Novel methodology for the solid-phase synthesis of phosphinic peptides

Article abstract of DOI:10.1039/b003848m

A novel, versatile strategy for the solid phase synthesis of phosphinic peptides is developed in which the phosphorus-carbon bond is formed on a polymer support during peptide synthesis. The formation of bis(trimethylsilyl) 1-(allyloxycarbonylamino)ethylphosphonite from 1-(allyloxycarbonylamino)ethylphosphinic acid is investigated, as well as the Michael addition of the former to a resin-bound acrylate. Conditions are also established for the clean, quantitative conversion of a resin-bound, N-terminus acryloylated peptide with bis(trimethylsilyl) 1-(allyloxycarbonylamino)-ethylphosphonite. Conventional peptide synthesis is then employed to obtain a phosphinic undecapeptide in high yield and purity.

Full text of DOI:10.1039/b003848m

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Novel methodology for the solid-phase synthesis of phosphinic
peptides†
Jens Buchardt and Morten Meldal*
1
Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Valby,
Denmark. E-mail: mpm@crc.dk
Received (in Cambridge, UK) 15th May 2000, Accepted 26th July 2000
First published as an Advance Article on the web 4th September 2000
A novel, versatile strategy for the solid phase synthesis of phosphinic peptides is developed in which the phosphorus–
carbon bond is formed on a polymer support during peptide synthesis. The formation of bis(trimethylsilyl) 1-(allyloxy-
carbonylamino)ethylphosphonite from 1-(allyloxycarbonylamino)ethylphosphinic acid is investigated, as well as the
Michael addition of the former to a resin-bound acrylate. Conditions are also established for the clean, quantitative
conversion of a resin-bound, N-terminus acryloylated peptide with bis(trimethylsilyl) 1-(allyloxycarbonylamino)-
ethylphosphonite. Conventional peptide synthesis is then employed to obtain a phosphinic undecapeptide in high
yield and purity.
Introduction
Phosphorus-backbone isosteres of peptides have received much
1–4
attention as inhibitors of various proteases, including matrix
5–7
metalloproteinases (MMPs).
In phosphinic peptides, the
conventional peptide bond is replaced by a phosphorus–carbon
bond, namely the -P(O)OH-CH - moiety. As a consequence this
2
particular kind of peptide isostere is more stable towards chem-
ical hydrolysis than the related phosphonic [-P(O)OH-O-] and
phosphonamidic [-P(O)OH-NH-] isosteres. The phosphinic
moiety has been introduced into peptides in a number of differ-
ent ways. Most recently phosphinic pseudo-dipeptide building
blocks have been used to prepare phosphinic peptides on solid
6–8
phase. The solid-phase technology is advantageous for the
preparation of peptides and peptidomimetics because of their
9
oligomeric structure. It is therefore desirable to establish reac-
tion conditions which allow the formation of the phosphinic
moiety to be conducted on solid support, thereby avoiding
the need for the 400 different phosphinic dipeptide building
blocks which would be necessary to represent the natural
amino acid side-chains. By contrast, a direct solid-phase
approach would require only 40 compounds (amino acid ana-
logues) to give synthetic access to the complete spectrum of
phosphinic peptides.
In the preparation of phosphinic peptides the P–C bond is
conveniently formed by a Michael-type addition of a bis(tri-
10,11
methylsilyl) phosphonite to an acrylate.
This transformation
has been restricted almost exclusively to solution-phase syn-
Scheme 1 Schematic presentation of the preparation of a complete
phosphinic peptide by solid-phase synthesis, including the coupling
of a bis(trimethylsilyl) 1-(allyloxycarbonylamino)alkylphosphonite to a
resin bound acrylate.
6,8
thesis of phosphinic dipeptide building blocks, except for one
example in which phosphinic dipeptoids were prepared on a
12
solid support. The present work describes the development of
a methodology for the assembly of a phosphinic peptide on a
solid support (Scheme 1), by a process which comprises con-
ventional solid-phase peptide synthesis (SPPS); acryloylation
of the peptide N-terminal; addition of a protected 1-amino-
alkylphosphinic acid [via the corresponding bis(trimethylsilyl)
phosphonite] to the N-terminal acrylamide, and finally elon-
gation of the pseudo-peptide by SPPS.
