Solid-Phase Synthesis of DOTA-Peptides

Article abstract of DOI:10.1002/chem.200305389

A general synthetic route to two DOTA-linked N-Fmoc amino acids (DOTA-F and DOTA-K) is described that allows insertion of DOTA at any endo-position within a peptide sequence. Three model pentapeptides were prepared to test the general utility of these derivatives in solid-phase peptide synthesis. Both DOTA derivatives reacted smoothly by means of standard HBTU activation chemistry to the point of insertion of the DOTA amino acid, but extension of the peptide chain beyond the DOTA-amino acid insertion required the use of pre-activated C-pentafluorophenyl ester N-α-Fmoc amino acids. Three Gal-80 binding peptides (12-mers) were then prepared by using this methodology with DOTA positioned either at the N terminus or at one of two different internal positions;the binding of the resulting GdDOTA-12-mers to Gal-80 were compared. The methodology described here allows versatile, controlled introduction of DOTA into any location within a peptide sequence. This provides a potential method for the screening of libraries of DOTA-linked peptides for optimal targeting properties.

Full text of DOI:10.1002/chem.200305389

FULL PAPER
Solid-Phase Synthesis of DOTA Peptides
Luis M. De LeÛn-Rodriguez,[a] Zoltan Kovacs,[a, b] Gregg R. Dieckmann,[a] and
A. Dean Sherry*[a, c]
Abstract: A general synthetic route to
two DOTA-linked N-Fmoc amino
acids (DOTA-F and DOTA-K) is de-
scribed that allows insertion of DOTA
at any endo-position within a peptide
sequence. Three model pentapeptides
were prepared to test the general utili-
ty of these derivatives in solid-phase
peptide synthesis. Both DOTA deriva-
tives reacted smoothly by means of
standard HBTU activation chemistry
to the point of insertion of the DOTA
amino acid, but extension of the pep-
tide chain beyond the DOTA-amino
acid insertion required the use of pre-
activated C-pentafluorophenyl ester N-
a-Fmoc amino acids. Three Gal-80
binding peptides (12-mers) were then
prepared by using this methodology
with DOTA positioned either at the N
terminus or at one of two different in-
ternal positions;the binding of the re-
sulting GdDOTA-12-mers to Gal-80
were compared. The methodology de-
scribed here allows versatile, controlled
introduction of DOTA into any loca-
tion within a peptide sequence. This
provides a potential method for the
screening of libraries of DOTA-linked
peptides for optimal targeting proper-
ties.
Keywords: gadolinium complexes ¥
macrocyclic ligands
solid-phase synthesis
¥ peptides ¥
Introduction
larly useful for screening peptide libraries for targets of spe-
cific cell types, tissues, or even individual macromole-
cules.[15,16] The rapid development of such procedures has
made metal-ion-conjugated peptides ideal agents for diag-
nostic and therapeutic biomedicine. Several types of ligands
including DOTA, DTPA, NOTA and TETA,[7] have been at-
tached to peptides. DOTA is of particular interest, since this
macrocyclic ligand forms complexes with a variety of metal
ions with exceptionally high binding affinities and kinetic
A number of low-molecular-weight peptides have been iden-
tified for targeting to specific cell receptors or macromole-
cules. Early applications of such systems have focused on
peptide radiopharmaceuticals for cancer diagnosis and ther-
apy,[1 but reports of peptide-based targeting vectors are
7]
beginning to appear in the molecular imaging literature, par-
ticularly those applications involving optical and magnetic
11]
resonance (MRI) imaging.[8
Peptide-based pharmaceuti-
stabilities.[17] DOTA peptides have been prepared by conju-
20]
cals offer some advantages over typical organic-targeting an-
alogues in that they can be prepared in reasonable quanti-
ties by using well-developed solid-phase peptide synthesis
(SPPS) methodologies.[12,13] Established combinatorial tech-
niques can then be used to create libraries of peptides for
screening purposes.[14] Phage display techniques are particu-
gating unprotected DOTA[18
or, more conveniently,
24]
DOTA-tris(tBu) esters[8,11,21
to the N terminus or a Lysꢀ
eꢀNH2 residue of resin bound peptides. In a different ap-
proach, DOTA peptides were obtained by synthesizing the
DOTA moiety in a stepwise manner on the N terminus of
peptides bound to the solid support.[25] A recent communica-
tion reported a DOTA peptide nucleic acid conjugate that
was synthesized by using a DOTA-Lys derivative that allows
the DOTA to be incorporated into any sequence position.[26]
Recently, we demonstrated[11] that MRI can detect the
binding event of a Gd3+ DOTA-labeled peptide to its
target protein (Gal-80 is a protein involved in regulation of
galactose metabolism[27]). DOTA was added to the N-termi-
[a] Dr. L. M. De LeÛn-Rodriguez, Dr. Z. Kovacs, Dr. G. R. Dieckmann,
Prof. A. D. Sherry
Department of Chemistry, University of Texas at Dallas
P.O. Box 830688, Richardson, TX 75083 (USA)
Fax : +(1)972-883-2925
E-mail: sherry@utdallas.edu
[b] Dr. Z. Kovacs
Macrocyclics, Inc., 17815 Davenport Road
Suite 120, Dallas, TX 75252 (USA)
nal
position
of
a
twelve
residue
peptide
(TFDDLFWKEGHR)[28,29] by treating the resin-bound pep-
tide with DOTA-tris(tBu) ester. Although the resulting
DOTA peptide displayed a ~5-fold lower affinity for Gal-
80, the binding event could still be detected at 25mm, sub-
stantially lower than the detection limits of typical Gd3+
[c] Prof. A. D. Sherry
Department of Radiology, Rogers Magnetic Resonance Center
University of Texas Southwestern Medical Center
5801 Forest Park Road, Dallas, TX 75235-9085 (USA)
E-mail: sherry@utdallas.edu
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DOI: 10.1002/chem.200305389
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER
based contrast agents. Given a
recent report of antibody rec-
ognition of simple LnDOTA
chelates,[30] it became evident
that one might be able to
lower the MRI detection limit
of a Gd peptide/protein recog-
nition process even further if
GdDOTA was located in an
endo-peptide position forming
part of the protein binding site.
