Trichloroethyl group as a protecting group for sulfonates and its application to the synthesis of a disulfonate analog of the tyrosine sulfated PSGL-143-50 peptide

Article abstract of DOI:10.1021/jo900122c

The trichloroethyl (TCE) group is shown to be a viable protecting group for sulfonates. TCE-protected sulfonates were found to be particularly stable to acid, a key characteristic that led to a straightforward enantioselective synthesis of L-FmocPhe(p-CH<

Full text of DOI:10.1021/jo900122c

have limitations.5,6a,b Due to the paucity of sulfonic acid
protecting groups, the development of additional protecting
groups, especially ones that exhibit chemical properties that are
distinct from those already developed, is highly desirable.
Trichloroethyl Group As a Protecting Group for
Sulfonates and Its Application to the Synthesis of
a Disulfonate Analog of the Tyrosine Sulfated
PSGL-143-50 Peptide
Ahmed M. Ali, Bryan Hill, and Scott D. Taylor*
Department of Chemistry, UniVersity of Waterloo, 200
UniVersity AVenue West, Waterloo, Ontario, Canada, N2L 3G1
s5taylor@sciborg.uwaterloo.ca
ReceiVed January 19, 2009
FIGURE 1. Structure of compounds 1-5.
Our interest in sulfonic acids and their protecting groups stems
from our desire to construct peptides bearing (sulfonometh-
yl)phenylalanine (1, SmP, Figure 1). Over the last 20 years,
there has been substantial interest in this amino acid as a stable
replacement of sulfotyrosine (sTyr) and as a monoanionic
phosphotyrosine mimic.1b,8,9a-h
In general, Smp is incorporated into peptides using solid phase
peptide synthesis (SPPS) methodology and Fmoc-protected
amino acid 2 in which the sulfonate group is unprotected (Figure
1).9e-g However, there are significant drawbacks to using 2 for
the synthesis of Smp-bearing peptides. First, an efficient
enantioselective synthesis of this amino acid has yet to be
developed.9e,g It is commercially available but extremely
expensive.10 Most importantly, the synthesis of Smp-bearing
peptides using amino acid 2 can sometimes be highly problem-
atic and low yielding.9g
Several years ago we reported the synthesis of peptides
bearing (difluoromethylsulfono)phenylananine (F2Smp) using
amino acid 3 as a building block (Figure 1). The sulfonate group
in compound 3 is protected with a neopentyl group (nPt).1c,6c
The desired peptides bearing F2Smp were obtained in good yield
upon removal of the neopentyl group from the sulfonate-
protected peptides using aq. acid or LiBr in refluxing butanone.
More recently, we have shown that sulfotyrosine-bearing
peptides can be readily synthesized in good yield employing
The trichloroethyl (TCE) group is shown to be a viable
protecting group for sulfonates. TCE-protected sulfonates
were found to be particularly stable to acid, a key charac-
teristic that led to a straightforward enantioselective synthesis
of L-FmocPhe(p-CH2SO3TCE)OH. This was used as a
building block for the solid phase synthesis of an octapeptide
corresponding to P-selectin glycoprotein ligand-1 residues
43-50 (PSGL-143-50) in which sulfotyrosine residues 46 and
48 were replaced with (sulfonomethyl)phenylalanine (SmP),
an important hydrolytically stable sulfotyrosine mimic.
The sulfonic acid group has been used as an isostere for the
phosphate and carboxylate groups in drug development1a-e and
is a vital constituent of antagonists of follicle stimulating
hormone2 and P2 purinergic receptors.3 Due to its highly polar
nature, the sulfonic acid moiety is usually protected during
multistep syntheses as this facilitates the handling and purifica-
tion of the sulfonylated intermediates and minimizes side
reactions. Sulfonic acids are most commonly protected as alkyl
esters (mainly isopropyl,2,4 isobutyl,5 and neopentyl1b,c,6,7). As
with any protecting group, these alkyl ester protecting groups
(6) (a) Truce, W. E.; Vrencur, D. J. J. Org. Chem. 1970, 35, 1226. (b) Roberts,
J. C.; Gao, H.; Gopalsamy, A.; Kongsjahju, A.; Patch, R. J. Tetrahedron Lett.
1997, 38, 335. (c) Liu, S.; Dockendorf, C.; Taylor, S. D. Org. Lett. 2001, 3,
1571–1574.
