Synthesis of sulfur-35 reagents for protein labeling

Article abstract of DOI:10.1002/jlcr.1163

Two ³⁵S reagents were developed to radiolabel proteins. The first reagent, a N-hydroxysuccinimide (NHS) ester (SMSB), acylates the ε-amino group of lysine residues in proteins. The second reagent, an aldehyde (MSAPPA), labels lysine residues vi

Full text of DOI:10.1002/jlcr.1163

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 395–398.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1163
Short Research Article
Synthesis of sulfur-35 reagents for protein labelingy
SUMEI REN*, PAUL MCNAMARA, DAVID KOHARSKI, DAVID HESK and SCOTT BORGES
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-4545, Kenilworth, NJ 07033, USA
Received 25 September 2006; Revised 1 November 2006; Accepted 17 November 2006
Abstract: Two 35S reagents were developed to radiolabel proteins. The first reagent, a N-hydroxysuccinimide (NHS)
ester (SMSB), acylates the e-amino group of lysine residues in proteins. The second reagent, an aldehyde (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. The biological activity of the proteins did
not change after labeling with these 35S reagents. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: sulfur-35; radiolabeled proteins; monoclonal antibodies
Introduction
used to label monoclonal antibodies (Mabs) in good to
excellent yields. Biological activity of the labeled Mabs
was unchanged.
Radiolabeled proteins are valuable tools for biochem-
ical and biomedical research. The most common
labeling methods use the g-emitting radionuclide 125I.
Direct labeling and conjugation are the most widely
used methods to radiolabel proteins.1 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 reagent (BH),2ꢀ6 reacts with the e-amino
group of lysine residues. There are also some limita-
tions 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 a high specific activity
(1485 Ci/mmol, t1/2=87 days) and is a viable alter-
native 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, SMSB and
MSAPPA, as shown in Scheme 1. Both reagents were
Results and discussion
Labeling proteins with activated
(SMSB)
[
35S]NHS ester
N-hydroxysuccinimide esters, such as the Bolton–
Hunter 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 esters of
aliphatic carboxylic acids.3 35S is most readily intro-
duced into organic molecules by sulfonylation of an
amine with [35S]methanesulfonyl chloride.7 Sulfonyla-
tion of alkyl amines proceeds in higher yields than aryl
amines. The simplest molecule incorporating these
structural features is [35S]SMSB. The synthesis of a
key intermediate, 4, is shown in Scheme 2.
The amine group of benzoic acid 1 was protected with
the Cbz group to give 2 in 56.1% yield. Carboxylic acid
2 was converted to the t-butyl ester 3 in 74.5% yield.
The Cbz protecting group is removed by hydrogenolysis
in 93.4% yield to give amine 4. The synthesis of
*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
yProceedings of the Ninth International Symposium on the Synthesis
and Applications of Isotopically Labelled Compounds, Edinburgh,
16–20 July 2006.
[
35S]SMSB from 4 and protein labeling with [35S]SMSB
are shown in Scheme 3.
Commercially available
[
35S]methane sulfonate
(5–10 mCi, specific activity: 1400 Ci/mmmol) was
Copyright # 2007 John Wiley & Sons, Ltd.

396 S. REN ET AL.
O
O
O
O
N
H
O
HO
O
O
O
125I
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
Scheme 1
O
O
Cl
OH
, NaHCO3, NaOH
H
EDCI, DMAP, CH2Cl2
OH
OH
Cbz
N
H2N
Dioxane, water
56.1%
Cbz
2
1
74.5%
O
O
Pd/C, H2, CH3OH
93.4%
O
O
H
N
H2N
Cbz
3
4
Scheme 2
O
O
O
35 S
O
35S
H2N
CO2Cl2, CH2CH2, DMF
4
H3C
OH
H3C
Cl
O
Et3N,CH2Cl2
O
~10 mCi (~10-6 mmol)
59.2% for two steps
5
O
O
O
O
HO
N
O
OH
H
N
O
H
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
N
H
O
N
35S
Me
pH=9.0, 0.1M sodium borate
50%
O
Protein
SMSB
H3C
NH
O
35
S
N-Succinimidyl-4-(methane
sulfonylamino-methyl)-benzoate
O
8
Scheme 3
7
converted to methane [35S]sulfonyl chloride 5
and
7 in 70% yield. Treatment of the carboxylic acid 7 with
N-hydroxysuccinimide and EDCI, gave [35S]SMSB in
38% yield after NP-HPLC purification. [35S]SMSB was
then reacted with 4 to give sulfonamide 6 in about 59%
yield. Acidolysis of the t-butyl ester gave carboxylic acid
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 395–398
DOI: 10.1002.jlcr

