Derivatization of phosphopeptides with mercapto- and amino-functionalized conjugate groups by phosphate elimination and subsequent Michael addition

Article abstract of DOI:10.1039/b505573c

Kinetics of the β-elimination of the phosphate group from H-Tyr-Ser(PO₃H₂)-Phe-OH and H-Tyr-Thr(PO₃H ₂)-Phe-OH and subsequent addition of thiols and amines to the dehydroalaninyl and β-methyldehydroalaninyl resi

Full text of DOI:10.1039/b505573c

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A R T I C L E
Derivatization of phosphopeptides with mercapto- and
amino-functionalized conjugate groups by phosphate elimination
and subsequent Michael addition
Kati Mattila,* Jaana Siltainsuu, Lajos Balaspiri, Mikko Ora and Harri Lo¨nnberg
Department of Chemistry, University of Turku, FIN-20014, Turku, Finland
Received 21st April 2005, Accepted 17th June 2005
First published as an Advance Article on the web 13th July 2005
Kinetics of the b-elimination of the phosphate group from H–Tyr–Ser(PO3H2)–Phe–OH and
H–Tyr–Thr(PO3H2)–Phe–OH and subsequent addition of thiols and amines to the dehydroalaninyl and
b-methyldehydroalaninyl residues formed, were followed by RP HPLC under alkaline conditions in the absence and
presence of Ba2+ ions. By this reaction sequence, the phosphoserinyl peptide was conjugated with
mono-N-(2-mercaptoethyl)amide of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (4), a
mercapto-functionalized pentapeptide, H–His–Gly–Gly–His–Gly–NH(CH2)4SH, and an amino-functionalized
fluorescent dye, 5-dimethylaminonaphthalene-1-[N-(5-aminopentyl)]sulfonamide (dansyl cadaverine). The
b-methyldehydroalanine residue was, in turn, observed to be a poor Michael acceptor.
The present study is aimed at providing a solid chemical basis
Introduction
for the exploitation of base-catalyzed phosphate elimination in
chemical tagging of phosphopeptides by intermediary formation
of a dehydroalanyl peptide. For this purpose, the kinetics of
phosphate elimination from two tripeptides, H–Tyr–Ser(PO3-
H2)–Phe–OH (1) and H–Tyr–Thr(PO3H2)–Phe–OH (2), and
subsequent Michael addition of various mercapto and amino
nucleophiles have been studied. As an application, mono-N-
(2-mercaptoethyl)amide of 1,4,7,10-tetraazacyclododecane-1,4,
7,10-tetraacetic acid (4) has been prepared and introduced in
1 by sequential phosphate elimination and Michael addition.
Since the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTA) moiety may be expected to exhibit a reversible
pH-dependent binding to metal-ion-loaded solid supports,
conversion of phosphopeptides to DOTA-conjugates offers a
viable method for their enrichment. In addition, they can
possibly be used as element-coded tags in mass spectrometry.24
Such conjugates may additionally find applications in biomed-
ical research as carriers of radioactive or paramagnetic metal
ions in various imaging techniques25 or radiopharmaceutical
applications.26 In addition to DOTA group, the phospho-
serinyl peptide (1) has been derivatized with a mercapto-
functionalized pentapeptide, H–His–Gly–Gly–His–Gly–NH-
(CH2)4SH (3), a mimic of metal-ion chelating peptides,27 and
with a fluorescent dye, 5-dimethylaminonaphthalene-1-[N-(5-
aminopentyl)]sulfonamide (dansyl cadaverine).
Reversible phosphorylation of serine, threonine and tyrosine
residues in proteins is a post-translational modification which
plays a major role in intracellular signaling.1–3 Accordingly, en-
richment and identification of phosphoproteins from the protein
pool of the cell is one of the central issues of the development of
chemical tools for cell biology.4 Traditional 32P-radiolabeling,5
phosphospesific antibodies6 and various MS-techniques com-
bined with affinity chromatography,7 while highly useful in
numerous cases, still suffer from some limitations. In particular,
quantification of low abundance phosphoproteins would benefit
from effective enrichment to a solid support, which allows
removal of non-phosphorylated proteins by simple washing.
