Evaluating the reducing power of 3-coordinate T-shaped Ni

The reactivity of (PNP)Ni^I, where PNP = (^tBu₂PCH₂SiMe₂)₂N, with oxidants was evaluated. Towards the nitroxyl TEMPO, a 1:1 adduct is formed which was shown to have η²-TEMPO bound through both N and O, with the consequence that one P of the PNP ligand is displaced, leaving the pincer ligand bidentate to Ni^II. DFT calculations show that the bidentate character of TEMPO is due to steric clash between ^tBu and TEMPO ring methyl groups. Reaction of (PNP)Ni with I₂, Br₂, C₂Cl₆ and even CH₂Cl₂ all yield (PNP)Ni^IIX, but never (PNP)Ni^IIIX₂. Excess Br₂ instead oxidizes one phosphorus, yielding the zwitterion [(Br^tBu₂PCH₂SiMe₂)N(SiMe₂CH₂P^tBu₂)]NiBr₂, whose structure is determined. DFT calculation of the species (PNP)Ni^III(Br)₂ yields reaction thermodynamics which show the reason for its absence, and also shows the low BDE of its Ni-Br bond. (PNP)Ni slowly catalyzes the polymerization of HCCR (R = H or Ph), but gives no detectable conversion to a new alkyne-derived nickel complex.     

Synthesis and reactivity of tin amide complexes

A terminal tin amide and a terminal tin anilide complex, (BDI)SnN(iPr₂) and (BDI)SnN(H)Ar (Ar = 2,6-ⁱPr₂C₆H₃), respectively, have been synthesized utilizing the bulky β-diketiminate ligand, [{N(2,6-ⁱPr₂C₆H₃)C(Me)}₂CH]⁻, or BDI, to stabilize the low coordinate divalent tin metal center. Only (BDI)SnN(iPr₂) reacts with phenylacetylene to yield (BDI)SnCCPh, but both species react with methyl triflate to give (BDI)SnOTf and carbon dioxide, resulting in the formation of (BDI)SnOC(O)N(iPr₂) and (BDI)SnOC(O)N(H)Ar.     

Selectivity of attack on a Si-C(sp) sigma bond coordinated to Ni

(PNP)Ni⁺ (as its ( salt) adds PhCN to Ni, but HX cleaves the Si-CH₂ bond to form Ni[η²⁻(^tBu₂PCH₂SiMe₂)N(H)(SiMe₂X)][η²-CH₂^tBu₂P]⁺, for X = OMe, piperidyl, N(H)CH₂Ph, N(H)Ph, morpholinyl. The diprotic reagent H₂O gives (η²-^tBu₂PCH₂SiMe₂OSiMe₂NH₂)(η²-^tBu₂CH₂P)Ni⁺. RCCH (R = Ph, SiMe₃, ^tBu) reacts, through three detected intermediates, to form (^tBu₂PCH₂SiMe₂)N(H)(SiMe₂CH₂^tBu₂PCCR)Ni⁺, a product where one P has been oxidized and Ni reduced, each by two electrons. This shows the dominant influence on reactivity of Si-C bond activation by its unconventional donation to nickel in the structure of (PNP)Ni⁺.     

Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy

We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual ¹⁵N–T ₁ timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s⁻¹. Backbone amide ¹⁵N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D₂O is employed as a solvent for sample preparation. Due to the intrinsically long ¹⁵N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.     

Utilization of the amide groups of asparagine and 2-hydroxysuccinamic Acid by young pea leaves

The fate of nitrogen originating from the amide group of asparagine in young pea leaves (Pisum sativum) has been studied by supplying [¹⁵N-amide]asparagine and its metabolic product, 2-hydroxysuccinamate (HSA) via the transpiration stream. Amide nitrogen from asparagine accumulated predominantly in the amide group of glutamine and HSA, and to a lesser extent in glutamate and a range of other amino acids. Treatment with 5-diazo,4-oxo-L-norvaline (DONV) a deamidase inhibitor, caused a decrease in transfer of label to glutamine-amide. Virtually no ¹⁵N was detected in HSA of leaves supplied with asparagine and the transaminase inhibitor aminooxyacetate. When [¹⁵N]HSA was supplied to pea leaves, most of the label was also found in the amide group of glutamine and this transfer was blocked by the addition of methionine sulfoximine, which caused a large increase in NH₃ accumulation. DONV was not specific for asparaginase, and inhibited the deamidation of HSA, causing a decrease in transfer of ¹⁵N into glutamine-amide, NH₃, and other amino acids. It is concluded from these results that use of the amide group of asparagine as a nitrogen source for young pea leaves involves deamidation of both asparagine and its transamination product HSA (possibly also oxosuccinamate). The amide group, released as ammonia, is then reassimilated via the glutamine synthetase/glutamate synthase system.     

