Synthesis of a-factor peptide from Saccharomyces cerevisiae and photoactive analogues via Fmoc solid phase methodology

Article abstract of DOI:10.1016/j.bmc.2010.11.006

a-Factor from Saccharomyces cerevisiae is a farnesylated dodecapeptide involved in mating. The molecule binds to a G-protein coupled receptor and hence serves as a simple system for studying the interactions between prenylated molecules and their cognate

Full text of DOI:10.1016/j.bmc.2010.11.006

Bioorganic & Medicinal Chemistry 19 (2011) 490–497
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of a-factor peptide from Saccharomyces cerevisiae
and photoactive analogues via Fmoc solid phase methodology
Daniel G. Mullen a, Kelly Kyro a, Melinda Hauser b, Martin Gustavsson c, Gianluigi Veglia a,c
,
Jeffery M. Becker b, Fred Naider d, Mark D. Distefano a,e,
⇑
a Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
b Department of Microbiology, University of Tennessee, Knoxville, TN 37996, United States
c Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, MN 55455, United States
d Department of Chemistry, College of Staten Island, CUNY, Staten Island, NY 10314, United States
e Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
a-Factor from Saccharomyces cerevisiae is a farnesylated dodecapeptide involved in mating. The molecule
binds to a G-protein coupled receptor and hence serves as a simple system for studying the interactions
between prenylated molecules and their cognate receptors. Here, we describe the preparation of a-factor
and two photoactive analogues via Fmoc solid-phase peptide synthesis using hydrazinobenzoyl AM
NovaGel™ resin; the structure of the synthetic a-factor was confirmed by MS–MS analysis and NMR;
the structures of the analogues were confirmed by MS–MS analysis. Using a yeast growth arrest assay,
the analogues were found to have activity comparable to a-factor itself.
Received 9 August 2010
Revised 29 October 2010
Accepted 2 November 2010
Available online 12 November 2010
Keywords:
a-Factor
Benzophenone
C-Terminal methyl ester
Farnesyl
Ó 2010 Elsevier Ltd. All rights reserved.
Peptide synthesis
Photoaffinity labeling
Prenylation
O
H
1. Introduction
YIIKGVFWDPA
YIIKGVFWDPA
N
OCH3
In the budding yeast, Saccharomyces cerevisiae, mating between
the two haplotypes involves the participation of two secreted
peptide pheromones.1 Each of these molecules binds to a specific G-
protein coupled receptor and activates a signal transduction cascade
resulting in a series of complex cellular events. One of these peptides
is a-factor (1, Fig. 1), a dodecapeptide that incorporates farnesylcys-
teine methyl ester as its C-terminal residue.2 The a-factor/receptor
systemisa usefulmodel for studyingtheinteractionsbetween preny-
lated molecules and proteins.3 Due to their widespread occurrence,
S
1
O
S
H
N
OCH3
2
O
O
Abbreviations: Boc, tert-butyloxycarbonyl;Br-BC5, [(E)-(3-((4-bromo-2-methylbut-
2-enyloxy)methyl)phenyl)-(phenyl)methanone]; Br-BC10, [(3-(((2E,6E)-8-bromo-2,6-
dimethylocta-2,6-dienyloxy)methyl)-phenyl)(phenyl)methanone]; t-Bu, tert-butyl;
CLEAR, cross-linked ethoxylate acrylate resin; Dde, 1-(4,4-dimethyl-2,6-dioxocyclo-
hexylidene) ethyl; DI, deionized; DIEA, diisopropylethylamine; DMF, N,N-dimethyl-
formamide; DMSO, dimethyl sulfoxide; ESI-MS, electrospray ionization mass
spectrometry; FA, formic acid; Fmoc, 9-fluorenylmethyloxycarbonyl; Fr, farnesyl; Fr-
Br, farnesyl bromide; HPLC, high pressure liquid chromatography; OAc, acetate; PBS,
phosphate buffered saline; PEG, polyethylene glycol; RP-HPLC, reversed-phase high
pressure liquid chromatography; SPPS, solid-phase peptide synthesis; TFA, trifluoroace-
tic acid; TFP, tetrafluorophenyl; Trt, trityl.
O
S
H
N
YIIKGVFWDPA
OCH3
3
O
O
⇑
Figure 1. Structures of a-factor (1) and photoactive analogues with 5-carbon (2)
and 10-carbon (3) isoprenoid spacers.
