Synthesis and NMR Characterization of the Prenylated Peptide, a-Factor

Article abstract of DOI:10.1016/bs.mie.2018.09.025

Protein and peptide prenylation is an essential biological process involved in many signal transduction pathways. Hence, it plays a critical role in establishing many major human ailments, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), malaria, and Ras-related cancers. Yeast mating pheromone a-factor is a small dodecameric peptide that undergoes prenylation and subsequent processing in a manner identical to larger proteins. Due to its small size in addition to its well-characterized behavior in yeast, a-factor is an attractive model system to study the prenylation pathway. Traditionally, chemical synthesis and characterization of a-factor have been challenging, which has limited its use in prenylation studies. In this chapter, a robust method for the synthesis of a-factor is presented along with a description of the characterization of the peptide using MALDI and NMR. Finally, complete assignments of resonances from the isoprenoid moiety and a-factor from COSY, TOCSY, HSQC, and long-range HMBC NMR spectra are presented. This methodology should be useful for the synthesis and characterization of other mature prenylated peptides and proteins.

Full text of DOI:10.1016/bs.mie.2018.09.025

ARTICLE IN PRESS
Synthesis and NMR
Characterization of the Prenylated
Peptide, a-Factor
1
Taysir K. Bader, Todd M. Rappe, Gianlugi Veglia, Mark D. Distefano
University of Minnesota, Twin Cities, Minneapolis, MN, United States
1
Corresponding author: e-mail address: diste001@umn.edu
Contents
1
2
. Introduction
. Synthesis of a-Factor
2
4
2
2
2
2
2
2
2
.1 Fmoc-Cys-OMe Synthesis
4
6
7
9
12
16
17
20
20
21
28
30
30
.2 Loading of Trityl Chloride Resin
.3 Quantification of Loading Using Fmoc-Assay
.4 SPPS of a-Factor Precursor
.5 HPLC Purification of Unfarnesylated a-Factor
.6 Farnesylation of a-Factor
.7 HPLC Purification of Farnesylated a-Factor
3
4
. Analysis of a-Factor
3
3
. Summary
Acknowledgments
References
.1 MALDI-MS Analysis
.2 NMR Analysis
Abstract
Protein and peptide prenylation is an essential biological process involved in many sig-
nal transduction pathways. Hence, it plays a critical role in establishing many major
human ailments, including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS),
malaria, and Ras-related cancers. Yeast mating pheromone a-factor is a small dode-
cameric peptide that undergoes prenylation and subsequent processing in a manner
identical to larger proteins. Due to its small size in addition to its well-characterized
behavior in yeast, a-factor is an attractive model system to study the prenylation path-
way. Traditionally, chemical synthesis and characterization of a-factor have been chal-
lenging, which has limited its use in prenylation studies. In this chapter, a robust method
for the synthesis of a-factor is presented along with a description of the characterization
of the peptide using MALDI and NMR. Finally, complete assignments of resonances from
Methods in Enzymology
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2018.09.025
# 2018 Elsevier Inc.
All rights reserved.
1

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Taysir K. Bader et al.
the isoprenoid moiety and a-factor from COSY, TOCSY, HSQC, and long-range HMBC
NMR spectra are presented. This methodology should be useful for the synthesis
and characterization of other mature prenylated peptides and proteins.
1. INTRODUCTION
Protein and peptide prenylation is an essential biological process
involved in many signal transduction pathways (Kassai & Fukada, 2011).
It involves the covalent attachment of an isoprenoid moiety to the cysteine
residue of a C-terminal CaaX box sequence via a thioester linkage (Casey &
Seabra, 1996). The attached isoprenoid unit can be either a 15-carbon farne-
syl group or a 20-carbon geranylgeranyl group, and facilitates anchoring in
the lipid membrane (Kassai & Fukada, 2011). Prenylated proteins and
changes in the levels thereof are at the heart of many significant human ail-
ments including malaria (Chakrabarti et al., 2002; Suazo, Schaber,
Palsuledesai, Odom John, & Distefano, 2016), Alzheimer’s disease (Eckert
et al., 2009), amyotrophic lateral sclerosis (ALS) (Li et al., 2016), as well
as many different cancers (Ochocki & Distefano, 2013). One noteworthy
lipidated protein is the oncogenic Ras protein, which is involved in more
than 30% of all human cancers. As such, understanding and inhibiting the
prenylation pathway have been a prime research target (Ochocki &
Distefano, 2013). The prenylation of the Ras protein (as well as other
prenylated proteins) with an isoprenoid group typically involves a three-step
pathway and three distinct enzymatic activities. It begins in the cytosol
where protein farnesyltransferase (PFTase) recognizes the CaaX sequence
and attaches a farnesyl group to the cysteine’s thiol. This leads to anchoring
in the lipid bilayer of the endoplasmic reticulum, where a membrane-
attached protease proteolytically cleaves off the “aaX” sequence. Finally,
the cysteine’s newly exposed C-terminal carboxylate becomes methylated
by a membrane-bound carboxymethyl transferase to yield the fully modified
protein (Diaz-Rodriguez & Distefano, 2017).
Significant strides in understanding the prenylation pathway have
emerged as a result of studying the Saccharomyces cerevisiae mating pheromone
a-factor (Sinensky, 2000). a-Factor is a hydrophobic dodecapeptide that is
processed through the prenylation pathway (Caldwell, Naider, & Becker,
1
995). The small size of a-factor makes it an attractive system to probe var-
ious aspects of the prenylation pathway. However, availability of a-factor has
traditionally been limited due to its significant hydrophobicity as well as

