Whole-body imaging with single-cell resolution by tissue decolorization.

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.

Lipid nanoparticle-mediated lymph node-targeting delivery of mRNA cancer vaccine elicits robust CD8+ T cell response.

The targeted delivery of messenger RNA (mRNA) to desired organs remains a great challenge for in vivo applications of mRNA technology. For mRNA vaccines, the targeted delivery to the lymph node (LN) is predicted to reduce side effects and increase the immune response. In this study, we explored an endogenously LN-targeting lipid nanoparticle (LNP) without the modification of any active targeting ligands for developing an mRNA cancer vaccine. The LNP named 113-O12B showed increased and specific expression in the LN compared with LNP formulated with ALC-0315, a synthetic lipid used in the COVID-19 vaccine Comirnaty. The targeted delivery of mRNA to the LN increased the CD8+ T cell response to the encoded full-length ovalbumin (OVA) model antigen. As a result, the protective and therapeutic effect of the OVA-encoding mRNA vaccine on the OVA-antigen-bearing B16F10 melanoma model was also improved. Moreover, 113-O12B encapsulated with TRP-2 peptide (TRP2180-188)-encoding mRNA also exhibited excellent tumor inhibition, with the complete response of 40% in the regular B16F10 tumor model when combined with anti-programmed death-1 (PD-1) therapy, revealing broad application of 113-O12B from protein to peptide antigens. All the treated mice showed long-term immune memory, hindering the occurrence of tumor metastatic nodules in the lung in the rechallenging experiments that followed. The enhanced antitumor efficacy of the LN-targeting LNP system shows great potential as a universal platform for the next generation of mRNA vaccines.

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein.

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.

Cu-Catalyzed Amination of Base-Sensitive Aryl Bromides and the Chemoselective N- and O-Arylation of Amino Alcohols.

We report a general and functional-group-tolerant method for the Cu-catalyzed amination of base-sensitive aryl bromides including substrates possessing acidic functional groups and small five-membered heteroarenes. The results presented herein substantially expand the scope of Cu-catalyzed C-N coupling reactions. The combination of L8, an anionic N1,N2-diarylbenzene-1,2-diamine ligand, along with the mild base NaOTMS leads to the formation of a stable yet reactive catalyst that resists deactivation from coordination to heterocycles or charged intermediates. This system enables the use of low catalyst and ligand loadings. Exploiting the differences in nucleophile deprotonation in C-O and C-N coupling reactions catalyzed by Cu·L8 we developed a method to chemoselectively N- and O-arylate a variety of amino alcohol substrates. Employing NaOt-Bu as the base resulted exclusively in C-O coupling when the amino alcohols featured primary alcohols and more hindered amines or aniline groups. Utilizing NaOTMS enabled the ability to override the steric-based selectivity of these reactions completely and exclusively promoted C-N coupling regardless of the structure of the amino alcohol. The ability to invert the observed chemoselectivity is distinct from previously described methods that require protecting group manipulations or rely entirely on steric effects to control reactivity. These results substantially improve the scope of Cu-catalyzed C-N coupling reactions using N1,N2-diarylbenzene-1,2-diamine ligands and introduce a new chemoselective method to arylate amino alcohols.

Self-Assembly of Ultrasmall 3D Architectures of (l)-Acyclic Threoninol Nucleic Acids with High Thermal and Serum Stability.

The primary challenge of implementing DNA nanostructures in biomedical applications lies in their vulnerability to nuclease degradation and variations in ionic strength. Furthermore, the size minimization of DNA and RNA nanostructures is limited by the stability of the DNA and RNA duplexes. This study presents a solution to these problems through the use of acyclic (l)-threoninol nucleic acid (aTNA), an artificial acyclic nucleic acid, which offers enhanced resilience under physiological conditions. The high stability of homo aTNA duplexes enables the design of durable nanostructures with dimensions below 5 nm, previously unattainable due to the inherent instability of DNA structures. The assembly of a stable aTNA-based 3D cube and pyramid that involves an i-motif formation is demonstrated. In particular, the cube outperforms its DNA-based counterparts in terms of stability. We furthermore demonstrate the successful attachment of a nanobody to the aTNA cube using the favorable triplex formation of aTNA with ssDNA. The selective in vitro binding capability to human epidermal growth factor receptor 2 is demonstrated. The presented research presents the use of aTNA for the creation of smaller durable nanostructures for future medical applications. It also introduces a new method for attaching payloads to these structures, enhancing their utility in targeted therapies.

