Download Mechanisms of Aspartimide Formation: The Effects of Protecting

yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Document related concepts

Biochemistry wikipedia, lookup

Nucleic acid analogue wikipedia, lookup

Proteolysis wikipedia, lookup

Fatty acid metabolism wikipedia, lookup

Genetic code wikipedia, lookup

Amino acid synthesis wikipedia, lookup

Fatty acid synthesis wikipedia, lookup

Metalloprotein wikipedia, lookup

Peptide synthesis wikipedia, lookup

Metabolism wikipedia, lookup

Biosynthesis wikipedia, lookup

Oligonucleotide synthesis wikipedia, lookup

Citric acid cycle wikipedia, lookup

Hepoxilin wikipedia, lookup

Butyric acid wikipedia, lookup

Specialized pro-resolving mediators wikipedia, lookup

15-Hydroxyeicosatetraenoic acid wikipedia, lookup

Ribosomally synthesized and post-translationally modified peptides wikipedia, lookup

silica gel and eluted with 250 ml of
ethylacetate to remove slow moving
impurities. After evaporation of the
combined solvent, a waxy solid (m.p.
68-69° C; similarly Boc-Glu(O-Hex)OBzl m.p. 74-76° C) was obtained.
Hydrogenation of this solid over 5%
Pd/BaS04 (1.0 g, prewashed with 50
ml of 95% EtOH) in 60 ml of 95%
EtOH for 2-4 h resulted in a solid, after
workup. Longer hydrogenation time
produced Boc-Asp-OH as a side product. Crystallization was effected in
cyclohexane-hexane (1:6, v/v) to obtain Boc-Asp(O.QHex)-OH in 85%
yield. m.p. 93-95° C, TLC (CA, Rf
0.67) Anal. (C1sH2sN06). Calcd C
57 .13, H 7 .99, N 4.44; found C 57 .22,
H 8.04, N 4.36.
Direct esterification with aspartic
acid and cyclohexanol. H2S04 (50
ml) was added to ethyl ether (500 ml:
CAUTION) and cyclohexanol (270
ml). The mixture was concentrated to a
constant volume under reduced pressure and 75 g of aspartic acid was then
added. The colloidal solution was
stirred at 50° C and became homogenous after 18 h. The reaction was
stopped after 24 h by pouring the mixture into crushed ice and 2 1 of 2 N
NaOH. The biphasic solution was
separated into the upper and lower
phases. The basic aqueous layer (containing mostly Asp) was extracted
twice with 200 ml of ether. The combined organic phase (containing the a-,
[3-, and di-esters) was washed once
with 0.1 N NaOH and then twice with
water. Upon storage in cold, the Asp
(O.QHex) crystallized. The crystalline
material contained 1 to 5% of diester.
TLC in CMA 85:10:5 gave an Rf of
0.14 (diester Rf 0.44; a-ester 0.1; Asp,
0). Final purification of Asp (O.QHex)
was achieved by ion exchange chromatography. Asp( O.QHex) (15 g). was
loaded onto a Dowex SOW-X-4 (2.5 x
30 em) column. It was eluted by 0.2 M
pH 3.1 pyridine acetate buffer. The
order of elution was Asp, Asp(O~Hex)
and AspO.QHex. A broad peak of Asp
(O~Hex) was collected between fraction 22 to 35 (5 ml fractions). After
lyophilization, 12.5 g of Asp(0£Hex)
was obtained.
Syntheses of Test Pcptides 10-13
and 24
Boc-Thr(Bzl)-OCH2-resin (30 g,
0.31 mmol/g resin, based on: Picric
acid titration; nitrogen analysis; HF
cleavage of resin; amino acid analysis
after 6 N HCI hydrolysis of resin) was
obtained from potassium fluoride esterification ( 10) of chloromethyl resin
(Lab Systems copoly-(styrene-1 %-divinylbenzene) resin, 200-400 mesh,
0.32 Cl/g substitution). Preparations of
tetrapeptides 10 (1 g), 11 (5 g), 12 (5 g)
and 24 ( 10 g) were accomplished using
Boc-Glu(OBzl)-OH, Boc-Glu(0£Hex)
-OH, Boc-Asp (OBzl)-OH, Boc-Asp
(O~hex)-OH or Boc-Asp-OBzl as Glu 1
and Asp 2 to form the tetrapeptide. The
essential protocol for one synthetic
cycle was: (1) De protection with trifluoroacetic acid/ methylene chloride
(1:1, v/v) for 1 and 20 min, (2) neutralization with diisopropylethylamine/
