A new method for the synthesis of tri-tert-butyl diethylenetriaminepentaacetic acid and its derivatives

Full text of DOI:10.1021/jo991453t

1562
J . Org. Chem. 2000, 65, 1562-1565
Sch em e 1. Im p r oved P r oced u r e for th e Syn th esis
A New Meth od for th e Syn th esis of
of DTP A-Mon op ep tid e Con ju ga tes
Tr i-ter t-bu tyl
Dieth ylen etr ia m in ep en ta a cetic Acid a n d
Its Der iva tives
Samuel Achilefu,* R. Randy Wilhelm,
Hermo N. J imenez, Michelle A. Schmidt, and
Ananth Srinivasan
Discovery Research, Mallinckrodt, Inc.,
St. Louis, Missouri 63042
Received September 14, 1999
Diethylenetriaminepentaacetic acid (DTPA) ligands
and other polyaminocarboxylates are widely used in
fundamental research as chelating agents1,2 and in the
pharmaceutical industry for diagnostic purposes. In
particular, they are useful chelators in magnetic reso-
nance imaging (MRI),3 nuclear medicine,4 and most
recently, in optical imaging5 and radiation therapy with
radioactive metals capable of destroying tumors.6,7 Such
applications are more effective when the radionuclide is
delivered to tumors by specific targeting mechanisms.
Advances in molecular biology and peptide chemistry
have facilitated the use of peptides to target tumors by
receptor-mediated uptake. This approach necessitates the
development of efficient methods for the synthesis of
peptide-DTPA conjugates. Traditionally, these conju-
gates are formed by the reaction of DTPA dianhydride
with peptides, which results in a mixture of the mono-
and the bis-peptide-DTPA derivatives.8,9 Studies have
shown that metal complexes of bispeptide-DTPA con-
jugates have high levels of liver and kidney uptake9 and
are less stable in vivo10,11 and in vitro12 than the
monopeptide-DTPA analogues. They must then be sepa-
rated from the more stable monoconjugates and dis-
carded. In view of the high cost of peptides, low yield of
the desired monopeptide-DTPA conjugates is highly
undesirable.
Although Arano13 described a method for the prepara-
tion of monofunctional DTPA intermediates, the proce-
dure is cumbersome and gives a low yield (20%) of the
final compound. Further, Arano’s method is not suitable
for selective functionalization of more than one DTPA
carboxyl group, and it is not amenable to large-scale
production due to the formation of ditrifluoroacetamides
at higher reactant concentrations. Taking advantage of
the procedure described by Rapoport14 to prepare differ-
entially protected DTPA derivatives, Srinivasan15 devel-
oped an efficient procedure for the synthesis of DTPA-
monopeptide conjugates, as shown in Scheme 1.
This method optimized the use of expensive peptides
in the DTPA-conjugation step and simplified the purifi-
cation process. However, it also introduced a different
kind of problem. In the synthesis of tri-tert-butyl DTPA,
1, from tert-butyl glycinate and N,N′-bis(tert-butylcar-
boxylmethyl)aminoethyl bromide, less than 15% of the
desired product was isolated. This is due to lack of
selectivity, resulting in the concomitant formation of 3,
lactams and some intermolecular amide side products,
in addition to the target secondary amine, 4 (Figure 1).
Thus, an alternative method for the synthesis of this
important ligand precursor is needed. We report a new
method for the synthesis of tri-tert-butyl DTPA and its
use in the preparation of monopeptide-DTPA deriva-
tives.
* To whom correspondence should be addressed. Phone: 314-654-
3485. Fax: 314-654-5337. E-mail: samuel.achilefu@mkg.com.
(1) Powell, H. D.; Ni Dhubhghail, O. M.; Pubanz, D.; Helm, L.;
Lebedev, Y.; Schlaepfer, W.; Merbach, A. E. J . Am. Chem. Soc. 1996,
118, 9333.
