Insight into peptide bond formation from 3D-structural chemistry

A large body of valuable methods for –CO–NH– bond formation is currently available. However, poor yields and outcome of undesired chemical and optical side products sometime still plague this fundamental reaction in peptide chemistry to a different extent. Therefore, a further improvement of this admittedly generally favorable situation, through a complete understanding of the details of the mechanisms operative in peptide bond formation, seems required.

In our 1996 review article,¹  we firmly established the extraordinary role played by the information extracted from 3D-structural analyses of C-activated species of protected (blocked) α-amino acids and peptides, in conjunction with kinetic experiments and theoretical investigations, in making this fundamental issue more clear. Among the physico-chemical techniques available to determine the geometry and conformation of the electrophilic reactive groups at amino acid and peptide C-termini, which will interact with an amino nucleophile to generate the novel –CO–NH– functionality, X-ray diffraction of crystalline products by far stands out as the most informative. More specifically, unique details, such as bond lengths, bond angles, torsion angles and non-covalent interactions (in particular, H-bond formation), are typically provided by this technique. However, it is fair to unambiguously state that for many years application of this powerful tool was dramatically restricted by the extremely poor crystallinity of most of the classes of C-reactive derivatives. Luckily enough, in recent years suitable combinations of N^α-protecting (blocking) moieties and specific families of amino acids have permitted peptide structural chemists to almost entirely overcome this serious drawback. In a limited number of C-activated compounds, some correlated data in solution, obtained by use of spectroscopic techniques (in particular, NMR and FT-IR absorption) were also found to be useful.

Because in the last 20 years no review article appeared in the literature on this topic, in this editorial effort we felt appropriate to update information on where we are now and what knowledge we feel is still missing. Although the content of this chapter would arise from an exhaustive search in the literature since 1997, particular emphasis is given to results published in recent years. Interestingly, 3D-structural information, although scarce, originated also from X-ray diffraction studies of C-activated amino acid derivatives in complexes with their protein counterparts.

Here, we review the electronic and steric properties of five classes of C-carboxyl reactive derivatives of α-amino acid and peptides. They are: (a) fluorides, (b) anhydrides, (c) esters, (d) azides, and (e) amides. The contributions of the available data to our understanding of amino acid and peptide reactivity, regiospecificity, and propensity to racemize (or epimerize) are presented. In any case, it is anticipated in particular that more information is urgently expected on the most popular group of additives and reagents currently employed in peptide synthesis, namely that of hydroxylamine-based esters. We are confident that in future investigations greater attention will be paid to this issue. Specifically, this review article will hopefully stimulate organic chemists, peptide specialists, and 3D-structural biochemists to work jointly in this exciting area to validate or disprove our current views.

Acyl fluorides from appropriately N^α-protected α-amino acids exhibit good chemical and optical stabilities.²  Moreover, these C-activated compounds, currently widely exploited in both solution and solid-phase peptide syntheses, are excellent electrophiles, reacting very fast with the amino components. The X-ray diffraction structures of the N^α-protected α-amino acyl fluorides solved in the last 20 years are: (i) Trt-(S)-pGlu-F (Trt, triphenylmethyl; pGlu, pyroglutamic acid),³  (ii) Tos-Aib-F (Tos, para-toluenesulphonyl; Aib, α-aminoisobutyric acid),⁴  and (iii) Tos-MeAib-F (MeAib, N-methyl Aib)⁴  (Scheme 1).

Trt-(S)-pGlu-F was the first acyl fluoride of this type which could be obtained in a suitable crystalline form for X-ray diffraction analysis (Fig. 1).⁵  In this compound, two independent molecules are present in the asymmetric unit, with the two five-membered pyrrolidine rings being folded into slightly different conformations. Moreover, one of the sides of the –CH–C(O)–F moiety is screened by one of the three phenyls of the Trt group. This 3D-structural disposition leaves the other side fully susceptible to the attack of the incoming amino nucleophile in peptide synthesis. Along with the effective electron-withdrawing property and the small size of the fluorine atom, this peculiarity appears to be responsible for the particularly high reactivity of this C-activating functionality.

The typical “propeller-like” conformation of the Trt group⁶  is seen in both independent molecules of Trt-(S)-pGlu-F. This arrangement allows reducing the unfavorable steric interactions among the three phenyl rings in this bulky moiety.

The relevant bond lengths, bond angles, and torsion angles for the three X-ray diffraction structures are given in Table 1. The bond lengths and bond angles agree well among each other. These data indicate that the CO bond length for acyl fluorides is longer than the corresponding one for acyl chlorides¹  and comparable to that for carboxylic esters.⁷  The observed range for the Csp²-F separation in aromatic and olefinic molecules reported in the literature is 1.33–1.36 Å.⁷  Significantly, the bond angle C^α–C′–F at the C′sp² atom is 6–10° narrower than the related OC′–F bond angle. Interestingly, the relative orientation of the N and F atoms in the N–C^α–C′–F moiety differs markedly between those of Trt-(S)-pGlu-F (transoid) and Tos-Aib-F/Tos-MeAib-F (cisoid) (Fig. 1). This latter disposition is consistent with the known propensity of Aib and MeAib to adopt folded/helical conformations, whereas the 3D-structural arrangement of the C-terminal part of the former amino acid derivative, beside minimizing the unfavorable interactions between the C(O)F group and the closest phenyl ring, might be associated with a possibly operative, favorable C^α–H^α⋯F interaction, the H^α⋯F separations being 2.40 Å and 2.46 Å, respectively, in the two independent molecules.

Anhydrides from amino acids are among the most effective intermediate in peptide synthesis. They are easily synthesized, exhibit excellent reactivity, are relatively poorly prone to racemization, and are chemically stable at room temperature over a period of days.^8,9  They can be classified as carboxylic–carboxylic symmetrical anhydrides, mixed anhydrides (either carboxylic–carboxylic or carboxylic–carbonic type), and N-carboxy anhydrides (NCA) (either with free NH group or with substituted NH group). In the last 20 years, X-ray diffraction structures of only carboxylic–carboxylic symmetrical anhydrides and the two types of NCAs have been published (Table 2).^10–19 

The non-coded α-amino acid residues from which they derive are reported in Scheme 2 and four representative examples of their 3D-structures in the crystal state are shown in Fig. 2.

