Multigram-scale synthesis of short peptides via a simplified repetitive solution-phase procedure

Article abstract of DOI:10.1021/jo902116p

(Chemical Equation Presented) A rapid repetitive solution-phase synthesis of peptides is described. The procedure involves coupling of amino acids and peptide acids, instead of the usual amino esters and peptide esters, to slight excesses of pentafluoroph

Full text of DOI:10.1021/jo902116p

pubs.acs.org/joc
Multigram-Scale Synthesis of Short Peptides via a Simplified Repetitive
Solution-Phase Procedure
ꢀ
Celia Meneses, Sarah L. Nicoll, and Laurent Trembleau*
School of Natural and Computing Sciences, University of Aberdeen, Meston Building, Aberdeen, AB24 3UE
l.trembleau@abdn.ac.uk
Received February 9, 2009
A rapid repetitive solution-phase synthesis of peptides is described. The procedure involves coupling
of amino acids and peptide acids, instead of the usual amino esters and peptide esters, to slight
excesses of pentafluorophenyl active esters in a THF/water solvent mixture. Due to their poor
solubility, peptide acid intermediates are easily isolated in high purity by acidification under
controlled conditions and removal of excess active esters by selective extraction. Contrary to modern
repetitive solution-phase peptide synthesis procedures, our approach does not require time-consum-
ing neutralization reactions and reduces significantly the number of operation units that are
necessary to obtain peptide intermediates. Efficiency of the method was demonstrated by the rapid
synthesis of short hydrophobic and hydrophilic peptides, the antimalarial cycloheptapeptide
mahafacyclin B, and a protected form of the hydrophilic pentapeptide GRGDS.
Introduction
peptide Atosiban, an antagonist of the oxytocin receptor,
utilizes five different coupling methods.3 The second syn-
thetic method, Merrifield “solid-phase peptide synthesis”
(SPPS), is the fastest way of producing peptides.4 However,
when applied to the production of multiton quantities the
method is costly and generates large amounts of chemical
waste.5 Recently, SPPS was used in combination with CSPS
for the large-scale production of Fuzeon, a 36-amino acid
long peptide that inhibits cell infection by the HIV virus. The
peptide fragments were produced on solid phase and subse-
quently assembled in solution.6 The third synthetic method
known as “repetitive (or continuous) solution-phase peptide
The development of rapid and efficient synthesis protocols
has been a key factor in allowing the pharmaceutical poten-
tial of peptides to be realized. Development has proceeded
such that in 2004 there were 40 peptide based drugs on the
market with a further 200 in clinical phases.1
Peptide drugs possess a significant advantage over other
small synthetic molecules as they are usually highly potent
and decompose into safe metabolites in vivo. Recent ad-
vances in synthetic and drug delivery technologies have
proved critical to lowering their production cost and coun-
teracting their poor oral bioavailability.2 Today, there are
three distinct chemical methods used for the manufacture of
peptides. The “classical solution-phase peptide synthesis”
(CSPS) involves the use of optimized coupling reactions and
purification techniques for each step. Consequently, deve-
lopment of synthetic routes for new peptides and the sub-
sequent scale-up can be a very time-consuming process.
For instance, the industrial production of the nine-residue
(3) Johansson, C.; Blomberg, L. Hlebowicz, W.; Nicklasson, H. Nilsson,
B. Andersson, L. Industrial production of an oxytocin antagonist (Atosiban):
Synthetic approaches to the development of multi-kilogram scale solution
synthesis. In HLS Maia, ed. Peptides; Proceedings of the 23rd European
Peptide Symposium, 1995; pp 34-35.
(4) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149. Merrifield, R. B.
Solid-Phase Pept. Synth. 1997, 289, 3–13. Barany, G.; Merrifield, R. B. Solid-
phase peptide synthesis. In The Peptides; Gross, E., Meienhofer, J., Eds.;
Academic Press: New York, 1979; Vol. 2, pp 1-284. Alsina, J.; Albericio, F.
