Design and synthesis of sulfur-35 agents and their applications for protein labeling

Article abstract of DOI:10.1002/jlcr.1606

Two new ³⁵S reagents were developed to radiolabel proteins. The first reagent, N-succinimidyl-4-(methane [³⁵S]sulfonylamino-methyl)- benzoate (SMSB), acylates the e-amino group of lysine residues in proteins. The second reagent, 4-(m

Full text of DOI:10.1002/jlcr.1606

Research Article
Received 21 January 2009,
Revised 18 March 2009,
Accepted 3 April 2009
Published online 28 May 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1606
Design and synthesis of sulfur-35 agents and
their applications for protein labeling
Ã
Sumei Ren, Paul McNamara, David Koharski, David Hesk,
and Scott Borges
Two new 35S reagents were developed to radiolabel proteins. The first reagent, N-succinimidyl-4-(methane [35S]sulfony-
lamino-methyl)-benzoate (SMSB), acylates the e-amino group of lysine residues in proteins. The second reagent, 4-(methane
[
35S]sulfonylamino-methyl)-phenylpropylaldehyde (MSAPPA), labels lysine residues via reductive alkylation. Comparing the
two methods, the reductive alkylation method labeled proteins over a broader pH range with higher overall radiochemical
yield. More than ten monoclonal antibodies (mAbs) have been labeled with these 35S labeling reagents, the biological
activity of the mAbs was unchanged. Part of this work was presented in the Ninth International Symposium on the
Synthesis and Applications of Isotopically Labelled Compounds, Edinburgh, 16–20 July 2006.
Keywords: sulfur-35; radiolabeled proteins; monoclonal antibodies
esters of aliphatic carboxylic acids.4 35S is most readily
introduced into organic molecules by sulfonylation of an amine
Introduction
with methane [35S]sulfonyl chloride.9 Sulfonylation of alkyl
Radiolabeled proteins are valuable tools for biochemical and
biomedical research.1 The most common labeling methods use
amines proceeds in higher yields than aryl amines. The simplest
the g-emitting radionuclide 125I. Direct labeling and conjugation
are the most widely used methods to radiolabel proteins.2 Direct
labeling uses sodium [125I]iodide and an oxidant to label
tyrosine and histidine residues in proteins. This method exposes
proteins to harsh oxidation conditions and the label is partially
lost in vivo. In the conjugation method, a small, radioiodinated
reagent, such as the Bolton–Hunter (BH) reagent,3–7 reacts with
the e-amino group of lysine residues. There are also some
limitations for BH-type reagents: in vivo de-iodination is reduced
but still occurs and acylation of lysine residues in proteins is
carried out at pH 8.5 or above where some proteins are not
stable. In both methods, extra safety procedures are needed
when handling a g-emitting isotope such as 125I. 35S has high
specific activity (1485 Ci/mmol, t1/2 = 87 days) and is a viable
alternative to 125I (2169 Ci/mmol, t1/2 = 60 days). Since it is a
weak b-emitter, radiation protection procedures are simpler. We
designed two 35S reagents, N-succinimidyl-4-(methane [35S]sul-
fonylamino-methyl)-benzoate (SMSB) and 4-(methane [35S]sul-
fonylamino-methyl)-phenylpropylaldehyde (MSAPPA), as shown
in Figure 1. Both reagents were used to label monoclonal
antibodies (mAbs) in good to excellent yields. Preliminary
in vitro experiments have shown good label stability.8
molecule incorporating these structural features is [35S]SMSB.
The synthesis of a key intermediate, 4, is shown in Scheme 1.
The amine group of benzoic acid 1 was protected with the
Cbz group to give 2 in 74.2% yield. Carboxylic acid 2 was
converted to t-butyl ester 3 in 74.5% yield. The Cbz protecting
group is removed by hydrogenolysis to give amine 4 in 93.4%
yield. Synthesis of [35S]SMSB from 4 and protein labeling with
[35S]SMSB are shown in Scheme 2.
Commercially available methane [35S]sulfonate (5–25 mCi,
specific activity: 1400 Ci/mmol) was converted to methane
[35S]sulfonyl chloride 5 using a literature procedure9 and then
reacted with 4 to give sulfonamide 6 in 59% overall yield.
Acidolysis of the t-butyl ester gave carboxylic acid 7 in 70%
yield. Treatment of the carboxylic acid 7 with NHS and N-ethyl-
N0-(3-dimethylaminopropyl)carbodiimide (EDCI) gave [35S]SMSB
in 38% yield after Normal-Phase High-Performance Liquid
Chromatography (NP-HPLC) purification. [35S]SMSB was reacted
with mAb in pH 9.0 sodium borate buffer for 30 min and then
quenched with glycine. The reactant was passed through a short
gel-filtration column. Usually, about 50% of SMSB was covalently
bound to the protein after initial purification. About 20% of the
labeled mAb was larger MW aggregated protein. The aggre-
gated protein was removed by size-exclusion HPLC.
Results and discussion
Labeling proteins with activated [35S]NHS ester (SMSB)
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-4545,
Kenilworth, NJ 07033, USA
N-hydroxysuccinimide (NHS) esters, such as the BH reagent, are
the most commonly used reagents for labeling proteins.
We designed a [35S]NHS ester as follows. Esters of aryl carboxylic
acids acylate amines in aqueous media with higher yields than
*Correspondence to: Sumei Ren, Schering-Plough Research Institute, 2015
Galloping Hill Road, K-15-4545, Kenilworth, NJ 07033, USA.
