Peptide Charge Derivatization as a Tool for Early Detection of Preeclampsia by Mass Spectrometry-A Comparison with the ELISA Test.

Early detection of any preeclampsia biomarkers may lower the risk of mortality, both for a mother and a child. Our study focuses on techniques for preeclampsia biomarker identification by comparing the results of a method using liquid chromatography mass spectrometry in multiple reaction monitoring mode (LC-MS/MS) with those by the enzyme-linked immunosorbent assay (ELISA) test, as well as by comparing the obtained results with clinical data. In the proposed LC-MS/MS method a tryptic digest peptide charge derivatization strategy was used as a tool for sensitive detection of podocin, i.e., a previously discovered preeclampsia biomarker present in urine samples from pregnant women. Urine samples from pregnant women with diagnosed preeclampsia were collected at different stages of pregnancy and from healthy subjects, and then were analyzed by ELISA test and the proposed method with LC-MS/MS. Charge derivatization of the ε amino group of C-terminal lysine residues in tryptic digests by 2,4,6-triphenylpyrylium salt was performed to increase the ionization efficiency in the LC-MS/MS mode. Podocin was identified at the early stage of pregnancy, while its detection using an ELISA test was not possible. The protocol for urine sample preparation was optimized. Our results show that the proposed method by LC-MS/MS in combination with peptide charge derivatization, provides an ultrasensitive tool for diagnosis of preeclampsia, and provides earlier detection than a clinical diagnosis or ELISA test. The proposed solution may revolutionize medical diagnostics.

Analysis of Fragmentation Pathways of Peptide Modified with Quaternary Ammonium and Phosphonium Group as Ionization Enhancers.

Peptide modification by a quaternary ammonium group containing a permanent positive charge is a promising method of increasing the ionization efficiency of the analyzed compounds, making ultra-sensitive detection even at the attomolar level possible. Charge-derivatized peptides may undergo both charge remote (ChR) and charge-directed (ChD) fragmentation. A series of model peptide conjugates derivatized with N,N,N-triethyloammonium (TEA), 1-azoniabicyclo[2.2.2]octane (ABCO), 2,4,6-triphenylopyridinium (TPP) and tris(2,4,6-trimetoxyphenylo)phosphonium (TMPP) groups were analyzed by their fragmentation pathways both in collision-induced dissociation (CID) and electron-capture dissociation (ECD) mode. The effect of the fixed-charge tag type and peptide sequence on the fragmentation pathways was investigated. We found that the aspartic acid effect plays a crucial role in the CID fragmentation of TPP and TEA peptide conjugates whereas it was not resolved for the peptides derivatized with the phosphonium group. ECD spectra are mostly dominated by cn ions. ECD fragmentation of TMPP-modified peptides results in the formation of intense fragments derived from this fixed-charge tag, which may serve as reporter ion.

Selective ESI-MS detection of carbonyl containing compounds by aminooxyacetic acid immobilized on a resin.

There are numerous examples of bioactive compounds containing carbonyl groups including modified proteins with oxidation of side chain of amino acid residues to aldehyde/ketone groups which are frequently considered as markers of oxidative stress. The carbonyl unit can be also distinguished as a substructure in many illegal drugs including anabolic steroids as well as cations derivatives. Based on chemoselective formation of oximes by solid phase immobilized hydroxylamine derivatives we proposed the protocol for derivatization and selective detection of carbonylated compounds in human serum albumin hydrolysate as a complex peptide mixture and of testosterone in urine samples. This allowed for the removal of the matrix and the qualitative and quantitative analysis of the derivatized analyte by LC-MS/MS (or LC-MRM). Herein we report the preparation and chemical characterization of a novel, ChemMatrix - based resin functionalized with aminooxyacetic acid (AOA). The hydroxylamine moiety in this resin is combined with a peptide linker (GRG) containing an arginine residue to enhance the ionization efficiency. Application of an isotopically labeled carbonylated peptide ((H-Leu-Val-Thr(O)-Asp-Leu-Thr-Lys [13C6,15N2]-OH and testosterone-d3 allowed us to carry out quantitative analyses of detected compounds. Our method is general and may be applied for analysis of carbonylated compounds in biological samples. Our method based on application of functionalized resin allowed to quantify the level of free testosterone in small sample (0.5 mL) of urine, while the non-derivatized testosterone from urine sample was not detected during direct LC-MRM analysis.

Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells.

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent N-hydroxysuccinimide esters of dihydro-[1,2,4]triazolo[4,3-a]pyridin-2-ium carboxylates (Safirinium P dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support; thus, they can be applied for derivatization as ionization enhancers. Safirinium tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom-up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, Safirinium NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as E. coli Top10 bacterial strain.

Evaluation of podocin in urine in horses using qualitative and quantitative methods.

No sensitive method for diagnosing early kidney dysfunction in horses has been identified so far. Many studies carried out in humans and small animals show that podocin can be useful to diagnose various kidney diseases, mainly affecting the glomeruli. The aim of this study was to perform a qualitative and quantitative analysis of podocin in urine samples obtained from healthy horses, horses with clinical kidney dysfunction and horses at risk of acute kidney injury. The study objectives aimed to assess: (1) whether the selected podocin tryptic peptide for LC-MS-MRM allows for podocin detection in horse; and (2) whether the species-specific ELISA test makes this detection possible as well;, (3) whether the chosen methods are sensitive enough to detect kidney dysfunction and glomerular injury, (4) whether the results of the tests applying both methods correspond with one another, (5) whether the results correlate with the hematological and biochemical data. The signals that may indicate the presence of trypsin fragments of podocin were found in three healthy horses, all the horses diagnosed with kidney dysfunction and half of the animals at risk for acute kidney injury. The concentration of podocin, diagnosed with the ELISA test was as follows: from 0.19 to 1.2 ng/ml in healthy animals, from 0.19 to 20.0 ng/ml in AKI horses, from 0.29 to 5.71 ng/ml in horses at risk for acute kidney injury. The results of both methods corresponded significantly. Podocin may be a potential biomarker of clinical kidney disease in horses and may be used in the detection of glomerular injury. However, its use is limited by the possibility of physiological podocyturia. LC-MS-MRM seems to be a more sensitive method to evaluate the presence of podocin than the ELISA test, whilst selected tryptic peptides of podocin appear to apply to horses. The ELISA test showed greater effectiveness in excluding the disease than in confirming it.

Intramolecularly stapled amphipathic peptides via a boron-sugar interaction.

Amadori products (deoxyfructosyllysine derivatives) that can selectively interact with phenylboronic acids and borate ions were synthesized. The intramolecular interactions between the fructosyl moiety and phenylboronic acid incorporated into various positions of the peptide chain were investigated using high-resolution mass spectrometry (HR-MS), circular dichroism (CD), and nuclear magnetic resonance (NMR).

Detection of Podocin in Human Urine Sediment Samples by Charge Derivatization and LC-MS-MRM Method.

Detection of podocytes in urine might serve as a useful diagnostic tool in both primary and secondary glomerular diseases. The utility of podocyturia has been confirmed for both pre-eclampsia and glomerulonephritis. Here, we present a new and sensitive method for qualitative LC-MS-multiple-reaction-monitoring (MRM) analysis of podocin, serving as a podocyturia biomarker in urine sediments. The following podocin tryptic peptides with the 169LQTLEIPFHEIVTK182, 213AVQFLVQTTMK223, 240SIAQDAK246, and 292MIAAEAEK299 sequences were applied as a model. The selective chemical derivatization of the ε amino group of C-terminal lysine residue in tryptic peptides, by 2,4,6-triphenylpyrylium salt (TPP) as a fixed charge tag, was employed to increase the ionization efficiency, in routine ESI-MS analysis. Additionally, the generation of a reporter ion, in the form of a protonated 2,4,6-triphenylpyridinium cation, makes the derivatized peptide analysis in the MRM mode unambiguous. Identification of derivatized and non-derivatized peptides were performed, and the obtained results suggest that the peptide with the 292MIAAEAEK299 sequence may serve as a marker of podocyturia.

