C-terminal Proline Research Articles

κB-Ras Binds to the Unique Insert within the Ankyrin Repeat Domain of IκBβ and Regulates Cytoplasmic Retention of IκBβ·NF-κB Complexes

The IkappaBalpha and IkappaBbeta proteins inhibit the transcriptional potential of active NF-kappaB dimers through stable complex formation. It has been shown that inactive IkappaBalpha x NF-kappaB complexes shuttle in and out of the nucleus, whereas IkappaBbeta x NF-kappaB complexes are retained exclusively in the cytoplasm of resting cells. The biochemical mechanism underlying this functional difference and its consequences are unknown. Although the two IkappaB proteins are significantly homologous, IkappaBbeta contains a unique 47-amino acid insertion of unknown function within its ankyrin repeat domain. In this study, we assess the role of the IkappaBbeta insert in regulating cytoplasmic retention of IkappaBbeta.NF-kappaB complexes. Deletion of the IkappaBbeta insert renders IkappaBbeta x NF-kappaB complexes capable of shuttling between the nucleus and cytoplasm, similar to IkappaBalpha x NF-kappaB complexes. A small Ras-like G-protein, kappaB-Ras, participates with the IkappaBbeta insert to effectively mask the NF-kappaB nuclear localization potential. Similarly, a complex between NF-kappaB and a mutant IkappaBbeta protein containing four serine to alanine mutations within its C-terminal proline, glutamic acid, serine, and threonine-rich sequence exhibits nucleocytoplasmic shuttling. This suggests a phosphorylation state-dependent role for the C-terminal proline, glutamic acid, serine, and threonine-rich sequence of IkappaBbeta in proper localization of IkappaBbeta x NF-kappaB complexes. These results are consistent with structural studies, which predicted that binary IkappaBbeta x NF-kappaB complexes should be capable of nuclear translocation, and with previous observations that hypophosphorylated IkappaBbeta.NF-kappaB complexes can reside in the nucleus.

Oct2 transcription factor of a teleost fish: activation domains and function from an enhancer

Oct2 transcription factors of the catfish ( Ictalurus punctatus) are expressed as alternatively spliced α and β isoforms. Functional analysis revealed an N-terminal glutamine (Q)-rich transactivation domain common to both isoforms of catfish Oct2. A C-terminal proline, serine, threonine (PST)-rich activation domain was identified exclusively in the β isoform. Activation domains of fish and mammalian Oct2 showed cell type- and species-specific activity correlated with their biochemical composition (Q-rich vs PST-rich). In contrast the activation domains of the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator of fish and mammals showed no correlation of activity with biochemical composition or species of origin. Although isolated catfish Oct2 activation domains were unable to drive transcription from a site 1.9 kb distal to the promoter, Oct2β activated transcription from both an IgH enhancer and an array of octamer motifs at this distal position. The properties of catfish Oct2 activation domains differ depending on whether they are studied in isolation or as components of the intact transcription factor.

Cloning of a prolidase gene from Aspergillus nidulans and characterisation of its product

Using EST sequence information available from the filamentous fungus Aspergillus nidulans as a starting point, we have cloned the prolidase-encoding gene, designated pepP. Introduction of multiple copies of this gene into the A. nidulansgenome leads to overexpression of an intracellular prolidase activity. Prolidase was subsequently purified and characterised from an overexpressing strain. The enzyme activity is dependent on manganese as a cofactor, is specific for dipeptides and hydrolyses only dipeptides with a C-terminal proline residue. Although these proline dipeptides are released both intracellularly and extracellularly, prolidase activity was detected only intracellularly.

Combination of silyl carbamate and amino acid fluoride for solid-phase peptide synthesis

Diketopiperadine (DKP) formation is an often encountered side reaction in the synthesis of peptide having C-terminal proline ester. A novel strategy for the avoidance of this side reaction was developed, which utilizes Pfp ester of Tsoc-amino acid and Fmoc-amino acid fluoride as the second and the third amino acid, respectively. This strategy was applied to the synthesis of 37–53 fragment of the β-chain of human chorionic gonadotropin (hCG).