Results and discussion
Prior to the synthesis of a phosphinic peptide, conditions were
established for addition of a protected 1-aminophosphinic
acid to a resin-bound acrylate (Scheme 2). As a model system,
1
-aminoethylphosphinic acid (alanine analogue) and resin-
bound acrylic ester 1 (glycine analogue) were selected. The
13,14
PEG-based resin POEPS-3
was used because of its chemical
1
inertness and was acylated with acryloyl chloride. The quanti-
tative formation of the acrylic ester on the solid support was
confirmed from a single bead Nano-probe Magic Angle
†
H NMR spectra for 9 and 11 after cleavage are available as supple-
mentary data. For direct electronic access see http://www.rsc.org/
suppdata/p1/b0/b003848m/
3
306
J. Chem. Soc., Perkin Trans. 1, 2000, 3306–3310
This journal is © The Royal Society of Chemistry 2000
DOI: 10.1039/b003848m

View Article Online
Scheme 2 Preparation of resin 1 and building blocks for the investi-
gation of the solid-phase Michael additions. Protection of the
phosphinic amino acids was performed with Alloc-Cl, Cbz-Cl,
Fmoc-OSu and Boc O, respectively, in basic aqueous solution.
2
1
Spinning (MAS) H NMR spectrum. In contrast, attempts to
couple acrylic acid to the resin using 2,4,6-mesitylsulfonyl-3-
15
nitro-1,2,4-triazole (MSNT) were unsuccessful. After reac-
tion, the hydroxy groups of the resin were blocked. However,
the acrylic ester was not formed, as determined by Nano-probe
1
H NMR spectroscopy.
The alanine 1 analogue 1-aminoethylphosphinic acid is
6
readily available in the benzhydryl-protected form, 2. It was
converted into the differently protected phosphinic alanine
analogues 3–6 by standard procedures. The Fmoc group was
anticipated to be the most convenient amine protection because
it can be removed under mild conditions as in conventional
SPPS. Since the Boc group is usually removed with acid it
would be limited to the preparation of peptides not containing
acid-labile side-chain-protective groups. In contrast, Cbz and
Alloc protective groups are stable to a variety of condi-
tions. Although Alloc protection is readily removed on solid
17
support using Pd(PPh ) , the Cbz group is cleaved either by
3
4
hydrogenolysis, which is not always easily achieved on solid
support, or by alternative conditions such as trimethylsilyl
iodide (TMSI), which is not compatible with many peptide
side-chain-protective groups.
Scheme 3 Reactions performed to (i) investigate the solid-phase
Bis(trimethylsilyl)acetamide (BSA) and a mixture of TMSCl
Michael addition of the phosphonite 8 to the resin-bound acrylic ester,
and (ii) prepare a phosphinic undecapeptide using only solid-phase
reactions.
and Et N were both investigated for the formation of bis-
3
(
trimethylsilyl) 1-(allyloxycarbonylamino)ethylphosphonite 8
(
Scheme 3). The former was found to be superior since TMSCl–
Et N partially cleaved the carbamate protective groups, espe-
3
cially at elevated temperatures. Furthermore, the Fmoc group
was slowly cleaved by BSA. Considering the general disadvan-
tages of using Boc and Cbz groups in Fmoc-based SPPS it
was decided to use the acryloylated resin 1 and 1-(allyloxy-
carbonyl)ethylphosphinic acid 3 together with BSA for further
investigations.
investigated. When reaction mixtures of 3 and increasing
31
amounts of BSA at room temperature were analysed by
P
NMR (CDCl ), the resonance originating from 3 (δ 33.4) was
3
P
seen to diminish and be replaced by multiple resonances around
δP 22.5, showing the formation of other tervalent phosphorus
species. However, on heating of the solutions to 100 ЊC for a
Previous work on this type of Michael addition both in solu-
short period in a closed container, a single resonance at δ 7.6
P
10
12
tion and on polymeric support has described the reaction
of phosphinic acids with acrylic esters using BSA at room tem-
perature. However, the reaction between 1, 3 and BSA (propor-
tions 1:3:9) for a period 20 h gave rise to less than 5% of the
desired product 7 although the product obtained was of high
purity. Increasing the reaction temperature to 60 ЊC resulted in
a higher yield of 7, but complete conversion of 1 was still not
achieved and the formation of the phosphonite 8 was therefore
appeared corresponding to complete conversion of 3 into 8.