Synthesis of Chiral 1,2-Amino Alcohol-Containing Compounds Utilizing Ruthenium-Catalyzed Asymmetric Transfer Hydrogenation of Unprotected α-Ketoamines.

Herein, we disclose a facile synthetic strategy to access an important class of drug molecules that contain chiral 1,2-amino alcohol functionality utilizing highly effective ruthenium-catalyzed asymmetric transfer hydrogenation of unprotected α-ketoamines. Recently, the COVID-19 pandemic has caused a crisis of shortage of many important drugs, especially norepinephrine and epinephrine, for the treatment of anaphylaxis and hypotension because of the increased demand. Unfortunately, the existing technologies are not fulfilling the worldwide requirement due to the existing lengthy synthetic protocols that require additional protection and deprotection steps. We identified a facile synthetic protocol via a highly enantioselective one-step process for epinephrine and a two-step process for norepinephrine starting from unprotected α-ketoamines 1b and 1a, respectively. This newly developed enantioselective ruthenium-catalyzed asymmetric transfer hydrogenation was extended to the synthesis of many 1,2-amino alcohol-containing drug molecules such as phenylephrine, denopamine, norbudrine, and levisoprenaline, with enantioselectivities of >99% ee and high isolated yields.

Expectations related to home-based telemonitoring of high-risk pregnancies: A qualitative study addressing healthcare providers' and users' views in Norway.

INTRODUCTION: A pregnancy can be evaluated as high-risk for the woman and/or the fetus based on medical history and on previous or ongoing pregnancy characteristics. Monitoring high-risk pregnancies is crucial for early detection of alarming features, enabling timely intervention to ensure optimal maternal and fetal health outcomes. Home-based telemonitoring (HBTM) is a marginally exploited opportunity in antenatal care. The aim of this study was to illuminate healthcare providers' and users' expectations and views about HBTM of maternal and fetal health in high-risk pregnancies before implementation. MATERIAL AND METHODS: To address diverse perspectives regarding HBTM of high-risk pregnancies, four different groups of experienced healthcare providers or users were interviewed (n = 21). Focus group interviews were conducted separately with midwives, obstetricians, and women who had previously experienced stillbirth. Six individual interviews were conducted with hospitalized women with ongoing high-risk pregnancies, representing potential candidates for HBTM. None of the participants had any previous experience with HBTM of pregnancies. The study is embedded in a social constructivist research paradigm. Interviews were analyzed using a thematic approach. RESULTS: The participants acknowledged the benefits and potentials of more active roles for both care recipients and providers in HBTM. Concerns were clearly addressed and articulated in the following themes: eligibility and ability of women, availability of midwives and obstetricians, empowerment and patient safety, and shared responsibility. All groups problematized issues crucial to maintaining a sense of safety for care recipients, and healthcare providers also addressed issues related to maintaining a sense of safety also for the care providers. Conditions for HBTM were understood in terms of optimal personalized training, individual assessment of eligibility, and empowerment of an active patient role. These conditions were linked to the importance of competent and experienced midwives and obstetricians operating the monitoring, as well as the availability and continuity of care provision. Maintenance of safety in HBTM in high-risk pregnancies was crucial, particularly so in situations involving emerging acute health issues. CONCLUSIONS: HBTM requires new, proactive roles among midwives, obstetricians, and monitored women, introducing a fine-tuned balance between personalized and standardized care to provide safe, optimal monitoring of high-risk pregnancies.

Facile Synthesis of Some New Peptidomimetic ß3 -and ß2,3 -Amino Alcohols Possessing Pyridyl Moiety via Reductive Ring Opening of Pyridyl-isoxazolidines.