E-mail: sumei.ren@spcorp.com
J. Label Compd. Radiopharm 2009, 52 316–323
Copyright r 2009 John Wiley & Sons, Ltd.

S. Ren et al.
O
O
O
O
N
H
O
HO
125I
O
O
O
N
H3C
NH
H3C
35S
NH
35
S
O
BH
Bolton-Hunter reagent
O O
O
O
MSAPPA
4-(Methanesulfonylamino
-methyl)-phenylpropylaldehyde
SMSB
N-Succinimidyl-4-(methane
sulfonylamino-methyl)-benzoate
N-Succinimidyl 3-(4-hydroxy-3-
Iodophenyl)propionate
Figure 1. Radioactive reagents for protein labeling.
O
O
Cl
OH
, NaHCO3, NaOH
EDCI, DMAP, CH2Cl2
OH 74.5%
OH
Cbz
H
N
H2N
Dioxane, water, 74.2%
Cbz
2
1
O
O
Pd/C, H2, CH3OH
93.4%
O
O
H
N
H2N
Cbz
3
4
Scheme 1. Synthesis of 4-aminomethyl-benzoic acid tert-butyl ester 4.
O
O
Cl
O
O
35 S
Cl
O
35 S
H2N
4
, CH2Cl2, DMF
O
H3C
OH
H3C
Cl
O
O
Et3N,CH2Cl2
~10 mCi (~10-6 mmol)
59.2% for two steps
5
O
O
O
O
HO
N
O
OH
H
O
H
N
TFA/CH2Cl2
70.9%
O
N
35S
35 S
Me
Me
EDCI, CH2Cl2
38.1%
O
O
6
7
HN
O
O
O
NH2
O
HN
N
O
O
H
O
35
N
H
O
N
S
pH=9.0, 0.1M sodium borate
50%
[35S]SMSB
Protein
Me
O
H3C
NH
O
35
S
N-Succinimidyl-4-(methane
sulfonylamino-methyl)-benzoate
O
8
Scheme 2. Synthesis of [35S]SMSB and its application for protein labeling.
J. Label Compd. Radiopharm 2009, 52 316–323
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

S. Ren et al.
O
O
H
Cl
OH
OH
S
*
H3C
O
S
O
S
O
NH
*
Oxidizing reagents
Low yield
NH
*
H3C
NH2
H3C
O
O
[
35S]MSAPPA
9
O
H
OEt
OEt
OEt
O
Cl
OEt
H+
S
*
H3C
O
O
O
*
NH
[
S
H3C
NH
*
S
H3C
35S]MSAPPA
NH2
O
O
Higher yield, cleaner reaction
14
Scheme 3. Synthetic design of [35S]MSAPPA.
O
H
O
Ph3P=CHCHO, THF, 50oC
64.2%
CH(OEt)3, EtOH, NBS, RT
95.1%
H
NC
NC
10
11
OEt
OEt
OEt
LiAlH4, Ether, 0oC-RT
64.1%
Pd/C, H2, EtOH, RT
95.7%
OEt
NC
12
13
NH2
OEt
OEt
NH2
14
Scheme 4. Synthesis of 4-(3, 3-diethoxy-propyl)-benzylamine.
Labeling proteins with [35S]SMSB has the following limitations:
35S-labeled aldehyde was designed as follows. The benzyl
amine moiety was used to introduce 35S, analogous to the SMSB
reagent. Aliphatic aldehydes alkylate amines in aqueous media
with higher yields than aryl aldehydes.11 [35S]MSAPPA incorpo-
rates these structural features. Two possible ways to prepare
[35S]MSAPPA were explored, and the route from protected
aldehyde 14 gave higher yield and cleaner reactions than the
route from alcohol substrate 9 as shown in Scheme 3.
ꢀ
ꢀ
SMSB reagent reacts with proteins at pH 8.5 and above;
some proteins may not stable to this pH range.
Acylated lysine residues are no longer charged at physiolo-
gical pH. This could change the activity of the protein.
Overall yield from methane [35S]sulfonate was low (ꢁ10%).
The reaction conditions led to some protein aggregation.
ꢀ
ꢀ
The synthesis of a key intermediate, 14, is shown in Scheme 4.
4-Cyanobenzaldehyde 10 was reacted with a Wittig-type
reagent to yield 64% of a, b-unsaturated aldehyde 11. Aldehyde
Labeling proteins with an [35S]aldehyde (MSAPPA) by
reductive alkylation
Proteins have been labeled by reductive alkylation using [14C]for- 11 was converted to diethyl acetal 12 in 95% yield. Reduction of
maldehyde, sodium [3H]borohydride,10 and [125I]aldehyde.11 This the cyano group gave amine 13 in 64% yield. Catalytic
chemistry has seen little use, even though it has some useful hydrogenation of 13 afforded 14 in 95% yield. Intermediate
features in contrast with the BH-type reagent, such as (1) the 14 was purified by Reversed Phase-High Performance Liquid
reductive alkylation of the lysine e-amino group occurs over a Chromatography (RP-HPLC) before reaction with methane
broad pH range (pH 6–9); (2) the modified lysine residues are still [35S]sulfonyl chloride.
charged at physiological pH with small pKa changes. A 35S aldehyde
Synthesis of [35S]MSAPPA from 14 and protein labeling with
labeling reagent will broaden the scope of 35S labeling chemistry. [35S]MSAPPA are shown in Scheme 5.
