Alpha N Protection
To prevent uncontrolled oligomerization of the activated amino acid during coupling, the alpha nitrogen of the amino acids must be protected with a temporary protecting group. Ideally, this temporary protecting group should be removable under conditions that do not affect the peptide-resin bond. Such a protecting group is referred to as â€œorthogonalâ€. A vast number of different amino protecting groups have been reported, but only two, t-butyloxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), currently have achieved widespread use in solid phase peptide synthesis.
Benzyloxycarbonyl, abbreviated Z or Cbz, has long been utilized in solution phase peptide synthesis, but has little application in solid phase synthesis.
In addition to alpha amine groups, reactive functional groups in amino acid side chains must also be protected to avoid undesired side reactions.
Generally the side chain protecting groups are orthogonal with the alpha amine protection and can be removed under the same conditions utilized to cleave the peptide from the resin. Special protecting groups have been developed for performing on-resin modification of peptides, such as cyclization, labeling or conjugation with lipids or sugars. These protecting groups can be removed without loss of the N-terminal protecting group or cleavage of the peptide from the solid phase support.
Common arginine side chain protecting groups used in Boc chemistry are NO2 and Tos. The NO2 group of Boc-Arg(NO2)-OH is removed during HF cleavage of the peptide from the resin, but it can undergo side reactions during cleavage leading to ornithine residues.1 The NO2 is stable to TFMSA, TMSOTf and HBr/AcOH and is useful for preparing protected peptide fragments for further condensation reactions. If the NO2 group is not removed during cleavage, it can be removed with stannous chloride2 or by hydrogeolysis.3
The Tos protecting group of Boc-Arg(Tos)-OH is also removed during HF cleavage from the resin, but it is not susceptible to side reactions as the NO2 protecting group is prone to. During cleavage the released Tos group can modify trytophan residues. This side reaction can be avoided by adding thioanisole in the cleavage mixture and using the Na-Boc-Nin-formyltryptophan derivative to introduce the tryptophan residues.
In Fmoc chemistry, the common arginine derivatives are Fmoc-Arg(Mtr)-OH, Fmoc-Arg(Pmc)-OH, and Fmoc-Arg(Pbf)-OH. The Mtr group is acid labile and can be removed with TFA/thioanisole.4 When there are multiple arginine residues in the peptide, complete removal of all Mtr groups becomes difficult. The prolonged reaction times or elevated reaction temperatures required can lead to undesired side reactions.5 The Pmc group is more acid labile than the Mtr group and is useful in preparing peptides with multiple arginine residues. The Pbf group is the most labile of these protecting groups and is especially useful in preparing peptides containing many arginine residues. During cleavage, side products can result from these protecting groups reattaching to tryptophan residues, though the Pbf group may be less prone to this side reaction than the other groups. One example reported in the literature showed a 3 hour cleavage and deprotection treatment with TFA resulted 46% of the desired peptide when Arg(Pmc) was used versus 69% when Arg(Pbf) was used.6 These side reactions can be suppressed by using Na-Fmoc-Nin-Boc tryptophan and thioanisole to scavenge the free sulfonyl groups.
Aspartic Acid and Glutamic Acid
In Boc chemistry, Boc-Asp(OBzl)-OH and Boc-Glu(OBzl)-OH are the common aspartic acid and glutamic acid derivatives. These residues can form sideproducts by cyclization, however. N-terminal glutamic acid residues can cyclize to pyroglutamte residues. Aspartic acid residues can cyclize to aminosuccinate moieties that in turn can reopen to produce a mixture of the desired alpha-coupled product and undesired beta-coupled isomer. Aminosuccinate formation is especially prevalent when an aspartic acid reside comes after glycine, serine, or phenylalanine. These undesired cyclizations could be minimized by blocking the side chains as cyclohexyl esters [Boc-Asp(OcHx)-OH and Boc-Glu(OcHx)-OH].
In Fmoc chemistry, these amino acid side chains are typically protected as tert-butyl esters [Fmoc-Asp(OtBu)-OH and Fmoc-Glu(OtBu)-OH]. If the aspartic acid or glutamic acid sidechain needs to be selectively deprotected, as for side chain cyclization of the peptide, 2-phenylisopropyl esters [Fmoc-Asp(O-2-PhiPr)-OH and Fmoc-Glu(O-2-PhiPr)-OH] or allyl esters [Fmoc-Asp(OAll)-OH and Fmoc-Glu(OAll)-OH] may be utilized. 2-phenylisopropyl esters can be removed with 1% TFA/DCM, conditions that do not affect t-butyl based protecting groups.7 Allyl esters are stable to both TFA and piperidine, but can be removed with palladium catalyst.
