Facile access to isotopically labelled valylleucyl anilides as biomarkers for the quantification of hemoglobin adducts to toxic electrophiles

Article abstract of DOI:10.1002/ejoc.200500429

An easy and efficient synthesis of valylleucyl anilide (HValLeuNHPh) and labelled HVal(¹³C₅,¹⁵N)LeuNHPh has been developed. Derivatization of these substances with oxirane, acrylonitrile, epichlorohydrin, glyceraldehyde an

Full text of DOI:10.1002/ejoc.200500429

FULL PAPER
DOI: 10.1002/ejoc.200500429
Facile Access to Isotopically Labelled Valylleucyl Anilides as Biomarkers for
the Quantification of Hemoglobin Adducts to Toxic Electrophiles
Vladimir N. Belov,[a,b] Michael Müller,[c] Oleg Ignatenko,[a,d] Ernst Hallier,[c] and
Armin de Meijere*[a,b]
Keywords: Isotopic labelling / Toxicology / Peptidomimetics / Biomarkers / Hemoglobin adducts
An easy and efficient synthesis of valylleucyl anilide (HVal-
LeuNHPh) and labelled HVal(13C5,15N)LeuNHPh has been
developed. Derivatization of these substances with oxirane,
acrylonitrile, epichlorohydrin, glyceraldehyde and other al-
dehydes gives a series of reference substances and internal
standards for the quantitative evaluation of human exposure
to toxic electrophiles by quantitative determination of their
hemoglobin adducts. Coupling of N-Z-N-Me-Val(13C5,15N)
OH with HLeuNHPh followed by hydrogenolysis affords N–
Me–Val(13C5,15N)LeuNHPh for quantification of the expo-
sure to methylating agents.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
(perfluoro)phenylthiohydantoins under basic conditions
followed by their extractive separation led to the develop-
ment of the so-called “N-alkyl” Edman procedure for GC-
MS quantification of Hb adducts.[4] This very helpful
method has been adopted in many laboratories around the
world and recommended for official use in Germany.[5]
However, up to now there was no uniform set of refer-
ence substances and internal standards for this method. For
this purpose, N-modified valine derivatives which mimic the
whole Hb molecule have been used.[6] To combine a refer-
ence substance and an internal standard in one chemical
entity, the method of isotopic dilution is often applied for
quantification. For example, a Swedish group reported the
chemical transformations of radioactively labelled (3H and
14C) and deuterated samples of epichlorohydrin followed by
their conjugation with globin to prepare standard glo-
bins.[1g] A similar methodology with sulfur mustard-[D8]
and [35S] sulfur mustard has also been reported by another
group.[4d] Incorporation of a (radioactive) label into the
“variable” part of the molecule attached to the terminal
valine residue is inconvenient for monitoring various toxic
electrophiles, and therefore this approach has not been pur-
sued any further. For routine use, it would be much better
to develop an analytical procedure in which isotopic di-
lution with stable isotopes incorporated into the valine resi-
due is adopted. Farmer et al. had reported about monitor-
ing the exposure to acrylonitrile. Their quantification was
based on N-(2-cyanoethyl)-[D8]Val-Leu-Ser-OH which may
be synthesized from the commercially available [D8]valine,
but a synthetic procedure was not published.[4g,4h]
Introduction
Human exposure to toxic electrophilic substances may
be monitored by measuring hemoglobin (Hb) adducts with
these compounds or their reactive metabolites.[1] In blood,
electrophilic toxicants may attack all possible nucleophilic
centers of Hb. All four chains of human Hb have the same
N-terminal valine residue. Therefore, detection and quanti-
tative determination of N-modified valine termini in Hb
may serve as a cumulative index for human exposure to
alkylating agents, Michael acceptors (acrylonitrile) and
other electrophilic pollutants. Classical Edman degrada-
tion[2] has been used as a simple and sensitive procedure for
cleaving off and analyzing N-terminal amino acid residues
in proteins. Modified terminal N-alkylated valine residues
in Hb were shown to be released easily in the reaction with
phenyl or perfluorophenyl isothiocyanate.[1a,1g,3] Spontane-
ous cyclization and cleavage of the corresponding N-alkyl-
[a] Institut für Organische und Biomolekulare Chemie, Georg-
August-Universität Göttingen,
Tammannstrasse 2, 37077 Göttingen, Germany
Fax: + 49-551-399475
E-mail: Armin.deMeijere@chemie.uni-goettingen.de
[b] KAdemCustomChem. GmbH,
Brombeerweg 13, 37077 Göttingen, Germany
Fax: + 49-551-23423
[c] Abteilung Arbeits- und Sozialmedizin, Georg-August-
Universität Göttingen
Waldweg 37, 37073 Göttingen, Germany
Fax: + 49-551-396184
