Amino Acid-Protecting Groups

Albert Isidro-Llobet,1 Mercedes Álvarez,1,2,3* Fernando Albericio1,2,4*

1
Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028-

Barcelona, Spain.

2
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine,

Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona, Spain.

3
Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028-

Barcelona, Spain.

4
Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1, 08028-

Barcelona, Spain.

E-mail: mercedes.alvarez@irbbarcelona.org; albericio@irbbarcelona.org

RECEIVED DATE

*Authors to whom correspondence should be addressed. Fax: 34 93 403 71 26. E-mail:

mercedes.alvarez@irbbarcelona.org; albericio@irbbarcelona.org;

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Contents

1. Introduction 4
2. α-Amino 5
2.1. General 5
2.2 Introduction of the protecting groups 5
2.3 Removal 7
2.3.1. Protecting groups removed by acid 7
2.3.2 Protecting groups removed by base 11
2.3.3 Other protecting groups 17
3. Lysine (Lys), Ornithine (Orn), Diaminopropionic acid (Dap) and
Diaminobutyric acid (Dab)
3.1. General 24
3.2 Introduction of the protecting groups 25
3.3 Removal 25
3.3.1. Protecting groups removed by acid 25
3.3.2 Protecting groups removed by base 27
3.3.3 Other protecting groups 29
4. α-Carboxylic acid
4.1. General 32
4.2 Introduction of the protecting groups 32
4.3 Removal 33
4.3.1. Protecting groups removed by acid 33
4.3.2 Protecting groups removed by base 34
4.3.3 Other protecting groups 35
5. Aspartic (Asp) and Glutamic (Glu) acids
5.1. General 40
5.2 Introduction of the protecting groups 41
5.3 Removal 41
5.3.1. Protecting groups removed by acid 41
5.3.2 Protecting groups removed by base 43
5.3.3 Other protecting groups 43
6. Amide backbone
6.1. General 45
6.2 Introduction of the protecting groups 46
6.3 Removal 46
6.3.1. Protecting groups removed by acid 46
6.3.2 Other protecting groups 48
7. Aspargine (Asn) and Glutamine (Gln)
7.1. General 50
7.2 Introduction of the protecting groups 51
7.3 Removal 51
7.3.1. Protecting groups removed by acid 51
8. Arginine (Arg)
8.1. General 54
8.2 Introduction of the protecting groups 55
8.3 Removal 55
8.3.1. Protecting groups removed by acid 55
8.3.2. Protecting groups removed by base 58
8.3.3. Other protecting groups 59

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 9. Cysteine (Cys)
9.1. General 60
9.2 Introduction of the protecting groups 61
9.3 Removal 62
9.3.1. Protecting groups removed by acid 62
9.3.2 Protecting groups removed by base 65
9.3.3 Other protecting groups 66
10. Methionine (Met)
10.1. General 70
10.2 Introduction of the protecting groups 70
10.3 Removal: sulfoxide reduction 70
11. Histidine (His)
11.1. General 72
11.2 Introduction of the protecting groups 73
11.3 Removal 73
11.3.1. Protecting groups removed by acid 73
11.3.2 Protecting groups removed by base 75
11.3.3 Other protecting groups 76
12. Serine (Ser), Threonine (Thr) and Hydroxyproline (Hyp)
12.1. General 77
12.2 Introduction of the protecting groups 77
12.3 Removal 78
12.3.1. Protecting groups removed by acid 78
12.3.2 Other protecting groups 79
13. Tyrosine (Tyr)
13.1. General 81
13.2 Introduction of the protecting groups 81
13.3 Removal 81
13.3.1. Protecting groups removed by acid 81
13.3.2 Other protecting groups 84
14. Tryptophan (Trp)
14.1. General 85
14.2 Introduction of the protecting groups 86
14.3 Removal 86
14.3.1. Protecting groups removed by acid 86
14.3.2 Protecting groups removed by base 87
14.3.3 Other protecting groups 88
15. Abbreviations 88
16. References 91

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 1. INTRODUCTION

Synthetic organic chemistry is based on the concourse of reagents and catalysts to achieve the
clean formation of new bonds and appropriate protecting groups are required to prevent the
formation of undesired bonds and side-reactions.1,2 Thus a promising synthetic strategy can be
jeopardized if the corresponding protecting groups are not properly chosen.
Emil Fischer was possibly the first to recognize the need to temporally mask a functional
group to allow regioselective bond formation in the synthesis of carbohydrates.3 However, the
first “modern” protecting group was the benzylozycarbonyl (Z) developed by Bergmann and
Zervas.4 Z fits with the main characteristics associated with a protecting group: (i) it is easily
introduced into the functional group; (ii) it is stable to a broad range of reaction conditions;
and (iii) it is safely removed at the end of the synthetic process or when the functional group
requires manipulation. Another cornerstone in this field was when Barany et al.5,6 described
the concept of orthogonality, in the sense that the two or more protecting groups belong to
independent classes and are removed by distinct mechanisms. The groups can be removed
therefore in any order and in the presence of the rest. Orthogonal protection schemes are
usually milder because selective deprotection is governed by alternative cleavage mechanisms
rather than by reaction rates. Since the pioneernig work of Bergmann and Zervas, the
development of new protecting groups has been deeply tied to peptide chemistry. Protection
is totally mandatory for the construction of these polyfunctional molecules, which contain up
to eight distinct functional groups in addition to indole and imidazole rings, which should also
be protected. Only the carbonyl function is absent from the natural amino acids, because even
phosphate-protecting groups have been developed for the synthesis of phosphopeptides.
Thus, the protecting groups first developed for peptide synthesis have been rapidily adapted
for the protection of building blocks used for the contruction of non-peptide molecules.1,2
Herein, we provide a concise but deep analysis of the protection of amino acids. The review
is divided into sections depending on the amino acid funcionalities protected. For each case,
methods for the introduction of the protecting groups as well as for their removal are
discussed. In each section, protecting groups are classified based on the following criteria: (i)
the most used in a Boc/Bn strategy; (ii) the most used in a Fmoc/tBu strategy; (iii) decreased
order of lability; and (iv) the most recently described, for which, in most cases, their potential
has not yet been explored. In all cases, families of protecting groups are classified together.
The compatibility of each protecting group with regard to the others is indicated in the
column “Stability to the removal of”, which shows which of the following α-amino-

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 protecting groups (Boc, Fmoc, Z, Trt, Alloc and pNZ) can be removed without affecting a
particular protector.
Special attention has been given to new protecting groups described in 2000-2008. Those
described in the literature earlier and that not have found a broad use have been omitted from
this review.

2. α-AMINO
2.1 General
Protection of the α-amino functionality of amino acids is one of the most important issues in
peptide chemistry and is mandatory to prevent polymerization of the amino acid once it is
activated.
As most peptide syntheses, both in solution and on solid phase, are carried out in the C to N
direction, α-amino-protecting groups (temporary protecting groups) are removed several times
during the synthesis and therefore removal must be done in mild conditions that do not affect
the remaining protecting groups (permanent, usually removed in the last step of the synthetic
process, and semi-permanent, usually at the C-terminus, removed in the presence of all other
protecting groups, when the peptide is to be coupled at its C-terminus) or even the peptidic
chain.
The α-amino-protecting group should confer solubility in the most common solvents and
prevent or minimize epimerization during the coupling, and its removal should be fast,
efficient, free of side reactions and should render easily eliminated by-products. Other
desired characteristics of α-amino-protected amino acids are that they are crystalline solids,
thereby facilitating manipulation, and stable enough.
The most common α-amino-protecting groups for solid-phase peptide synthesis (SPPS) are
the 9-fluorenylmethoxycarbonyl (Fmoc) and the tert-butyloxycarbonyl (Boc) groups, used in
the Fmoc/tert-butyl (t Bu) and Boc/benzyl (Bn) strategies respectively.
For solution synthesis, other α-amino-protecting groups used are the Z, the Nps (2-
nitrophenylsulfenyl) and the Bpoc [2-(4-biphenyl)isopropoxycarbonyl] in combination with
tBu-type side chain protection; or the Boc group in combination with Bn-type side chain
protection.
2.2. Introduction of the protecting groups
As there are several types of α-amino-protecting groups, there is a wide range of protection
methodologies. Most of these are based on the reaction of the free amino acids (side chain-
protected if necessary, see ω-amino protection part for selective Lys and Orn side chain

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 protection), with an haloformate7 or dicarbonate8,9 of the protecting group under Schotten
Baumann conditions (use of biphasic system: organic solvent-aqueous basic conditions) or Comentario [m1]: Refenecia Schotten
Baumann
with the corresponding halide in organic solvents.10 Nevertheless, in some cases the presence
of the free α-carboxylic acid can interfere in the reaction and lead, for instance, to the
formation of dipeptides (Figure 1).11,12 ,13,14,15,16,17,18
R O R
O a
a H2N COO Prot O N COO
H
Prot O Cl O O
OOC NHX b NHX
b Prot O O
R R R
X = H, Protoc
H2N COO
O R O R
H Protoc-Cl
Prot O N XHN
N COO N COO
H If X = H H
O R R
O

Protoc Prot O

Figure 1. Mechanism for the formation of protected dipeptides during the protection of
amino acids with haloformates. Adapted from 19.

The methodologies used to overcome this problem can be divided into two types: those that
involve a carboxylic acid-protecting group which is removed upon amino protection and those
that involve less reactive electrophiles on the reagent used to introduce the protecting group.
An example of the former is the use of trimethylsilyl esters of amino acids prepared in
situ,18,20 while an illustration of the latter is the use of N-hydroxysuccinimido (HOSu)
derivative or the corresponding azide, as in the case of the introduction of Fmoc where Fmoc-
OSu or Fmoc-N3 are used instead of Fmoc-Cl. However, the use of Fmoc-OSu can lead to
the formation of tiny amounts of Fmoc--Ala-OH or even of Fmoc--Ala-AA-OH (Figure
2), which can jeopardize the preparation of Fmoc-amino acids for the production of peptide-
based Active Pharmaceutical Ingredients (API).19,21

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 O Nu 1
O O
O
Fm O O N Fm O O N
Nu1
O O
Lossen Rearrangement
O
O
C Nu 2
Fm O + CO 2 + Nu 1 N

O
O O Nu 1 = OH
d
Nu 1 N Nu2 1 Nu2 = O N
O H
O
-Alanine Nu1 = Nu 2 = OH the succinimidyl
Nu1 = Nu 2 = O N
O carbamate
a c O should be very
O unstable as in a
Nu1 = O N
+
O O HO NH 2 CO2
O O Nu 2 = OH
O
N O N O N free -Alanine ready to
H b give Fmoc- -Ala
O O
SuO--Alanine-OSu Fmoc-OSu
according to Wilcheck and Miron
O Fmoc--Ala-OH
O
the succinimidyl N O NH2
carbamate 2
should be very
unstable O

-Alanine-OSu ready to react with AA to give -

Ala-AA-OH and with Fmoc-OSu to be protected

Fmoc-OSu and AA

Fmoc--Ala-AA-OH

Figure 2. Mechanism for the formation of Fmoc--Ala-OH and Fmoc--Ala-AA-OH during

the protection of amino acids. Adapted from 19.

2.3. Removal
2.3.1. Protecting groups removed by acid
- tert-Butyloxycarbonyl (Boc).22,23 Boc-amino acids are generally crystalline solids and their
particular suitability for SPPS has been clearly demonstrated.24,25 The Boc group has been
used for the solid-phase synthesis (SPS) of a number of relevant peptides using the so-called
Boc/Bn strategy. The most common removal conditions for Boc are 25-50% TFA in DCM
but other acids, such as 1 M trimethylsilyl chloride (TMS-Cl)-phenol in DCM,26 4 M HCl in
dioxane and 2 M MeSO3 H in dioxane,27 have been successfully used for solution and solid-

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 phase synthetic strategies. The Boc group is stable to bases and nucleophiles as well as to
catalytic hydrogenation.
- Trityl (Trt).28,29 It is removed with 1% TFA in DCM or 0.1M HOBt in 2,2,2-trifluoroethanol
(TFE) in solution. It can be removed in ever milder conditions such as 0.2% TFA, 1% H2O in
DCM30 or 3% trichloroacetic acid (TCA) in DCM,31 which are compatible with the TFA
labile 3-(4-hydroxymethylphenoxy)propionic acid (AB) linker or even with the more acid
labile Riniker handle,32 as well as with the synthesis of oligonucleotide-peptide conjugates.
Coupling yields of Trt-amino acids are lower than those of carbamate-protected amino acids.
An important application of the Trt group is for the protection of the second C-terminal amino
acid in order to prevent diketopiperazines (DKP) formation in a similar way as for the Boc
strategy.33,34 This procedure involves the coupling of the third amino acid with in situ
neutralization after the removal of the Trt group.30
Incorporation of Trt-amino acids is more difficult than that of carbamate-protected amino
acids, which implies the use of more powerful activating conditions. However, the bulkiness
of the Trt group protects the α-proton from the base abstraction and therefore makes Trt-AA-
OH more difficult to racemize.35
- α,α-Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz).36 Although Ddz is more acid-stable
than the Bpoc and the Trt groups, its removal with 1-5% TFA in DCM makes it compatible
with t Bu-type side chain protection.,37 It can also be removed by photolysis at wavelengths
above 280 nm,36 which makes it potentially very useful for SPS library screening procedures.
It has been used to prevent DKP formation in the Backbone Amide Linker (BAL) strategy in Comentario [m2]: Ref. BAL

38
a similar way as the Trt group. However, an advantage of Ddz- over Trt-amino acids is that
their incorporation is easier, which is a crucial factor when the corresponding amino acids are
to be incorporated on hindered amines.38
- 2-(4-Biphenyl)isopropoxycarbonyl (Bpoc).39 It is a highly acid-sensitive carbamate-type
protecting group, which is removed with 0.2-0.5% of TFA except when used in poly(ethylene
glycol)-based resins, in which more TFA is required because some of the acid is used to
protonate the oxymethyl moieties. 40 This is a common characteristic of several acid labile-
protecting groups.41 Most Bpoc-amino acids are oils and are unstable because the free α-
carboxylic acid is acidic enough to remove the Bpoc group. Thus, these amino acids are
usually stored either as DCHA salts or as pentafluorophenyl esters.42 In the early stages of
SPPS, before the introduction of Fmoc group, Bpoc-amino acids have been used in
combination with tBu-type side chain protection.40 Currently, Bpoc-amino acids are used

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 mostly for peptide derivatives containing phosphate groups such as phospopeptides or
peptide-oligonucleotide conjugates.43,44
- 2-Nitrophenylsulfenyl (Nps).45 It is removed most conveniently with diluted solutions of HCl
in AcOH.46 It is resistant to bases but can be removed by nucleophiles such as 2-
mercaptopyridine in combination with AcOH in MeOH, DMF or DCM.47 Removal using a Ni
48
Raney column and organic solvents, such as DMF, has also been described. Nps has been
applied in both solution and SPS. Its high acid lability requires similar precautions to the
Bpoc group in the presence of the free α-carboxylic acid.
- Benzyloxycarbonyl (Z). See “other protecting groups”

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Stability to
Name and Structure Removal conditions the removal Ref.
of
tert-Butyloxycarbonyl (Boc) 1) 25-50% TFA-DCM Fmoc, Z,a 22,23,
2) 4 M HCl in dioxane Trt, Alloc, 24,25,
3) 2 M MeSO3H in pNZ 26,27
dioxane
4) 1M TMS-Cl, 1M
phenol-DCM
Trityl (Trt) 1) 1% TFA-DCM Fmoc, Alloc 28,29,
2) 0.1M HOBt-TFE 30,31,
3) 0.2% TFA, 1% H2O- 33,35
DCM
4) 3% TCA-DCM

3,5-dimethoxyphenylisoproxycarbonyl 1-5% TFA-DCM Fmoc, Alloc 36,37,

(Ddz) 38
O

O
O
O

2-(4-Biphenyl)isopropoxycarbonyl 0.2-0.5%-TFA Fmoc, Alloc 39,40,

(Bpoc) 41,42,
43,44
O
O

2-Nitrophenylsulfenyl (Nps) 1) Diluted solutions of Fmoc 45,46,

HCl-CHCl3-AcOH 47,48
2) 2-Mercaptopyridine-
S
AcOH-MeOH, DMF or
NO2
DCM
3) Ni Raney column in
DMF
a
Catalytical hydrogenation removal.

10

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 2.3.2. Protecting groups removed by base
- 9-Fluorenylmethoxycarbonyl (Fmoc).49,50 It is removed by bases (mainly secondary
amines, because they are better at capturing the dibenzofulvene generated during the
removal), and is stable to acids. It is not completely stable to the catalytic
hydrogenolysis treatment required to remove benzyl esters when Pd/C or PtO2 are used
as catalysts The most selective catalyst is Pd/BaSO4.51 Solution removal: liquid NH3 (10
h) and morpholine or piperidine (within minutes), 10% diethylamine (DEA),
dimethylacetamide (DMA) (2 h),52 polymeric (silica gel or polystyrene) secondary
amines (i.e. piperazine, piperidine) in organic solvents.53,54 Applied for the first time for
SPPS by two different laboratories independently.55,56 Since then, several optimized
removal conditions for SPS have been described, the most relevant being: 20%
piperidine in DMF,55 which is the most common, 1-5% DBU in DMF,57,58 morpholine-
DMF (1:1)59 or 2% HOBt, 2% hexamethyleneimine, 25% N-methylpyrrolidine in
DMSO-NMP (1:1),60 the latter method leaving thioesters intact. The addition of a
relatively small amount of HOBt to the piperidine solution [0.1 M HOBt in piperidine-
DMF (2:8)] reduces the formation of aspartimide in the sequences prone to this side
reaction.61,62
Fmoc α-amino protection has been used for the SPS of several relevant peptides using
the so-called Fmoc/tBu strategy, the production in Tm scale of the T20 peptide being Comentario [a3]: Chan, Weng C.;
White, Peter D Editor(s). In Fmoc Solid
one of the most important examples.63 Nevertheless, the low solubility of some Fmoc Phase Peptide Synthesis (2000),
137-181. Oxford University Press,
Oxford, UK
derivatives in the most commonly used solvents for SPPS has stimulated the search for
new base-labile protecting groups.
- 2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc).64 It is considered the most promising
alternative to the Fmoc group.65,66,67,68 Nsc-amino acids are crystalline solids, more
soluble in common solvents than Fmoc amino acids, and can be deprotected with 20%
of piperidine or 1% DBU in DMF or preferably in DMF-dioxane (1:1).64,66
Nevertheless, the use of DBU accelerates aspartimide formation and other side
reactions.69 Nsc is 3-10 times more base-stable than the Fmoc group,66 thereby
preventing its undesired removal under slightly basic conditions. This is particularly
relevant in the synthesis of polyproline peptides in which the use of the Fmoc group
leads to deletions caused by premature Fmoc removal by the secondary amine of Pro
whereas no Pro insertions are observed when Nsc is used,67 Nsc is also important in
automated SPS, where amino acid solutions are stored for a long time. Further
advantages of the Nsc vs. Fmoc group are that the formation of the olefin-amine adduct

11

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 after removal is irreversible and faster for Nsc66 and Nsc protection reduces
racemization compared to Fmoc protection,67 which is particularly important in C-
terminal Ser, Cys and His.
- 1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc).70 It is the most
important of a series of protecting groups that are removed via a Michael addition.
Other protecting groups from the same family are the Bspoc (2-tert-butylsulfonyl-2-
propenoxycarbonyl)71 and the Mspoc (2-methylsulfonyl-3-phenyl-1-prop-2-
72
enyloxycarbonyl) and the Mspoc groups. The Michael addition removal mechanism
has several advantages over the β-elimination removal mechanism of Fmoc and Nsc: (i)
back alkylation by the β-elimination by-product is prevented because the deblocking
event is also a scavenging event;70 (ii) base-catalyzed side reactions, such as aspartimide
70,73
formation, are minimized as a result of lower concentrations of secondary amines
and (iii) the method can be applied to the rapid continuous solution synthesis technique.
73,74
Bsmoc-amino acids have been used to synthesize several model peptides in which
the Bsmoc group was removed with 2-5 % piperidine in DMF,70 and have shown better
performance than Fmoc amino acids in difficult couplings such as Aib-Aib.41
Furthermore, the Bsmoc group can be selectively removed with 2% of tris(2-
aminoethyl)amine (TAEA) in DCM in the presence of Fm esters.70
- (1,1-Dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α-Nsmoc).75 It is a novel
alternative to the Bsmoc group and is removed in the same way but slightly faster. α-
Nsmoc-amino acids are crystalline solids, thus they are a good alternative to Bsmoc in
the cases where Bsmoc-amino acids are oils.
- (1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) and 1-(4,4-Dimethyl-
2,6-dioxocyclohex-1-ylidene)-3-mehtylbutyl (ivDde). Both groups are removed by
hydranzinolysis and although can be used for α-amino protection,76 their principal
application is for the protection of Lys and Orn side chains. See Lys and Orn protection.
- 2,7-Di-tert-butyl-Fmoc (Fmoc*).77 It is removed in the same conditions as the Fmoc
group but is up to four times slower. Fmoc*-amino acid derivatives are more soluble
than the Fmoc ones.77,78 They have been recently used for the synthesis of cyclic
modular β-sheets.79
- 2-Fluoro-Fmoc (Fmoc(2F)).80 It is a more base-labile derivative of the Fmoc group
and has been used for the SPS of phosphopeptide thioesters. It is removed with a 4-min
treatment with 4% HOBt in 1-methylpyrrolidine-hexamethylenimine-NMP(1-
methylpyrrolidin-2-one)-DMSO (25:2:50:50).

12

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - 2-Monoisooctyl-Fmoc (mio-Fmoc) and 2,7-Diisooctyl-Fmoc (dio-Fmoc).81 Both are
novel protecting groups reported to show greater solubility than Fmoc* derivatives in
DCM-MeOH (100:4). Their removal with 20% piperidine in DMF is slower than Fmoc
removal; 2 times slower in the case of mio-Fmoc and 5 times slower for dio-Fmoc.
- Tetrachlorophthaloyl (TCP).82 It is a relatively new protecting group proposed for
SPPS. It is removed with hydrazine in DMF (15% of hydrazine, at 40 °C, 1 h for
repetitive deprotection) but stable to piperidine and to Boc removal conditions. It is also
used for side chain amino protection.
- 2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms).83 Pms-amino
acids are water-soluble. They have been developed relatively recently and allow SPPS
in water. Pms is removed with 5% aqueous NaHCO3, 2 x 3 min and 1 x 30 min for
SPS.83,84 Nevertheless, since Pms is an onium salt it is rather unstable compared to
conventional protecting groups.85
- Ethanesulfonylethoxycarbonyl (Esc).85 It is another relatively new protecting group for
peptide synthesis in water. The deriavatives of Esc are more stable than those of Pms. It
is removed either by 0.025 M NaOH in H2O-EtOH (1:1) or 0.05 M TBAF in DMF.
- 2-(4-Sulfophenylsulfonyl)ethoxycarbonyl (Sps).86 Developed parallelly to Esc at almost
the same time, it is also a protecting group for SPS in water. It is removed with 5%
aqueous Na2CO3. Sps-amino acids have a similar stability to Esc ones but with the
advantage that they absorb in the UV.

13

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal conditions Stability to Ref.
the
removal of
9-Fluorenylmethoxycarbonyl (Fmoc) Solid phase: Boc, Z,a 49,50,
1) 20% piperidine-DMF Trt, Alloc, 51,52,
2) 1-5% DBU-DMF pNZ a 53,54,
3) morpholine-DMF 55,56,
(1:1) 57,58,
4) 2% HOBt, 2% 59,60,
O
O hexamethyleneimine, 61,62
25% N-methylpyrrolidine
in DMSO-NMP (1:1)
Solution:
1) NH3 (10 h)
2) morpholine or
piperidine in organic
solvents (within minutes)
3) 10% DEA,DMA (2 h)
4) polymeric secondary
amines (i.e. piperidine,
piperazines) in organic
solvents
2-(4-Nitrophenylsulfonyl)ethoxycarbonyl 1) 20% of piperidine- Boc, Trt, 64,65,
(Nsc) DMF or DMF-dioxane Alloc 66,67,
O O (1:1) 68,69
O
O2N S O
2) 1% DBU-DMF or
DMF-dioxane (1:1)
1,1-Dioxobenzo[b]thiophene-2- 1) 2-5% piperidine-DMF Boc, Trt, 70,71,
ylmethyloxycarbonyl (Bsmoc) 2) 2% TAEA-DCM Alloc 72,73,
74
SO2 O
O

(1,1-Dioxonaphtho[1,2-b]thiophene-2- 1) 2-5% piperidine-DMF Boc, Trt, 75

yl)methyloxycarbonyl (α-Nsmoc) 2) 2% TAEA-DCM Alloc

O
SO2 O

14

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 1-(4,4-Dimethyl-2,6-dioxocyclohex-1- 2% N2H4·H2O-DMF Boc, Fmoc,
ylidene)-3-methylbutyl (ivDde) Z,a Trt,
Alloc

O O

2,7-Di-tert-butyl-Fmoc (Fmoc*) 20% piperidine,DMF Boc, Trt, 77,78,

t (solid phase) Alloc 79
Bu

O
O

t
Bu

2-Fluoro-Fmoc (Fmoc(2F)) 4% HOBt- Boc, Trt, 80

F 1-methylpyrrolidine- Alloc
hexamethylenimine-
NMP-DMSO
O
(25:2:50:50), 4 min.
O

2-Monoisooctyl-Fmoc (mio-Fmoc) and 2,7- 20% piperidine-DMF 81

Diisooctyl-Fmoc (dio-Fmoc)

Tetrachlorophthaloyl (TCP) 15% hydrazine-DMF, 1 Boc, Fmoc, 82

Cl O h, 40ºC Trt
Cl

Cl
Cl O

2-Phenyl(methyl)sulfonio)ethyloxycarbonyl 5% NaHCO3 (aq) Boc, Trt 83,84,

tetrafluoroborate (Pms) 85
O
S O

15

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Ethanesulfonylethoxycarbonyl (Esc) 0.025 M NaOH-H2O- Boc, Trt 85
O EtOH (1:1)
S
O O O

2-(4-sulfophenylsulfonyl)ethoxycarbonyl 5% Na2CO3 (aq) Boc, Trt 86

(Sps)
O O
O
HO3S S O

a
Except catalytical hydrogenation removal.

16

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 2.3.3. Other protecting groups
- Benzyloxycarbonyl (Z).4 It is one of the most widely used α-amino-protecting groups
for peptide synthesis in solution because of: (i) the easy preparation of Z-protected
amino acids; (ii) the high stability of protected amino acids and peptides, which are
stable to base and mild acid treatments (stability to Boc removal); (iii) the versatile
removal conditions: by catalytical hydrogenolysis during chain elongation or by strong
acids (HBr in acetic acid,87 TFA at high temperatures,88 TFA-thioanisole,89 liquid HF,90
BBr3)91 in the final deprotection of the peptide; and (iv) the supression of racemization
during peptide bond formation.92
- Allyloxycarbonyl (Alloc).93,94,95,96,97 It is removed by a palladium-catalyzed (usually
Pd(PPh3)4) transfer of the allyl unit to various nucleophiles/scavengers (preferably:
H3N·BH3, Me2NH·BH3 or PhSiH3)98,99 in the presence of a proton source. The use of
scavengers is mandatory to prevent allylation of the free amine upon Alloc removal. If
removed on solid phase, washings with sodium N,N-diethyldithiocarbamate (0.02 M in
DMF, 3 x 15 min) are carried out in order to remove Pd. Alloc-amino acids are oils but
can be stored as DCHA salts or pentafluorophenyl esters, both of which are crystalline
solids.100 The use of Alloc group is compatible with the Boc/Bn and Fmoc/tBu strategies
and allows tandem removal-acylation reactions when the palladium-catalyzed amino
deblocking is performed in the presence of acylating agents.101 This strategy has been
used to prevent DKP formation.102 Alloc has recently been applied as α-amino-
protecting group for a convergent synthesis of the anti-tumoral peptide Kahalalide F.103
- o-Nitrobenzenesulfonyl (oNBS) and p-nitrobenzenesulfonyl (pNBS).104 The most used
is oNBS. It is removed by a nucleophilic aromatic substitution mechanism using β-
mercaptoethanol and DBU when it is protecting N-alkyl derivatives but the deblcoking
of primary amines fails under these conditions and the cocktail used is 5% thiophenol in
DMF containing 2 eq. of K2CO3. The main advantage of oNBS- vs Fmoc-amino acids is
that the former do not form oxazolones and thus oNBS-amino acyl chlorides can be
used in difficult couplings with less risk of racemization.105 oNBS α-amino-protection
is also used for site-specific alkylation of amino acids on solid phase,106,107 making
these groups unique for the preparation of N-Me peptides (Figure 3).