Aspartamide formation can occur, especially in strongly basic conditions. Utilizing the more sterically hindered 2,4-dimethyl-3-pentyl ester can prevent or minimize aspartamide formation.8 Recently it was reported that adding a small amout of formic acid to the piperidine in the Fmoc removal reagent could suppress aspartimide formation.9 DBU, which is sometimes utilized in Fmoc-deprotection, often promotes aspartimide formation and should not be used if the peptide-resin contains aspartic acid residues.
Asparagine and Glutamine
Asparagine and glutamine can be used in either Boc or Fmoc chemistry without sidechain protection. There is some risk, however, of the amide moieties in the side chains reacting with carbodiimide reagents to form nitriles. This side reaction becomes a problem mostly in preparing long peptides where the asparagine or glutamine residue is repeatedly exposed to coupling reagents. This side reaction is minimized with a protecting group on the amide nitrogen. In Boc chemistry, the xanthyl (Xan) group is commonly used, while in Fmoc chemistry, the trityl (Trt) group is preferred. An added benefit of protecting the asparagine and glutamine sidechains is improved solubility characteristics of the protected asparagine and glutamine derivatives. Fmoc-Asn-OH and Fmoc-Gln-OH have very low solubility even in DMF and NMP. The Fmoc-Asn(Trt)-OH and Fmoc-Gln(Trt)-OH derivatives have solubility in DMF comparable to other Fmoc-protected amino acids. The trityl group normally is easily removed with trifluoroacetic acid (TFA). When an Asn(Trt) residue is the N-terminus of the peptide, removal of the trityl group is slow10 and deprotect time may need to be extended. Incomplete Asn(Trt) deprotection in the vicinity of reduced peptide bonds has also been reported.11
The cysteine sidechain must be protected during synthesis to prevent oxidation to form disulfide bonds. A number of cysteine protecting groups have been developed to allow selective disulfide cyclization between multiple cysteine residues, pre-cleavage cyclization and post-cleavage cyclization. In Boc chemistry, the acetamidomethyl (Acm), tert-butyl (But), benzyl (Bzl), 4-methylbenzyl (4-MeBzl), 4-methoxybenzyl (4-MeOBzl), trityl (Trt) and 9-fluorenylmethyl (Fm) groups are used. The Trt group may be removed with trifluoroacetic acid (TFA) and triisopropylsilane (TIS) and thus is useful in on-resin cyclization methods. The Trt group can also be removed, and at the same time cyclized, with iodine. The Acm group is cleaved by iodine, too, and can be used with Trt-protected cysteines in selective on-resin cyclization strategies. The Acm group is stable under peptide cleavage conditions so it is also used in post-cleavage cyclization schemes. The Acm group may also be cleaved without disulfide formation using mercury (II) acetate. The Fm group likewise is stable to peptide cleavage conditions, but is removed with piperidine. The Fm group is used in selective post-cleavage cyclizations. The tBu group is cleaved during HF cleavage and TMSOTf cleavage, but is only partially removed with 1M trifluoromethansulfonic acid (TFMSA). The Bzl, 4-MeBzl, and 4-MeOBzl groups are removed during HF cleavage. The 4-MeOBzl group is also removed during TFMSA cleavage and the 4-MeBzl group is removed in HBr cleavage conditions.
The major cysteine sidechain protecting groups used in Fmoc chemistry include Acm group, the tert-butyl (tBu) group, the tert-butylthio (t-Buthio) group, 4-MeOBzl group, Trt group and the 4-methoxytrityl (Mmt) group. Like the Trt group, the Mmt group can be cleaved with TFA or iodine. The Mmt group is more acid labile; it can be removed with 1% TFA in dichloromethane/TIS (95:5 v/v). This group is useful in selective on-resin cyclizations and for producing peptides with a free cysteine sidechain.12 The tBu group is stable to iodine oxidation and TFA cleavage conditions. Hence it is useful in selective deprotection and post-cleavage cyclization. The tBu group is removed using TFMSA, mercury (II) acetate, or TFA/dimethylsulfoxide/anisole.13 Recently, trimethylsilylbromide (TMSBr)-thioanisole/TFA was reported to cleave the tBu protecting group.14 The 4-MeBzl group is not removed in TFA cleavage and was used with tBu-protected cysteine residues in an elegant one-pot regioselective synthesis of the disulfide bridges in alpha-conotoxin SI. The t-Buthio group is acid stable but cleaved under reducing conditions, making this group useful for preparing peptides with a free cysteine residue. The t-Buthio group can also undergo exchange with thiols producing new disulfides.
