24 Peptides Synthesis of cystine-rich peptides - CHIMICA OGGI ...

Peptides 

24 

CYRIL BOULÈGUE 

HANS-JÜRGEN MUSIOL 

VIDYA PRASAD 

LUIS MORODER 

Synthesis of cystine-rich peptides 

Abbreviations used: Acm, acetamidomethyl; Boc, 

tert-butoxycarbonyl; Bzl, benzyl; Bzl(4-Me), 

4-methylbenzyl; DPDS, di(2-pyridyl)disulfide; DMSO, 

dimethylsulfoxide; Fmoc, 9-fluorenylmethoxycarbonyl; 

Mob, 4-methoxybenzyl; Npys, 3-nitro-2-pyridylsulfanyl; 

Pys, 2-pyridylsulfanyl; StBu, tert-butylsulfanyl; sec, 

selenocysteine; tBu, tert-butyl; TFA, trifluoroacetic acid; 

TfOH, trifluoromethanesulfonic acid; Trt, trityl; Xan, 

9H-xanthen-9-yl. 

Since the early days of peptide chemistry the synthesis 

of cysteine-containing peptides has been one of the 

most challenging tasks, primarily because of the 

difficulties involved in the formation of multiple 

regioselective disulfide bonds. To overcome these 

challenges, new protection strategies and selective thiol 

chemistry continue to be developed. Great advances 

have been achieved over the years as demonstrated by 

the numerous highly efficient syntheses of mono- and 

multiple stranded Cys-rich peptides. The state of the art 

Table 1. The most common thiol protecting groups used in peptide synthesis. 

Stabilities and cleavage conditions are taken from similar compilations (1, 4). 

in the field has been extensively reviewed in recent 

years (1-5). The main strategies that have evolved for 

the synthesis of Cys-rich peptides can be classified as 

follows: i) stepwise regioselective Cys pairings; ii) 

convergent strategies based on a combination of 

statistical oxidation of a minor number of Cys residues 

and regioselective disulfide formation; iii) oxidative 

folding of Cys-rich peptides by exploiting the structural 

information encoded in the sequence composition of 

the target peptides; iv) induction of disulfide 

connectivities with selenocysteine (Sec). In this 

minireview we will summarize the achievements to 

date of the research in this developing field, highlight 

new insights that have emerged and address new 

perspectives. It is important to note that the results 

reviewed here are based on prior work and aims to 

summarize achievements of the recent years. Due to 

the breadth of this topic, this review can by no means 

be comprehensive. We intend to draw more general 

criteria and principles as future perspectives from the 

conventional wisdom acquired in the field. 

SYNTHESIS OF LINEAR CYSTEINE- 

PEPTIDE PRECURSORS 

Among the large number of thiol-protecting 

groups commonly used nowadays (Table 1) only 

a restricted set fulfils the requirements of 

orthogonality in terms of the overall protection 

strategy (Fmoc/tBu or Boc/Bzl) and of reciprocal 

selectivity. The acid-sensitivity of many thiolprotecting 

groups and thus incompatibility with 

Boc/Bzl, has made Fmoc/tBu chemistry more 

applicable for larger combinatorial diversity. A 

major drawback is the repetitive piperidine 

treatments which are known to cause serious 

side reactions such as β-elimination and 

racemization. Racemization is particularly 

observed in the case when C-terminal Cys 

residues have to be directly coupled to the resin 

linker (3, 4, 6-12), e.g. relaxins and numerous 

toxins. In order to minimize these side reactions 

the thiol protecting groups Trt and Xan have 

proven to be more advantageous than Acm and 

StBu. An alternative strategy involves anchoring 

of the C-terminal Cys residues to the resin at the 

thiol function via the S-trityl (13) or the S-5-(9Hxanthen-9-yl-2-oxy)valeric 

acid anchor (14) as 

acid-labile linkers. This route provides effective 

access to free cysteine and cystine intermediates 

and/or products where both β-elimination and 

racemization are minimized. The former side 

reaction when occurring at the level of the sidechain 

anchored peptides leads to cleavage of 

the growing peptide chain from the resin and 

consequently removal of the side product in the 

washing steps of the stepwise synthesis. 

chimica oggi • Chemistry Today • Vol 24 nr 4 • July/August 2006

REGIOSELECTIVE DISULFIDE FORMATION 

Intra- and intermolecular multiple disulfide bonds with the 

correct Cys connectivities are generated either by simple 

oxidative refolding after removal of a single-type thiol 

protecting group from synthetic precursors or by 

regioselective methods based on orthogonal thiolprotection 

schemes. The success of the oxidative refolding 

procedures strongly depends on the structural information 

retained by the Cys-rich peptides that in vivo are 

generally derived post-translationally from larger 

precursor forms, and thus on the thermodynamic stability 

of the target peptide. For the synthesis of such naturally 

occurring Cys-rich peptides and proteins, the oxidative 

refolding strategy is most often applied. However, even in 

such cases the correct cystine-network is not always 

formed. Therefore, for the synthesis of these natural 

products as well as for non-natural isomers or for de novo 

designed constructs an efficient chemical control in the 

generation of multiple intra- and intermolecular disulfide 

bonds is required. Due to the disappointingly low yields 

in the early efforts to obtain correctly folded insulin from 

the direct oxidative assembly of its A and B chains (15- 

18), great efforts have been spent in the following 

decades to develop orthogonal thiol protection 

schemes and related cleavage/oxidation chemistries 

needed for the stepwise regioselective formation of 

disulfide bonds in single-, double- and even multiplestranded 

Cys-rich peptides. The advances in these 

rather complex multiple-step procedures have been 

comprehensively reviewed elsewhere (3, 4). 