17

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 O O
H
N H2N
Fmoc N N
H H R= alkyl
R1 R1
O R O R O
H
H2N N N HN
oNBS N oNBS N N
H H H
R1 R1 R1

Figure 3. oNBS protection for the synthesis of N-alkyl peptides. Reprinted with
permission from ref. 106 and 107. Copyright 1997, 2005, American Chemical Society.

- 2,4-Dinitrobenzenesulfonyl (dNBS).108 It is removed by treatment with HSCH2CO2H

(1.2 eq.) and TEA (3 eq.) in DCM for 30 min, leaving oNBS unaltered.
- Benzothiazole-2-sulfonyl (Bts).105,109 Used in solution in a similar way to NBS
groups. It is removed using thiophenol and base (K2CO3, DIPEA or tBuOK) in both
primary and secondary amines, NaBH4 in EtOH41 or HS(CH2)2CO2H, Na2CO3 in
DMF110 for secondary amines, and other reducing agents, such as Zn, H3PO2, Al/Hg, 105
which can be used for primary and secondary amines. However, in the latter case the
reaction is slower and highly concentration-dependent. Bts has been used for the
synthesis of the cyclosporin 8-11 tetrapeptide subunit, which contains three N-
methylamino acids,109 and more recently for the synthesis of macrocyclic antagonists of
the Human Motilin Receptor.111
- 2-Nitrophenylsulfanyl (Nps): See protecting groups removed by acid.
- 2,2,2-Trichloroethyloxycarbonyl (Troc).112 It is a classical protecting group that can be
removed selectively in the presence of Z, Boc, Fmoc and Alloc groups via a Grob
fragmentation using Zn dust in 90% aqueous AcOH or other reducing agents.112,113 It is
not stable to catalytic hydrogenolysis.
- Dithiasuccinoyl (Dts).114 It is removed with mild thiolysis using 0.5 M β-
mercaptoethanol and 0.5 M DIPEA in DCM or 0.5 M N-methyl mercaptoacetamide
(NMM) in DCM.115 It was used for α-amino protection in the first three-dimensional
orthogonal protection scheme suitable for the preparation of fully and partially protected
peptides, which also involved tert-butyl type groups for side chain protection and an o-
nitrobenzyl ester linkage.6 Although Dts is not commonly used for the synthesis of
peptides, it has proved useful for the synthesis of peptide nucleic acids (PNA)116 and O-
glycopeptides by protecting the 2-amino substituent in the corresponding glycosyl
donors.117
- p-Nitrobenzyloxycarbonyl (pNZ).118 It is a classical protecting group which has
recently found further applicability for the synthesis of complex peptides as well as for

18

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 minimizing side-reactions.119 It is much more stable to strong acids than the Z group
and is removed by reduction with tin(II) chloride in nearly neutral conditions (1.6 mM
HCl(dioxane)) in solid-phase and in solution synthesis,119,103 as well as by catalytic
120
hydrogenolysis or Na2S2O4 for solution synthesis. pNZ is orthogonal to the three
most important amino-protecting groups, Boc, Fmoc, and Alloc, thereby making it
highly suitable for the synthesis of cyclic complex peptides such as oxathiocoraline.121
If the second C-terminal amino acid in SPPS is introduced as pNZ derivative and the
pNZ group is removed using SnCl2 and catalytic ammounts of HCl, the formation of
DKP is prevented. The formation of aspartimides is also prevented using pNZ-amino
acids from the Asp residue to the N-terminus.119
- α-Azido carboxylic acids.122,123 Although not widely used because of the instability of
azides, there are examples of their successful application in SPPS.124,125 The azide is
reduced to amine using trimethylphospine in dioxane. α-Azido carboxylic acids can be
coupled as acyl chlorides without oxazolone formation.
- Propargyloxycarbonyl (Poc).126,127 It is removed by ultrasonic irradiation in the
presence of tetrathiomolybdate complexes such as [(PhCH2NEt3)2MoS4] in AcCN. It is
a relatively new and still not widely used protecting group for solution-phase peptide
synthesis. It is stable to Boc removal conditions and has been used to protect amino acid
chlorides to be used in couplings on hindered amines without racemization.
- o-Nitrobenzyloxycarbonyl (oNZ) and 6-Nitroveratryloxycarbonyl (NVOC).128 They
are removed by photolysis at wavelengths greater than 320 nm in the presence of
additives such as N2H4, NH2OH·HCl, or semicarbazide hydrochloride for several hours,
oNZ being the most easily removed. NVOC has been used for combinatorial library
production using the Affymax methodology. 129 Research effort is being made to
develop more easily removable photolabile protecting groups.
- 2-(2-Nitrophenyl)propyloxycarbonyl (NPPOC).130 It is a photolabile amino-protecting
group that is removed by UV light (λ=365 nm) about twice as fast as the classical
NVOC group.
- 2-(3,4-Methylenedioxy-6-nitrophenyl)propyloxycarbonyl (MNPPOC).131 It is removed
faster than the NPPOC and has been developed recently by the same research group.
- Ninhydrin (Nin). See Cys protection.
- 9-(4-Bromophenyl)-9-fluorenyl (BrPhF).132 It is a recently proposed safety-catch
amino-protecting group and has been tested only for solution synthesis. It prevents
epimerization and is more acid stable than the Trt group due to the the anti-aromatic

19

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 nature of the fluorenyl group. tBu esters can be selectively cleaved in its presence by
using ZnBr2 in DCM or trichloroacetic acid.133,132 BrPhF is removed by Pd-catalyzed
aminolysis with morpholine, followed by treatment of the resulting acid-labile
morpholine adduct with DCA and triethylsilane (TES) in DCM.
- Azidomethyloxycarbonyl (Azoc).134 It is a novel protecting group proposed for
solution- and solid-phase synthesis. It is removed by reduction of the azide with
phosphines. The removal is rapid when PMe3 or PBu3 (5 min on solid phase) are used,
and slower with polymer-bound PPh3 (30 min). Azoc is orthogonal to Fmoc and Mtt.
- Bidentate protecting groups.135Another possibility is the use of bidentate reagents such
as N-carboxy anhydrides (NCA) and the oxazolidinones derived from
hexafluoroacetone (HFA) or formaldehyde, which undergo heterocyclization with the
amino and the α-carboxylic group. In the heterocycle, the carboxylic group is
electrophilic, and a carboxy-derivatization is accompanied by N-deprotection (Figure 4).

CF3
F3C O O O
N O H2N
Nu F3C CF3
R R
Nu

Figure 4. Deprotection of a HFA-protected amino acid via nucleophilic attack

20

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal conditions Stability to Ref.
the removal
of
Benzyloxycarbonyl (Z) 1) H2 cat Boc, Fmoc, 4,87,88,89,
O 2) Strong acids such as: Trt, Alloc, 90,91,92
O HBr in AcOH, TFA at pNZ a
high temperatures, TFA-
thioanisole or liquid HF
3) BBr 3
Allyloxycarbonyl (Alloc) Pd(PPh) 3 cat., scavengers: Boc, Fmoc, 93,94,95,
a
O H3N·BH3, Me2NH·BH3 or Trt, pNZ 96,97,98,
O PhSiH3 in organic solvents 99,100,101,
102,103
o-Nitrobenzenesulfonyl 1) 5% thiophenol-DMF, 2 Boc, Fmoc, 104,105,
(oNBS) eq. of K2CO3 (primary Trt 106,107
O amines)
S
O
2) β-mercaptoethanol and
NO2
DBU (secondary amines)
2,4-Dinitrobenzenesulfonyl HSCH2CO2H (1.2 eq.), Boc, Trt 108
(dNBS) TEA (3 eq.) in DCM
O
O2N S
O
NO2

Benzothiazole-2-sulfonyl (Bts) 1) Al/Hg - 41,105,109,

N O 2) Zn 110,111
S
S O 3) H3PO2
4) PhSH and base (K2CO3,
DIPEA, potassium tert-
butoxyde)
5) NaBH4 in EtOH
2,2,2- Zn in 90% AcOH (aq) Boc, Fmoc, 112,113
Trichloroethyloxycarbonyl Trt
(Troc)
O

Cl3C O

21

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Dithiasuccinoyl (Dts) 1) 0.5 M β- Boc, Trt 6,114,115,
mercaptoethanol and 0.5 116,117
M DIPEA-DCM
2) 0.5 M N-
methylmercaptoacetamide-
NMM-DCM
p-Nitrobenzyloxycarbonyl 1) 1-6 M SnCl2, 1.6 mM Boc, Fmoc, 103,118,119,
(pNZ) HCl(dioxane) in DMF Trt, Alloc 120,121
O 2) H2 cat
O

O2N

α-Azidoacids PMe3 in dioxane - 122,123,124,

N3 COOH 125
R

Propargyloxycarbonyl (Poc) [(PhCH2NEt3)2MoS4] in Boc 126,127

O AcCN (ultrasonic
O
irradiation)
o-Nitrobenzyloxycarbonyl photolysis (λ>320 nm), Boc, Fmoc, 128
(oNZ) additives:N2H4, Trt, Alloc
NO2 O NH2OH·HCl, or
O semicarbazide·HCl
(several hours)
4-Nitroveratryloxycarbonyl photolysis (λ>320 nm), Boc, Fmoc, 128,129
(NVOC) additives: N2H4, Trt, Alloc
NO2 O NH2OH·HCl, or
O semicarbazide·HCl
MeO (several hours)
OMe

2-(2- photolysis (λ=365 nm), Boc, Fmoc, 130

Nitrophenyl)propyloxycarbonyl additives: 2.5 mM Trt, Alloc
(NPPOC) semicarbazide·HCl in
NO2 MeOH
O

2-(3, 4-Methylenedioxy-6- photolysis ((λ>350 nm)) Boc, Fmoc, 131

nitrophenyl) Trt, Alloc
propyloxycarbonyl (MNPPOC)
NO2
O
O
O
O

22

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 9-(4-Bromophenyl)-9- i) 2.5 mmol Pd(OAc)2 (0.05 - 132,133
fluorenyl (BrPhF) eq.), BINAP (0.05 eq.), dry
Br Cs2CO3 (5 eq.),
morpholine (1.2 eq.) in
toluene at reflux, 24 h.
(ii) DCA-TES-DCM
(14:3:83), 30 min.
Azidomethyloxycarbonyl 1) 1M PMe3 in THF-H2O Fmoc 134
(Azoc) (9:1), 2-5 min.
O 2) 1M PBu3 in THF-H2O
N3 O (9:1), 2-5 min.
3) Polymer-bound PPh3 (20
eq.) in THF-H2O (9:1), 30
min.
Hexafluoroacetone (HFA) Nucleophiles (i.e. alcohols, Boc, Trt, 135
amines, H2O) Allocb

a
Except catalytical hydrogenation removal.
b
Using PhSiH3 as scavenger.

23

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 3. LYSINE (Lys), ORNITHINE (Orn), DIAMINOPROPIONIC ACID (Dap) AND
DIAMINOBUTYRIC ACID (Dab)
3.1. General
The protection of the side chains of lysine (Lys) and ornithine (Orn) as well as
diaminopropionc acid (Dap) and diaminobutyric acid (Dab) (Figure 5) is essential in
peptide synthesis to prevent their acylation, which would lead to the formation of
undesired branched peptides.

Figure 5. Diaminopropionic acid (Dap), Diaminobutyric acid (Dab), Ornithine (Orn),

and Lysine (Lys)

Several groups used for the α-amino funcionality have found application for amino side
chain protection. It is worth commenting that ω-amino protection is more difficult to
remove than α-amino protection because of the higher basicity of the former. Thus, for
instance, in the case of trityl-type protection of the α-amino, the Trt group is used,
whereas for the ω-amino the more electron-rich 4-methyltrityl (Mtt) is preferred.
The most used permanent protecting groups for Orn and Lys side chains are the 2-
chlorobenzyloxycarbonyl (Cl-Z) and Z groups in the Boc/Bn strategy, and the Boc
group in the Fmoc/tBu strategy. In the synthesis of branched or cyclic peptides there are
several protecting groups orthogonal to Boc and Fmoc, Alloc being among the most
popular.
The N-Fmoc protecting group can be prematurely removed by a primary amine of
sufficient basicity, such as the -amino group of Lys and to a lesser extent the -amino
of Orn and the -amino of Dab, present in the peptide.136,137 This side-reaction is not
promoted by either the -amino side chain of the Dap residue or the -amino group.
These results are consistent with the pKa values of these amino functions in the model
compounds shown in Table 1. Thus, while the pKa values of the side amino functions of
Lys, Orn, and Dab are very close, the pKa of Dap is lower by one unit, making this

24

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 amino function less basic than the other derivatives. The same explanation applies for
the -amino function.

Table 1. pKa of amino function according to the pKalc module (PALLAS version 2.0,
CompuDrug).
O O
H
H 2N O O O N NH2
NH 2 O H H
H O N O N NH2 O
O N NH 2 NH2
O O
O
H2 N NH2
H2N NH2
O NH

pKa: 8.04 pKa: 8.49 pKa: 9.45 pKa: 10.00 pKa: 10.09

These pKa values must be taken into consideration when the -amino-protecting group
of Lys, Orn, or Dab is removed in the presence of an -amino protected by the Fmoc
group. An alternative is a change of strategy, e.g. use of Alloc or Mtt for -amino and
Fmoc for -amino protection, use of Mtt for -amino protection and a
coupling/neutralization protocol similar to that used to prevent DKP formation after Mtt
removal, or use of Alloc and a tandem deprotection-coupling reaction.136
3.2. Introduction of the protecting groups
For blocking the -amino function, a safe method is copper (II) complexation where
CuSO4·5H2O acts as a complexating agent with the α-amino and α-carboxylate groups,
thereby allowing the selective protection of the ω-amino funcionality.138,139,140 Another
alternative also based on complexation is the formation of boron complexes using
B(Et)3 as the complexating agent.141
In some cases (e.g: Z), side chain protection can be achieved by protecting both the α-
amino and the ω-amino funcionalities and then selectively deprotecting the former,
taking advantage of their higher lability.142
3.3. Removal
3.3.1. Protecting groups removed by acid
-2-Chlorobenzyloxycarbonyl (Cl-Z). It is removed with HF or TFMSA and is
preferentially used in the Boc/Bzl solid phase strategy over the Z group because Cl-Z
shows major resistance to the repetitive TFA treatments to remove Boc group.143 Both,
Z and Cl-Z, are stable to bases and can be removed by hydrogenolysis in solution.
- tert-Butyloxycarbonyl (Boc). It is removed with 25-50% TFA.144 It is used in the
Fmoc/tBu solid phase strategy and is resistant to bases and catalytic hydrogenation.

25

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - 4-Methyltrityl (Mtt). It can be used for temporary side chain protection in the Fmoc
strategy and is a better option than Boc in the presence of sensitive amino acids such as
Tyr, Met and Trp because it prevents side reactions during TFA cleavage because of the
low electrophilicity of the bulky trityl cation. As expected, ω-amino protection with Trt-
type groups is more stable than α-amino protection. Removal of Mtt (4-methyltrityl) is
performed selectively in the presence of Boc using 1% TFA in DCM for 30 min or with
AcOH-TFE-DCM (1:2:7) for 1 h.145 More acid-labile derivatives, like
monomethoxytrityl (Mmt) and dimethoxytrityl (Dmt), are more convenient when
hydrophilic resins (e.g. TentaGel) are used.146

26

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal Stability to the Ref.
conditions removal of
2-Chlorobenzyloxycarbonyl (Cl-Z) 1) HF, scavengers Boc, Fmoc, Trt, 143
O a
2) TFMSA-TFA Alloc, pNZ
O 3) H2 cat.
Cl

tert-Butyloxycarbonyl (Boc) 25-50% TFA-DCM Fmoc, Z, b Trt, 144

Alloc, pNZ
O
O

4-Methyltrityl (Mtt) 1) 1% TFA-DCM Fmoc, Alloc 145,146

2) AcOH-TFE-
DCM (1:2:7)

a
Except catalytical hydrogenation removal.
b
Catalytical hydrogenation removal.

3.3.2. Protecting groups removed by base

- 9-Fluorenylmethoxycarbonyl (Fmoc).141 See also α-amino section. It is usually
removed with 20% of piperidine in DMF or 1-5% DBU in DMF, its stability to acids
makes it useful for the synthesis of cyclic and branched peptides using the Boc/Bn
strategy. It is not completely stable to catalytic hydrogenation.
- 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-mehtylbutyl (ivDde).147 It is useful as
a temporary protecting group in the synthesis of cyclic and branched peptides.148 ivDde
is an improved derivative of Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-
ethyl),149,150,151 which is considerably less base-labile and therefore stable to Fmoc
removal conditions and can be removed by hydrazinolysis. An additional advantage of
ivDde is that its steric hindrance makes it is less prone to migrate to free Lys or Orn side
chains (Figure 6).147 To prevent the reduction of the allyl group by hydrazine, allyl
alcohol should be used when ivDde is removed in the presence of allyl-type protecting
groups.152

27

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Figure 6. Dde N→N’ migration. This side reaction is prevented using ivDde.

- Trifluoroacetyl (tfa). 153 It is removed by alkali treatment (0.2 N NaOH in 10 min), 154
155,156,157
aqueous piperidine or sodium borhydride.158 It is stable to strong acids and
therefore compatible with the Boc strategy. The basic conditions used for its removal
may promote aspartimide formation if aspartic residues are present or pyroglutamyl
formation in the case of N-terminal glutamine residues.
- 2-(Methylsulfonyl)ethoxycarbonyl (Msc).159 It is removed with 0.025-0.5 M Ba(OH)2,
or the 4N NaOH(aq)-dioxane-MeOH (0.25:7.5:0.25). It is highly stable to acids (TFA rt
160
and long reaction times, HF 0 ºC 30 min, HCl conc 40ºC 1h) and hydrogenolysis.
This reactivity allowed the use of ω-protection with Msc in combination with Boc and Z
α-protection. 161
- Tetrachlorophthaloyl (TCP).162 It is a relatively new protecting group proposed for
SPPS and also used for α-amino protection. TCP side chain protection is removed with
ethylenediamine-DMF (1:200) at 40°C, 1h in repetitive deprotections. Nevertheless,
hydrazine-based removal used for α-amino deprotection leads to a complex mixture of
compounds.162 TCP is stable to Fmoc, Boc and Alloc removal conditions.

28

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal Stability to Ref.
conditions the
removal of
9-Fluorenylmethoxycarbonyl (Fmoc ) 1) 20% piperidine- Boc, Z,a Trt 141
DMF Alloc,
2) 1-5% DBU-DMF pNZa
O
O (See also α-amino )

1-(4,4-Dimethyl-2,6-dioxocyclohex-1- 2% N2H4·H2O-DMF Boc, Fmoc, 147,148,149,

a
ylidene)-3-methylbutyl (ivDde) Z, Trt, 150,151,152
Alloc

O O

Trifluoroacetyl (tfa) 1) 0.2N NaOH(aq) Boc, Z,a 153,154,155,

O 2) 1 M piperidine(aq) Trt, Alloc 156,157,158
F3C 3) NaBH4 in EtOH
2-(Methylsulfonyl)ethoxycarbonyl 1) 0.025-0.5 M Boc, Z,b 159,160,161
(Msc) Ba(OH)2 Trt, Alloc
2) 4N NaOH(aq)-
dioxane-MeOH
(0.25:7.5:0.25)
Tetrachlorophthaloyl (TCP) Ethylenediamine- Boc, Fmoc, 162
Cl O DMF (1:200), 1 h, Trt, Alloc
Cl
40ºC
Cl
Cl O

a
Except catalytical hydrogenation removal.
b
Catalytical hydrogenation removal.

3.3.3. Other protecting groups

- Allyloxycarbonyl (Alloc).163,164,102 It is removed using a palladium catalyst in the
presence of a scavenger to capture the generated carbocation. It is compatible with the
Boc/Bn and Fmoc/tBu strategies. See also α-amino protection.
- 2-Chlorobenzyloxycarbonyl (Cl-Z). See protecting groups removed by acid.
- p-Nitrobenzyloxycarbonyl (pNZ). See also α-amino protection section for removal
details and references. pNZ protection of the side chains of Lys and Orn prevents the
undesired removal of the α-Fmoc group after side chain deprotection.165,166
- 2-Nitrobenzyloxycarbonyl (oNZ). See α-amino protection.

29

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - 6-Nitroveratryloxycarbonyl (NVOC).167 See α-amino protection.
- Phenyldisulphanylethyloxycarbonyl (Phdec) and 2-Pyridyldisulphanylethyloxy-
carbonyl (Pydec). These are recently developed protecting groups which have been used
either for solution or solid-phase synthesis.168 Both are removed by mild thiolysis using
dithiothreitol (DTT) or β-mercaptoethanol in Tris·HCl buffer (pH 8.5–9.0) for
deprotection in water or by treatment with β–mercaptoethanol and DBU in NMP for
deprotection in an organic medium.
- o-Nitrobenzenesulfonyl (o-NBS). It is widely used for the α-N-methylation of amino
acids. Because of its high stability to acids and bases, o-NBS has found application in
the side chain protection of secondary amines derived from Lys and Orn. It is removed
from secondary amines by mercaptoethanol in the presence of DBU.169,170

30

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal conditions Stability Ref.
to the
removal
of
Allyloxycarbonyl (Alloc) Pd(PPh)3 cat., Boc, 102,163,164

O
scavengers: Fmoc, Trt,
H3N·BH3, pNZ a
O
Me2NH·BH3 or
PhSiH3 in organic
solvents
p-Nitrobenzyloxycarbonyl 1) 1-6 M SnCl2, 1.6 Boc, 165,166
(pNZ) mM HCl(dioxane)-DMF Fmoc, Z,a
O 2) H2 cat Trt, Alloc
O 3) Na2S2O4
O2N

Phenyldisulphanylethyloxycarbonyl 1) DTT or β- Boc, 168

(Phdec) mercaptoethanol- Fmoc, Trt
S O Tris·HCl buffer (pH
S
O 8.5–9.0)
2) β–
mercaptoethanol,
DBU-NMP
2-Pyridyldisulphanylethyloxycarbonyl 1) DTT or β- Boc, 168
(Pydec) mercaptoethanol- Fmoc, Trt
S O Tris·HCl buffer (pH
S
N O 8.5–9.0)
2) β–
mercaptoethanol,
DBU-NMP
o-Nitrobenzenesulfonyl β-mercaptoethanol, Boc, 169,170
(o-NBS) (5eq.) DBU (10 eq.)- Fmoc, Trt
O DMF
S
O
NO2
a
Except catalytical hydrogenation removal.

31

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 4. α-CARBOXYLIC ACID

4.1. General
The protection of the C-terminal carboxylic acid is different in SPS to in solution
synthesis. In the former, the C-terminal is usually linked to the solid support and
therefore the linker/handle acts as a protecting group. There are excellent reviews
covering the linkers/handles used in SPPS and therefore they are out of the scope of the
present review. Nevertheless, in some synthetic strategies where the peptide is linked to
the resin by the backbone by an amino acid side chain, and also in the less frequent
synthesis in the reverse N-C direction, 38,171,172 C-terminal protection is required.
In the case of solution synthesis, C-terminal protection is not needed to form the
peptide bond. However, in other cases C-terminal protection is mandatory.
4.2. Introduction of the protecting groups 173
Protection of the α-carboxylic acid can be performed mainly by the following methods.
- Reaction of a α-amino free amino acid with an alcohol in acidic conditions (HCl and
p-TosOH are the most used acids).174
- tert-Butyl protection by reaction of an α-amino free or protected amino acid with
isobutene in acidic conditions (usually p-Tos-OH or H2SO4).175,176
- Reaction of an α-amino-protected amino acid in the presence of base or as a cessium
salt with the corresponding halide (usually bromide).177,178
- Reaction of an α-amino-protected amino acid with a condensating agent such as DCC
in the presence of DMAP and the alcohol derivative of the protecting group.179

For the particular case of aspartic (Asp) and glutamic (Glu) acids α-carboxyl protection,
two main strategies are possible:
- Protection of the α-carboxylic acid after selective protection of the side chain of H-
Asp-OH or H-Glu-OH either via acid catalyzed esterification or in the presence of a
copper chelate (see protection of side chain of Asp and Glu). Side chain deprotection
renders the desired protected derivative.180,181,141
- Selective protection of the α-carboxylic acid via formation of an intramolecular
anhydride between the two carboxylic acids and reaction with the corresponding alcohol
or via reaction with a halide in the presence of base. In both cases, N-protected Asp or
Glu acid are used as starting materials. In the first case, selective α-protection is
achieved as a result of the major electrophilicity of the α-carboxylic acid, whereas in the
second the selective protection is due to the major acidity of the α-carboxylic acid. 182,183

32

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 4.3. Removal
4.3.1. Protecting groups removed by acid.
- tert-Butyl (tBu).176 It is used in both solution- and solid-phase synthesis. It is removed
with high concentrations of TFA (solid phase and solution) or HCl in organic solvents
(solution). In the latter case, it is effectively used along with Bpoc Nα- protection and Trt
side chain protection or with Z group as Nα- protection. It is stable to base-catalyzed
hydrolysis and its bulkiness generally prevents DKP formation.184
- Benzyl (Bn): See “other protecting groups”
- 2-Chlorotrityl (2-Cl-Trt).185 It is removed with 1% TFA in DCM and is used as a
semi-permanent protecting group for the synthesis of large peptides using a convergent
approach.
- 2,4-Dimethoxybenzyl (Dmb).186 It is removed with 1% TFA in DCM (6 x 5 min). Due
to its high acid lability, it can be removed in the presence of tBu-type protecting groups
and also on Wang and PAL/Rink resins. It is used for Fmoc/tBu SPS of “head to tail”
cyclic peptides.
- 2-Phenylisopropyl (2-PhiPr).187 It is removed with 4% TFA in DCM for 15 min (Boc
group is stable to these conditions).
- 5-Phenyl-3,4-ethylenedioxythenyl derivatives (Phenyl-EDOTn).188 They have been
recently developed and are removed using very small concentrations of TFA (0.01-
0.5%), being the most acid labile derivative the 5-(3,4-dimethoxyphenyl)-3,4-
ethylenedioxythenyl.

33

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal conditions Stability to the Ref.
removal of
tert-Butyl ( tBu) 90% TFA-DCM (solid Fmoc, Z,a Trt 176,184
phase and solution) or Alloc, pNZ,
4 M HCl in dioxane
(solution)
2-Chlorotrityl (2-Cl-Trt) 1% TFA-DCM Fmoc, Alloc 185

Cl

2,4-Dimethoxybenzyl (Dmb) 1% TFA-DCM Fmoc, Alloc 186

MeO

OMe

2-Phenylisopropyl (2-PhiPr) 4% TFA-DCM Fmoc, Alloc 187

5-Phenyl-3,4-ethylenedioxythenyl 0.01%-0.5% TFA-DCM Fmoc

(Phenyl-EDOTn) and scavengers

a
Catalytical hydrogenation removal.

4.3.2. Protecting groups removed by base.

- 9-Fluorenylmethyl (Fm).189,190 It is removed with secondary amines such as piperidine
and DEA in DCM or DMF, and also by catalytical hydrogenation in solution.190 Used
for SPS in the reverse N-C direction,171 as well as for the preparation of “head to tail”
cyclic peptides.191
- 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino)benzyl
192
(Dmab). It is removed by 2% of hydrazine·H2O-DMF (1:1) within minutes. It is
stable to piperidine.
- Methyl (Me) and Ethyl (Et).193 Methyl esters are removed by saponification (usually
with LiOH), which can lead to epimerization and degradtion of Ser, Cys and Thr.

34

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Nevertheless, they have been used extensively in classical peptide synthesis in solution.
They are also a reasonable choice to obtain peptide amides by reaction of the methyl
ester with ammonia. Ethyl esters have a similar behaviour to methyl esters but are more
base-stable and therefore more prone to base- catalyzed side reactions.184
- Carbamoylmethyl (Cam).194,195 It is used for solution synthesis. It is removed by
saponification with NaOH or Na2CO3 in DMF. It is removed selectively in the presence
of Boc and Z. Nevertheless, it can not be selectively removed in the presence of side
chain Bn-protected Asp.

