Embed Size (px)
Citation preview
Chapter 5
Comparative Study of PS-TRPGGDA Supports with Merrifield, Pam and
Sheppard Resins
- -~ Comparative Study of PS-TRPGGDA . . ..97
5.1 Introduction
Solid phase peptide synthesis has been the subject of much
investigation ever since Merrifield introduced the technique in 1963. The
major problem that has been extensively investigated is the occurrence of
aggregation of the resin bo-md peptide within the resin matrix that could led to
very poor deprotection and coupling reaction rate which in turn results the
very low yield of the targc:t peptide.'-' Some peptide sequences after certain
stages of their synthesis, is found to be highly hindered due to internal
aggregation are known as the so called difficult sequences. The syntheses of
these peptide sequences or, a newly designed support are specifically used to
demonstrate the capability of new resins in SPPS. Reduced solvent
penetration within the polymeric support and incomplete solvation of the
peptidyl resin in the Merrifield type of supports may be the source of
difficulty of these sequences. Higher resin bound functional group substitution
and excess cross-linking can aggravate this problem.4 In order to evaluate the
efficiency of the new support PS-TRPGGDA and to test the effect of new
cross-linker on the polym(x, the so-called difficult peptide sequences were
synthesized and compared with commercially available resins under identical
reaction conditions. This can provide enough information about the ability of the
new resin to assist solvation and breaking of the peptide aggregates by direct
amphipathic interaction between polymer and peptide chaim5 The peptide
sequences that selected for the present study are Menifield's model peptide Leu-
Ala-Gly-Val, acyl canier protein fragment (ACP, 65-74), retro ACP (74-65) and
Ala-Arg-(Ala)6-Lys. 'The commercially available supports like Merrifield,
Sheppard and Pam resin were selected for the performance comparison with
PS-TRPGGDA using various chemistries. The efficiency of the new support
was further established tmy comparative synthesis of biologically active
peptide sequences syntide-:! and dermaseptin. The results clearly demonstrate
the positive influence of the hydrophilic cross-linker TRPGGDA on
polystyrene support that provides an optimum hydrophobiclhydrophilic
Comparative Study of PS-TRPGGDA .. ..98
balance to the polytneric support. Since the functional group on the cross-
linker is used as the growth point of the peptide the hydrophobic effect of the
polystyrene backbone is minimized in swollen state. The analysis of these
peptide sequences showed that the synthetic capability of the new support is
superior to some of the contmercially available resins.
5.2 Results and Disc:ussion
5.2.a Synthesis of peptides using Boc-amino acids
The synthetic protot:ol for the peptide chain elongation by the step
wise coupling of Boc-amirlo acids is shown in Scheme-1. The C-terminal
Boc-amino acid was attached to the PS-TRPGGDA support by Boc-amino
acid active ester method ising MSNT in presence of MeIm. The near
quantitative C-terminal attaclunent was found to take place within 25 min. of the
reaction. Deprotection of tenlporaly Na-Boc protecting group was camed out
using 30% TFA in DCM and the deprotected resin was neutralized with 5%
DIEA in DCM.
. . -. . .. Comparative Study of PS-TRPGGDA . . ..YY --
30 % TFA in DCM 5% DlEA in DCM
BO C--AA2I DCCI HOBT
iP 0 - 0 - C - - A A ~ - A A ~ - N H - B o c
I . De xotection 2. Ncltralisation
\. 3 . Coupling with respectiveAA
Scheme 1: Peptide synthetic protocol using Boc-chemistry
The acylation reactlon was carried out using DCC and HOBt using
NMP as a solvent. After : he conipletion of the synthesis, the peptide was
cleaved from the support uslng TFA and mixture of scavengers.
1. Comparative synthesis of retro acyl carrier (74-65) protein fragment
The synthesis of retro sequence (74-65) of acyl carrier protein is used
to demonstrate the efficiency of the new resin over cornlnercially available
Pam resin by following Boc-chemistry under identical synthetic conditions.
The C-terminal Boc-Val ur,ls attached to these resins via an ester bond using
MSNT in presence of MeIln under nitrogcn atnlosphere. The percentage of
incorporation of amino a c ~ d s to these resins was estinlated by picric acid
Comparative Study of PS-TRPGGDA . . . 1 0 0
titration method. Boc-deprotection was carried out with 30% TFA in DCM.
After neutralization of the resill with 5% DlEA respective amino acids were
attached by preformed HOBt active esters. After the synthesis, the peptide
was removed from the P:i-TRPGGDA resin using TFA in presence of
scavengers and standard trifluoromethanesulfonic acid procedure was
employed for the cleavage cf the peptide from the Pam resin. The yield of the
crude peptide obtained from PS-TRPGGDA and Pam resin is 21.58 mg and
22.5 mg respectively. HPLC analysis of peptides showed only a single major
peak in the case of PS-TRPGGDA, indicating the homogeneity of the peptide
synthesized (Fig. 1). Pam r-sin showed one addition peak in addition to the
major peak corresponding lo a deletion sequences. The comparative study
indicates that PS-TWGGDA can be used successively in the synthesis of
extremely difficult peptide sequences like retro ACP with high yield and
homogeneity. In PS-TRPGGDA support, peptides were synthesized on the
functional sites on the cross.-linker, which are proximal to the polymer matrix,
whereas in the Pam resin thf: growth site is from a spacer molecule attached to
the PS-DVB resin. Even then the enhanced reactivity and purity of peptide
synthesized on PS-TF.PGGII)A support compared to Pam resin validates the
hypothesis that the cross-linter provides a hydrophilic cavity for the synthesis
of peptide and thus optimizes the hydrophilic/hydrophobic balance of the
support, which is the prime :-equirement of an ideal polymeric support. Amino
acid analysis of the peptide from PS-TRPGGDA resin: Val, 1.1 1 (1); Glu,
0.93 (1); Ala, 2.18 (2); lle, 1.98 (2); Asp, 2.13 (2); Tyr, 0.81 (1); Gly 1.00 (1).
High value of Asp and Glu is due to the l~ydrolysis Asn and Gln to the
corresponding acids. .MALL)I-TOF-MS: m/z 1064.33 (M+H)+, C ~ ~ H ~ ~ O I ~ N I ?
requires M+ 1063.14 (Fig. 11:).
~- .- Comparative Study of PS-TRPGGDA . . .. I0 I
Figure I: HPLC tirne-course analysis of retro ACP fragment synthesised on (a) Pam iresin (b) PS-TRPGDDA support using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 80% acetonitrile in water, Gr:adient used; 0% B in 5 min and 100% B in 67 min
Figure l(c): MALDl TOF MS of retro ACP
~
Comparative Study of PS-TRPGGDA . . . . i 02
5.2.b Synthesis of peptities using Fmoc-chemistry
Solid phase :syntheses of peptides were carried out on PS-TRPGGDA
resin using Fnioc-chemistry. The general protocol for the synthesis of peptides
is shown in Scheme 2. TI-,is method pennits the use of peptide resin linkages
that can be cleaved using trifluoroacetic acid in a relatively short time period
with high yield and purity.