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 316–323

S. Ren et al.
OEt
OEt
O
Cl
, CH2Cl2, DMF
14
Et3N, CH2Cl2
O
35 S
Cl
O
35S
O
NH2
O
H3C
OH
H3C
Cl
O
65% for the two steps
O
~10 mCi (~10-6 mmol)
5
OEt
OEt
H
TFA/H2O
71%
35 NH
H3C
35 NH
S
S
H3C
15
[
35S]MSAPPA
O
O
O
O
4-(Methanesulfonylamino
-methyl)-phenylpropylaldehyde
HN
O
NH2
HN
Protein
O
N
H
NaCNBH3, pH=6-9, NaH2PO4, RT
70-98%
Protein
35S
O
NH
H3C
16
O
Scheme 5. Synthesis of [35S]MSAPPA and its application for protein labeling.
Methane [35S]sulfonate (5–20 mCi, specific activity: 1400 Ci/mmol) without further purification. Methane [35S]sulfonate was pur-
was converted to methane [35S]sulfonyl chloride 59 and then chased from PerkinElmer. All mAbs were from Schering-Plough
reacted with 14 to give sulfonamide 15 in 65% overall yield. Research Institute.
Hydrolysis of the acetal 15 gave [35S]MSAPPA in 71% yield after RP-
HPLC purification. [35S]MSAPPA was reacted with mAbs and Analytical methods
NaCNBH3 in pH 6–9 sodium hydrogen phosphate buffer overnight
Mass spectra were acquired on a JEOL MStation double-
at room temperature (RT). The reaction pH was chosen based on
focusing magnetic sector mass spectrometer at a Fast-Atom
the stability of the mAbs at the pH and the isoelectric point (pI) of
Bombardment (FAB) ionization mode.
1H-NMR and 13C-NMR (400 or 600 MHz) spectra were obtained
on a Varian spectrometer.
the mAbs, usually 1 pH unit away from pI of the mAbs to avoid
precipitation during reaction. The labeled mAbs was purified by
passing through a gel-filtration column (Bio-Gel, P-6). Usually, about
LC-MS: Waters Micromass with Waters 2695 Separation
70–98% of [35S]MSAPPA was covalently bound to the protein after
Module operating in the ES1 ionization mode. Supelcosil LC-
purification. Overall yield was ꢁ30% from methane [35S]sulfonate.
CN, 150 mm ꢂ 4.6 mm, 215 nm, isocratic, 0.1% HCO2H in
H2O:0.1% HCO2H in CH3CN (65:35), 1.0 ml/min.
Advantages of labeling proteins with [35S]aldehyde (MSAPPA)
by reductive alkylation are as follows:
ꢀ
ꢀ
Reaction is conducted at a broad pH range.
No aggregated proteins formed, and reactions gave cleaner
products.
Higher overall radiochemical yield was obtained.
Modified lysine residues are still charged at physiological pH
only with small pKa changes.
Analytical HPLC
Waters 600 Multisolvent Delivery System with Waters 2487
Absorbance Detector and Radiomatic 525TR Radioflow Detector,
Packard Flo-Scint III liquid scintillation cocktail (1:3). HPLC elute:
System 1: Zorbax extend C18, 5 mm, 150 mm ꢂ 3.0 mm,
254 nm, 0.05 M TEAA (pH = 9.0):CH3CN (65:35) for 15 min
followed by a step gradient to CH3CN, 0.5 ml/min.
ꢀ
ꢀ
System 2: Phenomenex Prodigy 5 mm silica, 150 mm ꢂ 3.2 mm,
254 nm. EtOAc:hexane:HOAc = 65:35:2, 0.5 ml/min.
Experimental section
General
System 3: TosoHaas TSK Gel G3000 SWXL column,
300 mm ꢂ 7.8 mm, 280 nm, 301C, 20 mM NaOAc/150 mM NaCl,
pH = 5.2, 1.0 ml/min.
Chemicals
Chemicals and solvents were purchased from standard com-
mercial sources (Aldrich, Fluka, Fisher, Acros) and were used
System 4: Bio-Rad Bio-Sil 250 5 SEC column, 300 mm ꢂ 7.8 mm
(or equiv.), 280 nm, 20 mM NaOAc/150 mM NaCl, pH 5.0, 1.0 ml/min.
J. Label Compd. Radiopharm 2009, 52 316–323
Copyright r 2009 John Wiley & Sons, Ltd.
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S. Ren et al.
Synthesis of 4-(benzyloxycarbonylamino-methyl)-benzoic acid tert-
butyl ester (3)
Semi-preparative HPLC
HPLC purification was conducted on a Waters Delta Prep 4000
with Waters 486 Tunable Absorbance Detector; the following
systems were used:
To a solution of 4-(benzyloxycarbonylamino-methyl)-benzoic
acid (2, 2.0 g, 7.0 mmol), DMAP (0.856 g, 7.0 mmol), and tert-
butanol (6.0 ml, 63.9 mmol) in anhydrous CH2Cl2 (6.0 ml) at 51C,
EDCI (3.35 g, 17.5 mmol) was added. The reactant was warmed
up to RT and stirred under N2 overnight. The reactant was
washed with H2O (2 ꢂ 50 ml), brine (50 ml), dried over Na2SO4,
filtered, and concentrated. The crude product was purified by
flash chromatography on silica gel (elution with 1–20% EtOAc/
hexanes with 1% Et3N) to give 1.79 g (74.5%) of compound 3 as
System a: Phenomenex Prodigy 5 mm silica, 250 mm ꢂ 10 mm,
254 nm. EtOAc:hexane:HOAc = 55:45:2, 4.0 ml/min.