E-mail: mmuelle3@gwdg.de
[d] Department of Organic Chemistry, St. Petersburg State
University,
To establish a new reference set, we incorporated another
commercially available labelled -valine [HVal(13C5,15N)
OH][7] into the “classical” HValLeuNHPh standards.[8]
Labelling with 13C and 15N is advantageous, because it al-
Universitetskii Pr. 26, 198504 St. Petersburg, Petrodvorets,
Russia
E-mail: ol20011@yandex.ru
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
5094
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Biomarkers for the Quantification of Hemoglobin Adducts to Toxic Electrophiles
FULL PAPER
ters physical properties of the standards (GC and HPLC valine terminus in hemoglobin (Scheme 2). Epichlorohydrin
retention times) much less than (per)deuteration, thus re- (ECH) which is an important monomer for the production
ducing the risk that the labelled standard will give a sepa- of epoxide resins, as an alkylating agent has been classified
rate peak in GC or HPLC traces and complicate the cali- as a category 2 carcinogen and allergen.[12] Alkylation of 7
bration procedure when applying the isotopic dilution tech- (as a free base) with ECH leads to the compound 10 which
nique. In addition, D-labelled substances tend to exchange mimics the initially formed Hb adduct of ECH. However,
D vs. H, especially in acidic or alkaline media which are another modified Hb was found in vivo after exposure to
often used in the extraction procedures prior to instrumen- ECH. It is represented by compound 11 with a hydroxy
tal analysis.
group instead of a chlorine atom. A plausible explanation
Here we report general procedures for the (reductive) al- for this fact is that in vivo, ECH also metabolizes to 3-
kylation and Michael addition of HVal(13C5,15N)LeuNHPh chloro-1,2-propanediol or glycidol. All three compounds
which may find wide use. Some unlabelled compounds of may react with the terminal valine residue of Hb, and their
this type are commercially available,[9] but their synthesis further transformations may end up with the single stable
has never been published. The easy production and product N-(2,3-dihydroxypropyl)valine.[1g] For a quantifica-
derivatization of labelled and unlabelled HValNHPh re- tion of ECH exposure, in addition to compound 10/10*,
ported here opens a toolbox for preparing a new series of the diol 11/11* was synthesized. Towards that, the reductive
internal standards for various biomonitoring procedures.
alkylation of 7/7* with racemic glyceraldehyde was ap-
plied,[13] to give a mixture of two diastereomers in 49%
yield. Similarly, reductive alkylations of 7/7* with benzalde-
hyde and butanal afforded the corresponding model com-
pounds 9 and 12 as mimics of the alkylation products of
Hb by benzyl and butyl halides.
Results and Discussion
The labelled and unlabelled dipeptide precursors 7*/7
were prepared according to the sequence summarized in
Scheme 1.[10]
In these reductive alkylations, sodium (triacetoxy)-
borohydride in dichloroethane consistently gave better re-
sults than Na(CN)BH3 in methanol. However, attempted
reductive alkylations of 7 with formaldehyde and acetalde-
hyde each gave inseparable mixtures of the starting com-
pound, N-monoalkyl and N,N-dialkyl derivatives. Similar
complications in the reductive alkylations with formalde-
hyde[6] and other aldehydes are known.[14] Nevertheless, re-
ductive alkylation is a much more reliable method for the
preparation of the analytical standards modelling the alkyl-
ated Hb termini. Alkyl and benzyl halides do not react se-
lectively enough, and always produce mixtures of N-alkyl-
and N,N-dialkylvalines, as well as N,N,N-trialkylammon-
ium salts.
Another very strong alkylating agent, oxirane (ethylene
oxide), produces the important reference compound 14.
The very toxic and reactive monomer acrylonitrile, readily
reacts with 7 and by way of a Michael addition gives N-
(2-cyanoethyl)valine 13. Preparative yields of the reference
substances in Scheme 2 are far from being quantitative. Our
goal was to get pure substances, and inevitable losses during
Coupling of BocLeuOH with 7-aza-1-hydroxybenzotri-
azole (HOAt) followed by the reaction with aniline (2) gave
the amide 3. Deprotection of 3 with a solution of HCl in
EtOAc produced the amine 4 as a hydrochloride, and subse-
quent amidation of the (labelled) BocVal*OH (5/5*)[11] with
4 afforded the intermediate 6/6* in excellent yield. Depro-
tection of the amino group in 6/6* resulted in the final di-
peptide 7/7*, which was isolated as the hydrochloride as
well, and it was used for further transformations as such.