When less than three molar equivalents of BSA with respect to
3 was used, the conversion at 100 ЊC remained incomplete and a
ratio of 3 to BSA of 1:3 was therefore selected to generate the
phosphonite 8.
A series of experiments in 1,2-dichloroethane (DCE)
showed that the Michael addition also required elevated tem-
peratures to proceed satisfactorily. Reaction of 6 equivalents
J. Chem. Soc., Perkin Trans. 1, 2000, 3306–3310
3307

View Article Online
Table 1 Conversion of resin-bound acrylate 1 with phosphinic acid
and BSA (1:6:18) at 100 ЊC for 30 min in various solvents
3
Solvent
Conversion (%)
Relative to DCM
DCM
DCE
MeCN
Ethyl acetate
Toluene
DMSO
CHCl3
57
52
36
30
28
26
26
16
16
1.00
0.91
0.63
0.53
0.49
0.46
0.46
0.28
0.28
THF
Neat BSA
1
Fig. 1 H NMR spectra of crude products formed in the reaction
between 1, 3 and BSA (1:6:18) at different periods of time and temper-
atures. A: rt, 18 h (40% conversion); B: 60 ЊC, 22 h (80% conversion); C:
1
00 ЊC, 1 h (100% conversion). Resonances a, b, c–e, and f–i originate
from acrylic acid, the Alloc group, and the phosphinic dipeptide
analogue, respectively.
of 8 with 1 at either room temperature for 18 h, or at 60 ЊC
for 22 h, both gave incomplete conversion of 1 (Fig. 1
A ϩ B). However, reaction at 100 ЊC for 1 h resulted in clean
and quantitative conversion into 7, with no detectable side-
products (Fig. 1C). Shorter reaction times at 100 ЊC led to
incomplete conversion of 1 and, moreover, a large excess of 8
(
1:3:BSA 1:6:18) was necessary, since TLC analysis of the
supernatant showed extensive degradation of 3 after 1 h (8
decomposed to 3 on the TLC plate). The degradation prod-
ucts did, however, not appear to react with the acrylate and
the crude product obtained after cleavage from the resin was
of high purity.
The reaction conditions were also applied to the analogues
Fig. 2 RP-HPLC chromatograms showing the high purity of the
crude compounds obtained at different stages of the synthesis sequence
during the preparation of the phosphinic undecapeptide 12 by solid-
phase reactions only.
4
–6. The Fmoc group did not tolerate these reaction conditions,
resulting in a complex mixture of products, whereas both the
Boc and the Cbz groups were unaffected though the reaction
rate was slower.
22
The rate of addition was investigated using a variety of
solvents ranging from acetonitrile to toluene in polarity (Table
uronium tetrafluoroborate (TBTU) and reaction with the N-
terminal resulted in a complex mixture of products (as deter-
mined by RP-HPLC and MALDI-TOF). Although the most
abundant component was the desired coupling product 9, the
mass spectrum of the mixture also indicated the presence of
the bis-adduct 10.
1
). Although the rate was highest in DCM the vapor pressure
of this solvent at 100 ЊC was too high for it to be useful and
reactions were therefore conducted in DCE. The rate of a
solid-phase reaction is known to be strongly influenced by the
18
properties of the solid support since this may be regarded as a
In the next step, the Michael addition of 8 to 9, the optimised
conditions from the model study failed to give complete conver-
19
co-solvent in the reaction. Therefore, the minimum reaction
time required is likely to be different if a resin other than
POEPS-3 is used.
1
sion of 9 into 11. H NMR spectroscopic analysis of the crude
cleavage products revealed that the amounts of both 3 and BSA
affected the yield of 11. The crude products contained only
varying amounts of the acryloylated peptide 9 in addition to 11.
When the addition reaction was run at 100 ЊC, using 12 equiv-
alents of 8 (1:3:BSA 1:12:36), conversion was complete in 4 h
Having established suitable reaction conditions for the model
system, the methodology was applied to the preparation
of a phosphinic peptide (Scheme 3). The peptide sequence
-
FAPFFG- was assembled on the POEPS-3 resin without the
1
use of a linker. The MSNT reagent was used to attach glycine
(as determined by H NMR, RP-HPLC (Fig. 2) and MALDI-
and standard Fmoc chemistry and pentafluorophenyl (Pfp)
TOF MS). Inconveniently long reaction times were required
when lower concentrations of 8 were used and the use of a
larger excess to BSA compared with 3 (1:4) increased the
conversion of 9 only marginally.
