N-tritioacetoxyphthalimide: A new high specific activity tritioacetylating reagent

Full text of DOI:10.1021/jo9616522

J . Org. Chem. 1996, 61, 9625-9628
9625
N-Tr itioa cetoxyp h th a lim id e: A New High
Sp ecific Activity Tr itioa cetyla tin g Rea gen t
Manouchehr Saljoughian,* Hiromi Morimoto,
Philip G. Williams,1 Chit Than, and
Stephen J . Seligman†
National Tritium Labelling Facility, Structural Biology
Division, Lawrence Berkeley National Laboratory,
One Cyclotron Road, Berkeley, California 94720, and
Department of Medicine and Department of Anatomy and
Cell Biology, Box 77, SUNY/ Health Science Center,
450 Clarkson Avenue, Brooklyn, New York 11203
Our aim was to develop a nonvolatile, stable, and facile
tritioacetylating reagent and to demonstrate its use on
simple peptides. Accordingly, we made the synthesis of
high specific activity N-(tritioacetoxy) derivatives of
succinimide, phthalimide, and naphthalimide our focus.
As our preferred approach, N-(tritioacetoxy)phthalimide
was prepared by radical dehalogenation of N-(iodo-
acetoxy)phthalimide using high specific activity tri-
butyltin tritide (Scheme 1). This tritiated acetylation
reagent was characterized by H and H NMR spectros-
copy and by radio-HPLC. Efficacy of the reagent was
investigated by tritioacetylation of several peptides at
their N-terminal amino group.
Received August 27, 1996
In tr od u ction
The limitations of tritiated acetic anhydride and tri-
tiated acetic acid as acetylating reagents are well known2-7
and include low tritium content, volatility, and poor
chemical selectivity. The activated imido esters (1, 2)
have been reported as useful N-acylating reagents8,9 and
are highly reactive at 0 °C. The analogous N-acetoxy
derivatives could be expected to be equally reactive, but
have been infrequently utilized.2,4,7,10-12 Specifically, N-
acetoxyphthalimide was used to prepare N-acetyl-
muramic acid,7,11 N-(iodoacetoxy)succinimide was used
for iodoacetylation of N6-(6-amino-n-hexyl)adenosine 5′-
phosphate,12 and tritiated acetyl derivatives have been
produced at low specific activity.2,10 De Groot10 reacted
[14C]phenylalanyl-soluble-RNA with N-acetoxysuccin-
imide and N-(tritioacetoxy)succinimide (no specific activ-
ity indicated), and Lindsay2 used N-(tritioacetoxy)suc-
cinimide at 21 GBq/mmol to tritioacetylate insulin. In a
single example, a high specific activity tritioacetyl group
was made by iodoacetylation of muramyl dipeptide with
N-(iodoacetyl)succinimide and subsequent catalytic tri-
tiodehalogenation.4 N-Succinimidyl [2,3-3H]propionate at
very high specific activity has been used to acylate
proteins,13,14 but propionylation may result in appreciable
loss of biological activity,15 may not show selectivity
between amino and thiol groups,16 or may require long
reaction times.17
3
1
Resu lts a n d Discu ssion
Ch oice of Acetyla tion Rea gen t. With the synthesis
of high specific activity N-tritioacetoxy derivatives of
succinimide, phthalimide, or naphthalimide as our goal,
our initial investigations centered on N-acetoxysuccin-
imide. Although highly reactive, it is a poorly-chromo-
phoric reagent and therefore difficult to analyze by
HPLC. Hence, while the N-acetoxysuccinimide reagent
may perform very well, we desired two reliable analytical
techniques for characterization of the highly tritiated
reagent and subsequently concentrated on the phthal-
imide and naphthalimide derivatives.
We extensively studied the N-acetoxyphthalimide pre-
cursor and found it to be readily synthesized and to have
excellent reactivity, solubility, and chromatographic char-
acteristics. In analogous studies of the naphthalimide
derivative, generation of the labeled reagent by the
radical dehalogenation approach was unacceptably slow,
so that line of investigation was discontinued.
