Marco Crisma Fernando Formaggio Alessandro Moretto Claudio Toniolo
Peptide Helices Based on a-Amino Acids
Institute of Biomolecular Chemistry, CNR, Department of Chemistry, University of Padova, 35131 Padova, Italy Received 13 May 2005; accepted 29 July 2005 Published online 25 August 2005 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20357
Abstract: Relevant parameters and stereochemical consequences of helices [a-helix, 310-helix, bbend ribbon spiral, g-helix, 2.05-helix, poly(Pro)n type-I and -II helices, and collagen triple helix] of peptides based on a-amino acids for use as templates in various branches of chemistry are briefly discussed. # 2005 Wiley Periodicals, Inc. Biopolymers 84: 3–12, 2006 This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at
[email protected] Keywords: a-amino acids; amphiphilicity; collagen; helices; intramolecular H-bonds; peptide conformations
INTRODUCTION In this paper we briefly review the most relevant parameters and stereochemical consequences of peptide helices formed exclusively by -amino acid building blocks. For recent review articles specifically devoted to some of these peptide helices as spacers or templates for applications in organic and physical chemistry, see Toniolo et al.1,2 As a significant part of the stabilization energy of peptide helices (at least for most of them) arises from intramolecular C¼ ¼O HN H-bonds, their nomenclature is usually described as nm, where n is the number of amino acid residues per helical turn, and m is the number of atoms involved in the (pseudo)cycle generated by the intramolecular H-bond. The most commonly found 3D-structures in a system of four linked peptide units are the 1/3, 1/4, and 1/5
intramolecularly H-bonded conformations depicted in Scheme 1.3 As they are (pseudo)cyclic forms and have different m values, these structures are also called C7, C10, and C13 conformations, respectively. An alternative nomenclature of common use for these structures is -turn, -turn (this conformation will be discussed in detail by Rotondi and Gierasch in this issue), and -turn, respectively. Helical structures originated from consecutively repeating these intramolecularly H-bonded conformations are called 2.27- (or -) helix, 3.010- (or more simply 310-) helix, and 3.613- (or -) helix, respectively, where the values 2.2, 3.0, and 3.6 correspond to their n parameter. These turns and helices are all characterized by a right-handed screw sense if based on amino acids with the usual L-configuration at the -carbon (as those discussed in this article).
Correspondence to: Claudio Toniolo; e-mail: claudio.toniolo@ unipd.it Biopolymers (Peptide Science), Vol. 84, 3–12 (2006) # 2005 Wiley Periodicals, Inc.
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SCHEME 1
DISCUSSION a-Helix and 310-Helix These two peptide helices will be both discussed in this section as they are strictly correlated. The classical -helix (Figure 1) is the most common regular secondary structure of peptides and proteins and, not surprisingly, the first to be experimentally authenticated (by X-ray diffraction analysis). The other principal long-range helical structure that occurs in peptides (particularly in peptaibols) and in proteins is the 310-helix4,5 (Figure 1). Interestingly, this structure was first proposed by Taylor6 as early as in 1941 (10 years before the -helix) owing to its integer number of amino acids per turn. This ternary helix is more tightly bound and more elongated than the -helix (Table I). The sets of , torsion angles of the - and 310-helices do not differ much, falling within the same region of the Ramachandran map (termed ‘‘helical’’ region). However, as mentioned above, their intramolecular C¼ ¼O H N H-bonding schemes are remarkably distinct, being of the 1/4 type (subtype III or helical -turn3,7,8) in the 310-helix, and of the 1/5 type (helical -bend9) in the -helix. In a long polypeptide chain formed by C-trisubstituted (e.g., protein) -amino acid residues, the 310helix is less stable than the -helix. Indeed, its van der Waals energy is less favorable (it has several close, although not forbidden, short contacts) and the H-bond geometry is not optimal. Thus, for many years it was considered unlikely that long stretches of 310-helix would be observed. However, there is no disallowed region of the , space completely separating these two regularly folded secondary structures. Thus, the -helix may be gradually transformed into a 310-helix (and vice versa) maintaining a nearly helical conformation of the chain throughout. Further, if one of the conformations should turn out to be impossible (say, as a result of side-chain interactions), the main chain may slip into the other conformation. In fact, the 310-helix appears to derive its