A first milestone in this regioselective approach was 

achieved with the successful synthesis of the doublestranded 

human insulin where condensation of 

fragments with preformed disulfide bridges was 

combined with regioselective cysteine-pairing 

procedures (19, 20). Subsequently the synthetic 

strategies have been further improved such that 

assembly of insulin and insulin-like peptides (21-23) 

and relaxins (24-26) from the two chains by 

regioselective intra- and interchain disulfide bridging 

methods can be carried out for both the Boc/Bzl and 

Fmoc/tBu synthetic strategies (Scheme 1). 

In the Boc/Bzl synthesis of the A and B chain of 

insulin-4 (placentin) the transient thiol protecting 

group, i.e. Bzl(4-Me), is removed during the resincleavage 

and deprotection step using HF at 0-4 °C, 

i.e. conditions where the Cys(tBu) derivative would 

be expected to be stable. However, partial removal 

of the latter protecting group was observed 

confirming the non-ideal orthogonality between the 

Cys(tBu) and Cys(Bzl(4-Me)) derivatives. The initial 

intrachain disulfide bridging was achieved using 

DPDS in aqueous solution. This was followed by the 

removal of the tBu protecting group with in situ 

activation of the resulting free thiol by treatment with 

TfOH in TFA and in the presence of DPDS as proposed by 

Maruyama et al. (21). Subsequent interchain disulfide 

bridgings were formed by thiolysis under slightly basic 

conditions, followed by I 2-mediated 

deprotection/oxidation of the pair of Cys(Acm) residues. 

Although these basic conditions are not optimal for the 

thiolysis step due to the possibility of scrambling of the 

preformed intrachain disulfide bridge, side reactions of 

this nature were not reported (23). Furthermore neither 

oxidation of the Met and Trp residues in the B chain of 

insulin-4 (both these sensitive residues are absent in 

human insulin) nor cyclization at the indole group via 

thioether formation by intermediately formed Cys 

S-iodide were reportedly observed. Conversely, 

adaptation of the protection scheme reported by Akaji et 

al. for the synthesis of human insulin (22) to the synthesis 

of relaxin-3 by the Fmoc/tBu chemistry met with little 

success (26). Deprotection of the pair of Cys(tBu) 

derivatives and simultaneous disulfide bond formation by 

treatment with MeSiCl 3/PhS(O)Ph in TFA (27) caused 

substantial destruction of the Trp residues present in each 

chain of relaxin. This result is not surprising considering 

the near quantitative chlorination of indole groups under 

such conditions (28). Additionally, the position of the 

Cys(Acm) derivatives relative to the Trp residues in both 

chains was found to be critical in the iodine-mediated 

disulfide formation. Placing this protecting group in the 

proximity of the Trp in the A chain iodolysis led 

predominantly to modification of the indole side chain, 

whereas placing the two Acm groups at C-terminal 

positions, as shown in Scheme 1 (right panel), improved 

the final product yields (26). This observation fully 

confirms previous reports about the decisive role of the 

ring size that favors backbone-to-side chain cyclization 

via 2-thioether formation (29). Correspondingly for 

I 2-mediated Cys(Acm) cleavage and oxidation in the 

presence of Met and particularly Trp residues, careful 

Scheme 1. Synthesis of human insulin-4 (placentin) (left) by the Boc/Bzl (23) 

and relaxin-3 (right) by the Fmoc/tBu chemistry (26). The thiol protection 

strategies were adapted from the previous syntheses of the insulin-like insect 

peptide bombyxin IV (21) and human insulin (22). Regioselective disulfide 

formation was achieved by successive reactions with (i) DPDS in aqueous 

solution (pH 8.5), (ii) TfOH/TFA (1:5)/PhSMe (1 per cent)/DPDS at 0 °C, 

(iii) thiolysis at pH 8.5 at rt, and (iv) I 2/aqueous AcOH. 

optimization of reaction conditions is required (30,31). 

Accordingly, for thiolysis of the Cys(Pys) derivatives in the 

presence of preformed disulfides, slightly acidic 

conditions are preferred over neutral or basic to prevent 

or at least to largely suppress undesired thiol-disulfide 

exchange reactions. 