System b: Zorbax extend C18, 250 mm ꢂ 9.4 mm, 254 nm,
0.05 M TEAA (pH = 8.9):CH3CN (65:35), 4.0 ml/min.
System c: Phenomenex Prodigy 5 mm silica, 250 mm ꢂ 10 mm,
254 nm. EtOAc:hexane:HOAc = 70:30:1, 4.0 ml/min.
System d: Zorbax extend C18, 250 mm ꢂ 9.4 mm, 254 nm,
0.05 M TEAA (pH = 9.0):CH3CN (60:40), 4.0 ml/min.
Radioactivity was measured on a Packard 2200CA liquid
scintillation analyzer using Scintiverse BD liquid scintillation
cocktail. TLC plates were scanned on a Bioscan 1000 linear
analyzer.
1
a colorless oil. H-NMR (DMSO-d6, 400 MHz, d): 7.92 (t, 1H, NH),
7.83 (d, J = 8.0 Hz, 2H), 7.34 (m, 7H), 5.03 (s, 2H), 4.25 (d, J = 6.4 Hz,
2H), 1.52 (s, 9H). 13C-NMR (DMSO-d6, d): 164.56 (1C), 156.16 (1C),
144.71 (1C), 136.88 (1C), 129.70 (1C), 128.93 (2C), 128.22 (2C),
127.68 (1C), 127.62 (2C), 126.81 (2C), 80.52 (1C), 66.45 (1C), 43.61
(1C), 27.86 (3C). MS m/z: 286 (Mꢃ(t-Bu)12H)1, 342 (M1H)1, 364
(M1Na)1. HRMS-FAB (m/z): [M1H]1 calcd. for C20H24NO4,
342.1705; found 342.1703.
Purification of protein
Bio-Spin columns with Bio-Gel P-6 were purchased from Bio-Rad
Laboratories. The column was used to initially clean up the
labeled proteins by using a swing bucket centrifuge. The
centrifuge (IEC Centra, CL2) was purchased from International
Equipment Company, and operated at 3.3 ꢂ 1000 RPM for 2 min
per each run.
The labeled proteins from SMSB route may be further purified
by the size-exclusion HPLC system e. The HPLC conducted was
on a Bio-Rad BioLogic Duo-flow protein purification system with
BioLogic QuadTec UV–Vis Detector. Superdex 200 column,
300 mm ꢂ 10 mm, 280 nm. Isocratic elution with 0.150 M NaCl,
0.020 M NaOAc, pH = 5.50 buffer, 0.5 ml/min.
Synthesis of 4-aminomethyl-benzoic acid tert-butyl ester (4)
To a solution of 4-(benzyloxycarbonylamino-methyl)-benzoic
acid tert-butyl ester 3 (1.77 g, 5.18 mmol) in CH3OH, Pd/C
(10% wt on dry, wet, 200 mg) was added. The flask was
degassed, and the reactant was stirred under an H2 balloon at
RT for 1 h. The mixture was filtered through a celite pad and
concentrated to dryness. The crude product was purified by
flash chromatography on silica gel (elution with 1–5% CH3OH/
CH2Cl2 with 1% Et3N) to give 1.00 g (93.4%) of compound 4 as a
1
colorless oil. H-NMR (DMSO-d6, 400 MHz, d): 7.81 (d, J = 8.0 Hz,
2H), 7.42 (d, J = 8.4 Hz, 2H), 3.75 (s, 2H), 1.97 (br, 2H, NH2), 1.52 (s,
9H). 13C-NMR (DMSO-d6, d): 164.70 (1C), 149.10 (1C), 129.07 (1C),
128.70 (2C), 126.83 (2C), 80.30 (1C), 45.27 (1C), 27.87 (3C). LC-MS
m/z: 191 (MꢃNH31H)1, 208 (M1H)1, 415 (2M1H)1. HRMS-FAB
(m/z): [M1H]1 calcd. for C12H18NO2, 208.1338; found 208.1332.
Synthesis of unlabeled corresponding compounds as
authentic standards
All corresponding unlabeled compounds were synthesized
and fully analyzed. The reaction conditions were different from
the labeled reactions due to the difference of the concentration
and the stoichiometry of the reagents in the reactions.
Synthesis of 4-(methane sulfonylamino-methyl)-benzoic acid tert-
butyl ester (6)
Labeling proteins with activated [35S]NHS ester (SMSB)
To a solution of 4-aminomethyl-benzoic acid tert-butyl ester
(4, 100 mg, 0.483 mmol) in anhydrous CH2Cl2 (5.0 ml), methane
sulfonyl chloride (38 ml, 0.489 mmol) and Et3N (100 ml,
0.754 mmol) were added. The reactant was stirred at RT under
N2 for 1 h, diluted with CH2Cl2 (20 ml), and washed with
saturated NaHCO3 (2 ꢂ 15 ml). The organic phase was dried
over Na2SO4, filtered, and concentrated. The crude product was
purified by flash chromatography on silica gel (elution with
0–0.5% CH3OH/CH2Cl2) to give 115 mg (83.9%) of compound 6
as a white solid. 1H-NMR (DMSO-d6, 400MHz, d): 7.87 (d,
J = 8.4 Hz, 2H), 7.66 (t, 1H, NH), 7.44 (d, J = 8.0 Hz, 2H), 4.21 (d,
J = 6.4 Hz, 2H), 2.86 (s, 3H), 1.53 (s, 9H). 13C-NMR (DMSO-d6, d):
165.04 (1C), 143.87 (1C), 130.56 (1C), 129.50 (2C), 127.96 (2C),
81.13(1C), 46.19 (1C), 40.51(1C), 28.40 (1C). LC-MS m/z: 230 (acid
1H)1, 459 (2acid1H)1, 571(2M1H)1, 593 (2M1Na)1. HRMS-
FAB (m/z): [Mꢃt-Bu12H]1 calcd. for C9H12NO4S, 230.0487; found
230.0495.