Tr itia tion of th e Acetyla tion Rea gen t. Preliminary
investigations with catalytic dehalogenation of N-(iodo-
acetoxy)succinimide using H3H gas (10%) in the presence
of Pd-C (10%) gave N-(tritioacetoxy)succinimide with
very poor radiochemical yield (7%) and very low tritium
incorporation into the acetoxy group. Analogous tritia-
tion of N-(bromoacetoxy)naphthalimide gave the tritiated
product with a high chemical yield (85%), but very low
specific activity (19 GBq/mmol, cf. 106 GBq/mmol theo-
retical incorporation).
† SUNY/Health Science Center.
(1) P.G.W. is also an Adjunct Faculty member with the Department
of Pharmaceutical Chemistry, University of California, San Francisco,
CA 94143-0446.
(2) Lindsay, D. G.; Shall, S. Biochem. J . 1971, 121, 737-745.
(3) Wallace, R. J . Br. J . Nutr. 1992, 68, 365-372.
(4) Baschang, G.; Brundish, D. E.; Hartmann, A.; Stanek, J .; Wade,
R. J . Labelled Compd. Radiopharm. 1983, 20, 691-696.
(5) Barnard, E. A.; Wieckowski, J .; Chiu, T. H. Nature 1971, 234,
207-209.
(6) Harding, C. R.; Scott, I. R. Anal. Biochem. 1983, 129, 371-376.
(7) Carroll, P. M. Nature 1963, 197, 694-695.
To improve the isotope incorporation, we discontinued
the catalytic dehalogenation approach and investigated
a radical dehalogenation reaction (Scheme 1). N-(Iodo-
acetoxy)phthalimide was reacted with commercial tri-
(8) Nefkens, G. H. L.; Tesser, G. I. J . Am. Chem. Soc. 1961, 83, 1263.
(9) Anderson, G. W.; Zimmerman, J . E.; Callahan, F. M. J . Am.
Chem. Soc. 1964, 86, 1839-1842.
(10) de Groot, N.; Lapidot, Y.; Panet, A.; Wolman, Y. Biochem.
Biophys. Res. Commun. 1966, 25, 17-22.
2
butyltin deuteride (100% H),18 and triethylborane was
(11) Osawa, T.; J eanloz, R. W. J . Org. Chem. 1965, 30, 448-450.
(12) Hampton, A.; Slotin, L. A.; Chawla, R. R. J . Med. Chem. 1976,
19, 1279-1283.
used as the radical initiator. The product N-(deuterio-
acetoxy)phthalimide was obtained in 40% chemical yield
with 60% deuterium incorporation. Fresh preparation
of tributyltin deuteride19 was shown by gas chromatog-
raphy to provide 67% chemical yield of the desired
(13) Fink, D. J .; Gainer, H. Science 1980, 208, 303-305.
(14) Dolly, O. J .; Nockles, E. A. V.; Lo, M. M. S.; Barnard, E. A.
Biochem. J . 1981, 193, 919-923.
(15) Caras, I. W.; Friedlander, E. J .; Bloch, K. J . Biol. Chem. 1980,
255, 3575-3580.
(16) Tang, Y. S.; Davis, A.-M.; Kitcher, J . P. J . Labelled Compd.
Radiopharm. 1983, 20, 277-284.
(18) Neumann, W. P. Synthesis 1987, 665-683.
(19) J aiswal, D. K.; Andres, H.; Morimoto, H.; Williams, P. G. J .
Chem. Soc., Chem. Commun. 1993, 907-909.
(17) Corringer, P. J .; Durieux, C.; Ruiz-Gayo, M.; Roques, B. P. J .
Labelled Compd. Radiopharm. 1992, 31, 459-468.