Regio- and stereoselective 1,3-dipolar cycloadditions of C-(3-pyridyl)-N-phenylnitrone (2) with variedly substituted dipolarophiles (3, 4) were carried out to obtain substituted pyridyl-isoxazolidines (5-8). Reductive cleavage of pyridyl-isoxazolidines (5-8) with ammonium formate, methanol-THF solvents, at ambient temperature, in the presence of Pd/C provided a facile route for the synthesis of ß3 -and ß2,3 -amino alcohols (9-12), with a substitution pattern having pronounced influence on torsional angles. The obtained compounds (9-12) are valuable scaffolds which can be utilized for peptidomimetics. Thus, the present methodology for reductive opening of isoxazolidine ring avoids the disadvantages of using expensive apparatus and hazards involved in the use of hydrogen gas. The preferential formation of amino-alcohols in case of bicyclic isoxazolidines (8a-c), which precludes any recyclization is rationalized by DFT calculations.

The Intramolecular Charge Transfer Mechanism by Which Chiral Self-Assembled H8-BINOL Vesicles Enantioselectively Recognize Amino Alcohols.

The chiral H8-BINOL derivatives R-1 and R-2 were efficiently synthesized via a Suzuki coupling reaction, and they can be used as novel dialdehyde fluorescent probes for the enantioselective recognition of R/S-2-amino-1-phenylethanol. In addition, R-1 is much more effective than R-2. Scanning electron microscope images and X-ray analyses show that R-1 can form supramolecular vesicles through the self-assembly effect of the π-π force and strong hydrogen bonding. As determined via analysis, the fluorescence of the probe was significantly enhanced by mixing a small amount of S-2-amino-1-phenylethanol into R-1, with a redshift of 38 nm, whereas no significant fluorescence response was observed in R-2-amino-1-phenylethanol. The enantioselective identification of S-2-amino-1-phenylethanol by the probe R-1 was further investigated through nuclear magnetic titration and fluorescence kinetic experiments and DFT calculations. The results showed that this mechanism was not only a simple reactive probe but also realized object recognition through an ICT mechanism. As the intramolecular hydrogen bond activated the carbonyl group on the probe R-1, the carbonyl carbon atom became positively charged. As a strong nucleophile, the amino group of S-2-amino-1-phenylethanol first transferred the amino electrons to a carbonyl carbocation, resulting in a significantly enhanced fluorescence of the probe R-1 and a 38 nm redshift. Similarly, S-2-amino-1-phenylethanol alone caused severe damage to the self-assembled vesicle structure of the probe molecule itself due to its spatial structure, which made R-1 highly enantioselective towards it.

Stereoselective Synthesis and Catalytical Application of Perillaldehyde-Based 3-Amino-1,2-diol Regioisomers.

A library of regioisomeric monoterpene-based aminodiols was synthesised and applied as chiral catalysts in the addition of diethylzinc to benzaldehyde. The synthesis of the first type of aminodiols was achieved starting from (-)-8,9-dihydroperillaldehyde via reductive amination, followed by Boc protection and dihydroxylation with the OsO4/NMO system. Separation of formed stereoisomers resulted in a library of aminodiol diastereoisomers. The library of regioisomeric analogues was obtained starting from (-)-8,9-dihydroperillic alcohol, which was transformed into a mixture of allylic trichloroacetamides via Overman rearrangement. Changing the protecting group to a Boc function, the protected enamines were subjected to dihydroxylation with the OsO4/NMO system, leading to a 71:16:13 mixture of diastereoisomers, which were separated, affording the three isomers in isolated form. The obtained primary aminodiols were transformed into secondary derivatives. The regioselectivity of the ring closure of the N-benzyl-substituted aminodiols with formaldehyde was also investigated, resulting in 1,3-oxazines in an exclusive manner. To explain the stability difference between diastereoisomeric 1,3-oxazines, a series of comparative theoretical modelling studies was carried out. The obtained potential catalysts were applied in the reaction of aromatic aldehydes and diethylzinc with moderate to good enantioselectivities (up to 94% ee), whereas the opposite chiral selectivity was observed between secondary aminodiols and their ring-closed 1,3-oxazine analogues.