REGIOSELECTIVE FORMATION OF MULTIPLE 

INTRAMOLECULAR DISULFIDES 

The strategy of judiciously positioning pairs of Cys 

residues such as S-Trt, S-tBu and S-Acm derivatives along 

the peptide chain is currently the most widely applied 

procedure for the stepwise regioselective formation of 


chimica oggi • Chemistry Today • Vol 24 nr 4 • July/August 2006 25


28 

three disulfide bonds in double-stranded and in singlestranded 

peptides (32). Thus, not only the peptide 

sequence itself, but also the distribution of the thiol 

protecting groups was found to strongly affect the success 

and the final yields of the desired disulfide isomers (33, 

34). Similar sequence dependencies were observed (34) 

when applying the DMSO/TFA-mediated 

deprotection/oxidation procedure (35,36) in a 

temperature-dependent mode for a one-pot regioselective 

disulfide formation between pairs of Cys(tBu) and 

Cys(Bzl(4-Me)) residues (37). This method was efficiently 

applied in the synthesis of bacterial enterotoxin ST 5-18 

with three disulfide bonds (38). In this one-pot procedure 

formation of the last disulfide using DMSO in TFA is 

carried out at 

higher 

temperatures 

(60 °C). 

Although such 

conditions may 

be acceptable 

in particular 

cases, there is 

a high 

propensity for 

sensitive 

residues such 

as Met and Trp 

to be oxidized, 

Table 2. Most common combinations of 

Cys protections for multiple regioselective 

stepwise disulfide formation in singlestranded 

Cys-rich peptides. 

given that the 

respective 

byproducts 

were already 

observed at 

room temperature (35). Additionally, cleavage of acid 

sensitive peptide bonds such as Asp-Pro can occur (34) 

and succinimide formation at Asp-Xaa sequences is 

expected to occur under these conditions. An alternative 

one-pot regioselective formation of two disulfide bridges 

is possible due to the significantly different rates of I 2mediated 

oxidation of Cys(Trt) and Cys(Acm) derivatives 

in different solvent systems (39). By using CHCl 3, CH 2Cl 2 

and 2,2,2-trifluoroethanol as solvents the Cys(Trt) 

derivative is cleaved within seconds, whereas for the 

Cys(Acm) derivatives the reaction takes hours to complete. 

The difference in reactivities have been successfully 

applied to the stepwise regioselective formation of two 

disulfides (1, 40, 41). 

Table 2 contains the most common combinations of 

pairwise Cys protections for stepwise regioselective 

disulfide formation in peptides prepared by the Boc/Bzl 

or Fmoc/tBu chemistry. In most cases for the first disulfide 

bond, thiol protections are selected which are removed in 

the resin cleavage/deprotection step. Alternatively, the 

Cys(StBu) protection is used which is cleaved reductively, 

preferentially in solution, to form the first disulfide bond, 

because under such conditions already preformed 

disulfides are also reduced. The procedure most 

commonly applied for the first disulfide formation is 

oxidation by either molecular oxygen in aqueous or 

aqueous/organic media under slightly basic conditions or 

alternatively by DMSO (36, 42). The method using 

DMSO can be applied over a wide pH range of 1 to 8, 

whereby faster oxidation rates are obtained with DMSO 

in acidic media. Since DMSO is known to disrupt 

aggregates, higher DMSO concentrations may 

additionally serve to solubilize peptides. Furthermore, 

modifications at sensitive amino acids like Met, Trp and 

Tyr are not observed by this oxidation method in aqueous 

solutions when operating at room temperature. More 

recently, polymer-bound oxidation agents such as the 

Ellman´s reagent have been proposed to facilitate the 

work-up procedures with the additional advantage of 

allowing oxidation under slightly acidic conditions (43, 

44). In acidic media di(2-pyridyl)disulfide, pH 4 (45) or 

di(4-pyridyl)disulfide, pH 1 (44) are more efficient. The 

latter reagent could possibly serve for intramolecular 

disulfide formation in the presence of an already 

preformed disulfide bond because thiol-disulfide 

exchange reactions are largely suppressed under acidic 

conditions. Therefore, oxidation of free thiols by di(4pyridyl)disulfide 

at pH values in the range of 1 to 4 could 

be advantageous over the DMSO/TFA procedure 

particularly in the presence of Met and Trp residues, 

thereby increasing the diversity of possible combinations 

of thiol-protecting groups. 

To our knowledge, there has been a limited number of 

reports of stepwise regioselective formation of more than 

three disulfide bonds. This is most likely due to the 

observation that with increasing number of disulfide 

bridges, oxidative refolding is found to be sufficiently 

successful as final synthetic step. Nevertheless, at least from 

an academic point of view, stepwise regioselective 

formation of a larger number of disulfides can well be 

envisaged from the knowledge accumulated on orthogonal 

combinations of thiol protecting groups, as shown in 

Scheme 2. Indeed, a similar protection scheme without the 

Cys(StBu) pair was used for the stepwise formation of four 

disulfides in an α-conotoxin dimer by exploiting the 

different rates of Cys(tBu) and Cys(Bzl(4-Me)) oxidation 

with DMSO/TFA at different temperatures (46). 

Extension of the number of disulfides to five would involve 

the critical temperature-dependent cleavage and 

oxidation of the Cys(Bzl(4-Me)) derivatives with 

DMSO/TFA discussed previously. But such an extensively 

disulfide-crosslinked peptide is unlikely to be a synthetic 

target. 

