Preparation of deuterated methyl and dimethyl substituted nicotinoylating agents for derivatization of the N-terminal of protein.

Article abstract of DOI:10.1248/cpb.51.1399

Methyl groups of 6-methylnicotinic acid and 2,6-dimethylnicotinic acid were deuterated by an H-D exchange reaction under conditions of 1% NaOD/D(2)O on heating. With a condensation reaction between the D-labeled nicotinic acid derivative and N-hydroxysuccinimide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, the nicotinoylating agents, 1-(6-methyl[D(3)]nicotinoyloxy)succinimide (2c) and 1-(2,6-dimethyl[D(6)]nicotinoyloxy)succinimide (2f) were prepared. Both D-labeled nicotinoylating agents and their unlabeled counterparts quantitatively modified the N-terminal of protein.

Full text of DOI:10.1248/cpb.51.1399

December 2003
Chem. Pharm. Bull. 51(12) 1399—1401 (2003)
1399
Preparation of Deuterated Methyl and Dimethyl Substituted
Nicotinoylating Agents for Derivatization of the N-Terminal of Protein
*
Hiroki TSUMOTO, Chie MURATA, Naoki MIYATA, Ryo TAGUCHI, and Kohfuku KOHDA
Graduate School of Pharmaceutical Sciences, Nagoya City University; Tanabedori, Mizuho-ku, Nagoya 467–8603, Japan.
Received July 31, 2003; accepted September 16, 2003
Methyl groups of 6-methylnicotinic acid and 2,6-dimethylnicotinic acid were deuterated by an H–D ex-
change reaction under conditions of 1% NaOD/D2O on heating. With a condensation reaction between the D-la-
beled nicotinic acid derivative and N-hydroxysuccinimide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride, the nicotinoylating agents, 1-(6-methyl[D3]nicotinoyloxy)succinimide (2c) and 1-(2,6-dimethyl-
[D6]nicotinoyloxy)succinimide (2f) were prepared. Both D-labeled nicotinoylating agents and their unlabeled
counterparts quantitatively modified the N-terminal of protein.
Key words nicotinoylating agent; N-terminal; protein; mass spectrometry; D-label; differential expression analysis
Proteins are cell components with a variety of important pound 1b, protons of the 6-CH3 exchanged so rapidly and the
functions such as serving as receptors, enzymes, hormones reaction was almost complete at 2 h. The 2-H proton was also
and immune factors. With a disruption of these functions, exchanged with time but the degree of the exchange was far
various diseases may develop. Therefore proteome analysis is lower than that of 6-CH3. In compound 1d, 2-CH3 protons
increasingly carried out in medical and pharmaceutical re- were exchanged with time, however, the degree of exchange
search. For analyses of proteins, mass spectrometry is widely was much lower than that of 6-CH3 protons of 1b and re-
used, and recently, there has been rapid development of this quired more than 8 h for a complete exchange. Protons of 6-
technology. At the same time, studies on agents for modify- H were also exchanged very slowly, as for 2-H of 1b. Protons
ing proteins (peptides) are also being conducted for the pur- of 4-H and 5-H of each of 1b and 1d were not exchanged
pose of facilitating ionization and sequence analysis in mass under these conditions. Figure 2 showed the H–D exchange
spectrometry. Using stable isotope-labeled modifying agents, reaction of 2,6-dimethylnicotinic acid (1e). Protons of the 6-
differential expression analysis of protein can be easily per- CH3 group exchanged more rapidly than those of 2-CH3 as
formed by mass spectrometry. The isotope-coded affinity tag
(ICAT) method1) reported by Gygi et al. is a well known
method of this type. Münchback et al. reported the effective
usage of the N-terminal-modifying agent, 1-(nicotinoyl-
oxy)succinimide; in allowing for easier analysis of the b-ion
of peptides in sequencing.2) Using the ring proton-D4-labeled
1-(nicotinoyloxy)succinimide, differential expression analy-
sis of protein was also made possible.2) However, D4-labeled
nicotinic acid, the starting material for the preparation of D4-
labeled 1-(nicotinoyloxy)succinimide, is extremely expen-
sive. In this study, we describe the ease of preparing deu-
terium-labeled 6-methyl[D3]nicotinic acid and 2,6-di-
methyl[D6]nicotinic acid, as well as 1-(6-methyl[D3]nicoti-
Chart 1
noyloxy)succinimide and 1-(2,6-dimethyl[D6]nicotinoyl-
oxy)succinimide, by the condensation of D-labeled nicotinic
acid derivatives and N-hydroxysuccinimide with 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).
Other 1-(nicotinoyloxy)succinimide derivatives and analogs
were also prepared using EDC, and their abilities in modify-
ing the N-terminal of peptides were compared.