Ion trap tandem mass spectrometry of intact GTP-binding protein γ-subunits

Tandem mass spectrometry (MS), including both MS2 and MS3 product ion scans, has been applied for the structural characterization of intact γ-subunits of heterotrimeric GTP-binding proteins (G-Proteins). Intact γ-subunits were ionized by electrospray ionization and fragmented by collision-induced dissociation in a quadrupole ion trap instrument. Extensive fragmentation was observed for the multiply charged precursor ions of these 7–8 kDa proteins. Spectral interpretation was facilitated by loss of the labile isoprenyl modification and fragmentation at C-terminal proline residues. General rules for interpretation of product ion spectra were developed for determination of sequence and posttranslational modification. Second generation product ion scans were shown to be valuable for assisting in spectral interpretation. Analysis of charge state effects on product ion spectra indicated nearly identical fragmentation patterns with differing charges on resulting product ions and provided confirmation of product ion structure assignments. This approach can be used to identify specific γ-subunits and to determine the structures of posttranslational modifications without the need for proteolytic peptide mapping.

Doxycycline-induced inhibition of prolidase activity in human skin fibroblasts and its involvement in impaired collagen biosynthesis

Several lines of evidence suggest that doxycycline, a semi-synthetic derivative of tetracycline, may be a useful agent in the treatment of osteoarthritis. It inhibits collagen synthesis and collagenase activity in hypertrophic chondrocytes, slowing the process of collagen turnover. However, the mechanism of doxycycline-induced inhibition of these processes has not been established. We considered prolidase, an enzyme involved in collagen metabolism, as a possible target for the doxycycline-induced inhibition of collagen synthesis. Cultured human skin fibroblasts, specialized for collagen synthesis, were used as model cells. Prolidase [E.C. 3.4.13.9] is a manganese-dependent cytosolic exopeptidase that cleaves imidodipeptides containing C-terminal proline, thus providing large amounts of proline for collagen resynthesis. Enzyme activity is regulated through the β 1 integrin receptor. Therefore, we compared the effect of doxycycline on prolidase activity and expression, collagen biosynthesis, gelatinolytic activity and β 1 integrin expression in 24-h treated cultured human skin fibroblasts. We found that doxycycline induced coordinately inhibition of prolidase activity and collagen biosynthesis (IC 50 at about 150 μg/ml) and gelatinolytic activity in cultured human skin fibroblasts. The inhibitory effect of doxycycline on the processes was not due to the cytotoxicity of this drug, as shown in the cell viability tetrazoline test. However, an inhibitory effect of the drug on DNA synthesis was observed (IC 50 at about 100 μg/ml). The decrease in prolidase activity in fibroblasts treated with doxycycline was not accompanied by any differences in the amount of prolidase or β 1 integrin recovered from these cells, as shown by Western immunoblot analysis. This suggests that the doxycycline-induced down-regulation of prolidase is a post-translational event. The data presented here raise the possibility that the doxycycline-induced decrease in collagen biosynthesis is mostly due to the inhibition of prolidase activity.

Self-Assembly of Dynamin

Dynamin is a large GTPase essential for various intracellular processes such as synaptic vesicle recycling, caveolae internalization and trafficking into and out of the Golgi. It is also involved in receptor-mediated endocytosis, and is believed to assemble at the necks of clathrin-coated pits and assist in pinching vesicles from the plasma membrane upon GTP binding and hydrolysis.Purified recombinant dynamin self-assembles into rings and spirals in low salt conditions [1]. A dynamin mutant lacking the c-terminal proline rich domain (APRD) also assembles into rings and spirals, however unlike wild type dynamin APRD constricts in the presence of GTP analogous such as GMP-PCP [2] or GTPγS. to explore differences in the behavior of the wild type and mutant dynamin we dialyzed them into different salt solutions containing various types of nucleotides and studied their assembly over time using negative staining and cryo-TEM.

Cutting edge: distinct motifs within CD28 regulate T cell proliferation and induction of Bcl-XL.

CD28 provides an important costimulatory signal in T cell activation that regulates multiple cellular processes including proliferation and survival. Several signal transduction pathways are activated by CD28; however, the precise biochemical mechanism by which CD28 regulates T cell function remains controversial. Retroviral gene transfer into primary T cells from TCR-transgenic, CD28-deficient mice was used to determine the specific sequences within CD28 that determine function. Discrete regions of the cytoplasmic domain of CD28 were identified that differentially regulate T cell proliferation and induction of the anti-apoptotic protein Bcl-X(L). Mutation of C-terminal proline residues abrogated the proliferative and cytokine regulatory features of CD28 costimulation while preserving Bcl-X(L) induction. Conversely, mutation of residues important in phosphatidylinositol 3-kinase activation partially inhibited proliferation but prevented induction of Bcl-X(L.) Thus the ability of CD28 to regulate proliferation and induction of Bcl-X(L) map to distinct motifs, suggesting independent signaling cascades modulate these biologic effects.

Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47phox in the association with p40phox.