Penicillamine is less widely used; only a few derivatives are available. For Boc chemistry, the thiol moiety in the penicillamine side chain is protected with the 4-MeBzl group [Boc-Pen(pMeBzl)-OH]. In Fmoc chemistry, the Trt group is used to protect the sidechain [Fmoc-Pen(Trt)-OH].
Histidine residues cause two problems in peptide synthesis. The imidazole moiety in the histidine sidechain, if unprotected, can react with activated acid moieties to form acylimidazoles during coupling. This seldom introduces sideproducts, for the acylimidazoles are reactive and the acyl group is removed from the histidine sidechain by the next coupling step. This reaction reduces the amount of activated acid available for coupling, however, so that more equivalents of acid are required to ensure rapid complete coupling. The more serious problem is that histidine is very prone to racemization during coupling and produces mixtures of enantiomeric peptides. The free N-pi in the imidazole moiety of the histidine sidechain catalyzes epimerization of the activated amino acid.
Sidechain acylation is prevented and racemization reduced by protecting the histidine side chain. In Boc chemistry, the commonly used protecting groups are Boc, 2,4-dinitrophenyl (Dnp) [Boc-His(Dnp)-OH], Tos [Boc-His(Tos)-OH] and benzyloxymethyl (Bom) [Boc-His(Bom)-OH]. The sidechain Boc group is removed when the N-terminal Boc group is removed, so Boc-His(Boc)-OH is mainly useful to prepare short peptides or to introduce a histidine residue near the N-terminal of a peptide. The Tos group is removed by HOBt, which is often added in coupling reactions to reduce racemization and is generated as a byproduct in coupling with BOP, HBTU and TSTU. Glycine insertion through Nim-NÎ± transfer on the deprotected histidine residue has been reported.15 Therefore, Boc-His(Tos)-OH is most useful for preparing short peptides or introducing histidine residues near the N-terminus of peptides.
The Dnp group is stable to most reaction and cleavage conditions, thus it is useful in preparing larger peptides. It may also be used to prepare protected peptide fragments for fragment coupling, but in typical peptide synthesis the Dnp group is removed prior to cleaving the peptide from the resin.
The previous protecting groups attach to the pi-nitrogen of the sidechain imidazole and suppress racemization to varying degrees, but racemization remains a problem. The Bom group, which is attached at the tau-nitrogen of the imidazole moiety, is very effective in suppressing racemization. Boc-His(Bom)-OH is more difficult to prepare than the other histidine derivatives and hence is more costly. This is why it is not commonly used in general peptide synthesis. It is invaluble, though, when racemization of histidine residues is a significant problem.
Histidine side chain protecting groups used in Fmoc chemistry include Fmoc and the trityl-based protecting groups, Trt [Fmoc-His(Trt)-OH], Mmt [Fmoc-His(Mmt)-OH], and 4-methyltrityl (Mtt) [Fmoc-His(Mtt)-OH]. The sidechain Fmoc group is removed when the N-terminal Fmoc protecting group is removed, so this histidine derivative is most useful in preparing short peptides and introducing a histidine residue near the N-terminal of a peptide. The trityl protecting groups are all acid labile. The general order of lability is Trt>Mtt>Mmt. The Trt protecting group is typically removed with 90% TFA and it can be used with 2-chlorotrityl resins to prepare protected peptide fragments. The Mtt and Mmt groups are completely removed with 15% TFA. Under the mild acetic acid conditions (1:1:8 acetic acid: trifluoroethanol: dichlormethane) used to cleave peptides from 2-chlorotritylchloride resins, 75% to 80% of the Mmt groups are cleaved within 30 minutes while only 3% to 8% of the Mtt groups are removed.16
The trityl based protecting groups do not prevent racemization during coupling.17 Modified coupling protocols have been developed, however, that minimize the extent of racemization.18
Lysine, Ornithine, 2,3-Diaminopropionic Acid, 2,4-Diaminobutanoic Acid
The sidechain protecting groups used in lysine derivatives need to withstand repeated N-terminal deprotection cycles to prevent branched peptide sideproducts from forming during peptide synthesis. In Boc chemistry, some of the lysine sidechain protecting groups are benzyloxycarbonyl (Z), 2-chlorobenzyloxycarbonyl (2-Cl-Z) and Fmoc. The 2-Cl-Z protected derivative Boc-Lys(2-Cl-Z)-OH is the lysine derivative commonly used in peptide synthesis by Boc chemistry. It is stable in 50% TFA, but is removed under the standard peptide cleavage conditions (e.g. HF, TFMSOTf, TFSMA, HBr/AcOH). The Fmoc group is acid stable and Boc-Lys(Fmoc)-OH is used to prepare protected peptide fragments for fragment coupling. It can also be selectively removed while the peptide is still attached to the resin, allowing selective modification of lysine residues (e.g. biotinylation or fluorescent labeling) on resin.