Preparation of Isotopically Labelled Standards of Creatinine Via H/D Exchange and Their Application in Quantitative Analysis by LC-MS.

Kidneys play a crucial role in maintaining metabolic homeostasis in a body. Serum creatinine concentration is a simple test used as an indicator of renal function. One of the known ways of quantifying creatinine concentration is the liquid chromatography-mass spectrometry (LC-MS) method, using an isotopically labeled analog of creatinine as an internal standard. Unfortunately, such isotope-labeled analogs are expensive and their synthesis is complex. Here we demonstrate a facile preparation of deuterated analogues of creatinine, via the H/D exchange of hydrogens located at the α-carbon (α-C) of the N-methylated amino acid part, under basic conditions. The stability of retrieved isotopologues was analyzed under both neutral or acidic conditions, and the results revealed that the introduced deuterons do not undergo back-exchange. In addition, the coelution of deuterated and non-deuterated forms under acidic and neutral conditions was observed. The prepared isotopologues were successfully applied in the quantitative LC-MS analysis of urine samples, and the results demonstrated that the presented strategy is novel and inexpensive, and that the quantification correlates with the commonly used Jaffe test method.

Enrichment of Cysteine-Containing Peptide by On-Resin Capturing and Fixed Charge Tag Derivatization for Sensitive ESI-MS Detection.

High complexity of cell and tissue proteomes limits the investigation of proteomic biomarkers. Therefore, the methods of enrichment of some chemical groups of peptides including thiopeptides are important tools that may facilitate the proteomic analysis by reducing sample complexity and increasing proteome coverage. Here, we present a new method of cysteine-containing tryptic peptide enrichment using commercially available TentaGel R RAM resin modified by the linker containing the maleimide group, allowing thiol conjugation. The captured tryptic peptides containing lysine residue were then tagged by 2,4,6-triphenylpyrylium salt to form 2,4,6-triphenylpyridinium derivatives, which increases the ionization efficiency during mass spectrometry analysis. This makes it possible to conduct an ultrasensitive analysis of the trace amount of compounds. The proposed strategy was successfully applied in the enrichment of model tryptic podocin peptide and podocin tryptic digest.

Functional derivatives of human dentin matrix protein 1 modulate morphology of calcium carbonate crystals.

Dentin matrix protein 1 (DMP1) is an acidic, extracellular matrix protein essential for biomineralization of calcium phosphate, in bone and dentin. It is proteolytically processed into two fragments, 44K and 56K. Recently, the presence of DMP1 was noticed in inner ear, specifically in otoconia, which are calcium carbonate biominerals involved in sensing of balance. In this study, the solution structure and biomineralization activity of otoconial 44K and 56K fragments toward calcium carbonate were investigated. The results of analytical ultracentrifugation, circular dichroism, and gel filtration indicated that DMP1 fragments are disordered in solution. Notably, 56K formed oligomers in the presence of calcium ions. It was also observed that both fragments influenced the crystal growth by in vitro biomineralization assay and scanning electron microscopy. In addition, they sequester the calcium ions during the calcite formation. Calcium carbonate crystals precipitated in vitro changed their size and shape in the presence of DMP1 fragments. Oligomerization propensity of 56K may significantly enhance this function. Our study indicates that intrinsically disordered DMP1 has a previously unknown regulatory function for biomineralization of otoconia.

Metal self-assembly mimosine peptides with enhanced antimicrobial activity: towards a new generation of multitasking chelating agents.