Anchoring of'the C-terminal amino acid on the support was carried out
as an active ester of MSN'T in presence of MeIm. The reaction was found to
proceed quantitatively within 25 niin. Fmoc-deprotection was carried out
using 20% piperidine in DIMF. The acylation reaction was carried out in DMF
by using a 3.5 mmol excess each of Fmoc-amino acid, HBTU, HOBt and
DIEA. Peptide chain elongation was carried out by repeating the deprotection
and acylation cycle. After (completion of the chain elongation cycle, protected
peptidyl resin was treated with piperidine to remove the N-terminal Fmoc-
group. The target peptide was then cleaved from the support by suspending
the resin in a mixture of TFA and scavengers. The reaction mixture was
filtered and filtrate concextrated under reduced pressure. The peptide was
precipitated by adding ice-cold ether and the precipitate was washed with
ether until the scavengers are removed and dried in vacuum.
. ~ ~- Comparative Study of PS-TRPGGDA . . . ,103 .
Hooc 8 -NH-FIIIO% MSNT, MeIm
I ) Couolinr with Respective . - Fmoc-anuno a c ~ d s
b
2) N-term~nal Fmoc Cleavage
1 Scavengers
PS-TRPGGDA 0 Side chain protection
Scheme 2: Genxal protocol for SPPS using Fmoc-amino acids
Comparative Study of PS-TRPGGDA . . .. 104 . -- -- --
1. Comparat ive synthesis of Leu-Ala-Gly-Val
Merrifield's model tetr-apeptide Leu-Ala-Gly-Val was chosen initially
fot- a comparative synthetic study to evaluate the efficiency of PS-TRPGGDA
!resin as a new solid suppo~ t for polypeptidc synthesis. This peptide was
synthesized sin~ultaneousl!~ on the new resin and the commercially available
Merrifield and Sheppard resins. All of these supports contain 4-(hydroxymethyl)
pl~enoxyacetic acid linker to enhance the final cleavage rate of the target
peptide from the suppot . The C-term~nal Val was Incorporated to the
respective support via all ester bond. The percentage of incorporation of
amino acids was estimated by measuring the optical density of the solution
formed when the pre-weighed resin was suspended in 20% piperidine in
DMF. The remaining amlno acids were incorporated to the support by using
the coupling reagent H13TU in presence of HOBt and DIEA. After the
synthesis, the peptlde was cleaved from the support using TFA in 4h. The
PS-TRPGGDA, PS-DVB and Sheppard resins yielded 8.4, 7.5 and 8.6 mg of
the crude peptide respe~tively. The peptides obtain from PS-TRPGGDA
resins and shepparcl resinis showed only a single peak in HPLC analysis where
as that obtained from Merrifield resin showed one small peak in addition to
the major peak (Fig. 2). Amino acid analysis o f the peptide from
PS-TRPGGDA resin: Leu, 0.97 (1); Ala, 1.01 (I); Gly, 1.01 (I); Val, 1.03 (1).
-. Comparati\~e ~~ Study of PS-TRPGGDA . . .. 105
(a)
Figure2: HPLC time-course analysis of Leu-Ala-Gly-Val synthesised on (a) merrifield resin (b) Sheppard resin and ( c ) PS-TRPGDDA support using the buffer (A) 0.5 mL TFA in 100 inL water; (B) 0.5 mL TFA in 80% acetonitrile in water, Gradient used; 0% B in 5 ~ n i n and 10046 B in 50 min
2. Comparative synthesis of acyl carrier protein (65-74) fragment
The synthesi:; of C-terminal region of acyl carrier protein (65-74) fragment
is well known for Illany :)f the sequence-dependent problems, which can be
encountered in the coursl: of the solid phase synthesis. Due to slow Fmoc-
deprotection and poor coupling reactions in vat-ious stages of its synthesis this
test peptide sequence is now become a standard to evaluate the efficiency of a
solid support and barious chemistry used dul-ing the synthesis. Its synthesis
regardless of the chemistry elnployed I-equired a number of sterilely hindered
amino acid couplings. Additionally there is a possibility of racemisation that
occur during the coupling of the isoleucine residue.
I11 order to establish the new polyliier as an alternative to most of the
co~iin~ercially avail:ible supports, a comparative synthesis of ACP fragment
was carried out sinlultaneously with PS-DVB and Sheppard Iresin (h'ovasyn K
KA 125) undel- identical iynthetic condition. The C-ter~ninal Fmoc-Gly was
incorporated to thc respective resins via an estel- bond. Tlic percentage of
Cornparahve Study of PS TRPGGDA 106 -- - -- pp --
incorporation of amino ; ~ ~ i d s was estimated from the optical density of the
solution formed when a ore-weighed resin was suspended in 20% piperidine
in DMF. A11 the atnide 301id formation is carried out by single amino acid
coupling in neat DMF it1 presence of 3.5 equivalents of HBTUlHOBt and
DIEA. The total amount of the coupling reagent required for the incorporation
of an Fmoc-amino acid into the three supports were weighed together,
dissolved in m i n i ~ n ~ m alr~ount of DMF and 113 of this solution was introduced
to each of the reacton vessel to make sure the same synthetic conditions were
met in all cases. 4fter the synthesis, the peptide was removed from the
individual supports under same cleavage conditions using TFA in presence of
scavengers. The HMPA derivatised PS-TRPGGDA, PS-DVB and Sheppard
resins yielded 25.2, 22 and 25 mg of crude peptide respectively. From the
HPLC profile, the peptide: obtained from PS-TRPGGDA showed sharp single
peak, which indicates the homogeneity of the peptide, synthesized (Fig. 3).
Figure3: liPLC tlrne-cc'urse analysis of ACP fragment synthesised on (a) Me~nfield rf:sin @) Sheppard resin and (c) PS-TRPGDDA support using the buffer (A) 0.5 IIIL TFA in 100 mL watel-; (B) 0.5 IIIL TFA in 80% aceto~litrilc in water, Gradient used; 0% B in 5 min and 100% B in 50 niin
-- Comparative Study of PS-TRPGGDA . . . ,107
The PS-DVB resir and Sheppard resin showed more than one major
peak corresponding to the aeletion sequences. The comparative study reveals that
PS-TRPGGDA resins can be used successfully for the synthesis of difficult
sequences. Amino acid analysis of the peptide from PS-TRPGGDA resin: Val,
1.01 (1); Glu, 0.91 (1); Ala, 2.16 (2); Ile, 2.08 (2); Asp, 1.96 (2); Tyr, 0.80 (1);
Gly 1.00 (1). High value of Asp and Glu is due to the hydrolysis Asn and Gln
to the corresponding am~ids. MALDI-TOF-MS: rnlz 1064.29 (M+H)+;
C47H74016NIZ requires M' 1063.14 (Fig. 3d).