Corresponding author. Tel.: +1 612 624 0544; fax: +1 612 626 7541.
E-mail address: diste001@umn.edu (M.D. Distefano).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.006

D. G. Mullen et al. / Bioorg. Med. Chem. 19 (2011) 490–497
491
their participation in cellular signaling and their pivotal role in
O
H
N
numerous diseases, prenylated proteins have been the focus of in-
tense study within the last 15 years.4,5 While initially prenylation
was thought to function only as a membrane targeting element, it is
nowclear that thislipidmodificationisalsoinvolved indirectlymedi-
ating certain protein–protein interactions.6 The binding of a-factor to
its receptor is one example of this latter phenomenon.7
Y(tBu)IIK(Boc)GVFW(Boc)D(tBu)PAC(Trt)
Reagent K
N
H
H
N
O
O
H
4
N
N
YIIKGVFWDPAC
H
H
N
SH
Given the membrane bound nature of Ste3p, the a-factor recep-
tor, photoaffinity labeling is an attractive method to probe the
interaction between the protein and the pheromone. Several labo-
ratories have developed analogues of the farnesyl moiety that
incorporate photoactivatable groups into the isoprenoids them-
selves, allowing specific interactions between prenyl groups and
their cognate protein receptors to be studied; those molecules in-
clude diazotrifluropropionates8–16, benzophenones17–27 and aryl
azides.28,29 Thus, we envisioned that a-factor analogues suitably
functionalized with photoactive farnesyl groups would be useful
for this purpose and accordingly, we sought to prepare such mole-
cules via solid-phase peptide synthetic methods.
O
O
Fr-Br, 5 eq.
DIEA, 12 eq.
DMF/N2
5
O
H
N
YIIKGVFWDPAC
S
N
H
N
H
6
Cu(OAc)2/O2
Pyridine/Acetic Acid
MeOH/CH2Cl2
YIIKGVFWDPAC-
OMe
1
Chemical synthesis using the Merrifield Boc/Bzl protecting group
strategy30 has been used extensively for the preparation of a-factor
and related analogues.31–34 That strategy has been the method of
choice due to the presence of the C-terminal ester. Recently, Wald-
mann and co-workers developed a method for generating C-termi-
nal methyl esters based on a hydrazine-containing resin.35,36 In
their approach, synthesis begins with attachment of the C-terminal
residue to hydrazinobenzoyl AM NovaGel™ resin via a hydrazide
linkage. Following standard synthetic protocols involving iterative
cycles of amide bond formation and deprotection, the completed re-
sin-bound fully protected peptide is then subjected to Cu(I) medi-
ated oxidation of the hydrazide linkage. This transformation yields
a diazo-ester intermediate that undergoes spontaneous alcoholysis;
global side-chain deprotection affords the desired C-terminal ester
product. An important feature of this strategy is that it is fully com-
patible with the Fmoc/t-Bu protecting group scheme37 whose popu-
larity has increased due to the elimination of the need to work with
HF in the final deprotection/resin cleavage step. Given that the syn-
thesis of cysteine-containing peptides can be problematic due to the
possibility of epimerization and b-elimination,38 the well prece-
dented hydrazide strategy appeared attractive. While other
methods such as the xanthenyl side-chain anchoring strategy devel-
oped by Barany et al. have great potential for the synthesis of pep-
tides containing C-terminal cysteine residues,39 the resin required
to implement that strategy is not commercially available. Thus, we
elected to explore the use of hydrazine-containing resin available
from Novabiochem for a-factor synthesis.
S
Scheme 1. Initial strategy for the synthesis of a-factor peptide precursor by
oxidative resin cleavage of an unprotected peptide.
groups used in Fmoc chemistry;40 exposure of isoprenoids to acidic
conditions typically leads to complex mixtures of solvolyzed and
rearranged products. Next, because the C-terminal methyl ester
is necessary for activity, standard amide and acid SPPS resin link-
ages are not suitable for use in this synthesis. Recent reports by
Waldmann et al.41–43 on the synthesis of lipidated peptides that
contain a C-terminal methyl ester by oxidative cleavage of peptides
from hydrazinobenzoyl AM NovaGel™ resin44 offered a possible
way forward. We envisioned a synthetic route in which all steps
would be done while the peptide was still attached to the solid
support (Scheme 1), which would minimize losses from handling
and from successive preparative HPLC purifications. In this reac-
tion sequence, after chain assembly by standard Fmoc-based SPPS,
the acid labile side-chain protecting groups in the fully protected
peptide 4 are first removed by Reagent K45 before alkylation of cys-
teine by farnesyl bromide. In the last step, oxidative cleavage of
alkylated peptide 6 would produce a-factor (1). Unfortunately,
after oxidative cleavage a complex mixture of products was ob-
served in which only a trace of desired product 1 could be found
(see Fig. 2). It is possible that unprotected tryptophan is not stable
to these oxidative conditions. Also, in the many examples of oxida-
tive cleavage published by Waldmann and co-workers, the side-
chains of trifunctional amino acids are generally protected during
this reaction.41–43 It is possible that unprotected side-chains che-
late copper and interfere with the cleavage reaction or undergo
oxidation themselves.