S0022-3263(96)01652-0 CCC: $12.00 © 1996 American Chemical Society

9626 J . Org. Chem., Vol. 61, No. 26, 1996
Notes
Sch em e 1
reagent. To mimic the tritiated reagent and tritiation
conditions, this deuterated reagent was used in several
N-deuterioacetylation reactions.
in the presence of triethylamine in dimethyl sulfoxide,
dioxane, methanol, or water at room temperature was
both rapid and specific. As an example, when 1 equiv of
L-cysteine in water was acetylated with 1 equiv of
N-acetoxyphthalimide in acetonitrile, N-acetyl-L-cysteine
was the sole product, as shown by NMR and mass
spectrometric analyses. Use of 2 equiv of the reagent in
the reaction generated N,S-diacetyl-L-cysteine. For N-
tritioacetylation reactions (Scheme 2), two peptides,
ACTH (1-4) (H2N-Ser-Tyr-Ser-Met-OH), and neuro-
tensin (8-13) (H2N-Arg-Arg-Pro-Tyr-Ile-Leu-OH), were
selected. The tritioacetylations were carried out by the
addition of N-(tritioacetoxy)phthalimide in acetonitrile
to a solution of the substrate in DMSO under mildly basic
conditions, as described fully in the Experimental Sec-
tion. The tritium NMR spectrum of the N-[3H]-acetylated
ACTH at the N-terminal serine (specific activity 680 GBq/
mmol) showed a single peak at δ ) 2.02 ppm. Similarly,
a single peak was observed at δ ) 2.03 ppm for neuro-
tensin N-[3H]-acetylated at the N-terminal arginine. The
chemical yield (37%) and specific radioactivity (420 GBq/
mmol) of N-(tritioacetyl)neurotensin (8-13) were deter-
mined by radio-HPLC, starting from the batch of tritiated
reagent with S.A. 480 GBq/mmol.
For the analogous tritiation reactions, high specific
activity tributyltin tritide19 was prepared and used for
the radical dehalogenation of the N-(iodoacetoxy)phthal-
imide. Two different batches of the (tritioacetoxy)-
phthalimide reagent were prepared. NMR and HPLC
analyses confirmed the desired product was present with
some byproducts that were formed during the radical
dehalogenation reaction. The tin byproducts and other
impurities were removed by dissolving the crude reaction
mixture in acetonitrile and repeated extraction of the
impurities into hexane.20 Radio-HPLC and proton and
tritium NMR analyses of the purified product revealed
a radiochemically pure reagent with chemical yields of
38 and 40% and specific activities of 480 and 680 GBq/
mmol.
Sch em e 2
The applicability of the reagent for N-tritioacetylation
reactions was further demonstrated with the labeling of
six different peptides (6-17 amino acid residues), pre-
pared by solid-phase techniques. In contrast to the two
examples given above, the C-terminus of each peptide
was still attached to a (hydroxymethyl)polystyrene (HMP)
resin, and all amino acid side chains were protected, with
only the N-terminus free for acetylation.21 After N-
tritioacetylation, the peptides were released from the
resin, deprotected, and purified by radio-HPLC to be used
in a peptide precipitation assay.22 The peptide with the
highest specific activity (Ile-Gly-Pro-Gly-Arg-Ala-Phe,
630 GBq/mmol) was also analyzed by tritium NMR
spectroscopy and showed a singlet at δ ) 2.04 ppm,
corresponding to the N-(tritioacetyl) group attached to
the N-terminal amino acid.
F igu r e 1. NMR spectra of N-(tritioacetoxy)phthalimide: (a)
320 MHz 3H NMR spectrum of N-(tritioacetoxy)phthalimide
in acetonitrile-d3 (δ 2.00-2.70 ppm); (b) 320 MHz 1H-decoupled
3H NMR spectrum of N-(tritioacetoxy)phthalimide; (c) 300
1
MHz H NMR spectrum of N-(tritioacetoxy)phthalimide.
The proton-coupled tritium NMR spectrum of the
purified N-(tritioacetoxy)phthalimide showed a triplet at
2.36 ppm for the tritioacetoxy group (Figure 1a, J HT
)
15.6 Hz), which collapsed to a singlet in the proton-
decoupled tritium spectrum (Figure 1b). The proton
NMR spectrum of the tritiated sample showed a singlet
for the CH3 species at 2.37 ppm and a doublet for the
CH23H species at 2.33 ppm. The downfield line of the
doublet is obscured by the CH3 singlet (Figure 1c).