Recently, two tagging methods applicable to this purpose have
been demonstrated.8 One of these is based on phosphate
group elimination from phosphoserine and phosphothreonine
residues and subsequent nucleophilic addition of the affinity
tag.9–11 A similar approach has been applied to introduce
mass spectroscopy tags12 and to convert phosphoserine and
phosphothreonine to lysine analogs that are recognized by
lysine-specific proteases.13 The other approach, in turn, consists
of a multistep conversion of the phosphate group to a tagged
phosphoramidate and it may, hence, be applied to enrichment
of all phosphoproteins, including the tyrosine-containing ones.14
Both approaches appear highly promising, but await thorough
chemical analysis for evaluation of their full potential.
In fact, exploitation of sequential phosphate elimination
and Michael addition for structural modification of peptides
dates back to the mid 1980s. In 1986 Meyer et al.15 converted
phosphoserine-containing peptides to their S-ethylcysteine
counterparts before sequencing by Edmans degradation. Re-
cently, glycosyl thiolates have been introduced to dehy-
droalaninyl peptides by Michael addition.16,17 In spite of this
rather long history and several applications, quantitative data on
the course of elimination and subsequent addition in aqueous
solution is limited to the early studies of Byford, concerning
the conversion of a phosphoserine residue to dehydroalanine
in saturated barium hydroxide and subsequent addition of
methylamine.18 Even the data on addition of amines and
thiols to monomeric dehydroalanine and its simple derivatives
is surprisingly scarce and it usually refers to non-aqueous
conditions.19–22 Only recently, a more extensive study on Michael
addition of amines and thiols to a dehydroalanine amide in
various aqueous solvents has been reported.23
Results and discussion
Preparation of peptides and mono-N-(2-mercaptoethyl)amide of
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
Phosphopeptides H–Tyr–Ser(PO3H2)–Phe–OH (1) and H–Tyr–
Thr(PO3H2)–Phe–OH (2) were prepared by manual synthesis
on a Wang resin applying the Fmoc-strategy. The phos-
phorylated amino acids were introduced as monobenzyl es-
ters to avoid elimination of a fully protected phosphate
group.28 Activation by 1-H-benzotriazolium-1-[bis(dimethyl-
amino)methylene)hexafluorophosphate N-oxide (HBTU) and
1-hydroxybenzotriazole (HOBt) in the presence of N,N-diiso-
propylethylamine (DIPEA) was used for coupling (Scheme 1).
Treatment with aqueous trifluoroacetic acid (TFA) containing
dithiotreitol resulted in final deprotection and cleavage from the
resin. The crude peptides were purified by reversed-phase HPLC
and characterized by NMR and mass spectroscopy.
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 3 9 – 3 0 4 4
3 0 3 9
©

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Scheme 1 Reagents and conditions: i) 20% piperidine–DMF; ii) Fmoc–Ser[PO(OBzl)OH]–OH; iii) HBTU, HOBt, DIPEA, DMF; iv) 20%
piperidine–DMF; v) Boc–Tyr(tBu)–OH, HBTU, HOBt, DIPEA, DMF; vi) TFA, DTT, H2O.
H–His–Gly–Gly–His–Gly–NH(CH2)4SH (3), used for tag-
ging of 1, was assembled on a commercially available (4-amino-
butanethio)-4-methoxytrityl support applying the Fmoc chem-
istry and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
[4,5-b] pyridinium hexafluorophosphate 3-oxide (HATU) cou-
pling. Acidolytic cleavage from the resin, followed by HPLC
purification, gave the mercapto functionalized peptide.
Commercially available 4,7,10-tris(2-tert-butoxy-2-oxoethyl)-
1,4,7,10-tetraazacyclododecane-1-acetic acid was converted to
its N-(6-amino-3,4-dithiahexyl)amide by HBTU/N-hydroxy-
succinimide (NHS) activated amidation with cystamine in DMF
(Scheme 2). Reduction with tris(2-carboxyethyl)phosphine
(TCEP) in aqueous dioxane, followed by acidolytic removal of
the tert-butyl groups with aqueous TFA containing triisopropy-
lsilane (TIPS) then gave the desired product (4).
material was disappeared, not even traces of any other products
could be observed. In 0.01 mol dm−3 sodium hydroxide, the elim-
ination product (5a) was still the only product detected during
the first half-life of the disappearance of 1, but, on prolonged
treatment, slow hydration of 5a to diasteromeric serinyl peptides
6a ([M + H]+ = 414.4, tR = 9.0 and 9.6 min) and isomerization
to several products having the same molecular mass as 5a ([M +
H]+ = 398.4, tR = 13.2, 14.7 and 27.0 min) took place (Fig. 1).