importance mainly from its proximity in the conformational energy map to the more stable -helix. Thus, the role of the 310-helix as an important intermediate in the mechanism of folding of -helical proteins may be envisaged.10 In 1988 Barlow and Thornton11 surveyed all helices that were found in 57 of the known globular protein crystal structures and showed that, in agreement with the above observations, 3.4% of the residues are involved in 310-helices (about 10% of the total helical residues). These 310-helices are generally irregular, the majority of them are short (the mean length is 3.3 residues, i.e., one turn of helix), and 24% of them occur as an N- or a C-terminal extension to an helix. More recently, significant improvements in atomic resolution have allowed protein crystallographers to detect a significant number of 310-helical segments, some of them as long as 7–12 residues. Thirty-five years ago Marshall12 used calculations of sterically allowed conformations to show that Aib (-aminoisobutyric acid or C,-dimethylglycine), the prototype of achiral C-tetrasubstituted -amino acids, can promote the onset of helices, due to steric interactions involving the gem-methyl groups linked to the -carbon. Since 1978, by taking advantage of the extremely high crystallinity of peptides rich in C-tetrasubstituted -amino acids, we and others have solved the X-ray diffraction structures of numerous Aib homo- and copeptides forming 310- and helical structures.5,13 In 1991 we carried out a general survey of the 32 310-helices experimentally observed in the high-resolution single crystal X-ray diffraction determinations of peptides, the atomic coordinates of which were available at that time.4 In parallel, a total of 22 -helical, Aib-rich peptides (to the hexadecapeptide level) were also analyzed. A number of conclusions were drawn from these data. The minimal main-chain length required for a peptide heavily based on Aib residues to form an -helix in the crystal state corresponds to seven residues (interestingly, about 13 amino acids are required to induce an Biopolymers (Peptide Science) DOI 10.1002/bip
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FIGURE 1 (a) The 3.613-helix (-helix) and its building block, the helical -turn (or C13-conformation). (b) The 3.010-helix and its building block, the helical (type-III) -turn (or C10-conformation).
helix in the solid state for a peptide with protein residues only). By contrast, there is no critical mainchain length dependence for 310-helix formation, i.e. incipient 310-helices are formed at the lowest possible level (an N-acylated tripeptide). An N- and Cblocked –(Aib–Ala)3– peptide gives a regular 310helix, but an –(Aib–Ala)4– peptide gives a predominant -helix. In peptides of eight or more residues the -helix is preferred over the 310-helix if the percentBiopolymers (Peptide Science) DOI 10.1002/bip
age of Aib residues does not exceed 50%. However, one or two 310-helical residues may be observed at either end of the -helical stretch (the short bits of 310-helix tighten up the ends of the -helix by moving the related peptide groups nearer the axis). The average number of -helical residues in undeca- and longer peptides is seven (2 turns). The average parameters for 310- and -helices obtained from our statistical analysis are listed in Table I. A comparison
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Table I Average Parameters for Peptide Helices Based on a-Amino Acids Parameter (8)b (8)c d n ˚ )e d (A ˚ p (A)f
-Helix
310-Helix
-Helix
2.05-Helix
Poly(Pro)n Ia
Poly(Pro)n IIa
63 42 3.63 1.56 5.67
57 30 3.24 1.94 6.29
70 70 2.20 2.80 6.16
180 180 2.0 3.70 7.40
70 160 3.30g 2.22 7.33
70 145 3.00g 3.12 9.36
In the poly(Pro)n I helix all ! (Ci C0i Niþ1 Ciþ1) torsion angles are cis (08), whereas in poly(Pro)n II they are all trans (1808). The C0i1 Ni Ci C0i torsion angle. c The Ni Ci C0i Niþ1 torsion angle. d Number of amino acid residues per helical turn (positive values refer to right-handed helices, whereas negative values refer to left-handed helices). e Axial translation (per residue). f Pitch or axial traslation (per helical turn). a
b
of the side-chain staggering for a 310- and an -helix, built up with the parameters given in Table I, is shown in Figure 2. Our crystallographic analysis has allowed us to characterize the peptide 310-helix in great detail (at atomic resolution). The number of residues per turn (3.24) is intermediate between those of the theoretical 3.010-helix and the theoretical/experimental (3.613-) helix. In a perfect 310-helix the side chains on successive turns are exactly eclipsed since there is an integer number of residues per turn. However, the experimentally observed, slightly fractional, number of residues per turn does not line up side chains, thereby inducing a slightly staggered, energetically more favorable, disposition. This property may have some implications if a relatively long, amphiphilic, 310-helix needs to be built (Figure 2). On the contrary, the -helix, with its largely fractional number of amino acids per turn, requires two turns (7 residues or a ‘‘heptad’’ repeat) to position two side chains exactly one on top of the other on the same helical face. Distinct hydrophobic/hydrophilic faces, in turn, are of paramount importance for a correct construction of peptide - and 310-helix coiled coils. Finally, it is particular worth mentioning that a fully developed, stable 310-helix in solution requires only about eight C-tetrasubstituted -amino acid (e.g., Aib) residues,14 but this figure is considerably higher (% 20 amino acids) for an -helix based on protein amino acids under the same experimental conditions.