Mimosine is a non-protein amino acid with various properties, such as antibacterial, anti-inflammatory, anti-cancer and anti-virus among others. Due to its structural similarity with deferiprone (DFP), mimosine is a potential excellent metal chelator. In the present work, we combine experimental and theoretical (DFT) approaches in order to investigate the properties of mimosine peptides. Six different peptides were synthesized and their complex stoichiometry and stability were characterized by means of UV-Vis spectrophotometry. Then, the binding mode and self-assembly features of the peptides were evaluated using a DFT approach, taking into account different number of mimosine amino acids and varying the length of the spacer between the mimosine residues, and there was good agreement between experimental data and computational calculations. Further elucidations of the structural properties of these peptides allowed us to propose improvements in the structure of the mimosine moiety which can lead to enhanced affinity for high-valent metals. Moreover, we demonstrate that these peptides show an anti-microbial activity against Gram positive bacteria that is enhanced by the formation of a complex with iron(iii) ions. The mimosine peptides could be an alternative to antimicrobial peptides (AMPs), which are expensive and susceptible to proteolytic degradation. In summary, in the present work, we propose a new generation of multipurpose mimosine-based peptides as new metal self-assembly chelators which could be a turning point in biomedical and nanotechnological applications.

An Optimised Di-Boronate-ChemMatrix Affinity Chromatography to Trap Deoxyfructosylated Peptides as Biomarkers of Glycation.

We report herein a novel ChemMatrix® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-α and N-ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.

Evaluation of Tryptic Podocin Peptide in Urine Sediment Using LC-MS-MRM Method as a Potential Biomarker of Glomerular Injury in Dogs with Clinical Signs of Renal and Cardiac Disorders.

The early asymptomatic stage of glomerular injury is a diagnostic challenge in the course of renal and extra-renal disease, e.g., heart insufficiency. It was found that podocin, a podocyte-specific protein present in the urine, may serve as a biomarker in the diagnosis of glomerular disease in humans and animals including glomerulonephritis, glomerulosclerosis, amyloidosis, or nephropathy. Therefore, there is a need of development of the sensitive and straightforward method of urinary podocin identification. In this work, we report our extended research under the glomerular injury investigation in dogs by application of clinical examination and LC-MS-MRM method in the identification of canine podocin in urine samples. The LC-MS-MRM method is based on the identification of podocin tryptic peptide with the 218H-AAEILAATPAAVQLR-OH232 sequence. The model peptide was characterized by the highest ionization efficiency of all the proposed model podocin tryptic peptides in a canine urine sediment according to the LC-MS/MS analysis. The obtained results revealed the presence of the model peptide in 40.9% of dogs with MMVD (active glomerular injury secondary to heart disease = cardiorenal syndrome-CRS) and 33.3% dogs with chronic kidney disease. The potential applicability of the developed methodology in the analysis of podocin in canine urine sediments was confirmed.

In vitro SOD-like activity of mono- and di-copper complexes with a phosphonate substituted SALAN-type ligand.

SALEN- and SALAN-based complexes with catalytically active metal centers are very promising small molecules to be utilized as part of antioxidant therapies. Here we discuss a modified SALAN-type molecule armed with two phosphonate groups that significantly increase its water solubility and aid to furnish mono- or dinuclear complexes with Cu2+ ions. The regulation of the SOD-mimicking (i.e., catalytic) disproportionation reaction of the superoxide radical anion (O2•-) at pH ~7.5 could be achieved by adjusting the metal-to-ligand stoichiometry as confirmed by McCord-Fridovich and pulse radiolysis tests. The higher antioxidant activity of the dicopper complex can be explained by the better access of O2•- to the copper centers and their more positive Cu(II)/Cu(I) redox potential. Simultaneously the analysis of in vitro effect on cells morphology indicates that cytotoxicity is also affected by the metal-to-ligand ratio, however, the active complex molecules do not show notable cytotoxicity that, together with the observed SOD-like activities, makes them potential candidates for antioxidant therapies.

Trends in the Design of New Isobaric Labeling Reagents for Quantitative Proteomics.