ARTICLE IN PRESS
Synthesis and NMR Characterization of a-Factor
3
having an esterified C-terminus, which complicates its synthesis using stan-
dard Fmoc-based solid phase peptide synthesis (SPPS). Earlier strategies for
a-factor synthesis involved solution state fragment condensation or
HF-based SPPS methods (Diaz-Rodriguez & Distefano, 2017). Addition-
ally, while in Vitro enzymatic farnesylation of CaaX substrates has been
described previously (Hosokawa et al., 2007; Palsuledesai et al., 2016;
Rowell, Kowalczyk, Lewis, & Garcia, 1997), we were not able to find evi-
dence of such a reaction applied to a-factor, likely due to the hydrophobic
nature of the peptides. Our group has developed more streamlined methods
for the synthesis of a-factor, first using hydrazine-containing resin followed
by oxidative resin cleavage (Mullen et al., 2011), and then more recently by
side-chain anchoring to trityl chloride resin (Diaz-Rodriguez, Ganusova,
Rappe, Becker, & Distefano, 2015; Diaz-Rodriguez, Mullen, Ganusova,
Becker, & Distefano, 2012). The later method showed greater versatility
and marked increases in yield with minimal epimerization observed after
prolonged exposure to basic conditions mimicking extended rounds of
SPPS coupling deprotection, and hence that procedure is described here.
In brief, Fmoc-Cys-OMe is synthesized and coupled to a trityl-chloride
resin via its thiol side-chain. The peptide is next extended via standard Fmoc
chemistry and is then cleaved from resin and side-chain-deprotected using
acidic conditions. The resulting peptide is subsequently purified and
farnesylated in solution, followed by HPLC purification to homogeneity.
This methodology has been employed to synthesize a-factor analogs and
incorporate farnesylated peptides with C-terminal methyl esters into whole
proteins using native chemical ligation (Zhang et al., 2017).
Once the final peptide is obtained and purified, it can be further analyzed
using mass spectrometry and NMR. Here, MALDI was used for mass
spectrometric analysis, as it resulted in better ionization of the peptide
and simpler spectra relative to ESI. In fact, MALDI typically produces singly
charged species and more predictable adducts (Nadler et al., 2017). Next,
NMR was used to study the chemical structure of a-factor at the atomic
level. Previous NMR studies showed that a-factor adopts a predominantly
disordered conformation in DMSO, which is largely unaffected by
farnesylation (Gounarides, Broido, Xue, Becker, & Naider, 1991). There-
fore, the NMR experiments presented here focus on the characterization of
the a-factor peptide using J coupling experiments to validate the chemical
structure of a-factor and its analogues. (Diaz-Rodriguez & Distefano,
2017; Diaz-Rodriguez et al., 2012; Vervacke et al., 2014). The experiments
and the methods described here can be used in structural studies of other
prenylated peptides of biological importance.

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Taysir K. Bader et al.
2. SYNTHESIS OF A-FACTOR
2
.1 Fmoc-Cys-OMe Synthesis
The first step in the synthesis of a-factor is to obtain Fmoc-protected cysteine
with a free side-chain thiol and C-terminal methyl ester. This can be
achieved through an acid catalyzed reaction of commercially available
Fmoc-L-Cys hydrate with methanol, as outlined in Scheme 1. This
reaction has also been employed to produce other alkyl esters of a-factor
(Diaz-Rodriguez et al., 2015).
2
•
•
•
•
.1.1 Equipment
TLC plates
Shortwave UV light
Standard synthesis labware
Rotary evaporator
2
•
•
•
•
•
.1.2 Reagents
Fmoc-L-cysteine hydrate
Reagent grade methanol
Concentrated hydrochloric acid
Acetone
50:50 Hexanes/ethyl acetate
2.1.3 Procedure
1
. Dissolve 361mg (1.00mmol) Fmoc-L-cysteine hydrate in 10.0mL of
methanol. The solution should be clear and colorless. Add 6–10 drops
of concentrated HCl and stir the solution overnight with a magnetic
stir bar.
2
. Within 24h, a white paste suspended in a slurry should be observed.
Gently shake to homogenize the solution then draw one drop, dilute
in acetone then spot on a TLC plate, and develop using 50:50
Scheme 1 Synthesis of Fmoc-L-CysOMe.

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Synthesis and NMR Characterization of a-Factor
5
hexane/ethyl acetate. Check for complete conversion of the starting
materials under shortwave UV light. The R of Fmoc-L-CysOMe is
f
0
.9, while the corresponding value for Fmoc-L-CysꢀH O is 0.3.
2
3
. Proceed to the next step regardless of the extent of completion of the
reaction. Dilute the solution with acetone until a clear, colorless solution
is observed. Transfer to a preweighed round-bottom flask, then remove
solvent in vacuo using a rotary evaporator. The heating bath temperature
should not exceed 30°C, since at elevated temperatures the product can
phase separate as an oil instead of a white precipitate powder. Once the
solvent is evaporated, remove from the heat and put the precipitate
under vacuum for several hours to remove any residual solvent.
4
5
. If the initial reaction did not proceed to completion, repeat steps 1 and 2
to convert any remaining starting material; otherwise proceed to step 5.
. Confirm the identity of the product by dissolving a small amount
1
(
ꢁ10mg) in 0.7mL CDCl and then acquire H 1D NMR spectrum.
3
Full assignment of the molecule resonances is shown in Table 1. Addi-
tionally, completion of the reaction can be further monitored by com-
1
paring the H spectrum of the product to that of the starting material; no
starting material should be observed. Furthermore, if any remaining
1
Table 1 H NMR Assignment of Fmoc-L-CysOMe in CDCl
3
was Carried Out With TMS as
an Internal Standard
Assignment
H–H (Hz) (From Scheme 1)
1
H Chemical Shift δ (ppm) Integration Multiplicity
J
4
1
3
3
4
4
4
5
7
7
7
7
.39
.03
.83
.26
.44
.67
.71
.35
.44
.64
.8
1
2
3
1
2
1
1
2
2
2
2
t
8.65
7.87
—
H
H
H
H
H
H
3
1
6
5
2
d
s
t
6.85
6.30
3.71
—
d
t
b
t
NH
8
7.20
7.48
7.48
7.6
H
9
t
H
7
d
d
H
1
0
H

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Taysir K. Bader et al.
1
solvent peaks are observed in the H NMR spectrum, the flask should be
subjected to vacuum for a few more hours to completely remove it.
. Weigh the flask after no solvent remains and calculate the final yield.
A 99% yield is typical for this protocol.
6
2
1
2
.1.4 Notes
. The final product should be stored at 2–8°C under nitrogen prior to use.
. The reaction can be scaled up or down as needed. It was successfully car-
ried out under the same conditions on a 0.1, 0.2, 0.4, 4, and 10mmol
scales. 15 Drops HCl were used when carried out on 4 and 10mmol
scales.
2
.2 Loading of Trityl Chloride Resin
Once Fmoc-L-CysOMe is obtained, it can be side-chain anchored to trityl
chloride resin as shown in Scheme 2. Trityl chloride resin is chosen for two
reasons; it offers high yields (Diaz-Rodriguez et al., 2015), and little cysteine
epimerization is observed during loading (Barany, Han, Hargittai, Liu, &
Varkey, 2003).
2
•
.2.1 Equipment
Rotator
•
Fritted polypropylene syringe
Syringe stopcock
•
2
•
.2.2 Reagents
Trityl chloride resin
Diisopropylethylamine
Synthesized Fmoc-L-CysOMe
•
•
3
Scheme 2 Loading of Fmoc-Cys-OCH onto trityl chloride resin.