Scheme 2. Potential thiol protection scheme for the 

regioselective formation of up to five intramolecular 

disulfide bonds: (i) reductive cleavage of the 

S-tert-butylsulfanyl groups and oxidation with 

di(2-pyridyl)disulfide or DMSO on resin or in 

aqueous/organic media upon cleavage of the fully 

protected peptide from the resin by mild acid 

conditions; (ii) deprotection of the peptide with TFA 

with concomitant removal of the S-trityl groups 

followed by HPLC purification and oxidation with 

DPDS under acidic conditions; (iii) iodine-mediated 

oxidation preferentially in aqueous AcOH; (iv) 

DMSO/TFA at rt, and (v) DMSO/TFA at 60 °C. 


Scheme 3. Disulfide crosslinking of collagenous peptides into 

heterotrimers by regioselective thiol chemistry. 

REGIOSELECTIVE FORMATION OF MULTIPLE 

INTERCHAIN DISULFIDES 

For homodimerization of a mono-cysteine peptide, the 

methodologies used for intramolecular cyclization of 

peptides by a disulfide bridge can be employed. In the 

case of hetero-dimerization and particularly hetero- 

oligomerization, selective thiol chemistries are required. 

This generally involves thiolysis of an unsymmetric 

disulfide such as the Cys(Pys), Cys(Npys) or the (5-nitro- 

2-pyridyl)sulfanyl derivative with an unprotected Cys thiol 

group of the second strand (Scheme 3). The unsymmetric 

disulfide species are obtained by reaction of an 

unprotected Cys residue of one peptide strand with di(2pyridyl)disulfide 

and di(3-nitro-2-pyridyl)disulfide or by 

the related sulfanylchlorides (Pys-Cl or Npys-Cl) (4). The 

sulfanylchlorides serve also to convert a Cys(Acm) residue 

of one of the disulfide crosslinked strands into a Cys(Pys) 

or Cys(Npys) derivative for a further oligomerization step 

by thiolysis as shown in Scheme 3, whereby these 

reactions have to be performed under acidic conditions to 

prevent thiol-disulfide exchanges at preformed disulfide 

bridges (47). Since the sulfanylchlorides react at high 

rates even with indole groups to form the related 2thioethers, 

such a cascade of reactions for regioselective 

disulfide crossbridging of peptide ladders cannot be 

applied in the presence of Trp residues. A possible 

alternative could be the direct conversion of Cys(Acm) 

residues into Cys(Pys) or Cys(Npys) derivatives by 

reaction with iodine in the presence of di(2pyridyl)disulfide 

or di(3-nitro-2-pyridyl)disulfide, which 

immediately trap the intermediately formed sulfanyl 

iodide (48). Disulfide crosslinking of collagen peptides 

into heterotrimers by artificial cystine-knots has been very 

successful (47, 49, 50), although yields were strongly 

dictated by conformational effects (51, 52). 

Conformational effects cannot be excluded for the failure 

of general strategies when applied to the production of 




30 

Scheme 4. Semiselective formation of three disulfides by a 

two-step procedure based on statistical oxidation of four thiols 

by air oxygen or DMSO in aqueous solution, followed by 

regioselective I 2-mediated oxidation of the two purposely 

placed Cys(Acm) derivatives. 

multiple disulfide bridges in Cys-rich peptides. These 

effects may not occur when disulfide formation is 

performed under non-aqueous conditions, but can be 

more pronounced when carrying out the selective Cys 

pairing reactions in aqueous solutions. In fact, even 

shorter peptide sequences may retain sufficient structural 

information for local conformational preferences in water. 

On the other hand, these locally folded peptides can 

advantageously be exploited for combining oxidative 

folding procedures and regioselective disulfide formation 

(vide infra). 

SEMISELECTIVE DISULFIDE FORMATION 

Despite the orthogonality of the thiol-protecting groups 

and the methods developed for their oxidative 

deprotection allow for regioselective formation of three 

and even more disulfide bridges, such syntheses require 

careful planning because of the multiple sequential 

deprotection/oxidation and purification steps. In 

principle, formation of three disulfide bonds by a 

combination of two different oxidative procedures, e.g. 

air oxidation of four Cys residues followed by one 

regioselective iodine-mediated disulfide formation can 

also be envisaged. However, in practice separation of 

Scheme 5. Two step oxidative refolding of synthetic hirudin 1-65 . 

isomers is inevitable because of the incomplete 

regioselectivity in the two disulfide bridging steps, as 

shown in Scheme 4 (32, 53). The advantage of this 

strategy is the relatively fast access to three well defined 

disulfide isomers instead of the 15 expected from random 

oxidation of six Cys residues. With the knowledge 

derived from families with common Cys-sequence patterns 

and hypothetically common disulfide connectivities, the 

two Cys(Acm) derivatives can be placed at defined 

positions to induce the desired isomers. These products 

can then be compared chromatographically with the 

natural compound. In the event that this material is not 

available, their bioactivities can be used to confirm or 

identify the native cystine frameworks. The procedure may 

also serve to resolve specific biological properties as well 

documented, e.g., in the case of β-defensins (54). 