The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox), and p40(phox) as well as by the Rac1-Rho-GDI heterodimer. The regulation is a consequence of protein-protein interactions involving a variety of protein domains that are well characterized in signal transduction. We have studied the behavior of the NADPH oxidase cytosolic factors in solution using small angle neutron scattering and gel filtration. p47(phox), two truncated forms of p47(phox), namely, p47(phox) without its C-terminal end (residues 1-358) and p47(phox) without its N-terminal end (residues 147-390), and p40(phox) were found to be monomeric in solution. The dimeric form of p67(phox) previously observed by gel filtration experiments was confirmed. Our small angle neutron scattering experiments show that p40(phox) binds to the full-length p47(phox) in solution in the absence of phosphorylation. We demonstrated that the C-terminal end of p47(phox) is essential in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich region of p47(phox). The radii of gyration observed for p47(phox) and the truncated forms of p47(phox) (without the C-terminal end or without the N-terminal end) show that all these molecules are elongated and that the N-terminal end of p47(phox) is globular. These results suggest that the role of amphiphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(phox)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).

The product of ORF III in cauliflower mosaic virus interacts with the viral coat protein through its C-terminal proline rich domain.

Using the yeast two-hybrid system, we show that the ORF III product of cauliflower mosaic virus (pIII) interacts through its C-terminus with the viral coat protein. The last five amino acids of pIII were essential for the interaction and virus infectivity. Deletion of the last three amino acids or the mutation F129A decreased the strength of the interaction by 90%. We further show that pIII is closely associated with virus particles found in the inclusion bodies of infected plants but not in viral particles released from the inclusion bodies by urea treatment.

The mechanism for anthracycline-induced inhibition of collagen biosynthesis

One of the recognized side effects accompanying anti-neoplastic anthracyclines administration is poor wound healing resulting from impairment of collagen biosynthesis. However, the precise mechanism of anthracyclines-induced inhibition of collagen synthesis has not been established. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracyclines-induced inhibition of synthesis of this protein. Prolidase [EC 3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen synthesis. Therefore, we compared the effect of daunorubicin and doxorubicin on prolidase activity and collagen biosynthesis in confluent cultured human skin fibroblasts. We have found that daunorubicin and doxorubicin coordinately induced the inhibition of prolidase activity (IC50=0.3 and 10 μM, respectively) and collagen biosynthesis (IC50=1 and 15 μM, respectively) in cultured human skin fibroblasts. The inhibitory effect of daunorubicin or doxorubicin on prolidase activity and collagen biosynthesis was not due to anti-proliferative activity of these drugs as shown by cell viability tetrazoline test. The decrease in prolidase activity due to the treatment of confluent cells with the anthracyclines was not accompanied by any difference in the amount of enzyme protein recovered from these cells as shown by Western immunoblot analysis. It may be suggested that the inhibition is a post-translational event. Since prolidase is metalloprotease, requiring manganese for catalytic activity, and anthracyclines are known as chelators of divalent cations, we considered that the chelating ability of anthracyclines might be an underlying mechanism for the anthracyclines-induced inhibition of prolidase activity. In order to determine the ability of daunorubicin or doxorubicin to form complexes with manganese (II), potentiometric method was employed based on the measurement of protonation constant by pH-metric titrated assay. We have found that both anthracyclines form stable complexes with manganese (II). The composition of the daunorubicin–Mn(II) complex was calculated as 3:1 while that of doxorubicin–Mn(II) complex was 2:1. The constant stability value for the investigated complexes were calculated as βav=(1.74±0.01)×1023 for daunorubicin, and βav=(1.99±0.025)×1011 for doxorubicin. The higher ability of daunorubicin vs. doxorubicin to chelate manganese and inhibit prolidase activity may explain the potential mechanism for its greater potency to inhibit collagen biosynthesis.

Early Endosomal Localization of Hrs Requires a Sequence within the Proline- and Glutamine-rich Region but Not the FYVE Finger

Hrs is an early endosomal protein that is tyrosine-phosphorylated in cells stimulated with growth factors. Hrs is thought to play a regulatory role in endocytosis of growth factor-receptor complexes through early endosomes. Early endosomal localization of Hrs seems to be essential for Hrs to exert its function in the endocytosis. Hrs has a FYVE finger domain that binds specifically to phosphatidylinositol 3-phosphate in vitro. The FYVE finger is a likely domain that mediates membrane association of endosomal proteins. In this study, we examined whether the FYVE finger participates in early endosomal targeting of Hrs. Hrs with a zinc binding-defective FYVE finger was still localized to early endosomes. In addition, the N-terminal FYVE finger-containing fragment of Hrs showed a cytosolic distribution in mammalian cells. These results indicate that the FYVE finger is not required for the localization of Hrs to early endosomes. Furthermore, by analyzing a series of deletion mutants of Hrs, we identified a sequence of about 100 amino acids within the C-terminal proline- and glutamine-rich region as a domain essential for the targeting of Hrs to early endosomes.