To make the method as versa-
tile as possible, we prepared
tris(tBu) ester DOTA deriva-
tives of N-a-Fmoc amino acids
that could be substituted for
standard N-a-Fmoc amino
acids in SPPS, making it possi-
ble to position DOTA any-
where in a peptide sequence.
The methods reported here
should be useful for generating
Ln3+-DOTA peptide libraries
for screening purposes.
Scheme 1. Synthesis of DOTA phenylalanine derivative (8). Reagents and conditions: a) benzyl chlorofor-
mate, Na2CO3, dioxane, water, 08C; b) benzylbromide, DIEA; c) Zn, EtOH, AcOH, 608C; d) bromoacetyl-
bromide, DIEA, CH2Cl2; e) DO3A-(tBu)3, K2CO3, CH3CN; f) H2, Pd on C, iPrOH; g) Fmoc-chloride,
Na2CO3, dioxane, water.
Results and Discussion
Preparation of DOTA-conju-
gated amino acids: Using the
amino acid sequence of the
Gal-80 binding peptide identi-
fied previously by phage dis-
play
(TFDDLFWKEGHR),
we first chose to prepare three
DOTA peptides differing only
in the location of DOTA
within this sequence. Conjuga-
tion of DOTA to the N termi-
nus of the resin-bound peptide
was easily accomplished using
Scheme 2. Synthesis of DOTA-lysine derivative 15. Reagents and conditions: a) benzyl bromide, DIEA,
MeCN; b) TFA, CH2Cl2; c) bromoacetyl bromide, DIEA, CH2Cl2; d) DO3A-(tBu)3, K2CO3, MeCN; e) H2, Pd
on C, iPrOH; f) Fmoc-Cl, Na2CO3, dioxane, water.
DOTA-tris(tBu) ester and standard Fmoc chemistry.[11] To
introduce DOTA into any endo-position within the se-
quence requires DOTA amino acid derivatives fully compat-
ible with SPPS conditions. This includes N-a-Fmoc protec-
tion, a free carboxyl group for coupling, and acid labile pro-
tection of the remaining groups on DOTA. Two model
amino acid derivatives (DOTA-F and DOTA-K) were
chosen to illustrate the method. Lysine and phenylalanine
were selected to test the feasibility of applying the proposed
chemistry to a charged/aliphatic versus an aromatic side-
chain. For both derivatives, the a-amino and carboxylic
groups of the parent amino acids were protected with the
carboxybenzyl (Cbz) and benzyl (Bn) protecting groups, re-
spectively. These two protecting groups can be removed
later by catalytic hydrogenation (Schemes 1 and 2). The flu-
orenylmethyoxycarbonyl (Fmoc) protecting group cannot be
introduced at this point due to its susceptibility to hydroge-
nation.[31] For DOTA-F (Scheme 1), reduction of the nitro
group of p-NO2-phenylalanine to the amine followed by
coupling with bromoacetyl bromide provided intermediate
5. This was then coupled to DO3A-tris(tBu) ester to give in-
termediate 6. Removal of the Cbz and Bn groups by catalyt-
ic hydrogenation and introduction of the Fmoc protecting
group yielded product 8 in approximately 70% overall
yield. Synthesis of DOTA-K was accomplished by using a
similar strategy starting with N-e-Boc-protected lysine
(Scheme 2). The resulting DOTA tris(tBu) esteramino acid
derivatives were then used to create DOTA peptides by
using SPPS methods.
Solid-phase peptide synthesis: To test the general reactivity
of DOTA-F, three different peptide sequences, each contain-
ing five residues but with a variable DOTA-F position, were
selected for synthesis. The reference peptides (GAADF,
GAFDG, FAADG) were prepared first, giving an average
yield of 65% after purification. Synthesis of (NH2-F(DO-
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DOTA Peptide Synthesis
1149 1155
TA)AADG-CONH2) using standard Fmoc-protected amino
acid chemistry proceeded smoothly but in those sequences
where F-DOTA was located at the C-terminus or near the
center of the sequence, the peptide sequence could not be
extended beyond the F-DOTA position. Furthermore, at-
tempts to add subsequent amino acids beyond F-DOTA
using either HATU[32] or TFFH[33] as coupling agents were
not successful. These coupling agents are reported[32,33] to
have superior coupling capabilities over HBTU for synthesis
of ™difficult∫ peptide sequences.
These unexpected results suggest that the bulky DOTA
tris(tBu) ester moiety on the peptide alters the coupling effi-
ciency when using HBTU-type activating agents. The fact
that N-acetyl (capped) derivatives (Ac-F(DOTA)-CONH2
and Ac-F(DOTA)DG-CONH2) were obtained as major
products demonstrated that the Fmoc group of DOTA-F
had been removed and that the N-terminal amino groups of
these derivatives were available for reaction with anhydride.
This suggested to us that a more reactive, pre-activated
amino acid might be necessary to couple additional amino
acids beyond the F-DOTA residue. Indeed, when N-a-Fmoc
pentafluorophenyl ester (OPfp) derivatives were used, the
remaining amino acids of the endo-DOTA peptides were
added with an average overall yield of 40% (Scheme 3).
Similar chemistry was then used to prepare an analogous
K-DOTA sequence (NH2-FWK(DOTA)LG-CONH2).
A recent report showed that N-a-Fmoc-DOTA-e-lysine
could be introduced into a peptide sequence by using stan-
dard HBTU-type activating agents when using a XAL-
PEG-PS resin.[26] PEG-PS solid supports are prepared by
grafting readily soluble polar PEG chains onto microporous
polystyrene-co-divinylbenzene and have been shown to be
superior to conventional resins for the synthesis of hydro-
phobic peptides.[35] This suggests that the DOTA tris(tBu)
estermoiety attached to peptides on the Rink amide resin
causes the growing peptide to aggregate and that this aggre-
gation may be prevented using a PEG-PS based resin.
analogues of the Gal-80 peptide binding sequence,
TFDDLFWKEGHR, shown in Table 1. After cleavage of
these peptides from Rink resin using trifluoroacetic acid and
Table 1. Derivatives of the Gal-80 binding peptide (BP).