ARTICLE IN PRESS
Synthesis and NMR Characterization of a-Factor
7
2
.2.3 Procedure
1
. Load 0.2mmol resin into fritted syringe, then wash with 5mL DMFꢂ3
times, followed by a wash with 5mL CH Cl ꢂ3 times. For the CH Cl
2
2
2
2
washes, place syringe on rotator for 5min each time to swell it. Drain the
resin after each wash.
2
3
. Dissolve 286mg (4.00M equiv.) of Fmoc-L-CysOMe in 3mL CH Cl2
2
and add 140μL (4.00M equiv.) of diisopropylethylamine to the solution.
Add this solution to the resin and leave overnight on rotating mixer.
. The next day, add 200μL methanol to the syringe containing the reac-
tion and place on the rotating mixer for 15min. This will cap any
unreacted sites, resulting in fewer side reactions. Do not leave the reac-
tion mixture on the rotating mixer for extended periods as the methanol
can displace the Fmoc-L-CysOMe.
4
. Drain the solution and then wash with 5mL DMFꢂ3 times followed by
5
mL CH Cl ꢂ3 times. Dry resin by pulling vacuum through the
2
2
syringe for at least 1h. Store the resin at 2–8°C under nitrogen.
2
1
.2.4 Notes
. Amount of resin used will vary from batch to batch depending on the
resin substitution.
2
. Unmodified trityl chloride resin should be stored at 2–8°C under nitro-
gen. Failure to do so can lead to hydrolysis, which will lead to lower cou-
pling efficiencies. If hydrolysis occurs, the resin can be regenerated using
HCl, acetyl chloride, or SOCl (Garc ´ı a-Mart ´ı n, Bayo, Cruz, Bohling, &
2
Albericio, 2006)
3. Loadings of Fmoc-L-CysOMe in the range of 0.7–1.2mmol/g are com-
mon. While it is possible to obtain even higher loadings (up to
1.8mmol/g) with extended coupling times, this procedure is undesired
as it leads to decreased yields during subsequent peptide synthesis and
side-product formation due to aggregation of the peptide on resin
surface.
2.3 Quantification of Loading Using Fmoc-Assay
Once the resin loaded with Fmoc-Cys-OMe is obtained, it is important to
quantify the loading efficiency to be able to proceed with SPPS, as it is the
basis for all subsequent calculations such as amount of resin to use as well as
amount of amino acids and coupling reagent. The most prevalent method
relies on a cuvette-based quantitative Fmoc assay where the Fmoc group
on the loaded amino acid is deprotected using piperidine, and the resulting

ARTICLE IN PRESS
8
Taysir K. Bader et al.
pyridine-Fmoc adduct is quantified using a known molar absorptivity con-
stant (ε) value (Chan & White, 2000; Eissler et al., 2017). This method,
however, was found to give inconsistent results. To address that issue, a
more accurate standard-curve-based assay was developed and used here.
2
•
•
•
•
•
•
.3.1 Equipment
UV/vis plate reader
Standard 96-well plate
Hamilton syringes
25mL Volumetric flasks
20mL Glass vials
Eppendorf tubes
2
•
.3.2 Reagents
Fmoc-OSu
•
Absolute ethanol
20% Piperidine in DMF
•
2.3.3 Procedure
1
. In triplicate, weigh ꢁ10mg resin aliquots in glass vials and record the
exact weight.
2
. Using a Hamilton syringe, add 0.5mL 20% piperidine in DMF to each
vial and incubate for 30min to deprotect Fmoc groups from Fmoc-L-
CysOMe on resin. Make sure most of the resin beads are at the bottom
of the vial and in contact with the solution. In the meantime, prepare a
stock of 1mM Fmoc-OSu in ethanol, then dilute it in Eppendorf tubes
or glass vials as described in Table 2 to make a series of standards and a
standard blank. Also make a sample blank consisting of 0.5mL 20%
piperidine in DMF diluted to 25mL in ethanol using a volumetric flask.
. Use ethanol to dilute the deprotected samples and transfer them to 25mL
volumetric flasks. Adjust the volume to 25mL with more ethanol.
. In triplicate, transfer 150μL of each sample, sample blank, and standard
into a 96-well plate. Read absorbance at 301nm.
3
4
5
. Construct a standard curve by first subtracting standard H (standard
blank) from all other standard absorbance values, then plotting average
absorbance measurements against Fmoc-OSu concentration.
. Subtract the average sample blank measurement from each average sam-
ple measurement and then calculate the Fmoc concertation using the
best line fit to the standard.
6

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Synthesis and NMR Characterization of a-Factor
9
Table 2 Dilutions of Fmoc-OSu in Ethanol Used for Fmoc Quantitative Assay
Amount of
Standard to
Amount of Final
Ethanol to Concentration Absorbance
Standard Add (μL)
Source
Add (μL)
(mM)
Range
A
B
C
D
E
300
200
300
300
300
300
300
0
1mM Stock 0
1
1.820–1.875
1.300–1.391
0.999–1.029
0.626–0.689
0.443–0.468
0.344–0.353
0.276–0.295
0.204–0.220
1mM Stock 100
1mM Stock 300
0.75
0.5
C
D
E
300
300
300
300
300
0.25
0.125
0.063
0.031
0
F
G
H
F
—
7. Calculate loading for each sample according the following equation
before averaging:
Fmoc concetrationmM ꢂ0:025L
Resin loading ðmmol=gÞ ¼
:
Amount of resin cleavegeðgÞ
2.3.4 Notes
1
. While it is possible to make a larger amount of standard stock and store it
for later use, more reproducible results are obtained if this standard is
made freshly before use.
2
.4 SPPS of a-Factor Precursor
With the loaded trityl resin in hand, the rest of the peptide
YIIKGVFWDPAC-OMe) can be extended using standard Fmoc-
(
chemistry as seen in Scheme 3. A potential consideration in this process is
epimerization of the C-terminal cysteine due to repeated exposure to piper-
idine during the synthesis (Han, Albericio, & Barany, 1997). However,
using the side-chain anchoring methodology described here, negligible
amount of epimerization was observed (Diaz-Rodriguez et al., 2015).
The synthesis was carried out using an automated peptide synthesizer
(PS3, Protein Technologies Inc., Memphis, TN). Sufficiently high yields