Formation of the isomeric products may tentatively be attributed
to intramolecular cyclizations by nucleophilic attack on the b-
carbon of the dehydroalanine residue. The structures of these
Elimination of the phosphate group
According to the results of Byford,18 elimination of the phos-
phate group from phosphoserinyl peptides in aqueous alkali
(Scheme 3) is catalyzed by group II metal ions, the catalytic
efficiency increasing in the order Ca2+ < Sr2+ < Ba2+. To
quantify the rate-accelerating effect of Ba2+ ion on elimination
of the phosphate group from both phosphoserinyl and phos-
phothreoninyl residues and to examine the occurrence of side
reactions at various hydroxide and barium ion concentrations,
elimination of the phosphate group from 1 and 2 was followed
as a function of pH (10.0–12.0) and barium ion concentration
(0.005–0.050 mol dm−3) at 60 ◦C. The composition of the
aliquots withdrawn at appropriate intervals from the reaction
mixture was determined by RP HPLC. The products were
identified by a mass spectrometric analysis (HPLC–ESI-MS).
In 0.1 mol dm−3 aqueous sodium hydroxide containing
barium nitrate 0.041 mol dm−3, the conversion of phosphoserinyl
peptide 1 (tR = 5.3 min) to the respective dehydroalaninyl peptide
5a (tR = 25.1 min) was quantitative. When 80% of the starting
Fig. 1 Time dependent product distribution for the conversion of phos-
phoserinyl peptide 1 to dehydroalaninyl peptide 5a in 0.010 mol dm−3
aqueous sodium hydroxide containing 0.041 mol dm−3 Ba(NO3)2 at
60 ◦C. 6a refers to diastereomeric serinyl peptides. The two other prod-
ucts, exhibiting retention times of 13.2 and 27.0 min, are unidentified
isomers of 5a.
Scheme 2 Reagents and conditions: i) cystamine, HBTU, NHS, DMF; ii) TCEP, dioxane–H2O; iii) TFA–TIPS–H2O.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 3 9 – 3 0 4 4
3 0 4 0

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Scheme 3 Reagents and conditions: i) NaOH, DMSO, EtOH, Ba(NO3)2; ii) RCH2CH2SH.
products were not, however, unambiguously determined. In less
basic solutions, the subsequent intramolecular reactions became
so fast compared to the elimination that several products were
observed already during the early stage of the disappearance of
5a.
With phosphothreoninyl peptide (2) (tR = 11.6 min), the
elimination was almost one order of magnitude slower than
with 1. Despite this, no additional products appeared during
the first two half-lives of the disappearance of 2 at hydroxide
ion concentrations higher than 0.010 mol dm−3. Evidently the
subsequent reactions of the elimination product (5b) ([M + H]+ =
412.4; tR = 33.0 min) are retarded by the presence of the
methyl group even more markedly than the elimination. This is
expected, since the methyl group protects the b-carbon against a
nucleophilic attack. Only at [OH−] < 0.001 mol dm−3 were traces
of additional products observed.
The elimination of both 1 and 2 was observed to be first-
order in hydroxide ion concentration at 0.001 mol dm−3
<
[OH−] < 0.100 mol dm−3 (Fig. 2) and first-order in barium ion
concentration at 0.005 mol dm−3 < [Ba2+] < 0.050 mol dm−3
(Fig. 3). In other words, one proton is removed on going to the
transition state and one metal ion is involved in this process.
One may tentatively assume that the rate-limiting step involves
abstraction of the phosphoserine a-proton by a hydroxo ligand
of the phosphate bound Ba2+.