Abbreviation
Peptide Sequence
Fluorescein-BP
N-acetyl-BP
Fluorescein-TFDDLFWKEGHR-CONH2
Ac-TFDDLFWKEGHR-CONH2
Exo-DOTA-BP
Endo-DOTA(K)-BP
Endo-DOTA(F)-BP
DOTA-TFDDLFWKEGHR-CONH2
Ac-TFDDLFW(K-DOTA)EGHR-CONH2
Ac-TFDDL(F-DOTA)WKEGHR-CONH2
purification by HPLC, the Gd3+ complexes were formed
and the resulting GdDOTA peptides were purified once
again by HPLC. The association constants of each
GdDOTA peptide with Gal-80 were determined by compet-
itive binding against fluorescein-TFDDLFWKEGHR-
CONH2 using fluorescence polarization.[11] A saturation ex-
periment was first performed to determine the KD for the
fluorescein-BP/Gal-80 interaction. A fit of these data to a
model that assumes 1:1 binding stoichiometry gave a KD of
173 ꢁ 13nm,which is similar to the value obtained previous-
ly from a different protein preparation.[11] Competition bind-
ing experiments were then performed by starting with 2nm
fluorescein-BP and 130nm Gal-80 and titrating in aliquots of
either N-acetyl-BP, exo-GdDOTA-BP, endo-GdDOTA(F)-
BP, or endo-GdDOTA(K)-BP. Competition binding curves
for the first three systems are presented in Figure 1. Addi-
tion of endo-GdDOTA(K)-BP gave similar results as that
shown for endo-GdDOTA(F)-BP. It was clear from these
data that both exo-peptides, N-acetyl-BP and exo-
GdDOTA-BP, compete effectively with fluorescein-BP,
while the two endo-GdDOTA peptides do not. The compet-
itive binding constants, KD’, for the two exo-peptides were
determined using Equation (1),[34] in which IC50 corre-
sponds to the concentration of competing peptide required
to decrease the fluorescence polarization by 50%. This fit-
DOTA conjugates of a Gal-80 binding peptide: This same
methodology was then used to synthesize the three DOTA
Scheme 3. Solid-phase peptide synthesis of three pentapetides with variable DOTA position. Reagents and conditions: a) Rink amide resin, Fmoc-amino
acids, HBTU, HOBT, DIEA; b) Fmoc-amino acid pentafluorophenyl esters, HOBT; c) CF3CO2H, thioanisole, 1,2-ethanedithiol, anisole.
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A. D. Sherry et al.
FULL PAPER
the native peptide are especially critical to the protein pep-
tide interaction and that introduction of the GdDOTA
moiety disrupts these crucial binding interactions. Even
though the exo-GdDOTA-BP/Gal-80 binding constant is
only ~26mm, this interaction has been shown to be strong
enough to allow detection of binding by MRI.[11]
Conclusion
The Gal-80 binding peptide, TFDDLFWKEGHR, was
modified by introducing a DOTA-chelating group at three
different residues (Thr1, Phe6, or Lys8). After addition of
Gd3+ to each peptide DOTA conjugate, competition bind-
ing experiments showed that the peptide labeled with
GdDOTA at the N-terminal (T) had a reasonable affinity
for Gal-80 (Ka =3.9î104 mꢀ1), while those peptides labeled
with GdDOTA at either Phe6 or Lys8 had no detectable
binding affinity for Gal-80. This demonstrates that location
of the GdDOTA moiety greatly affects the binding specifici-
ty of a peptide protein system in which structure activity
relationships have not been previously well defined, and il-
lustrates that screening libraries of DOTA-linked peptides
may be useful in identifying targeted metal-ion-based sys-
tems for new MRI diagnostic applications.
Experimental Section
General procedures: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroni-
um hexafluoro phosphate (HBTU), N-hydroxybenzotriazole (HOBT),
Fmoc-L-amino acids, Fmoc-L-amino acid pentafluorophenyl esters
(OPfp) and Rink amide resin were obtained from NovaBiochem (San
Diego, CA). Fmoc-L-threonine (OtBu)-OPfp was obtained by esterifica-
tion of Fmoc-L-threonine(OtBu) with pentafluorophenol (Lancaster) by
using DCC (Sigma). 2-(1H-Azabenzotriazole-1-yl)-1,1,3,3-tetramethy-
luronium hexafluorophosphate (HATU) and tetramethylfluoroformami-
dinium hexafluorophosphate (TFFH) were purchased from Applied Bio-
systems (Foster City, CA). 1,4,7-Tris(tert-butylacetate)-1,4,7,10-tetraaza-
cyclododecane (DO3A-tris(tBu)) ester was supplied by Macrocyclics
(Dallas, TX). Silica gel (200 400 mesh, 60 ä) for column chromatography
was purchased from Aldrich. TLC was conducted on Whatman precoated
silica gel on polyester plates. All other chemicals and solvents were pur-
chased from commercial sources and were used without further purifica-
Figure 1. Changes in fluorescence polarization (measured in mA) upon
incremental addition of either N-acetyl-BP (A), exo-GdDOTA-BP (B),
or endo-GdDOTA(F)-BP (C) to a solution of 2nm fluorescein-BP plus
130nm Gal-80. [ represents a data point where no competing peptide has
been added, and * represents a data point where a large excess of com-
peting peptide has been added and all fluorescein-BP is assumed to be
free.
ting procedure gave a KD’ of 6.4ꢁ0.3mm for N-acetyl-BP
and 25.9ꢁ0.9mm for exo-GdDOTA-BP. (This KD value is
~10-fold higher than the value reported in reference 11 due
to an error in the previous calculation.)