Kinetics of Strand Displacement Reaction with Acyclic Artificial Nucleic Acids.

Toehold-mediated strand displacement (TMSD) reaction, one of the DNA nanotechnologies, has great potential as s biological programmable platform in the cellular environment. Various artificial nucleic acids have been developed to improve stability and affinity for biological applications. However, the lack of understanding of the kinetics of TMSD reaction among artificial nucleic acids has limited their applications. We herein systematically characterized the kinetics of TMSD reactions with acyclic xeno nucleic acids (XNAs): serinol nucleic acid (SNA), acyclic D-threoninol nucleic acid (D-aTNA), and acyclic L-threoninol nucleic acid (L-aTNA). We found that the strand displacement reactions by D-aTNA and by L-aTNA were highly dependent on temperature. D-aTNA and L-aTNA systems were orthogonal to each other, and chirality of the input can be switched by using SNA as an interface. We also applied TMSD reactions of XNAs to a seesaw gate amplification system which utilizes the orthogonality. This work will contribute to the developments of thermoresponsive and bioorthogonal nucleic acid circuits.

Ring Opening of Glycerol Cyclic Phosphates Leads to a Diverse Array of Potentially Prebiotic Phospholipids.

Phospholipids are the primary constituents of cell membranes across all domains of life, but how and when phospholipids appeared on early Earth remains unknown. Pressingly, most prebiotic syntheses of complex phospholipids rely upon substrates not yet shown to have been available on early Earth. Here, we describe potentially prebiotic syntheses of a diverse array of complex phospholipids and their building blocks. First, we show that choline could have been produced on early Earth by stepwise N-methylation of ethanolamine. Second, taking a systems chemistry approach, we demonstrate that the intrinsically activated glycerol-2,3-cyclic phosphate undergoes ring opening with combinations of prebiotic amino alcohols to yield complex phospholipid headgroups. Importantly, this pathway selects for the formation of 2-amino alcohol-bearing phospholipid headgroups and enables the accumulation of their natural regioisomers. Finally, we show that the dry-state ring opening of cyclic lysophosphatidic acids leads to a range of self-assembling lysophospholipids. Our results provide new prebiotic routes to key intermediates on the way toward modern phospholipids and illuminate the potential origin and evolution of cell membranes.

Chemoselective Esterification of Natural and Prebiotic 1,2-Amino Alcohol Amphiphiles in Water.

In cells, a vast number of membrane lipids are formed by the enzymatic O-acylation of polar head groups with acylating agents such as fatty acyl-CoAs. Although such ester-containing lipids appear to be a requirement for life on earth, it is unclear if similar types of lipids could have spontaneously formed in the absence of enzymatic machinery at the origin of life. There are few examples of enzyme-free esterification of amphiphiles in water and none that can occur in water at physiological pH using biochemically relevant acylating agents. Here we report the unexpected chemoselective O-acylation of 1,2-amino alcohol amphiphiles in water directed by Cu(II) and several other transition metal ions. In buffers containing Cu(II) ions, mixing biological 1,2-amino alcohol amphiphiles such as sphingosylphosphorylcholine with biochemically relevant acylating agents, namely, acyl adenylates and acyl-CoAs, leads to the formation of the O-acylation product with high selectivity. The resulting O-acylated sphingolipids self-assemble into vesicles with markedly different biophysical properties than those formed from their N-acyl counterparts. We also demonstrate that Cu(II) can direct the O-acylation of alternative 1,2-amino alcohols, including prebiotically relevant 1,2-amino alcohol amphiphiles, suggesting that simple mechanisms for aqueous esterification may have been prevalent on earth before the evolution of enzymes.

Rapid Chemical Ligation of DNA and Acyclic Threoninol Nucleic Acid (aTNA) for Effective Nonenzymatic Primer Extension.

