Kinetic measurements
taken, diluted if necessary in pure water, and analysed by
HPLC, eluent water ϩ 0.05% TFA; tR/min = 4.5 (C-Thr), 5.0
Isocyanic acid degradation. A solution of 78 mg (0.96 mmol)
potassium cyanate in 60 mL water was stirred in a thermostated
reactor; the pH was regulated (5 to 6.5) using 0.2 M HNO3 by
means of the autotitrator. The reaction was followed by reverse
silver titration: at regular time intervals, 5 mL aliquots were
taken, cooled and added to 1 mL 0.1 M AgNO3. The AgOCN
precipitate was filtered off, washed with water, and the remain-
ing Agϩ in the filtrate was titrated with 0.01 M HCl, using silver
electrode potentiometry.
(C-ꢂ-Ala), 13.5 (α,ω-dicarbamoyllysine) at 1.0 mL minϪ1
.
Preparation of CAAs
N-Carbamoyl-L-valine C-Val. To a warm (50 ЊC) stirred solu-
tion of 175.5 g (1.5 mol) -valine in 1 L water was added 184.5 g
(2.25 mol) potassium cyanate. The pH was then regulated to 7.5
using the autotitrator to control continuous 6 M HCl addition.
Reaction completion was checked by HPLC, eluent water–
acetonitrile (95 : 5) ϩ 0.05% TFA; tR/min = 4.15 at flow rate 1
mL minϪ1. After completion (about 15 h), the reaction mixture
was cooled by means of an ice bath and acidified by adding 6 M
HCl until reaching pH 2. After filtration, the crude product was
recrystallised from boiling water and dried in a heating
desiccator, yield 84%. Mp 207–209 ЊC. δH 0.78 (3H, d, J = 7 Hz,
CH3), 0.82 (3H, d, J = 7 Hz, CЈH3), 1.93 (1H, m, Hβ), 3.95 (1H,
dd, Hα, Jαβ = 5 Hz, JαNH = 9 Hz), 5.55 (2H, s, NЈH2), 6.11 (NH,
d, J = 9 Hz), 12.45 (1H, s, COOH). δC 19.89 (Cγ), 21.05 (CγЈ),
29.90 (Cβ), 63.83 (Cα), 162.94 (NCONЈ), 179.69 (COOH).
Glycine N-carbamoylation. 20 ml of a 0.1–0.42 mM potas-
sium cyanate solution in water was stirred in a thermostated
reactor and the pH was adjusted with 4 M nitric acid. A solu-
tion of 126 mg (1.68 mmol) glycine in 10 mL water and
adjusted to the same pH was immediately added. The pH was
regulated throughout the reaction using 4 M nitric acid. At
regular time intervals, aliquots were taken and submitted to
FMOC derivatisation prior to HPLC analysis.
FMOC-derivatisation. (Adapted from Clapp et al.18): a 0.5
mL aliquot was taken, cooled and added to 0.5 mL of 0.2 M
borate buffer (pH 7.7) containing 1 mM -alanine (internal
reference). To half of this mixture 0.5 mL of a fluorenylmethyl
chloroformate solution (15 mM) in acetone was added and the
mixture was stirred for 30 s, then the solution was washed 3
times with 2 mL hexane. 0.1 mL of the aqueous layer was then
analysed by HPLC (eluent MeOH–MeCN–acetate buffer:
20 : 30 : 50. The acetate buffer was made from 3 mL acetic acid,
1 mL triethylamine in 0.9 L water, adjusted to pH 4.2 with
sodium hydroxide then made up to 1 L with water); tR/min = 8.5
N-Carbamoyl-L-alanine C-Ala. Same procedure as C-Val,
using 135 g (1.5 mol) -alanine in 1 L water and 184.5 g (2.25
mol) potassium cyanate, reaction at 50 ЊC, pH 7.5 for 15 h.
Eluent for HPLC analysis: water–acetonitrile (95 : 5) ϩ 0.05 %
TFA; tR/min = 8.4 at 0.8 mL minϪ1. The crude product was
recrystallised from boiling water to yield 240 g (82%) pure
material. Mp 165–167 ЊC. δH 1.22 (3H, d, Hβ, Jαβ = 7.24 Hz),
4.04 (1H, dq, Hα, JHα-NH = Jαβ = 7.35 Hz), 5.60 (2H, s, NЈH2),
6.23 (1H, d, NH, JNH-Hα = 7.7 Hz), 12.47 (1H, s, COOH).
δC 19.05 (Cβ), 48.79 (Cα), 159.06 (NCONЈ), 176.28 (COOH).
(FMOC-Gly), 11.1 (FMOC-Ala) at 0.9 mL minϪ1
.
3-Ureidopropionic acid C-ꢂ-Ala. Same procedure as C-Val,
using 13.35 g (150 mmol) β-alanine in 0.5 L water and 13.36 g
(165 mmol) potassium cyanate. pH regulated to 7.5, reaction
time 18 h. Eluent for HPLC analysis: water–acetonitrile
(80 : 20) ϩ 0.05% TFA; tR/min = 3.6 at 1.0 mL minϪ1. The
crude product was recrystallised from boiling water to yield
11.9 g (60%) pure material. δH 2.37 (2H, t, Hα, J = 6.0 Hz), 3.18
(2H, dt, Hβ, Jd = Jt = 5.9 Hz), 5.40 (2H, s, NЈH2), 6.00 (1H, t,
NH, Jt = 5.4 Hz), 12.20 (1H, s, COOH). δC 35.0 (Cα), 35.7 (Cβ),
159.0 (NCONЈ), 174.0 (COOH).