(2) Mortellaro, M. A.; Nocera, D. G. J . Am. Chem. Soc. 1996, 118,
7414.
(3) Wallace, R. A.; Haar, J . P.; Miller, D. B.; Woulfe, S. R.; Polta, J .
A.; Galen, K. P.; Hynes, M. R.; Adzamli, K. Magnetic Resonance in
Med. 1998, 40, 733.
(4) Fischman, A. J .; Babich, J . W.; Strauss, J . W. J . Nucl. Med. 1993,
34, 2253.
(5) Houlne, M. P.; Hubbard, D. S.; Kiefer, G. E.; Bornhop, D. J .
Biomed. Optics 1998, 3, 145.
(6) Bugaj, J . E.; Erion, J . L.; Schmidt, M. A.; Wilhelm, R. R.;
Achilefu, S. I.; Srinivasan, A. J . Nucl. Med. 1999, 40(Suppl.), Abstract
813.
(7) Otte, A.; J ermann, E.; Behe, M.; Goetze, M.; Buchner, H. C.;
Roser, H. W.; Heppeler, A.; Mueller, B.; Macke, H. R. Eur. J . Nucl.
Med. 1997, 24, 792.
(8) Edward, W. B.; Fields, C. G.; Anderson, C. J .; Pajeau, T. S.;
Welch, M. J .; Fields, G. B. J . Med. Chem. 1994, 37, 3749.
(9) Bard, D. R.; Knight, C. G.; Page-Thomas, D. P. Brit. J . Cancer
1990, 62, 919.
Commercially available benzylethylenediamine 5 was
alkylated with tert-butyl bromoacetate to give the tertiary
amine 7. Hydrogenolysis, followed by alkylation with the
bromide 8, gave orthogonally protected DTPA ester 9.
Subsequent hydrogenolysis of the benzyl ester gave tri-
(10) Brechbiel, M. W.; Gansow, O. A. Bioconjugate Chem. 1991, 2,
187. Our in vivo results will be published elsewhere.
(11) Anelli, P. L.; Fedeli, F.; Gazzotti, O.; Lattuada, L.; Lux, G.;
Rebasti, F. Bioconjugate Chem. 1999, 10, 137.
(12) Fossheim, R.; Dugstad, H.; Dahl, S. G. J . Med. Chem. 1991,
34, 819.
(13) Arano, Y.; Uezono, T.; Akizawa, H.; Ono, M.; Wakisaka, K.;
Nakayama, M.; Sakahara, H.; Konishi, J .; Yokoyama, A. J . Med. Chem.
1996, 39, 3451.
(14) Williams, M. A.; Rapoport, H. J . Org. Chem. 1993, 58, 1151.
(15) Srinivasan, A.; Schmidt, M. A. Proc. 15th Am. Peptide Symp.
1997, 267.
10.1021/jo991453t CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/12/2000

Notes
J . Org. Chem., Vol. 65, No. 5, 2000 1563
removal of the acid-labile tert-butyl esters. In method 2,
we used conventional catalytic hydrogenolysis of 7 under
hydrogen pressure. Contrary to literature reports,18,19 we
observed that the debenzylation was complete in less
than 2 h at room temperature and 3 atm of H2. Com-
parison of our result with the catalytic hydrogenolysis
of another tertiary benzylamine, N-benzyl-N,N-dipropyl-
amine, showed that the debenzylation of the latter is
sluggish, as expected, and required more than 12 h for
complete hydrogenolysis. Hence, removal of the N-benzyl
group may also be catalyzed by esters as was reported
for acids.19
Unlike most monofunctional DTPA intermediates,
compound 1 is a stable white powder at room tempera-
ture and is readily used in automated peptide synthesis
without the need for cumbersome postsynthetic coupling
reactions. Thus, reaction of 1 with peptides gave exclu-
sively the desired monoamide-DTPA derivatives (Table
1). The peptides were prepared by standard automated
Fmoc solid-phase peptide synthesis,20a and the DTPA
conjugation was carried out with the peptides on solid
support as described in the literature.15
F igu r e 1. Major reaction intermediates.