(7) Derivatives of the neopentyl ester protecting group, which are removed
using the safety-catch principle, have also been developed See: (a) Seeberger,
S.; Griffin, R. J.; Hardcastle, I. R.; Golding, B. T. Org. Biomol. Chem. 2007, 5,
132. (b) Reference 6b.
(8) Burke, T. R.; Lee, K. Acc. Chem. Res. 2003, 36, 426.
(1) For some examples see: (a) Hellmuth, K.; Grosskopf, S.; Lum, C. T.;
Wurtele, M.; Roder, N.; von Kries, J. P.; Rosario, M.; Rademann, J.; Birchmeirer,
W. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 7275. (b) Hussain, M.; Ahmed, V.;
Hill, B.; Ahmed, Z.; Taylor, S. D. Bioorg. Med. Chem. 2008, 15, 6764. (c) Leung,
C.; Lee, J.; Meyer, N.; Jia, C.; Grzyb, J.; Liu, S.; Hum, G.; Taylor, S. D. Bioorg.
Med. Chem. 2002, 10, 2309. (d) Bischoff, L.; David, C.; Roques, B.; Fournie´-
Zaluski, M.-C. J. Org. Chem. 1999, 64, 1420. (e) David, C.; Bischoff, L.; Meudal,
H.; Mothe, A.; De Mota, N.; DaNascimento, S.; Llorens-Cortes, C.; Fournie´-
Zaluski, M.; Roques, B. J. Med. Chem. 1999, 42, 5197.
(9) (a) Marseigne, I.; Roques, B. P. J. Org. Chem. 1988, 53, 3621. (b)
Marseigne, I.; Roy, P.; Dor, A.; Durieux, C.; Peleprat, D.; Reibaud, M.;
Blanchard, J. C.; Roques, B. P. J. Med. Chem. 1989, 32, 445. (c) Gonzalez-
Muniz, R.; Cornille, F.; Bergeron, F.; Ficheux, D.; Pother, J.; Durieux, C.;
Roques, B. P. Int. J. Peptide Protein Res. 1991, 37, 331. (d) Dorville, A.;
McCort,-Tranchepain, I.; Vichard, D.; Sather, W.; Maroun, R.; Ascher, P.;
Roques, B. P. J. Med. Chem. 1992, 35, 2551. (e) Miranda, M. T. M.; Liddle,
R. A.; Rivier, J. E. J. Med. Chem. 1993, 36, 1681. (f) Teresa, M.; Mirand, M.;
Craig, A. G.; Miller, C.; Liddle, R. A.; Riveir, J. E. J. Protein Chem. 1993, 12,
533. (g) Herzner, H.; Kunz, H. Tetrahedron 2007, 63, 6423. (h) Lam, S. N.;
Acharya, P.; Wyatt, R.; Kwong, P. D.; Bewley, C. A. Bioorg. Med. Chem. 2008,
16, 10113.
(2) Wrobel, J; Rogers, J.; Green, D.; Kao, W. L. Synth. Commun. 2002, 32,
2695.
(3) Yan, L.; Muller, C. E. J. Med. Chem. 2004, 47, 1031.
(4) Musicki, B.; Widlanski, T. S. J. Org. Chem. 1990, 55, 4231.
(5) Xie, M.; Widlanski, T. S. Tetrahedron Lett. 1996, 37, 4443.
(10) Amino acid 2 is commercially available from several companies for
$900-1000 USD for 1 g.
10.1021/jo900122c CCC: $40.75
Published on Web 03/30/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 3583–3586 3583

SCHEME 1
SCHEME 2
160 °C6b or LiBr in refluxing butanone.6c The TCE group has
never been examined as a protecting group for sulfonate esters;
however, we have found that this moiety is a highly effective
protecting group for sulfate esters and is removed under very
mild reducing conditions.14 Reaction of crude 7 with 5.0 equiv
of 2,2,2-trichloroethanol or with 5.0 equiv of neopentyl alcohol
in the presence of 4.0 equiv of 2,6-lutidine gave esters 8 and 9
in 70 and 59% yield, respectively (two steps). A key and
potentially problematic step in this synthesis is the required
selective deprotection of the carboxylic acid and amino func-
tionalities without loss of the sulfonate protecting group. We
were pleased to find that this could be achieved with compound
8 by refluxing 8 in 6 N HCl for 12 h, which gave amino acid
10 in a 91% yield. This reaction demonstrates the remarkable
stability of TCE-protected sulfonates to acid. However, subject-
ing neopentyl ester 9 to the same conditions resulted in
hydrolysis of the neopentyl ester to give Smp (1) in 68% yield.