All reactions, except the first one with inexpensive Boc-
LeuOH and aniline, were found to be high yielding. To fur-
ther reduce the costs of reagents, it is possible to perform
this coupling of 1 and 2 with inexpensive N-hydroxybenzo-
triazole (HOBt) or N-hydroxysuccinimide, instead of the
more expensive HOAt. In this case the yield was only insig-
nificantly lower (60 vs. 65%).
Reactions of 7/7* with various alkylating agents were
used to mimic the corresponding transformations of the N-
Scheme 1. Synthesis of the dipeptide precursor(s) 7/7*: a) 7-aza-1-hydroxybenzotriazole (HOAt), N-(3-dimethylamino)propyl-NЈ-ethylcar-
bodiimide hydrochloride (EDC·HCl), iPr2NEt, CH2Cl2; b) 4  HCl in EtOAc, CH2Cl2; c) di-tert-butyl pyrocarbonate (Boc2O), tBuOH,
aq. NaOH, 16 h, room temp.
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FULL PAPER
Scheme 2. Synthesis of (labelled) analytical standards from HCl·HVal(*)LeuNHPh: a) iPr2NEt, EtOH, room temp. 3 d; b) water/1,2-
dichloroethane, 4-Å molecular sieves, Et3N, then NaBH(OAc)3, 35 °C, 2–5 h.
Scheme 3. Synthesis of MeVal(*)LeuNHPh via oxazolidinone 16: a) camphor-10-sulfonic acid (CSA), toluene, reflux, 30 min; b) Et3SiH,
TFA, CHCl3, room temp., 3 d; c) EDC·HCl, iPr2NEt, CH2Cl2, HOBt, DMAP, room temp., overnight; d) H2, 10% Pd/C, EtOH.
the purification to some extent explain the low isolated and nitrogen were measured by physical methods, in which
yields which refer to thoroughly purified materials obtained the calibrations were made with unlabelled carbon dioxide
after column chromatography and recrystallization.
and nitrogen. For example, measuring thermal conductivi-
Another important reference is compound 18/18* with ties of carbon dioxide and nitrogen during their GC deter-
the N-methylated valine terminus. It may be used for the mination or IR intensities of the preselected CO2 and N2
evaluation of human exposure to hazardous methylating bands consistently gave values of carbon and nitrogen con-
agents such as diazomethane, iodomethane and dimethyl tents which were too low. Therefore, labelled standards were
sulfate. Direct synthesis of MeValLeuSerOH from HVal- characterized by mass and NMR spectrometry.
LeuSerOH[15] by reductive alkylation with formaldehyde
and Na(CN)BH3 has been reported to be inconvenient, be-
cause it was necessary to apply “semipreparative” HPLC
for the separation of the monomethylated product from by-
Conclusions
products and an unknown impurity which was present in
the starting material.[6] Therefore, another route was fol-
An easy and efficient synthesis of HValLeuNHPh and
lowed, along which N-Me–N-Z–ValOH (16) was first pre- labelled HVal(13C5,15N)LeuNHPh along with exemplified
pared in two steps from ZValOH via (S)-3-Z-4-isopropylox- procedures of their (reductive) alkylation with aldehydes
azolidin-5-one (15) applying
a
known protocol and their nucleophilic addition to reactive Michael ac-
(Scheme 3).[16] Coupling of N-Me–N-Z–ValOH with ceptors has made a variety of internal standards and refer-
HCl·HLeuNHPh followed by hydrogenolysis produces Me- ence substances easily available. Analytical chemists en-
ValLeuNHPh in high overall yield and purity. The same gaged in determinations of hemoglobin adducts to toxic
route was used for the synthesis of the labelled internal substances using the method of isotopic dilution may apply
standard MeValLeuNHPh.
this protocol in their own work and synthesize various new
The constitution and purity of the unlabelled final refer- reference sets depending on the nature of a hazardous elec-
ence compounds were confirmed by elemental analyses. trophile. Successful application of the compound 11 in a
Predictably, this method did not work for the labelled stan- highly sensitive analytical procedure for the determination
dards, if, after combustion, the contents of carbon dioxide of epichlorohydrin exposure has already been described.[17]
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Biomarkers for the Quantification of Hemoglobin Adducts to Toxic Electrophiles
FULL PAPER
2958, 2871, 1666, 1641, 1606, 1551, 1501, 1468, 1444, 1385, 1367,
1311, 1249, 1157, 1028. C24H33N3O2·0.25H2O (400.04): calcd. C
72.09.56, H 8.38, N 10.51; found C 72.22, H 8.39, N 10.42.