Sch em e 2. New Meth od for th e Syn th esis of
Tr i-ter t-bu tyl-DTP A
The free carboxyl group of the monopeptide-DTPA 16
is derivable with a variety of nucleophiles capable of
altering the physicochemical and biological properties of
the metal chelate complex. For example, we successfully
synthesized a somatostatin octapeptide analogue 18 on
solid support from 1 in order to decrease the renal uptake
of the tumor-targeting agent20b (Scheme 3).
A unique advantage of tri-tert-butyl DTPA over mono-
functional DTPA intermediates11,13 is the ease of using
the former as both linker and ligand for a variety of
applications. This is particularly useful for the introduc-
tion of different tumor specific agents on the same
molecule. Toward this end, we synthesized a DTPA-
bridged peptide derivative 19 (Figure 2) from the mono-
carboxylate 2 and monoprotected N-Fmoc ethylenedi-
amine. This reaction demonstrates the potential to use
tri-tert-butyl DTPA in the synthesis of a combinatorial
library of compounds.
In summary, tri-tert-butyl DTPA is a versatile inter-
mediate that can be used to synthesize a variety of
compounds for fundamental research and industrial
applications. It has several advantages over monofunc-
tional DTPA intermediates, including the ease of selective
functionalization of more than one carboxyl group, ame-
nability to automatic Fmoc solid-phase synthesis without
recourse to difficult postsynthetic coupling reactions, and
versatility in the modification of the physicochemical and
biological properties of the conjugates. We developed a
new and high-yielding method for the synthesis of this
ligand precursor and demonstrated its use as chelator
and linker in the preparation of DTPA derivatives. These
peptide conjugates are biocompatible as demonstrated by
the in vivo stability of the monopeptide-DTPA metal
chelate, 111In-DTPA-octreotide.21
tert-butyl DTPA 1 in about 70% overall yield. The new
procedure is summarized in Scheme 2.
Two methods were evaluated for the removal of the
benzyl protecting group of 7. In method 1, we employed
catalytic transfer hydrogenation with ammonium for-
mate,16 and the debenzylation was complete in 30 min.
We observed that a tenth of the recommended Pd
catalyst16 equally removed the benzyl group when the
benzylamine 7 and ammonium formate were refluxed in
methanol for 40 min. However, this approach also gives
side products that complicate purification of the second-
ary amine 4. Although formic acid is widely used in
catalytic transfer hydrogenation,17 we chose not to depro-
tect 7 with this reagent in order to avoid premature
(18) J ung, M. E.; Longmei, Z.; Tangsheng, P.; Huiyan, Z.; Van, L.;
J ingyu, S. J . Org. Chem. 1992, 57, 3528.
(19) Kocienski, P. J . Protecting Groups; Georg Thieme Verlag:
Stuttgart, 1994; pp 220-227.
(20) (a) Atherton, E. Fluorenylmethoxycarbonyl-polyamide solid-
phase peptide synthesis: General principles and development; Informa-
tion Press: Oxford, 1989. (b) Our biological results will be published
elsewhere.
(16) Ram, S.; Ehrenkaufer, R. E. Synthesis 1988, 85, 91.
(17) ElAmin, B.; Anantharamaiah, G. M.; Royer, G. P.; Means, G.
E. J . Org. Chem. 1979, 44, 3442.
(21) Kwekkeboom, D. J .; Krenning, E. P.; Kho, G. S.; Breeman, W.
A.; Van Hagen, P. M.Eur. J . Nucl. Med. 1998, 25, 1284 and references
therein.