The mechanism of Daunorubicin-induced inhibition of prolidase activity in human skin fibroblasts and its implication to impaired collagen biosynthesis

One of the recognized side effects accompanying antineoplastic anthracyclines administration is poor wound healing, resulting from impairement of collagen biosynthesis. However, the precise mechanism of anthracyclines-induced inhibition of collagen synthesis has not been established. We have suggested that prolidase, an enzyme involved in collagen metabolism may be one of the targets for anthracyclines-induced inhibition of synthesis of this protein. Prolidase [E.C. 3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen synthesis. We have found that daunorubicin (DNR) induced coordinately inhibition of prolidase activity (IC50 = 0.3 microM) and collagen biosynthesis (IC50 = 1 microM) in cultured human skin fibroblasts. The decrease in prolidase activity due to the treatment of confluent cells with DNR was not accompanied by any differences in the amount of the enzyme protein recovered from these cells as shown by western immunoblot analysis. Since prolidase is metaloprotease, requiring manganese for catalytic activity and anthracyclines are known as a chelators of divalent cations we considered that the chelating ability of anthracyclines may be an underlying mechanism for daunorubicin-induced inhibition of prolidase activity. In order to determine the ability of DNR to form complex with manganese (II), potentiometric method was employed based on the measurement of protonation constant by pH-metric titrated assay. We have found that DNR forms stable complex with manganese (II) and the composition of the complex of DNR with Mn (II) was calculated as 3:1. The constant stability value for the investigated complex was calculated as [beta(av) = (1.74 +/- 0.01) 10(23). The strong ability of DNR to chelate manganese may explain the potential mechanism for inhibition of prolidase activity, subsequently collagen biosynthesis and poor wound healing in patients administered DNR.

Domain Structure and Function of Dynamin Probed by Limited Proteolysis

Dynamin is a 100-kDa GTPase with multiple domains. Some of these have known functions, namely, the N-terminal GTPase domain, the PH domain that binds phosphatidylinositol lipids, and the C-terminal proline–arginine-rich domain (PRD) that binds to several SH3 domain-containing dynamin partners. Others, for example, the “middle” located between the GTPase domain and the PH domain and a predicted α-helical domain located between the PH domain and PRD, have unknown functions. Dynamin exists as a homotetramer in solution and self-assembles into higher-order structures resembling rings and helical stacks of rings. Dynamin self-assembly stimulates its GTPase activity. We used limited proteolysis to dissect dynamin's domain structure and to gain insight into intradomain interactions that regulate dynamin self-assembly and stimulate GTPase activity. We found that the PH domain functions as a negative regulator of dynamin self-assembly and stimulates GTPase activity and that the α-helical domain, termed GED for GTPase effector domain, is required for stimulated GTPase activity.

Recombinant Human DNA (Cytosine-5) Methyltransferase: I. EXPRESSION, PURIFICATION, AND COMPARISON OF DE NOVO AND MAINTENANCE METHYLATION

A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.

Spleen antibacterial peptides: high levels of PR-39 and presence of two forms of NK-lysin.

Antibacterial peptides were isolated from porcine spleen by acetic acid extraction, ion exchange chromatography and reverse-phase high-performance liquid chromatography. C-terminal ladder sequence analysis of a bioactive peptide with matrix-assisted laser desorption/ionization mass spectrometry after digestion with carboxypeptidases P and Y showed that it is identical to the antibacterial proline/arginine-rich intestinal peptide PR-39. It is present at high levels in granulocytes of the spleen, and peptides with C-terminal proline amide and internal adjacent Pro residues can be analyzed with this method. In addition, two forms of NK-lysin (NKL) were found. One, NKLi, is identical to that isolated from pig intestine, and the other, NKLbw, to a mature peptide deduced from a clone from a porcine bone marrow cDNA library.