Fig. 2 The effect of pH on the first-order rate constant of the conversion
of phosphoserinyl 1 (ꢀ) and phosphothreoninyl peptide 2 (᭹) to the
corresponding dehydropeptides 5a and 5b in the presence of Ba2+ (0.040
and 0.050 mol dm−3 with 1 and 2, respectively) at 60 ◦C. The pH
was adjusted with sodium hydroxide and a triethylamine buffer (I =
0.25 mol dm−3 with NaNO3). The pKa value of water is 12.82 under the
experimental conditions.29
To quantify the rate-accelerating effect of Ba2+ ion, the
elimination was additionally studied in the absence of the
metal ion. The elimination product (tR = 25.1 min) was still
clearly accumulated in 0.1 mol dm−3 aqueous sodium hydroxide
at 90 ◦C, but its subsequent hydration to the diastereomeric
serinyl peptides (6a tR = 8.8 and 9.2 min) and isomerization to
unidentified products mentioned above (tR = 13.2 and 27.6 min)
were so fast that the product mixture became complicated at a
rather early stage of the disappearance of the phosphopeptide
(Fig. 4). In 0.01 mol dm−3 aqueous sodium hydroxide, the
elimination product 5a was not any more markedly accumulated.
Comparison of the data in Fig. 2 and Fig. 3 indicates that
Ba2+ ion at a concentration 0.050 mol dm−3 accelerates the
elimination of the phosphate group by two orders of magnitude.
With the phosphothreoninyl peptide (2) for example, the rate
constants observed at [OH−] = 0.1 mol dm−3 and 60 ◦C are
2.5 × 10−4 s−1 and 2.1 × 10−6 s−1 in the presence and absence
of Ba2+ ion (0.050 mol dm−3), respectively. The subsequent
transformation reactions of the elimination products are not
similarly accelerated and, hence, the elimination product is
quantitatively accumulated in the presence of Ba2+ ion, but not
in its absence.
As suggested previously,10,13 addition of DMSO and ethanol
to aqueous alkali accelerates the elimination of the phosphate
group. When the elimination was carried out in an 8 : 3 : 1 (v/v/v)
mixture of water, DMSO and ethanol saturated with Ba(OH)2,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 3 9 – 3 0 4 4
3 0 4 1

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of the sulfide ion. This moderate rate acceleration may, however,
result from the fact that addition of the sulfide ion increased
the ionic strength from 0.1 to 0.2 mol dm−3. No elimination
product (5a) was accumulated, but the starting material (tR
=
5.3 min) was quantitatively converted to diastereomeric S-(2-
hydroxyethyl)cysteinyl peptides ([M + H]+ = 476.2, tR = 11.6
and 14.8). In other words, the attack of the sulfide ion on the
b-carbon of dehydroalaninyl residue was sufficiently fast as to
prevent all the transformation reactions of 5a that took place in
its absence.
The addition of thiols to the dehydroalaninyl peptide 5a
was then studied at 37 ◦C. The phosphoserinyl peptide 1
was first allowed to undergo complete elimination to 5a in
an 8 : 3 : 1 (v/v/v) mixture of water, DMSO and ethanol,
containing sodium hydroxide 0.13 mol dm−3 and Ba(NO3)2
0.040 mol dm−3. After 90 min, i.e. when 1 (1.5 × 10−4 mol dm−3)
was entirely converted to 5a, b-mercaptoethanol was added to
give the final concentration of 0.1 mol dm−3. In other words, the
hydroxide ion concentration was decreased from 0.13 mol dm−3
to 0.03 mol dm−3, owing to deprotonation of b-mercaptoethanol
to 2-hydroxysulfide ion. Under these conditions, the addition
reaction proceeded smoothly giving the diastereomeric S-(2-
hydroxyethyl)cysteinyl peptides (7a) as the only products. The
pseudo first-order rate constant for the disappearance of 5a
Fig. 3 The effect of Ba2+ ion concentration on the first-order rate
constant of the conversion of phosphoserinyl peptide 1 (ꢀ) and
phosphothreoninyl peptide 2 (᭹) to their dehydroalanine analogs 5a
and 5b at 60 ◦C. The data referring to 1 and 2 was obtained at pH 10.8
and 11.8 (I = 0.25 mol dm−3 with NaNO3), respectively.
was (1.31
0.02) × 10−2 s−1. Similar experiment with 2-
aminoethanethiol gave the rate constant (1.30 0.02) × 10−2 s−1.