Modern mass spectrometry is one of the most frequently used methods of quantitative proteomics, enabling determination of the amount of peptides in a sample. Although mass spectrometry is not inherently a quantitative method due to differences in the ionization efficiency of various analytes, the application of isotope-coded labeling allows relative quantification of proteins and proteins. Over the past decade, a new method for derivatization of tryptic peptides using isobaric labels has been proposed. The labels consist of reporter and balanced groups. They have the same molecular weights and chemical properties, but differ in the distribution of stable heavy isotopes. These tags are designed in such a way that during high energy collision induced dissociation (CID) by tandem mass spectrometry, the isobaric tag is fragmented in the specific linker region, yielding reporter ions with different masses. The mass shifts among the reporter groups are compensated by the balancing groups so that the overall mass is the same for all forms of the reagent. Samples of peptides are labeled with the isobaric mass tags in parallel and combined for analysis. Quantification of individual peptides is achieved by comparing the intensity of reporter ions in the tandem mass (MS/MS) spectra. Isobaric markers have found a wide range of potential applications in proteomics. However, the currently available isobaric labeling reagents have some drawbacks, such as high cost of production, insufficient selectivity of the derivatization, and relatively limited enhancement of sensitivity of the analysis. Therefore, efforts have been devoted to the development of new isobaric markers with increased usability. The search for new isobaric markers is focused on developing a more selective method of introducing a tag into a peptide molecule, increasing the multiplexicity of markers, lowering the cost of synthesis, and increasing the sensitivity of measurement by using ionization tags containing quaternary ammonium salts. Here, the trends in the design of new isobaric labeling reagents for quantitative proteomics isobaric derivatization strategies in proteomics are reviewed, with a particular emphasis on isobaric ionization tags. The presented review focused on different types of isobaric reagents used in quantitative proteomics, their chemistry, and advantages offer by their application.

Isobaric duplex based on a combination of 16O/18O enzymatic exchange and labeling with pyrylium salts.

Enzymatic 18O exchange, the well-established approach in comparative proteomics, has some disadvantages such as back exchange of labeled oxygen and overlapping the peak of a labeled peptide with isotopic peaks of an unlabeled one. Herein we demonstrated a simple procedure in which samples digested with a trypsin (with and without H218O) were reacted with unlabeled and quadrupled 13C-labeled pyrylium salt respectively which results in formation of pyridinium cations. Thus, each isobarically labeled peptide containing zero or four 13C atoms in the mass reporter group, during tandem MS/MS forms an unique reporter ion useful for a relative quantitation. Such a sample treatment improves the signal to noise ratio, reduces overlapping of the isotopic peaks and completely eliminates the back exchange problem.

The intrinsically disordered C-terminal F domain of the ecdysteroid receptor from Aedes aegypti exhibits metal ion-binding ability.

The dominant vector of dengue and Zika diseases is a female Aedes aegypti mosquito. Its reproduction is controlled by the formation of an active heterodimer complex of the 20-hydroxyecdysone receptor (EcR) and Ultraspiracle protein (Usp). Although EcR exhibits a structural and functional organization typical of nuclear receptors (NRs), the EcR C-terminus has an additional F domain (AaFEcR) that is rarely present in the NRs superfamily. The presence of F domains is evolutionarily not well conserved in the NRs. The structure-function relationship of EcR F domains in arthropods is unclear and enigmatic. To date, there have been no data concerning the structure and function of AaFEcR. Our results showed that AaFEcR belongs to a family of intrinsically disordered proteins (IDPs) and possesses putative pre-molten globule (PMG) characteristics. Unexpectedly, additional amino acid composition in silico analyses revealed the presence of short unique repeated Pro-His clusters forming an HGPHPHPHG motif, which is similar to those responsible for Zn2+ and Cu2+ binding in histidine-proline-rich glycoproteins (HPRGs). Using SEC, SV-AUC and ESI-TOF MS, we showed that the intrinsically disordered AaFEcR is able to bind metal ions and form complexes with these ions. Our studies provide new insight into the structural organization and activities of the F domains of NRs. This unique for the F domains of NRs ion-binding propensity demonstrated by the AaFEcR domain may be a part of the ecdysteroid receptor's mechanism for regulating the expression of genes encoding oxidative stress-protecting proteins.

Early Detection of Active Glomerular Lesions in Dogs and Cats Using Podocin.