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Taysir K. Bader et al.
Scheme 3 Synthesis of a-factor precursor peptide via standard solid phase peptide
synthesis starting with Fmoc-Cys-OCH anchored to trityl resin.
3
were obtained using the conditions described (>95% for crude peptide).
Based on these results, different types of automated synthesizers or manual
synthesis should also give similar results.
2
•
•
•
•
.4.1 Equipment
Peptide synthesizer
Fritted propylene syringe and syringe stopcock
Rotating mixer
Centrifuge
2
•
.4.2 Reagents
Fmoc-L-CysOMe-loaded trityl resin
•
Coupling
agent.
O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HCTU) was used
•
Standard Fmoc-protected amino acids with acid labile side-chain
protecting groups
•
•
•
•
•
Activator base (20% N-methylmorpholine in DMF used here)
20% Piperidine in DMF
DMF
Dry ice
Acetone
2.4.3 Procedure
1
. Carry out synthesis on a 0.1-mmol scale. Swell the resin in DMF for
10min. Extend the peptide using single couplings with 4.0equiv. of acti-
vator and amino acids in activator base. Each coupling step should be
allowed to proceed for at least 30min. Deprotect amino acid side chains
during the synthesis by treatment with 20% piperidine in DMF for
10min twice. Repeat for the final deprotection. Formation of the free

ARTICLE IN PRESS
Synthesis and NMR Characterization of a-Factor
11
amine can then be observed using a ninhydrin test (Kaiser, Colescott,
Bossinger, & Cook, 1970).
2
3
. Once free amine formation is confirmed, wash resin with 5mL
DMFꢂtimes, followed by CH Cl ꢂ3 times. Make 10mL Reagent
2
2
K (8.25mL TFA, 500mg phenol, 500μL H O, 500μL anisole, 250μL
2
1
,2 ethanedithiol) and sparge nitrogen through solution for 10min to
remove oxygen from it. Add to resin and place on rotary mixer for
h to cleave peptide.
2
. After 2h, fill two 50-mL falcon tubes with 30mL diethyl ether each,
then split the cleavage cocktail into both tubes. Wash resin with 5 more
mL of TFA, then split into falcon tubes again. Dilute to the top with
more ether, then cool tubes using acetone/dry ice bath. Spin down at
4000 RPM for 5min to pellet peptide. Decant ether, refill tubes with
fresh diethyl ether, and vortex tubes to resuspend pellet in solution.
Once the pellet is resuspended, centrifuge and repeat the wash one more
time for a total of three precipitation steps.
4
5
. Use ꢁ5 methanol to transfer peptide to preweighed glass vial, then dilute
with equal amount H O. Evaporate methanol and remaining ether using
2
a gentle stream of nitrogen until ꢁ5mL of solution remains. This could
take several hours. Flash-freeze using liquid nitrogen and then place on a
lyophilizer overnight.
. The correct product can be confirmed using MALDI (Section 2.4) after
solubilization as described in Section 2.5.3, step 6.
2
1
.4.4 Notes
. The synthesis was also performed using a microwave-assisted synthesizer
(CEM Liberty/Discover microwave synthesizer). In this case double
couplings were used. The coupling reagent was HBTU and the activator
base was 35% v/v DIEA in NMM. The synthesis with the CEM micro-
wave synthesizer gave similar results to the PS3 synthesizer. It is also
worth mentioning that very little epimerization of the C-terminal cys-
teine residue was observed as determined by NMR integration (<5%).
. Reagent K is extremely caustic, and so all handling should be done inside
a well-ventilated fume hood. Appropriate safety precautions should be
taken into consideration as well when handling of TFA, as it is highly
corrosive.
2
3
. It is possible to shorten step 4 in length by omitting the methanol transfer
and lyophilizing the peptide directly in the falcon tubes after the last
ether precipitation. This, however, could lead to inaccuracy when

ARTICLE IN PRESS
12
Taysir K. Bader et al.
measuring the mass of the crude material. To acomplish this, begin by
preweighing the falcon tubes before step 3. After decanting the ether
layer at the end of step 3, remove any remaining ether using a gentle
stream of nitrogen applied for at least 30min. Once the ether has evap-
orated completely, suspend the peptide in 5mL H O (solution will be
2
cloudy), flash-freeze, and place the suspension on a lyophilizer.
4
. Lyophilizing the peptide suspended in water as opposed to simply
removing the solvent in vacuo or using N stream results in a powder
2
rather than a residue, which helps with subsequent handling.
2
.5 HPLC Purification of Unfarnesylated a-Factor
The crude a-factor precursor peptide has low solubility in aqueous solutions
and many organic solvents. This can be due, in part, to adventitious disulfide
bond formation. Additionally, the solubilized peptide tends to precipitate
out remarkably fast, likely due to nonsolubilized material acting as nucle-
ation sites. A method has been developed here to solubilize the peptide
completely and reduce any disulfide bonds using DTT. One issue that
was encountered was that the peptide requires acidic conditions to dissolve,
but DTT is more reactive for disulfide reduction under basic conditions.
This issue was overcome by employing stepwise addition of organic solvents
to a NaHCO buffered aqueous DTT solution with extended sonication in
3
between, followed by the addition of a large excess of TFA.
2
•
•
•
•
•
.5.1 Equipment
HPLC instrument
Semi-prep RP C-18 HPLC column (9.4ꢂ250mm column used)
Analytical RP C-18 HPLC column (4.6ꢂ250mm column used)
Sonicator
Lyophilizer
2
•
•
•
•
•
•
•
.5.2 Reagents
HPLC grade water with 0.1% TFA (buffer A)
HPLC grade CH CN with 0.1% TFA (buffer B)
3
HPLC grade water buffered to pH 8.0 with NaHCO3
HPLC grade CH CN
3
TFA
HPLC grade methanol
0.5M DTT aqueous solution