The mono-N-(2-mercaptoethyl)amide of 1,4,7,10-tetraaza-
cyclododecane-1,4,7,10-tetraacetic acid (4) was efficiently con-
jugated to phosphoserinyl peptide 1 in a similar manner. A
0.0077 mol dm−3 solution of 1 in an 8 : 3 : 1 mixture of
H2O–DMSO–EtOH (100 mm3) containing sodium hydroxide
(0.13 mol dm−3) and Ba(NO3)2 (40 mmol dm−3) was allowed
to undergo elimination to 5a at 37 ◦C. After completion of
the elimination, 4 dissolved in 0.5 mol dm−3 aqueous sodium
hydroxide (120 mm3) was added, giving a final concentration
of 0.055 mol dm−3. Disappearance of 5a (tR 27.0 min) was
accompanied by formation of two products (tR 22.8 and 23.8
min) exhibiting the expected molecular ion mass of the desired
conjugate 8a ([M + H]+ = 861.7). In addition, only a minor
amount (< 3%) of disulfide of 4 (tR 15.0 min) was formed.
At this 15-fold excess of 4 compared to 5a, the disappearance
of 5a obeyed first-order kinetics, the pseudo first-order rate
constant being (1.69
0.02) × 10−3 s−1. At a one order of
Fig. 4 Time dependent product distribution for the conversion of
phosphoserinyl peptide 1 to dehydroalaninyl peptide 5a in 0.10 mol dm−3
aqueous sodium hydroxide at 90 ◦C. 6a refers to diastereomeric serinyl
peptides. The two other products, exhibiting retention times of 13.2 and
27.0 min, are unidentified isomers of 5a.
magnitude lower concentration of 4 (0.0055 mol dm−3) and 5a
(7.7 × 10−4 mol dm−3), the addition was one order of magnitude
slower (k = 1.8 × 10−4 s−1). Under these conditions, oxidative
dimerization of 4 was, however, more marked; about 15% of 4
was converted to a disulfide.
the first-order rate constant for the elimination reaction of 1
◦
Mercapto-functionalized peptide, H–His–Gly–Gly–His–
Gly–NH(CH2)4SH (3), was conjugated similarly to the
dehydroalaninyl peptide 5a. Upon addition of 3 (tR 16.2 min)
in 15-fold excess compared to 5a, gradual disappearance of 5a
was accompanied by appearance of the diastereomeric peptide
conjugates 9a at tR 23.4 and 23.7 min ([M + H]+ = 949.0).
In addition, the disulfide dimer of 3 was formed (tR 19.2 min,
[M + H]+ = 1100.1).
was 2.1 × 10−2 s−1 at 60 C and 3.0 × 10−4 s−1 at 25 ◦C, i.e.
10-fold faster than in 0.1 mol dm−3 aqueous NaOH containing
0.040 mol dm−3 Ba(NO3)2. To avoid possible precipitation of
BaCO3 during the reaction, the Ba2+ ion concentration was in
subsequent studies adjusted to 0.040 mol dm−3 with Ba(NO3)2
and the hydroxide ion concentration to 0.13 mol dm−3 with
NaOH in the same solvent mixture. In this solution, the first-
order rate constant for the elimination of 1 was (1.36 0.06) ×
10−3 s−1 at 37 ◦C, a temperature used in the subsequent conjuga-
tion studies. Under these conditions, the elimination proceeded
to completion without formation of any other products.
The b-methyldehydroalanininyl peptide (5b) was much less
susceptible than 5a to addition of thiols. It was not observed
to act as a Michael acceptor for 2-mercaptoethanol under
conditions where 5a was readily attacked. 1,2-Ethanedithiol
was observed to attack 5b, but 50 times less readily than
5a. Conjugation with 4 failed entirely under conditions where
the dehydroalaninyl peptide was quantitatively converted to a
DOTA conjugate.
