene to give 7 (2.14 g, 81%) as a fluffy white
4.3.3. H-Tyr-Tyr-Tyr-OtBu (10a). Compound 9a (2.27 g,
2.89 mmol) was suspended in CH2Cl2 (80 mL) and
piperidine (14.3 mL) was added. After stirring for 30 min,
toluene (50 mL) was added and the mixture was evaporated.
The residue was dissolved in CH2Cl2 and purified with flash
chromatography (toluene/MeOH 3:1, RfZ0.13) to give 10a
(1.42 g, 87%) as a white foam. [a]2D4ZK14 (c 0.5, MeOH);
1H NMR (MeOH-d4, 300 MHz) d 6.97 (m, 6H, Tyr-Hd),
6.67 (m, 6H, Tyr-H3), 4.56 (dd, JZ7.9, 6.0 Hz, 1H, Tyr-
Ha), 4.44 (t, JZ7.1 Hz, 1H, Tyr-Ha), 3.44 (dd, JZ8.0,
5.0 Hz, 1H, Tyr-Ha), 3.00–2.70 (m, 5H, 5!Tyr-Hb), 2.52
(dd, JZ13.8, 8.0 Hz, 1H, Tyr-Hb), 1.38 (s, 9H, OtBu);
HRMS calcd for C31H38N3O7 (MCH): 564.2701; found
564.2710.
powder. [a]2D2ZK41 (c 0.5, MeOH); H NMR (300 MHz,
1
DMSO-d6) d 7.83 (m, 4H, Bz-oC2!Fmoc-H), 7.76 (d, JZ
7.7 Hz, 2H, Bz-o), 7.65–7.30 (m, 11H, Bz-mCBz-pC
NHC4!Fmoc-H), 7.14 (q, JZ7.6 Hz, 2H, Fmoc), 5.50 (t,
JZ9.9 Hz, 1H, H3), 5.25 (t, JZ9.8 Hz, 1H, H4), 4.89 (t, JZ
5.7 Hz, 1H, OH), 4.64 (d, JZ8.4 Hz, 1H, H1), 4.18 (m, 2H,
Fmoc), 4.01 (t, JZ6.7 Hz, 1H, Fmoc), 3.72 (m, 2H, H2C
H5), 3.54 (m, 2H, 2!H6), 3.44 (s, 3H, OMe); HRMS (FAB)
calcd for C36H33NO9Na (MCNa): 646.2053; found
646.2059.
4.3.4. Fmoc-Tyr-Glu(OBzl)-OtBu (8b). The title com-
pound was prepared from Fmoc-Tyr-OH (1.26 g,
3.12 mmol) and H-Glu(OBzl)-OtBu$HCl (1.03 g,
3.12 mmol) using the method described in the synthesis of
8a, but using an additional equivalent of base to neutralize
the hydrochloride salt. The product was purified with flash
chromatography (toluene/EtOAc 3:1, RfZ0.19) to give 8b
(1.82 g, 86%) as a white foam. [a]2D2ZK16 (c 0.5, MeOH);
1H NMR (MeOH-d4, 300 MHz) d 7.77 (d, JZ7.5 Hz, 2H,
Fmoc), 7.55 (d, JZ6.6 Hz, 2H, Fmoc), 7.37 (d, JZ7.4 Hz,
2H, Fmoc), 7.27 (m, 7H, FmocCBzl), 7.06 (d, JZ8.4 Hz,
2H, Tyr-Hd), 6.68 (d, JZ8.3 Hz, 2H, Tyr-H3), 5.06 (m,
2H, Bzl), 4.30 (m, 3H, Tyr-HaCGlu-HaC1!Fmoc-H),
4.16 (m, 2H, Fmoc), 3.02 (dd, JZ13.9 Hz, JZ5.2 Hz, 1H,
Tyr-Hb), 2.77 (dd, JZ14.0, 9.4 Hz, 1H, Tyr-Hb), 2.43 (t,
4.3. Preparation of tripeptide tert-butyl esters 11a–c
4.3.1. Fmoc-Tyr-Tyr-OtBu (8a). Fmoc-Tyr-OH (700 mg,
1.74 mmol) was dissolved in THF (17 mL) and H-Tyr-OtBu
(412 mg, 1.74 mmol), HOBt (234 mg, 1.74 mmol),
EDC$HCl (349 mg, 1.82 mmol) and N-methylmorpholine
(0.380 mL, 3.47 mmol) were added. The mixture was stirred
overnight and then evaporated. The residue was dissolved in
MeOH and impregnated on silica. The product was purified
with flash chromatography (toluene/EtOAc 2:1, RfZ0.20)
to give 8a (948 mg, 88%) as a white amorphous solid.
[a]2D2ZK16 (c 0.5, MeOH); 1H NMR (MeOH-d4,

J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
869
JZ7.4 Hz, 2H, Glu-Hg), 2.15 (m, 1H, Glu-Hb), 1.90 (m, 1H,
Glu-Hb), 1.43 (s, 9H, OtBu); HRMS (FAB) calcd for
C40H43N2O8 (MCH): 679.3019; found 679.3014.
Tyr-Hd), 6.69 (d, JZ8.5 Hz, 2H, Tyr-H3), 6.65 (s, 1H, Mtr-
ArH), 4.48 (dd, JZ8.1, 6.5 Hz, 1H, Tyr-Ha), 3.82 (s, 3H,
Mtr-OMe), w3.28 (Arg-Ha, partially obscured by solvent
signal), 3.12 (br s, 2H, Arg-Hd), 2.98 (dd, JZ13.9, 6.4 Hz,
1H, Tyr-Hb), 2.86 (dd, JZ13.9, 8.2 Hz, 1H, Tyr-Hb), 2.66
(s, 3H, Mtr-Me), 2.60 (s, 3H, Mtr-Me), 2.12 (s, 3H, Mtr-
Me), 1.58 (m, 1H, Arg-Hb), 1.45 (m, 3H, 1!Arg-HbC2!
Arg-Hg), 1.38 (s, 9H, OtBu); HRMS (FAB) calcd for
C29H44N5O7S (MCH): 606.2961; found 606.2958.
4.3.5. Fmoc-Tyr-Glu(OBzl)-Tyr-OtBu (9b). The title
compound was prepared from 8b (1.73 g, 2.55 mmol) and
H-Tyr-OtBu (605 mg, 2.55 mmol) using the method
described in the synthesis of 9a. The product was purified
with flash chromatography (toluene/EtOAc 3:2, RfZ0.11)
to give 9b (1.77 g, 83%) as a white foam. [a]2D2ZK15 (c
0.5, MeOH); 1H NMR (MeOH-d4, 300 MHz) d 7.77 (d, JZ
7.5 Hz, 2H, Fmoc), 7.55 (dd, JZ7.3, 3.0 Hz, 2H, Fmoc),
7.37 (t, JZ7.4 Hz, 2H, Fmoc), 7.28 (m, 7H, FmocCBzl),
7.04 (d, JZ8.2 Hz, 2H, Tyr-Hd), 7.02 (d, JZ8.2 Hz, 2H,
Tyr-Hd), 6.69 (d, JZ8.5 Hz, 2H, Tyr-H3), 6.67 (d, JZ
8.4 Hz, 2H, Tyr-H3), 5.05 (m, 2H, Bzl), 4.41 (m, 2H, Tyr-
HaCGlu-Ha), 4.28 (m, 2H, 1!Tyr-HaC1!Fmoc-H), 4.25
(m, 2H, Fmoc), 2.92 (m, 3H, 3!Tyr-Hb), 2.75 (dd, JZ13.8,
9.5 Hz, 1H, Tyr-Hb), 2.40 (t, JZ7.7 Hz, 2H, Tyr-Hg), 2.07
(m, 1H, Glu-Hb), 1.89 (m, 1H, Glu-Hb), 1.35 (s, 9H, OtBu);
HRMS (FAB) calcd for C49H52N3O10 (MCH): 842.3653;
found 842.3646.