In the work reported here, we first describe the preparation of a-
factor via Fmoc SPPS using hydrazinobenzoyl resin; the structure of
the material prepared by this alternative synthetic route was con-
firmedbydetailed MS–MS analysis. Using a yeast growtharrestassay,
the synthetic material was found to be equipotent compared with the
a-factor prepared previously31–34 The resulting method was then
used to prepare two a-factor analogues containing photoactive ben-
zophenone groups incorporated within the isoprenoid moiety. Using
the same growth arrest assay, the photoactivatable analogues were
found to have activity comparable to that of a-factor itself.
2.2. Successful approach of a-factor and analogues via a fully
protected peptide
To circumvent the above problems, we decided to attempt oxi-
dative cleavage on a fully protected peptide sequence (Scheme 2).
Before resin cleavage, the amino terminus of the peptide was pro-
tected with the Boc group by treatment with di-tert-butyl dicar-
bonate in the presence of base to yield an on-resin fully
protected peptide 7. Oxidative cleavage by Cu(I) with methanol
as a cosolvent furnished fully protected methyl ester 8, which
was treated with Reagent K to cleave the acid labile protecting
groups. We were pleased to find only one main product by HPLC
analysis that corresponded to desired peptide 9. This result
2. Results and discussion
2.1. Attempted synthesis of a-factor peptide precursor by
oxidative resin cleavage of an unprotected peptide
The syntheses of a-factor (1) and benzophenone analogues 2
and 3 present several challenges. First, the all trans double bonds
of the prenyl groups are not stable to the acidic conditions neces-
sary to remove standard t-butyl-based side-chain protecting

492
D. G. Mullen et al. / Bioorg. Med. Chem. 19 (2011) 490–497
0.4
0.3
0.2
0.1
0
0.4
A
B
0.3
0.2
0.1
0
0
10 20 30 40 50 60 70
Time (min)
0
10
20
30
40
50
60
Time (min)
Figure 2. HPLC of crude reaction mixtures from the preparation of a-factor after oxidative resin cleavage Panel A: HPLC of crude reaction mixture of 1 after oxidative cleavage
of 6 as described in Scheme 1. The arrow indicates the peak containing 1. Panel B: HPLC of crude reaction mixture of 9 after oxidative cleavage and Reagent K treatment as
shown in Scheme 2. The arrow indicates the peak containing 9.
O
H
N
Y(tBu)IIK(Boc)GVFW(Boc)D(tBu)PAC(Trt)
Boc2O/DIEA
N
H
H
N
4
7
O
O
O
H
N
BocY(tBu)IIK(Boc)GVFW(Boc)D(tBu)PAC(Trt)
N
H
H
N
Cu(OAc)2/O2
Pyridine/Acetic Acid
MeOH/CH2Cl2
BocY(tBu)IIK(Boc)GVFW(Boc)D(tBu)PAC(Trt)- OMe
Reagent K
8
YIIKGVFWDPAC-OMe
9
SH
Fr-Br, 5 eq.
Zn(OAc)2 . 2H2O, 5 eq.
DMF/BuOH/0.1% aq. TFA
YIIKGVFWDPAC-OMe
1
S
Scheme 2. Successful strategy for the synthesis of a-factor peptide (1) precursor by oxidative resin cleavage of a fully protected peptide.
supported our hypothesis that a fully protected a-factor sequence
was necessary for successful oxidative cleavage. Alkylation of the
cysteine thiol of 9 with farnesyl bromide, catalyzed by Zn2+40 gave
a-factor (1) (Scheme 2). Benzophenone analogues 2 and 3 were ob-
tained by reaction of 9 with the corresponding allylic bromides un-
der the same alkylation conditions.