Experimental Section
General Remarks: Melting points (uncorrected) were determined in
capillaries using a Büchi 510 apparatus. Routine NMR spectra were
recorded with Varian MERCURY-300 and MERCURY-200 spec-
trometers at 300 (1H) and 75.5 MHz (13C and APT), as well as at
200 (1H) and 50.3 MHz (13C and APT), respectively. All spectra
are referenced to tetramethylsilane as an internal standard (δ =
0 ppm) using the signals of the residual protons of deuterated sol-
vents: 7.26 for CHCl3, 2.50 for [D5]DMSO and 3.30 for [D3]-
MeOH. Multiplicities of signals are described as follows: s = sing-
let, d = doublet, t = triplet, q = quadruplet, quint = quintuplet,
sept = septuplet, mc = centrosymmetrical multiplet. Coupling con-
stants (J) are given in Hz. EI-MS were recorded with MAT 95
(70 eV) and ESI-MS with LCQ spectrometers (Fa. Finnigan). IR:
Bruker IFS 66 (FT-IR) spectrophotometer, measured as KBr pel-
lets or oils between KBr plates. Analytical TLC was performed on
Macherey–Nagel ready-to-use plates AluGram Sil G/UV254; detec-
tion with UV light (254 nm), development with molybdatophosph-
oric acid solution (5% in EtOH) or 0.5% aq. KMnO4. Column
chromatography: Merck silica gel, grade 60, 230–400 mesh. Ele-
mental analyses were carried out at the Mikroanalytisches Labora-
torium des Instituts für Organische und Biomolekulare Chemie der
Georg-August-University of Göttingen. Solvents were purified ac-
cording to standard procedures. Organic solutions were dried over
MgSO4. All reactions were carried out with magnetic stirring, un-
less stated otherwise.
(2R/S)-N-(3-Chloro-2-hydroxypropyl)valylleucyl Anilide (10): To a
solution of compound 7 (342 mg, 1.00 mmol) and iPr2Net (129
mg, 1.00 mmol) in EtOH (10 mL), was added epichlorohydrin (92.5
mg, 1.00 mmol), and the reaction mixture was left to stand for 3
days. After concentration in vacuo, the residue was separated by
chromatography on silica gel (50 g), eluting with CH2Cl2/EtOH/
conc. aq. NH3 (250:5:1) to afford 10 (163 mg, 41%) as a mixture
of 2 diastereomers (1:1); m.p. 95 °C. 1H NMR (300 MHz, CD3OD,
ppm): δ = 0.9–1.0 (m, 12 H), 1.59–1.76 (m, 3 H, CH2 and CH in
Leu), 1.94 (mc, J = 6.9, 1 H, CHMe2 in Val), 2.58–2.65 (m, JAB
=
13, 1 H, CHHN), 2.72–2.79 (m, JAB = 13, 1 H, CHHN), 2.93 (t, J
= 7.5 Hz, 1 H, CHNH2 in Val), 3.58 (m, 2 H, ClCH2), 3.84 (m, 1
H, CHOH), 4.61 (m, 1 H, CHNH in Leu), 7.09 (t, J = 7.5 Hz, 1
H, 4-HAr), 7.24 (t, J = 7.5 Hz, 2 H, 3-HAr), 7.56 (d, J = 7.5 Hz, 2
H, 3-HAr). 13C NMR (75.5 MHz, [D4]MeOH, ppm): δ = 19.9, 19.0,
19.7, 21.8, 21.9, 23.5, 26.1, 32.7, 42.2 (CH2), 42.3 (CH2), 52.6
(CH2), 52.8 (CH2), 53.4 (CH), 53.5 (CH), 69.6 (CH), 70.1 (CH),
71.6 (CH), 121.5 (CH), 125.4 (CH), 129.8 (CH), 139.5 (Cipso), 141.0
(Cipso), 173.1 (CO), 176.3 (CO). MS (CI, NH3): m/z (%) = 400 (19)
and 398 (62) [M + H+], 242 (100), 209 (69). IR (KBr, cm–1): ν =
˜
3290, 2959, 1643, 1603, 1548, 1500, 1468, 1445, 1388, 1310, 1249,
1197, 1075. C20H32N3O3Cl (397.93): calcd. C 60.38, H 8.05, N
10.57; found C 60.65, H 7.90, N 10.51.