4.3.9. Fmoc-Tyr-Arg(Mtr)-Tyr-OtBu (13). The title
compound was prepared from Fmoc-Tyr-OH (854 mg,
2.12 mmol) and 12 (1.28 g, 2.12 mmol) using the method
described in the synthesis of 8a. The product was purified
with flash chromatography (toluene/EtOAc 1:4, RfZ0.16)
to give 13 (1.74 g, 83%) as a white foam. [a]2D2ZK8 (c 0.5,
1
MeOH); H NMR (300 MHz, MeOH-d4) d 7.76 (d, JZ
7.6 Hz, 2H, Fmoc), 7.55 (d, JZ7.1 Hz, 2H, Fmoc), 7.36 (t,
JZ7.4 Hz, 2H, Fmoc), 7.26 (t, JZ7.2 Hz, 2H, Fmoc), 7.04
(d, JZ7.8 Hz, 2H, Tyr-Hd), 7.01 (d, JZ8.2 Hz, 2H, Tyr-
Hd), 6.68 (d, JZ8.0 Hz, 2H, Tyr-H3), 6.67 (d, JZ8.1 Hz,
2H, Tyr-H3), 6.61 (s, 1H, Mtr-ArH), 4.42 (t, JZ7.2 Hz, 1H,
Tyr-Ha), 4.35 (dd, JZ8.3, 5.1 Hz, 1H, Arg-Ha), 4.31 (m,
2H, 1!Tyr-HaC1!Fmoc), 4.16 (m, 2H, Fmoc), 3.78 (s,
3H, Mtr-OMe), 3.12 (br s, 2H, Arg-Hd), 2.94 (m, 3H, Tyr-
Hb), 2.75 (dd, JZ13.8, 9.9 Hz, 1H, Tyr-Hb), 2.65 (s, 3H,
Mtr-Me), 2.59 (s, 3H, Mtr-Me), 2.08 (s, 3H, Mtr-Me), 1.74
(m, 1H, Arg-Hb), 1.58 (m, 1H, Arg-Hb), 1.48 (m, 2H, Arg-
Hg), 1.35 (s, 9H, OtBu); HRMS (FAB) calcd for
C53H63N6O11S (MCH): 991.4276; found 991.4250.
4.3.6. H-Tyr-Glu(OBzl)-Tyr-OtBu (10b). The Fmoc group
of 9b (1.74 g, 2.06 mmol) was removed using the same
method as in the synthesis of 10a. The product was purified
using flash chromatography (EtOAc/MeOH 40:1, RfZ0.21)
to give 10b (1.05 g, 88%) as a white foam. [a]2D2ZK19 (c
0.5, MeOH); 1H NMR (MeOH-d4, 300 MHz) d 7.32 (m, 5H,
Bzl), 7.03 (d, JZ8.5 Hz, 2H, Tyr-Hd), 6.99 (d, JZ8.5 Hz,
2H, Tyr-Hd), 6.68 (d, JZ8.5 Hz, 4H, Tyr-H3), 5.11 (s, 2H,
Bzl), 4.40 (m, 2H, Tyr-HaCGlu-Ha), 3.50 (dd, JZ7.5,
5.3 Hz, 1H, Tyr-Ha), 3.00–2.80 (m, 3H, Tyr-Hb), 2.68 (dd,
JZ13.6, 7.6 Hz, 1H, Tyr-Hb), 2.36 (t, JZ7.7 Hz, 2H, Glu-
Hg), 2.05 (m, 1H, Glu-Hb), 1.87 (m, 1H, Glu-Hb), 1.36 (s,
9H, OtBu). HRMS (FAB) calcd for C34H42N3O8 (MCH):
620.2972; found 620.2969.
4.3.10. H-Tyr-Arg(Mtr)-Tyr-OtBu (10c). The Fmoc group
of 13 (1.60 g, 1.61 mmol) was removed using the same
method as in the synthesis of 10a. The product was purified
using flash chromatography (EtOAc/MeOH 10:1C2%
Me2NEt, RfZ0.21) to give 10c (1.02 g, 83%) as a white
1
foam. [a]2D2ZK10 (c 0.5, MeOH); H NMR (300 MHz,
MeOH-d4) d 7.03 (d, JZ8.4 Hz, 2H, Tyr-Hd), 7.02 (d, JZ
8.4 Hz, 2H, Tyr-Hd), 6.71 (d, JZ8.4 Hz, 2H, Tyr-H3), 6.68
(d, JZ8.4 Hz, 2H, Tyr-H3), 6.65 (s, 1H, Mtr-ArH), 4.44 (t,
JZ7.2 Hz, 1H, Tyr-Ha), 4.37 (dd, JZ8.1 Hz, JZ5.7 Hz,
1H, Arg-Ha), 3.82 (s, 3H, Mtr-OMe), 3.67 (dd, JZ8.1,
5.4 Hz, 1H, Tyr-Ha), 3.12 (br t, JZ4.5 Hz, 2H, Arg-Hd),
2.92 (m, 3H, Tyr-Hb), 2.72 (dd, JZ14.0, 8.0 Hz, 1H, Tyr-
Hb), 2.66 (s, 3H, Mtr-Me), 2.61 (s, 3H, Mtr-Me), 2.12 (s,
3H, Mtr-Me), 1.73 (m, 1H, Arg-Hb), 1.59 (m, 1H, Arg-Hb),
1.48 (m, 2H, Arg-Hg), 1.37 (s, 9H, OtBu); HRMS (FAB)
calcd for C38H53N6O9S (MCH): 769.3595; found
769.3610.
4.3.7. Fmoc-Arg(Mtr)-Tyr-OtBu (11). The title compound
was prepared from Fmoc-Arg(Mtr)-OH (1.82 g, 2.99 mmol)
and H-Tyr-OtBu (709 mg, 2.99 mmol) using the method
described in the synthesis of 8a. The product was purified
with flash chromatography (toluene/EtOAc 1:3, RfZ0.17)
to give 11 (2.06 g, 83%) as a white foam. [a]2D2ZK6 (c 0.5,
1
MeOH); H NMR (300 MHz, MeOH-d4) d 7.78 (d, JZ
7.5 Hz, 2H, Fmoc), 7.64 (t, JZ5.6 Hz, 2H, Fmoc), 7.37 (t,
JZ7.4 Hz, 2H, Fmoc), 7.28 (t, JZ7.4 Hz, 2H, Fmoc), 7.00
(d, JZ8.4 Hz, 2H, Tyr-Hd), 6.67 (d, JZ8.3 Hz, 2H, Tyr-
H3), 6.62 (s, 1H, Mtr-ArH), 4.45 (t, JZ7.0 Hz, 1H, Tyr-Ha),
4.34 (m, 2H, Fmoc), 4.19 (t, JZ6.5 Hz, 1H, Fmoc), 4.05 (t,
JZ6.9 Hz, 1H, Arg-Ha), 3.78 (s, 3H, Mtr-OMe), 3.14 (br s,
2H, Arg-Hd), 2.01 (m, 2H, Tyr-Hb), 2.66 (s, 3H, Mtr-Me),
2.60 (s, 3H, Mtr-Me), 2.09 (s, 3H, Mtr-Me), 1.68 (m, 1H,
Arg-Hb), 1.54 (m, 1H, Arg-Hb), 1.47 (m, 2H, Arg-Hg), 1.35
(s, 9H, OtBu); HRMS (FAB) calcd for C44H54N5O9S (MC
H): 828.3642; found 828.3654.