IFigurr: 3(d): MALDI TOF MS of ACP
3. Comparative syntllesis of Ala-Arg-(Ala) 6-Lys peptide
The efficien'cy of the new support was further established by
comparing the synthetic yield and homogeneity of a well-known difficult
sequence Ala-Arg- (,41a) 6-Lys with commercial resins. This peptide sequence
bears an Arg residue in position 2 and Lys residue in position 9 within a
polyalanine framework, which are the minimal structural features required to
bind the MHC molecule E I L A - ~ 2 7 . ~ Therefore, in addition to its biological
implication, this particular sequence can serve as a suitable model to compare
the relative merits of'the nm-w support with commercially available Merrifield
and Sheppard resins.
-. Comparative Study of PS-TRPGGDA . . . ,108
This sequence is reported to have many of the sequence dependent
problems like ACP and retro ACP fragments. The synthesis of this sequence
encountered with irregular or a series of unacceptable Nu-acylation or
Nu-deprotection yields. The difficulty is arises from internal development of
secondary structure:$ which were competing with the desired amide bond
formation and from the ,ntermolecular assoc~ation of resin bound peptide
chams with extended P-sheet type structure during the sequent~al a ~ s e m b l ~ . ' ~ ~
The formation of this structure can result in a significant proportion of
Nu-amine group becoming inaccessible to acylation and deprotection. Resin
property is believeti to play an important role in determining the rate of
Na-deprotection and ~ ~ - a c ~ l a t i o n . ~
The test peptide was synthesized simultaneously on HMPA handle
incorporated PS-TRPGGDA, Memfield and Sheppard resins. The C-terminal
Fmoc-Lys was attached to the resin via an ester bond using MSNT in presence
of MeIm under nitrogen atmosphere. The quantitative incorporation is
achieved by a single step. The respective Fmoc amino acids were coupled by
using HBTU in presence of HOBt and DIEA. A total amount of reagent
required for the coupling of an individual Fmoc-amino acid in all the three
support were weighed together, dissolved in minimum amount of DMF and
1/3'* of this solution added to the respective resins to make sure that the
synthetic conditions used in all the resins were identical. After the synthesis
the peptide was removed from the corresponding resins under same cleavage
conditions using TlFA acd in presence of scavengers. The yield of crude
peptide obtained from the various resin by a four hour cleavage are 30 mg
from PS-TRPGGDA, 26.8 mg from PS-DVB and 29 mg from Sheppard
resins. From the HPLC profile, the peptide obtained from PS-TRPGGDA
showed sharp single peak, which reveal the high homogeneity of the peptide
(Fig. 4).
Comparative Study of PS-TRPGGDA . . .. 109
Figure 4: HPLC time-course analysis of Ala-Arg-(Ala)6-Lys synthesised on (a) Menifield resin (b) Sheppard resin and (c) PS-TRPGDDA support using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA. in 80% acetonitrile in water, Gradient used; 0% B in 5 min and 100% B in 67 min
Figure 4(d): MALDI TOF MS of Ala-Arg-(Ala)6-Lys
Whereas PS-DVE; resin and Sheppard resin showed some minor peaks
in addition to the major peak corresponding to the deletion sequences. The
comparative study indicates that PS-TRPGGDA resins can be used as an
efficient solid support for polypeptide synthesis than PS-DVB resin and
Sheppard resin. Amino a(:id analysis of the peptide from PS-TRPGGDA resin:
Comparative Study of PS-TRPGGDA . . . . I 10
Ala, 7.06 (7); Arg, 1.02 {:I); Lys, 1.03 (1). MALDI-TOF-MS: d z 801.10
(M+H)+; C33H610f01\113 requires M+ 799.86 (Fig. 4d).
4. Comparative syntl~esis of 15 residue syntide 2 peptide
A comparative synthetic study of Syntide 2 peptide was carried out on
a 4% cross-linked 4-(hydroxymethyl)phenoxyacetamido PS-TRPGGDA and
4-(hydroxy methy1)pheno:ryacteamido PS-DVB support. The C-terminal
Fmoc-Lys was attacbed to the resin via an ester bond and the deprotection and
coupling reactions were carried out just like the other comparative studies.
After the synthesis the peptide was, remove from the corresponding resins
under same cleavage conditions using TFA in presence of scavengers. The
synthetic yield of the crude peptide after 4h cleavage reaction from
PS-TRPGGDA and I'S-DVB are 68 and 60 mg respectively. From the HPLC
profile, the peptide obtained from PS-TRPGGDA showed sharp single peak,
which reveal the high homogeneity of the peptide (Fig. 5). Whereas PS-DVB
resin showed some minor peaks in addition to the major peak corresponding to
the deletion sequences. Tha comparative study indicates that PS-TRPGGDA
resins can be used as a more efficient solid support for polypeptide synthesis
than PS-DVB resin. Aminc! acid analysis of the peptide from PS-TRPGGDA
resin: Pro, 1.89 (2); Leu, 3.01 (3); Ala, 2.04 (2); Arg, 0.99 (1); Thr, 0.73 (1);
Ser, 0.68 (1); Val, 1.02 (1) Gly, 1.98 (2); Lys, 1.97 (2). Ser and Thr showed
low value due to its p,artial degradation during the hydrolysis
Comparative Study of PS-TRPGGDA . . .. 1 1 1
Figure 5: HPLC time-course analysis of Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val- Ala-Gly-Leu-Pro-Gly-Lys-Lys synthesised on (a) Merrifield resin (b) PS-TRLPGDDA support using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 80% acetonitrile in water, Gradient used; 0% B in 5 min and 100% B in 67 min
5. Comparative synthesis of 21 residue peptide amide of DermaseptinrA1a-L.eu-Trp-Lys-Asn-Met-Leu-Lys-GIy-Ile-Lys-Lys- Leu-Ala-Gly-Lys-A.la-Ala-Leu-Gly-Ala-NH2
The better effi.cienc!i of PS-TRPGGDA resin over the Merrifield resin
was hrther established by tne comparative synthesis of a medium size peptide
amide from dermaseptin peptides. The synthesis comparison is carried out on
Rink amide handle incorporated PS-TRPGGDA resin and PS-DVB resin
under identical synthetic condition. The C-terminal Fmoc-Ala was attached by
MSNT coupling method. The percentage of incorporation of the C-terminal
amino acid was estimared spectrophotometrically by measuring the
concentration of piperidine-dibenzofulvene adducts. The remaining amino
acids were coupled by HBTU chemistry as mentioned in the other
comparative studies. After the synthesis the peptide was removed from the
corresponding resins under same cleavage conditions using TFA in presence of
scavengers. The yield of th: crude peptide amide obtained after 4 h cleavage
is 130 and 121 mg from PS-TRPGGDA and PS-DVB. The HPLC profile of
Comparative Study of PS-TRPGGDA . . .. 1 12
the crude peptide obtained from PS-TRPGGDA showed sharp single peak,
which indicates the homogc:neity of the peptide, synthesized (Fig. 6). Whereas
PS-DVB resin showed additional peaks along with the major peak
corresponding to the deletion sequences. The comparative study confirms that
PS-TRPGGDA resin;j can be used successfully in the solid phase synthesis of
polypeptides. Amino acid analysis of the peptide from PS-TRPGGDA resin:
Ala, 5.16 (5); Leu, 4.02 (4); Asp, 0.95 (1); Met, 0.87 (1); Ile, 1.01 (1); Gly,
2.98 (3); Lys, 5.02 (5). Trp was destroyed during the hydrolysis.