Synthesis of 4-(benzyloxycarbonylamino-methyl)-benzoic acid (2)
A solution of 4-(aminomethyl)benzoic acid (3.0 g, 20.0 mmol) in
dioxane (30 ml) and 10% NaHCO3 (60 ml) was cooled to 51C, and
benzyl chloroformate (2.86 ml, 20.0 mmol) was added. The
reaction pH was adjusted to 8.0 with NaOH (1.0 N). Additional
NaOH may be added to keep the pH constant during the
reaction. The reactant was warmed to RT and stirred overnight.
The solvent was removed and H2O (100 ml) was added. The
solution was washed with Et2O (2 ꢂ 25 ml) and acidified to
pH = 1 with 2 N HCl. The resulting solid was filtered, washed with
H2O (2 ꢂ 5 ml), and dried under vacuum to give 4.2 g (74.2%) of
2 as a white solid. 1H-NMR (DMSO-d6, 400 MHz, d): 7.86–7.89 (m,
3H), 7.4 (m, 7H), 5.03 (s, 2H), 4.25 (d, J = 6.4 Hz, 2H). 13C-NMR
(DMSO-d6, d): 167.43 (1C), 156.70 (1C), 145.22 (1C), 137.42 (1C),
129.75 (2C), 129.68 (1C), 128.74 (2C), 128.20 (1C), 128.15 (2C),
127.34 (2C), 66.00 (1C), 44.16 (1C). LC-MS m/z: 286 (M1H)1, 571
(2M1H)1. HRMS-FAB (m/z): [M1H]1calcd. for C16H16NO4,
286.1079; found 286.1068.
Synthesis of 4-(methane sulfonylamino-methyl)-benzoic acid (7)
To the solution of 4-(methane sulfonylamino-methyl)-benzoic
acid tert-butyl ester (6, 852 mg, 3.0 mmol) in CH2Cl2 (30 ml), TFA
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J. Label Compd. Radiopharm 2009, 52 316–323

S. Ren et al.
(4.0 ml) was added. The reactant was stirred at RT under N2 for co-eluted with an authentic standard 6 by the HPLC system 1
6 h, and the solvent was removed under vacuum. The crude (tR = 11.0 min).
product was dried under vacuum to give 680 mg (99.4%) of
compound 7 as a white solid. The compound was directly used Synthesis of 4-(methane [35S]sulfonylamino-methyl)-benzoic acid
(
35S-7)
in the next step without further purification. 1H-NMR (DMSO-d6,
400 MHz, d): 7.90 (d, J = 8.0 Hz, 2H), 7.66 (t, 1H, NH), 7.45 (d,
J = 8.0 Hz, 2H), 4.21 (d, J = 6.4 Hz, 2H), 2.87 (s, 3H). 13C-NMR
(DMSO-d6, d): 167.41 (1C), 143.85 (1C), 129.98 (1C), 129.78 (2C),
127.96 (2C), 46.21 (1C), 40.50 (1C). LC-MS m/z: 212 (MꢃH2O1H)1,
230 (M1H)1. HRMS-FAB (m/z): [M1H]1 calcd. for C9H12NO4S,
230.0487; found 230.0482.
To the solution of 4-(methane [35S]sulfonylamino-methyl)-
benzoic acid tert-butyl ester (35S-6, 14.8 mCi) in CH2Cl2 (300 ml),
TFA (50 ml) was added. The reactant was stirred at RT in a sealed
vial overnight, and the solvent was removed under N2. The
crude product was purified by the NP-HPLC system c to give
10.5 mCi (70.9%) of 35S-7. The radiochemical purity was 96.6%
by the HPLC system 1. 35S-7 was co-eluted with an authentic
standard 7 by the HPLC system 1 (tR = 1.8 min).
Synthesis of N-succinimidyl-4-(methane sulfonylamino-methyl)-
benzoate (SMSB, unlabeled)
A solution of 4-(methane sulfonylamino-methyl)-benzoic acid 7 Synthesis of N-succinimidyl-4-(methane sulfonylamino-methyl)-
(100 mg, 0.436 mmol), NHS (251 mg, 2.18 mmol), and EDCI
benzoate (35S-SMSB)
(416 mg, 2.18 mmol) in anhydrous CH2Cl2 (5.0 ml) was stirred
A solution of 4-(methane [35S]sulfonylamino-methyl)-benzoic
at RT under N2 overnight. The reactant was diluted with CH2Cl2
acid 35S-7 (10.5 mCi), NHS (15 mg, 0.13 mmol), and EDCI (20 mg,
(10 ml), washed with H2O (2 ꢂ 10 ml), dried over Na2SO4, filtered,
0.10 mmol) in anhydrous CH2Cl2 (300 ml) was stirred at RT in a
and concentrated. The crude product was first purified by flash
sealed vial overnight. The solvent was removed under N2, and
chromatography on silica gel (elution with 0–40% EtOAc/
the crude product was purified by the NP-HPLC system c to give
hexanes in 1% HOAc), and then purified by NP-HPLC (HPLC
4.0 mCi (38.1%) of 35S-SMSB. The radiochemical purity was
1
system a) to give 130 mg (91.5%) of SMSB as a white solid. H-
97.2% by the HPLC system 2. 35S-SMSB was co-eluted with an
NMR (DMSO-d6, 400 MHz, d): 8.07 (d, J = 8.4 Hz, 2H), 7.74 (t, 1H,
authentic standard SMSB by the HPLC system 2 (tR = 5.2 min).