For Fmoc chemistry, lysine derivatives are available with the following protecting groups: allyloxycarbonyl (Aloc) [Fmoc-Lys(Aloc)-OH], Boc [Fmoc-Lys(Boc)-OH], Mtt [Fmoc-Lys(Mtt)-OH], Dde [Fmoc-Lys(Dde)-OH], ivDde [Fmoc-Lys(ivDde)-OH] and Z [Fmoc-Lys(Z)-OH]. Fmoc-Lys(Boc)-OHis the commonly used lysine derivative. The Boc group is removed when the peptide is cleaved from Wang resin, but is not removed under the milder cleavage conditions used with trityl chloride resins, Sieber resin and PAL resin. The tert-butyl carbonium ion that is generated in Boc deprotection can react with tyrosine and tryptophan residues if scavengers are not added. When this is a problem, the Mtt group can be used. Aloc and Z are stable to the acid conditions used to cleave peptides from Wang and Rink resins, so these groups are very useful for preparing protected peptide fragments on the resins. The Aloc group is removed with a palladium catalyst and a hydrogen donor. Z is removed by hydrogenolysis.
The Dde group is utilized when a lysine residue is to be selectively modified, as in a cyclization or a dye labeling. The Dde group is removed with hydrazine which does not affect t-butyl based protection groups.19 The deprotection byproduct is a strong UV absorption and the deprotection reaction can be monitored photometrically. Recently, it was shown that hydroxylamine hydrochloride/imidazole (1.3:1) in NMP could selectively remove Dde groups in the presence of Fmoc groups.20
Hydrazine will remove Fmoc groups, so the peptide should be protected with an N-terminal Boc group before the Dde group is removed. The N-terminal Boc group can be introduced by removing the N-terminal Fmoc group, then treating the peptide-resin with Boc anhydride or Boc-ON. Alternatively, the final N-terminal residue can be incorporated as an appropriate Boc-protected amino acid.
Problems with Dde migration and premature loss have been reported.21 The more sterically hindered ivDde overcomes most of these problems and may be used in place of Dde.22
Ornithine derivatives use the same sidechain protecting groups as lysine. In Boc chemistry, the Z [Boc-Orn(Z)-OH] and 2-Cl-Z [Boc-Orn(2-Cl-Z)-OH] groups are popular. For Fmoc chemistry, ornithine derivates with Aloc [Fmoc-Orn(Aloc)-OH], Boc [Fmoc-Orn(Boc)-OH] and Dde [Fmoc-Orn(Dde)-OH], are available.
2,3-Diaminoproanoic acid and 2,4-diaminobutanoic acid derivatives are commercially available. In Boc chemistry, Boc-Dap(Fmoc)-OH and Boc-Dab(Fmoc)-OH are available. In Fmoc chemistry, the Boc sidechain protected derivatives of these unusual amino acids are commonly used [Fmoc-Dap(Boc)-OH, Fmoc-Dab(Boc)-OH]. The Dde group can be utilized to protect the side chains of these amino acids, but precautions must be taken to prevent migration of the protecting group to the alpha nitrogen when N-terminal Dap residues [Fmoc-Dap(Dde)-OH] are deprotected for coupling. Fmoc-Dab(Mtt)-OH may be used when selective deprotection of the Dab sidechain is requeired.