The values of the C′–O–C″ bond angle for the three Ac_nc carboxylic–carboxylic symmetrical anhydrides¹⁰  are significantly greater than the sp³ value in dialkyl ethers (110°).^20,21  The –C′(O)–O–(O)C″ groups are remarkably out-of-planarity. Their overall conformations are of the anti,anti type a (Fig. 3). Interestingly, the anhydride moiety of the Ac₉c residue is extremely distorted. Indeed, this is the only one among these three compounds in which the molecules do not possess a C₂ symmetry with the anhydride C–O–C oxygen atom lying on the twofold axis. In any case, for all of them the global conformation is mainly dictated by the intramolecular steric repulsion between the two CO oxygens. All three symmetrical anhydrides from the C^α-tetrasubstituted α-amino acids Ac_nc with C^α⋯C^α side-chain to side-chain cyclization (see also ref. 1 for the anhydride from the n=6 residue) exhibit intramolecular (urethane) N–H⋯π (phenyl) interactions where the aromatic moiety is provided by the benzyloxycarbonyl N^α-protecting group (Fig. 2a).¹⁰  These H-bonds occur between the N–H group of one half of the molecule and the phenyl of the other half. Two such intramolecular interactions are seen in the anhydrides from Ac₇c and Ac₈c, but only one in the anhydride from Ac₉c. The non-symmetric disposition of this latter anhydride is associated with an unusual conformation for one of the two (urethane) C–O–CH₂–C (phenyl) moieties. In any case, these three X-ray diffraction structures have permitted us to define the parameters of that relatively new intramolecularly H-bonded conformation very precisely (at the level of atomic resolution).

In the orange-colored anhydride (Boc-NH-Fc-CO)₂O,¹¹  the N–H⋯OC intramolecular H-bond (Fig. 2b) generates a large ring structure containing the two Fc moieties (Scheme 2b) and the intervening anhydride group. The ring motif twists markedly the anhydride group and forces the Fc moieties to an almost perpendicular relative disposition. According to the IR absorption and NMR data, the intramolecular H-bonded ring structure does also occur in solution, albeit to some degree.

To synthesize homo-poly-α-amino acids, by far the most widely accepted approach relies on NCA (also termed Leuchs' anhydride or 1,3-oxazolidine-2,5-dione) polymerization, originally published at the beginning of last century.²²  For a good control of the polymerization process, the chemical purity of the NCA starting material is essential.

After 1996, the 3D-structures of six additional NCAs with free NH group were investigated by Kanazawa and coworkers^12–17  using X-ray diffraction (Table 2), namely those derived from the l-enantiomer (Fig. 2c)¹³  and the racemate¹²  of Phe, and from the l-enantiomer of Ile,¹⁴  Asp(OBzl),¹⁵  Glu(OMe),¹⁶  and Glu(OEt).¹⁷  A difference, albeit modest, is reported between the O–C′ and O–C″ bond distances. Conversely, the N–C(sp²) bond is markedly shorter (by about 0.10–0.12 Å) than the N–C(sp³) bond, possibly because of the resonance effect between the CO group and the N lone-pair electrons. Interestingly, in l-Asp(OBzl) NCA an intramolecular N–H⋯OC H-bond is seen, which involves the Asp β-carbonyl ester oxygen, generating a six-membered pseudo-ring motif.¹⁵  In all cases, an intermolecular N–H⋯OC H-bond is observed, the acceptor being a carbonyl oxygen of the ring system or the γ-carbonyl oxygen of the Glu(OMe)/Glu(OEt) side chain. The conformation of all –C′(O)–O–(O)C″ NCA functions is forced to be syn,syn type c (Fig. 3) by cyclization.

From previous studies on NCAs,¹  it was realized that the following conditions are favorable for polymerization: (a) Because of the sandwiched layer structure in the packing modes, polymerization should proceed within one sandwich and not destroy the molecular arrangement in the neighboring sandwiches. (b) Within a sandwich, the amino acid side chains should not hinder the NCA rings from approaching each other. (c) The side chain should be able to move easily and should not entwine with side chains of the neighboring molecules. Moreover, relatively loose contacts should occur between them. (d) In the process of polymerization, the crystals should be easily cloven between sandwiches, leaving a gap between them, with the consequence to allow CO₂ to go out through the gap without deteriorating the alignment of the NCA molecules. It may be concluded that: (i) intermolecular N–H⋯OC (closer to N) H-bond is more favorable for polymerization (taking into account the charge distribution of the NCA ring and facility of ring opening), and (ii) the sandwich structure accelerates the reactivity in the crystalline state.

In the crystals of d,l-Phe NCA,¹²  the two enantiomers occur as crystallographically independent molecules in a non-centrosymmetric space group. For each enantiomer, the N–H group of the five-membered ring forms intermolecular H-bonds with the OC (closer to N) along the a axis and the OC (closer to C^α) along the b axis of symmetry-related molecules of the same configuration. This 2D H-bonding network gives rise to layers parallel to the ab plane. Layers made of L molecules alternate with those made of D molecules along the c axis, generating a sandwich superstructure. This is the only NCA molecule investigated so far by X-ray diffraction which shows a 2D H-bonding network that forms layers. All other NCA crystals studied exhibit either a ribbon-type or a dimer-type H-bonding motif. Another feature of this crystal favorable to the solid-state polymerization is that the Phe side-chain benzyl aromatic groups are packed in a layer and the five-membered NCA rings packed in another layer (the two layers are aligned alternately).