4.4. Synthesis of the macrocycles 1a–c
4.4.1. Fmoc-SAA(di-O-Bz)-Tyr3-OtBu (14a). Compound
7 (1.60 g, 2.56 mmol) was dissolved in acetone (380 mL)
and the mixture was cooled to 0 8C. Jones’s reagent (4 M,
25.6 mL, prepared by dissolving 12.0 g CrO3 and 6.9 mL
concd H2SO4 in 23.1 mL water) was added. The solution
was stirred at room temperature for 1.5 h and then quenched
by addition of MeOH (100 mL). The mixture was carefully
evaporated (caution: bumping) and the residue was
dissolved in water (200 mL) and EtOAc (200 mL). The
phases were separated and the aqueous phase was extracted
with EtOAc (2!200 mL). The organic phases were
4.3.8. H-Arg(Mtr)-Tyr-OtBu (12). The Fmoc group of 11
(2.01 g, 2.42 mmol) was removed using the same method as
in the synthesis of 10a. The product was purified using flash
chromatography (EtOAc/MeOH 10:1, RfZ0.31) to give 12
(1.33 g, 90%) as a white foam. [a]2D2ZC7 (c 0.5, MeOH);
1H NMR (300 MHz, MeOH-d4) d 7.01 (d, JZ8.5 Hz, 2H,

870
J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
combined and washed with water (2!200 mL), dried over
Na2SO4 and evaporated. The crude oxidation product was
dissolved in THF (45 mL) and H-Tyr-Tyr-Tyr-OtBu 10a
(1.44 g, 2.56 mmol), HOBt (0.346 g, 2.56 mmol),
EDC$HCl (0.515 g, 2.69 mmol), and N-methylmorpholine
(0.56 mL, 5.12 mmol) were added. After 16 h, the mixture
was concentrated, dissolved in MeOH and impregnated on
silica. The product was purified with flash chromatography
(Toluene/EtOAc 2:3, RfZ0.10) to give 14a (1.37 g, 45%).
as a white amorphous solid [a]22DZK5 (c 0.5, MeOH); 1H
NMR (DMSO-d6, 400 MHz) d 9.22 (s, 1H, Tyr-OH), 9.13
(s, 1H, Tyr-OH), 9.12 (s, 1H, Tyr-OH), 8.29 (d, JZ7.3 Hz,
1H, NH), 8.12 (d, JZ8.2 Hz, 1H, NH), 7.97 (d, JZ8.2 Hz,
1H, NH), 7.83 (d, JZ6.7 Hz, 2H, Fmoc), 7.79 (d, JZ
7.9 Hz, 2H, Bz-o), 7.75 (d, JZ7.2 Hz, 2H, Bz-o), 7.62 (m,
3H, Bz-pCNH), 7.51 (d, JZ7.6 Hz, 1H, Fmoc), 7.42 (t, JZ
7.7 Hz, 4H, Bz-m), 7.38 (d, JZ8.8 Hz, 1H, Fmoc), 7.34 (td,
JZ7.5, 3.4 Hz, 2H, Fmoc), 7.15 (m, 2H, Fmoc), 7.01 (d, JZ
8.4 Hz, 2H, Tyr-Hd), 6.95 (d, JZ8.5 Hz, 2H, Tyr-Hd), 6.90
(d, JZ8.4 Hz, 2H, Tyr-Hd), 6.67 (d, JZ8.5 Hz, 2H, Tyr-
H3), 6.59 (d, JZ8.4 Hz, 2H, Tyr-H3), 6.55 (d, JZ8.4 Hz,
2H, Tyr-H3), 5.50 (t, JZ9.9 Hz, 1H, SAA-H3), 5.33 (t, JZ
9.6 Hz, 1H, SAA-H4), 4.68 (t, JZ8.3 Hz, 1H, SAA-H1),
4.43 (m, 2H, 2!Tyr-Ha), 4.27 (m, 3H, 1!Tyr-HaCSAA-
H5C1!Fmoc-H), 4.15 (dd, JZ10.6, 6.9 Hz, 1H, Fmoc),
4.02 (t, JZ6.5 Hz, 1H, Fmoc), 3.73 (q, JZ9.2 Hz, SAA-
H2), 3.42 (s, 3H, OMe), 2.78 (m, 5H, 5!Tyr-Hb), 2.58 (dd,
JZ14.7, 8.1 Hz, 1H, Tyr-Hb), 1.31 (s, 9H, OtBu); HRMS
(FAB) calcd for C67H66N4O16Na (MCNa): 1205.4372;
found 1205.4388.
MeOH and impregnated on silica. The product was purified
using flash chromatography (CH2Cl2/MeOH 10:1, RfZ
0.27) followed by size-exclusion chromatography to give
17a (347 mg, 65%) as a white amorphous solid. [a]2D2ZK8
1
(c 0.5, MeOH); H NMR (DMSO-d6, 400 MHz) d 9.20 (s,
1H, Tyr-OH), 9.10 (s, 2H, 2!Tyr-OH), 9.07 (s, 1H, Tyr-
OH), 9.06 (s, 1H, Tyr-OH), 9.06 (s, 1H, Tyr-OH), 8.33 (d,
JZ8.8 Hz, 1H, NH), 8.27 (d, JZ7.3 Hz, 1H, NH), 8.11 (d,
JZ8.7 Hz, 1H, NH), 7.93 (m, 4H, 4!NH), 7.74 (m, 10H,
Bz-oC2!Fmoc-H), 7.55 (m, 5H, Bz-pCNH), 7.39 (m,
12H, Bz-mC4!Fmoc-H), 7.12 (m, 2H, Fmoc), 6.99 (d, JZ
8.4 Hz, 2H, Tyr-Hd), 6.90 (m, 6H, Tyr-Hd), 6.78 (d, JZ
9.8 Hz, 2H, Tyr-Hd), 6.75 (d, JZ9.1 Hz, 2H, Tyr-Hd), 6.65
(d, JZ8.3 Hz, 2H, Tyr-H3), 6.51 (m, 10H, Tyr-H3), 5.51 (t,
JZ9.8 Hz, 1H, SAA-H3), 5.46 (t, JZ9.9 Hz, 1H, SAA-H3),
5.33 (t, JZ9.7 Hz, 1H, SAA-H4), 5.28 (t, JZ9.9 Hz, 1H,
SAA-H4), 4.71 (d, JZ8.3 Hz, 1H, SAA-H1), 4.64 (d, JZ
7.8 Hz, 1H, SAA-H1), 4.31 (m, 9H, 2!SAA-H5C1!
Fmoc-HC6!Tyr-Ha), 4.11 (m, 2H, SAA-H2C1!Fmoc),
3.99 (t, JZ6.4 Hz, 1H, Fmoc), 3.69 (q, JZ9.1 Hz, 1H,
SAA-H2), 3.39 (s, 3H, OMe), 3.37 (s, 3H, OMe), 2.82–2.20
(m, 12H, Tyr-Hb), 1.28 (s, 9H, OtBu); HRMS (FAB) calcd
for C115H112N8O29Na (MCNa): 2091.7433; found
2091.7444.
4.4.4. H-SAA(di-O-Bz)-Tyr3-SAA(di-O-Bz)-Tyr3-OtBu
(18a). The title compound was prepared from compound
17a (334 mg, 0.161 mmol) using the method described in
the synthesis of 15a to give 18a (285 mg, 95%) as a
yellowish amorphous solid. [a]2D1ZK11 (c 0.5, MeOH); 1H
NMR (MeOH-d4, 300 MHz) d 7.86 (m, 8H, Bz-o), 7.49 (m,
4H, Bz-p), 7.35 (m, 8H, Bz-m), 6.97 (m, 6H, Tyr-Hd), 6.90
(d, JZ8.7 Hz, 2H, Tyr-Hd), 6.81 (d, JZ8.8 Hz, 2H, Tyr-
Hd), 6.79 (d, JZ8.9 Hz, 2H, Tyr-Hd), 6.69 (d, JZ8.3 Hz,
6H, Tyr-H3), 6.61 (d, JZ8.5 Hz, 4H, Tyr-H3), 6.57 (d, JZ
8.5 Hz, 2H, Tyr-H3), 5.73 (t, JZ10.0 Hz, SAA-H3), 5.42
(m, 3H, SAA-H3C2!SAA-H4), 4.77 (d, JZ8.3 Hz, 1H,
SAA-H1), 4.50–4.30 ppm (m, 7H, SAA-H1C6!Tyr-Ha),
4.26 (d, JZ10.0 Hz, 1H, SAA-H5), 4.15 (dd, JZ10.6,
8.4 Hz, 1H, SAA-H2), 4.12 (d, JZ9.9 Hz, 1H, SAA-H5),
3.56 (s, 3H, OMe), 3.48 (s, 3H, OMe), 3.04 (dd, JZ10.2,
8.0 Hz, 1H, SAA-H2), 2.95-2.40 (m, 12H, Tyr-Hb), 1.34 (s,
9H, OtBu); HRMS (FAB) calcd for C100H102N8O27Na (MC
Na): 1869.6752; found 1869.6736.