Methionine derivatives are usually used in Boc and Fmoc chemistry without sidechain protection [Boc-Met-OH, Fmoc-Met-OH]. Methionine residues can oxidize to the sulfoxide during cleavage, however. The oxidation can be prevented if scavengers such as dimethylsulfide are added to the cleavage mixture. If oxidation does occur, the methionine sulfoxide moieties can be converted back to methionine by a post-cleavage reduction. In Boc chemistry, oxidation may occur during synthesis, too, leading to a mixture of oxidized and reduced peptides. Methionine sulfoxide [Boc-Met(O)-OH] is sometimes used in Boc synthesis. The peptide can be purified before the methionine residues are reduced; assuring that the peptide does not oxidize during isolation and purification.
Methionine can be replaced with the isosteric analog norleucine [Boc-Nle-OH, Fmoc-Nle-OH]. The norleucine side chain has nearly the same size and polarity as the methionine sidechain, but is not subject to oxidation. Peptide analogs in which methionine has been replaced with norleucine generally retain biological activity and are easier to isolate and purify. In addition, replacing the oxidizable methionine residues can increased shelf life of the peptide.
Serine, Threonine, and 4-Hydroxyproline
Serine and threonine can be incorporated into short peptides or the N-terminal of peptides without protection of the sidechains, but these amino acids are normally used with side chain protection. In Boc chemistry, the serine and threonine sidechains are protected most commonly as the benzyl ether [Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH]. In Fmoc chemistry, these amino acids are side chain protected as tert-butyl ethers [Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH] or trityl ethers [Fmoc-Ser(Trt)-OH]. The tert-butyl ether is removed under the conditions for cleaving peptides from Rink amide resin or Wang resin, but are stable under the mild conditions used to cleave peptides from 2-chlorotrityl resins and may be used to prepare protected peptide fragments. The trityl-protected derivatives can be selectively deprotected on resin, which is useful for preparing phosphoserine- and phosphothreonine-containing peptides by global phosphorylation methodology.
Boc and Fmoc hydroxyproline derivatives are available with or without protecting groups [Boc-Hyp-OH, Fmoc-Hyp-OH] on the alcohol moiety. As with serine and threonine, Bzl is the protecting group used in Boc chemistry [Boc-Hyp(Bzl)-OH] whereas the tert-butyl ether is used in Fmoc chemistry [Fmoc-Hyp(tBu)-OH].
In peptide synthesis by Boc and Fmoc chemistry, tryptophan can be used without protecting the indole moiety of the sidechain [Boc-Trp-OH, Fmoc-Trp-OH]. The tryptophan residue can be oxidized or can be modified by cationic species during cleavage, most notably the sulfonyl moieties released from arginine moieties. These problems can be greatly reduced by protecting the indole nitrogen. In Boc chemistry, tryptophan is protected with a formyl group on the indole nitrogen [Boc-Trp(CHO)-OH]. The formyl group is removed during HF cleavage, but it must be removed prior to cleavage with other cleavage reagents. In Fmoc chemistry, the tryptophan side chain is protected with a Boc group [Fmoc-Trp(Boc)-OH]. When used with Fmoc-Arg(Pbf)-OH, sulfonyl modification of tryptophan residues are nearly eliminated.
When the indole-Boc group is cleaved with TFA, the tert-butyl moiety leaves first leaving an indole-carboxy moiety which protects the trytophan sidechain from alkylation. This intermediate subsequently decarboxylates upon further treatment with dilute acetic acid.
In short peptides, tyrosine can be incorporated without side chain protection Boc-Tyr-OH, Fmoc-Tyr-OH]. The unprotected tyrosine sidechain can be acylated in coupling reactions, which could lead to side products and require more activated carboxylic acid to ensure complete coupling. In addition, the unprotected tyrosine side chain can be modified by cationic moieties that are released during deprotection and cleavage steps.