1564 J . Org. Chem., Vol. 65, No. 5, 2000
Ta ble 1. Mon oa m id e-DTP A Der iva tives
Notes
compd
structure
m/z
10
11
12
13
14
15
DTPA-Ile-Gly-Gly-Gly-Leu-Gly-Gly-Gly-NH2
960.4563
1423.5156
1551.6202
1543.6903
1378.6358
1120.4506
DTPA-Phe-cyclo(Cys-Tyr-Trp-Lys-Thr-Cys)-Thr-OH
R-DTPA-Lys-Phe-cyclo(Cys-Tyr-Trp-Lys-Thr-Cys)-Thr-OH
DTPA-Gly-Asp-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2
DTPA-Ala-Ala-Glu-Met-Ala-Pro-Trp-Lys-Thr-OH
DTPA-Gly-Pro-His-Trp-Ser-Tyr-OH
Combustion analyses were performed by Atlanta Microlab, Inc.,
Norcross, GA. Analytical (flow rate ) 0.5 mL/min) and semi-
preparative (flow rate ) 10 mL/min) RP-HPLC were performed
as described in the literature23 using either of two gradient
elution protocols: I (50 to 100% B in 30 min) and II (5 to 70% B
in 30 min) where A is 5% CH3CN in 0.1% aqueous TFA and B
is10% H2O in 0.1% TFA solution of CH3CN. Peak detection was
at 214 nm with a tunable absorbance detector.
N-Ben zyl-N,N′,N′-tr is(ter t-bu tyloxyca r bon ylm eth yl)eth -
ylen ed ia m in e (7). N-Benzylethylenediamine (5.0 g, 33.28
mmol) and K2CO3 (19.3 g, 139.64 mmol) were stirred in
anhydrous acetonitrile (200 mL) under Ar atmosphere. tert-Butyl
bromoacetate (39.2 g, 246.09 mmol) in 30 mL of anhydrous
acetonitrile was added dropwise to the reaction mixture over
90 min. The progress of the reaction was monitored by TLC and
was essentially complete in about 4 h but was left at room
temperature overnight (12 h). The insoluble residue was filtered
and washed with acetonitrile. The filtrate was evaporated to give
20 g of a yellow liquid that was triturated with hexane (100 mL),
and the resulting white precipitate was filtered. Evaporation of
the filtrate and purification of the crude product by DFC, eluting
with 10% Et2O in hexane, gave the pure triester 7 (13.8 g, 28.01
mmol, 85% yield; HPLC purity 100%, protocol II) as a colorless
liquid. 1H NMR (300 MHz, CDCl3) δ ppm: 7.28 (5H, m); 3.80
(2H, s); 3.44 (4H, s) 3.26 (2H, s); 2.84 (4H, m); 1.45 and 1.43
(27H, s). 13C NMR (75 MHz, CDCl3): 170.9; 170.7; 139.1; 129.0;
128.2; 127; 80.8; 80.6; 58.3; 56.2; 55.0; 52.3; 52.0; 28.2; 28.1. MS
(EI): 493.2 (M + H)+. Anal. Calcd for C27H44N2O6: C, 65.83; H,
9.00; N, 5.69. Found: C, 66.04; H, 9.06; N, 5.71
F igu r e 2. Bispeptide-linked DTPA.
Sch em e 3. Selective F u n ction a liza tion of
DTP A-P ep tid e Con ju ga tes
N ,N ′,N ′-T r i s (t er t -b u t y lo x y c a r b o n y lm e t h y l)e t h y l-
en ed ia m in e (4) b y Ca t a lyt ic Tr a n sfer H yd r ogen olysis.
N-Ben zyl-N,N′,N′-t r is(ter t-bu t yloxyca r bon ylm et h yl)et h yl-
enediamine 7 (6 g, 12 mmol) was added to a heterogeneous
mixture of 10% palladium on carbon (6 g, 1 weight equivalent)
in methanol (100 mL). Anhydrous ammonium formate (3.8 g,
60.26 mmol) was added to the reaction mixture in one bulk, and
the mixture was stirred for 2 h at room temperature. The product
was filtered over Celite, and the residue was washed with
chloroform. The filtrate was evaporated to dryness, and the
residue was triturated in chloroform. The insoluble solid was
filtered, and the filtrate was evaporated to give the pure
compound (4.6 g, 11.43 mmol, 96% yield) as a pale yellow liquid.