SYNTHESIS OF SULFUR-35 REAGENTS 397
reacted with Mab in pH 9.0 sodium borate buffer for
30 min and then quenched with glycine. The reaction
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
aggregated protein was removed by size-exclusion
HPLC.
Labeling proteins with the [35S]SMSB has the follow-
ing limitations:
*
The SMSB reagent reacts with proteins at pH 8.5 and
above; some proteins may not be stable at this pH range.
*
Acylated lysine residues are no longer charged at
physiological pH. This could change the activity of
the protein.
O
O
Ph3P=CHCHO, THF, 50oC
H
CH(OEt)3, EtOH, NBS, RT
H
64.2%
95.1%
NC
NC
9
10
OEt
OEt
OEt
OEt
LiAlH4, Ether, 0oC-RT
64.1%
Pd/C, H2, EtOH, RT
95.7%
NC
11
12
NH2
OEt
OEt
NH2
13
Scheme 4
OEt
OEt
13
O
35 S
O
35 S
CO2Cl2, CH2CH2, DMF
Et3N, CH2Cl2
H3C
OH
NH2
H3C
Cl
O
O
65% for the two steps
5
OEt
OEt
O
H
TFA/H2O
55-70%
35
S
NH
H3C
35
S
NH
H3C
14
O
O
MSAPPA
O
O
4-(Methanesulfonylamino
-methyl)-phenylpropylaldehyde
HN
O
NH2
HN
Protein
O
N
H
NaCNBH3, pH=6-9,
70-98%
NaH2PO4, RT
Protein
35
NH
H3C
S
15
O
O
Scheme 5
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 395–398
DOI: 10.1002.jlcr

398 S. REN ET AL.
The overall yield from [35S]methane sulfonate was
Advantages of labeling proteins with [35S] aldehyde
(MSAPPA) by reductive alkylation are as follows:
*
low (ꢁ10%).
*
The reaction conditions led to some protein
*
aggregation.
The reaction may be conducted over a broad pH
range;
Labeling proteins with an [35S]aldehyde (MSAPPA) by
reductive alkylation
*
No aggregated proteins were formed and the reac-
tions gave cleaner products;
*
Higher overall radiochemical yield was obtained;
*
Proteins have been labeled by reductive alkylation
Modified lysine residues are still charged at physio-
using
[
14C]formaldehyde, [3H]sodium borohydride8
logical pH only with small pKa changes.
and a [125I]aldehyde9. This chemistry has seen little
use, even though it has some useful features. Reductive
alkylation of the lysine e-amino group occurs over a
broad pH range (pH 6–9). In contrast with Bolton-
Hunter type reagent, the modified lysine residues are
still charged at physiological pH with small pKa
changes. A 35S-labeled aldehyde labeling reagent will
broaden the scope of 35S labeling chemistry.
35S labeled aldehyde was designed as follows. The
benzyl amine moiety was used to introduce 35S, in
analogous fashion to SMSB reagent. Aliphatic alde-
hydes alkylate amines in aqueous media with higher
yields than aryl aldehydes.9 [35S]MSAPPA incorporates
these structural features.
Conclusion
Two 35S reagents were developed to radiolabel proteins
in good to excellent yield. Five monoclonal antibodies
(Mabs) have been labeled with these 35S-reagents and
their bioactivity was unchanged. Reductive alkylation
(MSAPPA reagent) approach is our preferred method.
Acknowledgements
Special thanks to Mr Richard Ingram from Protein Eng.
& Biochem group for helpful instruction on handling
and purification of protein.
The synthesis of a key intermediate, 13, is shown in
Scheme 4. 4-Cyanobenzaldehyde 9 was reacted with a
Wittig type reagent to yield 64% of a, b-unsaturated
aldehyde 10. Aldehyde 10 was converted to diethyl
acetal 11 in 95% yield. Reduction of the cyano group
gave amine 12 in 64% yield. Catalytic hydrogenation of
12 afforded 13 in 95% yield. Intermediate 13 was
purified by RP-HPLC before reaction with [35S]metha-
nesulfonyl chloride.
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Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 395–398
DOI: 10.1002.jlcr