Biopolymers 2003, 71, 454–477.
(5) Bruckdorfer, T.; Marder, O.; Albericio, F. Curr. Pharm. Biotechnol.
2004, 5, 29–43.
(1) Albericio, F. Curr. Opin. Chem. Biol. 2004, 8, 211–221.
(2) For recent reviews, see: Adermann, K.; John, H.; Standker, L.;
Forssmann, W.-G. Curr. Opin. Biotechnol. 2004, 15, 599–606. Pomilio, A.
B.; Battista, M. E.; Vitale, A. A. Curr. Org. Chem. 2006, 10, 2075–2121.
Hamada, Y.; Shioiri, T. Chem. Rev. 2005, 105, 4441–4482.
(6) Schneider, S. E.; Bray, B. L.; Mader, C. J.; Friedrich, P. E.; Anderson,
M. W.; Taylor, T. S.; Boshernitzan, N.; Niemi, T. E.; Fulcher, B. C.; Whight,
S. R.; White, J. M.; Greene, R. J.; Stoltenberg, L. E.; Lichty, M. J. Pept. Sci.
2005, 11, 744–53.
564 J. Org. Chem. 2010, 75, 564–569
Published on Web 12/31/2009
DOI: 10.1021/jo902116p
r
2009 American Chemical Society

Meneses et al.
JOCArticle
SCHEME 1. Synthesis of Peptides by (a) Typical Repetitive Solution-Phase Peptide Synthesis (RSPS); (b) Proposed Simplified
Procedure
synthesis” (RSPS) is a methodology that aims to produce
peptides via a repetitive procedure without the need for
purification of intermediates.7 The advantage of this ap-
proach is that the same coupling and isolation procedures are
repetitively used, thus simplifying peptide production
(Scheme 1a). One of the key features of this method is the
utilization of superstoichiometric amounts of activated ami-
no acids to drive reactions to completion, an approach
similar to that utilized in SPPS. However, unlike SPPS,
excesses cannot be removed by simple washing and filtration
procedures. Instead, water-soluble nucleophilic reagents are
added to the reaction mixture to neutralize the excess of
activated species, after which the byproducts are removed by
acidic and basic aqueous extractions. The N-protecting
group is subsequently cleaved, and the pure deprotected
peptide obtained after additional aqueous washings. The
coupling cycles are repeated until the desired peptide se-
quence has been reached. Recently, some of these techniques
have been automated, most notably the DioRaSSP proce-
dure as reported by Eggen et al.8 In comparison to SPPS,
RSPS procedures use significantly lower quantities of ex-
pensive coupling reagents and produce less waste, which is
particularly advantageous for synthesis on a larger scale.
Additionally, RSPS techniques are less likely to lead to
truncated peptides when synthesizing challenging sequences,
but insertion sequences may arise if removal of activated
amino acids after the coupling step is incomplete.
In this paper, we describe a simplified repetitive solution-
phase synthesis of peptides (RSPS) using Boc-protected
pentafluorophenyl esters of amino acids as the activated
species for coupling reactions. The new method is illustrated
by the synthesis of small hydrophobic and hydrophilic
peptides and mahafacyclin B, a natural cycloheptapeptide
with antimalarial properties.9
Methodology
The main weaknesses of RSPS are 2-fold: (1) additional
reactions are necessary to neutralize the excess of coupling
reagent employed and (2) several acidic and basic aqueous
extractions are required to isolate the amino peptide esters in
high purity (Scheme 1a). These postsynthetic treatments are
time-consuming and undesirable. Our new procedure aims
to decrease significantly the number of unit operations that
are necessary for each coupling cycle, providing a rapid and
facile method of peptide synthesis.
A major problem in repetitive methods is the separation of
the peptide products from the excess of activated amino acids
and byproduct. We envisioned that this problem could be
easily solved if we used hydrophobic active esters and
produced hydrophilic peptide products (Scheme 1b). This
way, excess active esters would remain soluble in nonpolar
solvents but the peptide products would easily precipitate.