b-Bend Ribbon Spiral In a sequential peptide the alternation of a Pro residue, which disrupts the conventional H-bonding schemes observed in helices (lacking the usual NH donor group), and a helix-forming residue such as
Aib may give rise to a novel helical structure, called the -bend ribbon spiral15 (Figure 3). This structure may be considered a variant of the 310-helix, having approximately the same helical fold of the peptide chain and being stabilized by intramolecular C¼ ¼O HN H-bonds of the -turn type. The complete characterization of this peptide conformation, which may be of relevance in the development of models for peptaibol antibiotics (e.g., zervamicin) and for the numerous (Pro–X)n (with X = Pro) segments found in globular and fibrous proteins, was achieved by X-ray diffraction analyses of terminally blocked (Pro–Aib)n sequential peptides.16 The repeating –Pro–Aib– dipeptide unit shows on the average the sequence of backbone torsion angles (i, ’i and iþ1, ’iþ1) 788, 108 and 548, 408. The mean ˚ , and p ¼ helical parameters are n ¼ 3.43, d ¼ 1.94 A ˚ . Typically, the values for the –Pro–Aib– ! tor6.29 A sion angles deviate significantly (|D!| > 108) from the ideal trans 1808 value. The required energy for this structural change is partially regained by the for-
FIGURE 2 A comparison of the side-chain staggering for (a) a 310-helix and (b) an -helix. The two helices are viewed down the helix axis, with the C–C bonds projecting radially outwards, thus emphasizing the potential amphiphilic properties. Biopolymers (Peptide Science) DOI 10.1002/bip
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FIGURE 3 The -bend ribbon spiral (a variant of the 310-helix) generated by the –(Aib–Pro)n– repeating sequence.
mation of acceptable intramolecular C¼ ¼O HN H-bonds. The preferred conformation of the achiral Aib isostere Hib (-hydroxyisobutyric acid) has also been studied. This residue, although being a stronger promoter of -turn and helix than are protein amino acids, is less efficient than Aib. Interestingly, the (Aib–Hib)n depsipeptide sequence gives a -bend ribbon spiral more regular than that exhibited by the (Pro–Aib)n sequence.17 The Aib–Hib ester bonds were found to be trans.
c-Helix The 1/3 intramolecularly H-bonded peptide conformations (also called C7 forms or -turns)18,19 are ring
FIGURE 4
structures that are centered at a single residue and stabilized by an H-bond between the Hiþ2 and Oi atoms, as shown in Figure 4. The trans amide groups lie in two planes, which make an angle of about 1158. When R in –NH–CHR–CO– is not an H-atom, two different conformers (equatorial and axial) can exist, which are represented on the Ramachandran map by two centrosymmetric points, the coordinates of which (for an L-residue) are % 708, % 708 (for the more stable, equatorial form, called ‘‘inverse turn’’) and % 708, % 708 (for the less stable, axial form, called ‘‘-turn’’) (Table I). While the Hbond is strongly bent, it has a normal H O distance, and it still makes a sizable contribution to the stabilization of the folded structures. Actually, some variation of the values of the peptide bond torsion angle
The 2.27-helix (-helix) and its building block, the ‘‘inverse -turn.’’
Biopolymers (Peptide Science) DOI 10.1002/bip
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FIGURE 5 mation.
The 2.05-helix (fully-extended conformation) and its building block, the C5 confor-
(!) (|D!| % 108) is necessary for the stabilization of the 1/3 intramolecularly H-bonded peptide conformations. However, the small energy of torsional rotation is more than compensated for by the energy of the H-bond. The ‘‘inverse -turn’’ is quite common in cyclic tetra- and pentapeptides, whereas it has been only very rarely observed in linear peptides and in proteins. A series of consecutive -turns generates a - (or 2.27-) helix (Figure 4). This very tightly bound helix has never been experimentally authenticated in any peptide or protein. However, some time ago it was established that peptides rich in the C-tetrasubstituted amino acid 1-aminocyclopropane-1-carboxylic acid have a marked propensity for the ‘‘bridge’’ region of the , map ( % 08).13 Therefore, it is not surprising that quite recently the first unequivocal example of a peptide system with two consecutive turns (incipient 2.27-helix) directed by a cyclopropane residue with two bulky (phenyl) substituents on the same C atom was discovered.20 This so far unique -amino acid with a clear preference for the -turn conformation will be of great help for the construction of this novel 3D-structural motif in peptide templates or spacers.