Figure 6: HPLC time-course analysis of Ala-Leu-Trp-Lys-Asn-Met-Leu-Lys Gly-Ile-Lys-Ly:;-Leu-Ala-Gly-Lys-Ala-Ala-Leu-Gly-Ala-NH2 synthesised on (a) merrifield resin (b) PS-TRF'GDDA support using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 80% ac:etonitrile in water, Gradient used; 0% B in 5 min and 100% B in 157 min
5.3 Experimental
5.3.a Materials
4-(hydroxymethy1)phenoxyacetic acid (HMPA), p-[(R,S)-u(1-(9
H-fluorene-9-yl) melhoxy :bramido}-2, 4-dimethoxy benzyl] phenoxyacetic acid
(Rink amide handle), I.-butyl carbazate, Boc-azide, 2-(1 H-benzotriazol-l-yl) 1, 1,3,
Comparative Study of PS-TRPGGDA . . .. 1 13
3-tetramethyl uronium hexafluoro phosphate (HBTU),. 1- hydroxy benzahiazole
(HOBt), Boc and Fmoc-amino acids, Menifield resins, Boc-Val-Pam resin and
preloaded Sheppard resin ( ~ o v a s ~ n ~ KA 125) were purchased from Nova
Biochem Ltd. UK. Cliisopropylethyl amine (DIEA), L-amino acids, o-chloro
nitrobenzene piperidine, trifluoroacetic acid (TFA), ethanedithiol, phenol,
thioanisole, Sephade:~ G-111, G-25, and G-50 were purchased from Sigma-
Aldrich Corp., USA. All so!vents used were of HPLC grade purchased from E.
Merck (India), BDH (India) and SISCO Chemicals (Mumhai). HPLC was
done on a Pharmacia Akta purifier using C-I 8 reverse phase semi preparative
HPLC column. The aminc acid analysis was carried out on an LKB 4151
a-plus amino acid a.nalyzc:r. Mass spectra of peptides were recorded in a
Kratos PC Kompact MALDI-TOF-mass spectrometer.
5.3.b Preparation of Boc-azide 'O
t-Butyl carbazate (20 g) was dissolved in a mixture of glacial acetic
acid (27 mL) and water (37 5 mL). NaN02 (7.4 g) was added in small amounts
with vigorous stimng over a period of fifteen minutes by maintaining the
temperature at O'C. After 90 min. an oily layer was separated from the
aqueous layer. The aqueous layer was extracted with ether (3 x 50 mL) and
was mixed with oily layer The mixture was washed with water and 0.1 M
NaHC03 dried over dry Na2CO3. Boc-azide was obtained by evaporating the
ether under reduced pressure. It was used directly without further purification.
5.3.c Preparation of Boc-amino acids by Schnabel's method "
L-Amino acids (10 rnmol) were suspended in 1: 1 dioxane-water (10 mL)
and Boc-azide (1.6 ml, 10 mmol) was added to it. The mixture was stirred at
room temperature maintaining the pH in the alkaline range with 4 N NaOH.
After 24 h, water (25 mL) was added and solution was extracted with ether.
The aqueous layer vias cooled in an ice bath, acidified with 2 N HCI and
extracted with ethyl acetat: (3 x 20 mL). In the case of Boc-Leu ether was
used for extraction. It was then dried over anhydrous Na2S04 and the solvent
-- -- Comparative Study of PS-TRPGGDA .... I 14
was evaporated under vacuum. Purity of all protected amino acids was
checked by tlc on silica gel using CHC1,- MeOH-acetic acid (85:10:5) as
solvent system (Table 1). They were visualized by ninhydrin after 10 minute
exposure to HCI vapour.
5.3.d Preparation of 1-jHydroxy benzotriazole (HOBt)
r!id Lo Phenylalanine
Leucine
Isoleucine 10
Proline t:-9
o-Chloronitr,abenz~:ne (32 g) was dissolved in ethanol (100 mL),
hydrazine hydrate (20 g) was added. The solution was refluxed for 5 h, ethanol
was distilled off. The distillate obtained was diluted with water (100 mL) and
extracted with ether (4 x 50 mL). Aqueous layer was then acidified with HCI.
HOBt precipitated was then recrystallized from hot water (MP 157'~). Yield
22 g, (88%).
Table. 1 Preparation of Boc-amino acids
Yiel:y)
89
93
92
88
90
5.3.e Methods for purification and characterization of peptides
1. Column chromato:graphy
Melting Point
90
54
87
8 1
61
137
Sephadex G-10, G-25, and G-50 were used for the gel filtration
chromatography depending on the molecular weight of the peptides. Silica gel
60 (70-200 mesh size) was .~sed for column chromatography.
tlcRt
0.64
0.62
0.71
0.74
0.68
0.64
- Comparative Study of PS-TRPGGDA . ... I 15
2. Amino acid analysis
In a specific analysis, 10 mg of the peptidyl resin was transferred to a
sample tube. 6 N RCI and 200 pL of TFA (1:l) were added to the tube. The
tube was fixed under nitrogen and kept in a pre heated (1 10-120'~) heating
block for 6 h. The lube was then broken, open and the acid dried over NaOH
and P205 in a dest;icator under high vacuum. The amino acid mixture was
dissolved in a suitable buffer. Loading buffer and an aliquot loaded in the
analyzer capsule such that every amino acid was expected in 3-10 mmol range.
4. Matrix assisted laser desorption ionisation mass spectroscopy (MALDI)
MALDI-TOF-MS is a powerful tool that can accurately determine the
molecular weight of pepfides and protein using 5 pmole amounts of sample.
A saturated solution of the matrix was made by dissolving 10 mg of a-cyano-
4-hydroxycinnamic acid in 1 ml of a 1 : 1 solution containing 0.1% TFA in MQ
water: acetonitrile and the sample solution was prepared in MQ water
containing 1% TFA in such a way that 1p1 of the solution containing 5 pmole
of the sample. The samplg: solution was mixed with the matrix in the ratio 1 : 10
dilution and 2p1 of this s.olution is loaded to sample plate and the mixture is
then air dried. An aqueous solution containing 30 % acetonotrile and 0.1%
TFA. The instrument %as operated in the reflection mode, using 20 KV
accelerating voltage, wi1.h detection of positive ions. A 1pL of a solution
containing 5pmole of insulin and angiotoxin is taken as the reference.
5.3.f Preparation of Boc-Val- PS-TRPGGDA resin
2% PS-TR:PGGC'A (300 mg, 0.03 mmol) was swelled in dry DCM
(30 mL). After 1 h, excess DCM was removed; C-terminal Boc-Val (1 1 mg,
0.06 mmol), MSKT (0.06 mmol, 18 mg) and Melm (0.045 mmol, 3.6 pL)
mixture in dry DCIvI was added. The solution was kept for coupling for 30 min. in a
septum-stoppered flask under nitrogen atmosphere. Then the resin was washed with
dry DCM (5 x 15 rL), ethanol (5 x 15 mL), ether (5 x 15 mL) and dried under
Comparative Study of PS-TRPGGDA . . . ,116
vacuum. Roc-protection was removed by 30% TFA in DCM and the
percentage of incorporation of amino acid was determined by picric acid
method. The amino capacity of the resin: 0.09 mmolig.