NH), 7.60 (d, J = 8.4 Hz, 2H), 4.29 (d, J = 6.0 Hz, 2H), 2.91 (s, 3H),
2.87 (s, 4H). 13C-NMR (DMSO-d6, d): 170.61 (2C), 161.89 (1C),
General procedure for labeling protein (mAb) (35S-16) using 35S-
147.11 (1C), 130.54 (2C), 128.73 (2C), 123.58 (1C), 46.11 (1C),
SMSB
40.49 (1C), 26.16 (2C). LC-MS m/z: 212 (acidꢃH2O1H)1, 327 (M1
H)1, 349 (M1Na)1, 653 (2M1H)1. HRMS-FAB (m/z): [M1H]1 35S-SMSB (4.0 mCi) in CH2Cl2 (2.0 ml) was transferred to a 2 ml
calcd. for C13H15N2O6S, 327.0651; found 327.0635.
plastic vial, and the solvent was removed under N2. To the
residue, DMF (10 ml) was added and the vial was shaken for
5 min, and then a solution of mAbs (4.0 mg) in borate buffer
(300 ml, pH = 9.0, 0.1 M) was added. The reactant was incubated
at RT for 20 min and quenched by the addition of glycine (20 ml,
0.2 M). The labeled mAbs was initially purified by passing
through a gel-filtration column (Bio-Gel, P-6) to give 2.49 mCi
(62.3%) of 35S-16 with the radiochemical purity of 80.1% by the
HPLC system 3. The product was further purified by the size-
exclusion HPLC system e to give 1.1 mCi of 35S-16 with the
radiochemical purity of 96.7% by the HPLC system 3.
Synthesis of methane [35S]sulfonyl chloride (5)
An aliquot of methane [35S]sulfonate (25 mCi, 0.32 ml, specific
activity: 1400 Ci/mmol) was transferred to a 10 ml reaction vial
by a syringe, the syringe was washed with EtOH (6 ꢂ 50 ml), and
the washing solution was added to the vial. The mixture was
stirred for 5 min, and the solvent was removed under N2. The
residue was coevaporated with EtOH (2 ꢂ 0.8 ml), CH2Cl2
(2 ꢂ 0.8 ml) under N2, and dissolved in anhydrous CH2Cl2
(1.5 ml). To the solution, oxalyl chloride (0.20 ml, 2.3 mmol) was
added. After stirring at RT under N2 for 50 min, DMF (100 ml of
10% DMF in CH2Cl2) was added slowly and the reactant was
stirred at the same condition overnight. After dilution with
CH2Cl2 (1.5 ml), the reactant was cooled to 51C, and carefully
washed with NaHCO3 (1% solution, 3 ꢂ 2.0 ml) and NaHSO3 (2%
Labeling proteins with an [35S]aldehyde (MSAPPA) by
reductive alkylation
Synthesis of 4-cyano-cinnamaldehyde (11)
solution, 2 ꢂ 2 ml). The organic phase was dried over Na2SO4, To a solution of 4-cyanobenzaldehyde (1.02 g, 7.78 mmol) in
filtered, and concentrated by distillation at 601C under N2 THF (40 ml), (triphenyl phosphoramylidene)acetaldehyde
(1 atm). The crude product 5 was used immediately in the next (2.20 g, 6.94 mmol) was added. The suspension was stirred
step without further purification.
at 501C under N2 for 8 h and concentrated to dryness
under vacuum. The crude product was purified by flash
chromatography on silica gel (elution with 0–40% EtOAc/
hexanes) to give 0.70 g (64.2%) of compound 11 as a white
solid. 1H-NMR (DMSO-d6, 400 MHz, d): 9.70 (d, J = 7.6 Hz, 1H,
COH), 7.93 (s, 4H), 7.80 (d, J = 16.0 Hz, 1H), 7.01 (dd, J = 7.6 Hz,
16.0 Hz, 1H). HRMS-FAB (m/z): [M1H]1 calcd. for C10H8NO,
158.0606; found 158.0605.
Synthesis of 4-(methane [35S]sulfonylamino-methyl)-benzoic acid
tert-butyl ester (35S-6)
To the crude product of methane [35S]sulfonyl chloride 5,
a solution of 4-aminomethyl-benzoic acid tert-butyl ester
(4, 15 mg, 0.072 mmol) and Et3N (20 ml, 0.151 mmol) in anhydrous
CH2Cl2 (150 ml) was added. The reactant was stirred at RT in a
sealed vial for 1 h, and the solvent was removed under N2. The
crude product was purified by the RP-HPLC system b to give
14.8 mCi (59.2% for the first two steps) of 35S-6. The radio- To
Synthesis of 4-cyano-cinnamaldehyde diethyl acetal (12)
a
solution of 4-cyano-cinnamaldehyde (11, 695 mg,
chemical purity was 99.7% by the HPLC system 1. 35S-6 was 4.42 mmol) in EtOH (16 ml), triethyl orthoformate (1.15 ml,
J. Label Compd. Radiopharm 2009, 52 316–323
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S. Ren et al.