Name and Structure Removal conditions Stability to the Ref.

removal of
9-Fluorenylmethyl (Fm) 15% DEA or 20% Boc, Trt, Alloc 171,
piperidine-DMF or 189,
DCM 190,
191

4-(N-[1-(4,4-dimethyl-2,6- 2% hydrazine-H2O- Boc, Fmoc, Trt, 192

dioxocyclohexylidene)-3-methylbutyl]- DMF (1:1)
amino)benzyl (Dmab)

H
N

O O

Methyl (Me) and Ethyl (Et) LiOH, NaOH or KOH Boc, Z 184,
193
a b
Carbamoylmethyl (Cam) NaOH or Na2CO3- Boc, Fmoc Z 195,
O DMF-H2O 194
H2N
a
Diethylamine removal.
b
Only catalytical hydrogenation removal.

4.3.3. Other protecting groups

- Allyl (Al).163 It is removed using Pd(PPh3)4 (0.1 eq.) and PhSiH3 (10 eq.) as scavenger
in DCM within minutes or Pd(PPh3)4 and morpholine as nucleophile in THF-DMSO-
0.5 M HCl (2:2:1), both on solid phase and in solution.196 If removed on solid phase,
washings with sodium N,N-diethyldithiocarbamate (0.02 M in DMF, 3 x 15 min) are
carried out in order to remove Pd. Allyl C-terminal protection has been used for the
synthesis of C-terminal modified peptides using the backbone linker (BAL) strategy,38

35

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 and recently for the synthesis of peptide analogs where α-carboxyl protection is
necessary both in solution and on solid phase, such as the synthesis of cyclic peptides
via head-to-tail cyclization, among others.197198,199,200,201 In these cases, when the Al
group from the carboxyl group and the Fmoc from the amino group need to be removed,
it is preferable to first remove the Al and then the Fmoc. Removal of the Fmoc group
first could increase the risk of allylation of the amino function during the removal of the
Al.200,202
- Benzyl (Bn). It is used mostly in solution synthesis. It is usually removed by catalytic
hydrogenolysis. It can also be removed by saponification or hydrazinolysis to give the
corresponding C-terminal. Acidolytic removal is also possible but harsh conditions are
required. It is used in combination with the following Nα-protecting groups: Boc, Ddz,
Bpoc and Troc.184
- Phenacyl (Pac).203 It is used for synthesis in solution and removed by nucleophiles
such as sodium thiophenoxyde or by treatment with Zn in AcOH.203,204 It is degraded
and only partially removed by catalytical hydrogenation. It is more electrophilic than
the methyl ester, thereby making Pac-protected amino acids prone to racemization
during coupling because of a reversible cyclization mechanism (Figure 7).

R1 R1
O H NH2 N
H2N H
H O
O O O O
R1 O
O

R1 NH2
1
R N
R1 N
O O
O O O O O

Figure 7. Racemization mechanism of Pac-protected amino acids. Adapted from 205

- p-Nitrobenzyl (pNB). It is highly resistant to acids and removed using a variety of

reducing agents such as Na2S, Na2S2O4, SnCl2 or catalytical hydrogenation.206,207,208,209
Solid phase removal is performed by treatment with 8M SnCl2 in DMF containing 1.6
mM AcOH and 0.2% phenol for 5 h at 25°C or three treatments of 30 min at 60°C. 210
Washings with DMF, MeOH, DMF and treatment with 8M benzene sulfinic acid in
DMF for 30 min at 25°C and further washings with DMF and MeOH are performed to
211
eliminate the quinonimine methide formed during the removal. Use of a less
concentrated and more easy to handle 6 M SnCl2 in DMF solution, substitution of

36

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 AcOH by HCl in dioxane and alternative washings (DMF, DMF/H2O, THF/H2O, DMF
and DCM, 3×30 s each) have been described in the case of Glu side chain protection.165
These conditions should be easily adapted to the removal of the C-terminal protecting
group. Removal with TBAF in solution has also been proposed as an alternative to the
reductive removal.212
- 2-Trimethylsilylethyl (TMSE).213 It is removed with a quaternary ammonium fluoride
such as TBAF or tetraethylammonium fluoride (TEAF) in DMF. It is stable to
hydrogenolysis but unstable to anhydrous TFA. Nevertheless, Boc group can be
removed selectively in its presence when HCl solutions in organic solvents are used.
- (2-Phenyl-2-trimethylsiylyl)ethyl (PTMSE).214,215 It is removed by treatment with
TBAF·3 H2O in DCM in almost neutral conditions within 3-5 min. It is stable to the
hydrogenolytic cleavage of Z and Bn ester groups, base-induced removal of Fmoc
groups, palladium(0)-catalyzed removal of Alloc and even acidolytic cleavage of Boc
groups if carried out under special conditions (p-TsOH or 1.2 M HCl in 2,2,2-
trifluoroethanol (TFE). PTMSE esters are also stable under the conditions for amide
bond formation in peptide synthesis or peptide condensation reactions and therefore it is
considered a valuable novel carboxy protecting group. However, no studies on how the
use of PTMSE affects the formation of aspartimides have been performed to date.
- 2-(Trimethylsilyl)isopropyl (Tmsi).216 It is used for peptide synthesis in solution. It is
removed with TBAF (8 eq.) in THF in 1-1.5 h. It significantly reduces DKP formation
in comparison with TMSE.
- 2,2,2-Trichloroethyl (Tce).217 It is used mainly for solution synthesis. It is removed
with Zn dust in AcOH in similar conditions as Troc and therefore can be removed in the
presence of Z, Boc, Alloc and Fmoc. Tce is stable even at pH 1 and therefore Boc can
be removed selectively in its presence. It is not completely stable to hydrogenolysis.
- p-Hydroxyphenacyl (pHP).218 It is removed by photolysis (λ= 337 nm) and used as a
new phototrigger. It is stable to Boc removal.
4,5-Dimethoxy-2-nitrobenzyl (Dmnb).219 It is a photolabile protecting group analogous
to the NVOC group. It has been used for the synthesis of misacylated transfer RNAs,
and recently for the synthesis of caged peptides.220
- 1,1-Dimethylallyl (Dma).221 It is removed by treatment with Pd(PPh3)4 (10 mol %) in
THF at room temperature followed by dropwise addition of NMM (3 eq.) under
nitrogen. PhSiH3, potassium 2-ethyl hexanoate or p-toluene sulfonic acid sodium salt

37

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 can be used instead of NMM. It is orthogonal to the Fmoc group and can be removed in
the presence of Bn- and tBu-type groups but it is not stable to their acidolitic removal.
- Pentaamine cobalt (III).222 It was proposed as a C-terminal-protecting group for the
synthesis of side chain to side chain bicyclic peptides. It is described as orthogonal to
Fmoc and Boc and is removed in solution by mild reduction with DTT in the presence
of DIPEA in H2O-AcCN to the exchange labile Co (II) form. It has not been widely
used since then.

Name and Structure Removal conditions Stability to the Ref.

removal of
Allyl (Al) Pd(Ph3)4 (0.1 eq.) and Boc, Fmoc, 38,163,
a
scavengers (usually pNZ, Trt 196,197,
PhSiH3, 10 eq.)-DCM 198,199,
200,201,
202
Benzyl (Bn) 1) HF Boc, b Fmoc, 184
a
2) TFMSA pNZ, Trt, Alloc
3) H2 cat.
4) NaOH in aqueous
organic solvents
Phenacyl (Pac) 1) sodium Boc, Z,a Trt 203,204
O thiophenoxyde
2) Zn in AcOH

p-Nitrobenzyl (pNB) 1) SnCl2 in DMF Boc,Fmoc, Z,a 165,206

2) Na2S·9H2O-H2O, Trt, Alloc 207,208,
0-5ºC 209,210,
O2N
3 ) Na2S2O 4, Na2CO3- 212
H2O, 40ºC
4) H2 cat.
5) TBAF-THF, DMF
or DMSO
2-Trimethylsilylethyl (TMSE) TBAF or TEAF-DMF Zc 213

Me3 Si

(2-Phenyl-2-trimethylsilyl)ethyl TBAF·3 H2O-DCM Fmoc, Z,c Alloc 214,215

(PTMSE)

38

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Me3Si

2-(Trimethylsilyl)isopropyl (Tmsi) TBAF (8 eq.)-THF, 1- Zc 216

1.5 h
Me3Si

2,2,2-Trichloroethyl (Tce) Zn dust-AcOH Boc, Fmoc, Trt 217

Cl3C

p-Hydroxyphenacyl (pHP) Photolysis Boc, Trt 218

O (λ=337 nm)

HO

4,5-Dimethoxy-2-nitrobenzyl Photolysis Boc, Fmoc, Trt 219,220

(Dmnb) (λ>320 nm)
NO2

MeO

MeO

1,1-Dimethylallyl (Dma) Pd(PPh 3) 4 and Fmoc 221

scavengers: NMM,
PhSiH3, potassium 2-
ethyl hexanoate or p-
Tos-OK-THF
Pentaamine cobalt (III) DTT, DIPEA-H2O- Boc, Fmoc, Trt 222
Co(NH)5 ·Cl2 AcCN
a
Except catalytical hydrogenation removal.
b
Except repetitive removals.
c
Catalytical hydrogenation removal.

39

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 5. ASPARTIC (Asp) AND GLUTAMIC (Glu) ACIDS
5.1. General
The side chain carboxylic groups of Asp and Glu (Figure 8) must be protected in order
to prevent their activation during peptide synthesis, which would lead to undesired
branched peptides.
H2N COOH H2N COOH

COOH
COOH
Asp Glu

Figure 8. Aspartic (Asp) and Glutamic (Glu) acids.

Furthermore, in the case of Asp acid, the protecting groups used must also prevent or at
least minimize the formation of aspartimide. Hydrolysis of the aspartimide during
peptide synthesis renders two products: the α-peptide, which is the desired product, and
the β-peptide, which is usally the major compound. Aminolysis of aspartimide by
piperidine gives the corresponding α- and β-piperidides (Figure 9).
The same kind of intramolecular cyclization can also take place in the case of Glu,
thereby leading to pyroglutamic formation.223 However, in the case of Glu the reaction
is much less severe than with Asp.
O O O O
H H H H H H H
N N N N Hydrolysis N N N
N N N OH
H H
OX O O HO O O
O
O Aspartimide O
HN
Peptide (desired product)
piperidine
O

O Peptide
H H (undesired product)
N N
N
H N O
O O
O H
N
N N N
H H
O

Piperidide Piperidide

Figure 9. Aspartimide formation followed by piperidide formation upon piperidine

treatment or hydrolysis rendering the α- and β-peptides.

40

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Currently the most used protecting groups are tBu for the Fmoc/tBu strategy, and in the
Boc/Bn strategy the cyclohexyl (cHx) group, which is replacing the classical Bn group
because it is more effective at preventing the formation of aspartimide.
5.2. Introduction of the protecting groups
The protection of the side chain carboxylic acid can be achieved using several methods.
The simplest one is the acid-catalyzed esterification of the free amino acid, where
protonation of the amino group makes the α-carboxylic acid less reactive, thereby
allowing the selective protection of the side chain.224,225
Copper (II) and boron chelates used for the selective protection of the side chains of Lys
and Orn are also applied for the selective protection of the side chain of Asp and Glu.
After chelation and reaction with the appropiate protecting group halide, the chelate is
removed in the usual way.180,181,141 Another alternative is the formation of an
intramolecular anhydride between the two carboxylic acids, which leads to selective α-
protection thanks to the major electrophilicity of the α-carboxylic acid. This allows the
protection of the side chain with a distinct protecting group, followed by the removal of
the α-carboxylic acid protection.174,175
5.3. Removal
5.3.1. Protecting groups removed by acid
- Benzyl (Bn). It is the classical protecting group in Boc/Bn chemistry and is removed
with HF or TFMSA. However, it is more prone to acid-catalyzed aspartimide formation
than the cyclohexyl group. Other possible removal conditions are listed in the table.
- Cyclohexyl (cHx). It is removed with HF or TFMSA.226 227
It is widely used in the
Boc/Bn solid- phase strategy. It is superior to the benzyl group at preventing acid-
catalyzed aspartimide formation because of its major steric hindrance.228 In addition, it
is more resistant to acids than benzyl, thus making it more suitable for the synthesis of
long peptides using the Boc/Bn strategy.
- tert-Butyl (tBu). It is removed with 90% TFA in DCM (solid phase and solution) or 4
M HCl in dioxane(solution). It is the most used protecting group in Fmoc/tBu chemistry,
which is highly prone to aspartimide formation because of the reiterative use of
piperidine. The tBu group simply minimizes aspartimide formation because of its steric
hindrance compared to other less hindered protecting groups such as allyl. However,
although the tBu group is considered hindered in organic chemistry, it does not prevent
aspartimide formation in those sequences prone to it.229 See also α-amino protection.

41

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - β-Menthyl (Men).230 It is removed with HF or TFMSA and is resistant to TFA. It leads
to less base-catalyzed aspartimides than the cyclohexyl group, but is not widely used.
Sometimes diphenyl sulfide should be added as a scavenger to facilitate Men removal.
231

- β-3-Methylpent-3-yl (Mpe).232 It is removed with 95% TFA and is more sterically

hindered than the tBu group and therefore less prone to aspartimide formation.
- 2-Phenylisopropyl (2-PhiPr). It is removed with 1-2% TFA.233,187 It is used in the
Fmoc/tBu strategy mostly for the protection of Glu but also of Asp. 234
It can be
removed in the presence of tBu-type protecting groups and therefore it is useful for the
preparation of cyclic peptides.235
- 4-(3,6,9-trioxadecyl)oxybenzyl (TEGBz or TEGBn).236 It is a recently developed
protecting group which is removed with TFA-DCM. It has been used for the solid
phase synthesis of “difficult” peptide sequences (those very prone to aggregate) because
it minimizes chain aggregation during the synthesis.

Name and Structure Removal conditions Stability to the Ref.

removal of
Benzyl (Bn) 1) HF Boc, Fmoc,
2) TFMSA pNZ, a Trt,
3) H2 cat. Alloc,
4) NaOH in aqueous
organic solvents
Cyclohexyl (cHx) 1) HF Boc, Fmoc, 227,
2) TFMSA pNZ, Trt, Alloc 228

tert-Butyl ( tBu) 90% TFA-DCM (solid Fmoc, Z,b Trt, 229

phase and solution) or Alloc, pNZ
4 M HCl(dioxane)
(solution)
β-Menthyl (Men) HF, TFMSA-TFA Boc, Fmoc, Trt, 230,
Alloc, pNZ 231

β -3-Methylpent-3-yl (Mpe) 95% TFA-H2O Fmoc, Z, b Trt, 232

Alloc

2-Phenylisopropyl (2-PhiPr) 1-2 % TFA-DCM Fmoc, Alloc 187,

42

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 233,
234,
235
4-(3,6,9-trioxadecyl)oxybenzyl (TEGBz TFA-DCM Fmoc, Trt
or TEGBn)

a
Except catalytic hydrogenation removal.
b
Only catalytic hydrogenation removal.

43

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 5.3.2. Protecting groups removed by base
- 9-Fluorenylmethyl (Fm).141,237,238 It is removed with secondary amines such as
diethylamine or piperidine in DMF. It is stable to HBr in AcOH and TFA/thioanisole
and non-stable to catalytic hydrogenation and not completely stable to HF even at 0ºC.
It is used for the Boc/Bn strategy when orthogonal protection of the side chains is
required.
- 4-(N-[1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino)benzyl
192,239
(Dmab). It is removed with 2% hydrazine within minutes in DMF-H2O. It is stable
to 20% piperidine in DMF and TFA. Nevertheless, in some cases it can lead to
pyroglutamyl-terminated peptides.240

Name and Structure Removal conditions Stability to the Ref.

removal of
9-Flurorenylmethyl (Fm) Secondary amines: Boc, Trt, Alloc 141,
15% DEA or 20% 237,
piperidine-DMF or 238
DCM

4-(N-[1-(4,4-dimethyl-2,6- 2% hydrazine-DMF- Boc, Fmoc, 192,

dioxocyclohexylidene)-3-methylbutyl]- H2O Trt, Alloc 239,
amino)benzyl (Dmab) 240

H
N

O O

5.3.3. Other protecting groups

- Benzyl (Bn). See protecting groups removed by acid.
- Allyl (Al).163,164,241 It is removed with palladium and stable to TFA and bases. See also
α-carboxylic acid protection.
- p-Nitrobenzyl (pNB).242 It promotes aspartimide formation when used to protect Asp.
See also α-carboxylic acid protection.
- 2-(Trimethylsilyl)ethyl (TMSE).213,243 It is removed with fluorides, and is unstable to
acids and bases, and stable to hydrogenolysis. It is used for the protection of Asp acid
for cyclization on a Rink amide resin.244
- (2-Phenyl-2-trimethylsiylyl)ethyl (PTMSE). See α-carboxylic acid protection.

44

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - 4,5-Dimethoxy-2-nitrobenzyl (Dmnb). See α-carboxylic acid protection.

Name and Structure Removal conditions Stability to the Ref.

removal of
Allyl (Al) Pd(Ph 3) 4 (0.1 eq.) and Boc, Fmoc, 163,
scavengers (usually pNZ, a Trt 164,
PhSiH3, 10 eq.) in 241
DCM
p-Nitrobenzyl (pNB) 1) SnCl2 in DMF Boc,Fmoc, Z, a 242
2) Na2S·9H2O in H2O, Trt, Alloc
O2N
0-5ºC
3 ) Na2S2O 4, Na2CO3
in H2O, 40ºC
4) H2 cat.
5) TBAF-THF, DMF
or DMSO
Trimethylsilylethyl (TMSE) TBAF or TEAF-DMF Zb 213,
243,
Me3 Si
244
b
(2-Phenyl-2-trimethylsilyl)ethyl TBAF·3 H2O-DCM Fmoc, Z,
(PTMSE) Alloc
Me3Si

4,5-Dimethoxy-2-nitrobenzyloxycarbonyl Photolysis (λ>320 nm) Boc, Trt

(Dmnb)
NO2

MeO

MeO
a
Except catalytical hydrogenation removal.
b
Catalytical hydrogenation removal.

45

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 6. AMIDE BACKBONE
6.1. General
The NH backbone is usually unprotected in peptide synthesis. However, at least three
undesired interactions involving the NH backbone have been described.
First of all, peptide chains can aggregate during the synthesis as a result of intra- and
inter-molecular interactions, thereby significantly reducing coupling and deprotection
yields.245,246,247 Backbone protection (Figure 10) minimizes these aggregation
phenomena by preventing the formation of hydrogen bonds and also because of steric
hindrance. Thus, SPS of long peptidic sequences prone to aggregation is improved by
protecting some amides of the peptide.248,249,250,251
Secondly, nucleophilic attack of the amide NH of the amino acid before an Asp residue
(usually Gly, Ser or Thr)69,227,62,252,253,254,255 to the β-carboxyl group of Asp renders
aspartimide and the subsequent formation of β-peptide and other side products. (See
Asp and Glu side chain protection). Aspartimide formation is more severe in the
Fmoc/tBu strategy and with the Asp-Gly sequence but it can occur in many other cases.
Finally, although less frequent, internal DKP formation involving the NH and the
activated carboxylic acid of the previous amino acid has recently been described during
fragment coupling (Figure 11)121
The most used backbone protectors for the Fmoc/tBu strategy are pseudoprolines
(Figure 12),256,257,251 2-hydroxy-4-methoxybenzyl (Hmb),258 and 2,4-dimethoxybenzyl
(Dmb), and more recently 3,4-ethylenedioxy-2-thenyl (EDOTn) and 1-methyl-3-
indolylmethyl (MIM) 259 The pseudoproline concept is valid only for -hydroxy or thio
amino acids such as Ser/Thr or Cys. Although, the rest of protecting groups can be used
for all amino acids, practically they are only used for Gly because of the difficulty of
260
elongation of the peptide chain because of steric hindrance

Figure 10. Partially backbone-protected peptide. BPG= backbone-protecting group Comentario [m4]: No estoy segura del
cambio creo que quiere unificar la forma de
poner los grupos protectores GP o Prot
como en la figura 1

46

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 O O
O O N
Boc-HN N
Boc-HN N N O
N O N H
O R1
R4
H
O R3 N N O
HN
O
R2

Internal DKP Formation Desired Reaction

O O
O
Boc-HN N
Boc-HN O N O
N H
O R1
R4 N R4 O R3 O
O H
N N
R3 Boc-HN N O

+ O O R2
O O
Boc-HN N
N O
H
O R1
O
HN
O
R2

Figure 11. Internal DKP formation. Adapted from ref. 121

H
N COOH

O
R

Figure 12. Pseudoproline of Ser (R= H) and Thr (R= CH3).

6.2. Introduction of the protecting groups

Due to the steric hindrance of the protected amino acid, it is incorporated usually
through the corresponding dipeptides. Thus, pseudoproline dipeptides are prepared by
reaction of Fmoc-AA-Ser or Fmoc-AA-Thr with 2,2-dimethoxypropane.261 Most of the
other backbone protectors are introduced by reductive amination of the aldehyde of the
protecting group with the amine of the corresponding amino acid, followed by either α-
amino protection or dipeptide formation.262,258,259
6.3. Removal
6.3.1. Protecting groups removed by acid
- Pseudoprolines (ΨPro). The most used are dimethyloxazolidines (Ψ Me,Mepro) because
of their major acid lability (removed by TFA within minutes)261 Pseudoproline
derivatives have been extensively applied to the synthesis of difficult peptides.257,,263
However, they are limited to Ser and Thr. Dimethylthiazolidines (Cys pseudoprolines)

47

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 have also been described but they are not so widely used because of their major acid
stability (removed with TFA within hours).
- 2-Hydroxy-4-methoxybenzyl (Hmb).258 It is used mainly as Fmoc-(FmocHmb)AA1-
264 265
OH or as Fmoc-AA2-(Hmb)AA1-OH but also as Fmoc(Hmb)AA1-OH. It is
removed with TFA. The main advantage of the Hmb group compared with other
backbone protectors such as Dmb is that the coupling on Hmb-amino acids is easier.
Thus, Hmb is not restricted to Gly, and derivatives of more hindered amino acids can be
used. However, the presence of a free hydroxyl group can be a problem in depsipeptide
synthesis or in post-synthetic phosphorylations.
- 2,4-Dimethoxybenzyl (Dmb).266. It is removed with high concentrations of TFA. Its
major inconvenience is its bulkiness, which limits its use for non-sterically hindered
amino acids (mainly Gly),255 or for direct coupling of Dmb-protected dipeptides (Fmoc-
AA’-(Dmb)AA-OH). 267
- 2,4,6-Trimethoxybenzyl (Tmob).268 It is removed with TFA and has been used for the
Fmoc/tBu SPS of highly hydrophobic peptides.269 Although it is not as widely used as
Dmb, coupling on 2,4,6-trimethoxybenzyl amines of amino acids is described to be
faster than in the case of the less hindered 2,4-dimethoxybenzyl amines.258
- 1-Methyl-3-indolylmethyl (MIM) and 3,4-Ethylenedioxy-2-thenyl (EDOTn).259 These
are recently developed backbone protectors for the Fmoc/tBu strategy. They are
completely removed with TFA-DCM-H2O (95-2.5-2.5) in 1 h. Both are more acid-labile
than the 2,4-dimethoxybenzyl group, and EDOTn is less sterically hindered, thus
couplings on EDOTn amino acids are faster.

48

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal conditions Stability to the Ref.
removal of
Pseudoprolines (oxazolidines) 95% TFA and Fmoc, Alloc 257,
Pseudoprolines scavengers 261,
263
H
N COOH

O
R

R= H (Ser) or Me (Thr)

2-Hydroxy-4-methoxybenzyl (Hmb) 95% TFA and Fmoc, Alloc 258,

scavengers 264,
HO 265

OMe

2,4-Dimethoxybenzyl (Dmb) 95% TFA and Fmoc, Alloc 255,

scavengers 266,
MeO 267

OMe

2,4,6-Trimethoxybenzyl (Tmob) 95% TFA and Fmoc, Alloc 258,

scavengers 268,
MeO OMe 269

OMe

1-Methyl-3-indolylmethyl (MIM) TFA-DCM-H2O Fmoc, Alloc 259

(95:2.5:2.5)

N
Me

3,4-Ethylenedioxy-2-thenyl (EDOTn) TFA-DCM-H2O Fmoc, Alloc 259

(95:2.5:2.5)
O O

6.3.2. Other protecting groups

- 4-Methoxy-2-nitrobenzyl.270 It is removed by photolysis at λ=360 nm for more than 2 h
using Cys (200 mmol per 1 mmol of 4-methoxy-2-nitrobenzyl) as scavenger. This is a

49

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 backbone protector, fully compatible with Boc chemistry, thereby allowing the
obtention of backbone-protected peptides after HF cleavage.
- (6-Hydroxy-3-oxido-1,3-benz[d]oxathiol-5-yl)methyl 271,272 and 2-hydroxy-4-methoxy-
5-(methylsulfinyl)benzyl.273 These are safety-catch backbone protectors which become
unstable to TFA after reduction of the sulfoxide to sulfide. (6-Hydroxy-3-oxido-1,3-
benzoxathiol-5-yl)methyl is removed with 20 eq. each of NH4I and (CH3)2S in TFA at
0°C over 2 h, whereas 2-hydroxybenzyl-4-methoxy-5-(methylsulfinyl) is removed with
SiCl4-TFA-anisole-ethanedithiol, (5:90:2.5:2.5), for 2 h at room temperature. Acylation
as well as acyl migration is faster in the case of the latter.
- Boc-N-methyl-N-[2-(methylamino)ethyl]carbamoyl-Hmb (Boc-Nmec-Hmb).274 It is a
recently developed protecting group. It has been used for solid phase synthesis. After
the removal of the Boc group with TFA during the cleavage of the peptide from the
resin, the Nmec moiety is removed via an intramolecular cyclization in basic conditions
(N-methylmorpholine (10 eq) in DMF/H2O (3:7), 4 -8 h), leading to the Hmb protected
peptide. Then, Hmb is removed with 95% TFA and scavengers. The main advantage of
the Boc-Nmec-Hmb group is that after Boc removal, a cationic peptide is obtained
which increase the solubility of insoluble peptides making their purification easier.

Name and Structure Removal conditions Stability to the Ref.

removal of
4-Methoxy-2-nitrobenzyl Photolysis (λ=360 nm) Boc, Z, a Trt, 270
and Cys as scavenger Alloc
O2N

OMe

(6-Hydroxy-3-oxido-1,3- NH4I (20 eq.) and Boc, Fmoc,Trt 271,

benz[d]oxathiol-5-yl)methyl (CH3)2S (20 eq.)-TFA, 272
2 h, 0°C.
HO

S O
O

2-Hydroxy-4-methoxy-5- SiCl 4-TFA-anisole- Boc, Fmoc,Trt 273

(methylsulfinyl) benzyl ethanedithiol,
(5:90:2.5:2.5), 2 h, rt

50

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 HO

O
S
O Me
Me

Boc-N-methyl-N-[2- i) 25-50% TFA-DCM Fmoc, Trt

(methylamino)ethyl]carbamoyl-Hmb ii) N-methylmorpholine
(Boc-Nmec-Hmb) (10 eq) in DMF/H2O
(3:7), 4 -8 h
iii) 95% TFA and
scavengers

a
Except catalytical hydrogenation removal.

51

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 7. ASPARAGINE (Asn) AND GLUTAMINE (Gln)
7.1. General
Asn and Gln (Figure 13) are often used without side chain protection.
H2N COOH H2N COOH

CONH2
CONH2
Asn Gln

Figure 13. Asparagine (Asn) and Glutamine (Gln)

Nevertheless, unprotected derivatives show poor solubility and therefore slow coupling
rates. In addition, their free primary amides can undergo two main side reactions.
1) Dehydratation during the coupling (Figure 14), which is a base-catalyzed side
reaction and therefore more favoured in those coupling protocols that involve the use of
base. It can be minimized using the corresponding Nα-protected pentafluorophenyl
esters or carbodiimide-mediated couplings in the presence of HOBt.275,276 Dehydratation
is more important in the Fmoc/tBu strategy than in the Boc/Bn one because in the latter,
the use of HF apparently reverts the reaction, whereas in the former TFA is not acidic
enough to revert to the amide.276
O
H H O
N O
PG Act PG N H
O N
O PG OH

N N H C N
H H
B B

Figure 14. Dehydratation of Asn. Adapted from 277.

2) Pyroglutamyl (Figure 15) formation is a weak acid-catalyzed side reaction that occurs
on N-terminal Gln that leads to truncated peptidic chains. Being an acid-catalyzed
reaction, it has more importance in the Boc/Bn strategy and can be minimized by
reducing exposure to weak acids.278
O O
H2N H
H+ H N N N N
2 H O H
O
Pyroglutamyl

Figure 15. Pyroglutamyl formation.