It is worth noting that yields of alkylated products 1, 2, and 3,
appear low (8–19%) when calculated based on the masses of the
starting material and products; accordingly, we investigated this
by resynthesizing 1. However, in this second synthesis, the amount
of peptide 9 used was determined by DTNB titration while the
amount of product (1) was quantified via its tryptophan chromo-
phore. As was seen in the first synthesis of 1, HPLC analysis of
the alkylation reaction of 9 with farnesyl bromide (Fig. 3) revealed
nearly complete consumption of 9 and relatively clean conversion
to 1 with few side products formed, When the yield of 1 from this
reaction was calculated from the masses of the starting material
and product, the value was determined to be 19%. However, the
yield obtained via DTNB/UV analysis was calculated to be 65%. This
latter value is comparable to alkylation yields obtained using the
Zn(OAc)2 method on related peptides.7,46–48 In general, it is likely
that the lower yields of the peptide products quantified by mass
reflect the presence of H2O and/or TFA salts as well as uncertainties
inherent in the measurement of small (ꢀ1–2 mg) masses.
2.3. Mass spectrometric analysis of a-factor and two related
photoactive analogues
Since a new synthetic route was used to obtain a-factor and the
two photoactive analogues, the structures of these peptides were
examined in detail by mass spectrometry. ESI-MS–MS spectra of 1,
2, and 3 are shown in Figures 4–6. Similar fragmentation was ob-
served for all three peptides. In general, the results observed are
consistent with MS–MS data previously reported for a-factor by

D. G. Mullen et al. / Bioorg. Med. Chem. 19 (2011) 490–497
493
1
0.8
0.6
0.4
0.2
0
2
A
B
9
1
1.5
1
(alkylated
product)
9
(remaining)
0.5
0
0
5
10 15 20 25 30 35 40
Time (min)
0
10 20 30 40 50 60 70
Time (min)
Figure 3. HPLC analysis of starting material and crude reaction mixture from the alkylation of pure 9 to yield a-factor (1). Panel A: HPLC of purified starting material 9. Panel
B: HPLC of crude alkylation reaction mixture containing 9 and farnesyl bromide in the presence of Zn(OAc)2 to yield 1.
Compound 1
100
[M+2H]2+(-f)
[M+2H]2+
y3
y1
80
60
40
20
0
y4
b6
b8
b2
b9
b7
b3
b11
y6
[M+H]+(-f)
1400
b4
200
400
600
800
m/z, amu
1000
1200
Figure 4. ESI-MS-MS analysis of a-factor (1) produced by Fmoc SPPS. -f: loss of farnesyl (loss of C15H25).
Compound 2
250
[M+2H]2+(-d)
[M+2H]2+
y1
200
150
100
50
y3
y4
b8
b6
b
y1(-d)
2
b7
y2
b9
b3
y3(-d)
a2
y6
b4
b11
2+
y5
BP
b11
0
200
400
600
800
m/z, amu
1000
1200
1400
Figure 5. ESI-MS-MS analysis of a-factor analogue 2 incorporating a photoactive isoprenoid with a 5-carbon spacer. BP: benzophenone fragment, (3-benzoylphenyl)methy-
lium, (C14H11O+); -d: loss of (3-(hydroxymethyl)phenyl)(phenyl)menthanone (C14H13O2).
Anderegg et al.2 In that work, MS–MS data was obtained by FAB ion-
ization in lieu of ESI as was employed here. In their work, the frag-
ment ion (m/z = 1425.7) corresponding to the singly-charged
parent ion with loss of the isoprenoid group was identified, along
with several b-type ions. Interestingly, they reported the [M+H]+
parent ion (m/z = 1629.9) as the most abundant species in the FAB-
MS and fragmented that ion to obtain their MS–MS data; in contrast,
we observed the doubly-charged [M+2H]2+ parent ion (m/z = 815.5)
as the most abundant ion in the ESI-MS. Accordingly, fragmentation
was performed on that species for MS–MS analysis and yielded pri-
marily b- and y-type ions (see Table 1 in Supplementary data for
complete listings and assignments). Fragmentation corresponding
to cleavage of the thioether bond linking the isoprenoid moiety to
the cysteinyl thiol was observed in the MS–MS of a-factor, 1, and

494
D. G. Mullen et al. / Bioorg. Med. Chem. 19 (2011) 490–497
Compound 3
y1(-d)
BPH2O+
y1
[M+2H]2+
[M+2H]2+(-d)
400
300
200
100
0
y2
b8
y4
b6
y3
y3(-d)
[M+2H]2+(-e)
b2
[M+H]+(-e)
b9
BP
b7
b3
b4
b11
y6
b5
y5
200
400
600
800
1000
1200
1400
m/z, amu
Figure 6. ESI-MS-MS analysis of a-factor analogue 3 incorporating a photoactive isoprenoid with a 10-carbon spacer. BP: benzophenone fragment, (3-benzoylphe-
nyl)methylium, (C14H11O+); BPH2O+: (3-benzoylbenzyl)-oxonium, (C14H13O2þ); -d: loss of (3-(hydroxymethyl)phenyl)(phenyl)menthanone (C14H13O2); -e: loss of (3-
(((2E,6E)-2,6-dimethylocta-2,6-dienyloxy)methyl)phenyl)(phenyl)methanone, (loss of C24H28O2).
