Tetrahydropyranyl, a nonaromatic acid-labile cys protecting group for fmoc peptide chemistry

Article abstract of DOI:10.1021/acs.orglett.5b00444

Tetrahydropyranyl (Thp), which exploits the concept of being an S,O-acetal nonaromatic protecting group for cysteine, has been shown to be superior to Trt, Dpm, Acm, and StBu in solid-phase peptide synthesis using the Fmoc/tBu strategy. Thus, Cys racemiza

Full text of DOI:10.1021/acs.orglett.5b00444

Letter
pubs.acs.org/OrgLett
Tetrahydropyranyl, a Nonaromatic Acid-Labile Cys Protecting Group
for Fmoc Peptide Chemistry
Ivan Ramos-Tomillero,†,‡ Hortensia Rodríguez,*,†,§ and Fernando Albericio*,†,‡,§,∥,⊥
́
†Institute for Research in Biomedicine, 08028 Barcelona, Spain
‡Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain
§School of Chemistry, Yachay Tech, Yachay City of Knowledge, 100199 Urcuqui, Ecuador
∥CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, 08028 Barcelona,
Spain
⊥School of Chemistry, University of KwaZulu Natal, 4000 Durban, South Africa
S
* Supporting Information
ABSTRACT: Tetrahydropyranyl (Thp), which exploits the concept of being
an S,O-acetal nonaromatic protecting group for cysteine, has been shown to be
superior to Trt, Dpm, Acm, and StBu in solid-phase peptide synthesis using the
Fmoc/tBu strategy. Thus, Cys racemization and C-terminal 3-(1-piperidinyl)-
alanine formation were minimized when the Cys was protected with Thp. This
nonaromatic protecting group also improved the solubility of Cys-containing
protected peptides.
The Thp group has been widely used as a hydroxyl
lthough solid-phase peptide synthesis (SPPS) is highly
1
Aefficient, amino acid racemization is still an issue and protecting group due to the stability of the acetal derivative
toward strongly basic conditions, organometallics, hydrides, and
acylating and alkylating reagents. Thp elimination is usually
performed in acidic media through the hydrolysis or alcoholysis
of the acetal bond but also through various Lewis acids.10,11
To amplify the methodological spectrum of peptide
synthesis, especially for Cys-containing molecules, and taking
into account that the S,O-acetal is an excellent choice as a Cys
protecting group, we present Thp as a nonaromatic Cys
protecting group for the Fmoc/tBu strategy in SPPS. Although
Thp was introduced by Holland et al.12 as a Cys protecting
group a half-century ago for the synthesis in solution of insulin
peptides, to date, it has not been used in Fmoc chemistry.
The main drawback of using Thp in organic synthesis is the
formation of a new stereocenter that leads to diastereomeric
mixtures; however, when used as a protecting group, the chiral
center that forms is temporary. Consequently, the formation of
the stereocenter is not a shortcoming.
measures for its minimization are needed. Of the repertoire of
natural amino acids, Cys is the most problematic due to its
tendency to lose integrity. Furthermore, it has been shown that
the degree of racemization depends on the protection of the β-
thiol group, which can modulate the acidity of the α-proton.2
Associated with the same phenomenon, the SPPS of C-terminal
Cys-containing acid peptides can be contaminated with C-
terminal 3-(1-piperidinyl)alanine derivatives, which are formed
through β-elimination, followed by piperidine addition.3
Consequently, an interesting attempt to avoid this side reaction
has been done.4
While the most used thiol protection is in the form of
thioethers,5 the groups of Yajima and Nishiuchi developed S,O-
acetal protecting groups, such as the benzyloxymethyl (Bom)
for Boc chemistry6 and 4-methoxybenzyloxymethyl (MBom)
for Fmoc chemistry.7 Bom and MBom, whose syntheses are not
straightforward, result in a very low level of racemization;
however, their use can hamper the quality of the final product
because formaldehyde is formed as a side product during
cleavage and is accompanied by concomitant hydroxymethyla-
tion.