We were unable to find conditions that would allow us to
selectively remove the ethyl and acetyl groups without removing
the neopentyl group. Amino acid 5 was obtained in an 85%
yield from 10 by reacting 10 with Fmoc-Cl under standard
Schotten-Baumann conditions.15
Before attempting to prepare peptides using amino acid 5,
we determined conditions for removing the TCE group and
examined the stability of TCE sulfonate esters by subjecting
model sulfonate 11 (Scheme 2) to a variety of conditions. The
TCE group in 11 was readily removed using mild reducing
conditions such as Zn/ammonium formate in MeOH, Zn in
HOAc and hydrogenolysis (H2 or ammonium formate with 10%
Pd/C). Not surprisingly, compound 11 is stable to 100% TFA
with no detectable reaction occurring even after several days
and to TFA containing scavenging reagents such as triisopro-
pylsilane (TIPS), anisole and thioanisole. It is also stable to mild
reducing agents such as NaBH4. It is stable to sterically hindered
organic bases such as triethylamine but slowly decomposes in
the presence of an excess of stronger or less sterically hindered
organic bases. For example, 1H NMR studies in 20% piperidine/
DMF-d7 revealed that ester 11 undergoes nucleophilic attack
by piperidine on the sulfur atom to give compound 12 (Scheme
2) which was characterized by mass spectrometry and 1H NMR.
Compound 11 was completely consumed within 24 h. This is
in contrast to aryl sulfate 13 which we have shown first
undergoes a rapid elimination of HCl to give DCV ester 14
followed by a slower attack by piperidine on the sulfur atom
and subsequent formation of decomposition products (Scheme
2).11 We have also demonstrated that although 20% 2-MP in
DMF also converts sulfate ester 13 into DCV sulfate ester 14;
no further reaction between 2-MP and ester 14 occurs even after
FmocTyr(SO3DCV)OH (4, Figure 1) (DCV ) dichlorovinyl)
as a building block. To avoid loss of the DCV group during
Fmoc removal, 2-methylpiperidine (2-MP), a more sterically
hindered base than piperidine, the most commonly used base
for Fmoc removal, was used.11 The DCV group was removed
by hydrogenolysis after cleavage of the peptide from the
support.11
These ease with which sulfonate- and sulfate-protected amino
acids 3 and 4 were incorporated into peptides has prompted us
to examine whether a similar approach could be used for the
synthesis of peptides bearing Smp. Here we report an efficient
synthesis of a derivative of amino acid 2, in which the sulfonate
group is protected with a trichloroethyl (TCE) group (compound
5, Figure 1). We demonstrate that TCE group is a viable
protecting group for sulfonates and such protected sulfonates
are remarkably stable to acid, a key characteristic that led to a
straightforward synthesis of compound 5. We also show that
TCE-protected sulfonates exhibit different reactivity patterns
compared to TCE-protected sulfates when subjected to organic
bases. Finally, we show that amino acid 5 is an effective building
block for the solid phase synthesis of a disulfonate analog of
the tyrosine sulfated PSGL-143-50 peptide.
Our synthesis of a sulfonate-protected version of amino acid
2 is shown in Scheme 1. It begins with the oxidative chlorination
of readily prepared amino acid 6.12 We previously reported that
thioester 6 can be converted into sulfonyl chloride 7 by oxidative
chlorination using Cl2 gas in CH2Cl2-H2O.12 Very recently,
Nishiguchi et al. reported the synthesis of sulfonyl chlorides
from thioesters in high yield using N-chlorosuccinimide (NCS)
in aq. HCl-MeCN.13 This procedure has the advantage of not
requiring Cl2 gas, a toxic and difficult substance to work with.