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Synthesis and NMR Characterization of a-Factor
13
2
.5.3 Procedure
1
. Equilibrate the column with buffer A containing 1% buffer B. This
should be done for at least 45min to avoid that the peptide eluting pre-
maturely from the column due to the large amount of TFA utilized for
its solubilization. If an analytical column is used, a flow rate of
1
mL/min is appropriate. For a semi-prep column, it is advisable to
use a flow rate of 3mL/min.
2
. To a 10mg powder of crude peptide add 1mL of HPLC grade water
buffered to pH 8.0 with NaHCO and 30μL 0.5M DTT solution, then
3
sonicate for 15min.
3
4
5
. Add 1mL HPLC grade CH CN and sonicate for 15min.
3
. Add 1mL HPLC grade methanol and sonicate for 15min.
. Add TFA until solution becomes clear. Anywhere from 0.1 to 1mL can
be expected depending on the amount of peptide used as well as the
impurities present. Some particulates may still be floating in solution.
The least amount of TFA required should be used for solubilization
as it interferes with the purification where it will prevent the peptide
from binding the column resin.
6
7
. Centrifuge at 4000 RPM for 5min. This should be done immediately,
as a-factor can precipitate out of solution if particulates are present. If
this occurs, an additional sonication step and more TFA can be used to
resolubilize peptide. A small pellet may appear at the bottom.
. Transfer the supernatant to a new falcon tube being careful not to dis-
turb the pellet. Centrifuge again to ensure no particulates were trans-
ferred over. Dilute at least twofold with 50:50 buffer A/buffer
B solution. This is critical as the excess amount of TFA used can cause
the peptide to elute prematurely.
8
9
. Inject the desired amount into HPLC injection loop. For the analytical
column, 20μL peptide solution diluted to 100μL in 50:50 buffer with a
2
00-μL injection loop gave the best results. For the semi-prep column,
up to 4mL of solution can be used with a 5-mL injection loop.
. Run appropriate method. Fig. 1 legend outlines the event sequences
for analytical and semi-prep columns. One important consideration
is the extended period at beginning of the run where buffer
B remains constant at 1%. This is done to help prevent the peptide from
eluting prematurely. If that occurs, the peptide typically elutes during
this initial wash period and should be collected. The presence of pep-
tide can then be confirmed using MALDI (Section 2.4).

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Taysir K. Bader et al.
Fig. 1 (A) Crude semipreparative chromatogram of a-factor precursor peptide. The
absorbance was monitored at 254nm to observe aromatic groups and a flow rate of
3
mL/min. Gradient increased from 1% to 100% buffer B over 100min with 10-min delay
at the beginning of method. Fractions were collected between 40 and 60min at 1-min
intervals. (B) Analytical chromatogram of purified a-factor precursor. The absorbance
was monitored at 220nm to show all contaminants and a flow rate of 1mL/min. Gradi-
ent increased from 1% to 100% buffer B over 25min with 5-min delay at the beginning
of method.

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Synthesis and NMR Characterization of a-Factor
15
1
0. Unfarnesylated a-factor elutes between 60% and 80% buffer B,
depending on the size of the column, with larger columns having lower
retention times. Because of this, it is recommended to collect all frac-
tions in that region and keep the ones exhibiting the correct m/z ratio
(as measured by MALDI-MS). HPLC chromatograms of the crude
peptide and purified material are shown in Fig. 1A and B, respectively.
1. Pool together the fractions displaying the correct m/z ratio based on
desired purity as determined by MALDI or analytical HPLC integra-
tion (>90% required for NMR analysis, and>70% is required for
farnesylation reaction). For pure fractions, proceed to step 12. For
impure fractions, proceed to step 13.
1
1
2. Transfer pooled pure fractions to a preweighed glass vial and remove
excess organic solvent using a stream of N . This step can take several
2
hours. Once most of organic solvent is removed, the solution should be
cloudy white in appearance. Flash-freeze in liquid N and then place on
2
a lyophilizer overnight (2 days if NMR experiments are to be run on
sample to maximize water removal). Weigh the vial at the end to obtain
final mass of the purified material. The peptide should appear as a white
powder. 6–8mg of peptide can be obtained at the end depending on
purity of crude peptide. Final purified yields in the 60%–80% range
can be expected.
13. If the volume of impure fractions is sufficiently small, it can be directly
reinjected into the HPLC. Otherwise, remove the solvent using a gen-
tle stream of N and lyophilize as described in step 12, then dissolve the
2
resulting powder in 1:1 buffer A/buffer B and reinject into the HPLC.
2
.5.4 Notes
1
. The purified peptide should have improved solubility (relative to the
crude peptide) and can be dissolved in 1:1 buffer A/buffer B, DMSO,
or 4:2:1 DMF/1-BuOH/0.1% aqueous TFA in a concentration of up
to 5mg/mL.
2
3
4
. Once the peptide is solubilized, it is possible to accurately quantify its
amount using the Ellman’s assay for free thiols using previously published
procedure (Riddles, Blakeley, & Zerner, 1983).
. Multiple rounds of purification may be required to obtain highly pure
peptide. This can be determined via MALDI analysis as well as
analytical HPLC.
. The peptide should be stored at 2–8°C for short periods of time
(1–2 weeks), or at ꢃ20°C for extended periods.

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Taysir K. Bader et al.
5
. For semi-prep HPLC, monitoring at 254nm wavelength is rec-
ommended, as aromatic groups absorb at this wavelength. This simplifies
the spectrum and prevent oversaturating the detector.
6
7
. For analytical HPLC, monitoring at 220nm wavelength is
recommenced, as amide bonds absorbs in this region of the spectrum,
hence possible contaminants will be observed.
. Analytical chromatograms of HPLC fractions can be obtained by simply
injecting 100μL directly onto an analytical column and running the
analytical method.
2
.6 Farnesylation of a-Factor
This step is performed after the peptide synthesis and resin cleavage, since the
highly acidic conditions for global peptide deprotection and cleavage cause
isomerization and degradation of the isoprenoid group (Diaz-Rodriguez &
Distefano, 2017). The reaction employed here uses zinc acetate dihydride to
facilitate the nucleophilic reaction between the cysteinyl thiol moiety and
the alkyl halide of trans,trans-farnesyl bromide (Scheme 4). To overcome sol-
ubility problems and their effects on alkylation rate, a mixture of several dif-
ferent solvents was used (Diaz-Rodriguez et al., 2015). Those conditions
Scheme 4 Farnesylation of a-factor precursor peptide to yield a-factor.

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Synthesis and NMR Characterization of a-Factor
17
along with the use of partially purified a-factor precursor allowed the reac-
tion to proceed efficiently.
2
•
.6.1 Equipment
Standard synthetic chemistry labware
2
•
•
•
•
.6.2 Reagents
Purified or partially purified a-factor precursor
trans,trans-farnesyl bromide
Zn(OAc) ꢀH O
2
2
Degassed solvent blend: 4:2:1 DMF/1-BuOH/0.1% aqueous TFA
2
.6.3 Procedure
1
. Dissolve 10mg (7.0μmol) peptide in 3–10mL of solvent blend. The
amount required will vary depending on amount of impurities present.
The smallest amount possible should be used to simplify the subsequent
purification steps. The starting peptide needs to be at least partially puri-
fied (>70%), as crude peptide will likely have solubility issues.
. Add 6.5mg (5.0equiv.) of Zn(OAc) ꢀH O in 0.5mL H O containing
2
3
2
2
2
0.1% TFA to the solution.
. Dilute 9.5μL (5.0) equiv. of farnesyl bromide in 0.5mL of DMF, then
add it dropwise to the above solution while stirring. This step is critical to
ensure that the farnesyl bromide remains soluble. Watch closely for any
persisting undissolved materials. If observed, add more of the solvent
blend as needed in 0.1mL increments.
4
. Stir under nitrogen for 2–4h and then check for the reaction completion
using MALDI-MS or analytical HPLC. The reaction time may be
extended to obtain maximal conversion. Once the reaction is complete,
it can be purified using HPLC.
2
.6.4 Notes
1
. Farnesyl bromide is air and moisture sensitive, so its exposure to those
elements should be limited as much as possible. Unused material should
be stored under nitrogen at 2–8°C.
2.7 HPLC Purification of Farnesylated a-Factor
In this step, care must be taken to dilute the solution containing the crude
peptide appropriately to minimize the potential for premature elution. This
issue is similar to that noted in step 2.5. To avoid this, dilute the crude reaction