Torsional strain considerations in the mechanism of the proteolytic enzymes,with particular application to carboxypeptidase A

Torsional deformation of the peptide linkage by anti distortion of cis substituents (i.e., forcing groups attached to one side of an amide partial π bond out of plane in opposite directions) leads to rehybridization of the constituent atoms (nitrogen and carbonyl carbon) toward tetrahedral geometry. In consequence the partial π bond is uniquely activated toward trans (antarafacial) addition with defined steric orientation of addends. Application of these considerations to the known structure of an enzyme-substrate complex of carboxypeptidase A leads to a unique mechanistic hypothesis for proteolytic cleavage by this enzyme. Extant evidence concerning the mode of catalysis is considered in light of a mechanism involving electrostatically induced torsional activation of the scissile peptide bond, Lewis acid coordination of zinc to amide carbonyl, proton donation from Glu 270 to the amide nitrogen of the scissile bond, with concerted attack upon the amide carbonyl by solvent water.     

Protein Structure Validation and Refinement Using Amide Proton Chemical Shifts Derived from Quantum Mechanics

We present the ProCS method for the rapid and accurate prediction of protein backbone amide proton chemical shifts - sensitive probes of the geometry of key hydrogen bonds that determine protein structure. ProCS is parameterized against quantum mechanical (QM) calculations and reproduces high level QM results obtained for a small protein with an RMSD of 0.25 ppm (r = 0.94). ProCS is interfaced with the PHAISTOS protein simulation program and is used to infer statistical protein ensembles that reflect experimentally measured amide proton chemical shift values. Such chemical shift-based structural refinements, starting from high-resolution X-ray structures of Protein G, ubiquitin, and SMN Tudor Domain, result in average chemical shifts, hydrogen bond geometries, and trans-hydrogen bond (^h3 J_NC'') spin-spin coupling constants that are in excellent agreement with experiment. We show that the structural sensitivity of the QM-based amide proton chemical shift predictions is needed to obtain this agreement. The ProCS method thus offers a powerful new tool for refining the structures of hydrogen bonding networks to high accuracy with many potential applications such as protein flexibility in ligand binding.     

Amide Proton Spin-Lattice Relaxation in Polypeptides: A Field-Dependence Study of the Proton and Nitrogen Dipolar Interactions in Alumichrome

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the ¹H spinlattice relaxation times (T₁'s) are distinct in the Al³⁺-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of ¹H—¹H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T₁^-1 and Σr^-6 at the three magnetic fields. Considering the very sensitive r-dependence of T₁, the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T₁^-1 is proposed to mainly originate from the ¹H-¹⁴N dipolar interaction: this was supported by comparison with measurements on an ¹⁵N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {¹H}—¹H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the ¹H—¹H dipolar mechanism to cause NOE's. From the slope and intersect values of T₁^-1 vs. Σr^-6 linear plots, a number of independent estimates of τ_r, the rotational correlation time, were derived. These and the field-dependence of the T₁'s yield a best estimate <τ_r> ≈ 0.37 ns, in good agreement with 0.38 ns [unk] <τ_r> [unk] 0.41 ns, previously determined from ¹³C and ¹⁵N spin-lattice relaxation data.     

3D Triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins

Summary Two new 3D ¹H-¹⁵N-¹³C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the ²J_NC coupling to establish the sequential assignment of backbone NH and ¹⁵N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly ¹⁵N- and ¹³C-labelled ribonuclease T1.     