We applied Nishiguchi et al.’s procedure to thioester 6. 1H NMR
of the crude reaction mixture, after a brine wash, indicated that
sulfonyl chloride 7 was produced relatively cleanly with the
main contaminant being succinimide and this material was used
without purification for the next step.
We attempted to prepare derivatives of amino acid 2 with
the sulfonate moiety protected with either a neopentyl or a TCE
group. The neopentyl group is best removed under nucleophilic
conditions such as tetramethylammonium chloride in DMF at
(14) (a) Liu, Y.; Lien, I-F.; Ruttgaizer, S.; Dove, P.; Taylor, S. D. Org. Lett.
2004, 6, 209–212. (b) Ingram, L.; Taylor, S. D. Angew. Chem., Int. Ed. 2006,
45, 3503–3506.
(15) The enantiopurity of 5 was found to be >97% as determined by the
synthesis of diastereomeric peptides followed by HPLC analysis. See the
Supporting Information.
(11) Ali, A.; Taylor, S. D. Angew. Chem., Int. Ed. 2009, 48, 2024.
(12) Hill, B; Ahmed, V.; Bates, D.; Taylor, S. D. J. Org. Chem. 2006, 71,
8190.
(13) Nishiguchi, A.; Maeda, K.; Miki, S. Synthesis 2006, 4131.
3584 J. Org. Chem. Vol. 74, No. 9, 2009

several days.111H NMR studies of ester 11 in 20% 2-MP/DMF-
d7 revealed that although attack of 2-MP on the sulfur atom
still occurs, the reaction is considerably slower compared to
when piperidine is used and even after 8 h only 8% of 11 had
reacted. These results suggested to us that amino acid 5 could
be used in SPPS if 2-MP was used as the base for Fmoc
removal. To determine if this was possible we prepared an
amino acid 2 was used. We expect that the TCE group will be
useful as a protecting group for sulfonates in general and will
be especially effective in situations where stability to strongly
acidic conditions is required.
Experimental Section
AcPhe(p-CH2SO3TCE)OEt (8). To a mixture of CH3CN and
2N HCl (30 mL, 4:1) was added N-chlorosuccinimide (NCS, 4.7 g,
35.1 mmol), and the mixture was cooled to 10 °C and stirred until
most of the NCS dissolved. To this was added dropwise a solution
of thioester 612 (3.0 g, 9.3 mmol) in CH3CN (15 mL) over 10 min.
The solution was stirred for 40 min at 10 °C, diluted with Et2O
(350 mL) and washed with 12% NaCl (3 × 30 mL). The organic
layer was dried (Na2SO4) and concentrated to give 7 as an off-
white solid. The crude sulfonyl chloride was dissolved in dry THF
(60 mL). To this was added 2,2,2-trichloroethanol (4.45 mL, 46.5
mmol) and the mixture was cooled to 0 °C (ice bath). A solution
of 2,6-lutidine (8.09 mL, 37.2 mmol) in dry THF (60 mL) was
added dropwise over 10 min. The ice bath was removed and the
mixture was allowed to come to rt and stirred for 16 h. The mixture
was diluted with EtOAc (300 mL) and washed with 1.0 N HCl,
sat. brine, and the organic layer was dried (Na2SO4) and concen-
trated. The residue was subjected to flash chromatography (50%
EtOAc-50% hexane) which gave pure 8 as a white solid (3.08 g,
70%). Mp 96-98 °C; 1H NMR (300 MHz, CDCl3): δ 7.38 (2H, d,
J ) 8.1 Hz), 7.18 (2H, d, J ) 8.1 Hz), 6.08 (1H, d, J ) 7.6 Hz),
4.86 (1H, q, J ) 6.0 Hz), 4.50 (2H, s), 4.48 (2H, s), 4.17 (2H, dq,
J ) 7.1, 1.0 Hz), 3.14 (2H, dq, J ) 13.8, 6.0 Hz), 1.99 (3H, s),
1.25 (3H, t, J ) 7.1 Hz); 13C NMR (75 MHz, CDCl3): δ 171.3,
169.6, 137.6, 130.9, 129.9, 125.5, 93.4, 77.7, 61.5, 57.3, 53.0, 37.6,
23.0, 14.0; LREIMS m/z (relative intensity): 459 (M+, 2), 461 (M+,
2), 189 (100); HREIMS calculated for C16H20Cl3NO6S: 459.0077
found 459.0085
octapeptide,
Ac-Ala-Thr-Glu-Phe(p-CH2SO3-)-Glu-Phe(p-
CH2SO3-)-Leu-Asp-NH2 (15), which corresponds to residues
43-50 of P-selectin glycoprotein ligand-1 (PSGL-1) in which
the sTyr residues at positions 46 and 48 are replaced with Smp.