Addition of thiols to the dehydroalaninyl peptide 5a
Conversion of phosphoserinyl peptide 1 to diastereomeric S-(2-
hydroxyethyl)cysteinyl peptides 7a was first studied by carrying
out the elimination in an equimolar mixture of hydroxide
and 2-hydroxyethyl sulfide ions ([OH−] = [HOCH2CH2S−] =
0.1 mol dm−3) in the absence of Ba2+ ions. The 2-hydroxyethyl
sulfide ion did not markedly affect the rate of the disappearance
Addition of amines to the dehydroalaninyl peptide 5a
Addition of an amino-functionalized fluorescent dye, 5-
dimethylaminonaphthalene-1-[N-(5-aminopentyl)]sulfonamide
(dansyl cadaverine), to the dehydroalaninyl peptide 5a, obtained
◦
of 1. The first-order rate constant was 6.5 × 10−4 s−1 at 90 C,
i.e. approximately double to the value obtained in the absence
3 0 4 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 3 9 – 3 0 4 4

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as described above, was also attempted. Disappearance of 5a
was accompanied by formation two new products at 5.1 and
5.6 min longer retention times, respectively. Both products
exhibited the expected mass of [M + H]+ = 733.9 of the dansyl
conjugate. Unfortunately, the reaction was not quantitative, but
a marked side product having the same mass as 5a appeared
at a 2.5 min longer retention time than 5a. On using the
same 15-fold excess of the attacking nucleophile over 5a, as
used for preparing the DOTA conjugate, the first-order rate
DMF (1 : 1). The Fmoc group was removed by using 20% piperi-
dine in DMF (20 cm3) for 15 min. The treatment was repeated
(35 min), followed by washing with DMF, DMF–dioxane (1 : 1,
v/v) and DMF. The first coupling was achieved by suspending
the resin in DMF, then adding Fmoc–Ser[PO(OBzl)OH)–OH
(3.98 g, 8 mmol), HBTU, (3.03 g, 8 mmol), HOBt (1.23 g,
8 mmol) and DIPEA (3.4 cm3). After 3 h shaking, the resin was
filtered and washed with DMF, isopropanol and DMF. After
removal of the Fmoc group with the conventional piperidine
treatment, the second coupling with Boc-protected tyrosine
(2.70 g, 8 mmol) was carried out following the same procedure
described above. The deprotection and coupling was followed
by Kaiser’s test. The peptide was cleaved from support and
deprotected with a mixture of TFA–DTT–H2O (38 cm3: 1 mg
: 1 cm3) for 2 h at rt. The cleavage mixture was filtered and
evaporated to one third of the original volume. The crude
product was precipitated from cold diethyl ether (300 cm3),
collected by centrifugation and washed with cold ether (5 ×
20 cm3). The peptide was dissolved in water and lyophilized.
The crude peptide was purified by reversed-phase HPLC on
a Supelco LC-18 column (21.2 × 250 mm, 12 lm), using a
mixture of water and acetonitrile (18%) as an eluent (flow rate
6 cm3 min−1), containing 0.1% TFA. The combined fractions
were lyophilized to give 265 mg (27%) of pure product. The
authenticity of the product was verified by NMR and mass
spectroscopically. 31P NMR (dP) (162 MHz, D2O): 0.20. 1H
NMR (dH) (400 MHz, D2O): 7.11–7.25 (m, 5H, C6H5), 6.91
(d, 2H, J = 8.3 Hz, H3 and H5 of 4-OH–C6H4), 6.66 (d, 2H, J
= 8.3 Hz, H3 and H6 of 4-OH–C6H4), 4.45–4.52 (m, 2H, a-H
of Phe and Tyr), 4.07 (t, 1H, J = 7.3 Hz, a-H of Ser), 3.82–3.92
(m, 2H, b-H of Ser), 3.07 (dd, 1H, J = 5.4 and 8.3 Hz, b-H of
Phe or Tyr), 2.90 (dd, 1H, J = 8.3 and 8.0 Hz, b-H of Phe or
Tyr), 2.85 (d, 2H, J = 7.2 Hz, b-H of Phe or Tyr). 13C NMR
(dC) (100.5 MHz, DMSO-d6): 174.3, 169.5, 169.2, 155.1, 136.3,
130.8, 129.3, 128.8, 127.2, 125.3, 115.9, 64.2, 54.3, 54.1, 53.7,
36.4, 35.9. ESI−-MS: m/z 494.4 [M–H]−.
constant for disappearance of 5a was (3.4
0.2) 10−5 s−1
at 37 ◦C. In other words, the reaction was 50 times slower
than with the mercapto-functionalized DOTA, which explains
the appearance of additional reaction products formed by
intramolecular reactions of 5a.