Asparagine formation in soybean nodules

¹⁵NH₄⁺ and [¹⁵N](amide)-glutamine externally supplied to detached nodules from soybean plants (cv. Tamanishiki) were incorporated within nodule tissues by vacuum infiltration and metabolized to various nitrogen compounds during 60 minutes of incubation time. In the case of ¹⁵NH₄⁺ - feeding, the ¹⁵N abundance ratio was highest in the amide nitrogen of glutamine, followed by glutamate and the amide nitrogen of asparagine. In ¹⁵N content (micrograms excess ¹⁵N), the amide nitrogen of asparagine was most highly enriched after 60 minutes. ¹⁵NH₄⁺ was also appreciably assimilated into alanine.     

Conformation-Determining role for the N-acetyl group in the O-glycosidic linkage, α-GalNAc-Thr

An ¹H-nmr study of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-D-galactopyranose (AcGalNAc) glycosylated Thr-containing tripeptides in Me₂SO-d₆ solution reveals two mutually exclusive intramolecular hydrogen bonds. In Z-Thr(AcGalNAc)-Ala-Ala-OMe, there is an intramolecular hydrogen bond between the Thr amide proton and the sugar N-acetyl carbonyl oxygen. The strength of this hydrogen bond will be dependent on the amino acid residues on the Thr C terminal side to some undetermined distance. In Ac-Thr(AcGalNAc)-Ala-Ala-OMe, a different intramolecular hydrogen bond between the sugar N-acetyl amide proton and the Thr carbonyl oxygen exists. The choice of hydrogen bonds seems dependent on the bulkiness of the residues on the Thr N terminal side. The consequence of such strong hydrogen bonds is a clearly defined orientation of the sugar moiety with respect to the peptide backbone. In the former, the plane of the sugar pyranose ring is roughly oriented perpendicularly to the peptide backbone. The latter orientation is where the plane of the sugar ring is roughly in line with the peptide backbone. In both orientations, the sugar moiety can increase the shielding of the neighboring amino acid residues from the solvent. The idea that the amino acid residues near the glycosylated Thr influence orientation of the sugar moiety with respect to the peptide backbone and in turn possibly hinder peptide backbone flexibility has interesting implications in the conformational as well as the biological role of O-glycoproteins.     

Retardation of β-hydrogen elimination in PNP Pincer complexes of Pd

A series of new alkyl and alkoxide (^FPNP)Pd complexes have been synthesized. The alkyls and alkoxides containing β-hydrogens display remarkable thermal stability. Thermal decomposition of (^FPNP)PdOEt is very slow in pure C₆D₆ but is accelerated by the addition of EtOH co-solvent. It is proposed that the β-hydrogen elimination from (^FPNP)Pd-OCH₂R occurs via dissociation of the alkoxide anion.     

Proton uptake mechanism of bacteriorhodopsin as determined by time-resolved stroboscopic-FTIR-spectroscopy

Bacteriorhodopsin's proton uptake reaction mechanism in the M to BR reaction pathway was investigated by time-resolved FTIR spectroscopy under physiological conditions (293 K, pH 6.5, 1 M KCl). The time resolution of a conventional fast-scan FTIR spectrometer was improved from 10 ms to 100 μs, using the stroboscopic FTIR technique. Simultaneously, absorbance changes at 11 wavelengths in the visible between 410 and 680 nm were recorded. Global fit analysis with sums of exponentials of both the infrared and visible absorbance changes yields four apparent rate constants, k₇ = 0.3 ms, k₄ = 2.3 ms, k₃ = 6.9 ms, k₆ = 30 ms, for the M to BR reaction pathway. Although the rise of the N and O intermediates is dominated by the same apparent rate constant (k₄), protein reactions can be attributed to either the N or the O intermediate by comparison of data sets taken at 273 and 293 K. Conceptionally, the Schiff base has to be oriented in its deprotonated state from the proton donor (asp 85) to the proton acceptor (asp 96) in the M₁ to M₂ transition. However, experimentally two different M intermediates are not resolved, and M₂ and N are merged. From the results the following conclusions are drawn: (a) the main structural change of the protein backbone, indicated by amide I, amide II difference bands, takes place in the M to N (conceptionally M₂) transition. This reaction is proposed to be involved in the “reset switch” of the pump, (b) In the M to N (conceptionally M₂) transition, most likely, asp-85's carbonyl frequency shifts from 1,762 to 1,753 cm^-1 and persists in O. Protonation of asp-85 explains the red-shift of the absorbance maximum in O. (c) The catalytic proton uptake binding site asp-96 is deprotonated in the M to N transition and is reprotonated in O.     