With the same idea of exploiting the S,O-acetal protecting
group concept, we introduced tetrahydropyranyl (Thp) as a
Cys protecting group for SPPS. Thp has an advantage over
benzyl-based protecting groups (Trt, Dmp, Mmt, Bom) in that
it lacks aromaticity. In addition to producing more protected
hydrophobic peptides, the use of bulky aromatic protecting
groups in SPPS affects to the inter/intrachain interaction
during peptide elongation and therefore jeopardizes the purity
of the final product.8,9
Fmoc-Cys(Thp)-OH (2) was synthesized in good yield by
means of an acid-catalyzed reaction between Fmoc-Cys-OH
(1) and the versatile vinyl ether dihydropyran, using p-toluene
sulfonic acid as catalyst and dichloromethane (DCM) as
solvent (see Scheme 1).
To determine the acid stability of Thp as a protecting group,
2 was studied under a range of acidolytic conditions (see Table
1).
Thp remained stable in mild acidic conditions (entries 1−3).
On the contrary, the acid lability of the Thp group was strongly
Received: February 11, 2015
Published: March 12, 2015
© 2015 American Chemical Society
1680
DOI: 10.1021/acs.orglett.5b00444
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Organic Letters
Letter
Scheme 1. Synthesis of Fmoc-Cys(Thp)-OH (2)
Table 1. Acid Lability Studies of Fmoc-Cys(Thp)-OH (2)
“cocktail”
reaction
time
deprotected Cys,
a
entry
composition
ratio (%)
2:98
1 (%)
b
1
2
MES /H2O
48 h
48 h
48 h
48 h
48 h
24 h
24 h
2 h
0
b
MES /TIS/H2O
2:1.5:96.5
20:20:60
1:1:98
0
c
3
TFE /AcOH/DCM
4.7
d
4
TFA /H2O/DCM
28.5
93.7
27.4
89.0
98.5
>99.0
>99.0
>99.0
5
TFA/H2O/DCM
TFA/DCM
10:1:89
1:99
6
Figure 1. Fully protected synthesized Cys-containing tripeptides on
(A) Sieber amide resin and (B) 2-chlorotrityl chloride resin.
7
TFA/DCM
10:90
8
TFA/TIS/DCM
TFA/TIS/DCM
HCl/dioxane
1:1.5:97.5
10:1.5:88.5
12:88
9
5 min
2 h
10
11
HCl/TIS/dioxane
12:1.5:86.5
2 h
a
% w/v for MES (entries 1 and 2) and % v/v for the others (entries
b
c
3−11). MES = 2-(N-morpholino)ethanesulfonic acid. TFE =
trifluoroethanol. TFA = trifluoroacetic acid.
d
increased by the presence of triisopropylsilane (TIS) (entries 4,
6 vs 8 and 5, 7 vs 9) (Table 1). Interestingly, HCl in dioxane (4
M) removed Thp. Therefore, conventional cleavage conditions
such as TFA/TIS/DCM (10:2.5:87.5), TFA/TIS/H2O
(95:2.5:2.5), and 0.1 N HCl/HFIP-TIS (99:1)13 will ensure
complete elimination of Thp in short treatments.
To determine a broader compatibility of the Cys(Thp)-
containing peptide with continuous piperidine treatments and
coupling conditions, Fmoc-Ala-Cys(Thp)-Leu-X (X = NH2, 3a;
X = OH, 4a) was prepared by SPPS using Sieber amide 3a and
2-chlorotrityl chloride (2-CTC) (4a) resins as solid supports
(Figure 1). Moreover, the performance of Thp as a Cys
protecting group was studied by comparison of the synthesis of
3a and 4a with several Fmoc-Ala-Cys(PG)-Leu-X synthesized
using Cys protecting groups (PG): Trt (3b,c and 4b,c), Dpm
(3d), and Acm (4d) (Figure 1).2 To incorporate the protected
Cys derivatives, a 5 min preactivation procedure using Fmoc-
Cys(PG)-OH/DIPCDI/Oxyma Pure (2 equiv of each
compound) in dimethylformamide (DMF) for 90 min at
room temperature was used, which is described to minimize the
racemization.14
All synthesized tripeptides (3a−e, 4a−c) were cleaved from
the resin using low TFA concentrations [TFA/DCM (1:99)]
and analyzed by reverse phase high-performance liquid
chromatography (RP-HPLC). The chromatograms for the C-
terminal amides (3a−e, Figure 2) revealed that the Thp group
was stable during the tripeptide elongation and also during
cleavage from the resin. Similar behavior was observed for C-
terminal acid tripeptides (4a−c; see Supporting Information).