20,21
esters
were used in the subsequent couplings. The sequence
1
was selected such that reactions could be analysed by H NMR,
RP-HPLC and matrix-assisted laser desorption/ionisation
time-of-flight mass spectrometry (MALDI-TOF MS), after
cleavage of the crude product from the resin with NaOH. In
addition, amino acids containing side-chain-protective groups
were excluded to avoid competing reactions that could impair
structural analysis.
Removal of the Alloc group was effected with a solution of
17
Pd(PPh ) and N-ethylmorpholinium acetate in chloroform.
3
4
Subsequent amino acid couplings were performed using Fmoc-
protected amino acid Pfp esters with 3,4-dihydro-3-hydroxy-4-
oxo-1,2,3-benzotriazine (Dhbt-OH) as the catalyst. The coup-
ling of Fmoc-Phe-OPfp to the N-terminus of the phosphinic
peptide proved to be slow, but was complete within 16 h using 6
equivalents of Fmoc-Phe-OPfp, as determined by RP-HPLC
Acylation of the peptide N-terminal was carried out
using acryloyl chloride, since standard activation of acrylic
acid using O-(1H-benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyl-
3
308
J. Chem. Soc., Perkin Trans. 1, 2000, 3306–3310

View Article Online
analysis (50, 64, 84 and 100% complete after periods of ¼, ½,
and 16 h, respectively). Subsequent couplings with Fmoc-
protected amino acid pentafluorophenyl esters also required
extended reaction times.
The phosphinic undecapeptide 12 was cleaved from the
resin to afford a crude product which was essentially pure. Two
closely eluting peaks were visible by RP-HPLC, corresponding
to the two diastereomeric peptides resulting from the intro-
duction of the racemic Ala-Gly phosphinic dipeptide moiety
were 6 × 1 min unless otherwise stated. Minimum volume was
2
used for solid-phase reactions. Solid-phase reactions at elevated
temperatures were performed in 5 ml reaction vials (Pierce).
Amino (or hydroxy) group loadings were determined by
spectrophotometric determination of released piperidine–
9
dibenzofulvene adduct (290 nm) after coupling with Fmoc-
Gly-OPfp/DhbtOH (or Fmoc-Gly-OH/MSNT) and treatment
of a known sample with 20% piperidine in DMF.
(
Fig. 2). Interestingly, the two diastereomers were not resolved
Acryloylated POEPS-3 resin 1. 4-(Dimethylamino)pyridine
in the RP-HPLC chromatogram until after coupling of the
phenylalanine residue. Finally, purified phosphinic undeca-
peptide 12 was isolated by preparative RP-HPLC in 45% yield.
The yield and purity of undecapeptide 12 was comparable to
yields previously obtained by a more tedious building-block
(DMAP) (12.2 mg, 0.1 mmol) and Et N (277 µl, 2.0 mmol) were
3
dissolved in DCM (10 ml) and the solution was added to
13
POEPS-3 resin (500 mg, 0.1 mmol) which was allowed the
swell for 10 min. A solution of acryloyl chloride (163 µl, 2.0
mmol) in DCM (1 ml) was added to the swelled resin while it
was being stirred gently with a spatula. After a period of 2.5 h
the resin was drained and washed successively with DCM × 3,
MeOH × 3, DCM and lyophilised to obtain acryloylated resin
6
approach.
Conclusions
1
, δH,MAS(500 MHz; CDCl ; CHCl ) 4.30 [2 H, br t, J 4.7,
3 3
An efficient protocol for the solid-phase preparation of phos-
phinic peptides was developed. Conditions were established for
the reaction of a resin-bound acrylate or acrylamide with a
bis(trimethylsilyl) phosphonite derived from 1-(allyloxycarb-
onylamino)ethylphosphinic acid. The synthesis of a phosphinic
undecapeptide was performed in which all reactions were
conducted on solid support, including the Michael addition
reaction which generates the phosphinic pseudo-dipeptide
moiety. The final phosphinic peptide was isolated in high yield
and purity. Further studies will be directed towards applications
of this method for the combinatorial synthesis of phosphinic
peptides containing a large variety of both natural amino acids
as well as substitued acrylates and 1-aminoalkylphosphinic
acids.