In l-Phe NCA,¹³  the acceptor of the single intermolecular H-bond is the OC closer to N. The H-bonds are formed along the c axis. The benzene rings are almost parallel to each other and the five-membered NCA rings are packed in another layer. Here too, the layers are aligned alternately. Thus, the sandwiched five-membered rings are expected to react easily with one another within the layer along the c direction. In l-Ile NCA,¹⁴  again the molecules are linked via N–H⋯OC (closer to N) H-bonds, forming a tape structure along the a axis. The molecular arrangement along this axis seems to be preferable for formation of a polypeptide in the β-sheet structure, as in the case of poly-(l-Ile)_n. In l-Asp(OBzl) NCA,¹⁵  the overall packing mode is similar to that of l-Phe NCA.¹³  However, in the former compound the side-chain –CH₂–CO–O–CH₂–C₆H₅ benzyl ester group replaces the Phe –CH₂–C₆H₅ benzyl group in the aromatic layer. In the two NCAs from l-Glu γ-esters,^16,17  the potential intramolecular N–H⋯OC H-bond involving the side-chain carbonyl oxygen (which would have formed a seven-membered pseudo-ring motif) is not observed. An intermolecular N–H⋯OC H-bond is instead present, with the Glu(OMe) side-chain carbonyl oxygen as acceptor, forming a tape structure along the a axis. The tapes are linked by C–H⋯O interactions into a sheet parallel to the ac plane. The tapes are also stacked along the b axis with short C⋯O contacts between the five-membered rings, so that the NCA moieties are arranged in a layer parallel to the ab plane. The molecules of the Glu(OEt) NCA form a tape structure along the c axis. The NCA rings of adjacent tapes are arranged into a layer parallel to the ac plane. Detailed analyses of the kinetics and mechanism of solid-state amino acid NCA polymerization were published by Kanazawa, including a comparison with the same reaction in solution.^23,24 

The results of two X-ray diffraction studies on NCA molecules with substituted NH groups were reported.^18,19  The chemical stability of the H(PEt₃)₂Pt-l-Tle NCA addition product is promoted by the sterically bulky Tle side chain¹⁸  (Scheme 2c). The geometry around Pt is square-planar with the two phosphine ligands distorted toward the small hydride ligand. The Pt–N distance (2.15 Å) is typical for Pt–N single bonds and the NCA geometric parameters are only slightly distorted from those of NCA with a free NH group. The NCA IR absorption CO bands are significantly shifted to lower wavenumbers (by ca. 65 cm⁻¹) upon complex formation, indicating an increase in electron density in the anhydride functionality. When reacted with excess of l-Glu(OBzl) NCA in solution, the homo-polymer poly-[l-Glu(OBzl)]_n is formed in good yield. It was shown that the l-Tle NCA part of the complex is the one responsible for initiation of the polymerization, thus confirming that metallated NCAs (“activated monomers”) are indeed active initiating species in the NCA polymerization.

Silaproline (Sip) is a silicon-containing analog of Pro, exhibiting similar conformational properties (Scheme 2d).¹⁹  The presence of the 4,4-dimethylsilyl group confers to Sip a lipophilicity higher than that of Pro. The yield in the spontaneous cyclization from the N-carbamoyl intermediate producing Sip was higher than that affording Pro. It was concluded that this reaction is facilitated by the less constrained Sip five-membered ring (Fig. 2d). Preliminary results were also reported in a comparison of the polymerization of d-Sip NCA and l-Pro NCA to afford the corresponding homo-poly-α-amino acids, poly-(d-Sip)_n and poly-(l-Pro)_n, respectively, with an all-amide trans, semi-extended conformation, termed type-II poly-(l-Pro)_n, abbreviated as PPII, by the structural biochemists.

Linear carboxylic esters from ortho-nitrophenol (–ONP_o) (Scheme 3) of N^α-protected α-amino acids were first used in peptide synthesis 60 years ago by Bodansky.²⁵  The increase of electron-withdrawing properties of the aromatic nucleus by the strong effect of the ortho-nitro group^26–30  made these “active” esters useful intermediates in peptide bond formation. The high aminolysis rate of these compounds was explained by the effect of the ortho-nitro group being transferred to the ester carbonyl not only by resonance but also through a σ bond.²⁶  The relatively low sensitivity of these esters to solvent effects, their steric hindrance, high values of specific rotation ([α]_D), and particularly good reactivity in solid-state peptide synthesis were satisfactorily explained in terms of restricted rotation about bonds near the chiral α-carbon atom.³⁰  It was proposed that an intramolecular interaction of the nitro group would be responsible for the rigid geometry of the ortho-nitrophenyl esters. Having discarded the possibility of the presence of an intramolecular H-bond involving the protected α-NH function as the donor, initially suggested (Fig. 4a),³⁰  a dipole⋯dipole interaction between the nitro group and the ester moiety (Fig. 4b) was invoked as the stabilizing force.

This important conclusion was achieved by two crystallographic analyses.^31,32  In particular, the more recently published structure, Tos-Aib-ONP_o³²  (Table 3), confirmed the remarkable conformation of the ortho-nitrophenyl group with respect to the remainder of the molecule. The ONP_o phenyl group is almost perpendicular [77.9(2)°] to the plane of the adjacent ester moiety. One oxygen atom of the nitro group is wedged between the two ester oxygen atoms. The result of this insertion is that the potential rotation about the (phenyl)C–O bond is severely restricted. The nitro group is 11° out of the plane of the adjacent phenyl. The ester group is planar. The CO bond length, 1.199(3) Å, is typical for carboxylic esters, but the (phenyl) C–O bond, 1.399(3) Å, is markedly shorter than the average value found in alkyl esters^7,33  as a consequence of the conjugation of the nitroaromatic π system with the lone pair electrons of the ester oxygen atom. To summarize, the authors^31,32  attributed the proximity of the nitro group to the ester moiety, and the related rigid bending [the OC–O–C(phenyl) torsion angle is close to cis] of the latter, to coulombic attraction between the partially positively charged carbonyl carbon atom and the partially negatively charged oxygen atom of the nitro group (Fig. 4b). This explanation is based on the generally accepted electron distribution in “active” esters. The unique geometry and conformation of these –ONP_o esters provide a satisfactory motivation for their special chemical and spectroscopic properties. Because of the bulky and rigidly bent ester and the rotation around the nearby bond C^α–C′ (carbonyl) being at least partially frozen, few rotamers are expected for an –ONP_o moiety and, accordingly, a high optical rotation is reasonably associated with it. Moreover, the intramolecular involvement of the nitro group drastically reduces the interactions with solvent molecules and, as a result, limits the solvent effect on chemical reactivity. Finally: (i) these molecules, with their small solvated shell, can easily diffuse in a polymer 3D-matrix and perform well in solid-phase synthesis, and (ii) in spite of the crowded area occupied by the nitro and CO groups, one side of the latter is not encumbered and, consequently, it is fully exposed to the attack of the incoming amino nucleophile during peptide synthesis.