Results and Discussion
H–D Exchange of Nicotinic Acid Derivatives Hydro-
gen–deuterium exchange reactions of compounds 1b, d, e
(Chart 1) were carried out under conditions of 1% NaOD/
D2O at 180 °C, and the degrees of exchange at appropriate
times were estimated from the decrease in the height of the
peak for each proton of the compounds in the NMR spec-
trum. Figure 1 shows the degrees of exchange of 6-CH3, 2-H
and 4-H protons of 6-methylnicotinic acid (1b) and 2-CH3,
Fig. 1. H–D Exchange of 6-Methyl- and 2-Methylnicotinic Acids (1b, d)
4-H and 6-H protons of 2-methylnicotinic acid (1d). In com-
To whom correspondence should be addressed. e-mail: kohda@phar.nagoya-cu.ac.jp © 2003 Pharmaceutical Society of Japan

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Vol. 51, No. 12
Fig. 2. H–D Exchange of 2,6-Dimethylnicotinic Acid (1e)
Chart 2
already shown in Fig. 1. After 8 h, the exchange reactions of
both 6-CH3 and 2-CH3 groups were almost complete and
formed 1f. Under these conditions, protons of 4-H and 5-H
were not exchanged. From these results, we decided to use 1f
for subsequent preparation of 2f. For compound 1b, although
less than about 20% of the 2-H proton was also exchanged
when the exchange of the 6-CH3 was completed in a 3 h reac-
tion (Fig. 1), the deuterium-labeled compound 1c was also
considered to be effective and used in the preparation of 2c.
Preparation of 1-(Nicotinoyloxy)succinimide Deriva-
tives and Analogs The condensation reaction between the
carboxylic acid (1 and precursors of 2h, 2i, 3 and 4) and the
N-hydroxysuccinimide was carried out using EDC (Charts 1,
2). The reaction with 1a—f proceeded to a 56—85% yield.
In order to obtain the pure products of 2e, f, column chro-
matography was required. Products 2h,3) 2i,4) 35) and 45) were
obtained in a 40—66% yield. For the condensation reaction,
1,3-dicyclohexylcarbodiimide (DCC) is often employed,
however, the separation of the decomposed product, 1,3-dicy-
clohexylurea, is tedious. By using EDC, we could easily ob-
tain 2a6) in as good a yield as 85%.
N-Terminal Derivatization of Bradykinin Bradykinin
was treated with modifying agent 2 as described in Experi-
mental and its mass spectra were taken with a matrix-assisted
laser desorption ionization time-of-flight mass spectrometer
(MALDI-TOF/MS). As an example, MALDI-TOF mass
spectra of bradykinin before and after derivatization by 2e
and 2f are shown in Fig. 3 (mass m/z: 1060.18, 1193.25 and
1199.30, respectively). The degrees of the derivatization of
bradykinin by each modifying agent were calculated from the
peak height of the derivatized and underivatized peptides as
Fig. 3. MALDI-TOF Mass Spectra of Bradykinin
Before (A) and after derivatization by 1-(2,6-dimethylnicotinoyloxy)succinimide (2e)
(B) and by 1-(2,6-dimethyl[D6]nicotinoyloxy)succinimide (2f) (C).
Table 1. N-Terminal Derivatization of Bradykinin by Modifying Agent
Agent
2a (RϭH)
Modification (%)
90—100
90—100
90—100
90—100
90—100
90—100
0
2b (Rϭ6-CH3)
2c (Rϭ6-CD3)
2d (Rϭ2-CH3)
2e (Rϭ2,6-diCH3)
2f (Rϭ2,6-diCD3)
2g (Rϭ1-CH3)
2h (Rϭ5-Br)
2i (Rϭ6-Cl)
3
42
10
13
86
4
summarized in Table 1. The modifications by agents 2a—f
all proceeded almost quantitatively. Other agents examined
(2g—i, 3) did not show a stronger degree of modification,
except for compound 4 for which the degree was passable.
Since compound 2g has a cationic center, the derivatized
peptide was expected to have a capacity for very strong ion-
ization in mass spectrometry when this compound was intro-
duced at the N-terminal of the peptide. However, the reaction
for this peptide did not proceed under the several conditions
employed.
Utility of the Modifying Agents 2a—f As already de-
scribed above, the modifying agents 2a—f showed an almost

December 2003
1401
MS m/z: 237 (Mϩ).
quantitative ability to derivatize peptides. Therefore, by com-
binations of two desired modifying agents, one can select a
mass difference of 3 Da (2b, c), 6 Da (2e, f), 14 Da (2a, b, or
2b, e), etc. This also means that without using an expensive
deuterated modifying agent, differential expression analysis
can still be carried out. We already confirmed that more than
20% of the difference in protein expression can be detected
with these agents. The precise scope and limitation of these
nicotinoylating agents will be reported elsewhere.