ARTICLE IN PRESS
18
Taysir K. Bader et al.
mixture, equilibrate the column with buffer A, and add an extended hold
period in the HPLC method with buffer A to prevent premature elution.
2
•
•
•
•
•
.7.1 Equipment
HPLC instrument
Semi-prep RP C-18 HPLC column (9.4ꢂ250mm column used)
Analytical RP C-18 HPLC column (4.6ꢂ250mm column used)
Sonicator
Lyophilizer
2
•
.7.2 Reagents
HPLC grade H O with 0.1% TFA (buffer A)
2
•
HPLC grade CH CN with 0.1% TFA (buffer B)
3
•
HPLC grade CH OH
3
2.7.3 Procedure
1
. Using a Falcon tube, dilute the crude reaction mixture at least twofold
with a 50:50 buffer A/B mixture. This is done to prevent premature elu-
tion due to the large amount of DMF used in the farnesylation reaction.
. Centrifuge at 4000 RPM for 5min to remove any particulates in the
reaction mixture.
2
3
4
. Inject onto the HPLC column and run appropriate method. See
Section 2.5.3 steps 8 and 9.
. The farnesylated peptide typically elutes in the range of 75%–85% buffer
B. Collect all peaks in this region and confirm the presence of the desired
product by MALDI-MS. HPLC chromatograms of the crude peptide
and purified material are shown in Fig. 2A and B, respectively.
. After pooling the appropriate fractions (>90% purity as determined
by MALDI or analytical HPLC integration), evaporate the organic
5
solvent using a stream of N . The resulting solution should be cloudy
2
when all the solvent is removed. This typically requires several hours.
Once completely removed, flash-freeze using liquid N and then lyoph-
2
ilize for at least 2 days to remove all water for subsequent NMR analysis.
2.7.4 Notes
1
. More than one round of chromatography may be required to obtain
highly pure a-factor since some impurities elute with similar retention
times. If that is observed by MALDI-MS, partially pure fractions can
be combined, lyophilized, and rechromatographed. Some a-factor fails
to adsorb to the column and elutes in the 1% buffer B hold phase due

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Synthesis and NMR Characterization of a-Factor
19
Fig. 2 (A) Crude semipreparative chromatogram of the farnesylation reaction. The
absorbance was monitored at 254nm to observe aromatic groups with a flow rate of
2
mL/min. Fractions were collected between 80 and 100min at 1-min intervals.
(B) Analytical chromatogram of purified a-factor precursor. The absorbance was mon-
itored at 220nm to show all contaminants with a flow rate of 1mL/min.
to the DMF used for the farnesylation reaction. If a significant amount is
observed (via MALDI-MS analysis), it can also be recombined with
other impure fractions and then repurified.

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Taysir K. Bader et al.
3. ANALYSIS OF A-FACTOR
3
.1 MALDI-MS Analysis
MALDI-MS allows the presence of the desired compound to be determined
in a reliable manner. MALDI was chosen over ESI for two reasons; first,
it generally affords monocharged species, which simplifies the analysis,
especially when disulfide bonds were present (Nadler et al., 2017). Second,
MALDI-MS tends to give more reproducible ionization of a-factor
(compared with ESI).
3
•
•
•
•
.1.1 Equipment
MALDI Instrument (AB SCIEX MALDI-TOF 5800 was used)
MALDI-MS plate
Microcentrifuge
Micropipettes
3
•
.1.2 Reagents
50:50 buffer A/ buffer B mixture
•
α-Cyano-4-hydroxycinnamic acid
3.1.3 Procedure
1
. In a microcentrifuge tube, thoroughly mix a spatula-full of matrix in
1
mL of 50:50 buffer A/buffer B mixture, then centrifuge for 3min at
maximum speed. This will yield a saturated solution of matrix.
. If DMF is present in the peptide sample, dilute it fivefold in 50:50 buffer
A/buffer B mixture.
2
3
. Spot 0.5μL of the matrix solution on the MALDI-MS plate, followed by
0
.5μL of sample. Mix by pipetting up and down a few times. Add
another 0.5μL of matrix solution and pipette up and down repeatedly
until small crystals are observed. Leave for a few minutes to dry. If
DMF was present in solution a small drop of liquid (instead of crystals)
may still be present after 10min. If that is the case, add 1μL of the 50:50
buffer A/buffer B mixture, mix by pipetting up and down and then leave
to dry again. Repeat until spot appears completely dry. Alternatively,
dilute 0.5μL of initial sample with 2μL of 50:50 buffer A/buffer
B mixture and spot 0.5μL of newly diluted sample.