Binding of proline- and hydroxyproline-containing peptides and proteins to the capillary wall

Sticking of peptides (as demonstrated by peak distortion) containing a high proportion of glycine and proline residues to the capillary wall was investigated. At acid pH where the carboxy terminal proline (e.g. Gly–Pro) is protonated, distinct sticking of these peptides occurred. On the contrary, the Pro–Gly peptide yielded a well shaped peak. At alkaline pH, where the situation was reversed, the peptide sticking most to the wall was Gly–Gly, while the behaviour of the dipeptides possessing N- or C-terminal proline did not suffer from sticking to the wall. It was concluded that the separation of simple proline- and glycine-containing dipeptides can be partly optimized by manipulating the pH of the background electrolyte; particularly if cetyltrimethylammonium bromide, methanol or acetonitrile is added to the run buffer (reversed polarity mode with cetyltrimethylammonium bromide). However, all attempts to resolve more complex mixtures of proline- (and hydroxyproline-) containing di- and tripeptides failed; addition of an organic modifier, i.e. methanol and acetonitrile, hexylamine, triethylamine and cetyltrimethylammonium bromide was unsuccessful. Such separations can be materialized by using simple 25 mmol/l phosphate buffer at pH 10.5 provided that the sample is dissolved in (aqueous) 17.5 mmol/l Brij or 33 mmol/l sodium dodecyl sulfate. These systems are also applicable to large polypeptides rich in proline, hydroxyproline and glycine: typically parent collagen α-chains, their dimers, trimers and higher polymers were successfully separated.

Kinetic Properties of Human Erythrocyte Prolidase and Optimal Conditions for Prolidase Assay by Proline Colorimetric Determination

Prolidase (EC 3.4.13.9) is an iminodipeptidase that catalyses the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. it is involved in intracellular protein degradation, and apparently contributes to the conservation of iminoacides from endogenous and exogenous protein structures. Analysis of prolidase activity in human biological fluids and tissues has gained attention as a biochemical tool in acute and chronic liver diseases, adenocarcinoma of lungs and as a marker for fetal lung maturation and growth, in addition to long known genetic prolidase deficiency. in the present study, stability, activation and inhibition kinetics of human erythrocyte prolidase and optimal conditions for human erythrocyte prolidase assay were investigated. Level of proline, product of the enzymatic reaction was measured by Chinard's method. Human erythrocyte prolidase activity was stable tor six months at -10 °C. Preincubation of the enzyme at 37 °C with 1 mM MnCl2 provided appropriate activity. 
 Preincubation at 45 °C and 55 °C caused enzyme inactivation. After preincubation, prolidase activity was linearly related to incubation time up to at least two hours. Buffer ionic strength between 30-70 mM at pH 8.0 did not cause an obvious difference of prolidase activity. However pH changes between 7.0 and 8.5 caused significant differences with maximal activity at pH 8.0 The Km value tor Gly-L-pro dipeptide was approximately 7.0 mM. EDTA, iodo acetamide and some divalent metal ions (Cu2+, Fe2+, Zn2+, Hg2+) had inhibitory effect on human erythrocyte prolidase activity. According to our results, preincubation at 1 mM MnCl2 concentration for 2 hours at 37°C and incubation at 30 mM Gly-L-pro dipeptide concentration, 40 mM buffer ionic strength, pH 8.0 tor 30 minutes reveals optimal assay conditions for human erythrocyte prolidase. Presence of metal chelating agents and metal ions other than manganese in the reaction media can interfere results, thus should be avoided.

Dual function C-terminal domain of dynamin-1: modulation of self-assembly by interaction of the assembly site with SH3 domains.

Impairment of endocytosis by mutational targeting of dynamin-1 GTPases can result in paralysis and embryonic lethality. Dynamin-1 assembles at coated pits where it functions to cleave vesicles from donor membranes. Receptor endocytosis is modulated by SH3 (src homology 3) domain proteins, which directly bind to dynamin C-terminal proline motif sequences, affecting both the dynamin GTPase activity and its recruitment to coated pits. We have determined that dynamin-dynamin interactions, which are required for dynamin helix formation, involve these same SH3 domain-binding C-terminal proline motif sequences. Consequently, SH3 domain proteins induce the in vitro disassembly of dynamin helices. Our results therefore suggest the the dual function of the dynamin C-terminus (involving amino acids 800-840) permits direct regulation of dynamin assembly and function through interaction with SH3 domain proteins. Additionally, the N-terminal GTPase domain plays an important role in assembly. Finally, we show that the central PH (pleckstrin homology) domain exerts a strong inhibitory effect on the capacity for dynamin-1 self-assembly.

Synthesis of N-phosphoryl amino acids via phosphoramidite amine-exchange

N-Phosphoryl amino acids were prepared rapidly and in high yield via phosphoramidite amine-exchange in the presence of 1 H-tetrazole followed by oxidation with mCPBA. Formation of dibenzyl phosphoramidates incorporating the representative C-terminal protected amino acids leucine, phenylalanine, glutamic acid, and proline were demonstrated to be most successful with dibenzyl N, N-dimethylphosphoramidite although the N, N-diethyl and N, N-diisopropyl analogs also provided high yields.