Conclusions
In summary, both phosphoserinyl and phosphothreoninyl pep-
tides quantitatively undergo elimination to the corresponding
dehydroalaninyl and b-methyldehydroalaninyl peptides under
alkaline conditions in the presence of Ba2+. In the absence of
Ba2+, dehydroalaninyl residue undergoes subsequent hydration
and isomerization reactions so rapidly that side products may
be expected to be formed if the enone structure is not efficiently
trapped by an external nucleophile. The dehydroalaninyl residue
serves as a good Michael acceptor, which allows efficient attach-
ment of even relatively large conjugate groups, such as mercapto-
functionalized DOTA or pentapeptide, to the peptide backbone.
By contrast, b-methyldehydroalaninyl residue is not attacked
by thiols sufficiently to readily allow quantitative introduction
of conjugate groups. Attack of amino-functionalized conjugate
groups on a phosphoserinyl residue is 50 times slower than the
attack of a thiol of comparable size. Accordingly, side products
accumulate concurrent with formation of the peptide conjugate.
Experimental
The Fmoc-protected amino acids, HOBt and HBTU were
commercial products of Nova BioChem and 4,7,10-tris(2-tert-
buto‘xy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-acetic
acid a product of Macrocyclics. Wang resin purchased from
Nova BioChem was used for the solid-phase peptide synthesis.
The NMR spectra were recorded on a Bruker AM 200 or JEOL
400 spectrometer. The 1H NMR chemical shifts (400 MHz,
300 K) were referred to internal TMS and 31P NMR shifts
(162 MHz, 300 K) to external orthophosphoric acid. The mass
spectra were acquired using a Perkin Elmer Sciex API 365 triple
quadrupole LC/MS/MS spectrometer.
The progress of the phosphate elimination was followed by
RP HPLC (UV detection at 215 nm). The reaction solutions in
stoppered tubes were immersed in a thermostated water bath.
Ionic strength was adjusted to 0.1 M with sodium chloride
or sodium nitrate. Aliquots were withdrawn at appropriate
intervals, neutralized with acetic acid and cooled in an ice bath.
The initial peptide concentration was ca. 0.15 mmol dm−3. In the
absence of metal ions, the separations were carried out on a ODS
Hypersil column (4 × 250 mm, 5 lm), using water containing
18% (v/v) acetonitrile and 0.1% (v/v) TFA as an eluent. In
the presence of metal ions, the samples were analyzed on a
Hypersil HyPURITY C 18 column (4.6 × 150 mm, 5 lm). A
10 min isocratic eluation with 0.1% aqueous TFA was followed
by a linear gradient (30 min) up to 35% (v/v) of acetonitrile
(flow rate 1 cm3 min−1). The first-order rate constants were
calculated by applying the integrated first-order rate equation
to the diminution of the peak area of the starting material.
H–Tyr–Thr(PO3H2)–Phe–OH (2)
The phosphothreoninyl peptide 2 was prepared as described for
1. Yield (37%). 31P NMR (dP) (162 MHz, DMSO): −0.96. H
1
NMR (dH) (400 MHz, DMSO-d6): 9.32 (br s, 2H, NH2), 8.72 (d,
1H, J = 11.6 Hz, NH of Ser or Phe), 8.44 (d, 1H J = 12.6 Hz,
NH of Ser or Phe), 7.17–7.24 (m, 5H, C6H5), 7.01 (d, 2H, J =
8.3 Hz, H3 & H5 of 4-OH–C6H4), 6.66 (d, 2H, J = 8.3 Hz, H2
and H6 of 4-OH–C6H4), 4.43–4.49 (m, 2H, a-H of Phe and Tyr),
4.33–4.49 (m, 1H, b-H of Thr), 4.00 (t, 1H, J = 11.6 Hz, a-H of
Thr), 3.40 (br s, HDO), 3.05 (dd, 1H, J = 14.1 and 5.7 Hz, b-H
of Phe or Tyr), 2.89–2.97 (m, 2H, b-H of Phe and Tyr), 2.70 (dd,
1H, J = 14.5 and 8.1 Hz), 1.19 (d, 3H, J = 6.2 Hz, CH3). 13C
NMR (dC) (100.5 MHz, DMSO-d6): 172.8, 168.8, 168.4, 156.9,
137.7, 130.9, 129.6, 128.7, 126.9, 125.4, 115.8, 72.9, 58.3, 54.1,
54.0, 37.2, 36.2, 18.9. ESI−-MS: m/z 508.5 [M–H]−.