The uncommon color (deep orange) of the Z-l-Trp-ONP_o (Table 3) crystals, first noted by Bodansky and coworkers,^29,30  prompted us to investigate this ester by X-ray diffraction³⁴  (Fig. 5a). The geometry and conformation of the –ONP_o ester are remarkably close to those of the two esters of this type published previously.^31,32  The nitro group is on the same side of the backbone as the Trp side chain. The normal to the plane of the aromatic –ONP_o group is almost perpendicular to that of the aromatic Trp indole moiety. In the crystal packing, the Z-l-Trp-ONP_o molecules are held together by two types of intermolecular H-bonds, one of them involving the Trp indole NH as donor and one of the two –NO₂ oxygens as acceptor (Fig. 5b), linking molecules related through the twofold screw axis along the a direction. Interestingly, three short intermolecular O⋯N and O⋯C distances were observed between the two oxygens of the –ONP_o group and the nitrogen and two carbons of different, symmetry-related indole moieties. To summarize, our crystal-state analysis did not unravel any specific role for potentially operative intramolecular indole⋯nitroaromatic interactions. On the other hand, intermolecular interactions (including an H-bond) involving these two aromatic moieties were detected. In our view, an intermolecular charge-transfer complex is the most probable responsible for the onset of the observed orange color. The planarity and relatively limited bulkiness of the benzyl group of the Z-protection does not appear to hamper the indole⋯nitroaromatic interactions. A spectroscopic study in toluene solution revealed the presence of the charge-transfer band in the visible region (near 500 nm), but only at high concentrations.³⁴  This band is absent in other solvents and in other esters as well where either the Z-protecting group or the Trp residue has been replaced. Conversely, it does still occur in the case of the racemate, Z-d,l-Trp-ONP_o. A correct spatial separation between the donor ad acceptor groups in the charge-transfer complex is also an essential prerequisite, since the 500 nm band is not seen in the spectra of the di- and tripeptides Z-l-Trp-(Aib)_1,2-ONP_o.

Within the large body of N^α-protected α-amino acid oxylamino derivatives described in the peptide literature, one of the most extensively used method for activation of the α-carboxylic function involves formation of N-oxysuccinimido (–OSu) derivatives (Scheme 3).³⁵  The main advantages of this C-activation method are: (i) the water-soluble by-product 1-hydroxysuccinimide can be easily removed by extraction, and (ii) it allows peptide bond formation with almost no racemization (epimerization).³⁶  Since 1996, only one X-ray diffraction structure of an –OSu linear carboxylic ester was published (Tos-Aib-OSu)³⁷  (Table 3). The CO bond length is close to that typical for carboxylic esters.⁷  The penta-atomic succinimido ring is slightly puckered towards the ⁴T₃ (twist) disposition. The length of the characteristic O–N bond is 1.391(2) Å. The internal bond angles of the succinimido moiety have values close to 105°, as expected for a pentagonal ring, with the exception of the wider bond angle at nitrogen, 116.4(2)°. The exocyclic bond angles involving the two carbonyl oxygen atoms are remarkably expanded [in the range 124–131(2)°]. The dihedral angle between the average –C(O)–O– ester and succinimido planes is nearly orthogonal (Fig. 6), presumably to reduce potential lone-pair repulsions between the ester carbonyl oxygen and the two succinimido carbonyl oxygens. These geometric and conformational features reflect pretty well those previously reported for Boc-L-Val-OSu.³⁸  Five other X-ray diffraction structures were recently reported for –OSu esters^39,40  (Table 3). However, none of them is a carboxylic ester but rather they are sulfonyl esters, since the amino acid unit between the Tos (or the related Nas) protection and the –OSu (or ring mono- or bis-substituted –OSu) moiety is missing in these molecules.

The hydroxylamine-based, racemization-suppressive additives for peptide synthesis currently very extensively used are HOBt,^41,42  HOAt,^43,44  and HOOBt^45–48  (Scheme 4). An additional, interesting compound pertaining to a related class is 2-MBT^49,50  (Scheme 4), capable of avoiding a series of disturbing side reactions which occur with more reactive agents. The multifunctional reactivity of these additives may afford either O(S)– or N-derivatives (for HOBt, see Fig. 7II). Making this issue clear would allow us to obtain a deeper understanding of the role played by these compounds in peptide bond formation and racemization suppression.

The X-ray diffraction structures of the fifteen acylated HOBt, HOAt, HOOBt, and 2-MBT derivatives solved in the last 20 years^50–57  are listed in Table 3. Seven of them are linear –OBt esters, three are –OAt esters, one is an –OOBt ester, three are regioisomeric –(N)OBt amides or urethanes, and one is a 2-MBT thiolester. Four-OBt and one 2-MBT derivatives lack the aminoacyl moiety between the N-protecting (blocking) group and the C-activating functionality. Figs. 8 and 9 show six selected X-ray diffraction structures.

Common characteristics of the ten ester derivatives (see for example, Figs. 8a and 9b) are as follows: (i) The benzo(pyridino)triazolyl and benzotriazinyl systems are essentially flat. (ii) The ester group is trans. (iii) The plane of the ester group is almost perpendicular to that of the bicyclic aromatic moiety. (iv) The N–O distance is long (1.36–1.40 Å), close to that of an N–O single bond. (v) The (O)N–N(central) bond is remarkably longer than the (central)N–N(C) bond. (vi) The two external bond angles at the (O)N atom differ largely, the narrower always being the O–N–N(central). (vii) Among the bond angles internal to the penta-atomic ring of the triazolyl group, the bond angle at the (O)N atom is consistently larger than the normal pentagonal value.⁷  (viii) The relative positioning of the ester and benzotriazolyl planes, in correlation with the planarity at the (O)N atom, does not allow in any case the approach of the H-atom of the potentially chiral aminoacyl α-carbon to the (O)N atom. Moreover, the C^α H-atom, if present, would be located at a distance >4 Å from the basic (central)N atom. Therefore, the racemization observed in chiral aminoacyl –OBt esters, although in a limited number of cases, does not take place via formation of an H-bond between these two atoms. Most of these properties are typical for acyl derivatives of the 1-hydroxy tautomeric forms (Fig. 7IIa).

In the three –OAt ester derivatives investigated (Fig. 8b),^51,55  the internal bond angle at the pyridine N atom is markedly narrowed, while the external bond angle (pyridine)N–C–N(triazole), involving nitrogen atoms of two different rings, is significantly expanded. In the single –OOBt ester derivative studied (Fig. 9a),⁵¹  the O–N–C–O torsion angle is close to 0°, reflecting the cis disposition of these two oxygen atoms. The (central) N–(O)N–C bond angle, internal to the six-membered triazinyl ring, is markedly larger than 120°.