methylene chloride (1:19, v/v) for 2 x 5
min and (3) double coupling with 3
equivalent of preformed symmetrical
anhydride of Boc-amino acid for 1 h.
Amino acid analysis (HCI: HOAc:
phenol, 2:1:1, v/v/v; 120° C, 24 h) of
aU peptide resins after the completion
of the syntheses revealed that
Glu:Asp:Gly:Thrratios were 1:1:1:1 (±
Peptide 13 was obtained from peptide-resin 10 by hydrogenolysis (l M
concentration of Pd(0Ac)2 in dimethylformamide at 30° C for 24 h).
Phenol (0.1%) was added to the solution to prevent imide formation. The
yield was 21%, and <1% of aspartimide 15 was detected by ion-exchange chromatography. The crude
product was precipitated from ethylacetate-hexane. In the absence of
phenol, 3.9% of aspartimide 15 was
detected at 30° C, 48% at 50° C.
However, the cleavage yield at 50° C
was raised to 70%. The crude product
in all cases contained approximately
30-40% of Boc-Glu-Asp-Giy-Thr
Tritluoroacetic Acid Stability of
Cyclohexyl and Benzyl Esters
Boc-Glu(OBzl)-OH and
Boc-Glu(O.QHex)-OH (l mmol each)
were dissolved separately in 40 ml of
trifluoroacetic acid at 55° C. Boc-AlaOH (0.1 mmol) was included as the internal standard. At various time intervals, 1 ml aliquots of each solution
were withdrawn, evaporated to dryness, dissolved in pH 2.2 citrate buffer
and analyzed immediately for Glu or
Asp on a Beckman 120B AA-15
column. The rate of acidolytic Joss of
the protecting group was calculated according to the equation of lnXo/lnX1 kt, where Xo is the concentration of
either benzyl or cyclohexyl ester at the
beginning of the reaction, Xt is the concentration of the ester at time t, and k is
the rate constant.
Deprotection and cleavage of
amino acids and peptides in HF. The
deprotection of the side chain protected
amino acids, or the cleavage of the
resin-bound amino acids and peptides
to the free, unprotected amino acids
and peptides were carried out in a
fluorocarbon HF-Reaction Apparatus
(Type I, Peptide Institute, Japan). A
typical procedure was as follows: Peptide-resin (1 00 mg, 0.31 mmol/g of
peptide) was charged with 0.5 ml of
anisole (10% v/v) and then chilled by
dry ice-acetone bath to -78° C for 10
min. HF (4.5 ml, 90% v/v) was then
added and the temperature was quickly
brought up to the desired temperature
by the appropriate solvent bath (-15°,
0° or 25° C). After the appropraite time
treatment (0.5 ·- 4 h), HF was rapidly
removed under high vacuum at -10° C
to 0° C. The peptide-resin was extracted thrice with dry ether (3 ml) to
remove the remaining anisole, dried in
high vacuum, extracted with 10-25%
HOAC-H20 (v/v). The aqueous HOAc
mixture was collected and lyophilized
to obtain the peptide.
Cooling baths. Cooling baths of
-15° C could be attained using an
NaCl-ice mixture (23:77 w/w); NaCl:
H20: acetone (12:2:50, w/v/v) or carbon tetrachloride slush. The baths were
precooled by dry ice-acetone bath to 15° C and insulated with a layer of a
highly porous material such as glassfibers or styrofoam chips encased in
another beaker. To maintain the temperature, small pieces of dry ice were
added. Alternatively, a low temperature bath (Ultra Kryomat TK30,
MeOH as circulating solvent) was
used. Deprotection of protected amino
acids were as follows: A mixture of 2-5
J.lrnol each of protected amino acids:
Boc-Ser-(Bzl)-OH, Boc-Thr(Bzl)-OH,
Boc-Tyr (2,6-Ch-Bzl)-OH, Boc-Lys
(2-Cl-Z)-OH, Aoc-Arg(Tos)-OH, BocCys(4-Me-Bzl)- OH, Boc-His(Tos)OH, Boc-Asp(OBzl)-OH, Boc-Glu
(Bzl)-OH, Boc-Val-OH and Boc-AlaOH, was treated with HF:anisole (9:1,
v/v) at -15° C for 1 h, -15° C for 2 h, 0°
C for 1 hand 25° C for 1 h. Separately,
two samples of this mixture were
hydrolyzed in 6 N HCl at 120° C for 24
Vol. I, No. 1 (1988)