the related analogue, 3; this type of fragmentation has also been
previously noted in analyses of a-factor via FAB-MS.49 Both the
singly and doubly-charged parent ions with loss of isoprenoid
(m/z = 1425.7 and m/z = 713.4) (Figs. 4 and 6) were observed; this
was not observed for analogue 2. Cleavage of the ether bond linking
the benzophenone group to the isoprenoid spacer was noted in the
MS–MS of both 2 and 3 indicated by the doubly-charged parent ions
(m/z = 746.4 and m/z = 780.4 for 2 and 3, respectively) (Figs. 4 and 5)
and a few y-type ions showing loss of (3-(hydroxymethyl)phenyl)
(phenyl)menthanone (–BPH2O). A charged BPH2O+ fragment (m/
z = 213.1) and the related benzophenone (BP) fragment (3-benzoyl-
phenyl)methylium (m/z = 195.1) were also observed for both 2 and
3. In aggregate, these data clearly confirm the successful preparation
of a-factor and related analogues.
data on 1, prepared via the new route reported here. Accordingly,
a 2D 1H–1H TOCSY NMR spectrum was obtained; a portion of the
spectrum from the fingerprint region is shown in Figure 7. Compar-
ison of this spectral data with data previously reported for a-factor
(prepared via a different route employing Boc/Bzl protection)
shows excellent agreement. Hence, these results provide compel-
ling corroborating evidence that the methodology presented here
can be used to produce a-factor.
2.4. 1H NMR analysis of a-factor (1) produced and photoactive
analogues
While the MS–MS experiments described above provided
strong evidence for the formation of a-factor and the desired pho-
toactive analogues, we felt it would be useful to obtain 1H NMR
Figure 8. Biological assay of pheromones. Growth arrest in response to pheromone
was determined for the a-factor responsive strain RC757 (A–D) or the
responsive strain LM102 (E–H). The upper template indicates the amount of control
synthetic a-factor that was spotted on plates A and E. A disk impregnated with
factor (0.2 g) was applied to the bottom of the plate E. Compounds were applied to
the remaining plates as indicated in the lower template: B and F, synthetic a-factor
(1), C and G 3, D and H, 2. At the top of each plate (A–H) cells secreting a-factor
a-factor
Figure 7. Fingerprint region from a 2D 1H–1H TOCSY NMR spectrum of a-factor (1)
in DMSO-d6. Assignments are indicated adjacent to each cross peak. A peak arising
from exchange of the amide proton of Ile 2 with residual H2O is marked with an
asterisk in the spectrum. The peak corresponding to Ala 11 could be detected at a
threefold lower contour level as shown in the inset.
a-
l
(MATa) or a-factor (MATa) were applied to the lawn as indicated in the templates.

D. G. Mullen et al. / Bioorg. Med. Chem. 19 (2011) 490–497
495
2.5. Biological assay of synthetic a-factor and photoactive
analogues
based on 1% per minute linear gradients starting at 0% B (CH3CN).