In the three regioisomeric –(N)OBt derivatives (see for example Fig. 9c),^51,52,54  the bond lengths are consistent with the 1-oxide cationic form (Fig. 7 IIc). In particular, the N–O bond length is significantly shorter than in the ester derivatives, and the (O)N–N(central) bond is shorter than the (central)N–N(C) bond. The O–N–N bond angle is remarkably more compressed than the O–N–C bond angle. Among the bond angles internal to the penta-atomic ring, the one at the (central)N is very small, whereas the bond angle at the (O)N is markedly expanded. The C–N–C bond angle, external to the triazole ring, is very large. Finally: (i) The OC–N–N torsion angle is trans. (ii) The –C(O)–N benzotriazolyl moiety adopts an overall planar disposition, which is in sharp contrast to the 3D-structure of the acyl ester derivatives discussed above where the ester function is perpendicular to the benzotriazolyl plane. This latter disposition appears to make the carbonyl Csp² atom more accessible to external nucleophiles and might be responsible, at least in part, for the much faster rate of reaction of esters as compared to that of the amide forms.⁵⁸  (iii) In the only chiral –(N)OBt derivative examined,⁵²  the proximity of the basic oxygen atom O(N) on the triazole ring and the H-atom of the l-Glu(OMe) chiral α-carbon is precluded. However, this H-atom is positioned close to the (central)N basic atom of the same ring. In this amide, the extent of racemization via direct intramolecular H-abstraction seems therefore to depend not only on pure electronic consideration but also on steric factors that force the (aminoacyl) H^α⋯N(central) proximity.

Intriguing observations of intramolecular H-bonding were reported for the six acylated –OBt, –N(O)Bt, and –OAt derivatives based on the Fc ω-amino acid.^53–57  In Ac-NH-Fc-CO-OBt,⁵³  the triazole and ester planes are almost perpendicular, as commonly observed in –OBt esters, but they point to opposite directions. This orientation allows for an intramolecular H-bond from the acetamido NH to the (central)N of the triazole group. In Boc-NH-Fc-CO-OAt,⁵⁵  the benzotriazole moiety is oriented in a fashion which encourages intramolecular H-bond formation between the –OAt 7-pyridyl nitrogen and Boc-NH urethane hydrogen atoms, generating a 9-membered ring. In the dark red crystalline Fc-CO-OBt,⁵⁶  stable in the air and in common organic solvents, the benzotriazole moiety is remarkably rotated out of the ester plane, thus not allowing an efficient interaction with the π-system of the cyclopentadiene ring. A similar finding was reported for the related ester MeOCO-Fc-CO-OBt.⁵⁷  The C–O single bond of Fc-CO-OBt is longer than those of other Fc-COOR esters.This crystallographic result fits nicely with the known easy amidation of esters of this type in the presence of amines. Being a long ester bond, it is per se weak and hence it will rapidly react with the nucleophile to afford the amide product. The two 3D-structures of Boc-NH-Fc-CO-OBt and Boc-NH-Fc-CO-(N)OBt⁵⁴  are interesting examples of regioisomerism in this family of compounds (Fig. 9b and c). The ester isomer exhibits geometric and conformational properties similar to those discussed above for the related Fc-CO-OBt ester, while the features of the amide isomer reflect closely those of the related –(N)OBt derivatives mentioned earlier in the text. In contrast to the intramolecular H-bond present in the crystal of Ac-NH-Fc-CO-OBt (where the donor is the acetamido NH proton), the Boc-NH-Fc-CO-OBt analog does not display any intramolecular bond (where the donor should have been the Boc-urethane NH proton). However, in chloroform solution an NMR analysis highlighted the occurrence of an intramolecular H-bond in the latter compound. Interestingly, at variance with the amide regioisomers, the ester regioisomer was shown to react with N^α-deprotected α-amino acid and peptide esters.

Although the 2-MBT active species would be usually described as a mixture of S- and N-regioisomers, the crystal structure of its Fmoc-protected derivative unambiguously shows it is solely consistent of the S-regioisomer (Scheme 4 and Fig. 9d).⁵⁰ 

In 1996, when we published the first review article on this same topic,¹  only four X-ray diffraction structures of underivatized additives or reagents in peptide synthesis had been already published. They are: (i and ii). The two prototropic tautomers of HOBt (Scheme 4, Fig. 7I), (a rare case of desmotropy) which crystallize from solvent mixtures of different polarities.^59,60  (iii) and (iv) N-HBTU (benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium) and the related N-HATU (7-aza-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium), both of which occurring in the guanidinium N-oxide tautomer in the crystalline state.⁶¹ 

In the last 20 years, the list of X-ray diffraction structures of this class of compounds was greatly expanded:

• (A) HOAT and HOOBt (both shown in Scheme 4) in the 1-hydroxy form.⁵¹ 

• (B) HBPyU, 1-(1-pyrrolinidyl-1H-1,2,3-benzotriazol-1-ylmethylene)-pyrrolidinium hexafluorophosphate N-oxide, and HAPyU, 1-(1-pyrrolinidyl-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene) pyrrolidinium hexafluorophosphate N-oxide, in the guanidinium form.^62,63 

• (C) HOCt, ethyl-1-hydroxy-1H-1,2,3-triazolo-4-carboxylate, and its 5-methyl derivative (MeHOCt), in the 1-hydroxy form.⁶⁴ 

• (D) NBTU, the 6-NO₂ derivative of HBTU, in the guanidinium N-oxide form.⁶⁵ 

• (E) The O-bis-substituted reaction product of HOBt with CH₂Cl₂ in the presence of a tertiary amine.^66–68 

• (F) BOMY, N-(1H-benzotriazol-1-ylmethylene)-N-methylmethanaminium hexachloroantimonate N-oxide, and BDMP, 5-(1H-benzotriazol-1-yl)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate N-oxide, in the guanidinium form.^69,70 

• (G) O-HATU (HATU in the O-form).⁷¹ 

• (H) COMU, a combination of a morpholinium-based immonium moiety and Oxyma, ethyl-2-cyano-(hydroimino)acetate, in the O-form.⁷² 

• (I) DEPBT, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one, in the O-form.⁷³ 