52

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Adequate protection of Asn and Gln side chains prevents both side reactions. As for
dehydratation, it is not necessary for the protecting group of choice to be stable during
the whole peptide synthesis, but only during the coupling step. Furthermore, protection
of Asn and Gln side chains also increases coupling yields by confering more solubility
to the corresponding Asn and Gln derivatives and probably reducing the formation of
hydrogen bonds that stabilize secondary structures.
Removal of the protecting groups is usually easier in Gln than in Asn, being particularly
difficult in N-terminal Asn because of the proximity of the free and therefore protonated
α-amino group.281,279,280
Currently, the most used protecting groups are Xan (9-xanthenyl) and Trt, which are
compatible with both Boc/Bn and Fmoc/tBu strategies. In the case of the former, the
Xan group protects Asn and Gln side chains only during the coupling and is removed
during TFA treatments for Boc removal.
7.2. Introduction of the protecting groups
Protection is usually performed via acid-catalyzed reaction of the corresponding alcohol
with Z-Gln or Z-Asn, followed by catalytic hydrogenolysis to eliminate the Z group and
Fmoc or Boc Nα protection. 281,282 In the case of 9-xanthenyl, the direct protection of the
Fmoc-Asn and Fmoc-Gln has also been described. 283
7.3. Removal
7.3.1. Protecting groups removed by acid- 9-Xanthenyl (Xan).282 It is removed by
90% TFA and scavengers. In contrast to Trt, no extra reaction time is required when the
α-amino of Asn is free.279 Xan is used in both the Boc/Bn and Fmoc/tBu strategies.282,283
In the case of the Boc strategy, Xan is eliminated during TFA treatments to remove the
Boc group; however, Asn or Gln residues can undergo dehydratation only during the
coupling and thus, Xan elimination after it is a minor problem.283
- Trityl (Trt).280,281 It is removed with TFA-H2O-EDT (90:5:5), and used in both the
Boc/Bn and Fmoc/tBu strategies. The time required for removal increases from 10 min
to more than 4 h when the α-amino of Asn is free. Scavengers must be used to prevent
Trp alkylation. It is stable to bases and catalytic hydrogenolysis.
- 4-Methyltrityl (Mtt).284,280 It is a more acid-labile alternative to the Trt group (95%
TFA, 20 min), and is particularly useful when the α-amino of Asn is free.
- Dimethylcyclopropylmethyl (Dmcp) or Cyclopropyldimethylcarbinyl (Cpd).285,286 It is
removed with TFA-thioanisole-EDT-anisole (90:5:3:2), being another more acid labile

53

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 alternative to Trt, especially when the α-amino of Asn is free. It is more soluble and
coupling rates are better than with the Trt group.
- 4,4’-Dimethoxybenzhydryl (Mbh).287,288 It is used mainly in the Boc/Bn strategy but
also in the Fmoc/tBu one. Its removal using TFA is slow and requires scavengers to
prevent alkylation of Trp.275
- 2,4,6-Trimethoxybenzyl (Tmob).289 It is removed with 95% TFA and scavengers. It is
more acid-labile, more soluble and gives less side reactions during coupling than Mbh-
protected derivatives. However, it is not currently widely used because it can cause
alkylation of Trp and is reported to give worse results than the Trt group.290,281

54

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal conditions Stability to the Ref.
removal of
9-Xanthenyl (Xan) 90% TFA-scavengers Fmoc, Trt, 279,
Alloc 282,
283
O

Trityl (Trt) TFA-H2O-EDT Fmoc, Trt, 280,

(90:5:5) Alloc 281

4-Methyltrityl (Mtt) 95% TFA Fmoc, Trt, 280,

Alloc 284

Dimethylcyclopropylmethyl (Dmcp) or TFA-thioanisole-EDT- Fmoc, Alloc 285,

Cyclopropyldimethylcarbinyl (Cpd) anisole (90:5:3:2) 286

4,4’-Dimethoxybenzhydryl (Mbh) 1M TMSBr- Fmoc, Alloc 275,

thioanisole-EDT-m- 287,
cresol in TFA (2 h at 288
O O
0ºC)
2,4,6-Trimethoxybenzyl (Tmob) 95% TFA-DCM and Fmoc, Alloc 281,
scavengers 289,
O O 290

55

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 8. ARGININE (Arg)
8.1. General
Protection of the guanidino group of Arg (Figure 16) is required to prevent
deguanidination, which renders Orn (Figure 17) 291 and δ-lactam formation (Figure 18)
277
as a result of the nucleophilicity of the guanidino group. Arg side chain protection
remains unsolved in peptide synthesis because of the difficulty to remove the protecting
groups.
H2N COOH

 HN

HN NH2
' 

Figure 16. Arginine (Arg)

Since the guanidino group is basic (pKa= 12.5), it remains protonated in most of the
conditions used for peptide synthesis.292,293 To prevent deprotonation in Fmoc/tBu SPS,
washings with 0.25 M HOBt are carried out between Fmoc removal and the next
coupling.294 However, if deprotonation takes place, deguanidination occurs after
acylation of the neutral guanidino group. This drawback stimulated research into
protecting groups for Arg.
O O O O
H H H O O
H2N PG N C N H H
peptide PG N C
OX peptide PG N C N
peptide
R R Base R PG
PG-HN N
PG-N PG-HN R
PG-HN N
HN NH-PG N NH-PG
H O
PG N Deguanidilated peptide
O
R
Figure 17. Acylation of the side chain of Arg during amino acid coupling, followed by
base-catalyzed deguanidination. Adapted from 291

Arg derivatives tend to be worse acylating reagents compared with other amino acid
derivatives, mainly because of the formation of the -lactam from the activated species
(Figure 18). In a solid-phase mode, the presence of the -lactam does not translate in
an impurity in the crude reaction, because it is not reactive but it is translated in a less
active species to be coupled.

56

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 H O
PG N O NHR
OX H
PG N
N NH

NH
-lactam
RHN NH

Figure 18. Mechanism of δ-lactam formation. R= H or protecting group. Adapted from

277.

In principle, protection of all the nitrogens of the guanidino group is required to fully
mask its nucleophilicity. However, diprotection and monoprotection are easier to
achieve and minimize side reactions when bulky and electron-withdrawing protecting
groups are used.
The most used protecting strategy is sulfonyl protection of the -amino function. For
the Boc/Bn strategy, the most used group is Tos while for the Fmoc/tBu strategy the
most popular protecting groups are Pbf (pentamethyl-2,3-dihydrobenzofuran-5-
sulfonyl) and Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl). However, both, but
particularly Pmc, are too acid stable and their removal in peptides with multiple Args is
especially problematic.
8.2. Introduction of the protecting groups
It depends on the nature of the protecting group, in the case of sulfonyl-protecting
groups, which are the most used ones, they are usually introduced by reaction of the
corresponding sulfonyl chloride with Z-Arg-OH in H2O-acetone using NaOH as a base.
To obtain the corresponding Fmoc/Boc derivative, the Z group is removed by catalytic
hydrogenolysis and the Fmoc/Boc group is incorporated under regular conditions.295
8.3. Removal
8.3.1. Protecting groups removed by acid.
Arylsulfonyl ω-protection: Although this kind of protection does not fully prevent δ-
lactam formation, this process can be minimized by using carbodiimides in the presence
of HOBt derivatives to decrease the activity of the active O-acylisourea.184
- Tosyl (Tos). It is removed with HF, TFMSA-TFA-thioanisole or Na/NH3.296 It is the
most used protecting group in the Boc/Bn solid phase strategy.297
- 2,2,5,7,8-Pentamethylchroman-6-sulfonyl (Pmc).295 It is widely used in the Fmoc/tBu
solid phase strategy. It is removed by TFA-scavengers. Currently, it is being replaced
by the Pbf group.

57

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - 2,2,4,6,7-Pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf).298 It is removed by
TFA-scavengers and is more acid-labile than the Pmc group. Currently, it is the best
Arg-protecting group for the Fmoc/tBu solid phase strategy, although it is still too acid-
stable in peptides with multiple Arg-containing peptides where long reaction times are
required.
- Mesitylyl-2-sulfonyl (Mts).299,300 It is removed with TFMSA-TFA-thioanisole. It is
used in the Boc/Bn solid phase strategy and is more acid-labile than the tosyl group.
- 4-Methoxy-2,3,6-trimethylphenylsulfonyl (Mtr).301 It is removed by TFA-thioanisole.
Although it is still used, it has been mostly replaced by the more acid-labile Pmc or Pbf
in Fmoc/tBu chemistry.
- 1,2-Dimethylindole-3-sulfonyl (MIS).302 It is a recently developed protecting group,
which is much more TFA-labile than Pbf. It is completely removed with 50% TFA in
30 min, even in multiple Arg-containing peptides.
Other kinds of Arg protection
- ω,ω’-bis-tert-Butyloxycarbonyl (bis-Boc).303 It is removed with 90-95% TFA in the
presence of scavengers, prevents deguanidination but does not completely prevent δ-
lactam formation.304,305 The coupling rates of bis-Boc-protected Arg are low.
- ω-5-Dibenzosuberenyl (Suben), 5-Dibenzosuberyl (Sub) and 2-Methoxy-5-
306
dibenzosuberyl (MeSub). They are the most acid-labile derivatives (removed with 25-
50% TFA) and are reported to minimize δ-lactam formation and deguanidination
because of their steric hindrance. Although they look very promising, they have not
been widely used.
- ω-Nitro (NO2).307 It prevents δ-lactam formation and deguanidination in most cases. It
can be removed with HF (SPS) or catalytic hydrogenolysis. In both cases long reaction
times are required, which is an inconvenience in the case of sensitive peptides. For
instance, in the case of hydrogenolysis, partial hydrogenation of Trp or even Phe can
occur.308 Due to the clean removal of the nitro group by hydrogenolysis and its low cost,
nitro protection is still used for large-scale solution synthesis of peptides 309,310 and even
for SPS, where the nitro group is removed by hydrogenolysis after the cleavage from
the resin.311

58

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal conditions Stability to the Ref.
removal of
Protonation - - 294

NH

H2N NH2

p-Toluenesulfonyl (Tos) 1) HF Boc, Fmoc, Trt, 296,

2) TFMSA-TFA- Alloc 297
HN
O thioanisole
S N NH
H 3) Na/NH3
O

2,2,5,7,8-Pentamethylchroman-6- 90% TFA-scavengers Fmoc, Trt, 295

sulfonyl (Pmc) (H2O and TIS) several Alloc
hours.
HN
O
O S N NH
H
O

2,2,4,6,7-Pentamethyl-2,3- 90 TFA-scavengers Fmoc, Trt, 298

dihydrobenzofuran-5-sulfonyl (Pbf) (H2O and TIS) 1 h Alloc
(longer times in
HN
O multiple arginine
O S N NH
H containing peptides)
O

Mesitylyl-2-sulfonyl (Mts) TFMSA-TFA- Boc, Fmoc, Trt, 299,

thioanisole Alloc 300
HN
O
S N NH
H
O

4-Methoxy-2,3,6- 95% TFA-thioanisole Fmoc, Trt, 301

trimethylphenylsulfonyl (Mtr) Alloc

HN
O
O S N NH
H
O

1,2-Dimethylindole-3-sulfonyl (MIS) 50% TFA and Fmoc, Alloc 302

O scavengers, 30 min
S O

59

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 ω,ω’-bis-tert-Butyloxycarbonyl (bis-Boc) 90- 95% TFA and Fmoc, Alloc 303,
scavengers 304,
O NH O
305
O N N O
H

5-Dibenzosuberenyl (Suben) 25-50% TFA Fmoc, Alloc 306

HN
H
N
HN

5-Dibenzosuberyl (Sub) 25-50% TFA Fmoc, Alloc 306

HN
H
N
HN

2-Methoxy-5-dibenzosuberyl (MeSub) 25-50% TFA Fmoc, Alloc 306

HN
H
N
HN

Nitro (NO2) HF (solid phase) Boc, Fmoc, 307,

H2 cat. (solution) Alloc 308,
NH
309,
HN N NO2
H 310,
311

8.3.2. Protecting groups removed by base

- Trifluoroacetyl (tfa). It has been applied recently for the protection of guanidines used
in solution Boc peptide synthesis and Fmoc/tBu SPPS. However, although there are
references of tfa-protected Arg derivatives,312,313,314 to date it has not been implemented
for Arg protection in peptide synthesis.

60

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal conditions Stability to the Ref.
removal of
Trifluoroacetyl (tfa) 1) K2CO3-MeOH-H2O Boc, Fmoc, Z, a 312,
(solution) Trt, Alloc 313,
2) K2CO3-MeOH-DMF- 314
H2O (solid phase)
a
Catalytical hydrogenation removal.

8.3.3. Other protecting groups

- Nitro (NO2). See protecting groups removed by acid.
- ω,ω’-Bis-benzyloxycarbonyl (bis-Z).315 Its removal by catalytic hydrogenation requires
long reaction times. It is used mostly in Boc/Bn chemistry but also in the Fmoc/tBu
strategy.
- ω,ω’-bis- allyloxycarbonyl (Alloc).164 It is removed with Pd(PPh3)4 and scavangers
(dimethylbarbituric acid)316 and is compatible with the Boc/Bn solid phase strategy.
The base treatment required to remove the Fmoc group also eliminates one of the Alloc
groups.

Name and Structure Removal conditions Stability to the Ref.

removal of
ω,ω’-bis-benzyloxycarbonyl (Z) H2 cat. (long time) Boc, Fmoc, Trt 315

O NH O

O N N O
H

ω,ω’-bis- allyloxycarbonyl (Alloc) Pd(PPh3)4, barbituric Boc, Fmoc, Z, a 164,

acid Trt 316
O NH O

O N N O
H
a
Except catalytical hydrogenation removal.

61

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 9. CYSTEINE (Cys)
9.1. General
Protection of the side chain of Cys (Figure 19) is mandatory in peptide synthesis
because the nucleophilic thiol can otherwise be acylated, alkylated or oxidized to
disulfide by air.
H2N COOH

SH
Cys

Figure 19. Cysteine (Cys)

Nevertheless, even protected Cys can undergo several side reactions. The most relevant
are listed here.
- Oxidation and alkylation of the thioether. Although less critical than in the case of
Met, it can also occur.317,318,319 Oxidation of the Cys residues during global
deprotection can be minimized using 10% of H2O as scavenger.121
- β-Elimination (Figure 20) occurs when protected Cys is exposed to strong bases, such
as sodium in liquid ammonia (required to remove the Benzyl group), alkaline conditions
or hydrazylnolysis, but also to strong acids such as HF. This side reaction is particularly
critical in the case of C-terminal Cys, which in the Fmoc/tBu strategy undergoes β-
elimination followed by piperidine addition to give piperidylalanine residue. The extent
of β-elimination also depends strongly on the protecting group used, StBu being the
worst case followed by Acm and Trt.320,321 The Bn group can also produce β-
elimination.
O
NH O O
H H
N N N
H H H N
N H N
S H
PG H N
N

Piperidyl alanine

Figure 20. Base-catalyzed β-elimination of protected Cys followed by piperidine

addition leading to piperidyl alanine.
- Reaction with carbocations resulting from the elimination of protecting groups: after
its deprotection, Cys can react with the cations generated in acidic conditions. For
instance, S-tert-butylated Cys has been observed after the removal of the Boc group or
after global deprotection in a Fmoc/tBu strategy. 322

62

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - Reattachment to the resin: resin bound carbocations generated in the acidolytic
cleavage from resins can react with both protected and unprotected Cys, thus causing
reattachment of the peptide to the resin.323
- Transfer of Acm (Acetamidomethyl) group to Ser, Thr, Gln and Tyr during Acm
removal. 324 325 326
- Formation of thiazolidines of N-terminal Cys (Figure 21) can take place if His-
protecting groups such as Bom (benzyloxymethyl) or Bum (tert-butyloxymethyl),
which generate formaldehyde when removed, are present. It can be minimized using
Cys as scavenger. 327,328
O O O
H2N H
N N
H H H N
H
HS S
Thiazolidine

Figure 21. Thiazolidine formation by reaction of N-terminal Cys with formaldehyde.

- Racemization: Cys is highly prone to racemize during the anchoring to the solid
support or during the couplings.329 330
The extent of the racemization also depends on
the S-protecting groups (S Bu > Trt > Acm > MeBn > tBu)331,332,333,334,335 and coupling
t

methods used (favoured if pre-activation in the presence of base is performed and in the
coupling methods involving the use of base). Epimerization of the Cys linked to a
hydroxyl resin can even take place during the synthesis as a result of the repetitive base
treatments to remove the Fmoc group, 2-chlorotrityl resin being the least prone to this
process.331,336
The most used protecting groups for the Fmoc/tBu strategy are the Acm or Trt groups,
when the desired product is the disulfide, and the Trt group when the desired product is
the free thiol. For the Boc/Bn strategy, Bn, and Meb (p-methylbenzyl) are the most
used to obtain the free thiol and Acm to obtain disulfides.
9.2. Introduction of the protecting groups
The Cys thiol shows high nucleophilicity, therefore Cys thiol protection is usually
carried out using fully unprotected Cys as starting material. The S-protecting agents
used can be alkyl halides or tosylates, under acidic or basic conditions, or alcohols,
which are dehydrated under acidic conditions. Benzyl-type protection can also be
performed via reduction of the thiazolidine formed with the corresponding
benzaldehyde.337

63

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 9.3. Removal
The classification of the protecting group of Cys is particularly complex because most
of the protecting groups used can be removed either by oxidation to the disulfide bridge
or by other mechanisms. The following classification has drawn up taking into account
these other mechanisms but also indicating the conditions for the oxidative removal in
each particular case.
9.3.1. Protecting groups removed by acid.
- p-Methylbenzyl (Meb).338 More acid-labile than the Bn, it is removed with HF and
scavengers at low temperatures.339 340
It is gradually replacing the Bn in the Boc/Bn
solid phase strategy. It can also be removed with Tl(III) trifluoroacetate or with
MeSiCl3 in the presence of diphenylsulfoxide to yield disulfide bridges. However, p-
methoxybenzyl (Mob) is usually a cleaner option.341
- p-Methoxybenzyl (Mob).338 It is more acid-labile than Meb and is also used in the
Boc/Bn solid phase strategy. However, it is partially removed in the repetitive
treatments to remove the Boc group when long peptide sequences are synthesized.338 It
is completely removed by HF at 0ºC and scavengers, TFMSA/TFA342 and Hg (II)
acetate or trifluoroacetate in TFA or AcOH respectively.343 It can be selectively
removed in the presence of Meb using Ag(I) trifluoromethanesulfonate in TFA.344 An
intramolecular disulfide bridge between two Cys(Mob)-protected residues can be
formed by removing the Mob group with MeSiCl3 or SiCl4 in TFA in the presence of
diphenyl sulfoxide at 4ºC in 30 min.345 In addition, oxidative removal with Tl(III)
trifluoroacetate also leads to the formation of a disulfide bridge by reaction with a free
Cys side chain.
- Trityl (Trt).346 It is removed with TFA and scavengers, such as triisopropylsilane (TIS)
to prevent retritylation, or AgNO3.347 It is used for the Fmoc/tBu strategy although
Fmoc-Cys(Trt) can undergo racemization in basic carboxyl activation conditions.332 It
can also be removed by oxidation with iodine, thereby leading to a dilulfide bridge by
reaction with a free Cys side chain. Other oxidative removals are listed in the table.348
- Monomethoxytrityl (Mmt).349 It is removed with diluted TFA and scavengers. It is
considerably more acid-labile than the S-trityl group and can be removed selectively in
its presence as well as in the presence of tBu-protecting groups. Oxidative removal is
similar to the case of the Trt group.
- Trimethoxybenzyl (Tmob).350 It is another more acid-labile alternative to the Trt group
for the Fmoc/tBu strategy. It is removed with diluted TFA (5-30%) and scavengers;

64

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 however, the trimethoxybenzyl cation resulting from its cleavage can alkylate Trp
residues.
- 9-Xanthenyl (Xan).332 It has similar stability features to Mmt, thus, it can also be
removed selectively in the presence of S-trityl and tBu-protecting groups or Rink and
PAL handles.
- 2,2,4,6,7-Pentamethyl-5-dihydrobenzofuranylmethyl (Pmbf).351 It is a relatively new
highly acid-labile protecting group (Fmoc/tBu chemistry). It is removed with TFA-TES-
DCM (0.5-5-94.5) in 2 h to render the free thiol. Alternatively, treatment with I2 yields
the disulfide bridge. This protecting group has been succesfully applied to obtain
oxytocin.
- Benzyl (Bn).352 It is removed with HF at 25ºC or Na in liquid ammonia. However,
although still used, it is being replaced by other benzyl derivatives that do not require
such harsh conditions for their removal.
- tert-Butyl (tBu) and 1-Adamantyl (1-Ada).353 Both are fully stable to TFA and can
therefore be used in the Boc/Bn strategy. They are also quite stable to HF at low
temperatures but cleaved at higher temperatures in the presence of scavengers.335 They
are also stable to Ag (I) trifluoromethanesulfonate in TFA,344 which quantitatively
removes the S-Mmt group, and also to iodine oxidation. Other possible cleavage
conditions are listed in the table.343

65

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Removal conditions Stability to Compatibility
Name and Structure (final S form) the removal with the most Ref
of important sulfur
protecting groups
p-Methylbenzyl (Meb) 1) HF, scavengers Boc, Fmoc, Trt, a Acm, StBu, 338,
(SH) Trt, Alloc Npys, Fmb 339,
2) MeSiCl3 or SiCl4, 340,
TFA, Ph 2SO, (S-S) 341
3) Tl(TFA) 3 (S-S)
p-Methoxybenzyl (Mob) 1) HF, scavengers Boc, c Fmoc, Trt, a Acm, StBu, 338,
b
EMBED ChemDraw.Document.6.0 (SH) Trt, Alloc Npys, Fm 342,
2) TFMSA-TFA (SH) 343,
O
3) Hg(OAc) 2 in TFA 344,
or Hg(TFA)2, in 345
AcOH (SH)
4) Ag (TFMSO)
(SH)
5) Tl(TFA) 3 (S-S)
6) MeSiCl3 or SiCl4
TFA, Ph 2SO, 4ºC, 30
min, (S-S)
Trityl (Trt) 1) 95% TFA, Fmoc, Alloc Meb/Mob, a Acm,d 332,
scavengers (SH) StBu, Npys, Fmb 346,
2) Hg(OAc) 2 (SH) 347,
3) AgNO3 (SH) 348
4) I2 (S-S)
5) Tl(TFA) 3 (S-S)
Monomethoxytrityl (Mmt) 1) 1% TFA, Fmoc, Alloc Meb/Mob, a Acm,d 349
scavengers (SH) StBu, Npys, Fmb
2) Hg(OAc) 2 (SH)
O
3) AgNO3 (SH)
4) I2 (S-S)
5) Tl(TFA) 3 (S-S)
Trimethoxybenzyl (Tmob) 5-30% TFA, Fmoc, Alloc 350
O scavengers (SH)
O 2) I2 (S-S)

O 3) Tl(TFA) 3 (S-S)

66

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 9-Xanthenyl (Xan) 1% TFA , scavengers Fmoc, Alloc 332
(SH)

2,2,4,6,7-pentamethyl-5- 1) TFA-TES-DCM Fmoc 351

dihydrobenzofuranylmethyl (Pmbf) (0.5-5-94.5) in 2 h
(SH)
2) I2 (S-S)

Benzyl (Bn) 1) HF (SH) Boc, Fmoc, 352

2) Na, NH3 (SH) Trt, Alloc

tert-Butyl (tBu) 1) HF (20ºC) Boc, Fmoc, 335,

scavengers (SH) Trt, Alloc 343,
2)TFMSA-TFA and 344,
scavengers (SH) 353
3) Hg(OAc) 2 in TFA
(SH)
1-Adamantyl (1-Ada) 1) HF (20ºC) Boc, Fmoc, 335,
scavengers (SH) Trt, Alloc 343,
2)TFMSA/TFA and 344,
scavengers (SH) 353
3)Hg(OAc)2 in TFA
(SH)
a
The Trt group should be removed first
b
The Fm should be removed first
c
Except for repetitive treatments
d
Trt should be removed first with TFA solutions

9.3.2. Protecting groups removed by base

- 9-Fluorenylmethyl (Fm).354 It is removed with base (i.e. 50% piperidine-DMF for 2 h
or 10% piperidine-DMF overnight)355 and is very stable to strong acids such as HF. It is
used in the Boc/Bn solid-phase strategy. It can be removed on solid phase or in solution,
thereby yielding a disulfide because of air oxidation unless reducing thiols are
employed. It is resistant to oxidative cleavage with iodine or Tl(TFA)3 of other Cys-
protecting groups.335
- 2-(2,4-Dinitrophenyl)ethyl (Dnpe).356 It is removed with bases such as piperidine-
DMF (1:1) in 30-60 min, thereby yielding the disulfide bridge, or in the presence of β-
mercaptoethanol to give the free thiol. It is a less sterically hindered alternative to the

67

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Fm group for the Boc/Bn strategy (specially suited to facilitate the cleavage of peptides
with C-terminal Cys), stable to strong acids such as HF and oxidative conditions to form
disulfide bridges with Acm (I2 or Tl(TFA)3 in TFA).
- Benzyl (Bn). See protecting groups removed by acid.
- 9-Fluorenylmethoxycarbonyl (Fmoc).357 Only preliminary solution studies are
available for Cys thiol protection with Fmoc. It seems to be more base-labile than the
Fm group. It is removed with TEA in the presence of I2 or benzenethiol in DCM to
yield the corresponding disulfide. These removal conditions do not affect the Nα -Fmoc
group.

Name and Structure Removal conditions Stability to Compatibility with Ref

the removal other sulfur
of protecting groups
9-Fluorenylmethyl (Fm) 1) 10-50% piperidine Boc, Z,a Trt, Meb/Mob/Trt, b 335,
b
in DMF (S-S) Alloc Acm, 354,
2) DBU in DMF 355
(S-S)

2-(2,4-Dinitrophenyl)ethyl (Dnpe) Piperidine:DMF (1:1) Boc, Z,a Trt, 356

(S-S) Alloc
O2N
in the presence of
NO2 mercaptoethanol: (SH)
9-Fluororenylmethoxycarbonyl (Fmoc ) TEA-benzenethiol or Boc, Z, a 357
I2 (S-S) Trt, Alloc

O
O

a
Except catalytical hydrogenolysis.
b
The Fm should be removed first

9.3.3. Other protecting groups

- Acetamidomethyl (Acm).358,359 Removed by oxidative treatment with I2 or Tl(TFA)3
to form disulfide bonds or with Hg (II) and Ag(TFMSO)344 to obtain the free thiol. It is
comptible with both the Boc/Bn and Fmoc/tBu strategies. Nevertheless, it is partially
360,326
removed with HF or even TFA depending on the scavengers used. In the latter
361
case, absence of water and use of TIS minimizes the removal.
- Phenylacetamidomethyl (PhAcm).362 It is an analog of Acm that can be removed in
similar conditions and also by treatment with the enzyme penicillin aminohydrolase.

68

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - tert-Butylmercapto (StBu).363 It is removed with thiols (benzenethiol, β-
mercaptoethanol or dithiothreitol),364 Na2SO3 in AcOH, 365 or phosphines (PBu3 or PPh3
in CF3CH2OH).366 It is compatible with the Boc and Fmoc strategies. It is partially
removed with HF but completely stable to TFA and to bases like piperidine.367
- 3-Nitro-2-pyridinesulfenyl (Npys). It is removed by reducing thiols and phosphines to
render the free thiol.368 It is stable to TFA and HF but it is not stable to the low-high
cleavage protocol or to bases.369 It is used in the Boc/Bn strategy mainly to obtain
disulfide bonds by nucleophilic displacement by the thiol of a free Cys. 370
- 2-Pyridinesulfenyl (S-Pyr).371 It is used in the Boc/Bn strategy and is useful when
orthogonal protection of unprotected fragments is required. Ligation of a free
thiocarboxylic peptide with an S-Pyr-protected N-terminal Cys occurs at pH 2, the
subsequent S to N migration at pH 7 and final treatment with DTT renders the final
ligated peptide with free Cys. S-Pyr is stable to 1 M TFMSA in TFA-anisole (10:1) at
0ºC for 2 h (cleavage conditions for the MBHA resin)
- Allyloxycarbonyl (Alloc).163 It is removed with tributyltin hydride catalyzed by Pd(0)
(usually Pd(PPh3)4). Because of its base lability, it is used only in the Boc/Bn solid
phase strategy.
- N-allyloxycarbony-N-[2,3,5,6-tetrafluoro-4-(phenylthio)phenyl]] aminomethyl
(Fsam).372 It is an allyl-type protecting group that can be removed by palladium to
render the free thiol both in solution and on solid phase, and is the only Cys- protecting
group that allows a selective and easy release of the thiol on solid phase. It is
completely stable to TFA and piperidine and can also be removed by iodine oxidation to
render a disulfide bridge.
- o-Nitrobenzyl (oNB).373,374 It is a protecting group removed by photolysis (λ= 300-400
nm) and is used mainly in the synthesis of caged peptides.
- 4-Picolyl.375 It is removed in solution with Zn dust in AcOH to render the free thiol. It
was initially proposed for the Boc/Bn strategy but more recently has been succesfully
applied to the Fmoc/tBu synthesis of dihydrooxytocin, which was further oxidized to
oxytocin.
- Ninhydrin (Nin).376 It has been proposed as a protecting group for N-terminal Cys. It
protects both the amino and the thiol groups by forming a thiazolidine. Stable to HF and
TFA, it is removed with 1M Cys-OMe, 1M DIPEA in DMF for 30 min (solid phase),
10% TFA in H2O and Zn dust (solution) as well as by reducing thiols such as Cys in
combination with tris-carboxymethylphosphine (TCEP) (solution). It is coupled to

69

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 amines linked to the solid phase without using further protection at the amino group. Its
main applications are in ligation and its combination with His(Bom) in the Boc/Bn
strategy, which prevents tiazolidine formation after Bom removal (see His protection).