Integration of the methyl peaks in this proton spectrum
of the tritiated sample allowed calculation of the specific
activity of the N-(tritioacetoxy)phthalimide at 620 GBq/
mmol, in reasonable agreement with HPLC analyses.
Acet yla t ion R ea ct ion s. Acetylation of muramic
acid,7 L-cysteine, ACTH (1-4), and neurotensin (8-13)
Con clu sion s
We have demonstrated the synthesis and use of N-
(tritioacetoxy)phthalimide at high specific activity (up to
680 GBq/mmol) as a new and selective reagent for the
(21) Seligman, S. J . Anal. Biochem. 1993, 211, 324-325.
(22) Seligman, S. J . J . Immun. Methods 1994, 168, 101-110.
(20) Berge, J . M.; Roberts, S. M. Synthesis 1979, 471-472.

Notes
J . Org. Chem., Vol. 61, No. 26, 1996 9627
0.85 g, 4.35 mmol) were added to dry ethyl acetate (EtOAc, 125
mL), and the mixture was stirred for 3 h at room temperature
and then filtered. The filtrate was dried (Na2SO4), and the
residue was crystallized from ethanol to give a white solid (0.82
g, 90%): mp 185 °C; 1H NMR δ (CDCl3) 7.76-7.89 (m, 4H), 2.39
(s, 3H).24
C. N-Acetoxyn a p h th a lim id e. The sodium salt of N-hy-
droxynaphthalimide (235 mg, 1 mmol) was suspended in benzene
(5 mL), and a solution of acetyl chloride (0.08 mL, 1.14 mmol)
in benzene (1 mL) was added dropwise. The reaction was
vigorously stirred at room temperature, and after 5 min a white
precipitate (NaCl) was formed. TLC of the reaction mixture
(hexane:ethyl acetate 90:10) showed a new product (Rf 0.7). The
reaction mixture was filtered, and the solvent was evaporated
under a stream of nitrogen gas to furnish a pale yellow product
(234 mg, 91%): mp 205 °C; 1H NMR δ (CDCl3) 8.64 (d, 2H), 8.29
(d, 2H), 7.79 (t, 2H), 2.47 (s, 3H).
facile tritium labeling of peptides and other molecules
containing a free amino group. The reagent is solid and
nonvolatile, and normally 1-1.5 equiv affords rapid and
complete acetylation. The chemical and radiochemical
stability of this reagent was not specifically studied.
However, in one case the tritiated reagent was prepared
and stored in acetonitrile, and portions were used for
labeling reactions over more than 3 weeks, without any
apparent loss of specific activity between reagent and
acetylated product. Its facile preparation and analysis
make N-(tritioacetoxy)phthalimide synthetically very
attractive and superior to either tritiated acetic anhy-
dride or tritiated acetic acid as a tritioacetylating reagent.
In comparison with N-succinimidyl [2,3-3H]propionate,
N-(tritioacetoxy)phthalimide is unlikely to affect biologi-
cal activity, gives rapid acylation, and shows high
selectivity between thiol and amino groups.
Syn t h esis of P r ecu r sor s t o t h e La b eled Acet yla t ion
Rea gen ts. A. N-(Iod oa cetoxy)su ccin im id e. This synthesis
was identical to published methods.10,12,26
We have also developed a simple and mild method of
tritioacetylation of amino acids and peptides in organic
solvents (dimethyl sulfoxide, methanol, dioxane) or water.
We are currently investigating the utility and selectivity
of N-(tritioacetoxy)phthalimide in the high specific activ-
ity tritioacetylation of several biologically important
compounds containing thiol and hydroxyl functional
groups.
Similar precursor and labeling chemistry would yield
the N-(tritioacetoxy) derivatives of succinimide and naph-
thalimide. If fully developed into tritioacetylation re-
agents, these offer the promise of a very useful range of
solubility and acetylation characteristics. The hazards
associated with the generation and use of highly tritiated
acetylation or tin-derived reagents should be carefully
assessed in planning experiments such as those described
in this work.