• (J) The complex in which six molecules of deprotonated HOBt are coordinated via O atoms to a central Fe³⁺ ion in the octahedral geometry.⁷⁴ 

The huge number of crystal structures (more than 100) of 2,4-substituted 5(4H)-oxazolones (OXL), commonly called azlactones as they also contain a nitrogen atom in the ring structure, solved by X-ray diffraction in the last 20 years represents an additional, excellent reason to devote a dedicated sub-section of this review article to this group of cyclic esters of great relevance in amino acid an peptide chemistry. They are typically divided into two sub-groups, namely those derived from: (i) N^α-acylated or N^α-urethanyl-α-amino acids or peptides with a C^α-tetrasubstituted (quaternary) atom (the C₄-atom in the OXL numbering) in the C-terminal residue, and (ii) N^α-acylated C^α,C^β-didehydro-α-amino acids. The X-ray diffraction structures (with the related references) of compounds pertaining to sub-group (i) are listed in Table 4, while those of sub-group (ii) are given in Table 5. Figures 10 and 12 report the mean values of bond lengths and bond angles for the OXL moiety of C^α-tetrasubstituted and C^α,C^β-didehydro-α-amino acids, respectively, extracted from our statistical analyses, and Figs. 11 and 13 show selected examples of crystal strucures of OXLs from the two sub-groups of α-amino acids. More specifically, Fig. 11a illustrates the X-ray diffraction structure of the OXL from an N^α-acylated, C^α-tetrasubstituted, achiral α-amino acid (but with a chiral acyl moiety),⁸⁵  Fig.11b of the OXL from an N^α-urethanyl-α-amino acid (devoid of any chiral center),⁷⁹  and Fig. 11c of the OXL from the longest peptide solved so far (entirely based on a residue with the same C^α-atom chirality).⁷⁷  The two examples shown in Fig. 13 refer to OXLs from N^α-acylated C^α,C^β-didehydro-α-amino acids with the overwhelmingly more common Z-configuration at the C^αC^β bond (Fig. 13a),¹⁵²  and with the E-configuration (Fig. 13b).¹⁵⁵ 

More than 65 years ago, synthetic peptide chemists reported that peptide OXLs from C^α-tetrasubstituted (achiral or chiral but non-racemizable) α-amino acids, although severely sterically hindered, provide access to longer peptides in excellent yields when allowed to react with α-amino acid or peptide esters.^171,172  However, the use of amino acid and peptide OXLs in synthesis, when they are based on a protein or in general on a C^α-trisubstituted α-amino acid, is dramatically limited by their great propensity to racemize (epimerize).^173,174  This loss of optical purity is associated with the anomalously high acidity (pK_a≅9) of their C^α-proton¹⁷⁵  which permits rapid equilibration between the two stereoisomers via the intermediacy of the achiral, aromatic oxazol enol (Scheme 5). In any case, OXLs from C^α-trisubstituted residues were extensively employed for the preparation of enantiomerically enriched α-amino acid derivatives¹⁷⁵  and C^α-tetrasubstituted α-amino acids^98,99  (in the latter experiments, also in the context of investigations in the prebiotic and exobiotic fields).^77,100  Moreover, organic chemists have often used OXLs as substrates for cycloaddition reactions on the ring CN bond or (if based on C^α,C^β-didehydro-α-amino acids) on the exocyclic CC bond. The interpretation of the stereochemical pathways of these latter reactions was facilitated by the known conformational and configurational properties of the OXL starting materials, as obtained by X-ray diffraction studies. The extremely important role played by OXLs from C^α-trisubstituted α-amino acids as isolated or transient compounds in peptide bond formation, particularly in connection with the mechanism involving partial or total loss of the initial C^α-configuration, was recently highlighted by El-Faham and Albericio in an encyclopedic review article on peptide coupling reagents.⁷⁵ 

The OXLs from the N^α-acylated, C^α-tetrasubstituted α-amino acids and peptides (Table 4 and Figs. 11a and c) are characterized by an approximately planar annular system. The C₁^β1 and C₁^β2 atoms, linked at C₁^α, protrude from opposite sides of the average plane of the cyclic moiety. The C₀′N double bond length is 1.27 Å (Fig. 10, top) as expected for this type of bond.⁷  This result is a clear evidence that the CN bond is not conjugated with the cyclic ester part of the OXL. The lengths of the two bonds from O₀ have values of 1.38 and 1.40 Å, suggesting that the electron delocalization is modest, albeit not completely absent. The lengths of the bonds from C₁^α to the neighboring trigonal ring atoms (N₁ and C₁′) have values typical for those including a tetrahedral atom.⁷  The two bond angles at the sp²-hybridized C₁′ atom outside the penta-atomic ring show values (121.8 and 131.4°) which largely differ (by almost 10°) (Fig. 10, bottom). The more expanded bond angle is that involving the O₁ and C₁^α atoms.^33,176  This finding is strongly in favor of the active role of unfavorable interactions between the O₁ atom and the two substituents on C₁^α. Another abnormal (external) bond angle widening (to 127.8°) is observed, which includes the additional sp²-hybridized C₀′ atom of the ring system. Typically, the set of torsion angles of the penultimate residue in a peptide OXL is not characteristic of helical structures (Fig. 11c), even if this residue is very helicogenic, such as the C^α-tetrasubstituted α-amino acids Aib and (αMe)Val^177,178  (as for the latter, see Fig. 11c). It is believed that this uncommon observation would be the consequence of unfavorable interactions between atoms of the OXL ring and of the two preceding amino acid residues.