Name and Structure Removal conditions Stability to Compatibility with Ref.

the removal other sulfur
of protecting groups
Acetamidomethyl (Acm) 1) I2 (S-S) Boc, Fmoc, Meb/Mob, Trt,a 326,
t b
O 2) Tl(TFA) 3 (S-S) Alloc S Bu, Npys, Fm, 344,
N 3) Ag(TFMSO) (SH) PhAcmc 358,
H
4) Hg (II) (SH) 359,
360,
361
t
Phenylacetamidomethyl (PhAcm) 1) Hg (II) (SH) Boc, Fmoc, Meb/Mob, S Bu, 362
d b c
O 2) penicillin Z, Alloc Npys, Fm , Acm
N aminohydrolase (SH)
H
3) Tl (III)
trifluoroacetate (S-S)
4) I2 (S-S)
t
5-tert-Butylmercapto (S Bu) 1) thiols (benzenethiol, Boc, Fmoc, Meb/Mob, Trt, 363,
β-mercaptoethanol or Trt Acm 364,
S
dithiothreitol) 365,
2) Na 2SO3 in AcOH 366,
3) PBu 3 or PPh 3 in 367
CF3CH2OH
3-Nitro-2-Pyridinesulfenyl (Npys) 1) Thiol exchange Boc, Z,d Trt, Meb/Mob, Trt, 368,
N with a free Cys (S-S) Alloc Acm 369,
S
2) Reducing thiols 370
O2N
(SH)
3) PBu 3 (1 eq.), H2O
(SH)
2-Pyridinesulfenyl (S-Pyr) Thiocarboxylic acids Boc Meb/Mob, Trt, 371
N and DTT (SH) Acm
S

70

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Allyloxycarbonyl (Alloc) Pd(PPh 3)4, Bu3SnH Boc, Trt 163
O (SH)
O

N-allyloxycarbony-N-[2,3,5,6- 1) Pd (0), scavengers 372

tetrafluoro-4-(phenylthio)phenyl]] (i.e. PdCl2(PPh 3)2,
aminomethyl (Fsam) Bu 3SnH or Pd(PPh 3)4,
F PhSiH3)
F S
O 2) I2 (S-S)
O N F
F

o-Nitrobenzyl (oNB) Photolysis (λ= 300- Boc, Fmoc, 373,

NO2 400 nm) e 374
Trt, Z

4-Picolyl Zn dust in AcOH (SH) Boc, Fmoc 375

Ninhydrin (Nin) 1) 1M Cys-OMe, 1M Z,d Boc 376

O
H
O DIPEA in DMF (solid
N
OH phase)
S
O
2) 10% TFA in H2O
and Zn dust (solution)
Nin
3) Reducing thiols
such as Cys in
combination with
TCEP (solution)
a
The Trt should be removed first with TFA solutions
b
The Fm should be removed first
c
The PhAcm should be removed first enzymatically
d
Except catalytical hydrogenation removal.
e
HF/anisole removal.

The mercaptopropionic acid (des-amino Cys), which acts as an N-terminal capping in

some peptides of therapeutic interest, can be introduced as a dimer. The free thiol is
obtained after reduction with -mercaptoethanol or Bu3P.377

71

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 10. METHIONINE (Met)
10.1. General
The thioether funcionality of Met (Figure 22) can undergo two side reactions, oxidation
to sulfoxide and S-alkylation. The latter can lead to the formation of homoserine lactone
in C-terminal Met (Figure 2).378 These side reactions are favoured in acidic conditions.

H2N COOH

Met

Figure 22. Methionine (Met)

Figure 23. Homoserine lactone formation after Met alkylation during HF cleavage in
the Boc/Bn solid phase strategy.

In the Fmoc/tBu strategy, Met is used unprotected in most of the cases. To prevent
oxidation during amino acid side-chain deprotection and cleavage from the resin,
ethylmethylsulfide or thioanisole are used.379,380
In contrast, in the Boc/Bn strategy, free Met may not be the best option because of the
strong acidic conditions applied mainly in the cleavage from the resin but also in the
removal of the Boc group. Therefore, very frequently, Nα-Boc protected Met sulfoxide
is directly used and is reduced at the end of the synthesis.
10.2. Introduction of the protecting groups
The sulfoxide derivatives of Met are commercially available and can be prepared via
oxidation with H2O2. 381
10.3. Removal: sulfoxide reduction

72

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 In the case of SPS, the reduction of Met(O) can be performed either during the cleavage
or after it. In the latter case, the sulfoxide funcionality confers extra polarity to protected
peptides, which facilitates its purification; however, it must be taken into consideration
that sulfoxides are chiral and therefore different diastereomers will be observed.
Several reduction methods have been used:
1) Reduction during the low-high HF or TFMSA cleavage in the Boc/Bn strategy.
DMS and p-thiocresol or anisole should be used as scavengers to prevent S-alkylation.
2) N-methylmercaptoacetamide in 10% aquous acetic acid.382,383,384 It requires long
reaction times and disulfide bridges may be reduced.
3) TFA-NH4I-DMS.385,386,387 This method of reduction does not affect disulfide bridges
and if there are free Cys residues, a disulfide bridge is formed during the reduction of
the Met sulfoxide. tert-Butyl-type groups are removed during the reduction.
Dimerization of Trp (see Trp section) can occur in the case of long reaction times as a
result of overexposure to acidic conditions.
4) TiCl4 (3 eq.)- NaI (6 eq.) in MeOH-acetonitrile-DMF (5:5:4).388 Although a very fast
reduction method, it can also lead to reduction of disulfide bridges or oxidation of Trp,
the latter caused by the I2 generated in the sulfoxyde reduction.
5) TFA-TMSBr-EDT.389,390 In this method the reduction is carried out by addition of
TMSBr and EDT at the end of the cleavage step. It appears to be compatible with Trp-
containing peptides. The peptide is isolated by precipitation in diethylether.
6) Bu4NBr in TFA. It is an alternative to method 5 in which the reduction is also carried
out during the cleavage step.391
7) Sulfur trioxide (5 eq.), EDT (5 eq.) in pyridine-DMF (2:8).392 In this method
protection of hydroxyl groups is required to prevent sulfonylation.

Met des-tert-butylation.
If tert-butylation occurs during the global deprotection step, reversion to the free Met
residue is accomplished by heating a solution of the peptide in 4% AcOH(aq) at 60-65
ºC.393,394

73

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 11. HISTIDINE (HIS)
11.1. General
The imidazole ring of His (Figure 24) has two nucleophilic points, the π and τ-
nitrogens.395
H2N COOH H2N COOH

NH  N
N HN
 
His His

Figure 24. Histidine (His) tautomers.

Unprotected His is highly prone to racemization during the coupling (Figure 25) and
acylation during peptide synthesis followed by Nτ to α-amino migration (Figure
26).396,397
The basic and nucleophilic π-nitrogen is the one involved in racemization mechanisms
and can be masked in two ways: (i) direct protection (ii) τ-nitrogen protection with
bulky or electron-withdrawing protecting groups which reduce the basicity of the π-
nitrogen
O O
O
H2N H2N
OX H2N OX
H OX
H Racemization H
N N
N
NH HN
NH

Figure 25. Proposed racemization mechanism of His during the coupling step. Adapted
from 398)
O
O
H2N H
N R N
H H N
O H H
N
N
N
R HN
O
Acylated His Terminated peptide

Figure 26. Nτ to α-amino migration after acylation of His during peptide synthesis

74

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Although a large number of protecting groups have been tested for His side chain
protection, either in the π or the τ-nitrogen, the problem has still not been fully resolved,
the situation being more critical in the case of the Boc/Bn solid phase strategy.
The most used protecting groups are Trt for the Fmoc/tBu solid phase strategy and Dnp
(2,4-dinitrophenyl), Bom (benzyloxymethyl) and Tos (tosyl) for the Boc/Bn solid phase
strategy.
11.2. Introduction of the protecting groups 395
Protection of the imidazole ring of His requires α-amino, and carboxylic acid protection
with orthogonal protecting groups. In cases such as Trt, the α-amino group can be used
unprotected and at the end of the synthesis the Nα-trityl is removed, thereby leaving the
Nim-trityl unalterated. Generally, the reaction of the imidazole ring of His with the
corresponding active species (halides in general) gives the N τ-protected imidazole as a
π
majoritary and sometimes single product. Nevertheless, N protection is preferred
π
because, as previously mentioned, the N is the one directly involved in His
π
racemization. Thus, when possible, N -Protection is performed by masking the τ-
nitrogen with an orthogonal protecting group, which is removed at the end of the
synthesis of the derivative.
11.3. Removal
11.3.1. Protecting groups removed by acid
N τ-protection
- Nτ-Tosyl (Tos).399 It is removed with HF. It minimizes racemisation by reducing the
basicity of the Nπ by inductive effect and also because of steric hindrance. Although it
is still quite commonly used in the Boc/Bn solid phase strategy, it is unstable in the
presence of Nα groups and HOBt.400,396
- Nτ-Trityl (Trt): It is the usual protecting group for the Fmoc/tBu strategy.20,401 It is
removed with 95% TFA but is much less acid-labile than the Nα-trityl group and cannot
be selectively removed in the presence of tBu groups.402 Using Nτ protection, the free Nπ
can still catalyze racemization. However, the bulkiness of the Trt group minimizes this
side reaction in most cases but it is still critical in particular cases such as the formation
of ester bonds or when the amino component is sterically hindered.395
- Nτ-Methyltrityl (Mtt) and Nτ-monomethoxytrityl (Mmt). These are more acid-labile
derivatives of the Trt group, they are removed with 15% and 5% TFA in DCM, 1 h. 402
- Nτ-tert-butyloxycarbonyl (Boc). It is only useful for the synthesis of short sequences
via Fmoc chemistry because of its instability to prolonged piperidine treatments.401 Its

75

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 slightly greater acid stability compared with Trt makes it highly suitable for the
preparation of His-containing protected peptides using a ClTrtCl resin.
- Nτ-2,4-Dimethylpent-3-yloxycarbonyl (Doc).403 It is removed with liquid HF and is
used in the Boc/Bn solid phase strategy. In contrast to other proposed carbamate-type
His-protecting groups, it is very resistant to nucleophiles because of its bulkiness,
thereby preventing Nim to Nα transfer. It is not stable to 2% hydrazine in DMF but is
more stable to piperidine than the 2,4-dinitrophenyl (Dnp) group (see “other protecting
groups”) (its half life in 20% piperidine in DMF is 84 h).
N π-protection
- Nπ-Benzyloxymethyl (Bom). It is removed by HF, TFMSA or hydrogenolysis and is
completely stable to bases and nucleophyles. It has been extensively used for the
Boc/Bn solid phase strategy. As formaldehyde is released during Bom cleavage,
appropiate scavengers should be used to prevent formylation, methylation or the
formation of thiazolidines when an N-terminal Cys is present.327,328 In addition, a recent
report shows that α-amino Boc removal of Bom-protected His requires harsher
conditions than those commonly used.404
- Nπ-tert-Butoxymethyl (Bum).405,406It is removed by TFA and resistant to
hydrogenolysis. Formylation during its removal can be prevented using appropiate
scavengers in the same way as for Bom. It prevents racemization of His in the Fmoc/tBu
strategy; however, it is not widely used because of the difficult synthesis of Fmoc-
His(π-Bum)-OH.

Name and Structure Removal conditions Stability to the Ref

removal of
Nτ-tosyl (Tos) HF, scavengers Boc, Trt 396,
O
N
399,
S N
O
400

Nτ-Trityl (Trt) 95% TFA Fmoc, Alloc. 20,

395,
401,
N
N 402

76

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Nτ-Monomethoxytrityl (Mtt) 15 % TFA, DCM, 1 h Fmoc, Alloc 402

N
O N

Nτ-methyltrityl (Mmt) 5 % TFA, DCM, 1 h Fmoc, Alloc 402

N
N

Nτ-tert-butyloxycarbonyl (Boc) TFA, scavengers Fmoc, a Alloc 401

O
N
O N

Nτ-2,4-dimethylpent-3-yloxycarbonyl HF, scavengers Boc, Z, b Trt 403

(Doc)
O
N
O N

Nπ-benzyloxymethyl (Bom) 1) HF, scavengers Boc, Fmoc,c Trt 327,

O 2) TFMSA-TFA 328,
N
N 3) Catalytical 404
Hydrogenation
π
N -tert-butoxymethyl (Bum) TFA, scavengers Fmoc, Zb 395,
O 396
N
N

a
Only stable to a few Fmoc removal cycles (partially labile to piperidine).
b
Catalytical hydrogenation removal.
c
Except catalytical hydrogenation removal.

11.3.2. Protecting group removed by base

Nτ-9-Fluorenylmethoxycarbonyl (Fmoc).407 It is removed with piperidine-DMF (2:8)
and has been used for the synthesis of peptide-oligonucleotide conjugates.234
Nτ-2,6-Dimethoxybenzoyl (Dmbz).408 It is a relatively recently developed protecting
group for the Fmoc/tBu strategy and therefore it has not been widely used. Removed
with ammonia solutions and stable to the removal of tert-butyl type groups, it

77

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 minimizes His racemization during the coupling to the same extent as Trt and also
reduces acyl migration.

Name and Structure Removal conditions Stability to the Ref

removal of
9-Fluorenylmethoxycarbonyl (Fmoc) Piperidine-DMF (2:8) Boc 234,
O 407
N
O N

2,6-Dimethoxybenzoyl (Dmbz) 1) 32% NH3 (aq)- Boc, Fmoc, Trt 408

O O dioxane (1:1), 6 h.
N 2) 32% NH3 (aq)-EtOH
N
O (3:1), 2 h.

11.3.3. Other protecting groups

- Nτ-2,4-Dinitrophenyl (Dnp).409 It is removed by thiolysis,410,411 and is stable to HF. It
is also commonly used in the Boc/Bn solid phase strategy. However, it also has some
drawbacks: incomplete removal can occur in sequences rich in His and it is labile to
nucleophiles. These features makes it incompatible with Lys(Fmoc) because after Fmoc
removal the Dnp group can migrate to the free amino of the Lys.412 In addition, it must
be removed before eliminating the last α-Boc group. 413

Name and Structure Removal conditions Stability to the Ref.

removal of
τ
N -2,4-dinitrophenyl (Dnp) Thiolysis (e.g. Boc, Z,a Trt 409,410,
N thiophenol, DBU) 411,412,
O2N N
413
NO2

a
Except catalytical hydrogenation removal.

78

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 12. SERINE (Ser), THREONINE (Thr) AND HYDROXYPROLINE (Hyp)
12.1. General
Amino acids containing unprotected hydroxyl funcionalities such as Ser, Thr and Hyp
(Figure 27) can undergo side reactions such as dehydratation or O-acylation followed
by O-N migration after amino deprotection (Figure 28).
H
H2N COOH H2N COOH N COOH

OH OH
HO
Ser Thr Hyp

Figure 27. Serine (Ser), Threonine (Thr) and Hydroxyproline (Hyp).

PGHN COOH PGHN COOH H

H2N COOH R N COOH
(1) (2) (3)
O R
HO O O
O OH
R X O
R O
Figure 28. O-acylation followed by O-N migration after amino deprotection. (1) O-
acylation (2) Amino protecting group (PG) removal (3) O-N migration.

Although, the protected derivatives are the safest way to incorporate Ser, Thr or Hyp
into the peptide sequence, they can also be used with the free hydroxyl functionality.
Protection is more necessary in SPS because an excess of acylating agents is used, and
for Ser, whose primary alcohol is more prone to acylation than the secondary alcohols
of Thr and Hyp, which have been successfully used without protection in several
syntheses, including solid phase.414,415 Nevertheless, there are also some reports of the
succesful use of unprotected Ser in solution phase synthesis, but care must be taken
when choosing the activating agents.416,417
In peptide synthesis, hydroxyl funcionalities are protected as ethers, which are more
stable than the corresponding carbamates and esters. The most used protecting groups
for the Boc/Bn and Fmoc/tBu strategies are Bn (benzyl) and tBu (tert-butyl)
respectively.
12.2. Introduction of the protecting groups
Distinct protection methods are used depending on the kind of protecting group.

79

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 t
Bu protection is carried out via addition of isobutylene in acidic conditions.418 Bn
protection is performed using benzyl bromide in basic conditions in the case of Ser,
419,420
and reaction with benzyl alcohol in acidic medium in the case of Thr.421
Bn and tBu protections can also be achieved via formation of 2,2-difluoro-1,3,2-
oxazaborolidin-5-ones by reaction of the lithium salt of Ser or the sodium salt of Thr
with BF3. Treatment with isobutylene (tBu protection) or benzyl 2,2,2-
trichloroacetimidate (Bn protection) followed by a base treatment to destroy the 2,2-
difluoro-1,3,2-oxazaborolidin-5-one generates the desired protected derivatives.422 Trt
and alkylsilane protection are achieved using the respective chlorides in the presence of
a base. 423,424
12.3. Removal
12.3.1. Protecting groups removed by acid- Benzyl (Bn).425 It is removed with HF in
the presence of scavengers, and is the most used protecting group for Ser and Thr in the
Boc/Bn solid phase strategy. When many benzyl ethers are present, appropiate
scavengers should be used to avoid benzylation of free amino acid side chains.
- Cyclohexyl (cHx).426 It is an alternative to the benzyl group for the protection of Ser in
the Boc/Bn solid phase strategy. It is more stable to acids and completely stable to
catalytical hydrogenation. However, it has not been widely used.
- tert-Butyl (tBu).418 It is removed with TFA and used mainly in the Fmoc/tBu solid
phase strategy. tBu ethers are less acid-labile than the Boc group and some reports
indicate that they can be used even as temporary protecting groups in the Boc/Bn solid
phase strategy.427
- Trityl (Trt).423 It is removed with 1% TFA. It has been shown that the same peptide
with all the hydroxyl groups protected by Trt or tBu is obtained with better purity in the
case of the former.428
- tert-Butyldimethylsilyl (TBDMS).424 It is more acid-labile than the tBu group and can
be removed selectively in the presence this group using AcOH-THF-H2O (3:1:1) or
TBAF.
- Pseudoprolines: See amide backbone protection.

80

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Name and Structure Removal conditions Stability to the Ref.
removal of
Benzyl (Bn) 1) HF, scavengers Boc, Fmoc, Trt, 425
a
2) TFMSA-TFA Alloc, pNZ

Cyclohexyl (cHx) TFMSA-TFA Boc, Fmoc, Trt, 426

Alloc, pNZ

tert-Butyl ( tBu) 90% TFA-DCM Fmoc, Z,b 418,

Alloc, pNZ 427

Trityl (Trt) 1% TFA-DCM Fmoc, Alloc 423,

428

tert-Butyldimethylsilyl (TBDMS) 1) TFA Fmoc 424

2) AcOH-THF-H 2O
Si
(3:1:1), 18 h (Ser), 2
h (Thr)
3) 0.1M TBAF in
DMF, 2h (Ser), 18 h
(Thr)
Pseudoprolines 95% TFA and Fmoc, Alloc
O scavengers
C OH
R
HN
O

R= H (Ser) or Me (Thr)
a
Except catalytical hydrogenation removal.
b
Catalytical hydrogenation removal.

12.3.2. Other protecting groups

- tert-Butyldimethylsilyl (TBDMS). See protecting groups removed by acid.
- tert-Butyldiphenylsilyl (TBDPS). 429,430 It is typically removed by TBAF but also by 2
M NaOH(aq) EtOH (1:1). It is more acid-stable than TBDMS and stable to the removal
of N-Trt, O-Trt, O-TBDMS and Boc.

81

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - 4,5-Dimethoxy-2-nitrobenzyloxycarbonyl (Dmnb).431 It is a photolabile protecting
group analogous to the corresponding Dmnb ester. Ser(Dmnb) has been used recently to
control protein phosporyltion.432
- Propargyloxycarbonyl (Poc).433 It is removed with [(PhCH2NEt3)2MoS4] in AcCN, 1h,
rt. These removal conditions do not affect Boc, Z, methyl or benzyl esters, It has
recently been applied to the protection of Ser and Thr for peptide synthesis in solution.

Name and Structure Removal conditions Stability to the Ref.

removal of
tert-Butyldiphenylsilyl (TBDPS) 1) 1 M TBAF (2-3 Boc, Fmoc, Trt 429,
eq.), THF, 1-5 h 430
2) 2 M NaOH(aq) EtOH
Si (1:1), 7h

4,5-Dimethoxy-2-nitrobenzyloxycarbonyl Photolysis (visible Boc, Fmoc, Trt 431,

(Dmnb) blue light) 432
NO2

MeO
O
MeO O

Propargyloxycarbonyl (Poc) [(PhCH2NEt3) 2MoS4] Boc, Fmoc, Trt

O in AcCN, 1h.
O

82

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 13. TYROSINE (Tyr)
13.1. General
Use of unprotected Tyr (Figure 29) can lead to acylation of the phenol group because of
the nucleophilicity of the phenolate ion under basic conditions. In addition, the electron-
rich aromatic ring can undergo alkylation at the ortho position.
H2N COOH

OH
Tyr

Figure 29. Tyrosine (Tyr)

The acidity of phenol group makes alkyl-type protecting groups less stable than in the
case of Ser, Thr and Hyp. The most used Tyr-protecting groups for the Boc/Bn and
Fmoc/tBu solid phase strategies are Bn and tBu group, respectively.
13.2. Introduction of the protecting groups
To protect the phenolic function of Tyr,346 both the amino and carboxylic groups must
be protected either by forming a copper (II) chelate or using orthogonal protecting
groups.
t
Bu-protected Tyr is obtained using isobutylene in acidic medium,418 whereas with the
other Tyr-protected derivatives the corresponding alkyl halide is used as the protecting
agent.434,435
13.3. Removal
13.3.1. Protecting groups removed by acid
- Benzyl (Bn). It is removed with HF, but can lead to benzylation of the aromatic ring of
Tyr, and it is not stable enough to the repetitive treatments with 50% TFA in DCM to
remove the Boc group.436 Milder removal conditions for the Boc group allow the
synthesis of long peptides using benzyl protection.26 In solution synthesis it is usually
removed by catalytic hydrogenation.
- tert-Butyl (tBu). It is removed with TFA and is the most used protecting group for the
Fmoc/ tBu strategy solid phase strategy. It is more stable than the tert-butyl ethers of
Ser, Thr and Hyp. It is also stable to fluoride ions (TBAF) 277
- 2,6-Dichlorobenzyl (Dcb).434 It is removed with HF and because of its major acid
stabitlity it is an alternative to the Benzyl group for the Boc/Bn solid phase strategy.

83

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 - 2-Bromobenzyl (BrBn).437 It is another more acid-stable derivative of the benzyl
group; however, it has not found as wide application as Dcb.
- Benzyloxycarbonyl (Z).338 It is removed with HF, and protects the phenol funtionalilty
by forming a carbonate. Although still used, it is too acid-labile to withstand repetitive
treatments with 50% TFA to remove the Boc group.
- 2-Bromobenzyloxycarbonyl (BrZ).434,435 It protects the phenol funcionalilty by forming
a carbonate but unlike with other carbonates, only minor amounts of O to N transfer are
observed. In contrast to the above mentioned Z group, BrZ is very stable to acidic
conditions (removed with HF) and widely used for the SPS of long peptides using the
Boc/Bn solid phase strategy.434,435 It cannot be used in the Fmoc/tBu strategy because
being a carbonate it is very sensitive to bases and nucleophiles.438
- 3-Pentyl (Pen).439 It is a relatively new protecting group, stable to 50% TFA, bases
and catalytic hydrogenation and readily removed with HF.
- tert-Butyloxycarbonyl (Boc).440 This carbonate has been used occassionally for Tyr
side chain protection in the Boc/Bn solid phase strategy but only protects the phenol
during the coupling and is removed with TFA along to Nα -Boc.
- Trityl (Trt) and 2-Chlorotrityl (2-Cl-Trt). They are very acid-labile and have the
advantage of the low electrophilicity of trityl cations. Thus, they are a better alternative
to tBu for the synthesis of peptides containing residues prone to alkylation such as Trp
and Met.423,441,428 Removal is carried out with 2% TFA in DCM294
- tert-Butyldimethylsilyl (TBDMS).424 Unlike the tBu ethers, the TBDMS ether of Tyr is
more acid-labile than the corresponding tBu ethers; however, it can be removed
selectively with TBAF.
- 4-(3,6,9-trioxadecyl)oxybenzyl (TEGBz or TEGBn). See 5.3.1.

Name and Structure Removal conditions Stability to the Ref.

removal of
Benzyl (Bn) 1) HF and scavengers Boc, Fmoc, Trt, 26,
a
2) H2 cat. Alloc, pNZ 436

tert-Butyl ( tBu) 35% TFA-DCM Fmoc, Z,b 277

Alloc, Trt, pNZ

84

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 2,6-Dichlorobenzyl (Dcb) HF and scavengers Boc, Fmoc, Trt, 434
a
Cl Alloc, pNZ

Cl

2-Bromobenzyl (BrBn) HF and scavengers Boc, Fmoc, Trt, 437

Br Alloc, pNZa

Benzyloxycarbonyl (Z) HF and scavengers Boc, Trt 338

O

2-Bromobenzyloxycarbonyl (BrZ) HF and scavengers Boc, Trt 434,

Br O 435,
O 438

3-Pentyl (Pen) HF and scavengers Boc, Fmoc, Z, b 439

Trt

tert-Butyloxycarbonyl (Boc) TFA-DCM 440

O
O

Trityl (Trt) 2% TFA-DCM Fmoc, Alloc 294,

423,
428,
441

2-Chlorotrityl (2-Cl-Trt) 2% TFA in DCM Fmoc, Alloc 294,

423,
Cl 428,
441

tert-Butyldimethylsilyl (TBDMS) 1) 35% TFA Fmoc 424

2) 0.1M TBAF-DMF,
Si
15 min.
4-(3,6,9-trioxadecyl)oxybenzyl (TEGBz TFA-DCM Fmoc, Trt
or TEGBn)

a
Except catalytical hydrogenation removal.

85

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 b
Catalytical hydrogenation removal.

86

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 13.3.2. Other protecting groups
- Benzyl (Bn). See protecting groups removed by acid.
- tert-Butyldimethylsilyl (TBDMS). See protecting groups removed by acid.
- Allyl (Al).442,97,163 Removed with Pd (0), it is strictly orthogonal to the most common
protecting groups. It is used both in solution strategies and SPS.
- o-Nitrobenzyl (oNB).443 A photolabile protecting group, it has the same properties as
the oNB ester. It has been used for the synthesis of Tyr caged peptides. 444
- Propargyloxycarbonyl (Poc).433 (MATEIXA QUE POC, SER, P82) It is removed
with [(PhCH2NEt3)2MoS4] in AcCN, 1h, rt. These removal conditions do not affect Boc,
Z, methyl or benzyl esters, It has recently been applied to the protection of Tyr for
peptide synthesis in solution.
- Boc-N-methyl-N-[2-(methylamino)ethyl]carbamoyl (Boc-Nmec).445 It is a recently
developed protecting group (see also Boc-Nmec-Hmb in 6.3.2). After removal of the
Boc group the Nme moiety is removed with N-methylmorpholine (10 eq) in DMF/H2O
(3:7), 4 h.