A systematic evaluation of different hydrogen bonding patterns in unsymmetrical 1,n′-disubstituted ferrocenoyl peptides

The synthesis and spectroscopic characterization of 21 l,l′-disubstituted ferrocenoyl peptides of the general formula [Fe(C₅H₄-CO-Aal-OR) (C₅H₄-CO-Aa2-OR′)] is reported, with Aal and Aa2 being different amino acids. The one-pot synthesis from activated ferrocene-l,l′-dicarboxylic acid and two different amino acid esters gives the unsymmetrical ferrocenoyl peptides in yields between 27% and 42%, which can be easily separated from their symmetrical byproducts by column chromatography. All new compounds are comprehensively characterized by mass spectrometry (El and FAB, including high-resolution EI-MS), ¹H and ¹³C NMR, and UV/Vis spectroscopy. CD spectroscopy in conjunction with ¹H NMR is used to elucidate the solution structures. Using the achiral glycine (Gly) as Aal permits to determine qualitatively the structure-determining influence of the different amino acids Aa2. Helically chiral structures in ferrocene amino acids in this study are stabilized by hydrogen bonds. If one hydrogen bond partner is systematically moved away by the introduction of methylene groups, then indeed the strength of the hydrogen bond decreases as indicated by ¹H NMR chemical shifts of the amide protons and the strength of characteristic CD bands. As proline (Pro) is the only naturally accuring secondary amino acid it cannot contribute any amide proton to intra-strand hydrogen bonding. DFT calculations on the compound [Fe(C₅H₄-CO-Gly-OMe)(C₅H₄-CO-Pro-OMe)] with one achiral and one secondary amino acid were therefore performed to quantify the more subtle influence of the relative orientations of the ferrocene carbonyl groups and the cis-/trans-conformation of both amide bonds. Not unexpectedly, the conformations with both amide bonds in cis orientation are highest in energy. Surprisingly, the calculations suggest the presence of a low-energy conformation with a non-classical hydrogen bond between the proline ester carbonyl oxygen and a glycine H_α atom. However, a second conformation with no apparent intra-strand contacts but optimal positioning of all relevant groups is similar in energy. Although two conformations were observed in solution for this compound, the experimental data did not permit to assign those two conformations.     

Synthesis and characterization of metallatranes with phenyl substituents in atrane cage

A new series of mono- and diphenylsubstituted silatranes and boratranes N(CH₂CH₂O)₂(CHR³CR¹R²O)MZ (M = Si, Z = CH₂Cl, CCPh, H, OMenth, R¹, R², R³ = H, Ph; M = B, Z = nothing, R¹, R², R³ = H, Ph) have been synthesized. Both transalkoxylation and stepwise modification of a preformed metallatrane skeleton were used. The chloromethyl derivatives N(CH₂CH₂O)₂(CHRCHRO)SiCH₂Cl (R = H, Ph) react with tert-BuOK under intramolecular cycle expansion to give 1-tert-butoxy-2-carba-3-oxahomosilatranes N(CH₂CH₂O)(CH₂CH₂OCH₂)(CHRCHRO)SiO^tBu (R = H, Ph). The treatment of boratranes N(CH₂CH₂O)₂(CH₂CR¹R²O)B (R¹,R² = H, Ph) with triflic acid and trimethylsilyl triflate results in the products of electrophilic attack at the nitrogen atom. The molecular structures of four silatranes and one boratrane bearing phenyl groups in the atrane skeleton were determined by the X-ray structure analysis.     

The synthesis, characterization, and alkylation of nacnac chromium triflate derivatives

A Cr(III) triflate coordinated by the bulky β-diketiminate ^MeL^iPr (^MeL^iPr = 2,4-pentane N,N′-bis(2,6-diisopropylphenyl)diketiminate) was synthesized from the corresponding bridging iodide complex [^MeL^iPrCr(μ-I)]₂ by ligand substitution and subsequent oxidation with silver triflate (AgOTf). ^MeL^iPr Cr^III(OTf)₂ exhibits rare trigonal bipyramidal geometry about Cr(III). Attempts to alkylate this triflate synthon with 1,4-dilithiobutane (Li(CH₂)₄Li) led to reduction, while reaction with dimethylzinc (ZnMe₂) led to a mono-alkylated product; only the reaction with methyl lithium (MeLi) was successful in generating a dialkyl.     