This was chosen as a model peptide since Herzner and Kunz
reported difficulties in preparing a protected version of 15 using
sulfonate-unprotected amino acid 2.9g Automated SPPS was
performed using the Rink amide resin and HBTU/HOBt for the
coupling reactions. After the coupling of each Fmoc amino acid
the peptide was subjected to 3 × 10 min of 20% 2-MP/DMF
as opposed to the standard 2 × 10 min protocol when piperidine
is used.16,17 The completed peptide was cleaved from the resin
using 98% TFA/2% TIPS then precipitated in ether. The HPLC
chromatogram of the crude peptide consisted of one major peak
plus a few minor peaks.18 The -ESI mass spectrum of the crude
peptide indicated that the major product in the crude mixture
was desired TCE-protected peptide Ac-Ala-Thr-Glu-Phe(p-
CH2SO3TCE)-Glu-Phe(p-CH2SO3TCE)-Leu-Asp-NH2 (16) and
no peaks corresponding to peptides that had undergone substitu-
tion of the TCE group with 2-MP were detected.19 After
subjecting crude peptide 16 to H2 (balloon), 30 wt. % of 10%
Pd/C, and 15 equiv of ammonium formate for 6 h, HPLC
analysis of the crude reaction mixture revealed one major peak
plus a variety of minor peaks. Purification by semipreparative
RP-HPLC gave pure peptide 15 in a 60% yield in 95% purity.
We also prepared peptide 15 using the identical procedure except
sulfonate-unprotected amino acid 2 was used as a building block.
Amino acid 2 was prepared in 94% yield from compound 5 as
its free acid by subjecting 5 to Zn/ammonium formate in MeOH.
HPLC analysis of the crude peptide showed many peaks18 and
after a challenging purification using semipreparative RP-HPLC
pure peptide 16 was obtained in only a 19% yield.
Phe(p-CH2SO3TCE)OH (10). Sulfonate 8 (2.70 g, 5.6 mmol)
was refluxed in 6 N HCl (50 mL) for 12 h. The mixture was allowed
to cool and concentrated by high vacuum rotary evaporation to
dryness. EtOH (100 mL) was added followed by 11 mL propylene
oxide. The mixture was stirred for 16 h and filtered to give pure
10 as a white solid (1.88 g). The filtrate was concentrated, and
CHCl3 was added. The mixture was stirred for 30 min then filtered,
which yielded an additional 0.157 g of pure 10. The total yield
was 2.03 g (91%). Mp 207 °C (dec.); 1H NMR (300 MHz, DMSO-
d6): δ 7.39 (2H, d, J ) 8.1 Hz), 7.31 (2H, d, J ) 8.1 Hz), 4.98
(2H, s), 4.90 (2H, s), 3.68-3.64 (1H, m), 3.14 (1H, dd, J ) 14.4,
4.9 Hz), 2.95 (1H, dd, J ) 14.3, 7.5 Hz); 13C NMR (75 MHz,
DMSO-d6): δ 169.9, 137.6, 131.0, 129.7, 126.2, 94.0, 78.0, 55.2,
54.6, 36.3; LRESI+MS m/z (relative intensity): 390 ([M+H]+, 100),
In summary, an efficient synthesis of amino acid 5 was
achieved in which the sulfonate group is protected with a
trichloroethyl group. A key step in this synthesis was the
selective hydrolysis of the acetamide and ethyl esters in the
presence of the TCE-protected sulfonate using refluxing 6 N
HCl which demonstrates the exceptional stability of TCE-
protected sulfonates to strong acid. We also showed that TCE-
protected sulfonates are stable to a variety of conditions but
are not stable to an excess of organic bases such as piperidine.