1H NMR (300 MHz, CDCl3) δ ppm: 3.71 (2H, s); 3.49 (4H, s);
3.14 (2H, t, J ) 2.5 Hz); 3.07 (2H, t, J ) 2.5 Hz); 1.51 (9H, s);
1.46 (18H, s). 13C NMR (75 MHz, CDCl3): 170.9; 171.1; 165.9;
83.2; 81.6; 56.4; 51.4; 48.6; 45.9; 27.7. MS (EI): 493.2 (M + H)+.
N ,N ′,N ′-T r i s (t er t -b u t y lo x y c a r b o n y lm e t h y l)e t h y l-
en ed ia m in e (4) by Con ven tion a l Ca ta lytic Hyd r ogen oly-
sis. N-Benzyl-N,N′,N′-tris(tert-butyloxycarbonylmethyl)ethyl-
enediamine 7 (1 g; 2.0 mmol) was added to a heterogeneous
mixture of 10% Pd/C (0.1 g) in methanol (50 mL). The mixture
was hydrogenated at 3 atm for 2 h, and the product was filtered
over Celite. After the cake was washed with MeOH, the solvent
was evaporated to give 4 (740 mg, 1.84 mmol, 92% yield) as a
pale yellow liquid. The spectral properties are shown above.
N,N′,N′-Tr is(ter t-b u t yloxyca r b on ylm et h yl)-N′′,N′′-b is-
(ben zyloxyca r bon ylm eth yl)d ieth ylen etr ia m in e (9). A mix-
ture of N,N′,N′-tris(tert-butyloxycarbonylmethyl)ethylenediamine
4 (4.4 g, 9.82 mmol), 2-[bis(benzyloxycarbonylmethyl)amino]ethyl
bromide 8 (5.3 g, 12.76 mmol), and ethyldiisopropylamine (3.8
g, 29.45 mmol) in acetonitrile (100 mL) was stirred at reflux for
24 h under nitrogen atmosphere. After evaporation of the
solvent, the residue was dissolved in dichloromethane and the
solution was washed twice with water (100 mL). Evaporation
Exp er im en ta l Section
Gen er a l Com m en ts. 2-[Bis-(benzyloxycarbonylmethyl)ami-
no]ethyl bromide (8) was prepared as described in the litera-
ture.14 Dry flash chromatography (DFC) was performed on TLC
standard grade silica gel without binder as described else-
where.22 Electrospray ionization mass spectrometry experiments
were performed on a triple quadruple mass spectrometer. The
electrospray interface was operated in positive-ion mode with a
spray voltage of 4.5 kV and a capillary temperature of 225 °C.
Samples were introduced into the spectrometer by flow injection
utilizing acetonitrile/water (7:3) containing 0.1%. trifluoroacetic
acid. HRMS (MALDI-TOF) analyses were performed at the
Washington University School of Medicine in St. Louis, MO.
(23) Edwards, W. B.; Fields, C. G.; Anderson, C. J .; Pajeau, T. S.;
Welch, M. J .; Fields, G. B. J . Med. Chem. 1994, 37, 3749.
(22) Harwood, L. M. Aldrichim. Acta 1995, 18, 25.

Notes
J . Org. Chem., Vol. 65, No. 5, 2000 1565
of the organic solvent and subsequent purification of the crude
product by DFC, eluting with 40% Et2O in hexane, gave the
tertiary amine 9 (6.5 g, 8.76 mmol, 90% yield; HPLC purity
100%, protocol I) as a colorless liquid. 1H NMR (300 MHz, CDCl3)
δ ppm: 7.35 (10H, m); 5.13 (4H, s); 3.65 (4H, s); 3.44 (4H, s);
3.32 (2H, s); 2.88 and 2.78 overlap (8H, m); 1.45 and 1.47 overlap
(27H, s). 13C NMR (75 MHz, CDCl3): 171.4; 171.0; 135.9; 128.7;
128.4, 80.8; 66.1; 56.0; 55.1; 52.6. MS (EI): 742.6 (M + H)+. Anal.