Previously, nonenzymatic primer extension reaction of acyclic l-threoninol nucleic acid (L-aTNA) was achieved in the presence of N-cyanoimidazole (CNIm) and Mn2+; however, the reaction conditions were not optimized and a mechanistic insight was not sufficient. Herein, we report investigation of the kinetics and reaction mechanism of the chemical ligation of L-aTNA to L-aTNA and of DNA to DNA. We found that Cd2+, Ni2+, and Co2+ accelerated ligation of both L-aTNA and DNA and that the rate-determining step was activation of the phosphate group. The activation was enhanced by duplex formation between a phosphorylated L-aTNA fragment and template, resulting in unexpectedly more effective L-aTNA ligation than DNA ligation. Under optimized conditions, an 8-mer L-aTNA primer could be elongated by ligation to L-aTNA trimers to produce a 29-mer full-length oligomer with 60% yield within 2 h at 4 °C. This highly effective chemical ligation system will allow construction of artificial genomes, robust DNA nanostructures, and xeno nucleic acids for use in selection methods. Our findings also shed light on the possible pre-RNA world.

Comparative Pharmacokinetic Investigation on Multiple Active Aminoalcohol-Diterpenoid Alkaloids after Single Oral Administrations of Monomers and Aqueous Extract of Fuzi (Aconiti Lateralis Radix Praeparata) by UFLC-MS/MS.

To further study the aminoalcohol-diterpenoid alkaloids (ADAs) in Fuzi (Aconiti Lateralis Radix Praeparata), a simple and sensitive UFLC-MS/MS method was established and validated for the determination of five ADAs, aconine, mesaconine, hypaconine, deoxyaconine and fuziline, in rat plasma to compare the pharmacokinetic characteristics of pure ADAs and Fuzi decoction. After precipitating protein with methanol, plasma samples were isolated at 0.5 mL/min flow rate on Waters Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm). The mobile phase was composed of 0.1% formic acid-water and methanol with gradient elution. Mass spectrometric inspection was conducted on a 5500 UFLC-MS/MS system with an electrospray ionization source in patterns of positive ion and multiple reaction-monitoring (MRM). All calibration curves were proved to have acceptable linearity (r2 > 0.99) in linear ranges. Intra-day and inter-day precision and the accuracy met the requirements. The matrix effects of all analytes were between 85% and 115% of three concentration levels. This method has been under verification for comparative pharmacokinetic research after oral administration between aqueous extract of Fuzi and single pure ADAs. The results demonstrated that there are evident pharmacokinetic discrepancies between them, and administration in the extract form instead of pure form may contribute to higher absorption.

New isoxazolidinyl-based N-alkylethanolamines as new activators of human brain carbonic anhydrases.

Carbonic anhydrases (CAs) are widespread metalloenzymes which catalyse the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and a proton, relevant in many physiological processes. In the last few years, the involvement of CA activation in different metabolic pathways in the human brain addressed the research to the discovery of novel CA activators. Here, a new series of isoxazoline-based amino alcohols as CA activators was investigated. The synthesis and the CA activating effects towards four human CA isoforms expressed in the human brain, that are hCAs I, II, IV and VII, were reported. The best results were obtained for the (methyl)-isoxazoline-amino alcohols 3 and 5 with KA values in the submicromolar range (0.52-0.86 µM) towards hCA VII, and a good selectivity over hCA I. Being hCA VII involved in brain function and metabolism, the newly identified CA activators might be promising hit compounds with potential therapeutic applications in ageing, epilepsy or neurodegeneration.

Multicomponent Domino Cyclization of Ethyl Trifluoropyruvate with Methyl Ketones and Amino Alcohols as A New Way to γ-Lactam Annulated Oxazacycles.