4.4.2. H-SAA(di-O-Bz)-Tyr3-OtBu (15a). Compound 14a
(400 mg, 0.338 mmol) was dissolved in THF (10 mL) and
N-(2-mercaptoethyl)aminomethyl polystyrene (2.0 mmol/g,
1.69 g) and DBU (76 mL, 0.507 mmol) were added. After
stirring the mixture for 6 h, the solid phase was filtered off
and washed with THF (2!8 mL) and MeOH (2!8 mL).
The filtrate and washings were combined and evaporated.
The residue was dissolved in CH2Cl2/MeOH 9:1 and filtered
through silica. Evaporation of the filtrate gave 15a (306 mg,
94%) as a yellowish amorphous solid. [a]2D2ZK13 (c 0.5,
1
MeOH); H NMR (MeOH-d4, 300 MHz) d 7.92 (d, JZ
7.8 Hz, 2H, Bz-o), 7.84 (d, JZ7.8 Hz, 2H, Bz-o), 7.50 (m,
2H, Bz-p), 7.35 (m, 4H, Bz-m), 6.99 (d, JZ8.5 Hz, 4H, Tyr-
Hd), 6.89 (d, JZ8.5 Hz, 2H, Tyr-Hd), 6.69 (d, JZ8.5 Hz,
2H, Tyr-H3), 6.68 (d, JZ8.5 Hz, 2H, Tyr-H3), 6.61 (d, JZ
8.5 Hz, 2H, Tyr-H3), 5.50 (t, JZ9.8 Hz, 1H, SAA-H3), 5.38
(t, JZ9.6 Hz, 1H, SAA-H4), 4.42 (m, 4H, SAA-H1C3!
Tyr-Ha), 4.20 (d, JZ9.8 Hz, 1H, SAA-H5), 3.56 (s, 3H,
OMe), 3.02 (dd, JZ10.1, 8.06 Hz, 1H, SAA-H2), 2.86 (m,
5H, 5!Tyr-Hb), 2.66 (dd, JZ13.7, 7.9 Hz, 1H, Tyr-Hb),
1.34 (s, 9H, OtBu); HRMS (FAB) calcd for C52H56N4O14Na
(MCNa): 983.3690; found 983.3687.
4.4.5. Cyclo[SAA(di-O-Bz)-Tyr3-SAA(di-O-Bz)-Tyr3]
(19a). Compound 18a (37.6 mg, 20.3 mmol) was dissolved
in CH2Cl2 (1.6 mL) and Et3SiH (8.1 mL, 51 mmol) and TFA
(0.8 mL) were added. The mixture was stirred for 4 h and
coevaporated with toluene. The crude product was dissolved
in THF (20 mL) and DIPEA (10 mL, 61 mmol) and HAPyU
(10.6 mg, 24.4 mmol) were added. The mixture was stirred
for 1 h at room temperature and then evaporated. The
product was purified by flash chromatography (CH2Cl2/
MeOH 6:1, RfZ0.29) followed by size-exclusion chroma-
tography to give 19a (13.3 mg, 37%) as a white amorphous
solid. [a]2D3ZK6 (c 0.5, MeOH); 1H NMR (DMSO-d6,
400 MHz, 150 8C) d 8.47 (s, 2H, Tyr-OH), 8.41 (s, 2H, Tyr-
OH), 8.34 (s, 2H, Tyr-OH), 7.86 (d, JZ8.4 Hz, 2H, NH),
7.80 (t, JZ7.2 Hz, 8H, Bz-o), 7.47 (m, 4H, Bz-p), 7.41 (d,
JZ8.0 Hz, 2H, NH), 7.34 (m, 10H, Bz-mC2!NH), 7.05
(d, JZ8.3 Hz, 2H, NH), 6.89 (d, JZ8.5 Hz, 4H, Tyr-Hd),
6.83 (d, JZ8.4 Hz, 4H, Tyr-Hd), 6.79 (d, JZ8.3 Hz, 4H,
4.4.3. Fmoc-SAA(di-O-Bz)-Tyr3-SAA(di-O-Bz)-Tyr3-
OtBu (17a). Compound 14a (313 mg, 0.265 mmol) was
dissolved in CH2Cl2 (4 mL) and Et3SiH (105 mL,
0.66 mmol) and TFA (2 mL) were added. The mixture
was stirred for 4 h and coevaporated with toluene. The crude
free acid was dissolved in THF (3 mL) and 15a (255 mg,
0.265 mmol), HOBt (35.8 mg, 0.265 mmol), and DIC
(50 mL, 0.32 mmol) were added. The mixture was stirred
for 16 h and then concentrated. The residue was dissolved in

J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
871
Tyr-Hd), 6.66 (d, JZ8.5 Hz, 4H, Tyr-H3), 6.60 (d, JZ
8.4 Hz, 4H, Tyr-H3), 6.52 (d, JZ8.5 Hz, 4H, Tyr-H3), 5.85
(t, JZ10.0 Hz, 2H, SAA-H3), 5.66 (t, JZ9.6 Hz, 2H, SAA-
H4), 5.10 (d, JZ8.0 Hz, 2H, SAA-H1), 4.49 (d, JZ9.7 Hz,
2H, SAA-H5), 4.35 (m, 4H, Tyr-Ha), 4.13 (q, JZ8.4 Hz,
2H, SAA-H2), 4.01 (q, JZ7.9 Hz, 2H, Tyr-Ha), 3.48 (s, 6H,
OMe), 2.9–2.6 (m, 12H, Tyr-Hb); HRMS (FAB) calcd for
C96H92N8O26Na (MCNa): 1795.6020; found 1795.6010.
9.6 Hz, 1H, SAA-H3), 5.40 (t, JZ9.5 Hz, 1H, SAA-H4),
5.08 (s, 2H, Bzl), 4.44 (d, JZ8.0 Hz, 1H, SAA-H1), 4.49 (t,
JZ6.6, 1H, Tyr-Ha), 4.40 (t, JZ6.9 Hz, 1H, Tyr-Ha), 4.25
(m, 2H, SAA-H5CGlu-Ha), 3.55 (s, 3H, OMe), 2.94 (m,
5H, SAA-H2CTyr-Hb), 2.26 (m, 2H, Glu-Hg), 1.95 (m, 1H,
Glu-Hb), 1.77 (m, 1H, Glu-Hb), 1.39 (s, 9H, OtBu); HRMS
(FAB) calcd for C55H60N4O15Na (MCNa): 1039.3953;
found 1039.3960.
4.4.6. Cyclo(SAA-Tyr3-SAA-Tyr3) (1a). Compound 19a
(89.1 mg, 50.3 mmol) was dissolved in MeOH (18 mL) and
NaOMe/MeOH (1 M, 180 mL) was added. The solution was
stirred for 18 h, then neutralised with AcOH and evaporated.
The residue was purified using size-exclusion chromato-
graphy on a short column to afford 1a (41.7 mg, 61%) as a
4.4.9. Fmoc-SAA(di-OBz)-Tyr-Glu(OBzl)-Tyr-SAA(di-
OBz)-Tyr-Glu(OBzl)-Tyr-OtBu (17b). The title com-
pound was prepared from 14b (604 mg, 0.487 mmol) and
15b (496 mg, 0.487 mmol) using the method described in
the synthesis of 17a. The product was purified with flash
chromatography (CH2Cl2/MeOH 15:1, RfZ0.18) followed
by size-exclusion chromatography to give 17b (767 mg,
72%) as a white amorphous solid. [a]2D2ZC4 (c 0.5,
DMSO); 1H NMR (DMSO-d6, 400 MHz) d 9.23 (s, 1H, Tyr-
OH), 9.16 (s, 1H, Tyr-OH), 9.13 (s, 1H, Tyr-OH), 9.10 (s,
1H, Tyr-OH), 8.36 (d, JZ8.5 Hz, 1H, NH), 8.25 (d, JZ
7.1 Hz, 1H, NH), 8.20 (m, 2H, 2!NH), 8.14 (d, JZ7.7 Hz,
1H, NH), 8.09 (d, JZ7.3 Hz, 1H, NH), 7.85 (m, 3H, 2!
