Determination of intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides in the absence of nearest‐neighbor or conformational effects

Abstract

Understanding the hydrophilicity/hydrophobicity of amino acid side chains in peptides/proteins is one the most important aspects of biology. Though many hydrophilicity/hydrophobicity scales have been generated, an "intrinsic" scale has yet to be achieved. "Intrinsic" implies the maximum possible hydrophilicity/hydrophobicity of side chains in the absence of nearest-neighbor or conformational effects that would decrease the full expression of the side-chain hydrophilicity/hydrophobicity when the side chain is in a polypeptide chain. Such a scale is the fundamental starting point for determining the parameters that affect side-chain hydrophobicity and for quantifying such effects in peptides and proteins. A 10-residue peptide sequence, Ac-X-G-A-K-G-A-G-V-G-L-amide, was designed to enable the determination of the intrinsic values, where position X was substituted by all 20 naturally occurring amino acids and norvaline, norleucine, and ornithine. The coefficients were determined by reversed-phase high-performance liquid chromatography using six different mobile phase conditions involving different pH values (2, 5, and 7), ion-pairing reagents, and the presence and absence of different salts. The results show that the intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides (proteins) is independent of pH, buffer conditions, or whether C(8) or C(18) reversed-phase columns were used for 17 side chains (Gly, Ala, Cys, Pro, Val, nVal, Leu, nLeu, Ile, Met, Tyr, Phe, Trp, Ser, Thr, Asn, and Gln) and dependent on pH and buffer conditions, including the type of salt or ion-pairing reagent for potentially charged side chains (Orn, Lys, His, Arg, Asp, and Glu).

Alpha-helical cationic antimicrobial peptides: relationships of structure and function

“…The nonpolar face of an amphipathic α-helical peptide represents a preferred domain for binding to the hydrophobic matrix of a reversed-phase column. The disturbance of helix by incorporation of D-amino acid will cause the change of overall hydrophobicity, which is reflected by the retention time, and the peptide hydrophobicity value is calculated by the side-chain hydrophobicity (Kovacs et al, 2006).…”

Mixed‐mode hydrophilic interaction/cation‐exchange chromatography: Separation of complex mixtures of peptides of varying charge and hydrophobicity

“…The separation of the peptides then further improves with increasing ACN concentration until, at 90% ACN, the two pairs of +3 peptides are resolved almost to baseline and only the Tyr analogues of the +2 peptides still exhibit only partial separation, albeit even these are mainly resolved. Note that the Phe analogues of the +2 and +3 peptides are eluted prior to the respective Tyr analogues due to the greater hydrophobicity (lesser hydrophilicity) of Phe relative to Tyr [34]. It is also interesting that, for the acetylated analogues (+2 peptides), the peptides (AcC- Salt gradient rates of 2 mM and 0.5 mM NaClO 4 /min.…”

Enantioselective synthesis of (2R, 3S)‐ and (2S, 3R)‐4,4,4‐trifluoro‐N‐Fmoc‐O‐tert‐butyl‐threonine and their racemization‐free incorporation into oligopeptides via solid‐phase synthesis

“…To this end, we employed the latest reversed-phase liquid chromatography (RPLC) method for amino acid hydrophobicity determination developed by Hodges and coworkers, who determined the relative hydrophobicity of 22 L-amino acids and their D-enantiomers. 31 As pointed out by Hodges and coworkers, a criterion for measuring true hydrophobicity of an amino acid is that the D/L enantiomers should give the same retention time, t R . D-tfT/allo-L-tfT (t R = 11.6 min), co-elute, meeting the criterion established by Hodges and coworkers.…”

“…D-tfT/allo-L-tfT (t R = 11.6 min), co-elute, meeting the criterion established by Hodges and coworkers. 31 In RPLC, the larger the retention time, the more hydrophobic a molecule is. The allo-D-tfT/allo-L-tfT pair is more retentive than the allo-D-Thr/allo-L-Thr pair (Δt R = 11.6 -7.2 = 4.2 min).…”

“…To put matters into perspective, the retention time difference between allo-D-tfT/allo-L-tfT and allo-D-Thr/allo-L-Thr (Δt R = 4.2 min) is larger than that between D-Ala/L-Ala and Gly (Δt R = 2.8 min), comparable to that between D-Cys/L-Cys and D-Ala/L-Ala (Δt R = 4.8 min), and smaller than that between D-Val/L-Val and D-Ala/L-Ala (Δt R = 10.6 min). 31 The impact on hydrophobicity brought by replacing Thr with tfT in unprotected peptides will be evaluated in the context of octreotide and the results will be presented in a future report.…”