P. Bjorup et al./Bioorg. Med. Chem. 6 (1998) 891±901
È
899
Fmoc-2,4-dimethoxy-40-(carboxymethyloxy)-benzhydryl-
amine-resin was used. All solvents that were used were
of analytical grade (methanol and N,N-dimethylform-
amide) or were locally puri®ed by distillation (dichloro-
methane). N,N-Dimethylformamide was dried and
stored over 3 A molecular sieves. It was ¯ushed with
nitrogen immediately before it was used in order to get
rid of volatile amines. The ®rst residues of the peptide
esters Pyr-His-Trp-Ser-Tyr-OCam, Pyr-Pro-Ser-Lys-
Asp-Ala-Phe-OCam, and Pyr-Pro-Ser-Lys-Asp-Ala-Phe-
OCaPhe-NH2 were incorporated according to a solid
phase procedure implemented in our laboratory for
Cam esters.17 Brie¯y, bromoacetic acid (4 equiv.) is
linked to the resin using 1,3-diisopropylcarbodiimide as
coupling reagent for 1 h. The ®rst amino acid of the
acyl-part of the ester is then coupled to the resin via the
cesium salt method.29 In the case of CaPheNH2, the ®rst
step of the synthesis was the coupling of Fmoc-Phe-OH
to the resin and the elimination of the N-protection
using standard solid phase methods followed by the
protocol described above.
30 min, at a ¯ow rate of 1.5 mL/min, with detection at
215 nm.
The crude products were loaded onto a preparative
PrePack1 (Waters) column (47Â300 mm) ®lled with
either VYDAC2 C18, 300 A, 15±20 mm stationary phase
for the puri®cation of the LH-RH fragments or Bonda-
pack C18, 300 A, 15±20 mm stationary phase for the
puri®cation of the eledoisin fragments. The peptides
were puri®ed by CH3CN gradients in TEAP pH 2.3±2.4
(slope 0.2% CH3CN/min) and desalted by fast CH3CN
gradients in 0.1% aqueous TFA. The ¯ow rate was
100 mL/min and the products were detected at either
215 or 225 nm. Analysis of the fractions was accom-
plished under isocratic conditions in 0.1% aqueous
TFA/CH3CN using the same analytical column, ¯ow
rate, and detection as previously described. The ®nal
products were obtained by lyophilization as the tri-
¯uoroacetic salts of Pyr-His-Trp-Ser-Tyr-OCam (69 mg,
ES-MS m/z: 760.6 [(M+1)+], C36H41N9O10 requires
759.8), H-Gly-Leu-Arg-Pro-Gly-NH2 (385 mg, ES-MS
m/z: 498.5 [(M+1)+], C21H39N9O5 requires 497.6), Pyr-
Pro-Ser-Lys-Asp-Ala-Phe-OCam (109 mg, ES-MS m/z:
832.5 [(M+1)+], C37H53N9O13 requires 831.9), Pyr-Pro-
Ser-Lys-Asp-Ala-Phe-OCaPheNH2, (62 mg, ES-MS m/
z: 979.5 [(M+1)+], C46H62N10O14 requires 979.1), and
H-Ile-Gly-Leu-Met-NH2 (240 mg, ES-MS m/z: 431.7
[(M+1)+], C19H37N5O4S1 requires 431.6).
Chain extension was accomplished by stepwise incor-
poration of the Fmoc-amino acids (2.5±5 equiv.) by 1,3-
diisopropylcarbodiimide/1-hydroxybenzotriazole mediated
couplings.
When the sequences were completed, test cleavages were
carried out on 3 mg samples and monitored by HPLC.
The following mixtures were found to be appropriate
for treating the dierent resins: TFA/anisole/EDT/
DCM (75/5/1/19): Pyr-His-Trp-Ser-Tyr-OCam, TFA/
thioanisole/water/DCM (75/10/1/14): H-Gly-Leu-Arg-
Pro-Gly-NH2, TFA/anisole/EDT/DCM/water (75/5/3/
16/1): H-Ile-Gly-Leu-Met-NH2, TFA/anisole/water/DCM
(85/5/1/9): Pyr-Pro-Ser-Lys-Asp-Ala-Phe-OCam and
Pyr-Pro-Ser-Lys-Asp-Ala-Phe-OCaPhe-NH2.
Enzyme immobilization
The enzyme was deposited on a solid support. A solu-
tion of a-chymotrypsin (63.6 mg in 2 mL of 50 mM Tris±
HCl buer pH 7.8) was mixed with celite (2 g), and the
preparation was dried under vacuum.
Enzymatic reactions
Free peptides were obtained by treating the peptidyl-
resins (1.8±3.0 g) with appropriate cleavage mixtures
(28 ml) at 37 ꢀC for a period of time ranging from 1 h
45 min to 3 h 30 min. The crude peptides were pre-
cipitated by adding cold tert-butyl methyl ether (70 mL).
In the case of H-Ile-Gly-Ser-Met-NH2, the mixture had
to be placed in the freezer overnight to promote the
precipitation. After centrifugation the pellet was washed
with cold tert-butyl methyl ether (3Â70 mL). The crude
was dissolved in 0.1 aqueous TFA and the resin was ®l-
tered o. The solution was freed of ether at reduced
pressure, and it was then lyophilized overnight.
Reactions in aqueous media at low concentrations of
fragments. Eledoisin and LH-RH were synthesized from
fragments (obtained by solid-phase synthesis) in dier-
ent aqueous buers or mixtures of 50 mM Tris±HCl
buer pH 7.8 and dimethylformamide, 0±70% of DMF
(v/v). All reactions were carried out at 25 ꢀC in 1.5 mL
Eppendorf tubes that contained 50 mL of reaction
mixture: acyl-donor (Cam ester or CaPheNH2 ester)
(2.1 mM), acyl-acceptor (a peptide amide) (2.5 mM),
and a-chymotrypsin (0.2±2.0 mM). The reaction
mixtures were prepared in the following way: The pep-
tide fragments were dissolved in pure water (1 mL)
(acyl-donor: 2.3 mM and nucleophile: 2.8 mM), and ali-
quoted (45 mL fractions) in Eppendorf tubes. Then, the
water was evaporated at reduced pressure. The residue
was dissolved in 45 mL of either aqueous buer or
50 mM Tris±HCl buer pH 7.8 containing dimethylform-
amide (0±77.8% v/v). The enzymatic reaction was
Crude products were analyzed by HPLC on
a
VYDAC2 C18, 5 mm, 4.6Â250 mm column, eluted with
a binary system, (A) 0.1% aqueous tri¯uoroacetic acid,
(B) 0.08% tri¯uoroacetic acid in water:acetonitrile (2:3)
under gradient conditions from 10 to 70% B over