Fmoc-HCNH), 7.77 (m, 4H, Bz-o), 7.70 (m, 4H, Bz-o),
7.55 (m, 6H, 4!Bz-pC1!NHC1!Fmoc-H), 7.37 (m,
21H, Bz-mC3!Fmoc-HC2!Bzl), 7.15 (m, 2H, Fmoc),
7.01 (d, JZ8.5 Hz, 2H, Tyr-Hd), 6.97 (d, JZ8.5 Hz, 2H,
Tyr-Hd), 6.97 (d, JZ8.6 Hz, 2H, Tyr-Hd), 6.77 (d, JZ
8.4 Hz, 2H, Tyr-Hd), 6.66 (d, JZ8.5 Hz, 2H, Tyr-H3), 6.60
(d, JZ8.4 Hz, 2H, Tyr-H3), 6.59 (d, JZ8.1 Hz, 2H, Tyr-
H3), 6.47 (d, JZ8.4 Hz, 2H, Tyr-H3), 5.51 (t, JZ9.8 Hz, 1H,
SAA-H3), 5.47 (t, JZ10.1 Hz, 1H, SAA-H3), 5.37 (t, JZ
9.8 Hz, 1H, SAA-H4), 5.31 (t, JZ9.7 Hz, 1H, SAA-H4),
5.06 (s, 2H, Bzl), 5.05 (s, 2H, Bzl), 4.71 (d, JZ8.2 Hz, 1H,
SAA-H1), 4.66 (d, JZ8.2 Hz, 1H, SAA-H1), 4.44 (m, 3H,
SAA-H5C2!Tyr-Ha), 4.29 (m, 5H, SAA-H5C2!Tyr-
HaC1!Glu-HaC1!Fmoc-H), 4.13 (m, 3H, SAA-H2C
1!Glu-HaC1!Fmoc-H), 4.02 (t, JZ6.5 Hz, 1H, Fmoc),
3.71 (q, JZ9.4 Hz, 1H, SAA-H2), 3.41 (s, 3H, OMe), 3.38
(s, 3H, OMe), 2.75 (m, 7H, 7!Tyr-Hb), 2.56 (dd, JZ15.3,
11.2 Hz, 1H, Tyr-Hb), 2.23 (t, JZ8.0 Hz, 2H, Glu-Hg), 2.10
(m, 2H, Glu-Hg), 1.83 (m, 1H, Glu-Hb), 1.70 (m, 2H, Glu-
Hb), 1.56 (m, 1H, Glu-Hb), 1.28 (s, 9H, OtBu); HRMS
(FAB) calcd for C121H120N8O31Na (MCNa): 2203.7957;
found 2203.7947.
1
white amorphous solid. [a]2D1ZK15 (c 0.5, MeOH); H
NMR (MeOH-d4, 300 MHz) d 6.97 (m, 12H, Tyr-Hd), 6.70
(m, 12H, Tyr-H3), 4.75 (d, JZ8.5 Hz, 2H, SAA-H1), 4.52
(dd, JZ9.4, 5.1 Hz, 2H, Tyr-Ha), 4.41 (dd, JZ8.5, 4.7 Hz,
2H, Tyr-Ha), 4.29 (t, JZ6.8 Hz, 2H, Tyr-Ha), 3.82 (d, JZ
9.7 Hz, 2H, SAA-H5), 3.78 (t, JZ9.4 Hz, 2H, SAA-H3),
3.41 (t, JZ9.4 Hz, 2H, SAA-H4), 3.33 (s, 6H, OMe), w3.3
(SAA-H2, obscured by solvent signal), 3.05–2.80 (m, 10H,
Tyr-Hb), 2.54 (dd, JZ14.1, 9.4 Hz, 2H, Tyr-Hb); HRMS
(FAB) calcd for C68H76N8O22Na (MCNa): 1379.4972;
found 1379.4955.
4.4.7. Fmoc-SAA(di-OBz)-Tyr-Glu(OBzl)-Tyr-OtBu
(14b). The title compound was prepared from 7 (1.09 g,
1.74 mmol) and 10b (1.08 g, 1.74 mmol) using the method
described in the synthesis of 14a. The product was purified
with flash chromatography (toluene/EtOAc 1:1, RfZ0.13)
to give 14b (1.39 g, 64%) as a white amorphous solid.
[a]2D2ZK12 (c 0.5, MeOH); 1H NMR (DMSO-d6,
400 MHz) d 9.19 (s, 1H, Tyr-OH), 9.11 (s, 1H, Tyr-OH),
8.19 (d, JZ7.0 Hz, 1H, NH), 8.15 (d, JZ7.9 Hz, 1H, NH),
8.14 (d, JZ8.0 Hz, 1H, NH), 7.80 (d, JZ7.8 Hz, 2H,
Fmoc), 7.75 (d, JZ7.3 Hz, 2H, Bz-o), 7.68 (d, JZ7.3 Hz,
2H, Bz-o), 7.52 (m, 4H, Bz-pC1!Fmoc-HC1!NH), 7.35
(m, 12H, 1!Fmoc-HCBz-mCBzl), 7.12 (m, 2H, Fmoc),
6.98 (d, JZ8.4 Hz, 2H, Tyr-Hd), 6.94 (d, JZ8.5 Hz, 2H,
Tyr-Hd), 6.64 (d, JZ8.5 Hz, 2H, Tyr-H3), 6.57 (d, JZ
8.4 Hz, 2H, Tyr-H3), 5.46 (t, JZ9.9 Hz, 1H, SAA-H3), 5.32
(t, JZ9.7 Hz, 1H, SAA-H4), 5.04 (s, 2H, Bzl), 4.65 (d, JZ
8.3 Hz, 1H, SAA-H1), 4.44 (dd, JZ12.5, 7.5 Hz, 1H, Tyr-
Ha), 4.31 (d, JZ10.0 Hz, 1H, SAA-H5), 4.22 (m, 3H, Tyr-
HaCGlu-HaC1!Fmoc-H), 4.13 (dd, JZ10.7, 6.9 Hz, 1H,
Fmoc), 3.99 (t, JZ6.7 Hz, 1H, Fmoc), 3.71 (q, JZ9.3 Hz,
1H, SAA-H2), 3.40 (s, 3H, OMe), 2.81 (m, 3H, 3!Tyr-Hb),
2.71 (dd, JZ14.2, 7.9 Hz, 1H, Tyr-Hb), 2.19 (m, 2H, Glu-
Hg), 1.81 (m, 1H, Glu-Hb), 1.64 (m, 1H, Glu-Hb), 1.26 (s,
9H, OtBu); HRMS (FAB) calcd for C70H70N4O17Na (MC
Na): 1261.4634; found 1261.4623.
4.4.10. H-SAA(di-OBz)-Tyr-Glu(OBzl)-Tyr-SAA(di-
OBz)-Tyr-Glu(OBzl)-Tyr-OtBu (18b). The title com-
pound was prepared from 17b (676 mg, 0.310 mmol)
using the method described in the synthesis of 15a to give
18b (599 mg, 99%) as a yellowish amorphous solid.