Given that identical Cys-sequence patterns generally 

lead to identical cystine frameworks even in the case of 

low sequence homology (vide infra), placing the 

unprotected Cys residues at positions for successive 

disulfide crosslinking of the most proximal thiols in the 

sequence and the two Cys(Acm) residues at positions for 

formation of a disulfide that crosses one of the 

preformed consecutive disulfide bridges (Scheme 5), the 

probability of preferential generation of a single isomer 

is significantly enhanced by the quasi-stochastic 

oxidative folding of Cys-rich peptides according to the 

“proximity rule” (55). With the power-law dependence 

on loop length during the fast early oxidation and 

reshuffling steps, the thiols encounter each other with a 

statistical probability determined primarily by the loop 

entropy, albeit possibly modified by local 

conformational biases in the unfolded state. Indeed, 

applying such knowledge-based design to the protection 

scheme, straightforward generation of the desired native 

framework has been achieved (33, 56-58). An example 

of the application potential of this rather simple synthetic 

strategy has been recently reported for the synthesis of 

hirudin (56). Detailed analysis of the oxidative folding 

pathways of hirudin had clearly revealed a sequential 

flow of the unfolded chain through an ensemble of 

equilibrated one-disulfide to ensembles of equilibrated 

two-disulfide intermediates, with no accumulation of 

preferred native or non-native isomers. From these 

ensembles the correct native isomer accumulates either 

directly by formation of the third disulfide bridge or by 

reshuffling under reducing conditions. In this way the 

thermodynamically most favoured native cystine 

framework is obtained in high yields (59-61). 

Accordingly, the exclusive formation of the correct bisdisulfide 

isomer with the sequential Cys 6 -Cys 14 , Cys 22 - 

Cys 39 disulfides by oxidation of the reduced synthetic 

Cys(Acm) 16 ,Cys(Acm) 28 -hirudin 1-65 in the presence of βmercaptoethanol 

(Scheme 5) was particularly surprising, 

albeit in agreement with the “proximity” rule. This 

intermediate was then converted in high yields by I 2oxidation 

of the Cys(Acm) pair into the native cystine 

framework of hirudin by formation of the crossing 

disulfide bridge. This strategy in other cases however, 

proved to be rather unsuccessful as e.g. in ref. (34). 

OXIDATIVE FOLDING 

Anfinsen proposed the concept that an intrinsic folding 

code determines the correct folding of proteins into the 

presumed thermodynamic ground states uniquely 

governed by the amino acid sequence (62). This basic 

concept also applies when folding is coupled to disulfide 

formation, because the native conformation is only 


stabilized, not specified by the disulfide bonds. The 

oxidation of Cys residues to disulfides is a priori 

undirected, rather it requires spatial proximity of these 

residues. While conformational preferences can promote 

contacts and, thus, pairing of Cys residues, the formation 

of a disulfide bond strongly stabilizes the threedimensional 

structure. Folding in vivo is supported by a 

wide variety of molecular chaperons and of folding 

catalysts. These do not determine the final conformation 

of the polypeptide chain, but rather increase the efficiency 

of the folding process by inhibiting off-pathway 

aggregation phenomena, catalyzing reshuffling of 

disulfides and rate-limiting isomerization steps. The 

critical interplay between the coupled processes of folding 

and oxidation becomes most evident in Cys-rich bioactive 

peptides and miniproteins. In fact, synthetic replicates 

often fold preferentially into the native disulfide isomers 

under optimized oxidative conditions, even though the 

reduced forms are mostly unstructured (63). 

An ever growing number of Cys-rich peptides were 

discovered and isolated in recent years from the most 

diverse kingdoms of life including mammalians. These 

peptides include hormones, growth factors, protease 

inhibitors, components of the innate immunity system, 

toxins, antimicrobial agents etc. In the biosynthesis, these 

Cys-rich peptides are produced ribosomally as precursor 

molecules containing N- and C-terminal prosequences 

that can act as intramolecular chaperones during the 

folding process and are cleaved in the post-translational 

maturation process from the prefolded precursors to 

produce single- and in selected cases double-stranded 

disulfide-rich peptides of varying lengths from about 10 

to 60-70 amino acid residues. Moreover, in addition to 

the conformationally restricting disulfide bonds, backbone 

cyclizations have also been observed in plant protease 

inhibitors or plant and mammalian antimicrobial peptides 

(64-66). 

The production of these bioactive Cys-rich peptides by 

recombinant technologies using different host organisms 

is a challenging task, because of major obstacles such as 

low expression rates, high susceptibility towards 

degradation by the host cell proteases and a significant 

toxicity for the host organisms. Hence, chemical synthesis 

still represents the main access route. However, as 

products of post-translational maturation processes these 

peptides may have partially or totally lost the essential 

structural information for correct oxidative refolding. 

Consequently, a drastically decreased efficiency is likely 

for such folding processes given the number of possible 

isomers formed by random oxidation with increasing Cys 

residues. Indeed with four, six or eight Cys residues in 

principle 3, 15, and 105 different intramolecular disulfide 

isomers can be generated. Furthermore, the complexity of 

possible isomers increases enormously when two or more 

polypeptide chains are crosslinked by interchain disulfide 

bridges as in the classical case of the insulin family of 

hormones (67). For these double-stranded polypeptides 

the theoretically expected yield from random oxidation of 

the two chains should therefore be close to zero; 

nevertheless under optimized conditions human insulin is 

obtained in yields of up to 25-30 percent (68, 69). This 

result suggests that the native fold represents the most 

stable among the possible isomers (67). Equally successful 

was the assembly of relaxin-2 from the A and B chain 

(70, 71). On the contrary, this oxidative refolding 

procedure failed completely in the case of relaxin-3 (26) 

or insulin-4 (23), suggesting fine-tuned conformational 

preferences to be responsible for success or failure. 