6.91 mmol) and N-bromosuccinimide (14 mg, 0.079 mmol) were purified by flash chromatography on silica gel (elution with
added. The reactant was stirred at RT under N2 for 4 h, quenched 0–20% EtOAc/hexanes) to give 200 mg (52.3%) of compound 15
with NaOH solution (10%, 30 ml), and extracted with Et2O as a colorless oil. 1H-NMR (DMSO-d6, 600 MHz, d): 7.48 (t, 1H,
(3 ꢂ 30 ml). The organic phase was washed with brine (20 ml), NH), 7.23 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 4.41
dried over Na2SO4, filtered, and concentrated. The crude (t, J = 5.6 Hz, 1H), 4.09 (d, J = 6.4 Hz, 2H), 3.57–3.53 (m, 2H),
product was purified by flash chromatography on silica gel 3.43–3.38 (m, 2H), 2.81 (s, 3H), 2.57 (t, J = 8.0 Hz, 2H), 1.79–1.76
(elution with 0–15% EtOAc/hexanes) to give 970 mg (95.1%) of (m, 2H ), 1.10 (t, J = 6.4 Hz, 6H). 13C-NMR (DMSO-d6, d): 140.89
1
compound 12 as a white solid. H-NMR (DMSO-d6, 400 MHz, d): (1C), 135.98 (1C), 128.60 (2C), 128.09 (2C), 101.92 (1C), 61.06 (2C),
7.78 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 16.4 Hz, 46.39 (1C), 39.67 (1C), 35.56 (1C), 30.66 (1C), 16.00 (2C). HRMS-
1H), 6.46 (dd, J = 5.2, 16 Hz, 1H), 5.06 (dd, J = 5.2, 0.8 Hz, 1H), FAB (m/z): [M1Na]1 calcd. for C15H25NO4SNa, 338.1402; found
3.67–3.51 (m, 2H), 3.55–3.45 (m, 2H), 1.13 (t, J = 7.2 Hz, 6H). 338.1396.
HRMS-FAB (m/z): [M1H]1 calcd. for C14H18NO2, 232.1338; found
232.1327.
Synthesis of 4-(methane sulfonylamino-methyl)-phenylpropylalde-
hyde) (MSAPPA)
Synthesis of 4-aminomethyl-cinnamaldehyde diethyl acetal (13)
N-[4-(3, 3-diethoxy-propyl)]-methane sulfonamide (15, 50 mg,
0.16 mmol) was dissolved in TFA/water (1:3, 1.2 ml). The
reactant was stirred at RT for 1 h, then diluted with CH2Cl2
(10 ml), and quenched slowly with NaHCO3 (saturated,
10 ml) for 10 min. The aqueous phase was backextracted
with CH2Cl2 (10 ml). The combined organic phase was
dried over Na2SO4, filtered, and concentrated under vacuum.
The crude product was purified by flash chromatography on
silica gel (elution with 20–60% EtOAc/hexanes) to give 30 mg
(78.9%) of the compound MSAPPA as a white solid. 1H-NMR
(DMSO-d6, 600 MHz, d): 9.68 (s, 1H, COH), 7.50 (t, 1H, NH), 7.23 (d,
J = 7.2 Hz, 2H), 7.18 (d, J = 7.6 Hz, 2H), 4.09 (d, J = 6.0 Hz, 2H),
2.85–2.82 (m, 5H), 2.75 (t, J = 6.8 Hz, 2H). 13C-NMR (DMSO-d6, d):
202.96 (1C, CHO), 140.08 (1C), 136.16 (1C), 128.62 (2C), 128.11
(2C), 46.35 (1C), 44.94 (1C), 40.25 (1C), 27.76 (1C). HRMS-FAB
(m/z): [M1Na]1 calcd. for C11H15NO3SNa, 264.06703; found
264.07012.
To a solution of 4-cyano-cinnamaldehyde diethyl acetal 12
(950 mg, 4.11 mmol) in Et2O (10 ml) at 01C, LiAlH4 (1.0 M, 25 ml,
25 mmol) was added dropwise for 30 min under N2. The reactant
was stirred at 01C for 30 min, warmed up to RT, and stirred
for 2 h. The reactant was diluted with Et2O (50 ml), cooled to 01C,
and quenched slowly with H2O (30 ml). The organic phase was
washed with NaOH (10%, 30 ml), brine (30 ml), dried over
Na2SO4, filtered, and concentrated. The crude product was
purified by flash chromatography on silica gel (elution with
0.5–4% CH3OH (7 N NH3)/CH2Cl2) to give 620 mg (64.1%) of
compound 13 as a colorless oil. 1H-NMR (DMSO-d6, 400 MHz, d):
7.39 (d, J = 7.2 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 6.62 (d, J = 16.0 Hz,
1H), 6.19 (dd, J = 16.0, 4.8 Hz, 1H), 5.06 (d, J = 5.2 Hz, 1H), 3.67 (s,
2H), 3.60–3.56 (m, 2H), 3.48–3.44 (m, 2H), 1.13 (t, J = 7.2 Hz, 6H).
HRMS-FAB (m/z): [M1H]1 calcd. for C14H22NO2, 236.1651; found
236.1642.
Synthesis of N-[4-(3, 3-diethoxy-propyl)]-methane [35S]sulfonamide
(
Synthesis of 4-(3, 3-diethoxy-propyl)-benzylamine (14)
35S-15)
To
a solution of 4-aminomethyl-cinnamaldehyde diethyl
To the crude product of methane [35S]sulfonyl chloride 5
(18.5 mCi, 1.28 ꢂ 10ꢃ5 mmol), a solution of 4-(3, 3-diethoxy-
propyl)-benzylamine (14, 12 mg, 0.050 mmol) and Et3N (10 ml,
0.072 mmol) in anhydrous CH2Cl2 (140 ml) was added. The
reactant was stirred at RT in a sealed vial for 1 h 30 min, and
the solvent was removed under N2. The crude product was
purified by RP-HPLC system b, and the collected fractions were
concentrated to dryness and then dissolved in CH2Cl2, dried
over Na2SO4, filtered, and concentrated. The residue was
dissolved in CH3CN (3.0 ml) to give 13.8 mCi (74.6%) of 35S-15.