Boc-protected pentafluorophenyl esters seemed the ideal
candidate for use as the activated amino acid component,
because they are hydrophobic, easily accessible in pure form,
(7) For examples see: Sheehan, J. C.; Preston, J.; Cruickshank, P. A. J.
Am. Chem. Soc. 1965, 87, 2492–2493. Nozaki, S.; Muramatsu, I. Bull. Chem.
Soc. Jpn. 1982, 55, 2165–2168. Carpino, L. A.; Cohen, B. J.; Stephens, K. E.;
Sadat-Aalaee, J.-H. T.; Langridge, D. C. J. Org. Chem. 1986, 51, 3734–3736.
Carpino, L. A.; Sadat-Aalaee, D.; Beyermann, M. J. Org. Chem. 1990, 55,
1673–1675. Carpino, L. A.; Ismail, M.; Truran, G. A.; Mansour, E. M. E.;
Iguchi, S.; Ionescu, D.; El-Faham, A.; Riemer, C.; Warrass, R. J. Org. Chem.
1999, 64, 4324–4338. Carpino, L. A.; Ghassemi, S.; Ionescu, D.; Ismail, M.;
Sadat-Aalaee, D.; Truran, G. A.; Mansour, E. M. E.; Siwruk, G. A.; Eynon,
J. S.; Morgan, B Org. Process Res. Dev. 2003, 7, 28–37.
(8) Eggen, I. F.; Bakelaar, F. T.; Petersen, A.; Ten Kortenaar, P. B. W.
Org. Process Res. Dev. 2005, 9, 98–101. For further examples see: Eggen, I.
F.; Bakelaar, F. T.; Peterson, A.; Kortenaar, P. B. W.; Ankone, N. H. S.;
Bijsterveld, H. E. J. M.; Bours, G. H. L.; El Bellaj, F.; Hartsuiker, M. J.;
Kuiper, G. J.; Ter Voert, E. J. M. J. Pept. Sci. 2005, 11, 633–641.
(9) Baraguey, C; Blond, A.; Cavelier, F.; Pousset, J.-L.; Bolo, B.; Auvin-
Guette, C. J. Chem. Soc., Perkin Trans. 1 2001, 2098–2103.
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SCHEME 2
reached completion after 5 h stirring at room temperature.
Excess amino acid and protonated base were conveniently
removed by washing the organic layer with a 10% aqueous
solution of citric acid (pH 3). The organic solvent was dried
and evaporated to give the crude peptide product, contami-
nated only by pentafluorophenol. While the dipeptide pro-
duct is insoluble in petroleum ether, pentafluorophenol is
soluble, thus allowing its facile removal after three sonica-
tion extractions with this solvent. Ultrasonic-assisted extrac-
tion (USE) has been shown to be an effective method for
analytical extractions; hence, it is used here to facilitate
sample purification.13 Samples were sonicated by indirect
sonication using a sonic bath. The product was isolated in
92% yield in high purity (entry 1). Importantly, NMR and
HPLC analyses indicated that no epimerization had taken
place during coupling. The encouraging results of this initial
experiment were short-lived: reaction of the same active ester
with glycine required 10 h reaction to reach completion
(entry 2), and almost no reaction was observed when the
more hydrophilic (and therefore less soluble) amino acid
threonine was used (entry 3). Attempts to increase the amino
acid solubility and reaction rates in various polar solvents
did not give satisfactory results. In a final attempt, the
reaction was carried out in a THF-water (∼2:1) solvent
mixture.14 In this homogeneous system, the amino acid
and active ester are highly soluble, even in the presence of
TABLE 1. Reaction Times and Yields of Peptides Synthesized under
Varying Solvent Conditions
reaction
time
(h)
yielda
(%)
entry
product
solvent
DCM
DCM
1
2
3
4
5
6
7
8
9
Boc-L-Phe-L-Phe-OH, 1
Boc-L-Phe-Gly-OH, 2
Boc-L-Val-L-Thr-OH, 3
Boc-L-Phe-L-Phe-OH, 1
Boc-L-Phe-Gly-OH, 2
Boc-L-Phe-L-Thr-OH, 4
Boc-L-Ser(Bzl)-Gly-OH, 5
5
92
89
traces
97
98
92
94
93
84
10
24
2
1.5
2
2
2
6
DCM
THF/H2O
THF/H2O
THF/H2O
THF/H2O
Boc-L-Cys(Bzl)-Gly-OH, 6 THF/H2O
Boc-L-Val-L-Thr-OH, 3 THF/H2O
aEpimerization level was <99.9% as demonstrated by chiral HPLC.