An increased number of disulfide bridges in relatively 

short polypeptide chains leads to compaction of the 

globular structures with the disulfides mainly buried in the 

nonpolar core. This implies that such mature protein 

fragments should represent even in the precursor 

molecules stable subdomains. Therefore, at least to some 

extent sufficient structural information may be retained in 

their sequence for correct folding, provided appropriate 

experimental conditions are applied in terms of peptide 

concentration to prevent oligomerization, redox reagents, 

pH values, temperature, additives for solubilization or 

detergents as well as aqueous/organic media for both 

solubilization and conformational stabilization (63). 

Due to the varying yields of oxidative refolding among 

families and even within families of these Cys-rich 

peptides, an important role was speculatively assigned to 

the propeptide sequence (72). Indeed, propeptidefacilitated 

oxidative folding has been observed for a 

number of Cys-rich molecules, but there is still debate as 

to whether the prosequences or more importantly 

particular intramolecular and/or intermolecular 

interactions with specialized structural elements promote 

appropriate conformations to extents that support the 

correct oxidative refolding into the native disulfide 

frameworks (72-82). The question about the role of the 

prosequences is particularly pertinent for peptides with 

identical Cys-sequence patterns that fold into identical 

disulfide frameworks despite their marked sequence 

variability (83-85). In the case of the “conotoxin folding 

puzzle”(83) the propeptide portion plays a role in the 

PDI-assisted folding. In addition, particular posttranslational 

modifications such as γ-carboxylation of 

glutamate residues or C-terminal amidation were found to 

affect yields of oxidative folding (84, 86). The 

experimental benefit observed with propeptides in vitro 

may well rely on the intramolecular chaperone-like 

activity of the prosequences in terms of increased 

solubility of folding precursors which suppresses 

aggregation phenomena and thus precipitation. Upon 

oxidative folding, generally an enhanced solubility is 

observed by burial of hydrophobic patches into the core 

of the usually compact globular folds of the Cys-rich 

micro- and miniproteins. 

Although the production of native disulfide isomers in 

acceptable yields often fails, surprisingly an equal 

number of high rates of success are reported. However, 

the intrinsic driving forces encoded in these short 

peptide sequences for the unidirectional folding into 

distinct disulfide frameworks still remain a biochemical 

and structural puzzle. Stabilization of preferred ordered 

structures such as α-helices or β-sheets in structural 

motifs by distinct disulfide bonds may well represent the 

Figure 1. Possible disulfide isomers of peptides containing four Cys residues 

(top) and the preferred isomers formed upon oxidative folding of apamin 

and endothelin-1 (bottom). 



32 

Figure 2. NMR structures 

of apamin and related 

selenocystine analogues: 

A) wild-type Cys 1-11 ,Cys 3-15 - 

apamin; 

B) Sec 1-11 ,Cys 3-15 -apamin with 

the wild-type connectivities 

(globule isomer); 

C) Cys 1-15 ,Sec 3-11 -apamin 

(ribbon isomer) as main 

conformer with trans Pro 6 ; 

D) Sec 1-11 ,Cys 3-15 -apamin 

(ribbon isomer) with cis Pro 6 

(20 percent); E) Sec 1-3 ,Cys 11-15 - 

apamin isomer (bead isomer). 

Figure 3: Primary structure of minicollagen-1 from Hydra consisting of a 

propeptide [1-9], the N-terminal Cys-rich domain [10-32], poly-Pro 

[33-55], Gly-Pro-Pro triplets [60-101], poly-Pro [103-108] and the 

C-terminal Cys-rich domain [108-130]. 

major driving force. Indeed, a clear correlation between Cys-sequence 

patterns, disulfide networks and consequently, overall folds has been 

observed despite the different origins and functions of these Cys-rich 

peptides. This observation is made even in cases of poor sequence 

homology in the non-cysteine residues (87-94). Among these recurrent 

folds the most common structural motifs are the cystine-stabilized αβ, the 

cystine knot (knottin) and the β-hairpin-like motifs. Therefore, from the large 

body of data collected with the Cys-rich peptides the general consensus 

evolved, that identical Cys-sequence patterns correlate with identical 

disulfide connectivities and thus identical structural folds. Consequently, at 

least within superfamilies of Cys-rich peptides with identical Cys-sequence 

patterns it is assumed that the disulfide connectivities are identical and the 

overall folds very similar despite the low degree of sequence homology. 

Unfortunately however, only few investigations were carried out to prove 

experimentally the validity of these assumptions by either determining 

directly the disulfide connectivities of the natural products or by comparison 

with synthetic replicates. Therefore, assignment of disulfide connectivities by 

simple analogy should still be handled with caution as clearly illustrated by 

the two examples discussed below. 