tion. 1H and 13C NMR spectra were taken using
a JEOL Eclipse
270 MHz spectrometer with tetramethylsilane as the standard. Chemical
shifts are reported as parts per million (ppm), with splitting patterns des-
ignated as singlet (s), doublet (d), triplet (t), multiplet (m), or doublet of
doublets (dd). Coupling constants are reported in Hertz (Hz). No special
IC50
ꢀ
ꢁ
KD0ðcompeting peptideÞ
¼
ð1Þ
1 þ ½flourescein-BPꢂ=KDð177 nmÞ
1
experiments were performed to obtain definitive H and 13C peak assign-
ments. Melting points were determined by using a Fisher Johns Melting
Point Apparatus and are uncorrected. HPLC analysis was performed
over a C18 reverse-phase column on an HP1100 HPLC instrument equip-
ped with an automated injector and 1100 Series diode array UV/Vis de-
tector. Peptide purification was performed on a semi-preparative HPLC
(Waters 600) using a C18 reverse-phase column. Products were eluted at
flow rates of 1 mLminꢀ1 (HP1100) or 10 mLminꢀ1 (Waters) using a
linear gradient of H2O/TFA(0.1%) and acetonitrile/H2O/TFA (90/10/0.1).
This illustrates that the chemical properties of the N-ter-
minal group (fluorescein versus acetyl versus GdDOTA) has
a dramatic effect on the resulting peptide/Gal-80 binding in-
teraction either as a result of altered peptide conformation
or by additional repulsive forces between the N-terminal
moiety and the protein. This binding order suggests that hy-
drophobic interactions at the N-terminal site are more fa-
vorable (fluorescein-BP) than hydrophilic interactions (exo-
GdDOTA-BP). Neither endo-GdDOTA(K)-BP nor endo-
GdDOTA(F)-BP could significantly displace fluorescein-BP
from Gal-80, even at very highly competitive concentrations.
This indicates that the phenylalanine and lysine residues in
N-a-Carbobenzyloxy-p-nitro-L-phenylalanine (2): p-Nitro-L-phenylala-
nine (1) (9.56 g, 45.5 mmol) was dissolved in a mixture of dioxane
(80 mL) and Na2CO3 solution (16.5 g, 3 equiv, in 145 mL water) and
cooled in an ice bath. Benzyl chloroformate (9.56 g, 95%, 1.05 equiv) in
dioxane (100 mL) was added dropwise while stirring. The reaction mix-
ture was allowed to stand at RT overnight. The solvent was removed by
rotary evaporation, and the residue was dissolved in ethyl acetate
1152
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Chem. Eur. J. 2004, 10, 1149 1155

DOTA Peptide Synthesis
1149 1155
(100 mL), washed with water (50 mL), brine (2î50 mL) and water (2î
50 mL). The solvent was evaporated to give a clear oil that crystallized
from pentane/CH2Cl2. The white solid was filtered off and dried under
vacuum overnight to yield 15.02 g (95.6%) of product. M.p.: 126 1278C;
1H NMR (270 MHz, CDCl3, 258C, TMS):: d=8.07 (d, 2H), 7.33 7.29 (m,
7H), 5.38 (d, 1H), 5.06 (m, 2H), 4.71 (t, 1H), 3.29 ppm (m, 2H);
13C NMR (67.5 MHz, CDCl3, 258C, TMS): d=174.8, 155.9, 147.2, 143.6,
135.8, 130.4, 128.7 128.3, 123.8, 67.5, 54.4, 37.8 ppm. Elemental analysis
calcd. (%) for C17H18N2O6: C 59.30, H 4.68, N 8.14. Found: C 59.01, H
4.60, N 8.11; MS-ESI: m/z: 367 (calcd.367) [M+Na]+, 343 (calcd.343)
[MꢀH]ꢀ.
N-a-Carbobenzyloxy-p-nitro-L-phenylalanine benzyl ester (3): Com-
pound (2) (14.90 g, 43.2 mmol) was suspended in acetonitrile (300 mL),
then diisopropyl ethyl amine (6.42 g, 1.1 equiv) was added to give a
yellow homogeneous solution. Benzyl bromide (8.30 g, 1.05 equiv) in ace-
tonitrile (100 mL) was added and the flask was purged with argon and
sealed. The reaction mixture was stirred at RT for 2 days. Solid formation
was observed during the reaction. When the reaction was complete as
monitored by TLC, the solvent was evaporated and the oily residue was
dissolved in dichloromethane (500 mL) and washed with water (3î
400 mL). The organic phase was collected, and dried over Na2SO4, and
the solvent was evaporated to yield a white solid. It was dried under
vacuum overnight to give 17.9 g (95.1%) of product. Rf =0.68 (SiO2
TLC; hexanes/ethyl acetate, 60:40); m.p. 84 858C; 1H NMR (270 MHz,
CDCl3 258C, TMS):: d=7.96 (d, 2H), 7.35 7.32 (m, 10H), 7.08 (d, 2H,)
5.34 (d, 1H), 5.18 (m, 2H) 5.06 (m, 2H), 4.71 (t, 1H), 3.17 ppm (m, 2H);
13C NMR (67.5 MHz, CDCl3, 258C, TMS):: d=170.7, 155.5, 147.1, 143.5,
136.1, 134.8, 130.6, 128.9 128.3, 123.6, 67.7, 67.2, 54.5, 38.2 ppm. Elemen-
tal analysis calcd. (%) for C24H22N2O6: C 66.35, H 5.10, N 8.45. Found: C
66.00, H 4.98, N 8.48; MS-ESI: m/z: 457 (calcd.457) [M+Na]+.