The RP-HPLC profiles also corroborate that the use of Thp as
a thiol protecting group (3a) rendered a more soluble
tripeptide, in comparison to that achieved with the commonly
Figure 2. RP-HPLC profiles of the synthesized C-terminal amide
tripeptides 3a−e. Linear gradient H2O/MeCN (50:50 to 0:100) over
8 min.
used Trt (3b,c) and Dpm (3d) groups. This parameter is
relevant during the synthesis of Cys-containing protected
peptides.
Furthermore, as expected given the particularity of Thp, a
mixture of diastereoisomers was observed in the isolated
tripeptide cleaved under low TFA concentrations (Figure 3A).
Nevertheless, using higher concentrations of TFA in the
presence of scavenger [TFA/TIS/DCM (95:2.5:2.5)] as
cleavage conditions, only a single pure product (3) was
obtained (Figure 3B).
Fmoc removal followed by peptide cleavage with TFA/TIS/
DCM (95:1.5:1.5) yielded H-Ala-Cys-Leu-X tripeptides (X =
NH2, 3g; X = OH, 4e). When Thp was used (3a), an
inappreciable level of racemization ([D-peptide/L-peptide] ×
100 = 0.74%) compared with Trt (3b, 3.3%) and Dpm groups
(3d, 6.8%) was observed. The model tripeptides were also
synthesized under the same coupling conditions, by using Rink
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Figure 4. C-terminal cysteine tripeptide racemization studies for Boc-
Ala-Leu-Cys(PG)-O-resin (2-CTC and Wang resins), using piper-
idine/DMF (1:4) for 24 h. Cys racemization ratio was defined as
(AUC of H-Ala-Leu-D-Cys-OH [5b]/AUC of H-Ala-Leu-L-Cys-OH
[5a]) × 100.
Figure 3. RP-HPLC profiles of (A) Fmoc-Ala-Cys(Thp)-Leu-NH2
(3a) and (B) Fmoc-Ala-Cys-Leu-NH2 (3), using TFA/DCM (1:99)
and TFA/TIS/DCM (95:2.5:2.5) as cleavage conditions, respectively.
Linear gradient H2O/MeCN (50:50 to 0:100) over 8 min.
amide resin, which yielded similar crude products and low
racemization levels: 3a (0.45%), 3b (2.3%), and 3d (1.6%).
As mentioned above, the synthesis of C-terminal Cys-
containing acid peptides showed increased difficulty due to
racemization and formation of 3-(1-piperidinyl)alanine deriva-
tives in response to the repetitive piperidine treatments for
Fmoc elimination.15
To determine the racemization level of the C-terminal
cysteine, Boc-Ala-Leu-Cys(PG)-O-resin (PG = Thp or Trt)
was synthesized; both peptides were immobilized in 2-CTC
and Wang resins.
Peptidyl resins were exposed to piperidine/DMF (1:4)
(standard basic conditions for Fmoc removal in SPPS), and
then a small amount was collected after 2, 4, 6, and 24 h. Each
sample was cleaved with TFA/TIS/H2O (95:2.5:2.5), and the
tripeptide crude products were then analyzed by RP-HPLC to
determine racemization. The racemization level was determined
by the analysis of the area under the curve (AUC)
corresponding to H-Ala-Leu-L-Cys-OH (5a) and H-Ala-Leu-
D-Cys-OH (5b) present in the tripeptides synthesized in the
two resins (Figure 4).