stable to storage and known to rapidly undergo aminolyzes
with amino acids to yield epimerization-free peptide pro-
ducts.10,11 Reacting pentafluorophenyl esters with free ami-
no acids or side-chain protected peptide acids would produce
Boc-protected peptide acid products, which should be hy-
drophilic enough to be poorly soluble in nonpolar organic
solvents like petroleum ether or diethyl ether. Excess active
esters and the hydrophobic byproduct could be then easily
removed from the precipitated peptide acid product. The
coupling cycle would be repeated after removal of the Boc-
protecting group until the desired peptide sequence has been
obtained.
€
the Hunig’s base. To our satisfaction, coupling reactions are
much faster than hydrolysis of active esters; hence, a series
of dipeptide products could be prepared in high yields
SCHEME 3. Approach for the Synthesis of Mahafacyclin B
Results and Discussion
Synthesis of Boc-Protected Dipeptide Acids/Optimization
of the Method. The coupling conditions were optimized for
the synthesis of a series of dipeptides (Scheme 2, Table 1).
The Boc-protected active esters were readily obtained by
coupling the corresponding Boc-amino acids with penta-
fluorophenol (PfpOH) in the presence of 1-[3[(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (EDC
3
HCl).12 We found that pure active esters could be readily
purified in excellent yields by filtration of the crude mixture
containing the urea byproduct through a short pad of silica
gel. The pentafluorophenyl esters were then reacted with a
variety of amino acids, and the reactions were monitored
by thin layer chromatography. In these experiments,
substoichiometric amounts of active esters were used as
excesses of amino acids would be easily removed by aqueous
extraction.
Amino acids are poorly soluble in organic solvents, so one
would expect coupling reactions to be very slow or non
existent. To our surprise, Boc-L-Phe-OPfp was found to
react with phenylalanine (suspension) in dichloromethane
€
(DCM) in the presence of Hunig’s base, and the reaction
(13) Priego-Capote, F.; Luque de Castro, M. D. Trends Anal. Chem. 2004,
23, 644–653. Santos, D., Jr.; Krug, F. J.; de Godoi Pereira, M.; Korn, M.
Appl. Spectrosc. Rev. 2006, 41, 305–321.
(10) Kovacs, J.; Kisfaludy, L.; Ceprini, M. Q. J. Am. Chem. Soc. 1967, 89,
183.
(11) Jones, J. In The chemical synthesis of peptides; International Series of
Monographs on Chemistry; Claredon Press: Oxford, 1994. Rajappan, V. P.;
Hosmane, R. S. Synth. Commun. 1998, 28, 753–764.
(14) For examples on the use of solvent mixtures in peptide synthesis see:
Hiskey, R.; Beacham, L. M.; Matl, V. G.; Smith, J. N.; Williams, E. B.;
Thomas, A. M.; Woters, E. T. J. Org. Chem. 1971, 36, 488–490. Ho, G.-J.;
Nozaki, S.; Muramatsu, I. Bull. Chem. Soc. Jpn. 1982, 55, 2165–2168.
Emerson, K. M.; Mathre, D. J.; Shuman, R. F.; Grabowski, E. J. J. J.