A new route to bicyclic γ-lactams was found, which was proposed as a three-component cyclization of ethyl trifluoropyruvate with methyl ketones and 1,2-, 1,3-amino alcohols. As a result, a series of trifluoromethyl-substituted tetrahydropyrrolo [2,1-b]oxazol-5-ones and tetrahydropyrrolo[2,1-b][1,3]oxazine-6-ones was synthesized, in which the substituent at the nodal carbon atom was varied. The introduction of a twofold excess of ethyl trifluoropyruvate in reactions with amino alcohols and acetone made it possible to obtain the same bicycles, but functionalized with a hydroxyester fragment, which are formed due to four-component interactions of the reagents. Transformations with 2-butanone and aminoethanol lead predominantly to similar bicycles, while an analogous reaction with aminopropanol gives N-hydroxypropyl-2,3-dihydropyrrol-5-one. Almost all bicycles are formed as two diastereomers, the structure of which was determined using 1H, 19F, 13C NMR spectroscopy, including two-dimensional experiments and XRD analysis. A domino mechanism for the formation of tetrahydropyrrolo[2,1-b]oxazacycles was proposed, which was confirmed by their stepwise synthesis through the preliminary preparation of the aldol and bis-aldol from ethyl trifluoropyruvate and methyl ketones.

Stereoselective Synthesis and Antiproliferative Activity of Steviol-Based Diterpene 1,3-Aminoalcohol Regioisomers.

A series of novel diterpene-type 1,3-aminoalcohols and their regioisomers have been synthesised from natural stevioside in a stereoselective manner. The key intermediate ß-keto alcohol was prepared using Wagner-Meerwein rearrangement of the epoxide derived from steviol methyl ester. The primary aminoalcohol was formed via Raney-nickel-catalysed hydrogenation of an oxime, and a versatile library of aminoalcohols was synthesised using a Schiff base with the primary amines. The aminoalcohol regioisomers were prepared from the mesylate of the ß-keto alcohols. The corresponding primary aminoalcohol was formed via the palladium-catalysed hydrogenation of hydroxyl-azide, and click reactions of the latter were also carried out. The new compounds were characterised using 1D- and 2D-NMR techniques and HRMS measurements. The in vitro investigations showed high inhibition of cell growth in human cancer cell lines (HeLa, SiHa, A2780, MCF-7 and MDA-MB-231) in the case of naphthalic N-substituted derivatives. The antiproliferative effects were assayed using the MTT method.

Dynamic Covalent Optical Chirality Sensing with a Sterically Encumbered Aminoborane.

A sterically encumbered aminoborane sensor is introduced and used for quantitative stereochemical analysis of monoalcohols, diols and amino alcohols. The small-molecule probe exhibits a rigid ortho-substituted arene scaffold with a proximate boron binding site and a triarylamine circular dichroism (CD) reporter unit which proved to be crucial for the observed chiroptical signal induction. Coordination of the chiral target molecule produces strong Cotton effects and UV changes that are readily correlated to its absolute configuration, enantiomeric composition and concentration to achieve comprehensive stereochemical analysis within a 5 % absolute error margin. The sensing method was successfully applied in the chromatography-free analysis of less than one milligram of a crude asymmetric reaction mixture and the advantages of this chiroptical sensing approach, which is amenable to high-throughput experimentation equipment and automation, over traditional methods is discussed.

Non-Covalent Interactions in Molecular Systems: Thermodynamic Evaluation of the Hydrogen-Bond Strength in Amino-Ethers and Amino-Alcohols.

The intramolecular hydrogen bond (intra-HB) is one of the best-known examples of non-covalent interactions in molecules. Among the different types of intramolecular hydrogen bonding, the NH⋅⋅⋅O hydrogen bond in amino-alcohols and amino-ethers is one of the weakest. In contrast to the strong OH⋅⋅⋅N intramolecular hydrogen bond, the strength of the NH⋅⋅⋅O bond can hardly be measured with conventional spectroscopic methods, even for simple amino-alcohols, since the band belonging to the NH⋅⋅⋅O conformer merges with the free OH band. In this work, we developed a combination of G4 calculations, and a method based on experimental vaporization enthalpies to determine the NH⋅⋅⋅O hydrogen bonding strength. The archetypal compounds for this study are 2-amino-1-ethanol and 3-amino-1-propanol as well as their respective methoxy analogs. Based on these molecules, different series were studied to investigate various factors influencing NH⋅⋅⋅O intra-HB strength. In the first series, the influence of alkylation near the hydroxy or methoxy group and the amino group in sterically hindered aminoalcohols was examined. In the second series, the influence of alkylation of the amino-group was investigated. In the third series, the effect of extending the alkyl chain between functional groups was studied.