Name and Structure Removal conditions Stability to the Ref.

removal of
Allyl (Al) Pd(Ph3) 4, scavengers Boc, Fmoc, Za 97,
163,
442
o-Nitrobenzyl (oNB): Photolysis (λ=350 nm),12 h Boc, Fmoc, Trt 443,
NO2 444

Propargyloxycarbonyl (Poc) [(PhCH2NEt3) 2MoS4] in Boc, Trt

O AcCN, 1h.
O

Boc-N-methyl-N-[2- i) 25-50% TFA Fmoc, Trt

(methylamino)ethyl]carbamoyl ii) N-methylmorpholine (10
(Boc-Nmec) eq) in DMF/H2O (3:7), 4 h

a
Except catalytical hydrogenation removal.

87

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 14. TRYPTOPHAN (Trp)
14.1. General
The indole group of Trp (Figure 30) can undergo oxidation and alkylation if it is not
protected.446
H2N COOH

N
H
Trp

Figure 30. Tryptophan (Trp)

Alkylation during acid treatments can be done by carbocations from released protecting
groups or from the resin, the latter leading to irreversible bonding of the peptide to the
support (Figure 31).447

N
N H
H

OH
OH
OH

Figure 31. Alkylation of Trp by the Wang linker side products

Dimerization of Trp caused by alkylation by another protonated Trp has also been
observed (Figure 32).448,449

H+ (1) (2)

N N N
H H H

(3)
N N N
N H H
H H

Figrue 32. Mechanism of Trp dimerization. (1) Protonation, (2) Nucleophilic attack (3)
Elimination.

In the Boc/Bn strategy, the higher risk of oxidation and alkylation in acidic media
makes the protection of Trp necessary. In addition, care must be taken when chosing the

88

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 scavengers in the final cleavage. For instance, thioanisole should be avoided because
thioanisole cation adducts can alkylate Trp, and TIS, which is mainly used in the
Fmoc/tBu strategy, should be used instead of TES to prevent reduction of the indole ring
of Trp to indoline.450 The most used protecting group for the Boc/Bn strategy is For
(formyl).
In contrast, in the Fmoc/tBu strategy unprotected Trp is often used. However, in many
cases protection is necessary. A critical example is when the peptidic sequences contain
Arg protected by either Mtr, Pmc or Pbf groups, which after removal can react the
indole ring in the 2 position.451,452 The most used protecting group for the Fmoc/tBu
strategy is Boc.
14.2. Introduction of the protecting groups
Carbamate protection of the tert-butyl, benzyl, or phenacyl esters of Nα Boc or Z-Trp is
easily carried out using di-tert-butyl-dicarbonate or an appropiate chloroformate in the
presence of a tertiaty base. After that, the carboxylic acid and/or amino-protecting
groups are removed and Nα derivatization yields the Boc and Fmoc derivatives of the
protected Trp.453,454,455,456,457 The formyl group is introduced using an excess of formic
acid.458
14.3. Removal
14.3.1. Protecting groups removed by acid
- Formyl (For).459 Removal with HF may be slow and the use of thiols (i.e. EDT) as
460
scavengers makes it faster In the case of base cleavage, care must be taken with the
reaction conditions in order to avoid free amine formylation.461,462
- tert-Butyloxycarbonyl (Boc).455,456 It is removed with high concentrations of TFA and
is the protecting group of choice for the Fmoc/tBu solid phase strategy. It is more stable
than Boc α-amino protection, which can be removed in the presence of protected Trp if
care is taken with the reaction conditions, but not as a routine procedure. Boc protection
avoids Trp alkylation during the removal of Mtr, Pmc and Pbf from the Arg side-
chain.463,464 The N-carboxylated compound can be detected after tert-butyl removal but
later becomes unstable thereby giving the free indole. The stability of this carbamic acid
makes Boc-protected Trp less prone to electrophilic additions during the final
cleavage.455,456
- Cyclohexyloxycarbonyl (Hoc).454 It is an alternative to the formyl group for the
Boc/Bn strategy. Its high resistance to bases makes it useful for the synthesis of
protected peptides on base-labile resins.465 Although it is generally removed with HF in

89

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 the presence of p-cresol, Trp alkylation by p-cresol can occur during the removal. A
proposed solution for this problem is the use of Fmoc-Leu or butanedithiol as
scavengers.466
- Mesitylene-2-sulfonyl (Mts).467Another alternative for the Boc/Bn strategy, Mts is
removed by 1M CF3SO3H/TFA or MeSO3H but not by HF. Although it has not been
widely applied, there are reports of its use.468

Name and Structure Removal conditions Stability to the Ref.

removal of
Formyl (For) 1) Strong acid (HF) Boc 459,
and scavengers (i.e. 460,
O EDT) (slow) 461,
H 2) piperidine-H2O or 462
DMF
3) 1 M NH2OH, pH 9,
2h

tert-Butyloxycarbonyl (Boc) 95% TFA and Fmoc, Alloc 455,

scavengers a 456,
O
O 463,
464
Cyclohexyloxycarbonyl (Hoc) HF, scavengers Boc, Fmoc, 454,
O Alloc, 465,
O 466

Mesitylene-2-sulfonyl (Mts) 1) CF3SO3H/TFA Boc, Fmoc, 467,

O 2) MeSO3H Alloc, 468

S
O

a
The carbamic acid resulting from tert-butyl removal is quite stable. Complete decarboxylation takes
place by treatment with 0.1M AcOH (aq) or more slowly during lyophilization in H2O.

14.3.2. Protecting groups removed by base

- Formyl (For): see protecting groups removed by acid.

90

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 14.3.3. Other protecting groups
- Allyloxycarbonyl (Alloc).457 Removed with Pd (0), its orthogonality to Boc and Fmoc
(when removed with DBU but not when removed with piperidine) makes it potentially
useful for both the Boc/Bn and the Fmoc/tBu solid phase strategies.

Name and Structure Removal conditions Stability to the Ref.

removal of
Allyloxycarbonyl (Alloc) Pd(PPh3) 4, Boc 457

O
methylanylin in

O
DMSO-THF-0.5 M
HCl (1:1:0.5), 8h

15. ABBREVIATIONS

AB linker 3-(4-Hydroxymethylphenoxy)propionic acid linker

Acm Acetamidomethyl Con formato: Español (alfab.
internacional)
AcOH Acetic acid
1-Ada 1-Adamantyl
Al Allyl
Alloc Allyloxycarbonyl
API Active Pharmaceutical Ingredients
Arg Arginine
Asn Asparagine
Asp Aspartic acid
Azoc Azidomethyloxycarbonyl
Bn Benzyl
BAL Backbone Amide Linker
Boc tert-Butyloxycarbonyl
Bom Benzyloxymethyl
Bpoc 2-(4-Biphenyl)isopropoxycarbonyl
BrBn 2-Bromobenzyl
BrPhF 9-(4-Bromophenyl)-9-fluorenyl
BrZ 2-Bromobenzyloxycarbonyl
Bsmoc 1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl
Bum tert-Butoxymethyl
Cam Carboxamidomethyl
cHx Cyclohexyl
Cl-Z 2-Chlorobenzyloxycarbonyl
Cpd Cyclopropyldimethylcarbinyl
Cys Cysteine
Dab Diaminobutyric acid
Dap Diaminopropionic acid
DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene
Dcb 2,6-Dichlorobenzyl
DCHA Dicyclohexylammonium
DCM Dichloromethane

91

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Dde (1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl)
Ddz α,α-Dimethyl-3,5-dimethoxybenzyloxycarbonyl
dio-Fmoc 2,7-Diisooctyl-Fmoc
DIPEA N,N-Diisopropylethylamine
DKP Diketopiperazine
Dma 1,1-Dimethylallyl
Dmab 4-(N-[1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-
amino)benzyl
Dmb 2,4-Dimethoxybenzyl
Dmcp Dimethylcyclopropylmethyl
DMF N,N-Dimethylformamide
Dmnb 4,5-Dimethoxy-2-nitrobenzyl/oxycarbonyl
DMSO Dimethylsulfoxide
dNBS 2,4-Dinitrobenzenesulfonyl
Dnp 2,4-Dinitrophenyl
Dnpe 2-(2,4-dinitrophenyl)ethyl
Doc 2,4-Dimethylpent-3-yloxycarbonyl
Dts Dithiasuccinoyl
DTT Dithiothreitol
EDOTn 3,4-Ethylenedioxy-2-thenyl
Esc Ethanesulfonylethoxycarbonyl
Fm 9-Fluorenylmethyl
Fmoc 9-Fluorenylmethoxycarbonyl
Fmoc(2F) 2-Fluoro-Fmoc
Fmoc* 2,7-di-tert-Butyl-Fmoc
For Formyl
Fsam N-allyloxycarbony-N-[2,3,5,6-tetrafluoro-4-(phenylthio)phenyl]]
aminomethyl
Gln Glutamine
Glu Glutamic acid
HFA Hexafluoroacetone
His Histidine
Hmb 2-Hydroxy-4-methoxybenzyl
Hoc Cyclohexyloxycarbonyl
HOBt 1-Hydroxybenzotriazole
HOSu N-Hydroxysuccinimido
Hyp Hydroxyproline
ivDde 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-mehtylbutyl
Lys Lysine
Mbh 4,4’-Dimethoxybenzhydryl
MBHA 4-Methylbenzhydrylamine
Meb p-Methylbenzyl
Men β-Menthyl
MeSub 2-Methoxy-5-dibenzosuberyl
Met Methionine
MIM 1-Methyl-3-indolylmethyl
mio-Fmoc 2-Monoisooctyl-Fmoc
MIS 1,2-Dimethylindole-3-sulfonyl
Mmt Monomethoxytrityl
MNPPOC 2-(3,4-Methylenedioxy-6-nitrophenyl)propyloxycarbonyl

92

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Mob p-Methoxybenzyl
Mpe β-3-Methylpent-3-yl
Msc 2-(Methylsulfonyl)ethoxycarbonyl
Mtr 4-Methoxy-2,3,6-trimethylphenylsulfonyl
Mts Mesitylene-2-sulfonyl
Mtt 4-Methyltrityl
NCA N-carboxy anhydrides
Nin Ninhydrin
NMM N-methyl mercaptoacetamide
NMP 1-Methylpyrrolidin-2-one
NPPOC 2-(2-Nitrophenyl)propyloxycarbonyl
Nps 2-Nitrophenylsulfanyl
Npys 3-Nitro-2-pyridinesulfenyl
Nsc 2-(4-Nitrophenylsulfonyl)ethoxycarbonyl
α-Nsmoc 1,1-Dioxonaphtho[1,2-b]thiophene-2-methyloxycarbonyl
NVOC 6-Nitroveratryloxycarbonyl
oNBS o-Nitrobenzenesulfonyl
oNZ o-Nitrobenzyloxycarbonyl
Orn Ornithine
Pac Phenacyl
Pbf Pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl
Pen 3-Pentyl
PhAcm Phenylacetamidomethyl
Phdec Phenyldithioethyloxycarbonyl
2-PhiPr 2-Phenylisopropyl
pHP p-Hydroxyphenacyl
Pmbf 2,2,4,6,7-Pentamethyl-5-dihydrobenzofuranylmethyl
Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl
Pms 2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate
PNA Peptide Nucleic Acid
pNB p-Nitrobenzyl
pNBS p-Nitrobenzenesulfonyl
pNZ p-Nitrobenzyloxycarbonyl
Poc Propargyloxycarbonyl
ΨPro Pseudoprolines
Pydec 2-Pyridyldithioethyloxycarbonyl
Ser Serine
SPPS Solid Phase Peptide Synthesis
Sps 2-(4-Sulfophenylsulfonyl)ethoxycarbonyl
SPS Solid Phase Synthesis
S-Pyr 2-Pyridinesulfenyl
StBu tert-Butylmercapto
Sub 5-Dibenzosuberyl
Suben ω-5-Dibenzosuberenyl
TAEA tris(2-Aminoethyl)amine
TBAF Tetrabuthylammonium fluoride
TBDMS tert-Butyldimethylsilyl
TBDPS tert-Butyldiphenylsilyl
t
Bu tert-Butyl
TCA Trichloroacetic acid

93

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Tce 2,2,2-Trichloroethyl
TCEP tris-Carboxymethylphosphine
TCP Tetrachlorophthaloyl
TEA Triethylamine
TEAF Tetraethylammonium fluoride
TFA Trifluoroacetic acid
tfa Trifluoroacetyl
TFE 2,2,2-Trifluoroethanol
TFMSA Trifluoromethanesulfonic acid
Thr Threonine
Tmob 2,4,6-Trimethoxybenzyl
TMS Trimethylsilyl
TMSE Trimethylsilylethyl
Tmsi 2-(Trimethylsilyl)isopropyl
Tos Tosyl
Troc 2,2,2-Trichloroethyloxycarbonyl
Trp Tryptophan
Trt Trityl
Tyr Tyrosine
Xan 9-Xanthenyl
Z Benzyloxycarbonyl

16. REFERENCES

(1) Green, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis. Ed. John
Wiley and Sons: New York, 1999.
(2) Kocienski, P. J. Protecting Groups. Ed. Georg Thieme Verlag: Stuttgart-New York,
2004.
(3) Fischer, E.; Bergmann, M.. Ber. Deut. Chem. Ges. 1918, 51, 1760.
(4) Bergmann, M.; Zervas, L. Ber. Deut. Chem. Ges. 1932, 65B, 1192.
(5) Barany G.; Merrifield R. B. J. Am. Chem. Soc. 1977, 99, 7363.
(6) Barany, G.; Albericio, F. J. Am. Chem. Soc. 1985, 107, 4936.
(7) Fmoc protection: Chang, C.-D.; Waki, M.; Ahmad, M.; Meienhofer, J.; Lundell, E.
O.; Haug, J. D. Int. J. Pept. Prot. Res. 1980, 15, 59.
(8) Z protection: Sennyey, G.; Barcelo, G.; Senet, J. P. Tetrahedron Lett. 1986, 27,
5375.
(9) Boc protection: Keller, O.; Keller, W. E.; Van Look, G.; Wersin, G. Org. Synth.
1985, 63, 160.
(10) Trt protection via intermediate trityl esters: Mutter, M.; Hersperger, R. Synthesis
1989, 3, 198.

94

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (11) Tessier, M.; Albericio, F. ; Pedroso, E.; Grandas, A.; Eritja, R.; Giralt, E. ; Granier,
C.; van Rietschoten, J. Int. J. Pept. Protein Res. 1983, 22, 125.

(12) Sigler, G. F.; Fuller, W. D.; Chaturvedi, N. C.; Goodman, M.; Verlander, M.
Biopolymers 1983, 22, 2157.
(13) Lapatsanis, L., Milias, G., Froussios, K.; Kolovos, M. Synthesis 1983, 671.
(14) Ten Kortenaar, P. B. W.; Van Dijk, B. G.; Peeters, J. M.; Raaben, B. J.; Adams, P.
J.; Hans, M.Tesser, G. I. Int. J. Pept. Protein Res. 1986, 27, 398.
(15) Paquet, A. Can. J. Chem. 1982, 60, 976.
(16) Milton, R. C.; Becker, E.; Milton, S. C.; Baxter, J. E. J.; Elsworth, J. F. Int. J.
Pept. Prot. Res. 1987, 30, 431.
(17) Fmoc-N3: Cruz, L. J.; Beteta, N. G.; Ewenson, A.; Albericio, F. Org. Proc. Res.
Dev. 2004, 8, 920.
(18) Bolin, D. R.; Sytwu, I. I.; Humiec, F.; Meienhofer, J. Int. J. Pept. Prot. Res. 1989,
33, 353.
(19) Isidro-Llobet, A.; Just-Baringo, X.; Ewenson, A.; Álvarez, M.; Albericio, F.
Biopolymers 2007, 88, 733.

(20) Barlos, K.; Papaioannou, D.; Theodoropoulos, D. J. Org. Chem. 1982, 47, 1324.
(21) Hlebowicz, E.; Andersen, A.J.; Andersson, L.; Moss, B.A. J. Pept. Res. 2005, 65,
90-

(22) Carpino, L. A. J. Am. Chem. Soc. 1957, 79, 4427.

(23) Anderson, G.W.; Alberston, N.F. J. Am. Chem. Soc. 1957, 79, 6180.
(24)Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
(25) Merrifield, R. B. Adv. Enzymol. 1969, 32, 221.
(26) Kaiser, E.; Picart, F.; Kubiak, T.; Tam, J. P.; Merrifield, R. B. J. Org. Chem. 1993,
58, 5167.
(27) Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis. 2nd ed., Pierce
Chemical Company: Rockford, IL, 1984.
(28) Barlos, K.; Mamos, P.; Papaioannou, D.; Patrianakou, S.; Sanida, C.; Schaefer, W.
Liebigs Ann. Chem. 1987, 12, 1025.
(29) Bodanszky M.; Bednarek M. A.; Bodanszky A. Int. J. Pept. Prot. Res. 1982, 20,
387.
(30) Alsina, J.; Giralt, E.; Albericio, F. Tetrahedron Lett. 1996, 37, 4195.

95

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (31) de la Torre, B. G.; Marcos, M. A.; Eritja, R.; Albericio, F. Solid-phase peptide
synthesis using N-α-tritylamino acids. Lett. Pepide Sci. 2001, 8, 331.

(32)Floersheimer, A.; Riniker, B. Peptides 1990: Proc. 21st European Peptide

Symposium, Giralt, E.; Andreu, D. eds., ESCOM Sci. Publ.: Leiden, 1990, 131.
(33) 2,5-Diketopiperazines can be formed after the removal of the α-amino group from
the second C-terminal amino acid due to nucleophilic attack of the free amine to the
carboxylate group of the C-terminal amino acid.
(34) Gairí, M.; Lloyd-Williams, P.; Albericio, F.; Giralt, E. Tetrahedron Lett. 1990, 31,
7363.
(35) Barlos, K.; Papaioannou, D.; Patrianakou, S.; Tsegenidis, T. Liebigs Ann. Chem.
1986, 11, 1950.
(36) Birr, C.; Lochinger, W.; Stahnke, G.; Lang, P.. Liebigs Ann. Chem. 1972, 763,
162.
(37) Birr, C. In Innovation and Perspectives in Solid Phase Synthesis; Epton, R., Ed.;
SPCC (UK) Ltd.: Birmingham, UK, 1990, 155-181.
(38) Jensen, K.J.; Alsina, J.; Songster, M.F.; Vágner, J.; Albericio, F.; Barany, G. J. Am.
Chem. Soc. 1998, 120, 5441.
(39) Wang, S. S.; Yang, C. C.; Kulesha, I. D.; Sonenberg, M.; Merrifield, R. B. Int. J.
Pept. Prot. Res. 1974, 6, 103.
(40) Mojsov S; Merrifield R.B. Biochemistry 1981, 20, 2950.
(41) Albericio, F. Biopolymers 2000, 55, 123.
(42) Carey, R. I.; Bordas, L. W.; Slaughter, R. A.; Meadows, B. C.; Wadsworth, J. L.;
Huang, H.; Smith, J. J.; Furusjo, E. J. Pept. Res. 1997, 49, 570.
(43) Attard, T. J.; Reynolds, E. C.; Perich, J. W. Org. Biomol. Chem. 2007, 5, 664-
670.
(44) Zaramella, S.; Yeheskiely, E.; Stroemberg, R. J. Am. Chem. Soc. 2004, 126,
14029.
(45) Zervas, L.; Borovas, D.; Gazis, E. J. Am. Chem. Soc. 1963, 85, 3660.
(46) Najjar, V. A.; Merrifield, R. B. Biochemistry 1966, 5, 3765.
(47) Tun-Kyi, A. Helv. Chim. Acta. 1978, 61, 1086.
(48) Meienhofer, J. Nature 1965, 205, 73.
(49) Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92, 5748.
(50) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404.

96

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (51) Rabanal, F.; Haro, I.; Reig, F.; García-Antón, J. M. An. Quim. 1990, 86, 84.
(52) Butwell, F. G. W.; Haws, E. J.; Epton, R. Makromol. Chem., Macromol. Symp.
1988, 19, 69.
(53) Carpino, L. A.; Mansour, E. M. E.; Cheng, C. H.; Williams, J. R.; MacDonald, R.;
Knapczyk, J.; Carman, M.; Lopusinski, A. J. Org. Chem. 1983, 48, 661.
(54) Carpino, L. A.; Mansour, E. M. E.; Knapczyk, J. J. Org. Chem. 1983, 48, 666.
(55) Atherton, E.; Fox, H; Harkiss, D.; Logan, C. J.; Sheppard, R. C.; Williams, B. J. J.
Chem. Soc., Chem. Commun. 1978, 13, 537.
(56) Chang, C.-D.; Meienhofer, J. Int. J. Pept. Prot. Res. 1978, 11, 246-249.
(57) Wade, J. D.; Bedford, J.; Sheppard, R. C.; Tregear, G. W. Pept. Res. 1991, 4, 194.
(58) Meldal, M.; Bielfeldt, T.; Peters, S.; Jensen, K. J.; Paulsen, H.; Bock, K. Int. J.
Pept. Prot. Res. 1994, 43, 529.
(59) Liebe, B.; Kunz, H. Angew. Chem. Int. Ed. in Eng. 1997, 36, 618.
(60) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669.
(61) Martinez, J.; Bodanszky, M. Int. J. Petide Protein Res., 1978, 12, 277.
(62) Doelling, R.; Beyermann, M.; Haenel, J.; Kernchen, F.; Krause, E.; Franke, P.;
Brudel, M.; Bienert, M. J. Chem. Soc., Chem. Commun. 1994, 853.
(63) Han, Y.-K.; Johnston, D. A.; Khatri, H. N. PCT Int. Appl. WO 2006069727 A2 ,
2006, Chem. Abstr. 2006, 145, 103960.
(64) Samukov, V. V.; Sabirov, A.; Pozdnyakov, P. I. Tetrahedron Lett. 1994, 35, 7821.
(65) Sabirov, A. N.; Kim, Y.-D.; Kim, H.-J.; Samukov, V. V. Protein Peptide Lett.
1997, 4, 307.
(66) Ramage, R.; Jiang, L.; Kim, Y.-D.; Shaw, K.; Park, J.-L.; Kim, H.-J. J. Pept. Sci.
1999, 5, 195.
(67) Carreño, C.; Mendez, M. E.; Kim, Y.-D.; Kim, H.-J.; Kates, S. A.; Andreu, D.;
Albericio, F. J. Pept. Res. 2000, 56, 63.
(68) Maier, T. C.; Podlech, J. Adv. Synth. Cat. 2004, 346, 727-.
(69) Lauer, J. L.; Fields, C. G.; Fields, G. B. Lett. Pept. Sci. 1995, 1, 197.
(70) Carpino, L. A.; Philbin, M.; Ismail, M.; Truran, G. A.; Mansour, E. M. E.; Iguchi,
S.; Ionescu, D.; El-Faham, A.; Riemer, C.; Warrass, R.; Weiss, M. S. J. Am. Chem. Soc.
1997, 119, 9915.
(71) Carpino, L. A.; Philbin, M. J. Org. Chem. 1999, 64, 4315.
(72) Carpino, L. A.; Mansour, E. M. E. J. Org. Chem. 1999, 64, 8399.

97

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (73) Carpino, L. A.; Ismail, M.; Truran, G. A.; Mansour, E. M. E.; Iguchi, S.; Ionescu,
D.; El-Faham, A.; Riemer, C.; Warrass, R. J. Org. Chem. 1999, 64, 4324.
(74) Carpino, L. A.; Ghassemi, S.; Ionescu, D.; Ismail, M.; Sadat-AAlaee, D.; Truran,
G. A.; Mansour, E. M. E.; Siwruk, G. A.; Eynon, J. S.; Morgan, B. Org. Process Res.
Dev. 2003, 7, 28.
(75) Carpino, L. A.; Abdel-Maksoud, A. A.; Ionescu, D.; Mansour, E. M. E.; Zewail,
M. A. J. Org. Chem. 2007, 72, 1729.
(76)Hillman, Jeffrey D.; Orugunty, Ravi S.; Smith, James L. Preparation of
orthogonally-protected lanthionines for the synthesis of Nisin A analog and other
intramolecularly bridged polypeptides. U.S. Pat. Appl. Publ. (2007), 23pp.
CODEN: USXXCO US 2007037963 A1 20070215 CAN 146:252110 AN
2007:175279 CAPLUS
(77) Stigers, K. D.; Koutroulis, M. R.; Chung, D. M.; Nowick, J. S. J. Org. Chem. 2000,
65, 3858.
(78) Chinchilla, R.; Dodsworth, D. J.; Najera, C.; Soriano, J. M. Bioorg. Med. Chem.
Lett. 2002, 12, 1817.
(79) Woods, R. J.; Brower, J. O.; Castellanos, E.; Hashemzadeh, M.; Khakshoor, O.;
Russu, W. A.; Nowick, J. S. J. Am. Chem. Soc. 2007, 129, 2548.
(80) Hasegawa, K.; Sha, Y. L.; Bang, J. K.; Kawakami, T.; Akaji, K.; Aimoto, S. Lett.
Pept. Sci. 2002, 8, 277.
(81) Wessig, P.; Czapla, S.; Moellnitz, K.; Schwarz, J. Synlett 2006, 14, 2235.
(82) Cros, E.; Planas, M.; Barany, G.; Bardaji, E.. Eur. J. Org. Chem. 2004, 17, 3633.
(83) Hojo, K.; Maeda, M.; Kawasaki, K. J. Pept. Sci. 2001, 7, 615.
(84) Hojo, K.; Maeda, M.; Kawasaki, K. Tetrahedron 2004, 60, 1875.
(85) Hojo, K.; Maeda, M.; Smith, T. J.; Kita, E.; Yamaguchi, F.; Yamamoto, S.;
Kawasaki, K. Chem. Pharm. Bull. 2004, 52, 422.
(86) Hojo, K.; Maeda, M.; Kawasaki, K. Tetrahedron Lett. 2004, 45, 9293.
(87) Ben-Ishai, D.; Berger, A. J. Org. Chem. 1952, 17, 1564.
(88) Mitchell, A. R.; Merrifield, R. B. J. Org. Chem. 1976, 41, 2015.
(89) Kiso, Y.; Ukawa, K.; Akita, T. J. Chem. Soc. Chem. Commun. 1980, 3, 101.
(90) Sakakibara, S.; Shimonishi, Y.; Kishida, Y.; Okada, M.; Sugihara, H. Bull. Chem.
Soc. Jap. 1967, 40, 2164.
(91) Felix, A. M. J. Org. Chem. 1974, 39, 1427.
(92) Podlech, J.; Gurrath, M.; Müller, G.; Lohof, E. Protection of the α-Amino Group.
In “Synthesis of Peptides and Peptidomimetics (Houben-Weyl E22a: Methods of

98

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 Organic Chemistry)”, Goodman, M.; Felix, A.M.; Moroder, L.; Toniolo, C. eds., Georg
Thieme Verlag: Stuttgart and New York, 2002, pp. 41-165.
(93) Stevens, C. M.; Watanabe, R. J. Am. Chem. Soc. 1950, 72, 725.
(94) Tsuji, J. Tetrahedron 1986, 42, 4361, and references cited therein.
(95) Trost, B. M.; van Vranken, D. L. Chem. Rev. 1996, 96, 395, and references cited
therein.
(96) Guibé, F. Tetrahedron 1997, 53, 13509.
(97) Guibé, F. Tetrahedron 1998, 54, 2967.
98 Me2NH·BH3 proved to be more efficient than PhSiH3 for Alloc deprotection of
secondary amines: Fernandez-Forner, D.; Casals, G.; Navarro, E.; Ryder, H.; Albericio,
F. Tetrahedron Lett. 2001, 42, 4471.
(99) Dessolin, M,; Guillerez, M.-G.; Thieriet, N.; Guibé, F.; Loffet, A. Tetrahedron
Lett. 1995, 36, 5741.
(100) Gómez-Martínez, P.; Dessolin, M.; Guibé, F. Albericio, F. J. Chem. Soc. Perk. 1.
1999, 22871. .
(101) Thieriet, N.; Gomez-Martinez, P.; Guibé, F. Tetrahedron Lett. 1999, 40, 2505.
(102) Thieriet, N.; Alsina, J..; Giralt, E.; Guibé, F.; Albericio, F. Tetrahedron Lett.
1997, 38, 7275.
(103) Gracia, C.; Isidro-Llobet, A.; Cruz, L. J.; Acosta, G. A.; Álvarez, M.; Cuevas, C.;
Giralt, E.; Albericio, F. J. Org. Chem. 2006, 71, 7196.
(104) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995, 36, 6373.
(105) Vedejs, E.; Lin, S.; Klapars, A.; Wang, J. J. Am. Chem. Soc. 1996, 118, 9796.
(106) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119, 2301.
(107) Biron, E.; Kessler, H. J. Org. Chem. 2005, 70, 5183.
(108) Fukuyama, T.; Cheung, M.; Jow, C.-K.; Hidai, Y.; Kan, T. Tetrahedron Lett.
1997, 38, 5831.
(109) Vedejs, E.; Kongkittingam, C. J. Org. Chem. 2000, 65, 2309.
(110) Marsault, E.; Benakli, K.; Beaubien, S.; Saint-Louis, C.; Deziel, R.; Fraser, G.
Bioorg. Med. Chem. Lett. 2007, 17, 4187.
(111) Marsault, E.; Hoveyda, H. R.; Peterson, M. L.; Saint-Louis, C.; Landry, A.;
Vezina, M.; Ouellet, L.; Wang, Z.; Ramaseshan, M.; Beaubien, S.; Benakli, K.;
Beauchemin, S.; Deziel, R.; Peeters, T.; Fraser, G. L. J. Med. Chem. 2006, 49, 7190.