Org. Chem. 1995, 60, 3569–3570.
(12) Maier, T. C.; Podlech, J. Eur. J. Org. Chem. 2004, 4379–4386.
566 J. Org. Chem. Vol. 75, No. 3, 2010

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SCHEME 4. Synthesis of Mahafacyclin B through Rapid Repetitive Solution-Phase Peptide Synthesis
and excellent purity in significantly shorter reaction times
(Table 1, entries 4-9). Reactions of active esters of Boc-
amino acids with high susceptibility to racemization15 af-
forded the corresponding dipeptides in good yields without
stereomutation (entries 7 and 8). In contrast to earlier reac-
tions in DCM, both hydrophilic and hydrophobic amino acids
reacted equally well in this solvent system (entries 4 and 9).
Total Synthesis of Mahafacyclin B. The results above
allowed us to design a new rapid repetitive solution-phase
synthesis of peptides (RRSPS). In this new methodology,
Boc-protected dipeptide acids are synthesized as before using
a superstoichiometric amount of free amino acid to drive the
coupling reactions to completion. Extension of the peptide
sequence then proceeds by reaction of the deblocked dipep-
tide with an excess of activated Boc-amino acid in a
THF-water mixture. After evaporation of the organic
solvent and acidification of the aqueous solution, the tripep-
tide acid product is precipitated during ultrasound-assisted
extraction of the remaining active ester. The procedure is
repeated until the desired peptide sequence is obtained.
The versatility and simplicity of the method was demon-
strated by the synthesis of the bioactive cycloheptapeptide
mahafacyclin B (Scheme 3). The natural cyclopeptide con-
tains two consecutive Phe-Phe-Gly fragments followed by a
threonine residue. We decided to synthesize the heptapeptide
precursor 12 (H-Thr-Phe-Phe-Gly-Phe-Phe-Gly-OH) via
fragment condensation of tripeptide H-Phe-Phe-Gly 7, with
Boc-protected tetrapeptide 9, which was to be obtained by
coupling of a threonine residue to 7. The peptide fragments
were synthesized using the new RRSPS (Scheme 4).
without protecting the alcohol side chain. This should not be
necessary if the side chains of polar amino acids were
protected with hydrophobic groups. This peptide was sub-
sequently activated using EDC HCl and PfpOH and
3
coupled to the deblocked tripeptide 7 in THF-water to
produce 87% of Boc-protected heptapeptide 11. Reversed-
phase HPLC and NMR analysis of the deblocked heptapep-
tide 12 (Figure 1) showed that even after four RRSPS cycles
no purification was required. Finally, mahafacyclin B was
obtained in 63% yield (27 mg, 24% overall yield) after
purification by preparative HPLC by cyclization using
HBTU in the presence of triethylamine in DMF. NMR
spectra of the cyclopeptide were identical to those described
for the natural product (see Supporting Information).16
FIGURE 1. HPLC analysis (reversed-phase column) of crude hep-
tapeptide 12, synthesized by RRSPS.
Multigram-Scale Synthesis of a Protected Form of the
Hydrophilic Pentapeptide GRGDS. Having successfully pre-
pared short hydrophobic peptides by RRSPS, we undertook
the multigram-scale synthesis of the Boc-protected hydro-
philic peptide model Boc-Gly-L-Arg(Cbz)2-Gly-L-Asp(Bzl)-
L-Ser(Bzl)-OH (BocGRGDS, 14), for which the side chain of
the arginine (R) residue is protected by Cbz groups, and the
aspartic acid (D) and serine (S) residues are protected with
benzyl groups (Scheme 2).17
The synthesis started with the hydrochloride salt of the
benzyl protected serine 15 and the desired pentapeptide
sequence BocGRGDS 14 was obtained in over 90% purity
(Figure 2) after four cycles of RRSPS using only 1.1 equiv of
appropriate pentafluorophenyl amino esters (Scheme 5). The
Dipeptide acid 2 was obtained in 98% yield by reaction of
glycine with Boc-Phe-OPfp. Deblocking of the Boc group
using HCl 4 M in dioxane followed by reaction with excess of
the same active ester afforded Boc-protected tripeptide 8 in
90% yield after ultrasound-assisted extraction with petro-
leum ether. The Boc group was then cleaved and the resulting
peptide reacted with activated Boc-threonine to produce 9 in
76% yield. The lower yield obtained in this coupling is
explained by the fact that a more polar petroleum ether/
ethyl acetate solvent system was required for the ultrasound
extractions of Boc-Thr-OPfp, because we chose to use it
(16) Sayyadi, N.; Sropeta, D.; Jolliffe, K. A. Org. Lett. 2005, 24, 5497–
5499.