C(O)OCH CH -OPEG], 5.82 (1 H, d, J 10.4, cis CH ᎐CHCO ),
2
2
2
2
6
.14 (1 H, dd, J 10.4 and 17.6, CH ᎐CHCO ), 6.41 (1 H, d,
2 2
J 17.6, trans CH ᎐CHCO ). Resonances corresponding to the
2
2
1
resin were also observed in the H NMR MAS spectrum.
1
-(Allyloxycarbonylamino)ethylphosphinic acid 3. 1-(Benz-
16
hydrylamino)ethylphosphinic acid 2 (5.00 g, 18.2 mmol) was
mixed with 48% aq. HBr (16.7 ml, 8.2 eq.) and brought to 126 ЊC
for 15 min. After cooling to room temperature the mixture was
concentrated in vacuo and mixed with water (25 ml) and DCM
(
25 ml). Disregarding the precipitate present at this time,‡ the
aqueous layer was washed with DCM (3 × 25 ml), adjusted to
pH 7 with aq. NaOH (10 M; 3 ml), stirred for 30 min at 0 ЊC,
and filtered. Solid Na CO (3.85 g, 2 eq.) was added to the
2
3
filtrate and allyl chloroformate (2.30 ml, 1.2 eq.) was added
dropwise at room temperature with vigorous stirring. Reaction
was complete in 30 min after which the pH was adjusted to
between 0 and 1 with aq. HCl (10 M; 4 ml). The mixture was
stirred 30 min at 0 ЊC, filtered, and NaCl (approx. 30 g) was
added to supersaturate the solution with NaCl. Extraction with
ethyl acetate (12 × 25 ml) was carried out while keeping the
aqueous phase at pH 0–1 at all times. The combined extracts
were dried with MgSO4 and concentrated to dryness. The
resulting partly crystallised product was re-dissolved in ethyl
acetate (50 ml) and the compound was precipitated by addition
of light petroleum (75 ml); cooling overnight to give phosphinic
acid 3 (2.47 g, 70%) as a white powder, mp 74–76 ЊC (Found: C,
Experimental
General
Most anhydrous solvents were obtained by storing analytical
quality solvents over 3 or 4 Å activated molecular sieves after
which the water content was verified to be below 30 ppm by
Karl Fischer titration. Anhydrous DMF was obtained by frac-
tionally distillation at reduced pressure and stored over 4 Å
molecular sieves. All commercial starting materials were used
without purification. Solution NMR data were acquired on a
Bruker 250 MHz Avance DRX 250 spectrometer and were ref-
1
1
erenced to CHCl (δ 7.24, H), HDO (δ 4.75, H) or H PO (δ 0,
3
3
4
P
3
7
7.6; H, 6.1; N, 6.9. C H NO P requires C, 37.3; H, 6.3; N,
31
6 12 4
P, external), and MAS NMR spectra were aquired on a Varian
.3%); δ (250 MHz; 0.1 M NaOD in D O; HDO) 1.23 (3 H, dd,
1
H
2
Inova 500 MHz with a 4 mm H-observe nano-probe and a
spinning rate of 2.1 kHz. Coupling constants J are given in Hz.
Analytical RP-HPLC was performed on a Waters system (490E
detector, using two 510 pumps with a gradient controller and
J 15.5 and 7.4, CH ), 3.62 (1 H, m, NHCHCH ), 4.57 (2 H, d,
3
3
J 4.6, CH ᎐CHCH O), 5.24 (1 H, d, J 10.6, cis CH ᎐CHCH O),
2
2
2
2
5
.31 (1 H, d, J 17.4, trans CH ᎐CHCH O), 5.94 (1 H, m,
2 2
CH ᎐CHCH O), 6.79 (1 H, d, J 517, PH); δ (250 MHz; 0.1 M
2
2
P
8
mm diameter RCM C18 column). Preparative RP-HPLC
NaOD in D O; H PO ) 27.0.
2
3
4
purification of phosphinic undecapeptide 12 was carried out on
a Waters system (991 photodiode array detector and 600 E sys-
tem controller) connected to a Waters 25 mm diameter RCM
C -column. All RP-HPLC procedures were carried out with a
Reaction of acryloylated resin 1 with 1-(allyloxycarbonyl-
amino)ethylphosphinic acid 3. BSA (45 µl, 180 µmol) was added
to a suspension of 3 (11.6 mg, 60 µmol) in degassed, anhydrous
solvent (1 ml, DCE) and the resulting solution was purged for 2
min with Ar. The solution was added to resin 1 (50 mg, 10
µmol) in a 5 ml reaction vial and purged with Ar for 1 min. The
vial was placed in a heating block at the reaction temperature
1
8
linear gradient. Buffers: A (0.1% TFA in water) and B (0.1%
TFA in MeCN–water 9:1), 50 min gradient of 2% B min .