5.3.g Comparative synthesis of retro acyl carrier (74-65) protein fragment
The synthesis of iexceedingly difficult retro acyl carrier protein was
carried out manually using Boc-chemistry on Roc-Val-2% PS-TRPGGDA
resin (280 mg, 0.025 mn~ol) and Roc-Val-Pam resin (210 mg, 0.025 mmol).
The resins were swelled in DCM for 1 h. Boc-protection of C-terminal amino
bound resin was removed by suspending these resins in 30% TFA in DCM for
30 min. The successive amino acids were coupled by HOBt active ester
method. The H O B active ester was prepared by dissolving DCC (18 mg,
0.0875 mmol), HOBt (I2 mg, 0.0875 mrnol) in minimum amount of NMP and
stirred with respective amino acid (0.0875 mmol) for 40 min. DCU formed
was filtered off and the active ester was added into the resin. The active ester
mixture of Boc-amino acid were made at triple the indicated scale and
apportioned equally to the three parallel synthesis. Each coupling steps were
monitored by semi quantitative ninhydrin test. After the synthesis, the
peptidyl resin was washed with NMP (5 x 10 mL), DCM (5 x 10 mL), MeOH
(5 x 10 mL), ether (5 x 10 mL) and dried under vacuum.
The target peptidc: from the PS-TRPGGDA support was cleaved from
the support by suspending the peptidyl resin in TFA (2.8 mL), thioanisole
(150 pL), ethanedithiol (150 pL), and water (150 pL) for 14 h at room
temperature. The solutior~ was filtered and filtrate concentrated under reduced
pressure. The peptide was precipitated by adding ice-cold ether and the
precipitate was washed with ether until the scavengers are removed and dried
in vacuum. In the case c ~ f Pam resin the peptidyl resin was transferred into a
flask equipped with a stimng bar, thioanisole (300 pL), and ethanedithiol
(300 pL), were added. The flask was then cooled in an ice bath, and TFA
(3ml) was added and stirred for 5-l0min. TFMSA (300 pL) was then added
.- Comparative Study of PS-TRPGGDA . . . . I 17
drop wise into the flask, with vigorous stirring to dissipate the heat generated
during its addition. The cleavage mixture was allowed to stand for 2 h at room
temperature. The resin was filtered using a sintered glass funnel, and washed
with small amounts of TI'A, and the combined filtrates were collected. Peptide
was precipitated by the addition of cold diethylether (5 x 10ml). The peptide
was then washed thoroughly with ether to remove the scavengers, and finally
desalted using a sephadex column. HPLC analysis of the peptide was carried
out by injecting a small amount of peptide dissolved in water to C-18 RPC and
eluting using a gradient of solvent A: nanopure water containing O.S%TFA
and solvent B 80% acetorlitrile in nanopure water containing 0.5% TFA.
5.3.h. Derivatization of the resin with HMPA linker
1. Preparation of PS-TRPGGDA-HMPA resin
Amino 2% PS-TIWGGDA resin (Ig, O.lmmo1) was swelled in DMF.
After 1 h the excess DFAF was removed. HMPA (64mg, 0.35mmol), HBTU
(132mg, 0. 35mrno:I) and HOBt (47.29mg, 0.35mmol), DIEA (61~1, 0.35mmol)
mixture in DMF (3 r L ) were added to the swollen resin and the mixture was shaken
for I h. The completion ofthe coupling reaction was monitored by ninhydrin test
The resin was filtered, washed with DMF (5 x 50 mL), MeOH (5 x 50 mL), ether
(5 x 50 mL) and dried in vacuum. The derivatization of HMPA linker with
4% PS-TRPGGDP, was also carried out using amino PS-TRPGGDA resin
(lg, 0.18mmol). The coupling reaction was carried using the same procedure
as mentioned above.
2. Preparation of PS-DVB-HMPA resin
Amino PS-DVB ~es in (1 g, 0.12mmol) was swelled in DMF. After 1 h
the excess DMF was removed. HMPA (76mg, 0.42rnmol), HBTU (160mg,
0.42 mmol) and HOBt (56mg, 0.42mmol), DIEA (73~1, 0.42mmol) mixture in
DMF (3 mL) were added lo the swollen resin and the mixture was shaken for 1 h.
Comparative Study of PS-TRPGGDA . . .. 1 18
The completion of the coupling reaction was monitored by ninhydnn test. The
resin was filtered, washed with DMF (5 x 50 mL), MeOH (5 x 50 mL), ether
(5 x 50 mL) and dried in vacuum. The HMPA denvatisation was also carried out
with 0.2 mmoUg of the resin using the same procedure as mentioned above.
5.3.i Anchoring of C-terminal Fmoc-Val
1. Preparation of Fmc~c-Val-HMPA-PS-TRPGGDA resin
2% PS-TRPCrGDA-HMPA resin (300 mg, 0.03 mmol) was swelled in
dry DCM (30 mL). After 1 h, excess DCM was removed and C-terminal
Fmoc-Val (20.36 mg, O.O6mmol), MSNT (17.7 nlg, 0.06 mmol) and MeIm
(3.6 p1, 0.045 mmol) mixture in dry DCM was added. The solution was kept for
coupling for 30 min. in a septum-stoppered flask under nitrogen atmosphere.
Then the resin was washed with dry DCM (5 x 15 mL), ethanol (5 x 15 mL),
ether (5 x 15 mL) and dried under vacuum. Fmoc-protection was removed by
20% piperidine in DMF and the percentage of incorporation of amino acid was
determined by the W abso~ption of the dibenzofulvene-pipendine adduct formed
at 290 nm. The amino capacity of the resin: 0.094 mmoYg.
2 . Preparation of Fmoc-Val-HMPA-PS-DVB resin
PS-DVB-HMPA resin (300 mg, 0.036 mmol) was swelled in dry DCM
(30 mL). After 1 h, excess DCM was removed; C-terminal Frnoc-Val (24.4
mg, 0.072 mmol), MSNT (21.3 mg, 0.072 mmol) and MeIm (4.3 pl, 0.054
mmol) mixture in dry DCh4 was added. The solution was kept for coupling for
30 min. in a septum-stoppered flask under nitrogen atmosphere. Then the resin
was washed with dry DCId (5 x 15 mL), ethanol (5 x 15 mL), ether (5 x 15
mL) and dried under vacuum. Fmoc-protection was removed by 20 %
piperidine in DMF and the percentage of incorporation of amino acid was
determined by the UV absorption of the dibenzofulvene-piperidine adduct
formed at 290 nm. The amino capacity of the resin: 0.1 lmmol/g.