[a]2D2ZK23 (c 0.5, MeOH); 1H NMR (MeOH-d4,
400 MHz) d 7.72 (d, JZ8.5 Hz, 2H, Bz-o), 7.67 (d, JZ
8.4 Hz, 2H, Bz-o), 7.63 (d, JZ8.5 Hz, 2H, Bz-o), 7.58 (d,
JZ8.4 Hz, 2H, Bz-o), 7.28 (m, 4H, Bz-p), 7.12 (m, 18H,
Bz-mCBzl), 6.81 (m, 6H, Tyr-Hd), 6.63 (d, JZ8.5 Hz, 2H,
Tyr-Hd), 6.48 (m, 6H, Tyr-H3), 6.33 (d, JZ8.5 Hz, 2H, Tyr-
H3), 5.51 (t, JZ10.0 Hz, 1H, SAA-H3), 5.29 (t, JZ9.8 Hz,
1H, SAA-H3), 5.24 (t, JZ9.7 Hz, 1H, SAA-H4), 5.20 (t, JZ
9.6 Hz, 1H, SAA-H4), 4.87 (s, 4H, Bzl), 4.55 (d, JZ8.4 Hz,
1H, SAA-H1), 4.30 (t, JZ6.5 Hz, 1H, Tyr-Ha), 4.23 (m, 3H,
SAA-H1C2!Tyr-Ha), 4.16 (dd, JZ9.1 Hz, JZ5.0 Hz,
Tyr-Ha), 4.06 (m, 3H, 2!SAA-H5CGlu-Ha), 3.93 (m, 2H,
SAA-H2CGlu-Ha), 3.34 (s, 3H, OMe), 3.25 (s, 3H, OMe),
4.4.8. H-SAA(di-OBz)-Tyr-Glu(OBzl)-Tyr-OtBu (15b).
The title compound was prepared from 14b (720 mg,
0.581 mmol) using the method described in the synthesis of
15a to give 15b (534 mg, 90%) as a yellowish amorphous
1
solid. [a]2D2ZK24 (c 0.5, MeOH); H NMR (MeOH-d4,
300 MHz) d 7.91 (d, JZ7.9 Hz, 2H, Bz-o), 7.81 (d, JZ
8.5 Hz, 2H, Bz-o), 7.50 (m, 2H, Bz-p), 7.32 (m, 9H, Bz-mC
Bzl), 7.01 (d, JZ8.4 Hz, 4H, Tyr-Hd), 6.69 (d, JZ8.5 Hz,
2H, Tyr-H3), 6.67 (d, JZ8.4 Hz, 2H, Tyr-H3), 5.48 (t, JZ

872
J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
2.83 (dd, JZ10.1 Hz, JZ8.0 Hz, 1H, SAA-H2), 2.72 (m,
6H, 6!Tyr-Hb), 2.55 (dd, JZ14.2, 5.0 Hz, 1H, Tyr-Hb),
2.26 (dd, JZ13.9, 9.4 Hz, 1H, Tyr-Hb), 2.07 (m, 2H, 2!
Glu-Hg), 1.94 (m, 2H, 2!Glu-Hg), 1.72 (m, 1H, Glu-Hb),
1.57 (m, 3H, 3!Glu-Hb), 1.14 (s, 9H, OtBu); HRMS (FAB)
calcd for C106H110N8O29Na (MCNa): 1981.7276; found
1981.7268.
with flash chromatography (toluene/MeOH 7:1, RfZ0.21)
followed by size-exclusion chromatography to give 14c
(837 mg, 1.39 g, 46%) as a white foam. [a]2D4ZK5 (c 0.5,
MeOH); 1H NMR (DMSO-d6, 400 MHz) d 9.20 (s, 1H, Tyr-
OH), 9.12 (s, 1H, Tyr-OH), 8.15 (m, 2H, NH), 8.11 (d, JZ
8.2 Hz, 1H, NH), 7.82 (d, JZ7.0 Hz, 2H, Fmoc), 7.77 (d,
JZ7.3 Hz, 2H, Bz-o), 7.73 (d, JZ7.3 Hz, 2H, Bz-o), 7.55
(m, 4H, Bz-pC1!Fmoc-HC1!NH), 7.37 (m, 7H, 3!
Fmoc-HCBz-m), 7.15 (m, 2H, Fmoc), 7.00 (d, JZ8.4 Hz,
2H, Tyr-Hd), 6.97 (d, JZ8.5 Hz, 2H, Tyr-Hd), 6.66 (s, 1H,
Mtr-ArH), 6.66 (d, JZ8.4 Hz, 2H, Tyr-H3), 6.59 (d, JZ
8.3 Hz, 2H, Tyr-H3), 5.49 (t, JZ9.9 Hz, 1H, SAA-H3), 5.34
(t, JZ9.7 Hz, 1H, SAA-H4), 4.68 (d, JZ8.3 Hz, 1H, SAA-
H1), 4.46 (dd, JZ12.5, 7.6 Hz, 1H, Tyr-Ha), 4.35 (d, JZ
9.9 Hz, 1H, SAA-H5), 4.25 (m, 3H, Arg-HaCTyr-HaC1!
Fmoc-H), 4.15 (dd, JZ10.7 Hz, JZ6.9 Hz, 1H, Fmoc),
4.02 (t, JZ6.7 Hz, 1H, Fmoc), 3.76 (s, 3H, Mtr-OMe), 3.73
(q, JZ9.3 Hz, 1H, SAA-H2), 3.42 (s, 3H, SAA-OMe), 2.92
(br m, 2H, Arg-Hd), 2.81 (m, 3H, Tyr-Hb), 2.73 (dd, JZ
14.3, 7.7 Hz, 1H, Tyr-Hb), 2.59 (s, 3H, Mtr-Me), w2.50
(Mtr-Me, obscured by solvent signal), 2.03 (s, 3H, Mtr-Me),
1.52 (m, 1H, Arg-Hb), 1.28 (m, 3H, 1!Arg-HbC2!Arg-
Hg), 1.28 (s, 9H, OtBu); HRMS (FAB) calcd for C74H81-
N7O18SNa (MCNa): 1410.5257; found 1410.5261.
4.4.11. Cyclo[SAA(di-OBz)-Tyr-Glu(OBzl)-Tyr-SAA-
(di-OBz)-Tyr-Glu(OBzl)-Tyr] (19b). The title compound
was prepared from 18b (314 mg, 0.166 mmol) using the
method described in the synthesis of compound 19a. The
product was purified with flash chromatography (CH2Cl2/
MeOH 12:1, RfZ0.25) followed by size-exclusion chroma-
tography to give 19b (166 mg, 53%) as a white foam.
[a]2D2ZK3 (c 0.5, MeOH); 1H NMR (DMSO-d6, 400 MHz,
120 8C) d 8.49 (br s, 2H, Tyr-OMe), 8.33 (br s, 2H, Tyr-
OMe), 7.80 (m, 10H, Bz-oC2!NH), 7.48 (m, 6H, Bz-pC
2!NH), 7.32 (m, 18H, Bz-mCBzl), 7.05 (d, JZ7.8 Hz,
2H, NH), 6.99 (d, JZ8.4 Hz, 4H, Tyr-Hd), 6.81 (d, JZ
8.4 Hz, 4H, Tyr-Hd), 6.68 (d, JZ8.4 Hz, 4H, Tyr-H3), 6.50
(d, JZ8.4 Hz, 4H, Tyr-H3), 5.78 (t, JZ9.7 Hz, 2H, SAA-
H3), 5.60 (t, JZ9.4 Hz, 2H, SAA-H4), 5.09 (s, 4H, Bzl),
5.05 (d, JZ8.1 Hz, 2H, SAA-H1), 4.46 (d, JZ9.8 Hz, 2H,
SAA-H5), 4.43 (dd, JZ14.9, 7.7 Hz, 2H, Tyr-Ha), 4.34 (m,
2H, Tyr-Ha), 4.15 (dd, JZ10.0, 8.6 Hz, 2H, SAA-H2), 3.91
(dd, JZ7.7, 6.6 Hz, 2H, Glu-Ha), 3.48 (s, 6H, OMe), 3.00
(m, 4H, Tyr-Hb), 2.87 (dd, JZ14.4, 5.1 Hz, 2H, Tyr-Hb),
2.63 (dd, JZ14.4, 8.3 Hz, 2H, Tyr-Hb), 2.22 (m, 4H, Glu-
Hg), 1.90 (m, 4H, Glu-Hb); HRMS (FAB) calcd for
C102H100N8O28 (MCH): 1907.6545; found 1907.6549.
4.4.14. H-SAA(di-OBz)-Tyr-Arg(Mtr)-Tyr-OtBu (15c).
The title compound was prepared from 14c (400 mg,
0.288 mmol) using the method described in the synthesis
of 15a to give 15c (334 mg, 99%) as a yellowish foam.