Figure 5. Formation of Ala-Leu-[3-(1-piperidinyl)alanine]-OH (5d)
during the piperidine/DMF treatment of Boc-Ala-Leu-Cys(PG)-O-
resin. Percentage of 5d was calculated considering the AUC of 5d, and
D/L-tripeptides in the crude product analyzed 5a,b.
(92.5:2.5:5) as the cleavage cocktail (Scheme 2, products
from the cleavage from Wang resin).
As expected, when tBu was used as a Cys protecting group,
the product from the cleavage reaction was the thiol (StBu)-
protected C-terminal Cys tripeptide (5c).20
Comparing the results between Boc-Ala-Leu-Cys(PG)-OH,
with the protecting groups of Thp, Trt, and StBu, we conclude
that Thp presented a C-terminal Cys racemization lower than
that of the tripeptide in which Trt was used as the protecting
group (Figure 4). Furthermore, less formation of adducts 5d
was detected when Thp was used as the C-terminal Cys
protecting group (Figure 5).
In summary, the use of the Thp group for cysteine in SPPS
allowed racemization levels lower than that of conventional
protecting groups for Cys (Trt, Dpm, and StBu), with Thp in a
peptide sequence (3a and 4a, <1%) or as C-terminal Cys
protecting group (5a, <6%). In addition, fewer side products,
such as the 3-(1-piperidinyl)alanine adducts, were detected
during the synthesis of C-terminal Cys carboxylic acid peptides.
Furthermore, the solubility of Thp-protected peptides was
improved with respect to strategies involving the commonly
used hindered aromatic protecting groups. Our results reveal
Thp as a useful protecting group for Cys when applied to the
Fmoc/tBu strategy in SPPS. Due to lower racemization levels,
decrease of side reactions, better solubility, and its atom
As reported in similar studies,15 the racemization ratio for
Wang was higher than that for 2-CTC resin. Moreover, for both
resins, a greater racemization level was obtained when the Trt
group was used to protect C-terminal Cys, and therefore, the
racemization of the Cys-containing Thp group in the C-
terminal was considerably lower than the same Trt-protected
tripeptide.
Furthermore, the formation of the Ala-Leu-[3-(1-
piperidinyl)alanine]-OH (5d) was also detected, and its
extension was monitored for the SPPS of C-terminal Cys
tripeptides 5a and 5c in both Wang and 2-CTC resins (Figure
5).3
In addition, the alkylation of the thiol group with a p-
hydroxybenzyl (5e,f) [M + 107]+ was detected in the SPPS
conducted on Wang resin under standard cleavage conditions
[TFA/TIS/H2O (95:2.5:2.5)]. This side product resulted from
the undesired cleavage of the Wang linker.16−19 The formation
of this side product was reduced using TFA/TIS/DMB
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Scheme 2. Obtained C-Terminal Cys Tripeptides from Wang
Resins 5a−c and Their Side Products 5d−f
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economy, Thp will be particularly useful for the effective
synthesis of natural Cys-rich peptides.
ASSOCIATED CONTENT
* Supporting Information
■
S
Materials and methods, experimental section, compound
characterization, and RP-HPLC chromatograms and their
analyses. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: hrodriguez@yachaytech.edu.ecu.
*E-mail: albericio@irbbarcelona.org.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
I.R-T. thanks the Generalitat de Catalunya for a predoctoral
fellowship. This work was funded in part by the following: the
CICYT (CTQ2012-30930), the Generalitat de Catalunya
(2014 SGR 137), and the Institute for Research in Biomedicine
Barcelona (IRB Barcelona) (Spain); the National Research
Foundation and the University of KwaZulu Natal (South
Africa); and SENESCYT (Ecuador).
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DOI: 10.1021/acs.orglett.5b00444
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