H–His–Gly–Gly–His–Gly–NH(CH2)4SH (3)
Peptide 3 was assembled on a commercially available (4-
aminobutanethio)-4-methoxytrityl support applying the Fmoc
chemistry and HATU coupling. Acidolytic cleavage from the
resin, followed by HPLC purification, gave the mercapto-
functionalized peptide. The purification was carried out on a
LiChrospher RP-18 column (10 × 250 mm,10 lm), using a linear
gradient from 0.1% aqueous TFA to MeCN in 30 min. The flow
rate was 3.5 cm3 min−1. ESI–MS: m/z 551.5 [M–H]−.
Mono-N-(2-mercaptoethyl)amide of 1,4,7,10-tetraazacyclodode-
cane-1,4,7,10-tetraacetic acid (4)
H–Tyr–Ser(PO3H2)–Phe–OH (1)
The synthesis was carried out starting from Fmoc-protected
phenylalanine linked to Wang resin (2.0 g, 1 mmol g−1). Free
amine groups on the support were capped by acetic anhydride in
4,7,10-Tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclodo-
decane-1-acetic acid (0.5 g, 0.87 mmol) and cystamine were
dissolved in 7 cm3 of DMF, and N-hydroxysuccinimide (NHS)
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 3 9 – 3 0 4 4
3 0 4 3

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(0.3 g, 2.6 mmol) and HBTU (0.55 g, 1.5 mmol) were added.
After the solution was stirred for 24 h at rt, the crude product
was isolated by a conventional aqueous workup. The product
was dissolved in a mixture of dioxane and H2O (10 cm3; 1 : 1,
v/v) and TCEP hydrochloride (0.49 g, 1.7 mmol) was added to
reduce the disulfide linkage. The pH of the reaction solution was
adjusted to 7 with 1.0 mol dm−3 sodium hydroxide. The reaction
mixture was stirred overnight and the solution was concentrated
in vacuo to a syrup. The product was dissolved in water and
extracted with 3 × 10 cm3 of CH2Cl2. The organic layer
was washed with brine and evaporated to dryness. Tert-butyl
protecting groups were removed with TFA–H2O–TIPS (95 :
2.5 : 2.5, v/v/v). After the solution was stirred for 20 h, the
product was isolated by precipitation from diethyl ether to yield
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3 (0.2 g, 10%). H NMR (dH) (400 MHz, DMSO): 3.10 (8H,
br s), 3.24 (4H, m), 3.32 (4H, br s), 3.59 (4H, br s), 3.86 (4H,
br s), 4.02 (4H, br s), 8.59 (1H, s). 13C NMR (dH) (50.3 MHz,
DMSO-d6): 171.6, 169.0, 158.3, 54.8, 53.8, 52.7, 50.6, 50.4,
48.6, 48.3, 42.2, 23.3. ESI+-MS: m/z 464.7 [M + H]+.
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H–Tyr–dhAla–Phe–OH (5a)
1H NMR (dH) (400 mHz, 0.1 mol dm−3 NaOD): 7.17–7.28 (m,
5H, C6H5), 6.87 (d, 2H, J = 8.4 Hz, H3 and H5 of 4-OH–C6H4),
5.53 (s, 1H, b-H of dhAla), 5.36 (s, 1H, b-H of dhAla), 4.43–4.46
(m, 2H, a-H of Phe), 3.49 (dd, 1H, J = 6.8 and 6.8 Hz, a-H of
Tyr), 3.13 (dd, 1H, J = 14.0 and 4.8 Hz, b-H of Phe), 2.93 (dd,
1H, J = 14,0 and 8.4 Hz, b-H of Phe), 2.68 (dd, 2H, J = 6.8 and
2.8 Hz, b-H of Tyr).
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3 0 4 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 3 9 – 3 0 4 4