L-Valine N-carbamoylation. The same protocol was used as
for glycine, except that FMOC derivatisation was not used: 60
mL of a 15.9–37.3 mM potassium cyanate solution in water
was stirred in a thermostated reactor. To this a solution of 126
mg (1.08 mmol) valine in 10 mL water and adjusted to the same
pH was immediately added. At regular time intervals, aliquots
were taken and analysed by HPLC, after dilution to 1 : 10 in
HPLC eluent: water–acetonitrile (90 : 10) ϩ 0.05% TFA; tR/
min = 4.8 (Val), 10.1 (C-Val) at 0.9 mL minϪ1
.
Nꢀ-Carbamoylation of Nꢁ-trifluoroacetyl-L-lysine. Into
a
N-Carbamoyl-L-leucine C-Leu. Same procedure as C-Val,
using 30 g (0.23 mol) -leucine in 160 mL water and 30 g (0.37
mol) potassium cyanate, at 50 ЊC, pH 7.5 for 18 h. The crude
product was recrystallised from boiling water to afford 30.45 g
(76%) pure material. Mp 216–218 ЊC. δH 0.89 (6H, dd, Hδ,
J1 = J2 = 6.0), 1.45 (2H, m, Hβ), 1.65 (1H, m, Hγ), 4.08 (1Hd, td,
Hα, JHα-NH = 8.4 Hz, Jαβ = 6.3 Hz), 5.55 (2H, s, NЈH2), 6.18 (1H,
d, NH, J = 8.4 Hz), 12.44 (1H, s, COOH). δC 22.42 (CЈδ), 23.71
(Cδ), 25.16 (Cγ), 41.89 (Cβ), 51.56 (Cα), 159.19 (NCONЈ), 176.12
(COOH).
stirred solution of 2.55 g (10.5 mmol) Nε-trifluoroacetyl--
lysine in 150 mL water, warmed to 50 ЊC and adjusted to pH 8
with solid NaOH, was dissolved 946 mg (11.5 mmol) potassium
cyanate at time 0. The pH was regulated to 8 throughout the
reaction with 6 M HCl using the autotitrator. At regular time
intervals, 0.1 mL aliquots were taken, diluted in pure water and
analysed by HPLC, eluent: water–acetonitrile (90 : 10) ϩ 0.05%
TFA; tR/min = 5.4 (Lys(TFA), used as internal reference), 9.7
(C-Lys(TFA)) at 1.0 mL minϪ1
.
pKA of Nꢁ-trifluoroacetyl-L-lysine. 25 mL of a 0.02 M solution
of Nε-trifluoroacetyl--lysine in pure water and warmed to
50 ЊC was titrated with 0.1 M sodium hydroxide, the pH
being measured by the autotitrator. The pKA (8.9 at 50 ЊC)
was directly read from the titration curve (pH vs. NaOH mol.
added). This value is consistent with that found in the liter-
ature19 at 25 ЊC.
N-Carbamoyl-L-phenylalanine C-Phe. Same procedure as for
C-Val, using 11 g (67 mmol) phenylalanine and 20 g (246 mmol)
potassium cyanate in 500 mL water; at 50 ЊC, pH 7 for 15 h.
10.8 g (78%) pure material was obtained after recrystallisation
from water. Mp 200 ЊC. δH 2.8 (2H, dd, Hβ), 4.35 (1H, m, Hα),
5.10 (2H, s, NЈH2), 6.25 (1H, d, NH), 7.25 (5H, m, HAr), 11.80
(1H, s, COOH).
ꢂ-Alanine and L-threonine N-carbamoylation. Into a stirred
solution of amino acid and α,ω-dicarbamoyllysine (8 mM,
internal reference) in 150 mL water, warmed to 50 ЊC and
adjusted to pH 8 with solid sodium hydroxide (for Thr) or 6 M
hydrochloric acid (for β-Ala), was dissolved potassium cyanate
at time 0. The pH was regulated to 8 with 4 M HCl using the
autotitrator. At regular time intervals, 0.1 mL aliquots were
Nꢀ-Carbamoyl-Nꢁ-trifluoroacetyl-L-lysine C-Lys(TFA). Same
procedure as for C-Val, using 20 g (82.6 mmol) Nε-trifluoro-
acetyl--lysine and 10 g (123.5 mmol) potassium cyanate in 150
mL water at 50 ЊC; the pH was regulated to 7.5 during the
reaction. After stirring at 50 ЊC for 1 h 30 min, another 3.35 g
(41.4 mmol) potassium cyanate was added to the mixture,
which was again stirred at regulated pH for 1 h 30 min. The
J. Chem. Soc., Perkin Trans. 2, 2001, 1247–1254
1253