(2R/S)-N-(3-Chloro-2-hydroxypropyl)valyl(13C5,15N)leucyl Anilide
(10*): The title product was obtained similarly to the unlabelled
compound 10 and isolated in 43% yield; m.p. 95 °C. 1H NMR
(300 MHz, CD3OD, ppm): δ = 0.97 (dm, 6 H, J = 126, 13CH3 in
Val), 0.99 (d×2, J = 6.5, 6 H, Me in Leu), 1.59–1.79 (m, 3 H, CH2
and CH in Leu), 1.96 (dm, J = 125, 1 H, 13CHMe2 in Val), 2.62
(m, JAB = 13, 1 H, CHHN), 2.74 (m, JAB = 13, 1 H, CHHN), 2.91
(dm, J = 125, 13CHNH2 in Val), 3.58 (m, 2 H, ClCH2), 3.84 (m, 1
H, CHOH), 4.61 (m, 1 H, CHNH in Leu), 7.09 (t, J = 7.5 Hz, 1
H, 4-HAr), 7.24 (t, J = 7.5 Hz, 2 H, 3-HAr), 7.56 (d, J = 7.5 Hz, 2
H, 3-HAr). 13C NMR (75.5 MHz, [D4]MeOH, only chemical shifts
of the labelled atoms are given, ppm): δ = 18.9, 19.1 (d×2, J = 36),
19.8 (br. d, J = 35), 32.8 (q, J = 36), 69.9 (m), 176.6 (d, J = 50).
MS (ESI, positive mode): m/z (%) = 829 (100) [2M + Na+], 426
(27) [M + Na+]; negative mode: 448 (100) [M + 2Na – H+].
The procedures used for the preparation of the compounds 3, 4, 6,
6*, 7, 7*, 17, 18, 18* are given in the Supporting Information to
this article and published electronically (for details see also the
footnote on the first page of this article).
Benzyl (S)-4-Isopropyl-5-oxooxazolidine-3-carboxylate (15/15*)[18]
and N-benzyloxycarbonyl-N-methyl-L-valine (N-Z-N-MeValOH, 16/
16*)[19] were synthesized starting from ZValOH (Fluka) or ZVa-
l*OH[20] according to a previously reported general method[6]
(Scheme 3).
N-Benzylvalylleucyl Anilide (9): To a solution of compound 7 (342
mg, 1.00 mmol) and iPr2Net (129 mg, 1.00 mmol) in 1,2-dichloro-
ethane (DCE, 20 mL), was added molecular sieves (4 Å, 15 g) fol-
lowed by freshly distilled benzaldehyde (212 mg, 2.00 mmol). After
stirring for 30 min at room temperature, NaBH(OAc)3 (424 mg,
2.00 mmol) was added, and the mixture was stirred at room tem-
perature overnight. Then it was filtered, the molecular sieves were
washed several times with dichloromethane, the combined filtrates
(200 mL) were washed once with saturated aq. solution of
NaHCO3 (100 mL) and dried. After evaporation of the solvent in
vacuo, the residue was separated by column chromatography on
silica gel (50 g) eluting with chloroform/methanol (50:1) to afford
(2R/S)-N-(2,3-Dihydroxypropyl)valylleucyl Anilide (11): To a solu-
tion of compound 7 (365 mg, 1.07 mmol) in water (5 mL) was
added racemic dimer of glyceraldehyde (Fluka, 340 mg, 3.78 mmol)
followed by 1,2-dichloroethane (DCE, 100 mL), Et3N (0.142 mL,
1.00 mmol) and molecular sieves (4 Å, 17 g). The round-bottomed
reaction flask was rotated on a rotary evaporator (without vacuum)
for 2 h, then NaBH(OAc)3 (1.20 g, 5.66 mmol) was added in one
portion, and rotation was continued at 35 °C (bath temp.) for 2 h.
TLC of the reaction mixture indicated complete consumption of
1
9 (180 mg, 46%), m.p. 131–133 °C. H NMR (300 MHz, CD3OD,
ppm): δ = 0.93 (d, J = 6.9 Hz, 3 H), 0.96 (d, J = 6.8, 3 H), 0.99
(br. d, J = 6.8, 6 H), 1.59–1.76 (m, 3 H, CH2 and CH in Leu), 1.89
(mc, J = 6.9, 1 H, CHMe2 in Val), 2.91 (d, J = 6.2, 1 H, CHNH2 the starting material and two new poorly resolved spots with lower
in Val), 3.59 (d, J = 12.5, 1 H, PhCHH), 3.79 (d, J = 13.1, 1 H,
PhCHH), 4.63 (dd, J = 5.0 and 9.4 Hz, 1 H, CHNH in Leu), 7.09
(tm, J = 7.5 and 1.2 Hz, 1 H, 4-HAr), 7.17–7.34 (m, 7 H), 7.29 (tm,
J = 7.5, 2 H, 3-HAr), 7.54 (dm, J = 7.5, 2 H, 2-HAr). 13C NMR
Rf values (CH2Cl2/MeOH/conc. aq. NH3, 100:7:1). The reaction
mixture was filtered through a fritted glass filter, and the filter cake
was washed with several portions of DCE (until the product spots
could not be detected any more on the TLC plate). The combined
(75.5 MHz, [D4]MeOH, ppm): δ = 19.2, 19.8, 22.0, 23.4, 26.1, 32.9, organic solutions (ca. 350 mL) were washed with sat. aq. NaHCO3
42.3 (CH2), 53.3 (CH2), 53.4 (CH), 68.4 (CH), 121.4 (CH), 125.4 and then with brine (100 mL each). After drying and removal of
(CH), 128.1 (CH), 129.4 (CH), 129.5 (CH), 129.8 (CH), 139.6
(Cipso), 141.0 (Cipso), 172.9 (CO), 176.6 (CO). MS (ESI, positive
the solvent in vacuo, the oily residue was subjected to chromatog-
raphy on silica gel (50 g) eluting with CH2Cl2/MeOH/conc. aq.