2.05-Helix (Fully Extended Conformation) The fully extended peptide conformation, or 2.05helix, with ¼ ¼ 1808 (Figure 5 and Table I), was proposed at an early stage in structural studies of proteins. In this 3D-structure H-bonding takes place between the N–H groups of one chain and the C¼ ¼O groups of the chains on either side, thus making a pla-
nar sheet held together by intermolecular H-bonds directed approximately perpendicular to the mainchain axis. Neighboring sheets are then held together by van der Waals forces. In 1951 Pauling and Corey21 investigated the possibility of small contractions of the peptide chains and proposed precise conformations for parallel and antiparallel, pleated, -sheet forms (with % 1408 and % 1408), which better satisfy stereochemical and H-bonding requirements and have main-chain repeat lengths nearer those found experimentally (for the -sheet structures, see the article by Rotondi and Gierasch in this issue). These authors were also able to show that steric hindrance between adjacent main chains prevents the onset of the planar sheet in case the side chain is anything but a hydrogen, that is, it could be formed only by –(Gly)n– homopeptides. The repeating motif of the fully extended peptide conformation is the intramolecularly H-bonded form depicted in Figure 5. The relative disposition of the two dipoles, N–H and C¼ ¼O, is such that there is obviously some interaction between them. Since these four atoms, together with the central C atom, are involved in a pentagonal (pseudo)cyclic structure, this conformation is also called the C5 structure.1–3 The influence of the bulkiness of the lateral substituent can easily be explained by considering the intramolecular nonbonded interactions between the side group R and the preceding C¼ ¼O and following N–H groups, which induce a warping of this nonsymmetric structure. Unequivocal verification of the occurrence of the C5 form has been obtained in the crystal state by X-ray diffraction analyses of a few, favorable compounds, i.e., Gly- and Ala-rich peptides with Biopolymers (Peptide Science) DOI 10.1002/bip
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short side chains. In globular proteins a repeating C5 motif has so far been authenticated only in the X-ray diffraction analysis of the –(Gly)4– sequence of His– tRNA-synthetase.22 Conformational energy computations indicated that the , space explorable by the ‘‘monopeptides’’ from the C-tetrasubstituted, achiral -amino acids with two side chains identical and larger than methyls is severely restricted and the minimum energy conformation corresponds to the C5 structure. This study has demonstrated inter alia the sensitivity of the conformational preferences to the geometry and has unraveled a connection between the narrowing of the NCC0 bond angle (induced by the two bulky substituents at the C atom) and the occurrence of the C5 conformation.3 The highly crystalline nature of peptides composed of these residues was exploited for an extensive X-ray diffraction analysis.13 The C5 conformation is a common observation for these peptides in the crystal state. Interestingly: (i) The C,-diethyl (and C,-di-npropyl) glycine homopeptides represent the first examples in which consecutive C5 forms (2.05-helices) have been experimentally observed. (ii) The N–H and C¼ ¼O groups characterizing this structure are not involved in the intermolecular H-bonding scheme. (iii) The amino acid side chains tend to be fully extended to relieve the unfavorable intramolecular side chain-to-main chain and side chain-to-side chain interactions. (iv) The bond angles show a clear-cut trend: the angles internal to the pentagonal ring tend to be smaller, while those involving atoms of the main chain, external to the ring system, tend to be larger than the corresponding average angles observed in peptides. Interestingly, the critical NCC0 bond angle is narrowed to % 1038. (v) The axial translation ˚ , the longest possible for a sinper residue is % 3.70 A gle amino acid, which makes this conformation extremely attractive for its use as a spacer. However, more recently, theoretical and experimental work clearly shows that in this family of peptides the essential prerequisite for C5 structure formation is not side-chain symmetry (i.e., amino acid achirality), but rather any kind of bulky substitution at both C atoms (i.e., even chiral amino acids can be involved). In conclusion, it seems safe to assume that a sequence of amino acid residues with both side chains longer than a methyl group in a linear peptide would result in a marked stabilization of the 2.05helix. In any case, it is worth pointing out that, in general, this helix is rather fragile in that apparently modest sequence modifications (e.g., incorporation of C-methylated -amino acids) or environmental changes may generate the more robust 310-helix. Biopolymers (Peptide Science) DOI 10.1002/bip
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Finally, we synthesized by solution methods the first homopeptide series based on the C,-didehydro -amino acid DAla to determine the preferred conformation of this residue, characterized by sp2 - and carbon atoms and the smallest side chain.23 Our investigation showed that a multiple, consecutive, fully extended conformation largely predominates for all DAla oligomers investigated. These peptide molecules are essentially flat, including the amino acid side chains, and form completely planar sheets. This novel peptide structure is stabilized by two types of intramolecular H-bonds, Ni–H Oi¼ ¼Ci (typical of the 2.05-helix) and Ciþ1–H Oi¼ ¼Ci (characteristic of DAla peptides). The molecules pack in layers, without any significant contribution from intermolecular N–H O¼ ¼C H-bonds. For this striking flat peptide sheet system we foresee a bright future as a active bridge in physicochemical studies.