The term ‘overall yield’ includes chain assembly, further on-resin
or in solution conversions, Reagent K cleavage, and RP-HPLC purifi-
cation steps. The benzophenone-containing isoprenoids Br-BC5
[(E)-(3-((4-bromo-2-methylbut-2-enyloxy)methyl) phenyl)(phe-
nyl)-methanone] and Br-BC10 [(3-(((2E,6E)-8-bromo-2,6-dimethyl-
octa-2,6-dienyloxy)methyl)phenyl)-(phenyl)methanone] were pre
pared as previously described.21,26
To verify that the a-factor produced using the new synthetic
route described here retained full biological activity, it was evalu-
ated in a yeast growth arrest assay. Cells expressing the STE3 a-fac-
tor receptor protein (MAT
presence of a-factor peptide. In this assay, a plate containing a
lawn of MAT S. cerevisiae cells (strain RC757) was treated with dif-
a cells) undergo growth arrest in the
a
ferent amounts of a-factor. If the cells detect a-factor, a clear zone
of inhibition will develop in the region where the compound is ap-
plied. Several controls were included in this experiment. First, S.
cerevisiae cells which secrete biologically-synthesized a-factor
4.2. Chemical synthesis
4.2.1. YIIKGVFWDPAC-OCH3 (9)
The linear sequence, Y(t-Bu)IIK(Boc)GVFW(Boc)D(t-Bu)-
PAC(Trt)-hydrazide-resin (4) was synthesized on 4-Fmoc-hydra-
zino–benzoyl AM NovaGelÒ resin on the ABI 433 peptide
synthesizer by standard FastMoc chemistry. To protect the N-ter-
(X2180-1A) or
clear zone of inhibition formed around the a-factor secreting cells
but not around those secreting -factor (Fig. 8A–D) indicating that
a-factor (X2180-1B) were applied to the lawn. A
a
the RC757 cells respond specifically to a-factor. A previously char-
acterized, synthetic a-factor with wild-type potency31–34 also stim-
ulated growth arrest with an endpoint of 0.12 ng; the synthetic
material produced in this study yielded an identical endpoint
(see Fig. 8A and B). Next the biological activity of the a-factor ana-
logues containing the photoactive isoprenoids was evaluated using
the same assay. Analogue 2 containing the shorter isoprenoid
spacer gave an endpoint of 0.25 ng (see Fig. 8D) while analogue 3
functionalized with the longer isoprenoid spacer gave an endpoint
of 0.50 ng (see Fig. 8C). To ensure that the observed growth arrest
was not simply due to general toxicity, control experiments were
minus with the Boc group, 11
in DMF. Di-tert-butyl dicarbonate (44
(88 mol, 153 L) were added, and the reaction tumbled for 4 h.
The resin was washed with DMF and CH2Cl2 and was dried in va-
cuo to yield 7. To the resin was then added Cu(II)acetate (11 mol,
20 mg), and the resin was then swollen with 2 mL anhydrous
CH2Cl2 under N2. Glacial acetic acid (6.0 mmol, 400 L), anhydrous
pyridine (3.5 mmol, 280 L), and anhydrous methanol (21 mmol,
880 L) were added to the resin. Oxygen was bubbled through
the reaction for about 1 min and the reaction was agitated under
an oxygen atmosphere for 2.5 h. The resin was removed by filtra-
tion and washed with CH2Cl2 and methanol. Most of the solvents
were removed by rotary evaporation, and the oily residue was
lyophilized from dioxane to produce crude 8. The solid that re-
sulted was treated with freshly prepared Reagent K (8 mL) for
30 min to remove protecting groups. The peptide was precipitated
by the addition of 80 mL ether. After centrifugation to form a pellet,
which was washed twice with ether, the peptide was dissolved in
about 5 mL methanol, filtered to remove copper salts, and purified
l
mol of peptide resin was swollen
lmol, 96 mg) and DIEA
l
l
l
l
l
l
performed using a MATa strain (LM102), which expresses the
factor receptor and thus does not respond to a-factor.
a-
Using LM102, no growth arrest was observed in response to
cell-secreted a-factor, but growth arrest was observed in response
to cell-secreted
impregnated with synthetic
a
-factor ( Fig. 8E–H) and in response to a disc
-factor (Fig. 8E), thus indicating that
a
these cells were sensitive to their cognate pheromone. The LM102
cells did not yield a zone of growth inhibition in response to any of
the a-factor peptides indicating that compounds 1, 2, and 3 are not
toxic and are mating type specific (Fig. 8E–H). Thus, based on the
endpoint determinations reported above, the a-factor produced
via this new route is equipotent with naturally-derived material
and the photoactive analogues retain most (25–50%) of the biolog-
ical activity of the parent a-factor pheromone.
by RP-HPLC. Yield 21.4 mg (14% from 110 lmol peptide resin), pur-
ity by HPLC: 90%. ESI-MS: calcd: 1424.7, found: 1424.6.
tR = 37.6 min.