(15) Li, H.; Jiang, X.; Ye, Y.-H.; Fan, C.; Romoff, T.; Goodman, M. Org.
Lett. 1999, 1, 91–93.
(17) The GRGDS peptide is an inhibitor of angiogenesis: Nicosia, R. F.;
Bonanno, E. Am. J. Pathol. 1991, 138, 829–833.
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SCHEME 5. Synthesis of BocGRGDS 14 through Rapid Repetitive Solution-Phase Peptide Synthesis
yields for each coupling step were in excess of 85%, and the
protected pentapeptide was obtained in 67% overall yield
(nonoptimized) after 7 steps.
ultrasound cleaning bath by indirect sonication at the frequency
of 40 kHz. Reactions were monitored by thin-layer chromato-
graphy using a mixture of 1% acetic acid in ethyl acetate.
Abbreviations: PfpOH, pentafluorophenol; EDC HCl, 1-[3-
3
[(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride;
DIEA, N,N-diisopropylethylamine; HBTU, (O-benzotriazol-
1-yl)-N,N,N0,N0- tetramethyluronium hexafluorophosphate;
Bzl, benzyl; Cbz, carboxybenzyl; USE, ultrasonic-assisted
extraction; NMR, nuclear magnetic resonance; HPLC, high
performance liquid chromatography; RRSPS, rapid repetitive
solution-phase peptide synthesis.
Synthesis of Pentafluorophenyl Esters (Procedure A). PfpOH
(1 equiv) was added to a stirred solution of Boc-amino acid/
peptide in dichloromethane (0.2 M) followed by EDC HCl
3
3
(1.2 equiv). After 2 h, silica (10 times the mass of EDC HCl
used) was added to the solution, and the resulting suspension
was filtered over a bed of silica and Celite. The solvent was
removed under reduced pressure and the crude pentafluorophe-
nyl ester used directly in the next step.
FIGURE 2. HPLC analysis (chiral column) of crude pentapeptide
BocGRGDS 14, synthesized by RRSPS.
Synthesis of Boc-AA2-AA1-OH (Procedure B). Boc-AA2-
OPfp (1 equiv) in THF (0.2 M) was added to an aqueous
solution (1 M) of AA1 (2 equiv) with constant stirring. DIEA
(2 equiv) was then added, and the reaction left to proceed. Once
the reaction had reached completion (TLC) the THF was
removed under reduced pressure, the aqueous mixture acidified
to pH = 3 using a 10% solution of citric acid and extracted with
ethyl acetate. The combined extracts were dried (MgSO4) and
the solvent evaporated under reduce pressure. The crude was
subjected to USE with petroleum ether or diethyl ether to give
the desired dipeptide.