MALDI-TOF mass spectra were acquired on a Bruker Reflex
III high-resolution mass spectrometer. Electrospray mass
spectra were obtained on a Fisons VG Quattro 5098 mass
Ϫ1
(
100 ЊC) for the required time (1 h). After reaction the resin was
Ϫ1
spectrometer (mobile phase 50% aq. MeCN, 8 µl min , sample:
washed successively DCM and MeOH and the product(s) was/
were cleaved by treatment with aq. NaOH (500 µl; 0.1 M) for
Ϫ1
1
0 µl, ≈20 pmol µl ). Light petroleum refers to the fraction
with distillation range 60–80 ЊC.
1
h. The solution was lyophilised and the product was dissolved
31 1
in D O and analysed by P and H NMR. The optimum reac-
2
Solid-phase synthesis procedures
α
Deprotection of N Fmoc was performed with piperidine (20%
in DMF) for 2 and 10 min. Volumes of washing solvent were
‡
The precipitate was an aromatic compound not related to the 1-amino-
1
–2-times the volume necessary to swell the resin and washings
ethylphosphinic acid.
J. Chem. Soc., Perkin Trans. 1, 2000, 3306–3310
3309

View Article Online
tion conditions/amounts are given in parentheses. Under these
conditions the following analytical data were obtained (Fig. 1).
δ (250 MHz; 0.1 M NaOD in D O; HDO) 1.24 (3 H, dd, J 13.2
and MeOH. The product was cleaved from the resin by treat-
ment with NaOH (400 µl; 0.1 M), and the resin was washed
successively with water and MeOH. Aq. HCl (400 µl, 0.1 M)
was added to the combined filtrate and the solvents were
removed in vacuo. The solid residue (10 mg) was re-dissolved in
50% aq. MeCN (2 ml) and purified by preparative RP-HPLC.
Fractions containing the product were collected, and concen-
trated to dryness in vacuo to obtain phosphinic undecapeptide
LMF-AlaΨ{PO H-CH }Gly-FAPFFG 12 as a solid mixture of
H
2
and 7.2, CH ), 1.76 (2 H, dd, J 12.2 and 6.0, PCH ), 2.31 (2 H,
3
2
dd, J 11.9 and 6.9, CH CO), 3.70 (2 H, m, CHMeP), 4.56 (2 H,
2
d, J 4.7, CH ᎐CHCH O), 5.24 (1 H, d, J 11.0, cis CH ᎐CH-
2
2
2
CH O), 5.30 (1 H, d, J 18.0, trans CH ᎐CHCH O), 5.97 (1 H,
2
2
2
m, CH ᎐CHCH O); δ (250 MHz; 0.1 M NaOD in D O; H PO )
2
2
P
2
3
4
ϩ
4
2
1.6; m/z (ES-MS) 265.9 ([M ϩ H] . C H NO P requires m/z
9 17 6
ϩ
66.1), 287.9 ([M ϩ Na] . C H NNaO P requires m/z 288.1).
2
2
two diastereomers (5 mg, 45% based on the initial resin load-
ing or 90% based on loading of Fmoc-FAPFFG-O-POEPS-3);
9
16
6
13
Phosphinic undecapeptide 12. POEPS-3 resin (1.5 g, 0.3
RP-HPLC (2 peaks, 36.7 and 37.0 min). δ (250 MHz; 0.1 M
P
mmol) was swelled in anhydrous DCM (50 ml) and drained
after a period of 10 min. A solution of Fmoc-Gly-OH (267 mg,
NaOD in D O; H PO ) 39.2 and 39.7; m/z (MALDI-TOF)
2
3
4
ϩ
1240.0 ([M ϩ H] . C H N O PS requires m/z, 1239.6),
62
84 10 13
ϩ
0
.9 mmol) and N-methylimidazole (54 µl, 0.675 mmol)
in anhydrous DCM (30 ml) was mixed with MSNT (267 mg,
.9 mmol) and added to the resin. After 60 min the resin
1262.0 ([M ϩ Na] . C H N NaO PS requires m/z, 1261.6),
62 83 10 13
ϩ
1278.0 ([M ϩ K] . C H KN O PS requires m/z, 1277.6).