In most peptide synthesis applications, the tyrosine side chain is protected. Derivatives with the side chain protected as the Bzl ether are used in both Boc and Fmoc chemistry [Boc-Tyr(Bzl)-OH, Fmoc-Tyr(Bzl)-OH]. The Bzl group is partially removed by TFA, so this side chain protection is more useful in Fmoc chemistry, although Boc-Tyr(Bzl)-OH is useful for preparing moderate size peptides. Two protecting groups with greater acid stability are very useful in Boc chemistry. 2,6-Dichlorobenzyl (2,6-Cl2Bzl) ether and 2-bromobenzylcarbonate (2-BrZ) [Boc-Tyr(2-Br-Z)-OH] are stable in 50% TFA and are readily removed in HF as the peptide is cleaved from the resin. The 2,6-Cl2Bzl group is also compatible with TMSOTf cleavage. The 2-BrZ group is compatible with TFMSA cleavage and HBr cleavage. The 2,6-Cl2Bzl protecting group may also be used in Fmoc chemistry to produce fully protected peptide fragments on Wang resin for fragment condensation synthesis. The 2-BrZ is removed by piperidine23 hence its use in Fmoc chemistry is limited to preparing small to medium peptides and to introducing a tyrosine residue near the N-terminal of peptides. In Fmoc chemistry, the preferred protecting group for the tyrosine sidechain is the tert-butyl (tBu) ether [Fmoc-Tyr(tBu)-OH]. The tBu group is removed when the peptide is cleaved from Wang resin or Rink amide resin. When used with more acid labile resins such as Seiber amide resin and 2-chlorotrityl resins, Fmoc-Tyr(tBu)-OH may be used to prepare protected peptide fragments. Boc-Tyr(tBu)-OH is useful in both Boc and Fmoc chemistries as a derivative to attach N-terminal tyrosine residues to peptides for 125I labeling.
1Yamashiro, D.; Blake, J.; Li, C. H. J. Am. Chem. Soc. 1972, 94, 2855-2859.
2Hayakawa, T.; Fujiwara, Y.; Noguchi, J. Bull. Chem. Soc. Jpn. 1967, 40, 1205-1208.
3ElAmin, B.; Anantharamaiah, G. M.; Royer, G.; Means, G. E. J. Org. Chem. 1979, 44, 3442-3444; Anwer, M. K.; Spatola, A. F. Synthesis 1980, 929-932.
4Fujino, M.; Wakimasu, M.; Kitada, C. Chem. Pharm. Bull. 1981, 29, 2825-2831.
5Sieber, P. Tetrahedron Lett., 1987, 54, 1637.
6Fields, C. G.; Fields, G. B. Tetrahedron Lett. 1993, 34, 6661-6664.
7Yue, C.; Thierry, J.; Potier, P. Tetrahedron Lett. 1993, 34, 323-326.
8KarlstrÃ¶m, A.H.; UndÃ©n, A.E. Tetrahedron Lett. 1995, 36, 3909-3912.
9Michels, T.; DÃ¶lling, R.; Haberkorn, U,; Mier, W., Org. Lett., 2012, 20, 5218-5221.
10Friede, M; Denery, S; Neimark, J; Kieffer, S; Gausepohl, H; Briand, JP, Pept. Res., 1992, 5, 145-147.
11Quesnel, A; Briand, J-P, J. Pept. Res.,1998, 52, 107-111.
12Barlos, K.; Gatos, D.; Hatzi, O.; Koch, N.; Koutsogianni, S. Int. J. Pept. Protein Res., 1996, 47, 148-153.
13Cuthbertson, A.; Indrevoll, B. Tetrahedron Lett. 2000, 41, 3661-3663.
14Wang, H.; Miao, Z.; Lai, L.; Xu, X. Synthetic Communications 2000, 30, 727-735.
15Kusonoki, M.; Nakagawa, S.; Seo, K.; Hamara, T.; Fukuda, T. Int. J. Pept. Protein Res., 1986, 28, 107.
16Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G.; Tsegenidis, T. Tetrahedron Lett. 1991, 32, 475-478.
17Han, Y.; Albericio, F.; Baranay, G. J. Org. Chem. 1997, 62, 4307-4312.
18Mergler, M.; Dick, F.; Sax, B.; Schwindling, J.; Vorherr, T. J. Pept. Sci., 2001, 7, 502-510.
19Bycroft, B. W.; Chan, W. C.; Chhabra, S. R.; Hone, N. D. J. Chem. Soc., Chem. Commun. 1993, 778-779.
20J.J. Diaz-Mochon, et al., Org. Lett. 2004, 6, 1127.
21Augustyns, K.; Kraas, W.; Jung, G. J. Pept. Res. 1998, 51, 127-133; Srinivasan, A.; et al. in â€œPeptides: Fr