4.2.2. YIIKGVFWDPAC(Fr)-OCH3 (1)
YIIKGVFWDPAC-OCH3 (9, 10 mg, 7.0
DMF/1-butanol (2:1 v/v, 6.0 mL). Farnesyl bromide (35
5 equiv, 10 L) was added to the reaction and an HPLC analysis
performed. Then, Zn(OAc)2ꢁ2H2O (35 mol, 5 equiv, 8 mg) dis-
lmol) was dissolved in
l
mol,
l
3. Conclusion
l
solved in 0.10% aqueous TFA (2.0 mL) was added to initiate the
alkylation reaction. The reaction was monitored by RP-HPLC, and
once judged complete (typically 1 h), the solution was filtered,
and purified by RP-HPLC. Yield 1.0 mg (9%), purity by HPLC: 93%.
ESI-MS: calcd: 1628.9, found: 1628.8. tR = 58.7 min. In a second
An efficient synthesis of a-factor peptide from S. cerevisiae using
Fmoc solid phase methods is reported here. This synthesis has been
used to produce a-factor as well as two benzophenone-containing
analogues. The photoactive probes retain near-wild type levels of
activity in a yeast growth arrest assay. The simplified route to a-
factor analogues described herein should facilitate investigation
of a-factor receptor and other proteins that interact with prenylat-
ed peptides and proteins.
synthesis, 9 (12.6 mg, 8.8 lmol) was dissolved in DMF/1-butanol
(2:1 v/v, 8.0 mL). It should be noted (see below) that subsequent
DTNB titration50 of the sample of 9 used for this reaction indicated
that the true peptide content of the material was only 5.8
due to the presence of water and/or TFA salts. Farnesyl bromide
(44 mol, 5 equiv, 12 L) was added to the reaction and an HPLC
mol, 5 equiv,
lmol
4. Experimental
4.1. General
l
l
analysis was performed. Then, Zn(OAc)2ꢁ2H2O (44
l
9.6 mg) dissolved in 0.10% aqueous TFA (1.0 mL) was added to ini-
tiate the thiol alkylation. The reaction was monitored by RP-HPLC
and once judged complete (within 1 h), the solution was filtered,
and purified by RP-HPLC. The mass of the product was 2.8 mg
All solvents were of HPLC grade. DIEA and TFA were of Sequalog/
peptide synthesis grade from Fisher. Hydrazinobenzoyl AM Nova-
Gel™ resin was obtained from Novabiochem. Farnesyl bromide
(>95% E,E) was obtained from Sigma–Aldrich. Standard Fmoc/HCTU
chemistry was used for SPPS of the peptides. Vydac 218TP54 and
218TP1010 columns were used for analytical and preparative RP-
HPLC, respectively. All analytical and preparative RP-HPLC solvents,
water and CH3CN, contained 0.10% TFA. Retention times (tR) are
(1.7
l
mol) but UV measurement based on tryptophan absorbance
indicated 3.8 mol present. Using these
(e280 = 6990 Mꢂ1 cmꢂ1 51
)
l
numbers, yields of 19% yield based on mass and 65% based on
DTNB/UV measurements were calculated. A purity of 98% was
determined by HPLC. ESI-MS: calcd: 1628.9, found: 1628.8.
tR = 58.7 min.

496
D. G. Mullen et al. / Bioorg. Med. Chem. 19 (2011) 490–497
4.2.3. YIIKGVFWDPAC(BC5)-OCH3 (2)
4.5. Biological assays
YIIKGVFWDPAC-OCH3 (9, 10 mg, 7.0
lmmol) was dissolved in
DMF/1-butanol (2:1 v/v, 6.0 mL). To purify Br-BC5 [(E)-(3-((4-bro-
Saccharomyces cerevisiae strains RC757 (MATa sst2-1 rme1 his6
mo-2-methylbut-2-enyloxy)methyl)phenyl)(phenyl)methanone]21
met1 can1 cyh2) and LM102 (MATa, bar1, his4, leu2, trp1, met1,
ura3, FUS1-lacZ::URA3, ste2-dl) were used to test biological activity
as previously described.34 The strain LM102 bears the plasmid
(35 lmol, 12 mg) before reaction, it was dissolved in 0.50 mL of
DMF and loaded onto a C18 SepPackÒ column that had been
equilibrated with 5% CH3CN in aq. 0.10% TFA. The column was
washed with 10 mL of the 5% CH3CN solution, followed by
10 mL of 30% CH3CN. The purified bromide, Br-BC5, was then
eluted from the column with 5.0 mL DMF directly into the reac-
tion flask that contained the peptide. Then, Zn(OAc)2ꢁ2H2O
pBEC253 encoding Ste2p, the
a-factor receptor. RC757cells were cul-
tured in YEPD (1% yeast extract, 2% peptone, 2% dextrose) while
LM102 cells were cultured in MLT54 to ensure maintenance of the
plasmid. Cells were grown overnight at 30 °C with shaking in liquid
medium. For use in the growth arrest assay, cells were harvested by
centrifugation (1000 g), washed twice with sterile water, and resus-
pended to a final concentration of 1 ꢃ 106 cells/mL in water. The cell
suspension (1 mL) was combined with 3 mL Noble agar (1.1% in
water) and overlaid onto solid medium (YEPD or MLT containing
(35 lmol, 5 equiv, 8 mg) dissolved in 0.10% aqueous TFA
(2.0 mL) was added to initiate the alkylation reaction. The reac-
tion was monitored by RP-HPLC, and once judged complete (typ-
ically 1 h), the solution was filtered, and purified by RP-HPLC.