12.9 mmol). The temperature was increased to 608C using an oil bath,
and the resultant slurry was stirred for 15 minutes. Compound (5) (2.68 g,
5.1 mmol) in acetonitrile (50 mL) was added dropwise over 20 minutes
and the solution was stirred at 608C for three days. After filtering out re-
sidual solids the solvent was evaporated to give a quantitative yield of
yellow oil. 1H NMR (270 MHz, CDCl3, 258C, TMS): d=10.40 (s, 1H)
7.64 (d, J=7.18 Hz, 2H), 7.31 (m, 10H), 6.98 (d, J=7.18 Hz, 2H), 5.58
(d, 1H), 5.14 5.08 (m, 4H), 4.66 (m, 1H), 3.32 2.65 (overlapping multip-
lets, 26H), 1.39 ppm (s, 27H, ꢀC(CH); 13C NMR (67.5 MHz, CDCl3,
258C, TMS): d=170.4, 169.9 169.5, 154.8, 136.8, 135.4, 134.3, 129.9,
128.8, 127.6 127.1, 118.8, 79.9, 66.1, 65.9, 59.3, 58.5, 55.9, 54.9, 54.2, 53.9,
51.7, 51.3, 50.9, 36.6, 27.3. Elemental analysis calcd. (%) for C52H74N6O11:
C, 65.11; H, 7.78; N, 8.76. Found: C 65.13, H 7.71, N 8.79; MS-ESI: m/z:
960 (calcd.960) [M+H]+.
1,4,7-tris(tert-butylacetate)-1,4,7,10-tetraazacyclododecane-10-N-a-(9-flu-
orenylmethoxy carbonyl)-p-acetylamide-L-phenylalanine (8): 1,4,7-tris-
(tert-butylacetate)-1,4,7,10-tetraaza
cyclododecane-10-N-a-carbobenzy-
loxy-p-acetylamide-L-phenylalanine benzyl ester (6), (3.01 g, 3.12 mmol)
was dissolved in isopropanol (25 mL) and 10% PdꢀC catalyst (0.6 g) was
added. The reaction mixture was hydrogenated in a Parr hydrogenation
apparatus at 40 psi for 3 days. The solution was filtered to remove the
catalyst and the solvent was removed by rotary evaporation to yield
2.29 g (99.3%) of 1,4,7-tris(tert-butylacetate)-1,4,7,10-tetraazacyclodode-
cane-10-p-acetylamide-L-phenylalanine (7). This product was used in the
next step without further purification. A mixture of 7 (2.29 g, 3.12 mmol)
in dioxane (35 mL) and aqueous Na2CO3 (0.99 g, 3 equiv, 35 mL) was
added over 2 h to a solution of 9-fluorenylmethylchloroformate (0.87 g,
1.05 equiv) in dioxane (10 mL) with cooling in an ice bath. After comple-
tion, the solution was allowed to stand overnight at RT. The solvent was
evaporated at 308C under a high vacuum to give a water insoluble
gummy residue. It was washed with water (2î10 mL) followed by ether
(2î15 mL), yielding 2.61 g (87.3%) of a yellow solid product. 1H NMR
(270 MHz, CDCl3, 258C, TMS): d=11.37 (s, 1H) 7.69 7.20 (m, 12H),
6.13 (s, 1H), 5.07 (bs, 1H), 4.41 (bs, 2H), 3.90 2.70 (overlapping multip-
lets, 26H), 1.41 (s, 27H). 13C NMR (67.5 MHz, CDCl3, 258C, TMS): d=
174.5, 172.2 170.6, 155.6, 145.4, 144.3, 137.3, 133.5, 129.9, 128.4, 128.2,
126.9.8, 125.5, 119.5, 81.6, 66.2, 56.9 47.3, 37.5, 27.9. Elemental analysis
calcd. (%) for C52H72N6O11.2H2O: C, 62.88; H, 7.71; N, 8.46. Found: C,
62.52; H, 7.87; N, 8.56. MS-ESI: m/z: 958 (calcd. 958) [M+H]+.
N-a-Carbobenzyloxy-p-amino-L-phenylalanine benzyl ester (4): Com-
pound (3) (5.56 g, 12.8 mmol) and zinc dust (<10 micron) were suspend-
ed in absolute ethanol (60 mL). Glacial acetic acid (60 mL) was added
and the reaction mixture was heated to 658C with an oil bath and stirred
for 2 h. The reaction mixture was cooled in an ice bath and crushed ice
(20 mL) was added. The pH of the solution was adjusted to 9.0 by slow
addition of a 9m NaOH solution. The resulting white thick slurry was di-
luted with water, and ethyl acetate (400 mL) was added. The solids were
filtered out from the mixture and washed with ethyl acetate (~200 mL).
The organic phase was washed with saturated NaHCO3 solution (2î
200 mL), and brine (2î200 mL), and dried over K2CO3. Removal of the
solvent gave a light yellow solid that was dried overnight under vacuum,
which yielded 4.7 g (90.9%) of product. Rf =0.38 (SiO2 TLC; hexanes/
ethyl acetate, 60:40); m.p. 87 898C; 1H NMR (270 MHz, CDCl3, 258C,
TMS): d=7.29 7.27 (m, 10H), 6.75 (d, J=8.16 Hz, 2H), 6.46 (d, 2H),
5.53 (d, 1H), 5.07 (m, 4H), 4.60 (t, 1H), 3.65 ppm (brs, 2H), 2.94 (m,
2H); 13C NMR (67.5 MHz, CDCl3, 258C, TMS): d=171.7, 155.9, 145.8,
136.5, 135.4, 130.2, 128.6 128.1, 125.0, 115.3, 67.1, 66.9, 55.3, 37.3. Ele-
mental analysis calcd. (%) for C24H24N2O4: C, 71.27; H, 5.98; N, 6.93.
Found: C 69.28, H 5.90, N 6.91; MS-ESI: m/z: 405 (calcd.405) [M+H]+.
N-e-(tert-butoxycarbonyl)-N-a-carbobenzyloxy-L-lysine benzyl ester (10):
N-e-(tbutoxycarbonyl)-N-a-carbobenzyloxy-L-lysine
(9)
(5.14 g;
13.5 mmol), di-isopropyl ethyl amine (1.83 g; 1.05 equiv) and benzyl bro-
mide (2.48 g; 1.05 equiv) were dissolved in acetonitrile (120 mL). The re-
action mixture was stirred under argon at RTfor two days. The solvent
was removed by rotary evaporation and the residue was dissolved in di-
chloromethane (250 mL) and washed with water (4x100 mL). The organic
phase was dried over Na2SO4, filtered and removal of the solvent gave a
clear oil which crystallized from ether/hexanes yielding 6.28 g (98.7%) of
a white solid. Rf =0.63 (SiO2 TLC; hexanes/ethyl acetate, 60/40). M. p.
