On-resin conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) congeners

Article abstract of DOI:10.1002/psc.1223

The Acm protecting group for the thiol functionality of cysteine is removed under conditions (Hg²⁺) that are orthogonal to the acidic milieu used for global deprotection in Fmoc-based solid-phase peptide synthesis. This use of a toxic heavy metal for deprotection has limited the usefulness of Acm in peptide synthesis. The Acm group may be converted to the Scm derivative that can then be used as a reactive intermediate for unsymmetrical disulfide formation. It may also be removed by mild reductive conditions to generate unprotected cysteine. Conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) derivatives in solution is often problematic because the sulfenyl chloride reagent used for this conversion may react with the sensitive amino acids tyrosine and tryptophan. In this protocol, we report a method for on-resin Acm to Scm conversion that allows the preparation of Cys(Scm)-containing peptides under conditions that do not modify other amino acids. Copyright

Full text of DOI:10.1002/psc.1223

Protocol
Received: 30 October 2009
Revised: 23 January 2010
Accepted: 1 February 2010
Published online in Wiley Interscience: 1 April 2010
(www.interscience.com) DOI 10.1002/psc.1223
On-resin conversion of Cys(Acm)-containing
peptides to their corresponding Cys(Scm)
congeners
Daniel G. Mullen, Benjamin Weigel, George Barany and Mark D. Distefano∗
The Acm protecting group for the thiol functionality of cysteine is removed under conditions (Hg2+) that are orthogonal to
the acidic milieu used for global deprotection in Fmoc-based solid-phase peptide synthesis. This use of a toxic heavy metal for
deprotectionhaslimitedtheusefulnessofAcm inpeptidesynthesis. TheAcm groupmaybeconvertedtotheScm derivativethat
can then be used as a reactive intermediate for unsymmetrical disulfide formation. It may also be removed by mild reductive
conditions to generate unprotected cysteine. Conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm)
derivatives in solution is often problematic because the sulfenyl chloride reagent used for this conversion may react with the
sensitive amino acids tyrosine and tryptophan. In this protocol, we report a method for on-resin Acm to Scm conversion that
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allows the preparation of Cys(Scm)-containing peptides under conditions that do not modify other amino acids. Copyright ꢀ
2010 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: acetamidomethyl; S-carbomethoxysulfenyl; Cys(Acm); Cys(Scm); on-resin conversion; methoxycarbonylsulfenyl chloride;
SPPS
REACTION SCHEME
O
O
NH
O
S
S
S
O
Cl
S
O
H2N
N
H
H2N
O
N
H
CH2Cl2
O
O
-Cys(Acm)-
-Cys(Scm)-
O
S
S
Reagent K
H2N
N
H
CO2H
O
-Cys(Scm)-
GENERAL OPTIMIZED PROCEDURE
Standard Fmoc SPPS is performed to yield fully protected, resin-bound peptide, and the N-terminal Fmoc protecting group is removed before
Acm to Scm conversion since the Scm group is not stable to piperidine. Globally side chain protected peptide resin (0.025 mmol) is swollen in
CH2Cl2 (2.5 mL), followed by the addition of methoxycarbonylsulfenyl chloride (0.030 mmol, 1.2 eq., 2.7 µL). The resin is allowed to tumble for 3 h,
washed with CH2Cl2, and dried in vacuo. Freshly prepared Reagent K [TFA-phenol-thioanisole-water-ethanedithiol (82.5:5:5:5:2.5)] (2.0 mL) is
added to the resin and the cleavage reaction is allowed to proceed for 1-2 h. The peptide is precipitated with Et2O (40 mL) and isolated by
centrifugation to form a pellet. The pellet is washed twice with Et2O, dissolved, and purified by RP-HPLC.
Scope and Comments
∗
Correspondence to: Mark D. Distefano, Department of Chemistry, University of
Minnesota, Minneapolis, MN 55455, USA. E-mail: distef001@umn.edu
The Acm protecting group for the side-chain thiol functionality of
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
cysteine [1] is useful because it is stable to a variety of conditions
used to cleave other protecting groups. The Acm group is stable
to the acidic conditions (HF and TFA) used for global deprotection
in Boc- and Fmoc-based chemistries. It is stable to the basic
conditions used to remove the Fmoc group, to hydrazine, and
Abbreviations used: tBu, tert-butyl; Dc, decyl; DTT, dithiothreitol; HCTU,
2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophos-
phate; Npys, 3-nitro-2-thiopyridine; Npys-Cl, 2-(2-nitropyridyl)sulfenyl chlo-
ride; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Snip, S-(N-
piperidylcarbamoyl)sulfenyl.