Calcd for C40H59N3O10: C, 64.76; H, 8.02; N, 5.66. Found: C,
64.54; H, 7.93; N, 5.66.
N,N′,N′-Tr is(ter t-b u t yloxyca r b on ylm et h yl)-N′′,N′′-b is-
(a cetic a cid )d ieth ylen etr ia m in e (1). A mixture of N,N′,N′-
tris(tert-butyloxycarbonylmethyl)-N′′,N′′-bis(benzyloxycarbonyl-
methyl)diethylenetriamine 9 (3.3 g, 4.45 mmol) and 10% Pd/C
(0.21 g) in 50 mL of methanol was hydrogenated at 3 atm for 2
h. The mixture was filtered over Celite, and the residue was
washed with methanol. Evaporation of the solvent gave 1 (2.4
g, 4.27 mmol, 96% yield; HPLC purity 100%, protocol II) as a
white powder. Mp: 37 °C (uncorrected). 1H NMR (300 MHz,
CDCl3) δ ppm: 4.13 (4H, s); 3.57 (6H, s); 3.44 (2H, m); 3.10 (2H,
m); 2.93 (2H, m), 2.88 (2H, m); 1.45 (9H, s); 1.42 (18H, s). 13C
NMR (75 MHz, CDCl3): 170.7; 169.7; 169.1; 82.3; 81.6; 56.4; 55.6;
55.3; 52.6; 50.3; 48.7; 28.1; 28.0. MS (EI): 562.3 (M + H)+. Anal.
Calcd for C26H47N3O10: C, 55.60; H, 8.43; N, 7.48. Found: C,
54.70; H, 8.36; N, 7.44.
P ep tid e-DTP A 11 (DTP A-P h e-cyclo(Cys-Tyr -Tr p -Lys-
Th r -Cys)-Th r -OH). After the synthesis was complete, the
dithiol residue was cyclized with thallium trifluoroacetate (23
mg, 42 µmol in 2 mL of DMF for 90 min), and the product was
cleaved from the solid support with reagent A (8.9 mg, 6.3 µmol,
25% yield; 100% HPLC purity; HRMS calcd for C63H85N13O21S2
1423.5424, found 1423.5156).
P ep tid e-DTP A 12 (r-DTP A-Lys-P h e-cyclo(Cys-Tyr -Tr p -
Lys-Th r -Cys)-Th r -OH) was prepared as described above (7.8
mg, 5.0 µmol, 20% yield; 100% HPLC purity; HRMS calcd for
C69H97N15O22S2 1551.6374, found 1551.6202).
P ep tid e-DTP A 13 (DTP A-Gly-Asp -Gly-Gln -Tr p -Ala -Va l-
Gly-His-Leu -Met-NH2) was cleaved with reagent B (13.5 mg,
8.8 µmol, 35% yield; 100% HPLC purity; HRMS calcd for
C65H97N19O23S 1543.6725, found 1543.6903).
P ep tid e-DTP A 14 (DTP A-Ala -Ala -Glu -Met-Ala -P r o-Tr p -
Lys-Th r -OH) was cleaved with reagent B (13.1 mg, 9.5 µmol,
38% yield; 100% HPLC purity; HRMS calcd for C59H90N14O22
1378.6074, found 1378.6358).
S
P ep tid e-DTP A 15 (DTP A-Gly-P r o-His-Tr p -Ser -Tyr -OH)
was cleaved with reagent A (6.5 mg, 5.75 mmol, 23% yield; 100%
HPLC purity; HRMS calcd for C50H64N12O18 1120.4461, found
1120.4506).