Yield 1.3 mg (8% by mass, note comments above concerning 1),
purity by HPLC: 90%. ESI-MS: calcd: 1702.8, found: 1702.8.
tR = 50.1 min.
2% agar). The peptides were dissolved in MeOH (10 ng/
luted in 0.5% bovine serum albumin (BSA) to a final concentration
of 0.8 ng/ L, then serially diluted in 0.5% BSA to generate solutions
lL) and di-
l
of the desired concentrations. Five microliters of each dilution was
spottedontotheoverlaycontainingRC757orLM102cells. Theplates
were spotted in triplicate, and incubated overnight at 30 °C. The
experiment was repeated twice with similar results. The endpoint
of the assay was determined to be the lowest concentration at which
a clear zone of inhibition, which indicates growth arrest, was ob-
served. Wild type S. cerevisiae strains X2180-1A (MATa) and
4.2.4. YIIKGVFWDPAC(BC10)-OCH3 (3)
YIIKGVFWDPAC-OCH3 (9, 10 mg, 7.0 lmol) was dissolved in
DMF/1-butanol (2:1 v/v, 6.0 mL). To purify Br-BC10 [(3-(((2E,6E)-
8-bromo-2,6-dimethylocta-2,6-dienyloxy)methyl)phenyl)(phenyl)-
methanone]26 (28
lmol, 4 equiv, 12 mg) before reaction, it was
dissolved in 0.50 mL of DMF and loaded onto a C18 SepPackÒ col-
umn that had been equilibrated with 5% CH3CN in aq. 0.10% TFA.
The column was washed with 10 mL of the 5% CH3CN solution, fol-
lowed by 10 mL of 30% CH3CN. Br-BC10 was then eluted from the
column with 5.0 mL DMF directly into the reaction flask that con-
X2180-1B (MAT
endogenous sources for a-factor and
tide WHWLQLKPGQPNle12Y55 impregnated onto sterile paper disks
a) were cultured on YEPD medium and used as
a
-factor, respectively. The pep-
was used as a source of synthetic a-factor.
tained the peptide. Then, Zn(OAc)2ꢁ2H2O (35
lmol, 5 equiv, 8 mg)
Acknowledgments
dissolved in 0.10% aqueous TFA (2.0 mL) was added to initiate
the alkylation reaction. The reaction was monitored by RP-HPLC,
and once judged complete (typically 1 h), the solution was filtered,
and purified by RP-HPLC. To separate the product from unreacted
Br-BC10 it was necessary to use a preparative C4 column. Yield
1.7 mg (14% by mass, note comments above concerning 1), purity
by HPLC: 97%. ESI-MS: calcd: 1770.9, found: 1770.8. tR = 45.5 min.
The authors thank Bruce Witthuhn at University of Minnesota
Center for Mass Spectrometry and Proteomics for assistance in per-
forming the mass spectrometry experiments. This research was
supported by the National Institutes of Health Grants GM58442
(M.D.D.) and GM22087 (J.M.B.).
Supplementary data
4.3. Mass spectrometric analysis of a-factor, 1, and photoactive
analogues 2 and 3
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.11.006.
A small amount of lyophilized powder from 1, 2 or 3 was dis-
solved in 10
lL of 0.1% TFA/CH3CN and further diluted 1:50 in
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