[a]2D4ZK14 (c 0.5, MeOH); 1H NMR (MeOH-d4,
300 MHz) d 7.91 (d, JZ8.5 Hz, 2H, Bz-o) 7.79 (d, JZ
8.5 Hz, 2H, Bz-o), 7.53 (t, JZ7.5 Hz, 1H, Bz-p), 7.45 (t, JZ
7.4 Hz, 1H, Bz-p), 7.38 (t, JZ7.6 Hz, 2H, Bz-m), 7.29 (t,
JZ7.7 Hz, 2H, Bz-m), 7.01 (d, JZ8.4 Hz, 4H, Tyr-Hd),
6.69 (d, JZ8.5 Hz, 2H, Tyr-H3), 6.67 (d, JZ8.5 Hz, 2H,
Tyr-H3), 6.63 (s, 1H, Mtr-ArH), 5.48 (t, JZ9.6 Hz, 1H,
SAA-H3), 5.41 (t, JZ9.7 Hz, 1H, SAA-H4), 4.46 (t, JZ
6.7 Hz, 1H, Tyr-Ha), 4.46 (d, JZ8.0 Hz, 1H, SAA-H1), 4.39
(d, JZ7.2 Hz, 1H, Tyr-Ha), 4.26 (d, JZ9.5 Hz, 1H, SAA-
H5), 4.19 (dd, JZ8.3 Hz, JZ5.5 Hz, 1H, Arg-Ha), 3.79 (s,
3H, Mtr-OMe), 3.56 (s, 3H, SAA-OMe), 3.01 (m, 3H, SAA-
H2CTyr-Hd), 2.89 (m, 4H, Tyr-Hb), 2.65 (s, 3H, Mtr-Me),
2.59 (s, 3H, Mtr-Me), 2.09 (s, 3H, Mtr-Me), 1.61 (m, 1H,
Arg-Hb), 1.35 (m, 3H, 1!Arg-HbC2!Arg-Hg), 1.35 (s,
9H, OtBu); HRMS (FAB) calcd for C59H71N7O16SNa (MC
Na): 1188.4576; found 1188.4596.
4.4.12. Cyclo[SAA-Tyr-Glu-Tyr-SAA-Tyr-Glu-Tyr]
(1b). Palladium black (75 mg) was suspended in MeOH
containing 5% formic acid (3 mL). Compound 19b
(149 mg, 78.8 mmol) was dissolved in MeOH containing
5% formic acid (9 mL) and added to the suspension. After
20 min, the catalyst was filtered off (caution: catalyst may
catch fire when filtered to dryness), toluene (5 mL) was
added, and the mixture was evaporated. The residue was
dissolved in MeOH (30 mL) and NaOMe/MeOH (1 M,
450 mL) was added. The solution was stirred for 22 h, then
neutralised with AcOH, and evaporated. The residue was
dissolved in water and applied to a C18 cartridge. The
column was washed with water and the compound was
eluted with 30% MeOH in water to afford 1b (82.3 mg,
81%) as a fluffy white powder after lyophilization.
[a]2D2ZK31 (c 0.2, water); H NMR (D2O, 400 MHz) d
1
4.4.15. Fmoc-SAA(di-OBz)-Tyr-Arg(Mtr)-Tyr-SAA(di-
OBz)-Tyr-Arg(Mtr)-Tyr-OtBu (17c). The title compound
was prepared from 14c (315 mg, 0.227 mmol) and 15c
(265 mg, 0.227 mmol) using the method described in the
synthesis of 17a. The product was purified with flash
chromatography (CH2Cl2/MeOH 12:1, RfZ0.29) followed
by size-exclusion chromatography to give 17c (271 mg,
48%) as a white foam. [a]2D2ZC7 (c 0.5, MeOH); 1H NMR
(DMSO-d6, 400 MHz) d 9.21 (s. 1H, Tyr-OH), 9.13 (s. 1H,
Tyr-OH), 9.10 (s. 1H, Tyr-OH), 9.06 (s, 1H, Tyr-OH), 8.35
(d, JZ8.8 Hz, 1H, NH), 8.16 (m, 3H, 3!NH), 8.06 (d, JZ
8.2 Hz, 1H, NH), 7.82 (d, JZ7.5 Hz, 2H, Fmoc), 7.78 (m,
5H, 4!Bz-oC1!NH), 7.72 (m, 4H, 4!Bz-o), 7.55 (m,
6H, 4!Bz-pC1!Fmoc-HC1!NH), 7.40 (m, 11H, 3!
Fmoc-HC8!Bz-m), 7.15 (m, 2H, Fmoc), 7.01 (d, JZ
8.5 Hz, 2H, Tyr-Hd), 6.97 (d, JZ8.6 Hz, 2H, Tyr-Hd), 6.97
7.03 (d, JZ8.4 Hz, 8H, Tyr-Hd), 6.78 (d, JZ8.4 Hz, 4H,
Tyr-H3), 6.69 (d, JZ8.4 Hz, 4H, Tyr-H3), 4.58 (dd, JZ10.0,
4.6 Hz, 2H, Tyr-Ha), 4.51 (t, JZ6.2 Hz, 2H, Tyr-Ha), 4.45
(d, JZ8.7 Hz, 2H, SAA-H1), 3.86 (t, JZ7.1 Hz, 2H, Glu-
Ha), 3.68 (d, JZ10.0 Hz, 2H, SAA-H5), 3.66 (t, JZ9.4 Hz,
2H, SAA-H2), 3.40 (t, JZ9.5 Hz, 2H, SAA-H3), 3.32 (s, 6H,
OMe), 3.20 (m, 4H, 2!SAA-H4C2!Tyr-Hb), 2.92 (m,
4H, Tyr-Hb), 2.16 (m, 4H, 2!Tyr-HbC2!Glu-Hb), 2.03
(m, 2H, Glu-Hb), 1.88 (m, 4H, Glu-Hg); HRMS (FAB) calcd
for C60H72N8O24Na (MCNa):1311.4557; found 1311.4581.
4.4.13. Fmoc-SAA(di-OBz)-Tyr-Arg(Mtr)-Tyr-OtBu
(14c). The title compound was prepared from 7 (809 mg,
1.30 mmol) and 10c (998 mg, 1.30 mmol) using the method
described in the synthesis of 14a. The product was purified

J. F. Billing, U. J. Nilsson / Tetrahedron 61 (2005) 863–874
873
(d, JZ8.6 Hz, 2H, Tyr-Hd), 6.75 (d, JZ8.5 Hz, 2H, Tyr-
Hd), 6.66 (m, 4H, Tyr-H3C2!Mtr-ArH), 6.60 (d, JZ
8.4 Hz, 2H, Tyr-H3), 6.59 (d, JZ8.1 Hz, 2H, Tyr-H3), 6.47
(d, JZ8.4 Hz, 2H, Tyr-H3), 5.52 (t, JZ9.8 Hz, 1H, SAA-
H3), 5.48 (t, JZ9.7 Hz, 1H, SAA-H3), 5.38 (t, JZ9.7 Hz,
1H, SAA-H4), 5.33 (t, JZ9.6 Hz, 1H, SAA-H4), 4.72 (d,
JZ8.4 Hz, 1H, SAA-H1), 4.68 (d, JZ8.2 Hz, 1H, SAA-H1),
4.46 (m, 2H, 2!Tyr-Ha), 4.42 (d, JZ10.0 Hz, 1H, SAA-
H5), 4.35 (m, 2H, SAA-H5CTyrHa), 4.26 (m, 3H, Arg-
HaCTyr-HaC1!Fmoc-H), 4.15 (m, 1H, Fmoc), 4.08 (m,
2H, SAA-H2CArg-Ha), 4.02 (t, JZ6.7 Hz, 1H, Fmoc),
3.77 (s, 3H, Mtr-OMe), 3.76 (s, 3H, Mtr-OMe), 3.72 (q, JZ
9.7 Hz, 1H, SAA-H2), 3.42 (s, 3H, SAA-OMe), 3.41 (s, 3H,
SAA-OMe), 2.86 (m, 11H, 4!Arg-HdC7!Tyr-Hb), 2.59
(s, 6H, Mtr-Me), w2.50 (2!Mtr-Me, obscured by solvent
signal), 2.25 (br dd, JZ12.8, 8.7 Hz, 1H, Tyr-Hb), 2.03 (s,
3H, Mtr-Me), 2.01 (s, 3H, Mtr-Me), 1.53 (m, 1H, Arg-Hb),
1.43 (m, 1H, Arg-Hb), 1.29 (s, 9H, OtBu), 1.28 (m, 6H, 2!