The radiochemical purity was 99.5% by the HPLC system 1. 35S-
15 was co-eluted with an authentic standard 15 by the HPLC
system 1 (tR = 12.5 min).
acetal (13, 590 mg, 2.51 mmol) in EtOH (25 ml), Pd/C (10% wt
on dry, wet, 240 mg) was added. The flask was degassed,
and the reactant was stirred under H2 balloon at RT for 5 h.
The mixture was filtered through a celite pad and concentrated
to dryness to give crude 4-(3, 3-diethoxy-propyl)-benzylamine
14 (570 mg, 95.8% yield) as a colorless oil. The product
may be purified by HPLC d before reacting with methane
[35S]sulfonyl chloride 5. 1H-NMR (DMSO-d6, 600 MHz, d):
7.20 (d, J = 7.6 Hz, 2H), 7.10 (d, J = 7.6 Hz, 2H), 4.14 (t, J = 5.6 Hz,
1H), 3.65 (s, 2H, CH2NH2), 3.57–3.53 (m, 2H), 3.42–3.38
(m, 2H), 3.31 (br, 2H, NH2), 2.54–2.51 (t, J = 7.6 Hz, 2H),
1.77–1.75 (m, 2H ), 1.10 (t, J = 7.2 Hz, 6H). 13C-NMR (DMSO-d6,
d): 141.51 (1C), 139.22 (1C), 127.82 (2C), 126.91 (2C), 101.43 (1C),
60.40 (2C), 45.26 (1C), 34.98 (1C), 29.93 (1C), 15.24 (2C). HRMS-
FAB (m/z): [M1H]1 calcd. for C14H24NO2, 238.1807; found
238.1805.
Synthesis of 4-(methane [35S]sulfonylamino-methyl)-phenylpropy-
laldehyde) (35S-MSAPPA)
An aliquot of N-[4-(3, 3-diethoxy-propyl)]-methane [35S]sulfona-
mide (35S-15) in CH3CN (0.43 ml, 2.0 mCi) was transferred to a
Synthesis of N-[4-(3, 3-diethoxy-propyl)]-methane sulfonamide (15)
To a solution of 4-(3, 3-diethoxy-propyl)-benzylamine (14, plastic vial (7 ml), and the solvent was removed under N2. To the
288 mg, 1.22 mmol), Et3N (0.87 ml, 6.10 mmol), and five seeds residue, TFA/water (1:3, 200 ml) was added. After stirring at RT for
˚
of 5 A molecular sieve in anhydrous CH2Cl2 (2.0 ml), methane 1 h, the reactant was diluted with CH2Cl2 (0.5 ml), cooled to 51C,
sulfonyl chloride 5 (0.14 ml in 0.6 ml of CH2Cl2, 1.46 mmol ) was and quenched slowly with NaHCO3 (saturated, 0.5 ml) for 10 min.
added slowly. The reactant was stirred at RT in a sealed vial for The aqueous phase was backextracted with CH2Cl2 (3 ꢂ 0.5 ml).
3 h, and then diluted with CH2Cl2 (20 ml). The reactant was The combined organic phase was dried over Na2SO4, filtered,
washed with saturated NaHCO3 (10 ml), brine (10 ml), dried over and concentrated under N2 to give 1.35 mCi (67.5%) of 35S-
Na2SO4, filtered, and concentrated. The crude product was MSAPPA with the radiochemical purity of 73.3% by the HPLC
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S. Ren et al.
system 1. 35S-MSAPPA was co-eluted with an authentic
standard 35S-MSAPPA by the HPLC system 1 (tR = 4.0 min).
The crude product was directly used in the next step without
further purification.
Acknowledgement
Special thanks to Mr Richard Ingram from Protein Engineering
and Biochem group for the helpful instruction on handling and
purification of proteins.
General procedure for labeling protein (mAb) using 35S-MSAPPA
To the vial containing 35S-MSAPPA (1.35 mCi), DMSO (10 ml),
and NaH2PO4 buffer (50 ml, pH = 7.5, different buffer solutions
may be used depending on the protein), a solution of
mAbs (2.0 mg) in the reaction buffer (120 ml) and NaCNBH3
(50 ml, 0.1 M) were added. The reactant was incubated at
RT for 20 h and purified by passing through a gel-filtration
column (Bio-Gel, P-6) to give 0.91 mCi (74%) of 35S-16.
The radiochemical purity of the protein was 95% by the HPLC
system 4.
References
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Conclusion
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G. Melillo, R. L. Ellsworth, L. H. T. Van Der Ploeg, A. A. Patchett, R. G.
Smith, J. Med. Chem. 1996, 39, 1767–1770.
Two 35S reagents were developed to radiolabel proteins in good
to excellent yield. More than ten mAbs have been labeled with
these 35S reagents and their bioactivity was unchanged.
Reductive amination (MSAPPA reagent) approach is our
preferred method.
[10] G. E. Means, R. E. Feeney, Anal. Biochem. 1995, 224, 1–16.
[11] J. R. Panuska, C. W. Parker, Anal. Biochem. 1987, 160, 192–201.
J. Label Compd. Radiopharm 2009, 52 316–323
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org