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J. Pept. Sci. 2010; 16: 219–222
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

MULLEN ET AL.
finally, to reductive (Zn in acetic acid) reaction conditions. The Acm
group is typically removed by mercuric (Hg2+) salts, conditions
that are orthogonal to those necessary to remove Fmoc groups
and tBu-based protection. The necessity of using a toxic heavy
metal and the difficulty in removing Hg2+ ions from product
peptides are the main reasons that the Acm group is not more
widely used in peptide chemistry.
The most common use of the Acm protecting group is
for the direct oxidation of bis Cys(Acm)-containing peptides
(or intermolecular variants) to the cyclic cystine derivatives by
treatment with iodine [2–4]. This oxidative cyclization reaction has
been shown to occur without scrambling other disulfides already
present in the peptide [5]. A caveat of this reaction sequence is
that the iodination of tyrosine and tryptophan residues may occur
if long reaction times are necessary [3].
the conversion to proceed cleanly with only a slight excess of
sulfenyl chloride reagent. These mild conditions for on-resin Acm
to Scm conversion with methoxycarbonylsulfenyl chloride did not
cause the modification of the Trp(Boc) and Tyr (tBu) residues of
peptide 1 (Scheme 2). Just as protected tyrosine, Z-Tyr(tBu)-OMe,
residues are not susceptible to modification by I2 while Z-Tyr-OMe
is rapidly iodinated [3], the bulky tBu-based protecting groups
protect tryptophan and tyrosine from sulfenylation.
A second use of the Acm protecting group is the conversion to
the S-carbomethoxysulfenyl (Scm) group with methoxycarbonyl-
sulfenyl chloride. The Scm group has been used for directed
unsymmetrical disulfide formation (Scheme 1) [2,6–9], and may
be removed by treatment with DTT. This latter reaction sequence,
conversion of Acm to Scm followed by treatment with DTT, is a
milder, more environmentally benign method for removal of the
Acm group. Finally, the conversion of Acm to Scm may be done
without affecting other sensitive amino acid derivatives present in
a peptide, such as prenylated cysteine [8].
O
O
S
S
Scheme 2. On-resin conversion of Cys(Acm) to Cys(Scm) and removal of
the Scm group in solution by reduction with DTT.
N
Path A
Disulfide Formation
Path B
Reduction
H
O
A similar on-resin conversion of Cys(Acm) to Cys(Npys) with
sulfenyl halide Npys-Cl has been reported [13]. Cys(Npys)
derivatives have similar reactivity as compared to Cys(Scm). The
advantage of Cys(Scm) over Cys(Npys) as an activated disulfide
is that upon removal, Cys(Scm) produces COS and methanol as
by-products, whereas Cys(Npys) releases 3-nitro-2-thiopyridine.
The latter by-product can be difficult to separate from the product
peptide by RP-HPLC. Methoxycarbonylsulfenyl chloride is also
substantially cheaper than Npys-Cl. For these reasons, Cys(Scm) is
a useful alternative over Cys(Npys).
R1
S
RSH
DTT
S
SH
N
N
H
H
O
O
Scheme 1. Conversion of Scm-containing peptides to unsymmetrical
disulfides (Path A) or reduction to the free thiol with DTT (Path B).
Scm-containing peptides are easily obtained after standard
acidic cleavage conditions with Reagent K [14], followed by RP-
HPLC purification. The lyophilized powders are stable solids that
need no special handling other than standard desiccated storage
in a freezer. The mild and convenient procedure described here
should simplify and potentially increase the utility of the Acm
protecting group as an orthogonal alternative in both Fmoc and
Boc SPPS.
The conversion of Acm to Scm in solution is often problematic.
Acm-containing peptides often have very different solubilities
than their Scm counterparts. We have found that methoxycar-
bonylsulfenyl chloride is not stable in methanol [8], a common
solvent used for this conversion in the literature [6]. A large excess
of sulfenyl chloride is usually used for the Acm to Scm conversion,
yet sulfenyl halides are known to sulfenylate both tyrosine and
tryptophan [3,10,11]. Finally, unless a peptide contains tryptophan
(or other chromophore), the peptide concentration is often un-
known, which makes controlling the exact stoichiometry of the
reaction difficult.
Experimental Procedures
A simple solution to these problems is to convert Acm to
Scm while a peptide is still globally side chain-protected on the
solid phase. On the solid support, peptide solubility is not an
issue and stoichiometry is easy to control. Previous reports of
on-resin Acm to Scm conversion in the literature have used large
excesses of methoxycarbonylsulfenyl chloride [12]. We have found
Materials and methods
Methoxycarbonylsulfenyl chloride was purchased from Aldrich.
All solvents were of HPLC grade. DIEA and TFA were of
Sequalog/peptide synthesis grade from Fisher. CLEAR resins
werepurchasedformPeptidesInternational.StandardFmoc/HCTU
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ON-RESIN CONVERSION OF CYS(ACM)-CONTAINING PEPTIDES
chemistry was used for SPPS of the peptides. Vydac 218TP54 and
218TP1010 columns were used for analytical and preparative RP-
HPLC, respectively. AllanalyticalandpreparativeRP-HPLCsolvents,
water and CH3CN, contained 0.10% TFA. Retention times (Tr) are
based on 1%/min 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
purification steps.
was cleaved from the resin with freshly prepared Reagent K for
2.5 h. After precipitation with Et2O and washing the pellet as
described earlier, the pellet was dissolved in about 5 mL of DMF,
filtered, and purified on the C18 column. Overall yield: 9%, 3.0 mg,
Tr = 43.7 min. Purity by HPLC: 95%. ESI-MS: calculated 1302.8,
found 1302.8 (Scheme 3).
Synthesis of YIIKGVFWDPAC(Scm)-NH2 (3)
Y(tBu)IIK(Boc)GVFW(Boc)D(OtBu)PAC(Acm)-CLEAR amide resin
(1) (0.025 mmol) was swollen in CH2Cl2 (2.5 mL). Then, methoxy-
carbonylsulfenyl chloride (0.030 mmol, 1.2 eq., 2.7 µL) was added.
The resin was allowed to tumble for 3 h, washed with CH2Cl2, and
dried in vacuo. Next, the peptide resin (2) was treated with freshly
prepared Reagent K (2.0 mL) for 1 h. The peptide was precipitated
from the cleavage solution by the addition of Et2O (50 mL). The
precipitate was centrifuged to form a pellet, which was washed
twice with diethyl ether (Et2o). The peptide pellet was dissolved in
CH3OH (5.0 mL), further diluted by the addition of 0.1% aqueous
TFA (25 mL), filtered, and purified by preparative HPLC on the C18
column. Overall yield: 26%, 10.4 mg, Tr = 37.4 min. Purity by HPLC:
90%. ESI-MS: calculated 1499.7, found 1499.6.
Scheme 3. On-resin unsymmetrical disulfide formation after Cys(Acm) to
Cys(Scm) conversion.
Removal of Scm in solution to produce
YIIKGVFWDPAC-NH2 (4)
To the crude peptide (3, obtained from 0.025 mmol peptide
resin) dissolved in CH3OH (5.0 mL) was added 2.0 mL of 0.20 M
phosphate buffer, pH 8.0. Sufficient H2O was added to dissolve
any precipitated phosphate. DTT (0.25 mmol, 10 eq., 38 mg) was
added and the solution stirred for 10 min. The pH was adjusted
to about 3 by the addition of neat TFA. The peptide solution
was filtered and purified by HPLC on the C18 column. Overall
yield: 18%, 6.2 mg, Tr = 35.0 min. Purity by HPLC: 94%. ESI-MS:
calculated 1409.7, found 1409.6.
Limitations
The data presented here, together with previously reported
examples [8], suggest that this is a robust and versatile procedure.
However, there are two issues which require discussion.
A reviewer of this work has pointed out that sulfenyl chlorides
are known to react with free amino groups to form sulfenamide
derivatives [4]. Any such resulting sulfenamides would be cleaved
by Reagent K, and not be found in the product. This side
reaction would consume the methoxycarbonylsulfenyl chloride
reagent, which our procedure uses only in a small excess. As
a result, one might expect a significant amount of unreacted
Cys(Acm)-containing peptide in the crude product isolated
after Reagent K cleavage. For the two examples of on-resin
Cys(Acm) to Cys(Scm) conversion discussed here, analytical HPLC
analysis of crude YIIKGVFWDPAC(Scm)-NH2 (3) revealed none
(<1%) of the corresponding YIIKGVFWDPAC(Acm)-NH2 peptide,
whereas crude AKKSRRC(Scm)VIA (7) did contain untransformed
AKKSRRC(Acm)VIA (about 25%). From these data, we conclude
that the sulfenyl chloride reagent, which is a soft electrophile,
is kinetically much more reactive toward the corresponding soft
sulfur atom of the Cys(Acm), as compared to the hard nucleophilic
N-terminal amino group. Previous examples of this conversion on
amino acids and peptides with unprotected amines have always
started with trifluoroacetate or hydrochloride salts [8,15]. For a
case such as peptide 7 where incomplete conversion of Cys(Acm)
to Cys(Scm) is observed, it may be useful to wash the peptide
resin with a dilute solution of acetic acid prior to treatment with
methoxycarbonylsulfenyl chloride, to ensure that the amine is
further deactivated by protonation.
Synthesis of AKKSRRC(Scm)VIA (7)
AK(Boc)K(Boc)S(tBu)R(Pbf)R(Pbf)C(Acm)VIA-Wang resin (5)
(0.025 mmol) was swollen in CH2Cl2 (2.5 mL). Then, methoxycar-
bonylsulfenyl chloride (0.030 mmol, 1.2 eq., 2.7 µL) was added.
The resin was allowed to tumble for 3 h, washed with CH2Cl2,
and dried in vacuo to yield compound 6. Next, the peptide resin
was treated with freshly prepared Reagent K (2.0 mL) for 2 h.
The peptide was precipitated from the cleavage solution by the
addition of Et2O (50 mL). The precipitate was centrifuged to form
a pellet, which was washed twice with Et2O. The peptide pellet
was dissolved in 0.1% aqueous TFA (25 mL), filtered, and purified
by preparative HPLC on the C18 column. Overall yield: 50%,
30.5 mg, Tr = 25.4 min. Purity by HPLC: 98%. ESI-MS: calculated
1220.6, found 1220.6.
On-resin directed disulfide formation, AKKSRRC(S-Dc)VIA (8)
AK(Boc)K(Boc)S(tBu)R(Pbf)R(Pbf)C(Scm)VIA-Wang resin (6)
(0.025 mmol), was swollen in DMF (1.6 mL). In a test tube
was dissolved Zn(OAc)2· 2H2O (0.125 mmol, 5 eq., 27 mg) in 0.1 M
acetate buffer, pH 5.0 (0.40 mL). This solution was added to the
peptide resin. Then, decanethiol (0.025 mmol, 1.0 eq., 5.1 µL) was
added, and the solution allowed to tumble for 48 h. The peptide
The outcomes of on-resin reactions of the Scm group, either
reduction with DTT or disulfide exchange with alkanethiols,
are variable. An attempt to reduce Y(tBu)IIK(Boc)GVFW(Boc)
D(OtBu)PAC(Scm)-CLEAR amide resin (2) with ten equivalents
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J. Pept. Sci. 2010; 16: 219–222 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

MULLEN ET AL.
of DTT in DMF followed by Reagent K cleavage gave the
Scm-containing peptide (3) as the main product. Initially, this
result was surprising as the related Snip protected cysteine
has been successfully reduced with DTT while still on resin
[16]. However, in that case the Cys(Snip) residue was located
at the N-terminus of the sequence, rather than at the C-
terminus as reported here. Attempts at directed on-resin formation
of unsymmetrical disulfides have also given variable results.
When peptide AK(Boc)K(Boc)S(tBu)R(Pbf)R(Pbf)C(Scm)VIA-Wang
resin (6) was reacted with decanethiol in the presence of Zn2+
to promote disulfide exchange [8,17], the desired AKKSRRC(S-
Dc)VIA peptide (8) was the main product found after Reagent
K cleavage. But when the Cys(Scm)-containing CLEAR amide
resin (2) was treated with decanethiol under the same reaction
conditions, YIIKGVFWDPAC-NH2 (4) was the main product found
after cleavage. The location of the Cys(Scm) residue in the
sequence, and possibly the flanking residues, has a strong
influence on the outcome of on-resin reactions of the Scm group.
For these reasons, we prefer to first cleave peptides from the resin,
andthenreacttheScmgroupinsolution. Aspreviouslyreportedfor
Cys(Snip) [16], the activated disulfides of Scm-protected peptides
are stable to Reagent K.
4
5
Moroder L, Musiol J, Schaschke N, Chen L, Hargittai B, Barany G. Thiol
group. In Methods of Organic Chemistry, Vol. E22a Goodman M (ed.).
Houben-Weyl: New York, 2001; 384–423.
Munson MC, Barany G. Synthesis of alpha-conotoxin SI, a bicyclic
tridecapeptide amide with two disulfide bridges: illustration of
novel protection schemes and oxidation strategies. J. Am. Chem. Soc.
1993; 115: 10203–10210.
Hiskey RG, Muthukumaraswamy N, Vunnam RR. Sulfur-containing
polypeptides. XVII. The S-carbomethoxysulfenyl derivative as a
protective group for cysteine. J. Org. Chem. 1975; 40: 950–953.
Annis I, Hargittai B, Barany G. Disulfide bond formation in peptides.
Methods Enzymol. 1997; 289: 198–221.
Wollack JW, Zeliadt NA, Mullen DG, Amundson G, Geier S, Falkum S,
Wattenberg EV, Barany G, Distefano MD. Multifunctional prenylated
peptides forlive cellanalysis. J.Am.Chem.Soc. 2009;131:7293–7303.
Wollack JW, Zeliadt NA, Ochocki JD, Mullen DG, Barany G,
Wattenberg EV, Distefano MD. Investigation of the sequence and
length dependence for cell-penetrating prenylated peptides. Bioorg.
Med. Chem. Lett. 2010; 20: 161–163.
6
7
8
9
10 Scoffone E, Fontana A, Rocchi R. Sulfenyl halides as modifying
reagents for polypeptides and proteins. I. Modification of tryptophan
residues. Biochemistry 1968; 7: 971–979.
11 Buku A, Altmann R, Wieland T. Peptide syntheses. LX. Synthesis
of dinor-S-deoxoamaninamide and of two diastereoisomeric 6ꢁ-
dehydroxyamanullines. Liebigs Ann. Chem. 1976; 417–431.
12 Chen L, Zoulikova I, Slaninova J, Barany G. Synthesis and
pharmacology of novelanalogues of oxytocin and deaminooxytocin:
directed methods for the construction of disulfide and trisulfide
bridges in peptides. J. Med. Chem. 1997; 40: 864–876.
13 Barth D, Musiol HJ, Schuett M, Fiori S, Milbradt AG, Renner C,
Moroder L. The role of cystine knots in collagen folding and stability.
Part I. Conformational properties of (Pro-Hyp-Gly)5 and (Pro-(4S)-
FPro-Gly)5 model trimers with an artificial cystine knot. Chem. Eur. J.
2003; 9: 3692–3702.
Acknowledgement
This work was supported by the National Institutes of Health
(GM058842).
14 King DS, Fields CG, Fields GB. A cleavage method which minimizes
side reactions following Fmoc solid phase peptide synthesis. Int. J.
Pept. Protein Res. 1990; 36: 255–266.
References
15 Rietman BH, Peters RFR, Tesser GI.
A facile method for the
1
Veber DF, Milkowski JD, Varga SL, Denkewalter RG, Hirschmann R.
Acetamidomethyl. A novel thiol protecting group for cysteine. J. Am.
Chem. Soc. 1972; 94: 5456–5461.
preparation of S-(alkylsulfenyl)cysteines. Synth. Commun. 1994; 24:
1323–1332.
16 Gross CM, Lelievre D, Woodward CK, Barany G. Preparation of
protected peptidyl thioester intermediates for native chemical
ligation by Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) chemistry:
considerations of side-chain and backbone anchoring strategies,
and compatible protection for N-terminal cysteine. J. Pept. Res. 2005;
65: 395–410.
2
Kamber B. Synthesis of open-chain asymmetrical cystine peptides
during thiol induced fragmentation of sulfenyl thiocarbonates.
Insulin fragments with an intact A20-B19 disulfide bridge. Helv.
Chim. Acta 1973; 56: 1370–1381.
Kamber B, Hartmann A, Eisler K, Riniker B, Rink H, Sieber P, Rittel W.
The synthesis of cystine peptides by iodine oxidation of S-
tritylcysteine and S-acetamidomethylcysteine peptides. Helv. Chim.
Acta 1980; 63: 899–915.
3
17 Naider FR, Becker JM. Synthesis of prenylated peptides and peptide
esters. Biopolymers 1997; 43: 3–14.
c
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