63 648C. 1H NMR (270 MHz, CDCl3, 258C, TMS): d=7.26 (s, 10H.),
5.94 (d, 1H,) 5.06 (m, 4H), 4.34 (m, 1H), 2.97 (m, 2H), 1.80 1.64 (m,
4H), 1.36 (s, 11H). 13C NMR (67.5 MHz, CDCl3, 258C, TMS): d=172.5,
156.3, 136.2, 135.5, 128.6 128.1, 78.9, 67.0, 66.9, 54.0, 40.0, 31.8, 29.5, 28.5,
22.5. Elemental analysis calcd. (%) for C26H34N2O6: C, 66.36; H, 7.28; N,
5.95. Found: C, 66.16; H, 7.28; N, 5.98. MS-ESI: m/z: 493 (calcd. 493)
[M+Na]+.
N-a-carbobenzyloxy-p-bromoacetylamide-L-phenylalanine benzyl ester
(5): Compound(4) (14.4 g, 35.6 mmol) was dissolved in CH2Cl2 (250 mL)
and diisopropylethylamine (4.83 g, 1.05 equiv) was added. The reaction
flask was purged with argon and cooled to ꢀ978C (the freezing point of
CH2Cl2) in liquid N2. Bromoacetyl bromide (7.70 g, 1.05 equiv) was
added when the mixture started to melt. The reaction mixture was al-
lowed to warm to RT and stirring continued overnight. The mixture was
washed with water (2î100 mL), 0.1m HCl (2î100 mL) and water (1î
100 mL), dried over K2CO3, and evaporated to yield 18.1 g (96.7%) of a
white solid. Rf =0.50 (SiO2 TLC; hexanes/ethyl acetate, 60:40). M.p. 134
1358C; 1H NMR (270 MHz, CDCl3, 258C, TMS): d=8.13 (s, 1H), 7.36
7.26 (m, 12H), 6.95 (d, J=7.92 Hz, 2H) 5.27 (d, 1H), 5.11 (m, 4H), 4.67
(t, 1H), 3.97 (s, 2H), 3.06 ppm (m, 2H); 13C NMR (67.5 MHz, CDCl3,
258C, TMS): d=171.3, 163.4, 155.7, 136.2, 136.1, 135.1, 132.5, 130.1,
128.7 128.2, 120.1, 67.4, 67.1, 54.9, 37.7, 29.5 ppm. Elemental analysis
calcd. (%) for C26H25BrN2O5: C 59.44, H 4.80, N 5.33. Found: C 59.65, H
4.93, N 5.51; MS-ESI: m/z: 524 (calcd.524) [MꢀH]ꢀ.
N-a-carbobenzyloxy-L-lysine benzyl ester (11): N-e-(tbutoxycarbonyl)-N-
a-carbobenzyloxy -L-lysine benzyl ester (10) (6.28 g; 13.4 mmol) was dis-
solved in a mixture of CH2Cl2 (50 mL) and TFA (50 mL). The solution
was stirred at RT for 5 h. The solvent was evaporated by rotary evapora-
tion and the product was dissolved in water (100 mL) and crushed ice
(20 mL). The pH was adjusted to 9.5 by addition of Na2CO3. The aque-
ous layer was extracted with CH2Cl2 (2î500 mL). The organic phases
were combined, washed with water (300 mL), dried over Na2SO4, filtered
and removal of the solvent gave 4.68 g (94.6%) of a clear oil. Rf =0.83
(Al2O3 TLC; CH2Cl2/MeOH, 85/15). 1H NMR (270 MHz, CDCl3, 258C,
TMS): d=7.26 (s, 10H), 5.79 (s, 1H) 5.11 (m, 4H), 4.37 (m, 1H), 2.54
(m, 2H), 1.80 1.64 (m, 4H), 1.35 1.17 (m, 2H). 13C NMR (67.5 MHz,
CDCl3, 258C, TMS): d=172.5, 156.2, 136.4, 135.4, 128.6 128.1, 67.0, 66.9,
54.0, 41.8, 33.1, 32.3, 22.5. Elemental analysis calcd. (%) for C21H26N2O4:
C, 68.09; H, 7.07; N, 7.56. Found: C, 68.15; H, 7.00; N, 7.53.
1,4,7-Tris(tert-butylacetate)-1,4,7,10-tetraazacyclododecane-10-N-a-carbo-
benzyloxy-p-acetylamide-L-phenylalanine benzyl ester (6): 1,4,7-tris(tert-
butylacetate)-1,4,7,10-tetraazacyclododecane (2.62 g, 5.1 mmol) was dis-
solved in acetonitrile (100 mL) and solid K2CO3 was added (1.78 g,
1153
Chem. Eur. J. 2004, 10, 1149 1155
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

A. D. Sherry et al.
FULL PAPER
N-e-bromoacetylamide-N-a-carbobenzyloxy-L-lysine benzyl ester (12): A
solution of N-a-carbobenzyloxy-L-lysine benzyl ester (11) (4.670 g;
12.6 mmol) and di-isopropyl ethyl amine (1.713 g; 1.05 equiv) in CH2Cl2
(280 mL) was added to a solution of bromoacetyl bromide (2.729 g;
1.05 equiv) over one hour while maintaining the temperature at ꢀ308C.
Then the solution was stirred at ꢀ308C for one hour and then allowed to
stand at RT overnight. The organic phase was washed with water (2î
200 mL), 0.1 N HCl (2î200 mL) and water (2î200 mL), dried over
Na2SO4, filtered and removal of the solvent gave 6.05 g (97.7%) of a
white solid. Rf =0.83 (Al2O3 TLC; CH2Cl2/MeOH, 85/15). M.p. 70 728C.