1-(2-Methylnicotinoyloxy)succinimide (2d): Yield 56%. Recrystallization
from AcOEt gave colorless plates. mp 174—177 °C. 1H-NMR (CDCl3,
400 MHz) d: 2.87 (3H, s, CH3), 2.93 (4H, br s, CH2ϫ2), 7.30 (1H, dd,
Jϭ4.9, 7.8 Hz, 5-H), 8.38 (1H, dd, Jϭ1.7, 7.8 Hz, 4-H), 8.73 (d, 1H, Jϭ1.7,
4.9 Hz, 6-H). EI-MS m/z: 234 (Mϩ). Anal. Calcd for C11H10N2O4: C, 56.41;
H, 4.30; N, 11.96. Found: C, 56.27; H, 4.27; N, 11.98.
1-(2,6-Dimethylnicotinoyloxy)succinimide (2e): The product was purified
by column chromatography (silica gel, AcOEt : hexaneϭ1 : 1). Yield 66%.
Recrystallization from AcOEt gave colorless plates. mp 134—136 °C. H-
NMR (CDCl3, 400 MHz) d: 2.61 (3H, s, 6-CH3), 2.82 (3H, s, 2-CH3), 2.92
(4H, br s, CH2ϫ2), 7.19 (1H, d, Jϭ8.1 Hz, 5-H), 8.28 (1H, d, Jϭ8.1 Hz, 4-
H). EI-MS m/z: 248 (Mϩ). Anal. Calcd for C12H12N2O4: C, 58.06; H, 4.87;
N, 11.29. Found: C, 57.98; H, 4.95; N, 11.14.
1
Experimental
1H-NMR spectra were recorded on a JEOL GSX 400 spectrometer, and
chemical shifts were expressed in ppm using Me4Si as the internal standard.
Mass spectra were obtained with a JEOL JMS-DX300 spectrometer. Melting
points were measured with a Yanagimoto micro-melting point apparatus and
are uncorrected. Silica gel (Merck) was used for column chromatography.
For analysis of peptides, a Voyager Elite MALDI-TOF/MS (Perseptive
Biosystems) was used. 2,6-Dimethylnicotinic acid (1e) was prepared as re-
ported.7) Other nicotinic acid derivatives and analogs were purchased.
Bradykinin (R-P-P-G-F-S-P-F-R, monoisotopic molecular weight 1059.56)
was purchased from the Peptide Institute, Inc.
1-(2,6-Dimethyl[D6]nicotinoyloxy)succinimide (2f): The product was pu-
rified by column chromatography (silica gel, AcOEt : hexaneϭ1 : 1). Yield
65%. Recrystallization from AcOEt/hexane gave colorless plates. mp 135—
137 °C. 1H-NMR (CDCl3, 400 MHz) d: 2.92 (4H, br s, CH2ϫ2), 7.14 (1H, d,
Jϭ8.1 Hz, 5-H), 8.27 (1H, d, Jϭ8.1 Hz, 4-H). EI-MS m/z: 254 (Mϩ).
Preparation of Other Modifying Agents (2g—i, 3, 4) Compound 2g
was prepared as reported.6) Compounds 2h,3) 2i,4) 35) and 45) were prepared
by a condensation procedure between carboxylic acid and N-hydroxysuccin-
imide using EDC as described for 2a—f. For compound 3, dimethylform-
amide (DMF) was used instead of THF as the solvent because of the low
solubility in THF. The yields of 2h, 2i, 3 and 4 were 58%, 66%, 40% and
52%, respectively.
N-Terminal Derivatization of Bradykinin Solutions of bradykinin
(100 pmol/ml in H2O) and the modifying agent (1 mg of modifying agent
was dissolved in 1 ml of 50 mM sodium phosphate buffer (pH 8.5)) were pre-
pared. The bradykinin solution (20 ml) and the solution containing the modi-
fying agent (60 ml) were mixed and left at room temperature for 20 min. An-
other 60 ml of modifying agent was added and the mixture was left for a fur-
ther 2 h. Then, 60 ml of hydroxylamine solution (0.5 M in 50 mM sodium
phosphate buffer (pH 8.5)) was added and the mixture was left for about
17 h. An aliquot was taken from this solution and subjected to MALDI-
TOF/MS analysis.