B. N-(Iod oa cetoxy)p h th a lim id e. This synthesis was based
on published preparations of N-(iodoacetoxy)succinimide.10,12,26
N-Hydroxyphthalimide (1.4 g, 8.7 mmol), iodoacetic acid (1.6 g,
8.7 mmol), and DCC (1.7 g, 8.7 mmol) were added to dry EtOAc
(250 mL), and the mixture was stirred for 5 h at room temper-
ature. The reaction mixture was then filtered, the filtrate was
dried, and the residue was crystallized from ethanol to give a
white solid (2.15 g, 75%): mp 120 °C; 1H NMR δ (acetone-d6)
8.01 (m, 4H), 4.32 (s, 2H). Anal. Calcd C10H6NO4I: C 36.2; H
1.8; I 38.4; Found: C 36.4; H 1.8; I 38.2.
C. N-(Iod oa cetoxy)n a p h th a lim id e. This synthesis was
based on published preparations of N-(iodoacetoxy)succin-
imide.10,12,26 The sodium salt of N-hydroxynaphthalimide was
acidified and yielded two products, with the major component
being the ring-opened product. N-Hydroxynaphthalimide (22
mg, 0.1 mmol), iodoacetic acid (18 mg, 0.1 mmol), and DCC (19.5
mg, 0.1 mmol) were added to dry EtOAc (2 mL), and the mixture
was stirred for 5 h at room temperature. The reaction mixture
was then filtered, the filtrate was dried, and the residue was
crystallized from ethanol to give a white solid (27 mg, 69%): mp
184 °C; 1H NMR δ (CDCl3) 8.66 (d, 2H), 8.31 (d, 2H), 7.78 (t,
2H), 4.07 (s, 2H).
Exp er im en ta l Section
La belin g of Acetyla tion Rea gen ts by Ca ta lytic Deh a lo-
gen a tion . A. N-(Tr itioa cetoxy)su ccin im id e. N-(Iodoac-
etoxy)succinimide (14 mg, 0.05 mmol) was dissolved in EtOAc
(1 mL), and Pd-C (10%, 10 mg) and triethylamine (4 µL) were
added. The reaction vessel was connected to the vacuum line,
and the C-I bond was hydrogenolyzed under 1 atm of 10%
tritium gas for 2 h. The reaction was then halted by removal of
the tritium gas, and methanol (1 mL) was added and removed
by evacuation. The residue was dissolved in EtOAc (1 mL), and
the catalyst was filtered off. EtOAc was removed, and THF-d8
(1 mL) was added for 1H and 3H NMR analyses. The total
radioactivity was assessed by liquid scintillation counting as 370
MBq (10 mCi, 7.2%): 1H NMR δ (THF-d8) 2.23 (s, 3H), 2.71 (s,
4H); [1H]3H NMR (THF-d8) 2.24 (s, 50%, -OCOCH23H), 4.05 (s,
50%, unknown).
Gen er a l P r oced u r es. Chemical reagents were purchased
and purified as previously described.23 Similarly, mass spec-
trometric analyses, NMR spectroscopic studies, and liquid
scintillation counting details have been published,23 with the
minor change that Opti-Fluor LSC cocktail was used in this
project.
High -P r essu r e Liqu id Ch r om atogr aph y. Analytical HPLC
was performed on a Chemco Pak silica column. The mobile
phase was hexane:ether (75:25) for the N-(tritioacetoxy)phthal-
imide analysis. Peptide analyses were performed on a LC-18
Vydac column, using a gradient mobile phase of acetonitrile/
water with 0.1% TFA from 2 to 52% acetonitrile (1-26 min) and
flow rate of 1.5 mL/min. UV detection was at 234 and 210 nm
on a Hewlett-Packard 1040A diode array spectrophotometer, and
radioactivity was monitored by an IN/US â-Ram HPLC flow
detector, using a lithium glass scintillant cell with an efficiency
of ca. 0.5%. The specific radioactivity of the reaction products
was determined by comparison of UV standards with the
analytical sample, combined with liquid scintillation counting
of the isolated HPLC peak effluents.