1H NMR (270 MHz, CDCl3, 258C, TMS): d=7.32 (s, 10H), 6.57 (s, 1H),
5.51 (s, 1H), 5.14 (m, 4H), 4.40 (m, 1H), 3.79 (s, 2H), 3.17 (m, 2H),
1.84 1.23 (m, 6H). 13C NMR (67.5 MHz, CDCl3, 258C, TMS): d=172.3,
165.70, 156.1, 136.4, 135.3, 128.7 128.2, 67.3, 67.1, 53.7, 39.8, 32.2, 29.3,
28.7, 22.4. Elemental analysis calcd. (%) for C23H27BrN2O5: C, 56.22; H,
5.54; N, 5.70. Found: C, 56.09; H, 5.57; N 5.68. MS-ESI: m/z: 490 (calcd.
490) [MꢀH]ꢀ.
1,4,7-tris(tert-butylacetate)-1,4,7,10-tetraazacyclododecane-10-N-e-acetyl-
amide-N-a-carbobenzyloxy-L-lysine benzyl ester (13): 1,4,7-tris(tbutylace-
tate)-1,4,7,10-tetraazacyclo dodecane (1.08 g, 2.1 mmol) was dissolved in
acetonitrile (50 mL) and K2CO3 (0.76 g, 5.53 mmol) was added. The reac-
tion mixture was stirred at 608C for 15 minutes. N-e-bromoacetylamide-
Na-carbobenzyloxy-L-lysine benzyl ester (12) (1.04 g, 2.1 mmol) was dis-
solved in acetonitrile (50 mL) and added dropwise over 20 minutes. The
solution was stirred at 608C for 3 days. The solids were filtered out and
the solvent was removed by rotary evaporation. The oily residue was
dried to a constant mass to give the product quantitatively. 1H NMR
(270 MHz, CDCl3, 258C, TMS): d=8.62 (s, 1H), 7.29 7.24 (m, 10H), 5.58
(d, 1H), 5.12 (s, 2H), 5.05 (s, 2H), 4.32 (m, 1H), 3.19, 3.16, 2.97, 2.80,
2.78, 2.64, 2.44 (overlapping multiplets, 26H), 1.84 1.50 (m, 6H), 1.38 (s,
27H). 13C NMR (67.5 MHz, CDCl3, 258C, TMS): d=172.4, 172.2, 170.7,
170.6, 156.1, 136.4, 135.5, 128.6 128.1, 80.9, 67.1, 66.9, 60.4, 58.2 , 56.8,
56.3, 55.0, 54.1, 53.6, 52.6, 52.1, 38.7, 31.9, 29.5, 28.3, 22.8. Elemental anal-
ysis calcd. (%) for C49H76N6O11: C, 63.61; H, 8.28; N, 9.06. Found: C,
63.31; H, 8.22; N, 9.10. MS-ESI: m/z: 926 (calcd. 926) [M+H]+.
Peptide Synthesizer following standard Fmoc protocols using Rink amide
resin. Peptides were manually synthesized on a 0.1 mmol scale using
single step couplings of two equivalent Fmoc-amino acids (or Fmoc-
amino acid pentafluorophenyl esters), two equivalents coupling agent
(HBTU/HOBT or HOBT with the pentafluorophenyl esters) and 6 equiv-
alents di-isopropyl ethyl amine in DMF at RT. Free unreacted amino
groups were capped with a 4.75%v/v acetic anhydride solution in NMP.
During manual synthesis, coupling and capping completion was moni-
tored by the ninhydride test. A 10-fold excess of reagents was used for
the automated synthesizer. Following linear peptide assembly, cleavage
from the resin and final deprotection was carried out with TFA: thioani-
sole: 1,2-ethanedithiol: anisole (9:0.5:0.3:0.2) at 258C for 2 h. The resin
was filtered off and the filtrate concentrated with a gentle nitrogen flow.
Peptides were precipitated with cold diethyl ether. The precipitate was
filtered, washed with cold ether, dried and redissolved in 10% acetic
acid. The crude peptides were purified by reverse phase HPLC, lyophi-
lized and characterized by MS-ESI.
Peptide Characterization
H2N-GAADF-CONH2, HPLC: Rt =16.7 min, MS-ESI: m/z: 479 (calcd.
479) [M+H]+.
H2N-GAFDG-CONH2, HPLC: Rt =16.0 min, MS-ESI: m/z: 465 (calcd.
465) [M+H]+.
H2N-FAADG-CONH2, HPLC: Rt =14.6 min, MS-ESI: m/z: 479 (calcd.
479) [M+H]+.
H2N-(F-DOTA)DG-CONH2, HPLC: Rt =9.3 min, MS-ESI: m/z: 738
(calcd. 738) [M+H]+, 736 (calcd. 736) [MꢀH]ꢀ.
CH3CONH-(F-DOTA)-CONH2, HPLC Rt =11.0 min, MS-ESI: m/z: 607
(calcd. 607) [M+H]+.
H2N-GAAD(F-DOTA)-CONH2, HPLC: Rt =10.5 min, MS-ESI: m/z:
880 (calcd. 880) [M+H]+, 878 (calcd. 878) [MꢀH]ꢀ.
H2N-GA(F-DOTA)DG-CONH2, HPLC: Rt =11.1 min, MS-ESI: m/z:
866 (calcd. 866) [M+H]+, 864 (calcd. 864) [MꢀH]ꢀ.
H2N-(F-DOTA)AADG-CONH2, HPLC: Rt =13.6 min, MS-ESI: m/z:
880 (calcd. 880) [M+H]+, 878 (calcd. 878) [MꢀH]ꢀ.
1,4,7-tris(tert-butylacetate)-1,4,7,10-tetraazacyclododecane-10-N-e-acetyl-
amide-L-lysine (14): 1,4,7-tris(tbutylacetate)-1,4,7,10-tetraazacyclodode-
cane-10-N-e-acetylamide-N-a-carbobenzyloxy-L-lysine benzyl ester (13)
(1.02 g, 1.1 mmol) was dissolved in absolute ethanol (25 mL) and 10%
PdꢀC catalyst (0.4 g) was added. The reaction mixture was hydrogenated
in a Parr hydrogenation apparatus at 50 psi for 2 days. The solution was
filtered to remove the catalyst and the solvent was removed by rotary
evaporation. The residual oil was dried under vacuum to give 0.77 g
(100%) of a light yellow oil. The product was obtained as a white solid
by dissolving the oil in CH2Cl2 and extracting it into water and lyophiliz-
ing the aqueous phase. Rf =0.21 (Al2O3 TLC; CH2Cl2/MeOH, 85/15).