In veterinary medicine, sensitive and specific markers of the early stages of renal failure still remain to be established. Podocytes could be a promising diagnostic tool in veterinary nephrology, especially in the differentiation of active pathological disease and glomerulopathies. Podocin is one of the robust proteins exploitable in detection of podocyturia. This article presents podocyte detection in urine for diagnostic purposes in veterinary medicine using a variety of methods. We describe the advantages and disadvantages of the immunohistochemical technique currently used, and of scanning microscopy, chromatography, and immunostaining. The identification of podocin-positive cells is a promising diagnostic tool in the detection of the early stages of glomerular basement membrane damage. The detection of renal failure prior to the occurrence of azotaemia is of high clinical importance from the clinical and scientific points of view.

Cyclic OmpC peptidic epitope conjugated to tetanus toxoid as a potential vaccine candidate against shigellosis.

In earlier works we have described that mice immunized with outer membrane protein OmpC survive the challenge with live Shigella flexnerii 3a. We have also identified conformational epitope of this protein, that was recognized by mice antibodies. The aim of current work was to investigate whether synthetic OmpC epitope homologs can elicit immunological response sufficient in protecting mice against shigellosis. Several linear peptides containing RYDERY motif were synthesized and conjugated to poly-lysine. These conjugates appeared to be poor immunogens and to boost the immunological response an addition of the adjuvant (MPL) was required. Unfortunately, the MPL alone caused a very high immunological reaction that was masking response to peptidic epitope. Under those circumstances we used tetanus toxoid (TT) as the carrier protein for the peptides and the agent stimulating immunological response. Series of cyclic peptides, homologs of the OmpC main epitope were synthesized and conjugated to TT. The loop size in cyclic peptides varied by number of glycine residues, i.e., 1-3 residues added to the GLNRYDERYIGK motif. The linear GLNRYDERYIGC-TT was also prepared as the control. The latter conjugate gave the highest immunological response, followed by the cyclic-GGLNRYDERYIGC-TT and cyclic-GLNRYDERYIGC-TT. The third peptide, cyclic-GGGLNRYDERYIGC-TT, gave a very low response, although it was the most resistant to proteolysis. However, antibodies obtained against cyclic-GGLNRYDERYIGC-TT were more potent to recognize both OmpC and Shigella flexnerii 3a cells than the antibodies against linear GLNRYDERYIGC-TT. Furthermore, the monoclonal antibodies raised against linear GLNRYDERYIGC-TT showed 20-fold lower dissociation constant (KD) than the naturally occurring polyclonal antibodies from umbilical cord sera. Monoclonal antibodies also gave a weaker signal in electron microscope than mice and human polyclonal antibodies. In overall, our results point to cyclic peptides as better candidates for a vaccine development, since they are eliciting production of the higher affinity antibodies against Shigella cells and OmpC.

Solid-phase synthesis of peptides containing aminoadipic semialdehyde moiety and their cyclisations.

Pathological levels of oxidative stress (OS) have been implicated in many diseases including diabetes mellitus, neurodegenerative diseases, inflammatory diseases, atherosclerosis, and cancer. Studies of oxidative stress are however complicated by the low concentration of oxidation products. To resolve this problem, we tested a new derivative of aminoadipic semialdehyde (Fmoc-Aea-OH) in the solid-phase synthesis of carbonylated peptides. We prepared a series of peptides with free and acetylated N-terminal amino groups using the Fmoc-Aea-OH reagent. LC-MS, ESI-MS, and MS/MS spectra confirmed the sequences of the modified peptides, although the LC-MS and ESI-MS spectra were dominated by signals corresponding to dehydration products. NMR studies of acetylated products revealed that the dominant product formed in this reaction contains a 1,2,3,4-tetrahydropyridine-2-carboxylic acid residue. Another side reaction in this system was the cleavage of the amide bond between the Aea residue and the amino acid moiety preceding it resulting in the formation of a side product with a six-membered ring at the N-terminus (2,3,4,5-tetrahydropyridine-2-carboxylic acid residue). We found that, depending on the peptide sequence, one of those side products is predominant. Our work suggests new methods for the solid-state synthesis of peptides containing unnatural amino acids.