Poly(Pro)n Helices and Collagen Triple Helix Amide bonds are usually found in the trans conformation (! ¼ 1808) in linear peptides, whereas cis amide bonds (! ¼ 08) are observed in constrained situations such as those occurring in cyclic peptides, particularly if they are characterized by small ring sizes. A trans amide bond in a secondary amide is energetically more stable than a cis bonding by % 2 kcal/mol, which explains the overwhelming occurrence in linear peptides of the trans bonding in the crystal state and its large preponderance in solution. However, the energy difference between the two conformations markedly decreases in tertiary amides. Thus, not surprisingly, the cis bondings reported in the literature for linear peptides in almost all cases involve a tertiary amide in an –Xxx–Yyy– sequence where Yyy is a Pro or an N-alkylated (Sar, MeAla, peptoid unit, etc.) residue. Pro–Pro bonds generally adopt the trans conformation but, in some instances, particularly when the peptide is short or the sequence is syndiotactic (L-D or 24 D-L), cis bonding does occur. Interestingly, the homopolymer poly(Pro)n is dimorphic in that, under appropriate experimental conditions, the ‘‘all-trans’’ peptide bond conformation (type II) may exhibit a transition (mutarotation) to the ‘‘all-cis’’ peptide bond conformation (type I). Both helices are left-handed (for L-residues) and show quite similar , values (semi-extended conformations) (Figure 6a and b, and Table I). The classical poly(Pro)n type II is a ternary helix, which, with appropriated side-chain replacements (e.g. an –OH function at position 4 of the ring), may be endowed with an amphiphilic character
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FIGURE 6 The left-handed poly(Pro)n type I (a) and type II (b) helices. (c) Side-chain staggering for a poly [Pro–Pro–4(R)–Hyp] type II helix, as viewed down the helix axis to emphasize its amphiphilic character.
(Figure 6c). The transition from poly(Pro)n type I to type II helix implies a remarkable increase in the long dimension of the 3D-structure (Table I). As for the , torsion angles in Pro-based peptides, the former is almost invariant (–70 6 208) owing to the restrictions imposed by the five-membered pyrrolidine ring (Ni $ Ci cyclization). The values accessible to Pro residues either correspond to the semi-extended region mentioned above (trans0 conformation) or to the 310-/-helical region (cis0 conformation). This latter 3D-structure, usually stabilized by 1/4 (or 1/5) intramolecular C¼ ¼O HN Hbonds, cannot be formed by Pro residues only. Pro residues, in fact, can exclusively occur at the first two (three) positions of a 310- (-) helix, respectively, because any following Pro residue would act as a helix breaker in that it lacks the H-bonding donor (NH) fuctionality. The presence of a single Pro in those helices normally induces a ‘‘kink’’ in the structure. In the large majority of published examples, the semi-extended conformation is that preferentially
adopted by Pro, indicating that this residue has an intrinsic propensity to be in the poly(Pro)n structures (and at position iþ1 of a type-II -turn as well). This finding is expecially verified in the longest homooligo(Pro)n and is related to unfavorable steric interactions originating between the -carbon of a Proi residue and the -carbon of a Proi–1 residue if both are folded in an -/310-helical conformation. As for the role of the Pro pyrrolidine ring, no rationale seems to correlate the type of puckering that each residue assumes with its backbone conformation. In other words, ring conformations seem to have little control over , . However, the flexibility of the pyrrolidine ring is expected to expand the range of , values available to the Pro residue, leading to a minimization of unfavorable short-range interactions. In summary, the available evidence strongly supports the view that short oligo(Pro)n spacers and templates cannot be acritically viewed as ‘‘rigid rods,’’ as usually considered, but rather that the cis Ð trans (! Biopolymers (Peptide Science) DOI 10.1002/bip
Peptide Helices Based on a-Amino Acids