-- Comparative Study of PS-TRPGGDA . . . . I 19
5.3.j Comparative synthesis of Leu-Ala-Gly-Val
The synthesis of lieu-Ala-Gly-Val was carried out manually using
Fmoc-Val-HMPA-2% PS--TRPGGDA (280 mg, 0.026 mmol), Fmoc-Val-
HMPA-PS-DVB (238 mg, 0.026 mmol) and ~ m o c - ~ a l - ~ o v a s ~ n ~ KA 125
(Sheppard resin) (260mg, 0.026 mg) resin. Fmoc-protect~on of these amino
acid bound resin was removed by 20% piperidine in DMF and the resins were
washed thoroughly .with IIMF (5 x 15 mL). For each acylation cycle the
respective Fmoc-amino acids (0.091 mmol), mixed with HBTU (34.5 mg,
0.091 mmol), HOBt (12 rng, 0.091 mmol) and DIEA (16 p1, 0.091 mmol)
dissolved in minimum amount of DMF were added to Fmoc-deprotected resin.
Coupling solutions were made at triple the indicated scale and apportioned
equally to the three parallel syntheses. The coupling reaction was canied out
for 40 min. at room temptxature. The coupling and deprotection steps were
monitored by ninhytirin test. After incorporation of all amino acids Fmoc-
protection of the target pep.:idyl resin was removed and resin was washed with
DMF (5 x 15 mL), inethatiol (5 x 15 mL), ether (5 x 15 mL) and dried in
vacuum. The target peptities were cleaved from the polymer supports by
suspending the peptidyl resin in TFA (3 mL), thioanisole (150 pL),
ethanedithiol (150 ILL), r~henol (200 pL) and water (150 pL) at room
temperature for 4 h. 'The solution was filtered and filtrate concentrated under
reduced pressure. The peptide was precipitated by adding ice-cold ether and
the precipitate was washed wlth ether until the scavengers are removed and
dried in vacuum. HPI-C andlysis of the peptide was carried out by injecting a
small amount of peptide dissolved in water to C-18 RPC and eluting using a
gradient of solvent A: nanopure water contain~ng O.S%TFA and solvent B
80% acetonitrile in nslnopure water containing 0.5% TFA.
Comparative Study of PS-TRPGGDA . . . ,120
5.3.k Anchoring of C-tel-minal Fmoc-Gly
1. Preparation (of Fmoc-Gly-HMPA-PS-TRPGGDA resin
2% PS-TRPGGDA-I-IMPA resin (300 mg, 0.03 mmol) was swelled in dry
DCM (30 mL). Aftel- 1 h, excess DCM was removed, C-terminal Fmoc-Gly
(18 mg, 0.06 mmol), MSNT (0.06 mmol, 17.7 mg) and MeIm (0.045 mmol,
3.6 pL) mixture in dry DCh.1 was added. The solution was kept for coupling for
30 min. in a septum-stcpperetf flask under nitrogen atmosphere. Then the resin was
washed with dry DCM (5 x 15 mL), ethanol (5 x 15 mL), ether (5 x 15 mL) and
dned under vacuum. Fmoc-protection was removed by 20% piperidine in DMF
and the percentage of incorporation of amino acid was determined by the UV
absorption of the dibenzofulvene-piperidine adduct formed at 290 nm. The
amino capacity of the resin: 0.093 mmollg.
2. Preparation of Fmoc-Gly- PS-DVB-HMPA resin
PS-DVB-HMPA resin (300 mg, 0.036 mmol) was swelled in dry DCM
(30 mL). AAer 1 h, excess DCM was removed; C-terminal Fmoc-Gly (21.4 mg,
0.072 mmol), MSNT (21.3 mg, 0.072 mrnol) and MeIm (4.3 pL, 0.054 mmol)
mixture in dry DCM wzs addt:d. The solution was kept for coupling for 30 min. in
a septum-stoppered flask under nitrogen atmosphere. Then the resin was washed
with dry DCM (5 x 15 mL), ethanol (5 x 15 mL), ether (5 x 15 mL) and dried
under vacuum. Fmoc-protection was removed by 20% piperidine in DMF and
the percentage of incorpora~ion of amino acid was determined by the UV
absorption of the dibenzofu,vene-piperidine adduct formed at 290 nm. The
amino capacity of the resin: C . 105 mmollg.
5.3.1 Comparative s,ynthe!iis of acyl carrier (65-74) protein fragment
The synthesis of acyl carrier protein (65-74) fragment was carried out
nlanually using Fmoc-Gly-HlvlPA-2% PS-TRPGGDA (269 mg, 0.025 mmol),
Fnioc-Gly-HMPA-Merrifield resin (238 mg, 0.025 mmol) and Fmoc-Gly-
~ o v a s ~ n ~ KA 125 (250 mg, 0.025 mmol) resin. Fmoc-protection of these
-. Comparative Study of PS-TRPGGDA . . . . I 2 1
amino acid bound resin was removed by 20% pipendine in DMF and the reslns
were washed thoroughly with DMF (5 x 15 mL). For each acylation cycle the
respective Fmoc-amino acids (0.0875 mmol), mixed with HBTU (33 mg, 0.0875
mmol), HOBt (12mg, 0.0875 mmol) and DIEA (15 pL, 0.0875 mmol) dissolved
in minimum amount of DM:F were added to Fmoc-deprotected resin. Coupling
solution were made at triple the indicated scale and apportioned equally to the
three parallel syntheses. The. coupling reaction was carried out 40 min at room
temperature. The coupling and deprotection steps were monitored by ninhydnn
test. After incorporation of all amino acids Fmoc-protection of the target peptidyl
resin was removed and resin was washed with DMF (5 x 15 mL), methanol
(5 x 15 mL), ether (5 x 15 mL) and dried in vacuum. The target peptides were
cleaved from the pol!imer mpports by suspending the peptidyl resin in TFA
(3 mL), thioanisole (150 pL), ethanedithiol (150 pL), phenol (200 pL) and
water (150 pL) at room temperature for 4 h. The solution was filtered and
filtrate concentrated under reduced pressure. The peptide was precipitated by
adding ice-cold ether and the precipitate was washed with ether until the
scavengers are removed and dried in vacuum. HPLC analysis of the peptide
was carried out by inject in^: a small amount of peptide dissolved in water to
C-18 RPC and eluting wing a gradient of solvent A: nanopure water
containing O.5%TFA and solvent B 80% acetonitrile in nanopure water
containing 0.5% TFA.
5.3.m Anchoring of C-terminal Fmoc-Lys
1. Preparation of Fmoc-Lys-HMPA-PS-TRPGGDA resin
4% PS-TRPGGDA-HMPA resin (700 mg, 0.126 mmol) was swelled in
dry DCM (30 mL). ARer 1 11, excess DCM was removed, C-terminal Fmoc-Lys
(118 mg, 0.252mmol:), MSIVT (74.7 mg, 0.252 mmol) and MeIm, (15.2 pL,
0.189 mmol) mixture in dy DCM was added. The solution was kept for
coupling for 30 min. in a septum-stoppered flask under nitrogen atmosphere.