mode): m/z (%) = 813 (100) [2M + Na+], 791 (27) [2M + H+], 418 NH3, 100:7:1. Compound 11 (Rf = 0.2) was isolated as an oil
(25) [M + Na+], 396 (19) [M + H+], negative mode: 440 (87) [M +
(0.22 g) which crystallized from a CHCl3/hexane mixture to give
–
HCO ], 394 (100) [M – H+]. IR (KBr, cm–1): ν = 3278, 3145, 3064, 0.18 g (44%) of a colorless solid (1:1 mixture of 2 diastereomers).
˜
2
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1
The diastereomer with the higher Rf value was isolated from the
yield 56%, m.p. 145 °C. H NMR (300 MHz, CD3OD, ppm): δ =
1
head fraction; its H NMR (300 MHz, CD3OD, ppm): δ = 0.96 d
0.9–1.1 (m, 12 H), 1.58–1.80 (m, 3 H, CH2 and CH in Leu), 1.90
(mc, J = 6.4, 1 H, CHMe2 in Val), 2.59 (t, JAX = JBX = 8, 2 H, X-
part of the ABX2-system, CH2CN/CH2NH), 2.78 (A-part of the
ABX2-system, JAB = 13, CHHNH/CHHCN), 2.83 (B-part of the
ABX2-system, JAB = 13, CHHCN/CHHNH), 2.93 (d, J = 8 Hz, 1
(J = 6.8), 0.97 d (J = 6.0), 0.98 d (J = 7.5), 0.99 d (J = 6.0) (Σ, 12
H), 1.60–1.78 (m, 3 H, CH2 and CH in Leu), 1.99 (mc, J = 6.4, 1
H, CHMe2 in Val), 2.58 (dd, JAB = 12, JAX = 6.7, 1 H, CHHN),
2.69 (dd, JAB = 12, JAX = 3.8, 1 H, CHHN), 2.89 (d, J = 6.4 Hz,
1 H, CHNH in Val), 3.54 (d, J = 6.0 Hz, 2 H, CH2OH), 3.72 (mc, H, CHNH in Val), 4.58 (dd, J = 5 and 10 Hz, 1 H, CHNH in Leu),
1 H, CHOH), 4.60 (dd, J = 4.5 and 10, 1 H, CHNH in Leu), 7.08
7.09 (t, J = 7.5 and 1, 1 H, 4-HAr), 7.28 (t, J = 7.8 Hz, 2 H, 3-
(tt, J = 7.5 and 1, 1 H, 4-HAr), 7.30 (tm, J = 7.8, 2 H, 3-HAr), 7.54 HAr), 7.55 (d, J = 7.8 Hz, 2 H, 3-HAr). 13C NMR (75.5 MHz, [D4]-
1
(dm, J = 7.8, 2 H, 3-HAr). H NMR (300 MHz, CDCl3+CD3OD, MeOH, ppm): δ = 18.9, 19.1 (CH2), 19.8, 21.8, 23.5, 26.2, 32.7,
mixture of 2 diastereomers, ppm): δ = 0.73–0.82 (Σ, 12 H, 4×Me),
42.0 (CH2), 45.2 (CH2), 53.5 (CH), 69.2 (CH), 120.4 (CN), 121.4
1.48 (m, 3 H, CH2 and CH in Leu), 1.86 (m, 1 H, CHMe2 in Val), (CH), 125.3 (CH), 129.8 (CH), 139.6 (Cipso), 172.9 (CO), 176.5
2.36 (dd, JAB = 12.5, JAX = 6.6, CHHN), 2.45 (m), 2.56 (dd, JAB
= 12.4, JAX = 3.4, Σ 2 H, CHHN), 2.69 and 2.72 (d×2, J = 5, Σ 1
H, CHNH in Val), 3.31–3.48 (m, 2 H, CH2OH), 3.55 (mc, 1 H,
CHOH), 4.20 (m, 1 H, CHNH in Leu), 6.91 (t, J = 7.5 Hz, 1 H,
(CO). MS (EI): m/z (%) = 358 (5) [M+·], 315 (8) [M+ – C3H7], 224
(9), 191 (8), 125 (100). IR (KBr, cm–1): ν = 3274, 3204, 3146, 3094,
˜
2957, 2929, 2872, 2851, 2247, 1667, 1637, 1607, 1552, 1494, 1468,
1443, 1386, 1368, 1313, 1277, 1243, 1178, 1157, 1109, 1081.