P ep tid e-DTP A Mon oa m id e 18 (DTP A(NH2)-P h e-cyclo-
(Cys-Tyr -Tr p -Lys-Th r -Cys)-Th r -OH). The free carboxylic acid
function of tri-tert-butyl-DTPA-octapeptide 11 on solid support
(25 µmol) was activated with (HBTU)/(HOBt) in DMSO (0.2 M,
250 µL, 50 µmol) and N-ethyl-N,N-diisopropylamine in DMSO
(0.2M, 250 µL, 100 µmol) for 30 min, and aqueous ammonia
solution (density ) 0.9 g ml-1, 19.5 µL, 500 µmol) was added to
the resin. The mixture was mixed for 3 h at room temperature,
and the resin was washed with DMF, THF, and dichloro-
methane. Cleavage and removal of side chain protecting groups
were carried out with reagent A (6.4 mg, 4.5 µmol, 18% yield;
100% HPLC purity; MS (EI) 1423.6 (M + H)+).
Syn th esis of P ep tid e-DTP A Der iva tives. All the peptides
synthesized in this study follow the procedure described below.
Exceptions are noted under specific peptides. The peptides were
prepared on solid support by standard automated 9-fluorenyl-
methoxycarbonyl (Fmoc) procedures.20a Rink amide and Wang
resins were used for the synthesis of C-terminal amide and
carboxyl peptides, respectively. Initial loading of each Fmoc
amino acid bound resin is 25 µmol. Automatic activation of the
carboxyl group with
a mixture of N-hydroxylbenzotriazole
(HOBt) and 2-(1H-benzotriazole-1-yl)-1,1,1,3-tetramethyluro-
nium hexafluorophosphate (HBTU) and coupling of subsequent
Fmoc-protected amino acids (75 µmol) and the tri-tert-butyl
DTPA 1 (placed as the last cartridge on the synthesizer) were
carried out in situ. Either of the following reagent mixtures was
used to cleave the peptide from the resin and remove the side-
chain protecting groups, unless otherwise stated: reagent A
(85% TFA/5% H2O/5% PhOH/5% thioanisole) for 6 h or reagent
B (85% TFA/5% H2O/5% ethanedithiol/5% thioanisole) for 3 h.
The crude peptides were precipitated with cold tert-butyl methyl
ether and purified by RP-HPLC using gradient elution protocol
II described in the general section above. All peptide-DTPA
derivatives were characterized by analytical HPLC, electrospray
spectrometric (EI), and HRMS (MALDI-TOF) analyses after
lyophilization in 67% H2O/33% CH3CN. The HRMS data are
presented in Table 1 above.
DTP A-Br id ged
Bisp ep tid e 19 (H2N-Asp -P h e-Gly-
N H C 2H 4N H -D T P A-G ln -T r p -Ala -Va l-G ly -H is -L e u -Me t -
NH2). The free carboxylic acid function of tri-tert-butyl-DTPA-
octapeptide 16 on solid support (25 µmol) was activated as
described above and reacted with FmocNHC2H4NH2 (24 mg, 75
µmol) for 2 h. After the resin was washed with DMF and THF,
it was dried and returned to the synthesizer to complete the
synthesis of the second peptide fragment. After reaction, the
peptide was cleaved from the resin with reagent A (5.5 mg,
3.3 µmol, 13% yield; 93% HPLC purity; HRMS calcd for
C74H110N21O22S 1676.7855, found 1676.7808).
Su p p or tin g In for m a tion Ava ila ble: 1H NMR, 13C NMR,
HPLC, and mass spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
P ep tid e-DTP A 10 (DTP A-Ile-Gly-Gly-Gly-Leu -Gly-Gly-
NH2) was cleaved from the resin with 95% aqueous TFA (12.3
mg, 12.3 µmol, 49% yield; 100% HPLC purity; HRMS calcd for
C38H64N12O17 960.4512, found 960.4563).
J O991453T