Comparative Study of PS-TRPGGDA . . . ,122
Then the resin was washed with dry DCM (5 x 15 mL), ethanol (5 x 15 mL),
ether (5 x 15 mL) and dried under vacuum. Fmoc-protection was removed by
20% pipendine in DhIF anti the percentage of incorporation of amino acid was
determined by the W absorption of the dihenzofulvene-pipendine adduct formed
at 290 nm. The amino capacity of the resin: 0.174 mmol/g.
2. Preparation of Fmoc-Lys-HMPA-PS-DVB resin
PS-DVB-HMPA resin (700 mg, 0.14 mmol) was swelled in dry DCM
(30 mL). After 1 h, excess DCM was removed; C-terminal Fmoc-Lys (131 mg,
0.28 mmol), MSNT (83 nlg, 0.28 mmol) and MeIm (16.7 pL, 0.21 mmol)
mixture in dry DCM was added. The solution was kept for coupling for 30 min. in
a septum-stoppered flask untier nitrogen ahnosphere. Then the resin was washed
with dry DCM (5 x 15 mL), ethanol (5 x 15 mL), ether (5 x 15 mL) and dned
under vacuum. Fmoc-protection was removed by 20% pipendine in DMF and
the percentage of incorporation of amino acid was determined by the UV
absorption of the dihenzofulvene-piperidine adduct formed at 290 nm. The
amino capacity of the resin: 0.185 mmollg.
5.3.11 Comparative synthesis of Ala-Arg- (Ala) cLys peptide
The synthesis of A:a-Arg-(Ala)a-Lys was carried out manually using
Fmoc-Lys-HMPA-4% PS-TRPGGDA resin (230 mg, 0.04 mmol), Fmoc-Lys-
HMPA-Merrifield re:iin (2 I5 mg, 0.04 mmol) and ~ m o c - ~ ~ s - ~ o v a s ~ n ~ KA
125 resin (280 mg, 0.04 n~mol) resin. Fmoc-protection of these amino acid
bound resin was removed by 20% pipendine in DMF and the resins were
washed thoroughly with DMF (5 x 15 mL). For each acylation cycle the
respective Fmoc-amino acids (0.14 mmol), mixed with HBTU (53 mg, 0.14
mmol), HOBt (19 mg, 0.14 mmol) and DIEA (24 pL, 0.14 mmol) dissolved in
minimum amount of DMF were added to Fmoc-deprotected resin. Coupling
solution were made at triple the indicated scale and apportioned equally to the
three parallel syntheses. The coupling reaction was carried out 40 min. at room
temperature. The coupling and deprotection steps were monitored by ninhydnn
Comparative Study of PS-TRPGGDA . . . .I23
test. After incorporation of all amino acids Fmoc-protection of the target peptidyl
resin was removed and re:jin was washed with DMF (5 x 15 mL), methanol
(5 x 15 mL), ether (5 x 15 mL) and dried in vacuum. The target peptides were
cleaved from the pcmlymer supports by suspending the peptidyl resin in TFA
(3 mL), thioanisole (150 pL), ethanedithiol (150 pL), phenol (200 pL) and
water (150 pL) at mom temperature for 4 h. The solution was filtered and
filtrate concentrated under reduced pressure. The peptide was precipitated by
adding ice-cold ether ant1 the precipitate was washed with ether until the
scavengers are removed and dried in vacuum. HPLC analysis of the peptide
was camed out by ~njecting a small amount of peptide dissolved in water to
C-18 RPC and eh~ting using a gradient of solvent A: nanopure water
containing O.S%TFA ant1 solvent B 80% acetonitrile in nanopure water
containing 0.5% TF,4.
5.3.0 Comparative synthesis of Syntide 2 Peptide
The synthesis of the 15 residue Syntide 2 peptide Pro-Leu-Ala-Arg-
Thr-Leu-Ser-Val-Ala-GlyLeu-Pro-Gly-Lys-Lys was canied out manually
using Fmoc-Lys-4%) PS-TRPGGDA resin (280 mg, 0.048 mmol), and Fmoc-
Lys-HMPA-Merrifield res,in (260 mg, 0.048 mmol). Fmoc-protection of these
amino acid bound resin was removed by 20% pipendine in DMF and the resins
were washed thoroughly with DMF (5 x 15 mL). For each acylation cycle the
respective Fmoc-amino acids (0.168 mmol), mixed with HBTU (64 mg,
0.168 mmol), HOBt (23 mg, 0.168 mmol) and DIEA (29 pL) dissolved in
minimum amount of DMF were added to Fmoc-deprotected resin. Coupling
solution were made at double the indicated scale and apportioned equally to
the two parallel syntheses:. The coupling reaction was carried out 40 min. at
room temperature. The coupling and deprotection steps were monitored by
ninhydrin test. After incc'rporation of all amino acids Fmoc-protection of the
target peptidyl resin was removed and resin was washed with DMF (5 x 15 mL),
methanol (5 x 15 mL), ether (5 x 15 mL) and dried in vacuum. The target
-- Comparative Study of PS-TRPGGDA . . . ,124
peptides were cleaved from the polymer supports by suspending the peptidyl
resin in TFA (3 mL:), thioanisole (150 pL), ethanedithiol (150 pL), phenol
(200 pL) and water (150 pL) at room temperature for 4 h. The solution was
filtered and filtrate concer~trated under reduced pressure. The peptide was
precipitated by adding ice-cold ether and the precipitate was washed with
ether until the scavengers are removed and dried in vacuum. HPLC analysis of
the peptide was carried out by injecting a small amount of peptide dissolved in
water to C-18 RPC and eluting using a gradient of solvent A: nanopwe water
containing O.5%TFA and solvent B 80% acetonitrile in nanopwe water containing
0.5% TFA.
5.3.p Derivatization of s~~pports with Rink amide linker
1. Preparation of PS-'TRPGGDA Rink amide resin
PS-TRPGGDA resin (1 g, 0.18mmol) was swelled in dry DCM. After 1 h
the excess DCM was re:moved. Rink handle (291mg, 0.54mmol) and
N-methylimidazole (33pL, 0.40mmol) was dissolved in dry DCM (5mL) and
few drops of dry THI?, and shaken with the resin in a septum-stoppered flask
attached with a N2 balloon MSNT (160mg, 0.54mmol) was dissolved in dry
DCM (5 mL) and injected to the reaction mixture. After 1 h, the resin was
washed with dry DCM (5 >: 15 mL) and dried under vacuum. The dried resin
(10 mg) was mixed with 3 mL 20% piperidine in DMF for 30 min. The
percentage incorporation c~f Rink handle was estimated by measuring the
UV-absorbance of the abc've solution containing dibenzofulvene-piperidine
adducts at 290 nm. The amino capacity of the resin: 0.174mmol/g.
2. Preparation of Merrifield Rink amide resin
Hydroxymethyl Merrifield resin ( lg, 0.2 mmol) was swelled in dry
DCM. After I h the excess KICM was removed. Rink handle (323mg, 0.54 mmol)
and N-methyl- imidazole (3tjpL, 0.4Ommol) was dissolved in dry DCM (5 mL)
Compamtive Study of PS-TRPGGDA . . . ,125
and few drops of dry THI;, and shaken with the resin in a septum-stoppered
flask attached with .a Nz balloon. MSNT (l77mg, 0.6mmol) was dissolved in
dry DCM (5 mL) and injected to the reaction mixture. After 1 h, the resin was
washed with dry DCM (5 x 15 mL) and dried under vacuum. The dried resin
(10 mg) was mixed with 3 nL 20 % piperidine in DMF for 30 min. The percentage
incorporation of rink handle was estimated by measuring the UV-absorbance of the
above solution containing dibenzofulvene-piperidine adducts at 290 nm. The amino
capacity of the resin: 13.185 mmol/g.