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Synthesis and NMR Characterization of a-Factor
21
4
5
. Acquire MALDI-MS spectrum using positive reflector mode. Make sure
that the mass range encompasses at least double the peptide’s mass to
observe any dimerized peptide (disulfide-linked dimer).
+
+
. a-Factor typically appears as a mixture of [M+H] , [M+Na] , and
+
M+K] ions. Other ions may be observed however including
[
[
[
+
+
+
M+CH OH+H] , [M+CH CN+H] , [M+2NaꢃH] , and
3
3
+
M+CH CN+Na] .
3
6
. The correct m/z ratio for a-factor can be calculated using programs such
as ChemDraw that compute the monoisotopic mass. Sample MALDI-
MS spectra of purified unfarnesylated and farnesylated a-factor are shown
in Fig. 3A and B, respectively.
3
1
.1.4 Notes
. A calibrant should always be used to ensure accurate measurements. The
+
following calibration mixture was used: Leucine enkephalin ([M+H]
+
m/z¼556.2771), Bradykinin 2–9 ([M+H] m/z¼904.4680), Angio-
+
+
tensin I ([M+H] m/z¼1296.6850), Glu-fibrinogen ([M+H]
+
m/z¼1570.6770), and ACTH 18–39 ([M+H] m/z¼2465.1990).
2
. The matrix solution can be stored on the benchtop and reused. How-
ever, a blank MALDI-MS spectrum of the matrix solution alone should
be acquired each time to ensure that it has not been contaminated, and
also to compare to the peptide’s spectrum to avoid misidentifying the
matrix peaks as contaminants. Special care should be taken to avoid
labeling matrix clusters as contaminations (Smirnov et al., 2004).
. In the case of DTT reduction, the crude peptide spectrum will show the
3
4
+
+
[
M+Na] ion as the predominant species while the other two ([M+H]
+
and [M+K] ) may not be observed or only be observed at low levels.
. A good practice is to spot each sample twice (at different locations on the
plate) and acquire spectra of both. This is to prevent false-negative results
where some compounds fail to ionize due to inconstant cocrystallization
with the matrix.
3.2 NMR Analysis
For the complete chemical characterization of the peptide, four homonu-
1
clear ( H, H) and heteronuclear ( H, C) 2D NMR experiments were
1
1
13
1
necessary in addition to the basic H 1D experiment. COSY and TOCSY
1
1
were used to analyze the short and long range H– H coupled spin systems,
while HSQC and HMBC were used to analyze the short and long range
1
13
H– C coupled spin systems. The full assignment of farnesylated a-factor

Fig. 3 (A) Example MALDI spectrum of purified a-factor precursor. (B) Example MALDI spectrum of purified farnesylated a-factor. Notice the
ionic charged states.

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Synthesis and NMR Characterization of a-Factor
23
was achieved, allowing unambiguous structural analysis of a-factor and
a-factor precursors including specific stereochemical properties. For exam-
ple, the TOCSY experiment allows for analysis and rough quantification of
the extent of epimerization of the C-terminal cysteine residue (Diaz 2015),
as the presence of L and D cysteine gives rise to significantly different chem-
ical shifts.
3
•
.2.1 Equipment
DMSO-d -matched Shigemi NMR tube
6
•
Hamilton syringe
3
•
.2.2 Reagents
DMSO-d6
3
.2.3 Sample Preparation
•
Dissolve 1.5mg peptide in 300μL DMSO-d for a final concentration of
6
3
mM. Concentrations as low as 0.7mM have been shown to give suf-
ficient NMR signal (Diaz-Rodriguez et al., 2015), but higher concen-
trations will yield better spectra with stronger signal.
3
.2.4 NMR Experiments
All acquisition for the experiments reported here was done using a Bruker
1
13
15
Avance 700-MHz spectrometer with 5mm H– C– N TXI cryoprobe.
TopSpin was used to analyze all spectra.
1
1
3
.2.4.1 One-Dimensional H NMR (1D H NMR)
Acquire 1D proton NMR data. This can be extremely useful as a final quick
analytical tool, since it can be compared to known spectra to judge the qual-
ity of the sample before acquiring more time-consuming two-dimensional
1
experiments. A sample H 1D NMR spectrum is shown in Fig. 4. Assign-
1
ment of the H 1D spectrum of a-factor has been previously reported
1
Gounarides et al., 1991), as well as the H 1D spectrum of the farnesyl moi-
(
ety bonded to a factor (Anderegg, Betz, Carr, Crabb, & Duntze, 1988).
3
.2.4.2 COSY
1
1
Acquire COSY spectrum to gain primary assignment of H– H coupled spin
systems. The COSY spectrum will only show proton–proton coupled

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Taysir K. Bader et al.
13
12
11
10
9
8
7
6
5
4
3
2
1
0
ppm
1
6
Fig. 4 1D H NMR spectrum of farnesylated a-factor in DMSO-d . The water peak is
observed at 3.33, and DMSO peak is observed at 2.5 and was used for all calibrations.
Comparison to known 1D spectra offers a quick and reliable analytical method for
determining the presence of correct peptide as well as purity. Spectra were acquired
with 64 scans using 1s recycle delay, 30-degree pulse, 3.1s acquisition time with a
sweep width of 15ppm. Data were Fourier transformed with 0.3Hz line broadening
applied and manually phase-corrected. All acquisitions were done on a Bruker Avance
700-MHz spectrometer with a 5-mm TXI cryoprobe.
signals through less than three bonds. The assigned spectrum of a-factor is
shown in Fig. 5. The parameters used in the experiment are provided in
1
the figure legend. Assignments are based on the 1D H spectrum.
3.2.4.3 TOCSY
1
1
Acquire TOCSY spectra to observe long range H– H coupling (Bax &
Davis, 1985). The TOCSY experiment shows long range interactions
between coupled spins up to five continuous bonds. Assignments are based
1
on the COSY spectrum and the 1D H spectrum. The parameters used in
the experiment are described in Fig. 6.
3.2.4.4 HSQC
Acquire phase sensitive HSQC using Echo\Antiecho-TPPI gradient selec-
tion to gain primary assignment of the backbone (Boyer, Johnson, &
Krishnamurthy, 2003; Palmer, Cavanagh, Wright, & Rance, 1991).

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Synthesis and NMR Characterization of a-Factor
25
1
1
Fig. 5 2D COSY NMR spectrum of farnesylated a-factor in DMSO-d
6
, showing H– H
short range coupled spins (i.e., three bonds or less). Assignments are shown for each
peak in the spectrum. Peaks labeled with asterisk are shown at a twofold to fourfold
higher contour level. (A) Assigned aliphatic region. (B) Assigned backbone region. Spec-
trum was acquired using 64 scans/increment, 16 dummy scans, 0.8s recycle delay, and a
spectral widths of 15ppm. 6300ꢂ256 points were collected in the direct and indirect
dimensions, respectively. Data were zero-filled with 8192ꢂ1024 points and Fourier-
transformed with a shifted sine-bell squared function with 1.00ꢂ0.30Hz line broaden-
ing applied. 2ꢂ Linear prediction was applied in the indirect dimension. Sine bell shift
was 0ꢂ0 and Gaussian max position 0ꢂ0.1.

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Taysir K. Bader et al.
1
1
Fig. 6 2D TOCSY NMR spectrum of farnesylated a-factor in DMSO-d showing H– H
6
long range coupled spins (up to five bonds). Assignments are shown adjacent to each
peak. Peaks labeled with asterisk are shown at a twofold to fourfold higher contour level.
(
A) Assigned aliphatic region. (B) Assigned backbone region. ROESY peaks appear as
negative resonances and are omitted from the spectrum for clarity. Spectral parameters
were identical to the COSY spectrum except that the sine bell shift was 2ꢂ2 and the
Gaussian max position was 1ꢂ1.