62
83
10 13
0
Amino acid analysis: Gly 1.04, Ala 1.02, Met 0.93, Leu 0.95,
was drained, washed with anhydrous DCM × 3, and another
coupling of Fmoc-Gly-OH/MSNT/N-methylimidazole was
performed for 60 min. The resin was drained and washed suc-
cessively with DCM, DMF × 3, (5% diisopropylethylamine in
Phe 4.10, Pro 0.95 (LMF-AlaΨ{PO H-CH }Gly-FAPFFG
requires Gly 1, Ala 1, Met 1, Leu 1, Phe 4, Pro 1).
2
2
Acknowledgements
DMF) × 2, DMF × 3, and DCM, and lyophilised. Loading:
Ϫ1
0
.20 mmol g . A fraction of the resin (840 mg, 0.167 mmol)
This work has been supported by VækstFonden through
Osteopro A/S (Denmark, www.osteopro.dk) and by the
SPOCC program under the Danish National Research
Foundation. Dr Charlotte Gotfredsen is kindly thanked for
was treated with piperidine to remove Fmoc, washed with
DMF, and treated with a solution of Fmoc-Phe-OPfp (278 mg,
0
1
.501 mmol) and Dhbt-OH (27 mg, 0.167 mmol) in DMF for
h. Similarly, Fmoc-protected amino acid Pfp esters of Phe,
1
acquiring MAS H NMR spectra.
Pro, Ala, and Phe were coupled using Dhbt-OH until a negative
Kaiser ninhydrin test was observed (amounts Fmoc-Pro-OPfp:
Ϫ1
2
53 mg, Fmoc-Ala-OPfp: 240 mg). Loading: 0.10 mmol g . A
References
small aliquot of the resin was treated with piperidine, washed
with DMF × 3 and with MeOH × 3 and treated with NaOH
0.1 M) to give hexapeptide FAPFFG. RP-HPLC (1 peak,
9.3 min); m/z (MALDI-TOF) 685.7 ([M ϩ H] . C H N O
37 45 6 7
ϩ
requires m/z, 685.3), 707.8 ([M ϩ Na] . C H N NaO requires
1
M. C. Allen, W. Fuhrer, B. Tuck, R. Wade and J. M. Wood, J. Med.
Chem., 1989, 32, 1652.
P. A. Bartlett, J. E. Hanson and P. P. Giannousis, J. Org. Chem.,
1990, 55, 6268.
(
2
2
ϩ
3 C. Verbruggen, S. De Craecker, P. Rajan, X.-Y. Jiao, M. Borloo,
K. Smith, A. H. Fairlamb and A. Haemers, Bioorg. Med. Chem.
Lett., 1996, 6, 253.
37
44
6
7
m/z, 708.3).
A fraction of the resin (200 mg, 40 µmol) was treated with
piperidine and washed successively with DMF and DCM. Neat
Et N (111 µl, 800 µmol) was added to the resin followed by a
solution of acryloyl chloride (33 µl, 400 µmol) in DCM. The
reaction was complete within 5 min according to Kaiser
ninhydrin test. The resin was drained, washed successively with
DCM and MeOH × 3, and lyophilised. A small aliquot of the
resin was treated with NaOH (0.1 M) to give acryloylated
hexapeptide Acr-FAPFFG 9 RP-HPLC (1 peak, 39.0 min);
4
I. Yiallouros, S. Vassiliou, A. Yiotakis, R. Zwilling, W. Stöcker and
V. Dive, Biochem. J., 1998, 331, 375.
3
5 M. Whittaker, C. D. Floyd, P. Brown and A. J. H. Gearing, Chem.
Rev., 1999, 99 , 2735.
6 J. Buchardt, M. Ferreras, C. Krog-Jensen, J.-M. Delaissé, N. T.
Foged and M. Meldal, Chem. Eur. J., 1999, 5, 2877.
7
S. Vassiliou, A. Mucha, P. Cuniasse, D. Georgiadis, K. Lucet-
Levannier, F. Beau, R. Kannan, G. Murphy, V. Knäuper, M.-C.
Rio, P. Basset, A. Yiotakis and V. Dive, J. Med. Chem., 1999, 42,
2
610.