5.3.q Anchoring of C-terminal Fmoc-Ala
1. Preparation of Fn~oc-Ala-Rink Amide PS-TRPGGDA resin
4% PS-TRPGGDA-Rink amide-resin (400 mg, 0.07 mmol) was swelled
in dry DCM (30 m:L). After 1 h, excess DCM was removed and C-terminal
Fmoc-Ala (43.5 mg,, 0.14 mmol), MSNT (41.4 mg, 0.14 mmol) and MeIm,
(8.3pL, 0.105mmol) mixture in dry DCM was added. The solution was kept for
coupling for 30 min. in a septum-stoppered flask under nitrogen atmosphere.
Then the resin was ,washed with dry DCM (5 x 15 mL), ethanol (5 x 15 mL),
ether (5 x 15 mL) and dried under vacuum. Fmoc-protection was removed by
20% piperidine in ClMF and the percentage of incorporation of amino acid was
determined by the U\i absorption of the dibenzohlvene-pipendine adduct formed
at 290 nm. The amino capa'sity of the resin: 0.17 mmollg.
2. Preparation of Fmoc-Ala-Rink amide-Merrifield resin
Fmoc-Ala-Rink amide-Merrifield resin (400 mg, 0.074 mmol) was
swelled in dry DClM (30 mL). After 1 h, excess DCM was removed and
C-terminal Fmoc-Ala (46 mg, 0.148mmol), MSNT (44 mg, 0.148 mmol) and
MeIm (16.7 pL, 0.1 1 1 mmol) mixture in dry DCM was added. The solution was
kept for coupling for 30 min. in a septum-stoppered flask under nitrogen
atmosphere. Then the resn was washed with dry DCM (5 x 15 mL), ethanol
(5 x 15 mL), ether 1:5 x 1 5 mL) and dried under vacuum. Fmoc-protection was
removed by 20% piperidin? in DMF and the percentage of incorporation of amino
- p~
Comparative Study of PS-TRPGGDA . . .. 126
acid was determined by the VJ absorption of the dibenzohlvene-piperidine adduct
formed at 290 nm. The amino capacity of the resin: 0.175 mmoWg.
5.3.r. Comparative synthesis of 21 residue dermaseptin peptide amide: Ala-Leu-Trp-I~ys-Asn-Met-Leu-Lys-Gly-IIe-Lys-Lys-Leu-Ala-G1y- Lys-Ala-Ala-Leu-GI y-Ala-NH2
The 21 residue peptide amide of dermaseptin (Ala-Leu-Trp-Lys-Asn-Met-
Leu-Lys-Gly-Ile-Lys-L~~s-Leu-Ala-Gly-Lys-Ala-Ala-Leu-Gly-Ala-~~) was
camed out on Fmoc-Ala-Rink amide-4% PS-TRF'GGDA m i n (376 mg, 0.064 rnmol)
and with Fmoc-Ala-Rink arrlide-PS-DVB resin (366 mg, 0.064 mmol) under
identical synthetic condition. Fmoc-protection of these aniino acid bound resin was
removed by 20% piperidine in DMF and the resins were washed thoroughly with
DMF (5 x 15 mL). For each acylation cycle the respective Fmoc-amino acids
(0.224 mrnol), mixed with HHTU (85 mg, 0.224 mrnol), HOBt (30.26 mg, 0.224
mmol) and DIEA (39 ,uL, 0.224 mmol) dissolved in minimum amount of DMF
were added to Fmoc-deprotected resin. Coupling solution were made at double the
indicated scale and apportioned equally to the two parallel syntheses. The coupling
reaction was carried out 43 min. at room temperature. The coupling and
deprotection steps were monitored by ninhydrin test. After incorporation of all
amino acids Fmoc-protection of the target peptidyl resin was removed and resin
was washed with DMF (5 x 15 mL), methanol (5 x 15 mL), ether (5 x 15 mL) and
dried in vacuum. The target peptides were cleaved kom the polymer supports by
suspending the peptidyl resin in TFA (3 mL), thioanisole (150 pL), ethanedithiol
(150 pL), phenol (200 pL) and water (150 pL) at room temperature for 4 h. The
solution was filtered and filtn~te concentrated under reduced pressure. The peptide
was precipitated by adding ice-cold ether and the precipitate was washed with ether
until the scavengers are removed and dried in vacuum. HPLC analysis of the
peptide was canied out by injecting a small amount of peptide dissolved in water to
C- 18 RF'C and eluting using a gradient of solvent A: nanopure water containing
O.5%TFA and solvent B 80% acetonitrile in nanopure water containing 0.5% TFA.
p~
Comparative Study of PS-TRPGGDA . . . ,127
References
Kent, S. B. H.; Clark-Lewis, I. in "Synthetic peptides in Biology and
Medicine" Alitalo, I<.; Partanen, P.; Vaheri, A. Eds.; Elsevier Science
Publishers B.V., Amsterdam, 1985, pp 29-57.
Kent, S. B. H. in "F'eptides: Structure and Function, Proceedings of the
9Ih American peptide symposium", Deber, C. M.; Hmby, V. J.; Kopple,
K. D. Eds.; Pierce Chemical Co. Rockford, IL, 1985, pp 407-414.
Tam, J. P.; Lu, Y. A . J Am. Chem. Soc. 1995,117, 12058
Sarin, V. K.; Kent, S. B. H.; Mitchel, A. R.; Menifield, R. B. J. Am.
Chem. Soc. 1984, 1j76, 7845.
Meldal, M. in "Peptides 1992, Proc. of the 22"d Eur. Pept. Symp.",
Schneider, C. H.; Eberle, A. N. Eds.; ESCOM, Leiden, 1993, p 61.
Rovero, P.; Rigarlelliu, D.; Fruci, D.; Vigano, S.; Pegoraro, S.;
Revoltella, R.; Greco, G.; Butter, R.; Clementi, S.; Tanigaki, N. Mol.
Immunol. 19!14,31, 549.
Quibell, M.; Johnson,T.; Tumell, W. G. Biomedical peptide, Protein
and nucleic acid 19195, 1, 3.
Meister, S. h4.; Kent, S. B. H. in "Proceedings of the eighth American
peptide symposiuni"; Hurby, V. J. Rich, D. H. Eds., Pierce Chemical.
Co., Rockford, IL, 1983, 103.
Tam, J. P.; Lu, Y. '4. J. Am. Chem. Soc 1995, 117, 12058-63.
Brenner, M.; Huber,W. Helv. Chem. Acta. 1953, 36,1109.
Schnabel, E. Ann. Chem. 1967, 702, 188