Conclusions
We have developed an efficient and rapid method for
solution-phase synthesis of short peptides. Compared to
other existing solution-phase methods, our approach has
the advantage of not requiring fastidious and time-consum-
ing neutralization and washing steps. The THF-water
solvent system utilized for coupling reactions seems advan-
tageous over most nonpolar and polar solvents used in
peptide synthesis as peptides appeared very well soluble,
and THF is easily removed by rotary evaporation. In this
study, peptides were produced in good to excellent yields on
a multigram-scale. Vitally, the repetitive method was shown
to be epimerization-free and provide peptide intermediates
of high purity. The method proved applicable to the synth-
esis of both hydrophobic and hydrophilic peptides se-
quences, as demonstrated by the rapid synthesis of the
cyclic heptapeptide mahafacyclin B and a protected form
of the pentapeptide GRGDS.
Synthesis of Mahafacyclin B 13 and Boc-Gly-L-Arg(Cbz)2-
Gly-L-Asp(Bzl)-Gly-L-Ser(Bzl)-OH 14
Boc Deprotection (Procedure C). Typically, Boc-peptide
(1.2 mmol) was added to an ice-cold solution of 4 M HCl in
dioxane (10 mL). The reaction mixture was allowed to warm to
room temperature and stirred for 2 h. The solvent was removed
in vacuo and the resulting ammonium chloride salt was washed
with dry diethyl ether (3 ꢀ 15 mL) and collected by filtration.
The deblocked peptide was used in the coupling step without
further purification.
Coupling Protocol (Procedure D). A solution (0.1 M) of the
Boc-protected amino acid (or peptide fragment) pentafluoro-
phenyl ester (1.1-1.5 equiv) in THF was added to an aqueous
solution (or suspension; 0.2 M) of deblocked peptide (1 equiv)
containing 2-2.1 equiv of DIEA (pH aqueous solution = 8.5-
9), resulting in a homogeneous mixture. The progress of the
reaction was monitored by TLC (silica gel). Once the reaction
had reached completion (ca. 3 h), the THF was removed in
vacuo, and the aqueous mixture acidified to pH = 3 using a 10%
citric acid solution resulting in the precipitation of the Boc-
protected peptide. The product was extracted three times with
ethyl acetate, the combined extracts were dried over MgSO4,
and the solvent was evaporated under reduce pressure. The
product, contaminated by PfpOH and the excess active ester,
was subjected to USE with petroleum ether or diethyl ether (3ꢀ)
to give the purified Boc-protected peptide acid, which was
collected by filtration.
Experimental Section
Chiral HPLC was performed using a cellulose tris-3,5-di-
methylphenyl carbamate-coated column (4.6 mm ID, 250 mm)
with a flow rate of 1 mL/min, using a 0.1% TFA þ 10%
isopropanol in hexane solvent system for Boc-protected dipep-
tides and a 0.1% TFA þ 50% isopropanol in hexane solvent
system for Boc-GRGDS pentapeptide 14. Analytical reversed-
phase HPLC was performed using a C18 column (4.6 mm ID,
150 mm) with a flow rate of 0.8 mL/min. Semipreparative
reversed-phase HPLC was performed using a C18 column
(10 mm ID, 250 mm) with a flow rate of 1.3 mL/min.
NMR spectra were recorded on 250 and 400 MHz spectro-
meters. Melting points are uncorrected and reported in degrees
Celsius. Ultrasound-assisted extraction was performed in an
568 J. Org. Chem. Vol. 75, No. 3, 2010

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Protocols C and D were repeated until the desired peptide
sequence had been reached.
Mass Spectrometry Service Centre in Swansea for some MS
analyses. The Carnegie Trust for the Universities of Scotland
is also acknowledged for funding a Vacation Scholarship for
S.L.N. C.M. and S.L.N. were funded by the School of
Natural and Computing Sciences, University of Aberdeen.
Acknowledgment. The authors thank Jioji Tabudravu
and Wael Abdelmageed for help with HPLC analysis, and
the Marine Biodiscovery Group for use of HPLC facilities.
We also thank John Callan of the School of Pharmacy and
Life Sciences at the Robert Gordon University Aberdeen for
some NMR analyses. We acknowledge the EPSRC National
Supporting Information Available: Experimental details and
analyses for all peptides. This material is available free of charge
via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 3, 2010 569