Syn t h esis of Un la b eled Acet yla t ion R ea gen t s. A. N-
Acetoxysu ccin im id e. This synthesis was identical to a pub-
lished approach.24
B. N-Acetoxyp h th a lim id e. Previous syntheses of this
compound were not well described.7,11,25 The current synthesis
is similar to early preparations of N-(iodoacetoxy)succinim-
ide.10,12,26 N-Hydroxyphthalimide (0.7 g, 4.35 mmol), acetic acid
(HOAc, 258 µL, 4.35 mmol), and dicyclohexylcarbodiimide (DCC,
B. N-(Tr it ioa cet oxy)n a p h t h a lim id e. N-(Bromoacetoxy)-
naphthalimide (10.3 mg, 0.03 mmol) was dissolved in EtOAc (1
mL), and Pd-C (10%, 10 mg) and triethylamine (5 µL) were
added. The reaction vessel was connected to a vacuum line, and
the compound was hydrogenated under 1 atm of 10% tritium
gas for 2 h. The reaction was then halted by removal of the
tritium gas, and methanol (1 mL) was added and removed by
evacuation. EtOAc (1 mL) was added, the catalyst removed by
filtration, and the product was analyzed by radio-HPLC to give
6.5 mg (85%), specific activity 19 GBq/mmol: 1H NMR δ (CDCl3)
8.65 (d, 2H), 8.31 (d, 2H), 7.80 (t, 2H), 2.47 (s, 2.86H); [1H]3H
NMR δ (CDCl3) 2.47 (s); (1H)3H NMR δ (CDCl3) 2.45 (t), J HT
15.6 Hz.
)
La belin g of Acetyla tion Rea gen ts by Ra d ica l Deh a lo-
gen a tion . A. N-(Deu ter ioa cetoxy)p h th a lim id e. A solution
of N-(iodoacetoxy)phthalimide (10 mg, 0.03 mmol) in dry THF
(0.3 mL) was added dropwise to a mixture of tributyltin
deuteride (13 µL, 0.045 mmol) and triethylborane (4 µL, 0.004
mmol) in dry THF (0.7 mL) under a nitrogen atmosphere. The
reaction was stirred at room temperature for 3 h. After reaction,
the solvent was removed under a flow of nitrogen gas, and the
(23) Than, C.; Morimoto, H.; Andres, H.; Williams, P. G. J . Org.
Chem. 1995, 60, 7503-7507.
(24) Grochowski, E.; J urczak, J . Synthesis 1977, 277-279.
(25) Neunhoeffer, O.; Gottschlich, R. Liebigs Ann. Chem. 1970, 736,
100-109.
(26) Slotin, L. A.; Hampton, A. Nucleic Acid Chem. 1978, 2, 843-
846.

9628 J . Org. Chem., Vol. 61, No. 26, 1996
Notes
residue was dissolved in acetonitrile (1 mL) and extracted with
hexane (1 mL, 5×). HPLC analysis of the acetonitrile phase
showed the desired product (2.36 mg, 38%): mp 190 °C; m/ z
206 (60% D); [1H]2H NMR δ (CHCl3) 2.40 (s), (1H)2H NMR 2.39
(t).
and the filtrate was analyzed by radio-HPLC and tritium NMR
spectroscopy. N-[3H]Acetyl-ACTH (1-4): [1H]3H NMR δ (D2O)
2.02 (s); (1H)3H NMR δ (D2O) 2.02 (t). N-[3H]Acetylneurotensin
(8-13): [1H]3H NMR δ (D2O) 2.03 (s); (1H)3H NMR δ (D2O) 2.03
(t).
B. N-(Tr itioa cetoxy)p h th a lim id e. N-(Iodoacetoxy)phthal-
imide (20 mg, 0.06 mmol) was dissolved in dry THF (0.6 mL),
and this solution was added to freshly prepared tributyltin
tritide19 (0.09 mmol) and triethylborane (8 µL, 0.008 mmol) in
dry THF (1.4 mL) under N2. The reaction mixture was then
stirred at room temperature for 3 h, after which time the solvent
was removed under a flow of nitrogen gas. The crude reaction
product was dissolved in acetonitrile and extracted with hexane
(1 mL, 5×). The acetonitrile was then removed under a flow of
nitrogen gas and the residue dissolved in deuteriated acetonitrile
(1 mL). Radio-HPLC analysis showed 40% yield of the desired
product with a specific activity of 640 GBq/mmol: [1H]3H NMR
δ (CD3CN) 2.36 (s); (1H)3H NMR 2.35 (t), J HT ) 15.6 Hz.