In the three crystal structures of the 2-alkoxy-5(4H)-oxazolones (obtained from N^α-urethanyl-α-amino acids) (Table 4 and Fig. 11b) solved,^78,79  the OXL annular system is close to planarity. Among bond lengths and bond angles, only the C₀′–O₀ bond length differs, being slightly (0.02 Å) shorter in the 2-alkoxy compounds (which supports the view that the intra-ring electron delocalization is more efficient, though modestly, in them) and the bond angles at C₀′ vary (to a limited extent). However, an interesting geometrical property of the 2-alkoxy OXLs is the significantly short length of the exocyclic C₀′–O(1) bond (1.32 Å) as compared to that of a (sp²-hybridized)C–O single bond (1.37–1.38 Å).⁷  The orientation of the urethane R(1)–O(1) moiety towards the nitrogen of the OXL ring permits a proper disposition of a lone pair of the O(1) atom for its effective interaction with the π-electron system of N₁C₀′. Interestingly, the first X-ray diffraction structure of a 5(4H)-thiazolone (TXL) (Table 4) was recently published.⁸⁴ 

The OXLs from C^α,C^β-didehydro-α-amino acids (ΔAAs) are efficient intermediates in the preparation of peptides containing these residues.¹⁷⁹  In the 3D-structures of OXLs from N^α-acylated ΔAAs (Table 5 and Fig. 13) the perfectly planar conformation of the OXL moiety and the extra-ring C^β-atom permits a strong electronic conjugation. In particular, the outcome of this phenomenon is a markedly short (1.35 Å)⁷  C^αC^β bond. Not unexpectedly, only bond lengths and bond angles involving the N₁ and C₁′ atoms with C₁^α and O₁ (and C₁^β as well) are remarkably different in the two sets of OXLs (compare data in Figs. 10 and 12). More specifically, the N₁–C₁^α and C₁^α–C₁′ bond lengths are much shorter (by 0.06–0.07 Å) in the OXLs from ΔAAs (note that in these compounds the C₁^α-atom is sp²-hybridized, at variance with those from C^α-tetrasubstituted α-amino acids where it is sp³-hybridized). Also, large increases (by about 2.5 and 5.0°) are observed for the C₁^α–C₁′–O₁ and N₁–C₁^α–C₁′ bond angles, respectively, in the OXLs from ΔAAs. The two external bond angles N₁–C₁^α–C₁^β and C₁′–C₁^α–C₁^β involving the C^α-atom in the latter type of OXLs are expanded (128.2 and 123.3°, respectively) as compared to the classical sp² value (120°), perhaps to compensate for the narrow internal N₁–C₁^α–C₁′ bond angle (108.4°) forced in this geometry by the requirements of being part of a pentacyclic structure. OXLs from ΔAAs may adopt either the Z- or the E-configuration about the exocyclic C^αC^β bond (X-ray diffraction analyses were used to solve this issue). To an overwhelming percentage, this group of OXLs adopt the Z-configuration (Fig. 13a), where the N–C^α–C^β–C^γ torsion angle is close to the cis (0°) disposition and the side chain is directed towards the N atom. In many of these compounds where the C^γ-atom is part of an aromatic moiety, it was demonstrated that the lower stability of the E-stereoisomer, with respect to its Z-stereoisomer, is not associated with an out-of-plane Ar–CC group. The almost co-planar steric arrangement of the OXL and the aromatic moiety may allow formation of an intramolecular (aromatic)C–H⋯N(OXL) H-bond.

Although very rarely employed in modern solid-phase peptide synthesis, the carboxylic azide method¹⁸⁰  was for many years celebrated as the most important procedure in the peptide segment condensation approach, particularly because it involves a minimal risk of epimerization. To explain the extremely low loss of optical purity in this peptide coupling reaction, Young and coworkers¹⁸¹  suggested that the electrostatic attraction between the positively charged central nitrogen atom of the azido group of the acylated α-aminoacylazido anion (the NH proton has been removed by base) and the negatively charged carbonyl oxygen of the N^α-blocking group or the preceding amide bond (Fig. 14b) would be operative in preventing this oxygen from attacking the carbonyl carbon of the α-amino acid residue, thereby blocking formation of the potentially dangerous 5(4H)-oxazolone intermediate. The X-ray diffraction structure of the single N^α-protected α-aminoacyl azide reported before 1996 (Tos-Aib-N₃),¹⁸²  despite offering some interesting geometric and 3D-structure information, was not conclusive in supporting or repudiating the Young and coworkers' proposal. The main reason for this lack of explanation is to be associated with the use of the sulfonamide protecting group (bearing two oxygens) of Tos-Aib-N₃, instead of an acyl group (bearing one oxygen). Unfortunately, after 20 years from the original review article, this long-time awaited, more stringent test is still missing.

The only useful piece of information in the area of covalently bonded organic azides was a statistical survey of their geometric and conformational features published in 1999,¹⁸³  as extracted from X-ray diffraction results retrieved from the Cambridge Structural Database. The RNNN geometry (although in this list a Csp² R moiety would represent a case less frequent than a Csp³ R moiety) exhibit some relevant general features: (i) A large preference for the trans C_s conformation. (ii) A slightly bent N–N–N unit [the related bond angle has an average value of 172.6(2)°], with two distinctly different N–N bond lengths (Δ≅0.1 Å). The (central)N–N(terminal) bond is significantly shorter than the (C-bonded)N–N(central) bond. (iii) In this nitrogen triatomic segment, the triple bond character of the (central)N–N(terminal) bond increases as the single bond character of the (C-bonded)N–N(central) bond decreases. This phenomenon may be associated with the largely prevailing, limiting resonance form shown in Fig. 14a. (iv) A covalently bonded azide participates in H-bonding only in a limited number of structures, where the acceptor is the terminal nitrogen. This behavior is associated to a redistribution of the electron density within the N–N–N group (with respect to the azide anion) which decreases its capacity for H-bonding. In the azide anion, the dominating factor is π-delocalization, whereas in the R–N–N–N system an important role is played also by a strong negative hyperconjugation, which donates electron density from the filled σ (R–N) orbital into the unfilled, antibonding π* (central)N–N(terminal) orbital.

Ephemeral, reactive intermediates in peptide synthesis typically suffer from low melting point and poor crystallinity. However, we discovered that by use of an appropriate combination of a high molecular weight (aromatic) N^α-protecting group, e.g. Tos or Z, and a conformationally restricted α-amino acid, e.g. the C^α-tetrasubstituted residues Aib or MeAib (known to be able to induce a high propensity to crystallize to the resulting derivatives),^1,184–186  this disadvantage can be overcome.

Three published X-ray structures of elusive, C-reactive amides have been discussed earlier in the text (in the Section 4: Carboxylic esters) because they were discovered serendipitously as structural isomers of the corresponding 1-oxy-1,2,3-benzotriazole esters. In addition to those compounds, four other reactive amides were analyzed by X-ray diffraction. They are: (i) Tos-MeAib-Im^1,187  (Im, imidazole) (Scheme 5), Tos-Aib-TT^1,187  (TT, 1,3-thiazolidine-2-thione) (Scheme 6), Tos-MeAib-TT,¹  and Z-l-Ala-TT.¹  Two representative crystallographic structures are shown in Fig. 15.