Organocobalt B12 Models. Structures of trans-bis(glyoximato)(alkyl)(pyridine)cobalt(III), with Alkyl = Me,Et, i-Pr

The crystal structures of the organocobalt complexes, pyCo(GH)₂Me(1), pyCo(GH)₂Et(2) and pyCo(GH)₂Prⁱ(3) (py = pyridine, GH = monoanion of glyoxime) are reported. Compound (1) crystallizes in the space group P2₁2₁2₁ with cell parameters a = 8.508(1), b = 13.586(2) and c = 11.614(6) Å; (2) crystallizes in the space group P2₁2₁2₁ with cell parameters a = 8.448(4), b = 12.164(2) and c = 13.651(2) Å; (3) crystallizes in the space group P2₁/c with cell parameters a = 8.443(7), b = 12.913(2), c = 14.341(2) Å and β = 92.86(4).The three structures have been solved by Patterson and Fourier methods and refined by least squares methods to final R values of 0.045(1), 0.068(2) and 0.057(3) using 1819(1), 1653(2) and 1582(3) independent reflections. The pyCoalkyl fragment shows significant variation of CoN and CoC bond lengths. The latter increase from 2.003(4) to 2.084(9) Å following the increase of the alkyl bulk. The CoN(py) distances increase from 2.064(3) to 2.101(6) Å with the increasing σ-donor power of the alkyl group trans to pyridine. In comparison with cobaloximes having the same axial ligands, pyCo(DH)₂alkyl (DH = monoanion of dimethylglyoxime) does not show significant differences on the pyCo alkyl fragment. CoN axial bond lengths and exchange rates of the axial neutral ligand are consistent for the two series, although changes in bond lengths are detected only when rate constants are from two to three orders of magnitude different.     

Fast hydrogen exchange affects 15N relaxation measurements in intrinsically disordered proteins

Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, ¹H–¹⁵N HSQC, is used to measure the ¹⁵N transverse relaxation rate (R₂), the measured R₂ rate is convoluted with the HX rate (k_HX) and has higher apparent R₂ values. Since the ¹⁵N R₂ measurement is important for analyzing protein backbone dynamics, the HX effect on the R₂ measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing ¹⁵N R ₂ ^CPMG experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R ₂ ^CPMG can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D₂O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R ₂ ^CPMG values be obtained by methods described herein.     

Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,³J_{HN.C 2}H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the C^αH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu⁵]- and [Met⁵]-enkephalinamides.     

N and C NMR determination of allantoin metabolism in developing soybean cotyledons

The metabolism of allantoin by immature cotyledons of soybean (Glycine max L. cv Elf) grown in culture was investigated using solid state ¹³C and ¹⁵N nuclear magnetic resonance. All of the nitrogens of allantoin were incorporated into protein in a manner similar to that of each other and to the amide nitrogen of glutamine. The C-2 of allantoin was not incorporated into cellular material; presumably it was lost as CO₂. About 50% of the C-5 of allantoin was incorporated into cellular material as a methylene carbon; the other 50% was presumably also lost as CO₂. The ¹³C-¹⁵N bonds of [5-¹³C;1-¹⁵N] and [2-¹³C;1,3-¹⁵N]allantoin were broken prior to the incorporation of the nitrogens into protein. These data are consistent with allantoin's degradation to two molecules of urea and one two-carbon fragment. Cotyledons grown on allantoin as a source of nitrogen accumulated 21% of the nitrogen of cotyledons grown on glutamine. Only 50% of the nitrogen of the degraded allantoin was incorporated into the cotyledon as organic nitrogen; the other 50% was recovered as NH₄⁺ in the media in which the cotyledons had been grown. The latter results suggests that the lower accumulation of nitrogen by cotyledons grown on allantoin was in part due to failure to assimilate NH₄⁺ produced from allantoin. The seed coats had a higher activity of glutamine synthetase and a higher rate of allantoin degradation than cotyledons indicating that seed coats play an important role in the assimilation and degradation of allantoin.