The Cys-Xaa-Cys/Cys-(Xaa) 3-Cys sequence pattern 

Independent of the disulfide connectivities which may be dictated by 

Figure 4. Disulfide connectivities and solution structures of the N- and 

C-terminal Cys-rich domain of minicollagen-1 from Hydra nematocysts. 



34 

additional Cys residues present in the sequence, 

almost all the peptides and proteins that contain the 

Cys-(Xaa) 1-Cys/Cys-(Xaa) 3-Cys pattern, show the 

characteristic cystine-stabilized αβ structural motif, 

except for the cystine-knot of the growth factor 

superfamily that exhibits a four-stranded irregular 

antiparallel β sheet where the ring formed by the two 

disulfide bridges is penetrated by a third disulfide (87). 

The bee venom toxin apamin and the human hormone 

endothelin-1 exhibit the identical Cys pattern Cys- 

(Xaa) 1-Cys-(Xaa) 7-Cys-(Xaa) 3-Cys (Figure 1). 

Considering the possible disulfide connectivities, the 

two peptide backbones can be crosslinked by two 

disulfides in a parallel (ribbon isomer), crossing 

(globule isomer) or in a sequential manner (beads 

isomer). Oxidative refolding of the apamin generates 

quantitatively the native globule isomer (95), while in 

the case of endothelin-1 a mixture is formed of the 

native ribbon and non-native globule isomer at a ratio 

of 3:1 (96). Despite the identical Cys pattern, two 

different disulfide frameworks are preferred which 

nonetheless lead to the identical αβ fold (87). In both 

cases a sequential disulfide pattern (bead isomer) has 

not been observed in the oxidative folding mixtures, a 

fact that fully agrees with the disfavoured ring sizes of 

disulfide-bridged loops with one or three residues 

spacing the cysteines (97). 

In order to disclose the structural factors responsible 

for the distinct oxidative folding behaviour of 

apamin and endothelin, the highly reductive redox 

potential of Sec (98) was exploited to replace 

isosterically at three defined positions one disulfide 

with a diselenide group. Using this procedure, all 

three possible apamin isomers were obtained by 

oxidation of the two residual Cys residues (Figure 2) 

(99, 100). The preferred 3D structure of the Sec 1- 

11 ,Cys 3-15 -apamin analogue as globule isomer fully 

confirms the isosteric character of a diselenide. This 

makes such disulfide replacements particularly suited 

for the synthesis of heavy metal analogues of 

peptides and proteins (101). In addition, it could 

serve to enhance the robustness of disulfide 

frameworks especially because the reduction of 

diselenides by thiols of redox potentials in the range 

of glutathione is difficult to achieve (102). In the 

ribbon isomer the main conformer retains almost the 

identical overall fold as the wild-type apamin isomer 

with the Pro residue of the trans Ala 5 -Pro 6 buried 

together with the hydrophobic diselenide in the core 

of the molecule. A second conformer present in 

solution (20 per cent), however, exhibits an 

endothelin-like structure which is apparently induced 

by the cis Ala 5 -Pro 6 conformation that exposes the 

Pro side chain to the bulk of the solvent. Recently, an 

interesting finding was reported where the 

preference of cis or trans Xaa-Pro bonds strongly 

depends on the size of disulfide loops (103). The 

readiness to form a disulfide loop is known to 

depend on the number of intervening residues m 

between the two cysteines which is more important 

than the type of residues at least up to m = 6, with 

odd values of m being disfavoured and even 

numbers favouring the ring closure (97). In terms of 

cis and trans aminoacyl-Pro conformations the 

opposite rule was observed, with odd m numbers 

favouring in distinct manner the cis conformation 

(103). This may well account for the presence of the 

second conformer of Cys 1-15 ,Sec 3-11 apamin with m 

= 7 for the Sec 3-11 loop, but the resulting less 

compact structure should be thermodynamically less 

favoured than the wild-type globule isomer and thus 

less populated. Finally, the structure of the bead 

isomer clearly confirmed that a transient formation 

of this isomer in the early fast steps of oxidative 

folding according to the proximity rule would induce 

formation of a distorted α-helix which relaxes into 

the α-helix of the wild-type isomer upon thioldisulfide 

exchange reactions involving the 

disfavoured N-terminal Cys-Xaa-Cys loop. Thus, the 

proline residue in apamin, which is absent in 

endothelin-1, may well be the principal cause of 

their different disulfide frameworks, providing an 

exception to the general rules discussed above. 

Cysteine-rich domains of minicollagen-1 

from hydra nematocysts 

A similar decisive role of a proline residue in 

dictating the disulfide connectivities in peptides with 

identical Cys-sequence pattern was recently observed 

for the Cys-rich subdomains of minicollagen-1 from 

Hydra nematocysts. As shown in Figure 3, the Nand 

C-terminal domains of this collagen, which most 

probably derive from gene duplication, are 

characterized by the identical Cys pattern and by 

one conserved proline, while all other constituent 

residues differ in the two domains (104). 

Although the oxidative folding rates of synthetic 

replicates of the two domains in the presence of 

oxidized/reduced glutathione (9:1) and at pH 8.0 

differ significantly, in both cases almost exclusively one 

disulfide isomer was obtained. The disulfide 

connectivities of these two main oxidation products 

were derived unambiguously from their NMR 

structures in solution (105,106). Despite the identical 

Cys-sequence pattern, two different disulfide 

frameworks were determined and correspondingly two 

differing solution structures, as shown in Figure 4. 