4.4.18. Cyclo(SAA-Tyr-Arg-Tyr-SAA-Tyr-Arg-Tyr)
(1c). Compound 19c (25.5 mg, 11.7 mmol) was dissolved
in TFA containing 5% thioanisole (2.5 mL). After 24 h,
toluene (2.5 mL) was added and the mixture was evapor-
ated. The residue was dissolved in MeOH (5 mL) and
NaOMe/MeOH (1 M, 75 mL) was added. After 24 h, the
mixture was acidified with AcOH and evaporated. The
product was purified using preparative HPLC (C18 column,
15/20% B in A over 60 min, A: H2O C0.1% TFA, B:
CH3CN C0.1% TFA, tRZ10 min) to afford 1c (9.3 mg,
59%) as a fluffy white powder after lyophilization.
[a]2D2ZK15 (c 0.2, MeOH); H NMR (D2O, 400 MHz) d
1
7.03 (d, JZ8.4 Hz, 4H, Tyr-Hd), 7.00 (d, JZ8.2 Hz, 4H,
Tyrd), 6.77 (d, JZ8.3 Hz, 4H, Tyr3), 6.68 (d, JZ7.8 Hz, 4H,
Tyr3), 4.57 (m, 4H, 4!Tyr-Ha, partially obscured by
solvent signal), 4.43 (d, JZ8.6 Hz, 2H, SAA-H1), 3.81 (t,
JZ7.0 Hz, 2H, Arg-Ha), 3.66 (m, 4H, SAA-H5CSAA-H2),
3.40 (t, JZ9.8 Hz, 2H, SAA-H3), 3.32 (s, 6H, OMe), 3.18 (t,
JZ9.5 Hz, SAA-H4), 3.11 (dd, JZ15.1, 7.4 Hz, 2H, Tyr-
Hb), 2.96 (m, 8H, 4!Tyr-HbC4!Arg-Hd), 2.32 (dd, JZ
12.3, 11.6 Hz, 2H, Tyr-Hb), 1.66 (m, 2H, Arg-Hb), 1.52 (m,
2H, Arg-Hb), 1.27 (m, 2H, Arg-Hg), 1.16 (m, 2H, Arg-Hg);
HRMS (FAB) calcd for C62H82N14O20Na (MCNa):
1365.5728; found 1365.5729.
Arg-HbC4!Arg-Hg); HRMS (FAB) calcd for C129H142
N14O33S2Na (MCNa): 2501.9203; found 2501.9209.
-
4.4.16. H-SAA(di-OBz)-Tyr-Arg(Mtr)-Tyr-SAA(di-
OBz)-Tyr-Arg(Mtr)-Tyr-OtBu (18c). The title compound
was prepared from 17c (291 mg, 0.117 mmol) using the
method described in the synthesis of 15a to give 18c
(254 mg, 96%) as a yellowish amorphous solid. [a]2D2Z
K16 (c 0.5, MeOH); 1H NMR (MeOH-d4, 400 MHz) d 7.91
(d, JZ8.5 Hz, 2H, Bz-o) 7.85 (d, JZ8.6 Hz, 2H, Bz-o), 7.82
(d, JZ8.5 Hz, 2H, Bz-o), 7.77 (d, JZ8.5 Hz, 2H, Bz-o),
7.47 (m, 4H, Bz-p), 7.31 (m, 8H, Bz-m), 7.00 (m, 6H, Tyr-
Hd), 6.85 (d, JZ8.5 Hz, 2H, Tyr-Hd), 6.67 (m, 6H, Tyr-H3),
6.62 (s, 1H, Mtr-ArH), 6.61 (s, 1H, Mtr-ArH), 6.56 (d, JZ
8.5 Hz, 2H, Tyr-H3), 5.74 (t, JZ9.9 Hz, 1H, SAA-H3), 5.47
(t, JZ9.7 Hz, 1H, SAA-H3), 5.42 (t, JZ9.6 Hz, 1H, SAA-
H4), 5.40 (t, JZ9.6 Hz, 1H, SAA-H4), w4.81 (SAA-H1,
partially obscured by solvent signal), 4.46 (t, JZ6.7 Hz, 1H,
Tyr-Ha), 4.45 (d, JZ8.1 Hz, 1H, SAA-H1), 4.40 (m, 3H,
3!Tyr-Ha), 4.33 (d, JZ9.8 Hz, 1H, SAA-H5), 4.25 (d, JZ
9.7 Hz, 1H, SAA-H5), 4.21 (dd, JZ8.4, 5.2 Hz, 1H, Arg-
Ha), 4.14 (m, 2H, Arg-HaCSAA-H2), 3.78 (s, 3H, Mtr-
OMe), 3.77 (s, 3H, Mtr-OMe), 3.55 (s, 3H, SAA-OMe),
3.47 (s, 3H, SAA-OMe), 3.02 (m, 3H, SAA-H2CArg-Hd),
2.92 (m, 8H, 2!Arg-HdC6!Tyr-Hb), 2.77 (dd, JZ14.0,
5.1 Hz, 1H, Tyr-Hb), 2.66 (s, 3H, Mtr-Me), 2.65 (s, 3H, Mtr-
Me), 2.59 (s, 6H, 2!Mtr-Me), 2.51 (dd, JZ14.5 Hz, JZ
9.7 Hz, 1H, Tyr-Hb), 2.08 (s, 3H, Mtr-Me), 2.06 (s, 3H, Mtr-
Me), 1.63 (m, 1H, Arg-Hb), 1.54 (m, 1H, Arg-Hb), 1.44 (m,
1H, Arg-Hb), 1.35 (m, 12H, 1!Arg-HbC2!Arg-HgC
OtBu), 1.19 (m, 2H, Arg-Hg); HRMS (FAB) calcd for
C114H132N14O31S2Na (MCNa): 2279.8522; found
2279.8530.
4.5. NMR titrations
All experiments were performed at 400 MHz in a deuterated
phosphate buffer (100 mM phosphate, pH 7.2). In the
titration experiments, a stock solution with 0.5 mM receptor
concentration was prepared. The ligand to be titrated was
dissolved in a portion of the stock solution to give a solution
with 0.5 mM receptor and 100 mM ligand. These two
solutions were mixed in different proportions to give a series
of solutions with 0.5 mM receptor and ligand concentrations
up to 80 mM. 1H NMR experiments were performed on the
solutions and the chemical shifts of receptor signals were
fitted to a 1:1 binding isotherm using non-linear
regression.31 Acidic and basic ligands were used as sodium
salts and hydrochloride salts, respectively.
Acknowledgements
We thank the Swedish Research Council, the Swedish
Strategic Research Foundation, and the program ‘Glyco-
conjugates in Biological Systems’ sponsored by the
Swedish Strategic Research Foundation for financial
support.
4.4.17. Cyclo[SAA(di-OBz)-Tyr-Arg(Mtr)-Tyr-SAA(di-
OBz)-Tyr-Arg(Mtr)-Tyr] (19c). The title compound was
prepared from 18c (89.3 mg, 39.5 mmol) using the method
described in the synthesis of compound 19a. The product
was purified with flash chromatography (CH2Cl2:MeOH
8:1, RfZ0.32) followed by size-exclusion chromatography
to give 19c (29.3 mg, 34%) as a white amorphous solid.
[a]2D4ZK5 (c 0.5, MeOH); HRMS (FAB) calcd for
C110H122N14O30S2Na (MCNa): 2205.7790; found
2205.7795. A resolved NMR spectrum could not be
obtained.
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