4-HAr), 7.11(t, J = 7.4 Hz, 2 H, 3-HAr), 7.33 (d, J = 7.4 Hz, 2 C20H30N4O2 (358.48): calcd. C 67.04, H 8.38, N 15.64; found C
H, 3-HAr). 13C NMR (75.5 MHz, CDCl3+CD3OD, mixture of 2 66.99, H 8.41, N 15.58.
diastereomers, ppm): δ = 17.4, 17.6, 18.9, 21.0, 22.5, 24.5, 31.0,
40.9 (CH2), 50.8 (CH2), 51.52 (CH2), 51.56 (CH), 64.1 (CH2), 64.3
N-(2-Hydroxyethyl)valylleucyl Anilide (14): This compound was
synthesized from 7 (342 mg, 1.00 mmol) and oxirane (44 mg,
(CH2), 68.2 (CH), 68.5 (CH), 70.4 (CH), 70.5 (CH), 120.0 (CH),
1.00 mmol, 237 mg of 18.6% solution in THF) in the presence of
124.19 (CH), 128.5 (CH), 137.4 (Cipso), 171.4 (CO), 174.7 (CO),
iPr2Net (1 mmol) in EtOH (10 mL) as described for the product
174.8. MS (CI, NH3): m/z (%) = 380 (100) [M + H+], 226 (10), 209
10, yield 84 mg (24%), m.p. 119 °C. 1H NMR (300 MHz, CD3OD,
(22). C20H33N3O4 (379.50): calcd. C 63.30, H 8.76, N 11.07; found
C 63.08, H 9.02, N 11.04.
ppm): δ = 0.9–1.0 (m, 12 H), 1.59–1.79 (m, 3 H, CH2 and CH in
Leu), 1.96 (mc, J = 6.4, 1 H, CHMe2 in Val), 2.63 (mc, 2 H,
(2R/S)-N-(2,3-Dihydroxypropyl)valyl(13C5,15N)leucyl Anilide (11*):
CH2NH), 2.91 (d, J = 5.6 Hz, 1 H, CHNH in Val), 3.64 (t, J =
5.6 Hz, 2 H, CH2OH), 4.60 (dd, J = 5 and 10 Hz, 1 H, CHNH in
Leu), 7.08 (tt, J = 7.4 and 1, 1 H, 4-HAr), 7.29 (t, J = 8.1 Hz, 2 H,
3-HAr), 7.54 (d, J = 8 Hz, 2 H, 3-HAr). 13C NMR (75.5 MHz, [D4]-
MeOH, ppm): δ = 18.9, 19.7, 19.8, 21.9, 23.5, 26.1, 32.7, 42.3
(CH2), 51.8 (CH2), 53.5 (CH), 62.2 (OCH2), 69.7 (CH), 121.4 (CH),
125.4 (CH), 129.8 (CH), 139.6 (Cipso), 173.2 (CO), 176.7 (CO). MS
(ESI, positive mode): m/z (%) = 771 (100) [2M + Na+], 699 (100)
[2M + H+], 350 (28) [M + H+]; negative mode: 394 (100) [M +
The title product was obtained as described for the unlabelled com-
pound 11 and isolated in 41% yield. 1H NMR (300 MHz, CD3OD,
ppm): δ = 0.96 (dm, J = 128, 6 H, 13CH3 in Val), 0.97 and 0.99
(d×2, J = 5.5, 6 H, Me in Leu), 1.62 and 1.71 (m, 3 H, CH2 and
CH in Leu), 1.96 (dm, J = 120, 1 H, 13CHMe2 in Val), 2.49–2.75
(m, 2 H), 2.92 (dm, J = 120, 13CHNH2 in Val), 3.57 (m, 2 H,
ClCH2), 3.67 (m, 1 H, CHOH), 4.60 (m, 1 H, CHNH in Leu), 7.08
(t, J = 7.5 Hz, 1 H, 4-HAr), 7.29 (t, J = 7.5 Hz, 2 H, 3-HAr), 7.53
(d, J = 8.2 Hz, 2 H, 3-HAr). 13C NMR (75.5 MHz, [D4]MeOH,
only chemical shifts of the labelled atoms are given, ppm): δ = 18.8,
19.0 (d×2, J = 35), 19.8 (d, J = 35), 32.7 (q, J = 35), 69.9 (m),
176.7 (d, J = 51). MS (ESI, positive mode): m/z (%) = 793 (100)
[2M + Na+], 771 (19) [2M + H+], 408 (8) [M + Na+], 386 (18) [M
+ H+]; negative mode: 430 (19) [M + 2Na – H+], 384 (100) [M –
H+].