Most surprising were the different conformations of the 

Xaa-Pro bond involving the conserved proline residue 

in the two domains. Detailed studies of the folding 

pathways clearly revealed an unidirectional folding 

pathway from the reduced to the correctly folded 

isomer with the trans Xaa-Pro 119 bond for the Cterminal 

domain without the transient formation of 

intermediates (107). Conversely, for the N-terminal 

domain accumulation of a fully oxidized intermediate 

was detected with all six Cys residues disulfide-linked 

in a sequential manner, i.e. with the two disfavoured 

Cys-(Xaa) 3-Cys loops and the even more disfavoured 

C-terminal vicinal Cys-Cys loop. The disulfide 

connectivities of this intermediate, the conversion of the 

trans to cis Ala-Pro 25 bond in the transiently formed 

Cys-Ala-Pro-Val-Cys ring structure (in agreement with 

ref. (103)) and additional information derived from 

related (4R)- and (4S)-FPro analogues allowed to 

unequivocally assign the decisive role in dictating the 

folding pathway to the conserved proline. 

PERSPECTIVES 

From the collective experience gained over the years 

in the synthesis of Cys-rich peptides by using in the 

last step either regioselective thiol chemistries or the 

sequence-encoded structural information to establish 

the desired disulfide connectivities, it can be 

concluded that the main drawback stems from the 

individual behaviour of each peptide which limits 

general procedures to be proposed and applied. 

Nevertheless, the oxidative folding approach remains 


attractive because of its simplicity compared to the 

synthetic strategies for regioselective disulfide bond 

formation. It is certainly indispensable if the number 

of Cys residues exceeds the presently available 

chemistry for site-directed Cys pairings. With careful 

attention paid to experimental conditions, the nativetype 

fold of the peptide can be accomplished; 

however, misfolded disulfide isomers are often 

produced as main products in spite of continuous 

efforts to optimize the reaction conditions. Therefore, 

great attention has to be paid to experimental 

conditions such as pH, buffer, peptide concentration, 

redox reagents, temperature, additives, denaturants 

and organic solvents to generate the desired disulfide 

connectivities in more satisfying yields (63). In this 

context, besides the traditional use of glutathione as 

redox agent, increasingly the cystine/cysteine pair is 

employed and other thiols have been investigated 

such as aromatic thiols (108,109) or dithiols (110). 

Moreover, additives like guanidinium hydrochloride 

at low concentrations or detergents (111) proved to 

be useful for preventing aggregation and thus, 

oligomerization. However, based on the assumption 

that within families or even superfamilies of Cys-rich 

peptides the predominant isomer formed by the 

oxidative folding procedures should represent the 

native isomer, the experimental proof of the Cys 

connectivities has been largely neglected, although 

with the fast advancements in mass-spectrometry 

such analytical problems can be readily resolved. 

Since nature ingeniously applies complex disulfide 

frameworks to stabilize the most diverse spatial 

arrangements of epitopes for efficient cross-talking 

with receptor molecules, it is highly tempting to mimic 

such natural scaffolds for the engineering of 

miniature proteins in which functional sequence 

portions of other bioactive peptides are suitably 

displayed for molecular recognition processes. 

Indeed, the structural robustness of the disulfide 

frameworks of natural Cys-rich families of peptide 

which is well demonstrated by the large diversity of 

the sequence compositions in the non-cysteine 

positions, has already been exploited with success for 

the generation of hybrid molecules (112, 113). Such 

new type of de novo design of bioactive molecules by 

the use of natural disulfide frameworks may possibly 

further advance with the selenocysteine approach. 

This promising strategy is based on replacement of 

Cys pairs involved in the formation of unproductive 

folding intermediates by Sec residues which force 

diselenide formation at critical disulfide positions, 

resulting in the suppression of unproductive 

kinetically trapped intermediates. By such an 

approach not only significantly improved yields of 

the desired disulfide/diselenide frameworks can be 

expected, but also the access to biopharmaceuticals 

of superior stability under biological reducing 

conditions, if all disulfides are replaced by 

diselenides. per-Sec peptides have been obtained by 

recombinant techniques (114-116) as well as by 

synthesis. Using Bzl(4-Me) for the selenol protection 

instead of the Mob group, which was applied in the 

synthesis of apamin analogues (99), and 

correspondingly the Boc chemistry, β-elimination at 

the Sec(Mob) residues was avoided and αselenoconotoxins 

were efficiently synthesized. These 

retained full bioactivities of the wild-type toxin and 

showed the expected high stability under reducing 

biological media where the wild-type toxin was fully 

deactivated (117). 

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CYRIL BOULÈGUE, HANS-JÜRGEN MUSIOL, 

VIDYA PRASAD AND LUIS MORODER* 

*Corresponding author 

Max-Planck-Institut für Biochemie 

Am Klopferspitz 18, 

D-82152 Martinsried, Germany

24 Peptides Synthesis of cystine-rich peptides - CHIMICA OGGI ...

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