MALDI-TOF/MS Analysis The sample was desalted using a ZipTip
C18 (Millipore). As the matrix, a-cyano-4-hydroxycinnamic acid (CHCA)
was used (1 mg of CHCA was dissolved in 100 ml of solution consisting of
60% ethanol–36% CH3CN–4% H2O v/v). The matrix solution (1 ml), sample
solution (1 ml) and standard solution (1 ml) were mixed, and 1 ml of the mix-
ture was taken and cocrystallized on a sample plate. The MALDI-TOF/MS
was operated in delayed extraction reflector mode using an accelerating volt-
age of 20 kV, a pulse delay time of 100 ns, a grid voltage of 90—94%, a
laser intensity of 1800, and a guide wire voltage of 0.05%. CHCA (dimer,
monoisotopic [2MϩH]ϩ 379.0930) and ACTH (monoisotopic [MϩH]ϩ
2465.1989) were used as internal standards.
Preparation of Deuterium-Labeled Nicotinic Acid Derivatives (1c, f)
Nicotinic acid derivative (1b, e 5.0 mmol) was dissolved in 10 ml of 1%
NaOD–D2O and heated at 180 °C in a sealed tube for an appropriate time.
The degree of H–D exchange was checked by means of NMR spectra. After
the reaction was completed, a small amount of Dowex 50W cation exchange
resin (Hϩ form) was added and the solution was stirred for 10 min. After the
resin was removed by filtration, the filtrate was evaporated to dryness. With-
out further purification, these D-labeled nicotinic acids (1c, f) were used for
subsequent synthesis of 1-(nicotinoyloxy)succinimide derivatives (2c, f) as
described below.
6-Methyl[D3]nicotinic Acid (1c): 6-Methylnicotinic acid (1b) was allowed
to react for 3 h. Under these conditions, less than 20% of 2-H proton was
also deuterated. Yield 76%. 1H-NMR (CDCl3, 400 MHz) d: 7.32 (1H, d,
Jϭ8.1 Hz, 5-H), 8.27 (1H, dd, Jϭ2.2, 8.1 Hz, 4-H), 9.22 (1H, d, Jϭ2.2 Hz,
2-H). Electron impact (EI)-MS m/z: 140 (Mϩ), 141 (Mϩ of 2-D derivative).
2,6-Dimethyl[D6]nicotinic Acid (1f): 2,6-Dimethylnicotinic acid (1e)7)
was allowed to react for 8 h. Yield 71%. 1H-NMR (CDCl3, 400 MHz) d:
7.14 (1H, d, Jϭ8.1 Hz, 5-H), 8.27 (1H, d, Jϭ8.1 Hz, 4-H). EI-MS m/z: 157
(Mϩ).
Synthesis of 1-(Nicotinoyloxy)succinimide Derivatives (2a—f) [Gen-
eral procedure] Nicotinic acid derivative (1, 4 mmol) was dissolved in
80 ml of dehydrated tetrahydrofuran (THF). N-Hydroxysuccinimide
(4.8 mmol) and EDC (5.2 mmol) were then added to the solution and the
mixture was stirred at room temperature for 2 d. The soluble part was sepa-
rated from the viscous residue and the solvent was removed by evaporation.
The residue was dissolved in 80 ml of CHCl3 and washed with 80 ml of
brine. After the CHCl3 layer was separated and dried over anhydrous magne-
sium sulfate, the solvent was removed by evaporation to dryness. Recrystal-
lization afforded the product.
References
1) Gygi S. P., Rist B., Gerber S. A., Turecek F., Gelb M. H., Aebersold
R., Nat. Biotechnol., 17, 994—999 (1999).
2) Münchback M., Quadroni M., Miotto G., James P., Anal. Chem., 72,
4047—4057 (2000).
1-(Nicotinoyloxy)succinimide (2a)6): Yield 85%.
1-(6-Methylnicotinoyloxy)succinimide (2b): Yield 66%. Recrystallization
from AcOEt gave colorless plates. mp 164—165 °C. 1H-NMR (CDCl3,
400 MHz) d: 2.68 (3H, s, CH3), 2.92 (4H, br s, CH2ϫ2), 7.32 (1H, d,
Jϭ7.9 Hz, 5-H), 8.27 (1H, dd, Jϭ2.1, 7.9 Hz, 4-H), 9.21 (1H, d, Jϭ2.1 Hz,
2-H). EI-MS m/z: 234 (Mϩ). Anal. Calcd for C11H10N2O4: C, 56.41; H, 4.30;
N, 11.96. Found: C, 56.50; H, 4.39; N, 11.99.
1-(6-Methyl[D3]nicotinoyloxy)succinimide (2c): Yield 67%. Recrystal-
lization from AcOEt/hexane gave colorless plates. mp 165—167 °C. 1H-
NMR (CDCl3, 400 MHz) d: 2.92 (4H, br s, CH2ϫ2), 7.32 (1H, d, Jϭ8.1 Hz,
5-H), 8.27 (1H, dd, Jϭ2.2, 8.1 Hz, 4-H), 9.22 (1H, d, Jϭ2.2 Hz, 2-H). EI-
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