We demonstrated that amino acid 5 is an effective building block
for the solid phase synthesis of Smp-bearing peptides when
using 2-MP as base and that this approach provided the targeted
peptide in higher yield compared to when sulfonate-unprotected
392 ([M + H]+, 95); HRESI+MS calculated for C12H15NO SCl3
5
(M + H)+ 389.9737 found 389.9744
FmocPhe(p-CH2SO3TCE)OH (5). To a solution of amino acid
10 (2.10 g, 5.38 mmol) in dioxane (30 mL) and 10% Na2CO3 (40
mL) was added Fmoc-Cl (2.18 g, 8.07 mmol) and the mixture was
stirred for 20 h. The reaction was acidified to pH 2-3 using 1 N
HCl and the mixture extracted with EtOAc, dried (MgSO4) and
concentrated. The crude material was subjected to gravity chro-
matography (1% MeOH-99% CH2Cl2 then 2.5% MeOH-97.5%
CH2Cl2 then 5% MeOH-95% CH2Cl2 then 10% MeOH-90%
CH2Cl2) which gave pure 5 as a white amorphous solid (2.81 g,
(16) We have been able to obtain 2.5 kg of 2-MP from a commercial supplier
for $180.00 USD, and this could be used without further purification.
(17) Hachmann and Lebl have shown that Fmoc deprotection of FmocIle
attached to chlorotrityl resin using 2-MP occurred with a half-life that was 1.5
times greater than when piperidine was used. Hence, we have increased the Fmoc
deprotection times by 1.5 times. See: Hachmann, J.; Lebl, M. J. Comb. Chem
2006, 8, 149.
1
85%). H NMR (300 MHz, CDCl3): δ 7.75 (2H, d, J ) 7.3 Hz);
7.53 (2H, d, J ) 6.8 Hz), 7.16-7.41 (10H, m), 5.19 (1H, d, J )
8.4 Hz), 4.69 (1H, bs), 4.33-4.52 (6H, m), 4.17 (1H, app. t, J )
7.3 Hz), 3.06-3.25 (2H, m); 13C NMR (75 MHz, CD3OD): δ 174.8,
158.1, 145.0, 142.4, 139.8, 132.1, 130.8, 128.7, 128.1, 127.3,
126.22, 126.17, 120.9, 95.0, 79.3, 67.9, 57.5, 56.5, 48.1, 38.2;
LR+ESIMS m/z (relative intensity): 614 (M + H, 100); HR+ESIMS
calculated for C27H25NO7SCl3 (M + H)+ 612.0419, found 612.0417.
FmocPhe(p-CH2SO3H)OH (2). To a solution of amino acid 5
(1.0 g, 1.63 mmol) in 3 mL of HPLC grade methanol was added
(18) See the Supporting Information.
(19) Hari and Miller have shown that resin-supported (Wang) sulfonates
exhibit superior stability to organic bases compared to their solution counterparts.
This “resin protection” may be an explanation as to why we do not see any
substitution of the TCE groups with 2-MP during SPPS. See: Hari, A.; Miller,
B. L. Org. Lett. 1999, 1, 2109.
J. Org. Chem. Vol. 74, No. 9, 2009 3585

ammonium formate (0.61 g, 9.78 mmol) followed by Zn dust (0.64
g, 9.78 mmol). After overnight stirring, the reaction mixture was
filtered through Celite and concentrated under Vacuum. Compound
2 was purified by flash chromatography using CH2Cl2/MeOH/
AcOH/H2O (50:8:1:1), and the solvent system was shifted to
(CH2Cl2/MeOH/ AcOH /H2O 7:3:0.3:0.6) when compound 2 started
to come off the column. This gave pure 2 as a white solid (0.73 g,
crude peptide 16 (when using building block 5) and 22.3 mg of
crude peptide 15 (when using building block 2).