1H NMR (270 MHz, CDCl3, 258C, TMS): d=9.14 (s, 1H), 4.08 (m, 1H),
3.69, 3.46, 3.27, 3.11, 2.90 (overlapping multiplets, 26H,), 1.97 1.56 (m,
6H), 1.44 (s, 27H). 13C NMR (67.5 MHz, CDCl3, 258C, TMS): d=172.1,
168.8, 168.3, 164.6, 80.2, 79.9, 56.1, 55.1, 54.3, 53.3, 51.6, 50.4, 48.9, 47.4,
37.2, 28.8, 26.7, 20.7.
H2N-FW(K-DOTA)EG-CONH2, HPLC: Rt =22.5 min, MS-ESI: m/z:
1052 (calcd. 1052) [M+H]+, 1050 (calcd. 1050) [MꢀH]ꢀ.
(DOTA-T)FDDLFWKEGHR-CONH2, HPLC: Rt =33.1 min, MS-ESI:
m/z: 1936 (calcd. 1937) [M+H]+, 1934 (calcd. 1935) [MꢀH]ꢀ.
CH3CONH-TFDDLFWKEGHR-CONH2, HPLC: Rt =36.9 min, MS-
ESI: m/z: 1593 (calcd. 1592) [M+H]+, 1590 (calcd. 1590) [MꢀH]ꢀ.
CH3CONH-TFDDLFW(K-DOTA)EGHR-CONH2, HPLC: Rt =36.6 min,
MS-ESI: m/z: 1979 (calcd. 1979) [M+H]+, 1976 (calcd. 1977) [MꢀH]ꢀ.
CH3CONH-TFDDL(F-DOTA)WKEGHR-CONH2, HPLC: Rt =30.1 min,
MS-ESI: m/z: 1995 (calcd. 1995) [M+H]+, 1993 (calcd. 1993) [MꢀH]ꢀ.
Gadolinium Complex Formation: Complexes were prepared by adding a
GdCl3 stock solution to
a DOTA peptide ligand solution, pH 7.4
(HEPES buffer) in stoichiometric amounts (1:1). The solution was stirred
at RTfor 24 h and then centrifuged to remove any precipitated Gd(OH)3.
The presence of free Gd3+ was evaluated by colorimetry using xylenol
orange as an indicator. In a solution containing the complex GDDOTA
peptide at pH 5.2, a deep purple color indicates free Gd3+. Resultant
peptide complexes were further purified by HPLC using the previously
described procedure. The purified complex solution was lyophilized to
give the gadolinium complex as a powder solid.
1,4,7-tris(tert-butylacetate)-1,4,7,10-tetraazacyclododecane-10-N-e-acetyl-
amide-N-a-(9-fluorenylmethoxycarbonyl)-L-lysine (15): 1,4,7-tris(tbutyl-
acetate)-1,4,7,10-tetraazacyclo
dodecane-10-N-e-acetylamide-L-lysine
(14) (0.887 g, 1.27 mmol) was dissolved in dioxane (8 mL) and an aque-
ous solution of Na2CO3 (0.403 g, 3 equiv in 8 mL of water) was added.
The reaction mixture was immersed in an ice bath and a solution of 9-flu-
orenylmethylchloroformate (0.328 g, 1.23 mmol) in dioxane (5 mL) was
added under argon atmosphere. The solution was allowed to warm to RT
and stirred overnight. The solvents were evaporated under high vacuum
at 30(C. The solid residue was washed with water and dried to a constant
mass to give 1.01 g (86.4%) of a white solid. Rf =0.67 (Al2O3 TLC;
CH2Cl2/MeOH, 85/15), 1H NMR (270 MHz, CDCl3, 258C, TMS): d=
7.64 7.11 (m, 8H), 4.17 (m, 1H), 3.20 2.42 (overlapping multiplets,
26H), 1.84 1.50 (m, 6H), 1.32 (s, 27H). 13C NMR (67.5 MHz, CDCl3,
258C, TMS): d=173.8, 170.6, 170.2, 169.3, 154.0, 143.9,139.3, 125.7, 125.3,
123.8, 118.0, 79.9, 64.5, 54.3.0 45.5, 37.1, 30.2, 26.3, 20.3. Elemental analy-
sis calcd. (%) for C49H74N6O11: C, 63.75; H, 8.08; N, 9.10. Found C, 63.51,
H, 8.05, N, 9.15. MS-ESI: m/z: 922 (calcd. 922) [MꢀH]ꢀ.
Protein Binding Studies: To measure the KD of the Gal-80-fluorescein N-
labeled peptide (provided by Dr. Kodadek, University of Texas South-
western Medical Center, Dallas, Texas), the indicated amounts of His6-
Gal-80 and 2nm of labeled peptide were mixed in 200 mL of buffer (PBS
with 0.2 mgmLꢀ1 bovine serum albumin). Each solution was equilibrated
at RT for 20 minutes prior to measuring the fluorescence polarization of
each sample. Competition binding studies were done by titrating a com-
peting peptide into a sample containing 2nm of Fluorescein-labeled pep-
tide with 130nm Gal-80 (75% of KD). The dissociation of the fluorescein-
peptide from Gal-80 was monitored by fluorescence polarization spectros-
copy.
Solid-phase Peptide Synthesis: Solid-phase peptide synthesis was carried
out either manually or in an Applied Biosystem ABI 433A Automatuzed
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DOTA Peptide Synthesis
1149 1155
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Acknowledgement
This work was supported in part by grants from the Robert A. Welch
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Received: July 25, 2003
Revised: October 31, 2003 [F5389]
1155
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim