In!. J . Peptide Protein Res.

36, 1990, 255-266

A cleavage method which minimizes side reactions following Fmoc

solid phase peptide synthesis
DAVID S . KING’, CYNTHIA G. FIELDS’, and GREGG B. FIELDS’

‘Department of Molecular and Cell Biology, University of California. Berkeley, ‘Applied Biosystems, Inc., Foster City
’Department of Pharmaceutical Chemistry, University of California, San Francisco, California, U S A

Received 4 December 1989, accepted for publication 25 March 1990

The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is
often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain
deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and
Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess
the relative effectiveness of various scavengers in suppressing these side reactions. We found that the
cleavage mixture 82.5% TFA : 5% phenol : 5% H,O : 5% thioanisole : 2.5% EDT (Reagent K) was
maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each
containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for
efficient synthesis of complex peptides.
Key words: 9-fluorenylmethoxycarbonyl (Fmoc) amino acids; side reactions; solid phase peptide synthesis; weak acid
c1eavage

Solid phase peptide synthesis utilizing 9- (1 8,19), and Mbh (19) side-chain protecting groups. as
fluorenylmethoxycarbonyl (Fmoc)-amino acids (re- well as by ester (20,21) and amide (22,23) resin linkers.
viewed in ref. 1) has been designed to employ a rela- Tyr and Met residues may also be t-butylated (24,25).
tively weak acid, usually trifluoroacetic acid (TFA), Liberated Pmc groups and sulfonic acid can modify
for simultaneous cleavage of the side-chain protecting Tyr residues (26). Reattachment of the Trt group to
groups as well as the peptide-resin bond (2-5). Unfor- Cys residues following TFA cleavage is well doc-
tunately, TFA cleavage generates several highly reac- umented (27,28). Met is subject to acid catalyzed oxi-
tive species, often resulting in covalent modification of dation (29,30).
susceptible residues. Trp can be modified by TFA- The introduction of scavengers* to TFA during pep-
liberated tBu (6-15), Mtr (16), Pmc (17), Tmob tide-resin cleavage greatly reduces these undesirable
side reactions. EDT has been shown to be a highly
Abreviations used for amino acids follow the rules of the IUPAC-
efficient tBu trifluoroacetate (7) and HMP linker (21)
IUB Commission of Biochemical Nomenclature in J. Biol. Chem. scavenger, while H,O quenches tBu cations (6). Oxi-
(1972) 247, 977-983. All amino acids are the L-configuration. Ad- dation of the thioether of Met is suppressed by the
ditional abbreviations used are: Acm, acetamidomethyl; Boc, presence of EMS (31) or thioanisole (32). The Mtr
tertiary-butyloxycarbonyl; BOP, benzotriazol-I-yloxy-tris (di- group is scavenged by EDT :4-methylmercaptophenol
methy1amino)phosphonium hexafluorophosphate; DCM, di- (16). Thiophenol (28), benzyl mercaptan (28), EDT
chloromethane; EDT, 1,2-ethanedithiol; EMS, ethyl methylsulfide; (33), triethylsilane (33), or triisopropylsilane (33)
FAB-MS, fast atom bombardment mass spectrometry; Fmoc, 9- prevent realkylation of Cys by Trt. Combinations of
fluorenylmethoxycarbonyl; HMP, 4-hydroxymethylphenoxy- scavengers may thus be employed during cleavages of
methyl; HPLC, high performance liquid chromatography; Mbh, peptides containing several different residues suscep-
4.4’-dimethoxybenzhydryl; Mtr, 4-methoxy-2,3,6-trimethylben-
zenesulfonyl; NMM, N-methylmorpholine; PD-TOF-MS, Cf?”
tible to these reactions. Examples of TFA plus mul-
plasma desorption-time of flight-mass spectrometry; Pmc, 2,2,5,7,8-
pentamethylchroman-6-sulfonyl; tBu, tertiary-butyl; TFA, trifluo- *Excellent reviews of individual scavenger properties are found in
roacetic acid; Tmob, 2,4,6-trimethoxybenzyl; Trt, triphenylmethyl. refs. 80 and 91.

255
D.S. King et al.
tiple scavenger cleavage mixtures include 90% TFA : Peptides 5-10, 13, and 14 were synthesized on an
5% thioanisole : 5% EDT (34), 93% TFA : 3% anisole Applied Biosystems 43 1A Peptide Synthesizer, using
: 3% EMS : 1 YOEDT (35), 90% TFA : 5% thioanisole the following modifications of previously described
: 3% EDT : 2% anisole (Reagent R) (23,36,37), 67% protocols (42): couplings were performed in N M P
TFA :2% 2-mercaptoethanol : 1 YOanisole : 30% DCM with 5-10 equiv. Fmoc-amino acid for 30-60min
(Reagent M) (38), 94% TFA : 2% phenol : 2% EDT : [Fmoc-Arg(Pmc) was coupled for 90 min]. Peptides 11
2% anisole (39), and 95% TFA : 3% anisole : 1YOEMS and 12 were synthesized on a MilliGen/Biosearch
: 1 % EDT (40). The effectiveness of these scavenger 9050 Peptide Synthesizer** using preformed Pfp esters
combinations in suppressing all the aforementioned [preformed Dhbt esters of Fmoc-Ser(tBu) and Fmoc-
cleavage side reactions has not been extensively exam- Thr(tBu) for solubility reasons, in situ equimolar
ined. Fmoc-ArgfPmc)/HOBt/BOP/NMM for kinetic
The work presented here has used four model pep- reasons] (45-48). All Fmoc-amino acid ester couplings
tides, one containing 1 each of 18 naturally occurring were performed in N M P using 4 equiv. for 30-60 min
amino acids, one containing 1 Trp and 2 Arg residues, [Fmoc-Arg(Pmc) was coupled for 90 min]. Peptides 1,
and two containing 2 Arg and 5 Trp residues, to study 3-10, 13, and 14 were synthesized on HMP polysty-
systematically the suppression of side reactions by a rene resin, peptides 11 and 12 on Pepsyn KA, and
variety of scavengers and scavenger mixtures. A pre- peptide 2 on Rink resin.
liminary account of this study has already appeared For peptide-resin cleavages, indole was reacted over-
(41). Once an optimum scavenger mixture was found. night with TFA before use, while all other scavengers
some 10 peptides containing a broad spectrum of were added to TFA just prior to use. Cleavage reac-
side-chain protecting groups and susceptible residues tions of peptide-resins 1 - 4 were carried out at a
were synthesized and cleaved, and the purity of concentration of lOmg of peptide-resin in 1 mL, for
products assessed. In this manner a scavenger mixture 1 h at room temperature unless otherwise indicated.
has been developed that effectively suppresses the side After filtering to remove the resin, the cleavage
reactions which occur during TFA cleavage of pep- mixture was diluted in 20mL of 30% acetic acid (per
tide-resins. mL of mixture) and then extracted with DCM. 10 pL
samples of the acetic acid layer were injected for
HPLC analysis. Peptide-resins 5 - 14 were cleaved with
MATERIALS A N D METHODS
Reagent K by manual stirring for 1-2.5 h, room tem-
All Fmoc-amino acids, piperidine, 4-dimethylami- perature, at concentrations of 50-200 mg of peptide-
no pyridine, D C M, N, N-d ime t h y l formamide, resin in 2mL. The resin was filtered over a medium
diisopropylcarbodiimide, I -h ydrox y benzotriazole fritted glass filter and rinsed with TFA. The combined
(HOBt), and HMP-linked polystyrene resin were ob- filtrate and TFA rinse was reduced to a syrup under
tained from Applied Biosystems, Inc. 4-(2',4'- reduced pressure. The syrup was dissolved in 400pL
dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy- of TFA and the peptide precipitated by injecting the
linked polystrene resin (Rink resin) was obtained from TFA solution into a centrifuge tube containing 12mL
Novabiochem AG, Switzerland. HMP-linked polya- of methyl tBu ether. After brief cooling and centrifug-
mide/kieselguhr resin (Pepsyn KA) and all Fmoc- ing at 3000 rpm for 2 min, the peptide pellet was dis-
amino acid pentafluorophenyl (Pfp) and 3-hydroxy- persed and washed thoroughly 5-6 times with methyl
2,3-dihydro-4-oxo-benzotriazine(Dhbt) esters were tBu ether and air dried overnight. 20pg samples of
purchased from MilliGen/Biosearch, Novato, CA. crude peptide (dissolved in either 15-50% acetic acid
Side-chain protection was as follows: tBu for Asp. : H z O or 50% acetonitrile:H,O containing 0.1%
Glu, Ser, Thr, and Tyr; Boc for Lys; Mtr or Pmc for TFA) were injected for reversed-phase HPLC analy-
Arg; Tmob or none for Asn and Gln; Trt for His; and sis.
Trt or Acm for Cys. Scavengers were obtained from HPLC analyses of peptides 1-4 were performed on
the following sources: EDT, thioanisole, EMS, indole, an Applied Biosystems 130A Separation Module/757
phenol, anisole, 3-dimethylaminophenol, penta- Variable Wavelength Detector equipped with an
methylbenzene, and 3-cresol from Aldrich and kyn- Aquapore RP-300 C-8 reversed-phase column
urenine from Chemical Dynamics. Scavenger concen- (2.1 x lOOmm, 7pm particle size, 300 A pore size),
trations are either YO volume/volume (EDT, thio- with detection at 220 nm. Samples were eluted with a
anisole, EMS, anisole, 3-creso1, HzO) or YOweight/ linear gradient of 0- 100% B in 60 min, where A was
volume (indole, phenol, 3-dimethylaminophenol, 0.1 YOaqueous TFA and B was 0.08% TFA : acetoni-
pen tamet hyl benzene). 1-Methyl-2-pyrrolidinone trile, at a flow rate of 0.25mL/min. HPLC elution
(NMP) was purchased from E.Merck or Aldrich.
The peptides synthesized for this study are shown in
Table 1. Peptides 1-4 were synthesized on an Applied **The syntheses performed on the MilliGen/Biosearch 9050 Peptide
Biosystems 430A Peptide Synthesizer, where the syn- Synthesizer and subsequent analyses were carried out at the Uni-
thetic protocols have been documented (17, 42-44). versity of California.
256
 TABLE 1
Peptides synthesized for cleavage studies

#
- Sequence
1 Fmoc-Cys(Trt)-Pro-Asp(tBu)-Phe-Gly-His(Trt)-lle-Ala-Met-Glu(tBu)-Leu-Ser(tBu)-Val-Arg(Pmc)-Thr(tBu)-Trp-Lys(Boc)-Tyr(tBu)-HMP
resin
2 Ser(tBu)-Arg(Pmc)-Glu(tBu)-Pro-X-Arg(Pmc)-X-Trp-Cys(Acm)-His(Trt)-Pro-X-Lys(Boc)-Rinkresin
3 Fmoc-x-x-x-Trp-Lys(Boc)-Trp-x-Trp-Trp-x-Trp-Arg(Pmc)-Arg(Pmc)-HMP resin
4 Fmoc-x-x-x-Trp-Lys(Boc)-Trp-x-Trp-Trp-x-Trp-Arg(Mtr)-Arg(Mtr)-HMP resin
5 His(Trt)-Arg(Pnic)-Ser(tBu)-Thr(tBu)-Val-AIa-Ser(tBu)-Cys(Trt)-Met-His(Trt)-Arg(Pmc)-Gln-GIu(tBu)-Ala-Val-Asp(tBu)-Cys(Trt)-Leu-Lys(Boc)-Lys(Boc)-Phe-Asn-AIa-
Arg(Pmc)-Arg(Pmc)-Lys(Boc)-Leu-Lys(Boc)-Gly-A~d-HMP resin
6 His(Trt)-Arg(Pmc)-Ser(tBu)-Thr(tBu)-Val-Ala-Ser(tBu)-Cys(Trt)-Met-His(Trt)-Arg(Pmc)-Gln-Glu(tBu)-Gly-Val-Asp(tBu)-Cys(Trt)-Leu-Lys(Boc)-Lys(Boc)-Phe-Asn-Ala-
Arg(Pmc)-Arg(Pmc)-Lys(Boc)-Leu-Lys(Boc)-Gly-Ala-HMP resin
I His(Trt)-Arg(Pmc)-Ser(tBu)-Thr(tBu)-Val-Ala-Ser(lBu)-Met-His(Trt)-Arg(Pmc)-Gln-Glu(t
Bu)-Thr(tBu)-Val-Asp(tBu)-Cys(Trt)-Leu-Lys(Boc)-Lys(Boc)-Phe-Asn-Ala-Arg(Pmc)-
Arg(Pmc)-Lys(Boc)-Leu-Lys(Boc)-Gly-Ala-HMP resin
8 His(Trt)-Arg(Pmc)-Ser(tBu)-Thr(tBu)-Val-Ala-Ser(tBu)-Cys(Trt)-Met-His(Trt)-Arg(Pmc)-Gln-Glu(tBu)-Thr(tBu)-Val-Asp(tBu)-Cys(Trt)-Leu-Lys(Boc)-Lys(Boc)-Phe-Asn-Ala-
Arg(Pmc)-Arg(Pmc)-Lys(Boc)-Leu-Lys(Boc)-Gly-Ala-HMP resin
9 Glu(tBu)-Leu-Val-Glu(tBu)-Thr(tBu)-Arg(Pmc)-Pro-Ala-Gly-Asp(tBu)-Gly-Thr(tBu)-Phe-Gln-Lys(Boc)-Trp-Ala-Ala-Val-Val-Val-Pro-Ser(tBu)-Gly-Gln-Glu(
tBu)-Gln-Arg(Pmc)-
Tyr(tBu)-HMPresin
10 Glu(tBu)-Leu-Val-Glu(tBu)-Thr(tBu)-Arg(Pmc)-Pro-Ala-Gly-Asp(tBu)-Gly-Thr(tBu)-Phe-Gly-Lys(Boc)-Trp-Val-Ala-Val-Val-Val-Pro-Ser(tBu)-Gly-Gln-Glu(tBu)-Gln-Arg(Pmc)-
Tyr(tBu)-HMP resin
11 Glu(tBu)-Arg(Pmc)-Ile-Thr(tBu)-Gln-Ile-Ala-Lys(
Boc)-Gly-His(Trt)-Glu(tBu)-Lys(Boc)-Trp-Phe-Arg(Pmc)-Val-Ser(tBu)-Leu-Arg(Pnic)-Lys(
Boc)-Leu-Leu-Gly-Tyr(tBu)-Tyr
(tBu)-HMP resin
12 Glu(tBu)-Arg(Pmc)-lle-Thr(tBu)-Gln-Ile-Ala-Lys(Boc)-Asp(tBu)-Asn-Glu(tBu)-Gln-Trp-Phe-Arg(Pmc)-Val-Asn-Leu-Arg(Pmc)-Thr(tBu)-Leu-Leu-Gly-Tyr(tBu)-Tyr(tBu)-HMP
resin
13 Asn(Tmob)-Trp-Asn(Tmob)-Val-Pro-Glu(tBu)-Pro-Ser(tBu)-HMP resin
14 Ala-Glu(tBu)-Tyr(tBu)-Asp(tBu)-Tyr(tBu)-Asp(tBu)-Ala-Ala-Glu(tBu)-Asp(tBu)-Asn-Glu(lBu)-Leu-Thr(tBu)-Phe-Val-Glu(tBu)-Asn-Asp(tBu)-Lys(Boc)-Ile-Ile-Asn-Ile-Glu(tBu)-
Phe-Val-Asp(tBu)-Asp(tBu)-Asp(tBu)-Trp-Trp-Leu-Gly-Glu(tBu)-Leu-Glu(tBu)-Lys(Boc)-Asp(tBu)-Gly-Ser(tBu)-Lys(
Boc)-Gly-Leu-Phe-Pro-Ser(tBu)-Asn-Tyr(tBu)-Val-HMP
resin
D.S. King et al.
1 f) results in more heterogeneous product profiles, pri-
aD 30 Y) 50
Time (rnin.)
FIGURE 1
Analytical HPLC elution profiles of peptide 1 after cleavage by (a)
Reagent K, (b) 87.5% TFA : 5% H 2 0 : 5% thioanisole : 2.5Yn EDT.
(c) 90% TFA : 5% H 2 0 : 3'10 EMS : 2% EDT. (d) 959'0 TFA : 3 O i ,
anisole : 1% EMS : 1% EDT, (e) 92.4% TFA : 5'1; H 2 0 : 2.590
EMS : 0.1% indole, or ( f )91.5% T F A ; 5% HzO: 2.5% thioanisole :
1 Yn 3-dimethylaminophenol. Conditions are given in Materials and
Methods.
profiles were integrated by a Hitachi D-2000. HPLC
analyses of peptides 5 - 14 were performed on a Hew-
lett-Packard 1090 Liquid Chromatograph using the
same column and eluents, flow rate 0.3 mL/min, with
258
detection at 210nm. A Vydac 218TP1010 C-18 re-
versed-phase column (10 x 250mm, 10pm particle
size, 300 8, pore size) was used for HPLC purification
of peptide 14. The specific gradients used for the
elution of peptides 5-14 are noted in Figure legends.
Products from cleavages of peptide-resins 1-4 and
13 were analyzed by Edman sequencing on an Applied
Biosystems 477A Protein Sequencer, using previously
described procedures (49-52) and/or tandem FAB-MS
(performed by Dr. Terry Lee at the Beckman Re-
search Institute of the City of Hope). UV and deriva-
tive UV spectroscopy and either FAB-MS or
PDTOF-MS were used to characterize products from
cleavages of peptide-resins 5-14.
RESULTS AND DISCUSSION
Peptide 1 contains 1 each of 18 naturally occurring
amino acids, including Trp, Tyr(tBu), Cys(Trt), and
Met residues, as well as four different types of side-
chain protecting groups (Table 1). The peptide-resin
was initially cleaved by a multicomponent scavenger
mixture, 83.5% TFA : 5% phenol : 5% H,O : 5%
thioanisole : 2.5% EDT (Reagent K). HPLC analysis
of the cleavage product (Fig. 1, panel a) shows pri-
marily one peak, at 33.0min, which proved to be the
desired peptide. Elimination of phenol (Fig. 1, panel
b) or replacement of thioanisole with 3% EMS (Fig.
1. panel c) has little effect on the homogeneity or
integrity of the cleaved product. Since Met oxidation
was insignificant in the latter scavenger mixture,
thioanisole or EMS appears equally efficient in sup-
pressing Met oxidation. Replacement of H,O with 3%
anisole results in a lowered yield of the desired
product and an increased yield of a peptide eluting at
42.0 min (Fig. 1, panel d). The later peak is the proper
peptide with the Pmc group still attached to the Arg
side-chain, thus demonstrating a need for HIO during
Arg(Pmc) deprotection. The product eluting at
19.5 min was a non-peptide anisole by-product. Re-
placement of EDT with either 0.1% indole (Fig. 1,
panel e) or I %O 3-dimethylaminophenol (Fig. 1, panel
marily due to Trp modification by the Pmc group.
Indole (non-peptide) by-products are seen eluting at
19.0min (Fig. I , panel e).
Peptide 2 contains 2 Arg(Pmc) residues, 1 Trp
residue, and a carboxy terminal Lys(Boc) amide-
linker. The X residues are aliphatic. Cleavage by
Reagent K yields two distinct peaks (Fig. 2, panel a):
the larger peak (70% of the overall product) eluting at
20.0 min is the desired peptide, while the smaller peak
(21%) eluting at 21.0min is the desired peptide con-
taining 1 Arg(Pmc) residue. 6.1 mg of the crude
peptide was cleaved from 1 1.6mg of the peptide-resin
(104% of the theoretical value; resin substitution level
was 0.6 mmol/g), demonstrating that little, if any, of
the peptide was covalently bound to the amide linker

----
I
al J) Y) 50
Time (min.)
FIGURE 2
Analytical HPLC elution profiles of peptide 2 after cleavage by (a)
Reagent K, (b) 95% TFA : 3% anisole : 1% EMS : 1% EDT, (c)
95.8% TFA : 3% anisole : 1% EMS : 0.2% indole, (d) 96.5% TFA
: 2.5% EDT : 1.0% kynurenine, or (e) 95% TFA : 4% H,O : 1%
EDT. Conditions are given in Materials and Methods.
via Trp alkylation (22,23). As in the prior cleavages of
peptide 1, phenol was removed, thioanisole replaced
with 1YOEMS, and H 2 0replaced with 3% anisole; the
resultant cleaved product showed a significant in-
Fmoc amino acids
crease of a product eluting at 27.0min (Fig. 2, panel
b). This product, which was 31 % of the overall yield,
contains the proper sequence, with the side-chain of
either Lys or Trp modified by the Pmc group. The use
of 0.2% indole instead of EDT (Fig. 2, panel c), or
TFA cleavage mixtures containing 2.5% EDT : 1%
kynurenine (Fig. 2, panel d) or 1% EDT : 4% H,O
(Fig. 2, panel e) do not prevent Lys/Trp modification
by Pmc. Cleavage by TFAcontaining 7% pentamethyl-
benzene (for 4 or 24h) yields none of the desired
product (data not shown).
Tridecapeptide 3 contains 2 Arg(Pmc) and 5 Trp
residues, and 1 Lys(Boc) residue (53). Of the remain-
ing amino acids, none is susceptible to side reactions
and none is side-chain protected. Cleavage by
Reagent K yields mainly the desired product (8 1OO/ of
the overall yield), eluting at 29.5min, along with a
small amount of Trp(Pmc) peptides eluting at later
retention times (Fig. 3, panel a). Elimination of phenol
20 30 40 50
Time (min.)
FIGURE 3
Analytical HPLC elution profiles of peptide 3 after cleavage by (a)
Reagent K , (b) 95% TFA : 4% H,O : I % EDT, (c) 95.8% TFA :
4% H,O : 0.2% kynurenine, or (d) 95% TFA : 3% anisole : 1%
EMS : 1% EDT. Conditions are give in Materials and Methods.
259
D.S. King et al.

(data not shown) or phenol and thioanisole increases
product heterogeneity (Fig. 3, panel b), due to in-
creased Pmc modification of Trp residues. Phenol
appears to be useful for preserving the integrity of this
multiple Trp containing peptide. Replacement of
EDT with either 0.2% kynurenine (Fig. 3, panel c) or
0.3% indole (data not shown) greatly increases Trp
modification. Neither indole nor kynurenine is as effi-
cient in scavenging Pmc groups as EDT, an effect that
was also seen during peptide 1 cleavage. Substitution
of 3% anisole for HzO (Fig. 3, panel d) also increases
Arg(Mtr) deprotection (54), pentamethylbenzene does
not appear to scavenge either the Mtr or Pmc group.
While both Mtr and Pmc were equally well scavenged
by Reagent K, the greater acid lability of the Pmc
group makes it preferable for Arg side-chain protec-
tion, particularly in peptides containing more than
one Arg residue.
,001
500-

Trp modification, further demonstrating the need for 400-

H 2 0 as a Pmc scavenger. As seen for peptide 1 clea-
vage (Fig. 1, panel d), anisole non-peptide by-
products elute at 19.5 min (Fig. 3, panel d).
Peptide 4 has the same sequence as peptide 3; only
the Arg side-chain protection differs (Mtr instead o f .
Pmc). Cleavage by Reagent K yields a virtually pure
desired product (Fig. 4, panel a). Trp modification by
the Mtr group does occur with Reagent K when the
cleavage time is extended to 2 h (Fig. 4, panel b). When
H,O is replaced with 3% anisole, Trp modification is
extensive (see ref. 17, Fig. 3a); H 2 0is therefore crucial
for Mtr scavenging. Replacement of EDT with 0.2%
indole reduces Trp modification (Fig. 4, panel c),
suggesting use of indole in Mtr scavenging. Indole and
anisole non-peptide by-products eluted at 19.0 and
20.0min, respectively (Fig. 4, panel c). No desired
product was obtained by cleavage for 4 or 24 h with
TFA containing 5% pentamethylbenzene (data not
shown). Although demonstrated to accelerate I I

a0 30 40 50

Time (min.)

FIGURE 4
Analytical HPLC elution profiles of peptide 4 after cleavage by (a)
Reagent K (1 h), (b) Reagent K (2 h), or (c) 95.9% TFA : 3% anisole
: 1% EMS : 0.1% indole. Conditions are given in Materials and
Methods.
FIGURE 5
Analytical HPLC elution profiles of peptides (a) 5, (b) 6, (c) 7,and
(d) 8 after cleavage by Reagent K. Eluents are given in Materials
and Methods. Elution gradients were 10-50% B in 60min for (a)
and (b), 10-40% B in 60min for (c) and (d).

260
 Fmoc amino acids

The designated Reagent K cleavage mixture (82.5% analogs by Reagent K results in highly homogeneous
TFA : 5% phenol : 5% H,O : 5% thioanisole : 2.5% product profiles, as seen in Fig. 5. For all four peptides,
EDT) appears to suppress a variety of side reactions the largest product peak in each elution profile corre-
effectively. EDT and H,O are crucial components for sponds to the desired peptide. Analysis of the cleaved
preserving Trp integrity, while the presence of either products showed no modification of Met or Lys and
thioanisole or EMS is necessary to inhibit Met oxida- complete side-chain deprotection of Cys residues; the
tion. Phenol need only be utilized during cleavage of minor apolar product peaks seen in Fig. 5a-d were
Arg(Mtr/Pmc) and multiple Trp and/or Tyr contain- peptides containing Arg(Pmc) residues.
ing peptide-resins. To further test the effectiveness of
Reagent K, we synthesized a series of larger peptides,
all containing the potential for a great variety of side
reactions. The possible side reactions include Trp
modification by tBu (peptides 9-14), Pmc (peptides
9-12), or Tmob (peptide 13) side-chain protecting
groups, Met t-butylation or oxidation (peptides 5-8),
Tyr modification by tBu (peptides 9-12 and 14) or
Pmc (peptides 9-12) side-chain protecting groups, and
reattachment of the Trt group to Cys (peptides 5-8
and 14).
Peptides 5-8, which are analogs of the autoinhibit-
ory domain of a Ca++/calmodulin-dependent protein
kinase, contain 4 Arg(Pmc), 4 Lys(Boc), 1 Met, and
either 1 or 2Cys(Trt) residues. Cleavage of these
400‘

300-
500-

2 200-

100-

i-----h..
‘OOL
0
0
600

500

400

2 300
E

2 00

I0 20
Time ( m i n . )
30 40 50
1 00

FIGURE 7
J . . . 10, . .
10
. . 20, .
20
. . .3 ,0
30
Tlnc ( n t n . 1 ’
. .L
40
40
50
50
60
60

FIGURE 6
Analytical HPLC elution profiles of peptides (a) 9 and (b) 10 after
cleavage by Reagent K. Eluents are given in Materials and
Methods. Elution gradients were 20-50% B in 60min.
Analytical HPLC elution profiles of peptides (a) 11, (b) 11, and (c)
12 after cleavage by Reagent K. For cleavages of the peptide-resins
in (a) and (c), 5 % H,O was used, while 2.5% H 2 0 was used in the
cleavage of the peptide-resin in (b). Eluents are given in Materials
and Methods. Elution gradients were 20-50% B in 60 min.
26 1
D.S. King et al.
Peptides 9 and 10, which are analogs of the a3 helix
of the human class I histocompatibility antigen A2,
contain 1 Trp, 1 Tyr(tBu), 1 Lys(Boc), and 2 Arg(Pmc)
residues. Analysis of the cleavage products following
treatment with Reagent K (Fig. 6) showed nearly
complete deprotection of Arg(Pmc) and no modifica-
tion of the Trp, Tyr, or Lys residues. The largest
product peak in each elution profile was the desired
peptide.
Peptides 11 and 12, which are analogs of the tl, helix
of the mouse class I histocompatibility antigens 4 2
and Ld, contain 3 Arg(Pmc), 1 or 3 Lys(Boc), 1 Trp,
and 2Tyr(tBu) residues. Peptide 11 cleavage by
Reagent K resulted in a major and a minor product,
eluting at 27.0 and 36.5rni1-1, respectively (Fig. 7a).
Both products have the correct sequence, with the
later eluting peak containing a Pmc group still at-
tached to one of the Arg residues. If Reagent K clea-
vage is repeated with a lower H,O concentration
(2.5% instead of 5%), the proportion of the Arg(Pmc)
900
800
700
600
111
a
500
4 00
300
200
I00
0 the most efficient scavengers. Modification of suscep-
Time ( m i n .
FIGURE 8
Analytical HPLC elution profile of peptide 13 after cleavage by
Reagent K for (a) 2 or (b) 18 h. Eluents are given in Materials and
Methods. Elution gradient was 10-35% B in 45 min.
262
peptide is increased (Fig. 7b). This demonstrates the
necessity of H20 in deprotecting Arg(Pmc) residues,
as previously seen during the cleavage and side-chain
deprotection of ubiquitin (55,56). Trp, Tyr, and Lys
integrity are completely preserved during cleavage of
peptides 11 and 12 (Fig. 7a-c).
Peptide 13, which contains I Trp and 2 Asn(Tmob)
residues, is a model peptide previously used to study
Trp modification by TFA-liberated Tmob groups
(19). Peptide 13 was cleaved by Reagent K for 1,2,3,5,
or 18 h. The first product, which eluted at 15.3min, is
the desired peptide with no side-chain protecting
groups attached (Fig. 8). Eluting at 32.3min is the
desired peptide with Trp alkylated by the Tmob
group; this product is a pink/purple color, as
previously noted (19). Modification is greatly reduced
by maximizing the cleavage time, as seen by compar-
ing Fig. 8a to b. This result suggests that Tmob mod-
ification of Trp is reversible. Quenching of the Tmob
side-chain protecting group apparently requires both
the multiplicity of scavengers present in Reagent K
and an extended cleavage time.
As a final test of the effectiveness of Reagent K, a
fragment of a yeast actin-binding protein (57) was
synthesized and cleaved. The 50 residue peptide 14
contains 2 Trp, 3 Tyr(tBu), and 3 Lys(Boc) residues.
Fmoc SPPS and cleavage by Reagent K yielded
500mg of crude product (75% of the theoretical
overall yield). The analytical HPLC profile (Fig. 9)
shows a main product peak, eluting at 35.5 min. This
product was purified in three steps by HPLC: (i) semi-
preparative reversed-phase, eluted at a flow rate of
2.0mL/min with a gradient of 2545% B in 60min,
where A was 0.08 M triethylammonium phosphate,
pH 7.2, and B was acetonitrile; (ii) size exclusion on a
TSK G2000SW (0.75cm x 0.6m), eluted at a flow
rate of 0.6mL/min with 50% acetonitrile : H,O con-
taining 0.1 YOaqueous TFA; and (iii) semipreparative
reversed-phase, eluted at a flow rate of 2.0mL/min
with a gradient of 30-5070 B in 60min, with the
eluents described in Materials and Methods. 20 mg of
purified product was isolated (3% of the theoretical
overall yield). PD-TOF-MS showed this product had
the proper molecular mass of the desired peptide with
no side-chain protecting groups attached.
The peptides and cleavage conditions reported here
demonstrate the relative effectiveness of a variety of
scavengers in inhibiting specific side reactions. A clea-
vage mixture (Reagent K) was designed to incorporate
tible residues by multiple tBu, Boc, Mtr, Pmc, and/or
Trt side-chain protecting groups and the HMP linker
is minimized by EDT and H,O. Phenol supplies ad-
ditional protection of Trp and Tyr. Thioanisole
inhibits Met oxidation, and also accelerates Arg(Mtr)
deprotection (58). In addition, the presence of thioa-
nisole helps remove a great variety of benzyl-based

side-chain protecing groups with side-chain linkages
that are stable to cleavage by TFA alone (reviewed in
ref. 1). However, some caution should be taken before
using thioanisole, or free thiol scavengers, in the pres-
ence of certain Cys side-chain protecting groups. For
example, although no deprotection of Cys(Acm) was
seen during cleavage of peptide 2 by Reagent K, it has
been demonstrated that thioanisole partially de-
protects Cys(Acm), as well as Cys(StBu) (59). In
general, thioanisole and free thiol scavengers should
be avoided in the presence of Cys(Acm), Cys(tBu),
and Cys(StBu) (59,60). For sequences incorporating
these Cys derivatives, EMS or DMS may be used for
preserving Met integrity (instead of thioanisole) and/
or Trp integrity (instead of EDT) (7), while phenol
should aid Arg(Mtr) deprotection (59).
The effectiveness of scavengers in suppressing Trp
modification by amide linkers and protecting groups
has also been considered in this study. The majority of
TFA labile amide linkers fall into one of two classes,
based on the amide (Asn, Gln) side-chain protecting
groups Tmob (61,62) and Mbh (63,64). Both the
Tmob side-chain protecting group and the Tmob-
based PAL handle (23,65) have been shown to modify
Trp extensively despite the presence of scavengers
(18,19,23). Of the individual scavengers tested, 10%
dimethylsulfide (DMS) most effectively prevented
alkylation of Trp by Tmob carbonium ions during a
I h treatment (60% of the desired peptide was ob-
tained; see ref. 19, Table 3). However, even cleavage
by 77% TFA : 20% DMS : 3% EDT for 15 min could
not prevent extensive Trp modification by the Tmob
group in certain sequences (see ref. 18, Fig. 2a). PAL
,001 I SO), trimethylsilyl bromide (TMSBr), or trimethyl-
FIGURE 9
Analytical HPLC elution profile of peptide 14 after cleavage by
Reagent K. Eluents are given in Materials and Methods. Elution
gradient was 10-50% B in 45 min.
Fmoc amino acids
handle alkylation was best suppressed by cleavage
with either Reagent K or 96% TFA : 4% H,O for 3 h
(see ref. 23, Table 3). We have found that cleavage of
peptide 13 (which contains two Tmob side-chain pro-
tecting groups) by Reagent K for 18 h resulted in little
modification of Trp. The Tmob group appears to be
well scavenged by the combination of EDT, H,O,
phenol, and thioanisole, provided that sufficient clea-
vage time is allotted. The Mbh side-chain protecting
group and Mbh-based amide linkers (66-78) also
alkylate Trp (19,22, H. Gausepohl, personal com-
munication), even though the Mbh group forms a less
stable carbonium ion than the Tmob group (19). Mbh
modification of Trp was previously found to be 70%
inhibited by 10% DMS (see ref. 19, Table 3), while
Mbh linker alkylation was minimized most efficiently
by a cleavage mixture of 65% TFA : 25% DMS : 5%
3-cresol : 5% EDT (22). Reagent K cleavage of
peptide 2 (which contains an Mbh linker) showed little
Trp modification; in addition, the amount of peptide
released from the resin was quantitative. As in the case
of the Tmob group, Mbh carbonium ions appear to be
well quenched by the multiplicity of scavengers.
Alkylation of susceptible residues by Tmob or Mbh
groups can be minimized or eliminated by other
means not explored here. For example, Asn and Gln
may be side-chain protected during Fmoc solid phase
peptide synthesis by the Trt group (1 8). TFA-liberated
Trt groups are more easily scavenged than Tmob
groups, resulting in a lowered likelihood of Trp alkyl-
ation (18). Modification of Trp by very acid labile
peptide amide-linkers may be avoided by first cleaving
with dilute TFA or acetic acid, followed by side chain
protecting group removal from the peptide amide by
50-90% TFA : scavenger mixtures (23,65,68-70,79. H.
Gausepohl, personal communication). Alternatively,
peptide-resin cleavage by stronger acid, such as 1 M
trifluoromethanesulfonic acid (TFMSA) (reviewed in
silylmethyl trifluoromethanesulfonate (TMSOTQ-
thioanisole : TFA, results in rapid (1-3 h) liberation of
multiple Mbh groups (81-84). These stronger acids,
plus appropriate scavengers, have been used for the
rapid cleavage and deprotection of peptides contain-
ing Trp and multiple Arg(Mtr) residues with no report
of side reactions (85-88).
One rather ambiguous result from this study
concerns the applicability of indole as a scavenger in
place of EDT. Although the indole moiety is a rela-
tively slow tBu trifluoroacetate scavenger (7), it has
been demonstrated to be useful for preserving Trp
integrity during TFA cleavage (23,60,89). Substituting
indole for EDT during cleavages of peptides 1, 2, or 3
resulted in increased covalent modification of Trp,
while Trp modification could be reduced by substitut-
ing indole for EDT during cleavage of peptide 4.
Other investigators (23) have reported that Trp in-
263
D.S. King et a(.
tegrity was well preserved by 2% indole during TFA
cleavage of gastrin I, but that low yields of other
Trp-containing peptides were obtained following clea-
vage by TFA containing 2.6 - 4% indole. Indole is
most probably useful for scavenging a narrow range
of cation types; its effectiveness is limited by a TFA-
induced dimerization reaction (90).
The great number of known side reactions that
occur during TFA cleavage has often limited the ap-
plicability of Fmoc solid phase peptide synthesis. Spe-
cific side reactions may now be suppressed, as the
role(s) of individual scavengers has been more clearly
elucidated. For sequences containing multiple sus-
ceptible residues, utilization of Reagent K permits
rapid peptide-linker cleavage and side-chain deprotec-
tion with a minimum of undesirable side reactions.
Crude peptides may be efficiently recovered by methyl
tBu ether precipitation. A general cleavage method
has thus been developed to complement Fmoc solid
phase peptide synthesis.
ACKNOWLEDGEMENTS
The authors wish to thank J. Harrison for synthesizing peptide 2
and R. Noble for synthesizing peptide 1.
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