The exploitation of an acyl moiety linked to a heterocyclic nitrogen (an N-acylated Im derivative) in peptide bond formation was first reported by Wieland and Schneider¹⁸⁸  as early as in 1953, followed by interesting developments from other research groups.^189,190  The reactivity of these intermediates is increased by the catalytic effect of the imidazole generated in the course of the reaction.^191,192  Moreover, in the presence of a carbodiimide, N^α-protected α-amino acids regiospecifically acylate the five-membered heterocycle 1,3-thiazolidine-2-thione at the nitrogen atom.¹⁹³  The resulting yellowish 3-acyl-TT products are valuable reagents in peptide coupling reactions.

The C′O bond length for the two aforementioned compounds is about 1.20 Å. The average CO bond length for unreactive, carboxylic amides is 1.234 Å,^7,193  which is significantly longer than the corresponding one for their reactive amide counterparts (and carboxylic esters⁷  as well). The C′–N(ring) bond lengths are close to 1.41 Å. The average C′–N bond length for unreactive tertiary amides is much shorter (1.346 Å),^7,194  while it is 1.389 Å for substituted carboxylic imides (TT is a monothio-imide). In these compounds, the acylated nitrogen has geometric properties indicative of an sp² hybridization.

The torsion angles about the C′–N (ring) bond of Tos-MeAib-Im highlighted the good planarity of this part of the molecule. In contrast, the corresponding angle in the TT derivatives deviate from planarity as much as by 27–42°. The (amino acid)N and (ring)N are in a cisoid arrangement in the TT derivatives of MeAib and Aib, while in a transoid arrangement in the derivative of Ala. While the C′O and the C′S groups of the Aib and Ala derivatives are aligned in the common trans orientation, presumably because of potential lone-pair repulsion between the two heteroatoms, this orientation is cis in the MeAib derivative. The five-membered TT ring of the three compounds has a twist conformation with one of the Csp³ atoms above and the other below the average plane of the heterocycle. In the mid 1990's, Yamada^195,196  demonstrated that very highly reactive, largely twisted amides are produced when the TT NH function is acylated by a C^α-tetrasubstituted carboxylic acid, such as pivalic acid. In those compounds, X-ray diffraction analyses indicated that the twist angle may be as high as 75°. These data were explained on the basis of the classical carboxylic amide resonance theory, with an increase in the twist angle reducing the statistical weight of the canonical form bearing oppositely charged oxygen and nitrogen atoms. Carboxylic amides dramatically distorted from planarity are the focus of much interest in organic chemistry (as efficient enantioselective acylating reagents¹⁹⁷  and mimics of the transition state for amide bond hydrolysis¹⁹⁸ ) and in structural biochemistry (as models to investigate the cis/trans peptide bond conformational interconversion¹⁹⁹ ).

The major conclusions extracted from our crystallographic study on reactive carboxylic amides can be summarized as follows:

• (i) The Im carboxylic amide deviates only slightly from planarity. Therefore, it is evident that the reactivity of imidazolides towards amine nucleophiles in peptide synthesis has to be ascribed to the properties of imidazole as the leaving group in this process and its catalytic effects^191,192  rather than a deviation from planarity of the carboxylic amide group.

• (ii) A much more remarkable deviation from planarity is shown by the TT carboxylic amide group. This property makes the (ring)N lone pair orbital located in such a way to permit a more efficient electron delocalization towards the thiocarbonyl CS π-system rather than towards the carbonyl CO group. Finally, it is worth pointing out that, although the Aib and MeAib derivatives would bear a C^α-tetrasubstituted atom linked to the acyl moiety similarly to the pivaloyl TT derivatives,^195,196  the deviation from planarity of the carboxylic amide group is much more significant in the latter compounds.

In this chapter, mainly via the results obtained from the numerous X-ray diffraction analyses available in the literature, we have in particular provided a summary of the very valuable and detailed 3D-structural information on geometry (bond lengths and bond angles) and conformation (torsion angles) of chemically reactive α-amino acid and peptide derivatives typically used in amide bond formation. These findings, in turn, are extremely useful in deepening our knowledge on reactivity, regiospecificity, and racemization (epimerization) tendency of these critical electrophilic components. More specifically, we have discussed electronic and steric properties of the extensively exploited carboxylic halides (fluorides), anhydrides (symmetrical linear and N-carboxy cyclic), linear esters (nitrophenyl, N-oxy-succinimido and N-oxylamino), cyclic esters [azlactones or 5(4H)-oxazolones from either C^α-tetrasubstituted or C^α,C^β-didehydro α-amino acids], azides, and cyclic amides.

We are pretty much convinced that synthetic and 3D-structural peptide scientists will be stimulated by the content of this review article to join forces with the purpose of better understanding this domain of bioorganic chemistry, in combination with theoretical investigations and kinetic experiments, to permit a full insight into the mechanisms operative in peptide bond formation and in unwanted side reactions. As an example, the combination of 3D-structural information with theoretical calculations, aimed at establishing the most likely reaction path for this reaction (which may vary depending on the specific type of activation) and the related energy landscape, already proved to be effective in explaining that 5(4H)-oxazolones derived from urethane-protected α-amino acids are less prone to give racemized/epimerized peptide products than their N^α-acylated counterparts, not due to a significantly higher acidity of the α-hydrogen of the latter, but because the former are more reactive toward the amine nucleophiles.⁷⁹  The application of a similar multidisciplinary approach to the other classes of carboxyl reactive derivatives of α-amino acid and peptides reviewed in this chapter, and its extension to recently introduced and currently widely used reagents in novel methodologies for the production not only of longer peptides but of proteins as well might be very rewarding. In this connection, in the process called “chemical ligation”,²⁰⁰  the peptide carboxylic thiolester electrophiles play a fundamental role as reactive intermediates both in the first step (intermolecular trans-thiolesterification) and in the second step (intramolecular S→N acyl shift), to afford a larger product with a native amide bond at the ligation site. Remarkably, to the best of our knowledge, no results from 3D-structural chemistry investigations on peptide carboxylic thiolesters have been published to date (only a limited number of X-ray diffraction structures of N-terminally protected or blocked α-aminoacyl thiolesters is known^201–203 ).