torsion angle) and cis0 Ð trans0 ( torsion angle) equilibria, typical of the Pro residue, may severely hamper reliable conclusions from the experimental data. Interestingly, recent calculations have shown that even long (Pro)n peptides may be quite flexible with a defined tendency to fold in a ‘‘worm-like’’ chain characterized by multiple bends.25 Collagen, one of the most important fibrous proteins, is known to be a triple-helical coiled coil in which each of the three strands has a left-handed helical conformation of the poly(Pro)n II type. The three strands wrap parallel and in register around a common helical axis forming a right-handed superstructure and are held together by interchain H-bonds. The close-packed nature of the triple helix strictly requires a Gly residue in every third position. These conditions are achieved by the repetitive ‘‘consensus’’ triplet –(Gly–Pro–Xxx)–n in which Xxx is any amino acid that can accomodate in the semi-extended conformation (including an additional Pro or a Hyp residue).26–28 Due to its elongated nature, no intramolecular C¼ ¼O H-N H-bonds are possible for this structure even if secondary amides do occur in the sequence. A stable collagen-type triple helix requires at least five triplets. Pro residues can be replaced by acyclic N-alkylated analogues.
CONCLUSION Rigid molecular platforms provide well-defined distances and orientations between appropriate probes or functional groups, thus greatly facilitating a reliable and correct interpretation of experimental results based on the 3D-structural dependence of organic and physical chemistry processes. Peptide-based systems of different lengths present a remarkable advantage over other types of derivatized skeletons because they are easily synthetically assembled. Oligopeptide 3D-structures based on C-trisubstituted (e.g. protein) -amino acids of variable length have already been used as templates. However, particularly in the case of relatively short peptides, only a partially restricted mobility has been achieved. The most commonly used oligopeptide series in this context are (Pro)n, followed by (Gly)n, (Ala)n, and -substituted (Glu)n. As mentioned above, extensive investigations of (Pro)n oligomers have clearly shown that different populations of multiple conformers arise from cis Ð trans (! torsion angle) and cis0 Ð trans0 ( torsion angle) equilibria. On the other hand, (Gly)n oligomers are known to fold either in the ternary helix poly(Gly)n I or in the antiparallel -sheet conformation poly(Gly)n II, while (Ala)n and -substituted (Glu)n oligomers may adopt either the -helical Biopolymers (Peptide Science) DOI 10.1002/bip
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or the -sheet conformation. In addition, statistically unordered forms occur largely in the complex conformational equilibria of this type of short oligopeptides, with their population being inversely proportional to the peptide main-chain length. As it is clear that none of the peptide series discussed above can produce truly rigid backbone templates, in the past few years many groups concentrated their efforts on oligopeptide series rich in the structurally severely restricted C-tetrasubstituted amino acids.13 An appropriate choice of specific members of this latter class of amino acids will allow one to tailor well-determined peptide 3D-structures endowed with exactly specified, intramolecular C C distances. However, in the search for rigid templates/spacers, additional problems may arise from rotations about amino acid side-chain single bonds.29 Indeed, most investigations have exploited as probes or reactive pendants: (i) flexible, unmodified, protein amino acids (His, Trp, Tyr, Cys, Met, Glu, Asp, Ser) or (ii) flexible, appropriately side-chain modified, protein amino acids (Cys, Ser, Lys, Glu, Asp, Pro, Phe). Although a limited side-chain flexibility might in general be tolerated, or may even be beneficial, that arising from protein amino acids is definitely too large, making any conclusion quite approximate. In other words, by utilizing this type of side chain, any investigation inevitably suffers a range of uncertainty even larger than that saved from the restrictions imposed by rigidification of the backbone. In conclusion, from the results discussed in the present review article it is quite clear that to further expand this research area (peptides as templates) a larger armamentarium of side-chain and main-chain constrained amino acids is needed.
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Biopolymers (Peptide Science) DOI 10.1002/bip