99

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (112) Woodward, R.B.; Heusler, K.; Gosteli, J.; Naegeli, P.; Oppolzer, W.; Ramage,
R.; Ranganathan, S.; Vorbrüggen, H. J. Am Chem. Soc. 1966, 88, 852.
(113) Carson, J. F. Synthesis 1981, 268.
(114) Barany, G.; Merrifield, R. B. J. Am. Chem. Soc. 1980, 102, 3084.
(115) Albericio, F.; Barany, G. Int. J. Pept. Prot. Res. 1987, 30, 177.
(116) Planas, M.; Bardají, E.; Jensen, K. J.; Barany, G. J. Org. Chem. 1999, 64, 7281.
(117) Jensen, K. J.; Hansen, P. R.; Venugopal, D.; Barany, G. J. Am. Chem. Soc. 1996,
118, 3148.
(118) Carpenter, F. H.; Gish, D. T. J. Am. Chem. Soc. 1952, 74, 3818.
(119) Isidro-Llobet, A.; Guasch-Camell, J.; Álvarez, M.; Albericio, F. Eur. J. Org.
Chem. 2005, 3031.
(120) Liao, W.; Piskorz, C. F.; Locke, R. D.; Matta, K. L. Bioorg. Med. Chem. Lett.
2000, 10, 793.
(121) Tulla-Puche, J.; Bayó-Puxan, N.; Moreno, J. A.; Francesch, A. M.; Cuevas, C.;
Álvarez, M.; Albericio, F. J. Am. Chem. Soc. 2007, 129; 5322.
(122) Meldal, M.; Juliano, M. A.; Jansson, A. M. Tetrahedron Lett. 1997, 38, 2531.
(123) Tornoe, C. W.; Davis, P.; Porreca, F.; Meldal, M. J. Pept. Sci. 2000, 6, 594.
(124) Lundquist, J. T., IV; Pelletier, J. C. Org. Lett. 2001, 3, 781.
(125) Lundquist, J. T.; Pelletier, J. C. Org. Lett. 2002, 4, 3219.
(126) Bhat, R. G.; Sinha, S.; Chandrasekaran, S. Chem. Commun. 2002, 8, 812.
(127) Sinha, S.; Ilankumaran, P.; Chandrasekaran, S. Tetrahedron Lett. 1999, 40, 771.
(128) Patchornik, A.; Amit, B.; Woodward, R. B. J. Am. Chem. Soc. 1970, 92, 6333.
(129) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D.
Science 1991, 251, 767.
(130) Bhushan, K. R.; DeLisi, C.; Laursen, R. A. Tetrahedron Lett. 2003, 44, 8585.
(131) Bhushan, K. R. Abstracts of Papers, 232nd ACS National Meeting, 2006.
(132) Surprenant, S.; Lubell, W. D. J. Org. Chem. 2006, 71, 848.
(133) Kaul, R.; Brouillette, Y.; Sajjadi, Z.; Hansford, K. A.; Lubell, W. D. J. Org.
Chem. 2004, 69, 6131.
(134) Pothukanuri, S.; Winssinger, N. Org. Lett. 2007, 9, 2223.
(135) Spengler, J.; Bröttcher, C.; Albericio, F.; Burger, K. Chem. Rev. 2006, 106, 4728.
(136) Farrera-Sinfreu, J.; Royo, M.; Albericio, F. Tetrahedron Lett. 2002, 43, 7813.
(137) Vig, B. S.; Murray, T. F.; Aldrich, J. V. Biopolymers 2003, 71, 620.

100

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (138) Kurtz, A. C. J. Biol. Chem. 1938, 122, 477.
(139) Wünsch, E. In „XV/1. Synthesis of Peptides, Protecting Groups I (Houben-Weyl:
VII. The Synthesis of Peptides”, Wünsch, E. ed., Georg Thieme Verlag: Stuttgart, p
468.
(140) Wiejak, S.; Masiukiewicz, E.; Rzeskotarska, B. Chem. Pharm. Bull. 1999, 47,
1489.
(141) Albericio, F.; Nicolás, E.; Rizo, J.; Ruiz-Gayo, E.; Pedroso, E.; Giralt, E.
Synthesis. 1990, 119.
(142) Bergmann, M.; Zervas, L.; Ross, W.F. J. Biol. Chem. 1935, 111, 245.
(143) Erickson, B.W.; Merrifield, R.B.; J. Am. Chem. Soc. 1973, 95, 3757.
(144) Schwyzer, R.; Rittel, W. Helv. Chim. Acta. 1961, 44, 159.
(145) Aletras, A.; Barlos, K.; Gatos, D.; Koutsogianni, S.; Mamos, P. Int. J. Pept. Prot.
Res. 1995, 45, 488.
(146) Matysiak, S.; Böldicke, T.; Tegge, W.; Frank, R. Tetrahedron Lett. 1998, 39,
1733.
(147) Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.; Chan, W.
C. Tetrahedron Lett. 1998, 39, 1603.
(148) Wittmann, V.; Seeberger, S. Angew. Chem. Int. Ed. Engl. 2000, 39, 4348.
(149) Bycroft, B, W.; Chan, W. C.; Chhabra, S. R.; Hone, N. D. J. Chem. Soc. Chem.
Commun. 1993, 9, 778.
(150) Bloomberg, G. B.; Askin, D.; Gargaro, A. R.; Tanner, M. J. A. Tetrahedron Lett.
1993, 34, 4709.
(151) Dumy, P.; Eggleston, I. M.; Cervigni, S.; Sila, U.; Sun, X.; Mutter, M.
Tetrahedron Lett. 1995, 36, 1255.
(152) Rohwedder, B.; Mutti, Y.; Dumy, P.; Mutter, M. Tetrahedron Lett. 1998, 39,
1175.
(153) Schallenberg, E. E.; Calvin, M.; J. Am. Chem. Soc. 1955, 77, 2779.
(154) Weygand, F.; Csendes, E.; Angew. Chem. 1952, 64, 136.
(155) Goldberger, R.F.; Anfinsen, C.B. Biochemistry 1962, 1, 401.
(156) Ohno, M.; Eastlake, A.; Ontjes, D.; Anfinsen, C.B. J. Am. Chem. Soc. 1969, 91,
6842.
(157) Moroder, L.; Filippi, B.; Borin, G.; Marchiori, F. Biopolymers 1975, 14, 2061.
(158) Weygand, F.; Frauendorfer, E. Chem. Ber. 1970, 103, 2437.

101

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (159) Tesser, G. I.; Balvert-Geers, I.C. Int. J. Pept. Protein Res. 1975, 7, 295-305.
(160) Podlech, J.; Musiol, H-J.; Lohof, E.; Moroder, L. Protection of the ω-Amino
Group. In “Synthesis of Peptides and Peptidomimetics (Houben-Weyl E22a: Methods of
Organic Chemistry)”, Goodman, M.; Felix, A.M.; Moroder, L.; Toniolo, C. eds., Georg
Thieme Verlag: Stuttgart and New York, 2002, pp. 166-192.
(161) Boon, P.J.; Mous, J.F.M.; ten Kortenaar, P.B.; Tesser, G. Int. J. Pept. Protein
Res. 1986, 28, 477.
(162) Monroc, S.; Feliu, L.; Serra, J.; Planas, M.; Bardaji, E. Synlett 2006, 17, 2743-.
(163) Loffet, A.; Zhang, H.X. Int. J. Pept. Prot. Res. 1993, 42, 346.
(164) Lyttle, M. H.; Hudson, D. Peptides Chemistry and Biology. Proceedings of the
12th American Peptide Symposium. Smith, J. A.; Rivier, J. E. Eds. ESCOM: Leiden,
1992, 583-584.
(165) Isidro-Llobet, A.; Álvarez, M.; Albericio, F. Terahedron. Lett. 2005, 46, 7733.
(166) López, P. E.; Isidro-Llobet, A.; Gracia, C.; Cruz, L. J.; García-Granados, A.;
Parra, A.; Álvarez, M.; Albericio, F. Tetrahedron. Lett. 2005, 46, 7737.
(167) Rusiecki, V. K.; Warne, S. A. Bioorg. Med. Chem. Lett. 1993, 3, 707.
(168) Lapeyre, M.; Leprince, J.; Massonneau, M.; Oulyadi, H.; Renard, P-Y.; Romieu,
A.; Trucatti, G.; Vaudry, H. Chem. Eur. J. 2006, 12, 3655.
(169) De Luca, S.; Della Moglie, R.; De Capua, A.; Morelli, G. Tetrahedron Lett.
2005, 46, 6637.
(170) Poreddy, A. R.; Schall, O. F.; Marshall, G. R.; Ratledge, C.; Slomczynska, U.
Bioorg. Med. Chem. Lett. 2003, 13, 2553.
(171) Thieriet, N.; Guibé, F.; Albericio, F. Org. Lett. 2000, 2, 1815.
(172) Visintin, C.; Aliev, A. E.; Riddall, D.; Baker, D.; Okuyama, M.; Hoi, P. M.;
Hiley, R.; Selwood, D. L. Org. Lett. 2005, 7, 1699.
(173) Kohlbau, H-J.; Thürmer, R.; Voelter, W. Protection of the α-Carboxy Group. In
“Synthesis of Peptides and Peptidomimetics (Houben-Weyl E22a: Methods of Organic
Chemistry)”, Goodman, M.; Felix, A.M.; Moroder, L.; Toniolo, C. eds., Georg Thieme
Verlag: Stuttgart and New York, 2002, pp. 193-237.
(174) Waldmann, H.; Kunz, H. Liebigs Ann. Chem. 1983, 10, 1712.
(175) Roeske, R. J. Org. Chem. 1963, 28, 1251.
(176) Anderson, G. W.; Callahan, F. M. J. Am. Chem. Soc. 1960, 82, 3359.
(177) Maclaren, J. A. Aust. J. Chem. 1971, 24, 1695.

102

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (178) Wang, S-S.; Gisin, B. F.; Winter, D. P.; Makofske, R.; Kulesha, I. D.; Tzougraki,
C.; Meienhofer, J. J. Org. Chem. 1977, 42, 1286-1290.
(179) Matthews, J. L.; Gademann, K.; Jaun, B.; Seebach, D. J. Chem. Soc. Perk. T. 1,
1998, 20, 3331.
(180) Ledger, R.; Stewart, F. H. Aust. J. Chem. 1965, 18, 1477.
(181) van Heeswick, W. A. R.; Eenink, M.J. D.; Feijen, J. Synthesis. 1982, 744.
(182) Deimer, K-H. In „XV/1. Synthesis of Peptides, Protecting Groups I (Houben-
Weyl: VII. The Synthesis of Peptides”, Wünsch, E. ed., Georg Thieme Verlag:
Stuttgart, p, 332.
(183) Taylor-Papadimitriou, J.; Yovanidis, C.; Paganou, A.; Zervas, L. J. Chem. Soc. C:
Organic 1967, 19, 1830.
(184) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to the
Synthesis of Peptides and Proteins. Ed. CRC Press, Boca Raton: New York, 1997.
(185) Gatos, D.; Athanassopoulos, P.; Tzavara, C.; Barlos, K. Peptides 1998: Proc 25th
European Peptide Symposium, Bajusz, S.; Hudecz, F. eds. Akademiai Kiado: Budapest,
1999, 146-147.
(186) McMurray, J. S. Tetrahedron Lett. 1991, 32, 7679.
(187) Yue, C.; Terry, J.; Potier, P. Tetrahedron Lett. 1993, 34, 323.
(188) Isidro-Llobet, A.; Álvarez, M.; Albericio, F. Tetrahedron Lett, 2008, 49, 3304.
(189) Kessler, H.; Siegmeier, R. Tetrahedron Lett. 1983, 24, 281.
(190) Bednarek M A; Bodanszky, M. Int. J. Pept. Prot. Res. 1983, 21, 196.
(191) Valero, M.-L.; Giralt, E.; Andreu, D. Peptides 1996: Proc 24th European Peptide
Symposium. Ramage, R.; Epton, R. (Eds.) Maryflower Scientific Ltd., 1996, 857.
(192) Chan, W. C.; Bycroft, B.W.; Evans, D. J.; White, P. D. J. Chem. Soc., Chem.
Commun. 1995, 2209.
(193) Bodanszky M. Int. J. Pept. Prot. Res. 1984, 23, 111.
(194) Martinez, J.; Laur, J.; Castro, B. Tetrahedron Lett. 1983, 24, 5219.
(195) Martinez, J.; Laur, J.; Castro, B. Tetrahedron 1985, 41, 739.
(196) Lloyd-Williams, P.; Jou, G.; Albericio, F.; Giralt, E. Tetrahedron Lett. 1991, 32,
4207.
(197) Alcaro, M. C.; Sabatino, G.; Uziel, J.; Chelli, M.; Ginanneschi, M.; Rovero, P.;
Papini, A. M. J. Pept. Sci. 2004, 10, 218.

103

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (198) Montero, A.; Albericio, F.; Royo, M.; Herradon, B. Eur. J. Org. Chem. 2007, 8,
1301.
(199) Guzman-Martinez, A.; Lamer, R.; VanNieuwenhze, M. S. J. Am. Chem. Soc.
2007, 129, 6017.
(200) Kates, S.; Solé, N. A.; Johnson, C. R.; Hudson, D.; Barany, G.; Albericio, F.
Tetrahedron Lett. 1993, 34, 1549.

(201) Kates, S.; Solé, N.; Albericio, F. Barany, G. Peptides: design, synthesis, and
biological activity. Basava C.; Anantharamaiah, G.M. eds., Birkhauser: Boston
(Massachusetts), 1994, 39-58.
(202) Kates, S.; Daniels, S. B.; Albericio, F. Anal. Biochem. 1993, 212, 303.

(203) Stelakatos, G. C.; Paganou, A.; Zervas, L. J. Chem. Soc. C 1966, 13, 1191.
(204) Hendrickson, J. B.; Kandall, C. Tetrahedron Lett. 1970, 5, 343.
(205) Kuroda, H.; Shigeru, K.; Naowoshi, C.; Terutoshi, K.; Sakakibara, S. Int. J. Pept.
Prot. Res. 1992, 40, 114.
(206) Lammert, R.; Ellis, A. I.; Chauvette, R. R.; Kukolja, S. J. Org. Chem. 1978, 43,
1243.
(207) Guibé-Jampel, E.; Wakselman, M. Synth. Commun. 1982, 12, 219.
(208) Romanovskis, P.; Spatola, A. F. J. Pept. Res. 1998, 52, 356.
(209) Schwarz, H.; Arakawa, K. J.; J. Am. Chem. Soc., 1959, 81, 5691.
(210) Royo, M.; Farrera-Sinfreu, J.; Solé, L.; Albericio, F. Tetrahedron Lett. 2002, 43,
2029.
(211) Hocker, M. D.; Caldwell, C. G.; Macsata, R. W.; Lyttle, M. H. Pept. Res. 1995, 8,
310.
(212) Namikoshi, M.; Kundu, B.; Rineheart, K. L. J. Org. Chem. 1991, 56, 5464.
(213) Sieber, P.; Andreatta, R., H.; Eisler, K.; Kamber, B.; Riniker, B.; Rink, H. In
Peptides, Proceedings of 5th American Peptide Symposium, Goodman, M.; Meienhofer,
J. Eds.; Wiley: New York. (1977). 543.
(214) Wagner, M.; Kunz, H. Synlett 2000, 3, 400.
(215) Wagner, M.; Kunz, H. Zeitschrifft für Naturforschung, B: Chemical Sciences
2002, 57, 928.
(216) Borsuk, K.; van Delft, F. L.; Eggen, I. F.; ten Kortenaar, P. B. W.; Petersen, A.;
Rutjes, P. J. T. Tetrahedron Lett. 2004, 45, 3585.
(217) Just, G.; Grozinger, K. Synthesis 1976, 457.

104

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (218) Givens, R. S.; Weber, J. F. W.; Conrad, P. G.; Orosz, G.; Donahue, S.L.; Thayer.
S.A. J. Am. Chem. Soc. 2000, 122, 2687.
(219) Lodder, M.; Golovine, S.; Laikhter, A. L.; Karginov, V. A.; Hecht, S. M. J. Org.
Chem. 1998, 63, 794.
(220) Bourgault, S.; Letourneau, M.; Fournier, A. Peptides 2007, 28, 1074.
(221) Sedighi, M.; Lipton, M. A. Org. Lett. 2005, 7, 1473.
(222) Taylor, J. W.; Reddy, P.; Patel, K.; Dineen, T.; Naqvi, S. Peptides: The Wave of
the Future, Proceedings of the Second International and the Seventeenth American
Peptide Symposium, Lebl, M.; Houghten, R. A. Eds. American Peptide Society: San
Diego, CA, 2001, 67-68.
(223) Feinberg, R. S.; Merrifield, R. B. J. Am. Chem. Soc. 1975, 97, 3485.
(224) Liu, G.; Zhang, S.-D.; Xia, S.-Q.; Ding, Z.-K. Bioorg. Med. Chem. Lett. 2000, 10,
1361.
(225) Borek, B. A.; Waelsch, H. J. Biol. Chem. 1953, 205, 459.
(226) DiMarchi, R. D.; Tam, J.P.; Merrifield, R. B. Int. J. Pept. Protein Res. 1982, 19,
270.
(227) Tam, J. P.; Riemen, M.W.; Merrifield, R.B. Pept. Res. 1988, 1, 6.
(228) Bodanszky, M.; Kwei, J. Z. Int.J. Pept. Protein Res. 1978, 12, 69.
(229) Mergler, M.; Dick, F.; Sax, B.; Weiler, P.; Vorherr, T. J. Pept. Sci. 2003, 9, 36.
(230) Yajima, H.; Futaki, S.; Otaka, A.; Yamashita, T.; Funakoshi, S.; Bessho, K.;
Fujii, N.; Akaji, K. Chem. Pharm. Bull. 1986, 34, 4356.
(231) Thürmer, R.; Kohlbau, H-J.; Voelter, W. Protection of the ω-Carboxy Group. In
“Synthesis of Peptides and Peptidomimetics (Houben-Weyl E22a: Methods of Organic
Chemistry)” (Goodman, M.; Felix, A.M.; Moroder, L.; Toniolo, C. eds.), Georg Thieme
Verlag, Stuttgart and New York, 2002, pp. 238-259.
(232) Kalström, A.; Undén, A. Tetrahedron Lett. 1996, 37, 4243.
(233) Dick, F., Fritschi, U.; Haas, G.; Hässler, O.; Nyfeler, R.; Rapp, E. Peptides 1996:
Proc. 24th European Peptide Symposium. Ramage, R.; Epton, R. eds. Maryflower
Scientific Ltd., 1998, 339-340.
(234) Ocampo, S. M.; Albericio, F.; Fernandez, I.; Vilaseca, M.; Eritja, R. Org. Lett.
2005, 7, 4349.
(235) Balvinder S.; Murray, T. F.; Aldrich, J. V. J. Med. Chem. 2004, 47, 446.
(236) Kocsis, L.; Bruckdorfer, T.; Orosz, G. Tetrahedron Lett. 2008, 49, 7015.

105

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (237) Felix, A. M.; Wang, C. T.; Heimer, E. P.; Fournier, A. Int. J. Pept. Prot. Res.
1988, 31, 231.
(238) Bolin, D. R.; Wang, C. T.; Felix, A. M. Org. Prep. Proc. Int. 1989, 21, 67.
(239) Chan, W. C.; Bycroft, B. W.; Evans, D. J.; White, P. D. Peptides 1994,
Proceedings of the 23rd European Peptide Symposium, Maia, H. L. S. ESCOM: Leiden,
1995, 153.
(240) Johnson, T.; Liley, M.; Cheeseright, T. J.; Begum, F. J. Chem. Soc., Perk. T. 1,
2000, 2811.
(241) Belshaw, P. J.; Mzengeza, S.; Lajoie, G. A. Synth. Commun. 1990, 20, 3157.
(242) Isidro-Llobet, A.; Álvarez, M.; Albericio, F. Unpublished results.
(243) Sieber, P. Helv. Chim. Acta 1977, 60, 2711.
(244) Marlowe, C. K.; Bioorg. Med. Chem. Lett. 1993, 3, 437.
(245) Meister, S. M.; Kent, S. B. H. In Peptides: Structure, Function, Proceedings to
the 8thAmerican Peptide Symposium, Hruby, V. J.; Rich, D. H., eds., Pierce Chemical
Company: Rockford, 1983, 103-106..
(246) Kent, S. B. H. Pept.: Struct. Funct. Proc. Am. Pept. Symp. 9th 1985, 407.
(247) Coin, I.; Beyermann, M.; Bienert, M. Nat. Prot. 2007, 2, 3247.
(248) Bedford, J.; Hyde, C.; Johnson, T.; Jun, W.; Owen, D.; Quibell, M.; Sheppard,
R.C. Int. J. Pept. Prot. Res. 1992, 40, 300.
(249) Hyde, C.; Johnson, T.; Owen, D.; Quibell, M.; Sheppard, R. C. Int. J. Pept. Prot.
Res. 1994, 43, 431.
(250) White, P.; Keyte, J. W.; Bailey, K.; Bloomberg, G. J. Pept. Sci. 2004, 10, 18.
(251) Abedini, Andisheh; Raleigh, Daniel P. Org. Lett. 2005, 7, 693.
(252) Nicolás, E.; Pedroso, E.; Giralt, E. Tetrahedron Lett. 1989, 30, 497.
(253) Yang, Y.; Sweeney, W. V.; Schneider, K.; Thornqvist, S.; Chait, B. T.; Tam, J. P.
Tetrahedron Lett. 1994, 35, 9689.
(254) Cebrian, J.; Domingo, V.; Reig, F. J. Pept. Res., 2003, 62, 238.
(255) Zahariev, S.; Guarnaccia, C.; Pongor, C. I.; Quaroni, L.; Cemazar, M.; Pongor, S.
Tetrahedron Lett. 2006, 47, 4121.
(256) Mutter, M.; Nefzi, A.; Sato, T.; Sun, X.; Wahl, F.; Wuhr, T. Pept. Res. 1995, 8,
145.
(257) Haack, T.; Mutter, M.; Tetrahedron Lett. 1992, 33, 1589.

106

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (258) Johnson, T.; Quibell, M.; Owen, D.; Shepard, R. C. J. Chem. Soc. Chem.
Commun. 1993, 369.
(259) Isidro-Llobet, A.; Just-Baringo, X.; Álvarez, M.; Albericio, F. Biopolymers. 2008,
90, 444.
(260) Johnson, T. Packman, L.C.; Hyde, C.B.; Owen, D.; Quibell, M. J. Chem. Soc.
Perk. T. 1 1996, 719.
(261) Wöhr, T.; Wahl, F.; Netzi, A.; Rohwedder, B.; Sato, T., Sun, X.; Mutter, M. J.
Am. Chem Soc. 1996, 118, 9218.
(262) Nicolás, E.; Pujades, M.; Bacardit, J.; Giralt, E.; Albericio, F. Tetrahedron Lett.
1997, 38, 9047.
(263) Garcia-Martin, F.; White, P.; Steinauer, R.; Cote, S.; Tulla-Puche, J.; Albericio, F.
Biopolymers 2006, 84, 566.
(264) Johnson, T.; Quibell, M.; Sheppard, R. C. J. Pept. Sci. 1995, 1, 11.
(265) Zeng, W.; Regamey, P-O.; Rose, K.; Wang, Y.; Bayer, E. J. Pept. Res. 1997, 49,
273.
(266) Weygand, F.; Steglich, W.; Bjarnason, J.; Akhtar, R.; Khan, N. M. Tetrahedron
Lett. 1966, 29, 3483.
(267) Zahariev, S.; Guarnaccia, C.; Zanuttin, F.; Pintar, A.; Esposito, G.; Maravic, G.;
Krust, B.; Hovanessian, A. G.; Pongor, S. J. Pept. Sci. 2005, 11, 17.
(268) Clausen, N.; Goldammer, C.; Jauch, K.; Bayer, E. Peptides 1996: Proc 14th
American Peptide Symposium. Ramage, R.; Epton, R. Eds.Maryflower Scientific Ltd.:
Kingswinford, UK, 1996, 71-72.
(269) Jauch, K.; Goldammer, C.; Clausen, N.; Bayer, E. In Peptides 1996: Proc 24th
European Peptide Symposium. Ramage, R.; Epton, R. eds. Maryflower Scientific Ltd.:
Kingswinford, UK 1998, 497-498,
(270) Johnson, E. C. B.; Kent, S. B. H. Chem. Commun. 2006, 1557.
(271) Offer, J.; Quibell, M.; Johnson, T. Innovation and Perspectives in Solid Phase
Synthesis & Combinatorial Libraries: Peptides, Proteins and Nucleic Acids-Small
Molecule Organic Chemical Diversity, Collected Papers, International Symposium 5th,
Epton, R. ed., Mayflower Scientific Ltd.: Kingswinford, UK
1999, 357-360.
(272) Offer, J.; Johnson, T.; Quibell, M. Tetrahedron Lett. 1997, 38, 9047.
(273) Howe, J.; Quibell, M.; Johnson, T. Tetrahedron Lett. 2000, 41, 3997.

107

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (274) Wahlstroem, K.; Planstedt, O.; Unden, A. Tetrahedron Lett. 2008, 49, 3921.
(275) Gausepohl, H.; Kraft, M.; Frank, R. W. Int. J. Pept. Prot. Res. 1989, 34, 287.
(276) Mojsov, S.; Mitchell, A. R.; Merrifield, R. B. J. Org. Chem. 1980, 45,, 555-.
(277) Doherty-Kirby, A.; Lajoie, G. A. Side-Chain Protecting Groups. Solid-Phase
Synthesis, A Practical Guide, Kates, S. A.; Albericio, F. eds., Marcel Dekker Inc.: New
York. 2000, 103-128.
(278) Dimarchi, R. D.; Tam, J. P.; Kent, S. B. H.; Merrifield, R. B. Int. J. Pept. Prot.
Res. 1982, 19, 88.
(279) Quesnel, A.; Briand, J.-P. J. Pept. Res. 1998, 52, 107.
(280) Friede M; Denery S; Neimark J; Kieffer S; Gausepohl H; Briand J. P. Pept. Res.
1992, 5, 145.
(281) Sieber, P.; Riniker, B. Tetrahedron Lett. 1991, 32, 739.
(282) Shimonishi, Y.; Sakakibara, S.; Akabori, S. Bull. Chem. Soc. Jpn. 1962, 35,
1966.
(283) Han, Y.; Sole, N. A.; Tejbrant, J.; Barany, G. Pept. Res. 1996, 9, 166.
(284) Sax, B.; Dick, F.; Tanner, R.; Gosteli, J. J. Pept. Res. 1992, 5, 245.
(285) Carpino, L. A.; Chao, H.-G. WO 9526976 . Chem. Abstr. 1995, 124, 146865.
(286) Carpino, L. A.; Shroff, H. N.; Chao, H.-G.; Mansour, E. M. E.; Albericio, F.
Peptides 1994, Proceedings of the 23rd European Peptide Symposium, Maia, H. L. S.
ESCOM: Leiden, 1995. 155-156.
(287) Konig, W.; Geiger, R. Chem. Ber. 1970, 103, 2041-2051.
(288) Funakoshi, S. ; Tamamura, H.; Fujii, N. ; Yoshizawa, K. ; Yajima, H. ; Miyasaka,
K. ; Funakoshi, A.; Ohta, M. ; Inagaki, Y. ; Carpino, L. A. J. Chem. Soc., Chem.
Comm. 1988, 24, 1588.
(289) Hudson, D. Eur. Pat. Appl. EP 292228 A2, 1988, Chem. Abstr. 1989, 110,
213367.
(290) Shah, D.; Schneider, A.; Babler, S.; Gandhi, R.; Van Noord, E.; Chess, E. Pept.
Res. 1992, 5, 241.
(291) Rink, H.; Sieber, P.; Raschdorf, F. Tetrahedron Lett., 1984, 25, 621.
(292) Du Vigneaud, V.; Gish, D. T.; Katsoyannis, P. G.; Hess, G. P. J. Am. Chem. Soc.,
1958,, 80, 3355.
(293) Jones, D. A.; Miculec, R. A.; Mazur, R. H. J. Org. Chem. 1973, 38, 2865.