C. N-(Deu ter ioa cetoxy)n a p h th a lim id e. N-(Iodoacetoxy)-
naphthalimide (10 mg, 0.026 mmol) was dissolved in dry THF
(0.3 mL), and the mixture was added dropwise to a solution of
tributyltin deuteride (13 µL, 0.045 mmol) and triethylborane (4
µL, 0.004 mmol) in dry THF (0.7 mL) under nitrogen. After the
reaction was stirred at room temperature for 4 h, monitoring
by TLC (hexane:ethyl acetate 5:1) showed only 10-20% product.
The reaction was continued for an additional 12 h, after which
time the solvent was removed under nitrogen and the residue
was dissolved in CDCl3 for 1H NMR analysis. The ratio of the
product to starting material was estimated to be approximately
4:1: 1H NMR δ (CDCl3) 8.66 (d, 2H), 8.31 (d, 2H), 7.78 (t, 2H),
2.47 (s, 2H); m/ z 256 (64% D).
Gen er a l P r oced u r e for th e N-Tr itioa cetyla tion of HMP -
P ep tid es. The H2N-Ile-Gly-Pro-Gly-Arg-Ala-Phe-HMP (8.3 mg,
3.96 µmol) was suspended in DMSO (100 µL) in a conical vial,
and triethylamine (2 µL) was added. N-(Tritioacetoxy)phthal-
imide (640 GBq/mmol, 3.65 GBq, 1.2 mg, 5.7 µmol) in EtOAc
(100 µL) was prepared and added to the HMP-peptide in DMSO.
The reaction vessel was vortexed at room temperature for 30
min. The supernatant (200 µL) was separated and the residue
was then centrifuged at 15000g for 2 min and washed and
centrifuged alternately with N-methylpyrrolidine (0.5 mL) and
dichloromethane:methanol (1:1, 0.5 mL) for a total of six washes.
The residue was lyophilized for 10 min, and the N-[3H]-
acetylated peptide was deprotected and released from the resin
by the addition of thioanisole (10 µL), ethanedithiol (10 µL), and
trifluoroacetic acid (80 µL) while it was intermittently vortexed
for 2 h. Water (0.5 mL) was added, and the vial was centrifuged
to separate the resin. The supernatant was removed, and the
pellet was washed with the addition of water (0.5 mL). The
combined supernatant was lyophilized overnight (the usual ether
precipitation step21 was omitted because of the availability of
high vacuum apparatus). The resultant peptide was dissolved
in deuteriated water (0.3 mL) and purified by HPLC to give the
desired radiochemically pure peptide with a specific activity of
630 GBq/mmol and chemical yield of 35%: [1H]3H NMR δ (D2O)
2.04 (s); (1H)3H NMR δ (D2O) 2.06 (t).
Gen er a l P r oced u r e for th e N-Tr itioa cetyla tion of P ep -
tid es. Freshly prepared N-(tritioacetoxy)phthalimide (0.58 mg,
2.8 µmol, 480-680 GBq/mmol) was dissolved in EtOAc or
acetonitrile (250 µL). This solution was added to a mixture of
the peptide (2 µmol) in DMSO (250 µL) and triethylamine (5
µL). The mixture was vortexed at room temperature for 1 h. A
slight yellow to orange color was observed after the reaction was
finished. The solvents were removed by lyophilization, the
residue was dissolved in deuteriated water (300 µL) and filtered,
Ack n ow led gm en t. This research was supported by
the Biomedical Research Technology Program, National
Center for Research Resources, U.S. National Institutes
of Health under Grant P41 RR01237, through Depart-
ment of Energy Contract DE-AC03-76SF00098 with the
University of California.
J O9616522