ϩ
m/z (MALDI-TOF) 761.8 ([M ϩ Na] . C H N NaO requires
8 A. Yiotakis, S. Vassiliou, J. Jirácek and V. Dive, J. Org. Chem., 1996,
61 , 6601.
40
46
6
8
m/z, 761.3).
9
G. Fields, Z. Tian and G. Barany, in Synthetic Peptides: A Users
Guide, ed. G. A. Grant, W. H. Freeman and Company, New York,
A fraction of the resin (50 mg, 10 µmol) was placed in a 5 ml
reaction vial and a solution of 3 (23.2 mg, 120 µmol) and BSA
90 µl, 360 µmol) in degassed DCE (1 ml) was added to the
1
992, p. 77.
(
1
0 J. K. Thottathil, D. E. Ryono, C. A. Przybyla, J. L. Moniot and
resin. The resin slurry was purged with Ar for 1 min, and the
vial was closed and heated to 100 ЊC for 4 h, after which it was
cooled to rt and washed with DCM and lyophilised. A small
aliquot of the resin was washed with MeOH and treated with
NaOH (0.1 M) to give phosphinic octapeptide Alloc-AlaΨ-
R. Neubeck, Tetrahedron Lett., 1984, 25, 4741.
11 E. A. Boyd, M. Corless, K. James and A. C. Regan, Tetrahedron
Lett., 1990, 31, 2933.
2 P. H. Dorff, G. Chiu, S. W. Goldstein and B. P. Morgan, Tetrahedron
Lett., 1998, 39, 3375.
3 J. Buchardt and M. Meldal, Tetrahedron Lett., 1998, 39, 8695.
4 M. Grötli, C. H. Gotfredsen, J. Rademann, J. Buchardt, A. J. Clark,
J. Duus and M. Meldal, J. Comb. Chem., 2000, 2, 108.
15 B. Blankemeyer-Menge, M. Nimtz and R. Frank, Tetrahedron Lett.,
1990, 31, 1701.
6 E. K. Baylis, C. D. Campbell and J. G. Dingwall, J. Chem. Soc.,
Perkin Trans. 1, 1984, 2845.
1
1
1
{
PO H-CH }Gly-FAPFFG 11. RP-HPLC (1 peak, 35.6 min);
2 2
ϩ
m/z (MALDI-TOF) 932.7 ([M ϩ H] . C H N O P requires
m/z, 932.4), 954.8 ([M ϩ Na] . C H N NaO P requires m/z,
9
46
59
7
12
ϩ
4
6
58
7
12
ϩ
54.4), 970.7 ([M ϩ K] . C H KN O P requires m/z, 970.4).
46 58 7 12
1
A solution of Pd(PPh ) (32 mg, 30 µmol) in CHCl –AcOH–
3
4
3
N-ethylmorpholine 92.5:5:2.5 (800 µl) was added to the resin
and left for 15 min. The resin was washed successively with
1
7 S. A. Kates, S. B. Daniels and F. Albericio, Anal. Biochem., 1993,
2
12, 303.
CHCl , DMF × 3, (0.5% Et NCS Na in DMF) × 3 and DMF.
3
2
2
18 M. Meldal, in Solid Phase Peptide Synthesis (Methods in
Enzymology Vol 289), ed. G. Fields, Academic Press, New York,
1997, p. 83.
A solution of Fmoc-Phe-OPfp (33 mg, 60 mmol) and Dhbt-OH
0.6 mg, 10 µmol) in DMF was added to the resin and allowed
(
1
2
9 W. Li and B. Yan, J. Org. Chem., 1998, 63, 4092.
0 E. Atherton and R. C. Sheppard, J. Chem. Soc., Chem. Commun.,
to react for a period of 16 h. Similarly, Fmoc-protected amino
acid Pfp esters of Met and Leu were coupled using Dhbt-OH
1
985, 165.
(
amounts Met: 16 mg, 30 µmol and Leu: 16 mg, 30 µmol). After
2
2
1 M. Bodansky and M. A. Bednarek, J. Protein Chem., 1989, 8, 461.
2 R. Knorr, A. Trzeciak, W. Bannwarth and D. Gillesen, Tetrahedron
Lett., 1989, 30, 1927.
the final coupling the resin was washed with DMF, treated with
piperidine to remove Fmoc, and washed successively with DMF
3
310
J. Chem. Soc., Perkin Trans. 1, 2000, 3306–3310