Synthesis of Peptide 15 by Removal of the TCE Groups
from Peptide 16. To a solution of crude peptide 16 (30 mg) in 3
mL of HPLC grade methanol was added ammonium formate (19.4
mg, 15 equiv), followed by 10% Pd/C (9 mg, 30% wt.). The reaction
was fitted with a balloon filled with H2, stirred at rt and the reaction
was monitored by HPLC for the disappearance of peptide 16
(CH3CN/H2O (0.1% TFA) as eluent, linear gradient from 5 to 95
CH3CN in 60 min). After 6 h, the reaction mixture was transferred
into an Eppendorf tube and centrifuged to pellet the Pd/C. The
supernatant was removed and the pellet washed two more times
with 3 mL of methanol. The combined supernatants were concen-
trated under reduce pressure. Peptide 15 was purified using
semipreparative RP-HPLC with a UV detector set at 220 λ. A linear
gradient of 1%CH3CN/99% 100 mM ammonium acetate (pH ) 9)
to 10% CH3CN/90% 100 mM ammonium acetate over 60 min was
used as eluent (tR ) 39.3 min). Fractions containing peptide 15
were pooled, concentrated by high vacuum rotary evaporation, and
the residue was dissolved in water and repeatedly lyophilized until
a constant weight was obtained. Peptide 15 was obtained as a
flocculent white powder (16.7 mg, 60% yield based on resin
loading). Peptide 15 was 95% pure as determined by analytical
RP-HPLC.18 HR-ESIMS: calculated for C49H68N6O22S2 (M - H)-
1198.3920, found 1198.3917.
1
94%). Mp ) 182-185 °C; H NMR (300 MHz, CD3OD): δ 7.76
(2H, d, J ) 7.5 Hz); 7.59 (2H, d, J ) 7.2 Hz), 7.38-7.25 (6H, m),
7.15 (2H, d, J ) 7.8 Hz), 4.35-4.27 (2H, m), 4.24-4.18 (1H, m),
4.15-4.11 (1H, m),3.97 (2H, s), 3.14 (1H, dd, J1 ) 9.3 Hz and J2
) 4.8 Hz), 2.94 (1H, dd, J1 ) 8.4 Hz and J2 ) 5.4 Hz); 13C NMR
(75 MHz, DMSO-d6): δ 174.08, 156.13, 144.33, 144.21, 141.08,
136.60, 133.56, 130.32, 128.83, 128.02, 127.50, 125.81, 125.69,
120.49, 65.98, 57.63, 56.52, 47.01, 36.89; LR-ESIMS m/z (relative
intensity): 480 ([M - H]-, 100), 481 (M, 32); HR-ESIMS
calculated for C25H22NO7S (M - H)- 480.1117, found 480.1104
Peptide Synthesis. Peptide syntheses were performed on Rink
resin (35 mg, 0.71 mmol/g, preswollen in DMF, 3 × 10min, before
use) using a Quartet automated peptide synthesizer from Protein
Technologies. The first amino acid was attached as a pentafluo-
rophenyl ester by adding a solution of Fmoc-Leu-OPfp (4.0 equiv)
and HOBt (4.0 equiv) in DMF manually to the resin and reacted
for 1.5 h. Subsequent amino acids were coupled as their free acids
(Fmoc-AA-COOH) using HOBt/HBTU/DIPEA (1 × 1.5 h). The
side chains of Asp and Glu were protected as their t-butyl esters.
The side chain of Thr was protected as a t-butyl ether. All Fmoc
deprotections were accomplished using 20% 2-methylpiperidine/
DMF (3 × 10 min). The last amino acid was acetylated using a
2:1 mixture of acetic anhydride/pyridine. The peptides were cleaved
from the resin by subjecting the resin supported peptides to TFA:
triisopropylsilane (2 mL, 98:2) for 2.5 h after which the mixture
was filtered and the resin was washed with the cleavage cocktail.
The filtrate was concentrated by rotary evaporation to half the
volume and cold t-butylmethyl ether was added to precipitate the
peptides. The mixture was centrifuged, the supernatant discarded
and the pellet suspended in ether and centrifuged again. The
supernatant was discarded, the pellet was dried under vacuum for
1 h and suspended in water and lyophilized. This gave 34.5 mg of
Acknowledgment. This research was supported by a Dis-
covery Grant from the Natural Sciences and Engineering
Research Council (NSERC) of Canada to SDT and by a
scholarship from the Egyptian Government to AMA.
Supporting Information Available: 1H and 13C NMR
spectra of compounds 2, 5 and 8-10 and the 1H NMR spectrum
of compound 12. Analytical HPLC chromatogram of peptides.
Experimental for the synthesis of dipeptides for determining
the enantiomeric excess of amino acid 5 and their analytical
HPLC chromatograms and 1H NMR’s. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO900122C
3586 J. Org. Chem. Vol. 74, No. 9, 2009