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Synthesis and NMR Characterization of a-Factor
27
Fig. 7 See figure legend on next page.

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28
Taysir K. Bader et al.
This type of experiment only observes one bond carbon–hydrogen coupling
Schleucher et al., 1994). Peaks arising from CH or CH groups appear in
(
1
3
the positive region, while peaks from CH appear in the negative region.
2
The spectrum can be manually phased. The assignments are based on the
1 13
D H spectrum and assisted by the C chemical shift values predicted using
1
ChemDraw. The assigned spectrum of a-factor is shown in Fig. 7. The
parameters used for the data acquisition are described in the figure legend.
3.2.4.5 HMBC
Acquire phase sensitive HMBC using an echo/antiecho gradient selection
with a threefold low pass J-filter to suppress single bond correlations and
obtain long range interactions (Cicero, Barbato, & Bazzo, 2001). In this
experiment, coupled spin systems up to three continuous carbon–carbon
1
bonds were observed. The assignments are based on the 1D H and HSQC
spectra. An assigned spectrum of a-factor is shown in Fig. 8. The parameters
used in the experiment are described in the figure legend.
4. SUMMARY
Using the side-chain anchoring method described here, it is possible to
obtain a-factor in good yield and high purity. Most of the difficulties in the
process arise from poor solubility, due to the presence of impurities and
disulfide-linked dimers in the crude a-factor precursor. Sequential treatment
with DTT and appropriate solvents as well as excess of TFA can be used to
reduce the disulfide bond and keep the peptide solubilized for partial puri-
fication using HPLC. The excess TFA used, however, does interfere with
adsorption of the peptide on the HPLC column. Sample dilution with aque-
ous solvent prior to injection, extended column equilibration, and an
1
13
Fig. 7 2D H, C-HSQC NMR spectrum of farnesylated a-factor in DMSO-d showing
6
1
3
1
C– H short range coupled spin systems (via single bond). Assignments are shown
adjacent to each peak. Peaks labeled with asterisk are shown at a twofold to fourfold
higher contour level. Peaks with positive phasing (black) indicate a CH or CH moiety,
3
1
while peaks with negative phasing (red) indicate a CH moiety. (A) Assigned aliphatic
2
region. (B) Assigned backbone region. (C) Assigned aromatic region. Spectrum was
acquired using 32 scans/increment, 32 dummy scans, 1.5s recycle delay, and a spectral
widths of 15ppmꢂ165ppm. 2048ꢂ1024 points were collected in the directly and indi-
rectly detected dimensions, respectively. Data were zero-filled with 4096ꢂ2048 points
and Fourier-transformed with a shifted sine-bell squared function with 1.00ꢂ0.30Hz
line broadening applied. 3ꢂ Linear prediction was applied in the indirect dimension.
Sine bell shift was 2ꢂ2 and Gaussian max position 0ꢂ0.1.

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1
13
Fig. 8 2D H, C HMBC NMR spectrum of farnesylated a-factor in DMSO-d showing
6
1
3
1
C– H long range coupled spin systems (up to five bonds). Assignments are shown
adjacent to each peak. (A) Assigned aliphatic region. (B) Assigned backbone region.
The spectrum was acquired using 32 scans/increment, 16 dummy scans, 0.8s recycle
delay, and a spectral widths of 15ppmꢂ222ppm. 5594ꢂ265 points were collected
in the directly and indirectly detected dimensions, respectively. Data were zero-filled
with 16,384ꢂ2048 points and Fourier-transformed with a shifted sine-bell squared
function with 1.00ꢂ1.00Hz line broadening applied. 3ꢂ Linear prediction was applied
in the indirect dimension. Sine bell shift was 4ꢂ4 and Gaussian max position was
0.5ꢂ0.5.

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30
Taysir K. Bader et al.
aqueous hold period all help ensure proper binding for purification. Partially
purified a-factor precursor can then be farnesylated under mildly acidic con-
ditions using farnesyl bromide and Zn(OAc) . Solubility here is again prob-
2
lematic, as farnesyl bromide is soluble only in organic solvents, while
Zn(OAc) requires aqueous conditions. Here a DMF/BuOH/aqueous
2
TFA solvent blend is used to maintain a homogeneous mixture while pre-
solubilized reactants are added sequentially. Finally, once a-factor has been
farnesylated, it can be purified to homogeneity via multiple cycles of HPLC.
Throughout the synthesis, MALDI-MS can be used to identify the presence
of the correct products, as well as supplementing analytical HPLC for deter-
mining purity. MALDI-MS was chosen over ESI-MS methods as it simpli-
fied the analysis due to the production of singly charged species, as well as
better ionization of a-factor and precursor. Once purified peptide is
obtained, 2D NMR analysis provides an unambiguous analytical tool that
can be used to inspect a-factor on the atomic level. COSY and TOCSY
1
1
experiments provide the assignment of H– H coupled spin systems, while
1
13
HSQC and HMBC provide the assignments of H– C coupled spin sys-
tems. While the methods described here are specifically for the preparation
and characterization of a-factor, they can be used to prepare essentially any
peptide that includes a C-terminal prenylated cysteine.
ACKNOWLEDGMENTS
Mass spectrometry analysis was performed at The University of Minnesota Department of
Chemistry Mass Spectrometry Laboratory (MSL), supported by the Office of the Vice
President of Research, College of Science and Engineering, and the Department of
Chemistry at the University of Minnesota, as well as The National Science Foundation
(NSF, Award CHE-1336940). The content of this chapter is the sole responsibility of the
authors and does not represent endorsement by the MSL or NSF. This work was
supported in part by the National Institutes of Health (RF1AG056976, GM084152, and
GM106082) and by the National Science Foundation (CHE-1308655). NMR analysis
was performed at Minnesota NMR center. Funding for NMR instrumentation was
provided by the Office of the Vice President for Research, the Medical School, the
College of Biological Science, NIH, NSF, and the Minnesota Medical Foundation.
REFERENCES
Anderegg, R. J., Betz, R., Carr, S. A., Crabb, J. W., & Duntze, W. (1988). Structure of Sac-
charomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a
structural component. The Journal of Biological Chemistry, 263(34), 18236–18240.
Barany, G., Han, Y., Hargittai, B., Liu, R. Q., & Varkey, J. T. (2003). Side-chain anchoring
strategy for solid-phase synthesis of peptid acids with C-terminal cysteine. Biopolymers—
Peptide Science Section, 71(6), 652–666.