HCOO–], 348(48) [M – H+]. IR (KBr, cm–1): ν = 3298, 3144, 3089,
˜
2960, 2871, 1668, 1642, 1606, 1551, 1501, 1468, 1444, 1387, 1368,
1313, 1176, 1155, 1059. C19H31N3O3 (349.47): calcd. C 65.33, H
8.88, N 12.03; found C 65.10, H 8.60, N 11.82.
Acknowledgments
The authors are grateful to Mr. R. Machinek and his co-workers
for recording NMR spectra, to Dr. H. Frauendorf, Mrs. G. Udvar-
noki and Mrs. G. Krökel for measuring of the numerous mass spec-
tra, to Mr. F. Hambloch for elemental analyses, and to Dr. B. Knie-
riem for his careful reading of the final version of this manuscript.
N-Butylvalylleucyl Anilide (12): The title compound was prepared
from 1 mmol of 7, 3 mmol of freshly distilled butanal and 3 mmol
of NaBH(OAc)3 according to the method described above for com-
pound 9, yield 86 mg (24%), m.p. 135–137 °C. 1H NMR (300 MHz,
CD3OD, ppm): δ = 0.9–1.0 (m, 15 H), 1.38 (mc, 2 H), 1.48 (mc, 2
H), 1.60–1.78 (m, 3 H, CH2 and CH in Leu), 1.82 (mc, J = 6.4, 1
H, CHMe2 in Val), 2.53 (m, 2 H, CH2N), 2.92 (d, J = 8 Hz, 1 H,
CHNH in Val), 4.62 (dd, J = 5 and 10 Hz, 1 H, CHNH in Leu),
7.09 (tm, J = 7.5 and 1, 1 H, 4-HAr), 7.28 (tm, J = 7.8, 2 H, 3-
HAr), 7.55 (dm, J = 7.8, 2 H, 3-HAr). 13C NMR (75.5 MHz, [D4]
MeOH, ppm): δ = 14.3, 19.4, 19.6, 21.5 (CH2), 22.0, 23.5, 26.1,
32.8, 33.0 (CH2), 42.2 (CH2), 52.6 (CH2), 53.4 (CH), 69.4 (CH),
121.3 (CH), 125.4 (CH), 129.8 (CH), 139.6 (Cipso), 172.8 (CO). MS
(ESI, positive mode): m/z (%) = 745 (73) [2M + Na+], 362 (100)
[1] For a review see: a) M. Törnqvist, C. Fred, J. Haglund, H.
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[M + H+]; negative mode: 360 (100) [M – H+]. IR (KBr, cm–1): ν
˜
= 3271, 3146, 3093, 2957, 2931, 2871, 1665, 1640, 1608, 1553, 1501,
1467, 1444, 1386, 1312, 1250, 1179. C21H35N3O2 (361.52): calcd. C
69.81, H 9.70, N 11.63; found C 69.87, H 9.93, N 11.54.
N-(2-Cyanoethyl)valylleucyl Anilide (13): The title compound was
synthesized from equivalent amounts of 7 and acrylonitrile in the
presence of iPr2Net (1 equiv.) in EtOH as described for product 10,
5098
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2005, 5094–5099

Biomarkers for the Quantification of Hemoglobin Adducts to Toxic Electrophiles
FULL PAPER
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[7] HVal(13C5,15N)OH with 98.5 atom.% 15N and 98.6 atom.%
13C (98.9% -form) is produced by ISOTEC Inc. (USA) and
was purchased from SIGMA-ALDRICH.
[8] The amino acid sequence of Hb at the N-terminus starts from
H-Val-Leu-Ser-Pro-Ala-Asp-Lys.
[20] Isotopically labelled ZVal*OH was prepared from HVal*OH
(ref.[7]) and ZOSu: I. M. Pastor, P. Västilä, H. Adolfsson,
Chem. Eur. J. 2003, 9, 4031–4045.
[9] E. g., from the BACHEM company (Switzerland).
[10] Throughout this manuscript, compound numbers marked with
an asterisk * denote substances with the Val(13C5,15N) frag-
ment.
Received: June 13, 2005
Published Online: October 12, 2005
[11] BocVal*OH was synthesized from HVal(13C5,15N)OH (ref.[7]
)
according to the general method described by O. Keller, W. E.
Eur. J. Org. Chem. 2005, 5094–5099
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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