108

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (294) Ponsati, B.; Canas, M.; Jodes, G.; Clemente, J.; Barcadit, J. PCT Int. Appl. 2000,
WO 2000071570 A1, 2000, Chem. Abstr. 134, 17728.
(295) Ramage, R.; Green, J.; Blake, A. J. Tetrahedron 1991, 47, 6353.
(296) Kiso, Y.; Satomi, M.; Ukawa, K.; Akita, T. J. Chem. Soc. Commun. 1980, 1063.
(297) Ramachandran, J.; Li, C. H. J. Org. Chem. 1962, 27, 4006.
(298) Carpino, L. A.; Shroff, H.; Triolo, S. A.; Mansour, El Sayed M. E.; Wenschuh,
H.; Albericio, F. Tetrahedron Lett. 1993, 34, 7829.
(299) Yajima, M.; Akaji, K.; Mitani, N.; Fujii, N.; Funakoshi, S.; Adachi, H.; Oishi, M.;
Akazawa, Y. Int. J. Pept. Prot. Res. 1979, 14, 169.
(300) Yajima, M.; Takeyama, M.; Kanaki, J.; Mitani, N. J. Chem. Soc. Chem.
Commun. 1978, 482.
(301) Atherton, E.; Sheppard, R. C.; Wade, J. D. J. Chem. Soc. Chem. Commun. 1983,
19, 1060. .
(302) Isidro-Llobet, A.; Álvarez, M.; Albericio, F. J. Org. Chem. 2008 (submitted).
(303) Lundt, B. F.; Johansen, N. L.; Volund, A.; Markussen, J. Int. J. Pept. Protein Res.
1978, 12¸ 258.
(304) Verdini, A. S.; Lucietto, P.; Fossati, G.; Giordani, C. Peptides, Chemistry and
Biology, Proceedings of the 12th American Peptide Symposium. Smith, J. A.; Rivier, J.
E. eds. ESCOM: Leiden, 1992, 562-563.
(305) Verdini, A. S.; Lucietto, P. ; Fossati, G.; Giordani, C. Tetrahedron Lett. 1992, 33,
6541.
(306) Noda, M.; Kiffe, M. J. Pept. Res. 1997, 50, 329.
(307) Bergmann, M.; Zervas, L.; Rinke, H. H-S Z. Physiol. Chem. 1934, 224, 40.
(308) Young, G. T.; Schafer, D. J.; Elliott, D. F.; Wade, R., J. Chem. Soc. C. 1971, 46.
(309) Tamura, S. Y.; Semple, J. E.; Ardecky, R. J.; Leon, P.; Carpenter, S.H.; Ge, Y.;
Shamblin, B. M.; Weinhouse, M. I.; Ripka, W. C.; Nutt, R. F. Tetrahedron Lett. 1996,
37, 4109.
(310) Semple, J. E.; Rowley, D. C.; Brunck, T. K.; Ha-Uong, T.; Minami, N. K.;
Owens, T. D.; Tamura, S.Y.; Goldman, E. A.; Siev, D. V.; Ardecky, R. J.; Carpenter, S.
H.; Ge, Y.; Richard, B. M.; Nolan, T. G.; Hakanson, K.; Tulinsky, A.; Nutt, R. F.;
Ripka, W.C. J. Med. Chem. 1996, 39, 4531.
(311) Krishnamoorthy, R.; Vazquez-Serrano, L. D.; Turk, J. A.; Kowalski, J. A.;
Benson, A. G.; Breaux, N. T.; Lipton, M. A. J. Am. Chem. Soc. 2006, 128, 15392.

109

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (312) Molnar-Perl, I.; Fabian-Vonsik, V. J. Chromatogr. 1988, 446, 231.
(313) Sarkar, S. K.; Malhotra, S. S. J. Chromatogr. 1979, 170, 371.
(314) Bartoli, S.; Jensen, K. J.; Kilburn, J. D. J. Org. Chem. 2003, 68, 9416-9422.
(315) Calimsiz, S.; Morales Ramos, A. I.; Lipton, M. A. J. Org. Chem. 2006, 71,
6351.
(316) Eisele, F.; Kuhlmann, J.; Waldmann, H. Chemistry-A European Journal. 2002,
8, 3362.
(317) Barany, G.; Merrifield, R. B. The Peptides Analysis, Synthesis, Biology, Vol. 2,
Special Methods in Peptide Synthesis, Part A, Gross, E.; Meienhofer, J. Eds. Academic
Press: New York, 1979, 1.
(318) Yajima, H.; Funakoshi, S.; Fujii, N.; Akaji, K.; Irie, H. Chem. Pharm. Bull. 1979,
27, 1060.
(319) Yajima, H.; Akaji, K.; Funakoshi, S.; Fujii, N.; Irie, H. Chem. Pharm. Bull. 1980,
28, 1942.
(320) Lukszo, J.; Patterson, D.; Albericio, F.; Kates, S. A. Lett. Pept. Sci. 1996, 3,
157.
(321) Eritja, R.; Ziehler-Martin, J. P.; Walker, P. A.; Lee, T. D.; Legesse, K.; Albericio,
F.; Kaplan, B. E. Tetrahedron 1987, 43, 2675.
(322) Nacagawa,Y.; Nishiuchi, Y.; Emura, J.; Sakakibra, S. In Peptide Chemistry 1980,
Okawa, K. Ed.: Protein Research Foundation: Osaka, 1981, 41.
(323) Musiol, H-J.; Siedler, F.; Quarzago, D.; Moroder, L. Biopolymers 1994, 34, 1553.
(324) Lamthanh, H.; Roumestand, C.; Deprun, C.; Menez, A. Int. J. Pept. Prot. Res.
1993, 41, 85.
(325) Lamthanh, H.; Virelizier, H., Frayssinhes. Pept. Res. 1995, 8, 316.
(326) Engebretsen, M.; Agner, E.; Sandosham, J.; Fischer, P. M. J. Pept. Res. 1997, 49,
341.
(327) Kumagaye, K. Y.; Inui, T.; Nakajima, K.; Kimura, T.; Sakakibara, S. Pept. Res.
1991, 4, 84.
(328) Gesquière, J. C.; Najib, J.; Diesis, E.; Barbry, D..; Tartar, A. Peptides, Chemistry
and Biology, Proceedings of the 12th American Peptide Symposium, Smith, J. A.;
Rivier, J. E. eds., ESCOM: Leiden, 1992, 641.
(329) Kaiser, E. T.; Nicholson, G. J.; Kohlbau, H. J.; Voelter, W. Tetrahedron Lett.
1996, 37, 1187.

110

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (330) Atherton, E.; Hardy, P. M.; Harris, D. E.; Mathews, B. H. Peptides 1990,
Proceedings of the 21st European Peptide Symposium. Giralt, E.; Andreu, D. eds.
ESCOM: Leiden, 1991, 243-244.
(331) Fujiwara, Y.; Akaji, K.; Kiso, Y. Chem. Pharm. Bull. 1994, 42, 724.
(332) Han, Y.; Albericio, F.; Barany, G. J. Org. Chem. 1997, 62, 4307.
(333) Angell, Y. M.; Alsina, J.; Albericio, F.; Barany, G. J. Pept. Res. 2002, 60, 292.

(334) Siedler, F.; Weyher, E.; Moroder, L. J. Pept. Sci. 1996, 2, 271.
(335) Moroder, L.; Musiol, H-J.; Schaschke, N.; Chen, L.; Hargittai, B.; Barany, G.
Protection of the Thiol Group. In “Synthesis of Peptides and Peptidomimetics (Houben-
Weyl E22a: Methods of Organic Chemistry)” (Goodman, M.; Felix, A.M.; Moroder, L.;
Toniolo, C. eds.), Georg Thieme Verlag, Stuttgart and New York, 2002, 384-424.
(336) Atherton, E.; Benoiton, N. L.; Brown, E.; Sheppard, R. C.; Williams, B. J. J.
Chem. Soc. Chem. Commun. 1981, 336.
(337) Richter, L. S.; Marsters, J. C.; Gadek, T. R. Tetrahedron Lett. 1994, 35, 1631.
(338) Erickson, B. W.; Merrifield, R. B. J. Am. Chem. Soc. 1973, 95, 3750.
(339) Heath, W. F.; Tam, J.; Merrifield, R. B. Int. J. Pept. Protein Res. 1986, 28, 498.
(340) Sakakibara, S. Biopolymers 1995, 37, 17.
(341) Fujii, N.; Otaka, A.; Funakoshi, S.; Bessho, K.; Watanabe, T.; Akaji, K.; Yajima,
H. Chem. Pharm. Bull. 1987, 35, 2339.
(342) Yajima, H.; Fujii, M.; Ogawa, H.; Kawatami, H. J. Chem. Soc. Chem. Commun.
1974, 107-
(343) Nishimura, O.; Kitada, C.; Fujino, M. Chem. Pharm. Bull. 1978, 26, 1576.
(344) Fuji, N.; Otaka, A.; Watanabe, T.; Okamachi, A.; Tamamura, H.; Yajima, H.;
Inagaki, Y.; Nomizu, M.; Asano, K. J. Chem. Soc. Chem. Commun. 1989, 283.
(345) Akaji, K. ; Tatsumi, T.; Yoshida, M. ; Kimura, T.; Fujiwara, Y.; Kiso, Y. J.
Chem. Soc. Chem. Commun. 1991, 3, 167.
(346) Akabori, S.; Sakakibara, S.; Shimonishi, Y.; Nobuhara, Y. Bull. Chem. Soc. Jpn.
1964, 37, 433.
(347) Zervas, L.; Photaki, I. J. Am. Chem. Soc. 1962, 84, 3887.
(348) Yajima, H.; Fujii, N.; Funakoshi, S.; Watanabe, T.; Murayama, E.; Otaka, A.
Tetrahedron 1988, 44, 805.
(349) Barlos, K.; Gatos, D.; Hatzi, O.; Koch, N.; Koutsogianni, S. Int. J. Pept. Prot.
Res. 1996, 47, 148.

111

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (350) Munson, M. C.; García-Echevarría, C.; Albericio, F.; Barany, G. J. Org. Chem.
1992,57, 3013.
(351) García, O.; Nicolás, E.; Albericio, F. Innovation and Perspectives in Solid Phase
Synthesis and Combinatorial Libraries: Peptides, Proteins and Nucleic Acids-Small
Molecule Organic Chemistry Diversity Collected Papers, International Symposium, 6th.
Epton, R. ed. Mayflower Scientific Ltd.: Kingswinford, UK, 2001, 289-290.
(352) Sifferd, R. H.; du Vigneaud, V. J. Biol. Chem. 1935, 108, 753.
(353) Pastuszak, J. J.; Chimiak, A. J. Org. Chem.1981, 46, 1868.
(354) Bodanszky, M.; Bednarek, M. A. Int. J. Pept. Prot. Res. 1982, 20, 434.
(355) Ruiz-Gayo, M.; Albericio, F.; Pedroso, E.; Giralt, E. J. Chem. Soc., Chem.
Commun. 1986, 20, 1501.
(356) Royo, M.; Garcia-Echeverria, C.; Giralt, E.; Eritja, R.; Albericio, F. Tetrahedron
Lett. 1992, 33, 2391.
(357) West, C. W.; Estirarte, M. A.; Rich, D. H. Org. Lett. 2001, 3, 1205.
(358) Veber, D. F.; Milkowski, J. D.; Varga, S. L.; Denkewalter, R. G.; Hirschmann, R.
J. Am. Chem. Soc. 1972, 94, 5456.
(359) Kamber, B. Helv. Chim. Acta 1971, 54, 927.
(360) Lyle, T. A.; Brady, S. F.; Ciccarone, T. M.; Colton, C. D.; Paleveda, W. J.; Veber,
D. F.; Nutt, R. F. J. Org. Chem. 1987, 52, 3752-3759.
(361) Singh, P. R.; Rajopadhye, M.; Clark, S. L.; Williams, N. E. Tetrahedron Lett.
1996, 37, 4117.
(362) Royo, M.; Alsina, J.; Giralt, E.; Slomcyznska, U.; Albericio, F. J. Chem. Soc.,
Perk. T. 1. 1995, 9, 1095.
(363) Weber, U.; Hartter, P. Hoppe-Seyler’s Z. Physiol. Chem. 1970, 351, 1384-1388.
(364) Wünsch, E. In „XV/1. Synthesis of Peptides, Protecting Groups I (Houben-Weyl:
VII. The Synthesis of Peptides”, Wünsch, E. ed., Georg Thieme Verlag: Stuttgart, p
789.
(365) Wünsch, E.; Spangenberg, R. Ger. Offen. 1971, DE 1923480 1971012. 1971,
Chem. Abstr. 74, 88309.
(366) Moroder, L.; Gemeiner, M.; Göhring, W.; Jaeger, E.; Wünsch, E. In Peptides
1980, Brundfeldt, K., Ed.; Scriptor: Copenhagen, 1981, 121.
(367) Atherton, E.; Sheppard, R. C.; Ward, P. J. Chem. Soc. Perk. T. 1. 1985, 2073.
(368) Matsueda, R.; Kimura, T.; Kaiser, E. T.; Matsueda, G. R. Chem. Lett. 1981, 737.

112

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (369) Albericio, F.; Andreu, D.; Giralt, E.; Navalpotro, C.; Pedroso, E.; Ponsati, B.;
Ruiz-Gayo, M. Int. J. Pept. Prot. Res. 1989, 34, 124.
(370) Bernatowicz, M. S.; Matsueda, R.; Matsueda, G. R. Int. J. Pept. Prot. Res. 1986,
28, 107.
(371) Huang, H.; Carey, R. I. J. Pept. Res. 1998, 51, 290.
(372) Gómez-Martínez, P.; Guibé, F.; Albericio, F. Lett. Pept. Sci. 2001, 7, 187.
(373) Tatsu, Y.; Endo, Y.; Yumoto, N. Pept. Sci. 2003, 405.
(374) Pan, P.; Bayley, H. FEBS Lett. 1997, 405, 81.
(375) Bland, L.; Ramage, R. Innovation and Perspectives in Solid Phase Synthesis and
Combinatorial Librharies: Peptides, Proteins and Nucleic Acids-Small Molecule
Organic Chemistry Diversity Collected Papers, International Symposium, 6th. Epton,
R. ed. Mayflower Scientific Ltd.: Kingswinford, UK, 2001, 247-248.
(376) Pool, C. T.; Boyd, J. G.; Tam, J. P. J. Pept. Res. 2004, 63, 223.
(377) Werbitzky, O.; Oehlers, D. Chim. Oggi, 2008, 26(4), 26.
(378) Gairí, M.; Lloyd-Williams, P.; Albericio, F.; Giralt, E. Tetrahedron Lett. 1994,
35, 175.
(379) Guttmann, S.; Boissonnas, R. A. Helv. Chim. Acta 1959, 42, 1257.
(380) Yajima, H.; Kanaki, J.; Kitajima, M.; Funakoshi, S. Chem. Pharm. Bull. 1980, 28,
1214.
(381) Iselin, B. Helv. Chim. Acta. 1961, 44, 61.
(382) Houghten, R.A.; Li, C.H. Peptides, Chemistry, Structure & Biology, Proc. 5th
American Peptide Symposium, Goodman, M.; Meienhofer, J. eds., Pierce Chemical
Company: Rockford, 1977, 458.
(383) Houghten, R.A.; Li, C.H. Int. J. Pept. Prot. Res. 1978, 11, 345.
(384) Houghten, R.A.; Li, C.H. Anal. Biochem. 1979, 98, 36.
(385) Ferrer, T.; Nicolás, E.; Giralt, E. Lett. Pept. Sci. 1999, 6, 165.
(386) Vilaseca, M.; Nicolás, E.; Capdevila, F.; Giralt, E. Tetrahedron. 1998, 54, 15273.
(387) Andreu, D.; Nicolas, E. Solid-Phase Synthesis, A Practical Guide, Kates, S. A.;
Albericio, F. eds., Marcel Dekker Inc.: New York, 2000, 365-375.
(388) Pennington, M. W.; Byrnes, M. E. Pept. Res. 1995, 8, 39.
(389) Beck, W.; Jung, G. L.I.P.S. 1994, 1, 31.
(390) Teixidó, M.; Altamura, M.; Quartara, L.; Giolitti, A.; Maggi, C. A.; Giralt, E.;
Albericio, F. J. Comb. Chem. 2003, 5, 760.

113

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (391)Taboada, L.; Nicolas, E.; Giralt, E. Tetrahedron Lett. 2001, 42, 1891.
(392) Fukaki, S.; Yagami, T.; Taike, T.; Akita, T.; Kitagawa, K. J. Chem. Soc. Perk. T.
I. 1990, 653.
(393) Noble, R. L.; Yamashiro, D.; Li, C. H. J. Am. Chem. Soc. 1976, 98, 2324.
(394) Riniker, B.; Brugger, M.; Kamber, B.; Rittel, W.; Sieber P. Progress in Peptide
Research, Vol. II, Proceedings of the Second American Peptide Symposium, Lande, S.
ed., Gordon and Breach: New York, 1972, 116.
(395) Jones, J. H. Protection of the Imidazole Group. In “Synthesis of Peptides and
Peptidomimetics (Houben-Weyl E22a: Methods of Organic Chemistry)”, Goodman, M.;
Felix, A.M.; Moroder, L.; Toniolo, C. eds., Georg Thieme Verlag: Stuttgart and New
York, 2002, pp. 334-346.
(396) Kusunoki, M.; Nakagawa, S.; Seo, K.; Hamana, T.; Fukuda, T. Int. J. Pept. Prot.
Res. 1990, 36, 381.
(397) Ishiguro, T.; Eguchi, C. Chem. & Pharm.l Bull. 1989, 37, 506.
(398) Jones, J. H.; Ramage, W. I.; Witty, M. J. Int. J. Pept. Res. 1980, 15, 301.
(399) Fujii, T; Sakakibara, S. Bull. Chem. Soc. Jpn. 1974, 47, 3146.
(400) Fujii, T; Kimura, T.; Sakakibara, S. Bull. Chem. Soc. Jpn. 1976, 49, 1595.
(401) Sieber, P.; Riniker, B. Tetrahedron Lett. 1987, 28, 6031.
(402) Barlos, K.; Chatzi, O.; Gatos, D.; Stravropoulos, G.; Tsegenidis, T. Tetrahedron
Lett. 1991, 32, 475.
(403) Karlström, A.; Undén, A. J. Chem. Soc. Chem. Commun. 1996, 959.
(404) Yoshizawa-Kumagaye, K.; Nishiuchi, Y.; Nishio, H.; Kimura, T. J. Pept. Sci.
2005, 11, 512-515.
(405) Colombo, R.; Colombo, F.; Jones, J. H. J. Chem. Soc. Chem. Commun. 1984, 292.
(406) Mergler, M.; Dick, F.; Sax, B.; Schwindling, J.; Vorherr, T. J. Pept. Sci. 2001, 7,
502.
(407) Atherton, E.; Cammish, L. E.; Goddard, P.; Richards, J. D.; Sheppard, R. C.
Pept., Proc. Eur. Pept. Symp., 18th 1984, 153.
(408) Zaramella, S.; Strömberg, R.; Yeheskiely, E. Eur. J. Org. Chem. 2003, 2454.
(409) Chillemi, F.; Merrifield, R. B. Biochemistry 1969, 8, 4344.
(410) Shaltiel, S.; Biochem. Phys. Res. Commun. 1967, 29, 178.
(411) Shaltiel, S.; Fridkin, M. Biochemistry, 1970, 9, 5122.

114

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (412) Gesquière, J. C.; Najib, J.; Latailler, T.; Maes, P.; Tartar, A. Tetrahedron Lett.
1993, 34, 1921.
(413) Stewart, J. M.; Knight, M.; Paiva, A. C. M.; Paiva, T. Progress in Peptide
Research. Lande, S. (Ed.) 1972, 59-64.
(414) Fischer, P. M; Retson, K. V.; Tyler, M. I.; Howden, M. E. H. Int. J. Pept. Prot.
Res, 1991, 38, 491.
(415) Reissmann, S.; Schwuchow, C.; Seyfarth, L.; Pineda De Castro, L. F.; Liebman,
C.; Paegelow, I.; Werner, H.; Stewart, J. J. Med. Chem. 1996, 39, 929.
(416) Shvachkin, Yu. P.; Girin, S. K.; Smirnova, A. P.; Shishkina, A. A.; Ermak, N. M.
Bioorg. Khim. 1980, 6, 187.
(417) Arold, H.; Reissmann, S. J. Prakt. Chem. 1970, 312, 1130.
(418) Adamson, J. G.; Blaskowitch, M. A.; Groenvelt, H.; Lajoie, G. A. J. Org. Chem.
1991, 56, 3447.
(419) Hruby, V. J.; Ehler, K. W. J. Org. Chem. 1970, 35, 1690.
(420) Sugano, H.; Miyoshi, M. J. Org. Chem. 1976, 41, 2352.
(421) Mizoguchi, T.; Levin, G.; Woolley, D. W.; Stewart, J. M. J. Org. Chem. 1968, 33,
903.
(422) Wang, J.; Okada, W. Li; Yokoi, T.; Zhu, J. J. Chem. Soc. Perk. T. I, 1997, 621.
(423) Barlos, K.; Gatos, D.; Koutsogianni, S.; Schäfer, W.; Stavropoulous, G.; Yenqing,
Y. Tetrahedron Lett. 1991, 32, 471.
(424) Fischer, P. M. Tetrahedron Lett. 1992, 33, 7605.
(425) Reid, G. E.; Simpson, R. J.; Anal. Biochem. 1992, 200, 301.
(426) Nishiyama, Y.; Kurita, K. Tetrahedron Lett. 1999, 40, 927.
(427) Arzeno, H. B.; Beinfenheimer, W.; Blanchette, R.; Morgans, D. J.; Robinson III,
J. Int. J. Pept. Prot. Res. 1993, 41, 342.
(428) Barlos, K.; Gatos, D.; Koutsogiammi, S. J. Pept. Res. 1998, 51, 194.
(429) Lalonde, M.; Chan, T. H. Synthesis 1985, 817.
(430) Davies, J. S.; Higginbotham, C. L.; Tremeer, E. J.; Brown, C.; Treadgold, R. C. J.
Chem. Soc., Perk. T. 1. 1992, 22, 3043.
(431) Pirrung, M. C.; Nunn, D. S. Bioorg. Med. Chem. Lett. 1992, 2, 1489.
(432) Lemke, E. A.; Summerer, D.; Geierstanger, B. H.; Brittain, S. M.; Schultz, P. G.
Nat. Chem. Biol. 2007, 3, 769.

115

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (433) Ramesh, R.; De, Kavita; Gupta, S.; Chandrasekaran, S. J. Chem. Sci. 2008, 120,
163.
(434) Yamashiro, D.; Li, C. H. J. Am. Chem. Soc. 1973, 95, 1310.
(435) Yamashiro, D.; Li, C. H. J. Org. Chem. 1973, 38, 591.
(436) Yamashiro, D.; Li, C. H. Int. J. Pept. Protein Res. 1972, 4, 181.
(437) Salem, E. M.; Schou, O. Indian J. Chem. Sect. B. 1980, 19, 62.
(438) Rosenthal, K.; Kalström, A.; Unden, A. Tetrahedron Lett. 1997, 38, 1075.
(439) Bódi, J.; Nishiuchi, Y.; Nishio, H.; Inui, T.; Kimura, T. Tetrahedron Lett. 1998,
39, 7117.
(440) Smith, C. W.; Ferger, M. F. J. Med. Chem. 1975, 18, 822.
(441) Barlos, K.; Gatos, D.; Kapolos, S.; Poulos, C.; Schäfer, W.; Yao, W. Int. J. Pept.
Protein Res.. 1991, 38, 555.
(442) Dangles, O.; Guibé, F.; Balavoine, G.; Lavielle, S.; Marquet, A. J. Org. Chem.
1987, 52, 4984.
(443) Amit, B.; Hazum, E.; Fridkin, M.; Patchornik, A. Int. J. Pept. Prot. Res. 1977, 9,
91.
(444) Tatsu, Y.; Shigeri, Y.; Sogabe, S.; Yumoto, N.; Yoshikawa, S. Biochem. Biophys.
Res. Commun. 1996, 227, 688.
(445) Wahlstroem, K.; Planstedt, O.; Unden, A. Tetrahedron Lett. 2008, 49, 3779.
(446) Fontana, A.; Toniolo, C. Fort. Chem. Org. Nat. 1976, 33, 309.
(447) Giraud, M. ; Cavelier, F.; Martinez, J. J. Pept. Sci. 1999, 5, 457.
(448) Omori,Y.; Matsuda, Y.; Aimoto, S.; Shimonishi, Y.; Yamamoto, M. Chem. Lett.
1976, 805.
(449) Andreu, D.; García, F. J. Lett. Pept. Sci. 1997, 4, 41.
(450) Pearson, D. A.; Blanchette, M.; Baker, M. L.; Guindon, C. A. Tetrahedron Lett.
1989, 30, 2739.
(451) Sieber, P.; Tetrahedron Lett. 1987, 28, 1637.
(452) Stierandova, A.; Sepetov, N.; Nikiforovich, G. V.; Lebl, M. Int. J. Pept. Prot. Res.
1994, 41, 31.
(453) Karlström, A.; Unden, A. J. Chem. Soc. Chem. Commun. 1996, 1471.
(454) Nishiuchi, Y.; Nishio, H.; Inui, T.; Kimura, T.; Sakakibara, S. Tetrahedron Lett.
1996, 37, 7529.

116

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
 (455) White, P. Peptides, Chemistry and Biology, Proceedings of the 12th American
Peptide Symposium. Smith, J. A.; Rivier, J.E. eds. ESCOM: Leiden, 1992, 537-538.
(456) Franzen, H.; Grehn, L.; Ragnarsson, U. J. Chem. Soc. Chem. Commun. 1984,
1699.
(457) Vorherr, T.; Trzeciak, A.; Bannwarth, W. Int. J. Pept. Prot. Res. 1996, 48, 553.
(458) Ohno, M.; Tsukamoto, S.; Makisumi, S.; Izumiya, N. Bull. Chem. Soc. Jpn. 1972,
45, 2852.
(459) Yamashiro, D.; Li, C. H. J. Org. Chem. 1973, 38, 2594.
(460) Matsueda, G. R. Int. J. Pept. Prot. Res. 1982, 20, 26.
(461) Geiger, R.; König, W. The Peptides. Analysis, Synthesis, Biology. Vol. 3,
Protection of Functional Groups in Peptide Synthesis. Gross, E. and Meienhofer, J. eds.
Academic Press: New York. 1981, 1.
(462) Merrifield, R. B.; Vizioli, L. D.; Boman, H. G. Biochemistry 1982, 21, 5020.
(463) Choi, H.; Aldrich J. V. Int. J. Pept. Prot. Res. 1993, 42, 58.
(464) Fields, C. G.; Fields, G. B. Tetrahedron Lett. 1993, 34, 6661.
(465) Nishiuchi, Y.; Nishio, H.; Inui, T.; Bodi, J. Kimura, T. Innovation and
Perspectives in Solid Phase Synthesis and Combinatorial Libraries : Peptides, Proteins
and Nucleic Acids-Small Organic Chemistry Diversity, Collected Papers, International
Symposium, 6th. Epton, R. ed. Mayflower Scientific Ltd.: Kingswinford, UK, 2001,
331-332.
(466) Nishio, H.; Nishiuchi, Y.; Inui, T.; Nakata, M.; Yoshizawa-Kumagaye, K.;
Kimura, T. Peptides: The Wave of the Future. Proceedings of the Seventeenth American
Peptide Symposium, Lebl, M.; Houghten, R. A. eds. American Peptide Society: San
Diego, CA, 2001, 248-249.
(467) Fujii, N.; Futaki, S.; Yasumura, K.; Yajima, H. Chem. Pharm. Bull. 1984, 32,
2660.
(468) Miyoshi, K.; Otaka, A.; Kaneko, M.; Tamamura, H.; Fujii, N. Chem. Pharm. Bull.
2000, 48, 1230.

117

Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)